Dual AAV-based PCDH15 gene therapy achieves sustained rescue of visual function in a mouse model of Usher syndrome 1F.

Mutations in the PCDH15 gene, encoding protocadherin-15, are among the leading causes of Usher syndrome type 1 (USH1F), and account for up to 12% USH1 cases worldwide. A founder truncating variant of PCDH15 has a ∼2% carrier frequency in Ashkenazi Jews accounting for nearly 60% of their USH1 cases. Although cochlear implants can restore hearing perception in USH1 patients, presently there are no effective treatments for the vision loss due to retinitis pigmentosa. We established a founder allele-specific Pcdh15 knockin mouse model as a platform to ascertain therapeutic strategies. Using a dual-vector approach to circumvent the size limitation of adeno-associated virus, we observed robust expression of exogenous PCDH15 in the retinae of Pcdh15KI mice, sustained recovery of electroretinogram amplitudes and key retinoid oxime, substantially improved light-dependent translocation of phototransduction proteins, and enhanced levels of retinal pigment epithelium-derived enzymes. Thus, our data raise hope and pave the way for future gene therapy trials in USH1F subjects.

Pretreatment of human retinal pigment epithelial cells with sterculic acid forestalls fenretinide-induced apoptosis.

The ratio of saturated to monounsaturated fatty acids, thought to play a critical role in many cellular functions, is regulated by stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. Previously, we observed a decrease in both SCD protein and enzymatic activity in apoptosis induced by fenretinide, a synthetic analog of retinoic acid, in the human retinal pigment epithelial (RPE) cell line ARPE-19. Here, we investigated the effect of pretreating ARPE-19 with sterculic acid, a cyclopropenoic fatty acid inhibitor of SCD, on preventing fenretinide-induced apoptosis, given the role of SCD in cell proliferation and apoptosis. We show that sterculic acid pretreatment prevents the effects of fenretinide-induced apoptosis shown by changes in cell morphology, viability, and caspase-3 activation. Analysis of endoplasmic reticulum (ER)-associated proteins shows that sterculic acid pretreatment reduced the fenretinide-induced upregulation of heme oxygenase-1, ATF3 and GADD153 expression that are in response to reactive oxygen species (ROS) generation. Sterculic acid is as effective as allopurinol in inhibition of xanthine oxidase (XDH), and this may play a role in reducing the potential role of XDH in fenretinide-induced ROS generation. Sterculic acid pretreatment also results in a reduction in SOD2 mRNA expression. Dihydroceramide accumulation, compared to ceramide, and ROS generation indicate that a ceramide-independent pathway mediates fenretinide-induced apoptosis, and ROS mediation is borne out by activation of the NF-κBp50 and NF-κBp65 downstream signaling cascade. Its prevention by sterculic acid pretreatment further indicates the latter's antioxidant/anti-inflammatory effect. Taken together, our results suggest that sterculic acid pretreatment can mitigate ROS-mediated fenretinide-induced apoptosis. Thus, sterculic acid may serve as a potential antioxidant and therapeutic agent. These effects may be independent of its effects on SCD activity.

Proposed therapy, developed in a Pcdh15-deficient mouse, for progressive loss of vision in human Usher syndrome.

Usher syndrome type I (USH1) is characterized by deafness, vestibular areflexia, and progressive retinal degeneration. The protein-truncating p.Arg245* founder variant of PCDH15 (USH1F) has an ~2% carrier frequency amongst Ashkenazi Jews accounts for ~60% of their USH1 cases. Here, longitudinal phenotyping in 13 USH1F individuals revealed progressive retinal degeneration, leading to severe vision loss with macular atrophy by the sixth decade. Half of the affected individuals were legally blind by their mid-50s. The mouse Pcdh15R250X variant is equivalent to human p.Arg245*. Homozygous Pcdh15R250X mice also have visual deficits and aberrant light-dependent translocation of the phototransduction cascade proteins, arrestin, and transducin. Retinal pigment epithelium (RPE)-specific retinoid cycle proteins, RPE65 and CRALBP, were also reduced in Pcdh15R250X mice, indicating a dual role for protocadherin-15 in photoreceptors and RPE. Exogenous 9-cis retinal improved ERG amplitudes in Pcdh15R250X mice, suggesting a basis for a clinical trial of FDA-approved retinoids to preserve vision in USH1F patients.

