A large team of vision science researchers at the Cole Eye Institute is committed to making breakthroughs in understanding the causes of many blinding retinal diseases and developing ways to prevent or treat these diseases. Many of their discoveries have already had world-wide implications.
The research staff works in teams identified by the Principal Investigator in whose laboratory they work. To learn more about our personnel and the projects they are working on, click on specific laboratory listings below.
Director: Joe G. Hollyfield, Ph.D.
Director, Department of Ophthalmic Research
Cole Eye Institute
9500 Euclid Ave., i32
Office telephone: 216.445.3252
Goals and projects
Research in the laboratory of Joe G. Hollyfield, Ph.D., seeks to develop a model in rodents that would mimic the conditions found in age-related macular degeneration AMD.
Oxidative damage and inflammation are postulated to be involved in AMD. However, the molecular signal(s) initiating AMD are unknown. We have been able to generate AMD-like lesions in mice following immunization with mouse serum albumin adducted with carboxyethylpyrrole, an oxidation fragment of docosahexaenoic acid previously found in eye tissue and plasma from AMD patients. Mice develop antibodies to this hapten; fix complement component 3 in Bruch's membrane; accumulate drusen below the retinal pigment epithelium during aging; and develop lesions in the retinal pigment epithelium mimicking the blinding end-stage atrophy characteristic of dry AMD. We hypothesize that these mice are sensitized to the generation of this hapten in the outer retina where docosahexaenoic acid is abundant and conditions for oxidative damage are permissive. This model provides a platform for dissecting the molecular pathology of oxidative damage and the immune response contributing to the initiation of this disease.
The Role of Drusen in Macular Degeneration
The goal of this research is to define in molecular terms the linkage between the accumulation of soft drusen below the retinal pigment epithelium (RPE) in the macula and the increased risk of developing age-related macular degeneration (AMD). The presence of soft drusen in the macula is the hallmark risk factor for developing AMD. Surprisingly little is known of the composition or origin of drusen. To this end, a novel method for drusen isolation has been developed that allows the collection of microgram quantities of drusen from donor eye tissue.
At the time of isolation, different drusen sub-types can be identified and separated for use in studies that will characterize their molecular composition. The diagnostic utility of drusen in AMD can be likened to that of blood levels of cholesterol in atherosclerosis. The presence and abundance of drusen, like the level of cholesterol in the blood, indicates the degree to which a patient is at risk for developing the disease.
Because of the relationship of drusen and AMD, understanding the composition of drusen sub-types will provide important information on possible pathways that are causally involved in drusen development. Novel proteins or common modifications of proteins present in drusen should provide insight as to potential drug targets of therapeutic agents to treat AMD. The current research is focused on exploiting this drusen isolation procedure to define the molecular composition, distribution and cellular origin of drusen sub-types in normal and AMD tissues.
The Function of Hyaluronan in IPM Organization and Macular Degeneration
The goal of this research is to define the structure-function relationships of specific molecules in the interphotoreceptor matrix (IPM), a unique matrix that surrounds the extensions of photoreceptors projecting from the outer surface of the retina. Collectively, molecules in this matrix and their interactions establish the microenvironment required for the maintenance of photoreceptor function. This matrix must be porous, allowing the movement of metabolites between photoreceptors and RPE, while at the same time serving as a structured scaffold that supports the alignment of the photoreceptors. The entire IPM complex also serves as an attachment bridge with a tensile strength permitting a physical link between the retina and RPE.
Our ultimate goal is to define the functional role of the molecules in this matrix and determine how they support the health and survival of photoreceptors and the RPE. Of primary importance for retinal function is the role of the IPM in the attachment of the retina to the RPE. Understanding the nature of these interactions and their breakdown is of fundamental importance in understanding retinal detachment. Because of the strategic location of this matrix, it can be anticipated that defects in molecules residing in this compartment may be causally involved in some forms of macular degeneration and retinitis pigmentosa.
Sears Lab staff, left to right: George Hoppe, PhD, and Jonathan Sears, MD
Director: Jonathan Sears, MD
Department of Ophthalmic Research
Cole Eye Institute
9500 Euclid Ave., i32
Office telephone: 216.444.8157
Our lab is interested in the regulation of retinal vascular permeability and angiogenesis in the severely premature infant. We have undertaken two approaches to understanding the basic biology of retinopathy of prematurity (ROP). First we are investigating the possibility of creating normal vascularization in the setting of hyperoxia (phase I of ROP), a process which usually causes vaso-obliteration and subsequent ischemia that leads to hypoxia of retinal tissues (phase II of ROP) and neovascularization. Our hypothesis is that replacing growth factors present in normal retinal development, but absent during hyperoxia can enable physiologic and sequential development of the retina despite the hyperoxic environment of the severely premature infant. We propose to activate the transcription factor(s) responsible for the induction of these key gene products, focusing on hypoxia inducible factors (HIF) 1 and 2.
