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. 2010 Dec 7;20(23):2131-6.
doi: 10.1016/j.cub.2010.10.057. Epub 2010 Nov 18.

Inhibition of respiration extends C. elegans life span via reactive oxygen species that increase HIF-1 activity

Seung-Jae Lee  1 , Ara B Hwang, Cynthia Kenyon
Affiliations

Inhibition of respiration extends C. elegans life span via reactive oxygen species that increase HIF-1 activity

Seung-Jae Lee et al. Curr Biol. .

Abstract

A mild inhibition of mitochondrial respiration extends the life span of many organisms, including yeast, worms, flies, and mice, but the underlying mechanism is unknown. One environmental condition that reduces rates of respiration is hypoxia (low oxygen). Thus, it is possible that mechanisms that sense oxygen play a role in the longevity response to reduced respiration. The hypoxia-inducible factor HIF-1 is a highly conserved transcription factor that activates genes that promote survival during hypoxia. In this study, we show that inhibition of respiration in C. elegans can promote longevity by activating HIF-1. Through genome-wide screening, we found that RNA interference (RNAi) knockdown of many genes encoding respiratory-chain components induced hif-1-dependent transcription. Moreover, HIF-1 was required for the extended life spans of clk-1 and isp-1 mutants, which have reduced rates of respiration. Inhibiting respiration appears to activate HIF-1 by elevating the level of reactive oxygen species (ROS). We found that ROS are increased in respiration mutants and that mild increases in ROS can stimulate HIF-1 to activate gene expression and promote longevity. In this way, HIF-1 appears to link respiratory stress in the mitochondria to a nuclear transcriptional response that promotes longevity.

