Myogenesis and RNA Biogenesis in a Mouse Model of OPMD

 描述（由申请人提供）：眼咽肌营养不良症（OPMD）是一种迟发性疾病，导致眼睑和咽部以及近端肢体肌肉无力。OPMD的一个主要症状是由于咽部肌肉减弱导致的吞咽困难（吞咽困难）。患者经常会出现窒息或吸入性肺炎，在疾病的后期可能需要喂食管。目前，病理学的确切分子机制尚不清楚。治疗OPMD患者吞咽困难的唯一可用选择是暂时的，通常是外科手术，以减少咽喉阻力，改善吞咽。常染色体显性OPMD患者在核mRNA加工蛋白PABPN 1的基因中具有突变，其导致PABPN 1的N-末端聚丙氨酸延伸从10个扩展到11-18个残基。PABPN 1在mRNA加工中起关键作用，包括核转录物的多聚腺苷酸化和替代的多聚腺苷酸化和切割途径。PABPN 1在所有组织中表达，但突变仅在骨骼肌的特定子集中引起迟发性病理学。这种普遍表达的蛋白质的突变如何导致特定组织中的迟发性病理学仍然是一个悬而未决的问题。骨骼肌组织具有再生能力。与成人肌肉中的多核、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。在肌肉损伤时，卫星细胞被激活以增殖、分化并彼此融合以及与现有肌纤维融合。这个过程，被称为肌生成，恢复肌肉结构和功能。有趣的是，一些受OPMD影响最严重的肌肉，包括咽肌，比其他骨骼肌再生能力更强。驱动这里提出的工作的假设是，突变的PABPN 1破坏mRNA生物发生并降低咽卫星细胞的生肌能力，导致由于咽肌中对肌发生的异常高需求而导致的病理学。我们将采用一种新的小鼠模型，采用体外和体内综合方法，该模型是第一个准确复制OPMD患者基因型的模型。该小鼠模型在所有组织中表达突变型PABPN 1。因为已知PABPN 1影响mRNA代谢，所以将分离来自咽肌的卫星细胞并测定RNA表型（Aim 1）。生肌程序可以在体外重现，并用于量化肌生成的特定阶段。因此，将从咽肌中分离被称为成肌细胞的原代活化卫星细胞，并将其用于定量突变体PABPN 1对体外肌发生的特定阶段的影响（目的2）。然后将该模型用于测定突变体PABPN 1对体内咽肌中肌发生的影响（目的3）。这项工作的长期目标是了解突变的PABPN 1如何影响咽卫星细胞生物学，并可能确定新的药物OPMD治疗的靶点。

 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.