RESUMEN
The accumulation of lipofuscin, an autofluorescent aging marker, in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD). Lipofuscin contains several visual cycle byproducts, most notably the bisretinoid N-retinylidene-N-retinylethanolamine (A2E). Previous studies with human donor eyes have shown a significant mismatch between lipofuscin autofluorescence (AF) and A2E distributions. The goal of the current project was to examine this relationship in a primate model with a retinal anatomy similar to that of humans. Ophthalmologically naive young (<10 years., N = 3) and old (>10 years., N = 4) Macaca fascicularis (macaque) eyes, were enucleated, dissected to yield RPE/choroid tissue, and flat-mounted on indium-tin-oxide-coated conductive slides. To compare the spatial distributions of lipofuscin and A2E, fluorescence and mass spectrometric imaging were carried out sequentially on the same samples. The distribution of lipofuscin fluorescence in the primate RPE reflected previously obtained human results, having the highest intensities in a perifoveal ring. Contrarily, A2E levels were consistently highest in the periphery, confirming a lack of correlation between the distributions of lipofuscin and A2E previously described in human donor eyes. We conclude that the mismatch between lipofuscin AF and A2E distributions is related to anatomical features specific to primates, such as the macula, and that this primate model has the potential to fill an important gap in current AMD research.
Asunto(s)
Lipofuscina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Retinoides/metabolismo , Envejecimiento/metabolismo , Animales , Humanos , MacacaRESUMEN
The bis-retinoid N-retinylidene-N-retinylethanolamine (A2E) is one of the major components of lipofuscin, a fluorescent material that accumulates with age in the lysosomes of the retinal pigment epithelium (RPE) of the human eye. Lipofuscin, as well as A2E, exhibit a range of cytotoxic properties, which are thought to contribute to the pathogenesis of degenerative diseases of the retina such as Age-related Macular Degeneration. Consistent with such a pathogenic role, high levels of lipofuscin fluorescence are found in the central area of the human RPE, and decline toward the periphery. Recent reports have however suggested a surprising incongruence between the distributions of lipofuscin and A2E in the human RPE, with A2E levels being lowest in the central area and increasing toward the periphery. To appraise such a possibility, we have quantified the levels of A2E in the central and peripheral RPE areas of 10 eyes from 6 human donors (ages 75-91 years) with HPLC and UV/VIS spectroscopy. The levels of A2E in the central area were on average 3-6 times lower than in peripheral areas of the same eye. Furthermore, continuous accumulation of selected ions (CASI) imaging mass spectrometry showed the presence of A2E in the central RPE, and at lower intensities than in the periphery. We have therefore corroborated that in human RPE the levels of A2E are lower in the central area compared to the periphery. We conclude that the levels of A2E cannot by themselves provide an explanation for the higher lipofuscin fluorescence found in the central area of the human RPE.
Asunto(s)
Epitelio Pigmentado de la Retina/química , Retinoides/análisis , Anciano , Anciano de 80 o más Años , HumanosRESUMEN
Lipofuscin, an aging marker in the retinal pigment epithelium (RPE) associated with the development of age-related macular degeneration, is primarily characterized by its fluorescence. The most abundant component of RPE lipofuscin is N-retinylidene-N-retinylethanolamine (A2E) but its exact composition is not known due to the complexity of the RPE extract. In this study, we utilized MALDI imaging to find potential molecules responsible for lipofuscin fluorescence in RPE tissue from Abca4(-/-) , Sv129, and C57Bl6/J mice aged 2 and 6 months. To assert relationships, the individual images in the MALDI imaging datasets were correlated with lipofuscin fluorescence recorded from the same tissues following proper registration. Spatial correlation information, which is usually lost in bioanalytics, pinpointed a relatively small number of potential lipofuscin components. The comparison of four samples in each condition further limited the possibility of false positives and provided various new, age- and strain-specific targets. Validating the usefulness of the fluorescence-enhanced imaging strategy, many known adducts of A2E were identified in the short list of lipofuscin components. These results provided evidence that mass spectrometric imaging can be utilized as a tool to begin to identify the molecular substructure of clinically-relevant diagnostic information.
