The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform
Krzysztof Palczewski - One of the best experts on this subject based on the ideXlab platform.
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Prolonged prevention of retinal degeneration with retinylamine loaded nanoparticles.
Biomaterials, 2015Co-Authors: Anthony Puntel, Akiko Maeda, Marcin Golczak, Song Qi Gao, Krzysztof PalczewskiAbstract:Retinal degeneration impairs the vision of millions in all age groups worldwide. Increasing evidence suggests that the etiology of many retinal degenerative diseases is associated with impairment in biochemical reactions involved in the visual cycle, a metabolic pathway responsible for regeneration of the visual chromophore (11-cis-retinal). Inefficient clearance of toxic retinoid metabolites, especially All-Trans-Retinal, is considered responsible for photoreceptor cytotoxicity. Primary amines, including retinylamine, are effective in lowing the concentration of All-Trans-Retinal within the retina and thus prevent retina degeneration in mouse models of human retinopathies. Here we achieved prolonged prevention of retinal degeneration by controlled delivery of retinylamine to the eye from polylactic acid nanoparticles in Abca4(-/-)Rdh8(-/-) (DKO) mice, an animal model of Stargardt disease/age-related macular degeneration. Subcutaneous administration of the nanoparticles containing retinylamine provided a constant supply of the drug to the eye for about a week and resulted in effective prolonged prevention of light-induced retinal degeneration in DKO mice. Retinylamine nanoparticles hold promise for prolonged prophylactic treatment of human retinal degenerative diseases, including Stargardt disease and age-related macular degeneration.
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Expansion of First-in-Class Drug Candidates That Sequester Toxic All-Trans-Retinal and Prevent Light-Induced Retinal Degeneration
Molecular pharmacology, 2014Co-Authors: Jianye Zhang, Krzysztof Palczewski, Zhiqian Dong, Sreenivasa R. Mundla, William L. Seibel, Ruben Papoian, Marcin GolczakAbstract:All-Trans-Retinal, a retinoid metabolite naturally produced upon photoreceptor light activation, is cytotoxic when present at elevated levels in the retina. To lower its toxicity, two experimentally validated methods have been developed involving inhibition of the retinoid cycle and sequestration of excess of All-Trans-Retinal by drugs containing a primary amine group. We identified the first-in-class drug candidates that transiently sequester this metabolite or slow down its production by inhibiting regeneration of the visual chromophore, 11-cis-retinal. Two enzymes are critical for retinoid recycling in the eye. Lecithin:retinol acyltransferase (LRAT) is the enzyme that traps vitamin A (all-trans-retinol) from the circulation and photoreceptor cells to produce the esterified substrate for retinoid isomerase (RPE65), which converts all-trans-retinyl ester into 11-cis-retinol. Here we investigated retinylamine and its derivatives to assess their inhibitor/substrate specificities for RPE65 and LRAT, mechanisms of action, potency, retention in the eye, and protection against acute light-induced retinal degeneration in mice. We correlated levels of visual cycle inhibition with retinal protective effects and outlined chemical boundaries for LRAT substrates and RPE65 inhibitors to obtain critical insights into therapeutic properties needed for retinal preservation.
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retinyl ester storage particles retinosomes from the retinal pigmented epithelium resemble lipid droplets in other tissues
Journal of Biological Chemistry, 2011Co-Authors: Tivadar Orban, Grazyna Palczewska, Krzysztof PalczewskiAbstract:Levels of many hydrophobic cellular substances are tightly regulated because of their potential cytotoxicity. These compounds tend to self-aggregate in cytoplasmic storage depots termed lipid droplets/bodies that have well defined structures that contain additional components, including cholesterol and various proteins. Hydrophobic substances in these structures become mobilized in a specific and regulated manner as dictated by cellular requirements. Retinal pigmented epithelial cells in the eye produce retinyl ester-containing lipid droplets named retinosomes. These esters are mobilized to replenish the visual chromophore, 11-cis-retinal, and their storage ensures proper visual function despite fluctuations in dietary vitamin A intake. But it remains unclear whether retinosomes are structures specific to the eye or similar to lipid droplets in other organs/tissues that contain substances other than retinyl esters. Thus, we initially investigated the production of these lipid droplets in experimental cell lines expressing lecithin:retinol acyltransferase, a key enzyme involved in formation of retinyl ester-containing retinosomes from all-trans-retinol. We found that retinosomes and oleate-derived lipid droplets form and co-localize concomitantly, indicating their intrinsic structural similarities. Next, we isolated native retinosomes from bovine retinal pigmented epithelium and found that their protein and hydrophobic small molecular constituents were similar to those of lipid droplets reported for other experimental cell lines and tissues. These unexpected findings suggest a common mechanism for lipid droplet formation that exhibits broad chemical specificity for the hydrophobic substances being stored.
