retinaldehyde and Macular-Degeneration

Retinaldehyde adducts (bisretinoids) accumulate in retinal pigment epithelial (RPE) cells as lipofuscin. Bisretinoids are implicated in some inherited and age-related forms of macular degeneration that lead to the death of RPE cells and diminished vision. By comparing albino and black-eyed mice and by rearing mice in darkness and in cyclic light, evidence indicates that bisretinoid fluorophores undergo photodegradation in the eye (Ueda et al. Proc Natl Acad Sci 113:6904-6909, 2016). Given that the photodegradation products modify and impair cellular and extracellular molecules, these processes likely impart cumulative damage to retina.

Topics: Albinism; Amines; Animals; Antioxidants; ATP-Binding Cassette Transporters; Darkness; Eye Color; Free Radical Scavengers; Light; Lipofuscin; Macular Degeneration; Melanosis; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Photochemistry; Retinal Pigment Epithelium; Retinaldehyde; Rod Cell Outer Segment; Stargardt Disease; Vitamin E

To test the hypothesis that delayed dark adaptation in patients with macular degeneration is due to an excess of free unliganded opsin (apo-opsin) and a deficiency of the visual chromophore, 11. A total of 50 human autopsy eyes were harvested from donors with and without macular degeneration within 2-24 hrs. postmortem. Protocols were developed which permitted dark adaptation of normal human eyes after death and enucleation. Biochemical methods of purifying rod outer segments were optimized and the concentration of rhodopsin and apo-opsin was measured with UV-visible scanning spectroscopy. The presence of apo-opsin was calculated by measuring the difference in the rhodopsin absorption spectra before and after the addition of 11. A total of 20 normal eyes and 16 eyes from donors with early, intermediate and advanced stages of macular degeneration were included in the final analysis. Dark adaptation was achieved by harvesting whole globes in low light, transferring into dark (light-proof) canisters and dissecting the globes using infrared light and image converters for visualization. Apo-opsin was readily detected in positive controls after the addition of 11. Methods have been developed to study dark adaptation in human autopsy eyes. Eyes with age-related macular degeneration do not show a deficiency of 11

Topics: Consensus; Dark Adaptation; Humans; Macular Degeneration; Ophthalmology; Opsins; Retinaldehyde; Rod Cell Outer Segment; Societies, Medical

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, and the prevalence of the disease increases exponentially with every decade after age 50 years. It is a multifactorial disease involving a complex interplay of genetic, environmental, metabolic, and functional factors. Besides smoking, hypertension, obesity, and certain dietary habits, a growing body of evidence indicates that inflammation and the immune system may play a key role in the development of the disease. AMD may progress from the early form to the intermediate form and then to the advanced form, where two subtypes exist: the nonneovascular (dry) type and the neovascular (wet) type. The results from the Age-Related Eye Disease Study have shown that for the nonneovascular type of AMD, supplementation with high-dose antioxidants (vitamin C, vitamin E, and β-carotene) and zinc is recommended for those with the intermediate form of AMD in one or both eyes or with advanced AMD or vision loss due to AMD in one eye. As for the neovascular type of the advanced AMD, the current standard of therapy is intravitreal injections of vascular endothelial growth factor inhibitors. In addition, lifestyle and dietary modifications including improved physical activity, reduced daily sodium intake, and reduced intake of solid fats, added sugars, cholesterol, and refined grain foods are recommended. To date, no study has demonstrated that AMD can be cured or effectively prevented. Clearly, more research is needed to fully understand the pathophysiology as well as to develop prevention and treatment strategies for this devastating disease.

Topics: Aged; Aged, 80 and over; Aging; Dietary Supplements; Drug Therapy, Combination; Female; Genetic Therapy; Humans; Injections; Life Style; Macular Degeneration; Male; Middle Aged; Randomized Controlled Trials as Topic; Retinaldehyde; Risk Factors; Vascular Endothelial Growth Factor A; Visual Acuity; Vitamins; Vitreous Body; Zinc

The retina exhibits an inherent autofluorescence that is imaged ophthalmoscopically as fundus autofluorescence. In clinical settings, fundus autofluorescence examination aids in the diagnosis and follow-up of many retinal disorders. Fundus autofluorescence originates from the complex mixture of bisretinoid fluorophores that are amassed by retinal pigment epithelial (RPE) cells as lipofuscin. Unlike the lipofuscin found in other cell-types, this material does not form as a result of oxidative stress. Rather, the formation is attributable to non-enzymatic reactions of vitamin A aldehyde in photoreceptor cells; transfer to RPE occurs upon phagocytosis of photoreceptor outer segments. These fluorescent pigments accumulate even in healthy photoreceptor cells and are generated as a consequence of the light capturing function of the cells. Nevertheless, the formation of this material is accelerated in some retinal disorders including recessive Stargardt disease and ELOVL4-related retinal degeneration. As such, these bisretinoid side-products are implicated in the disease processes that threaten vision. In this article, we review our current understanding of the composition of RPE lipofuscin, the structural characteristics of the various bisretinoids, their related spectroscopic features and the biosynthetic pathways by which they form. We will revisit factors known to influence the extent of the accumulation and therapeutic strategies being used to limit bisretinoid formation. Given their origin from vitamin A aldehyde, an isomer of the visual pigment chromophore, it is not surprising that the bisretinoids of retina are light sensitive molecules. Accordingly, we will discuss recent findings that implicate the photodegradation of bisretinoid in the etiology of age-related macular degeneration.

Topics: Animals; Cattle; Fluorescence; Humans; Lipofuscin; Macular Degeneration; Mice; Molecular Structure; Rats; Retinal Pigment Epithelium; Retinaldehyde; Retinoids; Spectrometry, Fluorescence

Genetic association studies and investigations of the constituents of subretinal deposits (drusen) have implicated complement dysregulation as one factor predisposing individuals to increased risk of age-related macular degeneration (AMD). Here we review evidence that molecular fragments released by photooxidation of the bisretinoids of retinal pigment epithelial lipofuscin, can activate complement. Complement activation by this mechanism is dependent on the alternative pathway. The diretinal conjugates comprising RPE lipofuscin accumulate in the cells throughout the lifetime of an individual. As such, these photooxidative processes, in a setting of complement dysregulation could contribute to chronic inflammation underlying AMD pathogenesis.

