November 2012
Volume 53, Issue 12
Free
Retinal Cell Biology  |   November 2012
Metallothionein-III Deficiency Exacerbates Light-Induced Retinal Degeneration
Author Affiliations & Notes
  • Kazuhiro Tsuruma
    From the Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Gifu, Japan; and
  • Hiroki Shimazaki
    From the Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Gifu, Japan; and
  • Yuta Ohno
    From the Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Gifu, Japan; and
  • Yuki Inoue
    From the Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Gifu, Japan; and
  • Akiko Honda
    Laboratory of Pharmaceutical Health Sciences, School of Pharmacy, Aichi Gakuin University, Aichi, Japan.
  • Shunsuke Imai
    From the Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Gifu, Japan; and
  • Jinyong Lee
    Laboratory of Pharmaceutical Health Sciences, School of Pharmacy, Aichi Gakuin University, Aichi, Japan.
  • Masamitsu Shimazawa
    From the Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Gifu, Japan; and
  • Masahiko Satoh
    Laboratory of Pharmaceutical Health Sciences, School of Pharmacy, Aichi Gakuin University, Aichi, Japan.
  • Hideaki Hara
    From the Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Gifu, Japan; and
  • Corresponding author: Hideaki Hara, Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan; hidehara@gifu-pu.ac.jp
Investigative Ophthalmology & Visual Science November 2012, Vol.53, 7896-7903. doi:10.1167/iovs.12-10165
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      Kazuhiro Tsuruma, Hiroki Shimazaki, Yuta Ohno, Yuki Inoue, Akiko Honda, Shunsuke Imai, Jinyong Lee, Masamitsu Shimazawa, Masahiko Satoh, Hideaki Hara; Metallothionein-III Deficiency Exacerbates Light-Induced Retinal Degeneration. Invest. Ophthalmol. Vis. Sci. 2012;53(12):7896-7903. doi: 10.1167/iovs.12-10165.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Retinal photoreceptor damage is a common feature of ophthalmic disorders, such as age-related macular degeneration and retinitis pigmentosa. Oxidative stress has a key role in these diseases. Metallothioneins (MTs) are a family of cysteine-rich proteins, and various physiologic functions have been reported, including protection against metal toxicity and antioxidative potency. We investigated the functional role of MT-III in light-induced retinal damage.

Methods.: The expression of retinal MT-I, -II, and -III mRNA was evaluated by real-time reverse-transcription PCR in retina exposed to light. Retinal damage in MT-deficient mice was induced by exposure to white light at 16,000 lux for 3 hours after dark adaptation. Photoreceptor damage was evaluated histologically by measuring the thickness of the outer nuclear layer (ONL) 5 days after light exposure and by electroretinogram recording. In an in vitro experiment, the MT-III siRNAs were tested for their effects on light-induced mouse photoreceptor cell (661W) damage.

Results.: The mRNAs of the MTs were increased significantly in murine retina after light exposure. The ONL in the MT-III–deficient mice was remarkably thinner compared to light-exposed wild-type (WT) mice, and a- and b-wave amplitudes were decreased; the damage induced in MT-I/-II–deficient mice was comparable to that observed in WT mice. MT-III knockdown by siRNA in 661W exacerbated the cell damage and increased the production of reactive oxygen species in response to light exposure.

Conclusions.: These findings suggested that MT-III can help protect against light-induced retinal damage compared to MT-I/II. Some of these effects may be exerted by its antioxidative potency.

