Master of Science
Dr. Michael J Strong
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive degeneration of motor neurons. Rho Guanine Nucleotide Exchange factor (RGNEF) like other RNA-binding proteins, has been observed to form inclusions in the spinal cord motor neurons of both sporadic and familial cases of ALS. RGNEF has been determined to be a pro-survival factor under stress conditions. When comparing expression of different constructs of RGNEF in HEK293T cells, a Leucine-rich domain containing fragment of RGNEF (L-Rich) was found to form aggregates under metabolic stress that co-aggregated with TDP-43, another ALS-linked RNA-binding protein.
In this thesis, I used both in vivo and in vitro techniques to further characterize the role of L-Rich in the pro-survival effect of RGNEF. In vitro analysis examined whether L-Rich was needed against osmotic or oxidative stress for improved cell survival. Immunofluorescence determined whether RGNEF, RGNEF lacking the Leucine-rich domain (RGNEFΔL), or L-Rich alone were capable of colocalizing with TDP-43 positive granules following stress. Immunoprecipitation examined whether the Leucine-rich domain was essential for the interaction between RGNEF and TDP-43. A Drosophila model was included to examine the impact of L-Rich in modulating the toxic effect of human wildtype TDP-43 in Drosophila. My findings support the hypothesis that L-Rich of RGNEF is important in the cytoprotective effect of full length RGNEF in vitro. This effect varies depending on the type of cellular insult present. My findings also support the hypothesis that L-Rich of RGNEF is a protective modifier of TDP-43 toxicity in Drosophila.
Withers, Benjamin Martin, "The Leucine-rich domain of RGNEF: a modifier of TDP-43 toxicity in Drosophila melanogaster." (2019). Electronic Thesis and Dissertation Repository. 6012.
Available for download on Sunday, March 01, 2020