Aβ_1–42oligomer‐induced leakage in an<i>in vitro</i>blood–brain barrier model is associated with up‐regulation of<scp>RAGE</scp>and metalloproteinases, and down‐regulation of tight junction scaffold proteins

Abstract

Abstract Accumulating evidence indicates that abnormal deposition of amyloid‐β (Aβ) peptide in the brain is responsible for endothelial cell damage and consequently leads to blood–brain barrier ( BBB ) leakage. However, the mechanisms underlying BBB disruption are not well described. We employed an monolayer BBB model comprising bE nd.3 cell and found that BBB leakage was induced by treatment with Aβ 1–42, and the levels of tight junction ( TJ ) scaffold proteins ( ZO ‐1, Claudin‐5, and Occludin) were decreased. Through comparisons of the effects of the different components of Aβ 1–42 , including monomer (Aβ 1–42 ‐Mono), oligomer (Aβ 1–42 ‐Oligo), and fibril (Aβ 1–42 ‐Fibril), our data confirmed that Aβ 1–42 ‐Oligo is likely to be the most important damage factor that results in TJ damage and BBB leakage in Alzheimer's disease. We found that the incubation of bE nd.3 cells with Aβ 1–42 significantly up‐regulated the level of receptor for advanced glycation end‐products ( RAGE ). Co‐incubation of a polyclonal antibody to RAGE and Aβ 1–42 ‐Oligo in bE nd.3 cells blocked RAGE suppression of Aβ 1–42 ‐Oligo‐induced alterations in TJ scaffold proteins and reversed Aβ 1–42 ‐Oligo‐induced up‐regulation of RAGE , matrix metalloproteinase ( MMP )‐2, and MMP ‐9. Furthermore, we found that these effects induced by Aβ 1–42 ‐Oligo treatment were effectively suppressed by knockdown of RAGE using small interfering RNA (si RNA ) transfection. We also found that GM 6001, a broad‐spectrum MMP inhibitor, partially reversed the Aβ 1–42 ‐Oligo‐induced inhibitor effects in bE nd.3 cells. Thus, these results suggested that RAGE played an important role in Aβ‐induced BBB leakage and alterations of TJ scaffold proteins, through a mechanism that involved up‐regulation of MMP ‐2 and MMP‐9. image To reveal the role of RAGE in blood–brain barrier (BBB) disruption in Alzheimer's disease (AD), we employed an monolayer BBB model comprising bEnd.3 cell and found that BBB leakage was induced by treatment with amyloid‐β (Aβ), and the levels of tight junction (TJ) scaffold proteins including ZO‐1, Claudin‐5, and Occludin were decreased. Using receptor for advanced glycation end‐products (RAGE) neutralizing polyclonal antibody and siRNA, we confirmed that RAGE played an important role in Aβ‐induced BBB leakage and alterations of TJ scaffold proteins and in the up‐regulation of matrix metalloproteinase‐2 (MMP‐2) and matrix metalloproteinase‐9 (MMP‐9). NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells.