Mind & Matter

Wide release: Available for free by August 28, 2026, on Apple, Spotify, YouTube, etc.

Early access: Available to paid subscribers here on Substack.

Not medical advice.

Nick talks with Dr. Christopher Walsh about what actually differs between a human brain and a chimpanzee or mouse brain, and why that is still hard to name as a phenotype. Walsh, a neurologist and geneticist at Boston Children’s, walks through scale and association cortex, then the genome-first path: human accelerated regions, genes such as ASPM that can shrink a human brain toward chimpanzee volume, and mosaic mutations that come with building a large cortex.

Forthcoming work he describes finds that about a quarter to a third of late-fetal cortical neurons carry genetic defects, most of them gone by birth, with smaller defects still edited out through adult life. The conversation also covers why the developing brain overproduces neurons, how that editing relates to autism and other highly heritable disorders, and why most human-specific change is in gene regulation rather than new proteins.

ABOUT THE GUEST: Christopher Walsh, MD, PhD is Chief of the Division of Genetics at Boston Children’s Hospital, a neurologist, and an investigator of the Howard Hughes Medical Institute. His lab studies how the human brain develops and how it evolved.

RELATED CONTENT:

* Podcast | Comparative Brain Evolution: Mammals, Primates & Humans | Robert Barton | #171

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PRACTICAL TAKEAWAYS:

* “The brain is done at 25” is an MRI-scale claim. Neurons are mostly in place much earlier. Myelin still comes in through the teens and twenties. Synapses, and some genetically imperfect neurons, keep changing after that.

* A mosaic chromosome finding on IVF or amniocentesis can be limited to the placenta. Walsh’s point is that early embryos are routinely mosaic, and several correction paths exist, including dumping abnormal cells into the placenta.

* Most of the 80–100 new mutations a child carries relative to the parents are inconsequential. Trouble is concentrated in a smaller set of dosage-sensitive genes, which is why most kids still turn out fine.

SUBSCRIBER CONTENT BELOW: Reference paper + episode transcript.

Reference Paper:

* Paper: Genomic approaches for understanding the evolution of the human brain

Full AI-generated transcript below. Beware of typos & mistranslations!

Nick Jikomes

02:17 - 02:25

It’s a pleasure to be here. Where are you calling in from today? And tell everyone a little bit about who you are and what your lab does. Sure.

Christopher Walsh

02:26 - 02:53

So I’m here in Boston, and I am the Chief of the Division of Genetics at Boston Children’s Hospital. And that’s one of about 15 different divisions that take care of all of the different organs in a kid’s body. We take care of kids with rare genetic diseases of all kinds. My background actually is that I am a neurologist.

Christopher Walsh

02:54 - 03:15

and a neuroscientist, so I have a PhD studying the development of the brain. And so I have this clinical role as this chief of genetics, and then I’m also an investigator of the Howard Hughes Medical Institute, so I spend most of my time running a lab of about 25 or 30 people, and we study all different aspects of how the brain develops and also how the brain might have evolved over history.

Nick Jikomes

03:16 - 03:34

Yeah. You’ve definitely been on my list for a while as someone to talk to because I was a pin student at the program in neuroscience over there and I didn’t meet you, but I always kept tabs on your work. But I started out in developmental and evolutionary genetics. So I’ve had this interest in how do genes build the organism and build the brain.

Nick Jikomes

03:35 - 04:05

And you are one of the best people to talk to about the nexus of those fields. And you’ve also got this interesting background where, as you mentioned, In many ways, you’re a basic researcher, but you’re also doing a lot of applied and translational stuff, and you’re at the children’s hospital. So you’re right at the middle of translational stuff, basic stuff, medical and therapeutic stuff, but also really basic and really interesting biology. So, I mean, what do you just think about that, where you sort of sit at the crossroads of all those things?

Christopher Walsh

04:06 - 05:02

Well, you know, I’ve been doing science for a long time, and so things have changed over the years, but some things haven’t changed. And so what I’ve always been fascinated about is this miracle that happens, that a bunch of cells that start out looking just like a bunch of other cells in your body somehow then become endowed with this property of intelligence that leads to our creativity, our sense of self, and our scientific method, our logic, and all of these things, that there’s this miracle where these cells get transformed into a brain and a mind. And so I’ve studied that various ways over the years, first as a developed, when I was a neuroscience student, first as a descriptive process of just where are the cells formed, how are they born, where do they go, how do they get connected, and then increasingly using genetic

Christopher Walsh

05:02 - 05:22

tools. And You can’t study the genetic aspects of the development of the human brain, which I think is the most interesting brain to study in terms of its development. You can’t get involved in that without ending up running into human disease. And my clinical training as a neurologist made me very interested in human disease.

Christopher Walsh

05:22 - 06:14

So I’ve really been at this nexus. For a while, I used to describe our work as using humans as a convenient genetic model system for the mouse, where we could understand, where we could identify genes and mechanisms that we could study then at a mechanistic level. But increasingly now, we’ve gotten interested in the internal dynamics of the developing human brain, taking advantage of people who donate their brains post-mortem to science to try to reconstruct how the human brain develops, and then always relating that to disease. Again, initially pediatric diseases, diseases in children, that’s why I was recruited to Boston Children’s Hospital, and now also looking at how the developmental pattern of our brain might set us up for age-related diseases as well.

Nick Jikomes

06:15 - 06:38

I want to start out pretty high level here, and I want to talk about the adult human brain first before we get into the developmental stuff. And I want to talk about it phylogenetically here. So what makes the human brain unique? I think we all have an intuitive sense for this, but as a neuroscientist studying this, what are the major standout features of the human brain?

Nick Jikomes

06:38 - 06:51

functionally, that we are interested in understanding how they arise in terms of the structures in the human brain that are different or unique or that somehow stand out compared to other species? What are the major things there that stick out?

Christopher Walsh

06:52 - 07:19

Well, Nick, that’s really the challenge of studying the development of the human brain, but particularly the evolution of the human brain. We all have a sense that our brain is unique, that we have cognitive things that we do that other animals don’t do in quite the same way. But we know really so little about the brain in general. So the question of the evolution of the human brain is a challenge in understanding what is the phenotype that we’re trying to understand?

Christopher Walsh

07:20 - 07:55

What are the essential, really the essential differences between human brains and non-human brains? Because there are so many, so there are obvious things. Our brain is three times larger than that of our nearest living relative, the chimpanzee. But in fact, the difficulty in trying to tackle the genetics of the evolution of the human brain is really understanding what these differences are, because we are still at a fairly primitive level at understanding the inner wiring patterns of any brain.

Christopher Walsh

07:57 - 08:20

You know, you could ask, What about the evolution of mice versus rats? You would think those animals have been studied in the lab for over a century. We have all of these genetic strains, but we actually don’t really have a sense of what are the essential phenotypic differences between mice and rats. And so it’s a compounded difficulty when we try to figure out what are the phenotypic differences, what are the unique aspects of the human brain?

Christopher Walsh

08:21 - 08:59

I mean, obviously it’s size. We have areas devoted to language. Other animals that don’t communicate in the same way don’t have such areas and we have a particular aspects of a particular wiring diagram that other animals don’t have but really in many senses every animal is most highly evolved to its own purpose and so As I say, the real challenge in understanding evolution is really trying to figure out, the challenge in genetics in general, is what is the phenotype that you’re trying to understand at a genetic level?

Christopher Walsh

09:00 - 09:22

And it’s that characterization of just the facts that really drives the genetics. And that’s why genetics likes things that are black and white differences, whereas we get into trouble with the human brain because we know so little about the unique aspects of our brain relative to even chimps, much less the brains of our extinct ancestors.

Nick Jikomes

09:23 - 09:55

Right, right. And of course, it’s natural and attractive for people to look at just the sheer size of a brain and say, oh, well, the human brain is bigger than a chimpanzee brain, and size is an easy thing to look at. One of the things that you learn when you study neuroscience that I think is really interesting is when you start to look under the hood, and you look at, say, the neocortex of a mouse versus a chimp versus a human, to the untrained eye, you know, when you dig down to the cytoarchitectural level, the cell level, they actually look quite similar.

Nick Jikomes

09:55 - 10:19

And this sort of brings us to the question of, well, is the human brain just sort of a scaled up version of the other mammalian brains? Is it really mostly about size and number of neurons and number of connections? Or is there other stuff there? And I know it’s a little bit of all the above here, but Can you give people a sense for how scientists like you today are thinking about the question of scale?

Nick Jikomes

10:19 - 10:24

Is it just a matter of scaling up to a human brain, or is there probably quite a bit more to it than that?

Christopher Walsh

10:25 - 10:37

Yeah, that’s a great question, Nick. And it’s certainly a question of scale. I mean, our brain is about 1,000 times the mass of a mouse’s brain. And so that’s a big question of scale.

Christopher Walsh

10:37 - 10:55

And we’re maybe 30, 40, 50 times as big as the brain of a cat or a dog or something like that. And as I say, three times as big as a chimp. So a certain aspect of it is scaling up. And a lot of that scaling up involves the neocortex, the new cortex, which is the largest part of the human brain.

Christopher Walsh

10:55 - 11:12

It makes up about 80% of the volume of our brain. But it’s not just scaled up relative to a mouse, because there’s also a change. So the neocortex basically functions by having different areas of specialization. There’s an area that’s specialized for vision.

Christopher Walsh

11:13 - 11:32

There’s an area that’s specialized for language, areas that are specialized for motor control, and so forth. And then in the human brain, there are a lot of areas that are not obviously so specialized. We call them association cortex. And if we have a stroke in one of our association cortices, many times that stroke might be relatively silent.

Christopher Walsh

11:32 - 12:01

Whereas if we have a stroke in our language area, that’s big trouble for making language. Or if we have a stroke in our motor area, we’ll typically have a very focal paralysis. But the human brain then is not just a bigger version of the mouse, because the mouse their smaller brain is mostly taken up with these primary cortical areas, as we call them. The ones that directly are involved in sensation of the body, particularly in the case of the mouse, or the ones directly involved in vision, ones directly involved in motor.

Christopher Walsh

12:01 - 12:33

Mouse has very little of this so-called association cortex, which is less specialized. And so I think it’s widely, I think I share the belief of many, that it’s the development of these association areas in the human that so dominate compared to other species that may also give us some of our cognitive enhancements and some of our ability to do some of these very unique things, these high-level cognitive functions that humans seem to do so well relative to other species.

Nick Jikomes

12:34 - 13:11

Yeah, I think when we start to think about these things, and you think about what the wiring diagrams look like, and you think about the number of processing steps from the sensory organs to the higher parts of the brain, there are essentially more layers of processing in certain organisms compared to others. So there are layers of processing in our brain in places like these association cortices that are not directly tied to just analyzing the one sensory channel they might be hooked up to, say, like the visual cortex is hooked up to the visual channel, other sensory areas are hooked up to the other channels.

Nick Jikomes

13:11 - 13:31

There’s layers of processing stacked above those sensory layers. And we like to think that this is somehow important for abstraction for taking us away from just pure sensation and bodily stuff and doing more and more abstract things that we know are going to be important for cognitive behavior and complex behavior.

Christopher Walsh

13:32 - 14:12

Yeah, that’s a great point, Nick. So I said that the human has a larger cortex in terms of surface area with these association areas added. However, like you say, most of the cortex is sort of like a great big layer cake with layers that we refer to typically as six layers, numbered from the bottom six to the top number one. And in fact, they mostly form a fairly stereotyped repetitive signaling unit, where the inputs tend to come in from the bottom, there are a series of synaptic relays, and then information goes back out to somewhere else.

Christopher Walsh

14:13 - 15:05

However, as you point out, as we ascend the sort of phylogenetic scale or as we get closer to humans, in fact, the lower layers of the cortex still look pretty similar to other species, and mostly what’s added are additional layers on the top of the cake, and these are actually the layers, the neurons that are formed at later stages of development, and they’re also the neurons that are added at the later stages of ontogeny, or phylogeny, rather. So it’s actually sort of ontogeny recapitulating phylogeny, where these upper layers, layers two, and three, become very large and elaborate in the great apes and in humans. And these then add additional layers of circuits, which are very poorly represented, if at all, in rodents like mice and rats.

Christopher Walsh

15:06 - 15:42

And so, in fact, we have not only a larger cortex with more of these less differentiated areas, we also have, within that same circuit diagram, we’re adding another layer of analysis. And in fact, interestingly, these upper layers, these layers two and three, are preferentially made up of neurons that actually all they do is project to other layers of the cortex. They don’t project out of the cortex to do something like move your arm or move your leg. They seem to just bounce back and forth between the cortex, between different parts of the cerebral cortex proper.

Christopher Walsh

15:42 - 16:01

And so in that sense, they also have the look of neurons that are more involved in these higher level aspects of analysis, which again are greatly elaborated in humans and which are relatively simple in animals where the cortex is both smaller in its surface area and also squatter in its layering.

Nick Jikomes

16:02 - 16:34

Yeah. So I think what you’re saying is there’s layers on layers here. So we can think, and for this whole discussion, it’s going to be important for us to think about individuals and one part of the brain processing one stream of information within one brain, but then how evolution sort of played with areas and layers of processing across species. And I think what you’re saying is you can look at even a species like a mouse and their neocortex or their cerebral cortex is going to have this canonical layered structure to it.

Nick Jikomes

16:35 - 17:01

And it really is, you can kind of analogize it to maybe the hardware in our computers. There are layers of processing. You might have, say, the eyeball sending the brain information about visual stuff, and it reaches the visual cortex, and it really does go through layers of processing, one transformation, another transformation, then another. As it goes through those layers of processing in one area, that whole output can then get sent to another part of the cortex where it might then go through other layers.

Nick Jikomes

17:02 - 17:10

And then when we think about other species, I think what you’re saying is evolution has sort of added more and more layers, mostly on the top side of that, so to speak.

Christopher Walsh

17:11 - 17:44

Yes, exactly right. And so this presumably then is giving functions that really mice don’t really seem to need. And a lot of that might have to do with the fact that we live in such complex groups. Many people have suggested that some of this elaboration of the cortex and the rapid growth of the cortex over the last couple of million years may have correlated with the fact that humans and our immediate ancestors might have been living in much more larger, complex social groups than

Christopher Walsh

17:44 - 17:45

a lot of animals like mice live in.

Nick Jikomes

17:46 - 18:14

So I want to focus a little bit on the adult brain and some developmental questions related to, let’s call it late development, when our brains become mature. And I want to ask you kind of what that even means. So in popular speak, people talk about when the brain matures and when it’s fully developed. And that has a lot of practical implications in terms of how we think about how we relate to each other, laws, and who should be allowed to do what and when.

Nick Jikomes

18:14 - 18:27

The number that I often hear is the human brain becomes fully mature or fully developed or finishes developing at around age 25. Is that true? And what exactly does that mean? Does the brain really stop developing?

Nick Jikomes

18:27 - 18:30

Is there really a time when we can say it’s fully matured?

Christopher Walsh

18:32 - 19:04

Well, I think as a matter of definition, I think that the brain is constantly changing. So in that sense, development is a continuous process that continues throughout life. On the other hand, when we think of development, we think of it somewhat idealistically in terms of Aristotle or something like that, where we go from an unformed embryo to some sort of final product. And so I think common parlance, the term development refers to the formation of something rather than the subsequent aging of that same thing.

Christopher Walsh

19:06 - 19:32

So just a couple of aspects of the development of the brain, you know, there are these, the principal cells are neurons and glial cells. The neurons in our brain are all non-dividing cells. So they all have a birthday just like we do. We define the birthday of a neuron as the day at which it undergoes its final cell division and becomes permanently post-mitotic for the rest of the life of that person.

Christopher Walsh

19:33 - 19:50

And most of our neurons are born actually before our birthday. They’re born during fetal gestation, most of them during the first half of fetal gestation. And then it’s the glial cells that continue to divide at various rates throughout life. And so one way of thinking about development is, when are the neurons born?

Christopher Walsh

19:50 - 20:09

Well, in that sense, development is mostly done in the neocortex by the time of birth. There are a few neurons that are still born after birth. There are a couple of places in the cerebral cortex, like the hippocampus, that form at least some neurons, at least for a few years, if not longer. But most of the neurons are formed relatively early.

Christopher Walsh

20:11 - 20:41

And then we have this phase of glial formation, where glia are developing for years after birth in major ways. And a major sort of a glial development that occurs after birth are glia referred to as oligodendrocytes, which form the myelin insulation on nerve fibers. And so these cells are developing for the first couple of decades still fairly actively. There’s still a lot of formation of myelin through the teenage years.

Christopher Walsh

20:42 - 21:11

And so then most people define development as not being done because a lot of the glia are not yet in place. And so I think that the idea of development being done in the mid-20s is a sense that, well, the neurons are long done, a lot of synapses are formed, the connections between neurons are already formed, and then a lot of the major glia seem to be in place. But there are ongoing changes that continue at all times. The synapses are constantly changing.

Christopher Walsh

21:12 - 21:24

In strength and in number, the glia continue to be added at slower levels, but it’s much harder to detect. So if you do an MRI scan on someone’s brain, it looks like things are pretty well done by the sort of 20s.

Nick Jikomes

21:26 - 21:40

Got it. So there’s different aspects of development unfolding and finishing in different phases. Most of the neurons of the brain are going to end up where they’re going to end up and be there relatively early. But then as you said, there’s these other cells like glia.

Nick Jikomes

21:40 - 22:02

They’re continuing to change and do stuff well into our 20s. And that’s going to include things like making the insulation around neurons. So even though most of the neurons have ended up getting where they’re going to be, there are still these refinements where The installation is changing, and therefore the efficiency of communication is changing. And things are still unfolding into our 20s.

Christopher Walsh

22:04 - 22:18

Yes. I mean, we have a paper that’s coming out at some point. It’s in preprint form and is, in principle, accepted at a good journal. So we think it’ll be out, hopefully, by the end of the year, where we actually look at the development of the neurons.

Christopher Walsh

22:20 - 23:07

And we find that if you look at the, brain of fetuses that have been terminated but were outwardly normal, 25 or 30 percent of the neurons actually have grossly abnormal genomes. And then there’s a wave of elimination of these abnormal neurons in the second half of fetal gestation so that by the time a baby is born, only about 5% of the neurons have these large-scale copy number variants, or gains or losses of large segments of a chromosome. But we find that, in fact, other neurons have small little defects, gains or losses of short segments of the genome, and that’s still pretty common, even in newborns. 10 or 15% of the neurons will have these smaller-level imperfections.

Christopher Walsh

23:08 - 23:40

And we find, in fact, that these imperfect neurons are being edited out of our brain as long as we live. As I say, there’s a wave of elimination of the most abnormal neurons before birth, and then a more gradual elimination of these less abnormal neurons that continues all the way through adult life. And so there seems to be an ongoing competition for survival between neurons. And the weak ones, the weak ones that have an abnormal genome are being constantly eliminated by a competitive process that’s fast prenatally, and that’s slower right on through aging.

Nick Jikomes

23:41 - 23:57

Yeah, yeah. Yeah. So there’s this fascinating aspect to brain development where we start out with more neurons than we end up with. We actually take away a lot of stuff at different phases of development, refine connections, completely get rid of certain neurons.

Nick Jikomes

23:57 - 24:37

And that’s an important part of maturation and the ability for the adult brain to actually end up doing what it’s gonna need to do as an adult brain. And that stuff’s very interesting. When we think about some of these refinements that happen relatively early in development, after birth, This is an area too where you talk about things like critical periods or sensitive periods where there are these waves of time or these spans of time where you either have waves of loss or refinement connections and or somehow some way there’s a period where the type of neuroplastic change and therefore the type of learning that’s possible is different in one phase of development compared to later.

Nick Jikomes

24:37 - 24:52

Probably the most famous example of this in humans is the so-called critical period for language acquisition. Can you just say a little bit at a high level on the idea of critical periods and sensitive periods and what those are and what may be the major ones in humans that are studied?

Christopher Walsh

24:54 - 25:35

Gosh, well, yeah, there’s a lot of work on these critical periods of development. And the terms were first really developed specifically in animal models, looking at the development of vision in animals like cats and mice, because I found that animals immediately after birth would have a great deal of plasticity. If you closed one eye, for example, then the open eye would take over most of the visual part of the brain. And then if you reopened the eye later on, in fact, the eye that had been closed would be really almost completely shut out and would have a great deal of difficulty being seen.

Christopher Walsh

25:35 - 26:23

And this mirrored experiences of ophthalmologists, kids born with amblyopia, where if it was not corrected at an early age, the amblyopic eye would become permanently blind, where if it was corrected early, in fact, that originally abnormal eye could do very well. And so the critical period for that sort of thing seems to last a couple of years in humans, but it’s not like a sharp demarcation between plasticity early on and then a complete lack of plasticity later on, and then critical periods for different types of plastic change in the brain seem to vary. For example, kids who have strokes in their left hemisphere.

Christopher Walsh

26:23 - 26:43

If they have severe damage to their left hemisphere at an early age, they’re able to develop language in the right hemisphere. And that sort of plasticity lasts for a few years. But then again, of course, that’s not true in an adult. If an adult has a stroke in their left hemisphere in the language area, they typically permanently lose language and have relatively little recovery of it.

Christopher Walsh

26:43 - 27:04

So there’s a critical period during which the right hemisphere is actually capable of developing a language center. Even though in most people, the language center only develops on the left hemisphere, but the right hemisphere can stand in if the left hemisphere becomes damaged. And again, that sort of critical period tends to be the first few years of life. Again, it’s not defined.

Christopher Walsh

27:05 - 27:32

These situations are sufficiently uncommon that it’s not defined with tremendous precision and varied, and also depends on a lot of other variables. So we know that the human brain is particularly plastic for these first two, three, four, five years of life. Some people wonder whether that’s why we tend to have very few memories of our early childhood, because our brain is so plastic at that age that we lose those memories. They get overwritten, essentially.

Christopher Walsh

27:32 - 28:00

Or it’s also a phenomenon where children can learn a second language if it’s spoken at their home when they’re very young. But then once they’re in school, they will often lose that language, their ability in that second language if they’re not constantly using it. And so that’s a kind of a plasticity where things can be overwritten. And also it’s a common thing that we have actually relatively few memories of our early childhood.

Christopher Walsh

28:01 - 28:29

So there’s a phenomenon referred to as childhood forgetting. where we know relatively little of what, we have relatively few explicit memories of the very early parts of our life. And so again, some people think that it’s the extreme plasticity of that early brain that gives us a lot of capabilities, like developing a language area and all that, but which also cause us to have some relatively transient memories from that time.

Nick Jikomes

28:29 - 28:58

Yeah, and so when we think about the development of the human brain, the size and the structure of the human brain compared to other species, naively, you might think, well, bigger is better. More synapses mean more information, more memory capacity, more abilities, and so forth. And yet, when we study development, we see that there’s this overabundance of neurons and connections that get pruned away, that get taken away. A lot of this stuff is tied to this early developmental sensitive period stuff.

Nick Jikomes

28:59 - 29:16

Before we dig into some of the mechanics and some of the details of your work and others’ work, at a high level, conceptually, why do we think that development sort of has this pattern of generating more than enough and then pruning it back or refining it away? Why not keep all of those connections? Yeah.

Christopher Walsh

29:16 - 29:40

Well, again, that’s a great question, and I’m not sure we know the answer for sure. I’ve been impressed in this work I was just telling you about, which is, again, is very new. And so it’s not published in final form yet. In fact, this over-generation of neurons involves a lot of neurons that are highly error-prone, and their genomes are not in good shape.

Christopher Walsh

29:40 - 30:36

So again, an area of big surprise in the last few years is how error prone the early development of the embryo turns out to be. A lot of this comes from the IVF literature where families would be undergoing in vitro fertilization and then the obstetricians would take one or two of the cells of the embryo and they would want to check it to make sure that it looked good and they would look at the chromosomes under the microscope and they would only want to implant it if that cell that they examined, that sample that they took had normal looking chromosomes. But it would occasionally be the case in IVF where they would examine one or two cells from the blastocyst for the very, very early embryo, and in fact they would find that the cells that they looked at had very abnormal chromosomes, gains or losses of chromosomes, and then

Christopher Walsh

30:36 - 30:53

they would have a puzzle. What do we do? if they had no choice, because if they didn’t have any embryos that looked good, they would implant the embryo anyway, despite the abnormal chromosomes. And in fact, most of those embryos develop perfectly normally, it turns out.

Christopher Walsh

30:54 - 31:13

And the reason is because the blastocyst is dozens of different cells, and they just happen to take cells that are mosaic, where a couple of maybe 10 or 20 percent of the cells in that embryo were very abnormal but most of them are normal and that the abnormal ones are eliminated later on.

Nick Jikomes

31:14 - 31:17

There’s an error correction mechanism baked right into the whole process.

Christopher Walsh

31:17 - 31:36

Yeah, so it looks like our bodies have these error correction mechanisms. I mean, the question for us is, why does nature do that? Why does nature create all those errors and then correct them? We don’t know if that’s a deliberate process or it’s impossible to be perfect.

Christopher Walsh

31:38 - 31:56

But we have then built into our body error correction mechanisms. Of course, you know, these abnormal cells would turn into cancer or something if they weren’t also eliminated during normal development. Turns out neurons then also have an error correction process. Their error correction process looks like it relies on the formation of connections with other neurons.

Christopher Walsh

31:57 - 32:30

Basically, neurons have to synapse or die. Nature overgenerates neurons, and then of the neurons that are formed, she selects the best ones. And it’s possible to see advantages of that process, where there’s a form of Darwinian evolution that’s affecting the development, particularly of our brain, but likely other tissues as well, where basically, instead of just trying to get it exactly right every time, nature finds it easier to just overgenerate and then select.

Nick Jikomes

32:31 - 32:55

Yeah, no, there’s an analogy there with evolution by natural selection at the population level of organisms, right? Where there’s an overabundance produced and only a subset of individuals in any population are actually going to reproduce and pass on their traits. And I want to circle back to some of that stuff. But I want to ask you a little bit about the genetics and the phylogenetics of the human brain compared to other brains.

Nick Jikomes

32:55 - 33:16

So we know the human brain is different in various respects. We’ve got a big neocortex. We’ve got these big brains that are different in various incompletely understood ways from chimps and other mammals. But when we look at the genes here, I want to start asking you about some of the genetic work that informs how the human brain develops compared to other creatures.

Nick Jikomes

33:17 - 33:36

One maybe starting point here for us is if we go back to sort of the early days of genome sequencing and people started to look at genome sequences, how many genes do we have? How big is the genome? And so forth. One of the surprises back in the early days for people was the just the number of genes, the protein coding genes that humans had compared to other creatures.

Nick Jikomes

33:36 - 34:10

A lot of the earlier predictions were, well, we’re humans, we’re going to have a bajillion genes, and we’re going to have way more than chimps and cucumbers and jellyfish and so forth. And one of the surprises there, I think, was that we found that the differences were actually much more modest than maybe we thought before. And so the thinking turned, at least in some schools of thought, to, well, it’s not so much about the number of protein coding genes you have, It’s really about how you regulate them, how you express them and control them throughout development. Can you say a little bit at a high level about what we know about the genetics of human brain development?

Nick Jikomes

34:11 - 34:22

And is it about how we use genes and the regulation of them? Do we have more genes involved in brain development than other creatures? What does the broad picture start to look like here?

Christopher Walsh

34:22 - 34:38

Yeah, right. Yeah, basically, like you said, our genome is about the same size as the genome of a mouse. We both have about 20,000 genes. We have about 3 million base pairs of DNA in the entire genome.

Christopher Walsh

34:39 - 35:27

And that’s only a few more genes than fruit flies or roundworms that have 16,000 or 18,000 genes. There’s a debate on exactly how you define a gene, but I’ll set aside that debate for the time being, because we could talk all day just about what you mean by the term gene. But yes, so most of the genes we have are you can point to the genome of a mouse and you can say, oh yeah, there’s, you know, RAS, there’s ASPM in the mouse, and it looks more similar than different in mice compared to humans. There are, you know, maybe a few, so there are maybe a few dozen, human-specific genes, depending again on a little bit on how you define them, whether they’re duplications of previous genes.

Christopher Walsh

35:27 - 35:43

But basically, in general, there are three ways that evolution can happen. So evolution can add new genes. And it turns out that seems to be relatively uncommon. And that’s probably because it’s hard.

Christopher Walsh

35:43 - 36:01

Nature doesn’t generally invent things out of whole cloth. It’s hard to make a whole new gene that came out of nowhere. And then the second way that evolution can happen is that the same gene can be altered. It can be altered typically in the protein that it makes.

Christopher Walsh

36:01 - 36:32

So you can have alterations to the part of the gene that actually makes the protein, so that you change one amino acid for another amino acid, or you change a run of amino acids. And so that can take an existing protein and change its biochemistry. It might cause it to interact with proteins that it ordinarily doesn’t, or it might add a new function to an existing protein. And that turns out to be also a relatively uncommon way in which evolution happens, although it’s more common than adding a gene outright.

Christopher Walsh

36:33 - 36:57

And then the third way in which evolution can happen, which seems, as far as we can tell, to be by far the most common, is that you can change the way genes are regulated. You can change the cell. You’re not changing what the gene does as much as putting it in a cell that might not ordinarily express that gene. And so by putting it in a cell that ordinarily doesn’t use that gene, it might change what that cell does.

Christopher Walsh

36:58 - 37:21

Or you can be changing the timing or the level of expression, how much gene there is, or when and where it’s expressed in the brain. And that seems to be the most common mechanism. And that’s probably the most common mechanism because if you start tinkering with, because basically most changes to a protein are bad. We call them mutations.

Christopher Walsh

37:21 - 37:45

They cause disease. That’s what we see all the time in our clinics. We see kids that have the unfortunate experience that they have a change in the coding sequence of a gene that encodes a protein, and so consequently they lose the function of that gene and that typically creates a disease. It’s hard, again, to alter a protein in just the perfect way that it does something better.

Christopher Walsh

37:46 - 38:19

And yet, by changing when and where a gene is turned on, you’re not compromising the function of that gene in all of the cells that already use it. The cells that already use it, they still have access to it, and you can just add something onto it. And so this sort of regulatory changes to genes is generally done in regulatory sequences of the gene, which don’t code for the protein itself, but they’re sort of on-off switches that define when and where the protein is provided to a cell. And that seems to be where the bulk of the evolution is happening.

Christopher Walsh

38:20 - 38:32

And, of course, that is the part of the genome about which we also know the least. And so that is what creates the challenge for trying to define what all these evolutionary changes actually are.

Nick Jikomes

38:32 - 38:57

Yeah, yeah. So I see the basic idea here would be, if I’ve got a protein that does an important job, maybe it’s a transcription factor that controls the expression of other genes. Maybe it’s an enzyme that’s critical for a core metabolic process. If I start messing with the protein sequence, the actual meat of that protein, I’m probably going to compromise it rather than improve it, just statistically speaking.

Nick Jikomes

38:57 - 39:23

And if I do that in the protein coding sequence, it’s going to have that effect in every cell. all the neurons it’s in, all of the kidney cells it’s in, all of the whatever cells it’s in. But if we’ve got all of these parts of the genome that are involved in regulation rather than the protein coding part, we can now tinker with those and start to make 10% more of the protein, but maybe just in this cell or keep it on a little bit longer in brain development, but not somewhere else.

Nick Jikomes

39:24 - 39:31

And we can play with things in a more tinkering fashion where we’re just making these finer adjustments, let’s say.

Christopher Walsh

39:32 - 40:00

Exactly. I mean, a typical gene might have 5, 10, 15 different regulatory sequences that control, it’s on and it’s off. And they can be tuned so that their regulatory sequences, like you say, just for kidney cells as opposed to just for neurons. And so then evolution can work on just the neuronal use of that gene and leave alone what it does in another part of the body so that you don’t damage some other organ while you’re trying to improve another.

Nick Jikomes

40:01 - 40:13

And if we kind of connect some of that to human development, human development is long. We develop for longer than other animals. We have these extended childhoods. Our gestation’s pretty long.

Nick Jikomes

40:14 - 40:29

Is there a sense that a lot of human brain development is tied to just extending how long things happen so that you can add more layers of control and layers of neurons? Is a lot of this about just timing and taking our time, so to speak?

Christopher Walsh

40:31 - 40:54

Well, again, it’s hard to untangle, but it seems like that may well be the case. Certainly, our development is much extended compared to that of other species, even chimps. You know, mice develop in three weeks until they’re born, and then humans, you know, it takes us nine months. And it’s a long time even after the neurons are formed before we’re born.

Christopher Walsh

40:55 - 41:16

And then, as you say, the postnatal development is so gradual, much more gradual than other animals. It’s not totally clear why. We have this neoteny, as we call it, which is basically this slowing and more gradual developmental timetable. It’s not totally clear what the evolutionary advantage of it is.

Christopher Walsh

41:16 - 41:45

This is a very hot area of debate because, of course, in many ways that slow development creates survival challenges because the baby has to be protected from predators for that much longer. And so it must be doing something very essential for the brain for it to have evolutionary value because of, as I say, the potential survival challenges that happens. And so this might... Slow development appears to be associated with a very high level of plasticity.

Christopher Walsh

41:45 - 41:59

These critical periods, again, are much longer in humans than in other species. And so it might be that that gives us advantages. This extended plasticity might give us other advantages in terms of long-term cognitive function.

Nick Jikomes

42:00 - 42:20

And when we think about human brain evolution and we line up the genomes across species, when we look at the human genome and compare it to the chimp genome or whatever other genome you might want to point us to, is there a sense that there’s an enrichment of change that happened in neural development-related genes as opposed to other genes?

Christopher Walsh

42:20 - 42:35

Yes, there certainly is. You know, this is the... So this is the challenge of studying evolution in terms of genomics. as I said at the outset, that we know so little about the phenotype.

Christopher Walsh

42:36 - 42:59

And what are the essential differences in brains that we’re trying to explain? This is the advantage of doing it from the genome-first sort of point of view, because the brain is so complicated. The genome is complicated, but it’s a lot less complicated than the brain. I think of the genome, it’s 20,000 genes, sort of like the size of a small town.

Christopher Walsh

42:59 - 43:17

linear, because it’s digital, it’s finite. Whereas the brain, it’s like 20 billion cells, trillions and trillions of synapses. We don’t know what the key ones are. Rather than a small town is the analogy, it seems more like you need a celestial analogy, like the stars.

Christopher Walsh

43:17 - 43:49

It’s so complicated. So we have very good methods for lining up genes and lining up genomes. And so then what we can do is we can align the species of so many different species now, and we can identify those parts of the genome that are most uniquely human, those that have shown the most accelerated change in DNA, in humans relative to non-human species. So there are a variety of ways of defining these areas that look like they’re particularly evolutionarily important.

Christopher Walsh

43:50 - 44:19

One definition are called human accelerated regions, because they’re areas of accelerated evolution in the human genome relative to others. And then every animal would have these accelerated regions. There would be chimpanzee-accelerated regions that showed accelerated evolution in chimps. There are mouse-accelerated regions that showed accelerated evolution in mice, presumably in each case making changes that are most essential to make that species what it is.

Christopher Walsh

44:20 - 44:46

And in the case of the human-accelerated regions, we know that about half of them have to do with the brain. Many of them having to do with the development of the brain, and then others look like they’re mostly involved in adult brain function. And almost a half of these human accelerated regions are, in fact, these regulatory regions that tend to turn genes on or off. And so that looks like that’s a particularly hot spot for evolution.

Nick Jikomes

44:46 - 45:02

So at a high level, it does look like a lot of where the evolutionary action is for the human brain is in playing with how genes involved in neurons, in some way, how they’re used, how they’re expressed, how much, when, exactly where, that stuff.

Christopher Walsh

45:03 - 45:18

Yeah. Yes, that seems to be the most important thing quantitatively. There are some interesting examples. of genes where there seems to have been evolution on the protein coding sequence.

Christopher Walsh

45:20 - 45:33

An interesting example is one that got me hooked on studying evolution in the first place, right? I mean, I’m a neurologist. I am a doctor. How did I get mixed up in studying evolution?

Christopher Walsh

45:34 - 46:01

Well, we studied kids that had a very small brain called microcephaly, where the size of their brain is reduced to that of chimpanzees. And in fact, not just the size, the shape of the brain in these kids with microcephaly, in fact, takes on a bit more of the shape of a primate-like brain. The sylvia fissure is not as well developed, the language area is not as large, the frontal cortex is not as large relative to the rest of the brain.

Nick Jikomes

46:01 - 46:11

So you’re talking about a genetic abnormality that, at least at the gross level, basically makes someone develop what looks much more like a chimpanzee brain in terms of its overall size and shape.

Christopher Walsh

46:12 - 46:30

Yeah, surprisingly. Like I say, the volume is very close. And the first one of these microcephaly genes that was identified was identified by Jeff Woods at Cambridge. And I was fortunate enough to collaborate with him on that.

Christopher Walsh

46:31 - 46:46

And it’s a gene called ASPM, that’s an abbreviation for abnormal spindles microcephaly gene. And it’s a gene that was first described in the fruit fly where it regulates cell division in stem cells. So that sort of makes sense.

Nick Jikomes

46:46 - 46:51

So we’re talking about one gene here and it’s a gene that other creatures even like fruit flies have.

Christopher Walsh

46:51 - 47:00

Oh, yeah, absolutely. Even nematodes, even C. elegans has this gene. And so this was more than 20 years ago that we identified this gene.

Christopher Walsh

47:00 - 47:30

But as soon as we identified it, we compared the human version to the mouse and the fruit fly and the C. elegans. And we found that, in fact, this ASPM, again, unique or unusual among genes, has very dramatic differences in the protein coding sequence. contains a repeated sequence and all of the repeats start with the same two amino acids, isoleucine and then glutamine.

Christopher Walsh

47:30 - 47:52

And isoleucine is abbreviated I, and glutamine is abbreviated Q. And so these are called IQ repeats. And the human version has 72 repeats, and the mouse version had 55 repeats, and the fly version had 21, and sea aliens had two. And so Jeff said, wow, I mean, now we know that God has a sense of humor.

Nick Jikomes

47:53 - 47:56

Because it’d be so simple. It’s the length of the IQ repeats.

Christopher Walsh

47:56 - 48:15

The length of the IQ repeats that determines how big your brain is. And so it turns out that humans, in fact, mice are a special case. Their short version of the ASPM is unique to the rodent branch of the lineage tree. It’s more conventional for mammals to have the same number of repeats as humans.

Christopher Walsh

48:16 - 49:04

But in fact, then also, if you look at the details of the sequence, In humans compared to non-human primates, ASPM shows an excess of various amino acid altering changes. We call them missense changes in the lineage leading from the great apes to humans. And so it seems like there was not only large scale changes in the encoded protein over the course of more ancient evolution, but actually finer scale changes in the evolution of the protein coding sequence in the last 10 to 15 million years in the evolution of the primates. And so this got me kind of hooked on studying evolution and illustrated to me the tremendous power that human medical genetics can bring to understanding these questions of origin.

Christopher Walsh

49:05 - 49:13

Because humans have mutations now we know in every possible gene and so whatever Aspects of the genome are essential for evolution.

Nick Jikomes

49:13 - 49:36

We can probably learn a lot about it by studying human disease Yeah, and you know this starts to at least get me thinking about some things that I think start to tie together an interesting way here, so you’re talking about this microcephaly phenotype people that are basically born with this unfortunate small brain that looks kind of like a chimpanzee brain. It’s much smaller. It’s not a fully functional human brain.

Nick Jikomes

49:36 - 49:56

It’s a one gene mutation. It’s a gene that we share in common with other creatures, not just primates, but all kinds of other creatures. And it kind of connects the dots a little bit to what you said before around mutations and the risk of mutations. It’s really risky to mutate genes because there’s probably more ways to break things and hurt the development of the organism than to help them.

Nick Jikomes

49:57 - 50:27

And it also gets me thinking about something that you often hear about in evolutionary genetics. So when you’re interested in any given phenotype, any different change you might want to explain, when you think about it in genetic terms, you can sort of imagine, well, there could be one gene of large effect, a super mutation that in that one gene gives you the whole phenotype. And maybe it hurts you like in microcephaly, or maybe in ultra rare cases, you can imagine like a super mutation that really built some cool structure in the human brain.

Nick Jikomes

50:28 - 51:01

On the other extreme, phenotypes are often due to many, many different tiny changes in many different genes. When you’re thinking about brain evolution and studying it in the lab, In general, do we think that it’s more about tinkering and small mutations across many genes to change and shift the developmental process with all of these little changes? Or do we think there could have been major adaptive changes in single genes that sort of suddenly gave us big changes in the human lineage?

Christopher Walsh

51:02 - 51:19

Yeah, so I think there’s an element of both. The big changes, like loss of ASPN, those are the ones that are easier to study. Right? Because loss of ASPM causes a very dramatic phenotype in humans.

Christopher Walsh

51:19 - 51:32

It causes a somewhat, it causes almost no phenotype in mice. If you knock it out, the brain is hardly smaller at all. They seem to rely much less on the gene than humans do. And then we actually knocked it out in ferrets.

Christopher Walsh

51:32 - 52:22

As an animal that has a much larger brain than the mice, it has a much more dramatic phenotype than the mouse, but again, less dramatic than the humans. But here, we, We have, again, dramatic changes in the gene, these subtler changes over the course of evolution, changing one amino acid for another, and then the dramatic disabling of the entire gene in disease states that show us what the gene is essential to do. So basically, we have several examples like that in the study of brain evolution, where the addition of a gene or changes in the gene have been related to dramatic changes in the development of the brain. And so those are the ones that you may have heard about because those are the ones that are particularly robust to study scientifically.

Christopher Walsh

52:23 - 53:11

But again, over the course of evolution, it’s very likely that the tinkering around the edges of many, many genes, hundreds or thousands of genes, is the more important mechanism over the eons. However, these are mechanisms that, at the moment, we’re very limited in being able to study in animal models, because we’re talking about changes between humans and the androthal, or changes between humans and chimps. But these are changes that probably have no meaning for a mouse, because the mouse has such a terribly small cortex to begin with, it probably doesn’t have, you know, these other genes are as likely as not to really have essential functions in mice, so they’re difficult to study in the lab, and so they’re a little harder to nail down in terms of their essential nature.

Christopher Walsh

53:12 - 53:47

And again, that’s where medical genetics comes in handy, because then what you can do is you can look, you know, as a population of eight billion people on the planet, basically we have someone somewhere in the world has a mutation at every single base pair in the human genome. We all have about 80 to 100 mutations relative to our parents. And so if you have 80 to 100 mutations relative to your parents and you have 8 billion people, you do the math, it turns out that means that probably every single base pair in our 3 billion base pair genome has been mutated more than 100 times just in

Christopher Walsh

53:47 - 54:15

the last generation. And so we can then identify the essential roles of these regulatory sequences in the human genome, perhaps most effectively by studying human disease, which tends to make dramatic changes to them associated with disease. And then, of course, if we never find someone with a disease that has an alteration in a particular part of the genome, it probably means that that part of the genome is just not doing anything very important.

Nick Jikomes

54:17 - 54:39

When we’re thinking about human development, human evolution, and we want to study either a disease, something that’s going wrong that has a genetic basis, or we want to study how the human brain became the human brain. What are the changes that led to our brains doing what they do? Let’s say you’re in the lab. You identify a really interesting gene or set of genes.

Nick Jikomes

54:39 - 54:58

You identify variants or mutations that are unique to the human lineage. That’s really interesting, but you can’t do the types of invasive mechanistic detailed studies you can do in model organisms. When you are looking at human variants, how do you actually study them and really tease apart what might be going on there functionally?

Christopher Walsh

54:59 - 55:27

Yeah, so I think that’s really obviously a big technical challenge. So again, we’ve run into that in the study of these human accelerated regions. And so as I mentioned, the human-accelerated regions are the ones which show the most rapid differences in humans compared to non-human primates. And there also tend to be parts of the genome that are otherwise conserved in mammals.

Christopher Walsh

55:27 - 55:51

So they’ll be very similar in many, many different mammals and then suddenly a lot different in humans. We thought, oh, OK, great. Well, maybe some of these human accelerated regions are essential for some of the cognitive activities that we pride ourselves on. So we sequenced human accelerated regions in thousands of kids that had intellectual disability and autism spectrum disorders.

Christopher Walsh

55:51 - 56:14

And we find, in fact, that they’re is an excess of rare mutations in kids with social and cognitive defects in these human axillary regions. It suggests then that some of them do have essential roles in our cognition. But the challenge then is how do we model that in a dish? How do we model that in mice?

Christopher Walsh

56:14 - 56:48

You know, it’s now very common and increasingly useful to take human stem cells and you can differentiate them into a a little piece of brain-like substance. We call them mini-brains or organoids, where you can get them to differentiate into a certain range of human neurons. And so this gives you the ability to study at least isolated neurons in a dish. And so that’s been useful for studying at least some of these changes, because you can see what they’re doing in differentiated human neurons.

Christopher Walsh

56:49 - 57:14

However, these cultured human organoids by no means recapitulate anything remotely like the beautiful complexity of the whole human brain. And so exactly So again, some genes can be well studied in that context, but some can’t because they may be having a function that requires these connections between different parts of the brain or something that can’t be reproduced in the dish.

Nick Jikomes

57:15 - 57:30

Interesting. So I know that some people do studies where they will, say, identify an interesting human variant. Maybe there’s a regulatory mutation in some gene that’s involved in something to do with neurons. And then you can actually put the humanized version of that, say, in a mouse.

Nick Jikomes

57:30 - 57:44

How do those studies work? And how do they work? And how do we interpret them? Do they sometimes, we think, tell us directionally what that variant might be doing in humans, even though we’re putting it in the context of a lab animal like a rodent?

Christopher Walsh

57:45 - 58:23

Yeah, there’s been some really lovely studies that have done that. There’s some beautiful studies by Debbie Silver from Duke, and Frank Poulot from Columbia, and Pierre van der Haag, and also from Belgium, where they’ve basically engineered the mice to have these human-like sequences. And sometimes they will actually cause dramatic changes in the neurons that you can see even in a subset of neurons in the mouse brain. And so again, that is When that is the case, that is a very powerful demonstration that that regulatory sequence or that HAR is doing something really important.

Christopher Walsh

58:23 - 58:52

So Debbie has done some experiments where she’s put one or two HARs, these human version of a regulatory sequence into the mouse. And in a couple of cases, the mouse brain will get bigger. And these are human accelerated regions that look like they’re active in the stem cells. And so it looks like it will be, it doesn’t get, hugely bigger, but it gets a little bit bigger, suggesting that these regulatory regions are programming the stem cells to keep going a little bit longer and generate more neurons.

Nick Jikomes

58:53 - 59:24

Again, the idea here is we’ve got a gene in us, there’s the equivalent gene in a mouse, If you swap in the human regulatory sequence into the mouse regulatory region, you’ve got the same protein being made in the mouse because they’ve got the same gene. You’re now just putting it in a different spot or changing the timing. And in some cases, it does cause more stem cells to grow or to migrate and the brain to get a little bit bigger. So tinkering with sort of when and where the gene is turned on is making the phenotype.

Nick Jikomes

59:24 - 59:24

That’s right.

Christopher Walsh

59:25 - 1:00:12

Yeah, and another approach that a former fellow of mine, Janet Song, who now has her own lab here at Harvard, she’s actually made tetraploid cells with two nuclei, a chimp and a human, and then differentiated them into neurons. And this allows you to have the two genomes in the same cell type. And so if you see changes in gene expression and things like that, you can localize it to, it helps you determine whether that’s due to the specific regulatory changes that are unique to the human evolutionary lineage or not. And as I said, this is a new and very powerful system where you can actually take the tetraploid cells, even though they have two copies of the genome, one chimp and one human, they actually develop into very good looking neurons.

Christopher Walsh

1:00:12 - 1:00:18

So that also gives us another way of looking at how evolutionary changes relate to one another.

Nick Jikomes

1:00:18 - 1:00:49

Yeah. And I want to ask you some questions that I think relate to the more clinical side of what you do in some ways, which is when we look at the human brain and we study its development and we study the ways it’s unique, the ways that it’s bigger and the connections we have and all of the stuff that gives us what we think of as the good things of being human, sometimes it seems like those aspects of our brains also leave us vulnerable to pathologies or psychiatric conditions that maybe other organisms don’t really have as much or aren’t expressed in the same way.

Nick Jikomes

1:00:50 - 1:01:04

Can you say something here at a high level about how some of the changes that were probably good for human evolution to sort of build our big brains the way they are, maybe sort of predisposed us to psychiatric illnesses that humans have to deal with much more than other creatures.

Christopher Walsh

1:01:05 - 1:01:15

Yeah, sure. Yeah. So there are a couple of examples of that. So making a big brain puts a challenge on the stem cells.

Christopher Walsh

1:01:16 - 1:01:38

Because every time a cell divides, it has to copy all those 3 billion base pairs on each copy of the genome. It’s actually one 3 billion from your mom, one from your father. You’ve got 6 billion base pairs of genome that has to be copied precisely every time a cell divides. And so it’s inevitable in that process that some errors will come about.

Christopher Walsh

1:01:39 - 1:02:20

And so every time a cell divides, it creates the two daughter cells have mutations that we call somatic mutations or mosaic mutations. And so these are much less of a problem in mice because mice only take a couple of cell divisions to generate the whole animal. But with our larger brain, we’re more subject to these mosaic mutations than other species are. And so we find that mosaic mutations are an important, if they occur in a particular gene, they can cause intractable childhood epilepsy due to just a little clone of cells that have developed an abnormal mutation in a cancer gene that causes those neurons to be excessively active.

Christopher Walsh

1:02:20 - 1:02:44

And you can actually do surgery to remove that patch of the brain to cure the epilepsy. And then an even more common type of epilepsy is in the temporal lobe. And this is a part of the brain called, it localizes to a part of the brain called the hippocampus. And this is the part of the cerebral cortex I mentioned that continues to undergo cell division and proliferation for several years after birth.

Christopher Walsh

1:02:45 - 1:03:15

And this temporal lobe epilepsy is the most common epilepsy in adults. and it arises just from the hippocampus. And so we find, in fact, those patients often will get surgery to remove one hippocampus to cure their epilepsy, and we find that those cases typically will have also a mosaic mutation in an activating cancer-like pathway. And as far as we can tell, these kinds of mosaic disorders don’t occur in other species.

Christopher Walsh

1:03:15 - 1:03:39

We have some reason to think that there might be mosaic causes that contribute to schizophrenia as well, which is, again, a disease that we’re not clear that it actually exists outside of humans. And then also, of course, age-related things like Alzheimer’s disease. They also involve these somatic mutations, particularly in the cells in the brain that continue to divide throughout life, like some of the glial cells.

Nick Jikomes

1:03:39 - 1:04:02

Yeah. So the double-edged sword here at a very high level is you’ve got so many neurons and so many connections, and you can use that for really useful stuff, all those connections, all those possibilities. The flip side of that might be that if you’ve got too many connections or they’re not pruned properly, or you don’t refine and mold them properly, that can leave you vulnerable to other things like epilepsy.

Christopher Walsh

1:04:03 - 1:04:32

Exactly right, yeah. And some of these epileptic mutations, they occur in genes that often will cause cancer in a dividing cell, but neurons are not dividing. So there’ll be familiar cancer mutations, like the RAS pathway that’s been in the news lately because of the new RAS inhibitors, or the mTOR pathway, another common cancer pathway. But the mutations occur in the neurons, and neurons don’t divide, so they can’t form cancer, they just become really bad neighbors.

Christopher Walsh

1:04:32 - 1:04:47

They become hyperactive and epileptogenic. And so, again, these are problems that only happen if the mutation occurs during the developmental process when the neurons are formed, which, as I said, is mostly going on early in life.

Nick Jikomes

1:04:47 - 1:05:01

Yeah. And I want to go back to that early development and talk about the mechanics underlying this refinement process that’s so important. So neurons have to be born. The neuron has to divide the number of times it’s going to divide until it gets to its mature state.

Nick Jikomes

1:05:02 - 1:05:15

That neuron has to get where it’s going to end up in a human brain region. It’s got to hook up to other neurons, not too much, not too little, just the right way, whatever that means. And it’s also got to be working properly, right? The cell can’t have extra chromosomes or copy number variants.

Nick Jikomes

1:05:16 - 1:05:27

It can’t be messed up in some other way. So it seems like there’s probably checkpoints that these neurons go through. And a lot of them don’t make it through, as you mentioned earlier. There’s this huge refinement that happens early in development.

Nick Jikomes

1:05:28 - 1:05:44

We lose whole neurons. We prune away connections. And if we don’t do that properly, that’s probably what underlies, at least in part, some of the neurological and psychiatric issues that humans can end up with. I want to dig into sort of how that refinement happens and what those checkpoints are.

Nick Jikomes

1:05:44 - 1:06:02

You mentioned earlier that this concept of copy number variance, the wrong number of chromosomes, the wrong number of genes. What are some of those things that can go wrong and when and where do they actually go wrong? And then from there, I guess we’ll talk about what the checkpoints are and how the brain selects the ones that are working right.

Christopher Walsh

1:06:03 - 1:06:30

Yeah, right, so as you said, there are genetic mutations that disrupt every step of the process of the development of the human brain. The formation of the neurons, we talked about that in the case of microcephaly. There are other conditions where the brain is too big, but not normally functioning. And then the neurons are formed in one place, and then they generally have to migrate to a different place, and there are genetic abnormalities typically associated with epilepsy that disrupt this migration.

Christopher Walsh

1:06:31 - 1:07:19

And then, as you say, all these connections have to be formed and refined. You know, there’s an ongoing theory of things like autism spectrum disorders and perhaps some of the intellectual disabilities as well, is that they can either be defects of the formation of the connections on the one hand, where the connections are not made in the right ways or in the right number, or perhaps more commonly, defects in this competitive elimination. There are studies from a long time ago in humans and then studies in mice as well that suggest that this, this normal pruning process and these critical periods are abnormal in kids on the autism spectrum, that the elimination of neurons is not happening in the right way.

Christopher Walsh

1:07:20 - 1:07:50

Now, exactly how that elimination process happens is really complex. And it involves a large number of genes. We estimate that there are hundreds, very likely more than a thousand different genes that can be mutated to cause what we call autism spectrum disorder. And the vast majority of these hundreds of different genes have roles either in the synapse or in regulating the level of proteins of the synapse.

Christopher Walsh

1:07:51 - 1:08:24

And it looks like you can have defects all up and down this pathway from the synapse into the nucleus to turn the right genes on, and then the genes have to get out of the, then the RNAs have to get out of the nucleus, they have to get to the ribosome, they have to get translated to the right protein at the right level, then those proteins have to be targeted to the right synapse to strengthen that synapse. And so it looks like basically all of those genes are essential, and mutations in many, many different genes can cause this very similar phenotype. And so there are all kinds of mysteries as to how that happens.

Christopher Walsh

1:08:24 - 1:08:50

How does a neuron pick exactly which of its thousands of synapses to strengthen selectively and which to let go? And how is this competitive process managed at a large level? And as I say, there’s a lot we really don’t understand about how that happens. And that’s so critical, really, to understanding how autism develops.

Christopher Walsh

1:08:51 - 1:09:27

Again, it’s a profound mystery about autism that you can have genetic mutations in hundreds of different genes that can cause a similar look in a kid, that can cause similar defects in a child. How do these hundreds of different genes have such a similar phenotype? And yet, then each one of these genes tends to have a range of phenotypes, so that sometimes a mutation in a particular gene or a copy number variant at chromosome 16P11 might cause intellectual disability in one child, autism in another child, might cause no defect in a third child, might cause schizophrenia in a fourth child.

Christopher Walsh

1:09:28 - 1:09:52

And so that’s another mystery, how a single genetic defect can cause a range of phenotypes in the human, or how multiple different genes can cause seemingly indistinguishable ones. And this relates mostly to the fact that our genome does not specify precisely how the brain is going to be made. It instead specifies a historic process. It specifies rules.

Christopher Walsh

1:09:53 - 1:10:05

that govern the interactions between neurons. And that process plays out over an extended period of time. It’s modified by the environment. It’s modified by the teaching of our parents.

Christopher Walsh

1:10:05 - 1:10:12

It’s modified by early intervention. And there are many things that modify it that are not genetic. So the genetics is really important, but it’s not everything.

Nick Jikomes

1:10:13 - 1:10:40

Yeah. And when we think about how this refinement actually happens, when we think about what is the brain actually looking at to decide whether to keep one neuron and not another, or to keep one connection and not another, how are we starting to understand how that happens? So you hinted earlier that You were talking about things like copy number variance. So somehow, you might have too many copies of a gene, you might have too much protein in a cell or something like that.

Nick Jikomes

1:10:41 - 1:11:00

There’s probably mechanisms by which the brain somehow is detecting if there’s too much or too little of something, and then deciding to get rid of the neurons or the connections that have too much or are not working properly. But what do we know there about clear mechanisms where something is being selected for?

Christopher Walsh

1:11:02 - 1:11:31

Again, there are several steps in that. So the early aspects of these connections are largely governed by genetics, gradients of transcription factors, gradients of signaling molecules. And then a second series of formation of connections is largely governed by patterns of spontaneous activity. that neurons tend to just, once they get hooked up, they tend to just start firing in waves.

Christopher Walsh

1:11:32 - 1:11:54

And so those waves tend to be coordinated by neighboring neurons. And so then neurons which are active together tend to mutually strengthen connections that those neurons will find. And then a third phase of connectivity is more governed by interactions with the environment. What things that we actually see that create patterns of activity that strengthen synapses.

Christopher Walsh

1:11:54 - 1:12:24

And so then exactly how the synapses get strengthened is basically a process where when the neurons depolarize, when the neurons are active, they depolarize, meaning their voltage across their membrane changes. And so then this stimulates a cascade. that feeds back, there’s a calcium signaling that feeds back to the nucleus. This turns on genes that are called immediate early genes, genes which CFOS is an example.

Christopher Walsh

1:12:24 - 1:12:53

And so these are active within 30 seconds of the depolarization and they are transcription factors that bind to the DNA and turn on a whole bunch of other downstream genes. And then those genes activate another wave. So there’s actually a series of waves of gene activation that eventually regulate the levels of the synaptic proteins that then strengthen particular synapses and weaken others. And so this whole process is very sensitive to dosage, to levels of gene expression.

Christopher Walsh

1:12:53 - 1:13:36

You have this depolarization, then a sudden shoot up in the level of C-Fos and then upregulation of other genes. And this is probably one reason why a lot of our psychiatric conditions, like autism and schizophrenia, actually don’t reflect complete loss of the gene, like I told you about with ASPN, where both copies are mutated to cause that small brain. But in fact, in autism or schizophrenia, it’s usually just loss of one of the two gene copies, so that the person still has one gene, still has one good copy, but you need to have both copies, and that might have to do with these waves of activation of genes, and these ranges and levels of activation that are so important for refining synaptic connections.

Nick Jikomes

1:13:36 - 1:13:55

Yeah, and I guess at a simple level you can start to think like, okay, dosage can be important because if you’ve got a bit too much protein, a bit too little protein, maybe the synapse that should get pruned is now a little bit too strong, or maybe the one that you wanna keep is a little bit too weak because you don’t have the right sticky connections there at the right dose.

Christopher Walsh

1:13:55 - 1:14:13

Yeah, exactly. And so for some of these dosage-sensitive genes where loss of one of the two copies can cause autism or intellectual disability, in fact, having three copies instead of two is just as bad for you. And so you have to have exactly two copies.

Nick Jikomes

1:14:13 - 1:14:32

Yeah, yeah. It’s very Goldilocks, not too much, not too little. Yes, exactly. When the refinement is actually happening, when the brain somehow decides to get rid of a neuron or to get rid of a synapse between two neurons, what does that disposal process actually look like?

Nick Jikomes

1:14:32 - 1:14:35

How has the synapse gotten rid of and cleared out of there?

Christopher Walsh

1:14:36 - 1:15:22

Yeah, well, so I think we’ve tended to think about, originally we thought of that as a process that was mostly inherent to the neuron, that it would pull in its connections and then maybe sort of shrivel up and die. Now we’re thinking of it as a much more interactive process where, particularly through the work of people like Beth Stevens, they found that some of the glial cells, like microglia, are very involved at nibbling away at the neurons and assaying them in terms of their connectivity, and then helping play a role in targeting certain neurons for either removal of some of their synapses or, in some cases, removal of the entire neuron. And so it’s a much more dynamic and interactive process than it was originally envisioned, I think, 10 or 20 years ago.

Nick Jikomes

1:15:24 - 1:15:43

And so what are some of the major diseases of the brain that you work on in particular that we think have a particularly strong genetic basis? And what does it mean? What does strong genetic basis actually mean? So for whatever example you guide us through here, is it an example where we think it’s all due to a genetic mutation in a gene?

Nick Jikomes

1:15:44 - 1:15:47

Or is it a gene environment interaction? And how do we start to tease those things apart?

Christopher Walsh

1:15:48 - 1:16:16

Right. Diseases have a range of genetic roles, and we often characterize them in terms of the concept of heritability. And heritability was originally defined, and is still most commonly defined, by the study of identical twins. We have identical twins who we think of have an essentially identical genome.

Christopher Walsh

1:16:16 - 1:16:45

There might be a few little changes, but they’re mostly identical. If one child is affected with autism, for example, what’s the risk to the identical twin? Well, it turns out if one child is affected with autism, the identical twin has more than a 50% chance, depending on the series, as high as a 70% or 80% chance of having a disorder that’s on the autism spectrum at least. And so we consider autism to be something with a very high level of heritability.

Christopher Walsh

1:16:46 - 1:16:58

based on those identical twin studies. And then, of course, if they’re fraternal twins, the risk is much lower. It’s more like 15 or 20 percent. And if they’re unrelated, then the rate is, you know, 2 percent or something like that, much lower still.

Christopher Walsh

1:16:59 - 1:17:23

And so basically then we can look at, so autism is one of the most highly heritable neuropsychiatric conditions, probably the most highly heritable. Intellectual disability is similarly highly heritable. Schizophrenia a little bit less so, maybe more like 40% to 60% depending on the particular series. And then things like bipolar or ADHD subsequently a little bit lower.

Christopher Walsh

1:17:23 - 1:17:58

So in something like autism then, we can expect that someday if we have perfect knowledge, we might be able to explain more than half of all kids on the autism spectrum with connecting it to some sort of specific genetic defect. At the moment, we’re nowhere close to being able to do that. If a kid walks into our clinic and has a diagnosis on the autism spectrum, we’ll typically send a whole genome sequence on the kid and on the parents. And the rate at which we’ll be able to identify a specific genetic change is more like 25%, 30% on a good day.

Christopher Walsh

1:17:59 - 1:18:40

And so of the heritability of autism, we are still relatively early on in explaining where that comes from. and we don’t presently know what is missing. A lot of people think it might be mutations in these regulatory sequences, which are not generally tested for clinically, because we know so little about them. other, it’s also likely that a lot of that heritability has to do with subtle variation at hundreds of different loci across the genome, the sorts of things that can regulate, you know, intellect more broadly and social behavior more broadly.

Christopher Walsh

1:18:40 - 1:19:20

And so there might be a part of autism which is not just a single, there is in fact definitely a part of autism which is not due to a single genetic mutation but due to the interacting changes at hundreds of different loci across the genome. If we think of actually autism in a population terms, these multiple alleles, these common alleles, contribute the largest effect size to autism in a population. But if we’re looking at a single kid who’s in the clinic and has autism, that’s where a single genetic mutation can have the largest effect in an individual child. And so anyway, that’s how we think of heritability in terms of psychiatric diseases.

Christopher Walsh

1:19:20 - 1:19:32

In terms of schizophrenia, you know, we’re less directly involved in those studies, but there’s been a much smaller fraction of schizophrenia that’s relatable to these single gene disorders compared to autism even.

Nick Jikomes

1:19:33 - 1:19:56

Yeah. And I guess I would ask you, how good are we today at predicting ahead of time whether someone’s going to have some kind of brain problem or psychiatric problem from just the genetics? So if you’re talking about, say, an IVF clinic, where you go into the IVF clinic, you’ve got a bunch of fertilized embryos that you’re looking at. Of course, you can look for gross chromosomal abnormalities.

Nick Jikomes

1:19:56 - 1:20:04

But how good are we today at telling prospective parents there’s an X percent chance of some kind of autism and a Y percent chance of this or that.

Christopher Walsh

1:20:05 - 1:20:23

Oh, yeah. Well, that’s, you know, how good are we at taking any child and telling the parents what that child is going to turn into? And so that’s that that illustrates the challenge. I mean, that’s the biggest question in every parent’s mind is what’s going to happen to happen to my child.

Christopher Walsh

1:20:24 - 1:21:11

And that’s the always the biggest challenge for the physician is counseling any parent about that. You know, then getting to specifics, when we do, I mean, this is one reason, this will be a challenge on the uptake of prenatal genetic diagnosis as a routine, because basically the more commonly this is done, the more difficult it becomes, you know, to have predictive value about the majority of it. I would say for, there are a good number of genes that we have pretty good information about prognosis where we can be pretty confident in telling the parents that the child will have a severe diagnosis, such that they might consider reproductive decisions based upon that information.

Christopher Walsh

1:21:12 - 1:21:51

In terms of autism, I told you we thought there might be a thousand genes. Of that, there’s maybe a hundred or so are 150 that are very well validated and that are relatively commonly mutated so that we recognize not only the gene but the particular change where, you know, we might be able to get a sense. So this is, if we see that mutation, and it’s absent in the parents, it’s relatively likely that the child will be affected. But again, that leaves then hundreds of genes where we know less, where we have a sense that the penetrance of the defect is lower.

Christopher Walsh

1:21:52 - 1:22:21

where we might find that there are certain genes associated with autism where there are some people walking around with mutations in those genes in the general population, particularly girls, because girls are relatively protected against autism spectrum disorders. And so those have reduced penetrance. And so there it becomes very difficult to give accurate information. And so I think ultimately, we’re getting to the point now where we’ve sequenced millions of normal people.

Christopher Walsh

1:22:22 - 1:22:46

And as we get more and more information about the range of the genomes in normal people, we get increasingly precise information about each of these specific changes and which ones are the most likely to cause a serious defect. And so this is, again, it’s evolving very quickly, but it’ll still take years before we are able to be as good as we’d like to.

Nick Jikomes

1:22:46 - 1:23:04

Yeah, yeah. So for any given embryo that’s formed, a human embryo, remind us, how many novel mutations will be unique to that individual? And are those typically consequential or inconsequential? Does disease risk more come from the standing variation in the population that you get a piece of?

Nick Jikomes

1:23:04 - 1:23:10

Or do you acquire novel mutations early on when the embryo is formed, and those are consequential?

Christopher Walsh

1:23:10 - 1:23:34

Yeah. So each of us has about, as I mentioned, 80 to 100 single nucleotide variants relative to our parents and most of those are inconsequential. An average person will only have about one change in an exon relative to their parents and even most of those are inconsequential. That’s why most of us aren’t walking around with a disease somewhere.

Christopher Walsh

1:23:35 - 1:24:15

A typical person will have one coding change but a lot of times those coding changes might, as I say, be functionally irrelevant. They might, for example, not change an amino acid, because sometimes two different codons will code for the same amino acid, so you’re changing, it’s called a silent mutation. Or it could change an amino acid in a protein, but it turns out that that protein can tolerate that change in the amino acid, so again, no big deal. Or it could even disrupt one of the two copies of that gene completely, but it might be that that gene doesn’t happen to be one of these dosage-sensitive ones that I was telling you about, and so it’s still no problem.

Christopher Walsh

1:24:15 - 1:24:43

And so it’s only when you disrupt a dosage-sensitive gene that you’re likely to go into problems. And that’s why this is still a relatively rare event that happens, you know, that only affects a couple of all embryos, you know, one or two percent of all embryos. And why, fortunately, even after knowing all this genetics, I still think it’s okay for my kids to think about having kids because it still seems to work out okay most of the time.

Nick Jikomes

1:24:43 - 1:25:00

Yeah. And that brings us back to something fascinating you mentioned early on, which is there seem to be really powerful error correction mechanisms baked into development. And obviously a lot can go wrong, but most people don’t have anything that’s obviously wrong with them. And there’s a lot of checkpoints in place.

Nick Jikomes

1:25:00 - 1:25:20

I think you said something like, When you go into an IVF clinic and they can look at a bunch of fertilized eggs, they can often find gross abnormalities of some of the embryos and that seems like a big problem and it could be, but you actually mentioned that a lot of those might just develop normally because of these error correction mechanisms baked into our developmental program. That’s right.

Christopher Walsh

1:25:21 - 1:25:42

Yes, as I said, basically, if you systematically study blastocysts, you find that all of them are mosaic for chromosome anomalies. It’s just a question of whether it’s most of the cells or only a smaller fraction of the cells. The average is about a quarter of the cells in any given early embryo will have grossly abnormal chromosome patterns. But there are multiple error correction mechanisms.

Christopher Walsh

1:25:43 - 1:26:10

In one of them, in fact, The placenta is particularly tolerant of copy number alterations, and so a lot of these abnormal cells will go into the placenta. And this can sometimes be manifested by an abnormal amniocentesis that shows chromosome abnormalities in the amniocentesis, but it turns out they’re limited to the placenta. This is called placental mosaicism. And the embryo is fine, even though the amnio looks abnormal.

Nick Jikomes

1:26:10 - 1:26:16

Oh, wow. So it could actually look like something’s wrong, but you’re actually seeing a manifestation of things working well. Yeah. Yeah.

Christopher Walsh

1:26:17 - 1:26:49

They’re right. But then also, every organ has its own error correction mechanisms. I told you about how the brain seems to be able to preferentially eliminate neurons that have abnormal genomes. Other organs do that in other ways, most typically because we have cell cycle-based mechanisms for error correction that basically check the genome modulated by, you know, P53, which is called the guardian of the genome for that matter, for that reason, because it basically checks for large-scale chromosome alterations

Christopher Walsh

1:26:49 - 1:27:13

with each cell cycle and then tends to target the cell for death if there is abnormal genome copy. We don’t know why all these neurons sort of escape that p53 mechanism and why 25 or 30 percent of our neurons are formed with these abnormal genomes to begin with and why they need to have their own mechanism, but that might be having something to do with the fact that neurons divide in different ways and are post-mitotic for most of their life.

Nick Jikomes

1:27:13 - 1:27:30

Yeah. It’s relatively easy for me to think about how some of these genome abnormalities, like copy number variants, form when neurons are still dividing. And before they get to their final location, before they’re mature, because with every cell division, right, there’s a chance for a mistake. Maybe the polymerase slips.

Nick Jikomes

1:27:31 - 1:27:57

Maybe you duplicate some genes or a whole chromosome. And I can imagine that that’s going to happen at some rate, and therefore you had to evolve a mechanism for the developing brain to, you know, see when those abnormalities arise and then get rid of those neurons and do things like that. When we’re talking about the mature brain, the adult brain, Are there things that can go wrong? You’ve got an adult neuron, it’s mature, it’s stable, it’s doing its thing, it’s got its connections.

Nick Jikomes

1:27:58 - 1:28:12

I would imagine that over time as we age and get older, we’re exposed to toxins, things start to break down in different ways. Can the genome start to become more unstable as an age-related thing, and then we’ve got that problem on that side of our lives?

Christopher Walsh

1:28:13 - 1:28:46

Oh, golly, yeah, you’ve just led me down into my favorite rabbit hole there, Nick. So it turns out the genome of no cell is static, that genomes of every cell in our body are changing. And that the genetic, I just told you about the genetic diversity of the world’s population, you know, these 8 billion people with mutations at every spot in the genome. Well, it turns out every one of us has a genetic variation within us that far exceeds the entire genetic variation of the world’s population that I was just telling you about.

Christopher Walsh

1:28:46 - 1:29:20

So that we have basically 30 trillion cells, each of us has a trillion new mutations every day. And then in our brains, we have a million new mutations every day. And the biggest surprise for me came about 10 years ago when we developed a method to take the genome of a single neuron and amplify it and sequence it, just as though we were sequencing the genome of your blood, and then compare it to the neuron next to it.

Christopher Walsh

1:29:20 - 1:29:40

the genome and its genome. And we found that neurons accumulate mutations at a rate of about 20 per year. And they’re not copy number variants. As you pointed out, the copy number variants generally only arise in dividing cells because of a mis-slipping of the polymerase or a mis-segregation of the chromosomes.

Christopher Walsh

1:29:40 - 1:30:01

But it’s these point mutations. It’s a substitution of a single nucleotide, like a C to a T or a G to an A. And so our neurons accumulate about 17 of these copy number variants per year. And then they also accumulate two or three short insertions or deletions, the removal of two or three base pairs.

Christopher Walsh

1:30:02 - 1:30:36

And it’s remarkably linear in humans. So that’s about 20 point mutations per year when you sum together these insertion, deletions, and the SNVs. And it’s 20 mutations the first year of life, and it’s 20 mutations the 100th year of life, completely linear, in a way that marks time like a clock, with great precision. And that rate is, surprisingly, equivalently fast to the rate at which many dividing cells accumulate mutations.

Christopher Walsh

1:30:36 - 1:31:00

That 20 mutations per year is about the same as our blood stem cells, which accumulate mutations at about 18 or 20 per year. It’s faster than our sperm accumulate mutations, which is only about 5 per year. It’s slower than other cells like our lung cells that develop more like 50 per year. But why would non-dividing neurons develop these?

Christopher Walsh

1:31:01 - 1:31:40

That’s a mystery that we don’t completely understand. But then this process is beautifully and sharply linear in on brains with no neurological diagnosis, and then it’s rapidly accelerated in every neurodegenerative disease we’ve studied. So we have a paper that’s online at Cell that’ll come out September 7th showing that Alzheimer disease and ALS and frontotemporal dementia all show a rapid acceleration of this damage to the genome, particularly these short indels, as some aspect probably of the degenerative process that we’re trying to understand.

Nick Jikomes

1:31:41 - 1:31:57

Yeah, and when we think about mature neurons, you’ve got this interesting situation where on the one hand, by definition, a neuron that’s mature is no longer dividing. So this is sort of like your final chance. You’ve got to preserve this neuron as it is. I’ve got a pyramidal cell in my neocortex.

Nick Jikomes

1:31:57 - 1:32:09

It needs to stay a pyramidal cell. There needs to be a certain level of stability there in its genome and in general. But at the same time, there needs to be some instability and some flexibility because there’s still plasticity. It needs to be able to tune its synapses and so forth.

Nick Jikomes

1:32:10 - 1:32:44

But, because neurons are post-mitotic, they’re not dividing anymore, and also they are metabolically so active, they’re so expensive, you need to constantly be making refinements, constantly making and spending ATP, that’s going to mean a lot of oxidative stress and a lot of potentially mutation-causing, damage-causing, metabolic stuff happening in the neuron. Are neurons equipped with special mechanisms to buffer themselves against the damaging and potentially mutagenic effects of an intense metabolism because they’re not going to divide again?

Christopher Walsh

1:32:45 - 1:33:04

So that’s a great question. As far as we know, they’re not. They don’t have anything special. In fact, I think the biggest thing that neurons have to prevent them from developing a lot of these somatic mutations is that they don’t divide.

Christopher Walsh

1:33:05 - 1:33:39

I mean, replicating the whole genome is probably the biggest driver of creating these mutations, and that might be a big part of why neurons have been evolved not to divide, because it might be that that tends to then safeguard the genome from this kind of constant somatic mutation, which is, again, much more of a problem in gut cells and lung cells that are turning over all the time. And it might be why most of our blood cells, in fact, the stem cells, don’t divide very much. Even though they’re stem cells, they’re quiescent stem cells.

Christopher Walsh

1:33:40 - 1:34:02

And so probably that’s the biggest thing. So how do these mutations arise then? We think it’s probably because, as you say, neurons, although they’re not dividing, they’re very metabolically active, they’re very transcriptionally active. I told you we have these cascades of gene expression where the level of expression of CFOS goes up 100-fold in connection with patterns of neuronal activity.

Christopher Walsh

1:34:03 - 1:34:26

And that gene expression requires that the DNA become unwound and open to allow RNA polymerase in to turn the gene on. And so that is probably what makes the DNA vulnerable to being damaged. And because, in fact, when we look at these mutations, these 20 SNVs per year, we ask, what part of the genome are they taking place in? They’re enriched.

Christopher Walsh

1:34:28 - 1:34:47

at genes, and they’re particularly enriched at genes that are expressed in exactly that neuronal type. And so it looks like the pattern of this process of transcription is, again, a little bit of a dangerous thing. And so that’s a real problem because that means as we get old, just the genes that are most important are the ones that are differentially mutated.

Nick Jikomes

1:34:47 - 1:35:00

Right. Again, it’s a double-edged sword. It’s like the ability to have plasticity and to change requires you to make those genes vulnerable to damage because you have to literally open them up and make them open to potentially bad things happening.

Christopher Walsh

1:35:00 - 1:35:27

That’s right, exactly. And so at the moment, we don’t know. I assume that these 20 mutations per year don’t have a normal function, that they’re just a byproduct, an unfortunate byproduct of the fact that the neurons are very active. And rather than, as I say, I don’t think that they are there for a purpose, but just a little bit of a challenge for the neurons to get around.

Christopher Walsh

1:35:27 - 1:35:53

But fortunately, that 20 per year It ends up, again, most of these 20 mutations per year are inconsequential. Most of them occur in non-coding parts of the genome or don’t alter the protein coding sequence of a gene. So fortunately, even at 20 per year, the damage to the genome is sufficiently modest that most neurons are still able to function fine.

Nick Jikomes

1:35:54 - 1:36:19

What are some of the things that you’re working on in the lab today or just that are going on in the field of developmental neurobiology in general, especially as it relates to things like psychiatric disease, propensity, developmental disorders, and the like? What are some things where you think we’re making a lot of traction now for whatever reason, and we’re going to start to have really good new insights in the next few years, say?

Christopher Walsh

1:36:19 - 1:36:44

Sure. Right, well, I’m very excited about our ability to sequence the genomes of single neurons. There’s a lot of neurons in our brain, 20 billion, so we’re not going to be able to, anytime soon, sequence all 20 billion of them. But sequencing the genomes of single neurons allows us to recognize that every neuron is different.

Christopher Walsh

1:36:44 - 1:37:07

It allows us to put different identities on the different neurons and study them in different ways. And it turns out that the genome is essentially a tablet that records the life of the neuron. Just as our genome is a tablet that records our whole evolutionary history, right? Our genome records who our parents are.

Christopher Walsh

1:37:07 - 1:37:20

It recorded who their parents are. It records the history of the human species over time. It records the history of how the human species relates to the history of other species. And so it is a tablet that records history.

Christopher Walsh

1:37:21 - 1:37:35

It turns out that that doesn’t stop with the cells of our body. that every cell has a genome that records the history of that cell. So I just told you that neurons accumulate 20 mutations per year. That means every neuron knows exactly how old it is from the moment it was born.

Christopher Walsh

1:37:36 - 1:37:59

I told you that stem cells develop mutations when they divide. That means that every neuron knows who its family is. It knows exactly how it’s related to all of the other cells in the entire brain, in fact, all of the other cells of the whole body. So our somatic mutations of our body create a permanent forensic post-mortem map of the entire development of our body.

Christopher Walsh

1:37:59 - 1:38:31

But also, I just told you that the genome of neurons changes if you have a neurological disease. Well, that means that, in fact, the genome is recording the environment of the neuron. It’s recording who its neighbors are, what it’s exposed to. We think that those extra mutations and degenerative diseases come about probably because of the effects of the neighbors of those neurons, particularly the microglia, which can become very activated in inflammatory and abnormal ways in Alzheimer’s disease and in other degenerative diseases.

Christopher Walsh

1:38:31 - 1:39:00

We think that those microglia are pummeling the neurons with cytokines and reactive oxygen and other things that is damaging the genome of the neurons. And so in fact, the genome knows where it’s living and its neighborhood. And then I finally, I told you also that 25 or 30% of the neurons are eliminated during fetal development. You have these abnormal neurons that are generated and then selectively removed.

Christopher Walsh

1:39:00 - 1:39:43

And so it was only by sequencing the genomes of single neurons that we’re able to even recognize this Darwinian competition, that we can see these forces of selection. We can use the same tools that were developed for studying the evolution of organisms. looking at genomic conservation and changes and areas that are selected positively and negatively, and we can do it within our brain to see how selection acts in our brain. And we think that this selective process, this selective removal of damaged neurons and the selective survival of non-damaged neurons, we think that this selective process is going to be abnormal in the neuropsychiatric diseases.

Christopher Walsh

1:39:44 - 1:40:02

that this pruning is abnormal. That’s already been proposed. That’s what autism relates to, is this abnormal pruning. Well, if abnormal pruning happens so that there’s a failure of the normal evolutionary selection and selective removal of damaged neurons, that would leave the brain with a whole bunch of damaged neurons.

Christopher Walsh

1:40:03 - 1:40:26

And that might be an important mechanism of disease. And so I think that we’ll be looking The looking at things this way has caused me to look at every disease that affects the brain in a different way because it’s basically a new tool that allows us to state problems that are familiar problems but in a completely different light.

Nick Jikomes

1:40:28 - 1:40:56

We talked a lot about brain development stuff today. On the one hand, we talked a lot about the sort of exquisite beauty of development, all of the checkpoints and regulatory controls, the error correction mechanisms, the robustness of it. It’s sort of amazing that development of a human being ever goes right when you think about how many cells are in all of our organs and in our brain, et cetera. And on the other hand, we talked about, you know, places where things can go awry and in that complexity.

Nick Jikomes

1:40:56 - 1:41:09

We live in a time where fertility rates are basically lower than they’ve ever been. People are having fewer children in general. They’re having children later. They’re doing things like IVF because they’re having children later and things like that.

Nick Jikomes

1:41:09 - 1:41:29

And there’s a lot of people who get, I think, easily stressed or scared about what can go wrong. What would you say to people, knowing everything that you know about development, about how they should consider the question of whether to have children and how long to wait, given all of the beauty of the development process and all the things that can both go right and go wrong?

Christopher Walsh

1:41:30 - 1:42:08

Well, you know, I would just say, I mean, kids are just the most wonderful thing. I mean, I’ve got the two, I’ve got two of the most wonderful kids in the world and it’s been, it’s the greatest blessing in my life other than perhaps meeting my wife, Ming Wei. So I think, you know, I think having kids is an individual decision, but I was like one of eight kids, none of whom had any, you know, major neurological condition. So I think it’s easy to lose sight of the fact that these issues, while serious and life-changing, are rare.

Christopher Walsh

1:42:09 - 1:42:52

And that the vast majority of kids just turn out just fine. And again, I was studying the development of the brain way back before I had kids, but there’s just no greater miracle than watching the development of the brain in one’s own kids, and watching that light go on, and that miracle of cognition as it develops. And worrying all the time about whether things are going to be okay, but then taking a great deal of joy in the fact that they usually do work out fine. But as I say, it is crazy.

Christopher Walsh

1:42:54 - 1:43:13

When Ming Wei was pregnant with our kids, we had these ultrasounds, and even back then, just with these non-genetic tools, there always seemed to be a problem. And yet, in the end, everything worked out just fine. And we were both doctors, and we were like, wow, did we really have to have all those tests?

Nick Jikomes

1:43:14 - 1:43:16

Is that really necessary?

Christopher Walsh

1:43:18 - 1:43:25

And so I would, again, for those people who are thinking about having kids, it’s been the biggest blessing in my life.

Nick Jikomes

1:43:25 - 1:43:33

All right, well, that’s a great spot to end it. Chris, this was fascinating. Thank you for taking the time. I know that you’re a busy guy out there, but I think people are going to learn a lot about this.

Nick Jikomes

1:43:33 - 1:43:37

And this is definitely a subject that people are really interested in. So thank you very much.

Christopher Walsh

1:43:38 - 1:43:42

Well, thank you very much, Nick. It’s been a real pleasure. Thanks again.

This is a public episode. If you'd like to discuss this with other subscribers or get access to bonus episodes, visit mindandmatter.substack.com/subscribe

Wide release: Available for free by August 19, 2026, on Apple, Spotify, YouTube, etc.

Early access: Available to paid subscribers here on Substack.

Not medical advice.

Nick speaks with Dr. Nephi Stella about the endocannabinoid system as a distinct lipid signaling network. Stella explains how anandamide and 2-AG are produced by separate enzymes, trafficked by specialized proteins and microvesicles, and act primarily as retrograde inhibitors through CB1 receptors. The conversation covers developmental risks of adolescent THC exposure, the failure of early FAAH inhibitors, promising new therapeutic approaches, and lessons from rimonabant for metabolism-targeted therapies.

TOPICS DISCUSSED:

* Anandamide vs 2-AG: The two major endocannabinoids with a shared arachidonic acid backbone but distinct head groups biological roles.

* On-demand production: Calcium-dependent enzymes release cannabinoids from membranes in response to neuronal activity patterns.

* Trafficking: Fatty acid binding proteins and extracellular microvesicles traffic endocannabinoids within and between cells.

* Adolescent THC: Global receptor activation during circuit wiring may contribute to cannabis use disorder and psychosis vulnerability in susceptible individuals.

* Enzyme inhibitors: Early FAAH clinical trial halted by off-target toxicity; MAGL inhibitors advancing in phase 2 for anxiety and addiction.

* Peripheral CB1: Liver receptors regulate metabolism; restricted antagonists aim to reduce weight without central psychiatric risks of the weight loss drug rimonabant.

ABOUT THE GUEST: Nephi Stella, PhD is a professor at the University of Washington School of Medicine. He has studied endocannabinoid signaling for more than 25 years, focusing on therapeutic strategies that separate beneficial effects from side effects of cannabinoid-system modulation.

RELATED CONTENT:

* Podcast | Plant Chemistry, Cannabinoids, Cannabis Terpenes & the Entourage Effect | Ryan Vandrey

KNOW YOURSELF:

* Visit the SUPPORT PAGE if you find value in this content. Includes discount codes + links to affiliate partners, including products to help you measure and manage your health.

Listen or watch on your favorite platform:

* Audio version: [Apple] [Spotify] [Elsewhere]

* Video version: [YouTube]

PRACTICAL TAKEAWAYS:

* High-potency THC products, especially concentrates that produce rapid high brain levels, raise cannabis use disorder risk more than lower-potency forms; vulnerability is highest during adolescence.

* Endocannabinoid “tone” may influence longer-term neuronal phenotype (including BDNF expression) while phasic release controls rapid transmission; dietary fatty acid changes could slowly alter tone, but this remains unproven.

SUBSCRIBER CONTENT BELOW: Reference paper + episode transcript.

Reference Paper:

* Paper | The endocannabinoid 2-arachidonoylglycerol is released and transported on demand via extracellular microvesicles

AI-generated transcript below. Beware of mistranslations!

Nephi Stella

02:15 - 02:16

Thank you. Nice to see you.

Nick Jikomes

02:16 - 02:20

Do you want to just tell everyone a little bit about who you are and what your background is?

Nephi Stella

02:20 - 02:59

Yes, absolutely. I might give a little bit of a longer version so that it kind of introduces the field and what we’re gonna be talking about today. So I did my PhD in Europe between Switzerland and France, and actually that’s where I started to study lipid signaling network. In Switzerland, my PhD was more into the physiology, of lipid signaling and therefore it was about energy dependence and from cell types to cell types, neurons and glial cells.

Nephi Stella

02:59 - 03:35

And then in France it was much more pharmacology and it was much more around how different stimuli will actually produce these lipid signaling. And it turns out that these lipid signaling I was studying is arachidonic acid, which is one of the constituents that formed the endocannabinoids. So it was a very opportunistic, a good foundation for me. And then what happens is that as I was getting ready to go to do my postdoc, The endocannabinoid field was starting.

Nephi Stella

03:35 - 03:54

It was the very beginning. They had just discovered the first endocannabinoid, anandamide, which also has this arachidonic acid moiety. And I had the opportunity to go do my postdoc in San Diego with Daniele Piumelli. And I remember his question.

Nephi Stella

03:54 - 04:22

He said, when we started, he said, you know, if there’s one endocannabinoid, there’s probably a second one. Because we know that a lot of neurotransmitter systems and neuromodulators have multiple ligands and multiple receptors. So that’s how I started into the endocannabinoid field during my postdoc. and it was very centered on 2-arachidonoylglycerol, again, the arachidonic acid moiety.

Nephi Stella

04:23 - 04:57

And then, that was the very beginning, so we had so many fundamental questions to ask, how is it produced and everything where? And then I had the opportunity to start my lab, and I was really interested in trying to develop novel therapeutics. And you know, the field back then, the cannabinoid field, was very much focusing on studying the side effects, Memory impairment would be a really big one. But I was really interested in trying to figure out how, if there had any therapeutic potential for the endocannabinoids.

Nephi Stella

04:58 - 05:27

So I started my lab in Seattle at the University of Washington in the School of Medicine. in 99, if I remember, 25 years ago. And then through all these years, what I’ve done is kind of navigate the field, trying to understand and identify the most promising therapeutic approach to target the cannabinoids. So initially, I was interested in neuroinflammation, so multiple sclerosis.

Nephi Stella

05:28 - 05:59

Then I got really interested in seizure maintenance, especially with the effect of cannabidiol and the FDA approval. That was very promising, so I studied quite a bit of seizures and how endocannabinoids can actually modulate those seizures. And then more recently, when you want to develop a therapeutic approach, what you have to do is you have to think about the benefit, but you have to think about the side effects. What is the potential side effects of a therapeutic approach?

Nephi Stella

05:59 - 06:22

So I’ve been really interested in trying to separate the side effects versus therapeutic effects of both THC and cannabidiol. And to do that, I might tell a little bit about the new technology that we’re using to monitor these things. So that’s what we’re doing in my laboratory. As you can see, it’s very broad.

Nephi Stella

06:23 - 06:33

It’s really, the goal is to find the most promising therapeutic approach and understand it at the molecular level so that we can go all the way to human clinical trials.

Nick Jikomes

06:34 - 06:50

And so what are the endocannabinoids? You’ve already sort of hinted at the answer here. They’re fatty molecules, and we’ll maybe talk about where they come from and how they relate to diet and things like that. But just as a class of signaling molecules, what is the characteristic of endocannabinoids?

Nick Jikomes

06:50 - 06:51

What do they generally do?

Nephi Stella

06:52 - 07:29

Well, endocannabinoids are really interesting and very special. They are actually quite different from most of the signaling mechanism that we have in the brain. You know, the field started with excitatory glutamate GABA inhibitors, and then the field got really interested, and when I say field is in neuroscience in general, going beyond these neurotransmitters and looking at neuromodulators. And that’s when the field started discovering and working on serotonin, noradrenaline, dopamine, and then the opioids showed up.

Nephi Stella

07:29 - 07:48

And then the opioids, you know, those are small peptides that modulates. And now we have yet another signaling mechanism, which is actually driven and carried by those lipid signaling molecules like the endocannabinoids. So they’re very special. They’re very different.

Nephi Stella

07:49 - 08:09

than the other signaling mechanisms. And we can already say, we can start by emphasizing that there’s two endocannabinoids. So how, like I mentioned before, there’s anandamide, the first one, which means in Sanskrit, means inner bliss. Very appropriate name.

Nephi Stella

08:09 - 08:39

And then there’s a second one that I was involved in discovering, which is 2-AG. And even, I think the field now has really recognized that these two signaling endocannabinoids, anandamide on one side and 2-AG on the other side, are actually doing very different things in the brain and have very different functions, even though they’re both endocannabinoids and they act on cannabinoids we’re going to be focusing on the cannabinoid CB1 receptors in the brain.

Nick Jikomes

08:41 - 08:54

Got it. So there’s a bunch of different stuff in the brain and body, different types of signaling molecules, different classes, different structures, different functions. Obviously they all have to be made out of something. Some of them are made out of things like amino acids and the building blocks of proteins.

Nick Jikomes

08:55 - 09:06

You mentioned, you know, the opioids are often peptides. They are basically little proteins. A lot of classic neurotransmitters or neuromodulators are basically amino acid like molecules. and so on and so forth.

Nick Jikomes

09:07 - 09:22

But the endocannabinoids are basically fat-derived molecules. They’re pieces of fats that ultimately come from fats that we consume. So they’re fatty substances. Where are they stored and where do they come from in our brain?

Nick Jikomes

09:23 - 09:32

When an endogenous cannabinoid is used by a neuron to signal something, to do something, where is the neuron getting it from, literally?

Nephi Stella

09:33 - 09:56

Okay, so let’s unpack this a little bit, because there’s quite a bit of concepts here. It is true that endocannabinoids are lipids, and therefore where they reside is in the lipid bilayer of the membranes, for example. And those are what we call hydrophobic, so fat-preferring molecules. So they have a hard time going into solution into water.

Nephi Stella

09:57 - 10:45

So they’re mainly into the membrane. It is true that diet will influence the composition of the lipids, but it’s not a direct relationship between I eat something fat and then I produce endocannabinoids. It’s just the overall over time. I think we need to think about endocannabinoids, that these small lipids signaling molecules that are released from the membrane in an activity-dependent manner, the cell, there’s actually two we can Emphasize already another difference or characteristic of the endocannabinoid signaling system is that some endocannabinoids are going to be

Nephi Stella

10:45 - 11:01

produced in a tonic manner. They’re going to be there all the time, whereas endocannabinoids can also be produced in an activity-dependent manner. on and off, just like neurotransmitter. I stimulate something and then I stop stimulating.

Nephi Stella

11:02 - 11:11

So, endocannabinoids are produced on and off over seconds, very small time periods, but they can also accumulate for a very long time.

Nick Jikomes

11:12 - 11:26

back. Go ahead. No, I think that’s important, right? So, when we say that these things are fat-derived and when we talk about certain fat-derived substances in the body, it’s not that, you know, if I go eat a meal right now and I’m eating some fat and I’m putting it in my mouth and it goes into my

Nick Jikomes

11:26 - 11:54

stomach, it’s not that those fat molecules literally get turned into endocannabinoids right now, right after I absorb it into my body. It’s rather that those fatty acids get incorporated into membranes, into adipose tissue, maybe burned for energy, they get distributed throughout the membranes of my body. And then later on, they may or may not become things like endocannabinoids, but that will only happen if a relevant stimulus comes afterwards.

Nephi Stella

11:54 - 11:54

Correct.

Nick Jikomes

11:54 - 12:08

What I eat is going to influence the fatty acid composition of my cells and my tissues. And what happens to me, the stimuli that are impinging on my body and what’s happening internally, that will determine if and when those things later on get turned into endogenous cannabinoids.

Nephi Stella

12:08 - 12:15

Correct. Absolutely. That’s a very good description. So coming back to the idea of how are they produced?