Introduction
Excessive light exposure leads to photoreceptor degeneration in many animals, 1,2 and can be a risk factor for the onset and progression of age-related macular degeneration (AMD), and possibly some forms of retinitis pigmentosa (RP). 3 The pathology of AMD shows photoreceptor degeneration similar to that observed following intense light exposure in albino rodents. 1,3,4 Therefore, animal models of retinal light damage are used widely as models of AMD. Light irradiation can have detrimental thermal, mechanical and photochemical effects on the retina, resulting in damage by mechanisms that involve, for example, an increase in the level of oxidative stress. 5,6 The retina consumes significant amounts of oxygen in the human body and readily produces reactive oxygen species (ROS), such as superoxide (O2 ) and hydrogen peroxide (H2O2). 7 The outer retina, including the RPE, is exposed to a relatively high oxygen tension close to that found in arterial blood. 8 Visible light and ROS also damage the RPE, 810 leading to disruption of photoreceptor. In addition, many investigators have reported the efficacy of antioxidants, such as dimethylthiourea and ascorbate, against light-induced damage. 11,12 These findings suggest that oxidative stress has a pivotal role in light-induced retinal damage. 
Metallothioneins (MTs) are low molecular weight (6–7 kilodaltons [kDa]) metal-binding proteins with a high cysteine content. 13 MT isoforms are known as MT-I, -II, -III, and -IV, 1416 and they serve as important regulators of metal homeostasis and as a source of zinc incorporated into proteins, including transcription factors. 17 MT-I and MT-II are expressed in all tissues, MT-III is expressed in the central nervous system, and MT-IV is detected in the skin epidermis. MT has a high content of sulfhydryl, which allows it to scavenge superoxide anion and hydroxyl radicals with an affinity more than 300-fold higher that of reduced glutathione (GSH). 18 There are many reports of the expression of MT in human and rodent retina. 1921 In our previous report, MT-I/-II deficiency exacerbated retinal damage induced by an intravitreal injection of N-methyl-D-aspartate (NMDA). 22  
However, there have been few reports of the involvement of MT-III in retinal degeneration. Hozumi et al. reported that the MT-III protein is present in the optic nerve, but they did not detect it in the retina by immunostaining. 23 On the other hand, MT-III mRNA has been detected in human photoreceptor cells, 24 and MT-III mRNA has been reported to be increased slightly in light-exposed retinas. 25 Taken together, these findings suggest that MT-III may be an important neuroprotective substance for retinal degeneration. 
Therefore, the purpose of our study was to clarify the involvement of MT-III in light-induced retinal degeneration. 
Materials and Methods
Materials
Hoechst 33342, propidium iodide (PI), and 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) were purchased from Invitrogen (Carlsbad, CA). All other chemicals were commercial products of reagent grade. 
Animals
Male and female MT-I/-II–deficient, MT-III–deficient, and wild-type (WT) mice (Aichi Gakuin University, Aichi, Japan), and male adult ddY mice (Japan SLC, Hamamatsu, Japan), which are albino mice and maintained as a closed colony, were kept under 12-hour light/12-hour dark conditions, and had ad libitum access to food and water. The background strain of MT-deficient mice was 129/SV, and this strain was used as the WT control. All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and were approved and monitored by the Institutional Animal Care and Use Committee of Gifu Pharmaceutical University. 
Exposure to Light
After dark adaptation for 24 hours, the pupils were dilated with 1% cyclopentolate hydrochloride eye drops (Santen, Osaka, Japan) at 30 minutes before exposure to light. Nonanesthetized 129/SV WT and MT-deficient mice were exposed to 16,000 lux, and ddY mice were exposed to 8000 lux of white fluorescent light (Toshiba, Tokyo, Japan) for 3 hours 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 in the normal light/dark cycle. 
Electroretinogram (ERG)
ERG was recorded at 5 days after light exposure (Mayo, Aichi, Japan). Mice were maintained in a completely dark room for 24 hours. They were anesthetized intraperitoneally 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) 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 in dim red light, and the mice were kept warm during the entire procedure. 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. 
Histologic Analysis
In mice under anesthesia produced by an intraperitoneal injection of sodium pentobarbital (80 mg/kg; Nakalai Tesque, Kyoto, Japan), each eye was enucleated and immersed for at least 24 hours at 4°C in a fixative solution containing 4% paraformaldehyde. Care was taken to mark the orientation of superior and inferior quadrants in each eye, this orientation was continued through the paraffin embedding. 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 then was 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 in a masked fashion by a single observer (HS). Data from three sections (selected randomly from the six sections) were averaged for each eye. 
Cell Culture
The 661W cell line, a transformed mouse cone-cell line derived from mouse retinal tumors, was provided by Muayyad R. Al-Ubaidi (University of Oklahoma Health Sciences Center, Oklahoma City, OK). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM, with 1000 mg/L glucose, L-glutamine, and sodium bicarbonate; Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin under a humidified atmosphere of 5% CO2 at 37°C. 
RNA Interference
