Project Summary: The blood-brain barrier (BBB) is the cellular barrier lining the lumen of blood vessels in the brain. Adhesive molecules attaching these endothelial cells (ECs) together protects the brain from toxic substances in the blood. Underneath the EC barrier exists a complex layer of proteins known as the extracellular matrix (ECM), or basal lamina. ECM proteins anchor ECs to the tissue, and transmit signals into ECs to regulate certain functions. One such function is enhancement of adhesion molecules between endothelial cell-cell (EC-EC) contacts known as tight junctions (TJs). TJs are important for sealing the paracellular gaps and forming the ‘barrier’ properties of the BBB. Under normal conditions, the ECM proteins contribute to enhancing the barrier properties of the BBB, but it is not fully understood how. The protein claudin-5 (CLDN5) is the major constitutive protein of TJs serving to restrict paracellular permeability to small molecules. The degree of CLDN5 expression throughout blood vessels of the body is positively correlated with endothelial barrier tightness. We have identified a signaling pathway that converges on removal of a transcription factor from a site on DNA known to block the production of CLDN5. Indeed, we have found that certain ECM molecules promote this pathway, leading to high levels of CLDN5 and enhanced barrier function. During inflammation, certain factors cause interference in that pathway and the transcription factor (FOXO1) returns to the nucleus and represses CLDN5 production leading to BBB dysfunction. Increasing evidence implicates BBB dysfunction in the onset and/or progression of multiple neuroinflammatory diseases, such as multiple sclerosis, traumatic brain injury, stroke, and Alzheimer’s disease. Therefore, determining the precise molecular mechanisms involved in initiating BBB dysfunction is of great importance. In efforts to determine various factors responsible for decreased CLDN5 during inflammation, we discovered a protein called decorin that is increased in the basement membrane of mice with neuroinflammation. In culture stimulating brain ECs with an inflammatory-mediator also increased decorin, which was associated with the onset of barrier dysfunction. Furthermore, growing brain ECs on decorin coated surfaces caused them to have significantly less CLDN5 and decreased barrier integrity. Thus, we hypothesize that endothelial-derived decorin is increased in the BBB ECM during neuroinflammation, which contributes to barrier dysfunction by interfering with ECM-dependent maintenance of CLDN5 expression. We propose a complementary approach to researching this hypothesis: 1) in vivo physiological analyses in genetically altered mice, with and without neuroinflammation, and 2) molecular experiments in cultured brain EC models on various ECMs. Innovative experimental genetic models and new molecular biology tools will be developed. Data derived from the proposed work will not only establish novel theories in endothelial and matrix biology, but also provide new mechanistic insights into the pathophysiology of BBB dysfunction with potential to provide the basis for development of new therapeutic targets.