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1.  Canine and Human Visual Cortex Intact and Responsive Despite Early Retinal Blindness from RPE65 Mutation 
PLoS Medicine  2007;4(6):e230.
Background
RPE65 is an essential molecule in the retinoid-visual cycle, and RPE65 gene mutations cause the congenital human blindness known as Leber congenital amaurosis (LCA). Somatic gene therapy delivered to the retina of blind dogs with an RPE65 mutation dramatically restores retinal physiology and has sparked international interest in human treatment trials for this incurable disease. An unanswered question is how the visual cortex responds after prolonged sensory deprivation from retinal dysfunction. We therefore studied the cortex of RPE65-mutant dogs before and after retinal gene therapy. Then, we inquired whether there is visual pathway integrity and responsivity in adult humans with LCA due to RPE65 mutations (RPE65-LCA).
Methods and Findings
RPE65-mutant dogs were studied with fMRI. Prior to therapy, retinal and subcortical responses to light were markedly diminished, and there were minimal cortical responses within the primary visual areas of the lateral gyrus (activation amplitude mean ± standard deviation [SD] = 0.07% ± 0.06% and volume = 1.3 ± 0.6 cm3). Following therapy, retinal and subcortical response restoration was accompanied by increased amplitude (0.18% ± 0.06%) and volume (8.2 ± 0.8 cm3) of activation within the lateral gyrus (p < 0.005 for both). Cortical recovery occurred rapidly (within a month of treatment) and was persistent (as long as 2.5 y after treatment). Recovery was present even when treatment was provided as late as 1–4 y of age. Human RPE65-LCA patients (ages 18–23 y) were studied with structural magnetic resonance imaging. Optic nerve diameter (3.2 ± 0.5 mm) was within the normal range (3.2 ± 0.3 mm), and occipital cortical white matter density as judged by voxel-based morphometry was slightly but significantly altered (1.3 SD below control average, p = 0.005). Functional magnetic resonance imaging in human RPE65-LCA patients revealed cortical responses with a markedly diminished activation volume (8.8 ± 1.2 cm3) compared to controls (29.7 ± 8.3 cm3, p < 0.001) when stimulated with lower intensity light. Unexpectedly, cortical response volume (41.2 ± 11.1 cm3) was comparable to normal (48.8 ± 3.1 cm3, p = 0.2) with higher intensity light stimulation.
Conclusions
Visual cortical responses dramatically improve after retinal gene therapy in the canine model of RPE65-LCA. Human RPE65-LCA patients have preserved visual pathway anatomy and detectable cortical activation despite limited visual experience. Taken together, the results support the potential for human visual benefit from retinal therapies currently being aimed at restoring vision to the congenitally blind with genetic retinal disease.
The study by Samuel Jacobson and colleagues suggests that retinal gene therapy can improve retinal, visual pathway, and visual cortex responses to light stimulation, even after prolonged periods of blindness and in congenitally blind patients.
Editors' Summary
Background.
The eye captures light but the brain is where vision is experienced. Treatments for childhood blindness at the eye level are ready, but it is unknown whether the brain will be receptive to an improved neural message. Normal vision begins as photoreceptor cells in the retina (the light-sensitive tissue lining the inside of the eye) convert visual images into electrical impulses. These impulses are sent along the optic nerve to the visual cortex, the brain region where they are interpreted. The conversion of light into electrical impulses requires the activation of a molecule called retinal, which is subsequently recycled by retinal pigment epithelium (RPE) cells neighboring the retina. One of the key enzymes of the recycling reactions is encoded by a gene called RPE65. Genetic changes (mutations) in RPE65 cause an inherited form of blindness called Leber congenital amaurosis (LCA). In this disease, retinal is not recycled and as a result, the photoreceptor cells cannot work properly and affected individuals have poor or nonexistent vision from birth. Previous studies in dog and mouse models of the human disease have demonstrated that the introduction of a functional copy of RPE65 into the RPE cells using a harmless virus (gene therapy) dramatically restores retinal activity. Very recently, a pioneering gene therapy operation took place in London (UK) where surgeons injected a functional copy of RPE65 into the retina of a man with LCA. Whether this operation results in improved vision is not known at this time.
Why Was This Study Done?
Gene therapy corrects the retinal defects in animal models of LCA but whether the visual pathway from the retina to the visual cortex of the brain can respond normally to the signals sent by the restored retina is not known. Early visual experience is thought to be necessary for the development of a functional visual cortex, so replacing the defective RPE65 gene might not improve the vision of people with LCA. In this study, the researchers have studied the visual cortex of RPE65-deficient dogs before and after gene therapy to see whether the therapy affects the activity of the visual cortex. They have also investigated visual pathway integrity and responsiveness in adults with LCA caused by RPE65 mutations. If the visual pathway is disrupted in these patients, they reasoned, gene therapy might not restore their vision.
What Did the Researchers Do and Find?
The researchers used a technique called functional magnetic resonance imaging (fMRI) to measure light-induced brain activity in RPE65-deficient dogs before and after gene therapy. They also examined the reactions of the dogs' pupils to light (in LCA, the pupils do not contract normally in response to light because there is reduced signal transmission along the visual pathway). Finally, they measured the electrical activity of the dogs' retinas in response to light flashes—the retinas of patients with LCA do not react to light. Gene therapy corrected the defective retinal and visual pathway responses to light in the RPE65-deficient dogs and, whereas before treatment there was no response in the visual cortex to light stimulation in these dogs, after treatment, its activity approached that seen in normal dogs. The recovery of cortical responses was permanent and occurred soon after treatment, even in animals that were 4 years old when treated. Next, using structural MRI, the researchers studied human patients with LCA and found that the optic nerve diameter in young adults was within the normal range and that the structure of the visual cortex was very similar to that of normal individuals. Finally, using fMRI, they found that, although the visual cortex of patients with LCA did not respond to dim light, its reaction to bright light was comparable to that of normal individuals.
What Do These Findings Mean?
The findings from the dog study indicate that retinal gene therapy rapidly improves retinal, visual pathway, and visual cortex responses to light stimulation, even in animals that have been blind for years. In other words, in the dog model of LCA at least, all the components of the visual system remain receptive to visual inputs even after long periods of visual deprivation. The findings from the human study also indicate that the visual pathway remains anatomically intact despite years of disuse and that the visual cortex can be activated in patients with LCA even though these people have very limited visual experience. Taken together, these findings suggest that successful gene therapy of the retina might restore some functional vision to people with LCA but proof will have to await the outcomes of several clinical trials ongoing or being planned in Europe and the USA.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040230.
General information on gene therapy is available from the Oak Ridge National Laboratory
Information is provided by the BBC about gene therapy for Leber congenital amaurosis (includes an audio clip from a doctor about the operation)
The National Institutes of Health/National Eye Institute (US) provides information about an ongoing gene therapy trial of RPE65-Leber congenital amaurosis
ClinicalTrials.gov gives details on treatment trials for Leber congenital amaurosis
The Foundation Fighting Blindness has a fact sheet on Leber congenital amaurosis (site includes Microsoft Webspeak links that read some content aloud)
The Foundation for Retinal Research has a fact sheet on Leber congenital amaurosis
Find more detailed information on Leber congenital amaurosis and the gene mutations that cause it from GeneReviews
WonderBaby, information for parents of babies with Leber congenital amaurosis
doi:10.1371/journal.pmed.0040230
PMCID: PMC1896221  PMID: 17594175
2.  Lentiviral Gene Transfer of Rpe65 Rescues Survival and Function of Cones in a Mouse Model of Leber Congenital Amaurosis 
PLoS Medicine  2006;3(10):e347.