Retbindin mediates light-damage in mouse retina while its absence leads to premature retinal aging.

Vision requires the transport and recycling of the pigment 11-cis retinaldehyde (retinal) between the retinal pigment epithelium (RPE) and photoreceptors. 11-cis retinal is also required for light-mediated photoreceptor death in dark-adapted mouse eye, probably through overstimulation of rod cells adapted for low light. Retbindin is a photoreceptor-specific protein, of unclear function, that is localized between the RPE and the tips of the photoreceptors. Unexpectedly, young Rtbdn-KO mice, with targeted deletion (KO) of retbindin, showed delayed regeneration of retinal function after bleaching and were strongly resistant to light-induced photoreceptor death. Furthermore, bio-layer interferometry binding studies showed recombinant retbindin had significant affinity for retinoids, most notably 11-cis retinal. This suggests that retbindin mediates light damage, probably through a role in transport of 11-cis retinal. In Rtbdn-KO mice, retinal development was normal, as were amplitudes of rod and cone electroretinograms (ERG) up to 4 months, although implicit times and c-waves were affected. However, with aging, both light- and dark-adapted ERG amplitudes declined significantly and photoreceptor outer segments became disordered, However, in contrast to other reports, there was little retinal degeneration or drop in flavin levels. The RPE developed vacuoles and lipid, protein and calcium deposits reminiscent of age-related macular degeneration. Other signs of premature aging included loss of OPN4+ retinal ganglion cells and activation of microglia. Thus, retbindin plays an unexpected role in the mammalian visual cycle, probably as an adaptation for vision in dim light. It mediates light damage in the dark-adapted eye, but also plays a role in light-adapted responses and in long term retinal homeostasis.

CIB2 regulates mTORC1 signaling and is essential for autophagy and visual function.

Age-related macular degeneration (AMD) is a multifactorial neurodegenerative disorder. Although molecular mechanisms remain elusive, deficits in autophagy have been associated with AMD. Here we show that deficiency of calcium and integrin binding protein 2 (CIB2) in mice, leads to age-related pathologies, including sub-retinal pigment epithelium (RPE) deposits, marked accumulation of drusen markers APOE, C3, Aß, and esterified cholesterol, and impaired visual function, which can be rescued using exogenous retinoids. Cib2 mutant mice exhibit reduced lysosomal capacity and autophagic clearance, and increased mTORC1 signaling-a negative regulator of autophagy. We observe concordant molecular deficits in dry-AMD RPE/choroid post-mortem human tissues. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to 'nucleotide empty' or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts downregulates hyperactive mTORC1 signaling. Thus, our findings have significant implications for treatment of AMD and other mTORC1 hyperactivity-associated disorders.

Aberrant RNA splicing is the major pathogenic effect in a knock-in mouse model of the dominantly inherited c.1430A>G human RPE65 mutation.

Human RPE65 mutations cause a spectrum of retinal dystrophies that result in blindness. While RPE65 mutations have been almost invariably recessively inherited, a c.1430A>G (p.(D477G)) mutation has been reported to cause autosomal dominant retinitis pigmentosa (adRP). To study the pathogenesis of this human mutation, we have replicated the mutation in a knock-in (KI) mouse model using CRISPR/Cas9-mediated genome editing. Significantly, in contrast to human patients, heterozygous KI mice do not exhibit any phenotypes in visual function tests. When raised in regular vivarium conditions, homozygous KI mice display relatively undisturbed visual functions with minimal retinal structural changes. However, KI/KI mouse retinae are more sensitive to light exposure and exhibit signs of degenerative features when subjected to light stress. We find that instead of merely producing a missense mutant protein, the A>G nucleotide substitution greatly affects appropriate splicing of Rpe65 mRNA by generating an ectopic splice site in comparable context to the canonical one, thereby disrupting RPE65 protein expression. Similar splicing defects were also confirmed for the human RPE65 c.1430G mutant in an in vitro Exontrap assay. Our data demonstrate that a splicing defect is associated with c.1430G pathogenesis, and therefore provide insights in the therapeutic strategy for human patients.

Proinflammatory cytokine interferon-γ increases the expression of BANCR, a long non-coding RNA, in retinal pigment epithelial cells.

