Aging Genes: Genes and Cells that Determine the Lifespan of the Nematode C. elegans
Transcript of Part 2: Regulation of Aging by Signals from the Reproductive System
00:00:01.17 Welcome to part two of my lecture series. When we finished the first lecture 00:00:06.15 what we were talking about was whether there might be environmental factors 00:00:11.24 that could change the level of hormones present in the worm 00:00:15.14 that would then change the activity of the insulin/IGF-1 system 00:00:19.05 which would then lead to a longer lifespan. 00:00:22.10 And we discussed the possibility, or the likelihood 00:00:24.29 that the response to caloric restriction, 00:00:27.10 that is, eating less than you want to eat, is actually 00:00:31.05 mediated at least in part, in some animals through this insulin/IGF-1 pathway. 00:00:36.15 Now what I'm going to do is to tell you something 00:00:38.20 else that's really interesting that we discovered 00:00:40.19 which is that in C. elegans, sensory perception 00:00:45.13 regulates the insulin/IGF-1 pathway. 00:00:48.02 In other words, something that the worm 00:00:49.16 is either smelling or tasting is affecting its lifespan. 00:00:52.15 So, let me introduce you first to the sensory apparatus of the worm. 00:00:57.02 This is the beautiful face of C. elegans. 00:00:58.19 You can see its lips here. It has six lips. Here. 00:01:01.24 And what you see is...see right here? There's a little nostril. 00:01:04.10 There's another one right over here. 00:01:05.16 This is the nose of the worm and this is where the sensory neurons are. 00:01:10.07 So here you can see the sensory neurons. 00:01:11.22 Here are the endings. So here's the outside, here. 00:01:13.25 And this is the inside. The worm is now standing up. 00:01:17.12 So its head is up and its tail is down. 00:01:19.18 And you see these sensory neurons here. These are the endings. 00:01:22.08 So these little neurons allow the worms to smell 00:01:25.11 and taste things that are in the environment. 00:01:26.27 And worms move towards things they like, like sugar, 00:01:29.29 and away from things they don't like, like garlic. 00:01:32.02 But what we discovered in our lab was that mutations 00:01:37.03 that prevent the worm from smelling or tasting 00:01:39.20 as well as it normally would extend the lifespan of the animal. 00:01:43.02 So what you see here in this slide are lifespan curves 00:01:46.08 of nine different mutants. Each mutant is 00:01:50.15 defective in one gene that encodes a protein 00:01:54.28 that's required for sensory perception. 00:01:57.19 And what you can see is all the mutants are long-lived in red. You see them all? 00:02:00.20 They're long-lived. So one thing you should know is that 00:02:03.24 these worms are not calorically restricted. 00:02:05.25 They're eating as much food as they want. 00:02:08.05 They eat a lot of food, as much food as normal worms eat. 00:02:10.02 And if you calorically restrict a worm it has delayed reproduction, 00:02:13.28 but these worms don't have delayed reproduction. 00:02:17.00 So they're not calorically restricted, yet they're living longer. 00:02:20.05 So, we also found that we could change genes that... 00:02:24.04 all these lifespan curves that I'm showing you here 00:02:27.15 are from the lifespans of worms with defective genes 00:02:32.19 that are needed for the structures of these neurons. 00:02:35.00 But we also found that we could make mutations 00:02:37.18 or knock-down genes whose protein products are actually 00:02:41.16 specific chemosensory receptors, proteins that are situated 00:02:44.26 right here at the very tip of these neurons 00:02:46.29 with one face open to the outside world and the other end inside the neuron. 00:02:52.15 And knocking down those kinds of proteins also could increase lifespan. 00:02:59.07 OK, so this is our model. I'm not going to go into all the details about it. 00:03:03.26 The model is that there's in the environment 00:03:07.02 that worms are smelling or tasting that's affecting their lifespan. 00:03:10.08 And we think that at least part of the way this works 00:03:12.27 is by regulating the activity of the insulin/IGF-1 pathway. 00:03:16.25 Now, why do we think that? First of all, I told you earlier 00:03:20.07 that if you inhibit the activity of the DAF-2 hormone receptor 00:03:24.18 or this pathway in various different places, worms live long, 00:03:29.18 and that they have to have DAF-16, the transcription factor, 00:03:32.09 in order to live long. 00:03:33.24 And we found that DAF-16 was also required in order 00:03:37.11 for these sensory mutants to live long. 00:03:40.17 In fact, if you make a mutation that only affects 00:03:42.24 a protein right here, in the tip of the worms nose, 00:03:44.26 DAF-16 protein accumulates in all the nuclei 00:03:47.24 of all the cells in the animal. It's amazing. 00:03:49.29 Also, if we take a daf-2 mutant that already lives long, 00:03:53.17 and we make changes in these sensory genes, the worms don't live any longer. 00:04:00.25 OK, so this is our model. The model is that 00:04:03.11 the cell bodies of these sensory neurons, 00:04:06.09 which are shown here, contain the hormone. 00:04:08.08 And they do. We know...C. elegans has about 30 or 35 genes 00:04:12.02 that encode insulin and IGF-1 like proteins 00:04:14.24 and many of them are expressed in these neurons. 00:04:17.03 So, the idea is that there's something in the environment 00:04:19.09 that is regulating the activity or the release of these hormones. 