RESUMEN

Phagocytosis is one of the key functions of retinal pigment epithelium (RPE) cells, which maintain photoreceptor health by removing photoreceptor outer segments (POSs) that are regularly shed. A deficiency in RPE function to phagocytose POSs may lead to vision loss in inherited retinal diseases and eventually to age-related macular degeneration (AMD) with geographic atrophy. Significant progress has been made in the field of cell replacement therapy for AMD using stem-cell-derived RPE. To test their function, RPE cells are incubated with purified bovine POSs for the demonstration of efficient binding, internalization, and digestion of POSs. Here, we present an image-based method to measure phagocytosis activity by using POSs labeled with a pH-sensitive fluorescent dye, which has low fluorescence at neutral pH outside of the cell and high fluorescence at low pH inside the phagosome. Further, we introduce a unique flow-cytometry-based method for the characterization of POSs by measuring specific markers for POSs such as rhodopsin and opsin. Using this method, we demonstrated a comparable quality of several bovine POS isolation batches and a reliable assessment of POS quality on RPE phagocytosis assay performance when subjected to different stress conditions. This work provides new tools to characterize POSs and insight into RPE phagocytosis assay development for the functional evaluation of RPE cells in the field of cell replacement therapy.

Asunto(s)

RESUMEN

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the developed world. Caucasians are eightfold more likely to develop AMD than any other race, indicating a racial bias in AMD incidence which is unexplained. We hypothesize that pigmentation of the retinal pigment epithelium (RPE) and choroid protects from AMD and underlies this peculiar racial bias. We investigated GPR143, a receptor in the pigmentation pathway, which is activated by a melanin synthesis by-product, l-dopa. In this model, greater pigmentation leads to greater l-dopa production and, in turn, greater GPR143 signaling. GPR143 activity upregulates PEDF and downregulates both VEGF and exosomes; all of which reduce the angiogenic potential in the retina. Moreover, we demonstrate that GPR143 signaling enhances the digestion of shed photoreceptor outer segments. Together, our data suggests a central role for GPR143 signaling in RPE-photoreceptor interaction which is critical to healthy vision.

Asunto(s)

RESUMEN

The retinal pigment epithelium (RPE) ensures different functions crucial for photoreceptor survival, and thus for vision, such as photoreceptor outer segments (POS) phagocytosis and retinal adhesion. Both follow a circadian rhythm with an activity peak occurring respectively 1.5-2 and 3.5 h after light onset. Interestingly, we showed that two rodent models, ß5-/- and Prpf31+/- mice, display distinct alterations in both functions leading to different phenotypes. Indeed, the phagocytic peak totally disappears in ß5 knockout mice but is attenuated and shifted in Prpf31+/- mice. Conversely, the retinal adhesion peak only attenuated in ß5-/- mice is lost in Prpf31+/- mice. These distinct alterations have different consequences on retinal homeostasis proportional to the observed defects: ß5-/- mice progressively lose vision and accumulate RPE lipofuscin deposits, while Prpf31+/- mice develop RPE metabolic dysfunctions and gradual structural modifications indicative of cellular stress. Hence, animal models are useful to understand the importance of the proper regulation of these functions.

Asunto(s)

RESUMEN

INTRODUCTION: Central serous chorioretinopathy (CSCR) as a clinical entity is potentially damaging and may significantly affect retinal morphology and function, especially in the chronic form. Our study aimed to determine the amount of deficit of best corrected visual acuity (BCVA) and individual retinal layers, including ganglion cells, in different types of CSCR and with reference to its duration. METHODS: The retrospective analysis included 69 eyes of patients with resolved CSCR managed in Dobry Wzrok Ophthalmological Clinic between 1 January 2019 and 30 June 2022. The diagnosis of CSCR was based on the criteria outlined by the Central Serous Chorioretinopathy International Group. The analysis included data obtained from medical history, BCVA testing, and spectral domain optical coherence tomography (SD-OCT) measurements, with specific thickness values for individual retinal layers. The results were compared among affected eyes, unaffected fellow eyes, and healthy controls. RESULTS: BCVA values were significantly lower in acute (0.08 ± 0.12 logMAR) and chronic (0.26 ± 0.19 logMAR) cases versus controls (0.0 logMAR). The thickness of all retinal layers (central subfoveal thickness, CST; inner retina with ganglion cell complex, GC; outer retina, ORT; and photoreceptor outer segments, POS) and macular volume (MV) were significantly decreased in chronic eyes versus controls (p < 0.01). Acute eyes had significant thinning of the outer retina and POS only compared to control eyes (p < 0.01). The amount of deficit in CST, ORT, GC, and MV was strongly correlated with poorer BCVA (p < 0.001), and the deficit of CST, ORT, and GC was correlated with disease duration (p < 0.05). The subfoveal choroidal thickness was significantly greater in affected and fellow eyes versus controls (p < 0.001). CONCLUSION: Damage to the outer retina and photoreceptors occurs early in the course of CSCR, with a deficit in ganglion cells noted adjunctively in chronic forms of the disease. Further studies are required to precisely determine correlation between visual loss in CSCR and deficits in individual retinal layers.

