First Long-Lived Mutants:
of the Insulin/IGF-1 Pathway for Aging
Kenyon played a role in the discovery that the aging process is subject to
genetic regulation. This section (written by Cynthia)
describes that discovery. (It will be published in the Proceedings of
the Royal Society, 2010, in press)
insulin/IGF-1 signaling extends lifespan and delays age-related disease in
species throughout the animal kingdom. This life-extension pathway, the first
to be defined, was discovered through genetic studies in the small roundworm C.
elegans. This discovery is described here.
In spite of
the fascinating qualities of the aging process, such as its remarkably
different pace in different species, until the last few decades aging was not
thought to be subject to any active regulation. Now we know that the rate of
aging is indeed subject to regulation, by classical signaling pathways. These
pathways link the aging rate to environmental and physiological cues, and may
even underlie its diversification during evolution. At the heart of these pathways
are stress and metabolic sensors such as insulin and IGF-1 hormones, TOR kinase and
AMP kinase, whose up- or down-regulation can trigger a variety of
cell-protective mechanisms that extend lifespan.
lifespan pathway to be discovered was the insulin/IGF-1/FOXO pathway. This
pathway is evolutionarily conserved: Mutations in many insulin and IGF-1 pathway
genes extend the lifespan of mammals and several have been linked to human
longevity. In particular, DNA variants in FOXO transcription-factor genes have
been linked to exceptional longevity in human cohorts from around the world.
These and other exciting findings grew out of basic genetic research in the
small nematode C. elegans, where the first long-lived mutants were isolated. In this article, I describe how some
of these discoveries came about. I am not a historian but rather a scientist
who played a role in these discoveries. Although the literature I cite
establishes an objective narrative of these findings, I will describe the
studies in which I participated from my own personal perspective, being part of
the story as it unfolded.
elegans lives only
a few weeks, but during its lifetime it undergoes a physical and behavioral
decline that anyone, even someone who has never seen the worm, can recognize as
aging (see online movie). I remember realizing this for the first time in the
early 1980s when I was a postdoctoral fellow with Sydney Brenner in Cambridge,
England and I was cultivating a mutant strain that had very few progeny. Normal
hermaphrodites produce 300 self-progeny during their first week of life. So a
single worm on a culture dish soon disappears into a sea of progeny and cannot
be found. I left culture dishes with my almost-infertile mutants in the
incubator for several weeks, and then looked at them. With so few progeny, the
original animals were still easy to find, and to my surprise, they looked old.
This concept, that worms get old, really struck me. I sat there, feeling a
little sorry for them, and then wondered whether there were genes that
controlled aging, and how one might find them.
around that time, Michael Klass was already screening for long-lived mutants.
Klass was a postdoc in David Hirsh's lab at the University of Colorado,
Boulder. His elegant early work set the stage for genetic studies. Klass
showed, for example, that C. elegans lives longer and has fewer progeny when subjected to
dietary restriction. This phenomenon had first been observed in rodents in the
1930s and had remained unexplained. Klass also showed that worms, which are
ectotherms, live longer at low temperature than at high temperature (Klass,
1997) (1). By doing temperature-shift experiments, he discovered that the
animals carry a memory of their childhood temperature that affects their adult
lifespan, a phenomenon that has yet to be explained molecularly. Then, to find
genes affecting aging, Klass carried out a screen for long-lived mutants,
noting that "Because many mutations in vital genes will lead to a decrease
in life span, it is potentially more interesting to obtain mutants with
significantly increased life spans." (Klass, 1983) (2) [also see (Johnson
and Wood, 1982) (3)]. Klass mutagenized a group of animals and looked among
their second-generation descendants for mutants that lived long. To curtail
reproduction while also ensuring that any long-lived mutants could be
propagated, he carried out his screen in animals harboring a
temperature-sensitive fer-15 mutation, which prevented reproduction at high temperature.
