Neuromuscular endplate pathology in autosomal dominant and recessive desminopathies

“desminopathies”一词代表一组家族性和散发性肌病和心肌病，它们是由人类desmin基因突变引起的。Desminopathies是一种罕见的人类疾病，每10000人中只有不到5人患病。大多数终末细胞病变遵循常染色体显性遗传特征。此外，罕见的常染色体隐性遗传病例以及越来越多的散发性末位细胞病变已被描述。常染色体隐性病例可进一步细分为维持突变的促球蛋白表达的病例和完全缺乏促球蛋白的病例。我们之前从人和小鼠中获得的数据表明，R349P desmin突变体的完全缺失和唯一表达损害了神经肌肉终板的结构和功能完整性，从而导致了desmin病中肌肉无力的临床症状。在本项目中，我们旨在更详细地阐明野生型和突变型desmin之间的关系以及desmin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适当活检标本的严重阻碍，我们将利用我们的模拟患者的神经鞘病小鼠模型（神经鞘蛋白缺失、异型和纯合子R349P神经鞘蛋白敲入）以及相应的永活神经鞘病成肌细胞培养，进行全面的、多层的分析。我们的项目将解决以下主要研究问题：1)(a)缺乏desmin， (b)突变型desmin R349P的单独表达，(c)突变型和野生型desmin的混合表达对NMJs的结构和完整性有什么影响？2) (a) desmin缺乏，(b)突变型desmin R349P的单独表达，(c)突变型和野生型desmin的混合表达如何影响神经肌肉传递？3)与非永生化野生型和无desmin细胞相比，永生化野生型和desmin病变成肌细胞培养在烟碱乙酰胆碱受体（AChRs）的农业蛋白依赖性聚集方面是否存在差异？4)去丝蛋白以何种方式参与神经肌肉交界处achr的聚集？它是否影响AChR聚集的预图案化、形成和维持？desmin是否直接与突触后蛋白相互作用？在R349P突变蛋白存在时，这些相互作用是否受到损害？聚丝蛋白的缺乏或R349P聚丝蛋白突变体的存在是否会干扰突触后蛋白的磷酸化模式？6) desmin如何参与与NMJs相关的信号通路？在没有desmin或存在突变R349P的情况下，这些途径是如何受到影响的？7)沙丁胺醇介导的NMJ形态修复的分子机制是什么？

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?