Current address: Department of Veterinary Parasitology, University of Glasgow, Garscube Estate, Bearsden Road, Glasgow, UK
Lifespan extension in C. elegans by a molecular chaperone dependent upon insulin-like signals
Article first published online: 26 MAR 2003
Volume 2, Issue 2, pages 131–139, April 2003
How to Cite
Walker, G. A. and Lithgow, G. J. (2003), Lifespan extension in C. elegans by a molecular chaperone dependent upon insulin-like signals. Aging Cell, 2: 131–139. doi: 10.1046/j.1474-9728.2003.00045.x
- Issue published online: 26 MAR 2003
- Article first published online: 26 MAR 2003
- Accepted for publication 16 January 2003
- C. elegans;
- heat shock protein;
Insulin-like signalling is a key determinate of lifespan in diverse species including mammals but the mechanism by which this pathway influences the rate of aging is unknown. In the roundworm Caenorhabditis elegans, mutations in the insulin-like signalling pathway extend adult lifespan and are associated with up-regulation of stress response genes including those for heat shock proteins (HSPs). We tested the hypothesis that the C. elegans insulin-like signalling pathway determines longevity through modulating HSP levels. We introduced extra copies of the gene encoding HSP-16 and this conferred stress resistance and longevity both in a wildtype and a long-lived mutant strain. The DAF-16 transcription factor is essential for maximal hsp-16 expression and for lifespan extension conferred by hsp-16. This demonstrates that lifespan is determined in part by insulin-like regulation of molecular chaperones.
The C. elegans insulin-like signalling pathway has considerable influence on adult lifespan (Guarente & Kenyon, 2000; Gems & Partridge, 2001). Insulin-like signalling is mediated by the receptor protein DAF-2 and mutation of the daf-2 gene doubles worm lifespan (Kenyon et al., 1993; Gottlieb & Ruvkun, 1994; Dorman et al., 1995; Kimura et al., 1997; Paradis et al., 1999). Mutation of the downstream age-1 gene, which encodes a protein similar to the mammalian p120 catalytic subunit of phosphatidylinositol 3-kinase, leads to a 65% increase in mean lifespan (Friedman & Johnson, 1988; Malone et al., 1996; Morris et al., 1996). These effects depend on the integrity of the protein DAF-16, which has similarity to a family of mammalian forkhead transcription factors (Kenyon et al., 1993; Gottlieb & Ruvkun, 1994; Dorman et al., 1995; Larsen et al., 1995; Lin et al., 1997; Ogg et al., 1997). An insulin-like signalling pathway also influences aging in Drosophila (Tatar et al. 2001; Clancy et al., 2001). In the mouse, loss of a single copy of the igf1r gene (encoding the insulin-like growth factor type-1 receptor) results in a 26% increase in lifespan (Holzenberger et al., 2002) and knockout of the insulin receptor specifically in adipose tissue increases lifespan by 18% (Blüher et al., 2003).
A number of mechanisms have been suggested to explain these effects. Prompted by our observation that mutation of daf-2 or age-1 confers intrinsic resistance to heat shock (Lithgow et al., 1994, 1995) we proposed that longevity is a consequence of the over-expression of stress response genes and an enhanced ability to cope with macromolecular damage (Johnson et al., 1996; Lithgow, 1996). In addition, we demonstrated that young adult worms with mutations promoting longevity over-accumulate the heat shock protein HSP-16 following thermal stress (Walker et al., 2001). We hypothesize that altered hsp-16 regulation contributes to the longevity of insulin-like mutants and more generally that molecular chaperones influence the rate of aging (Lithgow & Kirkwood, 1996). To test this model we increase the copy number of the hsp-16A locus in strains with and without mutations in the insulin signalling pathway and measure HSP-16 protein, thermotolerance and longevity.
We constructed multiple transgenic strains of worms that maintained multiple copies of the entire hsp-16A locus (‘extra copy hsp-16A’ strains) containing two pairs of HSP-16-encoding genes (Candido et al., 1989). These extra copies of hsp-16A are under the control of their native heat shock transcriptional promoters, permitting us to investigate the regulatory relationship between hsp-16A and insulin-like signalling mutations. In addition, since hsp-16 promoters are not active in developing worms in the absence of stress (Candido et al., 1989) our constructs avoid deleterious effects of transgene expression during development (Krebs & Feder, 1997). Indeed, under our normal culture conditions, maintenance of the hsp-16A array does not alter development time (Fig. 1A) or fertility (data not shown).
