TrkB.T1 signaling in astrocytes

星形胶质细胞参与了中枢神经系统(CNS)生理的许多方面，包括调节 神经递质和钾离子浓度、突触发育和突触稳定。这些 星形胶质细胞的功能主要在远端、细小、外周星形胶质细胞突起(PAPs)中起调节作用。正是在这些过程中， 星形胶质细胞与它们的邻居交流，调节离子和神经递质水平，并参与突触 发展稳定。尽管几十年的研究表明星形胶质细胞包裹或接触兴奋性 和抑制性突触成分，随着成熟突触覆盖率的增加，关于 将星形胶质细胞PAPs招募到突触结构的信号。我们生成的RNA测序数据(和 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。异构体特异性鉴定表明星形胶质细胞主要表达截短形式TrkB.T1。 在大脑皮层，TrkB.T1几乎只存在于星形胶质细胞中。全球和星形胶质细胞特异的基因缺失 TrkB.T1导致星形胶质细胞的体积和分支复杂性显著减少。缺少星形胶质细胞 TrkB.T1显示与成熟星形胶质细胞功能相关的两个突触周围基因的异常表达 和促突触基因。我们在体外和体内观察到TrkB.T1 KO星形胶质细胞不支持正常 通过评估突触前和突触后兴奋性成分来评估兴奋性突触发生或功能 神经元mEPSC分析。初步的体外数据也表明TrkB.T1 KO星形胶质细胞不能包裹 谷氨酸能突触，我们很容易在WT星形胶质细胞中观察到的一种表型。在当前的提案中，我们测试 假设BDNF通过星形细胞TrkB.T1受体作为一条关键的信号通路在 星形胶质细胞突触周围突触向谷氨酸能突触募集，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来 确定星形胶质细胞中的BDNF/TrkB.T1信号对星形胶质细胞的结构可塑性和功能是否必要 谷氨酸能突触(目标1)。我们评估星形胶质细胞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相结合的方法来确定关键信号转导 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信号与肌动蛋白细胞骨架的分子机制框架 重组、形态细化、突起生长和突触包裹。这些研究确定了 一种全新的星形胶质细胞结构可塑性信号通路 促进我们对星形胶质细胞-突触相互作用的理解。而中断的BDNF/TrkB信号是 BDNF/星形胶质细胞TrkB.T1信号与许多中枢神经系统疾病有关，但尚未被考虑。

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.