Neurobiology of Aging xxx (2014) 1e8
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Intracellular amyloid beta alters the tight junction of retinal pigment epithelium in 5XFAD mice Sung Wook Park a, b, Jin Hyoung Kim a, Inhee Mook-Jung b, Kyu-Won Kim c, Woo Jin Park d, Kyu Hyung Park e, f, *, Jeong Hun Kim a, b, e, ** a
Fight against Angiogenesis-Related Blindness Laboratory, Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Korea SNU-Harvard NeuroVascular Protection Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea d Department of Life Sciences, Life Sciences Concentration GIST (Gwangju Institute of Science and Technology), Gwangju, Korea e Department of Ophthalmology, College of Medicine, Seoul National University, Seoul, Korea f Department of Ophthalmology, Seoul National University Bundang Hospital, Seongnam, Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 August 2013 Received in revised form 21 January 2014 Accepted 11 March 2014
Extracellular deposit of amyloid beta (Ab) is a common pathologic feature in both age-related macular degeneration (AMD) and Alzheimer’s disease, but the role of intracellular Ab on the tight junction of the retinal pigment epithelium (RPE) is unknown. In this study, we investigated the intracellular Ab expression and its role on the outer blood retinal barrier in the retina of 5XFAD mice, a mouse model of Alzheimer’s disease. The retina of 5XFAD mice showed the pathologic features of AMD with intracellular Ab in the RPE. As intracellular Ab accumulated, zonular occludens-1 and occludin were markedly attenuated and lost their integrity as tight junctions in the RPE of 5XFAD mice. Also, Ab42 uptake by ARPE-19 cells induced the tight junction breakdown of zonular occludens-1 and occludin without cell death. These results implicate that intracellular Ab42 could play a role in the breakdown of the outer blood retinal barrier in 5XFAD mice. Thus, we suggested that 5XFAD mice could be a mouse model of dry AMD with regard to the Ab42 related pathology. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Aging Alzheimer’s disease Ab Age-related macular degeneration Outer blood retinal barrier Tight junction Retina Retinal pigment epithelium Transgenic mice
1. Introduction Both age-related macular degeneration (AMD) and Alzheimer’s disease (AD) increase dramatically with aging, and it has been suggested that they might share common risk factors and pathogenesis (Ohno-Matsui, 2011). The extracellular deposit of amyloid beta (Ab) forms senile plaque in the brain, and it is one of the major hallmarks of AD. These deposits are also localized within the drusen * Corresponding author at: Kyu Hyung Park, MD, Department of Ophthalmology, Seoul National University College of Medicine, Seoul National University Bundang Hospital, 166 Gumiro, Bundang-gu, Seongnam, Gyeonggi-do 463-707, Korea. Tel.: þ82 31 787 7373; fax: þ82 31 787 4057 ** Corresponding author at: Jeong Hun Kim, MD, Fight against AngiogenesisRelated Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, and Department of Ophthalmology and Biomedical Sciences, College of Medicine, Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul 110-799, Korea. Tel.: þ82 2 2072 2438; fax: þ82 2 741 3187 E-mail addresses: [email protected] (K.H. Park), [email protected] (J.H. Kim). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.03.008
between the basement membrane of the retinal pigment epithelium (RPE) and the Bruch membrane in the human retina of AMD (Johnson et al., 2002; Luibl et al., 2006) and the mouse retina of AD model (Alexandrov et al., 2011). Furthermore, Ab is only found in the drusen from the eyes of the AMD patients (Dentchev et al., 2003). The ε4 allele of apolipoprotein E (APOE), the major genetic risk factor for AD (Rosvall et al., 2009), may be also associated with AMD (Adams et al., 2012; Malek et al., 2005). In addition, complement factor H, the main genetic risk factor of AMD, has been detected in Ab plaques in the brain of AD patients (Strohmeyer et al., 2000) and decreased in the retina of AD mouse model (Alexandrov et al., 2011). Although the classical concept suggests that Ab is deposited extracellularly, growing evidence from AD patients and transgenic mice indicates that Ab can also accumulate intracellularly and it may contribute to disease progression (LaFerla et al., 2007). In the neprilysin gene disrupted mice, Ab was localized within the subretinal deposits as well as in the cytoplasm of RPE (Yoshida et al.,
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2005). It has been also shown that extracellular Ab could induce breakdown of the tight junction in RPE (Bruban et al., 2009). However, the role of intracellular Ab in RPE has not been elucidated yet. Recently, we reported that intracellular Ab occurred before the formation of extracellular amyloid plaques in the subiculum of 5XFAD mice (Moon et al., 2012). In addition, 5XFAD mouse brain showed alterations in cerebral tight junctions (Kook et al., 2012). These findings suggest that accumulation of intracellular Ab might be an early process that triggers neuronal damage and alterations in outer blood retinal barrier in AD retina. While previous studies revealed the characteristics of the AD mouse retina, the retinal characteristics of 5XFAD mice was not evaluated except for exclusively high level of Ab42 (Alexandrov et al., 2011; Ning et al., 2008; Yoshida et al., 2005). We postulated that intracellular Ab might have an important role in the retina of 5XFAD mice. In the present study, we focused on the role of intracellular Ab and demonstrated that Ab was intracellularly expressed in the RPE of 5XFAD mice and that it attenuated the integrity of outer bloodretinal barrier. Taken together, our results suggest that intracellular Ab could be important in the pathogenesis of dry AMD and retinal aging in AD. 2. Methods 2.1. Transgenic mice This study used 5XFAD mice that were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Mice (5XFAD) overexpress mutant human amyloid precursor protein 695 with the Swedish mutation (K670N, M671L: elevate the production of total Ab), Florida mutation (I716V: elevates the production of Ab42 specifically), and London mutation (V717I: elevates the production of Ab42 specifically) and human presenilin 1 with 2 FAD mutations (M146L and L286V: elevate the production Ab42 specifically) (Oakley et al., 2006). All 5XFAD transgenic mice (Tg6799, B6 ⁄ SJL hybrid background) used were heterozygotes with respect to the transgene, and nontransgenic wild-type littermate mice served as controls. Care, use, and treatment of all animals in this study were in strict agreement with the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research. 2.2. Cell cultures ARPE-19 cells (American Type Culture Collection, Manassas, VA, USA) were used for human RPE cells. The cells were routinely maintained in Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (DMEM/F12) containing 10% fetal bovine serum, 100 U/ mL penicillin, and 100 mg/mL streptomycin. All culture reagents were purchased from Invitrogen (Carlsbad, CA, USA).
hematoxylin and eosin (H&E) for histologic examination via light microscopy (Nikon, Tokyo, Japan). 2.4. Immunofluorescence staining For immunofluorescence of cross-section of the retina, sections were treated with proteinase K (Sigma Aldrich, St Louis, MO, USA) for 20 minutes at 37 C and 70% formic acid (Sigma Aldrich) for 30 minutes at room temperature (RT). Then they were rinsed with PBS and treated with 0.2% Triton-X100 for 20 minutes at 37 C. After getting rinsed with PBS, the sections were treated with blocking solution (Vector Laboratories, Inc, Burlingame, CA, USA) for 1 hour at 37 C. Slides were incubated with mouse anti-Ab (4G8 or 12F4, 1:100, Covance, Princeton, NJ, USA) for 90 minutes at 37 C. Alexa 594 donkey anti-mouse IgG (1:400, Molecular Probes, Inc, Eugene, OR, USA) were used as secondary antibodies. The slides were mounted with aqueous mounting medium (DAKO, Glostrup, Denmark) and observed under fluorescence microscope (Nikon) and confocal microscopes (LSM 510 Meta, Carl Zeiss, Germany, and Leica TCS STED, Leica Microsystems, Germany). For the flat mount of RPE and/or choroid complex, enucleated eyes were dissected to remove neural retina, the RPE and/or choroid complex were gently flat mounted and fixed in methanol for 15 minutes at 20 C. After washing with PBS, RPE and/or choroid complex were incubated in 0.2% Triton-X100 for 2 hours at 37 C. After blocking for 1 hour at 37 C, the RPE and/or choroid complex were incubated at 4 C overnight with primary antibody with rabbit antizonular occludens-1 (ZO-1) (1:100, Invitrogen) and for 1 hour at 37 C with mouse anti-Ab (4G8 or 12F4). After washing with PBS, the RPE and/or choroid complex were incubated for 2 hours at 37 C with secondary antibodies (Alexa 488 goat anti-rabbit IgG, 1:200 and Alexa 594 goat anti-mouse IgG, 1:300). After washing with PBS, flat mounts were counterstained with 10 mg/mL 4, 6-diamidino-2phenolindole (DAPI, Sigma Aldrich). After washing with PBS, the RPE and/or choroid complex were mounted with aqueous mounting medium and observed under fluorescence microscope (Nikon) and confocal microscope (Leica TCS STED, Leica Microsystems). 2.5. Preparation of oligomeric Ab42 solution Oligomeric Ab42 (OAb42) solution was generated as the previously described method (Moon et al., 2011). Ab42 and Ab42-1 (American peptide, Sunnyvale, CA, USA) were dissolved in hexafluoroisopropanol (Sigma Aldrich) to a final concentration of 1 mg/ mL at RT for 3 days. The peptide was aliquoted and dried under vacuum for 1 hour. The aliquoted peptide was dissolved in dimethyl sulfoxide to a final concentration of 1 mM. The protein concentration was measured using a BCA protein assay kit (Pierce, Rockford, IL, USA). The Ab42 stock in dimethyl sulfoxide was diluted directly into DMEM/F12 at 10 mM, and incubated at 4 C for 24 hours to make OAb42.
