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. Author manuscript; available in PMC: 2014 Feb 14.
Published in final edited form as: Cell. 2013 Feb 14;152(4):806–817. doi: 10.1016/j.cell.2013.01.020

A genetic program promotes C. elegans longevity at cold temperatures via a thermosensitive TRP channel

Rui Xiao 1, Bi Zhang 1,3,5, Yongming Dong 1,5, Jianke Gong 1,3,5, Tao Xu 3,4, Jianfeng Liu 3, X Z Shawn Xu 1,2
PMCID: PMC3594097  NIHMSID: NIHMS437620  PMID: 23415228

Summary

Both poikilotherms and homeotherms live longer at lower body temperatures, highlighting a general role of temperature reduction in lifespan extension. However, the underlying mechanisms remain unclear. One prominent model is that cold temperatures reduce the rate of chemical reactions, thereby slowing the rate of aging. This view suggests that cold-dependent lifespan extension is simply a passive thermodynamic process. Here, we challenge this view in C. elegans by showing that genetic programs actively promote longevity at cold temperatures. We find that TRPA-1, a cold-sensitive TRP channel, detects temperature drop in the environment to extend lifespan. This effect requires cold-induced, TRPA-1-mediated calcium influx and a calcium-sensitive PKC which signals to the transcription factor DAF-16/FOXO. Human TRPA1 can functionally substitute for worm TRPA-1 in promoting longevity. Our results reveal a new function for TRP channels, link calcium signaling to longevity, and importantly, demonstrate that genetic programs contribute to lifespan extension at cold temperatures.

Introduction

Aging can be modulated by both environmental and genetic factors (Fontana et al., 2010; Kenyon, 2010). Work in model organisms, such as yeast, worms, flies and mice, has identified a number of avenues that promote longevity. For example, restricting food intake (dietary restriction or DR), decreasing insulin/IGF-1 signaling (IIS), slowing mitochondrial respiration, reducing germ-line function, or lowering temperature can all extend lifespan (Kenyon, 2010). Over the past two decades, studies in model organisms have led to an increasingly clear understanding of how DR, IIS, mitochondrial function and reproductive system modulate longevity (Fontana et al., 2010; Kenyon, 2010). By contrast, very little is known about how temperature regulates lifespan (Conti, 2008).

The phenomenon that poikilotherms (e.g. worms, flies and fish) have a longer lifespan at lower temperatures was first documented nearly a century ago (Loeb and Northrop, 1916). Recent work demonstrates that lowering the core body temperature of homeothermic animals, such as mice, also increases lifespan (Conti et al., 2006), highlighting a general role of temperature reduction in lifespan extension in both poikilotherms and homeotherms. In C. elegans, raising the culturing temperature (e.g. to 25 °C) greatly shortens lifespan, which is counteracted by the AFD neuron via neuroendocrine signals (Lee and Kenyon, 2009). Conversely, rearing worms at low temperatures (e.g. 20 °C and 15 °C) greatly lengthens lifespan (Klass, 1977; Wu et al., 2009). However, the mechanisms underlying this cold-dependent lifespan extension remains unclear. One prominent model is that lowering the body temperature would reduce the rate of chemical reactions, thereby leading to a slower pace of living. This model suggests that the extended lifespan observed at low temperatures is simply a passive thermodynamic process. Indeed, it takes a longer time for worms to develop from embryos to adults at lower temperatures, a phenomenon seemingly consistent with this model.

Here, we sought to challenge this view in C. elegans. We reasoned that if genetic programs can actively promote longevity under other paradigms, they might also do so at cold temperatures. We found that TRPA-1, a cold-sensitive TRP channel, can act as a thermo-sensor to detect temperature drop in the environment to initiate a genetic program to extend lifespan. This program includes TRPA-1, TRPA-1-mediated calcium influx, PKC-2 (a calcium-sensitive PKC), SGK-1 (a DAF-16/FOXO kinase), and the FOXO transcription factor DAF-16 which is a key regulator of lifespan. We also show that the intestine, a non-excitable tissue known to be a signaling hub for lifespan regulation, is in fact cold-sensitive and responds to temperature decreases in the environment. This finding expands the repertoire of cold receptors from neurons to non-excitable cells. Interestingly, human TRPA1 can functionally substitute for worm TRPA-1 in lifespan extension in a temperature- and agonist-dependent manner. Our studies not only demonstrate that genetic programs actively contribute to lifespan extension at cold temperatures, but also identify an unexpected function for TRP family channels and intestinal cells, and reveal a functional link between calcium signaling and longevity.

Results

Loss of TRPA-1 shortens lifespan at cold but not warm temperatures

We hypothesized that genetic programs may actively promote longevity at cold temperatures. As a first step to test this idea, we envisioned that a cold-sensor(s) may detect temperature decreases in the environment to initiate a pro-longevity genetic program. The best known cold sensors in mammals are the TRP family channels TRPA1 and TRPM8, but the latter of which does not have a homologue in C. elegans (McKemy et al., 2002; Peier et al., 2002; Story et al., 2003; Venkatachalam and Montell, 2007). TRPA-1, the C. elegans orthologue of the mammalian TRPA1 channel, thus came to our attention (Kindt et al., 2007; Venkatachalam and Montell, 2007; Xiao and Xu, 2009).

