FoundMyFitness - #062 Dr. Steve Horvath on epigenetic aging to predict healthspan: the DNA PhenoAge and GrimAge clocks

Episode Date: December 22, 2020

Steve Horvath Steve Horvath, PhD, is a professor of human genetics and biostatistics at UCLA's Fielding School of Public Health Dr. Steve Horvath has analyzed large data sets of DNA methylation profiles to derive an algorithm that accurately predicts a person's chronological age across multiple cells, tissues, and organs, and even mammalian species. He built on this algorithm to develop second-generation clocks that could predict time-to-death among people of the same chronological age, as well as lifespan and healthspan. In this episode, we discuss: (00:00) Introduction and overview (20:24) Horvath Aging Clocks (26:36) Heredity determines aging (35:49) DNAm PhenoAge vs GrimAge (45:58) Slowing the epigenetic clock (01:10:43) Epigenetics: Cause or consequence of aging? (01:16:43) Vitamin D reverses epigenetic age (01:19:07) Omega-3 slows GrimAge clock (01:24:19) Ongoing research If you're interested in learning more, you can read the full show notes here. Join over 300,000 people and get the latest distilled information on aging straight to your inbox weekly: https://www.foundmyfitness.com/newsletter Become a FoundMyFitness premium member to get access to exclusive episodes, emails, live Q+A's with Rhonda and more: https://www.foundmyfitness.com/crowdsponsor

Hello, my friends. It's been a while. This is the last episode of 2020, and while 2020 has been a tough year, this episode is fantastic. Today's guest is Dr. Steve Horvath, a professor of genetics and biostatistics at UCLA, and the creator of the Horbath epigenetic aging clock, or should I say clocks, plural, as there are more than one. The development of ways to measure aging is very important because finding clever ways to quantitate aging is the first step we have to take if we actually hope to find any therapies that can improve human health span. That's what makes Dr. Horvath's work so important. His lab has found something truly incredible, a story of our aging writing atop our DNA as epigenetic marks. This measurement may be more fundamental, more interesting than many other markers, because, as we'll discuss in a moment, its predictive value.

this episode, we're going to talk about two generations of epigenetic clocks. The first generation, a chronological age detector. Well, the Horvath aging clock is what I sometimes call the so-called pan-tissue epigenetic clock. And so it is the most accurate molecular measure of age. It applies to all cells in the bodies. So we can take cells from basically any tissue in the body and predict true chronological age from the robust genetic marks that exist on the DNA. Interestingly, these marks are surprisingly robust. You can almost think of them as hard mechanical switches that toggle gene expression on or off. In fact, Dr. Horvath says the marks on DNA in blood samples exposed to high heat still produce beautiful, clear data. I want you to think about

that for a moment because it really says something about the usefulness of this investigational tool in terms of reliability. That's a rare thing in biology, where even the tiniest of factors can throw off an experiment entirely. But not only is this tool reliable, it's versatile. In fact, the methylation profile

of almost any mammalian cell, at least those with DNA, can be run through some optimized version of Dr. Horbeth's algorithm to predict the age of the organism with a surprising degree of accuracy. So if somebody provides, for example, DNA from blood or DNA from neurons,

or DNA extracted from saliva or urine, I can very accurately estimate their age. Imagine the implications for forensics. What if you have a forensic sample, you know? So let's say a blood stain, you know, that was, let's say, in a room for a couple of weeks or even, let's say, a bone sample collected, you know, a couple of months later.

all of these data, in my opinion, would still lead to very good methylation measurements for the purpose of measuring aging. When we think of other measures like measures of DNA damage, for example, the one I've worked with called Gamma H2AX, these say such interesting things, but God help you let the blood sample sit for two hours at room temperature. The ability to determine chronological age is just one of some of the exciting implications of Dr. Horbeth's work on epigenetic clocks.

We have some epigenetic clocks whose purpose is really to measure chronologic age, period. But then we have other epigenetic clocks that are really defined to be lifespan predictors. Or they are meant to predict time to death or time to major onset of a disease or what people call health span. How long are you healthy?

The second generation epigenesis, genetic clocks focus on determining health span and even lifespan. The multi-billion dollar life insurance industry is driven by the idea that chronological age is useful in predicting how long a person will live. Insurance companies do this every day when they look at actuarial tables in combination with other factors to give you a price on your life insurance plan. At least at a population level, deaths from cancer or a cardiovascular event are predictable on the basis of an understanding of the movement of these clockwork-like molecular gears on our DNA,

humming along in a probabilistic sense like a biological actuarial table. The pan-tissue epigenetic clock, as Dr. Horvath sometimes calls it, which looks exclusively at DNA methylation patterns, can be combined with biochemical markers, though, for an even greater insight. This is the approach taken in the pheno-age and grim age clocks. This shifts things over into a more biologically-referal-we. relevant world where disease, risk and mortality, rather than just chronology, become interesting. We can begin to talk about whether we're on track for death from all of those statistical age-related

causes. In other words, biological age can be teased out. But if we compared telomere length versus an epigenetic clock such as grim age when it comes to predicting lifespan, time to cancer, time to coronary heart disease, I mean, there would be no comparison. Harrison, you know, so in this sense, telomere length plays a very important role in certain disorders, you know, but it's just not a broad biomarker for aging. In the pursuit of biological age, scientists have, in the past, had to rely on proxy measures that reflect certain hallmarks of aging that can be challenging in terms of the utility of their overall predictive value. Take telomeres, for example, a true darling of the

aging community, if they're ever working. was one. As a biomarker, telomeres have the shortcoming that they are highly dependent on cell types, since telomerelankt is governed partly by artifacts of cellular division. They also behave differently between tissues and species. Look, telomeres are a measure of a type of biological aging. Probably. Maybe. Sometimes. But this molecular epigenetic clock is the real deal. It feels almost Star Trek level cool in more than a few ways. So that's the good news. you're up for a great discussion. Here's the challenging news.

While epigenetic age can be determined by both genetic and lifestyle factors, so far data suggests that a big driver of epigenetic age is in fact genetics. Although this may change in the near future with gene editing technologies like CRISPR, genetics is the hard to address stuff. It's the thing that allows certain ancestries

to have a slow epigenetic clock and better aging, even in the presence of bad clinical biomarkers, like elevated blood glucose and lipids, the stuff your doctor more commonly uses to determine your health. An example of this, Dr. Horvath tells us, is something called the Hispanic mortality paradox. People of Hispanic ancestry, unfortunately Hispanics often have higher risk for diabetes, the higher metabolic syndrome. However, according to the epigenetic clock, they actually age more slowly, you know. And so, so there's this really this disconnect. That paradox where bad clinical biomarkers don't translate to faster aging, Dr. Horvath's algorithm can peer right

past. Spanics, as I mentioned, have often a disadvantageous risk profile according to clinical biomarkers. But it turns out on average, they live much longer than expected. They actually live longer lives than people of European ancestry, you know. And that association is paradoxical to a clinician who looks at clinical biomarkers. But according to the epigenetic clock, it's not paradoxical because, as I mentioned, we have found that Hispanics age more slowly. He can see when a person might be aging well, even if markers of their long-term blood glucose levels like hemoglobin A1C are suboptimal.

While factors of inheritance play an outsized role in aging, accounting for about 40% of the difference between individuals in how they age, there are other factors that regulate epigenetic age as well. One of Steve's articles presents a table in which he lays out in beautiful detail how metabolic traits and lifestyle factors are associated with epigenetic age acceleration, which is when your biological age is older than your chronological age. We see things like smoking at the top. If you want to make a mess of your biological age, take up smoking. Then we see the opposite numerical representation for things like dietary carotenoids. Everything your grandmother ever told you about living a healthy lifestyle is kind of corroborated by our epigenetic clocks.

