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1
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Tao L, He D, Chen Y, Yang K, He B, Cai P, Cai B, Liao C, Liu Z, Li S, Chen J, Wu Y. Transferrin ameliorates retinal degeneration by mediating the dimerization of all-trans-retinal. J Biol Chem 2025; 301:108054. [PMID: 39653244 PMCID: PMC11742617 DOI: 10.1016/j.jbc.2024.108054] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2024] [Revised: 11/26/2024] [Accepted: 11/29/2024] [Indexed: 12/30/2024] Open
Abstract
High levels of all-trans-retinal (atRAL) in the retina is considered to be responsible for the development of autosomal recessive Stargardt's disease (STGD1) and dry age-related macular degeneration (dAMD). Two bisretinoids, all-trans-retinal dimer (atRAL-dimer) and N-retinyl-N-retinylidene ethanolamine (A2E), form from the dimerization of atRAL in the retina but they possess much lower toxicity and phototoxicity toward retinal pigment epithelium (RPE) cells than atRAL. Here, we introduced a novel function of transferrin (TRF) in mediating the conversion of atRAL into atRAL-dimer and A2E, which effectively protected the retina from damage by atRAL and prevented retinal function decline in mice, and rescued atRAL-loaded RPE cells. Moreover, TRF-mediated conversion of atRAL to atRAL-dimer and A2E required the help of bicarbonate ions (HCO3-). atRAL had the capacity to stimulate the expression of TRF in RPE and photoreceptor cells as well as RPE/choroid and neural retina of mice, reflecting that the elevation of TRF levels by atRAL is most likely to help defy level increase and cytotoxicity of atRAL through facilitating its dimerization and thereby serves as a mechanism of retinal self-protection. Our findings offer a promising avenue for the treatment of retinopathies characterized by disrupted clearance of atRAL.
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Affiliation(s)
- Lei Tao
- Department of Ophthalmology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China; Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Fujian Engineering and Research Center of Eye Regenerative Medicine, Eye Institute of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Danxue He
- Department of Ophthalmology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China; Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Fujian Engineering and Research Center of Eye Regenerative Medicine, Eye Institute of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Yuling Chen
- Department of Ophthalmology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China; Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Fujian Engineering and Research Center of Eye Regenerative Medicine, Eye Institute of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Kunhuan Yang
- Department of Ophthalmology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China; Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Fujian Engineering and Research Center of Eye Regenerative Medicine, Eye Institute of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Beiting He
- Department of Ophthalmology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China; Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Fujian Engineering and Research Center of Eye Regenerative Medicine, Eye Institute of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Peixin Cai
- Department of Ophthalmology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China; Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Fujian Engineering and Research Center of Eye Regenerative Medicine, Eye Institute of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Binxiang Cai
- Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Fujian Engineering and Research Center of Eye Regenerative Medicine, Eye Institute of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Chunyan Liao
- Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Fujian Engineering and Research Center of Eye Regenerative Medicine, Eye Institute of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Zuguo Liu
- Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Fujian Engineering and Research Center of Eye Regenerative Medicine, Eye Institute of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Shiying Li
- Department of Ophthalmology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Jingmeng Chen
- School of Medicine, Xiamen University, Xiamen, Fujian, China; Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong, China
| | - Yalin Wu
- Department of Ophthalmology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China; Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Fujian Engineering and Research Center of Eye Regenerative Medicine, Eye Institute of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China; Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong, China.
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Murata T, Tsutsui H, Shiina I. ( E)-Selective Weinreb Amide-Type Horner-Wadsworth-Emmons Reaction: Effect of Reaction Conditions, Substrate Scope, Isolation of a Reactive Magnesium Phosphonoenolate, and Applications. J Org Chem 2024; 89:15414-15435. [PMID: 39393081 PMCID: PMC11536377 DOI: 10.1021/acs.joc.4c01140] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2024] [Revised: 08/07/2024] [Accepted: 09/12/2024] [Indexed: 10/13/2024]
Abstract
An iPrMgCl-deprotonating Weinreb amide-type Horner-Wadsworth-Emmons (HWE) reaction was developed, and the effects of diverse reaction conditions, including the base, cation, solvent, and concentration, were investigated to broaden the substrate scope and achieve high (E)-selectivity. The Weinreb amide-type phosphonoenolate generated from iPrMgCl was found to be isolable, stable for at least over a half year, and applicable in the HWE reaction keeping high productivity and selectivity compared with the in situ generated phosphonoenolate. The results prompted us to perform an application study including successive elongation, synthesis of a biscyclopropane, and Weinreb ketone syntheses.
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Affiliation(s)
| | | | - Isamu Shiina
- Department of Applied Chemistry,
Faculty of Science, Tokyo University of
Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
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3
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Scortecci JF, Garces FA, Mahto JK, Molday LL, Van Petegem F, Molday RS. Structural and functional characterization of the nucleotide-binding domains of ABCA4 and their role in Stargardt disease. J Biol Chem 2024; 300:107666. [PMID: 39128720 PMCID: PMC11405800 DOI: 10.1016/j.jbc.2024.107666] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2024] [Revised: 07/29/2024] [Accepted: 08/06/2024] [Indexed: 08/13/2024] Open
Abstract
ABCA4 is an ATP-binding cassette (ABC) transporter that prevents the buildup of toxic retinoid compounds by facilitating the transport of N-retinylidene-phosphatidylethanolamine across membranes of rod and cone photoreceptor cells. Over 1500 missense mutations in ABCA4, many in the nucleotide-binding domains (NBDs), have been genetically linked to Stargardt disease. Here, we show by cryo-EM that ABCA4 is converted from an open outward conformation to a closed conformation upon the binding of adenylyl-imidodiphosphate. Structural information and biochemical studies were used to further define the role of the NBDs in the functional properties of ABCA4 and the mechanisms by which mutations lead to the loss in activity. We show that ATPase activity in both NBDs is required for the functional activity of ABCA4. Mutations in Walker A asparagine residues cause a severe reduction in substrate-activated ATPase activity due to the loss in polar interactions with residues within the D-loops of the opposing NBD. The structural basis for how disease mutations in other NBD residues, including the R1108C, R2077W, R2107H, and L2027F, affect the structure and function of ABCA4 is described. Collectively, our studies provide insight into the structure and function of ABCA4 and mechanisms underlying Stargardt disease.
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Affiliation(s)
- Jessica Fernandes Scortecci
- Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada
| | - Fabian A Garces
- Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada
| | - Jai K Mahto
- Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada
| | - Laurie L Molday
- Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada
| | - Filip Van Petegem
- Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada
| | - Robert S Molday
- Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
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Carozza G, Zerti D, Tisi A, Ciancaglini M, Maccarrone M, Maccarone R. An overview of retinal light damage models for preclinical studies on age-related macular degeneration: identifying molecular hallmarks and therapeutic targets. Rev Neurosci 2024; 35:303-330. [PMID: 38153807 DOI: 10.1515/revneuro-2023-0130] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2023] [Accepted: 11/19/2023] [Indexed: 12/30/2023]
Abstract
Age-related macular degeneration (AMD) is a complex, multifactorial disease leading to progressive and irreversible retinal degeneration, whose pathogenesis has not been fully elucidated yet. Due to the complexity and to the multiple features of the disease, many efforts have been made to develop animal models which faithfully reproduce the overall AMD hallmarks or that are able to mimic the different AMD stages. In this context, light damage (LD) rodent models of AMD represent a suitable and reliable approach to mimic the different AMD forms (dry, wet and geographic atrophy) while maintaining the time-dependent progression of the disease. In this review, we comprehensively reported how the LD paradigms reproduce the main features of human AMD. We discuss the capability of these models to broaden the knowledge in AMD research, with a focus on the mechanisms and the molecular hallmarks underlying the pathogenesis of the disease. We also critically revise the remaining challenges and future directions for the use of LD models.
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Affiliation(s)
- Giulia Carozza
- Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, 67100 L'Aquila, Italy
| | - Darin Zerti
- Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, 67100 L'Aquila, Italy
| | - Annamaria Tisi
- Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, 67100 L'Aquila, Italy
| | - Marco Ciancaglini
- Department of Life, Health & Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy
| | - Mauro Maccarrone
- Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, 67100 L'Aquila, Italy
- European Center for Brain Research (CERC)/Santa Lucia Foundation IRCCS, 00143 Rome, Italy
| | - Rita Maccarone
- Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, 67100 L'Aquila, Italy
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Zhao J, Kim HJ, Montenegro D, Dunaief JL, Sparrow JR. Iron overload and chelation modulates bisretinoid levels in the retina. FRONTIERS IN OPHTHALMOLOGY 2023; 3:1305864. [PMID: 38983013 PMCID: PMC11182296 DOI: 10.3389/fopht.2023.1305864] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/02/2023] [Accepted: 12/07/2023] [Indexed: 07/11/2024]
Abstract
Aim Iron dysregulation in conjunction with other disease processes may exacerbate retinal degeneration. We employed models of iron overload and iron chelation to explore the interactions between iron-catalyzed oxidation and photoreactive bisretinoid lipofuscin. Methods The mice were injected intravitreally with ferric ammonium citrate (FAC) or were treated using the iron chelator deferiprone (DFP) from birth to 2 months of age. Short-wavelength fundus autofluorescence (SW-AF) and spectral-domain optical coherence tomography (SD-OCT) scans were acquired. The bisretinoid levels were quantified using ultra performance liquid chromatography (UPLC) and in vivo through quantitative fundus autofluorescence (qAF). In histologic sections, the photoreceptor cell viability was assessed by measuring the thickness of the outer nuclear layer (ONL). Results The levels of bisretinoids, all-trans-retinal dimers, and A2PE were significantly increased in the FAC-injected eyes of C57BL/6J mice. Seven days after FAC injection, hyperautofluorescent foci were visible in fundus autofluorescence (488 nm) images, and in SD-OCT scans, aberrant hyperreflectivity was present in the outer retina and ONL thinning was observed. In FAC-injected Abca4-/- mice with pronounced RPE bisretinoid lipofuscin accumulation, the hyperautofluorescent puncta were more abundant than in the wild-type mice, and the extent of ONL thinning was greater. Conversely, the intravitreal injection of FAC in Mertk-/- mice led to a more modest increase in A2PE after 2 days. In contrast to the effect of iron accumulation, chelation with DFP resulted in significantly increased levels of A2E and A2-GPE and qAF due to the reduced iron-catalyzed oxidation of bisretinoids. In Mertk-/- mice, the A2E level was significantly lower and the ONL area was smaller than in DFP-treated mice. DFP chelation did not impair the visual cycle in BALB/cJ mice. Conclusion Iron accumulation was associated with progressive impairment in photoreceptor cells that was associated with the increased formation of a bisretinoid species known to form in photoreceptor outer segments as a precursor to A2E. Additionally, disease features such as the development of hyperautofluorescence puncta in fundus AF images, hyperreflectivity in the outer retina of SD-OCT scans, and ONL thinning were more pronounced when iron was delivered to Abca4-/- mice with a greater propensity for bisretinoid formation. Higher bisretinoid levels and enhanced qAF are indicative of lesser bisretinoid loss due to oxidation.
