Molecular Mechanisms Section

• Current Research
• Staff
• Selected Publications

Current Research

The retinal pigment epithelium (RPE) is a single layer of cells lining the back of the retina. The RPE plays a pivotal role in the development and function of the outer retina. Without these cells the retinal photoreceptor cells, and vision itself, could not function. In this group we are interested in RPE-specific mechanisms of vitamin A metabolism. The RPE-specific mechanism of major interest to us is the visual cycle, the cyclical process by which vitamin A (all-trans retinol) is converted to the form (11-cis retinal) required for vision. In the process of light absorption by retinal photoreceptors, the 11-cis retinal chromophore bound to visual pigment is photo-isomerized to the all-trans isomer. The all-trans retinol is returned to the RPE and enzymatically isomerized to the 11-cis isomer. This in turn is oxidized to 11-cis retinal and then secreted to the photoreceptors to regenerate the visual pigment. We are studying the role of RPE65, a highly expressed developmentally regulated RPE protein, in this process. Evidence from biochemical studies and from molecular genetics studies in both mouse models and human genetic eye disease show that RPE65 is essential to the operation of the visual cycle. Recently, we have established that RPE65 is in fact the key isomerase in the visual cycle and is part of a family of enzymes that are specialized in carotenoid metabolism- including the enzyme that converts ß-carotene into vitamin A. RPE65, thus, plays a central and irreplaceable role in vision. Our ongoing goals are to elucidate the mechanism of action of RPE65 and to determine how it is integrated into the overall visual cycle. Understanding these aspects may help in design of rational therapies for treating blindness due to visual cycle defects. The techniques employed in these studies include molecular biology, molecular genetics, transgenic and knockout animal models, biochemistry, protein chemistry and biophysical methods.

This lab discovered and cloned RPE65 in the early 1990s. The crucial nature of this protein in the process of vision is demonstrated by its involvement in genetic diseases causing blindness. Mutations in the human RPE65 gene (see also: http://www.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?180069) result in Leber's congenital amaurosis (LCA; see also:http://www.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?204100) and autosomal recessive childhood-onset severe retinal dystrophy (arCSRD). Over 60 separate mutations have been identified since 1997. Mutations in this gene may account for up to 15% of cases of LCA in North America. Common features of these patients include severe loss of vision from birth or early childhood, complete night-blindness, extinguished rod electroretinography and severely reduced cone responses, suggesting a crucial role for RPE65 in retinal function.

To define this role of RPE65, we made an Rpe65 knockout mouse. The phenotype of this mouse confirms the crucial role of RPE65 in RPE vitamin A metabolism with the finding that the Rpe65-deficient mouse lacks functional visual pigment (rhodopsin), though it expresses the non-functional opsin apoprotein in the rod photoreceptor outer segments. As a result, the rod and cone electroretinograms (a measure of photoreceptor electrical response to light) are essentially abolished. The almost complete lack (>99.9% absent) of 11-cis retinal coincides with accumulation in the RPE of all-trans retinyl esters, thought to be the immediate precursor to 11-cis retinol. We can conclude that RPE65 is necessary for the production of 11-cis retinoids by the RPE. We are using the Rpe65-deficient mouse model to study possible therapies for the RPE65-associated human LCA-like dystrophies

Recently, the Briard dog model of LCA that harbors a RPE65 mutation was treated with adenoassociated virus-mediated RPE65 gene therapy that successfully restored some functional vision. This provides hope for a future treatment of patients with RPE65 retinal dystrophy. Our lab is cooperating with the groups involved in this endeavor. Much progress has been made in forwarding human gene therapy and clinical trials are moving forward but some clinical hurdles remain to be cleared. Of possible relevance to the RPE65 retinal dystrophy is our recent finding, in collaboration with the Laboratory of Immunology, NEI, that RPE65 is highly uveitogenic in rats. Thus, RPE65 joins certain other retinal antigens (e.g., S-Antigen/Arrestin and Interphotoreceptor retinoid-binding protein) as a potent uveitogenic protein. Since immune processes are involved in age-related macular degeneration (AMD), of which RPE is a major target, it is possible also that RPE65 autoimmunity may be involved at some stage of etiology of AMD.

Two studies done in collaboration with other groups showed a direct relationship between the level of RPE65 expression in mice and susceptibility to retinal light damage. Firstly, Rpe65 knockout mice are completely resistant to high intensity light damage. Since lack of RPE65 prevents rhodopsin regeneration, this indicates that a functional visual cycle is required for light damage. Secondly, a naturally occurring variant of the RPE65 gene in albino mice of the C57BL/6 lineage, unlike other albino mice, is associated with resistance to light damage. C57BL/6 mice, and not other mice, have a mutation in RPE65 that affects its expression and/or activity, though they can still see. Thus, mutations in RPE65 that do not drastically affect its function could be somewhat protective. It is also of interest to note, from another study, that aging Rpe65 knockout mice accumulate only 10% of the amount of lipofuscin fluorophore, an age pigment of the RPE that has been associated with AMD in humans, as do normal age-matched mice. This confirms previous work that lipofuscin fluorophores are a normal, if unwelcome, byproduct of the retinoid visual cycle.