The inflammatory response may contribute to retinal pigment epithelial (RPE) dysfunction associated with the pathogenesis of age-related macular degeneration (AMD). We investigated whether the inflammatory response affects the expression of long coding RNAs (lncRNAs) in human RPE-derived ARPE-19 cells. This class of regulatory RNA molecules recently came to prominence due to their involvement in many pathophysiological processes. A proinflammatory cytokine mixture consisting of IFN-γ, IL-1ß and TNF-α altered the expression several lncRNAs including BANCR in these cells. The cytokine responsible for increasing BANCR expression in ARPE-19 cells was found to be IFN-γ. BANCR expression induced by IFN-γ was suppressed when STAT1 phosphorylation was blocked by JAK inhibitor 1. Thus, proinflammatory cytokines could modulate the expression of lncRNAs in RPE cells and IFN-γ could upregulate the expression of BANCR by activating JAK-STAT1 signaling pathway.

Inhibitory effects of fenretinide metabolites N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR) on fenretinide molecular targets ß-carotene oxygenase 1, stearoyl-CoA desaturase 1 and dihydroceramide Δ4-desaturase 1.

The therapeutic capacity of fenretinide (N-[4-hydroxyphenyl] retinamide; 4-HPR) has been demonstrated for several conditions, including cancer, obesity, diabetes, and ocular disease. Yet, the mechanisms of action for its pleiotropic effects are still undefined. We hypothesized that investigation of two of the major physiological metabolites of fenretinide, N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR), might begin to resolve the multifaceted effects of this synthetic retinoid. We analyzed the effects of fenretinide, MPR, 3-keto-HPR, and the non-retinoid RBP4 ligand A1120, on the activity of known targets of fenretinide, stearoyl-CoA desaturase 1 (SCD1) and dihydroceramide Δ4-desaturase 1 (DES1) in ARPE-19 cells, and purified recombinant mouse beta-carotene oxygenase 1 (BCO1) in vitro. Lipids and retinoids were extracted and quantified by liquid chromatography-mass spectrometry and reversed phase HPLC, respectively. The data demonstrate that while fenretinide is an inhibitor of the activities of these three enzymes, that 3-keto-HPR is a more potent inhibitor of all three enzymes, potentially mediating most of the in vivo beneficial effects of fenretinide. However, while MPR does not affect SCD1 and DES1 activity, it is a potent specific inhibitor of BCO1. We conclude that a deeper understanding of the mechanisms of action of fenretinide and its metabolites provides new avenues for therapeutic specificity. For example, administration of 3-keto-HPR instead of fenretinide may be preferential if inhibition of SCD1 or DES1 activity is the goal (cancer), while MPR may be better for BCO1 modulation (carotenoid metabolism). Continued investigation of fenretinide metabolites in the context of fenretinide's various therapeutic uses will begin to resolve the pleotropic nature of this compound.

Appropriately differentiated ARPE-19 cells regain phenotype and gene expression profiles similar to those of native RPE cells.

PURPOSE: The RPE cell line ARPE-19 provides a dependable and widely used alternative to native RPE. However, replication of the native RPE phenotype becomes more difficult because these cells lose their specialized phenotype after multiple passages. Compounding this problem is the widespread use of ARPE-19 cells in an undifferentiated state to attempt to model RPE functions. We wished to determine whether suitable culture conditions and differentiation could restore the RPE-appropriate expression of genes and proteins to ARPE-19, along with a functional and morphological phenotype resembling native RPE. We compared the transcriptome of ARPE-19 cells kept in long-term culture with those of primary and other human RPE cells to assess the former's inherent plasticity relative to the latter. METHODS: ARPE-19 cells at passages 9 to 12 grown in DMEM containing high glucose and pyruvate with 1% fetal bovine serum were differentiated for up to 4 months. Immunocytochemistry was performed on ARPE-19 cells grown on filters. Total RNA extracted from ARPE-19 cells cultured for either 4 days or 4 months was used for RNA sequencing (RNA-Seq) analysis using a 2 × 50 bp paired end protocol. The RNA-Seq data were analyzed to identify the affected pathways and recognize shared ontological classification among differentially expressed genes. RPE-specific mRNAs and miRNAs were assessed with quantitative real-time (RT)-PCR, and proteins with western blotting. RESULTS: ARPE-19 cells grown for 4 months developed the classic native RPE phenotype with heavy pigmentation. RPE-expressed genes, including RPE65, RDH5, and RDH10, as well as miR-204/211, were greatly increased in the ARPE-19 cells maintained at confluence for 4 months. The RNA-Seq analysis provided a comprehensive view of the relative abundance and differential expression of the genes in the differentiated ARPE-19 cells. Of the 16,757 genes with detectable signals, nearly 1,681 genes were upregulated, and 1,629 genes were downregulated with a fold change of 2.5 or more differences between 4 months and 4 days of culture. Gene Ontology analysis showed that the upregulated genes were associated with visual cycle, phagocytosis, pigment synthesis, cell differentiation, and RPE-related transcription factors. The majority of the downregulated genes play a role in cell cycle and proliferation. CONCLUSIONS: The ARPE-19 cells cultured for 4 months developed a phenotype characteristic of native RPE and expressed proteins, mRNAs, and miRNAs characteristic of the RPE. Comparison of the ARPE-19 RNA-Seq data set with that of primary human fetal RPE, embryonic stem cell-derived RPE, and native RPE revealed an important overall similar expression ratio among all the models and native tissue. However, none of the cultured models reached the absolute values in the native tissue. The results of this study demonstrate that low-passage ARPE-19 cells can express genes specific to native human RPE cells when appropriately cultured and differentiated.