Background
RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice.
Methods and Findings
Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease.
Conclusions
By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65−/− mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose.
In theRpe65-/- mouse model of Leber congenital amaurosis, injection of a lentiviral vector expressing the Rpe65 mouse cDNA was able to prevent cone degeneration and restore cone function.
Editors' Summary
Background.
Leber congenital amaurosis (LCA) is the name of a group of hereditary diseases that cause blindness in infants and children. Changes in any one of a number of different genes can cause the blindness, which affects vision starting at birth or soon after. The condition was first described by a German doctor, Theodore Leber, in the 19th century, hence the first part of the name; “amaurosis” is another word for blindness. Mutations in one gene called retinal pigment epithelium-specific protein, 65 kDa (RPE65)—so called because it is expressed in the pigment epithelium, a cell layer adjacent to the light-sensitive cells, and is 65 kilodaltons in size—cause about 10% of cases of LCA. The product of this gene is essential for the recycling of a substance called 11-cis-retinal, which is necessary for the light-sensitive rods and cones of the retina to capture light. If the gene is abnormal, the sensitivity of the retina to light is drastically reduced, but it also leads to damage to the light-sensitive cells themselves.
Why Was This Study Done?
Potentially, eyes diseases such as this one could be treated by gene therapy, which works by replacing a defective gene with a normal functional one, usually by putting a copy of the normal gene into a harmless virus and injecting it into the affected tissue—in this case, the eye. The researchers here wanted to see whether expressing wild-type RPE65 using a particular type of gene vector that can carry large pieces of DNA transcript—a lentiviral vector—could prevent degeneration of cone cells and restore cone function in a mouse model of this type of LCA—mice who had had this Rpe65 gene genetically removed.
What Did the Researchers Do and Find?
Injection of the normal gene into the retina of Rpe65-deficient mice led to sustained expression of the protein RPE65 in the retinal pigment epithelium. Electrical recordings of the activity of the eyes in these mice showed that Rpe65 gene transfer restored retinal function to a near-normal level. In addition, Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mice.
What Do These Findings Mean?
These findings suggest that it is theoretically possible to treat this type of blindness by gene therapy. However, because this study was done in mice, many other steps need to be taken before it will be clear whether the treatment could work in humans. These steps include a demonstration that the virus is safe in humans, and experiments to determine what dose of virus would be needed and how long the effects of the treatment would last. Another question is whether it would be necessary (or even possible) to treat affected children during early childhood or when children start losing vision.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030347.
The Foundation for Retinal Research has detailed information on Leber's congenital amaurosis
Contact a Family is a UK organization that aims to put families of children with illnesses in touch with each other
The Foundation for Fighting Blindness funds research into, and provides information about many types of blindness, including Leber's congenital amaurosis
This Web site provides information on gene therapy clinical trials, including those dedicated to cure eye diseases
This foundation provides information on diseases leading to blindness, including Leber's congenital amaurosis
doi:10.1371/journal.pmed.0030347
PMCID: PMC1592340  PMID: 17032058
3.  A Regulatory Loop Involving PAX6, MITF, and WNT Signaling Controls Retinal Pigment Epithelium Development 
PLoS Genetics  2012;8(7):e1002757.
The separation of the optic neuroepithelium into future retina and retinal pigment epithelium (RPE) is a critical event in early eye development in vertebrates. Here we show in mice that the transcription factor PAX6, well-known for its retina-promoting activity, also plays a crucial role in early pigment epithelium development. This role is seen, however, only in a background genetically sensitized by mutations in the pigment cell transcription factor MITF. In fact, a reduction in Pax6 gene dose exacerbates the RPE-to-retina transdifferentiation seen in embryos homozygous for an Mitf null allele, and it induces such a transdifferentiation in embryos that are either heterozygous for the Mitf null allele or homozygous for an RPE–specific hypomorphic Mitf allele generated by targeted mutation. Conversely, an increase in Pax6 gene dose interferes with transdifferentiation even in homozygous Mitf null embryos. Gene expression analyses show that, together with MITF or its paralog TFEC, PAX6 suppresses the expression of Fgf15 and Dkk3. Explant culture experiments indicate that a combination of FGF and DKK3 promote retina formation by inhibiting canonical WNT signaling and stimulating the expression of retinogenic genes, including Six6 and Vsx2. Our results demonstrate that in conjunction with Mitf/Tfec Pax6 acts as an anti-retinogenic factor, whereas in conjunction with retinogenic genes it acts as a pro-retinogenic factor. The results suggest that careful manipulation of the Pax6 regulatory circuit may facilitate the generation of retinal and pigment epithelium cells from embryonic or induced pluripotent stem cells.
Author Summary
The retinal pigment epithelium or RPE in the back of the eye is critical for the normal function of the retina, and its abnormalities can lead to retinal disorders such as adult-onset macular degeneration. Insights into the pathogenesis of such disorders, and potential therapies, may come from using RPE cells generated in vitro from induced pluripotent stem cells. To obtain authentic RPE cells in vitro, we need to thoroughly understand the normal process of their development in vivo. Here we find that the potent retina-inducing transcription factor PAX6 plays a critical anti-retinogenic role in the RPE of mice. But how can PAX6 be pro-retinogenic in the retina and anti-retinogenic in the RPE? To address this question, we used gene expression studies and combined them with chromatin immunoprecipitation assays, which analyze the interaction of transcription factors with chromatin in vivo. Our findings show that, in the RPE, PAX6 cooperates with either one (or both) of two related RPE transcription factors, MITF and TFEC, to suppress extracellular signals that in the normal retina induce a signaling cascade promoting retina formation. Hence, this study provides mechanistic insights into RPE development that may become important for the efficient generation of retina and RPE from induced pluripotent stem cells.
doi:10.1371/journal.pgen.1002757
PMCID: PMC3390378  PMID: 22792072
4.  Engineering a Blood-Retinal Barrier With Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium: Transcriptome and Functional Analysis 
To develop a culture model for drug development and tissue-engineering human retina, retinal pigment epithelia (RPE) were derived from human embryonic stem cells (hESCs), and their barrier properties were compared with those of a well-regarded model of RPE function, human fetal RPE isolated from 16-week-gestation fetuses (hfRPE). It was found that hESC-derived RPE is highly differentiated but may be less mature than RPE isolated from 16-week fetuses. The study also identified a panel of genes to monitor further maturation of RPE.
