ADF serotonergic neurons are necessary and sufficient to extend lifespan downstream of the hypoxic response.

(A) Diagram of the conserved hypoxic response and genetic approaches to activate it, vhl-1 knockdown and HIF-1 stabilization. (B-D) Survival curves of WT, hif-1(ia4) knockout, hif-1(ia4); ADF::HIF-1S (B), hif-1(ia4); NSM::HIF-1S (C), and hif-1(ia4); HSN::HIF-1S (D) worms. N ≥ 256 (B), N ≥ 188 (C), and N ≥ 188 (D) worms per condition. (E) Survival curves of WT and ADF:tph-1 knockout worms on empty vector (EV) or vhl-1 RNAi. N ≥ 143 worms per condition. (F) Survival curves of WT and NSM:tph-1 knockout worms on empty vector (EV) or vhl-1 RNAi. N ≥ 193 worms per condition. (G) Survival curves of WT and tph-1(mg280); ADF:tph-1 rescue worms on empty vector (EV) or vhl-1 RNAi. N ≥ 178 worms per condition. (H) Survival curves of WT and tph-1(mg280); NSM:tph-1 rescue worms on empty vector (EV) or vhl-1 RNAi. N ≥ 160 worms per condition. (I) Gene expression of fmo-2 in WT and hif-1(ia4); ADF:HIF-1S worms. Significance in panels B-H is from a log-rank test comparing median survival. Significance in panel I is from a Student’s t-test (unpaired, two-tailed). In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

tph-1 is required for vhl-1 mediated longevity.

(A) Gene expression of vhl-1 in worms on vhl-1 RNAi compared to control worms on empty vector (EV) RNAi for two generations. N ≥ 200 worms per replicate, or 1200 worms per condition. The top of the bar represents the mean of the population and error bars indicate standard error of the mean (SEM). (B) Survival curve of WT, vhl-1(ok161), tph-1(mg280), and vhl-1(ok161); tph-1(mg280) worms. N ≥ 338 worms per condition. Significance is from a log-rank test comparing median survival. In all panels, NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. Three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons.

ser-7 expression in the GABAergic RIS neuron is required for hypoxia to extend lifespan.

(A) Quantification of fmo-2p::mCherry (WT positive control), fmo-2p::mCherry; ser-7(tm1325) knockout (negative control), and fmo-2p::mCherry; ser-7(tm1325) knockouts with ser-7 rescued in the whole body (ser-7p::ser-7), interneurons (glr-1p::ser-7), bioaminergic neurons (cat-1p::ser-7), glutamatergic neurons (eat-4p::ser-7), GABAergic neurons (unc-47p::ser-7), GABAergic motor neurons (unc-25p::ser-7), sensory neurons (osm-6p::ser-7), cholinergic motor neurons (acr-2p::ser-7), cholinergic neurons (unc-17p::ser-7), M3 & M4 neurons (ceh-28p::ser-7), and the intestine (vha-6p::ser-7) on empty vector (EV, WT control shown) or vhl-1 RNAi (all strains shown). Bar height indicates the mean fluorescence on vhl-1 RNAi of each genotype normalized to the empty vector control value from that genotype. The dashed line indicates the vhl-1-mediated fmo-2 induction of WT positive control. N ≥ 45 worms per condition. Error bars indicate SEM. Significance is from a two-way ANOVA (fmo-2 induction ∼ genotype*RNAi) and post-hoc Tukey HSD test (unpaired, two-tailed). Stars signify results from this post-hoc test comparing the fmo-2 induction of each strain on EV RNAi the fmo-2 induction of that strain on vhl-1 RNAi. (B) A table showing the 10 ser-7 expressing candidate neurons and their expression patterns among the ser-7 cell-specific strains that successfully rescued fmo-2 induction in Fig. 2A. Pink rows indicate rescues that full restored vhl-1 mediated fmo-2 induction, orange rows indicate rescues that partially restored vhl-1-mediated fmo-2 induction, and gray rows signify unsuccessful rescue constructs from the data in Fig. 2A. (C) Survival curve of WT and RIS ablation (Ex[srsx-18p::caspase-3(p12)-nz]; Ex[srsx-18p::cz-caspase-3 (p17)]; srsx-18p::GFP) worms on empty vector (EV) and vhl-1 RNAi. N ≥ 192 worms per condition. (D) Survival curve of WT and ser-7 (tm1325); ser-7 rescue in the RIS neuron (flp-11p::ser-7) worms on EV and vhl-1 RNAi. N ≥ 144 worms per condition. Significance in panels C-D is from a log-rank test comparing median survival. In all panels, NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons.

ser-7 expression in each ser-7 expressing neuron is rescued by at least one construct, and ser-7 is required for vhl-1-mediated longevity.

