A phosphorylation-sensitive anti-rhodopsin monoclonal antibody reveals light-induced phosphorylation of rhodopsin in the photoreceptor cell body

Rho-1C5, a monoclonal antibody sensitive to phosphorylation of rhodopsin, bound to the retinal photoreceptor cell body region of dark-adapted but not light-adapted 8 to 13-day-old-rats. There was no cell body labeling visible either before or after this time, although the photoreceptor outer segments were labeled at all times from postnatal day 5 (PN5) onwards, in both light and dark adapted retinas. However, opsin was detectable in the photoreceptor cell body region from birth onwards using another rhodopsin antibody binding to a site unaffected by phosphorylation. Competitive inhibition radioimmunoassays also indicated light-dependent differences in Rho-1C5 binding at PN8 and adult. Biochemical studies showed light-dependent phosphorylation of rhodopsin at PN8, PN13 (just after eye opening) and adult. These data indicate that rhodopsin can be phosphorylated in a light-regulated manner early in development before eye opening and imply that photoactive chromophores can attach to opsin in the cell body as well as the outer segment.     

Immunoassay of rod visual pigment (opsin) in the eyes of rds mutant mice lacking receptor outer segments

In 020/A mice, homozygous for the retinal degeneration slow (rds) gene, the photoreceptor cells fail to develop outer segments, and in the absorption spectra of retinal extracts the rhodopsin peak is lacking. Application of an enzyme-linked immunoassay using antisera against bovine opsin shows, however, that opsin is present in the homozygous mutant retina (0.010 nmol/eye) at 3% of the level of the normal retina (0.38 nmol/eye) of Balb/c mice. In the retina of heterozygous mice the opsin level (0.19 nmol/eye) is about half of the normal. Detection of opsin in the rds mutant retina demonstrates the functional basis for the reported electroretinographic response and light-mediated reduction in cyclic nucleotide levels in this mutant.     

The pineal organ is the first differentiated light receptor in the embryonic salmon,Salmo salar L.

Summary The initial appearance of S-antigen, -transducin, opsin and 5-HT during embryogenesis of the pineal organ and retina was studied by means of immunocytochemistry in the Atlantic salmon, Salmo salar L. The presence of these substances may be taken as a good indication of photoreceptor differentiation; -transducin and S-antigen are involved in the phototransduction process, opsin is the proteinaceous component of the photopigment rhodopsin, and 5-HT is a neurotransmitter or neurohormone produced by pineal photoreceptors. Two days after the retinal pigment layer became visible in the eggs, the outer segments of a few pineal photosensory cells showed immunoreactivity to opsin and -transducin. At the same time S-antigen and serotonin were present in pineal cells of the photoreceptor type. The number of immunoreactive cells in the pineal organ increased up to hatching. In the differentiating retina of the salmon, no immunoreactivity to antibodies raised against the mentioned substances was detectable until after hatching. These results indicate that in ontogeny the developing pineal organ of the salmon embryo has the ability to perceive light information much earlier than the retina.A preliminary account of this work was presented at the Tenth European Neuroscience Congress, Marseille, France, September 14–18, 1986     

The heat-shock response co-inducer arimoclomol protects against retinal degeneration in rhodopsin retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.     

Accumulation of immunoreactive opsin on plasma membranes in degenerating rod cells of rd/rd mutant mice

Immunoreactive opsin was detectable in the apical portion of normally developing photoreceptor cells on postnatal day 3 by the indirect enzyme-labeled antibody method. Immunoreactivity increased and had extended from the central retina to the periphery by the advanced stages of development. In the rd mutant retinas, accumulated opsin was present in the apical portion and in the outer nuclear layer on postnatal day 8. Immunoreactive opsin mainly was present in the outer nuclear layer by day 14, even being detectable on day 28. No immunoreactivity was present in the remaining cones. Electron microscopic immunocytochemistry confirmed the association of immunoreactive opsin with the persistent rod cell plasma membrane. Molecular weight of immunoreactive opsin in 14-day-old rd mutant mouse retina, as estimated by gel filtration chromatography, was large and did not seem to be degraded. These findings indicate that accumulated rhodopsin continues to function in the plasma membrane because an electroretinogram could be made after day 14 for the rd mutant mouse retina.     

