Regulation of Spine Morphogenesis by NrCAM

描述(申请人提供)：该项目的长期目标是确定哺乳动物新皮质中调节突触连接的机制，并阐明它们的缺失如何导致与自闭症谱系障碍(ASDS)等神经发育障碍相关的异常大脑连接。这项应用旨在阐明涉及神经元-胶质相关细胞黏附分子(NrCAM)的脊髓和突触调节的新机制，NrCAM是ASD的一个危险因素，对新皮质兴奋回路的发展至关重要。一个 有待研究的新概念是，NrCAM通过与排斥性引导分子Semaphorin3F(Sema3F)、Neuropilin-2(Npn-2)和PlexinA3(PlexA3)相互作用来调节脊柱的发育，NrCAM缺乏导致新皮质回路的过度兴奋。有待研究的中心假设是，NrCAM/Npn-2/PlexA3包含Sema3F的受体复合体，该复合体约束或重塑新皮质中锥体神经元的树突棘，以实现适当的兴奋平衡。目的1：通过分析野生型(WT)和NrCAM缺失小鼠前额叶和感觉皮质锥体神经元的树突棘和突触，并将NrCAM定位于突触部位，研究NrCAM在抑制脊髓和兴奋性突触形成中的作用。将采用双重杂合子分析来研究NrCAM与Npn-2、PlexA3和Sema3F在体内调节脊柱形态发生的假设遗传相互作用。目的2：为了确定NrCAM的缺失是否会导致锥体神经元的过度兴奋性，将测量星状锥体神经元的兴奋性反应，星状锥体神经元是丘脑皮质视觉输入的主要目标 通过全细胞记录，观察WT和NrCAM基因缺失突变小鼠视觉皮层脑片(第四层)的兴奋性反应，并比较Sema3F处理对WT和NrCAM缺失突变小鼠视觉皮层脑片兴奋性反应的影响。目的：通过与Npn-2和PDZ适配器的相互作用，研究NrCAM在Sema3F诱导的脊髓形态发生中的细胞自主、突触后机制。在具有Npn-2和PDZ适配器的WT和NrCAM结合突变体的宫内电穿孔的NrCAM缺失胚胎的星形锥体神经元中，以及在Sema3F处理的神经元培养中，将分析脊柱形态发生的遗传挽救。这些研究的结果有望产生重要的积极影响，因为它将描绘控制感觉皮质连通性和功能的脊柱形态发生的新的分子机制，并可能为深入了解ASD的分子机制提供帮助。

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.