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Despite the importance of the retinal pigment epithelium (RPE) for vision, the molecular processes involved in its specification are poorly understood. We identified two new mutant alleles for the zebrafish gene chokh (chk), which display a reduction or absence of the RPE. Unexpectedly, the neural retina (NR) in chk is specified and laminated, indicating that the regulatory network leading to NR development is largely independent of the RPE. Genetic mapping and molecular characterization revealed that chk encodes Rx3. Expression analyses show that otx2 and mitfb are not expressed in the prospective RPE of chk, indicating that the retinal homeobox gene rx3 acts upstream of the molecular network controlling RPE specification. Cellular transplantations demonstrate that rx3 function is autonomously required to specify the prospective RPE. Though rx2 is also absent in chk, neither rx2 nor rx1 is required for RPE development. Thus, our data provide the first indication that, in addition to controlling optic lobe evagination and proliferation, chk/rx3 also determines cellular fate.
Culture experiments with eye anlages of mouse embryos were performed to study developmental traits of the neuroepithelial cells of the prospective pigment epithelium in the eye anlage of pigmented mice. Between the neural plate stage on embryonic day 8 (ED 8, developmental stage 12) and the neural tube stage on embryonic day 9 1/2 (stage 15), the cultured neuroepithelium of the eye generated neurons and glia, identified by morphological and immunocytochemical evidence, but no pigmented cells. In contrast, eye anlages did produce pigment epithelium when cultured in their natural position in a head tissue fragment. A minority of developing neurons displayed tyrosine hydroxylase immunoreactivity, whereas GABAergic, serotoninergic and substance P-ergic neurons, which are common in the mature neuroretina, were not observed. When neuroepithelial cells from embryonic eyes older than stage 15 (ED 9 1/2) were cultured, they differentiated into pigment cells but not into nerve cells or glia. This developmental sequence indicates that the pigment cells derive from the neural lineage. Pigment cell fate dominates over the neural fate beginning at about stage 15 (ED 9 1/2-10). That is at least 2 days before the pigment cell phenotype becomes apparent in vivo (ED 11 1/2-12).
The choroid plexus epithelium within the brain ventricles orchestrates blood-derived monocyte entry to the central nervous system under injurious conditions, including when the primary injury site is remote from the brain. Here, we hypothesized that the retinal pigment epithelium (RPE) serves a parallel role, as a gateway for monocyte trafficking to the retina following direct or remote injury. We found elevated expression of genes encoding leukocyte trafficking determinants in mouse RPE as a consequence of retinal glutamate intoxication or optic nerve crush (ONC). Blocking VCAM-1 after ONC interfered with monocyte infiltration into the retina and resulted in a local pro-inflammatory cytokine bias. Live imaging of the injured eye showed monocyte accumulation first in the RPE, and subsequently in the retina, and peripheral leukocytes formed close contact with the RPE Our findings further implied that the ocular milieu can confer monocytes a phenotype advantageous for neuroprotection. These results suggest that the eye utilizes a mechanism of crosstalk with the immune system similar to that of the brain, whereby epithelial barriers serve as gateways for leukocyte entry.
Recent studies highlighted a link between ionizing radiation exposure during neurulation and birth defects such as microphthalmos and anophthalmos. Because the mechanisms underlying these defects remain largely unexplored, we irradiated pregnant C57BL/6J mice (1.0 Gy, X-rays) at embryonic day (E)7.5, followed by histological and gene/protein expression analyses at defined days. Irradiation impaired embryonic development at E9 and we observed a delayed pigmentation of the retinal pigment epithelium (RPE) at E11. In addition, a reduced RNA expression and protein abundance of critical eye-development genes (e.g. Pax6 and Lhx2) was observed. Furthermore, a decreased expression of Mitf, Tyr and Tyrp1 supported the radiation-induced perturbation in RPE pigmentation. Finally, via immunostainings for proliferation (Ki67) and mitosis (phosphorylated histone 3), a decreased mitotic index was observed in the E18 retina after exposure at E7.5. Overall, we propose a plausible etiological model for radiation-induced eye-size defects, with RPE melanogenesis as a major determining factor.
