The Molecular Mechanisms Section 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 RPE, the retinal photoreceptor cells, and vision itself, could not function. We are interested in RPE-specific metabolic mechanisms, and especially that of the visual cycle, the process by which dietary vitamin A (all-trans retinol) is converted to the “chromophore” form (11-cis retinal) required for vision. When light is absorbed by retinal photoreceptors, 11-cis retinal bound to visual pigment is photo-isomerized to the all-trans isomer. All-trans retinol is returned to the RPE and enzymatically isomerized to the 11-cis isomer, which is oxidized to 11-cis retinal and secreted to the photoreceptors to regenerate 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 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, animal models, biochemistry, protein chemistry and structural methods.
This lab discovered RPE65 in the early 1990s and characterized it as a unique, highly conserved protein that was highly expressed in, and highly preferentially localized to, the RPE. Its crucial role in the process of vision was demonstrated by its involvement in genetic diseases causing blindness. Mutations in the human RPE65 gene (http://www.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?180069) result in Leber’s congenital amaurosis (LCA; http://www.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?204100) and autosomal recessive childhood-onset severe retinal dystrophy (arCSRD), with about 100 separate mutations identified since 1997. Mutations in RPE65 may account for up to 15% of cases of LCA in North America. Common features include severe loss of vision from birth or early childhood, complete night-blindness, extinguished cone and rod function, suggesting a crucial role for RPE65 in retinal function. To define this role, we made an Rpe65 knockout mouse. Its phenotype confirms RPE65’s essential role in the visual cycle. Rpe65-deficient mice lack functional visual pigment (rhodopsin), though they express non-functional opsin apoprotein in the rod photoreceptor outer segments. As a result, the rod and cone electroretinograms (measuring 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 its precursor all-trans retinyl esters, providing prima facie evidence that RPE65 is the visual cycle retinol isomerase. In 2001, a Briard dog LCA model harboring a RPE65 mutation was treated with adeno-associated virus (AAV)-mediated RPE65 gene therapy and functional vision was successfully restored. Following much preclinical research, human RPE65 gene therapy clinical trials were launched in 2008. We are currently studying knock-in mouse models for less severe forms of human LCA. Human RPE65 mutations cause a spectrum of disease from severe early-onset disease to milder forms. Patients with these milder mutations are inadequately modeled by Rpe65 knockout mice. One such, the RPE65 P25L missense mutation, though having only <10% of wild-type activity, causes a relatively mild disease. We made Rpe65P25L/P25L knockin mice and found they can, under dim light conditions, produce sufficient 11-cis RAL to give a near wildtype phenotype under regular light conditions. Only when they are exposed to high light stress is this hypomorphic mutation manifested at the physiological level. Its reduced activity provides reduced recycling of chromophore, and so they are completely protected from light damage. We will use this model to better determine minimal chromophore requirement for cone photoreceptor survival, an important goal of RPE65 gene therapy.
In 2005 we and others confirmed definitively that RPE65 is the long-sought all-trans:11-cis retinol isomerase of the visual cycle, the indispensable enzyme catalyzing conversion of dietary vitamin A into chromophore for visual pigment regeneration. This is consistent with the severe phenotype of human LCA and the Rpe65 knockout mouse. We developed a robust cell culture model for the visual cycle producing physiological levels of 11-cis retinoids and showed that RPE65 is essential for this production. Furthermore, mutation of iron-binding residues of RPE65 abolishes the isomerase activity of RPE65. Insertion of mutations causing LCA in humans also results in loss of activity consistent with their clinical effect. The function of RPE65 is related to its evolutionary lineage. 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 (abscisic acid synthesis), the bacteria (lignin degradation) and Drosophila, chicken, mouse and human BCO1s that are ß-carotene cleavage enzymes, crucial for the entry of vitamin A into animal systems from plant derived pro-vitamin A precursors. We have cloned and characterized the mouse BCO1. Though the overall homology is low in the family as a whole, they share features crucial to their common general function, including 4 absolutely conserved histidine residues and several acidic amino acids residues that together bind catalytic iron necessary for enzyme activity. Mutating any of these histidines and some of the acidic residues abolish the respective enzymes’ ability to cleave β-carotene or isomerize retinol. Complementing a crystal structure for bacterial apocarotenal oxygenase (ACO), published in 2004, a structure for RPE65 was solved by Palczewski and co-workers in 2009. Using these models, we are exploring the details of how RPE65 catalyzes isomerization. In this regard, we find that RPE65 is not inherently 11-cis specific, supporting the hypothesis that the chemical mechanism for visual cycle retinol isomerization is cation-mediated. Of the two possible avenues, a carbocation or a radical cation intermediate, our most recent findings support the latter alternative, suggesting that isomerization occurs early in the temporal sequence with O-alkyl cleavage occurring later, and supporting the notion that specificity of isomerization depends on a mass action exerted by downstream 11-cis specific binding proteins. 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 aromatic lipophilic spin traps effectively inhibit RPE65. Other aspects of RPE65’s complex mechanism are being addressed.
