Molecular and cellular mechanisms governing interneuron development and connectivity

控制中间神经元发育和连接的分子和细胞机制

• 批准号: 9765678
• 负责人: Linda Van Aelst
• 金额: $ 69.07万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9765678
• 关键词: ANK3 gene; Action Potentials; Address; Affect; Axon; Binding; Biochemical; Biochemical Genetics; Brain; Brain Diseases; Cell Adhesion Molecules; Cells; Cytoskeleton; Data; Defect; Development; Disease; Distal; Electroporation; Ephrins; Epilepsy; Expression Profiling; Goals; Interneurons; Knockout Mice; Knowledge; Light; Link; Maintenance; Mediating; Molecular; Morphology; Neocortex; Neural Cell Adhesion Molecule L1; Neurons; Neuropilin-1; Output; Pathway interactions; Play; Population; Presynaptic Terminals; Proteins; Pyramidal Cells; RNA Interference; RNA interference screen; Reporting; Role; Schizophrenia; Signal Pathway; Site; Specificity; Spectrin; Structure; Surface; Synapses; Technology; Testing; Time; United States; autism spectrum disorder; base; excitatory neuron; experimental study; genetic approach; high resolution imaging; hippocampal pyramidal neuron; in utero; in vivo; insight; knock-down; mutant; neocortical; nerve supply; netrin receptor; neuronal circuitry; novel; postnatal; presynaptic; scaffold; tool; transcriptome sequencing; virtual

PROJECT SUMMARY/ABSTRACT Proper assembly and functioning of neuronal circuits in the neocortex, a brain structure critical for all higher- order functions, relies on the formation of specific synaptic connections between excitatory pyramidal neurons (PyNs) and different types of inhibitory GABAergic interneurons. One subset of interneurons that exerts powerful control over PyN spiking is the chandelier cell (ChC), which forms connections specifically at the site of action potential initiation in PyNs, referred to as the axon initial segment (AIS). Due to the unique connections formed between the terminals of ChC axonal arbors and the AISs of large populations of PyNs, ChCs are ideally suited to control the output of excitatory cortical networks. Hence, it is not surprising that ChC connectivity defects are linked to brain disorders, such as schizophrenia and autism spectrum disorder. Despite the importance of ChCs, the molecular mechanisms underlying their subcellular innervation of PyN AISs are unknown. To approach this, we initiated an in utero electroporation (IUE)-based in vivo RNAi screen of known neocortical PyN-expressed axonal cell adhesion molecules (CAMs) and select Ephs/ephrins. Strikingly, of all the molecules tested, we found the CAM L1CAM to be the only protein required for PyN AIS innervation by ChCs. In addition, based on single- ChC RNAseq data, we investigated the role of ChC-expressed netrin receptor Unc5b in ChC/PyN AIS innervation and found that it plays a key role in ChC axon terminal development and connectivity. Our findings provide a unique entry point for studies on the molecular basis of ChC/PyN connectivity. This application aims to elucidate how PyN L1CAM governs selective AIS innervation, to identify L1CAM’s presynaptic binding partner(s) on ChCs, and to scrutinize the mechanism of Unc5b in ChC axon terminal development and connectivity. To this end, Aim 1 will use molecular tools to disrupt interactions between L1CAM and the AIS cytoskeleton to assess whether cytoskeleton-mediated L1CAM clustering at the AIS is required for proper ChC/PyN AIS innervation. High resolution imaging will be performed to investigate the distribution of surface L1CAM on the AIS and distal axon. Also, inducible RNAi constructs delivered by IUE to temporally regulate PyN L1CAM levels in vivo will be used to determine whether PyN L1CAM is required for the establishment and/or maintenance of ChC/PyN innervation. Aim 2 will identify the presynaptic binding partner(s) of L1CAM on ChC terminals. Our preliminary data suggest that neuropilin-1 (Nrp1) is the L1CAM partner on ChCs required for ChC/PyN innervation. This will be tested by depleting Nrp1 in ChCs, using RNAi technology and conditional Nrp1 knockout mice. The Nrp1 domain(s) required for L1CAM binding will also be defined. Aim 3 will determine whether Unc5b governs ChC cartridge development by regulating terminal axon branching. We will also test whether the LARG/RhoA/ROCK pathway mediates Unc5b’s effect on ChC axon terminal development and connectivity using molecular/cellular tools and ChC-targeting IUE. Together, our studies will provide first insight into the mechanisms governing ChC/PyN connectivity and shed new light on the connectivity defects underlying common brain disorders. !

项目摘要/摘要 在新皮层中，神经元回路的适当组装和功能，这是所有较高的大脑结构 顺序功能，依赖于兴奋性金字塔神经元之间特定突触连接的形成 （Pyns）和不同类型的抑制性GABA能中间神经元。发挥强大的中间神经元的一个子集 对Pyn峰值的控制是枝形吊灯细胞（CHC），该单元格是在动作部位专门形成的连接 Pyns中的潜在计划，称为轴突初始段（AIS）。由于形成了独特的连接 CHC轴突轴向末端和大量Pyns群体之间，CHC非常适合 控制兴奋性皮质网络的输出。因此，CHC连接性缺陷是 与脑部疾病有关，例如精神分裂症和自闭症谱系障碍。尽管CHC很重要，但 其亚细胞支配Pyn AISS的分子机制尚不清楚。为了解决这个问题， 我们启动了一个基于已知新皮层Pyn表达的基于子宫内电穿孔（IUE）的体内RNAi屏幕 轴突细胞粘附分子（CAM）和选择Ephs/ephrins。令人惊讶的是，在所有测试的分子中，我们发现 CAM L1CAM是CHCS神经支配所需的唯一蛋白质。此外，基于单个 CHC RNASEQ数据，我们研究了CHC表达的Netrin受体UNC5B在CHC/PYN AIS神经上的作用 并发现它在CHC轴突终端的发展和连通性中起关键作用。我们的发现提供了 基于CHC/PYN连接性分子基础的研究的独特入口点。该应用程序旨在阐明 Pyn L1CAM如何控制选择性AIS神经，以确定L1CAM在CHC上的突触前结合伴侣， 并仔细检查CHC轴突终端发育和连通性中UNC5B的机制。为此，目标 1将使用分子工具破坏L1CAM与AIS细胞骨架之间的相互作用，以评估是否是否 适当的CHC/PYN AIS神经需要在AIS处进行细胞骨架介导的L1CAM聚类。高的 将进行分辨率成像，以研究表面L1CAM在AIS和远端轴突上的分布。 此外，将使用IUE提供的诱导RNAi构建体，用于在体内暂时调节Pyn L1CAM水平 确定建立和/或维持CHC/PYN神经支配是否需要Pyn L1CAM。 AIM 2将确定CHC终端上L1CAM的突触前结合伴侣。我们的初步数据暗示 该Neuropilin-1（NRP1）是CHC/PYN神经支配所需的CHC的L1CAM合作伙伴。这将通过 使用RNAi技术和条件NRP1敲除小鼠在CHC中耗尽NRP1。 NRP1域 L1CAM结合所需的也将定义。 AIM 3将确定UNC5B是否控制CHC墨盒 通过调节末端轴突分支来开发。我们还将测试Larg/Rhoa/Rock Pathway是否 使用分子/细胞工具和 CHC靶向。总之，我们的研究将首先深入了解有关CHC/PYN的机制 连通性并为常见脑疾病的连通性缺陷提供了新的启示。 呢