Myotonic Dystrophy type 2

描述（由申请人提供）：2型肌强直性营养不良症（DM2）是一种常染色体显性、多系统疾病，主要影响骨骼肌，导致骨骼肌损失、肌肉无力和肌强直。DM2是由ZNF9基因内含子1中CCTG重复序列的扩增引起的。CCTG扩增通过RNA CCUG重复序列的积累导致RNA代谢失调，从而诱导DM2病理。肌肉萎缩是DM2患者的主要健康问题。CCUG重复引起骨骼肌萎缩的机制尚不清楚。我们最近的研究结果表明，突变CCUG重复序列降低了ZNF9蛋白。由于ZNF9控制着翻译装置蛋白质的合成，它在DM2成肌细胞中的减少降低了整体蛋白质合成的速度。因此，本应用的主要假设是突变的CCUG重复序列通过降低整体蛋白质合成速率导致骨骼肌损失。本应用的目的1将确定CCUG重复序列下调DM2中ZNF9蛋白的机制。由于CCUG重复序列不会改变ZNF9的mRNA水平，我们将确定CCUG重复序列是否会减少ZNF9的蛋白质合成，还是会增加ZNF9的降解。免疫荧光分析显示，在DM2肌纤维中，ZNF9易位到肌纤维膜上，这表明CCUG重复序列可能通过增强ZNF9与膜蛋白的相互作用来减少细胞质ZNF9。因此，我们将测试ZNF9重新分布到DM2肌纤维膜上是否会导致细胞质中ZNF9的减少。DM2患者肌肉活检中ZNF9的减少表明，DM2患者成熟肌肉中整体蛋白质合成速率降低。因此，Aim 2将确定突变CCUG重复序列是否会降低体内整体蛋白质合成速率，从而导致骨骼肌萎缩。为此，我们将利用DM2小鼠模型，CCTG转基因小鼠，具有低水平的ZNF9。将ZNF9在CCTG - TR小鼠中归一化，并检测ZNF9纠正对肌肉萎缩和肌肉功能的影响。在Aim 3中，我们提出阐明突变CCUG重复序列稳定性增加的机制，目的是消除CCUG重复序列的毒性。我们发现纯CCUG重复序列非常稳定。我们已经确定了五种ccug100结合蛋白，它们在突变CCUG重复序列和DM2成肌细胞的积累过程中改变活性。我们假设这些蛋白质活性的改变是导致突变CCUG重复序列稳定性和毒性增加的原因。其中一种蛋白质已被纯化并确定为p68解旋酶。我们将阐明p68和其他已鉴定的ccug100结合蛋白在调节突变CCUG重复序列稳定性中的相对作用。拟议的研究结果将确定DM2患者骨骼肌损失的分子机制，并将有助于开发降解突变CCUG重复序列的方法。

DESCRIPTION (provided by applicant): Myotonic Dystrophy type 2 (DM2) is an autosomal dominant, multisystemic disease, that primarily affects skeletal muscle causing skeletal muscle loss, muscle weakness and myotonia. DM2 is caused by expansion of CCTG repeats in the intron 1 of ZNF9 gene. CCTG expansion induces DM2 pathology through accumulation of RNA CCUG repeats that misregulate RNA metabolism. Muscle wasting is a major health problem in patients with DM2. The mechanisms by which CCUG repeats cause skeletal muscle wasting are not known. Our recent findings show that the mutant CCUG repeats reduce ZNF9 protein. Because ZNF9 controls synthesis of proteins of translational apparatus, its reduction in DM2 myoblasts decreases the rate of global protein synthesis. Therefore, the main hypothesis of this application is that the mutant CCUG repeats cause skeletal muscle loss through decrease of the rate of global protein synthesis. The Aim 1 of this application will determine mechanisms by which CCUG repeats down regulate ZNF9 protein in DM2. Since CCUG repeats do not change ZNF9 mRNA levels, we will determine if CCUG repeats reduce protein synthesis of ZNF9 or if the CCUG repeats increase degradation of ZNF9. Immunofluorescent analysis revealed that, in DM2 myofibers, ZNF9 translocates to the myofiber membrane suggesting that CCUG repeats might reduce cytoplasmic ZNF9 by enhancement of its interaction with membrane proteins. Therefore, we will test if the re- distribution of ZNF9 to the membrane of DM2 myofibers causes the reduction of ZNF9 in cytoplasm. Reduction of ZNF9 in muscle biopsies from DM2 patients suggests that the rate of global protein synthesis is reduced in mature muscle in DM2 patients. Therefore, Aim 2 will determine if the mutant CCUG repeats reduce the rate of global protein synthesis in vivo causing skeletal muscle wasting. For this goal, we will utilize the DM2 mouse model, CCTG transgenic mice, which have low levels of ZNF9. ZNF9 will be normalized in CCTG TR mice and the effect of correction of ZNF9 on muscle wasting and muscle function will be examined. In Aim 3, we propose to elucidate the mechanisms responsible for the increased stability of the mutant CCUG repeats with the goal to eliminate toxicity of CCUG repeats. We found that pure CCUG repeats are very stable. We have identified five CCUG100-binding proteins that alter activity during accumulation of the mutant CCUG repeats and in DM2 myoblasts. We hypothesize that alterations in activity of these proteins are responsible for the increased stability and toxicity of the mutant CCUG repeats. One of these proteins has been purified and determined as p68 helicase. We will elucidate the relative role of p68 and other identified CCUG100-binding proteins in the regulation of stability of the mutant CCUG repeats. The results of the proposed studies will determine the molecular mechanism of skeletal muscle loss in patients with DM2 and will help to develop approaches to degrade mutant CCUG repeats. PUBLIC HEALTH RELEVANCE: Patients with Myotonic Dystrophy type 2 (DM2) suffer from skeletal muscle loss and muscle weakness. The application proposes to apply basic science expertise to outline the mechanism of muscle wasting in DM2 and to outline the mechanism controlling degradation of the mutant CCUG repeats. Our basic science approaches will provide the ground work for the development of the clinical treatment of DM2.