Asunto(s)
Lipofuscina/química , Degeneración Macular/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Fluorescencia , Regulación de la Expresión Génica , Humanos , Lipofuscina/metabolismo , Degeneración Macular/patología , Ratones , Proteómica , Epitelio Pigmentado de la Retina/química , Retinoides/químicaRESUMEN
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.
Asunto(s)
Lipofuscina/química , Epitelio Pigmentado de la Retina/metabolismo , Retinoides/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Cromatografía Líquida de Alta Presión/métodos , Color , Luz , Degeneración Macular/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente/métodos , Modelos Biológicos , Retina/metabolismo , Retinaldehído/farmacología , Segmento Externo de la Célula en Bastón/metabolismoRESUMEN
The accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD) in humans. The exact composition of lipofuscin is not known but its best characterized component is N-retinylidene-N-retinylethanolamine (A2E), a byproduct of the retinoid visual cycle. Utilizing our recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS)-based technique to determine the spatial distribution of A2E, this study compares the relationships of lipofuscin fluorescence and A2E in the murine and human RPE on representative normal tissue. To identify molecules with similar spatial patterns, the images of A2E and lipofuscin were correlated with all the individual images in the MALDI-IMS dataset. In the murine RPE, there was a remarkable correlation between A2E and lipofuscin. In the human RPE, however, minimal correlation was detected. These results were reflected in the marked distinctions between the molecules that spatially correlated with the images of lipofuscin and A2E in the human RPE. While the distribution of murine lipofuscin showed highest similarities with some of the known A2E-adducts, the composition of human lipofuscin was significantly different. These results indicate that A2E metabolism may be altered in the human compared to the murine RPE.
Asunto(s)
Lipofuscina/química , Fosfatidiletanolaminas/química , Epitelio Pigmentado de la Retina/química , Retinoides/química , Animales , Humanos , Lipofuscina/metabolismo , Lipofuscina/fisiología , Ratones , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/fisiología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/fisiología , Retinoides/metabolismo , Retinoides/fisiología , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
S-palmitoylation is a conserved feature in many G protein-coupled receptors (GPCRs) involved in a broad array of signaling processes. The prototypical GPCR, rhodopsin, is S-palmitoylated on two adjacent C-terminal Cys residues at its cytoplasmic surface. Surprisingly, absence of palmitoylation has only a modest effect on in vitro or in vivo signaling. Here, we report that palmitoylation-deficient (Palm(-/-)) mice carrying two Cys to Thr and Ser mutations in the opsin gene displayed profound light-induced retinal degeneration that first involved rod and then cone cells. After brief bright light exposure, their retinas exhibited two types of deposits containing nucleic acid and invasive phagocytic macrophages. When Palm(-/-) mice were crossed with Lrat(-/-) mice lacking lecithin:retinol acyl transferase to eliminate retinoid binding to opsin and thereby rendering the eye insensitive to light, rapid retinal degeneration occurred even in 3- to 4-week-old animals. This rapid degeneration suggests that nonpalmitoylated rod opsin is unstable. Treatment of 2-week-old Palm(-/-)Lrat(-/-) mice with an artificial chromophore precursor prevented this retinopathy. In contrast, elimination of signaling to G protein in Palm(-/-)Gnat1(-/-) mice had no effect, indicating that instability of unpalmitoylated opsin lacking chromophore rather than aberrant signal transduction resulted in retinal pathology. Together, these observations provide evidence for a structural role of rhodopsin S-palmitoylation that may apply to other GPCRs as well.
