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

• 批准号: 10297694
• 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
• 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/10297694
• 关键词: Address; Alleles; Animal Testing; Bar Codes; Biology; Biotinylation; CRISPR/Cas technology; Cell Adhesion Molecules; Cell Death; Cell Survival; Cells; Complement; Complex; Dendrites; Detection; Development; Discrimination; Disease; Dyslexia; Electrophysiology (science); Electroporation; Event; Exhibits; Failure; Family; Gene Cluster; Gene Delivery; Goals; Maps; Mass Spectrum Analysis; Measurement; Mediating; Modeling; Molecular; Morphology; Motion; Mus; Mutant Strains Mice; Mutate; Mutation; Nervous system structure; Neuraxis; 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; base; cell type; in vivo; insight; interest; mutant; neural circuit; neurodevelopment; neuronal survival; overexpression; patch clamp; small hairpin RNA; starburst amacrine cell; visual information

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和特定的细胞识别步骤，许多由细胞介导 粘附分子（CAM）。实际上，破坏凸轮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。我们的模型是鼠标视网膜， 中枢神经系统，其中约有100种类型的神经元组织成专用电路，这些电路编码的特征 视觉世界。我们在这里专注于γ--蛋白酶蛋白（γ-PCDHS），22个CAM从单个基因簇表达， 产生数千种不同的同质识别复合物。 γ-PCDH是神经元的关键调节剂 Starburst团体细胞（SAC）和细胞存活以及视网膜中许多其他类型的神经元中的自我避免。 γ-PCDHs提供这些功能的机制尚不清楚，γ-PCDH同工型的重要性也是如此 多样性。我们使用CRISPR/CAS9方法来生成一系列公正的等位基因突变体，其中1至 21个完整的γ-PCDH同工型。从中，我们了解到一种同工型γC4对于神经元生存至关重要，表明 这与其他21种的功能不同。我们建议定义自我避免的机制和 神经元存活，并使用我们的Allic系列来确定正常神经元所需的同工型多样性水平 电路形成。我们的中心假设是：1）高水平的γ-PCDH同工型多样性使神经元能够 在“自我”和“非自我”之间区别介导自我避免，同时允许与邻近互动 通过所有同工型共有的机制神经元；相比之下，2）神经元生存需要相互作用 特定于γC4同工型。在特定的目标1中，我们将使用减少多样性突变体的战略子集进行 确定囊中自我/非自我歧视所需的同工型多样性的程度，神经元对 视网膜中的运动检测电路。我们将分两个级别分析该电路：a）之间的接触形态 囊和b）方向选择性残神经细胞的电生理功能，下游神经元中的下游神经元 电路。在特定的目标2中，我们将使用体内基因递送到自避免的分子机制 操纵候选途径并映射基本域。在特定目标3中，我们将通过 γC4促进神经元存活。我们将使用常规电穿孔来绘制关键蛋白质结构域， 通过基于发现的蛋白质组学方法完成，以找到γC4的同工型特异性蛋白相互作用。这些 研究将使我们能够更好地了解γ-PCDHS如何促进细胞识别和神经回路 在视网膜中形成，并洞悉因神经发育障碍而破坏的过程。