Central serous chorioretinopathy (CSCR) is a common condition typically affecting young and middle-aged individuals. In its chronic form, it can lead to changes in retinal morphology and significant visual impairment. This disorder occurs basically in two forms: acute and chronic, depending on its duration. The study aimed to determine the amount of morphological and functional deficit occurring in the course of acute and chronic CSCR and refer it to healthy controls. The analysis of 69 resolved cases of CSCR was performed, including results of visual acuity testing and spectral domain coherence tomography (SD-OCT) measurements. Visual acuity was significantly lower in both acute and chronic groups compared to healthy control eyes, with greater deficit in the chronic group. Analysis revealed also a significant thinning of the retina in the chronic group versus control group. Chronic group demonstrated substantial loss of ganglion cells, which was not noted in acute form of the disease. Acute eyes demonstrated only a partial deficit in the outer retina, with the ganglion cell layer remaining intact. The amount of deficit of all retinal layers was strongly correlated with poorer visual acuity and disease duration.

RESUMEN

Aging may significantly modify antioxidant and photoprotective properties of melanin in retinal pigment epithelium (RPE). Here, photoreactivity of melanosomes (MS), isolated from younger and older human donors with and without added zeaxanthin and α-tocopherol, was analyzed by electron paramagnetic resonance oximetry, time-resolved singlet oxygen phosphorescence, and protein oxidation assay. The phototoxic potential of ingested melanosomes was examined in ARPE-19 cells exposed to blue light. Phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was determined by flow cytometry. Irradiation of cells fed MS induced significant inhibition of the specific phagocytosis with the effect being stronger for melanosomes from older than from younger human cohorts, and enrichment of the melanosomes with antioxidants reduced the inhibitory effect. Cellular protein photooxidation was more pronounced in samples containing older melanosomes, and it was diminished by antioxidants. This study suggests that blue light irradiated RPE melanosomes could induce substantial inhibition of the key function of the cells-their specific phagocytosis. The data indicate that while photoreactivity of MS and their phototoxic potential increase with age, they could be reduced by selected natural antioxidants.

Asunto(s)

