Lipofuscin, Its Origin, Properties, and Contribution to Retinal Fluorescence as a Potential Biomarker of Oxidative Damage to the Retina.

Lipofuscin accumulates with age as intracellular fluorescent granules originating from incomplete lysosomal digestion of phagocytosed and autophagocytosed material. The purpose of this review is to provide an update on the current understanding of the role of oxidative stress and/or lysosomal dysfunction in lipofuscin accumulation and its consequences, particularly for retinal pigment epithelium (RPE). Next, the fluorescence of lipofuscin, spectral changes induced by oxidation, and its contribution to retinal fluorescence are discussed. This is followed by reviewing recent developments in fluorescence imaging of the retina and the current evidence on the prognostic value of retinal fluorescence for the progression of age-related macular degeneration (AMD), the major blinding disease affecting elderly people in developed countries. The evidence of lipofuscin oxidation in vivo and the evidence of increased oxidative damage in AMD retina ex vivo lead to the conclusion that imaging of spectral characteristics of lipofuscin fluorescence may serve as a useful biomarker of oxidative damage, which can be helpful in assessing the efficacy of potential antioxidant therapies in retinal degenerations associated with accumulation of lipofuscin and increased oxidative stress. Finally, amendments to currently used fluorescence imaging instruments are suggested to be more sensitive and specific for imaging spectral characteristics of lipofuscin fluorescence.

Scavenging of Cation Radicals of the Visual Cycle Retinoids by Lutein, Zeaxanthin, Taurine, and Melanin.

In the retina, retinoids involved in vision are under constant threat of oxidation, and their oxidation products exhibit deleterious properties. Using pulse radiolysis, this study determined that the bimolecular rate constants of scavenging cation radicals of retinoids by taurine are smaller than 2 × 107 M-1s-1 whereas lutein scavenges cation radicals of all three retinoids with the bimolecular rate constants approach the diffusion-controlled limits, while zeaxanthin is only 1.4-1.6-fold less effective. Despite that lutein exhibits greater scavenging rate constants of retinoid cation radicals than other antioxidants, the greater concentrations of ascorbate in the retina suggest that ascorbate may be the main protectant of all visual cycle retinoids from oxidative degradation, while α-tocopherol may play a substantial role in the protection of retinaldehyde but is relatively inefficient in the protection of retinol or retinyl palmitate. While the protection of retinoids by lutein and zeaxanthin appears inefficient in the retinal periphery, it can be quite substantial in the macula. Although the determined rate constants of scavenging the cation radicals of retinol and retinaldehyde by dopa-melanin are relatively small, the high concentration of melanin in the RPE melanosomes suggests they can be scavenged if they are in proximity to melanin-containing pigment granules.

Is There an Optimal Combination of AREDS2 Antioxidants Zeaxanthin, Vitamin E and Vitamin C on Light-Induced Toxicity of Vitamin A Aldehyde to the Retina?

Vitamins C and E and zeaxanthin are components of a supplement tested in a large clinical trial-Age-Related Eye Disease Study 2 (AREDS2)-and it has been demonstrated that they can inhibit the progression of age-related macular degeneration. The aim of this study was to determine the optimal combinations of these antioxidants to prevent the phototoxicity mediated by vitamin A aldehyde (ATR), which can accumulate in photoreceptor outer segments (POS) upon exposure to light. We used cultured retinal pigment epithelial cells ARPE-19 and liposomes containing unsaturated lipids and ATR as a model of POS. Cells and/or liposomes were enriched with lipophilic antioxidants, whereas ascorbate was added just before the exposure to light. Supplementing the cells and/or liposomes with single lipophilic antioxidants had only a minor effect on phototoxicity, but the protection substantially increased in the presence of both ways of supplementation. Combinations of zeaxanthin with α-tocopherol in liposomes and cells provided substantial protection, enhancing cell viability from ~26% in the absence of antioxidants to ~63% in the presence of 4 µM zeaxanthin and 80 µM α-tocopherol, and this protective effect was further increased to ~69% in the presence of 0.5 mM ascorbate. The protective effect of ascorbate disappeared at a concentration of 1 mM, whereas 2 mM of ascorbate exacerbated the phototoxicity. Zeaxanthin or α-tocopherol partly ameliorated the cytotoxic effects. Altogether, our results suggest that the optimal combination includes upper levels of zeaxanthin and α-tocopherol achievable by diet and/or supplementations, whereas ascorbate needs to be at a four-fold smaller concentration than that in the vitreous. The physiological relevance of the results is discussed.