(A) A table showing all ser-7 expressing candidate neurons, and which constructs in Fig. 2A restored ser-7 expression in these neurons. Pink rows indicate rescues that fully restored vhl-1 mediated fmo-2 induction, orange rows indicate rescues that partially restored vhl-1-mediated fmo-2 induction, and gray rows signify unsuccessful rescue constructs from the data in panel A. (B) Survival curve of WT, and ser-7(tm1325) worms on empty vector (EV) and vhl-1 RNAi. N ≥ 259 worms per condition. Significance is from a log-rank test comparing median survival. Cox Regression for an interaction between the effect of ser-7 (tm1325) knockout and vhl-1 RNAi knockdown on lifespan. p = 0.003, **. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. Three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons.

GABA and tyramine synthesis are required for the hypoxic response to extend lifespan.

(A) Summary table of the necessity of each C. elegans neurotransmitter for vhl-1 RNAi-mediated longevity. (B-E) Survival curves of WT and unc-25(e156) (GABA deficiency) (B), tdc-1(n3419) (tyramine + octopamine deficiency) (C), tbh-1(n3247) (octopamine deficiency) (D), and RIC ablation (Ex[tbh-1p::caspase-3(p12)-nz]; Ex[tbh-1p::cz-caspase-3 (p17)]; tbh-1p::GFP) (E) worms on empty vector (EV) and vhl-1 RNAi. N ≥ 310 (B), ≥ 201 (C), ≥ 258 (D), and ≥ 258 (E) worms per condition. Significance in panels B-E is from a log-rank test comparing median survival. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons.

Acetylcholine, glutamate, and dopamine synthesis are not fully required for vhl-1 RNAi to extend lifespan.

(A-C) Survival curves of WT and unc-17(e113) (acetylcholine deficiency) (A), eat-4(ky5) (glutamate deficiency) (B), and cat-2(n4547) (dopamine deficiency) (C) worms on empty vector (EV) and vhl-1 RNAi. N ≥ 213 (A), ≥ 274 (B), and ≥ 270 (C) worms per condition. (C) Cox Regression for an interaction between genotype and vhl-1 RNAi, p < 0.0001, ****. Significance in all panels is from a log-rank test comparing median survival. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons.

The RIM neuron and the tyramine receptor tyra-3 act in the hypoxic response-mediated longevity pathway.

(A-B) Survival curves of WT, RIM rescue (tdc-1 (n3419); Ex[ocr-4p::tdc-1]) (A), and uv1 tdc-1 rescue (tdc-1 (n3419); Ex[gcy-13p::tdc-1]) (B) strains on empty vector (EV) and vhl-1 RNAi. N ≥ 126 (A) and N ≥ 140 (B) worms per condition. (C) Survival curves of TU3311(unc-119p::sid-1) and vhl-1(ok161); TU3311(unc-119p::sid-1) worms on empty vector (EV) and tyra-3 RNAi. N ≥ 215 worms per condition. Significance in all panels is from a log-rank test comparing median survival. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons.

tyra-2 and ser-2 are not necessary for vhl-1-mediated longevity, while lgc-55 is partially required.

(A) Gene expression of tyra-3, tyra-2, ser-2, and lgc-55 in TU3311(unc-119p::sid-1) worms raised on RNAi targeting each gene compared to control worms raised on empty vector (EV) RNAi for two generations. N ≥ 300 worms per replicate, or 900 worms per condition. The top of the bar represents the mean of the population and error bars indicate standard error of the mean (SEM). (B-D) Survival curves of TU3311(unc-119p::sid-1) and vhl-1(ok161); TU3311(unc-119p::sid-1) worms on empty vector (EV) and tyra-2 (B), ser-2 (C), or lgc-55 (D) RNAi. Cox Regression for an interaction between lgc-55 knockdown and vhl-1 knockout on lifespan in the TU3311background strain. p < 0.0001, ****. N ≥ 215 worms per condition. Significance is from a log-rank test comparing median survival. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons.

