Ann. N.Y. Acad. Sci. ISSN 0077-8923 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Issue: Reproductive Aging A pathway that links reproductive status to lifespan in Caenorhabditis elegans Cynthia Kenyon Department of Biochemistry and Biophysics, University of California, San Francisco, California Address for correspondence: Cynthia Kenyon, Mission Bay Genentech Hall MC2200, 600 16th Street, Room S312A, San Francisco, California 94158-2517. cynthia.kenyon@ucsf.edu In the n ematode Caenorhabditis elegans and the fruit fly Drosophila, loss of the germline stem cells activates lifespan- extending FOXO-family transcription factors in somatic tissues and extends lifespan, suggesting the existence of an evolutionarily conserved pathway that links reproductive state and aging. Consistent with this idea, reproductive tissues have been shown to influence the lifespans of mice and humans as well. In C. elegans, loss of the germ cells activates a pathway that triggers nuclear localization of the FOXO transcription factor DAF-16 in endodermal tissue. DAF-16 then acts in the endoderm to activate dow nstream lifespan-extending genes. DAF-16 is also required for inhibition of insulin/insulin-like growth factor 1 (IGF-1) signaling to extend lifespan. However, the mechanisms by which inhibition of insulin/IGF-1 signaling and germline loss activate DAF-16/FOXO are distinct. As loss of the germ cells further doubles the already-long lifespan of insulin/IGF-1 pathway mutants, a better understanding of this reproductive longevity pathway could potentially suggest powerful ways to increase healthy lifespan in humans. Keywords: germ cells; DAF-12; nuclear hormone receptor; aging The aging process touches everyone’s life, and, as a scientific problem, it is fascinating in its own right. What determines the rate at which we age? Aging was once thought to “just happen.” We wear out, like old shoes. Yet, the aging process is not completely random and stochastic. Different species can have dramatically different lifespans, and, even within a single animal species, the rate of aging can be influ- enced by environmental factors, such as the level or quality of nutrients, various stressors, temperature, and sensory cues. 1 The mechanisms by which en- vironmental conditions influence lifespan are now under investigation in many labs, and in many cases, genetic pathways employing classical regu- latory proteins, such as kinases and transcription factors, play important roles. Moreover,some mech- anisms that influence lifespan have been conserved during evolution. For example, reducing the level of insulin/insulin-like growth factor 1 (IGF-1) hor- mone signaling extends the lifespan of worms, flies, and mammals. 1,2 The mechanismof this lifespanex- tension has been explored extensively, particularly in worms, 1,3–6 where inhibitionof insulin/IGF-1 sig- naling has been shown to activate specific transcrip- tion factors, including the FOXO-family member DAF-16/FOXO. DAF-16/FOXO, in turn, increases lifespan by up- or downregulating a wide vari- ety of metabolic, cell-protective, chaperone, and anti-immunity genes that appear to act cumula- tively to extend lifespan. Mutations that alter the activity of this pathway can extend Cae norhabdi- tis elegans’ lifespan up to 10-fold. 7 DNA variants in a FOXO gene have now been associated with increased longevity in seven human populations across several continents, 1,8–11 indicating that hu- man longevity, too, is likely susceptible to the effects of this transcription factor. In many organisms, re- ducing insulin/IGF-1 signaling delays the onset of age-related diseases and reduces their severity, sug- gesting that this pathway couples the normal aging process to age-related disease susceptibility. 6 The relationship between aging and reproduc- tion is particularly fascinating, given the signifi- cance of both processes in the life of an individual and the success of the species. For example, in hu- mans, female menopause may promote longevity by doi: 10.1111/j.1749-6632.2010.05640.x 156 Ann. N.Y. Acad. Sci. 1204 (2010) 156–162 c  2010 New York Academy of Sciences. Kenyon Control of aging by reproductive tissues eliminating the chance that an older woman will die during childbirth. Recently, a fascinating rela- tionship between reproduction and aging has been revealed in small organisms; specifically, in the ne- matode C. elegans and the fruit fly Drosophila. In both species, the germ cells; that is, the cells that give rise to sperm and oocytes, influence the aging of the whole animal. If the germline precursor cells are removed in either worms or flies, lifespan is ex- tended by 40–60%. 12,13 These animals not only live longer, they also remain youthful and active longer than normal, suggesting that loss of the germline precursor cells extends lifespan because it slows the animals’ rate of aging. Thus, in these animals, the germ cells do not only produce the next generation, they also influence the lifespan of the body in which they reside. This review will describe what we know so far about how this pathway affects the aging pro- cess in the animal in which it has been studied most extensively, C. ele gans. In C. elegans, the germline can be removed by killing the two germline precursor cells (Z2 and Z3; Fig. 1) with a laser microbeam at the time of hatching. 