Poldip2 and the Brain Endothelial Barrier Function: Understanding Mechanisms that Regulate the Blood Brain Barrier Integrity

Poldip2 和脑内皮屏障功能：了解调节血脑屏障完整性的机制

• 批准号: 10658239
• 负责人: Marina Sorrentino Hernandes
• 金额: $ 40.9万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-21 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10658239
• 关键词: Actins; Adaptor Signaling Protein; Address; Affect; Anatomy; Animal Model; Area; Astrocytes; Blood; Blood - brain barrier anatomy; Blood brain barrier dysfunction; Brain; Brain Diseases; Brain Edema; Brain Ischemia; Cell physiology; Cell-Cell Adhesion; Cells; Central Nervous System; Central Nervous System Diseases; Cerebral Ischemia; Cerebral hemisphere hemorrhage; Cerebrovascular system; Cessation of life; Clinical; Cytoskeletal Modeling; Cytoskeleton; Data; Dependovirus; Development; Down-Regulation; Edema; Endothelial Cells; Endothelium; Event; Extravasation; Gene Expression; Genetic; Genetic Transcription; Goals; Hemorrhage; Heterozygote; Homeostasis; In Vitro; Inflammation; Knock-out; Knockout Mice; Leukocytes; Link; Maintenance; Malignant Neoplasms; Measures; Mediating; Molecular; Molecular Weight; Motor; Multiple Sclerosis; Mus; Myosin Light Chains; NADPH Oxidase; Outcome; Pathology; Pathway interactions; Pericytes; Permeability; Phenotype; Phosphorylation; Physiological; Plasma Proteins; Play; Polymerase; Process; Property; Proteins; Publishing; Reactive Oxygen Species; Regulation; Role; Sampling; Series; Signal Pathway; Stress Fibers; Stroke; Structure; Tertiary Protein Structure; Testing; Therapeutic; Tight Junctions; Tissues; Tracer; Traumatic Brain Injury; Tyrosine Phosphorylation; Vascular Permeabilities; Water Movements; blood-brain barrier disruption; blood-brain barrier function; blood-brain barrier permeabilization; brain endothelial cell; cell type; cerebrovascular; cytokine; disability; improved; in vivo; in vivo evaluation; insight; mortality; motor function improvement; neuroinflammation; neuropathology; neurotransmission; novel; novel therapeutics; prevent; response; rho; single-cell RNA sequencing; small hairpin RNA; therapeutic target

PROJECT SUMMARY The blood–brain barrier (BBB) is a term used to describe a series of properties possessed by the vasculature of the central nervous system (CNS) that play a critical role in maintaining a precisely regulated microenvironment for optimal neuronal signaling. The essential physiological functions of the BBB in supporting brain homeostasis are manifested within its most critical component, brain microvascular endothelial cells (ECs). Diminished EC barrier function and the consequent increase in vascular permeability significantly contribute to tissue damage, intracerebral hemorrhage and edema formation, as evidenced in pathologies of the CNS including stroke, cancer and brain trauma. We recently discovered that heterozygous global deletion of Polymerase δ-interacting protein 2 (Poldip2), a protein that regulates important fundamental processes, significantly protects against BBB disruption, edema and neuroinflammation induced by cerebral ischemia. While informative, these studies were performed in mice with a global deletion of Poldip2, an approach that prevented us from determining the mechanism of action and the specific cell types responsible for the observed phenotype. Our exciting new preliminary data suggests that knockout of Poldip2 specifically in the endothelium remarkably prevents BBB disruption after cerebral ischemia. Our in vitro studies further demonstrate that several mechanisms linked to EC barrier integrity are affected, including stress fiber formation and distribution of the key junctional adaptor protein ZO-1. Based on our preliminary studies, we hypothesize that Poldip2 regulates EC cytoskeleton organization, junctional stability via ZO-1, and cell-cell adhesion to mediate EC barrier function and BBB permeability following brain ischemia, and using our novel animal models will test the in vivo relevance and therapeutic potential of these observations. To test this hypothesis, in the first aim we will investigate the mechanisms by which Poldip2 regulates the actin cytoskeleton, RhoA activity, myosin light chain phosphorylation and brain EC gap formation in vivo and in vitro. In the second aim, we will explore a potential NADPH oxidase Nox4/reactive oxygen species- driven mechanism by which Poldip2 regulates the stability of tight junction proteins such as ZO-1 at areas of cell–cell contact in brain endothelial cells in vivo and in vitro. Finally, in the third aim, to increase the rigor of our proposed studies and to begin to define the potential therapeutic value of Poldip2 inhibition, we will investigate if inhibition of endothelial Poldip2 in vivo using adeno-associated virus will prevent BBB dysfunction and edema, resulting in improved motor function and survival after cerebral ischemia. We will also use single cell RNA sequencing on brain samples isolated from EC specific Poldip2 knockout mice to examine how Poldip2 depletion in ECs affects transcriptional and cellular responses in additional components of the BBB leading to the anti- permeability phenotype observed following cerebral ischemia. Completion of these three aims will shed new light into mechanisms involved in brain EC permeability and BBB dysfunction and will allow us to gain new insights into potential approaches for regulating cerebral vascular leakage in clinically meaningful ways.

