Cynthia Kenyon

Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans

Ageing is a fundamental, unsolved mystery in biology. DAF-16, a FOXO-family transcription factor, influences the rate of ageing of Caenorhabditis elegans in response to insulin/insulin-like growth factor 1 (IGF-I) signalling. Using DNA microarray analysis, we have found that DAF-16 affects expression of a set of genes during early adulthood, the time at which this pathway is known to control ageing. Here we find that many of these genes influence the ageing process. The insulin/IGF-I pathway functions cell non-autonomously to regulate lifespan, and our findings suggest that it signals other cells, at least in part, by feedback regulation of an insulin/IGF-I homologue. Furthermore, our findings suggest that the insulin/IGF-I pathway ultimately exerts its effect on lifespan by upregulating a wide variety of genes, including cellular stress-response, antimicrobial and metabolic genes, and by downregulating specific life-shortening genes.

The genetics of ageing

The nematode Caenorhabditis elegans ages and dies in a few weeks, but humans can live for 100 years or more. Assuming that the ancestor we share with nematodes aged rapidly, this means that over evolutionary time mutations have increased lifespan more than 2,000-fold. Which genes can extend lifespan? Can we augment their activities and live even longer? After centuries of wistful poetry and wild imagination, we are now getting answers, often unexpected ones, to these fundamental questions.

Genetic pathways that regulate ageing in model organisms.

Searches for genes involved in the ageing process have been made in genetically tractable model organisms such as yeast, the nematode Caenorhabditis elegans , Drosophila melanogaster fruitflies and mice. These genetic studies have established that ageing is indeed regulated by specific genes, and have allowed an analysis of the pathways involved, linking physiology, signal transduction and gene regulation. Intriguing similarities in the phenotypes of many of these mutants indicate that the mutations may also perturb regulatory systems that control ageing in higher organisms.

Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling

The lifespan of Caenorhabditis elegans is regulated by the insulin/insulin-like growth factor (IGF)-1 receptor homolog DAF-2, which signals through a conserved phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. Mutants in this pathway remain youthful and active much longer than normal animals and can live more than twice as long. This lifespan extension requires DAF-16, a forkhead/winged-helix transcription factor. DAF-16 is thought to be the main target of the DAF-2 pathway. Insulin/IGF-1 signaling is thought to lead to phosphorylation of DAF-16 by AKT activity, which in turn shortens lifespan. Here, we show that the DAF-2 pathway prevents DAF-16 accumulation in nuclei. Disrupting Akt-consensus phosphorylation sites in DAF-16 causes nuclear accumulation in wild-type animals, but, surprisingly, has little effect on lifespan. Thus the DAF-2 pathway must have additional outputs. Lifespan in C. elegans can be extended by perturbing sensory neurons or germ cells. In both cases, lifespan extension requires DAF-16. We find that both sensory neurons and germline activity regulate DAF-16 accumulation in nuclei, but the nuclear localization patterns are different. Together these findings reveal unexpected complexity in the DAF-16-dependent pathways that regulate aging.

Signals from the reproductive system regulate the lifespan of C. elegans

Understanding how the ageing process is regulated is a fascinating and fundamental problem in biology. Here we demonstrate that signals from the reproductive system influence the lifespan of the nematode Caenorhabditis elegans. If the cells that give rise to the germ line are killed with a laser microbeam, the lifespan of the animal is extended. Our findings suggest that germline signals act by modulating the activity of an insulin/IGF-1 (insulin-like growth factor) pathway that is known to regulate the ageing of this organism. Mutants with reduced activity of the insulin/IGF-1-receptor homologue DAF-2 have been shown to live twice as long as normal, and their longevity requires the activity of DAF-16, a member of the forkhead/winged-helix family of transcriptional regulators,,,. We find that, in order for germline ablation to extend lifespan, DAF-16 is required, as well as a putative nuclear hormone receptor, DAF-12 (refs 6, 7). In addition, our findings suggest that signals from the somatic gonad also influence ageing, and that this effect requires DAF-2 activity. Together, our findings imply that the C. elegans insulin/IGF-1 system integrates multiple signals to define the animals rate of ageing. This study demonstrates an inherent relationship between the reproductive state of this animal and its lifespan, and may have implications for the co-evolution of reproductive capability and longevity.

Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans

Many conditions that shift cells from states of nutrient utilization and growth to states of cell maintenance extend lifespan. We have carried out a systematic lifespan analysis of conditions that inhibit protein synthesis. We find that reducing the levels of ribosomal proteins, ribosomal‐protein S6 kinase or translation‐initiation factors increases the lifespan of Caenorhabditis elegans. These perturbations, as well as inhibition of the nutrient sensor target of rapamycin (TOR), which is known to increase lifespan, all increase thermal‐stress resistance. Thus inhibiting translation may extend lifespan by shifting cells to physiological states that favor maintenance and repair. Interestingly, different types of translation inhibition lead to one of two mutually exclusive outputs, one that increases lifespan and stress resistance through the transcription factor DAF‐16/FOXO, and one that increases lifespan and stress resistance independently of DAF‐16. Our findings link TOR, but not sir‐2.1, to the longevity response induced by dietary restriction (DR) in C. elegans, and they suggest that neither TOR inhibition nor DR extends lifespan simply by reducing protein synthesis.

Tissue-Specific Activities of C. elegans DAF-16 in the Regulation of Lifespan

In C. elegans, the transcription factor DAF-16 promotes longevity in response to reduced insulin/IGF-1 signaling or germline ablation. In this study, we have asked how different tissues interact to specify the lifespan of the animal. We find that several tissues act as signaling centers. In particular, DAF-16 activity in the intestine, which is also the animals adipose tissue, completely restores the longevity of daf-16(-) germline-deficient animals, and increases the lifespans of daf-16(-) insulin/IGF-1-pathway mutants substantially. Our findings indicate that DAF-16 may control two types of downstream signals: DAF-16 activity in signaling cells upregulates DAF-16 in specific responding tissues, possibly via regulation of insulin-like peptides, and also evokes DAF-16-independent responses. We suggest that this network of tissue interactions and feedback regulation allows the tissues to equilibrate and fine-tune their expression of downstream genes, which, in turn, coordinates their rates of aging within the animal.

A Role for Autophagy in the Extension of Lifespan by Dietary Restriction in C. elegans

In many organisms, dietary restriction appears to extend lifespan, at least in part, by down-regulating the nutrient-sensor TOR (Target Of Rapamycin). TOR inhibition elicits autophagy, the large-scale recycling of cytoplasmic macromolecules and organelles. In this study, we asked whether autophagy might contribute to the lifespan extension induced by dietary restriction in C. elegans. We find that dietary restriction and TOR inhibition produce an autophagic phenotype and that inhibiting genes required for autophagy prevents dietary restriction and TOR inhibition from extending lifespan. The longevity response to dietary restriction in C. elegans requires the PHA-4 transcription factor. We find that the autophagic response to dietary restriction also requires PHA-4 activity, indicating that autophagy is a transcriptionally regulated response to food limitation. In spite of the rejuvenating effect that autophagy is predicted to have on cells, our findings suggest that autophagy is not sufficient to extend lifespan. Long-lived daf-2 insulin/IGF-1 receptor mutants require both autophagy and the transcription factor DAF-16/FOXO for their longevity, but we find that autophagy takes place in the absence of DAF-16. Perhaps autophagy is not sufficient for lifespan extension because although it provides raw material for new macromolecular synthesis, DAF-16/FOXO must program the cells to recycle this raw material into cell-protective longevity proteins.

