Molecular Mechanisms Section
Current Research
Retinal pigment epithelial (RPE) cells stained red by RPE65 antibody.
The research in this section covers two major areas:
1) Visual Cycle: RPE65 and retinal retinoid metabolism
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 small molecule and other therapies for treating blindness due to visual cycle defects. Recently, somatic RPE65 gene therapy trials have succeeded in partially restoring vision in patients with RPE65 mutations. The techniques employed in these studies include molecular biology, molecular genetics, transgenic and knockout animal models, biochemistry, protein chemistry and biophysical structural 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 provided hope for a future treatment of patients with RPE65 retinal dystrophy. This section has been collaborating with one of the groups involved in this endeavor. Following much preclinical research, safety and efficacy of human RPE65 gene therapy clinical trials were reported in 2008.
RPE65 is a member of an ancient, family of enzymes-the carotenoid oxygenases- that primarily cleave carotenoids. These have crucial and unique functions including, in plants an enzyme in the abscisic acid synthesis pathway, the bacterial enzyme lignostilbene dioxygenase which produces vanillin, 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. In this regard, we have found that RPE65 is not inherently 11-cis specific in its isomerase activity. This finding supports the hypothesis that the chemical basis for retinol isomerization in the visual cycle is a carbocation- or radical cation mediated- mechanism. Furthermore, it also supports the notion that specificity of isomerization depends on a mass action effect due to 11-cis specific binding proteins downstream of the isomerase. A prediction of a radical cation-mediated mechanism is that RPE65 could be inhibited by spin traps, chemical agents which capture radicals. Indeed, we have found that to be the case, demonstrating that a particular class, aromatic lipophilic spin traps including N-tert-butyl-alpha-phenylnitrone (PBN), effectively inhibit RPE65.
2) Signalling pathways in the RPE/retina
The RPE is exposed to variety of stresses, including exposure to light, inflammatory mediators, and reactive oxygen species. Apoptotic RPE cell death resulting from increased oxidative stress could hasten the onset of age-related macular degeneration (AMRD). Retinoic acid, derived from oxidation of vitamin A, affects many cellular functions including cell growth, differentiation, and apoptosis. This effect is mediated through transcriptional regulation by the nuclear hormone receptors RAR and RXR for which retinoic acids are ligands. Synthetic analogs of retinoic acid also have significant effects on cellular function. One such analog, fenretinide, N-(4-hydoxyphenyl) retinamide (4HPR), has long been used as a cancer preventive agent. Recently, it has been proposed as a therapeutic agent for lipofuscin-based retinal diseases. At low doses, we have shown that 4HPR induces neuronal differentiation of cultured ARPE-19 human retinal pigment epithelial cells. At higher doses it causes apoptosis. We are interested in how these effects of 4HPR are mediated. We have studied the role of MAPK signaling pathways in the 4HPR-induced neuronal differentiation of ARPE-19 cells, demonstrating the specific contribution of MAPK signaling cascades in this process. Our results further indicate that signaling through both the ERK1/2 and SAPK/JNK pathways converged in the transactivation of AP-1.
MicroRNAs (miRNAs) have received much attention as post-transcriptional regulators of gene expression in all cell/tissue types. The extent of their importance is just beginning to be realized. Given the likely importance of this level of regulation in the response of RPE cells to various signals we are determining changes in miRNA expression in ARPE19 cells due to a variety of agents.
While most, if not all, enzymatic or binding protein components of the visual cycle have been identified, signaling events in the visual cycle have received less attention. It is anticipated that visual cycle retinoid flux is regulated by such external stimuli as day/night status, ambient light level, as well as by relative levels of retinoid isomers. Receptor mediated uptake of all-trans retinol as well as secretion of 11-cis retinal, both perhaps involving interphotoreceptor retinoid binding protein (IRBP), are also not fully understood. The role of IRBP in regulation of visual cycle may require receptors for transfer of retinoids. A long term goal is to identify such receptors for IRBP on the RPE and photoreceptor membrane surfaces.
