Document Type
Dissertation
Degree
Doctor of Philosophy
Major
Biology, Molecular and Cellular Biology
Date of Defense
11-17-2022
Graduate Advisor
Wendy M. Olivas
Committee
Bethany K. Zolman
Lon M. Chubiz
Michael R. Nichols
Abstract
Parkinson’s disease (PD) is the second most common age-related, neurodegenerative disease. A small collection of genes has been linked to PD, including SNCA encoding the protein alpha-synuclein that aggregates in Lewy bodies, a hallmark of PD. Overexpression of these genes can lead to pathogenesis, yet the regulatory mechanisms that control protein production of the genes are not fully understood. The SH-SY5Y neuronal cell line is a common model for the study of neurodegenerative diseases, including PD. However, SH-SY5Y cells are difficult to transfect, a process necessary for genetic manipulations and downstream analysis of RNA and protein. Herein, I describe the assessment, successful optimization, and protocols for several techniques for use with SH-SY5Y cells. Research into the etiology of PD is a critical area of need. The research presented here provides evidence for the regulation of PD-associated genes by human Pumilio (PUM) proteins. PUM proteins belong to the PUF family of proteins, which are RNA-binding proteins that post-transcriptionally regulate gene expression through RNA binding motifs in the 3’ untranslated region (UTR) known as PUF Recognition Elements (PREs). The 3’UTRs of LRRK2, SNCA and SAT1 each contain multiple putative PREs. Knockdown (KD) of the two human PUF homologs, PUM1 and PUM2, in the SH-SY5Y neurodegenerative model cell line resulted in increased SNCA and LRRK2 mRNA, as well as alpha-synuclein levels, suggesting these genes are normally repressed by the PUM proteins. Some studies have indicated a relationship between PUM and microRNA activities on the same target, especially when their binding sites are close together. LRRK2, SNCA, and SAT1 3’UTRs each contain putative microRNA-binding sites, many near PREs. Small RNA-seq and microRNA qPCR assays were performed in wild type and PUM KD SH-SY5Y cells to analyze global and differential microRNA expression. Of the 1404 miRs determined to be expressed in SH-SY5Y, 21 microRNAs were differentially expressed, six of which were previously established to be altered in PD patient samples or research models. Collectively, these results demonstrate that PUMs and microRNAs play a multi-faceted role in regulating PD-associated genes.
Recommended Citation
Snoderly-Foster, Lisa J., "Regulation of Parkinson’s Disease-Associated Genes by Pumilio Proteins and MicroRNAs in the Human Neuroblastoma Cell Line SH-SY5Y" (2022). Dissertations. 1276.
https://irl.umsl.edu/dissertation/1276