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limited roles of rdh8 rdh12 and abca4 in all trans retinal clearance in mouse retina
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Akiko Maeda, Marcin Golczak, Tadao Maeda, Krzysztof PalczewskiAbstract:A major intermediate of the retinoid (visual) cycle, the ocular vitamin A recycling system required for vision, is All-Trans-Retinal.1 Regeneration of the chromophore, 11-cis-retinal, is essential for continuous renewal of light-sensitive visual pigments in the vertebrate retina. Whereas inadequate 11-cis-retinal production leads to congenital blindness in humans, accumulation of the photoisomerized chromophore All-Trans-Retinal also can be detrimental. Such is the case when this reactive aldehyde is not efficiently cleared from the internal membranes of retinal outer segment discs.2,3 Clearance of All-Trans-Retinal consists of two steps: (1) translocation of All-Trans-Retinal across the photoreceptor disc membranes by ATP-binding cassette transporter 4 (ABCA4), and (2) reduction of All-Trans-Retinal to all-trans-retinol by retinol dehydrogenase 8 (RDH8) expressed in the outer segments of photoreceptors and RDH12 located in photoreceptor inner segments.4–6 In humans, lipofuscin fluorophores accumulate with age in the retinal pigmented epithelium (RPE), especially in RPE cells underlying the macula.7,8 Such accumulation has been considered to constitute one of the major risk factors for age-related macular degeneration (AMD), the predominant cause of legal blindness in developed countries.9 Lipofuscin fluorophores also are especially abundant in Stargardt disease, the most common juvenile form of macular degeneration.10 Di-retinoid-pyridinium-ethanolamine (A2E), the major fluorophore of lipofuscin, is formed by condensation of phosphatidylethanolamine with two molecules of All-Trans-Retinal followed by oxidation and hydrolysis of the phosphate ester.11 Various mechanisms have been proposed to explain the toxicity of A2E. These include properties of A2E as a cationic detergent,12 physiologic interference with RPE function,13,14 and radical reactions induced by light-dependent A2E oxidation.15 Recently, we reported that genetic ablation of Rdh8 and Abca4, two important enzymes responsible for All-Trans-Retinal clearance from photoreceptors, caused severe retinal degeneration in mice.16 Even though this disease was associated with accumulation of All-Trans-Retinal condensation products such as A2E and All-Trans-Retinal dimer (RALdi), the mechanisms of RPE and photoreceptor death remain to be clarified. Although Rdh8 is the major dehydrogenase responsible for clearing All-Trans-Retinal from outer segments of photoreceptor cells,17 Rdh12 also contributes to this process in the inner segments of photoreceptor cells, and mutations in Rdh12 are associated with congenital blindness.18 Therefore, we used genetically engineered mice lacking Rdh8, Rdh12, and/or Abca4 alone or in various combinations together with cell culture experiments to investigate the roles of these enzymes in All-Trans-Retinal clearance and induction of progressive light-dependent severe retinal degeneration.
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Limited roles of Rdh8, Rdh12, and Abca4 in All-Trans-Retinal clearance in mouse retina.
Investigative ophthalmology & visual science, 2009Co-Authors: Akiko Maeda, Marcin Golczak, Tadao Maeda, Krzysztof PalczewskiAbstract:Although the retinoid cycle is essential for vision, All-Trans-Retinal and the side products of this cycle are toxic. Delayed clearance of All-Trans-Retinal causes accumulation of its condensation products, A2E, and All-Trans-Retinal dimer (RALdi), both associated with human macular degeneration. The protective roles were examined of the all-trans-RDHs, Rdh8 and Rdh12, and the ATP-binding cassette transporter Abca4, retinoid cycle enzymes involved in All-Trans-Retinal clearance. Mice genetically engineered to lack Rdh8, Rdh12, and Abca4, either singly or in various combinations, were investigated because All-Trans-Retinal clearance is achieved by all-trans-RDHs and Abca4. Knockout mice were evaluated by spectral-domain optical coherence tomography (SD-OCT), electroretinography, retinal morphology, and visual retinoid profiling with HPLC and MS. ARPE19 cells were examined to evaluate A2E and RALdi oxidation and toxicity induced by exposure to UV and blue light. Rdh8(-/-)Abca4(-/-) and Rdh8(-/-)Rdh12(-/-)Abca4(-/-) mice displayed slowly progressive, severe retinal degeneration under room light conditions. Intense light-induced acute retinal degeneration was detected by SD-OCT in Rdh8(-/-)Rdh12(-/-)Abca4(-/-) mice. Amounts of A2E in the RPE correlated with diminished All-Trans-Retinal clearance, and the highest A2E amounts were found in Rdh8(-/-)Rdh12(-/-)Abca4(-/-) mice. However oxidized A2E was not found in any of these mice, and A2E oxidation was not induced by blue light and UV illumination of A2E-loaded ARPE19 cells. Of interest, addition of All-Trans-Retinal did activate retinoic acid receptors in cultured cells. Rdh8, Rdh12, and Abca4 all protect the retina and reduce A2E production by facilitating All-Trans-Retinal clearance. Delayed All-Trans-Retinal clearance contributes more than A2E oxidation to light-induced cellular toxicity.
Joseph L. Napoli - One of the best experts on this subject based on the ideXlab platform.
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Multiple Retinol and Retinal Dehydrogenases Catalyze All-trans-retinoic Acid Biosynthesis in Astrocytes
The Journal of biological chemistry, 2010Co-Authors: Chao Wang, Maureen A. Kane, Joseph L. NapoliAbstract:All-trans-retinoic acid (atRA) stimulates neurogenesis, dendritic growth of hippocampal neurons, and higher cognitive functions, such as spatial learning and memory formation. Although astrocyte-derived atRA has been considered a key factor in neurogenesis, little direct evidence identifies hippocampus cell types and the enzymes that biosynthesize atRA. Here we show that primary rat astrocytes, but not neurons, biosynthesize atRA using multiple retinol dehydrogenases (Rdh) of the short chain dehydrogenase/reductase gene family and retinaldehyde dehydrogenases (Raldh). Astrocytes secrete atRA into their medium; neurons sequester atRA. The first step, conversion of retinol into retinal, is rate-limiting. Neurons and astrocytes both synthesize retinyl esters and reduce retinal into retinol. siRNA knockdown indicates that Rdh10, Rdh2 (mRdh1), and Raldh1, -2, and -3 contribute to atRA production. Knockdown of the Rdh Dhrs9 increased atRA synthesis ∼40% by increasing Raldh1 expression. Immunocytochemistry revealed cytosolic and nuclear expression of Raldh1 and cytosol and perinuclear expression of Raldh2. atRA autoregulated its concentrations by inducing retinyl ester synthesis via lecithin:retinol acyltransferase and stimulating its catabolism via inducing Cyp26B1. These data show that adult hippocampus astrocytes rely on multiple Rdh and Raldh to provide a paracrine source of atRA to neurons, and atRA regulates its own biosynthesis in astrocytes by directing flux of retinol. Observation of cross-talk between Dhrs9 and Raldh1 provides a novel mechanism of regulating atRA biosynthesis.
