Restoration of Lysosomal Hydrolase Activities by <scp>LRRK2</scp> Inhibition in <i>GBA1</i> ‐ and <i>LRRK2</i> ‐Associated Parkinson's Disease Patient‐Derived Cells

Mutations in the GBA1 gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), and the LRRK2 gene, encoding leucine-rich repeat kinase 2 (LRRK2) are the most common genetic risk factors for Parkinson's disease (PD). The potential use of LRRK2 inhibitors for treating not only LRRK2-associated PD (LRRK2-PD) but also GBA1-associated PD (GBA1-PD) is currently under discussion. In the present study, we aimed to evaluate whether LRRK2 inhibition affects lysosomal hydrolase enzymatic activities, autophagy, and alpha-synuclein levels in various cell types derived from LRRK2-PD and GBA1-PD patients, including macrophages derived from peripheral blood mononuclear cells (PBMC-derived macrophages), dopaminergic (DA) neurons derived from induced pluripotent stem cells (iPSC-derived DA neurons), and SH-SY5Y cells. We first characterized PBMC-derived macrophages from patients with LRRK2-PD. Confocal microscopy revealed reduced colocalization of GCase with the lysosomal marker LAMP2, indicating impaired lysosomal translocation of the enzyme. In parallel, we observed decreased activity of galactosylceramidase (GALC), increased levels of lysosphingolipids (HexSph, LysoGb3, LysoSM) as measured by LC-MS/MS, and decreased pro-form of cathepsin D, as assessed by Western blot analysis, compared to controls. However, no significant differences in GCase activity and protein levels were observed in PBMC-derived macrophages of LRRK2-PD patients and controls. In subsequent experiments, we found that LRRK2 inhibition not only increased GCase activity in both PBMC-derived macrophages and iPSC-derived DA neurons but also augmented the activity of other lysosomal enzymes, including acid sphingomyelinase (ASMase), alpha-galactosidase (GLA), and GALC. Notably, this enhancement was more pronounced in iPSC-derived DA neurons compared to PBMC-derived macrophages. Similar increases in lysosomal hydrolase activities (GCase, ASMase, GALC, and GLA) were also observed in the SH-SY5Y neuroblastoma cell line after LRRK2 inhibition. Interestingly, LRRK2 inhibition was more effective in GBA1-PD than in LRRK2-PD. We demonstrated that LRRK2 inhibition enhances both the protein levels of GCase and its translocation to lysosomes. This study provides the first evidence that inhibition of LRRK2 activity may increase GCase level and its translocation to lysosomes, thereby ameliorating lysosomal function. Moreover, LRRK2 inhibition also enhances the activity of other lysosomal hydrolases. Our findings expand the understanding of the molecular mechanism of LRRK2 inhibitors, offering valuable insights for further development of PD treatments.