scholarly journals Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration

2018 ◽  
Vol 115 (47) ◽  
pp. E11120-E11127 ◽  
Author(s):  
Tamara L. Lenis ◽  
Jane Hu ◽  
Sze Yin Ng ◽  
Zhichun Jiang ◽  
Shanta Sarfare ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Wild Type Mouse ◽  
Mouse Retina ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Rpe Cells ◽  
Photoreceptor Outer Segments ◽  
Quantitative Immunoblotting

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4−/− mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk−/− but not Abca4−/− mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4−/− background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4−/− mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.

scholarly journals Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-derived Factor Receptor (PEDF-R)

2021 ◽  
Author(s):  
Jeanee Bullock ◽  
Federica Polato ◽  
Mones Abu-Asab ◽  
Alexandra Bernardo-Colón ◽  
Elma Aflaki ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Phospholipase A2 ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Photoreceptor Outer Segment ◽  
Western Blots ◽  
Rpe Cells ◽  
Photoreceptor Outer Segments ◽  
Transmission Electron ◽  
Bromoenol Lactone

AbstractPurposeTo examine the contribution of PEDF-R to the phagocytosis process. Previously, we identified PEDF-R, the protein encoded by the PNPLA2 gene, as a phospholipase A2 in the retinal pigment epithelium (RPE). During phagocytosis, RPE cells ingest abundant phospholipids and protein in the form of photoreceptor outer segment (POS) tips, which are then hydrolyzed. The role of PEDF-R in RPE phagocytosis is not known.MethodsMice in which PNPLA2 was conditionally knocked out in the RPE were generated (cKO). Mouse RPE/choroid explants were cultured. Human ARPE-19 cells were transfected with siPNPLA2 silencing duplexes. POS were isolated from bovine retinas. The phospholipase A2 inhibitor bromoenol lactone was used. Transmission electron microscopy, immunofluorescence, lipid labeling, pulse-chase experiments, western blots, and free fatty acid and β-hydroxybutyrate assays were performed.ResultsThe RPE of the cKO mice accumulated lipids as well as more abundant and larger rhodopsin particles compared to littermate controls. Upon POS exposure, RPE explants from cKO mice released less β-hydroxybutyrate compared to controls. After POS ingestion during phagocytosis, rhodopsin degradation was stalled both in cells treated with bromoenol lactone and in PNPLA2-knocked-down cells relative to their corresponding controls. Phospholipase A2 inhibition lowered β-hydroxybutyrate release from phagocytic RPE cells. PNPLA2 knock down also resulted in a decline in fatty acids and β-hydroxybutyrate release from phagocytic RPE cells.ConclusionsPEDF-R downregulation delayed POS digestion during phagocytosis. The findings imply that efficiency of RPE phagocytosis depends on PEDF-R, thus identifying a novel contribution of this protein to POS degradation in the RPE.

scholarly journals THE CIRCADIAN CLOCK IN THE RETINAL PIGMENT EPITHELIUM CONTROLS THE DIURNAL RHYTHM OF PHAGOCYTIC ACTIVITY

2020 ◽  
Author(s):  
Christopher DeVera ◽  
Jendayi Dixon ◽  
Micah A. Chrenek ◽  
Kenkichi Baba ◽  
P. Michael Iuvone ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Circadian Clock ◽  
Phagocytic Activity ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Optomotor Response ◽  
Rpe Cells ◽  
Photoreceptor Outer Segments ◽  
Outer Segments ◽  
Old Mice

AbstractThe diurnal peak of phagocytosis by the retinal pigment epithelium (RPE) of photoreceptor outer segments (POS) is under circadian control, and it is believed that this process involves interactions from both the retina and RPE. Previous studies have demonstrated that a functional circadian clock exists within multiple retinal cell types and RPE cells. Thereby, the aim of the current study was to determine whether the circadian clock in the retina and or RPE controls the diurnal phagocytic peak of photoreceptor outer segments and whether selective disruption of the circadian clock in the RPE would affect RPE cells function and the viability during aging. To that aim, we first generated and validated an RPE tissue-specific KO of the essential clock gene, Bmal1, and then we determined the daily rhythm in phagocytic activity by the RPE in mice lacking a functional circadian clock in the retina or RPE. Then using electroretinography, spectral domain-optical coherence tomography, and optomotor response measurements of visual function we determined the effect of Bmal1 removal in young (6-month old) and old (18-month old) mice. RPE morphology and lipofuscin accumulation was also determined in young and old mice. Our data show that the circadian clock in the RPE controls the daily diurnal phagocytic peak of POS. Surprisingly, the lack of a functional RPE circadian clock or the diurnal phagocytic peak does not result in any detectable age-related degenerative phenotype in the retina or RPE. Thus, our results demonstrate that the loss of the circadian clock in the RPE or the lack of the daily peak in phagocytosis of POS does not result in deterioration of photoreceptors or the RPE during aging.