Here we report multiple lines of evidence for a comprehensive model of energy metabolism in the vertebrate eye. Metabolic flux, locations of key enzymes, and our finding that glucose enters mouse and zebrafish retinas mostly through photoreceptors support a conceptually new model for retinal metabolism. In this model, glucose from the choroidal blood passes through the retinal pigment epithelium to the retina where photoreceptors convert it to lactate. Photoreceptors then export the lactate as fuel for the retinal pigment epithelium and for neighboring Müller glial cells. We used human retinal epithelial cells to show that lactate can suppress consumption of glucose by the retinal pigment epithelium. Suppression of glucose consumption in the retinal pigment epithelium can increase the amount of glucose that reaches the retina. This framework for understanding metabolic relationships in the vertebrate retina provides new insights into the underlying causes of retinal disease and age-related vision loss.
Axotomised retinal ganglion cells (RGCs) die rapidly by apoptosis and fail to regenerate because of the limited availability of neurotrophic factors and a lack of axogenic stimuli. However, we have recently showed that pigment epithelium-derived factor (PEDF) promotes RGC survival and axon regeneration after optic nerve crush injury. PEDF has multiple fragments of the native peptide that are neuroprotective, anti-angiogenic and anti-inflammatory. Here we investigated the neuroprotective and axogenic properties of a fragment of PEDF, PEDF-34, in retinal neurons in vitro and when delivered by intravitreal injection and eye drops in vivo. We found that PEDF-34 was 43% more neuroprotective and 52% more neuritogenic than PEDF-44 in vitro. Moreover, in vivo, intravitreal delivery of 1.88nM PEDF-34 was 71% RGC neuroprotective at 21days after optic nerve crush compared to intact controls, whilst daily eye drops containing 1.88nM PEDF-34 promoted 87% RGC survival. After topical eye drop delivery, PEDF-34 was detected in the vitreous body within 30min and attained physiologically relevant concentrations in the retina by 4h peaking at 1.4±0.05nM by 14days. In eye drop- compared to intravitreal-treated PEDF-34 animals, 55% more RGC axons regenerated 250μm beyond the optic nerve lesion. We conclude that daily topical eye drop application of PEDF-34 is superior to weekly intravitreal injections in promoting RGC survival and axon regeneration through both direct effects on retinal neurons and indirect effects on other retinal cells.
Two-photon excitation microscopy can image retinal molecular processes in vivo. Intrinsically fluorescent retinyl esters in subcellular structures called retinosomes are an integral part of the visual chromophore regeneration pathway. Fluorescent condensation products of all-trans-retinal accumulate in the eye with age and are also associated with age-related macular degeneration (AMD). Here, we report repetitive, dynamic imaging of these compounds in live mice through the pupil of the eye. By leveraging advanced adaptive optics, we developed a data acquisition algorithm that permitted the identification of retinosomes and condensation products in the retinal pigment epithelium by their characteristic localization, spectral properties and absence in genetically modified or drug-treated mice. This imaging approach has the potential to detect early molecular changes in retinoid metabolism that trigger light- and AMD-induced retinal defects and to assess the effectiveness of treatments for these conditions.
