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. 2018 Nov 6;28(5):776-786.e5.
doi: 10.1016/j.cmet.2018.07.011. Epub 2018 Aug 16.

Mitohormesis in Mice via Sustained Basal Activation of Mitochondrial and Antioxidant Signaling

Carly S Cox  1 , Sharen E McKay  2 , Marissa A Holmbeck  3 , Brooke E Christian  4 , Andrew C Scortea  5 , Annie J Tsay  3 , Laura E Newman  5 , Gerald S Shadel  6
Affiliations

Mitohormesis in Mice via Sustained Basal Activation of Mitochondrial and Antioxidant Signaling

Carly S Cox et al. Cell Metab. .

Abstract

Transient mitochondrial stress can promote beneficial physiological responses and longevity, termed "mitohormesis." To interrogate mitohormetic pathways in mammals, we generated mice in which mitochondrial superoxide dismutase 2 (SOD2) can be knocked down in an inducible and reversible manner (iSOD2-KD mice). Depleting SOD2 only during embryonic development did not cause post-natal lethality, allowing us to probe adaptive responses to mitochondrial oxidant stress in adult mice. Liver from adapted mice had increased mitochondrial biogenesis and antioxidant gene expression and fewer reactive oxygen species. Gene expression analysis implicated non-canonical activation of the Nrf2 antioxidant and PPARγ/PGC-1α mitochondrial signaling pathways in this response. Transient SOD2 knockdown in embryonic fibroblasts from iSOD2-KD mice also resulted in adaptive mitochondrial changes, enhanced antioxidant capacity, and resistance to a subsequent oxidant challenge. We propose that mitohormesis in response to mitochondrial oxidative stress in mice involves sustained activation of mitochondrial and antioxidant signaling pathways to establish a heightened basal antioxidant state.

Keywords: NRF2; PPARγ; hormesis; mitochondria; mtDNA; oxidative stress; reactive oxygen species; signaling; superoxide dismutase.

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Conflict of interest statement

