Abstract
Temperature profoundly affects aging in both poikilotherms and homeotherms. A general belief is that lower temperatures extend lifespan while higher temperatures shorten it. Though this “temperature law” is widely accepted, it has not been extensively tested. Here, we systematically evaluated the role of temperature in lifespan regulation in C. elegans. We found that while exposure to low temperatures at the adult stage prolongs lifespan, low temperature treatment at the larval stage surprisingly reduces lifespan. Interestingly, this differential effect of temperature on longevity in larvae and adults is mediated by the same thermosensitive TRP channel TRPA-1 that signals to the transcription factor DAF-16/FOXO. DAF-16/FOXO and TRPA-1 act in larva to shorten lifespan, but extend lifespan in adulthood. DAF-16/FOXO differentially regulates gene expression in larva and adult in a temperature-dependent manner. Our results uncover unexpected complexity underlying temperature modulation of longevity, demonstrating that temperature differentially regulates lifespan at different stages of life.
Introduction
Both environmental factors and genes affect aging (Fontana et al., 2010; Kenyon, 2010). Temperature and food are the two primary environmental factors that modulate longevity (Fontana et al., 2010; Kenyon, 2010). Reduction in either food intake (dietary restriction) or temperature can extend lifespan (Conti, 2008). While the effect of diet on longevity has been extensively characterized, very little is known about how temperature regulates lifespan.
It was documented a century ago that with the exception of extreme temperatures which may threaten animal survival, lower environmental temperatures generally extend the lifespan of poikilotherms such as worms, flies and fish, while higher temperatures shorten their lifespan (Loeb and Northrop, 1916). Recent studies demonstrate that this also appears to be the case in homeothermic animals. For example, lowering the core body temperature of mice extends lifespan (Conti et al., 2006); exposing rats to lower environmental temperatures also promotes longevity (Holloszy and Smith, 1986), indicating that both body and environmental temperatures affect lifespan in rodents. Interestingly, lower body temperatures are also associated with longer human lifespan in the Baltimore Longitudinal Study of Aging (Roth et al., 2002). These observations highlight a general role of temperature in lifespan regulation in both poiklioterms and homeotherms.
Traditionally, the rate-of-living theory has been adopted to explain the effect of temperature on lifespan (Loeb and Northrop, 1916). Namely, low temperatures reduce the rate of chemical reactions, thereby slowing down the pace of aging, and vice versa. Recent work in C. elegans, however, shows that genes can actively regulate lifespan in response to temperature changes (Lee and Kenyon, 2009; Xiao et al., 2013). Specifically, the cold-sensitive TRPA-1 channel can detect temperature drop in the environment to initiate a pro-longevity signaling cascade to extend lifespan (Xiao et al., 2013). At high temperatures, the heat-sensitive neuron AFD antagonizes the detrimental effect of high temperature to increase lifespan via neuroendocrine signaling (Lee and Kenyon, 2009). Additional genes are also found to regulate lifespan in a temperature-dependent manner (Horikawa et al., 2015; Mizunuma et al., 2014). Despite these observations, the “temperature law” remains: low temperatures increase lifespan while high temperatures decrease it.
In this report, we re-visited the “temperature law” in lifespan regulation in C. elegans. Our data show that while low temperature exposure at the adult stage extends lifespan, similar treatment at the larval stage, remarkably, shortens lifespan. Interestingly, this differential effect of temperature on lifespan requires the cold-sensitive TRPA-1 channel and its downstream transcription factor DAF-16/FOXO which is a key regulator in lifespan control (Kenyon et al., 1993; Lin et al., 1997; Ogg et al., 1997). Both DAF-16/FOXO and TRPA-1 promote longevity at the adult stage, but surprisingly play an inhibitory role on longevity at the larval stage. We further demonstrate that DAF-16/FOXO differentially regulates gene expression at the larval and adult stages in a temperature dependent manner, which is consistent with its role in mediating the differential effect of temperature on longevity in larvae and adults. Our studies demonstrate that temperature modulation of longevity is more complex than previously thought, suggesting that caution needs to be exercised when applying the “temperature law” to aging studies. More importantly, we show that temperature exerts a differential effect on lifespan at different stages of life.
Results
Temperature treatment at different stages of worm life differentially affects lifespan
Previous work mainly focused on the impact of temperature on lifespan at the adult stage (Klass, 1977; Lee and Kenyon, 2009; Wu et al., 2009; Xiao et al., 2013). As a result, how temperature treatment at the larval stage influences lifespan has not been explored. The cultivation temperature for C. elegans in the laboratory spans from 15 to 25 °C, with three temperatures (15, 20 and 25 °C) being most commonly used. To isolate the effect of temperature on adults, we first let three groups of larval worms grow at the same temperature (20 °C) until they reached the last larval stage L4, and then shifted them to three different temperatures (15, 20 and 25 °C) throughout the adult stage (Figure 1A). By fixing the temperature at the larval stage, we were able to interrogate how exposure to different temperatures at the adult stage affects lifespan. We found that adult worms exposed to 15 °C and 25 °C lived the longest and shortest lifespan, respectively (Figure 1A). This data is consistent with the view that low temperatures extend lifespan while high temperatures shorten it.
