Photoreceptor Survival Is Regulated by GSTO1-1 in the Degenerating Retina

Retinal Cell Biology Photoreceptor Survival Is Regulated by GSTO1-1 in the Degenerating Retina

Nilisha Fernando,1 Yvette Wooff,1,2 Riemke Aggio-Bruce,1 Joshua A. Chu-Tan,1 Haihan Jiao,1 Catherine Dietrich,1 Matt Rutar,1 Melissa Rooke,1 Deepthi Menon,1,3 Janis T. Eells,4 Krisztina Valter,1,2 Philip G. Board,1 Jan Provis,1,2 and Riccardo Natoli1,2 1The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia 2The ANU Medical School, The Australian National University, Canberra, Australian Capital Territory, Australia 3School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland 4Department of Biomedical Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, United States

Correspondence: Riccardo Natoli, PURPOSE. Glutathione-S-transferase omega 1-1 (GSTO1-1) is a cytosolic glutathione transferase TheJohnCurtinSchoolofMedical enzyme, involved in glutathionylation, toll-like receptor signaling, and calcium channel Research, Building 131, Garran regulation. GSTO1-1 dysregulation has been implicated in oxidative stress and inflammation, Road, The Australian National Uni- and contributes to the pathogenesis of several diseases and neurological disorders; however, versity, Canberra ACT 2601 Australia; its role in retinal degenerations is unknown. The aim of this study was to investigate the role [email protected]. of GSTO1-1 in modulating oxidative stress and consequent inflammation in the normal and NF and YW contributed equally to the degenerating retina. work presented here and should / therefore be regarded as equivalent METHODS. The role of GSTO1-1 in retinal degenerations was explored by using Gsto1 mice authors. in a model of retinal degeneration. The expression and localization of GSTO1-1 were Submitted: April 20, 2018 investigated with immunohistochemistry and Western blot. Changes in the expression of Accepted: July 4, 2018 inflammatory (Ccl2, Il-1b, and C3) and oxidative stress (Nox1, Sod2, Gpx3, Hmox1, Nrf2, and Nqo1) genes were investigated via quantitative real-time polymerase chain reaction. Retinal Citation: Fernando N, Wooff Y, Aggio- function in Gsto1/ mice was investigated by using electroretinography. Bruce R, et al. Photoreceptor survival is regulated by GSTO1-1 in the de- RESULTS. GSTO1-1 was localized to the inner segment of cone photoreceptors in the retina. generating retina. Invest Ophthalmol Gsto1/ photo-oxidative damage (PD) mice had decreased photoreceptor cell death as well Vis Sci. 2018;59:4362–4374. https:// as decreased expression of inflammatory (Ccl2, Il-1b, and C3) markers and oxidative stress doi.org/10.1167/iovs.18-24627 marker Nqo1. Further, retinal function in the Gsto1/ PD mice was increased as compared to wild-type PD mice.

CONCLUSIONS. These results indicate that GSTO1-1 is required for inﬂammatory-mediated photoreceptor death in retinal degenerations. Targeting GSTO1-1 may be a useful strategy to reduce oxidative stress and inﬂammation and ameliorate photoreceptor loss, slowing the progression of retinal degenerations. Keywords: oxidative damage, inflammation, retinal degeneration, knockout animals, GSTO1-1

lutathione-S-transferase omega 1-1 (GSTO1-1) is a member pathway.10,11 GSTO1-1–deficient macrophages consequently G of a family of glutathione S-transferase enzymes, with many demonstrate lower levels of oxidative stress and inflamma- reported roles including protein protection via glutathionylation.10,11 Notably, GSTO1-1 plays a role in modulating caspase tion and modulating oxidative stress and inflammation.1–3 3–dependent apoptosis in the mouse retina, and in 661W Protein-S-glutathionylation is a posttranslational modification photoreceptor-like cell cultures during hypoglycemia-induced whereby mixed disulfides are formed between protein thiols oxidative stress.12 In the retina, GSH, along with the and glutathione (GSH), to protect these groups from being antioxidant enzyme glutathione peroxidase (GPx), is expressed oxidized to sulfonic acids, and the protein becoming irrevers- in photoreceptors and acts to detoxify peroxides.13 GSH and ibly inactivated.4 In contrast to this role, GSTO1-1 has also been glutathione reductase, which is required for the regeneration of shown to deglutathionylate certain intracellular protein thiols,2 GSH from its disulfide GSSG, are rapidly depleted during exposing them to permanent oxidative damage and initiating a oxidative stress–induced photoreceptor cell death,14,15 while proinflammatory cascade. GSTO1-1 has been implicated in the high plasma GPx is considered to be a strong indicator of age- regulation of oxidative stress and inflammation in various organ related macular degeneration (AMD).13,15 However, the role of systems, where both overexpression and polymorphisms in the GSTO1-1 in modulating oxidative stress and inflammation in encoding gene have been linked to pathological diseases, the normal and degenerating retina is currently unknown. including cancer, and neurological disorders.5–9 Photoreceptors have one of the highest oxygen consump- GSTO1-1 is required for lipopolysaccharide (LPS)–mediated tion levels of any cell type in the body,15,16 and are considered inflammatory responses in macrophages, inducing reactive the major site of ROS production in the retina and are highly oxygen species (ROS) production and proinflammatory cyto- prone to oxidative damage.17,18 This vulnerability is exacerbat- kine expression through the toll-like receptor 4 (TLR4) ed by a range of environmental and lifestyle factors, such as

