Figure 4

Alpha-synuclein (ASYN) oligomers induce the disruption of the mitochondrial network and impair mitochondrial membrane potential in dopaminergic neurons independently of endogenous ASYN: (A, B, C and D) show representative confocal images of TH-GFP WT and TH-GFP ASYN-KO neurons (*) treated with 647-ASYN oligomers (green) and stained with the live-dye TMRE (red). (A) TH-GFP WT untreated neurons. (B) TH-GFP WT neurons with low load of ASYN. (C) TH-GFP WT neurons with higher amount of ASYN forming inclusions with no mitochondrial activity (arrows) and (D) TH-GFP ASYN-KO neurons with high amounts of ASYN forming inclusions with no mitochondrial activity (arrows). Arrowheads in (C) and (D) mark TH-GFP expressing neurons. Scale bar: 20 µm. Dot-plot graphic in (E) shows the effect of 647 tagged ASYN-oligomers on mitochondrial membrane potential (TMRE signal). No significant difference in the TMRE signal ratio is observed between WT and ASYN KO for neurons without 647 tagged ASYN uptake (TMRE no ASYN vs. no ASYN) (1-way ANOVA followed by Newman-Keuls Multiple Comparison Test: mean diff. = 0.09206 ± 0.1099; P = 0.4239). On the other hand, 647 tagged ASYN -containing WT neurons have a higher TMRE signal ratio compared to 647 tagged ASYN -containing ASYN KO neurons (TMRE ASYN vs. ASYN, 1-way ANOVA followed by Newman-Keuls Multiple Comparison Test: mean diff. = − 0.1999 ± 0.06873; P = 0.0103). Finally, ASYN KO neurons with 647 tagged ASYN inside have a significantly decreased TMRE signal ratio compared to WT and ASYN KO neurons without 647 tagged ASYN inside (1-way ANOVA followed by Newman-Keuls Multiple Comparison Test: mean diff. = 0.6002 ± 0.06999; P = 0.0010) Dot-plot graphic in (F) shows the effect of 647-tagged ASYN oligomers on TH-GFP fluorescence intensity. No significant difference in the TH (GFP) mean fluorescence intensity is observed between WT and ASYN KO without ASYN uptake (no ASYN)(1-way ANOVA followed by Bonferroni’s Multiple Comparison Test: mean diff. = 949.5 ± 10,159; P = 0.9268). Similarly, no significant difference is observed between ASYN-containing WT and ASYN-containing ASYN KO neurons (1-way ANOVA followed by Bonferroni’s Multiple Comparison Test: mean diff. = 7976 ± 5225; P = 0.1464). However, there is a significant reduction in the TH signal in ASYN-containing ASYN KO and WT neurons compared to neurons without 647 tagged ASYN inside (1-way ANOVA followed by Bonferroni’s Multiple Comparison Test: mean diff. = 26,221 ± 8900; P = 0.0114). Each data point in the graphs represents the signal from 1 neuron.