Interphotoreceptor Retinoid‐Binding Protein Protects Retinoids from Photodegradation

Retinol degrades rapidly in light into a variety of photoproducts. It is remarkable that visual cycle retinoids can evade photodegradation as they are exchanged between the photoreceptors, retinal pigment epithelium and Müller glia. Within the interphotoreceptor matrix, all‐trans retinol, 11‐cis retinol and retinal are bound by interphotoreceptor retinoid‐binding protein (IRBP). Apart from its role in retinoid trafficking and targeting, could IRBP have a photoprotective function? HPLC was used to evaluate the ability of IRBP to protect all‐trans and 11‐cis retinols from photodegradation when exposed to incandescent light (0 to 8842 μW cm^?2); time periods of 0–60 min, and bIRBP: retinol molar ratios of 1:1 to 1:5. bIRBP afforded a significant prevention of both all‐trans and 11‐cis retinol to rapid photodegradation. The effect was significant over the entire light intensity range tested, and extended to the bIRBP: retinol ratio 1:5. In view of the continual exposure of the retina to light, and the high oxidative stress in the outer retina, our results suggest IRBP may have an important protective role in the visual cycle by reducing photodegradation of all‐trans and 11‐cis retinols. This role of IRBP is particularly relevant in the high flux conditions of the cone visual cycle.     

Retinal Photodamage Mediated by All‐trans‐retinal†

Accumulation of all‐trans‐retinal (all‐trans‐RAL), reactive vitamin A aldehyde, is one of the key factors in initiating retinal photodamage. This photodamage is characterized by progressive retinal cell death evoked by light exposure in both an acute and chronic fashion. Photoactivated rhodopsin releases all‐trans‐RAL, which is subsequently transported by ATP‐binding cassette transporter 4 and reduced to all‐trans‐retinol by all‐trans‐retinol dehydrogenases located in photoreceptor cells. Any interruptions in the clearing of all‐trans‐RAL in the photoreceptors can cause an accumulation of this reactive aldehyde and its toxic condensation products. This accumulation may result in the manifestation of retinal dystrophy including human retinal degenerative diseases such as Stargardt’s disease and age‐related macular degeneration. Herein, we discuss the mechanisms of all‐trans‐RAL clearance in photoreceptor cells by sequential enzymatic reactions, the visual (retinoid) cycle, and potential molecular pathways of retinal photodamage. We also review recent imaging technologies to monitor retinal health status as well as novel therapeutic strategies preventing all‐trans‐RAL‐associated retinal photodamage.     

Influence of Arrestin on the Photodecay of Bovine Rhodopsin

Continued activation of the photocycle of the dim‐light receptor rhodopsin leads to the accumulation of all‐trans‐retinal in the rod outer segments (ROS). This accumulation can damage the photoreceptor cell. For retinal homeostasis, deactivation processes are initiated in which the release of retinal is delayed. One of these processes involves the binding of arrestin to rhodopsin. Here, the interaction of pre‐activated truncated bovine visual arrestin (Arr^Tr) with rhodopsin in 1,2‐diheptanoyl‐sn‐glycero‐3‐phosphocholine (DHPC) micelles is investigated by solution NMR techniques and flash photolysis spectroscopy. Our results show that formation of the rhodopsin–arrestin complex markedly influences partitioning in the decay kinetics of rhodopsin, which involves the simultaneous formation of a meta II and a meta III state from the meta I state. Binding of Arr^Tr leads to an increase in the population of the meta III state and consequently to an approximately twofold slower release of all‐trans‐retinal from rhodopsin.     

pH‐dependent Interaction of Rhodopsin with Cyanidin‐3‐glucoside. 2. Functional Aspects†

