Molecular Tools to Study FLVCR2-mediated Heme Transport in Brain Angiogenesis

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PROJECT ABSTRACT 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) and restrict the movement of molecules, ions and cells between the blood and the brain. BBB dysfunction contributes to various CNS diseases and greatly impedes drug delivery. Despite its importance, little progress has been made in manipulating the BBB due to a major gap in our understanding, and a lack of tools to interrogate, constituents of the BBB at a molecular and cellular level. Our recent work and preliminary data begin to characterize a role for FLVCR2, a plasma membrane heme transporter, in brain vascular development and disease. Flvcr2 is specifically expressed in BBB endothelial cells and its mutation in humans causes Proliferative Vasculopathy and Hydranencephaly Hydrocephalus PVHH - a rare genetic condition characterized by defective brain angiogenesis leading to ischemia and ventriculomegaly. Our work links BBB heme transport to brain angiogenesis, and uncovers a central role for heme in these pathologies. However, the mechanisms by which FLVCR2 transports heme and controls brain angiogenesis are unknown, and we currently lack the tools to probe these mechanisms at a molecular level. In this proposal we will address this void through two specific Aims: In Aim 1, we will determine the structure of FLVCR2 using cryo-electron microscopy (cryoEM), so as to provide a model to generate structure-based functional hypotheses. In Aim 2, using CRISPR-Cas9 technology combined with innovative heme reporters and angiogenesis assays well-known to our lab, we will develop cell-based angiogenesis assays to allow rapid and efficient testing of structure-based hypotheses. Our preliminary data include cryoEM analysis of recombinant FLVCR2 and complex formation with FLVCR2-specific antibodies (Fabs); and the manipulation of Flvcr2 expression in cell culture angiogenesis assays utilizing mouse brain endothelial cells. These data demonstrate that we are well-poised for structural determination and the development of an adaptable cell-based assay for future structure/function studies on FLVCR2, and its role in BBB heme transport and CNS disease.