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

• 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
• 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

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.

内容和摘要 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）。血脑屏障在中枢神经系统稳态功能中起主要作用， 星形胶质细胞血脑屏障导致一系列神经系统疾病，从神经发育和 从退行性疾病到CNS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。 在健康和诸如中风的疾病中，BBB完整性的潜在维持仍然不清楚。 该提案的总体目标是阐明新的目标和相关的信号通路， 正常的星形胶质细胞-BBB功能，并利用这些知识来定义BBB破坏的关键机制 缺血性脑卒中后这项建议的重点是星形胶质细胞富集的钠-碳酸氢盐协同转运蛋白 1，Slc 4a 4，这是以前确定为细胞内和细胞外pH的胶质细胞特异性调节剂。 虽然pH稳态对脑功能至关重要，但携带Slc 4a 4变体的患者可能患有缺血性脑损伤， 在中风中，Slc 4a 4在星形胶质细胞-BBB完整性中的调节作用仍然未知。开始解决这个问题 在知识空白的情况下，我们产生了暂时消融星形胶质细胞中的Slc 4a 4的新的转基因小鼠系。使用此 在遗传小鼠模型中，我们发现Slc 4a 4的缺失显著降低了星形胶质细胞的形态复杂性 并产生具有破坏的内皮连接的扩大的血管。我们对星形胶质细胞 Slc 4a 4缺陷小鼠和Slc 4a 4消融星形胶质细胞的条件培养基显示CCL 2产生增加 和分泌再加上失调的精氨酸-一氧化氮（NO）代谢，进一步支持了关键作用， 对于Slc 4a 4在星形胶质细胞-BBB完整性中的作用。使用缺血性中风小鼠模型，我们发现Slc 4a 4的缺失加剧了 中风引起的运动功能障碍，增加梗死面积，再加上受损的反应性神经胶质增生， 在BBB破坏中，其通过在体内抑制CCL 2来挽救。根据这些初步数据，我们的中央 假设Slc 4a 4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CCL 2。为了解决我们的假设，我们将首先确定 Slc 4a 4如何调节成年大脑中星形胶质细胞的形态和生理学（目标1）。第二，我们将确定 CCL 2在成人脑中Slc 4a 4依赖性BBB维持中的作用（Aim 2）。完成后，这些 研究将确定Slc 4a 4缺陷型星形胶质细胞是否以及如何影响解剖和生理 星形胶质细胞的性质，并将阐明如何Slc 4a 4调节星形胶质细胞的调节内皮细胞， 通过NO代谢的BBB完整性驱动成人大脑中的星形胶质细胞CCL 2信号传导。最后，我们将定义 Slc 4a 4通路在缺血性卒中后维持BBB完整性中的作用（目的3）。共同阐明了 Slc 4a 4通路可以揭示控制星形胶质细胞形态、生理和 分泌分子，它将促进我们对中风后星形胶质细胞-BBB相互作用的理解。