Campus Only Senior Thesis
Bachelor of Arts
Dr. Derek Narendra
Dr. Patrick Ferree
Roughly 10-15% of all Parkinson’s patients have early-onset Parkinson’s disease (EOPD), which is often caused by loss of function mutations (American Parkinson’s Disease Association). Mutations in PRKN (encoding the protein Parkin) and PINK1 are the most common genetic causes of EOPD, and they can cause mitochondrial dysfunction by affecting the PINK1-Parkin-mediated mitophagy pathway. Mitochondrial dysfunction leads to damage and depolarization, which can spread among fused mitochondria. MFN2, a mitochondrial fusion protein, has been shown to participate in the PINK1-Parkin-mediated mitophagy pathway, and therefore may contribute to Parkinson’s disease. A genome-wide, FACS based screen that analyzed gene perturbation effects on MFN2 levels in cells yielded a list of genes that altered MFN2 levels. This study focused on a few genes for further analysis, using flow cytometry, biochemical assays, and RNA microarrays, to verify their effects on MFN2 levels and determine their influence on mitochondrial morphology and RNA expression. The flow cytometry and Western blot results showed an increase in MFN2 levels in untreated cells with knocked down SIN3B, GATAD1, PHF12, EMSY, and SLC25A46. Untreated cells with knockdowns of LETM1 decreased MFN2 levels, and cells treated with oligomycin/antimycin A with knockdowns of MCU increased MFN2 levels. Cells without Parkin decreased MFN2 levels when LETM1 was knocked down, suggesting the gene’s interaction with MFN2 is Parkin-independent. Confocal microscopy showed no changes in mitochondrial morphology in cells with knocked down SIN3B, GATAD1, PHF12, and EMSY. RNA microarray data showed that the four cell lines showed varied RNA expression, but shared 26 genes that were similarly regulated, providing potential avenues for future research projects.
Subramaniam, Aanya, "Genome-wide CRISPRi screen identifies novel regulators of Mitofusin-2 levels" (2022). Scripps Senior Theses. 1987.
Available for download on Monday, June 10, 2024
This thesis is restricted to the Claremont Colleges current faculty, students, and staff.