Extra-copy hsp-16 confers thermotolerance and extended lifespan
To demonstrate the capacity for hsp-16A transgene expression in extra-copy strains, populations of young adult wildtype and transgenic worms were subjected to mild heat shock (30 °C ‘pretreatment’). The worms maintaining extra-copies of hsp-16A accumulate elevated HSP-16 protein proportional to duration of pretreatment (Fig. 1B,C). At all pretreatment intervals, wildtype control adults induce less HSP-16 protein (at each time, P < 0.0001). Without pretreatment, HSP-16 is not detectable by Western blot in either wildtype or extra-copy stains (data not shown).
Extra-copy hsp-16A confers thermotolerance. Four-day-old hermaphrodite control and hsp-16A strains were pretreated at 30 °C for various times and shifted to 35 °C. Survival at 35 °C is correlated to length of pretreatment in both extra-copy hsp-16A adults and in wildtype adults (Fig. 1D), and at all times extra-copy hsp-16 increases thermal-survival to a greater degree (P < 0.001, log rank test; P < 0.0001, combined probability test).
We investigated the effect of extra-copy hsp-16A upon adult lifespan with and without heat pretreatment. Life expectancy of five independently generated extra-copy transgenic lines was compared to three independent transgenic control lines. At constant 20 °C, extra-copy hsp-16A increases lifespan by 11% (Table 1). Extra-copy hsp-16A also increases survival from the time when 25% of the population remains (P < 0.0001, combined probability test). Pretreatment of young adults with extra-copy hsp-16A (representative survival data in Fig. 2) did not increase lifespan more than occurs in transgenic adults without pretreatment (Tables 1 and 2).
|Trial||Control strain||Lifespan (days)||n||hsp-16A||Lifespan (days)||n|
|Constant 20 °C|
|mean = 22.0||mean = 24.4||P = 0.0013*|
|4 h pretreatment|
|mean = 22.7||mean = 24.5||P = 0.0088*|
|6 h pretreatment|
|mean = 22.7||mean = 25.2||P = 0.001*|
|Trial||Heat duration||Lifespan (days)||n||Lifespan (days)||n||P*|
hsp-16 depends on the insulin signal pathway for extended lifespan
To determine how HSP-16 interacts with insulin-like signalling we crossed extra-copy hsp-16 and control extrachomosomal transgenic arrays into strains with an age-1 mutant (hx546 allele), a daf-16 mutant (m27 allele) and a double mutant strain, age-1(hx546); daf-16(m27).
In the age-1 mutant background and without pretreatment, extra-copy hsp-16A increases lifespan (Fig. 3A). The effect of extra-copy hsp-16A is significant in three of four trials (Table 3, top panel) and increases lifespan on average by 20%. Without pretreatment, extra-copy hsp-16A also specifically increases thermotolerance at 35 °C in the age-1 background (Fig. 3B).
|Lifespan (days)||N||Lifespan (days)||N||P*|
|25 °C trial||age-1(hx546)||hsp-16A;age-1(hx546)|
|Mean = 26.5||Mean = 31.8|
|25 °C trial||age-1(hx546); daf-16(m27)||hsp-16A; age-1(hx546); daf-16(m27)|
|Mean = 18.5||Mean = 18.0|
|trial||Control strain||hsp-16A array strain|
|GL175(rfEx2); daf-16(m27)||14||50||GL174(rfEx12); daf-16(m27)||14||39||NS|
|GL175(rfEx2); daf-16(m27)||14||42||GL174(rfEx12); daf-16(m27)||14||37||NS|
Reduced activity of the DAF-16 transcription factor abolishes the longevity produced by extra-copy hsp-16. The daf-16(m27) mutation confers thermosensitivity on both control lines and extracopy hsp-16A and fully suppresses thermotolerance conferred by extra copy hsp-16A (Fig. 4A). The mutation also reduces HSP-16 accumulation following pretreatment (Fig. 4B). Most importantly, extra-copy hsp-16A does not increase lifespan of daf-16 mutant lines including those also mutant at age-1 (Fig. 4C,D; Table 3).
HSP-16 is a classical stress response protein. There is no expression in unstressed young adults (Candido et al., 1989) but HSP-16 is detected in worms greater than 16 days old (Walker et al., 2001). When hsp-16A over-expression is induced in young adults we observe increases in thermotolerance, consistent with the proposed role of the chaperone in preventing accumulation of aggregated protein (Leroux et al., 1997b). In contrast to thermotolerance, there is no evidence for a relationship between heat pretreatment of young adults and subsequent lifespan – extra-copy hsp-16A intrinsically improves lifespan without heat shock. We therefore propose that longevity is improved by extra-copy hsp-16A acting late in life when the native promoters of this gene are endogenously up-regulated.
