Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biology112 Animal Models of DiseaseSunday, May 05, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 264-298/D0213-D0247Organizing Section: Biochemistry/Molecular BiologyProgram Number: 264 Poster Board Number: D0213Presentation Time: 8:30 AM - 10:15 AMIdentification of a novel genetic model for retinalneovascularizationMariacarmela Allocca 1 , Barrett D. Leehy 1 , Joseph A. White 1 , Leo A.Kim 1 , Magali Saint-Geniez 1 , Pascal Escher 2, 3 , Dean Eliott 1 , Eric A.Pierce 1 , Margaret M. DeAngelis 4 , Neena B. Haider 1 . 1 MassachusettsEye and Ear/Schepens Eye Research Institute, Department ofOphthalmology, Harvard Medical school, Boston, MA; 2 IRO-Institute for Research in Ophthalmology, Sion, Switzerland;3 Department of Ophthalmology, University of Lausanne, Lausanne,Switzerland; 4 Moran Eye Center, University of Utah, Salt lake City,UT.Purpose: Retinal vascular disease is a hallmark of many blindingdisorders. This includes diseases such as diabetic retinopathy (DR)(that affects millions worldwide), age related macular degeneration(AMD) (that affects 1:3 over the age of 65) and childhood diseasessuch as retinopathy of prematurity. These diseases cause theformation of de novo extra blood vessels (referred asneovascularization) and lead to debilitating vision loss and eventuallyblindness. There are no long-term viable treatment options or curesfor these disorders. Further, for DR and AMD the genetic causes arenot known and no genetic model is available to date. Here we reportthe characterization of Nrv1 mouse, the only known genetic modelthat recapitulates the vascular disease observed in humans.Methods: The Nrv1 mouse was generated by ENU-inducedmutagenesis. For the clinical and phenotypic characterization of themouse model we performed the following analyses: ophthalmoscopy,fluoroangiography, histology and immunohistochemistry on flatmountretinas and retinal sections. A linkage analysis followed bynext generation sequencing was performed to identify the causativegene.Results: The Nrv1 mouse exhibits abnormal production of de novoblood vessels in the retina that innervate into the vitreous. The bloodvessels in the retina are disorganized and clumped. A disorganizedastrocytes network underlines the abnormal vascular topography. Theabnormal blood vessels into the vitreous originate from retinalvasculature and are preceded by the uncommon presence ofastrocytes into the vitreous. The overall data suggests misguidancedefects due to the astrocytes. Linkage analyses mapped the vasculardisorder to a 1Mb region on the chromosome 9. We are currentlysequencing this region in order to identify the mutation and thecausative gene.Conclusions: The Nrv1 mouse model is the first genetic model forretinal neovascularization described thus far and recapitulates thevascular phenotype observed in patient with DR. Characterization ofthis mouse model and its genetic cause will have profound impact inunderstanding the molecular causes of human neovascular diseasesand in the development of a viable treatment that can prevent,attenuate, or cure these diseases.Commercial Relationships: Mariacarmela Allocca, None; BarrettD. Leehy, None; Joseph A. White, None; Leo A. Kim, None;Magali Saint-Geniez, None; Pascal Escher, None; Dean Eliott,Genentech (C), Regeneron (C), Ophthotech (C), Alcon (C), Bausch& Lomb (C), Allergan (C), Alimera (C), Acucela (C), Arctic (C),Salutaris (C); Eric A. Pierce, None; Margaret M. DeAngelis, None;Neena B. Haider, NoneSupport: Chase Fund and Edward N. & Della L. Thome MemorialFoundationProgram Number: 265 Poster Board Number: D0214Presentation Time: 8:30 AM - 10:15 AMSurvey of Common Genetic Eye Diseases in Mouse StrainsBo Chang, Ron Hurd, Jieping Wang, Patsy M. Nishina. The JacksonLaboratory, Bar Harbor, ME.Purpose: As in human populations, in which founder mutations havebeen identified in groups of families, a number of founder mutationshave been observed across strains in mice. In this report, we providea phenotype and genotype survey of three common eye diseases inthe collection of JAX ® Mice strains at The Jackson Laboratory.These eye diseases are retinal degeneration 1 (Pde6b rd1 ), retinaldegeneration 8 (Crb1 rd8 ) and cone photoreceptor function loss 3(Gnat2 cpfl3 ).Methods: We have adapted the tools for examining the small size ofthe mouse eyes for ocular abnormalities. These tools includedindirect ophthalmoscopy, slit lamp biomicroscopy, fundusphotography, histopathology and electroretinography (ERG). Ocularlesions for rd1 and rd8 were evaluated by fundus examination,fundus photography, and histopathology, and the abnormal retinalfunction observed in mice homozygous for cpfl3 was assessed byERG. Genotyping protocols for rd1, rd8 and cpfl3 mutation wereperformed by PCR with appropriate primers.Results: We have actively screened retired breeders for surfacedysmorphologies, and for intraocular defects by indirectophthamoscopy, slit lamp biomicroscopy, and ERG to discover newspontaneous mutations in strains from the Genetic Resource Science(GRS) production colony. We have found that 99 strains have the rd1mutation, 85 strains have the rd8 mutation and 20 strains have thecpfl3 mutation.Conclusions: Of the 1000 number of strains screened during thisstudy, 204 carried one of three founder mutations in pde6b, crb1 orcpfl3. Since these three retinal mutations commonly occur in variousmouse strains, genotyping for these mutations and/or avoiding mousestrains or stocks carrying these mutant alleles when studying newretinal disorders would be recommended.Commercial Relationships: Bo Chang, None; Ron Hurd, None;Jieping Wang, None; Patsy M. Nishina, NoneSupport: NIH EY019943, EY11996Program Number: 266 Poster Board Number: D0215Presentation Time: 8:30 AM - 10:15 AMUp-regulation Of Growth Hormone Releasing Hormone (GHRH)Receptors In The Ischemic RetinaSagar Patel 1 , Folami Lamoke 1, 2 , Chaunte E. Stampley 3 , BabakBaban 4 , Maritza Romero 2 , Rudolf Lucas 5, 2 , Norman L. Block 6, 7 ,Andrew Schally 6, 7 , Manuela Bartoli 1, 2 . 1 Department ofOphthalmology, Georgia Health Sciences University, Augusta, GA;2 Department of Pharmacology and Toxicology, Georgia HealthSciences University, Augusta, GA; 3 Rollins School of Public Health,Emory University, Atlanta, GA; 4 Department of Oral Biology,Georgia Health Sciences University, Augusta, GA; 5 VascularBiology Center, Georgia Health Sciences University, Augusta, GA;6 Veteran Affairs Medical Center and South Florida Veterans AffairsFoundation for Research and Education, Miami, FL; 7 Miller Schoolof Medicine, University of Miami, Miami, FL.Purpose: Modulation of the growth hormone axis may have potentialtherapeutic applications for the treatment of ischemic retinopathiesand retinal neovascularization. Growth hormone releasing hormone(GHRH) is a hypothalamic hormone involved in the regulation ofgrowth hormone release from the pituitary gland. Recent evidence©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologysuggests that GHRH expression and activity has extra-pituitaryeffects. Here we have studied the localization and expression ofGHRH receptors (GHRH-R) in normal and ischemic retina.Methods: Western blotting and immunohystochemical analyses wereemployed to identify GHRH-R in retinas of streptozotocin-induceddiabetic rats (STZ-rats) and control age-matched normoglycemic rats.Human post-mortem diabetic donor retinas and normoglycemiccontrols were also analyzed. Expression and immunolocalization ofGHRH was also determined in mice subjected to oxygen inducedretinopathy (OIR) and age-matched control mice. Retinal endothelialcells (RECs) were exposed to hypoxia (Hx, pO2=1%) for 6, 12 and24 hours. The expression of GHRH-R was then assessed by Westernblotting and compared to that measured in cells cultured in normoxicconditions (Nx, PO2=23%).Results: In normal retinas, GHRH-R expression is very low andlocalized to ganglion cells. In the diabetic retinas of both rats andhumans, GHRH-R are up-regulated, initially in ganglion cells andsubsequently, more markedly, around blood vessels. We, therefore,measured GHRH-R expression in a model of ischemic retinopathywhere retinal neovascularization takes place. Western blottinganalysis showed up-regulated GHRH-R expression in the ischemicretina, at postnatal days 14 and 17, with immunohistochemistryrevealing clear co-localization of GHRH-R with blood vessels.Exposure of RECs to hypoxia promoted the up-regulation of GHRH-R after 12 hours treatment, as measured by Western blotting.Conclusions: Our studies reveal, for the first time, that GHRH-R areexpressed in the retina and their expression levels are increased byischemia/hypoxia particularly in blood vessels and in retinalendothelial cells. Our work suggests that modulation of GHRHsignaling in retinal blood vessels may be of potential therapeuticvalue for the management of ischemic retinopathies and retinalneovascularization.Commercial Relationships: Sagar Patel, None; Folami Lamoke,None; Chaunte E. Stampley, None; Babak Baban, None; MaritzaRomero, None; Rudolf Lucas, None; Norman L. Block, None;Andrew Schally, None; Manuela Bartoli, NoneProgram Number: 267 Poster Board Number: D0216Presentation Time: 8:30 AM - 10:15 AMDetermination of intraocular oxygen tension and temperature inretinal diseasesYoung-Hoon Park, So-Yoon Kim, Ji-Wook Yang. Ophthalmology &Visual Science, College of Medicine, The Catholic Univ of Korea,Seoul, Republic of Korea.Purpose: To determine the intraocular distribution of oxygen tensionand temperature in the various retinal diseases.Methods: Oxygen tensions (pO2) and temperatures were measuredbefore vitrectomy at sites in the mid-anterior chamber (AC), in theanterior chamber angle, in the mid-vitrous cavity, and at the innerretina surface using an fiber-optic oxygen/temperature sensor(Oxylab pO2/Temp Optode; Oxford Optronix, Oxford, UK) in 8subjects with proliferative diabetic retinopathy (PDR) who haddeveloped recurrent vitreous hemorrhage, 17 subjects withrhegmatogenous retinal detachment (RRD), and 19 subjects withepiretinal membrane (ERM). In addition, 9 subjects with intravitrealsilicone oil and 4 subjects with pseudophakia were included in thisstudy.Results: The mean pO2 in the mid-vitreous in PDR (16.99 ± 8.22mmHg) was significantly higher than those in ERM (6.66 ± 3.39mmHg) (P=.027). The mean pO2 in the mid-AC and at the innerretina surface in RRD were 13.77 ± 5.95 and 4.81 ± 2.95 mmHg,which were significantly different from those in ERM (5.30 ± 4.46and 10.24 ± 3.18 mmHg) (P=.037 and .001). The mean pO2 in themid-vitreous and inner retina surface in silicone oil filled eyes were8.63 ± 2.45 and 8.76 ± 2.37 mmHg, which were not significantlydifferent from those in ERM (P=.052 and .493). However, the meanpO2 in the mid-AC (15.03 ± 2.81 mmHg) and in the mid-vitreous(16.88 ± 1.0 mmHg) in pseudophakia were significantly higher thanthose in ERM (5.30 ± 4.46 and 6.66 ± 3.39 mmHg) (P=.006 and.025). The mean temperature in the mid-AC, the AC angle, the midvitreous,and at the near inner retina were respectively 28.99 ± 1.28,28.70 ± 3.24, 31.97 ± 1.20, and 33.73 ± 1.36 °C, and those weresignificantly different from each other (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyProgram Number: 269 Poster Board Number: D0218Presentation Time: 8:30 AM - 10:15 AMDownregulation of aldehyde detoxification enzymes in thediabetic retinaRosie Mcdowell, Timothy M. Curtis, Graham McGeown. Centre forVision and Vascular Science, Queens University Belfast, Belfast,United Kingdom.Purpose: Previous studies have demonstrated accumulation of theacrolein-derived advanced lipoxidation end-product, FDP-lysine, inretinal Müller cells during diabetes. The aim of the present study wasto begin to elucidate the mechanisms by which this adductaccumulates within the diabetic retina, including examination of theexpression of aldehyde detoxification enzymes and also enzymesinvolved in polyamine metabolism.Methods: RNA from the retinas of Sprague Dawley rats was isolatedto determine the expression of aldehyde detoxification genes by RT-PCR. For immunohistochemistry, eyes were subject to fixation in 4%PFA, cryo-sectioning and then labelling with anti-bodies againstALDH1a1, ALDH2, AKR1B1 and AKR7a2. In separateexperiments, male Sprague-Dawley rats were rendered diabetic by asingle injection of streptozotocin (65mg/kg) and eye tissue washarvested after 3 months disease duration. RNA was isolated fromthe retinas of six diabetic animals and six age matched sham controlsand then processed for RT-PCR. All animal care and experimentalprocedures strictly conformed to the ARVO Statement for the use ofAnimals in Ophthalmic and Vision Research.Results: RT-PCR indicated ALDH1a1, 2, 3a1, 3a2, 9, 16, 18a1AKR1b1 and 7a2 were expressed in the rat retina.Immunohistochemistry experiments revealed that ALDH1a1, 2,AKR1B1 and 7a2 are principally localised to retinal Müller cells. Thecomparison of mRNA transcripts between control and diabeticanimals indicated that ALDH1a1 7a2 and 3a2 were significantlydown-regulated in whole retinal extracts of diabetic animals, whilstpolyamine metabolic enzyme levels were unchanged.Conclusions: Down-regulation of detoxifying enzymes in retinalMüller cells may represent a major mechanism contributing to theselective accumulation of FDP-lysine adducts in these cells duringdiabetes.Commercial Relationships: Rosie Mcdowell, None; Timothy M.Curtis, None; Graham McGeown, NoneSupport: Fight for Sight UK grantProgram Number: 270 Poster Board Number: D0219Presentation Time: 8:30 AM - 10:15 AMTXNIP regulates neurotrophic factor expression and neuronalinjury in early diabetic retinopathyLalit P. Singh 1, 2 , Takhellambam S. Devi 1 . 1 Anatomy and CellBiology, Wayne State Univ Sch of Med, Detroit, MI;2 Ophthalmology, Wayne State Univ Sch of Med, Detroit, MI.Purpose: We have recently published that the pro-oxidantthioredoxin interacting protein (TXNIP) is significantly up-regulatedin the diabetic retina in vivo and mediates cellular oxidative stressand retinal inflammation. The purpose of this study is to investigatethe role TXNIP plays in neurotrophic factor expression and neuronalinjury in early diabetic retinopathy (DR).Methods: Streptozotocin (STZ)-induced diabetic and age-matchednormal rats were maintained for 8 weeks. Subsequently, retinas wereharvested and analyzed for gene expression by real time quantitativePCR and immunohistochemistry. To knock down TXNIP, scrambledRNA and siTXNIP were injected intravitreally in diabetic rat retinas.Results: We observe that TXNIP, iNOS, radial GFAP (Muller cell(MC) activation) and protein s-nitrosylation (SNO) are increased indiabetic rat retinas when compared with normal rats. These resultssuggest that TXNIP up-regulation correlates with retinaloxidative/nitrosative (ROS/RNS) stress and MC activation. Inaddition, we found that the mRNA level of brain-derivedneurotrophic factor (BDNF) is not altered; however, the expression ofits high affinity receptor TrkB and that of glia derived neurotrophicfactor (GDNF) are significantly increased. Conversely, the mRNAlevels of dendritic spine protein synaptopodin and tyrosinehydroxylase (TH), a marker for dopaminergic inter-neurons, aredown-regulated in DR indicating neuronal injury. Finally, knockdown of TXNIP by siRNA reduces several molecular abnormalitiesof early DR.Conclusions: We propose that TXNIP expression is critical in theinitiation and development of early DR. Therefore, TXNIP representsa novel therapeutic target to ameliorate blinding ocular complicationsof diabetes.Commercial Relationships: Lalit P. Singh, None; TakhellambamS. Devi, NoneSupport: WSU SOM Bridge Funds; NEI Core Grant; Mid-West EyeBanksProgram Number: 271 Poster Board Number: D0220Presentation Time: 8:30 AM - 10:15 AMPost-Translational Processing and Expression of Synaptophysinis Altered by Reduced α-Mannosidase Activity in DiabetesAlistair J. Barber, Travis S. D'Cruz, Brittany N. Weibley.Ophthalmology, Penn State Hershey College of Medicine, Hershey,PA.Purpose: Previous research has shown that the neurotransmittervesicle protein, synaptophysin, is depleted in the retinas of diabeticrats. We recently demonstrated that diabetes alters the posttranslationalprocessing of synaptophysin by increasing mannoseglycosylation leading to an accumulation of mannose-richsynaptophysin. In this study we hypothesized that accumulation ofmannose-rich synaptophysin is due to reduced activity of α-mannosidase, the enzyme responsible for de-mannosylation ofsynaptophysin, resulting in reduced expression of the mature form ofthe protein. We tested this hypothesis by determining the effect ofdiabetes on α-mannosidase activity, and investigating whetherinhibition of this enzyme in control retinas affects synaptophysinexpression and turnover similar to diabetes.Methods: Diabetes was induced in male Sprague-Dawley rats bystreptozotocin (STZ, 65 mg/kg, i.p.). After four weeks ofhyperglycemia the retinas were explanted for in vitro study. The drugswainsonine was used to inhibit α-mannosidase activity in explantedcontrol retinas and the relative amount of synaptophysin wasmeasured by western blot analysis. The relative amount of mannoserichsynaptophysin was also determined by western blotting afterincubating lysates with endoglycosidase H (endo-H). Relativedegradation of synaptophysin was measured by western blottingretinas treated with cycloheximide.Results: Retinas from STZ-diabetic rats had significantly moremannose-rich (endo-H sensitive) synaptophysin compared to controls(p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyprocessing. Reduced activity of α-mannosidase offers a mechanismfor the diminished expression of retinal synaptophysin in diabetes.Commercial Relationships: Alistair J. Barber, None; Travis S.D'Cruz, None; Brittany N. Weibley, NoneSupport: JDRF, ADA, PA LionsProgram Number: 272 Poster Board Number: D0221Presentation Time: 8:30 AM - 10:15 AMAutophagy in Modified LDL-induced Pericyte Loss in DiabeticRetinopathyDongxu Fu 1, 2 , Shihe Yang 1 , Mingyuan Wu 1 , Mei Du 1 , Junping Chen 1 ,Kenneth Wilson 1 , Timothy J. Lyons 1 . 1 Harold Hamm OklahomaDiabetes Ctr, Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK;2 Department of Immunology, Harbin Medical University, Harbin,China.Purpose: We hypothesise that extravasation of plasma lipoproteinsthrough damaged blood retinal barriers and their subsequentmodification (glycation, oxidation) are more important in thepropagation of diabetic retinopathy (DR) than circulatinglipoproteins. We previously showed that extravasated, modified LDL(modeled by highly-oxidized glycated LDL, HOG-LDL) wasassociated with human retinal capillary pericyte (HRCP) apoptosisand autophagy mediated by oxidative and ER Stress. Little is knownabout role of autophagy in modified LDL-induced pericyte loss.Methods: HRCP was exposed to HOG-LDL at differentconcentrations (up to 200mg/l) and times (up to 24h); native LDL (N-LDL) was used as control. Cells were transfected with plasmid GFP-LC3, si-RNA (si-CHOP or si-JNK) ,for 24-36h or pre-treated withinhibitors of autophagy (3-methyladenine and chloroquine) andcaspase (z-VAD-fm), ER stress (4-phenyl butyric acid), or JNK(sp600125), each for 1h before LDL treatment. Autophagy,apoptosis, JNK activity, cell viability, oxidative stress and ER stresswere determined by methods including immunocytochemistry,western blots, CCK-8 assay, TUNEL assay and cellular ROSmeasurement assay. To define the relevance of the results in vivo,immunohistochemistry was performed to detect LC3 and p-JNK from(a) mouse model of DR that mimics exposure of the retina to elevatedglucose and elevated LDL levels, and (b) human subjects with andwithout diabetes and DR. Markers above were also measured byWestern blot in human retinas.Results: Compared with N-LDL, HOG-LDL induced autophagy inpericytes, pJNK played an essential role. With lower concentrationsof HOG-LDL (0-50mg/l), autophagy inhibited apoptosis promotingcell survival, but with higher concentrations (50-200mg/l), autophagyinduced autophagic death and apoptosis. In diabetic hyperlipidemicmice (vs. animals with either condition alone), retinal autophagy wasenhanced. Autophagy was also enhanced in diabetic human retina,but was not correlated with severity of DR.Conclusions: In DR, extravasated and modified plasma lipoproteinsplay an important role once blood retinal barrier (BRB) leakage isestablished, and autophagy is implicated. The findings may lead tonew treatments to inhibit the development of DR.Commercial Relationships: Dongxu Fu, None; Shihe Yang, None;Mingyuan Wu, None; Mei Du, None; Junping Chen, None;Kenneth Wilson, None; Timothy J. Lyons, NoneSupport: COBRE Program of the National Center for ResearchResources P20 RR 024215Program Number: 273 Poster Board Number: D0222Presentation Time: 8:30 AM - 10:15 AMProfiling miRNAs in a hyperglycemic and hypoinsulinemicIns2Akita mouse modelAudrey Giocanti-Auregan 1, 2 , Lisa Ahn 1 , Javier Sancho-Pelluz 3, 4 ,Stephen H. Tsang 3 , Szilard Kiss 1 , Mark Rosenblatt 1 , Thomas Tuschl 5 ,John T. Pena 1 , Donald J. D’Amico 1 . 1 Ophthalmology, Weill CornellMedical College, New York, NY; 2 Ophthalmology, Avicennehospital, Bobigny, France; 3 Bernard & Shirlee Brown GlaucomaLaboratory. Department of Ophthalmology, Columbia University,New York, NY; 4 Facultad de Medicina, Universidad Católica SanVicente Mártir, Valencia, Spain; 5 Howard Hughes Medical Institute,The Rockefeller University, New York, NY.Purpose: Diabetic retinopathy (DR) is a leading cause of blindnessworldwide involving neoangiogenesis and blood retinal barrierbreakdown. Our goal is to identify novel genetic targets involved inDR. microRNAs (miRNAs) are non-coding RNAs that regulate geneexpression and modulate several pathologic diseases. The purpose ofthis study is to characterize the in vivo perturbations of retinalmiRNAs in a hyperglycemic and hypoinsulinemic mouse model.Here, we characterize the vascular and macroscopic anatomicproperties of the Ins2Akita mouse and then profiled the miRNAabundance longitudinally.Methods: Heterozygous male Ins2Akita with a mutation in theinsulin 2 gene, were age matched with C57BL6/J mice. We analyzedvascular permeability by Evans blue extravasation, identified retinalvascular macroscopic lesions by fundus photography and fluoresceinangiography, and surveyed for functional visual impairment byelectroretinography (ERG). We measured miRNA abundance in theretina and choroid/retinal pigmented epithelium (RPE) at several timepoints using multiplexed libraries of Ins2Akita and wild-type mice at1 and 3 months of age. We identified cell-type specificity of miRNAsin fresh frozen retina tissue sections using a modified in situhybridization (ISH) procedure.Results: Morphological changes and fluorescein angiogramabnormalities were identified in the retinas of Ins2Akita at 1 and 3months. We observed substantially increased vascular permeability inthe Ins2Akita retina at 3 months. However, no functional changeswere observed by ERG for up to 3 months of age. miRNA profileswere generated for Ins2Akita and control mice (1 and 3 months),which showed substantial down regulation of several miRNAsincluding miR-126 and miR-143 in the retina and choroid/RPE. ISHstudies showed cell-type specific staining in several retinal layers inwild-type retina tissue sections.Conclusions: We further characterized the Ins2Akita mouse modelof hyperglycemia and hypoinsulinemia. We found that despitesubstantial macroscopic morphological changes in the fundus andincrease in retinal vascular permeability of Ins2Akita mice, nofunctional change was yet detectable by ERG at 3 months. SeveralmiRNAs are deregulated in the Ins2Akita mouse and severalmiRNAs are cell type specific. The miRNAs identified in our screenoffer potential targets for genetic modulation of hyperglycemicinduced pathology in the eye.Commercial Relationships: Audrey Giocanti-Auregan, None;Lisa Ahn, None; Javier Sancho-Pelluz, None; Stephen H. Tsang,None; Szilard Kiss, Alcon (F), Alimera (F), Alimera (C), Alimera(R), Allergan (F), Allergan (C), Allergan (R), Genentech (F),Genentech (C), Genentech (R), Regeneron (F), Regeneron (C),Regeneron (R), Optos (F), Optos (C), Optos (R), Eytech (C),Merge/OIS (C), Merge/OIS (I); Mark Rosenblatt, None; ThomasTuschl, The Rockefeller University (P), MIT (P), max planckinstitute (P), alnylam pharmaceuticals (C); John T. Pena, TheRockefeller University (P); Donald J. D’Amico, Ophthotech, Inc (I),OptiMedica, Inc (I), Neurotech, Inc (I), Genentech, Inc (C), LuxBiosciences, Inc (C)Support: NIH CTSC grant, Margaret M. Dyson Vision Research andInstitute Research to Prevent Blindness.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyProgram Number: 274 Poster Board Number: D0223Presentation Time: 8:30 AM - 10:15 AMInfluence of Proliferative Diabetic Retinopathy Aqueous inCausing Defective CD34 cellsSankarathi Balaiya 1 , Maria B. Grant 2 , K V Chalam 1 .1 Ophthalmology, Univ of Florida College of Med, Jacksonville, FL;2 Pharmacology and Therapeutics, University of Florida College ofMedicine, Gainesville, FL.Purpose: Proliferative diabetic retinopathy (PDR), a majorcomplication of diabetes induces retinal microvascular endothelialdysfunction lead to decreased retinal perfusion, hypoxia andsubsequent induction of angiogenic factors. Endothelial progenitorcells, a subpopulation of the circulating mononuclear cells arerecruited to sites requiring vascular repair and can contribute to therepair and viability of the vasculature. However, in diabetes,dysfunctional EPCs may not repair this injury and promotedevelopment of acellular capillaries and sustained retinal ischemia. Inthis study, we investigated whether angiogenic cytokines present inaqueous of PDR subjects might affect the reparative nature of CD34cells.Methods: Aqueous fluid was obtained from individuals with PDRundergoing pars plana vitrectomies in accordance with Institutionalreview board at the University of Florida, Jacksonville. Aqueousfluid from healthy individuals who underwent cataract extractionwithout any ocular diseases served as controls. Mobilized healthyhuman CD34 cells were maintained in an undifferentiated state andtreated with 1% and 5% concentration of PDR and control aqueousfluid. The effect of PDR aqueous on CD34 cells were analyzed usingtrypan blue exclusion assay on day 4 and day 7. In addition,migration chamber assay was performed on aqueous enriched CD34cells (10,000 cells); conditioned medium was analyzed withnanoelectrospray ionization mass spectrometry (nESI-MS).Results: In presence of aqueous, CD34 cells showed increasedproliferation in a time-dependent manner. On day 4, they did notshowed increased proliferation compared to control (12.8x106 Vs20x106), whereas proliferation increased on day 7 (3.73x106 Vs3.06x106). Pretreated CD34 cells with varying concentration ofaqueous fluid from PDR patients did not cause any increase inmigratory response compared to aqueous fluid from healthy patients.However, the migratory response was significantly reduced to 12.2%in comparison to control after treatment with 5% concentration ofaqueous fluid from PDR patients (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyROC: AUC= 0.7837, P= 0.006). Those eyes with better VAdemonstrated significant correlation of MMP-9 with proinflammatorycytokines, while in those with worse VA MMP-9correlates with pro-angiogenic cytokines. Additionally, MMP- 9levels are higher in the vitreous of non-diabetic controls than in thevitreous from PDR patients (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyPurpose: Purpose: Pericyte degeneration is an early event in diabeticretinopathy (DR) and plays an important role in retinal vascularleakage. Fenofibrate, a Peroxisome Proliferator-Activated Receptor α(PPARα) agonist, has been shown to have robust protective effectsagainst DR. This study is to evaluate the efficacy and molecularmechanism of Fenofibrate on pericyte loss in DR.Methods: Methods: Fenofibrate was administrated to streptozotocin(STZ)-induced diabetic Wistar rats at dose of 160 mg/kg/day for 2months. Periodic acid schiff (PAS) and hematoxylin staining wereperformed to quantify pericyte loss and acellular capillaries in retinadigestion samples. Palmitate, a saturated fatty acid, was used as adiabetic stressor in cultured primary human retinal capillary pericytes(HRCP). Cell viability was quantified by MTT assay, and apoptoticcells were measured by TUNEL assay . Intracellular ROS levels weremeasured by CM-H2DCFHDA. Western blot analysis was employedto measure changes of protein levels.Results: Results: Fenofibrate significantly decreased retinal pericyteloss and acellular capillaries in diabetic rat retina. Fenofibrateimproved HRCP viability and protected the cells from palmitateinducedapoptosis. Fenofibrate decreased palmitate-induced ROSproduction and down-regulated NOX4 expression. Fenofibratesuppressed NF-kB activation and prevented the down-regulation ofplatelet-derived growth factor-B (PDGF-B) expression in HRCP afterpalmitate treatment. Furthermore, over-expression of PPARα hadantioxidant and anti-inflammation effects in pericytes, similar toFenofibrate.Conclusions: Conclusions: Fenofibrate has therapeutic potential onretinal pericyte loss in DR and the effect is, at least in part, throughactivating PPARα.Commercial Relationships: Lexi Ding, None; Rui Cheng, None;Yang Hu, None; Jian-Xing Ma, NoneSupport: NIH grants EY018659, EY012231, EY019309 andP20GM104934Program Number: 280 Poster Board Number: D0229Presentation Time: 8:30 AM - 10:15 AMIGF-1R EXPRESSION IN AN OXYGEN-INDUCEDRETINOPATHY (OIR) MODELMaria C. Sanchez 1 , Valeria E. Lorenc 1 , Luna D. Jose 2 , Gustavo A.Chiabrando 1 . 