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Mitochondrial ROS Produced via Reverse Electron Transport Extend Animal Lifespan.

Scialò F, Sriram A, Fernández-Ayala D, Gubina N, Lõhmus M, Nelson G, Logan A, Cooper HM, Navas P, Enríquez JA, Murphy MP, Sanz A - Cell Metab. (2016)

Bottom Line: Here we show that the site of ROS production significantly contributes to their apparent dual nature.Furthermore, preventing ubiquinone reduction, through knockdown of PINK1, shortens lifespan and accelerates aging; phenotypes that are rescued by increasing reverse electron transport.These results illustrate that the source of a ROS signal is vital in determining its effects on cellular physiology and establish that manipulation of ubiquinone redox state is a valid strategy to delay aging.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, Newcastle University Institute for Ageing, Newcastle University, Newcastle upon Tyne NE4 5PL, UK.

No MeSH data available.


Related in: MedlinePlus

Increased ROS Production in Aging Flies Correlates with Mitochondrial Dysfunction(A) Representative images of dissected fly brains stained with MitoSOX from wild-type flies of the indicated ages.(B) Quantification of (A) (n = 5).(C) Representative EM images of Dahomey flight muscle sections at 1,000x magnification dissected at the indicated ages (n = 10, 1 muscle per fly; red arrows indicated exemplar swollen, rounded mitochondria, see Figure S1C for quantification).(D) Mitochondrial respiration in Dahomey and Oregon R flies at the indicated ages (n = 6).(E) CI and CIII enzymatic activity in wild-type flies of the indicated ages (n = 5).(F) CI (NDUFS3), CII (SDHB), CIII (CYTOB), CIV (COX4), and CV (ATP5A) levels in wild-type flies. GAPDH is used as a loading control.(G) Quantification of (F).(H) ImpL3 expression in wild-type flies of the indicated ages.Values shown represent means ± SEM of at least three biological replicates, unless otherwise stated. See also Figure S1.
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fig1: Increased ROS Production in Aging Flies Correlates with Mitochondrial Dysfunction(A) Representative images of dissected fly brains stained with MitoSOX from wild-type flies of the indicated ages.(B) Quantification of (A) (n = 5).(C) Representative EM images of Dahomey flight muscle sections at 1,000x magnification dissected at the indicated ages (n = 10, 1 muscle per fly; red arrows indicated exemplar swollen, rounded mitochondria, see Figure S1C for quantification).(D) Mitochondrial respiration in Dahomey and Oregon R flies at the indicated ages (n = 6).(E) CI and CIII enzymatic activity in wild-type flies of the indicated ages (n = 5).(F) CI (NDUFS3), CII (SDHB), CIII (CYTOB), CIV (COX4), and CV (ATP5A) levels in wild-type flies. GAPDH is used as a loading control.(G) Quantification of (F).(H) ImpL3 expression in wild-type flies of the indicated ages.Values shown represent means ± SEM of at least three biological replicates, unless otherwise stated. See also Figure S1.

Mentions: Initially, we asked whether increased mtROS production is a general feature of aging in flies by measuring ROS production in fly brains using two fluorescent probes, MitoSOX (for mitochondrial matrix ROS) and H2DCF (for total cellular ROS levels), and a redox-sensitive GFP based reporter for in vivo mitochondrial H2O2 (mtH2O2) (mtORP1-roGFP) (Albrecht et al., 2011). We observed a consistent increase in ROS in old flies in two wild-type strains (Dahomey and Oregon R) (Figures 1A, 1B, S1A, and S1B). In Dahomey flies, we observed that with age, dorsal flight muscle mitochondrial ultrastructure became increasingly rounded and swollen with the appearance of perturbed cristae structure at 75 days (d) (Figures 1C, S1C, and S1D). Further, in both strains, high-resolution respirometry and enzymatic assays showed a decrease in CI-linked respiration (CI-respiration from here on) and in the enzymatic activity of CI and CIII (Figures 1D and 1E). Aconitase activity initially decreased from 5 to 25 d but remained constant as the flies continued to age (Figure S1E). At this age (25 d), no decrease in locomotive activity (Figure S1F) or increase in ROS (Figures 1A, 1B, S1A, and S1B) was observed. Western blot analysis showed that only the levels of CI and aconitase were significantly affected with age (Figures 1F, 1G, and S1G–S1J). However, CI concentration was decreased at very late (75 and 85 d) ages, suggesting a shift in mitochondrial metabolism supported by an increase in ImpL3 (lactate dehydrogenase A homologue) expression (Figure 1H).


