Project Summary The central nervous system (CNS) is protected by two major barrier systems, the blood brain-barrier (BBB) and the blood-cerebrospinal fluid barrier (B-CSFB). These essential barrier systems each have unique cellular properties that tightly regulate the molecules and cells that can enter (or exit) the brain and the cerebrospinal fluid (CSF). CNS barriers are essential for development and health but are vulnerable to breakdown in a variety of diseases, causing or exacerbating CNS pathology. CNS barriers are also an impediment to delivery of therapies to treat disease. The development and function of the B-CSFB at the level of the meninges, a trilayered structure that surrounds the brain and spinal cord, is poorly understood. This is despite evidence implicating the meninges as an early site of immune cell entry into the CNS in neuroinflammatory diseases and the growing recognition of brain waste removal systems where waste must traverse the B-CSFB before removal via the meningeal lymphatics. One of two barrier structures in the meninges is the arachnoid barrier, a tight junction containing epithelial- like layer that segregates the outer meningeal dura, which contains non-barrier blood vasculature, from CSF in the subarachnoid space. Unlike the BBB and other parts of the B-CSFB, there is nothing known about the developmental program underlying arachnoid barrier formation. Further, only a few studies have looked at arachnoid barrier function and breakdown in CNS diseases, the conclusions of which were limited to descriptive studies. We have combined our knowledge of CNS vascular and BBB development with our unique expertise in the meninges to develop several new tools to study arachnoid barrier development and function. We will apply these new tools to study an animal model of a disease with high relevance to the meninges and the arachnoid barrier, bacterial meningitis. Experiments proposed here will identify mechanisms that underlie arachnoid barrier cell development and maintenance, investigate arachnoid barrier function, and measure its response to insult. To do this we will: 1) utilize in vivo and culture models to uncover the cellular and molecular mechanisms of arachnoid barrier specification and maturation, 2) use our new model where we perturb arachnoid barrier formation to determine its role in establishing separate meningeal and immune and vascular compartments 3) identify the cellular and molecular mechanisms of arachnoid barrier breakdown in bacterial meningitis. Completion of this work will substantially advance the field of CNS barrier systems by providing new tools to study arachnoid barrier function. We will generate a comprehensive model of arachnoid barrier cellular properties that can be investigated for breakdown in other diseases that involve the meninges. This new knowledge about the arachnoid barrier has the potential to be exploited to design new ways to limit crossing of molecules and cells at the arachnoi...