Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin.

Daily phagocytosis by the retinal pigment epithelium (RPE) of spent photoreceptor outer segment fragments is critical for vision. In the retina, early morning circadian photoreceptor rod shedding precedes synchronized uptake of shed photoreceptor particles by RPE cells. In vitro, RPE cells use the integrin receptor alphavbeta5 for particle binding. Here, we tested RPE phagocytosis and retinal function in beta5 integrin--deficient mice, which specifically lack alphavbeta5 receptors. Retinal photoresponses severely declined with age in beta5-/- mice, whose RPE accumulated autofluorescent storage bodies that are hallmarks of human retinal aging and disease. beta5-/- RPE in culture failed to take up isolated photoreceptor particles. beta5-/- RPE in vivo retained basal uptake levels but lacked the burst of phagocytic activity that followed circadian photoreceptor shedding in wild-type RPE. Rhythmic activation of focal adhesion and Mer tyrosine kinases that mediate wild-type retinal phagocytosis was also completely absent in beta5-/- retina. These results demonstrate an essential role for alphavbeta5 integrin receptors and their downstream signaling pathways in synchronizing retinal phagocytosis. Furthermore, they identify the beta5-/- integrin mouse strain as a new animal model of age-related retinal dysfunction

Disease-causing mutations in BEST1 gene are associated with altered sorting of bestrophin-1 protein

Doumanov, Jordan A. et al.Mutations in BEST1 gene, encoding the bestrophin-1 (Best1) protein are associated with macular dystrophies. Best1 is predominantly expressed in the retinal pigment epithelium (RPE), and is inserted in its basolateral membrane. We investigated the cellular localization in polarized MDCKII cells of disease-associated Best1 mutant proteins to study specific sorting motifs of Best1. Real-time PCR and western blots for endogenous expression of BEST1 in MDCK cells were performed. Best1 mutant constructs were generated using site-directed mutagenesis and transfected in MDCK cells. For protein sorting, confocal microscopy studies, biotinylation assays and statistical methods for quantification of mislocalization were used. Analysis of endogenous expression of BEST1 in MDCK cells revealed the presence of BEST1 transcript but no protein. Confocal microscopy and quantitative analyses indicate that transfected normal human Best1 displays a basolateral localization in MDCK cells, while cell sorting of several Best1 mutants (Y85H, Q96R, L100R, Y227N, Y227E) was altered. In contrast to constitutively active Y227E, constitutively inactive Y227F Best1 mutant localized basolaterally similar to the normal Best1 protein. Our data suggest that at least three basolateral sorting motifs might be implicated in proper Best1 basolateral localization. In addition, non-phosphorylated tyrosine 227 could play a role for basolateral delivery. © 2013 by the authors; licensee MDPI, Basel, Switzerland.This work was supported by Agence Nationale de la Recherche (Chaire d’Excellence to Shomi S. Bhattacharya), Fondation Voir et Entendre (Young Investigator Grants to Christina Zeitz and Emeline F. Nandrot), Centre National de la Recherche Scientifique (CNRS) and Fondation Bettencourt Schueller (to Emeline F. Nandrot), Université Pierre et Marie Curie-Paris6 (Bonus Qualité Recherche to Christina Zeitz), Foundation Fighting Blindness (grant number CD-CL-0808-0466-CHNO to Isabelle Audo), Centre d’Investigation Clinique 503 recognized as a Foundation Fighting Blindness Center (grant number C-CMM-0907-0428-INSERM04), Fundacion Progreso y Salud (to Shomi S. Bhattacharya, Maria Luz Bellido Diaz, Abhay Krishna and Paloma Dominguez Gimenez), Instituto de Salud Carlos III (grant number CM06/00183 to Maria Luz Bellido Diaz) and Bulgarian National Science Fund (grant number DDVU 02/10). Additionally, the Institut de la Vision is funded by Institut National de la Santé et de la Recherche Médicale, Université Pierre et Marie Curie-Paris 6, Centre National de la Recherche Scientifique and Départment de Paris.Peer Reviewe

Retinal atrophy, inflammation, phagocytic and metabolic disruptions develop in the MerTK-cleavage-resistant mouse model

In the eye, cells from the retinal pigment epithelium (RPE) facing the neurosensory retina exert several functions that are all crucial for long-term survival of photoreceptors (PRs) and vision. Among those, RPE cells phagocytose under a circadian rhythm photoreceptor outer segment (POS) tips that are constantly subjected to light rays and oxidative attacks. The MerTK tyrosine kinase receptor is a key element of this phagocytic machinery required for POS internalization. Recently, we showed that MerTK is subjected to the cleavage of its extracellular domain to finely control its function. In addition, monocytes in retinal blood vessels can migrate inside the inner retina and differentiate into macrophages expressing MerTK, but their role in this context has not been studied yet. We thus investigated the ocular phenotype of MerTK cleavage-resistant (MerTKCR) mice to understand the relevance of this characteristic on retinal homeostasis at the RPE and macrophage levels. MerTKCR retinae appear to develop and function normally, as observed in retinal sections, by electroretinogram recordings and optokinetic behavioral tests. Monitoring of MerTKCR and control mice between the ages of 3 and 18  months showed the development of large degenerative areas in the central retina as early as 4 months when followed monthly by optical coherence tomography (OCT) plus fundus photography (FP)/autofluorescence (AF) detection but not by OCT alone. The degenerative areas were associated with AF, which seems to be due to infiltrated macrophages, as observed by OCT and histology. MerTKCR RPE primary cultures phagocytosed less POS in vitro, while in vivo, the circadian rhythm of POS phagocytosis was deregulated. Mitochondrial function and energy production were reduced in freshly dissected RPE/choroid tissues at all ages, thus showing a metabolic impairment not present in macrophages. RPE anomalies were detected by electron microscopy, including phagosomes retained in the apical area and vacuoles. Altogether, this new mouse model displays a novel phenotype that could prove useful to understanding the interplay between RPE and PRs in inflammatory retinal degenerations and highlights new roles for MerTK in the regulation of the energetic metabolism and the maintenance of the immune privilege in the retina

Retinal pigment epithelial cells: the unveiled component in the etiology of Prpf splicing factor-associated retinitis pigmentosa

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The Retinal Pigment Epithelium: Cells That Know the Beat!

International audienceThe 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, β5 −/− and Prpf31 +/− mice, display distinct alterations in both functions leading to different phenotypes. Indeed, the phagocytic peak totally disappears in β5 knockout mice but is attenuated and shifted in Prpf31 +/− mice. Conversely, the retinal adhesion peak only attenuated in β5 −/− mice is lost in Prpf31 +/− mice. These distinct alterations have different consequences on retinal homeostasis proportional to the observed defects: β5 −/− 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

Retinal pigment epithelial cells: super phagocytes with a rhythm

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Lack of v 5 Integrin Receptor or Its Ligand MFG-E8: Distinct Effects on Retinal Function

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Novel role for alphavbeta5-integrin in retinal adhesion and its diurnal peak

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