Synergism between soluble guanylate cyclase signaling and neuropeptides extends lifespan in the nematode Caenorhabditis elegans

Summary Oxygen (O2) homeostasis is important for all aerobic animals. However, the manner by which O2 sensing and homeostasis contribute to lifespan regulation is poorly understood. Here, we use the nematode Caenorhabditis elegans to address this question. We demonstrate that a loss‐of‐function mutation in the neuropeptide receptor gene npr‐1 and a deletion mutation in the atypical soluble guanylate cyclase gcy‐35 O2 sensor interact synergistically to extend worm lifespan. The function of npr‐1 and gcy‐35 in the O2‐sensing neurons AQR, PQR, and URX shortens the lifespan of the worm. By contrast, the activity of the atypical soluble guanylate cyclase O2 sensor gcy‐33 in these neurons is crucial for lifespan extension. In addition to AQR, PQR, and URX, we show that the O2‐sensing neuron BAG and the interneuron RIA are also important for the lifespan lengthening. Neuropeptide processing by the proprotein convertase EGL‐3 is essential for lifespan extension, suggesting that the synergistic effect of joint loss of function of gcy‐35 and npr‐1 is mediated through neuropeptide signal transduction. The extended lifespan is regulated by hypoxia and insulin signaling pathways, mediated by the transcription factors HIF‐1 and DAF‐16. Moreover, reactive oxygen species (ROS) appear to play an important function in lifespan lengthening. As HIF‐1 and DAF‐16 activities are modulated by ROS, we speculate that joint loss of function of gcy‐35 and npr‐1 extends lifespan through ROS signaling.


Introduction
To survive, animals must sense key environmental factors, including temperature, food, and oxygen (O 2 ). Sensory information then regulates homeostatic responses to maintain physiological parameters within a narrow range . O 2 and reactive oxygen species (ROS) are important for animal viability but may be toxic when misregulated (Halliwell & Gutteridge, 2006).
Previous studies showed that, in addition to sGC signaling, neuropeptide signaling plays a critical role in C. elegans O 2 sensing (Gray et al., 2004). N2 worms that express the gain-of-function allele of neuropeptide receptor 1, npr-1(215V), do not avoid hyperoxia on food. However, animals bearing the weaker natural allele npr-1(215F) or the loss-of-function npr-1(ad609) allele show strong 21% O 2 avoidance on food and aggregate on the bacterial lawn border, where there is a thicker growth of bacteria and, therefore, less O 2 (~13%) (Gray et al., 2004). The aggregation of worms may further decrease the O 2 concentration inside the clump and so create a preferable O 2 concentration of around 8%. Therefore, N2 worms may experience higher O 2 levels compared with npr-1(ad609) animals under standard laboratory growth conditions. In this study, we explored the function of O 2 sensing and neuropeptide signaling in lifespan regulation.

