Aging Genes: Genes and Cells that Determine the Lifespan of the Nematode C. elegans
Transcript of Part 1: Genes that Control Aging
00:00:03.04 Hi, I'm Cynthia Kenyon and I'm going to be talking about genes that control aging. 00:00:08.24 So, you all know what aging is. There's an example of it in the slide. 00:00:15.18 And for many years, people thought that aging was just something that happened. 00:00:18.16 You just wear out like an old car. But I guess in the early 1990s or so, in the late 1980s, 00:00:24.27 I started to think more and more that aging was going to turn out 00:00:27.27 to be subject to active control by the genes. 00:00:30.22 And the reason I thought that is because everything else that people think just sort of happens 00:00:36.00 in a haphazard way in biology turns out to be regulated in a very elaborate way by the genes. 00:00:42.14 For example, if you look in nature what you see is that 00:00:45.12 different animals can have really different lifespans. 00:00:48.21 A mouse lives only two years, but a canary lives 15 years, and a bat can live 50 years. 00:00:54.11 So, these animals have extremely different lifespans in spite of the fact that 00:00:58.02 they're about the same size, and, even if they're living in the same place, 00:01:01.15 they have very different lifespans. 00:01:02.20 And the reason they have different lifespans is because they have different genes. 00:01:05.28 So that says right off the bat that there’s something 00:01:08.22 about these genes that's influencing their lifespan. 00:01:11.28 So the idea that I had was, look, if there are genes that actually control aging, 00:01:16.06 then if we change these genes we ought to be able to produce an animal that lives longer. 00:01:21.06 And then if we study the genes in more detail, 00:01:24.07 we'll be able to understand how aging is controlled. 00:01:28.00 So we didn't study this problem in people. 00:01:30.14 Instead, we studied it in my favorite little animal, C. elegans, 00:01:34.15 which is shown here for you that this is an old individual. 00:01:37.14 Now, what C. elegans is, is a very tiny little round worm that lives in the soil. 00:01:42.17 It’s about the size of a comma at the end of sentence. Very tiny. 00:01:46.20 And they're really good for studying things like aging 00:01:50.04 because they grow old and die in just about two weeks. 00:01:55.06 And we wondered, "Could C. elegans teach us anything about aging in humans?" 00:01:59.06 Because obviously, we like our worms, but we don't really only want to learn about the worms. 00:02:03.26 We really want to learn about people and higher animals, mammals. 00:02:07.15 So the question of course is: could studying aging in one of these little round worms 00:02:11.19 teach us any thing about humans? And I thought that the chances were pretty good that it could 00:02:16.27 because the idea that I had was that aging was actually going to be controlled by genes- 00:02:21.02 a set of genes that would be controlling aging in all animals. And the reason I thought that 00:02:26.13 is that many biological mechanisms that control other aspects of biology, 00:02:30.27 like how a muscle cell differentiates 00:02:33.02 or how an egg is fertilized, or how a cell divides happen in the very same way in all animals. 00:02:39.18 And, in fact, lots of genes that are important for people 00:02:42.27 were first discovered in these little C. elegans. 00:02:46.18 OK, now, we were very optimistic starting out that we would be able to find genes that extend lifespan. 00:02:52.07 And the reason was that there already was an animal that had an altered gene that lived longer. 00:02:57.20 And this was a mutant that had been identified by Michael Klass 00:03:01.07 and studied by Tom Johnson for a long time. 00:03:03.28 These worms lived about 30-50% longer than normal. 00:03:07.18 So we set out to look for long lived mutants and amazingly, we found that mutations 00:03:12.02 that damage one single gene in the worm, a gene whose name is DAF-2, 00:03:17.00 doubled the lifespan of the worm. So here you see a diagram of the lifespans of these worms. 00:03:24.20 So what we did is we took a whole population of worms 00:03:26.22 and we just let them age and asked how long they live. 00:03:30.00 So these here in black are normal worms here. And you can see that by day 30, the end of a month, 00:03:36.01 they’re all dead. So the fraction alive, what you see is over here, is now 0. 00:03:41.15 Whereas at the same time, our mutant worms, the worms that have only one gene change, 00:03:46.20 all the other genes are the same, are almost all still alive. 00:03:50.21 And it’s not until about twice as long, until 70 days, when they're all dead. 00:03:57.04 So, it’s incredible really. We just changed one gene, all the other genes are the same, 00:04:01.16 and the whole animal lives twice as long as normal. 00:04:05.18 And the really magical thing about these worms is that it’s not that they, 00:04:09.05 you know, get old and just hang on. They actually age more slowly than normal. So here 00:04:14.03 you see a normal C. elegans worm, quite beautiful, crawling along on its bacteria. 00:04:20.27 So this is a movie of these worms. What you're going to see first are the normal worms. 00:04:24.23 Here it is. A normal worm when it’s about the age of a college student. 00:04:27.28 It’s three days old, so it’s a young adult. 00:04:29.20 And you can see that they're very healthy. Now what you see here is the mutant worm, 00:04:33.24 the one that's going to live twice as long, when it’s also a young adult. 00:04:37.29 And what you see is that it’s very healthy. That's important. 00:04:41.10 It’s not sick when it’s young. 00:04:45.16 Now here, prepare yourself because this is a little bit sad, is the normal worm in just 2 weeks. 00:04:51.25 You see? Now the head here is moving. See the head? See it move? 00:04:58.00 There. But otherwise, it’s just lying there. It's in the nursing home basically, the old folks' home. 00:05:05.08 You're going to see some more worms in just a second. This worm is dead. 00:05:09.10 And again, this one, you see its head is moving but otherwise it's just lying there. 