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

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.

SUBMITTER: Fernandez B 

PROVIDER: S-EPMC9398093 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature