Scientist Stories: Cynthia Kenyon, Genes From the Fountain of Youth

So I'd like to introduce our first speaker this morning, Professor Cynthia Kenyon, who is a professor at the Department of Biochemistry and Biophysics at the University of California in San Francisco and director of the UCSF Hillblom Center for the Biology of Aging. She's also an American Cancer Society research professor. Simply quoting from the Dan David book, I can tell you what most of you probably already know, that Professor Kenyon is recognized as the scientist most responsible for first showing that the aging of the C. elegans worm is under genetic control, for identifying key genes involved in the regulation of aging, and for demonstrating the mechanisms by which these genes control aging through visionary and rigorous studies. With C. elegans, she showed that the hormonal pathway controlled by insulin-like and IGF-1-like hormones is a major determinant of the rate of aging in the worm. Professor Kenyon has won many awards and honors, and I'll just mention a few of them. She is, of course, a member of the National Academy of Sciences of the United States. And of the American Academy of Arts and Sciences. She won an honorary doctorate from the University of Paris, the King Faisal International Prize for Medicine, and many more awards. And she will tell us this morning about genes from the fountain of youth.

Professor Cynthia Kenyon. Thank you. I'm so honored to be here and to be the recipient of the prize, and I thank the Dan David Foundation for that very much. I'm going to get my talk prepared now. All right, so I'm going to be talking about aging. This is the word, the bright side of aging, the Fountain of Youth. I just want to start by giving a little historical context for this. It really wasn't that long ago that people just thought that you just wore out, that's it. That there was no control over aging by the genes particularly. There were even evolutionary biologists who said there couldn't be control over aging by the genes. But then, you know, if you look around you in nature, you see that different organisms can have really, really different Lifespans. It's really amazing, the differences. And obviously the reason these animals are different from one another is because they have different genes. So at some level that tells you that genes control aging, or the rate of aging. And so I started thinking about this, and you know, I worked at the time on developmental biology, how the fertilized egg turns into an animal. And we, the people in my field at the time, this was in the 1980s, were We were realizing again and again that most, most, even the tiniest little aspects of making an animal were controlled by the genes, and they did so in an incredibly elaborate and complicated choreographed way. And it was also conserved evolutionarily. What happened in these animals that we studied was very similar to what happens in humans and all other animals. So it was very easy very easy for me with that kind of background to think maybe aging is regulated also. It was very simple for me to think that because of my history. And in fact, I had this idea that maybe there was some kind of a dial and, you know, it would be turned up in the mouse so the mouse would age really fast, but it would be turned down somehow in evolution in the bat so the bat would age very slowly. So I got really excited about the idea of looking for genes are genes that control aging. If there are genes for aging, then if you change them, you should be able to change the lifespan of the animal. And so we didn't study this in people, obviously. Instead, we chose my favorite little animal, C. elegans, which looks exactly like this. And they're very tiny, about the size of a comma in a sentence, and they live in the soil, they're harmless. Okay, so what we did was then to look for genes that when you change them could make the animal live longer. And there actually already had been a gene that was reported to make a gene change that could extend the lifespan, but not much was known about it. There was a lot of controversy about it. So, um, so we set out to do this, and we were really lucky because— oh, this is my slide about my hope, which was really at the time a huge hope. First of all, that there even would be genes for aging, and secondly, that they would be evolutionarily conserved so that we could learn something something about higher organisms from it. Anyway, we were really lucky. It took a long time actually for me to find anyone that wanted to study aging in my lab. They didn't in my lab, so I had to get incoming graduate students who hadn't yet chosen a lab and just were just floating through my lab for a few months before they could— I wouldn't even let them talk to my lab members before I asked them if they wanted to try to look for genes that control aging. And one of them said yes, and he amazingly enough found out that if you make a mutation— oh, by the way, for the scientists in here, I made a talk that hopefully someone who's not a scientist can understand at least somewhat, so you'll see words like this instead of hypomorph, you might see damage. Okay, so anyway, mutations that inhibit the function of this particular gene called daf-2 doubled the worm's lifespan. So this was an amazing, amazing result because First, you can see that in this lifespan curve, by the end of a month, all the normal worms are dead, but most of these altered worms, these mutant worms, are still alive, and in fact, it's quite a long time until they all die. And this was really amazing because these worms have 20,000 genes, and all the other 19,999 genes were exactly the same. Just one gene was changed, and yet the whole animal stayed young. Young and active and aged more slowly than normal and lived twice as long. So all the other genes, even though they would always be decaying after just a few weeks, were capable of keeping the animal alive as long as you changed this one gene. Okay, so that automatically suggests that aging is subject to some kind of control by the genes. I want to show you a movie so you can see how amazing these worms are. So this is— you see immediately why they call them elegans, because they're very beautiful here. And they really are. And so here is the long-lived mutant when it's young. That's about a graduate student age, maybe college student age. And this is the same thing with the long-lived mutant. And the reason I'm showing you this is because I don't want you to think that these worms are miserable. If they are, they don't show it, that's for sure, because they look really good and they can be completely fertile too. So here, just look at this, in just a couple weeks the normal worm is already on its deathbed. The little head is moving here, but the rest of the animal is just lying there. And that's— so they age really quickly, and this is great for studies of aging because you can do an experiment and then you can do another experiment and another experiment. Here's a dead worm and another old one. And also, even if you've never saw a C. elegans before, which probably you didn't, you can see that they're old. Which I think is very interesting, right? There's something kind of universal about the old phenotype. Now this is our champion worm, the daf-2 mutant, at the same time. See? And so it should be dead or almost dead, and it's not. In fact, it's quite young. Not as young, but still quite young and very youthful and moving around and everything. So I have a little analogy that I like to make, which is that suppose you're in your 40s 40s, and you're, um, the reason I make this analogy is because people usually think when you tell them that the worm, you know, when it's, it would be like a 90-year-old person who looks younger, they think, okay, I can imagine a 90-year-old who looks really good for 90, maybe healthy, walking around, but that's not what I mean. This is what I