For mouse MT-III and negative control siRNAs, we used a duplex (Flexitube siRNA; Qiagen, Valencia, CA) and a control duplex (Stealth RNAi Negative Control Duplex, No. 12935 to 110, Low GC Duplex; Invitrogen), respectively. Sense and antisense strands of mouse MT-III siRNA were: sequence #1, 5′-CUCGGACAAAUGCAAGUGCAA-3′ (sense) and 5′-UUGCACUUGCAUUUGUCCGAG-3′ (antisense); sequence #2, 5′-CUGCAAAUGCACGAACUGCAA-3′ (sense) and 5′-UUGCAGUUCGUGCAU-UUGCAG-3′ (antisense); sequence #3, 5′-CCGGAUGUGAGAAGUGUGCCA-3′ (sense) and 5′-UGGCACACUUCUCACAUCCGG-3′ (antisense); sequence #4, 5′-CCCACA-CAGCCUAUGUAAAUA-3′ (sense) and 5′-UAUUUACAUAGGCUG-UGUGGG-3′ (antisense). 
Transfection with siRNA
A total of 661W cells was seeded at a density of 1000 cells/well into 96-well plates (for light exposure) or at a density of 10,000 cells/well into 12-well plates (for assessment of expression MT-III), and cultured with antibiotic-free normal medium at 37°C for 24 hours. Then cells were washed twice with medium (Opti-MEM; Invitrogen) supplemented with 1% FBS without antibiotics and then were immersed in the same medium. Reagent (Lipofectamine RNAiMAX; Invitrogen) was used as the transfection agent. MT-III and control siRNA, each at a concentration of 50 nM, were transfected by incubation for 4 hours, according to the manufacturer's instructions. Subsequently, the medium containing siRNA and Lipofectamine RNAiMAX was replaced by DMEM supplemented with 10% FBS. At 24 hours after the transfection, the cells were irradiated with visible light or subjected to real-time reverse transcription PCR (RT-PCR). 
Real-Time RT-PCR
Total RNA was isolated from retinas of male ddY mice or male and female MT-I/-II–deficient, MT-III–deficient, and 129/SV WT mice at 6 hours after the light exposure and 661W cells at 24 hours after MT-III siRNA treated using an RNeasy Mini Kit (Qiagen). Quantitative real-time PCR, after reverse transcription, was performed to determine the time-course of changes in the gene expression of MT isoforms (MT-I, -II, and -III) and antioxidant proteins (superoxide dismutase 1 [SOD1] and peroxiredoxin 1 [Prdx1]). Briefly, 1 μg total RNA was used for first-strand cDNA synthesis (PrimeScript RT reagent Kit; Takara Bio, Tokyo, Japan), in accordance with the manufacturer's instructions. Real-time PCR was performed in 50 μL of reaction mixture containing each primer, template cDNA, and supermix (SYBR Green; Bio-Rad, Hercules, CA) using a single-color real-time PCR detection system (MyiQ; Bio-Rad) according to the manufacturer's instructions. Reactions were performed in 40 cycles consisting of 15 seconds' denaturation (94°C) and 60 seconds' elongation with annealing (60°C). GAPDH was used as the reference standard, and relative levels of MT isoforms, SOD1, and Prdx1 compared to that of GAPDH were calculated. Primers were used: MT-I forward: 5′-GGTCCTCTAAGCGTCACCAC-3′, reverse: 5′-GAGCAGTTGGGGTCCATTC-3′; MT-II forward: 5′-CCTGTGCCTCCGATGGAT-3′, reverse: 5′-ACTTGTCGGAAGCCTCTTTG-3′; MT-III forward: 5′-CTGAGACCTGCCCCTGTC-3′, reverse: 5′-TTCTCGGCCTCTGCCTTG-3′; SOD1 forward: 5′-AGCATTCCATCATTGGCCGTA-3′, reverse: 5′-TACTGCGCAATCCCAATCACTC-3′; Prdx1 forward: 5′-ACAGCTGTTATGCCAGATGGAC-3′, reverse: 5′-CAATCACTTGGCAGTTGAGTTTC-3′;GAPDH forward: 5′-TGTGTCCGTCGTGGATCTGA-3′, reverse: 5′-TTGCTGTTGAAGTCGCAGGAG-3′. 
Visible Light Irradiation in MT-III Knockdown 661W Cells
At 24 hours after the transfection, the medium was replaced with 1% FBS-DMEM and cultured at 37°C for 1 hour. After preincubation, the cells were exposed to 2500 lux of white fluorescent light (Nikon, Tokyo, Japan) for 24 hours at 37°C. 
Hoechst 33342 Staining
At the end of the light exposure treatment, Hoechst 33342 (excitation 360 nm, emission 490 nm) and PI were added to the culture medium (final concentrations of 8.1 and 1.5 μM, respectively) and incubated for 15 minutes. Microscopic images through fluorescence filters for Hoechst 33342 (U-MWU; Olympus Co., Tokyo, Japan) and PI (U-MWIG; Olympus) were captured by a CCD camera (DP30BW; Olympus). 
Measurement of ROS Production in 661W Cells
Intracellular radical activation within 661W cells was determined using CM-H2DCFDA. At the end of the light exposure period, CM-H2DCFDA was added to the culture medium and incubated at 37°C for 1 hour at a final concentration of 10 μM. The 96-well plate was loaded into a plate-holder in a fluorescence spectrophotometer. The reaction was performed at 37°C, and fluorescence was measured at 488 nm excitation and 525 nm emission. The number of cells was determined by Hoechst 33342 staining and used to calculate ROS production per cell. 26  
Statistical Analysis
Data are presented as the means ± SEM. Statistical comparisons were made using Student's t-test or Dunnett's multiple comparison test (using STAT VIEW version 5.0; SAS Institute, Cary, NC). P < 0.05 was considered to indicate statistical significance. 
Results
Expression of the mRNA Level in the MT Family and in Antioxidant Proteins
To clarify changes in MT mRNA levels in light-exposed retina, MT expressions in 129/SV mice were examined after 6 hours of light exposure in the retina by real-time RT-PCR. MT-I and MT-III mRNA were elevated significantly after light exposure, 2.4- and 1.7-fold, respectively (Figs. 1A, 1C). On the other hand, the MT-II mRNA level was increased dramatically 44-fold versus the nonirradiated (control) group (Fig. 1B). The mRNA expressions in ddY mice, which generally were used in a light-induced photoreceptor damage model, were similar to those of 129/SV mice (Figs. 1D–F). An antioxidant protein, SOD1 was increased significantly after 6 hours of light exposure in the retina of 129/SV WT, MT-I/II–deficient, and MT-III–deficient mice compared to nonexposed groups, and there were no significant differences between WT mice and MT-I/II– or MT-III–deficient mice (Fig. 1G). Prdx1, the other antioxidant protein, was not changed significantly in any of the groups (Fig. 1H). The MT-III expression level in MT-I/II–deficient mice was not altered compared to that in 129/SV WT mice (data not shown). 
Figure 1. 
 
