Address correspondence and reprint requests to Professor Hideaki Hara, Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan. E-mail: email@example.com
Exposure to excessive levels of light induces photoreceptor apoptosis and can be a causative factor in age-related macular degeneration (AMD). However, the cellular events that mediate this apoptotic response are poorly understood. Here, we investigated the roles of endoplasmic reticulum (ER) stress in light-induced cell death in the murine retina and murine photoreceptor cells (661W). Excessive light exposure induced retinal dysfunction, photoreceptor degeneration, and apoptosis. Furthermore, the accumulation of polyubiquitinated proteins and the transcriptional expression of ER stress-related factors, including 78-kDa glucose-regulated protein (GRP78)/immunoglobulin-binding protein (BiP) and C/EBP-homologous protein (CHOP), were increased in light-exposed retinas. Light exposure also induced both cell death and up-regulation of polyubiquitinated proteins, S-opsin aggregation, bip and chop mRNAs in 661W cells in vitro. Knock-down of chop mRNA inhibited photoreceptor cell death induced by light exposure. Furthermore, treatment with BiP inducer X (BIX), an ER stress inhibitor, induced bip mRNA and reduced both chop expression and light-induced photoreceptor cell death. These data indicate that excessive ER stress may induce photoreceptor cell death in light-exposed retinas via activation of the CHOP-dependent apoptotic pathway, suggesting that the ER stress may play a pivotal role in light exposure-induced retinal damage.
Photoreceptor degeneration is caused by excessive light exposure in many animals (Noell et al. 1966; Shahinfar et al. 1991), and it can be a common causative factor in age-related macular degeneration (AMD) (Hirakawa et al. 2008) and retinitis pigmentosa (RP) (Paskowitz et al. 2006). Photoreceptor cell death is an irreversible event accompanied by night blindness, constriction of visual field, and finally causes loss of vision. The loss of photoreceptor cells, which underlies these diseases, is thought to occur through apoptosis (Adler et al. 1999). Currently, no successful therapies are available for the treatment of photoreceptor degeneration, and the cellular events that mediate the apoptotic cascades are poorly understood. Elucidating the apoptotic process may provide clues to preventing or treating photoreceptor loss in these diseases. Exposure to excessive levels of light also induces photoreceptor apoptosis and has previously been used as a model for the study of retinal degeneration (Noell et al. 1966; Shahinfar et al. 1991; Nickells and Zack 1996). During the exposure to light, intracellular calcium levels are increased in photoreceptors (Donovan et al. 2001; Wenzel et al. 2005) and reactive oxygen species (ROS) are generated (Yang et al. 2003; Wenzel et al. 2005). Many researchers have reported the efficacy of antioxidants such as ascorbate (Li et al. 1985), dimethylthiourea (Organisciak et al. 1992), thioredoxin (Tanito et al. 2002), phenyl-N-tertbutylnitrone (Tomita et al. 2005), and 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Tanito et al. 2007) against light-induced retinal damage. In addition, our group has studied the mechanism of disease progression and efficacy of drugs, including antioxidants [edaravone (Imai et al. 2010a; Shimazaki et al. 2011), crocetin (Yamauchi et al. 2011), purple rice (Tanaka et al. 2011)], and a calpain inhibitor (SNJ-1945) (Imai et al. 2010b) against light-induced retinal damage. Oxidative stress is likely to be involved in the pathogenesis of light-induced retinal damage. Thus, Ca2+ and ROS are initiators of degeneration, but the cellular steps by which Ca2+ and ROS initiate retinal degeneration remains unclear. On the other hand, thapsigargin, which depletes Ca2+ stores in the ER lumen, can induce an ER stress, indicating that disruption of Ca2+ homeostasis causes ER stress (Yoshida et al. 2006). A high Ca2+ level in ER lumen is essential for protein folding and maturation within the ER (Lodish et al. 1992). Furthermore, it has been reported that continuous increases of intracellular Ca2+ are detected after light exposure in mouse retina, and this elevation of intracellular Ca2+ is attenuated by a nitric-oxide synthase (NOS) inhibitor, suggesting that elevated intracellular Ca2+ requires nitric oxide (NO) generation (Donovan et al. 2001). Recently, Yang et al. (2008) demonstrated that ER stress proteins are up-regulated in mouse retinas following exposure to light.
The ER is a cellular organelle involved in folding and processing of proteins destined for secretion or diverse subcellular localizations. ER stress is caused by a number of cellular stress conditions such as perturbed calcium homeostasis and generation of ROS, and results in the accumulation of misfolded or unfolded proteins in the ER lumen (Kaufman 1999). The production of large amounts of misfolded proteins that exceed the functional capacity of the ER triggers a physiological response in the cell, collectively known as the unfolded protein response (UPR), to overcome the critical status induced by ER stress (Harding et al. 2000; Travers et al. 2000; Schroder and Kaufman 2005). The UPR consists of the following four pathways: (i) inhibition of protein translation to prevent the generation of more unfolded proteins; (ii) promotion of refolding of unfolded proteins through the induction of ER chaperones; (iii) activation of ER-associated degradation (ERAD) to degrade the unfolded proteins through the ubiquitin-proteasome pathway; and (iv) induction of ER stress-induced apoptosis, if the former three strategies are unsuccessful (Zhang and Kaufman 2006; Ron and Walter 2007; Rutkowski and Kaufman 2007). Thus, ER stress has been implicated in various neurodegenerative diseases such as Alzheimer's, Huntington's, and Parkinson's disease (Katayama et al. 2001; Ryu et al. 2002; Vidal et al. 2011). Recent studies have shown that ER stress is also involved in a variety of retinal neurodegeneration conditions such as diabetic retinopathy (Roybal et al. 2004), RP (Rebello et al. 2004; Lin et al. 2007), AMD (Sauer et al. 2008; Libby and Gould 2010), and glaucoma (Joe et al. 2003). However, little remains known about the role of ER stress in retinal damage.
