A2E Accumulation Research Articles

A High Omega-3 Fatty Acid Diet Reduces Retinal Lesions in a Murine Model of Macular Degeneration

Age-related macular degeneration (AMD) is one of the leading cause of blindness among the elderly; however, current therapy options are limited. Epidemiological studies have shown that a diet that is high in omega-3 polyunsaturated (n-3) fatty acids can slow disease progression in patients with advanced AMD. In this study, we evaluated the effect of such a diet on the retinas of Ccl2(-/-)/Cx3cr1(-/-) mice, a model that develops AMD-like retinal lesions that include focal deep retinal lesions, abnormal retinal pigment epithelium, photoreceptor degeneration, and A2E accumulation. Ccl2(-/-)/Cx3cr1(-/-) mice that ingested a high n-3 fatty acid diet showed a slower progression of retinal lesions compared with the low n-3 fatty acids group. Some mice that were given high levels of n-3 fatty acids had lesion reversion. We found a shunted arachidonic acid metabolism that resulted in decreased pro-inflammatory derivatives (prostaglandin E(2) and leukotriene B(4)) and an increased anti-inflammatory derivative (prostaglandin D(2)). We also measured lower ocular TNF-alpha and IL-6 transcript levels in the mice fed a diet of high n-3 fatty acids. Our findings in these mice are in line with human studies of AMD risk reduction by long-chain n-3 fatty acids. This murine model provides a useful tool to evaluate therapies that might delay the development of AMD.

A2E, a component of lipofuscin, is pro‐angiogenic in vivo

A recent study in vitro demonstrated that a major lipofuscin component, A2E, serves as a retinoic acid receptor ligand. The current study investigated the effects of A2E on retinal pigment epithelial (RPE) cells in vivo and was performed to extend the understanding of the effects of A2E. Firstly, subretinal injection of A2E was performed and 3 weeks after the injection, and it was demonstrated that subretinal injection of A2E induced RPE cell death, and concomitant upregulation of vascular endothelial growth factor (VEGF) in the RPE and choroid. The upregulation of VEGF was attenuated by an RARalpha antagonist. Next we performed laser photocoagulation in mice that accumulated A2E either after subretinal injection, by Ccl2 gene knockout or by aging demonstrated that mice that accumulated A2E in the RPE, which showed higher rates of choroidal neobascularization (CNV) formation after weak laser injury than the controls and the formation of CNV was inhibited by an RARalpha antagonist in all models tested. The data suggest that A2E accumulation induces RPE cell death, and concomitant increase of VEGF. Accumulation of A2E alone is not sufficient to induce CNV in vivo, but induces the expression of VEGF in RPE and choroid. The mice that accumulated A2E in RPE cells are vulnerable to CNV development via RAR activation, at least in part.

Involvement of All-trans-retinal in Acute Light-induced Retinopathy of Mice

Exposure to bright light can cause visual dysfunction and retinal photoreceptor damage in humans and experimental animals, but the mechanism(s) remain unclear. We investigated whether the retinoid cycle (i.e. the series of biochemical reactions required for vision through continuous generation of 11-cis-retinal and clearance of all-trans-retinal, respectively) might be involved. Previously, we reported that mice lacking two enzymes responsible for clearing all-trans-retinal, namely photoreceptor-specific ABCA4 (ATP-binding cassette transporter 4) and RDH8 (retinol dehydrogenase 8), manifested retinal abnormalities exacerbated by light and associated with accumulation of diretinoid-pyridinium-ethanolamine (A2E), a condensation product of all-trans-retinal and a surrogate marker for toxic retinoids. Now we show that these mice develop an acute, light-induced retinopathy. However, cross-breeding these animals with lecithin:retinol acyltransferase knock-out mice lacking retinoids within the eye produced progeny that did not exhibit such light-induced retinopathy until gavaged with the artificial chromophore, 9-cis-retinal. No significant ocular accumulation of A2E occurred under these conditions. These results indicate that this acute light-induced retinopathy requires the presence of free all-trans-retinal and not, as generally believed, A2E or other retinoid condensation products. Evidence is presented that the mechanism of toxicity may include plasma membrane permeability and mitochondrial poisoning that lead to caspase activation and mitochondria-associated cell death. These findings further understanding of the mechanisms involved in light-induced retinal degeneration.