Topics: C-Reactive Protein; Complement Activation; Complement Factor B; Humans; Lipofuscin; Macular Degeneration; Oxidation-Reduction; Peptides, Cyclic; Pyridinium Compounds; Retinal Pigment Epithelium; Retinaldehyde; Retinoids

The central retina mediates high acuity vision, and its progressive dysfunction due to macular degeneration is the leading cause of visual disability among adults in industrialized societies. Here, we summarize recent progress in understanding the pathophysiology of macular degeneration and the implications of this new knowledge for treatment and prevention. The past decade has witnessed remarkable advances in this field, including the development of new, non-invasive retinal imaging technologies, the development of animal models for macular disease, and the isolation of many of the genes responsible for both early- and late-onset macular diseases. These advances have set the stage for the development of effective mechanism-based therapies.

Topics: Aging; Animals; Genetic Predisposition to Disease; Humans; Laser Therapy; Macular Degeneration; Nerve Tissue Proteins; Photoreceptor Cells, Vertebrate; Pigment Epithelium of Eye; Retina; Retinal Artery; Retinaldehyde

Clarification of the function of bestrophin, the gene product of VMD2, establishes a basis for the understanding of the pathomechanisms leading to Best's vitelliform macular degeneration. Studies of heterologously expressed bestrophin showed that bestrophin can function as a Cl(-) channel. All four known bestrophins were found to display Cl(-) channel activity. A loss in Cl(-) channel function would elegantly explain the development of the leading symptom for Best's disease, the reduction of the light peak amplitude in the patient's electro-oculogram. However, there are still gaps in the chain of evidence demonstrating that bestrophin is a Cl(-) channel, and this hypothesis is inconsistent with newly published follow-up observations. In an alternative hypothesis bestrophin appears as a regulator of voltage-dependent Ca(2+) channels assuming an indirect involvement of bestrophin in the generation of the light peak. Further studies on either bestrophin-deficient mice or transgenic mice will show that either one of the hypotheses is right or maybe both will be proven correct, showing bestrophin as a Cl(-) channel and Ca(2+) channel regulator.

Topics: Bestrophins; Biomarkers; Calcium Channels; Chloride Channels; Corneal Dystrophies, Hereditary; Eye Proteins; Humans; Ion Channel Gating; Macular Degeneration; Models, Biological; Retinaldehyde; Structure-Activity Relationship

A substantial portion of the lipofuscin that accumulates with age and in some retinal disorders in retinal pigment epithelial (RPE) cells, forms as a consequence of light-related vitamin A recycling. Major constituents of RPE lipofuscin are the di-retinal conjugate A2E and its photoisomers. That the accretion of A2E has consequences for the cell, with the adverse effects of A2E being attributable to its amphiphilic structure and its photoreactivity, is consistent with evidence of an association between atrophic age-related macular degeneration (AMD) and excessive lipofuscin accumulation.

Topics: Atrophy; Humans; Light; Lipofuscin; Macular Degeneration; Pigment Epithelium of Eye; Pyridinium Compounds; Retina; Retinaldehyde; Retinoids; Vitamin A

The visual cycle refers to a series of biochemical reactions of retinoids in ocular tissues and supports the vision in vertebrates. The visual cycle regenerates visual pigments chromophore, 11-cis-retinal, and eliminates its toxic byproducts from the retina, supporting visual function and retinal neuron survival. Unfortunately, during the visual cycle, when 11-cis-retinal is being regenerated in the retina, toxic byproducts, such as all-trans-retinal and bis-retinoid is N-retinylidene-N-retinylethanolamine (A2E), are produced, which are proposed to contribute to the pathogenesis of the dry form of age-related macular degeneration (AMD). The primary biochemical defect in Stargardt disease (STGD1) is the accelerated synthesis of cytotoxic lipofuscin bisretinoids, such as A2E, in the retinal pigment epithelium (RPE) due to mutations in the ABCA4 gene. To prevent all-trans-retinal-and bisretinoid-mediated retinal degeneration, slowing down the retinoid flow by modulating the visual cycle with a small molecule has been proposed as a therapeutic strategy. The present study describes RPE65-61, a novel, non-retinoid compound, as an inhibitor of RPE65 (a key enzyme in the visual cycle), intended to modulate the excessive activity of the visual cycle to protect the retina from harm degenerative diseases. Our data demonstrated that (±)-RPE65-61 selectively inhibited retinoid isomerase activity of RPE65, with an IC50 of 80 nM. Furthermore, (±)-RPE65-61 inhibited RPE65 via an uncompetitive mechanism. Systemic administration of (±)-RPE65-61 in mice resulted in slower chromophore regeneration after light bleach, confirming in vivo target engagement and visual cycle modulation. Concomitant protection of the mouse retina from high-intensity light damage was also observed. Furthermore, RPE65-61 down-regulated the cyclic GMP-AMP synthase stimulator of interferon genes (cGAS-STING) pathway, decreased the inflammatory factor, and attenuated retinal apoptosis caused by light-induced retinal damage (LIRD), which led to the preservation of the retinal function. Taken together, (±)-RPE65-61 is a potent visual cycle modulator that may provide a neuroprotective therapeutic benefit for patients with STGD and AMD.

Topics: Animals; ATP-Binding Cassette Transporters; cis-trans-Isomerases; Interferons; Lipofuscin; Macular Degeneration; Mice; Nucleotidyltransferases; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinal Pigments; Retinaldehyde; Retinoids

Oxidative stress-caused damage to the retinal pigment epithelium (RPE) underlies the onset and progression of age-related macular degeneration (AMD). Impaired mitochondrial biogenesis sensitizes RPE cells to mitochondrial dysfunction, energy insufficiency and death. Src-homology 2 domain-containing phosphatase (SHP)-1 is important in regulating immune responses and cell survival. However, its roles in cell survival are not always consistent. Until now, the effects of SHP-1 on RPE dysfunction, especially mitochondrial homeostasis, remain to be elucidated. We sought to clarify the effects of SHP-1 in RPE cells in response to atRAL-induced oxidative stress and determine the regulatory mechanisms involved.. In the all trans retinal (atRAL)-induced oxidative stress model, we used the vector of lentivirus to knockdown the expression of SHP-1 in ARPE-19 cells. CCK-8 assay, Annexin V/PI staining and JC-1 staining were utilized to determine the cell viability, cell apoptosis and mitochondrial membrane potential. We also used immunoprecipitation to examine the ubiquitination modification of stimulator of interferon genes (STING) and its interaction with SHP-1. The expression levels of mitochondrial marker, proteins related to mitochondrial biogenesis, and signaling molecules involved were examined by western blotting analysis.. We found that SHP-1 knockdown predisposed RPE cells to apoptosis, aggravated mitochondrial damage, and repressed mitochondrial biogenesis after treatment with atRAL. Immunofluoresent staining and immunoprecipitation analysis confirmed that SHP-1 interacted with the endoplasmic reticulum-resident STING and suppressed K63-linked ubiquitination and activation of STING. Inhibition of STING with the specific antagonist H151 attenuated the effects of SHP-1 knockdown on mitochondrial biogenesis and oxidative damage. The adenosine monophosphate-activated protein kinase (AMPK) pathway acted as the crucial downstream target of STING and was involved in the regulatory processes.. These findings suggest that SHP-1 knockdown potentiates STING overactivation and represses mitochondrial biogenesis and cell survival, at least in part by blocking the AMPK pathway in RPE cells. Therefore, restoring mitochondrial health by regulating SHP-1 in RPE cells may be a potential therapeutic strategy for degenerative retinal diseases including AMD.