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30 TABLE 1. List of Primary Antibodies Used for Immunohistochemistry days PD, as previously described. Mice were dark-adapted overnight before being anaesthetized with an intraperitoneal Catalogue injection of Ketamine (100 mg/kg; Troy Laboratories, Glenden- Antibody Dilution Company No. ning, NSW, Australia) and Xylazil (10 mg/kg; Troy Laborato- Rabbit anti–IBA-1 1:500 Wako, Osaka, 019-19741 ries). Both pupils were dilated with one drop each of 2.5% wt/ Japan vol phenylephrine hydrochloride and 1% wt/vol tropicamide Rabbit anti–GSTO1-1 1:200 Kindly gifted by (Bausch and Lomb, Rochester, NY, USA). A single- or twin-flash PGB paradigm was used to elicit a mixed response from rods and PNA 1:500 Abcam ab110262 cones, and an isolated cone response, respectively. Flash Mouse anti-rhodopsin 1:200 Merck Millipore MAB5356 stimuli for mixed responses were provided by an LED-based system (FS-250A Enhanced Ganzfeld; Photometric Solutions International, Huntingdale, VIC, Australia), over a stimulus intensity range of 6.3 log cd s m2 (range, 4.4 to 1.9 log cd s light exposure, a history of smoking, and obesity.19 In addition, m2). Amplitudes of the a-wave and b-wave were analyzed by photoreceptors are rich in polyunsaturated fatty acids, also 15 using LabChart 8 software (AD Instruments, Dunedin, New making them vulnerable to lipid peroxidation. Photochem- Zealand) and data were expressed as the mean wave amplitude ical injury resulting from light exposure is also known to induce photoreceptor damage, apoptosis, and hypopigmenta- 6 SEM (lV). tion of the retinal pigment epithelium.15,16 As the central retina has a high concentration of photoreceptors, oxidative damage Tissue Collection and Preparation tends to accumulate in this area, contributing to the Animals were euthanized with CO following functional ERG pathogenesis of retinal degeneration. Given the naturally high 2 analysis. The superior surface of the left eye from each animal oxidative state of the retina, it is logical that there are tightly was marked and enucleated, then immersed in 4% paraformal- regulated cellular defense mechanisms in place, to protect the dehyde (PFA) for 3 hours. Eyes were then cryopreserved in retina from oxidative damage and the ensuing inflammatory 15% sucrose solution overnight, embedded in OCT medium response.17 Oxidative stress and inflammation are implicated (Tissue Tek, Sakura, Japan), and cryosectioned at 12 lmina in the pathogenesis of many retinal degenerations, including parasagittal plane (superior to inferior) with a CM 1850 AMD, diabetic retinopathy, and retinitis pigmentosa.15,17,20–24 Cryostat (Leica, Mt. Waverley, Victoria, Australia). To ensure Here, we aimed to characterize the role of GSTO1-1 in the accurate comparisons were made for histologic analysis, only retina by using / mice, and investigate its effect on Gsto1 sections containing the optic nerve head were used for analysis oxidative stress and inflammation in the degenerating retina, (n ¼ 6 per experimental group). The retina from the right eye using a rodent model of photo-oxidative damage (PD) in which of each mouse used was excised through a corneal incision and oxidative stress and inflammation have been shown to placed into RNAlater solution (Thermo Fisher Scientific, contribute to progressive focal retinal degeneration.25–29 The Waltham, MA, USA) at 48C overnight and then stored at results indicate that GSTO1-1 has a damaging effect in retinal 808C until further use (n ¼ 6 per experimental group). Some degenerations and demonstrate that knocking out GSTO1-1 retinas were collected into Cellytic M buffer (Sigma-Aldrich reduces oxidative stress and inflammation, and related Corp., St. Louis, MO, USA) containing a Protease Inhibitor photoreceptor cell death in the retina in a model of retinal Cocktail (Sigma-Aldrich) to extract whole cell protein lysates degenerations. and then stored at 808C until further use (n ¼ 6 per experimental group). METHODS Immunolabelling Animal Handling and Photo-Oxidative Damage Immunohistochemical analysis of retinal cryosections was All experiments were conducted in accordance with the ARVO performed as previously described.27 Immunocytochemistry Statement for the Use of Animals in Ophthalmic and Vision was performed on the dim and PD chamber slides of 661W Research and with approval from the ANU Animal Experimen- cells by using a protocol previously described.28 Details of tation Ethics Committee (Ethics ID: A2014/56). Adult male and primary antibodies used are displayed in Table 1. Fluorescence female C57BL6 wild-type (WT) and Gsto1/ mice (aged was visualized and images obtained by using a laser-scanning between 60–90 postnatal days) were bred and reared under A1þ confocal microscope (Nikon, Tokyo, Japan). Images panels 12-hour light/dark-cycle conditions (5 lux) with free access to were analyzed by using ImageJ (http://imagej.nih.gov/ij/; food and water. Gsto1/ mice were supplied by Taconic provided in the public domain by the National Institutes of Biosciences (Derwood, MD, USA), model TF1210 Gsto1/ Health, Bethesda, MD, USA) and assembled with Photoshop NM_010362, and were subsequently backcrossed to the CS6 software (Adobe Systems, San Jose, CA, USA). C57BL6 strain. The origin and characterization of the Gsto1/ For IBA-1 immunohistochemistry, the number of IBA-1þ mice has been previously described in detail.11 Age-matched cells (a marker of retinal microglia and macrophages) was WT and Gsto1/ mice were randomly assigned to PD and dim- counted across the superior and inferior retina in retinal reared (DR) control groups (n ¼ 6 per group). Animals in the cryosections. This quantification was performed on two PD group were continuously exposed to 100 K lux white LED retinal sections per mouse and averaged. Retinal cryosec- light for a period of 5 days, as described previously,30 while DR tions were stained with the DNA-specific dye bisbenzimide mice were maintained in 12-hour light (5 lux)/dark-cycle (1:10,000; Sigma-Aldrich) to visualize the cellular layers. The conditions. thickness of the outer nuclear layer (ONL) was determined by counting the number of rows of nuclei (photoreceptor Measurement of Retinal Function Using cell bodies) in the area of retinal lesion development (1 mm Electroretinography superior to the optic nerve head), to quantify photoreceptor survival. In addition, ONL thickness was calculated as the Full-field scotopic electroretinography (ERG) was performed to ratio of the thickness of the ONL to the whole retinal assess the retinal function of DR controls and animals after 5 thickness (outer limiting membrane to the inner limiting

