The Peter Attia Drive - #303 - A breakthrough in Alzheimer’s disease: the promising potential of klotho for brain health, cognitive decline, and as a therapeutic tool for Alzheimer's disease | Dena Dubal, M.D., Ph.D.

Episode Date: May 27, 2024

View the Show Notes Page for This Episode Become a Member to Receive Exclusive Content Sign Up to Receive Peter’s Weekly Newsletter Dena Dubal is a physician-scientist and professor of neurology at UCSF whose work focuses on mechanisms of longevity and brain resilience. In this episode, Dena delves into the intricacies of the longevity factor klotho: its formation and distribution in the body, the factors such as stress and exercise that impact its levels, and its profound impact on cognitive function and overall brain health. Dena shares insights from exciting research in animal models showing the potential of klotho in treating neurodegenerative diseases as well as its broader implications for organ health and disease prevention. She concludes with an optimistic outlook for future research in humans and the potential of klotho for the prevention and treatment of Alzheimer’s disease. Disclosure: Peter is an investor in Jocasta Neuroscience, a company working to develop klotho as a therapy for people with Alzheimer’s disease. We discuss: Dena’s fascination with aging and how she came to study klotho [3:30]; Biological properties of klotho: production, regulation, decline with age, and factors influencing its levels [11:45]; Potential benefits of klotho on brain health [22:00]; The relationship between soluble klotho protein, platelet factors, and cognitive enhancement [33:45]; The role of platelet factor 4 (PF4) and it’s interaction with GluN2B in mediating cognitive enhancement [46:45]; Benefits of klotho observed in a mouse model of Parkinson’s disease [55:45]; Benefits of klotho observed in a mouse model of Alzheimer’s disease [1:03:00]; Promising results of klotho in primate models, and the importance of finding an appropriate therapeutic dose before moving to human trials [1:08:00]; Speculating why a single klotho injection has such long-lasting effects [1:25:30]; Potential cognitive benefits of klotho in humans, the impact of the KL-VS genetic variant on klotho levels, and the need for human trials to confirm these effects [1:27:45]; The interaction between the KL-VS genetic variant and APOE4 and how it impacts risk of Alzheimer’s disease [1:34:45]; The significance of klotho levels: studies linking lower levels to increased mortality and the broader implications for organ health and disease prevention [1:47:15]; Measuring klotho levels and determining an individual’s KL-VS status [1:52:15]; The promising potential of klotho for Alzheimer’s disease treatment, and the importance of philanthropy for funding research [1:58:00]; and More. Connect With Peter on Twitter, Instagram, Facebook and YouTube

Hey everyone, welcome to the Drive Podcast. I'm your host, Peter Attia. This podcast, my website, and my weekly newsletter all focus on the goal of translating the science of longevity into something accessible for everyone. Our goal is to provide the best content in health and wellness, and we've established a great team of analysts to make this happen. It is extremely important to me to provide all of this content without relying on paid ads. To do this, our work is made entirely possible by our members, and in return, we offer exclusive member-only content and benefits above and beyond what is available for free.

If you want to take your knowledge of this space to the next level, it's our goal to ensure members get back much more than the price of a subscription. If you want to learn more about the benefits of our premium membership, head over to PeterAtiyaMD.com forward slash subscribe. My guest this week is Dr. Dina Dubal. Dina is a physician scientist, professor of neurology at the University of California, San Francisco and holds the David Coulter Endowed Chair in Aging and Neurodegenerative Disease.

She is also an investigator with the Simmons Foundation and the Baccar Aging Research Institute. Her work is recognized for its significant potential towards therapies to help people live longer and better. She directs a laboratory focused on mechanisms of longevity and brain resilience that integrate genetic and molecular approaches to investigate aging, Alzheimer's disease, and Parkinson's disease. In my conversation with Dina, we focus around something called Clotho. Now, if you've heard me talk on this podcast and other podcasts,

you've probably heard me bring up Clotho, either the protein or the gene that codes for the protein. My interest in Clotho really started a couple of years ago when I became aware of some of the genetic data in humans about the relationship between clotho and Alzheimer's disease prevention, particularly in people who are carriers of the A4 gene. This of course has led me deeper and deeper down the clotho rabbit hole and really all roads lead to Dina if you want to have this discussion.

So we begin our discussion with an overview of clotho. What is it? How is it formed? How does it get around our body and what does it do? We talk about the mechanisms regulating clotho in the body and in particular in the brain. We also talk about things that impact clotho levels such as stress and exercise and to what extent they do. From there we look at the research that's being done in how clotho relates

to various cognitive functions as well as its role in brain health across different species and across different ages, as well as understanding how clotho treatment may be helpful in treating neurodegenerative disease, particularly Alzheimer's disease. We wrap up this discussion speaking about the broader impacts of clotho on organ health in addition to what its potential may hold for the treatment of Alzheimer's disease in the future. Before getting to this podcast, I'd like to mention a conflict of interest, which is that I am an investor in a company called Jocasta.

Jocasta is a company that is trying to bring Clotho, the protein, into human clinical trials for treatment of Alzheimer's disease. So, without further delay, please enjoy my conversation with Dina Dubaul. Dina, thanks so much for joining me today. This is a topic I've been very interested in for the better part of about a year and a half to two years. And obviously there's no better person to discuss this with than you. I also suspect that this is a topic not enough people know about given its potential implications

and significance vis-a-vis Alzheimer's disease, which we'll get to. But maybe before we get into this, let's give people a bit of a sense of your background. Tell me a little bit about your clinical work, your research work, what you did during your PhD and how that carried forward during your tenure. Sure, well, thanks again for the invitation. I'm delighted to join you today. By way of introduction, I'm a neurologist

and I'm a neuroscientist and I direct a group that is deeply involved in the discovery around clotho. I was thinking back to when my interest in aging actually began, what was this journey? I thought back to my undergraduate days at UC Berkeley when I was a 19-year-old that was oddly interested in aging, kind of obsessed with aging. I worked with the medical anthropologist at Berkeley, Lawrence Cohen, on what it meant to experience dementia in different cultures. What was it like in India versus the United

States? And then simultaneously, I took a class on the physiology of aging. I remember it so clearly in Dwanell Hall and I was at the edge of my seat learning about cellular senescence. And I remember thinking, this is amazing. This is happening to us day by day aging, yet we don't know so much about it. And we don't know much about brain aging per se. I was committed as an undergraduate to really learn more about brain aging and possibly to do something about it.

That led me to an MD PhD at the University of Kentucky. I trained with Willis Wise, a neuroendocrinologist who studied brain aging, who has an amazing PhD, learned so much, fell in love with the discovery process of science. And fast forward, I then trained as a neurologist at UCSF where I am now. And that was over 20 years ago, 2003. And I'm on the wards. It's a regular day of patient rounding. And the former chair of UCSF neurology, Steven Hauser,

turned to me and he said, Dina, when you go back to the lab, do things that are big and important and not incremental or mediocre, because they'll take you the same amount of time. And that really clicked with me. It became my mantra. And after an Alzheimer's fellowship, both clinical and basic science, I had the chance

to build a group and to really start my own scientific discovery. And we focused on clotho, the subject of what we'll talk about today, the Greek fate who spins the thread of life. And the idea of studying clotho was to understand whether factors that help us to live longer could help us to live better. Whether this longevity factor could actually help the brain, could help it stave off Alzheimer's disease. And that's where we began. So let's go back to the first time Clotho crossed your radar. Yes. Well, I was just beginning my junior faculty days and was thinking what were we going to focus

on? What was going to be our chance to do something big and important? I was very intrigued by a few decades of work that was emerging that aging itself was malleable. And the first work with this was Cynthia Kenyon in worms, where she demonstrated that tweaking genetics, aging in worms could dramatically increase lifespan. And I really wanted to know whether that could have effects in the brain. Clotho had emerged as a longevity factor and it was a chance to understand could clotho do something in the brain. Nothing was known about clotho. Very little was known about clotho when we started.

A colleague had observed that the levels decreased in the white matter of monkey brains, that a variant of clotho that we can talk about in a bit was associated with decreased stroke risk. And the person who discovered clotho had noted that mice without clotho move slowly and they were cognitively a little slow. But I was the right person at the right time and had the chance to really dig in. It was risky to start with something that not much was known about in the brain, but it was a chance to do something maybe big and important. Let's talk a little bit about the discovery of clotho. Was it discovered through the mice that were deficient? Was it deliberately knocked out? What was the process of discovery? Just? Was it deliberately knocked out?

What was the process of discovery? I mean, just contrasting it with Cynthia's work, obviously she knocked out a gene that is, I think, sort of the analog of one of the IGF genes, if I'm not mistaken, correct? Yeah, the DAF-16. Yeah. So how was clotho discovered? It was in 1997, Makoto Kuro-O, a Japanese scientist, accidentally discovered it. And it's such a story of serendipity.

He was studying hypertension, and he engineered a mouse to insert a gene, which I believe was a sodium proton channel. So he engineered mice, he inserted this gene, and then he noticed that in a few lines of these mice, maybe it was just one line of these mice, there was this premature aging phenotype. So the mice lived to about three months instead of about 30 months, and they developed normally, but around two weeks of age, they became progeroid. They looked like they were rapidly aging with osteoporosis, atherosclerosis, emphysema. They looked and

they moved slow. They looked really old. And so he went back to that mouse and he mapped out what had happened and he had accidentally caused a mutation by inserting his sodium proton pump. So he went back, he mapped what had been disrupted and that was clotho. That was the first time clotho was found and he named it after the Greek fate who spins the thread of life, daughter of Zeus. Clotho, the Greek fate, is C-L-O-T-H-O. He named it K-L-O-T-H-O in homage to his discovery. His name is Kuro-O, K-U-R-O. And a longevity factor isn't something that just causes premature aging if disrupted. So, it was very important for him to then go back and see what would happen if he overexpressed it. Then he engineered mice to overexpress clotho and those

mice lived 30% longer. So that was a longevity factor. That disruption caused premature aging and overexpression extended lifespan. And it's such a beautiful story. It's a great story for people, especially maybe who don't do or haven't done science because it illustrates the role of curiosity and serendipity. Look, there are some people who maybe wouldn't have even gone back and done the experiments that he did to understand what turned out to be the most relevant thing. I'm sure that whatever he was doing with a sodium proton pump exchanger seemed interesting at the time, and that what he observed was actually

kind of a disappointment in that it ruined one of his lines of mice, but obviously it led to the far more relevant finding over the long arc of time. He opened the field, he followed the science, he opened the field, he looked into the mistake, the oops of science, and here we are, maybe on the cusp of a new therapy.

