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

描述（由申请人提供）：树突棘重塑是突触发育和可塑性的关键组成部分，并且在神经发育和神经退行性疾病中受到深刻影响。脊柱形态学的变化需要精确协调的粘附，细胞骨架重组，和功能的调制。粘附在调节脊柱形态中的关键作用才刚刚开始阐明。N-钙粘蛋白是一种主要的突触粘附分子，通过α-N-连环蛋白、Rho GTPases和肌动蛋白重排的作用影响棘结构，但这种信号传导机制的具体细节仍不清楚。此外，被称为nectin的粘附分子与N-钙粘蛋白潜在的合作关系涉及突触形成;然而，这些途径之间的确切关系尚不清楚。AF-6是一种在粘附连接和中央突触中普遍存在的PDZ蛋白组分，已显示与α-N-连环蛋白和连接蛋白相互作用。我们实验室的初步数据表明，AF-6与Rac GEF kalirin-7相互作用，这是树突棘形态的关键调节因子，将AF-6置于整合粘附和树突棘结构可塑性的理想位置。这项工作的目的是确定N-钙粘蛋白粘附复合物和连接素粘附复合物在调节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，我们将通过突触体的共免疫沉淀来表征AF-6和N-钙粘蛋白或连接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测定来确定AF-6/N-钙粘蛋白复合物相互作用的结构基础，并将这些重要的结构域与AF-6与连接素的已知结合域进行比较。然后，我们将测量AF-6招聘的N-钙粘蛋白复合物或连接蛋白激活后的棘，以确定每个途径在调节AF-6功能的相对重要性。最后，我们将比较α-N-连环蛋白，nectin和AF-6在N-钙粘蛋白介导的脊柱重塑中的相对重要性，通过使用显性负结构，然后增强或阻断N-钙粘蛋白信号传导。这些目标的结果将使我们更好地理解AF-6对树突棘结构的调节粘附介导的重塑，并将为健康和疾病状态下的结构可塑性机制提供深入了解。公共卫生相关性：该项目的目标是了解大脑中神经细胞之间的连接是如何建立的。许多脑部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毁灭性疾病的药物提供潜在目标

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