The Age Lipid A2E and Mitochondrial Dysfunction Synergistically Impair Phagocytosis by Retinal Pigment Epithelial Cells

Accumulation of indigestible lipofuscin and decreased mitochondrial energy production are characteristic age-related changes of post-mitotic retinal pigment epithelial (RPE) cells in the human eye. To test whether these two forms of age-related impairment have interdependent effects, we quantified the ATP-dependent phagocytic function of RPE cells loaded or not with the lipofuscin component A2E and inhibiting or not mitochondrial ATP synthesis either pharmacologically or genetically. We found that physiological levels of lysosomal A2E reduced mitochondrial membrane potential and inhibited oxidative phosphorylation (OXPHOS) of RPE cells. Furthermore, in media with physiological concentrations of glucose or pyruvate, A2E significantly inhibited phagocytosis. Antioxidants reversed these effects of A2E, suggesting that A2E damage is mediated by oxidative processes. Because mitochondrial mutations accumulate with aging, we generated novel genetic cellular models of RPE carrying mitochondrial DNA point mutations causing either moderate or severe mitochondrial dysfunction. Exploring these mutant RPE cells we found that, by itself, only the severe but not the moderate OXPHOS defect reduces phagocytosis. However, sub-toxic levels of lysosomal A2E are sufficient to reduce phagocytic activity of RPE with moderate OXPHOS defect and cause cell death of RPE with severe OXPHOS defect. Taken together, RPE cells rely on OXPHOS for phagocytosis when the carbon energy source is limited. Our results demonstrate that A2E accumulation exacerbates the effects of moderate mitochondrial dysfunction. They suggest that synergy of sub-toxic lysosomal and mitochondrial changes in RPE cells with age may cause RPE dysfunction that is known to contribute to human retinal diseases like age-related macular degeneration.

Total rod ERG suppression with high dose compassionate Fenretinide usage

Fenretinide is a synthetic retinoid that interferes with the attachment of retinol to retinol binding protein. It may inhibit accumulation of A2E and lipofuscin, and is proposed as therapy for Stargardt disease. It is currently used for cancer therapy, and mild depression of rod function and dark adaptation is a side effect at standard dosage. We studied two youngsters (aged between 12 and 13) receiving high doses as compassionate treatment for neuroblastoma: 800 mg daily for 1 out of every 3 weeks, for roughly 2 years. Goldmann-Weekers dark adaptometry, ISCEV standard ERG and mfERG were performed, and blood was analyzed for vitamin A. Neither child complained of night blindness or showed retinal fundus abnormalities. On initial exam, dark adaptation thresholds were elevated by 3 log units, and there were no detectable rod ERG responses. However, cone responses and mfERG were normal. Retesting one subject 3 months after stopping the drug revealed normal rod thresholds (slightly delayed) and low normal rod ERG responses. Serum vitamin A levels were normal from both subjects, but there is no record of whether the samples were drawn during cycles on or off drug. Our study demonstrates that high dose Fenretinide can suppress rod function quite completely, although serum vitamin A and rod function apparently return to normal or near normal levels rapidly once the drug is stopped. It is intriguing that cone function and access to vitamin A seems largely independent of Fenretinide effects on retinol availability.