Declaration of Interests. The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Generation of transient mitochondrial oxidative stress via inducible knock-down of SOD2 during mouse embryogenesis
A) Timeline of SOD2 knock-down and recovery in iSOD2-KD mice. As indicated, doxycycline (DOX) was fed to pregnant mothers to achieve knock-down in embryos from day 8.5e to day 12.5e or from day 0.5e to day 12.5e. At day 12.5e, DOX was discontinued, and the pups were aged to 4 weeks. In all cases, negative controls were DOX-treated littermates only expressing the rtTA transgene. B) Western blot of SOD2 in day 12.5 embryos relative to negative controls. C) Mitochondrial aconitase activity (U/min) in day 12.5 embryos relative to negative controls. 100% equals aconitase activity of 5.720 nmol/min/ml. D) TBARS assay for malondialdehyde (MDA) in day 12.5 embryos relative to negative controls. E) Western blots for SOD2 in adapted liver relative to negative controls. GAPDH was probed as a control for loading. F) Mitochondrial aconitase activity (U/min) of adapted liver relative to negative controls. Error bars represent +/−SEM. 100% equals aconitase activity of 2.969 nmol/min/ml. All calculations for statistical significance were completed using a non-parametric, unpaired, two-tailed t-test. n=6.
Figure 2.
Figure 2.. Adaptive changes in liver of mice that experienced SOD2 knock-down only during embryogenesis
A) Venn Diagram depicting the differentially expressed genes from adapted livers (from two strains, each containing a different shRNA) after knock-down with each shRNA (bottom). Predicted transcription factors that drive the observed changes in gene expression based on analysis of cis-regulatory elements enriched in the promoters. PPARγ, Peroxisome Proliferator-activated receptor gamma; Nrf2, Nuclear factor, erythriod-2 like 2; PGC-1α, Peroxisome Proliferator-activated receptor gamma coactivator 1-alpha; PPARα, Peroxisome Proliferator-activated receptor alpha. B) RNA expression levels (normalized to tubulin RNA) of top upregulated genes in adapted liver from iSOD2 mice. Error bars represent +/− SEM. ARK1C18, aldo-keto reductase family 1, member C18; GSTT1, glutathione s-transferase 1; GSTT3, glutathione s-transferase theta 3; PTGR1, prostaglandin reductase 1; UCP2, uncoupling protein 2; PGC-1 α, Peroxisome Proliferator-activated receptor gamma coactivator 1-alpha. C) Western blots of NQO1 and PCG-1α in adapted liver relative to negative controls. Actin was probes as a loading control. D) Western blot of OXPHOS complexes, VDAC, and TFAM in adapted liver relative to negative controls. Actin was probed as a loading control. E) Hydrogen peroxide (Amplex Red Assay) present in adapted liver relative to negative controls. F) Total ROS (CellROX) in adapted iSOD2 hepatocytes relative to negative controls. G) Mitochondrial oxygen consumption rate (OCR; pmol O2/min) in adapted hepatocytes relative to negative controls. Error bars represent mean +/− SD of eight technical replicates. Inhibitors used in the Seahorse analysis were added as indicated VM oligomycin (a), 1 μM FCCP (b), and 0.5 μM antimycin A + 0.5 μM rotenone (c). n=3 rtTA controls, n=2 adapted hepatocytes H) Electron microscopy of adapted hepatocytes relative to negative controls. Scale bar = 2 μM. All calculations for statistical significance were completed using a non-parametric, unpaired, two-tailed t-test. n=2-12.
Figure 3.
Figure 3.. Inducible SOD2 knock-down in mouse embryonic fibroblasts (MEFs) causes mitochondrial and cellular oxidative stress
A) Western blot for SOD2 in SOD2 knock-down MEFs relative to negative controls. GAPDH was probed as a loading control. B) MitoSox staining in SOD2 knock-down MEFs relative to negative controls. C) Mitochondrial aconitase activity (U/min) in SOD2 knock-down MEFs relative to negative controls. 100% equals aconitase activity of 4.244 nmol/min/ml. D) Mitochondrial oxygen consumption rate (OCR) in SOD2 knock-down MEFs compared to negative controls. Error bars represent +/− SD of six technical replicates. Inhibitors used in the Seahorse analysis were added as indicated 1μM oligomycin (a), 1 μM FCCP (b) and 0.5 μM antimycin A + 0.5 μM rotenone (c).◻E) Western blots of the indicated mitochondrial proteins in SOD2 knock-down MEFs relative to negative controls. F) Relative mtDNA copy number in SOD2 knock-down MEFs relative to negative controls. Error bars represent +/− SEM. G) Expression of antioxidant enzymes (normalized to tubulin RNA) in SOD2-KD MEFs relative to negative controls. HMOX1, Heme oxygenase 1; GCLM, glutamate-cysteine ligase modifier; NQO1, NAD(P)H quinone dehydrogenase 1; GRX, glutathione reductase. All calculations for statistical significance were completed using a non-parametric, unpaired, two-tailed t-test. n=6-8.
Figure 4.
Figure 4.. Analysis of adaptive responses to transient SOD2 knock-down in MEFs from iSOD2-KD mice
A) Western blot of SOD2 after DOX withdrawal to visualize the kinetics of SOD2 recovery. Actin was probed as a loading control. B) MitoSox staining adapted MEFs (day 12) relative to negative controls. C) Hydrogen peroxide (Amplex Red assay) of adapted MEFs (day 12) relative to negative controls.D) Western blots of indicated mitochondrial proteins in adapted MEFs (day 12). Actin was probed as a loading control. E) Relative mtDNA copy number in adapted MEFs (day 12). F) Mitochondrial oxygen consumption rate (OCR) in adapted MEFs (day 12) compared to negative controls. Error bars represent +/− SD of six technical replicates. Inhibitors used in the Seahorse analysis were added as indicated 1μM oligomycin (a), 1 μM FCCP (b), and 0.5 μM antimycin A + 0.5 μM rotenone (c). G) RNA expression of antioxidant and mitochondrial enzymes (normalized to tubulin) in adapted MEFs (day 12). Error bars represent +/− SEM. GSTT1, glutathione s-transferase theta 1; GSTT3, glutathione s-transferase 3; UCP2, uncoupling protein 2; PGC1a, Peroxisome Proliferator-activated receptor gamma coactivator 1-alpha; HMOX1, Heme oxygenase 1; NQO1, NAD(P)H quinone dehydrogenase 1. H) Western blot for the indicated antioxidant enzymes in adapted MEFs (day 12). Actin was probed as a loading control. I) Percent viable cells after 24 hours treatment with 50 μM menadione in adapted MEFs relative to negative controls. All calculations for statistical significance were completed using a non-parametric, unpaired, two-tailed t-test. n=4-9.
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