Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Neuroscience

Supervisor

Michael J. Strong

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a progressive motor neurodegenerative disorder with an average life expectancy of 2-5 years post-diagnosis. Common pathological features associated with ALS are the formation of cytoplasmic inclusions of intermediate filaments and RNA-binding proteins within motor neurons. The formation of intermediate filament cytoplasmic inclusions is believed to be driven by a loss of stochiometric expression between five neuronal intermediate filament proteins—NFL, NFM, NFH, INA and PRPH—where there is a selective suppression of the steady-state levels of NEFL, INA and PRPH mRNA. Further, three RNA-binding proteins—TDP-43, FUS and RGNEF—have been shown to co-aggregate with each other in ALS motor neurons indicating a possible common mechanism that leads to their dysregulation.

In the last decade, microRNAs (miRNAs)—small RNA molecules generally responsible for post-transcriptional regulation of gene expression—were observed to be massively dysregulated in the spinal cord tissue of ALS patients, providing a possible explanation for the changes observed in intermediate filament steady-state mRNA levels and RNA-binding protein dysregulation in ALS. Further, TDP-43 and FUS regulate miRNA biogenesis, indicating there may be a regulatory network between RNA-binding proteins and miRNAs that is disrupted in ALS. I hypothesize that a regulatory network between specific RNA-binding proteins and miRNAs is disrupted in ALS leading to changes in miRNA processing which contributes to intermediate filament and RNA-binding protein pathology.

In this dissertation, I have examined: 1) whether ALS-linked miRNA(s) contribute to the selective suppression of NEFL, PRPH, and INA; 2) whether ALS-linked miRNAs regulate the expression of NEFM and NEFH; 3) whether ALS-linked miRNAs regulate the expression of RNA-binding proteins whose metabolism is dysregulated in ALS (TDP-43, FUS, and RGNEF); and, 4) whether TDP-43 and FUS are in a regulatory network with ALS-linked miRNAs. Overall, 12 ALS-linked miRNAs were identified to regulate either intermediate filament or RNA-binding protein expression, and further, a novel negative feedback loop between TDP-43 and two miRNAs (miR-27b-3p and miR-181c-5p) was identified. This dissertation highlights that changes to miRNA levels, as seen in ALS, would contribute to overall ALS pathology, making them viable avenues for potential therapeutics.

Summary for Lay Audience

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder defined by the progressive loss of motor function such as walking, reaching grabbing, standing, etc., eventually confining individuals to a wheelchair. ALS is generally fatal within 2-5 years of diagnosis. This disease results from the dramatic loss of a specific cell-type within the brain and spinal cord tissue called motor neurons. Decades of research has tried to understand what causes the loss of motor neurons in ALS, and while much progress has been made, there remains a considerable amount that is not understood regarding the underlying cause(s) of ALS. Recent research has shown that small molecules known as microRNAs (miRNAs) are reduced in ALS motor neurons. MiRNAs regulate the levels of gene expression within our cells which is critical for cells to efficiently respond to environmental cues (i.e. stress) and maintain overall cell health. Lack of these small molecules, as seen in ALS motor neurons, has been shown to cause motor neuron death in mice, indicating the importance of miRNAs to motor neuron function. In this dissertation, I explored the potential consequence of reduced miRNA levels, as seen in ALS, and how this may relate to what we already know about ALS pathology. Further, I explored potential molecular pathways that may explain why miRNA reduction occurs in ALS. I have found that the loss of a specific pool of miRNAs in ALS may contribute to overall ALS pathogenesis. In addition, I have identified a novel molecular network between miRNAs and TDP-43—a protein that is dysregulated in 97% of all ALS cases—which may explain the loss of miRNAs seen in ALS motor neurons. Overall, this thesis implicates miRNAs as major contributors to ALS pathogenesis and identifies a disrupted molecular network between miRNAs and TDP-43, providing new avenues to explore potential therapeutics for ALS.

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