The sights along route 65.

Regeneration of rod visual pigments after photobleaching requires a protein called Rpe65. Several studies clarify its role in visual physiology and pathology, including a new one that shows it is required pigment regeneration in cone cells.

Multiple phosphorylation of rhodopsin and the in vivo chemistry underlying rod photoreceptor dark adaptation.

Dark adaptation requires timely deactivation of phototransduction and efficient regeneration of visual pigment. No previous study has directly compared the kinetics of dark adaptation with rates of the various chemical reactions that influence it. To accomplish this, we developed a novel rapid-quench/mass spectrometry-based method to establish the initial kinetics and site specificity of light-stimulated rhodopsin phosphorylation in mouse retinas. We also measured phosphorylation and dephosphorylation, regeneration of rhodopsin, and reduction of all-trans retinal all under identical in vivo conditions. Dark adaptation was monitored by electroretinography. We found that rhodopsin is multiply phosphorylated and then dephosphorylated in an ordered fashion following exposure to light. Initially during dark adaptation, transduction activity wanes as multiple phosphates accumulate. Thereafter, full recovery of photosensitivity coincides with regeneration and dephosphorylation of rhodopsin.

The color difference in orbital fat.

OBJECTIVE: To identify and quantify carotenoids found in white and yellow orbital fat. METHODS: Specimens of nasal (white) and preaponeurotic (yellow) orbital fat were obtained from patients during upper eyelid blepharoplasty. Carotenoids and retinoids were extracted and subjected to spectral and high-performance liquid chromatography analyses. RESULTS: The chromophore content of extracts from unsaponified fat, as measured by absorbance at 425 nm per gram of fat, was 2- to 4-fold higher in preaponeurotic fat than in nasal fat. High-performance liquid chromatography analysis from enzymatically digested fat revealed large amounts of lutein, beta-carotene, and retinol and small amounts of other unidentified carotenoids. The amount of beta-carotene and lutein in preaponeurotic fat was approximately 4-fold higher than in nasal fat. CONCLUSIONS: The higher carotenoid content of preaponeurotic fat might cause it to be more yellow than other orbital fat, and lutein and beta-carotene might be selectively absorbed from plasma by preaponeurotic fat. CLINICAL RELEVANCE: The results provide baseline information for studies of the physiological features of orbital fat in normal and diseased conditions.

Visual cycle impairment in cellular retinaldehyde binding protein (CRALBP) knockout mice results in delayed dark adaptation.

Mutations in the human CRALBP gene cause retinal pathology and delayed dark adaptation. Biochemical studies have not identified the primary physiological function of CRALBP. To resolve this, we generated and characterized mice with a non-functional CRALBP gene (Rlbp1(-/-) mice). The photosensitivity of Rlbp1(-/-) mice is normal but rhodopsin regeneration, 11-cis-retinal production, and dark adaptation after illumination are delayed by >10-fold. All-trans-retinyl esters accumulate during the delay indicating that isomerization of all-trans- to 11-cis-retinol is impaired. No evidence of photoreceptor degeneration was observed in animals raised in cyclic light/dark conditions for up to 1 year. Albino Rlbp(-/-) mice are protected from light damage relative to the wild type. These findings support a role for CRALBP as an acceptor of 11-cis-retinol in the isomerization reaction of the visual cycle.

Kinetics of visual pigment regeneration in excised mouse eyes and in mice with a targeted disruption of the gene encoding interphotoreceptor retinoid-binding protein or arrestin.