13 Or, the germline can be removed genet- ically; 14 for example, by shifting animals carrying the temperature-sensitive mutation glp-1(e2141) to the nonpermissive temperature, which forces mi- totically dividing germline stem cells to exit mitosis and enter meiosis. Lifespan can also be extended when the germline stem cells are forced into meio- sis during adulthood. 14 At this time, the animals contain germ cells arrested in meiosis as well as mature sperm and oocytes, and they are produc- ing progeny. This finding, along with others, singles out the germline stem cells (as opposed to sperm, oocytes, or meiotic cells) as being especially im- portant in affecting aging. In addition, this finding indicates that adult tissues are susceptible to the lifespan-extending effects of germline removal. A similar situation exists in flies. 12 How this system might have arisen during evolution is unknown, but one hypothesis is that it might confer a selective advantage by allowing the somatic tissues to “wait” for the germline to mature before aging progresses too extensively. 13 Such a system might help to coor- dinate the timing of reproduction with aging. How does germline loss extend lifespan? Because reproduction is an expensive process metabolically, one could imagine that lifespan is extended sim- ply because in the absence of reproduction, more Figure 1. Removing the germline of C. elegans extends life- span. (A) At the time of hatching, the animal’s entire repro- ductive system contains only four cells, so using a laser beam to ablate either the precursors of the germline (Z2 and Z3) or the precursors of all of the reproductive tissues (Z1–Z4) with a laser microbeam is straightforward. Killing Z2 and Z3 in this way extends lifespan ∼60%.(B)Thediagramshowsthere- productive system of the animal when it is an adult. At this stage, it contains mature sperm (squares) and oocytes (large ovals), and progeny are being produced (not shown). In addi- tion, the germline contains cells arrested in meiosis (blue circles) and proliferating germline stem cells (yellow circles). When the germline stem cells are forced into meiosis during adulthood using a temperature-sensitive mutation that inhibits signaling required for germline stem cell proliferation, then lifespan is increased. resources can be devoted to cell and tissue main- tenance. This idea, that there is a “cost of repro- duction,” has been put forth by evolutionary biol- ogists, and there are many examples of reciprocal relationships between reproduction and longevity in nature and in the laboratory. 15 In our case, this may be part of the answer, but there seems to be more to it. When the entire reproductive system of C. elegans is removed [that is, the cells that give rise to the somatic reproductive tissues (Z1 and Z4) as well as the cells that give rise to the germline (Z2 and Z3)], lifespan is not increased. 13 Animals that lack their entire reproductive systems are also ster- ile, so these findings argue against models invoking a Ann. N.Y. Acad. Sci. 1204 (2010) 156–162 c  2010 New York Academy of Sciences. 157 Control of aging by reproductive tissues Kenyon simple cost of reproduction. Instead, these findings indicate that both the germline and the somatic re- productive tissues play an active role in influencing lifespan. Specifically, the germline and the somatic reproductive tissues exert counterbalancing influ- ences on lifespan, with the germline preventing, and the somatic gonad promoting, lifespan extension. If active signaling is required for loss of the germ cells to extend lifespan, then what are these signals and how are they communicated to nonreproduc- tive tissues? It seems likely that steroidal hormone signaling plays an important role. Reduction-of- function mutations in the gene daf-12, which en- codes a nuclear hormone receptor (NHR), or in genes like daf-9, which encode proteins that syn- thesize sterol ligands for DAF-12, prevent loss of the germline from extending lifespan. 13,16,17 (For a summaryofgenesinthispathway,seeTable1.) DAF-12/NHR is not the only transcription fac- tor required for loss of the germ cells to extend lifespan. The DAF-16/FOXO transcription factor, described above, is required as well. 13 Interestingly, DAF-16/FOXO appears to act in the intestine of C. elegans to extend lifespan when the germline is re- moved. 18 Under these conditions, DAF-16/FOXO accumulates primarily in intestinal nuclei. This tis- sue appears to play a central role in this pathway, as expression of DAF-16/FOXO exclusively in the intestine can completely rescue the long lifespan of germline-defective daf-16(-) mutants. 