Duchenne Muscular Dystrophy (DMD) is a disorder caused by mutations in the dystrophin gene, preventing the production of the functional dystrophin protein which assures maintenance of the myofiber integrity throughout muscle contraction. A lack of dystrophin results in severe muscle degeneration and regeneration accompanied by a loss of muscle function. Many pre-clinical and clinical studies are focused on developing strategies to counteract the detrimental effects of DMD; however, there is no cure. One such approach consists of upregulating the endogenous protein utrophin A in dystrophic muscle, which, once highly expressed at the sarcolemma, could functionally compensate for the lack of dystrophin. Recent evidence demonstrates that utrophin A expression is regulated at its 3’ and 5’UTR through post-transcriptional and translational events. Therefore, in the work presented here, we hypothesized that repurposing FDA-approved drugs that target the signaling pathways involved in post-transcriptional and translational regulation of utrophin A will be an efficient approach in rapidly bringing new therapeutic interventions for DMD. In this work, we repurposed four promising FDA-approved drugs able to stimulate utrophin A expression levels in dystrophic muscles: the anti-coagulant drug Heparin, the anti-inflammatory drug Celecoxib, the β-adrenergic receptor blocking agent Betaxolol and the cholesterol-lowering drug Pravastatin. These drugs induce significant improvements in the dystrophic phenotype of mdx mice. This includes amelioration of muscle fiber integrity and muscle function as well as promoting morphological and fiber type changes in mdx mice muscles. Collectively, this thesis describes the potential of a repurposing approach to activate key post-transcriptional and translational pathways involved in utrophin A’s regulation in the hopes of developing new therapeutics for the treatment of DMD.

Understanding the regulatory mechanisms controlling utrophin A expression at the sarcolemma of dystrophic muscles will facilitate the development of therapeutic strategies to ameliorate the pathophysiological features of Duchenne Muscular Dystrophy (DMD). The main goal of this study was to characterize the regulation of utrophin A IRES activity using a transgenic mouse model expressing the utrophin A 5’UTR bicistronic reporter and to identify trans-acting factors that could mediate IRES activity and endogenous expression of utrophin A. We found that utrophin A IRES activity is specifically expressed in skeletal muscles. Moreover, we identified eEF1A2 as a muscle-specific trans-acting factor that can interact with utrophin A and mediate IRES-dependent translation of utrophin A. Finally, we showed that eEF1A2 mediates endogenous utrophin A expression and localization in skeletal muscle. Identifying pharmacological compounds that would specifically target eEF1A2 and increase endogenous levels of utrophin A expression could serve as a drug-based therapy to treat DMD.

Understanding the regulatory mechanisms controlling utrophin A expression at the sarcolemma of dystrophic muscles will facilitate the development of therapeutic strategies to ameliorate the pathophysiological features of Duchenne Muscular Dystrophy (DMD). The main goal of this study was to characterize the regulation of utrophin A IRES activity using a transgenic mouse model expressing the utrophin A 5’UTR bicistronic reporter and to identify trans-acting factors that could mediate IRES activity and endogenous expression of utrophin A. We found that utrophin A IRES activity is specifically expressed in skeletal muscles. Moreover, we identified eEF1A2 as a muscle-specific trans-acting factor that can interact with utrophin A and mediate IRES-dependent translation of utrophin A. Finally, we showed that eEF1A2 mediates endogenous utrophin A expression and localization in skeletal muscle. Identifying pharmacological compounds that would specifically target eEF1A2 and increase endogenous levels of utrophin A expression could serve as a drug-based therapy to treat DMD.

Understanding the regulatory mechanisms controlling utrophin A expression at the sarcolemma of dystrophic muscles will facilitate the development of therapeutic strategies to ameliorate the pathophysiological features of Duchenne Muscular Dystrophy (DMD). The main goal of this study was to characterize the regulation of utrophin A IRES activity using a transgenic mouse model expressing the utrophin A 5’UTR bicistronic reporter and to identify trans-acting factors that could mediate IRES activity and endogenous expression of utrophin A. We found that utrophin A IRES activity is specifically expressed in skeletal muscles. Moreover, we identified eEF1A2 as a muscle-specific trans-acting factor that can interact with utrophin A and mediate IRES-dependent translation of utrophin A. Finally, we showed that eEF1A2 mediates endogenous utrophin A expression and localization in skeletal muscle. Identifying pharmacological compounds that would specifically target eEF1A2 and increase endogenous levels of utrophin A expression could serve as a drug-based therapy to treat DMD.