Photodegradation of Lipofuscin in Suspension and in ARPE-19 Cells and the Similarity of Fluorescence of the Photodegradation Product with Oxidized Docosahexaenoate.

Retinal lipofuscin accumulates with age in the retinal pigment epithelium (RPE), where its fluorescence properties are used to assess retinal health. It was observed that there is a decrease in lipofuscin fluorescence above the age of 75 years and in the early stages of age-related macular degeneration (AMD). The purpose of this study was to investigate the response of lipofuscin isolated from human RPE and lipofuscin-laden cells to visible light, and to determine whether an abundant component of lipofuscin, docosahexaenoate (DHA), can contribute to lipofuscin fluorescence upon oxidation. Exposure of lipofuscin to visible light leads to a decrease in its long-wavelength fluorescence at about 610 nm, with a concomitant increase in the short-wavelength fluorescence. The emission spectrum of photodegraded lipofuscin exhibits similarity with that of oxidized DHA. Exposure of lipofuscin-laden cells to light leads to a loss of lipofuscin granules from cells, while retaining cell viability. The spectral changes in fluorescence in lipofuscin-laden cells resemble those seen during photodegradation of isolated lipofuscin. Our results demonstrate that fluorescence emission spectra, together with quantitation of the intensity of long-wavelength fluorescence, can serve as a marker useful for lipofuscin quantification and for monitoring its oxidation, and hence useful for screening the retina for increased oxidative damage and early AMD-related changes.

Ligand-based rational design, synthesis and evaluation of novel potential chemical chaperones for opsin.

Inherited blinding diseases retinitis pigmentosa (RP) and a subset of Leber's congenital amaurosis (LCA) are caused by the misfolding and mistrafficking of rhodopsin molecules, which aggregate and accumulate in the endoplasmic reticulum (ER), leading to photoreceptor cell death. One potential therapeutic strategy to prevent the loss of photoreceptors in these conditions is to identify opsin-binding compounds that act as chemical chaperones for opsin, aiding its proper folding and trafficking to the outer cell membrane. Aiming to identify novel compounds with such effect, a rational ligand-based approach was applied to the structure of the visual pigment chromophore, 11-cis-retinal, and its locked analogue 11-cis-6mr-retinal. Following molecular docking studies on the main chromophore binding site of rhodopsin, 49 novel compounds were synthesized according to optimized one-to seven-step synthetic routes. These agents were evaluated for their ability to compete for the chromophore binding site of opsin, and their capacity to increase the trafficking of the P23H opsin mutant from the ER to the cell membrane. Different new molecules displayed an effect in at least one assay, acting either as chemical chaperones or as stabilizers of the 9-cis-retinal-rhodopsin complex. These compounds could provide the basis to develop novel therapeutics for RP and LCA.

Red Light Irradiation In Vivo Upregulates DJ-1 in the Retinal Ganglion Cell Layer and Protects against Axotomy-Related Dendritic Pruning.

