Fig. 4.

Description of the endo-lysosomal pathway in Lrrk2 striatal astrocytes. A Representative TEM image of primary striatal astrocyte section containing electron-dense lysosomal-like structures (arrows). Scale bar 2 μm. B, C Forty TEM images were acquired (n = 4 per genotype). Each cell was imaged by covering the entire cytoplasm and lysosomal-like structure number and area were measured using ImageJ. D Representative image of the staining using Lamp2A (green) as a marker for the endo-lysosomal pathway, DAPI (blue) for the nuclei, and F-actin (cyano) to define cells. Scale bar 20 μm. Inset shows a close-up of Lamp2A-positive structures. Quantifications of Lamp2A-positive structure number and area were analyzed using ImageJ. E, F Four images per cell culture were analyzed (n = 3 per genotype). G Measurement of lysosomal pH was done in primary striatal astrocytes from the three genotypes upon unlabeled α-syn PPF treatment (n = 3 per genotype). Bafilomycin has been applied as negative control. Fluorescence ratio of light acquired at 535 nm upon excitation at 340 and 380 nm is provided. H Neutral red assay was performed in primary striatal astrocytes from the three genotypes upon unlabeled α-syn PPF treatment (n = 4 per genotype). Bafilomycin has been applied as negative control. Absorbance at 540 nm measured upon cell lysates was normalized by total protein content. Statistical analysis in B, C, and F was made by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Statistical analysis in E, G, and H was performed with one-way ANOVA followed by Tukey’s multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001