Astrocyte-specific TLR4 signaling and Blood Brain Barrier permeability following acute focal cerebral ischemia

项目摘要/摘要 目前批准的中风治疗方法包括未充分利用、有时间限制的、全身溶栓和机械治疗 再通选项。因此，需要更负担得起和更灵活的治疗方法， 可以广泛传播。一种方法包括靶向强势表达的先天途径，如 Toll样受体(TLR)信号通路在局灶性脑缺血后被激活。缺少这样一个人 关于下游通路的时间和细胞特异性的知识，这些通路由以下途径激活 TLR4在星形胶质细胞和神经血管单位的其他成分中。在这份提案中，我们将确定关键 星形胶质细胞TLR4信号的下游靶点及星形胶质细胞特异性TLR4信号对血液的影响 急性局灶性脑缺血后的脑屏障通透性(BBB)和神经行为结果。我们 将使用临床相关的内源性危险相关分子模式(DAMP)，HMGB1，一种已知的 TLR4配体确定星形胶质细胞TLR4信号的关键下游靶点。我们还将在体外使用 缺血模型，如氧糖剥夺(OGD)，确定TLR4依赖 在星形胶质细胞中，下游通路被其他阻滞剂激活。下游效应器的特征 星形胶质细胞TLR4信号转导对降低血脑屏障通透性和继发性脑损伤有重要意义 中风后的损害和改善预后。这项提议的总体假设是中风引起的， 星形胶质细胞特异性TLR4信号诱导卒中急性期血脑屏障紊乱及其抑制作用 星形胶质细胞中与缺血相关的DAMP-TLR4信号将降低急性局灶性脑损伤后血脑屏障通透性 脑缺血和改善行为结果。这一中心假设将在以下方面得到检验 目标：目标1：我们将确定在TLR4反应性和非反应性中活跃的信号通路 大脑中动脉闭塞半暗带星形胶质细胞模型的建立及转录 目的2)用HMGB1刺激培养的星形胶质细胞建立急性脑缺血模型 脑缺血，OGD，我们将通过Western印迹鉴定星形胶质细胞TLR4信号的下游靶点 和磷酸蛋白质组学。目的3：使用带有可诱导的星形胶质细胞特异性TLR4缺失的小鼠，我们将 确定星形胶质细胞特异性TLR4缺失对血脑屏障通透性和神经行为结局的影响 在MCAO之后。在这些研究的最后，我们将对分子机制有更好的了解。 这是星形胶质细胞中TLR4信号的基础。这些研究的结果将为 开发可减少中风后脑损伤的新疗法。

PROJECT SUMMARY/ABSTRACT Currently approved stroke therapies include underutilized, time-limited, systemic thrombolysis and mechanical recanalization options. Therefore, there is a need for more affordable and flexible approaches to treatment that can be disseminated widely. One approach includes targeting robustly expressed innate pathways such as the Toll-like Receptor (TLR) signaling pathways activated after focal cerebral ischemia. There is a lack of knowledge regarding the timing and cellular specificity of downstream pathways activated by pathways such as TLR4 in astrocytes and other components of the neurovascular unit. In this proposal, we will determine the key downstream targets in astrocyte TLR4 signaling and the effect of astrocyte-specific TLR4 signaling on blood brain barrier permeability (BBB) and neurobehavioral outcomes following acute focal cerebral ischemia. We will use a clinically relevant, endogenous danger associated molecular pattern (DAMP), HMGB1, a known TLR4 ligand to determine the key downstream targets in astrocyte TLR4 signaling. We will also use in vitro models of ischemia, such as Oxygen Glucose Deprivation (OGD), to determine the TLR4-dependent downstream pathways activated by other DAMPs in astrocytes. Characterization of the downstream effectors of astrocyte TLR4 signaling has important implications for decreasing BBB permeability and secondary brain damage and improving outcomes after stroke. The overall hypothesis of this proposal is that stroke-induced, astrocyte-specific TLR4 signaling induces BBB disruption in the acute phase of stroke, and that inhibiting ischemia-relevant DAMP-TLR4 signaling in astrocytes will decrease BBB permeability following acute focal cerebral ischemia and improve behavioral outcomes. This central hypothesis will be tested in the following aims: Aim 1: We will determine the signaling pathways active in TLR4-reactive and TLR4 non-reactive penumbral astrocytes using a model of middle cerebral artery occlusion (MCAO) and transcriptomics following acute cerebral ischemia Aim 2) Using HMGB1 stimulation of cultured astrocytes and an in vitro model of cerebral ischemia, OGD, we will identify downstream targets of TLR4 signaling in astrocytes via Western blot and phosphoproteomics. Aim 3: Using mice with inducible, astrocyte-specific deletion of TLR4, we will determine the effect of astrocyte-specific TLR4 deletion on BBB permeability and neurobehavioral outcomes following MCAO. At the end of these studies, we will have a better understanding of the molecular mechanisms that underlie TLR4 signaling in astrocytes. Results from these studies will lay the foundation for the development of novel therapeutics that can decrease brain damage after stroke.