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（DM 1）是一种多系统疾病，其特征在于进行性骨骼肌萎缩， 肌肉无力、肌肉萎缩和肌强直。DM 1的患病率为1/8500，是最常见的 成人型肌肉萎缩症的病因DM 1是由3'端非翻译区CTG重复序列的扩增引起的。 肌强直性肌营养不良蛋白激酶（DMPK）基因的区域。从扩增的DNA中转录的RNA DMPK等位基因包含扩展的CUG重复序列（CUGexp RNA），其隔离肌盲的旁系同源物 类似于RNA结合蛋白（MBNL）家族，MBNL 1和MBNL 2，导致其功能丧失。此外，本发明还提供了一种方法， 第二种RNA结合蛋白，CUGBP Elav样家族成员1（CELF 1），被上调至显示的水平， 对DM 1骨骼肌有毒性。而MBNL通过隔离而丧失功能的机制是很好的 建立了导致骨骼肌中CELF 1上调的机制及其对肌肉的贡献， DM 1的缺陷仍然未知。 为了在小鼠中建立成年发病的DM 1模型，我们产生了具有floxed Mbnl 1和Mbnl 2等位基因的小鼠，并诱导了 条件性双敲除（MbnldcKO）。Mbnl dcKO小鼠类似于成年发作的 DM 1具有显著的剪接变化、显著的肌肉萎缩和预期的轻度组织病理学。有趣的是， 我们还发现CELF 1蛋白水平的增加，揭示了一种以前未知的调节联系， MBNL和CELF 1，不依赖于CUGexp RNA。这一发现表明CELF 1的上调和毒性 是DM 1中MBNL功能丧失的直接后果。 这项建议有两个目的。在目标1中，我将确定MBNL功能丧失如何导致 CELF 1蛋白的上调，并揭示了参与细胞凋亡的顺式调节基序和反式作用因子。 过程我们以前证明CELF 1可以通过蛋白激酶C（PKC）介导的 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 PKC和增加CELF 1的磷酸化，或者如果其他机制参与了 CELF 1.由于骨骼肌中MBNL 1和MBNL 2的成人敲除以前没有被表征， 目的2，首先对肌肉的生理、组织病理和分子生物学特性进行深入研究 Mbnl dcKO小鼠的表型，以建立稳健的定量测定，然后将用于确定 CELF 1上调的程度有助于MbnldcKO肌肉表型。 在完成这项研究后，我将解决DM 1领域长期存在的问题，即如何 CELF 1被上调，并确定这种上调对Mbn 1 dcKO肌肉的贡献程度 赤字我们发现CELF 1的上调是由MBNL功能丧失引起的，这为我们研究CELF 1的作用机制提供了新的视角。 DM 1发病机制的复杂组合机制，将有助于未来对DM 1疾病病因的研究 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.