Functional mechanisms underlying Dystroglycan-dependent and independent roles of protein O-mannosylation in the nervous system

这个项目的主要目标是阐明潜在的调节作用机制 通过蛋白质O-甘露糖基化(POM)作用于神经系统。POM是一种基本的O-糖基化类型，具有 对多种动物的发育和生理都有深远的影响，从果蝇到 人类。虽然POM影响的生物功能谱很广，但到目前为止唯一被研究得很好的 POM的靶点是营养不良蛋白聚糖(DG)。DG的POM修饰缺陷导致严重的肌肉 营养不良症称为血糖营养不良症。与POM缺陷相关的病理机制是复杂的 人们对此仍然知之甚少，尤其是神经系统。最近的研究表明，POM 修饰影响许多蛋白质的功能，这有助于致病机制 糖代谢不良症。然而，除了DG外，POM在蛋白质上的功能在很大程度上还不清楚。这个 糖基化的复杂性和体内方法的局限性给研究带来了巨大的挑战 哺乳动物体内的POM。在这里，我们提出了一个多学科项目，该项目利用 果蝇模型，包括强大的遗传方法，简化的糖基化和 POM和DG突变体的实验适应性，以阐明DG-1的分子和细胞机制 POM的依赖和DG非依赖功能，重点是神经系统和 神经肌肉发育和生理学。我们的初步研究表明，受体蛋白 酪氨酸磷酸酶(RPTPs)是重要的POM靶标，研究表明POM调节 通过影响感觉神经元和中枢神经系统之间的通讯来协调肌肉收缩。我们 将利用这些结果，同时侧重于三个具体目标：(1)分析POM在以下方面的作用 感觉神经元和协调肌肉收缩的调节。使用实时成像技术 结合遗传学和神经生物学方法，我们将全面调查 POM在感觉神经元、中枢神经系统细胞和肌肉之间的通讯。(2)调查 POM on RPTP功能。使用体内和体外方法，我们将研究POM如何影响 在分子、细胞和组织水平上发挥RPTP的作用。(3)揭示POM的新分子靶点 并阐明它们在神经系统中的功能。我们将使用糖蛋白组学方法来鉴定 具有POM修饰的蛋白质。我们将分析POM在体内新靶点上的功能，重点是 在神经系统中起作用的蛋白质。我们预计，该项目将建立新的 POM介导的神经系统调节，并将阐明新的进化保守的DG- POM功能的依赖和独立机制，将有助于阐明POM的发病机制 与POM异常相关的人类疾病。

The main objective of this project is to elucidate functional mechanisms underlying regulation of the nervous system by protein O-mannosylation (POM). POM is an essential type of O-glycosylation that has a profound effect on the development and physiology in a broad range of animals, from Drosophila to humans. Although the spectrum of biological functions affected by POM is wide, so far the only well-studied target of POM is Dystroglycan (Dg). Defects in POM modifications of Dg result in severe muscular dystrophies called dystroglycanopathies. Pathomechanisms associated with POM defects are complex and remain poorly understood, particularly in the nervous system. Recent studies suggested that POM modification affects functions of many proteins, which contributes to pathogenic mechanisms of dystroglycanopathies. However, functions of POM on proteins besides Dg are largely unknown. The complexity of glycosylation and limitations of in vivo approaches create significant challenges for studying POM in mammalian organisms. Here we propose a multidisciplinary project that uses advantages of Drosophila model, including powerful arsenal of genetic approaches, simplified glycosylation and experimental amenability of POM and Dg mutants, to elucidate molecular and cellular mechanisms of Dg- dependent and Dg-independent functions of POM, with the focus on the nervous system and neuromuscular development and physiology. Our preliminary studies suggested that Receptor Protein Tyrosine Phosphatases (RPTPs) are functionally important POM targets and revealed that POM regulates coordinated muscle contractions by affecting communication between sensory neurons and the CNS. We will capitalize on these results while focusing on three specific aims: (1) To analyze the role of POM in regulation of sensory neurons and coordinated muscle contractions. Using live imaging techniques combined with genetic and neurobiological approaches, we will comprehensively investigate the role of POM in communication between sensory neurons, CNS cells and muscles. (2) To investigate the effect of POM on RPTP function. Using in vivo and in vitro approaches, we will investigate how POM affects functions RPTPs at molecular, cellular, and organismal levels. (3) To reveal new molecular targets of POM and elucidate their function in the nervous system. We will use glycoproteomic approaches to identify proteins with POM modifications. We will analyze functions of POM on novel targets in vivo, focusing on proteins that function in the nervous system. We anticipate that this project will establish new paradigms of POM-mediated regulation of the nervous system and will elucidate new evolutionarily conserved, Dg- dependent and independent mechanisms of POM functions, which will shed light on pathomechanisms of human diseases associated with POM abnormalities.