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Tight junctions (TJs) seal brain endothelial cells (BECs) to form a restrictive blood-brain barrier (BBB) to selectively control the influx of circulating substances into the central nervous system. Critical to the integrity of BBB are pericytes, cells that physically interact with BECs through Neural (N)-cadherin adhesion. Using brain samples from donors of different ages, we observed a significant loss of N-cadherin junctions in middle-aged patients. This change in N-cadherin was associated with a significant disruption of occludin TJs, suggesting a potential important crosstalk between N-cadherin and TJs. Therefore, to better understand the role of N-cadherin function in the BBB, we generated an inducible, EC-specific knockout of N-cadherin in mice. We demonstrated that mutant mice lacking N-cadherin exhibited greater BBB permeability as well as impairments in TJ ultrastructure. Analyzing different TJ proteins in the microvasculature of the brain cortex, we also found significant disruption of occludin but not claudin TJs in these mutant mice. Furthermore, analysis of occludin kinetics in BEC monolayers lacking N-cadherin adhesion revealed significantly greater internalization rates of occludin from TJs, suggesting that N-cadherin signaling stabilizes occludin TJs. We showed that N-cadherin induces the activation of phosphoinositide 3-kinase (PI3K), leading to Akt phosphorylation. Depletion of PI3K p110b or Akt3, but not other p110 or Akt isoforms, increased occludin internalization from TJs in BECs with intact N-cadherin adhesion. Our data indicate that N-cadherin adhesion activates PI3K p110b-Akt3 signaling in BECs that is essential for the stabilization of occludin TJs to strengthen the BBB.

The blood‐brain barrier (BBB) is a specialized microvasculature integral for brain tissue‐fluid homeostasis that is comprised of brain endothelial cells (BECs) and pericytes. Disruption of the BBB and subsequent vascular leakage of protein‐rich fluids is toxic to surrounding neurons and is an underlying risk factor in neurodegenerative disorders. BECs and pericytes form adhesions through the transmembrane protein Neural (N)‐cadherin. Although BEC‐pericyte interactions are critical for maintaining the BBB, the role of N‐cadherin adhesions in regulating the BBB remain unclear. Our previous work demonstrated that mutant mice lacking Cdh2 (N‐cadherin) in ECs or pericytes exhibited a size‐dependent increase in BBB permeability without affecting vessel or pericyte coverage. These findings raise the possibility that N‐cadherin junctions activate outside‐in signaling to strengthen the BBB. Analysis of tight junction (TJ) proteins occludin and claudins 1 and 5 in microvascular BECs of the cortex demonstrated a significant reduction in the accumulation of occludin, but not claudins, at TJs of KO mice. To delineate the signaling mechanism by which N‐cadherin adhesion‐mediated signaling regulates occludin TJs, we utilize biomimetic surfaces (Ncdh‐BioS) bearing covalently linked N‐cadherin extracellular domain to induce N‐cadherin adhesion in BEC monolayers in vitro. Consistent with our observations in mice, assembly of N‐cadherin junctions induced the accumulation of occludin and ZO1 at TJs in BEC monolayers. Depletion of N‐cadherin reversed these events, suggesting that N‐cadherin adhesion‐induced signaling assembles or stabilizes occludin TJs. Analysis of occludin‐Dendra 2 kinetics revealed decreased internalization rates of occludin from TJs in BECs grown on Ncdh‐BioS as compared to collagen, suggesting that N‐cadherin signaling stabilized occludin TJs. Furthermore, formation of N‐cadherin adhesion complexes led to activation of phosphoinositide 3‐kinase (PI3K) signaling as evidenced by the spatial redistribution of Akt to the plasma membrane as well as Akt phosphorylation. Pharmacological inhibition of class I PI3K with Copanlisib or depletion of PI3K p110β, but not of other PI3K catalytic isoforms, abolished Akt activation and increased the rate of occludin internalization from TJs in BECs grown on Ncdh‐BioS. Cumulatively, these data demonstrates that N‐cadherin outside‐in signaling strengthens the BBB by stabilizing occludin TJs through PI3K p110β signaling.

Capillary endothelial cells express Vascular Endothelial (VE)‐ and Neural (N)‐cadherin, with overlapping functions. VE‐cadherin forms homotypic adhesion between endothelial cells whereas N‐cadherin forms heterotypic adhesion with the surrounding pericytes in capillary endothelia. Endothelial specific deletions of Cdh2 (N‐cadherin) or Cdh5 (VE‐cadherin) in mice demonstrated poorly formed leaky capillaries and in utero death at E9.5 due to defective angiogenesis. These findings raise the question of whether N‐ and VE‐cadherin function independently or whether N‐cadherin activated signaling regulates the assembly of VE‐cadherin and thereby the formation of adherens junctions. We investigated the role of N‐cadherin in the formation of VE‐cadherin junctions using mouse genetic models and identifying N‐cadherin signaling pathways in endothelial monolayers. We show that N‐cadherin functions by interacting with the RhoGEF Trio to activate the RhoGTPases Rac1 and RhoA in nascent adherens junctions, inducing the recruitment of VE‐cadherin. This N‐cadherin activated signaling pathway is essential for maximal VE‐cadherin assembly and the formation of the endothelial junctional barrier.Support or Funding InformationSupported by NIH grant R01 HL103922 to Y.A.K.; AHA AWARD 16PRE27260230 to K.K.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.