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

Abstract

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.

Keywords: Caenorhabditis elegans; NPR-1; lifespan; oxygen sensing; reactive oxygen species; soluble guanylate cyclase.

Figure 1

NPR‐1 and GCY‐35 regulate Caenorhabditis elegans lifespan. (A–E, G) Survival curves comparing the lifespans of worm strains. These experiments were performed at 21°C on live OP50. (F) Speed measurements. The speed of worms was measured at 21% and 7% O₂ in the presence of OP50. Asterisks indicate significance for comparisons with npr‐1 animals’ speed at 21% O₂, Kruskal–Wallis test with Dunn's post‐test. Number sign indicates significance between speeds, within a strain. Unpaired t‐test with Welch's correction. n = 6 or more assays performed over at least 3 days. ** P < 0. 01, *** P < 0.001, ##P < 0. 01, ####P < 0.0001, NS, nonsignificant, Error bars represent SEM.

Figure 2

GCY‐31 and GCY‐33 are important for gcy‐35;npr‐1(ad609) animals’ extended lifespan. (A–D, K) Survival curves comparing the lifespans of worm strains at 21°C on live OP50. (C) Survival curves comparing the lifespan of gcy‐35;npr‐1(ad609) mutants to gcy‐35;npr‐1(ad609) transgenic worms expressing gcy‐33 RNAi in AQR, PQR, and URX (under the gcy‐37 promoter) or in BAG (under the flp‐17 promoter). (E–I) Survival curves comparing the lifespans of worm strains at different growth conditions, as indicated by the labels above the graphs. (J) Circuit diagram of neurons with synaptic connections to RIA.

Figure 3

Neurotransmitter/neuropeptide signaling regulate the lifespan of gcy‐35;npr‐1(ad609) mutants. (A) Survival curves comparing the lifespans of worm strains at 21°C on live OP50 at 21% O₂. (B–D) Survival curves comparing the lifespans of worm strains at different O₂ levels, as indicated by the labels above the graphs. (E) Bar graph comparing mean lifespan at different O₂ concentrations. The mean lifespan data for 1%, 11%, and 35% O₂ were taken from C–E, respectively. Data for 21% O₂ lifespan measurements were taken from Fig. 1B. Asterisks indicate significance for comparisons with N2's lifespan at each O₂ level. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS (not significant). Error bars represent SEM.

Figure 4

NPR‐1 and GCY‐35 effects on behavior, development, O₂ consumption, and ATP levels. All experiments (apart from D) were performed on the first and fifth days of adulthood. (A, B) The speed and thrashing measurement were performed in the absence of bacteria. (C) Pharyngeal pumping behavior. (D) The development of worms was monitored from the L1 to the adult stage. Worms that had at least one egg in their bodies were counted as adults. Asterisks indicate significance for comparisons with N2 worms (one‐way ANOVA with Bonferroni post‐test). (E) Egg‐laying measurements. Asterisks indicate significance for comparisons with N2 worms at days 1 and 5 (two‐way ANOVA with Bonferroni post‐test). (F, G) O₂ consumption and ATP measurements. One‐way ANOVA with Tukey's multiple comparisons post‐test. All assays were performed over the course of 3 days. The number of assays and the number of worms tested in each assay are indicated in Table S11 (Supporting information). Error bars represents SEM. ****P < 0.0001, NS (not significant).

Figure 5

IIS and HIF‐1 signaling interact genetically with npr‐1 and gcy‐35. (A–D) Survival curves comparing the lifespans of worm strains at 21°C on live OP50. One‐way ANOVA with Tukey's multiple comparisons post‐test. NS (not significant). Error bars represents SEM.

Figure 6

Survival assays of N2, npr‐1(ad609), and gcy‐35;npr‐1(ad609) animals. (A) PA14 killing assays (B) Survival after UV exposure. The assays were performed on days 1 and 5 of adulthood. (C, D) Lifespan experiments in the presence of the antioxidants tempol (5 mM) and BHA (25 μm). (E) Lifespan experiments in the presence of 0.1 mm paraquat. (F) Bar graph comparing the levels of ROS in N2, npr‐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.