Second, we have recently demonstrated that an important corticosteroid, triamcinolone acetonide, decreases the secretion of vascular endothelial growth factor (VEGF) by destabilizing VEGF mRNA through the non-genomic steroid receptor. We hypothesize that this receptor is membrane bound and are currently isolating and characterizing it as a target for therapies that modulate vascular permeability and proliferation without the side effects attributed to the classical, cytosolic ligand dependent steroid receptor.
Finally, we have a continued interest in the post-translation, reversible redox modification of proteins by thiol/disulfide exchange, termed protein S-glutathionylation (PSSG). We have demonstrated that heat shock protein 70 (Hsp70) and high mobility group protein B1 (HMGB1) are substrates of the enzyme that facilitates glutathionylation. In the case of Hsp70, PSSG increases its chaperone activity. The function of HMGB1 is largely unknown, but Dr. Hoppe has recently identified key thiol residues that target HMGB1 to the nucleus and has reported its circadian based localization within the outer retina.
- Beck W.F., Sears JE., Brudvig G.W., Kulawiec R.J. and Crabtree R.H.: Oxidation of exogenous substrates by the 02 evolving center of Photosystem II and related catalytic air oxidation of secondary alcohols via a tetra nuclear manganese (IV) complex. Tetrahedron 45:4903 4911, 1989.
- Ricker KW, Sears JE, Beck WF, Brudvig GW. Mechanism of irreversible inhibition of 02 evolution in photosystem II by Tris (hydroxymethyl) aminomethane. Biochemistry 1991 30: 7888-94.
- Sears JE., Fikrig E., Nakagawa T.N, Marcantonio N., Deponte K., Kantor F. and Flavell R.A.: Molecular mapping of Osp A mediated immunity against Borrelia burgdorferi, the agent of Lyme disease. Journal of Immunology 147:1995 2000, 1991.
- Nakagawa T.Y., von Grafenstein H., Sears JE, Williams J., Janeway C.A. and Flavell R.A.: The use of the polymerase chain reaction to map CD4 + T cell epitopes. European Journal of Immunology 21:2851 2855, 1991.
- Sears JE, Fikrig E, Nakagawa TY, Deponte K, Marcantonio N, Kantor FS, Flavell R.A.: Molecular mapping of Osp-A mediated immunity against Borrelia burgdorferi, the agent of Lyme disease. J Immunol. 1991 Sep 15;147(6):1995-2000.
- Fikrig E., Barthold S.W., Sears JE., Telford S., Speilman A., Kantor F.S. and Flavell, R.A.: A recombinant vaccine for Lyme disease. Lyme Disease: Molecular and Immunologic Approaches, Cold Spring Harbor Press 37:716 28, 1992.
- Mead A., Sears JE. and Sears, M.L.: Transepithelial transport of ascorbic acid by the isolated intact ciliary epithelial bilayer of the rabbit eye. Journal of Ocular Pharmacology and Therapeutics 12(3): 253-8, 1996.
- Wan, X., Sears JE., Chen, S. and Sears, M. Circadian aqueous flow mediated by -arrestin induced homologous desensitization. Experimental Eye Research 64:1005, 1997.
- Sears, JE, Nakano, T. and Sears, M. Adrenergic mediated Connexin43 phosphorylation in the ocular ciliary epithelium. Current Eye Research 17: 104-107, 1998.
- Sears J, Nakano T, Sears M. Adrenergic-mediated connexin43 phosphorylation in the ocular ciliary epithelium. Curr Eye Res. 1998 Jan;17(1):104-7.
- Chai YC, Hoppe, G, Sears, JE. Reversal of protein S-glutathiolation by glutaredoxin in the retinal pigment epithelium. Exp Eye Res 2003; 76(2): 155-159.
- Hoppe, G, Chai YC, Sears, JE. Endogenous Oxidoreductase Expression is Induced by Aminoglycosides. Archives of Biochemistry and Biophysics 2003 414 19-23.
- Hoppe, G, Chai YC, Crabb, JW, Sears, JE. Protein S-glutathionylation in the retinal pigment epithelium converts heat shock protein 70 to an active chaperone. Exp Eye Res 2004, 78: 1085-92.
