Molecular mechanisms underlying LRRK2-mediated centrosomal cohesion deficits as biomarker for Parkinson’s disease

Abstract

Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the most common cause of autosomal dominant familial Parkinson’s disease (PD), and variants in this gene increase risk for the sporadic form of the disease. LRRK2 has been reported to regulate various intracellular membrane trafficking events, including endocytic and endolysosomal trafficking, autophagy and retrograde trafficking, as well as participate in cell signalling cascades. However, the precise mechanism(s) underlying these events remain largely unknown. Recently, LRRK2 has been reported to phosphorylate a subset of RAB GTPases including RAB8A and RAB10, which bind preferentially to RILPL1 and RILPL2 only when phosphorylated. Pathogenic LRRK2 has been reported to cause centrosomal cohesion deficits in dividing cells, including in peripheral patient derived cells, in a kinase dependent manner by phosphorylating RAB8A, which causes its accumulation in a pericentrosomal area. Additionally, LRRK2-phosphorylated RAB10 is recruited to centrosome localised RILPL1, and this has been reported to interfere with primary cilia biogenesis. In the present doctoral thesis, we demonstrate that the LRRK2-mediated centrosomal cohesion deficits are not only caused by accumulation of phospho RAB8A but also of phospho-RAB10. Using CRISPR Cas9 approaches, we identify that the LRRK2-mediated centrosomal cohesion deficits are crucially dependent on the presence of RAB8A, RAB10 and RILPL1. Previous studies have indicated that RILPL1 may be localised to the centrosome. Here we show for the first time that RILPL1 is localised to subdistal appendages of the mother centriole, which allows for the recruitment of the LRRK2-phosphorylated RAB proteins to cause the centrosomal defects. The pathogenic LRRK2-mediated ciliogenesis defects also correlate with the pericentrosomal accumulation of both phospho RAB8A and phospho RAB10. In addition, we show here that various LRRK2 variants that modify risk for PD, as well as currently described regulators of the LRRK2 signalling pathway (vps35 and PPM1H) impact upon centrosomal cohesion deficits. Our data suggest that the LRRK2-mediated centrosomal cohesion and ciliogenesis defects are two distinct cellular readouts of the same underlying phospho RAB8/RAB10/RILPL1 nexus and highlight the possibility that either centrosomal cohesion and/or ciliogenesis alterations may serve as cellular biomarkers for LRRK2-related PD. Indeed, we demonstrate that LRRK2-mediated centrosomal cohesion and ciliogenesis defects are observable under endogenous conditions in primary astrocytes from mutant LRRK2 knockin mice as compared to control, and reverted upon LRRK2 kinase inhibition. In addition, we describe centrosomal cohesion deficits in peripheral blood mononuclear cell derived lymphoblastoid cell lines from a larger sampling of G2019S LRRK2 mutant PD patients as compared to healthy controls, which can also be observed in a subset of sporadic PD patient samples, and which are reverted upon pharmacological LRRK2 kinase inhibition in all cases. Altogether, we describe a robust cell biological assay based on centrosomal cohesion defects which may allow for the stratification of PD patients who may benefit from LRRK2-related therapeutics in clinical settings.