The mechanism of CELF1 upregulation and its role in the pathogenesis of Myotonic Dystrophy Type 1

项目摘要 强直性肌营养不良1型(DM1)是一种以进行性骨骼为特征的多系统疾病 肌肉无力、肌肉萎缩和肌强直。DM1的患病率为1/8500，是最常见的 成人型肌营养不良症的病因。DM1是由CTG重复序列在3‘非翻译序列中的扩增引起的 Dystrophia Myotonica蛋白激酶(DMPK)基因的区域。从扩增的RNA转录而来 DMPK等位基因包含可隔离肌肉盲人近亲的CUG重复序列(CUGexp RNA) 与RNA结合蛋白(MBNL)家族一样，MBNL1和MBNL2导致其功能丧失。此外, 第二个RNA结合蛋白，CUGBP Elav样家族成员1(CELF1)被上调到显示出的水平 在DM1骨骼肌中有毒。而MBNL通过封存而丧失功能的机制是好的 骨骼肌中CELF1上调的机制及其对肌肉的作用 DM1的赤字仍不得而知。 为了在小鼠中建立成年起病的DM1模型，我们产生了MBNL1和MbnL2等位基因的小鼠，并诱导了 成人骨骼肌条件性双基因敲除(MBNL DcKO)MBNL dcKO小鼠类似于成年型 DM1有显著的剪接变化，显著的肌肉萎缩，预计组织病理学较轻。有趣的是， 我们还发现CELF1蛋白水平增加，揭示了以前未知的调控联系 MBNL和CELF1，不依赖CUGexp RNA。这一发现表明CELF1上调和毒性 是DM1中MBNL功能丧失的直接后果。 这项提议的目标有两个。在目标1中，我将确定MBNL功能丧失如何导致 上调CELF1蛋白，揭示参与CELF1蛋白表达的顺式调控基序和反式作用因子 进程。我们先前已经证明，CELF1可以被蛋白激酶C(PKC)介导的稳定 磷酸化。因此，一个特别的兴趣将是测试MBNL功能丧失是否导致激活 PKC并增加CELF1的磷酸化，或者如果有其他机制参与上调 CELF1。由于先前尚未发现成人在骨骼肌中敲除MBNL1和MBNL2的特征，因此在 目标2，我将首先对生理、组织病理和分子肌肉进行深入的描述 MBNL dcKO小鼠的表型建立稳健和定量的分析，然后将用于确定 CELF1上调的程度对MBNL dcKO肌肉表型有贡献。 在完成这项研究后，我将解决DM1领域中长期存在的问题：如何 CELF1被上调，并决定了这种上调对MBNL dcKO肌肉的贡献程度 赤字。我们的发现，CELF1上调是由MBNL功能丧失引起的，这为我们提供了新的见解 DM1发病机制的复杂组合机制将有助于进一步研究DM1的发病机制 和进展，并将为未来治疗方法的发展提供信息。

Project Summary Myotonic Dystrophy Type 1 (DM1) is a multisystemic disorder characterized by progressive skeletal muscle weakness, muscle wasting, and myotonia. With a prevalence of 1 in 8500, DM1 is the most common cause of adult-onset muscular dystrophy. DM1 is caused by the expansion of CTG repeats in the 3' untranslated region of the Dystrophia Myotonica Protein Kinase (DMPK) gene. The RNA transcribed from the expanded DMPK allele contains expanded CUG repeats (CUGexp RNA) that sequester the paralogs of the Muscleblind Like (MBNL) family of RNA binding proteins, MBNL1 and MBNL2, resulting in their loss of function. In addition, a second RNA-binding protein, CUGBP Elav-like family member 1 (CELF1), is upregulated to a level shown to be toxic in DM1 skeletal muscle. While the mechanism of MBNL loss of function through sequestration is well established, the mechanism leading to CELF1 upregulation in skeletal muscle and its contribution to the muscle deficits in DM1 remain unknown. To model adult-onset DM1 in mice, we generated mice with floxed Mbnl1 and Mbnl2 alleles and induced conditional double knockout (Mbnl dcKO) in adult skeletal muscle. The Mbnl dcKO mice resemble adult-onset DM1 with striking splicing changes, significant muscle wasting, and expected mild histopathology. Interestingly, we also found an increase in CELF1 protein levels, revealing a previously unknown regulatory link between MBNL and CELF1, independent of CUGexp RNA. This discovery suggests that CELF1 upregulation and toxicity are a direct consequence of MBNL loss of function in DM1. The goal of this proposal is two-fold. In Aim 1, I will determine how MBNL loss of function leads to the upregulation of CELF1 protein and uncover the cis-regulatory motifs and trans-acting factors involved in the process. We previously demonstrated that CELF1 can be stabilized by Protein Kinase C (PKC)-mediated phosphorylation. As such, one particular interest will be to test if MBNL loss of function leads to an activation of PKC and increases the phosphorylation of CELF1 or if other mechanisms are involved in the upregulation of CELF1. As adult knockout of MBNL1 and MBNL2 in skeletal muscle has not been previously characterized, in Aim 2, I will first perform an in-depth characterization of the physiological, histopathological and molecular muscle phenotypes of Mbnl dcKO mice to establish robust and quantitative assays that will then be used to determine the extent CELF1 upregulation contributes to Mbnl dcKO muscle phenotypes. Upon completion of this study, I will have resolved the long-standing question in the DM1 field of how CELF1 is upregulated and determined the extent to which this upregulation contributes to the Mbnl dcKO muscle deficits. Our finding that CELF1 upregulation is caused by MBNL loss of function provides new insight into the complex combinatorial mechanisms of DM1 pathogenesis and will help future studies on DM1 disease causation and progression and will inform the development of future therapeutic approaches.