As is the case with its mammalian counterparts (Karashima et al., 2009; Story et al., 2003), TRPA-1 is also a cold-sensitive channel, opening when temperature drops to ~20 °C (Chatzigeorgiou et al., 2010). Three temperatures (i.e. 15 °C, 20 °C and 25 °C) are common laboratory conditions for culturing C. elegans. If TRPA-1 is involved in promoting longevity at low temperatures, one would expect that mutant worms lacking TRPA-1 should have a shorter lifespan at 20 °C and 15 °C but not at 25 °C than do wild-type worms. This is because this cold-sensitive channel is expected to be functional at 20 °C and 15 °C but remains closed at 25 °C. Consistent with this prediction, we found that trpa-1 null mutant worms showed a significantly shorter lifespan than did wild-type at 20 °C (Figure 1B, Table S1). A similar phenomenon was also observed at 15 °C (Figure 1C). By contrast, at 25 °C, the lifespan of trpa-1 mutant worms was similar to that of wild-type (Figure 1A). No notable defect in development or fecundity was detected in trpa-1 mutant worms (Figure S1A-B). These results suggest that TRPA-1 may function to extend lifespan at cold but not warm temperatures. This cold-dependent pro-longevity effect is opposite to that of other sensory channels, such as TAX-2, TAX-4 and OCR-2, all of which appear to shorten lifespan at low temperatures (20 °C) (Apfeld and Kenyon, 1999; Lee and Ashrafi, 2008; Lee and Kenyon, 2009).

Figure 1. TRPA-1 promotes longevity at cold but not warm temperatures.

Figure 1

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(A–C) trpa-1 null mutant worms are short-lived at 20 °C (B) and 15 °C (C) but not at 25 °C (A).

(D–E) Overexpression of wild-type trpa-1 gene extends lifespan at 20 °C and 15 °C but not at 25 °C. Transgenic worms express full-length trpa-1 genomic DNA.

See also Figure S1 and Table S1

Transgenic expression of TRPA-1 extends lifespan at cold but not warm temperatures

A short-lived mutant phenotype, however, does not provide sufficient evidence that the gene of interest promotes longevity. We therefore performed the converse experiment by overexpressing wild-type TRPA-1 in worms. Transgenic expression of TRPA-1 under its own promoter increased lifespan at 20 °C and 15 °C, but not at 25 °C (Figure 1D–F). This provides further evidence that TRPA-1 can promote longevity at low temperatures. As TRPA-1 is functional at 20 °C, for simplicity, we focused on this temperature for further characterizations.

We next examined in which tissues TRPA-1 acts to promote longevity. TRPA-1 is known to be expressed in multiple tissues, including neurons, muscles, hypodermal cells, and the intestine (Dupuy et al., 2007; Kindt et al., 2007) (Figure S1C). Expression of TRPA-1 in intestinal cells or neurons was sufficient to extend the lifespan of wild-type worms, with intestinal expression showing the most robust effect (Figure 2A). By contrast, expression of TRPA-1 in muscles or hypodermal cells did not extend lifespan (Figure 2A). This suggests that TRPA-1 can function in both intestinal cells and neurons to modulate lifespan. Notably, the intestine and nervous system are the two tissues where the transcription factor DAF-16/FOXO, a key regulator of lifespan, acts to promote longevity (Libina et al., 2003; Lin et al., 1997; Ogg et al., 1997). As intestinal expression of TRPA-1 displayed the strongest effect in lifespan extension, we focused on this tissue for further characterizations.

Figure 2. TRPA-1-dependent lifespan extension requires DAF-16.

Figure 2

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(A) Transgenic expression of trpa-1 in the intestine and neurons, but not in muscles or hypodermal cells, extends lifespan. The ges-1, rgef-1, myo-3, and dpy-7 promoter was used to drive expression of trpa-1 cDNA in the intestine, neurons, muscles and hypodermal cells, respectively (Aamodt et al., 1991; Altun-Gultekin et al., 2001; Fire and Waterston, 1989; Gilleard et al., 1997).

(B) Loss of daf-16 fully suppresses the long-lived phenotype of trpa-1 transgenic worms.

Lifespan: 20 °C. See also Figure S2 and Table S1

TRPA-1 acts upstream of the FOXO transcription factor DAF-16

Clearly, as a cold sensor, TRPA-1 cannot promote longevity on its own. We thus sought to identify transcription factors that act downstream of TRPA-1 to promote longevity, as transcription factors are known to be the master regulators of lifespan in C. elegans (Kenyon, 2010). Loss of daf-16 abolished the ability of trpa-1 transgenes to extend lifespan (Figure 2B). In addition, daf-16 RNAi abrogated the temperature sensitivity of trpa-1 mutant worms (Figure S2A–B). Furthermore, overexpression of DAF-16 rescued the short-lived phenotype of trpa-1 mutant worms at low temperatures (Figure S2C). These findings together strongly suggest that TRPA-1 acts upstream of DAF-16. By contrast, trpa-1 transgene can still extend the lifespan of worms deficient in several other known pro-longevity transcription factors, such as SKN-1/Nrf, HSF-1, and PHA-4/FOXA (Figure S2D–F) (Bishop and Guarente, 2007; Hsu et al., 2003; Panowski et al., 2007; Tullet et al., 2008). Similarly, the pro-longevity effect of trpa-1 transgene was also independent of the transcription factor DAF-12 (a nuclear hormone receptor) and its regulator DAF-9 (Figure S2G–H) (Antebi et al., 2000; Jia et al., 2002). While these results do not exclude the involvement of other transcription factors, they demonstrate that DAF-16 is required for the function of TRPA-1 in lifespan extension, suggesting that DAF-16 acts downstream of TRPA-1.

TRPA-1-mediated lifespan extension requires calcium and a calcium-sensitive PKC

As an ion channel, TRPA-1 is unlikely to directly signal to DAF-16. We thus set out to identify additional components in the TRPA-1- and DAF-16-dependent genetic pathway. TRPA proteins are calcium-permeable nonselective cation channels (Jordt et al., 2004; Story et al., 2003), and their ion selectivity has been well characterized (Wang et al., 2008). A highly conserved D/E residue in the putative selectivity filter of TRPA channels is essential for calcium permeability (Wang et al., 2008). Mutating this residue to A does not affect the overall function of the channel, but selectively renders the channel impermeable to Ca2+ without affecting its permeability to Na+ or K+ (Wang et al., 2008). We found that TRPA-1 harboring this point mutation (i.e. E1018A) failed to extend lifespan in transgenic worms (Figure 3A). This hints at a critical role for calcium in TRPA-1-mediated lifespan extension.

Figure 3. TRPA-1-dependent lifespan extension requires calcium and PKC-2.