- Hoppe G, O'Neil J, Hoff HF, and Sears JE. Products of Lipid Peroxidation Induce Missorting of the Principal Lysosomal Protease in the retinal pigment epithelium. BiochimicaBiochimicaBiophysica Acta 2004, 1689: 33-41
- Hoppe G, O'Neil J, Hoff HF, and Sears JE. Accumulation of Oxidized Lipid-Protein Complexes alters phagosome maturation in the retinal pigment epithelium Cell Mol Life Sci 2004, 61: 1664-74.
- Hoppe G, Chai YC, Crabb JW, Sears J. Protein s-glutathionylation in retinal pigment epithelium converts heat shock protein 70 to an active chaperone. Exp Eye Res. 2004 Jun;78(6):1085-92.
- Sears JE, and Hoppe G. Triamcinolone Acetonide Destabilizes VEGF mRNA in Müller Cells under Continuous Cobalt Stimulation Invest Ophthalmol Vis Sci. 2005 Nov;46(11):4336-41
- Hoppe G, Talcott KE, Bhattacharya SK, Crabb JW, Sears JE. Molecular basis for the redox control of nuclear transport of the structural chromatin protein Hmgb1. Exp Cell Res. 2006 Aug 2.
- Yokoyama T, Yamane K, Minamoto A, Tsukamoto H, Yamashita H, Izumi S, Hoppe G, Sears JE, Mishima HK. High glucose concentration induces elevated expression of anti-oxidant and proteolytic enzymes in cultured human retinal pigment epithelial cells. Exp Eye Res. 2006 Sep;83(3):602-9.
- Ebrahem Q, Renganathan K, Sears J, Vasanji A, Gu X, Lu L, Salomon RG, Crabb JW, Anand-Apte B. Carboxyethylpyrrole oxidative protein modifications stimulate neovascularization: Implications for age-related macular degeneration. Proc Natl Acad Sci U S A. 2006 Sep 5;103(36):13480-4. Epub 2006 Aug 25.
- Hoppe G, Talcott KE, Bhattacharya SK, Crabb JW, Sears JE. Molecular basis for the redox control of nuclear transport of the structural chromatin protein Hmgb1. Exp Cell Res. 2006 Nov 1;312(18):3526-38. Epub 2006 Aug 2.
- Ebrahem Q, Minamoto A, Hoppe G, Anand-Apte B, Sears JE Triamcinolone acetonide inhibits IL-6- and VEGF-induced angiogenesis downstream of the IL-6 and VEGF receptors. Invest Ophthalmol Vis Sci. 2006 Nov;47(11):4935-41.
- Sears, JE, Gilman, J., and Sternberg, P., Jr. Familial retinal arterial tortuosity with retinal hemorrhages. Arch Ophthalmol. 1998; 116:1185-1188
- Sears, JE., Capone, S., Aaberg, T.M., Sr. and January, B. Ciliary body endophotocoagulation during pars plana vitrectomy in pediatric patients, American Journal of Ophthalmology 1998 126: 723-725.
- Sears JE., Capone A. Jr., Aaberg, T.M. Sr., Lewis H., Grossniklaus H., Sternberg P, Jr., DeJuan E. Surgical Management of foveal neovascularization in children. Ophthalmology. 106 (5) 920-924, 1999.
- Sears J, Capone A Jr, Aaberg T Sr, Lewis H, Grossniklaus H, Sternberg P Jr, DeJuan E. Surgical management of subfoveal neovascularization in children. Ophthalmology. 1999 May;106(5):920-4.
- Kaiser, P.K., Reimann, C.D., Sears, J.E., and Lewis, H. Macular Traction Detachment and Diabetic Macular Edema Associated with Posterior Hyaloidal Traction. Am J Ophthalmol 2001; 131:44-49.
- Sears, JE, Aaberg, T.M., Sr., Daiger, S.P., Moshfeghi, D. Splice Site Mutation in the Peripherin/RDS Gene-Associated With Pattern Dystrophy of the Retina. Am J Ophthalmol, 2001;132: 693-697.
- Sullivan, P., Philsecker, L., and Sears, JE. Limited Macular Translocation with Scleral Retraction Sutures. British Journal of Ophthalmology, 2002, 86; 434-439.
- Moshfeghi, DM, Kaiser, PK, Grossniklaus, HE, Sternberg, P., Jr., Sears, JE, Johnson, MW, Ratliff, N, Branco, A, Blumenkranz, MS, Lewis H. Clinicopathologic study after submacular removal of choroid neovascular membranes treated with verteporfin ocular photodynamic therapy. Am J Ophthalmol 2003 135: 343-350.
