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Cells May 2023Mutations in the gene cause inherited retinal disease; however, the pathogenic mechanisms associated with RCBTB1 deficiency remain poorly understood. Here, we...
Mutations in the gene cause inherited retinal disease; however, the pathogenic mechanisms associated with RCBTB1 deficiency remain poorly understood. Here, we investigated the effect of RCBTB1 deficiency on mitochondria and oxidative stress responses in induced pluripotent stem cell (iPSC)-derived retinal pigment epithelial (RPE) cells from control subjects and a patient with -associated retinopathy. Oxidative stress was induced with tert-butyl hydroperoxide (tBHP). RPE cells were characterized by immunostaining, transmission electron microscopy (TEM), CellROX assay, MitoTracker assay, quantitative PCR and immunoprecipitation assay. Patient-derived RPE cells displayed abnormal mitochondrial ultrastructure and reduced MitoTracker fluorescence compared with controls. Patient RPE cells displayed increased levels of reactive oxygen species (ROS) and were more sensitive to tBHP-induced ROS generation than control RPE. Control RPE upregulated and expression in response to tBHP treatment; however, this response was highly attenuated in patient RPE. RCBTB1 was co-immunoprecipitated from control RPE protein lysates by antibodies for either UBE2E3 or CUL3. Together, these results demonstrate that deficiency in patient-derived RPE cells is associated with mitochondrial damage, increased oxidative stress and an attenuated oxidative stress response.
Topics: Humans; Reactive Oxygen Species; Antioxidants; Retinal Diseases; Epithelial Cells; Mitochondria; Retinal Pigments; Guanine Nucleotide Exchange Factors
PubMed: 37408192
DOI: 10.3390/cells12101358 -
Discovery Medicine 2022Transepithelial/transendothelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell... (Review)
Review
Transepithelial/transendothelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell culture models of endothelial and epithelial monolayers. The value of TEER reflects the physical structure and characteristics of epithelial/endothelial cells. TEER value is a preferred physiological indicator reflecting transport rate of ions and macromolecules through the paracellular pathway, which is used to evaluate permeability of paracellular pathway. TEER value has a high specificity for the permeability of reactive tightly connected complex. TEER value is an effective indicator to evaluate the integrity of cell barrier. The cell barrier not only controls the diffusion penetration of various substances in adjacent intercellular spaces, but also regulates the transport of ions and macromolecules across. On one hand, the cell barrier protects the body from harmful substances; on the other hand, it restricts the entry of therapeutic drugs. Therefore, with the increase of permeability in paraepithelial pathway, the TEER value decreased, otherwise, it increased. In this review article, we compared the advantages and disadvantages of the existing methods for measuring TEER and summarized the factors affecting TEER accuracy, as well as the roles of TEER in mechanisms of retinal pigment epithelial barrier and retinal disorders such as proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa.
Topics: Humans; Electric Impedance; Retinal Pigments; Endothelial Cells; Tight Junctions; Permeability; Epithelial Cells; Retinal Diseases
PubMed: 36274257
DOI: No ID Found -
Developmental Biology Jul 2021Vertebrate rod and cone photoreceptors detect light via a specialized organelle called the outer segment. This structure is packed with light-sensitive molecules known... (Review)
Review
Vertebrate rod and cone photoreceptors detect light via a specialized organelle called the outer segment. This structure is packed with light-sensitive molecules known as visual pigments that consist of a G-protein-coupled, seven-transmembrane protein known as opsin, and a chromophore prosthetic group, either 11-cis retinal ('A') or 11-cis 3,4-didehydroretinal ('A'). The enzyme cyp27c1 converts A into A in the retinal pigment epithelium. Replacing A with A in a visual pigment red-shifts its spectral sensitivity and broadens its bandwidth of absorption at the expense of decreased photosensitivity and increased thermal noise. The use of vitamin A-based visual pigments is strongly associated with the occupation of aquatic habitats in which the ambient light is red-shifted. By modulating the A/A ratio in the retina, an organism can dynamically tune the spectral sensitivity of the visual system to better match the predominant wavelengths of light in its environment. As many as a quarter of all vertebrate species utilize A, at least during a part of their life cycle or under certain environmental conditions. A utilization therefore represents an important and widespread mechanism of sensory plasticity. This review provides an up-to-date account of the A/A chromophore exchange system.