Asunto(s)
Lipoilación , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Opsinas de Bastones/metabolismo , Animales , Macrófagos/metabolismo , Proteínas de la Membrana/deficiencia , Ratones , Ratones Noqueados , Mutación , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Fosfoproteínas/deficiencia , Retina/citología , Retina/metabolismo , Retina/efectos de la radiaciónRESUMEN
As cone photoreceptors mediate vision in bright light, their photopigments are bleached at a rapid rate and require substantial recycling of the chromophore 11-cis-retinal (RAL) for continued function. The retinal pigment epithelium (RPE) supplies 11-cis-RAL to both rod and cone photoreceptors; however, stringent demands imposed by the function of cones in bright light exceed the output from this source. Recent evidence has suggested that cones may be able to satisfy this demand through privileged access to an additional source of chromophore located within the inner retina. In this study, we demonstrate that the protein RPE65, previously identified in RPE as the isomerohydrolase of the RPE-retinal visual cycle, is found within cones of the rod-dominant mouse retina, and the level of RPE65 in cones is inversely related to the level in the RPE. The light sensitivity of cone ERGs of BALB/c mice, which had an undetectable level of cone RPE65, was enhanced by approximately threefold with administration of exogenous chromophore, indicating that the cones of these animals are chromophore deficient. This enhancement with chromophore administration was not observed in C57BL/6 mice, whose cones contain RPE65. These results demonstrate that RPE65 within cones may be essential for the efficient regeneration of cone photopigments under bright-light conditions.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Ojo/metabolismo , Regeneración/fisiología , Células Fotorreceptoras Retinianas Conos/metabolismo , Epitelio Pigmentado de la Retina/fisiología , Animales , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Estimulación Luminosa , Epitelio Pigmentado de la Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Especificidad de la Especie , cis-trans-IsomerasasRESUMEN
RPE65 is an abundantly expressed protein within the retinal pigment epithelium (RPE) of the eye that is required for retinoid metabolism to support vision. Its genetic mutations are linked to the congenital disease Leber congenital amaurosis Type 2 (LCA2) characterized by the early onset of central vision loss. Current gene therapy trials have targeted restoration of functional RPE65 within the RPE of these patients with some success. Recent data show that RPE65 is also present within mouse cones to promote function. In this study, we evaluated the presence of RPE65 in human cones and investigated its potential mechanism for supporting cone function in the 661W cone cell line. We found that RPE65 was selectively expressed in human green/red cones but absent from blue cones and mediated ester hydrolysis for photopigment synthesis in vitro. These data suggest that cone RPE65 supports human diurnal vision, potentially enhancing our strategies for treating LCA2.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Ojo/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Regeneración/fisiología , Células Fotorreceptoras Retinianas Conos/metabolismo , Anciano de 80 o más Años , Proteínas Portadoras/genética , Línea Celular , Niño , Proteínas del Ojo/genética , Femenino , Humanos , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/metabolismo , Masculino , Adulto Joven , cis-trans-IsomerasasRESUMEN
Cones function in constant light and are responsible for mediating daytime human vision. Like rods, cones use the photosensitive molecule 11-cis-retinal to detect light, and in constant illumination, a continuous supply of 11-cis-retinal is needed. A retina visual cycle is thought to provide a privileged supply of 11-cis-retinal to cones by using 11-cis-retinol generated in Müller cells. In the cycle, 11-cis-retinol is transported from Müller cells to cone inner segments, where it is oxidized to 11-cis-retinal. This oxidation step is only performed in cones, thus rendering the cycle cone-specific. Interphotoreceptor retinoid-binding protein (IRBP) is a retinoid-binding protein in the subretinal space that binds 11-cis-retinol endogenously. Cones in Irbp(-/-) mice are retinoid-deficient under photopic conditions, and it is possible that 11-cis-retinol supplies are disrupted in the absence of IRBP. We tested the hypothesis that IRBP facilitates the delivery of 11-cis-retinol to cones by preserving the isomeric state of 11-cis-retinol in light. With electrophysiology, we show that the cone-like photoreceptors of Nrl(-/-) mice use the cone visual cycle similarly to wild-type cones. Then, using oxidation assays in isolated Nrl(-/-)Rpe65(-/-) retinas, we show that IRBP delivers 11-cis-retinol for oxidation in cones and improves the efficiency of the oxidation reaction. Finally, we show that IRBP protects the isomeric state of 11-cis-retinol in the presence of light. Together, these findings suggest that IRBP plays an important role in the delivery of 11-cis-retinol to cones and can facilitate cone function in the presence of light.
