Fig. 6: WT and mutant α-syn targeting to the mitochondrial matrix differently affects mitochondrial ATP levels.

a Complementation of the mitochondrial matrix targeted GFP_1-10 non-fluorescent moiety upon co-expression of the targeted mt WT α-synS11. The mitochondrial localization was verified by mitotracker staining. Confocal images were acquired at 488 and 594 nm excitation wavelength. b, c, d Histograms showing mitochondrial ATP production upon histamine stimulation measured by mtLuc probe in HeLa cells overexpressing untargeted or mitochondrial matrix targeted α-syn in HeLa cells untreated b or treated with 10 μM rotenone c or d 10 μM rotenone + 10 μM antimycin for 12 h at 37 °C in a 5% CO₂ atmosphere. e Histograms showing mitochondrial ATP production upon histamine stimulation measured by mtLuc probe in HeLa cells overexpressing mitochondrial matrix targeted mt WT, mt A53T and mt A30P α-syn. Average values are shown as mean % calculated from the counts per second (cps) with respect to control cells. f Mitochondrial Ca²⁺ transients upon stimulation with 100 μM histamine were measured in HeLa cells by co-transfecting mtAEQ and empty vector, or mt WT, mt A53T and mt A30P α-syn. Bars represent mean Ca²⁺ peak values upon histamine stimulation. Data were collected from at least 10 coverslips/conditions from three independent experiments. (One-way ANOVA coupled with Dunnett’s multiple comparison test retrieved a statistically significant difference between the groups: **p < 0.01, *p < 0.05). At least 10 independent measurements for three independent transfections have been done for each construct. Scale bar is 20 μm