Photoisomerization of 11-cis-retinal to all-trans-retinal and reduction to all-trans-retinol occur in photoreceptor outer segments whereas enzymatic esterification of all-trans-retinol, isomerization to 11-cis-retinol, and oxidation to 11-cis-retinal occur in adjacent cells. The processes are linked into a visual cycle by intercellular diffusion of retinoids. Knowledge of the mechanistic aspects of the visual cycle is very limited. In this study, we utilize chemical analysis of visual cycle retinoids to assess physiological roles for components inferred from in vitro experiments and to understand why excised mouse eyes fail to regenerate their bleached visual pigment. Flash illumination of excised mouse eyes or eyecups, in which regeneration of rhodopsin does not occur, produced a block in the visual cycle after all-trans-retinal formation; constant illumination of eyecups produced a block in the cycle after all-trans-retinol formation; and constant illumination of whole excised eyes resulted in a block of the cycle after formation of all-trans-retinyl ester. These blocks emphasize the role of cellular metabolism in the visual cycle. Interphotoreceptor retinoid-binding protein (IRBP) has been postulated to play a role in intercellular retinoid transfer in the retina; however, the rates of recovery of 11-cis-retinal and of regeneration of rhodopsin in the dark in IRBP-/- mice were very similar to those found with wild-type (wt) mice. Thus, IRBP is necessary for photoreceptor survival but is not essential for a normal rate of visual pigment turnover. Arrestin forms a complex with activated rhodopsin, quenches its activity, and affects the release of all-trans-retinal in vitro. The rate of recovery of 11-cis-retinal in arrestin-/- mice was modestly delayed relative to wt, and the rate of rhodopsin recovery was approximately 80% of that observed with wt mice. Thus, the absence of arrestin appeared to have a minor effect on the kinetics of the visual cycle.

Preferential release of 11-cis-retinol from retinal pigment epithelial cells in the presence of cellular retinaldehyde-binding protein.

In photoreceptor cells of the retina, photoisomerization of 11-cis-retinal to all-trans-retinal triggers phototransduction. Regeneration of 11-cis-retinal proceeds via a complex set of reactions in photoreceptors and in adjacent retinal pigment epithelial cells where all-trans-retinol is isomerized to 11-cis-retinol. Our results show that isomerization in vitro only occurs in the presence of apo-cellular retinaldehyde-binding protein. This retinoid-binding protein may drive the reaction by mass action, overcoming the thermodynamically unfavorable isomerization. Furthermore, this 11-cis-retinol/11-cis-retinal-specific binding protein potently stimulates hydrolysis of endogenous 11-cis-retinyl esters but has no effect on hydrolysis of all-trans-retinyl esters. Apo-cellular retinaldehyde-binding protein probably exerts its effect by trapping the 11-cis-retinol product. When retinoid-depleted retinal pigment epithelial microsomes were preincubated with different amounts of all-trans-retinol to form all-trans-retinyl esters and then [3H]all-trans-retinol was added, as predicted, the specific radioactivity of [3H]all-trans-retinyl esters increased during subsequent reaction. However, the specific radioactivity of newly formed 11-cis-retinol stayed constant during the course of the reaction, and it was largely unaffected by expansion of the all-trans-retinyl ester pool during the preincubation. The absence of dilution establishes that most of the ester pool does not participate in isomerization, which in turn suggests that a retinoid intermediate other than all-trans-retinyl ester is on the isomerization reaction pathway.

Molecular characterization of the mouse gene encoding cellular retinaldehyde-binding protein.

PURPOSE: To clone and characterize the mouse gene encoding cellular retinaldehyde-binding protein (CRALBP). CRALBP appears to modulate enzymatic generation and processing of 11-cis-retinol and regeneration of visual pigment in the vertebrate visual cycle. Mutations in human CRALBP segregate with autosomal recessive retinitis pigmentosa. METHODS: A genomic clone encompassing the 5' end of the CRALBP gene through exon 6 was isolated from a mouse 129/Sv genomic DNA library. Exons 7 and 8 were PCR amplified from mouse eye cDNA and 129/SvJ genomic DNA. The gene structure was determined by automated DNA sequence analysis. RESULTS: The sequence of 6855 nucleotides was determined, including all 8 exons, 3 introns plus 3932 and 629 bases from the 5'- and 3'-flanking regions, respectively. The lengths of introns 3-6 were determined by PCR amplification. Northern analysis identifies a approximately 2.1 kb transcript in mouse eye; Southern analysis supports a single copy gene. CONCLUSIONS: The mouse CRALBP gene is similar to the human gene; the coding sequence is approximately 87% identical, the non-coding sequence approximately 65% identical. In contrast to the human gene, the mouse gene contains a consensus TATA box. One of two photoreceptor consensus elements important for CRALBP expression in human retinal pigment epithelium is also present in the mouse gene. Additional conserved and species-specific consensus sequences are identified. The mouse CRALBP genomic clones and structure provide valuable tools for developing an in vivo model to study protein function and gene regulation.

