Loss of NRF-2 and PGC-1α genes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration

Contents lists available at ScienceDirect Redox Biology journal homepage: www.elsevier.com/locate/redox Research Paper Loss of NRF-2 and PGC-1αgenes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration Szabolcs Felszeghy a,b,1 , Johanna Viiri c,1 , Jussi J. Paterno c,d,1 , Juha M.T. Hyttinen c , Ali Koskela c , Mei Chen e , Henri Leinonen f , Heikki Tanila f , Niko Kivinen c,d , Arto Koistinen g , Elisa Toropainen h , Marialaura Amadio i , Adrian Smedowski j , Mika Reinisalo c,h , Mateusz Winiarczyk k,l , Jerzy Mackiewicz l , Maija Mutikainen f , Anna-Kaisa Ruotsalainen f , Mikko Kettunen f , Kimmo Jokivarsi f , Debasish Sinha m , Kati Kinnunen d , Goran Petrovski n , Janusz Blasiak°, Geir Bjørkøy p , Ari Koskelainen q , Heli Skottman r , Arto Urtti h,s , Antero Salminen t , Ram Kannan u , Deborah A. Ferrington v , Heping Xu e , Anna-Liisa Levonen f , Pasi Tavi f , Anu Kauppinen h , Kai Kaarniranta c,d,⁎ a Institute of Dentistry, University of Eastern Finland, Kuopio, Finland b Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland c Department of Ophthalmology, University of Eastern Finland, Kuopio, Finland d Department of Ophthalmology, Kuopio University Hospital, Kuopio, Finland e The Wellcome-Wolfson Institute of Experimental Medicine Queen's University Belfast, Belfast, UK f A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland g SIB Labs, University of Eastern Finland, Kuopio, Finland h School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland i Department of Drug Sciences, Section of Pharmacology, University of Pavia, Pavia, Italy j Chair and Department of Physiology, School of Medicine in Katowice, Medical University of Silesia, Katowice, Poland k Department of Epizootiology, University of Life Sciences of Lublin, Poland l Department of Vitreoretinal Surgery, Medical University of Lublin, Poland m The Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA n Centre of Eye Research, Department of Ophthalmology, Oslo University Hospital, University of Oslo, Oslo, Norway Department of Molecular Genetics, University of Lodz, Lodz, Poland p Centre of Molecular Inflammation Research and Department of Cancer Research and Molecular Medicine; Norwegian University of Science and Technology and Department of Technology; University College of Sør-Trøndelag, Trondheim, Norway q Department of Neuroscience and Biomedical Engineering, Aalto University School of Science, Aalto, Finland r Faculty of Medicine and Life Sciences, BioMediTech Institute, University of Tampere, Tampere, Finland s Centre for Drug Research, Division of Pharmaceutical Biosciences, University of Helsinki, Helsinki, Finland t Department of Neurology, University of Eastern Finland, Kuopio, Finland u Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Los Angeles, CA, USA v Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, USA ARTICLE INFO Keywords: Aging Autophagy ABSTRACT Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to https://doi.org/10.1016/j.redox.2018.09.011 Received 13 July 2018; Received in revised form 6 September 2018; Accepted 13 September 2018 Abbreviations: ER, endoplasmic reticulum; AMD, age-related macular degeneration; RPE, retinal pigment epithelium; PUFAs, polyunsaturated fatty acids; ROS, reactive oxygen species; NRF-2, nuclear factor-erythroid 2-related factor-2; ARE, antioxidant response element; Keap1, kelch-like ECH-associated protein 1; PGC-1, peroxisome proliferator-activated receptor gamma coactivator-1; PRC, PGC-1-related coactivator; SOD2, superoxide dismutase 2; TRX1, thioredoxin; AMPK, AMPactivated protein kinase; SIRT1, sirtuin 1; UPS, ubiquitin-proteasome system; p62/SQSTM1, sequestosome 1; LC3, microtubule-associated protein 1A/1B light chain 3; dKO, double knockout; KO, knockout; WT, wild type; 4-HNE, 4-hydroxynonenal); ROI, region of interest; TEM, transmission electron microscopy; ATF4, activating transcription factor 4; GRP78, glucose-regulated protein 78; Iba-1, ionized calcium binding adaptor molecule 1; ERG, electroretinography; Rd8, retinal degeneration 8; MRI, magnetic resonance imaging; mtDNA, mitochondrial DNA ⁎ Correspondence to: Department of Ophthalmology, University of Eastern Finland and Kuopio University Hospital, Kuopio, Finland. 1 Equal contribution. E-mail address: kai.kaarniranta@uef.fi(K. Kaarniranta). Redox Biology 20 (2019) 1–12 Available online 14 September 2018 2213-2317/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). T Degeneration Oxidative stress Protein aggregation Proteasome oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1α(peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1αin the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC1αdKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1αdKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD. 1. Introduction Chronic oxidative and endoplasmic reticulum (ER) stress, impaired autophagy, mitochondrial dysfunction and inflammation are strongly linked to age-related macular degeneration (AMD) [1–6]. One clinical hallmark of AMD is the degeneration of retinal pigment epithelial (RPE) cells, a process that associates with the accumulation of oxidative stress–derived lysosomal lipofuscin, impairing lysosomal degradation, and