Yeah, exactly. So let's talk a little bit about what this gene does. How big is this gene? How preserved is this gene over other species? And tell us a little bit about the protein that it codes for, what regulates it? So clotho itself is a pretty big protein. It's about a thousand amino acids by weight, maybe 130 kilodaltons if you look at it on a Western blot. And it codes for the aficionados,

a type one transmembrane protein, which means that its N-terminus will sit in the extracellular space. It has one pass through the membrane and it sees terminus as inside the cell. Now, it's N-term, that part that sits outside the cell has two repeat domains, KL1 and KL2. And those domains have high homology to proteins that are found throughout mammalian worm and fly biology. So there are homologs of clotho throughout species and pretty conserved in mammals. So clotho is a big protein. It's a transmembrane protein. And then what happens is there are a few forms of clotho. It's made primarily in the kidney, also in the choroid plexus of the brain, but it's made in the cells of the kidney.

And then it gets transported into the membrane and then enzymes come across, base, atom 10, atom 17, and they clip that extracellular portion of clotho and release it into the blood. Or in the case of the brain, release it into the CSF. And that form of clotho is known as a soluble form of clotho or the secreted form of clotho. I call it a hormonal form of clotho, the clotho hormone, because it's released at one site and then can act at different sites at multiple organs.

So that's how it's made. Those are the major forms. So how big is that end terminus piece that gets clipped, that now goes and acts like a hormone basically? It's the majority of the protein itself. The part that's inside the cell and crosses the membrane is very small. So it's really the majority of the protein is the hormone itself. If we were to take a blood test and measure a person's clotho levels, what could we observe about it across people of the same age, people of different ages? What factors do we think might influence the expression? And does the expression and

production of the protein become the sole determinant of the soluble factor? In other words, is there also variability at the cleavage efficacy or are there other factors that lead to consumption of the protein or degradation of the protein in the periphery? So starting with levels of clotho across the lifespan. Well, first clotho, it circulates in us, it circulates in mice. And when we started our studies in mice, I really wanted to make sure that this was gonna be relevant

to the human condition. And it is very relevant to the human condition. So we have circulating levels of clotho. We're born with about six times the levels that we have now in our cord blood. And then throughout our lifespan, clotho levels decline. In our, from say 40 onwards, they can decrease by half during aging. So, we're born with very high levels and they decrease across our lifespan. They also have a circadian rhythm or a diurnal rhythm in that when we wake up, we have high

levels of clotho. And by 4 p.m., 3 p.m., when people are looking to grab a cup of coffee, the clotho levels are really starting to decline and then they nader by around midnight. It can be a 40% or so decrease. So there's a circadian rhythm to clotho. It changes across the lifespan. Sorry, Dina, just to make sure I understand that. What explains the daily variation?

Is it some sort of quote unquote consumption? Or is it being turned over that quickly and the supply of clotho is decreasing by the time of day. The answer to that is not known. What regulates the daily levels? Is it, how is it being degraded? Is the expression being increased? The half-life is short in mice. In mice, it's maybe seven to 10 minutes. But in humans, well, I would say in monkeys, it's much longer. It's at least a day or so. So it's unlikely that the production is really actually increasing and decreasing because when it's made, it's half-life will be longer in non-human primates like us. So I would speculate,

though I don't know Peter, that it may be sequestered in organs or degraded or not sure. I've seen a couple of reports of this diurnal variation and it's really not clear why it's increasing and decreasing, but it is known in aging, for example, this longer term decline and slow decline that we see. It's been observed, and this was work by Fabrizio and Brosio's lab, that there is a hypermethylation around the clothopromoter that occurs with aging that stops its transcription and essentially transcription and translation. So that's an interesting thing to think about if we wanted to preserve our clotho levels, how can we interfere with that methylation that happens with aging?

That's interesting. So more methylation there leads to inhibition of the promoter. A lot of times you'll see the opposite. It's the loss of methylation that shuts off the promoter, correct? I don't know about that. In general, or clotho at least, the methylation that shuts off the promoter, correct? I don't know about that. In general, or clotho at least, the methylation really turns things off. Off, OK. I could be wrong about that, but I think that that's definitely the case with clotho.

And is that believed to be the main mechanism driving the reduced production of clotho is the increase in the methylation of the promoter? Dr. Julie Kinn That's what's known so far. There are other possibilities like maybe organs make it less, maybe the kidney is less efficient at making it with aging. That's possible. I think that Ambrosio's lab is really onto something. They showed it in chondrocytes and aging chondrocytes, but this really the first molecular demonstration about why clotho may be decreasing. Now there are other things,

Peter, that decrease clotho. One big one is chronic stress. We did a study with Alyssa Epple and Eric Prather here at UCSF and mothers with neurodevelopmentally at UCSF and mothers with neurodevelopmentally typical or atypical children and the mothers with high levels of stress with children with autism spectrum disorder had much lower levels of clotho as well as shorter telomeres. Their level of stress, increasing stress, there was a decrease in the clotho level. Did you demonstrate in those folks a higher degree of methylation of the promoter relative to their age and did it basically look like they were older at the level of the promoter? I think that would be so neat to look at.

We haven't done that, but it would be really neat to look at. So in other words, we don't yet know the mechanism by which chronic stress could be mediating the reduction. Ryan Brown here at UCSF and Eric Prather and also Alyssa Epple and myself looked at whether there might be a relationship between clotho levels and telomere length in these same women. And there is a very direct relationship with a very tight correlation between shorter telomeres and lower clotho levels.

So there may be some convergence around these different hallmarks of aging, maybe some relationship of them regulating each other, or maybe they're changing in parallel, it's hard to say. But here's some really good news, that in talking and thinking about what changes clotho levels, we do know that stress associates with lower clotho levels. But one of the most robust interventions that increases clotho levels is exercise.

It's been shown in study after study and in meta-analysis, it's been done in about 12 studies, but the data are that after 12 weeks of what people call chronic exercise, that clotho levels increased by about 30%. And maybe even increased in a study with preliminary data, in mice, they can even double acutely after a 45-minute treadmill run.

And how it's increased is unknown, but it's beginning to be thought of as an exorkine, something that is produced and released in the body following exercise. How would this stack up in your mind compared to something else that we would think of along these lines, BDNF, where I've talked about this at length on the podcast. Of course, if you really look objectively at all of the modifiable behaviors around

dementia, routinely exercise is at the absolute top of the list. More potent even in its magnitude than lipid management, glucose management, and hypertension, all of which themselves are enormously powerful. But something about exercise seems even greater. It could be that it indirectly mediates at least two of those three. But of course, people point to something else that's going on and BDNF is often discussed. Do you think that maybe part of that effect is clotho? And do you think that the effect of clotho may be even more potent? I was recently asked the same question

at an Alzheimer's conference at the Salk Institute, and I don't know the answer, but I think it's a really important question. Is there a relationship between clotho and BDNF? Does clotho increase BDNF levels in the brain? I don't know. BDNF has a very striking effect in the brain. Maybe, Dina, just tell folks a bit about BDNF levels in the brain, I don't know. BDNF has a very striking effect in the brain. Maybe, Dina, just tell folks a bit about BDNF.

Sometimes I forget that maybe not everybody listening to us today has also listened to all of the other content over the years. So maybe just give folks a little bit of a primer on BDNF as well. So BDNF is brain-derived neurotrophic factor, and it has been shown in the brain to really be associated with even drive positive brain health.

So intermittent fasting increases BDNF in the brain, exercise increases BDNF. If you give BDNF to the brain, the neurons function better. It's a really good trophic factor for the brain. And it does remind one of clotho and effects of clotho. And in fact, intermittent fasting simultaneously increases clotho in the brains of mice, along with BDNF. How long do the mice need to fast to get that effect? So this work was done at the SOC. The mice fasted for 24 hours. So not really something we can translate to humans given that that's – I mean 24 hours in a mouse is that's half the distance to death. So that's –

That's interesting. But this is a real problem. This is a real problem with extrapolating from mice data, right? Is even a 12 hour fast in a mouse is an overwhelming feat of calorie deprivation. 24 hours is staggering. We don't have a way to translate that to humans, but it could be that that's on the order of weeks. So I just want to make sure people aren't listening to this thinking, hey, all I got to do is skip breakfast and I'm getting the same amount of BDNF or cloth though that I would get from a 45 minute workout. My guess is those two are not even in the same zip code.

I think differently about it. So when I think about mouse time and mouse lifespan, so they live to two to three years, but I believe that they're aging faster. They have a shorter lifespan, but I believe that they're aging faster. They have a shorter lifespan, but I think it's possible that their 24-hour lives are similar to our 24-hour lives. But 48 hours of fasting will usually kill a mouse, right? I don't know how long a mouse can go. I used to be more facile with this literature, but I pay less and less attention to mouse

literature now than I used to. Maybe someone listening to this could write in and correct me, but I don't think it's a stretch to say that 48 hours, put it this way, what I do remember is an IRB would really scrutinize an investigator who's trying to get to 36 hours of fasting in a mouse. It's a really big deal. We don't do fasting studies in mice, but I would agree that in general, we have to be careful when extrapolating from mice to humans. I do think that in the area of cognition and neurodegenerative diseases, that studying mice and mouse brains and their neural circuits and their hippocampi and their prefrontal cortices tell us fundamental

things about the human condition, particularly because they have to have very strong navigation strategies and memories for foraging, for coming back to their nest. And these memory circuits are very, very fundamental and quite preserved up to non-human primates, including us. But having said that, we do the majority of our work in mice. We are always aware of, are we doing something that is important to the human condition? Is it relatable to the human condition? Is it relevant?

Who cares? Will any of this matter? These are always on my mind, these questions, particularly as a physician scientist. And there are certainly limitations. I also think that there are many strengths, particularly in studying memory and neural circuits in the brain. But we don't stop there. And we have tested whether I haven't talked yet about what we have found in mice, but I'll just spill the beans and say that we found early on that clotho enhances cognition in mouse brains.