GRP78/BiP, a highly conserved member of the 70-kDa heat-shock protein family, is one of several chaperones localized to the ER membrane. The UPR is initiated by the binding of BiP to misfolded proteins, which increases the protein folding capacity of the ER. Previous studies have shown that induction of BiP prevents neuronal death induced by ER stress (Katayama et al. 1999; Yu et al. 1999; Rao et al. 2002; Reddy et al. 2003). These reports also suggest that a selective inducer of BiP might attenuate ER stress. Therefore, we previously conducted a screen for low molecular mass inducers of BiP by using high-throughput screening with a BiP reporter assay system and identified BiP inducer X (BIX) as a compound that preferentially induces BiP with limited induction of other ER-related genes (Kudo et al. 2008). We have reported that pre-treatment with BIX reduces tuncamycin-induced retinal ganglion cell death in murine retinas in vivo (Inokuchi et al. 2009).
In this study, we investigated whether ER stress-induced apoptosis was involved in light-induced retinal damage in mice in vivo and murine photoreceptor cells (661W) in vitro. In addition, we examined whether knockdown of chop, an inducer of ER-mediated apoptosis, or treatment with a BiP inducer could inhibit photoreceptor cell death induced by light exposure in 661W cells.
Materials and methods
Male albino ddY mice (Japan SLC, Hamamatsu, Japan) between 8 and 10 weeks of age were used in this study. They were housed under controlled light–dark environment (12 h:12 h light/dark). All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and they were approved and monitored by the Institutional Animal Care and Use Committee of Gifu Pharmaceutical University.
BIX, 1-(3,4-dihydroxyphenyl)-2-thiocyanato-ethanone, was purchased from Chemstep (Carbon-Blanc, France).
Exposure to light
Light exposure was performed in accordance with our previously described procedure (Imai et al. 2010a). Briefly, after dark adaptation for 24 h, the pupils were dilated with 1% cyclopentolate hydrochloride eye drops (Santen Pharmaceuticals Co. Ltd., Osaka, Japan) at 30 min before exposure to light. Non-anesthetized mice were exposed to 8000 lux of white fluorescent light (Toshiba, Tokyo, Japan) for 3 h in cages with reflective interiors. The temperature during the exposure to light was maintained at 25 ± 1.5°C. After the exposure to light, all mice were placed into the dark for 24 h and then returned to the normal light/dark cycle.
To determine the time-course of changes in genes related to the ER stress response after light exposure, non-treated mice retina and light-exposed mice retina at 0, 3, 6, 12, 24, 48, 72, and 120 h after the light exposure for 3 h were enucleated. Mice were killed using 80 mg/kg i.p. of sodium pentobarbital, and the eyeballs were quickly removed. The retinas were carefully separated from the eyeballs and quickly frozen in liquid nitrogen. RNA was isolated from retinas with High Pure RNA Isolation kit (Roche, Tokyo, Japan). RNA concentrations were determined spectrophotometrically at 260 nm. First-strand cDNA was synthesized in a 10-μL reaction volume using PrimeScript RT reagent kit (Perfect Real Time) (Takara, Shiga, Japan).
To determine the expression of ER stress-related gene after light exposure, real-time PCR (TaqMan; Applied Biosystems, Foster City, CA, USA) was performed. Quantitative real-time PCR was performed using a sequence detection system (ABI PRISM 7900HT; Applied Biosystems) with a PCR master mix (TaqMan Universal PCR Master Mix; Applied Biosystems), a bip probe (Assay ID Details: Mm00517691), a chop probe (Assay ID Details: Mm00492097), a calreticulin probe (Assay ID Details: Mm00482936), an edem probe (Assay ID Details: Mm00551797), an asns probe (Assay ID Details: Mm00803785), a grp94 probe (Assay ID Details: Mm00441926), and a p58ipk probe (Assay ID Details: Mm00515299) according to the manufacturer's protocol. mRNA expression was measured by real-time PCR using a Assays-on-Demand Gene Expression Product (Applied Biosystems). The thermal cycler conditions were as follows: 2 min at 50°C and then 10 min at 95°C, followed by two-step PCR for 50 cycles consisting of 95°C for 15 s followed by 60°C for 1 min. For each PCR, we obtained the slope value, R2 value, and linear range of a standard curve of serial dilutions. All reactions were performed in duplicate. The results are expressed relative to the gapdh (Assay ID Details: Mm99999915) internal control.
