Pathogenic LRRK2 regulates centrosome cohesion via Rab10/RILPL1-mediated CDK5RAP2 displacement

Mutations in LRRK2 increase its kinase activity and cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab proteins which allows for their binding to RILPL1. The phospho-Rab/RILPL1 interaction causes deficits in ciliogenesis and interferes with the cohesion of duplicated centrosomes. We show here that centrosomal deficits mediated by pathogenic LRRK2 can also be observed in patient-derived iPS cells, and we have used transiently transfected cell lines to identify the underlying mechanism. The LRRK2-mediated centrosomal cohesion deficits are dependent on both the GTP conformation and phosphorylation status of the Rab proteins. Pathogenic LRRK2 does not displace proteinaceous linker proteins which hold duplicated centrosomes together, but causes the centrosomal displacement of CDK5RAP2, a protein critical for centrosome cohesion. The LRRK2-mediated centrosomal displacement of CDK5RAP2 requires RILPL1 and phospho-Rab proteins, which stably associate with centrosomes. These data provide fundamental information as to how pathogenic LRRK2 alters the normal physiology of a cell.We are grateful to Erich Nigg and Francis Barr for providing a variety of constructs and antibodies, and to Dario Alessi for providing various A549 cell lines and MEF cells. We thank LauraMontosa for excellent technical assistance with confocal microscopy. This work was supported by The Michael J. Fox Foundation for Parkinson's research (to S.H.), intramural funding from Rutgers University (to S.H.), the Spanish Ministry of Economy and Competitiveness (SAF2017-89402-R to S.H.), the BBVA Foundation (to S.H., S.A.C., and R.W. M.), the Spanish Ministry of Education, Culture and Sport (FPU12/04367 to J.M. P., FPU15/05233 to A.J. L.O.), and the Spanish Ministry of Science, Innovation and Universities (EST18/00412 to A.J.L. O.)

Protocol to measure centrosome cohesion deficits mediated by pathogenic LRRK2 in cultured cells using confocal microscopy

Summary: The present protocol allows for quantification of inter-centrosome distances in G2 phase cells by confocal fluorescence microscopy to determine centrosome cohesion deficits. We describe transfection and immunofluorescence approaches followed by image acquisition and analysis of inter-centrosome distances. This protocol is for adherent A549 cells transiently overexpressing pathogenic LRRK2 and for immortalized murine embryonic fibroblasts endogenously expressing LRRK2 but is amenable to any other cultured cell type as well.For complete details on the use and execution of this protocol, please refer to Fdez et al.1 and Lara Ordóñez et al.2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

The LRRK2 signaling network converges on a centriolar phospho-Rab10/RILPL1 complex to cause deficits in centrosome cohesion and cell polarization

The Parkinson's-disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases including Rab8 and Rab10, which enhances their binding to RILPL1 and RILPL2. The nascent interaction between phospho-Rab10 and RILPL1 blocks ciliogenesis in vitro and in the intact brain, and interferes with the cohesion of duplicated centrosomes in dividing cells. We show here that regulators of the LRRK2 signaling pathway including vps35 and PPM1H converge upon causing centrosomal deficits. The cohesion alterations do not require the presence of other LRRK2 kinase substrates including Rab12, Rab35 and Rab43 or the presence of RILPL2. Rather, they depend on the RILPL1-mediated centrosomal accumulation of phosphorylated Rab10. RILPL1 localizes to the subdistal appendage of the mother centriole, followed by recruitment of the LRRK2-phosphorylated Rab proteins to cause the centrosomal defects. The centrosomal alterations impair cell polarization as monitored by scratch wound assays which is reverted by LRRK2 kinase inhibition. These data reveal a common molecular pathway by which enhanced LRRK2 kinase activity impacts upon centrosome-related events to alter the normal biology of a cell