the presence of extracellular protein/lipid deposits (drusen) between the basal lamina of the RPE and the inner collagenous layer of the Bruch's membrane [7–11]. AMD can be subdivided into dry and wet forms with 80% and 20% prevalences, respectively. There are no effective treatments for dry AMD. The quiescent RPE cells in the macula are constantly subjected to high oxidative stress due to the diurnal digestion of high concentrations of polyunsaturated fatty acids (PUFAs) originating from the phagocytosis of the retinal outer segments, exposure to the oxygen-rich choriocapillary circulation, and the light-induced production of reactive oxygen species (ROS) by cellular pigment granules [7]. The mitochondria also produce ROS as a by-product of respiration. During the aging of RPE cells, the cellular capacity to neutralize ROS diminishes due to a decreased production of ROS-scavengers and other antioxidants [12,13]. In RPE cells, the NRF-2/ARE (nuclear factor-erythroid 2-related factor-2/antioxidant response element) pathway plays the major role in the oxidative stress regulation [14,15]. In response to oxidative stress, the NRF-2 is released from NRF-2-Keap1 (kelch-like ECH-associated protein 1) complex allowing the translocation of NRF-2 from cytosol into the nucleus and the expression of several antioxidant defense system and autophagy -related genes and proteasomal subunits [16–20]. Mice models with NRF-2 deficiency have been shown to be more susceptible to oxidative damage and protein aggregation than WT (wild type) mice and to negatively regulate autophagy-related genes [21,22]. The peroxisome proliferator-activated receptor gamma coactivator1 (PGC-1) family, consisting of PGC-1α, PGC-1βand PRC (PGC-1-related coactivator), regulates a network governing the transcriptional control of mitochondrial biogenesis and respiratory function as well as targeting mitochondrial antioxidant defense system [23–25]. The role of PGC-1αis to serve as a switch between mitochondrial biogenesis and oxidative damage by controlling the mitochondrial levels of ROS. Lossof-function studies of PGC-1αhave shown burst of ROS and an increase in mitochondrial damage and degradation whereas gain-of-function enhanced mitochondrial biogenesis and the expression of mitochondrial antioxidant defense system-related genes, such as SOD2 (superoxide dismutase 2) and TRX1 (thioredoxin) [23,25]. However, both pathways, mitochondrial biogenesis and ROS control, strive for the preservation of mitochondrial homeostasis. Post-translational modification of the energy sensors, AMPK (AMP-activated protein kinase) and SIRT1 (sirtuin 1) that induce autophagy, are known to regulate PGC-1αactivities [26–28]. In RPE cells, PGC-1αhas been shown to drive mitochondrial biogenesis as well as activating the antioxidant defense system [29,30]. A high ROS production combined with impaired antioxidant systems results in detrimental protein aggregation [8,31]. The ubiquitinproteasome system (UPS) and the lysosomal/autophagosomal degradation system share the major responsibility of maintaining cellular proteostasis [8,32]. Both systems recognize and actively select the material destined for degradation and recycling in cells. The p62/ SQSTM1 (sequestosome 1) has a multifactorial role since it acts as a bridge shuttling proteins between UPS and autophagy clearance as well as regulating the antioxidant response in RPE cells during oxidative stress [9,33,34]. Proteins assigned to the degradation are often ubiquitinated and directed to the proteasomal degradation. However, the rate of protein degradation by proteasomes is limited, and when UPS is overwhelmed, for example under heavy oxidative stress, proteins form aggregates which cannot be removed by proteasomes. Autophagy is specialized for removing protein aggregates and large cell organelles, such as mitochondria. p62/SQSTM1 recognizes ubiquitinated perinuclear protein aggregates and the p62/SQSTM1 tagged material is then isolated from the cytosol in p62/SQSTM1-LC3 (microtubule-associated protein 1A/1B light chain 3) interaction-guided autophagosome formation [35]. In the final step of the autophagy process, a lysosome is fused to the autophagosome resulting in the formation of the autolysosome followed by the degradation of its contents including ubiquitin, p62/SQSTM1 and LC3. Thereby, these proteins can be exploited as biomarkers of protein aggregation and autophagy activity [35,36]. Moreover, p62/SQSTM1 interacts with the NRF-2/ARE pathway by disrupting the NRF-2-Keap1 complex leading to nuclear localization of NRF-2 [37]. The large and increasing number of individuals affected by AMD means that this represents an urgent global health emergency requiring effective primary prevention strategies as well as improvements in the current treatments for this disease. Therefore, new studies contributing to a better understanding of the mechanisms regulating antioxidant defense system, proteostasis, and mitochondrial function may lead to breakthroughs in AMD research. Since the NRF-2 and PGC-1αtranscription factors have a key role in cellular detoxification and mitochondrial function in response to oxidative stress, we generated and characterized the NRF-2/PGC-1αdouble knockout (dKO) mouse model to study retinal changes that occur in degradative