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Affiliation(s)
- Jin Zhao
- Departments of Ophthalmology, Columbia University Medical Center, New York, NY, United States
| | - Hye Jin Kim
- Departments of Ophthalmology, Columbia University Medical Center, New York, NY, United States
| | - Diego Montenegro
- Departments of Ophthalmology, Columbia University Medical Center, New York, NY, United States
| | - Josh L Dunaief
- F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, United States
| | - Janet R Sparrow
- Departments of Ophthalmology, Columbia University Medical Center, New York, NY, United States
- Pathology and Cell Biology, Columbia University Medical Center, New York, NY, United States
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Lieffrig SA, Gyimesi G, Mao Y, Finnemann SC. Clearance phagocytosis by the retinal pigment epithelial during photoreceptor outer segment renewal: Molecular mechanisms and relation to retinal inflammation. Immunol Rev 2023; 319:81-99. [PMID: 37555340 PMCID: PMC10615845 DOI: 10.1111/imr.13264] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2023] [Accepted: 07/18/2023] [Indexed: 08/10/2023]
Abstract
Mammalian photoreceptor outer segment renewal is a highly coordinated process that hinges on timed cell signaling between photoreceptor neurons and the adjacent retinal pigment epithelial (RPE). It is a strictly rhythmic, synchronized process that underlies in part circadian regulation. We highlight findings from recently developed methods that quantify distinct phases of outer segment renewal in retinal tissue. At light onset, outer segments expose the conserved "eat-me" signal phosphatidylserine exclusively at their distal, most aged tip. A coordinated two-receptor efferocytosis process follows, in which ligands bridge outer segment phosphatidylserine with the RPE receptors αvβ5 integrin, inducing cytosolic signaling toward Rac1 and focal adhesion kinase/MERTK, and with MERTK directly, additionally inhibiting RhoA/ROCK and thus enabling F-actin dynamics favoring outer segment fragment engulfment. Photoreceptors and RPE persist for life with each RPE cell in the eye servicing dozens of overlying photoreceptors. Thus, RPE cells phagocytose more often and process more material than any other cell type. Mutant mice with impaired outer segment renewal largely retain functional photoreceptors and retinal integrity. However, when anti-inflammatory signaling in the RPE via MERTK or the related TYRO3 is lacking, catastrophic inflammation leads to immune cell infiltration that swiftly destroys the retina causing blindness.
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Affiliation(s)
- Stephanie A. Lieffrig
- Center for Cancer, Genetic Diseases and Gene Regulation, Department of Biological Sciences, Fordham University, Bronx, NY
| | - Gavin Gyimesi
- Center for Cancer, Genetic Diseases and Gene Regulation, Department of Biological Sciences, Fordham University, Bronx, NY
| | | | - Silvia C. Finnemann
- Center for Cancer, Genetic Diseases and Gene Regulation, Department of Biological Sciences, Fordham University, Bronx, NY
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7
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Xu T, Molday L, Molday R. Retinal-phospholipid Schiff-base conjugates and their interaction with ABCA4, the ABC transporter associated with Stargardt Disease. J Biol Chem 2023; 299:104614. [PMID: 36931393 PMCID: PMC10127136 DOI: 10.1016/j.jbc.2023.104614] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2022] [Revised: 03/10/2023] [Accepted: 03/11/2023] [Indexed: 03/17/2023] Open
Abstract
N-retinylidene-phosphatidylethanolamine (N-Ret-PE), the Schiff-base conjugate formed through the reversible reaction of retinal (Vitamin A-aldehyde) and phosphatidylethanolamine, plays a crucial role in the visual cycle and visual pigment photoregeneration. However, N-Ret-PE can react with another molecule of retinal to form toxic di-retinoids if not removed from photoreceptors through its transport across photoreceptor membranes by the ATP-binding-cassette transporter ABCA4. Loss-of-function mutations in ABCA4 are known to cause Stargardt disease (STGD1), an inherited retinal degenerative disease associated with the accumulation of fluorescent di-retinoids and severe loss in vision. A larger assessment of retinal-phospholipid Schiff-base conjugates in photoreceptors is needed, along with further investigation of ABCA4 residues important for N-Ret-PE binding. In this study we show that N-Ret-PE formation is dependent on pH and phospholipid content. When retinal is added to liposomes or photoreceptor membranes, 40-60% is converted to N-Ret-PE at physiological pH. Phosphatidylserine and taurine also react with retinal to form N-retinylidene-phosphatidylserine (N-Ret-PS) and N-retinylidene-taurine, respectively, but at significantly lower levels. N-Ret-PS is not a substrate for ABCA4 and reacts poorly with retinal to form di-retinoids. Additionally, amino acid residues within the binding pocket of ABCA4 that contribute to its interaction with N-Ret-PE were identified and characterized using site-directed mutagenesis together with functional and binding assays. Substitution of arginine residues and hydrophobic residues with alanine or residues implicated in STGD1 significantly reduced or in some cases eliminated substrate-activated ATPase activity and substrate binding. Collectively, this study provides important insight into conditions which affect retinal-phospholipid Schiff-base formation and mechanisms underlying the pathogenesis of STGD1.
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Affiliation(s)
- Tongzhou Xu
- Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, B.C. V6T 1Z3 Canada
| | - LaurieL Molday
- Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, B.C. V6T 1Z3 Canada
| | - RobertS Molday
- Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, B.C. V6T 1Z3 Canada.
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Protein-Mediated Carotenoid Delivery Suppresses the Photoinducible Oxidation of Lipofuscin in Retinal Pigment Epithelial Cells. Antioxidants (Basel) 2023; 12:antiox12020413. [PMID: 36829973 PMCID: PMC9952040 DOI: 10.3390/antiox12020413] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2023] [Revised: 01/31/2023] [Accepted: 02/03/2023] [Indexed: 02/10/2023] Open
Abstract
Lipofuscin of retinal pigment epithelium (RPE) cells is a complex heterogeneous system of chromophores which accumulates as granules during the cell's lifespan. Lipofuscin serves as a source of various cytotoxic effects linked with oxidative stress. Several age-related eye diseases such as macular degeneration of the retina, as well as some severe inherited eye pathologies, are accompanied by a significant increase in lipofuscin granule concentration. The accumulation of carotenoids in the RPE could provide an effective antioxidant protection against lipofuscin cytotoxic manifestations. Given the highly lipophilic nature of carotenoids, their targeted delivery to the vulnerable tissues can potentially be assisted by special proteins. In this study, we demonstrate how protein-mediated delivery of zeaxanthin using water-soluble Bombyx mori carotenoid-binding protein (BmCBP-ZEA) suppresses the photoinducible oxidative stress in RPE cells caused by irradiation of lipofuscin with intense white light. We implemented fluorescence lifetime imaging of the RPE cell culture ARPE-19 fed with lipofuscin granules and then irradiated by white light with and without the addition of BmCBP-ZEA. We demonstrate that after irradiation the mean fluorescence lifetime of lipofuscin significantly increases, while the presence of BmCBP-ZEA at 200 nM concentration suppresses the increase in the average lifetime of lipofuscin fluorescence, indicating an approx. 35% inhibition of the oxidative stress. This phenomenon serves as indirect yet important evidence of the efficiency of the protein-mediated carotenoid delivery into pigment epithelium cells.
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Feldman T, Ostrovskiy D, Yakovleva M, Dontsov A, Borzenok S, Ostrovsky M. Lipofuscin-Mediated Photic Stress Induces a Dark Toxic Effect on ARPE-19 Cells. Int J Mol Sci 2022; 23:12234. [PMID: 36293088 PMCID: PMC9602730 DOI: 10.3390/ijms232012234] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2022] [Revised: 10/10/2022] [Accepted: 10/11/2022] [Indexed: 12/25/2023] Open
Abstract
Lipofuscin granules from retinal pigment epithelium (RPE) cells contain bisretinoid fluorophores, which are photosensitizers and are phototoxic to cells. In the presence of oxygen, bisretinoids are oxidized to form various products, containing aldehydes and ketones, which are also potentially cytotoxic. In a prior study, we identified that bisretinoid oxidation and degradation products have both hydrophilic and amphiphilic properties, allowing their diffusion through the lipofuscin granule membrane into the RPE cell cytoplasm, and are thiobarbituric acid (TBA)-active. The purpose of the present study was to determine if these products exhibit a toxic effect to the RPE cell also in the absence of light. The experiments were performed using the lipofuscin-fed ARPE-19 cell culture. The RPE cell viability analysis was performed with the use of flow cytofluorimetry and laser scanning confocal microscopy. The results obtained indicated that the cell viability of the lipofuscin-fed ARPE-19 sample was clearly reduced not immediately after visible light irradiation for 18 h, but after 4 days maintaining in the dark. Consequently, we could conclude that bisretinoid oxidation products have a damaging effect on the RPE cell in the dark and can be considered as an aggravating factor in age-related macular degeneration progression.
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Affiliation(s)
- Tatiana Feldman
- Department of Biology, Lomonosov Moscow State University, Leninskiye Gory 1, 119234 Moscow, Russia
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygin Street, 119334 Moscow, Russia
- Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Street, 119334 Moscow, Russia
| | - Dmitriy Ostrovskiy
- Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Street, 119334 Moscow, Russia
- Sv. Fyodorov Eye Microsurgery Complex, 59a Beskudnikovsky bld., 127486 Moscow, Russia
| | - Marina Yakovleva
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygin Street, 119334 Moscow, Russia
- Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Street, 119334 Moscow, Russia
| | - Alexander Dontsov
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygin Street, 119334 Moscow, Russia
- Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Street, 119334 Moscow, Russia
| | - Sergey Borzenok
- Sv. Fyodorov Eye Microsurgery Complex, 59a Beskudnikovsky bld., 127486 Moscow, Russia
| | - Mikhail Ostrovsky
- Department of Biology, Lomonosov Moscow State University, Leninskiye Gory 1, 119234 Moscow, Russia
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygin Street, 119334 Moscow, Russia
- Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Street, 119334 Moscow, Russia
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10
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Feldman TB, Dontsov AE, Yakovleva MA, Ostrovsky MA. Photobiology of lipofuscin granules in the retinal pigment epithelium cells of the eye: norm, pathology, age. Biophys Rev 2022; 14:1051-1065. [PMID: 36124271 PMCID: PMC9481861 DOI: 10.1007/s12551-022-00989-9] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2022] [Accepted: 07/26/2022] [Indexed: 01/10/2023] Open
Abstract
Lipofuscin granules (LGs) are accumulated in the retinal pigment epithelium (RPE) cells. The progressive LG accumulation can somehow lead to pathology and accelerate the aging process. The review examines composition, spectral properties and photoactivity of LGs isolated from the human cadaver eyes. By use of atomic force microscopy and near-field microscopy, we have revealed the fluorescent heterogeneity of LGs. We have discovered the generation of reactive oxygen species by LGs, and found that LGs and melanolipofuscin granules are capable of photoinduced oxidation of lipids. It was shown that A2E, as the main fluorophore (bisretinoid) of LGs, is much less active as an oxidation photosensitizer than other fluorophores (bisretinoids) of LGs. Photooxidized products of bisretinoids pose a much greater danger to the cell than non-oxidized one. Our studies of the fluorescent properties of LGs and their fluorophores (bisretinoids) showed for the first time that their spectral characteristics change (shift to the short-wavelength region) in pathology and after exposure to ionizing radiation. By recording the fluorescence spectra and fluorescence decay kinetics of oxidized products of LG fluorophores, it is possible to improve the methods of early diagnosis of degenerative diseases. Lipofuscin ("aging pigment") is not an inert "slag". The photoactivity of LGs can pose a significant danger to the RPE cells. Fluorescence characteristics of LGs are a tool to detect early stages of degeneration in the retina and RPE.
