303-A breakthrough in Alzheimer’s disease: potential of klotho for brain health & as a therapeutic

1. 0:00 – 1:19

   Intro

   1. DDDena Dubal0:00

      If we want to jump into clinical trials, and I think this is the time to really, uh, move klotho toward human clinical trials, wouldn't it be amazing if we had a cocktail for Alzheimer’s disease? In addition to, um, lecanemab or donanemab that's removing the amyloid beta from the brain, what about adding something like klotho that can really help shore up the functions of the cells, no matter... Because we know that Alzheimer’s disease is a multi-proteinopathy. It's not just one protein that's, um, that's causing the disease. We need factors in our treatment. We need cocktails that can really resist multiple protein toxicities. So I, I just have this, mm, dream that, um, that, that people might be able someday to benefit from klotho, this, this factor that naturally circulates in our body, that helps with, with longevity, that helps with other organ systems and enhances the brain.

   2. PAPeter Attia1:08

      [upbeat music] Hey everyone, welcome to The Drive Podcast. I'm your host, Peter Attia.

2. 1:19 – 11:00

   Dena’s fascination with aging and how she came to study klotho

   1. PAPeter Attia1:19

      Dena, thanks so much for joining me today. Um, this is a topic I've been very interested in for, um, the better part of about a year and a half to two years. Uh, and obviously there's no better person to discuss this with than you. Um, I also suspect that this is a topic, um, not enough people know about given its potential implications and significance, uh, vis-a-vis Alzheimer’s disease, which we'll, we'll get to. But maybe before we get into this, let's, let's give people a bit of a sense of your background. Um, tell, tell me, tell me a little bit about your clinical work, your, your research work, um, you know, what you did during your PhD and how that carried forward, um, uh, during your tenure.

   2. DDDena Dubal2:03

      Sure. Well, thanks again for the invitation. I'm delighted to join you today. And, um, I, by way of introduction, I'm a neurologist and I'm a neuroscientist, and I direct a group, um, that is deeply involved in the discovery around klotho. And, um, I was thinking back to when, when my interest in aging actually began. What was this journey? And I thought back to, um, my undergraduate days at UC Berkeley when I was, um, a nineteen-year-old that was oddly interested in aging, kind of obsessed with aging. I worked with a medical anthropologist, um, at Berkeley, Lawrence Cohen, on what it meant to experience dementia in different cultures. Um, what was it like in India versus the United States? And then simultaneously, I took a class on the physiology of aging. Um, I remember it so clearly in Dwinelle Hall, and I was at the edge of my seat-

   3. PAPeter Attia3:06

      Hmm

   4. DDDena Dubal3:06

      ... learning about cellular senescence. Um, and I remember thinking, "This is amazing. This is happening to us day by day, aging, um, yet we don't know so much about it. Um, and we don't know much about brain aging per se." And I, I was committed as an undergraduate to really learn more about brain aging and possibly to do something about it. Um, that led me to an MD PhD at the University of Kentucky. Um, I trained with Phyllis Wise, a neuroendocrinologist who studied brain aging, who was an amazing PhD. Learned so much. Fell in love with the discovery process of science. And fast-forward, um, I then trained as a neurologist at UCSF, where I am now, and that was, um, over twenty years ago, two thousand and three. And I'm on the wards. It's a regular day of patient rounding, and the former chair of UCSF, um, neurology, Steven Hauser, turned to me and he said, "Dena, 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, um, 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 klotho, um, the subject of what we'll talk about today, the Greek fate who spins the thread of life. And the idea of studying klotho 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.

   5. PAPeter Attia5:08

      So let's kind of go back to the first time klotho crossed your radar.

   6. DDDena Dubal5:14

      Yes. Well, I was, um, just beginning my junior faculty days and, um, was thinking, what, what were we gonna focus on? You know, what was going to be our chance to do something big and important? And, um, I was very intrigued by, um, by a few decades of work that was emerging that aging itself was malleable. And the, and the first work with this was Cynthia Kenyon in worms, where she demonstrated that, that tweaking, um, tweaking genetics, like aging in worms could dramatically increase lifespan. And I really wanted to know whether that could have effects in the brain. Um, klotho had emerged as a longevity factor. And, um, and it was a chance to understand could klotho do something in the brain. Noth- nothing was known about klotho. Very little was known about klotho when we started. A colleague had, um, had observed that it w- that the levels decreased in the white matter of monkey brains, um, that, um, a v- a variant of klotho that we can talk about in a bit was associated with decreased stroke risk. Um, and the person who discovered klotho had noted that, um, that mice without klotho moved slowly, and they, they were cognitively a little slow. Um, but, you know, I was the, at the, uh, the right person at the right time, um, and, and had the chance to really dig in. It was, um, it was risky, you know, 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.

   7. PAPeter Attia7:03

      Let's talk a little bit about the discovery of klotho. So was it discovered through the mice that were deficient? 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, of one of the IGF genes, if I'm not mistaken. Correct?

   8. DDDena Dubal7:25

      Hmm. Yeah, the DAF-16.

   9. PAPeter Attia7:27

      Yep.

   10. DDDena Dubal7:28

       Um, so how was klotho discovered? It was, um... 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, um, which I believe was a sodium, um, proton, um, channel. So he engineered mice. He inserted this gene, and then he noticed that in a few lines of these mice, um, maybe it was just one line of the mou- of these mice, that there was this premature aging phenotype. So the mice lived to, um, about three months instead of about 30 months, and they developed normally. But around two weeks of age, they became, um, sort of progeroid. They rapid- They became-- They looked like they were rapidly aging, with osteoporosis, atherosclerosis, um, emphysema. Um, 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'd accidentally, um, caused a mutation by, by inserting his, um, sodium proton, um, pump. So, so he went back. He mapped what had been dis- disrupted, and, um, and that was klotho. Um, that was the first time klotho was found, and he named it after the Greek fate who spins the thread of life, daughter of Zeus. And, um, Clotho, the Greek fate, is C-L-O-T-H-O, um, but he named it K-L-O-T-H-O, um, in homage, um, to his discovery. His name is Kuro-o, K-U-R-O-O. And, um, and, uh, a- and a longevity factor isn't something that just causes premature aging if disrupted. So it was very important for him to then, excuse me, to go back and see what would happen if he overexpressed it.

   11. PAPeter Attia9:45

       Hmm.

   12. DDDena Dubal9:45

       So he made-- Then he engineered mice to overexpress klotho, and those mice lived 30% longer. So that was a longevity factor, that disruption caused premature aging, and overexpression extended lifespan.

   13. PAPeter Attia10:02

       Y- yeah, no, that's-- I mean, it's such a beautiful story. Um, I, I think, [laughs] uh, you know, it's a great story for people, especially maybe who don't, don't do or haven't done science, because, um, it illustrates the role of curiosity and serendipity. And, you know, I think, 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, uh, proton pump exchanger, uh, you know, seemed interesting at the time, and that what he observed was, you know, actually kind of a disappointment in that it, you know, ruined one of his lines of mice. But obviously, it led to the far more relevant finding over the long arc of time.

   14. DDDena Dubal10:48

       He opened the field, you know? He followed the science. He opened the field. He looked into the mistake, um, the oops of science, and, and here we are, you know, maybe on the cusp of a new therapy.

3. 11:00 – 23:08

   Biological properties of klotho: production, regulation, decline with age, and factors influencing its levels

   1. PAPeter Attia11:00

      Yeah, exactly. So let's talk a little bit about what this gene does. How big is this gene? Uh, uh, you know, how preserved is this gene over other species? Um, and tell us a little bit about the protein that it codes for. What regulates it?

   2. DDDena Dubal11:15

      Sure. So klotho itself is a pretty big protein. Um, it's about 1,000 amino acids and, um, by weight, maybe 130 kilodaltons if you look at it on a Western blot. And it codes for, for the aficionados, a type I, um, transmembrane protein, which means that its N-terminus, um, will sit in the extracellular space. It has one pass through the membrane, and its C-terminus is inside the cell. Now, um, its N-term, it, the part that sits outside the cell, has two repeat domains, KL1 and KL2. Um, and those domains have high homology to, um, proteins that are found throughout mammalian, um, worm and fly biology. So they're homologs of klotho, um, throughout species and, um, and pretty conserved in mammals. So, um, so klotho is a big protein. It's a transmembrane protein. And then what happens is there are a few forms of klotho. It, 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, um, base ADAM10, ADAM17, and they clip that extracellular portion of klotho and release it into the blood. Or in the case of the brain, release it into the CSF. And that form of klotho is kn- known as a soluble form of klotho or the secreted form of klotho. I call it a hormonal form of klotho, the klotho hormone, because it's released at one site and then can act at different sites at multiple organs. Um, so that's how it's made. Those are the major forms.

   3. PAPeter Attia13:18

      So how big is that N-terminus piece that gets clipped, that now goes and acts like a hormone basically?

   4. DDDena Dubal13:24

      It's the, it's the majority of the protein itself. Um, the p- the part that's inside the cell and crosses the membrane is very small.

   5. PAPeter Attia13:33

      Okay.

   6. DDDena Dubal13:33

      So it's really the majority of the protein is, is the hormone itself.

   7. PAPeter Attia13:38

      If we were to, you know, take a blood test and measure a person's klotho levels-

   8. DDDena Dubal13:45

      Mm-hmm. Oh

   9. PAPeter Attia13:45

      ... um, what could we observe about it across people of the same age, uh, 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, uh, efficacy or, um, are there other factors that lead to consumption of the protein or degradation of the protein in the periphery?

   10. DDDena Dubal14:15

       Yeah, great questions. So starting with levels of klotho across the lifespan. Um, well, first, um, first, it, klotho is, um, it circulates in us. It circulates in mice. And, um, 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. And so we have circulating levels of klotho. We're born with about six times the levels that we have now, um, in our cord blood. And then throughout our lifespan, klotho levels decline. And into, in our, um, from, let's say 40 onwards, they can decrease by half during aging. So, um, we're born with very high levels, and they decrease across our lifespan. They also have a circadian rhythm, um, or a diurnal rhythm in that when we wake up, we have high levels of klotho. And by 4:00 PM, 3:00 PM, when people are looking to grab a cup of coffee, the klotho levels are really starting to decline, and then they nadir by around midnight. And that can be, it can be a 40% or so decrease. So there's a circadian rhythm to klotho. It changes across the lifespan.