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TABLE 2. TaqMan Hydrolysis Probes Used for qRT-PCR two 8-well chamber slides (5000 cells per well) in growth medium, and were incubated overnight in dim conditions in a Gene Symbols Gene Name Catalogue No. humidified atmosphere of 5% CO2 at 378C. The following day, Actb b-actin Mm01205647_m1 cells in the 24-well plate and 8-well chamber slides were Chac1 Glutathione-specific gamma- Mm00509926_m1 incubated in reduced-serum DMEM (supplemented with 1% FBS, L-glutamine, and antibiotic-antimycotic) and after 24 hours glutamylcyclotransferase 1 2 C3 Complement component C3 Mn00437858_m1 were exposed to 15,000 lux light (2.2 mW/cm , irradiance Ccl2 Chemokine (C-C motif) ligand 2 Mm99999056_m1 measured with PM100D optical power meter; THORLABS, Gapdh Glyceraldehyde-3-phosphatase Mm01536933_m1 Newton, NJ, USA) from two white fluorescent lamps (2 3 10W dehydrogenase T4 tri-phosphor 6500K daylight fluorescent tubes; Crompton, Gpx3 Glutathione peroxidase 3 Mm00492427_m1 Minto, NSW, Australia) for 5 hours with 5% CO2 at 378C. For Gsto1 Glutathione-S-transferase Mm00599866_m1 dim control cells, one plate and one chamber slide in the omega 1-1 incubator were completely wrapped in aluminum foil to avoid Hmox-1 Heme oxygenase 1 Mm01536933_m1 light exposure. For air/gas exchange, six small incisions were Il-1b Interleukin-1b Mm00434228_m1 cut on the aluminum foil. Following incubation, cells in both Nox1 NADPH oxidase 1 Mm00549170_m1 the dim and PD chamber slides were washed in PBS before Nqo1 NAD(P)H dehydrogenase, Mm01253561_m1 being fixed in 4% PFA for 2 hours at 48C and then were quinone 1 maintained in PBS at 48C until further use. Cells in each of the Nrf2 Nuclear factor, erythroid Mm00477784_m1 24 wells were washed with PBS and were then triturated either derived 2, like 2 in TRIzol (Thermo Fisher Scientific) and stored at 808C until Sod2 Superoxide dismutase 2 Mm01313000_m1 further use (n ¼ 6 per experimental group), or placed into CellLytic M buffer (Sigma-Aldrich) containing a Protease Inhibitor Cocktail (Sigma-Aldrich) and then stored at 808C membrane), to take into account any obliquely cut sections. until further use (n ¼ 6 per experimental group). The process of ONL photoreceptor cell row quantification was performed five times per retina, on two retinal sections Quantitative Real-Time Polymerase Chain Reaction at comparable locations per mouse, while the ONL thickness Total RNA was extracted from the retinal and 661W cell measurement from each retina was the average of two samples as described previously,32 using a combination of retinal sections at comparable locations (1 mm superior to TRIzol (Thermo Fisher Scientific) and an RNAqueous Micro the optic nerve head). Total RNA Isolation kit (Thermo Fisher Scientific). The concentration and purity of each RNA sample was assessed TUNEL Staining and Quantification by using the ND-1000 spectrophotometer (Nanodrop Technol- ogies, Wilmington, DE, USA). Terminal deoxynucleotidyl transferase (TdT)–deoxyuridine Following purification of RNA, cDNA was synthesized from triphosphate (dUTP) nick-end labeling (TUNEL) was used as 1 lg of each RNA sample by using a Tetro cDNA Synthesis Kit a measure of photoreceptor cell apoptosis. TUNEL in situ (Bioline, London, UK) according to the manufacturer’s labelling was performed on retinal cryosections by using a TdT protocol. Gene expression was measured by using mouse- enzyme (Cat No. 3333566001; Sigma-Aldrich) and biotinylated specific TaqMan hydrolysis probes (Thermo Fisher Scientific), dUTP (Cat No. 11093070910; Sigma-Aldrich) as previously as shown in Table 2. The TaqMan probes, cDNA, and TaqMan described.31 In each retinal section, the total number of þ Gene Expression Master Mix (Thermo Fisher Scientific) were TUNEL cells was counted along both the superior and inferior plated in a 384-well transparent plate. Each reaction was retina. This process of quantification was performed on two performed in technical duplicate and was carried out by using retinal sections per animal and was calculated as the average þ a QuantStudio 12 K Flex RT-PCR machine (Thermo Fisher number of TUNEL cells per retinal section. Images of TUNEL Scientific). Analysis was performed by using the comparative C staining were captured with the A1þ confocal microscope at t method (DDCt). Results were analyzed as a percentage change 310 magnification. relative to DR control samples, and normalized to two reference genes, glyceraldehyde-3-phosphate dehydrogenase 661W Cell Culture (Gapdh) and b-actin (Actb). Murine photoreceptor-derived 661W cells (kindly gifted by Muayyad R. Al-Ubaidi, Department of Cell Biology, University of Western Blot Oklahoma Health Sciences Center, Oklahoma City, OK, USA) Western blot was used to measure the protein expression of within five passages of authentication were used for these GSTO1-1 in retinas from WT and Gsto1/ mice. The blot was experiments. Validation of authenticity was performed by performed by using whole retinal and 661W cell protein using gene expression of green cone pigments and cone lysates according to previously described methods.28 Twenty arrestin. Cells were further validated for species authenticity micrograms of denatured protein was loaded into a 4% to 20% (CellBank, Sydney, Australia). These cells were cultured as Mini-Protean TGX Precast Protein gel (Bio-Rad, Hercules, CA, 28 previously published, in growth medium containing Dulbec- USA) followed by semidry transfer to a nitrocellulose co’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supple- membrane. To detect protein expression of GSTO1-1 in the mented with 10% fetal bovine serum (FBS; Sigma-Aldrich), 6 retina, a GSTO1-1 primary antibody (1:200; kindly gifted by mM L-glutamine (Thermo Fisher Scientific), and antibiotic- PGB) was used along with a human GST-ubiquitin protein antimycotic (100 U/mL penicillin, 100 lg/mL streptomycin; (1:100; also gifted by PGB), as well as a secondary rabbit anti- Thermo Fisher Scientific). Cells were maintained in dim horseradish peroxidase conjugate for visualization (Bio-Rad). conditions in a humidified atmosphere of 5% CO2 at 378C The protein was visualized with chemiluminescence using a and passaged by trypsinization every 3 to 4 days. Clarity Western ECL kit (Bio-Rad) and images were captured At passage two, 661W cells were split and seeded into two and analyzed by using a Chemidoc MP with Image Lab software 24-well plates (at a density of 2.5 3 104 cells per well) and into (Bio-Rad).

