Understanding LRRK2 kinase activity in preclinical models and human subjects through quantitative analysis of LRRK2 and pT73\xa0Rab10

Xiang Wang,Sarah Huntwork-Rodriguez,Anastasia G Henry,Annie Arguello,Ceyda Llapashtica,Hilda Solanoy,Nathan J Moerke,Shan V Andrews,Manuel Montalban,Elvira Negrou,Michael T Maloney,Romeo Maciuca,Vitaliy V Bondar,Hoang Nguyen,Laralynne Przybyla

doi:10.1038/s41598-021-91943-4

Xiang Wang, Sarah Huntwork-Rodriguez + Show 13 more

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https://doi.org/10.1038/s41598-021-91943-4

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Journal: Scientific Reports	Publication Date: Jun 18, 2021
Citations: 36	License type: open-access

Affiliation: Denali Therapeutics (United States)

Abstract

Variants in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with increased risk for familial and sporadic Parkinson’s disease (PD). Pathogenic variants in LRRK2, including the common variant G2019S, result in increased LRRK2 kinase activity, supporting the therapeutic potential of LRRK2 kinase inhibitors for PD. To better understand the role of LRRK2 in disease and to support the clinical development of LRRK2 inhibitors, quantitative and high-throughput assays to measure LRRK2 levels and activity are needed. We developed and applied such assays to measure the levels of LRRK2 as well as the phosphorylation of LRRK2 itself or one of its substrates, Rab10 (pT73 Rab10). We observed increased LRRK2 activity in various cellular models of disease, including iPSC-derived microglia, as well as in human subjects carrying the disease-linked variant LRRK2 G2019S. Capitalizing on the high-throughput and sensitive nature of these assays, we detected a significant reduction in LRRK2 activity in subjects carrying missense variants in LRRK2 associated with reduced disease risk. Finally, we optimized these assays to enable analysis of LRRK2 activity following inhibition in human peripheral blood mononuclear cells (PBMCs) and whole blood, demonstrating their potential utility as biomarkers to assess changes in LRRK2 expression and activity in the clinic.

Highlights

Variants in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with increased risk for familial and sporadic Parkinson’s disease (PD)
Consistent with effects observed with western blot analysis, LRRK2 was not detected in LRRK2 KO cells and pS935 LRRK2 levels were reduced in wildtype cells following treatment with a selective LRRK2 kinase inhibitor (MLi-2) for 2 h (Fig. 1C–E, Supplementary Fig. S1)
We describe the development of high-throughput and sensitive assays to quantify the levels and phosphorylation of LRRK2 and Rab[10] and apply these assays to better understand how LRRK2 is regulated in cellular models and in human subjects that carry LRRK2 variants

Summary

Introduction

Variants in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with increased risk for familial and sporadic Parkinson’s disease (PD). Capitalizing on the high-throughput and sensitive nature of these assays, we detected a significant reduction in LRRK2 activity in subjects carrying missense variants in LRRK2 associated with reduced disease risk We optimized these assays to enable analysis of LRRK2 activity following inhibition in human peripheral blood mononuclear cells (PBMCs) and whole blood, demonstrating their potential utility as biomarkers to assess changes in LRRK2 expression and activity in the clinic. We demonstrated that the LRRK2 N551K R1398H variant associated with reduced risk for PD and Crohn’s disease leads to a reduction in Rab[10] phosphorylation in both cellular models as well as in human subjects, suggesting that this variant may reduce disease risk through its effect on LRRK2’s kinase activity We used these assays to assess LRRK2 levels and activity in whole blood and PBMCs following LRRK2 kinase inhibition, supporting their use as biomarkers to assess target and pathway engagement following dosing with LRRK2 inhibitors in the clinic

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Conclusion