Nephi Stella

12:16 - 12:48

This is actually a very fascinating molecular machinery that’s quite specific. So, there are several aspects to that. So, they are in the cell membrane, so they are lipids, and then we have enzymes that are going to cut those lipids, hydrolyze those lipids that are complex, and release smaller lipids that are going to be the endocannabinoids. Those enzymes are going to be different if we’re talking about the first endocannabinoid anandamide versus the second cannabinoid 2-AG.

Nephi Stella

12:49 - 13:34

Yet another concept to emphasize that these two endocannabinoids are going to be produced under different conditions by enzyme number one which is NAPLD for anandamide, and enzyme number two, which is daglycase for 2-AG. So two different enzymatic pathway are going to release those lipids from the membranes. So that’s already important to emphasize. Because lipids do not like to interact with water, there’s actually very recent studies studies that have discovered some mechanism by which you can actually shuttle these lipids from one cell to the other.

Nephi Stella

13:34 - 14:11

Because from one cell, it needs to go through the cytosol, which is hydrophilic water. So it needs something to help it. And I know you had Steve Laviolette that actually interviewed, and he talked about the fatty acid binding proteins. So those are actually really fundamental mechanism by which these proteins that have hydrophobic greasy pockets The endocannabinoids are gonna go into this greasy pocket of the protein, and like that, there’s gonna be a shuttle that’s gonna bring this endocannabinoid from one cell to the other.

Nephi Stella

14:11 - 14:42

Now let’s go deeper, let’s double-click on the cell itself. You can actually have endocannabinoids that are released from the plasma membrane, and then they will go intracellularly, because there’s also some intracellular receptors, and they’re gonna be doing a lot of things. You had You interviewed Giovanni Marzicano, so he discovered the cannabinoid CB1 receptors on mitochondria. So now we’re talking about an intracellular signaling pathway.

Nephi Stella

14:43 - 15:11

where endocannabinoids are produced from the plasma membrane, needs to go intracellularly and the shuttle is also gonna be a fatty acid binding protein. So that’s one mechanism that is actually very special. There’s no other signaling mechanism that uses these shuttles. And this is actually really important because we’ll probably talk about this, we will become a very important and interesting therapeutic target to those fatty acid proteins.

Nephi Stella

15:12 - 15:53

The second mechanism that has been recently discovered, because the question is, since these lipids do not like to interact with water, how are they going from one cell to the other? I mean glutamate, GABA, dopamine, it’s easy, they’re hydrophilic, they just go through water. But in the endocannabinoids, so there’s actually new research that’s shown that in an activity-dependent matter, again, the cell actually releases these extracellular microvesicles, tiny vesicles of lipid that are fascinating because they actually travel from one cell to the other, and they contain inside quite a bit of information. They might even have mRNA.

Nephi Stella

15:54 - 16:17

that will actually change their phenotype from the next cell. But they also have, because they’re micro-physical, they have a composition of their lipids. that is very specific and it turns out that they actually accumulate endocannabinoids. So now you have a packet of endocannabinoids that goes from one cell to the other, fuses with the next membrane.

Nephi Stella

16:17 - 16:43

You have more endocannabinoids in the next cell and therefore the cannabinoid receptors are gonna be activated. So that’s how endocannabinoids are thought to travel from one cell to the other via those fatty acid binding protein or microvesicles. or intracellularly also via those endofatty acid binding proteins. So those are very different and unique mechanism.

Nick Jikomes

16:43 - 17:11

Yeah. So these endocannabinoids, they are lipid molecules, they are little pieces of fat, and basically there are shuttles. So you can quite literally think of this almost like a city planner, like there’s buses. Like, like shuttles, like a protein has a water soluble end and a fat soluble end and a greasy molecule, a lipid, like an endocannabinoid can get onto the shuttle and be shuttled from one cell to a completely different cell or within a cell from one place to another,

Nick Jikomes

17:11 - 17:25

such as from the outer membrane to the mitochondria. there’s these little shuttles moving all over in different cells, moving these greasy molecules around to and from all of the different membranes on the outer side of the cell and even within the cell in places like the mitochondrial membrane.

Nephi Stella

17:26 - 18:05

Absolutely, and the trafficking map is actually a really good analogy because you have, I don’t know, I think we already have like 10 different fatty acid binding proteins. They’re gonna have different abilities to transport either anandamide versus 2-AG, so there’s another difference. There might be different shuttling mechanisms for anandamide versus 2AG that go at different speed. And all of this tight regulation of anandamide versus 2AG signaling is going to determine how much of the endocannabinoid is going to activate the cannabinoid receptor and where.

Nick Jikomes

18:06 - 18:17

And what are these receptors? Let’s go through them one at a time. So the main one, cannabinoid receptor one, what kind of receptor is it? How common is it?

Nick Jikomes

18:17 - 18:25

Where is it? Is it mostly in the cell membrane? Is it mostly in mitochondria? Is this in very specific parts of the brain or is this something that’s all over the place?

Nephi Stella

18:26 - 18:49

Yes, so the cannabinoid CB1 receptors is what we call the G-protein coupled receptor. So it’s part of the family of those receptors that are typically modulated, for example, dopamine, serotonin. So it’s a modulator receptor called G-protein coupled receptor. So it’s gonna modulate cellular activity.

Nephi Stella

18:50 - 19:07

The... Pretty much, let’s go at each level. So at the molecular level, what it does is, where is it expressed is actually very important. So cannabinoid CB1 receptors are predominantly expressed presynaptically on neurons.

Nephi Stella

19:08 - 19:17

And what they do there is they regulate neurotransmitter release, and they are typically inhibitors. of neurotransmitter release.

Nick Jikomes

19:18 - 19:36

So in other words, what you’re saying is at the point of contact between two neurons, you know, the first neuron is sending a signal to the second neuron. The second neuron is receiving that signal from the first neuron. The first neuron, the one sending the general neurotransmitter signal, that’s the one where the CD1 receptor will generally be.

Nephi Stella

19:40 - 19:48

So let’s start again. So we have a neuron number one, which we’re gonna be calling the postsynaptic neuron.

Nick Jikomes

19:48 - 19:52

Okay, so yeah, you’re saying postsynaptic is number one, the receiver of the message, yeah.

Nephi Stella

19:52 - 20:17

Correct, the receiver of the message. And then what it’s going to do is the endocannabinoid is gonna go back to the presynaptic. and say slow down, that’s enough, shut off, which is why the endocannabinoids are considered retrograde, so from postsynaptic to presynaptic inhibitors of transmission because they slow down transmission. That’s enough glutamate.

Nephi Stella

20:18 - 20:23

Endocannabinoids come on CB1 receptors and reduce glutamate release.

Nick Jikomes

20:25 - 20:37

Yeah, so if a neuron’s getting too much input or it’s gotten enough input already, it’s sending a signal back in the form of an endocannabinoid to the neuron, giving it that input, and it’s saying, that’s enough, no more needed.

Nephi Stella

20:38 - 20:55

Feedback, that’s enough negative, absolutely. And actually, so now let’s go, so that’s at the subcellular level, and that’s the most predominant is CB1 receptor. There’s a couple of more location where CB1 receptors are expressed. We already mentioned on mitochondria.

Nephi Stella

20:56 - 21:17

So that’s the intracellular CB1 receptor, and there its function is going to be to regulate mitochondrial function. And mitochondria does many things, including producing energy and substrates. So CB1 receptor has a role in regulating how much energy the cell will utilize. And then there’s also...

Nephi Stella

21:18 - 22:06

although much less studied, there’s also some CB1 receptors on the soma, like on the main cell. And there they have a slightly different function according to the initial results is that there they will actually signal all the way to the nucleus and change the phenotype of the neuron. And that’s actually another very predominant role of the endocannabinoid signaling system is to regulate the phenotype of the neuron, what kind of neurons are we gonna have, and where are they actually connecting? Because CB1 receptors, I believe, is already expressed at the stage two cell after, like, you know, you, really at the very beginning.

Nephi Stella

22:06 - 22:26

And actually the CB1 receptors regulate cell proliferation and cell migration. So the endocannabinoid signaling system, these lipids, when they activate and interact with the CB1 receptor during development, they’ll actually regulate the phenotype of the neuron.

Nick Jikomes

22:27 - 22:35

Yeah, yeah. So these things are very, very involved in not just what mature neurons are doing, but actually in the development and the setting up of cells and circuits itself.

Nephi Stella

22:36 - 23:16

Correct. Which is why this is a good moment to say, which is why it’s a major concern about adolescent consuming THC, because now the THC is going to flood the brain that is still developing and all the CB1 receptors are going to be interacting with THC instead of having a very precise directed endocannabinoid that tells the CB1 receptor go this way. So this is one of the hypothesis of cannabis use disorder is when adolescents are consuming THC.

Nephi Stella

23:16 - 23:51

THC is activating all these neurons and therefore the wiring of those neurons is not precise enough anymore because instead of following the endocannabinoids, the CB1 receptors are flooded with THC. And to the most vulnerable adolescent, this could actually It results in cannabis use disorder, but some of these patients actually have schizophrenic episodes, psychotic episodes, because of these neurons that have been miswired.

Nick Jikomes

23:52 - 24:10

And so when an endogenous cannabinoid, such as an andamine, gets used for signaling purposes, so it’s this little fatty substance that ultimately comes from fatty acids in the cell membrane, how exactly is the cell producing it? Is it literally clipping off a piece of the cell membrane?

Nephi Stella

24:10 - 24:26

Correct, yeah. Yes, it’s clipping off a subunit, a small piece of... Imagine the... The plasma membrane has a bunch of Legos and then it’s taking off one of the Legos and it’s cutting it off and giving it to the next cell.

Nephi Stella

24:27 - 25:10

Absolutely. One thing that’s really interesting to think about in the dichotomy between anandamide and 2AG, now the field is realizing how different these small molecules, there’s just a new paper that just came out by a very predominant lab, showing that different behaviors, so now we’re actually able to, to, this might be a good time to actually introduce the new tools we have to actually study endocannabinoids. So there’s actually new technology that came up from some labs that are allowed to sense or detect endocannabinoids production in mouse brain in real time.

Nephi Stella

25:10 - 25:51

And basically the technology is based on the cannabinoid CB1 receptor. And then in the intracellular part of the CB1 receptor, instead of having that G protein that does the signal transduction, they attach the fluorescent green protein, GFP. And now each time the endocannabinoid hits that sensor, the green protein flashes a green fluorescence. So now we’re actually able to take these sensors, which are proteins, virally infect some mouse brains so that these sensors are expressed in specific neurons or specific brain areas.

Nephi Stella

25:52 - 26:18

And now when a little mouse goes and does its little behavior, we can actually see in real time endocannabinoids being produced. You have no idea the The emotion I had the first time I saw that sensor go up and down in real time from a molecule that I discovered during my postdoc 25 years ago, you know, in a tube. And now I see that molecule being produced in real time. in a mouse.

Nephi Stella

26:19 - 26:58

So now let’s come back to, now that we know that we have this new tool to sense endocannabinoids, and there’s tools that can sense either the first endocannabinoids, anandamide, or the second endocannabinoids, 2-AG, and now we know that actually different behaviors will produce one endocannabinoid versus the other. It’s a very novel concept. For example, aversive stimuli, something that will create fear like, you know, shocking, an electrical shock for a little mouse, that will actually create endocannabinoid 2AG. as detected by the sensor.

Nephi Stella

26:58 - 27:44

By contrast, if you give a psychoactive drug, for example, nicotine or cocaine or ethanol, these psychoactive drugs are also going to produce endogenous cannabinoids, but with different dynamics. Some of them will produce more anandamide, some of them will produce more 2-AG. And therefore now the field is starting to realize that these different behaviors and these different stimuli differentially activate endocannabinoids, anandamide versus 2-AG, and therefore most likely different cellular responses. different CB1 activation on different cell types, depending on the brain area.

Nephi Stella

27:45 - 27:59

So that’s where the field is at now. We’re trying to understand how endocannabinoids are related to different behaviors and different stimuli so that we can actually foster that and maybe develop novel therapeutics.

Nick Jikomes

28:03 - 28:29

So people often say that Endocannabinoids are use dependent or they’re made on demand. So there’s mechanisms that allow the cell to say, OK, we need some endogenous cannabinoid right now. We can pull some from the membrane and use it right now for whatever the stimulus that’s calling for it is. And so what are the main signals that start to determine when and where and how much of these things are made?

Nick Jikomes

28:30 - 28:39

Are these things highly dynamic? Can different stimuli drive very different levels of production? And give us a sense for the dynamics here.

Nephi Stella

28:40 - 28:48

Absolutely. It’s a great question. And we’re at the cutting edge of science right now. These are actually the kind of questions that the fields are asking.

Nephi Stella

28:49 - 29:25

So, these endocannabinoids are produced by different enzymes, so it will really depend on where that enzyme is located in the cell, in the subcellular compartment. So, if this enzyme is only in cell type 1, For example, the enzyme that produces anandamide, NAPE-PLD, will be expressed only in neurons A. And by contrast, neuron B will only express the diglyphase, which produces the second endocannabinoid, 2AG. You already there have a dichotomy of one cell versus the other.

Nick Jikomes

29:25 - 29:45

Got it. So depending on the concentration and pattern of proteins, such as these enzymes in any given neuron, it can determine the cocktail of cannabinoids that might be produced. One neuron might produce only anandamide, or it might produce only 2-AG, or it might produce a combination.

Nephi Stella

29:45 - 29:52

That’s correct. Absolutely. And that’s the first level. So the enzymatic activity that produces the lipid.

Nephi Stella

29:53 - 30:26

So now how are we going to regulate the enzymatic activity? It turns out that most of these enzymes are actually calcium dependent. Calcium is a fundamental signal transduction mechanism in cells, that when a neuron, let’s focus on neuron, when a neuron is activated, when it’s depolarized, you have this influx of calcium that gets into the neuron, and that rise in intracellular calcium then will go and activate multiple enzymes, including enzyme that will produce the endocannabinoids.

Nephi Stella

30:26 - 30:58

So that’s the second level of regulation let’s say of endocannabinoid one the first one is where is the enzyme the second one is how much calcium is reaching that enzyme, and actually for how long? So that’s the third level of regulation. So now let’s stimulate the neurons with two different types of receptors. One receptor that will be on and off very quickly, or even three receptors.

Nephi Stella

30:58 - 31:37

One that’s on and off very quickly, one that goes on for minutes, and one that goes on for hours. like a glutamate metapotropic receptor for minutes, and NMDA receptors for almost, you know, for plasticity. So then you’ll have the third level of regulation, which is, does this neuron express this receptor, nicotinic and NMDA? And for how long is that receptor going to be activated, which will result in the increase in calcium, which will activate the enzyme that will release the endocannabinoid.

Nick Jikomes

31:37 - 32:20

And for the non-neurobiology people that listen to this, One of the things to connect here is when neurons are active, depending on how much they’re active, the pattern they’re active with, depending on the ion channels they have, calcium is tied to that activity. When the neurons are active, some ion channels allow calcium to come in. The important piece here is that some of these enzymes that produce endocannabinoids are calcium dependent. And so what that means is the level of activity, the pattern of activity that a given neuron is experiencing is going to determine then the pattern of endocannabinoids it makes because calcium is sort of the dot that connects the enzymes that make the endocannabinoid to the activity levels

Nick Jikomes

32:20 - 32:22

that neuron is experiencing overall.

Nephi Stella

32:23 - 32:35

Absolutely. Now let’s go to the other end of the signaling network. So we’ve produced the endocannabinoids now in an activity-dependent manner. These endocannabinoids are gonna activate specific receptors.

Nephi Stella

32:36 - 33:07

And now we need to talk about how we’re gonna stop that signal with enzyme that actually inactivate those endocannabinoids. So there’s another here, very nice diversity of enzyme that are going to stop endocannabinoid signaling. For anandamide, we have this enzyme which is called FA, which is a fascinating enzyme. So each time anandamide is produced and activates the CB1 receptor, then it unbinds the CB1 receptor and it’s inactivated or hydrolyzed by FA.

Nephi Stella

33:07 - 33:34

So that’s the first endocannabinoid. And then we have another mechanism for the second endocannabinoid 2AG. 2AG is produced, activates the CB1 receptor, and then it’s actually hydrolyzed or inactivated by two different enzymes that are expressed in two different parts of the cells, most likely because they have different functions. So, the most famous one is monoacylglycerol lipase, MAG-lipase.

Nephi Stella

33:34 - 33:59

This enzyme that hydrolyzes and inactivates 2AG is expressed close to the CB1 receptor in the presynaptic terminal. That means that MAG-lipase is really regulating the amount of endocannabinoid 2AG at the level of the target. There’s a second enzyme close to the CB1 receptor. There’s a second enzyme, which is actually postsynaptic, ABHD6.

Nephi Stella

34:00 - 34:14

And that enzyme hydrolyzes 2EG, where 2EG is produced postsynaptically. So you can see how these enzymatic pathway from one cell to the other are gonna regulate the signaling network very precisely.

Nick Jikomes

34:14 - 34:37

Yeah, so the production of endocannabinoids from the fatty cell membrane is coupled to the activity of a neuron. How active it is is going to be related to how much calcium comes in. That determines how much these enzymes start clipping the membrane to make endocannabinoids, and then those get deployed. And then there’s another set of enzymes that basically turn off the signal and get rid of those endocannabinoids.

Nick Jikomes

34:38 - 34:58

So what all of this basically means is on both ends, you’ve got enzymes, you’ve got protein-based regulation that allows you to have pretty precise spatial and temporal regulation of this endocannabinoid signal. The cell can turn on and turn off the signal in very particular locations on demand as it’s needed.

Nephi Stella

34:58 - 35:20

Correct. It’s very similar to other neurotransmitter and neuromodulator systems. Glutamate is produced in an activity dependent manner, and then it’s removed from the synaptic cleft by a transport mechanism. noradrenaline is produced, and then there’s an enzyme that, or even an uptake system that removes it.

Nephi Stella

35:21 - 35:38

So the signaling system in the brain is always an on-off. You need to produce, activate the receptor, and stop. And the endocannabinoids, that’s how they do it. There’s another aspect that’s really fascinating in lipid signaling, is that actually these lipids, when I say...

Nephi Stella

35:41 - 35:57

When I say that the enzyme is going to inactivate these endocannabinoids, that is correct. That means that for, let’s take the example of, 2-AG. So let’s take 2-AG. So 2-AG is arachidonic acid, the fatty acid, and then there’s glycerol.

Nephi Stella

35:58 - 36:12

The enzyme, what it’s going to do is going to cut this lipid and then release arachidonic acid. Therefore, it’s not an endocannabinoid anymore. But now it’s arachidonic acid. Arachidonic acid is very bioactive.

Nephi Stella

36:13 - 36:28

It will actually produce prostaglandins and be involved in inflammation. Now, let’s go before endocannabinoids. Diacylglycerol is the precursor of 2AG. Diacylglycerol is actually bioactive also.

Nephi Stella

36:28 - 36:59

It regulates protein kinases. So, the way to think about these lipids signaling is fascinating because there’s multiple As a lipid is transformed into another lipid, it becomes something else. Before an endocannabinoid, it regulates kinase, then an enzyme changes it, it becomes an endocannabinoid, it activates cannabinoid receptor. Then another enzyme changes it, it becomes arachidonic acid, and now it’s involved in inflammation.

Nick Jikomes

36:59 - 37:21

So you can have a piece of a cell membrane and it becomes another type of lipid and then another type of lipid and then another type, but they’re not merely intermediates. At each step, that molecule is now a new molecule that does something. And so it becomes very important now to think about, well, how many steps are there, but also how long do you spend at each step?

Nephi Stella

37:21 - 37:30

Correct. And therapeutically, when you block one enzyme, you’re not going to only block one thing. What happens? You’re blocking the next step, too.

Nephi Stella

37:30 - 37:32

You have to think about those things.

Nick Jikomes

37:32 - 37:48

Yeah. And this will probably tie into why, as I’m sure we’ll get to, why designing drugs and designing therapies isn’t as easy as we might conceptually like to think, because it’s not just this one thing does one thing. Lots of things are pleiotropic. They do multiple things.

Nick Jikomes

37:48 - 37:57

They can turn into another thing very quickly. And there’s regulation at all of these steps. And it’s, you know, it’s complex, literally complex. Absolutely.

Nick Jikomes

37:57 - 38:35

And it moves all the time. So, how do we start to think about the way that the overall fatty acid composition of our membranes and our neurons or anywhere else relates to the overall pattern of signaling molecules that get produced, including cannabinoids? And how do we think about how that relates to things like our pattern of dietary fatty acid intake? So, if I start eating a different level of polyunsaturated fat in my diet tomorrow, and I continue doing that for weeks and weeks, is that meaningfully gonna shift the membrane composition of the fatty acids in my neurons ultimately?

Nick Jikomes

38:36 - 38:42

And is that gonna in turn affect sort of the overall levels or the dynamics of things like endocannabinoid signaling?

Nephi Stella

38:43 - 38:56

I think there’s some initial evidence that that might be the case, but I would say that’s probably not the case. the main molecular mechanism that regulates the endocannabinoid signaling system.

Nick Jikomes

38:58 - 39:18

And so let’s talk about, well, I guess let’s just focus on anandamide and 2AG separately and together, but let’s just take them one at a time. How similar are they exactly? And then what are the differences in terms of their actual structure and then the general functional differences that they tend to have?

Nephi Stella

39:20 - 39:49

So the chemical structure is similar at one level, like this fatty lipid, the fatty portion of these endocannabinoids is the same. They both have arachidonic acid. And there’s a little piece of additional, molecule to arachidonic acid. In anandamide it’s ethanolamine, so it’s a small, and then in 2-Ag it’s arachidonic glycerol.

Nephi Stella

39:49 - 40:28

That little piece of molecule at the tip of arachidonic acid is what is going to clearly differentiate anandamide from 2-Ag. because they have the same arachidonic acid moiety. That little piece of molecule will make a big difference in the way the endocannabinoids are interacting, binding, with stimulating the cannabinoid CB1 receptor. Here’s another very big difference between anandamide and 2-AG, is that anandamide, as it interacts with the cannabinoid receptor, it’s only able to partially activate to the CB1 receptor.

Nephi Stella

40:30 - 40:59

which is actually very similar to what THC does. THC is a partial agonist. It doesn’t completely stimulate CB1 receptor, which is probably why it stayed around, because if you completely, fully stimulate CB1 receptor, you become cataleptic, way too strong. By contrast, endocannabinoid 2AG, the second one there is actually a full agonist at the endocannabinoid CB1 receptor.

Nephi Stella

40:59 - 41:06

So that’s another difference. Partial activation, anandamide, full activation, 2AG.

Nick Jikomes

41:06 - 41:20

Yeah, yeah. And this would actually correspond to, so activation of the CB1 receptor actually has an inhibitory effect on a neuron. So partial versus full activation would then correspond to a somewhat decreasing activity versus more strongly decreasing activity.

Nephi Stella

41:20 - 41:47

Correct. So now let’s actually look a little bit further in the difference also when we compare to THC. Because when somebody consumes or inhales THC, THC is going to flood the brain and therefore activates CB1 receptor everywhere that it finds it. Whereas endocannabinoids are going to be produced in an activity-dependent manner physiologically where it’s supposed to be.

Nephi Stella

41:48 - 42:09

That’s their role. The best example I have is they’re going to be produced when I go for a jog because it’s hurting, right? So, it’s going to be actually controlling my pain. and I’m a Neanderthal and being, you know, a T-Rex is behind me, I need to continue even though it hurts so that I can survive.

Nephi Stella

42:10 - 42:44

So endocannabinoids are actually also very parallel to the endorphins, the opioid system, which also are endogenous painkillers. So that’s how one can actually think about the big difference between THC activating all the neurons and inducing psychotropic effects, the high, versus endocannabinoids being produced exactly where they’re supposed to be produced, and therefore we don’t get high because these endocannabinoids are just doing what they’re supposed to do, which is, for example, regulate pain.

Nick Jikomes

42:44 - 43:01

Yeah, yeah. I think that’s important. That’s often people often ask this type of question or they have of me in the past where it’s like, why aren’t we high all the time if we’re filled with endogenous cannabinoids? But basically what you’re saying is, you might have one synapse in one part of your brain that needs more anandamide and it cooks it up right then and there.

Nick Jikomes

43:02 - 43:19

That’s not going to affect how much gets made at some other synapse in another neuron. It’s this point to point synapse by synapse regulation of these things, whereas when you consume an exogenous cannabinoid like THC, you’re now putting it into your bloodstream and it’s kind of flooding everywhere and hitting receptors all over the place all at once.

Nephi Stella

43:20 - 43:40

Absolutely. Here’s an interesting example that I think about sometimes is in the hippocampus, so this part of the brain that’s involved in memory formation consolidation. There’s quite a bit of CB1 receptors that are there. So what is the role of the cannabinoid CB1 receptor in the hippocampus?

Nephi Stella

43:41 - 44:06

Well, let’s think about what happens when somebody takes so much THC all the time during adolescence. One of the first impairment is memory impairment for people that consume THC. They forget things. their energy level might not be as high.

Nephi Stella

44:07 - 44:30

That’s because the THC is actually activating all these CB1 receptors in the hippocampus and is messing up the memory formation. Now, what is the role of the endocannabinoids in the hippocampus? Well, during a day, I think My hobby is photography, so I kind of hear about that sometimes. During a day, we see about 2,000 pictures.

Nephi Stella

44:36 - 44:44

But we don’t want to remember everything. I want to remember you. I want to remember how you look. So I’m going to consolidate that.

Nephi Stella

44:44 - 44:56

My noradrenaline is going to take that memory and consolidate that. But there’s many things that happened today that I saw that I don’t want to remember. I don’t need it. And the idea is that the endocannabinoids are going to go.

Nephi Stella

44:57 - 45:11

and eliminate this memory. I don’t need it. I don’t need to remember everything. So endocannabinoids could actually remove some selective memory, which is why the side effect of THC is memory impairment.

Nick Jikomes

45:12 - 45:26

Yeah. And that’s, you know, it’s always important to remember that memory The flip side of remembering is forgetting. You need both things in order for the memory and the memory consolidation to actually be functional. You simply can’t and don’t want to remember every detail of everything.

Nick Jikomes

45:26 - 45:27

That’s actually going to be an impairment.

Nephi Stella

45:28 - 45:51

Correct. Which brings us to post-traumatic stress disorder. all the individuals that have such strong fears that they actually have a hard time to forget. And one of the idea is that by using the self-medicate with THC to maybe forget those memories that are so hard to remember.

Nick Jikomes

45:52 - 46:11

Yeah, yeah. And people, I mean, people often talk about cannabis and cannabinoids in the context of PTSD. Maybe someone like a veteran has these traumatic memories. The idea is, right, you have the memory, you have the episodic memory, like you remember what happened, but the emotional power is sticking with you.

Nick Jikomes

46:11 - 46:29

You actually want to kind of forget or dampen that component. And the failure to forget that, so to speak, is what the PTSD is in some sense. And so the idea would be, well, how do you actually dampen the emotional salience of the memory? And perhaps cannabinoids are one way to do that.

Nephi Stella

46:29 - 46:58

Could be one way to do that. The memory was so salient that were so much nor adrenaline, it was so stressful that it was consolidated and remembered, and there was not enough endocannabinoids to forget it, and therefore you have post-traumatic stress disorder. The issue with treating with THC is that it has a very tight, narrow therapeutic window. It might have some medical properties, but it will have some side effects.

Nephi Stella

46:59 - 47:05

And therefore really it’s a decision, medical decision with the doctor usually to be able to find a good regimen.

Nick Jikomes

47:06 - 47:21

Well, so how do we think about, how do we think about things? You know, we’ve already said that this endocannabinoid system, the system of endocannabinoids, receptors and so forth, it’s ubiquitous. It’s all over the brain. It’s not like in one part.

Nick Jikomes

47:22 - 47:38

And it’s spatially and temporally specific, right? Sort of a point-to-point regulation to this. You can use it in a synapse-specific manner. Given that someone with PTSD, let’s say they have a traumatic memory that is bothering them and causing them issues in their lives.

Nick Jikomes

47:39 - 48:08

You want to dampen the emotional salience of that memory that they’re failing to forget, so to speak. But you don’t want to interfere with memory consolidation generally. You don’t want to disrupt other memories in their brain. How do you start to think about playing with this endocannabinoid system so that you’re going to selectively help with the forgetting and the remembering in the one circuit that’s problematic, or I should say the one memory engram that’s problematic, without scrambling memory or disrupting memory more generally?

Nephi Stella

48:08 - 48:30

Absolutely. That’s where actually the small molecules or medicine that will modulate the endocannabinoid signaling enzymes might come in. The big difference again, between the THC that is going to activate all the receptors throughout the brain. And actually let’s have one little precision.

Nephi Stella

48:30 - 48:56

Not every neuron in the brain expresses CB1 receptor. There are some, There are some neuronal population that don’t. And one of the most salient one to think about is the dopaminergic neurons from the VTA. And therefore, even though there is actually a drug addiction component to cannabinoids, and then cannabinoids increase dopamine, right?

Nephi Stella

48:56 - 49:08

It’s pleasurable. There’s an addiction. Actually, those dopamine neurons do not express CB1 receptors. The other ones close to the dopamine neurons that regulate dopamine neurons.

Nephi Stella

49:08 - 49:32

that stimulate dopamine production. But coming back to what you were saying, I think that’s where the field has a lot of promises is to actually target the enzyme that regulate the endocannabinoid signaling system instead of stimulating all the receptors. Because, for example, for memory, memory is actually a process. You have your short-term memory and then you have your long-term memory.

Nephi Stella

49:33 - 50:14

So one, It would be an interesting testing to do would be to actually provide, give some inhibitors of the enzyme that inactivate the endocannabinoids. So if I take an inhibitor of that enzyme, I’m gonna have more endocannabinoids that are gonna accumulate, and therefore maybe it’ll help me forget. And I’ll do it in a very specific time period where I have the short-term memory And therefore that short-term memory is gone and it’s not sent to the long-term memory and consolidated. That would be one way.

Nephi Stella

50:14 - 50:14

Yeah.

Nick Jikomes

50:15 - 50:53

And I guess if I play with this a little bit, the way that I’m imagining that would go would be... If we remember the fact that memories are consolidated and reconsolidated, every time we recall things, the memory sort of becomes a little bit more labile and can be changed, can be weakened, strengthened, changed in different ways. I guess the idea here might be that if you have someone with PTSD, say, they’ve got a traumatic memory, they’re failing to dampen that memory, to forget the emotional intensity of it. You might have them, say, take a therapeutic, like an endocannabinoid-based therapeutic, specifically when they’re recalling that memory, but not all of the time.

Nick Jikomes

50:54 - 50:59

And that might help it sort of be dampened down or forgotten, at least to some extent.

Nephi Stella

50:59 - 51:02

Absolutely. That would be nice. It’d be nice to try.

Nick Jikomes

51:03 - 51:20

Now, how much of this is... theoretical, like these ideas kind of all fit together in a conceptually nice way, but have we seen much success with developing things like endocannabinoid enzyme inhibitors to actually show these kinds of benefits? Is that something people are working on today?

Nephi Stella

51:22 - 51:41

That’s a good question. There’s a lot of hypotheses out there, very little theory. Theory will become, that’s when we have something that’s written in the textbook and we know it works like that. We have a lot of hypothesis and we are gonna be testing that it’s working like this.

Nephi Stella

51:42 - 52:31

So now this might be a good place to start talking about preclinical testing of novel endocannabinoid therapeutics. So, The cannabinoid field is actually quite young as compared to the opioid field because we only discovered in the 90s the endocannabinoids and the receptor compared to the opioids, for example. So we know much less on the endocannabinoid signaling system compared to the opioid signaling system. So there is, and then there’s also, there’s been some challenges to do preclinical research in the cannabinoid, not much funding, and also the schedule one limitation to have access to THC.

Nephi Stella

52:32 - 53:06

All of this has really slowed down the field to make progress. Now things are a little bit better, so there’s actually much more that we understand. So the preclinical field now, and we have the tools also to actually do some nice thorough preclinical testing. So the field is actually now testing multiple things, multiple therapeutic indication if we either The most logical one to start with is the one that we talked about, is to target the enzyme that degrade endocannabinoids.

Nick Jikomes

53:06 - 53:30

Just so people are clear, there’s enzymes that naturally degrade endocannabinoids when they’re released. So they’re controlling sort of when the signal gets turned off. One of the things that is attractive about making that a therapeutic target is you can regulate how much of the endocannabinoid signal there is, but still maintain sort of the natural patterns of endocannabinoid production.

Nephi Stella

53:31 - 53:37

Only where they’re produced physiologically, you make it stronger. Absolutely. Absolutely. So there’s some...

Nephi Stella

53:39 - 54:04

There’s some things that, so we’re gonna be talking mainly about two enzymes. We’re gonna be talking about FA, the fatty acid amidohydrolyzed that inactivates anandamide, endocannabinoid number one. And then we’re gonna be talking about MAG-Lipase, monoacetylglycerol lipase that inactivates the second endocannabinoid, 2-AG. So the first one targeting the fatty acid amidohydrolyzed, unfortunately, the...

Nephi Stella

54:06 - 54:29

the field went, in my opinion, a little bit too fast in two humans. We didn’t really have enough information. The two limits There’s two critical things that you have to think about when you develop a new therapeutic approach. One, you have to make sure that your molecule, your new medicine, is gonna do the job that you expect.

Nephi Stella

54:30 - 55:01

It’s gonna go and inhibit fatty acid aminohydrase only, and not do something else that could be toxic. The second challenge is to actually find the actual disease that we’re gonna try to temper or even cure. And that’s where the preclinical testing in our little mice, we have mice models of different diseases. And therefore what we’re gonna be doing the field is to take that fine inhibitor and test it in our little mouse for certain indication.

Nephi Stella

55:01 - 55:34

What is it for pain, for anxiety, fear, anxiety, for depression, all these indications. Unfortunately, there was actually a first molecule that was a fine inhibitor that went into a human clinical trial in Europe. And unfortunately, that clinical trial, or fortunately, was stopped right away because of tremendous side effects. Tremendous side effects.

Nick Jikomes

55:34 - 55:35

What were they?

Nephi Stella

55:37 - 55:54

I believe there were some patients that were even considered brain dead. They had no more neuronal activity in their brain. A handful, obviously, when you have that kind of side effect, you stop the clinical trial right away. Right.

Nick Jikomes

55:54 - 56:05

And I guess, so the idea here, right, if it’s a PHA inhibitor, you’re inhibiting the enzyme that degrades anandamide. So you would expect to have more anandamide generally, which means more inhibition of neural activity.

Nephi Stella

56:06 - 56:35

And basically you’re saying, if you take that to the extreme, you basically get... Well, actually, so there’s a second part to this story. which is what I said first, is you need to make sure that your molecule is clean and it’s doing what it’s supposed to do. And what was discovered a couple years later in a science paper by my colleague Mario van der Stelt, he actually, how would you say, he profiled that molecule.

Nephi Stella

56:35 - 57:03

He looked at what are the other proteins that are inhibited by this inhibitor that we thought were specific to pho. And he found this other protein, I forget what the name was, but it was a critical protein involved in neuronal viability. And therefore, this first molecule, unfortunately, that was given into humans stopped the clinical trial right away. The entire field freaked out.

Nephi Stella

57:04 - 57:15

Obviously, this is a bad therapeutic approach. But then now we understand it’s probably an okay therapeutic approach if you have the right molecule that only inhibits FAL.

Nick Jikomes

57:16 - 57:29

Yeah. So for some reason, people thought this was a specific pha inhibitor, an inhibitor of just this enzyme, but it wasn’t. How does that like, is that an easy thing to miss? Or was this just like a weird case?

Nick Jikomes

57:29 - 57:31

Why would they, why was it thought that this was specific?

Nephi Stella

57:32 - 57:55

I wish I was on the field to understand the details. But in drug development, that is a really big component that you do up front, is you kinda look at what are the off targets, what are the other proteins. Typically what we do is we look at the off targets, the proteins that are critical for life. Some proteins in the heart.

Nephi Stella

57:56 - 58:13

Don’t give this molecule because you’re gonna have a heart attack. That we know right away. I think this was a more, less understood enzyme that was targeted by this molecule. And therefore it was not on the radar.

Nephi Stella

58:13 - 58:28

People were not thinking about it. And it’s only when Mario did this unbiased approach that he discovered, oh no, there’s this enzyme. So now obviously we know molecules should not target that enzyme. They’re going to be brain dead.

Nick Jikomes

58:29 - 58:46

And I guess that’s one of the strengths of being able to do unbiased approaches in preclinical animal models is you don’t have, you know, there’s known unknowns, there’s unknown unknowns. These unbiased approaches allow you to sort of see everything even if you don’t already know something about it.

Nephi Stella

58:47 - 59:06

Correct. So that’s the story of the inhibition of pha. I think the field is still working on it and trying to have cleaner, more selective by inhibitors, and maybe find the right indication. What are we going to try to cure anxiety?

Nephi Stella

59:06 - 59:38

And so, and now that we know in our little mice that different endocannabinoids do different things, that might help the field to find the right indication. And now there’s other really promising therapeutic approaches that now are moving forward. based on preclinical evidence and cleaner molecules, and they’re targeting the monoacylglycerol lipase, the second enzyme that regulates 2AG. And those are moving forward nicely.

Nephi Stella

59:38 - 1:00:00

There’s multiple indications, including anxiety and addiction. And I believe they’re already in phase two human clinical trials, which means that their toxicity level is pretty good. There’s not much side effects if they’re moving forward. So, maglipase, I think that’s a very promising area.

Nephi Stella

1:00:00 - 1:00:50

And then there’s also, we mentioned the fatty acid binding proteins, those little shuttles that There’s some inhibitors now that are in clinical trials also based on strong preclinical mouse studies. They found the right indications and they’re treating these patients with these inhibitors of fatty acid binding proteins and boosting the endocannabinoids. The endocannabinoids often are I like to call them the help me signal or the repair signal. They’re produced when you need to slow down things and things and like, and therefore that’s the way I think about it in the therapeutic approach is what you’re doing is you’re boosting the endocannabinoids and therefore you’re boosting the help me signal and you’re helping your body to deal

Nephi Stella

1:00:50 - 1:00:51

with the disease.

Nick Jikomes

1:00:54 - 1:01:06

Oftentimes, the nervous system gets the most attention when we talk about endogenous cannabinoids. But of course, these things are used elsewhere. They’re used in the immune system. They’re used outside the nervous system in a bunch of tissue systems.

Nick Jikomes

1:01:06 - 1:01:31

What are some of the bigger examples that help us understand what this system of regulation is doing outside of the nervous system. Why do we have these receptors and these endocannabinoids in various different cell and tissue types? And what does that sort of tell us about the core function? I think you were kind of just starting to allude to it a little bit, but can you maybe give some examples of say immune system regulation by endocannabinoids or somewhere else to help illustrate this?

Nephi Stella

1:01:33 - 1:02:09

Absolutely, so it is true that the cannabinoid receptors are expressed by multiple cells in the peripheral tissue. I would say that if we start with the cannabinoid CB1 receptor, the level of expression of those CB1 receptors is much lower in those other cells in the periphery compared to neurons. So when we think about THC, we know that when you take THC, there’s gonna be some psychotropic effects. And THC will also modulate some physiological responses in the periphery.

Nephi Stella

1:02:09 - 1:02:55

Immune cells is one, vasculature, blood pressure is another one, which is why individual have red eyes when they’re high, because there’s a hypotension and therefore dilation of the blood vessels. There’s quite a bit of cannabinoid receptors in the liver, and they’re really involved in liver metabolism, which is why there’s this really interesting inverse correlation between the individuals that take cannabinoids, THC and actually lose weight, and they eat more. They take THC, they have the munchies, and actually they do not gain weight. There’s this dichotomy.

Nephi Stella

1:02:56 - 1:03:21

It’s because those CB1 receptor in the liver, for example, are going to regulate liver metabolism and energy expenditure and metabolism. So the liver, here is one organ that doesn’t have cannabinoid receptor is the kidney. The muscle also doesn’t really have, the heart doesn’t really have many CB1 receptor. Good thing, right?

Nephi Stella

1:03:21 - 1:03:38

We’re not gonna die from a heart attack. The immune system is fascinating because, so one of the function of the CB1 receptor that we mentioned, so in neurons it regulates neurotransmitter release, okay. and their phenotype. What do they look like?

Nephi Stella

1:03:38 - 1:04:26

In immune cells, it’s a little bit the same also. You have those CB1 receptors that are going to regulate the immune cell transmission, their ability to release immune molecule, those fatty acids we talked about, those prostaglandins, some cytokines, And here, the CB2 receptors are also very dominant. So, immune cells have both CB1 and CB2 receptor. And therefore, the endocannabinoid really regulates tightly the immune system, not only by regulating their phenotype, what kind of cytokines, the release, they even regulate how they It’s called phagocytosis, how they gobble up those bugs when they’re fighting those infections.

Nephi Stella

1:04:26 - 1:04:52

They regulate how the immune cells travel in the body. That’s similar into neurons, developing neurons. They’re involved. So they’d have these really fun, and they regulate, I don’t know, actually Giovanni, should check out if immune cells have CB1 receptors in their mitochondria, because that would be fascinating, because we know mitochondria are so important for immune responses, you need the energy.

Nephi Stella

1:04:53 - 1:05:34

So truly, endocannabinoids have this fundamental function. They regulate cellular function at multiple levels, somewhat similarly between cell types, proliferation, migration. Selectivity of functions, neuron, neurotransmitter, they have a lot of CB1 receptors. So yes, that opens up actually a new question and there’s an entire field there, which is, do we have, there’s many diseases in the periphery, that can we actually develop therapeutics that are targeting the endocannabinoid signaling system and can help with those diseases.

Nephi Stella

1:05:35 - 1:06:14

Liver, you know, cirrhosis or things like that. Immune, autoimmune disease, that’s how I started. working on neuroinflammation when I started my lab is because patients that have multiple sclerosis, they back in the 90s, that was the one of the diseases that was the most reported beneficial effect of cannabis use was the patients with multiple sclerosis. And therefore, that’s how I started thinking, well, maybe it regulates the immune system, it dampens, because multiple sclerosis is an autoimmune disease.

Nephi Stella

1:06:15 - 1:06:28

The disease is too strong and therefore let’s try to dampen it. And that was the idea. Let’s try to target the endocannabinoid system, CB1, CB2, to calm these immune cells in those dramatic autoimmune diseases.

Nick Jikomes

1:06:29 - 1:06:48

And we’ve talked about how these endocannabinoids are deployed in a activity-dependent manner. They’re made on demand. They can be regulated in a way that, you know, you can use it at this synapse over here when it’s needed, but not the synapse over here. And so there’s this really nice spatial and temporal regulation, this on-demand regulation of when these things get deployed.

Nick Jikomes

1:06:49 - 1:07:22

At the same time, we’ve talked about things like FAA inhibitors, inhibiting these enzymes, which sort of determine the rate at which these signals are broken down. So even though there’s this kind of point-to-point regulation and this very dynamic regulation of endocannabinoid signaling, there’s also sort of just a background level or background tone that we can talk about. Endocannabinoid tone is a concept that sometimes you hear people talk about. What is that and how much does the overall tone of endocannabinoids differ moment to moment in an individual or between, say, two individuals in a population?

Nephi Stella

1:07:23 - 1:08:00

So while we know much more about on and off production of endocannabinoids because that’s where neuroscience pay attention and how does it regulate neurotransmission, we know much less about the endocannabinoid tone. The way to think about it is that it’s going to have a very different biological function. Endocannabinoids are produced in an activity-dependent manner, and they’re gonna regulate the cellular function over a small amount of time. I’m gonna shut off this neuron for a while, because it’s too active.

Nephi Stella

1:08:01 - 1:08:34

whereas the endocannabinoid tone is going to tonically activate those receptors and therefore really change. So, it’s really, I think the only difference is basically the temporal activation of the cannabinoid receptor. Does our physiology want it on and off because we need to regulate neurotransmission now? Or are we going to regulate the tone and therefore change the overall phenotype of the neurons on a longer term?

Nephi Stella

1:08:34 - 1:09:07

I’m gonna activate these receptors maybe on the cell soma directly. So for example, there’s a really fascinating, again, another, function of endocannabinoids to help the brain, for example. So when you activate those CB1 receptors that we talked about on the main neurons, not the presynaptic, just on the cell body, they go and they change the phenotype, they change gene expression. One of the genes that they actually upregulate is brain-derived growth factors, BDNS.

Nephi Stella

1:09:08 - 1:09:50

That’s very valuable for the health of the brain. So here’s actually one situation where you would imagine, although it’s not been demonstrated, but if you put the building blocks together, that’s the most likely hypothesis is, for example, a higher tone, of endocannabinoids will activate more CB1 receptors on neuron cell soma and therefore have more BDNF and faster neuronal connectivity and things like that. So that would be one biological function that would separate the tone of endocannabinoid versus the on-off and I regulate neurotransmission.

Nephi Stella

1:09:50 - 1:10:16

The tone probably regulates much more the phenotype of the neuron over a long term. And that’s where the concept that you were referring to changing our diet. Right? I would imagine that changing the diet will have an effect predominantly actually on the tone and you might have more endocannabinoids and maybe a healthier brain.

Nephi Stella

1:10:17 - 1:10:22

This is all me putting pieces together. We’ve never proven this, but it’d be a cool experiment to test.

Nick Jikomes

1:10:22 - 1:10:47

Yeah. At any given moment, of course, there’s going to be highly dynamic changes that are highly context dependent in where endocannabinoids are being deployed and how many are being used. That’s all riding on top of this general background tone that we’re talking about. And you’re saying that perhaps, it’s not proven, but perhaps changes in diet and things like that might slowly change that background level of tone over time.

Nick Jikomes

1:10:47 - 1:10:53

But on top of that, you’re always going to have these sort of bigger, faster changes that are more dynamically regulated.

Nephi Stella

1:10:53 - 1:11:07

Correct. And you know, the parallel is dopamine. Dopamine has also a phasic activation and then a tonic activation. And those two things actually do different things on the dopaminergic receptor.

Nephi Stella

1:11:07 - 1:11:22

So I think this is a concept or a mechanism that biology uses quite often. Am I going to produce this neurotransmitter for a long time or just on and off? Probably both. Because they have different functions.

Nick Jikomes

1:11:22 - 1:11:25

Right, right. You can use the same signaling molecules,

Nephi Stella

1:11:27 - 1:11:27

Absolutely.

Nick Jikomes

1:11:28 - 1:12:02

And you can change how they function or what they do by playing with either a slower timescale or a faster timescale changes in the signal. Yeah. What about, so like, just based on your knowledge as a basic researcher and the general, you know, what we know generally about endocannabinoid signaling and all of the complexities involved, what’s your general take on the rise in THC consumption, exogenous cannabinoids that we’ve seen for a number of years now.

Nick Jikomes

1:12:02 - 1:12:26

I forget some of the exact statistics and stuff, but we know that cannabis use is up at least in certain some populations. I believe some populations are now consuming cannabis more often or more overall than even alcohol. What are the pros and cons here, both in terms of the overall amount people are using and also sort of the differences between adults and younger individuals?

Nephi Stella

1:12:27 - 1:12:42

Absolutely. So that’s a really good question. There’s a lot to say about this. I was actually involved in the task force to implement I-502, the legalization of cannabis in Washington State.

Nephi Stella

1:12:43 - 1:13:29

And so I was able to actually be really in the field, understanding how new legislation is actually put together. The biggest aspect to think about are, there’s several aspects, and we need to differentiate THC versus CBD. I’m not sure if we wanna go after CBD, but if we focus on Delta-9 tetrahydrocannabinol, THC, The first thing to consider is the age of consumption. And because we know, there’s actually really no doubt that there is a vulnerable population, a subset of adolescents that are very vulnerable to using cannabis during adolescence.

Nephi Stella

1:13:30 - 1:14:01

Now the question always becomes how much and how often? because everything at very high dose and all the time will actually be bad on your body, especially THC. So the fact there, there’s either a legal system, you make it illegal for adolescents, that’s one way to think about it, absolutely. The other way is to educate adolescents and let them understand that this is actually detrimental for their development.

Nephi Stella

1:14:02 - 1:14:27

The good news is that there’s actually an age where the detrimental effect is not as strong. I would say after 25 years old, when your brain is fully developed, then people start using cannabis and there’s less impact on brain development because the brain is already developed. And maybe there, there might actually have some positive effects. It might reduce stress levels a little bit.

Nephi Stella

1:14:28 - 1:14:55

It might temper inflammation sometimes, help you sleep for some individuals. So there’s really a period during development where we know it’s gonna be bad for the brain, and then a period where we can actually start thinking about good regimen, how much am I gonna take, how often, and that might become medical. So that’s one aspect, the regimen and how much. And now we can get into the products.

Nephi Stella

1:14:57 - 1:15:13

the cannabis-based products. So what has happened, oh yes, that’s right, because you also interviewed Ryan Vandrie for the PK. That is excellent. And he’s asking very important questions.

Nephi Stella

1:15:13 - 1:15:36

So depending on the product, are you going to smoke it and therefore it’s going to go into your brain on and off very quickly? Are you going to only smoke a joint or are you actually going to dab? 35% THC versus 95% THC. Plus dabbing is going to go up and down very quickly into your brain.

Nephi Stella

1:15:37 - 1:16:06

That is the one first criteria that will increase the likelihood of getting addicted to a drug. If your dopamine goes up fast, up nicotine, up and down very quickly. So dabbing THC, high concentration of THC very quickly will induce a very high increase in dopamine and their potential addiction, cannabis use disorder.

Nick Jikomes

1:16:06 - 1:16:27

Yeah. And let’s define and talk about that just a little bit for people because it’s important and there are very different viewpoints and biases that people have out there. When I worked in the marijuana industry, this was a very polarizing subject and very difficult to address because some people would insist You know, cannabis is not addictive. It’s not addictive like other drugs.

Nick Jikomes

1:16:27 - 1:16:45

And some people would say, well, yes, it is. It is habit forming addiction dependency, habit forming. Like, what do these terms mean to you? And from your perspective, you know, is what is the habit, habit forming potential of THC in particular relative to other drugs that we can talk about?

Nephi Stella

1:16:48 - 1:17:02

So the first thing is that both are right. It is true that some people get addicted and it’s true that some people don’t get addicted. Thus the concept of vulnerable population. Some people are more vulnerable to drug addiction than other ones.

Nephi Stella

1:17:03 - 1:17:22

The precise reason why they’re more vulnerable is something that the field is working on. Genotyping, is there a genetic, makeup that makes this poor individual more likely to become addicted or not. So I think both are right. I think some people get addicted and some people don’t.

Nephi Stella

1:17:24 - 1:17:46

What is the term of addiction in the field of cannabis? It’s related to cannabis use disorder. So in the DSM-5 now, there’s some criteria. It’s basically a new disease, a new syndrome that has been discovered because of the rise in the consumption of products that are very potent in THC.

Nephi Stella

1:17:47 - 1:18:10

The more potent, The more you consume, the more likely you’re going to have the cannabis use disorder. Within the cannabis use disorder, there’s multiple criteria that the patient needs to have to be considered having cannabis use disorder. One is not being able to stop. That’s the definition of addiction.

Nephi Stella

1:18:11 - 1:18:31

Even though I know that it’s not good for me, I’d rather continue smoking. That would be one criteria. How addictive is it compared to other drugs? It comes back to how much dopamine is produced in the brain and how quickly.

Nephi Stella

1:18:31 - 1:18:55

So it is going to, so nicotine is super addictive because you have a very quick rise. Cocaine is super addictive because you have a big rise. Cannabis is more, THC is more and more addictive as you get, as you start using the dabs, for example, because now you have 95% of THC on and off. So that’s how I think about it.

Nephi Stella

1:18:55 - 1:19:02

It’s like really the, the amount of THC, how often, and only of a subpopulation of vulnerable population.

Nick Jikomes

1:19:02 - 1:19:18

Yeah. And I think it’s worth dwelling on and really emphasizing this idea of vulnerability in discrete subpopulations, because it really is true. I’ve done a couple episodes with Christian Lucher, who does excellent work here. And just to give people a sense for this, you can give rats cocaine.

Nick Jikomes

1:19:19 - 1:19:44

And some of them get addicted, but some of them don’t. And it’s actually, it’s not just a random subsample for reasons that are still somewhat mysterious. There are discrete subsamples of individuals, whether it’s rats or humans that are just more prone to addiction to virtually anything it seems in some cases than others. So it’s not that know, everyone becomes addicted if you consume enough or no one becomes addicted because it’s a non-addictive substance.

Nick Jikomes

1:19:44 - 1:19:54

It can be that if you consume enough and on top of that, you’re this sensitive individual rather than someone else, you can develop a dependence or something like that.

Nephi Stella

1:19:55 - 1:20:11

Absolutely. And the fact that we actually see that in the preclinical rodents and rats is actually a power for us to understand what is the difference because we can actually do some gene sequencing and try to understand that.

Nick Jikomes

1:20:11 - 1:20:31

What’s kind of fascinating about that work is in rats and in people, the numbers kind of shake out the same. It’s like something like roughly one in five rats will become addicted to cocaine if you just give them a bunch of cocaine over and over again and nothing else to do. So it’s like one in five. But why that 20% and not the other 80%, nobody knows.

Nick Jikomes

1:20:31 - 1:20:40

But it turns out that the numbers are kind of similar in humans too. It’s like this one in five-ish percentage of the population for that substance. I agree. at least, that’s sensitive.

Nick Jikomes

1:20:40 - 1:20:45

And is it a birth order thing? Is it the way you grow up? I don’t think anyone really knows yet, but...

Nephi Stella

1:20:45 - 1:20:52

I would say, I would argue it’s probably really fundamental biology because rats in a lab are so similar.

Nick Jikomes

1:20:52 - 1:20:53

Right, right.

Nephi Stella

1:20:53 - 1:20:59

And then we have exactly the same thing in this really diverse population of humans. So it’s probably something very fundamental.

Nick Jikomes

1:20:59 - 1:21:08

Yeah. Christian Lucher had the same intuition because that is a huge difference. These rats are almost genetically identical, but humans obviously aren’t. So I don’t know what the answer is.

Nick Jikomes

1:21:08 - 1:21:18

I think he’s working on some hypotheses that have to do with birth order and how much of the nutrition you get early in life as the last pup born or whatever, but TBD on that.

Nephi Stella

1:21:19 - 1:21:25

Yeah, I’m excited. He’s an excellent scientist. I can’t wait to see what happens.

Nick Jikomes

1:21:25 - 1:21:35

So what are some of the things that you’re working on today or that just the field in general is working on maybe using some of these new sensors that you think are going to produce some exciting new insights relatively soon in the future?

Nephi Stella

1:21:37 - 1:22:17

Well, yes, so one of the biological questions that we’re testing in my lab is really to try to better understand the effect of THC on mouse behavior. So we have a couple of papers coming out now that have been accepted. One, I’m excited, coming out in Nature, that actually will describe multiple things. The first thing is that we actually studied a very specific behavior in the mouse, which is the mouse approaching a sucrose bottle so that it’s a reward.

Nephi Stella

1:22:17 - 1:22:58

So the mouse is very excited, it comes close to the sucrose bottle and sips a little bit of sucrose and that’s a reward. And you can actually train the mouse in the Pavlovian reward training so they get actually excited by the reward coming up because there’s a little light that opens up. So what actually drives this behavior, which is really fundamental, especially if you think about reward and addiction, how can we understand this behavior? So we actually looked at the endocannabinoid signaling system in specific brain areas in the thalamus that projects to actually the nucleus accumbens in the brain.

Nephi Stella

1:22:58 - 1:23:32

The nucleus accumbens is really an area of the brain that’s involved in drug addiction. And what we discovered is that these neurotransmitters that go from the thalamus and project to the nucleus accumbens, the neurotransmitter release of those is regulated by endocannabinoids, also CB1 receptors. So, we actually discovered a new function for the endocannabinoid signaling system in REWARD, which is to simply control the approach and going after. So, that’s the first part that we discovered.

Nephi Stella

1:23:32 - 1:24:01

How did we discover that? We developed a machine learning pose estimation algorithm. So in this time of AI computing, I love new technology. So we got really interested in monitoring those little mice in the linear track and tracking them, just like when you do, uh, facial recognition, where you know where the eyes are.

Nephi Stella

1:24:01 - 1:24:16

We have little dots on the mice, on the tail, on the nose, and we look at them as they travel in this linear track. They walk, they stop, they groom, they rear, they explore. It’s fascinating. And then there’s the sipper, and they go and they sip.

Nephi Stella

1:24:17 - 1:24:37

the sucrose. So we can actually, with a video camera, monitor these mice, and then with this AI machine learning approach, basically analyze what they’re doing. And we can say exactly how quickly it approaches the bottle of sucrose, how quickly it disengages, and all of these behaviors. I see.

Nick Jikomes

1:24:37 - 1:24:57

So using these types of tools, you can start to ask questions like, OK, if we put a bunch of mice in the chamber, they might all approach the sugar water sometimes, but some do it more often. Some do it more quickly. Some are more interested in doing other things. And you can sort of ask, how do things like endocannabinoid levels relate to those individual differences?

Nephi Stella

1:24:57 - 1:25:25

Correct. So the way we know that is now we can actually add those sensors that I talked about in these mouse brain, specifically, for example, in the thalamus, that brain that I talked about, or the nucleus accumbens, and look at how these endocannabinoids are produced. in an activity-dependent manner. And it turns out that as the mouse approaches the sucrose bottle, there’s these endocannabinoids that are produced for a couple seconds.

Nephi Stella

1:25:25 - 1:26:16

And the idea, and that was another part that we kind of did in the paper by electrophysiology, is that these endocannabinoids in this brain area are going to activate those CB1 receptors. with specific dynamics and regulate neurotransmitter release, reduce a little bit of the GABA transmission, reduce a little bit of the glutamate, and that will participate in the neuronal network that will control this behavior of approach. So that was the punchline of this paper, is really discovering the fundamental function of, in this case, it’s 2-AG, the second endocannabinoids that’s mainly produced, one new function of 2AG in the brain, which is to actually control reward approach. And that obviously brings up a new question.

Nephi Stella

1:26:17 - 1:26:29

Can we use this new understanding to develop therapeutics for drug addiction or for cannabis use disorder? Now that we know that these endocannabinoids are produced there at that moment.

Nick Jikomes

1:26:30 - 1:26:53

Yeah, yeah. And so it’s just worth emphasizing the tools you’re talking about. So you not only have the machine learning-based video tracking tools that allow you to quantify and record what the animal’s doing at the overall behavioral level, but some of these new sensors are allowing you to very specifically record endocannabinoid dynamics, how quickly our endocannabinoid level is going up and down in particular parts in the brain.

Nick Jikomes

1:26:53 - 1:26:58

So you’re not just looking at some sort of very, very fuzzy high-level signal, you’re actually getting quite specific with this.

Nephi Stella

1:26:59 - 1:27:22

and very temporally specific in a very time dependent. And at the same time, we’re also measuring neuronal activity. So like that, we can actually connect what we talked about. Ah, there’s more neuronal activity, there’s probably more calcium, and therefore more endocannabinoids that are produced that are going to regulate those CB1 receptors on nearby neurons.

Nick Jikomes

1:27:23 - 1:27:34

What... What about, so cannabinoids have powerful effects. Exogenous cannabinoids have powerful effects. Endogenous cannabinoids have powerful effects.

Nick Jikomes

1:27:35 - 1:27:50

They’re tapping into this sort of homeostatic regulation that’s really fundamental. And this is why cannabinoids have so many different effects. One of the major side effects of THC, as we mentioned earlier, is the munchies, right? Animals tend to get hungry, they eat more.

Nick Jikomes

1:27:50 - 1:28:22

And yet, there’s this interesting and seemingly paradoxical situation that you mentioned earlier where, yes, they stimulate feeding behavior in the munchies, but long-term THC users tend to have lower BMIs, lower body weight. So what’s going on there? Well, that might connect to something that I also think is interesting, which is that There used to be, for a little while in Europe at least, this CB1 receptor-based therapy for obesity, basically. It was a drug called remanubant, which antagonized the CB1 receptor and caused people to eat less.

Nick Jikomes

1:28:23 - 1:28:38

This was before GLP-1s were developed. You’ve probably never heard of remanubant, most of you, because it got pulled from the market. What was the remanubant story and what does that sort of tell us about how powerful things like the CB1 receptor actually are and how difficult it is to play with them?

Nephi Stella

1:28:39 - 1:29:10

Yeah, the hypothesis there was very simple back then, Sanofi Pharmaceuticals in France. The idea was, well, if THC is an agonist that CB1 receptors and produces munchies, we’re going to do an antagonist and it’s going to block the munchies. And therefore, that was rimonabun or SR1 that people talk about in the field. And that molecule actually went all the way to being accepted in Europe.

Nephi Stella

1:29:10 - 1:29:13

So it passed all the clinical trials, the safety.

Nick Jikomes

1:29:14 - 1:29:16

But it actually made people eat less.

Nephi Stella

1:29:17 - 1:29:29

It actually made people eat less. You would be on this drug and you would show the patient a cookie and they wouldn’t eat it. When does that happen? you know, only when your CB1 receptors are blocked.

Nephi Stella

1:29:29 - 1:29:45

So then it’s accepted. And then what happens, you know, when you do a phase three, you have thousands of people. And then when it’s accepted, now you have hundreds of thousands of people. And therefore the likelihood of identifying a side effect is increased because more people are using it.

Nephi Stella

1:29:46 - 1:29:58

And what happened, and actually it didn’t happen right away. It happened after a couple of years, if I remember well. Some patients were getting depressed. because actually CB1 receptor controls mood.

Nephi Stella

1:29:59 - 1:30:34

And therefore, if you break the CB1 receptor, you don’t have the, you know, the reward aspect of going after your cookie, you might actually become more depressed. That would be to the point. Some patients were so depressed that they committed suicide. And that’s when I think it was like after the third or fourth suicide, then they took the drug out of the market, because the side effect is the too dramatic if people make, and that shut down so many drug development projects, trying to target the CB1 receptor in

Nephi Stella

1:30:34 - 1:30:51

the brain for society to stop eating. But then what happened is the field continued to work. And then what we discovered is the CB1 receptors on the liver. and the CB1 receptors on the liver are the ones that regulate metabolism.

Nephi Stella

1:30:53 - 1:31:22

Obesity, and therefore now there’s a very simple trick that you can do in drug discovery is make a CB1 antagonist that does not go to the brain. That’s very easy. You put a molecule and then it won’t pass the blood-brain barrier. So now you’re avoiding the depression, the suicide, because you’re not targeting the CB1 receptors in the brain, but you’re still targeting the CB1 receptors on your liver, that actually controls metabolism.

Nephi Stella

1:31:22 - 1:31:24

So that’s where the field is at.

Nick Jikomes

1:31:24 - 1:31:25

Have people actually done that?

Nephi Stella

1:31:25 - 1:31:34

Yeah, that’s happening right now. There’s some molecules that are getting closer to human clinical trials, and those CB1 receptors do not pass the blood-brain barrier.

Nick Jikomes

1:31:34 - 1:31:44

So we’ve tested those in animals, CB1 antagonists that do not cross the blood-brain barrier. Correct. And what is the basic overall effect in those animals?

Nephi Stella

1:31:44 - 1:31:47

Well, they eat less. They eat less, absolutely.

Nick Jikomes

1:31:47 - 1:31:56

So you get a similar Ramanubandh-like effect in terms of eating less, but they don’t show any indications of anxiety or depression-like behavior. Correct.

Nephi Stella

1:31:56 - 1:32:11

Okay. And actually, this is a field that I follow on the sides only, because then the question is, I understand how if you activate CB1 receptors on the liver, you’re gonna change metabolism. Okay, that’s fine. How I’m gonna digest food.

Nephi Stella

1:32:12 - 1:32:37

But what about the... the motivation to get that cookie. Is it really only the CB1 receptors in the brain? Well, no, there’s actually some research that shows that when you get hungry, there’s actually another one of those lipid signaling molecules that is produced by the liver or the digestive system, goes into the blood.

Nephi Stella

1:32:37 - 1:33:03

It’s a lipid, it passes the blood-brain barrier, goes to your brain, activates its own receptors and say, go eat. So now the CB1 receptor antagonists that are peripherally restricted, they regulate the metabolism in the liver. But at the same time, those lipids can still go to the brain and tell you, oh, go ahead and eat. What are those lipids?

Nephi Stella

1:33:04 - 1:33:07

Oh, I forgot what they are, but they’re also signaling lipids. Yeah.

Nick Jikomes

1:33:07 - 1:33:20

Yeah. Signaling lipids, not unlike cannabinoids, but they’re different ones. They’re made in the liver. They go through the bloodstream and they actually pass the blood brain barrier and they can affect motivation to eat, even though the signal arises in the liver.

Nick Jikomes

1:33:20 - 1:33:30

Fascinating. Absolutely. Wow. So as a cannabinoid biologist, knowing the history here with remanubant and how cannabinoids work, we’ve got the GLP-1s now.

Nick Jikomes

1:33:31 - 1:33:57

Lots of people are using them at this point. We’re coming out with new versions basically every month, it seems. So this is going to be a huge area of growth, both in terms of the number of people using them, but also the diversity of the specific versions coming out. Are there any things that we should look out for as we go into the second, third, fourth, fifth year of people using GLP-1s in terms of them potentially having effects on mood and on motivation akin to what the cannabinoid-based drugs did in the past?

Nick Jikomes

1:33:57 - 1:34:02

Or are they likely working in such a different way that those types of side effects are unlikely to be seen?

Nephi Stella

1:34:04 - 1:34:30

I think they’re working in a very different mechanism and therefore the results are going to be very different. I think those GOP1 field is fantastic. It’s so interesting and it has a lot of potential indication. We started with obesity and food intake, but actually it turns out that that might be also potential beneficial for some addictive drugs like nicotine and ethanol.

Nephi Stella

1:34:30 - 1:35:11

So, it has that parallel with the cannabinoid signaling system that it regulates food intake and reward. But I think they’re going to be, because the receptors are very different, because the receptors are expressed in different parts of the brain compared to the CB1 receptor, I think the GLP-1 drugs are going to be having different effects. And plus, it’s only the beginning of this entire field. Now they’re optimizing, like every field in medicine, transformative, it always starts and then we have a little bit of toxicity and then we optimize and we can actually develop a completely different therapeutic approach.

Nephi Stella

1:35:11 - 1:35:44

One thing that I’ve, this is anecdotal, but I thought it was fascinating. So anecdotal, N equal one, this individual in California was taking GLP-1 drugs to lose weight. And then it’s the birthday of their kid and there’s a beautiful cake and they want to eat the cake of their birthday and they don’t have the motivation at all. And that individual actually used cannabis, got the munchies and was able to actually enjoy the cake.

Nephi Stella

1:35:45 - 1:35:57

So that to me is fascinating because it really tells me that those signaling system are actually very different and might be, you know, it’s just an interesting, an interesting tidbit. I’m not sure. Yeah. Yeah.

Nick Jikomes

1:35:57 - 1:36:05

Because, you know, the person wants to eat the cake. They want to lose weight, obviously, but they want to participate in the ritual of the birthday.

Nephi Stella

1:36:05 - 1:36:06

That one day is okay.

Nick Jikomes

1:36:06 - 1:36:11

Yeah. So you might have a temporary tool in this case, cannabis. Exactly. An exit.

Nick Jikomes

1:36:13 - 1:36:35

So what do you want people to know? What do you want people to take away about cannabinoids, generally speaking? And I’ll let you decide what you want to say. Endogenous cannabinoids, THC and exogenous cannabinoids, but what’s something that you want to really emphasize or reiterate from people related to the biology that we discussed today that are listening and that are interested in cannabinoid-based therapies or substances of any kind?

Nephi Stella

1:36:36 - 1:37:10

Absolutely. I think the field has come a very long way and that’s 20, well, a couple of things. The first thing is when I started studying the cannabinoids 25 years ago, signaling system, I spent a lot of my time explaining to people that it has some medical properties and not only the high, the psychotropic effect. Now the field has recognized and understood that cannabis has medical properties.

Nephi Stella

1:37:11 - 1:37:36

And I spend quite a bit of time telling people that it’s not gonna cure everything. It’s gonna be very specific. So that’s one thing that I think I’d like to people to understand is that cannabis, we used to talk about it as the drug for the potheads. you wanna smoke out and watch a movie and things like that, that’s fine, or mind altering.

Nephi Stella

1:37:37 - 1:38:06

That’s one way to think about it. But I think the future, we will maybe even forget that aspect and talk about cannabis as a transformative medicine. Because it was that the research that we are doing right now resulted in medicines that are beneficial or might even cure some diseases where we had nothing. The first example that comes to mind is the epilepsy field with the Dravet syndrome.

Nephi Stella

1:38:07 - 1:38:33

When cannabidiol was accepted by the FDA, that was a landmark, because these poor little kids, they have really pretty much nothing to help with those seizures. And then you have the CBD that transform a little young girl that has hundreds of seizures per week to only a couple seizures per week. That’s a life-changing experience. So I think we’re at the very beginning.

Nephi Stella

1:38:33 - 1:39:01

And I’ve been in the field for so many years that I’ve seen it evolved. And the beginning was, okay, let’s figure out the receptors, okay? And then let’s figure out the enzymes, okay? And now where we’re at is small molecules in clinical trials that are very promising for specific diseases, which is why I think the cannabinoid field is gonna generate some transformative new medicines that are gonna help people.

Nick Jikomes

1:39:03 - 1:39:05

All right, Professor Nefi Stella, thank you very much for your time.

Nephi Stella

1:39:06 - 1:39:10

That was great, thank you.

This is a public episode. If you'd like to discuss this with other subscribers or get access to bonus episodes, visit mindandmatter.substack.com/subscribe

Wide release: Available for free by August 12, 2026, on Apple, Spotify, YouTube, etc.

Not medical advice.

Nick speaks with Dr. Pinchas Cohen about mitochondrial-encoded microproteins, including humanin and MOTS-c. Cohen discusses how these small peptides regulate metabolism, muscle mass, innate immunity, and neurodegeneration, and how natural genetic variants influence disease risk and longevity across populations.

TOPICS DISCUSSED:

* Mitochondria Beyond ATP: Mitochondria act as signaling hubs that regulate cell fate, innate immunity, and inter-organ communication.

* Hidden Genes in “Junk” DNA: Small genes in mitochondrial and nuclear DNA encode functional peptides overlooked by classical genomics.

* Humanin Discovery & Function: Independently identified by multiple labs; binds several receptors and shows neuroprotective and metabolic effects.

* Centenarian Humanin Variant: A specific peptide variant is highly enriched in Ashkenazi centenarians and appears to neutralize APOE4-related cognitive decline.

* MOTS-c as Exercise Mimetic: Induced by exercise; preclinical data show muscle preservation and metabolic benefits; limited human trial data exist.

* Ethnicity-Specific Variants: Genetic studies reveal “thrifty-gene” variants (e.g. MENTSH) that were adaptive historically but raise diabetes and obesity risk today.

* Peptides vs Small Molecules: Naturally occurring peptides may offer favorable safety profiles within physiological ranges, though most require injection.

* Emerging Regulatory Landscape: FDA compounding recommendations and voluntary registries aim to improve safety data collection for peptides such as MOTS-c.

ABOUT THE GUEST: Pinchas Cohen, MD is Dean of the USC Leonard Davis School of Gerontology. An endocrinologist by training, he leads research on mitochondrial microproteins and their roles in aging, metabolism, and neurodegeneration.on mitochondrial peptides in aging, metabolism, and neurodegeneration.

RELATED CONTENT:

* Article | Metabolic Stress & the Peptide MOTS-c: How Much is Too Much?

* Podcast | MOTS-c & Mitochondrial Peptides in Health & Aging | David Lee

KNOW YOURSELF:

* Visit the SUPPORT PAGE if you find value in this content. Includes discount codes + links to affiliate partners, including products to help you measure and manage your health.

Listen or watch on your favorite platform:

* Audio version: [Apple] [Spotify] [Elsewhere]

* Video version: [YouTube]

PRACTICAL TAKEAWAYS:

* Endogenous MOTS-c rises with exercise; trained individuals show higher circulating levels than sedentary ones.

* Unregulated “research-only” peptides often contain inaccurate doses or zero active compound; purity and sterility risks are real.

* When the science supports it, combining muscle-preserving agents with GLP-1 therapies is a rational direction, though long-term human data remain limited.

* Observational data link “Mediterranean diet” patterns with higher levels of certain mitochondrial peptides; causality is not yet established.

SUBSCRIBER CONTENT BELOW: Reference paper + episode transcript.

Reference Paper:

* Paper | Acute endurance exercise stimulates circulating levels of mitochondrial-derived peptides in humans

AI-generated transcript below. Beware of mistranslations!

Nick Jikomes

02:18 - 02:22

Do you want to start off by telling everyone a little bit about who you are and what your background is and what your lab works on?

Pinchas Cohen

02:24 - 02:52

Absolutely. So, my day job is that I’m the Dean of the School of Gerontology in the University of Southern California. That is a school that’s devoted to healthy aging, longevity, and infrastructure of the aging society in the United States and abroad. In addition, I run a lab, a research lab, working on so-called mitochondrial peptide or mitochondrial microproteins.

Pinchas Cohen

02:53 - 03:09

And I’ve been doing that for about 20 years now. Prior to that, I was at UCLA. I ran the Diabetes Research Center over there. That’s where my interest in these peptides started.

Pinchas Cohen

03:10 - 03:23

So, I was trained as an endocrinologist. and while I no longer see patients regularly, my focus is still on developing interventions that will improve human health.

Nick Jikomes

03:25 - 03:59

Yeah, and I think one of the things that we’ll talk about today is this question of thinking about therapies from the perspective of treating diseases, fixing problems after they’ve started, versus promoting healthspan or health or the prevention of disease from even arising in the first place. But you’ve done a lot of interesting research for a number of years on mitochondrial peptides. I’ve talked to other people in this field, including people who came out of your lab recently. So we’re going to discuss this from multiple angles and sort of build on what was on that recent episode.

Nick Jikomes

03:59 - 04:20

But let’s start with some of the basics. If you sort of step back and think about your career and mitochondrial peptides and everything you’ve done so far, Beyond ATP, beyond energy production, what has the work in your lab said about what mitochondrial do beyond their famous textbook role in producing ATP for the cell? What else are they doing, broadly speaking?

Pinchas Cohen

04:21 - 04:44

Yeah, the fifth grade mitochondria is a powerhouse of the cell. Let me say, first of all, when I went to fifth grade, which was a long time ago, I don’t think we even knew that. But I think the big transition scientifically is on two levels. The first level is the recognition, like you said, that there’s more to mitochondria than producing energy.

Pinchas Cohen

04:45 - 05:26

And the roles of the mitochondria that have evolved over the last 20 or 30 years is, number one, regulation of cell fate. So mitochondria involved in apoptosis, or how the cells end up dying in a programmed fashion. Also, the innate immunity that people have recognized mitochondria is very critical for. And finally, the role of the mitochondria, not just as a factory, but as a communication hub, sending signals to the nucleus, to other organelles, and to other cells and other organs.

Pinchas Cohen

05:27 - 05:59

So, that is conceptually the big advance in our thinking as a result of the mitochondrial peptide discovery. The other big change in our thinking as the result of this work is the expansion of the human genome. And if you Google right now how many genes there are in the human genome, You might get 20,000, sometimes it comes up with 25,000, but these are all traditional large genes that encode large proteins. Maybe you add in the so-called long non-coding RNAs.

Pinchas Cohen

06:00 - 06:51

But when we cloned the first peptide that wasn’t a fragment of another protein, and that was humanin 25 years ago, That really created a new class of molecules, which are these small peptides encoded from their own little genes that typically are located in places that there shouldn’t be, what was previously known as junk DNA, the dark genome. Now that we recognize that in fact there are so many potential peptides, hiding in our DNA, and only 3% of our DNA were thought to contain these classical genes. The 97% that we didn’t understand, you know, is where the action is gonna be in the coming years regarding research, and these peptides are a key part of that.

Pinchas Cohen

06:52 - 07:05

And by understanding that we have an order of magnitude more molecules to study, to explore, and to utilize for health, I mean, that is a real revolution in science.

Nick Jikomes

07:06 - 07:33

Yeah. And when I think back on my start in science, when I started in molecular genetics, and when I started learning about the history of genetics, broadly speaking, I remember learning about, you know, when we first sequenced the human genome and going into that in the 90s, the expectation was, well, we’re humans, we’re so much more complex than all these other organisms, we must have hundreds of thousands or millions of genes. And then, you know, it came back that we only had tens of thousands.

Nick Jikomes

07:34 - 07:51

And one of the One of the ways people thought about that was, well, actually, even though that’s surprising, we’ve got all of this, what was called junk DNA, that we don’t know what it’s doing. Some of it’s regulatory. So it’s not about encoding proteins. It’s about deciding where these proteins get made and how much in regulation.

Nick Jikomes

07:52 - 08:18

What you’re saying is there’s at least one more big layer here, which is... the traditional genes that we think about in defining the genome, let’s call them regular genes or big genes, there’s also these sort of smaller genes that we didn’t think of as genes originally. And one of the things they seem to be encoding are these small peptides or mini proteins, which are something that we’re sort of just starting to appreciate the significance of.

Pinchas Cohen

08:19 - 08:42

Exactly. You’ve nailed it. And I do also want to say something about peptides. which became sort of a general term, and people hear it all the time, you know, they’re in the New York Times every day, and, you know, people talk about peptides as if it’s a group of molecules that we understand, there might be 50 of them or whatever, and

Pinchas Cohen

08:42 - 08:58

we know what they are. In fact, I think peptides are very complex, and I divide them into three categories. The first are molecules that are peptides, but are fragments of larger proteins. Insulin is an example.

Pinchas Cohen

08:59 - 09:21

It’s a fragment of pro-insulin. GLP-1 is a peptide, but it’s really a component of the pro-glucagon gene. Even another very popular peptide in the hacker community called BPC-157. That is a fragment of a bacterial protein that was isolated from gastric juice.

Pinchas Cohen

09:22 - 09:41

So, there’s that group of peptides that are fragments. Then there’s the new generation of peptides that are peptides or microproteins encoded by the small genes we call small open reading frames. And that’s a really rapidly expanding group of peptides. MOTC is one of them.

Pinchas Cohen

09:41 - 10:14

There’s multiple others. But I think the newest category that is really going to represent what we’re going to see in coming years in drug development is entirely artificial designer peptides. that with the aid of AI and other tools, we’ll be able to create new peptides that don’t exist in nature and design them to do specific roles, bind certain receptors or other molecules. So that’s a very exciting direction in drug development.

Nick Jikomes

10:15 - 10:15

Yeah, yeah.

Pinchas Cohen

10:16 - 10:19

I just think this is a super interesting field these days.

Nick Jikomes

10:19 - 10:34

Yeah. So, you know, there’s much more in the genome, the nuclear genome, and the mitochondrial genome, as we’ll discuss, than we once thought. Peptides are basically small proteins or mini proteins. So they’re a sequence of amino acids.

Nick Jikomes

10:35 - 10:47

How small is small? And where’s the cutoff between a protein and a peptide? And then can you maybe talk about what the relationship is between the size of the peptide or protein and its specificity.

Pinchas Cohen

10:50 - 11:04

Right, so nobody agrees on the numbers. The current convention that’s being proposed is to define peptides as less than 100 amino acids. That’s quite large. Other people used to say 50.

Pinchas Cohen

11:04 - 11:25

I think that 100 is probably going to be the dominating number. When you go down to the lowest cutoff, we have peptides that are three amino acid long, including several. One that’s recently been approved by the FDA was originally known as SS31. That’s a tripeptide.

Pinchas Cohen

11:25 - 11:51

There’s several other naturally occurring tripeptides and also fragments that are being designed that are tripeptides. SS31, by the way, is really an artificially designed peptide that was discovered to have important biological activities. So, I think, I guess, one amino acid is an amino acid. Two, I guess, conceptually, is a peptide, and three are definitely legit peptides.

Nick Jikomes

11:51 - 12:05

So we have examples of peptides that are only three amino acids long. They’re very short. So they can get down to at least three peptides, even though you could imagine maybe dipeptides. And they go up to dozens and dozens.

Nick Jikomes

12:06 - 12:08

And eventually, we just start calling them proteins at some point.

Pinchas Cohen

12:09 - 12:10

Exactly.

Nick Jikomes

12:11 - 12:49

And so I would imagine that, you know, if I imagine a tripeptide, that’s just three amino acids, and I imagine something that’s 50 or 100, as the length of a polypeptide increases, and we add more and more amino acids, you go from just having a chain of amino acids to something that can be folded on itself and have a secondary and tertiary structure. And so I would imagine there’s a relationship between the size of the polypeptide, the size of the mini protein or protein that you are working with, and its specificity in terms of its overall structure and the types of interactions it could have with DNA, with other proteins and so forth.

Pinchas Cohen

12:52 - 13:29

Right, so the word specificity, I’m not sure that really applies necessarily. You’re absolutely right that the larger a chain of amino acid is, whether it’s a large peptide or a protein, the more defined its structure. And one of the things that we’ve been struggling with in the peptide research community is the fact that when you just look at them, let’s say Moxie, 16 amino acids and several other peptides of similar size, they don’t really have a structure when they’re in solution. So, they’re thought to be just floating chains of amino acids.

Pinchas Cohen

13:29 - 14:13

but when they actually are in a physiological setting and they interact with their partners, so whether it’s a GLP-1 that interacts with its receptor, it’s a very specific binding of a particular pocket in the receptor that then activates signal transduction. So, these peptides find a way to form a structure. Same thing with MOTC. When it identifies its binding partner, which in this case is a molecule called CK2, it fits exactly into a particular pocket with one or two critical amino acids from each side being involved in this interaction.

Pinchas Cohen

14:14 - 15:08

So I think that what we’ve found is that there is a general movement in the drug development community that when you develop, when you discover a peptide, say GLP-1, and you realize that it has important biological effect and it’s gonna be a successful drug, the first thing people tell you is try to find a small molecule that mimics the action of this peptide. And for GLP-1, that was recently done when Eli Lilly developed a small molecule that activates the GLP-1 receptor so it can be taken orally, which is different than the oral version of Ozempic that Novo Nordisk creates. So that’s because they could distill the structure of the business end of GLP-1 into a small molecule that fits exactly in the binding packet of the receptor for GLP-1.

Nick Jikomes

15:08 - 15:14

Got it. So it’s a completely different molecule, but it was designed based on knowing the structure and the interactions of peptide.

Pinchas Cohen

15:14 - 15:16

Both sides of the interaction.

Nick Jikomes

15:17 - 15:17

Yeah.

Pinchas Cohen

15:17 - 15:56

From peptides that we work on, sometimes we identify the fact that A, they seem to have more than one binding partner. So, the first peptide we discovered was discovered by three different groups in 2001. and that was called humanin. And since then, people have identified at least three different binding partners for humanin, each one completely different, each one mediates totally different effects, sometimes in a coordinated way.

Pinchas Cohen

15:57 - 16:04

and we realize there’s no way we can design a single small molecule that will have all the functions of that peptide.

Nick Jikomes

16:04 - 16:13

Yeah, so you can design a small molecule drug that might mimic the interaction with one binding partner, but not all of them if there are many.

Pinchas Cohen

16:14 - 16:51

Exactly. So I think that, and it will remain to be determined if this new orally available small molecule version of GLP-1 is really going to have the same biological effect as the natural molecule. I think it’s an experiment that we’re doing right now at the community in health. The concept that you need small molecules to create successful products that people will be willing to take for their health, I think that has been challenged over the last three or four years, much to my surprise.

Pinchas Cohen

16:52 - 17:26

there appear to be hundreds of thousands of people who have no hesitation injecting themselves with peptides. And I think that demonstrates that it’s no longer a critical part of drug development to create orally available versions of these products. So, that allows the peptide research community to really focus on other aspects of the modifications of these peptides to make them active products that have benefits.

Nick Jikomes

17:26 - 17:51

Yeah, yeah. And I think one of the things you’re talking about here is, you know, by nature, if you swallow most of these peptides, with certain exceptions, they’re going to be degraded by your stomach and GI tract. So they’re not orally active, in contrast to small molecule drugs. So there’s probably a history here where It was just sort of presumed, well, if you have to inject something to get around that issue, like a peptide, people just aren’t going to want to do this.

Nick Jikomes

17:52 - 18:04

So let’s maybe not work on it or not focus on it. Add to that the fact that a lot of these peptides that you and others have discovered and worked on were effectively invisible. We kind of didn’t know they were there. They’re encoded by smaller genes until recently.

Nick Jikomes

18:05 - 18:16

That probably has a lot to do with the history here and why peptides are sort of seeing a resurgence in popularity, both on the research side and among people who apparently are willing to inject therapeutics.

Pinchas Cohen

18:18 - 19:00

Exactly. You know, I think that, and I’m sure you’re aware, The FDA convened a committee called PCAC that reviewed six, seven peptides, six of which this committee recommended for legal compounding that will probably begin in a few months. and that completely changes the opportunity to do additional development, additional clinical research, and I think will also encourage classical pharma drug companies to invest in next-generation peptides that will end up as approved drugs. It’s a very exciting time to be in this field.

Pinchas Cohen

19:01 - 19:15

And I think that we can now explore the biology, the physiology, and the clinical benefits and, of course, the concerns related to these peptides in a much more organized fashion.

Nick Jikomes

19:15 - 19:26

Yeah. So let’s talk about some of the endogenous peptides and some of the peptides you’ve worked on. You mentioned one briefly called humanin. So talk a little bit about that one.

Nick Jikomes

19:26 - 19:34

What was the discovery process like there? When did that happen? And what did we start to learn about this peptide after you figured out it was there?

Pinchas Cohen

19:35 - 19:52

Right. So humanin was discovered simultaneously by three different groups. My lab actually cloned it when we were back in Philadelphia in the late 90s. And to be honest, when I saw it for the first time, I said, this cannot be right.

Pinchas Cohen

19:53 - 20:15

It’s obviously an artifact. And I told my team, go back and figure out what’s really happening here. And the story was that we were working, so I started my career working on growth hormone and IGF-1. and growth hormone-related peptides, like growth hormone-releasing hormone and other growth hormone secretogogues, some of which, by the way, are now part of the peptide universe.

Pinchas Cohen

20:17 - 21:07

And we were very interested in the biology of how growth hormone works, which is A, to stimulate something called IGF-1, which promotes many of the effects of growth hormone, but also other activities, including making something called IGF-BP3, which binds to IGF and regulates its activities, and also, we thought, is involved in some processes related to diabetes and Alzheimer’s disease. And using various approaches, we recognized that IGF-PP3 acts beyond binding IGF-1. because when we eliminate the IGF system form models or cells, we still get a biological effect of IGF-PP3.

Pinchas Cohen

21:07 - 21:47

So we told ourselves it must interact with another protein to do that, and we utilized a technique called the yeast-to-hybrid system. which is really a fishing expedition. You take a molecule, that’s the bait, in this case it was IGFPP3, you transfect it into a yeast cell, and you also transfect a library of a cDNA, which is really a universe of molecules, proteins, that you can put into the same yeast. And if your protein and one of the proteins in the library interacts, It allows the yeast to survive.

Pinchas Cohen

21:48 - 21:54

And then you can pinpoint what proteins bind to your protein of interest. Yeah.

Nick Jikomes

21:54 - 21:59

It’s like you’re fishing. You have a protein. Yeah. You want to see what sticks to this protein.

Nick Jikomes

21:59 - 22:04

Is there anything out there? And you can put it into a yeast cell and something will stick or not. And then you can see it.

Pinchas Cohen

22:05 - 22:26

Exactly. And, you know, it’s a complicated technique. And, you know, we turned out that IGF-B3 binds more than IGF. We published a couple of solid observations with biological significance, including a molecule, a nuclear receptor called RXR, and another receptor known as the transferrin receptor.

Pinchas Cohen

22:27 - 23:01

But we kept cloning a piece of DNA or RNA encoded from it that comes from a region of the mitochondrial DNA known as the 16S ribosomal RNA, which is a structural RNA involved in translating proteins into mitochondria. And that was never supposed to encode an actual protein. It was supposed to encode a structural RNA. So I was like telling myself, how could this be something isn’t quite right.

Pinchas Cohen

23:01 - 23:32

What are we looking at here? And then at the same time, two other groups were using completely different visions to identify new peptides, new proteins. One was a gentleman named John Reed in the Burnham Institute, who was a very famous Bax scientist, cell death scientist. And he also believed that Bax and similar molecules have new protein partners that regulate the process of apoptosis.

Pinchas Cohen

23:32 - 24:11

and he pulled out humanin using a yeast two hybrid system with Bax as bait. And he didn’t know what to do with it for a couple of years. And then the gentleman who ended up naming humanin was a Japanese scientist called Iku Nishimoto, working in Keio University. And he was an Alzheimer’s researcher, and he believed that there would be endogenous proteins that protect brain cells from dying in response to amyloid beta, which is the classic business end of the Alzheimer’s process.

Pinchas Cohen

24:12 - 24:53

And he used another screening tool, which was a death trap, where you throw amyloid beta on cells, neuronal cells, they all die, then you transfect the library into these cells prior to that, and if some cells survive, it’s because they express a protein that protects them. and he cloned the same thing, the human region from the mitochondrial DNA. And while all of us were beginning to consider the possibility of a small open reading frame that encodes a protein peptide, he actually, you know, said, this is what’s happening here. He really innovated the field by recognizing this new phenomenon.

Pinchas Cohen

24:54 - 25:20

and ended up publishing the first paper on humanin and getting the right to name it. And immediately, I contacted him and his team, and we started working together. And the other folks at the Burnham Institute also jumped in, and we recognized that we got a new phenomena here. Small peptides encoded by new open reading frames that weren’t previously recognized as real genes.

Nick Jikomes

25:20 - 25:31

Yeah. So you find a gene, effectively, that didn’t really look like a normal gene. It’s in the mitochondrial genome, not the nuclear genome. And you’re working in yeast, it sounds like.

Nick Jikomes

25:32 - 25:34

Why is the name of this thing humanin?

Pinchas Cohen

25:38 - 25:54

who, you know, who unfortunately passed away from cancer a few years ago. He had this vision that when people develop Alzheimer’s disease, they lose their humanity. So, Humanin was supposed to restore their humanity. Nice name.

Pinchas Cohen

25:54 - 26:08

I liked it. I ended up naming a bunch of other peptides completely differently. We can talk about that. But, of course, the yeast-to-hybrid system was a tool to identify this molecule.

Pinchas Cohen

26:09 - 26:48

But since then, humanin has been the most studied microprotein. There are, I think, about 500 papers now published on humanin from, you know, 50 and more different laboratories around the world. All the work being done today is either in human cells or mammalian models like mice, and there’s some remarkable activities for humanin, including promoting longevity, improving heart disease, really effectively preventing Alzheimer’s, cardiovascular benefits. Really, it’s a very potent molecule with significant effects.

Pinchas Cohen

26:49 - 27:40

And I think you might be aware that about 12 years ago, I started a biotech company, which was called Cobar, together with my college roommate, Nir Barzilai, who is also a big longevity researcher. And we recognized the potential of human in the company, Cobar, really started out hoping to develop human analogs as drugs. We ended up changing course and developing MOXIE analogs, but we believe that humanin will have substantial benefits as an intervention. That didn’t happen, and I think it’s a shame because I think there’s a lot of potential for humanin We have several patents of humanin altogether.

Pinchas Cohen

27:40 - 28:01

There are more than 60 patents related to humanin filed that you can see online. Most of them are expired. So I think there’s less interest in the pharma community to work on humanin because the IP landscape is problematic for them. But I believe it does have potential to become a therapeutic.

Pinchas Cohen

28:03 - 28:14

There were issues related to human in terms of formulation and stability that really sent us into a different direction about 10 years ago.

Nick Jikomes

28:14 - 28:26

Yeah. Well, just thinking about the biology for a moment, can you give us a basic sense of the structure and function of human and how big is this thing and what does it look like? And what are some of the salient findings that have been out there in terms of what it’s doing for cells?

Pinchas Cohen

28:27 - 28:43

Right, so it’s a 24-amino acid. It’s a naturally occurring peptide. It’s reasonably well conserved. There is a version in fish There isn’t quite aversion in flies.

Pinchas Cohen

28:44 - 29:10

Maybe there is something in worms, maybe not. But all mammals have human in and it’s very well conserved. Interestingly, there’s a phenomena called numpt. I don’t know if you’ve heard that word, Nick, but over the course of evolution, fragments of mitochondrial DNA, large fragments of mitochondrial DNA, got incorporated into the nuclear DNA.

Pinchas Cohen

29:10 - 29:31

Now there’s nuclear mitochondrial DNA transfer. Why that happens is not clear, but it happened multiple times. So what that created are analogs of humanin, which is a mitochondrial microprotein, found a nucleogenome. And there’s over 20 versions of humanin in the nucleogenome.

Nick Jikomes

29:32 - 29:35

So it probably got duplicated and expanded multiple times.

Pinchas Cohen

29:35 - 29:49

Exactly. So I don’t know about expanded. a fragment came out of the mitochondria of DNA gets incorporated in the human, in the nuclear DNA. And then over evolution, it becomes degenerate.

Pinchas Cohen

29:49 - 30:06

So none of those versions of human in the nuclear DNA are exactly identical to human. Yeah, yeah. But some of them do exist. And we identified using mass spec, mass spec approaches that they’re found in tissues.

Pinchas Cohen

30:07 - 30:30

and there are some methylation sites next to some of them that seem to regulate their biology. So, humanin seems to be a very important fundamental molecule, and it’s not just, I think, a coincidence that it was the first one discovered and discovered simultaneously by several groups. It is a very important functionally. There are several receptors for humanin.

Pinchas Cohen

30:31 - 31:14

There’s one which is a trimeric receptor, similar to the IL-6 family of receptors, that probably regulates a lot of the effects on the neuronal system and the Alzheimer’s prevention effects of humanins. There’s another set of receptors called formal peptide-related receptors, or FPRL receptors, that HumanIN also binds. HumanIN interacts with IgF-BP3 and modulates both the growth hormone-IgF axis, and IgF-BP3 carries HumanIN into circulation. There might be additional proteins that HumanIN interacts with.

Pinchas Cohen

31:15 - 32:09

With all these different pathways, human inregulates multiple signaling pathways in cells, including classical pathways like AKT, as well as ERK pathways and others, and has really interesting profound effects in multiple systems, both in vivo and in vitro. I think the most exciting things in the human field for me was that about four or five, three or four years ago, again, working with my colleague Nir Barzilai, who has put together a cohort of centenarians, and 500 or so centenarians, all of them happen to be Ashkenazi Jews. And he’s been studying this population for 20 years now trying to identify genetic explanations to their exceptional longevity.