2.3. Histology
2.6. Cell viability assay
Mice were sacrificed at the age of 8 months (n ¼ 6) and 12 months (n ¼ 4). Mice were deeply anesthetized using a mixture of Zoletil 50 (Virbac, Carros, France) and Rompun (Bayer Korea, Seoul, Korea) (3:1 ratio, 1 mL/kg, intraperitoneally) and perfused transcardially with a fresh prepared 4% paraformaldehyde solution in phosphate-buffered saline (PBS). Enucleated eyes were fixed in 4% paraformaldehyde for 24 hours and embedded in paraffin for histologic analysis and cross section. Four-mm-thick serial sections were prepared from paraffin blocks. Sections were deparaffinized and hydrated by sequential immersion in xylene substitute and graded alcohol solutions. Then sections were stained with
Cell viability was determined by WST-1 (2-(4-Iodophenyl)-3(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt) assay. ARPE-19 cells were seeded into each well of 96well plates at a concentration of 5 103 cells/well. After incubation for 24 hours, cells were treated with either 1 mM Ab42-1 as a control or various concentration of Ab42 (0.01e10 mM) for 24 hours. Then, 10 mL of WST-1 solution was added into each well. After incubation at 37 C for 2 hours, absorption at 450 nm was measured using a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Three independent experiments were performed for each experimental condition.
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2.7. Immunocytochemistry
2.8. Western blotting
ARPE-19 cells with confluence were incubated with OAb42 (10 mM) for 24 hours. For the controls, ARPE-19 cells were incubated with Ab42-1 (10 mM). After removal of the medium, cells were intensively washed with PBS and fixed with 4% paraformaldehyde for 20 minutes at RT. Cells were washed with 0.05 % BSA in PBS. For the permeablization, 0.2% Triton X-100 in PBS was treated for 10 minutes. After washing, the cells were incubated in blocking solution for 1 hour at RT. Cells were incubated overnight at 4 C with rabbit anti-ZO-1 (1:100, Invitrogen), mouse anti-Ab (4G8 or 12F4, 1:200, Covance), and Alexa 488 conjugated phalloidin (1:500, Molecular Probes). After washing, cells were incubated for 1 hour at RT with secondary antibodies (Alexa 488 donkey anti-rabbit IgG, Alexa 594 donkey anti-mouse IgG, 1:200, Molecular Probes). Nucleus was counterstained with DAPI for 10 minutes at RT. After washing, the slides were mounted with aqueous mounting medium (DAKO) and observed under fluorescence microscope (BX50, OLYMPUS).
ARPE-19 cells were incubated with 10 mM OAb42 and Ab42-1 for 24 hour. Cell proteins were extracted with RIPA buffer (Tris 50 mM pH 7.4; NaCl 150 mM; SDS 0.1%; NaDeoxycholate 0.5%; Triton X-100 1%) with a complete protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes (GE healthcare, Piscataway, NJ, USA). After blocking in 5% skim milk in PBST (0.1% Tween 20 in PBS), the membranes were incubated with primary antibodies for ZO-1 (1:1000), ZO-2 (1:1000), occludin (1:2000), claudin-1 (1:1000), and actin (1:5000). The band intensity analyzed using ImageJ 1.40 software (National Institutes of Health, Bethesda, MD, USA). 2.9. Transepithelial electrical resistance measurement The measurement of transepithelial electrical resistance (TER) was performed by impedance analysis using cellZscope (NanoAnalytics GmbH, Munster, Germany) (Bornhorst et al., 2012).
Fig. 1. Retinal sections of 12-month-old 5XFAD mice show the features of dry AMD in the outer retina and intracellular Ab in RPE. The outer segment/retinal pigment epithelium (RPE)/choroid interface of cross sections are evaluated in 12-month old control and transgenic mice with 5 familial autosomal dominant mutations (5XFAD). In hematoxylin and eosin (H&E)-stained retinal sections, (A) WT mice show normal RPE layer and Bruch membrane. (B) Those of 5XFAD mice show large vacuoles (arrows), hypopigmentation and thick Bruch membrane (white arrowheads). Black arrowheads indicate sub-RPE drusen-like deposit. In retinal sections immunostained with Ab (red), (C) WT mice show no Ab accumulates in the RPE layer. (D) Intracellular Ab accumulates in the RPE layer of 5XFAD mice. There is a significant increase in Ab immunoreactivity in RPE layer (arrows). White arrowheads indicate Bruch membrane with Ab. Nuclei were counterstained for DAPI. (A, B) Magnification 1000. Scale bar ¼ 5 mm. (C, D) Magnification 1600. Scale bar ¼ 20 mm. Abbreviations: Ab, amyloid beta; AMD, age-related macular degeneration; DAPI, 4, 6-diamidino-2-phenolindole; OS, outer segment; Tg, 5XFAD transgenic mice; WT, wild type.
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Briefly, the cell covered transwell filters (0.4 mm, Corning Inc, NY, USA) were placed in this setup using 12-well plate. After stabilization of TER value on plateau, TER was measured with 10 mM OAb42 and Ab42-1 over 24 hours. The TER value just after treatment was normalized to 100% for relative analysis. 2.10. Statistical analysis Statistical analyses were performed using SPSS software version 18.0 (SPSS Inc, Chicago, IL, USA). p < 0.05 were considered to be statistically significant. Figures are depicted as mean SEM. 3. Results 3.1. The 5XFAD mice show the features of dry AMD in the outer retina with intracellular Ab in RPE It is known that Ab deposits lead to retinal degeneration similar to dry AMD in AD mouse retina (Ning et al., 2008). Among AD mouse models, 5xFAD mice showed the highest retinal abundance of Ab42 (Alexandrov et al., 2011). However, the retinal pathology focusing on the RPE of 5XFAD mice has not been described yet. To
evaluate whether 5XFAD mice show the features of dry AMD, light microscopic findings with H&E were evaluated in the 5XFAD mice retina. Retinas of 12-month-old 5XFAD mice were different from those of control littermate mice. Similar to senescent neprilysin geneedisrupted mice (Yoshida et al., 2005), 5XFAD mice had large vacuoles, hypopigmentation in the RPE layer, and thickened Bruch membrane (Fig. 1A and B). Of importance, 5XFAD mice had drusenlike deposit between RPE and Bruch membrane (Fig. 1B). In addition to thickened Bruch membrane with Ab deposits beneath the RPE layer, we found that retinal Ab accumulated in the RPE layer of 5XFAD mice (Fig. 1C and D). 3.2. Intracellular Ab accumulates in RPE and attenuates the tight junction of RPE in 5XFAD mice To visualize typical tight junction integrity of RPE cells, the flat mounts of RPE and/or choroid complex were performed and immunostained for the tight junction proteins and Ab (Fig. 2, Supplementary Fig. 1). In 8-month-old 5XFAD mice, abundant intracellular Ab (Fig. 2B and D) were detected in the cytosol of RPE compared with control mice (Fig. 2A and C). With increasing intracellular accumulation of Ab in the RPE cell, ZO-1 labeled tight
Fig. 2. Intracellular Ab accumulates in the retinal pigment epithelium (RPE) and attenuates RPE tight junction integrity in 5XFAD transgenic mice. (A, B) Representative flat mounts of RPE of WT (A) and Tg (B) mice stained for Ab42 (red) and ZO-1 (green). There is a significant increase in Ab42 immunoreactivity in the RPE of Tg retina with an attenuated and disorganized expression in ZO-1 (arrows). (C, D) Representative flat mounts of RPE of WT (C) and Tg (D) mice stained for Ab42 (red) and occludin (green). Occludin integrity is severely hampered where Ab42 immunoreactivity is detected in the RPE of Tg retina (arrows). All sections were counterstained for DAPI. Magnification 400. Scale bar ¼ 20 mm. Abbreviations: Ab, amyloid beta; DAPI, 4, 6-diamidino-2-phenolindole; Tg, 5XFAD transgenic mice; WT, wild type.
