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Retinal Pigment Epithelium: Molecular Mechanisms of Homeostasis and Function

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pathology, Diagnostics, and Therapeutics".

Deadline for manuscript submissions: closed (30 June 2022) | Viewed by 26099

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Prof. Dr. Silvia C. Finnemann

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Department of Biological Sciences, Center for Cancer, Genetic Diseases, and Gene Regulation, Fordham University, Bronx, NY 10458, USA
Interests: retinal pigment epithelium; retina; retinal degeneration; phagocytosis; integrins; signaling; cytoskeleton; aging
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Special Issue Information

Dear Colleagues,

The retinal pigment epithelium (RPE) is a specialized monolayer epithelium located in the posterior vertebrate eye that has unique epithelial polarity. At their basal surface, RPE cells face the highly vascularized choroid connective tissue. The microvilli at the apical surface of the RPE instead extend into the interphotoreceptor matrix that surrounds the outer segment aspects of rod and cone photoreceptor neurons and are thus an integral part of the neural retina. Barrier and selective transport functions of the RPE control flux of molecules into and out of the outer retina. As result, RPE cells are essential providers to and gate keepers for the neural retina. Moreover, RPE cells perform essential functions that maintain photoreceptor homeostasis and long-term functionality including a key role in photopigment regeneration in the visual cycle and their essential role in the daily outer segment renewal program through phagocytosis of spent outer segment tips. Deficiencies in the RPE that impacts its numerous activities in support of photoreceptors and the neural retina often cause or contribute to retinal dysfunction and degeneration. This is of particular relevance in the mammalian eye, where both RPE and neighboring photoreceptor are post-mitotic and thus interact in a functional unit for life. Many of the underlying regulatory pathways remain poorly understood. This Special Issue invites contributions on the fascinating, diverse molecular mechanisms governing the functions of the RPE in health and disease.

Prof. Dr. Silvia C. Finnemann
Guest Editor

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Keywords

cell communication
phagocytosis
signaling
membrane receptors
metabolism
cell junctions

Published Papers (9 papers)

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23 pages, 3504 KiB

Open AccessArticle

Single-Cell RNA Sequencing Reveals Molecular Features of Heterogeneity in the Murine Retinal Pigment Epithelium

by Ravi S. Pandey, Mark P. Krebs, Mohan T. Bolisetty, Jeremy R. Charette, Jürgen K. Naggert, Paul Robson, Patsy M. Nishina and Gregory W. Carter

Int. J. Mol. Sci. 2022, 23(18), 10419; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231810419 - 08 Sep 2022

Cited by 3 | Viewed by 3345

Abstract

Transcriptomic analysis of the mammalian retinal pigment epithelium (RPE) aims to identify cellular networks that influence ocular development, maintenance, function, and disease. However, available evidence points to RPE cell heterogeneity within native tissue, which adds complexity to global transcriptomic analysis. Here, to assess cell heterogeneity, we performed single-cell RNA sequencing of RPE cells from two young adult male C57BL/6J mice. Following quality control to ensure robust transcript identification limited to cell singlets, we detected 13,858 transcripts among 2667 and 2846 RPE cells. Dimensional reduction by principal component analysis and uniform manifold approximation and projection revealed six distinct cell populations. All clusters expressed transcripts typical of RPE cells; the smallest (C1, containing 1–2% of total cells) exhibited the hallmarks of stem and/or progenitor (SP) cells. Placing C1–6 along a pseudotime axis suggested a relative decrease in melanogenesis and SP gene expression and a corresponding increase in visual cycle gene expression upon RPE maturation. K-means clustering of all detected transcripts identified additional expression patterns that may advance the understanding of RPE SP cell maintenance and the evolution of cellular metabolic networks during development. This work provides new insights into the transcriptome of the mouse RPE and a baseline for identifying experimentally induced transcriptional changes in future studies of this tissue. Full article

(This article belongs to the Special Issue Retinal Pigment Epithelium: Molecular Mechanisms of Homeostasis and Function)

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20 pages, 3275 KiB

Open AccessArticle

Oxidation of DJ-1 Cysteines in Retinal Pigment Epithelium Function

by Sanghamitra Bhattacharyya, Johnathon Sturgis, Arvydas Maminishkis, Sheldon S. Miller and Vera L. Bonilha