Understanding the regulatory mechanisms controlling utrophin A expression at the sarcolemma of dystrophic muscles will facilitate the development of therapeutic strategies to ameliorate the pathophysiological features of Duchenne Muscular Dystrophy (DMD). The main goal of this study was to characterize the regulation of utrophin A IRES activity using a transgenic mouse model expressing the utrophin A 5’UTR bicistronic reporter and to identify trans-acting factors that could mediate IRES activity and endogenous expression of utrophin A. We found that utrophin A IRES activity is specifically expressed in skeletal muscles. Moreover, we identified eEF1A2 as a muscle-specific trans-acting factor that can interact with utrophin A and mediate IRES-dependent translation of utrophin A. Finally, we showed that eEF1A2 mediates endogenous utrophin A expression and localization in skeletal muscle. Identifying pharmacological compounds that would specifically target eEF1A2 and increase endogenous levels of utrophin A expression could serve as a drug-based therapy to treat DMD.

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Characterized by the severe progressive wastage of skeletal muscle, Duchenne muscular dystrophy (DMD) is a crippling X-linked recessive disease that is caused by the absence of the protein dystrophin. This thesis aimed to critically evaluate the potential of different therapeutic options to combat this disease. Utrophin is a paralogue of dystrophin. The Fiona mouse is an mdx (dystrophin-deficient) transgenic mouse that overexpresses the full-length utrophin protein in skeletal muscle, and various studies have shown that it does not display a dystrophic phenotype. However, these studies have only been performed on sedentary mice. In this work it is demonstrated that utrophin’s protective effect is partially diminished after a sustained period of exercise-induced stress, highlighting for the first time a functional difference between dystrophin and utrophin. This thesis also presents results of two mdx mouse drug trials testing the ameliorative effects of the administration of the drugs GW501516 and C1100, which show that treatment with both drugs results in partial amelioration of the dystrophic phenotype. GW501516 administration results in a beneficial fast-to-slow fibre type switch and an in vivo increase in utrophin protein levels. We have also shown that C1100 treatment results in a significant increase in utrophin A promoter activity in vitro, and the mechanism of action of this drug on this promoter has been deciphered. The global dysregulation of microRNAs in skeletal muscle of mdx and dko (dystrophin- and utrophin-deficient) mice was evaluated by microarray analysis to identify microRNAs involved in the dystrophic pathological cascades. The results of detailed expression analyses of miR-31, miR-206 and miR-503 are presented, and two therapeutically-relevant predicted targets of miR-503 were validated. Overall, this thesis evaluates the potential of different and possibly complementary therapeutic options to combat DMD.

Duchenne Muscular Dystrophy (DMD) results from the absence of a functional dystrophin protein. Among its possible therapeutic options is the upregulation of dystrophin’s autosomal analogue, utrophin A. This can be achieved by a pharmacologically induced shift towards a slower, more oxidative skeletal muscle phenotype, which has been shown to confer morphological and functional improvements on models of DMD. Whether these improvements are a result of the utrophin A upregulation or other beneficial adaptations associated with the slow, oxidative phenotype, such as improved autophagy, has not been determined. To understand the importance of utrophin A to the therapeutic value of the slow, oxidative phenotype, we used the utrophin/dystrophin double knockout (dKO) model of DMD. We found the dKO mouse to have a similar skeletal muscle signaling capacity and phenotype to mdx mice. When treated with the adenosine monophosphate activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), both dKO and mdx mice expressed a shift towards a slower, more oxidative phenotype. In the mdx mice, this shift caused improvements in muscle fiber central nucleation, IgM penetration, damage from eccentric contractions, and forelimb grip strength. These morphological and functional benefits were not seen in the AICAR treated dKO mice. This study highlights the importance of utrophin A upregulation to the benefits of the slow, oxidative myogenic program to dystrophic mice. It confirms utrophin A as a therapeutic target in DMD and the slow, oxidative myogenic program as clinically relevant avenue towards treatment of the disease.

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive neuromuscular disorder caused by mutations or deletions in the dystrophin gene. Utrophin up-regulation therapy is among the various therapeutic strategies that are being investigated to treat DMD. In this strategy utrophin, a dystrophin homologue, is up-regulated along the entire length of the sarcolemma to replace the absent dystrophin protein. Previous studies have revealed that utrophin A expression can be controlled by various transcriptional, post-transcriptional and translational mechanisms and pharmacological modulation of these pathways can stimulate its expression in muscle. In the present study we screened several FDA approved and natural pharmacological compounds that can potentially activate utrophin A expression in muscle. We found that AICAR (AMPK activator) and heparin (p38 activator) were most effective in stimulating utrophin A expression in our C2C12 muscle cell system. Next, we analyzed the effect of combining these activators on utrophin A expression in muscle cells and preclinical mdx mouse model of DMD. Our findings revealed that combinatorial treatment of AICAR and heparin instigated an additive effect on utrophin A expression both in C2C12 muscle cells and mdx mice. Further characterization of treated mdx mice revealed that combinatorial treatment of AICAR and heparin caused improvements in the dystrophic phenotype as indicated by decreased central nucleation, decreased fiber size variability and improved sarcolemmal integrity in dystrophic muscle. Together these findings established that combinatorial treatment of AICAR and heparin ameliorates the dystrophic phenotype in mdx mice and may serve as an effective therapeutic strategy for DMD.