AbstractAbnormal protein aggregation within neurons is a key pathologic feature of Parkinson’s disease (PD). The spread of protein aggregates in the brain is associated with clinical disease progression, but how this occurs remains unclear. Mutations in the gene glucosidase, beta acid 1 (GBA), which encodes the lysosomal enzyme glucocerebrosidase (GCase), are the most penetrant common genetic risk factor for PD and dementia with Lewy bodies, and also associate with faster disease progression. To explore the mechanism by which mutations in GBA influence pathogenesis of these diseases, we previously created a Drosophila model of GBA deficiency (Gba1b) that manifests neurodegeneration, motor and cognitive deficits, and accelerated protein aggregation. Proteomic analysis of Gba1b mutants revealed dysregulation of proteins involved in extracellular vesicle (EV) biology, and we found altered protein composition of EVs from Gba1b mutants. To further investigate this novel mechanism, we hypothesized that GBA may influence the spread of pathogenic protein aggregates throughout the brain via EVs. We found that protein aggregation is reduced cell-autonomously and non-cell-autonomously by expressing wildtype GCase in specific tissues. In particular, accumulation of insoluble ubiquitinated proteins and Ref(2)P in the brains of Gba1b flies are reduced by ectopic expression of GCase in muscle tissue. Neuronal expression of GCase also cell-autonomously rescued protein aggregation in brain as well as non-cell-autonomously rescued protein aggregation in muscle. Muscle-specific GBA expression rescued the elevated levels of EV-intrinsic proteins and Ref(2)P found in EVs from Gba1b flies. Genetically perturbing EV biogenesis in specific tissues in the absence of GCase revealed differential cell-autonomous effects on protein aggregation but could not replicate the non-cell-autonomous rescue observed with tissue-specific GBA expression. Additionally, we identified ectopically expressed GCase within isolated EVs. Together, our findings suggest that GCase deficiency mediates accelerated spread of protein aggregates between cells and tissues via dysregulated EVs, and EV-mediated trafficking of GCase may partially account for the reduction in aggregate spread.Author’s SummaryParkinson’s disease (PD) is a common neurodegenerative disease characterized by abnormal clumps of proteins (aggregates) within the brain and other tissues which can lead to cellular dysfunction and death. Mutations in the gene GBA, which encodes glucocerebrosidase (GCase), are the strongest genetic risk factor for PD, and are associated with faster disease progression. GCase-deficient mutant flies display features suggestive of PD including increased protein aggregation in brain and muscle. We found that restoring GCase protein in the muscle of mutant flies reduced protein aggregation in muscle and the brain, suggesting a mechanism involving interaction between tissues. Previous work indicated that GBA influences extracellular vesicles (EVs) – small membrane-bound structures released by cells to communicate and/or transport cargo from cell to cell. Here, we found increased aggregated proteins within EVs of mutant flies, which was reduced by restoring GCase in muscle. In addition, we found GCase within the EVs, possibly explaining how GCase in one tissue such as muscle could reduce protein aggregation in a distant tissue like the brain. Our findings suggest that GCase influences proteins within EVs, affecting the spread of protein aggregation. This may be important to understanding PD progression and could uncover new targets to slow neurodegeneration.