Retinal degenerations are a major cause of impaired vision in the elderly. Degenerations originate in either photoreceptors or the retinal pigment epithelium (RPE). RPE forms the outer blood-retinal barrier and functions intimately with photoreceptors. Animal models and cultures of RPE are commonly used to screen potential pharmaceuticals or explore RPE replacement therapy, but human RPE differs from that of other species. Human RPE forms a barrier using tight junctions composed of a unique set of claudins, proteins that determine the permeability and selectivity of tight junctions. Human adult RPE fails to replicate these properties in vitro. To develop a culture model for drug development and tissue-engineering human retina, RPE were derived from human embryonic stem cells (hESCs). Barrier properties of RPE derived from the H1 and H9 hESC lines were compared with a well-regarded model of RPE function, human fetal RPE isolated from 16-week-gestation fetuses (hfRPE). A serum-free medium (SFM-1) that enhanced the redifferentiation of hfRPE in culture also furthered the maturation of hESC-derived RPE. In SFM-1, the composition, selectivity, and permeability of tight junctions were similar to those of hfRPE. Comparison of the transcriptomes by RNA sequencing and quantitative reverse transcription-polymerase chain reaction revealed a high correlation between the hESCs and hfRPE, but there were notable differences in the expression of adhesion junction and membrane transport genes. These data indicated that hESC-derived RPE is highly differentiated but may be less mature than RPE isolated from 16-week fetuses. The study identified a panel of genes to monitor the maturation of RPE.
doi:10.5966/sctm.2012-0134
PMCID: PMC3697821  PMID: 23734062
Retinal pigmented epithelium; Blood-retinal barrier; Claudins; Embryonic stem cells
5.  Defining the Human Macula Transcriptome and Candidate Retinal Disease Genes UsingEyeSAGE 
Purpose
To develop large-scale, high-throughput annotation of the human macula transcriptome and to identify and prioritize candidate genes for inherited retinal dystrophies, based on ocular-expression profiles using serial analysis of gene expression (SAGE).
Methods
Two human retina and two retinal pigment epithelium (RPE)/choroid SAGE libraries made from matched macula or midperipheral retina and adjacent RPE/choroid of morphologically normal 28- to 66-year-old donors and a human central retina longSAGE library made from 41- to 66-year-old donors were generated. Their transcription profiles were entered into a relational database, EyeSAGE, including microarray expression profiles of retina and publicly available normal human tissue SAGE libraries. EyeSAGE was used to identify retina- and RPE-specific and -associated genes, and candidate genes for retina and RPE disease loci. Differential and/or cell-type specific expression was validated by quantitative and single-cell RT-PCR.
Results
Cone photoreceptor-associated gene expression was elevated in the macula transcription profiles. Analysis of the longSAGE retina tags enhanced tag-to-gene mapping and revealed alternatively spliced genes. Analysis of candidate gene expression tables for the identified Bardet-Biedl syndrome disease gene (BBS5) in the BBS5 disease region table yielded BBS5 as the top candidate. Compelling candidates for inherited retina diseases were identified.
Conclusions
The EyeSAGE database, combining three different gene-profiling platforms including the authors’ multidonor-derived retina/RPE SAGE libraries and existing single-donor retina/RPE libraries, is a powerful resource for definition of the retina and RPE transcriptomes. It can be used to identify retina-specific genes, including alternatively spliced transcripts and to prioritize candidate genes within mapped retinal disease regions.
doi:10.1167/iovs.05-1437
PMCID: PMC2813776  PMID: 16723438
6.  Image Defocus and Altered Retinal Gene Expression in Chick: Clues to the Pathogenesis of Ametropia 
Based on transcriptome responses of chick retina/RPE to unilateral spectacle lens wear, plus or minus lenses induce markedly different retinal responses, with initial responses quite different from those when growth patterns are well established. The lists of altered genes identify promising signaling candidates and regulatory pathways for future study, including the hypothesis that retinal circadian and clock genes may participate in the mechanisms governing refractive development.
Purpose.
Because of the retina's role in refractive development, this study was conducted to analyze the retinal transcriptome in chicks wearing a spectacle lens, a well-established means of inducing refractive errors, to identify gene expression alterations and to develop novel mechanistic hypotheses about refractive development.
Methods.
One-week-old white Leghorn chicks wore a unilateral spectacle lens of +15 or −15 D for 6 hours or 3 days. With total RNA from the retina/(retinal pigment epithelium, RPE), chicken gene microarrays were used to compare gene expression levels between lens-wearing and contralateral control eyes (n = 6 chicks for each condition). Normalized microarray signal intensities were evaluated by analysis of variance, using a false discovery rate of <10% as the statistical criterion. Selected differentially expressed genes were validated by qPCR.
Results.
Very few retina/RPE transcripts were differentially expressed after plus lens wear. In contrast, approximately 1300 transcripts were differentially expressed under each of the minus lens conditions, with minimal overlap. For each condition, low fold-changes typified the altered transcriptome. Differentially regulated genes under the minus lens conditions included many potentially informative signaling molecules and genes whose protein products have roles in intrinsic retinal circadian rhythms.
Conclusions.
Plus or minus lens wear induce markedly different, not opposite, alterations in retina/RPE gene expression. The initial retinal responses to defocus are quite different from those when the eye growth patterns are well established, suggesting that different mechanisms govern the initiation and persistence or progression of refractive errors. The gene lists identify promising signaling candidates and regulatory pathways for future study, including a potential role for circadian rhythms in refractive development.
doi:10.1167/iovs.10-6727
PMCID: PMC3176046  PMID: 21642623
7.  Complement gene expression and regulation in mouse retina and retinal pigment epithelium/choroid 
Molecular Vision  2011;17:1588-1597.
Purpose
To understand the expression of genes involved in different complement pathways in the retina and retinal pigment epithelium (RPE)/choroid under physiologic conditions and how their expression is regulated by inflammatory cytokines.
Methods
The expression of complement components of the classical pathway (CP), mannose-binding lectin (MBL) pathway, alternative pathway (AP), and terminal pathway in the retina and RPE/choroid was determined by conventional reverse transcription polymerase chain reaction (RT–PCR). The effect of inflammatory cytokines, tumor necrosis factor-alpha (TNF-α, 20 ng/ml), interleukin (IL)-6 (10 ng/ml), interferon-gamma (IFN-γ, 100 ng/ml) or lipopolysaccharides (LPS, 1 μg/ml) on the expression of these complement component genes was tested in vitro in primary cultured RPE cells and a microglial cell line (BV2 cells) and quantified by real-time RT–PCR.
Results
In the CP, complements C1qb, C1r, C1s, C2, and C4 were constitutively expressed by retina and RPE/choroid. Complement factor H and factor B of the AP as well as C3 were also detected in the retinal and RPE/choroidal tissues. In the MBL pathway, low levels of mannose-binding lectin (MBL)-associated serine protease (MASP)-1 in the retina and RPE/choroid and MASP2L in the retina were detected. Other components, including mannose-binding lectin 1 (MBL1), mannose-binding lectin 2 (MBL2), complement factor I (CFI), complement component 5 (C5) and complement factor H-related protein 1 (CFHR1), were not detected in either the retina or the RPE/choroid. The expression of CP- and AP-complement component genes in RPE and microglial cells was upregulated by interferon (IFN)-γ treatment. Treatment with TNF-α selectively upregulated the expression of C1s and C3 genes but downregulated complement factor H gene expression in RPE and microglial cells. The expression of genes involved in the MBL pathway was not affected by the inflammatory cytokines tested in this study.
Conclusions
Retina and RPE/choroid express a variety of complement components that are involved mainly in the CP and AP. RPE and microglial cells are the main sources of retinal complement gene expression. Retinal complement gene expression is regulated by inflammatory cytokines, such as IFN-γ and TNF-α.
