Investigating mechanisms of skeletal muscle degeneration in Myotonic Dystrophy

描述(由申请人提供)： 强直性肌营养不良(DM)是成人起病的最常见的原因。这种多系统疾病的特点是骨骼肌功能的衰弱和进行性丧失。糖尿病的主要形式是由位于DMPK基因3‘端非编码区的CTG重复序列扩大引起的。现在很清楚，在转录后，扩展的重复序列仍然被困在细胞核中，并导致选择性剪接的反式显性错误调节。虽然糖尿病的许多症状，如肌强直和胰岛素抵抗，可以通过特定的错误剪接事件来解释，但骨骼肌退行性变的发生机制仍然不清楚。这项提案将使用赞助商实验室产生的一种新的转基因小鼠模型来调查肌肉退化的原因。在特定的目标1中，将培育两个小鼠系来产生双转基因动物，经他莫昔芬诱导后，在骨骼肌中特异地表达960个CUG重复。利用一些细胞、分子和功能分析，双转基因小鼠将被测试，看看它们是否很好地概括了DM的表型。在具体目标2中，将研究缺陷卫星细胞在糖尿病肌肉退行性变中的作用。卫星细胞是肌肉细胞的一个亚群，它可以增殖、分化并与现有的肌管融合，以修复受损的肌肉。众所周知，运动、肌肉损伤和退行性疾病可以激活卫星细胞；但在糖尿病患者中，它们无法被激活。我们将从我们的双转基因小鼠中分离出卫星细胞，并将进一步研究导致缺陷激活的机制。在具体目标3中，将研究一种剪接因子CUGBP1，它调节出生后发育中的发育选择性剪接模式。尽管CUGBP1水平在成年后下降，但在糖尿病患者中仍然出人意料地高，并与某些选择性剪接事件的错误调节有关。在他莫昔芬治疗后，双转基因小鼠的CUGBP1稳态水平将被检测。通过PKCα途径的磷酸化增加CUGBP1蛋白水平的潜在机制也将被研究。这项拟议的工作旨在阐明成人型肌营养不良症-强直性肌营养不良症的主要原因中骨骼肌退化的致病机制。从这个项目中获得的信息也可能提供对其他三核苷酸扩张性重复疾病的洞察，如亨廷顿舞蹈症、弗里德里希共济失调、脊髓小脑性共济失调和脆性X综合征。

DESCRIPTION (provided by applicant): Myotonic dystrophy (DM) is the most common cause of adult-onset muscular dystrophy. This multi-systemic disorder is marked by a debilitating and progressive loss of skeletal muscle function. The major form of DM is caused by expanded CTG repeats located in the 3' UTR of the DMPK gene. It is now clear that after transcription the expanded repeats remain trapped in the nucleus and lead to a trans-dominant misregulation of alternative splicing. Although many symptoms in DM, such as myotonia and insulin resistance, are explained by particular misregulated splicing events, the mechanism by which skeletal muscle degeneration arises remains unknown. This proposal will investigate the cause of muscle degeneration using a novel transgenic mouse model generated in the sponsor's lab. In Specific Aim 1 two mouse lines will be bred to produce bi-transgenic animals which upon tamoxifen induction express 960 CUG repeats specifically in skeletal muscle. Using a number of cellular, molecular and functional assays bitransgenic mice will be tested to see how well they recapitulate the DM phenotype. In Specific Aim 2, the role of defective satellite cells in DM muscle degeneration will be examined. Satellite cells are a subpopulation of muscle cells which proliferate, differentiate and fuse with existing myotubes in order to repair injured muscle. Exercise, muscle damage and degenerative disorders are known to activate satellites cells; however in DM they fail to be activated. Satellite cells will be isolated from our bi-transgenic mice, and mechanisms leading to defective activation will be further studied. In Specific Aim 3 a splicing factor, CUGBP1, which regulates developmental alternative splicing patterns in post-natal development, will be investigated. Although CUGBP1 levels decrease in adulthood, they remain unexpectedly high in DM patients and correlate with misregulation of certain alternative splicing events. CUGBP1 steady state levels will be assayed in bi-transgenic mice after tamoxifen treatment. A potential mechanism for increased CUGBP1 protein levels via phosphorylation through the PKC alpha pathway will also be examined. The proposed work aims to elucidate the pathogenic mechanisms responsible for skeletal muscle degeneration in the leading cause of adult-onset muscular dystrophy, Myotonic Dystrophy. Information gained from this project may also provide insight into other tri-nucleotide expansion repeat disorders, such as Huntington's Chorea, Friedreich's Ataxia, Spinal Cerebellar Ataxia and Fragile X syndrome.