00:05:13.17 So these are what worms look like when they're old which is just when they're 2 weeks old. 00:05:17.07 And here is our long lived mutant. One gene change, that's all. And look at it! See? It looks healthy. 00:05:22.29 It’s moving around actively. They look much younger 00:05:25.23 than the worms... and this is like actually looking 00:05:28.23 at someone who's 90 and thinking that they're 45. 00:05:32.02 That's what it’s like. So it's like a miracle, 00:05:34.11 but it isn't a miracle. It’s science. 00:05:37.15 OK, so we want to know everything we possibly can about how changing one gene 00:05:42.20 can produce this miraculous appearance-a worm that doesn't get old on time. 00:05:49.03 The gene was cloned in the lab of Gary Ruvkun at Harvard. 00:05:52.16 And Gary's lab showed that the DAF-2 gene encodes a hormone receptor. 00:05:57.08 So here I've drawn for you a cell. This circle here's a cell. 00:06:00.11 And here we have the DAF-2 receptor, which is situated in the membrane of the cell 00:06:05.24 with one part out in the environment and the other part inside the cell. 00:06:09.17 And here are hormones that it's receiving here in green. 00:06:13.29 OK, so, what we have found was that the normal function of this hormone receptor 00:06:19.01 is to speed up aging. That's what this arrow means. 00:06:21.25 It means it promotes the aging process 00:06:23.24 because when we damage the gene with a mutation, the animals live long. 00:06:28.09 So the normal function is to speed up aging. 00:06:31.05 So, together our finding, along with the Ruvkun lab's findings, demonstrate that aging 00:06:36.23 is controlled and it's controlled by hormones. 00:06:39.28 Specifically, there are hormones in the worm that are speeding up the aging process. 00:06:45.01 They're making the worm get old faster. 00:06:49.03 Now, the really cool thing about this hormone receptor 00:06:51.14 is that it's similar to two hormone receptors in humans, 00:06:54.09 the receptors for insulin and IGF-1. These are two very well known hormones. 00:07:00.00 They're known to do the following. Insulin is known to promote 00:07:04.07 the uptake of nutrients into the tissues after a meal 00:07:06.23 and IGF-1, the IGF-1 receptor, is known to promote growth. 00:07:12.18 And so what our findings suggested in these little worms was that 00:07:16.02 maybe these hormones had another function that nobody knew about, 00:07:19.29 which is to speed up aging. Remember I told you that a lot of processes that happen 00:07:24.28 in these little worms happen the same way in higher animals. 00:07:28.07 OK, so the idea was that if these hormones are speeding up aging in worms 00:07:32.09 maybe they would be speeding up aging in other animals as well. 00:07:35.23 And that actually turns out to be the case as shown here in this slide. 00:07:41.15 First, over here we have the worm. This is the situation in C. elegans. 00:07:45.24 So we have the insulin and IGF-1 hormone activating (that's what this arrow means) the receptor. 00:07:51.25 And when the receptor's active it blocks longevity. That what this little cross bar is. 00:07:57.08 It means "blocks" longevity. So people who work on fruit flies, the Tater and Partridge labs 00:08:03.08 made the same kind of change in the gene that 00:08:06.00 encodes the fly hormone receptor for insulin and IGF-1. 00:08:09.21 And what they showed was that the flies lived longer. 00:08:12.27 That was true if you changed the insulin/IGF-1 receptor or 00:08:16.05 genes that act downstream of the pathway, down here. 00:08:20.09 And in mice, there are separate genes for the insulin receptor and the IGF-1 receptor. 00:08:25.16 There's one gene that encodes the insulin receptor and another one for the IGF-1 receptor. 00:08:29.10 And it turns out, amazingly enough, that if you change either one of these genes 00:08:33.14 mice can live longer. So first of all, the Halzenberger lab showed that if you 00:08:37.15 make a mutation in the IGF-1 receptor, in other words what you really do is… 00:08:41.26 A normal mouse has two copies of the gene, one from its mother and one from its father. 00:08:46.05 But if you make a mouse that has only one copy, so it's a heterozygous mouse, 00:08:50.14 it has half as much receptor. And what they found was that 00:08:54.02 these mice live long, about 20% longer than normal. 00:08:58.29 They're very healthy. They were completely fertile, and they had a normal metabolic rate. 00:09:04.27 The Kohn lab showed that if you remove the insulin receptor specifically from the fat tissue 00:09:10.05 the whole mouse lived longer and these mice were very lucky. 00:09:13.13 If you fed them a high fat diet, they didn't get fat. 00:09:15.23 OK, so it's really quite amazing because what this tells you 00:09:19.28 is that the insulin/IGF-1 hormone system is controlling 00:09:26.16 aging in all three of these very different kinds of animals 00:09:29.07 which suggests that it was actually controlling aging 00:09:31.18 during evolution in a common ancestor of these three animals. 00:09:36.00 And that common ancestor also gave rise to humans. 00:09:39.29 So, it suggests the possibility that maybe these genes also control aging in us. 00:09:45.11 So what about higher organisms? Do we know anything? 00:09:47.26 Well, very recently we learned something about dogs. 00:09:50.22 Now, dogs as you know come in different sizes. 00:09:53.02 Here's a Great Dane and here's a little Chihuahua here. 00:09:55.05 And it turns out that small dogs live a lot longer than large dogs. 00:10:01.04 So large dogs like a Great Dane live only 5-7 years, 00:10:06.01 whereas these little small guys can live up to twenty years. So it's very different. 00:10:10.13 And what was shown very recently was that 00:10:12.25 the reason that these small dogs are small is because they 00:10:16.12 have a mutation in the gene that encodes IGF-1, 00:10:19.27 which is the hormone that we've been talking about. 00:10:22.16 So that makes them small and as I say, small dogs are long lived, so it makes them long-lived as well. 00:10:28.01 So, this is really interesting for lots of reasons. First of all, these small dogs, they're real animals. 00:10:35.14 I mean the mutants are real animals, but they're laboratory animals. 