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URL: https://dx.doi.org/DOI:10.17504/protocols.io.bnhtmb6n

Authors: Ying Fan, Andrew J.M. Howden, Dario R. Alessi, Esther M. Sammler

Group: asap

Summary: We describe a fast and efficient immunomagnetic negative isolation method to purify peripheral blood neutrophils for LRRK2 kinase pathway analysis in humans .justify:after { content: ""; display:inline-block; width: 100%; } Gain of kinase function mutations in the Leucine rich repeat kinase 2 (LRRK2) are associated with causing Parkinson’s disease. LRRK2 phosphorylates a subgroup of Rab GTPases and their phosphorylation levels mirror LRRK2 kinase activation status. Here, we describe a facile and robust method for isolating peripheral blood neutrophils by immunomagnetic negative selection, subsequent treatment with and without the specific LRRK2 kinase inhibitor MLi-2 to ensure that any effect seen is LRRK2 kinase dependent and cell lysis. Neutrophil lysates can then be used to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of its endogenous Rab GTPase substrates either by quantitative immunoblotting or targeted mass-spectrometry. The benefit of using peripheral blood neutrophils is that they are an abundant and homogenous white blood cell population that express relatively high levels of LRRK2, Rab10 and other Rab GTPases. The downside is their high intrinsic elastase serine protease activity that necessitates using the neurotoxic, but efficient serine protease inhibitor Diisopropyl fluorophosphate (DIFP) when lysing the cells. Using this assay, we have demonstrated significantly elevated LRRK2 kinase pathway activity in human peripheral blood neutrophils of patients carrying specific mutations such as LRRK2 R1441G (doi:https://doi.org/10.1101/2021.01.28.21249614) and VPS35 D620N (PMID:29743203). This assay can also be used for pharmacodynamic and target engagement studies of small molecule LRRK2 kinase inhibitors. .justify:after { content: ""; display:inline-block; width: 100%; }

Affiliations: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK, Division of Cell Signaling and Immunology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK, Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK, Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK

Version: 1

Publication Date: 2021

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