Extended Data Fig. 7: Related to main Fig. 3 & 4.
From: Pathological structural conversion of α-synuclein at the mitochondria induces neuronal toxicity
(a) The hydrodynamic diameter (dh) of 150 mM lipid vesicles was determined by dynamic light scattering. (ai) Correlation function of the sample (aii) Size distribution of lipid vesicles determined by mass unweighted fitting which shows the distribution of lipid vesicles centered at 89, 92 and 104 nm for 15%, 40% and 100% cardiolipin in the vesicles, respectively. (b) No increase in ThT was observed in the control experiments with either A53T alone (black line) or liposomes alone (red line). (c) Kinetics of amyloid formation by α-Syn (50 µM) in the presence of DMPS (curve 1-4) and in the presence of DMPC (curve 5-8). Measurements were performed at 37 °C and pH 7.4. Protein concentration was 50 µM and lipid vesicle concentration was 400 µM. Thioflavin T (ThT) fluorescence was excited at 450 nm, and the emission wavelength was 482 nm. (d) NAO is co-localized mainly with a mitochondrial indicator, TMRM. (ei & ii) Single-molecule light microscopy (SMLM) images showing tom20-labeled mitochondria (dSTORM) and aggregates imaged with the aptamer (aptamer-DNA PAINT) in cyan, in WT and A53T oligomer treated iPSC-derived cortical neurons. Panel highlighted shows higher magnification. (eii) Quantification of the number of single-molecule localizations detected per aggregate in WT and A53T oligomer treated conditions (n = 70-310 aggregates per condition, *** p < 0.0005, Mann-Whitney t-test). (f) Contacts between α-Syn aggregates (labeled with amytracker) and cardiolipin (labelled with NAO) (fi) Representative images. (fii) Amytracker exhibits a higher fluorescent signal in SNCA-A53T than iso-CTRL neurons after the application of recombinant WT (99% monomer, 1% oligomer) (n = 3 fields imaged over 2 independent experiments). (fiii) Amytracker co-localization with NAO is higher in SNCA-A53T neurons than iso-CTRL (n = 3 fields imaged over 2 independent experiments). Note. Data are represented as Data ± SEM (box) unless mentioned. *p < 0.05, ***p < 0.0005. Detailed statistical information is provided in Supplementary Table 1.