Regulation of the renin expression in the retinal pigment epithelium by systemic stimuli

2010 ◽  
Vol 299 (2) ◽  
pp. F396-F403 ◽  
Author(s):  
Vladimir M. Milenkovic ◽  
Marisa Brockmann ◽  
Christian Meyer ◽  
Michael Desch ◽  
Frank Schweda ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Enzyme Inhibitor ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Renin Angiotensin System ◽  
Mouse Retina ◽  
Ang Ii ◽  
Rpe Cells ◽  
Renin Synthesis ◽  
Short Time

The retina expresses a local renin-angiotensin system (RAS). This study aimed to investigate the influence of systemic modulation of renin synthesis on the expression of renin in the retinal pigment epithelium (RPE), which forms part of the blood/retina barrier. Freshly isolated RPE cells showed expression of renin 1A, which is the secreted isoform of renin. Systemic administration of the angiotensin-converting enzyme inhibitor enalapril in mice increased the renin expression in both the kidney and the retina. Systemic infusion of ANG II led to a decrease in the renin expression in the kidney and in the retina and RPE. The ANG II-dependent down-regulation of renin expression in the RPE was prevented by systemic application of the AT1 receptor blocker losartan. However, water deprivation lead to an increase of the renin expression in the kidney but unexpectedly to a decrease of the renin expression in the retina. In sections of the mouse retina, the ANG II receptor AT1 was found in the RPE and localized at the blood side of the epithelium. Short-time cultured RPE cells showed increases in intracellular free Ca2+ in response to stimulation by ANG II that were sensitive to losartan. In summary, we conclude that the renin expression in cells of the blood/retina barrier is influenced by the systemic RAS. ANG II circulating in the plasma is likely a mediator of this influence.

scholarly journals SLC26A7 constitutes the thiocyanate-selective anion conductance of the basolateral membrane of the retinal pigment epithelium

2020 ◽  
Vol 319 (4) ◽  
pp. C641-C656
Author(s):  
Xu Cao ◽  
Manoocher Soleimani ◽  
Bret A. Hughes
Keyword(s):  
Retinal Pigment Epithelium ◽  
Ph Regulation ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Subretinal Fluid ◽  
Wild Type Mouse ◽  
Biologically Active ◽  
Rpe Cells ◽  
Anion Conductance

Anion channels in the retinal pigment epithelium (RPE) play an essential role in the transport of Cl− between the outer retina and the choroidal blood to regulate the ionic composition and volume of the subretinal fluid that surrounds the photoreceptor outer segments. Recently, we reported that the anion conductance of the mouse RPE basolateral membrane is highly selective for the biologically active anion thiocyanate (SCN−), a property that does not correspond with any of the Cl− channels that have been found to be expressed in the RPE to date. The purpose of this study was to determine the extent to which SLC26A7, a SCN− permeable-anion exchanger/channel that was reported to be expressed in human RPE, contributes to the RPE basolateral anion conductance. We show by quantitative RT-PCR that Slc26a7 is highly expressed in mouse RPE compared with other members of the Slc26 gene family and Cl− channel genes known to be expressed in the RPE. By applying immunofluorescence microscopy to mouse retinal sections and isolated cells, we localized SLC26A7 to the RPE basolateral membrane. Finally, we performed whole cell and excised patch recordings from RPE cells acutely isolated from Slc26a7 knockout mice to show that the SCN− conductance and permeability of its basolateral membrane are dramatically smaller relative to wild-type mouse RPE cells. These findings establish SLC26A7 as the SCN−-selective conductance of the RPE basolateral membrane and provide new insight into the physiology of an anion channel that may participate in anion transport and pH regulation by the RPE.

