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. 2023 Aug 8;120(32):e2216141120.
doi: 10.1073/pnas.2216141120. Epub 2023 Jul 31.

Mitochondrial sulfide promotes life span and health span through distinct mechanisms in developing versus adult treated Caenorhabditis elegans

Adriana Raluca Vintila  1 , Luke Slade  1   2 , Michael Cooke  1   3 , Craig R G Willis  4 , Roberta Torregrossa  2 , Mizanur Rahman  5 , Taslim Anupom  6 , Siva A Vanapalli  5 , Christopher J Gaffney  7 , Nima Gharahdaghi  2 , Csaba Szabo  8 , Nathaniel J Szewczyk  3   9 , Matthew Whiteman  2 , Timothy Etheridge  1
Affiliations

Mitochondrial sulfide promotes life span and health span through distinct mechanisms in developing versus adult treated Caenorhabditis elegans

Adriana Raluca Vintila et al. Proc Natl Acad Sci U S A. .

Abstract

Living longer without simultaneously extending years spent in good health ("health span") is an increasing societal burden, demanding new therapeutic strategies. Hydrogen sulfide (H2S) can correct disease-related mitochondrial metabolic deficiencies, and supraphysiological H2S concentrations can pro health span. However, the efficacy and mechanisms of mitochondrion-targeted sulfide delivery molecules (mtH2S) administered across the adult life course are unknown. Using a Caenorhabditis elegans aging model, we compared untargeted H2S (NaGYY4137, 100 µM and 100 nM) and mtH2S (AP39, 100 nM) donor effects on life span, neuromuscular health span, and mitochondrial integrity. H2S donors were administered from birth or in young/middle-aged animals (day 0, 2, or 4 postadulthood). RNAi pharmacogenetic interventions and transcriptomics/network analysis explored molecular events governing mtH2S donor-mediated health span. Developmentally administered mtH2S (100 nM) improved life/health span vs. equivalent untargeted H2S doses. mtH2S preserved aging mitochondrial structure, content (citrate synthase activity) and neuromuscular strength. Knockdown of H2S metabolism enzymes and FoxO/daf-16 prevented the positive health span effects of mtH2S, whereas DCAF11/wdr-23 - Nrf2/skn-1 oxidative stress protection pathways were dispensable. Health span, but not life span, increased with all adult-onset mtH2S treatments. Adult mtH2S treatment also rejuvenated aging transcriptomes by minimizing expression declines of mitochondria and cytoskeletal components, and peroxisome metabolism hub components, under mechanistic control by the elt-6/elt-3 transcription factor circuit. H2S health span extension likely acts at the mitochondrial level, the mechanisms of which dissociate from life span across adult vs. developmental treatment timings. The small mtH2S doses required for health span extension, combined with efficacy in adult animals, suggest mtH2S is a potential healthy aging therapeutic.

Keywords: H2S; health span; longevity; mitochondria; transcriptomics.

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Conflict of interest statement

M.W. and R.T. have intellectual property (patents) on sulfide delivery molecules and their use. M.W. is a co-founder and CSO of MitoRX Therapeutics, Oxford. S.A.V. and M.R. are co-founders of NemaLife Inc., and the microfluidic devices used in this study have been licensed for commercialization. S.A.V., M.R., and T.A. are named inventors on the microfluidic devices.