Proinflammatory cytokines decrease the expression of genes critical for RPE function.

PURPOSE: Proinflammatory cytokines interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1ß) secreted by infiltrating lymphocytes or macrophages may play a role in triggering RPE dysfunction associated with age-related macular degeneration (AMD). Binding of these proinflammatory cytokines to their specific receptors residing on the RPE cell surface can activate signaling pathways that, in turn, may dysregulate cellular gene expression. The purpose of the present study was to investigate whether IFN-γ, TNF-α, and IL-1ß have an adverse effect on the expression of genes essential for RPE function, employing the RPE cell line ARPE-19 as a model system. METHODS: ARPE-19 cells were cultured for 3-4 months until they exhibited epithelial morphology and expressed mRNAs for visual cycle genes. The differentiated cells were treated with IFN-γ, TNF-α, and/or IL-1ß, and gene expression was analyzed with real-time PCR analysis. Western immunoblotting was employed for the detection of proteins. RESULTS: Proinflammatory cytokines (IFN-γ + TNF-α + IL-1ß) greatly increased the expression of chemokines and cytokines in cultured ARPE-19 cells that exhibited RPE characteristics. However, this response was accompanied by markedly decreased expression of genes important for RPE function, such as CDH1, RPE65, RDH5, RDH10, TYR, and MERTK. This was associated with decreased expression of the genes MITF, TRPM1, and TRPM3, as well as microRNAs miR-204 and miR-211, which are known to regulate RPE-specific gene expression. The decreased expression of the epithelial marker gene CDH1 was associated with increased expression of mesenchymal marker genes (CDH2, VIM, and CCND1) and epithelial-mesenchymal transition (EMT) promoting transcription factor genes (ZEB1 and SNAI1). CONCLUSIONS: RPE cells exposed to proinflammatory cytokines IFN-γ, TNF-α, and IL-1ß showed decreased expression of key genes involved in the visual cycle, epithelial morphology, and phagocytosis. This adverse effect of proinflammatory cytokines, which could be secreted by infiltrating lymphocytes or macrophages, on the expression of genes indispensable for RPE function may contribute to the RPE dysfunction implicated in AMD pathology.

Mouse model of human RPE65 P25L hypomorph resembles wild type under normal light rearing but is fully resistant to acute light damage.

Human RPE65 mutations cause a spectrum of blinding retinal dystrophies from severe early-onset disease to milder manifestations. The RPE65 P25L missense mutation, though having <10% of wild-type (WT) activity, causes relatively mild retinal degeneration. To better understand these mild forms of RPE65-related retinal degeneration, and their effect on cone photoreceptor survival, we generated an Rpe65/P25L knock-in (KI/KI) mouse model. We found that, when subject to the low-light regime (∼100 lux) of regular mouse housing, homozygous Rpe65/P25L KI/KI mice are morphologically and functionally very similar to WT siblings. While mutant protein expression is decreased by over 80%, KI/KI mice retinae retain comparable 11-cis-retinal levels with WT. Consistently, the scotopic and photopic electroretinographic (ERG) responses to single-flash stimuli also show no difference between KI/KI and WT mice. However, the recovery of a-wave response following moderate visual pigment bleach is delayed in KI/KI mice. Importantly, KI/KI mice show significantly increased resistance to high-intensity (20 000 lux for 30 min) light-induced retinal damage (LIRD) as compared with WT, indicating impaired rhodopsin regeneration in KI/KI. Taken together, the Rpe65/P25L mutant produces sufficient chromophore under normal conditions to keep opsins replete and thus manifests a minimal phenotype. Only when exposed to intensive light is this hypomorphic mutation manifested physiologically, as its reduced expression and catalytic activity protects against the successive cycles of opsin regeneration underlying LIRD. These data also help define minimal requirements of chromophore for photoreceptor survival in vivo and may be useful in assessing a beneficial therapeutic dose for RPE65 gene therapy in humans.