Topics: Adenosine Monophosphate; AMP-Activated Protein Kinases; Annexin A5; Apoptosis; Humans; Interferons; Macular Degeneration; Mitochondria; Organelle Biogenesis; Oxidative Stress; Phosphoric Monoester Hydrolases; Reactive Oxygen Species; Retinal Pigment Epithelium; Retinaldehyde

Bis-retinoids are a major component of lipofuscin that accumulates in the retinal pigment epithelium (RPE) in aging and age-related macular degeneration (AMD). Although bis-retinoids are known to originate from retinaldehydes required for the light response of photoreceptor cells, the relative contributions of the chromophore, 11-cis retinal, and photoisomerization product, all-trans retinal, are unknown. In photoreceptor outer segments, all-trans retinal, but not 11-cis retinal, is reduced by retinol dehydrogenase 8 (RDH8). Using Rdh8-/- mice, we evaluated the contribution of increased all-trans retinal to the formation and stability of RPE lipofuscin.. Rdh8-/- mice were reared in cyclic-light or darkness for up to 6 months, with selected light-reared cohorts switched to dark-rearing for the final 1 to 8 weeks. The bis-retinoid A2E was measured from chloroform-methanol extracts of RPE-choroid using HPLC-UV/VIS spectroscopy. Lipofuscin fluorescence was measured from whole flattened eyecups (excitation, 488 nm; emission, 565-725 nm).. Cyclic-light-reared Rdh8-/- mice accumulated A2E and RPE lipofuscin approximately 1.5 times and approximately 2 times faster, respectively, than dark-reared mice. Moving Rdh8-/- mice from cyclic-light to darkness resulted in A2E levels less than expected to have accumulated before the move.. Our findings establish that elevated levels of all-trans retinal present in cyclic-light-reared Rdh8-/- mice, which remain low in wild-type mice, contribute only modestly to RPE lipofuscin formation and accumulation. Furthermore, decreases in A2E levels occurring after moving cyclic-light-reared Rdh8-/- mice to darkness are consistent with processing of A2E within the RPE and the existence of a mechanism that could be a therapeutic target for controlling A2E cytotoxicity.

Topics: Animals; Chromatography, High Pressure Liquid; Disease Models, Animal; Female; Lipofuscin; Macular Degeneration; Male; Mice; Retinal Pigment Epithelium; Retinaldehyde; Retinoids

Continuous regeneration of the 11-

Topics: Alcohol Oxidoreductases; Animals; ATP-Binding Cassette Transporters; Diterpenes; Light; Macular Degeneration; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Opsins; Protective Agents; Retina; Retinal Degeneration; Retinaldehyde; Retinoids

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the

Topics: Animals; ATP-Binding Cassette Transporters; c-Mer Tyrosine Kinase; Cells, Cultured; Disease Models, Animal; Lipofuscin; Lysosomes; Macular Degeneration; Mice; Mice, Inbred BALB C; Mice, Knockout; Phagocytosis; Photoreceptor Cells; Retina; Retinal Degeneration; Retinal Pigment Epithelium; Retinaldehyde; Rhodopsin; Stargardt Disease

Although currently available treatment options for age-related macular degeneration (AMD) are limited, particularly for atrophic AMD, the identification of predisposing genetic variations has informed clinical studies addressing therapeutic options such as complement inhibitors and anti-inflammatory agents. To lower risk of early AMD, recommended lifestyle interventions such as the avoidance of smoking and the intake of low glycemic antioxidant-rich diets have largely followed from the identification of nongenetic modifiable factors. On the other hand, the challenge of understanding the complex relationship between aging and cumulative damage leading to AMD has fueled investigations of the visual cycle adducts that accumulate in retinal pigment epithelial (RPE) cells and are a hallmark of aging retina. These studies have revealed properties of these compounds that provide insights into processes that may compromise RPE and could contribute to disease mechanisms in AMD. This work has also led to the design of targeted therapeutics that are currently under investigation.

Topics: Animals; Humans; Macular Degeneration; Molecular Targeted Therapy; Retinal Pigment Epithelium; Retinaldehyde; Risk Factors

Effective clearance of all-trans-retinal (atRAL) from retinal pigment epithelial (RPE) cells is important for avoiding its cytotoxicity. However, the metabolism of atRAL in RPE cells is poorly clarified. The present study was designed to analyze metabolic products of atRAL and to compare the cytotoxicity of atRAL versus its derivative all-trans-retinal dimer (atRAL-dimer) in human RPE cells. We found that all-trans-retinol (atROL) and a mixture of atRAL condensation metabolites including atRAL-dimer and A2E were generated after incubating RPE cells with atRAL for 6h, and the amount of atRAL-dimer was significantly higher than that of A2E. In the eyes of Rdh8

Topics: Alcohol Oxidoreductases; Animals; ATP-Binding Cassette Transporters; Cell Line; Cell Survival; DNA, Complementary; Epithelial Cells; Humans; Macular Degeneration; Membrane Potential, Mitochondrial; Mice; Mice, Knockout; Mitochondria; Oxidative Stress; Reactive Oxygen Species; Retinal Pigment Epithelium; Retinaldehyde

Emixustat is a visual cycle modulator that has entered clinical trials as a treatment for age-related macular degeneration (AMD). This molecule has been proposed to inhibit the visual cycle isomerase RPE65, thereby slowing regeneration of 11-cis-retinal and reducing production of retinaldehyde condensation byproducts that may be involved in AMD pathology. Previously, we reported that all-trans-retinal (atRAL) is directly cytotoxic and that certain primary amine compounds that transiently sequester atRAL via Schiff base formation ameliorate retinal degeneration. Here, we have shown that emixustat stereoselectively inhibits RPE65 by direct active site binding. However, we detected the presence of emixustat-atRAL Schiff base conjugates, indicating that emixustat also acts as a retinal scavenger, which may contribute to its therapeutic effects. Using agents that lack either RPE65 inhibitory activity or the capacity to sequester atRAL, we assessed the relative importance of these 2 modes of action in protection against retinal phototoxicity in mice. The atRAL sequestrant QEA-B-001-NH2 conferred protection against phototoxicity without inhibiting RPE65, whereas an emixustat derivative incapable of atRAL sequestration was minimally protective, despite direct inhibition of RPE65. These data indicate that atRAL sequestration is an essential mechanism underlying the protective effects of emixustat and related compounds against retinal phototoxicity. Moreover, atRAL sequestration should be considered in the design of next-generation visual cycle modulators.