1 CIBICI-Dpto de Bioquimica Clinica, Fac de CienciasQuimicas UNC, Cordoba, Argentina; 2 Clinica de Ojos Romagosa-Funadacion Ver, Cordoba, Argentina.Purpose: Various eye diseases, including proliferative diabeticretinopathy (PDR) and retinopathy of prematurity (ROP), are theresult of a pathological neovascularization. Different animal modelssuch as oxygen-induced retinopathy (OIR) have been developed inorder to understand the cellular and molecular mechanisms of thesediseases. One of the growth factors involved in both physiologicaland pathological neovascularization is the Insulin-like growth factor-1 (IGF-1). Previously, we have demonstrated the IGF-1 effect onretinal cell migration via IGF-1R and the expression of this receptorin retina of animals with and without OIR treatment (ARVO Meeting2012). Herein, we examine the IGF-1R expression, tissue distributionand cellular localization in relationship with the retinal cell death.Methods: Neonatal C57BL/6J mice were subjected to 75% oxygenfrom postnatal day 7 (P7) to P12 and then returned to room air forfive days. Control mice were exposed to room air from birth untilP17. Retinal blood vessel patterns were visualized by GSA labeling.Retinas from animals with and without OIR treatment were analyzedfor IGF-1R expression and tissue distribution at selected time points(P3, P12, P15, P18, P21 and P27). The IGF-1R localization wasexamined using cell-type specific markers (GFAP, GS, Brn3a, PKCalpha and calbindin among other) by immunofluorescence andconfocal microscopy. In order to identify apoptotic cells in the sameretinas, TUNEL assay (Roche) was used.Results: In retinas without OIR we first observed IGF-1R expressionin endothelial cells which was confirmed by using the GSA. Thisreceptor was also expressed at level of ganglion cells as well as in theend feet of Muller glial cells. In addition, we visualized staining forIGF-1R in inner and outer nuclear layers. When the IGF-1Rexpression in the OIR model was analyzed, we observed a change inthe distribution due to an alteration in the structure of the neuralretina. Considering that this model produces Muller glial cellsactivation and retinal cell death, then the expression of GFAP wasanalyzed. Increased GFAP expression was detected in Müller cells inOIR retinas from P15. Finally, in agreement with GFAP expression,TUNEL possitive cells in the retinas were also observed at P15.Conclusions: The IGF-1R distribution and localization in retinaswere modified after OIR treatment coinciding with the onset ofGFAP expression and the retinal cell death.Commercial Relationships: Maria C. Sanchez, None; Valeria E.Lorenc, None; Luna D. Jose, None; Gustavo A. Chiabrando, NoneSupport: FONCyT, CONICET, SeCyT-UNC.Program Number: 281 Poster Board Number: D0230Presentation Time: 8:30 AM - 10:15 AMPhotoreceptors are a main generator of superoxide in retinas ofdiabetic miceTimothy S. Kern 1, 2 , Yunpeng Du 1 , Krzysztof Palczewski 3 , Bruce A.Berkowitz 4 . 1 Medicine, Case Western Reserve University, Cleveland,OH; 2 Stokes Veterans Administration Hospital, Cleveland, OH;3 Pharmacology, Case Western Reserve University, Cleveland, OH;4 Anatomy/Cell Biol, Wayne State Univ Sch of Med, Detroit, MI.Purpose: Prior work by us and others has provided strong evidencethat oxidative stress and inflammatory processes play important rolesin the development of the vascular lesions of early diabeticretinopathy. Oxidative stress is known to regulate expression of proinflammatoryproteins, thus making it important to understand thesource of the oxidative stress. We have investigated the contributionof photoreceptors to the retinal oxidative stress and induction ofproinflammatory ICAM-1 in diabetes.Methods: Retinal oxidative stress was assessed histologically usingdichlorofluorescein staining, and quantitatively using luciginenluminescence in C57Bl/6J mice (nondiabetic and diabetic for 2 mos).Two models that caused degeneration of photoreceptors (a geneticmodel (rhodopsin knockout) and a chemically-induced degenerationof photoreceptors (iodoacetic acid)) were studied after 2 mosdiabetes.Results: In the diabetic mice, dichlorofluorescein stain indicated thatthe majority of oxidative stress was localized in the photoreceptorlayer. Both models of photoreceptor degeneration resulted inessentially total obliteration of photoreceptors. The diabetes-inducedincrease in superoxide observed in wildtype C57Bl/6J mice wassignificantly inhibited in both of the diabetic models lackingphotoreceptors. The diabetes-induced induction of ICAM-1 (used asa marker of inflammation) was significantly inhibited in diabeticanimals missing photoreceptors (rhodopsin knockout).Conclusions: These findings demonstrate a critical role ofphotoreceptors in the diabetes-induced oxidative stress in the retina,and presumably in the pathogenesis of other lesions of diabeticretinopathy.Commercial Relationships: Timothy S. Kern, Bausch & Lomb (F),PamLab (F); Yunpeng Du, None; Krzysztof Palczewski, QLT Inc(F), Polgenix Inc (E), Visum Inc (P), Amegen Inc (F); Bruce A.Berkowitz, NoneSupport: EY00300 and VA Merit grant©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyProgram Number: 282 Poster Board Number: D0231Presentation Time: 8:30 AM - 10:15 AMAn Xbp1-GFP-rhodopsin reporter transgene for assessing ERstress in Xenopus laevis models of retinitis pigmentosaBeatrice M. Tam 1 , Jonathan H. Lin 2 , Orson L. Moritz 1 .1 Ophthalmology and Visual Sciences, University of BritishColumbia, Vancouver, BC, Canada; 2 Pathology, UCSD School ofMedicine, La Jolla, CA.Purpose: We have previously demonstrated that in transgenic X.laevis, P23H rhodopsin is significantly retained in the ER, and thatlight exposure exacerbates this ER retention and promotes retinaldegeneration. Here we examine whether accumulation of the mutantprotein induces ER stress in vivo by monitoring the splicing of anXbp1-GFP reporter construct.Methods: The DNA binding domain of murine Xbp1 was deletedand an out of frame cDNA encoding a GFP-rhodopsin fusion proteininserted downstream of the unconventional splice site. Upon ERstress, splicing of the mRNA produces an in-frame Xbp1-GFPrhodopsin(XGR) fusion protein. This engineered cDNA was used togenerate transgenic X. laevis expressing the fusion protein under thecontrol of the opsin promoter. An XGR frog was mated with atransgenic frog expressing bovine P23H rhodopsin (bRhoP23H). Theresulting tadpoles were raised for 14 days in constant dark, whichprevents retinal degeneration in this animal model. Half the tadpoleswere then exposed to two 12:12 light:dark cycles and returned to thedark. The other half were maintained in constant dark. On day 21, alltadpoles were killed and their eyes used for dot blot analysis or forfluorescence microscopy.Results: In our line of XGR animals, GFP fluorescence peakedshortly after the onset of rod opsin expression (~d5) and thereafterdeclined and was virtually undetectable by d21. Interestingly, XGRdistributed to both inner and outer segment membranes. In contrast, asimilar protein (GFP-RhoCT44) which does not include Xbp1, isalmost exclusively delivered to the outer segment. In cryosections ofeyes that were not expressing bRhoP23H, little or no GFPfluorescence was observed regardless of whether the animals hadreceived light exposure or not. Similarly, dark reared bRhoP23Hretinas expressed minimal XGR. In contrast, light exposedbRhoP23H retinas exhibited higher levels of fluorescence.Furthermore, nuclear XGR localization was observed in this group.As expected, retinas expressing bRhoP23H underwent retinaldegeneration when exposed to light.Conclusions: At d21, significant levels of Xbp-GFP-RhoCT44 wereobserved only in light exposed animals expressing bRhoP23H. Thisis consistent with a pathogenic role for light-induced P23H rhodopsinmisfolding and ER stress in this model of retinitis pigmentosa.Commercial Relationships: Beatrice M. Tam, None; Jonathan H.Lin, None; Orson L. Moritz, NoneSupport: CIHR-64400, Foundation Fighting Blindness Canada,NIH/NEI RO1-EY020846Program Number: 283 Poster Board Number: D0232Presentation Time: 8:30 AM - 10:15 AMAnalysis of microRNA expression changes following explantationof bovine RPET. Michael Redmond 1 , Cynthia Jaworski 1 , William Samuel 1 , R K.Kutty 1 , Todd Duncan 1 , Toral P. Parikh 1, 2 . 1 LRCMB Bldg 6 Rm117A, National Eye Inst/NIH, Bethesda, MD; 2 Howard HughesMedical Institute, Bethesda, MD.Purpose: An enduring challenge in studying retinal pigmentepithelium (RPE) is the limited quantity of tissue available. Althoughcultured RPE cells are widely used, the phenotypes of cultured cells,with time, differ substantially from native tissue. For example, manycultured RPE cells do not express RPE65 protein, a distinguishingfeature of RPE. To better understand this alteration in geneexpression, in particular the loss of key visual cycle proteins, weasked whether the expression pattern of microRNAs (miRs) is alteredafter explantation, in concert with the changes in gene and proteinexpression.Methods: RPE was harvested from freshly dissected bovine eyes andplaced in tissue culture. RNA and protein were extracted by standardmeans from fresh tissue and from cells harvested after 4 or 8 weeksin culture. MiR expression was assessed using the miRCURY LNAmiR Array (Exiqon Services). Quantified signals were normalized.Expression levels of selected miRs were validated by real time RT-PCR.Results: Principal component analysis indicates that samples clusteraccording to their time in culture. Hierarchical clustering reveals thespecific miRs comprising each cluster. Nineteen miRs weresignificantly different (p-value less than 0.05 with Bonferronimultiple testing) between native tissue and 4-week cultures; 78differences were found between native tissue and 8-week cultures.There were 28 microRNAs that differed between the 4 and 8 weekculture samples. Several miRs show evidence of a transient change ofexpression at 4 weeks, returning to levels similar to that of nativetissue upon more prolonged culture time. Interestingly, the “sensorycluster” of miR-96, -182, and -183 all had diminished expressionwith culture, as did mir-204 and -211, reported to be characteristic ofRPE. Real time RT-PCR of selected miRs corroborated theexpression profiles. Expression of miR-21 and -184 rose markedlywith growth in culture; sensory cluster miR levels fell.Conclusions: The pattern of microRNA expression changessignificantly when bovine RPE cells are explanted and grown inculture, suggesting that this class of regulatory molecules may beimportant in producing the altered culture phenotype and/or inmaintaining the characteristics that define a functional in situ RPEcell. Analysis of potential gene targets showing increase or decreasewill help us discriminate between miRs regulating these opposingtrends.Commercial Relationships: T. Michael Redmond, None; CynthiaJaworski, None; William Samuel, None; R K. Kutty, None; ToddDuncan, None; Toral P. Parikh, NoneSupport: NIH NEI Intramural Research Program; Howard HughesMedical InstituteProgram Number: 284 Poster Board Number: D0233Presentation Time: 8:30 AM - 10:15 AMAccumulated Increase of mtDNA Damage Contribute toProgressive Loss of RGC in a Rat Model of GlaucomaJihong Wu, Shenghai Zhang. Eye & ENT Hospital, Fudan University,Shanghai, China.Purpose: To investigate the molecular mechanisms for theprogressive RGCs loss even after IOP elevation return to normal in aglaucomatous rat model.Methods: Wistar rat was used to induce glaucomatous modelthrough episcleral vein cauterization (EVC). IOP elevation sustainedfor 6 weeks and we continued the experiment till 6 months when IOPhad already returned to normal to mimic the clinical feature ofglaucoma. RGC loss was detected by retrograde labeling of FG.Fresh RGCs was isolated with magnetic beads coated by CD 11b/cand Thy-1 antibodies. Mitochondria isolation, DNA and RNAisolation were carried out in order to detect mtDNA damage ormutations, mtDNA copies, expression of mtDNA repair enzymes,and mitochondrial function. Cultured astrocytes were given 30mmHg to study the whether pressure induce mitochondria©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologydysfunction and possible mechanisms.Results: We have reported that there was a significant loss of RGCsat 2 and 4 weeks (15.26±1.57% and 22.35±2.01%) after EVC. Here,we found that the number of RGCs in the EVC- eyes was32.3±1.24% (p< 0.01) and 41.7±2.26% (p< 0.01) lower than in thecontralateral control eyes at 2, and 6 months, indicating theprogressive RGCs loss in the EVC-eyes even after IOP elevationreversal. mtDNA damage and mutations occurred as early as 2 weekafter EVC and continued to increase in the EVC-RGCs even whenthe IOP elevation return to normal and up to 17.8-folds than thecontrol at 6 months. mtDNA copies of RGC continued to decrease(by49% at 6 months compared to control) and mtDNA repair enzymedecreased during all the observation period. ATP production rate ofmitochondria in RGC was reduced by 47% and ROS increased by38% at 6 months after EVC. In vitro results shown that pressureresulted in mtDNA damage, mtDNA copies decreased, expression ofmtDNA repair enzyme decreased, mitochondrial membrane potentialdecreased and eventually cell death. Interestingly, we foundmitochondrial fission was induced as early as 2h pressure-treated andfission itself leaded to mtDNA abnormalities.Conclusions: Progressive RGCs loss in glaucomatous rat model evenafter IOP elevation reversal. Accumulated increase of mtDNAdamage contributed to the progressive loss of RGCs in theglaucomatous model. Pressure induced mitochondria abnormality atleast partly by inhibited mitochondria fusion.Commercial Relationships: Jihong Wu, None; Shenghai Zhang,NoneSupport: National Natural Science Foundation:81170839,81070726.Shanghai Natural Science foundation:11ZR1405800Program Number: 285 Poster Board Number: D0234Presentation Time: 8:30 AM - 10:15 AMConditional mutant analysis of Dnmts in murine retinal ganglioncellsRaymond Enke 1 , Zhiyong Yang 1 , William W. Hauswirth 2 , Sanford L.Boye 2 , Vince Chiodo 2 , Donald J. Zack 1 , Shannath L. Merbs 1 .1 Ophthalmology, Johns Hopkins University, Baltimore, MD;2 Ophthalmology, University of Florida, Gainesville, FL.Purpose: We hypothesize that epigenetic mechanisms may modulatethe onset and progression of disease in the retina. As a prelude tostudying the role of DNA methylation in glaucoma and optic nervedegeneration, we set out to determine the expression pattern of DNAmethyltransferase (Dnmt) enzymes in mature murine retinal ganglioncells (RGCs). We also use conditional Dnmt mutant lines todemonstrate RGC survival following optic nerve crush.Methods: Eyes from adult wt mice were fixed in 4% PFA,equilibrated in a sucrose gradient, and cryopreserved. Retinal crosssections were cut and used for immunohistochemical (IHC) analysisof Dnmts. Conditional Dnmt1, Dnmt3a, and Dnmt3b mutants werecreated by intravitreally injecting an adeno-associated virus serotype2 (AAV2) encoding Cre recombinase into transgenic mouse linesharboring lox P sites in each respective allele. Optic nerve crush wasperformed in adult mice to induce RGC degeneration. For RGCviability analysis, eyecups were fixed in 4% PFA and used for wholemount IHC quantification of Brn3 and Tuj1. Corresponding opticnerve cross sections were also stained for phosphorylatedneurofilament (pNF).Results: IHC analysis of the adult mouse retina demonstrated thatDnmt1 and Dnmt3b are expressed in postmitotic RGCs whileDnmt3a expression is limited to amacrine cells in the ganglion celllayer (GCL) of the retina. Intravitreal delivery of AAV2-Cre resultsin efficient transduction of the mouse GCL cells and proves to be aneffective technique for producing conditional Dnmt mutant mice.Dnmt1 mutant RGCs were partially protected from degenerationfollowing optic nerve crush. Surviving Dnmt1 mutant RGCs alsodisplayed more persistent expression of Brn3 following optic nervecrush compared to controls.Conclusions: Expression of Dnmt1 and Dnmt3b in postmitoticRGCs suggests that DNA methylation may be involved in RGChomeostasis. Our current experiments further address this question byassaying the role of Dnmts in RGC survival following optic nerveinjury. A conditional mutation in Dnmt1 imparts partial protectionagainst RGC degeneration following optic nerve crush. This findingindicates that DNA methylation may be an important epigeneticsignal in response to optic neuropathy. Ongoing analysis willdetermine if Dnmt3b conditional mutants also have improved RGCviability following optic nerve crush.Commercial Relationships: Raymond Enke, None; ZhiyongYang, None; William W. Hauswirth, AGTC (I), Bionic Sight (I),AGTC (C), Syncona (C), RetroSense (C); Sanford L. Boye,PCT/US2012/062478 (P); Vince Chiodo, None; Donald J. Zack,Alcon (C), Merck (F), Allergan (C); Shannath L. Merbs, NoneSupport: American Health Assistance Foundation NationalGlaucoma Research Grant G2012033Program Number: 286 Poster Board Number: D0235Presentation Time: 8:30 AM - 10:15 AMAdenosine triphosphate (ATP) Signaling Pathways Trigger GlialActivation in the Mouse RetinaCaitlin E. Mac Nair 1, 2 , Cassandra Schlamp 2 , Robert W. Nickells 2 .1 Cellular and Molecular Pathology Graduate Program, Univeristy ofWisconsin, Madison, WI; 2 Ophthalmology and Visual Sciences,Univeristy of Wisconsin, Madison, WI.Purpose: Optic nerve injury causes RGC death and activation of theretinal macroglia and microglia. Bax-deficient RGCs are resistant tothis acute injury and display an attenuated glial activation response,indicating a relationship between cell death and glial activation.Several studies suggest that injured neurons release ATP as a distresssignal, and we investigated the potential role of ATP in triggeringglial activation, the consequence of which may lead to cytokineproduction that damages additional ganglion cells.Methods: Wild type mice were intravitreally injected with 1μl of a250μM ATP receptor agonist, either ATPγS or BzATP, and mRNAand protein levels of glial activation markers (GFAP for macrogliaand AIF1 for microglia) were monitored by qPCR andimmunofluorescence. RGC gene markers Thy1, Nrn1, and Sncg werealso monitored by qPCR. Additional experiments will include Baxdeficientmice injected with either BzATP or ATPγS, and wild typemice treated with an ATP receptor antagonist, either oxATP orPPADS, to inhibit ATP signaling from crush injury. The effects onglial activation and RGC survival will be reported.Results: Bax-deficient RGCs were completely resistant to optic nervecrush and displayed an attenuated macroglial and microglialactivation response, suggesting that cell death is required for glialactivation. A single intravitreal injection of BzATP, a specific P2X 7receptor agonist, caused a significant spike in macroglial activationby 24 hours with prominent labeling in the Müller cells. By 48 hoursmacroglial activation returned to basal levels and remainedunchanged by 72 hours. Contrary, macroglial activation by the broadspectrum P2X receptor agonist, ATPγS, showed a moderate increaseat 24 hours above vehicle-injected eyes but continued to rise at 48and 72 hours, with strong labeling of the Müller cells by 72 hours. Adecline in RGC gene markers, indicative of RGC damage, was alsoapparent at 72 hours in ATPγS-injected eyes. Microglial activationwas not greatly affected by either treatment.Conclusions: These results indicate that ATP may contribute to©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologymacroglial activation during glaucoma. The sustained elevation ofATPγS over BzATP at 72 hours suggests that purinergic receptors inaddition to P2X 7 may be involved in triggering activation andsubsequent injury to RGCs.Commercial Relationships: Caitlin E. Mac Nair, None;Cassandra Schlamp, None; Robert W. Nickells, NoneSupport: NEI R01 EY012223, P30 EY016665, Research to PreventBlindnessProgram Number: 287 Poster Board Number: D0236Presentation Time: 8:30 AM - 10:15 AMWhirlin proteins localize at the outer limiting membrane andsubapical region of zebrafish retinaSabrina Toro, Jennifer B. Phillips, Monte Westerfield. Institute ofNeuroscience, University of Oregon, Eugene, OR.Purpose: Mutations in DFNB31 cause Usher syndrome type 2D.DFNB31 encodes Whirlin (Whrn), a scaffold protein thought tomediate interactions with other Usher proteins. We previouslyreported the discovery of two dfnb31 genes in zebrafish, each withmultiple splice variants. Here, we investigate the subcellularlocalizations and functions of WhrnA and WhrnB in the zebrafishretina.Methods: Immunohistochemistry on cryosectioned tissues wasvisualized by confocal microscopy. Antibodies were generated tounique regions of WhrnA and WhrnB, and the third PDZ domain(Whrn_PDZ3) common to both proteins. Antibodies againstHarmonin (ush1c), Usherin (ush2a), Gpr98 (ush2c), GlutamineSynthetase, Acetylated Tubulin and Calretinin were also used. Wholeembryos, larvae and adult eyes were analyzed from wild type (wt)and dfnb31a mutants. Splice blocking morpholinos for dfnb31a anddfnb31b were used to disrupt gene function, and optokinetic responseassays (OKR) were performed to test visual function.Results: WhrnA and WhrnB localize in a ring pattern at the base ofthe connecting cilium (CC), suggesting localization in the periciliaryregion. WhrnA is also observed both pre- and post-synaptically in theouter plexiform layer. Localization of Whrn_PDZ3 is restricted to theouter limiting membrane (OLM) and subapical region (SAR) where itlargely overlaps with Müller cell labeling. Morpholino knockdown ofdfnb31a or dfnb31b results in a reduced OKR. dfnb31a mutants aremorphologically normal, viable and fertile. No differences inabundance or localization of Whrn or other Usher proteins weredetected in mutant tissues.Conclusions: Whrn localization at the base of the CC and atsynapses is consistent with other species. However, localization at theOLM/SAR is a novel finding. Zebrafish Crumbs (Crb) proteins alsolocalize at the OLM/SAR, and Whrn was previously shown tointeract with the Crb pathway. Our data indicate conserved roles forzebrafish Whrn proteins at the CC and potential interaction betweenWhrn and Crb at the lateral cell contacts of photoreceptors. The lackof phenotype in dfnb31a mutants may indicate functional overlapwith dfnb31b. However, the mutation is not predicted to affect alldfnb31a isoforms, so persistence of functional splice variants isanother possibility under investigation.Commercial Relationships: Sabrina Toro, None; Jennifer B.Phillips, None; Monte Westerfield, NoneSupport: NIH Grants: DC004186, DC010447, HD22486Program Number: 288 Poster Board Number: D0237Presentation Time: 8:30 AM - 10:15 AMAge-related Upregulation of MicroRNA miR-34a In the MouseRetina and RPE/choroidZeljka Smit-McBride, Leonard M. Hjelmeland. Vitreo-RetinalResearch Lab, Univ of California, Davis Sch of Med, Davis, CA.Purpose: Age-related upregulation of miR-34a in blood and in brainhas recently been identified as a senescence marker for brain andliver tissue in mice. In order to test the hypothesis that miR-34a canbe considered as a senescence marker in retina and RPE/choroid, weconducted a real-time PCR analysis to dissect further 2mo, 18mo,24mo and 32mo old RPE/choroid and retina of male and femalemouse.Methods: Mice were obtained from The JAX and from NIA.Research was conducted in compliance with the ARVO Statement forthe Use of Animals in Ophthalmic and Vision Research. TotalmicroRNA from the tissue was isolated using the Qiagen miRNeasykit. Samples of miRNA were then analyzed using Taqman assaysusing preamp primer mix set A, miR-34a and three control smallRNAs - sno-135, sno-202 and U6. MicroRNA assays were run at1:1000 dilutions, in triplicate, with 3 biological replicas for each timepoint. Raw data was normalized with the geometric mean of the 3control small RNAs and calibrated to the average value for youngmice.Results: MiR-34a showed upregulation with age in RPE/choroid atall time points examined, 18mo, 24mo and 32mo in comparison withto 2mo old levels. Differences between 2mo and 24mo old expressionlevels were statistically significant in males (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologybundles oriented perpendicular to the vertical axis, and to Tuj1labeled axons (not shown), in a pattern most consistent with theorientation of astrocytic processes in the glial lamina (Figure 1a).Additionally, F-actin labeled the walls of the ONH vascularcomponents. F-actin partially co-labeled with Aqp4, furthersuggesting the astrocytic origin of the dense F-actin bundles (Figure2). Preliminary labeling intensity analysis of 8 glaucoma model eyeswith axonal injury grades from 1 to 4.9 (mean and peak IOP±SDfrom 19.2±1.6 to 29.6±3.9 and 22±3.2 to 50.4±1.3 mmHg,respectively) suggested that astrocytic structural changes, as indicatedby reduced F-actin labeling in eyes with minimal injury (Figure 1b),may precede axonal degeneration. With greater injury (Figure 1c),the ONH actin cytoskeleton became exceedingly disorganized withan apparent increase in F-actin fluorescence intensity, possibly due toastrocytic remodeling in response to injury. In contrast, in transectedONH, F-actin remained relatively preserved, suggesting that thechanges in the F-actin cytoskeleton with IOP elevation were notsimply secondary to non-specific axonal injury.Conclusions: The actin cytoskeleton of the rat ONH is a densestructure that may have a role in early and late ONH remodeling inresponse to glaucomatous injury. F-actin labeling within the ONHappears to highlight the glial lamina more effectively when comparedto Aqp4 labeling. In addition, F-actin labeling highlights the vascularcomponents of the ONH.Rat ONH actin cytoskeletal response to elevated IOPspots on SD-OCT and increased vascular permeability andpathologies of retinal blood vessels on FA. The pathologies of retinalblood vessels include micro- and macroaneurysms, pulling andtraction, dilated capillaries, capillary dropout and tortuosity. TEMrevealed neovascularization in the normally avascular retinal pigmentepithelium (RPE). TEM also showed thickening of the basementmembrane in these blood vessels. Abnormal lipid accumulation wasdetected throughout the retina and in retinal blood vessels byhistochemistry staining with oil red O, then confirmed by TEM.Areas with pathological blood vessels and structural abnormalitieshad increased staining with the markers for activated macrophagesF4/80 and Iba-1. Retinal blood vessels, the photoreceptor innersegments (PIS) and the outer plexiform layer (OPL) also showedstaining for iso[4]levuglandin E2 and carboxyethylpyrrole adducts aswell as 7-ketocholesterol, indicators of elevated oxidative stress.TEM also revealed mitochondrial swelling, degeneration andaggregation. These changes were observed in the RPE, PIS, OPL andinner nuclear layers. Significant alterations were found in the retinalexpression of a number of genes including the cholesterol effluxtransporter Abcg1, the lipoprotein Apob, the cholesterogenic enzymeHmgcr, the regulatory protein Insig1, and the inflammatorycytokines/chemokines Ccl1, Cxcl1, Cxcl2 and IL-6.Conclusions: We propose that combined deficiency of Cyp27a1 andCyp46a1 decreases activation of Lxrβ leading to decreasedexpression of target genes like Abcg1 and almost complete blockageof cholesterol elimination from retinal macrophages and vascularendothelium. The latter causes lipid accumulation, inflammation, andoxidative stress that drive the development of the abnormalities in theCyp27a1 -/- Cyp46a1 -/- retinas.Commercial Relationships: Aicha Saadane, None; Casey Charvet,None; Natalia Mast, None; Wenchao Zheng, None; Timothy S.Kern, Bausch & Lomb (F), PamLab (F); Suber Huang, UniversityHosp Case Medical Center (E), Sequenom (C), Notal Vision (C),Bausch and Lomb (C), Second Sight (C); Irina A. Pikuleva, NoneSupport: NIH RO1 EY018383Co-labeling of rat ONH actin cytoskeleton with aquaporin 4Commercial Relationships: Shandiz Tehrani, None; Elaine C.Johnson, None; William Cepurna, None; Matthew R. Bald, None;John C. Morrison, NoneSupport: NIH Gant EY010145Program Number: 290 Poster Board Number: D0239Presentation Time: 8:30 AM - 10:15 AMMechanistic insights into the link between impaired cholesterolelimination and vascular abnormalities in mouse retinaAicha Saadane 1 , Casey Charvet 1 , Natalia Mast 1 , Wenchao Zheng 1 ,Timothy S. Kern 2 , Suber Huang 3, 1 , Irina A. Pikuleva 1 .1 Ophthalmology and Visual Sciences, CWRU, Cleveland, OH;2 Medicine, CWRU, Cleveland, OH; 3 University Hospital, CWRU,Cleveland, OH.Purpose: To elucidate the mechanisms underlying development ofstructural and vascular abnormalities in the retina of mice lackingCyp27a1 and Cyp46a1, enzymes important for cholesteroleliminationMethods: Evaluations by high-resolution spectral-domain opticalcoherence tomography (SD-OCT), fluorescein angiography (FA), andtransmission electron microscopy (TEM); analysis of gene expressionby PCR arrays and quantitative RT-PCR; immuno- and histochemicalstaining; and sterol profiling by mass spectrometryResults: Cyp27a1 -/- Cyp46a1 -/- retinas have multiple hyperreflectiveProgram Number: 291 Poster Board Number: D0240Presentation Time: 8:30 AM - 10:15 AMTHERAPEUTIC EFFICACY OF MELATONIN IN REDUCINGRETINAL DAMAGE IN AN EXPERIMENTAL MODEL OFEARLY TYPE 2 DIABETES IN RATSRuth E. Rosenstein, Melina P. Bordone, Monica S. Chianelli, MaríaI. Keller Sarmiento, Damian Dorfman, Magdalena Miranda,Ezequiel M. Salido. Dept Human Biochem-Sch Med, University ofBuenos Aires, Buenos Aires, Argentina.Purpose: Diabetic retinopathy is a leading cause of acquiredblindness in adults, mostly affected by type 2 diabetes mellitus(T2DM). We have developed an experimental model of early T2DMin adult rats by combining diet-induced insulin resistance and a slightβ-cell secretory impairment, which mimics some features of humanT2DM at its initial stages, and provokes significant retinal alterations.The aim of the present work was to analyze the effect of melatoninon retinal changes induced by a moderate metabolic derangement.Methods: Adult male Wistar rats received a control diet or 30%sucrose in the drinking water ad libitum. Three weeks after thistreatment, animals were injected with vehicle or streptozotocin (STZ,25 mg/kg). One day after vehicle or STZ injection, animals weresubcutaneously implanted with a pellet of melatonin, which wasreplaced every 15 days. At 12 weeks of treatment, fasting andpostprandial glycemia, and glucose and insulin tolerance tests wereanalyzed. Retinal function (scotopic elctroretinograms), retinal lipidperoxidation (thiobarbituric acid reactive substance levels), NOSactivity (using 3H-arginine), TNFα (enzyme-linked immunosorbent©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyassay), retinal morphology (optical microscopy), and Müller cellsglial fibrillary acidic protein (GFAP) and vascular endothelial growthfactor levels (VEGF) (immunohistochemistry) were evaluated.Results: Animals which received a sucrose-enriched diet and STZshowed significant differences in metabolic tests, as compared withcontrol groups. Melatonin, which did not affect glucose metabolismin control or diabetic rats, prevented the decrease in theelectroretinogram a-and b-wave, and oscillatory potential amplitude,and the increase in retinal lipid peroxidation, NOS activity, TNFαand Müller cell GFAP and VEGF levels.Conclusions: These results indicate that melatonin protected theretina from the alterations observed in an experimental model ofdiabetic retinopathy associated with type 2 diabetes.Commercial Relationships: Ruth E. Rosenstein, None; Melina P.Bordone, None; Monica S. Chianelli, None; María I. KellerSarmiento, None; Damian Dorfman, None; Magdalena Miranda,None; Ezequiel M. Salido, NoneSupport: PICT 1623 (ANPCyT), PIP 112-201101-00446(CONICET), 20020100100678 (UBA)Program Number: 292 Poster Board Number: D0241Presentation Time: 8:30 AM - 10:15 AMFOXOs in the Mouse Visual SystemJullia A. Rosdahl, Guorong Li, Jianming Qiu, Pedro Gonzalez,Pratap Challa. Ophthalmology, Duke Eye Center, Durham, NC.Purpose: The FOXO family of transcription factors regulates genesinvolved in aging, oxidative stress, and metabolism, processesinvolved in the pathogenesis of glaucoma. The purpose of this studyis to determine whether the expression profile of the FOXO familymembers is consistent with a role in glaucomatous vision loss.Methods: Antibodies for FOXO1, FOXO3, and FOXO4 were usedto identify protein expression and localization in retinal and brainsections of pup, 2 month old, 6 month old, and 12 month old mice,using Immunohistochemistry and Western Blotting techniques. Agedand glaucomatous human retinas were also analyzed. Real time PCRwas used to assess RNA expression of the FOXO family membersand their known regulators, in mouse tissues.Results: FOXO1 and FOXO3 proteins are expressed in the retina andbrain in pup, 2 month old, 6 month old, and 12 month old mice. Verylow levels of FOXO4 expression were detected by both WesternBlotting and PCR techniques. In the retina, the highest expression ofFOXO1 and FOXO3 is seen in the retinal ganglion cell layer. In thebrain, the highest expression is seen in the cerebellum. These proteinslocalize to a subset of cells in the cerebellum, the cortex, the superiorcolliculus and inferior colliculus. Using real time PCR to quantify theexpression at the time points tested, the highest expression is seen in2-month-old mice. In human retina, FOXO1 and FOXO3 expressionis highest in the retinal ganglion cell layer, in both aged andglaucomatous tissue.Conclusions: FOXO1 and FOXO3, but not FOXO4, are expressed inthe visual system of mice. The FOXO proteins localize to the retinalganglion cell layer, in mice as well as humans. Their expressionpattern supports a role for this family of transcription factors inregulating aging, oxidative stress, and metabolism in the cellsaffected by Glaucoma.Commercial Relationships: Jullia A. Rosdahl, None; Guorong Li,None; Jianming Qiu, None; Pedro Gonzalez, None; Pratap Challa,NoneSupport: NIH Grant K12EY016333, Research to Prevent BlindnessGrant to DukeProgram Number: 293 Poster Board Number: D0242Presentation Time: 8:30 AM - 10:15 AMCharacterization of Genome-Wide DNA Methylation in theMouse RetinaVerity F. Oliver 1 , Jun Wan 1 , Saurabh Agarwal 3, 4 , Donald J. Zack 1, 2 ,Jiang Qian 1 , Shannath L. Merbs 1 . 1 Department of Ophthalmology,Johns Hopkins University, Baltimore, MD; 2 Department ofMolecular Biology and Genetics, Department of Neuroscience, andInstitute of Genetic Medicine, Johns Hopkins University School ofMedicine, Baltimore, MD; 3 Ludwig Institute for Cancer Research, LaJolla, CA; 4 Division of Biological Sciences, University of California,San Diego, La Jolla, CA.Purpose: Growing evidence suggests that DNA methylation plays arole in tissue-specific differentiation. We have shown that retinaspecificgenes (Rho and Rbp3) are hypomethylated in the expressingcells of mouse retina. Little is known about DNA methylation in theregulation of other retina-specific genes. We aimed to characterizegenome-wide DNA methylation patterns in the mouse retina.Methods: We developed a novel method to characterize global DNAmethylation: MBD2b/MBD3L1-enrichment of DNA followed bykinase ligation-mediated-PCR and microarray analysis (MeKL-chip).DNA was extracted from retina and brain samples of three 8 weekold male C57BL/6J mice. Enrichment was performed on 250 ngDNA using the MethylCollector Ultra kit (Active Motif). Enrichedand unenriched DNA was amplified using KLM-PCR. Qualitycontrol was performed pre- and post-amplification using QPCR forRho, Rbp3 and H19 (equally methylated in retina and brain). TheCHARM (comprehensive high-throughput arrays for relativemethylation) 2.1M probe NimbleGen microarray platform was usedto examine CpG sites throughout the genome. Tissue-specificdifferentially methylated regions (tDMRs) were identified. Validationof tDMRs was performed using bisulfite pyrosequencing of a secondmouse cohort. The lower limits of input DNA for the MeKL-chipmethod were explored.Results: 2498 tDMRs were identified between mouse retina andbrain. The top 5 tDMRs were successfully validated bypyrosequencing and included genes that were more methylated in theretina (Rgs20, Hes2,Cckbr and Six3os1) and more methylated in thebrain (Nfic). The top tDMR covered exon 3 of Rgs20 at an alternativetranscription start site (TSS). Exon 3 is included in the brain-specificisoform and excluded from the retina-specific isoform; supportingrecent evidence that intragenic DNA methylation may mediate tissuespecificexpression through alternative TSS regulation. MeKL-chipenables genome-wide screening of DNA methylation using a samplecontaining as little as 50 ng DNA.Conclusions: MeKL-chip is optimized for small samples and can beadapted for sequencing protocols. MeKL-chip on mouse retina hasenabled novel characterization of tDMRs important for understandingretinal development and disease. MeKL-chip experiments arecurrently being undertaken on the rd1 mouse model to determinewhether aberrant DNA methylation is implicated in the conedegeneration that follows loss of rods.Commercial Relationships: Verity F. Oliver, None; Jun Wan,None; Saurabh Agarwal, None; Donald J. Zack, Alcon (C), Merck(F), Allergan (C); Jiang Qian, None; Shannath L. Merbs, NoneSupport: NEI R21EY018703, NEI 5P30EY001765, Unrestrictedfunds from Research to Prevent Blindness, Generosity of AgnesNixonProgram Number: 294 Poster Board Number: D0243Presentation Time: 8:30 AM - 10:15 AMExpression Patterns Of Histone Deacetylases In Murine andChick Optic Nerve During DevelopmentMahesh Shivanna 1 , Teri L. Belecky-Adams 2 . 1 School of Optometry,Massachusetts College of Pharmacy and Health Sciences, Worcester,©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyMA; 2 Department of Biology, Center for Regenerative Biology andMedicine, IUPUI, Indianapolis, IN.Purpose: Optic nerve astrocytes play a crucial role in the survival ofretinal ganglion cells as well as retinal homeostasis. Duringdevelopment, astrocytes exhibit changes in expression profiles as aresult of chromatin reorganization. Chromatin reorganization can beinduced through histone modifying enzymes. We examined theexpression of histone deacetylases (HDACs), a class of histonemodifying enzyme in murine and chick optic nerve (ON) as a firststep towards understanding the epigenetic influence on astrocytelineage development.Methods: Expression of Class I, II and IV HDACs were examinedby Western blotting and quantitative real-time polymerase chainreaction (QPCR) in murine ON whereas Western blotting was used toconfirm the expression patterns in Chick ON. ONs were isolatedfrom chick embryos at E5 (embryonic day 5), E8 and E18 stageswhereas from mouse at E16, PN5 (post natal day 5) and PN30 stagesto coincide with various phases of astrocyte development. Theprotein from isolated ONs was quantified and resolved by SDS-PAGE using specific antibodies. Total RNA was isolated from mouseONs, cDNA synthesized and QPCR was performed using specificprimers to assess changes in gene expression.Results: The expression of Class I HDACs namely HDAC1, 2, 3 and8 decreased with development in mouse optic nerve as confirmed byboth western blotting and QPCR. Among class II HDACs, mRNAexpression of HDAC4, 6, 7 and 10 decreased whereas mRNAexpression of other class II HDACs, HDAC5 and 9, and HDAC 11, aclass IV HDAC showed a fluctuating pattern in mouse optic nervewith development. Western blot analysis showed increasedexpression of HDAC4 and 5 and decreased expression of HDAC 9,10 and 11 in mouse optic nerve. The expression of most of theHDACs in the mouse ON decreased significantly with development.However, the protein expression of acetylated lysine significantlyincreased with development. In chick ON, protein expression ofHDAC1, 2, 3, 4, 5 and 9 decreased with development whereasHDAC8 showed a fluctuating patternConclusions: Decreasing levels of expression of majority of HDACsin mouse and chick optic nerve and increasing levels of acetylatedlysine suggest an increase in transcriptional activity during the courseof development. The fluctuating pattern or increased expressionobserved in some of the HDACs suggests differential regulation ofsome of the developmental genes.Commercial Relationships: Mahesh Shivanna, None; Teri L.Belecky-Adams, NoneSupport: EY019525; EY020816Program Number: 295 Poster Board Number: D0244Presentation Time: 8:30 AM - 10:15 AMAblation of the X-linked Retinitis Pigmentosa 2 (Rp2) gene inmice results in opsin mistrafficking and photoreceptordegenerationLinjing Li 1 , Naheed W. Khan 2 , Toby Hurd 3 , Amiya K. Ghosh 2 ,Christiana Chang 4 , Robert S. Molday 4 , John R. Heckenlively 2 , AnandSwaroop 5 , Hemant Khanna 1 . 1 UMASS Medical School, Worcester,MA; 2 University of Michigan, Ann Arbor, MI; 3 University ofMichigan, Ann Arbor, MI; 4 Centre for Macular Research, Universityof British Columbia, Vancouver, BC, Canada; 5 National EyeInstitute, Bethesda, MD.Purpose: X-linked Retinitis Pigmentosa (XLRP) is a debilitatingdisorder of the eye characterized by degeneration of rod and conephotoreceptors. Mutations in the RP2 gene are associated with 10-15% of XLRP cases. However, the molecular mechanism ofpathogenesis of RP2-mediated photoreceptor degeneration is unclear.Methods: We utilized the Cre/loxp system to delete exon 2, amutational hotspot in humans, of the Rp2 gene in mice usingtransgenic mice expressing the Cre under the control of theubiquitously expressing CAG promoter. RP2 expression wasexamined by RT-PCR, immunoblotting, and immunohistochemistry.Histology and transmission electron microscopy (EM) wereemployed to find the morphological changes. Retinal function wastested by electroretinography (ERG).Results: The mutant retina (Rp2-conditional knock out; Rp2 CKO )exhibited undetectable RP2 protein levels, as determined byimmunoblotting and immunofluorescence analyses. The Rp2 CKO miceshowed progressive decline in photopic (cone) and scotopic (rod)ERG, starting at 2 months of age. Histological analysis revealedprogressive degeneration of the photoreceptor layer in the mutantretina. Deletion of Rp2 resulted in disorganized outer segment discsin rods and cones, while sparing outer segment development.Degeneration of both dorsal and ventral cones and mislocalization ofcone opsins to the nuclear and synaptic layers prior to the onset ofdegeneration were detected in the mutant retina. There was nodetectable defect in the expression and localization of rod and conearrestin, cone transducin subunits or RP2-interacting protein ARL3.Conclusions: Our results suggest that RP2 is involved in themaintenance of photoreceptor function and that cone opsinmisliocalization plays a critical role in the pathogenesis of RP2-associated disease.Commercial Relationships: Linjing Li, None; Naheed W. Khan,None; Toby Hurd, None; Amiya K. Ghosh, None; ChristianaChang, None; Robert S. Molday, None; John R. Heckenlively,None; Anand Swaroop, None; Hemant Khanna, NoneSupport: NIH Grant EY022372, Foundation Fighting Blindness,NIH Intramural SupportProgram Number: 296 Poster Board Number: D0245Presentation Time: 8:30 AM - 10:15 AMASSESSMENT OF RETINAL STRUCTURE FOLLOWINGREPEATED LIGHT EXPOSURE IN THE LIGHT SENSITIVET4R RHODOPSIN MUTANT DOG RETINASimone Iwabe, Kendra McDaid, Gustavo D. Aguirre, William A.Beltran. Section of Ophthalmology, University of Pennsylvania,Philadelphia, PA.Purpose: We previously reported that a 60 sec white light exposureto 0.1 mW/cm2 (170 lux) is a sub-threshold dose that does not causeretinal damage in T4R RHO dogs, while a corneal irradiance of 0.3mw/cm2 (551 lux)-or supra-threshold dose-leads to mild ONLthinning 2 weeks post exposure. Here, we evaluated the effect ofrepeated exposures to sub and supra-threshold doses in the retinas ofT4R/+ RHO mutant dogs.Methods: Group A: T4R/+ dogs #1, #2 had the right eye exposedonce at T0 and the left eye exposed 4 times at T0, T1, T2 and T3 witha 2 wk interval between exposures. Group B: T4R/+ dogs #3, #4 hadthe right eye shielded and the left eye exposed 4 times(T0, T1, T2and T3). Within each group, one dog was exposed to sub-thresholddoses (dogs #1, #3) while the other received supra-threshold doses(dogs # 2, #4). A non-mutant dog was used as a control (the right eyewas shielded and the left eye was exposed 4 times at same time pointto supra-threshold doses). ONL thickness was measured along the 4cardinal meridians by non-invasive cSLO/sdOCT imaging before,and every 2 wks after each light exposure; as well as by histologicexamination at termination.Results: No significant changes in ONL thickness were observedalong any of the 4 meridians when comparing the shielded eye of dog#3 to the eyes of dogs #3 and # 1 that were exposed up to 4 times to asub-threshold doses. Dog #2 that received a supra-threshold dose had©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologya similar decrease in ONL thickness in the superior area 8 weeks aftera one or 4 exposures (82,1 % and 86.2%, respectively). However, adifference was seen in the temporal meridian where a single exposureled to a 14.3% loss of ONL, while 4 exposures caused a more severe(63.4%) thinning. Dog #4 that also received a supra-threshold doseshowed only a 16.1% decrease in ONL thickness in the superiormeridian in the shielded eye vs. a 73.6% thinning in the eye exposed4 times. No significant changes were observed in this dog in thetemporal, nasal and inferior meridians.Conclusions: Repeated light exposure to sub-threshold doses of lightdoes not cause damage to T4R RHO retinas. However, repeatedexposure to retinotoxic light intensities causes cumulative retinaldamage that is worse than that achieved by a single exposure. Theseresults may be relevant to the suggested cumulative light damage thatoccurs in human patients with some RHO mutations.Commercial Relationships: Simone Iwabe, None; KendraMcDaid, None; Gustavo D. Aguirre, None; William A. Beltran,NoneSupport: NIH/NEI EY06855, EY17549, 2PNEY018241,R24EY021126, R24EY022012, Foundation Fighting Blindness.Program Number: 297 Poster Board Number: D0246Presentation Time: 8:30 AM - 10:15 AMGene expression changes under cyclic mechanical stretching inrat retinal glial (Müller) cellsXin Wang, Gezhi Xu. Department of Ophthalmology and VisionSciences, Eye and ENT Hospital, Shanghai Medical College, FudanUniversity, Shanghai, China.Purpose: To identify patterns of gene expression changes induced bycyclic mechanical stretching in Müller cells.Methods: Rat Müller cells were seeded onto flexible bottom cultureplates and subjected to a cyclic stretching regimen of 15% equibiaxialstretching for 1 and 24 h. RNA was extracted and amplified, labeled,and hybridized to rat genome microarrays. The expression profileswere analyzed using GeneSpring software, and the KyotoEncyclopedia of Genes and Genomes (KEGG) Pathway analysis wasused to find the association with biological functions. The selectedgenes of interest were further validated by RT-qPCR.Results: An analysis of microarray data showed that at 1 and 24 h,the expression of 532 and 991 genes in the Müller cells significantly(T-test, p < 0.05) differed between the mechanically stretched and theunstretched groups. At 1 h, 52 genes, which are involved in responseto stimuli, developmental processes, transcription regulator activity,and regulation of biological processes, showed at least a twofoldchange in expression. At 24 h, 63 genes, which are involved indevelopmental processes, anatomical structure formation, andextracellular regions, showed at least a twofold change in expression.KEGG canonical pathway analysis showed significant changes inMAPK signaling, ErbB signaling, TGF-beta signaling, and Wntsignaling at both time points.Conclusions: Cyclic mechanical strain induces extensive changes inthe gene expression in Müller cells through multiple molecularpathways. These results indicate the complex mechanoresponsivenature of Müller cells, and they may help in excluding possible novelmolecular mechanisms that would account for many retinal diseasesin which the retina is often subjected to mechanical forces, such asdegenerative axial myopia and proliferative vitreoretinopathy.Commercial Relationships: Xin Wang, None; Gezhi Xu, NoneSupport: NSFC Grant 81170857Program Number: 298 Poster Board Number: D0247Presentation Time: 8:30 AM - 10:15 AMA retinal phenotype in Usher IIIA mouse modelsRachel M. Stupay, W Clay Smith, Wen-Tao Deng, Ping Zhu, WilliamW. Hauswirth, Astra Dinculescu. Ophthalmology, University ofFlorida, Gainesville, FL.Purpose: Mutations in the Clarin-1 (CLN1) gene are the causativefactor for Usher Syndrome Type III (USH3A), an autosomalrecessive disorder that presents with progressive deafness and retinaldegeneration (RD) in humans. Currently, there is no apparent retinalphenotype in the existing USH3A mouse models, with both CLN1knock-out and the N48K knock-in mutant mice displaying earlyhearing loss, but normal retinal morphology and function. In thisstudy we attempt to uncover a retinal phenotype by comparing thepattern of light-driven arrestin1 translocation in the above mousemodels for USH3A and their isogenic wild-type controls.Methods: Adult CLN1 knock-out, CLN1 N48K knock-in, and controlmice were dark adapted overnight, then exposed to light undercontrolled conditions. Retinal sections were analyzed byimmunostaining for arrestin1 and postsynaptic density protein 95(PSD 95). Quantification of arrestin1 translocation was performed bymeasuring the staining intensity in retinal cross-sections from WTand Ush3A mice using NIH Image J software.Results: Arrestin1 distribution in response to light displays asubstantial movement towards the outer segments in both wild-typeand Ush3A photoreceptors. However, comparison of the pattern ofarrestin1 translocation between normal and mutant retinas is differentwith respect to the retention of arrestin1 in outer plexiform layer(OPL), with uniform and persistent arrestin1 immunostainingremaining within the OPL of mutant USH3A photoreceptors afterlight exposure. Quantification of arrestin1 staining in the OPLdemonstrated that the signal was significantly greater in mutant micecompared to wild-type, suggesting that arrestin1 movement from theOPL was delayed in the Ush3A mice.Conclusions: These results document a retinal phenotype for Ush3Amice with respect to arrestin1 translocation in response to light. Thisphenotype will be used to test potential retinal therapies for USH3A,including AAV-mediated delivery of the missing wild-type CLN1cDNA to the knock-out and N48K knock-in retinas.Commercial Relationships: Rachel M. Stupay, None; W ClaySmith, None; Wen-Tao Deng, None; Ping Zhu, None; William W.Hauswirth, AGTC (I), Bionic Sight (I), AGTC (C), Syncona (C),RetroSense (C); Astra Dinculescu, NoneSupport: Usher III Initiative, Inc., Foundation Fighting Blindness,Research to Prevent Blindness, Inc., and Macula Vision ResearchFoundation.131 Retina/RPE Biochemistry: Proteins and MetabolismSunday, May 05, 2013 10:30 AM-12:15 PMExhibit Hall Poster SessionProgram #/Board # Range: 691-708/D0248-D0265Organizing Section: Biochemistry/Molecular BiologyProgram Number: 691 Poster Board Number: D0248Presentation Time: 10:30 AM - 12:15 PMRetinal metabolic pathways: a comparative study withcerebellum, hippocampus and olfactory bulbJianhai Du 1 , Whitney M. Cleghorn 1 , Guy C. Chan 3 , JonathanLinton 1 , Martin Sadilek 2 , Viren Govindaraju 1 , James B. Hurley 1 .1 Biochemistry, University of Washington, Seattle, WA; 2 Chemistry,University of Washington, Seattle, WA; 3 Pharmacology, Universityof Washington, Seattle, WA.Purpose: To compare how retinas use different types of fuels forenergy metabolism and to compare the fuel preferences of retinas andother types of neurons.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyMethods: Mouse retina, cerebellum, hippocampus and olfactory bulbwere isolated and cultured in Krebs-Ringer bicarbonate (KRB)buffer. The retina and other neurons were incubated with 5 mM of avariety of 13C labeled fuel sources. Rat retinas were isolated andincubated in KRB buffer and sectioned at 50 micron thickness.Metabolites from both the tissue and the medium were extracted andmeasured by GC-MS. Results were corrected for the naturalabundance of isotopes. The overall enrichment and the distribution oflabeled carbons in each metabolite were calculated.Results: Retinas converted glucose to lactate and pyruvate andreleased them into the medium ~4 times higher than other neurons.Unlike other neurons retinas did not release glutamate. Retina had thehighest rate of 13C enrichment of the mitochondrial TCA cycleintermediates from glucose among all the neurons. By incubatingretinas and other neurons with the same concentration of 13Cglucose, 13C glutamine, 13C pyruvate or 13C lactate for 2 hours, wefound the 13C glutamine produced the highest 13C enrichment ofsuccinate, fumarate and malate. In the retina, glutamine robustlycompeted with glucose for enrichment of all mitochondrialintermediates except citrate. By incubating with 12C glucose togetherwith 13C glutamine, we found that retinas had the highest rate of 13Cenrichment in mitochondrial intermediates of all the types of neuronswe tested. Furthermore, rat retina sections showed that glutaminelabeling was localized primarily in the mitochondrial intermediates inthe photoreceptor layer.Conclusions: The retina has much higher metabolic rate from bothglucose and glutamine than other neurons. The retina, particularly thephotoreceptor layer, selectively uses glutamine over glucose,pyruvate and lactate to fuel mitochondria.Commercial Relationships: Jianhai Du, None; Whitney M.Cleghorn, None; Guy C. Chan, None; Jonathan Linton, None;Martin Sadilek, None; Viren Govindaraju, None; James B.Hurley, NoneSupport: NIH EY017863Program Number: 692 Poster Board Number: D0249Presentation Time: 10:30 AM - 12:15 PMUnique expression and regulation of glycolytic enzyme PKM2 inPhotoreceptor cells and the role of enzymatic activity modulatingmetabolism of the retinaKen Lindsay, Jonathon Linton, James B. Hurley. Biochemistry,University of Washington, Seattle, WA.Purpose: The photoreceptor cell is one of the most energydemanding cells in the body. While being a post-mitotic cell, thedaily turnover of Rod Outer Segment Discs imposes a need forprotein and lipid synthesis that rivals that of a rapidly dividing cell.This intense metabolic demand suggests that the expression ofglycolytic enzymes associated with increases in anabolism, typicallyassociated with tumors and other rapidly dividing cells may beexpressed in the retina and be regulated to fuel these processes. Initialobservations demonstrated that the retina expresses a form ofpyruvate kinase, PKM2, previously thought to be primarilyassociated with cancer cells. The purpose of this study was to analyzethe localization and possible regulation of PKM2 in retinas.Methods: We analyzed the distribution of PKM2, by enzymaticactivity assays and immunoblotting of serial sections of light anddark adapted rat retinas. Western Blot analyses of serial sections usedRhodopsin, Recoverin and Synaptotagmin as landmarks fororientation of enzymes throughout the retina. An antibody specific toPKM2 was used to localize this isoform in the retina.Results: Serial Sectioning revealed that PKM2 is present only in thephotoreceptor layer of the retina. While total expression of PKM2 isinsensitive to light, the fraction of enzymatic activity in thephotoreceptor layer increased significantly in Dark Adapted retinas (n= 7). Immunoblot analyses showed that phosphorylation of Y105 onPKM2, typically associated with decreased PKM2 enzyme activity,occurs independently of light.Conclusions: Our findings show that the PKM2 isoform of pyruvatekinase is present specifically in the photoreceptor layer of rat retinas.We also found that the fraction of pyruvate kinase activity that is inthe outer retina is diminished by light adaptation. Additional studieswill address how PKM2 helps photoreceptors to meet their unusualmetabolic demands.Commercial Relationships: Ken Lindsay, None; Jonathon Linton,None; James B. Hurley, NoneSupport: EY017863Program Number: 693 Poster Board Number: D0250Presentation Time: 10:30 AM - 12:15 PMMetabolic pathways of photoreceptors in light and darkWhitney M. Cleghorn 1 , Jianhai Du 1 , Martin Sadilek 2 , VirenGovindaraju 1 , James B. Hurley 1 . 