He established little families from several hundred individual potential
mutants at low (permissive) temperature, and then tested members of each family
at high temperature, where they could not reproduce, to ask which families, if
any, were long lived. Eight of Klass' families were long-lived, but, after
observing their additional phenotypes, he concluded that they probably did not
harbor interesting lifespan mutations. For example, several mutants were
feeding-defective, and Klass concluded that these animals were probably
long-lived because of dietary restriction. He wrote "The high correlation
of the decreased rate of food ingestion of these mutants with their increased
longevity is interpreted as indicating that the increased longevity is most
likely due to reduced caloric intake. These results appear to indicate that
specific life span genes are extremely rare or, alternatively, life span is
controlled in a polygenic fashion" (Klass, 1983) (2).
mutants were not abandoned, however. Another researcher, Tom Johnson, continued
to study them. Previously, Tom had been exploring the genetic basis of aging in
another way (Johnson, 1987, Johnson and Wood, 1982) (3, 4). Working in Bill
Wood's lab, which was also at the University of Colorado, Tom had crossed two
different strains of worms, the normal laboratory strain Bristol and a French
strain with a similar lifespan called Bergerac, and established new
"recombinant-inbred" lines from their descendants. These different
lines should contain new combinations of polymorphic alleles present in the
parental Bristol or Bergerac strains. Tom found something very interesting:
some recombinant-inbred lines lived much longer than others. This meant that
there were genetic polymorphisms in these strains that could lengthen or
Subsequently, Tom outcrossed some of Klass' mutants and
recovered a strain that ate perfectly well and yet still lived long (Friedman
and Johnson, 1988) (5). He wrote: "age-1(hx546) is a recessive mutant allele in Caenorhabditis
results in an increase in mean life span averaging 40% ... at 20 degrees; at 25
degrees age-1(hx546) averages a 65% increase in mean life span..." This was a very interesting
finding. However, the mutant displayed a phenotype that
raised the possibility of a more trivial explanation for its longevity: it had
sharply reduced fertility. Evolutionary theory predicted that animals with
reduced fertility would live longer, as they would be able to divert resources
that would otherwise be used for reproduction into somatic maintenance. Thus it
was possible that the primary effect of age-1(hx546) was to affect fertility, not aging.
In Johnson's words: "age-1(hx546) is associated with a 75% decrease in hermaphrodite
self-fertility...It is likely that the action of age-1 in lengthening life results not
from eliminating a programmed aging function but rather from reduced
hermaphrodite self-fertility or from some other unknown metabolic or
physiologic alteration." Subsequently, Johnson went on to characterize the
phenotype of this mutant in more detail, showing, for example, that age-1(hx546)
exponential increase in mortality rate that occurs with age (Johnson, 1990)
(6). Interestingly, later, in 1993, he crossed away the fertility defect of the age-1
mutant, and it
still lived long (7).
Around the time that Johnson first described age-1, I had become very interested in
studying aging. To me, aging seemed like unexplored territory likely to be full
of interesting surprises. I was fascinated by the "Hayflick limit"
(Hayflick, 1965; Hayflick, 1989) (8, 9) which raised the possibility of an
intrinsic life timer, and by human progeria diseases (Brown, 1979; Thomson and
Forfar, 1950) (10, 11), which suggested that at least some aspects of aging
could be accelerated. Because of my previous scientific experience, I had come
to think that there would be universal, evolutionarily-conserved regulatory
mechanisms for aging. This had recently been shown to be the case for
development, and my lab had played a role in this realization, discovering that
Hox (Antennapedia-like homeotic) genes patterned the bodies of a much broader
spectrum of species than had been anticipated (Costa et al., 1988) (12). In general, this was the time of a
great paradigm shift in biology, when we all began to realize that organisms
from yeast to humans utilized highly similar molecular mechanisms, albeit with
variation, to carry out the fundamental processes of life. Even if we didn't
know why we age, aging is a nearly ubiquitous phenomenon, and something so
universal seemed to me likely to be regulated. Furthermore, the remarkable
differences in lifespan that one sees between different species could
potentially have arisen by changes in regulatory genes. There are long- and
short-lived insects, birds, and mammals; thus the rate of aging appeared to be
highly "evolvable". This diversity could arise rapidly if it were
driven by changes in regulatory genes, which, like changes in the Hox genes
(which, for example, can convert the antennae of flies to legs), could produce
large transformations all at once. Eventually I came to hypothesize that there
would be some kind of universal mechanism for aging, controlled by regulatory
genes whose activities could be dialed up or down to lengthen or shorten
lifespan (Kenyon, 1996; Kenyon, 1997) (13, 14).
At the time, aging was generally thought to be a hopelessly
intractable, even futile, problem to study. We just wear out; that's it.