mean. Suppose you're in your 40s and you're single and you're dating and you find someone that you really like. Let's say that you're a man and you find a woman that you really like. And you go to a restaurant and pretty soon you ask her, "How old are you?" and she says, "I'm 80." That's what it's like. It really is. And see how it's kind of a shock, a jolt to the system because we're not used to the possibility of anything like that even existing. So what we're talking about here is something that no one even really imagined could happen, really. Although if you think about it, it had to happen in evolution, right? Because the little— assuming that we had very short lifespans lifespan back many, many, many years ago, the increase in lifespan had to come through changes in the genes. So, and that's why, you know, a person who's a month old doesn't look old like the worm does, it looks quite young. So it's been with us, if we just thought about it that way, we would realize that, you know, it would be possible to be young when you should be old. But I think this is It's because this is one species and you make this change, it's much more vivid. Okay, so now of course we want to know every possible thing we can about this gene DAF-2. How does it make an animal live longer and stay young longer? So the first thing I want to tell you is that the DAF-2 gene was already known. We weren't the first ones to discover this gene. It was known because it played an important role in the childhood of the worm, in its journey to adulthood. And it turns out that little sea elegans has a very interesting way of growing up. If it has a lot of food, then it just grows up through these 4 little molds to an adult and then it lays its eggs and so forth. So that's if there's a lot of food. But if there's not a lot of food around and the animals crowd together, then what happens is they exit from this growth half here, and they have a kind of a timeout where they enter a stage called the dour stage, which looks kind of like this. So dowers are about the same size as their counterparts here, but they're skinnier, and they're also, they have these little kind of shells on them. They can move around, but they have these kind of armor on them, and they can't eat, and they can't grow, and they're protected, they don't die if they dry out, and they live for a long time. And then if food appears suddenly, they can sense it and they come out of the dour stage and then they grow up and then they become normal adults. So it's like a timeout or a checkpoint or something that they have that allows them to wait if conditions in the environment don't really favor the idea of having a lot of progeny, like not having food. Okay. So it turns out that our gene, DAF2, controls the entry, the decision to go into the dour state. If you have a mutation that completely knocks out the DAF2 gene, like it deletes it, then what happens is the animals just become dowers, even if there's food around. So the DAF2 gene, the normal function of the gene, is required to allow the animals to become adults. Now, in our DAF2 gene change that I just described, I told you about. We didn't completely knock out the function of the gene, we just made it a little less robust than normal. So the animals, our mutants, could grow right up to become adults and they lived longer. Okay. And actually we showed, later on we showed using a technique which I would call timed RNAi for scientists, we showed that DAF2 acts exclusively in the adult. To affect the lifespan of the animal. So obviously it acts at this time in the childhood to determine whether the animal becomes a dour. But then it has another job to play in the adult where it controls aging. But the dour of— so it's not that the gene has two different functions, but its connection to the dour pathway is an interesting one, and I'm going to come back to that when we think about evolution of aging programs. In a little while. Okay, so, oh yeah, I forgot to say Gary Ruvkin's lab is one of the labs that have been studying the dour pathway for a long time, and when we discovered that the daf-2 mutants lived long, Gary was already cloning the daf-2 gene to figure out what it was doing, what it was. And what Gary discovered was that the daf-2 gene encodes a hormone receptor. And okay, so now here's a cell, And this is a hormone. I hope most of you should know what a hormone is. It's like testosterone or estrogen are very well-known hormones. They circulate in the body and they come in contact with the tissues, and the way that the tissues know that they're there is because they have receptors. So one end is sticking out, which grabs the hormone, and the other end is inside the cell or the tissue, and this end of the receptor can send signals into the cell to tell the cell what to do in response to the hormone. So what we had showed was that the normal function of the normal gene that's not damaged is to speed up aging, the normal DAF2 gene. Because remember, when we inhibit it, we live longer. So that's actually really interesting. It's like the animal has the Grim Reaper inside of itself. DAF2's normal function is to say, get old, get old. You would think that why would an animal have that, but it does. So anyway, so together our These findings I think were really, really, really important because they say that hormones control aging. And actually in this case, the hormones are speeding up the aging process. So when you clone any gene in C. elegans and determine its DNA sequence, the first thing that you do is compare it to all the other genes that have been sequenced in the whole world. And what you find immediately is that the DAF-2 gene, the hormone receptor gene, is very very similar to two hormone receptors that we have. One is the receptor for insulin and the other is a receptor for a hormone called IGF-1. So in worms, this receptor is called DAF-2. In humans, it's called insulin receptor, IGF-1. There are two different genes for these receptors in humans, whereas there's only one gene in the worm. So there was a duplication during evolution. But anyway, these hormones have been known for a long time. Insulin is known obviously to allow your tissues to take up the food that you eat, the nutrients that you eat, and it also tells the tissues to store the food or use it for energy. And IGF-1 is a little less well known, but IGF-1 promotes growth. So for example, pregnant women have a lot of IGF-1 that's allowing their unborn child to grow. So these were the known functions of, um, insulin and IGF-1, but what our results suggested was maybe they have another function, a third function that nobody knew about, which was to control aging. So this is a question that comes up immediately the second that you know from Gary's work that the DAF-2 gene encodes a hormone receptor like these human receptors. So I'm going to come back to this question of evolution in a little while, but first I'm going to talk a little bit more about how hormones ultimately affect the rate of aging by studying the worm. Okay, so the first really important entry point into understanding how the DAF-2 gene works came from our discovery that there was another gene that was needed for the DAF-2 mutant to live so long, and this was a gene called FOXO. FOXO. Now, for those of you who work on skelegens, it has another name called DAF16. It's the same thing, but I'm going to call it FOXO in this talk. So a gene called FOXO is activated in the DAF2 mutant, and this FOXO gene is like a fountain of youth gene. It extends lifespan. So here you see this long lifespan again of the DAF2 mutant in red here, but now you see what happens if you take away the FOXO gene. They have actually a slightly shorter lifespan, lifespan. They actually get old slightly faster than normal. They age a little quickly. And if you have a DAF— I mean, a FOXO— an animal that has no FOXO gene, and now you introduce a DAF2 mutation, nothing