INVESTIGATING THE ROLE OF GBA1 and LIMP2 IN LIPID HOMEOSTASIS: IMPLICATIONS FOR LYSOSOMAL DYSFUNCTION and NEURODEGENERATION

Lysosomal dysfunction and altered lipid metabolism are increasingly recognised as key contributors to neurodegenerative disorders, including Parkinson's disease. Mutations in GBA1, encoding the lysosomal enzyme β-glucocerebrosidase (GCase), represent the strongest genetic risk factor for Parkinson's disease and are associated with impaired lysosomal lipid degradation. GCase trafficking to the lysosome depends on the lysosomal integral membrane protein type-2 (LIMP-2), encoded by SCARB2. However, the contribution of GBA1 and LIMP-2 dysfunction to lipid-droplet homeostasis remains incompletely understood. This study investigated the roles of GBA1 and LIMP-2 in cellular lipid homeostasis using wild-type, GBA1-knockout (11F) and SCARB2-knockout (F6) HEK293 cells, characterised in parallel by Western blotting (PLIN2, GBA1, LAMP1), BODIPY-based lipid-droplet imaging, and targeted lipidomics by liquid chromatography–mass spectrometry. The two knockouts produced distinct, mechanism-specific alterations in lipid-droplet biology. GBA1 deficiency was associated with accumulation of glucosyl/galactosylceramides and glucosylated cholesterol, together with significantly smaller lipid droplets, reduced triglycerides and lower PLIN2, consistent with loss of GCase hydrolytic activity. In contrast, LIMP-2 deficiency drove an increase in lipid-droplet number enriched in free cholesterol and cholesteryl esters, with the lowest PLIN2 and triglyceride levels, consistent with the GCase-independent role of LIMP-2 in lysosomal cholesterol export. Together, these findings indicate that GBA1 and LIMP-2 contribute to lipid-droplet homeostasis through GCase-dependent and GCase-independent pathways, respectively, providing mechanistic insight into how lysosomal dysfunction reshapes cellular lipid storage in Gaucher disease and GBA1-associated neurodegeneration.