Decrease of opsin content in the developing rat photoreceptor cells by systemic administration of L-glutamate.

L-Glutamate, a putative photoreceptor cell neurotransmitter, causes thinning of the inner layers of the retina and has been used for preparing biologically fractionated photoreceptor cells. However, it is possible that absence of the inner retinal layers may affect the remaining retina, and/or glutamate may directly affect photoreceptor cells. We evaluated quantitatively the effects of L-glutamate on the developing photoreceptor cells by measuring the rod photoreceptor cell-specific protein, opsin. We purified rat rhodopsin and used it as the standard for measuring opsin content of rat retinas with competitive enzyme-linked immunosorbent assay. Various concentrations of glutamate were injected into 7-day-old rats, and the effects of the amino acid concentration on opsin expression were determined on postnatal day 14. Inner layers of the retina degenerated when 10 microliters or 15 microliters of 2.4 M glutamate/gram body weight was administered subcutaneously. Opsin content of these glutamate-treated retinas decreased significantly compared with control retinas. We administered glutamate to rats at various stages of development and determined the effects by light microscopy on postnatal day 14. The administration of glutamate resulted in no degeneration of the inner retina if injected on postnatal day 1 or 2, degeneration of the inner retina between day 3 to 7, and again, no degeneration after postnatal day 13. Opsin content decreased significantly when glutamate was administered between postnatal day 1 to 7, but not after day 13, the day the blood-retinal barrier seems to reach maturity. Our findings indicate that systemic administration of L-glutamate affects the expression of opsin in the developing rod photoreceptor cells.     

A naturally occurring mutation of the opsin gene (T4R) in dogs affects glycosylation and stability of the G protein-coupled receptor

Rho (rhodopsin; opsin plus 11-cis-retinal) is a prototypical G protein-coupled receptor responsible for the capture of a photon in retinal photoreceptor cells. A large number of mutations in the opsin gene associated with autosomal dominant retinitis pigmentosa have been identified. The naturally occurring T4R opsin mutation in the English mastiff dog leads to a progressive retinal degeneration that closely resembles human retinitis pigmentosa caused by the T4K mutation in the opsin gene. Using genetic approaches and biochemical assays, we explored the properties of the T4R mutant protein. Employing immunoaffinity-purified Rho from affected RHO(T4R/T4R) dog retina, we found that the mutation abolished glycosylation at Asn(2), whereas glycosylation at Asn(15) was unaffected, and the mutant opsin localized normally to the rod outer segments. Moreover, we found that T4R Rho(*) lost its chromophore faster as measured by the decay of meta-rhodopsin II and that it was less resistant to heat denaturation. Detergent-solubilized T4R opsin regenerated poorly and interacted abnormally with the G protein transducin (G(t)). Structurally, the mutation affected mainly the "plug" at the intradiscal (extracellular) side of Rho, which is possibly responsible for protecting the chromophore from the access of bulk water. The T4R mutation may represent a novel molecular mechanism of degeneration where the unliganded form of the mutant opsin exerts a detrimental effect by losing its structural integrity.     

Embryonic appearance of rod opsin in the urodele amphibian eye

Notophthalmus (Triturus) viridescens, a urodele amphibian (newt) common to the Eastern United States, is a promising subject for developmental and regeneration studies. We have available a monoclonal antibody shown to be specific in many vertebrates for rod opsin, the membrane apoprotein of the visual pigment rhodopsin. This antibody to an N-terminal epitope, by rigorous biochemical and immunological criteria, recognizes only rod photoreceptor cells of the retina in light-and electron-microscopic immunocytochemistry. To determine the ontogeny and localization of rhodopsin in developing rods as an indicator of function in the embryonic urodele retina, we have utilized this antibody in the immunofluorescence technique on sections of developing N. viridescens. It was applied to serial sections of the eye region of Harrison stage 28 (optic vesicle) through stage 43 (most adult retina histology complete) embryos, and subsequently visualized with biotinylated species antibody followed by extravidin fluorescein isothiocyanate. The first positive reaction to rhodopsin could be detected in two to four cells (total) of the stage 37 embryonic eye, in the region of the central retinal primordium where the photoreceptors will be found. Some indications of retinal outer nuclear and inner plexiform layers could be seen at this time. Later embryonic stages demonstrated increasing numbers of positive cells in the future photoreceptor outer nuclear layer and outer and inner segments, spreading even to the peripheral retina. Nevertheless, by stale 43, no positive cells could be found at the dorsal or ventral retinal margins. Thus, biochemical differentiation of a photoreceptor population in the urodele retina occurs at a stage before retinal histogenesis is complete. The total maturation of retinal rods occurs topographically over a long period until the adult distribution is achieved. Correspondence to: D.S. McDevitt     