Anthocyanins are a class of phytochemicals that confer color to flowers, fruits, vegetables and leaves. They are part of our regular diet and serve as dietary supplements because of numerous health benefits, including improved vision. Recent studies have shown that the anthocyanin cyanidin‐3‐O‐glucoside (C3G) increased regeneration of the dim‐light photoreceptor rhodopsin (Matsumoto et al. [2003] J. Agric. Food Chem., 51 , 3560–3563). In an accompanying study (Yanamala et al. [2009] Photochem. Photobiol.), we show that C3G directly binds to rhodopsin in a pH‐dependent manner. In this study, we investigated the functional consequences of C3G binding to rhodopsin. As observed previously in rod outer segments, regeneration of purified rhodopsin in detergent micelles is also accelerated in the presence of C3G. Thermal denaturation and stability studies using circular dichroism, fluorescence and UV/visible absorbance spectroscopy show that C3G exerts a destabilizing effect on rhodopsin structure while it only modestly alters G‐protein activation and the rates at which the light‐activated Metarhodopsin II state decays to opsin and free retinal. These results indicate that the mechanism of C3G‐enhanced regeneration may be based on changes in opsin structure promoting access to the retinal binding pocket.     

Melanopsin and the Non‐visual Photochemistry in the Inner Retina of Vertebrates

Melanopsin (Opn4), a member of the G‐protein‐coupled receptor family, is a vitamin A‐based opsin in the vertebrate retina that has been shown to be involved in the synchronization of circadian rhythms, pupillary light reflexes, melatonin suppression and other light‐regulated tasks. In nonmammalian vertebrates there are two Opn4 genes, Opn4m and Opn4x, the mammalian and Xenopus orthologs respectively. Opn4x is only expressed in nonmammalian vertebrates including reptiles, fish and birds, while Opn4m is found in a subset of retinal ganglion cells (RGCs), the intrinsically photosensitive (ip) RGCs of the inner retina of both mammals and nonmammalian vertebrates. All opsins described utilize retinaldehyde as chromophore, photoisomerized from 11‐cis‐ to all‐trans‐retinal upon light exposure. Visual retinal photoreceptor cones and rods, responsible for day and night vision respectively, recycle retinoids through a process called the visual cycle that involves the retinal pigment epithelium or glial Müller cells. Although Opn4 has been characterized as a bistable photopigment, little is known about the mechanism/s involved in its chromophore regeneration. In this review, we will attempt to shed light on the visual cycle taking place in the inner retina and discuss the state of the art in the nonvisual photochemistry of vertebrates.     

Lipofuscin and A2E Accumulate with Age in the Retinal Pigment Epithelium of Nrl−/− Mice†

Lipofuscin is a fluorescent material with significant phototoxic potential that accumulates with age in the retinal pigment epithelium (RPE) of the eye. It is thought to be a factor in retinal degeneration diseases. The most extensively characterized lipofuscin component, N‐retinylidene‐N‐retinylethanolamine (A2E), has been proposed to be a byproduct of reactions involving the visual pigment chromophore. To examine the impact of the visual pigment and photoreceptor cell type on lipofuscin accumulation, we analyzed the RPE from Nrl^?/? mice of various ages for lipofuscin fluorescence and A2E levels. The photoreceptor cells of the Nrl^?/? retina contain only cone‐like pigments, and produce cone‐like responses to photostimulation. The cone‐like nature of these cells was confirmed by the presence of RPE65. Lipofuscin was measured with fluorescence imaging, whereas A2E was quantified by UV/VIS absorbance spectroscopy coupled to HPLC. The identity of A2E was corroborated with tandem mass spectrometry. Lipofuscin and A2E accumulated with age, albeit to lower levels compared with wild type mice. The emission spectra of RPE lipofuscin granules from Nrl^?/? mice were similar to those from wild type mice, with λ_maxca 610 nm. These results demonstrate that cone visual pigments can contribute to the production of lipofuscin and A2E.     