Fortunately, because of my experience I had come to expect that biological
phenomena that seemed to happen haphazardly might well turn out to be
controlled by the genes. For example, as a graduate student at MIT, I had
worked on a gene that, amazingly enough, was required for UV light to cause
mutations (Bagg et al., 1981) (15). Not only was aging thought to be merely a
passive, entropic process, evolutionary biologists had argued forcefully that
aging could not
be regulated. For example, they felt that mechanisms for regulating aging would
have no way to evolve, as aging takes place after reproduction. These theories
were thought provoking, but to my mind, they had the effect of discouraging
searches for regulatory genes. It seemed to me, a molecular geneticist from the
outside, that one should keep an open mind and just have a look. So I saw the
analysis of aging as a fantastic opportunity to explore the unknown and perhaps
discover something new and important.
I heard Tom Johnson speak at several local genetics and C.
in the 1980s, as we were both working in California. I told Tom after a meeting
at Lake Arrowhead that I thought age-1 was extremely exciting. I was skeptical of the
interpretation that age-1 mutants lived long only because their reproduction was
impaired. The idea that resources saved by not reproducing would automatically
be shunted into longevity pathways seemed too simplistic to me. I remember
suggesting to Tom that he test the trade-off theory directly, by laser-ablating
the reproductive precursor cells of normal worms and asking whether the animals
lived longer. Later, I even invited him to our lab to use our laser. By this
time, I was chomping at the bit to study aging myself. I decided not to work on
age-1 for two
in our friendly C. elegans culture, it was not polite to study someone else's gene, and
to Tom. But mainly, I wanted to carry out my own screen for long-lived mutants,
to see what came out.
It had been very easy for me to attract students to my
laboratory to study pattern formation, but the situation with aging was
completely different. It took several years to find someone interested in
looking for long-lived mutants. At the University of California, San Francisco,
where I worked, new graduate students spend a few months "rotating"
in each of several labs, and I tried to interest these students. However, the
aging field at the time was considered a backwater by many molecular
biologists, and the students weren't interested, or were even repelled by the
idea. Many of my faculty colleagues felt the same way. One told me that I would
fall off the edge of the earth if I studied aging. However, at last, in the
spring quarter of 1992, a wonderful, risk-taking rotation student, Ramon
Tabtiang, agreed to come study aging.
Ramon had three projects in the lab. The first was to do the
experiment I described above, to test the reproductive trade-off theory by
laser-ablating the cells that give rise to the reproductive system. The second
was to screen for long-lived mutants. The third was to ask what effect retinoic
acid might have on Hox gene expression in the worm, a project related to my
lab's general research effort.
The Hox-gene project didn't go anywhere, but the two aging
projects went fantastically well. Ramon sat down at our laser-equipped
microscope, located newly-hatched worms' reproductive precursor cells, and
killed them with the laser. The worms grew up and were sterile, but they had a
completely normal lifespan. I was delighted. There was no reproductive trade-off.
C. elegans hermaphrodites
contain 959 somatic cells and 2000 germ cells, yet the resources freed up by
removing all those germ cells were not redirected to longevity. (This result
was consistent with Klass' and Johnson's earlier findings that sterile fer-15
mutants have a
normal lifespan. It was more definitive, however, as fer-15 mutants, whose sperm are defective,
still produce the massive germline and they lay unfertilized oocytes.)
For the mutant screen, we were so lucky that it is still hard
to believe. As I mentioned above, to look for long-lived mutants, one needed to
control reproduction. To do this, we decided to use a
"dauer-constitutive" mutation. Dauer (German for
"enduring") is a state of diapause, analogous to a bacterial spore. Dauer
formation is essentially a checkpoint that arrests the growth of developing
animals at a specific larval stage (an alternative L3 stage) if they encounter
low food levels or crowding (Figure 1).