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Affiliation(s)
- T. B. Feldman
- Department of Biology, Lomonosov Moscow State University, Moscow, Russia
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia
| | - A. E. Dontsov
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia
| | - M. A. Yakovleva
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia
| | - M. A. Ostrovsky
- Department of Biology, Lomonosov Moscow State University, Moscow, Russia
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia
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11
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Kotnala A, Senthilkumari S, Wu G, Stewart TG, Curcio CA, Halder N, Singh SB, Kumar A, Velpandian T. Retinal Pigment Epithelium in Human Donor Eyes Contains Higher Levels of Bisretinoids Including A2E in Periphery than Macula. Invest Ophthalmol Vis Sci 2022; 63:6. [PMID: 35671050 PMCID: PMC9187938 DOI: 10.1167/iovs.63.6.6] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Purpose With age, human retinal pigment epithelium (RPE) accumulates bisretinoid fluorophores that may impact cellular function and contribute to age-related macular degeneration (AMD). Bisretinoids are comprised of a central pyridinium, dihydropyridinium, or cyclohexadiene ring. The pyridinium bisretinoid A2E has been extensively studied, and its quantity in the macula has been questioned. Age-changes and distributions of other bisretinoids are not well characterized. We measured levels of three bisretinoids and oxidized A2E in macula and periphery in human donor eyes of different ages. Methods Eyes (N = 139 donors, 61 women and 78 men, aged 40–80 years) were dissected into 8 mm diameter macular and temporal periphery punches. Using liquid chromatography – electrospray ionization – mass spectrometry (LC-ESI-MS) and an authentic synthesized standard, we quantified A2E (ng). Using LC-ESI-MS and a 50-eye-extract of A2E, we semiquantified A2E and 3 other compounds (eye extract equivalent units [EEEUs): A2-glycerophosphoethanolamine (A2GPE), dihydropyridine phosphatidyl ethanolamine (A2DHPE), and monofuranA2E (MFA2E). Results A2E quantities in ng and EEEUs were highly correlated (r = 0.97, P < 0.001). From 262 eyes, 5 to 9-fold higher levels were observed in the peripheral retina than in the macula for all assayed compounds. A2E, A2DHPE, and MFA2E increased with age, whereas A2GPE remained unaffected. No significant right-left or male-female differences were detected. Conclusions Significantly higher levels were observed in the periphery than in the macula for all assayed compounds signifying biologic differences between these regions. Levels of oxidized A2E parallel native A2E and not the distribution of retinal illuminance. Data will assist with the interpretion of clinical trial outcomes of agents targeting bisretinoid-related pathways.
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Affiliation(s)
- Ankita Kotnala
- Ocular Pharmacology & Pharmacy Division, All India Institute of Medical Sciences, New Delhi, India
| | - Srinivasan Senthilkumari
- Department of Ocular Pharmacology, Aravind Medical Research Foundation (AMRF), Dr. G. Venkataswamy Eye Research Institute, #1, Anna Nagar, Madurai -20, Tamilnadu, India
| | - Gong Wu
- Department of Biostatics, Vanderbilt University Medical Centre, Nashville, Tennessee, United States
| | - Thomas G Stewart
- Department of Biostatics, Vanderbilt University Medical Centre, Nashville, Tennessee, United States
| | - Christine A Curcio
- Department of Ophthalmology and Visual Sciences, University of Alabama at Birmingham, Birmingham, Alabama, United States
| | - Nabanita Halder
- Ocular Pharmacology & Pharmacy Division, All India Institute of Medical Sciences, New Delhi, India
| | | | - Atul Kumar
- Department of Ophthalmology, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India
| | - Thirumurthy Velpandian
- Ocular Pharmacology & Pharmacy Division, All India Institute of Medical Sciences, New Delhi, India
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12
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Dontsov A, Yakovleva M, Trofimova N, Sakina N, Gulin A, Aybush A, Gostev F, Vasin A, Feldman T, Ostrovsky M. Water-Soluble Products of Photooxidative Destruction of the Bisretinoid A2E Cause Proteins Modification in the Dark. Int J Mol Sci 2022; 23:ijms23031534. [PMID: 35163454 PMCID: PMC8836230 DOI: 10.3390/ijms23031534] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2021] [Revised: 01/21/2022] [Accepted: 01/26/2022] [Indexed: 01/06/2023] Open
Abstract
Aging of the retina is accompanied by a sharp increase in the content of lipofuscin granules and bisretinoid A2E in the cells of the retinal pigment epithelium (RPE) of the human eye. It is known that A2E can have a toxic effect on RPE cells. However, the specific mechanisms of the toxic effect of A2E are poorly understood. We investigated the effect of the products of photooxidative destruction of A2E on the modification of bovine serum albumin (BSA) and hemoglobin from bovine erythrocytes. A2E was irradiated with a blue light-emitting diode (LED) source (450 nm) or full visible light (400–700 nm) of a halogen lamp, and the resulting water-soluble products of photooxidative destruction were investigated for the content of carbonyl compounds by mass spectrometry and reaction with thiobarbituric acid. It has been shown that water-soluble products formed during A2E photooxidation and containing carbonyl compounds cause modification of serum albumin and hemoglobin, measured by an increase in fluorescence intensity at 440–455 nm. The antiglycation agent aminoguanidine inhibited the process of modification of proteins. It is assumed that water-soluble carbonyl products formed as a result of A2E photodestruction led to the formation of modified proteins, activation of the inflammation process, and, as a consequence, to the progression of various senile eye pathologies.
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Affiliation(s)
- Alexander Dontsov
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 119334 Moscow, Russia; (M.Y.); (N.T.); (N.S.); (T.F.); (M.O.)
- Correspondence: ; +7-495-939-7422
| | - Marina Yakovleva
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 119334 Moscow, Russia; (M.Y.); (N.T.); (N.S.); (T.F.); (M.O.)
| | - Natalia Trofimova
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 119334 Moscow, Russia; (M.Y.); (N.T.); (N.S.); (T.F.); (M.O.)
| | - Natalia Sakina
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 119334 Moscow, Russia; (M.Y.); (N.T.); (N.S.); (T.F.); (M.O.)
| | - Alexander Gulin
- N.N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, 119991 Moscow, Russia; (A.G.); (A.A.); (F.G.); (A.V.)
| | - Arseny Aybush
- N.N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, 119991 Moscow, Russia; (A.G.); (A.A.); (F.G.); (A.V.)
| | - Fedor Gostev
- N.N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, 119991 Moscow, Russia; (A.G.); (A.A.); (F.G.); (A.V.)
| | - Alexander Vasin
- N.N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, 119991 Moscow, Russia; (A.G.); (A.A.); (F.G.); (A.V.)
| | - Tatiana Feldman
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 119334 Moscow, Russia; (M.Y.); (N.T.); (N.S.); (T.F.); (M.O.)
- Department of Biology, Lomonosov Moscow State University, Leninskiye Gory 1, 119234 Moscow, Russia
| | - Mikhail Ostrovsky
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 119334 Moscow, Russia; (M.Y.); (N.T.); (N.S.); (T.F.); (M.O.)
- Department of Biology, Lomonosov Moscow State University, Leninskiye Gory 1, 119234 Moscow, Russia
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13
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Lipofuscin Granule Bisretinoid Oxidation in the Human Retinal Pigment Epithelium forms Cytotoxic Carbonyls. Int J Mol Sci 2021; 23:ijms23010222. [PMID: 35008647 PMCID: PMC8745408 DOI: 10.3390/ijms23010222] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2021] [Revised: 12/16/2021] [Accepted: 12/22/2021] [Indexed: 01/06/2023] Open
Abstract
Age-related macular degeneration (AMD) is the primary cause of central blindness among the elderly. AMD is associated with progressive accumulation of lipofuscin granules in retinal pigment epithelium (RPE) cells. Lipofuscin contains bisretinoid fluorophores, which are photosensitizers and are phototoxic to RPE and neuroretinal cells. In the presence of oxygen, bisretinoids are also oxidized, forming various products, consisting primarily of aldehydes and ketones, which are also potentially cytotoxic. In a prior study, we identified that in AMD, bisretinoid oxidation products are increased in RPE lipofuscin granules. The purpose of the present study was to determine if these products were toxic to cellular structures. The physicochemical characteristics of bisretinoid oxidation products in lipofuscin, which were obtained from healthy donor eyes, were studied. Raman spectroscopy and time-of-flight secondary ion mass spectrometry (ToF–SIMS) analysis identified the presence of free-state aldehydes and ketones within the lipofuscin granules. Together, fluorescence spectroscopy, high-performance liquid chromatography, and mass spectrometry revealed that bisretinoid oxidation products have both hydrophilic and amphiphilic properties, allowing their diffusion through lipofuscin granule membrane into the RPE cell cytoplasm. These products contain cytotoxic carbonyls, which can modify cellular proteins and lipids. Therefore, bisretinoid oxidation products are a likely aggravating factor in the pathogenesis of AMD.
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14
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Molday RS, Garces FA, Scortecci JF, Molday LL. Structure and function of ABCA4 and its role in the visual cycle and Stargardt macular degeneration. Prog Retin Eye Res 2021; 89:101036. [PMID: 34954332 DOI: 10.1016/j.preteyeres.2021.101036] [Citation(s) in RCA: 45] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Revised: 12/07/2021] [Accepted: 12/13/2021] [Indexed: 12/17/2022]
Abstract
ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) transporters that is preferentially localized along the rim region of rod and cone photoreceptor outer segment disc membranes. It uses the energy from ATP binding and hydrolysis to transport N-retinylidene-phosphatidylethanolamine (N-Ret-PE), the Schiff base adduct of retinal and phosphatidylethanolamine, from the lumen to the cytoplasmic leaflet of disc membranes. This ensures that all-trans-retinal and excess 11-cis-retinal are efficiently cleared from photoreceptor cells thereby preventing the accumulation of toxic retinoid compounds. Loss-of-function mutations in the gene encoding ABCA4 cause autosomal recessive Stargardt macular degeneration, also known as Stargardt disease (STGD1), and related autosomal recessive retinopathies characterized by impaired central vision and an accumulation of lipofuscin and bis-retinoid compounds. High resolution structures of ABCA4 in its substrate and nucleotide free state and containing bound N-Ret-PE or ATP have been determined by cryo-electron microscopy providing insight into the molecular architecture of ABCA4 and mechanisms underlying substrate recognition and conformational changes induced by ATP binding. The expression and functional characterization of a large number of disease-causing missense ABCA4 variants have been determined. These studies have shed light into the molecular mechanisms underlying Stargardt disease and a classification that reliably predicts the effect of a specific missense mutation on the severity of the disease. They also provide a framework for developing rational therapeutic treatments for ABCA4-associated diseases.
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Affiliation(s)
- Robert S Molday
- Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, B.C., Canada; Department of Ophthalmology & Visual Sciences, University of British Columbia, Vancouver, B.C., Canada.
| | - Fabian A Garces
- Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, B.C., Canada
| | | | - Laurie L Molday
- Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, B.C., Canada
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15
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Yakovleva MA, Feldman TB, Lyakhova KN, Utina DM, Kolesnikova IA, Vinogradova YV, Molokanov AG, Ostrovsky MA. Ionized Radiation-Mediated Retinoid Oxidation in the Retina and Retinal Pigment Epithelium of the Murine Eye. Radiat Res 2021; 197:270-279. [PMID: 34879150 DOI: 10.1667/rade-21-00069.1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2021] [Accepted: 10/15/2021] [Indexed: 11/03/2022]
Abstract
The present study evaluated the effects of proton and gamma-ray ionizing radiation on the mouse eye. The aim of this work was to analyze radiation-mediated retinoid oxidation in the retina and retinal pigment epithelium (RPE). The findings from this analysis can be used to develop a noninvasive method for rapid assessment of the effects of ionizing radiation. Comparative fluorescence and chromatographic analyses of retinoids before and after irradiations were performed. The fluorescent properties of chloroform extracts from irradiated mouse retina and RPE exhibited an increase in fluorescence intensity in the short-wave region of the spectrum (λ < 550 nm). This change is due to increased retinal and RPE retinoid oxidation and degradation products after radiation exposure. Comparative analyses of radiation effects demonstrated that the effect of proton exposure on the retina and RPE was higher than that of gamma-ray exposure. The present study revealed a new approach to assessing the level of radiation exposure in ocular tissues.