00:10:38.09 But these small dogs are fully functional, happy, little, intelligent, little creatures. 00:10:42.13 So, that's one thing. You can have this low level of IGF-1 and they have much 00:10:48.00 lower levels of the IGF-1 hormone and be very healthy. But it also raises a question. 00:10:53.18 The question is: Would they have to be small to be long-lived? 00:10:59.14 So in other words, the IGF-1 gene is promoting two things, growth to be a big dog 00:11:05.20 number one and number two, long life. So can they be separated from one another? 00:11:11.01 Or would be you have to small to be long lived if you're a dog? 00:11:15.03 Well, I think the answer is you would not have to be small, and I'll tell you why I think that. 00:11:20.06 First of all, if you go back to this chart, the worms that we study 00:11:23.22 are not small. The fruit flies, if you make mutations 00:11:28.04 in this gene here, in the insulin/IGF-1 receptor 00:11:30.09 the flies are small and long lived, but if you just perturb the pathway slightly just a little bit 00:11:37.17 not too much, then you get flies that are still long lived, 00:11:41.02 but they're not small. They're big and long-lived. 00:11:43.26 Same with these mice. These mice here are not small. 00:11:47.08 They don't get fat, but they're not particularly small. 00:11:49.04 And these mice here, the IGF-1 receptor mice, the heterozygous mice 00:11:53.15 are almost completely the normal size. They're just a tiny bit smaller, almost completely normal. 00:11:57.29 And yet the mice live long. OK, so in all these animals it’s possible to uncouple the two of them. 00:12:04.19 And the second thing is if you think about it when would the hormone 00:12:08.03 be needed in the life of the animal 00:12:09.19 to make it large? Of course it would be needed during childhood, when it's developing into an adult. 00:12:16.09 When would the gene be needed for aging? Well, maybe not until it's an adult. 00:12:21.11 So, we did this experiment. We asked, "When is the 00:12:24.14 gene needed to control aging in our little worms?" 00:12:28.17 OK, this was done by Andrew Dillin when he was a post-doc in the lab. 00:12:31.15 So the question is: When does the DAF-2 receptor gene affect lifespan? 00:12:35.13 So what we did was to turn the activity of the gene down in different times in the animal's life. 00:12:45.02 And the way we did this was to subject the animals to something called RNAi. 00:12:49.26 Now if you don't know what it is, don't worry to much about it. Basically, all you have to know 00:12:53.09 is that it’s a way of inhibiting the function of any gene that you want. This is how it works. 00:13:01.12 If you feed a worm. Well, let me start over. 00:13:04.29 If you introduce double stranded RNA for a worm's gene or any gene into an animal 00:13:12.22 or into a cell, the double stranded RNA will initiate 00:13:16.27 a process that leads to the destruction of all of the mRNA 00:13:21.02 messengers in the cell (or lots of them anyway) for that particular gene. 00:13:25.27 And with worms, what's really cool is that you can have bacteria 00:13:28.25 express a worm gene in the form of double stranded RNA 00:13:32.10 and then you can feed the bacteria to the worms. 00:13:34.28 The bacteria go into the worms. They eat bacteria. 00:13:37.17 They go into the worms and then somehow the double stranded RNA 00:13:39.20 gets out of the bacteria and into the worm's cells and it catalyzes this break down 00:13:44.16 of messenger RNA inside the worm which essentially 00:13:47.22 does the same thing as making a mutation in the gene. 00:13:50.22 It knocks down the activity of the gene. So we did our timing experiments 00:13:55.15 in the following way, where we just took our worms and we grew them 00:13:58.25 on bacteria, normal bacteria until we wanted to turn the gene down 00:14:02.29 and then we took the worms off that bacteria 00:14:05.10 and put them on bacteria expressing double stranded RNAi 00:14:08.08 sorry, double stranded RNA for the gene and let them eat that bacteria. 00:14:13.27 So here's what we found. We found that if we turned the activity of that gene down throughout life, 00:14:18.21 that is if we put the worms on these RNAi bacteria 00:14:21.10 from the time of hatching, they had a long lifespan. 00:14:24.14 So now, here, the control are normal worms that have the DAF-2 gene completely active. 00:14:28.22 And here is what happens if you subject the animals to this RNAi from the time of hatching. 00:14:34.06 And sure enough...so they have the gene down when 00:14:36.28 they're growing up and when they're aging 00:14:39.11 and they live long. So what happens if we just turn it down only during adulthood? 00:14:44.12 Look, they live just as long. You see? So that tells us that the DAF-2 gene acts during adulthood 00:14:50.08 to affect lifespan because if you don't have it on when the animal's an adult, 00:14:55.21 if you turn it down when it's an adult, 00:14:57.09 if you don't have it on it doesn't...live correctly, it lives too long. 00:15:02.11 OK, and we did other experiments where we turned the gene down during development 00:15:06.15 and then we turned it back up when it was an adult, 00:15:09.15 and those experiments told us that DAF-2 acts 00:15:11.14 exclusively during adulthood to affect lifespan, OK? 00:15:16.05 So this gene is acting during development, you know, to do what ever it has to do. 00:15:22.15 For example, promote growth in these dogs. 00:15:24.25 But then, at least in worms it’s acting in the adult to control aging. 00:15:28.18 And there are hints that it's also acting in mice to control aging in the adult as well. 00:15:34.08 OK, so basically, it would be really interesting to take a tiny little dog like a Chihuahua 00:15:40.00 that's going to live, say, 15 or 20 years and give it IGF-1 00:15:43.25 when it’s a puppy and let it become a big dog and lower the IGF-1 levels 00:15:48.02 when it’s a adult and see if it lives long, and I bet it would based on these experiments. 00:15:55.11 OK, so this is all very good for our pets, but what about people? 00:15:59.13 Could this little worm, C. elegans actually lead us to the fountain of youth? 