Mechanisms of synapse formation and axon termination in C. elegans

线虫突触形成和轴突终止的机制

基本信息

批准号：
10431783
负责人：
Brock Grill
金额：
$ 62万
依托单位：
SEATTLE CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
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/10431783
关键词：
Address Affect Alzheimer&apos s Disease American Autophagocytosis Autophagosome Axon Binding Binding Proteins Biochemical Biochemistry Biology CDK5 gene CRISPR/Cas technology Caenorhabditis elegans Cells Code Development Disease Emotional Engineering Event Foundations Funding Genetic Goals Growth Health system Human Integrins Knowledge Ligase Link Mass Spectrum Analysis Mediating Molecular Nature Nematoda Nervous system structure Neurodegenerative Disorders Neurodevelopmental Disorder Neurons Organism Orthologous Gene Outcome Phosphorylation Phosphorylation Site Phosphotransferases Play Preventive therapy Process Protein Family Proteins Proteomics Public Health Receptor Signaling Research Role SKP Cullin F-Box Protein Ligases Sampling Signal Transduction Signaling Protein Structure Synapses Synaptic plasticity System Technology Testing Trauma Ubiquitination Work axonal degeneration base design experimental study genetic approach genetic testing in vivo inhibitor insight interest knock-down loss of function member multicatalytic endopeptidase complex mutant neuron development novel novel therapeutics programs synaptogenesis ubiquitin ligase ubiquitin-protein ligase

项目摘要

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。系统的建立可以为治疗神经发育障碍提供机会，并且将会得到应用。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，例如阿尔茨海默氏病（AD）。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之间的保守调节因子。 PHR 信号传导与神经发育障碍和神经退行性疾病（包括 AD）有进一步的研究对理解 PHR 信号网络有浓厚的兴趣在这里，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；以及新型泛素化“陷阱” 破译了秀丽隐杆线虫 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我们还评估了 RPM-1 对这种激酶的影响。神经元中自噬体的形成将有助于了解 PHR 蛋白是否调节。自噬，并解决体内神经系统中自噬如何受到抑制的问题。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我们将确定 RPM-1 如何抑制这种激酶，以及这是否会影响轴突终止和突触形成我们还致力于解决哪个下游问题。尽管该激酶很重要，但该激酶利用其影响轴突和突触发育的机制。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。第三个目标，我们关注三个特别引人注目的切入点 1) 最突出的 RPM-1 结合蛋白。 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的功能是我们对这些问题的兴趣。由一个简单的主题驱动：PHR 蛋白在任何系统中如何调节，仍然是黑暗的生物学。