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Affiliation(s)
- Marina A Yakovleva
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia
| | - Tatiana B Feldman
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia.,Department of Molecular Physiology, Biological Faculty, Moscow State University, Moscow, Russia
| | - Kristina N Lyakhova
- Laboratory of Radiation Biology, Joint Institute for Nuclear Research, Dubna, Moscow region, Russia
| | - Dina M Utina
- Laboratory of Radiation Biology, Joint Institute for Nuclear Research, Dubna, Moscow region, Russia
| | - Inna A Kolesnikova
- Laboratory of Radiation Biology, Joint Institute for Nuclear Research, Dubna, Moscow region, Russia
| | - Yuliya V Vinogradova
- Laboratory of Radiation Biology, Joint Institute for Nuclear Research, Dubna, Moscow region, Russia
| | - Alexander G Molokanov
- Laboratory of Radiation Biology, Joint Institute for Nuclear Research, Dubna, Moscow region, Russia
| | - Mikhail A Ostrovsky
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia.,Department of Molecular Physiology, Biological Faculty, Moscow State University, Moscow, Russia.,Laboratory of Radiation Biology, Joint Institute for Nuclear Research, Dubna, Moscow region, Russia
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16
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Zhang D, Mihai DM, Washington I. Vitamin A cycle byproducts explain retinal damage and molecular changes thought to initiate retinal degeneration. Biol Open 2021; 10:273577. [PMID: 34842275 PMCID: PMC8649638 DOI: 10.1242/bio.058600] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2021] [Accepted: 09/03/2021] [Indexed: 01/24/2023] Open
Abstract
In the most prevalent retinal diseases, including Stargardt disease and age-related macular degeneration (AMD), byproducts of vitamin A form in the retina abnormally during the vitamin A cycle. Despite evidence of their toxicity, whether these vitamin A cycle byproducts contribute to retinal disease, are symptoms, beneficial, or benign has been debated. We delivered a representative vitamin A byproduct, A2E, to the rat's retina and monitored electrophysiological, histological, proteomic, and transcriptomic changes. We show that the vitamin A cycle byproduct is sufficient alone to damage the RPE, photoreceptor inner and outer segments, and the outer plexiform layer, cause the formation of sub-retinal debris, alter transcription and protein synthesis, and diminish retinal function. The presented data are consistent with the theory that the formation of vitamin A byproducts during the vitamin A cycle is neither benign nor beneficial but may be sufficient alone to cause the most prevalent forms of retinal disease. Retarding the formation of vitamin A byproducts could potentially address the root cause of several retinal diseases to eliminate the threat of irreversible blindness for millions of people. Summary: During the vitamin A cycle, byproducts of vitamin A form in the eye. Using a rat model, we show that the byproducts alone can explain several retinal derangements observed in the prodromal phase of human retinal disease. Retarding the formation of these byproducts may address the root cause of the most prevalent retinal diseases.
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Affiliation(s)
- Dan Zhang
- Columbia University Medical Center, Ophthalmology, New York, NY 10032, USA
| | - Doina M Mihai
- Columbia University Medical Center, Ophthalmology, New York, NY 10032, USA
| | - Ilyas Washington
- Columbia University Medical Center, Ophthalmology, New York, NY 10032, USA.,biOOrg3.14, Buffalo, WY 82834, USA
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17
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Kim HJ, Montenegro D, Zhao J, Sparrow JR. Bisretinoids of the Retina: Photo-Oxidation, Iron-Catalyzed Oxidation, and Disease Consequences. Antioxidants (Basel) 2021; 10:antiox10091382. [PMID: 34573014 PMCID: PMC8467448 DOI: 10.3390/antiox10091382] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2021] [Revised: 08/25/2021] [Accepted: 08/26/2021] [Indexed: 01/06/2023] Open
Abstract
The retina and, in particular, retinal pigment epithelial cells are unusual for being encumbered by exposure to visible light, while being oxygen-rich, and also amassing photoreactive molecules. These fluorophores (bisretinoids) are generated as a byproduct of the activity of vitamin A aldehyde-the chromophore necessary for vision. Bisretinoids form in photoreceptor cells due to random reactions of two molecules of vitamin A aldehyde with phosphatidylethanolamine; bisretinoids are subsequently transferred to retinal pigment epithelial (RPE) cells, where they accumulate in the lysosomal compartment with age. Bisretinoids can generate reactive oxygen species by both energy and electron transfer, and they become photo-oxidized and photolyzed in the process. While these fluorescent molecules are accrued by RPE cells of all healthy eyes, they are also implicated in retinal disease.
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Affiliation(s)
- Hye Jin Kim
- Department of Ophthalmology, Columbia University Medical Center, 635 W., 165th Str., New York, NY 10032, USA; (H.J.K.); (D.M.); (J.Z.)
| | - Diego Montenegro
- Department of Ophthalmology, Columbia University Medical Center, 635 W., 165th Str., New York, NY 10032, USA; (H.J.K.); (D.M.); (J.Z.)
| | - Jin Zhao
- Department of Ophthalmology, Columbia University Medical Center, 635 W., 165th Str., New York, NY 10032, USA; (H.J.K.); (D.M.); (J.Z.)
| | - Janet R. Sparrow
- Department of Ophthalmology, Columbia University Medical Center, 635 W., 165th Str., New York, NY 10032, USA; (H.J.K.); (D.M.); (J.Z.)
- Department of Pathology and Cell Biology, Columbia University Medical Center, 635 W., 165th Str., New York, NY 10032, USA
- Correspondence: ; Tel.: +1-212-305-9944
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18
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Vitamin A cycle byproducts impede dark adaptation. J Biol Chem 2021; 297:101074. [PMID: 34391781 PMCID: PMC8427233 DOI: 10.1016/j.jbc.2021.101074] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2021] [Revised: 08/02/2021] [Accepted: 08/11/2021] [Indexed: 11/21/2022] Open
Abstract
Impaired dark adaptation (DA), a defect in the ability to adjust to dimly lit settings, is a universal hallmark of aging. However, the mechanisms responsible for impaired DA are poorly understood. Vitamin A byproducts, such as vitamin A dimers, are small molecules that form in the retina during the vitamin A cycle. We show that later in life, in the human eye, these byproducts reach levels commensurate with those of vitamin A. In mice, selectively inhibiting the formation of these byproducts, with the investigational drug C20D3-vitamin A, results in faster DA. In contrast, acutely increasing these ocular byproducts through exogenous delivery leads to slower DA, with otherwise preserved retinal function and morphology. Our findings reveal that vitamin A cycle byproducts alone are sufficient to cause delays in DA and suggest that they may contribute to universal age-related DA impairment. Our data further indicate that the age-related decline in DA may be tractable to pharmacological intervention by C20D3-vitamin A.
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19
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Kim HJ, Sparrow JR. Bisretinoid phospholipid and vitamin A aldehyde: shining a light. J Lipid Res 2021; 62:100042. [PMID: 32371567 PMCID: PMC7933493 DOI: 10.1194/jlr.tr120000742] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2020] [Revised: 04/29/2020] [Indexed: 12/17/2022] Open
Abstract
Vitamin A aldehyde covalently bound to opsin protein is embedded in a phospholipid-rich membrane that supports photon absorption and phototransduction in photoreceptor cell outer segments. Following absorption of a photon, the 11-cis-retinal chromophore of visual pigment in photoreceptor cells isomerizes to all-trans-retinal. To maintain photosensitivity 11-cis-retinal must be replaced. At the same time, however, all-trans-retinal has to be handled so as to prevent nonspecific aldehyde activity. Some molecules of retinaldehyde upon release from opsin are efficiently reduced to retinol. Other molecules are released into the lipid phase of the disc membrane where they form a conjugate [N-retinylidene-PE (NRPE)] through a Schiff base linkage with PE. The reversible formation of NRPE serves as a transient sink for retinaldehyde that is intended to return retinaldehyde to the visual cycle. However, if instead of hydrolyzing to PE and retinaldehyde, NRPE reacts with a second molecule of retinaldehyde, a synthetic pathway is initiated that leads to the formation of multiple species of unwanted bisretinoid fluorophores. We report on recently identified members of the bisretinoid family, some of which differ with respect to the acyl chains associated with the glycerol backbone. We discuss processing of the lipid moieties of these fluorophores in lysosomes of retinal pigment epithelial cells, their fluorescence characters, and new findings related to light- and iron-associated oxidation of bisretinoids.
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Affiliation(s)
- Hye Jin Kim
- Department of Ophthalmology, Columbia University, New York, NY, USA
| | - Janet R Sparrow
- Department of Ophthalmology, Columbia University, New York, NY, USA; Department of Pathology and Cell Biology, Columbia University, New York, NY, USA.
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20
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Furso J, Zadlo A, Szewczyk G, Sarna TJ. Photoreactivity of Bis-retinoid A2E Complexed with a Model Protein in Selected Model Systems. Cell Biochem Biophys 2020; 78:415-427. [PMID: 32920760 PMCID: PMC7567710 DOI: 10.1007/s12013-020-00942-1] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
The bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E) is formed as a byproduct of visual cycle in retinal pigment epithelium (RPE). It contributes to golden-yellow fluorescence of the age pigment lipofuscin, which accumulates in RPE. Lipofuscin can generate a variety of reactive oxygen species (ROS) upon blue-light excitation. Although in model systems photoreactivity of A2E has been determined to be low, this bis-retinoid exhibited significant phototoxicity in RPE cells in vitro. Although the mechanism of A2E-mediated phototoxicity remains mostly unknown, we hypothesize that formation of A2E-adducts with different biomolecules may play an important role. In this study, we investigated the photochemical reactivity of A2E and its complex with bovine serum albumin (BSA) using UV-Vis absorption and emission spectroscopy, EPR-spin trapping, EPR-oximetry, time-resolved singlet oxygen phosphorescence, and the fluorogenic CBA probe. Our data show that A2E after complexation with this model protein photogenerated an increased level of ROS, particularly singlet oxygen. We also demonstrated the ability of A2E to oxidize BSA upon excitation with blue light in aqueous model systems. The data suggest that pyridinium bis-retinoid could oxidatively modify cellular proteins under physiological conditions.
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Affiliation(s)
- Justyna Furso
- Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387, Krakow, Poland
| | - Andrzej Zadlo
- Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387, Krakow, Poland
| | - Grzegorz Szewczyk
- Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387, Krakow, Poland
| | - Tadeusz J Sarna
- Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387, Krakow, Poland.
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21
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Baeza Moyano D, Baeza Moyano S, Gómez López M, Salcedo Aznal A, González Lezcano RA. Nominal risk analysis of the blue light from LED luminaires in indoor lighting design. OPTIK 2020; 223:165599. [DOI: 10.1016/j.ijleo.2020.165599] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
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22
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Yakovleva MA, Radchenko AS, Feldman TB, Kostyukov AA, Arbukhanova PM, Borzenok SA, Kuzmin VA, Ostrovsky MA. Fluorescence characteristics of lipofuscin fluorophores from human retinal pigment epithelium. Photochem Photobiol Sci 2020; 19:920-930. [PMID: 32441276 DOI: 10.1039/c9pp00406h] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Lipofuscin granules accumulate in the retinal pigment epithelium (RPE) with age, especially in patients with visual diseases, including progressive age-related macular degeneration (AMD). Bisretinoids and their photooxidation and photodegradation products are major sources of lipofuscin granule fluorescence. The present study focused on examining the fluorescence decay characteristics of bisretinoid photooxidation and photodegradation products to evaluate the connection between fluorescence lifetime and spectral characteristics of target fluorophore groups. The primary objective of the study was to apply experimental spectral analysis results of lipofuscin granule fluorescence properties to interpretation of fluorescence lifetime imaging ophthalmoscopy data. Fluorescence analysis of the lipofuscin granule fluorophores in RPE collected from cadaver eyes was performed. The fluorescence lifetimes were measured by picosecond-resolved time correlated single photon counting technique. A global analytical method was applied to analyze data sets. The photooxidation and photodegradation products of bisretinoids exhibited a longer fluorescence lifetime (average value approximately 6 ns) and a shorter wavelength maximum (530-580 nm). Further, these products significantly contributed (more than 30%), to total fluorescence compared to the other fluorophores in lipofuscin granules. Thus, the contribution of oxidized lipofuscin bisretinoids to autofluorescence decay kinetics is an important characteristic for fluorescence lifetime imaging microscopy data analysis. The higher average fluorescence lifetime in AMD eyes was likely due to the higher abundance of oxidized bisretinoids compared with non-oxidized bisretinoids. Because higher level of oxidized bisretinoids is indicative of pathological processes in the retina and RPE, the present findings have the potential to improve fluorescence lifetime imaging approaches for early diagnosis of degenerative processes in the retina and RPE.