Figure 3

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(A) TRPA-1E1018A fails to extend lifespan. Lifespan: 20 °C.

(B) Loss of pkc-2 fully suppresses the long-lived phenotype of trpa-1 transgenic worms. Lifespan: 20 °C.

(C–E) pkc-2 mutant worms are short-lived at 20 °C and 15 °C but not at 25 °C.

(F) Overexpression of pkc-2 cDNA extends lifespan, which can be fully suppressed by loss of daf-16. Lifespan: 20 °C.

(G) Loss of sgk-1 fully suppresses the long-lived phenotype of trpa-1 and pkc-2 transgenic animals. Lifespan: 20 °C.

(H) Overexpression of sgk-1 extends lifespan, which can be fully suppressed by loss of daf-16. Lifespan: 20 °C. sgk-1 cDNA was expressed as a transgene in the intestine, a tissue in which endogenous sgk-1 gene is enriched (Hertweck et al., 2004). Lifespan: 20 °C.

See also Figure S3 and Table S1

Then how might calcium transmit signals to DAF-16? The function of DAF-16 is best known to be regulated by kinases. We thus asked whether any calcium-sensitive kinase can transmit calcium signals from TRPA-1 to DAF-16.

To meet the first requirement, the candidate gene should act downstream of TRPA-1. The best known calcium-sensitive kinases are probably CaMK. C. elegans encodes two CaMKs: CMK-1 and UNC-43 (Reiner et al., 1999; Satterlee et al., 2004). We therefore checked cmk-1 and unc-43 mutant worms, but found that null mutations of these two CaMKs failed to suppress the long-lived phenotype conferred by trpa-1 transgene (Figure S3AB), indicating that both CaMKs are not required for the function of TRPA-1 in promoting longevity. Another major class of calcium-sensitive kinases is PKC. The C. elegans genome encodes four PKCs; however, PKC-2 represents the sole classical PKC (cPKC) and also the only PKC equipped with a C2 domain that is known to confer calcium sensitivity to this type of kinases (Islas-Trejo et al., 1997). Importantly, PKC-2 has been demonstrated to be directly activated by calcium in vitro (Islas-Trejo et al., 1997). We found that loss of PKC-2 fully suppressed the long-lived phenotype of trpa-1 transgenic animals, indicating that PKC-2 is required for the function of TRPA-1 in the pathway (Figure 3B). These data suggest that PKC-2 may act downstream of TRPA-1 and calcium to promote longevity.

PKC-2 acts downstream of TRPA-1 but upstream of DAF-16 to extend lifespan

If PKC-2 indeed resides downstream of TRPA-1 and calcium to promote longevity, animals lacking PKC-2 should be short-lived. We thus scored the lifespan of pkc-2 mutant worms at different temperatures. Remarkably, worms lacking PKC-2 lived significantly shorter than wild-type at low (20 °C and 15 °C) but not warm (25 °C) temperatures (Figure 3C–E). No notable deficit in development or fecundity was detected in these mutant animals (Figure S1A-B). This temperature-dependent lifespan phenotype is very similar to that observed in trpa-1 mutant worms, providing additional evidence that PKC-2 transmits signals from TRPA-1 and calcium.

We further reasoned that if PKC-2 truly acts in the same pathway to promote longevity, its overexpression ought to extend lifespan. Similar to TRPA-1, PKC-2 is expressed in multiple tissues, including intestinal cells and neurons (Islas-Trejo et al., 1997). Indeed, transgenic expression of PKC-2 in these two tissues can both extend lifespan, with intestinal expression showing a more robust effect (Figure 3F). Unlike TRPA-1, this effect is temperature independent (not shown). This can be explained by the fact that PKC-2 is not the initial thermo-sensor, and it is common that over expression of a downstream effect or often bypasses its upstream genes. Indeed, pkc-2 transgene can fully suppress the short-lived phenotype of trpa-1 mutant worms, consistent with the view that PKC-2 acts downstream of TRPA-1 (Figure S3C). Moreover, the short-lived phenotype of trpa-1; pkc-2 double mutant at low temperatures was similar to that of trpa-1 and pkc-2 single mutants (Figure S3D-F). Thus, the effect of TRPA-1 and PKC-2 on lifespan is not additive, providing further evidence that they act in the same pathway. These observations together strongly suggest that PKC-2 acts downstream of TRPA-1 and calcium to promote longevity at low temperatures.

We then asked whether PKC-2 acts upstream of DAF-16 to extend lifespan. We found that loss of daf-16 abolished the long-lived phenotype of pkc-2 transgenic worms, indicating that DAF-16 is required for the function of PKC-2 in promoting longevity (Figure 3F). As was the case with trpa-1 mutant, daf-16 RNAi also abrogated the temperature sensitivity of pkc-2 mutant worms (Figure S2A-B). Furthermore, overexpression of DAF-16 rescued the short-lived phenotype of pkc-2 mutant worms at low temperatures (Figure S2C). These observations together strongly suggest that PKC-2 functions upstream of DAF-16 to extend lifespan.

TRPA-1 and PKC-2 dependent lifespan extension requires SGK-1, a DAF-16 kinase

How does PKC-2 signal to DAF-16? Given that DAF-16 is known to be regulated by phosphorylation, the simplest model would be that PKC-2 may signal to DAF-16 by directly phosphorylating DAF-16. However, we failed to detect phosphorylation of DAF-16 by PKC-2 in in vitro kinase assays (not shown). Though this does not rule out the possibility that PKC-2 may phosphorylate DAF-16 in vivo, it strongly suggests that PKC-2 probably regulates DAF-16 indirectly.