In a very recent study, we collaborated with biotech and Yale University and found that actually clotho enhances learning and memory and cognition in old monkeys. In a very complex brain, genetically diverse, anatomically diverse, functionally complex, that in a brain like ours that clotho had very similar effects to what we see in mice. There's so much I want to come back to on that topic because I've seen all of these data of course and truthfully, they seem overwhelmingly positive. Let's go back and use some of the mice data to allow us to speak about maybe greater insights around the causal relationship between clotho and brain health. So we've already established a few facts here. So you're going back to the discovery of clotho and brain health. We've already established a few facts here. You're going back to the

discovery of clotho in 97, which was based on effectively a knockout. We saw that that is incompatible with a long life. We saw that the reverse when clotho was enhanced, I believe you said it enhanced lifespan by about 30%. Again, that's really remarkable in a mouse. There aren't many things that enhance a mouse lifespan by that much. That's up there with the most draconian caloric restriction, rapamycin administration under certain settings. That's a very reproducible and robust finding. Let's talk a little bit about specifics to brain health, what you're talking about with respect to not a disease state per se, but just enhancing cognition in an aging mouse or even in a middle-aged mouse.

This is where we started. And we started with the hypothesis that in these mice that over express clotho that live longer, that maybe these mice would be resilient to Alzheimer's toxins. That was our starting point. And so we back-crossed Alzheimer's models with clotho overexpressing models to create mice who have mouse-heimers and then have mouse-heimers plus lots of clotho. And then we had normal mice and normal mice with lots of clotho. So there were four experimental groups.

And to our complete surprise, the normal groups, we're not talking about the mice that had Alzheimer's, modeled Alzheimer's, but in the normal groups that had higher levels of clotho versus normal levels of that group showed a remarkable difference, a remarkable statistically significant difference across multiple cognitive tasks and across different ages. And that was that the mice that overexpressed clotho that live longer were smarter. So you put them in a maze.

They could map the room better, escape quicker, not because they were swimming faster, not because they could see better, but because they were remembering and learning more efficiently and better. And that was a complete surprise. I remember the day unblinding the data, analyzing it. It was unimaginable.

Dina, I just want to add a point. Sorry to interrupt, but it's so powerful. You a moment ago said this was statistically significant, which of course it was. And too often, I think people hear the term statistically significant. And I think they can be forgiven for confusing it with clinically significant. But as you know, those are not necessarily the same thing. And I almost think that in this case, Dina, stating that it is statistically significant, which it is, almost undersells what I think the magnitude of the differences are. I have a friend who likes to point out that the really powerful stuff in biology is the stuff that you don't actually need to do the statistics on.

When the delta is so big that you don't need to whip out the student t-test and calculate the p-value to make sure you're not being fooled, which by the way is very important and you should be doing it every time, when it's actually just a formality because the answer is so clear looking at the data, that's the really big aha moment. And I think I'd be safe in saying that that was the case here, wasn't it? You're right. We tend to be really conservative. We don't want to make an error.

But having said that, you're totally right. I mean, I can imagine the graphs now, and there were non-overlapping data points. I didn't need to run the repeated measures between subjects and NOVA to really know that it was statistically significant. It was that type of a finding. So it's a point well made. Sometimes experiments will show you those differences and then the next time you do it, they don't. Someone who runs the water maze is wearing a new cologne and the mice get stressed out. They don't always repeat

and are not always replicable. This is a case that it was amazing, but was it going to replicate? Was it going to be true in different cognitive tasks? What would happen in aging mouse? All of these questions opened up, and we iterated and we did experiment after experiment, and the data held strong in mice. That clotho overexpression really enhanced their cognition in young mice, in the aging mice, in mice that modeled Alzheimer's disease, and also in mice in a publication that's in peer review now, but also in mice that model Parkinson's disease, that overexpression of clotho enhanced cognition. It is really a remarkable finding. I think it's maybe one of the most important findings in my professional career.

I would go one step further. I feel like it's one of the most important findings in brain health period. I mean, on some levels, it's a bit of a mystery to me why this isn't known by everyone. It's part of why I'm really, really happy to be sitting down with you and why I've wanted to sit down with you for the better part of six months. I know that between my schedule and yours, it took a minute to get us on the schedule. This is such a big deal.

I also want to reiterate something you said a moment ago, and I say this and we'll be linking to all of these studies so people can see the data for themselves. But the reproducibility coupled with the magnitude of the effect, so the consistent, enormous magnitude of difference across studies, across labs, across investigators, that signals something is going on here. Again, not to bring up rapamycin again, which people hear me talk about a lot, but people say why are you so confident in Rappamycin's Giroprotective effects? The magnitude of benefit is enormous and it doesn't seem to matter who does the study. If it's done in this part of the country or this

part of the world or with this animal or with that animal, it always seems to work. Now that doesn't guarantee it's going to work in humans. I'd be the first to admit that, but boy, I feel a lot better when it works in every model system, virtually every time. And when it passes the eyeball test, you don't need the error bars and the P values. So let's talk a little bit about before we go to primates and ultimately to the epidemiology

in humans, let's talk about mechanism of action. Tell me what you think is happening with this soluble clothoprotein. I assume it's crossing the blood-brain barrier or it wouldn't be having this effect. Has that been easily demonstrated? It is a winding story, Peter. And one that's exciting and has led us to very unanticipated findings. I first wanted to mention, as we think about how different interventions may converge upon the same biology or relate to the same biology, it is interesting that mice treated with rapamycin have increased clotho levels. So rapamycin either directly or indirectly will increase clotho levels,

just thinking about the convergence of biology. Now, how does clotho enhance cognition in a young, aging, and diseased brain? And that immediately became a major question for my lab and many other labs. And the very first thing that we had turned to was a component of NMDA receptors. So NMDA receptors are really key in connections between neurons. And when they're stimulated, they let in calcium into a neuron and they allow essentially a potentiation of a neuron for connections to form, for functional connections to form, and really for the neural substrate of memory to form. So NMDA receptors are really key. And there's a component of NMDA receptors called gl Gluen2B. It's a subunit. I'm really going tiny and molecular here, but it's very important because I had remembered that there

was something called a Doogie Howser mouse, affectionately called a Doogie Howser mouse. We're kind of dating ourselves here because there's going to be a lot of listeners who don't know who Doogie Howser is, but suffice it to say this was a prodigious mouse. Yes. It was a mouse that was smarter. There are very few model systems where you can actually pivot the system to make an engineer a mouse to be smarter. There are very, very few things. Clotho is one of them. This Gluen2B overexpressing mouse or the Doogie Howser mouse showed a very similar phenotype to the Clotho overexpressing mouse. When I looked at the behavioral data of the Gluen2b overexpressing mouse, in the very

same test, it was performing very well just like the Clotho overexpressing mice. I just wondered whether Clotho was acting throughuN2B to enhance cognition. That was the hypothesis. In this case, the hypothesis, we gathered data that supported the hypothesis. There wasn't a big surprise in that over-expressing clotho caused an increase in gluN2B at the synapse, at the area where brain cells connect,

and led to a more efficient connection and memory formation synaptic plasticity at the level of the neural cell. So this gluen to be was really important when we blocked gluen to be with multiple pharmacologic inhibitors, we abrogated clotho's ability to enhance cognition. So that was one clue, but here's the major head scratcher. So Peter, clotho is expressed in the kidney. It's also expressed in the brain secreted through the choroid plexus, which also makes the fluid for the brain that the brain sits in. But clotho does not cross the blood brain barrier. We have looked, others have looked through auto radiography, through IP and Western blot,

we've looked through many immunohistochemistry, we've looked through many different methods and can see very clearly that it doesn't cross into the brain. However, when we give a shot of clotho to mice, to monkeys, in their arm or their belly, doesn't matter, if we give them a shot of clotho, within four hours, there is cognitive enhancement. And that cognitive enhancement lasts for at least a couple of weeks.

So how is it that giving peripheral clotho is enhancing brain functions if it's not actually crossing into the brain. That's been a major question that we and others have been working on. And we have some clues that led us in unexpected places. I'm happy to talk about one of them if this is a good- I am all ears. To me, this is, I mean, I didn't want to get to this point yet, but this is

the natural time to talk about this. And then I think we should step back and talk about some other things. But yes, to me, this is the jugular question, Dina. Why is it that when we inject a monkey peripherally with clotho, it spends the next 14 to 19 days in a state of cognitive superiority. And that clotho isn't crossing the blood-brain barrier. This is, to me, the jugular question. You're right. And we have been working on this. I think I'll step back and say, it's actually amazing that you give something peripherally. It has a central action. It's not crossing into the brain.

And it's something physiologic that our bodies are used to, that we have seen high levels of upon birth and development. It does not have known side effects at physiologic levels that have been observed yet. It's very remarkable. So what is it doing? We hypothesize. So I'll just say we don't know yet. This is an area of fervent investigation for us and others, but we have one clue in a study that we published recently in nature aging. And this is what we did. We started with the premise that maybe Clotho was doing something in the blood that then would send a messenger into the brain. So maybe there was this messenger of

Clotho that was actually going into the brain when you gave it peripherally. And so we did a very simple experiment. We injected mice with Clotho. And four hours later, at the time of cognitive enhancement, we did an agnostic profiling of the proteins in their plasma, in their blood. And what we found was sort of unimaginable. It was unimaginable in that we found that all of these platelet factors were increased with clotho injection in the blood. And Peter, I have to be honest, when I saw this agnostic data set, I turned to my postdoc and I said, I'm really not interested in this.

I am not interested in looking at platelets. They are involved in wound healing, their coagulation factors. This is weird. I had a post-doc, Kena Park, who is very persistent and said, let me try, let me just study this for a little bit. I said, go for it, I could see your passion. One question, Dina, just to ask, in parallel,

did you do the same multi-omic observation of the CSF in the mice in parallel to see if there was something new that was showing up in the CSF to parallel the platelet factors in the plasma? Yes, we did. We haven't published this yet, but we did. In the same mice, we took their blood and we took their CSF. And what we found is that Clotho increased these platelet factors. It was totally bizarre. I didn't believe it. I tend not to believe data. I need to see things over and over again, need to see functional

studies. I need to see it matters. I tend not to believe data, but there it was. She repeated the findings in many different ways. but ultimately what she did was a series of studies demonstrating that this biology is real, that clotho is actually activating platelets very modestly. And then when a platelet is activated, so let's first just step back. What is a platelet? A platelet is an a nuclear cell in our blood and it contains little compartments of bioactive chemicals, chemokines. And when a platelet is stimulated or activated, it actually releases these factors and it does so in a context dependent way. So when there is a cut or a wound, there is a huge activation of platelets and they release all sorts of factors that help with the clotting and help with the wound healing.