Topics: Alcohol Oxidoreductases; Animals; ATP-Binding Cassette Transporters; Catalytic Domain; Cattle; cis-trans-Isomerases; Crystallography, X-Ray; Enzyme Inhibitors; Female; Free Radical Scavengers; Macular Degeneration; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Models, Molecular; Phenyl Ethers; Propanolamines; Retinal Degeneration; Retinaldehyde; Schiff Bases; Stereoisomerism

Age-related macular degeneration (AMD) is a neurodegenerative disease that causes adult-onset blindness. There are 2 forms of this progressive disease: wet and dry. Currently there is no cure for AMD, but several treatment options have started to emerge making early detection critical for therapeutic success. Analysis of the eyes of Abca4(-/-)Rdh8(-/-) mice that display light-induced retinal degeneration indicates that 11-cis-retinal and docosahexaenoic acid (DHA) levels were significantly decreased as compared with the eyes of control dark-adapted C57BL/6J mice. In addition, exposure to intense light correlated with higher levels of prostaglandin G2 in the eyes of Abca4(-/-)Rdh8(-/-) mice. Intense light exposure also lowered DHA levels in the eyes of wild-type C57BL/6J mice without discernible retinal degeneration. Analysis of human serum from patients with AMD recapitulated these dysregulated DHA levels and revealed dysregulation of arachidonic acid (AA) levels as well (∼32% increase in patients with AMD compared with average levels in healthy individuals). From these observations, we then built a statistical model that included levels of DHA and AA from human serum. This model had a 74% probability of correctly identifying patients with AMD from controls. Addition of a genetic analysis for one of the most prevalent amino acid substitutions in the age-related maculopathy susceptibility 2 gene linked to AMD, Ala(69)→Ser, did not improve the statistical model. Thus, we have characterized a reliable method with the potential to detect AMD without a genetic component, paving the way for a larger-scale clinical evaluation. Our studies on mouse models along with the analysis of human serum suggest that our small molecule-based model may serve as an effective tool to estimate the risk of developing AMD.

Topics: Adult; Aged; Alcohol Oxidoreductases; Amino Acid Substitution; Animals; ATP-Binding Cassette Transporters; Docosahexaenoic Acids; Female; Humans; Macular Degeneration; Male; Mice; Mice, Knockout; Middle Aged; Models, Biological; Mutation, Missense; Retinaldehyde

Gene and drug therapies are being developed to alleviate vision loss in patients with Stargardt's disease and age-related macular degeneration (AMD). To evaluate the therapeutic effects of these treatments, organic solvents are routinely used to extract and quantify bisretinoid lipofuscin constituents, such as N-retinylidene-N-retinyl-ethanolamine (A2E) and all-trans-retinal dimer (ATR-dimer). By high-performance liquid chromatography (HPLC), we found that A2E and ATR-dimer were both altered by tetrahydrofuran (THF) and chloroform, but were stable in dimethyl sulfoxide (DMSO) or methanol (MeOH). In addition, cyclohexane and ethanol (EtOH) did not alter ATR-dimer, whereas an alteration of A2E occurred in EtOH. On the basis of these findings, we designed processes II-IV, generated by modifications of process I, a routine method to measure bisretinoid compounds in vivo. Extra amounts of either ATR-dimer or A2E in mouse eyecups were released by processes II-IV versus process I. Efforts to clarify the effects of organic solvents on lipofuscin pigments are important because such studies can guide the handling of these fluorophores in related experiments.

Topics: Animals; Chromatography, High Pressure Liquid; Lipofuscin; Macular Degeneration; Mice; Mice, Inbred C57BL; Pigment Epithelium of Eye; Retinaldehyde; Solvents; Stargardt Disease

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by delayed clearance of all-trans-retinal from the retina, and in a retinitis pigmentosa mouse model with impaired retinal pigment epithelium (RPE) phagocytosis. Mouse microglia displayed RPE cytotoxicity and increased production of inflammatory chemokines/cytokines, Ccl2, Il1b, and Tnf, after coincubation with ligands that activate innate immunity. Notably, phagocytosis of photoreceptor proteins increased the activation of microglia/macrophages and RPE cells isolated from model mice as well as wild-type mice. The mRNA levels of Tlr2 and Tlr4, which can recognize proteins as their ligands, were elevated in mice with retinal degeneration. Bone marrow-derived macrophages from Tlr4-deficient mice did not increase Ccl2 after coincubation with photoreceptor proteins. Tlr4(-/-)Abca4(-/-)Rdh8(-/-) mice displayed milder retinal degenerative phenotypes than Abca4(-/-)Rdh8(-/-) mice. Additionally, inactivation of microglia/macrophages by pharmacological approaches attenuated mouse retinal degeneration. This study demonstrates an important contribution of TLR4-mediated microglial activation by endogenous photoreceptor proteins in retinal inflammation that aggravates retinal cell death. This pathway is likely to represent an underlying common pathology in degenerative retinal disorders.