00:04:24.02 So, for example, the idea is that when the worms 00:04:27.23 are in their normal environment 00:04:29.02 these hormones are being released rapidly 00:04:31.17 and so they bind to the receptor and speed up aging. 00:04:34.07 But when we make a mutation that knocks down the activity 00:04:37.13 of this pathway by screwing up these sensory neurons, 00:04:39.27 then there's less hormone so the activity of the receptor 00:04:43.20 is down so the worms live longer. 00:04:45.22 So that's our model. 00:04:48.07 So now we've been asking...it's clever I should say. 00:04:49.28 It's clever. I forgot to say this. 00:04:52.18 What the worms seem to be doing, possibly, 00:04:54.26 is responding in advance to a changing environment. 00:04:59.06 You can imagine, caloric restriction extends lifespan 00:05:01.25 but in order for that to happen you have to actually be hungry. 00:05:05.01 But if you just have to smell or taste the difference 00:05:06.29 you may be able to get a head start on a response to a poor environment. 00:05:12.08 OK, so the question is: Which neurons influence lifespan? 00:05:17.05 There's two possible models you can imagine. 00:05:19.09 One is that one neuron or one pair of neurons, one in each nostril 00:05:24.24 affects lifespan and that's it. 00:05:26.11 The other possibility is that all of those neurons in the nostrils are affecting lifespan. 00:05:31.19 Each one...there's 12 in each so each one would have 00:05:34.16 about one twelfth of the total amount of effect. 00:05:37.23 So we decided to see which was the case or if maybe there was 00:05:40.20 a third possibility by changing... 00:05:46.21 we decided to do this by killing individual neurons with a laser. 00:05:51.03 And so what we found was really interesting. 00:05:53.18 We found that killing one gustatory neuron or taste neuron 00:05:57.08 extends the lifespan of the animal. So let me show you how we did this. 00:06:00.22 This is a picture here of part...it's really the worm's throat here. 00:06:05.20 The nose is out here in this region. 00:06:09.00 And this is where the cell bodies are. 00:06:11.08 So we can take a laser and point it at one particular cell and kill that cell. 00:06:17.01 And we can do that in lots of animals and then see if the animals live longer. 00:06:21.07 And we found that if we kill this one particular cell, 00:06:23.14 they live long as you can see here in green. 00:06:28.21 And then what we found was the following. 00:06:30.03 That if we killed a different gustatory neuron 00:06:32.18 called J, which is located right there, called J, 00:06:37.01 as well as the first one (we killed both), they don't live any longer. 00:06:41.25 OK, isn't that interesting? So here we have one neuron that promotes longevity 00:06:46.26 and one neuron that inhibits longevity. 00:06:51.10 OK, so here's another way of just telling you what I already told you. 00:06:53.26 If we kill this one taste neuron, we have a long lifespan. 00:06:58.11 If we kill that same taste neuron plus J, 00:07:00.10 we live a normal life. We have a normal lifespan. 00:07:04.25 OK, now there are also neurons that mediate the response to smell. 00:07:08.00 So we can...to smells, they're olfactory neurons. So we can kill those neurons also. 00:07:12.15 So if you kill one of these smell neurons, in many cases, the worms live longer. 00:07:16.19 And actually, if you kill a taste neuron and a smell neuron, 00:07:20.06 both in the same animal, then they live even longer. 00:07:24.22 OK, so now what happens if we take an animal and we kill a smell neuron 00:07:28.15 plus J, plus this neuron? 00:07:32.27 Smell neuron plus J. Do we have a normal lifespan, 00:07:36.20 the way we did when we killed the taste neuron plus J? 00:07:40.01 Or do we have a long lifespan? The answer is we have a long lifespan. 00:07:43.25 OK, so killing J can affect the lifespan of an animal that lacks 00:07:50.09 the taste neuron but not an animal that lacks the smell neuron. 00:07:54.22 So, in other word, J is talking to this neuron but not this neuron. 00:08:01.11 You see? So they're having little, private conversations with each other. 00:08:04.05 So this is really interesting. We also find that if we kill 00:08:06.16 certain other neurons, nothing happens. 00:08:08.29 So it looks as though neither one of our two models is correct. 00:08:13.22 It's not just one neuron matters, 00:08:15.03 but it's also not the case that all the neurons have the same function. 00:08:18.23 It looks as though there's a lot of sensory discrimination 00:08:21.04 going on in the brain of this worm that's affecting its lifespan. 00:08:23.17 So, these and other findings I don't have time to 00:08:26.01 tell you about suggest that the lifespan of C. elegans is 00:08:29.04 influenced by its perception of both soluble compounds 00:08:33.14 that it tastes and volatile compounds that it smells in the environment. 00:08:38.00 So, of course, we always ask the question: 00:08:40.28 If smell and taste affects the lifespan of worms, 00:08:44.03 is this true in other organisms as well? And very recently it was shown that... 00:08:49.24 well, I'm going to get to humans in a minute. 00:08:51.22 Very recently it was shown that the fruit fly Drosophila 00:08:57.01 that its lifespan is also regulated by sensory perception. 00:09:00.27 In other words, if you change the ability of the fly to smell, it can live longer. 00:09:05.11 So what about humans? We don't know. 00:09:06.29 And it's kind of a scary thought that something that you smell or taste 00:09:09.09 could affect your lifespan. But, I'll just leave you with one thought, 00:09:12.12 which is that if you eat some food, 00:09:15.11 your insulin levels rise. But, if you also smell the food 00:09:19.28 that you're eating, they rise even more. 00:09:24.04 Alright, what about reproduction and aging? 