Correction of the disease phenotype in the mouse model of Stargardt disease by lentiviral gene therapy

Autosomal recessive Stargardt disease (STGD1) is a macular dystrophy caused by mutations in the ABCA4 (ABCR) gene. The disease phenotype that is most recognized in STGD1 patients, and also in the Abca4-/- mouse (a disease model), is lipofuscin accumulation in retinal pigment epithelium. Here, we tested whether delivery of the normal (wt) human ABCA4 gene to the subretinal space of the Abca4 -/- mice via lentiviral vectors would correct the disease phenotype; that is, reduce accumulation of the lipofuscin pigment A2E. Equine infectious anemia virus (EIAV)-derived lentiviral vectors were constructed expressing either the human ABCA4 gene or the LacZ reporter gene under the control of the constitutive (CMV) or photoreceptor-specific (Rho) promoters. Abca4-/- mice were injected subretinally with 1 microl ( approximately 5.0 x 10(5) TU) of each EIAV vector in one eye at postnatal days 4 and 5. An injection of saline, an EIAV-null vector, or an uninjected contralateral eye served as a control. Mice were killed at various times after injection to determine photoreceptor (PR) transduction efficiency and A2E concentrations. EIAV-LacZ vectors transduced from 5 to 20% of the PRs in the injected area in mice. Most importantly, a single subretinal injection of EIAV-CMV-ABCA4 to Abca4-/- mouse eyes substantially reduced disease-associated A2E accumulation compared to untreated and mock-treated control eyes. Treated eyes of Abca4-/- mice accumulated 8-12 pmol per eye (s.d.=2.7) of A2E 1 year after treatment, amounts comparable to wt controls, whereas mock-treated or untreated eyes had 3-5 times more A2E (27-39 pmol per eye, s.d.=1.5; P=0.001-0.005). Although extrapolation to humans requires caution, the high transduction efficiency of both rod and cone photoreceptors and the statistically significant reduction of A2E accumulation in the mouse model of STGD1 suggest that lentiviral gene therapy is a potentially efficient tool for treating ABCA4-associated diseases.

A2E, a Pigment of the Lipofuscin of Retinal Pigment Epithelial Cells, Is an Endogenous Ligand for Retinoic Acid Receptor

Lipofuscin contains fluorophores, which represent a biomarker for cellular aging. Although it remains unsubstantiated clinically, experimental results support that the accumulation of lipofuscin is related to an increased risk of choroidal neovascularization due to age-related macular degeneration, a leading cause of legal blindness. Here, we report that a major lipofuscin component, A2E, activates the retinoic acid receptor (RAR). In vitro experiments using luciferase reporter assay, competitional binding assay, analysis of target genes, and chromatin immunoprecipitation (ChIP) assay strongly suggest that A2E is a bona fide ligand for RAR and induces sustained activation of RAR target genes. A2E-induced vascular endothelial growth factor (VEGF) expression in a human retinal pigment epithelial cell line (ARPE-19) and RAR antagonist blocked the up-regulation of VEGF. The conditioned medium of A2E-treated ARPE-19 cells induced tube formation in human umbilical vascular endothelial cells, which was blocked by the RAR antagonist and anti-VEGF antibody. These results suggest that A2E accumulation results in the phenotypic alteration of retinal pigment epithelial cells, predisposing the environment to choroidal neovascularization development. This is mediated through the agonistic function of A2E, at least in part. The results of this study provide a novel potential therapeutic target for this incurable condition.