Redshifted and Near‐infrared Active Analog Pigments Based upon Archaerhodopsin‐3

Archaerhodopsin‐3 (AR3) is a member of the microbial rhodopsin family of hepta‐helical transmembrane proteins, containing a covalently bound molecule of all‐trans retinal as a chromophore. It displays an absorbance band in the visible region of the solar spectrum (λmax 556 nm) and functions as a light‐driven proton pump in the archaeon Halorubrum sodomense. AR3 and its mutants are widely used in neuroscience as optogenetic neural silencers and in particular as fluorescent indicators of transmembrane potential. In this study, we investigated the effect of analogs of the native ligand all‐trans retinal A1 on the spectral properties and proton‐pumping activity of AR3 and its single mutant AR3 (F229S). While, surprisingly, the 3‐methoxyretinal A2 analog did not redshift the absorbance maximum of AR3, the analogs retinal A2 and 3‐methylamino‐16‐nor‐1,2,3,4‐didehydroretinal (MMAR) did generate active redshifted AR3 pigments. The MMAR analog pigments could even be activated by near‐infrared light. Furthermore, the MMAR pigments showed strongly enhanced fluorescence with an emission band in the near‐infrared peaking around 815 nm. We anticipate that the AR3 pigments generated in this study have widespread potential for near‐infrared exploitation as fluorescent voltage‐gated sensors in optogenetics and artificial leafs and as proton pumps in bioenergy‐based applications.     

Studies on the Stability of the Human Cone Visual Pigments†

The retina of vertebrates contains two kinds of photoreceptor cells, rods and cones, which contain their specific visual pigments that are responsible for scotopic and photopic vision, respectively. In cone photoreceptor cells, there are three types of color pigments: blue, green and red, each with a distinctive absorption maximum. The goal of this investigation was to identify optimal conditions under which these pigments could be obtained and isolated in a stable form, thereby facilitating structural studies using high‐resolution approaches. For this purpose, all three human cone opsins were initially expressed in mammalian cells, reconstituted with 11‐cis retinal, detergent solubilized, purified and their stability compared with rod rhodopsin. As all three pigments showed dramatically reduced stability relative to rhodopsin, site‐directed mutagenesis was used in an attempt to engineer stability into the green cone pigment. The mutations introduced some structural motifs and sites of posttranslational modification present in rhodopsin, as well as amino acid substitutions that have been found to stabilize the rod opsin apo‐protein. We also modified the hydrophobic environment of the green cone pigment by varying the detergent and detergent/lipid composition used during solubilization and purification, and compared them with the retinal reconstituted pigment in membranes. Our results show that these changes do not significantly improve the inherent instability of the human cone pigments, and in some cases, lead to a decrease in stability and protein aggregation. We conclude that further efforts are required to stabilize the human cone pigments in a form suitable for high‐resolution structural studies.     

RPE65 and the Accumulation of Retinyl Esters in Mouse Retinal Pigment Epithelium

The RPE65 protein of the retinal pigment epithelium (RPE) enables the conversion of retinyl esters to the visual pigment chromophore 11‐cis retinal. Fresh 11‐cis retinal is generated from retinyl esters following photoisomerization of the visual pigment chromophore to all‐trans during light detection. Large amounts of esters accumulate in Rpe65^?/? mice, indicating their continuous formation when 11‐cis retinal generation is blocked. We hypothesized that absence of light, by limiting the conversion of esters to 11‐cis retinal, would also result in the build‐up of retinyl esters in the RPE of wild‐type mice. We used HPLC to quantify ester levels in organic extracts of the RPE from wild‐type and Rpe65^?/? mice. Retinyl ester levels in Sv/129 wild‐type mice that were dark adapted for various intervals over a 4‐week period were similar to those in mice raised in cyclic light. In C57BL/6 mice however, which contain less Rpe65 protein, dark adaptation was accompanied by an increase in ester levels compared to cyclic light controls. Retinyl ester levels were much higher in Rpe65^?/? mice compared to wild type and kept increasing with age. The results suggest that the RPE65 role in retinyl ester homeostasis extends beyond enabling the formation of 11‐cis retinal.     