So that's what they're traditionally known for. But it turns out Peter, that when we exercise, our platelets are activated and they're releasing factors that travel into the brain. I mean, who would have thought that platelets could be messengers of brain health and could take center stage as a messenger of brain health? So when we exercise, this was found by an Australian group led by Tara Walker, that when we exercise platelets are activated and certain platelet factors, one of them being platelet factor four, is released and travels to the brain and actually causes neurogenesis or the production of new neurons in the brain. That was very new data. And with that data in mind, I felt too, like maybe there's something to this Clotho platelet

connection. Maybe there's something to it. So Kana isolated platelets. She put Clotho on them. They released PF4, this platelet factor 4, the same one that the Australian group had shown. And then she gave this platelet factor four to mice, young, aging mice. She gave it to them as a shot in the belly, just like we have given clotho. And she

found that that platelet factor four enhanced cognition in a young mouse and in an aging mouse, and it reversed cognitive deficits, and it was totally remarkable. This is where it gets weirder and wilder. So I go to my colleague and close friend, Saul Valeta here at UCSF. He's the young blood and old brain scientist. So he's really built a really nice body of experiments

and literature showing that if you give young blood to an old mouse, it rejuvenates their brain. So Saul is studying brain rejuvenation through blood and I'm having coffee with him. I said, Saul, we've found something so interesting. I have to tell you about it. And I go on to tell him about PF4. Clotho stimulates platelets. Platelets release PF4.

PF4 enhances the brain. And he said, well, he was silent for a second. And he said, Dina, we found the same thing with young blood and that when we give young blood to old mice, that young blood is enriched for platelet factor four, which declines in aging. And then we give platelet factor four, we rejuvenate the old brain. And if you think that's a remarkable convergence of biology, wait till I tell you that Tara Walker in Australia had found the same thing with exercise. And this is all at the same time, that exercise was increasing PF4 and that when she gave PF4 to her old mice, it enhanced their cognition.

All of us who are close friends and colleagues now, have an incredible convergence of biology where clotho, young blood, and exercise, we're activating platelets, releasing PF4. PF4 is enhancing the brain. And as an aside, the biology is just amazing. Each of us had our own unique approaches and unique way of digging into the biology.

But a practical question, Peter, was like, what are we gonna do? Are we competitors now? Or are we going to hold hands and go through the publication process together? Are you going to screw me? Am I going to screw you? How is this going to come out? And what we did is I think a really nice example and model in biology where we held hands and we said, let's just stick together. We went to editors, we went through a couple of years of revisions and reviews, but at the end of the day, all three papers came out in the Nature

family of journals on the same day. It made for an impactful splash and it also just highlighted the convergence of the biology, the reproducibility of the biology. Again, this will never happen again in my lifetime, but this was amazing and a complete surprise that platelet factors could play a role here. So, let's talk a little bit more about this. So, platelet factor four is increased by all of these three things. Do we believe that it's platelet factor four that is directly acting on gluN2B, then? If so, what do you think is happening at the level of substrate and receptor? Dr. Lacey Krofman That's where we went next with the biology and the experiments.

One question was, does platelet factor four actually cross into the brain and we demonstrated that it does. We gave it peripherally. We immediately looked into the brain and saw that it was ending up near neural cells or even within neural cells. So it's crossing into the brain. And then does it act directly in the brain was the next question. And what one of my lab members did is take hippocampal slices. So the hippocampus is the area of the brain that is really executing the learning and memory and is targeted by aging and diseases of aging.

So he took hippocampus and then he put platelet factor four on it and saw immediately within seconds, actually, there was a, well, I don't know within seconds, but quite immediately, maybe seconds, maybe minutes, there was a change in the membrane potential. And more calcium was being let in. And we thought maybe this would be gluon2b acting.

And so we put on gluon2B inhibitors and platelet factor four no longer potentiated the synapse or enhanced the neural function. So we do know that platelet factor four, like Clotho, is working through gluN2B. And maybe Clotho is working through platelet factor 4 to change gluen2b. Sorry, has that same experiment been done, Dina, to look at the clotho that is derived from the chloride plexus to say when you dump clotho directly onto gluen2b, you see an influx of calcium. When you block gluen2b, you abrogate the effect as well. You now have two

things, platelet factor four and Clotho that can independently do the same thing. Is that a correct statement? We haven't done that experiment yet. We've done transgenic overexpression in the brain and we've done Clotho peripherally. We haven't't dumped clotho itself onto the hippocampus to see whether there can be a direct effect. Other groups have overexpressed clotho specifically in the brain and seen cognitive enhancement, but I haven't seen studies seeing whether clotho is actually directly influencing GLU-N2b. We know that

it indirectly influences gluN2b. We know that we can give it peripherally and it'll increase synaptic plasticity through gluN2b, but we don't know whether it can actually directly do that too. It may, based on other people's studies, it may. We don't know. But here's something else that was actually quite disappointing, but the biology is the biology. We then asked the question, does clotho rely on PF4 to enhance cognition? Is PF4 required for clotho-mediated cognitive enhancement? And to do that, we generated a colony of mice with PF4 knockout. They just don't have PF4. And then we gave them Clotho versus vehicle, always blinded, always randomized through

a series of cognitive tests, water maze, large Y-maze, one maze after another, because you'd like to see the same effect reproduce. And what we found was that Clotho continued to enhance cognition even in the absence of platelet factor 4. To the same extent, Dina? To the same extent. You might think there would have been a diminution, but to the same extent. It just tells us that there's more than one factor.

It has to be such an important pathway, and it is so conserved that there is no way biology is going to let one thing be the messenger. I'm making all this up, of course, but hypothesizing that if clotho is so important, it can't be limited to one messenger. If it gets to the gate and it can't get through, it has to have multiple messengers that it can deliver the message to PF4 being one of them. But clearly this experiment would suggest that there's at least one other messenger, right?

Right. That's the interpretation and conclusion because PF4 is sufficient to recapitulate also mediated cognitive enhancement, but not necessary. When you go back and look at the first experiment you did, the unbiased, was that just a proteomic assessment or did you look at all omics? How broadly did you sample the serum way back and was there something else you missed because the PF4 was such a big signal? Was there a smaller signal that was there perhaps as

well? There were many signals. There are many other platelet factors. There are many other proteins. We are systematically marching through those. We're marching through not just proteins, but metabolites. We are also asking in a cell type specific way through a system called turbo ID that labels the protein as it's secreted from the liver, the kidney, the heart, from lymphocytes. It's a really unique and elegant genetic manipulation that does a biotin label once a protein is secreted, let's say from the liver. And we're asking when we inject clotho, what gets secreted out of the liver and into the plasma. So we're doing like higher resolution studies to understand how clotho really changes the systemic circulation in a cell type specific way. And then asking

which one of those factors that comes from the kidney, the liver, the lymphocytes, the heart, which one of those factors is necessary and required for cloth-immediated cognitive enhancement? And the answer at the end of the day may be what you alluded to, that there may be many factors that have overlapping functions of cognitive enhancement. Maybe they work together, maybe they work independently. But you're right. If this is a very important biologic function, it really shouldn't rely on just one factor. I think that's a really important point to underline. Soterios Johnson Tell me a little bit more about this assay,

the in vitro assay that you were able to basically sprinkle on PF4, sprinkle on Clotho to the gluen2b subparticle and witness the massive influx of calcium. It seems to me that if that assay is reasonable, it could also become a great screening tool to identify rather than having to do the experiments to see if you can enhance cognition, at least do your first screen there. Now you run the risk that if Clotho is working through secondary mechanisms, you'll miss it. But at least as a first screen, in as much as Clotho is working through an MDA via GLUEN2B, let's screen a whole bunch of molecules on it, look for the activation, and then go back and search for those in a biased way in our serum sample and sort of triangulate back

and forth like that. I love it. Let's do it. And we are doing it and we're doing it with synaptic plasticity as a measure, as a substrate of cognition. And we're also doing it in vitro at the cellular level using live cell imaging, where we can isolate neurons from the brain, from a mouse brain, and then they grow beautifully on a dish. They connect with each other and they survive for weeks on end, actually. And you can do live cell imaging to see whether if you put clotho on, I have a graduate student

doing this right now, Barbara Shariva, who is putting clotho onto neurons and these other factors that we're seeing from the omics onto neurons and seeing whether there is an increase in the neurite outgrowth and the physical connections between neurons. Again, this isn't cognition itself, but it's a substrate of cognition. It may be a distant biomarker for it, but it's a really smart way to, when you're asking a question of this many proteins, which ones are important. It's a way to screen. So with synaptic plasticity and with neurite connections, with the outgrowth of the neurons connections.

So all of this makes a lot of sense for a mouse model of Alzheimer's disease, where the primary deficit is a cognitive deficit that seems disproportionately focused in memory. But you mentioned before I distracted us down this rabbit hole that you are also seeing positive signals in a mouse model of Parkinson's disease. Now are you seeing them in the mild cognitive impairment that can accompany Parkinson's disease or are you seeing it in the movement disorder?

Are you seeing an improvement in the primary issue associated with Parkinson's disease? Or are you seeing it in the movement disorder? Are you seeing an improvement in the primary issue associated with Parkinson's disease? Part of this is published. Part of this is in the peer review process. But it's really, really exciting to share. So these are the experiments. We took mice that overexpress alpha-synuclein, which is a pathogenic player in Parkinson's disease.