Redundant and unique roles of retinol dehydrogenases in the mouse retina

Highly abundant short-chain alcohol dehydrogenases (RDHs) in the retina were assumed to be involved in the recycling of 11-cis-retinal chromophore in the visual cycle. Mutations in human RDH genes are associated with Fundus albipunctatus, a mild form of night blindness (RDH5) and an autosomal recessive, childhood-onset severe retinal dystrophy (RDH12). Rdh12 knockout mice were found to be susceptible to light-induced photoreceptor apoptosis, whereas Rdh5 and Rdh8 knockout mice displayed only delayed dark adaptation. However, each knockout mouse eventually regenerated normal levels of visual pigments, suggesting that RDHs compensate for each other in the visual cycle. Here, we established RDH double knockout (Rdh8(-/-)Rdh12(-/-)) and triple knockout (Rdh5(-/-)Rdh8(-/-)Rdh12(-/-)) mice generated on various genetic backgrounds including a rod alpha-transducin knockout to test cone function. RDH activity was severely reduced in Rdh8(-/-)Rdh12(-/-) retina extracts, whereas Rdh8(-/-) RDH activity was intermediate and Rdh12(-/-) RDH activity was reduced only slightly. Surprisingly, all multiple knockout mice produced sufficient amounts of the chromophore to regenerate rhodopsin and cone pigments in vivo. Three-month-old Rdh8(-/-)Rdh12(-/-) mice characteristically displayed a slowly progressing rod-cone dystrophy accompanied by accumulation of N-retinylidene-N-retinylethanolamine (A2E), a toxic substance known to contribute to retinal degeneration. A2E accumulation and retinal degeneration were prevented by application of retinylamine, a potent retinoid cycle inhibitor. The results suggest that RDH8 and RDH12 are dispensable in support of the visual cycle but appear to be key components in clearance of free all-trans-retinal, thereby preventing A2E accumulation and photoreceptor cell death.

Neurotrophins enhance retinal pigment epithelial cell survival through neuroprotectin D1 signaling.

Integrity of retinal pigment epithelial cells is necessary for photoreceptor survival and vision. The essential omega-3 fatty acid, docosahexaenoic acid, attains its highest concentration in the human body in photoreceptors and is assumed to be a target for lipid peroxidation during cell damage. We have previously shown, in contrast, that docosahexaenoic acid is also the precursor of neuroprotectin D1 (NPD1), which now we demonstrate, acts against apoptosis mediated by A2E, a byproduct of phototransduction that becomes toxic when it accumulates in aging retinal pigment epithelial (RPE) cells and in some inherited retinal degenerations. Furthermore, we show that neurotrophins, particularly pigment epithelium-derived factor, induce NPD1 synthesis and its polarized apical secretion. Moreover, docosahexaenoic acid (DHA) elicits a concentration-dependent and selective potentiation of pigment epithelial-derived factor-stimulated NPD1 synthesis and release through the apical RPE cell surface. The bioactivity of signaling activated by pigment epithelium-derived factor and DHA uncovered synergistic cytoprotection with concomitant NPD1 synthesis when cells were challenged with oxidative stress. Also, DHA and pigment epithelium-derived factor synergistically modify the expression of Bcl-2 family members, activating antiapoptotic proteins and decreasing proapoptotic proteins, and by attenuating caspase 3 activation during oxidative stress. Thus, our findings demonstrate that DHA-derived NPD1 protects against RPE cell damage mediated by aging/disease-induced A2E accumulation. Also, our results identify neurotrophins as regulators of NPD1 and of its polarized apical efflux from RPE cells. Taken together, these findings imply NPD1 may elicit autocrine actions on RPE cells and paracrine bioactivity in cells located in the proximity of the interphotoreceptor matrix.

Enhanced accumulation of A2E in individuals homozygous or heterozygous for mutations in BEST1 ( VMD2)