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reduction of all trans retinal in the mouse liver peroxisome fraction by the short chain dehydrogenase reductase rrd induction by the ppar alpha ligand clofibrate
Biochemistry, 2003Co-Authors: Zhen Lei, Weiguo Chen, Min Zhang, Joseph L. NapoliAbstract:The mouse liver 16,000 g fraction, which contains peroxisomes, reduces All-Trans-Retinal, but has limited ability to dehydrogenate retinol enzymatically. Feeding mice for 2 weeks with a diet containing clofibrate (0.5%, w/w), a PPAR alpha ligand and peroxisome proliferator, increased the 16,000 g fraction approximately 2-fold in protein, approximately 2-fold in specific activity of retinal reduction, and approximately 4-fold in retinal reductase units compared to controls, and caused a 50% decrease in liver retinol. An increase in both reductase specific activity and units indicates that clofibrate/PPAR alpha induced expression of retinal-reducing enzymes(s), in addition to increasing reductase(s) content. We expressed a cDNA from the NCBI data bank that encodes a peroxisome short-chain dehydrogenase/reductase. The enzyme, mouse retinal reductase (RRD, also known as human 2,4-dienoyl-CoA reductase), reduces All-Trans-Retinal [V(m) = 40 nmol min(-1) (mg of protein)(-1); K(0.5) = 2.3 microM] and has 4- and 60-fold less activity with 13-cis-retinal and 9-cis-retinal, respectively. Recombinant RRD functions with both unbound and CRBP(I) (cellular retinol-binding protein)-bound retinal, but apo-CRBP(I) inhibits the reductase. RRD mRNA expression was initiated on embryo day 7. Most adult tissues assayed expressed the mRNA. Liver, kidney, and heart had the most intense expression, with much less intense expression in brain, spleen, and lung. Clofibrate feeding increased the amount of RRD protein in the 16,000 g fraction of liver, consistent with the clofibrate-induced increase in reductase activity. These data relate retinoid metabolism, PPAR alpha, peroxisomes, and RRD, and are consistent with a further function of CRBP(I) in retinoid metabolism.
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Reduction of All-Trans-Retinal in the mouse liver peroxisome fraction by the short-chain dehydrogenase/reductase RRD: induction by the PPAR alpha ligand clofibrate.
Biochemistry, 2003Co-Authors: Zhen Lei, Weiguo Chen, Min Zhang, Joseph L. NapoliAbstract:The mouse liver 16,000 g fraction, which contains peroxisomes, reduces All-Trans-Retinal, but has limited ability to dehydrogenate retinol enzymatically. Feeding mice for 2 weeks with a diet containing clofibrate (0.5%, w/w), a PPAR alpha ligand and peroxisome proliferator, increased the 16,000 g fraction approximately 2-fold in protein, approximately 2-fold in specific activity of retinal reduction, and approximately 4-fold in retinal reductase units compared to controls, and caused a 50% decrease in liver retinol. An increase in both reductase specific activity and units indicates that clofibrate/PPAR alpha induced expression of retinal-reducing enzymes(s), in addition to increasing reductase(s) content. We expressed a cDNA from the NCBI data bank that encodes a peroxisome short-chain dehydrogenase/reductase. The enzyme, mouse retinal reductase (RRD, also known as human 2,4-dienoyl-CoA reductase), reduces All-Trans-Retinal [V(m) = 40 nmol min(-1) (mg of protein)(-1); K(0.5) = 2.3 microM] and has 4- and 60-fold less activity with 13-cis-retinal and 9-cis-retinal, respectively. Recombinant RRD functions with both unbound and CRBP(I) (cellular retinol-binding protein)-bound retinal, but apo-CRBP(I) inhibits the reductase. RRD mRNA expression was initiated on embryo day 7. Most adult tissues assayed expressed the mRNA. Liver, kidney, and heart had the most intense expression, with much less intense expression in brain, spleen, and lung. Clofibrate feeding increased the amount of RRD protein in the 16,000 g fraction of liver, consistent with the clofibrate-induced increase in reductase activity. These data relate retinoid metabolism, PPAR alpha, peroxisomes, and RRD, and are consistent with a further function of CRBP(I) in retinoid metabolism.
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Molecular characterization of a mouse short chain dehydrogenase/reductase active with all-trans-retinol in intact cells, mRDH1.
The Journal of biological chemistry, 2001Co-Authors: Min Zhang, Susan M. Smith, Weiguo Chen, Joseph L. NapoliAbstract:Abstract Metabolic activation of retinol (vitamin A) via sequential actions of retinol and retinal dehydrogenases produces the active metabolite all-trans-retinoic acid. This work reports cDNA cloning, enzymatic characterization, function in a reconstituted path of all-trans-retinoic acid biosynthesis in cell culture, and mRNA expression patterns in adult tissues and embryos of a mouse retinol dehydrogenase, RDH1. RDH1 represents a new member of the short chain dehydrogenase/reductase superfamily that differs from other mouse RDH in relative activity with all-trans and cis-retinols. RDH1 has a multifunctional catalytic nature, as do other short chain dehydrogenase/reductases. In addition to retinol dehydrogenase activity, RDH1 has strong 3α-hydroxy and weak 17β-hydroxy steroid dehydrogenase activities. RDH1 has widespread and intense mRNA expression in tissues of embryonic and adult mice. The mouse embryo expresses RDH1 as early as 7.0 days post-coitus, and expression is especially intense within the neural tube, gut, and neural crest at embryo day 10.5. Cells cotransfected with RDH1 and any one of three retinal dehydrogenase isozymes synthesize all-trans-retinoic acid from retinol, demonstrating that RDH1contributes to a path of all-trans-retinoic acid biosynthesis in intact cells. These characteristics are consistent with RDH1 functioning in a path of all-trans-retinoic acid biosynthesis starting early during embryogenesis.