- Moshfeghi D, Laier PK, Scott IU, Sears JE et al. Acute endophthalmitis following intravitreal triamcinolone acetonide injection. Am J Ophthalmol 2003, 136:791-6.
- Cahill MT, Kaiser PK, Sears JE, Fekrat S. The effect of arteriovenous sheathotomy on cystoid macular oedema secondary to branch retinal vein occlusion. Br J Ophthalmol. 2003 Nov;87(11):1329-32.
- Al-Khayer K, Hagstrom S, Pauer G, Zegarra H, Sears JE, Traboulsi EI. 30 year follow up of a patient with leber congenital amaurosis and novel RPE65 mutations. Am J Ophthalmol. 2004, 137:375-7.
- Cahill MT, Kaiser PK, Sears JE, Fekrat S. The effect of arteriovenous sheathotomy on cystoid macular edema secondary to branch retinal vein occlusion. Br J Ophthalmol 2003 87: 1329-32.
- Moshfeghi D, Sears JE, Lewis H. Submacular surgery for choroidal neovascularization following nocardial endophthalmitis. Retina 2004 24:161-4.
- Roth D, Sears JE, Lewis H. Removal of retained subfoveal perfluoro-n-octane liquid. Am J Ophthalmol 2004 138: 287-89.
- Singh AD, Kaiser PK, Sears JE, et al. Photodynamic therapy of circumscribed choroidal hemangiomas. Br J Ophthalmol 2004 88: 1414-18.
- Moshfeghi D, Kim BY, Kaiser PK, Sears JE, Smith SD. Appositional suprachoroidal hemorrhage: a case control study. Am J Ophthalmol 2004 138: 959-63.
- Al-Khayer K, Hagstrom S, Pauer G, Zegarra H, Sears J, Traboulsi EI. Thirty-year follow-up of a patient with leber congenital amaurosis and novel RPE65 mutations. Am J Ophthalmol. 2004 Feb;137(2):375-7.
- Moshfeghi D, Kaiser P, Bakri SJ, Kaiser RS, Maturi RK, Sears JE. Presumed sterile endophthalmitis following intravitreal triamcinolone acetonide. Ophthal Surg Lasers Imaging 2005 36: 24-29.
- Singh AD Kaiser PK, Sears JE. Choroidal hemangioma Ophthalmol Clin North Am 2005 18:151-161.
- Bakri SJ, Sears JE, Singh AD. Transient closure of a retinal capillary hemangioma with verteporfin photodynamic therapy. Retina. 2005 Dec;25(8):1103-4.
- Singh RP, Patel C, Sears JE. Management of subretinal macular haemorrhage by direct administration of tissue plasminogen activator. Br J Ophthalmol. 2006 Apr;90(4):429-31.
- Saavedra E, Singh AD, Sears JE, Ratliff NB. Plexiform pigmented schwannoma of the uvea. Surv Ophthalmol. 2006 Mar-Apr;51(2):162-8.
- Taban M, Kosmorsky GS, Singh AD, Sears JE. Choroidal folds secondary to parasellar meningioma. Eye. 2006 Jun 16.
- Ou JI, Moshfeghi DM, Tawansy K, Sears JE. Macular hole in the shaken baby syndrome. Arch Ophthalmol. 2006 Jun;124(6):913-5.
- Singh RP, Sears JE. Retinal pigment epithelial tears after pegaptanib injection for exudative age-related macular degeneration. Am J Ophthalmol. 2006 Jul;142(1):160-2.
- Bakri SJ, Sears JE, Lewis H. Management of macular hole and submacular hemorrhage in the same eye. Graefes Arch Clin Exp Ophthalmol. 2006 Jul 27; [Epub ahead of print] PMID: 16871381.
- Taban M, Ufret-Vincenty RL, Sears JE. Endophthalmitis after 25-gauge transconjunctival sutureless vitrectomy. Retina. 2006 Sep;26(7):830-1.
- Taban M, Sears JE, Singh AD.Ciliary body naevus. Eye. 2006 Oct 13; [Epub ahead of print].
- Brasil OF, Smith SD, Galor A, Lowder CY, Sears JE, Kaiser PK. Predictive factors for short term visual outcome after intravitreal triamcinolone acetonide injection for diabetic macular edema: an OCT study.Br J Ophthalmol. 2006 Nov 15; [Epub ahead of print]
Front row, left to right: Jiayin Gu, graduate student, Case Western Reserve University; John W. Crabb, PhD, professor and staff; Sanjoy Bhattacharya, PhD, project scientist; Bharathi Govindarajan, graduate student, Case Western Reserve University; Xiaorong Gu, PhD, postdoctoral fellow; and Tanuja Chaudhary, lead research technologist.
