C.A.G.G. Driessen

Retinoic acid delays transcription of human retinal pigment neuroepithelium marker genes in ARPE-19 cells

&NA; The effect of retinoic acid on the differentiation of a human retinal pigment epithelium‐derived cell line ARPE‐19 was studied. Differentiation of ARPE‐19 cells is delayed by retinoic acid. The minimum all‐trans‐retinoic acid concentration needed for delay of ARPE‐19 differentiation is 1 μM. A delay of differentiation was also observed using 1 μM 9‐cis or 13‐cis‐retinoic acid. Differentiation at the molecular level was studied by analyzing transcription of two RPE‐marker genes, RPE65 and peropsin. In the presence of 1 μM retinoic acid the onset of transcription of both genes was delayed by 2–3 weeks. We conclude that alltrans‐, 9‐cis‐, and 13‐cis‐retinoic acid delay differentiation of ARPE‐19 cells into cells that phenotypically resemble cells from the human retinal pigment epithelium.

Dual-substrate Specificity Short Chain Retinol Dehydrogenases from the Vertebrate Retina

Retinoids are chromophores involved in vision, transcriptional regulation, and cellular differentiation. Members of the short chain alcohol dehydrogenase/reductase superfamily catalyze the transformation of retinol to retinal. Here, we describe the identification and properties of three enzymes from a novel subfamily of four retinol dehydrogenases (RDH11–14) that display dual-substrate specificity, uniquely metabolizing all-trans- andcis-retinols with C15 pro-Rspecificity. RDH11–14 could be involved in the first step of all-trans- and 9-cis-retinoic acid production in many tissues. RDH11–14 fill the gap in our understanding of 11-cis-retinal and all-trans-retinal transformations in photoreceptor (RDH12) and retinal pigment epithelial cells (RDH11). The dual-substrate specificity of RDH11 explains the minor phenotype associated with mutations in 11-cis-retinol dehydrogenase (RDH5) causing fundus albipunctatus in humans and engineered mice lacking RDH5. Furthermore, photoreceptor RDH12 could be involved in the production of 11-cis-retinal from 11-cis-retinol during regeneration of the cone visual pigments. These newly identified enzymes add new elements to important retinoid metabolic pathways that have not been explained by previous genetic and biochemical studies.

Disruption of the 11-cis-Retinol Dehydrogenase Gene Leads to Accumulation of cis-Retinols and cis-Retinyl Esters

ABSTRACT To elucidate the possible role of 11-cis-retinol dehydrogenase in the visual cycle and/or 9-cis-retinoic acid biosynthesis, we generated mice carrying a targeted disruption of the 11-cis-retinol dehydrogenase gene. Homozygous 11-cis-retinol dehydrogenase mutants developed normally, including their retinas. There was no appreciable loss of photoreceptors. Recently, mutations in the 11-cis-retinol dehydrogenase gene in humans have been associated with fundus albipunctatus. In 11-cis-retinol dehydrogenase knockout mice, the appearance of the fundus was normal and punctata typical of this human hereditary ocular disease were not present. A second typical symptom associated with this disease is delayed dark adaptation. Homozygous 11-cis-retinol dehydrogenase mutants showed normal rod and cone responses. 11-cis-Retinol dehydrogenase knockout mice were capable of dark adaptation. At bleaching levels under which patients suffering from fundus albipunctatus could be detected unequivocally, 11-cis-retinol dehydrogenase knockout animals displayed normal dark adaptation kinetics. However, at high bleaching levels, delayed dark adaptation in 11-cis-retinol dehydrogenase knockout mice was noticed. Reduced 11-cis-retinol oxidation capacity resulted in 11-cis-retinol/13-cis-retinol and 11-cis-retinyl/13-cis-retinyl ester accumulation. Compared with wild-type mice, a large increase in the 11-cis-retinyl ester concentration was noticed in 11-cis-retinol dehydrogenase knockout mice. In the murine retinal pigment epithelium, there has to be an additional mechanism for the biosynthesis of 11-cis-retinal which partially compensates for the loss of the 11-cis-retinol dehydrogenase activity. 11-cis-Retinyl ester formation is an important part of this adaptation process. Functional consequences of the loss of 11-cis-retinol dehydrogenase activity illustrate important differences in the compensation mechanisms between mice and humans. We furthermore demonstrate that upon 11-cis-retinol accumulation, the 13-cis-retinol concentration also increases. This retinoid is inapplicable to the visual processes, and we therefore speculate that it could be an important catabolic metabolite and its biosynthesis could be part of a process involved in regulating 11-cis-retinol concentrations within the retinal pigment epithelium of 11-cis-retinol dehydrogenase knockout mice.