Figure 1: The life cycle of C. elegans. Under replete conditions (green arrows), the C. elegans
hermaphrodite hatches from the egg, passes through four developmental stages
(L1-L4), and becomes a fertile adult. Under harsh environmental conditions (red
arrows), including low food availability, crowding and elevated temperature,
the animals enter the dauer diapause instead of becoming L3 larvae. When
environmental conditions improve, the dauers exit from the dauer state to
become L4s and then fertile adults.
tiny, growth-arrested juveniles that have their own special morphology, do not
feed or reproduce, and are quiescent and long-lived. Dauer formation
essentially allows the juvenile to outlast harsh environmental conditions
before reproducing. When food is restored, dauers resume development and become
fertile adults. Only young juveniles can become dauers; once the animals go
through puberty and become adults they no longer have this option. Mutations in
many genes were known to produce a dauer-constitutive phenotype, in which
juveniles enter the dauer state even in the presence of food. [In fact, because
dauers are long-lived, Klass had recovered dauer-constitutive mutants in his
screen for longevity (Klass, 1983) (2).]
Our plan was to EMS-mutagenize
animals that harbored a temperature-sensitive dauer-constitutive mutation.
After culturing the animals for several generations at low (non-dauer-inducing)
temperature (20íC), so that new mutations could become homozygous via
hermaphrodite self-fertilization, we would put individual worms, each a
potential long-lived mutant, on separate culture plates and allow each to lay
~20 eggs. At this point, we would remove the animal, and allow its progeny to
develop past the dauer decision-point. Then we would shift the plates to high
temperature (25íC), where, having escaped dauer formation, the worms would
continue growth to adulthood. At high temperature, these adults would have
progeny, but all the progeny would become dauers, which are small and
inconspicuous. Then we would wait until most of the normal adults should have
died. If we found plates containing long-lived mutants, we could propagate the
strain by shifting their dauer progeny back to the low temperature, where they
would exit the dauer stage, grow and reproduce.
Many genes affect dauer formation, and, as the entire
process is facilitated by high temperature, many dauer-constitutive mutants
become dauers at high but not low temperature (Riddle et al., 1981; Vowels and
Thomas, 1992) (16, 17). We chose the mutant daf-2(e1370) because it was a very
"tight" allele: when grown at high temperature all the hatchlings
became dauers, and when grown at low temperature, they all behaved like wild
type and grew to adulthood. To know when to examine the plates for long-lived
mutants, we had to determine when most or all of the control daf-2(e1370) animals would be dead. Ramon came
into my office one day and said: "Guess what, they're not dying". And
we had our first long-lived mutant.
Being a careful scientist, Ramon was not convinced. He
suggested that there might be a rogue age-1 mutation in the background. He
finished his rotation and joined another lab for his Ph.D. I went on to measure
the lifespans of two other daf-2 mutants, isolated independently in a different lab. They
were also long-lived, so the longevity must be caused by daf-2 mutation. daf-2 mutants were the most amazing things
I had ever seen. They were active and healthy and they lived more than twice as
long as normal (Kenyon et al., 1993) (18). It seemed magical but also a little
creepy: they should have been dead, but there they were, moving around.
This was a thrilling discovery scientifically, and it was
also important from a practical point of view. Now it was easy to attract
rotation students to work on aging. [Several years later, we re-identified daf-2
in real genetic
screens for longevity, first using EMS (Garigan et al., 2002) (19) and then
RNAi (Hansen et al., 2005) (20).]
We characterized the daf-2 mutants in more detail. For example,
we found that they lived long if we cultured them continuously at 20íC, where
they grew normally to adulthood. So the animals did not have to grow under
dauer-inducing conditions to live long. The simplest interpretation of these
results, confirmed later by additional mutant (Gems et al., 1998; Larsen et
al., 1995) (21, 22) and RNAi (Dillin et al., 2002) (23) analysis, was that
severe reductions in daf-2 activity triggered dauer formation, but milder reductions
that permitted growth to adulthood (or severe reductions following the dauer
decision point) extended adult lifespan. Later we showed using RNAi that the
wild-type daf-2 gene
acts exclusively during adulthood to affect aging (Dillin et al, 2002) (23), so
it acts twice: once to affect dauer formation, which can only take place during
development, and again later, to affect aging.