Electroretinogram (ERG) was recorded at 5 days after light exposure in accordance with our previously described procedure (Imai et al. 2010a). Briefly, mice were maintained in a completely dark room for 24 h. They were intraperitoneally anesthetized with a mixture of ketamine (120 mg/kg) (Daiichi-Sankyo, Tokyo, Japan) and xylazine (6 mg/kg) (Bayer Health Care, Tokyo, Japan). The pupils were dilated with 1% tropicamide and 2.5% phenylephrine (Santen). Flash ERG was recorded in the left eyes of the dark-adapted mice by placing a golden-ring electrode (Mayo, Aichi, Japan) in contact with the cornea and a reference electrode (Nihon Kohden, Tokyo, Japan) through the tongue. A neutral electrode (Nihon Kohden) was inserted subcutaneously near the tail. All procedures were performed under dim red light, and the mice were kept on a heating pad to maintain a constant body temperature during the ERG recording. The amplitude of the a-wave was measured from the baseline to the maximum a-wave peak, and the b-wave was measured from the maximum a-wave peak to the maximum b-wave peak. The a-wave shows the function of the photoreceptors, and the b-wave reflects bipolar and Müller cell functions.
Histological analysis was performed in accordance with our previously described procedure (Imai et al. 2010a). In mice under anesthesia produced by an intraperitoneal injection of sodium pentobarbital (80 mg/kg) (Nakalai Tesque, Kyoto, Japan), each eye was enucleated and kept immersed for at least 24 h at 4°C in a fixative solution containing 4% paraformaldehyde. Six paraffin-embedded sections (thickness, 5 μm) cut through the optic disc of each eye were prepared in the standard manner, and stained with hematoxylin and eosin. The damage induced by light exposure was then evaluated, with six sections from each eye used for the morphometric analysis described below. Light-microscope images were photographed, and the thickness of the outer nuclear layer (ONL) from the optic disc was measured at 240-μm intervals on the photographs. Data from three sections (selected randomly from the six sections) were averaged for each eye.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed according to the manufacturer's protocols (In Situ Cell Death Detection kit; Roche Biochemicals, Mannheim, Germany) to detect the retinal cell death induced by exposure to light (see in the Supplemental Materials and Methods).
The mice were anesthetized with 80 mg/kg i.p. of pentobarbital sodium at 12, 24, or 48 h after exposure to light for 3 h. The eyes were enucleated, fixed, embedded, and cut as described in the TUNEL staining section. Immunohistchemical staining was performed according to the following protocol: tissue sections were washed with phosphate-buffered saline (PBS) three times, and then endogenous peroxidase was quenched by treating the sections with 0.3% hydrogen peroxide in absolute methanol for 30 min, followed by pre-incubation with mouse-on-mouse blocking reagent (M.O.M. immunodetection kit, Vector Laboratories, Burlingame, CA, USA) for 1 h. Sections were subsequently incubated for 30 min with mouse anti-polyubiquitin monoclonal antibody (1 : 1000 dilution) (FK2; Nippon Biotest Laboratories Inc., Tokyo, Japan) in an M.O.M. diluent solution (M.O.M. immunodetection kit, Vector). The sections were then washed with PBS and incubated with secondary antibody (M.O.M. biotinylated anti-mouse) for 10 min. The avidin/biotinylated horseradish peroxidase complex (ABC Elite kit; Vector Laboratories) was applied for 30 min, and the sections were allowed to develop the chromogen in 3,3-diaminobenzidine (DAB) solution for 2 min. The density of polyubiquitin stained cells were measured in the ONL. In the DAB-labeled areas of anti-polyubiquitin (Nippon Biotest Laboratories Inc.) in the ONL at a distance between 480 and 920 μm from the optic disc, retinal DAB-labeled cell density was evaluated by means of appropriately calibrated computerized image analysis, using median density in the range of 0 to 255 as an analysis tool (Image Processing and Analysis in Java, Image J; National Institute of Mental Health, Bethesda, MD, USA). The data lie within the dynamic range of these assays.
661W cells were a kind gift from Dr. Muayyad R. Al-Ubaidi (University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA). 661W cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) containing 10% foetal bovine serum (FBS), 100 U/mL penicillin (Meiji Seika Kaisha Ltd., Tokyo, Japan), and 100 μg/mL streptomycin (Meiji Seika) under a humidified atmosphere of 5% CO2 at 37°C. 661W were passaged by trypsinization every 2 to 3 days.
Light irradiation and RNA isolation (in vitro)
In 661W culture, excessive light exposure reached a high temperature in CO2 incubator, so to avoid hyperthermia we used a prolonged light exposure with the mild light intensity at 2500 lux. In this condition, temperature in CO2 incubator and culture medium could be kept at 37°C. 661W cells were seeded in 96-well plates at a density of 3 × 103 cells per well. Twenty-four hours after the seeding, the entire medium was replaced with fresh medium containing 1% FBS, then they were exposed to 6-, 12-, or 24-h light exposure at 2500 lux. BIX at 3 μM were treated in the culture medium for 3, 6, or 30 h, or for 6 h followed by 6- or 24-h 2500 lux light exposure. Finally, total RNA was extracted by SYBR Green Cells-to-CT kit (Applied Biosystems), according to the manufacture's protocol.
Real-time PCR (in vitro)
SYBR Green real-time PCR was performed as described below. Single-stranded cDNA was synthesized from total RNA using a SYBR Green Cells-to-CT kit (Applied Biosystems). Quantitative real-time PCR was performed using the TaKaRa Thermal Cycler Dice® Real Time System TP800 with a SYBR Green PCR Master Mix (Applied Biosystems), according to the manufacturer's protocol (see in the Supplemental Materials and Methods).
