Interphotoreceptor retinoid-binding protein removes all-trans-retinol and retinal from rod outer segments, preventing lipofuscin precursor formation.

Interphotoreceptor retinoid-binding protein (IRBP) is a specialized lipophilic carrier that binds the all-trans and 11-cis isomers of retinal and retinol, and this facilitates their transport between photoreceptors and cells in the retina. One of these retinoids, all-trans-retinal, is released in the rod outer segment by photoactivated rhodopsin after light excitation. Following its release, all-trans-retinal is reduced by the retinol dehydrogenase RDH8 to all-trans-retinol in an NADPH-dependent reaction. However, all-trans-retinal can also react with outer segment components, sometimes forming lipofuscin precursors, which after conversion to lipofuscin accumulate in the lysosomes of the retinal pigment epithelium and display cytotoxic effects. Here, we have imaged the fluorescence of all-trans-retinol, all-trans-retinal, and lipofuscin precursors in real time in single isolated mouse rod photoreceptors. We found that IRBP removes all-trans-retinol from individual rod photoreceptors in a concentration-dependent manner. The rate constant for retinol removal increased linearly with IRBP concentration with a slope of 0.012 min-1 µm-1 IRBP also removed all-trans-retinal, but with much less efficacy, indicating that the reduction of retinal to retinol promotes faster clearance of the photoisomerized rhodopsin chromophore. The presence of physiological IRBP concentrations in the extracellular medium resulted in lower levels of all-trans-retinal and retinol in rod outer segments following light exposure. It also prevented light-induced lipofuscin precursor formation, but it did not remove precursors that were already present. These findings reveal an important and previously unappreciated role of IRBP in protecting the photoreceptor cells against the cytotoxic effects of accumulated all-trans-retinal.

Mass spectrometry provides accurate and sensitive quantitation of A2E.

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.

Effective and sustained delivery of hydrophobic retinoids to photoreceptors.

PURPOSE: Delivery of hydrophobic compounds to the retina/RPE has been challenging. The purpose of this study was to develop an effective method for the sustained delivery of retinoids to rod and cone photoreceptors of young mice lacking a normal supply of 11-cis retinal. METHODS: Solubilized basement membrane matrix (Matrigel; BD Biosciences, San Jose, CA) loaded with 9-cis retinal was administered subcutaneously into Rpe65(-/-) mouse pups for assessment of delivery to rods and cones and to Rpe65(-/-)Rho(-/-) mouse pups for assessment of delivery to cones. Intraperitoneal injections of 9-cis retinal were used for comparison. Cone density and opsin localization were evaluated with immunohistochemistry. Cone opsin protein levels were assayed with immunoblots, and cone function was analyzed by electroretinography (ERG) recordings. Retinoid content was determined by high-performance liquid chromatography analysis of retinal extracts. Pigment levels were quantified in homogenized retinas by absorption spectroscopy before and after light exposure. RESULTS: Single administration of Matrigel loaded with 9-cis retinal to Rpe65(-/-) mice increased cone densities in all analyzed regions of the retina compared with mice treated using intraperitoneal delivery. Cone opsin levels increased to near wild-type levels. Similar treatment in Rpe65(-/-)Rho(-/-) mice increased b-wave ERG amplitudes significantly, indicating the maintenance of cone function. Matrigel was shown to continuously release 9-cis retinal for periods up to 1 week. CONCLUSIONS: As a method for sustained drug delivery, subcutaneous administration using Matrigel proved more efficacious than intraperitoneal injection for in vivo delivery of retinoids to cone photoreceptors. These experiments are the first to show a sustained delivery of retinoids in mice and suggest a strategy for potential clinical therapeutic development.

11-cis- and all-trans-retinols can activate rod opsin: rational design of the visual cycle.