Back row, left to right: Kutralanathan (Nathan) Renganathan, graduate student, Case Western Reserve University; Jack Crabb, research technologist; Bogdan Gugiu PhD, postdoctoral fellow; and Suresh Annangudi, graduate student, Case Western Reserve University.
Director: John W. Crabb, PhD
Department of Ophthalmic Research
Cole Eye Institute
9500 Euclid Ave., i31
Office telephone: 216.445.0425
Goals and Projects
Proteomic Studies of Age-related Macular Degeneration
Age-related macular degeneration (AMD) is the most common cause of legal blindness in the elderly population of developed countries. Genetic and environmental factors both contribute to the disease, however, the cause of AMD is unknown and there are no cures. We hypothesize that similar mechanisms of oxidative damage are involved in AMD and retinal light damage and are identifying proteins and protein chemical modifications associated with AMD and light-damaged rat retina as an approach to defining these pathways. Major risk factors for developing AMD are extracellular deposits termed drusen, which accumulate with age beneath the retinal pigment epithelium on Bruch’s membrane. Methods used for proteomic characterization of drusen and retinal tissues include 1D and 2D chromatography and/or electrophoresis, bioinformatics and mass spectrometry. Oxidative protein modifications identified in AMD tissues include apparent crosslinks, carboxymethyl lysine and carboxyethyl pyrrole (CEP) protein adducts. CEP adducts are more abundant in AMD than in normal Bruch’s membrane, stimulate angiogenesis in vivo in chorioallantoic membrane and corneal implant assays and may contribute to choroidal neovascularization in late stage AMD. CEP immunoreactivity and CEP autoantibody titer are also significantly elevated in plasma from AMD donors relative to that from age-matched normal donors, and may be of diagnostic utility as biomarkers for predicting AMD susceptibility. Oxidative protein modifications identified in rat retina following intense in vivo light exposure include CEP adducts, argpyrimidine and nitrotyrosine. These data directly link oxidative injury with AMD and retinal light damage. We anticipate this protein chemical approach will provide insights into the etiology of AMD and new opportunities for finding cures for the disease.
Visual Cycle Studies
The process by which all-trans-retinal released from rhodopsin during bleaching is enzymatically isomerized to 11-cis-retinal in the retinal pigment epithelium (RPE), then shuttled back to the rod photoreceptor cells for visual pigment regeneration is known as the rod visual cycle. While the molecular details of this multicomponent process are not completely understood, the cellular retinaldehyde-binding protein (CRALBP) appears to serve multiple roles, a central function being as an 11-cis-retinol acceptor for the RPE isomerization of all-trans- to 11-cis-retinol. We are probing CRALBP structure-function relationships using a combination of protein chemical, nuclear magnetic resonance and molecular biological approaches. The structure of the CRALBP ligand binding pocket is of interest because it determines the specificity of ligand binding and influences the timely release of 11-cis-retinoid from CRALBP. CRALBP protein interactions are also critical in visual cycle processes and a functional interaction has now been unequivocally demonstrated with homogeneous recombinant 11-cis-retinol dehydrogenase. Other visual cycle protein-protein interactions have been sought in bovine RPE microsomes. Using reciprocal immunoprecipitations, immunoaffinity purification and mass spectrometry methods we have identified an RPE visual cycle protein complex. Current efforts focus upon identifying additional components of this RPE retinoid metabolizing protein complex and molecular details of the visual cycle.
Dr. Sanjoy Bhattacharya»
Dr. Sanjoy Bhattacharya, Project Scientist, firstname.lastname@example.org
- BS 1987, Banaras Hindu University, Banaras, India - Chemistry
- MS 1990, Banaras Hindu University, Banaras, India - Biotechnology
- MTech 1992, Institute of Technology, Banaras Hindu University, Banaras, India - Biochemical Engineering
- PhD 1997, Indian Institute of Technology-Delhi, Delhi, India - Biochemical Engineering & Biotechnology
Dr. Xiaorong Gu»
Dr. Xiaorong Gu, Postdoctoral Fellow, email@example.com
- BS 1993, Sichuan University, Sichuan, P.R. China - Chemical Engineering
- MS 1996, Sichuan University, Sichuan, P.R. China - Organic Chemistry
- PhD 2002, Case Western Reserve University, Cleveland, Ohio, USA, Department of Chemistry
Dr. Bogdan G. Gugiu»
Dr. Bogdan G. Gugiu, Postdoctoral Fellow, firstname.lastname@example.org
- BS 1995, Polytechnic University Bucharest, Department of Organic Chemistry, Romania.