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junction integrity was attenuated and disorganized (Fig. 2B) compared with the typical hexagonal shape of RPE tight junction in control mice (Fig. 2A). In addition, occludin labeled tight junction integrity was significantly attenuated in 5XFAD mice (Fig. 2D) than those in controls (Fig. 2C). 3.3. Exogenous oligomeric Ab42 is taken up by RPE and intracellular Ab42 attenuate tight junction integrity of ARPE-19 cells To investigate the role of Ab on the tight junction in RPE cells, we performed in vitro study. First, WST-1 assay was performed to determine the concentration of Ab with sublethal cytotoxicity. OAb42, which is known to have more potent cytotoxic effect than Ab42 monomer did not affect the viability of ARPE-19 cells up to 10 mM (Supplementary Fig. 2). Then, we performed immunocytochemistry for Ab and tight junction protein ZO-1 and occludin in ARPE-19 cells. After 24 hours of exogenous OAb42 treatment, Ab was abundantly detected in RPE cells, mainly in the intracellular area under confocal microscopic evaluation (Supplementary Fig. 3). Furthermore, we confirmed that both ZO-1 and occludin were decreased and lost their integrity as tight junction in ARPE-19 cells with intracellular OAb42 (Fig. 3B and D). On the other hand,
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ARPE-19 cells treated with reverse Ab42-1, an inactive control peptide, showed well-organized tight junction with no detectable Ab42-1 accumulation. RPE took up exogenous oligomeric Ab42 time dependent manner (Fig. 4AeD). RPE cells showed relatively conserved ZO-1 integrity at 6 hours (Fig. 4E and F). Intracellular Ab42 dramatically increased after 12 hours and Ab-laden RPE cells showed disrupted tight junction after 12 hours (Fig. 4G and H). Intracellular Ab42 induced tight junction breakdown without cell retraction (Fig. 4IeL). 3.4. Oligomeric Ab42 alters the tight junction protein in ARPE19 cells We also performed the Western blot assay to evaluate the change of tight junction protein with OAb42. ARPE-19 cells were treated with OAb42 10 mM or inactive reverse control peptide Ab42-1 as a control. After 24 hours of OAb42 10 mM treatment, while claudin-1 level was not changed, tight junction proteins including ZO-1, ZO-2, and occludin were decreased (Fig. 5A and B). In addition, OAb42 decreased occludin time dependent manner (Fig. 5C). In accordance with this data, TER with OAb42 10 mM was sustained for 6 hours, then declined abruptly (Fig. 5D).
Fig. 3. Exogenous oligomeric Ab42 is taken up by RPE and intracellular Ab42 attenuate tight junction integrity of ARPE-19 cells. Immunocytochemistry of Ab42 (red) and tight junction proteins (green) ZO-1 (A, B) and occludin (C, D) in ARPE-19 cells. ARPE-19 cells were incubated for 24 hours with (A, C) reverse Ab42-1 10 mM, and (B, D) OAb42 10 mM. (A, C) RPE cells show integrated and organized hexagonal shape with ZO-1 (A) and Occludin (C) expression as tight junctions without intracellular uptake of Ab. (B, D) RPE cells show intracellular distribution of Ab42 (red) 24 hours after exogenous OAb42 treatment. Ab uptaken RPE cells show disintegrated and disorganized hexagonal shape with attenuated ZO-1 and occludin expression as tight junctions. Arrows indicate disrupted tight junction. Magnification 400. Scale bar ¼ 20 mm. Abbreviations: Ab, amyloid beta; RPE, retinal pigment epithelium.
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Fig. 4. RPE takes up exogenous oligomeric Ab42 time dependent manner and intracellular Ab42 attenuate tight junction integrity. Immunocytochemistry were performed for Ab, tight junction protein ZO-1, and cytoskeleton F-actin. ARPE-19 cells were incubated for 24 hours with OAb42 10 mM. (A, B, C, D) Z-stack images were reconstructed with X and Y-axis orthogonal views under confocal microscopy. RPE takes up exogenous oligomeric Ab42 time dependent manner. Intracellular Ab42 increases after 12 hours. (E, F, G, H) RPE cells show relatively conserved ZO-1 integrity at 6 hours (F). (G, H) Ab-laden RPE cells show disrupted tight junction after 12 hours. Arrows indicate disrupted tight junction. (I, J, K, L) Ab induces tight junction breakdown without cell retraction. Arrows indicate disrupted tight junction. Magnification 1000. Scale bar ¼ 20 mm. Abbreviations: Ab, amyloid beta; RPE, retinal pigment epithelium.
4. Discussion In this study, we demonstrated that intracellular Ab was expressed in the RPE layer of 5XFAD mice, which showed characteristic features of dry AMD. With intracellular Ab42, tight junction of RPE in 5XFAD mice was attenuated. Intriguingly, exogenous Ab could be taken up by RPE cells and subsequently induce breakdown of the tight junction. Thus, intracellular Ab42 could play an important role in the pathogenesis of dry AMD. The 5XFAD mice could be used as an animal model for the study of the relationship between dry AMD and AD with regard to Ab related pathogenesis. AMD is the leading cause of vision loss in adults older than 65 years in the developed countries. Both AD and AMD share common risk factors and suggestive pathogenesis. Aging is a major risk factor of both AD and AMD. Despite controversial debates in the role of ApoE4 and complement factor H as common risk factors in both AD and AMD (Adams et al., 2012; Le Fur et al., 2010; Malek et al., 2005; Proitsi et al., 2012), Ab has been consistently considered as a pathologic molecule. Ab42 is more fibrillogenic than shorter Ab40,
and elevated Ab42 and conversion of Ab from monomeric form to oligomeric or aggregated form in the brain are considered key events in the pathogenesis of AD. Among the AD mice line, we used 5XFAD (Tg6799), the line with the highest Ab42 levels and intraneural Ab in the brain than others (Oakley et al., 2006). The 5XFAD mice generated Ab42 almost exclusively in the brain and retina (Alexandrov et al., 2011). Intracellular Ab42 accumulated in 5XFAD mice brain before plaque formation, implying potential role of intracellular Ab in the pathogenesis of AD (Alexandrov et al., 2011; Moon et al., 2012). In this study, we found intracellular Ab in the RPE and some characteristic features of dry AMD such as hypopigmentation, large vacuoles, Bruch membrane thickening, and drusen like sub-RPE deposit in aged 5XFAD mice. Although 5XFAD mice did not show all the dry AMD features, there is no model that recapitulated all the features of human AMD yet (Pennesi et al., 2012). Thus, 5XFAD mice could be used as an animal model for dry AMD, especially studying the Ab related pathogenesis in AMD. Regarding to differentiate Ab42 from amyloid beta precursor protein (AbPP) or other Ab peptides, consistent with previous
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Fig. 5. Oligomeric Ab42 alters the tight junction protein in ARPE-19 cells. (A, B) ARPE-19 cells were incubated for 24 hours with 10 mM oligomeric Ab42 (OAb42) and inactive reverse peptide Ab42-1 as a control. (A) Tight junction proteins (ZO-1, ZO-2, occludin, and claudin-1) were evaluated by Western blot. b-actin was used as an internal control. (B) Quantitative analysis was performed by measuring the densitometry of each band relative to b-actin. Each value represents the mean (SEM) of 3 independent experiments (* p < 0.05, # p > 0.05). (C) OAb42 induced significant decrease in occludin levels in a time-dependent manner in ARPE-19 cells. Cells were incubated for 6, 12, and 24 hours with 10 mM OAb42 and inactive reverse peptide Ab42-1 as a control. Occludin expression was evaluated by Western blot. b-actin was used as an internal control. (D) Transepithelial electrical resistance (TER) was measured and normalized to the TER value just after Ab treatment. Abbreviations: Ab, amyloid beta; SEM, standard error of the mean.