NPR-1 activity reduces Caenorhabditis elegans lifespan
The gain-of-function npr-1(215V) allele appears to have arisen during domestication of the N2 strain to laboratory conditions (McGrath et al., 2009). As npr-1(215V) activity suppresses the natural hyperoxia avoidance response on bacteria, we asked whether N2 worms suffer from misregulated O 2 -homeostatic responses and therefore have a shorter lifespan than the aggregating strain npr-1(ad609). We measured the lifespans of N2 and npr-1(ad609) worms on food. The lifespan of N2 worms was slightly but significantly shorter than that of npr-1(ad609) animals ( Fig. 1A; P = 0.0023), suggesting that NPR-1(215V) activity is harmful to C. elegans longevity.
A previous study showed that the tax-4 (ky89) and tax-4(p678) lossof-function alleles significantly lengthen the lifespan of N2 worms at 20°C (Apfeld & Kenyon, 1999). However, our experiments show that tax-4(ks28) mutants and N2 worms have similar lifespans (P = 0.6112, Fig. S1D, Supporting information). Apart from using a different loss-of-function allele of tax-4, a significant difference between our experiments and previous studies is that we did not use the thymidylate synthetase inhibitor FUDR in our lifespan assays. Therefore, we investigated whether the function of tax-4 in lifespan regulation is modulated by FUDR. FUDR significantly increased the lifespan of tax-4(ks28) mutants compared with control tax-4(ks28) mutants that grew without FUDR (P < 0.0001, Fig. S1D, Supporting information). In addition, a significant lengthening of the lifespan of N2 worms was observed (Fig. S1E, Supporting information). However, the lifespans of tax-4(ks28) mutants and N2 worms that grew on FUDR were not significantly different (P = 0.6091). Therefore, while our results show that FUDR significantly affects the function of tax-4 in lifespan regulation, they do not recapitulate previous results. As FUDR inhibits both DNA synthesis in worms and bacterial proliferation (Portal-Celhay et al., 2012), we explored whether the effect of tax-4 on lifespan is modulated by bacterial viability. We repeated the lifespan experiments using UV-killed bacteria as a food source. The lifespan of tax-4(ks28) mutants that were grown on dead bacteria was similar to tax-4(ks28) mutants that grew on live bacteria and FUDR (P = 0.0858), suggesting that FUDR extends the lifespan of tax-4(ks28) mutants by suppressing bacterial toxicity. Finally, under this experimental condition the lifespan of N2 worms was significantly lengthened (P < 0.0001) and was similar to the lifespan of tax-4(ks28) (Fig. S1F, Supporting information), suggesting that tax-4 may be important in the defense mechanism against bacterial toxicity.
Temperature and food quality affect the function of npr-1 and gcy-35 in lifespan regulation A previous study, conducted on the N2 background, showed that gcy-33 deletion extends lifespan, whereas deletion of gcy-35 shortens lifespan (Liu & Cai, 2013). However, in our experiments, the same gcy-33 and gcy-35 deletion alleles did not affect N2 worm lifespan significantly ( Fig. 2A). As Liu & Cai used different experimental conditions from ours (our assays were performed on live OP50 bacteria at 21°C, whereas Liu & Cai's experiments used UV-killed OP50 bacteria at 25°C), we asked whether temperature and bacterial viability could explain the difference between the results. We measured lifespan in three growth conditions: 21°C/UV-killed OP50; 25°C/live OP50; and 25°C/UV-killed OP50. All strains lived longest on UV-killed OP50 at 21°C (Fig. 2E). These results support previous studies that showed that live OP50 shortens C. elegans lifespan (Garigan et al., 2002). Importantly, under this experimental condition, gcy-33 and gcy-35 mutants had similar lifespans to N2 controls. However, N2 worms lived significantly longer compared with npr-1(ad609) worms (P = 0.0098), suggesting that thriving on dead OP50 bacteria requires NPR-1(215V) activity. Growing the worms on live OP50 at 25°C shortened the lifespan of all strains (Fig. 2F). The lifespans of gcy-33, gcy-35, and npr-1(ad609) mutants grown in this environment were similar to N2 controls. Finally, gcy-33 and gcy-35 mutants had shorter lifespans than N2 controls on dead OP50 at 25°C (Fig. 2G). Notably, gcy-35;npr-1(ad609) worms lived longer (compared with the other strains) on live and dead OP50 at 21°C (Fig. 2B,E) and on live OP50 at 25°C (Fig. 2F). However, when grown at 25°C on dead OP50, their lifespan was similar to N2 animals (Fig. 2G). Intriguingly, in this growth condition, npr-1(ad609) animals lived significantly longer compared with the other strains, indicating that gcy-35 activity is essential to thriving in this environment. Taken together, our results show that the synergistic effect of npr-1 and gcy-35 loss of function on lifespan is modulated by both temperature and bacterial viability. Moreover, the discrepancy between our data and that of and Liu & Cai could be attributed to experimental conditions in the case of gcy-35 mutants, as our findings at 25°C on dead OP50 are in agreement with theirs. However, the discrepancy between the gcy-33 mutant lifespan data could not be explained by differences in temperature or bacteria viability, as the gcy-33 deletion did not lengthen the lifespan of N2 worms (nor npr-1(ad609) or gcy-35;npr-1(ad609) mutants) in any condition we tested.
Joint loss of function of gcy-35 and npr-1 does not extend lifespan at 15°C Our results show that joint loss of function of gcy-35 and npr-1 affects lifespan in a temperature-and food quality-dependent way. To further explore this observation, we measured the lifespan of N2, npr-1(ad609), and gcy-35;npr-1(ad609) worms while feeding on either live or UV-killed OP50 at 15°C. The three strains had similar lifespans at 15°C when grown on live OP50 (Fig. 2H). Notably, at 15°C the lifespans of N2 and npr-1(ad609) worms were significantly increased (compared with 21°C, P < 0.0001), but that of gcy-35;npr-1(ad609) animals was not affected, suggesting that low temperature may recapitulate the effect of gcy-35 and npr-1 joint loss of function on lifespan. The lifespan of npr-1(ad609) worms on UV-killed OP50 at 15°C was significantly longer than both N2 and gcy-35;npr-1(ad609) worms ( Fig. 2I, P = 0.0002 and P = 0.0003, respectively), further supporting our conclusion that gcy-35 and npr-1 function in lifespan regulation is modulated by temperature and bacterial viability.
