TrkB.T1 signaling in astrocytes

星形胶质细胞有助于“正常”中枢神经系统（CNS）生理学的许多方面，包括调节 神经递质和K+离子浓度、突触发育和突触稳定性。这些 这些功能主要在远端、精细的外周星形胶质细胞过程（PAP）中介导。正是在这些过程中， 星形胶质细胞与它们的邻居沟通，调节离子和神经递质水平，并有助于突触 发展和稳定。尽管几十年的研究表明星形胶质细胞包裹或接触兴奋性 和抑制性突触元件，随着成熟突触覆盖率的增加， 将星形胶质细胞PAP募集到突触结构的信号。我们生成的RNA测序数据（以及 使用多种公共资源证实）表明星形胶质细胞表达非常高水平的BDNF受体， TrkB。同种型特异性鉴定表明星形胶质细胞主要表达截短形式TrkB.T1。 在皮质中，TrkB.T1几乎仅在星形胶质细胞中发现。整体和星形胶质细胞特异性基因缺失 TrkB.T1导致星形胶质细胞的体积和分支复杂性显著降低。星形胶质细胞缺乏 TrkB.T1显示与成熟星形胶质细胞功能相关的两种突触周基因的表达失调 和促突触基因。在体外和体内，我们观察到TrkB.T1 KO星形胶质细胞不支持正常的 通过评价突触前和突触后兴奋性元件评估兴奋性突触发生或功能 和神经元mEPSC分析。初步的体外数据还表明TrkB.T1 KO星形胶质细胞不能包裹 神经元能突触，我们在WT星形胶质细胞中容易观察到的表型。在目前的提案中，我们测试了 假设BDNF信号通过星形胶质细胞TrkB.T1受体作为一个关键的信号通路， 募集星形胶质细胞突触周突起形成神经元突触，从而促进肌动蛋白介导的结构 可塑性在目前的工作中，我们使用WT和星形胶质细胞特异性TrkB.T1 KO小鼠的超微结构成像， 确定星形胶质细胞中的BDNF/TrkB.T1信号传导是否是星形胶质细胞结构可塑性和功能所必需的。 神经元突触（Aim 1）。我们评估了星形胶质细胞TrkB. T1的缺失对神经元突触发育的影响， 和功能（目标2），我们使用体外和体内方法的组合来识别关键信号传导 BDNF与星形胶质细胞TrkB.T1受体结合的机制参与下游信号传导机制， 提供连接星形胶质细胞BDNF/TrkB.T1信号传导至肌动蛋白细胞骨架的分子机制框架 重组、形态学细化、突起生长和突触包裹。这些研究表明， 星形胶质细胞结构可塑性中的一种全新的信号通路，并有可能显着 推进我们对星形胶质细胞-突触相互作用的理解。虽然破坏BDNF/TrkB信号转导是 在许多CNS疾病中涉及BDNF/星形胶质细胞TrkB.T1信号传导的相关性尚未被考虑。

Astrocytes contribute to many facets of ‘normal’ central nervous system (CNS) physiology, including regulation of neurotransmitters and K+ ions concentration, synaptic development, and synapse stabilization. These functions are largely mediated at distal, fine, peripheral astrocyte processes (PAPs). It is at these processes that astrocytes communicate with their neighbors, regulate ion and neurotransmitter levels and contribute to synapse development and stabilization. Despite decades of research indicating astrocytes enwrap or contact excitatory and inhibitory synaptic elements, with increased coverage of mature synapses, there is little known regarding signals that recruit astrocyte PAPs to synaptic structures. RNA sequencing data we have generated (and confirmed using multiple public resources) indicate astrocytes express very high levels of the BDNF receptor, TrkB. Isoform specific identification demonstrates astrocytes predominately express the truncated form, TrkB.T1. In cortex, TrkB.T1 is found almost exclusively in astrocytes. Global and astrocyte specific genetic deletion of TrkB.T1 results in astrocytes with significantly reduced volume and branching complexities. Astrocytes lacking TrkB.T1 show dysregulated expression of both perisynaptic genes associated with mature astrocyte function and pro-synaptogenic genes. In vitro and in vivo we observed that TrkB.T1 KO astrocytes do not support normal excitatory synaptogenesis or function as assessed by evaluation of pre and post synaptic excitatory elements and neuronal mEPSC analysis. Preliminary in vitro data also indicate that TrkB.T1 KO astrocytes fail to enwrap glutamatergic synapses, a phenotype we readily observe in WT astrocytes. In the current proposal we test the hypothesis that BDNF signaling through the astrocytic TrkB.T1 receptor serves as a key signaling pathway in recruiting astrocyte perisynaptic processes to glutamatergic synapses thus facilitating actin mediated structural plasticity. In the current work we use ultrastructural imaging in WT and astrocyte specific TrkB.T1 KO mice to determine if BDNF/TrkB.T1 signaling in astrocytes is necessary for astrocyte structural plasticity and function at glutamatergic synapses (Aim 1). We evaluate the loss of astrocyte TrkB.T1 on neuronal synapse development and function (Aim 2) and we use a combination of in vitro and in vivo approaches to identify the key signaling mechanisms by which BDNF binding to astrocyte TrkB.T1 receptors engage downstream signaling mechanisms, providing a molecular mechanistic framework linking astrocyte BDNF/TrkB.T1 signaling to actin cytoskeletal reorganization, morphological refinement, process outgrowth and synapse enwrapment. These studies identify a completely novel signaling pathway in astrocyte structural plasticity and have the potential to significantly advance our understanding of astrocyte-synapse interactions. While disrupted BDNF/TrkB signaling is implicated in many CNS disorders the relevance of BDNF/astrocytic TrkB.T1 signaling has not been considered.