Real-time PCR analysis of MT isoforms (I–III) and antioxidants mRNA levels in light-damaged retina of 129/SV, ddY, and MTs-deficient mice. The expression levels of MTs mRNA in the retina were measured using a quantitative real-time RT-PCR assay 6 hours after light exposure in 129/SV (16,000 lux, AC) and ddY (8000 lux, DF) mice, and in respective unexposed controls. (A, D) MT-I, (B, E) MT-II, (C, F) MT-III expression levels. The expression levels of SOD1 (G) and Prdx1 (H) mRNA in the retina at 6 hours after light exposure (16,000 lux) in 129/SV WT, MT-I/II–deficient and MT-III–deficient mice. The data are shown as means ± SEM, n = 7 to 10. *P < 0.05, **P < 0.01 versus the control or nonirradiated matched group.
Figure 1. 
 
Real-time PCR analysis of MT isoforms (I–III) and antioxidants mRNA levels in light-damaged retina of 129/SV, ddY, and MTs-deficient mice. The expression levels of MTs mRNA in the retina were measured using a quantitative real-time RT-PCR assay 6 hours after light exposure in 129/SV (16,000 lux, AC) and ddY (8000 lux, DF) mice, and in respective unexposed controls. (A, D) MT-I, (B, E) MT-II, (C, F) MT-III expression levels. The expression levels of SOD1 (G) and Prdx1 (H) mRNA in the retina at 6 hours after light exposure (16,000 lux) in 129/SV WT, MT-I/II–deficient and MT-III–deficient mice. The data are shown as means ± SEM, n = 7 to 10. *P < 0.05, **P < 0.01 versus the control or nonirradiated matched group.
Light-Induced Retinal Damage was Exacerbated in MT-III–, but Not in MT-I/-II–Deficient Mice
The participation of MT in light-induced retinal degeneration was examined by comparing the MT-I/-II–deficient mice and the MT-III–deficient mice with the WT mice. Firstly, the participation of MT was examined with electrophysiologic analyses. In MT-III–deficient mice, the a- and b-wave amplitudes were reduced significantly 5 days after 16,000 lux white light exposure for 3 hours, compared to the light-exposed WT mice, with a reduction of 47% and 30%, respectively, (Figs. 2A, 2B) at 0.98 log cds/m2
Figure 2. 
 
Measurement of the dark-adapted ERG amplitudes 5 days after light exposure in the retina of the MT-deficient mice. (A) Typical traces of dark-adapted ERG responses measured 5 days after exposure to light. (B) Amplitudes of a- and b-waves of light-exposed (16,000 lux) WT mice versus light-exposed MT-deficient mice. Stimulus flashes were used from −1.92 to 0.98 log cds/m2. The data are shown as means ± SEM. n = 8 to 13. *P < 0.05 versus the light-exposed WT mice group.
Figure 2. 
 