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Glutathione Assay clotransferase 1 (Chac1), a glutathione degradation enzyme (Fig. 2G), as well as the total ROS/reactive nitrogen species A glutathione assay was conducted to compare the levels of (ROS/RNS) levels, were measured in WT and Gsto1/ mice, reduced glutathione (GSH) and oxidized glutathione (GSSG) in before (DR) and after PD (Fig. 2H). A significant increase in the order to determine the oxidative state of both DR and PD WT / gene expression of Nrf2 (P < 0.05; Fig. 2E) and Nqo1 (P < and Gsto1 retinas. The assay was performed with whole 0.05; Fig. 2F), as well as in the total ROS/RNS levels, was retinal protein lysates and a De-proteinising Sample Prepara- detected in WT PD retinas compared to WT DR retinas (P < tion Kit – TCA (ab204708; Abcam, Cambridge, UK) and a GSH/ 0.05; Fig. 2H). Chac1 expression was also found to decrease GSSG Ratio Detection Assay Kit (Fluorometric – Green, between both WT DR and Gsto1/ DR mice and between WT ab138881; Abcam), according to the manufacturer’s protocols. DR and WT PD mice (P < 0.05; Fig. 2G). While there was a significant increase in the gene expression of oxidative stress Statistical Analysis markers Hmox-1 (P < 0.05; Fig. 2A), Gpx3 (P < 0.05; Fig. 2B), Nrf2 (P < 0.05; Fig. 2E), and Nqo1 (P < 0.05; Fig. 2F), All graphing and statistical analyses were performed with Prism following PD in Gsto1/ mice compared to Gsto1/ DR 6 (GraphPad Software, San Diego, CA, USA). An unpaired controls, a significant decrease was seen in the gene Student’s t-test, 1-way ANOVA, or 2-way ANOVA with Tukey’s expression of Nqo1 (P < 0.05; Fig. 2F) as well as in the total multiple comparison post test was used to determine the ROS/RNS levels in Gsto1/ PD retinas compared to WT PD statistical outcome; a P value of <0.05 was considered retinas (P < 0.05; Fig. 2H). This result overall indicates a statistically significant. reduced oxidative state in GSTO1-1–deficient mice following PD. RESULTS GSTO1-1 Deficiency Reduces Inflammation and In Vivo and In Vitro Expression and Localization Activated Microglia/Macrophage Accumulation in of GSTO1-1 in the Retina the Retina Following Photo-Oxidative Damage GSTO1-1 protein and Gsto1 gene expression were absent from The number of microglia/macrophages accumulating in the Gsto1/ mice retinas (Fig. 1). In WT animals, GSTO1-1 was outer retina (photoreceptor layer and subretinal space) of WT detected at 28 kDa by Western blot, but was not found in and Gsto1/ mice was determined by using IBA-1 immunore- Gsto1/ retinas (Fig. 1A). Quantification of gene expression activity following PD. There was a significant increase in the levels by quantitative real-time polymerase chain reaction (qRT- total number of outer retinal IBA-1þ cells following PD in both PCR) confirmed an absence of Gsto1 gene expression in WT and Gsto1/ retinas compared to respective DR controls Gsto1/ DR retinas and in Gsto1/ retinas subjected to 5 days (P < 0.05; Fig. 3A). No significant difference in outer retinal PD (P < 0.05; Fig. 1B). These data indicate a complete IBA-1þ cell counts was observed between WT and Gsto1/ PD functional knockout of GSTO1-1 in the Gsto1/ mouse retina. groups (P > 0.05; Fig. 3A). However, analysis of IBA-1þ cell There was no change in GSTO1-1 protein or gene expression morphology (ramified versus amoeboid) indicated a signifi- between WT PD mice and WT DR controls (P > 0.05; Figs. 1A, cantly reduced number of amoeboid cells in the outer retina of 1B). Gsto1/ PD retinas compared to WT PD retinas (P < 0.05; Fig. Localization of GSTO1-1 in the WT retina by immunohisto- 3B). chemistry indicated that GSTO1-1 was strongly expressed in To investigate the effect of GSTO1-1 deficiency on key retinal cone photoreceptors (GSTO1-1 and peanut agglutinin inflammatory markers known to be upregulated in the PD [PNA] colabelling; Fig. 1D, inlet window), but not rod model,26,29,33 inflammatory gene expression was investigated photoreceptors (no colabelling with rhodopsin; Fig. 1K, inlet by using qRT-PCR. Results showed a significant downregula- window) and was seen specifically within the inner segment of tion in the expression of Ccl2 (P < 0.05; Fig. 3G), Il-1b (P < cones, highly expressed along the superior retina, and weakly 0.05; Fig. 3H), and C3 (P < 0.05; Fig. 3I) in Gsto1/ PD retinas in these cells along the inferior retina (Figs. 1C–J). In Gsto1/ compared to WT PD retinas. While there was a statistically mice, cone inner segments were weakly labelled with GSTO1-1 significant increase in the expression of all three inflammatory antibodies in DR and PD animals (Figs. 1F, 1J). GSTO1-1 genes between WT DR and WT PD groups (P < 0.05; Figs. 3G– immunoreactivity was also detected in inner nuclear layer I), there was only a statistically significant upregulation of C3 (INL) cells in DR and PD WT retinas, which we tentatively between Gsto1/ DR and Gsto1/ PD mice (P < 0.05; Fig. 3I). identify as amacrine cells (Figs. 1C, 1D, 1G, 1H). This labelling No statistical difference in Ccl2 or Il-1b expression between / / pattern was not observed in either DR or PD Gsto1/ retinas Gsto1 DR and Gsto1 PD mice (P > 0.05; Figs. 3G, 3H) (Figs. 1E, 1F, 1I, 1J). In vitro studies of 661W mouse was observed. Overall, these data indicate that Gsto1/ photoreceptor-like cell line also showed increased Gsto1 levels deficiency reduces inflammation and activated microglia/ following 5 hours PD by qRT-PCR (Fig. 1M) and immunocyto- macrophage accumulation in PD. chemistry (Fig. 1N). Gene expression of oxidative stress markers heme oxygenase-1 (Hmox1); nuclear factor, erythroid Retinal Function Is Increased and Cell Death derived 2, like 2 (Nrf2); and NAD(P)H quinone dehydrogenase Decreased in GSTO1-1/ Retinas Following Photo- 1(Nqo1) also increased in 661W cells after 5 hours PD (Fig. 1M). Oxidative Damage The level of photoreceptor cell death was measured and Changes in Oxidative Stress in Gsto1/ Retinas compared in the retinas of WT and Gsto1/ mice by using a Following Photo-Oxidative Damage TUNEL assay. Following PD, analyses showed that there was a significantly decreased number of TUNELþ photoreceptors (P To investigate the effect of GSTO1-1 deficiency on oxidative < 0.05; Fig. 4A) and a significantly increased number of stress production in the retina, the gene expression changes of photoreceptor cell rows (P < 0.05; Fig. 4B) in Gsto1/ mice oxidative stress markers Hmox-1, Gpx3, Nox1, Sod2, Nrf2, and compared to WT mice. This change in photoreceptor cell Nqo1 (Figs. 2A–F) and glutathione-specific gamma-glutamylcy- survivability was also reflected by using ERG, which demon-