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Affiliation(s)
- Marina A Yakovleva
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygin st. 4, 119334, Moscow, Russia
| | - Alexandra Sh Radchenko
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygin st. 4, 119334, Moscow, Russia
| | - Tatiana B Feldman
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygin st. 4, 119334, Moscow, Russia.,Department of Molecular Physiology, Biological Faculty, Lomonosov Moscow State University, Leninskie Gory 1, 119991, Moscow, Russia
| | - Alexey A Kostyukov
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygin st. 4, 119334, Moscow, Russia
| | - Patimat M Arbukhanova
- Sv. Fyodorov Eye Microsurgery Complex, Beskudnikovsky bld. 59a, 127486, Moscow, Russia
| | - Sergey A Borzenok
- Sv. Fyodorov Eye Microsurgery Complex, Beskudnikovsky bld. 59a, 127486, Moscow, Russia
| | - Vladimir A Kuzmin
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygin st. 4, 119334, Moscow, Russia
| | - Mikhail A Ostrovsky
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygin st. 4, 119334, Moscow, Russia.,Department of Molecular Physiology, Biological Faculty, Lomonosov Moscow State University, Leninskie Gory 1, 119991, Moscow, Russia
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A2E Distribution in RPE Granules in Human Eyes. Molecules 2020; 25:molecules25061413. [PMID: 32244898 PMCID: PMC7144568 DOI: 10.3390/molecules25061413] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2020] [Revised: 03/11/2020] [Accepted: 03/17/2020] [Indexed: 12/30/2022] Open
Abstract
A2E (N-retinylidene-N-retinylethanolamine) is a major fluorophore in the RPE (retinal pigment epithelium). To identify and characterize A2E-rich RPE lipofuscin, we fractionated RPE granules from human donor eyes into five fractions (F1–F5 in ascending order of density) by discontinuous sucrose density gradient centrifugation. The dry weight of each fraction was measured and A2E was quantified by liquid chromatography/mass spectrometry (LC/MS) using a synthetic A2E homolog as a standard. Autofluorescence emission was characterized by a customer-built spectro-fluorometer system. A significant A2E level was detected in every fraction, and the highest level was found in F1, a low-density fraction that makes up half of the total weight of all RPE granules, contains 67% of all A2E, and emits 75% of projected autofluorescence by all RPE granules. This group of RPE granules, not described previously, is therefore the most abundant RPE lipofuscin granule population. A progressive decrease in autofluorescence was observed from F2 to F4, whereas no autofluorescence emission was detected from the heavily pigmented F5. The identification of a novel and major RPE lipofuscin population could have significant implications in our understanding of A2E and lipofuscin in human RPE.
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Ellestad G, Zask A, Berova N. The enduring legacy of Koji Nakanishi's research on bioorganic chemistry and natural products. Part 1: Isolation, structure determination and mode of action. Chirality 2020; 32:632-651. [PMID: 32157754 DOI: 10.1002/chir.23214] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2020] [Revised: 02/11/2020] [Accepted: 02/12/2020] [Indexed: 01/09/2023]
Abstract
In this brief review on Koji Nakanishi's remarkable career in natural products chemistry, we have highlighted a number of his accomplishments that illustrate the broad diversity of his interests. These include the isolation, structure determination, and biological mechanism of action of many natural products including the triterpenoid pristimerin; the diterpenoid ginkgolides; insect and crustacean molting hormones; phytoalexins; the toxic red tide principle brevetoxin; the vanadium tunicate pigments; philanthotoxin from killer wasps; antisickling agents; mitomycin DNA adducts; insect antifeedants; a mitotic hormone, the small molecule fish attractants from the sea anemone; new isolation and purification technologies; molecular chemistry of vision; age-related macular degeneration; and the development of the exciton circular dichroism (CD) chirality method for microscale determination of absolute configuration of natural products and chirality of other chiral molecules and supramolecular assembly.
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Affiliation(s)
- George Ellestad
- Department of Chemistry, Columbia University, New York, New York
| | - Arie Zask
- Department of Biological Sciences, Columbia University, New York, New York
| | - Nina Berova
- Department of Chemistry, Columbia University, New York, New York
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25
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Pharmacotherapy for metabolic and cellular stress in degenerative retinal diseases. Drug Discov Today 2019; 25:292-304. [PMID: 31809750 DOI: 10.1016/j.drudis.2019.11.013] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2019] [Revised: 11/08/2019] [Accepted: 11/26/2019] [Indexed: 12/13/2022]
Abstract
Retinal photoreceptors continually endure stresses associated with prolonged light exposure and the metabolic demands of dark adaptation. Although healthy photoreceptors are able to withstand these stresses for several decades, the disease-affected retina functions at a reduced capacity and is at an increased risk for dysfunction. To alleviate cellular and metabolic stressors in degenerative retinal diseases, a new class of drugs that modulate the metabolic activity of the retina have been developed. A clinical candidate in this class (emixustat) has been shown to reduce retinal pathology in various animal models of human retinal disease and is currently under clinical study. Here, we describe the pharmacological properties of emixustat, its mechanisms of action, and potential for use in the treatment of specific retinal diseases.
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Klemm M, Sauer L, Klee S, Link D, Peters S, Hammer M, Schweitzer D, Haueisen J. Bleaching effects and fluorescence lifetime imaging ophthalmoscopy. BIOMEDICAL OPTICS EXPRESS 2019; 10:1446-1461. [PMID: 30891358 PMCID: PMC6420301 DOI: 10.1364/boe.10.001446] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/31/2018] [Revised: 12/02/2018] [Accepted: 12/02/2018] [Indexed: 05/13/2023]
Abstract
This study investigates the influence of photopigment bleaching on autofluorescence lifetimes in the fundus in 21 young healthy volunteers. Three measurements of 30° retinal fields in two spectral channels (SSC: 498-560 nm, LSC: 560-720 nm) were obtained for each volunteer using fluorescence lifetime imaging ophthalmoscopy (FLIO). After dark-adaptation by wearing a custom-made lightproof mask for 30 minutes, the first FLIO-measurement was recorded (dark-adapted state). Subsequently, the eye was bleached for 1 minute (luminance: 3200 cd/m2), followed by a second FLIO-measurement (bleached state). Following an additional 10 minute dark adaptation using the mask, a final FLIO-measurement was recorded (recovered state). Average values of the fluorescence lifetimes were calculated from within different areas of a standardized early treatment diabetic retinopathy study (ETDRS) grid (central area, inner and outer rings). The acquisition time in the bleached state was significantly shortened by approximately 20%. The SSC did not show any significant changes in fluorescence lifetimes with photopigment bleaching, only the LSC showed small but significant bleaching-related changes in the fluorescence lifetimes τ1 and τ2 from all regions, as well as the mean fluorescence lifetime in the central area. The fluorescence lifetime differences caused by bleaching were by far less significant than pathological changes caused by eye diseases. The magnitudes of fluorescence lifetime changes are <10% and do not interfere with healthy or disease related FLIO patterns. Thus, we conclude that bleaching is not a relevant confounder in current clinical applications of FLIO.
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Affiliation(s)
- Matthias Klemm
- Institute of Biomedical Engineering and Informatics, Technische Universität Ilmenau, POB 100565, 98694 Ilmenau, Germany
| | - Lydia Sauer
- University Hospital Jena, Department of Ophthalmology, Am Klinikum 1, 07743 Jena, Germany
- John A. Moran Eye Center, University of Utah, Salt Lake City, UT, USA
| | - Sascha Klee
- Institute of Biomedical Engineering and Informatics, Technische Universität Ilmenau, POB 100565, 98694 Ilmenau, Germany
| | - Dietmar Link
- Institute of Biomedical Engineering and Informatics, Technische Universität Ilmenau, POB 100565, 98694 Ilmenau, Germany
| | - Sven Peters
- University Hospital Jena, Department of Ophthalmology, Am Klinikum 1, 07743 Jena, Germany
| | - Martin Hammer
- University Hospital Jena, Department of Ophthalmology, Am Klinikum 1, 07743 Jena, Germany
- University of Jena, Center for Biomedical Optics and Photonics, 07740 Jena, Germany
| | - Dietrich Schweitzer
- University Hospital Jena, Department of Ophthalmology, Am Klinikum 1, 07743 Jena, Germany
| | - Jens Haueisen
- Institute of Biomedical Engineering and Informatics, Technische Universität Ilmenau, POB 100565, 98694 Ilmenau, Germany
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Gao Z, Liao Y, Chen C, Liao C, He D, Chen J, Ma J, Liu Z, Wu Y. Conversion of all- trans-retinal into all- trans-retinal dimer reflects an alternative metabolic/antidotal pathway of all- trans-retinal in the retina. J Biol Chem 2018; 293:14507-14519. [PMID: 30049796 DOI: 10.1074/jbc.ra118.002447] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2018] [Revised: 07/02/2018] [Indexed: 01/05/2023] Open
Abstract
Free all-trans-retinal (atRAL) and retinal pigment epithelium (RPE) lipofuscin are both considered to play etiological roles in Stargardt disease and age-related macular degeneration. A2E and all-trans-retinal dimer (atRAL-dimer) are two well characterized bisretinoid constituents of RPE lipofuscin. In this study, we found that, after treatment of primary porcine RPE (pRPE) cells with atRAL, atRAL-dimer readily formed and accumulated in a concentration- and time-dependent manner, but A2E was barely detected. Cell-based assays revealed that atRAL, the precursor of atRAL-dimer, significantly altered the morphology of primary pRPE cells and decreased cell viability at a concentration of 80 μm regardless of light exposure. By contrast, atRAL-dimer was not cytotoxic and phototoxic to primary pRPE cells. Compared with atRAL and A2E, atRAL-dimer was more vulnerable to light, followed by the generation of its photocleaved products. Moreover, we observed the presence of atRAL-dimer in reaction mixtures of atRAL with porcine rod outer segments (ROS), RPE/choroid, or neural retina. Taken together, we here proposed an alternative metabolic/antidotal pathway of atRAL in the retina: atRAL that evades participation of the visual (retinoid) cycle undergoes a condensation reaction to yield atRAL-dimer in both ROS and RPE. Translocation of atRAL, all-trans N-retinylidene-phosphatidylethanolamine (NR-PE), atRAL-dimer, and photocleavage products of atRAL-dimer from ROS into RPE is accomplished by phagocytosing shed ROS on a daily basis. Without causing damage to RPE cells, light breaks up total atRAL-dimer within RPE cells to release low-molecular-weight photocleavage fragments. The latter, together with ROS-atRAL-dimer photocleavage products, may easily move across membranes and thereby be metabolically eliminated.
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Affiliation(s)
- Zhan Gao
- From the Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Eye Institute of Xiamen University, College of Medicine, Xiamen University, Xiamen 361102, China
| | - Yi Liao
- From the Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Eye Institute of Xiamen University, College of Medicine, Xiamen University, Xiamen 361102, China
| | - Chao Chen
- From the Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Eye Institute of Xiamen University, College of Medicine, Xiamen University, Xiamen 361102, China
| | - Chunyan Liao
- From the Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Eye Institute of Xiamen University, College of Medicine, Xiamen University, Xiamen 361102, China
| | - Danxue He
- From the Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Eye Institute of Xiamen University, College of Medicine, Xiamen University, Xiamen 361102, China
| | - Jingmeng Chen
- the Clinical Skills Training Center, College of Medicine, Xiamen University, Xiamen 361102, China, and
| | - Jianxing Ma
- the Department of Physiology, Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104
| | - Zuguo Liu
- From the Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Eye Institute of Xiamen University, College of Medicine, Xiamen University, Xiamen 361102, China
| | - Yalin Wu
- From the Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Eye Institute of Xiamen University, College of Medicine, Xiamen University, Xiamen 361102, China,
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Kim HJ, Sparrow JR. Novel bisretinoids of human retina are lyso alkyl ether glycerophosphoethanolamine-bearing A2PE species. J Lipid Res 2018; 59:1620-1629. [PMID: 29986955 DOI: 10.1194/jlr.m084459] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2018] [Revised: 06/26/2018] [Indexed: 12/30/2022] Open
Abstract
Bisretinoids are a family of fluorophores that form in photoreceptor cells' outer segments by nonenzymatic reaction of two vitamin A aldehydes (A2) with phosphatidylethanolamine (PE). Bisretinoid fluorophores are the major constituents of the lipofuscin of retinal pigment epithelium (RPE) that accumulate with age and contribute to some retinal diseases. Here, we report the identification of a previously unknown fluorescent bisretinoid. By ultra-performance LC (UPLC) coupled to photodiode array detection, fluorescence (FLR), and ESI-MS, we determined that this novel bisretinoid is 1-octadecyl-2-lyso-sn-glycero A2PE (alkyl ether lysoA2PE). This structural assignment was based on molecular mass (m/z 998), UV-visible absorbance maxima (340 and 440 nm), and retention time (73 min) and was corroborated by biomimetic synthesis using all-trans-retinal and glycerophosphoethanolamine analogs as starting materials. UPLC profiles of ocular extracts acquired from human donor eyes revealed that alkyl ether lysoA2PE was detectable in RPE, but not neural retina. LysoA2PE FLR spectra exhibited a significant hyperchromic shift in hydrophobic environments. The propensity for lysoA2PE to undergo photooxidation/degradation was less pronounced than A2E. In mechanistic studies, A2PE was hydrolyzed by phospholipase A2 and plasmalogen lysoA2PE was cleaved under acidic conditions. The characterization of these additional members of the bisretinoid family advances our understanding of the mechanisms underlying bisretinoid biogenesis.