Best vitelliform macular dystrophy (BMD) is an autosomal dominant inherited macular degenerative disease caused by mutations in the gene BEST1 (formerly VMD2). Prior reports indicate that BMD is characterized histopathologically by accumulation of lipofuscin in the retinal pigment epithelium (RPE). However, this accumulation has not been quantified and the chemical composition of lipofuscin in BMD has not been examined. In this study we characterize the histopathology of a donor eye from a rare individual homozygous for a mutation (W93C) in BEST1. We find that this individual's disease was not any more severe than has been described for heterozygotes. We then used this tissue to quantify lipofuscin accumulation by enriching intracellular granules from RPE cells on sucrose gradients and counting the granules in each density fraction. Granules from the homozygous donor eye as well as a donor eye from an individual heterozygous for the mutation T6R were compared with age-matched control eyes. Interestingly, the least dense fraction, representing classical lipofuscin granules was either not present or significantly diminished in the BMD donor eyes and the autoflourescence associated with lipofuscin had shifted to denser fractions. However, a substantial enrichment for granules in fractions of higher density was also noted in the BMD samples. Inspection of granules from the homozygous donor eye by electron microscopy revealed a complex abnormal multilobular structure. Analysis of granules by HPLC indicated a ∼1.6- and ∼fourfold overall increase in A2E in the BMD eyes versus age-matched control eyes, with a shift of A2E to more dense granules in the BMD donor eyes. Despite the increase in A2E and total intracellular granules, the RPE in the homozygous donor eyes was relatively well preserved. Based on these data we conclude that the clinical and histopathologic consequences to the homozygous donor were not any more severe than has been reported previously for individuals who are established or presumptive heterozygotes. We find that A2E is a component of the lipofuscin accumulated in BMD and that it is more abundant than in control eyes suggesting that the etiology of BMD is similar to Stargardt's disease and Stargardt-like macular dystrophy. Finally, the changes we observe in the granules suggest that the histopathology and eventual vision loss associated with BMD may be due to defects in the ability of the RPE to fully degrade phagocytosed photoreceptor outer segments.

Accumulation of A2-E in mitochondrial membranes of cultured RPE cells

Lipofuscin occurs in association with various blinding diseases, including ARMD. Formation of lipofuscin is considered to be initiated by the inability of the RPE lysosome to degrade constituents of phagocytosed material resulting in its intralysosomal accumulation. Thus, the deposition of abnormal retinoid adducts causing the autofluorescent properties of RPE lipofuscin originates from abnormal products of the retinoid cycle contained in phagocytosed photoreceptor outer segments. The major lipofuscin retinoid conjugate A2-E was previously shown to exert toxic effects on RPE cells by directly damaging lysosomal function and structure. However, A2-E was also proposed to severely harm extralysosomal RPE cell structures during the pathogenesis of ARMD. This would require release or leakage of A2-E from the lysosomal compartment with subsequent targeting of other cellular compartments. We therefore now investigated intralysosomal accumulation, possible biodegradation, release from the lysosomal compartment and intracellular spreading of (14)C-labelled A2-E in cultured human RPE cells. We specifically loaded lysosomes of cultured human RPE cells with [(14)C]A2-E. A linear increase of intracellular radioactivity was observed during the 4-week loading period. Cell fractionation experiments indicated that more than 90% of loaded A2-E was specifically accumulating in the lysosomes. After loading, the fate of the radioactive label was chased over a period of an additional 4 weeks. No metabolism or secretion of A2-E to the medium was detectable. Subcellular fractionation revealed that during the chase period, about 13% were shifted from the lysosomes to mitochondrial fractions. This effect was strikingly intensified when after loading the cells with the labeled retinoid, its intralysosomal concentration was boosted by an additional load with non-labeled A2-E. Thus about 44% of the label were located in mitochondria at the end of the chase period. No significant spreading to other cell compartments was detectable. Since A2-E was suggested to act as a proapoptotic molecule via a mitochondrial pathway, we postulate that upon reaching a critical intralysosomal concentration, A2-E is released from the lysosome and then specifically targets the outer mitochondrial membrane thereby initiating apoptosis of the RPE cell. This may also apply correspondingly to other lipofuscin-associated molecules that cause leakage of the lysosomal membrane.

Reductions in Serum Vitamin A Arrest Accumulation of Toxic Retinal Fluorophores: A Potential Therapy for Treatment of Lipofuscin-Based Retinal Diseases