00:16:03.06 And I don't have the answer for you, but I can tell you that 00:16:05.05 there are some interesting unpublished data 00:16:07.20 floating around so keep your eyes open. 00:16:10.29 OK, so now, how do these hormones ultimately affect the rate of aging? 00:16:16.09 How does a hormone coursing around through the circulation affect the aging of an animal? 00:16:20.27 Wrinkles, grey hair, the nursing home, the whole shebang? 00:16:24.27 Well, our first clue came when we discovered that another gene, 00:16:28.28 a gene called DAF-16, is required for these daf-2 mutations to extend lifespan. 00:16:35.15 So, here in this graph you can see what happens if we take away the DAF-16 gene in a daf-2 mutant. 00:16:41.02 So in red here you see the long lifespan of the daf-2 mutant 00:16:44.03 and what you see in green here is the mutant that...it still has the daf-2 mutation 00:16:49.02 so it should live twice as long but we took away the daf-16 gene and now 00:16:53.01 you see it doesn't live long anymore. 00:16:55.08 So, DAF-16 is like a fountain of youth gene. It’s a gene whose normal function let's you live long. 00:17:01.09 In fact, we call it "sweet 16" for youthfulness. 00:17:08.03 OK, so what is DAF-16? Well, we cloned 00:17:10.07 the gene, and it was also cloned in the Ruvkun lab, 00:17:12.10 and it encodes a transcription factor. That is, it makes a protein that goes in 00:17:15.29 the nucleus and binds DNA and switches genes on and off. 00:17:20.03 So, if there ever were a regulatory protein, that's it. 00:17:22.25 In other words, there's no question that aging is subject to regulation 00:17:26.18 or to control because in order for these animals to live long, 00:17:30.05 they have to be expressing genes at different levels. 00:17:32.23 OK, so there's definitely a control system for aging. 00:17:37.03 OK, so what is it that the DAF-16 transcription factor 00:17:41.12 is controlling that lets the animals live long? 00:17:45.20 First of all, before I go into that let me just tell you a little bit more about the DAF-2 pathway. 00:17:52.04 So basically, I showed you before the DAF-2 receptor. 00:17:55.03 And what I'm showing you here in this slide is a summary 00:17:59.19 of information that was gathered from a lot of different laboratories, 00:18:03.10 primarily the laboratory of Gary Ruvkun but with important contributions from the Riddle lab, 00:18:07.15 the Thomas lab, our lab and Johnson's lab. 00:18:10.01 So what you see if that the way the hormones affect gene expression 00:18:18.10 is that they activate a highly conserved phosphorylation cascade or a kinase cascade 00:18:25.00 which ends up phosphorylating..these little yellow circles here are phosphate groups 00:18:30.10 attached to the DAF-16 transcription factor. 00:18:33.03 And when this happens the DAF-16 transcription factor 00:18:35.27 is not able to accumulate in the nucleus. 00:18:39.21 But, if you make mutations in DAF-2 or any of these downstream genes 00:18:43.09 here then the transcription factor no longer 00:18:45.23 gets phosphorylated, and it does accumulate in the nucleus 00:18:48.18 where it regulates genes that affect lifespan. 00:18:53.02 So, we needed to know: What are those genes? What are the genes that affect lifespan? 00:18:57.09 So nowadays there are really very good ways of asking 00:19:01.02 what genes in the animal are changed under a certain condition. 00:19:04.24 So worms have about 20,000 genes and you can actually profile all these 20,000 genes 00:19:11.24 using a technique called microarray analysis 00:19:14.13 to find out which genes are expressed at a higher level 00:19:17.21 or more active or which genes are less active in the long lived mutants. 00:19:21.05 So Colleen Murphy, a post-doc in the lab did that. She subjected these worms to 00:19:27.15 microarray analysis. What she found is that DAF-2 00:19:31.22 controls the expression of many different downstream genes. 00:19:35.07 OK, so here what this slide shows is the DAF-2 receptor 00:19:41.01 when it’s turned down by a mutation let's say. 00:19:44.05 The DAF-16 transcription factor becomes more active so that up arrow means more active 00:19:49.07 and as a consequence the expression of a lot of different genes changes. 00:19:53.05 Some go up, some go down. 00:19:56.02 OK, so that's interesting. Now, just because a gene 00:19:59.17 is more or less active doesn't mean it has anything to do with lifespan. 00:20:02.11 It could just be more or less active and not doing anything. 00:20:05.12 So we had to test that. So the way we tested this idea that these genes that were changing were 00:20:09.24 doing something to lifespan was again we used this RNAi technique. 00:20:13.25 So we took...we just made a list of all our genes 00:20:17.03 and at the top of the list we had the genes whose expression changed the most 00:20:20.14 in the long lived animal and at the bottom we had the ones that changed the least. 00:20:24.21 And we just started marching down the list, testing the activity of each individual gene with RNAi. 00:20:30.09 So we went to the refrigerator, opened it up, 00:20:32.05 got the bacteria out of it that were...or the freezer I guess, 00:20:35.11 got the bacteria out that expressed each one of these genes 00:20:40.12 whose expression changed in the long lived animal 00:20:42.13 and then we fed the long lived mutants those bacteria and we asked, "OK, if you knock down 00:20:48.12 like, this particular gene here, if you knock it down, 00:20:50.20 if it can't go up anymore, can that worm still live long? 00:20:54.05 And what about this one and what about this one?" And that's what we did. 00:20:57.06 And what we found was that lots of different genes affected lifespan. 00:21:01.04 So this shows you that inhibiting the activity of many of the genes that are turned up 00:21:06.03 in the long-lived daf-2 mutants shortens their lifespan. 00:21:09.15 OK, what you see here in black is the long lifespan 00:21:13.19 of the daf-2 mutant, and here as a control in this line 00:21:18.06 you see what happens if we subject these animals to RNAi for DAF-16, the transcription factor. 