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Affiliation(s)
- Hye Jin Kim
- Departments of Ophthalmology Columbia University Medical Center, New York, NY 10032
| | - Janet R Sparrow
- Departments of Ophthalmology Columbia University Medical Center, New York, NY 10032; Pathology and Cell Biology, Columbia University Medical Center, New York, NY 10032.
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Spectral analysis of fundus autofluorescence pattern as a tool to detect early stages of degeneration in the retina and retinal pigment epithelium. Eye (Lond) 2018; 32:1440-1448. [PMID: 29786089 DOI: 10.1038/s41433-018-0109-0] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2017] [Revised: 02/27/2018] [Accepted: 03/30/2018] [Indexed: 01/14/2023] Open
Abstract
PURPOSE The aim of this work is the determination of quantitative diagnostic criteria based on the spectral characteristics of fundus autofluorescence to detect early stages of degeneration in the retina and retinal pigment epithelium (RPE). METHODS RPE cell suspension samples were obtained from the cadaver eyes with and without signs of age-related macular degeneration (AMD). Fluorescence analysis at an excitation wavelength of 488 nm was performed. The fluorescence lifetimes of lipofuscin-granule fluorophores were measured by counting time-correlated photon method. RESULTS Comparative analysis of fluorescence spectra of RPE cell suspensions from the cadaver eyes with and without signs of AMD showed a significant difference in fluorescence intensity at 530-580 nm in response to fluorescence excitation at 488 nm. It was notably higher in eyes with visual pathology than in normal eyes regardless of the age of the eye donor. Measurements of fluorescence lifetimes of lipofuscin fluorophores showed that the contribution of photooxidation and photodegradation products of bisretinoids to the total fluorescence at 530-580 nm of RPE cell suspensions was greater in eyes with visual pathology than in normal eyes. CONCLUSION Because photooxidation and photodegradation products of bisretinoids are markers of photodestructive processes, which can cause RPE cell death and initiate degenerative processes in the retina, quantitative determination of increases in these bisretinoid products in lipofuscin granules may be used to establish quantitative diagnostic criteria for degenerative processes in the retina and RPE.
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Kotnala A, Senthilkumari S, Halder N, Kumar A, Velpandian T. Microwave assisted synthesis for A2E and development of LC-ESI-MS method for quantification of ocular bisretinoids in human retina. J Chromatogr B Analyt Technol Biomed Life Sci 2018; 1073:10-18. [PMID: 29232606 DOI: 10.1016/j.jchromb.2017.11.021] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2017] [Revised: 11/13/2017] [Accepted: 11/17/2017] [Indexed: 01/05/2023]
Abstract
PURPOSE To develop a microwave assisted method for the rapid synthesis of A2E and also to develop a method to quantify N-retinylidene-N-retinylethanolamine(A2E), all-trans retinal dimer (ATRD), A2-glycerophospho ethanolamine (A2GPE), dihydropyridine phosphatidyl ethanolamine (A2DHPE) and monofuran A2E (MFA2E) in age matched retina. METHODS The development of microwave assisted synthesis of A2E, its purification and characterization for its utility in quantification in human retina. The semi-quantitative method development using LC-ESI-MS, LC-ESI-MS/MS and LC-APCI-MS/MS from pooled macula and peripheral retina for the bisretinoid analysis has been done. RESULTS Maximum A2E conversion using microwave assisted process took place at 80°C for 45min with a yield of 55.01%. Highly sensitive and specific mass spectrometric method was developed using reverse phase C-18 separation with positive electrospray ionization and positive atmospheric phase chemical ionization of tandom mass spectrometry. A gradient mobile phase separation was achieved using water and methanol with 0.1% TFA. Multiple reaction monitoring acquisition for ESI and APCI was performed at ATRD m/z 551.2/522.2, A2GPE m/z 746.4/729.5, A2DHPEm/z 594.4/576.5, MFA2E m/z 608.2/591.2, A2E m/z 592.4/418.2. Method was validated using LC-ESI-SIM mode to determine selectivity, linearity, sensitivity, precision and accuracy. CONCLUSION An attempt towards optimization of the synthetic procedure of A2E was made so as to reduce the lengthy reaction time without compromising the yield. Developed method was capable enough for the detection of low level of bisretinids in retina.
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Affiliation(s)
- A Kotnala
- Ocular Pharmacology & Pharmacy Division, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India
| | - S Senthilkumari
- Department of Ocular Pharmacology, Aravind Medical Research Foundation, Madurai, Tamilnadu, India
| | - N Halder
- Ocular Pharmacology & Pharmacy Division, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India
| | - A Kumar
- Department of Ophthalmology, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India
| | - T Velpandian
- Ocular Pharmacology & Pharmacy Division, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India.
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31
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Dontsov A, Koromyslova A, Ostrovsky M, Sakina N. Lipofuscins prepared by modification of photoreceptor cells via glycation or lipid peroxidation show the similar phototoxicity. World J Exp Med 2016; 6:63-71. [PMID: 27909686 PMCID: PMC5114433 DOI: 10.5493/wjem.v6.i4.63] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/29/2016] [Revised: 09/19/2016] [Accepted: 10/18/2016] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the effect of two ways of lipofuscin production (lipid peroxidation and glycation) on lipofuscin fluorescence characteristics and phototoxicity and to compare them with the properties of natural lipofuscin.
METHODS Model lipofuscins were prepared on the basis of bovine photoreceptor outer segments (POS) with bisretinoid A2E addition. One set of samples was prepared from POS modified by lipid peroxidation, while another set from POS modified by glycation with fructose. Fluorescent properties and kinetics of photoinduced superoxide generation of model lipofuscins and human retinal pigment epithelium (RPE) lipofuscin were compared. The fluorescence spectra of samples were measured at 365 nm excitation wavelength and 380-650 emission wavelength.
RESULTS The fluorescence spectra of model lipofuscins are almost the same as the spectrum of natural lipofuscin. Visible light irradiation of both model lipofuscins and natural lipofuscin isolated from RPE cells leads to decrease of a fluorescence maximum at 550 nm and to appearance of a distinct, new maximum at 445-460 nm. The rate of photogeneration of reactive oxygen forms by both model lipofuscins was almost the same and approximately two times less than that of RPE lipofuscin granules.
CONCLUSION These data suggest that fluorescent characteristics and phototoxicity of lipofuscin granules depend only to an insignificant degree on the oxidative modification of POS proteins and lipids, and generally are defined by the bisretinoid fluorophores contained in them.
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Yakovleva MA, Gulin AA, Feldman TB, Bel’skich YC, Arbukhanova PM, Astaf’ev AA, Nadtochenko VA, Borzenok SA, Ostrovsky MA. Time-of-flight secondary ion mass spectrometry to assess spatial distribution of A2E and its oxidized forms within lipofuscin granules isolated from human retinal pigment epithelium. Anal Bioanal Chem 2016; 408:7521-8. [DOI: 10.1007/s00216-016-9854-8] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2016] [Revised: 07/24/2016] [Accepted: 08/01/2016] [Indexed: 11/25/2022]
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Pallitto P, Ablonczy Z, Jones EE, Drake RR, Koutalos Y, Crouch RK, Donello J, Herrmann J. A2E and lipofuscin distributions in macaque retinal pigment epithelium are similar to human. Photochem Photobiol Sci 2016. [PMID: 26223373 DOI: 10.1039/c5pp00170f] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The accumulation of lipofuscin, an autofluorescent aging marker, in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD). Lipofuscin contains several visual cycle byproducts, most notably the bisretinoid N-retinylidene-N-retinylethanolamine (A2E). Previous studies with human donor eyes have shown a significant mismatch between lipofuscin autofluorescence (AF) and A2E distributions. The goal of the current project was to examine this relationship in a primate model with a retinal anatomy similar to that of humans. Ophthalmologically naive young (<10 years., N = 3) and old (>10 years., N = 4) Macaca fascicularis (macaque) eyes, were enucleated, dissected to yield RPE/choroid tissue, and flat-mounted on indium-tin-oxide-coated conductive slides. To compare the spatial distributions of lipofuscin and A2E, fluorescence and mass spectrometric imaging were carried out sequentially on the same samples. The distribution of lipofuscin fluorescence in the primate RPE reflected previously obtained human results, having the highest intensities in a perifoveal ring. Contrarily, A2E levels were consistently highest in the periphery, confirming a lack of correlation between the distributions of lipofuscin and A2E previously described in human donor eyes. We conclude that the mismatch between lipofuscin AF and A2E distributions is related to anatomical features specific to primates, such as the macula, and that this primate model has the potential to fill an important gap in current AMD research.
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Affiliation(s)
- Patrick Pallitto
- Department of Ophthalmology, Storm Eye Institute, Medical University of South Carolina, USA.
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Abstract
Visual systems detect light by monitoring the effect of photoisomerization of a chromophore on the release of a neurotransmitter from sensory neurons, known as rod and cone photoreceptor cells in vertebrate retina. In all known visual systems, the chromophore is 11-cis-retinal complexed with a protein, called opsin, and photoisomerization produces all-trans-retinal. In mammals, regeneration of 11-cis-retinal following photoisomerization occurs by a thermally driven isomerization reaction. Additional reactions are required during regeneration to protect cells from the toxicity of aldehyde forms of vitamin A that are essential to the visual process. Photochemical and phototransduction reactions in rods and cones are identical; however, reactions of the rod and cone visual pigment regeneration cycles differ, and perplexingly, rod and cone regeneration cycles appear to use different mechanisms to overcome the energy barrier involved in converting all-trans- to 11-cis-retinoid. Abnormal processing of all-trans-retinal in the rod regeneration cycle leads to retinal degeneration, suggesting that excessive amounts of the retinoid itself or its derivatives are toxic. This line of reasoning led to the development of various approaches to modifying the activity of the rod visual cycle as a possible therapeutic approach to delay or prevent retinal degeneration in inherited retinal diseases and perhaps in the dry form of macular degeneration (geographic atrophy). In spite of great progress in understanding the functioning of rod and cone regeneration cycles at a molecular level, resolution of a number of remaining puzzling issues will offer insight into the amelioration of several blinding retinal diseases.