Rhodopsin is the photosensitive pigment in the rod photoreceptor cell. Upon absorption of a photon, the covalently bound 11- cis-retinal isomerizes to the all- trans form, enabling rhodopsin to activate transducin, its G protein. All -trans-retinal is then released from the protein and reduced to all -trans-retinol. It is subsequently transported to the retinal pigment epithelium where it is converted to 11- cis-retinol and oxidized to 11- cis-retinal before it is transported back to the photoreceptor to regenerate rhodopsin and complete the visual cycle. In this study, we have measured the effects of all -trans- and 11- cis-retinals and -retinols on the opsin's ability to activate transducin to ascertain their potentials for activating the signaling cascade. Only 11- cis-retinal acts as an inverse agonist to the opsin. All -trans-retinal, all -trans-retinol, and 11- cis-retinol are all agonists with all -trans-retinal being the most potent agonist and all -trans-retinol being the least potent. Taken as a whole, our study is consistent with the hypothesis that the steps in the visual cycle are optimized such that the rod can serve as a highly sensitive dim light receptor. All -trans-retinal is immediately reduced in the photoreceptor to prevent back reactions and to weaken its effectiveness as an agonist before it is transported out of the cell; oxidation of 11- cis-retinol occurs in the retinal pigment epithelium and not the rod photoreceptor cell because 11- cis-retinol can act as an agonist and activate the signaling cascade if it were to bind an opsin, effectively adapting the cell to light.

Breaking the covalent bond--a pigment property that contributes to desensitization in cones.

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.

Collagen XVIII/endostatin is essential for vision and retinal pigment epithelial function.

Age-related macular degeneration (ARMD) with abnormal deposit formation under the retinal pigment epithelium (RPE) is the major cause of blindness in the Western world. basal laminar deposits are found in early ARMD and are composed of excess basement membrane material produced by the RPE. Here, we demonstrate that mice lacking the basement membrane component collagen XVIII/endostatin have massive accumulation of sub-RPE deposits with striking similarities to basal laminar deposits, abnormal RPE, and age-dependent loss of vision. The progressive attenuation of visual function results from decreased retinal rhodopsin content as a consequence of abnormal vitamin A metabolism in the RPE. In addition, aged mutant mice show photoreceptor abnormalities and increased expression of glial fibrillary acidic protein in the neural retina. Our data demonstrate that collagen XVIII/endostatin is essential for RPE function, and suggest an important role of this collagen in Bruch's membrane. Consistent with such a role, the ultrastructural organization of collagen XVIII/endostatin in basement membranes, including Bruch's membrane, shows that it is part of basement membrane molecular networks.

Structure-function analysis of rods and cones in juvenile, adult, and aged C57bl/6 and Balb/c mice.

To determine whether the photoreceptors change structurally and functionally during aging, and to analyze whether pigmentation in the retinal pigment epithelium might be a contributing factor. Young, adult, and aged C57BL/6 and Balb/c mice (1, 4, and 17 months of age) were housed under a 12-h light/12-h dark cycle, with an ambient light intensity at the eye level of the mice of 85 +/- 18 lux. Scotopic single-flash and photopic-flicker electroretinograms (ERGs) after complete dark adaptation were used to assess rod and cone function, respectively. Numbers of rod photoreceptors were counted in plastic sections, and rhodopsin levels were measured using absorption difference spectrophotometry. Numbers and types of cones were determined using lectin staining in retinal flatmounts and cone-specific antibodies in radial frozen sections. Young pigmented C57BL/6 and nonpigmented Balb/c mice had similar numbers of rods. In both mouse strains, there was an overall decline in rod photoreceptor number during aging, which was more pronounced in albino mice. Rod cell numbers correlated with a drop in the overall amount of rhodopsin and a reduction in the maximum a-wave of the rod ERG. The number of short-wavelength cones was unaffected by age and pigmentation, whereas an age-related decline was observed in mid-wavelength (MWL) cones in albino, but not in pigmented mice. In contrast, MWL cone function was reduced during aging in both strains. Flicker-fusion frequency was determined to be approximately 10 Hz lower in albino animals, which is due to prolonged b-waves in these ERGs. Age-related changes were found in both photoreceptor systems, rods and cones, and in both pigmented and nonpigmented mice. However, rod photoreceptors appear to be more susceptible to both aging and the lack of pigmentation, when compared to cones. These results may help as we begin to understand certain age-related retinal diseases.

