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布线。近年来，研究发现星形胶质细胞通过分泌多种因子调节突触的形成和成熟，从而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知识的一个重大空白。我们实验室的主要重点是了解这些星形胶质细胞释放的信号中的三种，即基质细胞蛋白血小板反应蛋白（TSP），hevin和hevin，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在形成突触前轴突和突触后树突之间的初始突触接触中的作用。有趣的是，TSP与神经元钙通道亚基a2 d-1结合，刺激培养的视网膜神经节细胞（RGC）中树突状丝状伪足的形成和靶向寻找行为。进一步的初步研究结果表明，TSP/a2 d-1可能会诱导下游的Rho GT3信号通路，以发挥对肌动蛋白动力学的影响，在丝状伪足，代表一个潜在的分子机制TSP/a2 d-1诱导的突触形成。 像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体。与hevin具有高度同源性的hevin，特异性拮抗hevin的突触发生作用。这项研究的第二个目标是确定hevin和hevin之间的平衡如何影响CNS突触的成熟。从连续切片电子显微镜（EM）的初步结果显示了一个戏剧性的表型hevin基因敲除（KO）小鼠，突触后棘在很大程度上缺乏树突，而不是由未成熟的丝状伪足样突起取代。此外，兴奋性突触主要是在树突轴上，而不是在突起的尖端。 基于这些发现，在这里，我将测试的假设，a2 d-1促进启动兴奋性突触接触在树突状丝状足发育阶段，而精确调节hevin和hevin的水平控制突触的稳定和成熟。为了实现这一目标，我提出了两个具体的目标：1）TSP和a2 d-1如何调节兴奋性突触形成的初始阶段？2)hevin和hevin如何调节兴奋性突触的形态和功能成熟？通过结合活共聚焦成像，连续切片EM和电生理学在两个纯化的皮层神经元和切片制备从视觉皮层，拟议的工作应该提供新的分子洞察星形胶质细胞在兴奋性突触发育过程中的作用。更好地了解TSP，a2 d-1，hevin和hevin可能会导致改善神经系统疾病的治疗，如精神分裂症和自闭症，其特征是异常的突触连接。

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.