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Photoreceptor Outer Segments (photoreceptor + outer_segment)
Selected AbstractsRetina expresses a novel variant of the ryanodine receptorEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2007Varda Shoshan-Barmatz Abstract Calcium released from intracellular stores via the ryanodine receptor (RyR) mediates a variety of signalling processes. We previously showed that retina expresses the three known types of RyR, but retinal membrane preparations exhibit unique characteristics such as Ca2+ -independent [3H]ryanodine-binding and inhibition by caffeine. We have heretofore suggested that the major retinal RyR isoform is novel. The present study aimed to identify this receptor isoform and to localize RyR in mammalian retina. Immunoblotting with specific and pan-antibodies showed that the major retinal RyR has a mobility similar to that of RyR2 or RyR3. Real-time PCR revealed that the major type is RyR2, and RT-PCR followed by sequencing showed a transcript that encodes a protein with ~ 99% identity to RyR2, yet lacking two regions of seven and 12 amino acids and including an additional insertion of eight amino acids. An antibody against RyR2 localized this type to somas and primary dendrites of most retinal neurons. An antibody against RyR1 localized RyR to most somas but also revealed staining in photoreceptor outer segments, concentrated on the disk membranes at their rim. The ryanodine-binding properties and the electrophoretic mobility of RyR from the outer segments were similar to those of the whole retinal preparation. The results thus identify a novel variant of RyR2 which can contribute to regulating photoreceptor Ca2+ concentrations. The restricted localization of the outer segment RyR to the disk rim suggests that its activation mechanism involves a coupling between retinal RyR and the cGMP-gated channel. [source] Pigment epithelium-derived factor supports normal Müller cell development and glutamine synthetase expression after removal of the retinal pigment epitheliumGLIA, Issue 1 2001Monica M. Jablonski Abstract In conditions in which the retinal pigment epithelium (RPE) is dystrophic, carries a genetic mutation, or is removed physically, Müller cells undergo degenerative changes that contribute to the retinal pathology. We previously demonstrated that pigment epithelium-derived factor (PEDF), a glycoprotein secreted by the RPE cells with neuroprotective and differentiation properties, protects against photoreceptor degeneration induced by RPE removal. The purpose of the present study was to analyze the putative gliosupportive activity of PEDF on Müller cells of RPE-deprived retinas and assess whether protection of Müller cells was correlated with improved photoreceptor outer segment assembly. Eyes were dissected from Xenopus laevis tadpoles, and the RPE was removed before culturing in medium containing purified PEDF, PEDF plus anti-PEDF, or medium alone. Control eyes matured with an adherent RPE or in medium containing PEDF plus nonimmune serum. Müller cell ultrastructure was examined. Glial fibrillary acidic protein (GFAP) and glutamine synthetase were localized immunocytochemically, and the corresponding protein levels were quantified. In control retinas, Müller cells were structurally intact and formed adherens junctions with neighboring photoreceptors. In addition, they did not express GFAP, whereas glutamine synthetase expression was high. RPE removal dramatically altered the ultrastructure and biosynthetic activity of Müller cells; Müller cells failed to form adherens junctions with photoreceptors and glutamine synthetase expression was suppressed. PEDF prevented the degenerative glial response; Müller cells were ultrastructurally normal and formed junctional complexes with photoreceptors. PEDF also preserved the expression of glutamine synthetase at near-normal levels. The morphogenetic effects of PEDF were blocked by the anti-PEDF antibody. Our study documents the glioprotective effects of PEDF and suggests that maintenance of the proper Müller cell ultrastructure and expression of glutamine synthetase may be necessary to support the proper assembly of photoreceptor outer segments. GLIA 35:14,25, 2001. © 2001 Wiley-Liss, Inc. [source] Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy-mediated proteolysis in human retinal pigment epithelial cellsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 9b 2009Tuomas Ryhänen Abstract The pathogenesis of age-related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, but the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells is poorly understood. Prior to proteolysis, heat shock proteins (Hsps), agents that function as molecular chaperones, attempt to refold misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. In the present study, the roles of the Hsp70 molecular chaperone and proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE-19). The Hsp70 and ubiquitin protein levels and localization were analysed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. Hsp70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. The proteasome inhibitor MG-132 evoked the accumulation of perinuclear aggregates positive for Hsp70, ubiquitin-protein conjugates and the lysosomal membrane protein LAMP-2. Interestingly, the hsp70 mRNA depletion significantly increased cell death in conjunction with proteasome inhibition. We found that the accumulation of lysosomes was reversible: a cessation of proteasome inhibition led to clearance of the deposits via a mechanism believed to include autophagy. The molecular chaperone Hsp70, proteasomes and autophagy have an important regulatory role in the protein turnover of human RPE cells and may thus open new avenues for understanding degenerative processes in retinal