The central nervous system (CNS) requires a tightly controlled environment free of toxins and pathogens to provide the proper chemical composition for neural function. This environment is maintained by the ‘blood–brain barrier’ (BBB), which is composed of blood vessels whose endothelial cells display specialized tight junctions and extremely low rates of transcellular vesicular transport (transcytosis)1,2,3. In concert with pericytes and astrocytes, this unique brain endothelial physiological barrier seals the CNS and controls substance influx and efflux4,5,6. Although BBB breakdown has recently been associated with initiation and perpetuation of various neurological disorders, an intact BBB is a major obstacle for drug delivery to the CNS7,8,9,10. A limited understanding of the molecular mechanisms that control BBB formation has hindered our ability to manipulate the BBB in disease and therapy. Here we identify mechanisms governing the establishment of a functional BBB. First, using a novel tracer-injection method for embryos, we demonstrate spatiotemporal developmental profiles of BBB functionality and find that the mouse BBB becomes functional at embryonic day 15.5 (E15.5). We then screen for BBB-specific genes expressed during BBB formation, and find that major facilitator super family domain containing 2a (Mfsd2a) is selectively expressed in BBB-containing blood vessels in the CNS. Genetic ablation of Mfsd2a results in a leaky BBB from embryonic stages through to adulthood, but the normal patterning of vascular networks is maintained. Electron microscopy examination reveals a dramatic increase in CNS-endothelial-cell vesicular transcytosis in Mfsd2a−/− mice, without obvious tight-junction defects. Finally we show that Mfsd2a endothelial expression is regulated by pericytes to facilitate BBB integrity. These findings identify Mfsd2a as a key regulator of BBB function that may act by suppressing transcytosis in CNS endothelial cells. Furthermore, our findings may aid in efforts to develop therapeutic approaches for CNS drug delivery.
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We thank M. Karnovsky, E. Raviola and T. Reese for advice and discussion; S. R. Datta, C. Weitz, M. Greenberg, Q. Ma, C. Harvey and members of the Gu laboratory for comments on the manuscript; D. Sabatini and J. Reeling for sharing unpublished data; C. Betsholtz and C. Olsson for providing Pdgfbret/ret mouse brain samples; T. Schwarz and A. Oztan for discussion and advice on cell trafficking; W.-J. Oh for help with graphic illustrations; the Flow Cytometry Facility in the department of Systems Biology at Harvard Medical School for cell sorting; the Microarray Core at Dana-Farber Cancer Institute for Affymetrix assay; HSPH Bioinformatics Core, Harvard School of Public Health, for assistance with microarray analysis and Gene Expression Omnibus (GEO) submission; the Enhanced Neuroimaging Core at Harvard NeuroDiscovery Center for helping with confocal imaging and image analysis; the HMS Electron Microscopy Core Facility, the Neurobiology Imaging Facility for consultation and instrument availability that supported this work (this facility is supported in part by the Neural Imaging Center as part of an NINDS P30 Core Center grant no. NS072030); R. Polakiewicz and J. Xie from CST for generating Mfsd2a antibodies. This work was supported by the Harold Perlman postdoctoral fellowships, the Goldenson postdoctoral fellowship, and the Lefler postdoctoral fellowship (A.B.-Z.); the DFG-German Research Foundation postdoctoral fellowship (E.K.); the Mahoney postdoctoral fellowship (B.L.); NIH training grant 5T32MH20017-15 (B.J.A.); and the Sloan research fellowship, Armenise junior faculty award, the Genise Goldenson fund, the Freudenberger award, and NIH grant R01NS064583 (C.G.).
This file contains Supplementary Data, a Supplementary Discussion and additional references.
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Postnatal development of the astrocyte perivascular MLC1/GlialCAM complex defines a temporal window for the gliovascular unit maturation
Brain Structure and Function (2019)