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A CRISPRi screen to identify pathways of Leucine Rich Repeat Kinase 2 protein degradation in vivo

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Parkinson’s disease(PD) is a neurodegenerative movement disorder that affects roughly 1% of the population over 60. Point mutations in the LRRK2 gene are the most common cause of late-onset familial PD. Various studies have implicated LRRK2 kinase activity and LRRK2 protein level as key drivers of LRRK2 toxicity. Moreover, LRRK2 kinase inhibition can drive LRRK2 proteasomal degradation; however, the pathways by which this process occurs are unidentified. Therefore, delineating the mechanism controlling LRRK2 protein degradation should give valuable insights into understanding PD pathogenesis as well as possible new therapeutic approaches. The main goal of this thesis is to identify novel proteins that mediate LRRK2 turnover. Chapter 1 introduces LRRK2 and provides background into what is known about its cellular function and degradation. For Chapter 2, I tested the hypothesis that PJA2 and BIRC2, two proteins previously identified to regulate LRRK2 levels, catalyze LRRK2 turnover after kinase inhibition; however, I was unable to validate the result, which suggested that LRRK2 turnover after kinase inhibition occurs through another unidentified mechanism. Therefore, for chapter 3, we conducted a CRISPRi screen utilizing protein-coding genes relevant to the ubiquitin-proteasome system to identify pathways regulating LRRK2 protein levels in the presence and absence of kinase inhibition. From the screen, we identified two ubiquitination-like pathways, SUMOylation and neddylation, as potential pathways that regulate LRRK2 levels. I have validated single-gene members of these pathways as regulator of LRRK2 protein levels in an overexpression system. Next steps will test the effects of these regulators on endogenous LRRK2 levels. In summary, this thesis identifies novel potential regulatory pathways that drive LRRK2 protein degradation in the presence and absence of LRRK2 kinase inhibition.

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This item is under embargo until June 27, 2024.