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cdna cloning and expression of a human aldehyde dehydrogenase aldh active with 9 cis retinal and identification of a rat ortholog aldh12
Journal of Biological Chemistry, 2000Co-Authors: Min Lin, Joseph L. NapoliAbstract:This report describes the isolation of a heretofore uncharacterized aldehyde dehydrogenase (ALDH) with retinal dehydrogenase activity from rat kidney and the cloning and expression of a cDNA that encodes its human ortholog, the previously unknown ALDH12. The human ALDH12 cDNA predicts a 487-residue protein with the 23 invariant amino acids, four conserved regions, cofactor binding motif (G209 XGX3G), and active site cysteine residue (Cys287) that typify members of the ALDH superfamily. ALDH12 seems at least as efficient (Vm/Km) in converting 9-cis-retinal into the retinoid X receptor ligand 9-cis-retinoic acid as two previously identified ALDHs with 9-cis-retinal dehydrogenase activity, rat retinal dehydrogenase (RALDH) 1 and RALDH2. ALDH12, however, has ∼40-fold higher activity with 9-cis- retinal than with All-Trans-Retinal, whereas RALDH1 and RALDH2 have equivalent and ∼4-fold less efficiencies for 9-cis-retinal versusAll-Trans-Retinal, respectively. Therefore, ALDH12 is the first known ALDH to show a preference for 9-cis-retinal relative to All-Trans-Retinal. Evidence consistent with the possibility that ALDH12 could function in a pathway of 9-cis-retinoic acid biosynthesis in vivoincludes biosynthesis of 9-cis-retinoic acid from 9-cis-retinol in cells co-transfected with cDNAs encoding ALDH12 and the 9-cis-retinol/androgen dehydrogenase, cis-retinoid/androgen dehydrogenase type 1. Intense ALDH12 mRNA expression in adult and fetal liver and kidney, two organs that reportedly have relatively high concentrations of 9-cis-retinol, reinforces this notion.
Yiannis Koutalos - One of the best experts on this subject based on the ideXlab platform.
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Photooxidation mediated by 11-cis and all-trans retinal in single isolated mouse rod photoreceptors
Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology, 2020Co-Authors: Chunhe Chen, Masahiro Kono, Yiannis KoutalosAbstract:Retinal, the vitamin A aldehyde, is a potent photosensitizer that plays a major role in light-induced damage to vertebrate photoreceptors. 11-Cis retinal is the light-sensitive chromophore of rhodopsin, the photopigment of vertebrate rod photoreceptors. It is isomerized by light to all-trans, activating rhodopsin and beginning the process of light detection. All-trans retinal is released by activated rhodopsin, allowing its regeneration by fresh 11-cis retinal continually supplied to photoreceptors. The released all-trans retinal is reduced to all-trans retinol in a reaction using NADPH. We have examined the photooxidation mediated by 11-cis and all-trans retinal in single living rod photoreceptors isolated from mouse retinas. Photooxidation was measured with fluorescence imaging from the oxidation of internalized BODIPY C11, a fluorescent dye whose fluorescence changes upon oxidation. We found that photooxidation increased with the concentration of exogenously added 11-cis or all-trans retinal to metabolically compromised rod outer segments that lacked NADPH supply. In dark-adapted metabolically intact rod outer segments with access to NADPH, there was no significant increase in photooxidation following exposure of the cell to light, but there was significant increase following addition of exogenous 11-cis retinal. The results indicate that both 11-cis and all-trans retinal can mediate light-induced damage in rod photoreceptors. In metabolically intact cells, the removal of the all-trans retinal generated by light through its reduction to retinol minimizes all-trans retinal-mediated photooxidation. However, because the enzymatic machinery of the rod outer segment cannot remove 11-cis retinal, 11-cis-retinal-mediated photooxidation may play a significant role in light-induced damage to photoreceptor cells.
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RPE65 and the Accumulation of Retinyl Esters in Mouse Retinal Pigment Epithelium
Photochemistry and Photobiology, 2017Co-Authors: Colleen Sheridan, Nicholas P. Boyer, Rosalie K. Crouch, Yiannis KoutalosAbstract:The RPE65 protein of the retinal pigment epithelium (RPE) enables the conversion of retinyl esters to the visual pigment chromophore 11-cis retinal. Fresh 11-cis retinal is generated from retinyl esters following photoisomerization of the visual pigment chromophore to all-trans during light detection. Large amounts of esters accumulate in Rpe65-/- mice, indicating their continuous formation when 11-cis retinal generation is blocked. We hypothesized that absence of light, by limiting the conversion of esters to 11-cis retinal, would also result in the build-up of retinyl esters in the RPE of wild-type mice. We used HPLC to quantify ester levels in organic extracts of the RPE from wild-type and Rpe65-/- mice. Retinyl ester levels in Sv/129 wild-type mice that were dark adapted for various intervals over a 4-week period were similar to those in mice raised in cyclic light. In C57BL/6 mice however, which contain less Rpe65 protein, dark adaptation was accompanied by an increase in ester levels compared to cyclic light controls. Retinyl ester levels were much higher in Rpe65-/- mice compared to wild type and kept increasing with age. The results suggest that the RPE65 role in retinyl ester homeostasis extends beyond enabling the formation of 11-cis retinal.