Because DAF-16 is best known to be regulated by kinases and PKC-2 is probably not a DAF-16 kinase, we reasoned that another kinase may be present in the TRPA-1 pathway to regulate DAF-16, probably by acting downstream of PKC-2. We therefore considered those known DAF-16 kinases, including AKT-1, AKT-2, JNK-1, and SGK-1 (Hertweck et al., 2004; Lin et al., 2001; Oh et al., 2005; Paradis and Ruvkun, 1998). trpa-1 transgene can further extend the lifespan of akt-1, akt-2 and jnk-1 mutant worms, suggesting that these three DAF-16 kinases are probably not in the TRPA-1 pathway (Figure S3G-I). By contrast, loss of SGK-1 completely suppressed TRPA-1- and PKC-2-dependent lifespan extension (Figure 3G), suggesting that SGK-1 is probably a member of the TRPA-1 pathway and acts downstream of TRPA-1 and PKC-2.

SGK-1 acts downstream of TRPA-1 and PKC-2 but upstream of DAF-16 to extend lifespan

A previous study reported that RNAi knockdown of sgk-1 extends lifespan, suggesting that the function of this gene is to shorten lifespan (Hertweck et al., 2004). More recent studies using a sgk-1 deletion mutant, however, demonstrated that loss of sgk-1 shortens lifespan (Alam et al., 2010; Soukas et al., 2009). We also found that sgk-1 mutant animals were short-lived (Figure 3G). The short-lived phenotype of sgk-1 mutants suggests that the function of SGK-1 is to promote longevity. ImportantlyIndeed, overexpression of SGK-1 promoted extended lifespan (Figure 3H). No temperature dependence was observed in sgk-1 worms (not shown), consistent with the notion that SGK-1 is not a temperature sensor on its own. SGK-1 has been suggested to act upstream of DAF-16 (Alam et al., 2010; Hertweck et al., 2004). Indeed, loss of DAF-16 completely suppressed the long-lived phenotype of sgk-1 transgenic worms (Figure 3H). Conversely, overexpression of DAF-16 rescued the short-lived phenotype of sgk-1 mutant worms (Figure S2C). These data support the view that SGK-1 acts upstream of DAF-16 to positively regulate its activity.

Taken together, our data suggest a mode in which TRPA-1, calcium, PKC-2, SGK-1, and DAF-16 form a signaling pathway to promote longevity at low temperatures.

The TRPA-1 pathway does not promote nuclear translocation of DAF-16

How does the TRPA-1 pathway regulate the function of DAF-16? DAF-16 kinases, such as AKT-1, AKT-2 and JNK-1, are known to regulate nuclear translocation of DAF-16 (Henderson and Johnson, 2001; Lin et al., 2001; Oh et al., 2005; Paradis and Ruvkun, 1998), while some other DAF-16 regulators (e.g. HCF-1, EAK-7 and SMK-1) exert their effects by modulating DAF-16 nuclear activity (Alam et al., 2010; Li et al., 2008; Wolff et al., 2006). Using three different quantification methods, we found that the subcellular localization pattern of DAF-16::GFP fusion, which is encoded by the transgene zIs356 (Henderson and Johnson, 2001), was not affected by trpa-1, pkc-2 or sgk-1 transgenes (Figure 4A–F, Figure S4A-G). We also did not detect any notable change in DAF-16 subcellular localization pattern in sgk-1 mutant worms (Figure 4F). These data demonstrate that unlike other DAF-16 kinases, SGK-1 does not regulate DAF-16 localization. Thus, the TRPA-1 pathway does not appear to promote nuclear translocation of DAF-16.

Figure 4. The TRPA-1 pathway does not alter the subcellular localization of DAF-16 but instead promotes its nuclear activity.

Figure 4

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(A) DAF-16::GFP is localized to the cytoplasm as well as the nucleus. Sample images. Left: DIC. Right: DAF-16::GFP. See Figure S4 for images of higher magnification.

(B) trpa-1 transgene does not alter the localization pattern of DAF-16::GFP. Sample images. Left: DIC. Middle: DAF-16::GFP. Right: mCherry fluorescence marking trpa-1 transgene. This trpa-1 transgene (xuEx785) also extended lifespan: 23.1 ± 0.5 days vs. WT control: 18.9 ± 0.6 days; p<0.001 (log-rank).

(C) pkc-2 transgene does not alter the localization pattern of DAF-16::GFP. This pkc-2 transgene (xuEx911) also extended lifespan: 26.6 ± 0.5 days vs. WT control: 19.8 ± 0.7 days. p<0.001 (log-rank).

(D) sgk-1 transgene does not alter the localization pattern of DAF-16::GFP. This transgene is the same as that used in Figure 3H.

(E) Nuclear translocation of DAF-16::GFP in daf-2(RNAi) worms. The fluorescent puncta in the image label the cell nuclei of the worm (Henderson and Johnson, 2001). See Figure S4B for an image of higher magnification.

(F) Table summarizing the data in Figure 4A–D. We grouped animals into four categories based on the localization pattern of DAF-16::GFP in intestinal cells: 0% cells nuclear, <50% cells nuclear , >50% cells nuclear, and All nuclear, with each representing the percentage of cells showing nuclear enrichment of DAF-16::GFP. There is no significant difference between WT (daf-16::gfp) and trpa-1 or pkc-2 transgenic worms (ANOVA test).

(G–I) TRPA-1, PKC-2 and SGK-1 promote the lifespan of worms expressing DAF-16AM::GFP to a greater extent than that of worms expressing DAF-16::GFP. Extension of mean lifespan by trpa-1 transgene in DAF-16AM::GFP and DAF-16::GFP worms is: 38% vs. 16%, respectively. Extension of mean lifespan by pkc-2 transgene in DAF-16AM::GFP and DAF-16::GFP worms is: 41% vs. 26%, respectively. Extension of mean lifespan by sgk-1 transgene in DAF-16AM::GFP and DAF-16::GFP worms is: 34% vs. 13%, respectively. Lifespan: 20 °C. All scale bars: 100 µm.

