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

CELF1上调机制及其在强直性肌营养不良1型发病机制中的作用

• 批准号: 10752274
• 负责人: Larissa Nitschke
• 金额: $ 7.18万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752274
• 关键词: 3' Untranslated Regions; Adult; Affect; Alleles; Alternative Splicing; Biological Assay; CCAAT-enhancer-binding protein-delta; CUG repeat; Cardiac; Cell Culture Techniques; Cell Line; Complex; Data; Development; Disease; Disease Progression; Etiology; Family; Family member; Fetal Proteins; Future; Genes; Genetic Transcription; Goals; Heart; Histologic; Histopathology; Individual; Knock-out; Knockout Mice; Life Expectancy; Link; LoxP-flanked allele; Mass Spectrum Analysis; Mediating; Messenger RNA; Modeling; Molecular; Molecular Biology Techniques; Mus; Muscle; Muscle Weakness; Muscular Atrophy; Muscular Dystrophies; Mutation; Myopathy; Myotonia; Myotonic Dystrophy; Myotonic dystrophy type 1; Natural regeneration; Open Reading Frames; Pathogenesis; Pattern; Phenocopy; Phenotype; Phosphorylation; Phosphorylation Site; Physiological; Prevalence; Process; Protein Isoforms; Protein Kinase; Protein Kinase C; Proteins; RNA; RNA Processing; RNA Splicing; RNA-Binding Proteins; Research; Role; Signal Transduction; Skeletal Muscle; Testing; Therapeutic; Tissues; Toxic effect; Trans-Activators; Translations; Ubiquitination; Up-Regulation; Work; autosome; combinatorial; design; fetal; gain of function; insight; interest; knock-down; loss of function; mutant; overexpression; paralogous gene; protein activation; skeletal muscle weakness

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.

项目概要 强直性肌营养不良 1 型 (DM1) 是一种多系统疾病，其特征是进行性骨骼肌萎缩 肌肉无力、肌肉萎缩和肌强直。 DM1 的患病率为 8500 分之一，是最常见的 成人发病的肌营养不良症的原因。 DM1 是由 3' 非翻译区 CTG 重复序列的扩展引起的 肌强直营养不良蛋白激酶 (DMPK) 基因的区域。扩增后转录的RNA DMPK 等位基因包含扩展的 CUG 重复序列 (CUGexp RNA)，可隔离 Muscleblind 的旁系同源物 与RNA结合蛋白（MBNL）家族一样，MBNL1和MBNL2导致它们功能丧失。此外， 第二个 RNA 结合蛋白 CUGBP Elav 样家族成员 1 (CELF1) 被上调至如下水平： 对 DM1 骨骼肌有毒。虽然 MBNL 通过隔离而丧失功能的机制已得到很好的证实。 建立了导致骨骼肌中 CELF1 上调的机制及其对肌肉的贡献 DM1 的缺陷仍然未知。 为了在小鼠中模拟成年发病的 DM1，我们生成了具有 floxed Mbnl1 和 Mbnl2 等位基因的小鼠，并诱导 成人骨骼肌中的条件性双敲除（Mbnl dcKO）。 Mbnl dcKO 小鼠类似于成年发病 DM1 具有显着的剪接变化、显着的肌肉消耗和预期的轻度组织病理学。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节联系 MBNL 和 CELF1，独立于 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。 该提案的目标有两个。在目标 1 中，我将确定 MBNL 功能丧失如何导致 CELF1 蛋白的上调并揭示参与该过程的顺式调节基序和反式作用因子 过程。我们之前证明 CELF1 可以通过蛋白激酶 C (PKC) 介导来稳定 磷酸化。因此，一个特别感兴趣的是测试 MBNL 功能丧失是否会导致激活 PKC 并增加 CELF1 的磷酸化，或者是否有其他机制参与上调 CELF1。由于成人骨骼肌中 MBNL1 和 MBNL2 的敲除先前尚未被表征，因此 目标 2，我将首先对肌肉的生理学、组织病理学和分子分子进行深入的表征 Mbnl dcKO 小鼠的表型，以建立稳健的定量分析，然后用于确定 CELF1 上调对 Mbnl dcKO 肌肉表型的影响程度。 完成这项研究后，我将解决 DM1 领域长期存在的问题：如何 CELF1 上调并确定这种上调对 Mbnl dcKO 肌肉的贡献程度 赤字。我们发现 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，将有助于未来对DM1疾病因果关系的研究 和进展，并将为未来治疗方法的发展提供信息。