Role of TRIM46 in neuronal polarity in vivo

TRIM46 在体内神经元极性中的作用

基本信息

批准号：
10747819
负责人：
Allison Jacqueline Melton
金额：
$ 4.77万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10747819
关键词：
ANK3 gene Action Potentials Axon Axonal Transport Behavior Behavioral Brain Cells Complex Cytoskeletal Filaments Cytoskeleton Data Defect Dendrites Dependovirus Development Diffusion Environment Exhibits Fascicle Foundations Gatekeeping Impairment In Vitro Injections Injury Ion Channel Knock-out Knockout Mice Label Memory Methods Microscopy Microtubules Molecular Morphology Movement Mus Muscle Muscle fasciculation Nerve Crush Nervous System Neurodegenerative Disorders Neurodevelopmental Disorder Neurons Play Population Process Proteins Publishing Reporting Resolution Role Specific qualifier value Structure TRIM Motif Testing Thinness Toxic effect Training Transmission Electron Microscopy Work axon regeneration density experience experimental study immunoreactivity in utero in vivo insight knock-down loss of function migration molecular polarity prevent protein distribution recruit sciatic nerve small hairpin RNA superresolution microscopy trafficking transmission process

项目摘要

PROJECT SUMMARY. From movement to memory, the complex and wide-ranging tasks performed by the nervous system depend on the polarization of billions of neurons into two functionally distinct subcellular compartments: dendrites, which receive most of the input into the neuron, and the axon, which transmits information to other cells in the form of an action potential. Two powerful contributors to neuronal polarity are 1) the axon initial segment (AIS), a domain at the beginning of the axon that initiates action potentials, and 2) microtubules (MTs), cytoskeletal filaments involved in axon outgrowth and cargo trafficking1,2. Disrupting either of these impairs the other and causes broad downstream defects in neuronal polarity and function3–6, suggesting that the interplay between these structures is a key regulator of neuronal polarity. Intriguingly, MTs within the AIS form bundles, called “fascicles”, that are rarely observed outside the AIS7,8. This unique organization supports the idea that the AIS and MTs work together to control neuronal polarity. Therefore, investigating MT fasciculation could unlock new insights into how neurons establish and maintain polarity, as well as how that process might go awry in neurodevelopmental and neurodegenerative disorders. Though AIS MT fasciculation was first observed over 50 years ago7,8, little progress has been made in determining its function, largely because a lack of candidates for the driver of MT fasciculation made loss-of-function studies impossible. However, a recent breakthrough occurred with the discovery of the protein Tripartite Motif Containing 46 (TRIM46) at the AIS9. TRIM46 localizes to AIS MTs, and TRIM46-deficient cultured neurons lack AIS MT fasciculation9,10. They also exhibit impaired AIS formation and a range of defects in axonal outgrowth, protein distribution, and MT orientation, suggesting that TRIM46 is required for both AIS MT fasciculation and neuronal polarity more broadly9. However, these exciting claims are based on TRIM46 knockdown by shRNA, mostly in cultured neurons, so the observed defects could be attributed to shRNA toxicity or the artificial culture environment. No work has been done to investigate TRIM46 knockout in vivo. This project will leverage the Rasband lab’s extensive experience with the in vivo study of the AIS to fill this gap. The objective of this proposal is to use TRIM46 knockout mice to determine the role of TRIM46 in the nervous system and test the specific hypothesis that TRIM46 is required for AIS MT fasciculation and neuronal polarity in vivo. The proposed experiments will use immunostaining and high-resolution microscopy methods to determine the consequences of TRIM46 knockout from neuronal ultrastructure to behavior and in the contexts of development and injury. Aim 1 will determine the role of TRIM46 in AIS formation, ultrastructure, and MT fasciculation. Aim 2 will determine the role of TRIM46 in neuronal morphological, molecular, and MT polarity. Aim 3 will determine the consequences of TRIM46 knockout on neuronal migration, axon regeneration, and behavior. Completion of this project will reveal the function of TRIM46 at the convergence of the AIS and MTs from which neuronal polarity emerges.

项目摘要。从运动到记忆，神经系统执行的复杂而广泛的任务取决于数十亿个神经元极化成两个功能不同的亚细胞区室：树突，接收大部分输入到神经元和轴突，轴突以以下形式将信息传输到其他细胞：神经极性的两个重要贡献者是 1) 轴突初始段 (AIS)，一个域。位于启动动作电位的轴突起始处，以及 2) 微管 (MT)、细胞骨架丝参与轴突生长和货物贩运1,2。破坏其中任何一个都会损害另一个并导致广泛的后果。神经极性和功能的下游缺陷3-6，表明这些结构之间的相互作用有趣的是，AIS 内的 MT 形成束，称为“束”。在 AIS 之外很少观察到7,8 这个独特的组织支持 AIS 和 MT 协同工作的想法。因此，研究 MT 束颤可以解锁神经元如何控制的新见解。建立和维持极性，以及该过程如何在神经发育和发育过程中出错尽管 AIS MT 束震在 50 多年前首次被观察到7,8，但很少见。其确定功能取得了进展，很大程度上是因为缺乏 MT 驾驶员候选人肌束使功能丧失研究变得不可能。然而，最近出现了突破。在 AIS9 上发现含有 46 的三重基序 (TRIM46)，TRIM46 定位于 AIS MT，以及 TRIM46 缺陷的培养神经元缺乏 AIS MT 束化9,10，它们还表现出 AIS 形成受损和 AIS 形成受损。轴突生长、蛋白质分布和 MT 方向的一系列缺陷，表明需要 TRIM46 然而，这些令人兴奋的主张是基于更广泛的 AIS MT 束颤和神经极性9。 TRIM46 通过 shRNA 敲低，主要发生在培养的神经元中，因此观察到的缺陷可能归因于 shRNA 尚未开展研究体内 TRIM46 敲除的毒性或人工培养环境。该项目将利用 Rasband 实验室在 AIS 体内研究方面的丰富经验来填补这一空白。该提案的目的是使用 TRIM46 敲除小鼠来确定 TRIM46 在神经系统中的作用并测试TRIM46是体内AIS MT束颤和神经极性所必需的具体假设。拟议的实验将使用免疫染色和高分辨率显微镜方法来确定 TRIM46 敲除从神经超微结构到行为以及发育背景的后果目标 1 将确定 TRIM46 在 AIS 形成、超微结构和 MT 束颤中的作用。将决定 TRIM46 在神经元形态、分子和 MT 极性中的作用。 TRIM46 敲除对神经迁移、轴突再生和行为完成的影响。该项目将揭示 TRIM46 在 AIS 和 MT 汇合处的功能，其中神经元极性出现。