Fig. 2

Defects in mitochondrial structure and function in α-syn mutant hNs. a, b hiPSC-derived A53T hNs (a) and hESC-derived A53T and E46K hNs (b) have more highly fragmented mitochondria compared to corrected hNs and WT hNs respectively as shown by mitoDSRed expression; scale bar: 10 μm. c Percentage of total hNs that have fragmented mitochondria. Data represent mean ± s.e.m. **P < 0.01 by ANOVA followed by Tukey’s post hoc test, for hiPSCs, n = 12 coverslips, for hESCs, n = 6 coverslips from 3 independent differentiations, DIV: 60. d–f Transmission electron micrographs of hiPSC-derived corrected and A53T hNs (d) or hESC-derived WT, A53T and E46K hNs (e) show that mutant cells contain smaller mitochondria, with a lower average diameter (f), **P < 0.01 by ANOVA followed by Tukey’s post hoc test, n = 6 independent cultures from 3 independent differentiations. g Micrographs of neurites from corrected (left panels) and A53T-mutant (right panels) hNs expressing mitoDSRed and labeled for α-syn phosphoserine 129 (PS129). Arrows show areas where PS129 colocalizes with MitoDSRed. Boolean operation was employed to pseudo-label regions of colocalization in blue. Boxes show high magnification of the regions indicated. Scale bar: 10 μm. h Quantification of hNs with α-syn PS129 on fragmented mitochondria. Data represent mean ± s.e.m. **P < 0.0001 by t-test, n = 6, DIV: 60. i, j Proximity of Tom20 and α-syn as assessed by proximity ligation assay (PLA) (i) showed significantly more α-syn within 40 nm of the OMM in A53T relative to corrected hNs (j). **P = 0.0055 by Student's t-test, n = 6 coverslips from 3 independent differentiations, DIV: 60, scale bar: 10 μm