happens. It doesn't budge. It still has this lifespan. Not this one. Okay, so the hope then is by understanding what FOXO is doing, we can understand how changing the DAF2 gene can make the animal live longer, because FOXO FOXO is needed. So what is FOXO? The FOXO gene encodes what's called, for the scientists, a transcription factor or a gene regulator protein, which is a protein that turns genes on and off. So here's a picture of this. Here's a little bit of DNA here, and here you see one of these gene regulator proteins sitting down on the DNA. So what this says is that in the DAF2 mutant, that is in an animal that basically thinks it has lower levels of insulin and IGF hormone signaling, the FOXO protein is turning on a whole bunch of genes in the DNA that are making the animal live longer. So I'll show that here. So basically, here we have an abridged version of the DAF-2 pathway. It's more complicated than this. But the hormones bind to DAF-2 receptor. Activates a highly conserved set of events that happen in the cell. Okay, this was shown— the identities of these proteins were shown by Gary's lab. And then our lab and Gary's lab and Tom Johnson's lab all showed that in normal conditions, most of the— this is Daf-16, which is the worm name for FOXO. Most of it is normally out here, not in the nucleus where the DNA is. Just a little bit is where the DNA is. Where the DNA is. And that's why the animal has a normal lifespan. If you take it all away, there's nothing in the nucleus and then the animal gets short. But you want the gene regulator protein to be where the DNA is, here. And that's what happens, it turns out, when you inhibit either the DAF2 gene function or the functions of these signaling genes here. Now all the DAF16 FOXO protein goes into the nucleus and all these genes are turned on. Okay, so there's a little— it's kind of like a switch. There's a— the outside of the cell, there are these hormones, and they are the signals, the ultimate signals. And when they change, or when we experimentally change the ability of the receptor to respond to the signals, okay, then things happen inside the cell. And the main thing is this little fellow scoots into the nucleus and binds to lots of genes that it turns on. So what are these genes? Well, there are these magical lifespan-extending genes. There are very nice ways now, as most of you probably know, to figure out what genes are active or inactive under certain conditions. And so all we had to do is use this technology called microarray technology to ask what genes are either more or less active in the daf-2 mutant compared to wild type, to normal worms. And what we found— I'm going to start calling it FOXO again— that FOXO, the gene regulator protein, DAF-16 protein, influences lifespan by controlling a wide variety of genes. So here are some of the genes that we found and other people found control lifespan and are regulated by the DAF-2 gene. Let's start over. Let's start here. One set of genes that is regulated by DAF-2 that's more active are genes that control the animal's immune system. And those genes make the animal actually resistant to pathogens, Gary's lab showed that, and we showed that they actually allow it to live longer under normal conditions. There were, let's hop over here, there are these genes called chaperones. So chaperone proteins are proteins that actually take care of other proteins. So what proteins— they're in cells. Proteins are most of what cells are. They can be things like the hair or the skin. They're structures, but they also carry out functions like they're hormone receptors or gene regulator proteins. But in order for them to function, they have to have the right shape. And the chaperones maintain the shape of these proteins. They help them to assemble correctly and then maintain themselves. And actually, if a protein gets damaged,— then the chaperones basically escort them to the cell's garbage can so they can be recycled. And there are more chaperones in the long-lived DAF-2 mutant, and we show that those promote the longevity of the DAF-2 mutant. There are also changes in metabolic genes. There are genes, for example, that move cholesterol around the animal, and these genes are actually turned down when the animal— in the long-lived mutant. And if you take a normal worm and turn that gene down, it looks lives longer. So we don't know why, but this is important for the longevity of the animal. And there are other metabolic genes, like there are certain channels that let actually glycerol go through the cells of the animal. We have no idea why this affects lifespan, but they're more active in the DAF2 mutants, and it matters. And then let's hop over here. A lot of you have heard about reactive oxygen species, and there are proteins that detoxify these reactive oxygen species. Species, and they're called antioxidant proteins, and they're switched on in the long-lived animal. And we found in our lab that they actually promote the longevity of the animal, although now, from work from a lot of labs, has suggested that their role in longevity in general may be more controversial than originally thought. And finally, I just want to mention this one. I don't know if Gary's going to talk about it, but Gary's lab showed in another study that cells that are normally expressed in the germline gets switched on in the soma. And the germline is immortal, you know, that's the sperm and the oocytes, that's an immortal lineage. But genes that are normally only on there get switched on in the whole body in the daphnia, and they also contribute to the long lifespan of the daphnia. Okay, so what I want to impress on you really right now is that there's not just one magic reason why these worms live long. They don't live long because of one thing. They live long because of a combination or accumulation of many different protective mechanisms that are activated in the long-lived worms. And they seem to act cumulatively. In other words, if we have a daf-2 mutant, a mutant that would live long, and we knock down any one of these genes, one of the ones that normally promotes long life, the animal lives almost as long but not quite as long. So they have on their own pretty very small effects on lifespan, really suggesting that they act in a cumulative way. So it's this idea that you could actually have one gene regulator protein, FOXO, that could unite and/or coordinate the activities of many different genes in the DNA. That's a powerful concept. Let's put it another way. These little animals have the latent ability to live much longer than they normally do. It's all written into the DNA, this ability, through things like what FOXO does and these genes and what they do. It's all encoded in the DNA, but normally they don't express that ability. It's not until you come along and you start mucking around with the DACH2 gene like we did that you allow this program to be expressed in the animal convivium, but it was already there. And we will talk in a little while about why it might be there. So, but first I want a little analogy. You can think of FOXO as a building superintendent. And it's a great analogy, actually. So suppose you have a building. You know if you do nothing to it, if you move out and it's just there, it's going to deteriorate. If it's not vandalized by, for example, the analogy to a pathogen. But anyway, it's not vandalized. But if you have a building superintendent, that's what— so suppose your building has a superintendent. Normally he's a little lazy. But he does take reasonable care of the building. This is like FOXO in the normal animal. Remember, it's on, making it live a little longer than it would normally live otherwise. So, right, so, but then suddenly the phone rings and it's the owner of the building is coming, or it's the weather report saying that there's going to be a hurricane. So immediately the FOXO building superintendent he springs into action and he actually