Myotonic Dystrophy: Molecular Pathophysiology and CNS Effects

描述（由申请人提供）：我们先前证明强直性肌营养不良2型（DM 2）是由ZA/F9基因内含子1中的CCTG扩增引起的。DM 1和DM 2扩增突变之间的相似之处和与扩增的CUG和CCUG重复序列相互作用的RNA结合蛋白的表征揭示了一种涉及RNA功能获得效应的新型机制。在该模型中，CUG或CCUG扩增转录物的积累导致RNA结合蛋白的失调，所述RNA结合蛋白通常参与控制一组特定前mRNA转录物的选择性剪接中的发育变化。这组正常发育剪接变化的失败被认为是发生的，至少部分是因为DM 1和DM 2中CUG和CCUG扩增转录本的积累导致核MBNL 1的螯合和耗尽。值得注意的是，Swanson博士的小组最近进行的研究现在表明，AAV注射过表达MbnII的构建体可以挽救肌肉表型并逆转DM 1的骨骼肌多聚（CUG）模型中的剪接改变。虽然在了解骨骼肌和心肌中RNA发病的病理学和分子机制方面取得了重大进展，但对DM 1和DM 2中一组临床重要但知之甚少的CNS效应的分子变化知之甚少。成人发病的DM 1和DM 2患者具有惊人相似的多系统疾病，初步数据表明相似的大脑和小脑白色物质异常以及执行功能缺陷。受基因影响的DM 1患者有更严重的CNS异常，包括智力迟钝。先天性DM 1的一个可能机制是，在先天性病例中DM 1基因座的甲基化与DMPK表达增加相关，导致更高水平的含有CUG的转录物和更严重的先天性表型。本提案的重点是使用转基因模型来检验CUG和CCUG重复毒性在细胞和生物体水平上具有可比性的假设，即RNA扩增转录物的时空表达的改变导致DM 1和DM 2的表型相似性和差异，包括CNS效应，并且该疾病的许多多系统特征是可逆的。我们的具体目标是：1）DM 1和DM 2之间的临床相似性和差异是由CUG/CCUG扩增转录本的时间和空间表达模式的变化引起的，并且与重复基序和侧翼序列无关。2)强直性肌营养不良中发现的CNS特异性分子变化是由RNA获得功能效应引起的，并且DM 1和DM 2之间的分子和表型相似性和差异是由脑中CUG/CCUG扩增的时间和空间表达模式的变化引起的。3)通过产生MbnII过表达的诱导型转基因小鼠模型来评估骨骼肌和CNS中MbnII表达增加的作用。

Description (provided by applicant): We demonstrated previously that myotonic dystrophy type 2 (DM2) is caused by a CCTG expansion in intron 1 of the ZA/F9gene. Parallels between the DM1 and DM2 expansion mutations and the characterization of RNA binding proteins that interact with expanded CUG and CCUG repeats have uncovered a novel type of mechanism involving RNA gain-of-function effects. In this model, the accumulation of CUG or CCUG expansion transcripts results in the dysregulation of RNA binding proteins normally involved in controlling developmental changes in alternative splicing of a specific set of pre-mRNA transcripts. The failure of this set of normal developmental splicing changes is thought to occur, at least in part, because the accumulation of CUG and CCUG expansion transcripts in DM1 and DM2 leads to sequestration and depletion of nuclear MBNL1. Remarkably, recent studies performed by Dr. Swanson's group now show that AAV injection of constructs overexpressing Mbnll can rescue the muscle phenotype and reverse the splicing alterations in a skeletal muscle poly(CUG) model of DM1. Although significant progress has been made in understanding the pathology and the molecular mechanisms of RNA pathogenesis in skeletal and cardiac muscle, little is known about the molecular changes underlying a clinically important, but poorly understood, set of CNS effects in DM1 and DM2. Patients with adult onset DM1 and DM2 have strikingly similar multisystemic diseases, with preliminary data suggesting similar cerebral and cerebellar white matter abnormalities as well as executive function deficits. Congenially affected DM1 patients have more severe CNS abnormalities including mental retardation. A possible mechanism for congenital DM1 is that methylation at the DM1 locus in congenital cases is associated with increased expression of DMPK, resulting in higher levels of CUG containing transcripts and a more severe congenital phenotype. The focus of this proposal is to use transgenic models to test the hypothesis that CUG and CCUG repeat toxicity is comparable at the cellular and organismal levels, that alterations in temporal and spatial expression of RNA expansion transcripts cause phenotypic similarities and differences in DM1 and DM2, including CNS effects, and that many of the multisystemic features of the disease are reversible. Our specific aims are: 1) Clinical similarities and differences between DM1 and DM2 result from variations in temporal and spatial expression patterns of the CUG/CCUG expansion transcripts and are independent of both the repeat motif and flanking sequence. 2) CNS specific molecular changes found in myotonic dystrophy are caused by RNA gain of function effects and that molecular and phenotypic similarities and differences between DM1 and DM2 result from variations in temporal and spatial expression patterns of the CUG/CCUG expansions in the brain. 3) To evaluate the effects of increased Mbnll expression in skeletal muscle and the CNS by generating an inducible transgenic mouse model of Mbnll over-expression.