BB-2014

The blood‐brain barrier (BBB), a selective barrier formed by endothelial cells and dependent on the presence of tight junctions, is compromised during neuroinflammation. A detailed study of tight junction dynamics during transendothelial migration of leukocytes has been lacking. Therefore, we retrovirally expressed green fluorescent protein (GFP) fused to the N‐terminus of the tight junction protein occludin in the rat brain endothelial cell line GP8/3.9. Confocal microscopy analyses revealed that GFP‐occludin colocalized with the intracellular tight junction protein, ZO‐1, localized at intercellular connections, and was absent at cell borders lacking apposing cells. Using live cell imaging we found that monocytes scroll over the brain endothelial cell surface toward cell‐cell contacts, induce gap formation, which is associated with local disappearance of GFP‐occludin, and subsequently traverse the endothelium paracellularly. Immunoblot analyses indicated that loss of occludin was due to protein degradation. The broad spectrum matrix metalloproteinase (MMP) inhibitor BB‐3103 significantly inhibited endothelial gap formation, occludin loss, and the ability of monocytes to pass the endothelium. Our results provide a novel insight into the mechanism by which leukocytes traverse the BBB and illustrate that therapeutics aimed at the stabilization of the tight junction may be beneficial to resist a neuroinflammatory attack.—Reijerkerk, A., Kooij, G., van der Pol, S. M. A., Khazen, S., Dijkstra, C. D., de Vries, H. E. Diapedesis of monocytes is associated with MMP‐mediated occludin disappearance in brain endothelial cells. FASEB J. 20, E1901–E1909 (2006)

Under normal conditions, the cellular prion protein (PrP(c)) undergoes a proteolytic attack between amino acids 111 and 112 which gives rise to the N-terminal secreted N1 fragment and its C-terminal membrane-tethered counterpart C1. Importantly, this cleavage precludes the integrity of the neurotoxic 106-126 sequence. Here, we describe an original and reliable assay based on a quenched fluorimetric substrate (JMV2770) encompassing the 111/112 sequence of PrP(c). In whole brain homogenate, the JMV2770-hydrolysing activity is optimal at neutral pH and sensitive to the metalloprotease inhibitor BB3103 but not to acidic and serine protease blockers. JMV2770 is efficiently cleaved by intact HEK293 cells and fibroblasts in culture, consistent with an hydrolysis by a typical ectoprotease. Overexpressions of alpha-secretases a disintegrin and metalloprotease-9 (ADAM9), ADAM10 or TACE (ADAM17) in human cells increase BB3103-sensitive JMV2770 hydrolysis, while invalidation of ADAM10 and TACE or reduced expression of ADAM9 by an antisense approach significantly reduced its cleavage. Finally, analysis of JMV2770 hydrolysis following transient transfection of ADAM10 or ADAM9 cDNA in ADAM10(-/-) fibroblasts allowed to confirm our previous data establishing that ADAM9 does not behave as a genuine alpha-secretase but rather acts as an important upstream regulator of ADAM10 activity.