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

急性局灶性脑缺血后星形胶质细胞特异性 TLR4 信号传导和血脑屏障通透性

基本信息

批准号：
10572987
负责人：
Bolanle Famakin
金额：
$ 23.95万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-01 至 2028-02-29
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10572987
关键词：
Acute Adhesives Affect Antibodies Astrocytes Attenuated Award Blood - brain barrier anatomy Brain Brain Injuries Cell Survival Cells Cerebral Ischemia Complement Data Development Equipment Excision Extravasation Fluorescence Foundations Funding Gelatinase B Gene Expression Genetic Recombination Glucose Goals HMGB1 gene Hypoxia Immune Immune system Immunoglobulin G Immunohistochemistry Incubated Inflammation Ischemia Knowledge Lead Ligands Macrophage Mechanics Mediating Microglia Middle Cerebral Artery Occlusion Modeling Molecular Mus Natural Immunity Outcome Oxygen Pathway interactions Patients Pattern Permeability Phase Process Rattus Recombinants Research Role Serum Signal Induction Signal Pathway Signal Transduction Specificity Stains Stroke TLR4 gene Tamoxifen Testing Therapeutic Time Tissues Toll-like receptors Validation Vascular Permeabilities Walking Western Blotting behavioral outcome blood-brain barrier permeabilization career cerebral microvasculature clinically relevant deprivation flexibility foot immune activation immunoregulation improved improved outcome in vitro Model in vivo injured neurobehavioral neuron loss neurovascular neurovascular unit novel therapeutics phosphoproteomics post stroke response stroke outcome stroke therapy thrombolysis tissue injury transcriptome sequencing transcriptomics

项目摘要

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.

项目概要/摘要目前批准的中风疗法包括未充分利用的、有时间限制的、全身溶栓和机械疗法再通选项。因此，需要更实惠、更灵活的治疗方法可以广泛传播。一种方法包括针对稳健表达的先天途径，例如局灶性脑缺血后 Toll 样受体 (TLR) 信号通路激活。缺乏有关由以下途径激活的下游途径的时间和细胞特异性的知识星形胶质细胞和神经血管单元其他成分中的 TLR4。在本提案中，我们将确定关键星形胶质细胞 TLR4 信号传导的下游靶点以及星形胶质细胞特异性 TLR4 信号传导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，这是一种已知的 TLR4 配体确定星形胶质细胞 TLR4 信号传导的关键下游靶标。我们还将在体外使用缺血模型，例如氧糖剥夺 (OGD)，以确定 TLR4 依赖性星形胶质细胞中其他 DAMP 激活的下游通路。下游效应器的表征星形胶质细胞 TLR4 信号传导对降低 BBB 通透性和次级大脑具有重要意义损害和改善中风后的结果。该提案的总体假设是中风引起的，星形胶质细胞特异性 TLR4 信号传导在中风急性期诱导 BBB 破坏，并抑制星形胶质细胞中与缺血相关的 DAMP-TLR4 信号传导将降低急性局灶性血脑屏障通透性脑缺血并改善行为结果。这个中心假设将在下面进行检验目标：目标 1：我们将确定 TLR4 反应性和 TLR4 非反应性中活跃的信号传导途径使用大脑中动脉闭塞（MCAO）模型和转录组学研究半暗星形胶质细胞急性脑缺血目标 2) 使用 HMGB1 刺激培养的星形胶质细胞和体外模型脑缺血、OGD，我们将通过Western blot鉴定星形胶质细胞中TLR4信号传导的下游靶点和磷酸蛋白质组学。目标 3：使用可诱导的、星形胶质细胞特异性缺失 TLR4 的小鼠，我们将确定星形胶质细胞特异性 TLR4 缺失对 BBB 通透性和神经行为结果的影响继MCAO之后。在这些研究结束时，我们将对分子机制有更好的了解是星形胶质细胞中 TLR4 信号传导的基础。这些研究的结果将为开发可以减少中风后脑损伤的新疗法。