00:21:23.03 So, now we don't have the transcription factor so they can't live long. 00:21:26.08 But here what you see in color here are the lifespan curves of 00:21:30.03 lots of different populations of worms that have been subjected to RNAi 00:21:34.18 for any one of a number of those genes that were more active in the long lived mutant. 00:21:38.25 And you can see that now they don't live as long. 00:21:42.16 So, all of these genes here and more are needed for the long lifespan of the daf-2 mutant. 00:21:48.29 And there were some genes that were turned down in the long-lived mutants. 00:21:52.16 So we asked, "OK, are those genes preventing long lifespan? 00:21:57.18 If so, what would happen if you turned them down in the normal worm?" 00:22:00.15 So, we did that and what we found is that many genes that were turned down in the daf-2 mutants 00:22:05.10 also affect lifespan. And what we did here is we turned them down in normal animals. 00:22:10.21 So, here we have a control. It has a normal lifespan, OK? And each one of these lines here 00:22:16.23 corresponds to a set of normal worms with a good DAF-2 gene, 00:22:22.25 in which all we've done is to turn down 00:22:25.08 one of these many genes that are less active 00:22:28.19 in the long-lived mutant and you can see that they're living longer. 00:22:31.16 So, it’s really interesting. Both the genes that are turned up and the genes that are turned down 00:22:36.05 in the long-lived mutants make a difference. 00:22:39.16 OK, so what are these genes? Well, it turns out they do many different things. 00:22:43.14 Some encode antioxidant proteins. Some of these had already 00:22:47.22 been shown to be more active in the long lived mutants 00:22:49.27 by the lab of Gordon Lithgow and others, and we discovered some new ones. 00:22:54.29 But all together they include genes like superoxide dismutase, 00:22:58.18 metallothionine, glutathione S-transferases, 00:23:01.12 catalases, a whole variety of anti-oxidant proteins and 00:23:05.04 as I say inhibiting the function of these genes shortened the lifespan of the long lived mutant. 00:23:10.15 There were also genes that encode proteins called chaperones. 00:23:13.13 Now, what's a chaperone? A chaperone is a protein that just like the name suggests 00:23:17.21 takes care of other proteins. A chaperone protein will 00:23:20.21 bind to another protein physically and it will help it 00:23:24.06 assume the right shape, or if the protein is damaged it will actually escort 00:23:27.15 it to the cell's garbage can so the cell can get rid of it and make a new protein. 00:23:31.08 So, these genes encoding chaperones were more active in the long lived animals 00:23:36.02 and that made a difference because when we turn 00:23:38.04 the activity of these genes back down with RNAi, 00:23:41.00 the worms didn't live as long. 00:23:44.06 We also found a set of genes that are part of the worms innate immunity system, 00:23:48.24 genes whose protein products kill microorganisms. 00:23:52.25 These genes were much more active in the long-lived mutants. 00:23:56.06 And that's very interesting because we showed... 00:23:59.09 before that we had shown that if you feed worms 00:24:02.24 bacteria that can't divide, that can't proliferate, the worms live longer 00:24:08.14 suggesting that they're actually dying from infections and sure enough these long lived animals, 00:24:12.21 the daf-2 mutants, actually have more active anti-bacterial genes. 00:24:16.11 And actually the Ausubel and Ruvkun labs showed that 00:24:19.02 these long lived animals are resistant to pathogenic bacteria. 00:24:22.09 And then there were metabolic genes whose activities were changed. 00:24:26.19 So, for example, there are some genes that 00:24:28.08 whose normal function is to make proteins that transport 00:24:31.05 fat around the animal from place to place and these genes were less active in the long lived animals. 00:24:36.09 And when we made the genes less active in normal worms, they live longer. 00:24:40.05 And that's interesting because genes that transport fat or whose protein products transport fat 00:24:45.28 around the animal have been implicated in the ability of people to live to be a hundred. 00:24:52.03 People who live to be a hundred are called centenarians and it turns out that a lot of centenarians 00:24:56.28 seem to have mutations in genes whose function is to transport fat around the body. 00:25:02.00 And the mutations cause the genes to be less active just like these long-lived worms. 00:25:06.25 So there may be a link between this part of the worm pathway and centenarians. 00:25:12.00 And that was discovered by Nir Barzilai and other people. 00:25:16.12 OK, other labs, also, using different techniques identified 00:25:19.18 individual genes that are controlled by DAF-2 and DAF-16. 00:25:23.02 And again, they found that when they inhibited their activities 00:25:27.12 in many cases they affected the lifespan of the animal. 00:25:30.20 OK, so now let's look at the big picture here. 00:25:32.20 What we've seen is that these two genes, DAF-2 and DAF-16 00:25:37.20 together control a wide variety of subordinate genes, lots of them. 00:25:41.13 See, all these genes here, not just one but many. 00:25:45.29 OK? So, it's pretty neat. It’s actually kind of like a regulatory circuit or a little cassette 00:25:54.10 in which, you know, these control genes up here say, 00:25:59.10 you know, "Dance!" and all these genes down here say "OK, I will." 00:26:04.03 So it’s kind of like an orchestra where here we have the flutes 00:26:07.25 and the violins and the cellos and 00:26:09.21 the French horns and so forth. Each doing something different, 00:26:12.12 but all doing...everybody doing it at the same time. 00:26:15.24 And actually, I should point out...I didn't really emphasize this, 00:26:18.13 but it’s important, when you change any one of these genes 00:26:21.09 you get an affect on lifespan that is not as big as 00:26:25.05 the effect that you get when you change daf-16 or daf-2 00:26:28.05 suggesting that they act in a cumulative or additive way 00:26:31.00 to produce these huge effects on lifespan. 00:26:34.29 I just wanted to point out that DAF-16/FOXO, the transcription factor, 00:26:38.06 is actually a really important regulator of lifespan. 