Skip to main content
. Author manuscript; available in PMC: 2020 Oct 16.
Published in final edited form as: Sci Transl Med. 2019 Oct 16;11(514):eaau6870. doi: 10.1126/scitranslmed.aau6870

Figure 3. Activation of wild-type GCase with S-181 partially rescues pathogenic phenotypes in GBA-PD iPSC-derived dopaminergic neurons.

Figure 3.

(A and B) Heterozygous 84GG GBA1 mutant iPSC-derived dopaminergic neurons (GBA-PD1 clone #1 and #2) were treated with either DMSO (vehicle) or S-181 (5, 15, and 25μM) for 10 days. Triton-soluble lysates were obtained at day 60 of differentiation and analyzed for (A) GCase protein amount by immunoblotting with quantification of post-ER/ER GCase (N=3 independent experiments) and (B) GCase activity assessed by in vitro enzyme activity assay (N=3 independent experiments). (C and D) Heterozygous 84GG GBA1 mutant iPSC-derived dopaminergic neurons (GBA-PD2, clone #1 and #2) were treated with either DMSO (vehicle) or S-181 (5, 15, and 25μM) for 10 days. Triton-soluble lysates were analyzed at day 60 of differentiation for (C) GCase protein amounts by immunoblotting with quantification of post-ER/ER GCase (N=3 independent experiments) and (D) GCase activity assessed by in vitro enzyme activity assay (N=3 to 6 independent experiments). (E) Heterozygous 84GG GBA1 mutant iPSC-derived dopaminergic neurons (GBA-PD2) were treated with either DMSO (vehicle) or a nonactivating control compound (5, 15, and 25μM) for 10 days. GCase activity was analyzed in Triton-soluble lysates at day 60 of differentiation (N=3 independent experiments). (F) Quantification of intracellular glucosylceramide (GluCer) by mass spectrometry normalized to internal phosphate (Pi) was performed at day 70 of differentiation in heterozygous 84GG GBA1 mutant iPSC-derived dopaminergic neurons [GBA-PD1 (left), N=3 to 4 independent experiments and GBA-PD2 (right), N=3 independent experiments] treated with either DMSO (vehicle) or S-181 (5 and 15μM) for 10 days. (G) Lysosomal proteolysis of long-lived proteins was quantified at day 80 of differentiation by radioactive pulse-chase of heterozygous 84GG GBA1 mutant iPSC-derived dopaminergic neurons [GBA-PD1 (left), N=3 independent experiments and GBA-PD2, N=3 to 5 independent experiments] treated with either DMSO (vehicle) or S-181 (5 and 15μM) for 10 days. (H and I) Detection and quantification of oxidized dopamine (DA) was performed by near-infrared fluorescence assay at day 60 of differentiation of heterozygous 84GG GBA1 mutant iPSC-derived dopaminergic neurons (H) GBA-PD1 clone #1 and #3 (N=3 to 4 independent experiments), (I) GBA-PD2 clone #1 and #2 (N=4 to 6 independent experiments) treated with either DMSO (vehicle) or S-181 (5, 15, and 25μM) for 10 days. Error bars, mean ± SEM. *P<0.05 and **P<0.01, one-way ANOVA with Tukey post hoc test.