Abstract
BackgroundMutations in LRRK2 are a common genetic cause of Parkinson’s disease (PD). LRRK2 interacts with and phosphorylates a subset of Rab proteins including Rab8a, a protein which has been implicated in various centrosome-related events. However, the cellular consequences of such phosphorylation remain elusive.MethodsHuman neuroblastoma SH-SY5Y cells stably expressing wildtype or pathogenic LRRK2 were used to test for polarity defects in the context of centrosomal positioning. Centrosomal cohesion deficits were analyzed from transiently transfected HEK293T cells, as well as from two distinct peripheral cell types derived from LRRK2-PD patients. Kinase assays, coimmunoprecipitation and GTP binding/retention assays were used to address Rab8a phosphorylation by LRRK2 and its effects in vitro. Transient transfections and siRNA experiments were performed to probe for the implication of Rab8a and its phosphorylated form in the centrosomal deficits caused by pathogenic LRRK2.ResultsHere, we show that pathogenic LRRK2 causes deficits in centrosomal positioning with effects on neurite outgrowth, cell polarization and directed migration. Pathogenic LRRK2 also causes deficits in centrosome cohesion which can be detected in peripheral cells derived from LRRK2-PD patients as compared to healthy controls, and which are reversed upon LRRK2 kinase inhibition. The centrosomal cohesion and polarity deficits can be mimicked when co-expressing wildtype LRRK2 with wildtype but not phospho-deficient Rab8a. The centrosomal defects induced by pathogenic LRRK2 are associated with a kinase activity-dependent increase in the centrosomal localization of phosphorylated Rab8a, and are prominently reduced upon RNAi of Rab8a.ConclusionsOur findings reveal a new function of LRRK2 mediated by Rab8a phosphorylation and related to various centrosomal defects.
Highlights
Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson’s disease (PD)
Distinct pathogenic LRRK2 mutants cause centrosomal cohesion deficits in a kinase activity-dependent manner In dividing cells, centrosomes duplicate in S phase, but are held together by a flexible linker which gradually elongates during S and G2, allowing the duplicated centrosomes to mature by accumulating pericentriolar material
Whilst future studies will be required to address whether the centrosomal cohesion deficits remaining upon Rab8a knockdown are mediated by remnant Rab8a, by other functionally redundant Rab protein LRRK2 kinase substrates such as Rab8b or Rab10 [3], or by other, non-Rab-related LRRK2 substrates, these results indicate that a significant part of the phenotype is dependent on the presence of Rab8a
Summary
LRRK2 interacts with and phosphorylates a subset of Rab proteins including Rab8a, a protein which has been implicated in various centrosome-related events. Various pathogenic LRRK2 mutations have been described which all seem to converge on causing increased phosphorylation of select kinase substrates in intact cells [3], indicating that LRRK2 kinase activity may represent a therapeutic PD target. The downstream event(s) associated with abnormal LRRK2-mediated substrate phosphorylation remain unknown. A recent phosphoproteomics study has conclusively identified a subset of Rab proteins including Rab8a as LRRK2 kinase substrates [3]. Rab8a is a small GTPase localized to various intracellular compartments including Golgi, pericentrosomal recycling endosomes and centrosomes, and is known to regulate centrosome-related events [20,21,22]. The cellular consequences of LRRK2mediated Rab8a phosphorylation are currently unknown
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