00:26:41.13 You can get C. elegans to live long as I said by changing the DAF-2 pathway, 00:26:47.06 the insulin/IGF-1 hormone pathway, 00:26:49.06 but you can also get them to live long by changing other genes. 00:26:51.25 You can get them to live long if you over-express the gene 00:26:55.24 encoding a protein called Heat Shock Factor 00:26:57.20 which is a stress response protein that protects worms from heat, 00:27:02.12 worms and other animals from heat. 00:27:04.05 Another stress response protein called Jun kinase 00:27:06.28 or a histone deacetylase protein called SIR-2. 00:27:10.23 Over-expressing any of these proteins in the worm extends lifespan. 00:27:15.10 And interestingly, in each case the lifespan extension requires DAF-16/FOXO. 00:27:20.25 OK, so while the drawing that I just showed you has, 00:27:23.11 you know, DAF-16 and DAF-2 up at the top 00:27:25.26 and then it branches down at the bottom, 00:27:28.15 maybe it’s more like a network where you have lots of inputs- 00:27:31.07 one from DAF-2, one from SIR-2, one from Jun kinases and so forth into DAF-16 00:27:36.16 which is like a node in a regulatory circuit in a way and then you have another bifurcation 00:27:42.17 where you regulate all the downstream genes. OK, so DAF-16 is a key regulator. 00:27:47.06 So, what does it all mean? Why should insulin and IGF-1, 00:27:52.12 which are essential hormones, why should inhibiting them extend lifespan? 00:27:58.01 Insulin and IGF-1 are very important, and they're very good for you. 00:28:01.23 If you don't have them you die. If you're a worm, 00:28:04.00 if you're a mouse, if you're a dog, 00:28:05.23 or a person, anybody--everybody dies. 00:28:09.18 So, they're very important because they promote growth and food storage. 00:28:14.19 So, again, why would inhibiting their activities extend lifespan? 00:28:19.24 Well, I think this is the way to think about it. 00:28:21.29 I think that what happens is that when you 00:28:23.26 lower the level of insulin or IGF-1 you actually shift the metabolism of the animal 00:28:28.26 from one that favors growth and storage of food, 00:28:33.18 and things like that, to one that favors maintenance. 00:28:37.10 So, low insulin/IGF-1 signaling or high heat shock factor or high Jun kinase or high SIR-2 activity 00:28:44.13 promotes cell maintenance and kind of resistance to stress. 00:28:48.23 And actually these long lived animals are very resistant to lots of environmental stresses. 00:28:53.06 This was shown by Tom Johnson's lab, first by Pam Larson's lab actually a long time ago 00:28:58.19 and more recently also to other stresses by Gordon Lithgow's lab. 00:29:01.22 But basically, they're resistant to heat, 00:29:04.00 to UV, to hydrogen peroxide, to paraquat, to all sorts of things. 00:29:09.26 And it may be that the same proteins 00:29:13.03 that make them resistant to these environmental insults 00:29:16.14 also allow them to be resistant to the toxic 00:29:18.23 products that build up say from reactive oxygen species 00:29:22.18 generated by the mitochondria during normal lifespan. 00:29:25.18 So there may be a connection between the resistance that an animal has to environmental stress 00:29:31.20 and its ability to live long. 00:29:34.07 And, like I said, some of those downstream genes that I told you about do both. 00:29:38.18 They make the animals resistance to environmental stress and to aging. 00:29:45.02 OK, so the way to think about it is that you can shift the physiology from one that favors growth 00:29:49.11 to one that favors stress resistance and maintenance. OK, and then there are lots of different 00:29:54.22 ways I think to accomplish this shift-by lowering insulin/IGF-1 levels, by activating SIR-2, 00:30:01.01 heat shock factor, lots of ways. OK. ` 00:30:07.05 So what are the implications for this? Well, the implication again, as I said, is that 00:30:10.09 a longevity regulatory module exists. So, this is a regulatory module for lifespan. 00:30:15.14 This is a little set of gene interactions that's built into the cell 00:30:20.07 that allows the animal to live longer. We didn't have to introduce something from Mars to 00:30:25.13 get these animals to live longer. We just briefly perturbed genes that they already have. 00:30:31.08 And because they are connected to one another 00:30:32.28 in this way functionally we get the this big affect on lifespan. 00:30:36.26 So this actually brings up an interesting question, 00:30:39.01 which is how could this regulatory module evolve? 00:30:42.23 How could it have come around in evolution? 00:30:44.28 Well, it could be that there's an advantage for the worms to get old. 00:30:50.03 So they have, you know, genes that allow them to get old. 00:30:53.18 For example, maybe it prevents an older animal from 00:30:56.23 competing with its progeny which in the case of the worm has the exact same genes because 00:31:00.22 C. elegans is a hermaphrodite so it reproduces by self-fertilization. So that's one possibility. 00:31:07.00 But there's another possibility and in order for me to explain this other possibility 00:31:10.20 to you I have to tell you a little more about the lifespan, or, sorry... 00:31:13.18 I have to tell you a little more about the lifecycle of C. elegans. 00:31:16.24 And I'll do that here in this slide. Now, what you see up here is the egg. 00:31:21.13 This is...C. elegans hatches from an egg and then it grows up to be an adult. 00:31:26.23 And it goes through these four different stages 00:31:28.22 called L1, L2, L3 and L4 and then it becomes an adult. 00:31:31.26 Now, that's what it does if there's a lot of food. 00:31:34.20 But, if you take a C. elegans egg and you put it in an 00:31:39.00 environment where there's not a lot of food 00:31:41.28 and where the animals are all crowded together, 00:31:44.03 what happens is the animals don't grow up. 00:31:46.07 Instead of becoming normal L2d's here they actually, oh sorry, normal L2 animals here. 00:31:52.11 They become L2d animals here. 00:31:54.20 And then they enter a state called dauer. Now, what's a dauer? 00:31:59.17 Dauer is a German word that means "enduring." 00:32:02.15 And this is a kind of...it's like a hibernation kind of state except it’s not really hibernation, 00:32:08.07 it's also sort of like a bacterial spore. 