See also Figure S4 and Table S1

As a positive control, inactivation of daf-2, which encodes the worm insulin/IGF-1 receptor homologue (Kimura et al., 1997), resulted in a marked translocation of DAF-16 to the nucleus (Figure 4E–F, Figure S4B, S4F-G). This data is expected, as IIS is well known to regulate nuclear translocation of DAF-16 (Henderson and Johnson, 2001; Lin et al., 2001). This also raises the possibility that the TRPA-1 pathway and IIS may act in parallel to regulate DAF-16. Consistent with this model, transgenic expression of TRPA-1 can further extend the lifespan of daf-2(e1370) mutant worms (Figure S4H), as well as worms lacking akt-1 and akt-2, two key components of IIS (Paradis and Ruvkun, 1998) (Figure S3G-H). On the other hand, because daf-2 null alleles are lethal, we are unable to further test this model; thus, the possibility that the two act in an overlapping or the same pathway cannot be formally ruled out. Nevertheless, these data demonstrate that the TRPA-1 pathway does not alter the subcellular localization of DAF-16, suggesting that this pathway may regulate DAF-16 function by promoting its nuclear activity.

The TRPA-1 pathway promotes the nuclear activity of DAF-16

To gather additional evidence to support a role for the TRPA-1 pathway in promoting the nuclear activity of DAF-16, we performed further experiments. In wild-type worms, DAF-16 protein is distributed in both the cytosol and nucleus at resting states (Henderson and Johnson, 2001; Lin et al., 2001) (also see Figure S4A and S4F), and it is known that nuclear translocation alone is insufficient to activate DAF-16 (Lin et al., 2001). We reasoned that if the TRPA-1 pathway promotes DAF-16 nuclear activity, then increasing the amount of DAF-16 protein inside the nucleus should potentiate the effect of the TRPA-1 pathway in extending lifespan. To test this, we took advantage of DAF-16AM::GFP, an genetically engineered form of DAF-16 which is functional, but which IIS fails to sequester in the cytosol (Lin et al., 2001). Consequently, DAF-16AM::GFP constitutively translocates to the nucleus, leading to an enrichment of DAF-16 within the nucleus (Lin et al., 2001). Because of this nuclear enrichment of DAF-16, one would expect that TRPA-1, PKC-2 and SGK-1 should be more potent in promoting lifespan in this strain. Indeed, trpa-1, pkc-2 and sgk-1 transgenes extended the lifespan of worms containing DAF-16AM::GFP to a greater extent than that of worms expressing DAF-16::GFP (Figure 4G–I), further suggesting that the TRPA-1 pathway promotes the nuclear activity of DAF-16. Interestingly, in the absence of trpa-1, pkc-2 and sgk-1 transgenes, DAF-16AM::GFP itself did not extend lifespan when compared to DAF-16::GFP (Figure 4G–I), consistent with the view that nuclear translocation alone is insufficient to activate DAF-16 (Lin et al., 2001). This data provides additional evidence that the TRPA-1 pathway promotes DAF-16 nuclear activity.

To provide further evidence, we assayed DAF-16 targets. If the TRPA-1 pathway promotes DAF-16 nuclear activity, one would expect that this pathway should regulate the expression level of DAF-16 target genes. We first examined the expression level of sod-3 which is a direct transcriptional target of DAF-16 and also a widely used molecular reporter for DAF-16 activity (Lee et al., 2003; Murphy et al., 2003; Oh et al., 2006). We found that SOD-3::GFP fusion protein, which is encoded by the transgene muIs84 (Libina et al., 2003), was markedly upregulated in trpa-1, pkc-2 and sgk-1 transgenic worms (Figure 5A–E). We then examined the mRNA level of endogenous sod-3 gene as well as five other DAF-16 target genes, including dod-3, mtl-1, dod-24, dod-22, and ins-7 (Lee et al., 2003; Murphy et al., 2003). Among these 6 target genes, three (sod-3, mtl-1 and dod-3) are known to be up-regulated by DAF-16, while the other three (dod-24, dod-22 and ins-7) are down-regulated by DAF-16. We found that overexpression of trpa-1, pkc-2 or sgk-1 significantly potentiated the mRNA level of sod-3, mtl-1 and dod-3, while loss of trpa-1, pkc-2 or sgk-1 led to a significant reduction in the expression of these genes (Figure 5F), indicating that the TRPA-1 pathway regulates the expression level of DAF-16 targets that are up-regulated by DAF-16. Likewise, dod-24, dod-22 and ins-7, which are known to be repressed by DAF-16, were down-regulated in trpa-1, pkc-2 and sgk-1 transgenic worms (Figure 5G). Conversely, in trpa-1, pkc-2 and sgk-1 mutant worms, these three DAF-16 target genes became up-regulated (Figure 5G). Thus, the TRPA-1 pathway also regulates the expression level of DAF-16 target genes that are repressed by DAF-16. Furthermore, regulation of DAF-16 target genes by trpa-1, pkc-2 and sgk-1 transgenes were DAF-16-dependent (Figure S5). These results together provide further evidence supporting that the TRPA-1 pathway promotes DAF-16 nuclear activity.

Figure 5. The TRPA-1 pathway regulates the expression level of DAF-16 target genes.

Figure 5

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(A) SOD-3::GFP is expressed at low levels in wild-type worms. Left panel: DIC. Right panel: SOD-3::GFP. Scale bars: 100 µm.

(B)trpa-1 transgene potentiates the expression of SOD-3::GFP. Left: DIC. Middle: SOD-3::GFP. Right: mCherry fluorescence marking trpa-1 transgene.

(C) pkc-2 transgene potentiates the expression of SOD-3::GFP. Left: DIC. Middle: SOD-3::GFP. Right: mCherry fluorescence marking pkc-2 transgene.

(D) sgk-1 transgene potentiates the expression of SOD-3::GFP. Left: DIC. Middle: SOD-3::GFP. Right: mCherry fluorescence marking sgk-1 transgene.

(E) Quantification of SOD-3::GFP fluorescence intensity. n≥15. Error bars: SEM. **p<0.0001 (ANOVA with Bonferroni test).

