The Role of Clustered Protocadherins in Neurite Self-avoidance

簇状原钙粘蛋白在神经突自我回避中的作用

• 批准号: 8964441
• 负责人: THOMAS P MANIATIS
• 金额: $ 38.33万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-15 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8964441
• 关键词: Address; Adult; Affinity; Alleles; Alternative Splicing; Autistic Disorder; Axon; Binding; Biological Neural Networks; Birth; Cell Adhesion Molecules; Cell membrane; Cell surface; Cells; Chimera organism; Chromosomes; Chromosomes, Human, Pair 18; Complex; Defect; Dendrites; Disease; Embryo; Employee Strikes; Etiology; Financial compensation; Gene Cluster; Generations; Genes; Goals; Individual; Interneurons; Invertebrates; Knock-out; Knockout Mice; Label; Locomotion; Mediating; Mediator of activation protein; Mental disorders; Methods; Molecular; Mus; Mutation; Neonatal; Nervous system structure; Neurites; Neurons; Pattern; Phenotype; Play; Process; Protein Isoforms; Proteins; Purkinje Cells; Reagent; Research; Retina; Role; Schizophrenia; Sister; Source; Spinal; System; Technology; Testing; Time; Vertebrates; Work; cell type; central pattern generator; combinatorial; design; fly; gene function; genetic analysis; insight; loss of function; mRNA Precursor; mutant; nervous system disorder; neural circuit; neurodevelopment; neuron apoptosis; neuronal patterning; novel; promoter; public health relevance; research study; retinal neuron; starburst amacrine cell; tool

 DESCRIPTION (provided by applicant): A key principle of neural circuit assembly is neurite self-avoidance, a process in which sister axons or dendrites of the same neuron repel each other but interact freely with those from other neurons, indicating they can discriminate "self" from "non-self". In flies the cell adhesion molecule Dscam1 has been shown to be a key mediator for neurite self-avoidance. Alternative splicing of Dscam1 pre-mRNA generates a large number of distinct protein isoforms that interact in a strictly homophilic manner, which mediate contact-dependent repulsion that leads to self-avoidance. However, the vertebrate Dscam genes do no encode significant diversity. Rather, this diversity and function appear to be provided by the clustered protocadherins (Pcdhs). Studies in our lab and others have shown that 1) Stochastic expression of clustered Pcdhs by alternative promoter choice generates single cell diversity; 2) Combinatorial homophilic interactions between clustered Pcdhs at the cell surface generate single neuron identity; 3) Pcdh- γ proteins are required for dendritic self-avoidance in mouse starburst amacrine cells and Purkinje cells. These observations have led to the hypothesis that clustered Pcdhs function similarly as Dscam1 proteins to mediate self-avoidance in vertebrates. In this application, we propose to carry out a comprehensive genetic analysis to determine whether clustered Pcdhs play a central role in neurite self-avoidance in the vertebrate nervous system, whether Pcdh diversity is required for this process, and whether different Pcdh clusters functionally compensate each other in specific cellular contexts. To accomplish these goals, we will generate single cell knockouts of Pcdh- α, - β, and - γ gene clusters individually or altogether, and study the loss of function effects in neurite arborizationof individual neurons. In Aim 1, we will establish and validate two complementary single cell knockout methods, Mosaic Analysis with Double Markers (MADM) and Single Neuron Analysis in Chimeras (SNAC). Both methods sparsely generate and label mutant cells in the otherwise normal neural network, allowing robust morphological analysis and rigorous assessment of cell autonomous gene function in single neurons. In Aim 2, we will use single cell knockouts of the Pcdh- γ gene cluster to validate previously identified dendritic self-avoidance and arborization phenotypes, address the diversity requirement for these processes by generating a Pcdh- γ allele lacking all alternate isoforms, and identify spinal interneuron lineages that display neurit patterning defects that may be associated with a striking phenotype in the Pcdh- γ cluster deletion mice - complete loss of central pattern generator (CPG) for locomotion. In Aim 3, we propose to study the consequences of loss-of-function of all clustered Pcdhs on neural development, and perform a CNS-wide screen for neuronal cell types in which axonal or dendritic self-avoidance is disrupted. The proposed studies should provide significant new insights into mechanisms of neural circuit assembly in the vertebrate nervous system, as well as into etiologies of diverse neurological disorders resulting from altered neural circuitries.

 描述(申请人提供)：神经回路组装的一个关键原理是轴突自我回避，在这个过程中，同一神经元的姐妹轴突或树突相互排斥，但与来自其他神经元的姐妹轴突或树突自由相互作用，表明它们可以区分“自我”和“非我”。在果蝇中，细胞黏附分子Dscam1已被证明是轴突自我避免的关键媒介。Dscam1前-mRNA的选择性剪接产生了大量不同的蛋白质异构体，这些蛋白质异构体以严格的同嗜性方式相互作用，介导了导致自我回避的接触依赖排斥。然而，脊椎动物的Dscam基因并不编码显著的多样性。相反，这种多样性和功能似乎是由聚集的原钙粘附素(Pcdhs)提供的。本实验室和其他实验室的研究表明：1)通过选择性启动子选择的方式随机表达成簇的Pcdhs可产生单细胞多样性；2)成簇的Pcdhs在细胞表面的组合同源性相互作用产生单一神经元的特性；3)Pcdh-γ蛋白是小鼠星状突起无长突细胞和浦肯野细胞树突状自我避免所必需的。这些观察结果导致了一种假设，即聚集型Pcdhs的功能类似于Dscam1蛋白，在脊椎动物中介导自我回避。在这一应用中，我们建议进行一项全面的遗传分析，以确定聚集性Pcdh是否在脊椎动物神经系统的轴突自我回避中发挥核心作用，这一过程是否需要Pcdh多样性，以及不同的Pcdh簇在特定的细胞环境中是否在功能上相互补偿。为了实现这些目标，我们将单独或共同产生Pcdh-α，-β和-γ基因簇的单细胞敲除，并研究单个神经元突起树枝的功能丧失效应。在目标1中，我们将建立并验证两种互补的单细胞基因敲除方法，即双标记嵌合体分析(MADM)和嵌合体单神经元分析(SNAC)。这两种方法都在正常的神经网络中稀疏地产生和标记突变细胞，从而允许对单个神经元中的细胞自主基因功能进行稳健的形态分析和严格的评估。在目标2中，我们将使用Pcdh-γ基因簇的单细胞敲除来验证先前发现的树突状自我回避和树枝形成的表型，通过产生缺乏所有替代异构体的Pcdh-γ等位基因来满足这些过程的多样性要求，并鉴定显示神经元图案缺陷的脊髓神经元间谱系，该缺陷可能与Pcdh-γ簇缺失小鼠中的一个显著表型有关-运动中央模式生成器的完全丧失。在目标3中，我们建议研究所有聚集性Pcdh功能丧失对神经发育的影响，并对轴突或树突自我回避中断的神经细胞类型进行中枢神经系统范围的筛查。这项拟议的研究将为脊椎动物神经系统中神经回路组装的机制以及由神经回路改变引起的各种神经疾病的病因提供重要的新见解。