Retinal ganglion cells (RGCs) undergo dendritic pruning in a variety of neurodegenerative diseases, including glaucoma and autosomal dominant optic atrophy (ADOA). Axotomising RGCs by severing the optic nerve generates an acute model of RGC dendropathy, which can be utilized to assess the therapeutic potential of treatments for RGC degeneration. Photobiomodulation (PBM) with red light provided neuroprotection to RGCs when administered ex vivo to wild-type retinal explants. In the current study, we used aged (13-15-month-old) wild-type and heterozygous B6;C3-Opa1Q285STOP (Opa1+/-) mice, a model of ADOA exhibiting RGC dendropathy. These mice were pre-treated with 4 J/cm2 of 670 nm light for five consecutive days before the eyes were enucleated and the retinas flat-mounted into explant cultures for 0-, 8- or 16-h ex vivo. RGCs were imaged by confocal microscopy, and their dendritic architecture was quantified by Sholl analysis. In vivo 670 nm light pretreatment inhibited the RGC dendropathy observed in untreated wild-type retinas over 16 h ex vivo and inhibited dendropathy in ON-center RGCs in wild-type but not Opa1+/- retinas. Immunohistochemistry revealed that aged Opa1+/- RGCs exhibited increased nitrosative damage alongside significantly lower activation of NF-κB and upregulation of DJ-1. PBM restored NF-κB activation in Opa1+/- RGCs and enhanced DJ-1 expression in both genotypes, indicating a potential molecular mechanism priming the retina to resist future oxidative insult. These data support the potential of PBM as a treatment for diseases involving RGC degeneration.

Comparison of Antioxidant Properties of Dehydrolutein with Lutein and Zeaxanthin, and their Effects on Cultured Retinal Pigment Epithelial Cells.

Dehydrolutein accumulates in substantial concentrations in the retina. The aim of this study was to compare antioxidant properties of dehydrolutein with other retinal carotenoids, lutein, and zeaxanthin, and their effects on ARPE-19 cells. The time-resolved detection of characteristic singlet oxygen phosphorescence was used to compare the singlet oxygen quenching rate constants of dehydrolutein, lutein, and zeaxanthin. The effects of these carotenoids on photosensitized oxidation were tested in liposomes, where photo-oxidation was induced by light in the presence of photosensitizers, and monitored by oximetry. To compare the uptake of dehydrolutein, lutein, and zeaxanthin, ARPE-19 cells were incubated with carotenoids for up to 19 days, and carotenoid contents were determined by spectrophotometry in cell extracts. To investigate the effects of carotenoids on photocytotoxicity, cells were exposed to light in the presence of rose bengal or all-trans-retinal. The results demonstrate that the rate constants for singlet oxygen quenching are 0.77 × 1010, 0.55 × 1010, and 1.23 × 1010 M-1s-1 for dehydrolutein, lutein, and zeaxanthin, respectively. Overall, dehydrolutein is similar to lutein or zeaxanthin in the protection of lipids against photosensitized oxidation. ARPE-19 cells accumulate substantial amounts of both zeaxanthin and lutein, but no detectable amounts of dehydrolutein. Cells pre-incubated with carotenoids are equally susceptible to photosensitized damage as cells without carotenoids. Carotenoids provided to cells together with the extracellular photosensitizers offer partial protection against photodamage. In conclusion, the antioxidant properties of dehydrolutein are similar to lutein and zeaxanthin. The mechanism responsible for its lack of accumulation in ARPE-19 cells deserves further investigation.

Products of Docosahexaenoate Oxidation as Contributors to Photosensitising Properties of Retinal Lipofuscin.