We also measured the brood size of daf-2 mutants. We found that at 20íC, the daf-2(e1370)
mutant had 20%
fewer progeny than normal. Even though we had shown that loss of the whole
reproductive system did not extend lifespan, we wanted to test this
significance of this reduced brood size for longevity. A skeptic could argue
that daf-2 mutations
caused a particular type of
change in reproduction that in turn caused longevity. To test this possibility,
we killed the reproductive precursor cells of daf-2 mutants. The animals still lived
long (Kenyon et al., 1993) (18). Thus wild-type animals and daf-2 mutants had different lifespans in
the complete absence of their reproductive systems. Therefore they had
different lifespans for reasons other than differences in fertility. Later,
others showed that some daf-2 mutants had essentially normal reproduction (Gems et al.,
1998; Larsen et al., 1995; Tissenbaum and Ruvkun, 1998) (21, 22, 24), and, we
were able to uncouple daf-2's roles in aging and reproduction temporally, using RNAi
(Dillin et al., 2002) (23).
The daf-2 gene had been known to influence dauer formation since the
early 1980s (Riddle et al., 1981) (17). Don Riddle, and also Jim Thomas' lab,
had shown that the ability of daf-2 mutants to become dauers required another gene, daf-16 (Riddle et al., 1981; Vowels and
Thomas, 1992) (16,
17). Of course, I immediately wanted to know whether daf-16 was also required for the extended
lifespans of daf-2(-) adults. We still had only rotation students studying aging. (Other than
myself, all of the five authors on our 1993 paper were rotation students, and
none joined the lab for their Ph.D.s.) I tried to interest someone in testing
the role of daf-16, but no one agreed, so I did it myself. I found that daf-16 was completely required for daf-2
mutants to live long
(Kenyon et al., 1993) (18). Thus these two genes had analogous effects on dauer
formation and adult aging: Wild-type daf-2(+) prevented wild-type daf-16(+) from promoting dauer formation
during development and from extending the lifespan of the adult (Figure 2).
Because it kept animals youthful and extended their lifespans, we nicknamed
wild-type daf-16 "Sweet
Figure 2: daf-2 and daf-16 regulate dauer formation and
lifespan. When conditions are favorable during development, wild-type daf-2
inhibits the activity of daf-16, allowing growth to adulthood. Under harsh
environmental conditions, daf-2 activity levels fall, allowing daf-16 activity
to promote dauer formation. During adulthood, reducing daf-2 activity allows
daf-16 to promote longevity. This genetic pathway was inferred from the mutant
phenotypes of daf-2 and daf-16. Long-lived adults carrying relatively weak
daf-2 mutations do not look like dauers, move actively and can be completely
fertile. We now know that these genes act exclusively during adulthood to
regulate adult lifespan, whereas they act during development to regulate dauer
formation. daf-2 activity in the adult can be influenced by environmental
signals, as the pathway can mediate the longevity effects of caloric
restriction and it can extend lifespan in response to altered sensory cues.
daf-2 encodes an insulin/IGF-1-receptor that inhibits DAF-16/FOXO
transcriptional activity via a conserved protein kinase cascade that acts
directly on DAF-16/FOXO. Active DAF-16/FOXO, in turn, influences lifespan by
regulating a variety of cell protective and metabolic genes.
In 1992, the dauer pathway was known to comprise three
groups of genes [see (Riddle and Albert, 1997) (25)]. One group was later shown
to encode a TGF-beta signaling pathway. The second group turned out to encode
members of a guanylate-cyclase signaling pathway. The third group contained daf-2
and daf-16. We examined the lifespans of animals
carrying mutations in genes from these other two groups (Kenyon et al., 1993)
(18), but none was long lived. Larsen and Riddle also tested the lifespans of a
large variety of dauer-constitutive mutants, and, like us, found that daf-2 mutations increased lifespan, but
that dauer-constitutive mutations affecting these other branches did not
(Larsen et al., 1995) (22). (See Footnote #1.)