Figure 1. Temperature treatment at different stages of worm life differentially affects lifespan but not fertility.
(A) Exposure to low and high temperatures at the adult stage extends and shortens lifespan, respectively (p<0.001, log-rank test). The top panel describes the protocol of temperature treatment.
(B) Exposure to low and high temperatures at the larval stage shortens and extends lifespan, respectively (p<0.001, log-rank test). The top panel describes the protocol of temperature treatment.
(C–D) Exposure to different temperatures at the larval stage does not have a notable effect on fertility in adulthood. (C) Overall fertility. (D) Egg-laying pattern. The percentage of eggs laid every 12 hours (half a day) was plotted. Larvae were reared at 15, 20, and 25 °C, and their adults were moved to 20 °C to lay eggs. n=30. Error bars: SEM.
Also see Figure S1. Representative data are shown here, and replicates and detailed statistics are shown in Table S1.
To characterize how temperature treatment at the larval stage affects longevity, we exposed eggs at three different temperatures until they developed as L4 larvae and then shifted them to the same temperature to score adult lifespan (Figure 1B). Surprisingly, larvae developed at 15 °C exhibited the shortest lifespan, while those grown at 25 °C lived the longest life (Figure 1B). Similar results were obtained with worms that had been maintained at different temperatures for several generations (Figure S1A), and with other temperature treatment protocols (Figure S1B–D). Apparently, low temperature treatment at the larval stage shortened adult lifespan, while exposure to high temperature at this stage extended adult lifespan. We also checked fertility, but found that similar temperature treatments at the larval stage did not have a notable effect on fertility in adulthood (Figure 1C–D). These results demonstrate that temperature differentially affects longevity at different stages of worm life. This also shows that temperature treatment at the larval stage has a long-lasting effect on lifespan in adulthood.
The temporal window critical for temperature modulation of lifespan
We decided to focus on characterizing the effect of temperature treatment at the larval stage, as much is known about how such treatment at the adult stage affects lifespan. Since 15 °C and 25 °C treatment elicits the greatest difference, for simplicity we mainly focused on these two temperatures. We first asked whether the effect of temperature is uniform across all the larval stages or there is a specific time window during which temperature treatment elicits the greatest effect.
To test this, we employed two strategies. In the first protocol, we shifted eggs and different stages of larvae (i.e. L1, L2 and L3) to 15 °C and 25 °C until they reached L4, followed by assaying their adult lifespan at the same temperature (Figure 2A). We found that if animals were shifted after the L1 stage, temperature can no longer elicit a notable effect, indicating that the L1-L2 stage is a critical time window (Figure 2A). In the second protocol, we let eggs hatch at 15 °C and 25 °C and then shifted different stages of larvae and adults to 20 °C (Figure 2B). Similarly, if animals were shifted to 20 °C before the L2 stage, we no longer observed a notable effect of temperature (Figure 2B). This protocol also revealed the L1–L2 stage as a critical window. Interestingly, if larvae were allowed to develop into adulthood at 15 and 25 °C and then shifted to 20 °C, the temperature effect at the larval stage began to fade and finally disappeared at day 3 of the adult stage (Figure 2B). This is consistent with the notion that temperature treatment in the larval and adult stages elicits opposite effects and antagonizes each other. These experiments revealed a specific temporal window during which temperature elicits the greatest effect on longevity, demonstrating that the impact of temperature is not uniform across all the larval stages.
Figure 2. The temporal window critical for temperature modulation of lifespan.
(A) The L1–L2 stage is a critical window for temperature modulation of lifespan at the larval stage. The protocol was described in the upper panel. Hermaphrodite mothers were allowed to lay eggs at 20 °C. The specific stage (egg, L1, L2, and L3) at which animals were up- or down-shifted from 20 °C to 25 °C or 15 °C was labeled in each panel. At the L4 stage, animals were all shifted back to 20 °C to score adult lifespan. (egg: p<0.001, log-rank test; L1: p<0.001, log-rank test; L2: p=0.066, log-rank test; L3: p=0.198, log-rank test)
(B) A second protocol also shows that the L1–L2 stage is a window critical for temperature modulation of lifespan at the larval stage. The protocol was described in the upper panel. Eggs were allowed to hatch at 15 and 25 °C and developed to different stages of larvae or adults, which were shifted to 20 °C to score lifespan. The specific stage [L1, L2, L3, L4, D1 (Day 1 adult), D2 (Day 2 adult), and D3 (Day 3 adult)] at which animals were shifted to 20 °C were labeled in each panel (L1: p=0.399, log-rank test; L2: p=0.013, log-rank test; L3: p=0.001, log-rank test; L4: p<0.001, log-rank test; D1: p<0.001, log-rank test; D2: p=0.007, log-rank test; D3: p=0.788, log-rank test).
Representative data are shown here; replicates and detailed statistics are described in Table S1.