PMCID: PMC3123163  PMID: 21738388
8.  Otx2 ChIP-seq Reveals Unique and Redundant Functions in the Mature Mouse Retina 
PLoS ONE  2014;9(2):e89110.
During mouse retinal development and into adulthood, the transcription factor Otx2 is expressed in pigment epithelium, photoreceptors and bipolar cells. In the mature retina, Otx2 ablation causes photoreceptor degeneration through a non-cell-autonomous mechanism involving Otx2 function in the supporting RPE. Surprisingly, photoreceptor survival does not require Otx2 expression in the neural retina, where the related Crx homeobox gene, a major regulator of photoreceptor development, is also expressed. To get a deeper view of mouse Otx2 activities in the neural retina, we performed chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-seq) on Otx2. Using two independent ChIP-seq assays, we identified consistent sets of Otx2-bound cis-regulatory elements. Comparison with our previous RPE-specific Otx2 ChIP-seq data shows that Otx2 occupies different functional domains of the genome in RPE cells and in neural retina cells and regulates mostly different sets of genes. To assess the potential redundancy of Otx2 and Crx, we compared our data with Crx ChIP-seq data. While Crx genome occupancy markedly differs from Otx2 genome occupancy in the RPE, it largely overlaps that of Otx2 in the neural retina. Thus, in accordance with its essential role in the RPE and its non-essential role in the neural retina, Otx2 regulates different gene sets in the RPE and the neural retina, and shares an important part of its repertoire with Crx in the neural retina. Overall, this study provides a better understanding of gene-regulatory networks controlling photoreceptor homeostasis and disease.
doi:10.1371/journal.pone.0089110
PMCID: PMC3928427  PMID: 24558479
9.  Microarray Analysis of Murine Retinal Light Damage Reveals Changes in Iron Regulatory, Complement, and Antioxidant Genes in the Neurosensory Retina and Isolated RPE 
Purpose.
The purpose of this study was to investigate light damage–induced transcript changes within neurosensory retina (NSR) and isolated retinal pigment epithelium (RPE). Similar studies have been conducted previously, but were usually limited to the NSR and only a portion of the transcriptome. Herein most of the transcriptome, not just in the NSR but also in isolated RPE, was queried.
Methods.
Mice were exposed to 10,000 lux cool white fluorescent light for 18 hours and euthanized 4 hours after photic injury. NSR and isolated RPE were collected, and RNA was isolated. DNA microarray hybridization was conducted as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Microarray analysis was performed using probe intensity data derived from the Mouse Gene 1.0 ST Array. For the genes of interest, confirmation of gene expression was done using quantitative real-time PCR. Immunofluorescence assessed protein levels and localization.
Results.
Numerous iron regulatory genes were significantly changed in the light-exposed NSR and RPE. Several of these gene expression changes favored an iron-overloaded state. For example, the transferrin receptor was upregulated in both light-exposed NSR and RPE. Consistent with this, there was stronger transferrin receptor immunoreactivity in the light-exposed retinas. Significant changes in gene expression following light damage were also observed in oxidative stress and complement system genes.
Conclusions.
The concept of a photooxidative stress–induced vicious cycle of increased iron uptake leading to further oxidative stress was introduced.
We investigated the response of the mouse retina to light damage. Our data suggest that following photic injury the neural retinal and retinal pigment epithelium alter expression of iron regulatory genes favoring cellular iron accumulation. Increased labile iron may cause additional oxidative stress promoting cell death.
doi:10.1167/iovs.12-10204
PMCID: PMC4159963  PMID: 22736611
10.  L-DOPA Is an Endogenous Ligand for OA1 
PLoS Biology  2008;6(9):e236.
Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of β-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.
Author Summary
Albinism is the loss of pigmentation caused by mutations in one of several different genes that alter pigment synthesis by different mechanisms. In the eye, albinism impairs sensory retina development and causes significant vision problems. Regardless of the genetic mutation that causes albinism, the associated vision problems are the same. Interestingly, none of the pigmentation genes are expressed by the sensory retinal cells affected by albinism but by neighboring, retinal pigment epithelial cells (RPE). Furthermore, loss of pigmentation in RPE somehow leads to imprecise retinal development. To investigate this cellular relationship, we studied OA1, which is encoded by a gene in which mutations cause ocular albinism. OA1 is unique among proteins involved with albinism because OA1 is a potential receptor that could participate in signal transduction rather than being a direct member of the pigment synthesis machinery. We show that the ligand for OA1 is L-DOPA, thus removing OA1 from orphan G-protein coupled receptor (GPCR) status. L-DOPA is a by-product of pigment synthesis, indicating that pigment synthesis and OA1 signaling are intertwined. OA1 signaling is highly selective for L-DOPA, and we show that two closely related molecules, dopamine and tyrosine, bind to OA1 but fail to stimulate signaling. We also show that OA1 signaling controls secretion of a potent neuron survival factor. Taken together, our data suggest that all forms of albinism produce the same retinal defects because of a final common pathway through OA1 signaling with downstream effects on RPE neurotrophic factor secretion.
Albinism produces retinal defects, and OA1 is an orphan G-protein-coupled receptor that leads to albinism without acting directly on melanin synthesis. Here the ligand is identified and a mechanism is proposed by which the various forms of albinism signal through OA1, resulting in the same retinal phenotype.
doi:10.1371/journal.pbio.0060236
PMCID: PMC2553842  PMID: 18828673
11.  Induction of Ectopic Retina-Like Tissue by Transgenic Expression of neurogenin 
PLoS ONE  2015;10(1):e0116171.
Degeneration of retinal neurons is an underlying cause of several major types of blinding diseases, and effective therapies remain to be developed. The suppositive strategy of repopulating a degenerative retina with new cells generated onsite faces serious challenges, because the mammalian retina seems to lack the ability to regenerate itself or replace its lost neurons. We investigated the possibility of using a transcriptional factor with proneural activities to reprogram ocular tissue with regenerative capability to give rise to retinal cells. Transgenic mice were generated with DNA constructs that targeted the expression in the retinal pigment epithelium of proneural gene neurogenin1 from the promoter of Bestrophin1, or neurogenin3 from RPE65 promoter. Here we report the presence of ectopic retina-like tissue in some of the transgenic mice, young and aged. The ectopic retina-like tissue contained cells positive for photoreceptor proteins Crx, recoverin, red opsin, and rhodopsin, and cells positive for proteins that label other types of retinal neurons, including AP2α and Pax6 for amacrine cells, Otx2 for bipolar cells, and Brn3A for ganglion cells. The retina-like tissue often co-existed with darkly pigmented tissue positive for RPE proteins: cytokeratin 18, Otx2, and RPE65. The ectopic retina-like tissue was detected in the subretinal space, including two retinae co-existing in the same eye, and/or in the optic nerve or in the vicinity of the optic nerve head. On rare occasions, it was detected in the choroid and in the vicinity of the ciliary body. The presence of ectopic retina-like tissue in the transgenic mouse supports the possibility of inducing retinal regeneration in the mammalian eyes through gene-directed reprograming.
doi:10.1371/journal.pone.0116171
PMCID: PMC4312083  PMID: 25635399
12.  Expression profiling of the RPE in zebrafish smarca4 mutant revealed altered signals that potentially affect RPE and retinal differentiation 
Molecular Vision  2014;20:56-72.
