RAGE mediated intracellular Aβ uptake contributes to the breakdown of tight junction in retinal pigment epithelium

Abstract

Intracellular amyloid beta (Aβ) has been implicated in neuronal cell death in Alzheimer's disease (AD). Intracellular Aβ also contributes to tight junction breakdown of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). Although Aβ is predominantly secreted from neuronal cells, the mechanism of Aβ transport into RPE remains to be fully elucidated. In this study, we demonstrated that intracellular Aβ was found concomitantly with the breakdown of tight junction in RPE after subretinal injection of Aβ into the mouse eye. We also presented evidence that receptor for advanced glycation end products (RAGE) contributed to endocytosis of Aβ in RPE. siRNA-mediated knockdown of RAGE prevented intracellular Aβ accumulation as well as subsequent tight junction breakdown in RPE. In addition, we found that RAGE-mediated p38 MAPK signaling contributed to endocytosis of Aβ. Blockade of RAGE/p38 MAPK signaling inhibited Aβ endocytosis, thereby preventing tight junction breakdown in RPE. These results implicate that intracellular Aβ contributes to the breakdown of tight junction in RPE via the RAGE/p38 MAPK-mediated endocytosis. Thus, we suggest that RAGE could be a potential therapeutic target for intracellular Aβ induced outer BRB breakdown in AMD.

Keywords: Gerotarget; age-related macular degeneration; amyloid β; endocytosis; receptor for advanced glycation end products; tight junction.

Figure 1. Subretinal injection of Aβ leads to intracellular Aβ uptake and subsequent breakdown of tight junction in RPE

Effect of subretinally injected OAβ₄₂ (1 μg) on tight junction in retinal pigment epithelium (RPE) flat mount was evaluated at 1 week post injection. A.-E. RPE flat mounts with immunofluorescence staining against Aβ₄₂ (green), tight junction protein ZO-1 (red) and nucleus (DAPI, blue) are shown. A. RPE flat mount after subretinal vehicle injection (Con) shows tight junction with typical hexagonal shape. B. RPE flat mount after subretinal OAβ₄₂ injection (Subretinal OAβ₄₂) shows intracellular Aβ and disrupted irregular expression of ZO-1. Orthogonal images indicates intracellular position of Aβ. C., D. Optical zoom: ×2.4, a magnified portion of image a, b (enclosed in the white dotted box) to indicate intracellular Aβ and tight junction breakdown. D. Arrows indicate disrupted tight junctions. E. Representative images with 3-D reconstruction using Imaris software shows intracellular Aβ in RPE layer after subretinal injection of FITC-labeled OAβ_42. Arrows indicate intracellular Aβ (green). Magnification, ×1000. Scale bar = 20 μm. Figures were selected as representative data from three independent experiments.

Figure 2. Extracellular Aβ translocates into intracellular space via RAGE-mediated endocytosis in RPE

ARPE-19 cells were exposed to vehicle control A., C. or OAβ₄₂ 10 μM (B, D) for 60 min, fixed in 4% PFA, and stained by anti-human Aβ₄₂ (green) and anti-biotin (red) or anti-RAGE (red). A. RPE cells shows basal level of endocytosis of cell membrane proteins (red). B. RPE cells treated with OAβ₄₂ 10 μM shows increased level of endocytosis of cell membrane proteins (red) in orthogonal view with a 0.49 μm Z-step interval. A magnified portion of image B (enclosed in the white dotted box) indicates intracellular Aβ merged with biotinylated membrane proteins. Arrow indicates intracellular colocalization of Aβ and biotin. C. RPE cells shows RAGE expression. D. RPE cells treated with OAβ₄₂ 10 μM shows internalized RAGE (red) in orthogonal view. A magnified portion of image D (enclosed in the white dotted box) indicates intracellular Aβ merged with internalized RAGE. Arrow indicates colocalization of Aβ and RAGE. E., F. ARPE-19 cells were exposed to vehicle control E. or OAβ₄₂ 10 μM F. for 24 h, fixed in 4% PFA, and stained by anti-human Aβ₄₂ (green) and anti-ZO-1 (red). E. RPE cells show typical hexagonal shape tight junction. F. RPE cells show disintegrated and disorganized ZO-1 with intracellular Aβ. Magnification, ×1000. Scale bar = 20 μm. Figures were selected as representative data from three independent experiments.