- MS 1996, Polytechnic University Bucharest, Department of Organic Chemistry, Romania
- PhD 2004, Case Western Reserve University, Department of Chemistry, Cleveland, Ohio, USA, May 2004.
Tanuja Chaudhary, Lead Research Technologist, email@example.com
- BS 1982, University of Delhi, Delhi, India - Chemistry ( Hons.)
- MS 1984, University of Delhi, Delhi, India - Physical Chemistry
Jack Crabb, Research Technologist, firstname.lastname@example.org
- BA 2004, Case Western Reserve University, Computer Science
Suresh Annangudi, Graduate Student, email@example.com
- BSc 1997, Regional Institute of Education (NCERT), Mysore, India – Chemistry, Botany & Zoology.
- MSc 2000, Indian Institute of Tech, Bombay, India – Organic Chemistry
- PhD Program 2000-Present, Case Western Reserve University, Cleveland, Ohio, USA, Department of Chemistry
Jiayin Gu, Graduate Student, firstname.lastname@example.org
- BS 2000, Nanjing University, Nanjing. China - Chemistry
- MS 2002, Nanjing University, Nanjing. China - Chemistry
- PhD Program 2002-present, Department of Chemistry, Case Western Reserve University, Cleveland, Ohio, USA,
Bharathi Govindarajan, Graduate Student, email@example.com
- BSc 2000, Stella Maris College, Chennai, India - Chemistry
- MSc 2003, India Institute of Technology-Madras, Chennai, India -Chemistry
- Ph. D Program 2003-present, Department of Chemistry, Case Western Reserve University, Cleveland, Ohio, USA,
Renganathan Kutralanathan, Graduate Student, firstname.lastname@example.org
- BSc 1996, Loyola College, Chennai India - Chemistry.
- MSc 1998, Indian Institute of Tech, Chennai, India - Chemistry
- MS 2001, Eastern Michigan University, Ypsilanti, MI - Chemistry
- PhD Program 2002-Present, Case Western Reserve University, Cleveland, Ohio, USA, Department of Chemistry
From left: Quansheng Xi, PhD, Gayle T. Pauer, Stephanie A. Hagstrom, PhD
Not pictured: Andrea Crabb, Alison Szabo and Alyssa Wiener
Director: Stephanie A. Hagstrom, PhD
Department of Ophthalmic Research
Cole Eye Institute
9500 Euclid Ave., i32
Office telephone: 216.445.4133
Goals and projects
Genetic Analysis of Inherited Retinal Diseases
One of the main objectives of our lab is to identify and analyze genes responsible for inherited retinal degenerations such as retinitis pigmentosa, Leber congenital amaurosis and juvenile and age-related forms of macular degeneration. These objectives are met through a candidate gene approach involving the collection of DNA samples from patients with inherited retinal diseases, the selection of candidate genes based on a well-established set of criteria and large-scale mutation screening of the DNA samples using high-throughput, semi-automated molecular genetic techniques.
Retinitis pigmentosa (RP) is a genetically and phenotypically heterogeneous family of inherited retinal diseases. These diseases are characterized by progressive night blindness and peripheral visual loss, which then progresses to loss of central vision. Similar to RP, Leber congenital amaurosis (LCA) is the earliest and most severe form of inherited photoreceptor degeneration and is usually recognized at birth or shortly after. Both disorders feature attenuated retinal vessels, retinal pigmentary deposits and a reduced or nondetectable electroretinogram.
Macular degeneration is a separate heterogeneous group of retinal disorders characterized by progressive central vision loss. Age-related macular degeneration (AMD), the most common form of the disease, is the leading cause of visual impairment in the United States and in many developed countries. Based on clinical evaluation, AMD may be divided into two major subtypes. Approximately 80% of patients have atrophic or “dry” AMD, which is associated with drusen within or under the retinal pigment epithelium (RPE), irregularities in RPE pigmentation, and geographic atrophy of the posterior pole. The remaining 20% of AMD patients have the “wet” form, characterized by choroidal neovascularization (CNV) and/or RPE detachment. The most marked visual losses are associated with the presence of geographic atrophy or CNV. Stargardt disease is the most common form of juvenile macular dystrophy and shares many important clinical and histological features with AMD.
To date, mutations in more than 85 genes have been identified in hereditary retinal degeneration, including those diseases listed above, and it is estimated that an additional 50 disease-causing genes remain to be identified (www.sph.uth.tmc.edu/Retnet). The numerous genes and gene defects that have been identified suggest that many different mechanisms may lead to a common end point, photoreceptor cell death, and motivate our plan to continue to screen candidate genes causing inherited retinal degenerations.