   11. PAPeter Attia15:38

       Sorry, Dena, just to make sure I understand that.

   12. DDDena Dubal15:40

       Yeah.

   13. PAPeter Attia15:41

       What explains the daily variation? Is it some sort of, quote-unquote, "consumption"?

   14. DDDena Dubal15:46

       Yeah.

   15. PAPeter Attia15:46

       Or is it being turned over that quickly and the supply of klotho is decreasing by the time of day?

   16. DDDena Dubal15:54

       Yeah. I think that's a great question. Um, I-- the answer to that is not known.

   17. PAPeter Attia16:00

       Mm.

   18. DDDena Dubal16:00

       Um, what regulates the daily levels? Is it, how is it being degraded? Um, you know, is it, is the expression being increased? Um, the half-life is, is short in mice. Um, and in, um, in mice it's maybe seven to ten minutes. But in humans-- Well, I would say in monkeys, it's much longer. Um, it's at least a day or so. So, um, it's unlikely that the production is really actually increasing and decreasing because when it's made, its half-life is, will be longer in non-human primates like us. Um, so I would speculate, though I don't know, Peter, um, that, um, that it may be sequestered in organs or degraded or... Not sure. Um, I've, I've seen a couple of reports of this diurnal variation and, um, 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 has been, um, it's been observed, and this was work by, um, Fabrizia and, and Ambrosio's lab, that there is a hypermethylation around the klotho promoter that occurs with aging that stops its transcription and essentially transcription and translation. Um, and so that's an interesting thing to think about if we wanted to, um, if we wanted to preserve our klotho levels. How can we interfere with that methylation that happens with aging?

   19. PAPeter Attia17:45

       That's interesting. So more methylation there leads to inhibition of the promoter. Um, yeah. Y- and a lot of times you'll see the opposite, right? It's the loss of methylation that shuts off the promoter, correct?

   20. DDDena Dubal17:59

       I don't know. I, I don't know about that. In general, um, for klotho at least, the methylation really, really turns things off.

   21. PAPeter Attia18:08

       Off. Okay.

   22. DDDena Dubal18:10

       I could, I could be wrong about that, but I think that that's, that's definitely the case, um, in, with klotho. I think that's definitely the case. I-

   23. PAPeter Attia18:19

       And is that believed to be the main mechanism driving the reduced production of klotho, is the increase in the methylation of the promoter?

   24. DDDena Dubal18:29

       That's what's known so far.

   25. PAPeter Attia18:31

       Mm.

   26. DDDena Dubal18:32

       Um, there are other possibilities, like, um, maybe organs make it less. Maybe the kidney is less efficient at making it with aging. That's possible. Um, but I think that this, I think that, um, that Ambrosio's lab is really onto something. They showed it in chondrocytes, in aging chondrocytes, but this is really the first kind of molecular demonstration about why klotho-

   27. PAPeter Attia18:55

       Mm

   28. DDDena Dubal18:55

       ... may be decreasing. Now, there are other things, Peter, that decrease klotho, and, um, one big one is, um, is chronic stress.

   29. PAPeter Attia19:06

       Mm.

   30. DDDena Dubal19:07

       Um, we did a study with, um, Elissa Epel and Eric Prather here at UCSF in mothers, um, with neurodevelopmentally, um, typical or atypical children. And, um, the mothers with high levels of stress with, um, children with autism spectrum disorder, um, had much lower levels of klotho, as well as shorter telomeres. And, um, their level of stress, with increasing stress, there was a decrease in the klotho level.
4. 23:08 – 36:18

   Potential benefits of klotho on brain health

   1. DDDena Dubal23:08

      Um, BDNF has a very striking effect in the brain.

   2. PAPeter Attia23:13

      May- maybe, Dena, just tell folk, tell folks a bit about BDNF because I, sometimes I forget that maybe not everybody listening to us today has also listened to all of the other content over the years. Uh, so maybe just-

   3. DDDena Dubal23:24

      Sure

   4. PAPeter Attia23:24

      ... give folks a little bit of a primer on BDNF as well.

   5. DDDena Dubal23:27

      Sure. So BDNF is brain-derived neurotrophic factor. And, um, and it has been shown in the brain to really be associated with even drive, um, like, positive brain health. So, um, uh, i- intermittent fasting increases BDNF in the brain. Exercise increases BDNF. BDNF, if you give BDNF to the brain, uh, the neurons function better. Um, it's a, it's a really good trophic factor for the brain. And, um, and it does remind one of, of klotho and effects of klotho. And in fact, intermittent fasting simultaneously increases klotho, um, in the brains of mice, along with, uh, with BDNF.

   6. PAPeter Attia24:12

      How long do the mice need to fast to get that effect?

   7. DDDena Dubal24:15

      So this work was done, um, at the Salk and, um, the mice fasted for 24 hours.

   8. PAPeter Attia24:23

      Wow.

   9. DDDena Dubal24:23

      Uh, yeah.

   10. PAPeter Attia24:24

       So, so not really something we can translate to humans given that that's... I mean, 24 hours in a mouse is, you know, that's half the distance to death. So-

   11. DDDena Dubal24:34

       [laughs]

   12. PAPeter Attia24:35

       ... that's, uh, you know, it, it- no, but this is a real problem.

   13. DDDena Dubal24:39

       Interesting.

   14. PAPeter Attia24:39

       This is a real problem with extrapolating from mice data, right? Is ev- even a 12-hour fast in a mouse is a, is an overwhelming feat of calorie deprivation. Uh, 20, 24 hours is staggering. I mean, that's... I don't, we don't have a way to translate that to humans, but that, that, it could be that that's on the order of weeks. Um, so I just wanna make sure people aren't listening to this thinking, "Hey, all I gotta do is skip breakfast and I'm getting the same amount of BDNF or klotho that I would get from-

   15. DDDena Dubal25:11

       Mm-hmm

   16. PAPeter Attia25:11

       ... a 45-minute workout." My guess is those two are not even in the same zip code, right?

   17. DDDena Dubal25:17

       Yeah. Well, I, I mean, so I, I would think, I think differently about it. So when I think about, um, mouse time and mouse lifespan, I think, I believe that they are... So they live to two to three years. Um, but I believe that they're aging faster, that they just have, they have a shorter lifespan.

   18. PAPeter Attia25:36

       Sure.

   19. DDDena Dubal25:37

       But, but, um, but I think it's possible that their 24-hour lives are similar to our 24-hour lives.

   20. PAPeter Attia25:46

       But they'll usually, but th- but, you know, tw- 48 hours of, of fasting will usually kill a mouse, right?

   21. DDDena Dubal25:52

       Would 48 hour... I don't know how long a mouse can go.

   22. PAPeter Attia25:56

       I u- I used to be more facile with this literature, but I pay less and less attention to mouse literature now than I used to. But I, I, and, and maybe, you know, obviously someone listening to this could, could, could, could write in and, and correct me, but, but I don't think it's a stretch to say that 48 hours... Put, put it this way, what I do remember is, um, an IRB would, would really scrutinize an investigator who's trying to get to 36 hours of, of fasting in a mouse. It's a really big deal.

   23. DDDena Dubal26:28

       You know, we don't do fasting studies in mice, but, but I would agree that in general, it, you know, we have to be careful when extrapolating from, from mice to humans. I do think that in the area of cognition and neurodegenerative diseases, that, um, that s- that studying mice and mouse brains and their neural circuits and their hippocampi and their prefrontal cortices tell us fundamental things-

   24. PAPeter Attia26:55

       Yeah

   25. DDDena Dubal26:55

       ... about the human condition. Um, they, uh, particularly because they have to have very strong navigation strategies and memories for foraging, for coming back to their nests, and these memory circuits are very, very fundamental and, and, and quite preserved, um, up, up to non-human primates, including us. But having said that, um, and we do the majority of our work in mice, [laughs] and, um, we are always aware of, you know, are we doing something that is important to the human condition? Is it relatable to the human condition? Is it relevant? Um, who cares? Does, will any of this matter? These are always on my mind, these questions, particularly as a physician scientist. And there are certainly limitations, but I also think that there are many strengths, particularly in studying, uh, memory and neural circuits in the brain. But we don't stop there and, um, we have tested whether... I haven't talked yet about, um, what we have found in mice, but I'll, I'll just spill the beans and say that we found early on that klotho enhances cognition in mouse brains. And, um, and in a very recent study, we, um, collaborated with Biotech and Yale University and found that actually klotho enhances, uh, learning and memory and cognition in old monkeys.

   26. PAPeter Attia28:25

       Mm-hmm.

   27. DDDena Dubal28:25

       Um, in a very, um, complex brain, genetically diverse, anatomically diverse, functionally complex, that in a brain like ours, that klotho had very similar effects to what we see in mice.

   28. PAPeter Attia28:39

       Well, I, I, 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. Um, but let's go back and, and kind of use some of the mice data to, you know, allow us to speak about, um, maybe greater insights around the, the, the causal relationship between klotho and brain health. So we've already established a, a few facts here. So you're going back to the discovery of klotho in '97, which was based on effectively a knockout. Um, we saw that that, that is incompatible with a long life. Um, and we saw that the reverse when, uh, kl- klotho was enhanced, I believe you said it enhanced lifespan by about 30%. Uh, again, that's really remarkable in a mouse. Um, there aren't many things that enhance a mouse lifespan by that much. That's, you know... But again, that's, that's, that's up there with the most draconian caloric restriction, uh, rapamycin administration under certain settings. So that's, that's a very reproducible and robust finding. Um, l- let's talk a little bit about, um, specifics to brain health, kind of what you're talking about with respect to, um, not a disease state per se, but just enhancing cognition in an aging mouse, uh, or even in a, in a middle-aged mouse.

   29. DDDena Dubal30:12

       This is where we started, and we started with the hypothesis that- That in these mice that overexpress klotho 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 klotho overexpressing models to create mice who have, um, mouse-heimers and then have mouse-heimers plus lots of klotho. And then we had normal mice and normal mice with lots of klotho. So there were four experimental groups. And to our complete surprise, the normal groups, that means the m- m- we're not talking about the mice that had, um, Alzheimer's, modeled Alzheimer's. But in the normal groups that had higher levels of klotho versus normal levels of... That group showed a remarkable difference, a remarkable statistically significant difference across multiple cognitive tasks, um, and across different ages. And that was that the mice that overexpressed klotho that lived 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, um, and better. Um, and it was a-- That was a complete surprise. I remember the day, unblinding the data, analyzing it. It was, it was unimaginable.