Excessive accumulation of lipofuscin is observed in numerous degenerative retinal diseases. A toxic vitamin A-based fluorophore (A2E) present within lipofuscin has been implicated in the death of RPE and photoreceptor cells. Here, we used an animal model that manifests accelerated lipofuscin accumulation (ABCA4-/- mutant) to evaluate the efficacy of a therapeutic approach based on reduction of serum retinol. N-(4-hydroxyphenyl)retinamide (HPR) potently and reversibly reduces serum retinol. The interaction of HPR with retinol binding protein (RBP) and transthyretin was studied by spectrofluorometry and size-exclusion chromatography. To assess the effects of HPR on visual cycle retinoids and A2E biosynthesis, HPR was chronically administered to ABCA4-/- mice. Mice were evaluated using biochemical, electrophysiological, and morphologic techniques. Administration of HPR to ABCA4-/- mice caused immediate, dose-dependent reductions in serum retinol and RBP. Chronic administration produced commensurate reductions in visual cycle retinoids and arrested accumulation of A2E and lipofuscin autofluorescence in the RPE. Physiologically, HPR treatment caused modest delays in dark adaptation. Chromophore regeneration kinetics, light sensitivity of photoreceptors, and phototransduction processes were normal. Histologic examinations showed no alteration of retinal cytostructure or morphology. These findings demonstrate the vitamin A-dependent nature of A2E biosynthesis and validate a novel therapeutic approach with potential to halt the accumulation of lipofuscin fluorophores in the eye.

Characterization of Peroxy-A2E and Furan-A2E Photooxidation Products and Detection in Human and Mouse Retinal Pigment Epithelial Cell Lipofuscin

The nondegradable pigments that accumulate in retinal pigment epithelial (RPE) cells as lipofuscin constituents are considered to be responsible for the loss of RPE cells in recessive Stargardt disease, a blindness macular disorder of juvenile onset. This autofluorescent material may also contribute to the etiology of age-related macular degeneration. The best characterized of these fluorophores is A2E, a compound consisting of two retinoid-derived side arms extending from a pyridinium ring. Evidence indicates that photochemical mechanisms initiated by excitation from the blue region of the spectrum may contribute to the adverse effects of A2E accumulation, with the A2E photooxidation products being damaging intermediates. By studying the oxidation products (oxo-A2E) generated using oxidizing agents that add one or two oxygens at a time, together with structural analysis by heteronuclear single quantum correlation-NMR spectroscopy, we demonstrated that the oxygen-containing moieties generated within photooxidized A2E include a 5,8-monofuranoid and a cyclic 5,8-monoperoxide. We have shown that the oxidation sites can be assigned to the shorter arm of A2E, to the longer arm, or to both arms by analyzing changes in the UV-visible spectrum of A2E, and we have observed a preference for oxidation on the shorter arm. By liquid chromatography-mass spectrometry, we have also detected both monofuran-A2E and monoperoxy-A2E in aged human RPE and in eye cups of Abca4/Abcr-/- mice, a model of Stargardt disease. Because the cytotoxicity of endoperoxide moieties is well known, the production of endoperoxide-containing oxo-A2E may account, at least in part, for cellular damage ensuing from A2E photooxidation.

Light‐induced Damage to the Retina: Role of Rhodopsin Chromophore Revisited

The presence of the regenerable visual pigment rhodopsin has been shown to be primarily responsible for the acute photodamage to the retina. The photoexcitation of rhodopsin leads to isomerization of its chromophore 11-cis-retinal to all-trans-retinal (ATR). ATR is a potent photosensitizer and its role in mediating photodamage has been suspected for over two decades. However, there was lack of experimental evidence that free ATR exists in the retina in sufficient concentrations to impose a risk of photosensitized damage. Identification in the retina of a retinal dimer and a pyridinium bisretinoid, so called A2E, and determination of its biosynthetic pathway indicate that substantial amounts of ATR do accumulate in the retina. Both light damage and A2E accumulation are facilitated under conditions where efficient retinoid cycle operates. Efficient retinoid cycle leads to rapid regeneration of rhodopsin, which may result in ATR release from the opsin "exit site" before its enzymatic reduction to all-trans-retinol. Here we discuss photodamage to the retina where ATR could play a role as the main toxic and/or phototoxic agent. Moreover, we discuss secondary products of (photo)toxic properties accumulating within retinal lipofuscin as a result of ATR accumulation.