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Figures

Fig. 1
Fig. 1. Inhibiting respiration increases HIF-1 activity
(A) Animals expressing the HIF-1-regulated Pnhr-57::GFP transgene displayed low levels of GFP when grown on control bacteria carrying the empty RNAi vector. (B–F) In contrast, RNAi of respiratory-chain or ATP synthase genes cyc-1 (B), cco-1 (C), nuo-1 (D), atp-3 (E), or atp-5 (F) induced the expression of Pnhr-57::GFP. RNAi treatment of cyc-1 or cco-1 only during adulthood did not increase the level of Pnhr-57::GFP (Fig. S1D-H). (G–I) Mutations in clk-1 (H) and isp-1 (I), which reduce respiration, elevated Pnhr-57::GFP expression (G). (J) Quantification of fluorescence in A to F (n>30); and (K), in G to I (n>42). mRNA levels of nhr-57 (L) and F22B5.4 (M), another HIF-1-regulated gene, were significantly increased in clk-1(qm30) and isp-1(qm150) mutants [Please see Fig. S1A-C for qRT-PCR data of other hypoxia-responsive genes]. Data were obtained from 3 independent quantitative RT-PCR analyses and error bars represent s.e.m (*P<0.01, **P<0.001, ***P<0.0001, two-tailed Student’s t-test compared to wild type).
Fig. 2
Fig. 2. The lifespan extension conferred by respiration mutants requires hif-1
(A, B) hif-1(ia4) loss-of-function mutations decreased the longevity of clk-1(qm30) (A) and isp-1(qm150) (B) mutants significantly (in three out of four trials and four out of four trials, respectively; See Supplemental Table S2). (C, D) The long lifespan of clk-1(qm30) (C) and isp-1(qm150) (D) mutants was significantly shortened by aha-1 [HIF1β] RNAi. Neither the hif-1(ia4) mutation nor aha-1 RNAi affected the lifespan of wild type (WT). (See Supplemental Table S2 for statistical analysis.) [We note that whereas Mehta et al. and we both found that hif-1 mutations do not affect the lifespans of wild type [19], Chen et al. and Zhang et al. reported that hif-1 mutants live longer than wild type [–38]. We carried out additional experiments to resolve this discrepancy, which are described in supplemental material (Fig. S2M, N)] (E, F) The increased mRNA levels of the HIF-1-dependent genes nhr-57 (E) and F22B5.4 (F) in clk-1(qm30) and isp-1(qm150) mutants were significantly decreased by hif-1(ia4) mutation. Error bars represent s.e.m (n=3, *P<0.05, **P<0.01, two-tailed Student’s t-test). See Fig. S2G, H for quantitative RT-PCR data of other hif-1-dependent hypoxia-inducible genes.
Fig. 3
Fig. 3. Activation of HIF-1 by vhl-1 or egl-9 mutations does not further lengthen the lifespan of respiration mutants
(A, B) Mutations in vhl-1 increased the lifespan of wild type but did not further extend the lifespan of clk-1(qm30) (A) or isp-1(qm150) (B) mutants. (C, D) The egl-9(sa307) mutation did not further increase the lifespans of clk-1(qm30) (C) or isp-1(qm150) (D) mutants. (E) Consistent with previous reports [14, 39], mRNA levels of nhr-57 were significantly increased by vhl-1(ok161) and egl-9(sa307) mutations. (F, G) The increased mRNA levels of nhr-57 in vhl-1(ok161) (F) or egl-9(sa307) (G) mutants were not further augmented by clk-1(qm30) and isp-1(qm150) mutations (control, Ctrl.). (See Supplemental Table S3 for statistical analysis.) (H) Expression of F22B5.4 was highly induced in vhl-1(ok161) and egl-9(sa307) mutants as reported previously [14, 39]. (I, J) this induction was not significantly further increased by clk-1(qm30) and isp-1(qm150) mutations. Note that the increased F22B5.4 mRNA levels in egl-9(sa307) mutants by clk-1(qm30) mutation was marginally not significant (P=0.06). Error bars represent s.e.m (n=3, *P<0.01, **P<0.001, two-tailed Student’s t-test).
Fig. 4
Fig. 4. Increased ROS causes HIF-1 to promote longevity in respiration-defective mutants
(A) ROS levels, measured using a 2′,7′-dichlorofluorescein diacetate (DCF-DA) fluorescence assay, were significantly increased in clk-1(qm30) and isp-1(qm150) mutants (n=5). See also Fig. S4A for data using mev-1(kn1) mutant animals, which were previously shown to have increased ROS levels [40]. (B–C) A low dose (0.25 mM) (B) and high dose (4 mM) (C) of paraquat significantly increased the level of DCF-DA fluorescence. 4 mM paraquat was introduced from L4 to day 3 of adulthood because worms arrest as larvae if treated with 4 mM paraquat from hatching (n=3). Error bars represent s.e.m (*P<0.05, **P<0.01, two-tailed Student’s t-test). (D) Low doses of paraquat (0.125 mM, 0.25 mM, 0.5 mM and 1 mM) lengthened lifespan, whereas higher concentrations (4, 16, and 64 mM) shortened lifespan. Paraquat was introduced during adulthood. The lifespan measurements for 0.125 mM and 0.5 mM paraquat treatment were performed separately and therefore shown with different controls. See also Table S4. (E, F) Compared to untreated control Pnhr-57::GFP animals (E), animals treated with low levels of paraquat (0.25 mM) displayed increased GFP levels (F). Paraquat treatment further increased Pnhr-57::GFP levels in clk-1(qm30) and isp-1(qm150) mutant animals (Fig. S4J, K), suggesting that the induction was not saturated by either of the mutations or by the paraquat treatment. (G, H) The induction of Pnhr-57::GFP by paraquat treatment was significantly diminished in the hif-1(ia4) mutant. (I) Quantification of fluorescence in E to H (n >15). (J) The increased nhr-57 mRNA abundance caused by paraquat treatment, assayed using quantitative RT-PCR, was significantly decreased by hif-1(ia4) mutation. Data analysis was done from 10 independent quantitative RT-PCR experiments. [In two of our data sets, the levels of nhr-57 mRNA in paraquat-treated wild-type animals were increased by a very-large 681 and 755 fold compared to those in control animals. We excluded these two datasets from our analysis by using rejection analysis of QP-test for outliers (confidence level: 0.99) [41]]. (K) Mutations in hif-1 significantly decreased the longevity caused by paraquat treatment. Some long-lived mutants require the daf-16/FOXO transcription factor gene for their longevity, but respiration mutants do not [1, 4, 10]. We found that 0.25 mM paraquat treatment increased the lifespan of daf-16(mu86) null mutants (Fig. S4L). See Supplemental Table S4 for statistical analysis. Error bars represent s.e.m (*P<0.05, **P<0.01, two-tailed Student’s t-test). Previously, Rea et al. observed an increased trend in protein carbonylation levels at doses of respiratory-chain RNAi that increased lifespan, and also at higher RNAi doses, which did not extend lifespan [17]. On this basis, they concluded that ROS did not play a role in this longevity. One way to reconcile their findings with ours is to suggest that a sharp reduction in respiration prevents lifespan extension in spite of elevated ROS because it compromises the animals’ health.
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