pathways and document features previously associated with AMD. S. Felszeghy et al. Redox Biology 20 (2019) 1–12 2 2. Material & methods 2.1. Animals All animal protocols were approved by the Animal Experiment Board of Finland and conducted in compliance with the European Community Council Directives 2010/63/EU and ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Mice were group-housed in the Laboratory Animal Centre of University of Eastern Finland, Kuopio, Finland on a 12-h light/dark cycle and given food and water ad libitum. The mice strains deficient in NRF-2 or PGC-1α(C57BL/6J background) used in this study have been described earlier [38,39].To obtain mice lacking both NRF-2 and PGC-1α, homozygous-null NRF-2 and PGC-1αstrains were crossed first, and the resultant double heterozygotes were crossed together. Female and male KO (knockout) and dKO mice at six weeks, three months and one year of age were used for this study, together with age-matched WT (wild type) controls. 2.2. The genotyping of NRF-2 and PGC-1αknockout mice The genomic mouse DNA was extracted from small ear samples by heating the tissues for 1 h at 95 °C in 100 μl of 25 mM NaOH + 0.2 mM EDTA. The solutions were neutralized by adding 100 μl of 40 mM TrisHCl pH 7.5, and centrifuged for 10 min at 2000 rpm. The upper halves of the supernatants (100 μl) were collected to be used directly as templates in the PCR reactions. The genotypes of PGC-1αwere detected with a 4-primer PCR. The primer sequences were: WTA,5′-CCA GTT TCT TCA TTG GTG TG; WTB, 5′-ACC TGT CTT TGC CTA TGA TTC; KOA,5′-TCC AGT AGG CAG AGA TTT ATG AC; KOB,5′-CCA ACT GTC TAT AAT TCC AGT TC. The NRF-2 genotypes were studied with the following three primers: LacZ,5′-GCG GAT TGA CCG TAA TGG CAT AGG; Nrf2–5′,5′-TGG ACG GGA CTA TTG AAG GCT G; Nrf2–3’, GCC GCC TTT TCA GTA GAT GGA CG. Both reactions included 4 μl of DNA extracted from mouse ears, reaction buffer, 100 μM of each dNTPs, 1.5 mM of MgCl 2, 1μM of each primers, and 1.2 U of DreamTaq DNA polymerase (Thermo Fisher Scientific Waltham, MA USA), in a volume of 30 μl. For the PGC-1α, samples were denatured at 95 °C for 5 min, followed by 39 cycles at 95 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 7 min. For the NRF-2 genotyping, the reaction conditions were: denaturation at 95 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, at 59 °C for 30 s, and at 72 °C for 45 s, and a final extension at 72 °C for 7 min. All PCR products were separated using 1% agarose (Seakem LE, Cambrex) gel electrophoresis in standard Tris-acetate-EDTA buffer, visualized in UV light with ethidium bromide, and photographed. The amplicon sizes the for wild type allele of PGC-1αare 600 bp and for the KO allele 400 bp. Their sizes for the NRF-2 alleles are 700 and 400 bp, respectively (Supplementary Fig. 1). 2.3. Immunomapping for detection of protein aggregation, autophagy and oxidative stress markers: Ubiquitin, p62/SQSTM1, Beclin-1, LC3 and 4HNE The mice were sacrificed with cervical dislocation and the eyes were immediately carefully enucleated and placed in PBS (pH 7.4). Before dehydrating in graded series of ethanol and embedding in paraffin, the eyes were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 24–48 h. Five μm thick parasagittal serial sections were cut from embedded blocks with a microtome (SM2000 R, Leica, Heidelberg, Germany). After a random selection of slides from each of the individual specimens, the sections were dewaxed and rehydrated and the labeling of the retina was performed according to previously published methods, with minor modifications [36]. Briefly, the sections were treated with blocking solution (#IHC-101B Bethyl laboratories, Montgomery, TX, USA) for 30 min at room temperature to prevent the possibility of non-specific binding of the primary antibodies; the samples were then immunostained with different primary antibodies as follows: anti-ubiquitin (1/200; Dako, Glostrup, Denmark), antiSQSTM1/p62 (1/100; Abcam, Cambridge, UK), anti-Beclin 1 (1/160; Novus Biologicals, CO, USA), anti 4-HNE (4-hydroxynonenal) (1/200; LifeSpan BioSciences, Inc, Seattle, WA, USA), anti LC3B (1/200; Cell Signaling Technology, Danvers, MA, USA) overnight, at 4 °C. After rinsing the primary antibodies in TBS, the samples were then incubated with a secondary antibody anti mouse Alexa Fluor 594 (1/500; Thermo Fisher Scientific Waltham, MA USA) for 3 h at room temperature and rinsed with TBS. A fluorescent nuclear marker DAPI (4′,6-diamidino-2phenylindole dihydrochloride, #D9542, Sigma, St. Louis, MO, USA) diluted 1:10 000 in TBS was added and incubated for 30 min at room temperature. The sections were rinsed with TBS and covered with Mowiol mounting media. Immunofluorescence was evaluated independently of randomly selected sections by three researchers and no signal was recorded from technical negative controls (TBS instead of primary antibody). After the staining, the samples were analyzed as described above, photomicrographs were taken in a Zeiss AX10 Imager A2 (Zeiss, Göttingen, Germany) microscope. Images were captured sequentially from the green, red, and far-red channels on slices using a 63× oil immersion objective (NA:1.42, Plan Apochromat). The microscope settings were identical for all scans and kept constant during imaging. Representative scans were taken with a Jenoptik ProgRes C5 (Zeiss, Göttingen, Germany) digital camera mounted onto the microscope. In all imaging procedures, gamma adjustment was made on the whole image in order to maintain appropriate contrast and images were processed using Adobe Photoshop for documentation. The control sections originated from the same eyes and the validity of the sequential staining was then verified with the same protocol as described above except that one of the primary antibodies was omitted. The negative control samples displayed only a sub-minimal autofluorescence signal. 