Purpose
The purpose of this study was to develop a framework for analyzing retinal pigment epithelium (RPE) expression profiles from zebrafish eye mutants.
Methods
The fish model we used was SWI/SNF-related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (smarca4), a retinal dystrophic mutant with a previously described retinal phenotype and expression profiles. Histological and Affymetrix GeneChip analyses were conducted to characterize the RPE defects and underlying differential expression, respectively.
Results
Histological analysis revealed that smarca4 RPE was formed, but its differentiation was abnormal. In particular, ultrastructural analysis of smarca4 RPE by transmission electron microscopy demonstrated several defects in melanogenesis. The nature of these defects also suggests that the cytoskeletal dynamics, which are tightly linked with melanogenesis, were impaired in smarca4 RPE. To compare the expression profile of normal wild-type (WT) and smarca4 RPE, the gene expression profiles of microdissected retinas and RPE-attached retinas were measured with Affymetrix GeneChip analysis. The RPE expression values were then estimated from these samples by subtracting the retinal expression values from the expression values of the RPE-attached retinas. A factorial analysis was conducted using the expression values of the RPE, retinal, and whole-embryo samples. Specific rules (contrasts) were built using the coefficients of the resulting fitted models to select for three groups of genes: 1) smarca4-regulated RPE genes, 2) smarca4-regulated retinal genes, and 3) smarca4-regulated RPE genes that are not differentially expressed in the retina. Interestingly, the third group consists of 39 genes that are highly related to cytoskeletal dynamics, melanogenesis, and paracrine and intracellular signal transduction.
Conclusions
Our analytical framework provides an experimental approach to identify differentially-regulated genes in the retina and the RPE of zebrafish mutants in which both of these tissues are affected by the underlying mutation. Specifically, we have used the method to identify a group of 39 genes that can potentially explain the melanogenesis defect in the smarca4 RPE. In addition, several genes in this group are secreted signaling molecules. Thus, this observation further implicates that the smarca4 RPE might play a role in the retinal dystrophic phenotype in smarca4.
PMCID: PMC3888495  PMID: 24426776
13.  A New Strategy to Identify and Annotate Human RPE-Specific Gene Expression 
PLoS ONE  2010;5(5):e9341.
Background
To identify and functionally annotate cell type-specific gene expression in the human retinal pigment epithelium (RPE), a key tissue involved in age-related macular degeneration and retinitis pigmentosa.
Methodology
RPE, photoreceptor and choroidal cells were isolated from selected freshly frozen healthy human donor eyes using laser microdissection. RNA isolation, amplification and hybridization to 44 k microarrays was carried out according to Agilent specifications. Bioinformatics was carried out using Rosetta Resolver, David and Ingenuity software.
Principal Findings
Our previous 22 k analysis of the RPE transcriptome showed that the RPE has high levels of protein synthesis, strong energy demands, is exposed to high levels of oxidative stress and a variable degree of inflammation. We currently use a complementary new strategy aimed at the identification and functional annotation of RPE-specific expressed transcripts. This strategy takes advantage of the multilayered cellular structure of the retina and overcomes a number of limitations of previous studies. In triplicate, we compared the transcriptomes of RPE, photoreceptor and choroidal cells and we deduced RPE specific expression. We identified at least 114 entries with RPE-specific gene expression. Thirty-nine of these 114 genes also show high expression in the RPE, comparison with the literature showed that 85% of these 39 were previously identified to be expressed in the RPE. In the group of 114 RPE specific genes there was an overrepresentation of genes involved in (membrane) transport, vision and ophthalmic disease. More fundamentally, we found RPE-specific involvement in the RAR-activation, retinol metabolism and GABA receptor signaling pathways.
Conclusions
In this study we provide a further specification and understanding of the RPE transcriptome by identifying and analyzing genes that are specifically expressed in the RPE.
doi:10.1371/journal.pone.0009341
PMCID: PMC2866542  PMID: 20479888
14.  Systems-level analysis of age-related macular degeneration reveals global biomarkers and phenotype-specific functional networks 
Genome Medicine  2012;4(2):16.
Background
Age-related macular degeneration (AMD) is a leading cause of blindness that affects the central region of the retinal pigmented epithelium (RPE), choroid, and neural retina. Initially characterized by an accumulation of sub-RPE deposits, AMD leads to progressive retinal degeneration, and in advanced cases, irreversible vision loss. Although genetic analysis, animal models, and cell culture systems have yielded important insights into AMD, the molecular pathways underlying AMD's onset and progression remain poorly delineated. We sought to better understand the molecular underpinnings of this devastating disease by performing the first comparative transcriptome analysis of AMD and normal human donor eyes.
Methods
RPE-choroid and retina tissue samples were obtained from a common cohort of 31 normal, 26 AMD, and 11 potential pre-AMD human donor eyes. Transcriptome profiles were generated for macular and extramacular regions, and statistical and bioinformatic methods were employed to identify disease-associated gene signatures and functionally enriched protein association networks. Selected genes of high significance were validated using an independent donor cohort.
Results
We identified over 50 annotated genes enriched in cell-mediated immune responses that are globally over-expressed in RPE-choroid AMD phenotypes. Using a machine learning model and a second donor cohort, we show that the top 20 global genes are predictive of AMD clinical diagnosis. We also discovered functionally enriched gene sets in the RPE-choroid that delineate the advanced AMD phenotypes, neovascular AMD and geographic atrophy. Moreover, we identified a graded increase of transcript levels in the retina related to wound response, complement cascade, and neurogenesis that strongly correlates with decreased levels of phototransduction transcripts and increased AMD severity. Based on our findings, we assembled protein-protein interactomes that highlight functional networks likely to be involved in AMD pathogenesis.
Conclusions
We discovered new global biomarkers and gene expression signatures of AMD. These results are consistent with a model whereby cell-based inflammatory responses represent a central feature of AMD etiology, and depending on genetics, environment, or stochastic factors, may give rise to the advanced AMD phenotypes characterized by angiogenesis and/or cell death. Genes regulating these immunological activities, along with numerous other genes identified here, represent promising new targets for AMD-directed therapeutics and diagnostics.
Please see related commentary: http://www.biomedcentral.com/1741-7015/10/21/abstract
doi:10.1186/gm315
PMCID: PMC3372225  PMID: 22364233
15.  Diffusible retinal secretions regulate the expression of tight junctions and other diverse functions of the retinal pigment epithelium 
Molecular Vision  2008;14:2237-2262.
Purpose
The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier. It is unclear how culture conditions might alter barrier properties of isolated RPE. We examined whether retinal secretions that increase the barrier functions of tight junctions in vitro also make gene expression in general more in vivo-like.
Methods
Chick RPE from embryonic day 7 (E7) and E14 were cultured on filters. Media conditioned by organ culture of E14 neural retinas was added to the apical medium chamber. RNA was isolated to probe the chick genome on Affymetrix microarrays, and expression was compared to native E14 RPE. Expression was further analyzed by quantitative real-time PCR immunoblotting and immunocytochemistry.