doesn't do anything, he just gets on the telephone. Just like all FOXO does is to go into the nucleus and bind the genes, you know, to turn them on. But anyway, he gets on the telephone and he calls up the window person, the person who fixes the roof, the floor person, paints the house, all these different people with different jobs to do, but they all descend on the building and they fix it and they make it, they make it look good and they make it They protect it and obviously they're going to make the building last a lot longer. Okay, so they do two things. They protect it from the hurricane, but they also make its normal lifespan even without hurricanes last longer. And that's like what DAF-2 and DAF-16 does. So I'm going to come to that right now. How do we connect this idea of the building superintendent to the big picture? So what does it all mean? Okay, so normally insulin and IGF-1 are hormones that are abundant under good conditions. When you eat food, the sugar in your food causes your insulin level to rise, and that, like I said, you know, promotes the use of the food or the storage of the food. And IGF-1, again, is a hormone whose level is high under good conditions when the baby is growing, something like this. So these are hormones for good times. Um, if you— like I said, if you completely knock out insulin or IGF-1 genes in any animal the animal never makes it to adulthood. So they're essential genes. But the trick, the missing link here, is that under, in bad conditions, like let's suppose there isn't a lot of food or there's some kind of stress condition in the environment, then the level of insulin or IGF-1 could be a little lower than normal. And so even if the receptor is normal, if there's not very much hormone, it won't register a lot of a signal, and what happens then I think is that the animal interprets that as danger. This is the way the animal senses danger. And as a consequence, it activates FOXO, just like the building superintendent getting that phone call, which triggers cell protection and repair, but the same mechanisms that repair the cells and protect them may be the same mechanisms that can allow the cells to just last longer, so the animal lives longer. And in fact, the long-lived Daf2 mutants are incredibly resistant to stresses. We've never put them in hurricanes, but I bet you that they would do better than a normal worm because they are better at everything that has ever been tested, to my knowledge. They're resistant to oxidative stress like hydrogen peroxide or Agent Orange, to high temperature, to pathogens, to xenobiotics— these are poisons in the environment— tunicamycin, which generates ER stress and actually My former postdoc Sivan, who's in the audience, is studying this here in Israel. Anyway, they also are resistant to hypoxia and other harmful conditions. So these little guys live long and they are imperturbable. They are really resistant to everything. So this brings me to an interesting idea for evolution. Okay, so you might ask, how would you ever evolve of genes that can control aging, especially if aging happens after reproduction. And there actually are lots of models that make sense, but I want to talk about one that comes specifically from this work. And I think there's probably a combination of answers to the question. But remember that the DAF2 gene controls dour formation. So when an animal is a child, when it's growing up, if conditions are bad, bad, DAP2 activity will fall, insulin levels will fall, DAP2 activity will fall, and it will become a dower. So what happens? It's resistant to everything in the world, it won't die, and not only that, it will stay in this undead state for a long time. A dower can live a long time. Even a normal dower can live for months, unlike the normal worm where they're all dead in a month. So anyway, these dowers, then whatever horrible thing is in the environment has passed, the animals can exit from the dour stage and grow up and the sun is shining, rain is falling, and they can raise a family. So now you can easily imagine that there would be great selective pressure in evolution for that to evolve because an animal, if you have two worms and one has that ability and one doesn't, the one that has it will do what I just said and the one that doesn't have that will die, right? They'll start to have progeny and everything when there isn't food for them and the progeny will just die. So there's a huge selective advantage to having this. But if these genes that protect the animal and allow it to outlive bad environmental conditions, those genes are there. This whole little regulatory circuitry is written into the DNA. And if it's expressed at a basal level, so in other words, even in a normal worm, there's some of this, some of these protective functions and longevity functions, then there you are, you have already a cassette. And if a change in one of these genes, either FOXO might bind more tightly to the genes and make bigger effects on them, or some of the individual downstream genes could be strengthened, these kinds of changes in evolution could change the normal aging of an animal even that isn't a dour, you see. So the point is, the whole system The system could evolve for the dower, but there it is, and it's expressed at a low level all the time in dowers and non-dowers, allowing them to live, you know, a certain rate. And then you can change the— in evolution, you could change the activities of these things, which would allow a lifespan to get longer. And actually, I just learned something really interesting the other day. There's a study done by someone in Canada, and what they did was they looked at the level of insulin and IGF-1 of IGF-1 in mammals. Now, if you don't have a lot of IGF-1 when you're a child, you become small. So for example, small dogs are actually IGF-1 mutants and they live much longer than large dogs. But if you look at adults of all species of mammals and you compare the level of IGF-1, you find something really interesting. Now I have to say first of all that this study was done by comparing many different publications done in different labs using different conditions, so it has to be taken with a grain of salt, but what they found was that larger mammals actually have lower levels of IGF-1. So they would be a little bit like a DAF2 mutant compared to a small— I'm sorry, not larger— well, it is larger mammals, yes, when they're adults, and they also tend to live longer. So for example, an elephant For the most part, larger mammals live smaller— live longer than smaller mammals. So what I'm trying to say is that in grown-up mammals, there's a nice correlation between the level of IGF-1 and the lifespan, for the most part, although there are caveats to this study. Okay, so I hope that made sense. Anyway, I think this dour connection really is interesting, even though the long-lived worms are not dowers, just like small dogs are not dowers. So the fact that this same pathway, when you push it harder, can trigger the dower pathway makes a nice link here. How are we doing? Okay. So now what I wanna tell you is that FOXO does a lot more than just respond to insulin levels. So lots of labs are now studying aging, many, many labs. Our lab and other labs have discovered other ways of getting worms to live long besides just changing the insulin IGF-1 receptor. For example, we found that if you overexpress this protein, a heat sensor called heat shock factor, worms live long. And other labs showed that, listed down here, showed that overexpressing AMP, an energy sensor called AMP kinase, another stress sensor called gene kinase, those would also make the animal live longer. This is a really interesting one right here. This is a little— this is not actually a protein, this is a little RNA called lin-4, and I'm going to talk a little bit about this. Lin-4 has been known for a long time, and what lin-4 normally does, and what it was known to