00:32:10.14 Anyway, these animals can move around, 00:32:12.18 but they don't eat, and they don't grow, 00:32:15.03 and they don't reproduce. They're arrested. They're sort of suspended in time. 00:32:18.21 And if you then give them food again, they exit from this dauer stage 00:32:23.04 and then they grow up and become L4s. 00:32:26.23 OK, so I should also tell you the only time an animal can become a dauer is before puberty. 00:32:33.10 Puberty is when the reproductive system matures and that happens at this time. 00:32:36.29 So, if you take an adult animal and you restrict its food, 00:32:40.12 it doesn't become a dauer-only at this time right here. 00:32:44.03 OK, so what does this have to do with the evolution of aging? 00:32:47.13 Well, let me just tell you this, if you turned the DAF-2 gene off 00:32:51.08 instead of just down (we turned it down 00:32:53.08 when we got these animals that live long) 00:32:54.28 but if you turn it off what happens is the worms hatch... 00:32:58.24 well, if you turn it completely off it’s likely that they die, 00:33:01.17 but if you turn it down really far what happens is that they hatch from an egg here 00:33:07.16 and then they grow up to become dauers. They don't grow up, they become dauers. 00:33:16.01 OK. And then they just stay there. They never grow up. 00:33:19.03 So that means that you need the normal function of the DAF-2 gene to grow to be an adult. 00:33:25.18 Now remember I told you that we found out from doing timing experiments 00:33:29.06 that DAF-2 acts during the adult to affect aging. 00:33:32.17 Of course, it acts during development to affect the dauer 00:33:35.15 because it has to. It has to be on at this time in order for the animal not to become a dauer. 00:33:40.12 That is, to be able to grow up to become an adult you have to have the gene on at this time. 00:33:44.12 And then we show, like I told you, that you have to have it on again in the adult to age normally. 00:33:49.28 OK, what the DAF-2 gene is doing is two things: 00:33:53.07 during development, it's preventing the animal 00:33:56.25 from becoming a dauer, 00:33:57.28 and during the adult it's preventing the animal from living longer than it would otherwise live. 00:34:04.01 OK, so we know already that a lot of the same genes that are...whose expression is changed 00:34:12.14 in the long-lived adults that allows the worm to live long, that those same genes 00:34:16.10 have a different expression in the dauer. 00:34:18.05 They're turned either up or down, same as the adult in the dauer. 00:34:21.26 And dauers also are resistant to all sorts of stresses. 00:34:24.04 Like, if you take a dauer and you heat it up, 00:34:25.26 it doesn't die. If you put hydrogen peroxide or paraquat on it, it doesn't die. 00:34:30.03 If you shine UV on it, it doesn't die. 00:34:31.20 So they're very stress resistant just like the long-lived adults. 00:34:35.15 OK, so it's possible that this lifespan module that I've been telling you about 00:34:40.00 didn't evolve to control the lifespan of the adult. 00:34:44.05 Maybe instead it evolved along with other dauer specific functions 00:34:49.12 to allow the dauer to live for a long time. So think about this. 00:34:53.29 If...what this means...the fact that the animal can go into dauer is very beneficial for it. 00:34:57.09 Because it means that if food is limiting it doesn't have children that will all die. 00:35:02.29 It just stops and waits for conditions to improve and then it grows up and has children. 00:35:08.06 So, it’s obviously very advantageous for a worm to be able to become a dauer. 00:35:11.22 You can see that there's great survival benefit and that would be selected for during evolution. 00:35:16.18 But, once you have the regulatory system up and running, 00:35:19.18 so that it can extend lifespan of the dauer 00:35:22.23 (dauers can live a very long time) 00:35:23.28 well, there it is. It exists. So, it seems like it’s possible then 00:35:29.01 to elicit at least part of this program in the adult 00:35:33.13 so the animals can live long. Now, I should say, the long-lived adults-they're not dauers. 00:35:37.22 They're very active. They eat, unlike a dauer. They can be completely fertile, unlike a dauer. 00:35:42.27 So, they're not completely dauers. Just like little dogs aren't dauers, they're normal little animals. 00:35:48.10 OK. but I think that the same...like I said the same 00:35:52.25 regulatory module that can allow the animal 00:35:55.24 to become.. to live long 00:35:58.12 can also be used to protect it. 00:36:01.00 In fact, it would be interesting to study mammals when they're hibernating 00:36:04.26 to see if they have low levels of insulin/IGF-1 activity or high levels of DAF-16 activity. 00:36:10.06 That would be very interesting. 00:36:12.15 OK, so it could have evolved to permit survival 00:36:16.19 in response to environmental conditions of the dauer. 00:36:20.01 But, once it’s already up and running the same system 00:36:22.27 is there so it will automatically influence aging in the adult. 00:36:26.19 And this also leads me to suggest that changes in either the regulators, 00:36:30.14 like DAF-16 or DAF-2 or SIR-2 or heat shock factor, these other regulators 00:36:35.19 or in the downstream genes like the chaperones 00:36:37.27 and other genes may be responsible for increasing lifespan during evolution. 00:36:42.21 So, in other words maybe the bat lives a lot longer than the mouse because bats have 00:36:49.12 either lower, less active regulators or more active regulators or less or more active downstream genes. 00:36:57.20 OK, the next question I want to ask is a very interesting one having to do with hormones. 00:37:01.19 The question is: Could some kind of environmental signal affect the activity of this DAF-2 pathway? 00:37:08.05 Now, one thing about hormones is that...the cool thing about them 00:37:12.08 is that they don't have to be there all the time. 00:37:13.27 They can be...a hormone can be present under some circumstances but not others. 00:37:17.23 So, for example, the hormone testosterone is present in a developing XY human embryo. 