scholarly journals Absence of iron-regulatory protein Hfe results in hyperproliferation of retinal pigment epithelium: role of cystine/glutamate exchanger

2009 ◽  
Vol 424 (2) ◽  
pp. 243-252 ◽  
Author(s):  
Jaya P. Gnana-Prakasam ◽  
Muthusamy Thangaraju ◽  
Kebin Liu ◽  
Yonju Ha ◽  
Pamela M. Martin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Critical Role ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Mrna Levels ◽  
Wild Type ◽  
Rpe Cells

Haemochromatosis is an iron-overload disorder with age-dependent oxidative stress and dysfunction in a variety of tissues. Mutations in HFE (histocompatability leucocyte antigen class I-like protein involved in iron homoeostasis) are responsible for most cases of haemochromatosis. We demonstrated recently that HFE is expressed exclusively in the basal membrane of RPE (retinal pigment epithelium). In the present study, we used Hfe−/− mice to examine ferritin levels (an indirect readout for iron levels) and morphological changes in retina. We found increased ferritin accumulation in retina in 18-month-old, but not in 2-month-old, mice with considerable morphological damage compared with age-matched controls. The retinal phenotype included hypertrophy and hyperplasia of RPE. RPE cells isolated from Hfe−/− mice exhibited a hyperproliferative phenotype. We also compared the gene expression profile between wild-type and Hfe−/− RPE cells by microarray analysis. These studies showed that many cell cycle-related genes were differentially regulated in Hfe−/− RPE cells. One of the genes up-regulated in Hfe−/− RPE cells was Slc7a11 (where Slc is solute carrier) which codes for the ‘transporter proper’ xCT in the heterodimeric cystine/glutamate exchanger (xCT/4F2hc). This transporter plays a critical role in cellular glutathione status and cell-cycle progression. We confirmed the microarrray data by monitoring xCT mRNA levels by RT (reverse transcription)–PCR and also by measuring transport function. We also found increased levels of glutathione and the transcription factor/cell-cycle promoter AP1 (activator protein 1) in Hfe−/− RPE cells. Wild-type mouse RPE cells and human RPE cell lines, when loaded with iron by exposure to ferric ammonium citrate, showed increased expression and activity of xCT, reproducing the biochemical phenotype observed with Hfe−/− RPE cells.

scholarly journals Polyphenol Metabolite Pyrogallol-O-Sulfate Decreases Microglial Activation and VEGF in Retinal Pigment Epithelium Cells and Diabetic Mouse Retina

2021 ◽  
Vol 22 (21) ◽  
pp. 11402
Author(s):  
Daniela F. Santos ◽  
Mariana Pais ◽  
Cláudia N. Santos ◽  
Gabriela A. Silva
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Diabetic Mouse ◽  
Mouse Retina ◽  
Rpe Cells ◽  
Glut 1 ◽  
Diabetic Retina ◽  
The Impact ◽  
Epithelium Cells

(Poly)phenol-derived metabolites are small molecules resulting from (poly)phenol metabolization after ingestion that can be found in circulation. In the last decade, studies on the impact of (poly)phenol properties in health and cellular metabolism accumulated evidence that (poly)phenols are beneficial against human diseases. Diabetic retinopathy (DR) is characterized by inflammation and neovascularization and targeting these is of therapeutic interest. We aimed to study the effect of pyrogallol-O-sulfate (Pyr-s) metabolite in the expression of proteins involved in retinal glial activation, neovascularization, and glucose transport. The expression of PEDF, VEGF, and GLUT-1 were analyzed upon pyrogallol-O-sulfate treatment in RPE cells under high glucose and hypoxia. To test its effect on a diabetic mouse model, Ins2Akita mice were subjected to a single intraocular injection of the metabolite and the expression of PEDF, VEGF, GLUT-1, Iba1, or GFAP measured in the neural retina and/or retinal pigment epithelium (RPE), two weeks after treatment. We observed a significant decrease in the expression of pro-angiogenic VEGF in RPE cells. Moreover, pyrogallol-O-sulfate significantly decreased the expression of microglial marker Iba1 in the diabetic retina at different stages of disease progression. These results highlight the potential pyrogallol-O-sulfate metabolite as a preventive approach towards DR progression, targeting molecules involved in both inflammation and neovascularization.

