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Fig. 4 | Acta Neuropathologica Communications

Fig. 4

From: Exosome release and neuropathology induced by α-synuclein: new insights into protective mechanisms of Drp1 inhibition

Fig. 4

Drp1 inhibition prevents autophagy flux impairment induced by α-syn in the autophagy reporter cells. a Schematic diagram illustrating the autophagy flux pathway and b the construct used to create the mRFP-GFP-LC3 stable reporter HeLa cells. With this cell model, autophagosomes appear yellow due to the colocalization RFP and GFP signals. Red signal indicates the flux is functional because the green signal is quenched by the acidic environment of the lysosome, which fuses with autophagosome. c These stable Hela cells were co-transfected with human α-syn-wild type plasmid and siRNA-Drp1 or with scramble (scr) control. Representative images of cells transfected with empty vector (EV) control, α-syn, α-syn plus scramble siRNA, and α-syn plus siRNA-Drp1 were captured using confocal microscopy. d The numbers of autophagosomes (green vesicles) and autolysosomes (red vesicles minus green vesicles) were quantified using ImageJ. e & f As a complementary genetic approach, these reporter cells were co-transfected with plasmids expressing α-syn plus either Drp1- K38A (HA-tagged) or empty vector control. After 48 h, cells were fixed and immunostained with anti-α-syn and anti-HA antibodies and subsequently quantified for autophagosomes and autolysosomes. g Hela cells were transfected with α-syn as described above and treated with the putative Drp1 inhibitor mdivi-1 (10 μM) or vehicle control 24 h later. Next day, cells were fixed and immunostained for α-syn. h. Quantitative analysis of autophagosomes and autolysosomes using Image J. i Cells were transfected with either scramble or siRNA-Drp1 for 24 h before the addition of α-syn pre-formed fibrils (PFF, 8 μg/well) for 48 h, changed media for 24 h and then fixed and immunostained for α-syn. j Quantitative analysis of autophagosomes, autolysosomes and α-syn puncta was performed using image J. All data represent mean ± SEM, n = 3–4 independent experiment with ~ 30 cells analyzed per group, using one-way ANOVA followed by the Newman--Keuls post hoc test. *p < 0.05. Scale bar: 20 μm

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