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All-trans retinal levels and formation of lipofuscin precursors after bleaching in rod photoreceptors from wild type and Abca4-/- mice
Experimental eye research, 2017Co-Authors: Leopold Adler, Chunhe Chen, Yiannis KoutalosAbstract:The accumulation of lipofuscin in the cells of the retinal pigment epithelium (RPE) is thought to play an important role in the development and progression of degenerative diseases of the retina. The bulk of RPE lipofuscin originates in reactions of the rhodopsin chromophore, retinal, with components of the photoreceptor outer segment. The 11-cis retinal isomer is generated in the RPE and supplied to rod photoreceptor outer segments where it is incorporated as the chromophore of rhodopsin. It is photoisomerized during light detection to all-trans and subsequently released by photoactivated rhodopsin as all-trans retinal, which is removed through reduction to all-trans retinol in a reaction requiring metabolic input in the form of NADPH. Both 11-cis and all-trans retinal can form lipofuscin precursor fluorophores in rod photoreceptor outer segments. Increased accumulation of lipofuscin has been suggested to result from excess formation of lipofuscin precursors due to buildup of all-trans retinal released by light exposure. In connection with this suggestion, the Abca4 transporter protein, an outer segment protein defects in which result in recessive Stargardt disease, has been proposed to promote the removal of all-trans retinal by facilitating its availability for reduction. To examine this possibility, we have measured the outer segment levels of all-trans retinal, all-trans retinol, and of lipofuscin precursors after bleaching by imaging the fluorescence of single rod photoreceptors isolated from wild type and Abca4-/- mice. We found that all-trans retinol and all-trans retinal levels increased after bleaching in both wild type and Abca4-/- rods. At all times after bleaching, there was no significant difference in all-trans retinal levels between the two strains. All-trans retinol levels were not significantly different between the two strains at early times, but were lower in Abca4-/- rods at times longer than 20 min after bleaching. Bleaching in the presence of lower metabolic substrate concentrations resulted in higher all-trans retinal levels and increased formation of lipofuscin precursors in both wild type and Abca4-/- rods. The results show that conditions that result in buildup of all-trans retinal levels result in increased generation of lipofuscin precursors in both wild type and Abca4-/- rods. The results are consistent with the proposal that Abca4 facilitates the reduction of all-trans retinal to retinol; absence of Abca4 however does not appear to be associated with higher all-trans retinal levels compared to wild type.
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Reduction of All-Trans-Retinal in Vertebrate Rod Photoreceptors Requires the Combined Action of RDH8 and RDH12
The Journal of biological chemistry, 2012Co-Authors: Chunhe Chen, Debra A. Thompson, Yiannis KoutalosAbstract:In vertebrate rod cells, retinoid dehydrogenases/reductases (RDHs) are critical for reducing the reactive aldehyde All-Trans-Retinal that is released by photoactivated rhodopsin, to all-trans-retinol (vitamin A). Previous studies have shown that RDH8 localizes to photoreceptor outer segments and is a strong candidate for performing this role. However, RDH12 function in the photoreceptor inner segments is also key, because loss of function mutations cause retinal degeneration in some forms of Leber congenital amaurosis. To investigate the in vivo roles of RDH8 and RDH12, we used fluorescence imaging to examine all-trans-retinol production in single isolated rod cells from wild-type mice and knock-out mice lacking either one or both RDHs. Outer segments of rods deficient in Rdh8 failed to reduce All-Trans-Retinal, but those deficient in Rdh12 were unaffected. Following exposure to light, a leak of retinoids from outer to inner segments was detected in rods from both wild-type and knock-out mice. In cells lacking Rdh8 or Rdh12, this leak was mainly All-Trans-Retinal. Wild-type rods incubated with All-Trans-Retinal reduced moderate loads of retinal within the cell interior, but this ability was lost by cells deficient in Rdh8 or Rdh12. Our findings are consistent with localization of RDH8 to the outer segment where it provides most of the activity needed to reduce All-Trans-Retinal generated by the light response. In contrast, RDH12 in inner segments can protect vital cell organelles against aldehyde toxicity caused by an intracellular leak of All-Trans-Retinal, as well as other aldehydes originating both inside and outside the cell.
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All‐Trans Retinal Mediates Light‐Induced Oxidation in Single Living Rod Photoreceptors†
Photochemistry and Photobiology, 2012Co-Authors: Kosuke Masutomi, Kei Nakatani, Chunhe Chen, Yiannis KoutalosAbstract:All-trans retinal is a potent photosensitizer that is released in photoreceptor outer segments by the photoactivated visual pigment following the detection of light. Photoreceptor outer segments also contain high concentrations of polyunsaturated fatty acids, and are thus particularly susceptible to oxidative damage such as that initiated by light via a photosensitizer. Upon its release, all-trans retinal is reduced within the outer segment to all-trans retinol, through a reaction requiring metabolic input in the form of NADPH. The phototoxic potential of physiologically generated all-trans retinal was examined in single living rod photoreceptors obtained from frog (Rana pipiens) retinas. Light-induced oxidation was measured with fluorescence imaging using an oxidation-sensitive indicator dye from the shift in fluorescence between the intact and oxidized forms. Light-induced oxidation was highest in metabolically compromised rod outer segments following photoactivation of the visual pigment rhodopsin, and after a time interval, sufficiently long to ensure the release of all-trans retinal. Furthermore, light-induced oxidation increased with the concentration of exogenously added all-trans retinal. The results show that the all-trans retinal generated during the detection of light can mediate light-induced oxidation. Its removal through reduction to all-trans retinol protects photoreceptor outer segments against light-induced oxidative damage.
Akiko Maeda - One of the best experts on this subject based on the ideXlab platform.
