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

Abstract

The endolysosomal phospholipid, bis(monoacylglycerol)phosphate (BMP), is aberrantly elevated in the urine of Parkinsons patients with mutations in genes encoding leucine-rich repeat kinase 2 (LRRK2) and glucocerebrosidase (GCase). Because BMP resides on and regulates the biogenesis of endolysosomal intralumenal membranes that become extracellular vesicles (EVs) upon release, we hypothesized that elevated urinary BMP may be driven by increased exocytosis of BMP-enriched EVs. To test this hypothesis, we analyzed BMP metabolism and EV-associated release of BMP in wild type (WT) and R1441G LRRK2-expressing mouse embryonic fibroblast (MEF) cells. Using immunofluorescence microscopy and transmission electron microscopy we detected structural alterations in endolysosomes and antibody-accessible BMP pool, indicating that mutant LRRK2 affects endolysosomal homeostasis. Biochemical analyses of isolated EV fractions confirmed the effect on endolysosomes by showing an increase in LAMP2-positive EVs in mutant cells, which was partially restored by LRRK2 kinase inhibition but further augmented, albeit to a more variable extent, by GCase inhibition. Using mass spectrometry, we detected an overall increase in total di-22:6-BMP and total di-18:1-BMP in cell lysates from mutant LRRK2 MEFs compared to WT cells. Inhibition of LRRK2 kinase partially restored cellular BMP levels, whereas inhibition of GCase further increased the BMP content. In isolated EVs from LRRK2 mutant cells, LRRK2 inhibition decreased BMP content whereas GCase inhibition tended to increase it. Using metabolic labeling experiments, we demonstrated that the increase in cellular BMP content is not due to an increase in BMP synthesis, even though we observed an increase in BMP synthesizing enzyme, CLN5, in LRRK2 mutant MEFs and patient-derived fibroblasts. Finally, pharmacological modulation of EV release and live total internal reflection fluorescence (TIRF) microscopy of endolysosomal exocytosis in human G2019S LRRK2 fibroblasts, further confirmed that BMP release is associated with EV secretion. Together, these results establish LRRK2 as a regulator of BMP levels in cells and its release through EVs and suggest that GCase activity further modulates this process in LRRK2 mutant cells. Mechanistic insights from these studies have implications for the potential use of BMP-positive EVs as a biomarker for Parkinsons disease and associated treatments.