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Heat Shock Protein Accumulation Is Upregulated in a Long-Lived Mutant of Caenorhabditis elegans

^a School of Biological Sciences, University of Manchester, United Kingdom
^b Department of Biochemistry, University of British Columbia, Vancouver, Canada
^c Institute of Behavioral Genetics, University of Colorado, Boulder

Gordon J. Lithgow, The Buck Institute for Age Research, Novato, CA 94949 E-mail: glithgow{at}buckinstitute.org.

Decision Editor: John Faulkner, PhD

	Abstract

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We present evidence for elevated levels of heat shock protein 16 (HSP16) in an intrinsically thermotolerant, long-lived strain of Caenorhabditis elegans during and after heat stress. Mutation of the age-1 gene, encoding a phosphatidylinositol 3-kinase catalytic subunit, results in both extended life span (Age) and increased intrinsic thermotolerance (Itt) in adult hermaphrodites. We subjected age-synchronous cohorts of worms to lethal and nonlethal thermal stress and observed the accumulation of a small (16–18 kd) heat-shock-specific polypeptide detected by an antibody raised against C. elegans HSP16. Strains carrying the mutation hx546 consistently accumulated HSP16 to higher levels than a wild-type strain. Significantly, overaccumulation of HSP16 in the age-1(hx546) strain following heat was observed throughout the adult life span. A chimeric transgene containing the Escherichia coli ß-galactosidase gene fused to a C. elegans HSP16-41 transcriptional promoter was introduced into wild-type and age-1(hx546) backgrounds. Heat-inducible expression of the transgene was elevated in the age-1(hx546) strain compared with the wild-type strain under a wide variety of heat shock and recovery conditions. These observations are consistent with a model in which Age mutations exhibit thermotolerance and extended life span as a result of elevated levels of molecular chaperones.

LIFE span of Caenorhabditis elegans is determined, in part, by a pathway resembling the insulin signaling pathway in mammals. Mutations in some genes encoding components of the pathway cause increased mean and maximum life span (Age phenotype), slow the rate of aging, and also enhance resistance to stress ^(1)(2)(3).

Almost all Age mutations are pleiotropic, affecting early life history traits as well as longevity ⁽¹⁾⁽³⁾. Many of the proposed mechanisms of life-span extension by Age mutations come from studies of these associated phenotypes. During development, the insulinlike signaling pathway is one of several pathways that regulate the formation of a specialized larval diapause stage, called the dauer larvae ⁽⁴⁾. Dauer larvae develop from L1 worms during nutritional deprivation and in response to a pheromone signal. The dauer larva is nonfeeding, nonreproducing, stress resistant, and capable of surviving four to eight times longer than the adult ⁽⁵⁾. Loss-of-function mutations of either daf-2, which encodes a protein similar to vertebrate insulin-receptor ⁽⁶⁾, or age-1, which encodes a potential catalytic subunit of phosphatidylinositol-3-kinase (PI3K) ⁽⁷⁾, extend adult hermaphrodite life span by up to 100% ⁽⁸⁾⁽⁹⁾. Loss-of-function mutations of daf-16 (which encodes a forkhead transcription factorlike protein) ^(10)(11) suppress both the Age phenotype and Daf-c phenotypes of age-1 and daf-2 mutations ^(12)(13)(14). These mutations are also suppressed by mutations in daf-18, which encodes a protein similar to the human PTEN, which exhibits phosphatidylinositol 3,4,5-trisphosphate (PIP3) 3-phosphatase activity ^(15)(16). Additionally, genes encoding homologues of mammalian Akt/PKB and PDK-1 proteins act in this signaling pathway between age-1 and daf-16 ⁽¹⁷⁾. Taken together, an insulinlike signaling pathway is necessary for normal development and normal life span of C. elegans. The cell and tissue specificity of the action of this pathway has been investigated by using a series of daf-2 genetic mosaics ⁽¹⁸⁾. It appears that daf-2 may function in many cell types, but its activity in neurones and support cells is sufficient to determine development and life span.

