Biochemistry and Molecular Pharmacology
First Thesis Advisor
Daryl A. Bosco, PhD
Amyotrophic Lateral Sclerosis, DNA Damage, DNA Repair, Motor Neurons, RNA-Binding Protein FUS
Dissertations, UMMS; Amyotrophic Lateral Sclerosis; DNA Damage; DNA Repair; Motor Neurons; RNA-Binding Protein FUS
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the death of motor neurons, generally leading to paralysis and death within 3-5 years of onset. Over 50 different mutations in the gene encoding FUS/TLS (or FUS) will result in ALS, accounting for ~4% of all inherited cases. FUS is a multifunctional protein with important functions in DNA/RNA processing and stress response. How these mutations affect the structure or function of FUS protein and ultimately cause ALS is not known. The fact that mutations cause the protein to mislocalize from the nucleus to the cytoplasm of cells suggests that ALS pathogenesis may occur through a loss of nuclear function, gain of toxic cytoplasmic function, or both. Several FUS knockout animal models have been utilized for investigating a loss of function hypothesis and show phenotypes such as early lethality, reduced lifespan, and locomotor defects.
To uncover cellular pathways affected by loss of FUS function, I have characterized the knockdown of FUS in a motor neuron-like cell line, NSC-34. In NSC-34 cells, the depletion of FUS severely impacts cellular proliferation and potentially causes increased levels of DNA damage. A quantitative proteomics analysis performed on cells undergoing various degrees of FUS knockdown revealed protein expression changes for known RNA targets of FUS, consistent with a loss of FUS function with respect to RNA processing. Proteins that changed in expression as a function of FUS knockdown were associated with vii multiple processes, some of which influence cell proliferation including cell-cycle regulation, cytoskeletal organization, oxidative stress and energy homeostasis. Importantly, cellular proliferation could be rescued by the re-expression of FUS and by treatment with the small-molecule, rolipram, indicative of potential therapeutic approaches.
Collectively, the work presented in this dissertation demonstrates the importance of FUS for cell health and homeostasis, is suggestive of a role for FUS in DNA damage repair and identifies additional cellular pathways influenced by FUS depletion. Overall, this work provides mechanistic insight into ALS pathogenesis through loss of FUS/TLS function.
Ward, CL. The Cellular Consequences of FUS/TLS Depletion: A Loss of Function Model for Amyotrophic Lateral Sclerosis: A Dissertation. (2014). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 738. DOI: 10.13028/M2F607. http://escholarship.umassmed.edu/gsbs_diss/738
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