Measurement of the dark-adapted ERG amplitudes 5 days after light exposure in the retina of the MT-deficient mice. (A) Typical traces of dark-adapted ERG responses measured 5 days after exposure to light. (B) Amplitudes of a- and b-waves of light-exposed (16,000 lux) WT mice versus light-exposed MT-deficient mice. Stimulus flashes were used from −1.92 to 0.98 log cds/m2. The data are shown as means ± SEM. n = 8 to 13. *P < 0.05 versus the light-exposed WT mice group.
Figure 3 shows the results of the histologic evaluation, depicting the representative retinal images between 480 and 720 μm from the optic nerve in the inferior and superior area 5 days following light exposure. The ONL was slightly thinner in the WT and in the MT-I/-II–deficient mice (Figs. 3A–C, 3E). In contrast, photic damage was exacerbated in the MT-III–deficient mice (Figs. 3D, 3E). The ONL thickness in the light-exposed MT-III–deficient mice was reduced by 21% (average of points 240–1920 μm from the optic nerve) compared to the light-exposed WT retina (Fig. 3E). The result of the ERG was consistent with that of morphology. 
Figure 3. 
 
Retinal damage after light exposure in MT-deficient mice. Control retina from WT mice (A), light-exposed (16,000 lux) retina from WT mice (B), from MT-I/II–deficient mice (C), and from MT-III–deficient mice (D). (E) The ONL thickness 5 days after light exposure in the retina. The data are shown as means ± SEM, n = 8 to 13. *P < 0.05, **P < 0.01 versus the light exposed-WT mice. Scale bar: 50 μm.
Figure 3. 
 
Retinal damage after light exposure in MT-deficient mice. Control retina from WT mice (A), light-exposed (16,000 lux) retina from WT mice (B), from MT-I/II–deficient mice (C), and from MT-III–deficient mice (D). (E) The ONL thickness 5 days after light exposure in the retina. The data are shown as means ± SEM, n = 8 to 13. *P < 0.05, **P < 0.01 versus the light exposed-WT mice. Scale bar: 50 μm.
MT-III Knockdown Exacerbated Light-Induced 661W Cell Damage
As shown in Figure 4, each of the MT-III siRNAs (#1–4) that recognize nonoverlapping sequences of murine MT-III decreased the MT-III mRNA expression. MT-III siRNA #3 and #4 led to significant decreases in the MT-III mRNA level compared to control siRNA, whereas MT-III siRNA #1 and #2 did not produce a significant decrease (Fig. 4A). In our study, we used MT-III siRNA #3 and #4 (high efficiency), and MT-III siRNA #1 (low efficiency). Representative fluorescence staining of nuclei with Hoechst 33342 and PI are shown in Figure 4B. Hoechst 33342 stains all cells (live and dead cells), whereas PI stains only dead cells. The control siRNA-treated cells showed normal nuclear morphology and negative staining with PI. PI-positive cells were increased by light exposure, and the MT-III siRNA #3 and #4 treatment plus light exposure group showed increased light-induced damage compared to the control siRNA plus light exposure group. On the other hand, there were no changes in cell damage between the MT-III siRNA #1–treated group and the control siRNA group (Fig. 4C). 
Figure 4. 
 
Light-exposed photoreceptor cell damage in MT-III knockdown 661W cells. (A) Cells were transfected with either the control siRNA or with MT-III siRNAs (MT-III siRNA #1–#4). At 24 hours after transfection, the total RNA was extracted from the cells and analyzed using real-time PCR. The data are shown as means ± SEM, n = 3. **P < 0.01 versus control siRNA. (B) Representative fluorescence microscopy of Hoechst 33342 and PI staining after light exposure. The number of PI-positive cells increased after light exposure. (C) The number of cells exhibiting PI fluorescence was counted, and positive cells were expressed as the percentage of PI-positive to Hoechst 33342-positive cells. The data are shown as mean ± SEM, n = 6. ## P < 0.01 versus the control siRNA without light exposure, **P < 0.01 versus light-exposed control siRNA. Scale bar: 50 μm. Control: control siRNA.
Figure 4. 
 