cells. [source] 2131: Human pluripotent stem cells provide excellent source of functional pigment epithelial cellsACTA OPHTHALMOLOGICA, Issue 2010H SKOTTMAN Purpose Defined differentiation of functional RPE cells from human embryonic (hESC) or induced pluripotent stem cells (iPSC) is a prerequisite for their use in individualised disease modelling, drug discovery and transplantation for retinal diseases. In this study we report differentiation of RPE cells from hESC and iPSC in condition enabling easy translation to clinical quality cell production. Methods Pluripotent stem cells were produced on human fibroblast feeder cells in serum-free medium. The differentiation of the cells was induced using bFGF and feeder cell removal approach under serum-free conditions. The pigmentation and RPE morphology of the cells were analysed and the expression of genes and proteins characteristics for RPE cells were studied. The in vitro functionality of the cells was analysed using ELISA measurements and phagocytosis of photoreceptor outer segments. The integrity of the generated RPE layer was analysed using transepithelial electric resistance (TEER) measurements. Results With our differentiation method, we were able to generate RPE cells with satisfying efficiency. The typical pigmented cobblestone-like morphology and expression of RPE specific markers were confirmed at gene and protein level. The differentiated cells were able to phagocytose and secreted growth factors typical for RPE cells. In addition, cells formed a well polarized epithelium with high integrity, exhibiting good TEER values. Conclusion We have developed progressive differentiation protocol for production of functional RPE cells from hESC and iPSC. The developed production method is currently translated to defined and animal component free conditions enabling clinical grade cell production. [source] Choroidal neovascularization associated with cancer-associated retinopathyACTA OPHTHALMOLOGICA, Issue 5 2010Giuseppe Querques Abstract. Purpose:, To report an unusual association between cancer-associated retinopathy (CAR) associated with invasive thymoma and choriodal neovascularization (CNV), treated by photodynamic theraphy (PDT). Methods:, A 39-year-old man affected with thymoma and paraneoplastic syndrome (myasthenia gravis and diarrhoea) was observed between October 1997 and September 2007. The patient developed progressive visual dysfunction including bilateral visual acuity loss and concentric constriction of visual fields. Ophthalmological, immunological and systemic examinations were performed. Immunological evaluations included an assessment of antibody activity by indirect immunohistochemistry on sectioned rhesus monkey eye, and Western blot reactions upon an extract of pig retina. Results:, Fundus ophthalmoscopy and fluorescein angiography revealed retinal vessel attenuation and retinal pigment epithelium degeneration. Electroretinogram suggested both rod and cone dysfunction. Indirect immunohistochemistry identified antibody activity within the photoreceptor outer segments. Western blots on the retina revealed that most of the patient's antibody activity was focused upon a retinal protein antigen approximating 145 kD. These findings share the commonalities of size and retinal distribution of the interphotoreceptor retinoid-binding protein (IRBP), a recognized autoantigen. The surgically resected mediastinal tumour was diagnosed as invasive thymoma. No other malignancy has since been found throughout nearly 10 years of follow-up. In March 2006, the patient developed a subfoveal CNV in his left eye, which was treated by PDT. Conclusion:, We describe the third case of paraneoplastic retinopathy associated with invasive thymoma. This is the first example of CAR involving autoantibodies reactive with a retinal protein having the characteristics of the IRBP, and is also the first complicated by CNV treated by PDT. [source] Complement factor H and factor B expression in RPE cellsACTA OPHTHALMOLOGICA, Issue 2008Purpose Age-related macular degeneration (AMD) is the leading cause of untreatable blindness in the developed world. The pathogenesis of AMD is not fully understood. Recent evidence suggests that local inflammation in particular complement activation plays an important role. We aim to understand how complement activation is regulated at retina/choroidal interface. Methods The expression and distribution of complement factor H (CFH) and factor B (CFB) in mouse ocular tissues were examined by immunohistochemistry. Regulation of CFH and CFB gene expression by various cytokines or photoreceptor outer segments (POS) was investigated in vitro in cultured RPE cells. Changes in CFH or CFB gene expression after treatment were evaluated by RT-PCR. Results In normal mouse eyes, CFH was detected in corneal epithelial cells, ciliary body, RPE cells, Bruch's membrane and choroidal vessels. There is no significant change in either the expression level or the distribution pattern of CFH in ocular tissues of different ages of mice. CFB was exclusively detected in RPE cells in normal mice. The expression of CFB in RPE cells increases with age. In vitro in RPE cultures, the expression of CFH was negatively regulated by cytokine TNF-alpha and IL-6, whereas the expression of CFB was positively regulated by TNF-alpha and IFN-gamma. Short-term incubation of RPE cells with POS did not alter the expression of CFH or CFB, whereas long-term incubation of RPE cells with POS significantly down-regulated CFH expression but up-regulated CFB expression. Conclusion Complement regulatory factors CFH and CFB are produced locally in the retina/choroidal interface by RPE cells. The production of CFH and CFB in RPE cells is regulated differently by various cytokines and oxidized POS. [source] | |||||||||||||