studies in 5XFAD mice (Jawhar et al., 2012; Oakley et al., 2006), we showed the accumulation of 4G8-labeled intracellular Ab in the early stages of plaque formation in this mouse model (Moon et al., 2012). Although 4G8 is known to react not only with Ab but also with AbPP and its proteolytic derivatives, 4G8 antibody was needed to detect intracellular Ab, and thereof compare our results clearly with previous studies on intracellular Ab that used this antibody. We previously demonstrated that 4G8-stained hollow type plaques with nuclear remnants result from neuron-derived Ab, not from cross-reactivity with AbPP using an end-specific Ab antibody (Ab42) (Moon et al., 2012). Furthermore, 5XFAD specifically increased the expression of Ab42 and we used exogenous Ab42 in in vitro experiments. Thus, both 4G8 antibody and 12F4 antibody effectively detected intracellular Ab in this study. In the brain of AD patients, uptake of extracellular Ab into neurons and intracellular production of Ab could lead to the accumulation of intracellular Ab, which in turn may gradually compromise neuronal function, by disrupting the Ab-degrading pathway or by imparing the Ab secretion of the neurons. As a result, highly Ab-laden neurons may undergo death and lysis, releasing intracellular Ab into the surrounding extracellular space, and this becomes a seed for extracellular deposition. Same as in the brain
neuronal cells, we suggest that intracellular Ab accumulation could occur in the RPE cells as well by taking up extracellular Ab, or producing Ab intracellularly. This would disrupt the outer brain retinal barrier and potentially cause AMD. Although the mechanism of intracellular uptake of exogenous Ab in RPE cells is not well established yet, several endocytotic pathways were suggested as possible neuronal uptakes of soluble Ab. They include a7 nicotinic acetylcholine (a7-NAch) receptor, N-methyl D-asparatate receptor, low-density lipoprotein receptorrelated protein 1 and/or APOE, and receptor for advanced glycosylated end products (RAGE) (Omtri et al., 2012). The RAGE, a member of the immunoglobulin superfamily, is expressed ubiquitously in brain (Neeper et al., 1992; Schmidt et al., 2001). It is known to be present in RPE cells (Zhou et al., 2005) and is up-regulated in AMD (Howes et al., 2004). RAGE mediates Ab transport from the periphery to the central nervous system across the blood-brain barrier (Deane et al., 2003; Mackic et al., 1998), and the interaction between Ab and RAGE is believed to regulate the pathogenesis of AD (Lue et al., 2001). Ab and/or RAGE-mediated signaling contributes to Ab transport and Ab internalization in neurons (Takuma et al., 2009). Ab induced occludin down-regulation is mediated by MAPK activation in hCMEC/D3 cells (Tai et al., 2010). Thus, we
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postulated that Ab uptake might be mediated by RAGE-mediated endocytosis in RPE. However, the precise mechanism of RPE uptake of Ab should be investigated in the further study. If Ab uptake is mediated by RAGE, anti-RAGE therapy could block the intracellular accumulation of Ab and subsequently block the breakdown of tight junction in RPE. In the present study, we for the first time demonstrated that intracellular Ab contributed to the breakdown of outer blood retinal barrier in 5XFAD mice. Thus, intracellular Ab might play an important role in the pathogenesis of dry AMD. Also, we suggested that 5XFAD mice could be a mouse model of AMD with regard to the Ab42 related pathology and used for the study of the relationship between AD and AMD. Disclosure statement The authors declare no actual or potential conflicts of interest. All animals used in this study were cared and treated in strict agreement with the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research. Acknowledgements This work was supported by the Bio-Signal Analysis Technology Innovation Program (2009e0090895), the Pioneer Research Program (2012e0009544), the Global Research Laboratory Program of NRF/MEST (2011e0021874) of NRF/MEST, and the Seoul National University Research Grant (800e20130338). Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging. 2014.03.008. References Adams, M.K., Simpson, J.A., Richardson, A.J., English, D.R., Aung, K.Z., Makeyeva, G.A., Guymer, R.H., Giles, G.G., Hopper, J., Robman, L.D., Baird, P.N., 2012. Apolipoprotein E gene associations in age-related macular degeneration: the Melbourne Collaborative Cohort Study. Am. J. Epidemiol. 175, 511e518. Alexandrov, P.N., Pogue, A., Bhattacharjee, S., Lukiw, W.J., 2011. Retinal amyloid peptides and complement factor H in transgenic models of Alzheimer’s disease. Neuroreport 22, 623e627. Bornhorst, J., Wehe, C.A., Huwel, S., Karst, U., Galla, H.J., Schwerdtle, T., 2012. Impact of manganese on and transfer across blood-brain and blood-cerebrospinal fluid barrier in vitro. J. Biol. Chem. 287, 17140e17151. Bruban, J., Glotin, A.L., Dinet, V., Chalour, N., Sennlaub, F., Jonet, L., An, N., Faussat, A.M., Mascarelli, F., 2009. Amyloid-beta(1-42) alters structure and function of retinal pigmented epithelial cells. Aging Cell 8, 162e177. Deane, R., Du Yan, S., Submamaryan, R.K., LaRue, B., Jovanovic, S., Hogg, E., Welch, D., Manness, L., Lin, C., Yu, J., Zhu, H., Ghiso, J., Frangione, B., Stern, A., Schmidt, A.M., Armstrong, D.L., Arnold, B., Liliensiek, B., Nawroth, P., Hofman, F., Kindy, M., Stern, D., Zlokovic, B., 2003. RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain. Nat. Med. 9, 907e913. Dentchev, T., Milam, A.H., Lee, V.M., Trojanowski, J.Q., Dunaief, J.L., 2003. Amyloidbeta is found in drusen from some age-related macular degeneration retinas, but not in drusen from normal retinas. Mol. Vis. 9, 184e190. Howes, K.A., Liu, Y., Dunaief, J.L., Milam, A., Frederick, J.M., Marks, A., Baehr, W., 2004. Receptor for advanced glycation end products and age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 45, 3713e3720. Jawhar, S., Trawicka, A., Jenneckens, C., Bayer, T.A., Wirths, O., 2012. Motor deficits, neuron loss, and reduced anxiety coinciding with axonal degeneration and intraneuronal Abeta aggregation in the 5XFAD mouse model of Alzheimer’s disease. Neurobiol. Aging 33, 196.e29e196.e40. Johnson, L.V., Leitner, W.P., Rivest, A.J., Staples, M.K., Radeke, M.J., Anderson, D.H., 2002. The Alzheimer’s A beta -peptide is deposited at sites of complement activation in pathologic deposits associated with aging and age-related macular degeneration. Proc. Natl. Acad. Sci. U.S.A 99, 11830e11835.
Kook, S.Y., Hong, H.S., Moon, M., Ha, C.M., Chang, S., Mook-Jung, I., 2012. Abeta(1)(-)(4)(2)-RAGE interaction disrupts tight junctions of the blood-brain barrier via Ca(2)(þ)-calcineurin signaling. J. Neurosci. 32, 8845e8854. LaFerla, F.M., Green, K.N., Oddo, S., 2007. Intracellular amyloid-beta in Alzheimer’s disease. Nat. Rev. Neurosci. 8, 499e509. Le Fur, I., Laumet, G., Richard, F., Fievet, N., Berr, C., Rouaud, O., Delcourt, C., Amouyel, P., Lambert, J.C., 2010. Association study of the CFH Y402H polymorphism with Alzheimer’s disease. Neurobiol. Aging 31, 165e166. Lue, L.F., Walker, D.G., Brachova, L., Beach, T.G., Rogers, J., Schmidt, A.M., Stern, D.M., Yan, S.D., 2001. Involvement of microglial receptor for advanced glycation endproducts (RAGE) in Alzheimer’s disease: identification of a cellular activation mechanism. Exp. Neurol. 171, 29e45. Luibl, V., Isas, J.M., Kayed, R., Glabe, C.G., Langen, R., Chen, J., 2006. Drusen deposits associated with aging and age-related macular degeneration contain nonfibrillar amyloid oligomers. J. Clin. Invest. 116, 378e385. Mackic, J.B., Stins, M., McComb, J.G., Calero, M., Ghiso, J., Kim, K.S., Yan, S.D., Stern, D., Schmidt, A.M., Frangione, B., Zlokovic, B.V., 1998. Human blood-brain barrier receptors for Alzheimer’s amyloid-beta 1-40. Asymmetrical binding, endocytosis, and transcytosis at the apical side of brain microvascular endothelial cell monolayer. J. Clin. Invest. 102, 734e743. Malek, G., Johnson, L.V., Mace, B.E., Saloupis, P., Schmechel, D.E., Rickman, D.W., Toth, C.A., Sullivan, P.M., Bowes Rickman, C., 2005. Apolipoprotein E alleledependent pathogenesis: a model for age-related retinal degeneration. Proc. Natl. Acad. Sci. U.S.A 102, 11900e11905. Moon, M., Choi, J.G., Nam, D.W., Hong, H.S., Choi, Y.J., Oh, M.S., Mook-Jung, I., 2011. Ghrelin ameliorates cognitive dysfunction and neurodegeneration in intrahippocampal amyloid-beta1-42 oligomer-injected mice. J. Alzheimers Dis. 23, 147e159. Moon, M., Hong, H.S., Nam, D.W., Baik, S.H., Song, H., Kook, S.Y., Kim, Y.S., Lee, J., Mook-Jung, I., 2012. Intracellular amyloid-beta accumulation in calcium-binding protein-deficient neurons leads to amyloid-beta plaque formation in animal model of Alzheimer’s disease. J. Alzheimers Dis. 29, 615e628. Neeper, M., Schmidt, A.M., Brett, J., Yan, S.D., Wang, F., Pan, Y.C., Elliston, K., Stern, D., Shaw, A., 1992. Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins. J. Biol. Chem. 21, 14998e15004. Ning, A., Cui, J., To, E., Ashe, K.H., Matsubara, J., 2008. Amyloid-beta deposits lead to retinal degeneration in a mouse model of Alzheimer disease. Invest. Ophthalmol. Vis. Sci. 49, 5136e5143. Oakley, H., Cole, S.L., Logan, S., Maus, E., Shao, P., Craft, J., Guillozet-Bongaarts, A., Ohno, M., Disterhoft, J., Van Eldik, L., Berry, R., Vassar, R., 2006. Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation. J. Neurosci. 