   30. PAPeter Attia31:54

       C- Can, can I add a point here? Uh, Dena, I just wanna add a point. Uh, uh, sor- sorry to interrupt, but it's so powerful. You a m- 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, um, 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, Dena, stating that it is statistically significant, which it is, almost undersells what I think the magnitude of the differences are. I have a friend, um, 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. You know, 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. But 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, I think I'd be safe in saying that that was the case here, wasn't it?
5. 36:18 – 52:31

   The relationship between soluble klotho protein, platelet factors, and cognitive enhancement

   1. PAPeter Attia36:18

      up rapamycin again, which people hear me talk about a lot, but people say, "Why are you so confident in rapamycin's gero-protective effects?" The magnitude of benefit is enormous.

   2. DDDena Dubal36:29

      Mm-hmm.

   3. PAPeter Attia36:29

      And it doesn't seem to matter who does the study, right?

   4. DDDena Dubal36:32

      Mm-hmm.

   5. PAPeter Attia36:32

      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.

   6. DDDena Dubal36:40

      Mm-hmm.

   7. PAPeter Attia36:40

      Now, that doesn't guarantee it's gonna 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, [laughs] and 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-

   8. DDDena Dubal37:06

      Mm-hmm

   9. PAPeter Attia37:06

      ... let's talk about mechanism of action.

   10. DDDena Dubal37:09

       Hmm.

   11. PAPeter Attia37:09

       Tell me what you think is happening with this soluble klotho protein. Um, I assume it's crossing the blood-brain barrier, or it wouldn't be having this effect. Uh, has that been easily demonstrated?

   12. DDDena Dubal37:23

       It's, um, it is a winding story, Peter. [laughs]

   13. PAPeter Attia37:28

       [laughs]

   14. DDDena Dubal37:29

       And, um, and one that's exciting and has led us to very, um, unanticipated findings. I first wanted to mention, as we think about how different interventions may converge, um, upon the same biology or relate to the same biology, it is interesting that mice treated with rapamycin have increased klotho levels. Um, so rapamycin either directly or indirectly will increase klotho levels, just thinking about the convergence of biology. Now, um, how does klotho enhance cognition in a young, aging, and diseased brain? And, um, that immediately became, um, a major question for my lab and many other labs. And, um, the very first thing that we had turned to was, um, was a component of NMDA receptors. So NMDA receptors, um, are really key in connections between neurons. And when they're stimulated, they let in calcium into a neuron, and they, um, allow essentially a potentiation of a neuron for connections to form, for functional connections to form, and really for, like, the, the neural substrate of memory to form. So NMDA receptors are really key. And there's a component of NMDA receptors called GluN2B. It's a subunit. Um, I, I'm really, um, I'm really going tiny and molecular here, but it's re- it's very important because I had remembered that there was something called a Doogie Howser mouse, kind of affectionately called a Doogie Howser mouse. And that, in that mouse-

   15. PAPeter Attia39:15

       We're, we're kinda dating ourselves here 'cause there's gonna be a lot of listeners-

   16. DDDena Dubal39:18

       [laughs]

   17. PAPeter Attia39:19

       ... who don't know who Doogie Howser is. But suffice it to say, this was a prodigious mouse.

   18. DDDena Dubal39:25

       Yes. So it was a mouse that was smarter. So very, there are very few model systems where you can actually, you can, um, really pivot the system to make a, engineer a mouse to be smarter. There are very, very few things. Klotho's one of them. So this GluN2B overexpressing mouse or the Doogie Howser mouse, um, showed a very similar phenotype to the klotho overexpressing mouse. So when I looked at the behavioral data of the GluN2B overexpressing mouse, um, in the very same test, it was performing very well, just like the klotho overexpressing mice. So I just wondered whether klotho was acting through GluN2B to enhance cognition. That was the hypothesis. In this case, um, the hypothesis turn-- We, we gathered data that supported the hypothesis. There wasn't a big surprise in that overexpressing klotho caused a, it caused an increase in GluN2B at the synapse, at the area where brain cells connect, and led to a more efficient, um, connection and memory formation, synaptic plasticity, um, at the level of the neural cell. So this GluN2B was really important. When we blocked GluN2B with multiple pharmacologic inhibitors, we, um, abrogated klotho's ability to enhance cognition. So that was one clue. But here was, here was the, um, here is the major head scratcher. So Peter, klotho is ex- expressed in the kidney. It's also expressed in the brain, um, secreted through the choroid plexus, which also makes the fluid for the brain that the, the brain sits in. But, but klotho does not cross the blood-brain barrier. Um, we have looked, others have looked, um, through autoradiography, through IP and Western blot. We've looked through many immunohistochemistry. We looked through many different methods and, and, um, and can see very clearly that it doesn't cross into the brain. However, when we give a shot of klotho to mice, to monkeys, um, in their arm or their belly, doesn't matter. If we give them a shot of klotho, 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 klotho is enhancing brain functions if it's not actually crossing into the brain? That's been a major question, um, that we and others have been working on. And, um, and we have some clues, um, that led us in unexpected places. Um- I'm happy to talk about one of them if this is a good-

   19. PAPeter Attia42:35

       I, I am, I am all ears.

   20. DDDena Dubal42:38

       [laughs]

   21. PAPeter Attia42:38

       To me, this is-- I mean, I didn't wanna get to this point yet, but w- I think we're-- this is the natural time to talk about this, and then I think we should, you know, step back and talk about some other things. But yes, to me, this is the jugular question, Dena. Why is it that when we inject a monkey s- peripherally with klotho, it spends the next fourteen to nineteen days in a state of cognitive superiority, and that klotho isn't crossing the blood-brain barrier? This is, to me, the jugular question.

   22. DDDena Dubal43:16

       You're right. And, um, and we have been working on this. Um, I think, I think I'll step back and say, um, it's actually amazing, right? That some- that you give something peripherally, it has a central action, it's not crossing into the brain, um, and it's something physiologic that our bodies are used to, that we have seen high levels of upon birth and development. Um, it's not a small-- It's, it, it does not have known side effects at physiologic levels that have been observed yet. Um, 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, of fervent investigation for us, um, and others. Um, but we have one clue, um, in a study that we published recently, um, in Nature Aging. And this is what we did. We said-- We started with the premise that, that maybe klotho was doing something in the blood that then would send a messenger into the brain. So maybe there was this m- messenger of klotho that was actually going into the brain when you, when you gave it peripherally. And so we did a very simple experiment. Um, we injected mice with klotho, 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, um, what we found was, was, um, was sort of unimaginable. [laughs] It was unimaginable in that we found that all of these platelet factors were increased with klotho injection in the blood. And Peter, I have to be honest, when I saw this, um, agnostic data set, I, um, turned to my postdoc and I said, "I'm really not interested in this. [laughs] I am not interested in looking at platelets." You know, they are involved in wound healing. They're coagulation factors. I just like, "This is weird." Um, I had a postdoc, uh, Cana Park, who was very persistent and said, "Let me try. Like, let me just study this for a little bit." I said, "Go for it." I could see her passion.

   23. PAPeter Attia45:52

       Sorry, one, one question, Dena, 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?

   24. DDDena Dubal46:11

       Yes, we did, and it's a great question. We haven't published this yet, um, but we did. In the same mice, we took their blood, and we took their CSF. Um-

   25. PAPeter Attia46:22

       Okay

   26. DDDena Dubal46:22

       ... and, um, and what we found is that klotho increased these platelet factors. It was, um, totally bizarre. Um, I didn't believe it. I tend not to believe data. I, like, 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, um, many different ways. She took, um-- She did, she repeated the finding in many different ways. But ultimately, what she did was a series of studies, um, demonstrating that this biology is real, that klotho is actually activating platelets very modestly and relea-- And then when a platelet is activated, so let's say, let's first just step back and, and what is a platelet? A platelet is an anuclear cell in our blood, um, and it contains little compartments of bioactive chemicals, um, 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, um, 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, 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, who would have thought that platelets could be messengers of brain health-

   27. PAPeter Attia48:07

       Mm-hmm

   28. DDDena Dubal48:07

       ... and could take center stage as, 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 4, is released and travels to the brain and actually, um, 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 klotho platelet connection. Maybe there's something to it. So Kana isolated platelets. She put klotho 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 4 to mice, young aging mice. She gave it to them as a shot in the belly, just like we us- we have given klotho, and she found that that platelet factor 4 enhanced cognition in a young mouse and in an aging mouse, and it reversed cognitive deficits, and it was totally remarkable. And, um, this is where it gets weirder and wilder. So I go to my colleague and close friend, Saul Villeda, here at UCSF. He's the, um, young blood and old brain, um, scientist, so he's, he's really built a, 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 and I'm-- 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. Klotho stimulates platelets. Platelets release PF4. PF4 enhances the brain. And he said-- Well, he was silent for a second, and he said, "Dena, we found the same thing with young blood, in that when we give young blood to old mice, that young blood is enriched for platelet factor 4-

   29. PAPeter Attia50:27

       Mm

   30. DDDena Dubal50:27

       ... which declines in aging, and then we give platelet factor 4, we rejuvenate the old brain."
6. 52:31 – 1:02:52

   The role of platelet factor 4 (PF4) and it’s interaction with GluN2B in mediating cognitive enhancement

   1. PAPeter Attia52:31

      So, so let's talk a little bit more about this. So, um, platelet factor 4 is increased by all of these three things. Do we believe that it's platelet factor 4 that is directly acting on GluN2B, then?

   2. DDDena Dubal52:47

      Mm.

   3. PAPeter Attia52:48

      Is that the actual... And, and, and if so, what do you think is happening at the, at the level of s- substrate and receptor?

   4. DDDena Dubal52:57

      Yes. It-- That's where we went next with the biology and the experiments. And when we-- So one question was: Does platelet factor 4 actually cross into the brain? And we demonstrated that it does. We gave it peripherally. We, um, immediately looked into the brain and saw that it was ending up near neural cells or even within neural cells. Um, so it's crossing into the brain. And then does it act directly into, in the brain was the next question. And what, um, one of my lab members did is take hippocampal slices. So the hippocampus is the area of the brain, um, 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 4 on it and saw immediately, within seconds actually, there was a-- Well, I don't know within seconds, but, but quite immediately, maybe seconds, maybe minutes, um, there was a change in the membrane potential, and more calcium was being let in.