Retinal lipofuscin which accumulates with age in the retinal pigment epithelium (RPE) is subjected to daily exposures to high fluxes of visible light and exhibits potent photosensitising properties; however, the molecules responsible for its photoreactivity remain unknown. Here, we demonstrate that autooxidation of docosahexaenoate (DHE) leads to the formation of products absorbing, in addition to UVB and UVA light, also visible light. The products of DHE oxidation exhibit potent photosensitising properties similar to photosensitising properties of lipofuscin, including generation of an excited triplet state with similar characteristics as the lipofuscin triplet state, and photosensitised formation of singlet oxygen and superoxide. The quantum yields of singlet oxygen and superoxide generation by oxidised DHE photoexcited with visible light are 2.4- and 3.6-fold higher, respectively, than for lipofuscin, which is consistent with the fact that lipofuscin contains some chromophores which do contribute to the absorption of light but not so much to its photosensitising properties. Importantly, the wavelength dependence of photooxidation induced by DHE oxidation products normalised to equal numbers of incident photons is also similar to that of lipofuscin-it steeply increases with decreasing wavelength. Altogether, our results demonstrate that products of DHE oxidation include potent photosensitiser(s) which are likely to contribute to lipofuscin photoreactivity.

New avenues for therapy in mitochondrial optic neuropathies.

Mitochondrial optic neuropathies are a group of optic nerve atrophies exemplified by the two commonest conditions in this group, autosomal dominant optic atrophy (ADOA) and Leber's hereditary optic neuropathy (LHON). Their clinical features comprise reduced visual acuity, colour vision deficits, centro-caecal scotomas and optic disc pallor with thinning of the retinal nerve fibre layer. The primary aetiology is genetic, with underlying nuclear or mitochondrial gene mutations. The primary pathology is owing to retinal ganglion cell dysfunction and degeneration. There is currently only one approved treatment and no curative therapy is available. In this review we summarise the genetic and clinical features of ADOA and LHON and then examine what new avenues there may be for therapeutic intervention. The therapeutic strategies to manage LHON and ADOA can be split into four categories: prevention, compensation, replacement and repair. Prevention is technically an option by modifying risk factors such as smoking cessation, or by utilising pre-implantation genetic diagnosis, although this is unlikely to be applied in mitochondrial optic neuropathies due to the non-life threatening and variable nature of these conditions. Compensation involves pharmacological interventions that ameliorate the mitochondrial dysfunction at a cellular and tissue level. Replacement and repair are exciting new emerging areas. Clinical trials, both published and underway, in this area are likely to reveal future potential benefits, since new therapies are desperately needed. Plain language summary: Optic nerve damage leading to loss of vision can be caused by a variety of insults. One group of conditions leading to optic nerve damage is caused by defects in genes that are essential for cells to make energy in small organelles called mitochondria. These conditions are known as mitochondrial optic neuropathies and two predominant examples are called autosomal dominant optic atrophy and Leber's hereditary optic neuropathy. Both conditions are caused by problems with the energy powerhouse of cells: mitochondria. The cells that are most vulnerable to this mitochondrial malfunction are called retinal ganglion cells, otherwise collectively known as the optic nerve, and they take the electrical impulse from the retina in the eye to the brain. The malfunction leads to death of some of the optic nerve cells, the degree of vision loss being linked to the number of those cells which are impacted in this way. Patients will lose visual acuity and colour vision and develop a central blind spot in their field of vision. There is currently no cure and very few treatment options. New treatments are desperately needed for patients affected by these devastating diseases. New treatments can potentially arise in four ways: prevention, compensation, replacement and repair of the defects. Here we explore how present and possible future treatments might provide hope for those suffering from these conditions.

Discovery of Novel 2-Aniline-1,4-naphthoquinones as Potential New Drug Treatment for Leber's Hereditary Optic Neuropathy (LHON).

Leber's hereditary optic neuropathy (LHON) is a rare genetic mitochondrial disease and the primary cause of chronic visual impairment for at least 1 in 10 000 individuals in the U.K. Treatment options remain limited, with only a few drug candidates and therapeutic approaches, either approved or in development. Recently, idebenone has been investigated as drug therapy in the treatment of LHON, although evidence for the efficacy of idebenone is limited in the literature. NAD(P)H:quinone oxidoreductase 1 (NQO1) and mitochondrial complex III were identified as the major enzymes involved in idebenone activity. Based on this mode of action, computer-aided techniques and structure-activity relationship (SAR) optimization studies led to the discovery of a series naphthoquinone-related small molecules, with comparable adenosine 5'-triphosphate (ATP) rescue activity to idebenone. Among these, three compounds showed activity in the nanomolar range and one, 2-((4-fluoro-3-(trifluoromethyl)phenyl)amino)-3-(methylthio)naphthalene-1,3-dione (1), demonstrated significantly higher potency ex vivo, and significantly lower cytotoxicity, than idebenone.