RIA is important for the extended lifespan of gcy-35;npr-1 (ad609) mutants Previous studies suggested that the RIA interneuron integrates information about temperature from the AFD and AWC neurons, and information about O 2 from the URX and BAG neurons ( (Kimata et al., 2012;Luo et al., 2014) for illustration, see Fig. 2J). Therefore, we hypothesized that RIA is important for the extended lifespan of npr-1 (ad609) and gcy-35;npr-1(ad609) mutants. To explore this, we genetically ablated RIA by expressing the death activator egl-1 with the promoter region of the RIA-specific gene glr-3 (Brockie et al., 2001). The ablation of RIA shortened the lifespan of gcy-35;npr-1(ad609) mutants so it was similar to that of npr-1(ad609) worms (P = 0.6392, Fig. 2K), suggesting that RIA function is essential for the synergistic effect of gcy-35 and npr-1 loss of function on lifespan. Moreover, the lifespans of RIA genetically ablated N2 and npr-1(ad609) transgenic worms were similar (P = 0.6906) and significantly shorter than control worms (P < 0.0001), suggesting that RIA is also important for the individual effect of npr-1 loss of function on lifespan. Notably, the lack of difference between the lifespans of N2 and npr-1(ad609) worms with ablated RIA cannot be attributed to suppression of behavioral O 2 responses, because the ablation of RIA did not reduce the accumulation of npr-1(ad609) worms on the bacterial lawn border (Fig. S2F,  Supporting information).
These results show that the RIA interneurons play an important role in lengthening the lifespan of npr-1(ad609) and gcy-35;npr-1(ad609) worms and so suggest that the effect of joint loss of function of npr-1 and gcy-35 on lifespan regulation is mediated by neuronal communication.
The extended lifespan of npr-1(ad609) and gcy-35;npr-1 (ad609) mutants cannot be explained by decreased metabolism Some longed-lived mutants, such as clk-1 and daf-2 (mutants with reduced mitochondrial activity and insulin signaling, respectively), have reduced metabolic rates compared with N2 worms (Van Voorhies & Ward, 1999). Therefore, we asked whether the extended lifespan of gcy-35;npr-1(ad609) mutants is associated with decreased metabolic activity. We measured the locomotory activity of worms on both solid medium and liquid medium (speed and thrashing assays respectively), feeding (pumping assays), development, egg laying rate, O 2 consumption, and ATP levels (Fig. 4). These parameters (apart from development) were measured on the first and fifth days of adulthood, which precede the rapid decline of N2 worm viability. The locomotory activity and pharyngeal pumping of N2, npr-1(ad609), and gcy-35; npr-1(ad609) worms were similar on days 1 and 5 ( Fig. 4A-C). By contrast, N2 worms developed slightly faster compared with the other strains ( Fig. 4D) and had a higher egg laying rate on day 1, but not on day 5 (Fig. 4E). Notably, npr-1(ad609) worms developed slightly faster than gcy-35;npr-1(ad609) worms, but laid fewer eggs on day 1. Therefore, although our results suggest that NPR-1(215V) and GCY-35 signaling is important for both development and egg-laying regulation, the small differences between the strains probably cannot explain the lengthened lifespan of gcy-35;npr-1(ad609) mutants. To directly compare the metabolic activity of N2, npr-1(ad609), and gcy-35;npr-1(ad609) animals, we measured O 2 consumption and ATP levels in the three strains (Fig. 4F,G). Consumption of O 2 and ATP levels of the N2, npr-1(ad609), and gcy-35;npr-1(ad609) were similar on both days 1 and 5, indicating that the metabolic activity of these strains is similar.
The extended lifespan of gcy-35;npr-1(ad609) mutants is modulated by the activity of DAF-16 and HIF-1. As these transcription factors are important for protection against unfolded protein toxicity, heat, oxidative damage, and pathogenic bacteria (Back et al., 2012), we asked whether these animals live longer because they are more stress resistant. To address this question, we exposed N2, npr-1(ad609), and gcy-35;npr-1(ad609) worms on their first and fifth days of adulthood to various stresses and measured survival. Exposure to Pseudomonas aeruginosa PA14 bacteria induced rapid death in all three strains. However, the survival of gcy-35;npr-1(ad609) mutants was higher in both day 1 and day 5 of adulthood compared with N2 and npr-1(ad609) worms (Fig. 6A), indicating that joint loss of function of gcy-35 and npr-1 provides protection against PA14 toxicity. Similarly, gcy-35;npr-1(ad609) mutants were more resistant to UV stress than N2 and npr-1(ad609) worms (Fig. 6B). Interestingly, a previous study showed a striking similarity between the transcription of genes involved in DNA damage and innate immunity (Ermolaeva et al., 2013), suggesting that joint loss of function of gcy-35 and npr-1 induces an innate immunity response that extends the lifespan of gcy-35;npr-1(ad609) mutants. The increased resistance of gcy-35;npr-1(ad609) mutants to stress was specific to PA14 and UV. gcy-35;npr-1(ad609) mutants were significantly more sensitive to heat stress than N2 and npr-1(ad609) worms on the first day of adulthood (Fig. S4A, Supporting information). However, on day 5 of adulthood the three strains showed similar survival rates, which were significantly higher than day 1, suggesting that older worms are generally more resistant to heat stress than young worms. Tunicamycin prevents the first step of N-linked glycosylation of proteins in the ER so inducing accumulation of unfolded proteins and activating the unfolded protein response of the endoplasmic reticulum (UPR ER ) (Taylor & Dillin, 2013). N2, npr-1(ad609), and gcy-35;npr-1(ad609) worms showed similar survival in response to tunicamycin (both on days 1 and 5 of adulthood, Fig. S4B, Supporting information). Finally, to explore whether gcy-35;npr-1(ad609) mutants are more resistant to mitochondrial oxidative stress, we exposed the three strains to 200 mM paraquat, a superoxide generator (Halliwell & Gutteridge, 2006), and measured their survival over 24 h. The survival of N2 and npr-1(ad609) worms (on days 1 and 5) was similar to gcy-35;npr-1(ad609) mutants (Fig. S4C, Supporting information). Therefore, our results show that gcy-35;npr-1 (ad609) mutants are not generally resistant to stress, but rather show specific resistance to both PA14 and UV toxicity.