Figures

Fig. 1.
Fig. 1.
Lower doses of mitochondrion-targeted H2S extend life span. (A) C. elegans life span is significantly increased with higher (100 µM), but not lower (100 nM) treatment with the untargeted H2S donor, NaGYY4137 when administered from L1 larval stage across the entire lifecourse. (B) Conversely, lower doses (100 nM) of mitochondrion-targeted H2S (AP39) extend life span. Life span curves represent the average of three biological replicates (total ~300 to 600 animals per condition). **** denotes significant difference vs. untreated (0.01% DMSO) wild-type controls (P < 0.0001). ns, nonsignificant.
Fig. 2.
Fig. 2.
Mitochondrion-targeted H2S extends movement rate and maximal strength indices of health span. (A) Animal movement rate is increased across the entire lifecourse with both lower dose (100 nM) mitochondrion-targeted H2S (AP39) and higher dose (100 µM) untargeted H2S (NaGYY4137) when administered from L1 larval stage until death. Movement rates as a % change from day 0 baselines, across days 0, 2, 4, 8, 12, and 16 postadulthood, are presented as area under the curve. (B) Lower dose (100 nM) mitochondrion-targeted H2S maintains C. elegans maximal strength producing ability in later life (day 10 postadulthood), measured using our microfluidic NemaFlex device. Data presented are mean ± SD, n = 90 per condition, across 3 biological replicates. *P < 0.05, **P < 0.01 and ****P < 0.0001 denotes significant difference vs. untreated (0.01% DMSO) wild-type controls.
Fig. 3.
Fig. 3.
mtH2S prolongs mitochondrial integrity and content. (A) The percentage of well-networked and (B) moderately fragmented mitochondria during C. elegans aging is significantly improved with mtH2S (AP39), and for a longer duration than untargeted H2S (NaGYY4137) treatments. Data represent two biological replicates (total ~80 animals per time point/ condition and 450 muscle cells). (C, D) Representative green fluorescent protein-tagged mitochondrial images for normally arrayed (Left) and moderately fragmented (Right) mitochondria. White dashed boxes and corresponding magnified panels (Right) highlight each structural phenotype. (E) Citrate synthase activity with mtH2S at young adulthood (day 0) and day 4 postadulthood with mtH2S treatment, but not with untargeted H2S. Data represent two biological replicates, each with technical triplicates (total ~50 animals per time point/condition). All data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 denote significant difference from untreated (0.01% DMSO) wild-type controls.
Fig. 4.
Fig. 4.
Adult-onset treatment with mitochondrion-targeted H2S extends health span but not life span. (A–C) Survival curves are unaffected vs. wild-type (P > 0.05) with 100 nM mtH2S and 100 µM untargeted H2S treatments beginning at day 0, 2 or 4 of adulthood. (D–F) mtH2S significantly increases health span when administered from day 0, 2, or 4 of adulthood, and untargeted H2S improves health span when administered from day 2 or 4 postadulthood. Health span data presented movement as a % change from day 0 baselines across all time points postadulthood, analyzed as area under the curve, n = 360 per condition, across 3 biological replicates and 18 technical replicates. Life span data are ~300 animals per condition, across three biological replicates. *P < 0.05, ***P < 0.001 and ****P < 0.0001) denotes significant difference vs. untreated (0.01% DMSO) wild-type controls.
Fig. 5.
Fig. 5.
Effects of age and mtH2S on the C. elegans transcriptome. (A) Principal component (PC) analysis plot of all analyzed samples. (B) Differential gene quantities with time and between conditions. (C) Truncated violin plots depicting time/condition expression trends (represented as Z-score of gene abundance) for clusters of differentially expressed genes >200 genes in size. * = cluster genes have median FDR < 0.05 for given comparison with day 0 untreated (0.01% DMSO) wild-type animals, Φ = cluster genes have median FDR < 0.05 for direct comparison between treatments at given time point. (D) Representative term enrichments for each cluster shown in panel C. (E) Expression heatmap for top connected PPI network components for each gene cluster shown in panel C. Data represent ~60 animals across biological triplicates, per condition and time point. For all panels, WT = wild-type. D0, D4 and D10 = days 0, 2 and 4 postadulthood, respectively.
Fig. 6.
Fig. 6.
Adult-onset mtH2S preserves health span through the ELT-6 GATA transcription factor circuit. (A) ELT-6 expression increases with aging and is significantly repressed with AP39 treatment compared to untreated animals in later-life. (B) Representative images of ELT-6 expression (elt-6::mCherry). (C) Preservation of age-related declines in mitochondrial integrity by AP39 correspond with attenuated ELT-6 expression (using elt-6::mCherry + mito::GFP coexpression reporter strain). (D) AP39-induced improvements in aging movement capacity is confirmed in elt-6::mCherry + mito::GFP animals, and correspond with lowered ELT-6 and improved mitochondrial integrity. RNAi against elt-3 (E) and elt-6 (F) prevents the health span–promoting effects of AP39. Panels A–D employed transgenic animals coexpressing elt-6::mCherry + mito::GFP in body-wall muscle, across 25 to 45 animals and two biological repeats. Panels (E and F) employed wild-type N2 animals. Movement rates are from 80-120 animals per condition, per time point. # denote significant effect of aging compared to untreated day 0 animals (#P < 0.05; ###P < 0.001). * denote significant effect of treatment for within-day comparisons against untreated animals (*P < 0.05; **P < 0.01; ***P < 0.001).
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