Figure 2

Mitochondrial axonal transport is differentially affected by αSyn variants. (A) Neuron co-transfected with mito-GFP and WT αSyn. Scale bar, 100 μm. (B) Mitochondria (mito-GFP) in axonal projections from a transfected neuron used for live-imaging, magnified projection, and kymograph plotting. Kymograph obtained from a 150-s time-lapse movie at 3-s intervals. Left panel scale bar: 30 μm, horizontal: 10 μm. (C) Representative kymographs for αSyn transfected neurons obtained from supplementary movies (Sup Movies 1–4). Scale bar, 10 μm. (D) Overall anterograde, retrograde, or stationary axonal mitochondrial proportion does not change upon αSyn overexpression. Data represented as median plus interquartile range (n > 50 axons per condition from 10 independent experiments). Kruskal-Wallis ANOVA for each type of movement (p > 0.05). (E) Anterograde to retrograde moving time differences observed in control mitochondria are lost in A53T transfected neurons but not in WT and A30P αSyn. (F) Anterograde to retrograde mean segmental velocity differences are lost in WT and A53T αSyn-transfected neurons (n = 60 per condition from 10 independent experiments). (G) Anterograde to retrograde mitochondrial flux (traveled distance per mitochondria normalized to acquisition time (nm/seg)) is impaired in all αSyn overexpression conditions compared with control (n = 60 per condition from 10 independent experiments). Data represented as median plus interquartile range. Mann-Whitney U test between anterograde and retrograde movements for each parameter. ***p < 0.001, **p < 0.01, *p < 0.05. (H) αSyn overexpression does not impair mitochondrial membrane potential. Mitochondrial membrane potential was analyzed in transduced DIV14 neurons using JC1. Low and high (green and red emission, respectively) membrane potential was quantified by fluorescence intensity and Manders’ colocalization coefficient was calculated. Mitochondrial ionophore FCCP was used as positive control. Data represented as mean ± SEM (n = 7 independent experiments). Two-way ANOVA followed by Sidak’s corrected comparison of Manders’ coefficient for each condition, ***p > 0.001. Scale bar, 200 μm. See also Movies M1–M4, Figure S4.