(F–G) qPCR analysis of target genes regulated by DAF-16. qPCR reactions were run in triplicates for each genotype. Each experiment was repeated three times. Worms were reared at 20 °C. Error bars: SEM. *p<0.05; **p<0.005 (ANOVA with Bonferroni test). See also Figure S5

Cold evokes a robust TRPA-1-dependent calcium response in the intestine, a signaling hub for lifespan regulation

Having obtained genetic evidence supporting a role for TRPA-1 and calcium signaling in cold-dependent lifespan extension, we then sought to gather physiological evidence for this model. Though it has been well established that certain types of neurons can detect temperature drop in many organisms (Chatzigeorgiou et al., 2010; Gallio et al., 2011; Story et al., 2003), whether the intestine, a non-excitable tissue, can function as a thermo-receptor has not been explored. Notably, worm intestine is known to be a signaling hub that integrates multiple pro- and anti-aging cues to regulate lifespan (Kenyon, 2010). The observation that expression of TRPA-1 in the intestine or neurons was sufficient to extend lifespan prompted us to test whether intestinal cells can function as a cold receptor as do neurons. As it has not been possible to patch the intestine in vivo, we took a calcium imaging approach to examine the sensitivity of this tissue to cold temperatures. To do so, we expressed G-CaMP, a genetically-encoded calcium sensor (Nakai et al., 2001), as a transgene in the intestine. Remarkably, intestinal cells exhibited a robust increase in calcium level in response to cooling (Figure 6A–B). This cold-evoked calcium response was also found in the intestine dissected out of the animal, indicating that the response originated from the intestine and thus shall be an intrinsic feature of this tissue (Figure S6). Importantly, such a calcium response was greatly reduced in trpa-1 mutant worms, consistent with an important role for TRPA-1 in cold-dependent lifespan extension (Figure 6A–B). In addition, expression of wild-type TRPA-1 in the intestine under an intestine-specific promoter was sufficient to rescue the calcium defect in trpa-1 mutant worms, consistent with our genetic data that TRPA-1 can act in the intestine to promote longevity (Figure 6A–B).

Figure 6. Cold evokes a robust calcium response in the intestine.

Figure 6

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(A) Intestine responds to cooling, and TRPA-1 is important for this response. G-CaMP1.3 was expressed as a transgene in the intestine under the lfe-2b promoter (Clandinin et al., 1998). DsRed was co-expressed to enable ratiometric imaging. Shown on the top are the calcium ratio traces. Shades along the traces represent error bars (SEM). Shown on the bottom is the temperature trace.

(B) Bar graph summarizing the data in (A). Error bars: SEM. n≥6. **p<0.0003 (ANOVA with Bonferroni test). See also Figure 6

We then characterized the calcium-impermeable form of TRPA-1 (TRPA- 1E1018A) that has lost the capacity to extend lifespan (Figure 3A). Expression of this “calcium-free” TRPA-1 in the intestine failed to restore the cold-evoked calcium response in trpa-1 mutant worms, providing physiological evidence supporting a critical role for calcium signaling in lifespan extension at cold temperatures (Figure 6A–B). These calcium imaging data not only complemented our genetic analysis of aging, but also revealed a cellular correlate for cold-induced lifespan extension. In addition, they unveiled an unexpected role of intestinal cells as cold receptors in thermosensation, expanding the repertoire of cold receptors from neurons to non-excitable cells.

Human TRPA1 and a worm TRPA-1 variant can extend lifespan in C. elegans in a temperature- and agonist-dependent manner

As human TRPA1 shares a strong homology with worm TRPA-1 (Venkatachalam and Montell, 2007; Xiao and Xu, 2009), we wondered if it can functionally substitute for its worm homologue in promoting longevity. Remarkably, transgenic expression of human TRPA1 in worms extended lifespan at cold temperature (Figure 7A), but failed to do so at warm temperature (Figure 7B). This temperature-dependent effect on lifespan is similar to that observed with worm TRPA-1.

Figure 7. Human TRPA1 and a worm TRPA-1 variant can extend lifespan in C. elegans in a temperature- and agonist-dependent manner.

Figure 7

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(A) Transgenic expression of human TRPA1 (hTRPA1) extends lifespan at cold temperature. Lifespan was assayed at 15 °C.

(B–C) Human TRPA1 (hTRPA1) does not extend lifespan at warm temperature but can do so in the presence of its agonist AITC (B), and such lifespan extension depends on pkc-2, sgk-1 and daf-16 (C). AITC (10 µM) was included in NGM plates. Lifespan was assayed at 27 °C to suppress cold-induced activity of human TRPA1, as this channel is known to have a broad range of activation temperatures (8 – 28 °C) (Story et al., 2003).

(D) Transgenic expression of TRPA-1(Cys), a worm TRPA-1 variant incorporated with three Cys residues extends lifespan at cold temperature. Lifespan: 15 °C.

(G) A schematic model illustrating a genetic pathway that promotes longevity at cold temperatures in C. elegans.

See also Table S1

It might be argued that the inability of TRPA channels to extend lifespan at warm temperatures could be due to some non-specific effects; for example, these channels might not be folded properly or expressed well at high temperatures. To address this concern, it would be necessary to activate these channels at warm temperatures to assess whether they are then able to extend lifespan just like they did at cold temperatures. However, we are unable to do so with worm TRPA-1, as no specific agonists have been found to activate this worm channel. Unlike worm TRPA-1, human TRPA1 can be activated by pungent chemicals, such as AITC (allyl isothiocyanate) which is the active ingredient of wasabi and mustard oil (Bandell et al., 2004; Jordt et al., 2004). The three Cys residues responsible for AITC activation of mammalian TRPA1 somehow are not conserved in worm TRPA-1 (Hinman et al., 2006; Macpherson et al., 2007). Nevertheless, this property of mammalian TRPA1 offers us an opportunity to address the aforementioned concern. We found that, while AITC itself did not notably affect the lifespan of wild-type worms, this agonist extended the lifespan of transgenic worms expressing human TRPA1 at warm temperature (Figure 7B). Remarkably, this agonist-triggered, human TRPA1-dependent lifespan extension at warm temperature also depended on PKC-2 and SGK-1, as well as DAF-16 (Figure 7C), suggesting that such lifespan extension is mediated by the same genetic pathway as that recruited by worm TRPA-1 to promote longevity at low temperatures. Thus, though human TRPA1 failed to promote longevity at warm temperature, it was nevertheless functionally expressed, can be activated with chemical agonists to extend lifespan, and apparently does so by recruiting the same genetic pathway as that employed by worm TRPA-1.

