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: 9108445
• 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/9108445
• 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; Health; 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 therapeutics; promoter; research study; retinal neuron; single cell technology; 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（由适用提供）：神经元电路组装的关键原理是神经自我避免，在这种过程中，同一神经元的姐妹轴突或树突相互抑制，但与其他神经元的人自由相互作用，表明他们可以歧视“自我”与“非自身”。在苍蝇中，细胞粘附分子DSCAM1已被证明是神经元自我避免的关键介体。 DSCAM1前MRNA的替代剪接会产生大量不同的蛋白质同工型，这些蛋白质同工型以严格的均高均质方式相互作用，从而介导了接触依赖性的排斥，从而导致自我避免。但是，脊椎动物DSCAM基因没有编码明显的多样性。相反，这种多样性和功能外观由聚类协议蛋白（PCDHS）提供。我们实验室和其他人的研究表明，1）通过替代启动子选择的聚类PCDH的随机表达产生单细胞多样性； 2）细胞表面聚类的PCDH之间的组合均电相互作用会产生单个神经元同一性； 3）在小鼠starburst amacrine细胞和珀kinje细胞中，需要PCDH-γ蛋白来避免树突状自我避免。这些观察结果导致了以下假设：聚集的PCDH的功能与DSCAM1蛋白相似，可以介导脊椎动物中的自我避免。在此应用中，我们建议进行全面的遗传分析，以确定聚类的PCDHS在脊椎动物神经系统中是否起着核心作用，该过程是否需要PCDH多样性，以及不同的PCDH簇是否在特定的细胞环境中相互补偿。为了实现这些目标，我们将产生单个细胞敲除pCDH -α，-β和-γ基因的单独或完全或完全的组合，并研究AIM 1中神经蛋白轴承术在功能效应中的丧失，我们将建立和验证两种完整的单细胞敲除方法，具有双重标记（MADM）和单神经元分析（MADM）和单个神经元分析（SNAC）（SNAC）（SNAC）（SNAC）（SNAC）（SNAC）。两种方法都稀疏地产生和标记突变细胞在原本正常的神经元中，允许对单个神经元中细胞自主基因功能的强大形态分析和严格评估。在AIM 2中，我们将使用PCDH-γ基因簇的单细胞敲除来验证先前鉴定的树枝状自我避免和纵横化表型，通过产生缺乏所有替代性同工型的PCDH-γ等位基因来解决这些过程的多样性需求，并与脊柱间的缺陷相关联，这些链接可能与PRETS相关，使现有的表现出色的表现量很强。删除小鼠 - 运动发生器（CPG）的完全损失。在AIM 3中，我们建议研究所有聚类PCDH对神经元发育的功能丧失的后果，并对神经元细胞类型进行CNS范围的筛查，其中轴突或树突状自我避免是残疾的。拟议的研究应提供对脊椎动物神经系统中神经元电路组装机制的重要新见解，以及因神经元电路改变而导致的潜水神经系统疾病的病因。