19 The intes- tine of C. elegans appears to be the animal’s en- tire endoderm. C. ele g ans does not have a distinct adipose tissue, liver or pancreas; but the intestine stores fat (as does adipose), produces yolk (as does the liver), and produces some important insulin-like peptides,suchasINS-7 20 (as does the pancreas). It is Table 1. Some genes required for loss of the germline to extend the lifespan of C. elegans. For references, see text. Gene General function Response to germline loss Role in insulin/IGF-1 pathway daf-16 FOXO-family transcription factor Localizes to intestinal nuclei during adulthood. Acts in the intestine to extend lifespan Required for insulin/IGF-1-pathway mutants to live long. Localizes to nuclei in many tissues throughout life daf-12 Nuclear hormone receptor Is partially required for DAF-16 nuclear localization, but has another, unknown, function in this pathway Not required for lifespan extension daf-9 Cytochrome P450 required for DAF-12-ligand biosynthesis Partially required for DAF-16 nuclear localization Not required for lifespan extension tcer-1 Ortholog of the human transcription elongation/splicing factor TCERG1 Intestinal expression increases. Required for the upregulation of some, but not all DAF-16-dependent target genes Not required for lifespan extension kri-1 Ortholog of the human disease gene KRIT1. Contains protein interaction domains (ankyrin repeats) Intestinal protein required for DAF-16 nuclear localization and tcer-1 upregulation upon germline loss Not required for lifespan extension K04A8.5 Fat lipase Upregulated by DAF-16 in the intestine. Could potentially produce a downstream lifespan-extending signal from the intestine to other tissues Partially required for lifespan extension 158 Ann. N.Y. Acad. Sci. 1204 (2010) 156–162 c  2010 New York Academy of Sciences. Kenyon Control of aging by reproductive tissues possible that DAF-16/FOXO has a conserved lifespan-extending function in the intestine/adipose tissue, as overexpression of the Drosophila DAF- 16/FOXO ortholog only in adipose tissue extends fly lifespan. 12 Loss of the mouse insulin receptor specif- ically in adipose tissue also extends lifespan. 21 This intervention would be predicted to activate mouse FOXO proteins in this tissue. How DAF-16 activ- ity in the C. elegans intestine extends the lifespan of the entire animal is not known, but a fat lipase called K04A8.5 may be involved. 22 The gene encod- ing this lipase is upregulated by DAF-16/FOXO in the intestines of germline-defective animals, and its function is required for lifespan extension. It is pos- sible that this lipase is involved in the synthesis of downstream signals from the intestine that influ- ence the lifespan of the other tissues in the animal. The apparently conserved ability of adipose tissue to produce lifespan extending signals in response to FOXO activity makes this issue particularly interesting. How does loss of the germ cells activate DAF- 16/FOXO? One obvious question, given DAF- 16/FOXO’s central role in the insulin/IGF-1 path- way, is whether loss of the germ cells activates DAF-16/FOXO by inhibiting the activity of the insulin/IGF-1 pathway. Also, is the DAF-12 steroid signaling system involved? Is the somatic gonad required? The answers to these questions are not known in detail; but at least part of the story has begun to emerge (and is summarized in Figs. 2 and 3). First, the pathway that triggers DAF-16/FOXO nuclear localization is at least partially distinct from the pathway that localizes DAF-16 to nuclei when insulin/IGF-1 signaling is inhibited. In long- lived daf-2 (insulin/IGF-1-receptor) mutants, DAF- 16/FOXO accumulates in most or all somatic cell nuclei throughout life. 23–25 In contrast, when the germline is removed, DAF-16/FOXO does not ex- hibit nuclear accumulation until adulthood, and then it localizes primarily to nuclei in the intestine. Second, several genes that are required for DAF- 16/FOXO nuclear localization and lifespan exten- sion in response to loss of the germ cells are not required for lifespan extension in response to re- duced insulin/IGF-1 signaling. daf-12/NHR is one such gene 13,26 (discussed further). Another is kri- 1, the C. elegans homolog of the human disease gene KRIT1, which encodes an intestinal ankyrin- Figure 2. Loss of the germline precursor cells at the time of hatching (circles with Xs) triggers i mportant changes in intesti- nal cells of the adult. Killing the germ cells at the time of hatch- ing triggers several important changes in the intestine of the adult. The transcription factor DAF-16/FOXO accumulates in nuclei,andtheleveloftheputativetranscription-elongationfac- tor TCER-1 rises. Both of these events are completely dependent on kri-1. DAF-16 nuclear localization is partially dependent on the DAF-12 steroid signaling pathway,but TCER-1 upregulation is independent of DAF-12. Therefore, there must be a second gonad-to-intestine signaling pathway. The site of action of DAF- 12 itself is not known (question mark). It could potentially act in the intestine. domain containing protein. kri-1 is required for loss of the germline to mediate DAF-16/FOXO nu- clear localization and to extend lifespan. 