Roxana A. Radu

Recessive Stargardt's macular degeneration is an inherited blinding disease of children caused by mutations in the ABCR gene. The primary pathologic defect in Stargardt's disease is accumulation of toxic lipofuscin pigments such as N-retinylidene-N-retinylethanolamine (A2E) in cells of the retinal pigment epithelium. This accumulation appears to be responsible for the photoreceptor death and severe visual loss in Stargardt's patients. Here, we tested a therapeutic strategy to inhibit lipofuscin accumulation in a mouse model of recessive Stargardt's disease. Isotretinoin (Accutane) has been shown to slow the synthesis of 11-cis-retinaldehyde and regeneration of rhodopsin by inhibiting 11-cis-retinol dehydrogenase in the visual cycle. Light activation of rhodopsin results in its release of all-trans-retinaldehyde, which constitutes the first reactant in A2E biosynthesis. Accordingly, we tested the effects of isotretinoin on lipofuscin accumulation in abcr(-/-) knockout mice. Isotretinoin blocked the formation of A2E biochemically and the accumulation of lipofuscin pigments by electron microscopy. We observed no significant visual loss in treated abcr(-/-) mice by electroretinography. Isotretinoin also blocked the slower, age-dependent accumulation of lipofuscin in wild-type mice. These results corroborate the proposed mechanism of A2E biogenesis. Further, they suggest that treatment with isotretinoin may inhibit lipofuscin accumulation and thus delay the onset of visual loss in Stargardt's patients. Finally, the results suggest that isotretinoin may be an effective treatment for other forms of retinal or macular degeneration associated with lipofuscin accumulation.

PURPOSE: Excessive accumulation of lipofuscin is observed in numerous degenerative retinal diseases. A toxic vitamin A-based fluorophore (A2E) present within lipofuscin has been implicated in the death of RPE and photoreceptor cells. Here, we used an animal model that manifests accelerated lipofuscin accumulation (ABCA4-/- mutant) to evaluate the efficacy of a therapeutic approach based on reduction of serum retinol. METHODS: N-(4-hydroxyphenyl)retinamide (HPR) potently and reversibly reduces serum retinol. The interaction of HPR with retinol binding protein (RBP) and transthyretin was studied by spectrofluorometry and size-exclusion chromatography. To assess the effects of HPR on visual cycle retinoids and A2E biosynthesis, HPR was chronically administered to ABCA4-/- mice. Mice were evaluated using biochemical, electrophysiological, and morphologic techniques. RESULTS: Administration of HPR to ABCA4-/- mice caused immediate, dose-dependent reductions in serum retinol and RBP. Chronic administration produced commensurate reductions in visual cycle retinoids and arrested accumulation of A2E and lipofuscin autofluorescence in the RPE. Physiologically, HPR treatment caused modest delays in dark adaptation. Chromophore regeneration kinetics, light sensitivity of photoreceptors, and phototransduction processes were normal. Histologic examinations showed no alteration of retinal cytostructure or morphology. CONCLUSIONS: These findings demonstrate the vitamin A-dependent nature of A2E biosynthesis and validate a novel therapeutic approach with potential to halt the accumulation of lipofuscin fluorophores in the eye.

PURPOSE: Dietary supplementation with vitamin A is sometimes prescribed as a treatment for retinitis pigmentosa, a group of inherited retinal degenerations that cause progressive blindness. Loss-of-function mutations in the ABCA4 gene are responsible for a subset of recessive retinitis pigmentosa. Other mutant alleles of ABCA4 cause the related diseases, recessive cone-rod dystrophy, and recessive Stargardt macular degeneration. Mice with a knockout mutation in the abca4 gene massively accumulate toxic lipofuscin pigments in the retinal pigment epithelium. Treatment of these mice with fenretinide, an inhibitor of vitamin A delivery to the eye, blocks formation of these toxic pigments. Here the authors tested the hypothesis that dietary supplementation with vitamin A may accelerate lipofuscin pigment formation in abca4(-/-) mice. METHODS: Wild-type and abca4(-/-) mice were fed normal or vitamin A-supplemented diets. Tissues from these mice were analyzed biochemically for retinoids and lipofuscin pigments. Eyes from these mice were analyzed morphologically for lipofuscin in the retinal pigment epithelium and for degeneration of photoreceptors. Visual function in these mice was analyzed by electroretinography. RESULTS: Mice that received vitamin A supplementation had dramatically higher levels of retinyl esters in the liver and retinal pigment epithelium. Lipofuscin pigments were significantly increased by biochemical and morphologic analysis in wild-type and abca4(-/-) mice fed the vitamin A-supplemented diet. Photoreceptor degeneration was observed in 11-month-old albino, but not pigmented, abca4(-/-) mice on both diets. CONCLUSIONS: Vitamin A supplementation should be avoided in patients with ABCA4 mutations or other retinal or macular dystrophies associated with lipofuscin accumulation in the retinal pigment epithelium.

Recessive Stargardt's macular degeneration is a blinding disease of children caused by mutations in the ABCA4 (ABCR) gene. Mice with a knockout mutation in abcr accumulate toxic lipofuscin pigments in ocular tissues, similar to affected humans. The major fluorophore of lipofuscin is the bis-retinoid, N-retinylidene-N-retinylethanolamine (A2E). In the current study, we sought to define the effect of increasing light on A2E accumulation. We crossed the abcr(-/-) mutation onto an albino background. The retinoid profiles in albino mice indicated higher retinal illuminance than in pigmented mice exposed to similar ambient light. Unexpectedly, A2E levels were not higher in the albino mice. Also, A2E levels in abcr(-/-) mice reared under cyclic light at 30, 120, or 1,700 lux were similar. Thus, increased retinal illuminance was not correlated with higher A2E. A2E has been shown to undergo light-dependent oxidation to yield a series of A2E epoxides or oxiranes. These oxiranes react with DNA in vitro, suggesting a potential mechanism for A2E cytotoxicity. We analyzed ocular tissues from abcr(-/-) mice for A2E oxiranes by mass spectrometry. Unlike A2E, the oxiranes were more abundant in albino vs. pigmented abcr(-/-) mice, and in abcr(-/-) mice exposed to increasing ambient light. These observations suggest that both the biosynthesis of A2E and its conversion to oxiranes are accelerated by light. Finally, we showed that the formation of A2E oxiranes is strongly suppressed by treating the abcr(-/-) mice with Accutane (isotretinoin), an inhibitor of rhodopsin regeneration.