do for a long time, was to control the stages of growth from the fertilized— well, from the newly hatched worm to the adult. When the newly hatched The hatchworm goes through these different stages, as I showed you before, and in order to go from one stage to another, it needs LIN-4. In fact, if you don't have LIN-4, it's like a broken record, it keeps skipping and repeating one stage after another. So this is a timer, essentially. This little guy allows progression from one stage in development to another. Well, it turns out the work of the Slack lab showed that this little guy acts, again in the adult, control the rate of aging, which makes you think that maybe there's something like a timer for aging. Okay, but we don't know that. We don't actually know how it controls aging in detail, but the fact that it does it all is really interesting given what it does in development. Anyway, the way that it controls aging, at least partly, requires FOXO, because if you don't have FOXO or DAF-16 anymore, then it doesn't matter what you do to this little guy, it doesn't affect aging. Okay, so, um, all right, so there. And then this one here is a germline sensor. I'm going to talk about this a little bit more in a second, but the point is that there are a whole lot of different ways of getting lifespan to be increased, uh, through FOXO. You can do it with the insulin changes, but you could do it this way or this way or this way or this way or this way. So I think that's, um, important if for humans, if you think about how we would ever want to to capitalize on this knowledge to increase our own youthfulness, maybe it wouldn't work to change insulin or IGF-1 genes. It might, but maybe it wouldn't. But then that's okay because there would be other ways to try, and all you would need is one way. Or maybe two ways would work better than one, and actually I'll come back to that. If you change more than one of these, you can actually get an even longer lifespan. I want to mention that other gene regulators regulators or transcription factors also influence lifespan. They have names like SKIN1 and other things, but I'm not going to talk about them just because of time. But FOXO has a very, very important central function. So now what I want to say in this little slide here, and I want to talk about this for just a minute, is I want to talk about how conditions in the environment or conditions inside the animal itself can engage some of these pathways and influence the lifespan. So this gets to the question of why there would be this longevity-regulating system in the worm if it's just not on. But it can be turned on. So one pretty famous now way of increasing lifespan, I'm sure you've all heard of, is called caloric restriction, which means eating less than you want. And if you're, say, a mouse or a rat, or a fruit fly or a nematode or even a yeast cell, and you eat less than you want, you live longer. And actually this was discovered in the Depression, the Great Depression of the 1930s. And it was very interesting because people were actually really hungry for long periods of time. It was a real problem. And so scientists started asking, what's the effect of long-term hunger? And what they found was that, so they expected of course, that there would be all these bad effects, but it was the exact opposite. They found that the rats, which were the ones that they studied, lived about half again as long, 50% longer than they would normally live. And not only that, they were resistant to a whole bunch of different diseases like tumors and heart disease and things like that. So it was really quite remarkable, and it's been replicated in many different species. So a lot of people want to know, obviously, how does it work? How can caloric restriction restriction, not eating enough, make you live longer. And many people thought that, well, if you don't eat enough, then you're not metabolizing all that food, so you're not producing free radicals and other kinds of damage that would just age you. But it looks like it's more complicated than that. For example, if you feed an animal every other day, it will live longer. And when it's under those conditions, often on the day that they can eat, they eat more than normal. So they end up eating the normal number of calories, but they still have all the benefits of caloric restriction. So it's not just, it looks like some kind of regulated process. So the surprise has been, number one, it is regulated, which I'll show you in a minute, and number two, how it's regulated depends on how you decide to calorically restrict yourself, at least if you're a worm. So, and I'll show you two examples on this slide and one example on another slide. So you could take a worm like I just told you and you can You can feed it every other day, and if you do, it will live longer. Uh-oh, the lab that showed that is not written here, and I can't remember. I'm sorry, it's not written here, it should be. I think it's the Honjo Lab from Japan. But anyway, they showed that feeding every other day extends lifespan, and it probably goes through this pathway. It seems to be in the same pathway as insulin receptor, and it's largely dependent on foxa. So that's, that's one pathway. But you can also calorically restrict a worm by letting it become middle-aged and then restricting its food intake here. And if you do that, the Brunet lab showed that you activate this energy sensor, AMP kinase, and that extends lifespan through foxa. So this is one pathway that's activated by eating in one way, and this is by eating in another way. And there's a third way I'll tell you I'll tell you about it in a little, in a minute. But I want to tell you about several, two other ways of extending lifespan in this slide. So here, this one says sensory cues. It turns out, so these little worms, they actually are quite intelligent in their way. They can respond to many different smells and tastes, odors and tastes in the environment. And they, there's certain things they like and some things like garlic that they don't like at all. But anyway, it turns So what we did in our lab is we looked at animals that just had defects in the sensory neurons because of a mutation, and they couldn't smell or taste anything very well. And what we found is that they lived long. And we did some more studies and we found that the reason they lived long is because of this pathway here, because there were probably changes in the insulin receptor activity and FOXO was needed. That was what our genetics told us. So that's actually very interesting. These worms can live longer if they think, or if things in the environment— I mean, the implication here is that there are things in the environment that the worms normally taste or smell that control their lifespan, and we've just mimicked those by damaging the neurons themselves. But actually, we know a little bit. We know that there are some sensory neurons that promote longevity and others that inhibit longevity. They're opposite And what we think is that having the ability to— so the worms care about the environment, and maybe if they can sense a change in the environment by smelling or tasting it before they have to actually be hungry by not having enough to eat, they can get a head start on, you know, like the building superintendent, it's better off if he knows about the hurricane 2 days in advance than 20 minutes in advance. Or when it hits. So that's, yeah, this would be like having a phone call, having the sensory cues rather than having to hear the winds. Okay, so then the last one I want to mention, oh, I want to say one more thing, two more things about this. It's very cool. If you take a fruit fly and you calorically restrict it, it lives longer. But if you let it smell the food, then it doesn't live as long. So you have to think about that if you decide calorically restrict yourself. You have to stay away from people that are baking bread. Okay, and