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）是用于描述脑血管系统所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发挥关键作用 最佳的神经信号。血脑屏障在维持脑内环境稳定中的重要生理功能 表现在其最关键的组成部分，脑微血管内皮细胞（EC）。EC降低 屏障功能和随后的血管渗透性的增加显著地导致组织损伤， 脑内出血和水肿形成，如CNS病理学所证明，包括中风、癌症 和脑外伤我们最近发现聚合酶δ相互作用蛋白的杂合性全缺失， 2（Poldip 2）是一种调节重要基础过程的蛋白质，可显著保护BBB 脑缺血引起的破坏、水肿和神经炎症。虽然信息量大，但这些研究 在Poldip 2整体缺失的小鼠中进行，这种方法阻止了我们确定 作用机制和负责观察到的表型的特定细胞类型。我们激动人心的新闻 初步数据表明，特异性敲除内皮细胞中的Poldip 2可显著防止BBB， 脑缺血后破坏。我们的体外研究进一步证明了与EC相关的几种机制， 屏障完整性受到影响，包括应力纤维的形成和关键连接接头蛋白的分布 ZO-1基于我们的初步研究，我们假设Poldip 2调节EC细胞骨架组织， 通过ZO-1的连接稳定性和细胞-细胞粘附来介导EC屏障功能和BBB通透性， 脑缺血，并使用我们的新的动物模型将测试在体内的相关性和治疗潜力， 这些观察。为了验证这一假设，在第一个目标中，我们将研究Poldip 2 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 在体内和体外。在第二个目标中，我们将探索潜在的NADPH氧化酶Nox 4/活性氧- Poldip 2调节紧密连接蛋白如ZO-1在细胞膜区域的稳定性的驱动机制。 在体内和体外脑内皮细胞中的细胞-细胞接触。最后，在第三个目标中，为了提高我们的 为了开始确定Poldip 2抑制的潜在治疗价值，我们将研究 如果使用腺相关病毒在体内抑制内皮Poldip 2将防止BBB功能障碍和水肿， 从而改善运动功能和脑缺血后的存活率。我们也将使用单细胞RNA 对分离自EC特异性Poldip 2敲除小鼠的脑样品进行测序，以检查Poldip 2缺失如何影响脑细胞的发育。 影响BBB其他成分的转录和细胞反应，导致抗- 脑缺血后观察到的通透性表型。这三个目标的完成将为我们提供新的启示。 研究脑EC通透性和BBB功能障碍的机制，将使我们获得新的见解 以有临床意义的方式调节脑血管渗漏的潜在方法。