Int. J. Mol. Sci. 2022, 23(17), 9938; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23179938 - 01 Sep 2022

Cited by 2 | Viewed by 1507

Abstract

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. Full article

(This article belongs to the Special Issue Retinal Pigment Epithelium: Molecular Mechanisms of Homeostasis and Function)

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16 pages, 3161 KiB

Open AccessArticle

PEDF Deletion Induces Senescence and Defects in Phagocytosis in the RPE

by Ivan T. Rebustini, Susan E. Crawford and S. Patricia Becerra

Int. J. Mol. Sci. 2022, 23(14), 7745; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23147745 - 13 Jul 2022

Cited by 4 | Viewed by 6456

Abstract

The retinal pigment epithelium (RPE) expresses the Serpinf1 gene to produce pigment epithelium-derived factor (PEDF), a retinoprotective protein that is downregulated with cell senescence, aging and retinal degenerations. We determined the expression of senescence-associated genes in the RPE of 3-month-old mice that lack the Serpinf1 gene and found that Serpinf1 deletion induced H2ax for histone H2AX protein, Cdkn1a for p21 protein, and Glb1 gene for β-galactosidase. Senescence-associated β-galactosidase activity increased in the Serpinf1 null RPE when compared with wild-type RPE. We evaluated the subcellular morphology of the RPE and found that ablation of Serpinf1 increased the volume of the nuclei and the nucleoli number of RPE cells, implying chromatin reorganization. Given that the RPE phagocytic function declines with aging, we assessed the expression of the Pnpla2 gene, which is required for the degradation of photoreceptor outer segments by the RPE. We found that both the Pnpla2 gene and its protein PEDF-R declined with the Serpinf1 gene ablation. Moreover, we determined the levels of phagocytosed rhodopsin and lipids in the RPE of the Serpinf1 null mice. The RPE of the Serpinf1 null mice accumulated rhodopsin and lipids compared to littermate controls, implying an association of PEDF deficiency with RPE phagocytosis dysfunction. Our findings establish PEDF loss as a cause of senescence-like changes in the RPE, highlighting PEDF as both a retinoprotective and a regulatory protein of aging-like changes associated with defective degradation of the photoreceptor outer segment in the RPE. Full article

(This article belongs to the Special Issue Retinal Pigment Epithelium: Molecular Mechanisms of Homeostasis and Function)

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17 pages, 4590 KiB

Open AccessArticle

The Circadian Clock in the Retinal Pigment Epithelium Controls the Diurnal Rhythm of Phagocytic Activity

by Christopher DeVera, Jendayi Dixon, Micah A. Chrenek, Kenkichi Baba, Yun Z. Le, P. Michael Iuvone and Gianluca Tosini

Int. J. Mol. Sci. 2022, 23(10), 5302; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23105302 - 10 May 2022

Cited by 10 | Viewed by 2159

Abstract

The diurnal peak of phagocytosis by the retinal pigment epithelium (RPE) of photoreceptor outer segments (POS) is under circadian control and believed that this process involves interactions from the retina and RPE. Previous studies have demonstrated that a functional circadian clock exists within multiple retinal cell types and RPE. Thereby, the aim of this study was to determine whether the clock in the retina or RPE controls the diurnal phagocytic peak and whether disruption of the circadian clock in the RPE would affect cellular function and the viability during aging. To that, we generated and validated an RPE tissue-specific KO of the essential clock gene, Bmal1, and then 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 of visual function we determined the effect of Bmal1 removal in young (6 months) and old (18 months) mice. RPE morphology and lipofuscin accumulation was determined in young and old mice. Our data shows that the clock in the RPE, rather than the retina clock, controls the diurnal phagocytic peak. Surprisingly, absence of a functional RPE clock and phagocytic peak does not result in any detectable age-related degenerative phenotype in the retina or RPE. Thus, our results demonstrate that the circadian clock in the RPE controls the daily peak of phagocytic activity. However, the absence of the clock in the RPE does not result in deterioration of photoreceptors or the RPE during aging. Full article

(This article belongs to the Special Issue Retinal Pigment Epithelium: Molecular Mechanisms of Homeostasis and Function)

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15 pages, 4069 KiB

Open AccessArticle

Visualizing Cell Death in Live Retina: Using Calpain Activity Detection as a Biomarker for Retinal Degeneration

by Soumaya Belhadj, Nina Sofia Hermann, Yu Zhu, Gustav Christensen, Torsten Strasser and François Paquet-Durand