So for example, people who eat vegetables, people who exercise also actually educational level, you know, to some extent, even alcohol consumption, you know, show a beneficial effect. Sleep is important, too. Looked at women who have sleep disturbances in the women's health initiative, and sure enough, their epigenetic age of blood was slightly accelerated. While I'm disappointed that Steve didn't come on and say, Rhonda, because you exercise and take vitamins, you won't age. Instead, he emphasizes the role of inheritance.

If you have a parent who lived until age 100 or 105, then chances are that your blood is actually younger than the blood of a person of the same age, same gender, same everything, but whose parents didn't live until age 100, you know. So if you analyze the blood from a centenarian or super centenarian, it's true our age estimates are really way below their chronologic ages. could be 15 years younger.

It is a relief to me that we do have some control. When it comes to any intervention, including dietary intervention, that prevents anything related to metabolic syndrome or diabetes, that will be detectable. So in other words, if you have an intervention that takes an very obese person to a lean person, in my opinion, grim age will pick that up. It has to, you know. So yes, you want to avoid diabetes and all of that, you know, and certainly you don't want to smoke.

So let's say you have a friend who smokes and is obese, then yes, tell your friend to adopt a healthier lifestyle. Because for this person, it will have a huge effect. Some conditions may show up as tissue-specific accelerated aging, suggesting that the treatments may also be tissue-specific. And so I think a lot of stress factors really have an organ-specific effect. Conversely, anti-aging interventions also have an organ-specific effect. Something I should mention about this interview. We recorded it a while ago, pre-pandemic. The good news is that science doesn't always move as fast as you think, maybe especially in a pandemic.

But since we had this conversation, Dr. Horvath's group published a new study. A study that I know we would have discussed if we had this conversation a little more recently. This pilot study showed that a cocktail of human growth hormone, metformin, and DHA. taken for 12 months, reversed epigenetic age by 1.5 years in a small cohort of men. These are still early days. With earth-shatteringly cool and robust algorithms like Dr. Horbath's epigenetic aging clocks, I have no doubt that the promise of actually finding interventions that lead to robust age reversal stand a whole lot better chance of being discovered now than without this new method of inquiry. The reason why we have these different tools is that

we of course plan to use them in a human clinical trials of anti-aging interventions, you know, and in that context you want to see whether an intervention actually resets an epigenetic clock, and that resetting then has a benefit in terms of delaying risk for various diseases. And you mentioned heart disease. A grimace is a pretty good predictor of time to coronary heart disease. Surprisingly, these clocks, even predict time to cancer. These are no doubt exciting times for the aging field.

I want to tell you about two really profound experiments that tell us very interesting things about some of the drivers of epigenetic age. One is an accidental human experiment happening inadvertently in the clinic every single day. The other, an animal experiment, notable especially for its ingenuity. Scientists have long known that paracrine signaling, the molecular and cellular niche created from cell-to-cell interactions is extremely crucial in maintaining tissue health and integrity, and even as it turns out, youth maintenance. In other words, keeping our tissues young.

But what happens if you can bring an older animal into a younger animal's network of cellular signals on a daily minute-to-minute basis? That's the question. In animals, the experimental surgical union of their vascular systems known as paribiosis, leads to the transference of blood-borne factors from the younger animal into the older animal. Some evidence has shown that this can lead to a youthful phenotype and rejuvenating effect in certain tissues. While these experiments have been the excitement of the aging field for a while, despite their somewhat clinical impracticality, the results of Dr. Horvath's lab, he says, have been somewhat mixed.

However, humans have been doing a somewhat modified version of this experiment too, but inadvertently. Surprisingly, at least insofar as epigenetic clocks are concerned, the results here are even more exciting than animal research. Let's say you take a 50-year-old and you give this person a bone marrow transplant from a 20-year-old. And so after the transplant, the blood in the recipient recall. constitutes itself. The person has now new blood. And the question is, what's the age of the blood? Is that blood, does the blood have the age of the 20-year-old donor or the age of the 50-year-old recipient? There are now several scientific papers that really give an unequivocal answer, which is the reconstituted blood in the recipient has the age of the donor, you know. And that effect persists for

decades, you know. Bone marrow transplants are different from paribiosis. For example, in leukemia, it is sometimes necessary to irradiate, destroy, and ultimately replace the entire hematopoetic system via a bone marrow transplant. This ultimately results in the recipient having cells that no longer track with their own epigenetic clock. Rather, they track with the donors. That means if you're unlucky enough to have leukemia, but lucky enough to have a younger donor, you might ultimately be able to enjoy younger T cells, macrophages, white blood cells, and natural killer cells. A tissue-specific epigenetic age reversal effect that last, as you heard a moment ago, for decades. Unfortunately, cancer treatment as a whole does accelerate epigenetic age.

So that's on some level a very exciting finding because it kind of hints to an idea that you could possibly rejuvenate people through transplantation. The reason why this is not yet a viable strategy is because people who get a transplant often get so-called graft versus host disease. So there are all sorts of complications. It's a dangerous procedure, but in theory, it could work. There are a few experimental ideas for how to interfere with the epigenetic clock or otherwise rejuvenate the methylome. One is using drugs to play with inhibiting the enzyme.

that directly interact with DNA methylation known as DNA methyl transferases. These drugs already exist and in mice with disorders of the epigenetic machinery, these drugs are able to reverse some effects of those conditions. The other avenue, which is a bit more promising in my opinion, is manipulating the expression of genes that encode for transcription factors already known to reverse epigenetic age. Right now, there is an idea in the aging field to rejuvenate people, by leveraging this fundamental insight.

It's called reprogramming. You can take an old cell, you administer certain factors. Called yamanaka factors, these factors can revert a differentiated somatic cell back in epigenetic age to an embryonic or near-embrionic state. Even more interesting, scientists know that by genetically engineering a switch, one that is turned on by a chemical, they have been able to add these yamanaka factors to animals to reverse even certain aspects of tissue aging by pulsing these factors. But don't switch them on for too long, says Dr. Horvath,

because too much of the Yamanaka factors may revert cells too far back, ultimately inducing malignancy. One of the biggest challenges in repeating this experiment in humans, however, is that genetic engineering is not without risk. That, however, is for another conversation. What we really need is really dozens, if not hundreds of clinics, trials. Why just to have a chance for serendipity? Maybe it is as simple as a vitamin D supplement, but maybe you need something much more radical, like for example, a modification of the Yamanaka

cocktail, you know, or it could be plasma transfusions or so. You know, we as a field, we need to experiment, you know, with what kind of interventions work. Could be hormones, by the way. It could be a hormone intervention. Age-associated methylations are commonly found near genes involved in development. If you had asked an aging researcher five years ago whether developmental processes matter in aging, they would have said no. You know, many people think of aging as noise or wear and tear, you know. But these epigenetic clocks have really linked development to tissue dysfunction.

in a direct manner. An epigenetic clock is a continuous readout that links prenatal tissues directly to very old samples. Dr. Horvath issues a caution about using the epigenetic clock as a clinical biomarker. When I predict, for example, that you will develop heart disease in 15 years, you know, there would be a big arrow bar associated with it, plus, minus six years or so. Okay, so that is our long-winded intro. For a shorter intro, head to my YouTube channel while you will find a three-minute

primer on epigenetic clocks. You couldn't have a better pair to this interview. Find that by searching Found My Fitness YouTube or by going to foundmyfitness.com forward slash YouTube. It's just three minutes long. Let's get on to the actual podcast with Dr. Steve Horbath, where we talk all things epigenetic aging clocks. Hello, everyone.

