Molecular Biology Of Outer Retina-specific Proteins

National Eye Institute

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Abstract

The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels. To this end we have been studying the function and regulation of RPE65, a gene whose expression is restricted to the RPE and mutations in which cause severe blindness in humans. Disruption of the RPE-based vitamin A visual cycle metabolism of all-trans-retinyl esters to 11-cis-retinal appears to underlie the phenotype of the Rpe65 knockout mouse. The function of RPE65 thus appears to be associated with that of the retinol isomerase, the crucial enzyme in visual pigment regeneration. We have also continued studies on beta-carotene 15,15'-dioxygenase (beta-CD). Beta-CD is closely related to RPE65 and both are members of a newly emerging diverse family of carotenoid-cleavage enzymes. We postulate that beta-CD and RPE65 may share a similar mechanism of action. In the past year we have made the following progress: a) One year-old Rpe65 knockout mice show a dramatically lowered accumulation of lipofuscin fluorophores (an irreversible by-product of vitamin A associated with age-related macular degeneration) compared to wildtype. This, in parallel with an over-accumulation of retinyl esters in the RPE, shows that a functioning visual cycle is required for the generation of lipofuscin fluorophores. b) A collaborative effort using double knockout mice (Rpe65 knockout crossed with rhodopsin-knockout (pure cone response) or cyclic nucleotide gated channel alpha 3 knockout (pure rod response)) was used to study the origin of the reduced electroretinogram (ERG) response seen in Rpe65-deficient mice. In contrast to the original conclusion, it was determined that the ERG is actually due to severely desensitized rod photoreceptors rather than to cone photoreceptors. The rods in Rpe65-deficient mice, because of extreme chromophore starvation, have so little rhodopsin that they perceive far less light than is administered and, masquerading as cones, respond to bright light stimuli that would suppress normal rods. These findings also explain features of RPE65-associated retinal dystrophy in human patients and may be useful in therapy. c) By immunofluorescence microscopy and RT-PCR, beta-CD was found to be expressed in the mouse inner retina and not in the mouse RPE. RT-PCR of various RPE cell lines revealed expression in a monkey RPE cell line but not in 3 human RPE cell lines. d) We have analyzed the genomic structure of the beta-CD gene and are beginning transient transfection in a variety of cell lines, including monkey RPE, COS-7 and Caco-2, of beta-CD gene promoter-reporter constructs to analyze promoter function of the beta-CD gene. e) Experimental autoimmune uveoretinitis (EAU) induced by retina-specific antigens (e.g., arrestin, IRBP and rhodopsin) is a model for human ocular inflammatory diseases such as uveitis. In collaboration with LI, we have found that a moderate immunopathogenic response is induced in Lewis rats immunized with the RPE-specific RPE65 antigen. The disease is more active in the posterior segment and does not affect the anterior segment to the same extent as arrestin-induced EAU. Adoptive transfer of the disease from immunized to naive animals indicates that the disease is cell-mediated, like other forms of EAU. f) Rescue of the Rpe65 knockout mouse phenotype by AAV-mediated gene transfer has resulted in increased sensitivity of electrophysiological and behavioral responses to light in treated mice.