then another thing is that in humans even, our insulin levels are controlled partly by our sense of smell. If you smell the food that you're eating, your insulin levels rise even more. Now I don't mean to say that what you smell or taste is determining your rate of aging, although I can't say that it's not possible. Possible. It might be, but it could be because we do have the capacity to, you know, to respond via insulin signaling to odors in the environment. And the last thing I want to mention on this slide is this one. So the reproductive system obviously is crucial because it's in charge of the next generation, and you have to coordinate aging with reproduction. And there are all sorts of evolutionary theories about how the reproductive system would interact with the rest of the animal. And one very popular theory for a long time was this reproductive trade-off theory. And the idea was that if you didn't reproduce, then all of the energy that you would have used to reproduce could be channeled instead into protective mechanisms that would allow you to live longer. So there was there should be this trade-off. And when we found that DAF2 mutants live long, we asked how many progeny do they have. And I told you that some of them can have the normal number, but the mutant that we were looking at had 20% fewer progeny. So we were really worried about this because I was afraid if we published the paper, the reviewers would say, ah, of course it lives longer, it just doesn't have enough progeny, there's a reproductive trade-off. So what we did was we took a laser and we killed the cells the cells that give rise, 4 cells that give rise to the reproductive system, we killed them all. And what we found was that daf-2 mutants still live long and that the normal worms still had a normal lifespan. So that said, right off the bat, there was no reproductive trade-off because these normal worms weren't using all this, worms have 300 progeny and they were having none. So they weren't just channeling all that saved energy into longevity. Instead, they were— I don't know what they were doing with it, probably eating less, but they weren't living longer. But, okay, so that's the first thing, no reproductive trade-off. But it doesn't mean that the reproductive system doesn't have any relationship to the aging of the animal, because what we also found was that if we took our same laser and we just killed only the cells that give rise to the germline, but we left all the other tissues like the spermatheca and the uterus and all those other tissues intact, so we had an empty gonad in a sense. We had a gonad, a reproductive tissue that didn't have anything in it, didn't have any germ cells. Then we got the most amazing result. The animals lived about 60% longer than normal. And that's this pathway here. And it goes through this guy called TCR1 is needed for it, but FOXO is also needed for this to happen. And I'm not going to talk about the mechanism. It's really interesting though. We study it in the lab and it involves steroid hormone signaling and it involves signaling by a particular kind of pathway called the Wnt pathway. So there is a whole elaborate set of signals by which the reproductive system tells the body how fast to age. It's really neat because it's in control of the next generation, but it's also in control of the aging of the soma. It's a very interesting system. Okay, so the bottom line from this slide is that is there are lots of different ways that environmental signals or internal physiological signals like empty gonads can access this FOXO longevity system to change the rate of aging. So it's really very, very plastic. Not only does it not just happen passively, and not only is there a cassette that regulates it, but this cassette is kind of the slave of many masters. Ah, so I mentioned before that if you change two of these things, in particular this one plus this one, in the same animal, you can get a really long lifespan. So we found that if we had ADAPT2 mutant and we also changed the reproductive system in the same animals, that they could live 6 times as long as normal. And that's— now you're really getting serious here about lifespan extension. So I want to show you a movie. I have to show you. Where is my movie? Maybe it's down here. Okay, I've got to find the movie. It's buried under all this stuff here. But it's a nice movie, it's worth waiting for. There it is, okay, here it is. Okay, so these two worms were photographed when they were 144 days old. Amazing, huh? Remember that movie that I showed you at the beginning where the worms were 13 days old and on their deathbeds. These ones are 10 times that old and they're just moving around. So Nuno, this graduate student who discovered this, actually took the little petri dish where we keep our worms and these were the worms and he showed people in the lab and asked, and asked, how old do you think these worms are? And they said, oh, they're probably 5 days old. That was all, 5 days old, but they were really 144. So it— and actually though, if you think about it, evolution has gone way— done way better. We are— we live 2,000 times as long as the worm and probably as our distant ancestors lived. So by changing genes, you can go way beyond sixfold, you know, to create a longer lifespan. I mean, we do live— we look different from these little worms, but the aging mechanisms, probably there are probably gene changes that affect maybe specifically aging during this process. Okay, so back to the talk. How are we doing? So what about other organisms? This is a fountain of youth and I like it too, it's pretty cute. Okay, so it turns out that yes, the answer is yes, Changing these genes in other animals does extend their lifespan. So if you change the DAF2 gene, the insulin receptor, IGF-1 receptor in fruit flies, they live longer. And if you make these same kinds of changes in mice, or you change genes that control IGF-1 or insulin, or make genes that change genes that respond to insulin or IGF-1 in this little pathway, the mice live longer. So this means that the ability of of the DAF2 gene, and FOXO probably too, to affect lifespan is ancient. It was up and running probably in the common ancestor of all these three animals, which is a very ancient organism. And recently Nir Barzilai, who by the way is from Israel, and now he's working at Einstein in New York, but he studies Ashkenazi Jewish populations in New York to look for genes that affect aging in people. He's studying now people, not worms. And what he finds is that among his population, the members that live to be 100, that are centenarians, are more likely to carry mutations that reduce the activity of DAC2, the IGF-1 receptor, than are people who die earlier. And he knows that these altered genes carried by these centenarians are less functional because he's tested them. In biochemical studies. And even more amazing, I think, are these little stars. So each one of these stars represents a group of people, or the origin of a group of people, that was studied by these human gene mappers. And in every— each one of these cases, it was found that variants in one of the human FOXO genes, the DAF-16 gene regulator proteins, one called FOXO1, 3A, the DNA variants in that gene were more frequent in people who lived to be 90 or 100. And this is all over the world. So, China? Yeah, yeah, there's China. People that were studied in Hawaii but are from Japan. Here are the Ashkenazi Jewish people. Actually, I put the star in Israel here, even though the studies were done in New York, because they were Ashkenazi Jews. That was a little bit of poetic liberty. These were European populations, California and New England. This is different from the study. But so that really says that, I mean, it's not like having these changes is sufficient to let you be 100. It's not. They are sufficient to give you a higher probability, but it's not that much higher. But still, what