Cone cells fail to develop normally in transgenic mice showing ablation of rod photoreceptor cells

Transgenic mice were derived containing the cytotoxic dt-α gene driven by opsin promoter sequences. Mice expressing this construct showed progressive degeneration of rod photoreceptor cells commencing at birth, with obvious depletion of such cells by postnatal day 7. Ablation of rod photoreceptor cells in the transgenic retina was accompanied by the failure of developing cone cells to elaborate outer segments, although all other aspects of cone cell cytodifferentiation appeared normal. The results suggest that the 1.0-kb opsin promoter segment contains rod cell type specificity and that cone cells require maturation of rod cells to complete the late stages of their terminal differentiation and for their maintenance and cellular integrity.     

Expression of Drosophila rhodopsins during photoreceptor cell differentiation: insights into R7 and R8 cell subtype commitment

The R7 and R8 photoreceptor cells of the Drosophila retina are thought to mediate color discrimination and polarized light detection. This is based on the patterned expression of different visual pigments, rhodopsins, in different photoreceptor cells. In this report, we examined the developmental timing of retinal patterning. There is genetic evidence that over the majority of the eye, patterned expression of opsin genes is regulated by a signal from one subtype of R7 cells to adjacent R8 cells. We examined the onset of expression of the rhodopsin genes to determine the latest time point by which photoreceptor subtype commitment must have occurred. We found that the onset of rhodopsin expression in all photoreceptors of the compound eye occurs during a narrow window from 79% to 84% of pupal development (approximately 8 h), pupal stages P12-P14. Rhodopsin 1 has the earliest onset, followed by Rhodopsins 3, 4, and 5 at approximately the same time, and finally Rhodopsin 6. This sequence mimics the model for how R7 and R8 photoreceptor cells are specified, and defines the timing of photoreceptor cell fate decisions with respect to other events in eye development.     

A rod-dominated visual system in leptocephalus larvae of elopomorph fishes (Elopomorpha: Teleostei)

The nature and distributions of photoreceptor cell types were investigated in the retinas of 12 species (5 families) of elopomorph anguilliform leptocephalus larvae. Anti-opsin immunofluorescence, light microscopy and transmission electron microscopy (TEM) were used to assess opsin distribution across the retinas and to associate photoreceptor morphology and opsin content. Retinas of all species were immunoreactive with anti-rhodopsin throughout, while anti-cone opsin immunoreactivity was restricted only to the ventral region of the retina in all specimens. Rod and cone photoreceptors were morphologically indistinguishable at low magnifications; TEM revealed that nearly all photoreceptors had rod-like ultrastructure, with only rare examples of cone-like cells identified in the ventral retina. These results indicate a rhodopsin/rod-dominated retina in leptocephalus larvae of anguilliform eels in the teleost subdivision Elopomorpha, contrasting with the cone-dominated retinas of nearly all other species of teleost larvae. This distinctive developmental pattern shared among elopomorph larvae has important evolutionary and ecological implications, indicating a shared ancestor and/or ecological characteristics that are very different from most other teleost larvae.     

