An Assessment of LRRK2 Serine 935 Phosphorylation in Human Peripheral Blood Mononuclear Cells in Idiopathic Parkinson’s Disease and G2019S LRRK2 Cohorts

Abstract

The phosphorylated form of LRRK2, pS935 LRRK2, has been proposed as a target modulation biomarker for LRRK2 inhibitors. The primary aim of the study was to characterize and qualify this biomarker for therapeutic trials of LRRK2 inhibitors in Parkinson's disease (PD). To this end, analytically validated assays were used to monitor levels of pS935 LRRK2 and total LRRK2 in peripheral blood mononuclear cells (PBMCs) from the following donor groups: healthy controls, idiopathic PD, and G2019S carriers with and without PD. Neither analyte correlated with age, gender, or disease severity. While total LRRK2 levels were similar across the four groups, there was a significant reduction in pS935 LRRK2 levels in disease-manifesting G2019S carriers compared to idiopathic PD. In aggregate, these data indicate that phosphorylation of LRRK2 at S935 may reflect a state marker for G2019S LRRK2-driven PD, the underlying biology for which requires investigation in future studies. This study also provides critical foundational data to inform the integration of pS935 and total LRRK2 levels as biomarkers in therapeutic trials of LRRK2 kinase inhibitors.

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

“…Thus far, three assays have been published (Delbroek et al, 2013;Henderson et al, 2015;Scott et al, 2017), each utilizing a sandwich-ELISA approach by capture with a total LRRK2 antibody, followed by detection with a specific pS935-LRRK2 antibody. The latter two studies have facilitated accurate IC 50 measurements for LRRK2 kinase inhibitors from treated mouse tissues (brain and kidney) (Henderson et al, 2015), LRRK2 G2019S SH-SY5Y cell lysates (Scott et al, 2017), and human PBMC lysates (Padmanabhan et al, 2020). Notably, Meso Scale Discovery (MSD) has made a pS935-LRRK2 sandwich-ELISA-based assay commercially available, alongside a comparable assay that measures total LRRK2 protein levels.…”

“…In addition, the SIMOA assay is able to use sample volumes often <5 µL and run up to 400 samples per shift. A recent MJFF-led study (Padmanabhan et al, 2020) utilizing the SIMOA platform, reported levels as low as 19 pg/mL for total LRRK2 and 4.2 pg/mL for pS935-LRRK2 using full-length recombinant human LRRK2; and subsequently applied this approach to human PBMC lysates. Altogether, ELISA-based assays offer a more sensitive, high-throughput alternative to Western blotting with multiplex potential for measurement of pS935-LRRK2 biomarker levels.…”

“…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).…”

The Michael J. Fox Foundation’s Strategies for Accelerating Translation of LRRK2 into Therapies for Parkinson Disease

“…In addition, MJFF provided the group standardized clinical samples to enable head-to-head comparison of various high-throughput detection platforms. These efforts identified changes in LRRK2 activation in G2019S subjects by employing a highly sensitive pS935 LRRK2 assay [23] and demonstrated the potential to observe genotype-dependent shifts in LRRK2 inhibitor potency (based on pS935) in human peripheral blood mononuclear cells (PBMCs) that are likely chemotype specific (www.michaeljfox.org/mjff-scientific-publications-clinician-articles). The Consortium is now discussing methods to detect LRRK2 and its activation in the cerebrospinal fluid (CSF) and in immune cells such as monocytes and neutrophils given the high expression of LRRK2 in these cells [24].…”