Asunto(s)
Proteínas del Ojo/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Proteínas de Unión al Retinol/metabolismo , Vitamina A/metabolismo , Animales , Proteínas Portadoras/genética , Cromatografía Líquida de Alta Presión , Electrorretinografía , Proteínas del Ojo/genética , Luz , Ratones , Ratones Noqueados , Oxidación-Reducción , Retinoides/química , Retinoides/metabolismo , Visión Ocular/fisiología , cis-trans-IsomerasasRESUMEN
Rapid regeneration of the visual pigment following its photoactivation is critical for the function of cone photoreceptors throughout the day. Though the reactions of the visual cycle in the retinal pigment epithelium (RPE) that recycle chromophore for rod pigment regeneration are well characterized, the corresponding mechanisms that enable rapid regeneration of cone pigment are poorly understood. A key remaining question is the relative contribution of the recently discovered cone-specific retina visual cycle and the classic RPE-dependent visual cycle to mammalian cone pigment regeneration. In addition, it is not clear what role, if any, the abundant interphotoreceptor retinoid-binding protein (IRBP) presumed to facilitate the traffic of chromophore, plays in accelerating mammalian cone pigment regeneration. To address these issues, we used transretinal recordings to evaluate M/L-cone pigment regeneration in isolated retinas and eyecups from control and IRBP-deficient mice. Remarkably, the mouse retina promoted M/L-cone dark adaptation eightfold faster than the RPE. However, complete cone recovery required both visual cycles. We conclude that the retina visual cycle is critical for the initial rapid regeneration of mouse M/L-cone pigment during dark adaptation, whereas the slower RPE visual cycle is required to complete the process. While the deletion of IRBP reduced the amplitude and slowed the kinetics of mouse M/L-cone photoresponses, cone adaptation in bright, steady light and the kinetics of cone dark adaptation were not affected in isolated retina or in intact eyecup. Thus, IRBP does not accelerate cone pigment regeneration and is not critical for the function of mouse M/L-cones in bright light.
Asunto(s)
Opsinas de los Conos/biosíntesis , Proteínas del Ojo/biosíntesis , Estimulación Luminosa/métodos , Células Fotorreceptoras Retinianas Conos/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Pigmentos Retinianos/biosíntesis , Proteínas de Unión al Retinol/biosíntesis , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Retina/metabolismo , Factores de Tiempo , Percepción Visual/fisiologíaRESUMEN
Even in healthy individuals, aging leads to deterioration in visual acuity, contrast sensitivity, visual field, and dark adaptation. Little is known about the neural mechanisms that drive the age-related changes of the retina and, more specifically, photoreceptors. According to one hypothesis, the age-related deterioration in rod function is due to the limited availability of 11-cis-retinal for rod pigment formation. To determine how aging affects rod photoreceptors and to test the retinoid-deficiency hypothesis, we compared the morphological and functional properties of rods of adult and aged B6D2F1/J mice. We found that the number of rods and the length of their outer segments were significantly reduced in 2.5-year-old mice compared with 4-month-old animals. Aging also resulted in a twofold reduction in the total level of opsin in the retina. Behavioral tests revealed that scotopic visual acuity and contrast sensitivity were decreased by twofold in aged mice, and rod ERG recordings demonstrated reduced amplitudes of both a- and b-waves. Sensitivity of aged rods determined from single-cell recordings was also decreased by 1.5-fold, corresponding to not more than 1% free opsin in these photoreceptors, and kinetic parameters of dim flash response were not altered. Notably, the rate of rod dark adaptation was unaffected by age. Thus, our results argue against age-related deficiency of 11-cis-retinal in the B6D2F1/J mouse rod visual cycle. Surprisingly, the level of cellular dark noise was increased in aged rods, providing an alternative mechanism for their desensitization.