TRPA-1 is required for temperature modulation of longevity at the larval stage
The observation that low temperature exposure shortens rather than extends lifespan is inconsistent with the “temperature law”. This also suggests that such a phenomenon is probably regulated by genes. We therefore set out to identify genes that regulate this process.
At the adult stage, temperature modulation of longevity is regulated by the cold-sensitive channel TRPA-1 which presumably detects temperature drop in the environment to initiate a pro-longevity genetic program (Xiao et al., 2013). We thus tested whether TRPA-1 plays a role in mediating temperature modulation of longevity at the larval stage. We found that loss of trpa-1 gene nearly eliminated the effect of temperature (Figure 3A), indicating that TRPA-1 is also important for mediating temperature modulation of longevity at the larval stage.
Figure 3. TRPA-1 is required for temperature modulation of longevity at the larval stage.
(A) Loss of trpa-1 nearly eliminated the effect of larval temperature treatment on lifespan (p=0.104, log-rank test).
(B–C) trpa-1 mutant worms show a normal lifespan following treatment at 15 °C at the larval stage (B) (p=0.945, log-rank test), but are short-lived if their larvae were treated at 25 °C (C) (p<0.001, log-rank test). Temperature treatment was performed as shown in Figure 1B, and the adult lifespan was scored at 20 °C.
(D–E) trpa-1 mutant worms are short-lived when adult animals are reared at 15 °C (D) (p<0.001, log-rank test), but show a normal lifespan when reared at 25 °C (E) (p=0.934, log-rank test). Temperature treatment was performed as shown in Figure 1A, and the adult lifespan was scored at 15 °C and 25 °C in (D) and (E), respectively.
(F–G) trpa-1 mutant worms are long-lived following treatment at 15 °C at the larval stage (F) (p<0.001, log-rank test), but show a normal lifespan if their larvae were treated at 25 °C (G) (p=0.878, log-rank test). Temperature treatment was performed as shown in Figure 1B, but the adult lifespan was scored at 25 °C instead of 20 °C.
Also see Figure S2. Representative data are shown here; replicates and detailed statistics are described in Table S1.
TRPA-1 shortens lifespan at the larval stage while promoting lifespan at the adult stage in a temperature-dependent manner
How might TRPA-1 regulate lifespan at the larval stage? TRPA-1 is a cold-sensitive channel that is active at 15 °C (opens at ≤20 °C) but remains closed at 25 °C (Chatzigeorgiou et al., 2010; Xiao et al., 2013). At the adult stage, TRPA-1 extends lifespan at 15 °C but has no effect on lifespan at 25 °C (Xiao et al., 2013). Since low temperature treatment at the larval stage shortens lifespan, one would expect that cold activation of TRPA-1 at the larval stage shall shorten lifespan, but that at high temperatures this channel should have no effect on lifespan since it is inactive. If so, trpa-1 mutant worms should be long-lived or exhibit a normal lifespan when their larvae were reared at low or high temperatures, respectively. However, this does not appear to be the case (Figure 3B–C). In fact, the opposite was observed (Figure 3B–C). We realized that TRPA-1 also functions in adulthood to prolong lifespan at low temperatures (Xiao et al., 2013). This adult effect of TRPA-1 may obscure our results when we examined its role at the larval stage. To overcome this difficulty, we treated larvae under 15 °C and 25 °C, but scored their adult lifespan at 25 °C instead of 20 °C. As TRPA-1 shows no effect on lifespan at 25 °C at the adult stage (Figure 3D–E), this protocol should selectively restrict the effect of TRPA-1 to the larval stage. Using this protocol, we found that trpa-1 mutants were long-lived when their larvae were reared at low temperatures (Figure 3F), but showed a normal lifespan when their larvae were grown at high temperatures (Figure 3G). Though HSF-1 is best known to be activated at ≥30 °C in most organisms, it could be turned on in C. elegans when animals are up-shifted to 25 °C (Sugi et al., 2011). Thus, we knocked down hsf-1 by RNAi at the adult stage to exclude its potential contribution to lifespan, and obtained a similar result (Figure S2A–B). These data suggest that TRPA-1 shortens lifespan at low temperatures at the larval stage, a function opposite to that observed at the adult stage.
To provide further evidence, we expressed TRPA-1 as a transgene in wild-type worms to ascertain whether its overexpression would enhance the effect of temperature treatment on lifespan at the larval stage. As TRPA-1 is expressed in multiple tissues, including neurons, intestine, muscle and cuticle (Chatzigeorgiou et al., 2010; Xiao et al., 2013), we selectively expressed TRPA-1 in different tissues using tissue-specific promoters. While treatment at low and high temperatures at the larval stage elicited ~20% difference in lifespan in wild-type (Figure 4A and 4F), a neuronal trpa-1 transgene augmented the difference to ~38% (Figure 4B and 4F). By contrast, transgenic expression of TRPA-1 in other tissues had no obvious effect (Figure 4C–F), indicating that TRPA-1 may act in neurons to mediate its temperature-dependent longevity effect at the larval stage. The observation that overexpression of TRPA-1 enhanced the temperature effect further supports a role of TRPA-1 in regulating lifespan at the larval stage.
