Regulation Of GTPase Activity Of LRRK2 By MgcRacGAP And Its Implication In The Pathogenesis Of Parkinson's Disease

Parkinson's disease(PD)is a common neurodegenerative disease.Numerous data have shown that several pathogenic mutations of leucine-rich repeat kinase 2(LRRK2)are involved in the familiar and sporadic PD pathogenesis.It is widely believed that elevated endogenous Roc-GTPase and kinase activity of PD mutant LRRK2 may contribute to the selective and progressive loss of dopaminergic neurons in PD.However,how the LRRK2 kinase is regulated by its endogenous Roc-GTPase through a recently defined intramolecular mechanism is poorly studied.More importantly,how the LRRK2 Roc-GTPase activity is molecularly regulated remains elusive.By screening for potential regulator of the LRRK2 GTPase using a neuronal cell based yeast two hybrid library,we identified a novel interacting protein MgcRacGAP(MRG).We have examined the specificity of the interaction through a set of biochemical approaches such as co-immunoprecipitation,pull-down assay and GTP binding required protein-protein interaction analysis.We have shown that the specific interaction between LRRK2 and MRG depends on the GAP domain of MRG and Roc-GTPase domain of LRRK2.We have further examined the GAP activity of MRG on Roc-GTPase biochemically.The data shows that MRG significantly promotes the GTP hydrolysis of Roc-GTPase with increased Vmax but not GTP binding affinity(Km)of Roc GTPase,suggesting a specific GAP activity of MRG on LRRK2.We then investigate how MRG regulates the pathogenic effects of mutant LRRK2 on a set of functional assays including neurite shortening of differentiated neuronal SH-SY5Y cells,autophagic abnormality of HeLa Swiss and planned functional vertification using Drosophila LRRK2 model in vivo.We have shown that overexpressed MRG can rescue the neurite shortening and the elevated level of autophagy induced by pathogenic mutations of LRRK2,strongly supporting that MRG could function as a specific GAP for LRRK2 through inactivation of the Roc-GTPase of LRRK2 and intramolecularly inhibit its pathogenic kinase activity.Thus,the interaction of MRG with LRRK2 and the GAP activity of the novel interacting protein MRG may provide an important regulatory mechanism for understanding PD pathogenesis and a novel drug target for clinical therapeutic applications.