1 Biochemistry, University ofWashington, Seattle, WA; 2 Chemistry, University of Washington,Seattle, WA.Purpose: Rod and cone photoreceptors (PRs) need to produce bothATP and anabolic precursors to survive. The metabolic demands ofPRs are different in light and dark: dark adapted PRs use energy tofuel ion exchange, while PRs in the light undergo anabolicmetabolism to regenerate rhodopsin and synthesize new membranes.The purpose of this study is to understand how the metabolicpathways differ in PRs under light and dark conditions.Methods: Retinas were isolated from C57BL mice (6-8 weeks old)and cultured in 2 ml KRB buffer with either 5 mM 13C glucose or 5mM 13C glutamine in 37oC CO2 incubator. The metabolites wereanalyzed by GC-MS (Agilent 5973 MSD/6890 GC). Mass spectrawere collected and isotopomer distributions were corrected fornaturally-occurring heavy isotopes using the software, Isocor. Themean enrichment (ME) of each metabolite was calculated.Results: Carbons from uniformly labeled 13C glucose areincorporated rapidly into glycolytic intermediates and into citrate, butare incorporated more slowly into other TCA cycle intermediates.Interestingly, two of the TCA cycle metabolites, succinate andfumarate, show lower incorporation than surrounding intermediates,suggesting the carbons enter from multiple points into the TCA cycle.The overall incorporation of glucose is qualitatively similar in thedark, however accumulation of labeled intermediates happens faster.Incorporation of 13C labeled glutamine is well isolated fromglycolytic intermediates. Carbons from glutamine are incorporatedinto TCA cycle intermediates at the same rate as glucose, and showhigher incorporation in the dark. Interestingly, malate shows thebiggest increase in 13C in dark adapted retinas, regardless of the fuelused. In addition, our experiments show that the overall redox state ofthe retina is stable in glucose but not glutamine, but the reducingpower (lac/pyr ratio) is used up faster in the dark.Conclusions: Glutamine is the major source for formation of TCAcycle intermediates, whereas glucose provides cytosolic reducingpower for the photoreceptors. Generally each of these fuels is usedsimilarly in light and dark, but all of the metabolic reactions occurfaster in darkness than in light. In particular, pyruvate carboxylaseand malic enzyme activity appear to be faster in darkness.Commercial Relationships: Whitney M. Cleghorn, None; JianhaiDu, None; Martin Sadilek, None; Viren Govindaraju, None;James B. Hurley, NoneSupport: NEI grants EY017863 and EY06641Program Number: 694 Poster Board Number: D0251©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyPresentation Time: 10:30 AM - 12:15 PMNitric Oxide Leads to Cytoskeletal Reorganization and Apoptosisin Retinal Pigment EpitheliumO'Donnell Sylvester 1 , Srinivas R. Sripathi 2 , Weilue He 3 , TrevorMoser 2 , Paul S. Bernstein 4 , Folami Lamoke 5 , Manuela Bartoli 5 ,Megan C. Frost 3 , Wan Jin Jahng 1 . 1 Retina Proteomics Laboratory,Petroleum Chemistry, American University of Nigeria, Yola, Nigeria;2 Biological Sciences, Michigan Technological University, Houghton,MI; 3 Biomedical Engineering, Michigan Technological University,Houghton, MI; 4 Ophthalmology and Visual Sciences, University ofUtah, Salt Lake City, UT; 5 Ophthalmology, Georgia Health SciencesUniversity, Augusta, GA.Purpose: The molecular mechanism of oxidative stress-inducedapoptosis in the RPE remains elusive. Our proteomics approachdemonstrated that early biosignatures, including prohibitin, PP2A,vimentin, and nitric oxide synthase might be involved in the initiationof apoptosis as a positive or negative signal. Our quantitative analysisinvestigates how nitric oxide reorganizes cytoskeletal proteins in theRPE under oxidative stress and how the reorganization affectsapoptotic signaling in the RPE.Methods: Quantitative analysis using Chemiluminescence detectionwas performed to measure nitric oxide released from ARPE-19 cellsexposed to bright fluorescent light (7000 lux), oxidative stress (200µM tert-butyl hydroperoxide), reoxygenation (2 hrs hypoxiccondition by N2 then 21% O2) and lipopolysaccharide (5-10 µg/mL).Early biomarkers, including inducible nitric oxide synthase (iNOS),tyrosine nitration, vimentin, and PP2A were analyzed by SDS-PAGEand Western blotting. Phosphorylations and nitrations were analyzedby mass spectrometry and phospho/Y-NO2 Western blotting,respectively. Immunocytochemistry and scanning electronmicroscopy were performed to visualize protein localization and cellmorphology.Results: Light, oxidative stress and reoxygenation play a positiveregulatory role in the production of nitric oxide in the RPE. LPS,oxidative stress and light treatment significantly upregulated iNOSexpression in RPE cells in concentration or time-dependent manner.Western blotting showed increased nitration in various proteins underoxidative stress. Immunostaining demonstrated vimentin filamentdisorganization in the RPE under light induced stress conditions.Scanning electron microscopy revealed cell surface changes duringoxidative stress-induced RPE apoptosis. Analysis of AMD donoreyes, diabetic, and aged model demonstrated that tyrosine nitrationincreased compared to age-matching control.Conclusions: Oxidative stress-induced disassembly of vimentinintermediate filaments may cause vimentin phosphorylation in theRPE. The study implies dual roles of NO that 1-10 nmole/(min xcm2) may protect RPE cell by PP2A and vimentin phosphorylation,whereas >100 nmole/(min x cm2) generated under oxidative stressmay induce apoptosis, leading to AMD. Modulation of nitric oxidemight be assigned as a therapeutic intervention to maintain thebalanced phosphorylation status for cytoskeletal proteins andapoptosis.Commercial Relationships: O'Donnell Sylvester, None; SrinivasR. Sripathi, None; Weilue He, None; Trevor Moser, None; Paul S.Bernstein, Kalsec (C), Kemin Health (R), Science Based Health (C),Abbott Nutrition (F), Genentech (C), DSM (R), Sequenom (R),NuSkin/Pharmanex (P), Aciont (C), Thrombogenics (C); FolamiLamoke, None; Manuela Bartoli, None; Megan C. Frost, None;Wan Jin Jahng, NoneSupport: Research Excellence Fund from Michigan TechnologicalUniversityProgram Number: 695 Poster Board Number: D0252Presentation Time: 10:30 AM - 12:15 PMProhibitin Depletion Leads to RPE Apoptosis in Age-RelatedMacular DegenerationWan Jin Jahng 1 , O'Donnell Sylvester 1 , Trevor Moser 2 , FolamiLamoke 3 , Paul S. Bernstein 4 , Manuela Bartoli 3 , Srinivas R. Sripathi 2 .1 Retina Proteomics Lab, Petroleum Chemistry, American Universityof Nigeria, Yola, Nigeria; 2 Biological Sciences, MichiganTechnological University, Houghton, MI; 3 Ophthalmology, GeorgiaHealth Sciences University, Augusta, GA; 4 Ophthalmology andVisual Sciences, University of Utah, Salt Lake City, UT.Purpose: Understanding the molecular mechanisms that mediateoxidative stress-induced proteome changes may provide insight intopathogenesis of retinal degeneration. Our proteomic studydemonstrated that prohibitin is involved in oxidative stress signalingin the retina and the RPE. The current study was designed to examinethe altered interactions of prohibitin-PIP3/cardiolipin, prohibitinbindingproteins under oxidative stress and how these interactionsaffect macular degeneration.Methods: Immunoprecipitation and immunocytochemistry wereperformed to identify protein interactions and visualize proteinlocalization and expressions from ARPE-19 cells. Two dimensionalSDS-PAGE and Western blotting were used to identify proteins inAMD eyes. Proteomic data from aged and diabetic mouse/rat modelwere examined to identify prohibitin downstream signaling. AMDproteome were analyzed by, 2D SDS-PAGE, MALDI-TOF andtandem ESI/MS/MS. Protein-lipid interactions were analyzed toexamine protein binding to cardiolipin, cholesterol, and phosphatidylinositides.Results: Our study demonstrated that prohibitin-lipid binding switchmediated by cardiolipin and phosphatidylinositol may controldynamic translocalization of prohibitin between mitochondria and thenucleus. Decreased levels of prohibitin in the RPE from AMD eyes,compared to age-matching control, suggest that it may have an antiapoptosisfunction in RPE cells, especially under chronic oxidativestress conditions. Prohibitin expression in both macular andperipheral region in the retina increased in AMD, however, prohibitinis down-regulated in the RPE. The lipid interaction assaydemonstrated that mitochondrial prohibitin has a strong affinity atlow cardiolipin concentrations (6−10 nmol).Conclusions: We investigated oxidative stress-induced proteomechanges in RPE cells using the differential gel electrophoresis(DIGE) technique to identify early biomarkers in AMD. Underoxidative stress, the level of cardiolipin decreased and the level ofcardiolipin-prohibitin interactions were altered in RPE cells.Decreased levels of prohibitin in RPE cells of AMD eyes imply thatRPE cell death by mitochondrial dysfunction may happen prior toapoptosis of the retina. Anti-apoptotic and transcriptional regulatorfunction of prohibitin might be the crucial step for controllingapoptotic signaling as a cell death determinant from uncontrolledproliferation.Commercial Relationships: Wan Jin Jahng, None; O'DonnellSylvester, None; Trevor Moser, None; Folami Lamoke, None;Paul S. Bernstein, Kalsec (C), Kemin Health (R), Science BasedHealth (C), Abbott Nutrition (F), Genentech (C), DSM (R),Sequenom (R), NuSkin/Pharmanex (P), Aciont (C), Thrombogenics(C); Manuela Bartoli, None; Srinivas R. Sripathi, NoneSupport: Research Excellence Fund/MTUProgram Number: 696 Poster Board Number: D0253Presentation Time: 10:30 AM - 12:15 PMRhodopsin ADRP Mutant Ter349Glu Causes RetinalInflammation and Early-Onset Degeneration©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyT J Hollingsworth, Alecia K. Gross. Vision Sciences, University ofAlabama at Birmingham, Birmingham, AL.Purpose: The Ter349Glu rhodopsin mutant causes a severe retinalphenotype characterized by early-onset and rapid degeneration. Priorin vitro assays showed that Ter349Glu rhodopsin is able to fold andfunction similarly to wild type (WT) while mislocalizing in polarizedcells. To determine the origin of the severe phenotype, we examinedthe retina of a knock-in mouse bearing the mutation for receptorlocalization, rod cell morphogenesis and degeneration, andinflammation.Methods: Retinas from Ter349Glu heterozygote and homozygotealbino mice were examined for receptor localization, photoreceptordegeneration, and inflammation using fluorescentimmunohistochemistry, and the in vivo retinal condition usingfunduscopy and optical coherence tomography (OCT).Results: Rods of the Ter349Glu homozygote mice degenerate rapidlyand lack a clear delineation between inner and outer segments. Inheterozygote animals, the degeneration seen is slowed withapproximately two nuclei lost in the outer nuclear layer. In theserods, mislocalization of both WT and the mutant receptor wasobserved. Labeling for both phospho-STAT3 and F4/80 antigen, eacha marker of retinal inflammation, show activation of both theJAK/STAT pathway and macrophages, respectively. Funduscopyreveals hallmarks of retinitis pigmentosa including attenuated retinalvessels and patches of light reflection indicating a loss of neuralretina. Examination by OCT reveals detachments between thephotoreceptors and the pigmented epithelium.Conclusions: The rapid degeneration and RP phenotypes observed inhomozygote animals are consistent with what is seen in humanpatients. The lack of obvious rod outer segments is indicative of aloss of proper morphogenesis, possibly due to the occlusion of the C-terminal sorting motif. Mislocalization of the photoreceptor proteincould be due to aggregate formation and/or endoplasmic reticulumretention of both the mutant and WT proteins. The presence ofactivated macrophages in the retinas of homozygote knock-in animalsindicates an inflammatory response possibly associated with aninfiltration of blood-borne macrophages from the choroid. Theactivation of the JAK/STAT pathway is indicative of retinalinflammation and may be responsible for lowering the expressionlevels of rhodopsin through cytokine signaling, possibly in aninterleukin-6 dependent manner.Commercial Relationships: T J Hollingsworth, None; Alecia K.Gross, NoneSupport: NIH/NEI R01 EY019311Program Number: 697 Poster Board Number: D0254Presentation Time: 10:30 AM - 12:15 PMUNC119B, an UNC119/RG4 paralog expressed in retina andRPECecilia D. Gerstner 1 , Houbin Zhang 1 , Jeanne M. Frederick 1 ,Wolfgang Baehr 1, 2 . 1 Ophthalmology, University of Utah, Salt LakeCity, UT; 2 biology, University of Utah, Salt Lake City, UT.Purpose: UNC119B is related closely to UNC119/HRG4, a homologof C. elegans Unc-119, and more distantly to the prenyl-bindingprotein, PrBP/δ. Each of these (UNC119, UNC119B and PrBP/δ)forms an immunoglobulin-like β-sandwich fold for lipid binding. Ourpurpose was to identify expression and function of the UNC119Bisoform in retina and RPE.Methods: GST-pulldowns, preparation of monospecific antibodiesraised in rabbit and chicken, western blotting, immunohistochemistry,generation of an Unc119b knockout mouse.Results: By gene and EST blasting, UNC119B was determined to beexpressed in both vertebrates and invertebrates. Polyclonal antibodieswere generated using N-terminal peptide and recombinant full-lengthUNC119B as antigens. By multiple mouse tissue immunoblot, weshow that UNC119B is expressed strongly in testis, ovary, lung, brainand thymus, but weakly in whole eye, heart and muscle. UNC119B isexpressed prominently in the RPE and weakly in retina.Immunohistochemistry using frozen mouse and monkey (Macacamulatta) retina/RPE sections show partial colocalization with RDH5in RPE cell peripheral margins. Both GST-UNC119B and GST-UNC119/RG4 pull down Tα from retina lysates, suggesting closelyrelatedfunctions as acyl binding proteins. Both UNC119 isoformsreveal perfect conservation of all amino acids known to interact withthe acyl side chain and N-terminal residues of Tα, and predict nearlyidentical structures. An Unc119b-/- mouse was generated using aMMRCC (UC Davis) genetrap. The trap, located in exon3, blocksUNC119B protein expression. The Unc119b-/- mouse did not reveala retina degeneration phenotype at one-month postnatally.Conclusions: UNC119B is detected prominently in the RPE andweakly in the retina. UNC119B may function as an acyl bindingprotein with specificity for a novel G protein α-subunit expressed inthe RPE.Commercial Relationships: Cecilia D. Gerstner, None; HoubinZhang, None; Jeanne M. Frederick, None; Wolfgang Baehr, NoneSupport: EY08123; EYEYo14800A2;EY019298Program Number: 698 Poster Board Number: D0255Presentation Time: 10:30 AM - 12:15 PMIn Depth Analysis of Retinal Proteins using a CombinatoryHPLC based Mass Spectrometric PlatformSebastian Funke, Dominik Wolters, Lukas Weiler, Francis K. Mwiiri,Katharina Bell, Corina Wilding, Norbert Pfeiffer, Franz H. Grus.Experimental Ophthalmology, University Medical Center, Mainz,Germany.Purpose: The retina proteome with special focus on retinal ganglioncell proteins is of important interest regarding clinical research onneurodegenerative diseases like glaucoma. To intensivelycharacterize retinal proteins we developed protein extraction andfractionation techniques coupled to LC MALDI and ESI methods toincrease the sensitivity towards retinal sample species. Detailedreference maps should be generated by use of the developedworkflow providing new insights to the understanding of the retinaproteome.Methods: Retinal sample species were used as study material.Different extraction techniques were developed encircling non-ionicdetergent extraction, stepwise differential extraction and methanolchloroformextraction for sample fractionation. Extract analysisincludes gel-based RP-RP-2D-capillary-LC MALDI TOF/TOF andRP-capillary LC ESI-LTQ Orbitrap mass spectrometry. The mediummolecular weight range was directly investigated by top downmicrobore-LC MALDI TOF MS. Results were used to generatereference protein maps of so far identified retinal proteins.Results: By use of non-ionic detergent extraction more than 1000proteins per lysate could be identified with high reproducibility bythe LC ESI approach in a single sample lysate.The gel-based MALDIapproaches detected more than 300 proteins. More than 856 mediummolecular weight components per sample were detected by top downMALDI analysis. Furthermore ESI and MALDI methods incombination could distinctly increase the output emphasizing thecomplementary power of MALDI and ESI techniques. By use ofstepwise differential extraction and chloroform-methanol extraction adistinct and reproducible fractionation of proteins could be achieved.Moreover post-translational modification sites of retinal proteinshave been determined by mass spectrometric analysis.Conclusions: The use of the developed platform allowed us to get a©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologydeep view to the retina proteome providing detailed reference maps.Accordingly we could demonstrate the promising use of fractionationtechniques coupled to HPLC based mass spectrometric approachesfor retina related sample species. We think that the providedreference maps as well as the developed workflow will contribute toclinical retina research.Commercial Relationships: Sebastian Funke, None; DominikWolters, None; Lukas Weiler, None; Francis K. Mwiiri, None;Katharina Bell, None; Corina Wilding, None; Norbert Pfeiffer,Sensimed AG (F), Sensimed AG (R), MSD (F), MSD (R), Alcon (F),Allergan (F), Novartis (F), Novartis (R), Bayer (F), HeidelbergEngineering (F), Bausch&Lomb (F), Boehringer-Ingelheim (F), CarlZeiss Meditech (F), Chibret (F), Nidek (F), Pfizer (F), Santen (F),Santen (R), Topcon (F), Ivantis Inc (F), Ivantis Inc (R); Franz H.Grus, NoneProgram Number: 699 Poster Board Number: D0256Presentation Time: 10:30 AM - 12:15 PMRegulation of Synaptic and Gap Junctional Proteins in the Retinaby Rhodopsin-Regulated Protein Kinase DJesse Peterson 1 , Amitavo Mitra 2 , Minzhong Yu 3 , Edward J. Dudek 2 ,Jinsong Yang 2 , Neal S. Peachey 3, 4 , Janis Lem 2 . 1 Cell, Molecular &Developmental Biology, Tufts University Sackler School of GraduateBiomedical Sciences, Boston, MA; 2 Ophthalmology & MolecularCardiology, Tufts Medical Center, Boston, MA; 3 Cole Eye Institute,Cleveland Clinic Foundation, Cleveland, OH; 4 Research Service,Louis Stokes VA Medical Center, Cleveland, OH.Purpose: Protein kinase D (PKD) in the retina forms complexes withα-catenin, β-catenin and cadherin in a rhodopsin- and light-dependentmanner. These proteins colocalize primarily to synaptic layers of theretina, namely the outer limiting membrane, outer and innerplexiform and ganglion cell layers. Cadherin/catenin complexes areimplicated in regulation of synaptic strength at chemical synapses(Murase S, Neuron 2002) and in electrical gap junctions (Shaw RM,Cell 2007). We test the hypothesis that PKD regulates synapticchanges in response to light.Methods: Synaptic changes were assessed in synaptosomalpreparations isolated from photoreceptor-specific PKD knockout,PKD kinase-dead mutant mice and WT control retinas. Changes in α-catenin, β-catenin, cadherin and the synapse proteins PSD95, ribeye,synaptophysin and synaptic vesicle 2 (SV2) were analyzed byWestern blot analysis. Gap junctional changes were assessed byneurobiotin labeling after cut-loading (Choi HJ, JOVE 2012). ERGswere used to test for physiological changes.Results: Western blot analysis of synaptosomal preparations showedstatistically significant decreased levels of α-catenin, β-catenin andcadherin in PKD KO and kinase-dead retina compared to controlretina. Furthermore, the synaptic proteins PSD95, ribeye,synaptophysin and SV2 were significantly decreased. Levels ofprotein were unchanged in total retina lysate. Neurobiotin tracerstudies revealed a significant decrease in light-mediated amacrine cellcoupling in PKD KO and kinase-dead retinas compared to WTretinas. ERGs of PKD mutant mice were normal.Conclusions: Our data suggest that PKD acts downstream ofrhodopsin to alter the distribution of synaptic adherens proteins andaffects both chemical and electrical synapses. This suggests thatrhodopsin modifies synaptic plasticity in the retina. Further studiesare required to determine the precise molecular mechanism of thislight-regulated event as well as its physiological consequence.Commercial Relationships: Jesse Peterson, None; Amitavo Mitra,None; Minzhong Yu, None; Edward J. Dudek, None; JinsongYang, None; Neal S. Peachey, None; Janis Lem, NoneSupport: NIH Grant EY12008, FFB, RPBProgram Number: 700 Poster Board Number: D0257Presentation Time: 10:30 AM - 12:15 PMMapping the Receptor-binding Site on PEDF: Implications for aNeurotrophic Role of PEDF-RJason Kenealey 1 , Preeti Subramanian 1 , David Hoover 2 , S PatriciaBecerra 1 . 1 Retinal Cell & Molecul Biol, NEI, Bethesda, MD; 2 Centerfor Information Technology, National Institutes of Health, Bethesda,MD.Purpose: Pigment epithelium-derived factor (PEDF) protects theretina by blocking pathogenic angiogenesis, and by protecting theneural retina against degeneration. Two biologically active PEDFderivedpeptides have been discovered, an antiangiogenic 34mer anda neurotrophic 44mer, which bind distinct receptors; not yetelucidated. Our lab has identified a PEDF receptor, PEDF-R andmapped its ligand binding domain to amino acid positions 203-232 ofthe human PEDF-R. However, the region on PEDF that binds PEDF-R has not been identified. Therefore, this study is designed to map theregion on PEDF that binds PEDF-R to exert biological activity.Methods: Peptides designed from the human PEDF sequence, 34mer(44-77), 44mer (78-121), 17mer (98-104) and alanine scan of 17merpeptides, and from human PEDF-R, P1 (210-249) were chemicallysynthesized and purified (Biosynthesis, Inc.). Binding wasdetermined by ligand blotting, pull-down, surface plasmon resonance(SPR), and fluorescence anisotropy. The structure of P1 waspredicted in an ab initio model, and the resultant structure was thenused in a global docking search using the Rosetta software. Retinasurvival assays were performed with rat retina precursor R28 cellsinduced to cell death by serum starvation, and measuring apoptosisby TUNEL staining.Results: The 44mer peptide bound specifically to P1 peptide byligand blot, SPR, and anisotropy assays; whereas, the 34mer did notbind. The 44mer, and not the 34mer, bound to full-length PEDF-R ina pull-down assay. The affinity of the 44mer:P1 interaction calculatedfrom ligand blot data was KD=70±7.7 nM compared to 9.1±1.1 nMfor that of PEDF:P1. The 17mer bound P1 with a KD=410±80 nM.Alanine scanning of the 17mer demonstrated that R99 of 17mer wascritical for P1 binding, and that 17mer[H105A] variant bound P1more tightly than the unmodified peptide. Molecular modelingrevealed that P1 can fold into three alpha helices and dock in the44mer region of the PEDF protein. Like PEDF, the 44mer peptide,protected R28 cells from apoptosis induced by serum deprivation, butthe 34mer had no neuroprotective effect.Conclusions: Our results identify a novel PEDF-R binding site onPEDF in its neurotrophic region. Further, these data support the roleof PEDF-R as a neurotrophic receptor for PEDF.Commercial Relationships: Jason Kenealey, None; PreetiSubramanian, None; David Hoover, None; S Patricia Becerra,NoneSupport: NIH Intramural Research Program (EY000306)Program Number: 701 Poster Board Number: D0258Presentation Time: 10:30 AM - 12:15 PMMonitoring Kinetic Changes of Proper and Improper RhodopsinTransport ex vivoJoshua Sammons 1 , Alecia K. Gross 2, 1 . 1 Cell Biology, University ofAlabama at Birmingham, Birmingham, AL; 2 Vision Sciences,University of Alabama at Birmingham, Birmingham, AL.Purpose: We have generated knock-in mice expressing humanrhodopsin fused to photoactivatable green fluorescent protein (rhopaGFP)with the C-terminal targeting epitope appended to the C-terminus of the fusion protein (rho-paGFP-1D4) as a real-time probeof rhodopsin transport and mediator of proper rod outer segment©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyformation. Rho-paGFP-1D4 mice were bred to mice expressingrhodopsin (WT) or the rhodopsin C-terminal truncation autosomaldominant retinitis pigmentosa mutant, Q344ter. We havecharacterized the fusion protein, monitored localization underdifferent environments, and performed real time ex vivo traffickingstudies.Methods: To test for proper folding, rho-paGFP-1D4 was expressedin COS cells, reconstituted, solubilized, and subjected to UV/Visspectroscopy. Activation of the G-protein transducin was tested byuptake of GTPγ 35 S whereas rhodopsin phosphorylation was tested forby using ATPγ 32 P. Polarized mouse Inner Medullary Collecting Duct(IMCD) cells were transfected with rho-paGFP-1D4 cDNA toobserve in vitro localization relative to a primary cilium. TransgenicXenopus laevis were created expressing rho-paGFP-1D4 under theXenopus opsin promoter to examine in vivo localization in transgenicanimals and knock-in mice were generated expressing rho-paGFP-1D4 to monitor in vivo localization and real time rhodopsin transport.Results: Rho-paGFP-1D4 folds, activates transducin, and isphosphorylated similarly to WT rhodopsin. Rho-paGFP-1D4localizes similarly to WT rhodopsin in vitro and in vivo unless therhodopsin trafficking mutant Q344ter is expressed in the samesystem. This has allowed us to monitor the differences in proper andimproper transport of rhodopsin in live mouse retinas.Conclusions: Rho-paGFP-1D4 functions and localizes similarly toWT, despite the addition of 258 amino acids to the C-terminus. Assuch, it is being used as a real time probe of rhodopsin transport whenexpressed in retinas containing wild type properly-localizingrhodopsin and the mislocalizing mutant Q344ter.Commercial Relationships: Joshua Sammons, None; Alecia K.Gross, NoneSupport: EY019311Program Number: 702 Poster Board Number: D0259Presentation Time: 10:30 AM - 12:15 PMCharacterisation of a novel antibody against the loop betweenTMD 3 and 4 of human Bestrophin1Caroline Pasquay, Annabella Janise, Birgit Lorenz, Markus N.Preising. Dept. of Ophthalmology, Justus-Liebig-University,Giessen, Germany.Purpose: Purpose: Bestrophin1 is part of an integral membraneprotein complex located in the basolateral membrane of the retinalpigment epithelium. The gene is mutated in Best vitelliform maculardystrophy (VMD), an autosomal dominant macular degeneration withhighly variable expressivity and reduced penetrance. We want toinvestigate whether mutant bestrophin1 subunits form complexeswith wildtype protein subunits to get further information about thefunction of mutant protein complexes which is not well understoodup to now. For this purpose we constructed a novel antibody againstthe loop between transmembrane domains (TMD) three and four andconfirmed its applicability in Western blotting.Methods: Methods: An alignment with the four isoforms of humanbestrophin (1-4) was performed. A human bestrophin1 specificsequence stretch (AA204-220: PILLQSLLNEMNTLRTQ) within theloop between TMD 3 and 4 was chosen as antigen and anoligopeptide was synthesized. The antibody was raised against theoligopeptide in chicken and affinity purified by a commercialsupplier. The antibody specificity was confirmed by an ELISAagainst the oligopeptideMDCK II cells were seeded on transwell filters and cultured in highglucose DMEM supplemented with 10% FCS and 5 timespenicillin/streptomycin at 37°C and 5% CO 2 . His-tagged BEST1cloned into a pcDNA3(-) vector was transfected into MDCKII cellsusing RotiFect® according to the manufacturers’ instructions atsubconfluency. Cells were harvested 24 h after transfection andlysed. After lysis proteins were extracted and protein preparation tookplace after a modified protocol according to Bordier (JBC 1981).SDS-PAGE and Western blotting were performed following astandard protocol.Results: Results: The antibody showed a single specific bandcorresponding to a molecular weight of approx. 