Contents

πŸ“’ Scialo 2016

Mitochondrial ROS Produced via Reverse Electron Transport Extend Animal Lifespan

Sciwheel1

Mitochondrial ROS Produced via Reverse Electron Transport Extend Animal Lifespan

Graphical abstract1 https://els-jbs-prod-cdn.literatumonline.com/cms/attachment/c9396c09-532a-42b8-9452-25208213af1c/fx1_lrg.jpg

Introduction

  • ROS as harmful => ROS as secondary messenger
  • use of alternative respiratory enzymes, absent from mammals and flies, to modulate ROS generation at specific sites of the ETC
    • Alternative oxidase (AOX) of Ciona intestinalis bypassing CIII and CIV
    • NDI1 : rotenone-insensitive alternative NADH dehydrogenase, bypassing CI

Results and discussion

Aging: ROS increased, CI resp. decreased

https://els-jbs-prod-cdn.literatumonline.com/cms/attachment/6d88352a-d724-4663-b61f-bd0b33a45658/gr1_lrg.jpg

  • 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.

Over-Reduction of the CoQ Pool Increases ROS Production and Extends Lifespan

  • Extension of lifespan accompanied by increase in ROS (!!)
    • NDI1 + CI (NDI1 reduces Q, increased CI-ROS)
  • No extension of lifespan
    • AOX + CI (AOX oxidizes Q)
    • AOX + NDI1 + CI (AOX oxidizes Q)
    • NDI1 only (CI knock down, less reduced Q and less CI-ROS)
    • Added mitochondrial Catalase (Decreased ROS
  • Where does the ROS comes from?
    • Rotenone (specific CI Q-site inhibitor) significantly decreases ROS formation in ND1 expressing flies but increases ROS formation in wildtype flies. RET is the source of NDI1-mediated ROS production
    • FCCP (Uncoupler) depleted NDI1-mediated ROS production
    • myxothiazol (a CIII Qo site inhibitor): no effect
    • During ischemia reperfusion RET is the main source of superoxide in vivo 2

https://els-jbs-prod-cdn.literatumonline.com/cms/attachment/6f46fe3b-89d5-488f-9b17-b4a7e123ea48/gr2_lrg.jpg

  • A: Representative images of dissected brains from indicated genotypes stained with MitoSOX.
  • B: Quantification of (A) (n = 5).
  • C: Survival curves for the indicated genotypes (n = 200).
  • D: Schematic diagram illustrating effects of expressing two different alternative respiratory enzymes on electron transport: (i) NDI1 generates ROS by over-reducing the CoQ pool; (ii) AOX reverts the effects of NDI1 by re-oxidizing the CoQ pool; (iii) decrease in the levels of CI can prevent reduction of CoQ and subsequent ROS production; (iv) ectopic expression of mtCAT reduces ROS levels without altering mitochondrial respiration or the redox state of CoQ.
  • E: Representative images of brains from the indicated genotypes stained with MitoSOX.
  • F: Quantification of (E) (n = 5).
  • G: Survival curves for the indicated genotypes (n = 160).
  • H: In vivo ROS measurements from indicated genotypes in brains dissected from flies expressing a mitochondrially localized redox-sensitive GFP-based reporter.
  • I: Quantification of (H) (n = 5–7).
  • J: Quantification of brains dissected from flies of the indicated genotypes stained with MitoSOX (n = 5).
  • K: Survival curves for the indicated genotypes (n = 200).
  • L: Diagram illustrating using metabolic poisons to dissect ROS production: rotenone (ROT), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), or myxothiazol (MYX). Green dashed arrows indicate the possible flow of electrons following CoQ reduction.
  • M: Quantification of brains dissected from NDI1 flies fed with metabolic poisons, stained with MitoSOX (n = 4).

CoQ-Mediated ROS Signaling Can Rescue Pathology Induced by Oxidative Stress

  • SOD2 (mitochondrial SOD) knockdown : increase in superoxide and decrease in hydrogen peroxide, dramatically shortened lifespan
  • High level of superoxide inhibits CI and aconitase activity
  • AOX decreased superoxide and hydrogen peroxide, but little improvement
  • NDI1 expression partially rescued lifespan along with CI respiration and activity ( increasing ROS levels by RET)
    • disappeared when RET was prevented by depletion of CI levels by GeneSwitch system
  • RET may be necessary for the maintenance of mitochondrial function and lifespan extension

https://els-jbs-prod-cdn.literatumonline.com/cms/attachment/77bb0397-c807-4dc2-bb63-cba25b5733d7/gr3_lrg.jpg

  • A: Representative images of fly brains from indicated genotypes stained with MitoSOX.
  • B: Quantification of (A) (n = 5).
  • C: Survival curves of the indicated genotypes (n = 180–380).
  • D: Mitochondrial respiration in flies of the indicated genotypes (n = 6).
  • E: CI, CII, and aconitase enzymatic activities in flies of the indicated genotypes (n = 7).

Loss of CoQ-Mediated ROS Signaling Accelerates Aging

https://els-jbs-prod-cdn.literatumonline.com/cms/attachment/eb612782-1e7c-46c9-8b83-1b9b7ebfb2f4/gr4_lrg.jpg

Summary

  • mtROS production increases with age
  • un-detoxified ROS can be detrimental to Drosophila lifespan
  • But increasing ROS production specifically from reduced CoQ, possibly via RET acts as a signal to maintain mitochondrial function (notably CI) and extend lifespan
  • Manipulation of the redox state of CoQ may be a strategy for the extension of both mean and maximum lifespan

Reference


  1. ScialΓ² F, Sriram A, FernΓ‘ndez-Ayala D, et al. Mitochondrial ROS Produced via Reverse Electron Transport Extend Animal Lifespan. Cell Metab. 2016;23(4):725-34. PMC4835580 ↩︎

  2. Chouchani ET, Pell VR, Gaude E, et al. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature. 2014;515(7527):431-435. PMC4255242 ↩︎