Encouraged by the above results, we engineered a worm TRPA-1 variant TRPA-1(Cys) by introducing three Cys residues at the same positions as those found in human TRPA1. While expression of TRPA-1(Cys) extended lifespan at low temperature (Figure 7D), the same transgene did not promote longevity at warm temperature (Figure 7E). This is in line with the data obtained with wild-type form of worm TRPA-1, indicating that the three Cys residues introduced did not globally affect the function of TRPA-1. Interestingly, though TRPA-1(Cys) failed to extend lifespan at warm temperature, the agonist AITC can promote the lifespan of the same transgenic worms at warm temperature (Figure 7E). Furthermore, this AITC-triggered lifespan extension at warm temperature also depended on PKC-2, SGK-1 and DAF-16 (Figure 7F), the same set of genetic components as those recruited by TRPA-1 to extend lifespan at low temperatures. These data indicate that the genetic pathway recruited by TRPA- 1 to extend lifespan at low temperatures is a build-in program. In other words, the fact that TRPA-1 cannot extend lifespan at warm temperatures is not because this genetic program is not present or defective at warm temperatures, but rather because the cold-sensitive channel TRPA-1 remains closed at warm temperatures. These data, together with the intrinsic temperature sensitivity of these channels reported by others, strongly suggest that the observed temperature dependence of TRPA channels in lifespan regulation is specific. Furthermore, the observation that human TRPA1 can functionally substitute for worm TRPA-1 in lifespan extension also raises the intriguing possibility that mammalian TRPA1 channel could be a potential target for developing anti-aging agents.

Discussion

The past two decades have witnessed a rapid progress in our understanding of how IIS, DR, mitochondria, and germ-line regulate lifespan (Fontana et al., 2010; Kenyon, 2010). By contrast, very little is known about how temperature reduction promotes longevity. In this study, we have characterized cold-dependent lifespan extension in C. elegans. Our results indicate that genetic programs actively contribute to lifespan extension at low temperatures. Apparently, at least in C. elegans, the extended lifespan observed at low temperatures cannot be simply explained by a reduced rate of chemical reactions. Thus, cold-dependent lifespan extension is not simply a pure, passive thermodynamic process in C. elegans. Notably, lifespan is also regulated at high temperatures (Lee and Kenyon, 2009). Given that the mechanisms regulating lifespan show striking conservation across phylogeny (Fontana et al., 2010; Kenyon, 2010), our work raises the possibility that a similar phenomenon may also occur in other organisms.

TRP channels and calcium signaling in lifespan regulation

Our data suggest a model in which low temperatures activate the cold-sensitive TRPA-1 channel, and the ensuing calcium influx via TRPA-1 then stimulates the calcium-sensitive PKC-2 which signals via SGK-1 to DAF-16/FOXO, leading to an increase in longevity (Figure 7G). It should be noted that this model proposes a genetic rather than biochemical pathway through which cold temperatures regulate lifespan. Future studies are needed to elucidate the detailed biochemical mechanisms. Notably, our model for the first time links calcium signaling to longevity. Though calcium is well known as a key second messenger implicated in a wide range of physiological processes, surprisingly, a direct role of calcium signaling in regulating lifespan has not been appreciated. As numerous neurotransmitters, neuropeptides and hormones, as well as many signaling pathways modulate calcium dynamics, it would be interesting to investigate whether they also regulate lifespan.

Our results suggest that TRPA-1 acts a thermo-sensor to detect temperature drop in the environment to initiate a pro-longevity genetic program. This uncovers a novel function for TRP family channels. Expression of TRPA-1 in the intestine or neurons is sufficient to promote lifespan, consistent with the view that the intestine and nervous system are the two key tissues in lifespan regulation (Kenyon, 2010). Interestingly, human TRPA1 can functionally substitute for its worm homologue in lifespan extension. Similar to worm TRPA-1, mammalian TRPA1 is also expressed in neurons as well as several nonexcitable tissues (Earley et al., 2009; Stokes et al., 2006). It will be interesting to test whether this channel regulates lifespan in mammals. As feeding the TRPA1 agonist AITC to transgenic worms expressing human TRPA1 can extend lifespan, one simple experiment would be to test whether AITC or other TRPA1 agonists can extend lifespan in mice.

Nevertheless, our studies do not exclude the contribution of additional mechanisms to lifespan extension at low temperatures. trpa-1 mutant worms, though short-lived at low temperatures, exhibit a lifespan comparable to that of wild-type worms at warm temperatures, suggesting that additional mechanisms must contribute. For example, some yet-to-be identified cold-sensitive proteins may function in parallel to TRPA-1. In addition, the classic mechanism, i.e. a reduced rate of chemical reactions at low temperatures, may also contribute. Future studies are needed to uncover other unknown genetic programs and identify additional components in the TRPA-1 pathway, which ultimately may lead to a thorough understanding of how cold temperatures promote longevity in C. elegans.

Intestine, a signaling hub in lifespan regulation, is a cold receptor

We showed that cold evokes a robust TRPA-1-dependent calcium response in the intestine, providing a cellular correlate for cold-induced lifespan extension. This result also identifies the intestine as a cold receptor, uncovering an unexpected role for the intestine in thermosensation. While neurons are well known to act as cold receptors in various organisms (Chatzigeorgiou et al., 2010; Gallio et al., 2011; Story et al., 2003), no non-excitable cells have been reported to respond to cold. This finding thus expands the repertoire of cold receptors from neurons to non-excitable cells. Though both the nervous system and intestine are considered as key tissues in lifespan regulation, the intestine plays a special role in that it integrates multiple pro- and anti-aging cues and also signals to other tissues to coordinate a body-wide response (Kenyon, 2010; Libina et al., 2003). The finding that the intestine responds to cold highlights the role of this tissue as a signaling hub in lifespan regulation.

