Role of CUGBP1 in skeletal muscle wasting in myotonic dystrophy

描述（由申请人提供）：肌强直性营养不良（DM）是最常见的成人发病肌肉营养不良。1型糖尿病（DM1）是一种多系统神经肌肉疾病，由DMPK基因3'非翻译区CTG扩增引起。保留在细胞核中的含有重复序列的RNA隔离了MBNL1剪接因子并导致CUGBP1剪接因子的上调。选择性剪接失调在疾病机制中起关键作用，并直接导致肌强直和胰岛素抵抗两种疾病症状。含有重复序列的RNA诱导其他疾病症状的能力尚不清楚，如骨骼肌萎缩，这是患者死亡的主要原因。在Specific Aim 1中，将使用来自6种肌肉萎缩症小鼠模型的组织来确定不同肌肉萎缩症中剪接变化的流行程度和剪接调节因子水平的改变。在Specific Aim 2中，我们将研究CUGBP1在骨骼肌萎缩中的作用。CUGBP1在DM1患者和具有强消耗表型的DM1小鼠模型中异常上调。为了确定在DM1小鼠模型中CUGBP1的增加是否对消耗是必要的，我们将通过腺相关病毒传递shRNA来敲低CUGBP1。然后，将通过运动试验、肌肉重量和肌肉组织学来测试肌肉的完整性。为了确定CUGBP1的增加是否足以诱导消瘦，我们产生了强力霉素诱导的CUGBP1骨骼肌特异性表达的小鼠，并将对其进行肌肉功能和消瘦检查。在Specific Aim 3中，将确定PKC参与CUGBP1上调。在DM1心脏组织和诱导型DMl心脏模型中，CUGBP1响应CUG重复扩增的上调是通过pkc依赖性磷酸化事件发生的，导致CUGBP1过度磷酸化和稳定。PKC活性和CUGBP1过度磷酸化将通过DMl小鼠模型在骨骼肌中测定。在DM1小鼠模型中，将探索Bis-IX治疗PKC抑制作为抑制CUGBP1上调和防止肌肉萎缩的潜在治疗方法。这些实验提出了一个非常重要的问题，即肌强直性营养不良患者肌肉萎缩的原因。其作用机制可能更广泛地适用于其他神经肌肉疾病和三核苷酸重复疾病。公共卫生声明：肌强直性营养不良患者死亡的主要原因是严重的骨骼肌萎缩。本研究旨在利用肌强直性营养不良小鼠模型确定骨骼肌萎缩的原因，并利用化学抑制剂潜在地防止骨骼肌萎缩。

DESCRIPTION (provided by applicant): Myotonic dystrophy (DM) is the most common adult onset muscular dystrophy. DM type 1 (DM1) is a multi-systemic neuromuscular disease caused by a CTG expansion in the 3' untranslated region of the DMPK gene. The repeat-containing RNA retained in the nucleus sequesters the MBNL1 splicing factor and leads to up-regulation of the CUGBP1 splicing factor. Misregulated alternative splicing plays a critical role in the disease mechanism and has been directly attributed to two disease symptoms, myotonia and insulin resistance. The ability of repeat-containing RNA to induce other disease symptoms such as skeletal muscle wasting, the leading cause of death in patients, is not known. In Specific Aim 1, the prevalence of splicing changes and alterations in the levels of splicing regulators in different muscular dystrophies will be determined using tissue from six muscular dystrophy mouse models. In Specific Aim 2, the role of CUGBP1 in skeletal muscle wasting will be investigated. CUGBP1 is aberrantly up-regulated in DM1 patients and a DM1 mouse model with a strong wasting phenotype. To determine if increased CUGBP1 is necessary for wasting in this DM1 mouse model, CUGBP1 will be knocked down by delivery of shRNA via an adeno-associated virus. Then, the muscle integrity will be tested by exercise tests, muscle weight, and muscle histology. To determine if increased CUGBP1 is sufficient to induce wasting, mice with doxycycline-inducible and skeletal muscle specific expression of CUGBP1 have been generated and will be examined for muscle function and wasting. In Specific Aim 3, the involvement of PKC in CUGBP1 up-regulation will be determined. In DM1 heart tissue and an inducible DMl heart model, CUGBP1 up-regulation in response to expanded CUG repeats occurs through a PKC-dependent phosphorylation event resulting in hyperphosphorylated and stabilization of CUGBP1. PKC activity and CUGBP1 hyperphosphorylation will be determined in skeletal muscle from the DMl mouse model. PKC inhibition by Bis-IX treatment will be explored as a potential therapeutic approach to inhibit CUGBP1 up-regulation and prevent muscle wasting in the DM1 mouse model. These proposed experiments address a very important question in the field about the cause of muscle wasting in myotonic dystrophy. The mechanisms at play may be more broadly applicable to other neuromuscular diseases and trinucleotide repeat diseases. Public health statement: The primary cause of death in myotonic dystrophy patients is due to the severe skeletal muscle wasting. This proposal aims to determine the cause of skeletal muscle wasting using a myotonic dystrophy mouse model and potentially prevent the wasting using a chemical inhibitor.