00:09:26.16 It's a fascinating question of great interest to evolutionary biologists 00:09:30.03 and to everyone. 00:09:33.00 What is the link between longevity and reproduction? 00:09:36.13 I already sort of mentioned in lecture number one 00:09:38.08 that there's not a necessary decrease 00:09:42.29 in reproduction in order for daf-2 mutants to live long 00:09:46.27 And, in fact, there are a lot of long lived mutants now 00:09:49.02 that have even more progeny than normal. 00:09:52.16 So there doesn't have to be a direct trade off 00:09:55.04 between the length of life that you have 00:09:57.03 and the number of offspring that you can have, which is good. 00:09:59.29 However, I should tell you that if you lower the activity 00:10:02.17 of the daf-2 receptor a fair amount, 00:10:05.07 then you get a long-lived worm that does have fewer progeny. 00:10:09.22 OK, so the same gene, the daf-2 gene, controls reproduction and aging both. 00:10:15.16 But you can uncouple them from one another. 00:10:18.02 In other words, if you just lower the level of the gene a little bit, 00:10:20.13 you live long and you reproduce perfectly well. If you lower it a little bit more, 00:10:24.23 you still live long, but now you have problems reproducing. 00:10:30.08 So now we've talked actually about three things this gene does: 00:10:32.20 it controls reproduction; it controls growth, and it controls aging. 00:10:40.11 Now what I want to tell you is something really interesting 00:10:41.28 about the reproductive system and aging. 00:10:44.21 It turns out that the reproductive system 00:10:47.01 actually controls the aging of the worm. 00:10:50.24 So to tell you that first I have to introduce you to it. 00:10:53.00 So what you see here is the reproductive system of the worm 00:10:56.03 when it's just hatched from an egg. 00:10:58.13 It has four cells in it. These two blue cells here give rise to the germ line. 00:11:03.18 And these two green cells, here, give rise to the somatic reproductive tissues 00:11:09.16 like the uterus and the ovary and so forth. 00:11:12.19 Well, what we found several years ago was that removing the germ line, 00:11:16.13 again with a laser, extends lifespan. 00:11:18.28 So here in black you see a normal worm and in blue you see the lifespan 00:11:22.14 of a worm that doesn't have any germ cells 00:11:24.19 and they live a lot longer. 00:11:26.20 It's amazing, 60% longer. So you might think, 00:11:29.29 "Alright, are they living long because they're sterile?" 00:11:34.29 But, that's not the case and the reason we know that is because 00:11:38.07 if you take a laser and you kill all four cells, 00:11:40.24 they don't live long and now they have a normal lifespan. 00:11:43.17 So they're still sterile but they don't live long. 00:11:47.18 So what this means then is that both the germ cells 00:11:51.08 and the somatic cells are affecting lifespan. 00:11:54.18 So we have a new signaling pathway here: 00:11:56.25 from the reproductive system to the rest of the animal. 00:11:58.23 The germ cells somehow, these two blue cells, 00:12:01.29 are making something that is inhibiting longevity because 00:12:05.14 when we kill the germ cells the worms are living longer. 00:12:08.14 Whereas the green cells are making something that is promoting longevity 00:12:12.10 because when you kill those then you shorten the lifespan. 00:12:18.16 OK. So, we have sort of equal and opposite effects here. 00:12:22.10 So, the germs cells are kind of in charge. It's really interesting. 00:12:26.02 The germ cells are giving rise to the next generation. 00:12:29.08 They become the next generation, 00:12:30.19 but they're also controlling the aging of the body of the animal. 00:12:36.26 So they're in charge of everything. 00:12:37.24 So you might say, "Well, why is that?" 00:12:40.12 Is that so that an animal that lacks germ cells can live long, 00:12:43.21 which seems kind of odd? 00:12:44.24 But, so we thought, "Well, why might this have evolved, 00:12:47.07 a system like this?" 00:12:48.28 And our favorite idea is the following. 00:12:50.16 The idea is that maybe having the germ cells control 00:12:54.28 the aging of the body allows the animal to coordinate 00:12:58.21 the process of aging with reproduction. 00:13:03.05 OK. It's obviously important that an animal reproduce when it's in its prime. 00:13:06.26 So, let's just see what would happen, according to this model, 00:13:09.00 if something happened to the germ cells. 00:13:10.25 So, suppose the development of the germ cells were delayed somehow. 00:13:14.08 Well, then of course the animal would also reproduce later. 00:13:18.01 Its production of progeny would be delayed. 00:13:21.00 So you might worry. You might think, "Uh oh, I hope the 00:13:22.26 worm isn't going to be too old to have progeny." 00:13:25.12 But it wouldn't because you would also be slowing down the aging of the body. 00:13:31.26 So, that would keep the animal in its prime when it's ready to produce progeny. 00:13:35.27 So, you see how potentially this system could allow the animal to coordinate 00:13:40.06 two really important things it has to do; its rate of aging with its reproduction. 00:13:47.13 OK, so how does germ line removal extend lifespan? 00:13:50.10 Well, that same transcription factor DAF-16/FOXO 00:13:53.24 is required because in animals lacking that gene 00:13:57.02 if you kill the germ cells the worms don't live long. 00:13:59.16 They still have normal lifespan. 