Fig. 5.

ER–mitochondria Ca²⁺ exchange is disrupted in SH-SY5Y cells stably expressing α-synuclein and overexpression of VAPB to increase ER–mitochondria contacts rescues disrupted Ca²⁺ exchange. a, b Reduced mitochondrial Ca²⁺ uptake and delayed ER–mitochondrial Ca²⁺ exchange following IP3 receptor-mediated release from ER-stores in α-synuclein, α-synucleinA53T and α-synucleinA30P expressing cells. IP3 receptor-mediated Ca²⁺ release was triggered with oxotremorine-M (OxoM) and mitochondrial and cytosolic Ca²⁺ levels detected by Rhod2 and Fluo4 fluorescence, respectively. a Representative Rhod2 fluorescence traces are shown with normalized peak values (F/F ₀) in the bar chart. Data were analysed by one-way ANOVA and Tukey’s post hoc test. N = 29–44 cells from 3 different experiments; error bars are SEM, ***p < 0.001. b Delayed mitochondrial Ca²⁺ uptake following IP3 receptor-mediated release from ER-stores in α-synuclein, α-synucleinA53T and α-synucleinA30P expressing cells. Representative Fluo4 (cytosolic; Cyto) and Rhod2 (mitochondria; mito) fluorescence traces are shown. Bar chart shows the time-lag between peak cytosolic and mitochondrial Ca²⁺ signals. Data were analysed by one-way ANOVA and Tukey’s post hoc test. N = 85–115 cells from 4 independent experiments; error bars are SEM, **p < 0.01 and ***p < 0.001. c Expression of VAPB to increase ER–mitochondria contacts increases mitochondrial Ca²⁺ levels following IP3 receptor-mediated release and rescues defective Ca²⁺ uptake induced by α-synuclein. Representative Rhod2 fluorescence traces are shown with normalized peak values (F/F ₀) in the bar chart. Data were analysed by one-way ANOVA and Tukey’s post hoc test. N = 16–72 cells from three different experiments; error bars are SEM, **p < 0.01, ***p < 0.001