it tells you is important. It tells you, I think, that humans— oh, the other thing is we don't yet know how these DNA changes affect the function of FOXO? Hopefully, we would think they make it work better, because if it works better in animals, they live longer. But we don't know that yet. But anyway, the point is, I think what it really says is that we humans are susceptible to the effects of this lifespan control pathway, to the insulin IGF-1 and FOXO pathway. We're susceptible to it. And so now the question is, to what extent are we susceptible? And if we turn it up a little higher, could we stay young longer and live longer? And this has made a big effect on my lab because we, since these other labs, not our lab, discovered that FOXO forms in humans can affect lifespan, we started to now, a new project without worms, we started a project to look for small molecules that activate FOXO3A in human cells. So this is humans. Trying to find small molecules that can reproduce some of the beneficial effects that these changes have on animals in people. So we're real excited about that. And okay, so now I'm going to say a few more things. I'm going to just mention, so that you all know, that it's possible to extend lifespan without any FOXO at all. So if you— this is the third form, this guy is skinny, skinny because it's been calorically restricted its whole entire lifespan. And if you do that, so instead of middle age or every other day, now in this case, you calorically restrict the animal its whole lifespan. Now it lives long and it no longer needs FOXO to live long. Instead, it uses another mechanism, which I think is on the next slide. And I'll just mention here that worms have a shorter lifespan at high temperature than they have at low temperature, and our lab showed that This is due to not to FOXO but to steroid hormone signaling, at least partly. Part of this change is controlled by steroid hormone signaling. And then here, these little guys here are the energy producers of the animal called mitochondria. And they're, as scientists here will know, they're not exactly this large in the animal, but I just wanted you to be able to see them. But anyway, it turns out, actually Gary's lab and my lab and one more lab all at the same time discovered independently that inhibiting the activity of these mitochondria would increase lifespan. And that actually is true not only in the worm but also the fruit fly and the mouse. So this is another very interesting longevity mechanism, and this is completely independent of the FOXO gene too. So I'm telling you this to impress upon you the multitude of ways now that scientists have figured out to extend lifespan in animals. And this is more about that other caloric restriction pathway. The reason that those animals that had been calorically restricted their whole life live long is because they had less activity of a different nutrient sensor, one that's called TOR. And TOR inhibition extends lifespan in worms, but also in flies and mice. And the neat thing about TOR is that there's a drug called rapamycin that inhibits TOR activity. And if you give rapamycin, this drug, to mice, even when they're middle-aged, they live longer. So we already have a drug, at least for mice, that can make them live longer, which is amazing. Now, humans— this is an FDA-approved drug in the US, and it's used, and people use it for two things at least. They use it for suppressing the immune system, which may not be good for you, so that's important to think about. They suppress depress the immune system if they've had an organ transplant. But these people take rapamycin their whole life. And it's also used, actually, in cancer therapies too, as well, rapamycin. So I don't know if this is a drug that people would want to take because of this immune problem. There are also some other side effects that people have. But I do think it's just very significant and amazing that right now in 2011, there is a drug whose action on aging came out of all this work, these studies in C. elegans about aging, and that can be right now given to a mouse to increase its lifespan. And the other thing that's really, really remarkable about these worms that is very important is not only do they stay young longer, but they are resistant to age-related diseases. And this is a whole bunch of different diseases, and some of them are shown here. I won't read them all, but they're listed under the animal in which they were shown to be affected. But basically all of these diseases diseases are postponed in these long-lived animals, and even when they get the diseases, they're often not as severe. So this suggests a whole new therapeutic strategy. It suggests that we can, by finding, making pills or drugs that control the rate of aging, we could all actually have beneficial effects on protection against many different kinds of diseases all at once. So you don't have to only study cancer or only study Alzheimer's. If you study aging, you hopefully you can have an effect on all the diseases. And I just want to mention a worm tumor. It's possible to make a worm get a tumor, and I won't describe that. It's a germline tumor. But if you make a normal worm in orange here get this tumor, its lifespan is cut in half because the tumor just takes over the whole animal. But if you have a DAF2 mutant and now it gets the same tumor, the same tumor mutations in this animal, the The tumor, there is a tumor, but it's a very small tumor and it doesn't affect the lifespan at all. So it has a huge effect on the growth of these tumors in the worm. And the same kind of thing is seen in animals, and actually the tumor effects of the DAF-2 pathway also extend to humans, and that's been known for a while. So there may be someday a pill, but in the meantime, I'm out of time, so I'm just gonna make this really quick. Glucose, or sugar, stimulates insulin. Insulin in people. So insulin would be predicted to shorten lifespan. So we wondered, if we gave worms glucose, maybe we could hyperactivate the DAF-2 gene, and maybe that would turn FOXO down, which would shorten lifespan. And sure enough, we add a little sugar to the plate, not a lot, and they live shorter. And other labs found that too, and we explored the mechanism in our lab,, and we found that glucose does not further shorten the lifespan of the Daf16 mutant. So if the way glucose shortens lifespan is to inhibit Daf16 FOXO, then if you already knock out FOXO, sugar should have no effect, no more effect, and it doesn't. That's shown in this slide here. So I just want to jump ahead here and end with a very interesting question: could a low-sugar diet maybe extend your lifespan? And now I'm going to skip over this because I'm out of time. Sure, sure. Oh, oh, wait, wait, wait, wait. I have to thank the people. Please resume the webcast. This is the most important part of the whole slide. I actually talked— I didn't just talk about one thing my lab does. I talked about lots of things that we've done over the years, and the people I've mentioned here are people who initiated these various projects. So Ramon was this rotation student who was willing to look for long-lived mutants, and he found that Daf-2 mutants live long, and that really cemented the aging project in our lab. Colleen did the work showing, like the building superintendent work, all the different things that the FOXO protein could do to extend lifespan. Javier and Joy worked on the sensory perception. Honor is the one who discovered that killing the germ cells extends lifespan, the empty gonad extends lifespan. And Dylan showed that respiration, the mitochondria defects extend lifespan, also that the DAF2 pathway only works in the adult. You know, it's the one with the worms that were 144 years old. Sungjae studied the rest, uh, sorry, the glucose, the last thing I talked about, and Julie did the work on the tumors that I mentioned. So I'm sorry, now I can take a question.