Topics: Animals; Opsins; Photoreceptor Cells, Vertebrate; Retina; Retinal Cone Photoreceptor Cells; Retinal Pigment Epithelium; Retinal Pigments; Retinal Rod Photoreceptor Cells; Rod Opsins; Vitamin A
PubMed: 33684435
DOI: 10.1016/j.ydbio.2021.03.002 -
Cell Reports May 2024Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment...
Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment epithelium (RPE) and Müller glia (MG), but the relative functional importance of these two cellular pools is debated. Here, we report RPE- and MG-specific CRALBP knockout (KO) mice and examine their photoreceptor and visual cycle function. Bulk visual chromophore regeneration in RPE-KO mice is 15-fold slower than in controls, accounting for their delayed rod dark adaptation and protection against retinal phototoxicity, whereas MG-KO mice have normal bulk visual chromophore regeneration and retinal light damage susceptibility. Cone pigment regeneration is significantly impaired in RPE-KO mice but mildly affected in MG-KO mice, disclosing an unexpectedly strong reliance of cone photoreceptors on the RPE-based visual cycle. These data reveal a dominant role for RPE-CRALBP in supporting rod and cone function and highlight the importance of RPE cell targeting for CRALBP gene therapies.
Topics: Animals; Mice; Carrier Proteins; Ependymoglial Cells; Mice, Inbred C57BL; Mice, Knockout; Retinal Cone Photoreceptor Cells; Retinal Pigment Epithelium; Retinal Pigments; Retinal Rod Photoreceptor Cells; Male; Female
PubMed: 38676924
DOI: 10.1016/j.celrep.2024.114143 -
Proceedings of the National Academy of... Aug 2016It is a deeply engrained notion that the visual pigment rhodopsin signals light as a monomer, even though many G protein-coupled receptors are now known to exist and...
It is a deeply engrained notion that the visual pigment rhodopsin signals light as a monomer, even though many G protein-coupled receptors are now known to exist and function as dimers. Nonetheless, recent studies (albeit all in vitro) have suggested that rhodopsin and its chromophore-free apoprotein, R-opsin, may indeed exist as a homodimer in rod disk membranes. Given the overwhelmingly strong historical context, the crucial remaining question, therefore, is whether pigment dimerization truly exists naturally and what function this dimerization may serve. We addressed this question in vivo with a unique mouse line (S-opsin(+)Lrat(-/-)) expressing, transgenically, short-wavelength-sensitive cone opsin (S-opsin) in rods and also lacking chromophore to exploit the fact that cone opsins, but not R-opsin, require chromophore for proper folding and trafficking to the photoreceptor's outer segment. In R-opsin's absence, S-opsin in these transgenic rods without chromophore was mislocalized; in R-opsin's presence, however, S-opsin trafficked normally to the rod outer segment and produced functional S-pigment upon subsequent chromophore restoration. Introducing a competing R-opsin transmembrane helix H1 or helix H8 peptide, but not helix H4 or helix H5 peptide, into these transgenic rods caused mislocalization of R-opsin and S-opsin to the perinuclear endoplasmic reticulum. Importantly, a similar peptide-competition effect was observed even in WT rods. Our work provides convincing evidence for visual pigment dimerization in vivo under physiological conditions and for its role in pigment maturation and targeting. Our work raises new questions regarding a potential mechanistic role of dimerization in rhodopsin signaling.