Pinchas Cohen

32:10 - 32:47

And together we embarked on sequencing the mitochondrial genome in centenarians to identify maybe there’s something there that helps them become so old, healthily. We discovered that there’s a variant, I don’t want to call it a mutation, but it’s a naturally occurring variant of humanin, which now we call P3S humanin. It’s a one amino acid difference. it’s what we call a Jewish specific version of human in and found 1% of Ashkenazi Jews in general.

Pinchas Cohen

32:47 - 33:08

But when we look at centenarians, its frequency is 20 times higher. So it really is enriched in centenarians. And Those people who carry this version of humanin are completely protected, even at age 100, of getting any kind of cognitive decline, which is remarkable.

Nick Jikomes

33:08 - 33:15

So none of them that have that variant show signs of Alzheimer’s or anything like that, even at 100 years of age or older.

Pinchas Cohen

33:16 - 33:44

Yes. And even more exciting is the fact that I think most people are familiar with ApoE4, which is sort of the big genetic risk factor for Alzheimer’s disease. And when people have ApoE4, they’re much more likely, A, to die younger, but of course to develop Alzheimer’s disease. And by age 70, people with APOE4 often begin to experience the results of having that gene.

Pinchas Cohen

33:45 - 34:41

By age 80 and 90, you’d expect all of them to have some problem. Lo and behold, we found that among 100-year-olds, There are people who carry the APOE4 gene with no evidence of any biological issues, and almost all of them also carry the P3S human inversion, which neutralizes the APOE4. We had a very nice paper a couple years ago showing that there is a direct physical interaction that apoE4 and humanin are related both genetically, there’s a genetic kind of crosstalk, physical interaction, and biological relevance related to the protective effects. And, you know, there’s a whole science related to APOE4 resilience, because people recognize that some people do make it with APOE4.

Pinchas Cohen

34:42 - 34:58

And now, this is one example of a direct binding of human into APOE4 to negate its negative effects. And, you know, that’s another possibility to explore human as a potential intervention. So, it’s a fascinating molecule.

Nick Jikomes

34:58 - 35:09

Yeah. So APOE4, it’s connection to Alzheimer’s. People with APOE4, they’re predisposed to Alzheimer’s. They’re more likely to get it at all.

Nick Jikomes

35:09 - 35:40

They’re more likely to get it more severely earlier and die earlier. This subset of just Ashkenazi Jews has this variant of humanin, a mitochondrial peptide that is associated with their exceptional longevity and appears to prevent the development of Alzheimer’s even in APOE4 individuals within that subpopulation. So there’s some kind of connection here between neurodegeneration and humanin and what it’s doing. If we step back a little, what else do we know about its basic function in the cell?

Nick Jikomes

35:40 - 35:42

Does this connection make sense to you?

Pinchas Cohen

35:45 - 36:54

You know, that observation came after 20 years of human research, where the focus was on human and binds one of two receptors, there’s signaling that’s important for cell function, including in neuronal cells in the Alzheimer’s setting. And I should point out that we show that mice that are engineered to express APOE4 and therefore develop brain dysfunction, somewhat similar to Alzheimer’s disease in humans, you can ameliorate that by injecting these mice with this version of humanin called P3S-humanin. But prior to that, multiple groups have shown that just taking classical Alzheimer’s mice models that triple transgenic and 5x fad and such models of Alzheimer’s disease that don’t involve the APOE4, even it still has very profound effects on delaying the progression of Alzheimer’s-like symptoms. So, it seems to have multiple ways of preventing dementia, the client.

Pinchas Cohen

36:54 - 37:47

And I should point out that You know, a lot of people are working in this field, particularly in the human field, are biologists, cell biologists, physiologists, doing important work. We introduced a new angle to this whole peptide research field by incorporating genetics. and by that I mean we started doing these versions of GWAS, and I think, Nick, your audience is familiar with the term, genome-wide association studies, where you take large populations of people, all of which had genotyping or gene sequencing performed, then you look at a subpopulation with a certain condition, whatever disease you like, and compare them to another type of population that’s otherwise similar.

Pinchas Cohen

37:48 - 38:39

And then you see if there’s any particular genetic variant that’s represented in one of those groups that can be linked to the condition or disease you’re studying. And we looked, so we’ve been employing this approach using mitochondrial genetics for a decade now, using multiple cohorts. Our favorite, which is based in our school, led by a woman called Aileen Crimmins, is called the Health and Retirement Study. 30,000 people followed now for 40 years, and some of them have passed, and we have DNA and RNA information about them, and that’s our big playing field, but we’ve also worked on the so-called UK Biobank, in other cohorts, like the MESA cohort.

Pinchas Cohen

38:41 - 39:25

Many of them are in my area of interest, which is diabetes, but also in Alzheimer’s disease, we also looked at other cohorts. And in all those cohorts, we employed a version of GWAS that we call MIWAS, which is mitochondrial-wide association study. The reason for that is because, you know, When GWAS became popular, and it was transformative in science, the folks who worked on GWAS, mostly statisticians who applied their skills to biology, decided to ignore the mitochondria. leaving all the mitochondrial information on the table, which we gladly took over.

Pinchas Cohen

39:25 - 39:53

The reason for that was that mitochondrial DNA is really interesting, Nick, in the sense that you only get it from your mother, as opposed to all the other genes that you get from both parents. These nuclear genes undergo recombination, and you also have two sets of them, one for your dad and one for your mom, and you can have multiple combinations of these genes. not so for mitochondrial DNA. You only get it from your mom.

Pinchas Cohen

39:53 - 40:25

And while there are many mitochondria in every cell and many chromosome, mitochondrial chromosome in each mitochondria, but they’re all the same, theoretically. There’s no double versions, no recombination. So, each ethnicity has very specific variations in the nomadic country of DNA. I’ll give you an example of P3S in Jews, but we just published a paper last week, Nick, on a new peptide called Mench, It’s a fascinating peptide.

Pinchas Cohen

40:26 - 41:06

And we were looking for variations in mitochondrial DNA associated with diabetes. And we looked at the health and retirement study, and we found something. And then we subdivided our analysis by ethnicity. And lo and behold, this variant, which predisposes people to diabetes and obesity, in an open reading frame encoding a peptide that we named mensch, that variant is only found in really Mexican and Mexican-Americans, or as my colleague Kelvin Yen, who discovered this, likes to say people native to

Pinchas Cohen

41:06 - 41:07

the Americas.

Nick Jikomes

41:07 - 41:14

Right. Indigenous American populations, which includes a large percentage of people that are Mexican or Latin American.

Pinchas Cohen

41:14 - 41:32

Right. So really, when we say native to the American, the immediate thought is we’re talking about Native Americans, who, of course, by the way, also carry this variant. But they’re a very small community, relatively speaking. There are many, many millions of Mexicans and Mexican-Americans who carry this gene.

Pinchas Cohen

41:33 - 42:05

But, of course, so do Native Americans. What this happens to be is it’s a classic thrifty gene. And by that, people who coined this term many years ago mean that When you live in an environment where there’s very limited nutrients and you really need to hold on to every calorie to survive, if you have a gene that burns a lot of calories, that’s not a very efficient way to manage nutrients.

Pinchas Cohen

42:05 - 42:37

So, up until a hundred years ago, evolution favored thrifty genes. And the mutation that we discovered in mensch, and mensch helps you burn fat. So, when you have very limited calories, when people migrated from Asia through Alaska into North America and into South America, the mutation that inactivates mensch may have been beneficial, right? Because it helped you hold on to calories.

Pinchas Cohen

42:38 - 43:14

Now, when everybody has a pickup truck and there’s a McDonald’s in every corner, it’s no longer a beneficial gene to have the mutation in mensch. So, we discovered this new variant in a new open reading frame encoding a new peptide that is associated with much higher risk of diabetes and obesity. And 20% of Mexican and Mexican-Americans carry that. Certain Indian communities, like the Pima Indians, it’s over 50%.

Pinchas Cohen

43:14 - 43:47

It clearly correlates with diabetes risk. And that SNP is only found in that population. But then we take this peptide, inject it into mice or treat cells with it, and it has clear evidence that it reduces weight, it improves blood sugar, and works in many ways like, I don’t know, GLP-1, perhaps, although quite differently. So, that’s an example of how we utilize genetics to discover new microproteins.

Pinchas Cohen

43:47 - 44:40

By focusing first on the disease. We’ve done it a different time with a peptide we call Schmooze, where my colleague, Brendan Miller, who is now working at the Salk Institute, was interested in Alzheimer’s disease and believed that beyond humanin there might be other mitochondrial microproteins that are involved with Alzheimer’s. And he conducted this genetic screen called MIWAS on a bunch of cohorts that are focused on Alzheimer’s disease. And he cloned this SNP, he cloned this peptide called schmooze, that he named schmooze, which is also neuroprotective, has the mutation and activates the protein, and it, when administered to mouse model for Alzheimer’s disease, it improves the outcome.

Pinchas Cohen

44:41 - 44:53

And we’ve done it another time with MOTC. Now, when we discovered MOTC about 15 years ago, and my colleague David Lee was a postdoc in my lab, worked on it, and

Nick Jikomes

44:55 - 44:58

he basically guessed what it’s going to do.

Pinchas Cohen

44:59 - 45:22

He threw it here, he threw it there, tried in this model, tried in that model, he noted this has metabolic activity, did some more stuff, really honed in on exactly what’s going on, showed that you administer it to mice, you get weight loss and improved metabolism. Great story. but no origin story. And I love origin stories.

Pinchas Cohen

45:23 - 46:08

So later we went back, and in collaboration with some folks in Japan, including a gentleman called Hirokuma Gai, who spent some time in my lab and is now back in Japan, we looked at the Japanese cohort. that was focused on aging. And we looked for variants in this MIWAS approach that are associated with muscle mass, because Hiro is an exercise physiologist originally. Lo and behold, we identified a mutation in the MOTC gene that renders it completely inactive, which is found in 8% of Japanese people.

Pinchas Cohen

46:09 - 46:35

These 8% of people in Japan have a high risk of developing diabetes and also have low muscle mass or sarcopenia. That makes complete sense in the context of what we now believe MOTC could be useful for, which is a combination of prevention of metabolic dysfunction, maintaining weight, and most importantly, maintaining muscle mass.

Nick Jikomes

46:36 - 46:50

Yeah. And if people are interested in MOTC more, I’m sure we’ll talk about it here. But as you may or may not know, I just did a discussion with Dr. Li where we did a lot of discussion of MOTC. So I would encourage people to listen to that one in conjunction with this one.

Nick Jikomes

46:50 - 47:25

But Professor Cohen, one of the things I want to emphasize here for people that I think is really important comes from your genetics perspective that you’re bringing on all of this, because people often overlook this, even scientists oftentimes, which is for everything that you’ve talked about so far, all of these mitochondrial peptides, MOTC, MENSH, SMUS, et cetera, there’s natural variation in all of them. We all have different variants and those can be substantial in the sense that they protect or predispose us to disease. They help our metabolism in certain ways.

Nick Jikomes

47:26 - 48:02

or they tell us something about our ancestry and our origins. What’s important about this from a health perspective, in my view, is when you talked about that variant of mensch, I believe, that’s found in certain indigenous American populations, If you connect that back to where people migrated from and where populations came from, there’s this idea that, well, if these are thrifty genes, they may have helped in an ancestral environment, but they predispose us to disease in the environment of today, which is an environment of abundance. And anytime we think about ancestral environments, we also have to ask, whose ancestors?

Nick Jikomes

48:02 - 48:17

Which part of the world? Which environments? What I’m sort of queuing up here for our later discussion is when we start talking about the exact the use of exogenous peptides among people today, we also have to ask, which variants are they taking? And I think that’s interesting.

Nick Jikomes

48:18 - 48:35

But going back to the MOTC stuff, I talked a lot with David about MOTC and animals and some of the work that you had done with him on the preclinical side. What do we know about MOTC in humans today? Have you done any clinical work on it? Do we know how it affects human beings?

Pinchas Cohen

48:36 - 49:06

Yes, so again, to be honest, when we formed this company, Cobar, over a decade ago, We relied on the observation that David Lee and I made in mice, and there were A, that it prevented weight gain in a high-fat diet context, and B, that it really potently, completely prevented the development of fatty liver in these mice that, like humans, develop it when you give them a bad diet.

Nick Jikomes

49:06 - 49:07

Yeah, it was striking.

Pinchas Cohen

49:08 - 49:57

And it was striking in mice. So we formed this company, we raised enough money to initially looked at human and unsuccessfully, we transitioned to MOTC, we did some analoging of MOTC, and came up with a variant that was one amino acid different, that just extended the half-life somewhat, but otherwise was basically MOTC. We took it through IND-enabling studies and toxicity studies of multiple species, sailed right through. We conducted phase 1A clinical trials in 60-some people with very high doses of, you know, 200 milligrams per person with no evidence of side effects whatsoever.

Pinchas Cohen

49:57 - 50:37

Then we conducted a phase 1B clinical trial on 23 people that were randomized to placebo or the 4211 MOTC analog to dose of 20 milligrams per day for four weeks. And these were all obese people with fatty liver, pre-diabetics, if you will. And we’ve shown that the four weeks of MOTC, daily MOTC injections, resulted in some weight loss, improvements in blood sugar and improvements in liver enzymes that are representative of the fatty liver status.

Nick Jikomes

50:37 - 50:39

So, that was once daily injections for four weeks.

Pinchas Cohen

50:40 - 50:57

Correct. So, this was great on one level. It really proved that we had no drug-related side effects. You know, the dose, semi-arbitrary, as sometimes that is the way it goes in drug development.

Pinchas Cohen

50:57 - 51:36

You have to pick a dose, It wasn’t a bad dose in any sense of the word. People who are taking MOTC in the biohacking community often use lower doses, and that may emerge as the right approach. But what happened in 2020 when we were conducting these studies is that Ozempic came out and exploded. And we realized that our modest improvement in weight and blood sugar could match the incredible benefits of potent GLP-1s.

Pinchas Cohen

51:37 - 52:46

And our investors decided to not continue developing MOTC because it appeared not to offer greater benefits than GLP-1. What we didn’t understand at the time, we started really getting that about six or seven years ago, is that moxie sure has these metabolic effects on weight, but its main action is on muscle, and the key benefits of MOXIE are in maintaining muscle mass, in preventing sarcopenia, as noted by the genetic variant, multiple animal models, and most of this work has been done by Hirokuma Gai, whom I’ve mentioned, who really is a muscle biologist and an exercise physiologist, and he showed in multiple studies that you can take mice, For example, mice subjected to cast immobilization, which you basically, like anybody who broke a leg or an arm, you get put in a cast for a couple months to let the bone heal.

Pinchas Cohen

52:47 - 53:03

When you do that, you get major atrophy of the muscles in that limb, which can be a problem. Hero treated mice that we put a cast on with MOTC and completely protected them from muscle mass.

Nick Jikomes

53:03 - 53:17

So you put a little cast on mice, and normally the muscle would atrophy after some time, as you’d expect. But if you did that while giving them MOTC, probably once daily, it prevented that muscle atrophy. Exactly. Wow.

Pinchas Cohen

53:19 - 53:20

A short term, a few weeks.

Nick Jikomes

53:21 - 53:35

Wow. So we see that in animals. You’ve done some of those small initial trials, which didn’t proceed because of the GLP-1s coming to the forefront of everyone’s minds. What about endogenous MOTC in human beings?

Nick Jikomes

53:35 - 53:46

It’s being called an exercise memetic, and it’s supposedly induced by physical activity. What have people seen with regard to how endogenous MOTC changes with physical activity in human beings?

Pinchas Cohen

53:46 - 54:22

So the exercise connection. That is now where our focus is, and this is where we think there’s potential utility of MOTC as an emerging therapeutic. And the word exercise mimetic is something we came up with right from the start. because that’s a great concept to have, but it’s also exercise-induced, so the endogenous levels, so we also, under the strength of my lab, is that we develop assays for all these peptides, including humanin, MOTC, and others.

Pinchas Cohen

54:23 - 55:00

So, when we measure MOTC levels in people, in humans, as well as mice, before and after exercise, we see a dramatic increase in MOTC within minutes, but also in people who are trained versus sedentary, we see much higher levels in the trained people. So, it’s exercise-mimetic, exercise-induced, but also exercise-enhancing. So, this is David Lee’s work. you know, I was involved with that, but really with him and his postdoc, Joe Reynolds, or his students.

Pinchas Cohen

55:01 - 55:55

And what we’ve seen is that if you take mice, particularly old mice, and I’m sure David talked about it in the podcast we did with him, it enhances the exercise capacity, it allows mice to run significantly longer through And we’ve done other studies that are not published yet that shows that there’s clear improvement in strength in response to administering MOTC. The whole thinking on MOTC shifted for us from the weight metabolism regulation that it is involved in to the muscle biology aspect, which is the key effect. And now when we think about how could we harness that with MOTC to advance it into clinical development, we think of those situations where you have muscle loss.

Pinchas Cohen

55:56 - 56:29

So I gave castor mobilization as an example because it’s an acute, quick way to show the effect. But the biggest issues with muscle in today’s society is what we call sarcopenia of aging. You’re a young guy, Nick, but I’m, you know, well into my 60s, and I can tell you, you lose muscle mass when you age, even if you try to exercise, which I do, but to keep your muscle mass, you have to exercise and lift weight many hours a day, and frankly, no one can do that.

Pinchas Cohen

56:30 - 56:55

So, the chase, the race for new drugs that promote muscle growth, that’s the biggest thing in drug development today. If you think about what was the biggest problem we were facing 15 years ago, it was obesity. It was the main cause for so much of what ails Western society. We conquered obesity, Nick.

Pinchas Cohen

56:55 - 57:04

GLP-1 did the job. One out of eight Americans is on it now. It’s a $60 billion industry. It’s effective.

Pinchas Cohen

57:04 - 57:37

It reduces mortality. It’s an incredibly successful drug. Our new problem as a society is sarcopenia, and particularly in the GLP-1 context, as great a drug as it is, The biggest issue with GLP-1 is that one-third of the weight you lose is muscle. And even though the overall benefits are clear, that muscle loss might come back to haunt us later.

Pinchas Cohen

57:38 - 57:45

Of course, you can tell people you should lift weight every day for a couple hours and that will slow it down. Most people don’t do it.

Nick Jikomes

57:45 - 57:54

Yeah, yeah. And for those, I mean, for those who don’t lift weights, gaining one or five pounds of muscle is a lot of focused work, believe me.

Pinchas Cohen

57:55 - 58:20

Right? So, I think that utility of agents like Mod-C in the context of companion drugs for GLP-1 is very exciting in the context of acute events like gastrointubation or post-surgery. where you lose muscle mass, that’s compelling. And as a treatment for sarcopenia of aging is also a very exciting direction.

Pinchas Cohen

58:21 - 59:13

Now, clearly there are people out there in the biohacking community who take illegal moxie that’s labeled for research use only because they think it will enhance their exercise capacity. We don’t have data for that in humans. But I think there’s a legitimate important medical use for MOTC and MOTC analogs in the future for maintaining muscle mass, and secondarily, bone density. So, there’s a number of papers, not from our group, but from other groups, showing that in models of osteoporosis, MOTC ameliorate that prevents that, and the osteoporosis, which is a byproduct of aging as well as muscle loss, can also be protected by MOTC.

Pinchas Cohen

59:14 - 59:37

And so there’s all these emerging new roles for MOTC that I think still make it a viable candidate for drug development. And now that it’s going to be approved as a compounded peptide, We hope to see what are going to be the results that people will see and perhaps do some more clinical studies to demonstrate specific effects.

Nick Jikomes

59:37 - 59:47

Yeah. So it sounds like endogenous MOTC is induced under states, particular states. Exercise will do it. Other forms of metabolic stress will do it.

Nick Jikomes

59:48 - 1:00:04

So there’s a pattern of induction. It’s not necessarily cranked up all of the time. In the studies that have been done in animals and humans, there is some kind of administration protocol. Some amount of MOTC is given either once daily or intermittently.

Nick Jikomes

1:00:04 - 1:00:21

Let’s say someone out there was really excited about MOTC. They’re listening to your podcast here and they’re saying, wow. prevent sarcopenia, other metabolic benefits, might enhance my exercise, I’m going to start injecting MOTC every single day. It sounds like there’s only upside.

Nick Jikomes

1:00:21 - 1:00:31

Based on what we know about the basic biology and the effects on cells and metabolism, any potential downside to taking too much MOTC for too long for an animal or a human?

Pinchas Cohen

1:00:32 - 1:00:48

Let’s start with the fact that A, it’s illegal right now. B, the peptide that people are buying on the internet. We’re not sure what they are. Their purity, their sterility, their packaging is unknown.

Pinchas Cohen

1:00:49 - 1:01:39

And while, as far as we know, there hasn’t been any reports of problems related to that, there were a couple of deaths a few years ago in people who were injecting BPC-157 that weren’t related to the peptide itself, but they were contaminated and people died from sepsis. So, I am strongly against using any peptide that’s illegal and formulated in ways that we don’t know. Let’s start there. The FDA advisory panel that met a couple of weeks ago recommended that six peptides, including MOTC, would become legal to manufacture and compound under a physician’s supervision with a prescription.

Pinchas Cohen

1:01:39 - 1:02:24

And of course, right now, most of the people who appear to be taking MOTC don’t have a real doctor involved, there’s no prescription. So we’re moving into a new era where this will actually be legal reasonably safe from the perspective of the material, that doesn’t imply automatically that it’s going to be safe long-term at any dose, but at least you know you’re not going to get septic shock. And it will allow us to learn more about this. It’s not something that FDA approved as a drug for any kind of specific indication, and we do not have long-term data on the safety of MOTC.

Pinchas Cohen

1:02:25 - 1:02:42

Having said that, There are 250 papers published on the MOTC, well over 30 labs all over the world, mostly administering it to mouse models, and there’s been no safety signals whatsoever. None.

Nick Jikomes

1:02:42 - 1:02:46

No negative issues have clearly emerged. No negative issues.

Pinchas Cohen

1:02:46 - 1:03:31

And, you know, the benefits that people have shown in animal models really were typically in limited studies in three weeks, four weeks, six weeks, with the exception of one study, again, led by Dr. Lee and Dr. Reynolds, with my lab being involved in, which administered MOTC to mice three times a week for the entire length of their life, starting at middle age. And we demonstrated a 10% increase in lifespan. So I think that’s compelling that at least it’s unlikely to cause any kind of hidden problem in the future.

Pinchas Cohen

1:03:32 - 1:03:59

We also have the opposite effect that people who don’t make mochi, that 8% of the Japanese community, they seem to have at least decreased healthspan. We don’t know the lifespan yet. But clearly, not having mochi isn’t good. getting MOTC supplementation in mice seems to be beneficial for health men and potentially lifespan.

Nick Jikomes

1:03:59 - 1:04:22

It sounds like the main things we have are natural variants of MOTC in humans. There’s one that you see in certain Japanese people. It’s a non-functional or a differently functional variant, and they have problems, sarcopenia, et cetera. We know from preclinical research that using the protocols in mice and rodents with MOTC, no obvious problems emerge.

Nick Jikomes

1:04:22 - 1:04:39

Those are usually protocols where you’re giving it once a day for maybe three days per week, but sometimes for many, many weeks, which for a mouse’s life is quite a long time. And there’s very, very limited human data for MOTC, so we don’t really know much from the realm of randomized control trials.

Pinchas Cohen

1:04:41 - 1:05:24

So, that’s only one randomized control trial that I mentioned, which was four weeks, really small numbers, but still, it was a real study. Now, with all these peptides, there is this concept that literally hundreds of thousands of people are taking peptides now for several years, and for the most part, I don’t know what people are taking. By the way, so multiple friends of mine who take peptides, including Moxie, gave me the syringes that they inject that contain Moxie, and my team measured the level of Moxie. It’s never what it’s advertised to be.

Pinchas Cohen

1:05:24 - 1:05:29

Really? With these, you know... questionable producers.

Nick Jikomes

1:05:30 - 1:05:39

So you guys have taken the actual MOTC that people have bought online for research purposes only, and you’ve tested it in your lab. Have you found contaminants? Was the potency off? What were the actual issues?

Pinchas Cohen

1:05:40 - 1:06:06

Well, in my case, a syringe that was a stack, whatever that means, that included MOTC, supposedly there was zero MOTC. Wow. In another case, or two other cases, the amount of Moxie was between 20 to 80% of what was advertised. The reason for that could be A, that the manufacturers cheat, you know, there’s no regulation to this.

Pinchas Cohen

1:06:07 - 1:06:51

B, that the stability of the product they made because of how it was made and how it’s formulated results in degradation over time. Now, there are ways to address that, but I don’t know that people look to that in this illegal market that’s out there. So we really can’t say anything about this experience of the 1000s of people who’ve been taking it. But the expectation is that in the new world that we are entering, where it’s going to be legal, and manufactured and compounded in certified facilities, that we will begin to collect more information.

Pinchas Cohen

1:06:52 - 1:07:36

Yeah, there’s no mandated way to do that. I, together with some colleagues at Stanford, are planning to start a registry for peptides, particularly MOXIE, but we’ll probably try to incorporate other peptides as well. And this registry will be voluntary. HIPAA-compatible and privacy-protected, but we’ll try to get as many people as possible who are using peptides, particularly MOTC, to enter their information into this large database to allow us to collect data long-term, first and foremost for safety, which by far is the most important thing.

Pinchas Cohen

1:07:37 - 1:07:59

But then on What potential benefits people are seeing or not seeing and we’re still developing the concept and the structure and the. online data collection tools. But I think this is going to be a very important aspect of dealing with the new reality. And as you know, there are some people who are very concerned about this.

Pinchas Cohen

1:08:00 - 1:09:01

This is something that really has never happened before in this country under FDA, which is an organization with very, very strict approach to new drugs. And, you know, they are not happy with this new system, but it’s here. And I think it’s critical for a responsible community of physicians and scientists to do what we can to try to ensure safety, to collect data in a real way and to understand where are we going with this new universe of peptides that are legal. And most importantly for me, I consider this 503A compounding story a stepping stone toward developing newer, more potent, and fully FDA-approved versions of MOTC and other peptides.

Pinchas Cohen

1:09:01 - 1:09:02

That’s how I see it.

Nick Jikomes

1:09:03 - 1:09:28

I want to ask you something that’s relevant to the question of formulations and stability of these things. As you mentioned, regardless of what the rules and regulations are, people are not only taking these things more and more every day, they’re stacking them, as you said. And that happens in two ways, as far as I can tell. Sometimes people will take two or three separate peptides, separate syringes, and inject them, you know, one after the other.

Nick Jikomes

1:09:29 - 1:09:52

There also seem to be an increasing number of stacks that are pre-purchased. So one syringe or one vial will contain two or three peptides in the same solution. From the perspective of the peptide biochemistry and biophysics and stability, are there any concerns that you would have knowing that biology about combining multiple peptides in one solution? Could they aggregate?

Nick Jikomes

1:09:52 - 1:09:56

Could they interact and change each other? Are there any potential issues there?

Pinchas Cohen

1:09:57 - 1:10:09

There are many concerns. You’ve named several of them right now. The bottom line is that we have no information whatsoever. My lab, at least, has never put two peptides in the same syringe.

Pinchas Cohen

1:10:10 - 1:10:33

We have done experiments where we injected with two different syringes mice. with several peptides. Our key approach recently has been to combine MOTC and GLP-1 to demonstrate the benefits on muscle preservation. It’s kind of a stack, if you will, and I think it’s a very rational one.

Pinchas Cohen

1:10:34 - 1:11:11

That does not mean that you can mix them together. Maybe there’s a way to do it, but we haven’t demonstrated that. It will require real chemists, which not many of them are involved in this field right now, to look at this, weigh in, and really develop the right formulation for these peptides, one by one, and potentially as stacks. I think stability in solution is a key aspect of this whole field, and it’s not trivial.

Pinchas Cohen

1:11:11 - 1:12:00

And I don’t know that what people are using in the black market today are really stable preparations and that the stacked combinations may be problematic. I don’t think there’s any real evidence for serious safety concerns. But I think there’s many reasons to think maybe you’re not getting the fully active forms in the concentrations that you’re expecting in these stacks. I think that the new era of compounded peptides is a bit of a mystery to me, whether legitimate American pharmacies will compound combination of peptides.

Pinchas Cohen

1:12:00 - 1:12:37

I don’t think they will. I think that the six peptides that have been recommended for compounding, which include Moxie and BPC-157, may not represent what stacks in the black market are thought to be. So I personally would argue against stacking in the same syringe for sure. We have no information about this, and it will take a lot of effort to demonstrate safety as well as stability.

Pinchas Cohen

1:12:38 - 1:13:09

I think that the conceptual use of combining two agents, one of them or two of them being peptides, is not unreasonable conceptually. And again, The GLP-1-MOTC combination makes complete sense to be explored and studied. I know there’s a very popular combination of BPC-157 and TB500. There’s zero publications on that combination.

Pinchas Cohen

1:13:10 - 1:13:40

And indications for that are also not particularly clear. And I’m not sure that there are real benefits to stacking beyond the potential value of using a single peptide in any particular scenario. So I think that we don’t have enough information to allow someone like me to comment on this other than a cautionary approach.

Nick Jikomes

1:13:42 - 1:14:09

At a high level, Does there seem to be any fundamental difference between mitochondrial peptides, those encoded in the mitochondrial genome secreted by the mitochondria versus peptides that are in the nuclear genome and that come from the nucleus? Does the fact that they come from mitochondria, that there’s many mitochondria per cell and they can be located in different compartments of the cell, is there any significance to that in terms of the types of functions we see among the mitochondrial peptides compared to other peptides?

Pinchas Cohen

1:14:14 - 1:15:01

Peptidome is expanding and emerging and probably has very diverse activities that we have not fully discovered. I think of this from a functional perspective, the mitochondria has several key roles, energy production, innate immunity, healthy aging, among others, protection of involvement in apoptosis. And it’s particularly important biologically in the process of aging. So the nuclear genome has many, many elements that protect it from damage over time.

Pinchas Cohen

1:15:02 - 1:15:55

DNA repair and other biological processes that were developed evolutionarily to protect you when you age from damage to your genes and chromosomes. The mitochondria doesn’t have that, that there’s no DNA repair, there’s no protection, that the mitochondria really relies on eliminating damage mitochondria and making new ones. But over the process of aging, there is very clear damage and depletion and mutations in the mitochondrial genome that occur in old people. So I think that phenomena, the functionality, the potential value of mitochondrially encoded peptides is to address the fact that there are probably deficiencies that occur in older age.

Pinchas Cohen

1:15:56 - 1:16:39

The nuclear microproteins, I think, will emerge as much more important in processes related to early development, cell differentiation, cellular processes that involve, you know, normal function of cells. And so far, from what I’m learning, and this is way early, and I’m not committing to this, it appears that many of the nuclear peptides and microproteins are structural. They stay inside cells. They might get incorporated into membranes inside the cell.

Pinchas Cohen

1:16:39 - 1:17:12

They serve important functions. Not many of them are secreted, while at least the mitochondrial microproteins that we work on probably because I’m an endocrinologist and that’s kind of what I congregate towards, but these are essentially hormones. So, if we didn’t circulate this as a function, so you can think of them in the same context as you do in GLP-1 and insulin and growth hormone and other testosterone or what have you, and think of them in the context of measuring the levels with age, and showing decline and whatever.

Pinchas Cohen

1:17:13 - 1:17:59

So, none of that has been done for the new class of nuclear peptides that are being discovered. And that doesn’t mean that There aren’t potentially nuclear peptides that could be developed as drugs, and there are several drug companies who are exploring nuclear microproteins and, you know, emerging interest in developing mitochondrial microproteins. And I’m always raising money for a new company, Nick. And if any of your listeners are interested, there’s a lot of potential in developing ventures around these emerging class of molecules and pharmaceuticals.

Pinchas Cohen

1:17:59 - 1:18:10

So I think this is just gonna be so many opportunities to explore in this field. And I love the way it’s expanding so rapidly.

Nick Jikomes

1:18:11 - 1:18:49

Yeah, and there’s definitely a lot of excitement. We might be able to talk separately offline about helping you out with some of your ventures. But as a class, a therapeutic class, when you think about peptides compared to, say, small molecule drugs, Do you think there’s any fundamental differences there in terms of the types of therapies that will emerge on the peptide side as opposed to the small molecule drug side? Are these things going to be better or worse at doing certain things or come with a different risk reward profile based on the fundamental biological differences between a peptide therapy and a small molecule drug therapy?

Pinchas Cohen

1:18:52 - 1:19:29

Several differences that I can think of. So first of all, the peptides we’re talking about that doesn’t necessarily include all of the peptides that people are using in the black market, but peptides that I work on are all naturally occurring molecules. So, in terms of safety, if I make MOTC and you make MOTC, and we live with that molecule inside our body forever, you have a reasonably good sense that it’s safe within the right concentration range. So insulin, if you give somebody too much insulin, they can die from low blood sugar.

Pinchas Cohen

1:19:30 - 1:20:07

But insulin in general is relatively safe. Moxie and the rest of these naturally occurring peptides at least have this guarantee that there’s not going to be some crazy side effects that will kill you because it’s a foreign molecule. For small molecules that are essentially designed, they’re not naturally occurring, there’s always the risk that there will be off-site side effect, that there will be some unexpected phenomena that’s associated with that. So, I think that naturally occurring peptides have a higher likelihood of being safe.

Pinchas Cohen

1:20:08 - 1:20:41

On the other hand, people don’t want to inject themselves. And, you know, when you look at GLP-1, it’s a $60 billion industry that millions of Americans are enjoying the fruits of. But as soon as both Eli Lilly and Novo Nordisk came up with the oral versions, boom, people congregate to that because people don’t want to inject themselves. Now, there are emerging ways to administer peptides, for example, intranasally, which might overcome the injection issue.

Pinchas Cohen

1:20:42 - 1:21:16

But I think for the most part, most of the peptides will require injection. And the small molecules, of course, can be given orally. And that’s the most fundamental difference that people who are looking for the benefits will consider as a decision maker. Having said that, if you look at the GLP-1 universe, The injectable peptides versus the oral versions, the peptides are much more potent.

Pinchas Cohen

1:21:16 - 1:21:26

So if you look at the new oral version of a Zempik, it’s less than half as potent as injected version of Zempik.

Nick Jikomes

1:21:26 - 1:21:27

Yeah, I mean, it makes sense.

Pinchas Cohen

1:21:27 - 1:22:13

And also, moving from a peptide to a designed small molecule is very, very difficult, very expensive, a lot of work, and many years. So, I think that the emergence of developing peptides as drugs will rely first and foremost on maybe analogued mild modifications of these peptides, but early on keeping them as injectable peptides during the development and characterization phases. Yeah. And I also think that people are much more open today compared to 10 years ago for taking injections.

Nick Jikomes

1:22:14 - 1:22:26

Yeah, that’s obviously changing. I mean, as more and more people see their mom and their sister and their brother take an injection, people become more comfortable with it. I want to ask you about designer peptides. You hinted at this earlier.

Nick Jikomes

1:22:27 - 1:22:46

There’s a lot of endogenous peptides, obviously, MOTC and the other ones we talked about. And obviously it is reassuring that, hey, our bodies produce these things. So within the natural physiological range of concentrations, we know that our bodies can handle it. There’s probably nothing that’s gonna blow up inside of us from taking some supplemental peptides, assuming we’re getting the right thing at the right dose.

Nick Jikomes

1:22:47 - 1:22:58

What about designer peptides, novel peptides that are amino acid sequences that aren’t encoded in our genome and don’t occur naturally? What do you see as the future there in terms of the potential risks and benefits?

Pinchas Cohen

1:22:59 - 1:24:13

So, you know, there’s a lot of discussions these days about how AI is going to transform drug development, or, you know, transform everything. But I think that in the field of peptides, the concept that we can now really understand structure much better, especially for large proteins, and we can try to design sequences peptides that will interact with specific binding pockets in large peptides. That’s a compelling reason to employ these AI tools to try to develop completely new peptides. I think there are people already doing that at the drug development stage, and in various conditions, including cancer, where there are some undruggable proteins that are involved in cancer, where you could develop a peptide that will interact with molecules like B53 or the androgen receptor, all these large

Pinchas Cohen

1:24:13 - 1:25:09

proteins involved in cancer that you target instead of with a small molecule, which sometimes just can’t fit perfectly in that particular large protein, you could design completely artificial novel peptide that will fit just right and block the effect of this negative protein in cancer, for example, in metabolism or new generation. Similarly, you know, so we identified a human in analog that interacts with APOE4, but you could design a universe of peptides that could interact with APOE4 to block its effect, to prevent it from aggregating and killing cells. So all of this science relies on a really good understanding based on actual experiments and data to understand the structure of large proteins.

Pinchas Cohen

1:25:10 - 1:25:36

And for that matter, also, to some degree, understanding the structure of peptides. But this is a really exciting new direction in science and drug development. There’s several Nobel Prizes that have been indirectly awarded towards this area. And I think that’s where you look at where the really brilliant people are going to in science today.

Pinchas Cohen

1:25:36 - 1:26:09

It is an AI-driven understanding of biological processes and harnessing them for drug development. So, I think that that’s the future, but it’s always going to be informed by observations on natural molecules and the natural peptides, whether it’s MOTC or something else, really will set the direction for designing designer peptides. So these two fields will cross talk to each other and inform each other.

Nick Jikomes

1:26:09 - 1:26:37

Yeah, yeah. I want to ask you a little bit more about diversity and variation. So I think for all of the natural endogenous peptides you’ve worked on that we mentioned, at least, there’s natural variants that are out there, different versions and different people. What’s the significance of that, both in terms of, you know, helping us think about the underlying biology here, and in thinking about how we develop treatments that might, say, work in one population but not another, or help people in different ways, and things like that?

Pinchas Cohen

1:26:39 - 1:27:23

That’s a great question, Nick, and I’ve been thinking about it a lot. So let’s start with the observation that the variability in mitochondrial peptides is so much more common than the variability in nuclear genes. Much more, just because what I said about protecting cells and DNA from mutations, there’s much less variation. The mutation rate in the nuclear genome is one in I don’t know how many millions of base pairs per generation, the mutation rate in the mitochondria from one generation to the next is more than 10 times higher than in the nuclear genome.

Pinchas Cohen

1:27:24 - 1:27:56

So, you get a lot more diversity within the mitochondrial genome that’s transferred down from mother to children and to the next generation, and many of these are random. And they might be found in a person, and if that particular variant has no positive value, it will disappear. But if somebody, so we have several gain-of-function variants that we identified. One is the UMP3S.

Pinchas Cohen

1:27:57 - 1:28:29

Another is a SHLEP2, which is another peptide we discovered some years ago. There’s a SHLEP2 gain-of-function mutation that protects from Parkinson’s disease. And there might be, there appear to be other Gamma-function mutations that enhance the function of peptides. Then there’s all the negative loss-of-function mutations, like in MOTC and SHMOOZ and MENSH, again, that reduce the activity, which might be a good thing in certain situations.

Pinchas Cohen

1:28:30 - 1:29:07

So, first and foremost, there’s a lot of diversity in these peptides in human beings, which is ethnospecific. And that certainly facilitates discovery. So, if it wasn’t for our, you know, the Hispanic or the Mexican-American population that we studied, we never would have discovered dementia mutation. That way we have, the question that you’re leading me to is, should MENSH, for example, be considered a precision medicine approach for Hispanic Americans with diabetes?

Pinchas Cohen

1:29:08 - 1:29:55

or could it be universally used potentially as a drug for all people with diabetes? We don’t know yet, but the bottom line is that if you look at MOTC, it is deficient in 8% of Japanese. We don’t know, I don’t think there’s a lot of peptide use in Japan right now, and I’m not familiar with any Japanese Americans who reported any benefits who are genotypes. Theoretically, I would love to see at some point a targeted clinical trial comparing people of Japanese extraction with and without the MOTC mutation treated with MOTC to see if they benefit more because they don’t make their own.

Pinchas Cohen

1:29:56 - 1:30:45

But I think that there’s generally a well-accepted concept that even if you don’t have a mutation in MOTC or MENSH or what have you, you can benefit from exogenous administration of this peptide, just like you do with insulin and GLP-1 and all of those. So the potential for precision medicine approach based on this is exciting, not been proven yet. the generability of any particular peptide that was discovered by the fact that there is a population with a deficiency that exhibits a disease state. may or may not limit the development of such a peptide in a similar conditions with people who don’t have a deficiency.

Pinchas Cohen

1:30:46 - 1:31:13

But the bottom line is, the other thing that’s happening in modern medicine is the concept that everything is moving toward a precision approach. And that certainly diagnostically is important, targeting particular interventions to people with particular genetic structures that might benefit from that. It’s compelling. It’s already happening in certain areas in medicine.

Pinchas Cohen

1:31:15 - 1:31:27

It has not yet been applied in the peptide world. I hope that we will get to a place that we have the capacity to scientifically demonstrate the value of that, but it hasn’t happened yet.

Nick Jikomes

1:31:29 - 1:31:39

What are one or two of the things you’re working on in the lab today to do with peptide biology that you think are going to give us some exciting insights in the next, say, one to two years? What’s coming next?

Pinchas Cohen

1:31:41 - 1:32:29

So without spilling the beans on secret projects that my students have that are going to make their careers and I don’t want to steal their thunder, and there are a bunch of interesting things going on, I am beyond the fact that MOTSE is going to be legal in a few months, and there’s a lot of possibilities there to explore its utility in muscle preservation. The peptide that I’m most excited about at the moment, beyond that, is schmooze. Schmooze is effective in animal models to reduce the development of cognitive decline and Alzheimer’s symptoms.

Pinchas Cohen

1:32:30 - 1:33:01

We are successfully administering schmooze in mice intranasally, which is sort of the emerging tool in the field. And I think that That is the peptide I hope will get more attention. Maybe we can secure funding to develop it into a drug. But I think that that is where I think a lot of the new discoveries that are not yet published are happening in our lab.

Pinchas Cohen

1:33:01 - 1:33:58

We now linked schmooze remarkably, not just to Alzheimer’s disease, but to lung disease. Who knew, just some collaborator of mine who’s into the lung, physician did the screen, the genetic screen, and lo and behold, Shmue’s mutation might be related to lung disease and provides an opportunity for intervention there. We’re also looking at new disease states and new cohorts where we hope to discover new peptides. I think I always start with normal physiology, so energy and metabolism is the main goal of mitochondrial function, and several of the peptides work on that, motzy, mensch, perhaps humanin.

Pinchas Cohen

1:34:00 - 1:34:40

Immunity, innate immunity, is important, is an important part of mitochondrial function. We’re now discovering new peptides involving immunity, one that was just published this week, is called Muzzle, and it’s been discovered by one of my former students, Donna Silverstein, who was interested in immunity, and she did a screen, instead of diabetes or Alzheimer’s, for abnormal immune markers, like IL-1 and IL-6. Of course, elevated IL-1 indicates inflammation.

Pinchas Cohen

1:34:40 - 1:34:58

It’s not good. It’s associated with unhealthy aging and disease. So she discovered a mutation in this new muzzle peptide that’s associated with elevated IL-1 and IL-6. And that paper was also just published.

Pinchas Cohen

1:34:58 - 1:35:38

And the potential of intervening in immune or inflammation situations is very exciting as well. But I like to drive the discovery by thinking back, what is the mitochondria really doing, and how potentially could mitochondrial microproteins be involved in that? And I also am a big believer that the most important thing for healthy aging, Nick, is lifestyle. diet, exercise, stress reduction, avoiding inflammation, and so on.

Pinchas Cohen

1:35:39 - 1:35:49

And by using those guidelines to try to discover new mitochondrial microproteins, I think you’re going to get the biggest bunk for your back.

Nick Jikomes

1:35:51 - 1:36:10

Very briefly, we know that endogenous mitochondrial peptides like MOTC are modulated by exercise. We can exercise and affect how much MOTC is produced by our mitochondria. Is there any evidence that other lifestyle factors like diet or dietary components of various kinds affect MOTC or other mitochondrial proteins?

Pinchas Cohen

1:36:10 - 1:36:45

Yes, so exactly the point that I’m leading to. We do believe that diet regulates...we know diet regulates mitochondria for sure, but humanin is induced by certain diets, as do other peptides, but we just published a paper on Mediterranean diets. Now, again, I come from a Mediterranean extraction. I don’t know about you, but the Mediterranean diet is the one diet that has definitively been associated with healthy aging.

Nick Jikomes

1:36:46 - 1:36:48

And define that for us so people know concretely what that means.

Pinchas Cohen

1:36:49 - 1:37:45

So the Mediterranean diet is based on unprocessed food, limited carbs, a lot of fish, a lot of vegetables and fruits, and has been eaten in the Mediterranean for many centuries. And people who eat Mediterranean diet are much more likely to age well and have longevity compared to people who don’t. And my colleague, Roberto Di Sinanza, who is a big expert on Mediterranean diet, has shown, like many other investigators, the benefits of Mediterranean diet, particularly comparing people who adhere to Mediterranean diet versus those who don’t in Italy, where that’s like one of the birthplaces of the diet. And he’s shown that if you adhere to Mediterranean diet, there’s a lot of things that look better on your blood tests.

Pinchas Cohen

1:37:46 - 1:38:23

We then took a whole bunch of people that either adhere or don’t adhere to Mediterranean diet, measured monoclonal peptides, and shown that both human and particularly schmooze are much higher in people who eat the Mediterranean diet, and specifically olive oil, and I’m a big olive oil guy, olive oil increases schmooze levels substantially, not in an intervention trial, but in an observational trial. So we hope to find ways to demonstrate what specific foods elevate which peptides that is related to health. Wow.

Nick Jikomes

1:38:23 - 1:38:29

Well, yeah, one of my grandparents was Greek, so I’ve been eating olives all my life. So hopefully that’s good news for me.

Pinchas Cohen

1:38:30 - 1:38:56

Absolutely. And the Greece is also where many of the research studies on Mediterranean diets originate. But that brings me to a point you alluded to earlier. which is, if you’re from the Mediterranean, Greece, Italy, Spain, Israel, whatever, and you’re eating Mediterranean diet, there’s a very good chance you’re doing something good for yourself.

Pinchas Cohen

1:38:57 - 1:39:17

But what if you’re Swedish, or Chinese, or from Nigeria? Is there evidence that a Mediterranean diet is really better for you then? It’s not entirely clear. And for example, in Sweden, somebody did a study on the Mediterranean diet, couldn’t show any benefits.

Pinchas Cohen

1:39:17 - 1:39:43

But the Nordic countries actually have a healthy diet, sometimes referred to as the Nordic diet. A lot of fish, you know, whole grain stuff. I’m not an expert, but that diet has been shown to be beneficial in that part of the world. We know that in Asia, where many people have longevity, they eat very certain diets that are quite different.

Pinchas Cohen

1:39:43 - 1:40:11

So the whole blue zone field, which you probably have heard of, was trying to find what are the similarities between all these very different places? What are the habits and the foods that they all share? And it’s a limited number of things. So, I think that it’s very likely that people from different ethnicities might benefit from slightly different modalities of diet and exercise.

Pinchas Cohen

1:40:11 - 1:40:47

And similarly, might have different profiles of peptides in their blood and potentially might benefit from different types of interventions with pharmaceuticals based on peptides. And for example, GLP-1, the peptides that is the ultimate stars of the field, different ethnicities respond differently. And the fact of the matter is that almost every drug that’s being developed until now, has been developed for a European audience. That’s where most of the studies are done.

Pinchas Cohen

1:40:47 - 1:41:03

That’s how they’re tested. And after they’re approved and demonstrated to be effective in white people, then they’re tested in other races. And in many other ethnicities, GOP1 is not as effective as it is in Europeans.

Nick Jikomes

1:41:04 - 1:41:05

Really? What are examples?

Pinchas Cohen

1:41:06 - 1:41:20

You know, I think that both Hispanics and Asians show a 20 or 30% lesser response to GLP-1. They still respond, don’t get me wrong, but if you just look at percent weight loss.

Nick Jikomes

1:41:20 - 1:41:22

Yeah, the magnitude is less in those populations.

Pinchas Cohen

1:41:22 - 1:41:47

It’s less in those populations, which could be explained by different biological subtle differences between these two, between these different populations. There might be a way to understand that and develop interventions specifically to those types of people. And you don’t have to say, if you’re white, you get this, if you’re black, you get that. Especially, by the way, in this country.

Pinchas Cohen

1:41:49 - 1:42:09

The admixture is everywhere. There’s so much intermarriage. So you could be classifying yourself as Greek, but you might have some Asian grandfather that determines your susceptibility to disease and response. So doing a genetic analysis, don’t call it ethnicity.

Pinchas Cohen

1:42:09 - 1:42:35

Just get a gene sequencing thing, 23 million, what have you. And based on that information, say you should go on a GOP1, or you should go on Metformin to address your issues with metabolism and diabetes. So this precision medicine approach, we’ve been talking about it for a couple of decades. It hasn’t quite reached this very practical level, but it’s coming there.

Nick Jikomes

1:42:35 - 1:42:36

It’s coming. Yeah.

Pinchas Cohen

1:42:36 - 1:42:37

Not bad.

Nick Jikomes

1:42:38 - 1:42:43

Any final thoughts or things you want to reiterate from our discussion as it relates to peptides and the research you’ve done?

Pinchas Cohen

1:42:45 - 1:43:02

So first, I always want to install a level of caution in people. There’s a lot of enthusiasm about peptides, but at the same time, there’s so much we don’t know. Everything that’s out there right now is illegal and unproven. So let’s start there.

Pinchas Cohen

1:43:03 - 1:44:21

But now when some peptides are going to be legal to prescribe, I hope that a responsible community of physicians will oversee this and will try to limit whatever use of these peptides might happen to sensible, safe indications, and that the community of physicians and prescribers and healthcare professionals will try to incorporate whatever peptide use is going to happen into what we’re doing already with people’s health, and particularly the concept of registries and data collection and some clinical trials that can be developed will teach us a lot more about how to use these substances safely, and hopefully there’ll be an investment and an interest from pharma in taking some of these to the next step, developing high-potency versions. Now, if you look at GLP-1, the first one was produced and marketed twenty years ago with minimally effective the next one, five or ten years later with more more effective fifteen years later was even more effective.

Pinchas Cohen

1:44:22 - 1:44:49

A few years ago, Munjaro is even more effective, and soon, retatratide is super effective. So, the evolution of these analogs can be replicated for other peptides, like MOTC, where we are already in our lab developing new analogs that are more potent. So, I think that this is not going to be a static field. It’s always going to be improvements, better versions, better ways to use it.

Pinchas Cohen

1:44:50 - 1:44:57

And I hope that we’ll all do it responsibly. I think I’d like to finish there.

Nick Jikomes

1:44:57 - 1:45:03

All right. Well, Professor Hasse Cohen, thank you very much for your time. I really enjoyed this. I’ve been following your work for a little while now.

Nick Jikomes

1:45:04 - 1:45:09

So there’s a lot to follow. This field is really blowing up. But yeah, thank you very much for your time.

Pinchas Cohen

1:45:10 - 1:45:10

My pleasure.

This is a public episode. If you'd like to discuss this with other subscribers or get access to bonus episodes, visit mindandmatter.substack.com/subscribe

Wide release: Available for free by August 5, 2026, on Apple, Spotify, YouTube, etc.

Not medical advice.

Nick speaks with Harvard neuroimmunologist Dr. Isaac Chiu about bidirectional signaling between nociceptors and immune cells. Dr. Chiu describes how pain-sensing neurons directly detect bacterial toxins, release CGRP to suppress neutrophils, and how this crosstalk regulates barrier defenses, allergy priming, and the resolution—or failure—of inflammation.

TOPICS DISCUSSED:

* Nociception & Pathogens: Sensory neurons detect bacterial toxins within seconds, enabling rapid host-defense coordination ahead of immune-cell recruitment.

* CGRP & neutrophils: Nociceptor-release the peptide CGRP, which blocks neutrophil (immune cell) recruitment; pathogens exploit this in necrotizing fasciitis and meningitis.

* Pain sensitization: Inflammation can lower neuronal thresholds for pain, which sometimes becomes a chronic problem.

* Gut barrier protection: CGRP from gut nociceptors drives goblet-cell mucus secretion, supporting the protective “weep and sweep” response.

* Skin-to-gut allergy priming: Early skin exposure to allergens plus scratching may promote food allergies.

* Vagus nerve circuits: Distinct vagal sensory neurons selectively suppress TNF or induce IL-10, allowing precise inflammatory control.

* Brain inflammatory memory: Insular neurons encode site-specific immune responses that can later be recalled by reactivating those circuits. The brain remembers past inflammatory events.

ABOUT THE GUEST: Isaac Chiu, PhD is a professor in the Department of Immunology at Harvard Medical School. His lab studies nervous-immune system crosstalk in pain, itch, infection, and inflammatory disease.

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PRACTICAL TAKEAWAYS:

* Avoid habitual daily NSAID or anti-histamine use. Chronic use of anti-inflammatory drugs like NSAIDs may actually promote chronic inflammation in the long run.

* Early oral exposure to potential food allergens may promote tolerance, while repeated skin exposure risks sensitization. Be weary of exposing young children to artificial skin care products early in life.

* CGRP-blocking migraine drugs can slow wound healing and alter gut function; effects extend beyond headache relief. Drugs may produce chronic side-effects that are not apparent with intermittent or short-term use.

SUBSCRIBER CONTENT BELOW: Reference paper + episode transcript.

Reference Paper:

* Paper | Nociceptor Sensory Neuron–Immune Interactions in Pain and Inflammation

AI-generated transcript below. Beware of mistranslations!

Isaac Chiu

02:17 - 02:19

I’m happy to be here, Nick.

Nick Jikomes

02:20 - 02:23

Do you want to start off by just telling everyone a little bit about who you are and what your lab does?

Isaac Chiu

02:24 - 02:47

Sure. So I am a professor at Harvard Medical School, and my lab is in the Department of Immunology. And we actually study crosstalk between the brain, the nervous system, and the immune system. We’re very interested in this bidirectional crosstalk between these systems in different aspects of inflammation and medicine.

Isaac Chiu

02:47 - 03:20

So we’re interested in how this impacts pain and itch, which are two sensations we all are familiar with, and also how the nerve system actually affects the immune response. So how does it affect our ability to fight infections? And also how this also might impact diseases where inflammation is a big part of the disease, like skin inflammation, dermatitis, or gut inflammation, colitis. So I’m a neuro-immunologist, if you want to call me any specific term.

Nick Jikomes

03:20 - 03:45

Yeah, and I think your research in particular is a really nice illustration of the fact that, on the one hand, as humans and as scientists, we like to make distinctions. We like to draw boundaries between things, and that’s a necessary part of learning and understanding things. And it can be very convenient, even for just talking about them. But we have immunology textbooks, and then we have separate nervous system textbooks.

Nick Jikomes

03:45 - 04:14

And even within any given domain, we like to separate pain and itch. We like to separate the somatosensory system from the visual or other systems. And there are distinctions to be made there to be sure, but also this is biology and the boundaries can be fuzzy and there’s a lot of crosstalk and a lot of things that we once maybe thought were separate or discrete or completely compartmentalized actually aren’t. And there’s a lot of overlap and blurry lines and fuzzy boundaries, if you will.

Nick Jikomes

04:15 - 04:27

Yeah, what have you just at a high level, how did you sort of get into the realm of neuroimmunology yourself? And how is this sort of the interface between these two fields changed over the last few years?

Isaac Chiu

04:28 - 05:00

Oh, so I think you gave a great introduction to my field of neuroimmunology because I think traditionally these two systems were studied separately, right, as separate entities. But like you said, biology developed through evolution, not because they say the brain is the brain and the immune system is the immune system, but functions evolved in a common way. For example, both you know, our organism needs to be able to fight infections, right? And the brain actually, or the nervous system in primitive organisms like C.

Isaac Chiu

05:00 - 05:38

elegans actually is used to sense potentially harmful bacteria versus helpful ones, right? So discrimination is not just immune cells that help discriminate what’s good or bad. And similarly, things that we think neurons use like neurotransmitters or things like that have actually been used by immune cells to communicate as well. So what we think about as like okay this is an immune molecule or this is a function of the nervous system or immune system traditionally, actually there’s roles for the other cell type in that or other system in that function.

Isaac Chiu

05:38 - 06:05

I personally got interested in this partly because as an undergraduate, I was at Harvard College, I was fascinated by both subjects. I actually thought these were the two coolest systems, the most complex, both have memory and complex components. And actually, it was in my undergrad research lab. I was in Professor Jack Strominger’s lab.

Isaac Chiu

06:05 - 06:23

He’s one of the leaders in immunology who discovered MHC molecules. He said, Isaac, you should think about the brain. Because recently, at that time, it was early 2000s, the molecules he discovered, which were involved in the immune system, were also found in the brain. Work from Carla Schatz’s lab.

Isaac Chiu

06:24 - 06:51

showed that MHC actually was involved in memory and long-term potentiation. So I said, well, there has to be something fascinating that’s connecting these two systems. And so I decided after undergrad to pursue a PhD in immunology, but in that process learn about neuroimmunology? So how does the immune system act in the nervous system in the context of neurological diseases?

Isaac Chiu

06:54 - 07:08

So this kind of theme of the two systems has always been in my interest. And what is exciting is seeing that, indeed, now these two fields are really interested in each other because it’s very clear that they’re intimately communicating.

Nick Jikomes

07:09 - 07:32

Yeah, yeah. And I think, naively, One may think, OK, when I get an infection or I’m injured, that’s a problem that the immune system handles. The bacteria come in, the immune system does all of the sensing and handles the problem. And maybe the nervous system is affected by that in some way as kind of an innocent bystander.

Nick Jikomes

07:33 - 07:38

But that’s not really how it works. The immune system is involved. The immune system is detecting the pathogen. But so is the nervous system.

Nick Jikomes

07:38 - 08:00

And actually, the communication is bidirectional. with each component here. The infection or the injury is going to directly interact with neurons in the nervous system and immune cells in the immune system, and then the neurons and the immune cells are going to communicate in both directions. And so, again, there’s bidirectional communication across the board here, and we’re going to get into that with what you’ve discovered and some of the stuff that you work on.

Nick Jikomes

08:01 - 08:39

let’s give people a basic sense for how some of the key nuts and bolts here work on the nervous system side with respect to pain sensation. And I guess the cue that I’ll give people is obviously we feel pain if we get a bump or a bruise or a cut, but as everyone can probably sympathize, we also get pain signals when we get infections and things like this. So we’ll connect the dots here for people, but just on the pain sensation side, when I feel pain in my periphery, in my body somewhere, what are the neurons that are actually responsible for sensing the initial steps in that pain process?

Isaac Chiu

08:40 - 08:58

This is a great question, Nick. So the pain is sensed, pain actually is an unpleasant sensation, right, that we, our brain processes. But the initial step that initiates pain is called nociception. So the detection of potentially noxious, harmful stimuli.

Isaac Chiu

08:58 - 09:43

And there are specialized nerves or sensory neurons that innervate our skin, our joints, our GI tract that can be tuned to these harmful things. So for example, heat or cold, extreme cold temperatures, or something like you said, tissue injury, something that’s forceful. These nerves have abilities or receptors on their surface that can detect these specific noxious stimuli and that induces firing of nerves through action potentials and ion channels that are like, for example, what’s called trip channels. But in the context of inflammation or infection, what we found is that these nerves can also directly sense pathogens and what they secrete.

Isaac Chiu

09:43 - 10:16

So a bacterial pathogen secretes things that could damage tissues, like a toxin. We found a particular bacteria, Staph aureus, which is one of the most common types of bacteria that affects our skin, secretes a toxin which acts on these sensory neurons, these nociceptor sensory neurons, and it causes them to start firing because that toxin pokes holes in the membrane of the nerves. So this is just one example where the nerves sense directly a bacteria, which you might think the immune system would need to combat, right?

Isaac Chiu

10:16 - 10:33

And why do you think, why do we think that’s important is because, well, neurons have some advantages in terms of mediating host defense. They respond immediately within seconds, right? Or at least within minutes of detecting something. The immune system will take several longer than that.

Isaac Chiu

10:33 - 11:06

It might take hours, right, to be recruited to that site. So your neuron detecting that bacteria might be able to coordinate the host response to defend against the pathogen faster than actually traditional immune cells. In fact, one of their roles could be to modulate the immune cells that come in, right? Another reason is that actually some of these neuronal reflexes that help us, we think about expelling things that are harmful, may also help us fight against the pathogen.

Isaac Chiu

11:06 - 11:56

So you think about things like itch right where you’re scratching the surface maybe that is some way to get rid of things on the surface of our skin or coughing right coughing is something that’s activated by nociceptors that reside in our vagus nerve and the that reflex can help get rid of something harmful vomiting these kind of reflexes that are i would say generally nociceptive in nature are also activated when we’re sick, right? Because that might help us remove something that’s harmful in a much bigger way than maybe the immune response can do locally, right? So I think that, so the coupling of the sensory nerves that mediate pain or other noxious sensations to infection, I think evolutionarily evolved for that reason.

Nick Jikomes

11:57 - 12:17

Yeah, yeah. So nociceptors, so neurons that are nociception neurons, they’re detecting a potentially problematic stimulus. It might be causing a problem right now, or it might be causing a future problem, like an infection that’s nascent and just getting started. But through mechanisms, we don’t necessarily need to unpack completely.

Nick Jikomes

12:17 - 12:39

They can detect the pressure that comes with a cut. They can detect the heat that comes with burning yourself on the stove. And as you said, they can actually detect the presence of pathogens, they can detect molecules secreted from or on the surface of bacteria or whatever, that is sort of like an immune, that’s like, you know, you would think of that as being an immune system thing. And indeed, the immune cells do detect those things.

Nick Jikomes

12:40 - 13:01

But some of these nociceptor neurons do as well. So in other words, the same neurons in the periphery that detect pressure pain and itching and other noxious pain, they’re also directly detecting things like pathogens. So right from the start, the immune side of things is built right into the nervous system.

Isaac Chiu

13:02 - 13:18

That’s correct. And I think so that in terms of a broader point, right, like we need to fight things that are harmful or dangerous. And pathogens are one of the leading things that organisms have to deal with, right? Mammals have to deal with.

Isaac Chiu

13:18 - 13:57

And so having neurons be able to detect things from the cell wall of a bacteria like lipopolysaccharide or toxins, like I mentioned, I think makes a lot of sense evolutionarily. And then, you know, the sensation of pain also is protective in the sense that you then avoid putting pressure on that area, right? So that you have a wound or infected wound, you may want to leave that area alone to allow not just the immune system come in to clear the pathogens, but also wound healing afterwards, right? So pain is a protective response.

Isaac Chiu

13:58 - 14:38

And acutely, if we don’t have pain, this is a big problem because you’ll injure yourself, right? You can also cause, like there are people who have congenital insensitivities to pain because they have mutations and specific ion channels. And they often don’t live very long because they get into accidents or they have, you know, issues with that. And so I think pain, acute pain is absolutely critical for us to be able to survive as organisms, as mammals, and then to be able to couple that to inflammation or sickness, I think makes sense as well, because it might induce behavioral changes as well as cellular changes

Isaac Chiu

14:38 - 14:43

that might help facilitate our ability to fight pathogens.

Nick Jikomes

14:43 - 15:02

Yeah. And something that comes to mind here that is an interesting common denominator between the immune system, the immune cells per se, and the nervous system is plasticity and the ability to change and adapt. This happens in immune cells. They can form memories and change themselves.

Nick Jikomes

15:02 - 15:27

This happens in neurons as well. Can you maybe start to talk about examples where you get sick or you get an injury, the animal has some immune problem it has to take care of, it’s infected with a pathogen that causes some amount of pain, so the immune system and the nervous system are both involved at different levels and in different ways, but there’s constant crosstalk there. But then there has to be some sort of memory.

Nick Jikomes

15:27 - 15:48

of that injury or that infection. And we can probably, I’ll let you steer us into a particular example that gets into this, but we all have probably examples of we got sick or we got injured. And maybe if you’re used to a certain type of injury, like you’re a football player or whatever, you sort of get used to the pain and it’s not as painful anymore. So you become desensitized to the pain.

Nick Jikomes

15:49 - 16:03

But on the flip side, you can also become sensitized to pain. You can become more sensitive to a painful stimulus. And there are instances where this is adaptive and you kind of learn how to deal with something better because it’s not going to kill you. So you don’t need to freak out.

Nick Jikomes

16:03 - 16:22

But also where it’s maladaptive, where you become hypersensitive to pain after an infection, injury, and it gets in the way of life. So on the general topic of I guess, plasticity and how the sensation of pain can change in an experience-dependent manner. What are some examples of how that change actually happens?

Isaac Chiu

16:23 - 17:09

This is a great, very good, important question, actually, because like you mentioned, the memory system of this peripheral nervous system is actually called sensitization, this idea that some stimulus, it could be inflammation or injury, makes that neuron, which in the beginning took, let’s say, a small amount, sorry, a large amount of force or heat to induce that pain, then later on, you only need to have a small amount of force or the same temperature to activate that nerve. And we’ve experienced this, right? For example, if you, just as a simple example, if you get a sunburn, so there’s been inflammation there on the skin, and then you go take a shower that normally feels

Nick Jikomes

17:10 - 17:15

You know, like it’s just a normal temperature now feels like so hot and burning, right?

Isaac Chiu

17:15 - 17:39

Those nerves in the skin have now been sensitized. So they’re going to fire much easier with a smaller stimulus. And this is actually in terms of a term. There’s this term allodynia, which means normally innocuous touch or cold or heat causing now a dynia, a painful response.

Isaac Chiu

17:39 - 18:03

And how does that work? So there are multiple ways by which a nerve can be sensitized. One is that actually signaling pathways within the nerve terminal causes ion channels to be modulated. So I mentioned these, for example, TRIP channels, which detect the chili pepper component that’s in chili peppers that causes that burning sensation.

Isaac Chiu

18:03 - 18:28

There’s one called TRIPv1. This ion channel also senses heat. But when there’s inflammation, there are signaling pathways that from other receptors, for example, from a cytokine receptor, that once it detects a cytokine, then leads to the phosphorylation or change in this ion channel so that now it opens easier. So the next time you stimulate it with heat, it will open faster and easier.

Isaac Chiu

18:29 - 19:05

Just as an aside, like TRIBV1 was a very important ion channel because it was the first one to be discovered that detects a noxious stimuli, and that was discovered by David Julius at UCSF, and he won a Nobel Prize in Physiology and Medicine 2021 based on that discovery. So these ion channels can be modified by inflammation and now they’re opening easier, right? But there are also other ways by which peripheral sensitization could occur. For example, the neuron themselves in the skin or in these other tissues might change their innervation pattern.

Isaac Chiu

19:05 - 19:32

Like you mentioned plasticity, right? So maybe the nerve now sprouts into the tissue so that there are more branches and more fibers that can detect the stimulus. And that’s another, you know, the question is how does, for example, do immune cells make things that cause them to sprout or do injured tissues release growth factors? And then also there’s, of course, a lot of changes molecularly at the transcriptional level within these nerves.

Isaac Chiu

19:32 - 19:58

So they might turn on genes or change their expression of genes that relate to pain. So then, So you need to have this response, like I said earlier, to avoid further injury, right? Maybe the sensitization is adaptive in the sense of during the course of wound healing, you want to avoid touching that area or putting pressure there. However, there has to be resolution of pain, right?

Isaac Chiu

19:58 - 20:26

Actually, more broadly, there has to be resolution of inflammation. And this is a very important aspect of actually the immune system as well as the nervous system. that process has been going because of you’ve got an infection or you got injury, you have to be able to turn that down after the healing process, right? However, many, many people have maladaptive pains or chronic pain.

Isaac Chiu

20:26 - 21:04

And by definition, chronic pain is anything that lasts longer than three months after the initial injury that, you know, with healing, you should actually go away. But these people still experience, whether it is, you know, chronic back pain or chronic nerve injury-induced pain. And part of that big question is why that occurs and how do we shut down those nerves that are now overly sensitized? And so it’s a big question in the pain field, you know, developing ways of, you know, blocking sensitization.

Isaac Chiu

21:05 - 21:47

And we think, at least our lab and many others also thinks, one of the components that drives that long-term pain could be overactive immune cells, because they produce factors like cytokines or mediators that can cause the nerves to keep being sensitized. So the classic example actually of what an immune cell could make that causes a neuron to, or induces pain or causes is prostaglandins. So yeah, I mean, I think that we all at some point probably took a non-steroidal anti-inflammatory drug, NSAIDs, and these block actually they act by blocking prostaglandins, so like ibuprofen or...

Nick Jikomes

21:47 - 22:03

Right, right. And as many listeners might know, because I’ve covered some of this biology quite a bit, so prostaglandins are basically a type of fat. They’re a omega-6 polyunsaturated fat derived lipid and they are inflammatory. So these are common when we have like injuries and stuff.

Nick Jikomes

22:04 - 22:10

Prostaglandins are a major piece of the inflammation and the pain and like that side of it.

Isaac Chiu

22:11 - 22:35

That’s right, exactly. So that’s, and you know, when we eat certain types of foods that have a lot of omega-6 fatty acids, you know, or versus omega-3 fatty acids, right? So the balance of prostaglandin production versus pro-resolving mediators might be out of balance. But definitely prostaglandins are made by many immune cells like macrophages.

Isaac Chiu

22:36 - 23:17

and, you know, cells that come into tissues when there’s injury, and they release the prostaglandins which then act on the nerves and make them decrease that firing threshold that I mentioned earlier, right, so they’re more easily able to be activated and cause pain. So, I mean, prostaglandins are part of the equation, right? But there are many other factors that immune cells make that nerves can sense, which just shows how intimate these two systems are connected. So this language, like, so whether it’s prostaglandins or cytokines, immunologists have studied cytokines for a long time.

Isaac Chiu

23:17 - 23:46

Certain cytokines have been linked to autoimmune diseases like IL-17 certain cytokines might cause gut inflammation like TNF-alpha. Well, it turns out these cytokines, their receptors are also on the pain, the nociceptive neurons. So they can detect these cytokines and then that also signals within the neurons to upregulate pain. And I think that it could be context dependent, right?

Isaac Chiu

23:46 - 23:59

So certain conditions of chronic pain or inflammation induced pain is caused by certain types of cytokines. So we need to understand which ones are being produced in each of these situations to block them.

Nick Jikomes

24:00 - 24:29

Yeah, and sort of at all levels here, right, balance is key or homeostasis is key. You want inflammation to turn on when you need it and where you need it, but then you want it to turn off when it’s no longer needed. You want your nociceptors to be activated so that you feel pain so that you don’t touch the inflamed tissue and mess up the wound healing process, but then you want to turn it off after the problem has been handled. And this concept of balance arises at multiple levels.

Nick Jikomes

24:29 - 24:54

You can talk about imbalances between, as you said, omega-6 and omega-3 fats, which might cause imbalances between inflammatory lipids like prostaglandins and resolving lipids like some other ones we could talk about. You might talk about the balance between different immune cells, different white blood cells and T cells that we might talk about. They can be in balance so that they’re a little bit too aggressive or a little bit under aggressive. And then you can talk about balance at the level of the pain neurons, right?

Nick Jikomes

24:54 - 25:21

You want sensitization to go up and to go down in a way that’s appropriate for the context, but not to get stuck in a one configuration and sort of, you said, Basically, when we think about things like chronic pain or chronic inflammation, what we mean there is the inflammation and or the pain are lasting too long. They’re showing up at inappropriate times or places because there’s some kind of underlying imbalance of some kind or another.

Isaac Chiu

25:22 - 25:44

That’s correct, yes. I think that you’re exactly right. Actually, I also want to point out that inflammation, which we usually think about as tissue redness and things, well, pain is actually part of inflammation. It was defined by 2,000 years ago, actually, inflammation, this term came up, and it was defined by this Roman physician called Celsus.

Isaac Chiu

25:45 - 26:27

as having four signs of which one is pain the other three are redness heat and swelling right so so pain like you said pain is part of inflammation. It’s always been part of inflammation and it needs to have that proper kinetics where it gets induced acutely that helps protect us, but then it is resolved. And the body’s ability to resolve inflammation and pain is a really key area of research. And I want to highlight a bit of how In immunology, this has really also a key part and maybe the things that we’re learning in the immune system could also be applied to pain and the nervous system.

Isaac Chiu

26:27 - 27:09

So there are special T cells called regulatory T cells, which So T-cells are a branch of the immune system, the adaptive immune system, right? So we know that we need T-cells to induce an immune response against infections and pathogens, but also different branches of the different types of T-cells promote different types of inflammation. But there’s a specialized one called regulatory T-cells that then shut down all of these other T-cells, right? And they produce cytokines like IL-10, or other, you know, TGF-beta, these actually shut down the ability of other cytokines to work.

Isaac Chiu

27:10 - 27:27

And they’re very important to resolve the immune response, right? So they were discovered and now people are trying to harness them to treat autoimmune diseases. And so, you know, that discovery also won the Nobel Prize. Tregs were last year, actually.

Isaac Chiu

27:27 - 28:02

So, actually, just two days, last week, there was a symposium here at Harvard, which the winner of the Nobel Prize, Sakaguchi, who discovered Tregs, was here giving a talk. But I want to bring that up because there’s also been recent papers showing that regulatory T-cells might be harnessed to block pain. And that’s really interesting because... They produce not just IL-10, but also endogenous opioid peptides, proenkephalin, which is the peptide precursor to opioids.

Isaac Chiu

28:02 - 28:19

And so if they’re there next to a pain fiber, they actually secrete things that shut the pain down, right? So you could also not only shut down the immune response there, but also block that pain. So people are trying to see if we can direct Tregs into areas to treat pain as well.

Nick Jikomes

28:20 - 28:52

Yeah, so let’s talk about some examples of neuroimmune communication, some concrete examples, and talk about some of the neuropeptides that you study, like CGRP and others. And I’ll let you choose the specific examples here. But what people should imagine is, if we talk about, say, an infection, a pathogen like a bacteria gets into an animal, there’s going to be communication from the pathogen to the immune cells and vice versa, from the pathogen to neurons, like nociceptor pain or noxious detecting neurons and vice versa.

Nick Jikomes

28:52 - 29:16

And then also between the neurons and immune cells in both directions. So you can pick the example here, but let’s say you get a bacterial infection, the animal has to deal with it somehow. How is that infection initially detected by the nociceptor neurons? And what does some of that communication start to look like between the nociceptor neuron and the pathogen, and then the neuron and the immune cells that will get called in?

Isaac Chiu

29:17 - 29:42

Yeah, this is a great, so this is definitely at the heart of what my lab studies. We’re really interested in what molecules the neurons produce that could affect the immune system and vice versa, as well as what are they sensing from pathogens. So you mentioned neuropeptides and neuropeptides are really important in this communication. So neuropeptides are, peptides that are actually used by neurons to communicate.

Isaac Chiu

29:43 - 29:59

Classically, think about that in the brain, how they communicate with other neurons. But there are many types of neuropeptides. One called CGRP, calcitonin gene-related peptide, which is highly expressed in pain-related neurons, CGRP. Another one called Substance B.

Isaac Chiu

29:59 - 30:27

So these are two of the best-known neuropeptides that nociceptor neurons produce. And it turns out that the neuropeptides, their receptors, which we, again, was first discovered in the brain, are also expressed on immune cells. So these neuropeptides can act directly on immune cells. And so it depends on the type of immune cell, but the action of neuropeptides during infection could have various effects.

Isaac Chiu

30:27 - 31:05

So we found, which is somewhat surprising, actually, that in several types of infection, for example, infection caused by the strep pyogenes, the pathogen that causes strep throat. So when you think of it that, you know, when we get this really painful infection when we were young, but also it causes really very terrible bacterial infections of the skin. For example, necrotizing fasciitis, which is flesh eating disease, extremely painful. Well, it turns out that in that type of infection, it causes the nociceptors to be activated and then they release CGRP.

Isaac Chiu

31:07 - 31:23

They release them from the nerve terminal. So they’re actually stored in these dense core vesicles, which are at the endings of these nerves. And as soon as they’re activated, they release these CGRP out into the tissues. And then they acted, we found that they act on neutrophils.

Isaac Chiu

31:23 - 31:34

Neutrophils are really important immune cells that actually come and eat bacteria. So they’re circulating in the blood. They’re kind of like our army of white blood cells. They’re some of the most abundant immune cells.

Isaac Chiu

31:35 - 31:50

when you have an infection, they rush into the tissues to try to fight the bacteria, right? So they’re also the ones that produce pus. Like when they die, they actually, what pus is, it’s like neutrophils fighting bacteria.

Nick Jikomes

31:50 - 31:58

So- Yeah, it’s like when you go to the battlefield after the battle’s over, it’s all of the dead bodies- Yeah, exactly.

Isaac Chiu

31:58 - 32:24

Our immune cells have sacrificed themselves, but at the end, that’s good because they’ve also killed the bacteria. But we found almost a little bit counterintuitively that CGRP, the nociceptor-derived neuropeptide, actually shuts down the ability of these neutrophils to kill bacteria. They block their ability to clear bacteria, as well as block their recruitment into the tissues. So this is actually a really bad thing, right?

Isaac Chiu

32:24 - 32:30

Because if you block these neutrophils, then the bacteria can then expand and grow more.

Nick Jikomes

32:31 - 32:42

So you would not want to release a bunch of CGRP early in an infection. You’d want to maybe use it as a turn-off signal later on or something.

Isaac Chiu

32:42 - 33:07

Yes, exactly. So you’ve hit the nail on the head. So it turns out that CGRP we think is actually, or actually not just my work, but other labs have found that it’s quite important in inducing that tissue healing, wound healing part, which is part of resolution of tissue injury. So if it’s on too early, it actually suppresses the type of immune response that you need to fight the bacteria.

Isaac Chiu

33:08 - 33:43

So the bacteria in this case hijacked this neuropeptide for its advantage. And we’ve seen similar roles of this in also when we have brain infections. So meningitis is a really horrible type of bacterial infection that is life-threatening. So bacteria in the meninges, which is the outer part of the, you know, it’s a wrapping outside the brain, Actually, there are lots of pain fibers there, and these nerves are activated by the bacteria, they produce CGRP, which then again shuts down these neutrophils.

Isaac Chiu

33:44 - 34:07

So then the bacteria can get through that meninges into our brain. So we found, I think this was surprising, but also it gave us a therapeutic angle. there are now really good drugs that people are using to actually block CGRP. So CGRP has been shown to really be a key mediator of migraine headaches.

Isaac Chiu

34:08 - 34:32

And so there are multiple drugs on the market right now that target either neutralize CGRP, the neuropeptide, or block the receptor called RAMP1. And so we found if we took these drugs and gave them to animals that were infected with either necrotizing fasciitis bacteria or meningitis bacteria, this actually boosts the neutrophil response and helps the mice survive.

Nick Jikomes

34:32 - 34:36

I see. So hold on. Let’s back up for a second. CGRP is a neuropeptide.

Nick Jikomes

34:36 - 35:01

It’s a peptide released from neurons. It’s affecting the immune system. It is Basically turning down the neutrophil response and and turning it down. So There are there are infections you can get like necrotizing fasciitis where if i’m hearing you correctly The bacteria is sort of turning up the cgrp response so that the immune system is not called in and therefore the infection Is more robust and it spreads and it takes

Nick Jikomes

35:01 - 35:08

root and so again cgrp is a neuropeptide But it’s it’s sort of suppressing the immune system, basically

Isaac Chiu

35:08 - 35:50

That’s correct, that’s correct. So, you know, I was telling you earlier about how nociceptors detecting pathogens might be beneficial for us, but in this case, it’s actually the pathogen which is exploiting this system for its advantage. So the pain fiber shutting down neutrophils is definitely harmful in these situations where the bacteria are the main cause of damage and you really need neutrophils to kill them. But there are also counterexamples, which this is then why we think the system is there, where actually the immune response or overactive immune response is actually what kills you as the host.

Isaac Chiu

35:50 - 36:09

So one salient example of that is influenza infections. So when you have a flu, very severe lung infections caused by viruses like COVID-19 or flu, it’s not necessarily the virus itself that damages your lung. It’s actually the overactive immune system.

Nick Jikomes

36:09 - 36:16

Right, right. The immune response is too strong and the collateral damage is basically so great that you destroy yourself.

Isaac Chiu

36:17 - 36:38

Yes, exactly. And that’s where we found that the same nociceptive neurons actually are required for the host to survive because they shut down this same cell type, neutrophils. Neutrophils, you know, in the flu context, they come in and they’re not protective because first of all, they don’t really do that much in killing flu. But in fact, they damage the lung.

Isaac Chiu

36:38 - 36:51

So if you have now these neurons that are activated, they shut down neutrophils. that helps you suppress or resolve the inflammation in a way that allows you to survive.

Nick Jikomes

36:51 - 37:20

So, yeah. And this is, so of course, you know, we need to talk about these things one at a time, but it’s always a complex mixture and cocktail of signals that is regulating what’s happening in our tissues. So, you know, CGRP is one peptide that needs to go up and down in the right way so that the immune system is not over or under activated at the right phase of the infection, right? When the infection is manifesting and growing, you want to call in the troops to deal with it.

Nick Jikomes

37:20 - 37:36

But then as they do their job and they finish it, you want to turn that signal off. We talked about the prostaglandins earlier, right? When you get a bruise or a bump or whatever, you want some of those inflammatory lipids to come in, but you also want them to go away. You don’t want that inflammation to be chronic.

Nick Jikomes

37:37 - 38:05

So there’s a balance between many different things and all phases of an infection or an injury process. And all of these things need to sort of wax and wane in the appropriate relative amounts to have a healthy response that doesn’t turn into a pathological or chronic response. So CGRP is one example of a key player here. Can you say a little bit about, I just want to back up one more time before digging into that a little bit more.

Nick Jikomes

38:06 - 38:26

So CGRP is a neuropeptide, a peptide released from neurons. Peptides are basically miniature proteins. Of course, there’s other molecules involved here, small molecules, bigger proteins, like they’re all involved in different ways. But what are sort of the advantages or the uses of a peptide as a signal as opposed to other things?

Nick Jikomes

38:26 - 38:33

Are they faster or slower? Are they more specific or less specific? What’s nice about peptides as like a class of signaling molecules?

Isaac Chiu

38:34 - 38:57

It’s a great question. So I’ll contrast peptides to, let’s say, a neurotransmitter. So when we think about neurons that signal in the brain, we often think about classic neurotransmitters like you know, glutamate, acetylcholine, or adrenaline. So these things are very small molecules, and they are at the synapse, right?

Isaac Chiu

38:57 - 39:29

So in the synapse, which are two nerves really, really close together, they’re rapidly released, and then they’re rapidly acting on the next neuron, but they also degrade really quickly. So, Peptides, on the other hand, are larger than these neurotransmitters that we think about, so they’re more stable. They can be released and they can actually act a little bit more in the long distance, right? So they can, you don’t necessarily have to have a synapse, which is within really small distances between cells for a neuropeptide to act.

Isaac Chiu

39:29 - 39:54

neuropeptides can diffuse in tissues. That might be good in the sense of talking to immune cells or talking to blood vessels because they can diffuse and let’s say also affect recruitment of these cells or affect signaling in a longer term. These signals don’t degrade as much. And I mean, people are still trying to understand the logic of different neuropeptides.

Isaac Chiu

39:55 - 40:28

Actually, I mentioned two, CJRP, Substance P, but there are many other neuropeptides that sensory neurons make. In fact, I want to point out something that I didn’t mention earlier. If you look at all sensory neurons that innervate our skin, and there’s probably about 18 to 20 different types of these, and each of them express different types of neuropeptides and different types of receptors. So the complexity of sensation, whether it’s pain or touch or itch, is still being understood.

Isaac Chiu

40:29 - 40:50

And each of these neuropeptides may have a different role in talking to tissue cells, right? It could be maybe one of them is talking very specifically to one type of immune cell, whereas others are more broadly active. So there’s that advantage as well. They’re longer lasting, they’re maybe giving you a code of acting on their targets.

Isaac Chiu

40:50 - 41:08

I just want to highlight one interaction. So Substance P is another neuropeptide that’s not CGRP, right? It’s receptor... There’s been only in the last few years, a discovery of this, it’s another name, MRGB2, only found in mast cells.

Isaac Chiu

41:08 - 41:21

Mast cells are a type of immune cell that are full of granules themselves. And substance P can really potently induce a mast cell to empty its contents, right? So mast cells produce things like histamine.

Nick Jikomes

41:21 - 41:28

Yeah, when we talk about mast cells, we basically mean histamine. So when people think about allergies and allergy medicine, those are the cells we’re talking about.

Isaac Chiu

41:28 - 41:39

That’s right, exactly. So you might think, OK, in some conditions, I want. mass cells to be activated, but not neutrophils. I don’t want to affect neutrophils, right?

Isaac Chiu

41:40 - 42:01

So there could be some logic of why certain neuropeptides are in certain nerves and only activated by certain stimuli, for example, during allergies, so that can amplify the allergic response. Whereas when you have an infection, you don’t necessarily want an allergic response, right? It might have a different set of neural signals, neuropeptides that are released.

Nick Jikomes

42:02 - 42:18

Yeah. And so again, just to like hammer this home, we have to talk about these things one at a time, but it’s not like the nociceptors release substance P or CGRP and that’s it. It’s not like the immune cells release prostaglandins and that’s it. They’re all releasing a complex cocktail of stuff.

Nick Jikomes

42:18 - 42:44

And it’s the full mixture that determines the spatial, the temporal, dynamics for how inflammation plays out. It’s like this really complex balance of things that interact with each other. We talk about them one at a time. These components are important individually, but we always have to remember that these things fit back into a really complex and dynamic soup of stuff.

Nick Jikomes

42:44 - 42:45

Yes, yes.

Isaac Chiu

42:45 - 43:09

I think that’s the exciting thing or maybe difficult thing of science, right, is to understand the complexities in a real life situation, right? There’s all these different types of mediators that are coming out of each neuron subtype and then there are different immune cells which also produce other mediators. And I also want to point out the bacteria too. The bacteria have their own lots of things being produced, right?

Isaac Chiu

43:09 - 43:28

So I think there’s, there’s the understanding this crosstalk between systems is fascinating. I think as a scientist, we tend to try to be reductionist. So we say, okay, we’re going to look at our one, our one interesting molecule and receptor, but we have to take it into context of all the complexities that you just mentioned. Right.

Isaac Chiu

43:28 - 43:50

And I think, and, um, you know, in the course of infection, also there’s, like you said, kinetic changes in each of these parts. The bacteria is also trying to adapt, right? They have evolved to avoid, you know, each of these things. So they might make something that will target one type of nerve and one type of immune cell.

Isaac Chiu

43:50 - 43:56

And so that also adds another interesting level of complexity.

Nick Jikomes

43:56 - 44:33

Yeah. And then sort of another Another topic of discussion here that I think is really interesting is the tuning or the appropriateness of the immune response and how the nervous system informs the immune system in terms of how it needs to come in and deal with the problem, such as an infection. So let’s say, again, you get an infection, a pathogen is taking root, you’ve got to call in the troops, but there’s many different types of soldiers that can be in the army, and you want to call in specific soldiers for specific problems, depending on the pathogen, or if

Nick Jikomes

44:34 - 44:55

it’s an injury, and so on and so forth. You know, one way to talk about this is, right, there’s many different types of white blood cells. There’s many different types of T cells, regulatory T cells, other T cells, different subtypes. And every infection is going to, you know, call in some of those cells and also cause them to change, right?

Nick Jikomes

44:55 - 45:23

The immune system is sort of dynamically changing and cells are transforming into different types. in response to the particular pathogen or injury or problem that we’re experiencing. Can you start to talk about maybe some examples of where an animal gets an infection, there’s a pathogen, and then the neurons are communicating to the immune cells and sort of informing them about how to develop, how to change, which soldiers need to be called in. How does that communication look like?

Isaac Chiu

45:23 - 46:00

Yeah, I think this is a very interesting question. And I like that you’ve framed it that way, that the nervous system is ideally poised to coordinate the response, right? So I also want to talk maybe more broadly, the peripheral nervous system, right, which is these pain nociceptors are part of that larger peripheral nervous system has set up all these different neural circuits, right? That, for example, you have beyond sensory, you have sympathetic nerves that are part of our fight or flight response or parasympathetic nerves that are, you know, mediating other functions.

Isaac Chiu

46:00 - 46:31

We know from organ function, right? You have to have each of these branches coordinated to modulate, let’s say heart rate or modulate digestion, but the immune system is no exception. So it makes sense that the coordinated activation of different branches of the peripheral nervous system will induce different types of immune cells to come in or to leave in it or to dampen those kind of immune cells. And this is, I have to confess, it’s still early days.

Isaac Chiu

46:31 - 46:59

We’re trying to understand how these different branches of the nervous system, including sensory neurons that produce CGRP, are affecting the immune response in different conditions. I wanted to highlight a study that we did, which was on an infection, but actually in the gut, we were interested in the gut brain axis. So we did a very, I would say agnostic type of experiment. So we said, okay, there are so many different nerves in the gut, right?

Isaac Chiu

46:59 - 47:16

There are ones that are coming from sensory neurons, like the pain fibers. There’s also ones that come from the vagus nerve, which is a nerve that goes from the gut to the brainstem. And there’s also neurons that, sit in the gut itself, right? They’re called enteric neurons.

Isaac Chiu

47:16 - 47:32

They cause us to have peristalsis. So what we did is scientifically, we took a approach called chemogenetics, where we can activate neurons using a synthetic molecule. And we activated eight different types of neurons that innervate the gut. They’re of different types.

Isaac Chiu

47:32 - 48:00

And we just said, what happens to the immune system? Like unbiased, we said, okay, well, we’re gonna activate them, each of these, and then just profile immune cells in the gut. and we’ve discovered things that were unexpected like for example this one type of neuron when we activated them cholinergic neurons caused one type of immune cell to change in one part of the gut and or you know there’s kind of a matrix of like nerves controlling different immune cells.

Isaac Chiu

48:01 - 48:30

It turns out nociceptors, because that’s the one that we really know the best, had the strongest effect, and it acted on multiple types of immune cells. So macrophages, which are phagocytic immune cells change, and also regulatory T cells, the Tregs that I mentioned to you earlier. One of the most striking changes was if when we activated the pain fibers, nociceptors, the Treg population in the gut went down in number. So I told you that Tregs are important for controlling inflammation.

Isaac Chiu

48:31 - 48:53

Here, when we activated pain fibers or TRPV1-expressing nociceptors over the course of a week, or actually over a week, the Treg numbers went down. So maybe it’s a counterbalancing effect where now you have chronic inflammation. so to speak, chronic pain, that signal actually decreases Tregs. So now inflammation will even go up.

Isaac Chiu

48:53 - 49:22

So I think it’s extremely complex. We’re still trying to understand how, you know, the logic of all this, like how, why do certain nerves control certain immune cell types? how it’s all, you know, coordinated during inflammation or different conditions related to infection, but also autoimmune diseases, maybe cancer, things like that. So there’s lots of really interesting interactions between these two systems that are still starting, only starting to be understood.

Nick Jikomes

49:22 - 49:37

Yeah, yeah. I think it could be useful to do a kind of compare and contrast of the gut, the gastrointestinal tract as a barrier and a surface, and the skin. So on the one hand, there’s clear differences, right? That are sort of the intuitive differences, right?

Nick Jikomes

49:37 - 50:03

My skin’s touching the outside world. My gut is dealing with food absorption and all these things. But on the other hand, they’re both barriers, they are both interfaces between the true internal world inside of ourselves and things from the external world, whether it’s the skin directly in constant contact with the literal external physical world, or food, pathogens, stuff from the external world that we put into our mouth that goes through the GI tract.

Nick Jikomes

50:03 - 50:14

So there has to be a barrier there. It has to be selectively permeable. Stuff’s got to get in, but not certain things. And obviously, the immune, excuse me, the nervous system has to innervate it.

Nick Jikomes

50:15 - 50:40

It has to talk to the barrier in different ways. And then, of course, the immune the immune cells have to be there or be able to get there very easily because it is an interface, because it’s also a site where pathogens can get in and so forth. What are some of the key similarities and differences between the skin and the gut as it relates to just being barriers of the body that need to be selective and highly regulated?

Isaac Chiu

50:41 - 50:54

Great question. So these are two of my most favorite organs, right? So they’re both highly exposed to the environment. But you’re right that there are unique features, both in terms of the nervous system there, as well as the immune system.

Isaac Chiu

50:55 - 51:18

So in the skin, we have a lot of nerves that can sense, you know, mechanical stimuli to induce touch, right? Touch sensation, and there’s also pain that we talked about, but also itch, right? So itch is, at least in terms of what we think about scratching, an itch is actually pretty specific to the skin, right? If you think about that sensation, it causes you to scratch the skin.

Isaac Chiu

51:20 - 51:48

So in terms of nerves, that’s one thing, that there are only itch fibers in the skin, and maybe it’s because we need to remove things that are at the surface before they get in, right? And then in terms of The gut, the gut has, it’s a mucosal surface in the classical sense. So it produces mucus, mucus, this term, mucus, right? Which actually is a very important part of our barriers like the gut.

Isaac Chiu

51:48 - 52:21

So because the mucus lining separates the barrier, the epithelial cells from things in the lumen of the gut, like for example, bacteria that could be damaging that surface, right? So you need to, you need to separate. It’s kind of like an offense, but it’s really important how it’s produced, like the composition and whether that’s regulating homeostasis in the gut, right? So we actually found that nociceptors, pain fibers, induce mucus production.

Isaac Chiu

52:21 - 52:44

So they directly talk to cells called goblet cells, which are not in the skin, but in the gut. Goblet cells make mucus. So the neurons in the gut that actually express CGRP, this again, this neuropeptide I mentioned, CGRP directly acts on goblet cells. So as soon as you activate these nociceptors, these Zigaba cells start making mucus.

Isaac Chiu

52:44 - 53:08

And experientially, actually, one of the things that we showed is that capsaicin, which is the, you know, that active ingredient of chili peppers that causes spiciness. Capsaicin, if you give it to animals, it causes the immediate production of mucus. And it’s because of CGRP and these neurons. So when you eat spicy foods, you start getting secretions, right?

Nick Jikomes

53:08 - 53:19

And it’s the basic idea here, the body’s detecting something that’s potentially noxious, like a spicy substance, and the mucus is meant to neutralize it or trap it or prevent it from getting in.

Isaac Chiu

53:20 - 53:34

Yes, I think that’s exactly right. So it’s preventing something potentially harmful and it’s usually coordinated too with something like motility, right? So the gut starts speeding up and you make mucus. So this is, actually immunologists use this term weep and sweep.

Isaac Chiu

53:35 - 53:54

So you get rid of things by making production of secretions and then you sweep it out. And so it’s another protective mechanism. I also want to point out the gut is very also unique in the sense that there’s a huge amount of microbes that live there, right? There’s a microbiota of which many of them are actually good for us.

