The BBB protects the brain from toxins and pathogens and maintains homeostasis and proper function of the CNS1,2. To form the BBB, endothelial cells (ECs) lining brain blood vessels acquire a series of features that control the movement of ions, molecules and cells between the blood and the brain3. CNS ECs express specialized tight junctions that prevent paracellular exchange of small molecules4,5. They lack fenestrae and exhibit a low rate of transcytosis that limits transcellular exchange of large molecules6,7, and they express transporters that shuttle essential nutrients into and out of the brain8,9,10. ECs acquire BBB properties through their interactions with mural cells and glial cells in the CNS microenvironment and are modulated by neuronal activity11,12,13,14.
The canonical Wnt/β-catenin signaling pathway is a key regulator of BBB development and maintenance. CNS specific Wnt7a, 7b and Norrin ligands produced by glial cells bind to multiprotein receptor complexes including Frizzled4 and LRP6 on brain ECs15,16,17,18,19,20,21. Receptor activation causes β-catenin stabilization and nuclear translocation to induce activation of TCF and LEF1 transcription factors, which control expression of a BBB-specific gene expression repertoire by inducing tight junction, solute transporter and efflux transporter expression, and by repressing expression of the permeability protein PLVAP that forms the diaphragm in EC fenestrae, caveolae and transcytotic vesicles15,22,23,24. Whether Wnt/β-catenin signaling could be modulated to open the BBB “on-demand” or to restore its integrity when damaged, is unknown.
Unc5 was initially discovered in a C. elegans screen for motor dysfunctions, and controls axon guidance in all species examined25,26. Among the four vertebrate Unc5 family members, only Unc5B is expressed in ECs in mice and humans27. Unc5B binds Netrin-126,28, Robo429,30 and Flrt231,32 via its extracellular domain (ECD). Unc5B signaling is mediated by its intracellular domain (ICD), which encompasses a membrane-proximal ZU5 domain (named for its homology to ZO1), followed by a UPA domain (named for its conservation in Unc5B, PIDD and Ankyrin33) and a death domain (DD) that mediates apoptosis in the absence of ligand33,34. These domains form a supramodule in which ZU5 binds to both UPA and DD suppressing Unc5B biological function, while ligand binding to Unc5B triggers a conformational change such that ZU5 loses its interaction with DD and exposes the UPA domain to activate Unc5B signaling33. Global homozygous Unc5B knockout in mice is embryonically lethal due to placental vascular defects27,31, demonstrating that Unc5B has important functions in vascular development. To address biological roles of this receptor during postnatal and adult life, we generated mice with temporally inducible, endothelial-specific Unc5B deletions.
Blood-brain barrier (BBB) integrity is critical for proper function of the central nervous system (CNS). Here, the study shows that the endothelial Unc5B receptor controls BBB integrity by maintaining Wnt/β-catenin signaling. Inducible endothelial-specific deletion of Unc5B in adult mice leads to BBB leak from brain capillaries that convert to a barrier-incompetent state with reduced Claudin-5 and increased PLVAP expression. Loss of Unc5B decreases BBB Wnt/β-catenin signaling, and β-catenin overexpression rescues Unc5B mutant BBB defects. Mechanistically, the Unc5B ligand Netrin-1 enhances Unc5B interaction with the Wnt co-receptor LRP6, induces its phosphorylation and activates Wnt/β-catenin downstream signaling. Intravenous delivery of antibodies blocking Netrin-1 binding to Unc5B causes a transient BBB breakdown and disruption of Wnt signaling, followed by neurovascular barrier resealing. These data identify Netrin-1-Unc5B signaling as a ligand-receptor pathway that regulates BBB integrity, with implications for CNS diseases.
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