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RPE Visual Cycle and Biochemical Phenotypes of Mutant Mouse Models.
Methods in Molecular Biology, 2018Co-Authors: Bhubanananda Sahu, Akiko MaedaAbstract:The retinal pigmented epithelium (RPE) is a single layer of polarized epithelial cells which plays many important roles for visual function. One of such roles is production of visual chromophore, 11-cis-retinal through the visual cycle. The visual cycle consists of biochemical processes for regenerating chromophore by a collective action of the RPE and photoreceptor. Photoreceptors harbor the G protein-coupled receptors, opsin which enables to receive light when it bounds to 11-cis-retinal. With absorption of a photon of light, 11-cis-retinal photoisomerizes to All-Trans-Retinal. All-Trans-Retinal reduces to all-trans-retinol in the photoreceptor and further recycles back to 11-cis-retinal in the RPE. Acyltransferases and isomerohydrolase(s) along with retinol dehydrogenases sequentially convert all-trans-retinol to 11-cis-retinal in the RPE. Dysfunctions of any retinoid cycle enzymes in the RPE can cause retinal diseases. Phenotyping RPE functions by the use of mutant mouse models will provide great detailed biochemical insights of the visual cycle and further manipulative strategies to protect against retinal degeneration. Here, we describe biochemical analyses of the visual cycle in mouse models using RPE cells.
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Prolonged prevention of retinal degeneration with retinylamine loaded nanoparticles.
Biomaterials, 2015Co-Authors: Anthony Puntel, Akiko Maeda, Marcin Golczak, Song Qi Gao, Krzysztof PalczewskiAbstract:Retinal degeneration impairs the vision of millions in all age groups worldwide. Increasing evidence suggests that the etiology of many retinal degenerative diseases is associated with impairment in biochemical reactions involved in the visual cycle, a metabolic pathway responsible for regeneration of the visual chromophore (11-cis-retinal). Inefficient clearance of toxic retinoid metabolites, especially All-Trans-Retinal, is considered responsible for photoreceptor cytotoxicity. Primary amines, including retinylamine, are effective in lowing the concentration of All-Trans-Retinal within the retina and thus prevent retina degeneration in mouse models of human retinopathies. Here we achieved prolonged prevention of retinal degeneration by controlled delivery of retinylamine to the eye from polylactic acid nanoparticles in Abca4(-/-)Rdh8(-/-) (DKO) mice, an animal model of Stargardt disease/age-related macular degeneration. Subcutaneous administration of the nanoparticles containing retinylamine provided a constant supply of the drug to the eye for about a week and resulted in effective prolonged prevention of light-induced retinal degeneration in DKO mice. Retinylamine nanoparticles hold promise for prolonged prophylactic treatment of human retinal degenerative diseases, including Stargardt disease and age-related macular degeneration.
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autophagy protects the retina from light induced degeneration
Journal of Biological Chemistry, 2013Co-Authors: Yu Chen, Tadao Maeda, Osamu Sawada, Hideo Kohno, Carlos S Subauste, Akiko MaedaAbstract:Autophagy is a conserved feature of lysosome-mediated intracellular degradation. Dysregulated autophagy is implicated as a contributor in neurodegenerative diseases; however, the role of autophagy in retinal degeneration remains largely unknown. Here, we report that the photo-activated visual chromophore, All-Trans-Retinal, modulated autophagosome formation in ARPE19 retinal cells. Increased formation of autophagosomes in these cells was observed when incubated with 2.5 μm All-Trans-Retinal, a condition that did not cause cell death after 24 h in culture. However, autophagosome formation was decreased at concentrations, which caused cell death. Increased expression of activating transcription factor 4 (Atf4), which indicates the activation of oxidative stress, was recorded in response to light illumination in retinas of Abca4−/−Rdh8−/− mice, which showed delayed clearance of All-Trans-Retinal after light exposure. Expression of autophagosome marker LC3B-II and mitochondria-specific autophagy, mitophagy, regulator Park2, were significantly increased in the retinas of Abca4−/−Rdh8−/− mice after light exposure, suggesting involvement of autophagy and mitophagy in the pathogenesis of light-induced retinal degeneration. Deletion of essential genes required for autophagy, including Beclin1 systemically or Atg7 in only rod photoreceptors resulted in increased susceptibility to light-induced retinal damage. Increased photoreceptor cell death was observed when retinas lacking the rod photoreceptor-specific Atg7 gene were coincubated with 20 μm All-Trans-Retinal. Park2−/− mice also displayed light-induced retinal degeneration. Ultra-structural analyses showed mitochondrial and endoplasmic reticulum impairment in retinas of these model animals after light exposure. Taken together, these observations provide novel evidence implicating an important role of autophagy and mitophagy in protecting the retina from All-Trans-Retinal- and light-induced degeneration.
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Retinal Photodamage Mediated by All-Trans-Retinal
Photochemistry and Photobiology, 2012Co-Authors: Tadao Maeda, Marcin Golczak, Akiko MaedaAbstract:Accumulation of All-Trans-Retinal (all-trans-RAL), reactive vitamin A aldehyde, is one of the key factors in initiating retinal photodamage. This photodamage is characterized by progressive retinal cell death evoked by light exposure in both an acute and chronic fashion. Photoactivated rhodopsin releases all-trans-RAL, which is subsequently transported by ATP-binding cassette transporter 4 and reduced to all-trans-retinol by all-trans-retinol dehydrogenases located in photoreceptor cells. Any interruptions in the clearing of all-trans-RAL in the photoreceptors can cause an accumulation of this reactive aldehyde and its toxic condensation products. This accumulation may result in the manifestation of retinal dystrophy including human retinal degenerative diseases such as Stargardt’s disease and age-related macular degeneration. Herein, we discuss the mechanisms of all-trans-RAL clearance in photoreceptor cells by sequential enzymatic reactions, the visual (retinoid) cycle, and potential molecular pathways of retinal photodamage. We also review recent imaging technologies to monitor retinal health status as well as novel therapeutic strategies preventing all-trans-RAL-associated retinal photodamage.
