Pathogenesis of Myopathy in Models of Myotonic Dystrophy

描述(申请人提供)：强直性肌营养不良1型[DM1]导致发育不良、肌强直和骨骼肌萎缩。DM1是由DMPK基因3‘端非翻译区不稳定的CTG重复序列扩增所致。我们的中心假设是，在DM1中发现的骨骼肌是重复扩增转录本的毒性作用的结果。支持这一假设的研究来自对HSALR转基因小鼠的研究，这些小鼠在肌肉中表达CUG扩展RNA。(Cug)n转录本积累在核灶中，导致类似于DM1的强直性肌病。我们的工作模型假设了以下一系列事件：CUG扩展RNA-&GT的表达；(CUG)n RNA在核灶-GT中的积累；[MBNL]蛋白在核灶-GT中的隔离；选择性剪接-GT的异常调节；不适当剪接亚型的表达-GT；DM的症状。我们现在有证据表明，这个模型可以解释DM1的某些方面，如氯通道病和肌强直。我们计划扩展这一模式，并定义其局限性。首先，我们将比较HSALR、Mbn11基因敲除小鼠和野生型小鼠的选择性剪接模式，并测试CUG扩展RNA妥协依赖于MBNL1的选择性剪接特定发育程序的假设，MBNL1是肌肉中表达的主要MBNL蛋白。异常剪接的一个显著例子涉及肌浆网中的钙再摄取泵Serca1。错误剪接Serca1的生理意义将通过钙成像来确定。其次，关于(CUG)n转录本的代谢信息很少。我们已经获得了可诱导表达CUG扩展转录本的转基因小鼠。这些小鼠将被用来比较有或没有扩展的CUG重复序列的转录本的积累和降解。我们还将检验核mRNA降解酶过表达可以加速聚CUG RNA清除的假设。第三，我们将评估HSA(LR)小鼠的肌核形态和溴脱氧尿苷掺入，以验证CUG扩展RNA积累导致核死亡的假设。在相关实验中，我们将研究当停止生长因子时，HSA(LR)成肌细胞发生细胞死亡的机制。第四，我们获得了带有cre激活等位基因的转基因小鼠，以建立DM1相关的发育和消瘦模型，并验证了CUG扩展RNA干扰肌肉分化的假说。

DESCRIPTION (provided by applicant): Myotonic dystrophy type 1 [DM1] leads to maldevelopment, myotonia, and wasting of skeletal muscle. DM1 is caused by an unstable CTG repeat expansion in the 3' untranslated region of DMPK. Our central hypothesis is that skeletal muscle findings in DM1 result from a toxic effect of repeat expansion transcripts. Support for this hypothesis comes from studies of HSALR transgenic mice that express CUG expansion RNA in muscle. (CUG)n transcripts accumulate in nuclear foci, leading to a myotonic myopathy that is similar to DM1. Our working model postulates the following sequence of events: expression of CUG expansion RNA -> accumulation of (CUG)n RNA in nuclear foci -> sequestration of muscleblind [Mbnl] proteins in nuclear foci -> abnormal regulation of alternative splicing -> expression of inappropriate splice isoforms -> symptoms of DM. We now have evidence that this model can explain certain aspects of DM1, such as, chloride channelopathy and myotonia. We plan to extend this model and define its limits. First, we will compare patterns of alternative splicing in HSALR, Mbnll knockout, and wild-type mice and test the hypothesis that CUG expansion RNA compromises a specific developmental program of alternative splicing that depends on Mbnl1, the predominant Mbnl protein expressed in muscle. A striking example of aberrant splicing involves Serca1, the calcium re-uptake pump in sarcoplasmic reticulum. The physiologic significance of mis-splicing Serca1 will be determined by calcium imaging. Second, there is little information about metabolism of (CUG)n transcripts. We have derived transgenic mice for inducible expression of CUG expansion transcripts. These mice will be used to compare the accumulation and degradation of transcripts with or without an expanded CUG repeat. We also will test the hypothesis that overexpression of nuclear mRNA-degradases can accelerate clearance of poly-CUG RNA. Third, we will assess myonuclear morphology and bromodeoxyuridine incorporation in HSA(LR) mice to test the hypothesis that accumulation of CUG expansion RNA leads to nuclear demise. In related experiments we will investigate the mechanism of cell death that occurs in HSA(LR) myoblasts when growth factors are withdrawn. Fourth, we have derived transgenic mice with cre-activation alleles to develop models for DM1-related maldevelopment and wasting and test the hypothesis that CUG expansion RNA interferes with muscle differentiation.