Pathogenesis of Myopathy in Models of Myotonic Dystrophy

描述（由申请方提供）：强直性肌营养不良1型[DM 1]导致发育不良、肌强直和骨骼肌萎缩。DM 1是由DMPK的3'非翻译区中不稳定的CTG重复扩增引起的。我们的中心假设是，骨骼肌DM 1的结果从重复扩增转录的毒性作用。支持这一假设的研究来自HSALR转基因小鼠，表达CUG扩增RNA在肌肉中。（CUG）n转录物在核灶中积累，导致类似于DM 1的强直性肌病。我们的工作模型假定以下事件顺序：CUG扩增RNA的表达->（CUG）n RNA在核灶中的积累->肌盲[Mbnl]蛋白在核灶中的隔离->选择性剪接的异常调节->不适当的剪接异构体的表达-> DM的症状。我们现在有证据表明，这个模型可以解释DM 1的某些方面，如氯离子通道病和肌强直。我们计划扩展该模型并定义其限制。首先，我们将比较HSALR，Mbnll基因敲除和野生型小鼠的选择性剪接模式，并测试CUG扩增RNA妥协的选择性剪接的特定发育程序，取决于Mbnl 1，肌肉中表达的主要Mbnl蛋白的假设。异常剪接的一个引人注目的例子涉及Serca 1，肌浆网中的钙再摄取泵。将通过钙成像确定错误剪接Serca 1的生理意义。其次，关于（CUG）n转录物的代谢的信息很少。我们已经获得了诱导表达CUG扩增转录本的转基因小鼠。这些小鼠将用于比较具有或不具有扩增的CUG重复的转录物的积累和降解。我们还将检验核mRNA降解酶的过表达可以加速多聚CUG RNA的清除的假设。第三，我们将评估HSA（LR）小鼠的肌球蛋白形态和溴脱氧尿苷掺入，以检验CUG扩增RNA积累导致核死亡的假设。在相关的实验中，我们将研究生长因子撤出时HSA（LR）成肌细胞中发生的细胞死亡机制。第四，我们已经获得了转基因小鼠与cre激活等位基因开发模型DM 1相关的发育不良和浪费和测试的假设，CUG扩增RNA干扰肌肉分化。

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.