Staff
| Name | Title | |
|---|---|---|
T. Michael Redmond |
Section Chief | redmond@helix.nih.gov |
| Eugenia Poliakov | Staff Scientist | poliakove@nei.nih.gov |
| William Samuel | Staff Scientist | samuelw@nei.nih.gov |
| Krishnan Kutty | Biologist | kuttyk@nei.nih.gov |
| Yan Li | Biologist | liyan2@nei.nih.gov |
| Todd Duncan | Biologist | tduncan@helix.nih.gov |
| Preethi Chander | IRTA Fellow | chanderp@nei.nih.gov |
| Danielle Gutierrez | IRTA Fellow | danielle.gutierrez@nih.gov |
Selected Publications
Poliakov E, Parikh T, Ayele M, Kuo S, Chander P, Gentleman S, Redmond TM. Aromatic lipophilic spin traps effectively inhibit RPE65 isomerohydrolase activity. Biochemistry 50: 6739-41, 2011. PubMed
Samuel W, Kutty RK, Vijayasarathy C, Pascual I, Duncan T, Redmond TM. Decreased expression of insulin-like growth factor binding protein-5 during N-(4-hydroxyphenyl)retinamide-induced neuronal differentiation of ARPE-19 human retinal pigment epithelial cells: Regulation by CCAAT/enhancer-binding protein. J Cell Physiol, 224:827-836, 2010. PubMed
Kutty RK, Nagineni CN, Samuel W, Vijayasarathy C, Hooks JJ, Redmond TM. Inflammatory cytokines regulate microRNA-155 expression in human retinal pigment epithelial cells by activating JAK/STAT pathway. Biochem Biophys Res Commun. 402: 390-395, 2010. PubMed
Simonelli F, Maguire AM, Testa F, Pierce EA, Mingozzi F, Bennicelli JL, Rossi S, Marshall K, Banfi S, Surace EM, Sun J, Redmond TM, Zhu X, Shindler KS, Ying GS, Ziviello C, Acerra C, Wright JF, McDonnell JW, High KA, Bennett J, Auricchio A. Gene Therapy for Leber's Congenital Amaurosis is safe and effective through 1.5 years after vector administration. Mol Ther, 18: 643-650, 2010. PubMed
Redmond TM, Poliakov E, Kuo S, Chander P, and Gentleman S. RPE65, visual cycle retinol isomerase, is not inherently 11-cis specific: Support for a carbocation mechanism of retinol isomerization. J. Biol. Chem. 285: 1919-1927, 2010. PubMed
Poliakov E, Gentleman S, Chander P, Cunningham FX Jr., Grigorenko BL, Nemuhin AV, and Redmond TM. Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse Beta-carotene 15, 15’-monooxygenase. BMC Biochemistry 10:31, 2009. PubMed
Lorenz B, Poliakov E, Schambeck M, Friedburg C, Preising MN, Redmond TM. A novel RPE65 hypomorph expands the clinical phenotype of RPE65 mutations. A comprehensive clinical and biochemical functional study. Invest Ophthalmol Vis Sci. 49: 5235-5242, 2008. PubMed
Maguire AM, Simonelli F, Pierce EA, Pugh EN Jr, Mingozzi F, Bennicelli J, Banfi S, Marshall KA, Testa F, Surace EM, Rossi S, Lyubarsky A, Arruda VR, Konkle B, Stone E, Sun J, Jacobs J, Dell'Osso L, Hertle R, Ma JX, Redmond TM, Zhu X, Hauck B, Zelenaia O, Shindler KS, Maguire MG, Wright JF, Volpe NJ, McDonnell JW, Auricchio A, High KA, Bennett J. Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med. 358: 2240-2248, 2008. PubMed
Samuel W, Kutty RK, Sekhar S, Vijayasarathy C, Wiggert B, Redmond TM. Mitogen-activated protein kinase pathway mediates N-(4-hydroxyphenyl)retinamide-induced neuronal differentiation in the ARPE-19 human retinal pigment epithelial cell line. J Neurochem. 106: 591-602, 2008. 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
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
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
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
Last Reviewed: January 2012
