Deciphering Mechanisms of Astrocyte-BBB Interaction in Normal and Ischemic Stroke

解读正常和缺血性中风中星形胶质细胞-BBB相互作用的机制

基本信息

批准号：
10585849
负责人：
Hyun Kyoung Lee
金额：
$ 45.68万
依托单位：
BAYLOR COLLEGE OF MEDICINE
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：
2023-01-01 至 2027-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10585849
关键词：
Ablation Address Adult Anatomy Area Arginine Astrocytes Blood - brain barrier anatomy Blood Vessels Brain Brain Injuries Brain region CCL2 gene Cause of Death Cell Communication Cell physiology Cells Cerebral Ischemia Conditioned Culture Media Coupled Data Degenerative Disorder Disease Endothelial Cells Endothelium Exhibits Extravasation Genetic Gliosis Goals Health Homeostasis Impairment In Vitro Infarction Intercellular Junctions Ischemic Stroke Knowledge Maintenance Malignant Neoplasms Mediating Metabolic Metabolism Molecular Morphology Mus Neurodevelopmental Disorder Neuroglia Oxides Paracrine Communication Pathogenesis Pathologic Pathway interactions Patients Physiological Physiology Play Production Property Role Signal Pathway Signal Transduction Sodium-Bicarbonate Symporters Stroke Testing Time Transgenic Mice Variant awake blood-brain barrier disruption blood-brain barrier function cell type central nervous system injury chemokine cytokine extracellular gain of function in vivo loss of function motor disorder mouse model multiple omics nervous system disorder novel pH Homeostasis post stroke prevent response stroke patient stroke risk

项目摘要

SUMMARY AND ABSTRACT Astrocytes are the most diverse glial cell type and maintain essential interactions with endothelial cells to form the blood-brain barrier (BBB). The BBB plays a major role in CNS homeostatic function, while dysregulation of the astrocytic BBB contributes to a spectrum of neurological disorders, ranging from neurodevelopmental and degenerative diseases to CNS injury and malignancy. Particularly, pathologic astrocyte-BBB interactions contribute to ischemic stroke, which is the 5th leading cause of death in the U.S. However, the mechanisms underlying maintenance of BBB integrity, both in health and in diseases such as stroke, remain poorly defined. The overarching goal of this proposal is to elucidate novel targets and associated signaling pathways influencing normal astrocyte-BBB function and to leverage this knowledge to define key mechanisms for BBB disruption after ischemic cerebral stroke. This proposal focuses on an astrocyte-enriched sodium-bicarbonate cotransporter 1, Slc4a4, which was previously identified as a glial-specific regulator of both intracellular and extracellular pH. While pH homeostasis is essential for brain function and patients carrying Slc4a4 variants can suffer ischemic stroke, a regulatory role of Slc4a4 in astrocyte-BBB integrity remains unknown. To begin addressing this knowledge gap, we generated new transgenic mouse lines that temporally ablate Slc4a4 in astrocytes. Using this genetic mouse model, we showed that loss of Slc4a4 significantly reduces astrocytic morphological complexity and generates enlarged blood vessels with disrupted endothelial junctions. Our profiling analyses of astrocytic Slc4a4-deficient mice and conditioned media of Slc4a4 ablated astrocytes revealed increased CCL2 production and secretion coupled with dysregulated arginine-nitro oxide (NO) metabolism, further supporting a crucial role for Slc4a4 in astrocyte-BBB integrity. Using an ischemic stroke mouse model, we found loss of Slc4a4 exacerbates stroke-induced motor dysfunction and increases infarct area coupled with impaired reactive gliosis which results in BBB disruption, which is rescued by inhibition of CCL2 in vivo. Based on these preliminary data, our central hypothesis is that Slc4a4 functions to maintain astrocyte-BBB interactions and prevent progressive BBB leakage in ischemic stroke, in part by inhibiting the chemokine CCL2. To address our hypothesis, we will first determine how Slc4a4 regulates astrocyte morphology and physiology in the adult brain (Aim 1). Second, we will determine the role of CCL2 in Slc4a4-dependent BBB maintenance in the adult brain (Aim 2). Upon completion, these studies will establish whether and how Slc4a4-deficient astrocytes influence anatomical and physiological properties of astrocytes, and will elucidate how Slc4a4 regulates astrocytic modulation of endothelial cell and BBB integrity via NO metabolism that drive astrocytic CCL2 signaling in the adult brain. Lastly, we will define the Slc4a4 pathway in the maintenance of BBB integrity following ischemic stroke (Aim 3). Together, elucidating the Slc4a4 pathway could reveal fundamental mechanisms controlling astrocyte morphology, physiology, and secreted molecules, and it will advance our understanding of astrocyte-BBB interactions after stroke.