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limited roles of rdh8 rdh12 and abca4 in all trans retinal clearance in mouse retina
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Akiko Maeda, Marcin Golczak, Tadao Maeda, Krzysztof PalczewskiAbstract:A major intermediate of the retinoid (visual) cycle, the ocular vitamin A recycling system required for vision, is All-Trans-Retinal.1 Regeneration of the chromophore, 11-cis-retinal, is essential for continuous renewal of light-sensitive visual pigments in the vertebrate retina. Whereas inadequate 11-cis-retinal production leads to congenital blindness in humans, accumulation of the photoisomerized chromophore All-Trans-Retinal also can be detrimental. Such is the case when this reactive aldehyde is not efficiently cleared from the internal membranes of retinal outer segment discs.2,3 Clearance of All-Trans-Retinal consists of two steps: (1) translocation of All-Trans-Retinal across the photoreceptor disc membranes by ATP-binding cassette transporter 4 (ABCA4), and (2) reduction of All-Trans-Retinal to all-trans-retinol by retinol dehydrogenase 8 (RDH8) expressed in the outer segments of photoreceptors and RDH12 located in photoreceptor inner segments.4–6 In humans, lipofuscin fluorophores accumulate with age in the retinal pigmented epithelium (RPE), especially in RPE cells underlying the macula.7,8 Such accumulation has been considered to constitute one of the major risk factors for age-related macular degeneration (AMD), the predominant cause of legal blindness in developed countries.9 Lipofuscin fluorophores also are especially abundant in Stargardt disease, the most common juvenile form of macular degeneration.10 Di-retinoid-pyridinium-ethanolamine (A2E), the major fluorophore of lipofuscin, is formed by condensation of phosphatidylethanolamine with two molecules of All-Trans-Retinal followed by oxidation and hydrolysis of the phosphate ester.11 Various mechanisms have been proposed to explain the toxicity of A2E. These include properties of A2E as a cationic detergent,12 physiologic interference with RPE function,13,14 and radical reactions induced by light-dependent A2E oxidation.15 Recently, we reported that genetic ablation of Rdh8 and Abca4, two important enzymes responsible for All-Trans-Retinal clearance from photoreceptors, caused severe retinal degeneration in mice.16 Even though this disease was associated with accumulation of All-Trans-Retinal condensation products such as A2E and All-Trans-Retinal dimer (RALdi), the mechanisms of RPE and photoreceptor death remain to be clarified. Although Rdh8 is the major dehydrogenase responsible for clearing All-Trans-Retinal from outer segments of photoreceptor cells,17 Rdh12 also contributes to this process in the inner segments of photoreceptor cells, and mutations in Rdh12 are associated with congenital blindness.18 Therefore, we used genetically engineered mice lacking Rdh8, Rdh12, and/or Abca4 alone or in various combinations together with cell culture experiments to investigate the roles of these enzymes in All-Trans-Retinal clearance and induction of progressive light-dependent severe retinal degeneration.
Gabriel H Travis - One of the best experts on this subject based on the ideXlab platform.
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non photopic and photopic visual cycles differentially regulate immediate early and late phases of cone photoreceptor mediated vision
bioRxiv, 2020Co-Authors: Rebecca Ward, Diego Cobice, Dionissia A Pepe, Eoghan M Mcgarrigle, Susan E Brockerhoff, Gabriel H Travis, Joanna J Kaylor, James B Hurley, Breandán N. KennedyAbstract:Cone photoreceptors in the retina enable vision over a wide range of light intensities. However, the processes enabling cone vision in bright light (i.e. photopic vision) are not adequately understood. Chromophore regeneration of cone photopigments may require the retinal pigment epithelium (RPE) and/or retinal Muller glia. In the RPE, isomerization of all-trans-retinyl esters (atRE) to 11-cis-retinol (11cROL) is mediated by the retinoid isomerohydrolase Rpe65. An alternative retinoid isomerase, dihydroceramide desaturase-1 (DES1), is expressed in RPE and Muller cells. The retinol-isomerase activities of Rpe65 and Des1 are inhibited by emixustat and fenretinide, respectively. Here, we tested the effects of these visual cycle inhibitors on immediate, early and late phases of cone photopic vision. In zebrafish larvae raised under cyclic light conditions, fenretinide impaired late cone photopic vision, whereas emixustat-treated zebrafish unexpectedly had normal vision. In contrast, emixustat-treated larvae raised under extensive dark-adaption displayed significantly attenuated immediate photopic vision concomitantly with significantly reduced 11-cis-retinaldehyde (11cRAL). Following 30 minutes of light, early photopic vision recovered, despite 11cRAL levels remaining significantly reduced. Defects in immediate cone photopic vision were rescued in emixustat- or fenretinide-treated larvae following exogenous 9-cis-retinaldehyde (9cRAL) supplementation. Genetic knockout of degs1 or retinaldehyde-binding protein 1b (rlbp1b) revealed that neither are required for photopic vision in zebrafish. Our findings define the molecular and temporal requirements of the non-photopic and photopic visual cycles for mediating vision in bright light.
