Elevated In Vitro Kinase Activity in Peripheral Blood Mononuclear Cells of Leucine‐Rich Repeat Kinase 2 G2019S Carriers: A Novel Enzyme‐Linked Immunosorbent Assay–Based Method

Abstract

The cross-sectional and longitudinal relationships between white matter hyperintensities and dementia in patients with Parkinson's disease: a retrospective analysis of 132 patients in a single center.

The Current State-of-the Art of LRRK2-Based Biomarker Assay Development in Parkinson’s Disease

“…LRRK2 S935 phosphorylation rates decrease in LRRK2 carriers with PD, while all other groups show no significant differences in levels of the tested analytes (Padmanabhan et al, 2020). In another study, however, pS935-LRRK2 levels were reported to be slightly elevated in iPD patients (Melachroinou et al, 2020). To be fair, for this purpose, one must consider the most relevant cell type in which to measure LRRK2 markers.…”

“…Secondly, "cellular" assays (measuring phosphorylation of LRRK2 or its substrates) provide a "snap-shot" of kinase activation that is the result of the coordinated action of myriad upstream and downstream regulatory factors, interacting proteins, and the general activation state of the cell. We have employed such an assay, initially in over-expression models (e.g., Leandrou et al, 2019), but more recently in a clinical study assessing LRRK2 in peripheral blood cells (Melachroinou et al, 2020). In this approach, LRRK2 is purified in an ELISA plate, capturing the protein with anti-LRRK2 (or epitope tag) antibodies, followed by an in-well kinase reaction in which the reaction mixture containing the peptide substrate is added directly to the well containing immobilized LRRK2.…”

“…Quantification of reduced pS935-LRRK2 by conventional ELISA and SIMOA assays can accurately reflect pharmacodynamic response following administration of LRRK2 kinase inhibitors and therefore is currently used as a surrogate biomarker, even though pS935-LRRK2 is not a direct measurement of kinase activity (see above). In fact, it has been reported that the ratio of pS935-LRRK2 to total LRRK2 is significantly reduced in human PBMC lysates from PD manifesting LRRK2 G2019S carriers compared to iPD samples and healthy controls [with and without G2019S mutations (Padmanabhan et al, 2020)], although an alternative in-house developed ELISA detected a slight but significant increase in pS935-LRRK2 in PBMCs of iPD, compared to healthy controls (Melachroinou et al, 2020). Measurement of the auto-phosphorylation site pS1292-LRRK2 would be a more ideal marker of LRRK2 kinase activity, but reliance on this biomarker has been hindered by low physiological stoichiometry (Sheng et al, 2012), and limited phospho-specific antibodies.…”

“…Thus far, we have only been considering measurement of LRRK2 in blood for the purposes of target engagement, but there has been considerable effort put into measurement of LRRK2 levels and LRRK2 function in blood for the purpose of patient stratification or testing the hypothesis that PD patients without LRRK2 mutations have elevated LRRK2 function that contributes to PD pathogenesis. Total LRRK2, pS935-LRRK2 and pT73-Rab10 have all been measured in PBMCs and in neutrophils in sporadic PD patients, non-PD controls, and LRRK2 carriers with and without PD (Dzamko et al, 2013;Atashrazm et al, 2018;Fan et al, 2018;Melachroinou et al, 2020;Padmanabhan et al, 2020). LRRK2 S935 phosphorylation rates decrease in LRRK2 carriers with PD, while all other groups show no significant differences in levels of the tested analytes (Padmanabhan et al, 2020).…”

Urinary proteome profiling for stratifying patients with familial Parkinson’s disease

“…The first cohort was recruited at Columbia University Irving Medical Center (hereinafter referred to as 'Columbia cohort' and color-coded with orange) [11,37]. Participants in the Columbia cohort included 35 HC, 16 NMC, 40 iPD, 28 LRRK2 PD individuals, and one PD patient with an unknown LRRK2 status.…”