总结和摘要星形胶质细胞是最多样化的神经胶质细胞类型，并与内皮细胞保持必要的相互作用以形成血脑屏障（BBB）。 BBB 在 CNS 稳态功能中发挥着重要作用，而 BBB 的失调星形细胞 BBB 会导致一系列神经系统疾病，包括神经发育和退行性疾病导致中枢神经系统损伤和恶性肿瘤。特别是病理性星形胶质细胞-BBB 相互作用导致缺血性中风，这是美国第五大死亡原因。无论是在健康还是在中风等疾病中，血脑屏障完整性的基本维持仍然不明确。该提案的总体目标是阐明影响的新靶点和相关信号通路正常星形胶质细胞-BBB 功能，并利用这些知识来定义 BBB 破坏的关键机制缺血性脑中风后。该提案重点关注富含星形胶质细胞的碳酸氢钠协同转运蛋白 1，Slc4a4，之前被鉴定为细胞内和细胞外 pH 值的神经胶质特异性调节剂。虽然 pH 稳态对于大脑功能至关重要，但携带 Slc4a4 变异的患者可能会遭受缺血 Slc4a4 在星形胶质细胞-BBB 完整性中的调节作用仍不清楚。要开始解决这个问题由于知识差距，我们产生了新的转基因小鼠品系，可以暂时消除星形胶质细胞中的 Slc4a4。使用这个在遗传小鼠模型中，我们发现 Slc4a4 的缺失显着降低了星形胶质细胞的形态复杂性并产生扩张的血管和破坏的内皮连接。我们对星形胶质细胞的概况分析 Slc4a4 缺陷小鼠和 Slc4a4 消除星形胶质细胞的条件培养基显示 CCL2 产量增加和分泌加上精氨酸一氧化氮（NO）代谢失调，进一步支持了关键作用星形胶质细胞-BBB 完整性中的 Slc4a4。使用缺血性中风小鼠模型，我们发现 Slc4a4 的缺失会加剧病情中风引起的运动功能障碍并增加梗塞面积，再加上反应性神经胶质增生受损，从而导致在 BBB 破坏中，通过体内抑制 CCL2 可以挽救这种破坏。根据这些初步数据，我们的中央假设 Slc4a4 的功能是维持星形胶质细胞 - BBB 相互作用并防止进行性 BBB 渗漏在缺血性中风中，部分通过抑制趋化因子 CCL2 来实现。为了解决我们的假设，我们首先确定 Slc4a4 如何调节成人大脑中的星形胶质细胞形态和生理学（目标 1）。其次，我们将确定 CCL2 在成人大脑中 Slc4a4 依赖性 BBB 维持中的作用（目标 2）。完成后，这些研究将确定 Slc4a4 缺陷型星形胶质细胞是否以及如何影响解剖学和生理学星形胶质细胞的特性，并将阐明 Slc4a4 如何调节内皮细胞和星形胶质细胞的调节通过 NO 代谢维持 BBB 完整性，驱动成人大脑中的星形胶质细胞 CCL2 信号传导。最后，我们将定义 Slc4a4 通路在缺血性中风后维持 BBB 完整性中的作用（目标 3）。共同阐明 Slc4a4 通路可以揭示控制星形胶质细胞形态、生理和功能的基本机制分泌分子，它将促进我们对中风后星形胶质细胞与血脑屏障相互作用的理解。