Figure 4. Overexpression of TRPA-1 enhances the effect of larval temperature treatment on lifespan.
(A) The effect of larval temperature treatment on lifespan in wild-type worms (p<0.001, log-rank test). Temperature treatment was performed as described in Figure 1B.
(B–F) Overexpression of TRPA-1 as a transgene in neurons (B) (p<0.001, log-rank test), but not in the intestine (C) (p<0.001, log-rank test), muscle (D) (p<0.001, log-rank test), or cuticle (E) (p=0.001, log-rank test) augments the effect of larval temperature treatment on lifespan. Data in (B–E) were summarized in (F). p values are listed (paired t test). The ges-1, rgef-1, myo-3, and dpy-7 promoter drives expression in the intestine, neurons, muscle, and cuticle, respectively (Aamodt et al., 1991; Altun-Gultekin et al., 2001; Fire and Waterston, 1989; Gilleard et al., 1997).
(G–H) Overexpression of TRPA-1 as a transgene shortens lifespan following temperature treatment at 15 °C at the larval stage (G) (p<0.001, log-rank test), but the same transgene has no effect on lifespan if larvae are treated at 25 °C (H) (p=0.637, log-rank test). Temperature treatment was performed as shown in Figure 1B, but the adult lifespan was scored at 25 °C instead of 20 °C.
Also see Figure S3. Representative data are shown here; replicates and detailed statistics are described in Table S1.
We wondered how TRPA-1 overexpression might augment the temperature effect on lifespan at the larval stage. If TRPA-1 regulates lifespan at the larval stage in a temperature-dependent manner, one would predict that its overexpression should enhance the temperature effect by shortening lifespan at low but not high temperatures. To test this, we restricted the contribution of trpa-1 transgene to the larval stage by treating larvae at 15 °C and 25 °C, and then scored adult lifespan at 25 °C, a temperature under which trpa-1 transgene is no longer active (Xiao et al., 2013). Indeed, we found that trpa-1 transgene shortened lifespan at low but not high temperatures at the larval stage in both wild-type and hsf-1(RNAi) worms (Figure 4G–H and Figure S3A–B). It is worth noting that the same transgene extended lifespan at low but not high temperatures at the adult stage, an effect opposite to that observed at the larval stage (Figure S3C–D). Thus, both mutant and overexpression data support that TRPA-1 shortens lifespan at the larval stage in a temperature-dependent manner.
DAF-16 and HSF-1 mediate the temperature effect at the larval stage
As a temperature sensor, TRPA-1 cannot regulate lifespan on its own. Typically, longevity signals converge on a small group of transcription factors (Fontana et al., 2010; Kenyon, 2010). We therefore searched for the transcription factors that may act downstream of TRPA-1 to mediate its effect on temperature modulation of longevity. To this end, we examined those well-characterized transcription factors known to regulate lifespan. We found that loss of DAF-16/FOXO, a master regulator of lifespan, nearly abolished the effect of temperature treatment at the larval stage on lifespan (Figure 5A) (p=0.021, Cox-proportional hazard regression analysis). Namely, daf-16 mutant worms exhibited similar lifespans following larval temperature treatment at 15 and 25 °C (Figure 5A), revealing a critical role of daf-16 in mediating the temperature effect at the larval stage. Consistent with this model, RNAi of daf-16 at the adult stage alone had no notable effect (p=0.804, Cox proportional hazard regression analysis), as the difference in adult lifespan between individuals developed as larva at 15 °C (mean lifespan 14.16±0.28 days) and 25 °C (mean lifespan 16.89±0.33 days) remained in these daf-16 RNAi animals (p<0.001, log rank). On the other hand, these animals were short-lived, indicating an effect of daf-16 RNAi (also see Figure 6C–D). These data suggest that daf-16 may act at the larval stage.
Figure 5. DAF-16 and HSF-1 mediate the temperature effect at the larval stage.
(A) Loss of daf-16 nearly eliminated the effect of larval temperature treatment on lifespan (p=0.021, Cox proportional hazard regression analysis). The temperature treatment protocol was performed as described in Figure 1B.
(B) RNAi of hsf-1 blocked the effect of larval temperature treatment on lifespan (p=0.002, Cox proportional hazard regression analysis). The temperature treatment protocol was performed as described in Figure 1B.
(C–F) SKN-1 (p=0.788, Cox proportional hazard regression analysis), PHA-4 (p=0.390, Cox proportional hazard regression), DAF-9 (p=0.179, Cox proportional hazard regression analysis), and DAF-12 (p=0.190, Cox proportional hazard regression analysis) are not required for larval temperature treatment to affect lifespan.
Representative data are shown here; replicates and detailed statistics are described in Table S1.
Figure 6. DAF-16 mediates the effect of TRPA-1 on lifespan at the larval stage.