Anthocyanins Protect Against A2E Photooxidation and Membrane Permeabilization in Retinal Pigment Epithelial Cells¶

ABSTRACTThe pyridinium bisretinoid A2E, an autofluorescent pigment that accumulates in retinal pigment epithelial cells with age and in some retinal disorders, can mediate a detergent‐like perturbation of cell membranes and light‐induced damage to the cell. The photodynamic events initiated by the sensitization of A2E include the generation of singlet oxygen and the oxidation of A2E at carbon‐carbon double bonds. To assess the ability of plant‐derived anthocyanins to modulate adverse effects of A2E accumulation on retinal pigment epithelium (RPE) cells, these flavylium salts were isolated from extracts of bilberry. Nine anthocyanin fractions reflecting monoglycosides of delphinidin, cyanidin, petunidin and malvidin were obtained and all were shown to suppress the photooxidation of A2E at least in part by quenching singlet oxygen. The anthocyanins tested exhibited antioxidant activity of variable efficiency. The structural characteristics relevant to this variability likely included the ability to form a stable quinonoidal anhydro base at neutral pH, a conjugated diene structure in the C (pyrane) ring, the presence of hydroxyl groups on the B (benzene) ring and the relative hydrophobicity conferred by the arrangement of substituents on the B ring. Cells that had taken up anthocyanins also exhibited a resistance to the membrane permeabilization that occurs as a result of the detergent‐like action of A2E.

Anthocyanins protect against A2E photooxidation and membrane permeabilization in retinal pigment epithelial cells.

The pyridinium bisretinoid A2E, an autofluorescent pigment that accumulates in retinal pigment epithelial cells with age and in some retinal disorders, can mediate a detergent-like perturbation of cell membranes and light-induced damage to the cell. The photodynamic events initiated by the sensitization of A2E include the generation of singlet oxygen and the oxidation of A2E at carbon-carbon double bonds. To assess the ability of plant-derived anthocyanins to modulate adverse effects of A2E accumulation on retinal pigment epithelium (RPE) cells, these flavylium salts were isolated from extracts of bilberry. Nine anthocyanin fractions reflecting monoglycosides of delphinidin, cyanidin, petunidin and malvidin were obtained and all were shown to suppress the photooxidation of A2E at least in part by quenching singlet oxygen. The anthocyanins tested exhibited antioxidant activity of variable efficiency. The structural characteristics relevant to this variability likely included the ability to form a stable quinonoidal anhydro base at neutral pH, a conjugated diene structure in the C (pyrane) ring, the presence of hydroxyl groups on the B (benzene) ring and the relative hydrophobicity conferred by the arrangement of substituents on the B ring. Cells that had taken up anthocyanins also exhibited a resistance to the membrane permeabilization that occurs as a result of the detergent-like action of A2E.

Rpe65 Leu450Met variant is associated with reduced levels of the retinal pigment epithelium lipofuscin fluorophores A2E and iso-A2E.

There is a growing body of evidence that the nondegradable fluorophores that accumulate as the lipofuscin of retinal pigment epithelium (RPE) are involved in mechanisms leading to the degeneration of RPE in macular degeneration. Most of the constituents of RPE lipofuscin are inadvertent products of the retinoid visual cycle, the enzymatic pathway by which the 11-cis-retinal chromophore of rhodopsin is generated. Indeed, a major constituent of RPE lipofuscin, the pyridinium bisretinoid A2E, is a diretinal conjugate that forms in photoreceptor cells and is deposited in RPE cells as a consequence of the phagocytosis of the outer segment membrane by RPE cells. Given the adverse effects of A2E, there is considerable interest in combating its deposition so as to protect against vision loss. These efforts, however, necessitate an understanding of factors that modulate its formation. Here we show that an amino acid variant in murine Rpe65, a visual-cycle protein required for the regeneration of 11-cis-retinal, is associated with reduced A2E accumulation.

