Fig. 1

Drp1 inhibition reduces mitochondrial fragmentation induced by α-syn in rat dopaminergic neuronal cells. a Stable rat dopaminergic N27 cells with ecdysone-inducible system containing human wild type SNCA were transfected with DsRed-Mito overnight, treated with 20 μM PonA for 48 h to induce α-syn expression, followed by immunostaining for α-syn (gray) and Drp1 (green). Representative images show the effects of α-syn on mitochondrial morphology and Drp1 colocalization with mitochondria. b Total levels of α-syn and Drp1 in stable cells expressing empty vector control or SNCA after 48 h of 20 μM PonA treatment were assessed using immunoblotting. c N27 cells were co-transfected with DsRed-Mito and rat siRNA-Drp1 or scramble control overnight, then induced with PonA for 48 h. Representative images showing siRNA-Drp1, but not scramble control, attenuated mitochondrial fragmentation induced by α-syn. d As a complementary genetic approach, N27 cells were co-transfected with the dominant-negative mutation Drp1-K38A (HA- tagged) and DsRed-Mito, then induced with PonA for 48 h before immunostained for HA and α-syn. Representative images showing mitochondrial morphology in Drp1-K38A expression (arrows pointing to the characteristic Drp1 puncta formation) cells versus α-syn overexpression alone. e N27 cells were transfected with DsRed-Mito, and induced with PonA plus mdivi-1 (10 μM) or vehicle control (DMSO) for 48 h. Scale bars: 20 μm. Imaging data from c-e were quantified for mitochondrial morphology using Image J and summarized quantitatively in f. Both the values of “roundness” and “aspect ratio” approach 1 as the particle becomes circular. Data represents mean ± SEM, analyzed by one-way ANOVA (n = 4 or 5 independent experiments with > 500 mitochondria /group quantified for each experiment), followed by Newman-Keuls post hoc test. *p < 0.05