(A) Loss of daf-16 blocks the effect of trpa-1 transgene on temperature modulation of lifespan at the larval stage (p<0.001, Cox proportional hazard regression analysis). Temperature treatment protocol was performed as described in Figure 1B.
(B) hsf-1 RNAi cannot block the effect of trpa-1 transgene on temperature modulation of lifespan at the larval stage (p=0.824, Cox proportional hazard regression analysis).
(C–D) RNAi of daf-16 in adulthood shortens lifespan, independently of the cultivation temperature at the larval stage. Larvae were cultured at 15 °C (C) (p<0.001, log-rank test)] and 25 °C (D) (p<0.001, log-rank test) until L4, and then shifted to 20 °C to score lifespan. daf-16 RNAi was delivered at the adult stage only. At the larval stage, the same worms were fed vector RNAi. Control: worms were fed vector RNAi throughout the larval and adult stages.
(E–F) RNAi of daf-16 in larvae extends lifespan in a temperature-dependent manner. Larvae were cultured at 15 °C (E) (p<0.001, log-rank test) and 25 °C (F) (p=0.877, log-rank test) until L4, and then shifted to 20 °C to score lifespan. daf-16 RNAi was delivered throughout the larval and adult stages (larva+adult) or adult stage only (adult). For those worms treated with daf-16 RNAi at the adult stage only (adult), their larvae were fed vector RNAi.
Also see Figure S4. Representative data are shown here; replicates and detailed statistics are described in Table S1.
Deficiency in HSF-1, another prominent longevity regulator (Hsu et al., 2003; Satyal et al., 1998), also eliminated the temperature effect (Figure 5B) (p=0.002, Cox-proportional hazard regression analysis). By contrast, other transcription factors, such as SKN-1 (p=0.788, Cox-proportional hazard regression analysis), PHA-4 (p=0.390, Cox-proportional hazard regression analysis), DAF-12 (p=0.190, Cox-proportional hazard regression analysis) and its regulator DAF-9 (p=0.179, Cox-proportional hazard regression analysis) (Antebi et al., 2000; Bishop and Guarente, 2007; Jia et al., 2002; Panowski et al., 2007; Tullet et al., 2008), were not required (Figure 5C–F). These experiments identify DAF-16 and HSF-1 as key regulators mediating the temperature effect at the larval stage.
DAF-16 is required for TRPA-1 to regulate lifespan at the larval stage
We then asked whether DAF-16 and/or HSF-1 mediate the function of TRPA-1 in temperature modulation of longevity at the larval stage. While TRPA-1 overexpression greatly enhanced the effect of larval temperature treatment (15 and 25 °C) on lifespan, loss of daf-16 abrogated such an effect (Figure 6A) (p<0.001, Cox-proportional hazard regression analysis). Namely, transgenic worms harboring a daf-16 mutation exhibited similar lifespans in response to larval temperature treatment at 15 vs. 25 °C (Figure 6A). By contrast, such a temperature effect remained unchanged in hsf-1 deficient worms (Figure 6B) (p=0.824, Cox-proportional hazard regression analysis). This data suggests that DAF-16, but not HSF-1, is required for TRPA-1 to regulate lifespan at the larval stage in a temperature dependent manner.
To provide further evidence, we selectively examined the role of DAF-16 in mediating TRPA-1-dependent temperature effect at the larval stage. Again, we restricted the effect of trpa-1 transgene to the larval stage by treating larvae at 15 °C and 25 °C, and then shifted them to 25 °C to score adult lifespan. We found that loss of daf-16 blocked the ability of trpa-1 transgene to shorten lifespan at low temperatures in wild-type and hsf-1(RNAi) worms (Figure S4), providing further evidence that DAF-16 is required for TRPA-1 to regulate lifespan at the larval stage. We also obtained a similar result with worms lacking sgk-1 (Figure S4E–G), a gene which has been previously shown to act upstream of DAF-16 but downstream of TRPA-1 to regulate lifespan in a temperature-dependent manner in adulthood (Mizunuma et al., 2014; Xiao et al., 2013). These experiments suggest that TRPA-1 acts upstream of DAF-16 to regulate lifespan at the larval stage.
DAF-16 shortens lifespan at the larval stage while promoting lifespan at the adult stage
How might DAF-16 mediate the effect of TRPA-1 on longevity at the larval stage? The finding that TRPA-1 shortens lifespan in response to low temperature treatment at the larval stage raises the possibility that DAF-16 may also shorten lifespan in larvae. This model, however, is confounded by the general view that DAF-16 promotes lifespan (Kenyon, 2010). Interestingly, a previous report showed that DAF-16 promotes lifespan by acting in adulthood (Dillin et al., 2002). This prompted an intriguing question: does DAF-16 play a different role in lifespan regulation at the larval stage?