ROS is required for the extended lifespan of gcy-35;npr-1 (ad609) mutants As ROS signaling appears to be important for tolerance to UV and pathogenic bacteria (Hideg et al., 2013;Schieber & Chandel, 2014), we explored whether ROS is needed for the extended lifespan of gcy-35;npr-1(ad609) mutants, by performing lifespan experiments in the presence of antioxidants. We used the superoxide scavenger tempol (5 mM), and butylated hydroxyanisole (BHA). Tempol treatment significantly reduced the lifespan of N2, npr-1(ad609), and gcy-35; npr-1(ad609) worms (Fig. 6C), suggesting that either excessive scavenging of superoxide is generally damaging to C. elegans or long exposure to 5 mM tempol induces a toxic effect that is not associated with ROS. That being said, tempol treatment affected the three strains differently. The lifespans of npr-1(ad609) and gcy-35;npr-1(ad609) mutants were similar, suggesting that ROS is required for the positive synergistic effect of npr-1 and gcy-35 loss of function on lifespan. However, npr-1(ad609) mutants lived longer than N2 worms in the presence of tempol (Fig. 6C), suggesting that the effect of npr-1 on lifespan is not mediated by ROS. BHA significantly increased the lifespan of N2 worms (Fig. 6D, P = 0.0394) compared with N2 worms living on NGM plates with ethanol (the concentration of ethanol was similar in all plates). However, npr-1(ad609) worms growing on BHA lived longer than N2 worms with BHA (P = 0.0272). These results are in agreement with the tempol experiments that suggested that the effect of npr-1 on lifespan is not mediated by ROS. The lifespan of gcy-35;npr-1(ad609) animals growing with BHA was longer than the lifespan of npr-1(ad609) worms with BHA. However, the difference was much smaller and barely reached significance (P = 0.0438). Therefore, although the BHA results are not as conclusive as the tempol results, they do suggest that the synergistic effect of gcy-35/ npr-1 loss of function on lifespan in regulated by ROS.
Paraquat extends the lifespan of N2 worms but reduces the lifespan of both npr-1(ad609) and gcy-35;npr-1(ad609) mutants Previous studies showed that low levels of the superoxide generator paraquat lengthen the lifespan of N2 worms (Yang & Hekimi, 2010). Intriguingly, either a small increase or decrease in paraquat concentration can diminish the beneficial effect, suggesting that ROS act within a narrow range of concentrations to lengthen lifespan. Our studies suggest that the extended lifespan of gcy-35;npr-1(ad609) mutants is mediated by ROS. Therefore, we hypothesized that low levels of paraquat will not further extend the lifespan of gcy-35;npr-1 (ad609) mutants and may even induce a toxic effect. We measured the lifespan of N2, npr-1(ad609), and gcy-35;npr-1(ad609) worms grown on NGM plates supplemented with 0.1 mM paraquat, a concentration shown to maximize the lifespan extension of N2 worms (Yang & Hekimi, 2010). As expected, paraquat treatment lengthened the lifespan of N2 worms significantly (Fig. 6E, P < 0.0001), supporting the results from previous studies. By contrast, it shortened the lifespans of npr-1(ad609) and gcy-35;npr-1(ad609) mutants to N2 control level, suggesting that the level of ROS in these worms exceeded the beneficial level and elicited a toxic effect. Together, these results further support the hypothesis that ROS act within a narrow range of concentrations to lengthen worm lifespan and that excessive ROS is damaging.
Nevertheless, the conclusion from these studies is that ROS levels do not correlate with extended lifespan of gcy-35;npr-1(ad609) mutants. As degradation of ROS could be different in N2, npr-1(ad609), and gcy-35;npr-1(ad609) worms, we measured the oxidation of proteins on the  -1(ad609), and gcy-35;npr-1(ad609) worms at days 1 and 5. Asterisks indicate significance for comparisons with N2 worms at days 1 and 5 (two-way ANOVA with Bonferroni post-test). (G) Protein oxidation measurements by Oxyblot. Quantification of Western blot analysis from at least four independent biological repeats, for each condition. Two-way ANOVA with Bonferroni post-test. (H) Gene expression measurements in N2, npr-1(ad609), and gcy-35;npr-1(ad609) worms that were either grown on regular NGM plates (left panel) or on NGM plates containing 5 mM Tempol (right panel). Gene expression was measured by qPCR. Each measurement represents, at least, three biological repeats.
Lifespan regulation by cGMP and NPR-1 signaling, R. Abergel et al. first and fifth days of adulthood. Protein oxidation, as monitored by carbonyl accumulation, was similar in all strains on days 1 and 5 (Figs 6G and S4D, Supporting information), suggesting that ROS do not accumulate to levels that cause this commonly used gauge of protein oxidation.
In conclusion, our experiments failed to detect any correlation between the extended lifespan of gcy-35;npr-1(ad609) mutants and ROS levels or protein oxidation. However, our antioxidant, paraquat, DAF-16, and HIF-1 experiments strongly suggest that ROS is important for the extended lifespan of gcy-35;npr-1(ad609) mutants. A potential explanation for this discrepancy is that ROS are needed at small quantities in specific cells (e.g., the O 2 -sensing neurons). However, future studies are needed to explore this hypothesis.