Asunto(s)
Envejecimiento/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Trastornos de la Visión/fisiopatología , Visión Ocular/fisiología , Envejecimiento/patología , Animales , Recuento de Células , Tamaño de la Célula , Sensibilidad de Contraste/fisiología , Adaptación a la Oscuridad/fisiología , Electrorretinografía , Femenino , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Microelectrodos , Visión Nocturna/fisiología , Opsinas/metabolismo , Estimulación Luminosa , Retina/patología , Retina/fisiopatología , Células Fotorreceptoras Retinianas Bastones/patología , Trastornos de la Visión/patología , Pruebas de VisiónRESUMEN
Rpe65(-/-) mice are unable to produce 11-cis-retinal, the chromophore of visual pigments. Consequently, the pigment is present as the apoprotein opsin with a minute level of pigment containing 9-cis-retinal as chromophore. Notably, a 10-20% fraction of this opsin is mono-phosphorylated independently of light conditions. To determine the role of rhodopsin kinase (GRK1) in phosphorylating this opsin and to test whether eliminating this phosphorylation would accelerate photoreceptor degeneration, we generated the Rpe65(-/-)Grk1(-/-) mouse. The retinae of Rpe65(-/-)Grk1(-/-) mice had negligible opsin phosphorylation, extensive degeneration with decreased opsin levels, and diminished light-evoked rod responses relative to Rpe65(-/-) mice. These data show that opsin phosphorylation in the Rpe65(-/-) mouse is due to the action of GRK1 and is neuroprotective. However, despite the higher activity of unphosphorylated opsin, the severe loss of opsin in the rapidly degenerating Rpe65(-/-)Grk1(-/-) mice resulted in lower overall opsin activity and in higher rod sensitivity compared with Rpe65(-/-) mice. In Rpe65(-/-)Grk1(-/-)Gnat1(-/-) mice where transduction activation was blocked, degeneration was only partially prevented. Therefore, increased opsin activity in the absence of phosphorylation was not the only mechanism for the accelerated retinal degeneration. Finally, the deletion of GRK1 triggered retinal degeneration in Grk1(-/-) mice after 1 month, even in the absence of apo-opsin. This degeneration was independent of light conditions and occurred even in the absence of transducin in Grk1(-/-)Gnat1(-/-) mice. Taken together, our results demonstrate a light-independent mechanism for retinal degeneration in the absence of GRK1, suggesting a second, not previously recognized role for that kinase.
Asunto(s)
Quinasa 1 del Receptor Acoplado a Proteína-G/deficiencia , Degeneración Retiniana/genética , Transducina/metabolismo , Visión Ocular/genética , Adaptación Ocular/genética , Animales , Biofisica/métodos , Proteínas Portadoras/genética , Proteínas del Ojo/genética , Subunidades alfa de la Proteína de Unión al GTP/deficiencia , Ratones , Ratones Noqueados , Opsinas/metabolismo , Fosforilación/genética , Estimulación Luminosa/métodos , Degeneración Retiniana/fisiopatología , Células Fotorreceptoras Retinianas Bastones/fisiología , cis-trans-IsomerasasRESUMEN
Retinal analogues have been used to probe the chromophore binding pocket and function of the rod visual pigment rhodopsin. Despite the high homology between rod and cone visual pigment proteins, conclusions drawn from rhodopsin studies should not necessarily be extrapolated to cone visual pigment proteins. In this study, the effects of full-length and truncated retinal analogues on the human red cone opsin's ability to activate transducin, the G protein in visual transduction, were assessed. The result with beta-ionone (6) confirms that a covalent bond is not necessary to deactivate the red cone opsin. In addition, several small compounds were found able to deactivate this opsin. However, as the polyene chain is extended in a trans configuration beyond the 9-carbon position, the analogues became agonists up to all-trans-retinal (3). The 22-carbon analogue (2) appeared to be neither an agonist nor an inverse agonist. Although the all-trans-C17 (5) analogue was an agonist, the 9-cis-C17 (11) compound was an inverse agonist, a result that differs from that with rhodopsin. These results suggest that the red cone opsin has a more open structure in the chromophore binding region than rhodopsin and its activation or deactivation as a G-protein receptor may be less selective than rhodopsin.