Light‐Induced Bending of Needle‐Like Crystals of Naphthylvinylbenzoxazole Triggered by trans–cis Isomerization

New diarylethene derivatives containing benzoxazole ( NBO ) and benzothiazole ( NBT ) have been synthesized. Light‐induced trans–cis isomerization of NBO and NBT took place in crystals, and only induced the needle‐like crystals of NBO to bend backwards away from the UV light source. The movement of the atoms was deemed to take place during the isomerization of NBO ; hence, strain would be produced and accumulated rapidly in the surface of crystals exposed to UV light. The uniform release of strain led to the bending of needle‐like crystals. The light‐induced trans–cis isomerization efficiency of NBT was too low to drive the motion of crystals, which might have originated from the large repulsion between naphthyl and benzothiazole. These results provide a new platform for the transformation of light energy into mechanical energy in molecular crystals through the unimolecular photochemical reaction of diarylethene derivatives.     

1,2‐Dithienyldicyanoethene‐Based,Visible‐Light‐Driven,Chiral Fluorescent Molecular Switch: Rewritable Multimodal Photonic Devices

Reported here is the first example of a 1,2‐dithienyldicyanoethene‐based visible‐light‐driven chiral fluorescent molecular switch that exhibits reversible trans to cis photoisomerization. The trans form in solution almost completely transforms into the cis form, accompanied by a 10‐fold decrease in its fluorescence intensity within 60 seconds when exposed to green light (520 nm). The reverse isomerization proceeds upon irradiation with blue light (405 nm). When doped into commercially available achiral liquid crystal hosts, this molecular switch efficiently induces luminescent helical superstructures, that is, a cholesteric phase. The intensity of the circularly polarized fluorescence as well as the selective reflection wavelength of the induced cholesteric phases can be reversibly tuned using visible light of two different wavelengths. Optically rewritable photonic devices using cholesteric films containing this molecular switch are described.     

1,2‐Dithienyldicyanoethene‐Based,Visible‐Light‐Driven,Chiral Fluorescent Molecular Switch: Rewritable Multimodal Photonic Devices

Reported here is the first example of a 1,2‐dithienyldicyanoethene‐based visible‐light‐driven chiral fluorescent molecular switch that exhibits reversible trans to cis photoisomerization. The trans form in solution almost completely transforms into the cis form, accompanied by a 10‐fold decrease in its fluorescence intensity within 60 seconds when exposed to green light (520 nm). The reverse isomerization proceeds upon irradiation with blue light (405 nm). When doped into commercially available achiral liquid crystal hosts, this molecular switch efficiently induces luminescent helical superstructures, that is, a cholesteric phase. The intensity of the circularly polarized fluorescence as well as the selective reflection wavelength of the induced cholesteric phases can be reversibly tuned using visible light of two different wavelengths. Optically rewritable photonic devices using cholesteric films containing this molecular switch are described.     

Functional and Photochemical Characterization of a Light‐Driven Proton Pump from the Gammaproteobacterium Pantoea vagans

Photoactive retinal proteins are widely distributed throughout the domains of the microbial world (i.e., bacteria, archaea, and eukarya). Here we describe three retinal proteins belonging to a phylogenetic clade with a unique DTG motif. Light‐induced decrease in the environmental pH and its inhibition by carbonyl cyanide m‐chlorophenylhydrazone revealed that these retinal proteins function as light‐driven outward electrogenic proton pumps. We further characterized one of these proteins, Pantoea vagans rhodopsin (PvR), spectroscopically. Visible spectroscopy and high‐performance liquid chromatography revealed that PvR has an absorption maximum at 538 nm with the retinal chromophore predominantly in the all‐trans form (>90%) under both dark and light conditions. We estimated the pK_a values of the protonated Schiff base of the retinal chromophore and its counterion as approximately 13.5 and 2.1, respectively, by using pH titration experiments, and the photochemical reaction cycle of PvR was measured by time‐resolved flash‐photolysis in the millisecond timeframe. We observed a blue‐shifted and a red‐shifted intermediate, which we assigned as M‐like and O‐like intermediates, respectively. Decay of the M‐like intermediate was highly sensitive to environmental pH, suggesting that proton uptake is coupled to decay of the M‐like intermediate. From these results, we propose a putative model for the photoreaction of PvR.     

Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle – <Emphasis Type="Italic">structure/function study of Xenopus IRBP</Emphasis>

Background  

Interphotoreceptor retinoid-binding protein's (IRBP) remarkable module structure may be critical to its role in mediating the transport of all-trans and 11-cis retinol, and 11-cis retinal between rods, cones, RPE and Müller cells during the visual cycle. We isolated cDNAs for Xenopus IRBP, and expressed and purified its individual modules, module combinations, and the full-length polypeptide. Binding of all-trans retinol, 11-cis retinal and 9-(9-anthroyloxy) stearic acid were characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer. Finally, the X-ray crystal structure of module-2 was used to predict the location of the ligand-binding sites, and compare their structures among modules using homology modeling.     

INTERPHOTORECEPTOR RETINOID-BINDING PROTEIN AND α-TOCOPHEROL PRESERVE THE ISOMERIC AND OXIDATION STATE OF RETINOL

Retinol decomposes rapidly into a number of products, including its aldehyde form, retinal, when introduced into buffer in phospholipid vesicles or ethanol. Interphotoreceptor retinoid-binding protein at low concentrations is found to protect retinol from isomerization and oxidation. The addition of alpha-tocopherol to either liposomes or an ethanolic-buffer solution also prevents decomposition. Neither of these agents interferes with the successful regeneration of pigment with 9-cis retinal in rod outer segment preparations or the restoration of sensitivity by retinoids in isolated rod photoreceptors.     

Rhodopsin phosphorylation suggests biochemical heterogeneities of retinal rod disks.

Frogs (Rana pipiens) were injected subcutaneously with (3H)-leucine and allowed to incorporate the radioactive amino acid into newly assembled disks in the retinal rod outer segment. The labeled disks served as a temporal marker for following the turnover of rod outer segments. Animals were killed at different times after injection and outer segments were isolated and phosphorylated with ATP in the light. The visual pigment (as isorhodopsin) was regenerated with 9-cis retinal, extracted, and chromatographed on epichlorohydrin triethanolamine cellulose so that phosphorylated pigment could be separated from unphosphorylated pigment. The ratio of (3H)-radioactivity of phosphorylated pigment to that of unphosphorylated pigment was then plotted against the time after injection. The ratio was high when (3H)-labeled disks were largely associated with the basal region of the rod and decreased as the labeled disks moved toward the rod apical region. The results were interpreted as suggesting that newer disks are phosphorylated preferentially to older disks. Papain digestion of (3H)-labeled disks indicated that rhodopsin in newer disks is more susceptible to proteolysis than that in older disks.     

Light‐Induced Retinal Degeneration Is Prevented by Zinc,a Component in the Age‐related Eye Disease Study Formulation†

Mineral supplements are often included in multivitamin preparations because of their beneficial effects on metabolism. In this study, we used an animal model of light‐induced retinal degeneration to test for photoreceptor cell protection by the essential trace element zinc. Rats were treated with various doses of zinc oxide and then exposed to intense visible light for as long as 8 h. Zinc treatment effectively prevented retinal light damage as determined by rhodopsin and retinal DNA recovery, histology and electrophoretic analysis of DNA damage and oxidized retinal proteins. Zinc oxide was particularly effective when given before light exposure and at doses two‐ to four‐fold higher than recommended by the age‐related eye disease study group. Treated rats exhibited higher serum and retinal pigment epithelial zinc levels and an altered retinal gene expression profile. Using an Ingenuity database, 512 genes with known functional annotations were found to be responsive to zinc supplementation, with 45% of these falling into a network related to cellular growth, proliferation, cell cycle and death. Although these data suggest an integrated and extensive regulatory response, zinc induced changes in gene expression also appear to enhance antioxidative capacity in retina and reduce oxidative damage arising from intense light exposure.     

