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. 2012 Jun 27;32(26):8845-54.
doi: 10.1523/JNEUROSCI.6102-11.2012.

Aβ₁₋₄₂-RAGE interaction disrupts tight junctions of the blood-brain barrier via Ca²⁺-calcineurin signaling

Sun-Young Kook  1 , Hyun Seok Hong, Minho Moon, Chang Man Ha, Sunghoe Chang, Inhee Mook-Jung
Affiliations

Aβ₁₋₄₂-RAGE interaction disrupts tight junctions of the blood-brain barrier via Ca²⁺-calcineurin signaling

Sun-Young Kook et al. J Neurosci. .

Abstract

The blood-brain barrier (BBB), which is formed by adherens and tight junctions (TJs) of endothelial cells, maintains homeostasis of the brain. Disrupted intracellular Ca²⁺ homeostasis and breakdown of the BBB have been implicated in the pathogenesis of Alzheimer's disease (AD). The receptor for advanced glycation end products (RAGE) is known to interact with amyloid β-peptide (Aβ) and mediate Aβ transport across the BBB, contributing to the deposition of Aβ in the brain. However, molecular mechanisms underlying Aβ-RAGE interaction-induced alterations in the BBB have not been identified. We found that Aβ₁₋₄₂ induces enhanced permeability, disruption of zonula occludin-1 (ZO-1) expression in the plasma membrane, and increased intracellular calcium and matrix metalloproteinase (MMP) secretion in cultured endothelial cells. Neutralizing antibodies against RAGE and inhibitors of calcineurin and MMPs prevented Aβ₁₋₄₂-induced changes in ZO-1, suggesting that Aβ-RAGE interactions alter TJ proteins through the Ca²⁺-calcineurin pathway. Consistent with these in vitro findings, we found disrupted microvessels near Aβ plaque-deposited areas, elevated RAGE expression, and enhanced MMP secretion in microvessels of the brains of 5XFAD mice, an animal model for AD. We have identified a potential molecular pathway underlying Aβ-RAGE interaction-induced breakage of BBB integrity. This pathway might play an important role in the pathogenesis of AD.