Mitochondrial ROS Produced via Reverse Electron Transport Extend Animal Lifespan.

Scialò F, Sriram A, Fernández-Ayala D, Gubina N, Lõhmus M, Nelson G, Logan A, Cooper HM, Navas P, Enríquez JA, Murphy MP, Sanz A - Cell Metab. (2016)

Increased ROS Production in Aging Flies Correlates with Mitochondrial Dysfunction(A) Representative images of dissected fly brains stained with MitoSOX from wild-type flies of the indicated ages.(B) Quantification of (A) (n = 5).(C) Representative EM images of Dahomey flight muscle sections at 1,000x magnification dissected at the indicated ages (n = 10, 1 muscle per fly; red arrows indicated exemplar swollen, rounded mitochondria, see Figure S1C for quantification).(D) Mitochondrial respiration in Dahomey and Oregon R flies at the indicated ages (n = 6).(E) CI and CIII enzymatic activity in wild-type flies of the indicated ages (n = 5).(F) CI (NDUFS3), CII (SDHB), CIII (CYTOB), CIV (COX4), and CV (ATP5A) levels in wild-type flies. GAPDH is used as a loading control.(G) Quantification of (F).(H) ImpL3 expression in wild-type flies of the indicated ages.Values shown represent means ± SEM of at least three biological replicates, unless otherwise stated. See also Figure S1.
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Related In: Results  -  Collection

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fig1: Increased ROS Production in Aging Flies Correlates with Mitochondrial Dysfunction(A) Representative images of dissected fly brains stained with MitoSOX from wild-type flies of the indicated ages.(B) Quantification of (A) (n = 5).(C) Representative EM images of Dahomey flight muscle sections at 1,000x magnification dissected at the indicated ages (n = 10, 1 muscle per fly; red arrows indicated exemplar swollen, rounded mitochondria, see Figure S1C for quantification).(D) Mitochondrial respiration in Dahomey and Oregon R flies at the indicated ages (n = 6).(E) CI and CIII enzymatic activity in wild-type flies of the indicated ages (n = 5).(F) CI (NDUFS3), CII (SDHB), CIII (CYTOB), CIV (COX4), and CV (ATP5A) levels in wild-type flies. GAPDH is used as a loading control.(G) Quantification of (F).(H) ImpL3 expression in wild-type flies of the indicated ages.Values shown represent means ± SEM of at least three biological replicates, unless otherwise stated. See also Figure S1.
Mentions: Initially, we asked whether increased mtROS production is a general feature of aging in flies by measuring ROS production in fly brains using two fluorescent probes, MitoSOX (for mitochondrial matrix ROS) and H2DCF (for total cellular ROS levels), and a redox-sensitive GFP based reporter for in vivo mitochondrial H2O2 (mtH2O2) (mtORP1-roGFP) (Albrecht et al., 2011). We observed a consistent increase in ROS in old flies in two wild-type strains (Dahomey and Oregon R) (Figures 1A, 1B, S1A, and S1B). In Dahomey flies, we observed that with age, dorsal flight muscle mitochondrial ultrastructure became increasingly rounded and swollen with the appearance of perturbed cristae structure at 75 days (d) (Figures 1C, S1C, and S1D). Further, in both strains, high-resolution respirometry and enzymatic assays showed a decrease in CI-linked respiration (CI-respiration from here on) and in the enzymatic activity of CI and CIII (Figures 1D and 1E). Aconitase activity initially decreased from 5 to 25 d but remained constant as the flies continued to age (Figure S1E). At this age (25 d), no decrease in locomotive activity (Figure S1F) or increase in ROS (Figures 1A, 1B, S1A, and S1B) was observed. Western blot analysis showed that only the levels of CI and aconitase were significantly affected with age (Figures 1F, 1G, and S1G–S1J). However, CI concentration was decreased at very late (75 and 85 d) ages, suggesting a shift in mitochondrial metabolism supported by an increase in ImpL3 (lactate dehydrogenase A homologue) expression (Figure 1H).

Bottom Line: Here we show that the site of ROS production significantly contributes to their apparent dual nature.Furthermore, preventing ubiquinone reduction, through knockdown of PINK1, shortens lifespan and accelerates aging; phenotypes that are rescued by increasing reverse electron transport.These results illustrate that the source of a ROS signal is vital in determining its effects on cellular physiology and establish that manipulation of ubiquinone redox state is a valid strategy to delay aging.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, Newcastle University Institute for Ageing, Newcastle University, Newcastle upon Tyne NE4 5PL, UK.

No MeSH data available.


Related in: MedlinePlus