It is worth noting that the intestine is also the fat tissue of C. elegans (Soukas et al., 2009). Interestingly, the fat tissue also plays an important role in lifespan control in mammals (Bluher et al., 2003), underscoring a general role of the fat/intestine tissue in aging. In mammals, both the white and brown fat tissues are distributed right underneath the skin and may undergo some level of fluctuations in temperature when exposed to cold environments, though the core body temperature likely remains constant. It is conceivable that such temperature fluctuations in the fat tissue may trigger aging-related signaling events. A similar phenomenon may occur in the sensory neurons with their nerve endings embedded in the skin, which can certainly sense temperature drop in the environment. Currently, lowering the whole body temperature of homeothermic animals, such as mouse, via transgenic approaches is known to extend lifespan (Conti et al., 2006); however, whether a simple cooling of their peripheral tissues (e.g. skin and fat) by exposing these animals to cold environments affects lifespan has not been examined. Our study may encourage investigators to explore this intriguing possibility.

Experimental procedures

Strains

Wild type: N2. TQ1516: trpa-1(ok999) x6 outcrossed. TQ2571: pkc-2(ok328) x6 outcrossed. TQ1697: xuEx619[trpa-1(genomic) + sur-5::gfp]. TQ1643: xuEx601[Pges-1::trpa-1::SL2::yfp + Punc-122::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). TQ2014: daf-16(mgDF47); xuEx601. TJ356: zIs356[daf-16::gfp + roller]. TQ2505: zIs356; xuEx785. TQ1939: xuEx676[Pges-1::trpa-1(E1018A)::SL2::yfp + Punc-122::DsRed]. TQ2674: pkc-2(ok328);xuEx601. TQ2230: pkc-2(ok328); xuEx619. TQ2789: xuEx913[Pges-1::pkc-2::SL2::mCherry]. TQ2845: daf-16(mgDf47); xuEx913. TQ2853: zIs356; xuEx911[Pges-1::pkc-2::SL2::mCherry]. BQ1: akt-1(mg306). TQ2212: akt-1(mg306); xuEx601. TQ2927: akt-2(ok393) x4 outcrossed. TQ2928: akt-2(ok391); xuEx601. TQ2746: cmk-1(oy21) x4 outcrossed. TQ2749: cmk-1(oy21); xuEx601. TQ2752: cmk-1(oy21); xuEx619. TQ1996: unc-43(n498,n1186) x4 outcrossed. TQ2351: unc-43(n498,n1186); xuEx601. All RNAi clones were from the Ahringer library and confirmed by sequencing.

Lifespan assay

Unless indicated otherwise, all lifespan studies were conducted at 20 °C (Hsu et al., 2009). In all experiments, the first day of adulthood was scored as day 1. For each lifespan assay, 80–100 worms were included and transferred every 2–3 days to fresh 60 mm NGM (nematode growth medium) plates at a density of 10 worms per plate. Worms were censored if they crawled off the plate, exploded or bagged. FUdR was included in assays involving sgk-1 mutant worms which show a defect in egg-laying. For RNAi experiments, NGM plates were supplemented with carbenicillin (25 µg/ml) and IPTG (1 mM). HT115 bacteria containing either empty vector L4440 or RNAi plasmid were seeded on RNAi plates 2 days before experiments. All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc.) and IBM SPSS Statistics 19 (IBM, Inc.). P values were calculated using the log-rank (Kaplan- Meier) method.

Calcium imaging

Calcium imaging was performed on an Olympus upright microscope (BX51WI) under a 60x objective using a protocol described previously (Feng et al., 2006; Li et al., 2006). Worms were glued on an agarose pad and incubated in bath solution: 10 mM Hepes [pH 7.4], 5 mM KCl, 145 mM NaCl, 1.2 mM MgCl2, 2.5 mM CaC12, and 10 mM glucose. Bath solution of varying temperatures was perfused towards the animal. Its temperature was controlled by a bi-polar in-line temperature controller (Warner Instruments) triggered by an EPC10 amplifier. Images were acquired with a Roper CoolSnap CCD camera and processed by MetaMorph (Molecular Devices, Inc.). G-CaMP and DsRed fluorescence was excited at 484 nm and 535 nm, respectively, and peak percentage change in the ratio of G-CaMP/DsRed fluorescence was quantified.

Quantitative RT-PCR

Total RNA was extracted using TRI Reagent (Life Technologies, CA) from ~150 worms. The relative amount of mRNA of daf-16 target genes was analyzed by qPCR using CYBR Green (Life Technologies, CA) according the protocol provided by the manufacturer. act-1 (actin) was used as an internal reference for normalization. ΔΔCt method was used to analyze the qPCR data.

Supplementary Material

01
02

  1. Cold-dependent lifespan extension is not a passive thermodynamic process

  2. A genetic program actively contributes to lifespan extension at cold temperatures

  3. This program includes a cold-sensitive TRP channel, Ca2+ influx, PKC, SGK, and FOXO

  4. The intestine, a non-excitable tissue, can also act as a cold receptor

Acknowledgments

We thank Ao-Lin Hsu for providing reagents and critically reading the manuscript, Jiandie Lin, Xiaowei Chen and Zhaoyu Li for technical assistance, and Sean Curran and Gary Ruvkun for daf-16 strains. Some strains were obtained from the CGC and Knockout Consortiums in the U.S.A. and Japan. R.X. was supported by an AHA postdoctoral fellowship. This work was supported by the Chinese 111 project (J.L.) and grants from the NIGMS (X.Z.S.X), NIA (A.-L.H.), NSFC (T.X. and X.Z.S.X), and Pew scholar program (X.Z.S.X).

Footnotes

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Supplementary Materials

01
NIHMS437620-supplement-01.pdf (2.4MB, pdf)
02
NIHMS437620-supplement-02.xls (52KB, xls)

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