Molecular characterization of a novel short-chain dehydrogenase/reductase that reduces all-trans-retinal.

The reduction of all-trans-retinal in photoreceptor outer segments is the first step in the regeneration of bleached visual pigments. We report here the cloning of a dehydrogenase, retSDR1, that belongs to the short-chain dehydrogenase/reductase superfamily and localizes predominantly in cone photoreceptors. retSDR1 expressed in insect cells displayed substrate specificities of the photoreceptor all-trans-retinol dehydrogenase. Homology modeling of retSDR1 using the carbonyl reductase structure as a scaffold predicted a classical Rossmann fold for the nucleotide binding, and an N-terminal extension that could facilitate binding of the enzyme to the cell membranes. The presence of retSDR1 in a subset of inner retinal neurons and in other tissues suggests that the enzyme may also be involved in retinol metabolism outside of photoreceptors.

Cellular retinaldehyde-binding protein ligand interactions. Gln-210 and Lys-221 are in the retinoid binding pocket.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the retinal pigment epithelium (RPE) and Müller cells of the retina and has been linked with autosomal recessive retinitis pigmentosa. Ligand interactions determine the physiological role of CRALBP in the RPE where the protein is thought to function as a substrate carrier for 11-cis-retinol dehydrogenase in the synthesis of 11-cis-retinal for visual pigment regeneration. However, CRALBP is also present in optic nerve and brain where its natural ligand and function are not yet known. We have characterized the interactions of retinoids with native bovine CRALBP, human recombinant CRALBP (rCRALBP) and five mutant rCRALBPs. Efforts to trap and/or identify a Schiff base in the dark, under a variety of reducing, denaturing, and pH conditions were unsuccessful, suggesting the lack of covalent interactions between CRALBP and retinoid. Buried and solvent-exposed lysine residues were identified in bovine CRALBP by reductive methylation of the holoprotein followed by denaturation and reaction with [3H]acetic anhydride. Radioactive lysine residues were identified by Edman degradation and electrospray mass spectrometry following proteolysis and purification of modified peptides. Human rCRALBP mutants K152A, K221A, and K294A were prepared to investigate possible retinoid interactions with buried or partially buried lysines. Two other rCRALBP mutants, I162V and Q210R, were also prepared to identify substitutions altering the retinoid binding properties of a random mutant. The structures of all the mutants were verified by amino acid and mass spectral analyses and retinoid binding properties evaluated by UV-visible and fluorescence spectroscopy. All of the mutants bound 11-cis-retinal essentially like the wild type protein, indicating that the proteins were not grossly misfolded. Three of the mutants bound 9-cis-retinal like the wild type protein; however, Q210R and K221A bound less than stoichiometric amounts of the 9-cis-isomer and exhibited lower affinity for this retinoid relative to wild type rCRALBP. Residues Gln-210 and Lys-221 are located within a region of CRALBP exhibiting sequence homology with the ligand binding cavity of yeast phosphatidylinositol-transfer protein. The data implicate Gln-210 and Lys-221 as components of the CRALBP retinoid binding cavity and are discussed in the context of ligand interactions in structurally or functionally related proteins with known crystallographic structures.

Reduction of all-trans-retinal limits regeneration of visual pigment in mice.

