Myotonic Dystrophy type 2

描述（由申请方提供）：强直性肌营养不良2型（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、肌无力和肌强直。DM 2是由ZNF 9基因内含子1中的CCTG重复序列扩增引起的。CCTG扩增通过错误调节RNA代谢的RNA CCUG重复序列的积累诱导DM2病理。肌肉萎缩是2型糖尿病患者的主要健康问题。CCUG重复序列导致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重复减少ZNF 9蛋白。由于ZNF9控制翻译装置的蛋白质合成，因此其在DM 2成肌细胞中的减少降低了整体蛋白质合成的速率。因此，本申请的主要假设是突变CCUG重复序列通过降低整体蛋白质合成速率而引起骨骼肌损失。本申请的目的1将确定DM2中CCUG重复序列下调ZNF9蛋白的机制。由于CCUG重复不会改变ZNF 9 mRNA水平，我们将确定CCUG重复是否减少ZNF 9的蛋白质合成或CCUG重复是否增加ZNF 9的降解。免疫荧光分析显示，在DM2肌纤维，ZNF9易位到肌纤维膜表明CCUG重复序列可能会减少细胞质ZNF9通过增强其与膜蛋白的相互作用。因此，我们将测试ZNF9重新分布到DM2肌纤维的膜是否导致细胞质中ZNF9的减少。来自DM 2患者的肌肉活检中ZNF9的减少表明DM 2患者中成熟肌肉中的整体蛋白质合成速率降低。因此，目标2将确定突变CCUG重复是否降低体内整体蛋白质合成的速率，从而导致骨骼肌消耗。为此，我们将利用DM2小鼠模型，即具有低水平ZNF9的CCTG转基因小鼠。ZNF9将在CCTG TR小鼠中正常化，并且将检查ZNF9的校正对肌肉消耗和肌肉功能的影响。在目标3中，我们提出阐明负责增加突变CCUG重复序列的稳定性的机制，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。我们已经确定了五个CCUG100结合蛋白，改变活动的积累过程中的突变CCUG重复和DM2成肌细胞。我们推测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。其中一个蛋白质已被纯化并确定为p68解旋酶。我们将阐明p68和其他已确定的CCUG100结合蛋白在突变CCUG重复序列稳定性调节中的相对作用。拟议研究的结果将确定DM 2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。 公共卫生相关性： 强直性肌营养不良2型（DM2）患者患有骨骼肌损失和肌无力。该申请提出应用基础科学专业知识来概述DM2中肌肉萎缩的机制，并概述控制突变CCUG重复序列降解的机制。我们的基础科学方法将为开发DM2的临床治疗提供基础工作。

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.