Localization of Brain Endothelial Luminal and Abluminal Transporters with Immunogold Electron Microscopy

Immunogold electron microscopy has identified a variety of blood-brain barrier (BBB) proteins with transporter and regulatory functions. For example, isoforms of the glucose transporter, protein kinase C (PKC), and caveolin-1 are BBB specific. Isoform 1 of the facilitative glucose transporter family (GLUT1) is expressed solely in endothelial (and pericyte) domains, and ∼75% of the protein is membrane-localized in humans. Evidence is presented for a water cotransport function of BBB GLUT1. A shift in transporter polarity characterized by increased luminal membrane GLUT1 is seen when BBB glucose transport is upregulated; but a greater abluminal membrane density is seen in the human BBB when GLUT1 is downregulated. PKC colocalizes with GLUT1 within these endothelial domains during up- and downregulation, suggesting that a PKC-mediated mechanism regulates human BBB glucose transporter expression. Occludin and claudin-5 (like other tight-junctional proteins) exhibit a restricted distribution, and are expressed solely within interendothelial clefts of the BBB. GFAP (glial fibrillary acidic protein) is uniformly expressed throughout the foot-processes and the entire astrocyte. But the microvascular-facing membranes of the glial processes that contact the basal laminae are also polarized, and their transporters may also redistribute within the astrocyte. Monocarboxylic acid transporter and water channel (Aquaporin-4) expression are enriched at the glial foot-process, and both undergo physiological modulation. We suggest that as transcytosis and efflux mechanisms generate interest as potential neurotherapeutic targets, electron microscopic confirmation of their site-specific expression patterns will continue to support the CNS drug discovery process.