Extra-copy hsp-16A also strongly increases lifespan without heat shock in adults mutant for age-1. Since trials were conducted at various temperatures, presently we cannot determine whether the magnitude of this effect is significantly greater in age-1 adults relative to adults with wildtype insulin signalling. It is unambiguous that mutation of daf-16 reduces the expression of HSP-16 upon heat shock in extra-copy hsp-16A and negates the lifespan benefit of extra-copy hsp-16 in both the wildtype and age-1 backgrounds. We conclude that insulin-like signalling influences lifespan through hsp-16 expression.
The added survival conferred by extra-copy hsp-16A is modest relative to the extended lifespan evident for some alleles of age-1 and daf-2 (Friedman & Johnson, 1988; Kenyon et al., 1993), although the magnitude is similar to that seen with heat shock or HSP70 overexpression in both worm and fly (Lithgow et al., 1995; Khazaeli et al., 1997; Tatar et al., 1997; Yokoyama et al., 2002). Perhaps HSP-16 chaperones are not the unique lifespan-determining factor altered by insulin signal mutation. Most likely a suite of stress genes is required for large increases in lifespan (Larsen, 1993; Vanfleteren, 1993; Honda & Honda, 1999; Barsyte et al., 2001), and mutation of the insulin-like signal system influences the co-ordinated regulation of these genes. Since the insulin pathway functions in neurones to determine C. elegans lifespan (Apfeld & Kenyon, 1998; Wolkow et al., 2000) and age-1 influences the expression of hsp-16 in many cell types (Walker et al., 2001), it seems likely that downstream hormonal factors influence these stress response mechanisms throughout adult tissues (Larsen et al., 1995; Gerisch et al., 2001; Jia et al., 2002). Notably, hsp-16 promoters lack obvious binding sites for DAF-16, further implicating indirect regulation of hsp-16 by DAF-16. The Drosophila homologue of hsp-16, hsp22, is in part regulated by the insect hormone ecdysterone (Rudolph et al., 1991) and like hsp-16 of C. elegans, D. melanogaster hsp22 is intrinsically up-regulated with age (Wheeler et al., 1995). Similar hormonal regulation of stress genes may exist in the worm.
Mechanistically, HSP-16 could affect lifespan in many ways. HSP-16 may reduce oxidative stress by raising the pool of reduced glutathione in the worm in a fashion similar to that observed for HSP27 in murine cell culture (Mehlen et al., 1996). Pharmacological agents that reduce oxidative stress in the worm also increase lifespan, demonstrating a role for oxidative stress in aging (Melov et al., 2000). Alternatively, HSP-16 may interact with nuclear hormone receptors (Morimoto, 2002) to influence transcription of genes that have effects on aging. In D. melanogaster, molecular chaperones interact with the ecdysone nuclear hormone receptor complex (Arbeitman & Hogness, 2000), and two components of this complex, the ecdyone receptor EcR and the deacetylase Rpd3, have recently been found to increase longevity when mutated in flies (Rogina et al., 2002; Simon et al., 2003). Finally, since HSP-16 is also known to prevent protein aggregation (Leroux et al., 1997a), over-expression at late ages may reduce the accumulation of protein aggregates.
Our results may extend beyond the concept of lifespan and toward the problem of specific age-related disease. The risk of many diseases associated with the accumulation of toxic aggregate proteins accelerates with age (Yankner, 2000). We find it intriguing that the onset of polyglutamate aggregation is delayed by mutation of age-1 in a C. elegans model of triplet repeat disease (Morley et al., 2002). As well, disrupting expression of the hsp transcriptional activator, the hsf heat shock factor, shortens lifespan and appears to accelerate the appearance of aging markers in C. elegans (Garigan et al., 2002). If, as suggested by our experiments on the insulin-like signalling mutants, protein aggregation affects the rate of normal aging, then some human age-related diseases may arise directly from aging processes rather than from the simple passage of time.
Generation of transgenic lines and maintenance of nematode strains
Plasmid pGW104 was constructed by restricting cosmid T27E4 and subcloning the hsp-16A locus containing BamH1 fragment into the polycloning site of pUC19. Transgenic animals were obtained through microinjection (Mello et al., 1991). pGW104 and pUC19 were transformed into wildtype (N2) C. elegans with the dominant rol-6 transgenic marker plasmid pRF4 (Mello et al., 1991). Multiple independent transgenic lines were generated in each case. N2 (wildtype), TJ1052[age-1(hx546)II] and DR27[daf-16(m27)I] were obtained from the Caenorhabditis Genetics Center (http://biosci.umn.edu/CGC/Strains/strains.htm).
