Neuromuscular endplate pathology in autosomal dominant and recessive desminopathies

“结缔组织病”一词代表一组由人类结蛋白基因突变引起的家族性和散发性肌病和心肌病。Desminopsis属于罕见的人类疾病组，每10,000人中有不到5人受到影响。大多数结缔组织病遵循常染色体显性遗传特征。此外，还描述了罕见的常染色体隐性遗传病例和越来越多的散发性脱发疾病。常染色体隐性遗传病例可进一步细分为保持突变结蛋白表达的病例和完全缺失结蛋白的病例。我们之前从人和小鼠身上获得的数据表明，突变的R349P结蛋白的完全缺乏以及唯一的表达损害了神经肌肉终板的结构和功能的完整性，从而导致了结缔组织疾病中肌肉无力的临床症状。在本项目中，我们旨在阐明野生型和突变的结蛋白之间的关系以及结蛋白的缺乏，以及NMJ的形态和功能。由于对人类骨骼肌组织的全面研究受到合适活检标本的严重阻碍，我们将通过全面、多水平的分析来开发我们的模仿患者的结缔组织病小鼠模型(结蛋白缺失、杂合和纯合的R349P结蛋白敲入)以及相应的永生化结缔组织病成肌细胞培养。我们的项目将解决以下主要研究问题：1)(A)缺乏结蛋白，(B)突变结蛋白R349P的单一表达，(C)突变和野生型结蛋白的混合表达，对NMJs的结构和完整性？2)缺乏结蛋白，(B)突变结蛋白R349P的单独表达，(C)突变结蛋白和野生型结蛋白的混合表达，影响神经肌肉的传递？3)永生化野生型和结缔组织病型成肌细胞在烟碱型乙酰胆碱受体(AChRs)的聚集方面与非永生化野生型和结蛋白缺失细胞不同？4)结蛋白参与神经肌肉接头AChRs聚集的方式是什么？它是否影响AChR聚集的形成、形成和维持？5)结蛋白是否直接与突触后蛋白相互作用？在突变的R349P结蛋白存在的情况下，这些相互作用中是否有任何一种受到影响？结蛋白的缺失或突变型R349P结蛋白的存在是否干扰突触后蛋白的磷酸化模式？6)结蛋白如何参与与NMJ相关的信号通路？在缺乏结蛋白或存在突变型R349P的情况下，这些通路是如何受到影响的？7)沙丁胺醇介导的挽救NMJ形态的分子机制是什么？

The term "desminopathies" stands for a group of familial and sporadic myopathies and cardiomyopathies, which are caused by mutations in the human desmin gene. Desminopathies belong to the group of rare human diseases with less than 5 affected individuals in 10,000. The majority of desminopathies follow an autosomal dominant trait of inheritance. In addition, rare autosomal recessive cases as well as an increasing number of sporadic desminopathies have been described. The autosomal recessive cases may further be subdivided into those with maintained expression of mutant desmin and others with a complete lack of desmin. We previously generated data from man and mouse demonstrating that the complete lack as well as the sole expression of mutant R349P desmin impair the structural and functional integrity of neuromuscular endplates thus contributing to the clinical sign of muscle weakness in desminopathies.In the present project we aim to clarify the relationship between wildtype and mutant desmin as well as the lack of desmin, and NMJ morphology and function in more detail. Since comprehensive studies on human skeletal muscle tissue are strongly hampered by the availability of appropriate biopsy specimens, we will exploit our patient-mimicking desminopathy mouse models (desmin-null, hetero- and homozygous R349P desmin knock-in) as well as the corresponding immortalized desminopathy myoblast cultures by a comprehensive, multi-level analysis.Our project will address the following major research questions: 1) What are the effects of (a) the lack of desmin, (b) the sole expression of mutant desmin R349P, (c) the mixed expression of mutant and wildtype desmin, on the structure and integrity of NMJs?2) How does (a) the lack of desmin, (b) the sole expression of mutant desmin R349P, (c) the mixed expression of mutant and wildtype desmin, affect neuromuscular transmission?3) Are immortalized wildtype and desminopathy myoblast cultures different regarding the agrin-dependent aggregation of nicotinic acetylcholine receptors (AChRs), in comparison with non-immortalized wildtype and desmin-null cells?4) In which way is desmin involved in the aggregation of AChRs at the neuromuscular junction? Does it influence prepattering, formation and maintenance of AChR aggregation?5) Does desmin directly interact with postsynaptic proteins? Are any of these interactions compromised in the presence of mutant R349P desmin? Does the lack of desmin or the presence of mutant R349P desmin interfere with phosphorylation patterns of postsynaptic proteins?6) How is desmin involved in signaling pathways related to NMJs? How are these pathways affected in the absence of desmin or in the presence of mutant R349P?7) What is the molecular mechanism of salbutamol-mediated rescue of the NMJ morphology?