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FIGURE 1. Localization and expression of GSTO1-1 in the retina and in 661W cells before (dim-reared, DR) and after photo-oxidative damage (PD). (A) GSTO1-1 expression was detected via Western blot, producing a 28-kDa band in WT but not Gsto1/ retinas. No change was seen in GSTO1-1 protein expression in WT mice after PD. (B) Gsto1 gene expression was significantly reduced in both the DR and PD Gsto1/ mice compared to their respective controls (n ¼ 6, P < 0.05), while no change was seen in Gsto1 gene expression in WT PD mice. (C–J) Representative images of GSTO1-1 immunolabelling showed GSTO1-1 (green) to be highly expressed on the inner segments of cone (PNA, red) photoreceptors (outlined arrows), on the superior retina (SR), and weakly in the inferior retina (IR) in WT DR (D) and WT PD (H) mice. GSTO1-1 was also found to be weakly expressed in the same locations in Gsto1/ DR (F) and Gsto1/ PD (J) mice. GSTO1-1 was also expressed in the INL (filled arrows) on the SR as well as IR in WT DR (C, D) and PD (G, H) mice. (K) GSTO1-1 did not colocalize with rod photoreceptors (no colabelling with rhodopsin, red). (L) Negative control. (M) Gsto1-1 expression was significantly increased after 5 hours PD as compared to dim conditions (P < 0.05) in 661W cells. An increase in Nrf2, Nqo1, and Hmox1, markers of oxidative stress, was also seen to significantly increase in 661W cells after 5 hours PD as compared to dim controls (n ¼ 6, P < 0.05) (K). (N) GSTO1-1 (green) was found in all 661W cells in both dim and PD conditions. n ¼ 6; results were analyzed with 1-way ANOVA followed by Tukey’s multiple comparison post test. Asterisks denote a significant change where P < 0.05. Scale bar:25lm(C– L) and 10 lm(N).

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/ FIGURE 2. Changes in oxidative stress gene (A–G) and protein (H) expression in Gsto1 retinas compared to WT in both DR and following 5 days PD damage. (A–G) A significant increase in gene expression was seen in the expression of Hmox-1 (A) and Gpx3 (B) between Gsto1/ DR and Gsto1/ PD mice (P < 0.05), and a significant decrease was found in the expression of Gpx3 between WT DR and Gsto1/ DR mice (B)(P < 0.05). No statistical differences were shown between any other groups for these genes, or between any group for the expression of genes Nox1 (C)or Sod2 (D)(P > 0.05). A significant increase was found in the expression of Nrf2 (E) between WT DR and WT PD mice (P < 0.05), and between Gsto1/ DR and Gsto1/ PD mice (P < 0.05); however, there was no difference seen in expression of Nrf2 between WT PD and Gsto1/ PD mice (P > 0.05). (F) Nqo1 gene expression was significantly downregulated in both DR and PD Gsto1/ mice compared to respective controls (P < 0.05). (G) Gene expression changes of glutathione-specific gamma-glutamylcyclotransferase 1 (Chac1), which degrades glutathione, showed a significant decrease in expression between WT DR and Gsto1/ DR mice (P < 0.05), and between WT DR and WT PD mice (P < 0.05). No significant change, however, was seen in Chac1 expression between Gsto1/ DR and Gsto1/ PD mice (P > 0.05). (H) The total levels of ROS/RNS were measured in the retinas with results showing a significant increase in the total ROS/RNS levels between WT DR and WT PD retinas (P < 0.05), as well as a significant decrease between WT PD and Gsto1/ PD retinas (P < 0.05). n ¼ 5; results were analyzed with 1-way ANOVA followed by Tukey’s multiple comparison post test. Asterisks denote a significant change where P < 0.05.