Results
More than 86% of the genes expressed in vivo were expressed in basal culture conditions, including RPE-specific markers such as RPE65 and bestrophin. E14 retinal conditioned medium affected 15% of the transcriptome in E7 cultures (24% if serum was included), but only 1.9% in E14 cultures (12% with serum). Examination of 610 genes important for RPE function revealed that mRNAs for 17% were regulated by retinal conditioned medium alone in E7 cultures, compared to 6.2% for E14. For tight junctions, retinal conditioned medium had the most effect on members of the claudin family. Besides regulating mRNA levels, immunoblotting and immunocytochemistry suggested additional mechanisms whereby retinal secretions regulated protein expression and localization.
Conclusions
Gene expression in primary cultures of embryonic RPE resembled the native tissue, but differentiation and the levels of gene expression became more in vivo-like when elements of the retinal environment were introduced into the medium bathing the apical side of the cultures. Albeit insufficient, retinal secretions promoted differentiation of immature RPE and helped maintain the properties of more mature RPE.
PMCID: PMC2593753  PMID: 19057659
16.  Gene Transcription Profile of the Detached Retina (An AOS Thesis) 
Purpose:
Separation of the neurosensory retina from the retinal pigment epithelium (RPE) yields many morphologic and functional consequences, including death of the photoreceptor cells, Müller cell hypertrophy, and inner retinal rewiring. Many of these changes are due to the separation-induced activation of specific genes. In this work, we define the gene transcription profile within the retina as a function of time after detachment. We also define the early activation of kinases that might be responsible for the detachment-induced changes in gene transcription.
Methods:
Separation of the retina from the RPE was induced in Brown-Norway rats by the injection of 1% hyaluronic acid into the subretinal space. Retinas were harvested at 1, 7, and 28 days after separation. Gene transcription profiles for each time point were determined using the Affymetrix Rat 230A gene microarray chip. Transcription levels in detached retinas were compared to those of nondetached retinas with the BRB-ArrayTools Version 3.6.0 using a random variance analysis of variance (ANOVA) model. Confirmation of the significant transcriptional changes for a subset of the genes was performed using microfluidic quantitative real-time polymerase chain reaction (qRT-PCR) assays. Kinase activation was explored using Western blot analysis to look for early phosphorylation of any of the 3 main families of mitogen-activated protein kinases (MAPK): the p38 family, the Janus kinase family, and the p42/p44 family.
Results:
Retinas separated from the RPE showed extensive alterations in their gene transcription profile. Many of these changes were initiated as early as 1 day after separation, with significant increases by 7 days. ANOVA analysis defined 144 genes that had significantly altered transcription levels as a function of time after separation when setting a false discovery rate at ≤0.1. Confirmatory RT-PCR was performed on 51 of these 144 genes. Differential transcription detected on the microarray chip was confirmed by qRT-PCR for all 51 genes. Western blot analysis showed that the p42/p44 family of MAPK was phosphorylated within 2 hours of retinal-RPE separation. This phosphorylation was detachment-induced and could be inhibited by specific inhibitors of MAPK phosphorylation.
Conclusions:
Separation of the retina from the RPE induces significant alteration in the gene transcription profile within the retina. These profiles are not static, but change as a function of time after detachment. These gene transcription changes are preceded by the activation of the p42/p44 family of MAPK. This altered transcription may serve as the basis for many of the morphologic, biochemical, and functional changes seen within the detached retina.
PMCID: PMC2814587  PMID: 20126507
17.  The expression of retinal cell markers in human retinal pigment epithelial cells and their augmentation by the synthetic retinoid fenretinide 
Molecular Vision  2011;17:1701-1715.
Purpose
In several species the retinal pigment epithelium (RPE) has the potential to transdifferentiate into retinal cells to regenerate functional retinal tissue after injury. However, this capacity for regeneration is lost in mammals. The synthetic retinoic acid derivative, fenretinide [N(4-hydroxyphenyl) retinamide], induces a neuronal-like phenotype in the human adult retinal pigment epithelial cell line (ARPE-19). These changes are characterized by the appearance of neural-like processes and the expression of neuronal markers not normally associated with RPE cells. Here we assess whether fenretinide can induce a neuroretinal cell phenotype in ARPE-19 cells, by examining retinal cell marker expression.
Methods
ARPE-19 cells were treated daily with culture medium containing either 3 μM fenretinide or dimethyl sulfoxide as a control for 7 days. Cells were processed for immunocytochemistry, western blotting, and for analysis by PCR to examine the expression of a panel of RPE, neural, and retinal-associated cellular markers, including classical and non-canonical opsins.
Results
Treatment with fenretinide for 7 days induced the formation of neuronal-like processes in ARPE-19 cells. Fenretinide induced the expression of the cone long wavelength sensitive opsin (OPN1lw) but not rhodopsin (RHO), while decreasing the expression of RPE cell markers. Many of the neuronal and retinal specific markers examined were expressed in both control and fenretinide treated cells, including those involved in photoreceptor cell development and the multipotency of neural retinal progenitor cells. Interestingly, ARPE-19 cells also expressed both photoreceptor specific and non-specific canonical opsins.
Conclusions
The expression of retinal-associated markers and loss of RPE cell markers in control ARPE-19 cells suggests that these cells might have dedifferentiated from an RPE cell phenotype under standard culture conditions. The expression of molecules, such as the transcription factors paired box 6 gene (PAX6), sex determining region Y-box 2 (SOX2), cone-rod homeobox (CRX), and neural retina leucine zipper (NRL), further implies that in culture these cells are predisposed toward a retinal progenitor-like state. The fenretinide-induced increase in photoreceptor cell markers, accompanied by a decrease in RPE cell markers, suggests that retinoids may play a role in the transdifferentiation of RPE cells. Importantly, our data show for the first time the expression of a vertebrate ciliary opsin (OPN1lw) and rhabdomeric-like opsin, opsin 4 (OPN4 also known as melanopsin) in a clonal cell line. Together these data suggest that ARPE-19 cells are primed for and possess the capacity to differentiate toward a retinal cell-like lineage.
PMCID: PMC3130725  PMID: 21738400
18.  Altered Expression of Retinal Molecular Markers in the Canine RPE65 Model of Leber Congenital Amaurosis 
The effect of the functional deletion of the RPE65 gene on the expression of molecular markers in the mutant dog retina was investigated. Some markers changed in the inner and outer retina, but the retinal structure was well preserved.
Purpose.
Leber congenital amaurosis (LCA) is a group of childhood-onset retinal diseases characterized by severe visual impairment or blindness. One form is caused by mutations in the RPE65 gene, which encodes the retinal pigment epithelium (RPE) isomerase. In this study, the retinal structure and expression of molecular markers for different retinal cell types were characterized, and differences between control and RPE65 mutant dogs during the temporal evolution of the disease were analyzed.
Methods.
Retinas from normal and mutant dogs of different ages were examined by immunofluorescence with a panel of 16 different antibodies.
Results.
Cones and rods were preserved in the mutant retinas, and the number of cones was normal. However, there was altered expression of cone arrestin and delocalization of rod opsin. The ON bipolar cells showed sprouting of the dendritic arbors toward the outer nuclear layer (ONL) and retraction of their axons in the inner nuclear layer (INL). A decreased expression of GABA, and an increased expression of intermediate filament glial markers was also found in the mutant retinas. These changes were more evident in the adult than the young mutant retinas.