Preparation of small interfering RNAs
The following small interfering RNA (siRNA) sequences specific to murine chop were used: #1, 5′-CCA GGA AAC GAA GAG GAA GAA UCA A-3′ (sence), 5′-UUG AUU CUU CCU CDD CGU UUC CUG G-3′ (antisense), #2, 5′-CCU CGC UCU CCA GAU UCC AGU CAG A-3′ (sense), 5′-UCU GAC UGG AAU CUG GAG ACG GAG G-3′ (antisense), #3, 5′-UAG CUG AAG AGA ACG AGC GGC UCA A-3′ (sense), 5′-UUG AGC CGC UCG UUC UCU UCA GCU A-3′ (antisense). Stealth™ RNAi Negative Control Medium GC Duplex #2 was used as a control. All siRNAs were obtained from Invitrogen. Additional details on materials and methods used in this study are provided in the Supplemental Materials and Methods.
Light irradiation-induced cell death assay
Nuclear staining assays were carried out at the end of light exposure treatment. Hoechst 33342 (λex = 360 nm, λem > 490 nm) and propidium iodide (PI) (λex = 535 nm, λem > 617 nm) were added to the culture medium for 15 min at final concentrations of 8.1 μM and 1.5 μM, respectively. Hoechst 33342 freely enters living cells and then stains the nuclei of viable cells, as well as those that have suffered apoptosis or necrosis. PI is a membrane-impermeable dye that is generally excluded from viable cells. Images were collected using an Olympus IX70 inverted epifluorescence microscope (Olympus, Tokyo, Japan). The total number of cells was counted and calculated the percent of PI-positive cells.
To examine the effects of BIX on the cell death induced by light exposure, 661W cells were seeded at 3 × 103 cells per well in 96-well plates and then incubated for 24 h. The entire medium was then replaced with fresh medium containing 1% FBS, and BIX were treated or pre-treated for 6 h; then the cells were exposed to 2500 lux of white fluorescent light (Nikon, Tokyo, Japan) for 24 h at 37°C. BIX was dissolved in dimethyl sulfoxide (DMSO) and diluted with DMEM containing 1% FBS (a final concentration, 0.1% DMSO). Nuclear staining assays were carried out at the end of light exposure treatment as above-mentioned method.
Western blot analysis
661W cells were seeded in 24-well plates at a density of 1.8 × 104 cells per well. After the cells had been incubating for 24 h, the entire medium was replaced with fresh medium containing 1% FBS, then they were exposed to 6-h light exposure at 2500 lux. 661W cells were lysed using a cell-lysis buffer (RIPA buffer) (Sigma-Aldrich) with protease (Sigma-Aldrich) and phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich). The lysate was centrifuged at 12 000 g for 20 min, and the supernatant was used for this study. Assays to determine the protein concentration were performed by comparison with a known concentration of bovine serum albumin using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). A mixture of equal parts of an aliquot of protein was solubilized in sodium dodecyl sulfate-sample buffer, separated on 5–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gels. The separated protein was then transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore Corporation, Billerica, MA, USA). Transfers were blocked for 1 h at 17 to 28°C with BlockAce in 10 mM Tris-buffered saline with 0.05% Tween 20 (TBS-T), then incubated overnight at 4°C with the primary antibody. The transfers were then rinsed with TBS-T and incubated for 1 h at 17 to 28°C in secondary antibody. For immunoblotting, the following primary antibodies were used: mouse anti-polyubiquithin monoclonal antibody (1 : 2000; Nippon Biotest Laboratories Inc., Tokyo, Japan) and mouse anti-β-actin monoclonal antibody (1 : 5000; Sigma-Aldrich). The secondary antibody used was goat anti-mouse horseradish peroxidase-conjugated (1 : 2000). The immunoreactive bands were visualized using a chemiluminescent substrate (SuperSignal West Femto Maximum Sensitivity Substrate; Pierce Biotechnology). The band intensity was measured using LAS-4000 mini (FUJIFILM, Tokyo, Japan).
To evaluate aggregation of S-opsin induced by light exposure, 661W were seeded in 100 mm dish (BD Falcon, Franklin Lakes, NJ, USA) at 1.0 × 106 cells/dish and incubated 24 h. Additional details on materials and methods used in this study are provided in the Supplemental Materials and Methods.
To evaluate accumulation of S-opsin induced by light exposure in ER, we examined immunofluorescent staining after light exposure. 661W cells were plated onto glass chamber slides (Laboratory-Tek; Life Technologies, Gaithersburg, MD, USA), and incubated 24 h. The medium was then replaced by DMEM (Sigma) with 1% FBS and incubated for 1 h. Thereafter, the cells were exposed to 2500 lux of light for 6 h. Then, fixed with 4% fresh paraformaldehyde at 17 to 28°C, blocked in 3% horse serum and 0.2% Triton X-100 in PBS, and incubated overnight at 4°C with primary antibodies [anti-S-opsin rabbit polyclonal antibody (Chemicon, Temecula, CA, USA) and anti-GRP78 (N-20) goat polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA)]. After being washed, the cells were incubated for 1 h with secondary antibodies [Alexa Fluor® 488 donkey anti-goat IgG and Alexa Fluor® 546 donkey anti-rabbit IgG, both from Invitrogen (Carlsbad, CA, USA)], washed, and counter-stained with Hoechst 33342 (Invitrogen); images were captured using a confocal fluorescence microscope (Olympus).