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/ FIGURE 3. The effect of Gsto1 on inflammation and the accumulation of microglia/macrophages in the retina following 5 days PD. (A) Following PD, a significant increase in the total number of IBA-1þ microglia/macrophages was seen in the outer retina (ONL and subretinal space) in both WT PD and Gsto1/ PD mice compared to their respective controls (P > 0.05). No difference in the total number of IBA-1þ cells was seen between WT DR and Gsto1/ DR mice or between WT PD and Gsto1/ PD mice. (B) Significant increase in the ratio of amoeboid (activated) to ramified (resting) macrophages was seen in WT PD compared to Gsto1/ PD retinas (P < 0.05). (C–F) Representative images showing IBA-1þ labelling (green) in the outer retina. Thin ramified processes of IBA-1þ microglia/macrophages (filled arrows) can be seen projecting into the ONL in WT DR (C) and Gsto1/ DR (D) retinas, with larger and more numerous ramified processes seen in Gsto1/ PD retinas (F). In comparison, in WT PD retinas (E), numerous rounded (amoeboid) IBA-1þ cells can be seen in the ONL and subretinal space (outlined arrows). (G–I) Inflammatory markers were quantified by qRT-PCR in whole retinas. Ccl2 (G), Il-1b (H), and C3 (I) expression significantly increased in WT PD mice compared to WT DR mice (P < 0.05) and was significantly reduced in Gsto1/ PD mice compared to WT PD mice (P < 0.05). In addition, C3 expression increased significantly in Gsto1/ PD mice compared to Gsto1/ DR mice (P < 0.05); however, there was no difference in the expression of Ccl2 or Il-1b between these groups (P > 0.05). n ¼ 6; results were analyzed with either an unpaired Student’s t-test, or 1-way ANOVA followed by Tukey’s multiple comparison post test. Asterisks denote a significant change where P < 0.05. Scale bar:20lm.

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/ þ FIGURE 4. The effect of Gsto1 on retinal function and cell death in the retina following 5 days PD. (A) The total number of TUNEL cells in the ONL was significantly increased in both the WT PD and Gsto1/ PD retinas compared to their respective WT DR and Gsto1/ controls (P < 0.05). In addition, the total number of TUNELþ cells was significantly reduced in the retinas of Gsto1/ PD mice compared to WT PD retinas (P < 0.05). (B) A significantly reduced number of photoreceptor cell rows was seen in WT DR and Gsto1/ DR retinas and their respective WT PD and Gsto1/ PD controls. There was a significant increase in photoreceptor cell nuclei survivability in Gsto1/ PD compared to WT PD retinas (P < 0.05). (C–F) Representative images show the immunolabelling of TUNELþ cells (red) in the ONL as well as the ONL thickness (white line). TUNELþ cells (filled arrows) can be seen in both the WT DR (C) and Gsto1/ DR (D) retinas, while there are significantly more TUNELþ cells in the WT PD retina (E), and only a slight increase in TUNELþ cells in the Gsto1/ PD retina (F) compared to DR controls. (G–J) Retinal function was measured before (DR) and after 5 days PD by using ERG. There was no statistical difference observed in either the a-wave (G) or b-wave (H) between WT DR and Gsto1/ DR mice (P < 0.05). Following PD, there was a significant improvement in both the a-wave (I) and b-wave (J) function between Gsto1/ PD and WT PD mice (P < 0.05). n ¼ 5–10; results were analyzed with 1-way ANOVA followed by Tukey’s multiple comparison post test or a 2-way ANOVA for ERG analysis. Asterisks denote a significant change where P < 0.05. Scale bar:20lm.

