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Proceedings of the National Academy of... Jan 2013The presence of a photopigment (melanopsin) within certain retinal ganglion cells was a surprising and significant discovery. This pigment is routinely described as...
The presence of a photopigment (melanopsin) within certain retinal ganglion cells was a surprising and significant discovery. This pigment is routinely described as "nonvisual" to highlight its signaling role in pupil dilation and circadian rhythms. Here we asked whether light absorbed by melanopsin can be seen by healthy human subjects. To answer this requires delivering intense (above rod saturation), well-controlled lights using four independent primaries. We collected detection thresholds to many four-primary stimuli. Threshold measurements in the fovea are explained by trichromatic theory, with no need to invoke a fourth photopigment. In the periphery, where melanopsin is present, threshold measurements deviate from trichromatic theory; at high photopic levels, sensitivity is explained by absorptions in four, not three, photopigment classes. We consider a series of hypotheses to explain the tetrasensitivity at high photopic levels in the human peripheral field. The most likely hypothesis is that in healthy human subjects melanopsin absorptions influence visibility.
Topics: Adult; Color Vision; Female; Fovea Centralis; Humans; Male; Models, Biological; Optical Phenomena; Photic Stimulation; Retina; Retinal Cone Photoreceptor Cells; Retinal Pigments; Retinal Rod Photoreceptor Cells; Rod Opsins
PubMed: 23256158
DOI: 10.1073/pnas.1214240110 -
Experimental Eye Research May 2022The antimalarial drug chloroquine (CQ) induces retinopathy, a disorder characterized by lysosomotropic alteration. In this study, we examined whether D4476...
The antimalarial drug chloroquine (CQ) induces retinopathy, a disorder characterized by lysosomotropic alteration. In this study, we examined whether D4476 (4-(4-(2,3-dihydrobenzo [1,4] dioxin-6-yl)-5-pyridin-2-yl-1H-imidazole-2-yl) benzamide), a specific casein kinase 1 inhibitor, alleviate CQ-induced retinopathy in adult retinal pigment epithelial (ARPE-19) cells. Cultured ARPE-19 cells were exposed to CQ with or without D4476 and cell death was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To examine autophagy flux, ARPE-19 cells were transfected with green fluorescence protein light chain 3 (GFP-LC3)-red fluorescence protein (RFP)-LC3ΔG plasmid DNA and co-stained with the lysosomal-associated membrane protein (LAMP)-1 antibody. Western blotting and fluorescence-activated cell sorting (FACS) showed apoptosis, whereas the fluorescence intensity of 2'-7'-dichlorofluorescein diacetate revealed levels of cellular oxidative stress. We then confirmed the effect of D4476 on the interaction between Beclin 1 and B-cell lymphoma-2 (Bcl-2) through immunoprecipitation with an anti-Bcl-2 antibody. Following CQ exposure, ARPE-19 cells accumulated autophagosomes because of defective lysosomal degradation. Furthermore, CQ trapped Beclin 1 with Bcl-2, disturbing autophagy initiation and autolysosome formation. However, D4476 alleviated CQ-induced effects by rescuing ARPE-19 cells from CQ-induced toxicity by modulating the association between Beclin 1 and Bcl-2. Therefore, D4476 controls autophagy and apoptosis simultaneously by upregulating autophagy flux, decreasing ROS formation, and triggering the expression of anti-apoptotic proteins through inhibition of mTOR, JNK, and p38 MAPK signals. We conclude that D4476 is a promising treatment strategy for CQ-mediated retinopathy.
Topics: Apoptosis; Autophagy; Beclin-1; Casein Kinase I; Chloroquine; Humans; Proto-Oncogene Proteins c-bcl-2; Retinal Diseases; Retinal Pigment Epithelium; Retinal Pigments
PubMed: 35219693
DOI: 10.1016/j.exer.2022.109004 -
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; Retinal Pigment Epithelium; Mice; Mice, Knockout; Carrier Proteins; Retinal Cone Photoreceptor Cells; Ependymoglial Cells; Retinal Rod Photoreceptor Cells; Mice, Inbred C57BL; Retinal Pigments
PubMed: 38676924
DOI: 10.1016/j.celrep.2024.114143 -
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 -
Human Molecular Genetics Feb 2022The retinal pigment epithelium of the vertebrate eyes acquires vitamin A from circulating retinol binding protein for chromophore biosynthesis. The chromophore...
