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

基本信息

批准号：
10088479
负责人：
Linda Van Aelst
金额：
$ 65.61万
依托单位：
COLD SPRING HARBOR LABORATORY
依托单位国家：
美国
项目类别：
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/10088479
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 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

项目摘要

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. !

项目概要/摘要新皮质中神经回路的正确组装和功能，新皮质是所有高级功能都至关重要的大脑结构。秩序功能，依赖于兴奋性锥体神经元之间特定突触连接的形成 (PyN) 和不同类型的抑制性 GABA 能中间神经元，是发挥强大作用的中间神经元的一个子集。对 PyN 尖峰的控制是枝形吊灯细胞 (ChC)，它专门在作用位点形成连接 PyN 中的潜在起始，由于形成的独特连接而被称为轴突初始段 (AIS)。在 ChC 轴突乔木的末端和大量 PyN 的 AIS 之间，ChC 非常适合因此，ChC 连接缺陷是不足为奇的。尽管 ChC 很重要，但与精神分裂症和自闭症谱系障碍等脑部疾病有关。 PyN AIS 的亚细胞神经支配的分子机制尚不清楚。我们启动了基于子宫内电穿孔 (IUE) 的体内 RNAi 筛选，对已知的新皮质 PyN 表达的令人惊讶的是，在所有测试的分子中，我们发现了轴突细胞粘附分子（CAM）和选择的 Ephs/ephrins。 CAM L1CAM 是 ChCs 的 PyN AIS 神经支配所需的唯一蛋白质。 ChC RNAseq 数据，我们研究了 ChC 表达的 netrin 受体 Unc5b 在 ChC/PyN AIS 神经支配中的作用并发现它在 ChC 轴突末端的发育和连接中发挥着关键作用。我们的研究结果提供了一个线索。 ChC/PyN 连接性分子基础研究的独特切入点该应用旨在阐明。 PyN L1CAM 如何控制选择性 AIS 神经支配，以识别 ChC 上 L1CAM 的突触前结合伙伴，并仔细研究 Unc5b 在 ChC 轴突末端发育和连接中的机制。 1 将使用分子工具破坏 L1CAM 和 AIS 细胞骨架之间的相互作用，以评估是否 AIS 处的细胞骨架介导的 L1CAM 聚集是适当的 ChC/PyN AIS 神经支配所必需的。将进行分辨率成像以研究 AIS 和远端轴突上表面 L1CAM 的分布。此外，将使用 IUE 提供的诱导性 RNAi 构建体来临时调节体内 PyN L1CAM 水平确定 ChC/PyN 神经支配的建立和/或维持是否需要 PyN L1CAM。目标 2 将识别 ChC 终端上 L1CAM 的突触前结合伙伴。我们的初步数据表明。 Neuropilin-1 (Nrp1) 是 ChC/PyN 神经支配所需的 ChC 上的 L1CAM 伙伴。使用 RNAi 技术和条件 Nrp1 敲除小鼠来消耗 ChC 中的 Nrp1。 L1CAM 结合所需的目标 3 将确定 Unc5b 是否控制 ChC 卡盒。我们还将测试 LARG/RhoA/ROCK 通路是否通过调节末端轴突分支来发育。使用分子/细胞工具介导 Unc5b 对 ChC 轴突末端发育和连接的影响我们的研究将共同提供对 ChC/PyN 控制机制的初步见解。连接性并为常见脑部疾病潜在的连接缺陷提供了新的线索。！