Our discovery that daf-2 and daf-16 affected aging allowed us to draw some
interesting inferences. First, daf-2 and daf-16 were known to be regulatory genes, as they regulated dauer
formation. Thus, even without knowing the mechanism, the finding that changing known
regulatory genes could double lifespan suggested that aging was subject to
regulation. Specifically we said in our paper (about daf-2 and daf-16): "Both genes also regulate
formation of the dauer larva...Our findings raise the possibility that the
longevity of the dauer is not simply a consequence of its arrested growth but
instead results from a regulated lifespan extension mechanism that can be
uncoupled from other aspects of dauer formation" (Kenyon et al., 1993)
Another important aspect of this work is that it was the
first clear indication that genes encoding nutrient sensors regulate aging. The
study of dauer formation indicated that, in the presence of food, daf-2(+) was active and promoted growth to
adulthood. Low food was thought to trigger dauer formation by reducing daf-2
activity. In our
paper (Kenyon et al., 1993) (18), we said: "Lifespan in mammals, and to a
lesser extent, C. elegans, can be increased by food limitation. It is possible that daf-2
mutations elicit an
internal signal also generated by food limitation, which can extend
lifespan...It would be interesting to learn whether lifespan extension caused
by food limitation requires daf-16." This does appear to be the case, for at least two
methods of dietary restriction. Intermittent (every-other-day) feeding extends
the lifespan of wild-type animals, but it does not further extend the long
lifespan of daf-2 mutants.
In addition, this lifespan extension requires daf-16 for its full effect (Honjoh et al.,
2009) (27). Furthermore, daf-16 is required for dietary restriction initiated in middle age
to extend lifespan (Greer et al., 2007) (28). [Curiously, daf-16 is not required for life-long food
limitation to extend lifespan (Lakowski and Hekimi, 1998) (29) so it is not
required for lifespan extension under all conditions of dietary restriction.]
Knowing about age-1 for so long, I really wanted to learn whether the
long lifespan of the age-1 mutant, like that of the daf-2 mutant, required daf-16. Jenny Dorman, a lab technician
planning to go to graduate school, addressed this question by building the double
daf-16; age-1 mutant.
She (and other labs working independently) found that the double mutant was not
long lived (Dorman et al., 1995; Larsen et al., 1995, Murakami and Johnson,
1996) (22, 30, 31). This was a striking and exciting result. It meant that age-1
was part of the
same pathway as daf-2 and daf-16.
All of these genes were likely to be working in a single pathway to influence
lifespan. The finding was also crucial from a practical standpoint, as it
enabled the subsequent cloning and molecular identification of age-1.
The fact that loss of daf-16, a gene required for dauer formation,
suppressed the long lifespan of the age-1 mutant, suggested that age-1(hx546) might be a weak allele of a dauer
gene. In fact, a candidate for such a dauer gene existed. This gene, called daf-23, behaved like daf-2 genetically. daf-23 was being analyzed by Gary Ruvkun's
lab, which had been focusing on the daf-2/daf-16 branch of the dauer pathway. Gary's
lab published a beautiful paper about the role of these three genes in dauer
formation in 1994 (Gottleib and Ruvkun, 1994) (32). [Later, they and others
showed that daf-23 mutants were long lived (Larsen et al., 1995, Malone et al., 1996,
Morris et al., 1996) (22, 33, 34)]. To me, the exciting thing about daf-23 was that its map position was close
to the map position that Tom Johnson had reported for age-1 (Johnson et al., 1993) (7). I wanted to test the idea that age-1
might actually be
an allele of daf-23, but we still had almost no one working on aging in the lab and
therefore could not carry out our own mapping experiments and the
The day we learned that the age-1 mutant's longevity was suppressed by
I called and told my friend Jim Thomas, who studied daf-16's role in dauer formation. I never
thought that Jim would start working on age-1. However, unbeknownst to me, Jim had
found that animals carrying very weak dauer-constitutive mutations could become
dauers at 27íC, a very high temperature for worms. Sure enough, age-1(hx546)
dauers at 27íC. With this obvious phenotype, it was very easy for Jim's group
to map age-1, by
scoring dauer formation rather than lifespan. The position of age-1 relative to other genes turned out to be slightly different
from that reported by Tom (Johnson et al., 1993) (7) and exactly the same as that of daf-23
(Malone et al.,
1996) (34). In addition, Jim's lab showed that age-1(hx546) failed to complement daf-23(-) mutants. Ergo, they were the same gene.