Resveratrol attenuates CXCL11 expression induced by proinflammatory cytokines in retinal pigment epithelial cells.

Dysfunction of the retinal pigment epithelium (RPE) resulting from chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). RPE cells adjacent to drusen deposits in the AMD eye are known to contain CXCL11, a chemokine involved in inflammatory cell recruitment. We investigated the CXCL11 production by the human RPE (ARPE-19) cells under inflammatory conditions and tested its response to resveratrol, a naturally occurring anti-inflammatory antioxidant. A proinflammatory cytokine mixture consisting of IFN-γ, IL-1ß and TNF-α highly increased CXCL11 mRNA expression and CXCL11 protein secretion by ARPE-19 cells. Resveratrol substantially inhibited the proinflammatory cytokines-induced CXCL11 production while partially blocking nuclear factor-κB activation. This inhibitory action of resveratrol was also observed for the cytokines-induced expression of chemokines CXCL9, CCL2 and CCL5. Our results indicate that resveratrol could potentially attenuate RPE inflammatory response implicated in the pathogenesis of AMD.

The Rpe65 rd12 allele exerts a semidominant negative effect on vision in mice.

PURPOSE: The rd12 mouse was reported as a recessively inherited Rpe65 mutation. We asked if the rd12 mutation resides in Rpe65 and how the mutation manifests itself. METHODS: A complementation test was performed by mating Rpe65(KO) (KO/KO) and rd12 mice together to determine if the rd12 mutation is in the Rpe65 gene. Visual function of wild-type (+/+), KO/+, rd12/+, KO/KO, rd12/rd12, and KO/rd12 mice was measured by optokinetic tracking (OKT) and ERG. Morphology was assessed by retinal cross section. qRT-PCR quantified Rpe65 mRNA levels. Immunoblotting measured the size and level of RPE65 protein. Rpe65 mRNA localization was visualized with RNA fluorescence in situ hybridization (FISH). Fractions of Rpe65 mRNA-bound proteins were separated by linear sucrose gradient fractionation. RESULTS: The KO and rd12 alleles did not complement. The rd12 allele induced a negative semidominant effect on visual function; OKT responses became undetectable 120 days earlier in rd12/rd12 mice compared with KO/KO mice. rd12/+ mice lost approximately 21% visual acuity by P210. rd12/rd12 mice had fewer cone photoreceptor nuclei than KO/KO mice at P60. rd12/rd12 mice expressed 71% +/+ levels of Rpe65 mRNA, but protein was undetectable. Mutant mRNA was appropriately spliced, exported to the cytoplasm, trafficked, and contained no other coding mutation aside from the known nonsense mutation. Mutant mRNA was enriched on ribosome-free messenger ribonucleoproteins (mRNPs), whereas wild-type mRNA was enriched on actively translating polyribosomes. CONCLUSIONS: The rd12 lesion is in Rpe65. The rd12 mutant phenotype inherits in a semidominant manner. The effects of the mutant mRNA on visual function may result from inefficient binding to ribosomes for translation.

Fenretinide induces ubiquitin-dependent proteasomal degradation of stearoyl-CoA desaturase in human retinal pigment epithelial cells.