   5. PAPeter Attia54:16

      Mm.

   6. DDDena Dubal54:16

      And we thought maybe this would be GluN2B, um, acting, and so we put on GluN2B inhibitors and platelet factor 4 no longer potentiated the synapse or enhanced the neural function. Um, so we, we do know that pla- platelet factor 4, like klotho, is working through GluN2B, and maybe klotho is working through platelet factor 4 To change GluN2B

   7. PAPeter Attia54:47

      And sorry, d- has that same experiment been done, Dena, to look at the klotho that is derived from the choroid plexus to say when you dump klotho directly onto GluN2B you see an influx of calcium. When you block GluN2B, you abrogate the effect as well. So you now have two things, platelet factor 4 and klotho, that can independently do the same thing.

   8. DDDena Dubal55:12

      Mm-hmm. Mm-hmm.

   9. PAPeter Attia55:12

      Is that, is that a correct statement?

   10. DDDena Dubal55:14

       Well, it's, you know, it's a... We haven't done that experiment yet. So all of our work on klotho has been through peripheral... Well, that's not true. Um, we've done transgenic overexpression in the brain, um, and we've done klotho peripherally. We haven't dumped klotho itself onto the hippocampus-

   11. PAPeter Attia55:37

       I see

   12. DDDena Dubal55:37

       ... to see whether there can be a direct effect. Um, other groups have overexpressed klotho specifically in the brain and seen cognitive enhancement. But I haven't seen studies seeing whether klotho is actually directly influencing GluN2B. We know that it indirectly influences GluN2B. We know that we can give it peripherally-

   13. PAPeter Attia56:02

       Yes

   14. DDDena Dubal56:02

       ... and it- 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, um, based on other people's studies, it may. We don't know. But here's, here's something else that was actually quite disappointing. Um, but the biology is the biology. We then asked the question, does klotho rely on PF4 to enhance cognition? Is it, is PF4 required for klotho-mediated cognitive enhancement? And to do that, we generated a colony of mice with PF4 knockout, so they, they just don't have PF4. And then we gave them klotho versus vehicle. Always blinded, always randomized, um, through a series of cognitive tests. Water maze, large Y maze, one maze after another, 'cause you like to see the same effect reproduced. And what we found, um, was that klotho continued to enhance cognition even in the absence of platelet factor 4. And, um-

   15. PAPeter Attia57:15

       And to the same extent, Dena?

   16. DDDena Dubal57:17

       To the same extent. You know, you might think there would've been a diminution, but to the same extent. And so I-

   17. PAPeter Attia57:23

       So it just, it just tells us that there's more than one factor. This is, it- it has to be-

   18. DDDena Dubal57:28

       There is

   19. PAPeter Attia57:28

       ... 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 m- I'm making all this up, of course, but hypothesizing that, you know, you know, if klotho is so important, it can't be limited to one messenger, right? It sort of, 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?

   20. DDDena Dubal58:00

       Right. That's, that's the interpretation and conclusion because it, um, it is, PF4 is sufficient to replicate-

   21. PAPeter Attia58:09

       But not necessary

   22. DDDena Dubal58:10

       ... klotho-mediated cognitive enhancement, but not necessary.

   23. PAPeter Attia58:14

       And so 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? Did you, did, how broadly did you sample the serum, uh, way back, and, 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?

   24. DDDena Dubal58:37

       Yeah. There, there were many signals.

   25. PAPeter Attia58:39

       Mm.

   26. DDDena Dubal58:39

       There were many other platelet factors. There were many other proteins. Um, we are systematically marching through those. Um, we're marching through not just proteins, but metabolites. Um, we are also asking in a cell type specific way, is, um, through a system called Turbo ID that labels the protein as it's secreted from the liver, the kidney, the heart, from lymphocytes. Um, it's a really unique and elegant, um, genetic manipulation that does a biotin label once a, a protein is secreted, let's say from the liver. And we're asking when we inject klotho, what gets secreted out of the liver and into the plasma. So we're doing like higher resolution studies-

   27. PAPeter Attia59:29

       Yeah

   28. DDDena Dubal59:29

       ... to understand how klotho, um, 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 klotho-mediated cognitive enhancement. And the answer at the end of the day may, 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. Like, if this is a, if this is a very important biologic function, it's not, it, it really shouldn't rely on just one factor. I think that's a, a really important point to underline.

   29. PAPeter Attia1:00:21

       So, so Dena, uh, tell me a little bit more about this assay. The, um, in vitro assay that you were able to basically sprinkle on PF4, sprinkle on klotho to the GluN2B, um, subparticle and witness the calcium, the massive influx of calcium, right? Um, it seems to me that if that assay is, 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 klotho is working through secondary mechanisms, you'll miss it, right? But at least as a first screen, hey, you know, in as much as klotho's working through NMDA via, you know, GluN2B, 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 y- y- you know, triangulate back and forth like that, right?

   30. DDDena Dubal1:01:23

       I love it. Let's do it. [laughs] And we are doing it, and, um, we're doing it with s- synaptic plasticity as a measure, as a substrate of cognition. We're also doing it in vitro at the cellular level using live cell imaging where we can, um, isolate neurons from the brain, from a mouse brain, and then they grow beautifully on a dish.
7. 1:02:52 – 1:11:22

   Benefits of klotho observed in a mouse model of Parkinson’s disease

   1. PAPeter Attia1:02:52

      so all of this makes a lot of sense for a, a mouse model of Alzheimer's disease, where the primary deficit is a cognitive deficit that seems disproportionately focused in memory. Um, 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.

   2. DDDena Dubal1:03:15

      Mm-hmm.

   3. PAPeter Attia1:03:15

      Now, are you seeing them in the mild cognitive impairment that it can accompany Parkinson's disease, or are you seeing it in the movement, uh, disorder?

   4. DDDena Dubal1:03:24

      Mm.

   5. PAPeter Attia1:03:24

      Are you seeing an improvement in the, in the primary issue associated with Parkinson's disease?

   6. DDDena Dubal1:03:30

      It's a great question. And again, this, part of this is published, part of this is in the peer review process. Um, 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. Um, it dis- it dis- disrupts the synapse and, and, and, um, has a causative role in Parkinson's disease because we know that people with mutations in causing overexpression of alpha-synuclein can, will develop Parkinson's disease.

   7. PAPeter Attia1:04:10

      It's sort of like the equivalent of APP in-

   8. DDDena Dubal1:04:13

      Yes

   9. PAPeter Attia1:04:13

      ... people with Alzheimer's disease-

   10. DDDena Dubal1:04:15

       Yes

   11. PAPeter Attia1:04:15

       ... where you, yeah, you have this r- you know, it's not necessarily the dominant driver of the disease, but it's, it's clearly playing a causal role based on these mutation studies.

   12. DDDena Dubal1:04:25

       That's right. And then the mice that I've mentioned that we've used for our Alzheimer's, our M- Mouseheimer's studies, were APP mutant overexpressers.

   13. PAPeter Attia1:04:35

       Okay.

   14. DDDena Dubal1:04:35

       Just to, just to pedal back to that. So we took these Parkinson's model mice, and they have both motor deficits, uh, meaning that, um, you put them acro- uh, they walk across a balance beam and they'll fall. Um, if they're put on a ro- on a spinning rod, they're discoordinated. They have-- and they'll fall. They have motor difficulty. They also have cognitive difficulties, um, which are not as severe-

   15. PAPeter Attia1:05:05

       Right

   16. DDDena Dubal1:05:05

       ... as the Alzheimer's model mice, but they do have cognitive difficulties in their ability to, um, to map a spatial environment and to hold memory in their mind with working memory. So they have these deficits. We, um, we did two things. We injected them with klotho, and remarkably saw that klotho treatment improved their cognition. It, um, it didn't normalize, but it, it improved by, 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 klotho did not help that. That was also true with the transgenic overexpression of klotho all over the body and brain for a lifetime, that that im- that thwarted the cognitive deficits induced by Parkinson's toxicity, but it did nothing for the motor problems. And I've spoken to my, um, Parkinson's colleagues about this. Um, there's a lot of interest in potentially, uh, using klotho as a treatment for Parkinson's disease because, you know, they tell me, 'In our clinics, Dena, we, you know, we can treat the tremor, um, and the rigidity, but people complain over and over again consistently about the cognitive deficits that we now know are a part of, of Parkinson's disease, not just later in the disease, but even as part of the disease.' And those deficits specifically are, um, problems with executive function, um- Which is the ability to, um, to focus, to shift attention, um, to make certain judgments, to think quickly. Um, and it is, it is, uh, mediated by the prefrontal cortex, um, an area that became important when we looked at human studies. So the bottom line was that klotho en- in mice, klotho really enhanced, again, cognition, but didn't do anything for motor functions.

   17. PAPeter Attia1:07:35

       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-

   18. DDDena Dubal1:07:45

       Hmm

   19. PAPeter Attia1:07:45

       ... some of that alpha-synuclein pathology, uh, but also has a much more significant cognitive component?

   20. DDDena Dubal1:07:53

       Um, there are so many mouse models, Peter. I, I think that, that, um, a good mouse model for Lewy body would be, um, alpha-synuclein and, and maybe more in the brain stem, um, and the hippocampus and cortex. I just, I, I, I, I bet there is one, but I'm not-

   21. PAPeter Attia1:08:13

       Mm-hmm

   22. DDDena Dubal1:08:13

       ... I'm not, I'm not clear.

   23. PAPeter Attia1:08:16

       But a reasonable hypothesis would be, again, that it would probably provide some relief of the cognitive symptoms, though not necessarily the movement symptoms.

