Investigating GWAS-nominated risk genes associated with Parkinson's disease using a CRISPR-based toolkit - ABSTRACT Parkinson's Disease (PD) is an age-related progressive neurodegenerative disorder primarily characterized by the loss of dopamine (DA) neurons in the midbrain, which is crucial for motor control and balance. Current treatments, such as L-DOPA and deep brain stimulation, provide symptomatic relief but crucially, do not halt the progression of neurodegeneration. While familial PD cases have highlighted mutations in approximately 20 genes, the majority of PD cases are sporadic, likely resulting from complex interactions between aging, genetics, epigenetics, and environmental factors. Recent Genome Wide Association Studies have identified 90 risk loci and 305 nearby candidate genes that might have a significant genetic component to sporadic PD, however, the specific roles of these genes in PD pathophysiology remain to be confirmed and elucidated biologically. In this proposal, I will investigate the hypothesis that one of these genes, CTSB, plays a critical role in PD. CTSB encodes cathepsin B, a lysosomal enzyme implicated in various neurological disorders, with evidence suggesting its downregulation may be associated with PD. I will utilize a CRISPR-based functional toolkit in mouse models to downregulate Ctsb specifically in DA neurons and examine its effects on neuronal survival, morphology, physiology, and behavior over time. To achieve this, I will employ a Cre-dependent dCas9-KRAB (CRISPRi) system in combination with a DAT-IRES-Cre mouse line, allowing for targeted downregulation of Ctsb in midbrain DA neurons via intracranial injection of a Ctsb sgRNA compared to a scrambled non-targeting control sgRNA. I have already shown co-localization of Ctsb and Th mRNA in midbrain DA neurons and successful Ctsb downregulation in primary neuronal cultures. This study aims to determine whether Ctsb downregulation reveals cell autonomous alterations in dopaminergic neuronal function and behavior at 3, 6, and 12 months of age. These experiments will provide insights into the role of Ctsb in sporadic PD, potentially identifying it as a true risk gene. Understanding how Ctsb affects DA neurons may lead to the development of treatments that address the underlying pathology of PD, offering a significant advancement over current therapies that primarily manage symptoms without altering disease progression.
