Somatic mtDNA mutations cause aging phenotypes without affecting reactive oxygen species production

Abstract

The mitochondrial theory of aging proposes that reactive oxygen species (ROS) generated inside the cell will lead, with time, to increasing amounts of oxidative damage to various cell components. The main site for ROS production is the respiratory chain inside the mitochondria and accumulation of mtDNA mutations, and impaired respiratory chain function have been associated with degenerative diseases and aging. The theory predicts that impaired respiratory chain function will augment ROS production and thereby increase the rate of mtDNA mutation accumulation, which, in turn, will further compromise respiratory chain function. Previously, we reported that mice expressing an error-prone version of the catalytic subunit of mtDNA polymerase accumulate a substantial burden of somatic mtDNA mutations, associated with premature aging phenotypes and reduced lifespan. Here we show that these mtDNA mutator mice accumulate mtDNA mutations in an approximately linear manner. The amount of ROS produced was normal, and no increased sensitivity to oxidative stress-induced cell death was observed in mouse embryonic fibroblasts from mtDNA mutator mice, despite the presence of a severe respiratory chain dysfunction. Expression levels of antioxidant defense enzymes, protein carbonylation levels, and aconitase enzyme activity measurements indicated no or only minor oxidative stress in tissues from mtDNA mutator mice. The premature aging phenotypes in mtDNA mutator mice are thus not generated by a vicious cycle of massively increased oxidative stress accompanied by exponential accumulation of mtDNA mutations. We propose instead that respiratory chain dysfunction per se is the primary inducer of premature aging in mtDNA mutator mice.

Fig. 1.

Analysis of levels of mtDNA mutations in tissues and characterization of ROS production and respiratory chain function in MEFs. (A) The levels of mtDNA mutations (mut; cytochrome b region) in wild-type (wt; blue bars) and mtDNA mutator (red bars) embryo heads and mouse brains at different time points. Bars show mean values, and error bars indicate the SD. The low background level of PCR-induced mutations has been subtracted. (B) FACS analysis of production after 30 min (thin lines) and 120 min (thick lines) incubation with dihydroethidium in primary cultures of MEFs from wild-type (blue lines) and mtDNA mutator (red lines) embryos. (C) FACS analysis of H₂O₂ production after 30 min incubation with carboxy-H₂DCFDA in wild-type (blue line) and mtDNA mutator (red line) primary MEF cultures. (D) FACS analyses of production after 30 min incubation with dihydroethidium in wild-type (blue line) and mtDNA mutator (red line) immortalized MEFs and primary MEFs from mtDNA mutator embryos (yellow line). The black line in all FACS analyses indicate negative control, i.e., wild-type cells analyzed without previous treatment with dihydroethidium or H₂DCFDA. FL1-H and FL2-H, fluorescence intensity in log scale (green and red fluorescence, respectively). (E) Polarographic investigation of respiratory chain function in immortalized MEFs from wild-type (blue bars) and mtDNA mutator (red bars) embryos. Measurements (mean values ± SD) were performed without addition of exogenous substrate (rotenone-sensitive respiration) and with addition of succinate, with or without uncoupler. Asterisks indicate level of statistical significance: *, P < 0.05; **, P < 0.01.

Fig. 2.

H₂O₂ induced necrosis and apoptosis in wild-type (blue bars) and mtDNA mutator (red bars) primary MEF cultures. Cells were stained with propidium iodide and annexin V and assayed for viability (mean values ± SD) by FACS analysis after 1 h incubation in culture media containing 0.5 mM (A), 2 mM (B), and 10 mM (C) H₂O₂ or in medium without H₂O₂ (D). Asterisks indicate level of statistical significance: *, P < 0.05; **, P < 0.01.

Fig. 3.

Oxidative damage to proteins in tissues of wild-type (blue bars) and mtDNA mutator (red bars) mice. (A) Relative amounts of carbonyl groups in liver total protein extracts, liver mitochondrial protein extracts, and heart total protein extracts at 40 weeks of age. (B) Aconitase enzyme activity in hearts of 12-, 25-, and 40-week-old mice. Bars show mean values, and error bars indicate SD. Asterisks indicate level of statistical significance: *, P < 0.05.

Fig. 4.

Expression of antioxidant defense enzymes. (A) Northern blot analyses showing transcript levels of GPX1 and SOD2 in hearts of 25- and 40-week-old mice. The levels of 18S ribosomal RNA was used as loading control. (B) Western blot analysis showing SOD2 protein levels in total protein extracts from heart, liver, and purified liver mitochondria at 40 weeks of age. Mut, mutants; wt, wild types.