67 kD on SDS-PAGE and Western blotting. This corresponds well with themonomer size of human bestrophin1. Counterstaining by acommercially available anti-His antibody labelled the identical band.Conclusions: Conclusion: A novel antibody (cab-BEST1) was raisedin chicken against the intracellular loop between TMD 3 and 4 ofhuman bestrophin1. The antibody was specific for humanbestrophin1 in Western blots and will be useful in testing the functionof the N-terminal half of bestrophin1. Further data on its applicabilityin immunohistochemistry will be available at the meeting.Commercial Relationships: Caroline Pasquay, None; AnnabellaJanise, None; Birgit Lorenz, Optos (F); Markus N. Preising, NoneSupport: Dr. Gertrud und Franz-Karl Portmann-Stiftung, GiessenerLichtblicke e.V., Marie-Louise Geissler StiftungProgram Number: 703 Poster Board Number: D0260Presentation Time: 10:30 AM - 12:15 PMThe presynaptic glutamate transporter in rods binds to a distinctsubset of PDZ domain containing proteinsSarah Hengel 1 , Joseph G. Laird 1 , Will Watkins 2 , Randy A. Hall 2 ,Sheila A. Baker 1 . 1 Department of Biochemistry, Carver College ofMedicine, University of Iowa, Iowa City, IA; 2 Department ofPharmacology, Emory University School of Medicine, Atlanta, GA.Purpose: Visual signaling depends on removal of glutamate from thesynaptic cleft between photoreceptors and bipolar cells. Excitatoryamino acid transporter 5 (EAAT5) is the glutamate transporter foundin rod inner segments and synapses. The location and activity ofEAAT5 is likely regulated by interacting proteins, however bindingpartners have yet to be identified. The C-terminus of EAAT5contains a PDZ binding motif which prompted us to screen for PDZdomain interactions.Methods: We generated a GST fusion protein containing the 30 C-terminal amino acids of EAAT5 and probed an array of 96 PDZdomains. GST pull-downs and fluorescence anisotropy assays wereused to confirm interactions. A membrane-associated GFP reporterfused to EAAT5’s C-terminus was expressed in the rods of transgenicfrogs and subcellular location was visualized with confocalmicroscopy.Results: The C-terminus of EAAT5 bound to 13 of the 96 PDZdomains on the array. Only three of these positive hits, β2-syntrophin, SAP97, and MAGI-1a, are known to be expressed in theouter plexiform layer of the retina along with EAAT5. All three ofthese interactions were validated by independent GST pull-downassays. The β2-syntrophin PDZ domain interacts with the EAAT5 C-terminal peptide with a calculated KD=4.3 +/-0.3 µM. In transgenictadpoles the membrane reporter accumulates in the outer segmentregardless of the presence of EAAT5’s C-terminus.Conclusions: We identified multiple novel interaction partners forthe glutamate transporter EAAT5. These interactions are unlikely tobe necessary for the trafficking of EAAT5, suggesting they maymodulate its activity.Commercial Relationships: Sarah Hengel, None; Joseph G.Laird, None; Will Watkins, None; Randy A. Hall, None; Sheila A.Baker, NoneSupport: EY020542-04Program Number: 704 Poster Board Number: D0261©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyPresentation Time: 10:30 AM - 12:15 PMDifferential Analysis of Retinal Guanylyl Cyclase GUCY2D(RetGC1) Activation and Binding with GCAP1 and RD3 UsingIn Vitro Activity Assay and Cell-based Binding AssayIgor V. Peshenko, Elena V. Olshevskaya, Alexander M. Dizhoor.Pennsylvania College of Optometry and Department of BasicSciences, Salus University, Elkins Park, PA.Purpose: Regulation of retinal membrane guanylyl cyclase,GUCY2D (RetGC1), plays important role in rod and cone physiologyand becomes the cause of retinal diseases when altered by mutationsin GUCY2D, GCAPs and RD3. With a variety of mutations causingdifferent forms of congenital blindness, it becomes important todistinguish between the two general mechanisms through whichdisease-causing mutations affect GUCY2D activity and/or regulation- suppressing the catalytic activity of the enzyme versus thoseaffecting cyclase binding with its regulatory proteins.Methods: Point mutations affecting GUCY2D activity and linked toLeber Congenital Amaurosis 1 (LCA1) [1, 2] were introduced inGUCY2D. GUCY2D was co-expressed in HEK 293 cells withfluorescently tagged GCAP1 or RD3 and their association was testedin cyto using confocal microscopy. GUCY2D activation by GCAP1expressed in E. coli was measured in vitro.Results: Mutations that suppressed RetGC activation by GCAP1produced different effects on the ability of the mutant GUCY2D tobind GCAP1-GFP in HEK 293 cells: some of them suppressedGCAP1 GFP binding while the others had no effect on the bindingand evidently caused the loss of GUCY2D activity in vitro throughaffecting its catalytic activity per se. In some cases, they alsodifferentially affected binding of GCAP1, a positive regulator, and ofRD3, a negative regulator of the cyclase activity [3]. Mutations inGCAP1 located in EF-hand 1 and in proximity to the interfacebetween the two lobes of the GCAP molecule were able tocompletely block GCAP1 binding to guanylyl cyclase.Conclusions: Comparative analysis of GUCY2D activity and itsability to bind GCAP1-GFP in a cell-based assay provides a usefultool for distinguishing between the direct suppression of GUCY2Dactivity versus that of GCAP1 or RD3 binding. The binding sites forRD3 and GCAP1 in RetGC1 are likely overlapped but not identicaland can become differentially affected by disease-related mutations.References: [1] S. Jacobson et al. (2012) Hum Mol Genet., in press;[2] E. Stone (2007) Am J Ophthalmol 144:791-811; [3] I.Peshenko etal. (2011) Biochemistry 50:9511-9519Commercial Relationships: Igor V. Peshenko, None; Elena V.Olshevskaya, None; Alexander M. Dizhoor, NoneSupport: NEI R01 EY11522, Pennsylvania Department of HealthCURE formula grantProgram Number: 705 Poster Board Number: D0262Presentation Time: 10:30 AM - 12:15 PMMultiple isoforms of TRIP8b, a regulator of HCN channels, areexpressed in retinaYuan Pan, Joseph G. Laird, Sheila A. Baker. Biochemistry,University of Iowa, Iowa City, IA.Purpose: Hyperpolarization-activated cyclic nucleotide-gatedchannels (HCN) are cation-selective channels present in retina, brainand heart. The activity of HCN channels contributes to signalintegration, cell excitability and pacemaker activity. Rods expresshomomeric channels composed of HCN1; these channels participatein keeping light responses transient. The subcellular localization ofHCN1 varies among cell types. In rods, HCN1 is found in the innersegments, to a lesser extent in the synaptic terminals, but excludedfrom the outer segments. In contrast, HCN1 in the hippocampus isconcentrated in a subset of dendrites. A key regulator of HCN1trafficking and activity is TPR-containing Rab8b interacting protein(TRIP8b). Up to nine splice isoforms of TRIP8b are expressedthroughout the brain and can differentially regulate surfaceexpression and activity of HCN1. The purpose of the present studywas to determine which isoforms of TRIP8b are expressed inphotoreceptors and to test if the C-terminus of HCN1, containingTRIP8b binding sites, could confer targeting in rods.Methods: RT-PCR using RNA isolated from total mouse retina orphotoreceptor inner segments collected by laser capture microdissectionwas used to test for the presence of specific TRIP8b spliceisoforms. Immunohistochemistry was used to test for the expressionof TRIP8b protein in mouse retina. To assess targeting, the C-terminus of HCN1 and deletions of this region, were fused to amembrane reporter and expressed in transgenic Xenopus rods.Localization of the transgenically expressed proteins was visualizedwith confocal microscopy.Results: We found that multiple splice isoforms of TRIP8b areexpressed in photoreceptors, including those with opposing effects onHCN1 trafficking. TRIP8b protein was found in the same retinacompartments as HCN1. In transgenic Xenopus rods, the membranereporter localized primarily to outer segments but addition ofHCN1’s C-terminus changed its localization to the inner segment.Deletions of HCN1’s C-terminus uncovered a strong synaptictargeting signal located in a stretch of amino acids in between the twoTRIP8b binding sites. In order to achieve the normal localizationpattern of HCN1, this synaptic targeting signal needs to besuppressed.Conclusions: HCN1 trafficking involves interactions among multiplesignals within its C-terminus, and TRIP8b is a possible regulator ofthis process in photoreceptors.Commercial Relationships: Yuan Pan, None; Joseph G. Laird,None; Sheila A. Baker, NoneSupport: NIH Grant EY020542-04Program Number: 706 Poster Board Number: D0263Presentation Time: 10:30 AM - 12:15 PMVAMP7 as a Regulator of Rhodopsin Transport Carrier Fusionin PhotoreceptorsBridgette Ray 1 , Beatrice M. Tam 2 , Orson L. Moritz 2 , DusankaDeretic 1 . 1 Surgery-Opthalmology, University of New Mexico,Albuquerque, NM; 2 Dept. of Ophthalmology and Visual Sciences,University of British Columbia, Vancouver, BC, Canada.Purpose: Our lab has previously identified Syntaxin3 and SNAP25as the Qa and Qbc-SNAREs that are present on the rod inner segment(RIS) plasma membrane. We have also shown that these SNAREs areinvolved in the regulation of Rhodopsin transport carrier (RTC)fusion (Mazelova et al, 2009). However, the R-SNARE present onthe RTCs that interacts with Syntaxin3 and SNAP25 on the RISmembrane has not been identified. In this study, we identifiedVAMP7 as a candidate for the R-SNARE present on RTCs.Methods: Post-nuclear supernatant (PNS) was isolated from frogretinas and fractionated on sucrose density gradients. VAMP7localization in control and propranolol treated retinas was studiedusing confocal microscopy. Full length wild type VAMP7 or an N-terminal fragment of VAMP7, which acts as a dominant negative,will be expressed specifically in the photoreceptors of X. laevis. Theretinas of the transgenic frogs will be analyzed by confocalmicroscopy to determine the effects of VAMP7 on the delivery ofrhodopsin to the ROS.Results: Our data show that VAMP7 is present in both the RTCs andGolgi/TGN. Interestingly, VAMP7 co-fractionates with ASAP1 inboth the Golgi/TGN and RTC fractions. Confocal microscopy revealsthe presence of VAMP7 within the RIS of frog photoreceptors. When©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologythe retinas were treated with propranolol, which causes membranetetheringdefects and inhibits RTC fusion, there was a markedaccumulation of VAMP7 on the RIS membrane.Conclusions: Our study suggests that VAMP7 is present on RTCs.When retinas were treated with propranolol, VAMP7 accumulated onthe RIS membrane in a manner similar to what has been reported forSNAP25 and Syntaxin3, suggesting an interaction (Mazelova, 2009).Taken together, our data suggests that VAMP7 might be the R-SNARE that interacts with Syntaxin3 and SNAP 25, resulting infusion of RTCs with the RIS membrane.Commercial Relationships: Bridgette Ray, None; Beatrice M.Tam, None; Orson L. Moritz, None; Dusanka Deretic, NoneSupport: NIH grant EY012421Program Number: 707 Poster Board Number: D0264Presentation Time: 10:30 AM - 12:15 PMArrestin inhibits AldolaseC activityJ Hugh McDowell, W Clay Smith, Susan N. Bolch, Donald R.Dugger. Ophthalmology, University of Florida, Gainesville, FL.Purpose: In a previous study, we showed that arrestin in a retinalhomogenate was crosslinked to enolase1 using a water-solublecrosslinker. Further study showed that arrestin enhanced the activityof enolase1. Here we followed the same crosslinking protocol butused a lipid soluble crosslinker to identify other binding partners forarrestin that may be involved with the light-induced translocation ofarrestin.Methods: A bovine retinal homogenate was crosslinked usingdisuccinimidyl glutarate, a lipid soluble crosslinker. The majority ofthe crosslinked arrestin products were found in the supernatant fromthe retinal homogenate as well as in the rod outer segments preparedfrom the retinal homogenate. After following arrestin purificationprotocols, SDS-PAGE gels were run, stained, and immunoblots weredeveloped with anti-arrestin antibodies. Coomassie-stained bands thatwere also reactive with the anti-arrestin antibody were submitted tomass spectrometric analysis to identify the proteins in the band.His(6)-tagged arrestin was expressed in yeast as previouslydescribed. AldolaseC was heterologously expressed in bacteria with aHis(6) tag. AldolaseC activity was determined following publishedprocedures using fructose-1,6-bisphosphate and hydrazine sulfate inthe presence and absence of his-tagged arrestin.Results: A gel band at approximately 125kD was observed in thepurified sample that was stained with Coomassie and was alsodetected in an immunoblot developed with an anti-arrestin antibody.AldolaseC and arrestin were found to be the principal components ofthis band. In an assay of aldolase enzymatic activity, the presence ofarrestin resulted in a small, but significant reduction in the activity ofaldolaseC.Conclusions: AldolaseC was found to be an interaction partner forarrestin in bovine retinas. Mass spectrum analysis demonstrated thatboth aldolaseC and arrestin were present in an approximately 125 kDgel band. The presence of arrestin has a small inhibitory effect on theactivity of aldolaseC. The binding of arrestin to aldolaseC appears tobe weak as additional experiments intended to show binding failed todo so (data not shown). This relatively weak interaction suggests thataldolaseC may not have a significant role in the light-inducedtranslocation of arrestin, but that arrestin may have a role inregulating glycolysis in the rod inner segment.Commercial Relationships: J Hugh McDowell, None; W ClaySmith, None; Susan N. Bolch, None; Donald R. Dugger, NoneSupport: NIH grant EY014864, NIH grant EY006225, NIH grantEY0121721, and RPBProgram Number: 708 Poster Board Number: D0265Presentation Time: 10:30 AM - 12:15 PMGlutathione S-Transferase Pi Isoform (GSTP1) Expression inMurine Retina Decreases with AgingWen-Hsiang Lee, Pratibha M. Joshi, Rong Wen. Ophthalmology-U ofMiami, Bascom Palmer Eye Inst, Miami, FL.Purpose: Glutathione S-transferase pi isoform (GSTP1) is anintracellular detoxification enzyme that catalyzes reduction ofchemically reactive electrophiles and is a zeaxanthin-binding proteinin the human macula. We have previously demonstrated that GSTP1levels are decreased in human age-related macular degeneration(AMD) retina compared to normal controls. We showed that GSTP1levels parallel survival of human retinal pigment epithelial (RPE)cells exposed to UV light, and GSTP1 over-expression protects themagainst UV light damage. We also showed that GSTP1 in murineretina increases with developmental maturity (from post-natal day 20to 2 months of age) and is induced by light exposure. In the presentwork, we determined the GSTP1 expression in the retina in agingmice and in response to light challenge.Methods: Eyes from BALB/cBy mice at 2 months, 12 months, and24 months of age were prepared for retinal protein extraction andcryo sectioning, and the GSTP1 levels in the retina were analyzed byWestern blot and immunohistochemistry (IHC). Another group ofBALB/cBy mice with the same age ranges was exposed to 1000 luxof white fluorescent light for 24 hours, and their retinas wereanalyzed for GSTP1 expression by Western blot and IHC in a similarmanner.Results: GSTP1 levels in the murine retina decreased with increasingage at 2 months, 12 months, and 24 months of age. In response tobrief light exposure, GSTP1 expression initially increased at age 2months then decreased and plateaued at 12 months and 24 months ofage compared to age-matched controls under normal condition.Conclusions: GSTP1 expression in murine retina decreases withaging. Brief exposure to light induces GSTP1 expression in themurine retina at younger developmental age but declines with aging.GSTP1 induction may be a protective response to light-inducedoxidative damage in the murine retina, and this protective responsemay decline with aging.Commercial Relationships: Wen-Hsiang Lee, None; Pratibha M.Joshi, None; Rong Wen, Neurotech USA (C)Support: In part by: K08EY20864; R01EY018586; Hope for Vision;SanBio, Inc.; P30EY14801; an unrestricted grant from Research toPrevent Blindness.144 Diabetic Retinopathy, Vein Occlusion, Retinal IschemiaSunday, May 05, 2013 1:00 PM-2:45 PMExhibit Hall Poster SessionProgram #/Board # Range: 1127-1160/D0031-D0064Organizing Section: Biochemistry/Molecular BiologyProgram Number: 1127 Poster Board Number: D0031Presentation Time: 1:00 PM - 2:45 PMImpact of Anti-VEGF Therapy on Visual Impairment fromMacular Edema Due to Branch Retinal Vein OcclusionMariana S. Lopes 1, 2 , Connie J. Chen 1 , Voraporn Chaikitmongkol 1, 3 ,Yulia Wolfson 1 , Susan B. Bressler 1 , Neil M. Bressler 1 . 1 RetinaDivision, Wilmer Eye Institute, Johns Hopkins University, Baltimore,MD; 2 Instituto Suel Abujamra, São Paulo, Brazil; 3 Department ofOphthalmology, Chiang Mai University Hospital, Chiang Mai,Thailand.Purpose: Until recently, observation or grid laser treatment was thestandard care for macular edema (ME) due to a branch retinal veinocclusion (BRVO). Recently, several randomized clinical trials©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologydemonstrated that treatment with anti-vascular endothelial growthfactor (anti-VEGF) therapy was superior to observation or grid laserfor the treatment of ME due to BRVO. This study evaluates themagnitude of the impact of anti-VEGF therapy on visual impairmentfrom ME due to BRVO to estimate its potential impact in a clinicalsetting.Methods: A retrospective cohort study of patients with ME due toBRVO from 2002 to 2004 and from 2006 to 2011 was identified frompatient records of 2 retina specialists at a university-based clinic(SBB and NMB). Eligibility included ME in one or both eyes fromBRVO and treatment with grid laser from 2002 to 2004 or anti-VEGF therapy from 2006 to 2011. Those patients with less than 6months of follow-up were excluded.Results: A total of 12 eyes (12 patients) from 2002 to 2004 and 16eyes (16 patients) from 2006 to 2011 with BRVO were included.Among the 12 patients from 2002 to 2004 receiving laser for ME, 3eyes (25%) had at least mild visual impairment (worse than 20/40 inthe better-seeing eye) including 1 eye (8%) with at least moderatevisual impairment (worse than 20/80 in the better-seeing eye) atpresentation. At the 6-month follow-up for this cohort, 4 eyes (33%)had at least mild visual impairment, and all 4 eyes (33%) had at leastmoderate visual impairment, although none were legally blind(20/200 or worse in the better-seeing eye). Among the 16 patients in2006 to 2011 receiving anti-VEGF therapy for macular edema, 3(19%) had at least mild visual impairment, including 2 eyes (13%)with at least moderate visual impairment. At the 6-month follow-upfor this cohort, 3 eyes (19%) had at least mild visual impairment, andonly 1 eye (6%) had at least moderate visual impairment, and nonewere legally blind.Conclusions: Although there are several limitations to this studyinherent to its retrospective design, the conclusions provide evidencethat the prevalence of at least moderate visual impairment due to MEfrom BRVO may be declining among people in the era of anti-VEGFtherapy.Commercial Relationships: Mariana S. Lopes, None; Connie J.Chen, None; Voraporn Chaikitmongkol, None; Yulia Wolfson,None; Susan B. Bressler, Novartis (F), Bausch and Lomb (F),Genentech (F), Thrombogenics (F), Lumenis (F), Notal vision (F),GlaxoSmithKline (C), allergan (F); Neil M. Bressler, AbbottMedical Optics, inc (F), Alimera Sciences (F), Allergan (F), Bausch&Lomb, Inc (F), Bayer (F), Carl Zeiss Meditec, Inc (F), ForSightLabs, LLC (F), Genentech, Inc (F), Genzyme Corporation (F),Lumenis, Inc (F), Notal VIsion (F), Novartis Pharma AG (F), Pfizer,Inc (F), Regeneron Pharmaceuticals, Inc (F), Roche (F),Thrombogencis (F)surgery and pars plana vitrectomy. AF (0.05-0.10cc) was obtained viaanterior chamber paracentesis. Cataract surgery was performed afterwhich ~1cc undiluted VF was obtained through the vitrector beforeinitiating infusion. All samples were stored at -80°F until batchprocessed. Proteomic analysis used 30μL of undiluted sample.Proteins were analyzed by tandem mass spectrometry and proteinlevels compared using peptide/spectral matches assigned usingX!Tandem.Results: 228 unique proteins were identified overall in AF and VFwith 110 (48%) found in both AF&VF, 16 (7%) in AF only, and 102(45%) in VF only. The percentage of proteins found in both AF&VFfor NPDR, PDR and QPDR groups was 50% (88), 48 % (80) and58% (67), respectively. VF proteins were present only in VF in 37%(64) of NPDR, 47% (79) of PDR, and 32% (37) of QPDR. Overall,52% (110) of VF detected proteins were also detectable in the AF.When proteins were present in both AF&VF, peptide counts betweenVF and AF for an individual protein were highly correlated (r=0.76-0.89) and correlations did not differ substantially between DR groups(r = 0.83-0.84). Peptide counts were higher in VF than AF in 87.3%of proteins for the combined group, 92% in NPDR, 92% in PDR and76% in QPDR. On average, peptide counts for VF predominantproteins were 5 fold higher in VF than AF for any specific protein. Apartial survey of specific proteins known to be predominantly vitrealhad VF/AF ratios >1.0 with peptide numbers changing by group asexpected for changes in disease severity (eg. carbonic anhydrase 1,C1 inhibitor).Conclusions: This study reveals that approximately 50% of theproteins found in vitreous can also be detected in aqueous. There wasalso a high correlation between proteins found in both the AF andVF, and AF findings correlated with known VF changes for at leastsome specific proteins. These data suggest that AF sampling mightallow proteomic assessment reflecting the vitreome in the diabeticeye and help evaluate specific vitreous proteins as biomarkers of DRprogression or treatment response.Commercial Relationships: Nour Maya N. Haddad, None;Jennifer K. Sun, Boston Micromachines (F), Abbott Laboratories(C), Novartis (C), Genentech (F); Michael Molla, None; Paul G.Arrigg, None; Sabera T. Shah, None; Deborah K. Schlossman,None; Timothy J. Murtha, None; Edward P. Feener, JoslinDiabetes Center (P), KalVista Pharmaceuticals (C); Lloyd P. Aiello,Genentech (C), Genzyme (C), Thrombogenetics (C), Ophthotech (C),Kalvista (C), Pfizer (C), Proteostasis (C), Abbott (C), Vantia (C),Optos, plc (F)Support: Research to Prevent Blindness, JDRF 17-2011-359,Massachusetts Lions Eye Research FundProgram Number: 1128 Poster Board Number: D0032Presentation Time: 1:00 PM - 2:45 PMCorrelation of the Aqueous and Vitreous Proteomes in DiabeticEye DiseaseNour Maya N. Haddad 1 , Jennifer K. Sun 1, 2 , Michael Molla 4, 3 , PaulG. Arrigg 1, 2 , Sabera T. Shah 1, 2 , Deborah K. Schlossman 1, 2 , TimothyJ. Murtha 1, 2 , Edward P. Feener 4, 3 , Lloyd P. Aiello 1, 2 . 1 Beetham EyeInstitute, Joslin Diabetes Center, Boston, MA; 2 Department ofOphthalmology, Harvard Medical School, Boston, MA; 3 HarvardMedical School, Boston, MA; 4 Research Division, Joslin DiabetesCenter, Boston, MA.Purpose: To compare proteomes between aqueous fluid (AF) andvitreous fluid (VF) obtained concurrently from the same eye ofpatients undergoing ocular surgery and to assess differences betweendiabetic retinopathy (DR) severity levels.Methods: AF and VF were obtained from 6 eyes (2 each NPDR,active PDR and quiescent PDR) undergoing combined cataractProgram Number: 1129 Poster Board Number: D0033Presentation Time: 1:00 PM - 2:45 PMRetrospective Study of Anti-Vascular Endothelial Growth FactorTherapy in the Treatment of Branch Retinal Vein Occlusion andPredictive Factors for Visual OutcomePeng Lei. Ophthalmology, University of Texas Southwestern, Dallas,TX.Purpose: To compare the efficacy of bevacizumab and ranibizumabintravitreal injection in the treatment of branch retinal vein occlusion(BRVO). To assess the prognostic value on visual outcome of theintegrity of the inner segment-outer segment (IS-OS) junction line onoptic coherence tomography (OCT) and the integrity of the fovealcapillary ring on fluorescein angiography (FA).Methods: A retrospective study with 8 patients diagnosed withBRVO; 5 patients received bevacizumab intravitreal injections; 3patients received ranibizumab intravitreal injections. Average followup times were 8 months and 6.5 months, respectively. Primary©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyoutcomes included difference between initial best corrected visualacuity (BCVA) and best BCVA during follow up, integrity of IS-OSjunction, and integrity of foveal capillary ring.Results: In the bevacizumab group, average improvement in BCVAwas 0.375 +/- 0.205 logMAR. Best BCVA was achieved on averageafter 2.2 +/- 1.6 months and 1.4 +/- 1.1 intravitreal injections. In theranibizumab group, average improvement in BCVA was 0.141 +/-0.151 logMAR. Best BCVA was achieved on average after 2.4 +/-2.2 months and 2.0 +/- 1.7 intravitreal injections. In the bevacizumabgroup, average improvement in CMT was 404 +/- 41 um with bestCMT occurring on average after 1.1 +/- 0.1 months and 1.0 +/- 0.0injections. In the ranibizumab group, average improvement in CMTwas 286 +/- 47 um with best CMT occurring on average after 2.7 +/-1.1 months and 2.5 +/- 0.7 injections.Conclusions: Bevacizumab may be superior to ranibizumab fortreating BRVO with greater improvement in BCVA, fewer injectionsto achieve best BCVA, fewer injections needed during follow upcourse, greater improvement in CMT, shorter time interval to bestCMT, and fewer injections needed to achieve best CMT. Theintegrity of the IS-OS junction correlated well with visual acuityresponse to anti-VEGF treatment. The integrity of the foveal capillaryring in relation to visual outcome was equivocal. Statisticalsignificance was limited by the small sample size.Commercial Relationships: Peng Lei, NoneProgram Number: 1130 Poster Board Number: D0034Presentation Time: 1:00 PM - 2:45 PMVEGF and TNF Interact to Induce Retinal Edema in an AtypicalPKC Dependent MannerCheng-mao Lin 1 , Paul M. Titchenell 2 , Arivalagan Muthusamy 1 ,David A. Antonetti 1 . 1 Ophthalmology and Visual Sciences, KelloggEye Center, University of Michigan, Ann Arbor, MI; 2 Cellular andMolecular Physiology, Penn State College of Medicine, Hershey, PA.Purpose: Changes in permeability of retinal blood vessels contributeto macular edema and the pathophysiology of diabetic retinopathy.Both vascular endothelial growth factor (VEGF) and tumor necrosisfactor (TNF) have been implicated in macular edema. We tested thehypothesis that VEGF and TNF interact to induce retinal edema invivo and require atypical PKC (aPKC) activation.Methods: Male Long-Evans rats weighing 200 to 250 g were used toevaluate retinal vascular permeability and retinal thickening inducedby VEGF and TNF treatments. Animals received a single intravitrealinjection of vehicle, aPKC inhibitor, VEGF, TNF, combined VEGFand TNF or VEGF/TNF plus aPKC inhibitor to yield finalconcentration as indicated. Blood-retinal barrier leakage wasmeasured with intravenous FITC-BSA injection. Retinal leakage wascalculated from the accumulation of fluorescent albumin in the retinaand normalized to the plasma levels. Also, 5 h after intraocularinjection of the same factors, eyes were imaged 10 min after FITC-BSA injection for retinal vascular leakage using a Micron III camera.Eyes were then enucleated, fixed and subjected to flat-mount stainingfor subsequent imaging. Tight junction proteins were examined byimmunohistochemistry. Retinal structure and layer thickness, ameasure of edema, were assessed with spectral domain opticalcoherence tomography. Baseline data were obtained before anytreatment and used for comparison with the later time points in thelongitudinal study.Results: Partial discontinuity of occludin and ZO-1 immunostainingwas observed in the VEGF-treated retinas, whereas loss of ZO-1wasobserved in the TNF-treated retinas. VEGF/TNF combinationtreatment dramatically induced tight junction disorganization andinteracted to increase retinal thickening. Increased retinal albuminleakage and retinal thickening induced by combined VEGF/TNFtreatment were effectively prevented by an aPKC inhibitor in a dosedependentmanner. In the longitudinal study, aPKC inhibitionattenuated the initial (24 h) edema response from VEGF/TNFadministration by 40%. The group treated with the aPKC inhibitorreturned to baseline more rapidly than VEGF/TNF group.Conclusions: These data demonstrate that VEGF and TNF interact topromote retinal edema in vivo and provide an effective model to testtherapeutic intervention to control edema. Co-injection of aPKCinhibitor dramatically reduces induction of edema in vivo.Commercial Relationships: Cheng-mao Lin, None; Paul M.Titchenell, None; Arivalagan Muthusamy, None; David A.Antonetti, NoneSupport: JDRFProgram Number: 1131 Poster Board Number: D0035Presentation Time: 1:00 PM - 2:45 PMThe Role of Apelin in the Retina of Diabetic RatsYanrong Jiang, Qiang Lu, Jing Feng. Peking University People'sHospital, Beijing, China.Purpose: The purpose of this study was to investigate the effect ofapelin-13 on proliferative potential in diabetic retinopathy (DR), andits antagonist inhibitory effects.Methods: Localization of apelin-13, GFAP, and VEGF weredetected using immunofluorescence in the retina of diabetic rats. ThemRNA and protein of apelin-13, GFAP, and VEGF in the retina ofdiabetic rats were measured using real-time PCR and western blot.Apelin-13 antagonist F13A was used to block apelin-13, and to studyits effects in vivo.Results: Strong staining of apelin-13, co-localized with GFAP andVEGF, was observed in the retina of diabetic rats. Apelin-13, GFAP,and VEGF mRNA and protein levels were significantly increased inthe sample’s retina. Moreover, exogenous apelin-13 promoted retinalMüller cell proliferation in vivo. Simultaneously, apelin-13 inducedGFAP and VEGF expression. F13A markedly reduced the retinalgliosis caused by diabetes. Furthermore, F13A suppressed bothGFAP and VEGF expression in vivo.Conclusions: Our results strongly suggest that apelin-13 is associatedwith the development of DR, and contributes to changes in the retinaof diabetic rats. Apelin-induced promotion of cell proliferation lendssupport to the possibility that apelin-13 may play a role in theprogression of DR to a proliferative phase. This possible roledeserves further investigation, which may open new perspectives inthe early prevention and treatment of DR.