A Conserved Regulatory System for Aging

The similarities between these regulatory systems in worms, flies, and yeast are striking. First, each serves a similar biological function, allowing animals to postpone reproduction during unfavorable environmental conditions (Figure 1(Figure 1). Second, each regulates a similar set of processes: oxidative stress resistance, food utilization pathways, reproduction, and lifespan. Third, the systems are composed, at least in part, of homologous genes and pathways (Figure 2(Figure 2).Together these similarities suggest that this regulatory system arose early in evolution (see Kenyon 1996xKenyon, C. Cell. 1996; 84: 501–504Abstract | Full Text | Full Text PDF | PubMed | Scopus (45)See all References, Tissenbaum and Guarente 2001xTissenbaum, H.A and Guarente, L. Nature. 2001; 410: 227–230Crossref | PubMed | Scopus (1205)See all References). Its selective value should be enormous. When food becomes limiting, an animal lacking this system would either die of starvation, or produce progeny that die of starvation. In contrast, with this food-sensing system in place, as food declines, the animal begins to build up fat and/or glycogen reserves, elaborates stress-resistance mechanisms, and delays or suspends reproduction until food is restored. It also activates pathways that extend lifespan, which increases the organisms chance of being alive and still youthful enough to reproduce if it takes a long time for conditions to improve. Each of the diapause-linked processes, such as production of food storage reserves, or the suspension of reproduction, would have selective value on its own. Enhanced stress resistance could protect the animal from heat or toxins that cause oxidative damage (Mahajan-Miklos et al., 1999xMahajan-Miklos, S, Tan, M, Rahme, L.G, and Ausubel, F.M. Cell. 1999; 96: 47–57Abstract | Full Text | Full Text PDF | PubMed | Scopus (424)See all References(Mahajan-Miklos et al., 1999). Because each has intrinsic value, there is no reason a priori for any one of these traits to cause lifespan extension. Thus, the fact that so many of these traits can be uncoupled from lifespan extension genetically in model organisms need not be surprising.It is possible that this conserved system also operates in mammals (see Guarente and Kenyon 2000xGuarente, L and Kenyon, C. Nature. 2000; 408: 255–262Crossref | PubMed | Scopus (869)See all References, Clancy et al. 2001xClancy, D, Gems, D, Harshman, L.G, Oldham, S, Stocker, H, Hafen, E, Leevers, S.J, and Partridge, L. Science. 2001; 292: 104–106Crossref | PubMed | Scopus (824)See all References, Tatar et al. 2001xTatar, M, Kopelman, A, Epstein, D, Tu, M, Yin, C, and Garofalo, R.S. Science. 2001; 292: 107–110Crossref | PubMed | Scopus (884)See all References). Mutant mice that lack the pituitary gland develop into long-lived dwarfs (Bartke, 2000xSee all References(Bartke, 2000). These mice have reduced levels of several hormones, including IGF-1 (Brown-Borg et al., 1996xBrown-Borg, H.M, Borg, K.E, Meliska, C.J, and Bartke, A. Nature. 1996; 384: 33–34Crossref | PubMedSee all References(Brown-Borg et al., 1996), which is produced in response to pituitary growth hormone. Interestingly, small dogs, which have low levels of IGF-1, live longer than large dogs. The finding that, in flies, the insulin/IGF-1 system regulates size and lifespan independently of one another suggests that these mutant mice and small dogs might be long-lived even if they were not small. Caloric restriction, too, may extend lifespan by impacting an insulin/IGF-1 pathway, since it reduces the level of circulating insulin and IGF-1. Rodents that are calorically restricted during development are small, but rodents that are calorically restricted during adulthood are normal in size but still live long. Thus, again, size and longevity can be uncoupled.Insulin/IGF-1 pathways have increased greatly in complexity during evolution. Worms and flies have only one insulin/IGF-1 receptor, whereas vertebrates have at least three. Thus, it is possible that a lifespan regulatory function might be distributed among different branches of this endocrine system. In this regard, it is interesting that loss of the insulin receptor in the neuroepidermis decreases mouse fertility (Bruning et al., 2000xBruning, J.C, Gautam, D, Burks, D.J, Gillette, J, Schubert, M, Orban, P.C, Klein, R, Krone, W, Muller-Wieland, D, and Kahn, C.R. Science. 2000; 289: 122–2125Crossref | Scopus (1240)See all References(Bruning et al., 2000). The same is true of worms (Apfeld and Kenyon, 1998xApfeld, J and Kenyon, C. Cell. 1998; 95: 199–210Abstract | Full Text | Full Text PDF | PubMed | Scopus (211)See all References(Apfeld and Kenyon, 1998), and possibly flies as well. It would be interesting to learn whether these mice, like the worms and flies, are long lived.Aging proceeds at different rates in different animals. A mouse lives roughly two years; a bat, about 30–50; and a parrot, 90. How did these differences evolve? There must have been many mutations that increased lifespan during evolution, since all animals evolved from a common precursor that probably did not live very long. Longer lifespans may have evolved through changes in genes that regulate the rate of aging. For example, changes in insulin/IGF-1 pathway components could play a role in the evolution of lifespan, and this is suggested by the longevity of small dogs. This model is attractive because there is evidence that much of evolution occurs by changes in regulatory genes. In general, evolutionary biologists have not embraced the idea that aging is regulated, because there can be no selection for longevity once reproduction is complete and the progeny are able to live independently. However, the benefit of combining lifespan extension with other traits during diapause (particularly delayed reproduction) provides a strong justification for the evolution of a mechanism that regulates lifespan. If such a mechanism did not exist, the chance of an animal in diapause outliving harsh conditions before producing its progeny would be reduced. Because these insulin/IGF-1-like systems can link lifespan with reproduction, a mechanism for regulating lifespan would have selective value, and could arise during evolution.

Regulation of lifespan by sensory perception in Caenorhabditis elegans.

Caenorhabditis elegans senses environmental signals through ciliated sensory neurons located primarily in sensory organs in the head and tail. Cilia function as sensory receptors, and mutants with defective sensory cilia have impaired sensory perception. Cilia are membrane-bound microtubule-based structures and in C. elegans are only found at the dendritic endings of sensory neurons. Here we show that mutations that cause defects in sensory cilia or their support cells, or in sensory signal transduction, extend lifespan. Our findings imply that sensory perception regulates the lifespan of this animal, and suggest that in nature, its lifespan may be regulated by environmental cues.