It disrupts the synapse and has a causative role in Parkinson's disease because we know that people with mutations in causing overexpression of alpha-synuclein will develop Parkinson's disease. It's sort of like the equivalent of APP in people with Alzheimer's disease where you have this... It's not necessarily the dominant driver of the disease, but it's clearly playing a causal role based on these mutation studies. That's right. And then the mice that I've mentioned that we've used for our Alzheimer's or Mousheimer's studies were APP mutant overexpressors, just to pedal back to that. So we took these Parkinson's model mice and they have both motor deficits, meaning that

they walk across a balance beam and they'll fall. If they're put on a spinning rod, they're dis-coordinated and they'll fall. They have motor difficulty. They also have cognitive difficulties, which are not as severe as the Alzheimer's model mice, but they do have cognitive difficulties and their ability to map a spatial environment and to hold memory in their mind with working memory. So they have these deficits. We did two things. We injected them with clotho and remarkably saw that clotho treatment improved their cognition. It didn't normalize, but it improved, if I'm thinking about the data correctly, by maybe 70% or so, their cognitive abilities. So nearly normalized their cognitive functions. It didn't do anything

to their motor functions, so they continued to have motor dysfunction and clotho did not help that. That was also true with the transgenic overexpression of clotho all over the body and brain for a lifetime that warded the cognitive deficits induced by Parkinson's toxicity, but it did nothing for the motor problems. And I've spoken to my Parkinson's colleagues about this. There's a lot of interest in potentially using Clotho as a treatment for Parkinson's disease because they tell me in our clinic, Stena, we can treat the tremor and the rigidity. But people complain over and over again consistently

about the cognitive deficits that we now know are a part of Parkinson's disease, not just later in the disease, but even as part of the disease. And those deficits specifically are problems with executive function, which is the ability to focus, to shift attention, to make certain judgments, to think quickly. And it is mediated by the prefrontal cortex, an area that became important when we looked at human studies.

The bottom line was that in mice, clotho really enhanced, again, cognition, but didn't do anything for motor functions. Is there a mouse model that fits between the Alzheimer's model and the Parkinson's model that is more akin to a Lewy body dementia model where it has some of that alpha synuclein pathology, but also has a much more significant cognitive component. There are so many mouse models, Peter. I think that a good mouse model for Lewy body would be alpha synuclein and maybe more in the brainstem and the hippocampus and cortex. I bet there is one, but I'm not clear.

But a reasonable hypothesis would be, again, that it would probably provide some relief of the cognitive symptoms, though not necessarily the movement symptoms. That's right. So that's what we're seeing consistently. One of my friends and colleagues had an interesting analogy of thinking about clotho as a helmet for neurons, that whatever came crashing the

cells way, whether it was alpha-synuclein, tau, amyloid beta, aging stresses, that the neuron was protected, it remained resilient against multiple toxicities. The effect of Clotho is really in cognition itself. In hippocampal and frontal cortical circuits that these neurons and glia and other cell types are really protected against multiple toxicities. If we want to jump into clinical trials, I think this is the time to really move clotho toward human clinical trials.

Wouldn't it be amazing if we had a cocktail for Alzheimer's disease in addition to leucanumab or dinanumab that's removing the amyloid beta from the brain? What about adding something like clotho that can really help shore up the functions of the cells? Because we know that Alzheimer's disease is a multi-proteinopathy. It's not just one protein that's causing the disease. We need factors in our treatment. We need cocktails that can really resist multiple protein toxicities. So I just have this dream that people might be able someday to benefit from clotho, this factor that naturally circulates in our body that helps with longevity, that helps with other organ systems and enhances the brain. As we know from

our monkey studies, for weeks at a time, at least a couple of weeks, if not more, there was funding for a certain amount of time, but it has a long acting action. We need to put helmets around the neurons to really stave off multiple toxicities. And this is clotho, this is what clotho does. This is the new job of the Greek fate that spins the thread of life.

It's to protect our brains. Yeah. You're preaching to the, not just the choir, but the fully converted and obviously in my other activities, this is something that a lot of my energy goes into. I want to come back to a couple other questions on the mouth study that are going to factor into, I think, when we start to talk about the humans more. Has the following experiment been done where you take a mouse that is genetically susceptible to Alzheimer's disease, so the equivalent of your APP mouse or something like that, and you take a control group, maybe just for my own knowledge, if you took an APP mouse, at what point in its life, how

many months old before it starts to develop a clinical disease? So maybe two questions. At what point would it start to be accumulating amyloid in the CSF? How many months of age? And then how many age when it starts to show cognitive impairment? What are those two numbers roughly? It depends on the mouse model. And there are so many mouse models of Alzheimer's, including APP models. The one that we've used consistently is called the J20 model. It expresses

human APP, which produces amyloid beta in mutant forms. And it's a more aggressive model, Peter, and it causes synaptic loss, that connection between cells, it really disrupts that connection between cells really early, before three months. And then it starts producing cognitive deficits at around three to four months. And that's the J10? The J20 model. J20. Okay. So here's my question, Dina. Has the experiment been done where you give those mice clotho starting at birth at a high dose and does it delay the onset of the inevitability?

We know that through over expression throughout the brain and body, that those mice, those APP mice will have almost normalized cognition, but we don't know whether it's delaying the onset. There are others that have done human population clinical studies. I want to make sure I understand what you just said. You're saying if you take the J20, the APP mutant, and you also cause a mutation that overexpresses clotho, are you saying that you've done that in the same mouse? Yes. Tell me about that mouse's lifespan. Is it normal and is it cognitively normal or does it still get Alzheimer's disease? That mouse that has the APP mutation

and overexpresses clotho by about three to four fold, that mouse will live much longer, number one, so it extends its lifespan. Number two, it will normalize its cognition. Across its lifespan? Across its lifespan. At three months, seven months, eight months, yes, it normalizes its cognition.

It's very remarkable. And then when you look in the brain, Peter, when you look at levels of amyloid beta and of tau, they're not different from the mice without high levels of clotho. That is unbelievable. In other words, their brains are still riddled with the Alzheimer's toxins, but they've been able to really thwart the effects of those toxins because they show normal cognition. And if you look at their synapses,

this work was done in collaboration with Leonard Mucki and Eliezer Mazlia, who's at the NIH now. When Mazlia looked at the synapses in these mice, he saw that the synapses were all preserved. So again, like this analogy where Clotho is really providing a helmet around each neuron, it really allowed the synapses to be preserved, but there was a whole bunch of amyloid and tau still there. And that is resilience. These toxins are present. They won't necessarily go away with Clotho, at least in mice. The story may be different in humans. But clotho provided resilience and thwarted the toxicities of Alzheimer's disease. Okay.

So, I'm going to plant the seed with you, which we'll come back to. But the reason I'm asking this question, Dina, is I do believe that at some point we will have clotho as a drug. I think it's going to come to market. We're absolutely going to be injecting people with clotho. Where we'll go is do we believe this is a drug we will only want to give to people once they have MCI for the listener, right? So early, early, early stages. Or do we believe that we'll take anybody who's susceptible and just be giving them clotho even before there is a demonstrated

disease risk? So that's where I want to go with this is kind of thinking three or four moves ahead on the chessboard. Now let's go back and talk about the primates because we've talked about this incredible body of literature in mice. Again, I think it's reasonable to be circumspect around mice literature. But again, when you start to talk about the volume of literature here and the breadth of mouse models and the number of investigators that consistently find the same results and the magnitude of the results, again, it passes the eyeball test.

You don't need to do ANOVA tables all day long to get the answer and squeak at it. This looks incredible, but it would be nice if we had a more convincing model. So let's talk about the single best thing we can ever do before we get to humans, and that's to look at primates. So let's talk a little bit about primate brains

and the body of literature around clotho in that brain. This is an area that I wanted so much to get into. I don't work with monkeys. I think it's a very, very specialized field to test cognition in monkeys. And so I'll just take us back a long time ago, maybe eight years ago, when I really wanted to know, Peter, whether clotho can enhance a more complex brain than a mouse brain. And like you said, mice are really important in scientific discovery,

but there are so many examples of what worked in mice and cured Alzheimer's disease failed in humans. And that's the valley of death. Works in mice doesn't translate to humans. It's not true for everything. And mice have been very, very important for fundamental scientific discovery and for medicines. But I didn't wanna spend my career on something

that was not going to be relevant to humans. and for medicines. But I didn't want to spend my career on something that was not going to be relevant to humans. And that might not be that big and important thing that Stephen Hauser challenged me on when I was a resident. I was pitching this one attest cloth and primates all across Silicon Valley to biotech and pharma. And people were curious and enthusiastic about it, but one individual really got this, Ned David, who had started a company, Unity Biotechnology, to attack aging.

And we joined forces. They fundraised, they found really great collaborators at Yale, and really made this happen to test clotho and cognitive enhancement in a brain like ours. When people would tell me, if it doesn't work, it's going to kill your research program, and I would say, let it die then. I don't want to spend my career on something that's not important. If it's done well, we'll know. And so this collaboration between biotech, me and Yale was one in which a lot of thought, a lot of money went into the design and the execution.

So why non-human primates? Rhesus macaques are incredible organisms. They have very complex brains. They are more genetically diverse than humans. And that genetic diversity is something that often trumps effects, can often really influence whether an effect is able to survive genetic diversity or not. So having a lot of genetic diversity is very important to really challenge and test the biology. Mice are inbred. They all have the same genetics, just to say. So the rhesus macaques are incredibly genetically diverse, more than humans. They have a functional complexity that's very similar to humans in terms of the even more of the neural circuits, the hippocampus, the prefrontal cortex, and they have an anatomic complexity that's pretty similar to us. So by testing clotho and non-human primates, the idea was to jump over this valley of death. If it worked

in primates, let's look toward humans. If not, let's do something else. I want to pause there for a second, Dina. I mean, I think obviously the listener can imagine where this story is going, but that's a huge burn your ships moment. I want to just spend a minute probing that a little bit more and you were a wildly successful investigator before this. I don't think it's an obvious step that you would take to push that much further because let's be clear, if Clotho had failed miserably in primates, how many PhDs and post-docs and students do you have in your lab? We're a group of around, it fluctuates between seven to 11. Yeah, and obviously you probably have a very well-oiled machine

at generating R1s and all sorts of grants. It's not easy to start all over again if all of that dries up, right? Well, that's why it was so risky. And some people just go to a human trial. You can do biomarkers, but this is risky. And it is risky if it's not done well, but it's super informative even if it's negative. If it's good negative data, and not because the experiments were done in a sloppy way,

it's good negative data, then it really tells you something. And we have other projects on longevity. We study why women live longer than men. What does the second X chromosome have to do with it? And there are other longevity factors. So it was really important to push forward and to push forward in a rigorous way. And also with the knowledge that the results could really change the direction of what we're doing. But we have times in our lives where we reflect on what we're doing, how we're spending our time. Is it with people that we appreciate? Is it doing things that are meaningful? And it may be deeply philosophical, but from day to day, I really want to be doing something

that I have a lot of faith in and that potentially has meaning for the human condition to improve human health. And if we've done a really great experiment that's negative, and there's really reason to believe that this isn't going to go forward, then that's fine. We will mourn it and we will still move forward with something else. But we just don't have lifetimes to devote to a hundred different scientific projects. So we have to be very choosy. And I really, really wanted to make sure that this is something really worth putting time, energy, resource, passion, constant thought, training, fundraising.