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyFig 1. Indirect immunofluorescence evaluation of apelin-13 andGFAP distribution in retina of normal rats (A), diabetic rats (B) andF13A-injected diabetic rats(C). Merged images contain three colorchannels representing apelin-13 (red), GFAP (green) and DAPI(blue). Densitometric quantification of GFAP activity (D), GCL,ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiformlayer; ONL, outer nuclear layer.F13A, F13A-injected group. **indicates p < 0.01. Scale bars: 50µm.Univesity of Oklahoma Health Sciences Center, Oklahoma City, OK;2 Department of Medicine, University of OKlahoma Health SciencesCenter, Oklahoma City, OK; 3 Harold Hamm Diabetes Center,Univesity of Oklahoma Health Sciences Center, Oklahoma City, OK.Purpose: Diabetic retinopathy is a microvascular complication ofdiabetes mellitus and is the leading cause of blindness in developedcountries. MicroRNAs (miRNAs) are small, no-coding RNAs whichnegatively regulate target gene expression at the post-transcriptionlevel. miRNAs play important roles in pathological processes ofmany diseases. This study is to investigate changes of the miRNAprofile in diabetic retinopathy and to elucidate the potential role of aspecific miRNA, miR-200b in a type 1 diabetic mouse model.Methods: The visual functions of both Akita and wild-type (WT)mice were examined by electroretinogram (ERG) recording. RNAswere extracted from the retina of Akita mice (9 months of age) andage-matched WT mice. miRNA expression profiles were comparedusing a miRNA-specific microarray, and the expression change ofmiR-200b was confirmed by Taqman qPCR. The level of miR-200bwas also examined in a human Müller cell line (MIO-M1) after thetreatment of 4-hydroxynonenal (4-HNE). The target gene(s) of miR-200b was predicted in silico and confirmed by luciferase assay. Thefunction and role of target gene(s) of miR-200b was tested andverified in MIO-M1 cells by Western blotting and TUNEL assay.Results: ERG recording showed progressive declines of ERGresponses in Akita mice when compared with age-matched WT mice.Microarray and real-time PCR indicated that miR-200b wassignificantly up-regulated in the retina of Akita mice. Computationalsequence analysis and luciferase assay identified that Oxidationresistant 1 (Oxr1) was a direct target gene of miR-200b. In the retinaof Akita mice and in MIO-M1 cells exposed to 4-HNE, the upregulationof miR-200b correlated with down-regulation of the Oxr1mRNA levels in the retina. In MIO-M1 cells, miR-200b mimictransfection down-regulated Oxr1 expression, while the inhibitor ofmiR-200b increased Oxr1 expression. Moreover, the over-expressionof recombinant Oxr1 enhanced cell survival and reduced the 3-nitrotyrosine levels in MIO-M1 cells exposed to 4-HNE.Conclusions: The up-regulation of miR-200b in diabetes reduces theexpression of Oxr1 and weakens the anti-oxidant function of Oxr1 indiabetic retinopathy.Commercial Relationships: Qian Chen, None; Anne Murray,None; Yusuke Takahashi, None; Kelu Zhou, None; KyoungminPark, None; Jian-Xing Ma, NoneSupport: IH grants (EY018659, EY012231, EY019309,P20GM104934) and OCAST (HR12-103)Fig 2. RT-PCR and Western blot analysis showed the mRNA (A) andprotein (B) expression of apelin-13, GFAP and VEGF in diabeticrats, and representative Western blots are shown in Fig.3C **indicates p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyResults: In eyes with diabetic macular edema, the Pearsoncorrelation coefficient for choroidal and macular thickness at thefovea was r=0.39 (p=0.002); at 500μm from the fovea, r=0.063(p=0.6); at 1000μm, r=-0.026 (p=0.85); at 1500μm; r=-0.20 (p=0.17);and at 3000μm, r=0.084 (p=0.55). In non-diabetics, the correlation atthe fovea was r= -0.22 (p=0.11); at 500μm, r=-0.17 (p=0.20); at1000μm, r=012 (p=0.40); at 1500μm, r=0.068 (p=0.62); and at3000μm, r=-0.11 (p=0.40).The average choroidal thickness in diabetics was thinner than in nondiabetics(282μm vs. 369μm, respectively, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyof intravitreal ranibizumab, there was no significant difference inchoroidal hypopermeability change when compared to low-fluencePDT (P=0.139).Conclusions: Using our novel method of analysis of change inchoroidal hyperpermeability with treatment in chronic CSC, greaterchange was found in eyes with good response, and the superioroutcome of low-fluence PDT over ranibizumab may be attributed togreater influence on choroidal hyperpermeability.Commercial Relationships: Jeong-Ah Kim, None; Joo YoungShin, None; So Hyun Bae, None; Jeeyun Ahn, None; Hum Chung,None; Jang won Heo, NoneClinical Trial: NCT01325181Program Number: 1136 Poster Board Number: D0040Presentation Time: 1:00 PM - 2:45 PMCentral serous chorioretinopathy associated with homeopathicadrenal medicationDerek Huang 1, 2 , Robert Millay 2 , Brian Kim 2 . 1 UCLA Olive View,Sylmar, CA; 2 Ophthalmology, University of Vermont College ofMedicine, Burlington, VT.Purpose: To report a case of central serous chorioretinpathy (CSC)associated with homeopathic adrenal medication. CSC ischaracterized by leakage of fluid into the subretinal space from thechoroid that can lead to serous retinal detachments. CSC is mostprevalent in males aged 20-50 years old. Its association with type Apersonality, stress, steroid use and hypercortisolism has beenreported. Elevated catecholamine levels have also been shown. Theprognosis is good with spontaneous resolution in weeks to months;however about 5% of patients fail to regain greater than 20/30 acuity.Methods: One case of central serous chorioretinopathy wasdescribed at the University of Vermont College of MedicineDepartment of Ophthalmology.Results: This is the first case report describing central serouschorioretinopathy in association with the use of homeopathic adrenalmedication. A 42 year old man presented with acute visualcomplaints characterized as “blurry, dark spot in his central vision” inhis left eye of two days duration. He acknowledged using 2 tablets ofADHS supplement for adrenal health. ADHS is a homeopathicadrenal support supplement, marketed to support desireddehydroepiandrosterone (DHEA), Secretory IgA and normal cortisollevels. Examination revealed best-corrected visual acuity was 20/20in the right eye (OD) and 20/30-2 in the left eye (OS). Slit lampexamination showed left retinal pigment epithelial mottling andsubretinal fluid. Angiographic examination revealed two areas ofleakage in the left eye. Optical Coherence Tomography (OCT) wasconsistent with a diagnosis of central serous chorioretinopathy.During a follow up examination (1 week later), he denied further useof his homeopathic medication and experienced improvement in thedark central spot. His visual acuity was 20/20 OD and 20/30+2 OS.OCT demonstrated improvement in his subretinal fluid.Conclusions: CSC has been linked to endogenous Cushing’ssyndrome, sympathomimetic use, and elevated catecholamine levels.ADHS supplies raw materials necessary for the synthesis of steroidhormones and epinephrine, the conversion of tyrosine tocatecholamines, and hormonal secretion. The ingredients and aminoacids contained in ADHS could play a role in its pathogenesis inCSC. Additional investigation into the mechanism of adrenalsupplementation would aid in establishing guidelines for their useand potential side effects.Commercial Relationships: Derek Huang, None; Robert Millay,None; Brian Kim, NoneProgram Number: 1137 Poster Board Number: D0041Presentation Time: 1:00 PM - 2:45 PMPlasma Kallikrein Deficiency is protective against diabetesinduced retinal vascular dysfunctionAllen Clermont 1, 2 , Qunfang Zhou 2 , Takeshi Kita 1, 2 , Jia Liu 2 , Lloyd P.Aiello 1, 3 , Edward P. Feener 2 . 1 Beetham Eye Institute, Joslin DiabetesCenter, Boston, MA; 2 Vascular Cell Biology, Joslin Diabetes Center,Boston, MA; 3 Ophthalmology, Harvard Medical School, Boston,MA.Purpose: Components of the kallikrein-kinin system (KKS)including plasma kallikrein (PK) were shown to increase in thevitreous of patients with diabetic macular edema (DME). Whilepharmacological inhibition of PK reduces retinal vascularpermeability (RVP) in diabetic rats the effects of PK gene deficiencyon retinal function and structure has not been reported. The currentstudy examines the effect of PK gene deletion upon systemicparameters, RVP, and retinal thickeness in diabetic and nondiabeticmice and on the neovascular response induced by oxygen-inducedretinopathy (OIR).Methods: Diabetes was induced in 3 month old male and femaleKlKb1 knockout (KO) and wildtype (WT) mice by injection ofstreptozotocin (45mg/kg) with age-matched non-diabetic controlgroups. Retinal scans were obtained by SD-OCT (Bioptigen) after 3months. RVP was determined by extravasation of Evans blue dye(90mg/kg). PK, FXII, and their endogenous inhibitor, C1 inhibitor,were measured from plasma by western blot. OIR was induced by75% oxygen for 5 days in p7 mice.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyResults: PPK deficiency in DM mice did not alter body weight gainand blood glucose. FXII was increased and C1-INH was decreased inDM-WT by 40% compared to NDM-WT, indicating KKS activationin diabetes. Retinal RVP was increased by 203% in DM-WT versusNDM-WT (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biology(CTRL= 6878±2144 MI, glaucoma= 9815±4786 MI, p=0.027) andelevated levels were detected for anti-β-Amyloid/APP 4G8 (CTRL:3066±1423 MI; glaucoma=4161±1786 MI, p=0.056). Semiquantitativeanalysis of the immunohistochemical stainings in humanretinae: mean values (MV) of β-Amyloid 1-40 and 1-42 (12F4) weresignificantly up-regulated in the glaucomatous group (anti-β-Amyloid 1-40: CTRL= 0.7±0.4 MV, glaucoma= 1.8±0.7 MV;p=0.001; anti-β-Amyloid 1-42 (12F4): CTRL= 0.6±0.5 MV,glaucoma= 1.8±1.2 MV; p=0.037), while β-Amyloid/APP showedonly a trend toward up-regulation (4G8: CTRL= 1.3±0.8 MV,glaucoma= 1.8±0.7 MV; p=0.18) and APP did not differ. Moreover,β-Amyloid was visualized intracellular around the soma of RGC, aswell as extracellular, presumably on single axons.Conclusions: Antibody microarray and immunohistochemistryrevealed an increase of M. Alzheimer specific β-Amyloid inglaucomatous subjects. Therefore, β-Amyloid might be involved inthe pathology of glaucoma.Commercial Relationships: Nadine von Thun und Hohenstein-Blaul, None; Oliver W. Gramlich, None; Maarten B. Ruitenberg,Merz Pharmaceuticals GmbH (E); Andreas Gravius, MerzPharmaceuticals (E); Franz H. Grus, NoneProgram Number: 1140 Poster Board Number: D0044Presentation Time: 1:00 PM - 2:45 PMActivation of Prolyl Hydroxylase-2 Prevented High Glucose-Induced Endothelial Barrier DysfunctionXiaoling Liang 1 , Liqing Wei 1 , Gang Sun 1, 2 , Cheng Yang 1 , HuanjiaoZhou 1 , Lu Yang 1 , Peirong Lin 1 . 1 Department of Ophthalmology, StateKey Lab of Ophthalmology, Zhongshan Ophthalmic Center, SYSU,Guangzhou, China; 2 Affiliated Hospital of Weifang MedicalUniversity, Weifang, China.Purpose: To investigate the role of prolyl hydroxylase-2 (PHD-2)which catalyzes degradation of HIF-1α in high glucose-inducedendothelial barrier dysfunction in human retinal vascular endothelialcells (HRECs).Methods: Cultured cells were pretreated with PHD2 activator(R59949) or inhibitor (DMOG) for 1 hour followed by treatment with30mM high glucose for 72 hours. Mannitol at 30mM was used asosmotic control. Paracellular permeability assay was measured byusing FITC- dextran 70 kDa. Protein levels and arrangement patternof tight junctions (TJs) including occludin and zona occludensprotein-1 (ZO-1) were observed by Western blot andimmunofluorescence. The expression of PHD-2, hypoxia-inducedfactor (HIF)-1α and vascular endothelial growth factor (VEGF) weredetected by Western blot or ELISA.Results: Compared with control, high glucose increased theparacellular permeability of HRECs by 30.8 % (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyDistinct protein complex formation evokes insolubility of OPTNand mislocalization in iPSC-derived neural cells from E50K-POAG patientsYuriko Minegishi, Takeshi Iwata. Natl Hosp Org, Tokyo Medical Ctr,Meguro-ku, Japan.Purpose: Optineurin (OPTN) is a multifunctional protein and itsE50K mutation responsible for primary open angle glaucoma(POAG) and amyotrophic lateral sclerosis (ALS). We havepreviously reported our E50K transgenic mouse (E50K -tg ) exhibitsthinner retina and retinal ganglion cell loss, while the underlyingmolecular mechanisms have been unclear. Together with additionalphenotypes of E50K -tg retina and over expression studies, theendogenous OPTN and E50K mutant protein dynamics wasinvestigated by iPS Cell (iPSC) from E50K-POAG patients toelucidate the fundamental pathoetiology.Methods: E50K -tg retina were examined by using anti-GFAP, anti-OPTN, and anti-HA antibodies. The intracellular localization andprotein property of endogenous OPTN in wild type control and inE50K carrier were also examined by iPSCs and iPSC-derived neuralcells. FLAG-tagged OPTN and E50K were expressed in HEK293Tcells and protein complex formation/oligimerization was examinedby Native-PAGE and LC-MS/MS proteomics. Interaction withobtained candidates and further biochemical analysis were examinedby general molecular biological techniques.Results: E50K -tg retina exhibited significant reactive gliosis. InE50K -tg retina, E50K mutant protein is accumulated in OPL whereexhibited the severe cell death and atrophy. Overexpressed OPTNand E50K exhibited different hydrophobicity and only E50K isaccumulated in endoplasmic reticulum (ER) prior to Golgi transition.These distinct protein properties were also consistent with underendogenous condition in iPSCs and iPSC-derived neural cells fromE50K-POAG patient. Proteomics revealed distinct complexformation between OPTN and E50K. The treatment with specificinhibitor for E50K specific binding partner rescued the abnormalinsolubility.Conclusions: Underlying pathoetiology of E50K-POAG originatesfrom the alteration of protein complex formation that deteriorates theOPTN/E50K intracellular dynamics. This alteration is the probablecause of other terminal E50K phenotypes such as Golgi deformation,intracellular transport failure and cell deaths. We further investigateE50K phenotypes with endogenous conditions and E50K knock-inmouse, as well as the possibility of plasticity of E50K-glaucomatousphenotypes.Commercial Relationships: Yuriko Minegishi, None; TakeshiIwata, NoneSupport: JSPS Grant-in-Aid for Young Scientists (B)Program Number: 1143 Poster Board Number: D0047Presentation Time: 1:00 PM - 2:45 PMRole of Dopamine Deficiency in Visual Dysfunction in EarlystageDiabetic RetinopathyMoe H. Aung 1 , Moon K. Han 1, 4 , Han na Park 1 , Jane Abey 1 , Peter M.Thule 2, 5 , P M. Iuvone 1, 3 , Machelle T. Pardue 1, 4 .1 Neuroscience/Opthalmology, Emory University, Atlanta, GA;2 Endocrinology, Emory University, Atlanta, GA; 3 Pharmacology,Emory University, Atlanta, GA; 4 Rehab R&D Center of Excellence,Atlanta VA Medical Center, Atlanta, GA; 5 Biomedical LaboratoryResearch & Development, Atlanta VA Medical Center, Atlanta, GA.Purpose: Studies in diabetic patients and rodents consistentlyobserve subtle deficits in vision prior to the onset of vasculopathy;however, the underlying causes remain unclear. At such early stages,disruption in the retinal dopaminergic system has also been found. Asdopamine (DA) has recently been identified as crucial for visualfunction, the purpose of this study was to examine the role of DAdeficiency in early visual defects in diabetic retinopathy (DR).Methods: Hyperglycemia was induced in pigmented Long-Evansrats and C57BL/6 mice with STZ injection, a model of Type 1diabetes mellitus. Diabetes was confirmed by serially elevated bloodglucose (>250 mg/dl). Control and diabetic rats were sustained foreither 4 weeks or 12 weeks to evaluate DA levels with HPLC. Visualfunction with optokinetic tracking of each rat was assessed at the endof the study. In addition, hyperglycemic mice were maintained for 8weeks and then tested for the effects of acute DA receptor agonisttreatments on visual function. All mice received i.p. injections of adopamine D1-receptor agonist (SKF38393, 1mg/kg), vehicle, and adopamine D4-receptor agonist (PD168077, 1mg/kg). Visual functionwas evaluated with optokinetic tracking beginning 30 min after eachdrug injection.Results: Significant reduction in DA level (25-30%) was observed indiabetic rats at both time-points (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologypreviously described. Each of these proteins was chosen because theyrepresent crucial components of biochemical pathways involved inDR: inflammation, apoptosis, hypoxia and angiogenesis.Results: 4 of 33 measured proteins showed statistically significantdifferences between NPDR and PDR. These proteins were MatrixMetalloproteinase 9 (MMP-9) [p=.044, AUC=.7078], MatrixMetalloproteinase 14 (MMP-14) [p=.0122, AUC=.7062], VascularEndothelial Growth Factor 1175 (VEGF R2 Tyr1175) [p=.0088,AUC=.7024], and Bcl-2 thr56[(p=.0083, AUC=.7064]. All four ofthe same proteins were significantly up-regulated in NPDR versusPDR groups: MMP-9 (48.7%), MMP-14(32.9%), VEGF R2 Tyr1175(43.1%), & Bcl-2 thr 56 (33.4%). VEGF-A showed some upregulationbut fell short of statistical significance.Conclusions: Up-regulation of MMP-9, MMP-14, Activated VEGFR2 Tyr1175 and Bcl-2 thr 56 in PDR patients suggests a role in theconversion from NPDR to PDR. Hyperglycemia induced MMP-9activation stimulates apoptotic activity among pericytes, capillarycells and endothelial cells. It should be noted that studies haveimplicated these same biochemical markers during the transformationof many non-metastatic to metastatic cancers. This is the first timethat human vitreous samples have been sequentially sampled andinvestigated for proteomic changes along with clinical progression indifferent subsets of DR patients.Commercial Relationships: Joshua C. Hines, Ocular Proteomics(E); Stephanie M. Ecker, Ocular Proteomics LLC (E); Bert M.Glaser, Ocular Proteomics, LLC (E)Program Number: 1145 Poster Board Number: D0049Presentation Time: 1:00 PM - 2:45 PMCan Adiponectin be a Therapeutic Target for ProliferativeDiabetic Retinopathy?sulochana natarajan 1 , Subbulakshmi Chidambaram 1 , VidhyaSrinivasan 1 , Karthikka Palanisamy 1 , Pukhraj Rishi 2 , RadhakrishnanSelvi 1 . 1 Biochemistry and Cell Biology, Vision Research Foundation,Chennai, India; 2 Vitreoretinal, Medical Research Foundation,Chennai, India.Purpose: Adiponectin(APN) is a protein hormone havingantidiabetic and anti inflammatory properties. Its presence, in oculartissues, has been postulated, to have escaped from circulation due tothe dysfunctional BRB in retinal diseases. The purpose of this studyis to understand the origin of intraocular APN and its receptors,precisely measure vitreous APN in PDR and macular hole (MH),study its role in, in vitro angiogenesis and to screen small molecules,which can stimulate APN synthesis.Methods: All human studies were done with approval from IRB andconsent of the participants. Tissues derived from donor eye ballswere used for qPCR, immuno staining and western blot analysis.Vitreous samples were obtained from patients who underwentvitrectomy surgery for PDR and MH and used for APN measurementby ELISA. Human retinal endothelial cells (hREC), human choroidalendothelial cells (hCEC) and HUVEC were used for in vitroangiogenesis studies. Human adipocytes, hCEC and hREC were usedfor screening small molecules including amino acids and fatty acids,in order to select the one which stimulates APN secretion.Results: Expression of APN, AdipoR1 and AdipoR2 in retina,choroid, iris more specifically in the neural retina, photoreceptors,and endothelium was unambiguously identified by qPCR,immunostaining and western blot analysis. APN level in vitreous ofpatients with PDR (n=29) was found to be significantly (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyPharmacology and Toxicology, Georgia Health Sciences University,Augusta, GA; 3 Department of Experimental Medicine and Pathology,University of Rome, La Sapienza, Italy; 4 Department of PetroleumChemistry, American University of Nigeria, Yola, Nigeria.Purpose: Enhanced expression and activity of TLR4 in the diabeticretina has been implicated in the pathogenesis of DiabeticRetinopathy (DR). Presently, it is not known how diabetes promotesTLR4 activity in retinal and peripheral blood cells. Here weinvestigated the role of thioredoxin-dependent peroxidases,peroxiredoxin 1 (Prx1) and 2 (Prx2), in modulating TLR4 signalingin the diabetic retina as well as in retinal cells in culture.Methods: Retinas of streptozotocin-induced diabetic rats (STZ-rats,4 weeks hyperglycemia) and non-diabetic rats (ND rats) as well aspost-mortem retinas of control and DR donors (obtained fromGeorgia Eye Bank), were examined. Interactions between Prxisoforms with thioredoxin-1 (Trx-1) were identified by coimmunoprecipitationand Western blotting in retinal tissues of theexperimental groups shown above. The expression and TLR4interaction of the oxidative stress-induced sulfonic forms of Prx(Prx1-SO3H and Prx2-SO3H) were also measured in retinal tissues,using specific antibodies. Retinal endothelial cells isolated frombovine eyes (BRECs) were transfected with siRNA against Trx-1 andanalyzed for the expression of Prx-SO3H and TLR4 association.Following stimulation of ECs with high glucose (HG, 25mM,48hours), TLR4 expression, interaction with Prx-SO3H, andsubcellular localization were analyzed by immunocytochemistry andco-immunoprecipitation.Results: Retinas of STZ-rats and donors with DR displayedaugmented protein levels of hyperoxized Prx isoforms, and increasedTLR4 association to both Prx-SO3H isoforms. HG stimulated thetranslocation of TLR4 to the plasma membrane as well as an increasein TLR4/Prx-SO3H interaction in ECs. Inhibition of Trx-1, achievedby transfection of the BREC with specific siRNAs, resulted inincreased expression of both isoforms of Prx-SO3H and promotedincreased TLR4/Prx-SO3H interactions. This effect was partiallyblunted with blockade of Trx-1.Conclusions: We have identified Prx-SO3H as a novel chaperone forTLR4 in the diabetic retina. This correlates with our findings that theanti-oxidant capacities of the Trx and Prx systems are compromisedin the diabetic milieu. This study highlights that inhibition of the Trxsystem leads to an accumulation of stable, oxidized Prx isoformswhich crosstalk with TLR4, a striking new pathway which maycontribute to the subclinical inflammation seen in DR.Commercial Relationships: Folami Lamoke, None; Sean Shaw,None; AnnaLisa Montemari, None; Wan Jin Jahng, None;Manuela Bartoli, NoneSupport: NIH/NEI NRSA F31EY022289Program Number: 1148 Poster Board Number: D0052Presentation Time: 1:00 PM - 2:45 PMMethylglyoxal Activates Chronic ER Stress Mediated EpigeneticLoss of Nrf2/Keap1 Gene Regulation in Diabetic CataractousLensesPalsamy Periyasamy 1 , Masahiko Ayaki 2 , Keshore R. Bidasee 3 ,Toshimichi Shinohara 1 . 1 Ophthalmology and Visual Sciences,University of Nebraska Medical Center, Omaha, NE;2 Ophthalmology, Mita Hospital, Tokyo, Japan; 3 Pharmacology andExperimental Neuroscience, University of Nebraska Medical Center,Omaha, NE.Purpose: Methylglyoxal (MGO) is a highly cytotoxic metaboliteproduced from glucose metabolism. Earlier studies indicate thatMGO is attributed to aging and diabetic complications throughformation of advanced glycation end products. But evidence forMGO-induced endoplasmic reticulum (ER) stress and epigenetic lossof Nrf2/Keap1 dependent antioxidant protection is emerging indiabetes. This study investigated the mechanism by which MGOinduced the ER stress and by what epigenetic mechanisms MGOinactivated the Nrf2/Keap1 dependent antioxidant protection in lensepithelial cells (LECs) during cataractogenesis.Methods: Human and Nrf2 knockout (KO) mouse LECs werecultured with MGO to study the ROS production and cell death.MGO was intraperitoneally administered to control and diabetic Nrf2KO mice. The expression profiles of markers of ER stress,Nrf2/Keap1 dependent antioxidant system and DNA methylation anddemethylation enzymes were analyzed by qPCR and Westernblotting. ER-Ca 2+ release was assessed by live cell imaging.Proteasomal degradation was studied by using an inhibitor, MG-132.Promoter DNA methylation status was sequenced by bisulfitegenomic DNA sequencing.Results: We found that MGO stimulates ROS production and celldeath in human and Nrf2 KO mouse LECs. MGO also reduced theER-Ca 2+ content of human LECs. The mRNA and proteinexpressions studies confirmed that MGO treatment significantlyactivates the ER stress-specific genes and suppresses the Nrf2/Keap1dependent antioxidant genes and DNA methyltransferases. Also, thelevels of glyoxalase-1 and DNA demethylation enzyme,methylcytosine dioxygenase were over-expressed by MGO. DNAsequencing results revealed significant demethylated DNA in theKeap1 promoter of diabetic cataractous lenses than clear lenses.Similarly, MGO treated human LECs resulted in significantdemethylation of Keap1 promoter and had a notable increase inKeap1 mRNA and protein.Conclusions: This study revealed a possible mechanistic rationale bywhich MGO induces ER stress mediated Nrf2/Keap1 dependentantioxidant system failure and altered expressions of DNAmethylation and demethylation enzymes. Also, epigeneticmodification of Keap1 promoter by MGO stimulates Keap1expression, which increases proteosomal degradation of Nrf2. So,redox-balance is altered towards lens oxidation and cataractformation.Commercial Relationships: Palsamy Periyasamy, None;Masahiko Ayaki, None; Keshore R. Bidasee, None; ToshimichiShinohara, NoneSupport: RPB, EY018172, HL085061Program Number: 1149 Poster Board Number: D0053Presentation Time: 1:00 PM - 2:45 PMTo determine changes in levels of cytokines in the anteriorchamber (AC) fluid of eyes in patients with diabetic macularedema (DME)whom have been treated with repeated injections ofranibizumab (RBZ)Yasir J. Sepah 1 , Mohamed A. Ibrahim 1 , Alyssa Morimoto 2 , AbeerAkhtar 1 , Mauricio Maia 2 , Kyu H. Hong 2 , Montserrat Carrasco-Triguero 2 , Diana V. Do 1 , Menno van Lookeren Campagne 2 , QuanDong Nguyen 1 . 