2.4. Spectral imaging analysis The immunohistochemical results were examined by three independent observers searching for immunoreactive RPE cells. The different markers distribution pattern were semi-quantitatively analyzed by computer imaging as follows. High power view RGB 8-bit images of single RPE cells were generated with a Jenoptik ProgRes C5 as it described above in fluorescent mode, using a 63× objective lens. Blue pseudocolor of the nuclei was assigned by the investigators to help segmentation processes of RPE cell nuclei, respectively. For quantitation of the different protein aggregation and autophagy related (Ubiquitin, p62/SQSTM1, Beclin-1, LC3B) marker expression and oxidative stress marker (4-HNE), 10 RPE cells per animal of interest (n = 3 WT and n = 3 NRF-2/PGC-1αdKO) were manually designated as regions of interest (ROI). Special care was taken to select two representative areas per section for collecting RPEs from each individual samples close to the vicinity of optic nerve. Sampling for RPE image analysis was performed without external knowledge on samples genotype to be able to compare without influences from testers' preferences or expectations. A spectral imaging containing the characteristic wavelength emission of Alexa Fluor 594 was created by sampling pure RED spectra from RGB images; these were converted to black and white images, i.e. only the positive signal of interest was analyzed further. Via this grayscale conversion, the original, true RED color spectral information was automatically converted into one grayscale image. This process is based on the calculation of the average intensity per channel, therefore one can keep the original staining intensity information. Furthermore, this conversion makes it possible to focus only on the RED channel of interest. Using the open source Image J software (http// :imagej.nih.gov; NIH, USA), we quantified the immunostaining in the ROI, the software measured the amount of target signal (intensity of B& W pseudo-color) within each ROI, by measuring the density per pixel and returning a nominal value between 0 (the limit of noise level i.e. S. Felszeghy et al. Redox Biology 20 (2019) 1–12 3 data point from a blank sample /=negative immunohistochemical reaction/ was determined below 20) and 255 (white maximal staining) for that pixel. The integrated average gray level used in our further analysis was the sum of all the separate pixels in the examined area. Final data were displayed as average gray value/cell. With this setup described briefly above, we believe that the risk of interpretation bias is avoided. However, obtaining accurate results with this semi-quantitative-immunohistochemical analysis depends upon the specificity of the antibodies used. 2.5. TEM analysis Prior to transmission electron microscopy (TEM), the mice eyes were pre-fixed with 3% paraformaldehyde in 0.075 M cacodylate buffer (pH 7.2) with 1% glutaraldehyde for 5–7 days at + 4 °C. After 2 × 10 min washing in 0.075 M cacodylate buffer, the samples were post-fixed in 1% osmium tetraoxide and 1.5% potassium-ferrocyanide in 0.1 M cacodylate buffer (pH 7.2) for 2 h, and again washed with cacodylate buffer for 2 × 10 min and 5 min in distilled water prior to standard ethanol dehydration. Subsequently, the samples were infiltrated and embedded in LX-112 resin (Ladd Research, Williston, VT, USA). Polymerization was carried out at 60 °C for 48–72 h. One-micron semi-thin sections were cut with a Reichert Ultracut E microtome (Leica Microsystems Inc, IL, USA), stained with 1% toluidine blue, and examined with a light microscope to find the localization of interest for further TEM sectioning. Next, the 70 nm thick sections (cut with Leica EM UC7, Wetzlar, Germany) were stained with 1% uranyl acetate for 30 min and with lead citrate for 2 min. The sections were examined with a JEM-2100F TEM (Jeol, Tokyo, Japan) at 200 kV. Lipofuscin-like aggregates and autophagic compartments were manually counted by three different individuals. Similarly as with the histological samples, representative areas per sample were selected for collecting RPEs from each individual sample close to the vicinity of the optic nerve. Six cells were randomly selected from each group (WT and dKO mice aged 12 months, n = 3) for counting. 2.6. Vacuole area fraction analysis The analysis technique used in this study objectively measured the morphometric parameter of the vacuoles at the basal cytoplasmic site of RPE from TEM images, to characterize area fractions of vacuoles in the WT and dKO RPE at 1 year of age. Using the open source Image J software (http//:imagej.nih.gov; NIH, USA), the software measured the size of vacuoles and data were expressed according to equal size ROI (200 µm 2 ). 