UC San Diego

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.