RESUMEN

STUDY QUESTION: Do human granulosa cells (GCs) ingest and destroy apoptotic oocytes? SUMMARY ANSWER: Somatic GCs ingest and destroy apoptotic oocytes and other apoptotic substrates through unconventional autophagy-assisted phagocytosis. WHAT IS KNOWN ALREADY: Most (99%) ovarian germ cells undergo apoptosis through follicular atresia. The mode of cleaning of atretic follicles from the ovary is unclear. Ovarian GCs share striking similarities with testicular Sertoli cells with respect to their origin and function. Somatic Sertoli cells are responsible for the elimination of apoptotic spermatogenic cells through unconventional autophagy-assisted phagocytosis. STUDY DESIGN, SIZE, DURATION: Human GCs were tested for the ability to ingest and destroy the apoptotic oocytes and other apoptotic substrates. A systemic study of the main phagocytosis steps has been performed at different time points after loading of apoptotic substrates into the GC. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary cultures of GC retrieved following controlled ovarian stimulation of five women for IVF/ICSI and a human granulosa KGN cell line were incubated with different apoptotic substrates: oocytes which underwent spontaneous apoptosis during the cultivation of immature germ cells for IVF/ICSI; apoptotic KGN cells; and apoptotic membranes from rat retinas. Cultured GC were analyzed for the presence of specific molecular markers characteristic of different steps of phagocytic and autophagy machineries by immunocytochemistry, confocal microscopy, transmission electron microscopy and western blotting, before and after loading with apoptotic substrates. MAIN RESULTS AND THE ROLE OF CHANCE: Incubation of human GC with apoptotic substrates resulted in their translocation in cell cytoplasm, concomitant with activation of the phagocytosis receptor c-mer proto-oncogene tyrosine kinase MERTK (P < 0.001), clumping of motor molecule myosin II, recruitment of autophagy proteins: autophagy-related protein 5 (ATG5), autophagy-related protein 6 (Beclin1) and the rise of a membrane form of microtubule-associated protein 1 light chain 3 (LC3-II) protein. Ingestion of apoptotic substrates was accompanied by increased expression of the lysosomal protease Cathepsin D (P < 0.001), and a rise of lysosomes in the GCs, as assessed by different techniques. The level of autophagy adaptor, sequestosome 1/p62 (p62) protein remained unchanged. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The number of patients described here is limited. Also the dependence of phagocytosis on reproductive hormone status of patients should be analyzed. WIDER IMPLICATIONS OF THE FINDINGS: Removal of apoptotic oocytes by surrounding GC seems likely to be a physiological mechanism involved in follicular atresia. Proper functioning of this mechanism may be a new strategy for the treatment of ovarian dysfunctions associated with an imbalance in content of germ cells in the ovaries, such as premature ovarian failure and polycystic ovary syndrome. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by Rennes Metropole (AIS 2015) and Agence de BioMédecine. This work was supported by funding from Université de Rennes1, Institut National de la Santé et de la Recherche Médicale (INSERM) and CHU de Rennes. A.B. is funded in part by the program Actions Concertées Interpasteuriennes (ACIP) and a research grant from the European Society of Pediatric Endocrinology. This work is supported by the Agence Nationale de la Recherche Grants ANR-17-CE14-0038 and ANR-10-LABX-73. The authors declare no competing interests.

Asunto(s)

RESUMEN

Maintenance of a healthy photoreceptor-retinal pigment epithelium (RPE) interface is essential for vision. At the center of this interface, apical membrane protrusions stemming from the RPE ensheath photoreceptor outer segments (POS), and are possibly involved in the recycling of POS through phagocytosis. The molecules that regulate POS ensheathment and its relationship to phagocytosis remain to be deciphered. By means of ultrastructural analysis, we revealed that Mer receptor tyrosine kinase (MERTK) ligands, GAS6 and PROS1, rather than αVß5 integrin receptor ligands, triggered POS ensheathment by human embryonic stem cell (hESC)-derived RPE. Furthermore, we found that ensheathment is required for POS fragmentation before internalization. Consistently, POS ensheathment, fragmentation, and internalization were abolished in MERTK mutant RPE, and rescue of MERTK expression in retinitis pigmentosa (RP38) patient RPE counteracted these defects. Our results suggest that loss of ensheathment due to MERTK dysfunction might contribute to vision impairment in RP38 patients.

Asunto(s)

RESUMEN

Although the primary biological function of retinal photoreceptors is to absorb light and provide visual information, extensive exposure to intense light could increase the risk of phototoxic reactions mediated by products of rhodopsin bleaching that might accumulate in photoreceptor outer segments (POS). The phototoxicity of POS, isolated from bovine retinas, was examined in cultured retinal pigment epithelium cells (ARPE-19) containing phagocytised POS and in selected model systems by determining POS ability to photogenerate singlet oxygen, and photoinduce oxidation of cholesterol and serum albumin. Bleaching of rhodopsin-rich POS with green light resulted in the formation of retinoid products exhibiting distinct absorption spectra in the near-UV. Irradiation of POS-fed ARPE-19 cells with blue light reduced their survival in a dose-dependent manner with the effect being stronger for cells containing prebleached POS. The specific and non-specific phagocytic activity of ARPE-19 cells was inhibited by sub-lethal photic stress mediated by phagocytised POS. The oxidising ability of POS photobleaching products was demonstrated both in a model system consisting of serum albumin and in ARPE-19 cells. Distinct photooxidation of proteins, mediated by POS, was observed using coumarin boronic acid as a sensitive probe of protein hydroperoxides. Irradiation of POS with blue light also induced oxidation of liposomal cholesterol as determined by HPLC-EC(Hg). Time-resolved singlet oxygen phosphorescence demonstrated the efficiency of retinoids, extracted from POS by chloroform-methanol treatment, to photogenerate singlet oxygen. The results indicate that photic stress mediated by POS photobleaching products could inhibit phagocytic efficiency of RPE cells and, ultimately, compromise their important biological functions.