项目摘要 血脑屏障（BBB）是一个用来描述脉管系统具有的一系列特性的术语 中枢神经系统（CNS）在维持精确调节的微环境中起着至关重要的作用 用于最佳神经元信号。 BBB在支持大脑稳态方面的基本生理功能 表现在其最关键的成分，脑微血管内皮细胞（EC）中。 EC减少 障碍功能和随之增加的血管通透性的增加显着导致组织损伤， 脑内出血和水肿形成，如中枢神经系统的病理，包括中风，癌症 和脑创伤。我们最近发现，聚合酶δ相互作用蛋白的杂合整体缺失 2（Poldip2），一种调节重要基本过程的蛋白质，可显着保护BBB 脑缺血引起的破坏，水肿和神经炎症。虽然信息丰富，但这些研究是 在poldip2全球删除的小鼠中进行的方法，这种方法阻止了我们确定 作用机理和负责观察到的表型的特定细胞类型。我们令人兴奋的新 初步数据表明，在原始植物中特别防止BBB敲除POLDIP2的敲除 脑缺血后的破坏。我们的体外研究进一步表明，几种与EC相关的机制 屏障完整性受到影响，包括压力纤维的形成和关键连接适配器蛋白的分布 ZO-1。根据我们的初步研究，我们假设Poldip2调节EC细胞骨架组织， 通过ZO-1的连接稳定性和细胞细胞粘合剂介导EC屏障功能和BBB渗透性之后 脑缺血，使用我们的新型动物模型将测试体内相关性和治疗潜力 这些观察。为了检验这一假设，在第一个目的中，我们将研究poldip2的机制 调节肌动蛋白细胞骨架，RhoA活性，肌球蛋白轻链磷酸化和脑EC间隙形成 体内和体外。在第二个目标中，我们将探索潜在的NADPH氧化物NOX4/活性氧气 - POLDIP2调节紧密连接蛋白（例如ZO-1）的驱动机制，在 细胞 - 细胞在体内和体外的脑内皮细胞中的接触。最后，在第三个目标中，增加了我们的严谨性 拟议的研究并开始定义Poldip2抑制的潜在治疗价值，我们将研究 如果使用腺相关病毒抑制体内内皮POLDIP2在体内将防止BBB功能障碍和水肿，则 导致大脑缺血后的运动功能和存活率提高。我们还将使用单细胞RNA 从EC特定Poldip2基因敲除小鼠中分离出的大脑样品的测序，以检查POLDIP2的部署 在EC中，EC会影响BBB的其他成分的转录和细胞反应，导致抗 脑缺血后观察到的渗透性表型。完成这三个目标的完成将使新的光芒 进入涉及大脑EC渗透性和BBB功能障碍的机制，并使我们能够获得新的见解 进入以临床意义的方式控制脑血管泄漏的潜在方法。