Topics: Animals; Chemokine CCL2; Eye Proteins; Interleukin-1beta; Macular Degeneration; Mice; Mice, Knockout; Microglia; Photoreceptor Cells, Vertebrate; Retinaldehyde; Retinitis Pigmentosa; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Autosomal recessive Stargardt macular dystrophy is caused by mutations in the photoreceptor disc rim protein ABCA4/ABCR. Key clinical features of Stargardt disease include relatively mild rod defects such as delayed dark adaptation, coupled with severe cone defects reflected in macular atrophy and central vision loss. In spite of this clinical divergence, there has been no biochemical study of the effects of ABCA4 deficiency on cones vs. rods. Here we utilize the cone-dominant Abca4(-/-)/Nrl(-/-) double knockout mouse to study this issue. We show that as early as post-natal day (P) 30, Abca4(-/-)/Nrl(-/-) retinas have significantly fewer rosettes than Abca4(+/+)/Nrl(-/-) retinas, a phenotype often associated with accelerated degeneration. Abca4-deficient mice in both the wild-type and cone-dominant background accumulate more of the toxic bisretinoid A2E than their ABCA4-competent counterparts, but Abca4(-/-)/Nrl(-/-) eyes generate significantly more A2E per mole of 11-cis-retinal (11-cisRAL) than Abca4(-/-) eyes. At P120, Abca4(-/-)/Nrl(-/-) produced 340 ± 121 pmoles A2E/nmol 11-cisRAL while Abca4(-/-) produced 50.4 ± 8.05 pmoles A2E/nmol 11-cisRAL. Nevertheless, the retinal pigment epithelium (RPE) of Abca4(-/-)/Nrl(-/-) eyes exhibits fewer lipofuscin granules than the RPE of Abca4(-/-) eyes; at P120: Abca4(-/-)/Nrl(-/-) exhibit 0.045 ± 0.013 lipofuscingranules/μm² of RPE vs. Abca4(-/-) 0.17 ± 0.030 lipofuscingranules/μm² of RPE. These data indicate that ABCA4-deficient cones simultaneously generate more A2E than rods and are less able to effectively clear it, and suggest that primary cone toxicity may contribute to Stargardt's-associated macular vision loss in addition to cone death secondary to RPE atrophy.

Topics: Analysis of Variance; Animals; ATP-Binding Cassette Transporters; Basic-Leucine Zipper Transcription Factors; Blindness; Dark Adaptation; Eye Proteins; Humans; Lipofuscin; Macular Degeneration; Mice; Mice, Knockout; Microscopy, Electron; Pyridinium Compounds; Retina; Retinal Cone Photoreceptor Cells; Retinal Pigment Epithelium; Retinal Rod Photoreceptor Cells; Retinaldehyde; Retinoids; Vision, Ocular

Compromised clearance of all-trans-retinal (atRAL), a component of the retinoid cycle, increases the susceptibility of mouse retina to acute light-induced photoreceptor degeneration. Abca4(-/-)Rdh8(-/-) mice featuring defective atRAL clearance were used to examine the one or more underlying molecular mechanisms, because exposure to intense light causes severe photoreceptor degeneration in these animals. Here we report that bright light exposure of Abca4(-/-)Rdh8(-/-) mice increased atRAL levels in the retina that induced rapid NADPH oxidase-mediated overproduction of intracellular reactive oxygen species (ROS). Moreover, such ROS generation was inhibited by blocking phospholipase C and inositol 1,4,5-trisphosphate-induced Ca(2+) release, indicating that activation occurs upstream of NADPH oxidase-mediated ROS generation. Because multiple upstream G protein-coupled receptors can activate phospholipase C, we then tested the effects of antagonists of serotonin 2A (5-HT(2A)R) and M(3)-muscarinic (M(3)R) receptors and found they both protected Abca4(-/-)Rdh8(-/-) mouse retinas from light-induced degeneration. Thus, a cascade of signaling events appears to mediate the toxicity of atRAL in light-induced photoreceptor degeneration of Abca4(-/-)Rdh8(-/-) mice. A similar mechanism may be operative in human Stargardt disease and age-related macular degeneration.

Topics: Alcohol Oxidoreductases; Animals; ATP-Binding Cassette Transporters; Calcium; Corneal Dystrophies, Hereditary; Humans; Inositol 1,4,5-Trisphosphate; Light; Macular Degeneration; Mice; Mice, Knockout; NADPH Oxidases; Photoreceptor Cells, Vertebrate; Reactive Oxygen Species; Receptor, Muscarinic M3; Receptor, Serotonin, 5-HT2A; Retinaldehyde; Serotonin 5-HT2 Receptor Antagonists; Signal Transduction; Type C Phospholipases

The age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been associated with the development of retinal diseases, particularly age-related macular degeneration and Stargardt disease. A major component of lipofuscin is the bis-retinoid N-retinylidene-N-retinylethanolamine (A2E). The current model for the formation of A2E requires photoactivation of rhodopsin and subsequent release of all-trans-retinal. To understand the role of light exposure in the accumulation of lipofuscin and A2E, we analyzed RPEs and isolated rod photoreceptors from mice of different ages and strains, reared either in darkness or cyclic light. Lipofuscin levels were determined by fluorescence imaging, whereas A2E levels were quantified by HPLC and UV-visible absorption spectroscopy. The identity of A2E was confirmed by tandem mass spectrometry. Lipofuscin and A2E levels in the RPE increased with age and more so in the Stargardt model Abca4(-/-) than in the wild type strains 129/sv and C57Bl/6. For each strain, the levels of lipofuscin precursor fluorophores in dark-adapted rods and the levels and rates of increase of RPE lipofuscin and A2E were not different between dark-reared and cyclic light-reared animals. Both 11-cis- and all-trans-retinal generated lipofuscin-like fluorophores when added to metabolically compromised rod outer segments; however, it was only 11-cis-retinal that generated such fluorophores when added to metabolically intact rods. The results suggest that lipofuscin originates from the free 11-cis-retinal that is continuously supplied to the rod for rhodopsin regeneration and outer segment renewal. The physiological role of Abca4 may include the translocation of 11-cis-retinal complexes across the disk membrane.

Topics: Animals; ATP-Binding Cassette Transporters; Chromatography, High Pressure Liquid; Color; Light; Lipofuscin; Macular Degeneration; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Models, Biological; Retina; Retinal Pigment Epithelium; Retinaldehyde; Retinoids; Rod Cell Outer Segment

Aging of retinal pigment epithelial (RPE) cells of the eye is marked by accumulations of bisretinoid fluorophores; two of the compounds within this lipofuscin mixture are A2E and all-trans-retinal dimer. These pigments are implicated in pathological mechanisms involved in some vision-threatening disorders including age-related macular degeneration (AMD). Studies have shown that bisretinoids are photosensitive compounds that undergo photooxidation and photodegradation when irradiated with short wavelength visible light. Utilizing ultra performance liquid chromatography (UPLC) with electrospray ionization mass spectrometry (ESI-MS) we demonstrate that photodegradation of A2E and all-trans-retinal dimer generates the dicarbonyls glyoxal (GO) and methylglyoxal (MG), that are known to modify proteins by advanced glycation endproduct (AGE) formation. By extracellular trapping with aminoguanidine, we established that these oxo-aldehydes are released from irradiated A2E-containing RPE cells. Enzyme-linked immunosorbant assays (ELISA) revealed that the substrate underlying A2E-containing RPE was AGE-modified after irradiation. This AGE deposition was suppressed by prior treatment of the cells with aminoguanidine. AGE-modification causes structural and functional impairment of proteins. In chronic diseases such as diabetes and atherosclerosis, MG and GO modify proteins by non-enzymatic glycation and oxidation reactions. AGE-modified proteins are also components of drusen, the sub-RPE deposits that confer increased risk of AMD onset. These results indicate that photodegraded RPE bisretinoid is likely to be a previously unknown source of MG and GO in the eye.

