Mechanisms of synapse formation and axon termination in C. elegans

线虫突触形成和轴突终止的机制

• 批准号: 10306139
• 负责人: Brock Grill
• 金额: $ 45.51万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-11-19 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10306139
• 关键词: Mechanisms; synapse; formation; axon; termination

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.

概括 如果我们要了解神经是如何产生的，那么破译轴突如何终止生长并形成突触是至关重要的。 系统已建成。这些知识可以提供治疗神经发育障碍的机会，并将 如果我们要利用发育中的神经系统的稳健性和弹性来设计新颖的神经系统，就需要 治疗神经退行性疾病的疗法，例如阿尔茨海默病（AD）。我们的长期目标是 了解控制轴突终止和突触形成的分子和细胞机制 体内使用线虫秀丽隐杆线虫。 Pam/Highwire/RPM-1 (PHR) 蛋白是重要的泛素连接酶和信号中枢 轴突终止、突触形成和轴突变性的保守调节因子。之间正在出现的联系 PHR 信号传导与神经发育障碍和神经退行性疾病（包括 AD）有进一步的关系 对了解 PHR 信号网络的兴趣增强。在这里，我们使用最新的高灵敏度质量 光谱测定技术；快速自动化蛋白质提取和纯化；和新颖的泛素化“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 的调控方式提供了众多依据。 我们的第一个目标集中于自噬启动激酶作为新型 RPM-1 泛素化底物。 CRISPR/Cas9 编辑和遗传学测试 RPM-1 泛素连接酶活性是否影响其稳定性和周转率 激酶影响轴突和突触发育。我们还评估了 RPM-1 对这种激酶的影响如何影响 神经元中自噬体的形成。结果将深入了解 PHR 蛋白是否调节 自噬，并解决体内神经系统如何抑制自噬。我们对这些问题感兴趣 自噬在包括 AD 在内的神经退行性疾病中发挥的重要作用进一步推动了这一趋势。 我们的第二个目标是评估另一种新型 RPM-1 泛素化底物，一种具有重要作用的激酶 突触发育、突触可塑性和AD。我们将确定 RPM-1 如何抑制该激酶，以及 这是否会影响轴突终止和突触形成。我们还旨在解决哪些下游 该激酶利用影响轴突和突触发育的机制。尽管这种激酶很重要 在神经系统健康和疾病中，它在任何生物体中如何被抑制仍然未知。 最后，蛋白质组学为理解 RPM-1 的调控方式提供了几个切入点。在我们的 第三个目标，我们关注三个特别引人注目的切入点。 1) 最重要的 RPM-1 结合蛋白 确定。 2) 整个受体信号系统的组成部分被确定为 RPM-1 结合蛋白。 3） 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 定位以及 RPM-1 在轴突和突触发育中发挥作用。我们对这些问题的兴趣是 由一个简单的主题驱动：在任何系统中，PHR 蛋白如何被调节，仍然是黑暗的生物学。