2.7. Detection of lipofuscin-like granules in the RPE cells During the regular dewaxing and rehydration, the processed WT and dKO samples of interest were washed with 0.3% w/v Sudan Black B (SBB, cat. no. 3545-12, EMD Chemicals, Gibbstown, NJ, USA) in 70% ethanol at 37 °C for 20 min, rinsed with PBS 3 times 10 min and then the sections were mounted with Mowiol mounting Media (Sigma, St. Louis, MO, USA) [40]. The samples were examined in a laser scanning confocal microscope (Zeiss AX10 Imager A2, Zeiss, Göttingen, Germany) with sequential scanning and detection of dot-like lipofuscin granules, respectively, followed by merging and saving of the images. 2.8. Apoptosis assay TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was performed using the ApopTag®Peroxidase in situ Apoptosis Detection Kit (Millipore S.A.S., Molsheim, France) according to the manufacturer's instructions in one-year-old samples. TUNEL stained sections were washed in PBS and counterstained with DAPI. The sections were dehydrated and mounted. Positive and negative controls were used to control staining quality. Fig. 1. NRF-2/PGC-1αdKO mice display increased protein aggregation, autophagy and oxidative stress markers in RPE cells. Representative images and comparative computer-aided densitometric assay focusing on ubiquitin (white), p62/SQSTM1 (brown), Beclin-1 (red), LC3B (green) and 4-HNE (yellow) immunofluorescences in RPE cells (DAPI-stained nuclei/blue) of one-year-old wild type (WT), PGC-1αKO, NRF-2 KO and NRF-2/PGC-1αdKO mice. The scale bar indicates 5 µm. The gray level intensities of WT, PGC-1αKO, NRF-2 KO and NRF-2/PGC-1αdKO mice represent average intensities. Combined results from three different animals per genotype and n = 30. *p < 0.001 one-way ANOVA followed by Games-Howell post hoc test (n = 30). Results are expressed as means ± SD. S. Felszeghy et al. Redox Biology 20 (2019) 1–12 4 2.9. Immunohistochemistry of ER stress markers One-year-old WT and dKO retinas were stained in triplicate with rabbit anti-ATF4 (activating transcription factor 4, Abcam Cambridge, MA) and rabbit anti-GRP78 (glucose-regulated protein 78, Abcam Cambridge, MA) antibodies, separately. Immunohistochemistry was performed on paraffin embedded sections after antigen retrieval using an Invitrogen AEC red (Carlsbad, CA) substrate kit. For accurate comparisons, all sections were stained simultaneously and slides were developed under the same conditions. Imaging was performed with the Leica-Aperio CS digital ScanScope (Leica Microsystems, Buffalo, IL) at 40× magnification. When measuring positive pixels, images were digitally scribed using Aperio ScanScope software to create a ROI that encircled the entire cross-sectional profiles of the retina on each slide. The positive pixel count algorithm Aperio v.9.1 was used to determine stained vs. unstained pixel counts for areas within each ROI. 2.10. Flat mount and RPE size analysis Mice eyes were collected and fixed in 2% PFA for 2 h. The samples were then processed for the RPE/choroid flatmount preparation using a protocol previously described [34,35]. Briefly, the anterior segment of the eye, lens and the neuronal retina were removed under a dissecting microscope. Four to five vertical cuts were made from the edge of the RPE/choroid/sclera cup to the equator. The samples were then washed and treated with 1% triton X-100 for 2 h, followed by incubation with rabbit anti-mouse Iba-1 (ionized calcium binding adaptor molecule 1, Abcam, Cambridge, MA) 1:100, at 4 °C for 16 h, followed by FITCFig. 2. Transmission electron microscopy (TEM) analysis revealed dry AMD-like pathology in one-year-old NRF-2/PGC-1αdKO mice. Representative TEM image of WT RPE indicates normal cellular organelles, while in one year old dKO RPE loss of basal infoldings and thickened Bruchs's membrane (arrowheads) were detected (A and B). (C) Increased amount of (D1) melanosomes, (D2) autolysosomes, (D3, D4) damaged mitochondria and (D5) lipofuscin were detected in dKO RPE. The scale bars indicate 2 µm. WT = wild type; dKO = double knockout; BM = Bruch's membrane; HM = healthy mitochondria, DM = damaged mitochondria Data are quantified as % ratio of dKO versus WT ± SD. Thickness of Bruch's membrane (BM) microns ± SD. *p < 0.05, Student's t-test (n = 18). S. Felszeghy et al. Redox Biology 20 (2019) 1–12 5 conjugated (fluorescein isothiocyanate) goat anti-rabbit IgG (1:200, Abcam) and Alexa Fluor 568 Phalloidin (1:100, Life Technologies) at room temperature for 2 h. The samples were washed and flatmounted on a glass slide for confocal microscopy investigation (Eclipse TE200-U; Nikon UK Ltd.). Z-stack confocal images of RPE flatmounts were reconstructed using the NIS Element (Nikon) software. The border of each RPE cell was outlined based on the Phalloidin (F-actin) staining, and the size of each RPE cell was measured manually using the same software. 