The Molecular Mechanisms Section of the Laboratory of Retinal Cell & Molecular Biology studies vitamin A and lipid metabolism, and signaling and regulation processes central to vision and function of the retina and retinal pigment epithelium (RPE). The RPE is a single layer of cells lining the back of the retina and 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. RPE dysfunction has serious repercussions on photoreceptor viability. Inherited diseases of the RPE as well as the effects of aging on the RPE result in loss of visual acuity and blindness, with major medical and economic impacts. The most important of these diseases is age-related macular degeneration (AMD), which is growing inexorably in prevalence with aging of our population.
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. This section is interested in RPE-specific metabolic mechanisms. 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. 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. 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 β-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. The techniques employed in these studies include molecular biology, molecular genetics, transgenic and knockout animal models, biochemistry, protein chemistry and 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 function 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. The Briard dog model of LCA that harbors a RPE65 mutation was treated with adeno-associated virus-mediated RPE65 gene therapy that successfully restored some functional vision. 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 β-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 cation-mediated mechanism. Of the two possible avenues, a carbocation or a radical cation intermediate, our most recent findings support the latter alternative. This suggests that isomerization occurs early in the temporal sequence with O-alkyl cleavage occurring later. 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. Remaining aspects of the complex mechanism of RPE65 are being addressed.
2) Signaling pathways in the RPE/retina
As a post-mitotic non-renewing epithelium, RPE is exposed to a variety of life-long stresses, including exposure to light, inflammatory mediators, and reactive oxygen species. Apoptotic RPE cell death resulting from chronic oxidative stress, and other stresses may play a role in the onset of AMD. Such additional stresses include accumulation of bisretinoid compounds, byproducts of the visual cycle that accumulate with age, also called lipofuscin. The synthetic retinoid analog N-(4-hydroxyphenyl) retinamide (4HPR; fenretinide) in long use as a cancer preventive agent has recently been proposed as a therapeutic agent for lipofuscin-based retinal diseases such as AMD and as a potential therapy for diabetes. 4HPR is rather pleiotropic in action and mimics or antagonizes many functions of retinoids, including retinoic acid, which affects many cellular functions including cell growth, differentiation, and apoptosis. We are interested in how these effects of 4HPR are mediated, given its proposed therapeutic use in AMD, in other retinal diseases, and in diabetes. We have found that 4HPR’s effect on cells may be mediated in part by its action on stearoyl CoA desaturase (SCD) an important rate-limiting enzyme in monounsaturated fatty acid biosynthesis.
In addition, we are investigating mechanisms of post-transcriptional regulation in the RPE, including by miRNAs. This is directed towards discerning how the native phenotype of RPE’s biochemical systems (lipid metabolism, retinoid metabolism, phagocytosis, etc.) is maintained. RPE is a highly specialized tissue and understanding its complexity is crucial towards a clear biochemical understanding of AMD. Many aspects of its importance to photoreceptor function are as yet poorly understood. Such insights will also be helpful in characterizing what is necessary to achieve sufficient differentiation of stem cells to RPE for use in regenerative biology.
|T. Michael Redmond
|Eugenia Poliakov||Staff Scientistfirstname.lastname@example.org|
|William Samuel||Staff Scientistemail@example.com|
|Abdulkerim Eroglu||Visiting Fellowfirstname.lastname@example.org|
Poliakov E, Gubin AN, Stearn O, Li Y, Campos MM, Gentleman S, Rogozin IB, and Redmond TM. Origin and evolution of retinoid Isomerization machinery in vertebrate visual cycle: hint from jawless vertebrates. PLoS One, 2012 7(11): e49975. doi:10.1371/journal.pone.00499752012, epub November 27, 2012.
Chander P, Gentleman S, Poliakov E, Redmond TM. Aromatic residues in the substrate cleft of RPE65 govern retinol isomerization and modulate its progression. J Biol Chem 287: 30552-30558, 2012.
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 β-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 β-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