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strated that WT PD mice had significantly reduced retinal reduced levels of antioxidant enzymes Gpx3 and Nqo1. function for both a-wave (P < 0.05; Fig. 4I) and b-wave (P < Reduced levels of Gpx3 and Nqo1 suggest that GSTO1-1 0.05; Fig. 4J), compared to Gsto1/ PD mice. No statistical deficiency decreases oxidative stress under normal conditions difference (P > 0.05) in the number of TUNELþ photoreceptor in the retina, consistent with previous findings from models of cells (Fig. 4A), photoreceptor cell rows (Fig. 4B), or ERG obesity.11 Glutathionylation and deglutathionylation of proteins function (Figs. 4G, 4H) was observed between WT DR and by GSTO1-1 has been well documented in many other Gsto1/ DR mice. tissues.2,34–36 Despite this we saw no change in GSH, GSSG, or total glutathione levels between Gsto1/ and WT control GSTO1-1 Increases Total Glutathione Levels in the retinas, indicating a novel role of GSTO1-1 in the normal and degenerating retina. Given the cone photoreceptor–specific Retina Following Photo-Oxidative Damage labelling demonstrated in this article, we suggest that GSTO1-1 As glutathione exists in both an antioxidant reduced (GSH) may act at the interface of oxidative stress and inflammatory state and an oxidized (GSSG) state, the ratio of GSH to GSSG in pathways and may therefore contribute to photoreceptor cell the retina can be used as a measure of cell health, as high levels death following light-induced PD. This potential role will be of GSSG accumulate during increased oxidative stress. A the focus of the remainder of the discussion. glutathione assay was used to determine what effect GSTO1- 1 had on the GSH to GSSG ratio in the retina before and after GSTO1-1 Is Primarily Localized to the Inner PD. There was a statistically significant increase in GSH (P < Segment of Cone Photoreceptors 0.05; Fig. 5A), GSSG (P < 0.05; Fig. 5B), and total glutathione (GSHþGSSG) levels (P < 0.05; Fig. 5C) in WT PD retinas In rodents, the central superior retina (1–3 mm from the optic compared to WT DR retinas. Levels of GSH were also found to nerve) is known to be highly susceptible to PD-induced focal 30,37,38 be significantly reduced in Gsto1/ PD mice compared to WT photoreceptor cell death, in a region functionally PD mice (P < 0.05; Fig. 3A); however, while there was a comparable to the human macula, known as the ‘‘area 39 reduction in the levels of GSSG and total glutathione in centralis.’’ GSTO1-1 appeared to be most highly expressed Gsto1/ PD mice, it was not statistically significant (P > 0.05; in this area, specifically localized to the inner segments of the Figs. 5A–D) from either control levels or WT PD at 5 days. In cones, with photoreceptor inner segments known to be rich in addition, there was no statistical difference in the ratio of GSH mitochondria to accommodate their high rates of metabolism 40 to GSSG between any of the groups ( > 0.05; Fig. 5D). There and energy usage, and in endoplasmic reticulum (ER), where P 41 was also no statistical difference in the levels of GSH, GSSG, calcium homeostasis is maintained and posttranslational total glutathione, or GSH to GSSG ratio between WT DR and modification of proteins occurs, including glutathionylation. Gsto1/ DR mice (P > 0.05; Figs. 5A–D). Although weak GSTO1-1 labelling was also seen in the retina of Gsto1/ mice, likely antibody binding to another compensa- tory GST, no expression was detected in Western blot analysis / DISCUSSION or qRT-PCR of Gsto1 retinas. In drosophila, GSTO1-1 is involved in regulating mitochon- This is the first study to determine a possible role of GSTO1-1 drial ATP synthase activity and modulating oxidative stress and in mediating retinal inflammation, which can contribute to the has been suggested as a contributor to the neurodegeneration pathogenesis of retinal degenerations. Firstly, our data show in Parkinson’s disease.1 Photoreceptors have the highest levels that GSTO1-1 is present in the mouse retina, localized to the of ROS production in the retina,17,18 a large source of which inner segment of cone photoreceptors, and is maximally comes from mitochondria in the photoreceptor inner seg- expressed in the region of the retina most susceptible to ments.42,43 In a healthy state, ROS, including free radicals, oxidative stress, the central superior retina. Secondly, Gsto1/ superoxide anion, singlet oxygen, and hydrogen peroxide, are mice had lower levels of markers of oxidative stress, a quenched by cellular defense mechanisms, for example, by reduction in photoreceptor cell death, and improved retinal antioxidants such as glutathione (GSH).17 However, an function as compared to WT controls. Thirdly, following PD- accumulation of ROS can overburden the antioxidant capabil- induced oxidative stress, Gsto1/ mice had a reduced ities of the cell, leading to cell death.44 It has also been inflammatory response, accompanied by a decrease in reported that ER can respond to oxidative stress in the retina, activated microglia/macrophage accumulation in the outer with previous studies showing that in both PD retinas and retina as compared to WT controls. Lastly, our results indicate 661W cells, intracellular Ca2þ levels increase in the ER, that while there were changes in the levels of reduced and producing ROS and ultimately leading to ER stress and oxidized forms of glutathione (GSH and GSSG, respectively) in photoreceptor cell death.45,46 GSTO1-1 has been shown to PD WT compared to DR WT and Gsto1/ retinas, the overall play a role in modulating the activity of the ER calcium release ratio of GSH to GSSG (an indicator of redox status) remained channel, ryanodine receptor (RyR),34,47 and could potentially unchanged between all experimental groups. Overall, these play a similar role in retinal cone cells during oxidative stress. data indicate a role for GSTO1-1 in the progression of retinal Although not investigated further in this study, the underlying degenerations and indicate that GSTO1-1 may be a potential mechanism of interactions with these cellular organs is likely to target for the treatment of retinal degenerations where explain the reduced oxidative stress response seen in the oxidative stress and inflammation are key features of disease GSTO1-1–deficient mice. As it is known that cones are very progression. susceptible to oxidative damage over time,48 the suggested cellular location of GSTO1-1, combined with the known role of The Role of GSTO1-1 in the Normal Retina GSTO1-1 as a modulator of oxidative stress, indicates a potential role of GSTO1-1 in modulating oxidative stress in Mice deficient in GSTO1-1 have been previously shown to be the retina. phenotypically normal, however display reduced monocyte In addition to cone labelling, GSTO1-1 expression was also and eosinophils counts, compared to controls.11 We observed seen in a large, rounded cell type in the INL in both the no difference in retinal structure, number of photoreceptors, superior and inferior sides of the retina of WT mice, possibly macrophage recruitment, retinal function, or oxidative stress amacrine cells. Further experiments are necessary to verify this gene expression in Gsto1/ mice, with the exception of labelling pattern and to determine the potential role of GSTO1-

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FIGURE 5. The effect of GSTO1-1 on glutathione levels in the retina following PD. (A–D) The levels of GSH (A), GSSG (B), and total glutathione (GSHþGSSG) (C) were shown to be increased in WT PD mice compared to WT DR mice (P < 0.05). In addition there was a significant decrease in the levels of GSH between WT PD and Gsto1/ PD mice (A)(P < 0.05). While there was a large reduction in the levels of GSSG (B) and total glutathione (C) between WT PD and Gsto1/ PD mice, it was not statistically significant (P > 0.05). In addition there was no statistical difference in the ratio of GSH to GSSG between any of the groups (D)(P > 0.05). n ¼ 5; results were analyzed by unpaired Student’s t-test. Asterisks denote a significant change where P < 0.05.

