Fig. 2.

MitoQ₁₀ prevented caspase-dependent and independent death induced by NGF deprivation. (a) To determine the effects of the mitochondria-targeted agents on apoptosis in sympathetic neurons, cultures were deprived of NGF alone or in the presence of the indicated concentrations of the broad spectrum caspase inhibitor BAF, dTPP, MitoQ₁₀, MitoE₂, or MitoE₂ + MitoQ₁₀ (E + Q, 250 nM of each). Neurons that were not committed to die by caspase- dependent death (Commitment 1) were rescued by NGF replacement at 48 h; five days later the cells were fixed, counted, and quantified as the percent of viable neurons in sibling cultures that were maintained in NGF from the time of plating. MitoQ₁₀ treatment (0.25–1 μM) and BAF inhibited caspase-dependent death of NGF-deprived neurons (*p < 0.001). (b) To determine the effect of MitoQ₁₀ (500 nM) and MitoE₂ (500 nM) on caspase-independent death, cultures were deprived of NGF alone or with MitoQ₁₀ or MitoE₂ from the time of deprivation. These cultures were rescued with NGF 72 h later and quantified as described above. Cultures treated with BAF (50 μM) continued to die after 72 h, despite NGF readdition, indicating that the neurons were committed to die by caspase-independent death (Commitment 2). Cultures receiving MitoQ₁₀ alone and MitoQ₁₀ plus BAF treatment escaped impending death to a similar degree (p = 0.76), indicating that MitoQ₁₀ treatment was neuroprotective against both caspase-dependent and-independent death after 72 h of deprivation (*p < 0.001 for MitoQ₁₀ and MitoQ₁₀ + BAF versus –NGF + BAF). MitoE₂ had no effect on caspase- independent death. Results are presented as ±SEM from at least four independent experiments (n = 12–34 cultures). Statistical analysis was performed by ANOVA on ranks, followed by Dunns or Holm-Sidak post hoc tests for multiple comparisons between groups. (c) Representative micrographs of cultures treated as indicated, rescued with NGF after 48 or 72 h and fixed. Easily distinguishable cellular outlines of plump, crystal violet-stained neurons highlight viable neurons over the dead cells that show little or no staining.