High- and low-oxygen sensing neurons are important for hypoxic response-mediated longevity.

(A) Diagram of oxygen sensing neurons in C. elegans. (B) Survival curves of WT and URX/PQR/AQR ablated worms (qaIs2241 [gcy-36::egl-1 + gcy-35::GFP + lin-15(+)]) on empty vector (EV) and vhl-1 RNAi. N ≥ 279 worms per condition. (C) Survival curves of WT and BAG ablated worms (Ex[gcy-31p::caspase-3(p12)-nz]; Ex[gcy-31p::cz-caspase-3 (p17)]; gcy-31p::GFP) on empty vector (EV) and vhl-1 RNAi. N ≥ 188 worms per condition. (D) Survival curves of WT, hif-1(ia4); ADF-HIF-1S, URX/PQR/AQR (qaIs2241 [gcy-36::egl-1 + gcy-35::GFP + lin-15(+)]) ablation, and URX/PQR/AQR (qaIs2241 [gcy-36::egl-1 + gcy-35::GFP + lin-15(+)]) ablation; hif-1(ia4); ADF-HIF-1S worms. N ≥ 150 worms per condition. (E) Survival curves of WT, hif-1(ia4); ADF-HIF-1S, BAG (Ex[gcy-31p::caspase-3(p12)-nz]; Ex[gcy-31p::cz-caspase-3 (p17)]; gcy-31p::GFP) ablation, and BAG (Ex[gcy-31p::caspase-3(p12)-nz]; Ex[gcy-31p::cz-caspase-3 (p17)]; gcy-31p::GFP) ablation; hif-1(ia4); ADF-HIF-1S worms. N ≥ 186 worms per condition. Significance in all panels is from a log-rank test comparing median survival. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons.

The neuropeptide nlp-17 and its receptors npr-37 and npr-43 are required for hypoxic response-mediated longevity.

(A) Survival curves of unc-31(e169) and WT worms on empty vector (EV) and vhl-1 RNAi. N ≥ 242 worms per condition. (B-C) Survival curves of TU3311 (unc-119p::sid-1) and vhl-1(ok161); TU3311 (unc-119p::sid-1) worms on empty vector (EV) and egl-3 (B) or egl-21 (C) RNAi. N ≥ 209 worms (B) and N ≥ 185 worms (C) per condition. (D) Summary table of two sets of neuropeptide ligands/receptors that blunted vhl-1- mediated lifespan. (E-G) Survival curves of TU3311 (unc-119p::sid-1) and vhl-1(ok161); TU3311 (unc-119p::sid-1) worms on empty vector (EV) and npr-37 (E), npr-43 (F), or nlp-17 (G) RNAi. N ≥ 288 worms (E), N ≥ 309 worms (F), and N ≥ 309 worms (G) per condition. Significance in panels A-C and E-G are from log-rank test comparing median survival. Cox Regression for a significant interaction between npr-43 knockdown and vhl-1 knockout on lifespan, p <0.01, **. Cox regression for a significant interaction between nlp-17 knockdown and vhl-1 knockout on lifespan, p < 0.05, *. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, three replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons.

qPCR validation of RNAi hits from Figure 5, and neuropeptide screen hits that did not validate.

(A-B) Gene expression of (A) egl-3, egl-21, npr-37, and npr-43 in TU3311 (unc-119p::sid-1) worms raised on RNAi targeting each gene compared to control worms raised on empty vector (EV) RNAi for two generations. N ≥ 300 worms per replicate, or 900 worms per condition. The top of the bar represents the mean and error bars represent SEM. (B-D) Survival curves of TU3311 (unc-119p::sid-1) and vhl-1(ok161); TU3311 (unc-119p::sid-1) worms on empty vector (EV) and npr-3 (B), flp-15 (C), or flp-21 (D) RNAi. N ≥ 191 worms (B), N ≥ 181 worms (C), and N ≥ 183 worms (D) per condition. (E) Survival curves of nlp-17(ok3461) on empty vector (EV) or vhl-1 RNAi. N ≥ 275 worms per condition. Significance in panels B-E are from log-rank test comparing median survival. NS. = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. In all panels, 2-3 replicates were plotted together, and all statistics include a Bonferroni correction for multiple comparisons.

Working model of the hypoxia-mediated longevity pathway.