Conclusions.
The structure of the retina is well preserved in the mutant retina, but several molecular changes take place in photoreceptors and in bipolar and amacrine cells. Some of these changes are structural, whereas others reflect a change in localization of the examined proteins. This study provides new information that can be applied to the interpretation of outcomes of retinal gene therapy in animal models and humans.
doi:10.1167/iovs.10-5213
PMCID: PMC3055778  PMID: 20671290
19.  The Aged Retinal Pigment Epithelium/Choroid: A Potential Substratum for the Pathogenesis of Age-Related Macular Degeneration 
PLoS ONE  2008;3(6):e2339.
Background
Although the statement that age is the greatest risk factor for Age-related macular degeneration (AMD) is widely accepted, the cellular and molecular explanations for that clinical statement are not generally known. A major focus of AMD research is the retinal pigment epithelium (RPE)/choroid. The purpose of this study was to characterize the changes in the RPE/choroid with age that may provide a background for the development of AMD.
Methodology/Principal Findings
We compared the transcriptional profiles, key protein levels and histology of the RPE/choroid from young and old mice. Using three statistical methods, microarray data demonstrated marked changes in the old mouse. There were 315 genes differentially expressed with age; most of these genes were related to immune responses and inflammatory activity. Canonical pathways having significant numbers of upregulated genes in aged RPE/choroid included leukocyte extravasation, complement cascades, natural killer cell signaling and IL-10 signaling. By contrast, the adjacent neural retina showed completely different age-related changes. The levels of proteins that participate in leukocyte extravasation and complement pathways were consistently increased in the normal, aged RPE/choroid. Furthermore, there was increased gene expression and protein levels of leukocyte attracting signal, chemokine ligand 2 (Ccl2) in aged RPE/choroid. In old animals, there was marked extravasation and accumulation of leukocytes from the choroidal circulation onto Bruch's membrane and into the RPE.
Conclusions/Significance
These phenotypic changes indicate that the RPE/choroid in the normal, old mouse has become an immunologically active tissue. There are signals from the normal, aged RPE/choroid which recruit leukocytes from the circulation and activate the complement cascade. These age-related changes that occur in the RPE/choroid with age, to the extent that they occur in the human retina, may provide the background for an error in regulation of immunological activity to cause AMD to appear in an elderly individual.
doi:10.1371/journal.pone.0002339
PMCID: PMC2394659  PMID: 18523633
20.  Immuno-Histochemical Analysis of Rod and Cone Reaction to RPE65 Deficiency in the Inferior and Superior Canine Retina 
PLoS ONE  2014;9(1):e86304.
Mutations in the RPE65 gene are associated with autosomal recessive early onset severe retinal dystrophy. Morphological and functional studies indicate early and dramatic loss of rod photoreceptors and early loss of S-cone function, while L and M cones remain initially functional. The Swedish Briard dog is a naturally occurring animal model for this disease. Detailed information about rod and cone reaction to RPE65 deficiency in this model with regard to their location within the retina remains limited. The aim of this study was to analyze morphological parameters of cone and rod viability in young adult RPE65 deficient dogs in different parts of the retina in order to shed light on local disparities in this disease. In retinae of affected dogs, sprouting of rod bipolar cell dendrites and horizontal cell processes was dramatically increased in the inferior peripheral part of affected retinae, while central inferior and both superior parts did not display significantly increased sprouting. This observation was correlated with photoreceptor cell layer thickness. Interestingly, while L/M cone opsin expression was uniformly reduced both in the superior and inferior part of the retina, S-cone opsin expression loss was less severe in the inferior part of the retina. In summary, in retinae of young adult RPE65 deficient dogs, the degree of rod bipolar and horizontal cell sprouting as well as of S-cone opsin expression depends on the location. As the human retinal pigment epithelium (RPE) is pigmented similar to the RPE in the inferior part of the canine retina, and the kinetics of photoreceptor degeneration in humans seems to be similar to what has been observed in the inferior peripheral retina in dogs, this area should be studied in future gene therapy experiments in this model.
doi:10.1371/journal.pone.0086304
PMCID: PMC3897682  PMID: 24466015
21.  The genomic response of the retinal pigment epithelium to light damage and retinal detachment 
The retinal pigment epithelium (RPE) plays an essential role in maintaining the health of the retina. The RPE is also the site of pathologic processes in a wide variety of retinal disorders including monogenic retinal dystrophies, age-related macular degeneration, and retinal detachment. Despite intense interest in the RPE, little is known about its molecular response to ocular damage or disease. We have conducted a comprehensive analysis of changes in transcript abundance (the “genomic response”) in the murine RPE following light damage. Several dozen transcripts, many related to cell-cell signaling, show significant increases in abundance in response to bright light; transcripts encoding visual cycle proteins show a decrease in abundance. Similar changes are induced by retinal detachment. Environmental and genetic perturbations that modulate the RPE response to bright light suggest that this response is controlled by the retina. In contrast to the response to bright light, the RPE response to retinal detachment over-rides these modulatory affects.
doi:10.1523/JNEUROSCI.2401-08.2008
PMCID: PMC2570851  PMID: 18815272
retinal pigment epithelium; transcription; micro-array; mouse; ocular disease; light damage; retinal detachment
22.  Hes4 Controls Proliferative Properties of Neural Stem Cells During Retinal Ontogenesis 
Stem Cells (Dayton, Ohio)  2012;30(12):2784-2795.
The retina of fish and amphibian contains genuine neural stem cells located at the most peripheral edge of the ciliary marginal zone (CMZ). However, their cell-of-origin as well as the mechanisms that sustain their maintenance during development are presently unknown. We identified Hes4 (previously named XHairy2), a gene encoding a bHLH-O transcriptional repressor, as a stem cell-specific marker of the Xenopus CMZ that is positively regulated by the canonical Wnt pathway and negatively by Hedgehog signaling. We found that during retinogenesis, Hes4 labels a small territory, located first at the pigmented epithelium (RPE)/neural retina (NR) border and later in the retinal margin, that likely gives rise to adult retinal stem cells. We next addressed whether Hes4 might impart this cell subpopulation with retinal stem cell features: inhibited RPE or NR differentiation programs, continuous proliferation, and slow cell cycle speed. We could indeed show that Hes4 overexpression cell autonomously prevents retinal precursor cells from commitment toward retinal fates and maintains them in a proliferative state. Besides, our data highlight for the first time that Hes4 may also constitute a crucial regulator of cell cycle kinetics. Hes4 gain of function indeed significantly slows down cell division, mainly through the lengthening of G1 phase. As a whole, we propose that Hes4 maintains particular stemness features in a cellular cohort dedicated to constitute the adult retinal stem cell pool, by keeping it in an undifferentiated and slowly proliferative state along embryonic retinogenesis. Stem Cells 2012;30:2784–2795
doi:10.1002/stem.1231
PMCID: PMC3549485  PMID: 22969013
Retina; Neural stem cells; Hes4/XHairy2; Wnt and Hedgehog signaling; Cell cycle kinetics
23.  Ectopic Mitf in the Embryonic Chick Retina by Co-transfection of β-Catenin and Otx2 
The present study shows, by electroporation of chick embryonic eyes, that co-transfection of activated β-catenin and Otx2 can convert retinal progenitor cells into presumptive RPE cells expressing Mitf.