I think the reason I'm pointing this out, Dina, is I want to publicly commend you for something that honestly I don't think most scientists would be willing to do. I wouldn't even hazard a guess at the fraction, but I think there are a lot of scientists who have lost sight of what you just said and they think the job is doing science. But it's not. The job is knowledge creation. The job is knowledge creation for the purpose of making lives better. And those are very different things. They overlap. The former is a process to the latter, but it's very easy and it's very easy to lose sight of that in the weeds. So, I only call it out to say I don't think everybody would have come to the same conclusion

you did. So, with that said, let's talk about how you and your colleagues at Yale and basically thought of the right question to ask and then the right design of an experiment to ask the right question. First, I just want to say thank you and that is very well said. Monkeys undergo cognitive decline in a very similar parallel fashion to humans. They have synaptic loss, the connection between neurons with aging, as do humans do. And the circuits that are affected in aging are really similar to the human circuits. So they are hippocampal frontal circuits, the hippocampus and the frontal lobes. And aging will preferentially target working

memory and also spatial memory and other types of memory. But this working memory holding something in your mind, you go to the refrigerator, you open the door and you think, why did I come here? What was holding that immediate memory in your mind is something that aging really targets. And monkeys undergo this very similar cognitive decline to humans. And there are ways to test monkeys that interrogate those circuits. In this case, a spatial delayed task was used. Graham Williams and Stacey Kastner conducted the studies.

And clotho actually preferentially targets those circuits that aging erodes. So here is a model system. It's genetically, anatomically, functionally complex. It undergoes cognitive aging parallel to humans. And there are really well-developed tests by these exceptional scientists who are very experienced and adept at conducting these tests. So it was a really excellent setup to ask, can Clotho enhance cognition in these aging monkeys? What was done is something called

a spatial delayed response. And the monkeys presented with a bin of multiple wells. And sometimes there's a few wells and sometimes there's a lot of wells. And it's harder when there's a lot of wells to find a treat. So a treat is placed in a well and they can see you place the treat and then a screen drops and then all the wells are covered. You cannot see where the treat is. And then the blind is opened and they are tested where to choose because they're basing this on spatial

memory and on working memory where that treat was. Again, it's a little easier if there's only three wells. It's easier to choose one out of three and get it correct. It's harder when there's nine wells to remember spatial and working memory where that treat was. It was done very rigorously, I must say. The study was largely blinded. And it was done in a way in which that injection of clotho wouldn't interfere with causing stress in memory so that everyone got a baseline.

They then got a vehicle. They then got a vehicle and then they got vehicle or clotho. It's a really well designed study. And just for folks listening, vehicle is placebo so that they understand what we mean by that. Thank you. So the monkeys that got a clotho treatment, again, largely blinded, performed better than the ones that got vehicle treatment. Sorry, Dina, how did you guys decide how to raise the dose? First of all, when you obviously did all the mice experiments, you're dosing like you probably do based on a certain number of milligrams per kilogram or milligrams per gram of animal. What did you use to dose escalate that? Did you just assume the same dose per

unit body weight or did you make other adjustments? Well, we went with two things in mind. One is we really wanted to stay at least with one dose in a physiologic range. So with something that the body has been exposed to and has seen over its lifetime. And so in one dose, we wanted to go somewhere up to like maybe four to five. Four X. Yep. Yep. You're still just below what they would have been born at. Right. A very useful level of clothodes, a rejuvenating dose. And then the other doses were higher and were meant to test, could you really push the system to improve cognition even more than what is observed with that physiologic dose? By the way, that physiologic dose, that sort of natural dose is what we have always

used in mice. I should say we've also given mice huge, huge doses that were way beyond what they would ever see or used to. And it still enhanced cognition in mice. That's going to be different than the monkey story. 06.05.00 06.05.00 By the way, when you gave the supraphysiologic doses to mice, so presumably doses to mice, so presumably tenfold and beyond. Did you run into any problems? For example, did the mice ever develop antibodies to the protein? Did anything else arise that would suggest that more is not always better?

Dr. Kirsten Krohman We didn't see signals in mice. But having said that, we didn't systematically study whether doses of a hundred micrograms per kilogram was doing something different. We didn't observe differences in their normal behaviors or like in their basic blood work, but it worked. It didn't work better. So I should say like those super low doses were just as well as the super high doses in mice. And super low doses mean well below 3x normal. So if we just call 6x the peak physiologic dose, what was the minimum effective dose in mice relative to that?

More than five times less. A very, very small dose that still worked. I'm thinking if we gave mice 10 micrograms per kilogram, I'm remembering something like 0.5 or one enhanced cognition, 32 micrograms enhanced cognition. So very, very low, just a touch more, just a boost enhanced cognition. Our strategy was to stay within a physiologic dose and make sure that that was represented in the monkey studies. I mean, I really thought about this, consulted with people that have taken drugs to market. Tom Boone is one of them and was very instructive and taught me a lot in terms of thinking about relevantly dosing in monkeys.

And then there were higher doses that created much higher doses than the body has seen, which is maybe 10 times higher and beyond, 10, maybe 20 times higher and beyond. And what we found is that the low physiologic, that natural dose of clotho enhanced cognition in the monkeys. And it did so within four hours. And then that cognition stayed better. Their ability to think, remember stayed better for at least three weeks, 14 to 21 days. And some were even tested out to a month. I saw 28 days. Yeah. And then at some point the study had to be stopped.

It's a million dollars or more and so, but it was remarkable that one sub-q dose, like a shot of ozempic would be given, a sub-q dose that was low physiologic had an immediate effect on cognitive enhancement that lasted for a very long time. Now, there were a few different types of tests done. One was for normal memory load and one was for high memory load. And the high memory load was a test with a lot of different bins where they had to remember

which one among seven or eight wells was the treat hidden in. It's just more taxing with more choices to remember. And they did even better in that study of high memory load. With a low dose. With the low dose. So the effect was particularly pronounced. They were particularly smarter when the task became harder. And again, why this task, why monkeys, why clotho? It all came together because clotho works on these circuits that are tested in the monkeys that decline with aging and with neurodegenerative diseases like Alzheimer's and Parkinson's. So what about the high dose? Right. So the high dose did not work. The high doses that gave the monkey's clotho way beyond what they've seen in their lifetime,

they didn't harm. They did not impair cognition, but they didn't help cognition. And it's not too much of a stretch to think that too much of something that does multiple things in the body and maybe multiple pathways that could just create an imbalance that doesn't support cognitive function. Again, it wasn't harmful with the measures that we tested, but it didn't help to take so much more than the body has seen and is used to. And that again was different from the mice.

So the mice, and this may be a big difference between mice and a more complex brain with the non-human primate. In mice, we saw continued cognitive enhancement. And in monkeys, we really got a window into, before going into clinical trials, that if we're giving clotho, probably should be thinking about a specific therapeutic window, one that the body knows, one that the body is used to. So Dina, why do you think

the effect lasts for so long? This is also a little counterintuitive when you consider the little counterintuitive when you consider the hourly variation of clotho naturally, when you consider the transient effects potentially of things like exercise where you see these large boosts over a short period of time following a bout of exercise, but it seems hard to imagine that the benefits of an hour of exercise persist three weeks later, the way a subcutaneous injection of clotho did. So, what do you think accounts for that? I don't know the answer, but I'll speculate. First, I'll just say it's remarkable, isn't it? It's particularly remarkable as we imagine therapeutics in that maybe this is the

type of treatment of rejuvenation that could be administered maybe once a month, once every three months as a shot in the arm, for example. So I think it has really important therapeutic implications. Mechanistically, this means that clotho has not just an acute effect of immediately enhancing NMDA receptor functions, synaptic plasticity, cognitive function at four hours, but it has an organizational effect. It's doing something to, in the longer term, really help to, for example, remodel a synapse. So I would imagine that, for example, with GLUE N2B being trafficked to the synapse and to promote better functions, that those sort of synaptic organizational effects are happening

for and staying put for a longer period of time. I think the biology of this organizational effect of clotho has yet to be discovered, but it's something that's happening at the synapse. I have a very strong sense of something that's happening organizationally, structurally at the synapse. 05. As much as I want to go deeper into that and talk about the conformational changes that could be occurring, we'll have to save that for another discussion because where we have

to really go next is what do we think about in humans? And what evidence do we have, because we do have some really interesting evidence, even absent a single experiment, that everything we've talked about so far might indeed also be relevant to the species of interest. And no disrespect to the mice and the macaques, but there aren't too many people listening to this whose mind isn't already wondering, okay, enough about the animals already. Tell me, is this going to make a difference for my mom or for my dad who are in the early stages of dementia? Is this going to make a difference for me because of my risk factors, even though it's 20 years from now?