The mechanism of life-span extension in the Age mutants remains unknown, but we have proposed that it results in part from the overaccumulation of molecular chaperone molecules ^(19)(20)(21). This was prompted by the observation that mutations leading to extended life span also confer intrinsic thermotolerance (Itt) ^(19)(20)(22). Organisms respond to thermal stress by accumulating heat shock proteins (HSPs), many of which exhibit molecular chaperone activity ⁽²³⁾. The HSPs are encoded by distinct gene families, and expression of individual genes can confer resistance to lethal heat stress ⁽²⁴⁾; for example, hsp104 confers tolerance to a number of environmental stresses in Saccharomyces cerevisiae ⁽²⁵⁾, and inducible hsp70 confers thermotolerance in Drosophila embryos ⁽²⁶⁾. The small HSP family, characterized by an -crystallin domain, also contributes to thermotolerance. The hsp27 accumulates and is phosphorylated in response to a number of stimuli, including heat shock ⁽²⁷⁾. The protein is an actin polymerization modulator and appears to be a key modulator of stress-activated apoptosis as a consequence of its role in F-actin accumulation following stress ⁽²⁸⁾. The relationship between hsp27 and thermotolerance has been investigated in a number of heterologous expression studies. Expression of human hsp27 in rodent cells confers thermotolerance ⁽²⁷⁾ and expression of Drosophila hsp27 in COS cells leads to tolerance of thermal and oxidative stress ⁽²⁹⁾.

We have investigated the expression of a small hsp gene in C. elegans encoding the hsp27 homologue, HSP16. As many as eight genes, at three loci, encode the members of the HSP16 family ⁽³⁰⁾. HSP16-2 is known to form large oligomeric complexes and to function as a molecular chaperone ⁽³¹⁾. We have measured the accumulation of HSP16s under a variety of heat shock and recovery conditions, in young through to late-life adults and in both wild-type strains and a long-lived age-1 mutant strain. This study demonstrates that the mutation, age-1(hx546), confers an upregulation of HSP16 genes in response to thermal stress and consequently that an insulinlike signaling pathway regulates at least one family of hsp genes in C. elegans.

	Methods

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Strains and Culture of Nematodes
The wild-type Bristol (N2) strain and TJ1052 [age-1(hx546)II] were obtained from the Caenorhabditis Genetics Center. All strains were routinely grown at 20°C on small nematode growth media (NGM) agar plates ^(32)(33). Synchronous populations of hermaphrodites were established by picking eggs from mixed populations onto new plates and allowing them to develop at 20°C.

Life Span and Thermotolerance Assays
Life span and thermotolerance assays were undertaken as previously described ^(19)(20). Briefly, to assess thermotolerance, two populations of twenty-five 3- or 4-day-old hermaphrodites were shifted from 20°C to 35°C and scored for touch-provoked movement and pharyngeal pumping. Animals not pumping or responding to touch were scored as dead. Survival was scored on two populations of age-synchronized hermaphrodites at 20°C on NGM agar plates. Statistical analysis was performed by standard methods ⁽¹⁹⁾.

Antibody Generation
Polyclonal antibodies to HSP16-2 were prepared in rabbits as previously described ⁽³⁴⁾.

Preparation of Worm Extracts and Western Blotting
Four-day-old adult hermaphrodites were transferred from 20°C and placed at either 30°C, 33°C, or 35°C for various lengths of time up to 24 hours. Sixty adult animals were counted and picked at each time point into S-basal ⁽³³⁾ in a siliconized eppendorf tube, washed once in S-basal, and frozen in liquid N₂. No larvae were present in these samples.

For quantitative Western analysis, two identical gels were prepared. One gel was incubated in 20 ml of Sypro-red protein stain (Molecular Probes; Eugene, OR) for 50 minutes. The stained gel was visualized by using an ultraviolet light source and was photographed with a Sypro-red photographic filter (Cat. #S-6656). Following transfer of the second gel, the membrane and the gel were stained with Ponceau S and Coomassie Blue, respectively, to verify successful transfer. The membrane was incubated with primary rabbit anti-C. elegans HSP16 antibody diluted 1:10,000 in blocking buffer and then with secondary, goat anti-rabbit IgG antibody/horseradish peroxidase conjugate (Sigma; Poole, Dorset, England), diluted 1:5000 in blocking buffer for 1.5 hours on a shaker. Detection was undertaken with chemiluminescent reagents (SuperSignal, Pierce; Rockford, IL) and standard autoradiography. For comparison of worms across life span, signals were normalized to that for heat shocked, young wild-type worms.

Transgene Copy Number Determination
Chromosomal DNA was prepared from TJ830, TJ831, and N2 (wild-type) strains and probed with the plasmid pBluescript, which contains the bacterial lacZ. Identical restriction fragments were observed for both transgenic strains. Copy number was assessed by probing with a control catalase gene. Autoradiographs were digitally scanned and the signal was quantified by using the Sigma Gel software package. The signal was normalized for length and specific activity of probe.

Generation of Aged Worms
Three-day-old hermaphrodites were transferred from NGM plates and then aged in liquid media. Hermaphrodites and their progeny were separated by culturing populations in 40 µm nylon sieves. At each age tested, three replicates of 60 adult worms were treated with a 2-hour exposure at 35°C with a 24-hour recovery at 20°C, and then 40 survivors were collected as above and frozen in liquid N₂. No larvae were present in these samples. Quantitative Western analysis was performed as described above.

Histochemical ß-Galactosidase Staining
Three- to four-day gravid hermaphrodite adults raised at 20°C were shifted to 30°C, 33°C, or 35°C. At indicated times, plates were removed and both adults and larvae were washed off in S-basal immediately or allowed to recover at 20°C for 30 minutes to 10 hours. After gentle centrifugation, the S-basal was removed and the worms were frozen with liquid N₂. Samples were histochemically stained to detect ß-galactosidase. For histochemical staining, worms were thawed on ice, methanol permeablized, and washed in 10 mM of Tris buffer. Staining solution was added [21 mM of NaH₂PO₄, 104 mM of Na₂HPO₄, 0.01 mM of MgCl₂, 5 mM of K₄Fe(CN)₆, 5 mM of K₃Fe(CN)₆, 40 µg per ml of sodium dodecyl sulfate (SDS), 240 µg per ml of XGal, and 1.2% N,N-dimethylformamide], and worms were incubated in a 37°C water bath overnight before examining. The worms were scored blind by two researchers for both the intensity and location of staining.

	Results

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Accumulation of HSP16 in Young Worms During Heat Shock
We examined the accumulation of HSP16 during heat shock at 30°C, 33°C, and 35°C in age-1(hx546) and wild-type worms. Hermaphrodite worms were grown at 20°C for 4 days and then were subjected to heat shock. Every 2 hours during the heat shock, subpopulations of worms were analyzed for HSP16 levels. Upon heat shock at 30°C, 33°C, or 35°C, rapid accumulation of a heat shock protein species of approximately 16–18 kd was detected in both wild-type and age-1(hx546) worms. Quantitation of signals on the Western blots indicates that HSP16 accumulates preferentially in age-1(hx546) worms at all heat shock temperatures tested. Fig. 1 illustrates the results of four replicate experiments in which, after 4 hours at 35°C, the age-1(hx546) worms accumulated approximately five times more detectable HSP16 than wild-type strains. By the eighth hour of the heat shock, the levels of HSP16 continued to rise in both strains, and wild-type worms were beginning to die. As previously reported, there was no detectable HSP16 at 20°C in young wild-type worms. ^(35)(36). Likewise, HSP16 was not detected in age-1(hx546) worms at 20°C (data not shown).

View larger version (39K): [in this window] [in a new window]   Figure 1. Long-lived, age-1(hx546) mutants have elevated levels of HSP16 during heat shock. Synchronous, 4-day-old populations of hermaphrodites were heat shocked at 35°C. At the time points indicated, subsamples of 60 worms were analyzed on quantitative western blots. A, Autoradiogram of heat-shocked worm extracts probed with polyclonal antibody raised against HSP16. B, Quantitation of relative HSP16 abundance (mean ± SEM for four experiments) in 4-day-old hermaphrodite wild-type and age-1(hx546) worms. The amounts of HSP16 are normalized to the highest HSP16 level detected across all four experiments. HSP16 accumulates preferentially in age-1(hx546) worms at 35°C. At 2, 4, and 6 hours, the differences are not significant (p = .06, .06, and .09, repectively; one-tailed t test). The difference is significant at 8 hours (p = .044). The combined probability test (37) on independent tests of significance showed that age-1 (hx546) worms accumulated significantly more HSP16 than wild-type worms (p < .005).

View larger version (39K): [in this window] [in a new window]   Figure 2. Long-lived age-1(hx546) mutants have elevated levels of HSP16 during recovery from a mild, nonlethal, heat shock. Synchronous, 4-day-old populations of hermaphrodites were heat shocked at 35°C for 2 hours and then returned to 20°C for periods up to 68 hours. Subsamples of 60 worms were analyzed on quantitative Western blots. A, Autoradiogram of recovering worm extracts probed with polyclonal antibody raised against HSP16. B, Relative HSP16 abundance (mean ± SEM for four experiments) in 4-day-old hermaphrodite wild-type and age-1(hx546) worms. The amounts of HSP16 are normalized to the highest HSP16 level observed across all four experiments. The long-lived mutant worms accumulated significantly more HSP16 at 22 hours (p = .016) and 45 hours (p = .003) of recovery when compared with wild-type levels (one-tailed t test).

