Fig. 3

Drp1 inhibition protects against impaired autophagy and protein aggregation induced by Mn in N27 dopaminergic neuronal cells. Stable N27 cells expressing inducible human wild-type α-synuclein were co-transfected with siRNA-Drp1 (KD) and LC3-mcherry (due to low endogenous LC3 levels in this cell type) for 24 h. (A) Cells were then treated for another 24 h with MnCl2 (125 µM) or vehicle controls (Con 1 & Con 2), in the presence or absence of PonA (20 µM) to induce α-synuclein expression. (B), Following confocal imaging, the number of green and red vesicles, representing respectively p62 and LC3 puncta was quantified using Imaris image analysis software. Data represent mean ± SEM (n = 3 independent experiments, with at least 15 cells for each condition). Kruskal-Wallis ANOVA was used to compare between groups with Dunn’s post-hoc test. Scale bar 20 μm. (C), Representative confocal images showing total α-synuclein immunostaining (green) with or without Proteinase-K (PK) treatment. (D), N27 cells were transfected with siRNA-Drp1 (10nM) for 24 h, followed by α-synuclein overexpression induction (PonA20 µM), with and without Mn (125 µM) for 48 h. After post-fixation, cells were incubated with PK and then incubated with an antibody that detects α-synuclein (Millipore, AB5038). (E) Imaris was used to quantify PK-resistant α-synuclein-positive puncta. The number of aggregates was normalized to cytoplasmic volume. Data are shown as mean ± SEM (n = 3 independent experiments, with a minimum of 40 cells per group). Scale bar 20 μm