Int. J. Mol. Sci. 2022, 23(7), 3892; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23073892 - 31 Mar 2022

Cited by 1 | Viewed by 2034

Abstract

Calpains are a family of calcium-activated proteases involved in numerous disorders. Notably, previous studies have shown that calpain activity was substantially increased in various models for inherited retinal degeneration (RD). In the present study, we tested the capacity of the calpain-specific substrate t-BOC-Leu-Met-CMAC to detect calpain activity in living retina, in organotypic retinal explant cultures derived from wild-type mice, as well as from rd1 and Rho^P23H/+ RD-mutant mice. Test conditions were refined until the calpain substrate readily detected large numbers of cells in the photoreceptor layer of RD retina but not in wild-type retina. At the same time, the calpain substrate was not obviously toxic to photoreceptor cells. Comparison of calpain activity with immunostaining for activated calpain-2 furthermore suggested that individual calpain isoforms may be active in distinct temporal stages of photoreceptor cell death. Notably, calpain-2 activity may be a relatively short-lived event, occurring only towards the end of the cell-death process. Finally, our results support the development of calpain activity detection as a novel in vivo biomarker for RD suitable for combination with non-invasive imaging techniques. Full article

(This article belongs to the Special Issue Retinal Pigment Epithelium: Molecular Mechanisms of Homeostasis and Function)

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19 pages, 8065 KiB

Open AccessArticle

Pleiotropic Roles of Scavenger Receptors in Circadian Retinal Phagocytosis: A New Function for Lysosomal SR-B2/LIMP-2 at the RPE Cell Surface

by Quentin Rieu, Antoine Bougoüin, Yvrick Zagar, Jonathan Chatagnon, Abdallah Hamieh, Julie Enderlin, Thierry Huby and Emeline F. Nandrot

Int. J. Mol. Sci. 2022, 23(7), 3445; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23073445 - 22 Mar 2022

Cited by 4 | Viewed by 2303

Abstract

The retinal phagocytic machinery resembles the one used by macrophages to clear apoptotic cells. However, in the retina, the permanent contact between photoreceptor outer segments (POS) and retinal pigment epithelial (RPE) cells requires a tight control of this circadian machinery. In addition to the known receptors synchronizing POS internalization, several others are expressed by RPE cells. Notably, scavenger receptor CD36 has been shown to intervene in the internalization speed. We thus investigated members of the scavenger receptor family class A SR-AI and MARCO and class B CD36, SR-BI and SR-B2/LIMP-2 using immunoblotting, immunohisto- and immunocytochemistry, lipid raft flotation gradients, phagocytosis assays after siRNA/antibody inhibition, RT-qPCR and western blot analysis along the light:dark cycle. All receptors were expressed by RPE cell lines and tissues and colocalized with POS, except SR-BI. All receptors were associated with lipid rafts, and even more upon POS challenge. SR-B2/LIMP-2 inhibition suggested a role in the control of the internalization speed similar to CD36. In vivo, MARCO and CD36 displayed rhythmic gene and protein expression patterns concomitant with the phagocytic peak. Taken together, our results indicate that CD36 and SR-B2/LIMP-2 play a direct regulatory role in POS phagocytosis dynamics, while the others such as MARCO might participate in POS clearance by RPE cells either as co-receptors or via an indirect process. Full article

(This article belongs to the Special Issue Retinal Pigment Epithelium: Molecular Mechanisms of Homeostasis and Function)

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Review

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11 pages, 1153 KiB

Open AccessReview

Probing Photoreceptor Outer Segment Phagocytosis by the RPE In Vivo: Models and Methodologies

by Jade A. Vargas and Silvia C. Finnemann

Int. J. Mol. Sci. 2022, 23(7), 3661; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23073661 - 27 Mar 2022