I'm sitting here with Dr. Steve Horbath, who is a professor of genetics and biostatistics at UCLA. Probably one of Steve's most well-known contributions to biology is the development of what's known as the Horvath-Horbath aging clock. And since then, he's gone on to develop even more accurate aging clocks, which I'm so excited to talk about. I've talked about your work multiple times on multiple podcasts to multiple scientists. So thank you for spending time to talk. with me today. Thank you for your interest. Thank you for visiting me. Well, Steve, maybe we could kind of start at the beginning with the, what is this Horvath aging clock that you had developed? Like,

can you explain what it is? Well, the Horvath aging clock is what I sometimes call the so-called pan-tissue epigenetic clock. And so it is the most accurate molecular measure of age. It applies to all cells in the bodies, certainly all cells that have DNA, all tissues, all organs. It measures age in prenatal samples, in children, all the way to supercentenarians, people who are over 110 years old. So it's, and it measures age. So if somebody provides, for example, DNA from blood or DNA from neurons or DNA extracted from saliva or urine, I can very accurately estimate their age. Their chronological age, like how old they are in years.

Exactly, exactly. And that's already a deep question. So the clock does measure chronological age. However, it's of course not a perfect measure of chronologic age. There's always an error. For example, if I analyze the blood from a 50-year-old, the epigenetic clock may say, well, this person is actually 55. or 45. And so there's a small error, and this error is actually biologically meaningful.

You know, it's not just noise, but rather it is in part related to what people call biological age. That's super interesting. There, that basically the error was actually related to biological aging because that was the next thing I was going to say was, you know, people age at different rates. Like even, you know, obviously chronologically, they could be the same age. Yes. But if you look at a variety of biomarkers, in fact, there was a paper published a few years ago in P&AS that looked at like 18 different biomarkers. Yes.

They looked at glycated hemoglobin, so HBA1C, V-O-2 max, triglycerides, telomere length, immunosinensensens, a lot of the, you know, typical biomarkers that are clinically used to, like, determine health status. Yes. And it was basically found that people, you know, aged at very different rates based on those biomarkers. So some people biologically appeared much younger than their chronological age. And some people appeared much older than their chronological age. Exactly.

So how does, so how now you've developed a different clock that can actually predict? That's right. Well, let me comment first on the term biologic age. It's a very intuitive term. Most people have a vague understanding of it. It somewhat relates to morbidity risk or mortality risk and also aging. But strictly speaking, it's not well defined because different researchers have different ways of measuring biological age. Some people use clinical markers that you mentioned, various markers of glucose levels or lipid levels and so on.

on. Now, my take to measuring biologic aging is based on a chemical modification of the DNA molecule. It's DNA methylation, you know. So, and I mention it because depending on how you measure biologic age, you might get different answers. So a person might look bad in terms of glucose levels. And you would say, well, they age faster than they should. However, it could. turned out that according to methylation, they are actually in pretty good shape, you know. So, yeah. Have you seen that before where you can see people have, for example, like other clinical biomarkers that are not, that are unhealthy, like higher fasting blood glucose or maybe elevated

triglycerized, elevated C-reactive protein, a marker of inflammation? Do you find that those typically correlate well with the epigenetic age? I wouldn't say it correlates well. It correlates, you know. So people who have higher levels of inflammation and what you mentioned, the epigenetic clock goes a little bit faster. But the word is there's a weak relationship, you know, because it is quite possible that somebody looks,

turns out to be in good shape according to epigenetic aging rates. And the number one example I want to mention in this context, are actually people of Hispanic ancestry. Unfortunately, Hispanics often have higher risk for diabetes, the higher metabolic syndrome. However, according to the epigenetic clock, they actually age more slowly, you know. And so there's really this disconnect.

And this is actually an interesting disconnect because there's something known as the Hispanic mortality. paradox, you know. Hispanics, as I mentioned, have often a disadvantageous risk profile according to clinical biomarkers. But it turns out, on average, they live much longer than expected. They actually live longer lives than people of European ancestry, you know. And that association is paradoxical to a clinician who looks at clinical biomarkers. But according to the epigenetic clock, it's not paradoxical, because as I mentioned, we have found that Hispanics age more slowly, according to the epigenetic clock. That's very interesting. So in this case,

the epigenetic clock correlates more closely with lifespan than with clinical biomarkers of health status. That's right. That's right. Well, in a way, that's kind of good, because, you mean... Well, it shows it adds something, you know, so the epigenetic clock is actually very much under genetic control. Some people just inherit a genome that makes or DNA that really allows the epigenetic clock to progress more slowly. And so the heritability is about 40%, you know. And yeah, so in this sense, it's not just lifestyle factors. By contrast, some of the clinical biomarkers you mentioned are very much under under the influence of lifestyle, you know. So, you know, so. you can probably cure high glucose levels by just avoiding carbs, right?

And also high lipid levels by avoiding or taking statins, you know. So there are a lot of clinical markers that can be influenced with lifestyle interventions and pills. By contrast, we don't have many interventions that allow us to reverse the epigenetic aging rate. But do we know that? Have we been, we haven't been testing that though, right? Yeah, I mean, not really. I mean, in my lab, we clearly want to find interventions that slow the epigenetic clock, and by now many people are working on it, you know, and it's a gold rush. Who comes up with an intervention that affects the epigenetic clock, you know? So you mentioned that the irritability was about 40% of that, is that of the epigenetic aging?

clock or um okay so you because i you know i recall from your looking at these the um the epigenetic clock of these semi super centenarians people that are like 105 years old yes they had epigenetic aging clocks that were like 8.6 years younger than yeah that's true so that's true that's a manifestation of that so if you have a parent who lived until age 100 or 105 um then chances are that your blood is actually younger than the blood of a person of the same age, same gender, same everything, but whose parents didn't live until age 100, you know. So the offspring of centenarians obviously have a genetic advantage, hopefully, but also that is manifested in the epigenetic clock.

Yeah, so you see that their epigenetic aging seems to be slower. Yes. So that's one line of evidence, but there's a number. So people have these longitudinal epidemiological studies and they may have collected a blood sample from a person when they were, let's say, 40 years old. And then 15 years later, they get a second blood sample, you know. And so you can then ask the question whether a person who was aging quickly at the first blood draw, did they still age quickly at the second blood draw, you know? And the answer is yes. And conversely, you observe the same for people who age more slowly.

And that, in my opinion, this could already be observed when you study, let's say, a person at a young age, age 20. Draw their blood. Then if you followed them for 60 years, you would find this consistency. that people who are slow ages at age 20, they are also slow ages at age 60 or 80. So that does seem to imply genetic. I mean, unless someone dramatically changed their lifestyle. How stable are these changes in methylation, these methylation patterns that are so-called, you know, the aging clock? How stable are they over a person's lifetime?

I mean, they're... I mean, they're remarkably stable. So when we compare to any other genomic measurement, I mean, they would be far more stable than anything I'm aware of. They're far more stable than gene expression, proteomics, metabolomics measurements. All the omics are less stable. And that's really the biological reason why these epigenetic clocks are the most accurate measures of aging. It's just that methylation is so stable. And it's stable not just in vivo, but also when people collect DNA, so we've collected DNA,

and then we didn't store the blood tubes properly, so they melted, you know. And we extracted DNA and the measurements were perfect, you know. So even on a technical level, they're very stable. That's interesting because I've done experiments intentionally. almost the same where we were collecting blood samples from participants in a trial, and I was measuring DNA damage as biomarked by gamma H2AX. And I wanted to know how long I could have blood at room temperature before I started to get artifactual DNA damage happening.

And so I did time course and found that after two hours of blood being at room temperature, there's just tons of DNA damage. that's scary that's right so it would that wouldn't be the case with DNA methylation I know because I hired a phlebotomist here in LA to visit families to collect blood and this person didn't have an air conditioner in his car and it was the hottest day in LA ever so the blood tube melted you know that was my experiment and then I just couldn't send this phlebotomist out to go back to the families to collect blood you know I felt sorry for the family. So that's why we did this experiment, you know. And all I can tell you, we got beautiful data.