Renewal of opsin in the photoreceptor cells of the mosquito

Mosquito rhodopsin is a digitonin-soluble membrane protein of molecular weight 39,000 daltons, as determined by sodium dodecyl sulfate gel electrophoresis. The rhodopsin undergoes a spectral transition from R515-520 to M480 after orange illumination. The visual pigment apoprotein, opsin, is the major membrane protein in the eye. Protein synthesis in the photoreceptor cells occurs in the perinuclear cytoplasm and the newly made protein is transported to the rhabdom. Light adaptation increases the rate of turnover of this rhabdomal protein. The turnover of electrophoretically isolated opsin is also stimulated by light adaptation. The changes observed in protein metabolism biochemically, are consistent with previous morphological observations of photoreceptor membrane turnover. The results agree with the hypothesis that the newly synthesized rhabdomal protein is opsin.     

Microenvironmental regulation of visual pigment expression in the chick retina

Visual pigment (VP) expression in the chick embryo retina was investigated in ovo, in dissociated and explant cultures, and in cDNAs from individual cells. While VP mRNA is not detectable by in situ hybridization until embryonic day (ED) 14-16 in ovo, analysis of VP expression by RT-PCR showed that VP messages are present in the retina as many as 7-10 days before they become detectable by in situ hybridization, and are also detected in other regions of the embryonic CNS. On the other hand, red opsin expression is markedly accelerated when cells are isolated from their intraocular microenvironment at ED 6, and placed in pigment epithelium-free dissociated or explant cultures. This acceleration occurs regardless of cell density, birth date, or serum presence in the medium, suggesting that many photoreceptors are already programmed to express red opsin on or before ED 6, and that microenvironmental inhibitory factors prevent implementation of this program until ED 14 in ovo. The selectivity of this phenomenon is suggested by the finding that other VPs are not observed by in situ hybridization in ED 6 cultures, although they are detectable in cultures of older retinas. Taken together, these findings suggest that red opsin expression may be constitutive for many developing photoreceptor cells in the chick.     

On a soluble system for studying light activation of rod outer segment cyclic GMP phosphodiesterase

Bovine rod outer segment (ROS) cyclic GMP phosphodiesterase (PDE) could be activated about 6-fold by light, an effect that could be simulated by isolated bleached rhodopsin. About 90% of PDE activity in ROS could be extracted with 10 mM Tris-HCl, pH 7.5, but light is ineffective in activating the soluble enzyme. However, bleached rhodopsin could activate it in the presence of a very low concentration of ATP, strongly suggesting the mediation of rhodopsin in the light activation of the enzyme in ROS. Direct evidence is presented to suggest that the phosphorylation of opsin (bleached rhodopsin) is unrelated to the activation of PDE by bleached rhodopsin and ATP. The reconstitution of the light activation of PDE in a soluble system presented here opens up a new direction to future investigations on the mechanism of light regulation of cyclic GMP levels in retina and its implication in the photoreceptor function.     

Immunocytochemical demonstration of visual pigments in the degenerate retinal and pineal photoreceptors of the blind cave salamander (Proteus anguinus)

Visual pigments in the regressed eye and pineal of the depigmented neotenic urodele, the blind cave salamander (Proteus anguinus anguinus), were studied by immunocytochemistry with anti-opsin antibodies. The study included light- and electron-microscopic investigations of both the eye and the pineal organ. A comparison was made with the black pigmented subspecies Proteus anguinus parkelj (black proteus), which has a normal eye structure. In the retina of the black proteus, we found principal rods, red-sensitive cones and a third photoreceptor type, which might represent a blue- or UV-sensitive cone. Photoreceptors in the regressed eye of the blind cave salamanders from the Planina cave contained degenerate outer segments, consisting of a few whorled discs and irregular clumps of membranes. The great majority of these outer segments showed immunolabelling for the red-sensitive cone opsin and only a few of them were found to be positive for rhodopsin. An even more pronounced degeneration was observed in the photoreceptors of the animals derived from the Otovec doline, which are completely devoid of an outer segment, most of them not even possessing an inner segment. Even in some of these highly degenerate cells, the presence of rhodopsin could be detected in the plasma membrane; however, immunoreactions with antibodies recognizing cone visual pigment were negative. In the pineals of all studied animals, the degenerate photoreceptor outer segments were recognized exclusively by the antibody against the red-sensitive cone opsin. The presence of immunopositive visual pigments indicates the possibility of a retained light sensitivity in the blind cave salamander photoreceptors.