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Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity. Journal of Parkinson's disease Fernandez, B., Chittoor-Vinod, V. G., Kluss, J. H., Kelly, K., Bryant, N., Nguyen, A. P., Bukhari, S. A., Smith, N., Lara Ordonez, A. J., Fdez, E., Chartier-Harlin, M., Montine, T. J., Wilson, M. A., Moore, D. J., West, A. B., Cookson, M. R., Nichols, R. J., Hilfiker, S. 2022

Abstract

BACKGROUND: Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson's disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10.OBJECTIVE: To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols.METHODS: Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers.RESULTS: Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated.CONCLUSION: Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution.

View details for DOI 10.3233/JPD-213128

View details for PubMedID 35599495