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The retinal pigment epithelium (RPE) is an essential component of the retina that plays multiple roles required to support visual function. These include light onset- and circadian rhythm-dependent tasks, such as daily phagocytosis of photoreceptor outer segments. Mitochondria provide energy to the highly specialized and energy-dependent RPE. In this study, we examined the positioning of mitochondria and how this is influenced by the onset of light. We identified a population of mitochondria that are tethered to the basal plasma membrane pre- and post-light onset. Following light onset, mitochondria redistributed apically and interacted with melanosomes and phagosomes. In a choroideremia mouse model that has regions of the RPE with disrupted or lost infolding of the plasma membrane, the positionings of only the non-tethered mitochondria were affected. This provides evidence that the tethering of mitochondria to the plasma membrane plays an important role that is maintained under these disease conditions. Our work shows that there are subpopulations of RPE mitochondria based on their positioning after light onset. It is likely they play distinct roles in the RPE that are needed to fulfil the changing cellular demands throughout the day.

Purpose: We aim to characterize the pathways required for autofluorescent granule (AFG) formation by RPE cells using cultured monolayers., Methods: We fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24 hours the cells started accumulating AFGs that were comparable to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and Western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking cathepsin D by gene editing., Results: AFGs seem to derive from incompletely digested POS-containing phagosomes and after 3 days are surrounded by a single membrane positive for lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation, and that impairment of lysosomal pH or catalytic activity, particularly cathepsin D activity, enhances AF accumulation., Conclusions: We conclude that lysosomal dysfunction results in incomplete POS degradation and enhanced AFG accumulation.

Purpose: To correlate drusen morphology and outer retinal status with autofluorescence (AF) imaging in patients with intermediate age-related macular degeneration., Methods: Drusen type and morphology were analyzed using color fundus photography and spectral-domain optic coherence tomography, whereas fundus AF was used for drusen AF evaluation. Additional structural changes on spectral-domain optic coherence tomography, such as disruption of external limiting membrane, ellipsoid zone, and retinal pigment epithelium/Bruch membrane complex, as well as the presence of choroidal hypertransmission at correspondent locations were also evaluated and correlated with fundus AF findings. Spearman's correlation coefficient was used to analyze the correlation between spectral-domain optic coherence tomography morphological characteristics of drusen and AF appearance of the corresponding drusen. Strength of correlation was calculated (r), and a P value < 0.05 was considered statistically significant., Results: Two hundred and twenty-eight drusen from 53 eyes of 53 patients were analyzed, 130 soft drusen (57.02%) and 98 cuticular drusen (42.98%). Sixty percent of the drusen were isoautofluorescent (n = 136), 35% hyperautofluorescent (n = 80), and 5% hypoautofluorescent (n = 12). We found positive correlation between drusen AF and hyperreflective foci (r = 0.4). Outer retinal layers morphology (external limiting membrane and ellipsoid zone status and hypertransmission) also correlates with autofluorescent findings (r = 0.3)., Conclusion: Multimodal imaging reveals a broad spectrum of ultrastructural changes, which may reflect different stages in the evolution of drusen. Our results suggest that drusen morphological characteristics and autofluorescent findings are correlated but other factors or cofactors may be involved. The described correlations will help us understand new progression biomarkers of nonexudative age-related macular degeneration.

Purpose: The basal surface of the retinal pigment epithelium (RPE) is folded into a complex basal labyrinth thought to facilitate solute and water transport. We aimed to analyze and define the structural organization of the basal labyrinth of the RPE to enable quantitative analysis of structural changes in age and disease and to better understand the relationship between basal labyrinth structure and efficiency of transepithelial transport., Methods: Conventional transmission and serial block-face scanning electron microscopy and electron tomography were used to examine the structure of the basal labyrinth in mouse eyes of different ages and genotypes and with and without osmotic shock before fixation., Results: We identified structurally distinct zones (stacked and ribbon-like) within the RPE basal labyrinth that are largely organelle free and cisternal elements that make contact with the endoplasmic reticulum (ER) and mitochondria. These zones are lost in a hierarchic fashion with age and prematurely in a model of the progressive retinal degenerative disease, choroideremia. Junctional complexes crosslink closely opposed infoldings. Spacing between the basal infoldings was affected by subtle osmotic changes while osmotic shock induced dramatic remodeling of the infoldings., Conclusions: The basal labyrinth has complex but ordered structural elements that break down with age and in choroideremia. The geometry of these elements and site of contact with ER and mitochondria likely facilitate the ion transport that drives water transport across the basal RPE surface. Changes in structure in response to local osmotic variation may allow transport to be modulated in order to maintain RPE volume.