Retinal dystrophy associated with Danon disease and pathogenic mechanism through LAMP2-mutated retinal pigment epithelium

2019 ◽  
Vol 30 (3) ◽  
pp. 570-578 ◽  
Author(s):  
Masaya Fukushima ◽  
Tatsuya Inoue ◽  
Takashi Miyai ◽  
Ryo Obata
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Intracellular Localization ◽  
Retinal Dystrophy ◽  
Wild Type Mouse ◽  
Biological Study ◽  
Mouse Retina ◽  
Cone Dystrophy ◽  
Danon Disease

Introduction: Lysosome-associated membrane protein 2 plays an important role in autophagy and lysosomal function and its mutation is responsible for pathogenesis of Danon disease, which can cause retinopathy, though its pathophysiological contribution to retinal dysfunction remains unclear. The purpose of our research is to report the first case of Japanese Danon disease retinopathy and to understand how LAMP2 dysfunction contributes to pathogenesis of retinopathy. Methods: One case underwent ophthalmic examination including slit-lamp exam, fundus imaging, visual field testing, and electroretinogram. In molecular biological study, relative messenger RNA expression levels of three splicing variants of Lamp2 or LAMP2 in wild type mouse retina and retinal pigment epithelium, human retinal pigment epithelium cell line adult retinal pigment epithelium-19 were quantified. LAMP2 was knocked down by small interfering RNA in adult retinal pigment epithelium-19 and its effect to LC3, an autophagy marker, was assessed by Western blotting. Intracellular localization of LAMP2 and LC3 in untreated and LAMP2-knocked-down adult retinal pigment epithelium-19 was analyzed by confocal microscopy. Results: Our case manifested cone dystrophy in both eyes. In mice, expression of Lamp2a and Lamp2b was significantly higher in retinal pigment epithelium than that in neural retina. Expression of Lamp2a and Lamp2b were significantly higher than that of Lamp2c in mouse retinal pigment epithelium. Adult retinal pigment epithelium-19 cells showed similar LAMP2 expression pattern to mouse retinal pigment epithelium. LAMP2 knockdown in adult retinal pigment epithelium-19 reduced LC3-II amount and the number and size of autophagosome. Discussion: We report a Japanese case of Danon disease retinopathy, and our study implies that LAMP2 plays an important role in autophagosome formation in retinal pigment epithelium.

scholarly journals Loss of melanoregulin (MREG) enhances cathepsin-D secretion by the retinal pigment epithelium

2013 ◽  
Vol 30 (3) ◽  
pp. 55-64 ◽  
Author(s):  
LAURA S. FROST ◽  
VANDA S. LOPES ◽  
FRANK P. STEFANO ◽  
ALVINA BRAGIN ◽  
DAVID S. WILLIAMS ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Cathepsin D ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Disease ◽  
Protective Role ◽  
Phagocytic Cells ◽  
Phagocytic Capacity ◽  
Photoreceptor Outer Segments ◽  
Age Dependent

AbstractCathepsin-D (Cat-D) is a major proteolytic enzyme in phagocytic cells. In the retinal pigment epithelium (RPE), it is responsible for the daily degradation of photoreceptor outer segments (POSs) to maintain retinal homeostasis. Melanoregulin (MREG)-mediated loss of phagocytic capacity has been linked to diminished intracellular Cat-D activity. Here, we demonstrate that loss of MREG enhances the secretion of intermediate Cat-D (48 kDa), resulting in a net enhancement of extracellular Cat-D activity. These results suggest that MREG is required to maintain Cat-D homeostasis in the RPE and likely plays a protective role in retinal health. In this regard, in the Mregdsu/dsu mouse, we observe increased basal laminin. Loss of the Mregdsu allele is not lethal and therefore leads to slow age-dependent changes in the RPE. Thus, we propose that this model will allow us to study potential dysregulatory functions of Cat-D in retinal disease.

The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo

1988 ◽  
Vol 91 (2) ◽  
pp. 303-312
Author(s):  
N.M. McKechnie ◽  
M. Boulton ◽  
H.L. Robey ◽  
F.J. Savage ◽  
I. Grierson
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cytokeratin 18 ◽  
Rpe Cells ◽  
Human Retinal Pigment Epithelium ◽  
Cytoskeletal Elements

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.

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