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light driven regeneration of cone visual pigments through a mechanism involving rgr opsin in muller glial cells
Neuron, 2019Co-Authors: Ala Morshedian, Joanna J Kaylor, Roxana A Radu, Avian Tsan, Rikard Frederiksen, Lily Yuan, Alapakkam P Sampath, Gordon L Fain, Gabriel H TravisAbstract:While rods in the mammalian retina regenerate rhodopsin through a well-characterized pathway in cells of the retinal pigment epithelium (RPE), cone visual pigments are thought to regenerate in part through an additional pathway in Muller cells of the neural retina. The proteins comprising this intrinsic retinal visual cycle are unknown. Here, we show that RGR opsin and retinol dehydrogenase-10 (Rdh10) convert all-trans-retinol to 11-cis-retinol during exposure to visible light. Isolated retinas from Rgr+/+ and Rgr-/- mice were exposed to continuous light, and cone photoresponses were recorded. Cones in Rgr-/- retinas lost sensitivity at a faster rate than cones in Rgr+/+ retinas. A similar effect was seen in Rgr+/+ retinas following treatment with the glial cell toxin, α-aminoadipic acid. These results show that RGR opsin is a critical component of the Muller cell visual cycle and that regeneration of cone visual pigment can be driven by light.
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RESEARCH ARTICLE Diacylglycerol O-Acyltransferase Type-1 Synthesizes Retinyl Esters in the Retina and Retinal Pigment Epithelium
2016Co-Authors: Joanna J Kaylor, Nicholas Bischoff, Jacob Makshanoff, Shannan Eddington, Tran Bianconi, Roxana A Radu, Marcia Lloyd, Dean Bok, Gabriel H TravisAbstract:Retinyl esters represent an insoluble storage form of vitamin A and are substrates for the retinoid isomerase (Rpe65) in cells of the retinal pigment epithelium (RPE). The major retinyl-ester synthase in RPE cells is lecithin:retinol acyl-transferase (LRAT). A second pal-mitoyl coenzyme A-dependent retinyl-ester synthase activity has been observed in RPE homogenates but the protein responsible has not been identified. Here we show that diacyl-glycerol O-acyltransferase-1 (DGAT1) is expressed in multiple cells of the retina including RPE and Müller glial cells. DGAT1 catalyzes the synthesis of retinyl esters from multiple ret-inol isomers with similar catalytic efficiencies. Loss of DGAT1 in dgat1-/-mice has no effect on retinal anatomy or the ultrastructure of photoreceptor outer-segments (OS) and RPE cells. Levels of visual chromophore in dgat1-/-mice were also normal. However, the normal build-up of all-trans-retinyl esters (all-trans-RE’s) in the RPE during the first hour after a deep photobleach of visual pigments in the retina was not seen in dgat1-/-mice. Further, total retinyl-ester synthase activity was reduced in both dgat1-/- retina and RPE
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Identification of the 11-cis-specific retinyl-ester synthase in retinal Müller cells as multifunctional O-acyltransferase (MFAT)
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Joanna J Kaylor, Nicholas Bischoff, Jacob Makshanoff, Jeremy D. Cook, Jennifer Yong, Gabriel H TravisAbstract:Absorption of a photon by a rhodopsin or cone-opsin pigment isomerizes its 11-cis-retinaldehyde (11-cis-RAL) chromophore to All-Trans-Retinaldehyde (all-trans-RAL), which dissociates after a brief period of activation. Light sensitivity is restored to the resulting apo-opsin when it recombines with another 11-cis-RAL. Conversion of all-trans-RAL to 11-cis-RAL is carried out by an enzyme pathway called the visual cycle in cells of the retinal pigment epithelium. A second visual cycle is present in Muller cells of the retina. The retinol isomerase for this noncanonical pathway is dihydroceramide desaturase (DES1), which catalyzes equilibrium isomerization of retinol. Because 11-cis-retinol (11-cis-ROL) constitutes only a small fraction of total retinols in an equilibrium mixture, a subsequent step involving selective removal of 11-cis-ROL is required to drive synthesis of 11-cis-retinoids for production of visual chromophore. Selective esterification of 11-cis-ROL is one possibility. Crude homogenates of chicken retinas rapidly convert all-trans-ROL to 11-cis-retinyl esters (11-cis-REs) with minimal formation of other retinyl-ester isomers. This enzymatic activity implies the existence of an 11-cis-specific retinyl-ester synthase in Muller cells. Here, we evaluated multifunctional O-acyltransferase (MFAT) as a candidate for this 11-cis-RE-synthase. MFAT exhibited much higher catalytic efficiency as a synthase of 11-cis-REs versus other retinyl-ester isomers. Further, we show that MFAT is expressed in Muller cells. Finally, homogenates of cells coexpressing DES1 and MFAT catalyzed the conversion of all-trans-ROL to 11-cis-RP, similar to what we observed with chicken-retina homogenates. MFAT is therefore an excellent candidate for the retinyl-ester synthase that cooperates with DES1 to drive synthesis of 11-cis-retinoids by mass action.
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isomerization and oxidation of vitamin a in cone dominant retinas a novel pathway for visual pigment regeneration in daylight
Neuron, 2002Co-Authors: Nathan L. Mata, Gabriel H Travis, Roxana A Radu, Richard S ClemmonsAbstract:The first step toward light perception is 11-cis to all-trans photoisomerization of the retinaldehyde chromophore in a rod or cone opsin-pigment molecule. Light sensitivity of the opsin pigment is restored through a multistep pathway called the visual cycle, which effects all-trans to 11-cis re-isomerization of the retinoid chromophore. The maximum throughput of the known visual cycle, however, is too slow to explain sustained photosensitivity in bright light. Here, we demonstrate three novel enzymatic activities in cone-dominant ground-squirrel and chicken retinas: an all-trans-retinol isomerase, an 11-cis-retinyl-ester synthase, and an 11-cis-retinol dehydrogenase. Together these activities comprise a novel pathway that regenerates opsin photopigments at a rate 20-fold faster than the known visual cycle. We suggest that this pathway is responsible for sustained daylight vision in vertebrates.