And so tell me a little bit about a particular SNP associated with the clotho gene called KLVS and what its significance is to this story. None of this matters, Peter, if it doesn't have potential to help the human condition. It just doesn't matter. It's interesting, but the big and important is if it's relevant and may work in humans. I don't know whether we've done anything big until we test clotho in humans. So, with that said, back in the early days in, I think it was 2012 or so, or 2011, 2012, when these first clotho studies, we were doing them in mice and discovering

these cognitive enhancing effects. I went to, and again, I'm a physician scientist. I always have humans in mind. I have my patients in mind, Alzheimer's disease, 50 million people around the world. This is going to triple by 2050. Cognitive decline is our biggest biomedical challenge. Always have humans in mind. I went to my friend and colleague, Jennifer Yokoyama,

at the Memorand Aging Center here at UCSF. And I told her that there was this genetic variant of clotho that had been found. We can talk more about what that is. But was there a way, because she's a geneticist, and she and others at the Memory and Aging Center, Joel Kramer, Bruce Miller, have built this incredible population of individuals and patients, Alzheimer's disease with normal aging, frontotemporal dementia,

and they really carefully characterized them, their genetics, their blood biomarkers, et cetera. So I went to Jennifer and I said, is there any way we can know whether this genetic variant of clotho, KLVS, has any association with cognition? Because that would mean that this clotho is important to brain health. It would mean that there was some link with humans and brain health. So what is KLBS that you mentioned and what does it associate with? As we've discussed, we all have clotho circulating in our blood and around our brain. But some of us, about one to four, one to five of us will carry a gene for clotho, a genetic code for Clotho that leads to higher levels of production. And KLVS refers to two single nucleotide polymorphisms that cause a difference in the coding of the

protein itself. Again, so about one to four, one to five of us will have KLVs. Other people are non-carriers. And those people with KLVs will genetically have higher levels of clothocerculating in their blood. Now, interestingly, it's only the people with one copy of KLVs, the heterozygotes, and very rarely there are homozygotes of KLVS, and those people end up having multiple disadvantages in lifespan and vulnerability to different diseases. So when we talk about KLVS, we're talking specifically about heterozygosity carrying

one allele of it. What are some of the health consequences of being homozygous and what is the prevalence of homozygosity? It's really rare. So if the prevalence of heterozygosity is around 25% in populations, homozygosity is so... It's probably about 1 to 2% because actually it would kind of mirror APOE4 where the heterozygosity is about the same.

It's about one in four and the homozygosity is about one in 50 to one in 100. Yeah, that's about right. It's pretty rare, but it exists and those people have shorter lifespans. They have much lower levels of clotho and they are at risk for anything that the heterozygosity helps with, the homozygosity hurts. But the heterozygosity has been a really interesting window into clotho and natural experiments. How much higher is their clotho level? Well, we did this study.

Jennifer and I took the serum from individuals here at the Memorand Aging Center, a few hundred, and tested the ones that were non-carriers that just have the typical clotho gene versus the KLBS heterozygote carriers. And the heterozygote carrier status increased clotho levels by about 15%, 15, maybe 20%. And just to give you context, if you're wondering am I carrier and some people are and some people aren't,

I don't want people to forget that exercise is thus far one of the more powerful modulators of clotho expression. And exercise increases clotho on average by 30%, so much more than what the genetic influence is. But nonetheless, KLVS increases clotho levels by about 15%. So for example, if an average number, let's say a non-carrier had 800 picograms per mil of clotho circulating in their serum, a KLVS carrier might have like 950 or so picograms per mil. That's what I'm remembering from our graphs. There's reason to believe that it increases clotho because those two amino acid changes that translate into a different amino acid in the protein itself influences secretion of clotho from cells. There's a biologic reason that this variant

is probably changing cloth levels. The other piece to this story, Dina, that's just so fascinating is now when you take those data, the prevalence of heterozygosity of KLVS, and you cross it with APOE4. So now we have listeners of this podcast are absolutely not strangers

to the population-based risk of APOE4. Of course, we always want to remind people that at the individual level, very difficult to make that statement. But at the population-based level, we know that having an APOE3 and an APOE4 is at least a doubling of risk, maybe even slightly more. risk, maybe even slightly more. And homozygosity for APOE4 could be an 8 to 12 fold increase, so call it a log order increase in risk. So tell me, what did you find in people who are homozygous and heterozygous for APOE4, who also happen to be heterozygous, i.e. have the favorable variant of KLVS single copy.

This is really remarkable data. I first want to go back to that original experiment that we published in 2014, I believe, with Jennifer, and that those people that carried the KLVS allele did better across cognitive testing. And this is a normal aging. This was not an Alzheimer's disease, but carrying that variant, carrying KLBS associated

with better cognition across the board. I'm often asked, well, how much smarter were those individuals? And I would be careful about smarter and happier, and we don't know, but the cognitive tests showed us that they did better. And they did better to the same extent that APOE4 carriers would do worse. So it's a sort of a similar amount of change in cognition in which KLBS was improving and APOE4 was decreasing. With that in the background, since then, many groups and many

people have looked at KLBS in their populations. And that association has held largely in most studies, not in all, but in most studies, there is an association of KLVS and better cognition with heterozygosity. So then the question comes that you asked about APOE4, Alzheimer's risk and clotho in KLVS. We initially did a study in collaboration actually led by Osiyama Konkwo and his group at Wisconsin that showed that in individuals with APOE4 that were carriers of KLBS, that they just had less of the effects of ApoE4 in terms of less A beta deposition, less cognitive problems. It was a smaller study of a few hundred people at risk for Alzheimer's disease, but it was a good study.

But I'll tell you the study that I'm most excited about that we were not a part of that came from Stanford led by Mikhail Beloy. He's one to watch. He just established a lab at Wash U and his senior PI, Michael Grecius, and their wizards at doing genetic population studies. So Peter, they did this remarkable study of 22 different cohorts that were normal cognition, MCI, mild cognitive impairment that you mentioned earlier, and Alzheimer's disease, over 20,000 individuals, a very large meta-analysis, and they looked at if there was a relationship between KLVS carriers and ApoE4. And the bottom line of what they found was that if an individual carried KLVS, the ApoE4 didn't matter. So let me break down what that actually meant in their experiment.

That meant that in those people that carry ApoE4, if they were also heterozygote for KLBS, they had a decreased risk for developing Alzheimer's disease that was pretty close to the normal population. They had a decreased conversion from MCI to AD, and they had decreased Alzheimer's biomarkers, both in their CSF and in their brain. They just had less amyloid beta. It's a really striking study. Again, because it has the power of statistical analysis, it holds across many, many cohorts and it's many, many people. KLVS essentially blocks and abrogates the ApoE4 toxicity by this genetic associations. Really remarkable. Did the double E4s, did the homozygotes reduce to a completely normal 3-3 risk? My recollection, but it's been a while since I looked, there's a graph that demonstrates

all of this that's a great summary. My recollection, which could be wrong, that's why I'm asking, was that the 3-4s were completely abrogated to a 3-3 risk and that the 4-4s ended up coming way down, but they still looked like more of a 3-4. Am I misremembering that? They didn't actually publish that. They excluded the homozygotes from the analysis, but it might be in a supplement somewhere, but I actually reached out to Mikhail and I asked him about the 4-4. I had the same question. And so this is just by verbal communication, he indicated that the 4-4 also associated with the protection from the KLBS heterozygosity.

So that the effect of having one ApoE4 wasn't different than having two ApoE4s in terms of KLBS protection. It protected in both heterozygosity and homozygosity of the APOE4 allele. Again, it's just through verbal communication, it might have been in a supplement somewhere. So, did they find anything negative? So going way back to the mechanism of this, which at least in part is communicated through a platelet factor. Was there anything that might have been unwarranted such as an increase in stroke risk? I know that it's actually the opposite, but I'm just trying to say, is there anything that with an increase in a platelet factor

that could have led to an increase in DVTs or something? When you have a sample size as large as they did, presumably you would find something that was negatively associated with the KLVS. I'm not aware of increase. As you were saying, it's actually the opposite. The KLVS, heterozygosity, actually associates with the protection against stroke risks, cardiovascular risks.

Even metabolic risk. And metabolic, yeah, metabolic diseases. I've searched for something that could be negative. And I did find that in the cancer literature, KLBS heterozygosity has a poor prognostic indicates a poor prognosis in BRCA1 carriers indicates a poorer prognosis in BRCA1 carriers with breast cancer. There are examples where it's not helpful and one is in BRCA1 positive breast cancer. Deana, going back to the human homozygotes for KLVS, do we know how much of an increase in clotho they produce? If the heterozygotes are producing 15 to maybe 20% more, how much more clotho is being produced by the homozygotes and does that give

us an insight into toxicity in humans that was not observed in mice and not even observed in the primates? Because in the primates when you gave too much, you just didn't get a benefit, but you didn't get a harm. In the mice, you actually got more and more benefit. As the organisms get more and more complicated, it seems that the therapeutic window is getting narrower and narrower. Do the homozygotes give us any insight into that? With Jen here at the Memorand Aging Center, we have access to homozygote serum, and we actually looked at several homozygotes compared to non-carriers and heterozygotes. And what we found is that their clotho levels are actually lower than normal.

And we think that has something to do with, I'm going to get a little deeper into this, because I think it's really important when we think about therapeutics actually, that many genetic variants won't cause a change in the protein sequence itself. If a nucleotide change is in an intron, it's just not going to change the structure or function or anything of the protein. But in KLVS, there are two nucleotide changes and they translate into different amino acids in two places of the clothoprotein. And so what we think is happening, this is speculation, but what we think is happening

based on in vitro studies that Arking did in 2002, that in the clophto heterozygote in which one allele is making a mutant protein, we think that there's an overcompensation for that mutant protein by increasing wild type levels. Because what that variant does is it mucks up clothose secretion from the cell. So just to be clear, carrying one variant is likely changing the structure and function of clotho, but there's probably an overcompensation from the wild type allele causing higher levels of the wild type clotho. First of all, that is an unbelievable, I would not have guessed that, and I can't believe I didn't think to ask

such an obvious question, which is, do the KLVS individuals produce the same protein? I took it for granted that they produced the same protein and just made more of it, but to be clear, if you assay those individuals, are you finding two proteins in them? A protein that mirrors the wild type, that's the one that's elevated 15 to 20% and an actual mutated protein or a protein that has two different amino acids, which again, to many people sounds like big deal. You've got a thousand amino acids in the full protein. You've changed two of them.

How much can it matter? Well, unfortunately in biology, it can always matter. Ask a patient with sickle cell anemia. So is that what you're seeing? You're seeing two different proteins and it's the wild type one that is over expressed. At this point, it's the hypothesis that the wild type one is really compensating, overcompensating by increasing levels

for what may be a mutant protein. But in reality, and we've done ELISAs on the enzyme linked to immunoassays on thousands and thousands of individuals, but the ELISA itself doesn't distinguish between a KLBS protein and a normal wild type clothoprotein. It doesn't distinguish between the two. And so the answer to that question is not known.

ELISA's just too blunt an instrument to figure that out. If we take that a step further and say, what's happening in a clothothopomozygote, they're expressing a mutant protein from one allele that mucks up secretion, and they're expressing a mutant protein from another allele that mucks up secretion. And so they don't have wild type clothe. All of their clothe is the KLBS form and it's very low. How much lower is it, Dina?