26, 10129e10140. Ohno-Matsui, K., 2011. Parallel findings in age-related macular degeneration and Alzheimer’s disease. Prog. Retin. Eye Res. 30, 217e238. Omtri, R.S., Davidson, M.W., Arumugam, B., Poduslo, J.F., Kandimalla, K.K., 2012. Differences in the cellular uptake and intracellular itineraries of amyloid beta proteins 40 and 42: ramifications for the Alzheimer’s drug discovery. Mol. Pharm 9, 1887e1897. Pennesi, M.E., Neuringer, M., Courtney, R.J., 2012. Animal models of age related macular degeneration. Mol. Aspects Med. 33, 487e509. Proitsi, P., Lupton, M.K., Dudbridge, F., Tsolaki, M., Hamilton, G., Daniilidou, M., Pritchard, M., Lord, K., Martin, B.M., Johnson, J., Craig, D., Todd, S., McGuinness, B., Hollingworth, P., Harold, D., Kloszewska, I., Soininen, H., Mecocci, P., Velas, B., Gill, M., Lawlor, B., Rubinsztein, D.C., Brayne, C., Passmore, P.A., Williams, J., Lovestone, S., Powell, J.F., 2012. Alzheimer’s disease and age-related macular degeneration have different genetic models for complement gene variation. Neurobiol. Aging 33, 1843.e9e1843.e17. Rosvall, L., Rizzuto, D., Wang, H.X., Winblad, B., Graff, C., Fratiglioni, L., 2009. APOErelated mortality: effect of dementia, cardiovascular disease and gender. Neurobiol. Aging 30, 1545e1551. Schmidt, A.M., Yan, S.D., Yan, S.F., Stern, D.M., 2001. The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses. J. Clin. Invest. 108, 949e955. Strohmeyer, R., Shen, Y., Rogers, J., 2000. Detection of complement alternative pathway mRNA and proteins in the Alzheimer’s disease brain. Brain Res. Mol. Brain Res. 81, 7e18. Tai, L.M., Holloway, K.A., Male, D.K., Loughlin, A.J., Romero, I.A., 2010. Amyloid-betainduced occludin down-regulation and increased permeability in human brain endothelial cells is mediated by MAPK activation. J. Cell Mol. Med. 14, 1101e1112. Takuma, K., Fang, F., Zhang, W., Yan, S., Fukuzaki, E., Du, H., Sosunov, A., McKhann, G., Funatsu, Y., Nakamichi, N., Nagai, T., Mizoguchi, H., Ibi, D., Hori, O., Ogawa, S., Stern, D.M., Yamada, K., Yan, S.S., 2009. RAGE-mediated signaling contributes to intraneuronal transport of amyloid-beta and neuronal dysfunction. Proc. Natl. Acad. Sci. U.S.A 106, 20021e20026. Yoshida, T., Ohno-Matsui, K., Ichinose, S., Sato, T., Iwata, N., Saido, T.C., Hisatomi, T., Mochizuki, M., Morita, I., 2005. The potential role of amyloid beta in the pathogenesis of age-related macular degeneration. J. Clin. Invest. 115, 2793e2800. Zhou, J., Cai, B., Jang, Y.P., Pachydaki, S., Schmidt, A.M., Sparrow, J.R., 2005. Mechanisms for the induction of HNE- MDA- and AGE-adducts, RAGE and VEGF in retinal pigment epithelial cells. Exp. Eye Res. 80, 567e580.
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Intracellular amyloid beta alters the tight junction of retinal pigment epithelium in 5XFAD mice Sung Wook Park a, b, Jin Hyoung Kim a, Inhee Mook-Jung b, Kyu-Won Kim c, Woo Jin Park d, Kyu Hyung Park e, f, *, Jeong Hun Kim a, b, e, ** a
Fight against Angiogenesis-Related Blindness Laboratory, Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Korea SNU-Harvard NeuroVascular Protection Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea d Department of Life Sciences, Life Sciences Concentration GIST (Gwangju Institute of Science and Technology), Gwangju, Korea e Department of Ophthalmology, College of Medicine, Seoul National University, Seoul, Korea f Department of Ophthalmology, Seoul National University Bundang Hospital, Seongnam, Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 August 2013 Received in revised form 21 January 2014 Accepted 11 March 2014
Extracellular deposit of amyloid beta (Ab) is a common pathologic feature in both age-related macular degeneration (AMD) and Alzheimer’s disease, but the role of intracellular Ab on the tight junction of the retinal pigment epithelium (RPE) is unknown. In this study, we investigated the intracellular Ab expression and its role on the outer blood retinal barrier in the retina of 5XFAD mice, a mouse model of Alzheimer’s disease. The retina of 5XFAD mice showed the pathologic features of AMD with intracellular Ab in the RPE. As intracellular Ab accumulated, zonular occludens-1 and occludin were markedly attenuated and lost their integrity as tight junctions in the RPE of 5XFAD mice. Also, Ab42 uptake by ARPE-19 cells induced the tight junction breakdown of zonular occludens-1 and occludin without cell death. These results implicate that intracellular Ab42 could play a role in the breakdown of the outer blood retinal barrier in 5XFAD mice. Thus, we suggested that 5XFAD mice could be a mouse model of dry AMD with regard to the Ab42 related pathology. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Aging Alzheimer’s disease Ab Age-related macular degeneration Outer blood retinal barrier Tight junction Retina Retinal pigment epithelium Transgenic mice
1. Introduction Both age-related macular degeneration (AMD) and Alzheimer’s disease (AD) increase dramatically with aging, and it has been suggested that they might share common risk factors and pathogenesis (Ohno-Matsui, 2011). The extracellular deposit of amyloid beta (Ab) forms senile plaque in the brain, and it is one of the major hallmarks of AD. These deposits are also localized within the drusen * Corresponding author at: Kyu Hyung Park, MD, Department of Ophthalmology, Seoul National University College of Medicine, Seoul National University Bundang Hospital, 166 Gumiro, Bundang-gu, Seongnam, Gyeonggi-do 463-707, Korea. Tel.: þ82 31 787 7373; fax: þ82 31 787 4057 ** Corresponding author at: Jeong Hun Kim, MD, Fight against AngiogenesisRelated Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, and Department of Ophthalmology and Biomedical Sciences, College of Medicine, Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul 110-799, Korea. Tel.: þ82 2 2072 2438; fax: þ82 2 741 3187 E-mail addresses: [email protected] (K.H. Park), [email protected] (J.H. Kim). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.03.008
between the basement membrane of the retinal pigment epithelium (RPE) and the Bruch membrane in the human retina of AMD (Johnson et al., 2002; Luibl et al., 2006) and the mouse retina of AD model (Alexandrov et al., 2011). Furthermore, Ab is only found in the drusen from the eyes of the AMD patients (Dentchev et al., 2003). The ε4 allele of apolipoprotein E (APOE), the major genetic risk factor for AD (Rosvall et al., 2009), may be also associated with AMD (Adams et al., 2012; Malek et al., 2005). In addition, complement factor H, the main genetic risk factor of AMD, has been detected in Ab plaques in the brain of AD patients (Strohmeyer et al., 2000) and decreased in the retina of AD mouse model (Alexandrov et al., 2011). Although the classical concept suggests that Ab is deposited extracellularly, growing evidence from AD patients and transgenic mice indicates that Ab can also accumulate intracellularly and it may contribute to disease progression (LaFerla et al., 2007). In the neprilysin gene disrupted mice, Ab was localized within the subretinal deposits as well as in the cytoplasm of RPE (Yoshida et al.,
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2005). It has been also shown that extracellular Ab could induce breakdown of the tight junction in RPE (Bruban et al., 2009). However, the role of intracellular Ab in RPE has not been elucidated yet. Recently, we reported that intracellular Ab occurred before the formation of extracellular amyloid plaques in the subiculum of 5XFAD mice (Moon et al., 2012). In addition, 5XFAD mouse brain showed alterations in cerebral tight junctions (Kook et al., 2012). These findings suggest that accumulation of intracellular Ab might be an early process that triggers neuronal damage and alterations in outer blood retinal barrier in AD retina. While previous studies revealed the characteristics of the AD mouse retina, the retinal characteristics of 5XFAD mice was not evaluated except for exclusively high level of Ab42 (Alexandrov et al., 2011; Ning et al., 2008; Yoshida et al., 2005). We postulated that intracellular Ab might have an important role in the retina of 5XFAD mice. In the present study, we focused on the role of intracellular Ab and demonstrated that Ab was intracellularly expressed in the RPE of 5XFAD mice and that it attenuated the integrity of outer bloodretinal barrier. Taken together, our results suggest that intracellular Ab could be important in the pathogenesis of dry AMD and retinal aging in AD. 2. Methods 2.1. Transgenic mice This study used 5XFAD mice that were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Mice (5XFAD) overexpress mutant human amyloid precursor protein 695 with the Swedish mutation (K670N, M671L: elevate the production of total Ab), Florida mutation (I716V: elevates the production of Ab42 specifically), and London mutation (V717I: elevates the production of Ab42 specifically) and human presenilin 1 with 2 FAD mutations (M146L and L286V: elevate the production Ab42 specifically) (Oakley et al., 2006). All 5XFAD transgenic mice (Tg6799, B6 ⁄ SJL hybrid background) used were heterozygotes with respect to the transgene, and nontransgenic wild-type littermate mice served as controls. Care, use, and treatment of all animals in this study were in strict agreement with the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research. 2.2. Cell cultures ARPE-19 cells (American Type Culture Collection, Manassas, VA, USA) were used for human RPE cells. The cells were routinely maintained in Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (DMEM/F12) containing 10% fetal bovine serum, 100 U/ mL penicillin, and 100 mg/mL streptomycin. All culture reagents were purchased from Invitrogen (Carlsbad, CA, USA).