And so I see that over and over. And people sometimes ask me, what if you have a forensic sample, you know, so let's say a blood stain, you know, that was, let's say, in a room for a couple of weeks or even, let's say, a bone sample collected, you know, a couple of months later. All of these data in my opinion would still lead to very good methylation measurements for the purpose of measuring aging. Yeah, because it's so stable. It's just so stable. Do you find that because these methylation patterns obviously are changing with age, you were able to predict first chronological age with pretty 96% or so accuracy. Yes. And so, you know, obviously they're, they, while they're stable at the same time, they are changing. Yes. But do they change, like, is there a pattern of change? Like, do they change

you know, every few years, all of a sudden things rearrange, or is there like a pattern you can see where things start to change every, like, block of time? Yeah. Do you know what I'm saying? Yeah, I do. It's a good question, you know. I, so our epigenetic, let me start slowly and say the epigenetic clocks typically track several hundred locations in the genome, you know. For example, the pan-tissue clock is based on 353 locations in the genome. And a question is whether each locus changes, each location gains methylation in a continuous fashion, you know. And probably not, you know. So I think what happens is some locations gain methylation, others lose methylation.

And it's a bit random. But on aggregate, once you average hundreds of sites, you know, you kind of average out the noise, the variability. And that's, that gives rise then to this very accurate age estimate. Okay. I definitely want to jump into some questions on mechanism and stuff too. But before I kind of go into the woods, this predicting the biological age, your DNA methylation, pheno age. was, if I recall from reading your papers, which I read recently, was able to predict all-cause mortality, disease-specific mortality, like cardiovascular disease-related mortality.

That's right. I mean, that was pretty interesting to me. Just to set the stage. So we have some epigenetic clocks whose purpose is really to measure chronologic age, period. But then we have other epigenetic clocks that are really defined to be lifespan predictors. They are meant to predict time to death or time to major onset of a disease or what people call health span. How long are you healthy? And as you mentioned, we have actually two biomarkers.

One is called DNA methylation phenoh-age, but also another one that was named. named after the Grim Reaper, so the called DNA methylation Grimm age. Now these biomarkers were developed really for that purpose of predicting health span and lifespan as opposed to measuring aging. And the reason why we have these different tools is that we of course plan to use them in human clinical trials of anti-aging interventions. And in that context you want to see whether an intervention actually resets an epigenetic clock and that resetting then has a benefit in terms of delaying

risk for various diseases and you mentioned heart disease a grimage is a pretty good predict of time to coronary heart disease surprisingly these clocks even predict time to cancer and that is surprising because it's a measurement based on blood you know and so you could ask why would a measurement in blood be predictive of the onset of various types of cancers in other solid tissues, you know? You can predict it before other clinical diagnostics in some cases? Yeah, I mean, let me start out by saying, I'm not sure whether this biomarker is clinically useful, okay, because I'm very scared. People think they can now measure their blood, and I will

predict you will get cancer in 10 years. It's not at that level. However, if you have, for example, a study of 1,000 women and you measure that and somebody collected their blood in the 1990s you know and so for each woman you have follow-up information whether she developed breast cancer or when she developed breast cancer and if you then analyze the data you will find that biomarkers such as grim age and other biomarkers actually do predict onset to cancer in a statistical fashion you know the P-Vos and the P-Vos and value would be quite significant, you know. But as I said, I wouldn't claim that this is right now ready for prime time in a clinical setting for finding high-risk individuals, you know, because

the effect sizes are too small, you know. When I predict, for example, that you will develop heart disease in 15 years, you know, there would be a big arrow bar associated with it, plus, minus, six years or so, you know. So for the individual, it might not be useful. significant amount of time for a person. Yes, exactly. Yeah. What about, so disease states, you mentioned cancer. People with cancer or Alzheimer's disease or Parkinson's disease, what does, like, have you, you know, measured the epigenetic age of these individuals and does it look like it's accelerated aging? Yes. So we looked at blood samples from Parkinson's cases and controls and there's no question. There's an age acceleration effect in blood. It's minor. It's

one or two years, you know, but it is there. Alzheimer's disease, we looked at prefrontal cortex samples from the religious order study, you know, and again we found age acceleration in the prefrontal cortex. When it comes to blood samples from Alzheimer's disease, I think there might be a signal, but if there is a signal, it's very weak, you know, so what other disease did you mention? Cancer. I'm wondering, you know, cancer is a beast. I mean, there's so many different types and Yeah, cancer is complicated. So the exciting insight is that, yes, blood methylation data indicate that blood samples collected before the person developed cancer show a slight epigestion. age acceleration. So that supports the view that faster epigenetic aging is predictive of future

onset of cancer. And that finding has been validated by many groups. Problem is this association is weak. You know, you need really a couple of thousand people to observe it. But the question is, what about if we, for example, measured tumor tissue? Well, tumor tissue, the signal is huge. So if I, for example, when I analyzed breast, malignant breast tissue samples from women with so-called luminal breast cancer, the epigenetic age acceleration is off the chart. So their breast tissue is much older than expected. But it's complicated. Have you compared it to their blood? Like, is it tumor tissue the same? No, no, it would be different. I mean, I would say that, I mean, yeah, let's say in breast tissue, we find 10, 15 years age acceleration.

malignant tissue, but in blood, I mean, that the fact would be much smaller, if at all. That's very, it's very interesting because you mentioned the Alzheimer's disease, blood also very weak, very weakening signal. But if you mentioned the, if you measured actual brain tissue, which is where this, you know, that's a neurological disorder, you find a signal. Yes. The interesting thing is you said Parkinson's disease, you do measure the signal in the blood. Yes, that's true.

It'd be really interesting to know if the immune system is playing a role in Parkinson's disease. Yeah, I can tell you the following. So we did this study of Parkinson's people. Why? Because I was interested in epigenetic aging. However, my software also produces estimates of blood cell counts. And so it turned out that the blood cell counts, in particular neutrophils, were really highly elevated in Parkinson's disease.

Huge effect, you know. And so in certain ways, this was... completely surprising to me. But this finding has now been validated over and over. So yes, PD cases have highly elevated neutrophil counts. That's very interesting. Yes. So yes, immune system plays a role. We don't know the causal direction, you know. Is it first an immune dysregulation that gives rise to PD or is it the other way around? Yeah. There's been some interesting links to gut, the origination of gut and the Parkinson's and, of course, the immune system is involved. and the gut and all that.

So the reason I asked about the cancer tissue, though, was because, you know, another biomarker of aging telomere length, the longer the telomers are, it's associated with, you know, basically better biological aging in a way because your telomers get shorter with age. So it's assumed that longer telomeres means younger, right? Yes. But some cancers find a way to, like, reactivate enzymes, like, involved in, you know, building telomeres like telomerase, for example.

And so some cancers have longer telomeres. Absolutely. If you just were looking at that one biomarker, you'd look at that tissue sample and think, oh, this is, you know. Yeah, I mean, let me make a few comments about telomere length. And so as you said, by now we know that there is a U-shape behavior. you don't want telomeres that are too short and you don't want to have telomeres that are too long, you know.