We do have time for 2 or 3 quick questions.

Shimon, please. First of all, I enjoyed your talk immensely. I would like to ask you, how can you give a whole lecture on anti-aging without mentioning telomerase? And then, what is the relationship between extending life and becoming cancerous? Okay, I can talk about both of those, actually. So he said, what about— how can I talk? I talk about telomeres? And telomeres are the ends of chromosomes, and they're like the little polished parts at the end of a shoelace. They protect the end of the chromosome from fraying, essentially, and getting shorter when the cells divide. And that's very important. And, okay, so in mice, if you— so during normal aging, the telomeres the telomeres do get shorter. And it turns out that in mice, if you— there's a protein called telomerase that prevents the shortening of the telomeres. It slows it down. And if you engineer a mouse so that it has more telomerase, so in other words, its telomeres are more protected and the chromosomes can't get so short, most likely, that mouse lives long. But unlike our worms here, or the mice that have low levels of insulin or IGF-1, that are not overexpressing telomerase signaling, which don't get cancer, which get way less cancer than normal, these mice get more cancer, probably because the tumor cells need these— they divide, they keep proliferating, and they need to have chromosomes that aren't shrinking. So these mice get tumors. So they actually— so a normal mouse, if you just overexpress telomerase, it actually dies of cancer. But if you engineer the mouse so it can't get cancer by changing its cancer genes, then the mouse lives longer. Okay, so it seems to be a— and we don't think— the work on the C. elegans suggests that changes in telomerase are probably not very important for the longevity of the worms. But there are connections between conditions that perturb the insulin IGF-1 pathway in such a way that it would promote old age. Would make you age faster and shorter telomeres. There's a connection like that in mice. But I think the thing that's, one thing that's really interesting though is this connection between cancer. So the FOXO protein will actually kill tumor cells. So it's a very, it has a very hefty anti-cancer function. And whereas the telomerase isn't that. So our, the pathway I was telling you about is a danger pathway. It senses danger and it responds and makes a protective response. And that's one— and that TOR pathway is part of that. Everything I told you about probably has that rationale, whereas telomerase probably isn't part of that. Telomerase is just something that animals need to, you know, for the cells' needs. So it does affect lifespan, but it's different. And I think it would be really interesting to take one of these mice that lives long, if you make it not be able to get cancer. Cancer and you give it telomerase, so it's already living long. Now it would be great to take that mouse and also change the insulin pathway. Now maybe you get a mouse that lives 5 times as long. That would be very interesting to see, or maybe not. But they're both interesting. I mean, it would be interesting. Maybe they wouldn't live 5 times as long, maybe they would die, who knows, but it would be interesting to see. Yes, okay, there's a question here. [INAUDIBLE] Or not sugar, or maybe we're wrong too. But anyway, okay, go ahead. Okay, thank you. No, why do bats live so much longer? This is a really interesting question. I mean, it's a huge diversity, and no one knows what people are doing now. It's hard to figure it out. What you can do is, it's easy to take one species and change a gene and see if that species has a different lifespan. So for example, you know, you can change the insulin pathway or these respiration genes, the mitochondrial genes, in any species it seems like, and get them to live longer. But is that the cause of what happened in evolution? Evolution. So people are looking at these long-lived species to see if they have higher defense capacity, and they, they tend to, um, for the most part, but there's not a perfect correlation. They also— mammals that, um, bigger mammals, which tend to live larger, live longer, also have lower metabolic rates. So it could be that changes in respiration contributed to the long life of the mammal, or the fact that the IGF-1 IGF-1 signaling seems to be lower in the adult phase, if that's true, that Canadian study. So, but we— it's really hard to know, and people don't know, and I think it's going to be very hard to figure out, really hard. Well, no one has— the FoxO proteins are present, but they may— but I think the thing that would be the— I think looking at the level, the sensitivity of the cells to IGF-1, the levels of signaling through IGF-1, that could be measured. And like I said, at least in this comparison of studies, there was a correlation. No one has looked at FOXO itself, and actually people in my lab want to do that. They want to take FOXO genes from, you know, from the centenarians and the non-centenarians, you know, the different forms, and put them in the worm and see if they act differently in the worm. That would be a very interesting thing to do. But, um, yeah, but it's not an easy problem because you can't just cross a a bat with a mouse and do genetics. You can't just do that. And there would be more than one lifespan gene in the cross, so it would be very hard to find out.

I'm afraid we have to move on.

Thanks so much, Cynthia. You're welcome. Wonderful presentation. Really wonderful.