Stearoyl-CoA desaturase (SCD, SCD1), an endoplasmic reticulum (ER) resident protein and a rate-limiting enzyme in monounsaturated fatty acid biosynthesis, regulates cellular functions by controlling the ratio of saturated to monounsaturated fatty acids. Increase in SCD expression is strongly implicated in the proliferation and survival of cancer cells, whereas its decrease is known to impair proliferation, induce apoptosis, and restore insulin sensitivity. We examined whether fenretinide, (N-(4-hydroxyphenyl)retinamide, 4HPR), which induces apoptosis in cancer cells and recently shown to improve insulin sensitivity, can modulate the expression of SCD. We observed that fenretinide decreased SCD protein and enzymatic activity in the ARPE-19 human retinal pigment epithelial cell line. Increased expression of BiP/GRP78, ATF4, and GADD153 implicated ER stress. Tunicamycin and thapsigargin, compounds known to induce ER stress, also decreased the SCD protein. This decrease was completely blocked by the proteasome inhibitor MG132. In addition, PYR41, an inhibitor of ubiquitin activating enzyme E1, blocked the fenretinide-mediated decrease in SCD. Immunoprecipitation analysis using anti-ubiquitin and anti-SCD antibodies and the blocking of SCD loss by PYR41 inhibition of ubiquitination further corroborate that fenretinide mediates the degradation of SCD in human RPE cells via the ubiquitin-proteasome dependent pathway. Therefore, the effect of fenretinide on SCD should be considered in its potential therapeutic role against cancer, type-2 diabetes, and retinal diseases.

Complementation test of Rpe65 knockout and tvrm148.

PURPOSE: A mouse mutation, tvrm148, was previously reported as resulting in retinal degeneration. Tvrm148 and Rpe65 map between markers D3Mit147 and D3Mit19 on a genetic map, but the physical map places RPE65 outside the markers. We asked if Rpe65 or perhaps another nearby gene is mutated and if the mutant reduced 11-cis-retinal levels. We studied the impact of the tvrm148 mutation on visual function, morphology, and retinoid levels. METHODS: Normal phase HPLC was used to measure retinoid levels. Rpe65(+/+), tvrm148/+ (T(+/-)), tvrm148/tvrm148 (T(-/-)), RPE65(KO/KO) (Rpe65(-/-)), and Rpe65(T/-) mice visual function was measured by optokinetic tracking (OKT) and electroretinography (ERG). Morphology was assessed by light microscopy and transmission electron microscopy (TEM). qRT-PCR was used to measure Rpe65 mRNA levels. Immunoblotting measured the size and amount of RPE65 protein. RESULTS: The knockout and tvrm148 alleles did not complement. No 11-cis-retinal was detected in T(-/-) or Rpe65(-/-) mice. Visual acuity in Rpe65(+/+) and T(+/-) mouse was -0.382 c/d, but 0.037 c/d in T(-/-) mice at postnatal day 210 (P210). ERG response in T(-/-) mice was undetectable except at bright flash intensities. Outer nuclear layer (ONL) thickness in T(-/-) mice was -70% of Rpe65(+/+) by P210. Rpe65 mRNA levels in T(-/-) mice were unchanged, yet 14.5% of Rpe65(+/+) protein levels was detected. Protein size was unchanged. CONCLUSIONS: A complementation test revealed the RPE65 knockout and tvrm148 alleles do not complement, proving that the tvrm148 mutation is in Rpe65. Behavioral, physiological, molecular, biochemical, and histological approaches indicate that tvrm148 is a null allele of Rpe65.

Differential regulation of microRNA-146a and microRNA-146b-5p in human retinal pigment epithelial cells by interleukin-1ß, tumor necrosis factor-α, and interferon-γ.

