Fig. 4
From: C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction
Calcium and alpha-synuclein levels mediate dopamine toxicity. a-c Ventral midbrain neurons were incubated with 100 µM dopamine in the presence or absence of 5 µM isradipine. dSTORM super-resolution microscopy of alpha-synuclein and synaptotagmin-1 after 72 h revealed an increase in the area of alpha-synuclein puncta, an increase in the size of synaptotagmin-1 puncta and an increased co-localization of alpha-synuclein with synaptotagmin-1 upon dopamine treatment. These effects were reversed by the Cav1.3 calcium channel antagonist isradipine, showing decreased size of alpha-synuclein and synaptotagmin-1 puncta and decreased co-localization. ****p < 0.0001 for synaptotagmin and alpha-synuclein, **p < 0.0066, *p < 0.0446 for co-localization, calculated using one-way ANOVA with Tukey’s post-hoc correction, graphs indicate mean ± s.e.m. N = 2251, 1154, 1987 for synaptotagmin, d.f. 5353, n = 5198, 3210, 6845 for alpha-synuclein, d.f. 15250, where n indicates individual clusters identified from 30, 30, 29 images from three biological repeats, n = 30, 30, 29 for co-localization, where n indicates number of images. d Dopamine toxicity in SH-SY5Y cells after 72 h incubation with 100 µM dopamine was rescued upon treatment with 5 µM isradipine and upon alpha-synuclein knockdown, showing that both, calcium and alpha-synuclein are necessary for toxicity to occur. ***p < 0.0007, nsp = 0.9935 for isradipine, n = 12 for all groups, where n indicates number of wells, d.f. 44; **p < 0.0062, nsp = 0.9934 for alpha-synuclein knockout, n = 8 for all groups, where n indicates wells, d.f. 28, calculated using one-way ANOVA with Tukey’s post-hoc correction, graphs indicate mean ± s.e.m. Three biological repeats