To test this, we first selectively knocked down daf-16 by RNAi in adults and found that RNAi of daf-16 in adulthood shortened adult lifespan upon larval temperature treatment (Figure 6C–D), consistent with a role of DAF-16 in promoting lifespan in adults. RNAi is fairly potent in worms, and once triggered, its effect is long-lasting and can be amplified through RdRP (Pak et al., 2012). As such, it is difficult to selectively knock down daf-16 by RNAi only in larva without affecting the later adult stage. To overcome this technical difficulty, we knocked down daf-16 by RNAi throughout the larval and adult stages (larva+adult RNAi). By comparing the outcome of this larva+adult RNAi and adult-only RNAi, it would provide an alternative means of examining the role of daf-16 in larvae (Figure 6E–F). Strikingly, worms deficient in daf-16 at both the larval and adult stages (larva+adult RNAi) lived longer than those deficient in daf-16 in adults only (adult-only RNAi), suggesting that loss of daf-16 at the larval stage extends lifespan (Figure 6E). This phenomenon is temperature-dependent, as it was only observed in worms subjected to low (15 °C) but not high (25 °C) temperature treatment at the larval stage (Figure 6E–F). These results suggest that DAF-16 shortens lifespan upon low temperature treatment at the larval stage.
DAF-16 differentially regulates gene expression in larvae and adults at different temperatures
How might the same transcription fact DAF-16 mediate the opposite effects of temperature on lifespan in larvae and adults? One possibility is that DAF-16 differentially regulates gene expression in larvae and adults in response to temperature changes. To test this model, we first conducted a genome-wide microarray analysis to determine whether temperature treatment differentially affects gene expression in larvae and adults. We compared two temperatures: 15 °C and 25 °C. Genes differentially regulated by temperature are rather diverse, including those involved in the synthesis, modification, and/or processing of lipids, carbohydrates, carboxylic acids, and peptides (Figures S5). Genes regulating temperature responses (e.g. HSPs), electron transport chain, ion transport, and body morphogenesis were also picked up (Figure S5). Overall, 303 genes were found to be up-regulated by low temperature in larvae (15 °C vs.25 °C) (Figure 7A and Suppl. Table 2). Among them, only ~15% (44 genes) were up-regulated by low temperature in adults; the rest ~85% genes, however, became down-regulated or remained unchanged in adults (Figure 7A and Suppl. Table 2). Similarly, 391 genes were down-regulated by low temperature in larvae (15 °C vs.25 °C), of which merely ~43% (167 genes) were down-regulated in adults and the remaining (~67%) genes were instead up-regulated or unchanged in adult animals (Figure 7A and Suppl. Table 2). Apparently, temperature differentially regulates gene expression in larvae and adults.
Figure 7. DAF-16-dependent genes are differentially regulated by temperature in larvae and adults.
(A) Genes are differentially regulated by temperature in larvae and adults of wild-type (WT). Genes that were up-regulated (15 °C vs. 25 °C) in wild-type larvae are shown in left panel, while those genes that were down-regulated in wild-type larvae are shown in the right panel. As a comparison, the relative expression levels of the same set of genes in wild-type adults are also shown. Only differentially-regulated genes are included in the panels. FDR<0.05; log2 FC>0.7.
(B) DAF-16-dependent genes are differentially regulated by temperature in larvae and adults (15 °C vs. 25 °C). The left panel and right panel lists up- and down-regulated genes in larvae, respectively. Specifically, the first lane in the left panel lists DAF-16-dependent genes that were up-regulated by low temperature (15 °C vs. 25 °C) in wild-type larvae. The relative expression levels of the same set of genes in daf-16(mgDf47) mutant background are listed in the second lane. Each gene showed significant difference in expression level (p<0.05) between wild-type and daf-16 mutant, indicative of DAF-16 dependence. The third and fourth lane lists the relative expression levels of the same set of genes in wild-type adults and daf-16 adults, respectively. Some of these genes become DAF-16 independent in adults. The lanes in the right panel show down-regulated genes and are organized in a similar manner. FDR<0.05; log2 FC>0.7.
(C) qPCR quantification of DAF-16-dependent larval genes up-regulated by low temperature (15 °C vs 25 °C) in wild-type and daf-16 mutant worms. The expression level of each gene (8/10) was significantly different in daf-16(mgDf47) mutant larvae compared to wild-type, indicative of DAF-16 dependence. Their expression (10/10) is also significantly different in wild-type adults. Some of these genes (5/10), however, are no longer DAF-16 dependent in adults, indicating a possible switch in regulatory mechanisms. These genes were picked from Supple. Table 2A based on their high FC values. Experiments were repeated three times. Error bars: SEM. *p < 0.05; **p < 0.005 (ANOVA).
We also performed a similar analysis on daf-16 mutant worms. By comparing data from daf-16 mutant and wild-type animals, we found that 135 DAF-16-dependent genes were up-regulated by low temperature (15 °C vs.25 °C) in larvae (Figure 7B and Suppl. Table 2). Among these DAF-16-dependent larval genes, only 17% (23 genes) were up-regulated in adults by low temperature, and the rest was, however, either down-regulated or unaffected (Figure 7B and Suppl. Table 2). Even among those 23 genes that were also up-regulated in adults by low temperature, 13 became DAF-16 independent, indicating a possible switch in regulatory mechanisms (Figure 7B and Suppl. Table 2). A similar result was obtained with DAF-16-dependent genes that were down-regulated by low temperature (Figure 7B and Suppl. Table 2). Apparently, a large portion of DAF-16-dependent genes were differentially regulated by temperature in larvae and adults. Lastly, we sampled a few such genes and verified the microarray data by qPCR analysis (Figure 7C). These results suggest that DAF-16 differentially regulates gene expression in larvae and adults at different temperatures.
