About our work
Specific biochemical and cellular mechanisms of the retina and retinal pigment epithelium (RPE) are central to proper maintenance of vision. The RPE is a single layer of cells lining the back of the retina and plays a crucial role in the development and function of the retina. Without RPE the retinal photoreceptor cells, and vision itself, could not function. RPE dysfunction has serious repercussions on photoreceptor viability and health. 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. Several retinal degenerations involve genes that are exclusively or primarily expressed by the RPE and cause blindness in younger people, including newborns. Among diseases of aging eyes involving the RPE, the most important is age-related macular degeneration (AMD), which is growing inexorably in prevalence with aging of our population. The Molecular Mechanisms Section (MMS) has two major goals: 1) understanding retina/RPE specific mechanisms of vitamin A metabolism and how they affect vision health; and 2) the role of retina/RPE specific mechanisms of signaling and gene regulation in retina/RPE function.
The research in this section covers two major areas.
Visual cycle: RPE65 and retinal retinoid metabolism
The “visual cycle” is the process by which dietary vitamin A (all-trans retinol) is converted to the “chromophore” form (11-cis retinal) required for vision. The metabolic process of vision begins when light converts or “isomerizes” 11-cis retinal bound to visual pigment in the retina’s photoreceptors to the all-trans form. To restore light sensitivity this all-trans form is recycled to the RPE where it is enzymatically isomerized back to the 11-cis isomer which is then returned to the photoreceptors to regenerate visual pigment. We and others have shown, from basic biochemical studies and from molecular genetics studies in both mouse models and human genetic eye disease, that the RPE-restricted protein called RPE65, discovered in the early 1990s, is essential to the operation of the visual cycle. RPE65 is the key isomerase in the visual cycle and is part of a family of carotenenoid oxygenase enzymes that specialize in carotenoid metabolism- including the enzyme b-carotene oxygenase (BCO1) that converts β-carotene into vitamin A. Mutations in RPE65 were shown to cause Leber congenital amaurosis type 2 (LCA2), a hereditary blinding disease resulting in severe loss of vision from birth or early childhood. Based on 25 years of foundational basic research and clinical studies, in 2017 LCA2 became the first inherited disease to be treated by an FDA-approved gene augmentation therapy (Luxturna).
Currently, MMS continues to investigate the function of RPE65 and overall retinoid and carotenoid metabolism in the retina. Using assay systems we have developed, and drawing on predictions based on the RPE65 crystal structures of Kiser and coworkers, we have continued to explore further aspects of the complex catalytic mechanism of RPE65. In particular, we have validated the hypothesis that retinol isomerization by RPE65 proceeds via a carbocation (or possibly a radical cation) mechanism, rather than a SN2’ mechanism of nucleophilic addition to the carbon-11 of the retinyl moiety, and we have identified several aromatic and non-aromatic binding cleft residues of RPE65 critical for retinyl moiety isomerization. Based on this we predicted that spin trap compounds might be effective inhibitors of RPE65 activity and found this to be the case. We have also found, unexpectedly, that inhibitors of lipid metabolism also inhibit RPE65. Further aspects of RPE65’s complex structure and function are under investigation. This includes the role played by palmitoylation in RPE65 structure and function.
In studying how the visual cycle (a key milestone in the evolution of the vertebrate eye) evolved, MMS determined that RPE65 likely arose (along with other key aspects of the visual cycle) in the last common ancestor of jawed and jawless (e.g., lampreys) vertebrates, and not earlier as had been hypothesized. Further investigation to discern how RPE65 could have arisen reveals that the BCO family has evolved in an “extremely high turnover” fashion, providing a rationale for the evolution of RPE65, a BCO family outlier, which neither uses carotenoids as substrate nor is an oxygenase.
In addition, MMS uses transgenic mouse models and RPE derived from human induced pluripotent stem cells (iPSCs) to generate and study interesting missense mutations for RPE65-related retinal dystrophies (P25L, D477G, etc.). We use these models to study mechanisms of human disease, and to determine requirements for chromophore in the development and maintenance of photoreceptors. especially cone photoreceptors. This has an important practical role in determining what is an adequate augmentation levels for human RPE65 gene therapy. We also generate knockouts of other genes highly expressed in the RPE.
RPE biology and non-coding RNAs in RPE/retina gene regulation.
MMS studies cultured RPE cells to understand the biochemical and metabolic underpinnings of the RPE phenotype. 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. While primary RPE cultures best retain native characteristics and physiology, ARPE-19 and other immortalized cell lines are useful in biochemical studies, and RPE cells derived from induced pluripotent stem cells (iPSCs) are crucial in the study of RPE diseases (“disease in a dish”). 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. Our studies are directed towards discerning how the native phenotype of RPE’s biochemical systems (lipid metabolism, retinoid metabolism, phagocytosis, etc.) is maintained. 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.