PURPOSE: The inflammatory response of the retinal pigment epithelium (RPE) is implicated in the pathogenesis of age-related macular degeneration. The microRNAs miR-146a and miR-146b-5p can regulate the inflammatory process by attenuating cytokine signaling via the nuclear factor-κB pathway. The aim of the present study is to investigate the expression of miR-146a and miR-146b-5p in human RPE cells and their response to proinflammatory cytokines. METHODS: Confluent cultures of RPE cells established from adult human donor eyes were treated with the proinflammatory cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß. The expression of microRNAs was analyzed by real-time PCR using total RNA fraction. The retinal pigment epithelial cell line ARPE-19 was employed to analyze the promoter activity of the genes encoding miR-146a and miR-146b-5p. STAT1-binding activity of oligonucleotides was analyzed by electrophoretic mobility shift assay. ARPE-19 cells were transiently transfected with miR-146a and miR-146b-5p mimics for the analysis of IRAK1 expression by western immunoblotting. RESULTS: Real-time PCR analysis showed that miR-146a and 146b-5p are expressed in RPE cells. The cells responded to proinflammatory cytokines (IFN-γ + TNF-α + IL-1ß) by highly increasing the expression of both miR-146a and miR-146b-5p. This was associated with an increase in the expression of transcripts for CCL2, CCL5, CXCL9, CXCL10, and IL-6, and a decrease in that for HMOX1. The miR-146a induction was more dependent on IL-1ß, since its omission from the cytokine mix resulted in a greatly reduced response. Similarly, the induction of miR-146b-5p was more dependent on IFN-γ, since its omission from the cytokine mix minimized the effect. In addition, the increase in MIR146B promoter activity by the cytokine mix was effectively blocked by JAK inhibitor 1, a known inhibitor of the JAK/STAT signaling pathway. The expression of IRAK1 protein was decreased when ARPE-19 cells were transiently transfected with either miR-146a mimic or miR-146b-5p mimic. CONCLUSIONS: Our results clearly show that both miR-146a and miR-146b-5p are expressed in human RPE cells in culture and their expression is highly induced by proinflammatory cytokines (IFN-γ + TNF-α + IL-1ß). The induction of miR-146a showed a dependency on IL-1ß, while that of miR-146b-5p on IFN-γ. Our results show for the first time that miR-146b-5p expression is regulated by IFN-γ, potentially via the JAK/STAT pathway. These two microRNAs could play a role in inflammatory processes underlying age-related macular degeneration or other retinal degenerative diseases through their ability to negatively regulate the nuclear factor-κB pathway by targeting the expression of IRAK1.

MicroRNA expression in human retinal pigment epithelial (ARPE-19) cells: increased expression of microRNA-9 by N-(4-hydroxyphenyl)retinamide.

PURPOSE: MicroRNAs (miRNAs) are important regulators of many cellular functions due to their ability to target mRNAs for degradation or translational inhibition. Previous studies have reported that the expression of microRNA-9 (miR-9) is regulated by retinoic acid and reactive oxygen species (ROS). We have previously shown that N-(4-hydroxyphenyl)-retinamide (4HPR), a retinoic acid derivative, induces ROS generation and apoptosis in cultured human retinal pigment epithelial (RPE) cells, known as ARPE-19 cells. The aim of the present study was to investigate the expression of miR-9 in ARPE-19 cells in response to 4HPR treatment, and to identify other miRNAs normally expressed in these cells. METHODS: ARPE-19 cells in culture were treated with 4HPR, the total RNA fractions were isolated, and the expression of various miRNAs and mRNAs was analyzed using real-time PCR. The miRNA expression profile of ARPE-19 cells was analyzed using microarray hybridization. RESULTS: Treatment of ARPE-19 cells with 4HPR resulted in apoptosis characterized by the increased expression of HMOX1 and GADD153 genes. A twofold increase in the expression of miR-9 was also observed during this response. Potential binding sites for the transcription factors encoded by CEBPA and CEBPB genes were found to be present in the putative promoter regions of all three genes encoding miR-9. 4HPR-induced miR-9 expression was associated with parallel increases in the expression of these transcription factor genes. 5-Aza-2'-deoxycytidine, a methyl transferase inhibitor, also increased the expression of miR-9 in ARPE-19 cells. Microarray hybridization analysis identified let-7b, let-7a, miR-125b, miR-24, miR-320, miR-23b, let-7e, and let-7d as the most abundant miRNAs normally expressed in ARPE-19 cells. These miRNAs are known to regulate cell growth, differentiation or development. The 4HPR treatment increased the expression of miR-16, miR-26b, miR-23a, and miR-15b in ARPE-19 cells, although these increases were modest when compared to the increase in the expression of miR-9. CONCLUSIONS: Our studies demonstrate that miR-9 is expressed in the RPE cell line ARPE-19, and its expression is increased by a retinoic acid derivative and by an inhibitor of promoter hypermethylation. Several miRNAs with inherent ability to regulate cell growth, differentiation and development are also normally expressed in ARPE-19 cells. Thus, miR-9 and other miRNAs could be important in maintaining RPE cell function.

Decreased expression of insulin-like growth factor binding protein-5 during N-(4-hydroxyphenyl)retinamide-induced neuronal differentiation of ARPE-19 human retinal pigment epithelial cells: regulation by CCAAT/enhancer-binding protein.