00:14:03.00 So DAF-16 is involved. And I'm not going 00:14:05.27 to go into all the details because I don't have time. 00:14:08.19 But what I'll just tell you is removing the germs cells activates 00:14:12.02 another kind of hormone pathway, a steroid hormone pathway. 00:14:15.11 And these hormone pathways activate DAF-16 which extends lifespan. 00:14:20.24 And this is work from two labs; from the Antebi lab 00:14:23.19 who's discovered a sterol hormone called dafachronic acid 00:14:28.29 that acts in this pathway and Etienne Baulieu's lab 00:14:32.18 in collaboration with our lab whose work implicates 00:14:35.21 a different steroid hormone, pregnenolone, in this pathway. 00:14:41.22 OK, so what happens now if we take a worm and we change the daf-2 gene 00:14:46.10 and the reproductive signaling system both in the same animal? 00:14:50.03 What we get is something quite amazing. 00:14:52.10 We get an animal that lives six times as long as normal. 00:14:55.02 So, normal worms in this experiment have a lifespan of 20 days on average. 00:14:59.12 These worms lived 126 days. And what's really spectacular 00:15:03.09 is that they stay young and healthy for a really long time. 00:15:06.28 So this is a movie of these worms when they were 144 days old. 00:15:10.21 And you can see that they're moving. 00:15:12.08 If you think back to the slide...the movie that I showed you 00:15:14.24 at the beginning of the first lecture series. 00:15:16.16 Remember that worms that were 13 days old, that were just lying there, 00:15:21.07 in the nursing home. These animals are ten times as old, 00:15:23.18 and they're still moving around. 00:15:25.09 So, it's absolutely remarkable that you could do this, 00:15:28.23 that you could make a few very small changes 00:15:30.22 and produce a six-fold extension of lifespan is just amazing. 00:15:35.11 And it really makes you...this would be like people that live to 500. 00:15:38.20 So it really makes you wonder just what's possible. 00:15:41.07 I'm not saying that people could live to 500. I don't know. We don't know. 00:15:46.01 But, in human terms that's what it would be like and they're on the golf course. 00:15:52.11 OK, what about mammals? 00:15:53.12 Could the reproductive system affect lifespan in mammals? 00:15:55.22 It does. The lab of Gary Anderson and Jim Carey have shown, 00:15:59.15 those two labs have shown 00:16:00.27 that if you take a mouse and you remove 00:16:04.02 its own reproductive organs, the female mouse, 00:16:06.23 and you let it get old and then you introduce 00:16:10.02 the reproductive tissues from a young mouse into an old mouse 00:16:14.23 the old recipient mouse lives longer. 00:16:18.24 So, in other words, putting young reproductive tissues into 00:16:21.25 an older mouse extends the lifespan of the older mouse. 00:16:24.27 It's amazing because somehow the reproductive system is sending signals 00:16:29.01 to the rest of the body telling it not to age as fast. 00:16:32.20 We don't know at all how this works or if it's 00:16:35.14 related to the system I've been telling you about. 00:16:37.08 But, it's pretty cool because it's the reproductive system. 00:16:42.15 OK, so now the last thing that I want to talk about in my lecture series here 00:16:47.06 is what links normal aging to age-related disease? 00:16:51.13 This is a really important question. 00:16:52.28 There are all sorts of diseases that you get at 00:16:56.07 a much higher frequency if you're old. 00:16:58.03 You're 100-times more likely to get a tumor at age, 00:17:01.09 say, 65 than you are at age 35, much more likely. 00:17:04.22 And the same with lots of other diseases like Alzheimer's disease, 00:17:07.00 cardiac malfunction, all sorts of things. 00:17:09.25 So, what is it? Why is it that an older individual is more susceptible. 00:17:15.00 Well, you can ask the question genetically. 00:17:18.11 You can say, "Here's a pathway, the DAF-16/DAF-2 pathway 00:17:21.14 that affects aging. So, does the same pathway affect age related disease?" 00:17:28.10 And the answer is yes. And it's quite remarkable just how pervasive this effect is. 00:17:33.19 So let's start here with worms. 00:17:34.26 You can cause worms to get Huntington's disease or Alzheimer's disease by 00:17:38.26 expressing the genes that cause those diseases 00:17:41.04 in the worm and it turns out that the long-lived daf-2 mutants 00:17:45.20 are resistant. They get the diseases but not until they're much older. 00:17:50.00 Worms, normal worms actually, develop a muscle condition 00:17:53.02 that's very similar to human sarcopenia 00:17:55.06 where the muscles start to deteriorate when they get old. 00:17:57.25 And the daf-2 mutants get that condition later. 00:18:02.28 In the case of flies they've done a very interesting experiment. 00:18:05.23 If you take an electrical shock and you 00:18:11.04 deliver it to the heart of a young fly at a certain strength, the fly is fine. 00:18:15.20 But if you give an old fly that same shock, its heart fails. 00:18:21.02 But if you take this long-lived insulin/IGF-1 receptor mutant fly 00:18:25.06 and you do the same thing to it, 00:18:27.12 you stress the heart, even when it's old, 00:18:29.10 even when it's on its deathbed, the heart's fine. 00:18:31.26 So, in this case the heart seems to be in better shape 00:18:34.03 than the rest of the animal. 00:18:38.09 So there's an affect on cardiac function. 00:18:39.26 In mice we know that cancer is delayed in these long-lived animals. 00:18:44.22 And there are unpublished reports now of a lot of 00:18:46.24 other diseases as well that are delayed. 