Computational Studies towards the Identification of Novel Rhodopsin-Binding Compounds as Chemical Chaperones for Misfolded Opsins.

Accumulation of misfolded and mistrafficked rhodopsin on the endoplasmic reticulum of photoreceptor cells has a pivotal role in the pathogenesis of retinitis pigmentosa and a subset of Leber's congenital amaurosis. One potential strategy to reduce rhodopsin misfolding and aggregation in these conditions is to use opsin-binding compounds as chemical chaperones for opsin. Such molecules have previously shown the ability to aid rhodopsin folding and proper trafficking to the outer cell membranes of photoreceptors. As means to identify novel chemical chaperones for rhodopsin, a structure-based virtual screening of commercially available drug-like compounds (300,000) was performed on the main binding site of the visual pigment chromophore, the 11-cis-retinal. The best 24 virtual hits were examined for their ability to compete for the chromophore-binding site of opsin. Among these, four small molecules demonstrated the ability to reduce the rate constant for the formation of the 9-cis-retinal-rhodopsin complex, while five molecules surprisingly enhanced the formation of this complex. Compound 7, 13, 20 and 23 showed a weak but detectable increase in the trafficking of the P23H mutant, widely used as a model for both retinitis pigmentosa and Leber's congenital amaurosis, from the ER to the cell membrane. The compounds did not show any relevant cytotoxicity in two different human cell lines, with the only exception of 13. Based on the structures of these active compounds, a series of in silico studies gave important insights on the potential structural features required for a molecule to act either as chemical chaperone or as stabiliser of the 11-cis-retinal-rhodopsin complex. Thus, this study revealed a series of small molecules that represent a solid foundation for the future development of novel therapeutics against these severe inherited blinding diseases.

Scavenging of Retinoid Cation Radicals by Urate, Trolox, and α-, ß-, γ-, and δ-Tocopherols.

Retinoids are present in human tissues exposed to light and under increased risk of oxidative stress, such as the retina and skin. Retinoid cation radicals can be formed as a result of the interaction between retinoids and other radicals or photoexcitation with light. It has been shown that such semi-oxidized retinoids can oxidize certain amino acids and proteins, and that α-tocopherol can scavenge the cation radicals of retinol and retinoic acid. The aim of this study was to determine (i) whether ß-, γ-, and δ-tocopherols can also scavenge these radicals, and (ii) whether tocopherols can scavenge the cation radicals of another form of vitamin A-retinal. The retinoid cation radicals were generated by the pulse radiolysis of benzene or aqueous solution in the presence of a selected retinoid under oxidizing conditions, and the kinetics of retinoid cation radical decays were measured in the absence and presence of different tocopherols, Trolox or urate. The bimolecular rate constants are the highest for the scavenging of cation radicals of retinal, (7 to 8) × 109 M-1·s-1, followed by retinoic acid, (0.03 to 5.6) × 109 M-1·s-1, and retinol, (0.08 to 1.6) × 108 M-1·s-1. Delta-tocopherol is the least effective scavenger of semi-oxidized retinol and retinoic acid. The hydrophilic analogue of α-tocopherol, Trolox, is substantially less efficient at scavenging retinoid cation radicals than α-tocopherol and urate, but it is more efficient at scavenging the cation radicals of retinoic acid and retinol than δ-tocopherol. The scavenging rate constants indicate that tocopherols can effectively compete with amino acids and proteins for retinoid cation radicals, thereby protecting these important biomolecules from oxidation. Our results provide another mechanism by which tocopherols can diminish the oxidative damage to the skin and retina and thereby protect from skin photosensitivity and the development and/or progression of changes in blinding retinal diseases such as Stargardt's disease and age-related macular degeneration (AMD).

