Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles: Potential Implications for Age-Related Macular Degeneration

Background

The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to develop new therapeutics. This requires further in-depth knowledge of the similarities and differences between mouse and human RPE.

Methods

We performed a microarray study to identify and functionally annotate RPE specific gene expression in mouse and human RPE. We used a meticulous method to determine C57BL/6J mouse RPE signature genes, correcting for possible RNA contamination from its adjacent layers: the choroid and the photoreceptors. We compared the signature genes, gene expression profiles and functional annotations of the mouse and human RPE.

Results

We defined sets of mouse (64), human (171) and mouse–human interspecies (22) RPE signature genes. Not unexpectedly, our gene expression analysis and comparative functional annotation suggested that, in general, the mouse and human RPE are very similar. For example, we found similarities for general features, like “organ development” and “disorders related to neurological tissue”. However, detailed analysis of the molecular pathways and networks associated with RPE functions, suggested also multiple species-specific differences, some of which may be relevant for the development of AMD. For example, CFHR1, most likely the main complement regulator in AMD pathogenesis was highly expressed in human RPE, but almost absent in mouse RPE. Furthermore, functions assigned to mouse and human RPE expression profiles indicate (patho-) biological differences related to AMD, such as oxidative stress, Bruch’s membrane, immune-regulation and outer blood retina barrier.

Conclusion

These differences may be important for the development of new therapeutic strategies and translational studies in age-related macular degeneration.     

Conditional Ablation of Retinol Dehydrogenase 10 in the Retinal Pigmented Epithelium Causes Delayed Dark Adaption in Mice

Regeneration of the visual chromophore, 11-cis-retinal, is a crucial step in the visual cycle required to sustain vision. This cycle consists of sequential biochemical reactions that occur in photoreceptor cells and the retinal pigmented epithelium (RPE). Oxidation of 11-cis-retinol to 11-cis-retinal is accomplished by a family of enzymes termed 11-cis-retinol dehydrogenases, including RDH5 and RDH11. Double deletion of Rdh5 and Rdh11 does not limit the production of 11-cis-retinal in mice. Here we describe a third retinol dehydrogenase in the RPE, RDH10, which can produce 11-cis-retinal. Mice with a conditional knock-out of Rdh10 in RPE cells (Rdh10 cKO) displayed delayed 11-cis-retinal regeneration and dark adaption after bright light illumination. Retinal function measured by electroretinogram after light exposure was also delayed in Rdh10 cKO mice as compared with controls. Double deletion of Rdh5 and Rdh10 (cDKO) in mice caused elevated 11/13-cis-retinyl ester content also seen in Rdh5^−/−Rdh11^−/− mice as compared with Rdh5^−/− mice. Normal retinal morphology was observed in 6-month-old Rdh10 cKO and cDKO mice, suggesting that loss of Rdh10 in the RPE does not negatively affect the health of the retina. Compensatory expression of other retinol dehydrogenases was observed in both Rdh5^−/− and Rdh10 cKO mice. These results indicate that RDH10 acts in cooperation with other RDH isoforms to produce the 11-cis-retinal chromophore needed for vision.     

The Polarity of the Retinal Pigment Epithelium

The diversity of epithelia in the body permits a multitude of organ-specific functions. One of the foremost examples of this is the retinal pigment epithelium. Located between the photoreceptors of the retina and their principal blood supply, the choriocapillaris, the retinal pigment epithelium is critical for the survival and function of retinal photoreceptors. To serve this purpose, the retinal pigment epithelium cell has adapted the classic Golgi-to-cell-surface targeting pathways first described in such prototypic epithelial cell models as the Madin-Darby canine kidney cell, to arrive at a unique distribution of membrane and secreted proteins. More recent data suggest that the retinal pigment epithelium also takes advantage of its inherent asymmetry to augment the classical pathways of Golgi-to-cell-surface traffic. As retinal pigment epithelium transplants and gene therapy represent potential cures for retinal degenerative diseases, understanding the basis of the unique polarity properties of retinal pigment epithelium cells will be a critical issue for the development of future therapies.     

