HIF-1 modulates longevity and healthspan in a temperature-dependent manner

Abstract

The hypoxia-inducible factor HIF-1 has recently been identified as an important modifier of longevity in the roundworm Caenorhabditis elegans. Studies have reported that HIF-1 can function as both a positive and negative regulator of life span, and several disparate models have been proposed for the role of HIF in aging. Here, we resolve many of the apparent discrepancies between these studies. We find that stabilization of HIF-1 increases life span robustly under all conditions tested; however, deletion of hif-1 increases life span in a temperature-dependent manner. Animals lacking HIF-1 are long lived at 25°C but not at 15°C. We further report that deletion or RNAi knockdown of hif-1 impairs healthspan at lower temperatures because of an age-dependent loss of vulval integrity. Deletion of hif-1 extends life span modestly at 20°C when animals displaying the vulval integrity defect are censored from the experimental data, but fails to extend life span if these animals are included. Knockdown of hif-1 results in nuclear relocalization of the FOXO transcription factor DAF-16, and DAF-16 is required for life span extension from deletion of hif-1 at all temperatures regardless of censoring.

Figure 1. Temperature influences the effect of HIF-1 deletion but not HIF-1 stabilization

Life span from hatching was determined for N2, vhl-1(ok161), hif-1(ia4), and hif-1(ia4); vhl-1(ok161) animals at (A) 15°C, (B) 20°C, and (C) 25°C. Mean life span is shown in parentheses. Deletion of vhl-1 significantly increased median life span at all three temperatures, while deletion of hif-1 only significantly increased life span at 25°C. A minimum of 350 animals and three replicate experiments were examined for each genotype at each temperature. Pooled data are shown. Summary data and statistics for individual and pooled experimental data are provided in Supplemental Table 1.

Figure 2. Loss of HIF-1 impairs healthspan due to a defect in vulval integrity

Panels A-D show a representative example of the vulval integrity defect (Vid) observed in hif-1(ia4) animals across their entire life time. The vulval integrity defect is typically first detected morphologically when (A) healthy worms begin to show a (B) protruding vulva. This usually progresses through stages of (C) early rupture and (D) late rupture prior to death. Panel E shows the total fraction of N2, hif-1(ia4), vhl-1(ok161), and hif-1(ia4); vhl-1(ok161) animals displaying a vulval integrity defect prior to death at 25°C, 20°C and 15°C, respectively. Panel F shows the increase in vulval integrity defect of hif-1(RNAi) treatment from egg at 20°C. A, B and C are viewed at 70× magnification, and D is viewed at 75× magnification. Error bars represent SEM. Summary data and statistics for individual and pooled experimental data are provided in Supplemental Table 2.

Figure 3. Loss of vulval integrity is an age-dependent healthspan phenotype

The fraction of animals with observable vulval integrity defects (Vid) are shown as a function of age from hatching for N2, hif-1(ia4), vhl-1(ok161), and hif-1(ia4); vhl-1(ok161) at (A) 20°C and (B) 15°C.

Figure 4. Deletion of hif-1 increases life span when animals exhibiting a vulval integrity defect are censored

Reanalysis of our longevity data removing animals displaying loss of vulval integrity. Censored life span from hatching is shown for N2 and hif-1(ia4) animals at (A) 20°C and (B) 15°C. Censoring results in a significant increase in life span of hif-1(ia4) animals at 20°C but not 15°C. Summary data and statistics for individual and pooled experimental data are provided in Supplemental Table 3.

Figure 5. DAF-16 is required for life span extension from deletion of hif-1

Life span from hatching was determined for N2, daf-16(mu86), hif-1(ia4), and daf-16(mu86); hif-1(ia4) animals at (A) 25°C, (B) 20°C, and (C) 15°C. Mean life span is shown in parentheses. Deletion of hif-1 failed to significantly increase life span of daf-16(mu86) animals at any temperature. Summary data and statistics for individual and pooled experimental data are provided in Supplemental Table 4.

Figure 6. DAF-16 is still required for life span extension from deletion of hif-1 when animals with vulval integrity defects are censored

Reanalysis of the longevity data from Figure 5 removing animals displaying loss of vulval integrity at (A) 20°C and (B) 15°C. Mean life span is shown in parentheses. Summary data and statistics for individual and pooled experimental data are provided in Supplemental Table 5.

Figure 7. hif-1 RNAi causes nuclear relocalization of DAF-16∷GFP

(A) Fraction of animals showing distinct DAF-16∷GFP puncta following 2 hour treatments of either empty vector RNAi (EV), 37° heat shock (heat), daf-2(RNAi), hif-1(RNAi), or vhl-1(RNAi) at 15°, 20°, and 25°. *denotes p-value of less than 0.05 relative to EV by one-way ANOVA. Representative images of animals are provided in Supplemental Figure 1.

Figure 8. Model for HIF-1 modulation of longevity in C. elegans

Stabilization of HIF-1 promotes longevity by a mechanism distinct from both insulin-like signaling and dietary restriction (DR). HIF-1 also negatively impacts longevity by repressing nuclear localization and activation of DAF-16, in a temperature-dependent manner. HIF-1 also interacts with TOR/Raptor and the DR pathway, but in a complex manner dependent on additional experimental variables that are currently unknown (represented by dashed lines).