Photostimulation of mitochondria as a treatment for retinal neurodegeneration.

Absorption of photon energy by neuronal mitochondria leads to numerous downstream neuroprotective effects. Red and near infrared (NIR) light are associated with significantly less safety concerns than light of shorter wavelengths and they are therefore, the optimal choice for irradiating the retina. Potent neuroprotective effects have been demonstrated in various models of retinal damage, by red/NIR light, with limited data from human studies showing its ability to improve visual function. Improved neuronal mitochondrial function, increased blood flow to neural tissue, upregulation of cell survival mediators and restoration of normal microglial function have all been proposed as potential underlying mechanisms of red/NIR light.

Lipofuscin-associated photo-oxidative stress during fundus autofluorescence imaging.

PURPOSE: Current standards and guidelines aimed at preventing retinal phototoxicity during intentional exposures do not specifically evaluate the contribution of endogenous photosensitizers. However, certain retinal diseases are characterized by abnormal accumulations of potential photosensitizers such as lipofuscin bisretinoids in the retinal pigment epithelium (RPE). We sought to determine these contributions by a numerical assessment of in-vivo photo-oxidative stress during irradiation of RPE lipofuscin. METHODS: Based on the literature, we calculated the retinal exposure levels, optical filtering of incident radiation by the ocular lens, media, photoreceptors, and RPE melanin, light absorption by lipofuscin, and photochemical effects in the RPE in two situations: exposure to short-wavelength (λ = 488 nm) fundus autofluorescence (SW-AF) excitation light and exposure to indirect (diffuse) sunlight. RESULTS: In healthy persons at age 20, 40, and 60, respectively, the rate of oxygen photoconsumption by lipofuscin increases by 1.3, 1.7, and 2.4 fold during SW-AF-imaging as compared to diffuse sunlight. In patients with STGD1 below the age of 30, this rate was 3.3-fold higher compared to age-matched controls during either sunlight or SW-AF imaging. CONCLUSIONS: Our results suggest that the RPE of patients with STGD1 is generally at increased risk of photo-oxidative stress, while exposure during SW-AF-imaging amplifies this risk. These theoretical results have not yet been verified with in-vivo data due to a lack of sufficiently sensitive in-vivo measurement techniques.

Red Light Treatment in an Axotomy Model of Neurodegeneration.

Red light has been shown to provide neuroprotective effects. Axotomizing the optic nerve initiates retinal ganglion cell (RGC) degeneration, and an early marker of this is dendritic pruning. We hypothesized that 670 nm light can delay axotomy-induced dendritic pruning in the retinal explant. To test this hypothesis, we monitored the effects of 670 nm light (radiant exposure of 31.7 J cm(-2) ), on RGC dendritic pruning in retinal explants from C57BL/6J mice, at 40 min, 8 h and 16 h post axotomy. For sham-treated retinae, area under the Sholl curve, peak of the Sholl curve and dendritic length at 8 h post axotomy showed statistically significant reductions by 42.3% (P = 0.008), 29.8% (P = 0.007) and 38.4% (P = 0.038), respectively, which were further reduced after 16 h by 40.56% (P < 0.008), 33.9% (P < 0.007), 45.43% (P < 0.006), respectively. Dendritic field area was also significantly reduced after 16 h, by 44.23% (P < 0.019). Such statistically significant reductions were not seen in light-treated RGCs at 8 or 16 h post axotomy. The results demonstrate the ability of 670 nm light to partially prevent ex vivo dendropathy in the mouse retina, suggesting that it is worth exploring as a treatment option for dendropathy-associated neurodegenerative diseases, including glaucoma and Alzheimer's disease.

