Objective: We aim to explore the pathogenesis of GBA1-associated synucleinopathies and discern the gain- vs. loss-of-function hypotheses by genetically engineering GBA1 in an iPSC line derived from a patient with both Gaucher disease (GBA1: N370S/N370S) and Parkinson’s disease to separate GCase mutant potentially with toxic function and lipid accumulation.

Background: GBA1 encodes β-glucocerebrosidase (GCase), a lysosomal enzyme responsible for breaking down glucosylceramide into glucose and ceramide. Biallelic pathological mutations in GBA1 lead to Gaucher disease (GD), characterized by deficient GCase activity and lipid substrate buildup. Furthermore, GBA1 mutations are common genetic risk factors for Parkinson disease (PD) and dementia.

Method: We first introduced tdTomato into the endogenous TH gene in the parental iPSC line HT809 to facilitate the assessment of DA neuron differentiation. Subsequently, we inserted TMEM192-GFP-3xHA into the CLYBL safe harbor site to enable LysoIP. Lastly, we utilized CRISPR/Cas9 and PiggyBac systems to edit GBA1, correcting N370S/N370S back to wild type (WT/WT) and knocking out the pathologic mutant GCase (KO/KO). Whole genome sequencing (WGS) and targeted long read sequencing of the three isogenic lines confirmed editing specificity with minimal off-target events.

Results: Dopaminergic (DA) neurons differentiated from the WT/WT line exhibited higher GCase protein levels than the N370S/N370S line, whereas the KO/KO line showed minimal GCase levels. Glycosylation analysis indicated that only a small fraction of GCase N370S was retained the endoplasmic reticulum (ER) in DA neurons. Consistent with the glycosylation analysis, immunofluorescence demonstrated lysosomal localization of GCase N370S, confirming the successful targeting of some GCase N370S to lysosomes. Live-cell GCase activity assays with LysoFQ-GBA revealed reduced lysosomal GCase activity in the N370S/N370S line, whereas the KO/KO line exhibited no activity. Lipidomic analysis demonstrated significant substrate accumulation in the KO/KO line. Proteomic analysis of lysosomes purified from isogenic DA neurons identified GPNMB as a promising candidate. Ongoing experiments aim to further elucidate the mechanistic implications of these findings.

Conclusion: The GBA1 isogenic iPSC lines may serve as useful tools to provide insights into the individual contribution by GCase mutant and lipid accumulation to PD pathogenesis.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/investigating-the-pathogenesis-of-gba1-associated-parkinsons-disease-using-gba1-isogenic-ipsc-lines/