Fig. 3: Loss of GCase activity disrupts mitochondria–lysosome contact untethering in GBA1-PD patient dopaminergic neurons.

a, b Western blot analysis of PD patient-derived mutant GBA1 dopaminergic neurons (∆GBA; het 84GG) and its CRISPR-corrected isogenic control (Corr) neurons. GCase level was significantly reduced in ∆GBA neurons (N = 4 independent experiments). Paired two-sided Student’s t-test; **p = 0.0083. c–f ∆GBA and Corr neurons were treated with either DMSO or BafA1 and subjected to live-cell GCase activity analysis. e Quantification of the area under each curve (AUC) demonstrates decreased total GCase activity in ∆GBA neurons. Paired two-sided Student’s t-test; *p = 0.0183. f Lysosomal GCase activity was calculated by subtracting BafA1 values from DMSO. Values are expressed as fold-change compared to isogenic controls (N = 3 independent experiments). Paired two-sided Student’s t-test; *p = 0.0352. g Representative time-lapse confocal images of contacts between mitochondria (red, Mito-RFP) and lysosomes (green, Lyso-GFP) in Corr (left) and ∆GBA (right) human neurons. Yellow arrows mark stable M–L contacts. White arrows mark the site of M–L contacts after contact untethering. Black line shows duration of contacts. Scale bar = 500 nm (see Supplementary Movies 7 and 8). h Quantification of average minimum duration (left) and relative frequency distribution of the duration of stable M–L contacts (right), showing increased duration of stable M–L contacts in ∆GBA neurons (n = 48 contacts from Corr and n = 71 contacts from ∆GBA, N = 3 independent experiments). X-axis of the histogram represents bin centers. Bin width = 60 s. Unpaired two-sided Student’s t-test; *p = 0.0204. i Percentage of lysosomes contacting mitochondria (for >20 s) (n = 12 Corr and n = 36 ∆GBA neurons, N = 3 independent experiments). Unpaired two-sided Student’s t-test. For all quantifications, data are means ± S.E.M.; *p ≤ 0.05, **p ≤ 0.01, ns: not significant.