Topics: Cells, Cultured; Chromatography, Liquid; Enzyme-Linked Immunosorbent Assay; Glycation End Products, Advanced; Glyoxal; Guanidines; Humans; Lipofuscin; Macular Degeneration; Phenylhydrazines; Pyridinium Compounds; Pyruvaldehyde; Retina; Retinal Drusen; Retinaldehyde; Retinoids; Spectrometry, Mass, Electrospray Ionization

The possible role of labile endogenous metabolites in the cause of various chronic debilitating diseases such as macular degeneration has not been adequately explored. In the metabolism of the various retinoids, namely retinal (vitamin A aldehyde), retinol (vitamin A alcohol) and retinoic acid, each has the potential for generating labile intermediates, such as their corresponding 5,6-epoxides by the action of various cytochrome P(450)s. Such retinoid epoxides may well have the capacity for acting as toxins upon the neurons in the macula unless they are rapidly hydrolyzed by epoxide hydrolases. Since the cytochrome P(450)s responsible for epoxide formation and the various epoxide hydrolases involved in their hydrolysis are determined genetically, this may serve to explain a genetic component being involved in the causation of age-related macular degeneration.

Topics: Biochemistry; Cytochrome P-450 Enzyme System; Epoxide Hydrolases; Epoxy Compounds; Humans; Hydrolysis; Macular Degeneration; Models, Chemical; Models, Genetic; Nervous System Diseases; Neurons; Oxidative Stress; Retinaldehyde; Vitamin A

Fluorescent bisretinoid compounds accumulate in retinal pigment epithelial (RPE) cells as a consequence of two processes: random reactions of vitamin A aldehyde in photoreceptor cell outer segments, and phagocytosis of discarded photoreceptor outer segment discs by RPE. The formation of bisretinoid is accentuated in some forms of retinal degeneration. The detection of a novel bisretinoid fluorophore that is a conjugate of all-trans-retinal and glycerophosphoethanolamine is reported.. Human RPE/choroid, eyes harvested from Abca4 (ATP-binding cassette transporter 4) null mutant mice, and biosynthetic reaction mixtures were analyzed by ultra performance liquid chromatography coupled to mass spectrometry and by nuclear magnetic resonance spectra and spectrofluorometry.. A fluorescent compound in mouse eyes and in human RPE/choroid corresponded to the product of the reaction between all-trans-retinal and glycerophosphoethanolamine (A2-GPE), as determined on the basis of molecular weight (m/z 746), absorbance (approximately 338,443 nm), and retention time. Nuclear magnetic resonance spectra were consistent with a pyridinium molecule with a glycerophosphate moiety. The emission maximum of A2-GPE was approximately 610 nm. A2-GPE accumulated with age in mouse eyes and was more abundant in Abca4(-/-) mice, a model of recessive Stargardt disease.. To date, several bisretinoids of RPE lipofuscin have been isolated and characterized, and for all of these, formation involves the membrane phospholipid phosphatidylethanolamine. Conversely, the bisretinoid A2-GPE is detected as sn-glycero-3-phosphoethanolamine (GPE) derivatized by two all-trans-retinal. The pathways by which A2-GPE may form under conditions of increased availability of all-trans-retinal, for instance in the Abca4(-/-) mouse, are discussed.

Topics: Animals; ATP-Binding Cassette Transporters; Chromatography, High Pressure Liquid; Disease Models, Animal; Diterpenes; Humans; Lipofuscin; Macular Degeneration; Magnetic Resonance Spectroscopy; Mass Spectrometry; Mice; Mice, Knockout; Phosphatidylethanolamines; Pyridinium Compounds; Retina; Retinal Pigment Epithelium; Retinaldehyde; Retinoids; Spectrometry, Fluorescence; Stargardt Disease; Vitamin A

Vertebrate vision is initiated by photoisomerization of the visual pigment chromophore 11-cis-retinal and is maintained by continuous regeneration of this retinoid through a series of reactions termed the retinoid cycle. However, toxic side reaction products, especially those involving reactive aldehyde groups of the photoisomerized product, all-trans-retinal, can cause severe retinal pathology. Here we lowered peak concentrations of free all-trans-retinal with primary amine-containing Food and Drug Administration (FDA)-approved drugs that did not inhibit chromophore regeneration in mouse models of retinal degeneration. Schiff base adducts between all-trans-retinal and these amines were identified by MS. Adducts were observed in mouse eyes only when an experimental drug protected the retina from degeneration in both short-term and long-term treatment experiments. This study demonstrates a molecular basis of all-trans-retinal-induced retinal pathology and identifies an assemblage of FDA-approved compounds with protective effects against this pathology in a mouse model that shows features of Stargardt's disease and age-related retinal degeneration.

Topics: Amines; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Macular Degeneration; Mice; Retinal Degeneration; Retinaldehyde; Schiff Bases; United States; United States Food and Drug Administration

To assess the safety and to quantify the effects of a single application of all-trans-N-retinylacetamide on the rat retina measured by electroretinography (ERG). Brown Norway rats were assigned to either a control group (n = 13) or to one of the three groups treated with a single intra-peritoneal dose of all-trans-N-retinylacetamide: 20 (n = 8), 5 (n = 7), or 1 mg/kg (n = 8). Full-field ERGs were performed 7 days before (baseline) and 12 h after treatment. Intensity-response relationship of b-wave amplitudes were evaluated in dark-adapted conditions using white stimuli (0.000003-0.3 cd.s/m(2)). Fast dynamics of rod sensitivity was assessed by a paired-flash paradigm; recovery dynamics of b-wave amplitudes after bleaching was followed for 70 min. Light-adapted ERGs were recorded for cone evaluation. No effects were found on either dark-adapted sensitivity or on fast rod recovery. However, drug treatment at 5 and 20 mg/kg significantly delayed ERG amplitude recovery after bleaching: 60 min after bleaching the b-wave amplitude was 21 + or - 9% (P < 0.05) and 66 + or - 10% (P < 0.05), respectively, compared to baseline. Recovery rates returned to normal 8 weeks after treatment. There were no changes in light-adapted ERG in any group. Systemic administration of a single dose of the visual cycle modulator all-trans-N-retinylacetamide reversibly delayed recovery of dark-adapted ERG amplitudes after bleaching, leaving other functions unchanged. This finding could make the compound potentially useful in experimental conditions or in specific diseases where the visual cycle is involved, such as retinitis pigmentosa or age-related macular degeneration.