And so that's the first statement. The second statement is telomere length per se is actually not a good biomarker for predicting onset of various diseases. Most diseases don't have a strong relationship with telomere length. In particular when it comes to predict, lifespan, you know, telomere length is actually a shockingly weak predictor of lifespan. For many years, people wrote articles where they claimed there is no relationship to lifespan. By now, the field has moved on to say, well, if you have very large data, you do see in a relationship to lifespan. But if we compared telomere length versus an epigenetic clock

such as grim age when it comes to predicting lifespan, time to cancer, time to coronary heart disease, I mean, there would be no comparison, you know, so in this sense, telomere length plays a very important role in certain disorders, you know, but it's just not a broad biomarker for aging. Right. How does the epigenetic clock, whether we're talking about the pheno age or DNA grim age, relate to other biomarkers of aging? So does it usually correlate, like if you have, you know, so there's immunosinessence,

which, you know, is associated with aging, DNA damage, inflammation, there's telomere length. Does it correlate typically, like in the same direction? Yeah, it would. So talking about grimage or pheno age, they would correlate in a consistent fashion. You know, they, so they would have a weak correlation with telomere length to give you a number, correlation point one. So it's actually a weak correlation, but yes, if you have a thousand people, you pick it up, you know. In general, telomere biology is really a different hallmark of aging compared to epigenetic changes, you know.

So they measure different aspects of aging. But, yeah, there's consistency. Tlemy biology has been, I interviewed Dr. Alyssa E.L. in the podcast, she works closely with Dr. Elizabeth Blackburn. Yes. She's at UCSF. And she has published some studies showing that, like, stress plays a big role in, I mean, big role. It plays a pretty good role in telomere biology.

So we can find that, like, different types of stress can actually affect telomere length. Yes. So lifestyle factors that affect the epigenetic clocks. So, for example, diet, exercise, smoking. or even education. Education. Exactly. So how do those lifestyle factors in general affect epigenetic aging?

Yeah. Everything your grandmother ever told you about living a healthy lifestyle is kind of corroborated by our epigenetic clocks. So for example, people who eat vegetables, people who exercise, also actually educational level, to some extent even alcohol consumption, you know, show a beneficial effect. Now, the problem is these effects are weak, you know. Again, you need a couple of thousand people, then you pick it up. In terms of statistical significance, there's no debate. These are, yeah, clearly these associations are there. But,

for the individual, the question is, what if I follow the perfect lifestyle? Do I make a big dent on epigenetic aging? And the answer is, unfortunately, not really, you know. And I mean, I'm as much of a health nut as many other people in Southern California, you know, so I'm trying to have a healthy lifestyle, but so yes, you want to avoid diabetes and all of that,

you know, and certainly you don't want to smoke, you know. But the truth is, a lifestyle intervention will never have a profound impact on aging at a population level. Because what I would like to do is I would like to increase health span by 10, 15 years. And in my opinion, lifestyle interventions won't get us there. In healthy people, okay? so let's say you have a friend who smokes and is obese then yes tell your friend to adopt a healthier lifestyle because for this person it will have a huge effect but let's say you take somebody like me who is reasonably slender doesn't smoke and now you tell me what about if you become a vegetarian

you know or what if you double the amount of exercise you do will you have a strong effect on my lifespan. And the answer is no, not really. According to the epigenetic... According to the epigenetic clocks. So your, the physical activity was only like a week. Yeah, physical activity. Yeah, exactly. Unfortunately, weak. So I want to say correlation 0.08 for people who know what that means, that's a very weak correlation. In blood, though. So because the question is maybe if we studied heart tissue, or muscle, maybe we would find a much more pronounced effect, you know,

but at least in blood, we didn't see it. Well, that sort of brings the question about tissue types too as well. I mean, you know. Yeah, example is the effect of obesity on epigenetic aging. Turns out obese people age faster in blood. However, the strongest effect can be found in liver tissue. So obesity greatly accelerates the epigenetic age of liver tissue. And so I think a lot of stress factors really have an organ-specific effect.

Conversely, anti-aging interventions also have an organ-specific effect. So, for example, when we evaluated the effect of post-menopausal hormone therapy in women, we found no beneficial effect in blood. However, interestingly, the Buckel epithelial cells, So the cells inside of your mouth, they actually revealed that women who took hormone therapy were aging more slowly in these cells. Oh, interesting. Yes. Many cell types are also epithelial cells.

I mean, many of your organs have epithelial cells. Blood cells are a little different. Yeah. And now the finding made sense because blood cells don't have as many estrogen receptors as buckle epithelial cells. So, yes. obviously if you have a hormone intervention you want to study cells that are susceptible to it you know but yeah right so that that really does um highlight the importance of of measuring different tissue types to see because i mean one well sometimes it's easy to kind of think

well whatever fundamental mechanism is regulating this aging process would do it in this all the tissues but it doesn't necessarily mean it will be the same either right exactly It's tricky. Yeah, I mean, it would be fantastic if a blood measurement is really a surrogate for all other tissues and organs, you know. And but it seems to be not the case, at least for stress factors, as I mentioned. Environmental stress. But also even genetic factors, you know. So some people inherit a genetic variant that accelerates the epigenetic age in blood, but not really

in brain tissue, you know? Oh, really? Even there, it's complicated, yeah. So there's genes that are regulating the epigenetics. Yes. Instead of vice versa. So I kind of lost track of, I was going to ask you something. Geez, it was important, too.

But, oh, I know what it was. I had read a study, I think it was one of your really good reviews that you published, where you talked about bone marrow transplants. Yes. Because the question is, you know, there's lots of these studies coming out with paribiosis where you can transplant young blood into, from, you know, animals into, you know, animals that are older and sort of have this rejuvenation effect. Yes.

So the question is, if you take cells from a younger recipient, and put it into an older bone, like, sorry, if you take cells from a younger donor and put it into a older recipient, so You're basically taking young cells and putting them into an older person. Yes. So the young cells will have a younger epigenetic age, obviously, than what was already there. Yes. The environment around the cells, the niche, so to speak, does that play a role? And does that, do those blood cells, like, have more of an accelerated aging?

Do they... Not really, yeah. So just to be clear, I want to distinguish paribiosis from these hematopoetic stem cell transplantations. Yes, yes. I can comment on both. But let me start out with the hematopoetic stem cell transplantation. Some people have a very severe form of leukemia, and therefore their bone marrow stem cells have to be removed.

And the procedure is dangerous. I'll start out with that. So that's why I say only severe forms of leukemia patients get that treatment. But yes, let's say you take a 50-year-old, and you give this person. a bone marrow transplant from a 20-year-old. And so after the transplant, the blood in the recipient reconstitutes itself. The person has now new blood. The question is, what's the age of the blood?

Is that blood, does the blood have the age of the 20-year-old donor or the age of the 50-year-old recipient? And you can make a case for both scenarios. But there are now several. scientific papers that really give an unequivocal answer, which is the reconstituted blood in the recipient has the age of the donor, you know, and that effect persists for decades, you know. So if you take, again, the 50-year-old, got a bone marrow transplant from a 20-year-old, you follow this 50-year-old 30 years, now he's 80.

Question is, how old is his blood? Well, the age would be now 50, because 30 years have passed. and you add that to the age of the donor, you know. And one would think that the stem cell niche in the bone marrow, you know, so could possibly affect the aging rate, you know, but it just isn't the case, you know. And so that's on some level a very exciting finding because it kind of hints to an idea

that you could possibly rejuvenate people through transplantation, The reason why this is not yet a viable strategy is because people who get a transplant often get so-called graft versus host disease. So there are all sorts of complications. You know, it's a dangerous procedure, but in theory, you know, it could work. And talking about paribiosis, where people have connected two mice, you know, where one my mouse is much older than the other one or much younger than the mouse. We just recently analyzed paribiosis mice, you know, it's unpublished.

