Alterations in the LRRK2-Rab pathway in urinary extracellular vesicles as Parkinson's disease and pharmacodynamic biomarkers.

Expression or phosphorylation levels of leucine-rich repeat kinase 2 (LRRK2) and its Rab substrates have strong potential as disease or pharmacodynamic biomarkers. The main objective of this study is therefore to assess the LRRK2-Rab pathway for use as biomarkers in human, non-human primate (NHP) and rat urine. With urine collected from human subjects and animals, we applied an ultracentrifugation based fractionation protocol to isolate small urinary extracellular vesicles (uEVs). We used western blot with antibodies directed against total and phosphorylated LRRK2, Rab8, and Rab10 to measure these LRRK2 and Rab epitopes in uEVs. We confirm the presence of LRRK2 and Rab8/10 in human and NHP uEVs, including total LRRK2 as well as phospho-LRRK2, phospho-Rab8 and phospho-Rab10. We also confirm LRRK2 and Rab expression in rodent uEVs. We quantified LRRK2 and Rab epitopes in human cohorts and found in a first cohort that pS1292-LRRK2 levels were elevated in individuals carrying the LRRK2 G2019S mutation, without significant differences between healthy and PD groups, whether for LRRK2 G2019S carriers or not. In a second cohort, we found that PD was associated to increased Rab8 levels and decreased pS910-LRRK2 and pS935-LRRK2. In animals, acute treatment with LRRK2 kinase inhibitors led to decreased pT73-Rab10. The identification of changes in Rab8 and LRRK2 phosphorylation at S910 and S935 heterologous phosphosites in uEVs of PD patients and pT73-Rab10 in inhibitor-dosed animals further reinforces the potential of the LRRK2-Rab pathway as a source of PD and pharmacodynamic biomarkers in uEVs.

Functional Analyses of Two Novel LRRK2 Pathogenic Variants in Familial Parkinson's Disease.

BACKGROUND: Pathogenic variants in the LRRK2 gene are a common monogenic cause of Parkinson's disease. However, only seven variants have been confirmed to be pathogenic. OBJECTIVES: We identified two novel LRRK2 variants (H230R and A1440P) and performed functional testing. METHODS: We transiently expressed wild-type, the two new variants, or two known pathogenic mutants (G2019S and R1441G) in HEK-293 T cells, with or without LRRK2 kinase inhibitor treatment. We characterized the phosphorylation and kinase activity of the mutants by western blotting. Thermal shift assays were performed to determine the folding and stability of the LRRK2 proteins. RESULTS: The two variants were found in two large families and segregate with the disease. They display altered LRRK2 phosphorylation and kinase activity. CONCLUSIONS: We identified two novel LRRK2 variants which segregate with the disease. The results of functional testing lead us to propose these two variants as novel causative mutations for familial Parkinson's disease. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.