Role of TRIM46 in neuronal polarity in vivo

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依赖于 将数十亿神经元极化成两个功能不同的亚细胞区室：树突， 接受大部分输入到神经元和轴突，轴突将信息以 动作电位两个强大的贡献者神经元极性1）轴突起始段（AIS），一个域 在轴突的起始处启动动作电位，以及2）微管（MT），细胞骨架丝 参与轴突生长和货物运输1，2.破坏其中任何一个都会损害另一个， 神经元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 是神经元极性的关键调节器有趣的是，AIS中的MT形成称为“束”的束， 在AIS之外很少观察到7，8。这个独特的组织支持AIS和MT一起工作的想法 来控制神经元的极性因此，研究MT成束可以揭示神经元如何 建立和维持极性，以及这个过程如何在神经发育和 神经退行性疾病虽然AIS MT肌束震颤是在50多年前首次观察到的7，8， 在确定其功能方面取得了进展，主要是因为缺少MT驱动程序的候选人 肌束震颤使功能丧失研究成为不可能。然而，最近的一个突破发生在 在AIS 9上发现了包含46的蛋白质三分基序（TRIM 46）。TRIM 46定位于AIS MT， TRIM 46缺陷培养的神经元缺乏AIS MT成束9，10。他们还表现出受损的AIS形成和 轴突生长、蛋白质分布和MT方向的一系列缺陷，表明TRIM 46是必需的 对于AIS MT成束和神经元极性更广泛9。然而，这些令人兴奋的说法是基于 TRIM 46被shRNA敲低，主要在培养的神经元中，因此观察到的缺陷可归因于shRNA 毒性或人工培养环境。尚未进行研究体内TRIM 46敲除的工作。这 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。的 本提案的目的是使用TRIM 46基因敲除小鼠来确定TRIM 46在神经系统中的作用 并检验TRIM 46是体内AIS MT成束和神经元极性所需的特定假设。 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 TRIM 46基因敲除对神经元超微结构、行为和发育的影响 和伤害。目的1将确定TRIM 46在AIS形成、超微结构和MT成束中的作用。目的2 将确定TRIM 46在神经元形态、分子和MT极性中的作用。目标3将决定 TRIM 46敲除对神经元迁移、轴突再生和行为的影响。完成 该项目将揭示TRIM 46在AIS和MT会聚中的功能， 极性出现了。

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.