Data are presented as the means ± SEM. Statistical comparisons were made by way of Dunnett's test or Student's t-test using statistical analysis software [using Stat View version 5.0 (SAS Institute, Cary, NC, USA)]. p <0.05 was considered statistical significant.
Effects of light exposure on retinal function, histology, and apoptosis in mice
Mice were exposed to 8000-lux light for 3 h, and retinal functions were recorded by ERG 5 days after light exposure. In the light-exposed group, the a-wave and b-wave decreased by approximately 50% and 30%, respectively, compared with the non-treated retinas at 0.98 log cds/m2 5 days after exposure to light (Fig. 1a–c). The effects of light-induced retinal damage were further examined by histological analysis. The thickness of outer nuclear layer (ONL) was decreased 5 days after light exposure (Fig. 1e) compared with non-treated retinas (Fig. 1d). Light exposure decreased ONL in the retinal superior area from 240 to 1200 μm and in the inferior area from 240 to 1680 μm (Fig. 1f). To evaluate light-induced apoptotic cell death, we investigated the presence of TUNEL-positive cells at 0, 12, 24, 48, 72, and 120 h after light exposure (Fig. 1g–n). TUNEL-positive cells were rarely seen in normal control retinas (Fig. 1g). In the photic injury model, TUNEL-positive cells were rarely seen immediately following 3-h 8000 lux light exposure (Fig. 1h), but seen in the ONL 12 h after light exposure (Fig. 1i). The number of TUNEL-positive cells was increased 24 h after light exposure (Fig. 1j), and reached a peak at 48 h (Fig. 1k). Subsequently, the number gradually decreased at 72 and 120 h after light exposure (Fig. 1l and m).
Induction of protein polyubiquitination after light exposure
To show light-induced accumulation of polyubiquitin, we investigated the density of polyubiquitin-positive cells at 12, 24, and 48 h after light exposure. A representative photograph of a non-treated retina is shown in Fig. 2a. Optical density analysis of polyubiquitinated protein immunoreactivity in ONL showed that light exposure increased the level of polyubiquitinated protein (Fig. 2b–e). The immunoreactivity for polyubiquitinated protein was also increased in the inner retinal layers including ganglion cell layer (GCL), inner plexiform layer (IPL) and inner nuclear layer (INL) as well as in ONL.
Expression of genes related to the ER stress response after light exposure
The time-courses of changes in mRNA expression of grp78/bip, chop, calreticulin, edem, asns, grp94, and p58ipk after light exposure were determined by quantitative real-time RT-PCR. Real-time PCR revealed significant elevation of mRNAs for grp78/bip, chop, edem, p58ipk, calreticulin, and grp94 after light exposure versus the non-treated normal group (Fig. 3). Specifically, it revealed significant elevations of mRNAs for grp78/bip at 0 and 12 to 120 h (peak at 0 h), chop at 0 to 24 h (peak at 6 h), edem at 72 and 120 h, p58ipk at 0 and 12 to 120 h (peak at 0 h), calreticulin at 0, 3, and 24 to 120 h (peak at 0 h) and grp94 at 0, 3, and 72 h (peak at 0 h) after 3-h light exposure versus the non-treated normal group (Fig. 3). However, asns mRNA did not show a significant increase after light exposure. Overall, the induction of mRNAs for grp78/bip, chop, p58ipk, calreticulin, and grp94 was divided into two phases: early UPR and late UPR.
Accumulation of polyubiquitinated proteins and S-opsin aggregation in ER after light exposure in 661W cells
To show light-induced accumulation of polyubiquitinated proteins in 661W cells, we investigated the expression of polyubiquitinated protein at 6 h after light exposure. A representative immunoblot image is shown in Fig. 4a. Optical density analysis of polyubiquitinated proteins showed that 6-h light exposure significantly increased the level of polyubiquitinated proteins (Fig. 4b).
S-opsin reported to be resistant to ER-associated degradation, resulting in aggregation/accumulation, which induces apoptosis (Zhang et al. 2011). Therefore, we measured cone S-opsins localization and aggregation after light exposure in 661W cells. Light exposure for 6 h at 2500 lux induced accumulation of S-opsin merged with BiP (used as an ER marker) in 661W cells (Fig. 4c), and S-opsin in 1% Triton-insoluble fraction was significantly increased at 6 h after the light exposure (Fig. 4d and e).
ER stress-related mRNA in 661W cells induced by light exposure
To investigate whether in vitro light exposure affects the expressions of genes related to the ER stress response, such as bip and chop, we performed real-time RT-PCR using primers specific for bip or chop mRNA. Real-time PCR revealed a significant but mild increase of bip mRNA at 6 h (Fig. 5a), and a marked increase of chop mRNA at 12 and 24 h (Fig. 5b). In 661W, excessive light exposure at 8,000 lux, which is the same condition as mice in vivo, reached a high temperature in CO2 incubator, so to avoid hyperthermia we used a continuous light exposure with the light intensity at 2500 lux. In this condition, temperature in CO2 incubator and culture medium could be kept at 37°C. Therefore, the level of light exposure was lower in 661W cells than that in mice at 8000 lux for 3 h, resulting in mild cell damage and lower bip mRNA expression in 661W cells.