And we found two results. One corroborates things and one refutes it, okay? But just to explain it. So we looked at cortex and also subventricular zone, deep white, um, matter in the brain and we found that mice that were a young mouse that was connected to an old mouse actually aged faster according to an epigenetic clock in mice so that part confirmed these parabiosis experiments so in other words you can age a young mouse but that's not what people are interested in they are interested in the opposite you take an old mouse and you connect it

to a young mouse and then you study the brain of the old mouse and you want to see that the brain is rejuvenated, you know. And for that scenario, actually our results were disappointing. We didn't see a rejuvenation effect, you know. And so now we're trying to get additional data because our first study was underpowered, but one of these months we will have a definitive answer. Yeah. For, you know, some of these animal studies are really good for trying to understand.

understand mechanism. And all of this data suggests, you know, you've got a clock that can predict chronological age. You've got a clock that can look at your biological age and also predict time to death, lifespan, the grim age. Yes. And, I mean, something clearly is changing these methylation patterns. Yes.

So the question is, what is that? Is there a chronic signal that's doing it? Or is it just completely under genetic control? like what is, what is, and maybe they're related, right? So what are your thoughts, when your thoughts on the aging process and even? Yeah, I mean, well, when it comes to these epigenetic clocks, this is the number one weakness of these clocks that we don't completely understand the molecular mechanism.

And coming back to telomere length, that's a great advantage of telomere biology. We really understand very well what regulates telomere length, you know. But yeah, with the epigenetic clocks, this is a very active area of research. Top biologists and labs are working on that very question, you know. And there are many theories. Some people think stem cell biology plays an important role, and that's probably true for many tissues. In certain ways, it could measure aspects of stem cells, for example,

how often a stem cell divided. The problem with that interpretation is that the epigenetic clocks work beautifully in neurons, which really don't rejuvenate over the lifespan. And yes, another group thinks that epigenetic clocks

might relate to circadian rhythm, so there have been some theories. Now, I believe that all the processes that play a role in development must be playing a role here. And the reason is because my original pan-tissue epigenetic clocks works actually beautiful in prenatal brain samples. It works beautifully in various in vitro studies of so-called three-dimensional brains,

you know, or in retina samples. So really it captures aging of gestational age. during development, you know. And that, and during development, there's really no noise, you know, this is a highly coordinated process. And so, so yes, these processes also must play a role, you know. Yeah, certainly one thing I had thought about, and I want to get back to development because of the stem cell thing, but one thing that came to my mind with this chronic signal is one particular gene that is methylated during early, early age. age, but then becomes demethylated as a person ages, is this P-16 ink 4A gene.

And it plays a role in cell cycle progression, meaning it basically stops the cell from going on to the next cycle. So obviously you don't want it to be active during development or early age because you want your cells are growing. But it also, so when it becomes... active, it stops stem cells, like hematopoetic stem cells, they aren't growing. Yes. So there's like this kind of role in aging where it's, you know, sort of basically stopping

stopping the stem cell from growing is going to have a negative impact on aging. Although it can be positive for cancer because, well, positive for the person that has cancer because it can stop a cancer cell from growing, right? Yes, yes. So the question is there's a group of demethylases that can take off the methyl group that become active and take it off of this gene. And they're activated by inflammation. So what I'm wondering is, is anyone looking at, obviously these methyl groups are changing.

And so the enzymes that are pulling off methyl groups and the demethylases, the enzymes that are putting them on there, methyl transferases, they must be doing something. Yeah, and people are looking at it. What's changing those enzymes? Like, is there a signal there or is it a gene, is it genetic control? or what is it, you know, that can change. Yeah, you make very good points. So if you want to understand the epigenetic clock,

clearly you start with so-called DNA-methal transferases or these tet enzymes. Why? Because they, on the one hand, add methyl groups or remove methyl groups. So that's a low-hanging fruit. And just recently, we and others, several groups have actually found no doubt when you interfere with these enzymes, you affect epigenetic age.

They're very exciting findings where people studied certain developmental disorders where mutations deactivated DNA methyl transfer rays or mutation rendered it overactive. And sure enough, all of these mutations in humans affect epigenetic age. And so at that level, we know it has an effect as expected. And the effect is pronounced. It could add five or ten years to a person or the opposite, you know. And does that correlate with the lifespan of what... See, we don't know. Exactly. That's the question.

So some of these children have a developmental disorder and there's various syndromes, you know. And so I want one is so-called SOTOS syndrome. Anyways, and so we see strong deviations in blood in both directions, plus five years, minus five years. So at that level, it's all confirmed. Plus we right now have, we do mouse crosses, you know, where we knock out these DNA methyl transfer raises and just to very carefully study it in a very controlled setting. But I can already tell you you'll find strong effects. Now, but the question you really ask is, well, on this, an upstream of those, you know,

what regulates the clockwork, you know, what tells the DNA methyl transfer rays go to this location, you know, and deposit and work your magic, you know. Right. And that's where we don't have an understanding yet. And that's why I mentioned the Jumanji demethylases, because that is, you know, just one, it's one group of demethylases that's particularly plays a role in taking off the methyl groups for the ink for a locus. And, and I remember trying to figure out, you know, inflammation. Yes.

And you just plays a role in aging. And so, yes. I had seen studies showing, sure enough, you know, TNF alpha, these things play a role in activating those demethylases. Yes. And so it's like, well, is that something that just over time, chronic, you know, that there's a threshold or what, you know, I don't know. Absolutely. So I don't. I don't know. I've never seen the experiment done with that particular. Yeah. No, I mean, I've seen results from other groups, you know, that look at that issue, chronic inflammation, or even looking at these, this, sorry, what are these?

Transposons, you know. So there's now some very exciting results that show that some transposons become active in older tissues, you know. And so I've seen some. preliminary data where people said this was associated with increased epigenetic aging. And also our finding that HIV is very much associated with accelerated epigenetic aging also points again to this idea of a viral component. So clearly there must be a connection, you know, and it will be very interesting to tease it out, you know.

I don't have a good sense how strong that. effect is, you know, does, for example, chronic inflammation or do these enzymes that you mentioned, you know, how strong is the effect if we perturb it? Do they explain 30% of the variability or just 10%? You know, so that's in certain ways the question. Have you measured the epigenetic age in stem cells versus already differentiated cells? Is there, is there a different pattern or a different Yeah, there is a different pattern. So if you take, for example, a stem cell, an IPS cell, and then differentiate it into a more mature cell,

for example, a more mature neuron, you will find that the epigenetic age increases. The issue is it doesn't increase by a lot, you know, so a more differentiated cell, a more mature cell, maybe maybe one or two years older than the stem cell. The question is how do we age a cell by 30 years, you know, because when you study neurons in a dish, you want to perhaps understand neurodegeneration. And so you want to study very old neurons, you know. And so when you deal with

neurons derived from stem cells, we don't have a good way to age them. But nowadays, people pursue this idea called transdifferentiation. So you take, for example, a skin cell. and you add certain factors, maybe microRNAs or what have you, and then turn the skin cell into a neuron. And this transdifferentiation protocol actually preserves the epigenetic age, you know. And so we have shown in collaboration with several groups that, yes, the resulting neuron has the epigenetic age of the skin cell. Oh, that's interesting.

Yes. So what about when you take a skin cell? and you mentioned induced pluripotent stem cells. And you basically can add, you know, like four transcription factors, I think even less now. But the original from Shinya Yaminaka, four transcription factors and make this into like a pluripotent stem cell that can become any type of cell in the body. The epigenome, what happens to the epigenome? You completely reset it. To a young, completely young.