Glucose Metabolism in Rat Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is the major transport pathway for exchange of metabolites and ions between choroidal blood supply and the neural retina. To gain insight into the mechanisms controlling glucose metabolism in RPE and its possible relationship to retinopathy, we studied the influence of different glucose concentrations on glycogen and lactate levels and CO₂ production in RPE from normal and streptozotocin-treated diabetic rats. Incubation of normal RPE in the absence of glucose caused a decrease in lactate production and glycogen content. In normal RPE, increasing glucose concentrations from 5.6 mM to 30 mM caused a four-fold increase in glucose accumulation and CO₂ yield, as well as reduction in lactate and glycogen production. In RPE from diabetic rats glucose accumulation did not increase in the presence of high glucose substrate, but it showed a four- and a seven-fold increase in CO₂ production through the mitochondrial and pentose phosphate pathways, respectively. We found high glycogen levels in RPE which can be used as an energy reserve for RPE itself and/or neural retina. Findings further show that the RPE possesses a high oxidative capacity. The large increase in glucose shunting to the pentose phosphate pathway in diabetic retina exposed to high glucose suggests a need for reducing capacity, consistent with increased oxidative stress.     

Receptor-Coupled Phosphoinositide Hydrolysis in Human Retinal Pigment Epithelium

Carbachol and histamine stimulated phosphoinositide (PPI) hydrolysis in cultured human retinal pigment epithelium (RPE), as reflected by an accumulation of 3H-inositol phosphates in the presence of 10 mM Li+. Carbachol increased PPI hydrolysis to greater than 600% of basal with an EC50 of 60 microM; stimulation was linear up to 60 min. This activation likely occurred via the M3 muscarinic cholinergic receptor based on the IC50 values for 4-diphenylacetoxy-N-methylpiperidine methiodide (0.47 nM), pirenzepine (280 nM), and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11- dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one (1.4 microM). Carbachol-mediated PPI hydrolysis was decreased by 80% in the absence of extracellular Ca2+. Histamine stimulated PPI turnover in a linear manner by 180% with an EC50 of 20 microM by the H1 histaminergic receptor. Serotonin, glutamate, norepinephrine, and dopamine were inactive. In human RPE, the resting cytoplasmic Ca2+ concentration, as determined by fura-2 fluorescence, was 138 +/- 24 nM. On the addition of carbachol, there was a 180% increase in peak intracellular Ca2+; addition of histamine increased intracellular Ca2+ by 187%. These results suggest receptor-mediated, inositol lipid hydrolysis is coupled to intracellular Ca2+ flux in human RPE.     

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.     

Autophagy and Exosomes in the Aged Retinal Pigment Epithelium: Possible Relevance to Drusen Formation and Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of loss of central vision in the elderly. The formation of drusen, an extracellular, amorphous deposit of material on Bruch''s membrane in the macula of the retina, occurs early in the course of the disease. Although some of the molecular components of drusen are known, there is no understanding of the cell biology that leads to the formation of drusen. We have previously demonstrated increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we found that drusen in AMD donor eyes contain markers for autophagy and exosomes. Furthermore, these markers are also found in the region of Bruch''s membrane in old mice. By in vitro modeling increased mtDNA damage induced by rotenone, an inhibitor of mitochondrial complex I, in the RPE, we found that the phagocytic activity was not altered but that there were: 1) increased autophagic markers, 2) decreased lysosomal activity, 3) increased exocytotic activity and 4) release of chemoattractants. Exosomes released by the stressed RPE are coated with complement and can bind complement factor H, mutations of which are associated with AMD. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients and may be associated with the pathogenesis of AMD in the elderly.     

MicroRNA Expression Profiles of Human iPS Cells,Retinal Pigment Epithelium Derived From iPS,and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.     

Phototoxic Action Spectrum on a Retinal Pigment Epithelium Model of Age-Related Macular Degeneration Exposed to Sunlight Normalized Conditions