Effects of chop siRNA on light-induced photoreceptor cell death
Light exposure markedly up-regulated the amount of chop mRNA, and the groups treated with chop siRNAs containing #1, #2, and #3 sequences showed significantly less induction in the amount of chop mRNA (40%, 33%, and 19% reduction, respectively) compared with negative siRNA-transfected cells at 24 h after light exposure (Fig. 6a). None of the dark control groups treated with chop siRNA showed any changes of chop mRNA compared with the negative siRNA-transfected group. To investigate the effect of chop siRNA on 661W cells' viability following light exposure, cells were stained with Hoechst 33342 and PI. Hoechst 33342 stains all cells (live and dead cells), whereas PI stains only dead cells. Typical images of Hoechst 33342 or PI staining are shown in Fig. 6b. All the dark control groups showed limited cell death, indicating that chop knockdown did not show any toxicity. However, light exposure significantly induced photoreceptor cell death. Both #1 and #2 siRNAs reduced cell death from light exposure (30% and 26% reduction, respectively), whereas siRNA containing the #3 sequence was less effective at reducing cell death (Fig. 6c).
Protective effect of BIX against light-induced cell death in 661W culture
To investigate whether BIX can prevent cell death induced by light exposure, 661W cells were treated or pre-treated for 6 h with BIX, and then exposed to light for 24 h. Typical images of Hoechst 33342 and PI staining are shown in Fig. 7a, respectively. Light exposure significantly induced cell death, and treatment with BIX significantly inhibited this cell death at 3 μM (Fig. 7b). To investigate whether BIX induces bip in 661W cells, we used real-time RT-PCR using a specific primer recognizing bip mRNA. The level of bip mRNA increased at 3, 6, and 30 h after treatment with BIX at 3 μM (Fig. 7c). Finally, we investigated whether BIX down-regulates the increased expression of chop mRNA following light exposure. BIX significantly inhibited the expression of chop mRNA that was induced by 24 h of light exposure (Fig. 7d).
Nitrite/nitrate production in the cultured medium of 661W cells after light exposure
To demonstrate for NO production after light exposure, we measured the nitrite (NO2−)/nitrate (NO3−) as an indicator of NO production in the cultured medium of 661W cells. NO2−/NO3− level in the medium was significantly increased at 24 h after the light exposure at 2500 lux (Figure S1a).
Protective effects of NOS inhibitor and Ca2+ release inhibitor from ER against light-induced cell death in 661W culture
To confirm the involvements of NO and intracellular Ca2+ release from ER after light exposure, we examined the effects of NOS inhibitor and Ca2+ release inhibitor from ER. A NOS inhibitor, NG-Nitro-l-arginine methyl ester (l-NAME), at 10 to 1000 μM concentration-dependently inhibited the 661W cell death at 24 h after the light exposure at 2500 lux (Figure S1b). An intracellular Ca2+ release inhibitor from ER, 8-(N,N-Diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), at 10 μM also inhibited the 661W cell death induced by the light exposure (Figure S1c).
Time-course of changes of blood glucose and metabolic activity in mice and 661W cells, respectively, after light exposure
BiP is unregulated under energy depletion such as serum deprivation and so on. Therefore, we measured the time-course of changes in blood glucose level after the light exposure at 8000 lux for 3 h. However, there were no changes in blood glucose just after, at 12, 24, 72 h after the light exposure compares with those at 1 h before starting the light exposure or each control mice without the light exposure (Figure S2a). On the other hand, metabolic activity, which measured the amount of the formazan dye generated from WST-8 by dehydrogenases in cells, was only reduced at 24 h in 661W cells exposed by the continuous light at 2500 lux (Figure S2b). The reduction of metabolic activity coincided with the course of cell death measured as PI-positive cells.
In this study, we demonstrated that ER stress-related genes and polyubiquitinated proteins were markedly up-regulated in light-exposed retinas in vivo. Both bip and chop mRNA were markedly up-regulated in light-exposed 661W photoreceptor cells in vitro. Furthermore, down-regulation of chop mRNA and treatment with BIX, an ER stress inhibitor, ameliorated light-induced photoreceptor cell death in 661W cells. These results suggest that excessive ER stress may induce photoreceptor cell death in light-exposed retinas via activation of the CHOP-dependent apoptotic pathway.
In 661W, light exposure at 8000 lux for 3 h, which is the same condition as mice in vivo, reached a high temperature in CO2 incubator, so to avoid hyperthermia we used a continuous light exposure with the light intensity at 2500 lux. On the other hand, mild light at 2500 lux needed a long- time exposure to induce retinal damages in mice, and it helped mice fall asleep. Therefore, the excessive light at 8000 lux was exposed to mice for a short duration (3 h). However, both light intensities at 2500 and 8000 lux are excessive, because albino rodents cause retinal damage when room light intensity is above 270 lux for 3 to 7 days (Semple-Rowland and Dawson 1987).
