Figure 5.

 Genetic knockout of Sirt3 increases SNc dopaminergic neuron degeneration induced by mitochondrial oxidative stress in mice. (A) Immunohistochemical staining of TH-expressing dopaminergic neuron in ventral midbrain of 5-month-old WT, DJ-1^−/−, Sirt3^−/− and Sirt3^−/−; DJ-1^−/− mice. (B,C) Bar graph summarizing numbers of dopaminergic neurons quantified by TH staining and total numbers of neurons by Nissl staining in 5-month-old mice. Number of SNc dopaminergic neurons in Sirt3^−/−; DJ-1^−/− double knockout mice (n = 6) was significantly (P < 0.002) reduced compared with the number in WT mice (n = 8), whereas there was no significant change in DJ-1^−/− mice (n = 8) or Sirt3^−/− mice (n = 6). Total number of neurons in Sirt3^−/−; DJ-1^−/− double knockout mice (n = 6) quantified by Nissl staining was also significantly reduced (P < 0.002) compared with WT (n = 8), DJ-1^−/− (n = 8), and Sirt3^−/− mice (n = 6). (D) Immunostaining of TH-positive dopaminergic neurons in the ventral midbrain of 15-month-old WT, DJ-1^−/−, Sirt3^−/− and Sirt3^−/−; DJ-1^−/− mice. (E,F) Bar graph summarizing the number of dopaminergic neurons and the total number of neurons in the substantia nigra of 15-month-old mice. Sirt3^−/−; DJ-1^−/− double knockout mice (n = 6) showed significantly (P < 0.002) decreased number of SNc dopaminergic neurons, compared with WT (n = 8), DJ-1^−/− mice (n = 8) and Sirt3^−/− mice (n = 6). By 15 months of age, the degeneration of SNc dopaminergic neurons in these double knockout mice were more pronounced than that in 5-month-old double knockouts (P < 0.002). Total number of neurons in Sirt3^−/−; DJ-1^−/− double knockout mice (n = 6) quantified by Nissl staining was also significantly reduced (P < 0.002) compared with WT (n = 8), DJ-1^−/− (n = 8), and Sirt3^−/− mice (n = 6). Data are mean ± SEM. **P < 0.01, one-way ANOVA with Tukey’s HSD post hoc analysis.