Topics: Animals; Endoplasmic Reticulum; Mice; Opsins; Protein Multimerization; Reactive Oxygen Species; Retinal Pigments
PubMed: 27462111
DOI: 10.1073/pnas.1609018113 -
Frontiers in Immunology 2023Age related macular degeneration (AMD) is the most common cause of blindness in the elderly. Oxidative stress contributes to retinal pigment epithelium (RPE) dysfunction...
Age related macular degeneration (AMD) is the most common cause of blindness in the elderly. Oxidative stress contributes to retinal pigment epithelium (RPE) dysfunction and cell death thereby leading to AMD. Using improved RPE cell model systems, such as human telomerase transcriptase-overexpressing (hTERT) RPE cells (hTERT-RPE), pathophysiological changes in RPE during oxidative stress can be better understood. Using this model system, we identified changes in the expression of proteins involved in the cellular antioxidant responses after induction of oxidative stress. Some antioxidants such as vitamin E (tocopherols and tocotrienols) are powerful antioxidants that can reduce oxidative damage in cells. Alpha-tocopherol (α-Toc or αT) and gamma-tocopherol (γ-Toc or γT) are well-studied tocopherols, but signaling mechanisms underlying their respective cytoprotective properties may be distinct. Here, we determined what effect oxidative stress, induced by extracellularly applied tBHP in the presence and absence of αT and/or γT, has on the expression of antioxidant proteins and related signaling networks. Using proteomics approaches, we identified differential protein expression in cellular antioxidant response pathways during oxidative stress and after tocopherol treatment. We identified three groups of proteins based on biochemical function: glutathione metabolism/transfer, peroxidases and redox-sensitive proteins involved in cytoprotective signaling. We found that oxidative stress and tocopherol treatment resulted in unique changes in these three groups of antioxidant proteins indicate that αT and γT independently and by themselves can induce the expression of antioxidant proteins in RPE cells. These results provide novel rationales for potential therapeutic strategies to protect RPE cells from oxidative stress.
Topics: Humans; Aged; Antioxidants; Proteome; Oxidative Stress; Tocopherols; Macular Degeneration; Epithelial Cells; Retinal Pigments
PubMed: 37153596
DOI: 10.3389/fimmu.2023.1138519 -
Proceedings of the National Academy of... Oct 2023Many neurons in the central nervous system produce a single primary cilium that serves as a specialized signaling organelle. Several neuromodulatory G-protein-coupled...
Many neurons in the central nervous system produce a single primary cilium that serves as a specialized signaling organelle. Several neuromodulatory G-protein-coupled receptors (GPCRs) localize to primary cilia in neurons, although it is not understood how GPCR signaling from the cilium impacts circuit function and behavior. We find that the vertebrate ancient long opsin A (VALopA), a G-coupled GPCR extraretinal opsin, targets to cilia of zebrafish spinal neurons. In the developing 1-d-old zebrafish, brief light activation of VALopA in neurons of the central pattern generator circuit for locomotion leads to sustained inhibition of coiling, the earliest form of locomotion. We find that a related extraretinal opsin, VALopB, is also G-coupled, but is not targeted to cilia. Light-induced activation of VALopB also suppresses coiling, but with faster kinetics. We identify the ciliary targeting domains of VALopA. Retargeting of both opsins shows that the locomotory response is prolonged and amplified when signaling occurs in the cilium. We propose that ciliary localization provides a mechanism for enhancing GPCR signaling in central neurons.