View larger version (17K): [in this window] [in a new window]   Figure 3. Induced HSP16 expression (35°C for 2 hours, followed by 24-hour recovery at 20°C) in aged wild-type and age-1(hx546) hermaphrodites relative to expression levels in 4-day-old heat-shocked wild-type worms (mean ± SEM for three replicates). The combined probability test (37) on independent tests of significance showed that age-1(hx546) worms accumulated significantly more HSP16 than wild-type worms across all ages (p < .005).

View larger version (40K): [in this window] [in a new window]   Figure 4. Construction and analysis of transgenic reporter strains for hps16 expression studies. A, Transgene for the assessment of expression from an HSP16 divergent transcriptional promoter. B, Survival at 20°C of transgenic strains. TJ831 [zIs1; age-1(hx546)] is longer lived (p < .001) than TJ830 (zIs1). C, Survival during heat shock at 35°C of transgenic lines containing the zIs1 transgene. Synchronous cohorts of N2, TJ830 (zIs1), and TJ831 [zIs1; age-1(hx546)]. TJ831 is Itt (p < .0001). D, Accumulation of HSP16 and survival of TJ831[zIs1;age-1(hx546)] (squares) and TJ830[zIs1] (circles) during heat shock at 35°C. Filled symbols represent the fraction surviving at the indicated time points during the heat shock. The TJ831 is Itt (p < .0001). The open symbols represent the relative abundance of HSP16.

Reporter Gene Expression in an Age-1(hx546) Strain
We carried out a series of heat shock and recovery protocols and examined ß-galactosidase activity. In the absence of heat shock, no ß-galactosidase activity (ß-gal) was detected in either TJ830 or TJ831 (data not shown). Heat shocks at 30°C, 33°C, and 35°C all induce ß-galactosidase activity in both strains as revealed by histochemical staining. Distinct tissue-specific expression patterns were observed for different heat shock conditions, and considerable variation in the extent of staining was observed between different experiments. We recorded the number of animals in a sample exhibiting staining in either the pharynx, developing embryos, or throughout the entire body of the worm. A series of heat shock and recovery conditions were identified in which TJ830 and TJ831 exhibited distinct responses. For example, after a 30-minute heat shock at 35°C, followed by a 1-hour recovery, age-1(hx546) worms exhibited ß-gal activity throughout but no activity was detectable in wild-type worms. A series of 30-minute heat shocks at 35°C followed by variable recovery periods were undertaken to test the reproducibility of this finding (Table 1 and Fig. 5). In six out of seven independent experiments, age-1(hx546)-specific elevation of ß-galactosidase activity was observed in that the proportion of worms exhibiting ß-gal activity was greater, as was the intensity of ß-gal staining. It should be noted that there was considerable variation between experiments in the degree of response from both strains. Fig. 5 illustrates a recovery time course following a 30-minute heat shock at 35°C. Worms were stained 1, 2, and 10 hours after the heat shock; only very faint staining was observed in all wild-type worms, whereas 48 of 50 age-1 mutant worms exhibited extensive ß-gal activity. Similar time courses were performed after heat shocks at 30°C and 33°C with similar results (data not shown).

View larger version (117K): [in this window] [in a new window]   Figure 5. Histochemical staining for ß-galactosidase activity in transgenic lines. Cohorts of TJ831[zIs1; age-1(hx546)] and TJ830(zIs1) transgenic worms were grown at 20°C until 4 days of age and then heat shocked at 35°C for 30 minutes. The worms were then returned to 20°C to recover and subpopulations were frozen on liquid N2. A, wild type, 1-hour recovery; B, wild type, 2-hour recovery; C, wild type, 10-hour recovery; D, age-(hx546), 1-hour recovery; E, age-(hx546), 2-hour recovery; F, age-(hx546), 10-hour recovery.

	Discussion

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We examined strains of nematode worms mutant in age-1 during thermal stress and recovery from thermal stress. Under both conditions, HSP16 accumulates in the age-1-mutant worms to levels up to eight times higher than in wild-type worms. We also examined worms at seven different ages across the life span and found that adult age-1 mutants, when challenged by heat shock, generally accumulated greater levels of HSP16 compared with wild-type strains of the same chronological age. Mutation of age-1 also influenced the levels of a reporter activity (ß-galactosidase) under the transcriptional control of an HSP16 promoter. Histochemical staining for ß-galactosidase activity of worms exposed to different heat shock and recovery regimes revealed a number of mild heat shock conditions in which age-1(hx546) conferred enhanced induction of the transgene. There was considerable variation in the response of both strains between experiments, but the age-1 background consistently led to higher levels of ß-galactosidase activity in individual worms and also to a greater proportion of worms showing activity. This was true in adults, larvae, and developing embryos visible within the hermaphrodite worms.