Completely young. So that's like the ultimate. Yeah, it's the other. reversing aging, right? It's true because in the past people were wondering, are there interventions that actually reset the epigenetic clock? And the number one proof of principal study is really the administration of these Yamanaka factors

because it completely resets the age, actually to a prenatal stage, you know. And that's completely genetic, right? I mean, you're doing your manipulating genes. Yeah, I'm not sure. Yeah, I mean, it's really, so also messenger RNA can do it. I guess what I mean is it's not, it's, it's not, it's something that's kind of under a genetic control. Yes, yes. Like, ultimately. I mean, right now, there's an idea in the aging field to rejuvenate people by leveraging this fundamental insight that you call, it's called reprogramming. You can take an old cell, you administer certain factors.

as you mentioned four factors or three factors or other variants you know and by doing by administering this cocktail of factors for just a few days not too long because if you administer it for too long you greatly increase the risk of cancer you know and because the the cells lose their identity the skin cell forgets that it's a skin cell it thinks it's a stem cell, you know. But if you do it briefly for, let's say, five days, you get the benefit of rejuvenation. You may have rejuvenated the cell by five years or ten years, you know, but it still remembers its identity, you know. So does the epigenome reset a little bit? Like it only Yes, yes. So there are a couple of groups that are working on it and have already shown that

effect, you know. Yes. So I analyzed fiboblasts and enderoblasts. And plethearial cells from from such an intervention. It's called sometimes interrupted reprogramming or transient reprogramming, you know, and sure enough, you know, that idea worked. It reset the age, but the cells still remembered their identity and therefore arguably will not become malignant. Right. Yeah, that's, that is super interesting. I, yeah, I have to say that's, And the epigenetic clock is the ideal biomarker for that kind of a study, in my opinion, because, as I said, it very much tracks, you know, the state of the cell, you know, and, but we will see, you know, hopefully in five or ten years, it turns out that this

strategy turns into a viable anti-aging intervention that can be used in the clinic, you know. But at this point, these are all proof of concept studies that have been done in the dish, or in mouse studies, you know. Do you personally think that the epigenetic clock plays a causal role in aging? Yeah, let me answer it in two ways. It clearly relates to a process that plays a causal role, you know. And so to use a metaphor, is it the face of the clock, you know, or is it the clockwork of aging, you know?

And no doubt the epigenetic clock must relate to at least, one causal process because it predicts lifespan, you know. If it didn't relate to a causal process, it wouldn't be able to predict how long you live. Now, but the real question is perhaps what if you changed the methelome, you know, if you had an intervention, you changed DNA methalerases, you kind of, I want to call it the superficial way of perturbing the clock. that, so would that have a benefit? And personally, I don't have an answer to it yet. Have you ever looked at how, so you mentioned obesity and how obesity is associated with

an accelerated epigenetic age? Yes. Have you ever come across data with respect to the opposite of that, like fasting or even caloric restriction, if that slows epigenetic aging? Yeah, I mean, In mice, the answer is clear-cut. Definitely, caloric restriction slows the epigenetic clock in mice. And we know that because several groups have looked at it, including my group, all of us arrive at the same answer. Conversely, by the way, high-fat diet, you know, accelerates the epigenetic age of mice, you know.

So that's all clear-cut, you know? The question, though, is really, what about humans, you know? And I'm not aware of a study where people really looked at caloric restriction versus epigenetic aging. And but let me share some thoughts. When it comes to any intervention, including dietary intervention that prevents anything related to metabolic syndrome or diabetes, that will be detectable, you know. So in other words, if you have an intervention that takes an very obese person to a lean person, in my opinion, grim age will pick that up. It has to, you know.

However, when you ask the question, what about if you take a relatively lean person, healthy body mass index 23, and you and this person is so motivated to live 10 years longer and they pursue now a life, where the BMI is, let's say, 17, you know, almost at the point of being unhealthy. And so if you compare a healthy person versus a semi-starved person, would then, would that have a benefit? And in my opinion, that may not be the case. And I'm just speculating here. But even in, according to other biomarkers, I personally, I haven't seen, um,

convincing studies, you know. Otherwise, I think my BMI would be 17 right now, you know, so I, yes. There's an acquaintance of mine who has, he runs a pretty popular aging blog. His name is Josh Middlethorf. He's reached out to me, I don't know, a few months ago, because he's trying to organize a study where people that are basically already practicing some type of fasting, whether it's an intermittent fasting, they're doing, you know, a 16 or 24-hour fast, or sometimes maybe they do prolonged fasting, which in some cases can be, you know, longer than 48 hours, 48 hours or more. Yes.

People are doing this stuff, you know, it's just, it's just happening. I'm trying to, too, trying to do this intermittent fasting, but I don't have much self-discipline, but yeah, we will now analyze mice, you know, from. the lab from Joe Takahashi, who did various interventions. And probably in a couple of weeks, we will have some answers, you know, whether these strategies have a strong effect in various tissues in mice, you know. But yeah, when it comes to human data, that's really the prize. It is.

And as you mentioned, you know, you have someone that's starting off with already what you would think is healthy, someone who's not obese, someone who exercises doesn't smoke, someone who, you know, is, you know, eating a relatively good diet, you know, if you take that person who is already pretty healthy and then do an intervention. So is that intervention is going to be, whether it's like an intermittent fasting. So I mentioned Josh Middorf because he wants to track. You know, there's a company that does, based off of your clock, I don't know which clock, the Horvath, original clock or the pheno age, but they, they're called my DNA.

my DNA age. I just ordered there. I just ordered it, so I'm going to get, it just arrived, I'm going to get my blood tested. It's the original clock. It's the original. Except they have modified it a little bit, you know. So that's probably better at predicting chronological age rather than biological age. Well, I think nobody knows, you know, because these biomarkers should really be evaluated in a prospective study. Yeah. Because so that's the question. It's like, well, if you have these types of interventions on already healthy people, will they make any difference? At least according to epigenetic aging, right? I mean that. I mentioned before we started filming, I mentioned to you a pretty recent randomized controlled clinical trial with vitamin D supplementation

in a population that started off unhealthy. So these were obese, African Americans, which were very low in vitamin D. African Americans tend to be the lowest in vitamin D. because they have a natural sunscreen. And so they were given 4,000 I use a vitamin D a day. And after, I can't remember how long the trial was, a certain period of time, their epigenetic clocks were measured at baseline. And after and their epigenetic clock, the horbath clock was used. It basically was reduced by 1.8 years or something like that.

And you mentioned the significance is a little, the sample size is small. And so it's kind of like, well, it's a start. finding. I think it was based on 51 people and it's a nice finding. It would be brilliant. A simple intervention as taking a vitamin D supplement actually affected the epigenetic age. I think I agree with the authors. We just need larger studies to validate it, you know. But in general, you know, as you know, clinical trials are very expensive, you know, and that's a real bottleneck in all sorts of anti-aging interventions. We really wish, on the one hand, private industry would find merit in investing in clinical trials. Obviously, also the government

plays a role, but what we really need is really dozens, if not hundreds, of clinical trials. Why, just to have a chance for serendipity? Maybe it is a sense. simple as a vitamin D supplement, but maybe you need something much more radical, like, for example, a modification of the Yamanaka cocktail, you know, or it could be plasma transfusions or so. You know, we as a field, we need to experiment, you know, with what kind of interventions work. Could be hormones, by the way. It could be a hormone intervention. Well, you mentioned, obviously.

Do you see, I'm going to change topics. here, but I don't want to because, you know, the intervention trials are important, you know, as you mentioned, it may have some effect, it may not have a big effect. But, you know, even if a combination of factors like, you know, getting your fish intake, which you've shown to also, you know, seems to be related to. Interestingly, according to grim age, we did find that people who used omega-3 supplements of fish oil, they were actually aging more slowly. And we thought this was a nice little insight. Oh, that's interesting. Because, I take fish oil supplements.