The retinal pigment epithelium of the vertebrate eyes acquires vitamin A from circulating retinol binding protein for chromophore biosynthesis. The chromophore covalently links with an opsin protein in the adjacent photoreceptors of the retina to form the bipartite visual pigment complexes. We here analyzed visual pigment biosynthesis in mice deficient for the retinol-binding protein receptor STRA6. We observed that chromophore content was decreased throughout the life cycle of these animals, indicating that lipoprotein-dependent delivery pathways for the vitamin cannot substitute for STRA6. Changes in the expression of photoreceptor marker genes, including a downregulation of the genes encoding rod and cone opsins, paralleled the decrease in ocular retinoid concentration in STRA6-deficient mice. Despite this adaptation, cone photoreceptors displayed absent or mislocalized opsins at all ages examined. Rod photoreceptors entrapped the available chromophore but exhibited significant amounts of chromophore-free opsins in the dark-adapted stage. Treatment of mice with pharmacological doses of vitamin A ameliorated the rod phenotype but did not restore visual pigment synthesis in cone photoreceptors of STRA6-deficient mice. The imbalance between chromophore and opsin concentrations of rod and cone photoreceptors was associated with an unfavorable retinal physiology, including diminished electrical responses of photoreceptors to light, and retinal degeneration during aging. Together, our study demonstrates that STRA6 is critical to adjust the stoichiometry of chromophore and opsins in rod and cone photoreceptors and to prevent pathologies associated with ocular vitamin A deprivation.
Topics: Animals; Cone Opsins; Membrane Proteins; Mice; Opsins; Retinal Cone Photoreceptor Cells; Retinal Pigments; Retinaldehyde; Rod Opsins; Vitamin A
PubMed: 34508587
DOI: 10.1093/hmg/ddab267 -
Ophthalmic Research 2022Retinal homeostasis is essential to avoid retinal pigment epithelium (RPE) damage resulting in photoreceptor death and blindness. Mesenchymal stem cells-based cell...
INTRODUCTION
Retinal homeostasis is essential to avoid retinal pigment epithelium (RPE) damage resulting in photoreceptor death and blindness. Mesenchymal stem cells-based cell therapy could contribute to the maintenance of the retinal homeostasis. We have explored the effect of human uterine cervical stem cells (hUCESCs)-conditioned medium (hUCESC-CM) on RPE cells under oxidative stress condition.
METHODS
ARPE-19 cells were treated with hydrogen peroxide (H2O2) in the presence or absence of hUCESC-CM. qRT-PCR and Western blot were used to evaluate the expression of oxidative stress-related (HO-1, GCLC, and HSPB1) and vasculogenesis-related (VEGFA, PDGFA, and PDGFB) factors. Also, we assessed in vitro effects of hUCESC-CM on endothelial-cell (HUVEC) tube formation.
RESULTS
mRNA expression of HO-1, GCLC, HSPB1, VEGFA, PDGFA, and PDGFB were significantly increased in ARPE-19 cells treated with H2O2 + hUCESC-CM compared to cells treated with H2O2 only. Regarding the tube formation assay, HUVEC treated with supernatant from ARPE-19 cells treated with H2O2 + hUCESC-CM showed a significant increase in average vessel length, number of capillary-like junctions, and average of vessels area compared with HUVEC treated with supernatant from ARPE-19 cells treated with H2O2 only.
CONCLUSION
Our results show potential therapeutic effects of hUCESC-CM on RPE, such as protection from damage by oxidative stress, stimulation of detoxifying genes, and a better vascularization.
Topics: Cell Survival; Culture Media, Conditioned; Endothelial Cells; Epithelial Cells; Humans; Hydrogen Peroxide; Neovascularization, Pathologic; Oxidative Stress; Proto-Oncogene Proteins c-sis; RNA, Messenger; Retinal Pigment Epithelium; Retinal Pigments; Stem Cells
PubMed: 35584686
DOI: 10.1159/000524484 -
International Journal of Molecular... Jun 2023Diabetic retinopathy (DR) is the leading cause of vision loss and a critical complication of diabetes with a very complex etiology. The build-up of reactive oxygen...