Absorption of photons by pigments in photoreceptor cells results in photoisomerization of the chromophore, 11-cis-retinal, to all-trans-retinal and activation of opsin. Photolysed chromophore is converted back to the 11-cis-configuration via several enzymatic steps in photoreceptor and retinal pigment epithelial cells. We investigated the levels of retinoids in mouse retina during constant illumination and regeneration in the dark as a means of obtaining more information about the rate-limiting step of the visual cycle and about cycle intermediates that could be responsible for desensitization of the visual system. All-trans-retinal accumulated in the retinas during constant illumination and following flash illumination. Decay of all-trans-retinal in the dark following constant illumination occurred without substantial accumulation of all-trans-retinal, generated by constant approximately equal to visual pigment regeneration (t1/2 approximately 5 and t1/2 approximately 7 min, respectively). All-trans-retinal, generated by constant illumination, decayed approximately 3 times more rapidly than that generated by a flash and, as shown previously, the rate of rhodopsin regeneration following a flash was approximately 4 times slower than after constant illumination. The retinyl ester pool (> 95% all-trans-retinyl ester) did not show a statistically significant change in size or composition during illumination. In addition, constant illumination increased the amount of photoreceptor membrane-associated arrestin. The results suggest that the rate-limiting step of the visual cycle is the reduction of all-trans-retinal to all-trans-retinol by all-trans-retinol dehydrogenase. The accumulation of all-trans-retinal during illumination may be responsible, in part, for the reduction in sensitivity of the visual system that accompanies photobleaching and may contribute to the development of retinal pathology associated with light damage and aging.

Cellular retinaldehyde-binding protein is expressed by oligodendrocytes in optic nerve and brain.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium and Müller glial cells of the retina, where it forms complexes with endogenous 11-cis-retinoids. We examined the distribution of CRALBP in extraretinal tissues using polyclonal antibodies (pAb) and monoclonal antibodies (mAb). A protein was detected by immunoblot analysis in extracts of bovine and rat brain and optic nerve but not in several other tissues. This protein had electrophoretic, chromatographic, and retinoid-binding properties identical to those of CRALBP from bovine retina. Comparison of the masses of tryptic peptides and of partial amino acid sequences derived from brain and retinal CRALBP indicated that the two proteins are probably identical. Immunoperoxidase cytochemistry and double labeling immunofluorescence revealed CRALBP(+) cells in brain that resembled oligondendrocytes and not astrocytes, microglial cells, or pinealocytes. In 11-day-old rat brain, approximately 11% of the CRALBP(+) cells were labeled with the Rip antibody, a marker for oligodendroglia. In developing rat optic nerve, the temporal appearance of CRALBP(+) cells corresponded to that of oligodendrocytes and not that of astrocytes. In adult rat and mouse optic nerves, the CRALBP(+) somata showed the same distribution as oligodendrocytes. No endogenous retinoids were associated with CRALBP isolated from dark-dissected adult bovine brain. The results suggest that CRALBP has functions in addition to retinoid metabolism and visual pigment regeneration.

Activation and inactivation steps in the visual transduction pathway.

Recent genetic, biochemical and electrophysiological evidence has provided insights into the molecular identity of the substance responsible for bleaching desensitization in vision. Studies examining the molecular defects that cause delayed dark adaptation suggest that the desensitizing substance is recognized by rhodopsin kinase and/or arrestin and, therefore, is probably a complex comprising all-trans-retinal and opsin.

Characterization of aldehyde dehydrogenase-positive amacrine cells restricted in distribution to the dorsal retina.