Asunto(s)
Opsinas de los Conos/metabolismo , Retinaldehído , Animales , Bovinos , Opsinas de los Conos/química , Humanos , Estructura Molecular , Retina/química , Pigmentos Retinianos/metabolismo , Retinaldehído/análogos & derivados , Retinaldehído/química , Retinaldehído/metabolismo , Rodopsina/metabolismo , Estereoisomerismo , Transducina/metabolismoRESUMEN
Visual opsins bind 11-cis retinal at an orthosteric site to form rhodopsins but increasing evidence suggests that at least some are capable of binding an additional retinoid(s) at a separate, allosteric site(s). Microspectrophotometric measurements on isolated, dark-adapted, salamander photoreceptors indicated that the truncated retinal analog, ß-ionone, partitioned into the membranes of green-sensitive rods; however, in blue-sensitive rod outer segments, there was an enhanced uptake of four or more ß-ionones per rhodopsin. X-ray crystallography revealed binding of one ß-ionone to bovine green-sensitive rod rhodopsin. Cocrystallization only succeeded with extremely high concentrations of ß-ionone and binding did not alter the structure of rhodopsin from the inactive state. Salamander green-sensitive rod rhodopsin is also expected to bind ß-ionone at sufficiently high concentrations because the binding site is present on its surface. Therefore, both blue- and green-sensitive rod rhodopsins have at least one allosteric binding site for retinoid, but ß-ionone binds to the latter type of rhodopsin with low affinity and low efficacy.
Asunto(s)
Células Fotorreceptoras Retinianas Bastones/metabolismo , Retinoides/metabolismo , Rodopsina/metabolismo , Animales , Bovinos , Cristalografía por Rayos X , Microespectrofotometría , Norisoprenoides/química , Norisoprenoides/metabolismo , Unión Proteica , Retinoides/química , Rodopsina/química , Segmento Externo de la Célula en Bastón/metabolismo , Urodelos/metabolismoRESUMEN
11-cis-retinal is the light-sensitive component in rod and cone photoreceptors, and its isomerization to all-trans retinal in the presence of light initiates the visual response. For photoreceptors to function normally, all-trans retinal must be converted back into 11-cis-retinal through a series of enzymatic steps known as the visual cycle. The interphotoreceptor retinoid-binding protein (IRBP) is a proposed retinoid transporter in the visual cycle, but rods in Irbp(-/-) mice have a normal visual cycle. While rods are primarily responsible for dim light vision, the ability of cones to function in constant light is essential to human vision and may be facilitated by cone-specific visual cycle pathways. We analyzed the cones in Irbp(-/-) mice to determine whether IRBP has a cone-specific visual cycle function. Cone electroretinogram (ERG) responses were reduced in Irbp(-/-) mice, but similar responses from Irbp(-/-) mice at all ages suggest that degeneration does not underlie cone dysfunction. Furthermore, cone densities and opsin levels in Irbp(-/-) mice were similar to C57BL/6 (wild-type) mice, and both cone opsins were properly localized to the cone outer segments. To test for retinoid deficiency in Irbp(-/-) mice, ERGs were analyzed before and after intraperitoneal injections of 9-cis-retinal. Treatment with 9-cis-retinal produced a significant recovery of the cone response in Irbp(-/-) mice and shows that retinoid deficiency underlies cone dysfunction. These data indicate that IRBP is essential to normal cone function and demonstrate that differences exist in the visual cycle of rods and cones.
