Mechanisms of Cell Adhesion Molecule Function in Retinal Development

视网膜发育中细胞粘附分子功能的机制

基本信息

批准号：
10650788
负责人：
Andrew Garrett
金额：
$ 38.5万
依托单位：
WAYNE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10650788
关键词：
Alleles Animal Testing Bar Codes Biology Biotinylation CRISPR/Cas technology Cell Adhesion Molecules Cell Death Cell Survival Cells Central Nervous System Complement Complex Dedications Dendrites Detection Development Discrimination Disease Dyslexia Electrophysiology (science)Electroporation Event Exhibits Failure Family Gene Cluster Gene Delivery Goals Learning Maps Mass Spectrum Analysis Measurement Mediating Modeling Molecular Morphology Motion Mus Mutant Strains Mice Mutate Mutation Nervous System Neurites Neurodevelopmental Disorder Neurons PTK2 gene Pathway interactions Process Protein Isoforms Proteins Proteomics Public Health Regulation Research Retina Retinal Ganglion Cells Role Schizophrenia Series Specificity Synapses Tertiary Protein Structure Testing Therapeutic Vision Visual adeno-associated viral vector cell type in vivo insight interest mutant neural circuit neurodevelopment neuronal survival overexpression patch clamp small hairpin RNA starburst amacrine cell visual information

项目摘要

ABSTRACT Neural circuit formation requires a series of highly diverse and specific cell-cell recognition steps, many mediated by cell adhesion molecules (CAMs). Indeed, mutations that disrupt CAMs or their regulation are associated with circuit level neurodevelopmental disorders from dyslexia to schizophrenia. Our model is the mouse retina, an extension of the central nervous system where ~100 types of neurons organize into dedicated circuits that encode the features of the visual world. We focus here on the gamma-protocadherins (γ-Pcdhs), 22 CAMs expressed from a single gene cluster that generate many thousands of distinct homophilic recognition complexes. The γ-Pcdhs are critical regulators of neuronal self-avoidance in starburst amacrine cells (SACs), and cell survival and in many other types of neurons in the retina. The mechanisms through which the γ-Pcdhs serve these functions are unknown, as is the importance of γ-Pcdh isoform diversity. We used a CRISPR/Cas9 approach to generate an unbiased allelic series of mouse mutants with between 1 and 21 intact γ-Pcdh isoforms. From these, we learned that one isoform, γC4, is essential for neuronal survival, suggesting that this isoform functions differently from the other 21. We propose to define the mechanisms of self-avoidance and neuronal survival, and to use our allelic series to determine the level of isoform diversity required for normal neural circuit formation. Our central hypotheses are that: 1) a high level of γ-Pcdh isoform diversity enables neurons to distinguish between “self” and “non-self” to mediate self-avoidance while permitting interaction with neighboring neurons through mechanisms common to all isoforms; and 2) neuronal survival, in contrast, requires interactions specific to the γC4 isoform. In Specific Aim 1, we will use a strategic subset of our reduced-diversity mutants to determine the extent of isoform diversity required for self/non-self discrimination in SACs, neurons essential for the motion detection circuit in the retina. We will analyze this circuit at two levels: A) morphology of contacts between SACs, and B) the electrophysiological function of direction-selective retinal ganglion cells, the downstream neurons in the circuit. In Specific Aim 2, we will define the molecular mechanisms of self-avoidance using in vivo gene delivery to manipulate candidate pathways and map essential domains. In Specific Aim 3 we will uncover the mechanisms through which γC4 promotes neuronal survival. We will use retinal electroporation to map critical protein domains, complemented by a discovery-based proteomics approach to find isoform-specific protein interactions for γC4. These studies will allow us to better understand how the γ-Pcdhs contribute to cell-cell recognition and neural circuit formation in the retina and provide insight into processes disrupted by neurodevelopmental disorders.

抽象的神经回路的形成需要一系列高度多样化和特异性的细胞间识别步骤，其中许多步骤是由细胞介导的事实上，破坏 CAM 或其调节的突变与电路水平有关。从阅读障碍到精神分裂症的神经发育障碍我们的模型是小鼠视网膜，是视网膜的延伸。中枢神经系统，约有 100 种神经元组织成专用电路，对神经元的特征进行编码我们在此重点关注 γ-原钙粘蛋白 (γ-Pcdhs)，这是由单个基因簇表达的 22 个 CAM。产生数千个不同的同源识别复合物，γ-Pcdhs 是神经元的关键调节因子。星爆无长突细胞（SAC）的自我回避、细胞存活以及视网膜中许多其他类型的神经元。 γ-Pcdh 发挥这些功能的机制尚不清楚，γ-Pcdh 亚型的重要性也是未知的我们使用 CRISPR/Cas9 方法生成了一系列无偏差的等位基因小鼠突变体，其数量在 1 到 1 之间。 21 种完整的 γ-Pcdh 同工型从这些中，我们了解到一种同工型 γC4 对于神经元存活至关重要。该亚型的功能与其他亚型不同 21. 我们建议定义自我回避和神经元存活，并使用我们的等位基因系列来确定正常神经所需的亚型多样性水平我们的中心假设是：1）高水平的 γ-Pcdh 异构体多样性使神经元能够区分“自我”和“非自我”以调解自我回避，同时允许与邻居互动神经元通过所有亚型共有的机制；2）相反，神经元的生存需要相互作用在具体目标 1 中，我们将使用多样性减少的突变体的一个战略子集来特定于 γC4 同工型。确定 SAC 中自我/非自我辨别所需的亚型多样性程度，SAC 是视网膜中的运动检测电路我们将从两个层面分析该电路：A）接触之间的形态。 SAC，B）方向选择性视网膜神经节细胞的电生理功能，下游神经元在具体目标 2 中，我们将定义利用体内基因传递进行自我回避的分子机制。在特定目标 3 中，我们将通过操纵候选途径并绘制关键域来揭示其机制。其中 γC4 促进神经元存活，我们将使用视网膜电穿孔来绘制关键蛋白质结构域，辅之以基于发现的蛋白质组学方法，以寻找 γC4 的亚型特异性蛋白质相互作用。研究将使我们更好地了解 γ-Pcdhs 如何促进细胞间识别和神经回路视网膜中的形成并提供对神经发育障碍破坏的过程的深入了解。