Reversal of the mitochondrial phenotype and slow development of oxidative biomarkers of aging in long-lived Mclk1+/- mice

Abstract

Although there is a consensus that mitochondrial function is somehow linked to the aging process, the exact role played by mitochondria in this process remains unresolved. The discovery that reduced activity of the mitochondrial enzyme CLK-1/MCLK1 (also known as COQ7) extends lifespan in both Caenorhabditis elegans and mice has provided a genetic model to test mitochondrial theories of aging. We have recently shown that the mitochondria of young, long-lived, Mclk1(+/-) mice are dysfunctional, exhibiting reduced energy metabolism and a substantial increase in oxidative stress. Here we demonstrate that this altered mitochondrial condition in young animals paradoxically results in an almost complete protection from the age-dependent loss of mitochondrial function as well as in a significant attenuation of the rate of development of oxidative biomarkers of aging. Moreover, we show that reduction in MCLK1 levels can also gradually prevent the deterioration of mitochondrial function and associated increase of global oxidative stress that is normally observed in Sod2(+/-) mutants. We hypothesize that the mitochondrial dysfunction observed in young Mclk1(+/-) mutants induces a physiological state that ultimately allows for their slow rate of aging. Thus, our study provides for a unique vertebrate model in which an initial alteration in a specific mitochondrial function is linked to long term beneficial effects on biomarkers of aging and, furthermore, provides for new evidence which indicates that mitochondrial oxidative stress is not causal to aging.

FIGURE 1.

Increased lifespan of Mclk1^+/− mice is confirmed in a mixed (129S6 x BALB/c) background and is independent of MCLK1 function in ubiquinone biosynthesis. A, Kaplan-Meier survival curves of Mclk1^+/+ (n = 19) and Mclk1^+/− (n = 63) mice are shown. Males are identified with red symbols, and the symbols corresponding to animals from the previous dataset are marked with an asterisk (18). Ubiquinone levels are normal at all ages in Mclk1^+/− mice. Quantification of UQ₉ levels in whole liver homogenates from 3-month-old (B) and 23-month-old (C) Mclk1^+/+ and Mclk1^+/− isogenic F1 males from a 129S6 x BALB/c cross was performed by HPLC analysis. UQ₁₀ is barely detectable in liver (not shown). Each dot in the graphs represents an individual mouse.

FIGURE 2.

Protection against age-dependent decrease in mitochondrial function in Mclk1^+/− animals. Oxygen consumption (state 3) levels of isolated liver mitochondria from young (3 months), middle-aged (12 months), and old (23 months) male (A and B) and female (C and D) isogenic F1 mice of both genotypes from a 129S6 x BALB/c cross. Oxygen consumption levels were measured with glutamate/malate (A and C) and succinate (B and D) as respiratory substrates. Mitochondrial function in young Mclk1^+/− mice is altered in both sexes and with both substrates. The Mclk1^+/+ mitochondria show the known age-dependent decrease in oxygen consumption, whereas the Mclk1^+/− mitochondria are only very mildly affected by the aging process. Each point in the graphs represents the mean ± S.E. of 10–12 animals. The asterisk denotes statistical significance of the difference between Mclk1^+/+ and Mclk1^+/− animals for a specific age; p < 0.05.

FIGURE 3.

Age-related changes in the levels of mitochondrial and cytosolic oxidative damage. Levels of protein carbonyls were quantified in mitochondrial (A) and cytosolic (B) extracts from young (3 months), middle-aged (12 months, cytosolic only), and old (23 months) Mclk1^+/+ and Mclk1^+/− isogenic F1 males from a 129S6 X BALB/c cross. Data are the means ± S.E. of 8–10 animals. The asterisk denotes statistical significance of the differences between 3- and 23-month-old Mclk1^+/+ as well as between 3- and 23-month-old Mclk1^+/− animals; p < 0.05.

FIGURE 4.

Reduction in age-dependent systemic oxidative stress. Biomarker of oxidative damage to DNA, 8-OHdG, and to membrane lipids, 8-isoprostanes, were quantified in plasma samples of young (3 months), middle-aged (12 months, 8-OHdG only), and old (23 months) male (A and B) and female (C and D) isogenic F1 mice of both genotypes from a 129S6 x BALB/c cross. Each point in the graphs represents the mean ± S.E. of 10–12 animals. The asterisk denotes statistical significance of the difference between Mclk1^+/+and Mclk1^+/− animals; p < 0.05.

FIGURE 5.

Mclk1 heterozygosity suppresses the increased mitochondrial oxidative stress and the loss of mitochondrial oxygen consumption of Sod2^+/− mutants. The partial loss of Mclk1 in Sod2^+/− Mclk1^+/− mutants suppresses the defective mitochondrial oxygen consumption (state 3) phenotype of Sod2^+/− mice with both complex I (A) and complex II (B) substrates and positively affects the Sod2^+/− phenotype by reducing the mitochondrial oxidative stress as shown by increased mitochondrial aconitase activity (C), decreased lipid peroxidation (D), and reduced levels of protein carbonyls (E). Mclk1 heterozygosity also increases the level of SOD2 enzymatic activity in Sod2^+/− Mclk1^+/− mutants (F). Each point or bar in the graphs represents the mean ± S.E. of 8–10 animals. The statistical significance of the differences between Sod2^+/− and Sod2^+/− Mclk1^+/− animals (*) is shown on the graphs; p < 0.05.

FIGURE 6.

2-Fold reduction of Mclk1 expression significantly reduces the levels of systemic oxidative damage observed in Sod2^+/− mice. Mclk1 heterozygosity reduced the levels of systemic oxidative stress as revealed by an increased mitochondrial aconitase activity (A) as well as by a reduction in the amounts of cytosolic lipid peroxidation (B), cytosolic carbonyls (C), and plasmatic 8-OHdG (D) observed in 15-month-old Sod2^+/− Mclk1^+/− mutants. Each point or bar in the graphs represents the mean ± S.E. of 8–10 animals. The statistical significance of the differences between 3- and 15-month-old Sod2^+/− Mclk1^+/− animals (A) or between Sod2^+/− and Sod2^+/− Mclk1^+/− animals (B–D) were shown on the respective graphs; p < 0.05.