Light-exposed photoreceptor cell damage in MT-III knockdown 661W cells. (A) Cells were transfected with either the control siRNA or with MT-III siRNAs (MT-III siRNA #1–#4). At 24 hours after transfection, the total RNA was extracted from the cells and analyzed using real-time PCR. The data are shown as means ± SEM, n = 3. **P < 0.01 versus control siRNA. (B) Representative fluorescence microscopy of Hoechst 33342 and PI staining after light exposure. The number of PI-positive cells increased after light exposure. (C) The number of cells exhibiting PI fluorescence was counted, and positive cells were expressed as the percentage of PI-positive to Hoechst 33342-positive cells. The data are shown as mean ± SEM, n = 6. ## P < 0.01 versus the control siRNA without light exposure, **P < 0.01 versus light-exposed control siRNA. Scale bar: 50 μm. Control: control siRNA.
MT-III Knockdown Increased Light-Induced Intracellular ROS Production
CM-H2DCFDA, a cell-permeant indicator of ROS, is nonfluorescent until removal of its acetate groups by intracellular esterase. Within the cell, esterases cleave CM-H2DCFDA to release CM-H2DCFDH, which is converted to a fluorescent product (CM-H2DCF), when exposed to ROS. Intracellular ROS production increased significantly in the MT-III siRNA #3 and #4 groups compared to the control siRNA plus light exposure group (159% and 144%, respectively), whereas ROS production was not increased by MT-III siRNA #1 (Fig. 5). 
Figure 5. 
 
ROS production induced by light exposure in MT-III knockdown 661W cells. Intracellular ROS levels were determined by measuring the fluorescence of CM-H2DCFDA (at excitation 488 nm/emission 525 nm) after light exposure at 24 hours in MT-III knockdown 661W. The data are shown as means ± SEM, n = 6. ## P < 0.01 versus control siRNA without light exposure, **P < 0.01 versus the light-exposed control siRNA group. Control: control siRNA.
Figure 5. 
 
ROS production induced by light exposure in MT-III knockdown 661W cells. Intracellular ROS levels were determined by measuring the fluorescence of CM-H2DCFDA (at excitation 488 nm/emission 525 nm) after light exposure at 24 hours in MT-III knockdown 661W. The data are shown as means ± SEM, n = 6. ## P < 0.01 versus control siRNA without light exposure, **P < 0.01 versus the light-exposed control siRNA group. Control: control siRNA.
Discussion
In our study, we assessed the roles of MT isforms (I–III) in light-induced retinal degeneration. Firstly, to understand the isoform-specific upregulation in the 129/SV and ddY mice retina upon light damage, we performed real-time RT-PCR with isoform-specific primers. Previous work has shown that MT transcription is upregulated by oxidative stress. 27 In this study, MT isoforms (I–III) were upregulated 2.4-, 44-, and 1.7-fold in 129/SV mice, and 1.6-, 8.9-, and 1.3-fold in ddY mice after 6 hours of light exposure, respectively (Figs. 1A–F). In a previous report, MT isoforms (I–III) were upregulated 10-, 23-, and 1.4-fold in Balb/c mice, respectively, 6 hours after light exposure of the retina compared to control retina. 25 Our results showed a similar tendency as noted in the previous report, although there are some differences in the strain, irradiation intensity, and irradiation time. 
To investigate the functional role of MT, we used MT-I/-II– and -III–deficient mice. 28,29 We evaluated the involvement of MT in light-induced retinal dysfunction using ERG. The a-wave shows the function of the photoreceptors, and the b-wave reflects bipolar cells and Müller cell function. In our study, the a- and b-wave amplitude reduction was greater significantly in the MT-III–deficient mice than in the WT mice following light exposure. ONL thinning also was exacerbated in the MT-III–deficient mice. On the other hand, there were no changes in the retinal function or ONL thickness in the MT-I/II–deficient mice. 
The main effects of light-induced cell death have been shown to involve ROS production and activation of apoptotic signaling pathways. 30 Excessive light exposure induces many ROSs, and ROS production can be overcome by a retinal defensive mechanism; that is, increasing antioxidative proteins, such as SOD. 31 It has been reported that SOD is induced in the rat retina after light exposure, 32 and mutant SOD1 mice also have been shown to be highly susceptible to environmental light-induced retinal degeneration. 33 These reports indicate that many superoxide radicals (O2 ) are induced by light exposure and that they are associated with retinal dysfunction or cell death, 34 although ROS have been implicated in the regulation of many important cellular events, including transcription-factor activation, 35 gene expression, 36 and cell proliferation. 37 Additionally, superoxide radicals (O2 ) can change hydroxyl radicals (·OH), which can critically injure the DNA and the cell membrane. 38 For this reason, it is conceivable that oxidative stress is associated with a light-induced retinal damage mechanism. In this study, expression levels of SOD1 and Prdx1, two antioxidant proteins, were not changed by disruption of MTs (Figs. 1G, 1H). The amount of antioxidant proteins may be altered at other time point, and these proteins may provide a compensatory mechanism in MT-I/II–deficient mice, whereas they could not compensate for MT-III deficiency. MT-III exhibits the most efficient protective effect against OH-induced DNA single-strand breaks compared to that of MT-I/-II. 39 MT-I/-II has been reported to be upregulated in rodent models of RP, rd1 mice, rds mice, and Royal College of Surgeons rat retinas, but this upregulation did not coincide with the onset of photoreceptor cell loss. 40 The inability to produce endogenous MT-I/-II is not associated with the loss of photoreceptors induced by hyperbaric oxygen exposure. 41 These results suggested that MT-I/-II does not have a pivotal role in protecting against light-induced retinal photoreceptor cell loss, whereas MT-III has a neuroprotective effect possibly due to its strong interaction with ROS. In addition, MT-III is known as a neuronal growth inhibitory factor that interacts with many proteins. 42 Taken together, our results indicated that MT-III may exert an important neuroprotective influence over retinal photoreceptor cells, with antioxidant activity and/or other protection mechanisms. Previously, we reported that MT-II was increased in NMDA-treated retina, especially ganglion cell layer (GCL) and inner plexiform layer, and that the GCL in MT-I/II–deficient mice exhibited increased vulnerability to NMDA. 22 Moreover, the present in vitro studies revealed that MT-III has an antioxidant effect and a protective effect against light-induced cell death in cultured photoreceptor cells (Figs. 4, 5). Taken together with previous and present data, MT-I/II may have an important role in GCL mainly, and MT-III may exert its protective effects in ONL and photoreceptor cells in acute models. Detailed studies of MT-III localization in retina will contribute to elucidate the functions of the different MT isoforms. 
Evidence has been presented that dietary zinc deprivation is associated with an increased risk for the development of some forms of AMD. 43 Zinc acts as an antioxidant through some mechanisms, including the acceleration of MT synthesis. 44 Similar to zinc, MT also is decreased with age in human macular retinal pigment epithelium. 45 Suppression of MT synthesis has been observed in the retina of early onset macular degeneration in monkeys. 46 Taken together, these previous reports suggest that MT reduction is one of the factors of AMD onset. 
In conclusion, we demonstrated that MT isoforms (I–III) mRNA are upregulated in the murine retina by light exposure, and that light-induced retinal damage is exacerbated in MT-III–deficient mice. Additionally, cell death and ROS production increased in MT-III knockdown in 661W cells. Hence, these findings suggested that MT-III has a pivotal role as an endogenous neuroprotectant against light-induced retinal damage. 
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Footnotes
2  These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Footnotes
 Disclosure: K. Tsuruma, None; H. Shimazaki, None; Y. Ohno, None; Y. Inoue, None; A. Honda, None; S. Imai, None; J. Lee, None; M. Shimazawa, None; M. Satoh, None; H. Hara, None
Figure 1. 
 