00:18:50.11 OK, so it looks like these animals really are young in every sense of the word. 00:18:55.25 They look young and they're young in the sense that 00:18:57.29 they're not susceptible to age-related diseases. 00:19:00.11 OK, so I'm going to tell you a little bit now about one particular 00:19:03.15 age-related disease that we've been studying in our lab. 00:19:05.24 Oh, first before I do, I meant to tell you that this ushers in the possibility 00:19:10.15 of a brand new therapeutic strategy where we go after 00:19:15.01 lots of age-related diseases all at once by going after aging. 00:19:19.01 In other words, if we can slow down aging, then we can slow down 00:19:22.07 cancer, heart disease, protein aggregation diseases, 00:19:24.23 all the diseases of aging potentially. 00:19:26.15 So it's...we don't know if this would really work, 00:19:29.06 but it seems to be working for worms at least 00:19:33.09 and these other animals as well so the potential is huge. 00:19:37.17 And actually I founded a biotech company called 00:19:40.12 Elixir Pharmaceuticals that's trying to 00:19:43.27 develop compounds like that, to go after age related diseases 00:19:46.10 by going after aging itself, pathways that control aging. 00:19:51.23 OK, so now what I want to do is to tell you about 00:19:53.21 some work that we've been doing in our lab 00:19:55.03 on tumors that worms get. So, worms normally don't get tumors. 00:20:00.01 But you can cause them to get tumors with a mutation. 00:20:03.04 This is what...and these tumors affect the germ cells. 00:20:06.12 So, now what I want to do is introduce you to the 00:20:08.25 reproductive system of the normal adult worm. 00:20:10.29 I showed you before the reproductive system of the animal 00:20:13.12 at hatching when it only had four cells, 00:20:15.24 but now I'll show you what they look like when they're adults. 00:20:19.20 So what you can see is the reproductive system is huge, 00:20:22.09 here and it has these green cells, here, are the oocytes. 00:20:24.25 And these red cells here are the stem cells, the germ line stem cells that keep dividing in the adult 00:20:30.00 to replenish the germ cell population. 00:20:33.19 OK, so now mutations in a gene called GLD-1 which stands for germ line 00:20:38.08 defective number 1 cause germ line tumors. 00:20:42.07 And I'll show you what these look like. 00:20:43.10 What happens...so this is what the normal reproductive system looks like, again. 00:20:47.28 Here in green you see the oocytes. 00:20:49.15 But now look at this. In the mutant these oocytes 00:20:53.10 change their mind and they start to proliferate again. 00:20:56.22 They become very similar to these stem cells here. 00:20:58.29 And they divide and divide and divide and pretty soon 00:21:02.28 they fill up the whole gonad 00:21:03.28 and they burst out of the gonad and they fill 00:21:05.22 the whole entire animal up and then the animal dies. 00:21:08.11 So its lifespan is about half of what it would be normally. 00:21:12.22 OK, so we wanted to know: Do mutations 00:21:15.11 that extend lifespan delay the gld-1 tumor phenotype? 00:21:20.00 OK, do they delay the tumor? 00:21:23.26 OK, now there are lots of different longevity pathways in C. elegans. 00:21:27.29 And I'm just going to remind you of the ones I told you about 00:21:31.18 and tell you about a couple new ones. 00:21:32.29 First of all there's the insulin/IGF-1/FOXO signaling pathway 00:21:36.08 that I've been telling you about. 00:21:37.13 There's also caloric restriction, you can get worms... 00:21:40.10 you can calorically restrict worms by using a mutation 00:21:42.24 that prevents the animal from eating food very well. 00:21:45.05 That's an eat-2 mutant and those worms live longer. 00:21:49.04 You can also get worms to live long by perturbing 00:21:52.04 the mitochondria in various ways. 00:21:53.20 If you make mutations that inhibit respiration, worms live long. 00:21:57.24 And one of these mutations is one that's been studied 00:21:59.24 in the Hekimi lab called isp-1. 00:22:02.02 And another mutation that affects the production of ubiquinone 00:22:04.29 also studied in the Hekimi lab is called clk-1. 00:22:07.27 And these mutants also live long. 00:22:09.22 And this pathway seems to be different from either caloric 00:22:13.16 restriction or the insulin/IGF-1 pathway. 00:22:15.23 OK, so the question is: What happens to these tumors in these mutants? 00:22:23.15 All these pathways are conserved so it seems like 00:22:25.29 a reasonable question to ask because 00:22:28.00 what we learn in worms may have relevance for higher organisms. 00:22:32.19 And as I already told you, both caloric restriction and insulin/IGF-1 signaling 00:22:38.03 are known to extend lifespan and delay tumors in mice. 00:22:42.08 OK, so we wanted to see what we could learn from the worm. 00:22:44.28 So there were several possible outcomes. I should tell you right away that 00:22:50.10 there are mutations in mice that have been described that affect proteins like 00:22:54.29 p53 and other proteins that have a kind of bad effect on the animal. 00:22:59.15 They suppress the formation of tumors, but they speed up aging. 00:23:02.03 So there's a trade off there. You benefit by not having a tumor, but 00:23:06.10 you don't really benefit because you're dead anyway 00:23:07.24 because you die of premature aging. 00:23:10.19 So one possibility would be that any or all of these pathways 00:23:15.11 could actually cause the tumor...if the worms would live long 00:23:20.05 without the tumor, but they might speed up aging. 