Age-related macular degeneration (AMD) is a common disease with a multifactorial aetiology, still lacking effective and curative therapies. Among the early events triggering AMD is the deterioration of the retinal pigment epithelium (RPE), whose fundamental functions assure good health of the retina. RPE is physiologically exposed to high levels of oxidative stress during its lifespan; thus, the integrity and well-functioning of its antioxidant systems are crucial to maintain RPE homeostasis. Among these defensive systems, the Nrf2-pathway plays a primary role. Literature evidence suggests that, in aged and especially in AMD RPE, there is an imbalance between the increased pro-oxidant stress, and the impaired endogenous detoxifying systems, finally reverberating on RPE functions and survival. In this in vitro study on wild type (WT) and Nrf2-silenced (siNrf2) ARPE-19 cells exposed to various AMD-related noxae (H2O2, 4-HNE, MG132 + Bafilomycin), we show that the Nrf2-pathway activation is a physiological protective stress response, leading downstream to an up-regulation of the Nrf2-targets HO1 and p62, and that a Nrf2 impairment predisposes the cells to a higher vulnerability to stress. In search of new pharmacologically active compounds potentially useful for AMD, four nature-inspired hybrids (NIH) were individually characterized as Nrf2 activators, and their pharmacological activity was investigated in ARPE-19 cells. The Nrf2 activator dimethyl-fumarate (DMF; 10 μM) was used as a positive control. Three out of the four tested NIH (5 μM) display both direct and indirect antioxidant properties, in addition to cytoprotective effects in ARPE-19 cells under pro-oxidant stimuli. The observed pro-survival effects require the presence of Nrf2, with the exception of the lead compound NIH1, able to exert a still significant, albeit lower, protection even in siNrf2 cells, supporting the concept of the existence of both Nrf2-dependent and independent pathways mediating pro-survival effects. In conclusion, by using some pharmacological tools as well as a reference compound, we dissected the role of the Nrf2-pathway in ARPE-19 stress response, suggesting that the Nrf2 induction represents an efficient defensive strategy to prevent the stress-induced damage.
The therapeutic potential of pluripotent stem cells is great as they promise to usher in a new era of medicine where cells or organs may be prescribed to replace dysfunctional tissue. At the forefront are efforts in the eye to develop this technology as it lends itself to in vivo monitoring and sophisticated non-invasive imaging modalities. In the retina, retinal pigment epithelium (RPE) is the most promising replacement cell as it has a single layer, is relatively simple to transplant, and is associated with several eye diseases. However, after transplantation, the cells may transform and cause complications. This transformation may be partially due to incomplete maturation. With the goal of learning how to mature RPE, we compared induced pluripotent stem cell-derived RPE (iPSC-RPE) cells with adult human primary RPE (ahRPE) cells and the immortalized human ARPE-19 line. We cultured ARPE-19, iPSC-RPE, and ahRPE cells for one month, and evaluated morphology, RPE marker staining, and transepithelial electrical resistance (TEER) as quality control indicators. We then isolated RNA for bulk RNA-sequencing and DNA for genotyping. We genotyped ahRPE lines for the top age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR) risk allele polymorphisms. Transcriptome data verified that both adult and iPSC-RPE exhibit similar RPE gene expression signatures, significantly higher than ARPE-19. In addition, in iPSC-RPE, genes relating to stem cell maintenance, retina development, and muscle contraction were significantly upregulated compared to ahRPE. We compared ahRPE to iPSC-RPE in a model of epithelial-mesenchymal transition (EMT) and observed an increased sensitivity of iPSC-RPE to producing contractile aggregates in vitro which resembles incident reports upon transplantation. P38 inhibition was capable of inhibiting iPSC-RPE-derived aggregates. In summary, we find that the transcriptomic signature of iPSC-RPE conveys an immature RPE state which may be ameliorated by targeting "immature" gene regulatory networks.
We assessed structural elements of the retina in individuals with Friedreich ataxia (FRDA) and in mouse models of FRDA, as well as functions of the retinal pigment epithelium (RPE) in FRDA using induced pluripotent stem cells (iPSCs). We analyzed the retina of the FRDA mouse models YG22R and YG8R containing a human FRATAXIN (FXN) transgene by histology. We complemented this work with post-mortem evaluation of eyes from FRDA patients. Finally, we derived RPE cells from patient FRDA-iPSCs to assess oxidative phosphorylation (OXPHOS) and phagocytosis. We showed that whilst the YG22R and YG8R mouse models display elements of retinal degeneration, they do not recapitulate the loss of retinal ganglion cells (RGCs) found in the human disease. Further, RPE cells differentiated from human FRDA-iPSCs showed normal OXPHOS and we did not observe functional impairment of the RPE in Humans.
