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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.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

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