Diabetic retinopathy (DR) is the leading cause of vision loss and a critical complication of diabetes with a very complex etiology. The build-up of reactive oxygen species (ROS) due to hyperglycemia is recognized as a primary risk factor for DR. Although spermidine, a naturally occurring polyamine, has been reported to have antioxidant effects, its effectiveness in DR has not yet been examined. Therefore, in this study, we investigated whether spermidine could inhibit high glucose (HG)-promoted oxidative stress in human retinal pigment epithelial (RPE) cells. The results demonstrated that spermidine notably attenuated cytotoxicity and apoptosis in HG-treated RPE ARPE-19 cells, which was related to the inhibition of mitochondrial ROS production. Under HG conditions, interleukin (IL)-1β and IL-18's release levels were markedly increased, coupled with nuclear factor kappa B (NF-κB) signaling activation. However, spermidine counteracted the HG-induced effects. Moreover, the expression of nucleotide-binding oligomerization domain-like receptor (NLR) protein 3 (NLRP3) inflammasome multiprotein complex molecules, including TXNIP, NLRP3, ASC, and caspase-1, increased in hyperglycemic ARPE-19 cells, but spermidine reversed these molecular changes. Collectively, our findings demonstrate that spermidine can protect RPE cells from HG-caused injury by reducing ROS and NF-κB/NLRP3 inflammasome pathway activation, indicating that spermidine could be a potential therapeutic compound for DR treatment.
Topics: Humans; Inflammasomes; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Reactive Oxygen Species; Spermidine; Oxidative Stress; Diabetic Retinopathy; Glucose; Epithelial Cells; Retinal Pigments
PubMed: 37445726
DOI: 10.3390/ijms241310550 -
Experimental Eye Research May 2022Age-related macular degeneration (AMD) has been associated with both complement activation and increased levels of circulating cytokines. Here, we sougth to investigate...
Age-related macular degeneration (AMD) has been associated with both complement activation and increased levels of circulating cytokines. Here, we sougth to investigate if cytokine-preexposure of retinal pigment epithelial (RPE) leads to increased complement activation and deposition of membrane attack complex (MAC). Primary human RPE and the ARPE19 cell line cultured in serum-free conditions were preexposed to 100 ng/ml interferon-gamma (IFNγ) and 20 ng/ml tumor necrosis factor-alpha (TNFα) for 48 h followed by exposure to diluted serum from healthy donors or complement factor B deficient (CFBd) serum for 70 min. Deposition of membrane attack complexes (MAC) was examined by use of a MAC-ELISA kit and by immunofluorescence. Eculizumab (anti-C5) was examined for its ability to prevent deposition of MAC on RPE cells exposed to serum. Lactatdehydrogenase (LDH) and thiazolyl blue tetrazolium bromide (MTT) assays were used to assess cellular metabolism and survival. MAC was deposited only on RPE preexposed to both IFNγ and TNFα. Lack of complement factor B or inhibition of C5 abrogated the MAC-deposition on RPE cells, while reconstitution of CFBd serum with CFB resulted in MAC-deposition. MAC-deposition resulted in RPE-release of LDH, but unaltered mitochondrial activity estimated by MTT. We conclude that preexposure of primary RPE and ARPE19 with inflammatory cytokines promoted alternative pathway activation of complement and deposition of MAC. This implies that circulating inflammatory mediators may increase susceptibility to local complement activation and MAC-deposition, which may represent an early event in the pathogenesis leading to AMD development.
Topics: Complement Activation; Complement Factor B; Complement Membrane Attack Complex; Humans; Interferon-gamma; Macular Degeneration; Retinal Pigment Epithelium; Retinal Pigments; Tumor Necrosis Factor-alpha
PubMed: 35183540
DOI: 10.1016/j.exer.2022.108982 -
Experimental Eye Research Feb 2024Human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) therapies are promising alternatives for the...