Asunto(s)

RESUMEN

Retinal pigment epithelial (RPE) cells are among the most actively phagocytic cells in nature. Primary RPE and stable RPE cell lines provide experimental model systems that possess the same phagocytic machinery as RPE in situ. Upon experimental challenge with isolated photoreceptor outer segment fragments (POS), these cells promptly and efficiently recognize, bind, internalize, and digest POS. Here, we describe experimental procedures to isolate POS from porcine eyes and to feed POS to RPE cells in culture. Furthermore, we provide experimental protocols to synchronize the POS binding and engulfment steps of phagocytosis. Finally, we describe three different and complementary methods to quantify total POS uptake by RPE cells and to discriminate surface-bound from engulfed POS.

Asunto(s)

RESUMEN

MerTK is required for photoreceptor outer segment (POS) phagocytosis by retinal pigment epithelial (RPE) cells, a diurnal function essential for vision maintenance. In vivo, MerTK is stimulated at the time of the phagocytic peak through an intracellular signaling pathway. However, MerTK ligands Gas6 and Protein S are expressed in both RPE cells and photoreceptors, and at least one of them required for phagocytosis to occur. Still, their exact role in the retina was not clear until recently. This review combines results from different studies to shed the light on a tissue-specific regulation of MerTK function by its ligands. Indeed, with opposite effects on RPE phagocytosis and changes in their expression levels around the time of POS uptake, Gas6 and Protein S may contribute to the tight control of the acute phagocytic peak in the retina.

Asunto(s)

RESUMEN

The phagocytosis of photoreceptor outer segments (POSs) by the retinal pigment epithelium (RPE) is essential for retinal homeostasis. Defects in this process can be caused by mutations in the photoreceptor cells, the RPE cells, or both cell types. This function can be experimentally investigated by performing an in vitro phagocytosis assay, in which cultured RPE cells are challenged with isolated POSs, and subsequently tested for their ability to degrade the POSs. A significant advantage of this approach is that mutant phenotypes can be attributed either to the photoreceptor or the RPE cells, by experimenting with different permutations of mutant and control photoreceptor and RPE cells. In this chapter, we detail the method for a double-immunofluorescence assay for analysis of the binding, ingestion, and subsequent degradation of isolated mouse POSs by cultured mouse primary RPE cells.

Asunto(s)

RESUMEN

Macroautophagy/autophagy is an intracellular stress survival and recycling system whereas phagocytosis internalizes material from the extracellular milieu; yet, both pathways utilize lysosomes for cargo degradation. Whereas autophagy occurs in all cells, phagocytosis is performed by cell types such as macrophages and the retinal pigment epithelial (RPE) cells of the eye where it is supported by the noncanonical autophagy process termed LC3-associated phagocytosis (LAP). Autophagy and LAP are distinct pathways that use many of the same mediators and must compete for cellular resources, suggesting that cells may regulate both processes under homeostatic and stress conditions. Our data reveal that RPE cells promote LAP through the expression of RUBCN/Rubicon (RUN domain and cysteine-rich domain containing Beclin 1-interacting protein) and suppress autophagy through the activation of EGFR (epidermal growth factor receptor). In the morning when photoreceptor outer segments (POS) phagocytosis and LAP are highest, RUBCN expression is increased. At the same time, outer segment phagocytosis activates the EGFR resulting in MTOR (mechanistic target of rapamycin [serine/threonine kinase]) stimulation, the accumulation of SQSTM1/p62, and the phosphorylation of BECN1 (Beclin 1, autophagy related) on an inhibitory residue thereby suppressing autophagy. Silencing Rubcn, preventing EGFR activity or directly inducing autophagy in RPE cells by starvation inhibits phagocytic degradation of POS. Thus, RPE cells regulate lysosomal pathways during the critical period of POS phagocytosis to support retinal homeostasis.

