Proteasomal regulation of the hypoxic response modulates aging in C. elegans

Abstract

The Caenorhabditis elegans von Hippel-Lindau tumor suppressor homolog VHL-1 is a cullin E3 ubiquitin ligase that negatively regulates the hypoxic response by promoting ubiquitination and degradation of the hypoxic response transcription factor HIF-1. Here, we report that loss of VHL-1 significantly increased life span and enhanced resistance to polyglutamine and beta-amyloid toxicity. Deletion of HIF-1 was epistatic to VHL-1, indicating that HIF-1 acts downstream of VHL-1 to modulate aging and proteotoxicity. VHL-1 and HIF-1 control longevity by a mechanism distinct from both dietary restriction and insulin-like signaling. These findings define VHL-1 and the hypoxic response as an alternative longevity and protein homeostasis pathway.

Fig. 1

VHL-1 and EGL-9 modulate proteotoxic stress and lifespan. RNAi knock-down of vhl-1 significantly enhances resistance to (A) polyglutamine toxicity (p<1×10₋₅) and (B) amyloid beta toxicity (p<1×10₋₅), relative to animals fed empty vector (EV) bacteria. RNAi knock-down of egl-9 significantly enhances resistance to (C) polyglutamine toxicity (p<1×10₋₅) and (D) amyloid beta toxicity (p<1×10₋₅), relative to animals fed EV bacteria. RNAi knock-down of (E) vhl-1 (p<1×10₋₅) or (F) egl-9 (p<1×10₋₅) significantly increased adult lifespan relative to the EV-fed control. Paralysis and lifespan statistics in Table S1 and S6.

Fig. 2

VHL-1 modulates longevity, age-pigment accumulation, and reproduction in a HIF-1-dependent manner. Gene structure of (A) known hif-1 splice variants, hif-1(ia4) deletion, and hif-1(RNAi), and (B) known vhl-1 splice variants, vhl-1(ok161) deletion, and vhl-1(RNAi). Black boxes represent exons; yellow * indicates a stop codon. Blue boxes indicate RNAi target sequences from Anringer (Ah) or Vidal (Vi) library clones. The RNAi clones used to knock-down hif-1 and vhl-1 target all known splice variants. (C) Vhl-1(ok161) animals are significantly longer-lived than wild type (N2) animals (p<1×10₋₅); vhl-1(ok161) hif-1(ia4) double mutant animals are not longer-lived than N2 (p=0.66). (D) Accumulation of autofluorescent age-pigment is significantly reduced by deletion of vhl-1 (p < 1×10₋₅). Autofluorescence is not significantly different in N2 versus vhl-1(ok161) hif-1(ia4) double mutant animals (p=0.17). (E) Vhl-1(ok161) animals produce significantly fewer progeny than N2 animals (p=3.6×10₋₃). No significant difference in brood size was observed for vhl-1(ok161) hif-1(ia4) double mutant animals (p=0.69) or hif-1(ia4) animals (p=0.43), relative to N2 animals. Data in D, E are mean ± SD of at least 9 animals per condition. Lifespan statistics provided in Table S6.

Fig. 3

VHL-1 and HIF-1 modulate longevity by a mechanism distinct from dietary restriction. (A) Lifespan extension from bacterial deprivation (BD) is not significantly different in N2 and hif-1(ia4) animals (p=0.97). (B) BD significantly increases the lifespan of vhl-1(ok161) animals (p<1×10₋₅) or (C) vhl-1(ok161); hif-1(ia4) double mutant animals (p<1×10₋₅). (D) Hif-1(RNAi) does not significantly alter the lifespan extension of eat-2(ad465) animals (p=0.6). (E) The eat-2(ad465) mutation significantly reduces pharyngeal pumping rate relative to N2 (p<1×10₋₅) or vhl-1(ok161) animals (p<1×10₋₅). Pharyngeal pumping rate is not significantly different in N2 and vhl-1(ok161) animals (p=0.06). Data are mean ± SD. (F) Relative to animals fed empty vector (EV) bacteria under normoxic conditions, vhl-1(RNAi) under normoxia or growth on EV under hypoxia (hyp, 0.5% oxygen) failed to significantly increase autophagy, as indicated by the presence of LGG-1::GFP puncta (p=0.6 and 0.5, respectively). 35 animals per condition were imaged for EV and vhl-1(RNAi). 7 animals were imaged for hypoxia. Data are mean ± SEM. Lifespan statistics provided in Table S6.

Fig. 4

Insulin/IGF-1-like signaling and VHL-1 modulate longevity by distinct mechanisms. Daf-2(RNAi) significantly increases the lifespan of (A) vhl-1(ok161) (p<1×10₋₅), (B) hif-1(ia4) (p<1×10₋₅), or (C) hif-1(ia4); vhl- 1(ok161) animals (p<1×10₋₅). (D) Vhl-1(RNAi) does not induce nuclear localization of DAF-16. Daf-2(RNAi) or heat shock significantly increases DAF-16 nuclear foci (p<1×10₋₅ in each case). DAF-16 nuclear foci per animal was quantified for 10 animals per group. (E) Daf-16(RNAi) does not prevent lifespan extension from deletion of vhl-1 (p<1×10₋₅). (F) Deletion of vhl-1 significantly reduces auto-fluorescence in animals fed empty vector bacteria (p<1×10₋₅) or daf-16(RNAi) (p=0.004), but does not reduce autofluorescence in animals fed hif-1(RNAi) (p=0.9). Median integrated pixel density shown for at least 10 randomly chosen animals per condition. Lifespan statistics provided in Table S6. Data in D, F are mean ± SEM.