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Liu Z, Ueda K, Kim HJ, Sparrow JR. Photobleaching and Fluorescence Recovery of RPE Bisretinoids. PLoS One 2015; 10:e0138081. [PMID: 26366866 PMCID: PMC4569084 DOI: 10.1371/journal.pone.0138081] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2015] [Accepted: 08/26/2015] [Indexed: 01/01/2023] Open
Abstract
The autofluorescence of the retina that originates primarily from lipofuscin fluorophores in retinal pigment epithelial cells, is observed to undergo photobleaching during the acquisition of fundus autofluorescence images. Bisretinoid fluorophores isolated from retinal pigment epithelial cells have the spectral characteristics consistent with their being the source of fundus autofluorescence. Clinically relevant experiments were designed to better understand conditions in the micromilieu of bisretinoid fluorophores that can influence fluorescence efficiencies, photobleaching, and subsequent fluorescence recovery of this fluorophore. The consumption of the bisretinoid A2E due to photooxidation-induced degradation was quantified in solvent systems of variable relative permittivity (formerly called dielectric constant), in micelles, and in phospholipid vesicles of varying composition. Reorganization within biphasic systems was also examined. A2E content was measured by high performance liquid chromatography (HPLC) and fluorescence intensity was quantified spectroscopically. As solvent polarity was increased, A2E fluorescent spectra exhibited red-shifted maxima and reduced intensity. A2E was depleted by light irradiation and the loss was more pronounced in less polar solvents, lower concentrations of anionic surfactant, and in gel- versus fluid-ordered phospholipid liposomes. Conditions that permit A2E aggregation promoted photooxidation/photodegradation, while movement of A2E between bisphasic systems was associated with fluorescence recovery after photobleaching. The fluorescence characteristics of A2E are subject to environmental modulation. Photooxidation and photodegradation of bisretinoid can account for fundus autofluorescence photobleaching. Return of fluorescence intensity after photobleaching likely occurs due to redistribution of A2E fractions amongst co-existing heterogeneous microdomains of the lysosomal compartment.
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Affiliation(s)
- Zhao Liu
- Department of Ophthalmology, Columbia University Medical Center, New York, New York, United States of America
| | - Keiko Ueda
- Department of Ophthalmology, Columbia University Medical Center, New York, New York, United States of America
| | - Hye Jin Kim
- Department of Ophthalmology, Columbia University Medical Center, New York, New York, United States of America
| | - Janet R. Sparrow
- Department of Ophthalmology, Columbia University Medical Center, New York, New York, United States of America
- Departments of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, United States of America
- * E-mail:
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Ferrington DA, Sinha D, Kaarniranta K. Defects in retinal pigment epithelial cell proteolysis and the pathology associated with age-related macular degeneration. Prog Retin Eye Res 2015; 51:69-89. [PMID: 26344735 DOI: 10.1016/j.preteyeres.2015.09.002] [Citation(s) in RCA: 187] [Impact Index Per Article: 18.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2015] [Revised: 08/29/2015] [Accepted: 09/01/2015] [Indexed: 12/12/2022]
Abstract
Maintenance of protein homeostasis, also referred to as "Proteostasis", integrates multiple pathways that regulate protein synthesis, folding, translocation, and degradation. Failure in proteostasis may be one of the underlying mechanisms responsible for the cascade of events leading to age-related macular degeneration (AMD). This review covers the major degradative pathways (ubiquitin-proteasome and lysosomal involvement in phagocytosis and autophagy) in the retinal pigment epithelium (RPE) and summarizes evidence of their involvement in AMD. Degradation of damaged and misfolded proteins via the proteasome occurs in coordination with heat shock proteins. Evidence of increased content of proteasome and heat shock proteins in retinas from human donors with AMD is consistent with increased oxidative stress and extensive protein damage with AMD. Phagocytosis and autophagy share key molecules in phagosome maturation as well as degradation of their cargo following fusion with lysosomes. Phagocytosis and degradation of photoreceptor outer segments ensures functional integrity of the neural retina. Autophagy rids the cell of toxic protein aggregates and defective mitochondria. Evidence suggesting a decline in autophagic flux includes the accumulation of autophagic substrates and damaged mitochondria in RPE from AMD donors. An age-related decrease in lysosomal enzymatic activity inhibits autophagic clearance of outer segments, mitochondria, and protein aggregates, thereby accelerating the accumulation of lipofuscin. This cumulative damage over a person's lifetime tips the balance in RPE from a state of para-inflammation, which strives to restore cell homeostasis, to the chronic inflammation associated with AMD.
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Affiliation(s)
- Deborah A Ferrington
- Department of Ophthalmology and Visual Neurosciences, 2001 6th St SE, University of Minnesota, Minneapolis, MN 55455, USA.
| | - Debasish Sinha
- Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Room M035 Robert and Clarice Smith Bldg, 400 N Broadway, Baltimore, MD, 21287, USA.
| | - Kai Kaarniranta
- Department of Ophthalmology, University of Eastern Finland and Kuopio University Hospital, P.O. Box 100, 70029 KYS, Finland.
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Sparrow JR, Duncker T. Fundus Autofluorescence and RPE Lipofuscin in Age-Related Macular Degeneration. J Clin Med 2015; 3:1302-21. [PMID: 25774313 PMCID: PMC4358814 DOI: 10.3390/jcm3041302] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Genes that increase susceptibility to age-related macular degeneration (AMD) have been identified; however, since many individuals carrying these risk alleles do not develop disease, other contributors are involved. One additional factor, long implicated in the pathogenesis of AMD, is the lipofuscin of retinal pigment epithelium (RPE). The fluorophores that constitute RPE lipofuscin also serve as a source of autofluorescence (AF) that can be imaged by confocal laser ophthalmoscopy. The AF originating from lipofuscin is excited by the delivery of short wavelength (SW) light. A second autofluorescence is emitted from the melanin of RPE (and choroid) upon near-infrared (NIR-AF) excitation. SW-AF imaging is currently used in the clinical management of retinal disorders and the advantages of NIR-AF are increasingly recognized. Here we visit the damaging properties of RPE lipofuscin that could be significant when expressed on a background of genetic susceptibility. To advance interpretations of disease-related patterns of fundus AF in AMD, we also consider the photochemical and spectrophotometric features of the lipofuscin compounds responsible for generating the fluorescence emission.
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Affiliation(s)
- Janet R. Sparrow
- Department of Ophthalmology, Columbia University Medical Center, 635 W. 165th Street, New York, NY 10032, USA; E-Mail:
- Department of Pathology and Cell Biology, Columbia University Medical Center, 630 168th Street, New York, NY 10032, USA
- Author to whom correspondence should be addressed; E-Mail: ; Tel.: +1-212-305-0044
| | - Tobias Duncker
- Department of Ophthalmology, Columbia University Medical Center, 635 W. 165th Street, New York, NY 10032, USA; E-Mail:
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Rescue of the Stargardt phenotype in Abca4 knockout mice through inhibition of vitamin A dimerization. Proc Natl Acad Sci U S A 2015; 112:8415-20. [PMID: 26106163 DOI: 10.1073/pnas.1506960112] [Citation(s) in RCA: 81] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Stargardt disease, an ATP-binding cassette, subfamily A, member 4 (ABCA4)-related retinopathy, is a genetic condition characterized by the accelerated accumulation of lipofuscin in the retinal pigment epithelium, degeneration of the neuroretina, and loss of vision. No approved treatment exists. Here, using a murine model of Stargardt disease, we show that the propensity of vitamin A to dimerize is responsible for triggering the formation of the majority of lipofuscin and transcriptional dysregulation of genes associated with inflammation. Data further demonstrate that replacing vitamin A with vitamin A deuterated at the carbon 20 position (C20-D3-vitamin A) impedes the dimerization rate of vitamin A--by approximately fivefold for the vitamin A dimer A2E--and subsequent lipofuscinogenesis and normalizes the aberrant transcription of complement genes without impairing retinal function. Phenotypic rescue by C20-D3-vitamin A was also observed noninvasively by quantitative autofluorescence, an imaging technique used clinically, in as little as 3 months after the initiation of treatment, whereas upon interruption of treatment, the age-related increase in autofluorescence resumed. Data suggest that C20-D3-vitamin A is a clinically amiable tool to inhibit vitamin A dimerization, which can be used to determine whether slowing the dimerization of vitamin A can prevent vision loss caused by Stargardt disease and other retinopathies associated with the accumulation of lipofuscin in the retina.
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Gao L, Smith RT. Optical hyperspectral imaging in microscopy and spectroscopy - a review of data acquisition. JOURNAL OF BIOPHOTONICS 2015; 8:441-56. [PMID: 25186815 PMCID: PMC4348353 DOI: 10.1002/jbio.201400051] [Citation(s) in RCA: 92] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/24/2014] [Revised: 07/10/2014] [Accepted: 07/12/2014] [Indexed: 05/20/2023]
Abstract
Rather than simply acting as a photographic camera capturing two-dimensional (x, y) intensity images or a spectrometer acquiring spectra (λ), a hyperspectral imager measures entire three-dimensional (x, y, λ) datacubes for multivariate analysis, providing structural, molecular, and functional information about biological cells or tissue with unprecedented detail. Such data also gives clinical insights for disease diagnosis and treatment. We summarize the principles underpinning this technology, highlight its practical implementation, and discuss its recent applications at microscopic to macroscopic scales. Datacube acquisition strategies in hyperspectral imaging x, y, spatial coordinates; λ, wavelength.
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Affiliation(s)
- Liang Gao
- Department of Biomedical Engineering, Washington University in St. Louis, MO, 63139.
| | - R Theodore Smith
- Department of Ophthalmology, NYU School of Medicine, New York, NY, 10016.
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40
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Penn J, Mihai DM, Washington I. Morphological and physiological retinal degeneration induced by intravenous delivery of vitamin A dimers in rabbits. Dis Model Mech 2014; 8:131-8. [PMID: 25504631 PMCID: PMC4314778 DOI: 10.1242/dmm.017194] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The eye uses vitamin A as a cofactor to sense light and, during this process, some vitamin A molecules dimerize, forming vitamin A dimers. A striking chemical signature of retinas undergoing degeneration in major eye diseases such as age-related macular degeneration (AMD) and Stargardt disease is the accumulation of these dimers in the retinal pigment epithelium (RPE) and Bruch's membrane (BM). However, it is not known whether dimers of vitamin A are secondary symptoms or primary insults that drive degeneration. Here, we present a chromatography-free method to prepare gram quantities of the vitamin A dimer, A2E, and show that intravenous administration of A2E to the rabbit results in retinal degeneration. A2E-damaged photoreceptors and RPE cells triggered inflammation, induced remolding of the choroidal vasculature and triggered a decline in the retina's response to light. Data suggest that vitamin A dimers are not bystanders, but can be primary drivers of retinal degeneration. Thus, preventing dimer formation could be a preemptive strategy to address serious forms of blindness.
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Affiliation(s)
- Jackie Penn
- Columbia University Medical Center, Ophthalmology, New York, NY 10032, USA
| | - Doina M Mihai
- Columbia University Medical Center, Ophthalmology, New York, NY 10032, USA
| | - Ilyas Washington
- Columbia University Medical Center, Ophthalmology, New York, NY 10032, USA.
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41
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Feldman TB, Yakovleva MA, Arbukhanova PM, Borzenok SA, Kononikhin AS, Popov IA, Nikolaev EN, Ostrovsky MA. Changes in spectral properties and composition of lipofuscin fluorophores from human-retinal-pigment epithelium with age and pathology. Anal Bioanal Chem 2014; 407:1075-88. [PMID: 25471291 DOI: 10.1007/s00216-014-8353-z] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2014] [Revised: 11/16/2014] [Accepted: 11/17/2014] [Indexed: 10/24/2022]
Abstract
Fundus autofluorescence mostly originates from bisretinoid fluorophores in lipofuscin granules, which accumulate in retinal-pigment-epithelium cells with age. The dynamics of accumulation, photo-oxidation, and photodegradation of bisretinoids during aging or in the presence of pathology have been insufficiently investigated. Changes in spectral properties and composition of human lipofuscin-granule fluorophores with age and pathology have now been investigated by a high-performance liquid chromatography method using spectrophotometric and fluorescent detectors connected in series. It was found that: (i) N-retinylidene-N-retinylethanolamine (A2E) fluorescence intensity is not predominant in the chloroform extract of human-cadaver-eye retinal pigment epithelium studied; bisretinoid photo-oxidation and photodegradation products have much higher fluorescent properties; (ii) the relative emission maximum in the fluorescence spectrum of suspended retinal-pigment-epithelium cells obtained from an individual human-cadaver eye without pathology is irrespective of donor age and falls within the range 575 ± 15 nm; in two cadaver eyes with signs of age-related macular degeneration, emission maxima were shifted by 23-36 nm towards the shortwave region; and (iii) the ratio of bisretinoid photo-oxidation and photodegradation products to unoxidized bisretinoids in the chloroform extract of cadaver-eye retinal pigment epithelium increases with donor age, from 0.69 ± 0.03 to 1.32 ± 0.04. The differences in fluorescence properties between chloroform extracts obtained from cadaver eyes with and without signs of age-related macular degeneration could be used to increase the potential of fundus autofluorescence imaging as a noninvasive diagnostic method.