Asunto(s)

RESUMEN

AIM: To investigate the effect of two ways of lipofuscin production (lipid peroxidation and glycation) on lipofuscin fluorescence characteristics and phototoxicity and to compare them with the properties of natural lipofuscin. METHODS: Model lipofuscins were prepared on the basis of bovine photoreceptor outer segments (POS) with bisretinoid A2E addition. One set of samples was prepared from POS modified by lipid peroxidation, while another set from POS modified by glycation with fructose. Fluorescent properties and kinetics of photoinduced superoxide generation of model lipofuscins and human retinal pigment epithelium (RPE) lipofuscin were compared. The fluorescence spectra of samples were measured at 365 nm excitation wavelength and 380-650 emission wavelength. RESULTS: The fluorescence spectra of model lipofuscins are almost the same as the spectrum of natural lipofuscin. Visible light irradiation of both model lipofuscins and natural lipofuscin isolated from RPE cells leads to decrease of a fluorescence maximum at 550 nm and to appearance of a distinct, new maximum at 445-460 nm. The rate of photogeneration of reactive oxygen forms by both model lipofuscins was almost the same and approximately two times less than that of RPE lipofuscin granules. CONCLUSION: These data suggest that fluorescent characteristics and phototoxicity of lipofuscin granules depend only to an insignificant degree on the oxidative modification of POS proteins and lipids, and generally are defined by the bisretinoid fluorophores contained in them.

RESUMEN

Oxidative stress is a main factor responsible for key changes leading to the onset of age-related macular degeneration (ARMD) that occur in the retinal pigment epithelium (RPE), which is involved in phagocytosis of photoreceptor outer segments (POS). In this study, hydrogen peroxide (H2O2), H2O2 and iron ions (Fe) or rose Bengal (RB) in the presence of NADH and Fe were used to model free radical mediated oxidative stress to test if free radicals and singlet oxygen have different efficiency to inhibit phagocytosis of ARPE-19 cells. Free radical mediated oxidative stress was confirmed by HPLC-EC(Hg) measurements of cholesterol hydroperoxides in treated cells. Electron paramagnetic resonance (EPR) spin trapping was employed to detect superoxide anion. Cell survival was analyzed by the MTT assay. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS and non-specific phagocytosis of fluorescent beads were measured by flow cytometry. HPLC analysis of cells photosensitized with RB in the presence of NADH and Fe indicated substantial increase in formation of free radical-dependent 7α/7ß-hydroperoxides. EPR spin trapping confirmed the photogeneration of superoxide anion in samples enriched with RB, NADH and Fe. For all three protocols sub-lethal oxidative stress induced significant inhibition of the specific phagocytosis of POS. In contrast, non-specific phagocytosis was inhibited only by H2O2 or H2O2 and Fe treatment. Inhibition of phagocytosis was transient and recoverable by 24 h. These results suggest that free radicals may exert similar to singlet oxygen efficiency in inhibiting phagocytosis of RPE cells, and that the effect depends on the location where initial reactive species are formed.

Asunto(s)

RESUMEN

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age-related retinal degenerative disorders particularly age-related macular degeneration. During aging RPE cells decline in number, suggesting an age-dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose-dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted 'postmitotic' status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long-standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age-related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells.

Asunto(s)

RESUMEN

Renewal of rod photoreceptor outer segments in the mammalian eye involves synchronized diurnal shedding after light onset of spent distal outer segment fragments (POS) linked to swift clearance of shed POS from the subretinal space by the adjacent retinal pigment epithelium (RPE). Engulfed POS phagosomes in RPE cells mature to acidified phagolysosomes, which accomplish enzymatic degradation of POS macromolecules. Here, we used an acidophilic fluorophore LysoTracker to label acidic organelles in freshly dissected, live rat RPE tissue flat mounts. We observed that all RPE cells imaged contained numerous acidified POS phagolysosomes whose abundance per cell was dramatically increased 2 h after light onset as compared to either 1 h before or 4 h after light onset. Lack of organelles of similar diameter (of 1-2 µm) in phagocytosis-defective mutant RCS rat RPE confirmed that LysoTracker live imaging detected POS phagolysosomes. Lack of increase in lysosomal membrane protein LAMP-1 in RPE/choroid during the diurnal phagocytic burst suggests that formation of POS phagolysosomes in RPE in situ may not involve extra lysosome membrane biogenesis. Taken together, we report a new imaging approach that directly detects POS phagosome acidification and allows rapid tracking and quantification of POS phagocytosis by live RPE -tissue ex situ.