A class 1 aldehyde dehydrogenase (ALDH) catalyzes oxidation of retinaldehyde to retinoic acid in bovine retina. We used immunocytochemistry and in situ hybridization to localize this enzyme in adult and fetal bovine retinas. Specific ALDH immunoreactivity was present in the cytoplasm of wide-field amacrine cells restricted in distribution to the dorsal part of the adult retina. The somata diameters ranged from approximately 8 microns to approximately 15 microns, and the cells increased in density from approximately 125 cells/mm2 near the horizontal meridian to approximately 425 cells/mm2 in the superior far periphery. The ALDH-positive cells had somata on both sides of the inner plexiform layer (IPL) and processes in two IPL strata. The majority of ALDH-positive cells were unreactive with antibodies against known amacrine cell enzymes and neurotransmitters, including GABA and glycine. The ALDH-positive amacrine cells also did not react with anti-cellular retinoic acid-binding protein, which was present in a subset of GABA-positive amacrine cells. In flat-mounted retinas processed by in situ hybridization, the larger ALDH-positive amacrine cells tended to be more heavily labeled. In addition to amacrine cells, Müller cell processes in the inner retina were weakly immunoreactive for ALDH; however, these glial cells did not contain ALDH mRNA. The pattern of ALDH expression in fetal bovine retinas was documented by immunocytochemistry. No ALDH reactivity was found before 5.5 months; for the remainder of the fetal period, ALDH immunoreactivity was present in amacrine cells similar to those in adult retina. The ALDH-positive amacrine cells in bovine retina are novel, being limited in distribution to the dorsal retina and unlabeled with other amacrine cell-specific markers. Identification of ALDH in amacrine cells provides additional evidence that cells of the inner retina are involved in retinoid metabolism.

Mechanisms of opsin activation.

Rhodopsin is constrained in an inactive conformation by interactions with 11-cis-retinal including formation of a protonated Schiff base with Lys296. Upon photoisomerization, major structural rearrangements that involve protonation of the active site Glu113 and cytoplasmic acidic residues, including Glu134, lead to the formation of the active form of the receptor, metarhodopsin II b, which decays to opsin. However, an activated receptor may be generated without illumination by addition of all-trans-retinal or its analogues to opsin, as measured in this study by the increased phosphorylation of opsin by rhodopsin kinase. The potency of stimulation depended on the chemical and isomeric nature of the analogues and the length of the polyene chain with all-trans-C17 aldehyde and all-trans-retinal being the most active and trans-C12 aldehyde being the least active. Certain cis-isomers, 11-cis-13-demethyl-retinal and 9-cis-C17 aldehyde, were also active. Most of the retinal analogues tested did not regenerate a spectrally identifiable pigment, and many were incapable of Schiff base formation (ketone, stable oximes, and Schiff base-derivatives of retinal). Thus, receptor activation resulted from formation of non-covalent complexes with opsin. pH titrations suggested that an equilibrium exists between partially active (protonated) and inactive (deprotonated) forms of opsin. These findings are consistent with a model in which protonation of one or more cytoplasmic carboxyl groups of opsin is essential for activity. Upon addition of retinoids, the partially active conformation of opsin is converted to a more active intermediate similar to metarhodopsin II b. The model provides an understanding of the structural requirements for opsin activation and an interpretation of the observed activities of natural and experimental opsin mutants.

Characterization and localization of an aldehyde dehydrogenase to amacrine cells of bovine retina.

An enzyme of bovine retina that catalyzes oxidation of retinaldehyde to retinoic acid was purified to homogeneity and a monoclonal antibody (mAb H-4) was generated. MAb H-4 recognized a single component (Mr = 55,000) in extracts of bovine retina and other bovine tissues. The antibody showed no cross-reactivity with extracts of rat, monkey, or human retinas. A 2067 bp cDNA was selected from a retina cDNA expression library using mAb H-4. The cDNA hybridized with a similarly sized, moderately abundant mRNA prepared from bovine retina. Nucleotide sequence analysis indicated that the cDNA contained a single open reading frame encoding 501 amino acids that have 88% sequence identity with the amino-acid sequence of human hepatic Class 1 aldehyde dehydrogenase. Amino-acid sequence analysis of purified enzyme demonstrated that the cDNA encodes the isolated enzyme. MAb H-4 specifically labeled the somata and processes of a subset of amacrine cells in bovine retinal sections. Labeled amacrine somata were located on both sides of the inner plexiform layer, and their processes ramified into two laminae within the inner plexiform layer. The inner radial processes of Müller (glial) cells were weakly reactive with mAb H-4. Weak immunostaining of amacrine cells was found in monkey retina with mAb H-4, but no signal was detected in rat or human retina. The results provide further evidence for metabolism and function of retinoids within cells of the inner retina and define a novel class of retinal amacrine cells.