I was disappointed that we were not able to show this ourselves, but at the
same time glad that Jim had moved the field forward so quickly. The Thomas lab
thanked us for "communicating unpublished results that motivated us to
study the Daf-c phenotype of age-1" (Malone et al., 1996) (34). The Ruvkun group also showed that age-1
and daf-23 were the same gene (Morris et al.,
What kinds of proteins did daf-2, daf-23/age-1 and daf-16 encode? During the early 1990s, there
was a frenzy of dauer-gene cloning going on in several labs [see (Riddle and
Alberts, 1997) (25)]. In fact, the Ruvkun lab was already in the process of
cloning daf-2, daf-23 and daf-16
when we discovered that daf-2 and daf-16 affected aging. We were of course very interested in
learning more about the molecular roles of these genes in aging. When I first
told Gary at a meeting that daf-2 mutants were long lived, he (like most members of the worm
field) did not seem to be interested in aging. "Aging? You mean, you look
at old worms?" he said. However, being a curious person and fantastic
scientist, he soon seemed to have a change of heart. A few weeks later, when we
asked him whether he would share sequence information prior to publication so
that we could study their molecular roles in aging, he said no, as he had now
become interested in the process. So we needed to clone the genes ourselves. We
didn't have enough people working on aging yet to go after both genes, so we
chose the more downstream gene, daf-16, thinking that it would be closer to the actual
mechanism for aging.
The first gene Gary and co-workers cloned was daf-23/age-1, which turned out to encode a
phosphatidyl inositol 3-kinase (PI 3-kinase) (Morris et al., 1996) (33). PI
3-kinases were known to signal downstream of the insulin and IGF-1 receptor
tyrosine-kinases, and nowadays, I sometimes hear that the cloning of daf-23/age-1
was the event that
revealed that insulin/IGF-1 signaling affected aging. However, to my knowledge,
no one at the time drew that conclusion. They really couldn't, as PI 3-kinases
were part of many different tyrosine kinase and other signaling pathways. [For
specific examples, see: (Auger et al., 1989; Bjorge et al., 1990; Calabretta
and Skorski, 1996; Comoglio and Boccaccio, 1996; Herman and Emr, 1990; Shimizu
and Hunt, 1996; Sjolander et al., 1991; Varticovski et al., 1989; Ward, 1996;
Weiss and Yabes, 1996) (35-44).] In his paper, Gary concluded only that daf-23/age-1 was part of a PI 3-kinase pathway
(noting presciently that it might involve tyrosine kinase receptors). He did
not mention insulin or IGF-1. Likewise, the next year, Tom Johnson wrote "daf-23
has recently been
shown to be a PI3 kinase suggesting interesting possibilities in the regulation
of the lifespan..." (Johnson, 1997) (45). Don Riddle, in 1997, wrote:
branch [of the dauer pathway] involves phosphatidylinositol signaling in
unknown cells that inhibit dauer formation and limit adult longevity"
(Riddle and Alberts, 1997) (25). Again, there was no mention of insulin or
IGF-1. The nature of this PI 3-kinase pathway was revealed later, by the
cloning of daf-2 and
The DNA sequences of daf-2 and daf-16 were determined in 1997. In August,
the Ruvkun lab reported in Nature that daf-2 encoded the C. elegans homolog of the human insulin and IGF-1 receptors
(Kimura et al., 1997) (46). This was a stunning finding: hormones,
evolutionarily-conserved hormones, controlled aging. The sequence of daf-2 made sense from the point of view
of dauer formation, as IGF-1 and insulin signaling were known to regulate
growth and the body's response to nutrients, both of which were integral to the
process of dauer formation. However, the idea that inhibiting these essential signaling pathways,
which are known for causing diabetes when dysfunctional, could slow aging and
extend lifespan was jarring to some. I remember giving a talk about daf-2 at a major diabetes meeting, and
being told by a rather forceful member of the audience that such mutations
would never extend the lifespan of a mammal. They would just cause diabetes.
This was Morris White, a world expert in insulin signaling, who later became my
friend and went on to report that mouse mutations disrupting the
insulin and IGF-1-pathway gene IRS2 extended lifespan (Taguchi et al., 2007) (47).
(Interestingly, these mutations also increased blood glucose levels. Reducing
insulin/IGF-1 signaling is now thought to extend lifespan, at least in part, by
generating a "danger signal" that shifts the animal's physiology
towards cell protection and maintenance.)
