Abstract/Summary

Parkinson’s Disease (PD) is a progressive neurodegenerative disorder that is characterized in part by a loss of dopamine neurons in the Substantia Nigra and affects the nigrostriatal pathway. Mutations in the gene encoding leucine�rich repeat kinase 2 (LRRK2) have been found to cause late onset PD through a gain of function of its kinase domain. Thus, LRRK2 has become an intriguing candidate for therapeutic intervention by kinase inhibition. While preclinical studies have shown that ablating the kinase activity of wildtype LRRK2 is safe with a mild and reversible lung phenotype, the molecular effects of chronic LRRK2 inhibition have not been examined in the context of mutant LRRK2. Using the potent LRRK2 kinase-specific inhibitor, Merck LRRK2 inhibitor 2 (MLi�2), hyperactive G2019S LRRK2 was reduced to wildtype levels chronically in G2019S knock-in (KI) mice and autophosphorylation of LRRK2 and phosphorylation of direct substrates Rab10, Rab12, and Rab29 was assessed. Unbiased total and phospho-proteomics revealed alterations in endolysosomal proteins similar to those found in LRRK2 knockout (KO) animals after 10 weeks of treatment. LRRK2 has been shown to play roles in a number of pathways within the endolysosomal system and studies have reported LRRK2 presence on many organelles from the trans-Golgi network (TGN) to the lysosome. Based on the current study’s proteomic results, eight different trap plasmids were generated to evaluate LRRK2 kinase activity at distinct endolysosomal membranes. As a result, LRRK2 kinase activity was found to be enhanced after being trapped to all membranes and downstream Rab10 and Rab12 phosphorylation were increased in vitro, but recruitment of these Rabs revealed differential patterns in localization specifically when targeting LRRK2 on lysosomes compared to other membranes. Evaluation of lysosomal position through manipulation of various motor proteins showed phosphorylated Rab10 was preferentially restricted to a subset of perinuclear lysosomes, whereas pRab12 was present at most LRRK2-positive lysosomes regardless of their position. This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular pathways that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients. Complementary in vitro data provides novel insight into the differences in LRRK2-dependent Rab localization that can help elucidate the role of LRRK2 at the lysosome which may be relevant to PD pathogenesis. The work presented in this thesis additionally contributes to our knowledge on the utility of pS1292 LRRK2 and pS106 Rab12 as robust biomarkers of both kinase hyperactivity and inhibition in G2019S LRRK2 KI mice in brain and peripheral tissues that is worth assessment in patients with PD harboring the G2019S mutation.