Increased mitochondrial oxidative stress in the Sod2 (+/-) mouse results in the age-related decline of mitochondrial function culminating in increased apoptosis

Abstract

To determine the importance of mitochondrial reactive oxygen species toxicity in aging and senescence, we analyzed changes in mitochondrial function with age in mice with partial or complete deficiencies in the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD). Liver mitochondria from homozygous mutant mice, with a complete deficiency in MnSOD, exhibited substantial respiration inhibition and marked sensitization of the mitochondrial permeability transition pore. Mitochondria from heterozygous mice, with a partial deficiency in MnSOD, showed evidence of increased proton leak, inhibition of respiration, and early and rapid accumulation of mitochondrial oxidative damage. Furthermore, chronic oxidative stress in the heterozygous mice resulted in an increased sensitization of the mitochondrial permeability transition pore and the premature induction of apoptosis, which presumably eliminates the cells with damaged mitochondria. Mice with normal MnSOD levels show the same age-related mitochondrial decline as the heterozygotes but occurring later in life. The premature decline in mitochondrial function in the heterozygote was associated with the compensatory up-regulation of oxidative phosphorylation enzyme activity. Thus mitochondrial reactive oxygen species production, oxidative stress, functional decline, and the initiation of apoptosis appear to be central components of the aging process.

Figure 1

Western blot analysis of MnSOD expression in Sod2^tm1Cje mouse liver mitochondria. Neonate Sod2^tm1Cje (−/−) and controls on the left (+/+). Adult Sod2^tm1Cje (+/−) and controls (+/+) on the right. Expression was detected with antisera raised against MnSOD.

Figure 2

Mitochondrial oxygen consumption in neonatal Sod2^tm1Cje (−/−) and (+/+) livers. Liver mitochondrial respiration was measured on mitochondria from 8- to 10-day-old mice. State III (ADP-stimulated), state IV, and uncoupled respiration rates were determined for Sod2^tm1Cje (−/−) (▴) and control (●) animals. The data shown are the means and standard errors for four independent experiments.

Figure 3

Measurement of the mitochondrial membrane potential (ΔΨ) in aging Sod2^tm1Cje (+/−) and (+/+) mouse livers. ΔΨ was measured by the uptake of tetraphenylphosphonium cation (TPP⁺) in Sod2^tm1Cje (+/−) (▴) and in control (●) animals. The data shown are the means and standard errors for four to six independent experiments.

Figure 4

Mitochondrial oxygen consumption in aging Sod2^tm1Cje (+/−) and (+/+) mouse livers. (A) State IV rates of aging Sod2^tm1Cje (+/−) (▴) and controls (●). (B) State III (ADP-stimulated) rates of aging Sod2^tm1Cje (+/−) (▴) and controls (●). Data shown are the means and standard errors for four to six independent experiments.

Figure 5

Lipid peroxidation measurements in aging Sod2^tm1Cje (+/−) and (+/+) mouse livers. (A) Levels of lipid peroxides in liver mitochondria from Sod2^tm1Cje (+/−) (▴) and controls (●). (B) Total liver cytoplasmic lipid peroxide measurements from Sod2^tm1Cje (+/−) (▴) and controls (●). Data shown are the means and standard errors from four independent experiments performed in triplicate.

Figure 6

Calcium-induced high-amplitude swelling (t_1/2) in aging Sod2^tm1Cje (+/−) and (+/+) mouse livers. The opening of the mtPTP was measured by cyclosporin A-inhibitable high-amplitude swelling in Sod2^tm1Cje (+/−) (▴) and controls (●). The t_1/2 is defined as the time in seconds until the mitochondria are half maximally swollen. The data shown are the means and standard errors from six independent experiments.

Figure 7

TUNEL analysis of old Sod2^tm1Cje (+/−) and (+/+) mouse livers. Livers were snap frozen, sectioned, and analyzed for apoptotic hepatocytes. (A) The percentages of TUNEL-positive hepatocytes were determined from six separate livers. The data and the means and standard errors are shown. (B) Liver images from an old Sod2^tm1Cje (+/−) and control mouse.

Figure 8

Mitochondrial OXPHOS enzymology measurements of Sod2^tm1Cje (+/−) and (+/+) mice. (A) Measurements of the changes of cytochrome c oxidase (COX) and citrate synthase (CS) with age in Sod2^tm1Cje (+/−) (▴) and controls (●). (B) Relative enzyme activities of liver submitochondrial particles in old Sod2^tm1Cje (+/−) (■) and controls (□). The data shown are the means and standard errors of four independent experiments performed in duplicate.