Me too. Yeah. Because what happened is I want to say six months ago, there was a publication, really very large-scale clinical trials, looking at fish oil supplementation. And they did not observe any benefits. And I said, oh my God,

I wasted all these years eating fish oil. But then we analyzed really an observational study. And that's our problem. Our study was an, epidemiological study. I want to say the Women's Health Initiative. And there we did see this association that women who took fish oil supplements were aging more slowly according to grim age. And I'll just add a note to that study. You mentioned the big Vital D study, which was a vitamin D and omega-3. They did a huge study. There was no effect on the primary outcome, which was looking at all

combined cardiovascular related events. However, when you specifically looked, at heart, like heart attack versus stroke, there was a strong effect. So it's always this, you know, the secondary outcome, like whatever, however you're designing the trial, you know, there's a primary outcome. If that primary outcome is negative, then it's like, well, you know. The problem with the primary outcome is that it's far removed from the processes that are being targeted by the molecules, you know. I agree.

And that is really the benefit of having what I call a surrogate marker, you know, something like Grimm age is arguably much closer to the mechanisms, is perhaps less susceptible to confounding factors, you know. And so maybe that's why these surrogate markers pick it up, you know. Right. Yeah, I mean, obviously the methylation patterns, these are molecular changes that happen before someone has a heart attack, before someone has high blood pressure. I mean, these are accumulated, you know, changes at the molecular level that are occurring. And also, when you think about heart disease, it probably relates to many confounders such as psychosocial stress. You go through a divorce, you know, you get fired, various substance abuse, you know.

A lot of processes that are in certain ways stress factors, but they are not part of what I would call innate aging processes, you know. Whereas these biomarkers, such as an epigenetic clock, is hopefully much closer to an innate ageing, process, you know. And when we want to cure aging, we want to cure innate aging. We don't want to, it's not my goal to reduce people's stress levels, you know, for that they should do yoga or any other intervention, you know. Right. Sleep. I mean, obviously sleep is another thing associated with heart attacks and I don't know if sleep is even usually one of the confounding factors that's adjusted for, right? I don't even think that's something I usually see. You know, we looked at people

women who have sleep disturbances in the women's health initiative. And sure enough, their epigenetic age of blood was slightly accelerated. That makes sense. Yeah. I talked to a sleep expert not long ago. Dr. Matthew Walker, he runs the human sleep center at UC Berkeley. And he just talked about all the, you know, basically various diseases and, you know, all cosmortality and how everything just goes up when sleep quality goes down.

And so I certainly feel like, you know, I always, I always hate these studies because I don't sleep well. I like the studies where they study the so-called super sleepers, you know, who sleep only five hours at night and still are perfectly healthy. So I like more the optimistic spin on things. But also, just to tell you, so the effect of sleep quality, you know, versus epigenetic aging in blood, these associations were statistically very significant, but again, they were weak. you know, and that makes me hopeful. Yeah, compared to the semi-supercentenarians,

which had an epigenetic age of 8.6 years younger than they're chronologically. That's pretty robust, right? That would be a robust or more strong, I guess. Yes. Yes. I want to say it was maybe not eight years. I want to say it was five years. So it depends on what you compare. Not the offspring, but the actual person.

Yeah, that's right. Yeah. So you're right on. So if you analyze the blood from a centenarian or super centenarian, it's true our age estimates are really way below their chronologic ages. Could be 15 years younger, you know. So there's a real leveling office effect, you know. Yeah.

What about studying how other, like there's other biological processes that at least in animals when you perturb them are known to regulate aging, for example, and you can mutate certain mitochondrial factors and have accelerated aging or, you know, cellular senescence. You can also have a certain mouse where you're, you can accelerate aging or the opposite where you lower growth hormone levels and they'll, you know, they live longer. Have you or anyone looked at the epigenetic aging, how that relates to these other? To some extent, yeah.

What you mentioned, these growth hormone knockout mice that are known to live longer, sure enough, according to the epigenetic clocks in mice, they really age more slowly. So it was a beautiful validation of the fact that epigenetic clocks measure biologic age. Because growth hormone receptor knock out mice, that's really a gold standard anti-aging intervention. And you want that a clock picks that up. And when it comes to other strategies, senescence, right now, it's a hot topic, the so-called xenogenes, that remove senescent cells, you know. And we are about to analyze data collected by James Clement, who looked at, who did

clinical trials of these xenolitic strategies. And hopefully we'll have an answer in a couple of weeks, you know, but I just don't know whether removing senescent cell has an effect on epigenetic age. In general, the relationship between senescence and epigenetic age is complicated. Because when it comes to inducing senescence, there are several ways of inducing senescence. One is simply what is known as replicative senescence. You passage cells, you split them and let them grow and grow and grow. And that form of senescence is somewhat related to epigenetic clocks, you know.

then there are other forms, so-called radiation-induced senescence. You radiate a cell, and that form of senescence doesn't seem to accelerate the epigenetic clocks, you know. So it's complicated. And conversely, there are ways of immortalizing cells by overexpressing the component of the telomerase, the turd. Now that, so immortalizing a cell actually doesn't see. stop epigenetic aging, you know. You can have immortalized cells that you can passage for decades,

but the epigenetic age keeps going up in the cells. Yes. So do you see, epigenetic clocks are not simply markers of cellular senescence. They really pick up a different aspect of biology. The radiation and DNA damage is kind of surprising because I think in one of your papers looking at, and this was another question I wanted to ask, was where are these methylation patterns occurring in the genome? Are there genes that are really particularly known to be involved in the aging process, you know, like, or, you know, basically in just health in general? Yes. Like, are these, like, you know, the metabolism genes or DNA repair and things like that? Yeah, I wouldn't make that claim. So let me start out by saying, so my

original clock used 353 loci. When we look at Grimmage from Akelu, my scientist in my lab, it uses over 1,000 locations in the genome. And now one question is, what if we remove these locations from our data and just build a new clock? Would we still get another good clock? And the answer is yes, you know, I could have built alternative clocks using other locations. in the genome, you know. On that level, these locations are not unique, you know. And when you look at the genome, we have, in principle, 28 million locations in the genome are cytosines, you know. And I want to say a quarter of them change with age. Some of them gain methylation, some of them lose methylation. So these methylation changes are almost global, you know. And in that sense,

epigenetic clocks look at perfect representatives of the entire what is known methalone they represent everything that's going on but you can see that maybe looking at only 300 locations is it's not ideal you know having said this we certainly did look at it and say are these locations enriched you know with certain pathways and no doubt there are, you know. So sites that gain methylation with aging are known to be located in so-called polycombe group protein target sites. So certain proteins that play a very important role in maintaining stem cells, you know, or conversely, sites that play a role in cell differentiation and development. So these sites tend to gain methylation with aging, you know. The sites that

loose methylation. Also, I enriched with certain themes, for example, often they're in so-called enhancer regions, you know, so the field of epigenetics has very much characterized the genome, which parts change with aging. There's wonderful review articles on it. And then the, I just got on the stem cell thing, that's just so interesting that a lot of those or regulating stem cell function because it's just... Coming back to the mechanism of the clock, that's really a profound insight, you know, that when you look at the data,

you keep seeing themes related to development, tissue differentiation, organ development. And it is a profound insight, because if you had asked an aging researcher five years ago whether developmental processes matter in aging, they would have said no, you know, Many people think of aging as noise or wear and tear, you know, but these epigenetic clocks have really linked development to tissue dysfunction in a direct manner.

An epigenetic clock is a continuous readout that links prenatal tissues directly to very old samples. It's really blowing my mind. It's actually that is, I never would have thought. field, you know? Because many people obviously study development, but these are not aging researchers, you know, but these clocks really point to commonalities, you know, these fields. Wow. It's just, it's amazing. It's very interesting. I just want to thank you so much for doing all the work that you're doing and we'll continue to follow, you know, your work. Thank you for your interest, yeah.

People that want to learn more about your research, probably the best place is Wikipedia. We talked about I mean, I've written a review article and nature reviews genetics. People have written Wikipedia pages. Then you mentioned the blog by Josh Middletorf, you know, so there are various forms of learning about them, you know. Well, thank you, Steve, so much. I really enjoyed this conversation. Great, thanks.

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