Discussion
ROS signaling is essential for the extended lifespan of gcy-35; npr-1(ad609) animals Harman's theory of aging (Harman, 1956) suggested that the creation of ROS via aerobic respiration underlies the development and progression of aging. However, studies from the last decade have challenged this theory. For example, C. elegans lacking all superoxide dismutases (sod-1sod-5) live longer than N2 animals, and chemical agents that enhance mitochondrial ROS increase lifespan (Van Raamsdonk & Hekimi, 2012). Our results suggest that tight regulation of ROS level is essential for the extended lifespan of gcy-35;npr-1(ad609) mutants (Fig. 6). Both ROS scavenging by antioxidants and increased ROS by sublethal level of paraquat shorten the lifespan of gcy-35;npr-1|(ad609) worms. This hypothesis is further supported by the requirement of DAF-16 and HIF-1 for the extended lifespan of gcy-35;npr-1(ad609) animals (Fig. 5), as these transcription factors are regulated by ROS (Hwang & Lee, 2011;Zou et al., 2013). Notably, the interpretation of our results should be made with caution, because we failed to detect any significant difference in either ROS accumulation or oxidative damage in gcy-35; npr-1(ad609) worms compared with both N2 and npr-1(ad609) animals. That being said, we suspect that ROS act in specific neurons to regulate lifespan. Therefore, more specific and sensitive methods are needed to detect these localized changes.
Despite the above caveats, we suggest the following working model. The function of NPR-1(215V) and GCY-35/GCY-36 in the O 2sensing neurons AQR, PQR, and URX inhibits lifespan extension. By contrast, GCY-33 signaling through TAX-2/TAX-4 triggers the secretion of neuropeptide/neurotransmitter that increases the level of ROS in either the O 2 -sensing neurons themselves, downstream neurons, or even non-neuronal tissues. The increase in ROS levels activates the HIF-1 and DAF-16 transcription factors which induce innate immunity. This induction of innate immunity increases lifespan. Although much of the details in this model are still not known, for example, the identity of its interacting partner of GCY-33 in AQR, PQR, and URX, and where DAF-16 and HIF-1 function is needed for the life extension, this model provides a framework for future studies to identify the 'anti-aging' neurotransmitter/neuropeptide and to elucidate how innate immunity genes prolong lifespan. In this respect, it is interesting to note that a previous study showed that the expression of the innate immunity genes dod-17, C32H11.4, and ZK6.11 is substantially downregulated in daf-10(m79) worms (Gaglia et al., 2012). Moreover, the high sensitivity of daf-10(m79) animals to PA14 is rescued by the gcy-35(ok769) mutation. DAF-10 is important for the activity of ciliated neurons, such as AQR, PQR, and BAG. Therefore, in the future it will be intriguing to explore whether daf-10 function in AQR, PQR, and BAG is important for the extended lifespan of gcy-35;npr-1(ad609) mutants and whether this function is ROS dependent.