18 In con- trast, kri-1 is not required for daf-2 inhibition to extend lifespan. 18 Moreover, apart from DAF- 16/FOXO’s nuclear accumulation, the requirements for DAF-16/FOXO-dependent gene expression are different between germline-defective animals and insulin/IGF-1-signaling mutants. A putative tran- scription elongation factor called TCER-1 (a C. elegans homolog of human TCERG1) is required for lifespan extension and for the increased ex- pression of many DAF-16-target genes in germline- deficient animals, but it is not required for lifespan extension or daf-16-dependent gene regulation in insulin/IGF-1 mutants. 27 It is not clear why TCER- 1 should be required for DAF-16 target-gene ex- pression in response to germline ablation, but (for at least some of the same genes) not in response to inhibition of insulin/IGF-1 signaling, and this is an interesting question. Whatever the answer, it is clear that many aspects of the reproductive longevity pathway distinguish it from the insulin/IGF-1 path- way. If loss of the germline extended lifespan sim- ply by inhibiting insulin/IGF-1 signaling, then the Ann. N.Y. Acad. Sci. 1204 (2010) 156–162 c  2010 New York Academy of Sciences. 159 Control of aging by reproductive tissues Kenyon Figure 3. The reproductive and insulin/IGF-1 pathways are distinct. The lifespan extension produced by loss of the germ cells requires KRI-1, DAF-12, and TCER-1, whereas the lifespan extension produced by daf-2(e1370) receptor mutations does not. (The question mark following “DAF-12” refers to our un- certainty about the site of action of DAF-12.) Moreover, TCER-1 is required for the increased expression of a set of DAF-16- regulated genes in response to germline loss, but TCER-1 is not required for lifespan extension or for the expression of these DAF-16-regulated genes in insulin/IGF-1 mutants (at least not for the genes that have been examined). However, it is impor tant to note that mutations in the insulin/IGF-1 pathway can affect the operation of the germline pathway (see text), so it is not yet clear whether the two pathways act completely independently of one another. The drawings depict working models: for example, TCER-1 and DAF-16 are hypothesized to interact on individual promoters, but this has not been shown directly. behavior of DAF-16/FOXO would be expected to be more similar in the two pathways than it actually is. Finally, removing the germlines of animals carry- ing daf-2/insulin/IGF-1-receptor mutations further doubles the already-long lifespans of the animals. 13 This effect is consistent with the idea that these two pathways are not the same. How is intestinal DAF-16/FOXO informed about the state of the germline? The daf-12-dependent steroid signaling pathway appears to play a role. In germline-deficient animals lacking daf-12 /NHR or the DAF-12-ligand-synthesizing genes daf-9 or daf-36, intestinal DAF-16/FOXO nuclear localiza- tion is incomplete. 17,18 Administering the DAF-12 ligand dafachronic acid to germline-deficient daf-9 mutants (which can not make dafachronic acid) re- stores full DAF-16 nuclear localization and lifespan extension. 17 This finding suggests that the DAF-12 signaling pathway plays a role in mediating commu- nication between the reproductive system and the intestine. In addition to promoting DAF-16/FOXO nu- clear localization, DAF-12 has another, unknown, function in this pathway. 18 It is possible to force DAF-16 nuclear localization by mutating the AKT- phosphorylation sites on DAF-16 through which insulin/IGF-1 signaling prevents DAF-16 nuclear accumulation in normal intact animals. This con- stitutively nuclear mutant DAF-16 protein can sub- stitute for wild-type DAF-16 and extend lifespan in germline-deficient animals. However, this lifespan extension is still dependent on DAF-12. This find- ing indicates that DAF-12 has another function that is essential for lifespan extension, in addition to its role in DAF-16 nuclear localization. The nature of this other function is unknown. The fact that some DAF-16/FOXO nuclear local- ization takes place when the germline is removed in daf-12(-) or daf-9(-) mutants suggests that there is a second pathway that informs the intestine about the status of the germline. This interpretation is consis- tent with another observation 27 : When the germline is removed, the level of TCER-1 rises in the intestine. This increase requires KRI-1, but it is completely in- dependent of DAF-12/NHR. It will be very interest- ing to learn the identity of this daf-12-independent gonad-to-intestine signaling pathway. Many aspects of this longevity system remain mysterious. Why is the somatic gonad required for loss of the germline to extend lifespan? The so- matic gonad is not required for germline loss to trigger DAF-16 nuclear localization 28 or TCER-1 upregulation, 27 butitisrequiredforgermlinelossto induce at least some DAF-16-dependent transcrip- tion. 28 Perhaps these DAF-16-dependent genes are essential for lifespan extension. Whetherthe somatic gonad has other functions that do not involve DAF- 16 is unknown. Curiously,in strong daf-2(-) mutants, thesomatic reproductive tissues are no longer required for loss of the germline to further extend lifespan. 