Fig. 3: Ultrastructural analysis of endo-lysosomal compartment and GCase function in fibroblasts from LRRK2 G2019S mutant compared with controls.

a Low magnification (2–5 µm) TEM micrographs showing electron-dense structures accumulating in the LRRK2 G2019S fibroblasts (Patient #1), while they are almost completely absent in control cells (Control #1). b High magnification (500 nm) TEM images three patient fibroblast cell lines and age and sex matched controls. Patient fibroblasts show accumulation of endo-lysosomal structures that resemble multilamellar bodies (MLBs). c Violin plots of the number of the MLBs per µm² for each patient and each control cell line, showing that the number of MLBs is larger in LRRK2 G2019S fibroblasts than in control cells (given a certain degree of variability across individuals) (one-way ANOVA, Tukey’s multiple comparisons test; **p < 0.01, ***p < 0.001, ****p < 0.0001). d Western blot analysis of GCase, LIMP2, pT731 RAB10 and total RAB10. e Quantification of ELISA assays for pS935 LRRK2, total LRRK2 (UDD3 antibody) and pLRRKtide (ng/ml LRRK2) (Mann–Whitney non-parametric test, p > 0.05; data are expressed as mean ± SEM). f Quantification of western blots in e (Mann-Whitney non-parametric test, controls vs. patients: pRAB10 p > 0.999, GCase p = 0.0095, LIMP2 p = 0.019; data are expressed as mean ± SEM). g CGase activity assessed with the in-lysate 4-MU method. G2019s patients display increased activity compared to controls while MLi-2 treatment has no effect in both genotypes (two-way ANOVA, genotype effect p = 0.001 F(1, 16) = 16.04, treatment effect p = 0.2891 F(1, 16) = 1.202). h GCase activity assessed with the in-cell method from Benz 2021 protocol is significantly increased in G2019S patients (Mann–Whitney non-parametric test, p = 0.0381; data are expressed as mean ± SEM). Each dot represents one subject and the value is the average of two biological replicates each with three technical replicates.