Asunto(s)
Proteínas del Ojo/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Proteínas de Unión al Retinol/deficiencia , Proteínas de Unión al Retinol/fisiología , Animales , Proteínas del Ojo/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión al Retinol/genética , Percepción Visual/genéticaRESUMEN
The isomerization of 11-cis retinal to all-trans retinal in photoreceptors is the first step in vision. For photoreceptors to function in constant light, the all-trans retinal must be converted back to 11-cis retinal via the enzymatic steps of the visual cycle. Within this cycle, all-trans retinal is reduced to all-trans retinol in photoreceptors and transported to the retinal pigment epithelium (RPE). In the RPE, all-trans retinol is converted to 11-cis retinol, and in the final enzymatic step, 11-cis retinol is oxidized to 11-cis retinal. The first and last steps of the classical visual cycle are reduction and oxidation reactions, respectively, that utilize retinol dehydrogenase (RDH) enzymes. The visual cycle RDHs have been extensively studied, but because multiple RDHs are capable of catalyzing each step, the exact RDHs responsible for each reaction remain unknown. Within rods, RDH8 is largely responsible for the reduction of all-trans retinal with possible assistance from RDH12. retSDR1 is thought to reduce all-trans retinal in cones. In the RPE, the oxidation of 11-cis retinol is carried out by RDH5 with possible help from RDH11 and RDH10. Here, we review the characteristics of each RDH in vitro and the findings from knockout models that suggest the roles for each in the visual cycle.
Asunto(s)
Retina/metabolismo , Retinal-Deshidrogenasa/fisiología , Visión Ocular/fisiología , Animales , Humanos , Ratones , Ratones Noqueados , Oxidación-Reducción , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Orange autofluorescence from lipofuscin in the lysosomes of the retinal pigment epithelium (RPE) is a hallmark of aging in the eye. One of the major components of lipofuscin is A2E, the levels of which increase with age and in pathologic conditions, such as Stargardt disease or age-related macular degeneration. In vitro studies have suggested that A2E is highly phototoxic and, more specifically, that A2E and its oxidized derivatives contribute to RPE damage and subsequent photoreceptor cell death. To date, absorption spectroscopy has been the primary method to identify and quantitate A2E. Here, a new mass spectrometric method was developed for the specific detection of low levels of A2E and compared to a traditional method of analysis. The new mass spectrometric method allows the detection and quantitation of approximately 10,000-fold less A2E than absorption spectroscopy and the detection and quantitation of low levels of oxidized A2E, with localization of the oxidation sites. This study suggests that identification and quantitation of A2E from tissue extracts by chromatographic absorption spectroscopy overestimates the amount of A2E. This mass spectrometric approach makes it possible to detect low levels of A2E and its oxidized metabolites with greater accuracy than traditional methods, thereby facilitating a more exact analysis of bis-retinoids in animal models of inherited retinal degeneration as well as in normal and diseased human eyes.
Asunto(s)
Ojo/química , Espectrometría de Masas/métodos , Compuestos de Piridinio/análisis , Retinoides/análisis , Anciano de 80 o más Años , Animales , Humanos , Ratones , Estructura Molecular , Oxidación-Reducción , Sensibilidad y EspecificidadRESUMEN
11-cis Retinal is the light-sensitive component in rod and cone photoreceptors, and its isomerization to all-trans retinal in the presence of light initiates the visual response. For photoreceptors to function normally, all-trans retinal must be converted back into 11-cis retinal through the visual cycle. While rods are primarily responsible for dim light vision, the ability of cones to function in constant light is essential to human vision and may be facilitated by cone-specific visual cycle pathways. The interphotoreceptor retinoid-binding protein (IRBP) is a proposed retinoid transporter in the visual cycle, but rods in Irbp ( -/- ) mice have a normal visual cycle. However, there is evidence that IRBP has cone-specific functions. Cone electroretinogram (ERG) responses are reduced, despite having cone densities and opsin levels similar to C57Bl/6 (WT) mice. Treatment with 9-cis retinal rescues the cone response in Irbp ( -/- ) mice and shows that retinoid deficiency underlies cone dysfunction. These data indicate that IRBP is essential to normal cone function and demonstrate that differences exist in the visual cycle of rods and cones.