The presence of a phagocytic peak of photoreceptor outer segments by the retinal pigment epithelium (RPE) one or 2 h after the onset of light has been reported for several diurnal and nocturnal species. This peak in phagocytic activity also persists under constant lighting conditions (i.e., constant light or dark) thus demonstrating that the timing of this peak is driven by a circadian clock. The aim of this study was to investigate the change in RPE whole transcriptome at two different circadian times (CT; 1 h before (CT23) and 1 h after (CT1) subjective light onset). C57BL/6J male mice were maintained in constant dark conditions for three days and euthanized under red light (<1 lux) at CT23 and CT1. RPE was isolated from whole eyes for RNA library preparation and sequencing on an Illumina HiSeq4000 platform. 14,083 mouse RPE transcripts were detected in common between CT23 and CT1. 12,005 were protein coding transcripts and 2078 were non-protein coding transcripts. 2421 protein coding transcripts were significantly upregulated whereas only 3 transcripts were significantly downregulated and 12 non-protein coding transcripts were significantly upregulated and 31 non-protein coding transcripts were significantly downregulated at CT1 when compared to CT23 (p < 0.05, fold change ≥ ±2.0). Of the protein coding transcripts, most of them were characterized as: enzymes, kinases, and transcriptional regulators with a large majority of activity in the cytoplasm, nucleus, and plasma membrane. Non-protein coding transcripts included biotypes such as long-non coding RNAs and pseudogenes. Gene ontology analysis and ingenuity pathway analysis revealed that differentially expressed transcripts were associated with integrin signaling, oxidative phosphorylation, protein phosphorylation, and actin cytoskeleton remodeling suggesting that these previously identified phagocytic pathways are under circadian control. Our analysis identified new pathways (e.g., increased mitochondrial respiration via increased oxidative phosphorylation) that may be involved in the circadian control of phagocytic activity. In addition, our dataset suggests a possible regulatory role for the identified non-protein coding transcripts in mediating the complex function of RPE phagocytosis. Finally, our results also indicate, as seen in other tissues, about 20% of the whole RPE transcriptome may be under circadian clock regulation.
Butterfly-shaped pigment dystrophy is an eye disease characterized by lesions in the macula that can resemble the wings of a butterfly. Here we report the identification of heterozygous missense mutations in the CTNNA1 gene (encoding α-catenin 1) in three families with butterfly-shaped pigment dystrophy. In addition, we identified a Ctnna1 missense mutation in a chemically induced mouse mutant, tvrm5. Parallel clinical phenotypes were observed in the retinal pigment epithelium (RPE) of individuals with butterfly-shaped pigment dystrophy and in tvrm5 mice, including pigmentary abnormalities, focal thickening and elevated lesions, and decreased light-activated responses. Morphological studies in tvrm5 mice demonstrated increased cell shedding and the presence of large multinucleated RPE cells, suggesting defects in intercellular adhesion and cytokinesis. This study identifies CTNNA1 gene variants as a cause of macular dystrophy, indicates that CTNNA1 is involved in maintaining RPE integrity and suggests that other components that participate in intercellular adhesion may be implicated in macular disease.
The retina and RPE cells are regularly exposed to chronic oxidative stress as a tissue with high metabolic demand and ROS generation. DJ-1 is a multifunctional protein in the retina and RPE that has been shown to protect cells from oxidative stress in several cell types robustly. Oxidation of DJ-1 cysteine (C) residues is important for its function under oxidative conditions. The present study was conducted to analyze the impact of DJ-1 expression changes and oxidation of its C residues on RPE function. Monolayers of the ARPE-19 cell line and primary human fetal RPE (hfRPE) cultures were infected with replication-deficient adenoviruses to investigate the effects of increased levels of DJ-1 in these monolayers. Adenoviruses carried the full-length human DJ-1 cDNA (hDJ) and mutant constructs of DJ-1, which had all or each of its three C residues individually mutated to serine (S). Alternatively, endogenous DJ-1 levels were decreased by transfection and transduction with shPARK7 lentivirus. These monolayers were then assayed under baseline and low oxidative stress conditions. The results were analyzed by immunofluorescence, Western blot, RT-PCR, mitochondrial membrane potential, and viability assays. We determined that decreased levels of endogenous DJ-1 levels resulted in increased levels of ROS. Furthermore, we observed morphological changes in the mitochondria structure of all the RPE monolayers transduced with all the DJ-1 constructs. The mitochondrial membrane potential of ARPE-19 monolayers overexpressing all DJ-1 constructs displayed a significant decrease, while hfRPE monolayers only displayed a significant decrease in their ΔΨm when overexpressing the C2S mutation. Viability significantly decreased in ARPE-19 cells transduced with the C53S construct. Our data suggest that the oxidation of C53 is crucial for regulating endogenous levels of ROS and viability in RPE cells.