Retinal degeneration and visual impairment are the first signs of juvenile neuronal ceroid lipofuscinosis caused by CLN3 mutations, followed by inevitable progression to blindness. We investigated retinal degeneration in Cln3(Δex1-6) null mice, revealing classic 'fingerprint' lysosomal storage in the retinal pigment epithelium (RPE), replicating the human disease. The lysosomes contain mitochondrial F0-ATP synthase subunit c along with undigested membranes, indicating a reduced degradative capacity. Mature autophagosomes and basal phagolysosomes, the terminal degradative compartments of autophagy and phagocytosis, are also increased in Cln3(Δex1) (-6) RPE, reflecting disruption to these key pathways that underpin the daily phagocytic turnover of photoreceptor outer segments (POS) required for maintenance of vision. The accumulated autophagosomes have post-lysosome fusion morphology, with undigested internal contents visible, while accumulated phagosomes are frequently docked to cathepsin D-positive lysosomes, without mixing of phagosomal and lysosomal contents. This suggests lysosome-processing defects affect both autophagy and phagocytosis, supported by evidence that phagosomes induced in Cln3(Δex1) (-) (6)-derived mouse embryonic fibroblasts have visibly disorganized membranes, unprocessed internal vesicles and membrane contents, in addition to reduced LAMP1 membrane recruitment. We propose that defective lysosomes in Cln3(Δex1) (-) (6) RPE have a reduced degradative capacity that impairs the final steps of the intimately connected autophagic and phagocytic pathways that are responsible for degradation of POS. A build-up of degradative organellar by-products and decreased recycling of cellular materials is likely to disrupt processes vital to maintenance of vision by the RPE., (© The Author 2015. Published by Oxford University Press.)

In a variety of cells, secretory processes require the activation of both Rab27a and L-type channels of the Ca(V)1.3 subtype. In the retinal pigment epithelium (RPE), Rab27a and Ca(V)1.3 channels regulate growth-factor secretion towards its basolateral side. Analysis of murine retina sections revealed a co-localization of both Rab27a and Ca(V)1.3 at the basolateral membrane of the RPE. Heterologously expressed Ca(V)1.3/β3/α2δ1 channels showed negatively shifted voltage-dependence and decreased current density of about 70% when co-expressed with Rab27a. However, co-localization analysis using α(5)β(1) integrin as a membrane marker revealed that Rab27a co-expression reduced the surface expression of Ca(V)1.3 only about 10%. Physical binding of heterologously expressed Rab27a with Ca(V)1.3 channels was shown by co-localization in immunocytochemistry as well as co-immunoprecipitation which was abolished after deletion of a MyRIP-homologous amino acid sequence at the II-III linker of the Ca(V)1.3 subunit. Rab27a over-expression in ARPE-19 cells positively shifted the voltage dependence, decreased current density of endogenous Ca(V)1.3 channels and reduced VEGF-A secretion. We show the first evidence of a direct functional modulation of an ion channel by Rab27a suggesting a new mechanism of Rab and ion channel interaction in the control of VEGF-A secretion in the RPE., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Analysis of melanosome biogenesis in the retinal pigment epithelium (RPE) is challenging because it occurs predominantly in a short embryonic time window. Here, we show that the zebrafish provides an ideal model system for studying this process because in the RPE the timing of melanosome biogenesis facilitates molecular manipulation using morpholinos. Morpholino-mediated knockdown of OA1 (also known as GPR143), mutations in the human homologue of which cause the most common form of human ocular albinism, induces a major reduction in melanosome number, recapitulating a key feature of the mammalian disease where reduced melanosome numbers precede macromelanosome formation. We further show that PMEL, a key component of mammalian melanosome biogenesis, is required for the generation of cylindrical melanosomes in zebrafish, which in turn is required for melanosome movement into the apical processes and maintenance of photoreceptor integrity. Spherical and cylindrical melanosomes containing similar melanin volumes co-exist in the cell body but only cylindrical melanosomes enter the apical processes. Taken together, our findings indicate that melanosome number and shape are independently regulated and that melanosome shape controls a function in the RPE that depends on localisation in the apical processes., (© 2015. Published by The Company of Biologists Ltd.)