Sub-lethal Photodynamic Damage to ARPE-19 Cells Transiently Inhibits Their Phagocytic Activity†

Efficient phagocytosis of photoreceptor outer segments (POS) membranes by retinal pigment epithelium (RPE) plays a key role in biological renewal of these highly peroxidizable structures. Here, we tested whether photodynamic treatment, mediated by merocyanine 540 (MC 540), rose Bengal or a zinc-substituted chlorophyllide inhibited phagocytic activity of ARPE-19 cells in vitro. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS isolated from cow retinas and nonspecific phagocytosis of fluorescent polystyrene beads were measured by flow cytometry. Photodynamic treatment, mediated by all three photosensitizers with sub-threshold doses, induced significant inhibition of the cell-specific phagocytosis. The nonspecific phagocytosis was inhibited by photodynamic treatment mediated only by MC 540. The inhibition of phagocytosis was a reversible phenomenon and after 24 h, the photodynamically treated cells exhibited phagocytic activity that was comparable with that of untreated cells. This study provides proof of principle that sub-threshold photodynamic treatment of ARPE-19 cells with appropriate photosensitizers is a convenient experimental approach for in vitro study of the effects of oxidative stress on specific phagocytic activity of RPE cells. We postulate that oxidative damage to key components of the cell phagocytic machinery may be responsible for severe impairment of its activity, which can lead to retinal degeneration.     

Cytotoxicity of All‐Trans‐Retinal Increases Upon Photodegradation†

All‐trans‐retinal (AtRal) can accumulate in the retina as a result of excessive exposure to light. The purpose of this study was to compare cytotoxicity of AtRal and photodegraded AtRal (dAtRal) on cultured human retinal pigment epithelial cells in dark and upon exposure to visible light. AtRal was degraded by exposure to visible light. Cytotoxicity was monitored by imaging of cell morphology, propidium iodide staining of cells with permeable plasma membrane and measurements of reductive activity of cells. Generation of singlet oxygen photosensitized by AtRal and dAtRal was monitored by time‐resolved measurements of characteristic singlet oxygen phosphorescence. Photodegradation of AtRal resulted in a decrease in absorption of visible light and accumulation of the degradation products with absorption maximum at ～330 nm. Toxicity of dAtRal was concentration‐dependent and was greater during irradiation with visible light than in dark. DAtRal was more cytotoxic than AtRal both in dark and during exposure to visible light. Photochemical properties of dAtRal indicate that it may be responsible for the maximum in the action spectra of retinal photodamage recorded in animals. In conclusion, photodegradation products of AtRal may impose a significant threat to the retina and therefore their roles in retinal pathology need to be explored.     

Solvent‐Enhanced Diastereo‐ and Regioselectivity in the PdII‐Catalyzed Synthesis of Six‐ and Eight‐Membered Heterocycles via cis‐Aminopalladation

The Pd^II‐catalyzed intramolecular oxidative cyclization of tosyl‐protected cis‐ and trans‐N‐allyl‐2‐aminocyclohexanecarboxamides was examined, and efficient syntheses of cyclohexane‐fused pyrimidin‐4‐ones and 1,5‐diazocin‐6‐ones were developed. In the course of the research, a marked solvent effect was observed on both the regio‐ and diastereoselectivity. Additionally, a novel Pd^II‐mediated domino oxidation, oxidative amination reaction was discovered. Our experimental and theoretical findings suggest that the reactions proceed via a cis‐aminopalladation mechanism.