In age-related macular degeneration (AMD) the retinal pigment epithelium (RPE) deteriorates, leading to photoreceptor decay and severe vision loss. New therapeutic strategies aim at RPE replacement by transplantation of pluripotent stem cell (PSC)-derived RPE. Several protocols to generate RPE have been developed where appearance of pigmentation is commonly used as indicator of RPE differentiation and maturation. It is, however, unclear how different pigmentation stages reflect developmental stages and functionality of PSC-derived RPE cells. We generated human embryonic stem cell-derived RPE (hESC-RPE) cells and investigated their gene expression profiles at early pigmentation (EP) and late pigmentation (LP) stages. In addition, we compared the hESC-RPE samples with human endogenous RPE. We used a common reference design microarray (44 K). Our analysis showed that maturing hESC-RPE, upon acquiring pigmentation, expresses markers specific for human RPE. Interestingly, our analysis revealed that EP and LP hESC-RPE do not differ much in gene expression. Our data further showed that pigmented hESC-RPE has a significant lower expression than human endogenous RPE in the visual cycle and oxidative stress pathways. In contrast, we observed a significantly higher expression of pathways related to the process adhesion-to-polarity model that is typical of developing epithelial cells. We conclude that, in vitro, the first appearance of pigmentation hallmarks differentiated RPE. However, further increase in pigmentation does not result in much significant gene expression changes and does not add important RPE functionalities. Consequently, our results suggest that the time span for obtaining differentiated hESC-RPE cells, that are suitable for transplantation, may be greatly reduced.
Retinal pigment epithelial (RPE) degeneration is potentially involved in the pathogenesis of several retinal degenerative diseases. mTORC1 signaling is shown as a crucial regulator of many biological processes and disease progression. In this study, we aimed at investigating the role of mTORC1 signaling in RPE degeneration. Methods: Western blots were conducted to detect mTORC1 expression pattern during RPE degeneration. Cre-loxP system was used to generate RPE-specific mTORC1 activation mice. Fundus, immunofluorescence staining, transmission electron microscopy, and targeted metabolomic analysis were conducted to determine the effects of mTORC1 activation on RPE degeneration in vivo. Electroretinography, spectral-domain optical coherence tomography, and histological experiments were conducted to determine the effects of mTORC1 activation on choroidal and retinal function in vivo. Results: RPE-specific activation of mTORC1 led to RPE degeneration as shown by the loss of RPE-specific marker, compromised cell junction integrity, and intracellular accumulation of lipid droplets. RPE degeneration further led to abnormal choroidal and retinal function. The inhibition of mTORC1 signaling with rapamycin could partially reverse RPE degeneration. Targeted metabolomics analysis further revealed that mTORC1 activation affected the metabolism of purine, carboxylic acid, and niacin in RPE. Conclusion: This study revealed that abnormal activation of mTORC1 signaling leads to RPE degeneration, which could provide a promising target for the treatment of RPE dysfunction-related diseases.