   24. DDDena Dubal1:08:28

       That's right. So that's what we're seeing consistently, that, that, uh, one of my friends and colleagues had a, an interesting analogy of thinking about klotho as a helmet for, for neurons, that, um, that whatever came crashing the cell's way, whether it was alpha-synuclein, tau, amyloid beta, aging stresses, that, um, the neuron was protected. It was re- it remained resilient against multiple toxicities. And the effect of klotho is, is really in cognition itself, um, in hippocampal and frontal cortical circuits that, um, these neurons and glia and other cell types are really protected against multiple toxicities. And if we wanna jump into clinical trials, and I think this is the time to really, uh, move klotho toward human clinical trials, wouldn't it be amazing if we had a cocktail for Alzheimer's disease in addition to, um, lecanemab or donanemab that's removing the amyloid beta from the brain? What about adding something like klotho that can really help shore up the functions of the cells? No matter, because we know that Alzheimer's disease is a multi-proteinopathy. It's not just one protein that's, um, that's causing the disease. We need factors in our treatment. We need cocktails that can really resist multiple protein toxicities. So I, I just have this, mm, dream that, um, that, that people might be able someday to benefit from klotho, this, this factor that naturally circulates in our body, that helps with, with longevity, that helps with other s- organ systems, and enhances the brain, as we know from our monkey studies, for weeks at a time, um, at least a couple of weeks if not more. Um, 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, um, to really stave off multiple toxicities. And, and this is klotho. This is what klotho does. This is what that, this is the new job of the Greek fate [laughs] that spins the thread of life. It's to protect our brains.

   25. PAPeter Attia1:10:59

       Yeah. Well, you're, you're preaching to the, uh, not just the choir, but the fully converted, and, and obviously in, in, in, in my, in my other activities, uh, this is, this is something that, that, uh, a lot of my energy goes into. Um, I wanna come back to a couple other questions on the mouse study that are gonna factor into, I think, when we talk, start to talk about the humans more.

8. 1:11:22 – 1:17:18

   Benefits of klotho observed in a mouse model of Alzheimer’s disease

   1. PAPeter Attia1:11:22

      So, uh, has the following experiment been done, where you, you take a mouse that is genetically susceptible to Alzheimer's disease, so the equivalent of your APP mouse or something like that? Um, and you take a control group. Uh, so, so maybe just for my own knowledge, if, if you took an APP mouse, um, at what it point in its life, uh, how many months old before it starts to develop, uh, a clinical disease? Uh, so, so maybe two questions. At what point would it start to be accumulating amyloid in the CSF, at how many months of age? And then how many aids when it, age when it starts to show cognitive impairment? What are those two numbers roughly?

   2. DDDena Dubal1:12:05

      It depends on the mouse model.

   3. PAPeter Attia1:12:06

      Okay.

   4. DDDena Dubal1:12:06

      And there are so many mouse models of Alzheimer's and including APP models.

   5. PAPeter Attia1:12:13

      Okay.

   6. DDDena Dubal1:12:13

      The one that we've used, um, consistently is, is called a J20 model. It's, um, it expresses human APP, which produces amyloid beta in mutant forms. And it is, um, it's a more aggressive model, Peter, and it causes synaptic loss, that connection between cells. It, um, really disrupts that connection between cells really early, like before three months, and then it starts producing cognitive deficits, um, at around three to four months.

   7. PAPeter Attia1:12:50

      And that's the J10?

   8. DDDena Dubal1:12:52

      The J20 model.

   9. PAPeter Attia1:12:53

      J20.

   10. DDDena Dubal1:12:54

       It is.

   11. PAPeter Attia1:12:54

       And, um, okay, so here's my question, Dena. Has the experiment been done where you give those mice klotho starting at birth at a high dose, and does it delay the onset of the inevitability?

   12. DDDena Dubal1:13:11

       Mm-hmm. The delaying the onset. Um, we know that through overexpression throughout the brain and body that those mice, those, those APP mice will have, um, almost normalized cognition. Um, what I-- but we don't know whether it's delaying the onset. Um, we, we have some, um, there are others that have done human population clinical studies that show-

   13. PAPeter Attia1:13:44

       Sorry, make sure, I wanna make sure I understand what you just said.

   14. DDDena Dubal1:13:46

       Yeah.

   15. PAPeter Attia1:13:47

       You're saying if you take the A, you take the, the J20-

   16. DDDena Dubal1:13:51

       Mm-hmm

   17. PAPeter Attia1:13:51

       ... the APP mutant, and you also cause a mutation that overexpresses klotho, are you, are you saying that you've done that in the same mouse?

   18. DDDena Dubal1:14:00

       Yes. Mm-hmm.

   19. PAPeter Attia1:14:01

       W- tell me about that mouse's lifespan. Is it normal, and is it cognitively normal, or does it still get Alzheimer's disease?

   20. DDDena Dubal1:14:09

       That mouse that has the APP mutation and overexpresses klotho by about three to fourfold-

   21. PAPeter Attia1:14:17

       Yes

   22. DDDena Dubal1:14:17

       ... that mouse will live much longer, um, number one, so it extends its lifespan, number one. Number two, it in, it will normalize its cognition-

   23. PAPeter Attia1:14:34

       Across its lifespan?

   24. DDDena Dubal1:14:35

       Remarkably across its lifespan. Um, well, let me s- uh, um, at, at three months, seven months, eight months, yes, it, 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 klotho.

   25. PAPeter Attia1:15:07

       I mean, that is unbelievable.

   26. DDDena Dubal1:15:10

       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, um, in collaboration with Leonard Muki and, um, Eliezer Masliah looked at s- at, he's at, who's at the NIH now. Um, when, when Masliah looked at the synapses in these mice, they saw, he saw that, that the synapses were all preserved. So again, like, like this analogy where klotho 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. You know, um, they won't necessarily go away with klotho, at least in mice. The story may be different in humans. But klotho provided resilience and thwarted the toxicities of Alzheimer's disease.

   27. PAPeter Attia1:16:26

       O- okay. So I'm gonna plant the seed with you, which we'll come back to, but the reason I'm asking this question, Dena, is I do believe that at some point we will have klotho as a drug. I do believe that-

   28. DDDena Dubal1:16:40

       I think it's gonna come to market

   29. PAPeter Attia1:16:40

       ... we're, we're absolutely going to be injecting people with klotho. Where I'm gonna start, you know, where, where we'll go is, do we believe this is a drug we will only want to give to people once they have MCI? Well, 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 klotho even before there is a demonstrated, uh, disease risk? So that's, that's where I wanna go with this, is kind of thinking, you know, sort of three or four moves ahead on the chessboard. Now let's go

9. 1:17:18 – 1:39:34

   Promising results of klotho in primate models, and the importance of finding an appropriate therapeutic dose before moving to human trials

   1. PAPeter Attia1:17:18

      back and talk about the primates because we've talked about this incredible body of literature in, in mice, and again, I think it's, it's reasonable to be, uh, um, y- you know, 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. Um, this looks incredible. But it would be nice if we had a more convincing model. So let's, let's talk about the single best thing we can ever do shy of before, you know, before we get to humans, and that's to look at, at primates. So let's talk a little bit about primate brains and the body of literature around klotho in the, in that brain.

   2. DDDena Dubal1:18:16

      This is, this is an area that I wanted so much to get into. Um, I don't work with monkeys, and, um, I think it's a very, very specialized field to test cognition in monkeys. And so I'll just take us back, um, almost, I, a long time ago, maybe s- eight years ago, when, um, I really wanted to know, Peter, whether klotho can enhance a more complex brain than a mouse brain. And like you said, mice are, um, are really important in scientific discovery, but there are so many examples of what worked in mice and cured mouse-heimer'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 that might not be that, you know, big and important thing that, that Steven Houser challenged me on when I was a resident. So I, um, I actually was, I was pitching this, like wanna test klotho in primates, wanna... And I went all across Silicon Valley, um, from biotech and pharma to biotech and pharma, and people were, um, people were curious and enthusiastic about it. But, um, but I'll tell you, one individual really got this, um, and that was, um, Ned David, um, who had started a company, Unity Biotechnology, um, to attack aging. And, um, we joined forces. They, um, fundraised. They found really great collaborators at Yale, and, and really made this happen to test klotho and, um, and cognitive enhancement in a brain like ours. You know, this-- And if this didn't-- When people would tell me, "Dena, this, like, if it doesn't work, it's gonna kill your research program," and I would say, "Let it die then." I just-- Like, I don't wanna 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, um, went into the design and, um, the execution. So why non-human primates? So, um, rhesus macaques are, um, are incredible organisms. Um, they sh- they, um, have very complex brains. They are more genetically diverse than humans, um, and that genetic diversity is something that often, um, that often trumps effects. So, um, 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. So rhesus m-- And our 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, um, complexity that's very similar to humans in terms of the even more of the neural circuits, the hippocampus, the prefrontal cortex, um, and they have an anatomic complexity that's pretty similar to us. So by testing klotho in 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.

   3. PAPeter Attia1:22:20

      So, so I just wanna, I wanna pause there for a second, Dena. I mean, I think obviously the listener can imagine where this story is going, but, um, that's a huge burn your ships moment.

   4. DDDena Dubal1:22:33

      [laughing]

   5. PAPeter Attia1:22:36

      Um, and um, I wanna, I wanna, I wanna just spend a minute probing that a little bit more. And, and, um, look, you're, you were a wildly successful investigator before this. Um, I don't think it's, um, an obvious step that you would take to push that much further, because let's be clear, if klotho had failed miserably in primates, you're w- like how many PhDs and postdocs and students do you have in your lab?

   6. DDDena Dubal1:23:08

      We're a group of around, it fluctuates between, um, seven to eleven.

   7. PAPeter Attia1:23:13

      Yeah. And obviously you probably have a very well-oiled machine at generating R1s and [clears throat] all sorts of grants. Um, it's not easy to start all over again if all of that dries up, right?

   8. DDDena Dubal1:23:29

      That's why it was so risky, and some people-

   9. PAPeter Attia1:23:30

      Yeah

   10. DDDena Dubal1:23:30

       ... told me this is like, you know, just go to a human trial, you know. Um, you can do biomarkers and, and but, but this is risky. And it is risky if it's not done well.

   11. PAPeter Attia1:23:44

       Mm-hmm.

