FIGURE 5.

Mitochondrial fragmentation by synuclein precedes neuronal death. Rat hippocampal neurons were transfected 5 days after plating with mRFP and either α-synuclein (syn) or vector control (con) (A–F), without or with mitoGFP (A, E, and F). MitoGFP (A) or mRFP (not shown) were then imaged on a daily basis using an automated microscope. Scale bar is 10 μm. B, cumulative risk of death curves indicate that primary neurons expressing synuclein have a significantly greater risk of death than control cells (***, p < 0.0001, log-rank test). n = 112–115 neurons per group. The experiment was repeated three times with similar results. C, mRFP fluorescence of individual neurons was measured 24 h after transfection and used as a surrogate for synuclein expression. Based on mRFP fluorescence, neurons were stratified into three expression groups (low, medium, and high). a.u., arbitrary units of fluorescence. Within each of these groups, synuclein (dark) and control (light) transfected neurons showed no significant difference in mRFP fluorescence. D, cumulative risk of death curves indicates that neurons expressing higher levels of synuclein (red and blue) have a dose-dependent greater risk of death than controls expressing equivalent mRFP. **, p < 0.005; ***, p < 0.0001; ns, not significant, n = 22–46 neurons per group. E, cumulative risk of death curves show that neurons with fragmented mitochondria at 48 h have a progressively greater risk of death than neurons displaying intermediate or nonfragmented mitochondria (*, p < 0.05; **, p < 0.005; ***, p < 0.001). E and F, neurons with fragmented mitochondria express progressively higher levels of synuclein (*, p < 0.05).