Protein misfolding and the pathogenesis of ABCA4-associated retinal degenerations.

Mutations in the ABCA4 gene are a common cause of autosomal recessive retinal degeneration. All mouse models to date are based on knockouts of Abca4, even though the disease is often caused by missense mutations such as the complex allele L541P;A1038V (PV). We now show that the PV mutation causes severe human disease whereas the V mutation alone causes mild disease. Mutant ABCA4 proteins expressed heterologously in mammalian cells retained normal cellular localization. However, basal and all-trans-retinal-stimulated ATPase activities were reduced substantially for P and PV but only mildly for V. Electron microscopy revealed marked structural changes and misfolding for the P and PV mutants but few changes for the V mutant, consistent with the disease severity difference in patients. We generated Abca4(PV/PV) knock-in mice homozygous for the complex PV allele to investigate the effects of this misfolding mutation in vivo. Mutant ABCA4 RNA levels approximated WT ABCA4 RNA levels but, surprisingly, only trace amounts of mutant ABCA4 protein were noted in the retina. RNA sequencing of WT, Abca4(-/-) and Abca4(PV/PV) mice revealed mild gene expression alterations in the retina and RPE. Similar to Abca4(-/-) mice, Abca4(PV/PV) mice showed substantial A2E and lipofuscin accumulation in their RPE cells but no retinal degeneration up to 12 months of age. Thus, rapid degradation of this large misfolded mutant protein in mouse retina caused little detectable photoreceptor degeneration. These findings suggest likely differences in the unfolded protein response between murine and human photoreceptors and support development of therapies directed at increasing this capability in patients.

Light-induced damage to the retina: current understanding of the mechanisms and unresolved questions: a symposium-in-print.

Light-induced injury to the retina resembles many features of several retinal degenerative diseases, particularly age-related macular degeneration. This Symposium-in-Print on Retinal Photodamage discusses the mechanisms involved and protective strategies to increase the retinal resistance to damage and/or to counteract its deleterious effects. Recent results help explaining the wavelength dependence of susceptibility of the retina to photodamage and different sites of the initial injury for shorter- and longer-wavelength light. Still, there are many unanswered questions pointing toward next directions in research so as to increase the understanding of the responses of the retina to photodamage and help to develop effective therapeutic approaches for retinal degenerations.

Concentration dependence of vitamin C in combinations with vitamin E and zeaxanthin on light-induced toxicity to retinal pigment epithelial cells.

The purpose of this study was to determine the effects of increasing concentration of ascorbate alone and in combinations with α-tocopherol and zeaxanthin on phototoxicity to the retinal pigment epithelium. ARPE-19 cells were exposed to rose bengal and visible light in the presence and absence of antioxidants. Toxicity was quantified by an assay of cell-reductive activity. A 20 min exposure to visible light and photosensitizer decreased cell viability to ca 42%. Lipophilic antioxidants increased viabilities to ca 70%, 61% and 75% for α-tocopherol, zeaxanthin and their combination, respectively. Cell viabilities were ca 70%, 56% and 5% after exposures in the presence of 0.35, 0.7 and 1.4 mm ascorbate, respectively. A 45 min exposure increased cell death to ca 74% and >95% in the absence and presence of ascorbate, respectively. In the presence of ascorbate, zeaxanthin did not significantly affect phototoxicity. α-Tocopherol and its combination with zeaxanthin enhanced protective effects of ascorbate, but did not prevent from ascorbate-mediated deleterious effects. In conclusion, there is a narrow range of concentrations and exposure times where ascorbate exerts photoprotective effects, exceeding which leads to ascorbate-mediated increase in photocytotoxicity. Vitamin E and its combination with zeaxanthin can enhance protective effects of ascorbate, but do not ameliorate its deleterious effects.

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.

Retinal pigment epithelium lipofuscin proteomics.

Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.