2.11. ERG recordings We tested the retinal function in three-month-old (referred to as “young”; WT, n = 6; dKO, n = 6) and one-year-old mice (referred to as “old”; WT, n = 6; dKO, n = 7) by ERG (electroretinography, Thorlabs PM100D, Thorlabs, NJ, USA). The ERG method has been described in detail previously [41]. The animals were dark-adapted overnight for at least 12 h. They were anesthetized with isoflurane (induction: concentration 4–4.5%; maintenance: concentration ~ 1.2%) and placed into a customized stereotaxic frame and kept warm with a physiological heating pad at 38 °C. The corneal ERG electrode was a silver wire constructed as a semi-circular loop. One stainless steel subcutaneous needle was attached into the cheek (reference) and another into the lower back (common ground). All animal manipulations were performed under a dim red light and animals were further dark-adapted in a completely dark room for 5 min. The dark-adapted light stimulation was performed in an ascending series with respect to the stimulus intensity (3.60, −2.90, −2.10, −1.65, −0.45, 0.50, 1.00, 1.60 and 2.00 log cd·s/m 2 ), and the inter-stimulus interval (ISI) was increased accordingly (2 s, 4 s, 4 s, 10 s, 10 s, 10 s, 20 s, 50 s and 50 s, respectively). Finally, a paired-flash paradigm was used to isolate the cone component from the mixed rod-cone waveform [42]. A strong flash (2.00 log cd·s/m 2 ) was delivered to evoke transient rod saturation and 400 ms later a second probe flash (2.00 log cd·s/m 2 ) was delivered to generate a cone-specific response. Rod-saturation at 400 ms after flash was assumed based on a previous ex vivo ERG saturation test [41], where the strongest flash was estimated to yield ~ 14,000 photoisomerizations (*R) per mouse rod. The light intensity was quantified with an energy meter (Thorlabs PM100D, Thorlabs, NJ, USA). In the waveform analysis, the baseline for ERG response was taken as an average amplitude between −100 and 0 ms before the stimulus onset. The a-wave amplitude was calculated from baseline to the trough of the first negative deflection after the stimulus onset. The b-wave was calculated from the a-wave trough to the peak of the first major positive wave. The ´cone´ response amplitude was calculated between the largest negativity and the major positive wave peaking around 40 ms after the probe flash. 2.12. Statistical analysis All statistical analysis was performed using SPSS statistics software (SPSS Inc., Chicago, Ill, USA). p-values < 0.05 were considered significant. Spectral imaging analysis: The statistical analysis of the data Fig. 3. The pathological changes of RPE in NRF-2/PGC-1αdouble knockout (dKO) mice. (A) The light microscopic images of WT and dKO samples of the toluidine blue-stained epoxy section from one-year-old mouse. The white dashed arrowheads indicate the dome-shaped extracellular deposits between the RPE and Bruch's membrane (upper panels). The cystic cytoplasmic alterations were observed in dKO samples (asterisks). (B) Excessive accumulation of lipofuscin-like material (white arrowhead) in the dKO RPE. In the inserts of Fig. B On the insert of figure B, the yellow dashed arrows indicate the RPE layer. The scale bar indicates 10 µm. (C) The red arrowhead indicates the ubiquitin positivity of drusen-like deposits in the close vicinity of Bruch's membrane. (D) Restricted apoptosis was detected within some cells in the RPE layer (green arrow) of the dKO retina with Apoptag®kit. PR: photoreceptor layer; RPE: retinal pigment epithelium; BM: Bruch's membrane. The scale bar indicates 5 µm. E (endothelial cell nucleus); N (RPE nucleus). S. Felszeghy et al. Redox Biology 20 (2019) 1–12 6 was conducted with a one-way analysis of variance (ANOVA), followed by Games-Howell post hoc test (n = 30). TEM analysis: The significance of differences between WT and dKO groups were analyzed with Student's t-test (n = 18). Vacuole area fraction analysis: The statistical analysis of the data was conducted with a Student's t-test (n = 25). Immunohistochemistry of ER stress markers: The average cell numbers from 5 different regions were calculated. Student's t-test was applied to assess statistical differences between WT and dKO groups (n = 4–7). Flat mount and RPE size analysis: Statistical significance was analyzed with Student's t-test (n = 4). ERG recordings: aand b-wave amplitude and b-a ratio statistical analyses were performed with a two-way repeated measured ANOVA using the genotype as the between-subject factor and stimulus intensity as the within-subjects factor, followed by Bonferroni post hoc test. The cone-flash amplitude was analyzed with a regular two-way ANOVA. 3. Results We investigated the effects of NRF-2/PGC-1αdKO on macroscopic anatomy and age-related RPE degeneration using light, confocal, and electron microscopy, as well as performing a functional electroretinography analysis (Supplementary Figs. 2 and 3). To ensure that there were no complications due to the spontaneous Rd8 (retinal degeneration 8) frameshift mutation c.3481delC in Crb1 (crumbs homolog 1) resulting retinal degeneration [43,44], screening for this mutation was performed for mouse lines used in this study: WT, NRF-2 KO, PGC1αKO and NRF-2/PGC-1αdKO. Our analysis revealed that these mice strains were negative for the Rd8 mutant allele (Supplementary Fig. 4). 