00:23:23.17 That's one possible outcome. The next possible outcome is that 00:23:26.14 there's no affect on the tumor. 00:23:27.16 In which case, the animals would still die from the tumor at a normal time. 00:23:30.12 And the third possibility is that these mutations would cause 00:23:34.28 the animals to get the tumors later in life. 00:23:38.00 So the animals would live longer. 00:23:40.20 And what we found is that all of the longevity mutations 00:23:43.12 extended the lifespans of the tumor mutants; 00:23:45.10 the caloric restriction mutations, the daf-2 mutations, 00:23:48.04 and also the mitochondrial mutations. 00:23:52.08 The daf-2 mutants were amazing. 00:23:53.22 They were completely immune to the lifespan shortening effects of the tumors. 00:23:57.03 This is normal worm here in gold. 00:23:58.20 That's its lifespan. They're all dead in this experiment by about 25 days. 00:24:05.14 And this is what happens if you just introduce the tumor 00:24:08.20 mutation into a normal worm. 00:24:10.08 You cut the lifespan in half. 00:24:12.09 And now out here you see the wonderful long daf-2 lifespan in red 00:24:16.10 and you see that it doesn't really shorten much at all, 00:24:18.26 if any. It's not statistically significantly different 00:24:22.03 if you introduce the gld-1 mutation. 00:24:26.14 In other words, in a daf-2 mutant the gld-1 mutation no longer kills the animal. 00:24:30.23 The tumor doesn't kill the animal. So, why not? 00:24:33.08 Here's what the animals look like. Let me just introduce you to this. 00:24:37.00 This is a little line drawing of the head of a worm. 00:24:39.07 Here's the pharynx, here. And this is the intestine here. 00:24:43.12 And what you see here in this drawing or this photograph are the nuclei 00:24:49.22 of cells that are stained with DAPI, which is a stain for DNA. 00:24:52.12 And now, look at this. This is the gld-1 tumor mutant. 00:24:56.04 And what you see is that the animal is just jam packed full of germ cells. 00:25:00.13 And that's why it dies very soon, very early. 00:25:04.06 But this is what the tumor looks like in the daf-2 mutant. It's much smaller. 00:25:09.16 There still...it doesn't look normal. You don't have the oocytes back, 00:25:13.00 but there are 50% fewer germ cells here. 00:25:16.24 And so it doesn't...it's no longer toxic or lethal to the animal. 00:25:20.12 OK, so how exactly do these longevity mutations inhibit the tumor? 00:25:25.10 What are they doing? 00:25:26.22 Well, first of all let's talk about what the gld-1 mutation does. 00:25:29.23 It does two things...it causes a tumor. 00:25:31.28 It does two things; it speeds up or it promotes cell division 00:25:37.16 and it prevents apoptosis. Apoptosis is programmed cell death. 00:25:42.00 So, in the gld-1 mutant...normally in a normal germ line some cells just die. 00:25:48.13 But in the gld-1 mutant they no longer die. 00:25:51.05 OK, so if you have fewer dead cells and you have more cell divisions, 00:25:55.04 you're going to get a tumor. 00:25:56.04 And that's what happens. 00:25:58.29 OK, so what do the daf-2 mutations do to delay the growth of the tumor? 00:26:04.13 Well, the first thing is they cause apoptosis to occur. 00:26:07.25 So here is the normal worm, called N2. 00:26:10.00 Here's the apoptosis that you see in the normal worm. 00:26:12.10 Here's the tumor mutant where it's gone. 00:26:14.12 And you see in the daf-2 mutant it comes back partly. 00:26:17.14 And here in this part you see these two little dots here 00:26:21.16 are little cells that are undergoing cell death. 00:26:24.15 They're stained with something that is specific for cells 00:26:28.22 that are undergoing apoptosis. 00:26:32.20 The daf-16 gene is required for this. 00:26:37.08 What about p53? 00:26:38.09 p53 is a famous tumor suppressor gene that's known 00:26:41.03 to play a role in apoptosis in mammals. 00:26:42.29 So is it required? 00:26:45.01 Yes, it is. Here what you see is a normal worm. 00:26:48.12 There's a certain amount of apoptosis. 00:26:52.04 Oh, I'm sorry, this is a worm treated with gld-1 RNAi so it has a tumor. 00:26:57.07 So the level is low. You have a little bit because the RNAi 00:27:00.01 is not quite as effective as the mutation. 00:27:01.24 It goes way up in a daf-2 mutant, and then it goes back down again if you take away p53. 00:27:08.17 Ok, now I have to tell you something interesting about this. 00:27:10.24 In C. elegans, p53 plays only a minor role 00:27:15.08 in germ cell death under normal conditions. 00:27:18.00 But, what we really see the effect of p53 is when you 00:27:23.03 take a worm and you shine...you expose it to gamma rays 00:27:28.03 or other agents that produce damage in DNA. 00:27:31.13 Then you get a lot of cell death, and that cell death 00:27:33.23 in the germ line requires p53. 00:27:36.29 OK, so I told you that cell death...sorry, I told you that 00:27:40.23 daf...sorry that p53 is required 00:27:43.12 for the increase in cell death that you get in a daf-2 mutant. 00:27:47.00 So we wondered, "Is it possible that a daf-2 mutant 00:27:49.16 is sort of similar to an animal that's been subjected to genic toxic stress. 00:27:53.22 So, in other words, are daf-2 mutations maybe inducing a response to stress? 00:27:58.14 And we think that's probably the case. 00:28:00.18 Part of the reason we think that is we found that DAF-16, 00:28:03.19 the transcription factor, is required for gamma rays 00:28:06.26 to trigger germ cell death in normal worms. 