J.J.M. Janssen

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.

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.

Derogation and distancing as terror management strategies: the moderating role of need for closure and permeability of group boundaries.

Previous research has revealed that when individuals are confronted with criticism of a personally relevant group, mortality salience can lead to either derogation of the source of criticism or distancing from the group. In this article, the authors investigated closure as a potential moderator of these reactions. In Study 1, mortality salience led to greater derogation of a critic of a relevant group among high-need-for-closure participants but led to distancing from the group among low need-for-closure participants. Study 2 showed that when a relevant group was criticized, mortality salience led to greater derogation among participants who were led to believe that the boundaries of that group were impermeable but led to greater distancing among participants who were made aware of the permeable nature of the group boundaries. These findings demonstrate that closure of group membership moderates reactions to criticism of a personally relevant group after mortality salience.

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.

Synthesis of functional bovine opsin in insect cells under control of the baculovirus polyhedrin promoter.

In vitro expression of cDNA encoding bovine opsin is accomplished using the baculovirus expression vector system. Full-length opsin was synthesized which was recognized by poly- and monoclonal antisera raised against bovine rhodopsin. Upon infection with a recombinant virus, 1×106 insect cells produced up to 3 μg opsin. Incubation of the in vitro synthesized opsin with 11-cis retinal produced a hydroxylamine-stable, photosensitive pigment.

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.

Development and degeneration of retina in rds mutant mice: immunoassay of the rod visual pigment rhodopsin

Development and loss of photoreceptor cells in mice, afflicted by the rds (retinal degeneration slow) gene, was analyzed by measuring the ocular visual pigment content as rhodopsin (spectroscopy) and opsin (immunoassay). With regard to the postnatal age, where opsin was just detectable, and to the initial rate of opsin synthesis, the mutants did not strongly deviate from the normal animals. The final maximal visual pigment level was, however, about half of normal for the heterozygous mutants and about 3% of normal for the homozygous mutants, both in the pigmented and in the albino strain. In the pigmented normal or heterozygous mutant the (rhod)opsin levels remain stable up to at least 1 year of age. For the corresponding albino animals this was only observed up to 9 months of age. Thereafter the level declines. In the homozygous mutants, maximal opsin levels were observed at about 3 weeks postnatal. Subsequently, this level gradually declined to about 40% in the pigmented and about 15% in the albino mutant. The results indicate that the rds gene does not directly affect the biosynthetic pathways of opsin. The physiological effect of the rds gene is aggravated by photodamage for which the albino animal is particularly susceptible.

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.