Recently, we investigated how the visual cycle, a key milestone in the evolution of the vertebrate eye, evolved. The vertebrate eye employs the visual cycle to regenerate chromophore enzymatically in the dark, maintaining light sensitivity at all light levels, unlike in all other animal groups, which rely on light to re-isomerize chromophore. It had been speculated that primitive chordates, such as tunicates and cephalochordates, anticipated the visual cycle. We hypothesized that the origin of the vertebrate visual cycle was linked to coordinated evolution of an ancestral carotenoid oxygenase to acquire a new retinyl ester isomerase function AND the evolution of lecithin:retinol acyltransferase (LRAT), which generates the all-trans retinyl ester substrate required by RPE65. We showed that neither true RPE65 nor LRAT exists in Ciona, a tunicate, or in cephalochordates. We identified both RPE65 and LRAT cDNAs for the first time in Sea Lamprey, a jawless vertebrate, and that these had appropriate enzymatic activities, but that Ciona BCMOa (previously described as Ciona RPE65) had carotenoid oxygenase activity but no RPE65 activity. We conclude that the key transition from carotenoid double bond cleavage (BCOs) to isomerase function (RPE65), along with LRAT origin, arose (~500 mya) in the last common ancestor of the jawless and jawed vertebrates, after divergence of primitive chordates (tunicates, etc.) from the line leading to vertebrates.
Visual cycle retinoid metabolism and daily phagocytosis of outer segments (OS) cause accumulation of autofluorescent lipofuscin in RPE such as the pyridinium bisretinoid. As much work has been published on the adverse effects of A2E and other bisretinoid byproducts of the visual cycle, we are interested in mechanisms RPE cells utilize to deal with a lifetime’s accumulation of bisretinoids. While bisretinoid accumulation is implicated in pathogenesis of several retinal degenerations such as Stargardt disease, Best Vitelliform Macular Dystrophy, and AMD, these also accumulate in RPE during normal aging, with most people not developing AMD. In our studies we find that ARPE-19 cells respond to A2E treatment and OS challenge by producing a melanin-containing lysosome fraction. We speculate that this prevents them from becoming impaired in OS processing. Melanosomes may protect ARPE-19 from oxidative stress, while exogenous melanin/melanosomes protect A2E from photooxidation inside RPE cells. Our results also suggest a protective effect on ARPE-19 lysosomes when melanin was induced by A2E.
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. MicroRNAs (miRNAs), which target many mRNAs for translational inhibition or degradation, are important regulators of many cellular functions. We have shown earlier by microarray analysis that many miRNAs are expressed in cultured RPE cells and that the expression of miR-9, known to regulate neuronal differentiation, is regulated by retinoic acid and promoter hypermethylation. We are continuing to study their roles in RPE pathophysiology. Ocular inflammation is often associated with the infiltration of lymphocytes and macrophages to the posterior compartment of the eye and their secretion of pro-inflammatory cytokines (PIC) such as IFN-γ, TNF-α and IL-1. We found an ~11-fold increase in miR-155 expression, validated by real-time PCR. miR-155 expression increased when cells were treated with IFN-γ, TNF-α or IL-1β separately, but combinations of the cytokines exaggerated the effect. miR-155 has the potential to modulate the response of the RPE cells to inflammatory stimuli and, therefore, it may serve as a target for therapeutic intervention in retinal degenerative diseases such as AMD. Two other miRNAs, miR-146a and miR-146b-5p, are known regulators of inflammatory responses and attenuate cytokine signaling via the NF-κB pathway. Therefore, we investigated their expression in ARPE-19 cells in response to IFN-γ, TNF-α and IL-1β. miR-146a expression was increased ~35-fold, and that of miR-146b ~6-fold by PIC treatment. Both microRNAs were found to target IRAKI, an essential mediator for the activation of NF-κB pathway by IL-1, for translational repression. Both microRNAs could modulate the response of the RPE cells to inflammatory stimuli by potentially targeting IRAK1 and thus could serve as potential therapeutic targets for arresting retinal degenerative diseases resulting from unregulated RPE inflammatory response.
In concert with our efforts to understand the biochemical and metabolic underpinnings of the RPE phenotype, we are studying how culture conditions affect RPE phenotype. Primary RPE cultures best retain native characteristics and physiology. ARPE-19 and other immortalized cell lines are useful in biochemical studies. However, during in vitro culture all RPE cells show a strong tendency to dedifferentiate, manifesting in morphological changes, towards a fibroblastic phenotype with epithelial-mesenchymal transition (EMT) playing an important role. These studies are 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 Scientistemail@example.com|
|William Samuel||Staff Scientistfirstname.lastname@example.org|
|Tingting Liu||Visiting Fellowemail@example.com|
Li Y, Yu S, Duncan T, Li Y, Liu P, Gene E, Cortes-Pena Y, Qian H, Dong L, Redmond TM. Mouse model of human RPE65 P25L hypomorph resembles wildtype under normal light rearing but is fully resistant to acute light damage. Hum Mol Genet. 24:4417-4428, 2015.
Poliakov E, Strunnikova NV, Jiang J-K, Martinez B, Parikh T, Lakkaraju A, Thomas C, Brooks, BP, Redmond TM. Multiple A2E treatments leads to melanization of rod outer segment-challenged ARPE-19 cells. Molecular Vision, 20:285-300, 2014.
Samuel W. Kutty RK, Duncan T, Vijayasarathy C, Kuo B, Chapa K, Redmond TM. Fenretinide induces ubiquitin-dependent proteasomal degradation of stearoyl-CoA desaturase in human retinal pigment epithelial cells. J. Cell Physiol. 229: 1028-1038, 2014.
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