13,28 Why inhibiting insulin/IGF-1 signaling removes the re- quirement for the somatic gonad is not clear. One could imagine that the insulin pathway is down- stream of the somatic gonad; that is, that the so- matic gonad extends lifespan in germline-deficient animals by inhibiting insulin/IGF-1 signaling. How- ever, this model does not explain the tissue-specific localization of DAF-16, or why kri-1 and tcer-1 are required for germline loss, but not insulin/IGF-1- pathway inhibition, to extend lifespan. It is possible 160 Ann. N.Y. Acad. Sci. 1204 (2010) 156–162 c  2010 New York Academy of Sciences. Kenyon Control of aging by reproductive tissues that the longevity requirement normally fulfilled by the somatic gonad can also be fulfilled, possi- bly in a different way, by inhibition of insulin/IGF-1 signaling. Another intriguing question is how the germline stem cells “tell” the animal that they are present. If the presence or loss of the germline activates a signaling pathway, what is the initiating event? How is it linked to germline stem cell proliferation? Finally, it is interesting to ask whether this same reproductive signaling pathway, or a molecular variant, might influence lifespan in higher ani- mals. There are several intriguing parallels with Drosophila that suggest a common evolutionary ori- gin. First, as mentioned above, forcing the germline stem cells to exit mitosis and enter meiosis dur- ing adulthood extends the lifespan of both adult flies and adult worms. 12,14 Moreover, in flies, as in worms, this treatment activates DAF-16/FOXO ac- tivity. 12 In contrast to the situation in worms, when the germline is removed in Drosophila during de- velopment, lifespan is not extended. 29 The reason for this is unknown, but it is tempting to speculate that lifespan is not extended because in flies (un- like in worms), early loss of the germline prevents the correct development of the somatic reproductive tissues. Less is known about mammals; however trans- planting the ovaries of young mice into old females extends lifespan. 30,31 Thus, signals from re- productive tissues can influence mammalian life- span. Whether there are any additional similarities between the worm pathway and the pathway trig- gered by this transplantation in mice is notknown. It is interesting to wonder whether the loss of oocytes might extend the lifespan of human females, who live longer than men. This is of course completely unknown, but, interestingly, if the ovaries as well as the germ cells are absent in postmenopausal women, then the rate of all-cause mortality (including age- related diseases) increases. 32 It will be fascinating to watch this interesting new field grow and begin to encompass higher organisms. Whether or not human lifespan is influenced in the same way by reproductive tissues, if we learn how the reproduc- tive systems of smaller animals, like worms and flies, regulate DAF-16/FOXO activity, it may be possible to use this information to intervene downstream of the reproductive pathway and extend healthy lifes- pan without affecting the germline itself. Consis- tent with this idea, in C. elegans, overexpressing the putative transcription-elongation factor gene tcer-1 extends the lifespan of worms that have an intact re- productive system and are fully fertile. 27 This lifes- pan extension correlates with the upregulation of many germline-specific DAF-16 target genes, and it is dependent on DAF-16 activity. The evidence that FOXO proteins influence human lifespan 1,8–11 makes the possibility of harnessing this informa- tion to influence human health and longevity seem increasingly possible. Acknowledgments I thank the members of my laboratory, and the re- viewers, for helpful comments and suggestions. Conflicts of interest The author declares no conflicts of interest. References 1. Kenyon, C.J. 2010. The genetics of aging. Na ture 464: 504– 512. Review. 2. Bartke, A. 2008. Insulin and aging. Cell Cycle 7: 3338– 3343. 3. Kleemann, G.A. & C.T.Murphy.2009.The endocrine regula- tion of aging in Caenorhabditis elegans. Mol. Cell. 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Ann. N.Y. Acad. Sci. ISSN 0077-8923 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Issue: Reproductive Aging A pathway that links reproductive status to lifespan in Caenorhabditis elegans Cynthia. Mutations that alter the activity of this pathway can extend Cae norhabdi- tis elegans lifespan up to 10-fold. 7 DNA variants in a FOXO gene have now been associated with increased longevity in. not make dafachronic acid) re- stores full DAF-16 nuclear localization and lifespan extension. 17 This finding suggests that the DAF-12 signaling pathway plays a role in mediating commu- nication