Isaac Chiu

53:54 - 54:07

They actually... immune system to help produce metabolites that really help us with energy. And also the other unique thing is we eat our food, right? Food goes through the gut.

Isaac Chiu

54:08 - 54:46

So the gut has all these foreign things there, food and microbes that our immune system has to tolerate. So it’s called tolerance. So the gut is very complicated because you have to have, in fact, there’s lots of Tregs in the gut, the regulatory T cells, because they cause our gut not to interact or overact, our immune system not to overact against food or against these bacteria, right? And so it’s interesting to also think about how nerves there interact with each of these things, like the food that we eat or the bacteria that reside there.

Isaac Chiu

54:47 - 55:17

We know from the immune response that it’s also quite different when you have, and one of the things we’re actually studying and many people in the immunology field studying is food allergies, like when you have reaction against food, which is normally supposed to benefit us, how does that occur? Well, there’s one theory that it’s because the food first went through the skin when the child is young, because when something goes through the skin, it’s thought to be, this is bad. We have to have an immune response against it.

Isaac Chiu

55:17 - 55:27

So there’s evidence that, for example, kids who are exposed to peanut in some form, peanut oil is often found in lotions.

Nick Jikomes

55:27 - 55:31

Right. Yeah. I was going to say like, yeah, food isn’t just found in food. We have all of these things.

Nick Jikomes

55:31 - 55:50

We’ve made lotions, moisturizers, shampoos. We put them on our surface. But if they’ve got some food component in them, You’re putting it through the skin route of entry as opposed to the gut route of entry. And as you’re implying, maybe the immune system therefore misrecognizes it.

Isaac Chiu

55:51 - 56:25

Exactly. So there’s this correlation between skin exposure to things early in life to future development of food allergies. And one of the things we’re interested in is whether itching is part of that process. So, you know, when kids get eczema, which is a very common, or atopic dermatitis, a very common skin disease in young kids or newborns, and they’re scratching their skin, if that then is co-exposed with something, and we’re using egg white as a model antigen, but that then induces

Isaac Chiu

56:25 - 56:41

a very strong immune response. So maybe the skin barrier, by opening that barrier, Now you start inducing immune response, and next time when it goes to the oral cavity, instead of tolerating that, or oral tolerance, so to speak, now you’re reacting against it.

Nick Jikomes

56:41 - 56:52

Right, right. And just, you know, it’s hopefully pretty easy for people to think about. what might happen here and why these things are so common. Kids are going to itch scratches.

Nick Jikomes

56:52 - 57:03

They’re going to open those wounds. That’s now an entry point. And just think of all of the things that we put on our body, lotions, soaps, whatever. They’ve got tens or even hundreds of components.

Nick Jikomes

57:04 - 57:36

And it’s not too difficult to think like, okay, yeah. the peanut molecule in the moisturizer or the fruit or whatever, you know, component is in your skincare product can now get in and potentially be recognized as a pathogen, essentially, as something that needs to get rid of. And then the next time you eat a food, a literal food that has that same substance inside of it, you’re now going to trigger an immune response. It’s not too surprising to suppose that this has a lot to do with the increase in allergies and autoimmunity that we’ve seen over time.

Isaac Chiu

57:36 - 57:54

Yeah, exactly. And I think that, you know, unfortunately, my son developed severe nut allergies. And, you know, it’s kind of also you see like generational, right? So when you’re not growing up in the US, but then your next generation grows up here, then these allergies start arising more and more.

Isaac Chiu

57:56 - 58:19

And part of it could be the way that we’re exposed to different skin products, like you mentioned. Another one is like the fact that actually we don’t expose our kids to the food orally early enough, right? So this idea that you actually should give your kids nuts when they’re really, really young. And so the immune system now recognizes that as food rather than something foreign.

Nick Jikomes

58:19 - 58:42

Right, right, right. The first, you know, you can imagine, right, the first interaction of the body with a substance, depending on how it gets in, if it goes in your mouth, it might say, okay, well, this, this must be food. And obviously, the immune system is plastic, it develops, it needs to learn about things. Versus if it goes in through the skin, you know, the body’s assumption, I would think is going to be well, if it comes in from the outside world through the skin, it’s probably a problem.

Isaac Chiu

58:43 - 59:09

Exactly. Yeah. So we, because we were very interested in how microbes affect this as well. So one of the things we had found a few years ago is that Staphylococcus aureus, which I mentioned earlier in my talk with you, that bacteria is very common on humans and actually it’s coating the skin in skin diseases like atopic dermatitis or other itchy skin diseases.

Isaac Chiu

59:10 - 59:48

So we’re we and it’s known that you know kids who develop peanut allergies actually there’s a correlation between Staph aureus on their skin and peanut allergy development. So we’re asking can these bacteria if you mix them in with these food allergens, could it trigger this skin allergic response, which then leads to gut, like skin to gut access, and whether nerves are a part of that as well. So we had found that Staph aureus can secrete a molecule called V8 protease, which stimulates itch fibers. And then the itch fibers, once they’re activated, then you scratch, right?

Isaac Chiu

59:48 - 1:00:09

So the bacteria actually may, again, use this in some way for its advantage to help spread, but it induces this itchy response. So if we block the itch, does that also block the ability of Staph aureus to induce this bystander allergy response? So this is one of the projects we’re working on right now in the lab.

Nick Jikomes

1:00:09 - 1:00:24

Yeah. So we’ve touched on a couple important things here that have to do with what causes immune reactions to go awry or become problematic. One key dimension here is when things happen. Obviously, hopefully it’s pretty intuitive for people.

Nick Jikomes

1:00:25 - 1:00:44

When things happen earlier in life or fail to happen early in life, the immune system might not be trained properly. It might mislearn or inappropriately learn something. So that’s why early life is important, right? It takes time for the immune system to learn and to develop and to recognize self versus non-self, pathogen versus food, and so on and so forth.

Nick Jikomes

1:00:45 - 1:01:14

The other dimension here that we mentioned that’s important is the spatial component, where things are happening. If something comes in through the mouth, the body is going to be predisposed or maybe make certain assumptions about it. They’re going to be different than if it comes in through the skin. So when you’re exposing someone through the skin versus through the GI tract, earlier versus later in development, all of these things are going to matter in terms of the immune system response and how the immune system gets trained to recognize and respond to things.

Nick Jikomes

1:01:15 - 1:02:06

The next thing I want to ask you more about is this sort of concept of chronic inflammation or inappropriate inflammation when inflammatory responses don’t resolve properly. It’s something I’ve discussed on the podcast before in the context of things like dietary lipids, like the omega-6s and omega-3s and the balance between them and how this can affect how long an inflammatory response lasts and make it maybe last a little bit too long if there’s an imbalance there, even though you wanted some inflammation for a while, you also want it to turn off when it’s no longer needed. When we start thinking about things like chronic inflammatory conditions, autoimmune conditions, things that have to do with this failure of inflammation resolution, What can you start to talk about there from your own lab or from stuff you’re familiar with in terms of how this actually happens or what’s

Nick Jikomes

1:02:06 - 1:02:09

causing the failure of inflammation to resolve?

Isaac Chiu

1:02:09 - 1:02:29

I mean, yeah, this is a really key question in immunology, right? So why are chronic inflammatory diseases, you know, actually rising? in society, and also how do we, you know, how do we solve that, right? I think there’s also many factors that could influence chronic inflammation.

Isaac Chiu

1:02:29 - 1:02:57

One, there’s this thing called inflammation, so aging itself, you were talking about you’re young versus old. Well, the old immune system may actually be more susceptible to inducing certain types of chronic inflammation. And that could then contribute to many diseases, including brain diseases, like we think about Alzheimer’s disease, or also chronic pain, like pain. So too much inflammation will contribute to damage to multiple organs.

Isaac Chiu

1:02:59 - 1:03:23

So we are trying to also understand, I guess the wrinkle in there is do peripheral nerves also play a role, right? Because neurons are, unlike immune cells, they last you their entire lifetime, right? So they’re not usually replaced. there are exceptions, there are parts in the hippocampus that get replaced, but these neurons that reside in our peripheral nerves actually last our lifetime.

Isaac Chiu

1:03:23 - 1:03:47

So could there be aspects of how they change that then also drive our chronic inflammation, right? So maybe they’re not, you said, we talked about sensitization earlier, right? Maybe they’re rewired in a way, due to injury or due to some other factor, right? That then causes that immune response to be overactive.

Isaac Chiu

1:03:48 - 1:04:21

I told you earlier about the flu example, for example, where we need nerves in our lung to control actually too much inflammation, right? So is it possible, as an example, that the nerves in the lung are not acting the way they should, and then you get like chronic lung inflammatory diseases, right? And so there’s a big interest in this because certain branches of the peripheral nervous system have especially been linked to modulating chronic inflammation. One particularly is the vagus nerve.

Isaac Chiu

1:04:21 - 1:04:40

So the vagus nerve I mentioned connects the gut to the brain, but also multiple other organs. It includes the lungs, the heart. A lot of our internal organs are connected to the brain via the vagus nerve. The vagus nerve is It’s called the vagus nerve because it’s a wandering nerve.

Isaac Chiu

1:04:40 - 1:05:11

It innervates so many parts of our internal organs. And what scientists have found that if you activate the vagus nerve, it actually has a very potent anti-inflammatory effect. Dr. Kevin Tracy was one of the first in New York to find that vagus nerve stimulation can suppress inflammation during sepsis, which is a big inflammatory condition. Many scientists are trying to understand how this works, right?

Isaac Chiu

1:05:12 - 1:05:19

So it’s possible that the vagus nerve and its dysfunction could lead to chronic inflammation as well.

Nick Jikomes

1:05:21 - 1:05:34

Yeah, it makes sense. I mean, the vagus nerve is talking to basically all of the organs in our body. And as you said, you know, I had Kevin Tracy on and I encourage people to go listen to that episode because I think it goes with this one quite nicely. But on the one hand, it’s like this remarkable result.

Nick Jikomes

1:05:34 - 1:05:56

You basically just stimulate the vagus nerve nonspecifically and you kind of just shut down inflammation, let’s say. But there’s probably a lot of fine tuning there that can happen. And of course, it’s not at all clear, you know, exactly how this is happening. Like, why would just generic, fairly generic stimulation of the vagus nerve just kind of shut down inflammation.

Nick Jikomes

1:05:56 - 1:06:10

And what is actually going on there in terms of the mechanics? And as you’re saying, like presumably there’s some kind of code, right? Like different parts of the vagus nerve probably firing different patterns, probably to turn up and down inflammation in complex ways.

Isaac Chiu

1:06:11 - 1:06:19

Yes. Yeah. So that’s almost certainly the case. So I mentioned earlier that sensory neurons are highly diverse in the skin.

Isaac Chiu

1:06:20 - 1:07:01

Well, the vagus nerve, there’s dozens of types of sensory neurons, and this is just looking at the molecules that they express. They’re not only anatomicals, some are innervating different organs, but some are clearly expressing different receptors for immune factors. Like for example, one, type of vagal neuron might express the receptor for TNF-alpha, which is a cytokine, and another one express one for IL-10. So actually, there was recently a really beautiful paper from another very well-known neuroscientist, Charles Zucker, who he’s actually the one who discovered all the taste receptors, but he’s now interested in the vagus nerve.

Isaac Chiu

1:07:01 - 1:07:37

And he found that if you activated one type of vagal neuron, that will induce one change in the immune system, very specific change in the immune system, for example, shutting down TNF production. And then another one, if you simulate that, will actually induce IL-10, which is this immunosuppressive cytokine. So he’s only focused so far on those two, but there are probably multiple other vagus nerve circuits that specifically talk to a certain branch of the immune system or certain type of inflammatory response. And it all goes through the brain.

Isaac Chiu

1:07:37 - 1:08:00

So the vagus nerve synapses in the brainstem from the body, and then in brainstem, there are also regions of the brainstem, for example, the NTS and DMV. So these neurons that map to other regions of the brainstem, and then they signal back to the body. And that back-to-the-body part, like you mentioned, is still being mapped. So what nerves actually take the signal back from the brain to the body?

Isaac Chiu

1:08:01 - 1:08:18

And how do they then control that very specific immune response? How do you tell immune cells only to turn on one type of cytokine or shut down another one? Very mysterious, I think. And I want to bring this to an even broader question.

Isaac Chiu

1:08:18 - 1:08:48

Is this body-brain axis with inflammation? There are theories that there might be very specific regions of the brain that are there just to encode inflammatory sensing. So, part of the cortex called the insula region has been shown that they might capture memory of inflammation from the body. And then if you reactivate those neurons, it can actually recall the inflammatory response in the body.

Isaac Chiu

1:08:48 - 1:08:59

So it’s amazing that you can store memory of something that happened, let’s say in your gut. And then you recall that and then the gut immune response gets reactivated when you start

Nick Jikomes

1:08:59 - 1:09:11

Right, right. And just so people know, so the insula is basically a part of our brain on the side of our head more or less. And it’s got a map of our internal organs. It’s sort of like a map of the internal body.

Nick Jikomes

1:09:11 - 1:09:20

And you’re saying that part of the insula at least can retain a memory of maybe like a gut infection we once had or something like that.

Isaac Chiu

1:09:22 - 1:09:50

Yeah, exactly. And this was really work from Asya Rolls in Israel who had a really interesting study where they found that, let’s say colitis, gut inflammation, can be encoded in insula. And then she used a different part of the body, like the peritoneal cavity, doing inflammation. And when she’s activated nerves due to one type of inflammation, it caused that specific anatomical part to then have immune cells go in later on, after the inflammation’s gone.

Isaac Chiu

1:09:51 - 1:09:54

You can recall that just by activating those neurons.

Nick Jikomes

1:09:54 - 1:10:04

So the brain can not only encode a memory of something like an infection, it can quite literally direct the immune system where to go. Go to that organ, but not that organ.

Isaac Chiu

1:10:05 - 1:10:24

And that’s the amazing thing about the brain, right? That the immune system really can’t do, right? To really have anatomical and kinetic specific cellular specificity encoded by memory. And I think that that is probably a place where neuroimmunology is, there’s so much to understand there.

Isaac Chiu

1:10:24 - 1:10:51

Like how is that memory, you know, encoded and how do at the cellular level, how do these nerves actually control the immune system very specifically in one site? And similarly, we’re interested in similarly, whether the peripheral nervous system memory. So like I mentioned, sensitization, right? That process that we usually think about as inducing increased pain, but maybe that’s also a mechanism to store an immune memory.

Isaac Chiu

1:10:52 - 1:11:16

So because let’s say our peripheral nerve that’s still very sensitive, if you activate that, does that also affect the next immune response at that same site? a logic to this that the nervous system may have an advantage, right? You can encode spatially and also long-term memory response. We know the immune system has memory too, right?

Isaac Chiu

1:11:16 - 1:11:30

So when you can reinfect it with a virus, that same immune cell will, like the T cell or B cell, will then proliferate and respond. But that doesn’t necessarily have, that may have the molecular specificity, but doesn’t have that spatial

Nick Jikomes

1:11:31 - 1:12:02

uh you know and or kinetic specificity that the nervous system can can supply yeah yeah interesting um i want to ask you a little bit about the connection between things like pain and itch um they’re distinct but they’re also related Maybe one fun way to get you talking about this as a scientist who knows this stuff well is we’re all familiar with pain and itch on our skin from experience. We’ve all gotten mosquito bites. We’ve all had rashes or contact dermatitis or whatever.

Nick Jikomes

1:12:02 - 1:12:22

We’ve all had bumps and bruises. When I was a child, and I think a lot of people experienced this, I was always told, don’t itch the rash, don’t itch the mosquito bite because it’ll spread it. And yet itch seems to be, in an ecological sense, it’s kind of like a behavioral impulse to remove something noxious from the skin. So let me ask you this.

Nick Jikomes

1:12:23 - 1:12:35

If you get an itch, like a full itch, maybe I’ve got poison ivy or I got an infection or a mosquito bite, but I’m really itchy somewhere on my body, what’s the appropriate action to take? And what is it really doing?

Isaac Chiu

1:12:37 - 1:12:46

I mean, I think this is a great question. So why do we itch, right? So that’s the, I think pain is a little bit more intuitive, right? You avoid injuring that site, right?

Isaac Chiu

1:12:46 - 1:12:58

And you avoid being exposed again to that hot, you avoid a fire, right? You avoid the source of danger, but itch, you’re actually scratching. So how is scratching protective? Well, there’s different theories, right?

Isaac Chiu

1:12:59 - 1:13:28

So one is this idea of back way back when we were living in the jungle or forest and you get something on your skin, including insects or parasites, that this itching would remove that. And there is also this phenomenon called mirror itch. So when you think about itching or you see somebody scratching, you also start scratching. So again, it could be like a protective behavior as a herd that you remove things that are bad on your skin.

Isaac Chiu

1:13:29 - 1:13:37

But there’s a counter argument to that in the sense that, as you know, with a mosquito bite, you’re itching way after the mosquito has left you, right?

Nick Jikomes

1:13:37 - 1:13:38

Right.

Isaac Chiu

1:13:38 - 1:13:47

You probably, you know, minutes or even hour before. And now the thing is becoming. So then why do you scratch? Like, how is scratching even helpful?

Isaac Chiu

1:13:48 - 1:14:08

Well. So actually one of my close friends and collaborators, Dan Kaplan at University of Pittsburgh, has been studying potentially an idea that itching is actually therefore inducing an immune response. So when you scratch, right, as we just talked about earlier, it induces a rash, it induces inflammation.

Nick Jikomes

1:14:09 - 1:14:18

Right. It’s almost like it’s almost like an adjuvant or something. It’s like you’re you’re you’re causing the immune response to be even stronger by actually agitating the surface of the skin.

Isaac Chiu

1:14:19 - 1:14:25

Exactly. And especially for if you want to induce a certain type of immune response, like, for example, allergic immune response. Right. So maybe.

Isaac Chiu

1:14:26 - 1:15:05

And there is a theory that for things like injected into you, like venoms, that, you know, that an allergic immune response actually helps wall off that venom or helps remove that thing. that let’s say the mosquito has injected. So if you can induce that by scratching, maybe locally in the skin where the mosquito has bitten you, there would be a protective response, right? So this is all very theoretical and we’re all trying to understand that, but that could also be why I mentioned earlier when you get a food allergen going through the skin and you’re scratching that that then induces the allergy response, which is not protective for you, right?

Isaac Chiu

1:15:06 - 1:15:40

But I also want to point out that other than, you know, scratching and itch, Just like with pain, there’s very complex mechanisms by which itch occurs. Like you have different nerve types that cause itch and they respond to different what’s called pruritogens, things that can activate an itch fiber. There’s at least three different types of itch fibers and so how they’re all activated and what they’re doing is potentially different. But there is also, like you mentioned earlier about pain, there’s adaptive itch, but also maladaptive itch.

Isaac Chiu

1:15:40 - 1:16:10

So there’s chronic itch, right? So there are people who have itchy conditions that don’t go away. There are many skin diseases, for example, like I mentioned, atopic dermatitis. which is probably one of the most common ones, but there’s also other ones like prurigo nodularis and there’s ones that are very much there’s even idiopathic itch which is unknown origin where people just keep scratching and they it’s very hard to treat that because we don’t yet understand

Isaac Chiu

1:16:11 - 1:16:30

you know, what is triggering that and how to address it, right? So there’s conditions where shutting that down, I mean, to be honest, people who have those conditions are miserable, right? That could be even worse than chronic pain where you’re just constantly wanting to scratch and you can’t relieve that itch.

Nick Jikomes

1:16:30 - 1:16:31

Yeah, yeah, yeah.

Isaac Chiu

1:16:32 - 1:16:55

Yeah, so I think I did want to point out the differences also between pain and itch. So it used to be, let’s say a decade ago, thought that itch was just a subset of pain where like maybe itch is like where there’s a lower amount of tickling of the same nerves that cause pain and that’s why you get an itch. But now it’s very clear they’re devoted nerves to each of these type of sensations.

Nick Jikomes

1:16:55 - 1:16:57

It’s a distinct type of sensation.

Isaac Chiu

1:16:58 - 1:17:09

Yes. And so the itch fibers have different receptors on them than pain fibers. So you mentioned prostaglandins earlier. So prostaglandins we know causes pain, right?

Isaac Chiu

1:17:09 - 1:17:28

Because prostaglandin receptors are in many of these nociceptors. For itch fibers, we had found and others that this other lipid called leukotrienes, cystine leukotriene, leukotriene C4, there’s a receptor specifically on itch fibers and this leukotriene is activated

Nick Jikomes

1:17:29 - 1:17:30

during allergies.

Isaac Chiu

1:17:50 - 1:18:17

But they cause different sensations. So we know, and logically, again, if there’s a logic to why these two systems are talking to each other, so during allergies, you want to itch, whereas during, you know, tissue injury, you want pain. So the mediators that are produced by immune cells, many of these lipids are produced by immune cells, are produced in different contexts. And so you have itchiness in one condition versus pain in another condition.

Isaac Chiu

1:18:18 - 1:18:27

And then the resolution modulators are also a hot topic. could resolve itch or pain, right? Yeah.

Nick Jikomes

1:18:28 - 1:18:48

Interesting. I want to ask you a little bit more about things like mast cells, histamine, itching and allergies. So I feel like, I don’t know how much of this is just me reading more about this stuff and noticing it more versus it actually changing in people’s lives. But I had seasonal allergies as a kid.

Nick Jikomes

1:18:48 - 1:19:16

Antihistamines definitely were effective for me. Antihistamines, if I take them preemptively, will definitely prevent me from having the cold-like reaction, the snot, the sneezing and stuff. They also seem to affect things like itch, which hopefully that starts to make sense for people now. When we think about things like chronic inflammatory conditions, autoimmunity, I’m hearing more, it seems, about things like mast cell disorders, histamine sensitivity, and so forth.

Nick Jikomes

1:19:16 - 1:19:29

Can you give us a sense for allergic responses, autoimmunity, especially as they relate to mast cells and histamine? Are these things going up? And is this like an area of, is this a hot topic right now in neuroimmunology at all?

Isaac Chiu

1:19:30 - 1:19:43

Yeah. So the mast cell nerve interaction is really intimate. In fact, if you look in most tissues, Where you find a mast cell, you’ll find a nerve next to it. So they’ve somehow co-evolved in some way.

Isaac Chiu

1:19:44 - 1:20:00

And so one is through it, for the reason of itch, right? So like you mentioned, histamine is one of the key things that mast cells make. And many of these nerves have histamine receptors, right? So when you activate these mast cells, then they start firing and you get this itch sensation.

Isaac Chiu

1:20:00 - 1:20:04

Mast cells are also chock full of cytokines and also proteases.

Nick Jikomes

1:20:04 - 1:20:21

Can we think of like mast cells? They’re basically a type of immune cell and they’re filled with these big bags, these big vesicles, and they can just dump a lot of histamine, cytokines, et cetera. So it’s like a big bucket gets dumped when something stimulates a mast cell.

Isaac Chiu

1:20:21 - 1:20:35

Yeah, exactly. And they’re very powerful, right? They’re few in number, but they are extremely powerful in inducing inflammation and inducing the downstream immune response. So, yes, there are definitely more and more interest in mast cells beyond just allergies.

Isaac Chiu

1:20:35 - 1:21:02

I mean, I think, like you mentioned, autoimmune diseases, you know, many, many inflammatory conditions, mast cells, there’s hints that they play a role. One of the things I mentioned earlier though, is the really interesting thing where the nerves can also activate the mast cells. So it’s not just, so the nerves could still be part of that equation, right? So they release this, I mentioned neuropeptide called substance P and substance P that induces a mast cell to degranulate.

Isaac Chiu

1:21:02 - 1:21:23

So, Classically, in allergies, you have antibodies like IgE, which is induced against, for example, the peanut, right? And that’s on the mast cell. And then when a peanut antigen comes in, it activates the mast cell. So that’s classic antibody-driven mast cell degranulation.

Isaac Chiu

1:21:23 - 1:21:55

But then there’s also neurogenic mast cell degranulation, which I mentioned is the nerves can also, independent of antibodies, induce the mast cell to release their contents. And so there’s still a question of, are these, when you’re activated by one thing or the other, do mast cells release the same things or do they release different things? And that’s very important because what they release could affect the downstream immune response very strongly, right? And then could we block some of these pathways to block mast cells from going awry?

Nick Jikomes

1:21:56 - 1:22:20

I want to step back for a minute and ask you kind of a general question. So we know that various forms of autoimmunity, allergies, chronic inflammation have been on the rise. A lot of people experience these things chronically, all the time, basically. One way that people often respond to having such an issue is they might start taking antihistamines like Zyrtec every day, just as a preemptive measure.

Nick Jikomes

1:22:20 - 1:22:45

They might start taking NSAIDs every day, just because they just get in the habit of it. It does help. It can help, at least in the right circumstance. Is there any, you know, so there’s a difference between treating the symptoms, treating, you know, the end result of the inflammation, the pain you might feel, the allergic, you know, stuffy nose response that you might have to an allergy to something.

Nick Jikomes

1:22:46 - 1:23:25

But there’s a difference between that and understanding the cause of that and the cause of the inappropriateness of it. Maybe it’s the balance of fatty acids in your diet affecting how inflamed you get when you get an injury. Maybe it’s not understanding that you’ve got a food allergy and you keep eating something that’s stimulating an immune response, but you just don’t realize it because it’s not so striking that you notice like, oh, hey, I must be allergic to peanuts or whatever. Is there anything you want to say or any downsides to chronically treating these immune responses, these inflammatory responses with things like NSAIDs or antihistamines day by day, week by week?

Isaac Chiu

1:23:26 - 1:23:48

Yes, I think this is a very good question because many people take these without, you know, there’s no side effects, right, in their mind. But you mentioned earlier one thing, right? Inflammation is there for a reason, right? So you need to have inflammation go in this way where it goes up after tissue injury and then goes away.

Isaac Chiu

1:23:48 - 1:24:23

So there was a really interesting study from a group in Montreal where they were a pain lab, this is Luda Dyatchenko’s lab, where they were thinking, OK, well, chronic pain, what we just talked about, right, is something that occurs in many people. But what’s the difference between somebody who gets it or not? And one of the striking things, which, again, very surprising, was the use of NSAIDs during the injury. So people who used NSAIDs I don’t, to be honest, I don’t remember exactly what kind of injury they were looking at, but they developed chronic pain more than people who didn’t use NSAIDs.

Isaac Chiu

1:24:24 - 1:24:40

So meaning that they were acutely blocking their pain, but maybe that inflammatory response caused by prostaglandins was required for proper tissue healing. But then long-term, they actually got worse pain because they used NSAIDs, right?

Nick Jikomes

1:24:40 - 1:25:10

And again, like people should probably remember here, There’s a whole pattern and chain of events that needs to play out. The start of the cleanup and the wound healing and the rejuvenation is literally tied to the inflammation part of this. There’s a sequence of things that should be happening. If you block one part of the sequence because it makes you more comfortable, it might make you more comfortable, but you might also be blocking the next steps that is actually going to be part of what we would call the healing side of it.

Isaac Chiu

1:25:10 - 1:25:31

Yes, yes. And I wanted to also bring up, um, more related to my research, right? So I mentioned to you that these nociceptors play such an important role in talking to different cell types, whether in the gut, they talk to goblet cells to make mucus, or they talk to immune cells to shut down there. And one of the, you know, mediators is CJRP, right?

Isaac Chiu

1:25:31 - 1:25:48

So I personally think There are lots of people taking CGRP medications now for migraine, like long-term. They’re taking it prophylactically, but that has an effect outside of migraine that I think one of the side effects that people already know is it affects the gut. There’s constipation.

Nick Jikomes

1:25:48 - 1:25:54

Remind us, are these medications blocking CGRP? CGRP is causing the migraine is the idea?

Isaac Chiu

1:25:54 - 1:26:15

Yes, yes. So they’re blocking either the neuropeptide itself, they’re antibodies that neutralize CGRP, or they’re small molecules that block the receptor for CGRP. And there are now millions of people on these drugs worldwide. So I would think that that will, in some cases, dysregulate things in the gut or in other cases in wound healing.

Isaac Chiu

1:26:15 - 1:26:22

Like I mentioned, there’s this Nature paper that showed that if you don’t have CGRP and you have a skin wound, it doesn’t heal as well.

Nick Jikomes

1:26:23 - 1:26:33

Wow. So there are things like that. And that’s, you know, that’s sort of insidious too, because you could imagine someone suffers from migraines and migraines suck. So they take a drug that blocks CRP and it helps with their migraines.

Nick Jikomes

1:26:33 - 1:26:39

But are they going to notice if their cuts heal 20% slower? Probably not.

Isaac Chiu

1:26:40 - 1:26:50

Exactly. And I think they’re being touted by the headache field as like a miracle drug because there’s no side effects. But I think that that’s, we’ll see. We’ll see what happens long term.

Isaac Chiu

1:26:51 - 1:27:08

The other thing is many of these, so I talk about neuroimmune interactions, right? There are so many people on different neuromodulators, right? It could be well, for pain, definitely things like opioids, right? And, you know, these things have effects beyond just pain.

Isaac Chiu

1:27:08 - 1:27:38

I think they will dysregulate your immune response or, you know, have effects on other systems, even like the gut, the microbiome gets dysregulated when you’re taking. So these kinds of things where, again, the neurologists are thinking mostly, okay, well, we’re gonna give this SSRI to treat depression. But that could be affecting our gut and other things where they’re not, uh, you know, I think these things need to be better, uh, more systematically studied.

Nick Jikomes

1:27:39 - 1:27:50

Yeah. Yeah. And it kind of goes back to the. The thing that we touched on at the very beginning where, you know, our, our minds like to compartmentalize things and categorize things and divide things.

Nick Jikomes

1:27:51 - 1:28:03

but there’s often blurry lines between all this stuff. In some sense, you know, basically everything’s pleiotropic. Everything’s multifunctional. There’s not, the endorphins aren’t just pain peptides and pain molecules.

Nick Jikomes

1:28:03 - 1:28:09

They do a bunch of other stuff. Serotonin is not just a mood molecule. It’s going to affect your gut and a bunch of other things.

Isaac Chiu

1:28:10 - 1:28:15

Exactly. Exactly. And I think that biology is, is that your, your baby?

Nick Jikomes

1:28:15 - 1:28:18

Okay. Oh, that’s actually the cat. Oh, the cat.

Isaac Chiu

1:28:18 - 1:28:58

Okay, sorry. Yeah, so I think there’s this this it’s one it’s a sobering point right to realize that every system is connected and the brain especially to to every system right so so understanding how um these affect other systems is something that doctors need to think more about however on the other side i think that it’s also exciting because if some of these same you know, modulators of, let’s say, neuroimmune communication can be harnessed to treat disease. I’ll give you one example where there’s kind of an exploding area, which is neuroimmune interactions in cancer.

Isaac Chiu

1:28:59 - 1:29:16

So, CGRP again. CGRP, as an example, has been shown to be activated in cancers. And in some cases, the tumor cells cause the CGRP to be released. And again, it’s like the bacteria I showed you earlier, they are talked about.

Isaac Chiu

1:29:16 - 1:29:36

It hijacks this system because then that CGRP shuts down the protective immune cells and kind of polarizes more of a wound healing response. But tumors are kind of like a wound that doesn’t heal, right? It’s just keep going. people are actually targeting CGRP to think about treating things like stomach cancer right now.

Nick Jikomes

1:29:37 - 1:29:53

Yeah. So again, the idea is whether it’s a pathogen like a bacteria or a cancer, ordinarily in a healthy state, the immune system would come in and get rid of those things. CGRP is basically turning down an immune response. It’s preventing immune cells from coming in.

Nick Jikomes

1:29:53 - 1:29:59

So by hijacking that system, turning up CGRP, a pathogen or a cancer cell can protect itself.

Isaac Chiu

1:30:00 - 1:30:07

Yes, exactly. You’ve got it right. So, and this extends beyond the pain system. There are people looking at stress signaling, right?

Isaac Chiu

1:30:07 - 1:30:34

Why do people who are stressed out have a dampened immune system because a lot of those molecules that the sympathetic nerves like adrenaline or cortisol, they’re there to shut down. I mean, acutely, this might be a good thing, but chronically, it’s really bad. And so in cancer, that’s one of the things that people are really looking into. If we block adrenergic signaling, can that increase the immune response against tumors?

Isaac Chiu

1:30:35 - 1:30:54

So, I think, We need to understand this brain-body or brain-immune-body interaction better because in some conditions, like you mentioned, like chronic diseases, this could be the culprit, right? But in other conditions, it may actually lead to new treatments for diseases that are like cancer and

Nick Jikomes

1:30:55 - 1:31:15

Yeah. And just to kind of riff a little bit more on this theme of bi-directional communication, balance, trade-offs, pleiotropy, all of that stuff. You mentioned two examples with CGRP where something bad happens. A pathogen or a cancer cell can hijack CGRP, turn up the CGRP signal to basically prevent itself.

Nick Jikomes

1:31:17 - 1:31:31

something like necrotizing fasciitis, something like cancer. These are bad things you don’t want, an infection and cancer. And higher CGRP can prevent your immune system from handling the problem. So CGRP high, immune system low, bad outcome.

Nick Jikomes

1:31:31 - 1:31:49

Are there potentially therapeutic applications to elevating CGRP where maybe you’ve got a chronic autoimmune or inflammatory condition and want that CGRP to turn up, or excuse me, to turn down so the immune system can, or you want it to go up so the immune system can turn off. Yes.

Isaac Chiu

1:31:49 - 1:32:14

Yeah, exactly. So that’s, that’s exactly the case that I mentioned earlier, wound healing, right? So that’s one place where it could accelerate. So that, that paper, the nature paper I mentioned, they also made a synthetic, longer lasting cgrp that they put on wounds and it actually accelerated wound healing right so that’s one place for autoimmune diseases that’s something of interest we did find so also this role of cgrp and

Isaac Chiu

1:32:14 - 1:32:44

the immune response is context dependent too so in the gut we mentioned that it makes mucus, right? So if you actually don’t have CGRP, the gut is much more susceptible to colitis, which is inflammatory condition, and also more susceptible to infection. Because what happens is, we looked at salmonella infection, the CGRP also maintains the balance of the microbiome. So then if you don’t have that, then the salmonella gets in more.

Isaac Chiu

1:32:45 - 1:33:06

So you need it there in some conditions to really, really facilitate wound healing and also fighting infection. In the gut, we haven’t thought about giving more of it, but that could be one thing to shut down or to cure some of these diseases. So I agree with you that harnessing that system and upping it in certain conditions could actually be protective.

Nick Jikomes

1:33:07 - 1:33:43

Yeah. And when we think about peptides more generally, especially because peptides are becoming very popular, more and more people are using exogenous peptides that they’re injecting into their body for various reasons, they’re becoming interested in this. On just the general theme of peptides on context dependent and context appropriate responses, you know, not too much, not too little. What would you say to people around peptides and experimenting with them in terms of how they should think about regulating their use and think about acute versus chronic use and things like that?

Nick Jikomes

1:33:43 - 1:33:58

Because it sounds like a general theme with this stuff is context dependence. You want to use not too much, but not too little. You want to use it in the appropriate context, but not in other contexts. So these things are probably not something you just want to use in a haphazard fashion all the time.

Isaac Chiu

1:33:59 - 1:34:11

I agree. Actually, you’ll have to educate me on what peptides are now being used in the market. But generally, I would say, you know, it should be rigorously studied by the FDA and by scientists. Right.

Isaac Chiu

1:34:11 - 1:34:33

Because like you said, too much of a good thing is actually could be really harmful. And, you know, our body uses these for different physiological and immune purposes. So if you if you’re supplementing it, just like anything, you know, it could have really nasty side effects. And Um, could you give me an example of, for example, like actually what are people using peptides?

Nick Jikomes

1:34:33 - 1:34:52

Oh, I mean, well, there’s, there’s a bunch. I mean, obviously you’ve got the various GLP one drugs, um, which are out there being used for weight loss and metabolism. Um, the last episode I did was with, uh, someone who studies a mitochondrial peptide called MOTC. Um, people are using this for its metabolic effects and it’s sort of exercise related effects.

Nick Jikomes

1:34:53 - 1:35:09

There are ones that affect the immune system that are people, you know, that are using for information purposes, uh, things like KPV. Um, there are other ones. I mean, there’s a whole, there’s a whole list we could talk about, but people are using them, uh, more and more and yeah, there’s, there’s a whole, there’s a whole list of them.

Isaac Chiu

1:35:10 - 1:35:19

Yeah, I mean, you’re right. I forgot about the GLP. That’s it. I mean, I wonder how society, you know, how health will be changed, right?

Nick Jikomes

1:35:19 - 1:35:48

I mean, that’s like, it’s kind of like what you said before, where obviously a lot of people are using the GLPs and they’re going to continue using them. What’s interesting to me and potentially concerning is It’s like we talked about before, where you can have an acute benefit to something like an NSAID. And then there can be a chronic problem that’s not obvious or not clear to detect right away. And you don’t even recognize it as a side effect because, you know, hey, if my wound healing is compromised by 10 or 20%, I’m probably not gonna notice that.

Nick Jikomes

1:35:48 - 1:36:01

But I am gonna notice that I’m in less pain when I bump my knee. If someone’s taking a GLP-1 for months, years, decades, What effects is that going to have beyond the obvious acute effect? And there’s probably going to be a whole lot.

Isaac Chiu

1:36:01 - 1:36:16

Yeah. Yeah. I agree with you. I think that, um, I mean, yeah, to GLP-1, there should be more studies on every system, right, and what’s going on in these patients who get years of treatment.

Isaac Chiu

1:36:17 - 1:36:50

But I mean, I think at least GLP-1 has been rigorously clinical trialed. I think it’s really important that people not just take the news of, oh, this one thing does one thing, right? trusting that, but then again, CGRP antagonists are in the same group, right? So I think that as a point, like we should be very, very careful and understand that every system is connected to each other.

Isaac Chiu

1:36:50 - 1:37:07

So there could definitely be drawbacks to everything. So, So yeah, I mean, I think chronic pain is one of these areas where there’s a huge clinical need, right? We need to find ways of dampening chronic pain. And there’s, people want more specificity, right?

Isaac Chiu

1:37:07 - 1:37:46

So I think that’s one of the things is there, if there’s a way to target just nociceptors and not other cells in the body, right? Maybe that would be better than opioids, which have all these effects on all reward systems and other places in the CNS. But also I think, to your point, if you can find a way that doesn’t take away the beneficial roles of these systems, but shuts down the overactive aspects of it, then I think that’s the way to go in the future, rather than just cutting something completely off or activating it to a huge extent that it’s not physiological.

Nick Jikomes

1:37:46 - 1:38:21

Yeah. And just to kind of ask it one more time in a slightly different way, when people are um, interfacing with various immune modulators, whether we’re talking about NSAIDs, non-steroidal anti-inflammatory drugs, ibuprofen, aspirin, things like this, or we’re talking about antihistamines, Zyrtec, you know, allergy medication. I’m guessing you would say it’s probably not a good idea to be taking these things all of the time, every day, but to be taking them in a more context-appropriate fashion, like only when you really think you need them, rather than getting in the habit of using them all the time.

Nick Jikomes

1:38:21 - 1:38:28

Even if it sort of seems completely innocuous, it probably isn’t. I agree, I agree.

Isaac Chiu

1:38:28 - 1:38:52

I think that if it was a severe medical condition where you need to take it all the time, then follow your doctor, right? But I think what I’m hearing you’re saying is like, these are over-the-counter drugs, and people might think, oh, I need to block my chronic inflammation, so I’m just gonna take an NSAID every day. I think that’s a bad idea. I think there’s, you know, if you don’t need it, I mean, pharmacology can do things.

Isaac Chiu

1:38:52 - 1:39:09

Not everything is also highly specific, right? Yes, they’re supposed to do this one thing, but drugs have other effects. And I think it’s just not good for us to kind of unknowingly going in and doing things because it seems like it’s harmless.

Nick Jikomes

1:39:09 - 1:39:24

Yeah. What are some projects in the lab right now or questions you’re working on that you’re really excited about in terms of neuroimmune interactions and the things we’ve discussed that you think we’re going to see some interesting and exciting results in the next one or two years?

Isaac Chiu

1:39:25 - 1:39:40

Yeah, thank you. I have a lot of interesting things going on in my lab. Related to chronic pain, one of the areas we’re interested in is how Infections might induce chronic pain. So as an example, we’re looking at Lyme disease right now.

Isaac Chiu

1:39:41 - 1:40:13

Lyme disease, as you may know, is a tick-borne bacterial pathogen, which a lot of people suffer from long-term effects of Lyme disease. So some people, you know, you get, actually most people, you get treated with antibiotics and then the symptoms go away. But there’s about 10 to 20% of patients who have this chronic, a lot of times neurological, issues, fatigue, pain, as well as neuropathy. So we have found that we think at least part of the mechanism is through immune cells.

Isaac Chiu

1:40:13 - 1:40:41

So these bacteria actually cause a long term immune response, which doesn’t resolve like you were just talking about resolution of inflammation. So we have a study that we’re really excited about is understanding what causes these immune cells, and they actually accumulate in the nerves that cause pain? And so why are they there and how do we shut them down? So that’s one of the exciting projects we’re working on.

Isaac Chiu

1:40:42 - 1:41:06

And let me think about other ones. My lab is also starting to get more into not just pain but also neurodegenerative diseases. So we’re interested in Alzheimer’s disease and also in ALS and frontal temporal dementia. So we have a specific interest in molecules and neurons that are actually related to the immune system.

Isaac Chiu

1:41:06 - 1:41:39

So again, this kind of separation of what we think about what is a neuron, what is an immune cell, but neurons also harbor innate immune receptors. So receptors inside their cell that need to protect them from viruses. So we’re interested in whether some of these pathways that the neuron needs, because every cell needs to be protected from viruses, are activated in the wrong way during neurodegenerative diseases. So those proteins that cause neurodegeneration, are they activating what the neuron thinks is the virus?

Isaac Chiu

1:41:40 - 1:41:47

And then that eventually leads to the neurons to die and degenerate. So we’re very interested in that area.

Nick Jikomes

1:41:49 - 1:41:56

Are there any final thoughts you want to leave people with or anything you want to reiterate from our discussion today just on the general subject of neuroimmune interactions?

Isaac Chiu

1:41:57 - 1:42:49

Yeah, I think that if people can take away the idea that these two systems are really intimately connected and that there’s powerful effects of each one on the other. And I think one of the most exciting points to take away is that in the future, if we understand some of these neuroimmune interactions, we might be able to really harness it to treat disease. So that includes blocking harmful immune molecules to treat chronic pain or, you know, actually activating or inducing neural responses to shut down the immune system and autoimmune diseases or to ramp it up and let’s say cancer and things like that. So I think this, the potential of understanding these two, how these systems talk to each other is tremendous in terms of finding new treatments for disease.

Nick Jikomes

1:42:50 - 1:43:01

All right, well, Professor Isaac Chu, thank you very much for your time. This is very, very interesting research that you do, and I love learning about it. So hopefully I can check back in a couple of years and see what else you’ve discovered.

Isaac Chiu

1:43:01 - 1:43:05

Thank you very much, Nick. It’s been my pleasure to be here, and thank you.

This is a public episode. If you'd like to discuss this with other subscribers or get access to bonus episodes, visit mindandmatter.substack.com/subscribe

Wide release: Available for free by July 30, 2026, on Apple, Spotify, YouTube, etc.

Not medical advice.

Nick speaks with Dr. David Lee about the mitochondrial-encoded peptide MOTS-c. Lee describes its discovery within the mitochondrial genome, its movement into the nucleus under metabolic stress to broadly reshape gene expression, and preclinical evidence that it blocks diet-induced obesity, improves treadmill performance in older mice, and modulates autoimmune responses.

TOPICS DISCUSSED:

* Mitochondrial Roles: Not just ATP production, but “speaker of the house” via communication molecules that “talk” to the rest of the cell.

* Origin of Communication: Mitochondrial peptides were likely co-opted from ancient antimicrobial factors to synchronize nuclear and mitochondrial genomes.

* MOTC Discovery: Identified in mitochondrial ribosomal RNA regions linked to interferon responses.

* Stress Induction: MOTS-c is produced in response to exercise, immune activation, and nutrient withdrawal; circulates as a mitokine.

* Nuclear Regulation: MOTS-c translocates to the nucleus under stress and acts as a nonspecific cofactor that shapes chromatin accessibility and gene expression.

* Metabolic Effects: Prevents diet-induced obesity and fatty liver in mice without altering baseline weight; engages AMPK and suppresses anabolic pathways.

* Immune Modulation: Shifts immune cell programs to reduce autoimmune attack in type 1 diabetes models.

* Aging Phenotypes: Daily or intermittent dosing doubles running capacity and preserves walking ability in old mice.

ABOUT THE GUEST: Changhan David Lee, PhD is Associate Professor at the University of Southern California Leonard Davis School of Gerontology. His laboratory investigates mitochondrial communication and microproteins in the biology of aging.

RELATED CONTENT:

* Article | MOTS-c & Cellular Energy Homeostasis

KNOW YOURSELF:

* Visit the SUPPORT PAGE if you find value in this content. Includes discount codes + links to affiliate partners, including products to help you measure and manage your health.

Listen or watch on your favorite platform:

* Audio version: [Apple] [Spotify] [Elsewhere]

* Video version: [YouTube]

PRACTICAL TAKEAWAYS:

* Intermittent rather than continuous metabolic stress aligns with the MOTS-c peptide’s natural production rhythm.

* Exogenous MOTS-c use should respect short half-life and cycling patterns observed in animal studies rather than continuous elevation.

SUBSCRIBER CONTENT BELOW: Reference paper + episode transcript.

Reference Paper:

* Paper | The Mitochondrial-Derived Peptide MOTS-c Promotes Metabolic Homeostasis and Reduces Obesity and Insulin Resistance

AI-generated transcript below. Beware of mistranslations!

Changhan David Lee

02:17 - 02:21

Thank you for having me here, Nick. It’s a pleasure to be on your podcast.

Nick Jikomes

02:21 - 02:26

Do you want to start off by just telling everyone a little bit about who you are, what your background is and what your lab studies?

Changhan David Lee

02:28 - 02:48

Yeah, my name is David Lee, Chang-Han David Lee. I’m an associate professor at the University of Southern California, Leonard Davis School of Gerontology. And as the name of our school implies, my main focus is studying the biology of aging. And we focus on mitochondria in our lab.

Changhan David Lee

02:49 - 03:15

More specifically, as I’ll talk about more later, my lab is very interested in understanding how mitochondria communicate to regulate things, in addition to being the so-called powerhouse of the cell, as well as what I call the slaughterhouse of the cell. We think mitochondria are what I call the speaker of the house that regulates things that are relevant to aging.

Nick Jikomes

03:17 - 03:20

So the slaughterhouse of the cell, what exactly does that mean?

Changhan David Lee

03:22 - 03:40

So it’s a play on words, powerhouse, slaughterhouse, speaker of the house. Powerhouse, you probably know, generates energy. It provides building blocks for life. The slaughterhouse is because mitochondria are very integral to what’s called program cell death.

Changhan David Lee

03:41 - 04:16

So when a cell deems that a certain damage is too much and it’s beyond repair, then it activates, it’s called apoptosis, which is a programmed cell death process. It’s also important for development and other processes like your digits, your fingers, the way they’re formed is through these programmed cell death to separate out certain structures. And therefore, mitochondria being one of the most important regulators of this process, I call it the slaughterhouse. And the speaker of the house, obviously, because it talks.

Nick Jikomes

04:17 - 04:36

Yeah. So mitochondria are at the epicenter of bioenergetics. They’re making most of the ATP for our cells. Obviously, that energy is used for building things and making things, but it’s also going to be an important part of tearing things down, cell death and things that you were just talking about.

Nick Jikomes

04:37 - 05:10

And whether we’re talking about building things up or tearing them down, ATP has to be used, energy has to go places and fuel different cellular processes. And the mitochondria, of which there are usually many inside of our cells, have to talk to other parts of the cell, so to speak. So you’ve been involved, and I think discovered, some of the amazing communication molecules that we’re going to talk about today. But give us, at a very general level, a basic rundown of how does something, an organelle like mitochondria, talk to other parts of the cell?

Nick Jikomes

05:10 - 05:11

What does that look like at a basic level?

Changhan David Lee

05:18 - 05:42

I kind of think about it as a mitochondrial language, of which the alphabets are many. And there are several that have been pretty well defined. One of the more fundamental ones are the metabolites themselves, such as aspects of the electron transport chain, like ATP. NAD you might’ve heard of.

Changhan David Lee

05:43 - 06:30

These things can communicate, act as a communication factor through epigenetics, which means it can influence how the nuclear genome regulates or operates. And then there are other proteins that are encoded in the nuclear genome that can live in the mitochondria and they go back to the nucleus to report back on certain situations. Mitochondria, I’ll touch upon a bit later more, being derived from bacteria, still retain a lot of these bacterial components. And the leakage or the export of some of these more bacterial-like compounds or molecules can stimulate the cells to recognize it as an immune response or inflammatory response.

Changhan David Lee

06:30 - 06:57

So there are many ways to do that. mitochondrial DNA itself can be is considered an inflammatory signal, a part of our immune system. But they will be studying our lab, our little proteins that are called microproteins, or peptides, depending on who you talk to, they’re encoded in the mitochondrial DNA, and are used as communication factors. So they’re very, they’re very versatile in the way they communicate.

Nick Jikomes

06:58 - 07:26

Yeah, so we’ve got a nuclear genome, the genome that we normally refer to is the one that’s in our nucleus, it contains most of our genes. We’ve also got these mitochondria, which used to be free living bacteria that became incorporated into larger cells. They have their own mitochondrial genome, their own mitochondrial DNA that encodes certain things. There has to be communication between both genomes because there’s actually things encoded in the nuclear genome that are part of the mitochondria.

Nick Jikomes

07:27 - 07:41

And there are things, as we’ll learn, encoded by the mitochondrial genome that can go into the nucleus or talk to other parts of our cells. So there’s a bidirectional communication at the level of genes and gene products between the mitochondria and the nucleus and even other parts of the cell.

Changhan David Lee

07:43 - 08:00

Yeah, no, you put it really well. That’s actually a very important aspect of this communication. Maybe you can go back to the very beginning of this relationship, perhaps. So the way we like to think about it in our lab is going back to the origin of eukaryotes.

Changhan David Lee

08:00 - 08:26

So rewind a billion to two billion years, depending on who you talk to. Before we had eukaryotes, there was the ancestral cell, a bigger cell, and then this really special bacteria that was good at extracting energy. And the bacteria, in a way, infects the bigger cell and invades it and enters the bigger cell. Some people call it a marriage, but it’s basically an infection.

Changhan David Lee

08:26 - 08:48

And it decides to live in there, right? So typically when an infection happens, one kills the other and one reigns supreme. But in this case, something weird happened where they figured out that not only is it beneficial to live together, it will be good enough to make it permanent. So the bacteria became a mitochondria, right?

Changhan David Lee

08:48 - 09:15

That’s called the endosymbiosis theory. That was first posited by Lynn Margulis. But what we think was really important here is the communication started from there. And that was the most important thing, because to coordinate something as complex as integration of two independent life forms, basically two sovereign countries coming together, must have had a lot of coordination required, which absolutely requires communication.

Changhan David Lee

09:16 - 09:30

So how they communicate, how they talk to each other, I think is where things started. And they probably didn’t invent a whole new language to start talking to each other. It’s an infection, right? So a lot of these will be tooth and nail kind of situation.

Changhan David Lee

09:30 - 09:54

A lot of these supposedly immune factors that they use to control other organisms may have been used. And There are those today, bacteria, those are called antimicrobial peptides. Basically every living form or kingdom on this planet has these very ancient immune peptides. We have it, flies have it, you know, birds have it.

Changhan David Lee

09:55 - 10:20

bacteria have it, plants have it, everybody has it. And they are used as the next generation antibiotics commercially. It’s one of the frontline developments for antibiotics. But we think those peptides might have been used to talk to each other, to control instead of kill, because a lot of these peptides have very specific functions.

Changhan David Lee

10:21 - 10:41

So the first line of attack would be bombarding the membrane and disrupting the membrane using its characteristics. It has to have a positive charge and it has to have a hydrophobic domain. And they use that to target the membranes. Once it gets in, many of them have intracellular targets that are vital.

Changhan David Lee

10:41 - 10:57

These are evolutionary selected ones, so they don’t fool around with minor processes. They go for the jugular. So DNA synthesis, RNA synthesis, ribosome function, cell wall synthesis. And these are very well defined today, in the ones that we know today.

Changhan David Lee

10:58 - 11:38

But if you think about it, the bacteria’s biggest fear would be dilution because in a way it’s paying rent inside this bigger cell and has this benefits in living inside it. But if the bigger cell decides to divide without them, If the division is not synchronized, after a couple of divisions, they’ll be diluted out. So the theory that we have is that somehow the bacteria had to have a way to control the cell division so they’re synchronized. And one way they could have done it is by using these, I guess, hostile communication factors, typically, to dial it down.

Changhan David Lee

11:39 - 12:04

So instead of bombarding it with high concentration, if you dial it down, DNA synthesis will be halted, but it won’t be halted forever to cause cell death. It’ll just slow down the division rate. So if you fast forward that another billion years today, we think this is a foundation of cell regulation. So when a cell starves, how does the cell know how to regulate its metabolism and slow down cell division?

Changhan David Lee

12:04 - 12:11

We think That is the foundation of how this so-called cell regulation. Yeah.

Nick Jikomes

12:11 - 12:29

Yeah. So even, you know, even pathogens, bacteria, viruses, our cells have to communicate with them and respond to them. And that should make kind of intuitive sense to people. Like even if we get infected with a bacteria, that bacteria is going to have an interest in shutting down our immune responses, modulating the inflammatory signals that our cells produce.

Nick Jikomes

12:30 - 13:06

Obviously, our cells are going to have an interest in getting around that, at preventing our own cell machinery from being hijacked. There’s going to be crosstalk there. And so a lot of that crosstalk will naturally involve things that we think of as immune functions. And so if we sort of think back to the endosymbiotic event by which bacteria that became mitochondria got permanent residence within our cells and eukaryotic cells, they had to modify this language or modify the communication so that they could cohabitate and they could play nice so that, you

Nick Jikomes

13:06 - 13:34

know, our immune reaction wouldn’t be destroying them. So that, you know, as you said, the cell cycle stuff was synchronized so that we retained mitochondria with every cell division. And then obviously, so that the bioenergetic stuff could be incorporated into the cell and put to good use. So it sort of makes good intuitive sense, there’s going to naturally be communication between these entities, the nuclear genome, the mitochondrial genome, that it’s going to involve things like inflammation and cytokines and stuff like that.

Nick Jikomes

13:35 - 13:49

and so on and so forth. What I want to talk to you about specifically are these microproteins or peptides. So let’s start at a basic level before diving into MOTC, which will be our focus today. What are peptides?

Nick Jikomes

13:49 - 14:00

And can you give people just a general sense for are these very numerous? Are they everywhere? Is there a whole bunch of them? Are they highly specialized, weird mitochondrial things?

Nick Jikomes

14:00 - 14:03

What are peptides and what does the landscape generally look like?

Changhan David Lee

14:04 - 14:34

So peptides at a very high level definition in my mind is just small proteins. But I guess the debate comes down to how small is small. And if you talk to a lot of academics, I guess right now there’s a broad consensus of roughly about 100 amino acids being the cutoff to qualify as a peptide. But of course, there are ones that are slightly bigger and very, very small.

Changhan David Lee

14:34 - 14:55

Like the ones that we study in our lab that we named MOD-C is only 16 amino acids. If you ask FDA, they define it as 40 amino acids or less. So 39 and below are considered peptides in the eyes of the FDA. So there really is no universal definition of what a peptide is.

Changhan David Lee

14:56 - 15:29

But there are many sources of these smaller proteins, they can be processed, smaller peptides that are processed from a bigger mother protein, like insulin, for example, is considered a peptide hormone, but the pro insulin is much bigger. But then there are the fragmented ones too, those that we’re not sure if they’re all processed intentionally or if they just broke off. They’re the smaller fragments of the bigger proteins and peptides. And then there are ones like the ones that we study that are encoded in a small gene.

Changhan David Lee

15:29 - 15:58

The gene itself is very small because it’s intended to encode for a small protein peptide. And many people now use the word microprotein to kind of differentiate between synthetic, artificial and broken pieces of proteins that are small peptides, and then ones that are gene encoded and ribosomally translated intentionally to be small. So different words.

Nick Jikomes

15:58 - 16:13

Yeah. And now when we think about natural endogenous peptides, are these things always explicitly encoded? So like a small gene encoding a small sequence of amino acids, or can they also result from like the breakdown of larger proteins?

Changhan David Lee

16:15 - 16:37

Right. So like I just said, insulin, for example, is a processed form of a bigger protein, pro-insulin. So that’s already very well studied. The smaller genes have recently emerged and for a long time, we didn’t know of the existence of these gene-encoded, small gene-encoded peptides or microproteins.

Changhan David Lee

16:38 - 17:12

And that kind of goes back to the human genome project, you know, 2000s, late 20th century, when we sequenced most of the human genome, we thought we had a map, and the string of ATCUs came out. And to map it, you have to annotate the genes, right? And during the process of annotation, there were many criteria that were involved, but for various reasons, these smaller genes were not included in the annotation. And that roughly kind of cuts off around 100 amino acids for many reasons.

Changhan David Lee

17:13 - 17:47

Go into detail if you want to, but for that reason, if you go and run all the proteins in a human sample, for example, using a mass spec, you would see the existence of these smaller peptides, but there was no gene to ping them back to. There was no way to identify them. But now with increased technology and the advent of all these informatics and tools that we have now, we’re starting to see these little genes and the little peptides that they make. So there’s a floodgate that’s opened up in the nuclear genome.

Changhan David Lee

17:48 - 17:51

Many, many have been discovered as well as the mitochondrial genome.

Nick Jikomes

17:51 - 18:03

Got it. So there’s a lot of peptides out there. By definition, they’re small proteins. And for historical and technological reasons to do with things like the resolution of our methods, these things have always been there.

Nick Jikomes

18:03 - 18:11

They’ve always been abundant and probably doing important things. But to some extent, we sort of missed them or couldn’t quite detect them until relatively recently.

Changhan David Lee

18:12 - 18:14

Exactly. Exactly. Yeah. Yeah.

Nick Jikomes

18:15 - 18:28

And so we’re going to spend most of the time today talking about a particular mitochondrial peptide called MOTC. It’s getting a lot of attention. It’s really interesting. Talk to us a little bit about what MOTC is and let’s maybe start with the discovery.

Nick Jikomes

18:28 - 18:36

I believe you were part of the group that actually discovered MOTC. How did you guys stumble into this? Were you looking for it explicitly or was it serendipitous?

Changhan David Lee

18:37 - 19:02

Yeah, so I discovered MODSI when I was a postdoc in Hasi Cohen’s lab at UCLA. And I was really interested in aging, of course. That was my main interest. And instead of going on to another type of postdoc, I met Hasi, who was working with the first one that was found, the first peptide that was found in the mitochondrial DNA called humanin.

Changhan David Lee

19:03 - 19:31

And it was very interesting. And the idea for me was, well, maybe there’s more than just human, because why would there just be one? And just to give a little bit of background, my first papers as first author, well, my first author paper, first first author paper, was in my undergraduate research lab, excuse me, in Kelly Hughes’ lab. I did UW.

Changhan David Lee

19:35 - 19:58

background was to look for genes in the bacterial genome. So regulatory genes, going hunting for genes in Salmonella was my first paper. So I already had this background of bacterial genetics, looking for genes that are regulators in the bacteria Salmonella. And I forgot about that for a while because I moved into humans and aging.

Changhan David Lee

19:58 - 20:19

And then I heard about these peptides. And so it kind of clicked with me, kind of a cruel twist of fate, maybe. I left the bacterial world, went into humans, and then I got attracted back into that semi, half bacterial, half mammalian world. And I thought it’d be interesting to look for more genes.

Changhan David Lee

20:21 - 20:38

I guess in a way, it’s a very interesting way that science careers can develop. So I came back and said, you know, let’s look for more genes. That was very intentional. But then if you start looking at the mitochondrial genome, even if it’s small, it’s only a 16 and a half kilo basis compared to the nuclear genome.

Changhan David Lee

20:38 - 20:59

It’s just very minor in size. But if you apply the possibility of genes that can be encoded inside other genes, right, or genes that can overlap and share codes. That’s very typical of bacterial genes, genomes. then the possibility was very, very large.

Changhan David Lee

20:59 - 21:23

So I started to look for papers to kind of help me guide in areas to look for it. Human was first discovered, the first discovery, it was encoded in one of the two ribosomal RNAs in the mitochondrial DNA. Mitochondrial DNA has two ribosomal RNAs, 13 proteins and 22 tRNAs that were known to have been encoded. So we were looking at the map and said, well, where do we go?

Changhan David Lee

21:24 - 21:55

And this is such a large thing if you consider this overlapping gene situation. And then there was a paper that was published in Cell in 2011, what is called mitochondrial transcriptomics. So going back to the textbook drawing of the mitochondrial DNA with a very simple layout of the genes, The transcriptomics paper said there’s tons of transcripts that are small in the mitochondria that we cannot map to genes again because it was not annotated. So there were hot spots here and there.

Changhan David Lee

21:56 - 22:24

So we’re looking for different areas. And then I came upon this paper that was published by Sidney Peska’s lab, who’s considered the father of interferon, he recently passed away, where they kind of published this paper out of frustration in 1983 in BBRC, basically saying, hey, now we know how to induce interferon. We know that it’s an immune protein. you infect a macrophage or monocyte-like cell with a virus, antivirus, and that induces interferon.

Changhan David Lee

22:24 - 22:41

They got to that part very well. And then they said, well, let’s look at all the genes that are induced after interferon goes up, and those might be interferon-induced or regulated genes. Very logical, right? And this is before RNA-seq or microarrays or any of that QPCR.

Changhan David Lee

22:41 - 23:21

So he went through restriction mapping, right, which is basically figuring out piecing together the sequences. And they said about over roughly about 80% of what they find that is induced by transcripts that are induced by interferon or induced after interferon goes up, map to the mitochondrial DNA. And there they kind of put a finger on a rough area in the mitochondria, a loci, a locus, which happened to be ribosomal RNA. So we have humanin that was in one of the two ribosomal RNAs, and then the other ribosomal RNA, there were several other potential possibilities.

Changhan David Lee

23:22 - 23:25

And that’s where we went to look for these genes first.

Nick Jikomes

23:26 - 23:38

Yeah. So a couple of things were going on here. Interferon, it’s an immune molecule. As we said before, you probably expect the mitochondria to be communicating with immune system stuff.

Nick Jikomes

23:39 - 24:10

So not too surprising, perhaps, the mitochondrial genome would light up, so to speak, in response to something like interferon. And also, despite the fact that the mitochondrial genome is like a bacterial genome, it’s small and it’s simple, sure, relative to the nuclear genome, you guys surmise that there’s probably more going on there than we know about yet or we appreciate because, you know, this is biology, things are messy, there can be lots of genes interdigitated with one another lying on top of each other. And so sure enough, you sort of discover that there is more going on here.

Nick Jikomes

24:10 - 24:13

And that brings you to the discovery of MOTC.

Changhan David Lee

24:14 - 24:22

Yeah, yeah, yeah. And it’s really interesting. I like the way you put it, this whole immune connection. I didn’t understand it in the beginning.

Changhan David Lee

24:22 - 24:56

Why is it an interferon-related peptide? That was somewhere in my brain, but I really didn’t grasp the importance of that until many, many years later when we started to develop these theories. And it clicked one day in one of the lab meetings. If the origin of this is in fact from the bacteria that infected and they did use these immune peptides, then it could have been the first immune system that the joint proto-eukaryotic cell had that we have now.

Changhan David Lee

24:57 - 25:22

But the nature of the communication must have been immunologic. So that’s consistent with the idea that immune systems and metabolic systems co-evolved for many, many reasons. And what we see now is that the MOTC is very immune related. In fact, we just recently published a paper describing it as an antimicrobial peptide.

Nick Jikomes

25:22 - 25:51

Yeah, yeah. And again, you might think naturally, like, mitochondria used to be bacteria, they’re generally going to probably want to have turned down immune responses in the host cell. And that will maybe connect to other things we talked about related to how MOTC is affecting inflammation and genes related to the regulation of inflammation. But these little bacteria, they wanted to live peacefully or become cohabitating inside of eukaryotic cells, what became eukaryotic cells.

Nick Jikomes

25:52 - 26:21

It makes sense. They’re going to respond to the immune response of the larger host cell and perhaps do a lot of stuff that will tune or turn down the immune response so that they’re not just killed off like we would kill off a pathogen. Let’s talk about what stimulates MOTC and then talk about where MOTC goes and does stuff within the cells. In general, in the literature that I’ve read from you and others, MOTC tends to be induced from metabolic stress.

Nick Jikomes

26:21 - 26:27

What exactly does that mean and what are the high-level stimuli that induce the mitochondria to produce MOTC?

Changhan David Lee

26:31 - 27:01

It depends on the cell type, I guess. I guess when we talk about mitochondria, and this is kind of what we talk a lot about within the academic ring, it’s easy to simplify mitochondria as one thing, but in fact, every different cell type, just like they have different energetic demands, the role of the mitochondria are many. and they respond to different things because they adapt to the local environment.

Changhan David Lee

27:01 - 27:39

For example, the mitochondria in the brain versus mitochondria in a sperm, for example, they have different roles. And what we know so far is that in the immune cells, like the macrophage, which I just mentioned, Sidney Peska’s lab already kind of published in 83, we kind of recreated that. We wanted to ask, is what Sidney Peska saw really the peptides we discovered? And so if you stimulate monocytes or macrophages, we do see a time-dependent increase of the peptide at the protein level.

Changhan David Lee

27:40 - 28:11

So immune responses could be one that could stimulate monterey production. And then another one that we published was exercise. So exercise is a combination of multiple different types of stress, right? It’s metabolic stress, but also heat stress that the mitochondria generate your your mitochondria can heat up to, according to some papers, over 60 degrees Celsius, which is basically, you know, a death situation.

Changhan David Lee

28:11 - 28:28

So there’s a lot of heat stress. And then there’s mechanical stress, you know, muscles contracting and shearing. So all these stressors will activate. So we don’t know exactly what the stimulation is that triggers MODSI activation, probably a composite of mitochondria.

Changhan David Lee

28:29 - 28:51

Yeah, so that’s another one that we know. Other than that, it’s still in progress. We’re trying to figure out what it does. In the brain, we also showed in a paper in 2021, In a cell metabolism paper, we showed that in the brain also, exercise will stimulate MOTC production in a place called hypothalamus.

Nick Jikomes

28:52 - 29:05

Yeah. So exercise will stimulate MOTC production. Other forms of stress, including immune reactions will as well. There’s probably what, hundreds to thousands, potentially even many thousands of mitochondria per cell.

Nick Jikomes

29:06 - 29:27

Throughout this conversation, I’ll let you sort of direct us to what cells and tissues we’re actually talking about for any given experiment or phenomenon we might be discussing. But in general, let’s say someone’s exercising, an animal, a human, whatever, that’s causing some amount of stress. The mitochondria are detecting that one way or another. They’re producing MOTC.

Nick Jikomes

29:28 - 29:34

Where does that MOTC go? Is it staying inside the mitochondria? Are they excreting it and it goes everywhere within the cell? Where does it go and what does it do from there?

Changhan David Lee

29:35 - 29:54

That’s the question. We’re still trying to figure that out. So in the 2021 Nature Con paper, we showed that in humans, this is a collaboration with Troy Mary at New Zealand. He had these 10 undergraduates who were very brave to give muscle and blood while on a stationary bike.

Changhan David Lee

29:54 - 30:10

So we measured MOTSI levels before, during, and after exercise. Of course, during exercise, we couldn’t get their muscle. So that was just the blood. And we do see a very significant increase of MOTC in the skeletal muscle as well as in circulation.

Changhan David Lee

30:10 - 30:40

So MOTC is secreted out in the circulatory system and it does act as an endocrine factor, if you will, or I see it more as a cytokine now. So it does circulate. The source has always been a question. When we first published the debut paper, the commentary kind of named moxie or referred to moxie as a mitochondrial hormone because it circulates.

Changhan David Lee

30:41 - 31:02

And then some people call it a cytokine or mitokine. We were leaning more towards the mitokine than a hormone, because by definition, a hormone has to have a specific cell that secretes it. And we didn’t think that it was one cell type or one tissue type. Many, many cells seem to be expressing MOTC from our studies.

Changhan David Lee

31:03 - 31:32

So the source of the circulating MOTC, During exercise, especially, we don’t know exactly where that’s coming from. We suspect that the muscle is involved, but we haven’t really found a way to detect that. In terms of the elevation of the skeletal muscle, that’s the same story. We’re not sure if it’s made in the muscles and kept inside the muscle tissue or skeletal muscle, or if it’s taking and accumulating it from the circulation.

Changhan David Lee

31:32 - 31:35

That we’re not sure. But what we know is that the levels do go up.

Nick Jikomes

31:36 - 31:48

Yeah. So levels of MOTC go up with exercise. They plausibly go up with other forms of stress as well, but they are definitely exercise inducible. And this is not a highly, highly localized signal.

Nick Jikomes

31:48 - 31:55

It’s not just staying within the mitochondria that produces it. It’s getting to other places. It’s getting into general circulation to some extent.

Changhan David Lee

31:56 - 32:00

Yeah. Yeah. Yeah. The source, we would really love to know where that comes from.

Nick Jikomes

32:00 - 32:14

Yeah. Yeah. And now we mentioned before that there is communication between mitochondria and the nuclear genome. What do you guys know about MOTC and when it’s produced by mitochondria, whether or not it’s regulating the nuclear genome?

Changhan David Lee

32:16 - 32:39

Yeah, so in the textbook still today, the nuclear genome is described as the keeper of all regulatory genes. Things that regulate the cell are exclusively in the nuclear genome. The 13 proteins in the mitochondria, the big proteins, were just trapped there to support the electron transport chain. That’s kind of how the textbooks describe this.

Changhan David Lee

32:40 - 33:09

In 2018, we published a paper in Cell Metabolism showing that MOTI actually does go into the nucleus upon stress. And this was worked by a postdoc in the lab, Kyung-Won Kim, who now has her own lab. But one day, you know, she came and said, and I keep imaging MOTC, and I’m looking in the microscope, and there are these little speckles in the nucleus, like, it’s so weird. And that doesn’t seem to be an artifact because it happens, you know, too frequently.

Changhan David Lee

33:10 - 33:52

And so we came up with this hypothesis, well, probably then maybe MOTC, something in the mitochondrial genome, could go into the nucleus. And it’s not new, it’s very consistent with the fact that the nuclear alphabets of the language regulate chromatin dynamics in the nucleus. So like I said, the histone regulations, the phosphorylations, the acetylations, these are mostly metabolites, acetyl-CoA, provides the substrate for acetylation, ATP phosphorylation, methylation comes from the methionine folate cycle, which a half of it almost runs in the mitochondria. So it’s not new.

Changhan David Lee

33:52 - 34:28

ROS, oxidative stress, is also known to regulate gene expression. So communicating with the nucleus was already an established route of, I guess, communication. And so we asked the question, well, does it go anytime? And when we fast forward, what we learned was if you stress a cell with glucose withdrawal or growth factor withdrawal, or even oxidative stress, about 30 minutes to one to two hours, you see MOTC just moving into the nucleus very significantly.

Changhan David Lee

34:28 - 34:44

And then it stays there, depending on the cell type. We use the generic cell type because we thought this was more of a fundamental biology. We use something called a hectonitrile cell, which is really a very generic cell type. In that cell type, it stays in there for about four hours, and then it comes back out or disappears.

Changhan David Lee

34:44 - 35:02

We don’t know if it’s degraded in the nucleus or if it gets shuttled back out, but the signal disappears from the nucleus. So it’s one of those rapid stress adaptive signals, MOTC, that goes in the nucleus. and it regulates nuclear gene expression.

Nick Jikomes

35:03 - 35:40

So some kind of stress happens, it could be something like a cell being deprived of glucose, that causes the mitochondria to make MOTC, MOTC levels go up, and then at least some of that MOTC goes into the nuclear genome, into the nucleus, where it is presumably causing some genes to turn up and turn down, it would make a lot of sense that this is going to be some kind of, to stimulate some kind of adaptive response to the stress, to make the cell better able to cope with that stress, whatever it may be. So another question that comes up at this point is, which genes get turned on and off? Is there a clear pattern as to how MOTC is regulating gene expression?

Changhan David Lee

35:41 - 36:10

Yeah, so that’s a really interesting question, and one that one of the PhD students in the lab is really digging deep for our thesis project. Long story short, MOTI is a very small protein, and it doesn’t have specificity. So bigger proteins have specificity because there are many bells and whistles. Smaller proteins have less, but they’re much more versatile in the way they can move around.

Changhan David Lee

36:10 - 36:23

They can find it easier, right? So what we think is going on is MOTC itself does not directly regulate gene expression. because it’s too small. It acts as a cofactor.

Changhan David Lee

36:23 - 36:53

It basically provides a flavor when the cell decides to change its policies upon, you know, facing some kind of stress. And the mitochondria will have a say in how this should be operated, right, the by genomic integration. What we think is going on is, and this is really interesting because it’s kind of related to histones from our observations. Histones many people believe are derived from antimicrobial peptides.

Changhan David Lee

36:54 - 37:07

So bacteria don’t have histones. Mitochondria don’t have histones. They just evolved in the middle somewhere. And if you look at these DNA binding proteins, they also operate in the same way that antimicrobial peptides work.

Changhan David Lee

37:08 - 37:26

I don’t know if you remember when I said antimicrobial peptides have a positive charge in the hydrophobic domain. The DNA is negative. So the positively charged residues will bind or get attracted and bind, interact with the DNA. And then the hydrophobic domains allow for interaction with other proteins.

Changhan David Lee

37:27 - 37:55

So when we talk about chromatin dynamics and histones opening and closing and sirtuins and NAD and acetylation and all of that, what basically is happening is an electromagnetic shift in the field. So histones are using their positive charge, are binding to the DNA. if you acetylate the histone tails, that changes the local electromagnetic field and it loosens that interaction so it can open or close. Kind of the same concept.

Changhan David Lee

37:56 - 38:20

MODSY goes in and it regulates various types of genes depending on the stress. This is an adaptive stress, so it’s not a generic stress. It’s specific to the type of stress that it’s trying to respond to. And the reason we think it’s so diverse is because the other transcription factors that come in in response to that particular type of stress, will bind to the DNA.

Changhan David Lee

38:20 - 38:24

And those transcription factors are the ones that start opening up to chromatin.

Nick Jikomes

38:24 - 38:24

Yeah, yeah.

Changhan David Lee

38:25 - 38:33

And that can go in, and in those exposed areas, it can bind and regulate the electromagnetic field there.

Nick Jikomes

38:33 - 38:48

Got it. Let me see if we can unpack this a little bit for people. Inside of the nucleus, we’ve got the nuclear genome, thousands and thousands of genes, lots of long stretches of DNA. DNA can be regulated.

Nick Jikomes

38:48 - 39:01

Genes can be turned on and off. in different ways at different levels. So the histones and the stuff that you’re referring to, they’re like these spools. People sometimes use the analogy of like a fishing reel, right?

Nick Jikomes

39:01 - 39:24

You have a bunch of line wrapped around the reel. The stuff that’s exposed, the string that’s exposed can be turned on or off. But if something’s wound around the spool, it’s completely inaccessible. You can change the transcription factors that are turning on and turning off.

Nick Jikomes

39:25 - 39:58

the open parts of the string that are not spooled around the reel. But then you can also change which segments of the chromosome are spooled up and tight versus accessible to these proteins. And without getting too much into the biophysical weeds here, a lot of this has to do with biophysics, positive and negative charge, things sticking to each other. A lot of peptides are biophysically well positioned to change some of these charge interactions and sort of maybe loosen up which segments of DNA are accessible to be turned on or not.

Changhan David Lee

40:00 - 40:23

Exactly, exactly. And we still see that today in our body. One of the antimicrobial peptides we have in the nuclear genome called LL37 also binds to DNA. And there’s evidence that if our LL37 antimicrobial peptide binds to our own DNA, it can present it to our immune system in an erroneous way.

Changhan David Lee

40:23 - 40:37

And there is some evidence suggesting that is the foundation for autoimmune conditions like psoriasis, where your body starts to attack your own because it’s misdisplayed. So yeah, so it’s kind of on the same context.

Nick Jikomes

40:37 - 40:54

Yeah, yeah. And you mentioned, right, these peptides are relatively non-specific by nature. Because they’re mini proteins, they are relatively short sequences of amino acids. They’re not as elaborate, and they don’t have as much refined structure in bells and whistles as a larger protein.

Nick Jikomes

40:55 - 41:17

And therefore, they have less specificity. So they’re not acting like a transcription factor, which might be highly specific to a very small number of genes that it specifically binds to and turns on or turns off. they have these sort of more generic interactions and they’re sort of more generally shaping which parts of the chromosomes are accessible to be played with by other things.

Changhan David Lee

41:18 - 41:35

Yeah, so if you think about these bigger proteins that are like the engine of a car, it’s fine tuning the response. So depending on the terrain, how do we fine tune this engine? Is it going to be an SUV? Is it going to go off-roading?

Changhan David Lee

41:35 - 41:46

Is it going to be on a track? Is it going to be in a limousine? Whatever it is, the engine is there, but fine tuning its use makes it much more adaptable.

Nick Jikomes

41:47 - 42:08

Yeah. But you know, at a high level, it’s like MOTC, or things like that, peptides like that, they’re not going to affect one gene, they’re going to affect a whole number of genes. So they’re because I mean, ultimately, what a cell is, right, a muscle cells, a muscle cell, and not a brain cell, because of all the the pattern of genes that are on and off. which determines the pattern of proteins and other things that are doing stuff in the cell.

Nick Jikomes

42:09 - 42:21

So if you’ve got something like a peptide, such as MOTC, and it affects many genes across many chromosomes, it’s going to reprogram, really reprogram the behavior of the cell potentially in a big way.

Changhan David Lee

42:23 - 42:55

Exactly, exactly. And these high-level regulators, so to speak, like sirtuins, for example, they regulate histones, but they don’t always regulate one histone in one region, one locus of a chromosome. It provides an overall shift. And that makes perfect sense because when a cell goes in to regulate nuclear genomes, and from the mitochondria’s perspective, It’s really one of the most sensitive sensors of the environment.

Changhan David Lee

42:55 - 43:10

And the mitochondria is trying to say, hey, look, our funding levels are going down. The economy has to shift. And to adapt to that, you can’t just go and pinpoint to one thing. It’s an overall code red that every department has to respond to, right?

Changhan David Lee

43:10 - 43:46

Because the overall economy is going down. So it makes perfect sense that we kind of say, you know, mitochondria talks to the proto mitochondria, the bacteria was trying to communicate and regulate the bigger cell, but the bigger cell was was was counting cash, the rent. So these ATP and AD, these are actually cash currencies in the cell that pay for processes. And so they directly read off of the economic state and incorporate it into their gene expression profile, which is very smart in a way, because you need to have an overall sense of the economy.

Changhan David Lee

43:46 - 43:57

And so pistons, instead of being specific, it’s much more strategically beneficial, increasing the fitness of the cell to make it a lot more broad. general regulation.

Nick Jikomes

43:57 - 44:20

Yeah. And then hopefully by now people are starting to appreciate how powerful this type of regulation could be because immediately you can think of all of the situations where there’s some kind of stress that requires a pretty wholesale transformation of what our cells are doing. Like if I get sick with an infection, right? I’ve got to have white blood cells that essentially transform into...

Nick Jikomes

44:20 - 44:49

They go from a dormant state where they’re not doing so much to a completely different state where they’re attacking and directing resources and dealing with the infection. If we exercise or we fast and our cells are running out of energy or energy demands are changing or we’re running out of fat stores or glycogen stores, our cells, whether they be muscle cells, fat cells, or whatever, they might have to completely change what they’re doing in many ways. So they’re going to need to turn on and turn off whole constellations of genes.

Nick Jikomes

44:50 - 45:04

And that will require some kind of signal, such as MOTC or one of the things that we’re talking about, that can have that sort of general reprogramming effect, rather than just turning on one gene or moving one protein around.

Changhan David Lee

45:05 - 45:34

Exactly, exactly. And also the component of time, time as a biological variable, I think is very important here. So because we’re more focused on aging, the way I see and many people in the field kind of look at bioresearch is that there’s an unbalanced focus on acute responses, for many reasons, you know, nobody wants to do a long experiment, you know, and you want to you want to understand the mechanism.

Changhan David Lee

45:34 - 46:01

So a strong induction is required to understand pathways in the beginning, right, when we don’t know anything. But in fact, what really happens in our daily lives, is more of these subtle, moderate stress that’s more chronic, that’s more relevant to us, unless you have a disease situation or some kind of infection or accident. most of the stress and adaptations are going to occur over time. So for example, exercise is what induces MOTC.

Changhan David Lee

46:01 - 46:29

But for the same reason, MOTC is not going to be a acute, you know, regulator, it requires time. So you need to go to the gym for six months plus to see some change in your body, because your body is adapting to that. And that’s very smart, because nobody wants to shift their policy dramatically, you know, with a with a cough or with a with a temporary transient stimulation. That’s just not good.

Nick Jikomes

46:29 - 46:49

Yeah. And before we discuss some of the phenomenology, the amazing phenomenology of MOTC and some of the early papers you did, I want people to also think about, you know, our cells need to transform what they’re doing and how they’re behaving all of the time. And that can happen in healthy or adaptive ways. Like you can turn on immune cells, they deal with the problem and then they turn off.

Nick Jikomes

46:50 - 47:17

You can shift the bioenergetics of your cell to do your exercise, and then you rest and recover afterwards, and things are constantly changing and waxing and waning. But then, of course, there are disease states, pathological states, age-related decline states where the cell might get stuck in a state it’s not supposed to be in or that is suboptimal. And we want to figure out how to unstick it. You mentioned things like autoimmune conditions like psoriasis.

Nick Jikomes

47:17 - 47:47

I actually have psoriasis. In a sense, you could think of it as the skin cells and or immune cells are somehow stuck in a state of hyperactivity that we would like to unstick them from. And then when we start to think about these peptides like MOTC as these sort of general purpose metabolic reprogramming signals, they start to become very interesting and potentially very powerful from a therapeutic standpoint when we start to think about shifting our cells and tissues out of pathological states and into healthy states.

Changhan David Lee

47:48 - 48:14

Yeah, I love the way you phrase that. So maybe I’ll start with this response to that comment. Drug development historically has been exactly that. It doesn’t cure the disease, but what they’re trying to do with these drugs is to be very specific in correcting what has been wrong with a goal of bringing back homeostasis, right, the balance.

Changhan David Lee

48:14 - 48:38

And of course, this country’s medical system is the same way. FDA approves drugs for disease indications only, right, with a very specific mechanism. But the problem, again, like you were saying, is bringing back the balance is a very generic thing, a general thing. If you don’t have a specific problem, right?

Changhan David Lee

48:38 - 49:10

So what do you do in this situation, where if you want to extend the person’s healthy lifespan, you don’t wait until you get a disease and try to treat that and then therefore artificially prolonging their life? what we want to do is to really increase our homeostatic balance. And that’s exactly what we do with exercise, right? Every time you go and exercise, you’re slowly increasing your capacity to maintain homeostasis in a very stressful condition, right?

Changhan David Lee

49:10 - 49:44

So that takes time. And for that reason, a lot of these regulators will be general and multi-component, multi-factorial shifts. It’s a big problem because it’s not going to be therapeutic in a sense that it’s going to target the traditional way of approaching the disease, but therapeutic in a way by increasing our youthfulness, which I define it as the capacity to maintain homeostasis, just like we do with exercise and diet.

Nick Jikomes

49:45 - 50:01

Yeah, yeah. Let’s talk a little bit now about some of the effects of MOTC that you’ve seen in animals. And, you know, people should keep in mind some of the mechanics we were kind of discussing before. MOTC is this peptide, it’s a microprotein, it’s made by mitochondria.

Nick Jikomes

50:02 - 50:19

It’s stress responsive. You know, when we exercise, when something happens like that, the mitochondria make MOTC, it can go into the nucleus. It’s changing the expression of a lot of genes to change what the cells are doing. So let’s now talk about some of the interesting experiments you did in mice related to obesity and insulin resistance.

Nick Jikomes

50:19 - 50:33

If we go back to that first MOTC paper that you did, where you’re looking at how MOTC affects metabolic homeostasis, broadly speaking, What were the basic results you saw in that study with respect to diet-induced obesity and insulin resistance?

Changhan David Lee

50:34 - 51:02

So when we first found MOTC, we had no idea what it did. There was a very loose expectation that it had to do something with metabolism because it’s from the mitochondria. And so the first cell culture in vitro experiments were to look at the effects of MOTC on respiration and, you know, all these metabolic parameters. And there were significant changes there.

Changhan David Lee

51:02 - 51:30

So that was convincing that it was a metabolic regulator of sorts. And then when we moved into mice, we still had no clue because how do you translate a cell culture experiment into a physiological condition? So we went with the thing that we knew best to regulate metabolism in mice and Hasi’s lab, Hasi’s an endocrinologist by training. And so diabetes, obesity, that was the first venue.

Changhan David Lee

51:30 - 51:44

So we just fed mice a high fat diet, we knew that was a metabolic stress, and that screws around with their insulin sensitivity and all that. And then we started to inject Moxie to see if it does anything, either make worse or make it better.

Nick Jikomes

51:45 - 52:05

So the basic setup here is you were doing diet-induced obesity models in animals, pretty similar to many, many other experiments that have been done. You give them a certain type of high-fat diet. We’ll not get into the details of the nutrients there in this podcast probably, but you give them a diet that makes them fat. They become bigger, they become fatter, they become insulin-resistant.

Nick Jikomes

52:06 - 52:10

And the experiment is, okay, if we do that, but we give them MOTC, what happens?

Changhan David Lee

52:11 - 52:23

Exactly. That’s what we did. And we just basically observed what happened. And it was really shocking because it reduced or it prevented diet-induced obesity very dramatically.

Changhan David Lee

52:23 - 52:54

And when we were dissecting the mice, you open it up and one of the characteristics of these high-fat diet mice, and the same for humans, is fatty liver. You open up the mouth and the liver is clearly pinkish because of the fat accumulation, where it should be very deep red. And when we opened up the Moxie ones, not only did they not gain as much weight, their livers were all red. And so that was the beginning of the studies that led to Moxie, to translation into clinics.

Changhan David Lee

52:54 - 53:22

So a company called Cobar, which was co-founded by Hasi Cohen, my postdoc advisor and the dean of our school right now, and also Nir Barzilai, who’s in New York. The indication that the company went for was fatty liver and obesity for that reason. So that was one of the first shocking things that we saw. And then everything else, you know, came history.

Nick Jikomes

53:23 - 53:29

Yeah. So you give mice this high fat diet, they get bigger. They get quite a bit bigger too. It’s not a subtle effect.

Nick Jikomes

53:29 - 53:43

They get quite a bit fatter than the mice skinny normal diet. They get a fatty liver. If you give them that high fat diet, but you’re also giving them MOTC at the same time or a little bit ahead of time, they basically don’t. It’s not that they don’t get as fat.

Nick Jikomes

53:43 - 53:48

You basically almost completely blocked the diet induced obesity. It was a dramatic effect. Yes.

Changhan David Lee

53:49 - 53:52

Yes, it wasn’t 100%, but it was very, very significant.

Nick Jikomes

53:53 - 54:04

Yeah. And then you open them up and you’re also blocking the fatty liver. What happened when you gave normal mice MOTC? Did they waste away and lose weight?

Changhan David Lee

54:06 - 54:31

No, in fact, nothing happened. And that’s one of the themes with MOTC. It doesn’t have a job unless there’s a job to do, which means in a peaceful condition, as best as we can define, even in cell culture, if you don’t perturb the cells and leave them in their happy state, we never saw anything happen to happy cells. It’s always some kind of stress that was required.

Nick Jikomes

54:31 - 54:49

So, so it’s a context dependent effect. It wasn’t, it could have been the case that MOTC was just causing, you know, the wasting away of fat tissue and lean mass or whatever, but that’s not the case. It was actually preventing diet induced obesity in the context where you’re stressing the animal with this high fat diet, but it wasn’t doing much of anything under normal baseline conditions.

Changhan David Lee

54:50 - 54:51

Nothing, nothing.

Nick Jikomes

54:51 - 55:09

Interesting. And then, you know, when you dug under the hood a little bit, what is going on that underlies this dramatic effect? I want to ask you about, you know, sort of the metabolic cell level effects of MOTC. And this is going to bring us to something called AMP kinase.

Nick Jikomes

55:09 - 55:12

What is AMP kinase and how does MOTC tie into that?

Changhan David Lee

55:13 - 55:36

So AMP kinase is also dubbed as the master metabolic regulator. The AMP part of AMPK is the monophosphate version of ATP. So the phosphate in ATP is the source of energy. When you cleave that, you get energy and you can do things, you pay for things.

Changhan David Lee

55:37 - 56:02

Once he uses the stored energy phosphates, it becomes ADP, diphosphate or monophosphate, AMP. What AMP kinase does, it’s a protein that senses the levels of AMP versus ADP. So how much of our cash currency have we used? If ANP levels go up, that means we’ve used up a lot of our resources.

Changhan David Lee

56:02 - 56:07

And that’s more consistent with a fasting state or energy depletion state. Got it.

Nick Jikomes

56:09 - 56:15

It’s like a fuel gauge or almost like a detector of when your bank account is running low on cash.

Changhan David Lee

56:15 - 56:27

Exactly, exactly. It’s like an alert system. I guess not an alert system, it’s a sensor of cash flow. And when it goes up, when AAP goes up, your cash is low.

Changhan David Lee

56:27 - 56:47

So they have to rearrange things and say, okay, look, we have to live with this new environment where we’re not as affluent as before, you know, strap down things. Yeah. And the kinase, K part is kinase. Kinase basically means a protein that goes and phosphorylates other proteins.

Changhan David Lee

56:48 - 56:49

So it’s a regulator in that sense. Yeah.

Nick Jikomes

56:50 - 57:11

So AMP kinase becomes active when ATP levels go down, which basically means you’re running low on cash. So if we just play with this analogy a little bit, if you’re running low on cash and your income’s constrained, you’re not going to be building a new kitchen in your house. You’re not going to be buying a new car. You’re going to want to preserve your cash flow and focus on new ways to generate income.

Changhan David Lee

57:13 - 57:36

Absolutely. Yes, yes. And so that ties in really importantly with aging for us, because, you know, one of the most important things that can regulate somebody’s lifespan, or health span is metabolism. And so there’s so many, there’s ocean of literature on how things like A&P kinase, there’s another master regulator called mTORC1.

Changhan David Lee

57:38 - 57:53

That’s another one that regulates more protein side. These have been studied extensively in the field of gerontology, aging. And if you activate those pathways, it typically leads to extended health span or lifespan. Yeah.

Changhan David Lee

57:53 - 57:55

For that reason, regulates metabolism.

Nick Jikomes

57:55 - 58:04

Yeah. What are some of the natural stimuli of AMP kinase? What makes AMP kinase go up at the level of behavior and macroscopic stuff?

Changhan David Lee

58:06 - 58:28

I mean, very clearly, energy deficiency. So dietary restriction will definitely increase it. But this is one of the things that I think most people that are not in our field kind of go over. and miss is that these are stress signals, and they’re not supposed to be activated for a prolonged period of time.

Changhan David Lee

58:28 - 58:50

So think of it as stress hormones like cortisol. Yeah, you need cortisol, but you don’t want cortisol levels up for extended period of time, it should spike, and then go back down, right? Because it activates downstream pathways, and then it has to shut off. Otherwise, if it’s constantly up, that causes your device instead of saving it.

Changhan David Lee

58:50 - 58:56

So it’s hard to say what activates and what goes, you know, but clearly P, energy deficit.

Nick Jikomes

58:57 - 59:22

But you know, the things that would cause that fasting, food deprivation, exercise, and you know, as you’re sort of implying here, these things can be good or they can stimulate this pathway in adaptive ways, but you don’t want these signals to be turned on chronically or for too long. You don’t want to exercise too hard for too long because then it can go from being a healthy thing to an unhealthy thing. Fasting can be great, but obviously if you go too long without food, then you’re in trouble.

Changhan David Lee

59:23 - 59:23

Malnutrition, yes.

Nick Jikomes

59:25 - 59:44

So AMP kinase basically gets activated more when MOTC goes up, which can be induced by exercise and other things. What happens after that? What happens to the mitochondria after that? What’s sort of the net result of turning up this AMP kinase pathway in terms of the mitochondria and what they’re doing and how many there are?

Changhan David Lee

59:45 - 1:00:04

So many, many things happen, including M-kinase activating a pathway through something called PGC1-alpha to increase mitochondrial biogenesis. So it’s an energy deficit situation. So you want to maximize your energy efficiency. So it could stimulate mitochondrial biogenesis.

Changhan David Lee

1:00:05 - 1:00:35

It regulates a lot of the proteins that are related to metabolism, like fatty acid metabolism, it starts to increase fatty acid metabolism, because it’s your storage, right? And in times of deficit, you want to start using your storage. So fatty, fatty acid oxidation can go up, your usage of glucose goes down. So a good manifestation or good example of this in a more translational way is a drug called metformin.

Changhan David Lee

1:00:36 - 1:01:04

It’s the number one frontline drug for diabetes, and it regulates your glucose production and in some ways consumption too as well. So that’s what, it’s a broad effect because it’s such a high level regulator. So if you switch around your glucose metabolism, your fatty acid metabolism, the mitochondria is at the center of that. And it’ll respond in many, many different ways.

Nick Jikomes

1:01:04 - 1:01:13

Yeah. Yeah. So we’re in a state of energy deficit because we’re exercising or food deprived or something. Moxie is being produced by the mitochondria.

Nick Jikomes

1:01:13 - 1:01:39

This is causing amp kinase to go up. This is sort of our, our sensor of energy deficit. We’re in a state of relative energy deficit, amp kinase is going up. One of the things this ultimately leads to is mitochondrial biogenesis, where basically the cells basically saying, we’re running low on ATP, let’s build up our ATP production capacity so that we can counteract this deficit we’re facing, what’s happening to other things the cell might be doing?

Nick Jikomes

1:01:39 - 1:01:45

Things like mTOR, the anabolic parts of what the cell is doing to build up other structures.

