Role of N-cadherin and AF-6 signaling in regulating dendritic spine morphology

描述(申请人提供)：树突棘重塑是突触发育和可塑性的关键组成部分，它在神经发育和神经退行性疾病中受到深刻影响。脊柱形态的改变需要黏附、细胞骨架重组和功能调节的精确协调。粘连在调节脊柱形态中的关键作用才刚刚开始被阐明。N-钙粘附素是一种主要的突触黏附分子，通过α-N-连环素、Rho GTP酶和肌动蛋白重排等作用影响脊柱结构，但其信号转导机制的具体机制尚不清楚。此外，被称为Nectins的黏附分子与N-钙粘附素潜在的协同作用参与了突触的形成；然而，这些途径之间的确切关系尚不清楚。AF-6是一种广泛存在于粘连连接和中央突触中的PDZ蛋白成分，已被证明与α-N-连环素和蜜环素相互作用。我们实验室的初步数据显示，AF-6与RAC全球环境基金Kalirin-7相互作用，Kalirin-7是树突状脊柱形态的关键调节因子，使AF-6处于整合粘附性和树突状脊柱结构可塑性的理想位置。这项拟议工作的目的是确定N-钙粘附素黏附复合体和明胶黏附复合体在调节AF-6的活性及其随后的树突棘结构重塑中的相对作用。首先，我们将通过共免疫沉淀法从突触小体中鉴定AF-6与N-钙粘蛋白或果胶蛋白之间的相互作用。我们还将通过突变型AF-6蛋白与突触体提取物的亲和结合分析来确定AF-6/N-钙粘素复合体相互作用的结构基础，并将这些重要的结构域与已知的AF-6与果胶蛋白的结合域进行比较。然后，我们将测量AF-6通过N-钙粘蛋白复合体或被激活的胶凝素重新募集到脊柱上，以确定每条途径在调节AF-6功能中的相对重要性。最后，我们将通过使用显性-负性结构，然后增强或阻断N-钙粘素信号，比较α-N-连环素、果胶凝集素和AF-6在N-钙粘素介导的脊柱重塑中的相对重要性。这些目标的结果将使我们更好地理解AF-6对树突棘结构黏附介导的重塑的调节，并将为健康和疾病状态下结构可塑性的机制提供洞察力。与公共卫生相关：这个项目的目标是了解大脑中神经细胞之间的联系是如何建立的。许多大脑疾病是由异常的连接强度和数量引起的，了解这些连接是如何形成的将为可能用于治疗这些破坏性疾病的药物提供潜在的靶点

DESCRIPTION (provided by applicant): Dendritic spine remodeling is a key component of synaptic development and plasticity, and it is profoundly affected in neurodevelopmental and neurodegenerative disorders. Spine morphological changes require precise coordination of adhesion, cytoskeletal reorganization, and functional modulation. The crucial role of adhesion in regulating spine morphology has only begun to be elucidated. N-cadherin, a major synaptic adhesion molecule, influences spine structure through the actions of alpha-N-catenin, Rho GTPases, and actin rearrangement, but the specifics of this signaling mechanism remain unclear. Additionally, adhesion molecules called nectins have been implicated in synapse formation in a potentially cooperative association with N-cadherins; however, the exact relationship between these pathways is unknown. AF-6, a PDZ protein component prevalent in adhesion junctions and in central synapses, has been shown to interact with both alpha-N-catenins and nectins. Preliminary data from our laboratory have shown that AF-6 interacts with the Rac GEF kalirin-7, a key regulator of dendritic spine morphology, placing AF-6 in an ideal position to integrate adhesion and dendritic spine structural plasticity. The aim of this proposed work is to determine the relative roles of the N-cadherin adhesion complex and the nectin adhesion complex in regulating the activity of AF-6 and its subsequent remodeling of dendritic spine structure. First, we will characterize the interactions between AF-6 and N-cadherin or nectins by coimmunoprecipitation from synaptosomes. We will also determine the structural basis for the AF-6/N-cadherin complex interaction by affinity binding assays of mutant AF-6 proteins with synaptosome extracts, and compare these important structural domains to the known binding domain of AF-6 with nectins. We will then measure AF-6 recruitment to spines by N-cadherin complexes or by nectins upon their activation, to determine the relative importance of each pathway in regulating AF-6 function. Finally, we will compare the relative importance of alpha-N-catenin, nectins, and AF-6 in N-cadherin-mediated spine remodeling by using dominant-negative constructs and then enhancing or blocking N-cadherin signaling. The results from these aims will allow a better understanding of the regulation adhesion-mediated remodeling of dendritic spine structure by AF-6, and will provide insight into the mechanisms of structural plasticity in health and in disease states. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand how connections between nerve cells in the brain are established. Many brain disorders are caused by abnormal connection strength and numbers, and understanding how these connections are formed will provide potential targets for drugs that might be used to treat these devastating disorders