hematoxylin and eosin (H&E) for histologic examination via light microscopy (Nikon, Tokyo, Japan). 2.4. Immunofluorescence staining For immunofluorescence of cross-section of the retina, sections were treated with proteinase K (Sigma Aldrich, St Louis, MO, USA) for 20 minutes at 37 C and 70% formic acid (Sigma Aldrich) for 30 minutes at room temperature (RT). Then they were rinsed with PBS and treated with 0.2% Triton-X100 for 20 minutes at 37 C. After getting rinsed with PBS, the sections were treated with blocking solution (Vector Laboratories, Inc, Burlingame, CA, USA) for 1 hour at 37 C. Slides were incubated with mouse anti-Ab (4G8 or 12F4, 1:100, Covance, Princeton, NJ, USA) for 90 minutes at 37 C. Alexa 594 donkey anti-mouse IgG (1:400, Molecular Probes, Inc, Eugene, OR, USA) were used as secondary antibodies. The slides were mounted with aqueous mounting medium (DAKO, Glostrup, Denmark) and observed under fluorescence microscope (Nikon) and confocal microscopes (LSM 510 Meta, Carl Zeiss, Germany, and Leica TCS STED, Leica Microsystems, Germany). For the flat mount of RPE and/or choroid complex, enucleated eyes were dissected to remove neural retina, the RPE and/or choroid complex were gently flat mounted and fixed in methanol for 15 minutes at 20 C. After washing with PBS, RPE and/or choroid complex were incubated in 0.2% Triton-X100 for 2 hours at 37 C. After blocking for 1 hour at 37 C, the RPE and/or choroid complex were incubated at 4 C overnight with primary antibody with rabbit antizonular occludens-1 (ZO-1) (1:100, Invitrogen) and for 1 hour at 37 C with mouse anti-Ab (4G8 or 12F4). After washing with PBS, the RPE and/or choroid complex were incubated for 2 hours at 37 C with secondary antibodies (Alexa 488 goat anti-rabbit IgG, 1:200 and Alexa 594 goat anti-mouse IgG, 1:300). After washing with PBS, flat mounts were counterstained with 10 mg/mL 4, 6-diamidino-2phenolindole (DAPI, Sigma Aldrich). After washing with PBS, the RPE and/or choroid complex were mounted with aqueous mounting medium and observed under fluorescence microscope (Nikon) and confocal microscope (Leica TCS STED, Leica Microsystems). 2.5. Preparation of oligomeric Ab42 solution Oligomeric Ab42 (OAb42) solution was generated as the previously described method (Moon et al., 2011). Ab42 and Ab42-1 (American peptide, Sunnyvale, CA, USA) were dissolved in hexafluoroisopropanol (Sigma Aldrich) to a final concentration of 1 mg/ mL at RT for 3 days. The peptide was aliquoted and dried under vacuum for 1 hour. The aliquoted peptide was dissolved in dimethyl sulfoxide to a final concentration of 1 mM. The protein concentration was measured using a BCA protein assay kit (Pierce, Rockford, IL, USA). The Ab42 stock in dimethyl sulfoxide was diluted directly into DMEM/F12 at 10 mM, and incubated at 4 C for 24 hours to make OAb42.
2.3. Histology
2.6. Cell viability assay
Mice were sacrificed at the age of 8 months (n ¼ 6) and 12 months (n ¼ 4). Mice were deeply anesthetized using a mixture of Zoletil 50 (Virbac, Carros, France) and Rompun (Bayer Korea, Seoul, Korea) (3:1 ratio, 1 mL/kg, intraperitoneally) and perfused transcardially with a fresh prepared 4% paraformaldehyde solution in phosphate-buffered saline (PBS). Enucleated eyes were fixed in 4% paraformaldehyde for 24 hours and embedded in paraffin for histologic analysis and cross section. Four-mm-thick serial sections were prepared from paraffin blocks. Sections were deparaffinized and hydrated by sequential immersion in xylene substitute and graded alcohol solutions. Then sections were stained with
Cell viability was determined by WST-1 (2-(4-Iodophenyl)-3(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt) assay. ARPE-19 cells were seeded into each well of 96well plates at a concentration of 5 103 cells/well. After incubation for 24 hours, cells were treated with either 1 mM Ab42-1 as a control or various concentration of Ab42 (0.01e10 mM) for 24 hours. Then, 10 mL of WST-1 solution was added into each well. After incubation at 37 C for 2 hours, absorption at 450 nm was measured using a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Three independent experiments were performed for each experimental condition.
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2.7. Immunocytochemistry
2.8. Western blotting
ARPE-19 cells with confluence were incubated with OAb42 (10 mM) for 24 hours. For the controls, ARPE-19 cells were incubated with Ab42-1 (10 mM). After removal of the medium, cells were intensively washed with PBS and fixed with 4% paraformaldehyde for 20 minutes at RT. Cells were washed with 0.05 % BSA in PBS. For the permeablization, 0.2% Triton X-100 in PBS was treated for 10 minutes. After washing, the cells were incubated in blocking solution for 1 hour at RT. Cells were incubated overnight at 4 C with rabbit anti-ZO-1 (1:100, Invitrogen), mouse anti-Ab (4G8 or 12F4, 1:200, Covance), and Alexa 488 conjugated phalloidin (1:500, Molecular Probes). After washing, cells were incubated for 1 hour at RT with secondary antibodies (Alexa 488 donkey anti-rabbit IgG, Alexa 594 donkey anti-mouse IgG, 1:200, Molecular Probes). Nucleus was counterstained with DAPI for 10 minutes at RT. After washing, the slides were mounted with aqueous mounting medium (DAKO) and observed under fluorescence microscope (BX50, OLYMPUS).
ARPE-19 cells were incubated with 10 mM OAb42 and Ab42-1 for 24 hour. Cell proteins were extracted with RIPA buffer (Tris 50 mM pH 7.4; NaCl 150 mM; SDS 0.1%; NaDeoxycholate 0.5%; Triton X-100 1%) with a complete protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes (GE healthcare, Piscataway, NJ, USA). After blocking in 5% skim milk in PBST (0.1% Tween 20 in PBS), the membranes were incubated with primary antibodies for ZO-1 (1:1000), ZO-2 (1:1000), occludin (1:2000), claudin-1 (1:1000), and actin (1:5000). The band intensity analyzed using ImageJ 1.40 software (National Institutes of Health, Bethesda, MD, USA). 2.9. Transepithelial electrical resistance measurement The measurement of transepithelial electrical resistance (TER) was performed by impedance analysis using cellZscope (NanoAnalytics GmbH, Munster, Germany) (Bornhorst et al., 2012).
Fig. 1. Retinal sections of 12-month-old 5XFAD mice show the features of dry AMD in the outer retina and intracellular Ab in RPE. The outer segment/retinal pigment epithelium (RPE)/choroid interface of cross sections are evaluated in 12-month old control and transgenic mice with 5 familial autosomal dominant mutations (5XFAD). In hematoxylin and eosin (H&E)-stained retinal sections, (A) WT mice show normal RPE layer and Bruch membrane. (B) Those of 5XFAD mice show large vacuoles (arrows), hypopigmentation and thick Bruch membrane (white arrowheads). Black arrowheads indicate sub-RPE drusen-like deposit. In retinal sections immunostained with Ab (red), (C) WT mice show no Ab accumulates in the RPE layer. (D) Intracellular Ab accumulates in the RPE layer of 5XFAD mice. There is a significant increase in Ab immunoreactivity in RPE layer (arrows). White arrowheads indicate Bruch membrane with Ab. Nuclei were counterstained for DAPI. (A, B) Magnification 1000. Scale bar ¼ 5 mm. (C, D) Magnification 1600. Scale bar ¼ 20 mm. Abbreviations: Ab, amyloid beta; AMD, age-related macular degeneration; DAPI, 4, 6-diamidino-2-phenolindole; OS, outer segment; Tg, 5XFAD transgenic mice; WT, wild type.
