Deubiquitinating enzyme A20 and its role in NF-kappaB regulation: Implications for treatment of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is one of the most common muscle disorders affecting about 1 in 3500 male births worldwide. It is caused by mutations in the gene dystrophin, the protein product of which is required for muscle structure and stability. Studies suggest that lack of dystrophin protein leads to dystrophic pathology in skeletal muscle; an important pathologic mediator is nuclear factor kappa B (NF-κB), a transcription factor that regulates several genes responsible for stress responses, cell survival and various inflammatory conditions. NF-κB is pathologically activated in dystrophic muscle in DMD patients and in the mdx mouse model for DMD. NF-κB activation in mdx mice is thought to activate protein degradation and cause chronic inflammation in skeletal muscle. Furthermore, pathological activation of NF-κB downregulates myogenic regulatory factors and interferes with muscle regeneration. Attenuation of NF-κB activation in mdx mice has been shown to improve muscle stability and strength. Thus, strategies to inhibit NF-κB activation are being actively pursued as a therapeutic option for DMD. In this thesis, I have characterized the role of A20, a deubiquitinating enzyme known to attenuate NF-κB activation, in skeletal muscle and examined its potential as a therapeutic target to attenuate NF-κB activation in DMD. I investigated the effect of A20 on NF-κB activation in cultured mdx and control C57BL/10 myotubes in vitro and observed its role in muscle regeneration and differentiation. Characterization of A20 localization in mdx muscle demonstrated expression predominantly in regenerating fibers; interestingly, most were fast- twitch muscle fibers. This correlation between expression of an NF-κB inhibitor attenuating NF- κB activation and pathology of DMD was a promising observation to further explore the efficacy of A20 as a therapeutic target. To this effect, I developed adeno-associated viral vectors carrying A20 expression cassettes to test the therapeutic benefit of A20 over-expression in the mdx model of DMD. Over-expression of A20 caused a significant decrease in NF-κB activation, an overall reduction in regeneration and number of inflammatory cells in skeletal muscle. These studies show that A20 could be pursued further as a therapeutic agent for inhibition of NF-κB activation, not only in DMD, but also in other disorders