Crystal structure of rhodopsin: a template for cone visual pigments and other G protein-coupled receptors

The crystal structure of rhodopsin has provided the first three-dimensional molecular model for a G-protein-coupled receptor (GPCR). Alignment of the molecular model from the crystallographic structure with the helical axes seen in cryo-electron microscopic (cryo-EM) studies provides an opportunity to investigate the properties of the molecule as a function of orientation and location within the membrane. In addition, the structure provides a starting point for modeling and rational experimental approaches of the cone pigments, the GPCRs in cone cells responsible for color vision. Homology models of the cone pigments provide a means of understanding the roles of amino acid sequence differences that shift the absorption maximum of the retinal chromophore in the environments of different opsins.

Retinol Dehydrogenase (RDH12) Protects Photoreceptors from Light-induced Degeneration in Mice

RDH12 has been suggested to be one of the retinol dehydrogenases (RDH) involved in the vitamin A recycling system (visual cycle) in the eye. Loss of function mutations in the RDH12 gene were recently reported to be associated with autosomal recessive childhood-onset severe retinal dystrophy. Here we show that RDH12 localizes to the photoreceptor inner segments and that deletion of this gene in mice slows the kinetics of all-trans-retinal reduction, delaying dark adaptation. However, accelerated 11-cis-retinal production and increased susceptibility to light-induced photoreceptor apoptosis were also observed in Rdh12-/- mice, suggesting that RDH12 plays a unique, nonredundant role in the photoreceptor inner segments to regulate the flow of retinoids in the eye. Thus, severe visual impairments of individuals with null mutations in RDH12 may likely be caused by light damage1.

Evaluation of the role of the retinal G protein-coupled receptor (RGR) in the vertebrate retina in vivo

The retinal G protein‐coupled receptor (RGR) is a protein that structurally resembles visual pigments and other G protein‐coupled receptors. RGR may play a role as a photoisomerase in the production of 11‐cis‐retinal, the chromophore of the visual pigments. As the proposed function of RGR, in a complex with 11‐cis‐retinol dehydrogenase (RDH5), is to regenerate 11‐cis‐retinal under light conditions and RDH5 is expected to function in the light‐independent part of the retinoid cycle, we speculated that the simultaneous loss of function of both proteins should more severely affect the rhodopsin regeneration capacity. Here, we evaluated the role of RGR using rgr–/– single and rdh5–/–rgr–/– double knockout mice under a number of light conditions. The most striking phenotype of rgr–/– mice after a single flash of light includes light‐dependent formation of 9‐cis‐ and 13‐cis‐retinoid isomers. These isomers are not formed in wild‐type mice because either all‐trans‐retinal is bound to RGR and protected from isomerization to 9‐cis‐ or 13‐cis‐retinal or because RGR is able to eliminate these isomers directly or indirectly. After intense bleaching, a transient accumulation of all‐trans‐retinyl esters and an attenuated recovery of 11‐cis‐retinal were observed. Finally, even under conditions of prolonged light illumination, as investigated in vitro in biochemical assays or in vivo by electroretinogram (ERG) measurements, no evidence of catalytic‐like photoisomerization‐driven production of 11‐cis‐retinal could be attained. These and previous results suggest that RGR and RDH5 are likely to function in the retinoid cycle, although their role is not essential and regeneration of visual pigment is only mildly affected by the absence of both proteins in rod‐dominated mice.

The visual cycle retinol dehydrogenase: possible involvement in the 9‐cis retinoic acid biosynthetic pathway

The 11‐cis‐retinol dehydrogenase (11‐cis‐RoDH) gene encodes the short‐chain alcohol dehydrogenase responsible for 11‐cis‐retinol oxidation in the visual cycle. The structure of the murine 11‐cis‐RoDH gene was used to reinvestigate its transcription pattern. An 11‐cis‐RoDH gene transcript was detected in several non‐ocular tissues. The question regarding the substrate specificity of the enzyme was therefore addressed. Recombinant 11‐cis‐RoDH was found capable of oxidizing and reducing 9‐cis‐, 11‐cis‐ and 13‐cis‐isomers of retinol and retinaldehyde, respectively. Dodecyl‐β‐1‐maltoside used to solubilize the enzyme was found to affect the substrate specificity. This is the first report on a visual cycle enzyme also present in non‐retinal ocular and non‐ocular tissues. A possible role in addition to its role in the visual cycle is being discussed.

