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.

PROJECT NARRATIVE The blood-brain barrier (BBB) formed by the cerebrovascular endothelial cells is necessary for normal brain function. Endothelial barrier opening and the consequent BBB disruption occurs in many brain diseases, leading to leakage of blood components into the brain, edema formation, and potentially death. Genetic deletion of the newly identified protein Poldip2 protects against endothelial barrier opening and brain edema formation, and this proposal investigates the underlying cellular and molecular mechanisms with a goal towards developing new therapies.