Real-time PCR analysis of MT isoforms (I–III) and antioxidants mRNA levels in light-damaged retina of 129/SV, ddY, and MTs-deficient mice. The expression levels of MTs mRNA in the retina were measured using a quantitative real-time RT-PCR assay 6 hours after light exposure in 129/SV (16,000 lux, AC) and ddY (8000 lux, DF) mice, and in respective unexposed controls. (A, D) MT-I, (B, E) MT-II, (C, F) MT-III expression levels. The expression levels of SOD1 (G) and Prdx1 (H) mRNA in the retina at 6 hours after light exposure (16,000 lux) in 129/SV WT, MT-I/II–deficient and MT-III–deficient mice. The data are shown as means ± SEM, n = 7 to 10. *P < 0.05, **P < 0.01 versus the control or nonirradiated matched group.
Figure 1. 
 
Real-time PCR analysis of MT isoforms (I–III) and antioxidants mRNA levels in light-damaged retina of 129/SV, ddY, and MTs-deficient mice. The expression levels of MTs mRNA in the retina were measured using a quantitative real-time RT-PCR assay 6 hours after light exposure in 129/SV (16,000 lux, AC) and ddY (8000 lux, DF) mice, and in respective unexposed controls. (A, D) MT-I, (B, E) MT-II, (C, F) MT-III expression levels. The expression levels of SOD1 (G) and Prdx1 (H) mRNA in the retina at 6 hours after light exposure (16,000 lux) in 129/SV WT, MT-I/II–deficient and MT-III–deficient mice. The data are shown as means ± SEM, n = 7 to 10. *P < 0.05, **P < 0.01 versus the control or nonirradiated matched group.
Figure 2. 
 