Most importantly, the identification of defective retinal genes has a number of potential clinical benefits for patients:
- It can have prognostic value since there are correlations between specific mutations and severity of visual loss;
- It can improve genetic counseling by refining the diagnosis to include the specific genetic defect, allowing specific molecular diagnostic assays to be applied to the patient’s family;
- It can have implications for therapy, since cataloguing the set of gene defects that cause retinal degeneration will help in understanding the pathogenic disease mechanisms. It is through this knowledge that agents might be developed that slow, stop or reverse these blinding diseases.
The Function of TULP1 in Normal Photoreceptors and in Photoreceptor Degeneration.
The long-term objectives of this project are to explore the physiologic properties of the TULP1 gene product in the retina and define the underlying pathogenic mechanism responsible for photoreceptor degeneration associated with TULP1 mutations. We previously identified mutations in TULP1 that cause a form of autosomal recessive RP, a group of progressive retinal degenerations leading to blindness.
TULP1 is a member of a family of four proteins named TULPs for tubby-like proteins, defined by the highly conserved C-terminal half of their primary sequences. This protein family includes TUB, TULP1, TULP2 and TULP3, all of which have very different N-termini. Database searches do not reveal any significant homology with known proteins or functional motifs. Their physiological functions are unknown but two (TULP1 and TUB) have been linked to photoreceptor degeneration.
We have begun to explore the role of TULP1 in the retina by analyzing the tissue distribution of the protein in normal mice and the photoreceptor disease phenotype in TULP1 knockout mice. We determined that the TULP1 protein is found exclusively in the photoreceptors, localizing predominantly in the inner segments and connecting cilium. In addition, TULP1 -/- mice develop early-onset, progressive photoreceptor degeneration with involvement of both rods and cones. At an early age, the rod and cone opsins, normally targeted to the outer segment, were aberrantly localized to the plasma membranes of inner segments, perinuclear cytoplasm and synaptic regions. At the same age, an abnormal accumulation of rhodopsin-bearing extracellular vesicles was found surrounding the ellipsoid region of the inner segments.
Based upon our data, we hypothesize that TULP1 is involved in the polarized transport of nascent opsin from its site of synthesis in the inner segment to its final destination in the outer segment. We are testing this hypothesis using several different approaches. We are using a proteomic approach to identify cellular proteins that interact with TULP1 and a cell biological approach to determine the subcellular localization of wild-type TULP1 and mutant versions of TULP1.
Lab staff members:
- Stephanie A. Hagstrom, PhD, Director
- Quansheng Xi, PhD, Postdoctoral Fellow
- Gayle T. Pauer, Lab Manager
- Andrea Crabb, Student
- Alison Szabo, Student
- Alyssa Wiener, Student
Front Row: Quteba Ebrahem, MD, Bela Anand-Apte, MD, MBBS, PhD, and Nina Moore, PhD Back Row: Phil Klenotic, PhD, and Jian Hua Qi, PhD
Director: Bela Anand-Apte, PhD, MBBS
Department of Ophthalmic Research
Cole Eye Institute
9500 Euclid Ave., i32
Office telephone: 216.445.9739
Goals and projects
Ocular Neovascularization Regulated by Extracellular Matrix
The clinical significance of ocular angiogenesis is enormous, due to the fact that in the Western hemisphere, retinal neovascularization resulting from diabetic retinopathy is the most common cause of new blindness in young patients. Choroidal neovascularization (CNV) is the chief cause of severe and irreversible loss of vision in elderly patients.
Much progress has been made in the field of angiogenesis research in recent years, fueled by the hypothesis that inhibition of angiogenesis would be a useful strategy to treat cancers. However, there are a number of other diseases in which pathologic angiogenesis plays a role. Retinal neovascularization involves the development of sprouts from retinal vessels, which usually penetrates the inner limiting membrane (ILM) and grows into the vitreous. Retinal neovascularization is observed in ischaemic retinopathies such as diabetic retinopathy, retinopathy of prematurity, central vein occlusion and branch retinal vein occlusion. Choroidal neovascularization (CNV) refers to the formation of new vessels in the subretinal or sub RPE space, which arises from the choriocapillaris. CNV is seen in ocular diseases such as AMD, presumed ocular histoplasmosis, high myopia and angioid streaks.