Null mutation in the human 11-cis retinol dehydrogenase gene associated with fundus albipunctatus

PURPOSE Recent studies show that mutations in the gene encoding 11-cis retinol dehydrogenase are associated with fundus albipunctatus. The authors wanted to investigate whether additional, more severe, mutations in the 11-cis retinol dehydrogenase gene might be responsible for more severe forms of hereditary retinal diseases. DESIGN Case-control molecular genetics study. PARTICIPANTS AND CONTROLS Two index patients, 7 relatives, and 50 control individuals. METHODS The authors screened two index patients diagnosed with fundus albipunctatus for mutations in exons 2 to 5 and exon/intron boundaries of the 11-cis retinol dehydrogenase gene by direct sequencing. Control individuals were screened for the presence of the mutations using allele-specific oligonucleotide hybridization. MAIN OUTCOME MEASURES Mutations in exons 2 to 5 and exon/intron boundaries of the 11-cis retinol dehydrogenase gene. RESULTS In a compound heterozygote, two novel mutations were found: a 4 bp insertion in exon 2 and a missense mutation Cys267Trp in exon 5. In a second pedigree, a homozygous frameshift mutation in codon 43 (Arg42ct[1-bpdel]) was detected. In both families, the mutations segregate with the disease. The mutations were not found in 50 control individuals. CONCLUSIONS On the basis of our observations, it is unlikely that mutations in the 11-cis retinol dehydrogenase gene are associated with other, possibly more severe, retinal pathologic conditions/dystrophies or syndromic diseases in which the retina is also affected.

Novel targeting strategy for generating mouse models with defects in the retinoid cycle.

In addition to RDH5, other enzymes capable of oxidizing 11-cis-retinol are present within the retinal pigment epithelium, Müller cells and/or photoreceptors. Candidate proteins have meanwhile been identified. To study the physiological and pathological aspects of these enzymes, mice in which these genes are no longer functional are being generated. A fast-targeting strategy for the disruption of genes was developed. Generation of double and triple knockouts will aid in determining if these retinol dehydrogenases are responsible for the remaining 11-cis-retinol oxidation observed in RDH5 knockout animals.

Cloning and Expression of a Cdna-Encoding Bovine Retinal-Pigment Epithelial 11-Cis Retinol Dehydrogenase

PURPOSE Identification of a 32-kd protein in the bovine retinal pigment epithelium. METHODS A bovine retinal pigment epithelium cDNA library was constructed in the bacteriophage lambda ZAP Express. A monoclonal antibody, designated 21-C3/AV, was used to isolate the cDNA encoding the 21-C3/AV antigen. A positive full-length clone, designated 21-C3RDH/CD, was sequenced. Northern blot analysis was used to determine the length of the mRNA and the tissue expression pattern. The entire open reading frame of clone 21-C3RDH/CD was used to isolate a recombinant baculovirus clone and expressed in Spodoptera frugiperda insect cells. Enzymatic activity toward 11-cis retinaldehyde was investigated. RESULTS The complete nucleotide sequence of 21-C3RDH/CD was obtained. The deduced amino acid sequence reveals homology with short-chain alcohol dehydrogenases. Northern blot analysis detected a 1.2-kb transcript. Although the monoclonal antibody used to isolate 21-C3RDH/CD also reacts with other ocular and nonocular tissues, the authors were unable to demonstrate any reactivity with RNA samples isolated from different (non)ocular tissues. Recombinant baculovirus-infected insect cells synthesized the 21-C3/AV antigen. This protein showed 11-cis retinol dehydrogenase activity. CONCLUSIONS Homology to the human D-beta-hydroxybutyrate dehydrogenase precursor and other alcohol dehydrogenases shows that 21-C3RDH/CD encodes a short-chain alcohol dehydrogenase. Furthermore, tissue specificity and molecular weight of the antigen suggest that 21-C3RDH/CD encodes the bovine retinal pigment epithelial 11-cis retinol dehydrogenase. Direct proof came from experiments in which we used the baculovirus-based expression system for in vitro synthesis of the protein encoded by 21-C3RDH/CD. Protein extracts obtained from recombinant baculovirus-infected insect cells were found capable of reducing 11-cis retinaldehyde.