Probing the Role of the LRRK2 GTPase in Parkinson's Disease

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Project Summary/Abstract Parkinson’s disease is a common neurodegenerative disorder that has been described clinically for at least 200 years. While treatments have advanced to manage patient symptoms, a fundamental understanding of its physiological underpinnings remains elusive, and no curative or progression modifying treatment is currently available. Since the completion of the Human Genome Project, LRRK2 (Leucine Rich Repeat Kinase 2) has been understood to form a strong genetic association with certain familial cases of Parkinson’s disease. Autosomal dominant mutations in LRRK2 of variable penetrance have been demonstrated in isolated cohorts, and GWAS studies have indicated the importance of LRRK2 SNPs in the development of idiopathic Parkinson’s disease as well. Thus, the study of LRRK2 could elucidate findings in the pathophysiological mechanisms of Parkinson’s as a whole. Current hypotheses postulate that LRRK2 kinase hyperactivity is responsible for specific cellular toxicity and resultant neurodegeneration, resulting in the development of LRRK2 kinase inhibitors in clinical and pre-clinical testing. One of the key catalytic domains of LRRK2, its Roc- COR family GTPase, is a site of autosomal dominant mutations of high penetrance that also demonstrate increased kinase activity. Despite this, this domain is relatively understudied compared to the kinase domain. Efforts to understand the GTPase domain of LRRK2 may lead to an alternative modality of therapy, as there are concerns about toxicity mechanisms in currently tested kinase inhibitors. I propose to study the LRRK2 GTPase using chemical genetic and chemical methods via the development of tool compounds and appropriate biochemical and biophysical assays to determine the effect of GTPase modulation on LRRK2 mediated physiology. Preliminary data indicates that the LRRK2 GTPase is surveyable using a variety of developed assay techniques, and can be recombinantly expressed in sufficient amounts to enable large scale screening campaigns. In Aim 1, I propose to study the LRRK2 GTPase via the development of an electrophile sensitive (ES) approach to conformationally lock the domain into either GDP- or GTP-bound states. I will then introduce this system into iPSC-derived dopaminergic neurons and measure the effects of G nucleotide on LRRK2 activity and localization. In Aim 2, I propose to execute complementary small molecule discovery campaigns against the LRRK2 GTPase to uncover tool compounds that can target either the orthosteric or disease mutation defined allosteric sites. I will then test these compounds in primary dopaminergic neuron cells for their effects on ameliorating LRRK2 mutant-mediated cellular toxicity. Ultimately, the findings from these studies will result in small molecule tool compounds and potential therapeutic leads that can be used to better understand the molecular basis of Parkinson’s disease and its avenues for treatment. Training under this fellowship will be supported by several collaborations including the Small Molecule Discovery center (SMDC) at UCSF.