Changhan David Lee

1:01:46 - 1:02:09

Right. So, AP kinase and mTOR1 are related, they’re networked, so they cross affect each other. mTORC1 is a different thing in terms of benefits. Well, I guess you want to turn on A-B kinase, but you want to turn off mTORC1 because mTORC1, like you said, is anabolic.

Changhan David Lee

1:02:09 - 1:02:29

it stimulates protein synthesis. And that’s not something you want to do when energy is low, because protein synthesis is one of the most expensive processes in a cell. It can take up to 30% of a cell’s energy source. So when AMP kinase goes up, typically it suppresses mTORC1 activation.

Nick Jikomes

1:02:30 - 1:02:45

Yeah. So again, if we play with our analogy, it’s like, If you’re running a household and you get demoted at work or you lose your job, income is going down. So you want to preserve the cash flow that you have. Think about ways of building up your income capacity.

Nick Jikomes

1:02:46 - 1:02:55

And you want to turn off the remodeling of the kitchen. You don’t want to hold off on buying a new car and building up these new structures because income is constrained and those things cost money.

Changhan David Lee

1:02:56 - 1:03:02

Exactly. Exactly. So you’re basically shutting down. Getting ready for war.

Nick Jikomes

1:03:02 - 1:03:13

Yeah, yeah, yeah. So, okay. So, AMP kinase stuff we just talked about. We mentioned earlier that MOTC goes into the nucleus and it affects gene expression in a big way.

Nick Jikomes

1:03:13 - 1:03:28

Many different genes, not just one or two genes. You’re reconfiguring what the cell is doing and how it’s using its energy and all of these things. What, where does the methionine folate cycle come into this? How is MOTC affecting that cycle?

Nick Jikomes

1:03:28 - 1:03:31

And how does that cycle relate to the gene expression stuff?

Changhan David Lee

1:03:34 - 1:03:52

Yeah, so that’s what we’re really trying to figure out right now. Um, what it does in the nucleus is, is just so diverse. Like I said before, it doesn’t have specificities. It really depends on the other cofactors that come in or the other transcription factors that come in.

Changhan David Lee

1:03:55 - 1:04:00

Yeah, I mean, that’s an ongoing project in the lab. We’re still trying to figure that out.

Nick Jikomes

1:04:01 - 1:04:22

Got it. But one of the things I wanted to get you talking about is you mentioned earlier the importance of some of these phenomena, some of these signals being acute and not chronic. Exercise is a stress, and it’s good to intermittently have that acute stress, but you don’t want to have chronic stress, too much stress for too long. That can lead to problems.

Nick Jikomes

1:04:23 - 1:04:51

When we start thinking about exogenous MOTC and we start thinking about, you know, the natural dynamics of endogenous MOTC, it goes up, it comes down because stress is intermittent. It’s not chronic usually. What might start to happen or what problems might start to arise if you were just say, giving an animal exogenous MOTC for a very long time, turning on this AMP kinase pathway, would it have benefits initially that maybe turn into problems with chronic use?

Changhan David Lee

1:04:53 - 1:05:23

I mean, I think theoretically it’s possible. What we did in the 2021 paper, in the Nature Account paper, was we injected mice, old mice and middle-aged mice, old equivalent to humans of roughly 65 years of age, 60, 65. We injected the mice with Moxie daily. And within a week, we saw a trend of physical capacity going on, meaning they were running much longer on the treadmill.

Changhan David Lee

1:05:24 - 1:05:34

And then, but it wasn’t, it wasn’t significant statistically. So we extended the protocol to two weeks, two weeks of daily injection, and then the older mice were doubling their running. Doubling? Time?

Changhan David Lee

1:05:34 - 1:05:44

Treadmill? Yeah, doubling. And they were out running their middle-aged friends, or I guess they’re not friends, they’re clones. So they were performing really well.

Changhan David Lee

1:05:45 - 1:05:56

And the middle-aged were doing well too. But these were daily injections. So what we did after that was the effect was much stronger than we anticipated. So we didn’t want to euthanize the older mice.

Changhan David Lee

1:05:57 - 1:06:16

precious resources. So we switched the protocol and got approved to do lifelong Monsi injection. And here is what you were just saying really comes into play about this stress signal that shouldn’t be up the whole time. And that’s true for a lot of these longevity interventions.

Changhan David Lee

1:06:17 - 1:06:57

For example, rapamycin, which is one of the most powerful lifespan-enhancing drugs out there, can be tested. There’s a very famous paper that shows intermittent rapamycin injection is very powerful, meaning you don’t want to yell stress the whole time, you want to provide that stress, give the cell time to recuperate, increase their capacity, just like exercise, little by little, and respond. So that’s, that’s the major theme of Mod C right now. In a paper we recently put up on bioRxiv, we try to make that point very clear.

Changhan David Lee

1:06:57 - 1:07:32

And there now we have a much more molecular description of that. The MOTC’s effect is not unimodality, meaning it’s not just one thing that it does, like A and B kinase activation. It seems to be activating a program, and the program has an acute phase, which is the cell type that we use, again, the generic hexanithrase or the fibroblast, it lasts about less than 24 hours. And that focuses on regulating protein translation.

Changhan David Lee

1:07:32 - 1:07:57

So if you take a step back and look at it, what we see, how we interpret it is, When moxie goes into the cells, it’s a stress condition, but you don’t know exactly what’s going on. The cell’s been hit with something and they’re trying to figure out what it is so they can mount the appropriate response. And they have to figure out if this is going to be transient or persistent. And so the first 24 hours is kind of like the cell trying to figure out what’s going on.

Changhan David Lee

1:07:57 - 1:08:16

And as we just talked about, the first thing it does is to reduce protein translation. So moxie goes in and translation gets shut down very, very fast. Very, very quickly. And that lasts about 24 before we see gene expression happen at around 24 hours.

Changhan David Lee

1:08:16 - 1:08:17

So it’s a phase.

Nick Jikomes

1:08:17 - 1:08:18

Yeah.

Changhan David Lee

1:08:18 - 1:08:32

We call it acute phase and the chronic phase. In the chronic phase, you have gene expression coming out. But this effect goes away if you don’t continuously give MONCY. It’s not one activation.

Changhan David Lee

1:08:32 - 1:08:47

It fades away if you don’t give it to it. And in this paper, we show something really crazy. And this was another crazy moment that gave me goosebumps. We use something called a PSM model, it’s progressive stress media.

Changhan David Lee

1:08:47 - 1:09:15

And going back to what we were discussing earlier, a lot of the cell culture models are geared towards acute stress, that are typically not physiological, meaning we take away glucose, we take away certain amino acids. And that’s very stressful, but that rarely happens in a living body, right? You don’t have no glucose and you’re dead at that point, or an amputation or something. So what we did was we had the cells use up the media the way they do naturally physiologically.

Changhan David Lee

1:09:15 - 1:09:33

And then we collected the spent media. But we had different gradients, you know, spent a little bit spent moderately and extensively different levels of stress. different levels of stress. And we identify that using mass spec metabolomics, we kind of know what changed and what went up and down.

Changhan David Lee

1:09:33 - 1:09:54

And in that media, it’s really comprehensive, because it’s what really happens in a body. It’s not just usage and depletion of nutrients, the cells while they’re responding to the nutrient deficit, start making other things because it’s metabolism, right? You metabolize things. So it’s a very, in our opinion, much more physiological stress.

Changhan David Lee

1:09:54 - 1:10:26

And the reason that’s important is because our cells have evolved to generate these programs that adapt to that situation. more so than removal of a single nutrient situation. So the programs that get activated in the chronic physiological state will not be activated by, you know, removal of certain nutrients was our thinking. And we kind of put that in the paper too, when we just dilute the media, not collected from a spent situation.

Changhan David Lee

1:10:27 - 1:10:49

Moxie does not protect the cells. It doesn’t have much role because I guess those are non-physiological situations that are wired into the cell to activate this response. But when we use the spent media, normally cells will start dying off in this moderate stress, physiological stress level in about three days. The cells can’t do more.

Changhan David Lee

1:10:49 - 1:11:05

They just go down and then die about three days. Motsi, if we treat them with Motsi, we kept them alive for over a month, which is crazy. If you think about it, we didn’t give them any food, any media. We just gave Motsi and they go for over a month.

Changhan David Lee

1:11:06 - 1:11:21

And we stopped in a month because the PhD student had to deliver our babies. She stopped there, but I don’t know how far it could have gone, but that’s where it is. But if you stop the treatment of MOTC, the curve starts to drop almost immediately the next couple of days after.

Nick Jikomes

1:11:22 - 1:11:28

MOTC is somehow reconfiguring the cells so that they can survive in those deprived conditions for much longer.

Changhan David Lee

1:11:28 - 1:11:50

Right. And that’s why I think it’s much more consistent with the exercise secretion. So exercise stimulates Mod C because it’s preparing for a more persistent stress condition to respond to. So if I do one bout of exercise, I’m not going to get physiological changes that I wish I did.

Changhan David Lee

1:11:51 - 1:12:01

But going to the gym once, nothing really happens. Going to the gym for three months, nothing seems to happen. Because for the body to respond to that immediately is a waste of energy, right? Right, right.

Changhan David Lee

1:12:01 - 1:12:10

If you grow muscle, then it better be something that you’re going to use or it’s a new environment that you have to adapt to that’s persistent, right?

Nick Jikomes

1:12:10 - 1:12:11

Yeah. So that makes sense.

Changhan David Lee

1:12:11 - 1:12:14

Yeah. That monster stress during those conditions.

Nick Jikomes

1:12:14 - 1:12:43

Yeah. A lot of this stuff at the molecular and cellular level can hopefully start to get people to make more sense out of what happens at the whole body level when we do things like exercise. You have to do things that are persistent, but also at the same time dynamic. If you start going to the gym three times a week for three months, and you exercise hard, and then you rest, and then you exercise hard, then you rest, that’s going to be different than not doing that at all.

Nick Jikomes

1:12:44 - 1:12:57

It’s also going to be different than going and exercising hard every single day, which might put you into this chronic stress mode that actually prevents you from building muscle, translating protein, turning on mTOR, all of the stuff that we’re talking about under the hood here.

Changhan David Lee

1:12:58 - 1:13:16

Exactly, exactly. So it’s not pressing a button to get to the next level. Our body is again, the whole focus is on homeostasis. And the only reason you would increase that capacity is when it’s necessary, because your body is very economic.

Changhan David Lee

1:13:16 - 1:13:32

It’s not going to build muscle if it doesn’t need it. So if you put a cast on, I don’t know if you had a cast before, for about three months, you take it off, it’s almost like your arm wilted. Atrophy happens and muscle is lost because you’re not using it. The body doesn’t need it.

Changhan David Lee

1:13:32 - 1:13:37

It gets rid of it. But if you want to build muscle, you better need it. So it has to be something that’s persistent.

Nick Jikomes

1:13:37 - 1:14:00

Yeah. If we go back to the diet-induced obesity animal model, you said that exogenous MOTC giving it to animals that are going to get a high-fat diet, it basically prevented most of the weight gain and fatty liver that that diet would otherwise induce. What other changes did you see in those animals with regard to things like energy expenditure and just their basal metabolic rate?

Changhan David Lee

1:14:01 - 1:14:27

So we put them in metabolic cages and nothing really popped out that could easily explain the prevention of weight gain. For example, we were hoping that there would be more activity, but their daily activity levels were about the same. What did change was their metabolism during night. So these are nocturnal animals, that’s when they feed.

Changhan David Lee

1:14:28 - 1:14:49

So when we’re asleep, they’re active. And during the night phase, when they’re actively running around, then their metabolism kind of differed from the controls. But you know, that was pretty much the only trend that we saw. We didn’t see much in body heat, but then the equipment was not really there to accurately measure body heat.

Changhan David Lee

1:14:49 - 1:15:10

There was a paper that came out that showed that MODSI increases the capacity to maintain body heat. It’s a very gruesome experiment. We were thinking of doing it, but we didn’t have the heart to do it. You put mice in a cold room, in a fridge, and the body temperature goes down fast, right?

Changhan David Lee

1:15:10 - 1:15:40

That group that published, they gave MODSI and they showed that the body temperature drop was much more retarded. It maintained body heat much more. So one hypothesis that we had was, there are many ways, many reasons that MODSI and that obesity study that we did prevented weight gain. One of them could be expelling the energy through heat, but it’s not going to heat up so we can detect it easily.

Changhan David Lee

1:15:40 - 1:15:50

It’s going to be a subtle increase over time, but it’s going to be difficult to detect. That’s our suspicion at this point, because of that study.

Nick Jikomes

1:15:51 - 1:16:03

Yeah, yeah. MOTC has been called, it’s being called an exercise memetic. Like, you know, you take exogenous MOTC and it’s going to be as if you exercise, but you didn’t actually exercise. Is that a fair description?

Nick Jikomes

1:16:03 - 1:16:09

Is it recapitulating most of what happens with actual exercise or just a piece of it? What do you think people should know?

Changhan David Lee

1:16:10 - 1:16:35

No, but I think, I mean, I hear that a lot too. I’m not a big fan of the word exercise pneumatic, just because I feel that discounts the complexity of exercise and kind of frames the way people think of exercise as a single dimension. Yeah, activity. If you think about it, exercise is not just about burning calories.

Changhan David Lee

1:16:35 - 1:17:00

It’s also about innervation of the nervous system. It’s also about connecting the nervous system with the muscle because one of the reasons you don’t grow muscle is not because of protein intakes of deficiency. It’s also your nervous system has to know that it’s being used to activate it. Like I said, if I don’t use my arm when I have a cast, I’m not using it, my brain is not connecting to it, so it gets rid of it.

Changhan David Lee

1:17:00 - 1:17:22

So the connection to the nervous system is one thing that’s totally missed. And then the complexity of the stress, like I said, it’s heat stress, it’s shear stress, it’s the entire universe stress, right? Everything that happens just, but not just biochemical. So to recapitulate all of that with one I think it’s going to be very difficult.

Changhan David Lee

1:17:23 - 1:17:43

Is MOTC a component? I definitely believe it’s a component and perhaps it falls into the category of extra kinds. These are those immune responses in response to exercise that triggers these physiological results. In 2021, we published a paper in cell metabolism showing that.

Changhan David Lee

1:17:43 - 1:18:19

So in the brain, the hypothalamus, which is the part that regulates metabolism, There are cells called POMC neurons that express this protein called POMC that regulates appetite and energy expenditure. What we see was, if you put the mouse on this exercise regimen, which is basically a treadmill, moderate exercise, this is not crazy athletic, this is just very moderate exercise. You do see these extra kinds like IL-6 go up and they end up in the brain. So the muscle is talking to the brain when you exercise for a while.

Changhan David Lee

1:18:20 - 1:18:47

And these are immune responses as well, interesting enough. And the brain receives that and it starts regulating things. MOTC levels go up in the POMC neurons, and it works with these other extra kinds, like IL-6. So IL-6 will signal through its receptor, and MOTC will interact with something called the STAT pathway, and it promotes the expression of these POMC genes, which then gets processed as regulators.

Changhan David Lee

1:18:48 - 1:19:13

The other interesting thing that we saw was when we inject MOTC intracranially, so if you go straight into the brain and inject MOTC into that part of the brain, What we see is a dramatic shift in the peripheral metabolism, especially with the white adipose tissue, beiging significantly. So you see an increase in beige fat when you inject Moxie into the brain, not peripherally.

Nick Jikomes

1:19:13 - 1:19:26

So you inject it into the brain and you’re seeing effects peripherally. So somehow the brain is changing and sending signals out to the body. What’s the effect of the beiging of the white adipose tissue? Or excuse me, what’s the significance of that effect?

Changhan David Lee

1:19:27 - 1:19:47

So, I mean, beige fat has many, many things that... change the physiology. So it’s changing, for example, white ass position would be your fast storage into a much more brown fat like condition. So brown fat is the fat that keeps you warm.

Changhan David Lee

1:19:48 - 1:20:09

You know, yeah, yeah, we don’t have much as adults, but babies have much more, but we still have it in certain areas like the heart where you have to have that energy source and heat. increases basal, I guess, usage of these lipids because of their characteristics, phage fat.

Nick Jikomes

1:20:10 - 1:20:23

Got it. So it’s like, yeah, MOTC can get into the, or it’s going into the brain or when it’s in the brain, it’s having effects throughout the body and basically stimulating changes in other tissues, even though it’s the origin of the signals in the brain.

Changhan David Lee

1:20:24 - 1:20:40

Right, right, right, right. And then the POMC neurons, they’re related to endorphins. So the POMC gets processed into endorphins. So we were kind of joking with the PhD student with the exercise study and he said, you know what, I don’t think we’re really doing that much to the mice other than making them high.

Changhan David Lee

1:20:40 - 1:20:57

So they’re runners high. I don’t know if you’ve heard that term, runners high. is basically endorphins, kind of easing the pain in these elite runners, so they don’t feel much. And we’re saying, you know, maybe the mice were running longer because it’s a pain management system.

Changhan David Lee

1:20:58 - 1:21:03

We’re activating this metabolic pain management system. So we were looking at that a little bit, but we haven’t gone far in that regard.

Nick Jikomes

1:21:03 - 1:21:12

Oh, interesting. I mean, quick side note, I believe there’s work out there showing that this effect in mice is actually mediated by endogenous cannabinoids.

Changhan David Lee

1:21:12 - 1:21:14

That’s the same pathway. Yeah. Yeah. Okay.

Changhan David Lee

1:21:15 - 1:21:16

Okay. Interesting.

Nick Jikomes

1:21:16 - 1:21:34

I want to ask you a little bit about autoimmunity. You know, we talked about immune system modulation by MOTC and other peptides. There’s one paper that looks quite remarkable that you were involved in using MOTC in an autoimmune diabetes model. Can you talk a little bit about what you guys saw there and how significant that was?

Changhan David Lee

1:21:34 - 1:21:48

Yeah, yeah. So type 1 diabetes model. So type 1 diabetes is an autoimmune disease, unlike type 2 diabetes. And those two shouldn’t be mingled too much in terms of, you know, models.

Changhan David Lee

1:21:50 - 1:22:11

Basically, it’s your own immune system attacking your beta cells, your pancreatic beta cells that produce insulin and then you run out of insulin. It’s a very painful disease, right? The only treatment for that is to keep injecting insulin. What we saw was in the model, we prevented the onset of type 1 diabetes, the autoimmunity.

Changhan David Lee

1:22:11 - 1:22:32

And when we looked at the pancreas, specifically in the beta cells, the infiltration of the immune cells that were typical, that come in to kill your beta cells, were not there. So the immune cells did not come. they were not recruited in the beta cells. And that’s why we think the pancreas was kept alive and it was making insulin as usual.

Changhan David Lee

1:22:33 - 1:23:17

What we kind of figured out was the involvement of the immune cells and the regulation of immune cells, more specifically in that paper, T cells. So the experiment that the first author did was if you take Mod C treated beta cells, immune cells, and if you give it to another animal, meaning you take a normal mice, and then you take the immune cells of a type one diabetes model. So the immune cells are now transferred into a normal mouse. So you’ll have antibodies or immune cells that recognize yourself as a target.

Changhan David Lee

1:23:18 - 1:23:36

If you do that in a normal cell, you’ll induce type one diabetes there too. But if you take the cells, immune cells from the mossy treated type one diabetes mice, right, and you transplant that, you transfer that to a normal mouse, nothing happens, which tells us that it suppressed the autoimmune recognition somehow.

Nick Jikomes

1:23:36 - 1:23:36

Yeah.

Changhan David Lee

1:23:37 - 1:23:59

And it turns out that has to do with something called regulatory T cells. So the T cells that actually regulate the autoimmunity. So if your antibody or if your T cells recognize your own self, these Treg cells will typically come and say, no, no, no, that’s you, don’t attack. Those cells were much more involved.

Nick Jikomes

1:24:01 - 1:24:33

So you’ve got an animal model, a mouse model, they will develop autoimmune diabetes, type 1 diabetes, their immune cells will attack their pancreas and destroy their insulin producing cells. If you give those mice MOTC, exogenous MOTC, it prevents the development of that autoimmune diabetes. And what seems to be happening is one way or another, MOTC is affecting how some of the white blood cells, some of the T cells are developing and changing so that they don’t develop this pathological tendency to attack the animal’s own pancreas.

Changhan David Lee

1:24:34 - 1:24:51

Exactly, exactly. And exactly how that happens, we’re not sure, but we believe it’s going to be not a direct action. Of course, there are direct actions on the actual T cells that go through the mTOR1 pathway, as we figured out. But I think, broadly speaking, it’s not just the T cells that are affected.

Changhan David Lee

1:24:51 - 1:25:20

We have evidence now that the macrophages are also affected. So these are antigen presenting cells, macrophages, you know, B cells. And what we think is going on is that it’s shutting down the activation of these immune cells, meaning it’s regulating how the cells decide whether or not to stimulate a further path or a further response. And that’s why it’s reducing overall autoimmunity.

Changhan David Lee

1:25:20 - 1:25:20

Wow.

Nick Jikomes

1:25:21 - 1:25:39

Does this potentially imply that One could change whether or not autoimmune diabetes develops based on the pattern of stress in early life. So for example, could an individual exercise more in early life and potentially affect the trajectory of such an autoimmune disease?

Changhan David Lee

1:25:40 - 1:26:06

And so that’s the idea. So with aging, autoimmunity goes up, especially in women. And so if you push back the process of aging or slow it down, yes, the idea is that you will also push back the onset of these age-related autoimmunity. And one of the projects, so that’s kind of what we call a new tolerance project that in our lab, there’s a PhD student who’s digging into that as well as that for a thesis.

Changhan David Lee

1:26:08 - 1:26:37

What MOTC seems to be doing is to again provide the flavor to these macrophages when they’re deciding whether or not to activate things. But what we found in the paper, we showed in the paper was the way that it does it is by shifting the way the macrophages mature. So if you rewind a little bit, macrophages, there are two types. There are the tissue resident ones that come from a different lineage.

Changhan David Lee

1:26:37 - 1:26:56

And there’s the monocyte-derived macrophages that come from your hematopoietic stem cells. like other immune cells, right? When the monocytes differentiate into macrophages, it’s taking cues from all over. For example, is this an infection or is this an exercise-induced repair that we need to do?

Changhan David Lee

1:26:56 - 1:27:16

You know, muscle shearing, that’s what happens after you exercise, the immune response is very dramatic and it’s required for muscle building. But that’s more of a repair kind of macrophage that needs to clear out the debris of the broken down muscles. versus an infection, your job is to go and kill. It’s a license to kill kind of situation.

Changhan David Lee

1:27:17 - 1:27:46

So the gradient of different types of macrophages is very, very broad. There are different types. What MOTC does, as we showed in the recent paper in eLife, when we give MOTC during that monocyte differentiation, it provides a different kind of macrophage, which is really interesting because if you go back to the story of MOTC discovery, we were basing a lot of that on that Sidney Peska paper with the interferon.

Changhan David Lee

1:27:46 - 1:28:00

And the most striking thing is the signature of the macrophages that are made with MOTC as a cue is defined by two things. One is interferon and the other is antigen presentation.

Nick Jikomes

1:28:01 - 1:28:08

Wow. Wow. Yeah. So you’re shifting, MOTC is shifting the pattern of white blood cell identities that show up.

Changhan David Lee

1:28:09 - 1:28:20

Yeah. So broadly put, and this is just my interpretation, instead of saying, Hey, there’s a, there’s a, there’s an infection. You have to go and kill it. It happens to be your pancreas.

Changhan David Lee

1:28:20 - 1:28:28

The mission changed because they’re saying, no, no, it’s more like a muscle repair system. Let’s not go and kill it, but let’s figure it out. You know, what’s going on.

Nick Jikomes

1:28:29 - 1:28:32

Yeah, a cleanup crew as opposed to the Navy SEALs.

Changhan David Lee

1:28:32 - 1:28:46

Right, because you can recognize, the SEALs can still recognize yourself as a foreign thing. I don’t think once it’s changing that, that’s already determined during development. But what it can do is to change the mission.

Nick Jikomes

1:28:47 - 1:28:51

Yeah. Change the mission and therefore who’s called in to achieve it. Right.

Changhan David Lee

1:28:51 - 1:28:58

You recognize it, but bring your tolerance out. This is not an infection situation. Let’s call it a repair environment.

Nick Jikomes

1:28:59 - 1:29:28

Yeah. So one of the things that people who are injecting themselves with exogenous Mott CR reporting, the most common thing that I see out there is I’m basically body composition slash exercise related changes. So people are saying that it’s helping them reduce or burn off body fat, but maintain muscle mass. So if they’re working out, lifting weights, they’re not going to lose their muscle mass, but they are going to lower their body fat a little bit.

Nick Jikomes

1:29:28 - 1:30:00

It seems like a lot of people are know, assuming they’re getting genuine MOTC, and I’m not sure how people are dosing this, but it also seems common that they’re cycling this, so they’re not taking it for weeks on end. As far as I can tell, they’re often taking it for a couple days on, a few days off. They’re doing things like this. Given all of what we know from the preclinical research and the mechanistic work that people like you have done, Is this a likely outcome for humans who might use exogenous MOTC, you know, saying nothing about whether they should or shouldn’t or what the rules and regulations currently state.

Nick Jikomes

1:30:00 - 1:30:05

Does that make sense to you that that would be preserving lean muscle mass, but helping them burn off body fat?

Changhan David Lee

1:30:06 - 1:30:28

Yeah. Yeah. Um, it’s very consistent with the preclinical data and, uh, you know, it’s difficult to ignore the fact that exercise is to the best of our knowledge, one of the strongest inducers of MOTC in humans. And therefore, a lot of the effects of exercise would be seen with Mod C as my expectation.

Changhan David Lee

1:30:28 - 1:30:59

And it’s not a mimetic, it’s part of the exercise response. And therefore, you expect, but like I said, the effect of exercise is many. Of those, I believe Mod C would have an impact on the metabolic, immunometabolic side of exercise. So the benefits that we see with immune system changes, as well as the metabolic, especially the conjunction of the two, the composite effect of the immunometabolic response is what I would expect to see.

Changhan David Lee

1:30:59 - 1:31:18

Yeah. Are you burning more fat is a good question. Our data indicates that moxie doesn’t really increase beta oxidation. But the way that it’s reducing fat accumulation, so there’s two different things.

Changhan David Lee

1:31:19 - 1:31:31

There’s a burning of the fat. And then there’s the reduction of synthesis of fat storage, right? Yeah. So what we think is happening with MOTC is more on the lipid regulation.

Changhan David Lee

1:31:31 - 1:32:06

And we put this in the bioarchive paper a little bit. Under stress, we learned that one of the most important, this is already known, we just learned recently, after learning about it in textbooks for a long time ago, one of the most important things with the cells during stress is to retain its membrane lipids. So other things can be you know, sourced from various nutrients, like energy can be from, you know, multiple different sources. But the one thing that the cells cannot give up or go wimpy on is the lipid membranes.

Changhan David Lee

1:32:06 - 1:32:28

So that includes internal organelle membranes, like if you want to increase autophagy, you need to have the vesicles you know, the autophagosomes, which are all lipid, right? And then the cell membrane is lipid. You can’t give that up. What we see is not moxie burning fat during the spent media condition.

Changhan David Lee

1:32:28 - 1:32:46

They’re not using the fat to generate energy. That is not there in our metabolomics. But what is very clear is that they’re shifting the lipids into phospholipids. which is not the type of white fat triglycerides storage for membranes as opposed to adipose tissue.

Changhan David Lee

1:32:47 - 1:32:54

Exactly. Exactly. So what we think is that it’s something like the ketogenic diet. Yeah.

Changhan David Lee

1:32:54 - 1:33:07

The fact that you don’t have carb, you don’t activate the mechanism of the light pieces to start putting in more triglycerides into your body. That’s why you lose fat on a high fat ketogenic diet. Right.

Nick Jikomes

1:33:07 - 1:33:14

Yeah. So it’s not the burning of fat for energy. It’s where the fat’s going and how it’s being utilized.

Changhan David Lee

1:33:14 - 1:33:20

That is, to our knowledge, what seems to be more so. I’m not saying it doesn’t burn any fat.

Nick Jikomes

1:33:22 - 1:33:48

Yeah, but in your model systems, that’s more so what seems to be going on. So you actually, you also mentioned earlier, you know, we talked about amp kinase and mitochondrial biogenesis. You’re basically boosting that your, how many mitochondria you have, um, in older animals and your guys’s hands, it could double their exercise capacity on the mouse treadmill stuff that you use. I suppose one effect in humans could be that to the extent that is increasing exercise capacity.

Nick Jikomes

1:33:48 - 1:33:54

Maybe people are just working out a little bit harder when they’re taking Moxie or something like that, rather than, you know, burning off that.

Changhan David Lee

1:33:55 - 1:34:12

So, yeah, I agree with that. And, you know, I met so many people that are taking Moxie recently. I actually sat down next to somebody in a plane who was taking Moxie and that person was looking at my presentation. I was doing my presentation in the plane and they asked, Oh, do you know Moxie?

Changhan David Lee

1:34:13 - 1:34:24

And I was like, well, yeah. And they told me their experience, which I think is consistent with what I’ve heard a lot from other people. But basically it doesn’t make you superhuman. It doesn’t make you stronger or faster.

Changhan David Lee

1:34:25 - 1:34:49

What people kind of feel is that the recovery time between the intervals or the exercise goes down, which means it’s easier to get back on, which is kind of what is consistent with the immunometabolic side of these things. the macrophages that go to your muscle, they have to signal and clear out the debris. Yes. And they have to resolve that.

Changhan David Lee

1:34:50 - 1:34:54

So you can go back and do it again. Yeah, yeah, yeah. That’s going on with Moxie.

Nick Jikomes

1:34:55 - 1:35:14

Got it. So it could be that. Yes, it’s allowing for sort of more efficient recovery. If all of these things are true and the results you had in the diabetes paper are what’s happening in humans who are exercising, it could be that you’re shifting the profile of immune cells that come in in the recovery phase so that you’re just cleaning up the debris

Nick Jikomes

1:35:14 - 1:35:20

more efficiently and not calling in some of those other types of immune cells that might not be aiding in the recovery.

Changhan David Lee

1:35:21 - 1:35:37

Right. So, so, uh, in a way clarifying the mission. Yeah. And that’s why in the older mice, when we injected them and they were running better, we were trying to figure out why, and it came down to the muscle that seemed to be the tissue that really responded, but also the liver as well, because it has to be,

Changhan David Lee

1:35:37 - 1:35:55

there has to be a metabolic cross communication. But in the muscle, what we found with all these unbiased omics is that it was regulating proteostasis. So we were really hoping to see a huge shift in metabolism or mitochondrial, you know, whatnot. But the signature was very clear.

Changhan David Lee

1:35:55 - 1:36:09

It was maintaining protein homeostasis as one of the main mechanisms. So it’s multiple things. Immune cells are involved. Like I said, Mod C being a mitochondrial thing will have multiple roles in different types of tissue.

Changhan David Lee

1:36:10 - 1:36:31

So from an immune perspective, yes, the recovery with the clean out and the signals to repair, but at the muscle level, again, the key concept is homeostasis. The protein, you know, protein is really important muscle, right? So maintaining the protein stability is a key thing. to maintain exercise.

Changhan David Lee

1:36:31 - 1:36:49

And that goes down with age. That’s why aging mice, even after a week of injection, we’re already improving in their running. It’s not because we’re making new cartilage or muscle or whatnot. We were actually bringing back the lost communication in the body with age for many, many reasons.

Changhan David Lee

1:36:50 - 1:36:55

We’re kind of bringing back that communication and activating their stress response.

Nick Jikomes

1:36:56 - 1:36:56

Yeah.

Changhan David Lee

1:36:56 - 1:37:01

Yeah. Maintaining their proteins better and their immune system to respond to it.

Nick Jikomes

1:37:01 - 1:37:10

I mean, so I think it is fair to say that, yeah, the aged mice, given MOTC, just had more youthful cellular stress responses.

Changhan David Lee

1:37:11 - 1:37:14

Yes. Yeah. Wow. More youthful endostatic capacity.

Nick Jikomes

1:37:15 - 1:37:27

Yeah. Yeah. Yeah. Given the fact that people are using MOTC, there’s an increasing number of people injecting themselves with MOTC, say what we will about the rules and regulations.

Nick Jikomes

1:37:28 - 1:37:47

The people are doing it. Let’s assume that we’re talking about people who are getting genuine MOTC. What would you have people really think carefully about if they are going to use these things? One thing that comes to mind for me, based on what I’ve heard from you so far, is you certainly wouldn’t want to use something like exogenous MOTC on a chronic basis.

Changhan David Lee

1:37:49 - 1:38:12

Yeah, if I were to do it, I would definitely not do it chronically. The effects are very different. So in the, in mice, we do daily injections and that’s when we see the strongest effect, but it’s a single dose. It’s a single dose a day.

Changhan David Lee

1:38:12 - 1:38:34

And the half-life is pretty short of this synthetic peptide. It disappears in a matter of minutes. And so it’s not like we have a constant surge of cortisol the whole time. It’s almost like that one injection in animals was like a spike, and then it disappears, which is what we want to do.

Changhan David Lee

1:38:34 - 1:38:49

So when we say chronic, I think the focus should be more on keeping that cycle going as any other stress does. But it’s hard to say, you know, how do you determine the regimen? That’s the clinical challenge.

Nick Jikomes

1:38:49 - 1:39:05

Yeah. So yeah, so you have to think about the timing of this on a couple levels. You could do one dose a day or multiple, and you were giving the mice one per day. You could also do many days in a row or a few days in a row, and probably both things are worth thinking about.

Changhan David Lee

1:39:05 - 1:39:29

Yeah. And then when we shifted to the chronic lifelong treatment to see the effect on lifespan, we did three times a week. Got it. Again, the whole idea of the time becoming a biological parameter, a real biological variable, an intensive two-week injection we hypothesized was going to be different from an accumulation of months.

Nick Jikomes

1:39:29 - 1:39:36

Yeah. And remind us, what was the basic result when you did the long-term three injections per week experiment?

Changhan David Lee

1:39:37 - 1:39:53

It was pretty dramatic. We didn’t extend lifespan because we extend that health span of the mice. And you know, lifespan studies take a lot of mice because as you go, mice are dropping. So we were not powered to do a lifespan study.

Changhan David Lee

1:39:53 - 1:40:16

We just did it. Even with a small number of mice, we saw a continuation or maintenance of physical capacity with age. Of course, changes in the body composition. For example, older mice in the really later years, so we’re talking about 30 months, and that’s kind of a human equivalent of today as almost something somewhat around 80.

Changhan David Lee

1:40:16 - 1:40:34

You can’t really run on the treadmill, even mice. So they walk. So we made up this one minute walking test, for example, and most of the control mice did not finish that one minute walking test at that age. But almost every mouse that was left with Mod C completed that.

Nick Jikomes

1:40:35 - 1:40:46

Wow. So they’re not as old and frail looking, and they perform much better on these walking tests. How early in their lives did you start that three times per week injection for those?

Changhan David Lee

1:40:46 - 1:41:17

I was about 24 months of age, so... Middle age. Around So the way we see it is when you take a lifespan curve, the moment when the lifespan starts to drop, because it looks like a waterfall for most species. If you compare that to humans, because it’s hard to correlate mouse age to human age, just using that stage where the mice start to drop and when humans start to drop, it’s right around that area.

Changhan David Lee

1:41:17 - 1:41:21

So we’re talking about, for humans today, somewhere around 60s.

Nick Jikomes

1:41:21 - 1:41:23

Yeah, so later middle age, yeah.

Changhan David Lee

1:41:23 - 1:41:27

Yeah, which now is later in middle age, because we live up to almost 90 now.

Nick Jikomes

1:41:27 - 1:41:33

Yeah, yeah. OK, so you’re improving. You may or may not have improved lifespan. You just weren’t empowered to see that.

Nick Jikomes

1:41:33 - 1:41:39

But you were definitely improving health span. They were behaving in a more youthful way much longer in their lives.

Changhan David Lee

1:41:40 - 1:41:49

Right, right. And it’s pretty amazing to see those 30-month mice on a treadmill still walking. They could go more than a minute. We just stopped it because we called it a minute test.

Nick Jikomes

1:41:49 - 1:42:02

Yeah, yeah. Wow. Little time that we have left, I want to ask you, so MOTC is very fascinating. You’ve done a lot of great work there, and I’m sure there’s more to come that we’ll be able to talk about before too long here.

Nick Jikomes

1:42:03 - 1:42:13

The world of mitochondrial peptides in general, how many of these things do we know about? Do you think there’s a lot more to be discovered? What do you think the landscape of mitochondrial peptides is going to start to look like?

Changhan David Lee

1:42:14 - 1:42:41

So we have another one that we’re preparing to publish soon. So for sure, there’s one more. But Hasi’s lab, Dr. Cohen’s lab has discovered many other ones. Right now, there’s about a dozen out there of these mitochondrial peptides, some that we have discovered, my lab and Hasi’s lab.

Changhan David Lee

1:42:42 - 1:42:54

But other people have found mitochondrial peptides as well. They’re a little bit bigger than ours, but still, you know, it falls into that category. But bioinformatically, there can be hundreds potentially.

Nick Jikomes

1:42:55 - 1:42:56

Yeah. Yeah.

Changhan David Lee

1:42:56 - 1:43:07

Which really is crazy because that almost doubles our proteome. If we start including these little peptides, yeah There’s a ton, right, right.

Nick Jikomes

1:43:07 - 1:43:25

Okay Yeah, so still probably a lot more to be discovered For those listening. So we just did a pretty deep dive on MOTC in this podcast. I’m actually talking to dr. Cohen next week So between those two, well, I’m trying to cover, you know almost everything that’s out there to be known about MOTC so far.

Nick Jikomes

1:43:26 - 1:43:43

So if you like this episode, I would pair those together because we’re going to talk a lot more about MOTC next week. What is on the horizon for you? What are some experiments that you guys are doing today that you want to alert us to where you think we’re going to learn some really interesting stuff about MOTC or other mitochondrial peptides in the near future?

Changhan David Lee

1:43:45 - 1:44:08

Um, well, there’s so many, I mean, my, my lab is almost like a collage of topics because, you know, we basically just follow the data and it takes us to these really wild places. Um, aging is the ultimate goal for us. And mitochondria are just mitochondrial communication is just a, an emerging field. And there’s so many going on.

Changhan David Lee

1:44:08 - 1:44:21

It’s a, it’s almost. more exciting than science fiction. George Lucas, who made Star Wars, if you’re a Star Wars fan, you would know midichlorians. It’s like the source of the force.

Changhan David Lee

1:44:23 - 1:44:40

There’s a scene, the story goes that George Lucas learned about mitochondria while he was making Star Wars, and he thought it was crazier than fiction. So he had to put it in a series, and they call it midichlorians. And it actually has Liam Neeson read from the textbook. And I think there was so much insight there.

Changhan David Lee

1:44:40 - 1:45:00

I don’t think he understood this, but he somehow said it. In the movie, Liam Neeson sits down and kneels down and reads from the textbook saying, midichlorians are intelligent life forms that live within ourselves. And these are words in the movie, in the cells. And we are symbionts with them.

Changhan David Lee

1:45:00 - 1:45:19

And he says something crazy. He says, they continuously talk to us to provide the source of the horse, basically. And when I heard that, I didn’t really realize it. But going back now, the communication the talking part and the intelligent form.

Changhan David Lee

1:45:19 - 1:45:38

I think that’s where we really want to focus on. How much does the mitochondria get involved in the computation of these environmental switches, right? And how do they process that? And how do they communicate that to the cell?

Changhan David Lee

1:45:38 - 1:45:48

That whole cycle, I think, is going to be really important to understand this. And it’s not just going to be metabolic. We are focusing more on the immunometabolic interface right now.

Nick Jikomes

1:45:49 - 1:46:08

Yeah. Are you generally what’s your general high level take on the realm of peptides as a sort of new? I don’t even want to say treatment modality because people aren’t necessarily using these only to treat problems. They’re using these to increase their youthfulness or vitality or whatever you might want to say.

Nick Jikomes

1:46:08 - 1:46:22

Are you generally excited about the prospect of peptides as an emerging class of substances that human beings are going to use for health-related reasons? Or do you think we’re getting a little overblown with the excitement on these things?

Changhan David Lee

1:46:23 - 1:46:45

No, I think peptides have so much potential. I mean, I don’t recommend taking Monsi right now, just because we don’t understand it fully. And there’s always dangers associated when you enter unknown territory. But I think what’s going on right now in this country is a reflection of that demand.

Changhan David Lee

1:46:46 - 1:47:25

people don’t want to wait until they get sick to be prescribed the drug. I think the supplement field has demonstrated very clearly that there is an intention in a market of preventative medicine, a sort of a vaccination against aging kind of situation, where you want to do something to slow down the process. And I think these peptides are great because for many reasons, but like I said before, smaller peptides don’t have a huge specificity. A lot of the drugs out there that are protein based are very specific and that’s how FDA approves it.

Changhan David Lee

1:47:26 - 1:48:09

But the specificity comes with a lot of side effects because you are going to do something dramatic, you know, go and clog some pathway very, very significantly. But like I said, these peptides typically won’t have that level of specificity, but they’ll be more involved in the general flavor of responses, which means it’s more going to be in the optimization, fine tuning of major processes rather than clogging or activating that process directly. And so therefore, I think from a longevity drug, a preventative drug, I think peptides for that reason, have a lot of potential. But of course, we need a lot of clinical trials to make sure we understand what we’re doing.

Changhan David Lee

1:48:09 - 1:48:14

So do no harm is the first thing, right? Yeah, we cross that barrier first.

Nick Jikomes

1:48:15 - 1:48:28

All right, well, Professor David Lee, this was a great conversation. You’ve done some excellent work in this realm that I’ve been reading for the last week or so. So thank you very much for your time. And I look forward to seeing what comes out of your lab in the next couple of years.

Changhan David Lee

1:48:29 - 1:48:32

Thank you. Thank you. I really enjoyed our conversation today.

This is a public episode. If you'd like to discuss this with other subscribers or get access to bonus episodes, visit mindandmatter.substack.com/subscribe

Wide release: Available for free by July 22, 2026, on Apple, Spotify, YouTube, etc.

Not medical advice.

Nick speaks with Dr. Rahi Sarbaziha about GHK copper, a naturally occurring tripeptide that binds copper and participates in skin wound repair and collagen regeneration. Sarbaziha notes that levels peak in young adulthood before declining, and she outlines how the compound modulates gene expression tied to repair processes in addition to exerting acute effects on oxidative stress. Central concepts include the molecular properties required for topical penetration, distinctions between immediate antioxidant actions and longer-term remodeling, and trade-offs between topical products and injectable administration.

TOPICS DISCUSSED:

* Endogenous Decline: GHK-copper levels peak in young adulthood then fall with age, reducing natural support for skin repair and collagen maintenance.

* Skin Penetration: Effective topical delivery depends on molecular size under and fat solubility, often improved by procedures such as microneedling that briefly disrupt the barrier.

* Mechanisms of Action: The peptide upregulates fibroblasts and vascular endothelial growth factor, stimulates glycosaminoglycan production, and alters expression of genes involved in tissue regeneration and oxidative stress reduction.

* Lasting Remodeling: Gene expression changes produce sustained collagen rebuilding that develops over weeks to months rather than delivering only transient effects.

* Administration Routes: Topical use is accessible and pairs well with hyaluronic acid for hydration; injectables may reach higher local concentrations but lack extensive human trial data and FDA approval for this purpose.

* Safety & Protocols: The compound is generally well tolerated with mild transient injection-site reactions; typical approaches use daily microgram doses for one to two months followed by off periods.

* Hair & Other Uses: Clinical observations indicate potential for hair shaft strengthening comparable to minoxidil with a favorable side-effect profile, alongside exploratory work in wound healing and neurological contexts.

* Research Needs: Expanded human studies are required to standardize dosing, confirm long-term safety, and evaluate value in neurodegeneration, lung repair, and related areas.

ABOUT THE GUEST: Rahi Sarbaziha, MD is a physician in Beverly Hills, California whose practice focuses on regenerative aesthetics, hormone therapies, peptides, and interventions that support vitality, tissue repair, and overall wellness.

RELATED CONTENT:

* M&M 300 | Peptides & Blood-Brain Barrier in Health & Disease | William Banks

KNOW YOURSELF:

* Visit the SUPPORT PAGE if you find value in this content. Includes discount codes + links to affiliate partners, including products to help you measure and manage your health.

Listen or watch on your favorite platform:

* Audio version: [Apple] [Spotify] [Elsewhere]

* Video version: [YouTube]

PRACTICAL TAKEAWAYS:

* Select topical GHK copper serums formulated as liposomal or combined with hyaluronic acid to aid absorption and support skin hydration.

* Use products for at least four weeks before evaluating changes in texture or fine lines, since collagen remodeling and gene-driven effects require time to appear.

* Pair topical application with professional microneedling when feasible to increase delivery into deeper dermal layers where repair activity occurs.

* Approach injectable forms cautiously due to absent dosing standardization and regulatory approval; cycle usage under clinician supervision if this route is considered.

SUBSCRIBER CONTENT BELOW: Reference paper + episode transcript.

Reference Paper:

* Paper | Copper Peptides in Regenerative Aesthetic Dermatology

AI-generated transcript below. Beware of mistranslations!

Rahi Sarbaziha

02:23 - 02:40

Yes, I’m a medical doctor based in Beverly Hills and I focus on beauty, health and wellness. So a lot of regenerative aesthetics and hormones, wellness, peptides, weight loss, muscle building, just overall vitality.

Nick Jikomes

02:41 - 02:58

Yeah, and so today we are going to talk mostly about peptides and GHK copper with respect to skin health and skin biology. So starting pretty basic here, can you just talk a little bit about the extent to which naturally occurring peptides are an important part of normal skin health and skin biology?

Rahi Sarbaziha

02:59 - 03:14

Yeah, sure. So peptides are naturally occurring in our bodies. And I know like the term peptide, now everyone’s talking about peptides, but they’ve existed in our bodies forever, obviously. And so we have naturally occurring peptides and then there’s synthetic peptides as well.

Rahi Sarbaziha

03:15 - 03:21

Peptides basically act as messengers. So they deliver signals to other cells to perform certain actions.

Nick Jikomes

03:24 - 03:40

And one of the peptides that people are talking a lot about these days, it’s becoming increasingly popular, is called GHK copper. This is apparently an endogenous peptide in our own bodies. But what exactly is this peptide and what is the significance of the copper part?

Rahi Sarbaziha

03:42 - 04:09

So yeah, the peptide part is the GHK and the copper is the ion. And then together is how they are transported throughout the body. So GHK-Cu, so there’s three amino acids and it is naturally occurring within our bodies, GHK-Cu. And studies have shown that it has so many different benefits for hair, skin, nails, but not just that, it goes even deeper.

Rahi Sarbaziha

04:09 - 04:30

There’s benefits associated with neurologic and even tissue repair internally, lung repair. So it’s not just an aesthetic thing, GHKCU. So GHK stands for glyceol histidyl lysine. So three amino acids together and then with the copper make up the peptide GHKCU.

Rahi Sarbaziha

04:30 - 04:30

Yeah.

Nick Jikomes

04:31 - 04:39

And so it’s an endogenous peptide, it’s inside of us. And my understanding is basically it peaks in young adulthood and then declines with age.

Rahi Sarbaziha

04:41 - 04:48

Exactly. And that’s just basically almost with every peptide that’s occurring in our bodies. It peaks and then it declines.

Nick Jikomes

04:48 - 04:56

And do we have a good sense for what endogenous GHK is actually doing specifically within skin in particular?

Rahi Sarbaziha

04:58 - 05:12

It participates in wound repair, participates in collagen regeneration, it helps with angiogenesis, which means blood vessel formation. So those are the main things that GHKCU participates in.

Nick Jikomes

05:15 - 05:28

There’s many different ways that people use skincare products and stuff. With the peptides in particular, there’s topical application of some of these peptides like GHK copper. There’s also injectable versions. I want to get to some of that.

Nick Jikomes

05:28 - 05:53

But stepping back for a second, when we just think about topicals for skin health, facial creams, moisturizers, all of that stuff, it’s a huge industry. Lots of people use lots of these things. When we put stuff on our skin, how much of that is going to get into the skin and what are the major factors that determine how much of the active ingredients can actually penetrate the skin barrier and get into our tissues?

Rahi Sarbaziha

05:55 - 06:20

Well, there’s so many different factors that can actually affect that. And it depends on a various person and also what you’re putting on your skin. So the size of the particle itself. So for example, if it’s less than 500 Daltons, it should be able to be penetrated within the skin and then the ability to be lipophilic.

Rahi Sarbaziha

06:20 - 06:33

And then other factors too, like how, you know, if there’s any medication affecting the skin, if you’ve put on any different type of solvents or anything that can help your skin penetrate it. So it varies for sure.

Nick Jikomes

06:33 - 06:41

Yeah. But basically the size of the matter, smaller things will tend to penetrate better. Yes. Solubility and how much they dissolve into fats matters.

Rahi Sarbaziha

06:42 - 06:50

Exactly. So exactly. And it varies person to person and it varies product to product. Exactly.

Nick Jikomes

06:51 - 07:01

For GHK copper in particular, when it’s topically applied, does that have good penetration or does it require like other factors to be alongside of it to help it penetrate the skin?

Rahi Sarbaziha

07:03 - 07:32

It’ll penetrate because it is a small enough molecule. But it is better if it’s combined with an aesthetic procedure, such as microneedling, fractional laser, really anything, even microcurrent, injection, which we’re talking about mostly topical right now. But it is better if it’s combined with a procedure that kind of compromises the skin barrier to allow the GHKCU to penetrate deeper.

Nick Jikomes

07:33 - 07:50

And so from your role, you know, working directly with people that are using GHK copper peptides topically and other related things in general, what are people using this for? And in general, where do we tend to see the benefit with respect to skin biology and skin health?

Rahi Sarbaziha

07:51 - 08:09

The thing about GHKCU, it has so many different benefits for skin and hair. It helps with fine lines, wrinkles, pigmentation. It’ll help thicken the skin. It’ll also help strengthen the hair shaft if used for hair growth.

Rahi Sarbaziha

08:09 - 08:19

So there’s multiple benefits. So in general, most people use it as an anti-aging tool in their skincare toolbox.

Nick Jikomes

08:21 - 08:29

And so this is how people are actually using it. Is there any clinical data on this where, you know, people have carefully studied it in a clinical trial setting or anything like that?

Rahi Sarbaziha

08:31 - 08:43

Actually, there’s so much. There’s so much on THKCU. When I was writing my article on basically, it was a meta-analysis of all the different articles out there on copper. And it was fascinating.

Rahi Sarbaziha

08:43 - 09:01

There’s so much out there. There’s so many studies that show how effective it is for any type of um, anti-aging, but also wound healing as well. There’s a ton of studies that support the use, um, either in conjunction with hyaluronic acid or on its own. It is extremely effective.

Nick Jikomes

09:02 - 09:12

Yeah. You know, I did, I did read that it can work in conjunction with hyaluronic acid. I’m familiar with that term. I’ve seen it, you know, on products and talked about, but I don’t really know what it is.

Nick Jikomes

09:12 - 09:15

What is hyaluronic acid and why is that in so many products?

Rahi Sarbaziha

09:16 - 09:34

Hyaluronic acid is an essential component of our skin, joints, and even our eyes. And it occurs, it’s located within the dermal layer of our skin. And it basically is what keeps our skin hydrated. It attracts water and it helps keep us hydrated.

Rahi Sarbaziha

09:35 - 09:57

So that plump looking you know, like a baby when they have that plump skin, that’s lots of hyaluronic acid. So hyaluronic acid is also used in traditional fillers, you know, in lips, cheeks, et cetera. So hyaluronic acid is a very important component to keeping us looking and feeling hydrated as well.

Nick Jikomes

09:58 - 10:18

Mm hmm. And so I want to get into a little bit sort of the mechanics of what we know is actually going on under the hood here. So GHK copper, when it gets into our skin, when it gets into our bodies, I know that it affects things like oxidative stress and inflammation. Do we know much about what it’s actually doing and what its major mechanisms of action are?

Rahi Sarbaziha

10:20 - 10:34

Well, there are so many different mechanisms of action of GHKT. That’s the thing. I’d have to actually review that because there’s just so many different things that it does. It helps stimulate glycosaminoglycan production.

Rahi Sarbaziha

10:34 - 10:45

It helps upregulate fibroblasts, vascular endothelial growth factor. It works through various different mechanisms. It’s not just one.

Nick Jikomes

10:46 - 10:48

Yeah.

Rahi Sarbaziha

10:48 - 11:09

And that’s why it has so many different effects in the body. And it has so many different benefits in the body because it just has such a wide range of actions. It also modulates gene expression as well with regards to tissue repair. And there’s just a list of different genes that it can affect.

Nick Jikomes

11:09 - 11:34

Yeah, I was doing a little bit of reading ahead of this. And yeah, it seems to have acute effects, like it’s acutely helping with oxidative stress, basically. But it’s also, like you said, affecting gene expression of a bunch of genes. And what’s interesting about that, or where I want to go next with that, is this would imply, I would think, that it has not just acute effects right now, but it could potentially have lasting effects that persist beyond the presence of the actual peptide itself.

Nick Jikomes

11:34 - 11:45

When you see people using peptide for skin care, Do they see effects that sort of outlast the actual peptide being present? Do they use it for a few days or a few weeks and then continue to see changes in their skin?

Rahi Sarbaziha

11:47 - 12:15

Absolutely. That is definitely what will happen when you use GHKCU because the whole point of regenerating collagen production or upregulating gene expression, it’s not going to be something that you apply it and you consistently apply it for, let’s say, a month. And then it’ll work to upregulate these genes and also stimulate this collagen. And that’s going to take months to actually come to fruition for you to notice the difference.

Nick Jikomes

12:15 - 12:33

I see. So this is the type of thing where Despite any acute effects it might have today, you’re going to need to use regular application or administration of the substance somewhere or another, because some of these changes, such as modulating gene expression, take days or even weeks to sort of unfold and reach a new steady state.

Rahi Sarbaziha

12:35 - 12:57

Yes. And that’s just how, that’s basically the whole basis of regenerative medicine and integrative aesthetics is. you know, doing things today and where you’ll see results in three months, four months, six months, up to a year. That’s basically just the premise of the, really like the way aesthetics is heading.

Nick Jikomes

12:58 - 13:09

And so GHK Copper is getting quite a bit of attention recently. It almost seems like a new thing, but apparently it’s been around for quite a while. How long has it been around and known?

Rahi Sarbaziha

13:11 - 13:25

Well, I mean, it’s in our bodies. So it’s been around as long as we humans have been here. But it was discovered in 1973 by scientist Lauren Picard. And he isolated it at GHKCU.

Rahi Sarbaziha

13:25 - 13:30

And that’s how we know of GHKCU. But it’s been occurring in our, it naturally occurs in our bodies.

Nick Jikomes

13:31 - 13:37

Yeah, yeah. So it’s naturally occurring. It’s always inside of us to some degree. It was discovered by scientists in the 70s.

Nick Jikomes

13:37 - 13:38

And so we’ve known about it for decades.

Rahi Sarbaziha

13:39 - 13:55

For sure. Yeah. And I guess maybe I can ask another question, like why is it becoming more popular now? And I think just the realm of regenerative medicine is becoming more popular and people are learning more about peptides and wellness and people are taking more charge of their health and wellness.

Rahi Sarbaziha

13:55 - 14:00

And that’s why things like GHK-C are becoming more talked about.

Nick Jikomes

14:00 - 14:13

Yeah, yeah. And then how critical is the copper ion? Is GHK always GHK copper or is GHK, can it stand alone and be used without the copper? Does that make a difference?

Rahi Sarbaziha

14:14 - 14:34

Well, the whole point is the copper, the GHK is what helps it transport throughout the body. Copper on its own is actually can, if it’s copper on its own without the GHK, they need to work together is what I’m trying to say. If it’s copper on its own without the GHK, then you can have actual oxidative stress with that. It can cause oxidative stress in the body.

Rahi Sarbaziha

14:34 - 14:58

So it’s kind of contradictory. It kind of almost makes no sense, but together as they provide a synergy that provides the benefits. But then there is another compound, it’s AHK-CU, which has been studied, alanil histidyl lysine copper. And that has been studied specifically for the hair benefits.

Rahi Sarbaziha

14:58 - 15:10

Although GHK can also provide a lot of hair benefits too. So I think they definitely overlap, but you want them together, not separate.

Nick Jikomes

15:10 - 15:23

Got it. And so people are using this topically in the form of like serums or creams that you just put on your skin. But they’re also using it in other ways. Peptides, including GHK apparently, are injected.

Nick Jikomes

15:23 - 15:32

Do you have any experience seeing these different routes of administration with patients? Do they make a difference? Do you see different effects if you inject it in the skin versus apply it topically?

Rahi Sarbaziha

15:33 - 15:55

Yes. So I must say, it is not FDA approved for injection. However, people do inject it. And although there aren’t many human studies out there, so this is just from my experience and from the experience of other providers that I know.

Rahi Sarbaziha

15:55 - 16:16

But in general, injectable is going to be a little bit more effective. Well, again, it depends. For example, if you’re doing hair and you inject the copper GHKCU in the scalp, it can be a little bit more effective than topically. But if you’re doing topical, you want to do something like microneedling prior.

Rahi Sarbaziha

16:18 - 16:35

However, unfortunately, there aren’t many human studies on this, or any really, when I was doing my research. There are very few to none. And in addition to that, it is not FDA approved, but are people doing it? Yes.

Nick Jikomes

16:35 - 16:48

Yeah. Yeah. People, yeah, people are definitely doing it. I would imagine, right, if you’re just injecting it under the skin, I mean, it’s, you’re getting more of it down into the skin, you know, topically applied, no matter how it is, it’s probably not all penetrating.

Nick Jikomes

16:48 - 17:10

So the next question I have about injections versus topical is, Is the effect highly localized or when you inject it, is it also going systemically? And is there any reason to think, is this working mostly in the skin or is it going to be having effects on other tissue systems that when you inject it, it’s going to go systemic and potentially have effects outside the skin and other tissue systems?

Rahi Sarbaziha

17:10 - 17:29

It’s for sure systemic as well as localized. A lot of times when you inject peptides, let me give you an example of a very well-known peptide that most people don’t even know is a peptide. For example, insulin, you know, a type one or even a type two diabetic will inject it. They don’t necessarily inject it into their pancreas.

Rahi Sarbaziha

17:30 - 17:49

They’ll inject it to the back of their arm or their abdomen, but then the effect is systemic, right? And so that’s the same for other peptides too. However, Some people will inject it in the area of concern because it will... first go through that area.

Rahi Sarbaziha

17:49 - 17:53

So for example, the hair, the scalp, you’ll inject it in the scalp, but it also goes systemically as well.

Nick Jikomes

17:53 - 18:00

Yeah. So yeah, it makes sense. It’s higher at the site of injection, but it will go systemic. Lots of peptides work this way.

Nick Jikomes

18:00 - 18:21

Makes perfect sense. And then when I kind of think about the basic biology that we briefly discussed, and I’ve looked into a little bit, GHK copper is having effects on oxidative stress pathways, on inflammatory pathways, and copper itself is really important for just metabolism in general in all of ourselves. So I would imagine that GHK copper is probably having effects anywhere that it goes in the body.

Rahi Sarbaziha

18:22 - 18:23

Anywhere, exactly.

Nick Jikomes

18:23 - 18:30

Yeah. Do we know about any specific effects or benefits it’s having outside the skin that are worth talking about?

Rahi Sarbaziha

18:32 - 19:08

There are actually a lot of studies showing that copper GHKCU can help with other conditions. I kind of went over it a little bit earlier. But certain cancers, specifically when I was writing this article, I really did focus on the aesthetics and wound healing portion of GHKCU. If you look at the research being done, there’s a lot of benefits for lung disease, specifically COPD, certain cancers, specifically colon cancer, but there are other cancers out there.

Rahi Sarbaziha

19:08 - 19:20

So there are a lot of different disease states that GHKCU can benefit, but obviously we have to be mindful not to overdo it as well, because then there could be copper overload.

Nick Jikomes

19:21 - 19:32

Right, right. Interesting. So yeah, I wanted to ask you about that. Is there any, is there any risk of side effects or negative effects from disrupting copper homeostasis or delivering too much copper via GHK?

Rahi Sarbaziha

19:33 - 20:07

I think it’s in, again, there’s really no studies showing, um, showing this because there just unfortunately are not very many human studies and there should be because there’s so much potential for peptides and there’s so much potential for specifically GHKCU. But there is a disease called Wilson’s disease that has an issue with copper metabolism. But I think that is you have to have this liver issue where you can’t metabolize it. Otherwise, I think your body will metabolize it.

Rahi Sarbaziha

20:08 - 20:25

And with peptides, you cycle on and off. So you’re not going to be injecting it every single day, 12 months out of the year. You’re going to cycle on for like one or two months and then you’ll cycle off, give your body a break. And so that So I haven’t really heard of anyone overdosing.

Rahi Sarbaziha

20:25 - 20:34

That’s just theoretical. And also knowing that there is that Wilson’s disease where people can’t process copper well in their bodies.

Nick Jikomes

20:34 - 20:41

Got it. Got it. So there’s a theoretical potential here, but no one’s really observed anything yet. but it’s also not particularly well studied in this regard.

Nick Jikomes

20:41 - 21:00

On the topic of cycling and injection protocols, can you just say a little bit more about that? It sounds like you said, typically people are going to be using this for one or two months at a time. What would be the approximate dose and number of injections per day and the total length that would be a typical cycle that someone like you sees with the people you work with?

Rahi Sarbaziha

21:03 - 21:32

So the dosing isn’t really standardized for copper. I would have to like review the dosing protocols to be honest, because I don’t have the dosing on the top of my head and is not standardized. But it is usually injected in micro doses, like micrograms daily, usually five days out of the week. You take a few days off and then you’ll cycle on for one to two months and then you’ll cycle off.

Rahi Sarbaziha

21:33 - 21:36

Give your body a break for a few months and then you can cycle back on again.

Nick Jikomes

21:37 - 21:37

Yeah.

Rahi Sarbaziha

21:37 - 21:39

That’s usually how it goes with peptides.

Nick Jikomes

21:39 - 21:46

Yeah. And so, yeah, this is pretty typical peptides, right? They’re basically degraded by the body quickly. So people are doing daily or near daily injections.

Rahi Sarbaziha

21:48 - 22:00

Yes. Injections, topical. I’ve heard of transdermal, although, I mean, not transdermal, sublingual, but I don’t have much experience with that.

Nick Jikomes

22:00 - 22:18

Yeah, I have seen, you know, apparently people are marketing oral GHK copper products. So they’re sublingual, if you let something absorb, you know, in your mouth. For GHK copper, or any other peptide, but GHK in particular, is it likely that the oral route is going to work really well, that GHK can actually survive the stomach and the gut and all that?

Rahi Sarbaziha

22:19 - 22:43

Well, if it’s sublingual, it actually bypasses the gut. It goes directly into the bloodstream because it’s sublingual. So if it’s a pill that you swallow, that goes through the stomach and that has very low bioavailability, just like any supplement that you take. Orally is very low bioavailability because you’re going through the gastric acids, the gastric juices in your stomach.

Rahi Sarbaziha

22:45 - 23:23

And that’s why supplements, you have to usually take them on a daily basis versus like IV, you can do like once a month, et cetera. But again, the issue is that there’s just no studies, no human studies. It’s just people will just create these products and other people will take them, but there’s really no studies to say how effective they are or not. It’s a shame because I know copper has so much potential and there should be, because it has a lot of potential for a lot of different, not just aesthetics, but cancer and diseases like chronic lung diseases.

Rahi Sarbaziha

23:23 - 23:35

Oh, and I forgot to mention, there’s a lot of studies on the neurological benefits, like diseases like Alzheimer’s, Parkinson’s, there’s a lot of studies out there talking about the benefits of GHKCU and these diseases.

Nick Jikomes

23:37 - 23:52

And so walk us through like, what would be like a typical example of someone who comes in and you recommend GHK Copper for them, you know, topically administered or otherwise, when would you sort of recommend GHK Copper rather than some other skin treatment?

Rahi Sarbaziha

23:56 - 24:08

I mean, I really haven’t seen any negative side effects from GHK, ACU, copper, and any skin care. It’s very well tolerated. And that’s the thing about peptides. They’re, in general, well tolerated.

Rahi Sarbaziha

24:08 - 24:18

I haven’t seen any allergic reactions. I haven’t seen. And this is just peptides in general. In general, they’re pretty well tolerated for almost everybody.

Rahi Sarbaziha

24:20 - 24:28

So I wouldn’t know why I would not recommend it. There’s really not a reason why you would not recommend GHK-CU.

Nick Jikomes

24:28 - 24:35

Okay, so you’re not saying a whole lot with respect to side effects, injection site reactions, things like that?

Rahi Sarbaziha

24:35 - 24:40

There is injection site reaction. If you do inject it, it does sting, but it goes away.

Nick Jikomes

24:40 - 24:41

Got it. So the initial, yeah.

Rahi Sarbaziha

24:41 - 24:42

Yeah, the initial.

Nick Jikomes

24:45 - 24:56

I want to ask you really concretely, too, when people inject this, what does it look like? What is actually being injected? What is the solvent that the peptide is actually dissolved in? Is it water?

Nick Jikomes

24:56 - 24:57

Is it something else?

Rahi Sarbaziha

24:58 - 25:03

Bacteriostatic water. You usually mix it with bacteriostatic water. And the peptide itself is usually blue.

Nick Jikomes

25:04 - 25:11

Got it. Yeah, it does have this quite interesting, vibrant blue color. I’ve seen it. So basically, bacteriostatic water, you’re basically just sterile water.

Rahi Sarbaziha

25:13 - 25:18

Yeah, bacteriostatic I think is a little bit different than serowater, but yeah, yes.

Nick Jikomes

25:18 - 25:22

So injection site reactions tend to be mild and they tend to be transient.

Rahi Sarbaziha

25:23 - 25:29

Yes, yes, but specifically out of all the injectable peptides, I think copper is the one that stings a little bit the most.

Nick Jikomes

25:30 - 25:32

Got it, so that’s relatively common, a little bit of a sting.

Rahi Sarbaziha

25:34 - 25:45

Yes, and then overall it’s well tolerated and no, I haven’t seen any systemic, But again, I haven’t done any studies on it. This is just what I’ve observed short term.

Nick Jikomes

25:46 - 26:01

Yeah. Yeah. And like, give us a sense for, I mean, how many people out there, I mean, are more, are you noticing a lot more people using GHA copper and peptides in general for skincare? Or is this a pretty niche thing that not that many people are doing?

Nick Jikomes

26:01 - 26:03

What is your sense of that as a clinical person?

Rahi Sarbaziha

26:05 - 26:24

Um, it, people are using it more and more and more every single day, especially in combination with other peptides. There’s a lot of different sort of combos that people are approaching me about like glow and glow. Let me see glow. See, I don’t even know all the combos because it’s just,

Nick Jikomes

26:25 - 26:29

Yeah, there’s just so many combinations of peptides and new ones all the time.

Rahi Sarbaziha

26:29 - 26:47

Yeah, like the GHK, BPC, TB500 combo, and then the CLO, which I recently, somebody told me about, which is the BPC, TB500, GHK, and KPV. So yes, there’s a lot, a lot of different, people are on it more and more every single day for sure.

Nick Jikomes

26:48 - 27:01

And how common is it for people to use this for and see benefit for hair growth purposes? Are people like literally using this to help their hair grow back in after they’ve started to go bald? Or how are people using it in that regard?

Rahi Sarbaziha

27:03 - 27:18

We’re not using it as much as they should be. They should definitely be using it more, I think. Studies show that it can be just as effective, if not more, than minoxidil. And minoxidil has probably more side effects than GHKCU.

Nick Jikomes

27:19 - 27:20

What is minoxidil?

Rahi Sarbaziha

27:22 - 27:42

Minoxidil is the medication that’s used for hair. You can take it orally. You could also do it topically and also it can treat blood pressure as well. It’s an oral FDA approved medication, but also it’s topical and they also have it in a foam option as well.

Rahi Sarbaziha

27:43 - 27:48

But actually I think it’s also known as Rogaine. I’m sure you’ve heard of that.

Nick Jikomes

27:48 - 27:50

Oh, yeah. That’s Rogaine. Okay. Got it.

Rahi Sarbaziha

27:51 - 27:59

Yeah. Yeah. Yeah. So I’m not still I guess it can cause like low blood pressure if you take it orally.

Rahi Sarbaziha

28:00 - 28:08

Both men and women can use it. But I think copper has less side effects, negative side effects.

Nick Jikomes

28:08 - 28:27

Yeah. And so, you know, we mentioned earlier, peptides generally have a short half-life. So, you know, when you’re cycling a peptide or using a peptide, you might be using it for some number of weeks, but injecting relatively small amounts on a daily basis or, you know, majority of days per week. Can you say a little bit more about a dose response and anything that we know there?

Nick Jikomes

28:28 - 28:38

You said the doses are not standardized, but do people use a wide range of doses or relatively narrow range? Do we see different effects with different doses or is that unknown?

Rahi Sarbaziha

28:40 - 29:04

I think for humans it’s unknown. There are some doctors that have created protocols that people follow, but I don’t think there’s studies specifically looking at human dosing. I think a lot of the studies are through mice and that’s the issue. But in general, it’s like micrograms per day, like 100 micrograms, 200 micrograms.

Rahi Sarbaziha

29:06 - 29:17

per day for like five or six days a week depends. Some protocols are a little bit more aggressive. They want you to inject it twice a day. And you’ll do it for like two months, five days a week, six days a week.

Rahi Sarbaziha

29:17 - 29:34

But there’s a lot of confusion out there because it is confusing because there’s no standardization for sure. Some people are injecting things daily into themselves and some, you know, are just continuously on it. So, I guess standardization would be very helpful.

Nick Jikomes

29:35 - 29:52

Yeah, yeah. So, you know, on the one hand, you know, you’ve said some of these things are not FDA approved, you know, injection of GHK, you know, peptides are a little bit up in the air right now. A lot of people are using them, even though they’re not, you know, officially approved in different ways. The laws and regulations around that might be changing in the near future.

Nick Jikomes

29:53 - 30:19

Given all that, but given also that GHK copper in particular is a natural peptide, our bodies make it, you know, they’re set up to handle it, so to speak, how much risk reward, like, how would you advise someone out there who’s curious about this type of thing in terms of risk reward? Is this a relatively low risk thing to try, because it’s an endogenous compound, because it’s broken down relatively quickly, and you can get, you know, quick feedback on it? Or is there some risk to these things right now?

Rahi Sarbaziha

30:21 - 30:28

It’s just, I’m scared to even give, because I feel like it would become medical advice if I say that, say anything specific.

Nick Jikomes

30:30 - 30:35

In general, I mean, how do I word it exactly?

Rahi Sarbaziha

30:37 - 31:17

People are injecting every single day, GHKCU. Would I say, in general, studies have shown peptides are Not all peptides, not all peptides, because there are some peptides out there that I would stay away from. And then there are some peptides that can have serious side effects. Based on the studies that I’ve evaluated, it appears to me that GHKCU can be considered one of the safer, less immunogenic, less side effects peptide out there.

Rahi Sarbaziha

31:18 - 31:23

But this is just based on the research that I’ve done, not based on studies.

Nick Jikomes

31:23 - 31:28

No, that sounds fair. That sounds totally fair. What are the ones that you would stay away from, clearly?

Rahi Sarbaziha

31:29 - 31:50

Well, the one that scares me the most is Melanotan too. The one that causes tanning in the skin. Some people swear by it and say it’s super safe, but it does darken the moles of your body as well. And there’s case studies linking it to melanoma.

Rahi Sarbaziha

31:51 - 31:52

That’s what I’m concerned about.

Nick Jikomes

31:52 - 31:57

Got it. Got it. So, you know, it’s kind of like the Wild West right now. A lot of people are using these things.

Nick Jikomes

31:57 - 32:09

There’s some research, but maybe not big clinical trials. It’s kind of a gray area. And, you know, there could be risk there. We don’t really know how these things work, at least some of them on a deep level.

Nick Jikomes

32:09 - 32:12

And some of them are potentially quite profound effects.

Rahi Sarbaziha

32:13 - 32:40

Yeah, exactly. And you hear things like, and I don’t know if it’s true, but I heard about people injecting peptides that were unregulated and people going into anaphylactic shock. I’ve never seen anyone experience that or had anyone or know of anyone, but I’ve read about it on the internet and I don’t know if it’s true because it wasn’t even, it was like more on social media than it was in the news. So I don’t know.

Nick Jikomes

32:40 - 32:41

Right, right.

Rahi Sarbaziha

32:42 - 33:19

Instead of the FDA banning it, there should be more regulation and studies because people are going to use it anyways. They’re going to use them anyways. So there’s no reason to start banning things and making things difficult to access. For example, I have been researching GHKCU for quite some time now and I have been posting about it on my social media with like sources backed up and lots of, you know, it’s all like from legitimate sources, legitimate scientific sources.

Rahi Sarbaziha

33:19 - 33:27

And for whatever reason, Instagram didn’t like it. And they’ve like kind of blocked part of my account, which makes no sense to me.

Nick Jikomes

33:29 - 33:46

So let’s think back about GHK Copper topically. And let’s just assume we’re talking about someone who’s going to buy a GHK Copper topical product, a cream of some kind online. So they’re not necessarily going to come in and get a procedure from someone like you, microneedling or whatever. They’re buying these things online.

Nick Jikomes

33:46 - 33:52

There’s many products with GHK Copper in them. There’s different ones out there, different concentrations, different formulations, and so forth.

Rahi Sarbaziha

33:53 - 33:53

Yes.

Nick Jikomes

33:53 - 34:10

How much variability do you think there is out there in the quality in terms of how well the GHK will penetrate? Could someone just kind of get any GHK Copper Cream and it will probably be reasonably well effective and absorbed? Or is that going to vary a lot depending on the particular product and brand that they actually use?

Rahi Sarbaziha

34:10 - 34:28

Yeah, of course. It’s going to definitely vary like the way that a product is made, if it’s made more lipid soluble, what other ingredients are in the product that can help the copper penetrate into your skin. So yes, for sure. I mean, there’s so many products out there.

Rahi Sarbaziha

34:29 - 34:42

It’s hard to, I wouldn’t even know where to begin to, you know, I just have to specifically look at the product itself and look at the ingredients and see if this product is good or not. But there’s so many products out there.

Nick Jikomes

34:42 - 34:53

Yeah. Are there any indicators in that regard? You hear about micronized products, you hear about liposomal products. Are these things people can look for and should look for on the packaging?

Rahi Sarbaziha

34:55 - 35:11

Liposomal, definitely there’s studies that show, and I think that was in my article too, where there’s for wound care, liposomal encapsulated GHKCU has been shown to help with wound healing. So that’s definitely a good term to look at.

Nick Jikomes

35:12 - 35:16

And that basically means like the GSK is sort of wrapped in a little bubble of fat, right?

Rahi Sarbaziha

35:17 - 35:30

Exactly. And then that can penetrate better into the skin. And I think a lot of the skincare out there has it like that, but I can’t be sure. I’d have to look at the labeling and see how it comes.

Nick Jikomes

35:31 - 35:39

Yeah, yeah. And just to repeat something I think we mentioned earlier, there is evidence in the literature apparently that GHK copper and hyaluronic acid pair well together basically.

Rahi Sarbaziha

35:40 - 35:52

They do pair well together, exactly. And then there’s studies that show that GHK-CU can actually be as effective if not more effective. But yes, they’re very synergistic. They work very well together.

Rahi Sarbaziha

35:53 - 36:07

So combining GHK-CU with a hyaluronic acid will be very effective. And I’ve actually used products like that. There are some K-Beauty skincare products out there that combine the two that I’ve used in my practice and on myself as well.

Nick Jikomes

36:07 - 36:29

And it makes a lot of sense that, you know, when you look at some of the mechanistic data that I read in your article that you referenced, you know, GHK copper is helping with oxidative stress. It’s basically acting as an antioxidant at some level. It’s tapping into gene expression and modulating pathways that are involved in inflammation. Obviously over time, our body, you know, experiences some amount of inflammation, it experiences oxidative stress.

Nick Jikomes

36:30 - 36:54

Those things are a key component of aging. And when we talk about skin, you know, we’re talking about you know, wrinkles, blemishes, all the stuff that starts to happen more and more as we get older. When you have people come into your clinic and, you know, they’re middle-aged, they’re older, they’re developing wrinkles, or they have a few wrinkles, things like that, and you’re giving GHK copper to them, either topically or an injection, what’s kind of like the time course where they start to see benefits?

Nick Jikomes

36:54 - 37:02

You mentioned earlier that people are gonna cycle on injections for one or two months at a time. Do they start to see effects within a few days or weeks, or does it take longer than that?

Rahi Sarbaziha

37:04 - 37:31

Well, it depends on what you’re talking about. Are you talking about like GHK, CU combined with like a microneedling procedure, for example? So usually that requires at least two to three sessions and it’ll like enhance your results of the microneedling versus if you did just GHK, sorry, if you just did microneedling without the GHK and you did it with just a basic serum, just like a gliding serum, like a hyaluronic acid.

Rahi Sarbaziha

37:32 - 37:43

But I can’t give you the percentage of how much more enhanced it would be. If you’re doing injectable GHKCU, it takes about probably four weeks of consistent use for you to notice a difference.

Nick Jikomes

37:44 - 37:50

Got it. So roughly speaking, you’re talking about a month or so before the typical person really sees the differences.

Rahi Sarbaziha

37:50 - 37:58

Yeah, you’re not going to see things immediately. It definitely takes some time for anything regenerative for it to come into effect.

Nick Jikomes

37:58 - 38:11

Yeah. I mean, it makes sense. Yeah. What else are you generally seeing on the ground in your clinic when it comes to GHK copper, but just peptide therapies more generally with regards to skin health?

Nick Jikomes

38:12 - 38:21

What are some of the trends you’re seeing in terms of what people are starting to use or request more and the types of specific skin conditions that the peptides seem to be beneficial for.

Rahi Sarbaziha

38:26 - 38:48

As I mentioned earlier, a lot of people are doing combo, like peptide stacks. Whether or not I think that’s a good idea, I’m not sure yet. I usually prefer my peptides individual so I know what’s doing what, what’s affecting what. what peptides.

Rahi Sarbaziha

38:48 - 39:00

I really like the growth hormone-releasing peptides like tessamoralin. I feel like they’re very effective for collagen stimulation as well in the skin and overall like vitality.

Nick Jikomes

39:01 - 39:10

So with like growth hormone, normally you might think that would be something like bodybuilder type guys are using, but people use it for skin as well.

Rahi Sarbaziha

39:11 - 39:27

Yeah. If you use it appropriately, you’re not going to look like a bodybuilder. You’re going to just be toned and your skin is going to look better. Your hair is going to be thicker, etc. So if you’re using it appropriately, not excessively, but the bodybuilders are doing other things too.

Rahi Sarbaziha

39:27 - 39:48

It’s not just like the... Some of them are injecting growth hormone on its own, which we don’t actually use anymore growth hormone. I mean, that’s maybe used for like pediatrics if they have growth hormone deficiency, but it’s usually like the growth hormone releasing hormones, growth hormone releasing peptides that we’re using in adults.

Nick Jikomes

39:48 - 39:55

Yeah. The difference is growth hormone itself being injected versus a peptide that modulates growth hormone, secreting hormone.

Rahi Sarbaziha

39:56 - 39:59

Right, you’re releasing your own growth hormone.

Nick Jikomes

39:59 - 40:19

Right, right. So it’s a more naturalistic pattern of growth hormone secretion. And like, obviously a lot of people use cosmetics and moisturizers and stuff for their body, but their face in particular. With GHK Copper, are people injecting it right into their face?

Nick Jikomes

40:19 - 40:21

Is that something people do?

Rahi Sarbaziha

40:23 - 40:34

I’ve heard of people doing that, but it’s more like microneedling and then putting it on topically afterwards. I’ve heard of people doing that. It’s not the standard. Yeah.

Nick Jikomes

40:35 - 40:48

Got it, got it. And so when it comes to things like wound healing and skin remodeling, are people noticing like effects there, like literally like scrapes and cuts healing faster or effects on scar tissue or anything like that?

Rahi Sarbaziha

40:48 - 41:16

I mean, yes, it should. And that’s what all the studies of GHKCU and wound care are. in the wound care world, because that’s like a whole separate world of medicine, but I’m hoping that they’re using GHKCU often because the studies show that it is so effective for wound healing. It helps regenerate collagen and angiogenesis, as I mentioned, and you need all of those things for wound healing.

Rahi Sarbaziha

41:16 - 41:31

You need blood vessels, you need more collagen, you need the hyaluronic acid, you need the vitamin C, you need glycosaminoglycans, proteoglycans, so you need all of that in order to heal the wound better.

Nick Jikomes

41:32 - 41:46

Yeah. And as we mentioned, you know, this is an endogenous peptide, GHK copper. And I mean, in this article that you cite some evidence that it does decline quite dramatically with age. You know, it’s going to go down by more than half comparing like a 20 year old to a 60 year old, say.

Nick Jikomes

41:46 - 42:01

And so, you know, it is one of these things, like so many things that decline with age. So in some sense, it’s the idea here that by using exogenous GHK copper, whether it’s injected topically applied or whatever, you’re basically just restoring some level of what your GHK levels used to be.

Rahi Sarbaziha

42:02 - 42:07

Exactly. That’s exactly what we’re trying to do.

Nick Jikomes

42:07 - 42:23

Yeah. We’ve kind of talked about this a little bit, but do you see any risk with, say, chronic, very long-term use of topical GHK for months or years at a time? Or in general, would you cycle it on and off, no matter how you’re actually administering it?

Rahi Sarbaziha

42:26 - 42:53

I don’t think there’s any harm with topical long-term use, although there’s, again, not too many like human studies, but it’s so well tolerated. I’ve never seen a reaction. Maybe there are reactions somewhere, someone’s seen one, but I have not, and I’ve not heard of any. In general, with peptides, it’s good to cycle on and off.

Rahi Sarbaziha

42:54 - 43:20

And it’s good to do that with skincare in general, with supplements, so your body doesn’t kind of get used to it and maybe not process it as much. But specifically peptides, you want to cycle on and off. But I don’t think that it’s not like a steroid cream, where if you’re chronically using it, it can thin your skin. If anything, it’ll probably maintain it.

Nick Jikomes

43:23 - 43:38

I mean, what would you like to know more about GTA Copper in terms of what clinical trials might show us, in terms of effectiveness outside the skin, in terms of how it’s working? In terms of the research direction that you would like to see, what do you think is top of mind there?

Rahi Sarbaziha

43:39 - 44:17

I really want to see more of the neurological benefits because there seems to be a lot. So things like Alzheimer’s, Parkinson’s disease, I’d like to know how it can benefit patients with cancer or potential cancer prevention. Those are really, really important areas that I think if we can use copper as a therapy, why wouldn’t we? It’s well tolerated, it’s easy to use and it has so many different benefits versus more traditional pharmaceuticals that usually have more side effects.

Rahi Sarbaziha

44:17 - 44:37

So it would be great if there were more studies on those areas. I mean, aesthetics are really important too, because your skin is your first line of defense from the outside world. And to me, aesthetics is not about just vanity. It really is like if you keep your skin healthy, the rest of your body remains healthy.

Rahi Sarbaziha

44:37 - 44:55

And that’s just like a fact. As we age, everything breaks down in the skin. So if we can keep the structure of the skin, then we’ll keep... external insults from entering the body and keep ourselves as strong and healthy as possible.

Nick Jikomes

44:55 - 45:17

Yeah. And it sounds like, in general, sort of what I’m getting from you is you are appropriately cautious but optimistic about peptides. GHK seems to be particularly interesting and low risk relative to some other things that you might advise people not to try quite yet. But what’s your sort of general take on the whole realm of peptides?

Nick Jikomes

45:17 - 45:24

Are you cautiously optimistic? Do you think there’s a lot of potential here? Is it overhyped? What’s your general take on this area?

Rahi Sarbaziha

45:25 - 45:48

I love peptides. I’m definitely optimistic, but I think there’s a lot of misinformation out there. I think that peptides have been, over the past few years, they’ve just blown up so much that they’re never going to leave. So a lot of times people, a lot of doctors out there don’t know about peptides and they’re very negative towards them.

Rahi Sarbaziha

45:49 - 46:07

That’s the wrong thing to do because Patients are going to be using peptides now indefinitely. So a lot of providers need to be more educated in what peptides are, the benefits, and which ones are actually safe versus not. And a lot of providers make it like a blanket statement, like peptides aren’t safe. They’re not FDA approval.

Rahi Sarbaziha

46:07 - 46:29

That’s wrong. So I just hope that there’s more research out there on human studies on these peptides so that there’s more of a general consensus and there’s more of an approval through the medical community so that people can use them and feel more safe.

Nick Jikomes

46:30 - 46:34

Yeah. For GHK Copper, have you used it yourself?

Rahi Sarbaziha

46:36 - 46:37

Yes, I have.

Nick Jikomes

46:38 - 46:42

How do you use it? Do you currently use it? Is it topically applied? How do you do it?

Nick Jikomes

46:42 - 46:42

Just out of curiosity.

Rahi Sarbaziha

46:44 - 47:05

I’ve done both injection and I’ve done topical and I’ve seen benefits with both. The most recent was Topical. It was a K-Beauty skincare brand that combined copper with hyaluronic acid and I believe niacinamide. And it was a serum and it was amazing.

Rahi Sarbaziha

47:05 - 47:10

It was excellent. I used it all. I kind of want more. I used it all super quickly.

Rahi Sarbaziha

47:10 - 47:14

It was just such a good serum. It really works really well. But yes, I’ve definitely used copper.

Nick Jikomes

47:15 - 47:24

Yeah. Is there anything that you want to reiterate from our discussion today or any final thoughts you want to leave people with about GHK copper peptide in particular, and then just skin health in general?

Rahi Sarbaziha

47:29 - 47:59

I guess more like about peptides in general, I’d like to say, you know, the world of peptides is definitely emerging and physicians need to get on board because patients are already there and they’re going to get the peptides. And no matter what, they’re here to stay. So we need more studies that are supporting its use in humans. GHK, you know, I encourage everyone to at least try out some skincare with GHK.

Rahi Sarbaziha

47:59 - 48:07

And there’s a lot out there. I don’t have any specific brand that, you know, I’m promoting at the moment, but there’s a lot and just try it out and see, cause it’s usually well tolerated.

Nick Jikomes

48:07 - 48:22

Yeah. So pretty well tolerated, not a lot of risk to topical application, maybe things like the liposomal encapsulated products that are probably going to penetrate a little bit better. And then it sounded like you want to try these things for at least about a month or so at a time. before making finals?

Rahi Sarbaziha

48:23 - 48:38

You definitely want to use it for a month or so. And if you’re doing topical, I encourage you to do it with microneedling or fractional laser or some sort of like skin procedure that kind of compromises the barrier so that the copper can penetrate deeper.

Nick Jikomes

48:40 - 48:48

All right, well, Dr. Rahi Sarbazieha, thank you very much for your time. I know I only had you for an hour because you’ve got a new baby at home, so congratulations. I do.

Rahi Sarbaziha

48:48 - 48:52

Aw, thank you. Thank you so much.

Nick Jikomes

48:52 - 48:53

All right, have a good day.

Rahi Sarbaziha

48:54 - 48:55

Take care. All right, bye-bye.

This is a public episode. If you'd like to discuss this with other subscribers or get access to bonus episodes, visit mindandmatter.substack.com/subscribe

Wide release: Available for free by July 15, 2026, on Apple, Spotify, YouTube, etc.

Not medical advice.

Nick speaks with Dr. William Banks about the blood-brain barrier. Banks, who has spent over 45 years studying it, shows that the barrier is a sophisticated, regulated interface that permits bidirectional communication between body and brain. Key concepts include how small lipid-soluble molecules and specific peptide transporters enable entry of signaling molecules, the role of efflux systems, and how barrier changes contribute to aging and neurodegenerative disease. They also discuss practical implications for peptide-based therapeutics and drug delivery to the central nervous system.

TOPICS DISCUSSED:

* Blood-Brain Barrier Structure: Capillary endothelial cells with tight junctions, absent fenestrae, and minimal vesicles create a selective interface; supported by astrocytes and pericytes.

* Crossing Mechanisms: Lipid-soluble small molecules diffuse passively; glucose, amino acids, and regulatory peptides use saturable transporters whose rates vary by need.

* Peptide Transport: Many peptides cross via lipid solubility or specific transporters; rate, not all-or-none presence, determines biological relevance; efflux systems remove some (e.g. certain enkephalins).

* Dynamic Regulation: Transporters for leptin and insulin are modulated by epinephrine, triglycerides, and brain demand; changes occur in aging and Alzheimer’s (reduced glucose uptake, impaired Aβ efflux).

* Circumventricular Organs: Leaky capillary regions allow rapid sampling of blood signals while tanycytes limit spread to surrounding brain tissue.

* Drug Delivery Challenges: Small lipid-soluble drugs, barrier disruption (e.g. focused ultrasound), and “Trojan-horse” strategies each have limitations; intranasal delivery shows rapid brain distribution.

* Peptides & Brain Effects: Gut peptides like GLP-1 analogs, insulin, and ghrelin influence cognition and memory; brain entry predicts central effects.

ABOUT THE GUEST: William Banks, MD is a physician-scientist and leading researcher on the blood-brain barrier. He has focused on humoral brain-body communication, peptide transport, and the barrier’s role in health and disease.

RELATED CONTENT:

* M&M 298 | GLP-1s & Novel Peptides in Obesity, Diabetes & Metabolic Health | Katrin Svensson

KNOW YOURSELF:

* Visit the SUPPORT PAGE if you find value in this content. Includes discount codes + links to affiliate partners, including products to help you measure and manage your health.

Listen or watch on your favorite platform:

* Audio version: [Apple] [Spotify] [Elsewhere]

* Video version: [YouTube]

PRACTICAL TAKEAWAYS:

* The brain receives many gut and fat-derived signals (leptin, GLP-1) that affect appetite, metabolism, and cognition—supporting metabolic health benefits brain function.

* Most small lipid-soluble molecules reach the brain; highly polar or large peptides usually require specific design or delivery methods.

* Aging and inflammation subtly alter barrier function; prioritize sleep, exercise, and metabolic health to support vascular integrity.

* When evaluating peptide supplements or drugs, consider whether they are formulated to cross or act at the barrier; brain effects depend on transport.

SUBSCRIBER CONTENT BELOW: Reference paper + episode transcript.

Reference Paper:

* Paper | Strategies for blood–brain barrier rejuvenation and repair

AI-generated transcript below. Beware of mistranslations!

William Banks

02:24 - 02:41

Sure. I grew up in Southeast Missouri, a country boy, and I fell in love with nature and biology. I admit I’m one of those geeky kids that set up a lab in my bedroom. And you can buy all kinds of chemicals then you can’t buy now.

William Banks

02:41 - 03:09

So I set up a microscopy lab of sorts. Really fell in love with biology and medicine as an undergraduate, and then was lucky enough to go to med school, but decided that science was really what I wanted to do. And so after med school, I did internship and residency and that kind of thing. under the auspices of two really great guys, Abba Kasten and also Andrew Shalley.

William Banks

03:11 - 03:43

By this time, I was interested in how the brain and the body communicated. I almost went into the vagus, but then I got interested in humoral communication. And just about this time, there were a lot of papers that were coming out that all of the peptides that had been found in the gastrointestinal tract were now being found in the brain, for example, cholecystokinin. And all the peptides that had been first found in the brain, like somatostatin, were now being found in the gut.

William Banks

03:44 - 04:08

And some of this work came out of a New Orleans lab with Abha Kastan and Andrew Shally. And Andrew Shally, in fact, had just won the Nobel Prize for the discovery of the hypothalamic hormones. And Abha was also interested, and that obviously connected the brain to the body in that direction by controlling the pituitary. And Abha was interested, did it go the other way as well?

William Banks

04:08 - 04:34

Because he would inject these hormones peripherally and find brain effects even in animals that didn’t have a pituitary. So there was some cross-communication there. So we wound up being interested in the same problem. And the problem is that it was the blood-brain barrier, and everyone said peptides could not get across the blood-brain barrier.

William Banks

04:34 - 05:06

If they could, then that would be a way that you could get signaling from the blood into the brain, or the brain into the blood, and so have brain-body communication. But people said, no, they’re too big. I have no idea why this was such a dogma against that particular topic, but it really was, and still is to some degree in some circles. But so we set it up as sort of a tautology, so that either way the answer would be important.

William Banks

05:06 - 05:41

And the idea was that if peptides could normally cross the blood-brain barrier, then that could be a way that the brain and the body normally communicate. But if they didn’t, then when the blood-brain barrier was disrupted in various diseases, then maybe they could leak across, and that would be a way of brain-body communication during disease states and explain some of the pathology. So with that, we endeavored to determine, do peptides cross? We started out with a small peptide known as peptide, which again, this is back in the 80s.

William Banks

05:44 - 06:44

it had the virtue of we could do various tricks and determine does the full length nine amino acid peptide really get into brain and does it do anything when it gets there and we answered the questions both were yes and then we did some enkephalin work and some other things so after about 10 years of slogging away we were able to say yes peptides can cross the blood-brain barrier and they can do something significant once they get into the brain And then once you accept that premise that there’s a humoral connection between the body and the tissues mediated by substances, circulating substances, you can study anything that you’re interested in. obesity, how do viruses get in, how do they affect the brain, sleep, almost appetite, anything that you can think of that involves peptides in the periphery that have functions, have effects in the brain, you can study all kinds of things.

William Banks

06:44 - 06:48

And so that’s sort of what I’ve been trying to do for the last 45 years.

Nick Jikomes

06:49 - 07:03

Yeah. So brain-body communication, obviously, we’re one integrated unit, the body is. The brain and the body have to communicate bidirectionally. One channel that can happen through is things like cranial nerves, like the vagus nerve.

Nick Jikomes

07:03 - 07:24

Exactly. So nerves that come down from the brain and touch and directly interact with our organs and the rest of the body. Another channel that we knew about a long time ago when you were getting started was the pituitary. So the brain, places like the hypothalamus, the pituitary gland, can secrete substances such as peptides that get into the blood and then go elsewhere.

Nick Jikomes

07:24 - 07:47

And so naturally, the question arose, it sounded like, well, does it go in the other direction? Can things get from the body and the periphery into the brain via the bloodstream? And, you know, that brings us to the topic of the blood brain barrier. So some people I guess thought, no, there’s probably nothing coming from the periphery into the brain because that barrier is a barrier and it doesn’t really let much of anything through.