P4-220 β-amyloid-induced inflammatory gene expression in human brain endothelial cells

Polyphenols exert beneficial effects on vision. We hypothesized that polyphenol components of Vaccinium uliginosum L. (V.U.) extract protect retinal pigment epithelial (RPE) cells against blue light–induced damage. Our aim was to test extracts containing polyphenol components to ascertain effects to reduce damage against blue light in RPEs. We measured the activity in fractions eluted from water, ethanol, and HP20 resin (FH), and found that the FH fraction had the highest beneficial activity. We isolated the individual active compounds from the FH fraction using chromatographic techniques, and found that FH contained flavonoids, anthocyanins, phenyl propanoids, and iridoids. Cell cultures of A2E-laden ARPE-19 exposed to blue light after treatment with V.U. extract fractions and their individual constituents indicated improvement. V uliginosum L extract fractions and constituent compounds significantly reduced A2E photo-oxidation–induced RPE cell death and inhibited intracellular A2E accumulation. Furthermore, Balb/c male mice were exposed to blue light at 10000 lux for 1 h/d for 2 weeks to induce retinal damage. One week after the final blue light exposure, retinal damage evaluated revealed that the outer nuclear layer thickness and nuclei count were improved. Histologic examination of murine photoreceptor cells demonstrated that FH, rich in polyphenols, inhibited the loss of outer nuclear layer thickness and nuclei. Our findings suggest that V.U. extract and eluted fractions are a potential source of bioactive compounds that potentially serve a therapeutic approach for age-related macular degeneration.

Lipofuscin and macular degeneration

The accumulation of the autofluorescent pigment lipofuscin in the retina that occurs with aging has been explained as a side effect of the visual cycle. It occurs when two molecules of all-trans-retinal condense with one molecule of phosphatidylethanolamine in the discs of the rod outer segments, and is followed by uptake into retinal pigment epithelium (RPE) and conversion to the stable A2E, a pyridinium bisretinoid that is toxic to RPE cells. The accumulation of A2E, the major component of lipofuscin causes RPE cell apoptosis, thereby explaining age-related macular degeneration and macular degeneration characteristic of Stargardt disease. The drug isotretinoin (13-cis-retinoic acid) prevents accumulation of A2E in mice by slowing down the visual cycle and might therefore be used to prevent macular degeneration.

Light exposure stimulates formation of A2E oxiranes in a mouse model of Stargardt's macular degeneration.

Recessive Stargardt's macular degeneration is a blinding disease of children caused by mutations in the ABCA4 (ABCR) gene. Mice with a knockout mutation in abcr accumulate toxic lipofuscin pigments in ocular tissues, similar to affected humans. The major fluorophore of lipofuscin is the bis-retinoid, N-retinylidene-N-retinylethanolamine (A2E). In the current study, we sought to define the effect of increasing light on A2E accumulation. We crossed the abcr(-/-) mutation onto an albino background. The retinoid profiles in albino mice indicated higher retinal illuminance than in pigmented mice exposed to similar ambient light. Unexpectedly, A2E levels were not higher in the albino mice. Also, A2E levels in abcr(-/-) mice reared under cyclic light at 30, 120, or 1,700 lux were similar. Thus, increased retinal illuminance was not correlated with higher A2E. A2E has been shown to undergo light-dependent oxidation to yield a series of A2E epoxides or oxiranes. These oxiranes react with DNA in vitro, suggesting a potential mechanism for A2E cytotoxicity. We analyzed ocular tissues from abcr(-/-) mice for A2E oxiranes by mass spectrometry. Unlike A2E, the oxiranes were more abundant in albino vs. pigmented abcr(-/-) mice, and in abcr(-/-) mice exposed to increasing ambient light. These observations suggest that both the biosynthesis of A2E and its conversion to oxiranes are accelerated by light. Finally, we showed that the formation of A2E oxiranes is strongly suppressed by treating the abcr(-/-) mice with Accutane (isotretinoin), an inhibitor of rhodopsin regeneration.