Experimental procedures
We used standard molecular biology and genetic protocols. A detailed description of strains and oligonucleotides used is found in Table S10 (Supporting information) and Supplemental Experimental Procedures.

Lifespan analysis
All lifespan assays were conducted at 21°C and started with synchronized young adults, unless otherwise mentioned. Statistical analysis on lifespan survival curves was performed using the log-rank (Mantel-Cox) test with Prism 6 software, and the results are displayed in Tables S1-S9 (Supporting information). Further details are found in Supplemental Experimental Procedures.

Supporting Information
Additional Supporting Information may be found online in the supporting information tab for this article.

Fig. S3
The function of NPR-1 in lifespan regulation is modulated by neuropeptide/neurotransmitter signaling (related to Figure 3).

Fig. S4
Joint loss-of-function of npr-1 and gcy-35 does not increase tolerance to heat, ER UPR, or mitochondrial UPR stress (related to Figure 6).

Table S1
Numerical values for data plotted in Fig. 1.

Table S2
Numerical values for data plotted in Fig. S1. Lifespan regulation by cGMP and NPR-1 signaling, R. Abergel et al.   Fig. S2.

Table S5
Numerical values for data plotted in Fig. S3.

Table S6
Numerical values for data plotted in Fig. 3.

Table S7
Numerical values for data plotted in Fig. 5.

Table S8
Numerical values for data plotted in Fig. 6.

Table S9
Numerical values for data plotted in Fig. S4. Table 10 List of primers used for npr-1, gcy-35 and egl-1 sequencing, primers for cell specific RNAi by PCR fusion, and quantitative RT-PCR experiments. Appendix S1 Supplemental Experimental Procedures.