   12. DDDena Dubal1:23:45

       But it's super informative even if it's negative. Um, really, really informative. I-- Negative data, if it's good negative data, and not because the experiments were done in a sloppy way, it's good negative data, then it's, it really tells you something. And, um, you know, 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? Um, and there are other longevity factors. Um, so it was really important to push forward and to push forward in a rigorous way, and also with the, with the knowledge that, um, that the results could really change the direction of, of what we're doing. But, you know, Peter, um, we have these, I don't know, we have times in our lives where we reflect on what we're doing, how we're spending our time. Um, is it with people that we appreciate? Is it with doing things that are meaningful? Um, and it may be deeply philosophical, but you know, from day to day, like I really wanna be doing something that I have a lot of faith in and that potentially has meaning, um, for the human condition, to improve human health. And if, and if we've done a really great experiment that's negative, um, and there's really reason to believe that this isn't gonna go forward, then that's fine. You know, we will mourn it, and we will still move forward with something else. But, um, but we just don't have, just don't have lifetimes, you know, to, to devote to all, you know, 100 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, um, fundraising. [laughs]

   13. PAPeter Attia1:25:49

       I think the reason I'm pointing this out, Dena, is I want to, uh, publicly commend you for something that honestly I don't think most scientists would be willing to do. And I, m- I, I, 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, um, they think the job is doing science. But it's not. The job is, is knowledge creation. The job is knowledge creation for the purpose of making lives better. And those are very different things. They overlap. The fir- the former is a process to the latter.

   14. DDDena Dubal1:26:29

       Mm-hmm.

   15. PAPeter Attia1:26:30

       But it's very easy, and I'm y- you know, it's very easy to, to lose sight of that in the, in the weeds. Um, so again, I, I just, I only call it out to say I do- I think... I, I don't, I don't think everybody would have made the, come to the same conclusion you did. So with that said, let's talk about how you and your colleagues, uh, at Yale and, um, uh, you know, basically thought of the right question to ask, and then the right-

   16. DDDena Dubal1:27:02

       Mm

   17. PAPeter Attia1:27:02

       ... design of an experiment to ask the right question.

   18. DDDena Dubal1:27:05

       First, I just wanna say thank you, um, and that is very well said. Um, and, and to the, to the monkeys. So, um, monkeys undergo cognitive decline in a very similar parallel fashion to humans. Um, they have synaptic loss, the connection between neurons, um, with aging, as do humans do. And the circuits that are affected in aging are really similar to the human circuits. So they are, um, the hippocampal frontal circuits, the hippocampus and the frontal lobes. And aging will preferentially target working memory, um, and, 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, why did I come here?" What was holding that, that immediate memory in your mind is something that t- that aging really targets. And monkeys undergo this, a very similar cognitive decline to humans. And there are ways to test monkeys that interrogate those circuits. Uh, in this case, um, a spatial delay task was used. Graham Williams and Stacy Castner conducted the studies. And, um, and the fa- and klotho actually preferentially targets those circuits that aging erodes. So here, 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, um, by these exceptional scientists, um, who are, who are, who are very, um, experienced and adept at conducting these tests. And it, so it was a really, uh, it was a excellent setup to ask, can klotho enhance cognition in these aging monkeys? And so what they did, what was done is, um, something called a spatial delayed, uh, response. And the monkey is presented with a, a bin of multiple wells. And one, 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 at some point, and then all the bins are covered, so you can't, now you cannot see where, all the wells are covered. You cannot see where the treat is. And then, um, and then the blind is opened, and they are tested where to choose, because they're bas- basing this on spatial memory and on working memory, where that treat was. Um, 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, to, to remember spatial and working memory where that treat was. So what was fa-- So it was done very rigorously, I must say. Some, um... The study was largely blinded. Um, and it was done in a way in which, um, injections w- uh, injection of klotho wouldn't interfere with causing stress and memory, so that everyone got a baseline. They then got a vehicle, and then they got vehicle or klotho. So like a, it's a really well-designed study in 18 monkeys.

   19. PAPeter Attia1:31:09

       And, and just for folks listening, vehicle is placebo, so that they understand what we mean by that.

   20. DDDena Dubal1:31:14

       Yes.

   21. PAPeter Attia1:31:14

       Yeah.

   22. DDDena Dubal1:31:14

       Thank you. Um, so the monkeys that, that, that got a klotho treatment, again, largely blinded Performed better than the ones that got vehicle treatment

   23. PAPeter Attia1:31:29

       Sorry, Dena. How did you guys decide how to raise the dose? So 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, of animal. Um, what, 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?

   24. DDDena Dubal1:31:55

       We went with, um... Well, we went with, with t- 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, been u- been exposed to and has seen over its lifetime. And so we-- so in one dose, we wanted to go somewhere up to, like, maybe four to fivefold.

   25. PAPeter Attia1:32:23

       Four X. Yep, yep. Exactly.

   26. DDDena Dubal1:32:25

       Yeah.

   27. PAPeter Attia1:32:25

       So, so sort of you're, you're still just below what they would have been born at.

   28. DDDena Dubal1:32:30

       Right.

   29. PAPeter Attia1:32:30

       Yep.

   30. DDDena Dubal1:32:31

       So a, a very youthful level of klotho.
10. 1:39:34 – 1:42:45

    Speculating why a single klotho injection has such long-lasting effects

    1. DDDena Dubal1:39:34

       to.

    2. PAPeter Attia1:39:35

       So Dena, why do you think the effect lasts for so long? This is also a little counterintuitive when-

    3. DDDena Dubal1:39:46

       Mm-hmm

    4. PAPeter Attia1:39:46

       ... you consider the hourly variation of klotho naturally, when you consider the transient effects potentially of things like exercise, where you see these large boosts over, you know, 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 klotho did. So what, what do you think accounts for that?

    5. DDDena Dubal1:40:21

       I don't know the answer, but I'll speculate. First, I'll just say it's remarkable, isn't it? Um, it's particularly remarkable as we imagine therapeutics in that maybe this is the type of treatment, um, of rejuvenation that could be administered maybe once a month, once every three months as, um, as a shot in the arm, for example. So I think it has really important therapeutic implications. Mechanistically, this means that klotho has not just an acute effect of immediately enhancing, 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 to help-- So I would imagine that, um, that, for example, with GluN2B being trafficked to the synapse and to, um, to promote better functions, that those sort of synaptic organizational effects, um, are happening for and staying put for a longer period of time. I think the biology of this organizational effect of klotho has yet to be, um, discovered. Um, but it's something that's happening at the synapse. I, I'm, I, I have a very strong, um, sense of, of it's something that's happening organizationally, structurally at the synapse.

    6. PAPeter Attia1:42:03

       You know, as, as much as I wanna go deeper into that and to talk about the, the confirmational changes that could be occurring, we'll, we'll have to save that for another discussion because where we have to really go next is, you know, 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, uh, be relevant to the species of interest. And no disrespect to the mice and the macaques, but-

    7. DDDena Dubal1:42:39

       [laughs]

    8. PAPeter Attia1:42:39

       There, there aren't too many people listening to this whose mind isn't already wondering, "Okay,

11. 1:42:45 – 1:50:10

    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. PAPeter Attia1:42:45

       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 gonna make a difference for me because of my risk factors, even though it's twenty years from now?" And so tell me a little bit about a particular SNP associated with the klotho gene called KLVS and what its significance is to this story.

    2. DDDena Dubal1:43:15

       Sure. Well, you, um, really described well why, and, and, like, none of this matters, Peter, if it doesn't have potential to help the human condition. It just doesn't matter. You know, it's interesting and, but, um, but the big and important Um, is if it's relevant and may work in humans. I don't, I don't know whether we've done anything big until we test-

    3. PAPeter Attia1:43:41

       Yep

    4. DDDena Dubal1:43:41

       ... klotho in humans. Um, so with that said, in the early-- back in the early days in, um, I, I think it was twenty twelve or so when these... or twenty eleven, twenty twelve, when these first klotho studies were-- we were doing them in mice and, 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, um, Alzheimer's disease, fifty million people around the world. This is gonna triple by twenty fifty. Uh, cognitive decline is our biggest biomedical challenge. Um, so always have humans in mind. And I went to my, um, my friend and colleague, Jennifer Yokoyama, at the Memory and Aging Center here at UCSF, and I told her that there, there was this genetic variant of klotho that had been found, and we'll-- we can talk more about what that is. But was there a way, 'cause 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, um, with Alzheimer's disease, with normal aging, with frontotemporal dementia, and they really carefully characterize them, their genetics, um, 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 klotho, KLVS, has any association with cognition?" Because that would mean that there-- that this klotho is, is important to brain-- Uh, it would mean that there is some link with humans and brain health. So what is KLVS that you mentioned, um, and what does it associate with? So, um, as we've discussed, we all have klotho circulating in our blood, um, and around our brain. But some of us, about one to four, one to five of us will have-- will carry a gene for klotho, a genetic code for klotho, that leads to higher levels of production. And that is, and KLVS refers to two, um, single nucleotide, um, polymorphisms that cause a difference in the coding of the protein itself. Um, 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 klotho circulating 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, um, end up having multiple disadvantages in lifespan and, um, vulnerability to different diseases. So when we talk about KLVS, we're talking specifically about heterozygosity, carrying one allele of it.

    5. PAPeter Attia1:47:01

       Sorry, what, what are some of the health consequences of being homozygous, and what is the prevalence of homozygosity?

    6. DDDena Dubal1:47:10

       It's really rare. So if the prevalence of heterozygosity is, um, around twenty-five percent in populations, homozygosity is so-

    7. PAPeter Attia1:47:22

       It's probably about one to two percent because actually-

    8. DDDena Dubal1:47:26

       Yeah

    9. PAPeter Attia1:47:26

       ... it would kinda mirror APOE4, where the heterozygosity is about the same, right? It's about one in four, and the homozygosity-

    10. DDDena Dubal1:47:36

        That's right

    11. PAPeter Attia1:47:36

        ... is about one in a h- one in fifty to one in a hundred.

    12. DDDena Dubal1:47:40

        Yeah. That's about right. Um, it, it is-- it's pretty rare, but it exists and, and, um, those people have shorter lifespans. They have much lower levels of klotho, um, and they are at risk for anything that the heterozygosity helps with, the homozygos- homozygosity hurts.

    13. PAPeter Attia1:48:01

        Wow.

    14. DDDena Dubal1:48:01

        So, um, so but the heterozygosity has been a really interesting window into klotho and, and natural experiments of, of humans-

    15. PAPeter Attia1:48:12

        And how, how much higher is their klotho level?