In our studies, it was, I'm trying to visualize the graph. If there was a 15% increase with heterozygosity compared to non-carriers, there was probably a 30% decrease with homozygosity compared to non-carriers. So to give you a sense of numbers, if a non-carrier was at 800, a KLVS homozygous carrier was below 600, something like that. This is why this matters. It matters for many reasons, but there have been some really large scale studies recently, Peter. One is called the N. Haines study published in 2022, I believe. And what this study showed us is that clotho levels as measured by this immunoassay by

Eliza, that the clotho levels really correlated with mortality. I want to make sure I get this right. So it was over 10,000 people in the United States. Clotho serum was measured on everyone. The mean age was about 56 years. And if the mean clotho level was around 800, then something less than what they defined as 666 by their study, picograms per mil, associated with a 30% mortality over five years.

And that was replicated in another study called Inchianti, that lower levels of clotho. And again, this isn't like half the levels. Maybe it's like 30% of a decrease of levels associated with a 30% mortality over five years. And that mortality was primarily in cancer and cardiovascular disease. I think levels are going to matter as we think about our human aging, as we think about our organismal health, heart health, brain health, cancer health, the levels, at least by association right now, matter and lower levels are really associated with more diseases of aging. It's just like, can we even scratch the surface of this? I mean, we've just sat here and talked

for two plus hours about the limits of our understanding of clotho and brain health and yet this NHANES study is actually looking at something totally different which is all-cause mortality and saying basically if you're in your 50s and your clotho levels are 30% below what would be expected for someone to your age, it's associated with a 30% increase in all-cause mortality over the next five years. If that turns out to be causal, and you're saying that the manner in which that death was distributed was cardiovascular and cancer, so it doesn't even have to do with more dementia, which of course wouldn't be kicking in, in your 50s, it suggests that clotho is doing even more than just protecting brain health, which if it did nothing else would still be arguably the most important thing

that we should be trying to get into humans for clinical trials. So I know that your area of expertise is not oncology or cardiovascular medicine, but I'm just curious as to what your thoughts are as to how that could be happening. I couldn't agree with you more. And my expertise is as a neurologist and neuroscientist is really on the brain. But having said that the side effects of clotho increasing it may be much more than in helping brain health.

It clearly has a very strong association with protection against cancer, cardiovascular disease and kidney disease. There is, Peter, a very, very, very large literature on clotho and organ health, a very strong one. In the cancer field, there are many preclinical models, again, in rats and mice, that show that giving clotho the soluble hormonal form actually stopped and reversed cancers in mice, like pancreatic cancer, for example.

And then there are association studies in humans too, but there is so much more to the biology of clotho than only what it's doing in the brain. In kidney disease, clothothe is gonna come to market and maybe the kidney people beat the brain people. If clothe comes to market, I think people are gonna benefit no matter what. But in chronic kidney disease, there is a decrease in clothe secretion

and decrease in clothe in the blood. And the kidney folks, the kidney specialists are really developing clotho as a diagnostic biomarker to understand when the kidney starts failing. The idea there is that right now the measures are glomerular filtration rate, urea, and these are not as sensitive as they would want biomarkers to be in detecting kidney dysfunction. As soon as the kidney starts having trouble, clotho declines. As soon as it starts having trouble, so there's a fervent interest in developing clotho as a biomarker for kidney function.

And I would say, again, if we think in a broader sense about clotho, maybe someday and maybe someday soon we would have our clotho levels checked just as we do our blood pressure and our cholesterol and we get breast exams and we have colonoscopies. Why not have a clotho level checked? Everyone's will be different. Now, how do we get around the issue that it has this diurnal effect? That's the only thing that jumps to my mind when you're talking about a marker of EGFR because I know in our practice, we're fanatical about monitoring EGFR.

We look at cystatin C, we look at creatinine, we're triangulating them. At least to our ability, these don't depend terribly on time of day. Do you think that the answer for clotho is you have to have it done 60 minutes after you wake up in the morning, it's non-negotiable, don't ever let somebody check your clotho level at two in the afternoon, otherwise we can't interpret it? Is that how you think it's going to factor in? I think that is going to give us the best and more accurate level of clotho. And in fact, I mentioned that we've done thousands and thousands of clotho

measurements and human serum and human CSF, cerebral spinal fluid. And I always make sure that whoever we're collaborating with and who has collected the specimen did it in a morning fasting manner. So the individual or patient hasn't eaten and it's first thing in the morning. Why do you know that eating impacts cloth levels? I don't know that and I don't know that it's really been tested per se, but in the spirit of what one does, for example, just being incredibly consistent and rigorous and with less confounding factors in the spirit of what one does, for example- Just being incredibly consistent and rigorous in a way.

Yeah, with less confounding factors in the background. But having said that, the decline over the course of a day isn't dramatic. The decline by age is more relevant than the decline by time of day? I would say that the decline by age is a consistent decline, but the time of day does matter because it can decrease by almost 40% by midnight. And this is done with, I have to say, I've looked through the literature, the diurnal rhythm of cloth has been shown in a small number of people, but the data look good. That it's not really statistically different until like afternoon, but it's starting to decline. I had to design the time and day and I would say that we need a clotho test

that people take in the morning, fasting like they do with their cholesterol, just get it along with the cholesterol, draw. And most importantly, we need a standardized assay. Yeah. I was about to say, is there a CLIA based assay for this? No, no. I think it's coming given how many people and how many samples levels haven't been coordinated between institutions and labs. So everything we do in our lab is consistent to our methods and standards and we standardize everything to, and I include my own samples in there, but it's all standardized. But then my lab may have slightly different values than what is going on in another lab. Then of course, so if I woke up in the morning

and I checked my level and I was at 800, is it picograms per milliliter? Is that the unit? Yeah. Yeah. If I did my test at 6 o'clock in the morning and I'm it picograms per milliliter? Is that the unit? Yeah. If I did my test at six o'clock in the morning and I'm 800 picograms per milliliter, and then from seven o'clock to nine o'clock in the morning, I'm in the gym, I could easily be 1200 picograms per milliliter after. So how does that impact our understanding of what's going on? I think that's a really good important point.

I do want to emphasize that the human studies show chronic exercise increases clotho by after a few months, after three months. And there's been a mouse study that shows an acute bout of exercise can really nearly double clotho. But I think we would be interested again, we're imagining a not so distant world. It would be important to get a baseline level.

Where are you living? So just to be clear, you're saying you do not have human data that demonstrate that acute bouts of exercise dramatically change you. I have not seen that. I have not seen that. I know a person who might be willing to do that experiment for you.

We could discuss that offline. Sure. Okay. So, final question on this, Dina, because I know it's come up for us a lot in our practice. So, I'm hoping you have a better solution for us. We do like to test our patients, especially those who are higher risk for Alzheimer's disease. We do like to test to see if they have the KLVS polymorphisms. It is very difficult to get that information. Are you guys doing that work yourselves? Are you outsourcing that?

How are you guys getting that information on your own? I realize that much of the research you're doing is on a database where that's been done, but are you aware of any commercially easy ways for individuals to determine their KLVS status, should they choose to know? I don't know the exact answer to this. I know that the information is represented somewhere on 23andMe. We can't seem to get it out of 23 and me, even when we use Prometheus. I may be wrong about that. Yeah, we can't seem to do it.

Hopefully somebody listening can say, no, you're wrong, Peter. You just got to do this, but we haven't figured it out. Yeah, I have to say that in our case, when we collaborate with the clinical researchers, they have their populations completely genotyped for Clopo ApoE4 and most other genes out there with the polymorphisms. There's been a really deep sequencing and genetic screen of all of those individuals, but I'll have to circle back to you about that. Okay. Well, Dina, this has been an, I mean, I think at the risk of being hyperbolic, just an insanely fascinating discussion. And I'm so happy to be able to have

discussed your work with you and then by extension to be able to put this in front of so many people because I do think that for a disease like Alzheimer's disease that if we're being brutally honest of all the what I call the four horsemen of death, if there's one that we have very, very little to offer patients, once they're in the throes of the disease, this is the sad poster child for it. It is unfortunately today, I don't need to tell you what you see every day, but I think people watching this understand that even with the advent of anti-emolloid therapies, they have barely, barely been able to put a dent in this enormous problem. It's for that reason that I'm very excited about this and just applaud you on your career.

I think this is only the beginning. We have to get this into human clinical trials. That's what's next. Thank you so much for this invitation. It's been really, really fun talking to you. Alzheimer's disease is one of our biggest biomedical challenges. Our entire world is aging. It used to be the US, Japan, Sweden, but it's the entire world. China, India, Africa, all continents are aging.

We're aging rapidly. Again, this is one of our biggest biomedical problems. We really do not have effective therapies. And I'm hopeful that with multiple shots on goal right now, something will come to market that provides that resilience. In addition to anti-amyloid therapies, that is a cocktail that provides resilience for the suffering that really erodes our memory. It destroys families, economies. It's just a devastating problem.

We're glad to be working on it and we hope that we can do something big and important, but that remains to be seen. I also just wanted to say I'm so lucky to be doing what I love to do. Really, really love to do. to be doing what I love to do, really, really love to do. And I am also lucky enough to have a very diverse portfolio of being funded by the NIH, by foundations, and in the past by biotech and also by philanthropy. I can't emphasize enough how much friends and supporters

of our lab have enabled really risky science, have enabled us to just take a leap, ask big questions, take a big risk, and see what happens and win or lose to really see what happens when we go down that route. And I think that's so important to progress in science is to do something risky, to take a risk. So I'm very grateful to be doing what I'm doing. I'm glad you mentioned that. I do think that philanthropy can have an enormous impact in science when philanthropists come in with the right attitude, which is,

I want to fund something that is asymmetric in the following way. It is too risky for, the NIH to fund or too risky for industry to come in and fund, but yet it has enough biologic plausibility and enough potential upside that if it works, it's a game changer. And look, the reality of it is, I think Clotho is a poster child for that type of work. At least as of this moment, it looks like it's actually paying off. I and countless others are grateful for what's been done.

Dina, thank you so much for your time today. Really, really appreciate it. Thank you. This was wonderful. Thanks. Thank you for listening to this week's episode of The Drive. It's extremely important to me to provide all of this content without relying on paid ads.

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