3.1. NRF-2/PGC-1αgene inactivation evokes severe axial skeleton and eye morphology alterations The radiographs reveal the abnormally small axial skeleton already present in six-week-old dKO mice (Supplementary Fig. 3). In comparison with WT controls, the dKO mice had lighter body weights and expressed prominent dysmorphic features, such as a shorter axial skeleton, smaller limbs and head parameters at all ages studied (Supplementary Fig. 3). The fur of the dKO mice was also more greyish than in the corresponding single KOs or WT mice. The macrophotography and micro-MRI (magnetic resonance imaging) revealed a smaller eyeball size in the dKOs. 3.2. NRF-2/PGC-1αdKO mice display increased protein aggregation and oxidative stress markers in RPE cells Chronic oxidative stress and impaired autophagy are strongly linked to RPE degeneration and the protein aggregation observed in AMD [1,3]. The content and localization of protein aggregates-conjugated marker ubiquitin, autophagy regulator Beclin-1, autophagy markers p62/SQSTM1 and LC3B, and oxidative stress marker 4-HNE (product of lipid peroxidation) were evaluated in RPE cells by immunostainings and confocal microscopy in one-year-old WT, PGC-1αKO, NRF-2 KO and NRF-2/PGC-1αdKO mice (Fig. 1). Removal of PGC-1αgene resulted in higher levels of Beclin-1 (p < 0.001), LC3B (p < 0.001) and 4-HNE (p < 0.001) compared to the WT mice. However, the marker of protein aggregation, ubiquitin, decreased significantly and the p62/SQSTM1 levels remained stable indicating an increase in oxidative stress but, as expected, functional UPS/autophagy appears capable to maintain mitochondrial/protein homeostasis. NRF-2 KO resulted in higher expression levels of all markers studied compared to the WT (p < 0.001). The marker of oxidative stress (4-HNE) as well as autophagy markers Beclin-1 and p62/SQSTM1 and protein aggregation marker ubiquitin were significantly elevated in NRF-2 KO compared to PGC-1αKO mice suggesting stronger oxidative stress, insufficient proteasome function and autophagic clearance. NRF-2/PGC-1αdKO mice had the highest Fig. 4. NRF-2/PGC-1αdKO mice display increased endoplasmic reticulum (ER) stress in RPE cells. (A) Representative transmission electron microscopy (TEM) image of WT RPE indicates normal cellular structures and melanosomes. (B) Increased number of melanosomes, the basal laminar deposits (arrow), the loss of basal infoldings and ER damage (asterisks) were detected in dKO samples. (C) Increase of the ER vacuolization (% per 200 µm2) was recorded in dKO RPE compared to aged matched WT samples. Increased immunohistochemical staining intensity for (D) GRP78 and (E) ATF4 was detected in RPE layer of dKO mice compared to WT. The scale bar indicates 10 µm (A, B) and 2 µm (C, D). *p < 0.05, **p < 0.01, Students t-test. Data are mean ± SD (A-D n = 4–7; E n = 25). S. Felszeghy et al. Redox Biology 20 (2019) 1–12 7 accumulation of all markers studied suggesting the highest degree oxidative stress and more severe defect in the UPS/autophagy clearance. 3.3. Ultrastructural analysis indicates an increase in autolysosomes, damaged mitochondria and melanosomes in NRF-2/PGC-1αdKO mice Since insufficient autophagic clearance and high oxidative stress were detected in dKO mice, we assessed ultrastructural changes of WT and dKO RPE cells from the TEM images (Fig. 2). Compared to intact normal cell structures in WT RPE cells, the examination of dKO revealed the presence of larger autolysomes, higher ratio of damaged mitochondria (p < 0.05), thicker Bruch's membrane (p < 0.05), the loss of basal infoldings and moderate increase in lipofuscin particles. Interestingly, the number of melanosomes was higher in dKO mice. Moreover, Bruch's membrane elastic and collagenous layers were not often observed, and increased thickness of Bruch's membrane was detected along with an accumulation of electron dense amorphous material in dKO animals. 3.4. Micro-structural changes in the RPE and Bruch's membrane of NRF-2/ PGC-1αdKO mice Our aim was then to evaluate whether the NRF-2/PGC-1αdKO tissue samples showed AMD-like pathological features at the histological level. Analysis of the epoxy semi-thin and wax sections revealed Bruch's membrane dysmorphology, increased accumulation of lipofuscin-like particles and cytoplasmic alteration in the dKO RPE (Fig. 3A). Drusen-like deposits between RPE and Bruch's membrane were positive for ubiquitin staining (Fig. 3B and C). The intracellular cystic degeneration of RPE, nuclear debris and apoptosis (Fig. 3D) were observed in dKO RPE cells. 3.5. NRF-2/PGC-1αdKO mice RPE cells display increased ER stress markers and loss of basal infoldings The involvement of ER stress was assessed by ultrastructural analysis and staining with anti-GRP78 and anti-ATF4. Ultrastructural analysis revealed statistically significant ER vacuolization as a marker of ER damage in dKO RPE cells compared to corresponding WT RPE cells (Fig. 4A-C). The retinas from one-year-old dKO exhibited more intense staining for GRP78 (p < 0.05) and ATF4 (p < 0.01) in the RPE layer when compared to the corresponding tissues in WT controls (Fig. 4D-E). 3.6. Iba-1 is upregulated and the size of RPE cell is increased in NRF-2/ PGC-1αdKO mice The innate immune system is activated in a number of degenerative and inflammatory retinal disorders, including AMD. Retinal mononuclear phagocytes are also present in most mouse models of retinal disease [45]. To evaluate whether Iba-1 expression is enhanced in dKO RPE due to inflammatory processes, Iba-1 analysis was performed in RPE flat mounts. Data in Fig. 5 show a statistically significant 400% increase in the Iba-1 staining intensity in RPE of one-year-old NRF-2/ PGC-1αdKO versus WT mice (p < 0.05). Notably, we also observed a clear RPE size increase in our dKO mice compared to their WT counterparts (p < 0.01). During normal aging, an increased RPE cell size in C57BL/6J mouse has been interpreted as a prognosis of cell death [46]. 