Molecular Mechanisms of the Myotonic Dystrophy Mutation

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DESCRIPTION (provided by applicant): Myotonic dystrophy (DM1), the most common muscular dystrophy in adults, is caused by an expanded (CTG)n tract in the 3' untranslated region (3'UTR) of the DM protein kinase (DMPK) gene resulting in nuclear entrapment of the "toxic" mutant RNA and interacting RNA-binding proteins (e.g. MBNL1) in ribonuclear inclusions. To address this, we generated transgenic mice expressing the DMPK 3'UTR as part of an inducible RNA transcript encoding green fluorescent protein (GFP) (ref). Surprisingly, we found that mice overexpressing a normal DMPK 3'UTR mRNA reproduced cardinal features of DM including myotonia, cardiac conduction abnormalities, DM histopathology and RNA splicing defects in the absence of detectable nuclear inclusions. Importantly, these effects were reversible in both mature skeletal and cardiac muscles by silencing transgene expression. These results represented the first in vivo proof of principle for a therapeutic strategy to treat DM through ablation of the toxic RNA or silencing its expression. Using this model, we have started to study the cardiac conduction defects. We have now found evidence for an unanticipated mechanism for key aspects of DM1. Transgene expression resulted in cardiac conduction defects exactly like those in DM, aberrant expression of the cardiac transcription factor Nkx2.5, and profound disturbances in connexin 40 (Cx40) and connexin 43 (Cx43), proteins that are targets of Nkx2.5 and crucial for normal cardiac conduction. In skeletal muscle, over-expression of the DMPK 3'UTR mRNA also unexpectedly induced transcriptional activation of Nkx2.5, normally a cardiac transcription factor postnatally. Our results suggest that expression of the DMPK 3'UTR mRNA induces Nkx2.5 transcription, by as yet unknown mechanisms. To study this further, the aims of this proposal are to understand the role of Nkx2.5 in modulating the DM1 phenotype, to create a transgenic mouse model of Nkx2.5 expression and to attempt to understand how the DMPK 3'UTR mRNA transcriptionally activates the Nkx2.5 gene. Myotonic dystrophy is the most common muscular dystrophy in adults. It is thought to be caused by a toxic RNA. We have developed a mouse model of RNA toxicity and hope that with these studies we may be able to understand how this toxic RNA causes DM, so that we can contemplate strategies for new therapies. PUBLIC HEALTH RELEVANCE: Myotonic dystrophy is the most common muscular dystrophy in adults. It is thought to be caused by a toxic RNA. We have developed a mouse model of RNA toxicity and hope that with these studies we may be able to understand how this toxic RNA causes DM, so that we can contemplate strategies for new therapies.