Regulation of Spine Morphogenesis by NrCAM

描述（由申请人提供）：该项目的长期目标是确定哺乳动物新皮层中调节突触连接的机制，并阐明它们的缺乏如何导致与神经发育障碍（如自闭症谱系障碍（ASD））相关的异常大脑布线。本申请试图阐明涉及NrCAM（神经元-胶质细胞相关细胞粘附分子）的棘和突触调节的新机制，NrCAM是ASD中的风险因子，其对于新皮质中兴奋回路的发展是重要的。一 待研究的新概念是NrCAM通过与驱避导向分子脑信号蛋白3F（Sema 3F）、神经纤毛蛋白-2（Npn-2）和丛蛋白A3（PlexA 3）相互作用来调节脊柱发育，并且NrCAM缺乏导致新皮层回路中的过度兴奋。待研究的中心假设是NrCAM/Npn- 2/PlexA 3包含Sema 3F的受体复合物，其约束或重塑新皮质中锥体神经元的树突棘以获得适当的兴奋性平衡。要解决的目的如下：目的1：NrCAM在约束棘和兴奋性突触形成的作用将通过分析树突棘和锥体神经元的突触在前额叶和感觉皮层区域的野生型（WT）和NrCAM无效小鼠，并定位NrCAM的突触位点。将进行双杂合子分析，以研究NrCAM与Npn-2、PlexA 3和Sema 3F的假定遗传相互作用，以调节体内脊柱形态发生。目标二：为了确定NrCAM损失是否诱导锥体神经元的过度兴奋性，将在星星锥体神经元中测量兴奋性反应，所述星形锥体神经元是丘脑皮质输入视觉刺激的主要靶标。 将比较通过全细胞记录的WT和NrCAM无效突变小鼠的视觉皮层切片中的兴奋性反应（层4），以及Sema 3F处理对WT和无效突变切片中的兴奋性反应的影响。目标3：将研究NrCAM的细胞自主突触后机制，用于通过与Npn-2和PDZ衔接子的相互作用介导的Sema 3F诱导的脊柱形态发生。将在NrCAM无效胚胎的星星锥体神经元中分析棘形态发生的遗传拯救，所述NrCAM无效胚胎在子宫内用Npn-2和PDZ衔接子的WT和NrCAM结合突变体电穿孔，并且在Sema 3F处理的神经元培养物中分析棘形态发生的遗传拯救。这些研究的结果预计将产生重要的积极影响，因为它将描绘控制感觉皮层连接和功能的脊柱形态发生的新分子机制，并可能提供对ASD靶向分子机制的深入了解。 公共卫生相关性：这项拟议中的研究与公共卫生有关，因为它试图定义调节哺乳动物大脑神经连接发育的分子决定因素。这项研究与NIH的使命有关，即阐明神经发育的基本机制，这对理解遗传性大脑疾病很重要，特别是与自闭症和相关综合征的感觉异常有关，这些综合征被称为“自闭症谱系障碍”，其中NrCAM，Neuropilin-2和Semaphorins是候选风险因素。

DESCRIPTION (provided by applicant): The long-range goal of this project is to identify mechanisms regulating synaptic connectivity in the mammalian neocortex and to elucidate how their deficiency causes abnormal brain wiring relevant to neurodevelopmental disorders such as autism spectrum disorders (ASDs). This application seeks to elucidate a novel mechanism for spine and synapse regulation involving NrCAM (Neuron-Glial Related Cell Adhesion Molecule), a risk factor in ASD, which is important for development of excitatory circuits in the neocortex. A new concept to be studied is that NrCAM regulates spine development by interacting with repellent guidance molecules Semaphorin3F (Sema3F), Neuropilin-2 (Npn-2), and PlexinA3 (PlexA3), and that NrCAM deficiency leads to hyperexcitability in neocortical circuits. The central hypothesis to be investigated is that NrCAM/Npn- 2/PlexA3 comprises a receptor complex for Sema3F that constrains or remodels dendritic spines of pyramidal neurons in the neocortex for appropriate excitatory balance. Aims to be addressed are as follows: Aim 1: A role for NrCAM in constraining spine and excitatory synapse formation will be investigated by analyzing dendritic spines and synapses of pyramidal neurons in prefrontal and sensory cortical areas of wild type (WT) and NrCAM null mice, and localizing NrCAM to synaptic sites. A double heterozygote analysis will be undertaken to investigate the postulated genetic interaction of NrCAM with Npn-2, PlexA3, and Sema3F to regulate spine morphogenesis in vivo. Aim 2: To determine if NrCAM loss induces hyperexcitability of pyramidal neurons, excitatory responses will be measured in star pyramidal neurons, the principal target of thalamocortical input to visual cortex (layer 4), in visual cortical slices of WT and NrCAM null mutant mice by whole cell recordings, and the effect of Sema3F treatment on excitatory responses in WT and null mutant slices will be compared. Aim 3: A cell autonomous, postsynaptic mechanism for NrCAM will be investigated for Sema3F-induced spine morphogenesis mediated by interaction with Npn-2 and PDZ adaptors. Genetic rescue of spine morphogenesis will be analyzed in star pyramidal neurons of NrCAM null embryos electroporated in utero with WT and NrCAM binding mutants for Npn-2 and PDZ adaptors, and in Sema3F-treated neuronal cultures. The outcome of these studies is expected to have an important positive impact, because it will delineate novel molecular mechanisms of spine morphogenesis that control sensory cortical connectivity and function, and may provide insight into molecular mechanisms targeted in ASDs. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health, because it seeks to define molecular determinants that regulate development of neural connectivity in the mammalian brain. The research is relevant to the mission of NIH in illuminating basic mechanisms of neurodevelopment important for understanding inherited brain disorders, and especially relevant to sensory abnormalities in autism and related syndromes, termed "Autism Spectrum Disorders," for which NrCAM, Neuropilin-2, and Semaphorins are candidate risk factors.