Measurement of the dark-adapted ERG amplitudes 5 days after light exposure in the retina of the MT-deficient mice. (A) Typical traces of dark-adapted ERG responses measured 5 days after exposure to light. (B) Amplitudes of a- and b-waves of light-exposed (16,000 lux) WT mice versus light-exposed MT-deficient mice. Stimulus flashes were used from −1.92 to 0.98 log cds/m2. The data are shown as means ± SEM. n = 8 to 13. *P < 0.05 versus the light-exposed WT mice group.
Figure 2. 
 
Measurement of the dark-adapted ERG amplitudes 5 days after light exposure in the retina of the MT-deficient mice. (A) Typical traces of dark-adapted ERG responses measured 5 days after exposure to light. (B) Amplitudes of a- and b-waves of light-exposed (16,000 lux) WT mice versus light-exposed MT-deficient mice. Stimulus flashes were used from −1.92 to 0.98 log cds/m2. The data are shown as means ± SEM. n = 8 to 13. *P < 0.05 versus the light-exposed WT mice group.
Figure 3. 
 
Retinal damage after light exposure in MT-deficient mice. Control retina from WT mice (A), light-exposed (16,000 lux) retina from WT mice (B), from MT-I/II–deficient mice (C), and from MT-III–deficient mice (D). (E) The ONL thickness 5 days after light exposure in the retina. The data are shown as means ± SEM, n = 8 to 13. *P < 0.05, **P < 0.01 versus the light exposed-WT mice. Scale bar: 50 μm.
Figure 3. 
 
Retinal damage after light exposure in MT-deficient mice. Control retina from WT mice (A), light-exposed (16,000 lux) retina from WT mice (B), from MT-I/II–deficient mice (C), and from MT-III–deficient mice (D). (E) The ONL thickness 5 days after light exposure in the retina. The data are shown as means ± SEM, n = 8 to 13. *P < 0.05, **P < 0.01 versus the light exposed-WT mice. Scale bar: 50 μm.
Figure 4. 
 
Light-exposed photoreceptor cell damage in MT-III knockdown 661W cells. (A) Cells were transfected with either the control siRNA or with MT-III siRNAs (MT-III siRNA #1–#4). At 24 hours after transfection, the total RNA was extracted from the cells and analyzed using real-time PCR. The data are shown as means ± SEM, n = 3. **P < 0.01 versus control siRNA. (B) Representative fluorescence microscopy of Hoechst 33342 and PI staining after light exposure. The number of PI-positive cells increased after light exposure. (C) The number of cells exhibiting PI fluorescence was counted, and positive cells were expressed as the percentage of PI-positive to Hoechst 33342-positive cells. The data are shown as mean ± SEM, n = 6. ## P < 0.01 versus the control siRNA without light exposure, **P < 0.01 versus light-exposed control siRNA. Scale bar: 50 μm. Control: control siRNA.
Figure 4. 
 
Light-exposed photoreceptor cell damage in MT-III knockdown 661W cells. (A) Cells were transfected with either the control siRNA or with MT-III siRNAs (MT-III siRNA #1–#4). At 24 hours after transfection, the total RNA was extracted from the cells and analyzed using real-time PCR. The data are shown as means ± SEM, n = 3. **P < 0.01 versus control siRNA. (B) Representative fluorescence microscopy of Hoechst 33342 and PI staining after light exposure. The number of PI-positive cells increased after light exposure. (C) The number of cells exhibiting PI fluorescence was counted, and positive cells were expressed as the percentage of PI-positive to Hoechst 33342-positive cells. The data are shown as mean ± SEM, n = 6. ## P < 0.01 versus the control siRNA without light exposure, **P < 0.01 versus light-exposed control siRNA. Scale bar: 50 μm. Control: control siRNA.
Figure 5. 
 
ROS production induced by light exposure in MT-III knockdown 661W cells. Intracellular ROS levels were determined by measuring the fluorescence of CM-H2DCFDA (at excitation 488 nm/emission 525 nm) after light exposure at 24 hours in MT-III knockdown 661W. The data are shown as means ± SEM, n = 6. ## P < 0.01 versus control siRNA without light exposure, **P < 0.01 versus the light-exposed control siRNA group. Control: control siRNA.
Figure 5. 
 
ROS production induced by light exposure in MT-III knockdown 661W cells. Intracellular ROS levels were determined by measuring the fluorescence of CM-H2DCFDA (at excitation 488 nm/emission 525 nm) after light exposure at 24 hours in MT-III knockdown 661W. The data are shown as means ± SEM, n = 6. ## P < 0.01 versus control siRNA without light exposure, **P < 0.01 versus the light-exposed control siRNA group. Control: control siRNA.
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