    16. DDDena Dubal1:48:17

        Well, we did this study. We, um, Jennifer and I, um, took the serum from individuals here at the Memory and Aging Center, um, a few hundred, and tested the ones that were non-carriers that just have the typical klotho gene versus the KLVS heterozygote carriers. And, um, the heterozygote carrier status increased klotho levels by about fifteen percent. Fifteen, maybe twenty percent. And just to give you context, if you're wondering, am I a carrier, and, you know, some people are and some people aren't. I don't want people to forget that exercise is some-- is thus far one of the more powerful modulators of klotho expression, and exercise increases klotho on average by thirty percent, so much more than what the genetic influence is. But nonetheless, um, KLVS increases klotho levels by about fifteen percent. So for example, if an average number, um, let's say a non-carrier had eight hundred picograms per mil of klotho circulating in their serum, um, a KLVS carrier might have, like, nine hundred and fifty or so. Um, picograms per ml. That's what I'm remembering from our graphs. Um, so it does increase klotho and, um, there's reason to believe that it increases klotho because, because those two, um, amino acid changes that translate, um, into a different, um, amino acid in the protein itself, um, influences secretion of klotho from cells. So there is a biologic reason, um, that this variant, um, is probably changing klotho levels.

12. 1:50:10 – 2:05:26

    The interaction between the KL-VS genetic variant and APOE4 and how it impacts risk of Alzheimer’s disease

    1. PAPeter Attia1:50:11

       The other piece to this story, Dena, that's just so fascinating is now when you take those data, the prevalence of heterozygosity of KLVS, and you cross it with APOE4.

    2. DDDena Dubal1:50:27

       Mm-hmm.

    3. PAPeter Attia1:50:28

       So now we have, you know, listeners of this podcast are absolutely not strangers to the population-based risk of APOE4. Of course, we always wanna remind people that at the individual level, very difficult to make that statement, but at the population-based level, we know that having, uh, an APOE3 and an APOE4 is at least a doubling of, of risk, maybe even slightly more. Um, and, and homozygosity for APOE4 could be an eight to twelve-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?

    4. DDDena Dubal1:51:16

       Yeah. This is really remarkable data. I first want to, um, to go back to that original experiment, um, that we published in twenty fourteen, I believe, with, with, um, Jennifer, and that those people with that, that carried the KLVS allele did better across cognitive testing. Just, and this is in normal aging, this was not in Alzheimer's disease. But carrying that variant, carrying KLVS associated with better cognition across the board. And if you look at-- I'm often asked, "Well, how much smarter does it, you know, were those individuals?" And I would be careful about, you know, smarter and happier and all. We don't know, but the cognitive test showed us that they did better, and they did, um, they did better to the same extent that APOE4 carriers would do worse. So it was a sort of a, a similar, um-- it was a s- a similar amount of change in cognition in which KLVS was improving and APOE4 was decreasing. So with that in the background, since then, um, many groups, um, and many people have exampled-- have looked at KLVS 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, um, heterozygosity. So then the question comes that you asked about APOE4 Alzheimer's risk and klotho in KLVS. And, um, we did, uh, we initially did a study in collaboration actually led by, um, Ozioma Okonkwo and his group at Wisconsin that showed that, um, that in individuals with, um, with APOE4 that were carriers of KLVS, that they just had, um, 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, um, were not a part of, and that is, that came from Stanford, um, led by, uh, Michael Beloy, um, he's one to watch. He just established a lab at WashU. And, um, his, um, senior PI, Michael Greicius, um, and they're wizards at doing genetic population studies. So Peter, they did this remarkable study, um, of twenty-two different cohorts that were normal cognition, MCI, mild cognitive impairment that you mentioned earlier, and Alzheimer's disease. Over twenty thousand individuals, um, a very large meta-analysis. And they looked at, um, and they looked at if there was a relationship between KLVS, um, 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, um, that in those people that carry APOE4, if they were also heterozygote for KLVS, they had, um, 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 Um, Alzheimer's biomarkers, both in their CSF and in their brain. They just had less amyloid beta. Um, it was, uh, it's a really striking study, again, because it has, uh, the power of statistical analysis. It holds across many, many cohorts, and it's many, many people. And, uh, you know, it's, um, KLVS essentially blocks and abrogates the APOE4 toxicity by this association, um, by this genetic association. It's really remarkable.

    5. PAPeter Attia1:56:18

       W- did the double E4s, did the homozygotes reduce to a completely normal 3/3 risk? My, my recollection, but it's been a while since I looked at the, the... I- there's a graph that, uh, you know, demonstrates all of this that's, that's a g- 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?

    6. DDDena Dubal1:56:50

       Well, uh, this is, this is why it's, um, it's... They didn't actually publish that. Um, they excluded the homozygotes from the analysis. Um, but, and it might be in a supplement somewhere. Um-

    7. PAPeter Attia1:57:05

       Got it

    8. DDDena Dubal1:57:05

       ... but I actually reached out to Mikael, and I asked him about the 4/4. I had the same question. Um, and so this is, um, this is just by verbal communication. He indicated that the 4/4 also associated with, with the protection from the KLVS heterozygosity, so that, that the effect of having one APOE4 wasn't different than h- um, having two APOE4s in terms of KLVS protection.

    9. PAPeter Attia1:57:38

       Mm.

    10. DDDena Dubal1:57:38

        It protected in both heterozygosity and homozygosity of the APOE4 allele. Again, it's, it's, um, again, just through verbal communication. It might have been in a supplement somewhere.

    11. PAPeter Attia1:57:50

        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, um, unwarranted, such as, you know, an increase in stroke risk? I mean, I know that it's actually the opposite, but, but I'm just trying to say, like, is there anything that with an increase in a platelet factor that, that could have led to an increase in DVTs or something? I mean, when you have a-

    12. DDDena Dubal1:58:18

        Sure

    13. PAPeter Attia1:58:18

        ... sample size as large as they did, presumably you would find something that was negatively associated with the KLVS.

    14. DDDena Dubal1:58:27

        I'm not aware of, of increase. A- a- as you were saying, it's actually the opposite. The KLB- VS heterozygosity actually prote- is associates with the protection against stroke risks, um, cardiovascular risks, um-

    15. PAPeter Attia1:58:43

        Even metabolic risk

    16. DDDena Dubal1:58:44

        ... and metabolic. Yeah, metabolic diseases. Um, it's very remarkable. I did. I, I, I searched, I searched [laughs] uh, for something that could be negative, and I did find some... I did find that, um, that in the cancer literature, that KLVS heterozygosity has a poorer prognostic, um, val- as, um, indicates a poorer prognosis in BRCA1 carriers with breast cancer. So, um, it's, there are examples where it's, um, not helpful, and one is in, in BRCA1 positive breast cancer.

    17. PAPeter Attia1:59:30

        Dena, going back to the human homozygotes for KLVS-

    18. DDDena Dubal1:59:35

        Mm

    19. PAPeter Attia1:59:35

        ... do we know how much of an increase in klotho they produce? So if the heterozygotes are producing fifteen to maybe twenty percent more, how much more klotho is being produced by the homozygotes?

    20. DDDena Dubal1:59:48

        Mm.

    21. PAPeter Attia1:59:48

        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. So as the, 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?

    22. DDDena Dubal2:00:15

        It's a great question. Um, with, um, Jen here at the Memory and Aging Center, we also, we h- we have access to homozygote serum, and we actually looked at, um, several homozygotes compared to non-carriers and heterozygotes. And what we found is that their klotho levels are actually lower than normal. And we think that has something to do with, um... And I'm gonna get a little deeper into this because I think it's really important when we think about therapeutics actually. That there's something about... So many genetic variants won't cause a change in the protein sequence itself. If it happen, if the variant, if the n- nucleotide change is in an intron, it's just not going to change the, um, the s- structure or function or anything of the protein. But in KLVS, the nucleotide changes, there are two nucleotide changes, and they translate into different amino acids in two places of the klotho protein. And so what we think is happening, this is speculation, but what we think is happening, um, based on in vitro studies that Arcane did in two thousand two, that- That in the klotho heterozygote in which one allele is making a mutant protein, we think that there's an overcompensation for that mutant protein-

    23. PAPeter Attia2:01:56

        [laughs]

    24. DDDena Dubal2:01:56

        ... by increasing wild-type levels. Because what that polymorph- what that variant does is it mucks up klotho secretion from the cell. So just to be clear, carrying one variant, this chain is likely changing the func- the structure and function of klotho, but there's probably a, uh, an, an overcompensation from the wild-type allele causing higher levels of the wild-type klotho.

    25. PAPeter Attia2:02:30

        And in the, in the... First of all, that is an unbelievable stor- like, I, I would not have guessed that and I'm, I, I can't believe I didn't think to ask such an obvious question, which is, do the KL-VS, uh, 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, a, 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 1,000 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, is that what you're seeing? You're seeing two different proteins, and it's the wild type one that is overexpressed?

    26. DDDena Dubal2:03:35

        At this point, it's the h- it's a hypothesis-

    27. PAPeter Attia2:03:38

        Mm

    28. DDDena Dubal2:03:38

        ... that the wild type one is really compensating, overcompensating by increasing levels for the, what may be a mutant protein. But in reality are... And we've done ELISAs on, um, the enzyme linked immunoassays on thousands and thousands of individuals, but the ELISA itself doesn't distinguish between a KL-VS protein and a normal wild-type klotho protein. It doesn't distinguish between the two. And so the answer to that question is not known.

    29. PAPeter Attia2:04:13

        Yeah. ELI- ELISA is just, ELISA is just too, too blunt an instrument to figure that out.

    30. DDDena Dubal2:04:18

        And if we take that a step further and say, "What's happening in a klotho homozygote?" Well, they have-- 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 klotho. Um, all of their klotho is the KL-VS form, and it's very low.
13. 2:05:26 – 2:11:48

    The significance of klotho levels: studies linking lower levels to increased mortality and the broader implications for organ health and disease prevention

    1. DDDena Dubal2:05:26

       is why this matters. [laughs] It matters for many reasons, but there have been some really large scale studies recently, Peter. Um, one is called the NHANES study, published in, in 2022, I believe. And what this study showed us is that klotho levels, as measured by the, this immunoassay, by the ELISA, that the klotho levels really correlated with mortality. So that, um, if there was, um... I wanna make sure I get this right. So it was over 10,000 people in the United States. Um, klotho serum was measured on everyone. The mean age was about 56 years. And if the mean klotho level was around 800, then something less than what they defined as 666 by their study picograms per ml, associated with a 30% mortality over five years. And that was replicated in another study called INCHIANTI, that lower levels of klotho... And again, this isn't like half the levels. This is, um, it's, it's... Maybe, maybe it's like 30% of a, a decrease of levels. Um, drove a 30-- Well, associated with a 30% mortality over five years, and that mortality was primarily in, um, cancer and cardiovascular disease. So the level... You know, I, I think levels are gonna matter as we think about our human aging, as we think about our, our organismal health, heart health, brain health, cancer health. The levels, um, at least by association right now, matter. Um, and lower levels are really associated with, with, um, with more diseases of aging.

