Levels of retinoic acid and retinaldehyde dehydrogenase expression in eyes of the Mitf-vit mouse model of retinal degeneration.

PURPOSE: Several reports have characterized the retinal degeneration observed in the Mitf(vit) mutant mouse. Despite these reports, the factor(s) that may cause or modulate the degeneration still are not well defined; however, it is known that the photoreceptors of Mitf(vit) mice die through an apoptotic mechanism. We reported previously that retinoid metabolism in the RPE of Mitf(vit)++ mice is perturbed. Retinoids regulate genes via the RAR and RXR nuclear receptor pathway that are involved in numerous cellular responses including apoptosis. It is possible that retinoic acid (RA) modulates the retinal degeneration observed in the Mitf(vit) mice. The purpose of this study was to evaluate the levels of RA in whole eyes, as well as its distribution between neural retina and RPE, of the Mitf(vit) mutant mouse model. An additional purpose was to examine the expression of the RA generating enzyme, retinaldehyde dehydrogenase (AHD2), in the eyes of mutant and control mice. METHODS: The distribution of AHD2 in eyes of pre- and postnatal Mitf(vit) and C57BL/6 wild-type mice was determined immunohistochemically. Quantitative and qualitative analyses of RA were performed using reversed-phase high performance liquid chromatography (HPLC). RESULTS: The distribution of AHD2 in ocular tissues was similar between pre- and postnatal Mitf(vit) and C57BL/6 control mice. At postnatal week 10, however, a marked increase in AHD2 immunoreactivity was noted in the central dorsal neural retina of Mitf(vit) mice. No differences in the level of total RA in whole eyes were noted between Mitf(vit) and control mice at early postnatal ages. By 10 weeks of age there was a significant elevation of RA that was localized to the neural retina. CONCLUSIONS: In this study, we show a high level of AHD2 and RA in the neural retina of Mitf(vit) mice relative to control mice. It is possible that this elevation of RAs contributes to the retinal degeneration observed in Mitf(vit) mice either by inducing apoptosis or by enhancing the effect of some other factor(s) involved in the apoptotic pathway.

Delayed rhodopsin regeneration and altered distribution of interphotoreceptor retinoid binding protein (IRBP) in the mi(vit)/mi(vit) (vitiligo) mouse.

Rhodopsin regeneration requires attachment between the retinal pigment epithelium (RPE) and rod outer segments; however, in experimentally induced retinal detachment, rhodopsin regeneration can be restored partially upon addition of IRBP (interphotoreceptor retinoid binding protein). The mi(vit)/mi(vit) (vitiligo) mutant mouse, a model of slowly progressing photoreceptor cell degeneration, has a marked elevation of IRBP at 4 weeks as well as progressive detachment of the retina. The purpose of this study was to determine whether this mutant is capable of regenerating rhodopsin within a few hours following an intense light bleach. Rhodopsin regeneration was determined spectrophotometrically in mice after an intense one hour light bleach followed by 0,1,2,4 or 24 h of dark recovery. IRBP was localized immunohistochemically in fixed frozen tissue at the light microscopic level and in LR Gold embedded tissue at the ultrastructural level. Rhodopsin regeneration experiments indicated that rhodopsin levels following 0,1,2 and 4 h dark-recovery were significantly less in mi(vit)/mi(vit) mutants compared with controls. Immunohistochemical detection of IRBP indicated an altered distribution of the protein in the mutant mice compared with controls. There was accumulation in the region of the inner segments in mutant retinas rather than distribution only to the RPE/OS apical regions as in controls. The data suggest that regeneration of rhodopsin is reduced by 4 weeks postnatally in the mi(vit)/mi(vit) mouse. There is partial detachment of the retina at this age; and IRBP, thought to be essential for proper functioning of the visual cycle, is aberrantly distributed in this mutant.

Control of Drosophila retinoid and fatty acid binding glycoprotein expression by retinoids and retinoic acid: northern, western and immunocytochemical analyses.

In Drosophila, thorough retinoid deprivation is possible, optimizing investigation of the effects of vitamin A metabolites and retinoic acid on the visual system. Retinoids had been found to control transcription and translation of Drosophila's opsin gene. To follow this line of inquiry, we examined the effect of retinoids on the translation and transcription of a Drosophila Retinoid and Fatty Acid Binding Glycoprotein. Western blots showed that this protein is high in retinoid replete flies and low in deprived flies. Flies grown on media capable of activating the opsin gene's transcription and which contain alternate transcription activators including retinoic acid yielded extracts containing significant amounts of Retinoid and Fatty Acid Binding Glycoprotein. Immunocytochemistry confirmed its absence in deprived flies and its presence in flies reared or replaced on these diverse media containing retinoids or general nutrients. Immunocytochemistry localized Retinoid and Fatty Acid Binding Glycoprotein to the Semper (cone) cells and the intraommatidial matrix (the interphotoreceptor matrix of the ommatidium). Positive staining of Semper cells in mutants of the opsin gene and a mutant lacking receptors suggests that Retinoid and Fatty Acid Binding Glycoprotein does not depend on presence of opsin and that it is not synthesized in receptor cells respectively. Northern blots demonstrated greatly diminished mRNA for Retinoid and Fatty Acid Binding Glycoprotein in flies grown on deprivation food relative to flies grown on normal food. Although the synthesis of Retinoid and Fatty Acid Binding Glycoprotein does not require chromophore precursors as does that of opsin, the control of Retinoid and Fatty Acid Binding Glycoprotein and opsin transcription by retinoids including retinoic acid might very well be the same. Our results suggest that Retinoid and Fatty Acid Binding Glycoprotein may be involved in retinoid transport. Also, Semper cells may be analogous to vertebrate retinal pigment epithelium in retinoid metabolism and/or delivery.

Choroideremia: a clinical, electron microscopic, and biochemical report.

An asymptomatic 19-year-old male with choroideremia had diffuse loss of retinal pigment epithelium (RPE) and choroid except for the periphery and macula. Fluorescein angiography of the arteriovenous phase showed absence of retinal pigment epithelium and exaggerated visualization of choroidal vessels in involved areas. The mother was a typical carrier with pigment stippling of the midperipheral retina. Histopathologic examination of affected areas of one eye showed marked degeneration of the outer and midretina with loss of retinal pigment epithelium and Bruch's membrane, absence of choriocapillaris, chorioretinal adhesions and gliosis. Atrophy of inner and mid-choroid was also observed. Pigmented macrophage-like cells had migrated into the outer and midretinal layers. Electron microscopy disclosed macrophage-like cells with trilaminar structures and photoreceptor phagosomes in the RPE and outer retina. Remnants of photoreceptor outer segments were adherent to the plasma membranes of the macrophage-like cells. Biochemical analysis of retinal tissue samples for interphotoreceptor retinoid-binding protein (IRBP) showed marked reduction in the 146K bands in the equator and posterior pole in the patient compared to controls. Cyclic nucleotide content was altered in the retinal equator. Cyclic AMP was several-fold higher in the RPE-choroid complex of the affected eye than in the control.