A major goal of our work has been to identify tissue-specific and developmentally regulated transcriptional and translational regulatory regions involved in the expression of RPE65. We found that a 700 base pair fragment of RPE65 gene promoter was sufficient to direct RPE-specific expression in transgenic mice. This also directed high reporter activity in the human RPE cell line D407, but weak activity in non-RPE cell lines. Functional binding of potential transcription factors to sites in this fragment was demonstrated. Mutations of these sites abolished binding and corresponding transcriptional activity. Current work in our group is directed towards identifying the transcription factors binding to the RPE65 promoter.

RPE65 is a member of a newly described, but ancient, family of enzymes-the carotenoid oxygenases- that primarily cleave carotenoids. These have crucial and unique functions including maize vp14, a 9-cis epoxycarotenoid cleavage enzyme in the plant abscisic acid synthesis pathway, the bacterial Pseudomonas lignostilbene dioxygenase, and Drosophila, chicken, mouse and human ß-carotene 15,15'-monooxygenases that are ß-carotene cleavage enzymes. These latter are crucial enzymes regulating the entry of vitamin A into animal systems from plant derived pro-vitamin A precursors. We have cloned and characterized the mouse ß-carotene 15,15'-monooxygenase. In the eye, ß-carotene 15,15'-monooxygenase is expressed in both retina and RPE, though in low and variable levels in both. We have also found that the transcriptional regulation of the ß-carotene 15,15'-monooxygenase gene is integrated into the overall regulation of vitamin A metabolism. Though the overall degree of homology is quite low among the various family members, they do share amino acid residues that are crucial to their common general function. Included among these are 4 absolutely conserved histidine residues as well as several acidic amino acids residues. These bind catalytic iron necessary for enzyme activity. Mutation of any of these conserved histidines and some of the acidic residues abolish the ability to cleave ß-carotene. All these features are consistent with a recently published crystal structure for apocarotenal oxygenase, a bacterial representative of the family.

Finally, the function of RPE65 is related to its evolutionary lineage. We have determined that RPE65 is the long-sought all-trans:11-cis retinol isomerase of the vitamin A visual cycle of the human and vertebrate retina, the indispensable enzyme that catalyzes the conversion of dietary vitamin A into the chromophore required for visual pigment regeneration. This finding is consistent with the severe phenotype observed in human LCA and in the Rpe65 knockout mouse. We accomplished this by developing a robust cell culture model for the visual cycle that is capable of producing physiological levels of 11-cis retinoids. We showed that RPE65 is essential for this production. Furthermore, mutation of the equivalent iron-binding residues of RPE65, as are found in ß-carotene 15,15'-monooxygenase, abolishes the isomerase activity of RPE65. Insertion of mutations found to cause LCA in humans also results in loss of activity consistent with their clinical effect. Currently we are investigating the details of how RPE65 catalyzes this crucial step in vision.

Staff

Name	Title	E-Mail
T. Michael Redmond	Section Chief	redmond@helix.nih.gov
Shirley Yu	Biologist	shirley@helix.nih.gov
Eugenia Poliakov	Staff Scientist	poliakove@nei.nih.gov
Alicia Acitores, PhD	Visiting Fellow	acitoresa@mail.nih.gov

Selected Publications

Redmond TM, Weber CH, Poliakov E, Yu S, Gentleman S. Effect of Leu/Met variation at residue 450 on isomerase activity and protein expression of RPE65 and its modulation by variation at other residues. Mol. Vis. 2007, 13:1813-1821. PubMed

Redmond, T.M., Poliakov, E., Yu, S., Tsai, J.-T., Lu, Z. and Gentleman, S. Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle. Proc Natl Acad Sci USA, 102 (38):13658-13663, 2005. PubMed

Poliakov, E., Gentleman, S.,Cunningham, F.X., Miller-Ihli, N.J. and Redmond, T.M. Key Role of histidines in Mouse ß-Carotene 15, 15'-Monooxygenase Activity. J Biol Chem 280(32):29217-29223, 2005. PubMed

Lyubarsky AL, Savchenko AB, Morocco SB, Daniele LL, Redmond TM, Pugh EN Jr. Mole Quantity of RPE65 and Its Productivity in the Generation of 11-cis-Retinal from Retinyl Esters in the Living Mouse Eye. Biochemistry. 44(29):9880-9888, 2005 PubMed

Dejneka, N.S., Surace, E.M., Aleman, T.S., Cideciyan, A.V., Lyubarsky, A, Savchenko, A., Redmond, T.M., Tang, W., Wei, Z., Rex, T.S., Glover, E., Maguire, A.M., Pugh, E.N. Jr, Jacobson, S.G., Bennett, J. In utero gene therapy rescues vision in a murine model of congenital blindness. Mol Ther. 9:182-8, 2004. PubMed