Retinyl esters are the substrate for isomerohydrolase.

Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.

Correlation of regenerable opsin with rod ERG signal in Rpe65-/- mice during development and aging.

PURPOSE: RPE65 has been shown to be essential for the production of 11-cis retinal by the retinal pigment epithelium. Mutations in RPE65 are known to be associated with severe forms of early-onset retinal dystrophy. This project was designed to determine the amount of regenerable opsin in Rpe65-/- mice during development and aging, and to examine the function of this rhodopsin by electroretinography (ERG). METHODS: Young and aged Rpe65-/- and wild-type (WT) mice were dark adapted. Endogenous rhodopsin and regenerable opsin were measured using absorption-difference spectrophotometry. Photoreceptor function was assessed with scotopic single-flash ERGs and photoreceptors were counted in histologic sections. Opsin's primary structure was analyzed by mass-spectrometric mapping. RESULTS: Unlike WT mice, amounts of regenerable opsin in Rpe65-/- mice decreased significantly with age, which correlated with a decrease in the number of photoreceptors and a decline in ERG amplitudes. Opsin structure, however, did not change. No endogenous levels of rhodopsin were measurable in the Rpe65-/- mice (detection limit: 0.225 pmol). 11-cis Retinal injections resulted in the regeneration of similar amounts of rhodopsin and improved rod function in a comparable way, irrespective of age. CONCLUSIONS: In the aged Rpe65-/- mouse, opsin levels decrease because of the loss of photoreceptors. The remaining opsin is structurally intact, and the components of the phototransduction cascade and the retinal circuitry remain functional, despite the absence of normal photoreceptor activity.

11-cis-retinal reduces constitutive opsin phosphorylation and improves quantum catch in retinoid-deficient mouse rod photoreceptors.

Rpe65(-/-) mice produce minimal amounts of 11-cis-retinal, the ligand necessary for the formation of photosensitive visual pigments. Therefore, the apoprotein opsin in these animals has not been exposed to its normal ligand. The Rpe65(-/-) mice contain less than 0.1% of wild type levels of rhodopsin. Mass spectrometric analysis of opsin from Rpe65(-/-) mice revealed unusually high levels of phosphorylation in dark-adapted mice but no other structural alterations. Single flash and flicker electroretinograms (ERGs) from 1-month-old animals showed trace rod function but no cone response. B-wave kinetics of the single-flash ERG are comparable with those of dark-adapted wild type mice containing a full compliment of rhodopsin. Application (intraperitoneal injection) of 11-cis-retinal to Rpe65(-/-) mice increased the rod ERG signal, increased levels of rhodopsin, and decreased opsin phosphorylation. Therefore, exogenous 11-cis-retinal improves photoreceptor function by regenerating rhodopsin and removes constitutive opsin phosphorylation. Our results indicate that opsin, which has not been exposed to 11-cis-retinal, does not generate the activity generally associated with the bleached apoprotein.

Mass spectrometric analysis of porcine rhodopsin.

Rhodopsin is the dim light photosensitive pigment of animals. In this work, we undertook to study the structure of rhodopsin from swine and compare it with bovine and rat rhodopsin. Porcine rhodopsin was analyzed using methodology developed previously for mass spectrometric analysis of integral membrane proteins. Combining efficient protein cleavage and high performance liquid chromatography separation with the sensitivity of mass spectrometry (MS), this technique allows the observation of the full protein map and the posttranslational modifications of the protein in a single experiment. The rhodopsin protein from a single porcine eye was sequenced completely, with the exception of two single-amino acid fragments and one two-amino acid fragment, and the gene sequence reported previously was confirmed. The posttranslational modifications, similar to the ones reported previously for bovine and rat rhodopsin, were also identified. Although porcine rhodopsin has a high degree of homology to bovine and rat rhodopsins and most of their posttranslational modifications are identical, the glycosylation and phosphorylation patterns observed were different. These results show that rhodopsin from a single porcine eye can be characterized completely by MS. This technology opens the possibility of rhodopsin structural and functional studies aided by powerful mass spectrometric analysis, using the fellow eye as an internal control.