Topics: Animals; Zebrafish; Receptors, G-Protein-Coupled; Signal Transduction; Opsins; Rod Opsins; Neurons; Cilia
PubMed: 37844228
DOI: 10.1073/pnas.2311131120 -
The Journal of Biological Chemistry 2021All that we view of the world begins with an ultrafast cis to trans photoisomerization of the retinylidene chromophore associated with the visual pigments of rod and... (Review)
Review
All that we view of the world begins with an ultrafast cis to trans photoisomerization of the retinylidene chromophore associated with the visual pigments of rod and cone photoreceptors. The continual responsiveness of these photoreceptors is then sustained by regeneration processes that convert the trans-retinoid back to an 11-cis configuration. Recent biochemical and electrophysiological analyses of the retinal G-protein-coupled receptor (RGR) suggest that it could sustain the responsiveness of photoreceptor cells, particularly cones, even under bright light conditions. Thus, two mechanisms have evolved to accomplish the reisomerization: one involving the well-studied retinoid isomerase (RPE65) and a second photoisomerase reaction mediated by the RGR. Impairments to the pathways that transform all-trans-retinal back to 11-cis-retinal are associated with mild to severe forms of retinal dystrophy. Moreover, with age there also is a decline in the rate of chromophore regeneration. Both pharmacological and genetic approaches are being used to bypass visual cycle defects and consequently mitigate blinding diseases. Rapid progress in the use of genome editing also is paving the way for the treatment of disparate retinal diseases. In this review, we provide an update on visual cycle biochemistry and then discuss visual-cycle-related diseases and emerging therapeutics for these disorders. There is hope that these advances will be helpful in treating more complex diseases of the eye, including age-related macular degeneration (AMD).
Topics: Animals; Humans; Isomerism; Retinal Cone Photoreceptor Cells; Retinal Pigments; Vertebrates; Vision, Ocular; cis-trans-Isomerases
PubMed: 33187985
DOI: 10.1074/jbc.REV120.014405 -
Journal of Visualized Experiments : JoVE Apr 2023The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed...
The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.
Topics: Animals; Humans; Lipofuscin; Retinal Pigments; Phagocytosis; Retinal Pigment Epithelium; Cell Line; Cells, Cultured
PubMed: 37125790
DOI: 10.3791/65242 -
Translational Vision Science &... Nov 2022The purpose of this study was to compare the performances of infrared (IR), fundus autofluorescence (FAF), and multicolor (MC) imaging in the characterization of...
PURPOSE
The purpose of this study was to compare the performances of infrared (IR), fundus autofluorescence (FAF), and multicolor (MC) imaging in the characterization of geographic atrophy, with a focus on the possibility to detect incomplete retinal pigmented and outer retinal atrophy (iRORA) on en face imaging.
METHODS
The ground truth was established by two graders evaluating atrophy on spectral-domain optical coherence tomography (SD-OCT) images. A score for visibility of foveal sparing and margins of atrophy was attributed. Measurement of the atrophic area and the fovea-to-margin distance were performed. Accuracy of detection of foveal sparing was evaluated through comparison with B-scan images ground truth, with/without the inclusion of patients with foveal iRORA.
RESULTS
Seventy patients were included in this study. Foveal sparing and atrophy's margins subjective visibility were significantly higher rated on MC images compared to IR and FAF (P < 0.005 and P < 0.001). Agreement with OCT B-scan assessed foveal sparing revealed a significantly higher area under receiver operating characteristic curves (AUROC) for MC images at the analysis performed both with (0.876) and without (0.853) inclusion of patients with foveal iRORA (P < 0.001 and P = 0.006). Quantitative measurements revealed lower atrophy extension (P = 0.026) and fovea-to-margin distance (P = 0.019) with MC imaging.
CONCLUSIONS
MC imaging performed better at foveal sparing assessment, especially in the setting of foveal iRORA. MC also resulted in higher visibility of atrophy's margins, lower atrophy extension measurements, and lower distance from the fovea to atrophy's margins compared to both FAF and IR.
TRANSLATIONAL RELEVANCE
MC rated significantly higher in foveal sparing and atrophy detection, higher visibility of atrophy's margins, lower atrophy extension measurements, and lower distance from the fovea to atrophy's margins, compared to FAF and IR.
Topics: Humans; Geographic Atrophy; Fovea Centralis; Optical Imaging; Retinal Pigments; Atrophy; Margins of Excision; Multimodal Imaging
PubMed: 36445699
DOI: 10.1167/tvst.11.11.21