1 Retinal Imaging Research and Reading Center,Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD;2 Bioanalytical Sciences, Genentech, Inc.,, San Francisco, CA.Purpose: To determine changes in levels of cytokines in aqueousfluid of eyes of patients with diabetic macular edema (DME) treatedwith serial injections of ranibizumab (RBZ).Methods: Aqueous levels of the pro-inflammatory cytokine IL6 andthe angiogenic chemokine IL8 were measured in serial samples from131 patients, collected at baseline (BL), months (M) 3, 6, 9 and 12from eyes with DME receiving treatment in the READ-3 Study.Patients received 6 monthly mandatory injections starting at BL.Thereafter, starting at M6, patients were retreated with RBZ if: 1)©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologycentral foveal thickness reached 250 µm on Stratus TD-OCT; and/or2) intraretinal fluid was detected on Spectralis SD-OCT. The aqueoussamples were tested undiluted in an antibody-based multipleximmunoassay. Levels of IL6 and IL8 in the samples were quantitatedfrom seven-point standard curves using recombinant IL6 and IL8.Minimum quantifiable concentration for both analytes was 0.61pg/mL.Results: A decreasing trend was noted in the level of IL-8, a proangiogenicchemokine, from BL to M12 upon RBZ treatment. Themean changes (decrease from BL) at Ms 3, 6, 9 and 12 were 2.4pg/mL (p< 0.19), 3.4 pg/mL (p< 0.07), 4.7 pg/mL (p< 0.02) and 5.0pg/mL (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyConclusions: It is suggested that aberrant activation of Wnt signalingpathway in the diabetic retina may cause pathogenesis of diabeticretinopathy. Current study suggests that down-regulation of miR-184in diabetes may lead to activation of Wnt signaling in the retina,which plays an important role in the pathogenesis, development andprogression of diabetic retinopathy.Commercial Relationships: Yusuke Takahashi, None; Qian Chen,None; Anne Murray, None; Jian-Xing Ma, NoneSupport: NIH grants (EY018659, EY012231, EY019309 andP20GM104934) and OCAST (HR12-103)Program Number: 1152 Poster Board Number: D0056Presentation Time: 1:00 PM - 2:45 PMTHE PATHOGENIC ROLE OF DOWN-REGULATION OFPPAR-ΑLPHA EXPRESSION IN DIABETIC RETINOPATHYYang Hu 1 , Ying Chen 1, 2 , Lexi Ding 1 , Xuemin He 1 , Yusuke Takahashi 2 ,Rui Cheng 1 , Yang Gao 1 , Wei Shen 1 , Qian Chen 1 , Jian-Xing Ma 1, 2 .1 Physiology, OUHSC, Oklahoma City, OK; 2 Harold Hamm DiabetesCenter, OUHSC, oklahoma, OK.Purpose: Diabetic retinopathy is a common cause of vision losswhich results from progressive pathological changes including retinalinflammation, vascular leakage, neovascularization (NV) andfibrosis. Recent clinical studies have shown that PPAR-α agonist hastherapeutic effect on diabetic retinopathy. The purpose of this studyis to investigate the role of PPAR-α in diabetic retinopathy.Methods: Adult Brown Norway rats, PPAR-α knock-out (KO) miceand C57/BL6 mice were injected with Streptozotocin (STZ) to inducediabetes. PPAR-α, PPAR-β and PPAR-γ expression levels in theretina were compared between STZ-induced diabetic rats, Akita miceand db/db mice and their respective non-diabetic controls usingWestern blot analyses and real-time RT-PCR. PPARs protein andmRNA levels were measured in hTERT RPE cells and rat Müllercells treated with high glucose medium. Vascular permeability,leukostasis and trypsin digestion were performed in PPAR-α KOmice and C57/BL6 mice 3 months after the STZ injection.Adenovirus expressing PPAR-α was injected intravitreally intodiabetic rats, with adenovirus expressing GFP as control.Results: Both of mRNA and protein levels of PPAR-α weresignificantly decreased in the retinas of STZ-induced diabetic rats,Akita mice and db/db mice compared with non-diabetic controls, andin the hTERT RPE cells and rat Müller cells treated with highglucose. Retinal vascular permeability, adherent leukocytes,endothelial cell/pericyte ratio were significantly higher in STZinducedPPAR-α KO mice compared with diabetic C57/BL6 mice.Over-expression of PPAR-α significantly reduced vascularpermeability and attenuated over-expression of pro-inflammatoryfactors in the retinas of STZ-induced diabetic rats.Conclusions: PPAR-α defect plays an important role in diabeticretinopathy. PPAR-α is a novel therapeutic target for diabeticretinopathy.Commercial Relationships: Yang Hu, None; Ying Chen, None;Lexi Ding, None; Xuemin He, None; Yusuke Takahashi, None;Rui Cheng, None; Yang Gao, None; Wei Shen, None; Qian Chen,None; Jian-Xing Ma, NoneSupport: NIH grants EY018659, EY012231, EY019309,P20GM104934Program Number: 1153 Poster Board Number: D0057Presentation Time: 1:00 PM - 2:45 PMHigh Glucose and Diabetes Modulate Cellular ProteasomeFunction: Implications in the Pathogenesis of DiabetesComplicationsSaeed Yadranji-Aghdam 1 , Christine M. Sorenson 2 , Nader Sheibani 1 .1 Ophthalmology and Visual Sciences, University of Wisconsin,Madison, Madison, WI; 2 Pediatrics, University of Wisconsin,Madison, Madison, WI.Purpose: The precise link between hyperglycemia and its deleteriouseffects on the retinal and kidney microvasculature, and morespecifically loss of perivascular supporting cells including smoothmuscle cell/pericytes (SMC/PC), are not completely understood. Wehypothesized that differential cellular proteosome activity contributesto sensitivity of PC to high glucose-mediated oxidative stress andvascular rarefaction.Methods: Retinal endothelial cells (EC), PC, and choroidal EC wereprepared and cultured as previously described. NIH3T3 and 661Wcells were obtained from ATCC. These cells were cultured undervarious glucose conditions for 5 days prior to any analysis. Wild typeor Akita/+ mice with different duration of diabetes were obtainedfrom Jackson laboratories. For in vitro proteasome peptidase assays,the cells or tissue lysates (50µl of 1µg/µl) per assay were loaded intoeach well of a dark 96-well microplate and were probed with 100µMof each of the synthetic fluorogenic peptide substrates for caspaselike,trypsin-like, and chymotrypsin- and calpain-like proteaseactivity. The expression levels and localization of various proteinswere analyzed by Western blotting and immunofluorescence,respectively. The mRNA expression levels were determined byquantitative real time PCR.Results: Here we show that retinal EC have significantly higherproteasome peptidase activity compared to PC. High glucosetreatment (HGT) increased the level of total ubiquitin-conjugatedproteins in cultured retinal PC and EC, but not photoreceptor cells. Inaddition, in vitro proteasome activity assays showed significantimpairment of proteasome chymotrypsin-like peptidase activity inPC, but not EC. The HGT-mediated rise in ubiquitination wasattenuated by treatment with N-acetylcysteine. Furthermore, HGTincreased the half life and protein levels of the members of the 11Sregulatory subunits of mammalian proteasomes (PA28-a/-b proteins)in retinal PC, but not EC or photoreceptor cells. The PA28-a/-bprotein levels were also higher in the retina and kidney glomeruli ofdiabetic mice.Conclusions: Together our results demonstrate that high glucose hasdirect biological effects on cellular proteasome function in a celltype-specific manner, and this modulation might be protective againstcellular stress or damage induced by high glucose.Commercial Relationships: Saeed Yadranji-Aghdam, None;Christine M. Sorenson, None; Nader Sheibani, NoneSupport: NIH Grants EY016695, EY021357, EY016665, ADA grant1-10-BS-160, RPB, RRFProgram Number: 1154 Poster Board Number: D0058Presentation Time: 1:00 PM - 2:45 PMApelin silencing exacerbated retinal edema in BRVO ratsWang Xinlei, Yong Tao, Yanrong Jiang. Department ofOphthalmology, People’s Hospital, Peking University, & KeyLaboratory of Vision Loss and Restoration, Ministry of Education,Beijing, China.Purpose: We recently reported the neuroprotection of apelin peptidefor primary rat retinal Müller cells under hypoxia or glucosedeprivation.Therefore we hypothesized that its administration mightbe a promising protection for ischemic retina. In the present study,we targeted that whether apelin silencing was resistantor susceptibleto retinal edema in branch retinal vein occlusion (BRVO) rats.Methods: We engineer recombinant HIV-1-based lentiviral vectors(LV) capable of highly efficient and sustained apelin gene-knockdown. Apelin-RNAi LV and GFP lentiviral control vectors were©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologydelivered by intravitreal injection to the right eyes of Sprague-Dawley rats, whose contralateral eyes served as normal control.Apelin silencing efficiency was confirmed by in vivo fluorescentfundus imaging and western blot 2 weeks after injection. In both LVtransfected eyes, BRVO was induced by Ar laser photocoagulation ofretinal veins. Also induced were normal right eyes (BRVO group).Optical coherence tomography and electroretinography wereperformed in above four groups as quantitative assessments of retinaledema and visual function impairment. Meanwhile apelin and itsreceptor APJ expression were analyzed by immunofluorescencelabeling, real time PCR and western blot.Results: Apelin mRNA showed down-regulated, however APJmRNA and protein both up-regulated (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyLaila A A. Eissa, None; Mamdouh El-Shishtawy, None; GregoryI. Liou, NoneSupport: Egyptian Cultural and Educational Bureau (NME andGIL), Department of Defense DM102155 (GIL) and VisionDiscovery Institute (GIL)Program Number: 1156 Poster Board Number: D0060Presentation Time: 1:00 PM - 2:45 PMPeripheral Retinal Perfusion and Functional Analysis of Patientswith Ischemic Central Retinal Vein OcclusionCharles Wykoff 1, 2 , David M. Brown 1, 2 , Daniel Croft 1 . 1 RetinaConsultants of Houston, Houston, TX; 2 Weill Cornell MedicalCollege, Methodist Hospital, Houston, TX.Purpose: Progressive retinal perfusion and peripheral visual field(VF) changes in eyes with central retinal vein occlusions (CRVO) areincompletely understood. Our purpose was to quantify long-termtrends in capillary non-perfusion and VF patterns in patients withischemic CRVO treated with ranibizumab over 36 months.Methods: Wide-field fluorescein angiography (FA) and GoldmannVF (GVF) were performed serially in patients with ischemic CRVOwho were treated in the 36-month prospective Rubeosis Anti-VEGF(RAVE) trial evaluating intravitreal ranibizumab. All patientsreceived ranibizumab injections for 9 months followed by pro re nataranibizumab in months 12-36. Serial wide-field FA was performedwith a Heidelberg Spectralis HRA utilizing the Staurenghi contactlens. Areas of perfusion/non-perfusion were graded by maskedgraders and pixels quantified. GVF were assessed every 6 monthsand changes in the I-4e isopter were analyzed.Results: Twelve patients had sequential wide-field FA for 36months. At baseline, mean perfusion was 36.7% (106.4 disc areas) ofthe gradable field (mean: 290 disc areas, range 178-452). The area ofnon-perfusion increased in all patients with a mean loss ofapproximately 5% of retina area (loss of 14.5 mean disc areas ofperfusion) per 6 month period. In comparison to progressive loss ofperfusion, 3 patterns of GVF progression were identified: early loss(n=6), early gain with late loss (n=3), and stability (n=3). Progressivenon-perfusion and changes in GVF were most pronounced in the firstyear of the trial. Despite capillary and GVF losses, vision improvedin these eyes from a baseline of 23.1 best corrected Early TreatmentDiabetic Retinopathy Study (ETDRS) letters to 30.5 ETDRS letters(mean gain: +7.4 letters) at month 36.Conclusions: In this prospective CRVO trial, progressive capillaryloss was quantified. Similarly, peripheral visual field loss asmeasured by GVF progressed in most eyes (n=9). Changes inperfusion and GVF were particularly evident in the first year oftreatment. Despite perfusion and GVF losses in many patients, visionimproved, as the majority of these anatomic and functional changeswere peripheral.Wide field fluorescein angiogram of ischemic CRVO at baselineWide field fluorescein angiogram of the same ischemic CRVO after 2years of intravitreal ranibizumab treatment showing extensive loss ofretinal perfusionCommercial Relationships: Charles Wykoff, Genentech (R),Regeneron (R), Bayer (C); David M. Brown, RegeneronPharmaceuticals, Inc. (F), Regeneron Pharmaceuticals, Inc. (C),Regeneron Pharmaceuticals, Inc. (R), Bayer HealthCare (F), BayerHealthCare (C), Bayer HealthCare (R), Genentech (C), Roche (C),Alimera (C), Alcon (C), Novartis (C), Thrombogenics (C),Genentech (F), Roche (F), Thrombogenics (F), GSK (F), Alimera (F),Alcon (F), Allergan (F), Eli Lilly (F); Daniel Croft, NoneClinical Trial: NCT00406471Program Number: 1157 Poster Board Number: D0061Presentation Time: 1:00 PM - 2:45 PMThe Aqueous Humor Proteome in Patients with DiabeticRetinopathyRachida Bouhenni 1 , Deepak P. Edward 2, 3 , Sandeep Grover 4 , KakarlaChalam 4 , Abby L. Sewell 1 , Khaled K. Abu-Amero 5 . 1 Ophthalmology,Summa Health System, Akron, OH; 2 Ophthalmology, King KhaledEye Research Hospital, Riyadh, Saudi Arabia; 3 Ophthalmology,Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD;4 Ophthalmology, University of Florida, Jacksonville, FL; 5 Genetics,King Saud University, Riyadh, Saudi Arabia.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyPurpose: To identify differences in the protein composition of theaqueous humor (AH) of patients with diabetic retinopathy (DR)compared to patients without history of diabetes undergoing cataractsurgery (control).Methods: AH samples (n=6 for control and n=7 for DR) wereanalyzed by Liquid Chromatography/Tandem Mass Spectrometry(LC-MS/MS) and relative protein abundances were determined byspectral counting. G test followed by post hoc Holm Sidak was usedfor statistical analyses to determine significance in the differentialexpression of proteins between DR and control groups. Proteins wereclassified into functional groups using Scaffold 3.0.Results: Using stringent filtering criteria, seven proteins weresignificantly altered in the DR AH (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyAventix Animal Helth (F), Aventix Animal Helth (R), TherapeuticVision, Inc (F), Therapeutic Vision, Inc (F), Therapeutic Vision, Inc.(R), Threapeutic Vision, Inc (S), US 20090105269 (P)Support: NIH grant EY016730Program Number: 1160 Poster Board Number: D0064Presentation Time: 1:00 PM - 2:45 PMThe significant relationships between Fibronectin, Tenascin-Cand Periostin in the eyes of the patients with diabetic retinopathyYukio Sassa 1, 2 , Shigeo Yoshida 2 , Keijiro Ishikawa 2 , Ryo Asato 2 ,Mitsuru Arima 2 , Masanori Miyazaki 2 , Hiroshi Enaida 2 , Yuji Oshima 2 ,Toshihiro Kono 1 , Tatsuro Ishibashi 2 . 1 Ophthalmology, FukuokaUniversity Chikushi Hospital, Fukuoka, Japan; 2 Ophthalmology,Kyushu University, Fukuoka, Japan.Purpose: Periostin (PN) , Tenascine-C (TN) and Fibronectin (FN)are extracellular matrix (ECM)proteins, which compose fibrovascularmembrane (FMV) in ocular proliferative diseases. We havepreviously reported that PN expression increased in the vitreous ofpatients with proliferative diabetic retinopathy (PDR).We examinedthe correlations among TN, FN and PN in the vitreous of patientswith macular hole (MH), diabetic macular edema (DME) and PDR.Methods: Vitreous fluid was obtained from 198 individuals (MH of28 eyes in 28 individuals, DME of 20 eyes in 20 individuals, PDR of163eyes in 150 individuals) undergoing pars plana vitrectomy atFukuoka University Chikushi Hospital and Kyushu University (fromOctober 2007 to March 2011) in accordance with Human DiscardedSpecimen Research Protocol approved by institutional review boards.Undiluted samples were collected at the time of surgery, immediatelystored at -80 °C until used. The concentrations of PN/ TN/ FN weredetermined with ELISA method at the same time.Results: The concentrations of PN/ TN/ FN significantly increased inpatients with DME (average 1.48/ 10062/ 0.64 ng/ml) and PDR(average 12.16/ 4131/ 0.92 ng/ml) compared to those in MH (0.19/749/ 0.36 ng/ml) (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyProtein localization was assayed by immunohistochemistry fromretinal sections and by production of transgenic Xenopus laevistadpoles expressing EGFP-rab11a and/or mCherry-NUDC.Results: NUDC was identified as a protein that bound rhodopsin inassociation with rab11a. Reciprocal pull-down experimentsconfirmed the association between rhodopsin, rab11a, and NUDC.Rhodopsin C-terminal mutants Q344Ter and rhodopsin-EGFP boundneither rab11a nor NUDC. Immunohistochemistry revealed NUDClocalization in the inner segment of photoreceptors. Examination oftransgenic mCherry-NUDC expressing tadpoles revealed a punctatedistribution in the inner segment with minimal outer segmentlocalization. In tadpoles expressing EGFP-rab11a and mCherry-NUDC, there is overlap between EGFP and mCherry positive punctain the inner segment, in a distribution between the Golgi apparatusand the base of the outer segment.Conclusions: NUDC was shown to interact with rab11a andrhodopsin in preparations from mouse retina. This interaction wasabolished in two mouse lines expressing either truncated or extendedrhodopsin C-termini. Immunohistochemical and transgenicexpression experiments reveal a role for NUDC in rab11a positivevesicular trafficking in the inner segment.Commercial Relationships: Alecia K. Gross, None; Nicholas J.Reish, NoneSupport: NIH Grant EY019311, The EyeSight Foundation ofAlabama, The Karl Kirchgessner Foundation, E. Matilda ZieglerFoundationProgram Number: 1225Presentation Time: 9:00 AM - 9:15 AMAddressing the Role of PhLP in the Folding and Assembly of G-protein βγ dimerMaxim Sokolov, Satyabrata Sinha, Marycharmain Belcastro, XueliGao. Ophthalmology, West Virginia Univ Eye Institute,Morgantown, WV.Purpose: To get better understanding of the roles of the cytosolicchaperonin containing T-complex protein 1 (CCT) and its co-factorphosducin-like protein (PhLP) in the folding and assembly of theheterotrimeric G-protein βγ subunits complex.Methods: The interaction of PhLP and its short splice isoform(PhLPs) with CCT and G protein subunits was studied in both mousephotoreceptors and cell culture. The levels of mRNA and proteinswere determined by quantitative real-time PCR and Western blotting,respectively. The assembly of epitope-tagged Gβ1 and Gγ1 subunitswas monitored using a pull down assay.Results: We found that both PhLP and PhLPs form a complex withCCT, however, only PhLP facilitated the assembly of the Gβ1γ1dimer. Interestingly, while doing so, PhLP interacted primarily withGβ1 and not with the Gβ1γ1 complex. In contrast, PhLPs, whichlacks an important Gβ-binding domain, showed a tendency to form astable and apparently inactive tertiary complex with both CCT andthe Gβ1 trapped inside. Such a mode of action was consistent withthe pronounced cytotoxicity of PhLPs in rod photoreceptorsexpressing high levels of Gβ1. As a result, the levels of Gβ1 and,contingent on it, Gγ1 in rods became significantly reduced. Rathersurprisingly, Gαt1 became down-regulated under this circumstanceon the transcriptional level.Conclusions: Our data suggest that PhLP assists G proteinbiosynthesis by shuttling newly folded Gβ1 away from thechaperonin, priming it for binding with Gγ1, which in turn, triggersPhLP’s own release. A splice isoform of PhLP, however, hinders thisprocess via a complex mechanism.Commercial Relationships: Maxim Sokolov, None; SatyabrataSinha, None; Marycharmain Belcastro, None; Xueli Gao, NoneSupport: NIH Grant EY019665, RPBProgram Number: 1226Presentation Time: 9:15 AM - 9:30 AMLight-Dependent Phosphorylation of BBS5 in Photoreceptorsand Its Interaction with Arrestin1Tyler S. Smith, Donald R. Dugger, Susan N. Bolch, J HughMcDowell, W Clay Smith. Ophthalmology, University of Florida,Gainesville, FL.Purpose: We have previously demonstrated that Bardet-BiedlSyndrome 5 (BBS5) protein is one member of the phosphoproteomein photoreceptors. Here we provide an analysis of the kinase involvedin BBS5 phosphorylation and investigate the functional effect ofBBS5 phosphorylation on its interaction with other proteins.Methods: BBS5 was cloned from murine retinal mRNA, andheterologously expressed in bacteria with either a His(6) tag or as afusion protein with glutathione-S-transferase (GST). BBS5 wasphosphorylated in situ by endogenous kinases; phosphorylation ofBBS5 was tested in vitro using a panel of six kinases. Interaction ofBBS5 with arrestin was assessed in vitro via immunoprecipitation.Tissue localization of BBS5 was examined in cryosections ofXenopus laevis retina using a monoclonal antibody prepared againstheterologously expressed BBS5.Results: BBS5 is phosphorylated in a light-dependent manner,showing a threshold of phosphorylation that correlates with theinitiation of arrestin1 translocation. Phosphorylation of BBS5 canalso be stimulated in vivo by phorbol-12,13-diacetate, a proteinkinase C agonist. In vitro analysis demonstrates that BBS5 isphosphorylated by PKC, but not by protein kinase A, cGMPdependentprotein kinase, casein kinase I or II, orcalcium/calmodulin-dependent protein kinase II. Arrestin1 coimmunoprecipitateswith BBS5/GST; phosphorylation of BBS5reduces the interaction between arrestin1 and BBS5. Under identicalconditions, arrestin1 shows negligible co-immunoprecipitation withBBS8. In photoreceptors, BBS5 principally localizes to the axonemesof rods and cones, photoreceptor inner segments, and synapticregions.Conclusions: Phosphorylation of BBS5 in retina is initiated by light,apparently through PKC activation based on in vitro studies.Activation of PKC in situ using phorbol ester leads to BBS5phosphorylation, supporting this conclusion. The three-foldobservation that (1) BBS5 directly interacts with arrestin1, (2) thatphosphorylation of BBS5 reduces the affinity of arrestin1 for BBS5,and (3) that BBS5 principally localizes to the axoneme suggests thatphosphorylation of BBS5 may play a role in regulating lightdependenttranslocation of arrestin1.Commercial Relationships: Tyler S. Smith, None; Donald R.Dugger, None; Susan N. Bolch, None; J Hugh McDowell, None; WClay Smith, NoneSupport: NIH Grants EY014864, EY006225, EY021721 andResearch to Prevent BlindnessProgram Number: 1227Presentation Time: 9:30 AM - 9:45 AMThe Essential Role of CCT in Outer Segment MorphogenesisSatyabrata Sinha 1 , Marycharmain Belcastro 1 , Seongjin Seo 2 , MaximSokolov 1 . 1 Ophthalmology, West Virginia University, Morgantown,WV; 2 Ophthalmology and Visual Sciences, University of IowaCollege of Medicine, Iowa, IA.Purpose: Previously, we demonstrated that the development of therod outer segment, a sensory cilia responsible for light detection, iscrucially dependent on the normal function of the cytosolicchaperonin containing T-complex protein 1, CCT. Our goal here was©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyto identify the underlying molecular mechanism for such aphenotype.Methods: The activity of CCT in photoreceptors and cell culture wassuppressed by overexpressing a short splice isoform of phosducinlikeprotein, PhLPs. Cellular morphology was analyzed by lightmicroscopy and electron microscopy. Protein-protein interactions ofCCT were determined using an immunoprecipitation assay coupled toLC/MS/MS. Protein expression levels were studied byimmunofluorescence and Western blotting.Results: We identified several proteins that were severely downregulatedin response to CCT suppression and involved in outersegment morphogenesis. Those included Bardet-Biedl Syndromeproteins 2, 5 and 7, essential subunits of the cilia BBSome complex,and oxysterol-binding protein 2 (OSPB2), implicated in non-vesicularlipid trafficking. Interestingly, the reduction of rhodopsin andperipherin appeared to be secondary event, as both proteins were stilltargeted to the outer segment remnants.Conclusions: Our data provide evidence that CCT may potentially beinvolved in posttranslational processing of BBS 2, BBS5, BBS7, andOSPB2, which thus underlines its crucial importance in outersegment morphogenesis.Commercial Relationships: Satyabrata Sinha, None;Marycharmain Belcastro, None; Seongjin Seo, None; MaximSokolov, NoneSupport: NIH Grant EY019665, RPBProgram Number: 1228Presentation Time: 9:45 AM - 10:00 AMThe farnesylated small GTPase RAB28 is mutated in autosomalrecessive cone-rod dystrophySusanne Roosing 1, 2 , Klaus Rohrschneider 3 , Avigail Beryozkin 4 ,Nicole Weisschuh 5 , Susanne Kohl 5 , Bernd Wissinger 5 , Eyal Banin 4 ,Frans P. Cremers 1, 2 , Anneke I. Den Hollander 1, 6 . 1 Human Genetics,Radboud University Nijmegen Medical Centre, Nijmegen,Netherlands; 2 Nijmegen Centre for Molecular Life sciences,Nijmegen, Netherlands; 3 Department of Ophthalmology, Universityof Heidelberg, Heidelberg, Germany; 4 Department ofOphthalmology, Hadassah-Hebrew University Medical Center,Jerusalem, Israel; 5 Molecular Genetics Laboratory, Institute forOphthalmic Research, Centre for Ophthalmology, Tuebingen,Germany; 6 Department of Ophthalmology, Radboud UniversityNijmegen Medical Centre, Nijmegen, Netherlands.Purpose: The majority of genetic causes for autosomal recessive (ar)cone-rod dystrophy (CRD) are currently unknown. Therefore, weemployed a combined approach of homozygosity mapping andexome sequencing to identify new genes for arCRD.Methods: In a German arCRD family with three affected siblings,homozygosity mapping was performed using Affymetrix 250K SNPmicroarrays. DNA samples of two affected individuals underwentexome sequencing using Agilent’s SureSelect Human All Exon v.2Kit on a SOLiD4 sequencing platform. Sanger sequencing of theRAB28 gene was performed in 617 additional unrelated individualswith CRD or cone dystrophy, and in families with conspicuouslylarge homozygous regions assessed in SNP data available through theEuropean Retinal Disease Consortium. Identified mutations werescreened in ethnically matched controls. Ophthalmic examinationsincluded ERG, perimetry, OCT, FAF, and fundus photography.Results: Exome sequencing revealed a homozygous nonsensemutation in RAB28 (c.565C>T;p.Q189*) in all three affectedindividuals of the German arCRD family. In addition, a homozygousnonsense mutation (c.409C>T;p.R137*) was identified in twoaffected members of a consanguineous arCRD family of MoroccanJewish ancestry. Both mutations were not identified in 176 and 118ethnically matched controls, respectively. The five affectedindividuals of both families presented with hyperpigmentation in themacula, progressive loss of the visual acuity, atrophy of the retinalpigment epithelium, and severely reduced cone and rod responses onthe electroretinogram.Conclusions: RAB28 encodes a member of the Rab subfamily of theRAS-related small GTPases. Alternative RNA splicing yields threepredicted protein isoforms with alternative C-termini, which are alltruncated by the nonsense mutations identified in this study. Opposedto other Rab GTPases which are generally geranylgeranylated,RAB28 is predicted to be farnesylated. Interestingly, mutations inAIPL1, encoding a chaperone of farnesylated proteins, werepreviously found in individuals with ar Leber congenital amaurosisand autosomal dominant CRD. Analogous to the function of otherRAB family members, RAB28 might be involved in ciliogenesisand/or opsin transport in photoreceptor cells. This study reveals acrucial role for RAB28 in photoreceptor function, and suggests thatmutations in other Rab proteins may also be associated with retinaldystrophies.Commercial Relationships: Susanne Roosing, None; KlausRohrschneider, None; Avigail Beryozkin, None; NicoleWeisschuh, None; Susanne Kohl, None; Bernd Wissinger, None;Eyal Banin, None; Frans P. Cremers, None; Anneke I. DenHollander, NoneSupport: Foundation Fighting Blindness USA (grants BR-GE-0510-04890RAD and C-GE-0811-0545-RAD01)235 Ocular Disease Expression, Proteomics, Biomarkers, andPolymorphismsMonday, May 06, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 1574-1595/D0001-D0022Organizing Section: Biochemistry/Molecular BiologyProgram Number: 1574 Poster Board Number: D0001Presentation Time: 8:30 AM - 10:15 AMNew Potential Biomarker of Neurofibromatosis Type I,discovered with Multi-Spectral Imaging (MSI) of the RetinalPigment Epithelium (RPE) and ChoroidDorothy Hitchmoth 4, 1 , Jerome Sherman 2, 3 . 