Cited by 7 | Viewed by 2756

Abstract

In the vertebrate retina, the light-sensitive photoreceptor rods and cones constantly undergo renewal by generating new portions of the outer segment and shedding their distal, spent tips. The neighboring RPE provides the critical function of engulfing the spent material by phagocytosis. RPE phagocytosis of shed rod outer segment fragments is a circadian process that occurs in a burst of activity shortly after daily light onset with low activity at other times, a rhythm that has been reported for many species and over 50 years. In this review, we compare studies on the rhythm and quantity of RPE phagocytosis using different in vivo model systems and assessment methods. We discuss how measurement methodology impacts the observation and analysis of RPE phagocytosis. Published studies on RPE phagocytosis investigating mice further suggest that differences in genetic background and housing conditions may affect results. Altogether, a comparison between RPE phagocytosis studies performed using differing methodology and strains of the same species is not as straightforward as previously thought. Full article

(This article belongs to the Special Issue Retinal Pigment Epithelium: Molecular Mechanisms of Homeostasis and Function)

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11 pages, 1126 KiB

Open AccessReview

Circadian Regulation of Retinal Pigment Epithelium Function

by Kenkichi Baba, Varunika Goyal and Gianluca Tosini

Int. J. Mol. Sci. 2022, 23(5), 2699; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23052699 - 28 Feb 2022

Cited by 12 | Viewed by 2945

Abstract

The retinal pigment epithelium (RPE) is a single layer of cells located between the choriocapillaris vessels and the light-sensitive photoreceptors in the outer retina. The RPE performs physiological processes necessary for the maintenance and support of photoreceptors and visual function. Among the many functions performed by the RPE, the timing of the peak in phagocytic activity by the RPE of the photoreceptor outer segments that occurs 1–2 h. after the onset of light has captured the interest of many investigators and has thus been intensively studied. Several studies have shown that this burst in phagocytic activity by the RPE is under circadian control and is present in nocturnal and diurnal species and rod and cone photoreceptors. Previous investigations have demonstrated that a functional circadian clock exists within multiple retinal cell types and RPE cells. However, the anatomical location of the circadian controlling this activity is not clear. Experimental evidence indicates that the circadian clock, melatonin, dopamine, and integrin signaling play a key role in controlling this rhythm. A series of very recent studies report that the circadian clock in the RPE controls the daily peak in phagocytic activity. However, the loss of the burst in phagocytic activity after light onset does not result in photoreceptor or RPE deterioration during aging. In the current review, we summarized the current knowledge on the mechanism controlling this phenomenon and the physiological role of this peak. Full article

(This article belongs to the Special Issue Retinal Pigment Epithelium: Molecular Mechanisms of Homeostasis and Function)

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Other

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10 pages, 1253 KiB

Open AccessBrief Report

Differences in Diurnal Rhythm of Rod Outer Segment Renewal between 129T2/SvEmsJ and C57BL/6J Mice

by Jade A. Vargas and Silvia C. Finnemann

Int. J. Mol. Sci. 2022, 23(16), 9466; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23169466 - 22 Aug 2022

Cited by 2 | Viewed by 1478

Abstract

In all mammalian species tested to date, rod photoreceptor outer segment renewal is a circadian process synchronized by light with a burst of outer segment fragment (POS) shedding and POS phagocytosis by the adjacent retinal pigment epithelium (RPE) every morning at light onset. Recent reports show that RPE phagocytosis also increases shortly after dark onset in C57BL/6 (C57) mice. Genetic differences between C57 mice and 129T2/SvEmsJ (129) mice may affect regulation of outer segment renewal. Here, we used quantitative methods to directly compare outer segment renewal in C57 and 129 mouse retina. Quantification of rhodopsin-positive phagosomes in the RPE showed that in 129 mice, rod POS phagocytosis after light onset was significantly increased compared to C57 mice, but that 129 mice did not show a second peak after dark onset. Cone POS phagosome content of RPE cells did not differ by mouse strain with higher phagosome numbers after light than after dark. We further quantified externalization of the “eat me” signal phosphatidylserine by outer segment tips, which precedes POS phagocytosis. Live imaging of retina ex vivo showed that rod outer segments extended PS exposure in both strains but that frequency of outer segments with exposed PS after light onset was lower in C57 than in 129 retina. Taken together, 129 mice lacked a burst of rod outer segment renewal after dark onset. The increases in rod outer segment renewal after light and after dark onset in C57 mice were attenuated compared to the peak after light onset in 129 mice, suggesting an impairment in rhythmicity in C57 mice. Full article

(This article belongs to the Special Issue Retinal Pigment Epithelium: Molecular Mechanisms of Homeostasis and Function)

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