Discussion
Temperature has long been thought to regulate lifespan by globally altering the rate of chemical reactions and hence the pace of aging (Loeb and Northrop, 1916). It is generally believed that lower temperatures extend lifespan while higher temperatures shorten it. With the exception of extreme temperatures which may jeopardize an animal’s survival, this “temperature law”, thus far, has been consistent with empirical observations, particularly in poikliotherms. Although recent work demonstrated that genes play an active role in temperature modulation of longevity (Lee and Kenyon, 2009; Xiao et al., 2013), the “temperature law” remains valid. In the current study, we systemically interrogated how temperature affects lifespan in C. elegans. By examining the commonly used cultivation temperature range (15–25 °C), we found with surprise that exposure to low temperatures at the larval stage in fact shortens adult lifespan while higher temperature treatment prolongs it. This observation is inconsistent with the “temperature law”. Apparently, temperature differentially regulates lifespan at different stages of worm life, unveiling an unexpected layer of complexity underlying temperature modulation of longevity.
The fact that temperature treatment at the larval stage affects adult lifespan also uncovers an interesting phenomenon: temperature experience in the early life of an animal can induce a long-lasting effect on its late life. We found that the cold-sensitive channel TRPA-1 plays a key role in regulating lifespan at the larval stage. Interestingly, recent work shows that thermosensitive TRP channels also have a role in longevity in mammals (Riera et al., 2014). As a well-characterized temperature-sensitive channel, TRPA-1 may act a thermosensor to detect temperature drop in the environment to regulate lifespan at low temperatures through DAF-16, a master regulator of longevity. TRPA-1 is probably not the only thermosensor that mediates temperature modulation of lifespan at least at the larval stage. Indeed, we found that HSF-1, another master regulator of lifespan, also mediates the effect of temperature on lifespan in larvae. HSF-1 is not required for TRPA-1 to regulate lifespan, suggesting that HSF-1 acts independently of and probably in parallel to TRPA-1 and DAF-16. As it has been well established that HSF-1 promotes longevity and can be activated by high temperatures through heat shock signaling (Baird et al., 2014; Hsu et al., 2003; Satyal et al., 1998), it may potentially act as a heat-sensor, albeit indirectly, to detect temperature rise in the environment to promote lifespan at the larval stage. This points to an interesting model that TRPA-1 acts as a cold-sensor to shorten lifespan while HSF-1 responds to heat to extend lifespan at the larval stage.
Interestingly, TRPA-1 also regulates lifespan at the adult stage, although the outcome is the opposite to that observed at the larval stage (Xiao et al., 2013). Specifically, TRPA-1 acts through DAF-16 to prolong lifespan in adults while reducing lifespan in larvae at low temperatures. Notably, SGK-1, which is known to act upstream of DAF-16 to mediate the temperature effect of TRPA-1 in adulthood (Mizunuma et al., 2014; Xiao et al., 2013), is also required for TRPA-1 to regulate lifespan at the larval stage. Apparently, the TRPA-1 genetic pathway is employed to regulate lifespan in both larvae and adults through DAF-16.
An intriguing question is how DAF-16 might produce two opposite lifespan outcomes in larvae and adults? As a master regulator of lifespan in C. elegans, DAF-16 is known to activate/repress the expression of hundreds of genes directly and indirectly (Lee et al., 2003; Murphy et al., 2003). One possibility is that DAF-16 differentially regulates gene expression at the larval and adult stages in response to temperature. This indeed appears to be the case, as shown by gene profiling and qPCR analysis. Such DAF-16-dependent differential regulation of gene expression in larvae and adults may contribute to the distinct effects of temperature on lifespan at the larval and adult stages.
DAF-16 has long thought to be a pro-longevity transcription factor. Our results suggest that DAF-16 can also inhibit lifespan, depending on the stage of worm life (larva vs. adult) and context (low vs. high temperatures). Interestingly, a separate study showed that overexpression of DAF-16 in hypodermis results in tumorigenic activity and shortens lifespan (Qi et al., 2012). Overexpression of mammalian FOXO1 in myocytes during development causes lethality (Evans-Anderson et al., 2008). These observations uncover a multifaceted role of DAF-16/FOXO in lifespan regulation. In summary, our studies demonstrate for the first time that temperature differentially regulates lifespan, establishing a framework for investigating this interesting phenomenon in a powerful genetic model organism.
