Mechanisms Underlying the Suppression of Transcytosis at the Blood Brain Barrier

Harvard Medical School, Boston MA

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Abstract

DESCRIPTION (provided by applicant): The tightly controlled chemical environment of the CNS required for proper synaptic transmission is maintained by the 'blood brain barrier' (BBB), which is composed of highly specialized blood vessels whose endothelial cells act as a physiological barrier to seal the CNS and control substance influx and efflux. Two unique features of CNS endothelial cells determine BBB integrity, namely specialized tight junctions between the endothelial cells lining the CNS capillaries and extremely low rates of vesicular trafficking between the luminal and abluminal plasma membranes, a process termed transcytosis. A solid understanding of how BBB function is regulated to ensure brain homeostasis is missing in the field, as there are little to no molecular insights into how CNS endothelial cells acquire and maintain their specialized properties. Furthermore, the most promising strategies for delivering therapeutic agents across the BBB involve the manipulation of transcytotic pathways, an avenue of research which requires mechanistic understanding of how this process is normally regulated. Previous work in our lab has identified MFSD2A as the first molecule to maintain BBB integrity specifically by suppressing transcytosis. MFSD2A is exclusively expressed in CNS endothelial cells, and genetic ablation of Mfsd2a results in both extravasation of exogenous tracer from the vessel lumen to brain parenchyma and an increased density of vesicles within CNS endothelial cells, while intercellular tight junctions remain normal The goal of this study is to understand the molecular mechanism whereby MFSD2A suppresses transcytosis. My preliminary data show that MFSD2A is localized to the luminal plasma membrane of CNS endothelial cells and that loss of Mfsd2a results in increased immunoreactivity of Cav-1. Therefore, I hypothesize that MFSD2A acts as a suppressor of caveolae-mediated transcytosis in CNS endothelial cells, possibly by interacting with transcytotic machinery to inhibit caveolae dynamics at the luminal plasma membrane. To this end, I have developed a rigorous experimental plan with three aims: I will (1) use in vivo tools and an in vitro cell-based system I have developed to determine which specific transcytotic pathway is suppressed by MFSD2A in CNS endothelial cells, (2) perform structure-function experiments to dissect how structural domains of MFSD2A contribute to its function in suppressing transcytosis, and (3) test the hypothesis that MFSD2A interacts with well-known molecular determinants of caveolae-mediated transcytosis to elicit its function. I expect that these experiments will provide novel insight into how BBB function is regulated, leading to new pathways for BBB manipulation for therapeutic purposes.