3.7. NRF-2/PGC-1αdKO mice show age-related visual loss and photoreceptor dysmorphology Given the observed structural changes noted above, we subsequently assessed the in vivo function of rod and cone pathways by performing ERG in the WT and dKO mice. As presented in Fig. 6, the ERGs amplitudes were lower in dKO mice even at a young age, but the difference reached statistical significance only in b-wave amplitudes (awave: p = 0.13; b-wave: p < 0.05). The cone response, as determined by the paired-flash paradigm, was comparable between young dKO and WT mice (Fig. 6E). With age, the rod-function significantly decreased in WT mice (aand b-wave: p < 0.001), while cone-function remained stable (Fig. 6B-E). Group-wise comparison revealed significant differences between old dKO and WT mice in dark-adapted aand b-wave amplitudes (a-wave: p < 0.05, b-wave: p < 0.01). There was a clear tendency towards age-genotype interaction in cone-flash amplitude (p = 0.06), indicating that also cone-function tends to decrease in dKO mice with age (see Fig. 6E). The ratio between band a-wave amplitudes remained unchanged regardless of genotype or age (Fig. 6D, all group-wise comparisons p > 0.28), indicating that a photoreceptor defect was the cause for the ERG amplitude changes. Since impaired photoreceptor function was found in the retina of dKO mice, the next step was to study the photoreceptor layer in more detail. The photoreceptor cells of dKO mice showed decreased thickness of the outer nuclear layer (ONL) and disorganization in the outer portion of the ONL (Fig. 6F-I). The staining of protein aggregation, autophagy and Fig. 5. Increased immune cell infiltration and the enlargement of RPE cells are present in NRF-2/PGC-1αdKO mice. Representative confocal Iba-1 (green) and Phalloidin (red) immunohistochemical images of retinal flat mounts samples of one-year-old (A) WT and (B) NRF-2/PGC-1αdKO mice. Insets show the low magnification view from RPE layer. The scale bar indicates 20 µm. (C) There was a 400% increase in Iba-1 positive cells in the NRF-2/PGC1αdKO RPE compared to the WT flat mount samples. (D) The RPE cell size difference in NRF-2/PGC-1αdKO and WT mice. The horizontal black lines represent the average size of the RPE cell. *p < 0.05, Student's t-test (n = 4). S. Felszeghy et al. Redox Biology 20 (2019) 1–12 8 oxidative stress markers and cellular morphology of photoreceptors in one-year-old mice were similar to those observed in RPE cells (Supplementary fig. 5). Electron micrographs, in addition to RPE cells, revealed focal photoreceptor layer degeneration in dKO mice (Supplementary fig. 5). 3.8. NRF-2/PGC-1αdKO mouse model shows a dry AMD-like phenotype Taken together, our dKO mice show pathological signs that are consistent with those observed in patients with dry AMD. The pathological changes observed in these dKO mice include age-related RPE degeneration, intraand extracellular deposit accumulation, Bruch's membrane dysmorphology, immune cell invasion, damaged mitochondria, impaired autophagy, altered pigmentation and visual loss consistent with a defect in photoreceptors (Fig. 7). 4. Discussion Recently, impaired proteolysis in response to chronic and overt oxidative stress has been suggested as a key contributor to age-related retinal degeneration, particularly in AMD [1,8]. To study proteolysis of age-related retinal degeneration, we sought to develop an animal model that would provide the most robust protein aggregation in RPE cells. Previously, genetic ablation of the NRF-2 and PGC-1αhas been shown to induce RPE degeneration [15,30]. Our findings reveal that knockout of both, NRF-2 and PGC-1α, resulted in the highest increase of markers Fig. 6. Dysmorphology of photoreceptors coincides with impaired retinal function in NRF-2-PGC1αdKO mice. (A) Dark-adapted ERG waveforms at 1.60 log cd·s/ m 2 flash. Thin lines represent individual mouse responses and thick lines group-averaged waveforms. (B) ERG b-wave declined already in three-month-old (3 M) dKO mice (n = 6) as compared to their age-matched WT (n = 6) littermate mice, and the difference became more significant in one-year-old (1Y) mice (dKO: n = 7; WT: n = 6). (C) ERG a-wave tended to decline at both ages but reached statistically significant level only at older age. (D) The ratio between band a-wave amplitudes did not change regardless of the genotype or the age. (E) The double-flash paradigm revealed a tendency towards age-related decline in the cone-dominant function in dKO mice. (F) Thin unstained wax section, (G) DAPI staining of outer nuclear layer (ONL) in WT samples compared with (H,I) one-year-old (y) dKO mice, respectively. Photoreceptor atrophy of the ONL (red vertical arrow) is seen in DAPI-labeled dKO retinas cut from sections located 250–350 µm from the optic nerve head. Retinas show disorganization in the outer portion of the ONL (yellow asterics). The scale bar indicates 5 µm. **p < 0.01, *p < 0.05, two-way ANOVA. S. Felszeghy et al. Redox Biology 20 (2019) 1–12 9