Fig. 4.

Bioenergetic and oxidative stress consequences of the ND6 mutation in neuronal tissue. (A) Oxygen consumption rate (OCR) of synaptosomes in the presence of 1 µg⋅mL⁻¹ oligomycin, 5 µM veratridine, and 4 µM FCCP (n = 6). The reported results are the OCR of synaptosomes from the ND6 mutant mice expressed as a percentage of the rate of synaptosomes isolated from control mice. (B) Ability of synaptosomes from control and ND6 mutant to maintain ATP levels under energetically demanding conditions. The ATP levels of synaptosomes were determined before and after 15 min of incubation without (control) or with plasma membrane–depolarizing agents, and the results are expressed as the percentages of the initial ATP level (n = 7). In ND6 P25L mice, the initial ATP levels were 126,535 ± 14,871 relative light units (RLU)/25 µg of synaptosomes, and in B6 control mice, the initial ATP levels were 126,663 ± 10,264 RLU/25 µg. (C) ability of synaptosomes to maintain ATP levels in the presence of 100 nM rotenone (n = 3). (D) Rate of hydrogen peroxide production by nonsynaptosomal brain mitochondria measured using the Amplex red system (n = 5). The rate is expressed as the percentage difference between mitochondria isolated from the ND6 mutant and control mice at the same time. (E) Rate of hydrogen peroxide generation from synaptosomes in the presence and absence of rotenone (4 µM) using glucose as a substrate (n = 7). The rate is expressed as the percentage. (F and G) Immunoblotting of 3-nitrotyrosine (F) and GFAP (G) levels in whole-brain lysates (n = 5). N refers to independent preparations of mitochondria, synaptosomes, or whole-cell lysates. Assays were performed at least in triplicate for each preparation.