Purpose.
Development of the retinal pigment epithelium (RPE) is controlled by intrinsic and extrinsic regulators including orthodenticle homeobox 2 (Otx2) and the Wnt/β-catenin pathway, respectively. Otx2 and β-catenin are necessary for the expression of the RPE key regulator microphthalmia-associated transcription factor (Mitf); however, neither factor is sufficient to promote Mitf expression in vivo. The study was conducted to determine whether Otx2 and β-catenin act in a combinatorial manner and tested whether co-expression in the presumptive chick retina induces ectopic Mitf expression.
Methods.
The sufficiency of Wnt/β-catenin activation and/or Otx2 expression to induce RPE-specific gene expression was examined in chick optic vesicle explant cultures or in the presumptive neural retina using in ovo-electroporation. Luciferase assays were used to examine the transactivation potentials of Otx2 and β-catenin on the Mitf-D enhancer and autoregulation of the Mitf-D and Otx2T0 enhancers.
Results.
In optic vesicles explant cultures, RPE-specific gene expression was activated by lithium chloride, a Wnt/β-catenin agonist. However, in vivo, Mitf was induced only in the presumptive retina if both β-catenin and Otx2 are co-expressed. Furthermore, both Mitf and Otx2 can autoregulate their own enhancers in vitro.
Conclusions.
The present study provides evidence that β-catenin and Otx2 are sufficient, at least in part, to convert retinal progenitor cells into presumptive RPE cells expressing Mitf. Otx2 may act as a competence factor that allows RPE specification in concert with additional RPE-promoting factors such as β-catenin.
doi:10.1167/iovs.09-5015
PMCID: PMC3066625  PMID: 20463321
24.  An Autonomous Circadian Clock in the Inner Mouse Retina Regulated by Dopamine and GABA 
PLoS Biology  2008;6(10):1-18.
The influence of the mammalian retinal circadian clock on retinal physiology and function is widely recognized, yet the cellular elements and neural regulation of retinal circadian pacemaking remain unclear due to the challenge of long-term culture of adult mammalian retina and the lack of an ideal experimental measure of the retinal circadian clock. In the current study, we developed a protocol for long-term culture of intact mouse retinas, which allows retinal circadian rhythms to be monitored in real time as luminescence rhythms from a PERIOD2::LUCIFERASE (PER2::LUC) clock gene reporter. With this in vitro assay, we studied the characteristics and location within the retina of circadian PER2::LUC rhythms, the influence of major retinal neurotransmitters, and the resetting of the retinal circadian clock by light. Retinal PER2::LUC rhythms were routinely measured from whole-mount retinal explants for 10 d and for up to 30 d. Imaging of vertical retinal slices demonstrated that the rhythmic luminescence signals were concentrated in the inner nuclear layer. Interruption of cell communication via the major neurotransmitter systems of photoreceptors and ganglion cells (melatonin and glutamate) and the inner nuclear layer (dopamine, acetylcholine, GABA, glycine, and glutamate) did not disrupt generation of retinal circadian PER2::LUC rhythms, nor did interruption of intercellular communication through sodium-dependent action potentials or connexin 36 (cx36)-containing gap junctions, indicating that PER2::LUC rhythms generation in the inner nuclear layer is likely cell autonomous. However, dopamine, acting through D1 receptors, and GABA, acting through membrane hyperpolarization and casein kinase, set the phase and amplitude of retinal PER2::LUC rhythms, respectively. Light pulses reset the phase of the in vitro retinal oscillator and dopamine D1 receptor antagonists attenuated these phase shifts. Thus, dopamine and GABA act at the molecular level of PER proteins to play key roles in the organization of the retinal circadian clock.
Author Summary
The circadian clock in the mammalian retina regulates many retinal functions, and its output modulates the central circadian clock in the brain. Details about the cellular location and neural regulation of the mammalian retinal circadian clock remain unclear, however, largely due to the difficulty of maintaining long-term culture of adult mammalian retina and the lack of an ideal experimental measure of the retinal clock. We have circumvented these limitations by developing a protocol for long-term culture of intact mouse retinas to monitor circadian rhythms of clock gene expression in real time. Using this protocol, we have localized expression of molecular retinal circadian rhythms to the inner nuclear layer. We find molecular retinal rhythms generation is independent of many forms of signaling from photoreceptors and ganglion cells, or major forms of neural communication within the inner nuclear layer, and have characterized light-induced resetting of the retinal clock. Retinal dopamine and GABA, although not necessary for the generation of molecular retinal rhythms, were revealed to regulate the phase and amplitude of retinal molecular rhythms, respectively, with dopamine participating in light-induced resetting. Our data indicate that dopamine and GABA play prominent roles in the organization of the retinal circadian clock.
Long-term culture of mouse retinas reveals a circadian clock in the inner retina that can be reset by light and is regulated by the neurotransmitters dopamine and GABA.
doi:10.1371/journal.pbio.0060249
PMCID: PMC2567003  PMID: 18959477
25.  A Degenerative Retinal Process in HIV-Associated Non-Infectious Retinopathy 
PLoS ONE  2013;8(9):e74712.
HIV retinopathy is the most common non-infectious complication in the eyes of HIV-positive individuals. Oncotic lesions in the retinal nerve fiber layer, referred to as cotton wool spots (CWS), and intraretinal (IR) hemorrhages are frequently observed but are not unique to this pathology. HIV-positive patients have impaired color vision and contrast sensitivity, which worsens with age. Evidence of inner–retinal lesions and damage have been documented ophthalmoscopically, however their long term structural effect has not been investigated. It has been hypothesized that they may be partially responsible for loss of visual function and visual field. In this study we utilized clinical data, retinal imaging and transcriptomics approaches to comprehensively interrogate non-infectious HIV retinopathy. The methods employed encompassed clinical examinations, fundus photography, indirect ophthalmoscopy, Farmsworth-Munsell 100 hue discrimination testing and Illumina BeadChip analyses. Here we show that changes in the outer retina, specifically in the retinal pigment epithelium (RPE) and photoreceptor outer segments (POS) contribute to vision changes in non-infectious HIV retinopathy. We find that in HIV-positive retinae there is an induction of rhodopsin and other transcripts (including PDE6A, PDE6B, PDE6G, CNGA1, CNGB1, CRX, NRL) involved in visual transduction, as well as structural components of the rod photoreceptors (ABCA4 and ROM1). This is consistent with an increased rate of renewal of rod outer segments induced via increased phagocytosis by HIV-infected RPE previously reported in culture. Cone-specific transcripts (OPN1SW, OPN1LW, PDE6C, PDE6H and GRK7) are uniformly downregulated in HIV positive retina, likely due to a partial loss of cone photoreceptors. Active cotton wool spots and intraretinal hemorrhages (IRH) may not affect photoreceptors directly and the interaction of photoreceptors with the aging RPE may be the key to the progressive vision changes in HIV-positive patients.
doi:10.1371/journal.pone.0074712
PMCID: PMC3775801  PMID: 24069333

Results 1-25 (704352)