00:37:25.17 And that's why the XY embryo develops into a male, but is not present in the XX embryo. 00:37:30.11 So that's an example of a hormone being present under some conditions but not others. 00:37:35.10 So, is it possible that there are some kind of 00:37:37.03 environmental conditions that affect the activity of 00:37:40.14 this DAF-2 pathway so that you could slow down aging? 00:37:44.23 I should just note that all the changes that we've made so far are changes where we actually 00:37:50.11 reach in and change the gene itself. We make a mutation in the gene. 00:37:54.23 But, what I'm trying to suggest here is that maybe 00:37:56.08 it would be possible to change the activity of the pathway 00:37:59.05 by changing something in the environment. 00:38:01.21 OK, so the first obvious idea is caloric restriction. So, this is a rat, a picture of a rat. 00:38:08.27 And if you...a normal rat lives about three years here. 00:38:12.23 But if you calorically restrict a rat, that is 00:38:14.24 if you give it less food than it wants to eat it will live a lot longer. 00:38:19.01 And not only that, it stays disease resistant, 00:38:21.03 they don't get cancer or a lot of other age related diseases. 00:38:23.25 It's kind of magical. It's really neat. 00:38:25.28 And...so you would think that the insulin/IGF-1 pathway would mediate the response 00:38:31.05 to caloric restriction because when you eat food your insulin levels rise. 00:38:39.07 And so, I just told you that if you keep the insulin level down, and IGF-1 levels down, 00:38:43.25 you live longer, at least in these animals. 00:38:46.29 So, it’s a nice model to think that if you...when you don't eat enough you lower the level of 00:38:54.03 these hormone pathways, the activity of these pathways and as a consequence you live longer. 00:38:59.21 It’s a very pleasing idea and it seems like it’s probably right. 00:39:03.02 It's not really clear actually yet whether it's true in the worm. 00:39:05.23 It may be and it may not be. There's some conflict there. 00:39:08.24 Or it may be in some conditions but not others. 00:39:10.14 But it is pretty clear that caloric restriction...that the response to caloric restriction 00:39:15.22 is mediated at least in part by the insulin/IGF-1 pathway in yeast. Yeast actually also have 00:39:21.20 a little insulin/IGF-1 pathway. They don't have the actual hormones, 00:39:24.28 but they have some of the genes that are downstream 00:39:27.17 of the receptor, one called AKT, here. 00:39:29.25 And if you change this gene, the yeast actually are small. 00:39:33.25 They're tiny little yeast and they live long. 00:39:36.19 And it turns out that that pathway, the group of Brian Kennedy and others showed 00:39:41.24 that pathway is required for the response to caloric restriction. 00:39:46.26 In fruit flies the Partridge lab showed that the same thing is probably true. 00:39:50.27 And in mice there's some really cool experiments recently from the Bartke lab. 00:39:55.16 Now, I didn't tell you this already, but the hormone IGF-1 00:39:59.06 is produced under the control of another hormone, growth hormone. 00:40:02.29 So growth hormone, which is made by the pituitary gland, stimulates the release of IGF-1. 00:40:09.06 And mice that lack the receptor for growth hormone are also long lived. 00:40:14.11 And what Andre Bartke showed was really interesting. 00:40:18.21 He showed that if you took these long lived mice 00:40:20.20 that don't have growth hormone receptor 00:40:23.08 and you calorically restrict them, they don't live any longer. 00:40:27.01 And it's pretty cool. You take a normal mouse 00:40:30.26 and a long lived growth hormone receptor mutant mouse. 00:40:36.29 One is already living long-the growth hormone receptor mutant mouse 00:40:39.27 and that mouse, its tissues are very sensitive to insulin already. 00:40:44.26 When you calorically restrict this mouse, the mutant mouse, it doesn't live any longer 00:40:49.10 and it doesn't become any more insulin sensitive. 00:40:51.19 But when you calorically restrict the normal mouse, it becomes just as insulin sensitive 00:40:56.20 as the mutant mouse, and it lives just as long as the mutant mouse. 00:41:00.25 OK, so it kind of turns into that mutant mouse in that physiologically sense. 00:41:04.14 Although, it's not a mutant, it's just a hungry mouse. 00:41:08.13 The cool thing is both lose weight. 00:41:11.00 In fact, these growth hormone receptor mice are 00:41:13.22 just a little bit on the chubby side to begin with. 00:41:16.00 But they lose weight. So, it looks as though these growth hormone receptor mutants 00:41:22.01 are actually reaping the benefits or caloric restriction without going hungry. 00:41:26.29 So, OK, now I get to the most important part of my talk 00:41:31.00 which is to acknowledge the people that did the work that I talked about. 00:41:33.18 Now, this list of names, these are people that did the work 00:41:36.18 in both part one and part two of my lecture series. 00:41:39.22 But the work I just talked about was done by...first it was started by Ramon Tabtiong. 00:41:45.09 Ramon was a rotation student who came to my lab 00:41:47.19 and discovered that daf-2 mutants were long lived. 00:41:50.21 And I was so happy, because it was extremely hard 00:41:53.24 to get anyone at the time to come and studying aging. 00:41:55.26 People generally thought that aging was something 00:41:57.28 that just happened and there was nothing to study. 00:42:00.12 So, I was very, very lucky that he came to the lab. 00:42:03.27 Colleen Murphy did the work on the lifespan regulatory module 00:42:09.06 that I talked about. She did the microarray analysis. 00:42:11.07 And she showed that some genes were turned up in the long lived mutants and others down. 00:42:14.24 And that that made a big difference. 00:42:16.28 Andy Dillin did the timing experiments I talked about 00:42:20.10 showing that the DAF-2 gene and DAF-16 also 00:42:23.05 act exclusively in the adult to affect aging. 00:42:25.24 And Kui Lin over here, Kui cloned the DAF-16 gene and showed 00:42:32.00 that the protein that is encoded by the DAF-16 gene 00:42:34.28 is a transcription factor that regulates gene expression. 00:42:38.24 OK, see you in part 2.