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

项目总结 血脑屏障(BBB)是一个术语，用来描述血管系统具有的一系列特性 中枢神经系统(CNS)在维持精确调节的微环境中起着关键作用 以获得最佳的神经元信号。血脑屏障在维持脑内稳态中的基本生理功能 表现在其最关键的组成部分--脑微血管内皮细胞(ECs)。缩减的EC 屏障功能和随之而来的血管通透性增加显著地促进了组织损伤， 脑内出血和水肿的形成，在包括中风、癌症在内的中枢神经系统的病理中得到证实 和脑部创伤。我们最近发现，聚合酶δ相互作用蛋白的杂合性全局缺失 2(Poldip2)，一种调节重要基本过程的蛋白质，显著防止血脑屏障 脑缺血所致的脑损伤、脑水肿和神经炎症。虽然这些研究提供了信息，但它们 在Poldip2基因全局缺失的小鼠身上执行，这种方法阻止了我们确定 作用机制和对观察到的表型负责的特定细胞类型。我们激动人心的新消息 初步数据表明，在内皮细胞中特异地敲除Poldip2可以显著防止血脑屏障 脑缺血后的脑电中断。我们的体外研究进一步证明了几种与EC有关的机制 屏障的完整性受到影响，包括应激纤维的形成和关键连接接头蛋白的分布 ZO-1。根据我们的初步研究，我们假设Poldip2调控EC细胞骨架组织， 通过ZO-1的连接稳定性，以及细胞-细胞黏附介导EC屏障功能和BBB通透性 脑缺血，并使用我们的新动物模型将测试体内相关性和治疗潜力 这些观察结果。为了验证这一假设，在第一个目标中，我们将研究Poldip2 调节肌动蛋白细胞骨架、RhoA活性、肌球蛋白轻链磷酸化和脑内皮细胞间隙形成 在体内和体外。在第二个目标中，我们将探索一种潜在的NADPH氧化酶NOX4/活性氧物种- Poldip2调控紧密连接蛋白稳定性的驱动机制 体内和体外脑内皮细胞的细胞-细胞接触。最后，在第三个目标中，增加我们的严谨 拟议的研究，并开始确定Poldip2抑制的潜在治疗价值，我们将调查 如果在体使用腺相关病毒抑制内皮细胞Poldip2可以防止血脑屏障功能障碍和水肿， 从而改善脑缺血后的运动功能和存活率。我们还将使用单细胞RNA EC特异性Poldip2基因敲除小鼠脑样本的测序研究Poldip2是如何耗尽的 在ECs中影响BBB附加成分的转录和细胞反应，从而导致抗- 脑缺血后观察到的通透性表型。完成这三个目标将带来新的曙光。 研究涉及脑内皮细胞通透性和血脑屏障功能障碍的机制，并将使我们获得新的见解 探索以临床有意义的方式调节脑血管渗漏的潜在方法。

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.