ER stress is caused by the accumulation of misfolded or unfolded proteins, and these proteins are rapidly polyubiquitinated and degraded via the ubiquitin-proteasome pathway (Uehara 2007). In fact, accumulation of polyubiquitinated proteins and increased levels of ER stress-related proteins have been observed in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and glaucoma (Uehara et al. 2006; Ito et al. 2011). In this study, the increase in polyubiquitinated proteins was preceded by an increase in TUNEL-positive cells in the retina after light exposure. These findings indicate that excessive ER stress induces retinal apoptotic cell death, suggesting that the ER stress pathway may play an important role in light-induced photoreceptor cell death. On the other hand, the immunoreactivity for polyubiquitinated protein was increased in the inner retinal layers, including GCL, IPL and INL as well as in ONL. Previously, Yang et al. (2008) also reported that BiP and caspase-12 immunoreactivities were increased in the INL in addition to the ONL after light exposure in mice. Furthermore, excessive light exposure has been reported to induce inner retinal damages at late stages, suggesting that light-induced retinal damage is not limited to the outer retina such as photoreceptors and retinal pigmented epithelium (Garcia-Ayuso et al. 2011). These findings suggest that ER stress is also activated in inner retina during photoreceptor degeneration.
The expression levels of bip, grp94, calreticulin, and p58ipk were up-regulated immediately after the end of light exposure and returned to basal levels at 3 h. Their expression was up-regulated again between 12 h and 120 h. It has been reported that increases in BiP, GRP94, calreticulin, CHOP, and p58IPK expression, mediated by the activating transcription factor 6 (ATF6) pathway, were accompanied by activation of ER stress response elements (ERSEs) (Kokame et al. 2001; van Huizen et al. 2003). These results suggest that the UPR, mediated by the ATF6 pathway, may be activated by excessive light exposure, and this activation is divided into early and late phases. Previous studies have reported that intracellular calcium levels in murine retinas are elevated during and following light exposure (Donovan et al. 2001), and disruption of ER Ca2+ homeostasis can induce ER stress (Verkhratsky 2005; Concannon et al. 2008). These findings indicate that elevated Ca2+ levels disrupt ER homeostasis and induce early UPR. Early UPR may suppress ER stress and restore ER homeostasis by 3 h after 3-h light exposure. On the other hand, the late UPR might be activated by other triggers such as nitric oxide (NO). In fact, the level of neuronal nitric oxide synthase (nNOS) was increased in the retina from 3 to 24 h and peaked 24 h after 2-h exposure to 5000 lux light (Donovan et al. 2001). nNOS catalyzes the conversion of arginine to citrulline and NO. Furthermore, it has previously been reported that light exposure increases protein nitrosylation in rat retina (Palamalai et al. 2006) and that systemic administrations of NG-Nitro-l-arginine methyl ester (l-NAME), a NOS inhibitor, and 7-nitroindazole, an nNOS inhibitor, both inhibit the light-induced photoreceptor apoptosis in mice (Donovan et al. 2001). In this study, l-NAME inhibited the 661W cell death at 24 h after the light exposure at 2500 lux, and NO2−/NO3− level in the medium was significantly increased at 24 h after the light exposure at 2500 lux. Furthermore, an intracellular Ca2+ release inhibitor from ER, TMB-8, at 10 μM also inhibited the 661W cell death induced by the light exposure. These findings suggest that NO production may participate in intracellular Ca2+ elevation and photoreceptor degeneration after light exposure. Interestingly, nitrosative stress caused by NO prevents normal function of the ER via S-nitrosylation of protein-disulfide isomerase (PDI), which is located in the ER lumen, and may contribute to the accumulation of misfolded proteins as well as sustained activation of the UPR pathway (Uehara 2007). These studies suggest that nNOS can induce the late UPR following light exposure. Taken together, these findings indicate that ER stress is induced by excessive light exposure in the retina and UPR is activated and divided into two phases. Early UPR may be induced by elevated Ca2+ levels following light exposure, and late UPR may result from nNOS activity induced by light exposure. However, further experiments will be needed to clarify the detailed mechanism in the retina.
BiP is a molecular chaperone and works as an ER stress sensor, binds Ca2+, and inhibits neuronal death induced by ER stress (Katayama et al. 1999; Yu et al. 1999; Rao et al. 2002; Reddy et al. 2003; Ni and Lee 2007). Yang et al. (2008) reported that BiP protein levels were up-regulated from 0 h to 3 days and peaked at 24 h after 24-h exposure to 3500 lux light. On the other hand, our present data showed significant up-regulation of bip mRNA at 0 and 12 to 120 h (peaked at 0 h) after 3-h exposure to 8000 lux light. These findings indicate that UPR is activated in the retina by excessive light exposure, and activated UPR is divided into two phases: early UPR and late UPR. GRP94 is a molecular chaperone similar to BiP that binds Ca2+ and inhibits apoptosis (Ni and Lee 2007). Calreticulin is a molecular chaperone that folds glycoproteins and binds Ca2+ (Ni and Lee 2007). p58IPK, a co-chaperone that assists other chaperones, inhibits the phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eukaryotic initiation factor 2 (eIF2α), and suppresses ER stress (van Huizen et al. 2003). PERK is one of the ER-resident transmembrane proteins from major UPR pathways that act as proximal signal sensors. The activation of PERK upon ER stress involves autophosphorylation of its serine/threonine kinase domain, and the activated PERK further phosphorylates eIF2α (the α subunit of translational initiation factor). Yang et al. (2008) showed that the expression of p-eIF2α was down-regulated 0 h to 6 h after 24-h light exposure, and the expression of p-PERK did not increase until 3 h after 24-h light exposure. In this study, p58ipk mRNA levels peaked after just 3-h light exposure. These results may indicate that p58IPK is up-regulated and inhibits the phosphorylation of PERK and eIF2α in early UPR induced by excessive light exposure.