00:28:09.27 Both p53 and DAF-16 are needed for this extra cell death in the daf-2 mutant 00:28:14.23 and in a normal worm that you treat with gamma rays. 00:28:18.27 So, we also tested another, different kind of a tumor. 00:28:22.15 This tumor has a different name, it's called glp-1 00:28:25.06 and this is a tumor in which what happens is that the germ line stem cells 00:28:29.15 just keep dividing. The oocytes are still oocytes. 00:28:34.01 This is affecting a different population of cells. 00:28:36.01 In this case, what happens is that normally there's a receptor 00:28:39.29 on the surface of the stem cells that 00:28:41.13 receives a signal from the environment, from nearby cells, telling them to divide. 00:28:45.23 And what happens is that when the germ cells move away 00:28:49.28 from the source of the signal they stop dividing. 00:28:51.25 But in this mutant, the receptor for this signal 00:28:55.06 is just on all the time even if there's no signal. 00:28:57.29 So these cells just divide and divide and divide, and they make a tumor. 00:29:00.10 So what happens in this tumor? 00:29:03.06 We found that daf-2 mutants or mutations 00:29:05.25 also extend the lifespan of this tumor mutant, 00:29:07.24 but they don't trigger germ line cell death so they're doing it a different way, 00:29:11.14 specifically by cell proliferation. I'll get to that in a minute. 00:29:14.07 But, keep this in mind because I'll come back to this later. 00:29:17.10 So what about cell division? We found that daf-2 mutations reduce mitosis, 00:29:22.20 cell division, in both tumor mutants, in both gld-1 and glp-1. 00:29:26.29 So here you see dividing cells in the normal animal, 00:29:30.03 lots of dividing cells in the tumor mutant 00:29:32.14 and many fewer dividing cells in the...when you put 00:29:35.15 daf-2 mutations into the tumor mutant. 00:29:41.28 The cool thing is that daf-2 mutations don't just willy-nilly block cell division. 00:29:49.00 They only block cell division within the tumor. 00:29:51.11 Here's what happens. So here's the tumor, with lots of cell division. 00:29:54.26 And here's when you put a daf-2 mutation into the tumor, 00:29:58.10 into the animal containing the tumors. 00:30:00.14 Cell division goes down, but look what happens if you put a daf-2 mutation 00:30:03.19 into a normal worm. 00:30:06.00 We still get the same amount of germ cell proliferation, no effect. 00:30:11.00 So that's really neat. Somehow it knows which cells are the tumor cells. 00:30:14.00 I mean it doesn't really know consciously but you know, 00:30:16.25 teleologically, you could say that. 00:30:18.23 So I just want to stop for a second and pause here 00:30:21.04 and say that this...we see effects both on cell death and on cell proliferation. 00:30:25.25 And there's some similarities here between the worms 00:30:28.22 and vertebrates, and I'll point them out now. 00:30:30.25 One is, in humans it's known that mutations 00:30:35.19 that activate insulin and IGF-1 signaling cause cancer. 00:30:41.04 So we've shown the opposite in the worms. 00:30:43.02 We've shown that if you turn down insulin/IGF-1 signaling, 00:30:46.10 lower than it would be normally, you actually sort of cure cancer. 00:30:51.07 I mean you don't die from it anyway. I shouldn't say cancer either. 00:30:53.22 These are tumors. And I should make this clear. 00:30:55.13 These tumors they...a tumor is a group of cells that 00:31:01.06 is proliferating too much. It's over proliferating. 00:31:03.25 And that's what happens. That's why they're called tumors. 00:31:05.28 But the cells don't undergo the late stages of human cancer 00:31:09.18 like angiogenesis where they attract blood vessels 00:31:13.00 and they don't actually actively metastasize. 00:31:15.22 So they're a good model possibly for the early events of human tumors 00:31:19.10 but not the later ones. I should have made that point clear before. 00:31:22.02 But anyway, the cool thing is that what we've shown is that if you 00:31:25.08 inhibit this pathway you can actually do the opposite of what you would do 00:31:30.14 of what would be happening in a human that has a mutation that 00:31:33.19 increases the level of signal. 00:31:36.23 The second thing is, I told you before that 00:31:39.06 in the long-lived mice that have defects in this pathway 00:31:43.25 it's known that cancer levels are down and 00:31:46.14 what our findings suggest is that maybe DAF-16 and p53 00:31:50.09 are involved in this. Maybe that's the pathway. 00:31:52.07 Nothing is known about the pathway in the mice, 00:31:54.12 but maybe that's the pathway and maybe that's 00:31:56.10 affecting cell death as well as mitosis. 00:32:00.03 What about the other mutants? 00:32:01.05 Well, caloric restriction and mitochondrial mutations 00:32:04.07 also inhibit the tumor growth. Here's a picture, again, 00:32:08.11 a diagram of the head of the worm. 00:32:09.28 And here's the DAPI staining and this is the animal packed full of germ cells 00:32:14.11 and you can see that it's a lot better 00:32:15.24 if you, in this case, introduce a mutation that causes caloric restriction and 00:32:20.12 you get the same effect if you introduce 00:32:21.21 a mutation that affects the mitochondria. 00:32:24.18 They don't affect apoptosis (cell death), 00:32:28.14 but they do reduce the number of mitotic germ cells. 00:32:31.19 And amazingly none of them affect mitosis 00:32:35.01 in the normal germ line, none of them. 00:32:37.27 The daf-2 mutation I told you before and these 00:32:40.10 mitochondrial mutations and the eat-2 mutation, 00:32:42.21 none of them affect normal mitosis, just...they all 00:32:45.02 can tell the difference between normal mitosis and the tumor. 