Molecular mechanisms contributing to the expression of utrophin at the mammalian neuromuscular synapse.

Abstract

Duchenne muscular dystrophy (DMD) is the most severe and prevalent primary myopathy. This disease is characterized by repeated cycles of muscle fiber degeneration and regeneration with an eventual failure to regenerate leading to the progressive replacement of myofibers by adipose and connective tissues. The genetic defects responsible for DMD are mutations in the short arm of the X chromosome which prevent the production of normal size dystrophin, a large cytoskeletal protein of 427 kDa. In contrast to the homogeneous distribution of dystrophin along muscle fibers, utrophin preferentially accumulates at the neuromuscular junction. Due to this sequence similarity between dystrophin and utrophin, it has been suggested that increased expression of utrophin into extrasynaptic regions of dystrophic muscle fibers may represent a therapeutic strategy for DMD. Recently, it has been confirmed that the upregulation of utrophin can, indeed, functionally compensate for the lack of dystrophin and alleviate the muscle pathology. In this context, it thus becomes essential to determine the cellular and molecular mechanisms presiding over utrophin expression in attempts to overexpress the endogenous gene product throughout skeletal muscle fibers. In this Thesis, I explore the mechanisms underlying the selective accumulation of utrophin at the postsynaptic membrane of the neuromuscular synapse. We determined by in situ hybridization that local transcription contributes to the accumulation of utrophin at the neuromuscular junction. Using direct injections of utrophin promoter-reporter constructs into skeletal muscle, we also defined the promoter elements involved in this local transcription and determined that the N-box element is a key consensus sequence that directs transcriptional control of utrophin expression at the neuromuscular junction. Furthermore, additional experiments revealed that utrophin gene transcription is dependent on the extracellular matrix proteins agrin and ARIA/heregulin, and this regulation is dependent upon the N-box element. Indeed, in vitro transfection assays and electromobility shift assays indicated that agrin and ARIA/heregulin may ultimately initiate a cell signaling cascade that activates the ETS-related transcription factor, GA-binding protein (GABP) which binds and activates the N-box element. In these experiments, we determined by RT-PCF, immunoblotting, and nuclear run on assays that, in contrast to the large changes in AChR, utrophin expression was only marginally increased under these conditions. (Abstract shortened by UMI.)