Mechanisms of endocytosis and transcytosis into endothelial cells of the blood-brain barrier

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OVERVIEW: The overall aim of the present program is to provide a much needed understanding of how the body communicates with the brain. More specifically, we will decipher the mechanisms underlying the endocytosis and intra-/intercellular trafficking of bioactive molecules circulating in the bloodstream and interacting with transporters/receptors located at the blood-brain barrier (BBB). BACKGROUND: It remains unclear to which extent large endogenous blood-borne substrates, like transferrin, undergo endocytosis, amass in endosomes and lysosomes, or are ferried across the BBB into the brain (transcytosis). One thing that is certain is that circulating compounds have to interact first with receptors expressed on the luminal side of brain microvessel endothelial cells (BMECs), forming the BBB. However, subsequent cellular routes and molecular mechanisms involved remain elusive. RECENT PROGRESS: In the last years, our group has developed a research platform with all the tools to accurately and quantitatively monitor compounds inside BMECs and in other brain cells, by combining in vitro and in vivo approaches with high-resolution technology. SPECIFIC OBJECTIVES: The subcellular fate of endogenous (substrates) or exogenous (monoclonal antibodies) ligands of vascular endothelial growth factor receptor 2 (VEGFR2), insulin-like growth factor 2 receptor (IGF2R) and ephrin type-A receptor 2 (ePHA2) will be determined after binding to their receptor in cultured BMEC. Mechanisms will be probed using changes in temperature and various inhibitors of endocytic pathways. Technical approaches will combine binding studies and immunofluorescence. Cellular and subcellular distribution and colocalization analyses will be performed with super-resolution STED fluorescence microscopy and scanning electron microscopy. Confirmatory experiments using quantitative and qualitative methods will be performed in vivo, using in situ cerebral perfusion in mice and intravenous injections. NOVELTY: The novelty of our general approach stems from the combination of quantitative and qualitative methodologies using both in vitro and in vivo studies and high-resolution technology. Consistent results from different receptor-mediated systems, with complementary methods, will strengthen each other to decipher endocytic molecular pathways. CONCLUSION: The main deliverables of the present grant application will be (i) the formation of graduate students with training on techniques pertaining to cellular transport mechanisms in the BBB, which are available in just a few laboratories in the world, (ii) the consolidation of a research platform for BMEC-internalizing ligands, (iii) a better understanding of endocytic mechanisms within BMECs triggered upon ligand binding to receptors and their impact on BBB function and brain homeostasis.