In 1997, insulin and IGF-1 signaling were known to activate
multiple downstream pathways, including PI 3-kinase pathways, but the mechanism
by which they might affect lifespan was not at all clear. The cloning of daf-16, by Gary's and my labs, was a major
breakthrough. As described above, my lab started cloning daf-16 soon after we found that it affected
aging. The gene was located in a region of the genome that proved difficult to
navigate. Both Gary's lab and our lab initially tried to clone daf-16 based on its map position. Gary was
successful with this approach eventually, while we switched to a different
approach, transposon tagging. Gary was the first to sequence the gene,
presenting his findings at the C. elegans meeting. We gave a poster at the meeting describing
our success with transposon tagging, and went on to complete our cloning,
independently of Gary's work, soon after. When we were ready to
submit our paper, we informed Gary. He quickly wrote his work up, and the two studies were published in
December (Lin et al., 1997; Ogg et al., 1997) (48, 49).
The sequence of daf-16 was incredibly informative. The DAF-16 protein was a
forkhead-family (FOXO) transcription factor. This was valuable information for
people studying mammalian diabetes and cancer, as it linked a specific
transcription factor to mammalian insulin and IGF-1 action for the first time.
For aging, it was monumental. A transcription factor could promote lifespan
extension. There was no doubt any more that the aging process was subject to
regulation, and now there was a clear path towards understanding mechanisms of
longevity at the molecular level, at least in part, via the identification and
analysis of DAF-16's targets.
Since 1997, the study of insulin/IGF-1 signaling and FOXO
(DAF-16) proteins in aging has exploded. [For additional references, please see
(Kenyon, 2005; Kenyon, 2010) (50, 51).] Worm lifespan was extended first by six
and now by ten fold, and we now know a lot about the underlying mechanism,
which involves DAF-16/FOXO's regulation of different types of cell protective and
metabolic genes that appear to act cumulatively to affect lifespan. Many
insulin/IGF-1-pathway mutations have been shown to influence lifespan in flies
and mice. There are even hints that changes in these regulatory genes can
influence lifespan during evolution, an idea that had motivated me to study aging
in the first place: small dogs, which are IGF-1 mutants, live longer than large
dogs, and the level of circulating IGF-1 is inversely correlated with lifespan
among ~30 strains of mice housed at the Jackson labs in Maine. At least in
worms and flies, FOXO proteins can extend lifespan in response to many inputs
(for example, AMP kinase and Jun kinase activity) not just reduced
insulin/IGF-1 signaling. Most exciting are the new links to human longevity.
For example, impaired IGF-1 receptor activity has been linked to centenarianism
in Ashkenazi Jews, and FOXO DNA variants have been linked to exceptional
longevity in Hawaiians of Japanese descent, Californians, New Englanders,
Germans, Italians, Ashkenazi Jews and the Chinese. (Though how these FOXO variants
affect gene activity, a key question, has not been determined.) Additional
transcription factors, such as the heat-shock factor HSF-1, the
xenobiotic-response factor SKN-1/NRF and the ER unfolded-protein-response
regulator XBP-1 have been shown to contribute to the longevity of daf-2 mutants. In 1999, the Guarente lab
discovered that Sir2, later shown to be an NAD-dependent protein deacetylase, can increase
lifespan in yeast (Kaeberlein et al., 1999) (52), and the roles of sirtuins in
aging have been studied intensively ever since. Additional nutrient, stress and
energy sensing pathways, many of which engage in cross-talk with the
insulin/IGF-1 pathway, are now known to influence aging in worms and higher
animals, including the TOR pathway, for which life-extending drugs are already
available (at least for mice). Perhaps best of all, many long-lived mutants are
resistant to age-related diseases, including cancer, heart disease and
protein-aggregation disease, suggesting the possibility of forestalling
multiple diseases all at once by targeting aging itself. Along with the many
new labs that have joined the aging field, the labs of Gary Ruvkun, Tom
Johnson, Don Riddle, Pam Larsen and myself have continued to make interesting
new contributions. It's all very exciting and wonderful to experience, and of
course it's not over yet.
I would like to thank my lab members, Jasper Rine, the
reviewers of the Proceedings of the Royal Society and especially Tom Johnson
and Gordon Lithgow for helpful comments on this piece.
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#1. Much later, Coleen Murphy's lab showed that dauer-constitutive mutations in
the TGF-beta pathway genes do extend lifespan, but only if the animals are
treated with a chemical that prevents the growth of un-laid eggs within the
animal (Shaw et al., 2007) (26). As neither Larsen et al (1995) nor our lab
observed a lifespan increase, it must be that animals that would have lived
long were killed prematurely by their own progeny.