Topics: Animals; Dark Adaptation; Dose-Response Relationship, Drug; Electroretinography; Female; Humans; Injections, Intraperitoneal; Macular Degeneration; Rats; Rats, Inbred BN; Retina; Retinaldehyde; Retinitis Pigmentosa; Retinoids; Time Factors; Visual Pathways

ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) transporters, which has been implicated in the clearance of all-trans retinal derivatives from rod and cone photoreceptor cells following photoexcitation as part of the visual cycle. Mutations in ABCA4 are known to cause Stargardt macular degeneration and related disorders, associated with a severe loss in vision. Recently, a solid-phase binding assay has been developed to identify retinoids that likely serve as substrates for this transporter. In this procedure, monoclonal antibodies directed either against an epitope within ABCA4 (Rim 3F4 antibody) or against the 9 amino acid 1D4 epitope tag engineered onto the C-terminus of expressed ABCA4 (Rho 1D4 antibody) are covalently bound to a Sepharose matrix. This immunoaffinity matrix is then used to isolate ABCA4 from photoreceptor outer segments or transfected cells. All-trans retinal is added to immobilized ABCA4 in the presence of a phospholipid mixture containing phosphatidylethanolamine. The bound retinoid is then analyzed directly by spectrophotometry or identified by HPLC and/or mass spectrometry following extraction with organic solvents. Using this procedure, it has been shown that unprotonated N-retinylidene-phosphatidylethanolamine binds with high affinity to ABCA4 and is released by the addition of ATP. These procedures and related radiometric assays using titrated retinal have been used to study the binding of N-retinylidene-PE to wild-type and mutant ABCA4 in the absence and presence of nucleotides for structure-function studies.

Topics: Amino Acid Sequence; Animals; ATP-Binding Cassette Transporters; Cattle; Cell Line; Chromatography, High Pressure Liquid; Humans; Macular Degeneration; Mutation; Photoreceptor Cells; Protein Binding; Recombinant Fusion Proteins; Retinaldehyde; Retinoids; Rod Cell Outer Segment; Scintillation Counting

Bisretinoid adducts accumulate as lipofuscin in retinal pigment epithelial (RPE) cells of the eye and are implicated in the pathology of inherited and age-related macular degeneration. Characterization of the bisretinoids A2E and the all-trans-retinal dimer series has shown that these pigments form from reactions in photoreceptor cell outer segments that involve all-trans-retinal, the product of photoisomerization of the visual chromophore 11-cis-retinal. Here we have identified two related but previously unknown RPE lipofuscin compounds. By high performance liquid chromatography-electrospray ionization-tandem mass spectrometry, we determined that the first of these compounds is a phosphatidyl-dihydropyridine bisretinoid; to indicate this structure and its formation from two vitamin A-aldehyde (A2), we will refer to it as A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE). The second pigment, A2-dihydropyridine-ethanolamine, forms from phosphate hydrolysis of A2-DHP-PE. The structure of A2-DHP-PE was corroborated by Fourier transform infrared spectroscopy, and density functional theory confirmed the presence of a dihydropyridine ring. This lipofuscin pigment is a fluorescent compound with absorbance maxima at approximately 490 and 330 nm, and it was identified in human, mouse, and bovine eyes. We found that A2-DHP-PE forms in reaction mixtures of all-trans-retinal and phosphatidylethanolamine, and in mouse eyecups we observed an age-related accumulation. As compared with wild-type mice, A2-DHP-PE is more abundant in mice with a null mutation in Abca4 (ATP-binding cassette transporter 4), the gene causative for recessive Stargardt macular degeneration. Efforts to clarify the composition of RPE lipofuscin are important because these compounds are targets of gene-based and drug therapies that aim to alleviate ABCA4-related retinal disease.

Topics: Age Factors; Animals; ATP-Binding Cassette Transporters; Cattle; Chromatography, High Pressure Liquid; Diterpenes; Humans; Lipofuscin; Macular Degeneration; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Molecular; Molecular Structure; Pigment Epithelium of Eye; Retina; Retinaldehyde; Spectroscopy, Fourier Transform Infrared; Tandem Mass Spectrometry; Vitamin A

The bis-retinoid pigments that accumulate in retinal pigment epithelial cells as lipofuscin are associated with inherited and age-related retinal disease. In addition to A2E and related cis isomers, we previously showed that condensation of two molecules of all-trans-retinal leads to the formation of a protonated Schiff base conjugate, all-trans-retinal dimer-phosphatidylethanolamine. Here we report the characterization of the related pigments, all-trans-retinal dimer-ethanolamine and unconjugated all-trans-retinal dimer, in human and mouse retinal pigment epithelium. In eyecups of Abcr(-/-) mice, a model of recessive Stargardt macular degeneration, all-trans-retinal dimer-phosphatidylethanolamine was increased relative to wild type and was more abundant than A2E. Total pigment of the all-trans-retinal dimer series (sum of all-trans-retinal dimer-phosphatidylethanolamine, all-trans-retinal dimer-ethanolamine, and all-trans-retinal dimer) increased with age in Abcr(-/-) mice and was modulated by amino acid variants in Rpe65. In in vitro assays, enzyme-mediated hydrolysis of all-trans-retinal dimer-phosphatidylethanolamine generated all-trans-retinal dimer-ethanolamine, and protonation/deprotonation of the Schiff base nitrogen of all-trans-retinal dimer-ethanolamine was pH-dependent. Unconjugated all-trans-retinal dimer was a more efficient generator of singlet oxygen than A2E, and the all-trans-retinal dimer series was more reactive with singlet oxygen than was A2E. By analyzing chromatographic properties and UV-visible spectra together with mass spectrometry, mono- and bis-oxygenated all-trans-retinal dimer photoproducts were detected in Abcr(-/-) mice. The latter findings are significant to an understanding of the adverse effects of retinal pigment epithelial cell lipofuscin.