Nick Jikomes

07:48 - 08:13

And of course, you know, your whole career and some of the things we’ll talk about it makes the answer much more interesting and complex and dynamic than that. But I want to give people some background on the basics as we understand it today before we get into some of the other work that you’ve done that’s been fascinating over the years. From today’s perspective and your understanding and everything that we’ve learned over the years, Let’s just start basic. What is the blood brain barrier?

Nick Jikomes

08:13 - 08:17

And can you just give people a very high level sense for its its general structure?

William Banks

08:18 - 08:58

Yeah, I think there’s two common definitions, a more generalized one and a very specific one, the more generalized one, I think you could say there’s a blood-brain barriers, and that these consist of all the processes you might think about that prevent circulating substances in the blood from getting into the brain. And then the more specific one, blood-brain barrier, is often used to distinguish it from the blood-cerebral-spinal-fluid barrier or the choroid plexus. And in that case, the blood-brain barrier is essentially the capillary bed of the brain.

William Banks

08:58 - 09:33

And some people, most people, would include the immediate arterioles and venules on either side. And as your audience knows, I’m sure very well that in most tissues, the capillary is a little bit leaky. And it’s by that leakage that those tissues get all of the nutrients that they need and that begins a lymphatic system that clears out toxins and all this kind of stuff. But in the capillary bed of the brain, there is no leakage.

William Banks

09:33 - 10:02

There is no production of an ultrafiltrate, a nourishing ultrafiltrate. And that’s great because that keeps all the circulating riffraff out of the brain and makes it a pristine shirt sleeve environment to do whatever it wants to do. But on the other hand, it creates a problem. If there’s no ultra-filtrate, nourishing ultra-filtrate being produced, how does the brain get all the vitamins and nutrients that it needs?

William Banks

10:02 - 10:39

How does it get all the glucose it needs? It’s the most metabolically active tissue in the body, 10 times more glucose being used per gram than almost any other tissues, what’s commonly cited. And the answer then, interestingly, are the blood-brain barriers, the choroid plexus, the vascular blood-brain barrier, and some of the others, like at the circumventricular organs, there’s a tanacitic barrier there, and there’s a couple of other barriers that are very commonly listed now, too. But the major one, and the one that most work has been done on, is the blood-brain barrier.

William Banks

10:39 - 11:20

And I often have to apologize when I’m giving talks that I don’t really know what’s going on with the choroid plexus. I just had it focused someplace in my career, and so I’ve sort of ignored the choroid plexus, although I think that many of the processes that have been found at the blubbering barrier, particularly at the principal level, are also occurring there. And some future reincarnation, maybe I can come back, or some other great scientists can take on and compare and contrast these two barriers. That was very popular in the 70s, but no one’s really doing that right now, I think.

Nick Jikomes

11:20 - 11:32

Well, before we dig into the details on the blood-brain barrier proper in just a moment, let’s just give people a sense for some of the things that you just mentioned. Starting with the choroid plexus, what exactly is that on a very basic level?

William Banks

11:34 - 12:11

So if you think about your brain, you know, there’s a hollow area inside the brain where there’s a lot of CSF and there there’s little bags of tissue, the corte plexus, that produces the cerebrospinal fluid that bays the the outer rim of the brain, the interior, and goes down to the spinal cord. But it’s not just producing cerebrospinal fluid, it’s also dictating what the contents of that cerebrospinal fluid is, and in that way is delivering various substances or allowing various substances to cross.

Nick Jikomes

12:12 - 12:30

So, the choroid plexus is sort of the barrier that involves the cerebral spinal fluid as opposed to the blood-brain barrier, which is the blood vessels carrying literal blood. Yes, exactly. Got it. And then you said circumventricular organs, that might be something we circle back to.

Nick Jikomes

12:30 - 12:31

What are those?

William Banks

12:33 - 13:10

So imagine the blood vessels going through and the capillaries are there and for a very short distance those capillaries decide to have no barrier or a lower barrier. So those vessels become leaky and there’s seven of those in the mammalian brain and There’s all around the ventricles of the brain are near the ventricles hence the name circumventricular as near the ventricles. However, in most cases they are delimited by tannocytes. So they’re true pieces of the brain.

William Banks

13:10 - 13:36

They’re true pieces of the central nervous system. but they have two different modifications. One, that their vessels are leaky, and two, that those leaky areas are delimited by tannocytes, by a tannocytic barrier. So the things that leak into their interstitial fluid don’t necessarily very easily get into the surrounding interstitial fluid of the adjacent brain.

William Banks

13:36 - 13:59

What does that mean? It means you’ve got these little pockets where the central nervous system changes the rules. And they’re all of a sudden intimately exposed to what’s going on in the blood. And it’s interesting that many of these are involved in sort of emergency reactions.

William Banks

13:59 - 14:19

like blood pressure or if you swallow a poison. These are the emetic centers and things like that. I’m sure they do a lot of more things and the CBOs are being studied by a small group of very talented people. So I think that’s an area that’s going to really grow a lot over the next decades.

Nick Jikomes

14:20 - 14:42

So basically, there’s a blood-brain barrier that is regulating and limiting what can go from the blood into the brain. But these circumventricular organs are special or exceptional parts of the brain where that barrier is less strong. And the brain, specific parts of the brain, can more easily sample things that are in the bloodstream.

William Banks

14:42 - 15:13

Right, right. So you see that things get very interactive and complicated and sort of a yin and a yang and for every rule there’s an exception and for every exception to the rule there’s an exception to that rule and this makes studying the blood-brain barrier really intriguing because all of this complexity is fine-honed, you know, it’s beautifully put together. It’s not sort of, oh, Slipshot or something like that.

William Banks

15:13 - 15:42

So if you can wrap your mind around it you begin to see all these opportunities By which the plant brain can play one rule off against the other and get very very sophisticated and how it can have its cake and eat it too, and how it can respond elegantly to communication. And all of these are also opportunities for drug delivery to the brain. We probably will talk about that later.

William Banks

15:42 - 15:47

But the blood brain barrier is the biggest obstacle to developing drugs for the central nervous system.

Nick Jikomes

15:47 - 16:12

Yeah, yeah. So So a central conundrum here from the body’s perspective is how do we, on the one hand, protect the brain and not just let anything in the blood get into the brain, which could be very problematic. But on the other hand, if we build a barrier that prevents bad stuff that we don’t want to get into the brain from getting into the brain, how do we allow nutrients and all of the other stuff that we actually want to supply to the brain, how do we allow them to actually get through?

Nick Jikomes

16:12 - 16:26

And we’re going to dig into some of that. But if we just think very high level to start with here, and you said something interesting, there’s general rules here, and then there’s exceptions to rules. So we’re talking about tendencies. There’s going to be lots of exceptions.

Nick Jikomes

16:26 - 16:46

There’s nothing that’s like ironclad here necessarily. But as a general rule, how How do the basic properties of substances relate to how easily they can get through the blood-brain barrier? Their general size, their charge, their water solubility. How do these things start to factor into whether or not something is more or less likely to pass that barrier?

William Banks

16:46 - 17:21

Right, there are again general rules for which substances can get in and there’s different classes depending on what they use and the big two are saturable, those using transporters and those that aren’t saturable that don’t use transporters. So the first one is small lipid substances in terms of molecular size and molecular weight. Small substances that are lipid soluble can just get into the brain very easily. And we’re talking about things, endogenous things like steroids.

William Banks

17:22 - 17:45

And most of the drugs that we use that get into the brain and have a CNS effect are small lipid soluble molecules. But again, there’s exceptions to that rule. There’s two major exceptions. If you’re too lipid soluble, you’ll get hung up in the membranes, cell membranes, and you won’t be able to partition back into the interstitial fluid of the brain.

William Banks

17:46 - 18:18

So there’s a limit to how, if you’re going to build a drug or build a substance to get into the brain, there’s a limit to how lipid soluble you want to make it. And another big exception is there’s a brain-to-blood efflux system, brain-to-blood transport system, that’s actually in the cell membrane that faces the blood. And it can catch lipid-soluble drugs that are of a particular conformation and spit those out. So that’s sort of the exception to the rule.

William Banks

18:18 - 18:46

Lipid-soluble substances get in, unless they’re too lipid-soluble, or unless they’re a substrate for one of the efflux systems that keep you out. And then there’s exceptions to the efflux system. It’s a regulated system. Yeah, so if you have inflammation or things like that, you can turn it up, you can turn it off, and there’s lots and lots of disease situations where that comes into play and explains how drugs work in patients.

William Banks

18:46 - 18:53

Yeah, so we’ve got size, we’ve got fat or water solubility, in general, smaller things

Nick Jikomes

18:53 - 19:14

will tend to pass through more easily, but certainly not all of them. Small things that tend to be fat soluble are especially likely to diffuse passively through this barrier, but there are exceptions and, you know, it’s sort of like an intermediate level of solubility. You have to be fat soluble and small enough to be able to get through the cell membranes, but not so fat soluble that you get stuck in the cell membranes. Right.

Nick Jikomes

19:15 - 19:32

Perfectly said and so and so those things that can passively diffuse through some of them pass through and then that’s it They’re in other things pass through but they’re immediately pumped back out the brain presumably really doesn’t want those things to be in there Well, actually they don’t even mean depending on the efflux system.

William Banks

19:32 - 20:02

They may not even make it into the brain the The so to back up a bit the capillary bed is is one cell layer thick. It’s amazing this barrier this incredible barrier. It’s just one cell thick. And so that means that if you were in the bloodstream, you would go into the luminal cell membrane and then the cytoplasm for the endothelial cell and then this abluminal membrane.

William Banks

20:02 - 20:23

and then you would reach the interstitial fluid of the brain. So the efflux for P-glycoprotein, PGP, is actually the plasma membrane that faces the blood. So it doesn’t even get into the cytoplasm of the endothelial cell. So this is really a pretty cool setup, I think.

Nick Jikomes

20:23 - 20:34

Yeah, yeah. So depending on size, depending on fat solubility, things might diffuse in. If they diffuse in, they might stay in or they might not. They might get kicked right back out.

Nick Jikomes

20:34 - 20:48

And then there’s things that kind of get actively sensed and transported in. What do some of those mechanisms look like? How does the brain decide or sense something that can’t passively diffuse in, but needs to be actively shuttled in somehow?

William Banks

20:49 - 21:18

Yeah, there’s a whole variety of transporters. Of course, there’s a whole variety of transporters in any cell type. And most of these have been discovered by cell biologists not working on the blood-brain barrier. And so we’re sort of, I mean, if there’s one area I would say of the blood-brain barrier that has needed a lot more attention for decades, It’s cell biology of looking at the brain endothelial cell itself.

William Banks

21:19 - 21:53

But in general, the transporter systems are for substances that aren’t lipid soluble or that you can’t get enough in without some extra boost. And the most famous, of course, will be the transporter for glucose into the brain. And it is going at near max, theoretical max. There’s so many transporters of GLUT1 as it is that almost every molecule that could be transported from blood into brain is.

William Banks

21:54 - 22:26

And that’s one reason that we can look at how active a region of the brain is by doing a PET scan or some other scan and looking at blood flow. Because if a brain area becomes active and it needs more, therefore more glucose, you can’t do it by upregulating the transporters, you can only do it by increasing the blood flow. and therefore making more glucose available through increasing the flow rate. So if you measure flow rate, it’s usually very well coupled to metabolic rate in the brain.

William Banks

22:28 - 23:10

So then there are others. There’s amino acids, and some of those are used in abundance, and they’re not going in quite as fast as glucose, but they’re often going in maybe maybe a tenth of that rate, so the most desirable ones. And again, those are often what we call flow limited, so that if you want more in, you’ve got to really increase blood flow. Then you get to substances that are transported in a much lower rate and this tends to be the regulatory substances.

William Banks

23:10 - 23:53

They’re going in at a very low rate and therefore not affected by blood flow and so therefore have more of a constancy and can vary which region that they’re transported in most by need or something like that. And we’re talking about informational molecules like some peptides, cytokines, growth factors, insulin, GI hormones like leptin, ghrelin, things like that. And in many of these cases, all the work has been done and shown that the rate of entry, the ability to be transported across the blood-brain barrier has effects on brain function.

Nick Jikomes

23:53 - 24:20

Yeah. So various things can get past the blood-brain barrier by different means. They might passively diffuse in depending on their properties, they might be shuttled in through or transported in through some kind of special protein that is specifically designed, so to speak, to get just that thing in, whether it’s glucose or amino acids or some other nutrient. You can also have signaling molecules and regulatory factors, peptides, cytokines, hormones, things like this.

Nick Jikomes

24:20 - 24:28

And they can also get in. And these things have their own ways of getting in. They come through different transporters. They can get in at different rates.

Nick Jikomes

24:28 - 24:51

Something like glucose kind of goes in relatively in abundance and quickly because the brain needs to have some minimum supply of glucose for energy purposes, basically at all times. Amino acids come in a little bit more slowly, and then other things might come in more slowly still. But there’s a number of ways that the barrier can allow different things to come through. That’s true.

Nick Jikomes

24:51 - 25:23

Yes. And so when we think about sort of the basic structure here, so you’ve got a capillary, a tube with blood flowing through it, it’s coming to the brain. Should we imagine a tube that’s being surrounded by basically a very tight network of cells that are specialized to be close together so that not just anything can passively diffuse in and that each contain some pattern of these transporters that will dictate sort of what can get through and how quickly.

William Banks

25:24 - 25:58

Yeah, I think the way to sort of think about it is you imagine a flat endothelial cell and now we wrap it around so it becomes a tube, and now we stick these tubes together. Wherever these tubes come together, they’re cemented together by tight junctions. That’s one of three changes that occur, however. So if we go over and look at a capillary bed, say the liver, which is probably the leakiest capillary bed in the body, We see two other reasons why there’s fluids going across.

William Banks

25:59 - 26:23

There’s actual sort of holes piercing the endothelial cell proper, and those are called fenestrae from the Latin word windows. And indeed, if you look at an EM, they look like cathedral windows in some cases. And they have little membranes, they come in different sizes, you know, nothing simple in biology. There’s If there’s one form, there’s thousands of forms, it seems like.

William Banks

26:24 - 26:39

So there’s various plays on the fenestrae. But ultimately, they are sort of holes piercing through the endothelial membrane. And those things can leak through that. And then there’s vesicles that form and take things across.

William Banks

26:40 - 27:14

And those are missing in the brain endothelial cells. So we have these three modifications that you can that you can only see these, by the way, by electron microscopy. And it’s interesting that the idea of a blood-brain barrier originated in about the 1880s, 1890s, and there was a debate well into the 1960s if the blood-brain barrier truly existed as a physical barrier, or if there was some strange thing, like the brain cells just couldn’t bind dye or something like that.

William Banks

27:15 - 27:31

And the reason is because if you look at the capillary grossly or by light microscopic methods, it doesn’t look like anything special. So people started saying, well, they all look the same. It looks the same as in the liver. Maybe the capillary isn’t there.

William Banks

27:31 - 27:54

Maybe there isn’t a real blood-brain barrier. And in the late 60s, there a guy named Reese with colleagues found looked by IEM and he found all these changes and so okay there is actually a physical blood-brain barrier. So we were on the moon before we knew for sure if there was a blood-brain barrier and I think that’s kind of interesting.

Nick Jikomes

27:55 - 28:15

Yeah. And so, tell us a little bit about the history of this field in terms of what the original ideas are and how those evolved over time as it relates to some of your discoveries. It seemed like in the past, after we established that there was a blood-brain barrier, people often thought it was a very, very strict barrier. Almost nothing got through.

Nick Jikomes

28:15 - 28:30

And over time, you sort of kept discovering, well, actually, some things can get through, and there’s all these different mechanisms of many kinds that regulate this, and it’s actually much more complex and dynamic than we thought. What did sort of the general arc of that discovery history look like?

William Banks

28:33 - 29:06

Most histories will start with Paul Ehrlich. And Paul Ehrlich did some experiments supposedly when he was a student. He was a medical student in Germany and they had to do a thesis. And so it may be that his thesis is that he injected dyes into animals and he found that these dyes, there was a group of dyes, aniline dyes, that went everywhere and stained almost every tissue except for the brain.

William Banks

29:06 - 29:34

So A deeper reading shows that there were other people doing similar things with sort of different approaches. And so probably Ehrlich gets too much credit. But the reason I think Ehrlich gets so much credit is he later became very famous. He won one of the first Nobel Prizes and he’s considered the father of both pharmaceutical sciences and pharmacology.

William Banks

29:34 - 29:56

And famously, he did discover the drug for the first treatment of syphilis. And so he’s really a fascinating story. And so to add one more story onto the legend is so tempting. And I’ve recently I haven’t read this but recently that why is this guy so interested in dyes?

William Banks

29:56 - 30:18

why is it so interested in staining and there then I Think that the answer is this that he was he was developing drugs this was back in the time when when you know, the idea of having a pharmacopeia was sort of new and He wanted how did how do you? discover what a drug will do. Well, probably sight of action.

William Banks

30:19 - 30:46

So if a drug goes to the liver, it’s probably going to have effects on the liver. And the only way in his day that you could determine where a drug went, really, was to see it. because we didn’t have any way, we didn’t have mass spec or anything like that to otherwise detect the substance. So I think that his interest in where dyes go was first the trial of establishing the principle of sight of action.

William Banks

30:47 - 31:12

Anyway, so he found that the dyes weren’t taken up by the brain and the great Ehrlich got it wrong. He thought it wasn’t taken up by the brain just because the brain tissue could not hold on to the dye. He never thought that there might be a barrier. But some other people were injecting bile acids into the brain and said, look, you know, it doesn’t have any effect unless we inject it directly into the brain.

William Banks

31:12 - 31:31

Therefore, there’s something keeping the bile acids out when we inject them peripherally. And there’s some other studies like that. So even though Ehrlich wasn’t in favor of blood-brain barrier, there was some thought going on. And one of his students decided to do a simple experiment and inject the dye directly into the brain.

William Banks

31:31 - 31:48

And sure enough, the brain tissues took up the dye. So now we’re back to a barrier idea. But as I say, it was very difficult to see a physical barrier. And for some reason, people wanted to really see this to be convinced.

William Banks

31:49 - 32:17

And so there was a lot of going back and forth and some early studies from a Russian lab and a little bit later studies from an English lab, a guy named Davson. And they sort of said, well, you know, there’s the choroid plexus now also, and it’s important, and some things are coming through there. And at one point, it was believed that perhaps that was the only important thing. and the capillary bed was it was just mechanical just just a barrier.

William Banks

32:18 - 32:44

And then Daphson showed in the 50s that no it looks like both are important and it looks like that you know the the barrier is important and that it’s at the level of the capillary bed. And so then the studies of Reese and Karnofsky and others with the EM showed, yes, there are these modifications there. Then they looked at the chord plexus. They found tight junctions there as well.

William Banks

32:44 - 33:15

So by this time, by early 70s, the blood-brain barrier was established and it was established that it was these modifications that we’ve talked about. And Then there had already been a lot of work showing the lipid solubility, things crossing, but no one had really looked too much at transporters. There was a little bit of work with electrolytes. And so a guy named Oldendorf came up with a method called BUI.

William Banks

33:16 - 33:48

And this allowed him to look at things that are transported across very quickly. And he said, oh, glucose is transported very quickly and by a saturable system, and amino acids are, and those two are different transport systems. And this began the era of looking at transport systems and some of the early tools to do that. And then Pat Lack, Blasberg, and Fenstermacher came up with a mathematical way of looking at substances that cross very slowly.

William Banks

33:49 - 34:10

And this allowed us to do our work and look at the ability of informational molecules to cross. So that’s sort of the kinetic story. Then there’s a more of a cellular story. Astrocytes and pericytes were discovered, and they are the cells that tell the blood-brain barrier to be a barrier.

William Banks

34:10 - 34:29

So when they come into the brain, and they do so very early. It was a thought that the astrocyte was the main cell that did this, but now we know the pericyte’s at least as important and actually tells the blood-brain barrier to form in the neonate, in the fetus.

Nick Jikomes

34:30 - 34:39

What is this concept of saturable transport? How does that relate to passive diffusion versus active or regulated transport across the barrier?

William Banks

34:39 - 35:09

Yeah, so there’s various ways to be transported by a SATCHable system. So if it’s SATCHable, it says there’s a receptor-like protein or transport protein that’s there, as opposed to diffusion by lipid solubility, which is not SATCHable. So it comes in sort of two big flavors. Does it require energy, in which case that’s ATP or something like that?

William Banks

35:10 - 35:33

Or is it non-saturable, not requiring energy, and that would be a passive diffusion sort of thing? Glucose is actually passive diffusion. It doesn’t require energy to take glucose across. Of course, it takes energy to build a transporter and all that, but it doesn’t take energy to run the transporter once it’s in place.

William Banks

35:34 - 35:46

It’s still saturable, but just doesn’t require energy. And the active transporters are active. Active doesn’t mean saturable. Active means it requires energy.

William Banks

35:46 - 36:03

It is saturable, but it requires energy. Unfortunately, the terms get mixed up and many people who are either novices to the blood-brain barrier or experts but get sloppy often when they talk about transport, they talk about active. but that’s only one type of. Got it.

Nick Jikomes

36:04 - 36:32

So some substances can basically get through the blood brain barrier by some means. And all that’s really dictating that or limiting that is the concentration in the bloodstream versus in the brain and the nervous system. And some things are going through receptors that can be saturated. So there’s this sort of extra regulatory step that is limiting the rate by the rate at which something goes from the bloodstream into the nervous system.

William Banks

36:33 - 36:42

Right, right. And those can be energy requiring or not energy requiring. So that would be passive diffusion versus active transport.

Nick Jikomes

36:42 - 37:06

Yeah, yeah. And then as your career was going and new things were being discovered and debated initially, What were some of the early discoveries around some larger substances that actually do get through the blood-brain barrier, such as peptides, which are basically just multiple amino acids strung together? Are there any interesting examples where maybe there was a debate and some interesting discoveries early on there?

William Banks

37:07 - 37:58

Yeah, in the 80s and even into the early 90s, there was, particularly in America, not so much in Europe, there was a lot of pushback that substances like this could cross. And I’m still mystified why small peptides would have been thought to be too large, because it was pretty much agreed that thyroxin, which is the thyroid hormone that needs to get into the brain, saturated, saturated, transported is about 700 Daltons, about 774 Daltons. And there are peptides that we were looking at that were smaller than that and able to get in, tripeptides and things like that. And so I think it was just that the only example of a string of amino acids that we had was things like albumin.

William Banks

37:58 - 38:27

And we knew that albumin didn’t get into the brain. And so I think that it was just sort of an over-application of what little we knew about blood-brain barrier that was overly applied to another stream of amino acids, the peptides. But yeah, the first one we looked at was delta sleep-inducing peptide. And it had just been isolated from the cerebrospinal fluid of sleeping cats.

William Banks

38:28 - 38:44

And supposedly, if you took this and injected it into the brain of the cats, they went to sleep. I’ve never seen that. I’d love to see that because I’ve had many cats in my life. And, you know, anything you can do to control the behavior of a cat is a mystery to me.

William Banks

38:45 - 39:05

So that would have been a great thing, I think. But so here was a peptide that we knew was circulating in the blood, occurred in the brain as well. and that had a profound effect on cats, on the behavior. And it was nine amino acids long.

William Banks

39:06 - 39:47

as I said before there were tricks we could do so we could demonstrate that that substance would get in as an intact all nine amino acids and and so we showed that and then we had a guy who’s a peptide chemist very rare in the 80s to have peptide chemists and this guy was great David Coy I think he’s still active oh he’s and he did fantastic work And he had made all of these analogs of DSIP, Delta Sleep Inducing Peptide. And so we were able to label those radioactively. And if you use radioactive materials to look, you have really incredible sensitivity.

William Banks

39:49 - 40:10

And these peptides varied in size and composition, lipid solubility, molecular charge. So we did that thing. We looked to see, you know, what’s important here. And what we found is that this class of peptides, they tended to cross, they crossed at different rates, and the best predictor was their lipid solubility.

William Banks

40:11 - 40:37

So that said, okay, although they’re big, almost a thousand Daltons, they’re still able to use this lipid soluble area and get in at sizable amounts. And so then we said, well, what else can we do? So we went to the catalog and we ordered all of the radioactively labeled peptides that were available commercially then. There were about, I think, 18.

William Banks

40:37 - 40:59

And we looked at them with our method, which in those days was before the math of PATLAC. So it was a little crude, but we were able to do that. And we found that a lot of the substances, yes, agreed, the more lipid soluble they were, the better they got in. But there were a set of three that didn’t get in very well at all.

William Banks

41:01 - 41:24

And we looked at them more carefully and we said, oh, they all have a similar structure. They’re less than five amino acids and they have an N-terminal tyrosine. And so we speculated that there was a brain-to-blood efflux transporter for that, for them. And then we set up some methods, because there were no methods really to do this kind of work at that point.

William Banks

41:24 - 41:46

So we figured out a method. to look at that, and we found, yes, there was a brain-to-blood saturable transporter for small internal tyrosines. And that was an interesting group of peptides because it included the opiate peptide class of the enkephalins. And so that was sort of interesting in that way.

William Banks

41:49 - 41:53

So yeah, I can go into whatever happened after that, but I’ll stop there.

Nick Jikomes

41:54 - 42:19

So some peptides can get in, many peptides can get in. I would imagine here, this is an area where we’ve got some general rules, but there’s a lot of exceptions to the rules. You mentioned that the lipid or the fat solubility of the peptides is a major factor. The ones that are more fat soluble generally get in more easily, but there’s key exceptions like the ones you just mentioned that have certain amino acids at one end of the peptide.

Nick Jikomes

42:20 - 42:30

In general, do most peptides get through? Is it really a mix and it depends on the individual peptide? Do smaller ones generally get in more readily than larger ones?

William Banks

42:33 - 43:13

So I would say that most peptides get in, but the caveat is that besides our lab, there aren’t a lot of labs that do this kind of work, and therefore there’s lots and lots of peptides that haven’t been specifically examined. And of course, we’ve examined the ones that there’s a good bet they’re getting in because we we know that they have profound effects on CNS. So at least within that group, most peptides get in, and if they don’t, there’s usually an efflux system for it. So I think one misconception about the blood-brain barrier is that it’s all or none.

William Banks

43:14 - 43:38

It’s either open or it’s leaking, or that a substance gets in or doesn’t get in. But there’s no absolutes in biology. And so it’s much better to think about, and you said your audience likes to delve into the weeds, so I’m gonna go into the weeds here. It’s better to think about the rate at which a substance gets into the brain on a logarithmic scale.

William Banks

43:38 - 44:00

So if you’re talking about glucose, you’re talking about 10 to the minus six microliters per gram per minute, that would be the rate. And if you’re talking about albumin, you’re talking about something like one times 10 to the minus six. So even albumin, a little bit of albumin gets into the brain. It doesn’t get in at the capillary, it gets in.

William Banks

44:01 - 44:34

through another right but the point is it accumulates in the brain and that’s why there’s a little bit of albumin in the cerebrospinal fluid and that and some of the things that this choroid plexus does so that’s a that’s a million-fold difference and then there’s some things that get in even less than albumin have rates lower like IgG molecules and that’s because in addition to being big and not lipid soluble there’s also an efflux system for them so they get in about 10 to the minus 7.

Nick Jikomes

44:36 - 44:57

So instead of asking, does this or that peptide get in or not, perhaps a better way to think about this is, at what rate or to what extent does a given peptide or substance get in? It might be very, very, very slowly and very little, but it’s going to be a spectrum of answers depending on the peptide, depending on the molecule, and so forth.

William Banks

44:58 - 45:18

Right. And so the real question should probably be, Is the permeability biologically relevant? And if it is, under what circumstances? Because it may be important in a disease context, but not normal physiology, for example.

William Banks

45:18 - 45:32

So yeah, that’s really what it should be. It shouldn’t be an absolute, does it get in or not get in? If you have sensitive enough methods, you may very well find it. There’s very few substances that we’ve not found at least a trace, you know?

William Banks

45:35 - 45:41

And so it really has to, but we’ve found many substances that that trace is like irrelevant.

Nick Jikomes

45:41 - 46:11

Yeah, yeah. So this brings me to another set of questions I have, which is, you know, we can think about a substance’s ability to get through the blood-brain barrier, but that itself might change. I want to ask you about how dynamic the barrier is. Can it fine-tune or change how permeable it is to a given substance, both by deciding to do this, so to speak, through mechanisms that are easily to modulate, and also as a function of things like the integrity breaking down over time with age-related decline?

Nick Jikomes

46:11 - 46:24

So starting with how dynamic and regulated the healthy blood-brain barrier is, can you start to give us a sense for what that looks like? How easy it is for the brain to play with the permeability to any given substance?

William Banks

46:25 - 46:46

Yeah, I think this is one of the areas that I really enjoyed investigating the most, because it does show the blood-brain barrier. Probably we should think about it, first of all, to summarize everything we’ve done as an interface rather than a barrier. It’s clearly a barrier, but it’s a barrier plus. So the blood-brain interface.

William Banks

46:47 - 47:06

The blood-brain interface, yeah. So I’m not advocating changing 120 years of nomenclature, but it’s worth thinking about this is not just a brick wall. This is really a dynamic area. And so it’s not surprising then that many of the key transporters are regulated.

William Banks

47:06 - 47:28

And we already talked about how do you regulate glucose. because it’s going through so fast and that’s regulated by blood flow. But the substances that aren’t flow dependent, they get in much slower, they have the ability to be tweaked. And of course, we know that transporters and enzymes and other cell types, enzymes and transporters have very similar math.

William Banks

47:29 - 48:13

describing them and we know those are all you know have regulatory sites and all this kind of stuff so it’s logical blood-brain barrier transporters would too and indeed they do. leptin, for example, we found is which, which is a substance produced by your fat secreted from the fat into into blood. And the you can take a person’s blood level and the leptin level will correlate extremely well with the amount of fat that they have amount of adipose tissue. So, and then that crosses the blood-brain barrier and is one of the things that regulate body weight in a very interesting way.

William Banks

48:13 - 48:37

It doesn’t, it seems that what it does is when you’re starving and leptin levels, and therefore you have less fat, leptin levels drop, that absence of leptin becomes an emergency signal to the brain, you better start looking for food. It’s a little bit like your gas gauge on your car. You’re driving down the road and it’s on full. You got lots of leptin in the blood and in the brain.

William Banks

48:37 - 48:45

You don’t worry about it, but when you look, oh my gosh, it’s almost on empty, it’s the absence of that gas that makes you panic and so it’s the absence of the leptin.

Nick Jikomes

48:45 - 48:53

The absence of leptin is basically telling the brain that the body’s out of fat, i.e. stored energy, so we better get some more energy in and store it up.

William Banks

48:53 - 49:16

Exactly, exactly. So that turns out to be regulated by a couple of interesting things. One is epinephrine, and it tends to increase the transporter, and another one is triglycerides. Triglycerides interfere with the leptin transporter, And that actually makes it, I think, pretty cool evolutionary sense.

William Banks

49:16 - 49:53

We think of triglycerides in our modern world as being associated with high-fat diet and obesity. But actually, if you go back to the hunter-gatherer or something like that, triglycerides go up when you start to starve. Because you’re not eating, so you’re calling on your fat And your fat will break down to free fatty acids and triglycerides, both of which become elevated in the bloodstream. And so what triglycerides are doing then is preventing the leptin from crossing into the brain and accentuating the lack of leptin in the brain and therefore the starvation signal.

William Banks

49:55 - 50:25

So I think that’s an example of a transporter regulation that we can imagine why it’s important. And there are other regulatory systems going on and sort of similarly affecting a peptide transport and that kind of thing. Ghrelin is also affected by epinephrine. Insulin probably has a lot of regulators, but we haven’t really found those.

William Banks

50:25 - 50:36

It varies a lot from experiment to experiment. And so it’s probably having a lot of peripheral substances that are affecting that as well.

Nick Jikomes

50:38 - 51:08

Talk a little bit about how some of these things, leptin, ghrelin, these other peptides and hormones, they can cross the blood-brain barrier in a regulated fashion. To what extent is the brain accumulating those things from them crossing the blood-brain barrier versus sampling at the circumventricular organs? Is it some of both? And I guess related to that, when something like ghrelin or leptin or whatever gets through the blood-brain barrier, how far into the brain tissue can it actually go?

William Banks

51:08 - 51:25

Right. Yeah, I think it’s going to be integrated. And I think we need to study those integrations much better. Again, the circumventricular organs, again, It’s really, remember, it is part of the brain, it’s part of brain tissue.

William Banks

51:26 - 51:54

So there are nerves coming into and out of that and connecting it to other regions of the brain. And yet, so it’s connected by nerves to the rest of the brain. but its humoral interaction is delimited by the by the tannosites. So again a rather complicated system that’s both sequestered from the brain humorally but well connected by neuronally.

William Banks

51:55 - 52:22

And that think that this probably, if I could be imaginative, probably means that a given hormone, such as leptin, can come in through the blood-brain barrier or the choroid plexus, circumventricular organ. All of these rates of entry will be different. The immediacy will be in the circumventricular organ. And so probably that gives the opportunity for an immediate influence on brain function.

William Banks

52:22 - 52:40

which may be different from the later or more sustained entry through the chord plexus or the blood-brain barrier. So those kinds, I think with that kind of theoretical framework, we need to do more work sort of demonstrating that.

Nick Jikomes

52:41 - 53:03

And you mentioned earlier that there was a structural feature, I think you said a terminal tyrosine that some peptides have. And even though they can maybe get through the blood-brain barrier to some extent, they are effluxed. So they’re basically sent right back out. Are there any examples like that of structural features that tend to generally make peptides more or less permeable?

William Banks

53:04 - 53:33

I think that’s probably the best example. There’s, you can also, I think another example, there’s some somatostatin analogs we looked at. But the general principle is that you can make a peptide chain very lipid soluble if you circularize it and make it a cyclic peptide. But those are substrates for the P-glycoprotein system.

William Banks

53:33 - 54:02

So it’s an example of cheating and making something very lipid soluble but you get caught up with the PGP and so that trick doesn’t work. PGP itself, which has some peptides as its substrates, also has many other substances as its substrate, is actually a great example of blood-brain barrier regulation of a transporter. And remember, this is efflux, brain-to-blood. So you gotta think in reverse, you know, the enemy of my enemy is my friend kind of thing.

William Banks

54:03 - 54:36

But it’s, for example, is greatly increased activity with inflammation. One of the things that causes inflammation is seizure activity. And we try to control seizure activity with anti-seizure drugs, most of which are PGP substrates. So one mystery is that when people have status epilepticus, that is repeated seizures over and over and over again, it’s really hard to break that seizure cycle.

William Banks

54:37 - 55:00

And so as a result, the blubbering barrier is probably explaining this. The seizure drug is a substrate for PGP, but maybe not a very strong one. So enough gets in to affect seizures. But if the seizure activity sort of breaks through, inflammation, there’s more PGP activity, less drug is residing in the brain.

William Banks

55:01 - 55:21

So it becomes a catch-22, you know, the more seizures, the more inflammation, the more inflammation, the more PGP. So it’s really hard to break that. And there’s a couple of exceptions to that rule. There’s a couple of drugs that still work even instead of epileptics, at least in many cases, and neither of those are substrates for PGP.

William Banks

55:22 - 55:54

So the answer to status epilepticus is also sort of dictated in the blood brain barrier activity. And there’s one drug called Akinra, which is an interleukin-1 receptor agonist. And it is useful for controlling seizures in children who have developed seizures because they’ve had some sort of intercurrent illness and inflammation. And for some reason, they get these seizures that nothing seems to break them.

William Banks

55:54 - 56:25

And Akinra is able to do that. We haven’t looked at Anakinra specifically, but we have looked at the parent compound, interleukin-1 receptor agonist, and it is transported across the blood-brain barrier by the interleukin-1 transporter. And so it makes sense that Anakinra is independent of PGP, is able to get into brain by its transporter in there, it’s able to tamp down inflammation and treat seizures in these children.

Nick Jikomes

56:26 - 56:51

When it comes to the dynamic regulation of the blood-brain barrier’s permeability, either in general or to specific substances. Can you give us a sense for what that looks like in the healthy state? So for example, things I’m thinking of here, does the permeability of the barrier change according to, say, circadian rhythms or sleep-wake cycles? Does it become more permeable at night when we’re sleeping or anything like that?

Nick Jikomes

56:51 - 56:58

What is the sort of temporal dynamics of how tight the regulation of the permeability is for the blood-brain barrier?

William Banks

56:58 - 57:35

Yeah, I think if you talk about lipid solubility, the membrane composition, which is often the cell membrane permeability to lipid molecules for other tissues is often affected by diet. So if you change your diet and fats or something like that, a cell membrane might become more permeable to something. But in the blood-brain barrier, the brain endothelial cell, that liposolubility doesn’t seem to change. So liposoluble drugs will get in pretty much the same order and the same magnitude, say in a neonate versus an adult, that kind of thing.

William Banks

57:35 - 58:07

But the transporters are different. The transporters are highly regulated. key here to thinking about this is that the blood-brain barrier, and I’m talking now about the blood-brain barrier and probably on it will also include the choroid plexus and any other cell junction that has transporters like the tannosites probably, but the key here is that the barrier or barriers are subject slave to the needs of the brain.

William Banks

58:08 - 58:31

So if the brain, and the brain’s always changing, right? We have a different brain in the neonate, it’s growing. We have different brain with aging. And so all of these signals that are transmitted to the brain endothelial cell through the pericyte and the astrocyte, but also probably by every other cell in the brain as well.

William Banks

58:31 - 58:50

There’s all this crosstalk going on between all of these cells. They’re telling the blood-brain barrier what the brain needs. And as a result, the blood-brain barrier will adapt and try to give what the brain needs. If that breaks down, that could be a disease state.

William Banks

58:52 - 59:11

So, let’s take, for example, Alzheimer’s disease. Alzheimer’s has got a lot of blood-brain-barrier changes. And we have good ideas, or at least, I think, interesting hypotheses, we being the field, about what that means. But ultimately, we’re not sure.

William Banks

59:11 - 59:39

They’re still, I would say, at the testable stage. So, for example, glucose transport across the blood-brain-barrier of the Alzheimer’s disease brain is reduced. Now, we don’t know if that is a primary function at the blood-brain barrier. There’s a decreased ability for some reason for the glucose to be transported across, in which case the poor neurons are starving and that could be a very big problem in AD, maybe a causal.

William Banks

59:39 - 1:00:16

Or is it that the brain cells are less metabolically active to begin with, and so the endothelial cell is simply responding to a decreased need of a sort of sick cell, if you will? We don’t know, but we do know that the transporter is not working well. We also know one of the hallmarks of Alzheimer’s disease is its plaques and tangles, and its plaques consist of amyloid beta peptide. And that’s usually transported out of the brain by efflux systems, by other methods as well, degradation and things like that.

William Banks

1:00:17 - 1:00:37

But about 25% of the A-beta is gotten rid of by efflux systems. Two in particular, and neither one of those work in Alzheimer’s disease. They go offline for unknown reasons. And it’s pretty clear that going offline contributes to the plaque formation.

William Banks

1:00:38 - 1:00:53

In fact, we did an experiment. We made anti-senses against one of them, LRP1. PGP is the other one. LRP1 knocked it out and A-beta levels went up in the brain and the mouse had all kinds of behavioral learning problems.

William Banks

1:00:54 - 1:00:59

So those transporters seem to be really critical to controlling the level of A-beta in the brain.

Nick Jikomes

1:01:00 - 1:01:24

So, yeah. In general, with aging, setting aside individual disease states like Alzheimer’s or something else, as we get older, what does the integrity of the blood-brain barrier generally look like as we age? Does it just become more permeable to everything and more leaky? Does it just become dysregulated in terms of its ability to fine-tune exactly what comes in and to what extent?

Nick Jikomes

1:01:25 - 1:01:28

Is it all over the place or is there a general arc to that?

William Banks

1:01:29 - 1:02:04

Yeah, the change in transporters, because you have to look at every transporter individually to talk about that transporter, is a little mystery. We know that there are lots of changes in the blood-brain, oh, sorry, let me back up. We know that the few transporters that have been looked at, again, we’re talking about the field, not just our lab’s work, there are a lot of changes in blood-brain barrier. But in terms of the integrity of the blood-brain barrier, it looks like that there is an increased leakage in the hypothalamus with normal aging.

William Banks

1:02:05 - 1:02:37

And that occurs around 60. And, you know, I try to be dispassionate about science, but I really hate this fact because I’m over the age of 60. So, you know, to think that my hypothalamus isn’t just as good as it used to be really irritates me. But anyway, um but here’s the thing uh unfortunately uh one of the things about bbb there’s a lot of myths and there’s a lot of things that sort of get out and then they get sort of take on a life of their own so if you read a

William Banks

1:02:37 - 1:03:13

lot of the reviews now it sounds like the aging brain even the normal one or the AD brain, either one, it’s just it’s leaky. It’s just open everywhere. And the fact is that it’s though the aging brain has a few places where it looks like it’s gotten more leaky, but it’s not widespread and it’s not profound. And here I’m going to go off on a tangent about blood-brain barrier and disruption because we’re getting a huge list of diseases now where there’s probably disruptions of the blood-brain barrier.

William Banks

1:03:13 - 1:03:42

But these are not profound. These do not return the blood-brain barrier to that of a peripheral tissue. Even in even in cancers of the brain, which are probably much more leaky than anything we see in other states, the blood-brain barrier is still highly restrictive. You know, albumin might come in four times faster, but that’s still a far cry from, you know, it’s million times slower than glucose.

William Banks

1:03:42 - 1:03:58

So the barrier is just squeaked open a bit. Now is that relevant? Probably is because the brain is very sensitive to disruption and can cause inflammation and go forward. But they’re very subtle openings.

William Banks

1:03:59 - 1:04:16

So they’re hard to detect. In fact, the MRI detection methods that are used to show these disruptions, they’re really at the level of detection, detection limits.

Nick Jikomes

1:04:18 - 1:04:47

And you could imagine too, you could imagine a pathology coming from increasing the barriers permeability generally, because then it’s just more leaky and stuff’s getting through that you don’t want to. You could also imagine it turning pathological from going in the other direction. If you make it more impermeable, then you can’t get stuff that you might want to get into the brain. Give us a general sense for like what’s going on today in terms of how people are thinking about developing therapeutics that relate to modulating blood-brain barrier permeability.

Nick Jikomes

1:04:47 - 1:05:18

So for example, many small molecule drugs, as we said, can’t really cross the blood-brain barrier very well. And my understanding is there are people developing methods and potential approaches where you might say temporarily enhance the permeability of the barrier, to allow it to have a drug come in. To what extent is that happening today? And what do you see as sort of the general pros and cons at a high level of playing with the permeability of the barrier so as to allow things like medications to more readily cross it?

William Banks

1:05:19 - 1:05:40

Yeah, there’s three basic ways to get drugs into the brain or develop drugs that have taken up maybe 95 or even 99% of the effort. And the first one is production of small lipid soluble drugs. And we’ve talked a lot about that and it’s caveats and things like that.

Nick Jikomes

1:05:40 - 1:05:46

So the basic idea is you’ve got a small molecule drug, but you’re gonna make it lipophilic to a degree that makes it likely to just diffuse it.

William Banks

1:05:47 - 1:06:02

Yes, it’s probably more the other way. We’ve, you know, for decades and decades and decades been able to make small drugs. And those that wound up having effects on the brain in retrospect turned out to be lipid soluble. It’s probably serendipity.

William Banks

1:06:03 - 1:06:20

But now we know that. And so now, yes, we can actively engineer small drugs to follow the rules of Lipinski, for example. So that’s one way. And that’s, you know, proven it’s right to be there.

William Banks

1:06:20 - 1:06:39

The other two are disruption of the blood-brain barrier. And then the other one is I’ll call as a categories Trojan horse. And Trojan horse is basically anything you have a drug that, sorry, you have a substance that’s already crossing the blood-brain barrier. So you just put something on it that will drag it across.

William Banks

1:06:40 - 1:07:21

And so let’s talk about disruption. Oh, and I should also say that although that’s taken up the lion, those three have taken up the lion’s share, there are really a lot of other approaches that we could take. And we wrote a review in Nature Reviews Drug Discovery about 10 years ago, listing all the other approaches that one could have for getting therapeutic effects into the brain other than those three, because the last two, quite frankly, haven’t been very successful. So disruption The idea is that you have the drug in the blood already, but the barrier is there.

William Banks

1:07:21 - 1:07:53

And so if you open the blood-brain barrier, the drug will go in and that will be therapeutic effect. And the problems with that is that anything that is, that many things are able to go in. And even if you make the disruption small, so say, you know, less than 200 Daltons or so. A lot of things that are toxic to the brain, like monoamines, are smaller than that.

William Banks

1:07:53 - 1:08:17

So you get a lot of things that are meant to stay out of the blood-brain barrier. Inevitably, this causes inflammation in the brain. Focused ultrasound right now is the biggest area that is being looked at for disruption. Previously it was chemical agents like antihistamines and things like that that regulated tight junctions.

William Banks

1:08:18 - 1:08:39

And FUS is probably not only opening tight junctions but also producing increased vesicularization and things like that. And my fear is ultimately it will have the same problem of causing disruption and being non-specific. But I think there’s a lot of people working on that. So they may figure out how to tweak it.

William Banks

1:08:39 - 1:09:02

Who knows? But I think that’s sort of where that field is right now. The Trojan horse field is huge because people have tried all kinds of versions of this for about the last 40 years. And there are promising efforts, particularly in the last five years, I think there’s been some promising ideas.

William Banks

1:09:03 - 1:09:25

But it’s strange to think that we’ve been doing this for 40 years and we haven’t already solved the problem because it’s so intellectually gratifying. It seems so intellectually elegant. You know, something’s going across, just stick it on and let it get across. So why don’t we have lots of shuttles for that?

William Banks

1:09:26 - 1:09:43

And I think that there’s a couple of reasons. So the idea is that you take a molecule that’s already crossing, let’s say insulin, and you stick something on it, and it plugs into the insulin transporter and is transported across. That’s the way it should be. But it doesn’t seem like that’s what’s happening.

William Banks

1:09:44 - 1:10:11

And I think the reason is because It doesn’t route through the receptor any longer. It routes through another pathway called absorptive transcytosis or absorptive endocytosis. And that often gets routed to lysosomes, which break down everything and that kind of thing. And the blood-brain barrier seems very, very good at finding these molecules that have been altered.

William Banks

1:10:12 - 1:10:48

you know, and saying, well, that’s not really insulin, that’s something foreign, and routing it over to this different way. And I think that the reason the blood brain barrier is so good is because there’s been, you know, if you go back evolutionarily, as soon as you find an organism that has something that will pass for neuronal tissue, and something that will pass for hemolymph, there’s something in between that will pass for barrier function. So we have millions of years of the blood-brain barrier being faced with pathogens that are trying to get to that neuronal tissue and eat it up.

William Banks

1:10:48 - 1:11:36

So it’s had to be very clever about figuring out things that are trying to trick their way in and so we have these millions of years of the blood-brain barrier being faced with substances trying to trick their way into the brain because there you get this really great meal high in fats and everything you know and in fact the virus some viruses that most of the things that can cross the blood-brain barrier by tricking by tricking it are like HIV and rabies virus are able to survive the lysosomal compartment. And so they’re probably going to the lysosomes, most is being recycled back out into the blood, but some is able to get across in the other direction to the, to the luminal, abluminal membrane.

Nick Jikomes

1:11:38 - 1:12:14

One thing I want to ask you about, so, you know, if we’re thinking about what can and cannot get through the blood-brain barrier and to what extent, when we’re thinking about developing novel therapies, you know, one of the approaches is let’s try and play with the permeability of the barrier itself to allow something to get through that couldn’t get through so well before. Another approach is, well, maybe let’s play with something that can naturally get through to a pretty good extent or modifying something, maybe creating a new peptide using some of the principles that we discussed earlier to engineer something that can get through the blood-brain barrier without changing the integrity of the barrier itself.

Nick Jikomes

1:12:14 - 1:12:44

When we’re thinking about those things, we also come to an interesting question in my mind, which is, how do some of these things like peptides that are secreted affect tissues in different ways. So, for example, if I’ve got a gut peptide that’s secreted and gets to the brain, or I’ve got a brain peptide that’s secreted and gets to the periphery, do these peptides and other substances generally affect different organs in the same way at the cellular level? Or can they have very distinct effects in, say, neural tissue versus liver tissue or something else?

William Banks

1:12:44 - 1:13:02

Yeah, I think one of the reasons there was so much excitement when peptides were discovered is what we were then called pluripotents. They had all kinds of effects. And they tended to be sort of mild, and they tended to be sort of integrative. But this was sort of a new way of thinking.

William Banks

1:13:03 - 1:13:29

And so, yeah, leptin has a lot of effects in the periphery. It has a lot of effects on immune regulation and sort of energy usage in different cells. And I have to admit, I haven’t kept up on the leptin peripheral tissue literature, but it’s there and it’s really interesting. Other tissue, other peptides, there is one called PAYCAP.

William Banks

1:13:30 - 1:14:17

a pituitary adenylate cyclase activating polypeptide, PCAP, that was discovered in the 90s and it was very much in vogue and it did all kinds of different things. It was a difficult peptide to synthesize and to sort of put in a bottle sticky and things like that uh which sort of held back its progression but people are still working on this and i think that the age of peptides which was envisioned way back in the 80s is only now coming forward and we’re getting a number of peptide drugs so these really do have the potential of doing just that sort of treating a condition or a situation at multiple integrated levels

Nick Jikomes

1:14:25 - 1:14:41

You said that some of this peptide stuff, peptides as therapeutics, that was envisioned back in the 80s. Why were people envisioning that? What is it about peptides that makes them attractive from a therapeutic standpoint or just a general wellness standpoint?

William Banks

1:14:42 - 1:15:17

Yeah, they were sort of first discovered in the 70s and by the 80s they were established as being real and not some fragment or phenomenon that wasn’t really based in reality. And there was an explosion then because peptides were found everywhere to do everything and things like that. And there was a big push in the 80s and 90s by some pharmaceutical companies to develop peptides, including for central nervous system treatments. But the blood-brain barrier sort of waylaid those.

William Banks

1:15:17 - 1:15:42

But they do illustrate a very important point about the blood-brain barrier and how to get around that. And if you want to get a drug into the brain, and one presumes also something secreted in physiological action might have to think about this too, is that there are really three Ps. We’ve talked about permeability, But there’s two others. There’s potency, of course, in the brain.

William Banks

1:15:43 - 1:16:06

And if you only need one molecule in the brain to have an effect, or you need 10 molecules in the brain, that potency figures out how much permeability you need. And the other P is the pharmacokinetics. So one of the big problems with peptides and developing them therapeutically is they have very short half-lives. usually minutes.

William Banks

1:16:06 - 1:16:31

And so if you want to develop this, then you need to figure out a way to make them more stable in blood. And actually this is true of insulin. Some people consider insulin a peptide and some people don’t because it winds up having two parallel chains of amino acids linked together by sulfide bonds. And some purists say, no, you can only have one chain to be a peptide.

William Banks

1:16:31 - 1:17:08

But at any rate, let’s call it a peptide-like substance. We had the same problem, you know, Banting and Best discovered insulin could be used to treat diabetes. And so people started extracting it from pancreas and this is how Lilly came into existence. but amongst those problems was it had a very short half-life and so you were often treating and you didn’t know the potency because since it was biologically extracted some batches would be more potent than others and so it was really tough and so insulin had to be stabilized

William Banks

1:17:09 - 1:17:28

And eventually, of course, it was synthetically produced, but that wasn’t for decades. So that’s where the zinc insulins and things like this came in. And the peptide field never really went through that. It never really went through being able to produce peptides that were pharmacologically stable.

William Banks

1:17:29 - 1:17:52

And I’m not quite sure why there wasn’t more effort than that. But by making analogs and things like that, But there’s, as I say, there’s a rebirth of this. In fact, you know, the integrin receptor agonists are peptides. They’re derived from glucagon-like peptide 1.

William Banks

1:17:53 - 1:18:09

And they have this great history of treating diabetes, of treating ozempic weight loss, and maybe also affecting neurodegenerative diseases like Alzheimer’s and Parkinson’s disease.

Nick Jikomes

1:18:10 - 1:18:28

A lot of these peptides, the GLP-1s and many others, are injected, and so they can have both local and systemic effects. People are also talking about intranasal delivery of peptides. Other drugs are delivered that way sometimes. What does the blood-brain barrier look like in the nasal passage?

Nick Jikomes

1:18:28 - 1:18:35

Is it strong there? Is it relatively weak? Can things pass through the intranasal route that couldn’t get through otherwise?

William Banks

1:18:37 - 1:19:06

Yeah, so intranasal delivery to the brain is really interesting. So to save a lot of confusion that I had for about 10 years, let’s start off by saying there’s three ways of delivering things intranasally. One is you’ve got a sinus condition and you’re congested and you just put something there that relieves that, that’s a local thing. There’s other ways of giving it and you know those nasal passages are very vascular so it can be absorbed directly into the bloodstream.

William Banks

1:19:07 - 1:19:31

And calcitonin, for example, for the treatment of bone pain is delivered that way. But the final way is you give this very high up at the level of the cribriform plate. And for some reason, substances can then enter the brain. So a bit of anatomy, we have the olfactory bulb and the olfactory nerve, which is a cranial nerve and therefore part of the brain.

William Banks

1:19:32 - 1:19:52

coming through and going through a part of the skull called the cribriform plate. It looks like a flat plate and it’s got a lot of holes drilled in it. And the nerve goes through there. So you have, as I like to tell my students, you have a piece of your brain dangling out of the top of your nose, which is, I think, a very graphic way of thinking about it.

William Banks

1:19:53 - 1:20:20

If we can get substances to that level, those substances will enter the brain. And we understand that in part. We know that there can be retrograde transmission or retrograde absorption so that substances go up those nerves and into the brain. And there’s also the trigeminal nerve which is in that area so it can retrograde and go to those regions of the brain.

William Banks

1:20:21 - 1:20:48

But I think that only explains part of it, because there’s some interesting pharmacokinetics that nasal delivery does. And one of them is that whatever that distribution pattern will be in the brain, it achieves that within five or 10 minutes, at least in the mouse brain. And we have no way, I think, of explaining how we get that much diffusion, we’ll call it, that quickly so fast. So that’s a bit of a mystery.

William Banks

1:20:49 - 1:20:57

And the other is that it can indeed do that. It can distribute all the way from the site of entry all the way to the cerebellum.

Nick Jikomes

1:20:58 - 1:21:10

So things like peptides in animals, at least, can get pretty deep into the brain from the intranasal route. And that probably can’t just be explained by a passive diffusion mechanism.

William Banks

1:21:10 - 1:21:28

Yeah, I don’t think it can be explained by passive diffusion or any of the other mechanisms that we thought of. Those other mechanisms do explain part of the mystery, just not all of the mystery. So, I mean, that’s why science is fun. You know, if all the mysteries were we’re solved, it’s time to go to another field.

William Banks

1:21:28 - 1:22:07

But what this means is that this is really a possibility for treating conditions and it’s been shown to be active in humans. Suzanne Kraft has done some studies giving insulin to the brain and Here’s an here’s an aside everyone. I’m sure has heard of insulin and its ability to control glucose levels in the blood But when you get into the brain Insulin has a different life It doesn’t control blood sugar rate of entry into the brain or anything like that. It seems to, though, act as a mnemonic.

William Banks

1:22:07 - 1:22:32

It has effects on memory and that kind of thing. In fact, every GI peptide discovered starting with CCK in the 30s to anyone you can imagine, they all have profound effects on brain, on cognition, and memory, and that kind of thing. Yeah, it’s really, I can make up an evolutionary theory for that, sort of probably.

Nick Jikomes

1:22:32 - 1:22:54

Yeah, I mean, I think I could too, it makes a lot of sense intuitively. But so you’re saying it’s a general observation that a lot of these, what we think of as gut hormones or gut peptides, also have effects in the brain, which affect cognition, memory, things like this. So perhaps it’s not so surprising that people are observing brain effects like that for things like the GLP-1s that people are taking for weight loss.

William Banks

1:22:55 - 1:23:13

Exactly. I don’t think the GLP-1s are an exception to the class, because GLP-1, they’re really gut peptides. But it’s been shown for cholecystokinin, somatostatin, insulin, ghrelin. I can’t think of a GI peptide that, pancreatic polypeptide.

William Banks

1:23:13 - 1:23:24

They all have been shown to have either behavioral effects or effects on neuronal sprouting and all of these kinds of things. It’s a great class, untapped class.

Nick Jikomes

1:23:24 - 1:23:36

Yeah, it does. I mean, it makes a lot of sense to me. If you just think about an animal in nature, it’s going to have to learn and remember and behave in a lot of different ways in order to survive. And what’s a big part of survival?

Nick Jikomes

1:23:36 - 1:23:39

Well, it’s finding food, it’s responding to scarcity, all of that stuff.

William Banks

1:23:39 - 1:23:53

Exactly. You know, the GI tract is the first really, truly integrated thing from the first time you see food. So you start salivating and telling the stomach to get ready. Everything all the way to evacuation has to be controlled and monitored.

William Banks

1:23:53 - 1:24:04

And it’s got all these systems. And so it’s already a complex system, so why not use that complexity to help teach the brain how to get the next meal? Exactly, I think that’s it.

Nick Jikomes

1:24:05 - 1:24:39

Yeah. So given the decades of experience you have in this field, studying things like peptides, studying the blood-brain barrier, what do you generally make of the current boom in peptide biology? Everything from the GLP-1s that people are using to treat metabolic issues and address obesity, to the research peptides that an increasing number of people are experimenting with themselves, What’s most scientifically exciting about this general area to you? And what are some of the biggest gaps you see in the claims and the data that’s actually out there?

William Banks

1:24:40 - 1:25:03

Yeah, so obviously I’m biased towards brain and that kind of thing. And I’m sure there’s all kinds of peripheral tissue stuff that’s going to be great, and I don’t know anything about it. And so with that caveat of my limitations, I think treating central nervous system diseases are really exciting. And we have a lot.

William Banks

1:25:05 - 1:25:19

everything from depression to dementia. I mean, it just goes on and on and on. Parkinson’s, Alzheimer’s, of course, are big ones. And I think that this is really where it is, you know, for my interests.

William Banks

1:25:20 - 1:25:40

And, you know, there’s lots of things to think about, like, how do these really work in the brain? And what do they do? And do we, when we start to get down to it, will we need, some drugs will affect some types of diseases, rather than being universal? And will we need to, and other drugs affect others?

William Banks

1:25:41 - 1:26:22

Will we need multiple drugs? You know, if you look at any big diseases that we have, like hypertension or diabetes, we have multiple classes of diseases and we often have people on multiple drugs. And so it may be that when we finally get effective drugs for treating neurodegenerative diseases, it may have to be a little bit of a cocktail. We’re able to do that for hypertension because we understand hypertension so well, you know, that we’re doing salt and water here and we’re doing vasomotor stability there, you know, so we use beta blockers and diuretics and angiotensin 2 receptors and things like that.

William Banks

1:26:23 - 1:26:46

If we get that kind of level of understanding brain function, then we have a really rational way of developing drugs and using them in our patients. But those are probably decades away. So more immediately, what we need to think about in terms of the GLP and neurodegeneration is, are all these drugs going to work? Because we have a whole bunch of them now, right?

William Banks

1:26:47 - 1:27:05

And Ozempic was recently tested and didn’t do that well. in treating AD, but azimuth doesn’t cross the blood-brain barrier. And so the first question is, how are these substances working? We know that if we treat diabetes, the brain is protected against dementia, but that takes decades.

William Banks

1:27:06 - 1:27:24

These drugs seem to extend in and those sorts of things seem to work rather quickly. So I think they have to get into the brain. and work at the GLP or insulin receptors or whatever. And therefore, we need to look at the effect of drugs that can cross the blood-brain barrier very well.

William Banks

1:27:25 - 1:27:40

Zympic doesn’t cross very well. And so I think that we should consider the effect of GLP-1 and IRAS on cognition still untested in the human population.

Nick Jikomes

1:27:41 - 1:27:57

Yeah. So a lot of these classic gut peptides and hormones actually do affect the brain, they affect cognition, they affect behavior, all of that stuff. Are people working on other gut-derived hormones other than GLP-1 and thinking about the brain and things like that?

William Banks

1:27:59 - 1:28:20

Certainly at the basic science level, certainly in labs. What pharmaceutical companies are doing though, of course, we don’t know. Hopefully they are. The problem has always been in science is there can be so much energy and excitement and things can roll on and the first big disappointment or series of disappointments, people give up.

William Banks

1:28:20 - 1:28:43

And I always think that as scientists, we sometimes give up a little early in certain areas. Of course, I can also give examples where we get bogged down and we should give up, but we keep on plowing through. Because I lived through this era of peptides, it’s just when I was starting. Peptides were hot.

William Banks

1:28:43 - 1:29:09

Peptides failed as a therapeutic class. and although the decades of discovering all the different peptides and the different things they did continued. So, you know, I hope that we don’t get frustrated too early because none of these things are easy. and they really take an integration of things, particularly if you’re looking at blood-brain barrier penetration.

William Banks

1:29:09 - 1:29:30

A lot of blood-brain barrier is still experimental. It’s not theoretical, you know. Yes, we can get drugs, for example, we can get drugs in by making them small and lipid soluble, except, you know, then we discovered the efflux systems. So it remains an experimental science because we still know so little about blood-brain barrier.

William Banks

1:29:31 - 1:29:42

And so that makes it harder for a pharmaceutical company to put in the effort to get something past patents and into the clinic.

Nick Jikomes

1:29:43 - 1:30:01

You mentioned a peptide from history earlier that was kind of intriguing, Delta sleep inducing peptide, apparently isolated from cats and allegedly had something to do with sleep, although it sounded like that was never proven. Are there any peptides that have a clear effect on sleep and wakefulness or circadian rhythms?

William Banks

1:30:04 - 1:30:53

Yes, there’s a lot. Delta sleep-inducing peptide, we looked, had a circadian rhythm for getting in. Weihongpan, about 20 years ago, got very involved in chronobiology of the blood-brain barrier and worked with one of the great leading lights of the area and did some work, not recalling a whole lot about specific examples right now. But it’s probably, yes, that the other thing that seems to happen is not well documented, but that there may be other kinds of entrainments, not necessarily circadian rhythms, but, you know, ghrelin, for example, maybe is more permeable near a meal than not and things like that.

William Banks

1:30:53 - 1:31:14

That’s not really definitely worked out. But usually the changes in blood-brain barrier, again, are relatable to the needs of the brain, and so in an efficient way. So there’s probably a lot of examples like that, but still pretty much unexplored, I think.

Nick Jikomes

1:31:16 - 1:31:44

Can you talk a little bit more about the timescales on which peptides work compared to other things? So they have a relatively short half-life, usually on a minutes-long scale, so shorter than a lot of drugs that are out there. Can you say a little bit more about that and the extent to which that might be a feature or a bug, or we might want that or not want that from a therapy? Are there any potential benefits or downsides to something that lasts on that type of timescale compared to other therapies?

William Banks

1:31:45 - 1:32:13

Yeah, this has, I think, been the major problem of directly adapting them as drugs is that they’re there a very short time. And the other problem is, of course, that they’re by and large not orally absorbed. Again, using insulin as an example, there are a lot of studies trying to get in, how can we give insulin other than injecting it with a needle? And so orals were tried to be developed and those just haven’t worked.

William Banks

1:32:14 - 1:32:31

It’s just totally broken down. So there’s those issues as well. The action of a peptide, though, interestingly, lasts longer than minutes, particularly if it’s given peripherally and you’re looking at a brain function. Some mystery there.

William Banks

1:32:32 - 1:33:03

And we sort of understand some of the mystery. And again, using insulin as an example, you can almost look at the pathways of insulin. There’s the immediate effect, it plugs into the receptor, whatever it’s going to do is done for that tissue, you know. But then it triggers other things, and so there’s cytoplasmic events, and those may last for hours, and then it can actually have new levels at the nuclear level, altering mRNA production and that kind of thing, and those can last for days.

William Banks

1:33:03 - 1:33:21

And that actually has been mapped out for insulin. It was a great review in life sciences way back when I was a puppy, when I was a student. But I read it, and it was just intriguing how it could explain how these effects. And they’re not necessarily effects on glucose anymore.

William Banks

1:33:21 - 1:33:40

There are other effects on metabolism and things like that. So insulin has a very interesting evolutionary history. It didn’t start out as regulating glucose. It was more of a metabolic substance and had things to do with cell growth and things like that.

William Banks

1:33:41 - 1:34:05

And its cousin, IGF-1, still has all of that. And interestingly, it seems that insulin in the brain has a lot of IGF-1 effects, and that’s why it has effects on, really, on neuronal sprouting and things like that. It’s got its old metabolic effects, its old effects on cell growth. Still intact.

William Banks

1:34:06 - 1:34:07

Yeah, yeah.

Nick Jikomes

1:34:08 - 1:34:15

What sorts of things are you working on today, or what do you think is particularly interesting in the field of blood-brain barrier rejuvenation and or peptide biology generally?

William Banks

1:34:17 - 1:34:43

Thank you. I’m going to switch over to commercial mode now. There’s a lot of things, but there’s one thing that I’m really interested in. Recently, Peter Saracen and I wrote a review in Nature Reviews Drug Discovery, and we changed, we’d written one previously, I’d written one previously, about 10 years previously, about all the ways that are being neglected of getting drugs into the, across the blood-brain barrier.

William Banks

1:34:44 - 1:35:18

So the blood-brain barrier is a therapeutic conduit. But here we looked at the blood-brain barrier as a therapeutic target. And the reason is that in, since those 10 years, the blood-brain barrier as a site of disease has become increasingly realized. We talked about that there’s a lot of diseases that now seem to have blood-brain barrier disruption, but there’s a lot of diseases that have blood-brain barrier dysfunctions in the transporters or productions of other things or secretions and this kind of thing.

William Banks

1:35:20 - 1:35:46

There’s a lot of basic science and a few other things around that show that we can really auto-target the blood-brain barrier. And again, we talked a bit just passingly about how all the cells are talking. So this is a formal concept called the neurovascular unit. And basically what this does is it takes a slight, imagine a slice of the brain and we come very blood-brain barrier centric or endocentric.

William Banks

1:35:47 - 1:36:07

And we talk about all the cells that surround that, you know, the neurons and the microglia and the pericytes and the astrocytes and the mast cells and everything you can think of. And they’re all talking to each other. And so one of the things is this is how the blood-brain barrier knows to be a blood-brain barrier. But it works the other way, too.

William Banks

1:36:07 - 1:36:42

If we could fix the brain endothelial cell, if it’s the cause of other diseases within the brain, then we’re altering that communication. And so one way to treat a neuron might be by first treating the endothelial cell, letting the endothelial cell then affect affect the neuron. So we have that effect too, but mostly we were interested in diseases that have, you know, sort of a blood brain barrier story. And so that’s sort of my current passion is to sort of think about that and promote that and work on that.

William Banks

1:36:42 - 1:37:05

But we’re also interested very much in GLP-1. We’ve looked at about a dozen of them and how well they cross the blood-brain barrier. And it seems that that is not predictive of how good they are at regulating glucose, but it is predictive of how good they are at regulating brain functions.

Nick Jikomes

1:37:06 - 1:37:09

Which of the GLPs cross the blood-brain barrier the most?

William Banks

1:37:10 - 1:37:36

One that crosses very well is Xendin, which is one of the very early ones. Unfortunately, it has a short half-life and has to be given multiple times a day, or at least twice a day. There’s a group in China led by Horsher who has an analog, and it looks like it crosses very well. and it looks like it has CNS functions.

William Banks

1:37:36 - 1:37:57

And I’m sure there are other companies as well. But he’s recently published on that, so that’s how we know about it. So there’s, interestingly, most of the 12 that we looked at, I think eight, crossed to some degree. And the other four crossed very poorly, or even in one case, not at all.

William Banks

1:37:58 - 1:38:17

So unfortunately, the ones that don’t cross at all are the ones with really long half-lives and therefore very easy to give compared to the other ones. So it will take more effort to do a clinical trial if you have to talk people into giving themselves shots more than once a day.

Nick Jikomes

1:38:17 - 1:38:34

Got it. So there’s a lot of peptides out there for research use that people are buying and experimenting with and trying for various reasons. It sounds like in general, because for a lot of these things, we don’t have hard data. We won’t literally know for sure if it’s getting into the brain or not.

Nick Jikomes

1:38:34 - 1:38:53

But it sounds like in general, a lot of these peptides will cross the blood-brain barrier to some extent. So maybe it’s a safe assumption if anyone out there is experimenting with these things to just assume that it’s probably getting into your brain to some extent for good or for ill. Yeah, for good or for ill. Yeah, that’s exactly the way I would phrase it.

William Banks

1:38:53 - 1:39:14

That probably something’s getting in as a biologically relevant, that should be the real question we’re asking. Because on an absolutist scale, a sensitive enough method, you can often find something getting in. But yeah, I don’t know if you really want these things in your brain or not. I mean, it depends on what drug you’re doing.

William Banks

1:39:18 - 1:40:00

ran at one point I ran the hypertension clinics in St. Louis and my VA there and we had a group that got very interested in herbals and I was traveling a lot then into Germany and things like this and there was the ginkgo seed came up for the treatment of AD which it was well tested in Germany and was found, you know, not to have much. But we talked about how can you do things, you know, in the U.S. And the one person who is very knowledgeable in this area says, essentially, you can’t in America because if you go and buy echinacea at your local drugstore, the one thing you can be sure it

William Banks

1:40:00 - 1:40:29

does not contain is echinacea. So I always remember that and that you know because the FDA doesn’t regulate these things and so you can put anything in a bottle and sell it and So I really wonder about peptides which you know, if you’re if you’re buying a peptide, they’re not easy they’re not hard to make these days, but a lot harder than almost anything else and So it’d be much easier to put some powder in there. It could be whatever, calcium carbonate or something.

William Banks

1:40:29 - 1:40:35

Who would know? So I don’t have a trust of non-regulated stuff.

Nick Jikomes

1:40:35 - 1:40:44

Are there any final thoughts you want to leave people with or anything you want to reiterate from our discussion today about the blood-brain barrier, about peptide biology, or about any of the topics that we covered?

William Banks

1:40:45 - 1:41:08

Yeah, I think maybe that it’s the blood brain barrier is, without a doubt, the biggest obstacle to, to drug development. But I think it’s not that hard. You know, the amount of drug you need to get in, in most cases is very, very low. L-delta, only about 0.1% of the administered dose gets in the brain.

William Banks

1:41:09 - 1:41:25

Morphine, if we inject morphine into a mouse, only one molecule in 5,000 gets into the brain. You don’t need a lot of stuff in the brain. In fact, you don’t want a lot of stuff. There’s been examples where people made very permeable substances and they were just too toxic.

William Banks

1:41:26 - 1:41:48

For example, morphine, its cousin is diacetylated morphine, is heroin. Gets into the brain like a shot. It’s too powerful, you know. So I think that there’s a lot of hope if people can sort of step back and do a thoughtful approach to blood-brain barrier.

William Banks

1:41:49 - 1:42:22

And indeed, in our article that I’ve mentioned a couple of times is that we try to do that. We try to find examples where you can use the blood-brain barrier to facilitate a therapeutic effect and things like that. And I think the other thing I’d conclude is that it’s a very complicated field, but that’s where its beauty is intellectually, of course, but it’s also where its therapeutic potential is and its ability to do so many physiological things. There’s more than one way to skin this cat.

William Banks

1:42:22 - 1:42:42

There’s more than one way in which things fit together and its complexity and its elegance is also not the barrier to understanding how to use it, but it’s the key to understanding all the different ways that we can therapeutically improve drug effects in the brain.

Nick Jikomes

1:42:42 - 1:42:51

Yeah. I love the term that you used earlier, blood-brain interface. It’s not just a cement wall that lets a couple of things through. It really is an interface.

William Banks

1:42:51 - 1:43:09

Right, right. Yes, it’s very, for lack of a better word, very thoughtfully thought out. And the regulation, particularly by its chief customer, the brain, is able to regulate what it needs. It’s a very elegant system.

William Banks

1:43:10 - 1:43:20

And been a lot of fun studying it for 45 years. All right. Well, Dr. William Banks, thank you very much for your time. Thank you, sir.

William Banks

1:43:20 - 1:43:23

Thank you very much. Been a real pleasure talking with you.

This is a public episode. If you'd like to discuss this with other subscribers or get access to bonus episodes, visit mindandmatter.substack.com/subscribe

Wide release: July 8, 2026. Not medical advice.

Nick speaks with Dr. Pamela Maher, research professor at the Salk Institute, about ferroptosis and its contributions to neurodegenerative disease. Maher’s central argument is that chronic exposure to elevated iron or partial glutathione depletion, unlike acute insults, lowers glutathione peroxidase-4 levels, permits gradual lipid peroxide buildup, and leaves cells more vulnerable to secondary stressors. Key concepts include the Fenton reaction that links labile iron to membrane damage, distinctions among programmed cell death pathways, and evidence that markers of lipid peroxidation appear early in affected brain regions in Alzheimer’s and Parkinson’s disease.

TOPICS DISCUSSED:

* Cell Death Pathways: Apoptosis proceeds through regulated steps with surface “eat me” signals that enable non-inflammatory clearance by phagocytes, whereas ferroptosis and related pathways release cellular contents that can amplify local damage and inflammation.

* Ferroptosis Discovery: Work on glutamate toxicity in neuronal cell lines first identified a glutathione-depletion pathway termed oxytosis; parallel studies in cancer cells renamed and generalized it as ferroptosis to highlight iron’s catalytic role.

* Lipid Peroxidation: Polyunsaturated fatty acids in membranes are oxidized non-enzymatically when ferrous iron reacts with hydrogen peroxide in the Fenton reaction, generating hydroxyl radicals that produce toxic lipid peroxides and reactive carbonyls that modify proteins.

* GPX4 & Glutathione: Glutathione peroxidase-4 is an enzyme that reduces lipid peroxides within membranes and requires glutathione as cofactor; depletion of either component removes this critical line of defense.

* Chronoferoptosis: Nine-day treatment of differentiated neuronal cells with chronic iron or glutathione synthesis inhibitor reduced GPX4 protein, raised basal lipid peroxidation, and increased cell death after addition of otherwise sublethal secondary stressors.

* Protective Compounds: Screening identified flavonoids such as fisetin and sterubin plus synthetic molecules J-147 and CMS-121 that block lipid peroxidation directly or indirectly and, in several cases, also raise glutathione levels.

* Human Disease Evidence: Postmortem brain tissue from Alzheimer’s and Parkinson’s patients shows elevated lipid peroxidation markers and altered iron handling in vulnerable regions; similar changes appear in animal models of multiple diseases.

* Dietary & NRF2 Links: Compounds that activate the NRF2 transcription factor, including sulforaphane from broccoli and certain flavonoids, upregulate antioxidant enzymes including GPX4 and may support long-term cellular resilience.

ABOUT THE GUEST: Pamela Maher, PhD is a research professor at the Salk Institute for Biological Studies in La Jolla, California. Her laboratory studies ferroptosis mechanisms in neurodegenerative diseases and works to identify compounds that inhibit iron-dependent lipid peroxidation.

RELATED CONTENT:

* Article | Burning Down the House: Lipid Peroxidation, Oxidative Stress & Cell Death

KNOW YOURSELF:

* Visit the SUPPORT PAGE if you find value in this content. Includes discount codes + links to affiliate partners, including products to help you measure and manage your health.

Listen or watch on your favorite platform:

* Audio version: [Apple] [Spotify] [Elsewhere]

* Video version: [YouTube]

PRACTICAL TAKEAWAYS:

* Consume a variety of foods daily, such as broccoli and other cruciferous vegetables for sulforaphane and strawberries for fisetin, to activate NRF2-driven production of antioxidant enzymes that help neutralize lipid peroxides.

* Support glutathione status through consistent sleep, regular physical activity, and avoidance of unnecessary oxidative loads, because glutathione is required for GPX4 to detoxify membrane lipid peroxides.

* Recognize that low-grade chronic stressors can erode cellular defenses over days to years without immediate cell death, making sustained dietary patterns that supply protective phytochemicals relevant for long-term neuronal resilience.

SUBSCRIBER CONTENT BELOW: Reference paper + episode transcript.

Reference Paper:

* Paper | Sustained dysregulation of iron and glutathione homeostasis induces chronoferroptosis, a persistent ferroptotic adaptation in neuronal cells

AI-generated transcript below. Beware of mistranslations!

Pamela Maher

02:50 - 02:52

Oh, yeah, I’m happy to be here.

Nick Jikomes

02:52 - 02:56

Do you want to just remind everyone a little bit about who you are and what your lab studies?

Pamela Maher

02:57 - 03:39

Yeah. So I’m a research professor at the Salk Institute for Biological Studies in La Jolla, California. And my laboratory has been interested for a number of years in what was but originally described as a cell death pathway called, that’s now named ferraptosis and its potential role in neurodegenerative diseases. And we’ve both explored this at a basic research level, but also use this pathway as the basis for drug development or potential therapeutics for neurodegenerative diseases.

Nick Jikomes

03:40 - 04:11

So basically you study cell death, and in particular, this specific form of cell death called aferoptosis, which turns out to be relevant in the context of neurodegenerative disease. And if we think about it in very basic terms, neurodegeneration, when our neurons are born and they’re done maturing, those are the neurons we’re left with. With few exceptions, those neurons can’t be replaced. And so if they die, we have a problem because they can’t grow back and we get neurodegeneration, Alzheimer’s disease, and other things that people are familiar with.

Nick Jikomes

04:12 - 04:40

But if we kind of think about this at a high level and get people thinking about some of the basic biology here, Can you talk a little bit about the basic forms of cell death that are out there that we might read about in textbooks? What are the different ways that cells can die, both in the sense of when they’re supposed to die or healthy versions of cell death, and then versions where it’s not supposed to happen and it results in damage that can’t be undone?

Pamela Maher

04:41 - 05:20

Yeah, there’s a number of different kinds of cell death. The first one that was described is a process called apoptosis, which was originally described in the, I believe, in the early 1990s, and is a called a, it’s a regulated cell death pathway. It goes through specific steps and there are the specific markers for that pathway. The most commonly used is cleavage of an enzyme called caspase-3.

Pamela Maher

05:21 - 05:52

Apoptosis can be activated by several different inducers and it eventually, involves loss of proteins from the mitochondria and activation of this enzyme, Caspase-3. It plays a role in brain function. in development. So it’s been implicated, particularly since it was sort of the first cell death pathway described.

Pamela Maher

05:52 - 06:41

It was initially implicated in a variety of diseases. But as I said, it also plays an important role in development, particularly in tissues where you have excess cells and you that are produced during development and they need to be removed. The other thing that distinguishes apoptosis from most other cell death pathways is that when cells start to undergo this process, they put a signal on their surface of phosphatidylethanolamine that says essentially, eat me. And so they are cells that are undergoing apoptosis tend to be taken up by cells of the immune system, like like phagocytic cells, like macrophages.

Pamela Maher

06:41 - 07:25

And so they don’t release their contents into the extracellular environment. So unlike other cell death pathways, apoptosis is tends not to induce a major inflammatory response. I mean, because these cells eat them and they don’t, the cells, the phagocytic cells eat them and digest them rather than having the cells burst open and release their contents into the surrounding tissue or blood or whatever. And that can, it stimulate a major inflammatory response, which can be as detrimental as the loss of the initial cell.

Pamela Maher

07:26 - 08:09

There are other types of cell death, a number of necroptosis, which is a distinct pathway. that involves a set of enzymes that are activated when this pathway is turned on. Parthenotosis, which is yet another pathway that involves overactivation of an enzyme called PARP. And then there are several other cell death pathways that have been described that are activated under distinct situations.

Pamela Maher

08:11 - 09:06

There’s a recent one that’s received some attention that seems to be more related to cancer, or cancer-related diseases. consequences of chemotherapeutic drugs called cuproptosis that involves copper. But there’s a number of different pathways out there and they all are distinct in terms of the different steps that are involved. And they also, a number of them have clear markers that you can use to identify distinguish them, their activation in either cell culture models or even for pathways such as apoptosis and necroptosis and parthenotosis, I’m not sure I’m saying it right, the markers that you can also can

Pamela Maher

09:06 - 09:11

be used in tissue, in vivo, in like tissue sections.

Nick Jikomes

09:11 - 09:25

So there are multiple ways that cells can die. There’s apoptosis, there’s a number of other cell deaths that can happen. The end result of all of these is that the cell goes away, it gets cleared out, it dies. It’s not there anymore.

Nick Jikomes

09:25 - 10:08

But the specific steps, the causes or the inducers of all of these different forms of cell death can be different. And the steps and the level of regulation can differ between these forms of cell death in terms of what’s causing them and how they play out and how the cell might be, say, communicating with the immune system in order to be cleared out in the end. One of the things that you said that I think is important is in apoptosis, for example, programmed cell death, the cell is basically sending out a signal or putting a signal on its surface to tell the immune system to clear it out so that the cell can be removed safely, let’s say, without the cell leaking or bursting open and causing more problems.

Nick Jikomes

10:08 - 10:23

How important is it when a cell dies that it sends out a signal on its surface to be engulfed by a macrophage or something like that versus gets physically torn open and releases its insides to the external environment?

Pamela Maher

10:23 - 11:09

I think it depends on the situation where the cell is damaged or dying. I think in terms of certain types of injury, particularly in peripheral injuries, probably releasing some of those molecules can be important in terms of the actual healing process. So bringing in various cells that can in the long-term contribute to the resolution of the injury. In the brain, I think, particularly in the context of aging and neurodegeneration, then that’s probably makes things worse.

Pamela Maher

11:09 - 12:11

So I think this release can be, have been, in certain circumstances, beneficial. And as I said, in certain types of acute injuries, I think there’s growing evidence that actually allowing some of this inflammatory process to proceed is actually helpful. But in other contexts, particularly where the system is maybe not as healthy, then so in these injury conditions, you get a the inflammation and you get a process of resolution, but perhaps particularly in the brain, particularly in the context of aging and neurodegenerative disease, that resolution process doesn’t occur.

Pamela Maher

12:11 - 12:39

So if cells start releasing their contents, this leads to sort of a feed forward mechanism where you’re getting more and more release of various components of the cells that can both aggravate the inflammatory process, but also directly, potentially directly damage surrounding cells. So it’s, I think context is important.

Nick Jikomes

12:39 - 12:57

Yeah, yeah. So, in the abstract, what are the major ways, what are the major things or reasons why cells need to die? Obviously, one of them is just physical damage. If I bump my knee or get a cut or I’m burned, there’s a bunch of cells in some patch of tissue that get physically broken.

Nick Jikomes

12:57 - 13:28

They’re split open, there’s holes poked in them, all of their insides are spilling out. And all of that stuff needs to be vacuumed up basically by the immune system so that things can be rejuvenated, new cells can grow there and be put in place and that type of thing. What about cells that aren’t physically like opened up through physical injury. What are some of the major inducers of a cell that will cause it to undergo apoptosis, say, to actually decide to send a signal that says, come eat me immune system?

Nick Jikomes

13:29 - 13:35

Are we talking about infection here, or are we also just talking about something inside of the cell not functioning properly?

Pamela Maher

13:38 - 14:35

This isn’t really my area of expertise, but infections can cause that. they can lead to, so the release of DNA into the cytoplasm in cells, either from mitochondria or potentially from a DNA viral infection or something, or I suppose bacterial infection, potentially can activate, pathways that I believe can lead to cell death. The other, and RNAs as well, viral, different RNAs can induce those. in cells that can eventually lead to death.

Pamela Maher

14:35 - 14:38

As I said, this isn’t really my area of expertise.

Nick Jikomes

14:38 - 14:50

So there’s a bunch of ways that cells can die. There’s different pathways, different mechanisms. But ultimately, well, let’s just think about this next. a lot of cells can be replaced.

Nick Jikomes

14:50 - 15:08

So it’s sort of okay in a sense that they die in certain contexts, at least because you could build a new one, you can build a new skin cell, you can build a new whatever cell. But of course, there’s some cells that can’t be replaced. One of those is the neuron, most mature neurons can’t be replaced. And we’ll probably get into that in the context of neurodegeneration.

Nick Jikomes

15:08 - 15:27

But is there anything that we should talk about upfront about I don’t know, protective mechanisms that maybe are special to post-mitotic non-dividing cells like neurons. Do they have any special systems in place to try and prevent things from going wrong and trying to prevent cell death from happening because they can’t be replaced?

Pamela Maher

15:28 - 16:27

You know, that’s an area, that’s an interesting question, but it’s an area that really hasn’t received all that much attention. I think people are starting to think about that more, this concept of resilience. And so I was just reading an article a summary of some work that had been done recently where they’re trying to actually, in the context of a variety of particularly of genetic diseases, particularly those that seem to manifest very differently, even within a family say that people, multiple people carry the gene for this disease, but when they get it and how severe the symptoms are in certain diseases can vary quite a lot.

Pamela Maher

16:27 - 17:17

And I until people haven’t really understood why. And now they’re starting, because of all these big databases that are available, and how cheap DNA sequencing has, and readily available, has become, different groups have started to explore this to see if in the people who have this specific genetic mutation, but seem to manifest it either not at all, or the symptoms are very mild or occur only much, much later than say the kind of traditional picture of the disease. Trying to identify genes that might be protective in the context of the disease.

Pamela Maher

17:17 - 18:09

In this paper I was reading, there was an example with sickle cell anemia. And some people with it have, they recognized a number of years ago, have much milder symptoms. And it turns out that they are able, they don’t shut down the synthesis of a kind of an embryonic form of hemoglobin that is not susceptible to the same mutation that gives rise to the, the form of hemoglobin that leads to the damage in sickle cell disease. And that’s actually how the gene modification approach that they’re using to treat sickle cell disease relied on discovering this, this...

Pamela Maher

18:12 - 19:05

knocking down this protein that’s normally involved in turning off the sort of embryonic form of hemoglobin. So they’re taking this, trying to identify these genes for a number of of genetic diseases that might provide resilience. So yeah, there seem to be, at least in certain individuals, the ability to modulate the consequences in this context of genetic disease, but you could probably extend this to aging or exposure to various toxins or other things like that. that people, and it’s, some people are going to be much more resilient.

Pamela Maher

19:05 - 19:15

And so, but there hasn’t been until more recently much exploration of that concept.

Nick Jikomes

19:16 - 19:46

And so can you give us, so we’re going to talk about Feroptosis and some of the biology related to acute versus chronic stress. iron biology, polyunsaturated fats and oxidative stress and sort of tie all these things together for people so they can understand how this process unfolds. I want to ask you a little bit about some basic iron biology to give people a little bit of a background here. Probably the most famous role, I think, for iron in the body is its role in hemoglobin to help carry around oxygen in the blood.

Nick Jikomes

19:46 - 19:57

But when we’re thinking about individual cells and what’s going on inside of them, Can you give us a basic picture of what are some of the key things that iron is doing inside of our cells that makes it just so important?

Pamela Maher

19:59 - 20:42

Well, iron plays a key role in the function of a number of different proteins in the cell. Some of the most important of those proteins are in the mitochondria and they are involved in energy production as well as other functions of the mitochondria. And there’s a whole family of proteins called iron sulfur cluster proteins. So they have iron as part of their key catalytic activity.

Pamela Maher

20:42 - 21:18

And these are found both in mitochondria, but also in the cytoplasm. And when those, in the, when there isn’t enough iron, those proteins don’t function properly. And there’s quite a large number of proteins that involve iron enzymes that are lipoxygenases, which can produce, lipid peroxides, but also have important roles in immune response and other processes. Those are iron-containing proteins.

Pamela Maher

21:20 - 22:17

So there’s a number of different proteins that are important for cellular function that require iron for their activity. And... Yeah, so when there’s not enough iron or either there’s not enough iron or the iron handling is somehow messed up or actually the ability of these proteins to incorporate The formation of these iron-sulfur clusters is actually fairly complex, involves several enzymatic steps. And so when that is somehow altered detrimentally, that can also, the cell thinks it, I think, thinks it doesn’t have enough iron even when it does.

Pamela Maher

22:18 - 22:38

Because it can’t make, I think the cell is telling itself, I need these proteins, I need these. But because the process of making them is somehow disrupted, there becomes a problem. So the cell keeps bringing in iron to make them, but then it can’t do it.

Nick Jikomes

22:39 - 22:55

So in addition to helping us transport oxygen through our bloodstream, iron is important for mitochondria. And basically almost all of our cells have mitochondria. Most of our cells have a lot of mitochondria. And as people probably know, mitochondria are the epicenter of oxidative metabolism.

Nick Jikomes

22:55 - 23:07

This is where most of our ATP comes from. And iron ions have multiple states they can exist in. They can have a plus two or plus three charge. So basically they can lose or gain an electron.

Nick Jikomes

23:07 - 23:25

And so when we think about mitochondrial biology, there’s the electron transport chain. Mitochondria basically are moving electrons as part of the elaborate mechanism they have to make all of this ATP. And iron is involved in basically some of the mechanisms, some of the steps in that chain that are moving electrons around in order to generate ATP.

Pamela Maher

23:27 - 23:57

Yeah, yeah, at least in the function of the proteins that are involved in the different steps of the electron transport chain, yeah. It doesn’t... Yeah, I’m not totally sure that iron is... Changes in the redox of iron from iron two to iron three or back are directly involved in the electron transport chain, but it is involved in the function of proteins that form the different complexes of the electron transport chain.

Nick Jikomes

23:57 - 24:14

So yeah, iron’s important for mitochondrial function. And if we ignore the details, if the mitochondria break down in some way, then the cell’s got a real big problem. So this maybe starts to hint at how iron could potentially be a good signal to tell a cell whether or not its mitochondria are functioning properly.

Pamela Maher

24:19 - 24:20

Yeah, potentially.

Nick Jikomes

24:22 - 24:32

And so, how did you come to the discovery of feroptosis? Can you give us just a very basic summary of how that unfolded?

Pamela Maher

24:33 - 25:19

Well, this goes back quite a number of years. So, a long time ago, we were actually interested in the discovery of feroptosis So in nerve cells, there’ve been known for, there was a number of years that the activation of what are called excitotoxic glutamate receptors can be harmful to nerve cells and cause their death. But these receptors are also involved in synaptic transmission, so sending signals between neurons. But over, activation of these receptors, as I said, could result in nerve cell damage and death.

Pamela Maher

25:19 - 26:26

And people had been searching for ways to study this in cell culture outside the animal to be able to do more mechanistic type studies. And so there’d been a real search for particularly or potentially nerve cell lines that could be cultured indefinitely. Because all of the studies that were done, and actually still are done, are on primary neurons that have to be isolated frequently from rodent embryos. And so a long time ago now, Dave Schubert, my late collaborator, and I were interested in, he was particularly interested in trying to identify a nerve cell line that had, was responding, that displayed this excitotoxicity in cell culture.

Pamela Maher

26:26 - 26:55

And there was a publication he read that described a line. And so he got this line, and it turned out that they did not have excitotoxic receptors, but they were still quite sensitive to glutamate in that it was still able to kill them, but not at a high level. at a higher concentration that normally induced excitotoxicity. And also we, through PCR, showed the cells didn’t express excitotoxic receptors.

Pamela Maher

26:55 - 28:04

And about the same time, a group at the Johns Hopkins University described a high concentration also described glutamate as killing nerve cells through a process that didn’t seem to involve excitotoxic receptors. And so we were, became intrigued by this pathway because, you know, the cells, as I said, it was a higher concentration of glutamate that’s usually involved in excitotoxicity, but still at a levels that could be achieved in vivo under certain circumstances of glutamate. So we started exploring it a lot further. And based on this previous work from the group at Johns Hopkins, we confirmed that it involved loss of glutathione seem to involve the inhibition of a transporter which exchanges cysteine, the oxidized form of the amino acid cysteine, which is rate limiting for the synthesis of glutathione, which is

Pamela Maher

28:04 - 28:41

the major intracellular antioxidant. that set up a cascade of changes in the cells that eventually led to death. And so we decided to pursue the different steps in that process, and found that it involved the, you know, Following glutathione loss, you’ve got a major increase in reactive oxygen species production, particularly out of the mitochondria, and activation of lipoxygenases, which are these enzymes that can generate lipid peroxides.

Pamela Maher

28:42 - 29:09

and then influx of calcium and cell death. And we described this pathway as oxytocin. But it remained sort of a niche pathway because we described it specifically in nerve cells and it was associated with glutamate, which is an enzyme is more associated with nerve cells than other types of cells.

Nick Jikomes

29:09 - 29:12

You didn’t know at that point how general this form of death was?

Pamela Maher

29:13 - 29:43

Well, we did do some studies even before that. We showed that we could kill other types of cells, except it took quite a lot of higher levels of glutamate. the relevance to these other types of cells, at least in the physiological context, wasn’t entirely clear. But we did show in a number of studies, like with different types of fibroblasts, that we could induce this pathway.

Pamela Maher

29:43 - 30:32

And also, we did this with, I have a collaborator, Anne Hanekin, that works on the eye, is a retinal surgeon. And we had a number of papers showing that different types of cells in the eye that are impacted in macular degeneration, so retinal pigmented epithelial cells, and also the cells in glaucoma, retinal ganglion cells that also seem to be quite sensitive to this pathway. So we knew it was more general than just nerve cells, but we didn’t realize how general. And others, just to step out, others had also shown that other types of cells in the brain, such as oligodendrocytes, are very sensitive to this pathway.

Pamela Maher

30:32 - 31:32

But then in 2012, Brent Stockwell’s lab published a paper where they read They described this same pathway, but in the context initially of cancer cells, and they called it varroptosis because they highlighted the role of iron in the pathway. We knew that iron was involved, but because we had shown that iron chelators could protect from the activation of the pathway, but that hadn’t been a big focus of our work probably. mistakenly. But so the Stockwell lab showed, they used, and they did this through a synthetic molecule called arastin, which it turned out inhibits the same transporter that exchanges cysteine for glutamate, only the way glutamate blocks it is by directly, it’s a anti-porter, so it exchanges cysteine for glutamate, so

Pamela Maher

31:32 - 31:53

it’s just excess extracellular glutamate blocks the cysteine uptake, whereas this small molecule called arastin, irreversibly inhibits this exchanger, but it’s the same protein. And the consequences are the same in that you get a depletion of glutathione in the cells, which sets off this whole pathway.

Nick Jikomes

31:54 - 32:14

So glutathione is a major antioxidant. Antioxidants help protect our cells against oxidative stress, which comes from the use of oxygen in our cells. Obviously, a major site at which oxidative metabolism is happening and oxidative stress and reactive oxygen species are generated are the mitochondria, right? Because that’s where the electron transport chain is.

