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

• 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
• 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; 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

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.

项目摘要/摘要 目前批准的中风疗法包括未充分利用，限时的，全身溶栓和机械性的 重新制定选项。因此，需要采用更实惠和灵活的治疗方法 可以广泛传播。一种方法包括靶向强有力表达的先天途径，例如 局灶性脑缺血后激活的Toll样受体（TLR）信号通路。缺乏 关于由途径激活的下游途径的时间和细胞特异性的知识 神经血管单元的星形胶质细胞和其他成分中的TLR4。在此提案中，我们将确定钥匙 星形胶质细胞TLR4信号的下游靶标和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，脑屏障通透性（BBB）和神经行为结局。我们 将使用临床上相关的内源性危险相关的分子模式（DAMP），HMGB1，已知 TLR4配体确定星形胶质细胞TLR4信号传导中的钥匙下游靶标。我们还将在体外使用 缺血模型，例如氧葡萄糖剥夺（OGD），以确定依赖TLR4 星形胶质细胞中其他潮湿激活的下游途径。下游效应器的表征 星形胶质细胞TLR4信号传导对降低BBB渗透性和次级大脑具有重要意义 中风后的损害和改善结果。该提议的总体假设是中风引起的， 星形胶质细胞特异性TLR4信号传导在中风的急性阶段诱导BBB破坏，并抑制 星形胶质细胞中与缺血相关的潮湿-TLR4信号传导将降低急性局灶性的BBB渗透率 脑缺血并改善行为结果。该中心假设将在以下测试中进行检验 目的：目标1：我们将确定TLR4反应和TLR4无反应性的信号传导途径 使用脑动脉闭塞模型（MCAO）和转录组学的半星形胶质细胞随后 急性脑缺血目标2）使用HMGB1刺激培养的星形胶质细胞和一个体外模型 脑缺血，OGD，我们将通过蛋白质印迹鉴定星形胶质细胞中TLR4信号的下游靶标 和磷蛋白质组学。 AIM 3：使用具有诱导型，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渗透性和神经行为结果的影响 跟随MCAO。在这些研究结束时，我们将更好地了解分子机制 这是星形胶质细胞中TLR4信号传导的基础。这些研究的结果将为 开发新型治疗剂，可以减少中风后脑损伤。