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Briefly, the cell covered transwell filters (0.4 mm, Corning Inc, NY, USA) were placed in this setup using 12-well plate. After stabilization of TER value on plateau, TER was measured with 10 mM OAb42 and Ab42-1 over 24 hours. The TER value just after treatment was normalized to 100% for relative analysis. 2.10. Statistical analysis Statistical analyses were performed using SPSS software version 18.0 (SPSS Inc, Chicago, IL, USA). p < 0.05 were considered to be statistically significant. Figures are depicted as mean SEM. 3. Results 3.1. The 5XFAD mice show the features of dry AMD in the outer retina with intracellular Ab in RPE It is known that Ab deposits lead to retinal degeneration similar to dry AMD in AD mouse retina (Ning et al., 2008). Among AD mouse models, 5xFAD mice showed the highest retinal abundance of Ab42 (Alexandrov et al., 2011). However, the retinal pathology focusing on the RPE of 5XFAD mice has not been described yet. To
evaluate whether 5XFAD mice show the features of dry AMD, light microscopic findings with H&E were evaluated in the 5XFAD mice retina. Retinas of 12-month-old 5XFAD mice were different from those of control littermate mice. Similar to senescent neprilysin geneedisrupted mice (Yoshida et al., 2005), 5XFAD mice had large vacuoles, hypopigmentation in the RPE layer, and thickened Bruch membrane (Fig. 1A and B). Of importance, 5XFAD mice had drusenlike deposit between RPE and Bruch membrane (Fig. 1B). In addition to thickened Bruch membrane with Ab deposits beneath the RPE layer, we found that retinal Ab accumulated in the RPE layer of 5XFAD mice (Fig. 1C and D). 3.2. Intracellular Ab accumulates in RPE and attenuates the tight junction of RPE in 5XFAD mice To visualize typical tight junction integrity of RPE cells, the flat mounts of RPE and/or choroid complex were performed and immunostained for the tight junction proteins and Ab (Fig. 2, Supplementary Fig. 1). In 8-month-old 5XFAD mice, abundant intracellular Ab (Fig. 2B and D) were detected in the cytosol of RPE compared with control mice (Fig. 2A and C). With increasing intracellular accumulation of Ab in the RPE cell, ZO-1 labeled tight
Fig. 2. Intracellular Ab accumulates in the retinal pigment epithelium (RPE) and attenuates RPE tight junction integrity in 5XFAD transgenic mice. (A, B) Representative flat mounts of RPE of WT (A) and Tg (B) mice stained for Ab42 (red) and ZO-1 (green). There is a significant increase in Ab42 immunoreactivity in the RPE of Tg retina with an attenuated and disorganized expression in ZO-1 (arrows). (C, D) Representative flat mounts of RPE of WT (C) and Tg (D) mice stained for Ab42 (red) and occludin (green). Occludin integrity is severely hampered where Ab42 immunoreactivity is detected in the RPE of Tg retina (arrows). All sections were counterstained for DAPI. Magnification 400. Scale bar ¼ 20 mm. Abbreviations: Ab, amyloid beta; DAPI, 4, 6-diamidino-2-phenolindole; Tg, 5XFAD transgenic mice; WT, wild type.
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junction integrity was attenuated and disorganized (Fig. 2B) compared with the typical hexagonal shape of RPE tight junction in control mice (Fig. 2A). In addition, occludin labeled tight junction integrity was significantly attenuated in 5XFAD mice (Fig. 2D) than those in controls (Fig. 2C). 3.3. Exogenous oligomeric Ab42 is taken up by RPE and intracellular Ab42 attenuate tight junction integrity of ARPE-19 cells To investigate the role of Ab on the tight junction in RPE cells, we performed in vitro study. First, WST-1 assay was performed to determine the concentration of Ab with sublethal cytotoxicity. OAb42, which is known to have more potent cytotoxic effect than Ab42 monomer did not affect the viability of ARPE-19 cells up to 10 mM (Supplementary Fig. 2). Then, we performed immunocytochemistry for Ab and tight junction protein ZO-1 and occludin in ARPE-19 cells. After 24 hours of exogenous OAb42 treatment, Ab was abundantly detected in RPE cells, mainly in the intracellular area under confocal microscopic evaluation (Supplementary Fig. 3). Furthermore, we confirmed that both ZO-1 and occludin were decreased and lost their integrity as tight junction in ARPE-19 cells with intracellular OAb42 (Fig. 3B and D). On the other hand,
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ARPE-19 cells treated with reverse Ab42-1, an inactive control peptide, showed well-organized tight junction with no detectable Ab42-1 accumulation. RPE took up exogenous oligomeric Ab42 time dependent manner (Fig. 4AeD). RPE cells showed relatively conserved ZO-1 integrity at 6 hours (Fig. 4E and F). Intracellular Ab42 dramatically increased after 12 hours and Ab-laden RPE cells showed disrupted tight junction after 12 hours (Fig. 4G and H). Intracellular Ab42 induced tight junction breakdown without cell retraction (Fig. 4IeL). 3.4. Oligomeric Ab42 alters the tight junction protein in ARPE19 cells We also performed the Western blot assay to evaluate the change of tight junction protein with OAb42. ARPE-19 cells were treated with OAb42 10 mM or inactive reverse control peptide Ab42-1 as a control. After 24 hours of OAb42 10 mM treatment, while claudin-1 level was not changed, tight junction proteins including ZO-1, ZO-2, and occludin were decreased (Fig. 5A and B). In addition, OAb42 decreased occludin time dependent manner (Fig. 5C). In accordance with this data, TER with OAb42 10 mM was sustained for 6 hours, then declined abruptly (Fig. 5D).
Fig. 3. Exogenous oligomeric Ab42 is taken up by RPE and intracellular Ab42 attenuate tight junction integrity of ARPE-19 cells. Immunocytochemistry of Ab42 (red) and tight junction proteins (green) ZO-1 (A, B) and occludin (C, D) in ARPE-19 cells. ARPE-19 cells were incubated for 24 hours with (A, C) reverse Ab42-1 10 mM, and (B, D) OAb42 10 mM. (A, C) RPE cells show integrated and organized hexagonal shape with ZO-1 (A) and Occludin (C) expression as tight junctions without intracellular uptake of Ab. (B, D) RPE cells show intracellular distribution of Ab42 (red) 24 hours after exogenous OAb42 treatment. Ab uptaken RPE cells show disintegrated and disorganized hexagonal shape with attenuated ZO-1 and occludin expression as tight junctions. Arrows indicate disrupted tight junction. Magnification 400. Scale bar ¼ 20 mm. Abbreviations: Ab, amyloid beta; RPE, retinal pigment epithelium.
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Fig. 4. RPE takes up exogenous oligomeric Ab42 time dependent manner and intracellular Ab42 attenuate tight junction integrity. Immunocytochemistry were performed for Ab, tight junction protein ZO-1, and cytoskeleton F-actin. ARPE-19 cells were incubated for 24 hours with OAb42 10 mM. (A, B, C, D) Z-stack images were reconstructed with X and Y-axis orthogonal views under confocal microscopy. RPE takes up exogenous oligomeric Ab42 time dependent manner. Intracellular Ab42 increases after 12 hours. (E, F, G, H) RPE cells show relatively conserved ZO-1 integrity at 6 hours (F). (G, H) Ab-laden RPE cells show disrupted tight junction after 12 hours. Arrows indicate disrupted tight junction. (I, J, K, L) Ab induces tight junction breakdown without cell retraction. Arrows indicate disrupted tight junction. Magnification 1000. Scale bar ¼ 20 mm. Abbreviations: Ab, amyloid beta; RPE, retinal pigment epithelium.
