Mechanisms of synapse formation and axon termination in C. elegans

总结 如果我们要了解神经元是如何生长的，那么， 系统建立。这些知识可以提供治疗神经发育障碍的机会， 如果我们要利用发育中的神经系统的健壮和弹性来设计新颖的 用于治疗神经退行性疾病如阿尔茨海默病（AD）的疗法。我们的长期目标是 了解支配轴突终止和突触形成的分子和细胞机制 在体内使用线虫C.优雅 Pam/Highwire/RPM-1（PHR）蛋白是泛素连接酶和信号传导中心， 轴突终止、突触形成和轴突变性的保守调节因子。新出现的联系， PHR信号传导和神经发育障碍和神经退行性疾病（包括AD）已经进一步发展。 对理解PHR信号网络的兴趣增加。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;和新的泛素化“陷阱”， 破译C.线虫PHR蛋白，RPM-1。这揭示了两个假定的RPM-1 泛素化底物，并为破译RPM-1是如何调节的提供了许多立足点。 我们的第一个目标是将自噬启动激酶作为新型RPM-1遍在化底物。 CRISPR/Cas9编辑和遗传学测试RPM-1泛素连接酶活性是否会影响该基因的稳定性和周转率。 激酶影响轴突和突触发育。我们还评估了RPM-1对这种激酶的作用如何影响 神经元中的自噬体形成。结果将提供洞察PHR蛋白是否调节 自噬，并解决如何在体内神经系统中抑制自噬。我们对这些问题的兴趣 自噬在包括AD在内的神经退行性疾病中发挥的突出作用进一步推动了这一研究。 我们的第二个目标是评估另一种新的RPM-1泛素化底物，一种具有突出作用的激酶 突触发育、突触可塑性和AD。我们将确定RPM-1如何抑制这种激酶， 是否影响轴突终止和突触形成。我们还致力于解决下游 这种激酶利用影响轴突和突触发育的机制。尽管这种激酶 在神经系统健康和疾病中，它是如何被抑制的在任何生物体中仍然是未知的。 最后，蛋白质组学提供了几个切入点来了解RPM-1可能是如何调节的。在我们 第三个目标，我们侧重于三个特别引人注目的切入点。1)最突出的RPM-1结合蛋白 鉴定2)整个受体信号传导系统的组分被鉴定为RPM-1结合蛋白。第三章 RPM-1中的许多残基在体内被磷酸化。我们将评估这些机制如何影响 RPM-1的定位，以及RPM-1在轴突和突触发育中的功能。我们对这些问题的兴趣在于 由一个简单的主题驱动：在任何系统中，PHR蛋白如何被调节，仍然是黑暗生物学。

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.