Myogenesis and RNA Biogenesis in a Mouse Model of OPMD

 描述(申请人提供)：眼咽肌营养不良症(OPMD)是一种迟发性疾病，导致眼睑、咽部和四肢近端肌肉无力。OPMD的一个主要症状是吞咽困难(吞咽困难)，这是由于咽肌无力所致。患者经常经历窒息或吸入性肺炎，在疾病的后期可能需要喂养管。目前，其确切的发病分子机制尚不清楚。治疗OPMD患者吞咽困难的唯一可行选择是临时性的，通常是通过外科手术来减少喉部阻力以改善吞咽。常染色体显性遗传性OPMD患者核mRNA处理蛋白PABPN1的基因突变，导致PABPN1的N端多丙氨酸延伸从10个残基扩大到11-18个残基。PABPN1在核转录产物的多聚腺苷化以及交替的多腺苷和裂解途径的mRNA加工中起着关键作用。PABPN1在所有组织中都有表达，但突变只在骨骼肌的特定亚群中导致迟发性病理。这种无处不在的表达蛋白的突变如何导致特定组织的迟发性病理仍然是一个悬而未决的问题。骨骼肌组织能够再生。在成人肌肉中，与多核、有丝分裂后的肌纤维密切相关的是被称为卫星细胞的干细胞池。在肌肉受损时，卫星细胞被激活以增殖、分化，并与彼此和现有的肌纤维融合。这个过程被称为肌肉发生，恢复肌肉的结构和功能。有趣的是，一些受OPMD影响最严重的肌肉，包括咽部肌肉，比其他骨骼肌再生能力更强。推动这项工作的假设是，突变的PABPN1扰乱了mRNA的生物合成，降低了咽部卫星细胞的生肌能力，导致了病理，这是由于咽部肌肉对肌肉生肌的异常高需求。我们将采用体外和体内一体化的方法，使用一种新的小鼠模型，这是第一个准确复制OPMD患者基因的模型。这种小鼠模型在所有组织中都表达突变的PABPN1。由于PABPN1已知会影响信使核糖核酸的新陈代谢，因此将从咽部肌肉中分离出卫星细胞，并对其进行RNA表型分析(目标1)。生肌程序可以在体外重现，并用于量化肌肉发生的特定阶段。因此，将从咽部肌肉中分离出原代激活的卫星细胞，即成肌细胞，并将其用于量化突变的PABPN1在体外肌肉发生的特定阶段中的作用(目标2)。然后，该模型将被用于在体内测试突变的PABPN1对咽肌肌肉生成的影响(目标3)。这项工作的长期目标是了解突变的PABPN1如何影响咽卫星细胞生物学，并可能确定新的药物治疗OPMD的靶点。

 DESCRIPTION (provided by applicant): Oculopharyngeal muscular dystrophy (OPMD) is a late onset disorder causing weakness of eyelid and pharyngeal, and proximal limb muscles. A major symptom of OPMD is swallowing difficulty (dysphagia) due to weakened pharyngeal muscles. Patients often experience choking or aspiration pneumonia, and may require a feeding tube in later stages of disease. At present, the exact molecular mechanisms of pathology are unknown. The only available options to treat dysphagia in OPMD patients are temporary, usually surgical procedures to decrease resistance in the throat to improve swallowing. Autosomal dominant OPMD patients harbor a mutation in the gene for the nuclear mRNA processing protein PABPN1, which causes the N-terminal polyalanine stretch of PABPN1 to expand from 10 to 11-18 residues. PABPN1 plays key roles in mRNA processing including polyadenylation of nuclear transcripts and the alternative polyadenylation and cleavage pathway. PABPN1 is expressed in all tissues, but mutation causes late-onset pathology only in a specific subset of skeletal muscles. How mutation of this ubiquitously expressed protein leads to late-onset pathology in specific tissues remains an open question. Skeletal muscle tissue is capable of regeneration. Closely associated with multinucleated, post-mitotic myofibers in adult muscles is a pool of stem cells known as satellite cells. Upon muscle damage, satellite cells become activated to proliferate, differentiate, and fuse with each other and existing myofibers. This process, known as myogenesis, restores muscle architecture and function. Interestingly, some of the muscles most severely affected by OPMD, including pharyngeal muscles, are more highly regenerative than other skeletal muscles. The hypothesis driving the work proposed here is that mutant PABPN1 disrupts mRNA biogenesis and decreases the myogenic capacity of pharyngeal satellite cells, leading to pathology due to the unusually high demand for myogenesis in pharyngeal muscles. We will take an integrative in vitro and in vivo approach using a novel mouse model that is the first to accurately copy the genotype of OPMD patients. This mouse model expresses mutant PABPN1 in all tissues. Because PABPN1 is known to impact mRNA metabolism, satellite cells from pharyngeal muscles will be isolated and assayed for RNA phenotypes (Aim 1). The myogenic program can be recapitulated in vitro and used to quantify specific stages of myogenesis. Therefore, primary activated satellite cells, known as myoblasts, from pharyngeal muscles will be isolated and used to quantify the effects of mutant PABPN1 on the specific stages of myogenesis in vitro (Aim 2). This model will then be used to assay the effect of mutant PABPN1 on myogenesis in pharyngeal muscle in vivo (Aim 3). The long-term objective of this work is to understand how mutant PABPN1 affects pharyngeal satellite cell biology and potentially identify targets for new pharmacologic OPMD treatments.