4. Discussion In this study, we demonstrated that intracellular Ab was expressed in the RPE layer of 5XFAD mice, which showed characteristic features of dry AMD. With intracellular Ab42, tight junction of RPE in 5XFAD mice was attenuated. Intriguingly, exogenous Ab could be taken up by RPE cells and subsequently induce breakdown of the tight junction. Thus, intracellular Ab42 could play an important role in the pathogenesis of dry AMD. The 5XFAD mice could be used as an animal model for the study of the relationship between dry AMD and AD with regard to Ab related pathogenesis. AMD is the leading cause of vision loss in adults older than 65 years in the developed countries. Both AD and AMD share common risk factors and suggestive pathogenesis. Aging is a major risk factor of both AD and AMD. Despite controversial debates in the role of ApoE4 and complement factor H as common risk factors in both AD and AMD (Adams et al., 2012; Le Fur et al., 2010; Malek et al., 2005; Proitsi et al., 2012), Ab has been consistently considered as a pathologic molecule. Ab42 is more fibrillogenic than shorter Ab40,
and elevated Ab42 and conversion of Ab from monomeric form to oligomeric or aggregated form in the brain are considered key events in the pathogenesis of AD. Among the AD mice line, we used 5XFAD (Tg6799), the line with the highest Ab42 levels and intraneural Ab in the brain than others (Oakley et al., 2006). The 5XFAD mice generated Ab42 almost exclusively in the brain and retina (Alexandrov et al., 2011). Intracellular Ab42 accumulated in 5XFAD mice brain before plaque formation, implying potential role of intracellular Ab in the pathogenesis of AD (Alexandrov et al., 2011; Moon et al., 2012). In this study, we found intracellular Ab in the RPE and some characteristic features of dry AMD such as hypopigmentation, large vacuoles, Bruch membrane thickening, and drusen like sub-RPE deposit in aged 5XFAD mice. Although 5XFAD mice did not show all the dry AMD features, there is no model that recapitulated all the features of human AMD yet (Pennesi et al., 2012). Thus, 5XFAD mice could be used as an animal model for dry AMD, especially studying the Ab related pathogenesis in AMD. Regarding to differentiate Ab42 from amyloid beta precursor protein (AbPP) or other Ab peptides, consistent with previous
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Fig. 5. Oligomeric Ab42 alters the tight junction protein in ARPE-19 cells. (A, B) ARPE-19 cells were incubated for 24 hours with 10 mM oligomeric Ab42 (OAb42) and inactive reverse peptide Ab42-1 as a control. (A) Tight junction proteins (ZO-1, ZO-2, occludin, and claudin-1) were evaluated by Western blot. b-actin was used as an internal control. (B) Quantitative analysis was performed by measuring the densitometry of each band relative to b-actin. Each value represents the mean (SEM) of 3 independent experiments (* p < 0.05, # p > 0.05). (C) OAb42 induced significant decrease in occludin levels in a time-dependent manner in ARPE-19 cells. Cells were incubated for 6, 12, and 24 hours with 10 mM OAb42 and inactive reverse peptide Ab42-1 as a control. Occludin expression was evaluated by Western blot. b-actin was used as an internal control. (D) Transepithelial electrical resistance (TER) was measured and normalized to the TER value just after Ab treatment. Abbreviations: Ab, amyloid beta; SEM, standard error of the mean.
studies in 5XFAD mice (Jawhar et al., 2012; Oakley et al., 2006), we showed the accumulation of 4G8-labeled intracellular Ab in the early stages of plaque formation in this mouse model (Moon et al., 2012). Although 4G8 is known to react not only with Ab but also with AbPP and its proteolytic derivatives, 4G8 antibody was needed to detect intracellular Ab, and thereof compare our results clearly with previous studies on intracellular Ab that used this antibody. We previously demonstrated that 4G8-stained hollow type plaques with nuclear remnants result from neuron-derived Ab, not from cross-reactivity with AbPP using an end-specific Ab antibody (Ab42) (Moon et al., 2012). Furthermore, 5XFAD specifically increased the expression of Ab42 and we used exogenous Ab42 in in vitro experiments. Thus, both 4G8 antibody and 12F4 antibody effectively detected intracellular Ab in this study. In the brain of AD patients, uptake of extracellular Ab into neurons and intracellular production of Ab could lead to the accumulation of intracellular Ab, which in turn may gradually compromise neuronal function, by disrupting the Ab-degrading pathway or by imparing the Ab secretion of the neurons. As a result, highly Ab-laden neurons may undergo death and lysis, releasing intracellular Ab into the surrounding extracellular space, and this becomes a seed for extracellular deposition. Same as in the brain
neuronal cells, we suggest that intracellular Ab accumulation could occur in the RPE cells as well by taking up extracellular Ab, or producing Ab intracellularly. This would disrupt the outer brain retinal barrier and potentially cause AMD. Although the mechanism of intracellular uptake of exogenous Ab in RPE cells is not well established yet, several endocytotic pathways were suggested as possible neuronal uptakes of soluble Ab. They include a7 nicotinic acetylcholine (a7-NAch) receptor, N-methyl D-asparatate receptor, low-density lipoprotein receptorrelated protein 1 and/or APOE, and receptor for advanced glycosylated end products (RAGE) (Omtri et al., 2012). The RAGE, a member of the immunoglobulin superfamily, is expressed ubiquitously in brain (Neeper et al., 1992; Schmidt et al., 2001). It is known to be present in RPE cells (Zhou et al., 2005) and is up-regulated in AMD (Howes et al., 2004). RAGE mediates Ab transport from the periphery to the central nervous system across the blood-brain barrier (Deane et al., 2003; Mackic et al., 1998), and the interaction between Ab and RAGE is believed to regulate the pathogenesis of AD (Lue et al., 2001). Ab and/or RAGE-mediated signaling contributes to Ab transport and Ab internalization in neurons (Takuma et al., 2009). Ab induced occludin down-regulation is mediated by MAPK activation in hCMEC/D3 cells (Tai et al., 2010). Thus, we
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postulated that Ab uptake might be mediated by RAGE-mediated endocytosis in RPE. However, the precise mechanism of RPE uptake of Ab should be investigated in the further study. If Ab uptake is mediated by RAGE, anti-RAGE therapy could block the intracellular accumulation of Ab and subsequently block the breakdown of tight junction in RPE. In the present study, we for the first time demonstrated that intracellular Ab contributed to the breakdown of outer blood retinal barrier in 5XFAD mice. Thus, intracellular Ab might play an important role in the pathogenesis of dry AMD. Also, we suggested that 5XFAD mice could be a mouse model of AMD with regard to the Ab42 related pathology and used for the study of the relationship between AD and AMD. Disclosure statement The authors declare no actual or potential conflicts of interest. All animals used in this study were cared and treated in strict agreement with the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research. Acknowledgements This work was supported by the Bio-Signal Analysis Technology Innovation Program (2009e0090895), the Pioneer Research Program (2012e0009544), the Global Research Laboratory Program of NRF/MEST (2011e0021874) of NRF/MEST, and the Seoul National University Research Grant (800e20130338). Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging. 2014.03.008. References Adams, M.K., Simpson, J.A., Richardson, A.J., English, D.R., Aung, K.Z., Makeyeva, G.A., Guymer, R.H., Giles, G.G., Hopper, J., Robman, L.D., Baird, P.N., 2012. Apolipoprotein E gene associations in age-related macular degeneration: the Melbourne Collaborative Cohort Study. Am. J. Epidemiol. 175, 511e518. Alexandrov, P.N., Pogue, A., Bhattacharjee, S., Lukiw, W.J., 2011. Retinal amyloid peptides and complement factor H in transgenic models of Alzheimer’s disease. Neuroreport 22, 623e627. Bornhorst, J., Wehe, C.A., Huwel, S., Karst, U., Galla, H.J., Schwerdtle, T., 2012. Impact of manganese on and transfer across blood-brain and blood-cerebrospinal fluid barrier in vitro. J. Biol. Chem. 287, 17140e17151. Bruban, J., Glotin, A.L., Dinet, V., Chalour, N., Sennlaub, F., Jonet, L., An, N., Faussat, A.M., Mascarelli, F., 2009. Amyloid-beta(1-42) alters structure and function of retinal pigmented epithelial cells. Aging Cell 8, 162e177. Deane, R., Du Yan, S., Submamaryan, R.K., LaRue, B., Jovanovic, S., Hogg, E., Welch, D., Manness, L., Lin, C., Yu, J., Zhu, H., Ghiso, J., Frangione, B., Stern, A., Schmidt, A.M., Armstrong, D.L., Arnold, B., Liliensiek, B., Nawroth, P., Hofman, F., Kindy, M., Stern, D., Zlokovic, B., 2003. RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain. Nat. Med. 9, 907e913. Dentchev, T., Milam, A.H., Lee, V.M., Trojanowski, J.Q., Dunaief, J.L., 2003. Amyloidbeta is found in drusen from some age-related macular degeneration retinas, but not in drusen from normal retinas. Mol. Vis. 9, 184e190. Howes, K.A., Liu, Y., Dunaief, J.L., Milam, A., Frederick, J.M., Marks, A., Baehr, W., 2004. Receptor for advanced glycation end products and age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 45, 3713e3720. Jawhar, S., Trawicka, A., Jenneckens, C., Bayer, T.A., Wirths, O., 2012. Motor deficits, neuron loss, and reduced anxiety coinciding with axonal degeneration and intraneuronal Abeta aggregation in the 5XFAD mouse model of Alzheimer’s disease. Neurobiol. Aging 33, 196.e29e196.e40. Johnson, L.V., Leitner, W.P., Rivest, A.J., Staples, M.K., Radeke, M.J., Anderson, D.H., 2002. The Alzheimer’s A beta -peptide is deposited at sites of complement activation in pathologic deposits associated with aging and age-related macular degeneration. Proc. Natl. Acad. Sci. U.S.A 99, 11830e11835.
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