    2. PAPeter Attia2:07:24

       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, you know, the limits of our understanding of klotho 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 klotho levels are 30% below what would be expected for somebody 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 mitigated was, uh, or, or rather the, the, the manner in which that death was, was, um, 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. Um, uh, I mean, w- it, it, it suggests that klotho is doing even more than just protecting brain health, which if it did nothing else, would still be arguably the most important thing that, you know, we should be trying to get into humans for clinical trials. So, uh, w- I, I know that your area of expertise is not oncology or cardiovascular medicine, but I'm, I'm just curious as to what your thoughts are as to how that could be happening.

    3. DDDena Dubal2:08:48

       I couldn't agree with you more. [laughs] You know, my... and my expertise is as a neurologist and neuroscientist is really on the brain. Um, but having said that, I, you know, the side effects of klotho in, in, um, increasing it may be much more than im- um, helping brain health. It's, it clearly has, um, a very strong association with, um, with protection against cancer, cardiovascular disease, and kidney disease. And, um, there is, Peter, a very, very, very large literature, um, on klotho and organ health, um, a very strong one. Um, in the cancer field, there are many preclinical models, again, in, um, rats and mice, that show that giving klotho, um, the soluble hormonal form, um, actually stopped, um, and, and reversed, um, cancers in mice, like pancreatic cancer, for example. Um, and then there are association studies in humans too, but there's, um, there is so much more to the biology of klotho than, um, than only what it's doing in the brain. Um, so, and, and kidney disease, so there's a, there's, like, klotho's gonna come to market and, and maybe the kidney people will beat the brain people. [laughs] Um, if klotho comes to market, I, I think people are gonna benefit no matter what. But in chronic kidney disease, there is a decrease in klotho, um, secretion and decrease in klotho in the blood, and the kidney folks, the kidney specialists, are really developing klotho as a diagnostic biomarker, um, to understand, um, when the kidney starts failing. And the idea there is that right now the measures are, um, glomerular filtration rate, um, urea, um, and, and these are not as sensitive as, as they would want biomarkers to be in detecting kidney dysfunction. But klo- as soon as the kidney starts having trouble, klotho declines, as soon as it starts having trouble. So there is a lot of, um... there's a fervent interest in developing klotho as a biomarker for kidney function. And I would say, again, if we think in the broader sense about klotho, maybe someday, and maybe someday soon, we would have our klotho levels checked, you know, just as we do our blood pressure, um, and our cholesterol, um, and we get breast exams, um, and we have colonoscopies that, you know, why not have a klotho level

14. 2:11:48 – 2:18:32

    Measuring klotho levels and determining an individual’s KL-VS status

    1. DDDena Dubal2:11:48

       checked? Everyone's will be different.

    2. PAPeter Attia2:11:50

       N- now, how do we get around the issue that it has this diurnal effect? Um, that's the only thing that jumps-

    3. DDDena Dubal2:11:55

       Um

    4. PAPeter Attia2:11:55

       ... to my mind when you're talking about a marker of, of eGFR, because I know in our practice, I mean, we're fanatical about monitoring eGFR. I mean, we, we, you know, we look at cystatin C, we look at creatinine, we're triangulating them. Um, but at least to our ability, they, these don't mar- th- these don't depend terribly on time of day. So do you think that the answer for klotho 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 klotho level at 2:00 in the afternoon, otherwise we can't interpret it. Is that how you think it's going to factor in?

    5. DDDena Dubal2:12:35

       I think that is going to give us the best and more accurate level of klotho, and in fact, I mentioned that we do klo- we've done thousands and thousands of klotho, um, measurements in human serum, in human CSF, cerebrospinal fluid, and I always make sure that, um, whoever we're collaborating with and who has collected the specimen did it in a morning fasting manner so that the, um, the individual or patient hasn't eaten and it's first thing in the morning.

    6. PAPeter Attia2:13:07

       Why do you think, um, or why do you know that eating impacts klotho levels?

    7. DDDena Dubal2:13:13

       I don't know that, and I don't know that it's really been tested per se. But, um, in the spirit of what one does, for example, for-

    8. PAPeter Attia2:13:21

       Right, just being, just being incredibly consistent and rigorous early.

    9. DDDena Dubal2:13:24

       Yeah, with, with, um, just less confounding factors in the background. But having said that, um, the decline over the course of a day isn't dramatic.

    10. PAPeter Attia2:13:36

        So the decline by age is more relevant than the decline by time of day?

    11. DDDena Dubal2:13:43

        I would say that, um, the decline by, by age is, is a consistent decline. Um, and then the-- but the time of day does matter because it can decrease by almost forty percent by midnight. But the statistic-- And this is done with-- I have to say, I've looked through the literature. The diurnal rhythm of klotho has been shown in a small number of people, but the data look good, um, that it's not really statistically different until, like, after noon, but it's starting to decline.

    12. PAPeter Attia2:14:14

        Mm.

    13. DDDena Dubal2:14:14

        So I would, you know, if I had to design, um, a time and day and-- I would say that we need a klotho test that people take in the morning fasting like they do with their cholesterol, just get it along with the cholesterol draw. And, um, and most importantly, we need a standardized assay that, that-

    14. PAPeter Attia2:14:34

        Yeah, I was about to say, is there a CLIA-based assay for this?

    15. DDDena Dubal2:14:37

        Yeah. No. No. But, but I think, I, I think it's coming given how many people ha- and how many samples have been s- it just hasn't been-- You know, levels haven't been coordinated between institutions and labs and, um... So everything we do in our lab is consistent to our, um, is, is consistent to, to our methods and standards, and we standardize everything to, um, and I include my own, um, samples in there. But it's all standardized. But then my lab, um, may have slightly different values than-

    16. PAPeter Attia2:15:14

        Yep

    17. DDDena Dubal2:15:14

        ... what is going on in another lab.

    18. PAPeter Attia2:15:16

        And then of course, you know, so if I woke up in the morning and I checked my level and I was at eight hundred, is it picograms per milliliter? Is that the unit?

    19. DDDena Dubal2:15:25

        Yeah.

    20. PAPeter Attia2:15:25

        Yeah.

    21. DDDena Dubal2:15:26

        Mm-hmm.

    22. PAPeter Attia2:15:26

        So if we-- if I did my test at six clock in the morning and I'm eight hundred picograms per milliliter, and then from seven o'clock to nine o'clock in the morning I'm in the gym-

    23. DDDena Dubal2:15:36

        Mm-hmm

    24. PAPeter Attia2:15:36

        ... I could easily be-

    25. DDDena Dubal2:15:37

        Very tall

    26. PAPeter Attia2:15:37

        ... twelve hundred picograms-

    27. DDDena Dubal2:15:38

        Right

    28. PAPeter Attia2:15:38

        ... per milliliter after. Um, so, so how does that impact-

    29. DDDena Dubal2:15:43

        Yeah

    30. PAPeter Attia2:15:43

        ... our understanding of what's going on?
15. 2:18:32 – 2:23:23

    The promising potential of klotho for Alzheimer’s disease treatment, and the importance of philanthropy for funding research

    1. PAPeter Attia2:18:32

       Dena, this has, um, this has been an, I mean, I think at the risk of being hyperbolic, just an insanely fascinating discussion. Uh, and I'm so happy to be able to have discussed your work with, you know, with you, and, and then by extension to be able to, to kind of put this in front of so many people. Um, 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, um, this is, this is the sad poster child for it. Um, it, it is unfortunately today, um... It's, it's, you know, I, I don't need to tell you what you see every day, but I think, I think people watching this understand that even, even with the advent of anti-amyloid therapies, they have, they have barely, barely been able to, uh, put a dent in this, uh, enormous problem. And, uh, it's for that reason that I'm very excited about this and, uh, uh, just applaud you on your, on your career. Um, and I, I think this is only the beginning, right? Like, the, the-- we have to get this into human clinical trials.

    2. DDDena Dubal2:19:51

       That's what's next. Um, thank you so much for this invitation. Um, it's been really, really fun talking to you. Um, and, um, I, you know, Alzheimer’s disease is one of our biggest biomedical challenges. Um, our entire world is aging. It used to be the US, Japan, Sweden, but it's the entire world. Japan, uh, China, India, Ev- Afri- Africa, all continents are aging and we're aging rapidly. And, um, again, this is one of our biggest biomedical problems. We really do not have effective therapies. Um, and I'm, I'm hopeful that, um, that with multiple shots on goal right now, that something will come to market that, that provides that resilience, you know, in addition to anti-amyloid therapies, that is a cocktail that provides resilience, um, for the suffering that really erodes our, our memory. It, um, destroys families, economies. It's, it's, it's just a devastating problem. Um, we're, 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, um, I'm so lucky to be doing what I love to do, like really, really love to do. And, um, and I am also lucky enough to have a very diverse portfolio of being funded by the NIH, by foundations, um, and in the past by biotech, and also by philanthropy. And, um, I can't emphasize enough how much, um, 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, um, and see what happens. And, um, you know, 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, um, to take a risk. So, um, I'm just, I'm very grateful, um, to be doing what I'm doing.

    3. PAPeter Attia2:22:10

       Well, I, I'm glad you mentioned that. I, I do think that philanthropy, um, can have an enormous impact in science, um, 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, say, the NIH to fund or too risky for industry to come in and fund, um, 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 klotho is a poster child for that type of work. Um, and at least as of this moment, it looks like it's actually paying off. So, um, I, I think I'm, I'm, you know, I and countless others are grateful for, uh, for what's been done. Dena, thank you so much for your time today.

    4. DDDena Dubal2:23:08

       Thank you so much, Peter.

    5. PAPeter Attia2:23:09

       Really, really appreciate it.

    6. DDDena Dubal2:23:10

       Thank you. This was wonderful. Thanks. [outro music]

Episode duration: 2:23:23