Asunto(s)

RESUMEN

Zeaxanthin and α-tocopherol have been previously shown to efficiently protect liposomal membrane lipids against photosensitized peroxidation, and to protect cultured RPE cells against photodynamic killing. Here the protective action of combined zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells subjected to photodynamic (PD) stress mediated by rose Bengal (RB) or merocyanine-540 (MC-540) at sub-lethal levels. Stress-induced cytotoxicity was analyzed by the MTT assay. The peroxidation of membrane lipids was determined by HPLC-EC (Hg) measurements of cholesterol hydroperoxides using cholesterol as a mechanistic reporter molecule. The specific phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was measured by flow cytometry, and the levels of phagocytosis receptor proteins αv integrin subunit, ß5 integrin subunit and MerTK were quantified by Western blot analysis. Cytotoxicity measures confirmed that PD stress levels used for phagocytosis analysis were sub-lethal and that antioxidant supplementation protected against higher, lethal PD doses. Sub-lethal PD stress mediated by both photosensitizers induced the accumulation of 5α-OOH and 7α/ß-OOH cholesterol hydroperoxides and the addition of the antioxidants substantially inhibited their accumulation. Antioxidant delivery prior to PD stress also reduced the inhibitory effect of stress on POS phagocytosis and partially reduced the stress-induced diminution of phagocytosis receptor proteins. The use of a novel model system where oxidative stress was induced at sub-lethal levels enable observations that would not be detectable using lethal stress models. Moreover, novel observations about the protective effects of zeaxanthin and α-tocopherol on photodynamic damage to ARPE-19 cell membranes and against reductions in the abundance of receptor proteins involved in POS phagocytosis, a process essential for photoreceptor survival, supports the importance of the antioxidants in protecting of the retina against photooxidative injury.

Asunto(s)

RESUMEN

Autophagy regulates cellular homeostasis and response to environmental stress. Within the retinal pigment epithelium (RPE) of the eye, the level of autophagy can change with both age and disease. The purpose of this study is to determine the relationship between reduced autophagy and age-related degeneration of the RPE. The gene encoding RB1CC1/FIP200 (RB1-inducible coiled-coil 1), a protein essential for induction of autophagy, was selectively knocked out in the RPE by crossing Best1-Cre mice with mice in which the Rb1cc1 gene was flanked with Lox-P sites (Rb1cc1(flox/flox)). Ex vivo and in vivo analyses, including western blot, immunohistochemistry, transmission electron microscopy, fundus photography, optical coherence tomography, fluorescein angiography, and electroretinography were performed to assess the structure and function of the retina as a function of age. Deletion of Rb1cc1 resulted in multiple autophagy defects within the RPE including decreased conversion of LC3-I to LC3-II, accumulation of autophagy-targeted precursors, and increased numbers of mitochondria. Age-dependent degeneration of the RPE occurred, with formation of atrophic patches, subretinal migration of activated microglial cells, subRPE deposition of inflammatory and oxidatively damaged proteins, subretinal drusenoid deposits, and occasional foci of choroidal neovascularization. There was secondary loss of photoreceptors overlying the degenerated RPE and reduction in the electroretinogram. These observations are consistent with a critical role of autophagy in the maintenance of normal homeostasis in the aging RPE, and indicate that disruption of autophagy leads to retinal phenotypes associated with age-related degeneration.

Asunto(s)

RESUMEN

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.

Asunto(s)

RESUMEN

BACKGROUND AND OBJECTIVE: To investigate photoreceptor outer segment layer thickness measured with a manual technique on images from spectral domain optical coherence tomography (OCT) in healthy volunteers. MATERIALS AND METHODS: In 60 eyes of 30 healthy volunteers, a spectral domain OCT device (Spectralis, Heidelberg Engineering) was used to obtain cross-sectional images of the retina. For each volunteer, two images of each eye were obtained in one sitting. Images were digitally enlarged and the manual calipers feature of the device's software was used to measure, at the lowest point in the fovea, the thickness of the photoreceptor outer segment layer. All measurements were performed by the same investigator. Repeatability was evaluated with the Bland-Altman repeatability coefficient, and intersubject variability with Pearson's coefficient of variation. RESULTS: The mean values of measurements across all the volunteers were as follows: right eye first image 38.1 micrometers, right eye second image 37.9 micrometers, left eye first image 37.9 micrometers, left eye second image 37.9 micrometers. The repeatability coefficient, i.e. the difference between repeated measurements which would be exceeded in only 5% of cases, was 1.6 micrometers. Coefficients of variation for the right eye were 3.4% for the first images and 3.4% for the second images, and for the left eye they were 3.2 and 4.0% respectively. CONCLUSION: With a manual method based on spectral domain OCT, the thickness of the photoreceptor outer segment layer at the central fovea can be measured within a useful range of repeatability and appears to be relatively constant across healthy volunteers.

Asunto(s)