Molecular Mechanisms of the Myotonic Dystrophy Mutation

描述（由申请人提供）：肌强直性营养不良（DM1）是成人中最常见的肌肉营养不良，是由DM蛋白激酶（DMPK）基因3‘非翻译区（3’ utr）的n束扩大（CTG）引起的，导致“毒性”突变RNA和核包体中相互作用的RNA结合蛋白（如MBNL1）的核夹带。为了解决这个问题，我们培育了表达DMPK 3'UTR的转基因小鼠，作为编码绿色荧光蛋白（GFP）的诱导RNA转录物的一部分（参考文献）。令人惊讶的是，我们发现过表达正常DMPK 3'UTR mRNA的小鼠在没有可检测到的核内含物的情况下再现了糖尿病的基本特征，包括肌强直、心脏传导异常、糖尿病组织病理学和RNA剪接缺陷。重要的是，通过沉默转基因表达，这些作用在成熟骨骼肌和心肌中都是可逆的。这些结果首次在体内证明了通过消融有毒RNA或沉默其表达来治疗糖尿病的治疗策略的原理。利用该模型，我们开始了心脏传导缺陷的研究。我们现在已经发现了DM1关键方面的一种意想不到的机制的证据。转基因表达导致心脏传导缺陷与糖尿病完全相同，心脏转录因子Nkx2.5表达异常，连接蛋白40 （Cx40）和连接蛋白43 （Cx43）严重紊乱，这些蛋白是Nkx2.5的靶点，对正常心脏传导至关重要。在骨骼肌中，DMPK 3'UTR mRNA的过度表达也意外地诱导了Nkx2.5的转录激活，Nkx2.5通常是出生后的心脏转录因子。我们的研究结果表明，DMPK 3'UTR mRNA的表达诱导了Nkx2.5的转录，其机制尚不清楚。为了进一步研究这一点，本提案的目的是了解Nkx2.5在调节DM1表型中的作用，创建Nkx2.5表达的转基因小鼠模型，并试图了解DMPK 3'UTR mRNA如何转录激活Nkx2.5基因。肌强直性营养不良症是成人最常见的肌肉营养不良症。它被认为是由一种有毒的RNA引起的。我们已经开发了一种RNA毒性小鼠模型，希望通过这些研究，我们能够了解这种有毒RNA是如何导致糖尿病的，这样我们就可以考虑新的治疗策略。公共卫生相关性：肌强直性营养不良是成人最常见的肌肉营养不良。它被认为是由一种有毒的RNA引起的。我们已经开发了一种RNA毒性小鼠模型，希望通过这些研究，我们能够了解这种有毒RNA是如何导致糖尿病的，这样我们就可以考虑新的治疗策略。

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.