Exosome-mediated release of bis(monoacylglycerol)phosphate is regulated by LRRK2 and Glucocerebrosidase activity

Abstract

ABSTRACTBackgroundThe endo-lysosomal phospholipid, bis(monoacylglycerol)phosphate (BMP), is aberrantly high in the urine of Parkinson’s patients with mutations in genes encoding leucine-rich repeat kinase 2 (LRRK2) and glucocerebrosidase (GCase). Because BMP resides on and regulates the biogenesis of endo-lysosomal intralumenal membranes (exosomes when released), we hypothesized that elevated urinary BMP may be driven by increased exocytosis of BMP-enriched exosomes.ObjectiveOur aim was to explore the contribution of LRRK2 and GCase activities in the regulation of BMP metabolism and release.MethodsMicroscopy and biochemical assays were used to analyze antibody-accessible BMP and exosome release in wild type (WT) and R1441G LRRK2–expressing mouse embryonic fibroblast cells. Lipidomics analysis was conducted to measure BMP and lipid content in cells and isolated exosomes. In these experiments, we tested the effects of LRRK2 and GCase inhibitors, MLi-2 and conduritol β-epoxide respectively, to assess regulation of BMP by LRRK2 and GCase.ResultsAlterations in antibody-accessible BMP and endo-lysosome morphology were detected in R1441G LRRK2 cells. Lipidomics analysis revealed increased BMP content in mutant LRRK2 MEFs compared to WT MEFs. Inhibition of LRRK2 partially restored cellular and exosome-associated BMP levels; abrogation of GCase activity had the opposite effect. Metabolic labeling experiments confirmed that BMP synthesis is not influenced by LRRK2 or GCase activities. Pharmacological modulation of exosome release further confirmed exosome-mediated BMP exocytosis.ConclusionsLRRK2 regulates BMP in cells and its release in exosomes, which can be further modulated by GCase activity. These results have implications for the use of exosomal BMP as a Parkinson’s disease biomarker.