Experimental procedures
Strains
Wild-type: N2. TQ1516: trpa-1(ok999) X6 outcrossed. TQ11643: xuEx601[ges-1::trpa-1::SL2::yfp+Punc122::DsRed]. TQ1648: xuEx606[Prgef-1::trpa-1::SL2::yfp+Punc-122::DsRed]. TQ1657: xuEx610[Pmyo-3::trpa-1::SL2::yfp+Punc-122::DsRed]. TQ1658: xuEx611[Pdpy-7::trpa-1::SL2::yfp+Punc-122::DsRed]. TQ1654: daf-16(mgDF47). TQ2012: xuEx606[Prgef-1::trpa-1::SL2::yfp+Punc-122::DsRed]; daf-16(mgDF47). TQ6068: daf-9(rh50). TQ6069: daf-12(rh61rh411).
Lifespan
Lifespan was performed as described previously (Hsu et al., 2009; Liu et al., 2013). Wild-type (N2) worms were maintained at 20 °C unless indicated otherwise. To test the effect of larval temperature treatment, parents (L4 stage) reared at 20 °C were moved to 15, 20 and 25 °C to mature and lay eggs at these temperatures. Eggs were allowed to hatch and develop at these three temperatures until reaching L4, and then shifted back to 20 °C to score adult lifespan. To test the effect of adult temperature treatment, eggs were allowed to hatch and develop into L4 at 20 °C, and then shifted to 15, 20, and 25 °C to score adult lifespan. The first day of adulthood was recorded as day 1 in all experiments. Worms which crawled off the plate, exploded or bagged were censored at the time of the event. All lifespans were performed on OP50 bacteria with the exception of those involving RNAi, in which case were conducted on HT115 bacteria. It should be noted that different diets may affect lifespan differently (Mizunuma et al., 2014).
For the lifespan experiments involving RNAi, fresh single colonies of HT115 bacteria containing empty vector L4440 or RNAi plasmid were cultured overnight at 37 °C in LB with carbenicillin (100 μg/ml). Two days prior to the experiments, freshly grown RNAi bacteria were seeded on NGM plates containing carbenicillin (25 μg/ml) and IPTG (1 mM). Most RNAi clones were obtained from the Ahringer library (Kamath and Ahringer, 2003). hsf-1 RNAi clone was generated as described previously (Walker et al., 2003)
Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc.) and IBM SPSS Statistics 19 (IBM, Inc.). P values were calculated with the log-rank (Kaplan-Meier) method and Cox-proportional hazard regression as indicated.
Microarray procedure and data analysis
For microarray sample preparation, synchronous populations of worms were generated by making 25 to 30 adult worms to lay eggs on newly-seeded NGM plates for 2–3 hours. To obtain the larval sample, 300 hundreds worms were harvested when they reached late L4 stage. To obtain the adult sample, 120 worms were harvested when they reached 3-days old adult. Biological samples were prepared on separate days and total RNA was extracted with TRl Reagent (Life Technologies). The concentrations and quality of total RNA samples were checked with Thermo NanoDrop 2000c (Thermo Scientific, Wilmington, DE) and Agilent 2100 Bio-Analyzer (Agilent Technologies, Palo Alto, CA). Microarrays were done on Affymetrix C. elegans Gene 1.1 ST Array Strips. Preparation of cDNA, hybridization, quality controls and scanning of arrays were performed according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA) at the Microarray Core Facility of University of Michigan.
All microarray analysis was performed with bioconductor implemented in R statistical environment (Gentleman et al., 2004). Expression values of each gene are calculated using a robust multi-array average (RMA) method (Irizarry et al., 2003). Differentially expressed genes were identified using weighted linear models designed specifically for microarray analysis (Ritchie et al., 2006; Smyth, 2004). p-values were adjusted for multiple comparisons using false discovery rate (FDR) (Benjamini and Hochberg, 1995). Hierarchical clustering was performed with an uncentered correlation similarity metric by Cluster using Centroid linkage clustering method (de Hoon et al., 2004). Gene ontology analysis was analyzed using DAVID and functional clusters are identified using Functional Annotation Chart tool (Huang et al., 2008).
Quantitative RT-PCR
Total RNA was extracted from 100–300 worms with TRI Reagent (Life Technologies, CA). qPCR was carried out using CYBR Green (Life Technologies, CA) according the protocol provided by the manufacturer. We used act-1 (actin) as an internal reference for normalization, and ΔΔCt method was used to analyze qPCR data.
Supplementary Material
Acknowledgments
We thank S.J. Lee for communicating unpublished results. Some strains were provided by the Caenorhabditis Genetics Center and the Japan knockout consortium. R.X. is supported by an NIA T32 training grant. This work was supported by NSFC (31130028 and 31225011 to J.L.), the Program of Introducing Talents of Discipline to the Universities from the Ministry of Education (B08029 to J.L.), the Ministry of Science and Technology of China (2012CB51800 to J.L.), and grants from the NIH (X.Z.S.X. and A.L.H.).
Footnotes
Accession Number
The microarray data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (accession number: GSE62297).
Author contributions
B.Z., R.X., and E.A.R. performed the experiments and analyzed the data. Y.H. assisted B.Z. on data analysis. B.Z., A.L.H., J.L., and X.Z.S.X. wrote the paper.
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