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP5), an important member of the IGF axis involved in regulating cell growth and differentiation, acts by modulating IGF signaling and also by IGF-independent mechanisms. We identified IGFBP5 by microarray analysis as a gene differentially regulated during N-(4-hydroxyphenyl)retinamide (4HPR)-induced neuronal differentiation of human retinal pigment epithelial (RPE) cells. IGFBP5 is expressed in human RPE cells, and its expression, mRNA as well as protein, is greatly decreased during the 4HPR-induced neuronal differentiation. Exogenous IGFBP5 does not block the neuronal differentiation indicating that IGFBP5 down-regulation may not be a prerequisite for the neuronal differentiation. IGFBP5 down-regulation, similar to neuronal differentiation, is mediated by the MAPK pathway since U0126, an inhibitor of MEK1/2, effectively blocked it. The overexpression of transcription factor CCAAT/enhancer binding protein-beta (C/EBPbeta) inhibited the 4HPR-induced down-regulation of IGFBP5 expression and the neuronal differentiation of RPE cells. Interestingly, the binding of C/EBPbeta to the IGFBP5 promoter was decreased by the 4HPR treatment as indicated by gel shift and chromatin immunoprecipitation analyses. Further, the deletion of C/EBP response element from IGFBP5 promoter markedly decreased the basal promoter activity and abolished its responsiveness to 4HPR treatment in reporter assays, suggesting that the expression of IGFBP5 is regulated by C/EBP. Thus, our results clearly demonstrate that the IGFBP5 expression is down-regulated during 4HPR-induced neuronal differentiation of human RPE cells through a MAPK signal transduction pathway involving C/EBPbeta.

Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein.

NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P(270)KKRKAP(276)) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting.

Confocal immunolocalization of bovine serum albumin, serum retinol-binding protein, and interphotoreceptor retinoid-binding protein in bovine retina.

PURPOSE: Recently it has been shown that the transport as well as clearance of retinol from isolated rod photoreceptors requires an extracellular factor. Interphotoreceptor retinoid-binding protein (IRBP) is a component of the interphotoreceptor matrix (IPM) and is known to bind visual cycle retinoids. Serum albumin and serum retinol-binding protein (sRBP), proteins capable of binding retinoids, have also been reported to be components of the IPM. It is of interest to know the components present in the IPM that are capable of binding visual cycle retinoids and that also facilitate rhodopsin regeneration. The purpose of this study was to determine the localization of serum albumin, sRBP, and IRBP in bovine retina using immunofluorescence analysis. METHODS: Fresh bovine eyes, obtained from a local abattoir, were fixed immediately after enucleation. Tissue sections (100 microm) were incubated with primary antibodies to bovine serum albumin (BSA), sRBP, and IRBP. Sections were washed then incubated 4 h with 4'-6-Diamidino-2-phenylindole (DAPI), Alexa Fluor(R) 488 goat antimouse, and Alexa Fluor 568 goat antirabbit secondary antibodies. Sections were analyzed using a laser scanning confocal microscope equipped with Nomarski optics. Western immunoblot analysis of bovine retinal tissues and protein standards was performed using the primary antibodies to BSA, sRBP, and IRBP to show specificity to their respective antigens. RESULTS: Immunoblot analysis showed that monoclonal anti-BSA was highly specific for BSA detecting only a single band at about 67 kDa. Antihuman sRBP and antibovine IRBP were also highly specific, recognizing a single band at about 25 and about 133 kDa, respectively. No immunopositive bands were observed in bovine neural retinal when probed with the anti-sRBP antibody; however, a single immunoreactive band at about 67 and about 133 kDa was detected in bovine neural retina by the anti-BSA and IRBP antibodies, respectively. Immunofluorescence analysis showed labeling for IRBP throughout the IPM. IRBP labeling was especially associated with the outer segments of photoreceptors and also with the apical surface of the retinal pigment epithelium. Immunofluorescence labeling for serum albumin was associated only with the lumen of retinal and choroidal blood vessels. Staining for both serum albumin and sRBP in the IPM was negative. CONCLUSIONS: Immunofluorescence analysis of fresh bovine eyes using antibodies to BSA and sRBP clearly shows that serum albumin and sRBP are not components of bovine IPM. IRBP, on the other hand, is localized to the IPM where it is available for the binding and transport of visual cycle retinoids. From these data we conclude that serum albumin and sRBP are not factors that could participate in the binding as well as transport of visual cycle retinoids in the IPM of bovine retina.