CHOP is a member of the CCAAT/enhancer-binding protein family induced by ER stress and plays a pivotal role in ER-mediated apoptosis (Wang et al. 1996). The expression of chop mRNA was up-regulated 0 h to 6 h after 3-h light exposure, and then declined and returned to the basal level by 72 h. The difference of induction between chop and other genes of the ATF6 pathway suggested that sequences other than ERSEs may be involved in the induction of chop because CHOP is also elevated by the ATF4 pathway via the amino-acid-regulatory element (AARE) (Averous et al. 2004). On the other hand, the expression of EDEM was significantly up-regulated later than other genes of the ATF6 pathway after light exposure. EDEM is a molecular chaperone that recognizes and targets unfolded glycoproteins for ER-associated degradation (ERAD) (Ni and Lee 2007). In a previous study, EDEM was transcriptionally up-regulated by the IRE1/XBP-1 branch of the UPR (Ni and Lee 2007), and the active form of ATF6 was produced faster than XBP1 in ER-stressed cells (Yoshida et al. 2001). Therefore, the expression of EDEM might occur later than for other genes of the ATF6 pathway following light exposure. Yang et al. (2008) revealed that cleavage of caspase-12 was strongly up-regulated from 24 h to 3 days after 24-h light exposure. Caspase-12 is a mediator of ER stress-induced apoptosis that is localized on the cytoplasmic side of the ER (Nakagawa and Yuan 2000), and ER-stress-activated IRE1 can bind tumor necrosis factor receptor-associated factor 2 (TRAF2), which can mediate cleavage of caspase-12 (Yoneda et al. 2001). Their studies support the idea that the IRE1 pathway, including expression of EDEM and cleaved-caspase-12, is a late event in the UPR. Taken together, these findings indicate that UPR was activated in retinas by excessive light exposure, and activated UPR was divided into two phases: early UPR and late UPR. Our present data suggest that the ATF6 pathway, including BiP, GRP94, calreticulin, and p58IPK, is activated in early UPR, and the IRE1 pathway, including EDEM, is activated in late UPR. Collectively, early UPR signaling can suppress ER stress and restore ER homeostasis, but late ER stress may be so severe that UPR cannot restore ER homeostasis, thus resulting in apoptotic cell death. These findings are summarized in Fig. 8. Therefore, the activation of early UPR pathway proteins such as BiP might be able to inhibit apoptosis induced by ER stress.
661W cells serve as a useful in vitro model for photoreceptor cell responses to visible light, as the cells in culture showed light-induced cell death that is similar to that observed in photoreceptor cells in vivo (Krishnamoorthy et al. 1999; Tanaka et al. 2011). In this study, continuous light exposure at 2500 lux led to a transient increase of bip mRNA and a marked increase of chop mRNA. The rapid induction of bip is consistent with our in vivo study, but it was small compared with that in mice in vivo. However, the increased transcription of bip gene was evident, suggesting that an UPR response was activated during light exposure. Futhermore, polyubiquitinated proteins were increased at 6 h after continuous light exposure at 2500 lux. These results strongly suggest that misfolded/unfolded protein may be increased in ER after light exposure. Recently, Zhang et al. (2011) have reported that S-opsin is resistant to ER-associated degradation, resulting in aggregation/accumulation, which induces apoptosis. Furthermore, over-expression of S-opsin in COS-7 induces protein aggregation/accumulation, resulting in ER stress with CHOP expression (Zhang et al. 2011). Thus, it is possible that rhodopsin and cone opsins are folded correctly in the presence of 11-cis-retinal, but they are misfolded or unfolded without 11-cis-retinal, resulting that it promotes aggregation. Accordingly, excessive light exposure may induce accumulation of unfolded/misfolded opsin in ER. In this study, light exposure for 6 h at 2500 lux induced accumulation of S-opsin merged with BiP (used as an ER marker) in 661W cells, and S-opsin in 1% Triton-insoluble fraction was increased at 6 h after the light exposure. These data may provide evidence for ER-stress activation induced by light exposure in 661W cells. Next, we tried to determine whether chop knock-down would attenuate photoreceptor cell death induced by light exposure. Transfection with chop siRNA reduced both light-induced chop mRNA expression and photoreceptor cell death. These results indicate that light exposure can cause ER stress in photoreceptors, and that this neurotoxicity is due in part to a mechanism dependent on CHOP protein induction through excessive ER stress. Furthermore, our present data show that BIX led to increased bip expression and reduced both photoreceptor cell death and chop expression induced by light exposure in 661W cells. The data also indicate that ER stress may be involved in light-induced photoreceptor degeneration.
In conclusion, the present findings suggest that ER stress plays a pivotal role in light-induced photoreceptor degeneration, and that this effect is mediated by CHOP. Therefore, the modulation of ER stress may be a potential therapeutic strategy for the treatment of retinal degenerative diseases.
TN, MS, SS, TK, SI, YI, KT, and HH declare no conflicts of interest, financial, or otherwise. TN, MS, SS, TK, SI, YI, and KT contributed scientific experiments; TN and MS designed the experiments, and wrote the manuscript; HH supervised the experiments and performed the analyses.