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Relationship between myosin heavy chain fibre type and restoration of dystrophin expression and key components of the dystrophin-associated glycoprotein complex by Tricyclo-DNA mediated exon skipping
Exon skipping mediated by tricyclo-DNA (tc-DNA) antisense oligonucleotides has been shown to induce significant levels of dystrophin restoration in mdx, a mouse model of Duchenne Muscular Dystrophy. This translates into significant improvement in key disease indicators in muscle, cardio-respiratory function, heart and the central nervous system. Here we examine the relationship between muscle fibre type, based on Myosin Heavy chain profile, and the ability of tc-DNA to restore not only dystrophin but also other members of the dystrophin-associated glycoprotein complex (DAPC). We first profiled this relationship in untreated mdx muscle and found that all fibre types support reversion events to a dystrophin positive state, in an unbiased manner. Importantly, we show that only a small fraction of revertant fibres expressed other members of the DAPC. Immunoblot analysis of protein levels, however, revealed robust expression of these components, which failed to correctly localise to the sarcolemma. We then show that tc-DNA treatment leads to nearly all fibres expressing not only dystrophin but also other key components of the DAPC. Of significance, our work shows that MHC fibre type does not bias the expression of any of these important proteins. This work also highlights that the improved muscle physiology following tc-DNA treatment reported previously results from the complete restoration of the dystrophin complex in all MHCII fibres with equal efficiencies
Assembly of the Dystrophin-Associated Protein Complex Does Not Require the Dystrophin Cooh-Terminal Domain
Dystrophin is a multidomain protein that links the actin cytoskeleton to laminin in the extracellular matrix through the dystrophin associated protein (DAP) complex. The COOH-terminal domain of dystrophin binds to two components of the DAP complex, syntrophin and dystrobrevin. To understand the role of syntrophin and dystrobrevin, we previously generated a series of transgenic mouse lines expressing dystrophins with deletions throughout the COOH-terminal domain. Each of these mice had normal muscle function and displayed normal localization of syntrophin and dystrobrevin. Since syntrophin and dystrobrevin bind to each other as well as to dystrophin, we have now generated a transgenic mouse deleted for the entire dystrophin COOH-terminal domain. Unexpectedly, this truncated dystrophin supported normal muscle function and assembly of the DAP complex. These results demonstrate that syntrophin and dystrobrevin functionally associate with the DAP complex in the absence of a direct link to dystrophin. We also observed that the DAP complexes in these different transgenic mouse strains were not identical. Instead, the DAP complexes contained varying ratios of syntrophin and dystrobrevin isoforms. These results suggest that alternative splicing of the dystrophin gene, which naturally generates COOH-terminal deletions in dystrophin, may function to regulate the isoform composition of the DAP complex
Neural integrity is maintained by dystrophin in C. elegans
The dystrophin protein complex, an important regulator of muscle membrane integrity, also maintains neural organization through interactions with the L1CAM family member SAX-7
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