Mechanisms of synapse formation and axon termination in C. elegans

摘要 破译轴突如何终止生长和形成突触是至关重要的，如果我们要理解 神经系统是建立起来的。这些知识可以为治疗神经发育障碍提供机会， 如果我们要利用发育中的神经系统的强健和弹性性质来 设计治疗神经退行性疾病的新疗法，如阿尔茨海默病(AD)。我们的长- 学期目标是了解支配轴突终止和细胞分化的分子和细胞机制 利用线虫在体内形成突触。 Pam/Highwire/RPM-1(PHR)蛋白是重要的泛素连接酶和信号中枢 保守的轴突终止、突触形成和轴突变性的调节因子。两国之间正在形成的联系 PhR信号与神经发育障碍和神经退行性疾病(包括AD)进一步 对了解PHR信令网络的兴趣增强。在这里，我们使用最新的高灵敏度质量 光谱技术；快速自动化蛋白质提取和纯化；以及新的泛素化“陷阱” 破译线虫PHR蛋白RPM-1的信号网络。这揭示了两个假定的RPM-1 泛素化底物，并为破译RPM-1是如何调控的提供了许多立足点。 我们的第一个目标是研究一种新的RPM-1泛素化底物--自噬启动蛋白。 CRISPR/Cas9编辑和遗传学测试RPM-1泛素连接酶活性是否影响细胞的稳定性和周转 这种激酶影响轴突和突触的发育。我们还评估了RPM-1对该激酶的影响 影响神经元中自噬小体的形成。结果将为PHR蛋白是否调节 自噬，以及在体内神经系统中自噬是如何被抑制的。我们对这些产品的兴趣 自噬在包括阿尔茨海默病在内的神经退行性疾病中扮演的重要角色进一步加剧了人们的疑问。 我们的第二个目标将评估另一种新的RPM-1泛素化底物，一种具有突出作用的激酶 在突触发育中，突触可塑性和AD。我们将确定RPM-1是如何抑制该激酶的，并且 这是否影响轴突终止和突触形成。我们还致力于解决哪些下游 该激酶用于影响轴突和突触发育的机制。尽管这很重要 在神经系统健康和疾病中，它是如何被抑制的，在任何有机体中仍不清楚。 最后，蛋白质组学为理解RPM-1可能如何被调控提供了几个切入点。在我们的 第三个目标，我们将重点放在三个特别引人注目的切入点上。1)最显著的RPM-1结合 确定了蛋白质。2)被鉴定为RPM-1结合蛋白的整个受体信号系统的组成部分。 3)体内存在大量磷酸化的RPM-1残基。我们将评估这些机制是如何 影响RPM-1的定位，以及RPM-1在轴突和突触发育中的作用。我们对这些产品的兴趣 问题由一个简单的主题驱动：在任何系统中，PHR蛋白是如何受到调控的，仍然是黑暗的生物学。

Summary Deciphering how axons terminate growth and forms synapses is essential if we are to understand how a nervous system is built. Such knowledge could provide opportunities to treat neurodevelopmental disorders, and will be needed if we are to harness the robust and resilient nature of the developing nervous system to design novel therapies for treating neurodegenerative diseases, such as Alzheimer’s disease (AD). Our long- term goal is to understand the molecular and cellular mechanisms that govern axon termination and synapse formation in vivo using the nematode C. elegans. The Pam/Highwire/RPM-1 (PHR) proteins are ubiquitin ligases and signaling hubs that are important conserved regulators of axon termination, synapse formation and axon degeneration. Emerging links between PHR signaling and neurodevelopmental disorders and neurodegenerative diseases (including AD) have further heightened interest in understanding PHR signaling networks. Here, we use the latest high-sensitivity mass spectrometry technology; rapid automated protein extraction and purification; and novel ubiquitination ‘traps’ to decipher the signaling network of the C. elegans PHR protein, RPM-1. This has revealed two putative RPM-1 ubiquitination substrates, and provided numerous footholds for deciphering how RPM-1 is regulated. Our first aim focuses on an autophagy initiating kinase as a novel RPM-1 ubiquitination substrate. CRISPR/Cas9 editing and genetics test if RPM-1 ubiquitin ligase activity affects the stability and turnover of this kinase to influence axon and synapse development. We also evaluate how RPM-1 effects on this kinase affect autophagosome formation in neurons. Outcomes will provide insight into whether PHR proteins regulate autophagy, and address how autophagy is inhibited in the nervous system in vivo. Our interest in these questions is further fueled by the prominent role autophagy plays in neurodegenerative diseases, including AD. Our second aim will evaluate another novel RPM-1 ubiquitination substrate, a kinase with prominent roles in synapse development, synaptic plasticity and AD. We will determine how RPM-1 inhibits this kinase, and whether this affects axon termination and synapse formation. We also aim to address which downstream mechanisms this kinase utilizes to affect axon and synapse development. Despite the importance of this kinase in nervous system health and disease, how it is inhibited remains unknown in any organism. Finally, proteomics provided several entry points into understanding how RPM-1 might be regulated. In our third aim, we focus on three particularly compelling entry points. 1) The most prominent RPM-1 binding protein identified. 2) Components of an entire receptor signaling system identified as RPM-1 binding proteins. 3) Numerous residues in RPM-1 that are phosphorylated in vivo. We will evaluate how these mechanisms affect RPM-1 localization, and RPM-1 functions in axon and synapse development. Our interest in these questions is driven by a simple theme: How PHR proteins are regulated, in any system, remains dark biology.