Journal: Frontiers in Neuroscience

Article Title: LRRK2 Kinase Inhibition Rescues Deficits in Lysosome Function Due to Heterozygous GBA1 Expression in Human iPSC-Derived Neurons

doi: 10.3389/fnins.2020.00442

Figure Lengend Snippet: Broad lysosomal impairment is observed in GBA1 heterozygous-null neurons. High-content image analysis, as detected by Lysotracker TM staining normalized to number of cells, show (A) lysosome number in WT and two clones of GBA1 heterozygous-null iNs, (B) lysosomal number in cell bodies (proximal) vs. neurites (distal) and (C) average lysosomal area in these cells. Cy3 fluorescence from Lysotracker TM staining is observed in representative microscopy images. (D) Western blot analysis of LAMP1, nuclear TFEB, and actin proteins in WT and GBA1 heterozygous iNs. (E) Determination of lysosomal pH using LysoSensor TM in WT and GBA1 heterozygous iNs. (F) General lysosomal protease activity, as detected by DQ-BSA cleavage (green), in WT and GBA1 heterozygous iNs. Green fluorescence due to cleavage of DQ-BSA is observed in representative microscopy images. (G) Cathepsin B and (H) Cathepsin L activities, as detected by cleavage of Magic-Red substrate (red), in WT and GBA1 heterozygous iNs. Red fluorescence from Cathepsin B/L-specific substrate cleavage is observed in representative microscopy images. In all fluorogenic plate-based activity assays, nuclei were detected by Hoechst 33258 (blue) for normalization of fluorescent signal. All lysosomal analyses were collated from 2–3 independent experiments for each of three differentiations with 10–20 wells per genotype, per experiment, on 21-day old iNs ( N = 3, * p < 0.0001, ANOVA followed by Tukey’s post-hoc test).

Article Snippet: Blots were probed with primary antibodies to LAMP1 (abcam ab108597), GCase (abcam ab55080), pT73-Rab10 (abcam ab230261), Rab10 (cell signaling 8127S), pT72-Rab8a (abcam ab230260), Rab8a (abcam ab188574), pS935-LRRK2 (abcam 133450), LRRK2 (clone, 8629). αSyn oligomer (abcam ab209538).

Techniques: Staining, Clone Assay, Fluorescence, Microscopy, Western Blot, Activity Assay