Defects in phagocytosis and degradation of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) are associated with aging and retinal disease. The daily burst of rod outer segment (ROS) phagocytosis by the RPE provides a unique opportunity to analyse phagosome processing in vivo. In mouse retinae, phagosomes containing stacked rhodopsin-rich discs were identified by immuno-electron microscopy. Early apical phagosomes stained with antibodies against both cytoplasmic and intradiscal domains of rhodopsin. During phagosome maturation, a remarkably synchronised loss of the cytoplasmic epitope coincided with movement to the cell body and preceded phagosome-lysosome fusion and disc degradation. Loss of the intradiscal rhodopsin epitope and disc digestion occurred upon fusion with cathepsin-D-positive lysosomes. The same sequential stages of phagosome maturation were identified in cultured RPE and macrophages challenged with isolated POS. Loss of the cytoplasmic rhodopsin epitope was insensitive to pH but sensitive to protease inhibition and coincided with the interaction of phagosomes with endosomes. Thus, during pre-lysosomal maturation of ROS-containing phagosomes, limited rhodopsin processing occurs upon interaction with endosomes. This potentially provides a sensitive readout of phagosome-endosome interactions that is applicable to multiple phagocytes., (© 2014. Published by The Company of Biologists Ltd.)

The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm(Flox), Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch's membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5-6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm(Flox), Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.

Background: Choroideremia (CHM) is a progressive X-linked degeneration of three ocular layers: photoreceptors, retinal pigment epithelium (RPE) and choroid, caused by the loss of Rab Escort Protein-1 (REP1). As a recessive monogenic disorder, CHM is potentially curable by gene addition therapy. The present study aimed to evaluate the potential use of lentiviral vectors carrying CHM/REP1 cDNA transgene for CHM treatment., Methods: We generated lentiviral vectors carrying either CHM/REP1 cDNA or EGFP transgene under the control of the elongation factor-1α promoter (EF-1α) or its shortened version EFS. We transduced human (HT1080) and dog (D17) cells, CHM patient's fibroblasts and mouse primary RPE cells in vitro, as well as wild-type and CHM mouse retinas in vivo by subretinal injections. Transgene expression was confirmed by immunoblotting, fluorescence-activated cell sorting, immunofluorescence and confocal microscopy. CHM/REP1 transgene functionality was assessed by an in vitro prenylation assay., Results: Lentiviral vectors with CHM/REP1 and EGFP transgenes efficiently transduced HT1080, D17 and CHM fibroblast cells; CHM/REP1 transgene lead to an increase in prenylation activity. Subretinal injections of lentiviral vectors into mouse retinas resulted in efficient transduction of the RPE (30-35% of total RPE cells transduced after a 1-µl injection), long-term expression for at least 6  months and a decrease in amount of unprenylated Rabs in the CHM RPE. Transduction of neuroretinal cells was restricted to the injection site., Conclusions: Lentiviral CHM/REP1 cDNA transgene rescues the prenylation defect in CHM mouse RPE and thus could be used to restore REP1 activity in the RPE of CHM patients., (Copyright © 2012 John Wiley & Sons, Ltd.)

Purpose: Choroideremia (CHM) is a progressive X-linked degeneration of three ocular layers (photoreceptors, retinal pigment epithelium, and choroid), with a complex and still largely unclear pathogenesis. To investigate the pathophysiology of CHM, the authors engineered mice with a cell type-specific Chm/Rep1 knockout (KO)., Methods: A mouse line carrying a conditional allele Chm(Flox) was crossed with the transgenic line IRBP-Cre to achieve Chm KO, specifically in the photoreceptor layer, and Tyr-Cre to produce Chm KO, specifically in the retinal pigment epithelial and other pigmented cells. Chm(Flox), Tyr-Cre+ and Chm(Flox), IRBP-Cre+ mice were mated to produce mice with Chm KO in both layers. All mouse lines were studied by histology, electron microscopy, electroretinography (ERG), scanning laser ophthalmoscopy (SLO), and biochemical, Results: In Chm(Flox), IRBP-Cre+ mice the authors observed the progressive degeneration of photoreceptors in the presence of normal retinal pigment epithelium (RPE). Chm(Flox), Tyr-Cre+ mice exhibited coat color dilution and pigment abnormalities of the RPE in the presence of an intact outer nuclear layer. In 6- to 8-month-old Chm(Flox), Tyr-Cre+, IRBP-Cre+ mice, the degeneration of photoreceptors was accelerated compared with Chm(Flox), IRBP-Cre+ mice but became leveled with age, such that it was comparable at 12 to 14 months. Detailed ERG and SLO analysis supported the histopathologic findings., Conclusions: Defects in photoreceptors and RPE can arise because of intrinsic defects caused cell autonomously by the Chm KO. However, when both photoreceptors and RPE are diseased, the dynamics of the degenerative process are altered. Photoreceptor functional deficit and cell death manifest much earlier, suggesting that the diseased RPE accelerates photoreceptor degeneration.