Woodruff ML, Wang Z, Chung HY, Redmond TM, Fain GL, Lem J. Spontaneous activity of opsin apoprotein is a cause of Leber congenital amaurosis Nature Genetics 35: 158-164, 2003. PubMed

Boulanger, A., McLemore, P., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Gentleman, S., and Redmond, T.M.: Beta-carotene 15,15'-monooxygenase is a peroxisome proliferator activated receptor target gene. FASEB J 10.1096/fj.02-0690fje, 2003. PubMed

Narfstrom, K., Katz, M., Bragadottir, R., Seeliger, M., Boulanger, A., Redmond, T.M., Caro, L., Lai, C.-M., Rakozcy, E. Functional and structural recovery of the retina after gene therapy in the RPE65 null mutation dog. Invest Ophthalmol Vis Sci. 44:1663-1672, 2003. PubMed

Moiseyev, G., Crouch, R.K., Goletz, P., Oatis, J. Jr., Redmond, T.M., Ma, J.X. Retinyl esters are the substrate for isomerohydrolase. Biochemistry 42:2229-38, 2003. PubMed

Bhatti, R., Yu, S., Boulanger, A., Fariss, R.N., Guo, Y., Bernstein, S.L., Gentleman, S., and Redmond, T.M.: Expression of beta-carotene 15, 15' monooxygenase in retina and RPE/choroid. Invest Ophthalmol Vis Sci 44: 44-49, 2003. PubMed

Ham D.I., Gentleman S., Chan C.C., McDowell J.H., Redmond T.M., and Gery, I.: RPE65 is highly uveitogenic in rats. Invest Ophthalmol Vis Sci 43:2258-63, 2002. PubMed

Boulanger, A. Liu, S., Yu, S., and Redmond, T.M.: Sequence and Structure of the mouse gene for RPE65. Molecular Vision 7: 283-287, 2001. PubMed

Katz, M.L. and Redmond, T.M.: Rpe65 knockout prevents accumulation of lipofuscin fluorophores in the retinal pigment epithelium. Invest Ophthalmol Vis Sci 42: 3023-3030, 2001. PubMed /p>

Seeliger, MW, Grimm, C, Stahlberg, F, Friedburg, C, Jaissle, G, Zrenner, E, Guo, H, Reme, CE, Humphries, P, Hofmann, F, Biel, M, Fariss, RN, Redmond, TM, and Wenzel, A: New views on RPE65 deficiency: The rod system is the source of vision in a mouse model of Lebers congenital amaurosis. Nature Genetics, 29: 70-74, 2001. PubMed

Redmond, T.M., Gentleman, S., Duncan, T., Yu, S., Wiggert, B., Gannt, E., and Cunningham, F.X., Jr.: Identification, expression and substrate specificity of a mammalian ß-carotene 15,15'- dioxygenase. J Biol Chem 276:6560-6565, 2001. PubMed

Boulanger, A., Liu, S., Henningsgaard, A.A., Yu, S., and Redmond, T.M.: The upstream region of the RPE65 gene confers retinal pigment epithelium-specific expression in vivo and in vitro and contains critical octamer and E-box binding sites. J Biol Chem 275: 31274-31282, 2000. PubMed

Danciger, M., Matthes, M.T., Yasamura, D., Akhmedov, N.B., Rickabaugh, T., Gentleman, S., Redmond, T.M., Lavail, M.M., and Farber, D.B.: A QTL on distal Chromosome 3 that influences the severity of light-induced damage to mouse photoreceptors. Mammalian Genome 11: 422-427, 2000. PubMed

Grimm, C., Wenzel, A., Hafezi, F., Yu, S., Redmond, T.M., and Reme, C. E.: Protection of Rpe65-deficient mice identifies rhodopsin as mediator of light-induced retinal degeneration. Nature Genetics 25(1):63-66, 2000. PubMed

Redmond, T.M., Yu, S., Lee, E., Bok, D., Hamasaki, D., Chen, N., Goletz, P., Ma, J.-X., Crouch, R.K. and Pfeiffer, K.: Rpe65 is necessary for production of 11-cis-Vitamin A in the retinal visual cycle. Nature Genetics 20: 344-350, 1998. PubMed

Marlhens, F., Bareil, C., Griffoin, J.-M., Zrenner, E., Amalric, P., Eliaou, C., Liu, S.-Y., Harris, E., Redmond, T.M., Arnaud, B., Claustres, M. and Hamel, C.P.: Mutations in RPE65 cause Leber's congenital amaurosis. Nature Genetics 17: 139-141, 1997. PubMed

Hamel, C.P., Tsilou, E., Pfeffer, B.A., Hooks, J.J., Detrick, B. and Redmond, T.M.: Molecular cloning and expression of RPE65, a novel retinal pigment epithelium-specific microsomal protein that is post-transcriptionally regulated in vitro. J Biol Chem 268: 15751-15757, 1993. PubMed