Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity

• Published in: Journal of Parkinson's disease Vol. 12; no. 5; pp. 1423 - 1447
• Main Authors: Fernández, Belén, Chittoor-Vinod, Vinita G., Kluss, Jillian H., Kelly, Kaela, Bryant, Nicole, Nguyen, An Phu Tran, Bukhari, Syed A., Smith, Nathan, Lara Ordóñez, Antonio Jesús, Fdez, Elena, Chartier-Harlin, Marie-Christine, Montine, Thomas J., Wilson, Mark A., Moore, Darren J., West, Andrew B., Cookson, Mark R., Nichols, R. Jeremy, Hilfiker, Sabine
• Format: Journal Article
• Language: English
• Published: London, England SAGE Publications 01.01.2022; Sage Publications Ltd; IOS Press
• Subjects: Cell culture; Embryo fibroblasts; Kinases; Laboratories; LRRK2 protein; Lymphoblastoid cell lines; Michael J. Fox Foundation – Paper; Movement disorders; Mutants; Neural coding; Neurodegenerative diseases; Parkinson's disease; Phosphorylation; Western blotting; RAB protein; kinase; proximity ligation assay; phosphorylation; LRRK2
• ISSN: 1877-7171; 1877-718X
• DOI: 10.3233/JPD-213128

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.