Molecular Mechanisms of the Myotonic Dystrophy Mutation

描述（由申请人提供）：强直性肌营养不良（DM 1）是成人中最常见的肌营养不良，由DM蛋白激酶（DMPK）基因3'非翻译区（3'UTR）中扩展的（CTG）n道引起，导致“毒性”突变RNA和相互作用的RNA结合蛋白（例如MBNL1）在核糖核内含物中的核截留。为了解决这个问题，我们产生了表达DMPK 3 'UTR作为编码绿色荧光蛋白（GFP）的诱导型RNA转录物的一部分的转基因小鼠（参考文献）。令人惊讶的是，我们发现过表达正常DMPK 3'UTR mRNA的小鼠再现了DM的主要特征，包括肌强直、心脏传导异常、DM组织病理学和RNA剪接缺陷，而不存在可检测的核内含物。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。这些结果代表了通过切除毒性RNA或沉默其表达来治疗DM的治疗策略的原理的第一个体内证明。利用这个模型，我们已经开始研究心脏传导缺陷。我们现在已经发现了DM1关键方面的意外机制的证据。转基因表达导致心脏传导缺陷，与DM中的心脏传导缺陷完全一样，心脏转录因子Nkx2.5的异常表达，以及连接蛋白40（Cx40）和连接蛋白43（Cx43）的严重干扰，这些蛋白是Nkx2.5的靶蛋白，对正常心脏传导至关重要。在骨骼肌中，DMPK 3'UTR mRNA的过表达也意外地诱导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Nkx2.5转录，通过迄今未知的机制。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，建立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。强直性肌营养不良是成人中最常见的肌营养不良。它被认为是由有毒的RNA引起的。我们已经开发了一种RNA毒性的小鼠模型，并希望通过这些研究，我们能够了解这种有毒RNA如何导致DM，以便我们可以考虑新的治疗策略。公共卫生相关性：强直性肌营养不良是成人最常见的肌营养不良。它被认为是由有毒的RNA引起的。我们已经开发了一种RNA毒性的小鼠模型，并希望通过这些研究，我们能够了解这种有毒RNA如何导致DM，以便我们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.