Role of TRIM46 in neuronal polarity in vivo

TRIM46 在体内神经元极性中的作用

• 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
• 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

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。 从移动到记忆，神经系统执行的复杂而广泛的任务取决于 数十亿个神经元分为两个功能上不同的亚细胞室的极化：树突，它们 接收大多数输入到神经元和轴突中，并以 动作潜力。神经元极性的两个强大贡献者是1）轴突初始段（AIS），一个域 在发起动作电位的轴突开头，2）微管（MTS），细胞骨架细丝 参与轴突的生长和货物贩运1,2。破坏这两者都会损害对方并造成广泛 神经元极性和功能3-6的下游缺陷表明这些结构之间的相互作用 是神经元极性的关键调节剂。有趣的是，AIS形式内的MT捆绑着称为“束”，是 在AIS7,8之外很少观察到。这个独特的组织支持AIS和MTS共同努力的想法 控制神经元极性。因此，调查MT迷恋可以解锁对神经元如何的新见解 建立和维持极性，以及该过程如何在神经发育和 神经退行性疾病。尽管第50年前首次观察到AIS MT着迷7,8，但很少 在确定其功能方面取得了进展，主要是因为缺乏MT驾驶员的候选人 着迷使功能丧失研究不可能。但是，最近发生了一次突破 在AIS9处的蛋白质三方基序的发现。 TRIM46本地化为AIS MTS，并且 TRIM46缺陷培养的神经元缺乏AIS MT fasticulation9,10。他们还暴露了AIS组的障碍和 轴突产物，蛋白质分布和MT方向的缺陷范围，表明需要TRIM46 对于AIS MT诱惑和神经元极性，更广泛地9。但是，这些令人兴奋的主张是基于 shRNA的TRIM46敲低，主要是在培养的神经元中，因此观察到的缺陷可以归因于shRNA 毒性或人工文化环境。尚未做过研究体内Trim46淘汰赛的工作。这 项目将利用Rasband Lab的体内研究丰富的经验来填补这一空白。这 该建议的目的是使用TRIM46敲除小鼠确定Trim46在神经系统中的作用 并检验了特定假设，即在体内AIS MT诱体和神经元极性所必需的TRIM46。 提出的实验将使用免疫染色和高分辨率显微镜方法来确定 TRIM46敲除从神经元超微结构到行为和发展背景的后果 和受伤。 AIM 1将确定TRIM46在AIS形成，超微结构和MT诱惑中的作用。目标2 将确定TRIM46在神经元形态，分子和MT极性中的作用。 AIM 3将确定 TRIM46敲除对神经元迁移，轴突再生和行为的后果。完成 该项目将在AIS和MTS的收敛中揭示Trim46的功能 极性出现。