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Affiliation(s)
- Tatiana B Feldman
- Department of Molecular Physiology, Biological Faculty, Lomonosov Moscow State University, Leninskie Gory1, Moscow, 119991, Russia,
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42
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Retinal metabolism in humans induces the formation of an unprecedented lipofuscin fluorophore ‘pdA2E’. Biochem J 2014; 460:343-52. [PMID: 24712709 DOI: 10.1042/bj20140089] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
We identified a novel by-product of retinal metabolism called pdA2E in the lipofuscin of the retinal pigment epithelium, demonstrated the photoisomerization and oxidation of pdA2E, and proposed a non-enzymatic mechanism for its formation in the retina.
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43
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Wu YL, Li J, Yao K. Structures and biogenetic analysis of lipofuscin bis-retinoids. J Zhejiang Univ Sci B 2014; 14:763-73. [PMID: 24009196 DOI: 10.1631/jzus.b1300051] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Age-related macular degeneration (AMD) is still an incurable blinding eye disease because of complex pathogenic mechanisms and unusual diseased regions. With the use of chemical biology tools, great progress has been achieved in improving the understanding of AMD pathogenesis. The severity of AMD is, at least in part, linked to the non-degradable lipofuscin bis-retinoids in retinal pigment epithelial (RPE). This material is thought to result from the lifelong accumulation of lysosomal residual bodies containing the end products derived from the daily phagocytosis of rod outer segments by RPE cells. Here, we present previously recognized bis-retinoids with focus on structures and biosynthetic pathways. In addition to a brief discussion on the mutual conversion relationships of bis-retinoids, future perspectives and the medical relevance of such studies on these lipofuscin constituents are also highlighted.
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Affiliation(s)
- Ya-lin Wu
- College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
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44
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Ablonczy Z, Smith N, Anderson DM, Grey AC, Spraggins J, Koutalos Y, Schey KL, Crouch RK. The utilization of fluorescence to identify the components of lipofuscin by imaging mass spectrometry. Proteomics 2014; 14:936-44. [PMID: 24453194 DOI: 10.1002/pmic.201300406] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2013] [Revised: 12/16/2013] [Accepted: 12/21/2013] [Indexed: 11/08/2022]
Abstract
Lipofuscin, an aging marker in the retinal pigment epithelium (RPE) associated with the development of age-related macular degeneration, is primarily characterized by its fluorescence. The most abundant component of RPE lipofuscin is N-retinylidene-N-retinylethanolamine (A2E) but its exact composition is not known due to the complexity of the RPE extract. In this study, we utilized MALDI imaging to find potential molecules responsible for lipofuscin fluorescence in RPE tissue from Abca4(-/-) , Sv129, and C57Bl6/J mice aged 2 and 6 months. To assert relationships, the individual images in the MALDI imaging datasets were correlated with lipofuscin fluorescence recorded from the same tissues following proper registration. Spatial correlation information, which is usually lost in bioanalytics, pinpointed a relatively small number of potential lipofuscin components. The comparison of four samples in each condition further limited the possibility of false positives and provided various new, age- and strain-specific targets. Validating the usefulness of the fluorescence-enhanced imaging strategy, many known adducts of A2E were identified in the short list of lipofuscin components. These results provided evidence that mass spectrometric imaging can be utilized as a tool to begin to identify the molecular substructure of clinically-relevant diagnostic information.
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Affiliation(s)
- Zsolt Ablonczy
- Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA
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45
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Álvarez R, Vaz B, Gronemeyer H, de Lera ÁR. Functions, therapeutic applications, and synthesis of retinoids and carotenoids. Chem Rev 2013; 114:1-125. [PMID: 24266866 DOI: 10.1021/cr400126u] [Citation(s) in RCA: 157] [Impact Index Per Article: 13.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Affiliation(s)
- Rosana Álvarez
- Departamento de Química Orgánica, Centro de Investigación Biomédica (CINBIO), and Instituto de Investigación Biomédica de Vigo (IBIV), Universidade de Vigo , 36310 Vigo, Spain
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46
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Li J, Yao K, Yu X, Dong X, Gan L, Luo C, Wu Y. Identification of a novel lipofuscin pigment (iisoA2E) in retina and its effects in the retinal pigment epithelial cells. J Biol Chem 2013; 288:35671-82. [PMID: 24169698 DOI: 10.1074/jbc.m113.511386] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Lipofuscin accumulation in retinal pigment epithelial (RPE) cells of the eye implicates the etiologies of Stargardt disease and age-related macular degeneration, a leading cause of blindness in the elderly. Here, we have identified a previously unknown RPE lipofuscin component. By one- and two-dimensional NMR techniques and mass spectrometry, we confirmed that this compound is a new type of pyridinium bisretinoid presenting an unusual structure, in which two polyenic side chains are attached to adjacent carbons of a pyridinium ring. This pigment is a light-induced isomer of isoA2E, rather than A2E, referred to as iisoA2E. This pigment is a fluorescent lipofuscin compound with absorbance maxima at ∼430 and 352 nm detected in human, pig, mouse, and bovine eyes. Formation of iisoA2E was found in reaction mixtures of all-trans-retinal and ethanolamine. Excess intracellular accumulation of this adduct in RPE cells in vitro leads to a significant loss of cell viability and caused membrane damage. Phospholipase D-mediated phosphodiester cleavage of the A2PE series generated isoA2E and iisoA2E, in addition to A2E, thus corroborating the presence of isoA2PE and iisoA2PE that may serve as biosynthetic precursors of isoA2E and iisoA2E.
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Affiliation(s)
- Jie Li
- From the College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China and
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47
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Charvet CD, Saadane A, Wang M, Salomon RG, Brunengraber H, Turko IV, Pikuleva IA. Pretreatment with pyridoxamine mitigates isolevuglandin-associated retinal effects in mice exposed to bright light. J Biol Chem 2013; 288:29267-80. [PMID: 23970548 DOI: 10.1074/jbc.m113.498832] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
The benefits of antioxidant therapy for treating age-related macular degeneration, a devastating retinal disease, are limited. Perhaps species other than reactive oxygen intermediates should be considered as therapeutic targets. These could be lipid peroxidation products, including isolevuglandins (isoLGs), prototypical and extraordinarily reactive γ-ketoaldehydes that avidly bind to proteins, phospholipids, and DNA and modulate the properties of these biomolecules. We found isoLG adducts in aged human retina but not in the retina of mice kept under dim lighting. Hence, to test whether scavenging of isoLGs could complement or supplant antioxidant therapy, we exposed mice to bright light and found that this insult leads to retinal isoLG-adduct formation. We then pretreated mice with pyridoxamine, a B6 vitamer and efficient scavenger of γ-ketoaldehydes, and found that the levels of retinal isoLG adducts are decreased, and morphological changes in photoreceptor mitochondria are not as pronounced as in untreated animals. Our study demonstrates that preventing the damage to biomolecules by lipid peroxidation products, a novel concept in vision research, is a viable strategy to combat oxidative stress in the retina.
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48
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Ablonczy Z, Higbee D, Grey AC, Koutalos Y, Schey KL, Crouch RK. Similar molecules spatially correlate with lipofuscin and N-retinylidene-N-retinylethanolamine in the mouse but not in the human retinal pigment epithelium. Arch Biochem Biophys 2013; 539:196-202. [PMID: 23969078 DOI: 10.1016/j.abb.2013.08.005] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2013] [Revised: 08/08/2013] [Accepted: 08/10/2013] [Indexed: 01/08/2023]
Abstract
The accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD) in humans. The exact composition of lipofuscin is not known but its best characterized component is N-retinylidene-N-retinylethanolamine (A2E), a byproduct of the retinoid visual cycle. Utilizing our recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS)-based technique to determine the spatial distribution of A2E, this study compares the relationships of lipofuscin fluorescence and A2E in the murine and human RPE on representative normal tissue. To identify molecules with similar spatial patterns, the images of A2E and lipofuscin were correlated with all the individual images in the MALDI-IMS dataset. In the murine RPE, there was a remarkable correlation between A2E and lipofuscin. In the human RPE, however, minimal correlation was detected. These results were reflected in the marked distinctions between the molecules that spatially correlated with the images of lipofuscin and A2E in the human RPE. While the distribution of murine lipofuscin showed highest similarities with some of the known A2E-adducts, the composition of human lipofuscin was significantly different. These results indicate that A2E metabolism may be altered in the human compared to the murine RPE.
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Affiliation(s)
- Zsolt Ablonczy
- Department of Ophthalmology, Medical University of South Carolina, Charleston, SC 29425, United States.
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49
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Joshi D, Field J, Murphy J, Abdelrahim M, Schönherr H, Sparrow JR, Ellestad G, Nakanishi K, Zask A. Synthesis of antioxidants for prevention of age-related macular degeneration. JOURNAL OF NATURAL PRODUCTS 2013; 76:450-4. [PMID: 23346866 PMCID: PMC4069254 DOI: 10.1021/np300769c] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/01/2023]
Abstract
Photooxidation of A2E may be involved in diseases of the macula, and antioxidants could serve as therapeutic agents for these diseases. Inhibitors of A2E photooxidation were prepared by Mannich reaction of the antioxidant quercetin. These compounds contain water-solubilizing amine groups, and several were more potent inhibitors of A2E photooxidation than quercetin.
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Affiliation(s)
- Dharati Joshi
- Department of Chemistry, 3000 Broadway, Columbia University, New York, NY 10027, United States
| | - James Field
- Department of Chemistry, 3000 Broadway, Columbia University, New York, NY 10027, United States
| | - John Murphy
- Department of Chemistry, 3000 Broadway, Columbia University, New York, NY 10027, United States
| | - Mohammed Abdelrahim
- Department of Chemistry, 3000 Broadway, Columbia University, New York, NY 10027, United States
| | - Heike Schönherr
- Department of Ophthalmology, Columbia University, 630 West 168th Street, New York, NY 10032, United States
| | - Janet R. Sparrow
- Department of Ophthalmology, Columbia University, 630 West 168th Street, New York, NY 10032, United States
| | - George Ellestad
- Department of Chemistry, 3000 Broadway, Columbia University, New York, NY 10027, United States
| | - Koji Nakanishi
- Department of Chemistry, 3000 Broadway, Columbia University, New York, NY 10027, United States
| | - Arie Zask
- Department of Chemistry, 3000 Broadway, Columbia University, New York, NY 10027, United States
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50
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Abstract
The human eye is constantly exposed to sunlight and artificial lighting. Light transmission through the eye is fundamental to its unique biological functions of directing vision and circadian rhythm and therefore light absorbed by the eye must be benign. However, exposure to the very intense ambient radiation can pose a hazard particularly if the recipient is over 40 years of age. There are age-related changes in the endogenous (natural) chromophores (lipofuscin, A2E and all-trans-retinal derivatives) in the human retina that makes it more susceptible to visible light damage. Intense visible light sources that do not filter short blue visible light (400-440 nm) used for phototherapy of circadian imbalance (i.e. seasonal affective disorder) increase the risk for age-related light damage to the retina. Moreover, many drugs, dietary supplements, nanoparticles and diagnostic dyes (xenobiotics) absorb ocular light and have the potential to induce photodamage to the retina, leading to transient or permanent blinding disorders. This article will review the underlying reasons why visible light in general and short blue visible light in particular dramatically raises the risk of photodamage to the human retina.
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Affiliation(s)
- Albert R Wielgus
- Department of Ophthalmology, Duke University Medical Center, Durham, NC, USA
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