The broad, long-term goal of the laboratory is to gain an understanding of the mechanism(s) by which alterations in matrix integrity may regulate ocular neovascularization. We are continuing our studies on the role of Tissue Inhibitor of Metalloproteinases-3 in the regulation of choroidal neovascularization and are exploring its potential as a therapeutic agent. We have also identified a novel ADAM-TS like molecule which is expressed in the retina and may play a role in angiogenesis. We are attempting to identify other novel endogenous inducers and inhibitors of angiogenesis to understand the basic biology of neovascularization with a final goal of designing therapeutic approaches to combat this process in disease states.
Our ultimate goal is the prevention and/or reversal of this process in an effort to control the devastating consequences of ocular neovascularization.
Members of the Peachey lab, from left, are Sherry Ball, PhD., Elisa Bala, M.D., Jiang Wu, M.D., Neal Peachey, PhD., Minzhong Yu, M.D., PhD., and Ruth Yarnevic, B.S.
Director: Neal S. Peachey, PhD.
Department of Ophthalmic Research
Cole Eye Institute
9500 Euclid Ave., i32
Office telephone: 216.445.1942
Goals and projects
Mouse Retinal Electrophysiology
As the mouse has become the premiere laboratory model for retina research, it has become increasingly important to develop objective measures of retinal function that can be used to evaluate the function of different classes of retinal cells.
We have adopted a noninvasive technique that has been used to investigate the origins of visual dysfunction in human hereditary and acquired retinal disorders. The ERG (electroretinogram) is the mass electrical response of the retina to light. In the research laboratory, the response provides a sensitive means to evaluate experimental therapies for retinal disease, which can be repeated at different time points on the same animal. In addition, the ERG is used to characterize the effects of pharmacological manipulation or introduction of gene defects.
By controlling the conditions under which stimuli are presented, the activity of the rod or cone visual pathways can be monitored independently. Based on contributions from a number of investigators, it is now possible to relate the different components that comprise the rod-mediated ERG to the major cell types of the rod visual pathway. This knowledge has led to a comprehensive model of the rod ERG which finds wide application.
In comparison, the components that underlie the mouse cone ERG have not been identified. As a major focus of the CEI research program is macular degeneration, we are using pharmacological agents that block transmission from cone photoreceptors to second order neurons that comprise the cone pathway (cone depolarizing bipolar cells, and the cone hyperpolarizing bipolar cells) to determine the contribution of these cell types to the cone ERG. At the completion of this work, we will define a model capable of relating the components of the cone ERG to the cells that comprise the cone pathway.
In collaboration with Alan D. Marmorstein, PhD., we have also developed a noninvasive procedure for recording the electrical response of the retinal pigment epithelium (RPE) to light. In comparison to the rod and cone ERGs mentioned above, the RPE components are very slow, necessitating dc-recording. This procedure will be particularly useful in characterizing rodent models expressing mutant RPE genes.
Mouse Models of Congenital Stationary Night Blindness
For the past several years, we have been working with a naturally occurring mouse model of complete congenital stationary night blindness (CSNB1). This mutant, named nob, involves a defect in transmission from rod and cone photoreceptors to depolarizing bipolar cells. The nob mouse carries a mutation in the nyctalopin gene, which is also the gene involved in human CSNB1. As the function of nyctalopin is unknown, studies are under way to define the role of this protein in normal retinal function and development.
We have also identified a mouse model of another form of human disease, incomplete CSNB (CSNB2). The mouse model involves the CNS-specific deletion of the gene encoding a subunit of the L-type calcium channel that normally regulated release of glutamate at the photoreceptor terminal. These mice develop the same phenotype seen in patients with CSNB2, which involve mutations in the a 1F subunit. Studies are under way to define the role of L-type calcium channels in ribbon synapse formation.
Evaluation of the Retina with a Sub-retinal Microphotodiode Array
This project concerns the tissue compatibility of a subretinal microphotodiode array that has been developed in an attempt to restore vision in patients blinded by diseases causing photoreceptor degeneration. In these diseases, only the photoreceptors degenerate, sparing the inner retinal neurons. The microphotodiode approach relies on electrical stimulation of these inner retinal layers to propagate the visual signal centrally.
In the course of evaluating several implant designs, we have developed a body of data indicating that the implant has good biocompatibility and have found that the use of specific materials for implant fabrication results in a device that will respond consistently for up to 3 years following implantation. While the implant induces disorganization of the inner retinal cell layers, there is no loss of inner retinal neurons in the implanted retina. In addition, the use of cytochemical markers has identified subtle but reproducible changes in the distribution of inhibitory neurons in the inner retina.
We are trying to determine the time course over which these changes occur. The data derived from these studies will provide valuable information on how the inner retina responds to the implant, and may also define areas for implant design improvements.