Assay of thermotolerance
Thermotolerance was assayed as previously described (Lithgow et al., 1995). Following development at 20 °C, 4-day-old gravid adult hermaphrodites were placed on 30-mm NGM plates seeded with E. coli strain OP50 and warmed to 35 °C. Heat pretreatment was applied by the same procedure but with temperature set at 30 °C for durations of 2–5 h prior to exposure at 35 °C. Survival at 35 °C was assessed approximately every 60 min after the first 180 min. Plates were temporarily removed from the 35 °C incubator and worms were scored for motility, touch-provoked movement and pharyngeal pumping. Worms failing to display any of these traits were scored as dead. Survival at 35 °C was estimated by Kaplen–Meyer methods and compared among groups by log rank tests implemented in Prism (Graphpad Software, San Diego, CA, USA).
Synchonous populations of worms were placed as eggs on 50-mm NGM plates seeded with E. coli OP50 (40–50 per plate, 80–300 individuals per experiment) and grown at 20 °C, 25 °C or 27 °C. Each population was examined for synchrony at the beginning of the reproductive period. Adults were continuously maintained at the development temperature except when subjected to 30 °C pretreatments at adult age 4 days. Individuals were transferred to fresh plates daily from 3 to 10 days post-hatching and then every 3 days thereafter. The plates were examined daily for live, dead, and lost or bagged individuals (these censored data were excluded from analysis). No increase in the frequency of bagging was observed for any of the transgenic lines. Individuals were scored for touch-provoked movement and pharyngeal pumping. Mean survival is reported from the day of egg hatching. Survival during normal aging was estimated by Kaplen–Meyer methods and compared among groups by log rank tests implemented in Prism (Graphpad Software).
HSP-16 abundance determination
Four-day-old adult hermaphodites were transferred from 20 °C and placed at either 30 °C or 35 °C for various durations. At each time point, 60 adult animals were collected into S-basal (Sulston & Hodgkin, 1988) in a siliconized microfuge tube, washed once in S-basal and frozen in liquid N2. Quantitative Western analysis was applied as previously described (Walker et al., 2001). Briefly, two identical gels were run; one gel was used for total protein quantification using Sypro-Red protein stain (Molecular Probes, OR, USA), the second gel was transferred onto nitrocellulose for probing with a primary rabbit anti-C. elegans HSP-16 antibody (gift from E. P. M Candido, University of British Columbia). The anti-HSP-16 polyclonal antibody (Hockertz et al., 1991) was raised against a peptide fragment corresponding to amino acids 110–145 of the HSP-16-2 protein. It detects primarily a single polypeptide of 18 kDa, which corresponds to the 16-1/2 pair (HSP-16-1 and HSP-16-2 are highly similar to each other at positions 110–145). In addition, the anti-2/110-145 antibody faintly detects a 16-kDa band corresponding to the 16-41/48 pair (HSP-16-41 and HSP-16-48 are highly similar to each other, but are only 50% identical to HSP-16-2 at positions 110–145). Detection used chemiluminescent reagents (SuperSignal®, Pierce, Rockford) and signals were normalized to total protein.
Body size measurements
Four-day-old hermaphrodite worms were transferred in 10 µL medium onto a glass slide, covered with a glass coverslip which was sealed along the edge with wax to prevent evaporation. Worm images were obtained by video microscopy and worm area was measured from digitized images using Scion Image (Scion Corporation, MD, USA). The greyscale image was transformed using a Hat 17 × 17 convolution, and automatic edge detection was used to define area after calibration with a stage micrometer. Measurement error was ± 158.9 µm2. Area was compared, using unpaired Students t-test, assuming unequal variance.
We wish to thank the members of the Lithgow lab and the Biotechnology and Biosciences Research Council (BBSRC) Science of Aging Initiative (SAGE) grant holders, Tom Johnson for helpful discussions and to Julie K. Andersen, Matthew S. Gill, Nicole L. Jenkins, Gawain McColl, and for critical reading of the manuscript. G.W. was funded by a Medical Research Council studentship. Thanks to Matthew Gill for assistance with body size determination. The research was supported by the BBSRC's SAGE Initiative and G.J.L. is supported by NIH AG21069 and the Ellison Foundation.
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