00:32:48.21 And so how do they do that? Why should this be? 00:32:52.04 Well, we have an idea and I think a clue comes from the stem cell tumor, 00:32:56.03 the glp-1 mutant that I told you about. 00:32:57.27 Now, in that tumor, daf-2 mutations inhibit cell divisions and those tumors, 00:33:04.05 the glp-1 tumors, are thought to consist of lots of normal germ line stem cells. 00:33:09.10 In other words, those tumors, if you recall, are different from... 00:33:13.15 are caused because the receptor for 00:33:18.00 a growth factor is...thinks it's on all the time. 00:33:22.07 It's mutant so it's constitutively active. 00:33:24.14 And that's why the cells are dividing. 00:33:26.08 But that receptor is normally active in those cells at a normal time. 00:33:29.20 So basically, I think what we've done in that mutant 00:33:32.03 is just to make many more copies of normal cells. 00:33:34.28 The cells aren't, probably, any different 00:33:37.00 from a normal cell, but they're just more of them. 00:33:38.21 And yet, when there's a lot of them, 00:33:41.03 you need DAF-2 for cell division and these other genes for cell division. 00:33:46.10 Whereas when there's just a few of them you don't. 00:33:48.29 So the model then is a tumor is a large metabolic load. 00:33:53.14 In other words, having all these cells 00:33:55.07 dividing and dividing may require a lot of energy, a lot of nutrients. 00:33:59.15 And perhaps these longevity pathways, which as I told you 00:34:02.25 shift the physiology of an animal from 00:34:04.26 one that favors growth to one that favors maintenance, 00:34:07.01 maybe they just can't support the tumor. 00:34:09.21 They can...it's not a problem for them to support 00:34:12.08 the small little group of germ line cells that are dividing normally. 00:34:18.05 But when you put a real big metabolic load on they just can't stand it. 00:34:22.00 And that's why I think the tumor cells are probably affected. 00:34:24.10 At least that's the model. 00:34:27.03 OK, so to summarize: daf-2 mutations do two things: 00:34:29.28 they induce a stress response that triggers apoptosis 00:34:33.02 in a p53 dependent fashion, 00:34:34.29 and they also reduce mitosis within the tumor, 00:34:38.20 and the other mutations specifically reduce mitosis. 00:34:41.24 This raises an interesting question of whether these clk-1 mutations 00:34:46.06 are going to turn out to affect tumors in mammals and stay tuned. 00:34:50.07 So, a few thoughts about this: 00:34:52.08 first of all, we see a really strong correlation in these worms between 00:34:56.21 the rates of normal aging and tumor growth 00:34:58.12 and a variety of different longevity mutants. 00:35:01.04 In nature it's generally the older individuals that are susceptible to cancer. 00:35:05.21 And it's not...the animals aren't...it's not the number of days they've been alive. 00:35:09.19 So, for example, people get high rates of cancer 00:35:12.20 after many decades, 60, 70 so forth, at high rates. 00:35:16.06 Whereas a dog gets cancer at the same high rate when it's 10 years old 00:35:20.14 and a mouse when it's just a year and a half. 00:35:22.16 But all these animal have in common the fact that they're elderly. 00:35:26.08 OK, so there's this correlation that you see in all animals 00:35:29.20 between being elderly and being susceptible to cancer. 00:35:32.07 And so it's really interesting that all these mutations 00:35:35.25 that increase lifespan also delay tumors, 00:35:38.00 and it suggests to me that maybe single gene mutations 00:35:41.07 in the genes that I've been telling you about or 00:35:43.02 the downstream genes they control link these two processes during evolution. 00:35:46.27 In other words, you have an animal with a short lifespan 00:35:48.26 and there's a mutation that 00:35:50.11 increases the lifespan. Maybe that same mutation 00:35:52.22 also makes it more tumor resistant. 00:35:54.25 So that if you keep doing this, you know, 00:35:57.02 through evolution pretty soon you get a human 00:35:58.14 that has a really long lifespan and stays really resistant to cancer for a long time. 00:36:04.05 OK, alright so I'm going to end now my lecture series. 00:36:07.09 And I just want to tell you about the people who did the experiments 00:36:09.25 I talked about in part two of my series. 00:36:12.26 I started off by talking about sensory perception. 00:36:15.16 Javier Apfeld is the one who discovered that sensory mutants 00:36:18.28 extend the lifespan of worms. 00:36:20.14 Joy Alcedo did the experiments of killing individual neurons and showing 00:36:23.24 that there's a really an elaborate sensory processing going on here 00:36:27.11 in the brain of the worm. 00:36:28.08 Honor Hsin showed that killing the germ cells extends lifespan. 00:36:33.18 And Kui, Natasha, Jen have worked on the reproductive system. 00:36:39.20 And also I mentioned that in collaboration with Florence Broue, 00:36:43.05 who was a graduate student in Etienne Baulieu's lab 00:36:45.11 and our lab...this collaboration resulted in the knowledge that steroid hormones 00:36:52.01 can influence the lifespan of C. elegans via this pathway. 00:36:57.22 And I also mentioned that Adam Antebi's lab working independently showed 00:37:00.27 that dafachronic acid, a sterol hormone, was part of this pathway. 00:37:04.20 Nuno Arantes-Oliveira produced these worms 00:37:07.23 that lived 6-times as long as normal, these amazing worms. 00:37:10.13 And Julie Pinkston did all the work on the tumors that I talked about. 00:37:14.17 OK, thank you very much.
