Control of Excitatory Synapse Formation and Maturation by Astrocytes

描述（由申请人提供）：突触形成是大脑发育的关键步骤，需要精确调节以防止可能表现为神经功能障碍的错误连接。近年来，人们发现星形胶质细胞通过分泌多种调节突触形成和成熟的因子来诱导中枢神经系统的兴奋性突触发生。然而，为什么星形胶质细胞释放各种影响突触发生的信号的问题代表了神经发育领域的一个重大知识空白。我们实验室的主要重点是了解这些星形胶质细胞释放的三种信号，基质细胞蛋白血栓反应蛋白（TSP）， hevin和SPARC，对兴奋性突触的初始形成和成熟的贡献。本研究的第一个目标是确定TSP在突触前轴突和突触后树突之间形成初始突触接触中的作用。有趣的是，TSP与神经元钙通道亚基a2d-1结合，刺激培养视网膜神经节细胞（RGCs）树突状丝状足的形成和寻靶行为。进一步的初步研究结果表明，TSP/a2d-1可能诱导下游Rho GTPase信号通路，从而影响丝状足肌动蛋白动力学，这代表了TSP/a2d-1诱导突触形成的潜在分子机制。与TSP一样，基质细胞蛋白hevin在培养的神经元和体内都是突触前发生的。SPARC与hevin具有高度同源性，对hevin的突触形成作用具有特异性拮抗作用。该研究的第二个目标是确定hevin和SPARC之间的平衡如何影响中枢神经系统突触的成熟。序列切片电镜（EM）的初步结果显示，hevin基因敲除（KO）小鼠的突触后棘在树突中大部分缺失，取而代之的是未成熟的丝状足样突起。此外，兴奋性突触主要位于树突轴上，而不是突起的尖端。基于这些发现，本文将验证a2d-1在树突丝状发育阶段促进兴奋性突触接触的启动，而hevin和SPARC水平的精确调节控制突触的稳定和成熟的假设。为了实现这个目的,我提出了两个具体目标:1)TSP和a2d-1如何调节兴奋性突触形成的初始阶段?2) hevin和SPARC如何调控兴奋性突触的形态和功能成熟？通过对纯化的皮层神经元和视觉皮层的切片制备进行实时共聚焦成像、连续切片EM和电生理学的结合，本研究将为星形胶质细胞在发育过程中兴奋性突触发生中的作用提供新的分子视角。更好地了解TSP、a2d-1、hevin和SPARC可能会改善以突触连接异常为特征的精神分裂症和自闭症等神经系统疾病的治疗。

DESCRIPTION (provided by applicant): Synapse formation is a critical step in brain development that requires precise regulation to prevent miswiring that can manifest as neurological dysfunction. In recent years, it has come to light that astrocytes induce excitatory synaptogenesis in the central nervous system by secreting multiple factors that regulate synaptic formation and maturation. However, the question of why astrocytes release a variety of signals affecting synaptogenesis represents a significant gap in knowledge in the field of neurodevelopment. The primary focus of our lab is to understand the contributions of three of these astrocyte-released signals, the matricellular proteins thrombospondin (TSP), hevin and SPARC, to the initial formation and maturation of excitatory synapses. The first goal in this proposed study is to determine the role of TSP in forming the initial synaptic contacts between presynaptic axons and postsynaptic dendrites. Intriguingly, TSP binds to the neuronal calcium channel subunit a2d-1 to stimulate the formation and target-seeking behavior of dendritic filopodia in cultured retinal ganglion cells (RGCs). Further preliminary findings indicate that TSP/a2d-1 may induce downstream Rho GTPase signaling pathways in order to exert effects on actin dynamics in filopodia, representing a potential molecular mechanism for TSP/a2d-1-induced synapse formation. Like TSP, the matricellular protein hevin is prosynaptogenic both in cultured neurons and in vivo. SPARC, which shares a high degree of homology with hevin, is specifically antagonistic to hevin's synaptogenic effects. The second goal of the proposed study is to determine how the balance between hevin and SPARC affects the maturation of CNS synapses. Preliminary results from serial section electron microscopy (EM) revealed a dramatic phenotype in hevin knockout (KO) mice whereby postsynaptic spines were largely absent from dendrites, replaced instead by immature filopodia-like protrusions. Furthermore, excitatory synapses were primarily made onto the dendritic shaft rather than at the tips of protrusions. Based on these findings, here I will test the hypotheses that a2d-1 promotes the initiation of excitatory synaptic contacts during the dendritic filopodial stage of development, while precise regulation of the levels of hevin and SPARC controls the stabilization and maturation of synapses. To achieve this objective, I propose two specific aims: 1) how do TSP and a2d-1 regulate the initial phase of excitatory synapse formation? 2) How do hevin and SPARC regulate the morphological and functional maturation of excitatory synapses? Through a combination of live confocal imaging, serial section EM and electrophysiology in both purified cortical neurons and slice preparations from visual cortex, the proposed work should provide new molecular insight into the role of astrocytes in excitatory synaptogenesis during development. Better understanding of TSP, a2d-1, hevin and SPARC may lead to improvements in the treatment of neurological diseases such as schizophrenia and autism which are characterized by aberrant synaptic connectivity.