Pigment Epithelium-Derived Factor (PEDF) is a secreted glycoprotein belonging to the family of non-inhibitory serpins. It is known, that in cases of complicated myopia, the content of PEDF in aqueous humor of the anterior chamber is significantly reduced. Here we examined a bulk of Tenon's capsule samples obtained from various groups of myopes, to examine PEDF processing in progressive myopia. We have analyzed the distribution of full length PEDF50 and its truncated form PEDF45 in the soluble and insoluble fractions extracted from Tenon's capsule of myopic and control (non-myopic) patients using SDS-polyacrylamide gel electrophoresis, as well as monitored the proteolytic degradation of PEDF ex vivo by enzyme-linked immunosorbent assay. These results were complemented by PEDF mRNA analysis in correspondent tissues by using qPCR and immunohistochemistry analysis of PEDF distribution in normal and myopic specimens. We found that in the Tenon's capsule of patients suffering from a high myopia the level of "soluble" 45 kDa PEDF reduced by 2-fold, while the content of "insoluble" 50 kDa form of PEDF was increased by 4-fold compared to controls. Excessive amount of PEDF50 in myopic specimens have been shown to correlate with the abrogated PEDF processing rather than with an increase of its expression. Moreover, immunohistochemical staining of the myopic Tenon's capsule tissue sections revealed the halo of deposited PEDF50 in the fibroblast extracellular space. These findings suggest that in myopia limited proteolysis of PEDF is altered or abrogated. Accumulation of full-length PEDF insoluble aggregates in the fibroblast intercellular space may affect cell survival and consequently causes the destructive changes in the extracellular matrix of the eye connective tissues. As a result, the abrogation of full-length PEDF normal processing can be an important mechanism leading to biomechanical destabilization of the scleral capsule and myopia progression.
Retinal pigment epithelium (RPE) provides nourishment and protection to the eye. RPE dysfunction due to oxidative stress and inflammation is one of the major reason for many of the retinal disorders. Organophosphorus pesticides are widely used in the agricultural, industrial and household activities in India. However, their effects on the eye in the context of RPE has not been studied. In this study the defense of the ARPE19 cells exposed to Chlorpyrifos (1 nM to 100 µM) in terms of the enzyme paraoxonase (PON) was studied at 24 hr and 9 days of treatment. Chlorpyrifos was found to induce oxidative stress in the ARPE19 cells as seen by significant increase in ROS and decrease in glutathione (GSH) levels without causing cell death. Tissue resident Paraoxonase 2 (PON2) mRNA expression was elevated with chlorpyrifos exposure. The three enzymatic activities of PON namely, paraoxonase (PONase), arylesterase (PON AREase) and thiolactonase (PON HCTLase) were also found to be significantly altered to detoxify and as an antioxidant defense. Among the transcription factors regulating PON2 expression, SP1 was significantly increased with chlorpyrifos exposure. PON2 expression was found to be crucial as ARPE19 cells showed a significant loss in their ability to withstand oxidative stress when the cells were subjected to chlorpyrifos after silencing PON2 expression. Treatment with N-acetyl cysteine positively regulated the PON 2 expression, thus promoting the antioxidant defense put up by the cells in response to chlorpyrifos.
Glucocorticoids are amongst the most used drugs to treat retinal diseases of various origins. Yet, the transcriptional regulations induced by glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation in retinal pigment epithelium cells (RPE) that form the outer blood-retina barrier are unknown. Levels of endogenous corticoids, ligands for MR and GR, were measured in human ocular media. Human RPE cells derived from induced pluripotent stem cells (iRPE) were used to analyze the pan-transcriptional regulations induced by aldosterone-an MR-specific agonist, or cortisol or cortisol + RU486-a GR antagonist. The retinal phenotype of transgenic mice that overexpress the human MR (P1.hMR) was analyzed. In the human eye, the main ligand for GR and MR is cortisol. The iRPE cells express functional GR and MR. The subset of genes regulated by aldosterone and by cortisol + RU-486, and not by cortisol alone, mimics an imbalance toward MR activation. They are involved in extracellular matrix remodeling (CNN1, MGP, AMTN), epithelial-mesenchymal transition, RPE cell proliferation and migration (ITGB3, PLAUR and FOSL1) and immune balance (TNFSF18 and PTX3). The P1.hMR mice showed choroidal vasodilation, focal alteration of the RPE/choroid interface and migration of RPE cells together with RPE barrier function alteration, similar to human retinal diseases within the pachychoroid spectrum. RPE is a corticosteroid-sensitive epithelium. MR pathway activation in the RPE regulates genes involved in barrier function, extracellular matrix, neural regulation and epithelial differentiation, which could contribute to retinal pathology.
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