Regulation of blood-brain barrier formation and function by EHDs

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 DESCRIPTION (provided by applicant): The blood-brain barrier (BBB) is a term used to describe the unique properties of the blood vessels that vascularize the central nervous system (CNS). This barrier is crucial both to maintain brain homeostasis, which allows for proper neuronal function, and to protect the CNS from injury and disease. Many of the symptoms of stroke, multiple sclerosis, edema, and brain traumas are due to a breakdown of the BBB that accompanies the primary insult. Furthermore, the BBB provides an obstacle for the treatment of all neurological diseases as it greatly impedes drug delivery to the CNS. Therefore, understanding the mechanisms that regulate BBB function may identify novel targets to modulate the BBB, both to repair the BBB and to develop methods to bypass the BBB for drug delivery. Many of the properties of the BBB are manifested within the endothelial cells that make up the walls of the blood vessels, which allows them to restrict the movement of molecules, ions and cells between the blood and the brain. One important barrier property is that the CNS endothelial cells undergo transcytosis at extremely low rates, thus limiting the transcellular permeability. Although these are properties of the endothelial cells, studies have revealed that they are induced by interactions with the CNS microenvironment. In particular we have identified that CNS pericytes are important to induce BBB properties in endothelial cells during development, and they do so by inhibiting transcytosis through the endothelial cells. In preliminary experiments, we have identified that specific EHD family members are enriched in peripheral `leaky' vessels such as the liver and lung which undergo lots of transcytosis, compared to CNS vessels which undergo very low amounts of transcytosis. We have further demonstrated that genetic loss of pericytes, stroke and neuroinflammation all lead to up-regulation of EHD genes in CNS blood vessels that correlates with BBB leakage. We have used genetic mouse models to demonstrate that over-expression of EHD4 leads to leakage of the BBB in vivo, and conditional knockout of EHD1 and EHD4 in peripheral endothelial cells leads to less peripheral vascular leakage. Therefore we aim to test the hypothesis that: Pericytes regulate the BBB by inhibiting the expression of EHDs in CNS endothelial cells, and that up-regulation of EHDs is an important component of BBB dysfunction during neurological disease. We have developed transgenic mouse models to reversibly over-express EHD- GFP fusion proteins in endothelial cells, as well as conditional knockout models to delete EHDs from endothelial cells. Using these models we will address the following questions: How does up-regulation of EHDs lead to BBB dysfunction? Are EHDs required for BBB dysfunction during diseases? What leads to EHD upregulation at the BBB? Answering these questions will enable us to determine the role of EHDs in regulating the BBB and determine if they are a target to modulate the BBB to treat neurological diseases and aid drug delivery to the CNS.