Fig. 2

Drp1 inhibition improves mitochondrial function and reduces oxidative stress induced by α-syn. Drp1 inhibition was performed as described in Fig. 1 and the expression of α-syn was induced with 20 μM PonA for 48 h before the following experiments were performed: a & b Mitochondrial membrane potential (ΔΨm) was assessed using TMRM (50 nM), and fluorescent intensity was analyzed using flow cytometry. The uncoupler agent carbonyl cyanide 4- (trifluoromethoxy) phenylhydrazone (FCCP, 20 μM) was used as positive control to collapse ΔΨm to establish the threshold. Signal intensity (AU, arbitrary unit) was expressed as % above this threshold. c-g Mitochondrial respiration and glycolysis in live cells were assessed by measuring oxygen consumption rate and extracellular acidification rate using the XFe96 Extracellular Flux Analyzer. Sequential injections of oligomycin (to inhibit oxygen consumption mediated by ATP synthase), FCCP (an uncoupler to induce maximal OCR), Rotenone/Antimycin (to inhibit complex I and III, respectively). Spare Respiratory Capacity was calculated as % = (Maximal Respiration) / (Basal Respiration) × 100. α-syn overexpression reduced ATP production rate through oxidative phosphorylation, but not through glycolysis (d & e) as well as impaired spare respiratory capacity (f & g). Drp1 knockdown and mdivi-1 conferred protection. MitoSox red dye (h & i) and dihydroethidium (DHE, j & k) were used to measure mitochondrial and total cellular ROS, respectively, and signal intensity was quantified by plate reading and flow cytometry respectively. Data represent the mean ± SEM, one-way ANOVA (n = 4), followed by Newman-Keuls post hoc test. *p < 0.05 ** p ≤ 0.02, *** p < 0.001