Among the identified risk factors of age-related macular degeneration, sunlight is known to induce cumulative damage to the retina. A photosensitive derivative of the visual pigment, N-retinylidene-N-retinylethanolamine (A2E), may be involved in this phototoxicity. The high energy visible light between 380 nm and 500 nm (blue light) is incriminated. Our aim was to define the most toxic wavelengths in the blue-green range on an in vitro model of the disease. Primary cultures of porcine retinal pigment epithelium cells were incubated for 6 hours with different A2E concentrations and exposed for 18 hours to 10 nm illumination bands centered from 380 to 520 nm in 10 nm increments. Light irradiances were normalized with respect to the natural sunlight reaching the retina. Six hours after light exposure, cell viability, necrosis and apoptosis were assessed using the Apotox-Glo Triplex™ assay. Retinal pigment epithelium cells incubated with A2E displayed fluorescent bodies within the cytoplasm. Their absorption and emission spectra were similar to those of A2E. Exposure to 10 nm illumination bands induced a loss in cell viability with a dose dependence upon A2E concentrations. Irrespective of A2E concentration, the loss of cell viability was maximal for wavelengths from 415 to 455 nm. Cell viability decrease was correlated to an increase in cell apoptosis indicated by caspase-3/7 activities in the same spectral range. No light-elicited necrosis was measured as compared to control cells maintained in darkness. Our results defined the precise spectrum of light retinal toxicity in physiological irradiance conditions on an in vitro model of age-related macular degeneration. Surprisingly, a narrow bandwidth in blue light generated the greatest phototoxic risk to retinal pigment epithelium cells. This phototoxic spectrum may be advantageously valued in designing selective photoprotection ophthalmic filters, without disrupting essential visual and non-visual functions of the eye.     

Prohibitin as the Molecular Binding Switch in the Retinal Pigment Epithelium

Previously, our molecular binding study showed that prohibitin interacts with phospholipids, including phosphatidylinositide and cardiolipin. Under stress conditions, prohibitin interacts with cardiolipin as a retrograde response to activate mitochondrial proliferation. The lipid-binding switch mechanism of prohibitin with phosphatidylinositol-3,4,5-triphosphate and cardiolipin may suggest the role of prohibitin effects on energy metabolism and age-related diseases. The current study examined the region-specific expressions of prohibitin with respect to the retina and retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). A detailed understanding of prohibitin binding with lipids, nucleotides, and proteins shown in the current study may suggest how molecular interactions control apoptosis and how we can intervene against the apoptotic pathway in AMD. Our data imply that decreased prohibitin in the peripheral RPE is a significant step leading to mitochondrial dysfunction that may promote AMD progression.     

Ultimate Fate of Rod Outer Segments in the Retinal Pigment Epithelium

To investigate the degradation pathway of rod outer segments (ROS) in vivo, we injected gold-labeled ROS into the subretinal space of rabbits using a pars plana approach. Histology and electron microscopy performed on the specimens 72 hr after ROS injection revealed that the retina over the injection site was reattached, the retinal pigment epithelial (RPE) cells were intact, and gold granules were localized inside melanin granules and melanosomes. These results indicate that, in RPE, in vivo degradation of ROS is associated with melanosomes.     

Changes in the Composition and Fluorescent Properties of Bisretinoids in the Retina and the Retinal Pigment Epithelium of the Mouse Eye under Exposure to Ionizing Radiation

Biology Bulletin - Fluorescence and chromatographic analysis of bisretinoids from the retina and retinal pigment epithelium of mouse eyes was carried out before and after exposure to accelerated...     

Genetic Ablation of Afadin Causes Mislocalization and Deformation of Paneth Cells in the Mouse Small Intestinal Epithelium

Afadin is an actin filament-binding protein that acts cooperatively in cell adhesion with the cell adhesion molecule nectin, and in directional cell movement with the small G protein Rap1 in a nectin-independent manner. We studied the role of afadin in the organization of the small intestinal epithelium using afadin conditional gene knockout (cKO) mice. Afadin was localized at adherens junctions of all types of epithelial cells throughout the crypt-villus axis. Paneth cells were localized at the base of the crypt in control mice, but not confined there, and migrated into the villi in afadin-cKO mice. The distribution of other types of epithelial cells did not change significantly in the mutant mice. The Paneth cells remaining in the crypt exhibited abnormal shapes, were buried between adjacent cells, and did not face the lumen. In these cells, the formation of adherens junctions and tight junctions was impaired. Rap1 and EphB3 were highly expressed in control Paneth cells but markedly down-regulated in the afadin-deficient Paneth cells. Taken together, the results indicate that afadin plays a role in the restricted localization of Paneth cells at the base of the crypt by maintaining their adhesion to adjacent crypt cells and inhibiting their movement toward the top of villi.     

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.     

MERTK Interactions with SH2-Domain Proteins in the Retinal Pigment Epithelium

The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE). A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2)-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK_571–999), purified and phosphorylated. Ni²⁺-NTA pull downs were performed using 6xHis-rMERTK_571–999 in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK_571–999 and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α), VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS), siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.