New tools for understanding the blood brain barrier

The central nervous system (CNS) requires a tightly controlled environment freeof toxins and pathogens to provide the proper chemical conditions for synaptic transmission. Thisenvironment is maintained by the ‘blood brain barrier’ (BBB), which is composed of highly specializedblood vessels whose endothelium display specialized tight junctions and unusually low rates oftranscellular vesicular transport (transcytosis). While BBB breakdown has recently been associatedwith various neurological disorders, an intact BBB also poses a major obstacle for drug delivery to theCNS. Pharmaceutical companies spend billions of dollars to develop drugs that can penetrate the BBBto treat disease. However, little progress has been made on manipulating the BBB due to a significantknowledge gap in understanding how BBB function is regulated and identifying the essential molecularconstituents governing its processes. This limited understanding has also thwarted our ability totherapeutically manipulate the BBB.The major impediment to understanding the BBB is identifying its essential constituent and unravelingthe mechanism by which these key regulators control BBB function. However, the current in vitromodels rely on fully differentiated endothelial cells, which already contain unique properties that preventtheir use in reconstitution studies. Similarly, the main technique to study the BBB has been EM,however its static snapshots do not provide information on active and dynamic vesicular transport,directionality, or their specific routes to allow investigators to interrogate the key molecular mechanismsthat regulate BBB integrity.Recently, with our research background in developmental co-wiring of nervous and vascular systems,we used the traditional developmental approach, to first mapped the precise timing of BBB formationand then identified neural cues that induce CNS endothelium to acquire BBB properties, and moleculeswith possible roles in BBB function from simple transcriptome comparisons between CNS andperipheral endothelial cells. Surprisingly, we also found that instead of a physical buildup or disruptionof structurally important tight junctions as previously thought, transcytosis regulation seems to be themore likely the major mechanism underlying BBB integrity. In characterizing these developmentalproperties, I realized that these findings are just the tip of the iceberg and that truly fundamentalquestions remain in identifying the core pathway and understand how they regulate BBB function. New tools thus are needed for understanding the BBB. Here we propose first to develop a new stem cellbased system to allow reconstitution of a functional BBB in vitro, and then to develop a genetic-optical system for monitoring the functional integrity of the BBB in vivo at subcellular resolution in real time. This integrated approach will address fundamental questions about the regulation of the BBB, which will then lead to more effective therapeutic strategies and specific targets for BBB restoration and manipulation.