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Figures

Figure 1.
Figure 1.
Aβ1–42 alters ZO-1 distribution and disrupts TJs in bEnd.3 cells. A, bEnd.3 cells were incubated with various doses of Aβ1–42 for 24 h (red, ZO-1; blue, DAPI). B, 5 μm Aβ1–42-induced alterations (arrowheads) in ZO-1 distribution in a time-dependent manner. Scale bar, 50 μm. n = 8 for each group. C, D, ZO-1 levels in bEnd.3 cells were decreased significantly with Aβ1–42 (5 μm, 24 h) in total lysate. Tubulin is a loading control. E–G, Claudin-5 and occludin levels in bEnd.3 cells were decreased significantly with Aβ1–42 (5 μm, 24 h) in total lysate. β-actin is a loading control. H, Diffusion of FD-40 through the bEnd.3 monolayer under the same conditions as performed in C was increased significantly after Aβ1–42 (5 μm, 24 h). y-axis is the fluorescence signals from the bottom chamber of Transwells. Blank shows a high value when no cells were on the Transwell plate. Control (Ctrl) shows a low value when bEnd.3 cells were compacted on the Transwell.*p < 0.05, **p < 0.01 versus control sample.
Figure 2.
Figure 2.
Aβ1–42 increases intracellular calcium levels and secretion of MMPs in bEnd.3 cells. A, Fluo-4-loaded bEnd.3 cells were incubated with 5 μm Aβ1–42 or 1 μm A23187 (calcium ionophore). Live cell images were obtained on a fluorescent microscope at the indicated times. Intensity of Fluo-4-staining in cells was increased in a time-dependent manner after treatment of Aβ1–42 compared with control (DMSO). Scale bar, 50 μm. B, Fluorescence intensity in bEnd.3 cells had a tendency to increase to 1 h with Aβ1–42 and A23187. At 6 h, fluorescence intensity was increased significantly compared with control (Ctrl). *p < 0.05 versus control sample at 6 h. C, Gelatin zymography using conditioned medium from bEnd.3 cells showed an increase of MMP-9 (95 kDa) and MMP-2 (72 kDa) after treatment with 5 μm Aβ1–42 (lane 3) compared with control (DMSO). M, molecular weight marker.
Figure 3.
Figure 3.
Aβ1–42-induced alterations of TJs in bEnd.3 cells are attenuated by GM6001 and FK506. A, B, bEnd.3 cells were incubated with 5 μm Aβ1–42 for 24 h in the presence of GM6001 or FK506. ZO-1 immunoreactivity (red) in the presence of Aβ1–42 was rescued in a dose-dependent manner by both GM6001 (A) and FK506 (B). Three independent experiments were done for both A and B. Scale bar, 50 μm. C, D, Changes of diffusion for FD-40 through the bEnd.3 monolayer were measured at each dose of GM6001 (100 nm) and FK506 (10 μm). Data are represented as mean ± SEM of three independent experiments performed in triplicate. *p < 0.05, ***p < 0.001 versus control sample; #p < 0.05, ##p < 0.01 versus Aβ1–42-treated sample.
Figure 4.
Figure 4.
RAGE mediates Aβ1–42-induced disruptions in TJs. A, Aβ1–42-induced alterations in ZO-1 distribution are attenuated by an anti-RAGE antibody. Con, DMSO; RAGE-Ab, neutralizing anti-RAGE antibody; goat-IgG, IgG from goat used as a negative control. Red signal is ZO-1 staining. Three independent experiments were performed. Scale bar, 100 μm. B, C, Transiently transfected bEnd.3 cells with mock or full-length human RAGE (hRAGE) were incubated with 5 μm Aβ1–42 for 24 h. Representative Western blot images (B) and the densitometry results (C) are presented. Data are represented as mean ± SEM of three independent experiments performed in triplicate. #p < 0.05 versus Mock Aβ1–42-treated sample of RAGE Aβ1–42.
Figure 5.
Figure 5.
Cerebral capillaries are impaired in the brains of 5XFAD mice. A, Coronal serial sections of brains from 8-month-old mice (n = 3 for each from littermates and 5XFAD mice) were costained with anti-GLUT-1 (red) and anti-Aβ (green; 4G8) antibodies and imaged by confocal microscopy. Capillaries stained with anti-GLUT-1 antibody (red) showed long tubular-like form in littermate mice. 5XFAD mice displayed amyloid plaque deposition (green) and cut capillary forms (the parts shown in the yellow dotted circle). Capillaries adjacent to the amyloid plaques displayed disconnected tubular-like form in 5XFAD in the merged images (the parts shown in the white dotted circle). Scale bar, 40 μm. B, 3D-SIM images of the brains from littermates and 5XFAD mice. Brain slices were each recorded by 3D-SIM images in the z-direction with a thickness of 0.150 μm, reconstructed, and made into a 3D volume image with α blending function. Axial directions were represented on each image. Capillaries stained with anti-GLUT-1 antibody (red) and amyloid plaque stained with anti-Aβ (green; 4G8) antibody. Arrow, sectioned z-axis image; arrowhead, damaged microvessel. Scale bar, 2 μm. 3D depth, 4.65 μm in littermate and 5.45 μm in 5XFAD.
Figure 6.
Figure 6.
Expressions of RAGE and MMP are increased in the capillary from the brains of 8-month-old 5XFAD mice. A, Representative figure of RAGE expression in the cortex. RAGE-positive cells were increased in the cortex of 5XFAD mice. Red, anti-RAGE antibody staining. 1, 2, and 3 represent cortical layers 1, 2, and 3. Scale bar, 60 μm. B, Immunostainings of both RAGE (green) and GLUT-1 (red). RAGE expression was increased more in GLUT-1-positive capillaries of the brains of 5XFAD than in littermate brains. Red, GLUT-1; green, RAGE; blue, DAPI staining for nucleus. Scale bar, 30 μm. Boxed areas were magnified for each figure. Scale bar, 30 μm. C, Percentage of area occupied by RAGE was quantified. n = 4 for each from littermate and 5XFAD mice. D, 3D-SIM reconstruction images of RAGE expression on the capillaries. RAGE expression on the capillaries (arrows) was increased significantly more in 5XFAD than in littermate. The sectioned z-axis images represented that the capillaries were more impaired in the brains of 5XFAD mice than in the brains of their littermates. Each 3D-SIM image was taken in a z-direction with a thickness of 0.150 μm, reconstructed, and made into a 3D volume image with the α blending function. Axial directions were represented on each of the images. The first frame image showed on top of the reconstructed 3D-SIM image and its scale is represented on the image. Scale bars, 2 μm. 3D depth, 7.95 μm in littermate and 6.90 μm in 5XFAD. E, Immunoreactivity of MMP was increased around the cerebral capillaries of 8-month-old 5XFAD mice brains. n = 3 for each cortex from littermate and 5XFAD. Red, GLUT-1; green, RAGE (D) or MMP (E); blue, DAPI staining for nucleus. Scale bar, 20 μm.
Figure 7.
Figure 7.
5XFAD mice show alterations in cerebral TJs. A, EM from the brains of 5XFAD showed alterations of TJs compared with littermates. TJs of 5XFAD mice were shorter than the littermate's TJs. n = 3 for each cortex from littermate and 5XFAD. Arrow, TJs; RBC, red blood cells. Scale bar, 500 nm. B, Higher magnification of TJs in the littermate and 5XFAD mice brains. Arrow, TJs. Scale bar, 200 nm. C, Length of 15 TJs from littermate (1091.2 nm) and 5XFAD (538.1 nm) were examined. Graph shows an average of 12 capillaries. *p < 0.05.
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