Figure 3. siRNA-mediated knockdown of RAGE suppresses Aβ uptake in RPE

RAGE siRNA was transfected in ARPE-19 cells. Negative siRNA was used as a control. RPE cells were treated OAβ₄₂ 10 μM for 24 h. Intracellular Aβ uptake is decreased in RPE with RAGE siRNA compared to RPE with negative siRNA. A. Relative expression of AGER mRNA is decreased in RPE cells with RAGE siRNA. B. RAGE expression is decreased in RPE cells with RAGE siRNA. C. Immunocytochemistry of Aβ₄₂ (green) shows decreased intracellular Aβ in RPE cells with RAGE siRNA. D. Intracellular Aβ was evaluated by Western blot. E. Relative band density of Aβ was analyzed using ImageJ 1.42 software. β-actin was used as a loading control. Data are presented as mean ± SEM. in graphs. *p < 0.05 (two tailed, unpaired T-test). Figures were selected as representative data from three independent experiments.

Figure 4. RAGE-mediated p38 MAPK signaling contributes to endocytosis of Aβ in RPE

A. RPE cells were treated OAβ₄₂ 2 μM or 10 μM for 30 min. Phosphorylation of p38 MAPK is increased at 30 min. B. RAGE siRNA was transfected in ARPE-19 cells. Negative siRNA was used as a control. RPE cells were treated OAβ₄₂ 10 μM for indicated time (30 min and 24 h). Phosphorylation of p38 MAPK and intracellular Aβ uptake are decreased in RPE with RAGE siRNA compared to RPE with negative siRNA. C. RPE cells were pretreated with anti-RAGE neutralizing antibody (20 μg/ml, 2h) and SB 203580 (10 μM, 30 min) and were treated with OAβ₄₂ 10 μM for indicated time (30 min and 24 h). Anti-RAGE neutralizing antibody and SB 203580 decrease intracellular Aβ in RPE. β-actin was used as an internal control. Figures were selected as representative data from three independent experiments.

Figure 5. Blockade of RAGE inhibits intracellular Aβ-induced tight junction breakdown

A. RAGE siRNA was transfected in ARPE-19 cells. Negative siRNA was used as a control. RPE cells were treated OAβ₄₂ 10 μM for 24 h. Tight junction proteins (ZO-1 and occludin) were evaluated by Western blot. β-actin was used as an internal control. B. Relative band density was analyzed using ImageJ 1.42 software. C. RPE cells were pretreated with anti-RAGE neutralizing antibody (20 μg/ml, 2h) and SB 203580 (10 μM, 30 min) and were treated with OAβ₄₂ 10 μM for 24 h. Occludin was evaluated by Western blot. β-actin was used as an internal control. D. Transepithelial electrical resistance (TER) was measured for 48 h and normalized to the TER value just after Aβ treatment. TER values with anti-RAGE neutralizing antibody (triangle) are compared to TER with OAβ₄₂ treatment alone (square) at indicated time point. Data are presented as mean ± SEM. in graphs. *p < 0.05 (two tailed, unpaired T-test). E. RPE cells were pretreated with anti-RAGE neutralizing antibody (20 μg/ml, 2h) and SB 203580 (10 μM, 30 min), and were treated with OAβ₄₂ 10 μM for 24 h. Cells were stained by anti-human Aβ₄₂ (white) and anti-ZO-1 (green). Magnification, ×1000. Scale bar = 20 μm. Figures were selected as representative data from three independent experiments.