Topics: Animals; ATP-Binding Cassette Transporters; Carrier Proteins; Chromatography, High Pressure Liquid; cis-trans-Isomerases; Eye Proteins; Humans; Lipofuscin; Macular Degeneration; Mice; Phosphatidylethanolamines; Pigment Epithelium of Eye; Pyridinium Compounds; Retinaldehyde; Retinoids; Singlet Oxygen

Recessive Stargardt's macular degeneration is an inherited blinding disease of children caused by mutations in the ABCR gene. The primary pathologic defect in Stargardt's discase is accumulation of toxic lipofuscin pigments such as N-retinylidene-N-retinylethanolamine (A2E) in cells of the retinal pigment epithelium (RPE). This accumulation appears to be responsible for the photoreceptor death and severe visual loss in Stargardt's patients. Here, we tested a novel therapeutic strategy to inhibit lipofuscin accumulation in a mouse model of recessive Stargardt's disease. Isotretinoin (Accutane) has been shown to slow the synthesis of 11-cis-retinaldehyde (11cRAL) and regeneration of rhodopsin by inhibiting 11-cis-retinol dehydrogenase (11cRDH) in the visual cycle. Light activation of rhodopsin results in its release of all-trans-retinaldehyde (atRAL), which constitutes the first reactant in A2E biosynthesis. Accordingly, we tested the effects of isotretinoin on lipofuscin accumulation in abcr-/- knockout mice. Isotretinoin blocked the formation of A2E biochemically and the accumulation of lipofuscin pigments by electron microscopy. We observed no significant visual loss in treated abcr-/- mice by electroretinography. Isotretinoin also blocked the slower, age-dependent accumulation of lipofuscin in wild-type mice. These results corroborate the proposed mechanism of A2E biogenesis. Further, they suggest that treatment with isotretinoin may inhibit lipofuscin accumulation and thus delay the onset of visual loss in Stargardt's patients. Finally, the results suggest that isotretinoin may be an effective treatment for other forms of retinal or macular degeneration associated with lipofuscin accumulation.

Topics: Animals; Disease Models, Animal; Enzyme Inhibitors; Esters; Isotretinoin; Lipofuscin; Macular Degeneration; Mice; Retinaldehyde; Time Factors

Topics: Adenosine Triphosphatases; Animals; ATP-Binding Cassette Transporters; Cattle; Chromatography, Affinity; Humans; In Situ Hybridization; Kinetics; Macaca; Macular Degeneration; Mice; Protein Structure, Secondary; Proteolipids; Rats; Retinal Rod Photoreceptor Cells; Retinaldehyde; RNA, Messenger; Rod Cell Outer Segment

Many substrates for P-glycoprotein, an ABC transporter that mediates multidrug resistance in mammalian cells, have been shown to stimulate its ATPase activity in vitro. In the present study, we used this property as a criterion to search for natural and artificial substrates and/or allosteric regulators of ABCR, the rod photoreceptor-specific ABC transporter responsible for Stargardt disease, an early onset macular degeneration. ABCR was immunoaffinity purified to apparent homogeneity from bovine rod outer segments and reconstituted into liposomes. All-trans-retinal, a candidate ligand, stimulates the ATPase activity of ABCR 3-4-fold, with a half-maximal effect at 10-15 microM. 11-cis- and 13-cis-retinal show similar activity. All-trans-retinal stimulates the ATPase activity of ABCR with Michaelis-Menten behavior indicative of simple noncooperative binding that is associated with a rate-limiting enzyme-substrate intermediate in the pathway of ATP hydrolysis. Among 37 structurally diverse non-retinoid compounds, including nine previously characterized substrates or sensitizers of P-glycoprotein, only four show significant ATPase stimulation when tested at 20 microM. The dose-response curves of these four compounds are indicative of multiple binding sites and/or modes of interaction with ABCR. Two of these compounds, amiodarone and digitonin, can act synergistically with all-trans-retinal, implying that they interact with a site or sites on ABCR different from the one with which all-trans-retinal interacts. Unlike retinal, amiodarone appears to interact with both free and ATP-bound ABCR. Together with clinical observations on Stargardt disease and the localization of ABCR to rod outer segment disc membranes, these data suggest that retinoids, and most likely retinal, are the natural substrates for transport by ABCR in rod outer segments. These observations have significant implications for understanding the visual cycle and the pathogenesis of Stargardt disease and for the identification of compounds that could modify the natural history of Stargardt disease or other retinopathies associated with impaired ABCR function.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Amiodarone; Animals; ATP-Binding Cassette Transporters; Carbohydrate Sequence; Cattle; Digitonin; Hydrolysis; In Vitro Techniques; Kinetics; Macular Degeneration; Models, Chemical; Molecular Sequence Data; Molecular Weight; Norisoprenoids; Retinaldehyde; Rod Cell Outer Segment; Terpenes

Rim protein (RmP) is an ABC transporter of unknown function in rod outer segment discs. The human gene for RmP (ABCR) is affected in several recessive retinal degenerations. Here, we characterize the ocular phenotype in abcr knockout mice. Mice lacking RmP show delayed dark adaptation, increased all-trans-retinaldehyde (all-trans-RAL) following light exposure, elevated phosphatidylethanolamine (PE) in outer segments, accumulation of the protonated Schiff base complex of all-trans-RAL and PE (N-retinylidene-PE), and striking deposition of a major lipofuscin fluorophore (A2-E) in retinal pigment epithelium (RPE). These data suggest that RmP functions as an outwardly directed flippase for N-retinylidene-PE. Delayed dark adaptation is likely due to accumulation in discs of the noncovalent complex between opsin and all-trans-RAL. Finally, ABCR-mediated retinal degeneration may result from "poisoning" of the RPE due to A2-E accumulation, with secondary photoreceptor degeneration due to loss of the RPE support role.

Topics: Adaptation, Ocular; Animals; ATP-Binding Cassette Transporters; Darkness; Electroretinography; Genomic Library; Humans; Macular Degeneration; Metabolic Clearance Rate; Mice; Mice, Knockout; Phenotype; Phospholipids; Retina; Retinaldehyde; Rhodopsin; Rod Cell Outer Segment

Topics: Aged; Humans; Lipofuscin; Macular Degeneration; Retinaldehyde; Zinc