Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Pathology and Laboratory Medicine

Supervisor

Strong, Michael J.

Abstract

The presence of neuronal cytoplasmic inclusions (NCIs) composed of RNA-binding proteins (RBPs) and neurofilaments is considered to be ALS’s neuropathological hallmark. RGNEF has been previously shown to interact with TDP-43 and to have a regulatory effect on the expression levels of NEFL mRNA and NFL protein in vitro. Here, I examined the mechanism of the RGNEF N-terminus, leucine-rich domain (LeuR) domain’s interaction with TDP-43. I observed that the minimal domain required is 110 amino acids (LeuR110), that the Ankyrin domain adjacent to LeuR110 does not participate, and that LeuR110 forms of a high molecular weight complex with TDP-43 in vitro consistent with the co-aggregation of LeuR242 and TDP-43 in double transgenic drosophila melanogaster. I also observed that RGNEF interacts directly with and destabilizes the TARDBP mRNA 3’UTR. These findings support that RGNEF interacts with TDP-43 through the formation of a high molecular weight complex and the down-regulation of its expression.

Summary for Lay Audience

Amyotrophic Lateral Sclerosis (ALS) is a progressive, adult-onset disease characterized by the degeneration of motor neurons. In the cytoplasm of neuronal cells, the buildup of RNA-binding proteins (RBPs), neurofilaments and other proteins are considered to be the disease’s hallmark. Among these proteins, Rho guanine nucleotide exchange factor (RGNEF) has been identified as a key element in the development of ALS, where it accumulates in the cytoplasm of motor neurons. Previously, RGNEF has been shown to interact with another protein TDP-43 and to regulate a neurofilament protein NFL. Here, I focused on the mechanism of interaction between TDP-43 and a region of RGNEF, leucine-rich (LeuR). I observed that a minimal domain of LeuR interacts with TDP-43 by forming a complex of proteins, consistent with our observations in the brain and eye tissue of an ALS fly model. In addition to the interacting mechanism, I studied the role of RGNEF in the regulation of TDP-43. I observed that in the presence of the LeuR, there is a downregulation in TDP-43 levels. By defining the role of the interaction between two ALS-associated RBPs, RGNEF and TDP-43, we can get a step further into identifying a potential therapeutic target in ALS.

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