These reporter gene experiments suggest that altered HSP16 levels in Age worms arise from an alteration of gene expression, although we have not ruled out the possibility that the turnover rate of HSP16 protein is also altered in the mutant strain. As the lacZ reporter transgene contains the 5'-untranslated region of hsp16-41, the gene expression alteration may be at the level of translation or the rate of accumulation of the hsp16 mRNA. The regulation of HSP genes has not been extensively studied in the nematode, although the hsp-16 promoter does contain sequences that are similar to the binding site for the heat shock factor (HSF) transcriptional regulator ⁽⁴⁰⁾. Mutation of the age-1 does not seem to cause a constitutive derepression of hsp16 gene expression, but when hsp16 is induced, wild-type AGE1 appears to repress expression. Therefore, mutation of age-1 may increase the level of hsp16 gene transcription, perhaps by increasing the activity of HSF or by reducing the activity of a negative regulator such as heat shock factor binding protein (HSF-BP1) ⁽⁴¹⁾.

The results presented here are consistent with the observation that age-1 mutants are Itt ⁽²⁰⁾ and that an inhibitor of PI3K activity, LY294002, causes thermotolerance ⁽⁴²⁾. However, it is unlikely that expression of the hsp16 gene family alone accounts for all the stress phenotypes associated with these mutants. Mutation of age-1, for example, also results in resistance to oxidative stress ^(43)(44), ultraviolet radiation stress ⁽⁴⁵⁾ and high concentrations of heavy metals ⁽⁴⁶⁾. Consistent with these phenotypes, age-1 mutant worms have elevated levels of the antioxidant enzymes superoxide dismutase and catalase ^(43)(44) and also overexpress at least one metallothionein gene ⁽⁴⁶⁾.

Taken together, these results suggest that the insulinlike signaling pathway coordinately regulates the expression of a range of stress proteins. Furthermore, this is consistent with a role for this pathway in dauer formation, as dauers are also resistant to a range of environmental stresses ⁽⁴⁷⁾.

The results presented here support an emerging picture of the role of stress proteins during normal aging ^(2)(22). Acclimation of both C. elegans and Drosophila to heat shock also results in extended life span ^(20)(48). Drosophila carrying extra copies of the inducible hsp70 gene exhibit even greater heat-acclimation-associated increase in survival ⁽⁴⁹⁾. In addition, a series of publications illustrate that overexpression of antioxidant enzyme genes extends the life span of Drosophila ^(50)(51)(52).

We propose that the relationship between thermal stress and life span is based upon the rate at which conformationally altered protein accumulates both during thermal stress and during aging under nonstressed conditions ^{(53)(54)(55)(56)}. As molecular chaperones promote the refolding of protein from a nonnative to a native state and prevent aggregate formation, the presence of chaperones during both thermal stress and aging may be advantageous. Two additional observations are consistent with this view. The first is that HSP expression is induced during normal aging in Drosophila. Inducible forms of hsp22 and hsp70 are expressed in the absence of any environmental stress and, it is likely that this is due to the accumulation of oxidatively damaged proteins ⁽⁵⁷⁾. We have also observed the accumulation of small amounts of HSP16 at late ages in the worm (data not shown). The second observation is that caloric restriction, in which rodent life span is significantly increased by a controlled reduction in caloric intake, leads to a more robust heat shock response and overexpression of hsp70, hsp27, and hsp-90 ⁽⁵⁸⁾. Taken together, these findings suggest that molecular chaperones are candidates for longevity-associated proteins.

	Acknowledgments

 
Glenda Walker was supported by a Medical Research Council Studentship. This work was also supported by the Biotechnology and Biosciences Research Council Science of Ageing Initiative. We thank the members of the Lithgow laboratory and the Johnson laboratory for critical and helpful discussions.

Tiffany White is currently with the Department of Microbiology, University of Colorado, Denver. Glenda Walker is currently with the Department of Veterinary Parasitology, University of Glasgow, Scotland. Gawain McColl and Nicole Jenkins are currently with the Buck Institute for Age Research, Novato, California.

Received March 29, 2000

Accepted December 11, 2000

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