Mitochondrial Peroxiredoxin 6 Declines With Aging Concomitant With Elevated Hydrogen Peroxide Generation

Purpose: Oxidative eustress is the physiological range in which the tissues maintain redox homeostasis characterized by a metabolic steady state. The diminished ability to scavenge free radicals at the same rate from which they are generated has been proposed to be part of the aging process. Peroxiredoxins (PRDXs), a class of antioxidants generally known for regulating levels of hydrogen peroxide (H2O2) in multiple tissues and cellular compartments. Here, we hypothesized that PRDX6 localizes to the mitochondria targeting mitochondrially generated ROS, and that gradual loss of mitochondrial PRDX6 contributes to increased damage during aging. Methods: In this study, respiratory rates and complex activities in isolated skeletal muscle mitochondria obtained from young (3-6 months) and old (20-24 months) C57/BL6J mice. Amplex UltraRed was used to determine rates of mitochondrial H2O2 production. Immunoblots were used to quantify protein and ELISA was used to measure lipid peroxidation in mitochondrial fractions. Results: Our findings revealed that isolated mitochondria from skeletal muscle in old mice generated higher rates of H2O2 during state 3 and state 4 (S3; S4) derived respiration (3.93 ± 0.41; 3.56 ± 0.38 pmol H2O2..s-1.mg-1) compared to young mouse mitochondria (1.57 ± 0.48; 1.89 ± 0.59 pmol H2O2.s-1.mg-1) (p < 0.05). This was also found when saturating amounts of ADP were used to stimulate maximal rates of oxygen consumption (old: 3.65 ± 0.77; young 1.40 ± 0.21 pmol H2O2-1.mg-1, p < 0.05). Similarly, mitochondrial PRDX6 abundance was lower while levels of lipid peroxidation product malondialdehyde were higher in skeletal muscle from old animals (old: 2712 ± 1320 pg.mL-1; young: 489 ± 225 pg.mL-1 ,p < 0.05). Conclusions: These results show higher mitochondrial ROS generation rates and oxidative damage in old animals. These data also suggest that loss of PRDX6 in aging leads to mitochondrial dysfunction and membrane integrity. We are currently conducting mechanistic studies in cellular models with disrupted PRDX6 expression to confirm the effect of this protein in maintaining mitochondrial function. This study offers novel observations on the significance of mitochondrially localized PRDX6 in antioxidant defense as well as the sources and roles of ROS generation in contributing to an aging phenotype. NIH Grant #: 1 R01 AG059715-01