Novel Mechanisms of LRRK2-Dependent Neurodegeneration in Parkinson's Disease

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 DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a common neurodegenerative movement disorder caused primarily by the degeneration of dopaminergic neurons in the substantia nigra. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant PD, and LRRK2 genomic variation increases PD risk. LRRK2 has emerged has an important therapeutic target for treating PD and therefore it is critical to understand the molecular mechanisms that lead to LRRK2-dependent neurodegeneration. LRRK2 is a multi- domain protein containing Ras-of-complex (Roc) GTPase and C-terminal-of-Roc (COR) domains, in addition to a protein kinase domain. We have previously shown that familial LRRK2 mutations increase kinase activity (G2019S) or impair GTPase activity (R1441C/G/H or Y1699C) but commonly induce neuronal damage in cultured cells. Our studies have also highlighted an important role for the GTPase domain in regulating LRRK2 kinase activity and neuronal toxicity, highlighting the GTPase domain as a promising target for inhibiting LRRK2. We have also shown that the G2019S mutation, which produces a hyperactive kinase, can induce dopaminergic neuronal degeneration in rats via adenoviral-mediated gene transfer through an unknown mechanism. In the present application, we now propose to explore whether kinase activity is commonly required for dopaminergic neurodegeneration induced by familial PD mutations (R1441C, Y1699C and G2019S) in an adenoviral-based LRRK2 rat model (Aim 1). We hypothesize that certain familial mutations exert their detrimental effects through a kinase-dependent mechanism. Accordingly, genetic and pharmacological inhibition of LRRK2 kinase activity will be evaluated in this adenoviral model for disease- modifying effects. Authentic substrates of LRRK2 kinase activity have not yet been identified in vivo. We recently identified ArfGAP1 as a robust kinase substrate of LRRK2 that is critically required for LRRK2-induced neuronal toxicity in cultures. We now propose to identify the sites of ArfGAP1 phosphorylation by LRRK2 in vitro and in vivo in brain tissue, and evaluate the contribution of ArfGAP1 phosphorylation and expression to LRRK2-induced neuronal damage in primary neuronal and adenoviral-based rat models (Aim 2). Finally, our studies will explore the role of the Roc-COR tandem domain in regulating LRRK2 activity and toxicity (Aim 3). We hypothesize that LRRK2 functions as a GTPase activated by dimerization (GAD) and accordingly we will explore how intermolecular (i.e. COR domain-mediated dimerization) and intramolecular (Roc/COR interactions) interactions contribute to LRRK2 activity and toxicity. We will determine whether disrupting these unique Roc-COR interactions serve to attenuate LRRK2-mediated neurodegeneration. Our proposal is novel, innovative and timely and will provide critical mechanistic insight into the relative contributions of GTPase and kinase activity to LRRK2-mediated neurodegeneration. Our studies will have important implications for the identification of therapeutic strategies for PD based upon attenuating LRRK2 activity and neuronal toxicity.