Alzheimer’s Disease (AD) and Parkinson’s Disease (PD) are two of the most common neurodegenerative diseases affecting millions of people, especially those over 65 years old. Specific mutants of two protein kinases, PKC? and LRRK2, with increased kinase activity have been discovered to be genetic drivers underlying late onset AD and PD, respectively. To understand PKC?-driven AD, we are interested in identifying the substrates phosphorylated by PKC? mutants and how that differs from normal PKC?. By employing a chemical genetic strategy, I have generated “bump hole” versions of wildtype PKC? and PKC? mutant and established an in-vitro kinase assay for measuring the kinase activity of these kinases. Although LRRK2 mutants have implicated increased kinase activity, kinase inhibitors tested in animal models have resulted in phenotypic abnormalities and decreased protein levels suggesting a relationship between LRRK2 kinase activity and protein levels. Therefore, understanding how LRRK2 is degraded with and without kinase inhibition can provide insight into LRRK2-driven PD. We know that LRRK2 mainly degrades via the ubiquitin proteasome system (UPS), but the regulators involved are unknown so we conducted a CRISPRi screen that would provide us with a list of potential gene targets important for LRRK2 degradation via the UPS. From the CRISPRi screen, we found that components of the VCP-NPL4-UFD1L complex are within the top 30 hits and validated these targets except for the multifunctional VCP through knockdowns in overexpressed and endogenous LRRK2 lines. Understanding the regulators of these protein kinases can provide useful insight into potential therapeutics for treating neurodegeneration.

Parkinson’s Disease (PD) is a neurodegenerative movement disorder that affects a high percentage of the elderly population over 60. Autosomal dominant point mutations in the Leucine-Rich Repeat Kinase (LRRK2) gene mark the most common cause of familial PD. The main features of LRRK2 that drive PD include but are not limited to its kinase activity and binding partners. However, cellular functions and interactors of PD-driving and wild-type LRRK2 are only partially defined. Therefore, characterizing LRRK2 binding partners becomes one of the top priorities in learning about the disease. Chapter 1 provides a detailed background on LRRK2, its known functions, and interactors. Chapter 2 demonstrates Rab38 as a novel kinase regulator of LRRK2 under healthy and pathologic physiological conditions with LRRK2 PD mutants in murine melanocytes. Moreover, Rab38 also mediates membrane association and cellular localization of LRRK2. Rab38 and its more widely studied homolog Rab32 have been treated as proteins of the same functions; my experiments elucidate more distinct functions of Rab38 and Rab32 in murine melanocytes. Chapter 3 delineates a well-known LRRK2 differential binding partner, 14-3-3, while using it to calibrate an affinity purification mass-spectrometry (AP-MS) system for novel LRRK2 interactor screening. CLIMP63 is believed to be a novel binding partner of LRRK2 in our first LFQ (label-free quantification) MS screen. While investigating the binding between CLIMP63 and LRRK2 WT and R1441G, I found that exogenous Myc-CLIMP63’s binding to LRRK2 is mediated by LRRK2 residues independent from S910/935, which calls for further investigation into its precise binding sites.

Parkinson’s Disease (PD) is the second most common neurodegenerative disorder, currently affecting over 7 million individuals worldwide. Clinically, PD results in progressive cell death of dopaminergic neurons in the basal ganglia and substantia nigra, but what drives this cell death is still unclear. Point mutations to the Leucine-Rich Repeat Kinase 2 (LRRK2) gene are the most common cause of inherited PD and importantly, LRRK2-PD phenocopies sporadic PD. Thus, better understanding the roles of the LRRK2 protein could provide insights into the driving forces behind PD pathogenesis and progression. LRRK2 has several protein interaction domains and has been shown to be regulated by a number of interactors, yet adequate characterization of the LRRK2 protein interactome is still lacking. Chapter 1 will provide a comprehensive background on LRRK2 biology detailing its structure, function and interactions to inform topics explored in subsequent chapters. Chapter 2 identifies the E3 ubiquitin ligase TRIM1 as a novel regulator of LRRK2 microtubule localization and LRRK2 degradation by the proteasome. Chapter 3 comprises the first report of endogenous regulation of LRRK2 by a Rab protein. Endogenous Rab38 regulates LRRK2 subcellular localization in B16-F10 mouse melanocytes. Finally, Chapter 4 explores the effects of chloroquine and IFN-γ treatment on LRRK2 localization and activity in macrophages. This chapter reveals a novel interaction between LRRK2 and Rab32 in RAW 264.7 macrophages under IFN-γ treatment. These results highlight the need for further research into LRRK2 protein interactors which could regulate LRRK2 activity through a number of mechanisms in a cell-type specific manner.

Parkinson’s Disease (PD) is a progressive neurodegenerative disorder that affects more than 10 million people worldwide. While most cases of PD are sporadic, point mutations in Leucine-Rich Repeat Kinase (LRRK2) have been determined to be the most common cause of autosomal dominant late-onset PD. One key limitation within the field of LRRK2 biology lies within our inability to visualize endogenous LRRK2 due to its low expression. Without definitive evidence in endogenous systems, there remain questions surrounding how representative conclusions drawn from overexpression systems are regarding LRRK2 kinase activity, LRRK2 protein levels, and mechanisms underlying LRRK2-driven PD. Hence, it is crucial to develop a system to better understand endogenous LRRK2. Chapter I introduces LRRK2 biology by demonstrating the progress the field has made to unravel LRRK2’s complexities within the last 20 years. Chapter II tests the ability of novel HaloTag technology CRISPR-tagged to LRRK2 to better visualize endogenous LRRK2. Chapter III encompasses the use of a CRISPRi screen to identify pathways that potentially regulate endogenous LRRK2 degradation. Notably, the CRISPRi screen revealed two pathways modulating proteasomal degradation: SUMOylation and VCP/p97 system. Through validation of specific gene targets within a knockdown system, we found that the NPL4/UFD1L complex is involved in regulating endogenous LRRK2 degradation through an unknown mechanism. Future directions would include using microscopy to further corroborate the importance of the NPL4/UFD1L complex and establish its interaction to LRRK2 in the endogenous degradation pathway.