MOUSE MUSCLEBLIND MODEL FOR MYOTONIC DYSTROPHY

强直性肌营养不良(DM1和DM2)是最常见的成人型肌营养不良症，是临床表现相似的常染色体显性遗传病。值得注意的是，DM1和DM2是由两个不同基因DMPK和ZNF9的非翻译区中不稳定的微卫星扩张引起的。为了解释这些非编码扩展突变是如何导致显性遗传性神经肌肉疾病的，我们提出了一个针对强直性肌营养不良的有毒RNA模型。突变的DM1(CTG)n和DM2(CCTG)n等位基因的转录导致具有(CUG)n和(CCUG)n重复扩展的异常RNA转录本的产生。这些扩张折叠成稳定的双链(Ds)RNA结构，招募并隔离一系列dsRNA结合因子，即肌肉盲蛋白。因为这种有毒的RNA模型表明，DM1和DM2疾病是由于肌肉盲蛋白的丢失所致 功能，我们获得了肌肉盲1(MBNL1)基因敲除小鼠。这一建议旨在检验我们的工作假设，即MBNL1/-基因敲除小鼠将是一个有用的模型，用于研究涉及强直性肌营养不良疾病发病机制的潜在分子机制。首先，我们将表征MBNL1-/-肌肉的表型，并检验MBNL1是氯离子通道CIC-1正确的选择性剪接和功能所必需的假设。该离子通道的缺陷最近被认为是DM1和DM2相关性肌强直的原因。通过用不同水平表达(CUG)n RNA的转基因小鼠品系培育Mbnll基因敲除小鼠，将检验有毒RNA和结合蛋白之间的化学计量关系。其次，我们将研究盲肌蛋白通过与选择性剪接和/或其他因素相互作用来影响CIC-1氯通道水平的可能性。第三， 假设可以通过重组腺相关病毒介导的野生型成人CIC-1的表达来获得肌强直表型，这一假设将得到验证。最后，我们将调查额外的疾病相关表型是由于整个MBNL1基因的缺失，还是由于组织特异性的MBNL1表达，或者是由于所有三个肌盲基因(MBNL1，Mbnl2/Mbn11，Mbnl3/Mblx)的组合丢失造成的。

The myotonic dystrophies (DM1 and DM2), which are the most common form of adult-onset muscular dystrophy, are autosomal dominant diseases with similar clinical presentations. Remarkably, DM1 and DM2 are caused by unstable microsatellite expansions in the untranslated regions of two different genes, DMPK and ZNF9. To explain how these non-coding expansion mutations lead to dominantly inherited neuromuscular disorders, we have proposed a toxic RNA model for the myotonic dystrophies. Transcription of the mutant DM1 (CTG)n and DM2 (CCTG)n alleles leads to the production of unusual RNA transcripts with (CUG)n and (CCUG)n repeat expansions. These expansions fold into stable double-stranded (ds) RNA structures that recruit and then sequester a family of dsRNA-binding factors, the muscleblind proteins. Because this toxic RNA model suggests that DM1 and DM2 diseases are due to loss of muscleblind protein function, we have derived muscleblind 1 (Mbnl1) knockout mice. This proposal is designed to test our working hypothesis that Mbnl1-/- knockout mice will be a useful model to examine underlying molecular mechanisms involved in myotonic dystrophy disease pathogenesis. First, we will characterize the Mbnl1-/-muscle phenotype and test the hypothesis that Mbnl1 is required for proper alternative splicing and function of the chloride channel CIC-1. Deficiency of this ion channel has been recently implicated as the cause of DM1- and DM2- associated myotonia. The stoichiometric relationship between toxic RNA and binding protein will be examined by breeding Mbnll knockout mice with lines of transgenic mice that express (CUG)n RNA at different levels. Second, the possibility that muscleblind proteins influence CIC-1 chloride channel levels by interacting with alternative splicing, and/or other, factors will be examined. Third, the hypothesis that the myotonia phenotype can berescued using recombinant adeno-associated virus mediated expression of wild type adult CIC-1 will be tested. Finally, we will investigate if additional disease-associated phenotypes result from deletion of the entire Mbnl1 gene, from tissue-specific Mbnl1 expression or from combinatorial loss of all three muscleblind (Mbnl1, Mbnl2/Mbnll, Mbnl3/Mblx) genes.