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'-monooxygenase (beta-CM; formerly described as a dioxygenase). Beta-CM is closely related to RPE65 and both are members of a newly emerging diverse family of carotenoid-cleavage enzymes. We postulate that beta-CM and RPE65 may share a similar mechanism of action. In the past year we have made the following progress: a) A collaborative study by acute radiolabeling with all-trans-retinol of wildtype and Rpe65 knockout mice showed that liver and serum uptake was broadly similar in both, over the tested period of 48 hours. In the retina, however, uptake of label by this tissue as retinaldehyde was rapid in wildtype and not seen at all in the Rpe65 knockout. In the RPE, accumulation as retinyl ester was transient in the wildtype, but progressive over 48 hours in the knockout. Analysis of the retinoid influx data into the RPE, gave rise to the conclusion that a robust efflux that occurs in the wildtype does not occur in the Rpe65 knockout RPE. These data suggest that 11-cis retinoids stimulate the efflux of all-trans retinol at the basolateral membrane of the RPE. The data in wildtype retina further corroborate the ?last in/first out? hypothesis of all-trans retinol processing to 11-cis retinol, where retinoid in the active visual cycle flux does not traverse the stored pool of retinyl ester. b) The role of residues conserved in all members of the carotenoid-cleavage enzyme family (including beta-CM and RPE65) has been investigated by site-directed mutagenesis of beta-CM. The data show a crucial role in enzymatic activity for residues hypothesized to be involved in metal coordination. c) Beta-CM expression was found by immunofluorescence microscopy and RT-PCR to be low and variable in retina and RPE of a variety of species (mouse, bovine, monkey and human) 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. We conclude that its function in these tissues is not likely to be a retina- or RPE-specific one. d) We have analyzed the genomic structure of the mouse beta-CM gene and found it to have 11 exons. We have cloned the 5? flanking region of the gene into a luciferase reporter plasmid and generated a series of nested deletions of the putative promoter. Transient transfection of these into monkey RPE, ARPE19 and the TC7 and PF11 subclones of the Caco-2 human enterocytes cell line demonstrated that a consensus peroxisome proliferator activated receptor (PPAR) response element (PPRE) is required for optimal activation of the gene. Electrophoretic mobility shift experiments showed that nuclear proteins from monkey RPE cells and TC7 and cell line bind to the PPRE element. Supershift experiments with antibodies specific to PPARs demonstrated the binding of PPARgamma to the PPRE site in this promoter. Furthermore, we showed, by co-transfection with PPARgamma and RXRalpha and activation with PPARgamma agonists LY 17883 and Ciglitazone, that the PPRE element confers peroxisome proliferator responsiveness via the PPARgamma and retinoid X receptor (RXR) alpha heterodimer. 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. We have continued our collaboration with LI on EAU induced with 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, and, unlike the latter, there is no pinealitis. Adoptive transfer of the disease from immunized to naive animals indicates that the disease is cell-mediated, like other forms of EAU. f) Collaboration continues on the rescue of the Rpe65 knockout mouse phenotype and the Briard dog RPE65 dystrophy by AAV-mediated gene transfer. An increased sensitivity of electrophysiological and behavioral responses to light in treated mice and dogs has been noted. In spite of functional improvements, morphological rescue has been limited.
| Huber, Gesine; Beck, Susanne C; Grimm, Christian et al. (2009) Spectral domain optical coherence tomography in mouse models of retinal degeneration. Invest Ophthalmol Vis Sci 50:5888-95 |
| Maguire, Albert M; Simonelli, Francesca; Pierce, Eric A et al. (2008) Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med 358:2240-8 |
| Cortes, Lizette M; Mattapallil, Mary J; Silver, Phyllis B et al. (2008) Repertoire analysis and new pathogenic epitopes of IRBP in C57BL/6 (H-2b) and B10.RIII (H-2r) mice. Invest Ophthalmol Vis Sci 49:1946-56 |
| Lu, Zhongjian; Poliakov, Eugenia; Redmond, T Michael (2006) Identification of a KRAB-zinc finger protein binding to the Rpe65 gene promoter. Curr Eye Res 31:457-66 |
| Moiseyev, Gennadiy; Takahashi, Yusuke; Chen, Ying et al. (2006) RPE65 is an iron(II)-dependent isomerohydrolase in the retinoid visual cycle. J Biol Chem 281:2835-40 |
| Fan, Jie; Wu, Bill X; Sarna, Tadeusz et al. (2006) 9-cis Retinal increased in retina of RPE65 knockout mice with decrease in coat pigmentationt. Photochem Photobiol 82:1461-7 |
| Stoddart, Chris W; Yu, Meaghan J T; Martin-Iverson, Matthew T et al. (2006) Assessing the efficacy of gene therapy in Rpe65-/- mice using photoentrainment of circadian rhythm. Adv Exp Med Biol 572:239-45 |
| Redmond, T Michael; Poliakov, Eugenia; Yu, Shirley et al. (2005) Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle. Proc Natl Acad Sci U S A 102:13658-63 |
| Rohrer, Baerbel; Lohr, Heather R; Humphries, Peter et al. (2005) Cone opsin mislocalization in Rpe65-/- mice: a defect that can be corrected by 11-cis retinal. Invest Ophthalmol Vis Sci 46:3876-82 |
| Poliakov, Eugenia; Gentleman, Susan; Cunningham Jr, Francis X et al. (2005) Key role of conserved histidines in recombinant mouse beta-carotene 15,15'-monooxygenase-1 activity. J Biol Chem 280:29217-23 |
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