Asunto(s)
Proteínas del Ojo/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Retinoides/metabolismo , Proteínas de Unión al Retinol/metabolismo , Envejecimiento/metabolismo , Animales , Electrorretinografía , Humanos , Ratones , Ratones Endogámicos C57BL , Células Fotorreceptoras Retinianas Conos/citología , Proteínas de Unión al Retinol/deficiencia , Visión OcularRESUMEN
Retinal rod and cone pigments consist of an apoprotein, opsin, covalently linked to a chromophore, 11-cis retinal. Here we demonstrate that the formation of the covalent bond between opsin and 11-cis retinal is reversible in darkness in amphibian red cones, but essentially irreversible in red rods. This dissociation, apparently a general property of cone pigments, results in a surprisingly large amount of free opsin--about 10% of total opsin--in dark-adapted red cones. We attribute this significant level of free opsin to the low concentration of intracellular free 11-cis retinal, estimated to be only a tiny fraction (approximately 0.1 %) of the pigment content in red cones. With its constitutive transducin-stimulating activity, the free cone opsin produces an approximately 2-fold desensitization in red cones, equivalent to that produced by a steady light causing 500 photoisomerizations s-1. Cone pigment dissociation therefore contributes to the sensitivity difference between rods and cones.
Asunto(s)
Retina/citología , Células Fotorreceptoras Retinianas Conos/fisiología , Pigmentos Retinianos/fisiología , Retinaldehído/metabolismo , Opsinas de Bastones/metabolismo , Algoritmos , Animales , Proteínas Portadoras/farmacología , Células Cultivadas , Color , Adaptación a la Oscuridad , Diterpenos , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Electrofisiología , Larva , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Estimulación Luminosa/métodos , Fotoblanqueo/efectos de los fármacos , Fotoblanqueo/efectos de la radiación , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Retinaldehído/farmacología , Espectrofotometría/métodos , Factores de Tiempo , Urodelos , Visión Ocular/efectos de los fármacos , Visión Ocular/fisiologíaRESUMEN
Lecithin retinol acyl transferase (LRAT) and retinal pigment epithelium protein 65 (RPE65) are key enzymes of the retinoid cycle. In Lrat(-/-) and Rpe65(-/-) mice, models of human Leber congenital amaurosis, the retinoid cycle is disrupted and 11-cis-retinal, the chromophore of visual pigments, is not produced. The Lrat(-/-) and Rpe65(-/-) retina phenotype presents with rapid sectorial cone degeneration, and the visual pigments, S-opsin and M/L-opsin, fail to traffic to cone outer segments appropriately. In contrast, rod opsin traffics normally in mutant rods. Concomitantly, guanylate cyclase 1, cone T alpha-subunit, cone phosphodiesterase 6alpha' (PDE6alpha'), and GRK1 (G-protein-coupled receptor kinase 1; opsin kinase) are not transported to Lrat(-/-) and Rpe65(-/-) cone outer segments. Aberrant localization of these membrane-associated proteins was evident at postnatal day 15, before the onset of ventral and central cone degeneration. Protein levels of cone T alpha and cone PDE6alpha' were reduced, whereas their transcript levels were unchanged, suggesting posttranslational degradation. In an Rpe65(-/-)Rho(-/-) double knock-out model, trafficking of cone pigments and membrane-associated cone phototransduction polypeptides to the outer segments proceeded normally after 11-cis-retinal administration. These results suggest that ventral and central cone opsins must be regenerated with 11-cis-retinal to permit transport to the outer segments. Furthermore, the presence of 11-cis-retinal is essential for proper transport of several membrane-associated cone phototransduction polypeptides in these cones.