Nick Jikomes

32:14 - 32:33

That’s basically why we breathe oxygen. It’s got to go there. That’s a site of oxidative stress, but also that’s a lot of where the action is in terms of antioxidants, because the whole point of an antioxidant is to help protect the cell from oxidative stress. Before we dig into that a little bit more, I want to ask you a little bit more about iron.

Nick Jikomes

32:34 - 32:46

So we said it’s important for certain aspects of mitochondrial function. You often hear a lot about the concept of iron storage. Our bodies store iron in some organs. We store a lot of iron.

Nick Jikomes

32:46 - 32:55

At the level of organs and at the level of cells, how and why is actually iron stored? Can you give us just a basic sort of picture of that?

Pamela Maher

32:55 - 33:38

Yeah. So the major storage molecule for iron is a a complex called ferritin that’s made up of both two proteins, both ferritin heavy chain and ferritin light chain. And these come together in complexes of large numbers of these proteins, like up to 24 or more. And they, can store hundreds, if not more, or thousands of iron molecules in this complex, and then release it when it is set.

Pamela Maher

33:39 - 33:58

it’s it’s needed for say the synthesis of this um iron iron cluster proteins or or this active synthesis of other types of proteins that that require iron correct got it so basically ferritin there’s a protein complex that can stick to a bunch of iron atoms and hold on to them store them

Nick Jikomes

33:59 - 34:09

to use them if and when they’re needed. So there must be a lot of need inside of cells to have iron stored up and ready to go just in case.

Pamela Maher

34:10 - 34:30

Yeah, there seems to be. Well, ferritin can be very quickly induced by excess iron. So ferritin levels go up. in cells and at least in cell culture models within a few hours.

Pamela Maher

34:32 - 34:33

Yeah.

Nick Jikomes

34:33 - 34:37

So basically the cell can pretty quickly change its capacity to store iron.

Pamela Maher

34:37 - 34:38

Yes. Yeah.

Nick Jikomes

34:39 - 34:44

And is that related to problems that can come from too much iron being around?

Pamela Maher

34:46 - 35:48

It can be a marker of too much iron being around, but it can also be a marker, I mean, it can be good and bad. I mean, it can be good of, I mean, a marker of too much persistent, like too much iron persisting for an extended period of time, but it’s also an indicator that the cell or the tissue is responding. It can be, as I said, it can come up and it can go down as well. So it may be, changing just with various in peripheral organs such as the liver or something it I’m not actually don’t know this for sure but it could vary with your diet or or other environmental exposures such as that.

Nick Jikomes

35:49 - 36:03

So iron is important for mitochondrial function. The cell’s got ways to store it and change how much can be stored. Mitochondria are the site of oxidative metabolism in our cells. They’re also the site of a lot of the reactive oxygen species production.

Nick Jikomes

36:03 - 36:42

So the sparks flying off the engine that can potentially damage components of the mitochondria or the cell But we’ve got glutathione and antioxidants that can help contain those reactive oxygen species. And then obviously, it should be pretty intuitive that these things should be in good balance, right? You can handle a certain amount of oxidative stress because you’ve got antioxidant systems like glutathione, but obviously if those antioxidant systems for some reason get depleted or there’s just too much reactive oxygen species production, it overwhelms the antioxidant systems and then the mitochondria or other parts of the cell can take on damage and experience oxidative stress more.

Nick Jikomes

36:42 - 36:53

You’ve also said something once or twice about lipid peroxides. How do lipids and lipid peroxides come into the picture when we think about oxidative stress and cell function?

Pamela Maher

36:56 - 37:57

So lipid peroxides are generated from what are called polyunsaturated fatty acids, which are one of the major constituents of cell membranes, and they help to make them flexible. So because they have a double bonds, they are these lipids, these, fatty acids in the membrane go into the, yeah, the outside, what faces the outside of the cell or the cytoplasm are the head groups, which are called phosphatases. And then the lipid molecules themselves go into the membranes. And you want a membrane that is not entirely stiff, but has some flexibility for cells to move around and move around.

Pamela Maher

37:58 - 38:04

and if they’re a cell in the bloodstream, then to be able to squeeze through capillaries and blood.

Nick Jikomes

38:04 - 38:25

So you want your membrane to be fluid enough to be a little pliable and bendable so that it’s not so rigid. Basically what you’re saying here is there’s a trade-off. The more polyunsaturated fats in the membrane, the more fluid and elastic the membrane is, those red blood cells can squeeze through tight spaces and so on and so forth. But there’s a downside to having a lot of those polyunsaturated fats, right?

Pamela Maher

38:25 - 38:36

Yes. So they can be what are called oxidized. So oxygen molecules can bind to them. And that can happen two ways.

Pamela Maher

38:36 - 39:15

It can happen enzymatically through the activity of those enzymes I mentioned previously, like poxygenases. And probably more as importantly, it can happen non-enzymatically through a process called the Fenton reaction. And this involves, is the interaction of ferrous iron, which is Fe2, F plus two, with hydrogen peroxide. And hydrogen peroxide is produced by the mitochondria and sort of a, byproduct.

Pamela Maher

39:17 - 40:04

And it’s also produced by a couple of other enzymes within the cells, like NADP oxidases. So the hydrogen peroxide interacts with ferrous iron to generate what’s called a hydroxyl radical, which doesn’t move very far, but is extremely reactive, and particularly can react with these double bonds in unsaturated fatty acids. generate, oxidize them. So iron, excess iron, free iron, what are called labile iron, plays a key role in generating these lipid peroxides.

Pamela Maher

40:04 - 41:10

And the lipid peroxides themselves, not only does it alter the fatty acid chain of the the phospholipids in the membranes, but it also can lead to the production of what are called reactive carbonyl species, that these can break down and form a small molecule that, based on the lipid fatty acid chain, that can then interact with phospholipids. particularly with proteins, and when it does so, it can alter their function and it can also promote, in some cases, aggregation. the consequences of lipid peroxidation are not just alterations in the membranes themselves, but also potentially these modifications to various proteins, which can have additional consequences on top of affecting potentially membrane function.

Nick Jikomes

41:10 - 41:41

So reactive oxygen species and oxidative stress can cause unregulated oxidation of these polyunsaturated fats. And when this lipid peroxidation happens, it can generate highly reactive lipid peroxides. These are basically just molecules that come from the oxidation, the non enzymatic unregulated oxidation of these polyunsaturated fats. And these things will then just go on and react with something like a protein or some other cell component and break its function.

Nick Jikomes

41:42 - 41:59

So when we think about cell damage or cell dysfunction, this is sort of like where the rubber meets the road. We’re generating reactive molecules from oxidation of lipids that then go touch a protein or something else and break or hamper its ability to do its normal intended function.

Pamela Maher

42:00 - 42:01

Yes. Yeah.

Nick Jikomes

42:01 - 42:19

Okay, okay. And so reactive oxygen species can oxidize polyunsaturated fatty acids, and there’s a lot of them in the double membrane of mitochondria. And of course, that’s where a lot of the oxygen and oxidative metabolism is happening. Where exactly does iron come into the picture here?

Pamela Maher

42:19 - 42:53

You mentioned ferrous iron, but how is this sort of facilitating the- Well, because it interacts with the hydrogen peroxide that’s produced sort of by both comes from mitochondria, but also comes from several other enzymatic sources. And that through this chemical reaction called the Fenton reaction generates these hydroxyl radicals that directly react with the unsaturated bonds in phospholipids to oxidize them.

Nick Jikomes

42:53 - 43:08

I see. So basically this Fenton reaction generates some of these cell toxins by oxidizing lipids. And if there is too much of iron basically floating around, if you’ve got an overabundance of iron, you’re going to tend to get more of these toxins produced?

Pamela Maher

43:08 - 43:08

Yes.

Nick Jikomes

43:09 - 43:24

Yeah. Got it. And so when we think about the full set of steps to go from healthy cell to ferroptotic cell death. What is actually triggering the final phase of this when the cell actually dies?

Nick Jikomes

43:24 - 43:38

I would imagine there’s just an increased oxidative stress burden, you’re getting more of these lipid peroxides forming, they’re damaging proteins and stuff. At sort of the end of this causal chain, what is causing the ferroptosis and the actual cell death to happen?

Pamela Maher

43:38 - 44:12

That is actually still being debated. We showed a number of years ago and in other studies more recently, that there is an influx of calcium into cells that seems to be very shortly before the actual death. Not everybody agrees with us, but I still think that that plays an important role. Exactly what mediates that transport, There’s been a couple of, it’s not fully clear.

Pamela Maher

44:12 - 44:34

So that comes as a result of these changes in both the the cell membrane and also the mitochondrial function.

Nick Jikomes

44:35 - 45:08

But, you know, ignoring some of the gory details here, essentially what we’re talking about, if I’m hearing you correctly, is for some reason, there’s an increase in oxidative stress in the cell. Maybe antioxidant systems are depleted. Maybe you’re producing more ROS, maybe both. Iron is part of the picture here, you get more lipid peroxidation, you get more of these highly reactive toxins produced, they start breaking proteins and other things in the cell, and at some point the cell is basically just so broken and dysfunctional that it’s going to be killed.

Pamela Maher

45:09 - 45:11

Well, it’s going to die. Yeah.

Nick Jikomes

45:12 - 45:25

Yeah. It’s going to die. And when it dies this way, through ferraptosis, is it just that one cell is going to die and go away? Or can dying this way actually cause nearby cells to also have problems?

Pamela Maher

45:27 - 46:07

It can also cause nearby cells to have problems. I think there’s one group, at least, that showed that there, at least in some models, they’ve seen sort of waves of theroptosis. in tissues where all the cells are connected. So that, yes, I think there’s some idea that because the cells, unlike the apoptosis that I mentioned earlier, the cells release their contents and some of those contents can be damaging or activate various processes in other cells.

Nick Jikomes

46:07 - 46:25

When we think about this happening, ferraptosis, is this the type of thing that happens relatively quickly or does it play out over an extended period of time? Are you going to have some stressor that induces a bunch of oxidative stress and the cell dies pretty quickly or can this play out over much longer periods of time?

Pamela Maher

46:25 - 47:46

Well, this has been sort of a big question in the field, because it can happen very quickly, particularly in culture, which is where many people study it. But when you’re talking about it in the context of, neurodegenerative, age-related neurodegenerative diseases or other diseases that occur over even peripheral diseases that occur over a lengthy period of time, then that that’s sort of a an oxymoron. I mean it’s it’s hard to apparent contradiction because if it happens really quickly but that always happens really quickly, then how do you say, well, it’s involved in something that some disease or that happens over an extended period of time? And this is where we came up with this idea of therapeutic stress or chronotherapeutosis that we’re calling it, where some of these changes such as decreases in glutathione, increases in iron, increases in lipid peroxidation, accumulate slowly over time, leading to changes in cellular function and perhaps loss of

Pamela Maher

47:47 - 48:40

some level of resilience to various insults. But the cells can stay in this state. They’re viable and I mean, I guess would depend on the degree of changes functional, but they are not fully healthy. And so based on a lot of the studies I’ve read on on Feroptosis, often people, particularly that involve studies in animals, they see these changes like loss of, decreases in glutathione, increases in lipid peroxidation, changes in some enzymes involved in the, levels of enzymes involved in the therapeutosis

Pamela Maher

48:40 - 49:24

pathway, but they often don’t really see much cell death. And so I think that this, therapeutic stress or chronophereptosis actually may be as important or more important than the actual death because I think it sets the cells up for not functioning fully and also sets them up to be more sensitive to other types of insults, either directly related to therapeutosis or related to other types of stress that then make them more likely to die when exposed to those other stresses.

Nick Jikomes

49:24 - 49:51

So basically under different conditions, a cell’s sensitivity or propensity to be killed off like this can change. And I can imagine at least a couple of different ways that a cell’s sensitivity to these types of stressors could change. One of them would be antioxidant depletion. If for some reason your glutathione antioxidant system is going down, then you’re less able to cope with the normal levels of oxidative stress that a cell is going to have to deal with.

Nick Jikomes

49:52 - 50:08

Perhaps another one would be the amount of lipid peroxidation that can happen, which I imagine is going to be a function of how many of these polyunsaturated fats are actually in the membrane. Can you talk about what some of these levers are that actually make a cell more sensitive to oxidative stress and ultimately ferropetosis?

Pamela Maher

50:09 - 50:47

Well, one of the key enzymes that I haven’t brought up yet that sort of ties glutathione to lipid peroxidation is an enzyme called glutathione peroxidase-4. And there’s a family of glutathione peroxidases. I think there are seven or eight of these enzymes. and as the name suggests they use glutathione and they require glutathione for their activity and they remove various forms, various oxidants from cells.

Pamela Maher

50:47 - 51:30

Glutathione peroxidase-4 is the only one of this family that can remove lipid peroxides from lipids in membranes. So it converts them to lipid alcohols, which are non-toxic as opposed to the oxidized form. And The glutathione peroxidase-4 is absolutely dependent, as I mentioned, on glutathione for activity. So if glutathione levels, even if there’s plenty of glutathione peroxidase-4 around, if there’s a real shortage of glutathione, then this enzyme can’t perform its function.

Nick Jikomes

51:31 - 52:16

So there are antioxidant enzymes, like the ones you’re talking about, and they are proteins, they’re enzymes that can basically neutralize these toxins, these lipid peroxides that we’ve mentioned, but they need glutathione to be present. So you’ve got glutathione, you’ve got these peroxidases, they both need to work together. And even if you get some lipid peroxides formed from these polyunsaturated fats getting oxidized in the mitochondrial membrane, say, they can basically be handled and neutralized and taken care of by this antioxidant system, the peroxidases working together with glutathione. But if for some reason the peroxidase isn’t there or your glutathione’s been depleted, now there’s lower capacity to actually handle these highly reactive toxic lipid peroxides.

Nick Jikomes

52:16 - 52:23

And instead of removing them from the membrane and neutralizing them, they can cause damage and cause damage to spread.

Pamela Maher

52:24 - 52:40

Yes. Yeah. Yeah. And so the glutathione peroxidase is critical in the context of the asperoptosis pathway, because as I said, it’s the only enzyme that can remove lipid peroxides from lipids in membranes.

Pamela Maher

52:41 - 53:12

Some of the other glutathione peroxidases play important roles in removing other types of reactive oxygen species, and again, they are also impacted if glutathione levels go down. And there’s actually a form of glutathione peroxidase that seems to be specific for mitochondria. So that does highlight the need to protect mitochondrial membranes as well as the plasma membrane.

Nick Jikomes

53:13 - 53:40

And, you know, thinking about the causal chain here, can you have cells where say they’re experiencing an increase in oxidative stress, there’s more ferrous iron around and or there’s more reactive oxygen species being produced, They’ve got these peroxidase enzymes and this glutathione antioxidant system to handle that for some reason. Can you induce increased oxidative stress, increased iron overload, but then prevent the lipid peroxidation and prevent the cell from dying?

Pamela Maher

53:46 - 54:45

So the people, a number of labs have developed compounds that inhibit, that either directly inhibit lipid peroxidation, or can also, from through various processes that inhibit lipid peroxidation, indirectly inhibited. So there are some of the first paraptosis inhibitors that were described are direct inhibitors of lipid peroxidation. And we, several years ago now, we did a screen of a variety of natural products and found, interestingly, that many of them had no effect on actual just reactive oxygen species production, but they could block lipid peroxidation. And most of them did it directly.

Pamela Maher

54:45 - 55:37

So we could generate, test this in a synthetic, what are called liposomes, where we incorporated a dye that we can use to monitor the oxidation. And we could show that liposomes a variety of compounds that we identified that were protective against ferruptosis, were direct inhibitors of lipid peroxidation. And this has become a bit, can be a bit of a problem if you’re looking at pathways involved in ferruptosis, because, you know, some there have been molecules that suggested some signaling pathway was involved, but it actually turned out that the classical or standard inhibitor of that pathway was a direct inhibitor of lipid peroxidation.

Pamela Maher

55:37 - 56:07

So even though using this inhibitor blocked the baroptosis, it wasn’t because necessarily it was inhibiting this pathway, but it had, because of the nature of its structure, it could act as a direct inhibitor of lipid peroxidation. So that’s something that, yeah, if you’re proposing a novel pathway sometimes and you’re using only like a single inhibitor of that pathway, you gotta be a little careful.

Nick Jikomes

56:08 - 56:29

Yeah. So there are compounds out there that can prevent lipid peroxidation and help prevent cell death, ferrooptotic cell death, even if the cell is experiencing an increase in oxidative stress, it’s producing more reactive oxygen species. You’re just, this compound is just getting away in the way of the downstream step that would initiate lipid peroxidation that would then trigger the cell death.

Pamela Maher

56:29 - 57:09

Yeah, there’s also compounds that can reduce fatty acid synthesis. Fatty acids, for reasons that aren’t totally clear, because the production of unsaturated fatty acids is quite complicated, but it seems that compounds, some compounds that reduce fatty acid synthesis either directly or indirectly or promote lipid oxidation can also be protective. not acting as direct inhibitors of lipid peroxidation, but somehow it’s not fully clear, reducing the substrate load, I guess.

Nick Jikomes

57:09 - 57:28

So a couple of different ways to help prevent lipid peroxidation induced cell death. One would be to neutralize or take care of the lipid peroxides, the toxins that are formed as they’re being formed. Another way would be to decrease their production or the rate of production by, say, lowering polyunsaturated fatty acid production.

Pamela Maher

57:29 - 58:05

Yeah. And then there’s also reducing upstream reactive oxygen species production, like hydrogen peroxide production, that could lead to the generation of the hydrosyl radicals that induce the lipid peroxidation. And also it’s been shown, so as I said, lipid peroxides can also be generated enzymatically. And there are a number of inhibitors of the lipoxygenase enzymes that do this.

Pamela Maher

58:06 - 58:15

And so those inhibitors have also been shown to be fairly effective, at least in models of feral ptosis.

Nick Jikomes

58:18 - 58:41

In essence, what we’re talking about here is there are components of membranes like mitochondrial membranes, like polyunsaturated fats. And if you burn them, if you take an oxygen and do oxidation, they’re basically going to release toxic fumes. And there’s multiple ways that a cell might handle or regulate this problem. You can sort of quench or neutralize the fumes as they’re being formed.

Nick Jikomes

58:42 - 59:02

you could put less dry kindling in the membrane so that there’s less of it being lit on fire, or you can decrease the amount of sparks that are flying off your machines in the form of reactive oxygen species to light that on fire to begin with. Is that roughly like an idea how people can start to conduct it?

Pamela Maher

59:03 - 59:06

Yeah. Yeah, I think that’s fairly accurate, yeah.

Nick Jikomes

59:07 - 59:14

So what are some of the compounds that you guys have seen? What are some of the specific compounds that help prevent lipid peroxidation from happening?

Pamela Maher

59:16 - 1:00:30

Well, yeah, the original compounds, ones people use in a lot of research studies are two compounds called ferrostatin, which was developed by Brent Stockwell’s lab and leproxtatin, which was developed by Marcus Conrad’s lab. But in terms of therapeutics, those have not, they don’t, they have poor pharmacokinetics and they haven’t, they’ve kind of worked in animal models, in terms of actual drugs for humans, I don’t think they’re, I mean, they haven’t been pursued, I think, because of the poor pharmacokinetics. But we’ve, so we’ve identified a number of compounds and other, well, yeah, we’ve identified a number of compounds that, you know, are very effective against this pathway, mainly more by probably indirectly inhibiting lipid peroxidation. There’s several natural products that we’ve been working on.

Pamela Maher

1:00:32 - 1:01:19

for a number of years, a couple of flavonoids, one called Vicetin, another called Sterubin. And then we also have some synthetic molecules, one based on the polyphenol curcumin, that’s called J-147, and another one based on the flavonoid Vicetin called CMS-121. And both of those, so the flavonoids themselves work at like low micromolar concentrations, but the synthetic compounds work at nanomolar concentrations to block this pathway. And they seem to act at, I think the most effective compounds actually seem to act at multiple steps.

Pamela Maher

1:01:20 - 1:01:57

So both Fisetin and CMS-121 as well as Darubin are not only able to They are able to increase glutathione levels significantly, and they also can reduce lipid peroxidation, but in some cases indirectly. J147 doesn’t affect glutathione levels, but it very much indirectly reduces lipid peroxidation.

Nick Jikomes

1:01:59 - 1:02:34

And so set us up for understanding your new study. So it looked to me like one of the key questions here was this issue of time. If you increase the stress on a cell, iron overload and so forth, there’s a difference between an acute stressor, which a cell might be able to deal with to some extent right now, versus a chronic stressor where, you know, over time you’re wearing down antiaccident systems or something like this, so that even though the cell could deal with some of this oxidative stress for a while, at some point it can no longer keep up with it, and then, you know, you’ve got a problem.

Nick Jikomes

1:02:34 - 1:02:40

So what was sort of the basic setup and motivation for the recent paper on chronofaroptosis?

Pamela Maher

1:02:41 - 1:03:15

Yeah, well, it was what I mentioned that we and others have been saying for a long time that the therapeutosis is involved in neurodegenerative diseases and other... well, particularly neurodegenerative diseases. And we’ve been gotten pushback because people say, well, like in particularly say in Alzheimer’s disease, the nerve cells really don’t die until much, much later in the disease. But then you’re telling us it’s involved in potentially development and certainly the progression of the disease.

Pamela Maher

1:03:16 - 1:03:49

So, you know, the people were saying, we don’t buy this because you’re in culture, cells die very quickly from when you activate this pathway. And so we would expect to see massive, if it’s involved in AED, massive nerve cell death early on, and that’s not what’s seen. And that’s the case. Well, with Parkinson’s, there is more death, at least in the substantia nigra, probably, earlier on.

Pamela Maher

1:03:50 - 1:04:33

But nevertheless, this seemed to be a real contradiction that we needed to try and resolve. And so we from some, and we got some hints of this from some studies we’ve done with Salvador Soriano at Loma Linda University and his lab, one of the areas they’ve been interested in is a genetic peripheral neuropathy called Charcot-Marie-Tooth syndrome. And in this case, it’s actually not neurocells that, so this is a progressive neuropathy. It’s genetically inherited.

Pamela Maher

1:04:33 - 1:05:02

There’s a number of different mutations that can lead to this disease, but the most common form is a mutation in a protein involved in myelin synthesis by Schwann cells. called PMP 22. And that’s, so this occurs, it’s an age-dependent disease. So people, most people don’t get symptoms until they’re in their 40s or 50s.

Pamela Maher

1:05:02 - 1:05:49

And it, they, as I said, it’s a peripheral neuropathy. So they develop problems with, you know, walking and then also in their hands and arms, but it has no impact on cognitive function. And so his lab was, they were, knew about our work on, well, what we called originally oxytocin, but baroptosis, but they thought that it, based on what was known from the literature in this disease, that perhaps this therapeutosis was involved, but not as a death pathway, more as a therapeutic stress pathway.

Pamela Maher

1:05:49 - 1:06:29

Because the Schwann cells, which are the impacted cells here, not nerve cells. So Schwann cells are the cells in the periphery that make myelin, that coats the neurons. the axons. And they so they developed they based on some both some cell culture studies as well as more looking at transcriptomic data that was actually already out there developed evidence that this pathway might indeed be activated in the context of sarcoma or E2 syndrome, but again, not as a

Pamela Maher

1:06:29 - 1:07:24

death pathway, at least until much later, but again, more as a stress pathway where these changes that I told you about, increases in lipid peroxidation, decreases in glutathione, changes and other changes in iron lead to cellular dysfunction. And that’s really what contributes to the disease and only later cell death. And so based on that, we were interested in, well, With that idea, with some suggestion already that this might actually happen in vivo, we decided to see if we could develop a model in the lab where we could compare a cell-based model, acute versus chronic stress, and specifically focus on stressors associated with this pharaohsiosis pathway.

Nick Jikomes

1:07:26 - 1:07:32

Yeah, and so how are you actually stressing cells and putting them in this chronic stressed state?

Pamela Maher

1:07:34 - 1:08:24

So we used two approaches. One is to give them chronic excess iron, which is already known to be well changed in the context of mild cognitive impairment and more so in, in the AD, in the regions of the brain that are affected in Alzheimer’s disease. And we also used glutathione, partial glutathione depletion, which also glutathione levels have been known for, particularly in the context of Parkinson’s disease to be very much decreased in the region of the brain that’s most affected, the substantia nigra. And early on before there’s really significant loss of neurons in that region of the brain.

Pamela Maher

1:08:24 - 1:09:13

So both of these changes are seen in the context of increased iron and decreased glutathione in the context of neurodegenerative diseases. And with newer techniques they’ve shown, it used to be that you had to look at samples from autopsy brains, but now they have techniques to look at both glutathione as well as iron in the brains of living subjects. This has confirmed, the ability to look at this in living subjects has confirmed that these changes indeed do happen fairly early on in the disease and are likely to be contributing factors. So that was why we use these two insults and use them.

Pamela Maher

1:09:13 - 1:10:10

Nawab Dar, the postdoc in my lab that led this study, he used a cell line called SHSY-5Y cells, which can be differentiated into neurons. And then he treated these with either acute iron, the same concentrations or chronic iron levels, say for nine days to see if he could sort of mimic this idea or to see if he could see differences between how cells responded to an acute exposure versus long-term exposure, which, I mean, it’s only nine days, it’s not, you know, years, but it’s still, you know, we felt somewhat mimicked the different, potentially the difference between... Yeah, might be more relevant to what was happening.

Nick Jikomes

1:10:11 - 1:10:35

Yeah, so to kind of review so far, lipid peroxidation produces cell toxins that can ultimately kill the cell. One thing that can increase lipid peroxidation is excess iron. Another thing that can increase lipid peroxidation is depletion of glutathione antioxidant systems. So these are two different ways, they’re not mutually exclusive, that a cell can become more sensitive to lipid peroxidation.

Nick Jikomes

1:10:36 - 1:10:51

and this therapeutic state. And we know that in neurodegenerative diseases in humans, in certain parts of the brain, depending on the disease, you either see iron overload, basically signs of excess iron and or depletion of antioxidant systems like glutathione.

Pamela Maher

1:10:52 - 1:10:52

Yeah.

Nick Jikomes

1:10:54 - 1:11:08

Okay, so walk us through the next step of the experiments you guys just did. So you did this nine day long experiment. So it’s nine days long, as opposed to a few hours long, and the cells are in this more susceptible state. What did you observe after that?

Pamela Maher

1:11:09 - 1:11:38

Well, we looked at a number of changes. We first focused on proteins, some of the key proteins in the therapeutosis pathway. we were surprised, really surprised, actually. And we always compared the acute treatment with the chronic treatment, again, using the same concentrations of either iron or an inhibitor of glutathione biosynthesis.

Pamela Maher

1:11:42 - 1:12:36

Initially, when we did the studies, Nawab did the studies with iron, he was really surprised to find that while the acute treatment with iron had no effect on this enzyme that I told you about, glutathione peroxidase-4, is the key enzyme for removing lipid peroxides. Chronic treatment led to a reduction in GPX4 levels, a big reduction. It also led to changes in several iron handling proteins, as well as including ferritin. But the which was not unexpected, because when we actually looked at the levels of free iron in the cells, in the acute treatment, we saw a sharp increase in free iron over the course of the experiment.

Pamela Maher

1:12:36 - 1:14:01

But over the chronic treatment, we actually saw an increase a decrease in free iron, which correlated with the increase, a big increase in ferritin after a few days, in this case, a few days of treatment. But the decrease in GPX4, glutathione peroxidase 4, was very surprising because there was, there’s still, this is something we’re continuing to study, but why it seems why this chronic iron would change the expression of that protein, or the levels of that protein so dramatically is completely unclear, but suggests that chronic exposure to some of these insults, I guess you’d say, that are associated with the aging brain in the context of these neurodegenerative diseases, that they could make these changes, drive some changes in enzymes, or this enzyme in particular, that normally you would think that the cell would up-regulate as a protective mechanism was very surprising.

Nick Jikomes

1:14:01 - 1:14:32

So basically, you do some iron overload of the cell for a little while, and there’s more iron around, but the antioxidant systems that can deal with the lipid peroxides that might be forming are in place, and so it’s not that big of a deal. But if you overload the cell with iron for several days, now you’ve got iron overload And for some reason or another, you deplete this glutathione peroxidase enzyme, and now the cell’s actually got a weakened ability to deal with the lipid peroxidation byproducts that are gonna form and damage the cell.

Pamela Maher

1:14:33 - 1:15:20

Yeah, yeah, and so we looked at lipid peroxidation in the cells, and we found indeed that the acute treatment had no effect on the levels of lipid peroxidation, whereas the chronic treatment, we saw a dose-dependent increase in the levels of I guess you’d say chronic lipid peroxidation in the cells. And that correlated with some also alterations in the production of mitochondrial reactive oxygen species and changes in the mitochondrial membrane potential, which also correlated is can be a driver of reactive oxygen species production.

Nick Jikomes

1:15:20 - 1:15:45

So basically you can stress a cell with iron overload. If you do that a little bit for a little while, the cell’s basically fine, it’s viable, its antioxidant systems aren’t affected and it can deal with any lipid peroxidation that might happen. And you don’t really see that much lipid peroxidation being induced. But if you just extend that iron overload for longer, now the antioxidant systems become depleted and the cell is now more susceptible to damage from lipid peroxidation.

Pamela Maher

1:15:45 - 1:16:46

Yes, yeah, yeah. And so what we found was that that was indeed the case, because then when we added either a low dose of a compound that directly induces the ferruptosis pathway, or a general oxidant, hydrogen peroxide, to the outside of cells, which we know actually does not induce ferruptosis, we found that there were even bigger increases in lipid peroxidation, and also the cells were now more likely, there was increased cell death. So I didn’t mention, and this is important, that the levels of iron we used were not killing the cells, nor would the, the level of loss of glutathione, the cells weren’t dying until we added an additional insult.

Pamela Maher

1:16:46 - 1:17:22

And then we did see increased susceptibility to death. And what was the most interesting, it wasn’t necessarily surprising that we, given that with, particularly with the iron, that it’s already, known to help drive therapeutosis that when we added a therapeutosis inducer we did see some cell death but when we added hydrogen peroxide at a level which by itself wasn’t didn’t did kill cells under the acute condition, we did see increased cell death under the chronic condition.

Nick Jikomes

1:17:22 - 1:17:43

Yeah. So some iron overload, but not enough to kill the cell, isn’t killing the cell on its own, but it’s putting it into this more susceptible state where now if you add another stressor that also wouldn’t ordinarily kill the cell, now all of a sudden the cell’s more susceptible and that stressor that was relatively benign before is now causing lipid peroxidation and even cell death.

Pamela Maher

1:17:44 - 1:17:47

It’s increasing lipid peroxidation and yes, causing cell death. Yeah.

Nick Jikomes

1:17:47 - 1:18:01

Yeah. Are there any particular lipid peroxidation products like HNE or MDA, any specific ones that seem to be doing the damage here or is it kind of a general- We actually, that’s a good question, but we haven’t explored that yet.

Pamela Maher

1:18:01 - 1:18:34

We didn’t look at the specific lipid peroxides or products of lipid peroxidation. But yeah, I mean, we wanted to, get this paper out there and kind of establish the idea. And then going forward, we plan to explore various aspects, details of what’s exactly what’s going on. For instance, as I mentioned, what’s happening and why are glutathione peroxidase four levels going down?

Pamela Maher

1:18:35 - 1:19:14

And also, Are there specific, your question is a good one. Are there specific products of lipid peroxidation that are increasing and perhaps, you know, going further, are there specific, because as I mentioned, these, you know, the HNE and the maldenaldehyde can bind to proteins. So are there specific targets of this that might be particularly relevant to the changes that are occurring in the cells?

Nick Jikomes

1:19:16 - 1:19:36

Do we know anything about the polyunsaturated fats that are actually driving this lipid peroxidation? Are there specific polyunsaturated fatty acids that are predominantly driving it or is it all of them? And related to that, how much do the levels of polyunsaturated fats actually change in the cell? Can they go up and down by quite a bit or are they relatively stable?

Pamela Maher

1:19:42 - 1:20:08

From other people’s studies, the phospholipid that seems to be most susceptible to being altered in the context of pharoptosis is phosphatidylethanolamine. So that’s the head group, and then it has fatty acids. But phospholipids contain, phosphoethanolamine phospholipids seem to be the most susceptible. I don’t think anybody knows exactly why.

Pamela Maher

1:20:10 - 1:20:45

They’re fairly abundant in membranes, but I’m not sure they’re, I don’t think they’re the most abundant of the phospholipids. The cells can change the levels of phospholipids fairly, fairly dynamically in various membranes. And as I said, some of the compounds we’ve identified that seem to be protective against this pathway, modulate fatty acid synthesis. And so, and they’re protective pretty, quickly.

Nick Jikomes

1:20:45 - 1:21:01

So, um, yeah, you mentioned that there’s, there’s compounds that modulate fat acid synthesis, so you can protect a cell or make it less susceptible to lipid peroxidation simply by decreasing the amount of polyunsaturated fats that are being produced and put into the membrane.

Pamela Maher

1:21:02 - 1:21:19

We, well, we think that’s what’s happening. Yeah. But, um, we, we actually, we, We don’t know for sure if we’re actually decreasing the levels of the polyunsaturated fatty acids in the membranes.

Nick Jikomes

1:21:21 - 1:21:55

And to what extent, if we kind of step back from this particular body of work for a second, and just talk about lipid peroxidation in general, to what extent is this a phenomenon that we see mostly in in vitro studies in a dish? And to what extent do we think this is actually happening in vivo in human beings? Are there markers or things that we can measure in humans that tell us that this is happening to some degree? Or is this likely to be something that happens in a very different way or to a very different extent in model systems like the ones you’re using compared to in living, breathing animals?

Pamela Maher

1:21:55 - 1:22:56

Oh, no, there’s actually quite a body of literature going back, oh, maybe 20 years, more evidence of lipid, or maybe more, yeah, 20 years, evidence of lipid peroxidation in samples from autopsy brains generally. of particularly AD patients and also some other Parkinson’s disease patients and I believe some other neurodegenerative diseases. So this is something, so mostly people have relied on these modifications that I mentioned that occur subsequent to lipid peroxidation through either this molecule called hydroxynonanol or maldenaldehyde or a couple of other products of lipid peroxidation that react covalently with proteins. And so you can use them as, and their production is specific to lipid peroxidation.

Pamela Maher

1:22:56 - 1:23:10

So you can use them as markers of lipid peroxidation, which makes them quite suitable for looking at on autopsy tissues.

Nick Jikomes

1:23:39 - 1:23:40

lipid peroxidation was in fact happening?

Pamela Maher

1:23:42 - 1:24:02

No. Well, they bind actually covalently to proteins. So they specifically bind to lysine residues in proteins. And so whether that is the cause of potentially impacting the function of these proteins and whether every protein that binds these is altered.

Pamela Maher

1:24:02 - 1:24:38

I don’t believe it’s known, but because the binding is covalent, it’s very stable. And so that makes them particularly... And there are antibodies. There are various ways you can detect them by mass spectrometry, but there are also antibodies to these groups that can be used, say, for looking at sections using immunofluorescence microscopy or by a technique called Western blotting where you separate proteins on a gel by size and can then

Pamela Maher

1:24:38 - 1:24:42

probe the different proteins with these antibodies.

Nick Jikomes

1:24:42 - 1:25:02

Yeah, so lipid peroxidation, you know, it does happen in our own cells and tissues to some degree or another, it’s probably gonna happen to different degrees, depending on your relative state of, you know, health, metabolic health, whether you’re diseased, your total antioxidant, stress capacity, and so on and so forth. So it’s not just something that happens in, you know, in a dish under contrived circumstances.

Pamela Maher

1:25:02 - 1:25:43

Oh, well, I should say to that. There’s been numerous studies in animal models, looking at theraptosis in the context of a variety of different diseases, not just neurodegenerative disease, but also various liver toxicities, cardiac, different forms of ischemia, cardiac ischemia, or cardiac toxins, kidney toxins. from groups all over the place. And almost everybody, if they’re looking at ferritosis, has looked at lipid peroxidation.

Pamela Maher

1:25:43 - 1:26:13

If they can get fresh tissue, then they can use some of these other probes for lipid peroxidation. But usually, often they use these other markers. But yeah, in terms of it happening in vivo, in the context of various diseases, there’s, yeah, really, an overwhelming amount of data that suggests that indeed this occurs in a variety of different contexts.

Nick Jikomes

1:26:15 - 1:26:33

And when we look at the literature there and we look at the animal studies that have been done, are there any things that can, any manipulations that can be done, changes in the diet, giving exogenous compounds, perhaps some of the ones you mentioned before, they can actually prevent or reduce the amount of lipid peroxidation that’s happening in various tissues?

Pamela Maher

1:26:34 - 1:27:37

Yeah, well, as I think I mentioned earlier, some of these two kind of ferrostatin or liproxstatin, which are the direct inhibitors of lipid peroxidation, in experimental models, people have often seen effects with those, because they’re trying to show that this ferroptosis pathway is involved in whatever they’re... disease that they’re interested in. So they’ve had focused on these well, inhibitors that everybody agrees blocks this pathway. So, but there’s other, I mean, people have used other compounds to show that target, particularly say if they think a specific pathway might be involved, they’ve used compounds that modulate those specific pathways.

Pamela Maher

1:27:37 - 1:27:45

As I mentioned earlier, you gotta be a little careful with those in case the compound they’re using actually also inhibits lipid peroxidation.

Nick Jikomes

1:27:46 - 1:28:10

And can you give us a sense for like, where do you guys wanna go Next, with respect to this stuff, this chronophereoptotic state where the cells are more susceptible and how it ties into neurodegenerative disease, where do you see this going in terms of mapping out how all this works and developing ways to treat things like neurodegeneration by preventing or inhibiting in some way lipid peroxidation from happening?

Pamela Maher

1:28:11 - 1:29:25

Well, in terms of where it goes next, as I said, we want to use our culture model to understand why is, say, chronic iron or chronic glutathione depletion, or how mechanistically it’s driving some of the changes that we saw. But we also want to establish this more clearly in tissues and in animal models. So initially, we’re thinking of using like tissue slices to characterize it more fully in tissues like the brain or the brain tissue. as a peripheral tissue, say the liver, where we can see if different types of cells perhaps in these tissues are more susceptible to changes, say initially changes in lipid peroxidation, which there are good fluorescent dyes to be able to monitor that.

Pamela Maher

1:29:30 - 1:30:17

understand what types of cells, you know, are all cells susceptible, say in the brain, are the nerve cells particularly susceptible to this? And, or are other types of cells also susceptible? And Also, our idea is that the susceptibility may change in the context of aging. So, as I said, initially, we’re hoping to use just isolated tissues where we make slices from different aged mice, say, and look at the susceptibility to activation of this pathway in different cell types.

Pamela Maher

1:30:17 - 1:30:57

But we’d also, like to pursue this in in vivo models. I mean, there are definitely models of iron overload in animals. Advantage of using the tissue slices initially is that all of the cells are exposed to the same level of, would be say of iron or even an inducer, low levels of an inducer, a direct inducer of ferreptosis. Whereas when you administer it systemically, different cell types may be exposed differently.

Pamela Maher

1:30:57 - 1:31:41

And so that makes it a little harder to interpret that cell type sensitivity. But the goal, eventually, is to test some of these ideas in animal models and see if, indeed, we can, there’s a fair amount of literature on iron overload, and it does suggest that it can contribute to cognitive dysfunction, particularly in the context of aging. So, yeah. it would be more tying this more directly to this therapeutic stressor.

Nick Jikomes

1:31:42 - 1:32:07

And I think you mentioned earlier that when we look at tissues from autopsies from individuals who had Alzheimer’s and other neurodegenerative diseases, we see indications of iron overload in the parts of the brain that are degenerating. Obviously you guys can induce iron overload in your experimental systems, Um, do we know much about what causes iron overload to you know, people out in the world? Are there dietary factors here? Do certain diseases cause it just as part of the disease?

Nick Jikomes

1:32:08 - 1:32:11

Where does iron overload actually where do we actually see it in people?

Pamela Maher

1:32:11 - 1:32:19

Um The well there is one disease, uh chemo chromo

Nick Jikomes

1:32:21 - 1:32:22

hemochromatosis.

Pamela Maher

1:32:22 - 1:32:52

Yes. Okay. And that although that’s interesting, because Males with that disease, at least with certain mutations, not all, there’s a number of different mutations that can cause that disease, which is an iron overload, a systemic iron overload disease. Males with that disease do, and I said, specific mutations do tend to develop cognitive problems, but females don’t, so they don’t understand that.

Pamela Maher

1:32:52 - 1:33:42

So that’s an interesting area, perhaps, of looking at these Brazilian spiders that I was talking, potentially earlier. The other reason cells might develop iron overload, particularly in the context of aging, is changes in proteins that handle iron, either bring iron into cells or move it out. So a protein, I think you mentioned earlier, the transferrin receptor is the main protein for bringing iron into cells, that’s quite dynamically regulated. But if that And then there’s a protein, actually the only protein that’s involved in export of iron, that’s called ferroportin.

Pamela Maher

1:33:43 - 1:34:22

And that could also, I think there’s some evidence that that changes in the context of at least of Alzheimer’s disease. I don’t know if it’s explored, though it goes down, but exactly in which cells. And there also may be changes, and I don’t think this has been explored all that much, in how iron is passed in the context of the brain, particularly between cells in the brain, and whether some of those systems may also change in the context of aging. So I think there’s iron metabolism.

Pamela Maher

1:34:23 - 1:35:12

Metabolism is fairly complex. And so I think there’s further avenues for exploration in the context of, particularly just in the context of aging. I should say, however, that Iron chelators, at least the ones they have tried so far, have not worked in patients in either Parkinson’s or in Alzheimer’s disease patients. And so it’s not entirely clear why they have failed because people, given all this evidence for the increase in iron, that and the evidence that how iron can be detrimental.

Pamela Maher

1:35:13 - 1:35:51

So in both cases, it was very disappointing, both for the investigators and the patients, but just for the general community that’s interested in these diseases, that the iron chelators really failed quite well. dramatically in terms of providing any benefits. And it may be that the problem are they just chelate too much iron. As I mentioned, iron is important for these mitochondrial proteins and other, the function of other proteins in cells.

Pamela Maher

1:35:51 - 1:36:11

So it may be that most of these iron chelators are pretty non, are nonspecific and maybe they just took out both the bad iron, the iron that potentially could cause damage, but also the iron that’s needed for key cellular functions.

Nick Jikomes

1:36:11 - 1:36:15

Right, right. Yeah, as with most things in biology, you don’t want too much or too little.

Pamela Maher

1:36:15 - 1:37:04

Yeah, yeah. No, that’s, so I think I would, I would guess that some of the labs that are really interested, we’re working iron design, on iron chelation as a potential therapeutic, maybe they’re still exploring whether there are better iron chelators out there that might not be quite as specific. I mean, well, might be more specific to get rid of, say, labile iron, but might not chelate other forms of iron that might be needed for cellular functions, or whether they decided that moving into other approaches might be more appropriate.

Nick Jikomes

1:37:04 - 1:37:16

Yeah. Are there any final thoughts you want to leave people with or anything you want to reiterate from our conversation today as it relates to oxidative stress, cell health, and lipid peroxidation, especially in the context of neurodegenerative disease?

Pamela Maher

1:37:18 - 1:38:25

Well, I think There’s been one of the things that’s come out of numerous studies where people have looked at lifestyle factors, particularly in the context of dementia, Alzheimer’s or other types of dementia. One of the... points that often it comes out of these are diet and how a diet that’s rich in fruits and vegetables and other nuts and other products and lower in fats and can be beneficial in terms of reducing the risk of developing dementia. So that would fit with the ideas of kind of overall that having more antioxidants or more compounds that can activate.

Pamela Maher

1:38:25 - 1:39:10

I didn’t mention there’s a protective pathway called, that’s mediated by a transcription factor called NRF2, which regulates the synthesis of a variety of antioxidant enzymes, including this enzyme GPX, glutathione peroxidase-4. And there’s, quite a bit of data that activation of that pathway can be protective against the periptosis and periptonic stress. And a number of dietary compounds are able to activate that pathway as well. Well, what are those?

Pamela Maher

1:39:10 - 1:40:11

Well, there’s a compound in broccoli called sulforaphane that can activate the pathway of that compound I mentioned, the flavonoid, Fisetin, and the other one I mentioned, Stirubin, can activate the pathway. Those are in, well, Fisetin is in a variety of, particularly in strawberries, but probably not at concentrations that would be sufficient to activate the pathway. I think people, there’s another one, Quercetin, which is found in, a variety of, particularly it’s the yellow color and yellow onions, but it’s also found in other fruits and vegetables. And so I think just having a variety of fruits and vegetables that maybe these compounds individually aren’t sufficient, but in combination would be enough if you’re consuming these on a daily basis too.

Nick Jikomes

1:40:11 - 1:40:24

So there are dietary components, such as some of the examples you just shared, and these things can affect the transcription factor that’s responsible for upregulating the antioxidant enzyme that we talked about before.

Pamela Maher

1:40:24 - 1:41:04

Yeah, and others as well that seem to provide other checks and balances on this pathway. Yes. So I think that would be right now, you know, there’s a lot of other risk factors for dementia, but in relation to what specifically ties into this chronophereptosis and also that people can do something about. I think, yeah, modulating diet has a strong potential to be helpful.

Nick Jikomes

1:41:05 - 1:41:11

All right. Well, Dr. Pam Lamar, thank you again for joining me. I really enjoyed this. And yeah, again, thank you for your time.

Pamela Maher

1:41:11 - 1:41:12

Okay. Well, thanks for having me.

This is a public episode. If you'd like to discuss this with other subscribers or get access to bonus episodes, visit mindandmatter.substack.com/subscribe

Wide release: June 30, 2026. Not medical advice.

Nick speaks with Dr. Katrin Svensson, a Stanford physiologist, about peptide hormone biology and its applications to energy regulation and weight loss. Svensson explains the precursor processing that allows one gene to produce different peptides in different tissues and how this context specificity supports precise physiological responses. The discussion covers why engineered GLP-1 drugs achieve robust brain effects on satiety and food preference that endogenous GLP-1 does not, along with her team’s genomic approach to discovering additional peptides that act on distinct brain pathways.

TOPICS DISCUSSED:

* Peptide Structure & Production: Short amino acid chains (3-50 residues) cleaved from larger precursors by PCSK enzymes, stored in dense-core vesicles, and released upon stimuli rather than acting as folded enzymes like proteins.

* Tissue-Specific Processing: The same precursor yields different peptides by tissue; preproglucagon produces glucagon in pancreas but GLP-1 in gut L-cells due to distinct cleavage patterns.

* Signaling Timescales: Peptides act over minutes via GPCRs and can drive longer transcriptional changes such as FOS expression, yet are cleared rapidly by proteases and kidneys.

* Endogenous GLP-1: Meal-stimulated incretin from gut that boosts insulin secretion with a half-life of only minutes, limiting primary action to periphery rather than deep brain structures.

* GLP-1 Engineering for Brain Access: DPP4 site mutations plus lipid conjugation enable albumin binding, extend half-life, and raise brain exposure.

* Dose-Dependent Effects: Low GLP-1 doses mainly lower blood glucose via pancreas; higher doses engage hindbrain and hypothalamic neurons to suppress appetite and alter food preference.

* Novel Peptide Discovery: Computational scans across the genome flag hundreds of uncharacterized precursors, facilitating new peptide discovery.

* BRP Peptide: This newly discovered brain-derived peptide suppresses feeding in GLP-1 receptor knockout mice, activates non-overlapping neurons, and does not slow gastric emptying.

ABOUT THE GUEST: Katrin Svensson, PhD, is a biochemist and physiologist who leads a laboratory at Stanford University focused on peptide hormone physiology and the regulation of energy balance.

RELATED CONTENT:

* M&M 161: GLP-1, Weight Loss Drugs, Ozempic, Obesity, NMDA Receptors, Metabolism & Brain Health | Christoffer Clemmensen

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PRACTICAL TAKEAWAYS:

* GLP-1 drugs require higher doses to engage brain satiety circuits for meaningful weight loss; lower doses primarily improve glucose control with little effect on appetite.

* Response varies widely across individuals, with some losing over 25% body weight while others see minimal change.

* Emerging peptides under study may suppress appetite via brain pathways that avoid the gastric slowing responsible for common GI side effects of current GLP-1 drugs.

* Research peptides purchased online frequently show variable purity below pharmaceutical standards and may contain unsafe formulation components or aggregates.

SUBSCRIBER CONTENT BELOW: Reference paper + episode transcript.

Reference Paper:

* Paper | Prohormone cleavage prediction uncovers a non-incretin anti-obesity peptide

AI-generated transcript below. Beware of mistranslations!

Katrin Svensson

02:49 - 02:50

Thanks for having me.

Nick Jikomes

02:50 - 02:54

Do you want to start off by just telling everyone a little bit about who you are and what your lab does?

Katrin Svensson

02:55 - 03:17

Yeah, sure. Yeah, so I’m a biochemist physiologist with a PhD from Sweden. And then I did a postdoc in Boston in physiology. I started my lab at Stanford, where we’re studying physiology of peptide hormones and how they can regulate energy balance.

Nick Jikomes

03:19 - 04:05

And I wanna start, we’re gonna go do a pretty deep dive into some of this stuff today, but I wanna start out basic and talk about some foundational knowledge that I think will set us up for people to understand things that we’re gonna talk about later on. I wanna talk about what peptides are, compare and contrast them to other things, and then get into things like how they’re produced and synthesized and how they’re secreted, because I want people to understand the spatial and the temporal scale that peptides operate on. And I want them to understand something about how these things are made impacts, how we discover them and how much remains to be discovered. So starting out with a basic definition, just tell people what are peptides and how are they similar to or different than say proteins or small molecule drugs say.

Katrin Svensson

04:06 - 04:29

So peptides are short chains of amino acids. Typically you think of them as 3 to 50 amino acids in length. So they’re short and they act on cell surface receptors, typically GPCRs, to induce cell signaling pathways. and transcriptional changes in cells.

Katrin Svensson

04:29 - 04:57

So this is a broad class of molecules that have widespread functions. They’re different than proteins in that they’re typically not folded in a structure, and proteins can have many different functions as enzymes, scaffolding proteins, binding partners, growth factors. peptides are typically seen as signals by themselves.

Nick Jikomes

04:58 - 05:04

I see. So they’re relatively small. They’re smaller than proteins. They’re up to perhaps 50 amino acids.

Nick Jikomes

05:04 - 05:15

So it’s a sequence of amino acids, and it acts as a signaling molecule that might activate a receptor, but they’re typically not folded into an elaborate three-dimensional shape like a big protein.

Katrin Svensson

05:16 - 05:16

That’s right.

Nick Jikomes

05:17 - 05:39

And so can you talk a little bit about how these things are produced and synthesized and maybe contrast that with proteins a little bit? One of the things I want to get you talking about is there’s quite an elaborate set of mechanisms and processes for sort of pre-processing these peptides before they are fully active and functional in our cells.

Katrin Svensson

05:40 - 06:24

Yeah, you know, the process of making a peptide is such an interesting biological process because they are not just made in as small peptides and secreted and that’s it. They are typically made, they’re encoded in the genome, so they have an amino acid sequence, but they’re made as part of the processing of a larger precursor protein. So you have this large precursor protein that is made and then transported into the ER for sorting and secretion. And in there, there are enzymes, proconvertases, for example, the PCSK class of enzymes.

Katrin Svensson

06:25 - 07:02

that pretty literally degrade this peptide into multiple small fragments that are typically thought of as the bioactive fragments. After the processing, there’s also other enzymes that are chemically modifying them, so putting on C-terminal modifications, And then they’re stored typically in cord dense granule vesicles and then they can be released. So it’s quite an elaborate process of making these peptides and then they’re released up on different stimuli.

Nick Jikomes

07:03 - 07:37

So the sequence of amino acids that the peptide is, it’s encoded in the genome. There’s a DNA sequence that encodes the amino acid sequence, but it’s not merely that the DNA is transformed into the amino acid sequence of the peptide and that’s it. Typically you’re making like a large protein and then basically chopping it off in different places and then making some other modifications before the mature peptide is actually fully assembled. So there’s a whole sort of processing phase that goes through that’s in between the mature peptide and the DNA encoding its sequence.

Katrin Svensson

07:38 - 08:05

Right. And it also depends on where you have this preprocessing enzyme, and that is expressed differently in different tissues. So if you take, for example, preproglucagon, which is the primary sequence that is making GLP-1 and glucagon, It’s actually processed differently in different tissues. So in the pancreas, you get it processed in one way, where you get glucagon.

Katrin Svensson

08:06 - 08:26

And in the gut, you get it processed at the different sites. So you get primarily GLP-1. So from the same sequence, you have this energy conservation where you just start with one sequence, but you generate different peptides in different tissues. There’s also this process where you can make a combination of peptides.

Katrin Svensson

08:26 - 08:44

Oxentamodulin is an example like that, where you have two peptides that could be cleaved into two different forms, but they could also be combined together. And that has an entirely different receptor and different functions. So you can imagine the diversity of peptides from a single precursor protein.

Nick Jikomes

08:44 - 09:07

Right. So basically you can have one amino acid sequence, one protein, and depending on exactly where it’s cut, how it’s chopped up, what else is added to it, this can determine the actual peptide that’s made. You can get one peptide, you can get effectively a completely different peptide, or you could modify, say, aspects of like where it goes inside of the cell and how it’s actually functioning.

Katrin Svensson

09:08 - 09:08

Right.

Nick Jikomes

09:10 - 09:45

And so there’s this whole let’s call it pre-processing apparatus within cells in between the DNA sequence encoding these things and the mature peptides that actually go on to do something. And so I don’t think we need to go on all cover off on all of the details here, but maybe people should just appreciate something about how how elaborate this process can be. There’s essentially something like an Amazon sorting facility that takes care of packaging things, making sure they’ve got little barcodes, let’s say, and making sure they know where to go and how to function before they’re actually sent off to do something.

Katrin Svensson

09:46 - 09:47

Yes. Yeah.

Nick Jikomes

09:48 - 10:27

And so one of the things that I think we’ll come back to too is sort of the diversity and the context specificity of a lot of these peptides. You mentioned, I just want to ask you one more time about this, you mentioned that you can have the same sequence, the same precursor sequence, and it can be processed the same exact precursor sequence can be processed in two different ways in two different cells, such that the same starting material ends up being effectively a different thing in the pancreas versus, you know, some other tissues, say. What is sort of the significance of that, the ability to sort of process these precursor sequences in a different way in a tissue-specific manner?

Katrin Svensson

10:30 - 11:04

The significance of that is that different tissue can respond differently to different stimuli. So for this particular example, in the gut, the gut is responding to, for example, a glucose load. So when you eat, you want to have a rapid response where you have GLP-1 secretion from the gut, while the pancreas needs to make a glucagon to counteract this increased glucose levels. So it’s sort of a feedback, feed forward mechanism.

Nick Jikomes

11:05 - 11:41

And so these peptides are, you know, by definition, they’re much smaller than proteins, but they’re larger than other things like say a neurotransmitter. Can you talk a little bit about how the size and the structure of peptides relates to the time scales and the spatial scales they operate on? So, you know, in biology, we’ve got everything from like hormones that might go through the bloodstream and go to all of our tissues and have effects that last for hours and days. And then we’ve got things like fast neurotransmitters in the brain that are operating, you know, from one neuron to another on the timescale of milliseconds.

Nick Jikomes

11:41 - 11:50

How do peptides generally fit into this? And how do you start to think about the significance of how long these signals last and how far they travel?

Katrin Svensson

11:51 - 12:15

Yeah, so neurotransmitters are typically like seconds, milliseconds or seconds. Larger proteins are typically an hour’s timescale, like antibodies that are circulating. And peptides are minutes to hour, maximum one hour, I would say, endogenously. So the timescale is shorter because they’re degraded more quickly.

Katrin Svensson

12:16 - 12:43

But the effects on a particular pathway or cell can still be longstanding because they induce different transcriptional responses, for example, FOS in the brain. So it’s not entirely correlated with the actual half-life of the peptide. But you do have something in between, a small molecule neurotransmitter, and a protein.

Nick Jikomes

12:43 - 12:58

So they have a half-life that’s on the order of minutes. So they stick around longer than something like a fast neurotransmitter, but not as long as something like a hormone. But importantly, their effects can outlast the peptides themselves. So the peptides can be released.

Nick Jikomes

12:58 - 13:08

They can stick around and do things for minutes, say, but the actual signaling effects they have with themselves can outlast the peptide that triggered it.

Katrin Svensson

13:08 - 13:08

Yes.

Nick Jikomes

13:10 - 13:34

And so when we think about the regulation of these things, you sort of mentioned it a little bit there, they’re peptides, they’re sort of like mini proteins. So there’s lots of enzymes that can degrade them. Is that like a purely passive process or is the degradation of these things by proteases, is that part of the core regulation for how long these signals actually last?

Katrin Svensson

13:35 - 13:59

Yes, it’s definitely part of the core regulation. Enzymatic processing, for example, DPP4 is an enzyme that is cutting off part of GLP1, which is contributing to its short half-life. If you mutate that site so that enzyme no longer can do that function, you will extend the half-life. So for sure, that is one of the mechanisms.

Katrin Svensson

13:59 - 14:07

But these peptides are also very small. So even if you don’t have any degradation process in the body, they’re still going to be cleared by the kidney.

Nick Jikomes

14:08 - 14:21

OK. So just in virtue of their size, they get cleared out relatively quickly. That’s right. And so, um, a lot of the focus in peptide research and peptide biology is in the general area of energy homeostasis.

Nick Jikomes

14:22 - 14:32

Um, so we’re going to talk about the GLP ones. We’re going to talk about some other things, but high level to start out here. What is energy homeostasis? What does that term mean to a biologist like you?

Nick Jikomes

14:33 - 14:45

And what are some of the core concepts there that we want to convey to people before we dig into how GLPs and some other related peptides actually work to affect things like how energy is used in the body?

Katrin Svensson

14:49 - 15:13

the combination of energy intake and energy output. And that can be maintained in the body through different mechanisms, but that’s the core idea, that the energy you’re taking in needs to match the output, how much you expend. Otherwise, you’re going to either increase your body weight or lower your body weight.

Nick Jikomes

15:14 - 15:47

And so these things last for, let’s just call it intermediate time scales, as we said, minutes, they can have effects that outlast the peptides that instigated them. I want to talk a little bit about where these things are secreted from and the secretion process itself. I’ll let you steer us in terms of the examples that you think are the best to talk about. But when we think about how these things get released at the cellular level, in neuroscience, I often learned that you have different types of vesicles, different types of secretion mechanisms.

Nick Jikomes

15:48 - 16:04

Some things get sort of released right away after they’re made, and other things get stored and saved for the right time. Where, you know, do these peptides generally fall into one class there? Are they sort of secreted on demand? Are they stored up and saved for later?

Nick Jikomes

16:04 - 16:07

Or does it depend entirely on the tissue and the peptide?

Katrin Svensson

16:08 - 16:44

I think to some extent it depends on what the peptide is. So for some of them you have constitutive secretion, so they’re just secreted when they are made. But for the classical ones that we think about in our endocrine organs, pituitary or the endocrine tissues and cells, they are released upon certain stimuli. So for example, when you have a hyperpolarization or calcium mobilization, then you get this burst of secretion of these stored vesicles with different peptides.

Katrin Svensson

16:45 - 17:02

But I think the other constitutive secretion is a mechanism that we know less about. And there may be instances where you have regulated secretion through non-core vesicles as well. They’re just way less studied.

Nick Jikomes

17:03 - 17:25

Yeah, yeah, yeah. And so when we think about where they’re secreted from, you mentioned a couple key areas already, you know, the brain, certain parts of the brain secrete peptides and hormones. As we’ll get into, there’s gut hormones and gut peptides secreted from the gut. Are there a limited number of tissues in the body that are generally secreting these things or are they secreted all over the place?

Katrin Svensson

17:27 - 17:56

Yeah, so I think for a very long time, the field of endocrinology has really focused on the endocrine tissues. So all the classical ones that you would think of, the brain that got the adrenal gland pituitary. But I think we start to appreciate now that These peptides can actually be made from non-classical endocrine organs. Things can be secreted from adipose tissue, from the liver.

Katrin Svensson

17:57 - 18:02

These are major metabolic organs, but they’re not the classical endocrine organs.

Nick Jikomes

18:03 - 18:16

Okay. So there’s definitely organs that are at least more famous for secreting these things. But over time, we’ve also started to learn that other tissues that we previously didn’t maybe appreciate could secrete hormones or peptides actually do.

Katrin Svensson

18:17 - 18:17

Right.

Nick Jikomes

18:18 - 18:45

And what do you want to say about just sort of the general theme of, I guess you could say one of the purposes of these things is communication across tissues and organs, right? Like if my gut needs to communicate with my brain or vice versa, this is done via some of these molecules that get secreted and travel throughout the body. What do you want to say about that, just to sort of set up and contextualize the conversation that we’re going to go into about gut hormones and GLP-1s and that type of thing?

Katrin Svensson

18:46 - 19:16

that’s the classical endocrinology view that tissues need to communicate, the brain needs to know what the status is of the cell, how much adiposity do you have, and this is why you have leptin from the fat communicating to the brain, you should eat less because we have enough fat stores, right? The gut is communicating with the brain that we are full, you should stop eating. So it’s a very intuitive way of thinking about how these organs are communicating together or with each other.

Katrin Svensson

19:18 - 19:28

But I think that there’s a lot of inter-tissue communication too, like the brain itself communicates a lot within the cell types in the brain. And there’s a lot we don’t know.

Nick Jikomes

19:28 - 20:06

Yeah. And so when we think about neuropeptides and the brain, the hypothalamus is a major center that we talk about here. When we think about how hunger and satiety are regulated, can you say just a little bit about some of the major themes and how the hypothalamus is uh sensing what the rest of the body is doing you know sensing things like leptin from fat cells as you just mentioned and then using that to generate signals about what the animal should do with its behavior to meet its demands and we can keep it fairly high level here but i want to give people a sense for like you know, okay, so the hypothalamus is able to sense

Nick Jikomes

20:06 - 20:31

hormones and that tells it something about the periphery, how much fat there is, how much hunger should be generated by circuits in the hypothalamus and so forth. But, you know, give us like maybe some examples or a sense of how this works from, you know, the hypothalamus sensing the levels of leptin or something like this to generating behaviors such as say feeding behavior coming on or feeding behavior being turned off.

Katrin Svensson

20:32 - 21:03

Yeah, so I think the most well known, well understood process is the melanocortin system, which is a system where leptin acts through. So here you have MC4R neurons. So these are neurons in the brain that release alpha MSH, which is a peptide that suppresses feeding. So you have this loop where you have increased alpha MSH acts on neurons to suppress a feeding.

Katrin Svensson

21:04 - 21:09

And there are multiple such examples, but the melanocortin pathway is the most well established.

Nick Jikomes

21:09 - 21:39

Yeah. So even within the brain, within the hypothalamus, there’s sensing of what’s going on throughout the body, sensing things like gut hormones, leptin, as you mentioned. But then those signals from the periphery can be transformed into neural signals using transmitters, but also neuropeptides within the hypothalamus to shape the activity that’s going to happen in the brain that’s actually going to correspond to the animal, you know, going out and finding food or feeling full and satiated and things like that.

Katrin Svensson

21:40 - 21:41

Yeah.

Nick Jikomes

21:42 - 22:01

And so where do the GLP-1s come in? Let’s talk about endogenous GLP-1 first before we talk about some of the drugs that are being more and more widely used now. So endogenous GLP-1, what does that stand for? And can you give us a sense for the major site of production and secretion in the body?

Katrin Svensson

22:02 - 22:26

Yeah, so GLP-1 stands for glucagon-like peptide 1. It’s one of the bioactive peptides from the pre-proglucagon gene. It’s made in the L-cells in the gut in response to a glucose load or a meal. And it has functions throughout the periphery, but also in the brain.

Katrin Svensson

22:26 - 22:56

But I would say the consensus in the field is that endogenous GLP-1 primarily has functions in the periphery. so it’s an incretin so it acts on the pancreas to increase insulin secretion to lower blood glucose. So if you have endogenous GLP-1 or just slightly raised levels, you will lower blood glucose. And that’s one way of, you know, controlling glucose homeostasis in the body.

Nick Jikomes

22:57 - 23:06

So the basic idea is the gut is sensing how much glucose is coming in. And the more that’s coming in, the more you want to secrete insulin to then, you know, pull that glucose into cells and do stuff with it.

Katrin Svensson

23:07 - 23:07

That’s right.

Nick Jikomes

23:08 - 23:17

And so it doesn’t last very long. And so it can get to the brain. But largely, endogenous GLP-1 is thought to act mostly outside the brain, in the periphery.

Katrin Svensson

23:18 - 23:32

Right. And there are some ideas or data supporting that it can act on the vagus nerve locally, and thereby communicating to the brain. But it’s thought that endogenous GLP-1 is primarily peripheral. Yeah.

Nick Jikomes

23:32 - 23:50

Yeah. So there’s a couple different ways that something could actually communicate to the central nervous system in principle, right? It can get all the way through the blood-brain barrier and get up into the brain in our heads, but it could also, as you just mentioned, perhaps affect things through the vagus nerve or nerve terminals that are actually down in the periphery.

Katrin Svensson

23:50 - 23:51

Right.

Nick Jikomes

23:53 - 24:02

And so endogenous GLP-1, is that something that’s also lasting on the minutes time scale? Is it sort of an intermediate signal?

Katrin Svensson

24:02 - 24:05

Oh, it has a very short half-life. Within a couple minutes, it’s gone.

Nick Jikomes

24:06 - 24:12

Got it. So it’s secreted in response to basically eating something, and then it’s secreted, but it only lasts a couple of minutes.

Katrin Svensson

24:12 - 24:12

Right.

Nick Jikomes

24:13 - 24:29

And so one of the major innovations of the GLP-1 drugs had to do with that short half-life. Can you talk a little bit about how the half-life of the endogenous peptide is regulated, and then tell us a little bit about how the GLP-1 drugs were engineered to last longer?

Katrin Svensson

24:30 - 24:56

Yeah, so Endogenous GLP-1 has a recognition site for the DPP4 enzyme that is pretty much inactivating the peptide. And so that’s one mechanism by which it’s, I wouldn’t say degraded, but it’s inactivated. And then it’s cleared by the kidneys. And those are the two major modes of inactivation and clearance.

Katrin Svensson

24:57 - 25:34

So the way, I’m no pharmacology expert, and these are really people at the pharmaceutical companies that develop these drugs. But as far as I understand, you can mutate the site that is recognized by DPB4 and change some other amino acids to prevent further degradation and stabilizing the peptide. And then the major advance was actually putting on a lipid. conjugated to the actual peptide, which makes it bind albumin in the blood.

Katrin Svensson

25:35 - 25:55

And that albumin is typically 60 kilodalton, it’s large, it prevents renal clearance, which means that this peptide is hanging on to albumin and then secretes it or like being circulating for much, much longer time. which means that the brain also has high exposure to this peptide for a longer time.

Nick Jikomes

25:56 - 26:07

Okay, so endogenous GLP-1, it only lasts for a couple minutes. It’s secreted in the gut after a meal. That makes perfect sense. but it’s only lasting for a couple minutes.

Nick Jikomes

26:07 - 26:22

It’s going to get cleared out by the kidney. There were some modifications engineered by humans. You add that lipid, you maybe do a couple other things. And the overall effect of that is this thing basically travels and hangs around in the blood longer because it’s sticking to albumin.

Nick Jikomes

26:23 - 26:28

And that allows it to not just last longer, but last long enough to get up into the brain at a higher concentration.

Katrin Svensson

26:29 - 26:29

Yes.

Nick Jikomes

26:31 - 26:38

And so what do these things actually do in the brain to alter satiety?

Katrin Svensson

26:39 - 27:34

Yes, so different peptides act differently, and there’s still a debate exactly which sparing regions are responsible for the function of each of these peptides because it’s hard to determine the primary site of action. As you know, you can have a site where you have activity and then you have projections to other areas. So for GOP1, there have been a lot of studies suggesting that it acts on the hindbrain, where you see FOSS activation in the area postrema, in the NTS, in the hindbrain, and that is then projecting into the hypothalamus, the archid nucleus, MC4R neurons, and others. So that is the general idea, and that is how many peptide hormones work actually, either directly acting on the hindbrain or directly acting in the hypothalamus.

Nick Jikomes

27:35 - 27:48

Got it. So these things get into the brain and there are neurons in the hindbrain and hypothalamus that have receptors that these things act on. So they’re directly interacting with neurons and ultimately changing the pattern of activity of those neurons.

Katrin Svensson

27:49 - 27:56

Right. But it doesn’t get into the entire brain. I think this is a question that I get a lot. How do these peptides cross the blood-brain barrier?

Katrin Svensson

27:57 - 28:13

And in many of these cases, they enter through areas within the circumventricular organs, so the hindbrain and the arcuate nucleus in the hypothalamus. So it’s not like they’re occupying the entire brain. They actually have very specific access to some of these regions.

Nick Jikomes

28:13 - 28:23

Yeah. Yeah. And I think this is an important concept for people to understand. So the blood brain barrier is, as the name suggests, a barrier between the brain and the blood and the rest of the body.

Nick Jikomes

28:24 - 28:40

And some things are allowed to pass the blood-brain barrier and some things are allowed, but they’re sort of specially transported across. But then there are other parts of the brain that don’t really have a blood-brain barrier. Say a little bit more about that and just really define that for us and where those are at.

Katrin Svensson

28:40 - 29:12

Yeah, so there are ventricles in the brain that have direct access to the blood. So the hindbrain is close to the fourth ventricle and the hypothalamus is close to the third ventricle. And there you can have direct access to peptides that are normally not seen by other parts of the brain, for example, the cortex. So this is a way for the body to communicate directly with those areas in the brain.

Nick Jikomes

29:12 - 29:41

I see. So this is, this is basically a feature of this whole system is you have the blood brain barrier to kind of protect the brain from stuff you don’t want to get into the brain from getting in. But there are a couple spots where actually the brain barrier isn’t really there, near the hypothalamus, near the hindbrain. And it’s basically a way for certain parts of the brain to have access to the bloodstream and sample what’s in it, to help with brain-body coordination and communication.

Nick Jikomes

29:41 - 29:55

So on the one hand, most stuff in the brain is protected by the blood-brain barrier, and that’s a very tight regulation. But then you’ve got these sort of special spots that are allowed to sample from the periphery and effectively read what’s going on in the body.

Katrin Svensson

29:56 - 29:56

Right.

Nick Jikomes

29:58 - 30:35

And so when we think about these GLP-1 drugs that more people are taking, there’s all different versions of these things. I want to ask you a little bit about some of the new GLP-1s that are out there. So we already talked about one innovation in this realm, which is that the endogenous peptide was basically modified so that it lasts longer and can travel up to the brain and get to the brain at a higher concentration than endogenous GLP-1. Is there anything else that we should understand about how these new drugs based on GLP-1 are operating besides that modification?

Nick Jikomes

30:35 - 30:40

Is there anything else they’re doing in the brain, for example, that endogenous GLP-1 doesn’t do?

Katrin Svensson

30:41 - 31:23

Yes, we’re starting to understand more about the physiology and endogenous function of these peptides, not just GLP-1, but others too, where we have pharmacology that is good. And that the pharmacology sometimes can be very different than what you would understand by studying physiology, for example, the knockout mouse of GLP-1 receptor. When we look at the basic phenotype, if you’re lacking the ability to respond to endogenous GLP-1, you should, in principle, phenocopy a pharmacological action, but in the opposite way. But that’s actually not entirely the case.

Katrin Svensson

31:24 - 31:36

So if you remove the ability to respond, you would expect these mice to be obese because they can no longer respond to these anorexigenic signals. Yeah.

Nick Jikomes

31:36 - 31:40

You’ve taken away the ability to read the satiety signal and therefore they should just eat a lot.

Katrin Svensson

31:41 - 31:41

Right.

Nick Jikomes

31:41 - 31:42

That would be the expectation.

Katrin Svensson

31:43 - 32:06

That is the expectation. And there is some very weak signal that these mice eat a little bit more, but they’re not heavily, heavily obese, which means that You know, when you go in with pharmacology, you increase the levels to supra-physiological levels. You’re no longer in the endogenous range. And this means that you have effects that you would just never see by studying the endogenous biology.

Katrin Svensson

32:07 - 32:32

It’s true, it’s specific, because it binds this receptor. And if you remove the receptor, you don’t have these effects of the pharmacology, but you cannot see it by understanding or by looking only at the endogenous function. So these pharmacological agents, when they stick around for longer, will now have much different effects than just studying the physiology. And I think that that is very interesting.

Katrin Svensson

32:33 - 32:54

We see already signs that these GLP-1-based drugs can have other functions than just suppressing satiety. It’s increasing satiety. So they, for example, affect the reward system or addiction, or there are different signals that you wouldn’t see otherwise.

Nick Jikomes

32:54 - 33:30

Yeah, and this is maybe an important thing to dwell on for a minute. Simply in virtue of the fact that the dose is sufficiently high and the half-life is sufficiently long compared to the endogenous peptide, you can get effects that you wouldn’t see with the endogenous compound simply because there’s now enough time for it to, say, diffuse into places it doesn’t normally go to, or stick around long enough that it’s activating not just the endogenous receptors like GLP-1, but perhaps additional receptors that have lower affinity for the peptide that maybe the endogenous peptide just never sticks around long enough to act on.

Katrin Svensson

33:30 - 33:44

I don’t think there’s any evidence that it binds to other receptors. I think it’s still just one receptor, but you can imagine that there are projections that could happen now when you have a long-lasting signal that wouldn’t be there otherwise.

Nick Jikomes

33:45 - 34:07

OK, OK. And so you mentioned effects on reward processing and things like that. Can you say a little bit more about that and what the relevance is here for these new GLP-1 drugs? So how are these what we think of as hunger and satiety circuits hooked into reward processing systems in the brain?

Nick Jikomes

34:08 - 34:27

And what’s sort of the relevance to Should we think about hunger and satiety regulation as completely distinct from reward processing generally, or are these things intertwined in a way that we can’t unpack fully, that they’re really merged together?

Katrin Svensson

34:28 - 35:11

It’s super interesting, and I think we fully don’t know how. how these systems are related, and if there are pharmacology or even endogenous signals that can dissociate these things. But what we do see is that DLP1s, at least from our mouse work, and there’s also some data in humans suggesting this, that the preference for eating sugar type molecules, sucrose and saccharose, goes down when you are taking a GOP1. So this is not just appetite, this is actually a change in preference.

Katrin Svensson

35:12 - 35:39

So this is interesting because that ties into dopamine release, potential reward system. But there are other types of rewards that have been tested, for example, amphetamine and fentanyl, where it doesn’t seem to have an effect. So it’s really interesting that you, you know, I think different molecules may do different things and depending on what neuronal signals and projections that are being sent.

Nick Jikomes

35:40 - 35:51

Yeah. So, I mean, that’s interesting. It’s not just affecting satiety per se, it’s affecting food preference. So it’s not, you know, just scaling down how much an animal eats.

Nick Jikomes

35:51 - 35:55

It’s actually changing what the animal will choose to eat on top of that.

Katrin Svensson

35:56 - 36:00

Yes, that seems to be the overall signal that we see.

Nick Jikomes

36:01 - 36:27

And can you give us just sort of a basic sense of what are the major robust effects that these GLP-1 drugs seem to have both in animals and humans? Beyond the fact that food intake goes down, what are some of the other effects that we’re seeing on reward processing or reward-related behavior, on hormone release and other things going on that are more endocrine related? What’s happening beyond just the simple decrease in food intake?

Katrin Svensson

36:28 - 37:20

So it’s really hard to say what is primary and what is secondary, you can get a lot of secondary effects. And we know that there’s protection in cardiovascular disease, there’s, there’s lowering of, you know, blood sugar, obviously, there’s an on target effect is lowering cholesterol, there’s, all these secondary effects. So I think we need to think about what is primary made by, you know, the signal GOP1 on the GOP1 receptor, and then everything else that can be secondary to the weight loss or secondary to the lowering of diabetes, if that is the basal state. So I think in order to answer these questions, all of these experiments need to be done in preclinical models in mice, where you’re now doing it in the GLP1 receptor knockout mice, and also to see what is actually holding up in humans.

Katrin Svensson

37:20 - 37:34

We have done a lot of mouse work, and although the overall mechanism is highly, highly conserved, some of these behavioral effects may not be conserved between mice and human entirely.

Nick Jikomes

37:38 - 37:59

the basic effects on food intake and hunger satiety at a high level, how universal are they across individuals? Do we see this in more or less all individuals when we look at the animal data? When people take GLP-1s, roughly what percentage of people do they have the primary intended effect for?

Katrin Svensson

38:00 - 38:17

Yeah, so in humans there’s a huge heterogeneity. In our mouse studies and even non-rodent models, these are inbred animals, so it’s very, very low heterogeneity. Every single animal responds about the same. at the same dose.

Katrin Svensson

38:17 - 38:36

So that is extremely, extremely homogeneous and robust. In humans, there’s a huge range. Some people lose 50 pounds, they lose 25%, 30% of their body weight. Some people are so called like low responders and non responders to lose 5% of their body weight.

Katrin Svensson

38:37 - 39:01

And it’s not clear why they’re responding so poorly. There has been some studies showing that it correlates with the GLP-1 receptor expression, which makes a lot of sense. If they have less of the receptor, they may respond less, but that may not be the entire answer. And it’s unclear why some individuals respond better or worse.

Nick Jikomes

39:01 - 39:24

Yeah. I want to ask you a little bit about sort of dose dependent effects here, and how the effects of the GLP ones can start to change depending on dose. So I would presume, although I don’t actually know the answer to this, I would presume there’s been a lot of dose response studies in the animal literature, you know, what happens when you scale up the dose? Do you just get more of the same effect?

Nick Jikomes

39:24 - 39:44

Do you start to get different effects? Do you get any unwanted side effects when you go sufficiently high. And, you know, I want to have in mind when we have this discussion about the animal work, the fact that, you know, a lot of people are taking these GLP ones now. And I can only imagine that, you know, if I’m someone who’s struggled with weight loss my whole life, I start taking an injection, I’m like, wow, I lost 20 pounds.

Nick Jikomes

39:44 - 39:52

I should just double the dose. I should just triple the dose. What’s going to start to happen if and when people start scaling up the dose? So let’s start with the animal data.

Nick Jikomes

39:53 - 39:58

How much are the effects dose dependent? And do we see them change very much as we escalate the dose?

Katrin Svensson

39:59 - 40:22

Yes. So we both have animal data and human data for this. So in animals, it’s very dose dependent. So as far as glucose lowering, as far as appetite suppression, it’s clearly dose dependent to the degree that the mice will almost entirely stop eating if you are giving high enough dose.

Katrin Svensson

40:24 - 40:43

And once they come off, they rebound and they start to eat just normally. So it’s really an effect of the drug. In humans, we actually know that lower dose will just lower blood glucose, but will not cause weight loss. This is because GLP1s were first approved for diabetes at a much lower dose.

Katrin Svensson

40:44 - 41:09

So the Osempic drug is a diabetes drug. That’s a lower dose than Vigovi, which is just the same molecule, but at a higher dose for obesity as the indication. So if you go in with a lower dose, you will have glucose lowering, you will restore your diabetes, but you will not lose a lot of weight. So we clearly know that it’s dose dependent and generates different effects.

Katrin Svensson

41:09 - 41:31

So if you have a low dose, it’s primarily effect in the periphery and the pancreas. If you go high enough dose, you will have high enough levels entering the brain and then you get the appetite suppression. But at these higher doses, you will also have an effect on the gut. We haven’t talked much about this, but the GOP1 also acts on the gut to slow down gastric emptying.

Katrin Svensson

41:32 - 41:49

It acts to slow transit time in the colon. So this is what is causing GI upset and constipation. and or sometimes both in the same patients. So this is the major drawback of going too high in dose.

Nick Jikomes

41:50 - 42:15

So these things have central effects in the brain related to hunger and satiety. They also have peripheral effects outside the central nervous system in the gut. So, you know, if you’re taking a higher dose GLP-1 drug for weight loss, you’re going to be taking enough that it gets up to the brain, affects all of those hindbrain and hypothalamus circuits to make you, let’s say, want to eat less and maybe change your food preference and maybe lower the reward value of food and things like this.

Nick Jikomes

42:16 - 42:39

But it’s also physically slowing how quickly the food you do eat goes through your gut. So when we think about that at a fairly high level, Let’s divorce ourselves from the GLP-1s for a moment. When and why would the body want to modulate how quickly food goes through the gut? What are the advantages and disadvantages of speeding up or slowing down transit time?

Katrin Svensson

42:42 - 43:29

So lowering your gastric emptying time, that makes a lot of sense because if the stomach is not releasing the food into the intestine, you feel full longer. So that makes a lot of sense in terms of the colonic transit time, that would just reduce the time that it takes for the food to go through the intestine. I’m not entirely sure why GLP-1s would, what’s the evolutionary function of that, but the stomach gastric emptying makes a lot of sense to just increase your fullness. And maybe the colonic transit time is just a functional consequence of that.

Katrin Svensson

43:29 - 43:30

I’m not entirely sure.

Nick Jikomes

43:30 - 43:52

Got it. So when we think about the effects on the gut, The things that we can think about there in particular are how long it takes the stomach to release the food. The longer it sits in the stomach, the more it’s presumably gonna get chopped up by stomach enzymes, but also just the more full you’ll feel. And then there’s the time it actually takes to go through all of the intestines and how long it sits in the colon.

Nick Jikomes

43:52 - 44:01

And so I’m not sure how much is known here, but presumably that would affect things like nutrient absorption, the microbiome and all of that stuff.

Katrin Svensson

44:02 - 44:06

Yes, yes. And I think this is an area where it’s still intensely being studied.

Nick Jikomes

44:07 - 44:35

Yeah, yeah, yeah. And so when we think about escalating the dose, so at a relatively low dose, you mentioned, you know, things like ozempic are basically used as diabetes drugs, that’ll be more glucose homeostasis, blood sugar regulation, basically, the dose isn’t high enough for very much of it to get up to the brain to have central effects. But then the higher dose versions of these things, are high enough to get up to the brain, that’s where you have more direct effects on the central regulation of satiety, and you’re going to get more weight loss in general.

Nick Jikomes

44:36 - 44:51

What happens if you keep escalating the dose further? Is there just sort of a dose regime where you can just escalate somewhat and basically just get more satiety and less food intake before you start to get anything that you would call, say, a side effect?

Katrin Svensson

44:52 - 45:23

Yeah, and I think that’s very dose-dependent and it’s very personalized too, so different individuals will have different responses. But for sure, when you have central effects, you will experience side effects too, and you see this in individuals that have, you know, in the newspapers or you look at, they have gotten compounded GLP-1 and then misdosed or dosed 10 times higher, they just end up in the ER vomiting because the nausea is just too terrible.

Nick Jikomes

45:24 - 45:30

Yeah. Yeah. So actually, did you, there was an interview recently with Sam Altman from OpenAI. Did you see that?

Nick Jikomes

45:32 - 45:47

So for those who didn’t see it, Sam Altman, the CEO of OpenAI, he got one of the GLP drugs from somewhere. I don’t know where. And he either messed up or the compounding pharmacy messed up and he took like a mega dose. I don’t know how much, but it was like way more than it was supposed to be.

Nick Jikomes

45:48 - 46:11

And the basic effect was, well, he wasn’t hungry and he didn’t eat, but he also didn’t want to do anything. I think a neuroscientist would call it anhedonia. He was basically just laying in bed, didn’t want to work, didn’t want to socialize, didn’t want to do anything. Is that basically the risk of doing like a mega dose of one of these things that you’re basically just tapping into some of that general purpose reward circuitry and shutting all of it down?

Katrin Svensson

46:12 - 46:34

Yeah, I mean, that is something that is being studied. And the anhedonia is interesting because obviously that’s not what you want. You just want appetite suppression, but still have enough energy to do other things. And it’s unclear if it’s only linked to that lack of energy intake or if it’s something else.

Nick Jikomes

46:35 - 46:35

Yeah, yeah.

Katrin Svensson

46:35 - 46:56

But one thing that we have seen in mice is that we can inject them with high-dose semi-glutare GOP1 and then put them on a treadmill. And they can run just fine. There’s no issue with their energy regulation. They’re not exhausted in that they can no longer run.

Katrin Svensson

46:57 - 47:11

But if you ask them to do voluntary wheel running, so they have the option to run on a running wheel, they choose not to. Huge reduction. And why is that? That’s sort of an anhedonia response.

Katrin Svensson

47:11 - 47:20

And we still don’t understand what the mechanism is of this, but that’s a clear effect that they choose to run less on a treadmill. And I find that very interesting.

Nick Jikomes

47:20 - 47:30

Right. So yeah, that is interesting. At a high enough dose, they don’t choose to run, but they can run. There’s no energy deficit preventing them from having enough energy to run.

Nick Jikomes

47:30 - 47:44

There’s no overt motor impairment that’s preventing them from coordinating their muscles. They just choose not to run for whatever reason. But that does imply there’s some volitional effect there. We know that mice do like to run, right?

Nick Jikomes

47:44 - 48:09

It’s a rewarding thing for most mice. They like to run on little wheels and stuff. So that does start to imply that maybe you’re tapping into what I’ll just call reward circuitry beyond hunger and satiety regulation. And that does start to get me thinking about other things that people are reporting, which is, you know, apparently, and I don’t know how meticulously this has been studied yet, but apparently people are reporting spontaneous reductions in things like alcohol intake and drug intake.

Nick Jikomes

48:10 - 48:15

Do you know anything about that? And does that start to tell us something about what these drugs are doing beyond hunger and satiety per se?

Katrin Svensson

48:16 - 48:53

Yeah, so we don’t know enough about which areas in the brain are controlled by GLP-1. Those reward areas are typically not thought to be directly exposed to GLP-1, but they might very well be. induced or reduced through projections. This is not my area of expertise, but we’re working with some collaborators on this, where we’re looking at incretins and non-incretin peptides and how they can affect reward pathways and addiction pathways, and there seems to be different responses.

Nick Jikomes

48:53 - 49:16

Yeah, yeah, yeah. So, you know, there could be something really interesting happening here. That is also we’re thinking about from a clinical perspective, where you take the dose up high enough on these things, and you get weight loss, you get better hunger satiety regulation, we’ll call that an effect that people are after. But then maybe you go a little bit higher and you start getting less motivation in general.

Nick Jikomes

49:16 - 49:32

And if you go even higher, then you get anhedonia and things like that. So it could be that there’s a sweet spot here where you get enough satiety regulation to give you the weight loss effects, but not so much that it’s going to bleed into other domains of life like just your willingness to do other things.

Katrin Svensson

49:32 - 49:53

Right. But I think, importantly, for the GLP-1s, there isn’t a strong signal in inducing depression. It doesn’t seem to be inducing depression, which would be very serious, and that would be a very serious side effect. So it’s not just the dopamine system, there must be something else.

Katrin Svensson

49:53 - 49:55

We don’t understand what that is yet.

Nick Jikomes

49:56 - 50:13

Yeah, yeah. And there was Ramanuband years ago, it was sort of like an earlier type of weight loss drug that got released in Europe. And then it was pulled from the market because it was basically causing depression and psychiatric problems for people. And that was causing weight loss and less satiety, but it was acting through the cannabinoid system.

Nick Jikomes

50:13 - 50:19

So, so far, it does not seem like the GLP-1s are having that exact type of side effect.

Katrin Svensson

50:20 - 50:39

Yeah, that’s right. And the Romana band story really deterred people from going into this. Many people were convinced that you cannot get appetite suppression without affecting all these other systems. And the GOPs have shown that this is not true.

Katrin Svensson

50:40 - 50:52

So it’s really amazing and has opened up a new area where we can learn more about addiction, reward behavior, and and feeding and how they’re linked.

Nick Jikomes

50:53 - 51:16

Yeah, I want to talk a little bit more about the innovation and evolution of this general class of treatments. We talked already about one innovation, which was you modify endogenous GLP such that it basically lasts longer and therefore can get to the brain at a higher level. But now there’s new drugs that you can talk to us about. And they’re not just acting on GLP1, they’re acting on other things.

Nick Jikomes

51:16 - 51:31

Basically, we’re combining things so that we’re playing with multiple knobs in the brain and body at the same time. Can you talk about things like RETA and some of the newest versions of these peptide therapies and what they’re doing beyond GLP-1?

Katrin Svensson

51:32 - 51:57

Yes, so semaglutide is the GLP-1 molecule by itself. And then there are other approved drugs, tercepatide, which is a combination of GLP-1 agonism and GIP. So this is another infratin, they pretty much both come from the gut and they act in a similar way. It turns out when you combine them, you actually have even stronger appetite suppressing effects.

Katrin Svensson

51:58 - 52:34

It’s unclear whether all these other behaviors that we were talking about are also sustained now when you have the two molecules. And then the third molecule is beta-drutide, which you mentioned, which is a triple agonist. So this is GLP-1, GLP, and then glucagon. And this is very interesting because the glucagon component is kind of, it’s not obvious why this would really work to enhance weight loss even more, because by itself it doesn’t induce weight loss, but it increases energy expenditure.

Katrin Svensson

52:35 - 53:09

the equation of your suppressed food intake, but you also increase energy expenditure. So overall, you have a greater weight loss and other metabolic benefits. So it will be super interesting to see in the future which of these benefits and side effects will be maintained with all these different combinations. There are some indications that the tercepatide, for example, has lower side effects or reduced side effects in terms of the GI upset.

Nick Jikomes

53:09 - 53:24

Yeah, yeah, yeah. So basically you’re talking about drugs that affect one of these peptides and there’s drugs that affect two or even three of them. So we’re playing with now multiple peptide-based systems at the same time in parallel.

Katrin Svensson

53:24 - 54:10

Yes, but I think it’s not clear if the triple agonist, for example, has now some new pharmacology that is not seen with the three individual peptides by themselves. That is still being worked out and it seems to be some indication that it’s actually not just the three combined. Because you can imagine that when you have a triple agonist combined, you will not reach all the brain regions or the same brain regions as you would with the individual peptides by themselves. So you may restrict this triple agonist, restrict it to certain brain regions, now generating different functional outcomes.

Nick Jikomes

54:11 - 54:23

Yeah, yeah, yeah. So even the triple agonist, it’s hitting three different peptide receptors, but there’s also additional modifications such that it’s also doing things that none of these endogenous peptides do on their own.

Katrin Svensson

54:25 - 54:30

Yeah, not through different modifications, but just by the fact that they are fused in a single.

Nick Jikomes

54:30 - 54:35

I see. I see. Yeah, yeah. So it is literally like a fusion of three peptides.

Katrin Svensson

54:35 - 54:36

Yes, yes.

Nick Jikomes

54:36 - 54:47

Yeah. Interesting. And so peptides as a general area is getting a lot more attention lately. And there’s all sorts of peptides.

Nick Jikomes

54:47 - 55:05

One of the things that we talked about up front is, you know, these peptides are relatively small. They’re basically mini proteins. And there’s also a lot of pre-processing that goes into getting them to their final functional form. And those things sort of imply that there’s a huge possibility out there, like a huge diversity of possible peptides.

Nick Jikomes

55:06 - 55:20

Can you say a little bit about how how big do we think the world of endogenous peptides is? And what are, how much of it do you think we’ve mapped? Have we only discovered a small portion of what’s likely out there? And is there a lot more to be discovered?

Katrin Svensson

55:22 - 56:06

Yeah, I mean, this is what my lab studies and this is my entire research program. I think that there are many more that we have not discovered yet. But I would say that the ones that have been discovered starting from 1905 and 1909, where the first peptide hormone was discovered, I think we have found the most important ones for physiology, but there may be additional ones with functions that we started to uncover now. So I think it’s about a dozen or so that we know fairly well how they work, and they have receptors, and we have mapped out their functions in preclinical models.

Katrin Svensson

56:06 - 56:35

Some have already gone to humans, but that’s less than a handful, actually. So we still have a lot to learn from the ones that we know the identity of. And then my lab is interested in trying to see what else is out there. since we now have the genome, we can see that just by encoding, encoded in the genome based on where these enzymes are cleaving, we know that there should be additional ones that have not been characterized.

Katrin Svensson

56:36 - 56:41

Whether they’re gonna be important or not, we don’t know, but for sure they’re encoded.

Nick Jikomes

56:41 - 56:49

Okay, so you can tell or predict at least to some extent just from DNA sequence where a peptide is likely in the genome.

Katrin Svensson

56:50 - 56:50

Yes.

Nick Jikomes

56:51 - 57:05

And so can you talk about some of the work that’s being done there using computational methods to predict and characterize novel peptides? What does that look like? And have you guys discovered any new ones from, you know, just predicting it from the raw sequence?

Katrin Svensson

57:06 - 57:31

Yeah. So what we did started now a couple of years ago was to look across the entire human body. how many precursor proteins similar to pre-proglucagon may we have. So again, we know about a dozen, but we were pretty much using the PCSK cleavage site as a guide.

Katrin Svensson

57:31 - 57:50

And we’re just asking, We know that PCSK1 and 2 are cleaving many of the known bioactive hormones. What else might it cleave that would look similar? And when we did this mapping, we took the sequences in our genome. This is humans, but you could do the same thing in mice.

Katrin Svensson

57:51 - 58:21

And then just ask which ones have this characteristic signature. And it turns out that it’s about 300. Okay, so not all those will be bioactive, of course, but that will tell you where to first look. And then you can divide it up into different organs and see that actually the predominant class of those are in the brain, in the liver, and in the placenta, and then a few are in this other classical endocrine organs.

Katrin Svensson

58:22 - 58:43

So this would suggest that there are other precursors that can make peptides that are entirely understudied. And there are also new peptide fragments from known precursors. For example, POMC, there is a fragment that has not been studied. So that would also be interesting to uncover the function of.

Nick Jikomes

58:43 - 58:56

Yeah. So I guess at a high level, the world of peptides is much larger than we know right now. There’s much more that we probably don’t know than we do know, just in terms of the number and identity of all of the peptides that are endogenously present inside of us.

Katrin Svensson

58:57 - 58:57

Yes.

Nick Jikomes

58:59 - 59:16

And so I want to talk a little bit about some other peptides. But I think before we do that, we should talk about some other things. And maybe we can do this in the context of the GLP-1s and related drugs, some of those that you mentioned. When we think about weight loss and body weight regulation and obesity, Right.

Nick Jikomes

59:16 - 59:36

We’ve got the energy inside of the equation and the energy outside of the equation. I think the first half is more easy and intuitive for people to understand. Obviously, if you eat less food, that’s going to be part of why someone might lose weight on a drug like this. But you also mentioned that some of these can affect energy expenditure, basically just how much your body is burning through.