Human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) therapies are promising alternatives for the treatment of retinal degenerative diseases caused by RPE degeneration. The generation of autologous RPE cells from human adult donors, which has the advantage of avoiding immune rejection and teratoma formation, is an alternative cell resource to gain mechanistic insight into and test potential therapies for RPE degenerative diseases. Here, we found that limbal stem cells (LSCs) from hESCs and adult primary human limbus have the potential to produce RPE cells and corneal stromal stem cells (CSSCs). We showed that hESC-LSC-derived RPE cells (LSC-RPE) expressed RPE markers, had a phagocytic function, and synthesized tropical factors. Furthermore, during differentiation from LSCs to RPE cells, cells became pigmented, accompanied by a decrease in the level of LSC marker KRT15 and an increase in the level of RPE marker MITF. The Wnt signaling pathway plays a role in LSC-RPE fate transition, promotes MITF expression in the nucleus, and encourages RPE fate transition. In addition, we also showed that primary LSCs (pLSCs) from adult human limbus similar to hESC-LSC could generate RPE cells, which was supported by the co-expression of LSC and RPE cell markers (KRT15/OTX2, KRT15/MITF), suggesting the transition from pLSC to RPE cells, and typical polygonal morphology, melanization, RPE cell marker genes expression (TYR, RPE65), tight junction formation by ZO-1 expression, and the most crucial phagocytotic function. On the other hand, both hESC-LSCs and pLSCs also differentiated into CSSCs (LSC-CSSCs) that expressed stem cell markers (PAX6, NESTIN), presented MSC features, including surface marker expression and trilineage differentiation capability, like those in human CSSCs. Furthermore, the capability of pLSC-CSSC to differentiate into cells expressing keratocyte marker genes (ALDH3A1, PTGDS, PDK4) indicated the potential to induce keratocytes. These results suggest that the adult pLSC is an alternative cell resource, and its application provides a novel potential therapeutic avenue for preventing RPE dysfunction-related retinal degenerative diseases and corneal scarring.
Topics: Humans; Limbal Stem Cells; Induced Pluripotent Stem Cells; Retinal Pigment Epithelium; Cell Differentiation; Epithelial Cells; Retinal Pigments
PubMed: 38171475
DOI: 10.1016/j.exer.2023.109778 -
Molecules and Cells Jul 2023Age-related macular degeneration (AMD) is one of the leading causes of blindness in elderly individuals. However, the currently used intravitreal injections of...
Age-related macular degeneration (AMD) is one of the leading causes of blindness in elderly individuals. However, the currently used intravitreal injections of anti-vascular endothelial growth factor are invasive, and repetitive injections are also accompanied by a risk of intraocular infection. The pathogenic mechanism of AMD is still not completely understood, but a multifactorial mechanism that combines genetic predisposition and environmental factors, including cellular senescence, has been suggested. Cellular senescence refers to the accumulation of cells that stop dividing due to the presence of free radicals and DNA damage. Characteristics of senescent cells include nuclear hypertrophy, increased levels of cell cycle inhibitors such as p16 and p21, and resistance to apoptosis. Senolytic drugs remove senescent cells by targeting the main characteristics of these cells. One of the senolytic drugs, ABT-263, which inhibits the antiapoptotic functions of Bcl-2 and Bcl-xL, may be a new treatment for AMD patients because it targets senescent retinal pigment epithelium (RPE) cells. We proved that it selectively kills doxorubicin (Dox)-induced senescent ARPE-19 cells by activating apoptosis. By removing senescent cells, the expression of inflammatory cytokines was reduced, and the proliferation of the remaining cells was increased. When ABT-263 was orally administered to the mouse model of senescent RPE cells induced by Dox, we confirmed that senescent RPE cells were selectively removed and retinal degeneration was alleviated. Therefore, we suggest that ABT-263, which removes senescent RPE cells through its senolytic effect, has the potential to be the first orally administered senolytic drug for the treatment of AMD.
Topics: Animals; Mice; Retinal Degeneration; Senotherapeutics; Antineoplastic Agents; Macular Degeneration; Apoptosis; Epithelial Cells; Retinal Pigments; Cellular Senescence
PubMed: 37222160
DOI: 10.14348/molcells.2023.2188