MMS also investigates mechanisms of post-transcriptional regulation in the RPE by miRNAs and long non-coding (lnc) RNAs. MicroRNAs (miRNAs), short RNAs which target many mRNAs for translational inhibition or degradation, are important regulators of many cellular functions, and may be involved in RPE pathophysiology. For example, we have studied the role of miR-155, miR-146a and miR-146b-5p, known regulators of inflammatory responses. These, and other miRNAs, could serve as potential therapeutic targets for arresting retinal degenerative diseases resulting from unregulated RPE inflammatory response. We also study the role of lncRNAs in the regulation of RPE transcription. lncRNAs are generally untranslated RNA transcripts that are at least 200 nucleotides in length and whose roles in biology are just beginning to be discerned. Unlike protein-coding mRNAs, lncRNAs do not have a high degree of sequence conservation. Using RNAseq we have identified a number of lncRNAs that appear to play important roles in RPE differentiation. These lncRNAs appear to work in coordination with miRNAs. This is an area ripe for exciting new discoveries.
(Complete bibliography at: https://www.ncbi.nlm.nih.gov/sites/myncbi/thomas.redmond.1/bibliography/48047072/public/?sortby=pubDate&sdirection=descending)
Yang U, Gentleman S, Gai X, Gorin MB, Borchert MS, Lee TC, Villanueva A, Koenekoop R, Maguire AM, Bennett J, Redmond TM, Nagiel A. Utility of In Vitro Mutagenesis of RPE65 Protein for Verification of Mutational Pathogenicity Before Gene Therapy. JAMA Ophthalmol. 2019 Oct 3:1-9. doi: 10.1001/jamaophthalmol.2019.3914.
Uppal S, Liu T, Poliakov E, Gentleman S, Redmond TM. The dual roles of RPE65 S-palmitoylation in membrane association and visual cycle function. Scientific Reports, 2019 Mar 26;9(1):5218. doi: 10.1038/s41598-019-41501-w
Postnikova OA, Rogozin IB, Samuel W, Nudelman G, Babenko VN, Poliakov E, Redmond TM. Volatile Evolution of Long Non-Coding RNA Repertoire in Retinal Pigment Epithelium: Insights from Comparison of Bovine and Human RNA Expression Profiles. Genes 2019, Volume 10 (3): 205; DOI: 10.3390/genes10030205
Li Y, Furhang R, Ray A, Duncan T, Soucy J, Mahdi R, Chaitankar V, Gieser L, Poliakov E, Qian H, Liu P, Dong L, Rogozin IB, Redmond TM. Aberrant RNA splicing is the major pathogenic effect in a knock-in mouse model of the dominantly inherited c.1430A>G human RPE65 mutation. Human Mutation, Jan 10, 2019; doi: 10.1002/humu.23706
Poliakov E, Soucy J, Gentleman S, Rogozin IB, Redmond TM. Phylogenetic analysis of the animal BCO/RPE65 superfamily: an ancestral gene assemblage of BCO-like proteins in branchiostome (lancelet) and nematode genomes. Scientific Reports, 7:13192. DOI:10.1038/s41598-017-13521-x, 2017.
Samuel W, Jaworski C, Kutty RK, Duncan T, Poliakov E, Tan L, Lakkaraju A, and Redmond TM. Appropriately differentiated ARPE-19 cells regain phenotype and gene expression profile similar to native RPE cells. Molecular Vision, 23: 60-89, eCollection 2017.
Eroglu A, Gentleman S, Poliakov E, and Redmond TM. Inhibition of RPE65 retinol isomerase activity by inhibitors of lipid metabolism. J Biol Chem, 291: 4966-4973, 2016.
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.
Kutty RK, Nagineni CN, Samuel W, Vijayasarathy C, JaworskI C, Duncan T, Cameron JE, Flemington EK, Hooks JJ, Redmond TM. Differential Regulation of MicroRNA-146a and MicroRNA-146b-5p in Human Retinal Pigment Epithelial Cells by IL-1β, TNF-α and IFN-γ. Mol Vis 19: 737-750, 2013.
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
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
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
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
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
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
Molecular Mechanisms Section key staff
|Eugenia Poliakov, Ph.D.||Staff Scientistfirstname.lastname@example.org|
|Olga Postnikova||IRTA Fellowemail@example.com|
|Sheetal Uppal||Visiting Fellowfirstname.lastname@example.org|
|Susan Gentleman||Special Volunteeremail@example.com|
|William Samuel, Ph.D.||Staff Scientistfirstname.lastname@example.org|