Nick Jikomes

59:37 - 59:52

Um, can you talk just a little bit about energy expenditure at a high level, and some maybe basic principles there that we should think about? And then I want to get into like, do we actually know how some of these peptide therapies are affecting the energy expenditure side of the equation?

Katrin Svensson

59:53 - 1:00:03

You know, this is a very interesting area. And that’s part of you know what’s missing. So now we have drugs that can suppress appetite. We don’t have any approved drugs that can increase energy expenditure.

Katrin Svensson

1:00:04 - 1:00:24

There was a molecule back in the day, DNP, that actually was used in humans. It increases energy expenditure by uncoupling the ATP in the mitochondria. So it creates excessive heat. So you will pretty much sweat and lose your weight this way.

Katrin Svensson

1:00:25 - 1:00:34

But it’s also, you know, it cause death. So it’s not safe. So that’s the whole question. Can we increase energy expenditure in a safe way?

Katrin Svensson

1:00:34 - 1:01:02

One natural way of doing that is to exercise. So you can increase your energy expenditure by just using much more energy, using glucose in the muscle. But that’s something that it’s hard to make people exercise if they’re not motivated to do that. But that’s pretty much the only way we have currently to increase energy expenditure or increase your muscle mass.

Katrin Svensson

1:01:03 - 1:01:05

long term to have increased basal energy expenditure.

Nick Jikomes

1:01:06 - 1:01:22

Side question. So you mentioned earlier in the animal data for some of the GLP-1 drugs that they would run if they were forced to, but they wouldn’t do as much voluntary running. Is there any human data on if these drugs are affecting people’s exercise willingness?

Katrin Svensson

1:01:23 - 1:01:57

Yes, I think there are studies and I think it’s complicated to interpret in humans, because in a lot of the human data, the GOP1 drugs have been given to obese individuals. And as they’re losing weight, they start to move more, and they’re more motivated to exercise, or they have increased movements, just in general, because it’s easier to move around. So I don’t recall any strong signal in humans that it’s causing less voluntary exercise.

Nick Jikomes

1:01:57 - 1:01:58

Yeah.

Katrin Svensson

1:01:58 - 1:02:03

I imagine that’s actually pretty clear if that’s like just a mouse phenomenon or if this is really true in humans.

Nick Jikomes

1:02:03 - 1:02:16

I imagine it could be pretty difficult to parse that too because it’s like, well, is it a direct effect of the treatment or are they getting more positive feedback in their life because they’re starting to lose weight and they just feel more motivated? It might be difficult to tease that out.

Katrin Svensson

1:02:16 - 1:02:17

Yes.

Nick Jikomes

1:02:17 - 1:02:36

Yeah. And so there’s lots of other peptides that people are taking outside of the context of being prescribed them by their doctors. And there’s all kinds of peptides out there that people are talking about in different ways. Some of them that I’ve learned about on the podcast and elsewhere affect things like energy expenditure.

Nick Jikomes

1:02:37 - 1:03:09

But I do want to get your perspective as a peptide researcher on the social phenomenon of peptides right now. In thinking about the success and the wider spread use of GLP-1s, thinking about how many peptides are out there and the more attention that’s on them right now, and just thinking about the full landscape of how people are allowed to use these things, not allowed to use these things. They can order them online without a prescription, but they’re not supposed to be injecting them, but they are.

Nick Jikomes

1:03:11 - 1:03:29

Like, what is your sort of general take on this phenomenon as a peptide biologist? Is this relatively exciting? Is this kind of scary and dangerous? Are these things, I guess one question would just be like, how dangerous do you think it is to be playing around with peptides as an individual?

Katrin Svensson

1:03:30 - 1:04:06

Yeah. Overall, it’s quite scary because peptides can be very, very potent, both the on-target effect, the real effects of them, but also the way that they’re made and administered, it creates a lot of risk. But in general, I think the impression is that because peptides are endogenous molecules, they’re made by the body, they are generally safe. And I think Although that may be true that their physiological function is safe, we’re talking about pharmacology where you’re injecting a very high dose.

Katrin Svensson

1:04:06 - 1:04:30

So some of these newer peptides, for example, melanotone, is injected to suppress appetite, increase tanning. and have all sorts of other effects. It’s actually acting on all the MC receptors, MC4R, MC1R and MC3R. It’s a polyagonist.

Katrin Svensson

1:04:31 - 1:04:51

So it is an endogenous similar molecules synthetic peptide made from an endogenous molecule, it acts truly on those receptors is specific, but because it’s such a high dose acts on multiple different receptors, you will have a lot of side effects. And I think sometimes the general public, they don’t really appreciate that.

Nick Jikomes

1:04:52 - 1:05:07

So melanotan, named after the fact that it’s interacting with all of these melanocortin receptors. We talked about melanocortin-4 earlier, but there are a variety of these melanocortin receptors. They’re very important in the body. And its primary effect that people are after is that it makes them tan?

Katrin Svensson

1:05:09 - 1:05:09

Yeah. Yeah.

Nick Jikomes

1:05:11 - 1:05:17

And do we know anything else about known side effects or expected side effects from playing with all of those receptors?

Katrin Svensson

1:05:18 - 1:05:44

Yeah, so it changes the melanocyte production, so you can develop nevi or hyperpigmentation. It also suppresses appetite and can cause nausea, so a lot of side effects associated with the true effect of these of these peptides. There was some speculation that it can increase your risk for skin cancer. I’m not entirely sure if that is true.

Katrin Svensson

1:05:45 - 1:06:02

But again, it has not been tested like we don’t know what the long term effects are. So even if you manage to get an entirely pure peptide, it’s not a contaminant. in there and it’s purified in the correct way, even that, it needs to go through rigorous testing. I wouldn’t take it.

Nick Jikomes

1:06:03 - 1:06:17

Yeah. So yeah, there’s the question of, are you actually getting the pure peptide and assuming you are, then what is it doing? And then there’s just the simple fact that there’s basically no long-term human data on any of these more or less.

Katrin Svensson

1:06:18 - 1:06:18

Right.

Nick Jikomes

1:06:19 - 1:06:29

What are some of the other popular ones out there that people are talking about and taking more, such as Melanotan, that you’ve heard about or know anything about?

Katrin Svensson

1:06:30 - 1:06:56

there is this BPC-157 molecule which people take for muscle recovery and that has come up in multiple different scenarios and people have asked me about it. So I did a quick search in the genome and it actually does not have a precursor protein. It’s described as a gastric peptide identified in humans. It actually does not have a human sequence.

Katrin Svensson

1:06:57 - 1:07:14

I don’t think it exists. I could not find any homologue or anything similar to this. I think it’s just probably a contaminant that it has been reported in the literature, but I think it’s probably just a contaminant. You can make this peptide, you can inject it, maybe people see something or not.

Katrin Svensson

1:07:14 - 1:07:36

it does not seem to be reproducible, people still take it for various things. If it has some inflammatory effect or some off-target effect, I’m sure people can see something or observe what they think is a true effect, but that is pretty much entirely unfounded as far as the scientific evidence.

Nick Jikomes

1:07:37 - 1:07:53

I see. So BPC-157, body protection compound 157. If I was hearing you correctly, this was originally isolated from like a gastric extraction of some kind. And so it was speculated that perhaps it’s an endogenous peptide or derived from one.

Nick Jikomes

1:07:53 - 1:08:01

But you’re saying that you did a genome search using some of the tools you’ve developed, and there’s no obvious indication that there’s an endogenous version or precursor to this.

Katrin Svensson

1:08:01 - 1:08:03

There’s no endogenous version. Yeah.

Nick Jikomes

1:08:03 - 1:08:07

Okay. Okay. And that would be probably, would that be pretty clear for you to see if it was there?

Katrin Svensson

1:08:08 - 1:08:09

It’s absolutely clear. Yes.

Nick Jikomes

1:08:09 - 1:08:11

Yeah. Okay. Okay. So it’s a peptide.

Nick Jikomes

1:08:11 - 1:08:15

You can make it, people are using it, but it’s not an endogenous peptide.

Katrin Svensson

1:08:15 - 1:08:15

Right.

Nick Jikomes

1:08:16 - 1:08:37

Got it. Um, what about so one of the other ones I’ve heard a lot about, and I’ve done a little reading on in terms of the animal literature, which is very interesting, actually, was MOTS-C, this mitochondrial-derived peptide, which does seem to have, in rodents at least, an anti-obesity effect on food intake, and also does seem to affect energy expenditure. Is this something that you know much about?

Katrin Svensson

1:08:37 - 1:09:03

I don’t know much about that, the mitochondrial peptides, but I know that has been a big area of investigation. This is not the only one that has been identified. There are other ones too that are these cryptic peptides or peptides that are mitochondrial that have been missed sort of in the genomic mapping because they were typically shorter than what we would assign a gene.

Nick Jikomes

1:09:06 - 1:09:13

What are some of the major projects that you’re working on in the lab right now with regard to peptide biology that you’re particularly excited about?

Katrin Svensson

1:09:14 - 1:09:38

We are working primarily on trying to identify non-incretin peptides in biological activities. So things that don’t act on the pancreas to increase insulin secretion, but rather act through some other receptor or mechanism to control feeding or to control other aspects of metabolic regulation, but primarily we’re looking at feeding behavior.

Nick Jikomes

1:09:39 - 1:09:57

And you did, there is a paper that I glanced at where you discovered this thing called BRP. So can you talk a little bit about how you discovered that? And then maybe a little bit about what’s the motivation to find things like anti-obesity peptides that act through different mechanisms from the GLPs?

Katrin Svensson

1:09:58 - 1:10:39

Yeah, so the reason we were looking for non-incretin and non-GLP type peptides was we wanted to have a way where we modulate feeding without affecting the gut. So where you don’t have any effect on gastric emptying, or that would be one way where you can imagine that you would avoid the side effects on inducing nausea and GI upset. So that was our motivation for starting that. And we did first a computational search across the entire human genome to look for pre-prochormals.

Katrin Svensson

1:10:40 - 1:10:58

And then we focused on the brain. So this BRP peptide is the precursor is expressed in the brain. in the cortex, but also in the hypothalamus, and has pretty high expression. And it’s expressed in cells that also express the PCSK1 enzyme.

Katrin Svensson

1:10:59 - 1:11:25

So it’s possible that since it has these canonical cleavage sites, that it’s making small peptides. So we saw this, and what we decided to do was to generate small peptides and just test from this parent protein. It’s called BRI-NP2. And then we tested them by injecting them into mice and look at different physiological functions, including feeding, glucose regulation, and other things.

Katrin Svensson

1:11:26 - 1:11:43

And what we quickly saw was we tested, I think, 20 or so high potential candidates in mice. We injected them. And this BRP peptide really robustly reduced food intake. Within 30 minutes, the mice stopped eating.

Nick Jikomes

1:11:43 - 1:11:59

Okay, so this was an example where you basically predicted the existence of this from the DNA sequence and then you went and looked and indeed there’s this BRP peptide and it has an anti-obesity effect basically, but it’s acting through a different mechanism than GLPs.

Katrin Svensson

1:12:00 - 1:12:18

Yes, yes. So what we have done now, we did experiments, for example, in the GOP1 receptor knockout mice that no longer respond to GOP1, they still respond to BRP. So we know that it’s an independent pathway. And we can also look in the gut and we don’t see any gastric emptying effect.

Katrin Svensson

1:12:18 - 1:12:34

So there’s no slowing down of gastric emptying. So it only acts on the brain. And when we go into the brain, we can do whole brain false mapping. And we see that it activates different neurons that are non overlapping with the neurons that are activated by GLP one.

Katrin Svensson

1:12:35 - 1:12:42

So we Yeah, so it’s so we can we can clearly see it’s, it is an activity, there is an activity, but it’s just not in the same level.

Nick Jikomes

1:12:43 - 1:13:13

So the GLP-1s have central effects in the brain and peripheral effects in the gut, one of which is they’re slowing transit time of food, basically. This new BRP peptide that you guys discovered using computational methods has an anti-obesity effect, a weight loss effect in rodents. It seems to have no peripheral effect, or at least not the gastric emptying and transit time effects that GLP-1s have. And it does seem to act centrally in the brain, but it’s doing something very different in the brain than GLP-1s.

Nick Jikomes

1:13:13 - 1:13:14

It’s affecting different neurons in different ways.

Katrin Svensson

1:13:15 - 1:13:16

Yes. Yes.

Nick Jikomes

1:13:17 - 1:13:33

So I guess the hope here would be that you could discover peptides, develop new peptide therapies that either A, work for people that don’t really respond to GLP-1s for whatever reason, or B, do respond to GLP-1s, but maybe have side effects that they don’t like.

Katrin Svensson

1:13:34 - 1:13:42

Yes. And one idea, I mean, we’re not trying to compete with GLP-1s. GLP-1s are absolutely amazing. That would be first-line therapy for a very long time.

Katrin Svensson

1:13:43 - 1:14:03

And I think that’s an amazing discovery, an amazing drug. It doesn’t work for everybody. And you can imagine that some people need to go off of it and need to have other types of therapies. Or you can imagine that some individuals would need even more weight loss, where you can add on different other types of peptides.

Nick Jikomes

1:14:04 - 1:14:15

Mm-hmm. And so endogenous peptides, as we said, operate on intermediate time scales. So not seconds, not hours. They’re more operating on the scale of minutes.

Nick Jikomes

1:14:17 - 1:14:47

So they don’t stick around forever, but they last longer than some other super fast signals like neurotransmitters and things like that. The engineered weight loss drugs, like the GLPs, are engineered to basically last longer. Is sort of the timescale that endogenous peptides work on, to what extent is that a feature or a bug that we want to engineer away? Do you think a lot of these peptides might be best used as short-acting things?

Nick Jikomes

1:14:48 - 1:14:59

Or do you think we’re going to continue to need to modify them to make them last longer? And do you think that could pose any downside risk in terms of what these things are going to end up doing to people who use them for years and years?

Katrin Svensson

1:15:00 - 1:15:22

Yeah, so I think it really depends on what molecule it is and what the function is of that molecule. So we have had insulin for over 100 years. And this is a molecule that you want to have very short acting and people inject insulin, it does its thing after a meal or blood glucose. And they’re even looking to make even shorter acting insulins.

Katrin Svensson

1:15:22 - 1:15:54

So it depends on what kind of effect you’re looking for, it may not be beneficial to extend the half-life of all the molecules and all the peptides into a long-acting molecule. You may induce other types of effects or other side effects you wouldn’t see if it would be short-acting. But I think it’s more a practical question. So before semaglutide and ozempic came along, there was a molecule called liraglutide, and it had to be injected daily.

Katrin Svensson

1:15:54 - 1:16:22

And that’s just a thing that people don’t like to inject daily, if you can inject it weekly. So all these new drugs that are coming along, there are antibody conjugates, different things that are put on the peptide molecules to make it a monthly injection. or, you know, that typically sounds more attractive. but not even talking about the oral peptides that are coming along now.

Nick Jikomes

1:16:22 - 1:16:38

Yeah, yeah, yeah. Well, that’s actually, that’s great that you brought that up. That’s something I wanted to ask you about. So you mentioned DPC earlier, which was derived from a gastric secretion, which implies that it has some ability to withstand the stomach, basically.

Nick Jikomes

1:16:38 - 1:17:04

Given the fundamental structure of peptides here, they’re just small sequences of amino acids, How robust are these things or can they be made to withstand the GI tract so that oral routes of administration are viable? Are most of them going to be chopped up very easily in the stomach and inactivated? Or is it relatively easy to discover or engineer peptides that are going to be orally active and then there’s no need for injection?

Katrin Svensson

1:17:05 - 1:17:35

For a very long time, I think the the main hypothesis was that we cannot make peptides orally bioavailable. But it has now been proven that we can actually do this by engineering the actual peptide or by co-administered with things that protect them from degradation. But in general, it’s not an easy thing to do. It really depends on the molecule.

Katrin Svensson

1:17:35 - 1:17:49

You may not be able to do it at all with all the molecules that has been shown to you know, work for GOP1 may not be applicable for all these other duogonists and triagonists. We’ll just have to wait and see.

Nick Jikomes

1:17:51 - 1:18:15

And how long, so how long now do we have data for on long-term use of GLP-1s in humans? What’s sort of the longest timeframe people have been studied in a clinical setting who have taken these? Because one of the things that people talk about is you often need to stay on these things to maintain the weight loss. Some people are probably just gonna stay on them for years and years at a time.

Nick Jikomes

1:18:15 - 1:18:30

That’s likely for a large number of people. Is there any reason to believe that you might have negative consequences that only arise after very extended periods of use? And how long is the clinical data that we actually have in terms of the length of use right now?

Katrin Svensson

1:18:31 - 1:19:12

The clinical data for GLP1s at the dose where they’re treating obesity is not very long, but they have been used in individuals with diabetes for a very long time, 10, 15 years now. And it only seems to be positive so far, so there hasn’t been any direct long-term negative side effects. If anything seems to be protective, against cardiovascular disease. And there are some indications that it has a protective role against certain types of obesity-associated cancers even, although that data is still coming in.

Katrin Svensson

1:19:14 - 1:19:26

We will have to see what at this higher dose, whether it will be even larger benefit or whether there will be some other side effects. But in general, they are thought of as beneficial.

Nick Jikomes

1:19:28 - 1:19:32

And so what are some of the other projects that your lab is pursuing today beyond the BRP story?

Katrin Svensson

1:19:33 - 1:19:58

So we’re working on other modifications of peptides. So we have done a similar search where we look for potentially new cleavage fragments with imprinted modifications. Phosphorylation is a modification that is typically thought of as being put on proteins. And that is very well established that it affects the protein function.

Katrin Svensson

1:19:58 - 1:20:22

On peptides, it also is there, it’s detected. pretty much no functional value as far as we know. We think that there are some scattered papers showing that phosphorylation of a peptide can affect the bioactivity, but it’s not entirely clear how that works. And we find that there are many hundreds of peptides that are phosphorylated on a serine.

Katrin Svensson

1:20:23 - 1:20:46

And how does that affect the function? It’s entirely unclear. So we’re working a lot and trying to understand what is the rest of the landscape of these peptides, when are they made, where are they made, how are they modified in a sort of larger scale than what was done previously, you know, in the 50s and 60s, they went one by one purifying them and looking at what particular modifications were there.

Katrin Svensson

1:20:47 - 1:20:52

We’re trying to do this in a more systematic way, and then try to identify which ones of these are important.

Nick Jikomes

1:20:53 - 1:21:26

So, a lot of people are using a lot of different peptides at this point, as we’ve touched on. There’s also going to be some potential new regulatory changes coming up this summer that affect basically how easy it’s going to be for people to acquire these research peptides. As the type of scientist you are with your background in peptides, what is your general make of that in terms of what you’re going to be looking out for and what you think people should look out for when they think about accessibility and actual personal use of these things?

Nick Jikomes

1:21:26 - 1:21:44

I would love if you could do that in the context of just like the stability and the structure of these things and how easy it is to store them, to make sure they are what they say they are, and just how stable and safe they are as molecules that might be sitting in someone’s refrigerator.

Katrin Svensson

1:21:46 - 1:22:25

Yeah, I would be very careful just in general to take anything where you don’t have an analytical instrument to really make sure that what is in the vial is really what you think it is. I think for many of these research grade peptides, we buy them for research use. And the purity can be anything from like 85% up to 98%. But what is the rest of the, you know, even if it’s 98% pure, the rest of it could be something else that you have no idea what effect that could have injected at a high enough dose that could actually be significant.

Katrin Svensson

1:22:26 - 1:22:57

The other thing with research grade peptides is that they’re often dissolved in different types of salt. So there’s a TFA salt that is very often used when you make just general peptide synthesis. That is not safe for human injection and it’s pretty much impossible for a general person to, they don’t label on the vial, whether it’s made with TFA salt or something else. So it’s pretty much impossible to know what’s in the vial.

Katrin Svensson

1:22:58 - 1:23:37

And then again, if you dissolve it in sterile water, how do you know that it’s actually sterile? How do you know that you store it in the fridge that is not aggregated? That is a major concern that was the problem for amylin peptides for a long time, where the endogenous peptide is actually forming amyloids, aggregates. And if you inject this aggregate, it can accumulate in your tissues, in the pancreas, maybe in the brain, and cause some severe adverse effects that you don’t know what the functional consequences are.

Katrin Svensson

1:23:38 - 1:23:47

That we have no idea about for any of these new peptides. So this is short term. It may not cause any harm. You say, oh, this is safe.

Katrin Svensson

1:23:48 - 1:24:08

It’s fine. And I inject it and nothing happened. But you don’t know long term what this But the chemical structure of this peptide looks like you would need a very advanced instrument like an NLR to determine if this is aggregated or not. If it’s not sterile, that’s a whole other issue, right?

Katrin Svensson

1:24:08 - 1:24:20

You keep it in the fridge for some time, you take it in and out, maybe you’re not using sterile technique. to draw up in the needles. And then you’re injecting yourself with this. You can cause sepsis.

Katrin Svensson

1:24:20 - 1:24:22

So it’s just not safe.

Nick Jikomes

1:24:22 - 1:24:40

Yeah. So if I’m hearing you correctly, even when you guys in the lab order research-grade peptides for literal research purposes, they can be of variable purity. And it’s very common for them to be 98% pure or less. And you don’t even know it’s in there, the other 2%.

Katrin Svensson

1:24:42 - 1:24:49

Right. I mean, we do analysis. We do analysis of them to see what’s in there. So we use LC-MS or NMR to look and see what’s in there.

Katrin Svensson

1:24:50 - 1:24:55

But unless you know how to interpret that data or have access to this instrument, how would you know?

Nick Jikomes

1:24:55 - 1:25:08

Right, right, right. Yeah. So if someone buys peptides online and it’s 98% pure, maybe even the company that made it did test it, but they probably don’t even know what the other 2% is. And that’s just going to be a question mark.

Katrin Svensson

1:25:09 - 1:25:09

Right.

Nick Jikomes

1:25:10 - 1:25:26

Yeah, yeah. Um, one of the final topics I want to ask you about and this is something that people are also talking about with respect to the GLP ones is. what’s going on with the weight loss in terms of the tissue that’s lost? Is it mostly fat?

Nick Jikomes

1:25:26 - 1:25:46

Is there lean mass being lost as well? How does that compare from lean mass loss and fat loss that you would get from regular dieting and things like that? So can you just kind of give us a lay of the land on what the data says with the GLP-1s and related drugs in terms of fat loss and preservation of lean mass?

Katrin Svensson

1:25:48 - 1:26:31

Yes. So there’s been a lot of discussion in the field because there have been studies in both humans and mice showing that when you’re losing weight, when you’re on a GLP-1, you lose both fat mass and lean mass. And there’s a concern that the lean mass loss would lead to reduced muscle function or sarcopenia. And But I think the latest data would suggest that you don’t lose predominantly more lean mass when you’re on a GLP-1 versus if you’re just dieting and go on caloric restriction, you lose body weight, you lose the same proportion amount of fat mass and lean mass.

Katrin Svensson

1:26:32 - 1:26:48

A leaner person will have less lean mass. that’s not necessarily detrimental. So unless you have good markers or good ways of measuring actual muscle function, it’s not clear whether this is actually a problem or not.

Nick Jikomes

1:26:48 - 1:27:11

Got it. So if you’re going to lose a substantial amount of weight, you’re probably going to lose some combination of fat and lean mass, such as muscle mass, regardless of how you do it. So it’s really a matter of, are these drugs causing more lean mass loss than you would get from regular dieting weight loss, above and beyond what you would get from that? Or is it about what you would expect from doing that?

Nick Jikomes

1:27:12 - 1:27:17

And you’re saying basically so far, it doesn’t seem to be an obviously concerning amount of lean mass loss.

Katrin Svensson

1:27:17 - 1:27:40

Right. And when you look, so for the preclinical status in animals, if we look at muscle strength after GOP1 treatment, the muscle strength is actually higher per milligram of muscle tissue you have. So even though you lose muscle mass, you actually don’t lose muscle strength. So the strength is intact, which would suggest the muscle is working just fine.

Katrin Svensson

1:27:41 - 1:28:09

But this is in, you know, young animals, it may be different if they’re very aged. So one, and I think that this is true also in humans, that there’s no reduction in grip strength and there’s not really good measurements in humans. But it doesn’t seem like overall muscle function is reduced. But this is the average, I think, GLP-1 user is like a 45-year-old female.

Katrin Svensson

1:28:09 - 1:28:27

So it may be different in males. It may be different in very aged individuals that are losing a lot of weight that are not exercising. So we would need to really look at different populations and different amounts of weight loss. and maybe their protein intake.

Katrin Svensson

1:28:27 - 1:28:46

So that is something people talk about when you’re on GLP-1. You should also take extra protein to see if there’s any clinical detrimental effect. So far, it doesn’t seem to be that the evidence is suggesting that, but it could be a potential problem in elderly, sarcopenic individuals.

Nick Jikomes

1:28:46 - 1:29:06

Yeah. So I want to ask you a question from your perspective as a scientific expert on this stuff. But not as a physician, so we’re not giving medical advice or anything, and we never do on this podcast. But let’s imagine ourselves in the shoes of someone who’s kind of like that average user that you mentioned.

Nick Jikomes

1:29:06 - 1:29:20

You’re middle-aged, maybe you’re in your 40s or 50s. You’re overweight, but you’re not morbidly obese. You’re not catastrophically metabolically ill. You’re just hoping to lose some weight and get into somewhat better shape.

Nick Jikomes

1:29:20 - 1:29:40

You’ve struggled somewhat with that, but you’ve probably put in, say, a normal amount of effort. You’re sort of a normal potential candidate for one of these things. What would be the kind of calculus in your mind that you would go through to evaluate whether or not to try one of the GLP-1s that’s out there if you were such a person?

Katrin Svensson

1:29:42 - 1:30:36

I think it’s a personal preference, but also, unless you’re talking about research-grade drugs, which is not recommended for anybody, if a physician is prescribing these compounds, then I think following the advice of them would be the best way to go. I don’t have any strong opinions either way, you know, whether they should be used, whether you’re clinically obese or not. I think the BMI scale is different for different ethnicities and different populations. So you can, and you can also imagine that there are many scenarios where you’re not morbidly obese, but your values, your LDL, cholesterol, glucose levels, everything would be improved if you would just lose 10 pounds of body weight.

Nick Jikomes

1:30:37 - 1:30:51

Yeah. So you can imagine situations where it would make sense to take it from a metabolic health perspective, even if you weren’t morbidly obese. And then how would you, you know, let’s just talk a little bit more one more time about the side effects. What are the major side effects?

Nick Jikomes

1:30:52 - 1:30:57

And are those generally things that are going to be, you think, relatively mild for people or things they should really stop and think about?

Katrin Svensson

1:30:59 - 1:31:15

The side effects will depend on the dose and actually it will depend on the individual. Some individuals report very few side effects and some have to stop taking them because they cannot stand the side effects. So it’s hard to predict how you’re going to respond.

Nick Jikomes

1:31:17 - 1:31:29

Is there any risk that... in general, physicians might get a little bit too enthusiastic about prescribing these things as they perhaps had for other drugs in the past?

Katrin Svensson

1:31:33 - 1:31:40

It’s really hard for me to speculate on that. In general, I think they would prescribe according to the guidelines.

Nick Jikomes

1:31:41 - 1:31:49

OK, that’s an interesting answer. Any other exciting projects that you’re working on the lab that you want to talk about before we stop today?

Katrin Svensson

1:31:50 - 1:32:22

Yeah, so we’re working on weight loss drugs and their potential benefit for cancer and I’m really interested in this area just because of the clinical data that we see with GLP1s and that may be protective against certain types of obesity associated cancers. And we’re extending this now to look at other molecules that are reducing body weight. Is this directly correlated with the body weight loss or is this something else in general? Anything that is causing weight loss, would that be protective?

Katrin Svensson

1:32:22 - 1:32:51

Because that’s another layer that you know, where we could have a huge benefit for a large number of people. The other area where we’re also entering into, although it’s not my area of expertise, is the addiction and the reward pathways. I think many of these molecules are made in brain, and they are made in different areas, and they gotta have some function there. And can we identify some common circuits?

Katrin Svensson

1:32:51 - 1:33:00

Can we identify something where we can modulate them with pharmacology that is mimicking the endogenous function, I think that that would be extremely interesting.

Nick Jikomes

1:33:01 - 1:33:16

Yeah. So kind of similar to the idea of, can we develop therapies that speak to the hunger and satiety regulation without hitting reward processing more generally? This would be, can we develop something that might be good for addiction without bleeding into some of these other things?

Katrin Svensson

1:33:17 - 1:33:17

Yes. Yes.

Nick Jikomes

1:33:18 - 1:33:22

Yeah. Yeah. Interesting. Well, I want to thank you very much for your time.

Nick Jikomes

1:33:22 - 1:33:31

We covered a lot of ground. Is there anything else you want to say? Any final thoughts or anything you want to reiterate about peptides or GLP-1s generally before we sign off?

Katrin Svensson

1:33:32 - 1:33:34

No, this was a very interesting discussion. Thank you.

Nick Jikomes

1:33:35 - 1:33:38

All right. Well, Dr. Katrin Svensson, thank you very much for your time.

Katrin Svensson

1:33:39 - 1:33:39

Thank you.

This is a public episode. If you'd like to discuss this with other subscribers or get access to bonus episodes, visit mindandmatter.substack.com/subscribe

Wide release: June 18, 2026. Not medical advice.

Nick speaks with Gábor Somlyai, PhD about the biological role of deuterium, a heavier, non-radioactive isotope of hydrogen present in water and organic molecules. Somlyai’s key argument is that small shifts in the deuterium-hydrogen (D-H) ratio act as a fundamental regulator of cell growth, with deuterium-depleted water (DDW) inhibiting tumor progression. They explore mitochondrial production of naturally deuterium-depleted metabolic water, deuterium’s effects on biochemical reaction rates, evidence from cell culture and animal models, and human clinical data showing improved outcomes in cancer patients using DDW as an adjunct therapy.

TOPICS DISCUSSED:

* Deuterium Basics: Deuterium is twice as massive as regular hydrogen; natural water contains ~150 ppm, with stronger oxygen-deuterium bonds slowing reactions compared to oxygen-hydrogen.

* Mitochondrial Role: Healthy mitochondria produce deuterium-depleted metabolic water (~110 ppm from fat oxidation vs. ~150 ppm from carbs), helping maintain low cellular D:H ratios.

* Cell Growth Regulation: Lower deuterium slows cell division in vitro; cells sense small D:H changes, with higher levels (near/above 150 ppm) promoting growth.

* In Vitro Evidence: DDW (e.g., 25-125 ppm) can reduce proliferation and alters cancer-related gene expression.

* Animal Models: DDW caused tumor regression in mice with transplanted human cancers and showed possible efficacy in veterinary use for dogs and cats.

* Human Prostate Cancer: Phase 2 randomized study showed greater Prostate-Specific Antigen (PSA) reduction, tumor shrinkage, and better 1-year survival with DDW plus standard therapy vs. placebo.

* Dietary Modulation: Ketogenic/high-fat diets lower body deuterium from lipid oxidation.

* Broader Implications: Potential in neurodegeneration models; larger trials needed for registration as adjunct cancer therapy.

ABOUT THE GUEST: Gábor Somlyai, PhD is a molecular biologist who pioneered deuterium depletion studies at the National Institute of Oncology in Hungary. His primary work focuses on the role of deuterium in living organisms and the development of deuterium-depleted water for cancer therapy.

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PRACTICAL TAKEAWAYS:

* Drink deuterium-depleted water (DDW) at targeted concentrations (e.g., 85-105 ppm) for months may act as a potential adjunct to standard care in oncology, following established protocols.

* Prioritize high-fat, low-carbohydrate (ketogenic-style) eating to naturally reduce bodily deuterium levels through mitochondrial metabolism.

SUBSCRIBER CONTENT BELOW: Reference paper + episode transcript.

Reference Paper:

* Paper | Real-World Data Confirm That the Integration of Deuterium Depletion into Conventional Cancer Therapy Multiplies the Survival Probability of Patients

AI-generated transcript below. Beware of mistranslations!

Nick Jikomes

02:56 - 03:03

Do you want to start off by just telling everyone a little bit about who you are and what your background is and what you research?

Gábor Somlyai

03:06 - 03:46

I graduated as a molecular biologist very, very long ago. I worked with DNA during my study and even after I graduated. But the reason I want to become a biologist is because my aim was to do something on cancer research, hoping that I can find or figure out something which can be very important and useful for everybody. I started that type of research with deutium and deutium-depleted water after I came back from the United States where I was a postdoc position in a gene mapping work.

Gábor Somlyai

03:46 - 04:14

This is what I did. But finally I come back to Hungary and I looking for job and I went to work to the National Institute of Oncology. So I start, because I have a PhD, I had the right to do research what I wanted. The aim of the research was to figure out whether this deuterium has any role in the living organism.

Gábor Somlyai

04:15 - 04:58

and in the very first study at the Institute of Oncology I could prove that a lower deuterium concentration can cause very, very significant effect in tissue culture, or mice have been transplanted with tumor. So since that type, our job is to generate more and more research, to involve more and more scientists, and to test on a different field of the biology, the effect of deuterium in the living organism. So, and this is where we are now. I could say that The concept that the duty has a very important role in the living organism is well established.

Gábor Somlyai

04:59 - 05:40

This year we just organized the fifth international congress on duty depletion. People coming from certain countries, and we can see that searching on the PubMed for deuterium depleted water, the number of papers exponentially increasing since we published our first paper in 1993. So finally, my job is to start it with a concept, with an idea that the deuterium has an important role. After that, started the research, we published paper and now aim is to to get a cancer drug registered for human consumption.

Nick Jikomes

05:41 - 05:54

So we’re going to be talking about deuterium and deuterium depleted water today. This is a new area for me. I don’t know much about this at all. I have been hearing more about this, and I think people in general have started to hear more about this.

Nick Jikomes

05:54 - 06:09

But I think most people listening will know very, very little about this. So we have to start with the very basics today. So we’re talking about deuterium, which is a version of hydrogen. So there are different types of hydrogen that differ based on their physical properties.

Nick Jikomes

06:09 - 06:15

Let’s just start with the very bare bones basics here. What are the different types of hydrogen and what is deuterium?

Gábor Somlyai

06:16 - 06:35

So in nature, there are three different hydrogen. The hydrogen with mass number one, deuterium with mass number two, and the tritium with mass number three. This is radioactive. The deuterium is a non-radioactive version of the hydrogen.

Gábor Somlyai

06:35 - 07:03

But in that case, there are 100% difference in the mass between the hydrogen and deuterium. And that cause very big differences in the chemical reaction when we talk about hydrogen or deuterium. Typically, an oxygen-H bond split faster than an oxygen-deuterium bond. So it is called isotopic effect.

Gábor Somlyai

07:03 - 07:50

This is very known since the discovery of the heavy hydrogen, the deuterium. The reason the deuterium can be important that even everybody knows that deuterium is present, but in an surface water in a normal water, the ratio between hydrogen and deuterium is about 6,600 to one. So we can say that very rare, the deuterium. But if we explain the deconcentration in millimole, because we know that in a calcium level in the blood about 2.5 millimole, the potassium is about 3.55 millimole.

Gábor Somlyai

07:50 - 08:44

but the deuterium is 12 millimole or 13 millimole in our body. And considering that here is the deuterium in a so relevant concentration, and if you see that calcium, magnesium, potassium, sodium is so strictly regulated in the living organism, but the deuterium 10 times higher than magnesium and nobody investigated the possible role of the deuterium. So the breakthrough in my way of thinking and the research that there’s a question what will happen if we reduce the deuterium level and whether it cause any effect because if we see some significant effect we can suppose that during the evolution the deuterium some role of the deuterium.

Nick Jikomes

08:44 - 09:06

So that was the question. Our bodies are made out of, there’s lots of hydrogen in our bodies, there’s lots of water, and water is H2O, so two hydrogens, one oxygen. There’s three types of hydrogen that we know about in the universe. There’s hydrogen, what we call, let’s just call it regular hydrogen, and that’s basically just one proton in the nucleus.

Nick Jikomes

09:06 - 09:31

So then deuterium would be a proton and a neutron. So that nucleus is twice as massive in the deuterium compared to the regular hydrogen, Both of those are non-radioactive. And then there’s tritium, which is one proton, two neutrons. So it’s got three times the mass of the nucleus of regular hydrogen, which is that single proton.

Nick Jikomes

09:31 - 09:53

That one is radioactive. Today, we’re talking about regular hydrogen compared to deuterium. And basically what you said is, so the deuterium is way more rare than regular hydrogen if we just measure it out in the world and measured in our bodies, but it’s still more plentiful than some other things in our bodies that we know are important. Yeah.

Nick Jikomes

09:53 - 10:16

Yeah, exactly. Yeah. So when we think about this just in raw physical terms, so the deuterium is twice as massive as the regular hydrogen. So if we imagine a water molecule, most water molecules in our body and in the world are one oxygen and two regular hydrogens, but sometimes they can have an oxygen with one or two deuteriums.

Nick Jikomes

10:17 - 10:20

So they’re going to be heavy. It’s going to be a heavier, more massive water molecule.

Gábor Somlyai

10:21 - 10:56

Typically, at that low concentration, the water is in HDO form, so one hydrogen, one deuterium and one oxygen. It’s very rare at a very low level of deconcentration that two deuterium bind to the oxygen. If we talk about heavy water, in that case, the deconcentration is close to 99%. Yes, and that is to deuterium bind to the oxygen, but at a natural concentration, we can talk about HDO.

Gábor Somlyai

10:56 - 11:00

This is the way how the deuterium is present in a water form.

Nick Jikomes

11:00 - 11:22

Yeah, yeah. And I guess if we think about this, so there’s water and most of the water is going to be normal, regular water, how we would normally think about it. H2O, both of those hydrogens are the regular hydrogen. Sometimes you’ve got a deuterated heavy water and that’s H2O, but one of the hydrogens is this deuterium hydrogen, so it’s a little bit heavier.

Nick Jikomes

11:22 - 11:34

So I guess it’s literally gonna be heavier and that would presumably affect the water in terms of just like the physics of how it moves because its mass is different.

Gábor Somlyai

11:34 - 12:23

I would say yes, but the point is that in our body, one single cells, there are billions and billions chemical reaction every second and the hydrogen And the hydrogen bond has a very, very strict role in the DNA, within the DNA, when the messenger RNA is synthesized and the base pairing between the DNA and the RNA, when the ribosome is synthesizing the proteins. So it’s very crucial. So billions and billions of reactions are running with the hydrogen, splitting the hydrogen bond and forming a new hydrogen bond.

Gábor Somlyai

12:24 - 12:57

And the question is, when how frequently that reaction is running with the deuterium, which slows down that process, maybe stop the whole reaction, slow it down. And that, what is the effect for the next step of that chemical reaction? And how can it modify that whole process? So the reason we should think about when I talk about those who are not really expert.

Gábor Somlyai

12:59 - 13:25

We should understand how the life can be organized and how can be so sophisticated, so perfect. People should think about that there is a cell size which is, let’s say 40 micrometer. which is 25th part of a one millimeter. And there is a nucleus which is 10 micrometer.

Gábor Somlyai

13:27 - 13:48

Can you imagine how small is that? But within that nucleus, there is 1.8 meter length DNA. Everybody has the DNA packaged in the 10 micrometer size nucleus with a 3,200,000,000 base pair. and it’s so condensed information.

Gábor Somlyai

13:49 - 14:26

containing the story of the evolution, containing all the knowledge, everything about the people, the color of the hair, color of the eye, everything. And it is packaged. And the question is whether the life can be organized by big protein molecules. What we suppose, and we are building the drug development, that finding a mutation in the in the gene and hoping that if we can target that protein that we can get a cancer drug.

Gábor Somlyai

14:26 - 15:21

And this is how the world is trying to solve the cancer problem. when we talk about that how the life can be organized and how the life, the cell, regulate the cell division, then I say based upon our research it is organized on a sub-molecular level. So during the billions of years When finally the living organism could use out that there is a heavy hydrogen, they build up a regulatory system, changing the cell, they are able to modify the DH ratio in the cell, and after that changing the ratio simultaneously can send the message to all the molecules, all the proteins, all the nucleic acids within the cell.

Gábor Somlyai

15:22 - 15:54

So this is a change of paradigm. So far we believe that finding a molecule binding to one protein, a kinase or whatever, that can be so effective that we really can intervene and stop the cell growth and cure the cancer. But the aim is how can we control the whole system at the same time? And this is what we are saying.

Gábor Somlyai

15:54 - 16:47

So if we talk about only the prokaryotes, the bacteria, the bacteria don’t need the deuterium because they are so primitive organism that they can organize the cells, metabolize based on the molecular level. But when the mitochondria, which was a bacteria, targeted and moved into the aortic cell and they live together that give a chance for the living organism to set up a new system. And we say today that the life, the membrane transport can increase the deuterium hydrogen ratio. Meantime, everybody, every day, by the mitochondria we are synthesizing water.

Gábor Somlyai

16:47 - 17:04

So we effectively produce every day about 0.3-0.4 liter metabolic water. So we are burning the hydrogen to water. This is a metabolic water. and that way the mitochondria producing deuterium depleted metabolic water.

Gábor Somlyai

17:05 - 17:11

So, and this is a mechanism how it works, the heavy hydrogen, the semi-heavy hydrogen.

Nick Jikomes

17:12 - 17:45

So, okay, let me see if I’m following so far. So, you’ve got deuterium and you’ve got regular hydrogen. So, we can abbreviate those to H for regular hydrogen, D for deuterium, which is twice as massive. There is some water that is heavy water because it’s got a deuterium hydrogen as part of the H2O molecule and the presence of a regular hydrogen or reverses a deuterium hydrogen in a water molecule will change how easy it is for the bond to be broken between the

Nick Jikomes

17:45 - 18:29

hydrogen and the oxygen. And I guess what people should understand going into this is, you know, a big reason why water is essential for life is not only because it’s a solvent that we’re immersed in, but it’s participating in biochemical reactions. So what you’re saying is billions of times per second in all of our cells, we’re constantly breaking the hydrogen oxygen bonds of water as part of the biochemistry of life. And so if the strength of the bond between a hydrogen oxygen and a deuterium oxygen is different, then it would have to be the case that the concentration of heavy deuterium-containing water would affect things like the rate of all of the chemical reactions in a cell that depend

Nick Jikomes

18:30 - 18:32

on water.

Gábor Somlyai

18:32 - 19:09

So there is one more comment to you. The water is the only molecule in our body which has hydrogen and deuterium. All the molecules, all the amino acids, all the nucleic acids have hydrogen and deuterium. So when we are talking about deuterium depleted water, this is not the reason that So the key is that using deuterium-depleted water, we are able to modify the D-H ratio in all the molecules where there are the hydrogen in an exchangeable position.

Gábor Somlyai

19:09 - 19:45

So when we, and not chemical reaction is running only with the water, but there are 2000 different chemical reaction in one single cell, Typically, most of the reaction has a hydrogen or deuterium. So finally, when we talk about hydrogen deuterium, we talk about all the molecules, all the chemical agents, all the amino acids, everything which has either hydrogen or deuterium in a given bond or in a given position during the chemical reaction.

Nick Jikomes

19:45 - 20:01

Yeah. So if deuterium is present in a hydrogen, and that heavy hydrogen is bound to something, it takes more energy to break that bond. Is that what we’re saying? Yeah.

Nick Jikomes

20:02 - 20:05

So it should slow down the rate of reactions, yes?

Gábor Somlyai

20:06 - 20:12

Yeah, those chemical reaction, which is running with heavy hydrogen, it’s much slower.

Nick Jikomes

20:12 - 20:16

And that’s been measured, have people measured that directly and we know that for sure?

Gábor Somlyai

20:17 - 20:38

Yeah, sure, yeah, yeah. There are, it’s a long story that everybody knows that which chemical bond, how strong and how slow, depending on the hydrogen and deuterium. But in our case, Let me tell you one experiment which was published. This is related to diabetes.

Gábor Somlyai

20:39 - 21:16

One day, a patient with cancer and consuming DDW, this is diluting depleted water, and came back and he said that his blood sugar level went down because he was a diabetic, not only cancer patient. So, and that, I went to the Semmelweis University and we run several rats study. So we made the rats diabetic. And the question was whether the drinking water of the deuterium can reduce the blood sugar level of the rat.

Gábor Somlyai

21:17 - 21:59

At the first time we used 25 ppm and 150 as a normal. So the normal deconcentration is 150 ppm, which means there are 150 deuterium out of 1 million hydrogen. And we found that when the rats received insulin, then the 25 ppm reduced the blood sugar level of the rat, which is very good because we can say that With a lower deconcentration, we could reduce the blood sugar level of a diabetic rat. But then we ran other studies with 75, 105, 125, and we went up to 150 ppm.

Gábor Somlyai

21:59 - 22:44

And it turned out that the best deuterium level is between 125 ppm and 140. So not the lowest deconcentration reduce the best way the blood sugar level of the diabetic rat. So, and this is the reason I say, so there is a very, very specific signal system in the cell. They recognized that for the insulin signal, to send the message to the cell, the 125 and 140 ppm is the best to reduce the blood sugar level.

Gábor Somlyai

22:44 - 22:47

And I don’t know, do you know the meaning of GLUT4?

Nick Jikomes

22:48 - 22:52

I do. Yeah, but I think we should explain that for people. Yeah. Okay.

Gábor Somlyai

22:52 - 23:20

Yeah. So a glut4 is a protein which is responsible to pick up the glucose from the blood. So then we checked whether the deconcentration can modify the GLUT4 concentration in the membrane of the muscle. And when we checked it, it turned out that the GLUT4 concentration was the highest in the membrane at 125 ppm.

Nick Jikomes

23:20 - 23:38

So you’re saying you changed the deuterium levels in the water and across different concentrations and changing the amount of deuterated water in the system changed the amount or the activity of a protein that’s responsible for regulating glucose levels.

Gábor Somlyai

23:39 - 24:23

Yes, yeah, yeah. So when the insulin bind to the membrane and send the message by the receptor, they stimulate the glut4 translocation from the cytosol to the membrane. Based upon that result, it’s obvious that the insulin-signal system works much better within 125-140 ppm range than at 150 ppm. And then we run a human study, the people consuming 105 ppm for 90 days, and at the end of the third month, we could reduce the fasting glucose level, and at 30% of the population, we reduce the insulin resistance.

Gábor Somlyai

24:24 - 25:11

And the reason it’s very important, because there was a big change in the diet at the 70s, when in the United States, it was said, change the calorie intake from lipid to carbohydrate. So when we are eating carbohydrate, our mitochondria will synthesize metabolic water with 150 ppm. So that today’s population average D level is much higher than it was 60, 70 years ago, when the people were eating much more fat, because from the fat, when we burn the fat in the mitochondria, we produce 110 ppm metabolic water. And this is a big game.

Gábor Somlyai

25:12 - 25:24

So there are 400 diabetic patients in the world, because the average is close to 150 ppm. So the insulin system is not so good for that.

Nick Jikomes

25:24 - 25:45

Yeah. I want to get to carbohydrate versus fat metabolism after we talk about mitochondria, after we talk about some more basic stuff, but let’s back up even further. So you’ve said things like 150 ppm, 140 ppm. Let’s start with what is the deuterated water and the deuterium concentration of water out in the world?

Nick Jikomes

25:45 - 25:51

Like if I go scoop up some water from the lake or from the ocean, what is the concentration of this deuterated water?

Gábor Somlyai

25:53 - 26:22

In the ocean, in the Indian Ocean, it’s 155 ppm. And this is a standard. When the clouds appear in the equatorial area, and the clouds are moving to the North and South Pole, we could say that the heavy hydrogen, semi-hydrogen fall out earlier. And in, let’s say, I live in Europe, middle Europe, here it’s about 145 ppm, the deconcentration.

Gábor Somlyai

26:23 - 26:51

If in Norway or at the North Pole, it should be about 130 ppm. Even when we’re going up to the hillside, increasing the altitude, that way the deuterium concentration again decreasing. So there is, let’s say 20 ppm fluctuation or changing depending on where we are on the earth.

Nick Jikomes

26:51 - 27:25

I see. So around the equator, basically, in the ocean, you would find deuterium water at about 150 ppm. So that means 150 molecules of water have a deuterated hydrogen out of 1 million molecules of water? And then because deuterium is more massive than regular hydrogen, as you go up in altitude or as you go towards the poles away from the equator, you know, water is evaporating, water is falling, it’s moving around like water does just as a consequence of the physics of all

Nick Jikomes

27:25 - 27:41

of this. Because deuterium is more massive, the water closer to the poles is more deuterium depleted. The deuterium is sort of falling away and there’s less deuterium, there’s less concentration near the poles compared to the equator.

Gábor Somlyai

27:42 - 28:18

That’s the question of the evaporation. So because the semi-hydrogen, semi-heavy water is bigger, bigger mass, stronger bond, it means the boiling point is higher. So when you are boiling the water, the H2 molecule much easier can go to the atmosphere than the heavier, semi-heavy water. So this is how we can separate the heavy water, this is how we produce deuterium-depleted water.

Gábor Somlyai

28:18 - 28:28

We are producing tons of steam to get a couple of liters of deuterium-depleted water during that so-called fractional distillation.

Nick Jikomes

28:28 - 28:49

Got it. So that’s how people make deuterium-depleted water, water that has less deuterium than the water surrounding it. You’re basically just doing a lot of boiling, a lot of steam generation, and then you’re separating out the water from the steam. And because the regular hydrogen-containing water will evaporate more easily, you can fractionate the water.

Nick Jikomes

28:49 - 29:10

You can separate it and get deuterium-depleted water with less deuterium in it. Yeah, that’s it. Okay, so 150 PPMs roughly around the equator, that will change as you go to different parts of the earth. So as you go north away from the equator, the PPMs, the concentration of deuterium in the water will tend to go down.

Nick Jikomes

29:11 - 29:20

In a normal living, breathing animal, a normal living, breathing person, how much deuterium do our cells and tissues tend to have compared to the external water in the environment?

Gábor Somlyai

29:23 - 30:02

So typically we say that people should drink 1.52 liter of water per day. And the other water what we gain is coming from the food, and not the food which water is in the food, the food when the hydrogen is bounded to the carbon source, glucose, amino acids, or lipids. So when we’re talking about metabolic water, in that case, the hydrogen is coming from the glucose, hydrogen from the lipid hydrogen. And in that case, there is big difference in the DH ratio.

Nick Jikomes

30:03 - 30:10

Yeah, yeah. So let’s define this now clearly for people. You said metabolic water. Describe that in very basic terms for us.

Nick Jikomes

30:10 - 30:14

How does our body make what we call metabolic water? What is that?

Gábor Somlyai

30:15 - 31:04

The metabolic water is made by the mitochondria. So the mitochondria can produce the ATP that within the mitochondria from the inner part, the hydrogen is transferred to the, between the two layer and based upon the hydrogen concentration coming within the mitochondria and that type generating the ATPase which can finally synthesize the ATP. And the energy which was finally based on the sunlight the plant gain the energy from the sunlight. That way they make from carbon dioxide glucose.

Gábor Somlyai

31:04 - 31:42

So from carbon dioxide and water make the glucose. And when we oxidize that glucose, it’s going back to carbon dioxide and metabolic water. So that is the circle on the earth. And that energy which is coming from the sun, captured by the plant, we get it back when that hydrogen flows through the ATPase generator energy build into ATP, and that ATP is used up in the cell.

Nick Jikomes

31:42 - 31:55

Yeah, yeah. So basically, right, our cells are filled with mitochondria. This is the site where most of our ATP, the energy currency of the cell, is produced. It’s produced because the mitochondria have two membranes.

Nick Jikomes

31:55 - 32:20

They’re creating a proton or a hydrogen gradient, and those hydrogen ions, those protons are flowing through the ATPase enzyme, which is basically a little, molecular rotary motor. And so the flow of hydrogen through that little protein spins that motor and we’re making ATP, but we’re also making water molecules. And that would be like why our breath contains water vapor when we exhale.

Gábor Somlyai

32:20 - 32:33

Yeah. This is, we need brass because when the hydrogen is coming, need the oxygen and that they can’t get the electrons. And then we finally can synthesize the metabolic water.

Nick Jikomes

32:33 - 32:51

Yeah. Yeah. And the weight, what’s powering all of that is the proton gradient, but also we’re oxidizing or burning our food, our carbohydrates or fats. And that’s feeding into this process to help create that gradient and make the ATPase do what it does to eventually create this water.

Nick Jikomes

32:50 - 33:06

So, what else is going on in the mitochondria here that’s relevant? So this might be where we start to talk about where this deuterium depleted water comes from and where it is within the mitochondria.

Gábor Somlyai

33:09 - 34:04

So the TCS cycle is running in the mitochondria. So this is the basis that finally, that generate the free hydrogen, and that’s what you explained so properly. But based on the regulatory system, what we say exists, the big game is how the TCS cycle is running, what is the food we are feeding the mitochondria, so whether it’s carbohydrate, lipids, or whatever, and that will determine the final deconcentration of the metabolic water. And the key issue is everybody knows that there is one common feature of each cancer cells, and that is that the damaged mitochondria.

Gábor Somlyai

34:04 - 34:36

So if we say there are healthy cells and cancer cells, 90% of the cancer, the mitochondria is damaged. So we can say no metabolic water production in the cancer cells. It means no deuterium depleted water production in the cancer cells. And that is very important because on the other side, when a gross hormone bind to the receptor and stimulate the sodium hydrogen antiport.

Gábor Somlyai

34:36 - 35:00

So in that case, the cell pick up on sodium and transport on hydrogen out. We published more than 30 years ago, that process will prefer to eliminate the hydrogen. So when the growth hormone stimulate the sodium hydrogen antiport, that will cause an increase in the DH ratio. And then we talk about the key regulatory system.

Gábor Somlyai

35:00 - 35:40

The question is whether a cell are able to lift up the DH ratio to trigger the cell division. If you have a healthy mitochondria, producing dilutin-depleted water, then it’s very difficult because the mitochondria is producing the dilutin-depleted metabolic water, so it’s counteract with the increasing membrane transport. But in the cancer cells, the cells very easily lift up the DH ratio, and this is the reason that finally the people can get the cancer. So that is a key, as we say, very important.

Nick Jikomes

35:40 - 35:55

So, I want to really connect the dots here between the deuterium depleted water and the metabolic water. So, water is being produced as a consequence of mitochondrial metabolism. I get that. The hydrogens are flowing through the ATPase.

Nick Jikomes

35:55 - 36:15

We’re oxidizing our food, our fats, and or carbohydrates. and we’re producing water in the mitochondria as a consequence of that oxidative metabolism. Why is the water produced, this metabolic water produced inside the mitochondria, why is it deuterium depleted? Why does it contain less deuterium?

Nick Jikomes

36:15 - 36:18

Where is the deuterium leaving the equation here?

Gábor Somlyai

36:19 - 36:48

Good question. Yeah, so just because we talk about that the D bond is always stronger than the H bond. It means that in each chemical reaction, always the hydrogen will be preferred. So at that goes when the plant synthesizing the carbohydrate, that’s, we could say that there are six carbon in a glucose.

Gábor Somlyai

36:48 - 37:07

It’s very easy. But when the cells synthesizing lipids, which means they taking the glucose source and making lipids, two carbon, two carbon, two carbon binding.

Nick Jikomes

37:07 - 37:08

There’s more steps.

Gábor Somlyai

37:09 - 37:29

Yeah, there are 16, 18, whatever, 24 carbon in one lipid molecules. That means in that process, as I mentioned, always the hydrogen will be preferred. So finally, here will be a big depletion for the deuterium. This is one thing.

Gábor Somlyai

37:29 - 38:02

But there are more important thing that in the plants, there are three different pathway to freeze the carbon dioxide from the air. There are so-called C3, C4, and the CAMV. And based upon those plants, for example, the corn belongs to the C4 pathway. that is not so depleted than the wheat, which has a C3 pathway.

Gábor Somlyai

38:03 - 38:13

So even depending on what type of plants you eat, that can cause differences in the deconcentration.

Nick Jikomes

38:14 - 38:29

I see. So literally what you’re saying is, so Let me translate two things here. So there’s different types of plants, just like there’s different types of animals. C3 and C4 plants are plants with different ways of doing photosynthesis.

Nick Jikomes

38:30 - 39:07

So the biochemistry is just happening in a different way. The level of deuterium in C3 compared to C4 plants is different. And the reason the level of deuterium is gonna depend on C3 versus C4 in a plant or carbohydrate versus fat metabolism in our own cells has to do with the number of chemical reactions it takes to do stuff. Because the deuterium is heavier and the carbon-hydrogen bond is stronger and therefore more difficult to break, then the more chemical steps, the more chemical reactions you have in a system, each time the hydrogen is gonna be preferred to the deuterium.

Nick Jikomes

39:08 - 39:26

So statistically speaking, when you have many more reactions that need to happen to say, do lipid metabolism compared to high carbohydrate metabolism, you’re gonna have more of that deuterium falling away and therefore you will produce more deuterium depleted water. Yeah, exactly.

Gábor Somlyai

39:26 - 39:33

Do you know the capsaicin? The what? Capsaicin which makes the pepper hot.

Nick Jikomes

39:33 - 39:34

Yeah, capsaicin.

Gábor Somlyai

39:35 - 39:53

You know, it’s a very, very complicated molecule. The D-level is about 60 ppm. So just because synthesizing one capsaicin molecule is so different, so much biochemical step, so it brings down the concentration of that specific molecule.

Nick Jikomes

39:53 - 40:15

Yeah, yeah. So to summarize here again, just to repeat this, because it might be a little bit hard for people to keep track of all this if they’re new to it. there are more chemical steps involved in breaking down lipids than carbohydrates. And so that means for each of those steps, you’re gonna prefer the hydrogen to the deuterium, prefer the hydrogen to the deuterium and so on and so forth.

Nick Jikomes

40:15 - 40:46

So fat oxidation, lipid oxidation produces more deuterium depleted water, less deuterium in the water compared to carbohydrate oxidation. Similarly, and for similar physical reasons, different types of plants that do photosynthesis in different ways and involve different biochemistry, they have different levels of deuterium inside of them. And therefore, you know, eating wheat versus corn, as you said, is going to put a different amount of deuterated hydrogen in your own body.

Gábor Somlyai

40:47 - 41:10

Yeah, yeah. The pineapple that, for example, can enrich deuterium. So when somebody eating pineapple, that should be even over 150 ppm, because this is the third type of chemical reaction, chemical way to to fix the carbon dioxide.

Nick Jikomes

41:10 - 41:56

deuterium’s out there, it’s in the environment to some extent, its concentration depends on where you are in the globe, and its concentration depends on the type of biochemistry an animal’s doing, the type of photosynthesis a plant is doing, like C3 versus C4, the type of food that you’re eating, like carbohydrates versus lipids, and then at different locations in the cell, like in the mitochondria, you can get different levels of deuterium depletion based on how the biochemistry works. So there’s different concentrations of deuterated water in different parts of the world and in different parts of our body and in different organisms. What do we really know about the effects of different deuterium concentrations, different concentrations of deuterium depleted water on biology?

Nick Jikomes

41:56 - 42:18

And let’s start here with the basics. Let’s start with in vitro experiments. When you’re studying cells and culture in a petri dish where you can directly control the deuterium depleted water levels, what have you observed and what have others observed about how deuterium levels affect basic cell processes like gene expression, cell division, and that type of thing?

Gábor Somlyai

42:19 - 42:38

Oh, that’s a good question. So the very first study, of course, I did the simplest way. I prepared media for tissue culture with deuterium depleted water. And the very first paper we published, I used 25 ppm, 150, and I even tested

Nick Jikomes

42:41 - 42:56

So again, just for reference, 150 ppm, we might call that sort of the normal background level. Lower than 150 is deuterium depleted, and then higher would be deuterium enriched. Yeah. Okay.

Gábor Somlyai

42:56 - 43:29

And what we found that Reducing the D-level below 150 ppm that way we could slow down the cell growth, but increasing over 150 ppm that stimulated the cell growth. that the higher the level, close to the 150 ppm, it was very advantageous for the cell to start the cell division. So that was the very first. Later, there is a very extremely good technology recently.

Gábor Somlyai

43:30 - 43:53

In that case, the cells, we tested again, the cells were growing on a gold layer. So in that case, we didn’t need to manipulate with the cell, we just followed how the cells can modify the impotency of that gold layer. So it makes us very precise following what the deuterium can do.

Nick Jikomes

43:53 - 44:04

So hold on, you’re saying there’s literally cells growing on a layer of gold and it’s changing, the impedance can change, so you can kind of measure how well electrical currents are being conducted.

Gábor Somlyai

44:04 - 44:29

The faster they grow, the faster they modify the impedance of that gold layer. So in that case, we again could prove changing the D-level from whatever over and below 150. On those dependent, we reduce the growth rate. At 40 ppm, we completely over 150 ppm be stimulated.

Gábor Somlyai

44:30 - 45:08

But in another study, we investigated how sensitive for the deconcentration. In that case, we run a study that every eight hours, we change the deconcentration with one ppm. And we found that changing, reducing with one ppm every eight hours, that resulted a slower growth rate. So the cells are so sensitive and so precise and measuring the D level that one ppm decrease resulted a slower growth rate.

Gábor Somlyai

45:10 - 45:15

And that is very important to see that how precise system we are talking about.

Nick Jikomes

45:16 - 45:40

Yeah. So basically the concentration of deuterium in your water, you just have cells growing in a Petri dish and you’re giving deuterium, or you’re giving water with different levels of deuterium in it. When the deuterium levels are lower, under 150, that slows down growth. Cells grow slower, the less deuterated water is in the medium.

Gábor Somlyai

45:41 - 45:46

The more lower the D level, the more slows down or completely stop the cell growth.

Nick Jikomes

45:46 - 46:07

Yeah. So if you increase or you enrich the deuterium levels in the water, cell growth speeds up. That’s a little bit, I guess, counterintuitive to me, you know, as someone who doesn’t really know about this stuff, because if there’s more deuterium, there’s more of that heavy, heavy water, that bond should be stronger. I would intuitively think that that would slow things down.

Gábor Somlyai

46:08 - 46:51

Yeah, you are right. But it depends on. So when in the very first study, when it goes up to 900 ppm, that slows down. But within the range 150 ppm plus minus 30 or 30 ppm, The question is not the physical differences between hydrogen and deuterium, but this is a sensitive system for the D-H ratio, where very small changes in D-H ratio are amplified and enhanced by the enzymes, by the cells.

Gábor Somlyai

46:51 - 47:23

Because small changes, for example, there is a protein which is in a given conformation. If we change the DH ratio, that less or more deuterium can fix the conformation of the enzyme, which will be maybe more active or maybe inactive. So small changes in the D-H ratio, an enzyme could be even faster or slower, and that enhances the effect of the concentration.

Nick Jikomes

47:24 - 47:57

I think if I’m hearing you correctly, what I’m hearing is if you go across a very wide range, you can see differences that are different than if you’re in a narrower range. A cell on Earth will never see zero PPMs and it will never ever see 900 PPMs. But within the range that biology actually encounters out there, 150 PPMs and a bit lower, a bit higher, you’re saying in that range is sort of where the action is. It’s where cells have evolved and therefore what they’re sensitive to.

Nick Jikomes

47:58 - 48:12

Yeah, yeah, this is exactly. Interesting. So what else have you seen in in vitro experiments other than cell growth? What other things have been looked at that are sensitive to the deuterium depletion levels of the water?

Gábor Somlyai

48:14 - 48:57

So our recent paper, we have published a couple of paper, what will happen with the gene, with the genes, because this is one of the key issue. We know that We know hundreds of genes having key role in the cell growth, and the drug development is based on finding the mutated genes, and then based upon that, try to find the new drug. So the question is, what will happen with the gene expression when we modify the D-uterine-hydrogen ratio? And in that study, we used 40, 80 ppm as a diluted water, 150 and 300 ppm.

Gábor Somlyai

48:58 - 49:25

And we could check over 500 kinase gene and over 200 cancer-related genes. And the point was that 97% of the genes responded for the higher deconcentration. And why can be important? Because as I mentioned, when a gross hormone binds to the receptor, stimulate the sodium hydrogen, that will cause an increase in the DH ratio.

Gábor Somlyai

49:26 - 50:12

And if the DH ratio is increasing, that can simultaneously activate hundreds of genes, which has an important role to manage the cell growth. And this is a big difference between the cancer drug development and applying DW in the cancer therapy, because the cancer drug will target one gene, one mutated genes. By modifying the DH ratio, That way we will not allow all the 100 cells to be activated because we keep the D level at a much lower level. So the cells are even the sodium hydrogen transport is activated from the low D level.

Gábor Somlyai

50:12 - 50:36

They are not able to lift up the DH ratio to the threshold. So that is a reason that we not targeting only one signal pathway, but drinking DW, they simultaneously can impact the whole network of this cell growth regulation.

Nick Jikomes

50:36 - 51:12

Yeah. Well, I think, you know, I think one of the things, one of the points I think you’re trying to make is I’m already starting to change how I’m thinking a little bit in the sense of in what we might call normal biology or normal drug development, we think about one protein binding to one receptor or one drug binding to one receptor, and then this pathway turns on and then it goes to the gene. But what you’re talking about is basically the the weight and the viscosity of all of the water can change the speed and the patterns of all of the reactions everywhere all at once.

Nick Jikomes

51:12 - 51:24

This is more of a distributed biophysical thing, not a one gene, one molecule, one protein pathway way of thinking about biology. Yeah, that’s it.

Gábor Somlyai

51:24 - 51:26

This is what I’d like to explain.

Nick Jikomes

51:26 - 51:46

Yeah, let me see if this analogy works a little bit. I’m trying to play with this in my own mind right now. So we talked about the deuterium concentrations in the water, and we said the more deuterium depleted you are, the less deuterium, the more it slowed down growth relative to 150. And then if you increase it, it sped it up.

Nick Jikomes

51:46 - 52:05

But then you said, if you really increase it a lot, it will, in fact, slow things down. And I guess the analogy I’m thinking of here is like, I could go swimming in a pool, and it could be freshwater or saltwater. And they’ll have different levels of salt in the water, but I can still swim through it. And maybe I can kind of tell there’s a little bit of a difference, so it affects my behavior.

Nick Jikomes

52:06 - 52:23

But if I go try and swim through molasses or tar, it’s just too big and I can’t swim. But I’m also never going to go swim inside of molasses for that reason. So I mean, is this kind of maybe how to start thinking about it? It’s we’re talking about the the viscosity or the weight of the water.

Gábor Somlyai

52:25 - 52:58

So so based so. There is a limit and I suppose that for different cell lies are there are a different part of the limit. So within the range for I don’t know what type of cell. We can increase up to 300, 400, but another cell line, maybe we can even higher, and we do not see that flashback that the deter finally even inhibit.

Gábor Somlyai

52:59 - 53:22

So the point is, going back to the insulin, that there are receptors and there are indicators for this D-H ratio, which can multiply a very simple changes in the D-H ratio, and then they can multiply 10-fold, 100-fold.

Nick Jikomes

53:23 - 53:28

Can you give me an example of that? How is this D-H ratio actually being read out by certain proteins?

Gábor Somlyai

53:30 - 53:31

What do you mean?

Nick Jikomes

53:32 - 53:43

So describe how this amplification process is happening. I mean, are there actual literal sensors in the cell, like certain proteins that are directly sensing the DH ratio?

Gábor Somlyai

53:45 - 54:43

I suppose that each of the proteins recognize the changes in the DH ratio and must be very, very precise and key players of that whole protein system, which are more sensitive and more important role they have, how they react for a changing DNA ratio. So based on the gene expression study, we could see that there was a couple of genes, we can get over 100% increase in the gene expression, and there was a 50% increase. So it depends on the genes, for example, which and for example, those mTOR gene we could get a 100% increase in the expression at 300 ppm. We know that the mTOR is a very important key player in that signal system.

Nick Jikomes

54:43 - 54:59

So mTOR would be a key protein information pathway involved in growth. It’s often dysregulated in cancer. And you’re saying increasing deuterium levels in the water increases the expression of mTOR in certain cell cultures.

Gábor Somlyai

55:01 - 55:04

The other point should be, do you know the Herceptin?

Nick Jikomes

55:05 - 55:06

No.

Gábor Somlyai

55:06 - 55:45

The Herceptin is a very good cancer drug. At that, cover the receptor, so when the growth hormone bind to the membrane, cannot bind to the receptor. And it means that the cell will not stimulate the sodium hydrogen antiport, so they are not able to lift up the DH ratio. And what we see that those patients who are receiving the Herceptin plus consuming dilutin-depleted water, we’ve got a much better data, it’s much better results than only receiving the Herceptin because we are targeting the same system.

Gábor Somlyai

55:46 - 56:37

With dilutin-depleted water, we bring down the deconcentration, so that way we make it very difficult for the cell to increase, but at the same time the Herceptin not allow for the cell to stimulate the sodium hydrogen and that way try to increase the DH ratio. So, and there are other kinase that we saw that giving the drug, combining with BDW, we can get much better results. So sometimes patients receiving the drug, no real good response, combining the BDW, that way we can increase the efficacy of the drug, which means somehow in that case we’re targeting that given protein which is targeted by the drug.

Nick Jikomes

56:40 - 57:09

Okay, so you can change the deuterium levels in water, put it cells in a dish growing in this water with different concentrations of deuterium. And you see effects on cell cycle, cell growth rates, how often the cells are dividing, you can speed it up, you can slow it down. When you play within a certain range of deuterium depleted water, you can affect gene expression up or down depending on the concentration, depending on the gene, all of that cells growing in a dish. Let’s not talk about animals.

Nick Jikomes

57:09 - 57:23

When we do experiments in animals like lab rats or mice or something like that, has anyone done experiments where they’re supplying deuterium depleted water, increasing or decreasing the deuterium levels and looking at cancer rates and anything like that?

Gábor Somlyai

57:25 - 57:51

The first paper that we published in 1993, we mentioned the mice study. So in that case, we transplanted human breast cancer to the mice. and one group of the mice consumed 150 ppm, the other 30 ppm. 60% of the animals were still alive and they were healthy.

Gábor Somlyai

57:51 - 58:19

On the control group, only one mice survived somehow the tumor. So it was evident that when the mice consumed deuterium-depleted water, That way we could stop the tumor growth in the mice. And finally, the tumor disappeared, 60% of the mice. And after that, we studied other mice study, but the point is we’ve got a registered drug for veterinary use.

Gábor Somlyai

58:20 - 58:56

So we tested on dogs and cats. Then we replaced the drinking water with 25 ppm. In that case, we say roughly 50% of the animals, we can get a complete response, the tumor disappear, and the other 20, 30%, we can get a partial response. So that is on the market, and the pet’s owner like it, buy it, use it, and then sending the pictures about the dogs on the Facebook, and then showing that how happy they are that the animals are fine.

Nick Jikomes

58:56 - 59:20

Okay, hold on. In the rodent studies, you’re basically grafting human breast cancer tissue into a rodent, and then you’re saying, we’re going to give them regular water in one group, and then we’re going to give them deuterium depleted water, lower deuterium in the water for the other group. You said the deuterium depleted water group saw slower cancer growth and or lower cancer rates?

Gábor Somlyai

59:21 - 59:29

I would say in that study that we published in the first case, we eliminated the tumor from the mice.

Nick Jikomes

59:29 - 59:32

So the tumor went away? Yeah.

Gábor Somlyai

59:32 - 59:35

I wouldn’t say all, 60% of the mice survived.

Nick Jikomes

59:35 - 59:43

60%, yeah. Yeah. And what was the control group? What did the rate of death look like?

Gábor Somlyai

59:44 - 59:58

One might survive out of 11 in the control group. So I would say, In that mice, I suppose that the tumor couldn’t start, otherwise all of them would have died.

Nick Jikomes

59:58 - 1:00:20

Okay, and then you said, okay, you’ve also got a veterinary drug that’s given to cats and dogs when they have cancer. And you’re saying when you give deuterium depleted water to cats and dogs with that drug, they see better outcomes in terms of cancer? And has that been published?

Gábor Somlyai

1:00:23 - 1:00:48

So the drug itself, yeah, we published mainly in Hungary about, no, I have a couple of English paper published with docs showing the size of the primary tumor and the size was decreased. We published, I guess, two or three papers. We have a paper ready for publication with the animal study, but that latest one has not been published yet.

Nick Jikomes

1:00:48 - 1:01:01

Yeah. Do you, little side note here, do you publish papers yourself and just the scientific community in Hungary? Are there papers published that are not published in English, that are only in Hungarian?

Gábor Somlyai

1:01:02 - 1:01:07

No, I would say all the paper was published in English. Only a couple was published in Hungarian.

Nick Jikomes

1:01:07 - 1:01:09

Okay. Okay. Okay. Yeah.

Gábor Somlyai

1:01:09 - 1:01:31

So, and then, as I said, on the PubMed, There are about over 200 paper appear searching for DW. 100 is relating to the cancer and all this stuff. Most of them is not published, me or our group.

Nick Jikomes

1:01:32 - 1:01:48

Yeah. I want to back up a little bit and ask you something about more basic biology before we move on to some of the human studies you’ve been involved in. So we talked about mitochondria. Mitochondria are where a lot of the action is in terms of bioenergetics in our cells.

Nick Jikomes

1:01:48 - 1:02:13

We’ve got that proton gradient that the mitochondria set up. The hydrogen ions flow through the ATPase, the little motor that ends up creating ATP and also metabolic water. I would imagine that, you know, when I think about this biophysically, hydrogen ions are lighter, they can flow through at a different weight or maybe with different ease compared to deuterium because it’s going to be heavier. Is that the case?

Nick Jikomes

1:02:13 - 1:02:31

Do we know that if you compare the inner mitochondrial space and the mitochondrial matrix, and you look at the flow of hydrogen through the ATPase, does the DH ratio affect the actual ATPase in terms of how fast it’s spinning or its efficiency in generating ATP?

Gábor Somlyai

1:02:32 - 1:02:49

That’s again a very good question. So I would like to see, and I try to run study just directly with mitochondria. So far I couldn’t. But what we have done, we tested the effect of DW on athletes.

Gábor Somlyai

1:02:50 - 1:03:18

So we made two groups of athletes, and we made a loading test, and we took the blood samples. And then one group consumed DW for 44 days. And then we made another loading test to the blood samples. And then we found that after consuming DW, the lactic acid appeared much later during the loading test.

Gábor Somlyai

1:03:18 - 1:03:32

The anion gap was smaller and They regenerated much better. The glucose mobilization was better. What was the loading time exactly? They were rowing.

Nick Jikomes

1:03:33 - 1:03:33

Oh, I see.

Gábor Somlyai

1:03:34 - 1:04:18

and five minutes break, five minutes break, five minutes break. So four times we repeated and at each time we took the blood samples. So based upon that experiment that do you know it’s in a race, it’s critical whether the acidification, how it’s running and whether they can reach the 200 meters in a swimming pool. So this is what we found that as the lactic acid appeared later, it means that the mitochondria could work properly, and they could synthesize the, they had enough oxygen, they could synthesize the metabolic water, and they didn’t run out of the oxygen.

Gábor Somlyai

1:04:19 - 1:04:57

And the lactic acid, this is the reason it appeared later, because when no enough oxygen, and the mitochondria is not able to cope with that increasing hydrogen ion concentration, then it goes to the lactic acid, so they are not able to burn the glucose to carbon dioxide and metabolic water. So this is what we had. And I know that there are papers and talking about that, and it’s very probably possible that then the D is reaching the ATP is that stay maybe longer.

Gábor Somlyai

1:04:58 - 1:05:10

before flowing through on the membrane. It’s very probably that way we can interact and then modify the effect of the ATPase.

Nick Jikomes

1:05:10 - 1:05:21

Okay, but the basic result from the experiment you described is you got two groups of people and they’re rowing. They’re coming into the lab and they’re rowing. And obviously when you start rowing, you’re just gonna start to sweat. You’ll eventually get tired.

Nick Jikomes

1:05:21 - 1:05:44

Eventually your muscles will burn. All of the normal things will happen when you do that type of exercise. One group was just drinking regular water and one group was drinking deuterium depleted water, lower levels of deuterium for a number of many days leading up to the test. The group that drank the deuterium depleted water was able to basically row longer before lactic acid built up.

Nick Jikomes

1:05:44 - 1:06:07

And that would imply that they probably could have rowed further or stronger. Interesting. Okay, so you’ve done experiments in people, you’ve seen effects in cell culture and in animals with, you know, giving different levels of deuterium in the water. What studies have been done in human beings in a more clinical setting?

Nick Jikomes

1:06:07 - 1:06:29

Obviously, one of the things that people talk about when they get excited about deuterium and deuterium-depleted water is cancer. You mentioned the animal work already. Lower deuterium levels, meaning deuterium-depleted water, seem to go with lower rates of cancer or better outcomes in cancer in animals. Has any work been done with DDW in humans?

Nick Jikomes

1:06:29 - 1:06:31

Human cancer patients, I mean.

Gábor Somlyai

1:06:32 - 1:06:57

So we could run one face-to-double randomized clinical trial with prostate cancer patients. So the reason I choose the prostate, because one of the first paper person had an inoperable prostate cancer. He started drinking DW, and a month later, they couldn’t palpate the tumor. And then it will decrease in the PSA.

Gábor Somlyai

1:06:57 - 1:07:19

This is the reason I choose the prostate cancer. So in that, that was very, very long ago, even I didn’t know a lot of things about lutein depletion. But anyway, that was a double by randomized clinical trial. There were bottles with a number, five numbers means normal water, five numbers was 85 ppm.

Gábor Somlyai

1:07:20 - 1:07:45

And neither the MDs nor the patients know what number means the DW. And what we found that there was 80% decrease in the PSA level in the treated arm, and there was only 47% decrease in the PSA level on the placebo arm. And what is that? What is PSA?

Gábor Somlyai

1:07:46 - 1:08:06

The PSA, you know, prostate-specific antigen. I see, okay. That PSA is correlated with the size of the tumor. So we had 22 treated, 22 placebo person, and the cumulative number of the PSA was at the beginning, I don’t know, about 800.

Gábor Somlyai

1:08:06 - 1:08:36

And four months later, it was 60% less, whatever. And so there was a big sharp decrease in the PSA volume. But more important was that every month, prostate size was followed by rectal ultrasound. And after two months, the MDs know that that number means the DW, that number means the placebo, because it was so obvious.

Gábor Somlyai

1:08:37 - 1:08:47

There was 160 cubes with a decrease in the placebo one. So three times more.

Nick Jikomes

1:08:48 - 1:09:12

You’re basically saying you did an experiment, you gave two different groups of people with prostate cancer water. One of them got regular water, one of them got deuterium depleted water. After a couple months, and I’m guessing they were getting some other treatment here, you’re basically saying the physicians are used to seeing a certain rate of decrease in the tumor size, and it was so much bigger in the DDW group that they could actually guess which group was the placebo and which group was the treatment group.

Gábor Somlyai

1:09:12 - 1:10:01

Everybody received the commercial therapy plus placebo or DW. But the most important thing was that the study ran only for four months, and then we checked the survival after a year. So we lost two people out of 22 in the treated arm, but nine in the placebo arm, which So that was a face-to-double bind. And if we say that that was a prospective study, and if we check all the retrospective studies that we have done and we have published, this roughly we can say the survival is typically three to seven fold longer if the patients consuming DW are beside the commercial therapy

Gábor Somlyai

1:10:01 - 1:10:05

comparing to those who only receiving the commercial therapy.

Nick Jikomes

1:10:07 - 1:10:30

So basically when you’ve given people deuterium depleted water, in addition to whatever treatment they’re getting, basically it’s like an adjuvant therapy, it’s something you add to another treatment, you’ve generally seen better survival. So they last longer before dying and or the tumors shrink faster in the DDW group. Yeah, exactly.

Gábor Somlyai

1:10:31 - 1:10:52

Wow. So this, so we have, we have published with lung cancer, breast cancer, prostate cancer, glioblastoma, pancreatic cancer data. That should be about a thousand patients. But just a year ago, we evaluated all the patients we had.

Gábor Somlyai

1:10:52 - 1:11:23

In that case, we evaluated 2,649 patients were in the database. That patients representing the Hungarian cancer population, because we had lots of colon cancer, breast cancer, prostate cancer, and less number in the leukemia or whatever. And then the question was, what are the critical parameters to maximize the efficacy of DW? And we have three conclusions.

Gábor Somlyai

1:11:24 - 1:11:49

The patients should survive at least three, four months at the start of DW consumption. So those who are in a late stage, full with cancer, we cannot guarantee that we can show real efficacy. The other was the patients should drink DDW at least for three, four months. It’s obvious.

Gábor Somlyai

1:11:49 - 1:12:01

So within two weeks, we cannot get a real breakthrough. And the other important was the patient should start DW within nine months after the diagnosis.

Nick Jikomes

1:12:01 - 1:12:05

So you want to be consuming it long enough, early enough in the disease.

Gábor Somlyai

1:12:06 - 1:12:22

Of course. So in that case, for that population, the median survival time was 11.6 year. 11.6 year. Comparing to the Hungarian population is 2.4.

Gábor Somlyai

1:12:22 - 1:12:37

And the conclusion of that paper was integrating DW into the existing therapy, we can get 80% decrease in the cancer mortality. This is what we can offer.

Nick Jikomes

1:12:38 - 1:12:44

Can you imagine? Yeah. So I mean, basically, what you’re saying is, this is a big effect. Um, so this is true.

Nick Jikomes

1:12:44 - 1:13:04

And if this replicates, this is a big effect. And it’s not, um, it’s an adjuvant therapy, you’re adding it to the existing therapy, and it’s just water, right? So you have to get DDW. deuterium-depleted water, but you’re just splitting the population in two groups and giving one regular water and one deuterium-depleted water.

Nick Jikomes

1:13:04 - 1:13:25

It’s pretty straightforward to do that. If the effects are as big as you’re saying, and this is as powerful as the results you just shared indicate, I’m not aware that people are doing experiments all over the place, clinical trials in the US and everywhere else, looking at deuterium-depleted water. Why is that?

Gábor Somlyai

1:13:26 - 1:13:44

The, yeah, it’s a big question. The difficulties for us is the funding. So we are running, we are planning new clinical trials. You need five to 10 million euros to start a new clinical trial.

Gábor Somlyai

1:13:45 - 1:14:07

So we are lack of that money. So, and those who can do that first, they will control the market, I would say. To change the paradigm is never easy. I do not believe that the farming industry is against us.

Gábor Somlyai

1:14:08 - 1:14:39

I suppose that they have not recognized the potential of the ultimate depletion. If they recognize it, then the world will solve all the problems, the metabolic disease and the cancer problems, because they will put the money and people and the deuterium depletion will be part of the therapy. And because it’s so obvious. But to convince the pharma industry that they are making some kind of drug development.

Gábor Somlyai

1:14:40 - 1:15:24

I just read a paper that GSK bought a company for $10.6 billion just now because they have revenue, they will have a new cancer drug for lung cancer. Then I cited my paper that we can get a seven-fold increase for lung cancer population, integrating DW into the therapy. The only way that we can proceed is if we generate more data, if we involve more scientists, and if we publish more scientific papers. And one day, when the business recognizes the potential, then the money will come.

Gábor Somlyai

1:15:25 - 1:15:30

We will run several clinical trials and we will integrate DW into the therapy.

Nick Jikomes

1:15:31 - 1:15:46

Well, I would imagine, I mean, deuterium depleted water, you’ve got to make it by basically boiling water and doing a lot of steam generation. But, you know, as opposed to a novel drug, I’m guessing nobody can own a patent to deuterium depleted water or something like that.

Gábor Somlyai

1:15:47 - 1:16:20

No, we had a very strong position 30 years ago. Yeah, we have a patent worldwide for diluted depleted water indicating for the cancer therapy. But the point is that we talk about diluted depleted water, but there are very strict protocol that we recommend. What kind of PPM, what to change, how long drink, how to combining with radiotherapy, chemotherapy, hormone therapy.

Gábor Somlyai

1:16:21 - 1:17:00

So if you really want to maximize the potential DW, we need to integrate, we need to educate the patients and the oncologists. And so this is the way to move forward. If the people can buy it on the drug store or in the supermarket, they will lose the potential DW because we need to know how long, what type of concentration, when to stop, when to start again. So this is the reason we want to get a registered drug because then the insurance company should cover the cost.

Gábor Somlyai

1:17:01 - 1:17:19

They should spare billions of dollars because the people can go back to work and pay the tax and all the money. So simple water, but on the other hand, it requires very strict rules to follow.

Nick Jikomes

1:17:19 - 1:17:47

Yeah. So you’re saying, one thing you’re saying it sounds like is, you can’t just sort of drink deuterium depleted water whenever you want. You have to drink a certain concentration at a certain rate, and you have to control your intake of other water too, because that will dilute the level of deuterium that you’re consuming on a day-by-day basis. The research you’ve cited and the stuff you’ve observed is using a strict protocol of a known concentration of deuterium-depleted water.

Nick Jikomes

1:17:47 - 1:18:05

It’s a specific protocol. You’ve seen direct effects on the cell cycle and stuff at different concentrations of deuterium, so the concentration seems to matter. Did you also say that you do own a patent on something like a protocol? Used to be had.

Gábor Somlyai

1:18:05 - 1:18:15

Used to be had. Now it’s run out. But do you know that I wrote the book, my second book, it is duty and depletion. In that book, I wrote the protocols.

Gábor Somlyai

1:18:17 - 1:18:28

Can I show you? Yeah, sure. This is the book. People can get it on the Amazon in German also.

Gábor Somlyai

1:18:29 - 1:18:59

So what I try to do to sharing the knowledge, those who can get access to DW, to see how to use it, when to use it, and how to combine it. And we need to work a lot in the future to move forward, because it’s also very important. So integrating DW, it will not mean that we will cure everybody. So we don’t like the pancreatic cancer.

Gábor Somlyai

1:19:00 - 1:19:15

We don’t like the triple negative breast cancer. We need lots of more research to figure out why we can get good response for lung cancer, but not so good response for triple negative breast cancer.

Nick Jikomes

1:19:15 - 1:19:25

I see. So you’ve done experiments with different cancers. You see positive results in some cases, such as the colon cancer example we talked about, but you don’t see positive results for things like triple negative breast cancer.

Gábor Somlyai

1:19:26 - 1:19:39

It’s more, yeah, it’s more excessive or not so sensitive for lutein depletion. And of course, there are genetic background or some kind of reason there is behind that.

Nick Jikomes

1:19:40 - 1:19:57

I see. And so you can drink deuterium depleted water that a human being has created so that it has less deuterium than the other water in the environment. But you also mentioned that our own metabolism can create different levels of metabolic water. in our cells.

Nick Jikomes

1:19:57 - 1:20:18

So carbohydrate versus fat metabolism, for example, we talked a little bit about how oxidizing fats, you know, takes more steps. So it results in more deuterium depletion. So the metabolic water will have less deuterium in it compared to carbohydrate oxidation. Can you say a little bit more about that with respect to diet?

Nick Jikomes

1:20:18 - 1:20:34

So a lot of people, it’s becoming more and more common for people to talk about and study things like ketogenic diets, which are high in fat, low in carbohydrate. What do you sort of think about that general realm of science with respect to how it’s going to affect metabolic water and deuterium levels?

Gábor Somlyai

1:20:36 - 1:21:16

I would say that, so we always recommend people keep a ketogenic type diet when drinking DW. And you know that the pyramid, which was said at the 70s, that turned it upside down. So today, they changed it. So we are going back to the real world where more covering the calorie intake with more fat, that way we can reduce the average deconcentration of the metabolic water, and that way, the average deconcentration of the patient.

Gábor Somlyai

1:21:16 - 1:21:38

I have data for 142 ppm, for example, those who are on a ketogenic diet, and 148 or 150 ppm who are eating lots of sugar and carbohydrate. So being on a ketogenic diet can cause a six to eight in the deconcentration of the body.

Nick Jikomes

1:21:38 - 1:21:41

And how do you measure that? How do you actually measure that in people?

Gábor Somlyai

1:21:42 - 1:22:20

Yeah, I figured out a new small device that we precipitate the exhaled water and we collect it and we can measure that deconcentration of that water. We publish the paper, how it’s changing and how we can follow day by day the changes in the deconcentration depending on what type of people But in a human clinical study we made, in that case we measured the D-level of the blood, and at the beginning it was 148-150 ppm, and after three months it was 133 ppm. They consumed 105 ppm.

Gábor Somlyai

1:22:20 - 1:22:58

And so that way we just bring into the optimal range of the deconcentration for the insulin signal system because 133 ppm is the best for the insulin. And this is the reason that we could reduce the insulin resistance. So we highly recommend people stop eating sugar, reduce the carbohydrate intake, and increase the fat in the diet. That in general, they can cause a couple of ppm decrease of the average D level of the body.

Gábor Somlyai

1:22:59 - 1:23:04

And each ppm is count. Each ppm can be very important.

Nick Jikomes

1:23:04 - 1:23:32

Yeah, so based on the experiments that you described earlier, you would say that even a small change in the PPM and the concentration of deuterium, the level of deuterium depletion of the water, that can make a difference because you’ve seen examples in cell culture where the cells are sensitive to that. And you’ve done work in humans where presumably, remind us, what were the DDW concentrations in the water that you gave to cancer patients? Was it around this magnitude of change?

Gábor Somlyai

1:23:32 - 1:24:05

We have six different deconcentration, 125, 105, 85, 65, 45, and the lowest is 25 ppm. And the patients, depending on the cancer type, the staging, we recommend starting with 105 or 85, and we recommend to change it and going down after two, three months, just to always challenging the cancer cells. They need to adjust everything to the lowering the level. And this is what they cannot do.

Gábor Somlyai

1:24:05 - 1:24:06

Hopefully.

Nick Jikomes

1:24:07 - 1:24:24

And in those patients where you gave them deuterium depleted water at say 105 ppm, did you also measure the deuterium levels from the exhaled water throughout that intervention to see how much that actually shifted their deuterium levels in their body?

Gábor Somlyai

1:24:25 - 1:25:06

So sometimes we receive samples, even those who are buying our products and sending back samples, and we can measure it. But the point is, if they drink it, the D-level will decrease it. So the kinetics we have tested in my study, in dogs and cats, and then we tested lots of people. It’s good for them if they check it, whether it’s lowering, but the key issue is they should be very strict, drinking only DDW.

Gábor Somlyai

1:25:06 - 1:25:12

Those who are combining with beer and wine and Coca-Cola, we never can get the good results.

Nick Jikomes

1:25:13 - 1:25:29

I see, I see. What types of experiments and what types of clinical work are you working on or hoping to work on right now or in the near future? Can you talk to us a little bit about not just your own work, but anyone else that’s doing work that relates to deuterium depleted water?

Gábor Somlyai

1:25:32 - 1:26:14

Our company that HYD LLC is one of the company in a consortium in Europe. the university from Wurzburg, the Lisbon University, and Oxford is part of that consortium. And we received a fund from the EU to check the effect of deuterium for the central nervous system. So we used a my study, and we used six oxygen dopamine, we treated half part of the brain of the mice.

Gábor Somlyai

1:26:15 - 1:26:46

And the question was, if they’re drinking DW with 25 ppm or normal, whether we can protect the nervous system after checking and treating the dopamine. This is a Parkinson’s disease model system. and it works extremely good. So we could see that the movement and all the parameters, what he could follow, it was much better than the mice drinking DW.

Gábor Somlyai

1:26:47 - 1:27:30

And then checking the brain, we found that we protected the area of the brain with the 25 ppm. So we are pretty sure If the mitochondria are working properly, using a lower D-level, the ATP is working on a better way, synthesizing more ATP, then the whole central nervous system will be more healthy and can tolerate that type of therapy or treatment. So this is running. I hope that from Oxford the study will come and they will publish it.

Gábor Somlyai

1:27:30 - 1:27:52

Meantime, I’m working on getting funds. We are targeting investors. We are trying to get the funds to cover the cost of the next filgure. We are selling the products.

Gábor Somlyai

1:27:52 - 1:28:36

We are happy. and it’s getting stronger and more and more people get access, but without registering as a drug, that will not be part of the whole society and the tendency will not change. Just, I took part in a European Cancer Society Conference in Hungary, and the number means today there are 20 million new cancer cases and we lost 9.8 million and within 24 years that will be double. So this is the prognosis for 2050.

Gábor Somlyai

1:28:37 - 1:28:48

If we want to do that, we need to follow what we have done. If we want to turn it back and change the future, then we need to integrate DW into the therapy.

Nick Jikomes

1:28:48 - 1:29:08

So the results you’ve seen so far that you talked about earlier with regards to cancer in humans were pretty big from what I heard. And so that was a relatively small phase two trial. If those results replicate and they translate and you were to see them at a larger scale, that would be a major result. That would be very big news.

Nick Jikomes

1:29:08 - 1:29:18

The problem for you and anyone interested in this subject is receiving the funding to scale up and do a trial of that size because we’re talking about many millions of dollars.

Gábor Somlyai

1:29:18 - 1:29:44

So far, do you know, to register a drug, it takes about 1 to 2 billion dollars. We started with 1,500. This is how we started 33 years ago. And the reason that we are here, that we generated the revenue and that revenue was used up to do research and to proceed.

Gábor Somlyai

1:29:45 - 1:30:17

And this is how we could survive the last 30 years. Once we had a big investor with 1.3 million euros, in that case, we set up a facility according to the GMP rules. So we are the only company who are ready to produce DW for clinical trials. And on the other hand, we have all the documentation which is necessary to go and apply for ethical approval and to start a clinical study.

Gábor Somlyai

1:30:17 - 1:30:19

So the missing part is the funding.

Nick Jikomes

1:30:22 - 1:30:29

Are there any final thoughts you want to leave people with or anything you want to reiterate about deuterium depleted water or any of the biology that we discussed today?

Gábor Somlyai

1:30:31 - 1:31:09

So there are, if you check the literature, you can see that there are papers that DW was good in a long-term memory, in a RAD study. We have published a paper with Vanderbilt University, the anti-aging effect of diluting depleted water with a Sennorhub dis-elegance system. So what I try to say that I never wanted to find a cancer drug. I wanted to find the mechanism how the life is organized.

Gábor Somlyai

1:31:10 - 1:31:36

And that dutin-hydrogel ratio is a key mechanism. So all the process, all the disease, some way is coupled with the dutin-hydrogel ratio. And I guess, we will discover lots of new things in the future. And I hope that the scientists, more and more scientists will be involved and more and more data will come.

Nick Jikomes

1:31:38 - 1:31:46

All right. Well, I want to thank you again for your time, Gabor, especially joining me from all the way in Hungary with the time zone difference. I really appreciate it. Thank you.

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