1 in these cells; however, it is not the focus of this study. genes Nox1, Sod2, and Gpx3 between WT PD and Gsto1/ PD Nevertheless, given the specificity of GSTO1-1 labelling in the retinas, a large reduction in total ROS/RNS levels was detected cone photoreceptors, GSTO1-1 could be playing a role in in the Gsto1/ PD mice compared to PD controls, indicating modulating the effects of oxidative stress in these cells of the that GSTO1-1 may instead act at the protein level inducing the INL as well. production of ROS and RNS, potentially through the activation of TLR signaling and NF+B pathways.7 Glutathione is an intracellular antioxidant and is considered Oxidative Stress and Photoreceptor Cell Death Is 53 Promoted by GSTO1-1 in Retinal Degenerations to be one of the most important ROS scavengers. In normal healthy cells, approximately 90% of glutathione is in its We report that the gene expression of Gsto1-1 and oxidative reduced form, GSH54; however, following oxidative stress, stress markers Hmox1, Nrf2, and Nqo1 concurrently increased GSSG (oxidized form) accumulates and the ratio of GSH to in 661W photoreceptor-like cells, following PD. HMOX1 is an GSSG becomes an important indicator of oxidative stress in integral membrane protein in the ER and is a key regulator of tissues. However in this study, the individual levels of GSH and inflammatory processes,49 which is known to be induced by GSSG were both equally higher in WT PD than Gsto1/ PD high levels of oxidative stress.49 Although there was no mice, which resulted in no significant difference in the GSH to significant difference seen in the expression of Hmox1 in GSSG ratio between these groups, indicating no relative WT retinas compared to Gsto1/ retinas following PD, change in redox status. photoreceptor-specific changes may be masked by whole The increase seen in GSH and GSSG levels in WT PD mice is retinal changes.50,51 Recent results from Menon et al.11 show supported by the gene expression analysis of Chac1,an that expression levels of Nqo1, an antioxidant enzyme target of enzyme involved in the cleavage and degradation of glutathi- the NRF2 pathway, are significantly lower in liver lysates from one which, when overactivated, causes glutathione depletion Gsto1/ mice,11 indicating a reduced oxidative state in these and apoptosis.55,56 Results demonstrated that there was a Gsto1/ mice compared to controls. The present study significant reduction in Chac1 expression in Gsto1/ DR mice confirms these findings, with significantly lower levels of compared to WT DR mice, with expression remaining equally Nqo1 found in DR and PD Gsto1/ mice compared to their low in Gsto1/ DR controls following PD, suggesting that the respective WT controls. As GSTO1-1 is a downstream target of levels of glutathione degradation in the group was a NRF2, the expression of Nrf2 was not expected to change in consequence of GSTO1-1 deficiency and not PD. In contrast the Gsto1/ mice compared to WT and may be expressed at however, Chac1 was found to be significantly reduced in WT higher levels in an attempt to compensate for the lack of PD mice compared to WT DR controls, indicating that there is downstream GSTO1-1 in the deficient mice.52 While no a reduction in the degradation of glutathione in these mice, differences were noted in the expression of oxidative stress potentially to combat increased levels of ROS/RNS seen

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following PD. Despite no changes in the redox state of each of study, it has been reported that TLR4 is present on both retinal the experimental groups, we postulate that the increased levels photoreceptors and 661W cells.64,65 It is therefore possible of GSH and GSSG seen in the WT PD mice are due to an that GSTO1-1 is mediating inflammatory cascades via this increased oxidative state as supported by data in this study. It pathway, promoting the recruitment of macrophages to the remains unclear if the detrimental effects of GSTO1-1 on the damaged photoreceptors and consequently inducing photore- retina following PD are occurring through the glutathionyla- ceptor cell death. This concept is supported by the reduced tion cycle, or perhaps if GSTO1-1 is modulating the gluta- levels of ROS, inflammation, and photoreceptor cell death seen thionylation of a specific protein rather than the overall redox in Gsto1/ mice following PD and indicates that targeting 7,11 potential as previously described. GSTO1-1 may be beneficial in slowing the progression of retinal degenerations and warrants further investigations via GSTO1-1 Mediates an Inflammatory Response in the use of a GSTO1-1 inhibitor delivered to the retina. Retinal Degenerations

It has been well characterized in photo-oxidative retinal CONCLUSIONS degeneration that an increase in oxidative stress and photoreceptor cell death leads to the release of proinflammatory This study describes a damaging role for GSTO1-1 in retinal cytokines, macrophage accumulation, and complement depo- degenerations, through the promotion of oxidative stress and sition.25,26,29,33,57 Release of proinflammatory cytokines (in- inflammation, leading to photoreceptor cell death, with mice cluding IL-1b and CCL2) and complement production deficient in GSTO1-1 showing a reduction in the damaging (including C3) are characteristic features of many retinal levels of oxidative stress and inflammation that is central to the degenerations.24–26,29,58–62 Modulation of GSTO1-1 leads to the progression of retinal degenerations. While further research posttranslational modification and subsequent activation of IL- into the possible mechanisms by which GSTO1-1 acts in the 1b,11,63 a fundamental component in the early inflammatory retina is necessary, this study highlights that GSTO1-1 may be a response of many neurodegenerative diseases. Our results possible therapeutic avenue for slowing the progression of demonstrate that following PD, Gsto1/ retinas showed retinal degenerations. reduced levels of Il-1b, Ccl2, and C3, compared to WT controls. CCL2 is a potent chemokine known to recruit macrophages to areas of retinal damage.26,32 We observed that Acknowledgments Gsto1/ microglia/macrophages were less amoeboid with Supported by grants from the Gretel and Gordon Bootes Medical processes, but not cell bodies, extending into the ONL, Research and Education Foundation, and the National Health and indicating a reduced activation state as compared to WT Medical Research Council of Australia (NHMRC, Project Grant microglia/macrophages. Upon recruitment to the outer retina, APP1124673 to PGB). activated microglia and macrophages are known to produce IL- Disclosure: N. Fernando, None; Y. Wooff, None; R. Aggio- 1b and C3, which ultimately exacerbates photoreceptor cell Bruce, None; J.A. Chu-Tan, None; H. Jiao, None; C. Dietrich, 29 death. Therefore, the reduction in expression of these None; M. Rutar, None; M. Rooke, None; D. Menon, None; J.T. / inflammatory markers in the Gsto1 microglia/macrophages Eells, None; K. Valter, None; P.G. Board, None; J. Provis, None; also indicates a reduced inflammatory state. In addition, as high R. Natoli, None levels of Chac1 are known to initiate apoptosis via GSH depletion, a significant reduction in this gene suggests a lower apoptotic state in GSTO1-1–deficient mice. This hypothesis is References supported by results from this study demonstrating that 1. Kim K, Kim SH, Kim J, Kim H, Yim J. Glutathione s-transferase Gsto1/ mice had reduced photoreceptor cell death following þ omega 1 activity is sufficient to suppress neurodegeneration PD. While a significantly higher level of TUNEL cells were in a Drosophila model of Parkinson disease. JBiolChem. shown in WT PD mice compared to both controls and 2012;287:6628–6641. Gsto1/ PD mice, a correlative increase in Chac1 expression 2. Menon D, Board PG. A role for glutathione transferase Omega was not found. It is possible that as gene expression was only 1 (GSTO1-1) in the glutathionylation cycle. 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