1 Surgery, Department ofVeterans Affairs, White River Junction, VT; 2 Clinical Science,SUNY College of Optometry, New York, NY; 3 SUNY Eye Institute,State University of New York, New York, NY; 4 Affiliate ResidencyPrograms, New England College of Optometry, Boston, MA.Purpose: The purpose of our research was to document the natureand type of choroidal lesions that were detected with a novelcommercially available imaging device.Methods: A retrospective analysis of the examination records andimages of 210 consecutive patients who were imaged with theAnnidis RHA. Ten sequential images were obtained, each with adifferent color LED with a unique spectrum. Seven of 10 of theseLEDs were in the red and infra-red part of the spectrum. Visiblelesions greater than .5 DD on one or more of the 10 images wereidentified. Patient records of those with such documented lesionswere evaluated in detail. Ultra-widefield (UWF) color images(Optos) were available in each case. In select cases, UWF FA, AF,Spectralis OCT, AF, FA, Topcon OCT and color images were alsoavailable.Results: 1)There was one patient with approximately 15 lesions ineach eye with corresponding dermatological lesions consistent withNeurofibromatosis Type I (NFI-1). The lesions were highly definedby MSI with red and infra-red LEDs but were invisible on©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyophthalmoscopy, SD-OCT, and high resolution Magnetic ResonanceImaging of the orbits.2) Five patients had choroidal lesions which were visible withophthalmoscopy and hence typical of choroidal nevi 3) Three patientshad choroidal lesions that were invisible to ophthalmoscopy andhence not traditional nevi or other known choroidal lesions. One ofthese 3 patients had skin lesions highly suggestive of NF1.Conclusions: Multi-spectral imaging (MSI) is a novel way toexamine the retina and choroid. This technique revealed choroidallesions associated with Neurofibromatosis 1 which are invisible toophthalmoscopy and many other imaging devices. Such lesions havebeen rarely, if ever, documented in the world’s literature. Wehypothesize that these choroidal lesions revealed by MSI, that wereclinically invisible on SD-OCT and high resolution MRI, mayrepresent a new biomarker for Neurofibromatosis 1. There are at leasta dozen NFI -1 interventional drug studies presently underway.Whether these lesions change with treatment with any of theseexperimental drugs remains to be determined.were above or below the mean ≥ 1 SD and p values ≤ 0.05.Results: About 284 proteins were quantified per mouse strain. Moreproteins were altered in the choroid fraction (n = 44) than in theretina (n = 8), with the majority detected in TIMP3 KO mice. Moreproteins were elevated in KO mice than decreased while very fewproteins were elevated in KI mice and none were common to thoseelevated in KO mice. All TIMP3 KO and KI mouse strains exhibitedsignificantly reduced amounts of crystallins in both the retina andchoroid fraction, including α-crystallin B, β-crystallin B2 and α-crystallin A. Homozygous KI mice also exhibited significantlyreduced levels of several collagens, fibrilin-1 and TGFβ-induced igh3in the choroid fraction.Conclusions: Protein changes observed in both TIMP3 KO and KImice are more likely due to TIMP3 dysfunction than collateralgenetic alterations. Our results suggest lower crystallin levels are aconsequence of TIMP3 dysfunction. Crystallins are inhibitors ofapoptosis. Reduced crystallins may facilitate inflammatory responsesand cellular degeneration in TIMP3 dysfunctional animals, includingheterozygous TIMP3 KI mice which exhibit an SFD-like phenotype.Other proteins decreased in homozygous KI mice implicatedysregulation of extracellular matrix homeostasis.Commercial Relationships: Geeng-Fu Jang, None; Alecia Cutler,None; Lei Zhang, None; John S. Crabb, None; Heidi Stoehr,None; John W. Crabb, SKS Ocular (P), Allergan (C); Bela Anand-Apte, 7 183 256 B2 (P)Support: EY021840, EY022134, EY016490, CA10641505, BIMR1104, FFB Center Grant, RPB Challenge GrantAnnidis-RHA Multi-spectral ImagingCommercial Relationships: Dorothy Hitchmoth, Annidis, Corp.(C); Jerome Sherman, Optos, Inc. (F), Optos, Inc. (C), Optos, Inc.(R), Annidis (C), Annidis (R), Zeiss (R)Support: no supportProgram Number: 1575 Poster Board Number: D0002Presentation Time: 8:30 AM - 10:15 AMQuantitative Proteomic Analysis of TIMP3 DysfunctionGeeng-Fu Jang 1 , Alecia Cutler 1 , Lei Zhang 1 , John S. Crabb 1 , HeidiStoehr 2 , John W. Crabb 1 , Bela Anand-Apte 1 . 1 Cole Eye Institute &Lerner Research Institute, Cleveland Clinic, Cleveland, OH; 2 Instituteof Human Genetics, University of Regensburg, Regensburg,Germany.Purpose: Polymorphisms in tissue inhibitor of metalloproteinase 3(TIMP3) have been associated with Sorsby fundus dystrophy (SFD)and age-related macular degeneration. Toward a molecularunderstanding of TIMP3 dysfunction, we pursued quantitativeproteomic analyses of the retina and choroid from TIMP-3 knockout(KO) mice and knockin (KI) mice expressing the TIMP3 S156Cmutation that causes SFD.Methods: Soluble proteins from isolated retinas and choroidcontainingposterior globes from TIMP3 KO mice (n = 5 mice), KIhomozygotes (n = 5), KI heterozygotes (n = 4), and wild-type mice (n= 7) were quantified by iTRAQ technology. Protein was digestedwith trypsin, peptides labeled with iTRAQ tags, fractionated bystrong cation exchange chromatography, and peptides were analyzedby LC MS/MS. Proteins were identified using the Swiss-Proteindatabase and quantified using code written in R. Proteins quantifiedwith ≥ 2 unique peptides/protein in ≥ 3 mice/strain were consideredsignificantly altered if average ratios relative to wild-type tissuesProgram Number: 1576 Poster Board Number: D0003Presentation Time: 8:30 AM - 10:15 AMAssociation of the polymorphism of the 5' UTR region of theHBD1 gene in patients with infectious blepharitisHector J. Perez-Cano 1 , Ingrid V. Gonzalez Leon 2 , Oscar Fernandez-Vizcaya 2 , Justine Nolasco-Lopez 1 , Jean R. Clemenceau 1 , AtzinRobles-Contreras 1 . 1 Biomedical Research Center, HospitalFoundation "Nuestra Senora de la Luz", Mexico, Mexico; 2 cornea,hospital foundation "Nuestra Señora de la Luz", Mexico, Mexico.Purpose: Recently, there is a particular interest in the participation ofsingle nucleotide polymorphisms (SNPs) in DEFB1 gene andinvolvement in susceptibility to infections. Two polymorphisms havebeen associated in the 5'UTR region of HBD1 gene,-52G/A and -44C/G, and have been reported -44C and -55G alleles associated withincreased risk of HIV infection and respiratory infections withCandida in diabetic patients. However, little is known about theinvolvement of these polymorphisms in ocular infections. Wepropose that polymorphisms in the 5'UTR region in HBD1 gene isassociated with infectious blepharitisThere are not information about infectious blepharitis and itsrelationship to the 5'UTR polymorphism DEFB1 gene for that reasonafter conducting an epidemiological study to identify the mostcommon pathogens that occur in infectious blepharitis in patientsattending at Hospital Foundation Nuestra Senora de la Luz wasperformed an analysis looking association between infectiousblepharitis and 5'UTR DEFB1 gene polymorphism.Methods: One hundred patients with infectious blepharitis, and thirtyone not infected subjects, as controls, were studied. We obtainedsamples from scraping palpebral for DNA extraction. The 5'UTRregion of HBD1 gene was amplified by PCR, and then, the PCRproducts were purified and analyzed the presence of SNPs -52G/Aand -44C/G by direct sequencing.Results: The allelic frequency of -44C was 60% and -52G was 50%of the 5'UTR region of HBD1 gene in patients, and in controls 50%and 60%, respectively. Our results not showed significantly©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologydifferences between patients and control subjects (Chi-square 2.02,p=0.077; Chi-square 2.02, p=0.155 respectively).Conclusions: In this study of Mexican population on infectiousblepharitis and polymorphism of the gene 5'UTR DEFB1 noassociation was found, however, it is necessary to conduct severalstudies to find the risk factors for developing infectious blepharitiswhich might be involved systemic diseases affecting the immunemechanisms.Commercial Relationships: Hector J. Perez-Cano, None; IngridV. Gonzalez Leon, None; Oscar Fernandez-Vizcaya, None;Justine Nolasco-Lopez, None; Jean R. Clemenceau, None; AtzinRobles-Contreras, NoneProgram Number: 1577 Poster Board Number: D0004Presentation Time: 8:30 AM - 10:15 AMComparative Analysis of Donor Medical History and DiseaseAttributesKieron Torres, Verity F. Oliver, Raymond Enke, Jin Song, ShannathL. Merbs. Ophthalmology, Johns Hopkins Wilmer Eye Institute,Baltimore, MD.Purpose: Age-related macular degeneration (AMD) and primaryopen angle glaucoma (POAG) are among the leading causes ofblindness worldwide. We aim to identify DNA methylation patternsassociated with AMD and POAG through a genome-wide analysis ofaffected cells being collected from control or diseased human eyes.This purpose of this study was to determine the accuracy of the tissuebank diagnosis by photographic and histologic methods.Methods: Adult human eyes were obtained from NDRI and theMaryland Anatomy Board. Donor eyes were labeled as either control(no history of retinal disease, POAG or AMD), POAG or AMDbased on what was listed under diagnoses in the provided donormedical history. POAG or AMD eyes that had additional informationincluding duration of disease, type of AMD, or treatment details wereconsidered as confirmed, and eyes without as unconfirmed. Diagnosisof POAG was verified by grading optic nerve cross-sections todetermine the presence of axonal loss. AMD was verified by gradingretinal images taken during eye dissection for posterior funduspathology.Results: 24 control, 13 POAG and 14 AMD donors were identified.23 of 24 controls had normal fundus photos and no axonal loss in atleast 1 eye. The majority of the POAG (11) and AMD (10) donorswere confirmed by additional history from NDRI. 6 of 11 confirmedPOAG individuals were verified by axonal loss. 6 of 10 confirmedAMD donors were verified by photo grading, and of these, 5 had atleast one eye without axonal loss. Additional confirmation of AMDby histologic section is ongoing.Conclusions: To facilitate short preservation times, tissue banksoften do not have access to comprehensive donor medical histories.Even donor eyes with an accurate ocular history may not havedisease-associated pathology if obtained within early stages of thedisease or if treatment had slowed progression. Additionally, controleyes may have undiagnosed or unrecorded POAG or AMD, makingthem inappropriate as controls. Our study shows that diagnoses needto be verified by histologic and photographic methods.Commercial Relationships: Kieron Torres, None; Verity F.Oliver, None; Raymond Enke, None; Jin Song, None; Shannath L.Merbs, NoneSupport: NEI R01EY020406Program Number: 1578 Poster Board Number: D0005Presentation Time: 8:30 AM - 10:15 AMTreatment of Usher syndrome with antisense oligonucleotidesJennifer J. Lentz 1 , Mette Flaat 1 , Francine M. Jodelka 2 , AnthonyHinrich 2 , Yongdong Zhou 1 , Kate McCaffery 2 , Dominik M. Duelli 3 ,Nicolas G. Bazan 1 , Frank Rigo 4 , Michelle L. Hastings 2 .1 Neuroscience Center, LSUHSC, New Orleans, LA; 2 Cell Biologyand Anatomy, Rosalind Franklin University, North Chicago, IL;3 Cellular and Molecular Pharmacology, Rosalind FranklinUniversity, North Chicago, IL; 4 Isis Pharmaceuticals, Carlsbad, CA.Purpose: Usher syndrome (Usher) is the leading genetic cause ofcombined deafness and blindness. Type 1 Usher (Usher 1) ischaracterized by profound hearing impairment and vestibulardysfunction at birth, and the development of retinitis pigmentosa(RP) in early adolescence. The 216G>A (216A) mutation in USH1Ccreates a cryptic splice site that is used preferentially over the correctsite and results in a truncated harmonin protein. We created a mousemodel for Usher 1C by knocking in the 216A mutation. 216AA miceexhibit circling behavior indicative of severe vestibular dysfunctionand deafness, have retinal dysfunction by 1 month of age and beginto lose photoreceptors after 6 months of age. This mouse modelprovides an opportunity to test the feasibility of correcting thedisease-associated genetic defect using antisense oligonucleotides(ASOs).Methods: Antisense oligonucleotides (ASOs) were used to targetUsh1c mutations in vitro and in vivo by systemic or local injection.Correction of splicing was evaluated by RT-PCR and western blot.Immunofluorescence was used to analyze Harmonin expression andASO localization. Hearing and vestibular function were evaluated byauditory-evoked brain stem response (ABR) and open-field chamberanalysis, respectively. Visual function was evaluated byelectroretinogram (ERG) analyses and optical coherence tomography(OCT) imaging was used to examine ocular structures.Results: ASOs effectively corrected splicing of 216A RNA in anUsher patient cell line, and in the cochleae and retinas of 216AAmice when delivered systemically or locally. Cell-free and cellularassays also demonstrated that ASOs targeted to the Ush1c.238dupCmutation result in in-frame skipping of exon 3. Treatment with 216AtargetedASOs to neonate mice corrected protein expression with animprovement in harmonin localization in the hair cells andphotoreceptors; rescued vestibular and hearing function, anddemonstrated a small improvement in visual function.Conclusions: Our results show that ASOs can effectively targetUsh1c mutations both in vitro and in vivo. These results suggest thetherapeutic potential of ASOs in Usher syndrome and other diseasesof the ear and eye.Commercial Relationships: Jennifer J. Lentz, None; Mette Flaat,None; Francine M. Jodelka, None; Anthony Hinrich, None;Yongdong Zhou, None; Kate McCaffery, None; Dominik M.Duelli, None; Nicolas G. Bazan, None; Frank Rigo, None;Michelle L. Hastings, Isis Pharmaceuticals (P)Support: NIH P30 GM103340, NIH/NEI EY005121, Research toPrevent BlindnessProgram Number: 1579 Poster Board Number: D0006Presentation Time: 8:30 AM - 10:15 AMGenetic Heterogeneity Among Patients with Pericentral RetinitisPigmentosaJason Comander 1, 2 , Aliete Langsdorf 1 , Shyana Harper 2 , CarolWeigel-DiFranco 2 , Mark B. Consugar 1 , Michael Sandberg 2 , XiaowuGai 3 , Joseph A. White 1 , Eliot L. Berson 2 , Eric A. Pierce 1, 2 . 1 OcularGenomics Institute, Massachusetts Eye and Ear Infirmary, HarvardMedical School, Boston, MA; 2 Berman-Gund Laboratory for theStudy of Retinal Degenerations, Massachusetts Eye and EarInfirmary, Harvard Medical School, Boston, MA; 3 Center forBiomedical Informatics, Department of Molecular Pharmacology and©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyTherapeutics, Loyola University Chicago Health Sciences Division,Maywood, IL.Purpose: Pericentral retinitis pigmentosa (RP) is an atypical form ofRP that affects the near-peripheral retina first and tends to spare thefar periphery (Sandberg et al, AJO, 2005). Mutations in the TOPORSgene have been described in selected pericentral RP families (e.g.Selmer et al, Acta Ophthalmol, 2010). This study was performed tofurther define the overall mutation spectrum in patients with thisphenotype. It remains to be determined to what extent pericentral RPcould have similar genetic causes as typical RP, or whether thedifferent phenotype of pericentral RP is a reflection of mutations innovel genes or pathways.Methods: We identified a cohort of 34 patients from separatefamilies with pericentral RP based on a comprehensive analysis oftheir retinal phenotype. DNA samples were collected from thesepatients, along with 14 samples from affected and unaffectedrelatives. A multistage gene discovery approach was used, startingwith selective exon capture and next generation sequencing (NGS) tosequence 196 genes previously associated with inherited retinaldegenerations. Then the samples without mutations in known genesunderwent analyses of copy number variation (CNV) for deletiondetection, followed by whole exome sequencing for novel genediscovery.Results: The selective exon capture/NGS approach provided a meandepth of coverage of the targeted sequences of >124 fold, and >97%of the targeted sequences were covered with ≥10x depth. Mutationsprovisionally responsible for disease were found in 5 of the first 12patients analyzed. These include mutations in RHO (4 patients) andPDE6B (1 patient). No mutations were found in TOPORS. Twomutations have been previously identified in patients with morecommon forms of typical RP and four mutations appeared to benovel. Sequencing analyses in the remainder of the samples isongoing.Conclusions: Initial results suggest that some patients withpericentral RP have mutations in the same genes that cause morecommon forms of typical RP. Further molecular genetic subtyping ofthe entire cohort, including ongoing whole exome sequencing, willbetter define the genetic heterogeneity that appears to exist withinthis clinical phenotype.Commercial Relationships: Jason Comander, None; AlieteLangsdorf, None; Shyana Harper, None; Carol Weigel-DiFranco,None; Mark B. Consugar, Agilent Technologies, Inc. (R); MichaelSandberg, None; Xiaowu Gai, None; Joseph A. White, None; EliotL. Berson, None; Eric A. Pierce, NoneSupport: EY021910 (EAP), grants from the Foundation FightingBlindness (EAP and ELB), and NEI K12 EY16335 (JC)Program Number: 1580 Poster Board Number: D0007Presentation Time: 8:30 AM - 10:15 AMDistinct molecular mechanisms responsible for differentretinopathies caused by CRX mutations DNH and LFHAlan Zhang, Nicholas M. Tran, Xiaodong Zhang, Shiming Chen.Ophthalmology, Washington University St. Louis, st louis, MO.Purpose: Mutations in the transcription factor CRX are associatedwith dominant retinopathies of varying severity in humans. Wecreated two Knock-IN mouse models for CRX disease: Dominant-Negative High Expression (DNH) carries a 2-bp deletion at codon168 resulting in a truncated CRX protein lacking the transactivationdomains; Loss-of-function High Expression (LFH) carries an aminoacid substitution, R90W in the DNA binding domain, compromisingCRX’s ability to bind DNA. The Crx-DNH mouse models severedominant Cone-Rod Dystrophy, while Crx-LFH models a recessiveLeber Congenital Amaurosis. This study focuses on the distinctbiochemical mechanisms by which the DNH and LFH proteins affectCRX target gene expression to cause distinct forms of CRXassociateddisease.Methods: Quantitative real time PCR (QRT-PCR) detectedexpression changes for selected CRX target genes in mutant vs WTretinas. Changes in DNA binding and transactivation activities ofmutant vs WT CRX proteins were measured by retinal chromatinimmunoprecipitation (ChIP), electrophoretic mobility shift assays(EMSA), and Rhodopsin and Crx promoter dual luciferase reporterassays in transiently transfected HEK293 cells.Results: Crx-DNH/+ mice show severely decreased photoreceptorgene expression along with impaired rod and cone function and rapidretinal degeneration, while Crx-LFH/+ mice show normal retinalgene expression and function. Dual-luciferase assays revealed thatneither LFH nor DNH protein can activate target gene expression invitro and that DNH actively impairs WT CRX-mediatedtransactivation. EMSA showed DNH but not LFH binds DNA invitro. Surprisingly, ChIP showed that DNH and LFH proteins bothassociate with the chromatin of CRX target genes in vivo. Thissupports our QRT-PCR findings that DNH/DNH cannot activateCRX target gene expression but LFH/LFH allows a low level oftranscription.Conclusions: DNH causes dominant disease by binding to DNA andinterfering with WT CRX’s transactivation ability. LFH loses DNAbinding and transactivation activity in vitro but can still associatewith DNA in vivo to exert hypomorphic regulatory function. CrxDNH and LFH mice represent two mechanistically distinct models ofCRX disease with vastly different pathologies, providing excellentmodels for testing novel therapeutic approaches.Commercial Relationships: Alan Zhang, Howard Hugh MedicalInstitute (F), National Institute of Health (F), Washington Universityin St. Louis (E); Nicholas M. Tran, None; Xiaodong Zhang, None;Shiming Chen, NoneSupport: NIH EY012543-10S (to SC), EY02687 (to WU-DOVS),T32 Vision Sciences Pre-doctoral Training Grant (to NMT), WU-HHMI undergraduate student travel award (to AZ), RBP Lew R.Wasserman Award (to SC) and unrestricted funds (to WU-DOVS)and Foundation Fighting Blindness (to SC)Program Number: 1581 Poster Board Number: D0008Presentation Time: 8:30 AM - 10:15 AMComprehensive genetic analysis of an Usher I patient cohortKinga M. Bujakowska 1 , Emily Place 1 , Mark B. Consugar 1 , Daniel G.Taub 1 , Aliete Langsdorf 1 , Carol Weigel-DiFranco 2 , Shyana Harper 2 ,Xiaowu Gai 3 , Eliot L. Berson 1, 2 , Eric A. Pierce 1, 2 . 1 Ocular GenomicsInstitute, Massachusetts Eye and Ear Infirmary, Harvard MedicalSchool, Boston, MA; 2 Berman-Gund Laboratory for the Study ofRetinal Degenerations, Harvard Medical School, Massachusetts Eyeand Ear Infirmary, Boston, MA; 3 Department of MolecularPharmacology and Therapeutics, Loyola University Chicago HealthSciences Division, Maywood, IL.Purpose: Usher syndrome is the most frequently inherited dualimpairment of vision and hearing. Usher syndrome type 1 (USH1) isthe most severe form characterized by profound congenital deafness,vestibular dysfunction and prepubescent onset of retinaldegeneration. Currently, there are six genes associated with USH1:Myosin VIIa (MYO7A), Harmonin (USH1C), Cadherin-23(CDH23), Protocadherin-15 (PCDH15), Sans (USH1G) and recentlyidentified CIB2. The products of five of these genes have recentlybeen identified to be part of the calyceal processes of photoreceptors.The purpose of the study was to genetically characterize a cohort of49 USH1 probands.Methods: An USH1 cohort of 49 probands, partially pre-screened for©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyMYO7A mutations, was high-throughput sequenced for targetedexons of genes associated with inherited retinal degenerations.Genetically unsolved samples were subsequently screened fordeletions using a custom design comparative genomic hybridization(CGH) array. When possible, the likely pathogenic variants wereconfirmed by co-segregation in available family members withSanger sequencing.Results: Selective exon capture and Illumina sequencing providedexcellent coverage of the targeted exons, with >95% of exons having>10X sequence depth. With this sequencing approach, we were ableto solve 35% of 49 cases, where mutations in MYO7A were the mostfrequent (16%), followed by CDH23 (10%) and USH1C (6%). 37%of the samples contained one heterozygous likely pathogenic changein a USH1 gene. The remaining, 28% did not carry mutations in anyknown USH1 gene. CGH array analyses for unsolved cases is inprogress and will be reported.Conclusions: Targeted exon sequencing provides an effectiveapproach for genetic diagnostic testing of patients with Ushersyndrome. The results of this study show that there is a considerablenumber of USH1 patients with an unknown genetic cause of thedisease, indicating additional genetic loci for this condition.Acknowledgements:This work was supported by grants from the National Eye Institute(EY012910) and the Foundation Fighting Blindness.Commercial Relationships: Kinga M. Bujakowska, None; EmilyPlace, None; Mark B. Consugar, Agilent Technologies, Inc. (R);Daniel G. Taub, None; Aliete Langsdorf, None; Carol Weigel-DiFranco, None; Shyana Harper, None; Xiaowu Gai, None; EliotL. Berson, None; Eric A. Pierce, NoneSupport: National Eye Institute (EY012910); Foundation FightingBlindnessProgram Number: 1582 Poster Board Number: D0009Presentation Time: 8:30 AM - 10:15 AMEpigenetic factors in the pathogenesis of corneal dystrophyXiaohua Li, Xiaohua Li, Min Yuan, Ruijie Yin. Henan Eye Institute,Henan Provincial Eye Hospital, Zhengzhou, China.Purpose: Tumor suppressor gene Hypermethylated In Cancer 1(HIC1) regulates the expression of SIRT1 and in turn SIRT1deacetylates of P53 (AC-p53) leading to the response to DNA repair,stress and senesces, therefore, a HIC1, SIRT 1 and P53 loop havebeen implied in the pathogenesis of cancer, ageing and degenerativediseases. The current research sought to investigate the roles of HIC1,SIRT1 and P53 in the pathogenesis of corneal dystrophy and itsassociation with CTGF and TGF-beta expression.Methods: Formalin-fixed paraffin embedded cornea section from 25patients (ages 5-68; 17 male, 8 female) with corneal dystrophy wereprepared for the study.Five corneal specimens from normal adultwere included as control. H&E, PAS, Congo red, Masson andcolloidal iron staining were performed to confirm the diagnosis ofcorneal dystrophy. Sections were analyzed for the expressions ofHIC1, SIRT1, acetylated -P53, CTGF, TGF-beta, and α-SMA byimmunohistochemistry. The red chromagen color was developedusing amino ethyl carbazole. The Slides were examined using a digitmicroscope.Results: 14 cases were diagnosed with macular corneal dystrophyand 11 cases were diagnosed with lattice corneal dystrophy accordingto H&E and histochemical staining. In cases of macular dystrophy,the deposits were positive with colloidal iron staining, while in casesof lattice dystrophy, the deposits stained positively with Massontrichrome, PAS and Congo red staining. In corneal specimen bothmacular corneal and lattice corneal dystrophy, HIC1 was highlyexpressed compared with control especially in corneal epithelial cell .The expression of SIRT1 was much weaker compared with the HIC1and normal corneal specimens staining. Notably, Acetylated P53immnunoreactivity is abundant in the degenerated areas of thecornea. In addition, the increased expression of fibrotic inducer suchas CTGF and TGF-beta were seen in the corneal fibrosis lesion,striking, the expression of CTGF was much stronger than TGF-beta.α-SMA was also unregulated in the area where CTGF was detected.Conclusions: The high HIC1 expression is associated with increasedexpression of Ac-p53 and CTGF and the down regulation of SIRT1in the corneal tissue; the distinct expression of the epigenetic factorsHIC1, SIRT1, AC-p53 and their interaction with CTGF may play anessential role in pathogenesis of the corneal dystrophy.Commercial Relationships: Xiaohua Li, None; Xiaohua Li, None;Min Yuan, None; Ruijie Yin, NoneSupport: Grants 81100650, National Natural Science Foundation ofChinaProgram Number: 1583 Poster Board Number: D0010Presentation Time: 8:30 AM - 10:15 AMBiomarkers for Neuronal Injury following Blast Trauma to theEyeSteven G. Hart 1 , XiangDi Wang 1 , Tonia S. Rex 2 , Eldon E. Geisert 1 .1 Ophthalmology, University of Tennessee Health Science Center,Memphis, TN; 2 Ophthalmology, Vanderbilt University, Nashville,TN.Purpose: The pathogenic pathways triggered by blast injury to theeye and biomarkers that reflect the activation of these pathways arelargely unknown. The present study is the initial attempt to definepotential biomarkers that reflect the severity of the retinal injury.Methods: Blast injuries to the eye were produced by a paintball gunfitted with a shortened and narrowed barrel and an integrated pressureregulator. The mice were deeply anesthetized and secured in a PVCpipe. A 45-psi overpressure wave was delivered selectively to the eyeof C57BL/6 mice and DBA/2J mice. The animals were thensacrificed at 2 or 5 days after the blast injury. To aid in our initialsurvey of potential biomarkers of retinal injury, we examined ourOptic Nerve Crush Microarray Dataset and compared it to ourNormal Retinal Microarray Dataset in GeneNetwork.org. This workled to six potential biomarkers for blast injury, Gfap, Iba1, C1q,Aqp4, Sox11 and Hsp25. One set of retinas were removed and stainedby indirect immunohistochemical methods to assess the distributionand intensity of the staining compared to uninjured control retinas.For a second set of retinas, the animals were anesthetized; the retinaswere removed and placed in sample buffer. The level of proteinexpression was determined by semi-quantitative immunoblotmethods.Results: Immunostaining sections of retina revealed that two of themarkers, SOX11 and HSP25, were upregulated in the neurons of theinner retina following blast. Both SOX11 and HSP25 labeled cells inthe ganglion cell layer and the inner nuclear layer. In the ganglioncell layer SOX11 labeled approximately 90% of the cells, indicatingthat it was labeling most ganglion cells and displaced amacrine cells.Furthermore, amacrine cells in the inner nuclear layer were labeledby SOX11. The intensity of staining increased dramatically after blastinjury and on immunoblots there was approximately a 2-fold increasein the intensity of the SOX11 band. A similar pattern of staining wasobserved with HSP25. The increased staining after blast injury didnot appear to be as dramatic as it was for SOX11. On immunoblots,there was also an observed increase in the intensity of the HSP25band following injury.Conclusions: SOX11 and HSP25 are markers for blast injury to theretina, labeling both retinal ganglion cells and amacrine cells. Thebetter of the two markers appears to be SOX11.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyCommercial Relationships: Steven G. Hart, None; XiangDiWang, None; Tonia S. Rex, PCT/US2012/021247 (P); Eldon E.Geisert, NoneSupport: DOD-TATRC W81XWH-12-1-0255; Unrestricted Grantfrom Research to Prevent Blindness, Inc.Program Number: 1584 Poster Board Number: D0011Presentation Time: 8:30 AM - 10:15 AMColorimetric Image Analysis in Detection of Leukocoria fromRetinoblastoma in Snapshots Taken by Standard DigitalPhotographyKatherine E. Talcott 1 , Elizabeth Shaw 4 , Rebecca Holden 4 , BrandonW. Taylor 4 , Erich J. Baker 3 , Greg Hamerly 3 , Alex Kentsis 2 , ShizuoMukai 1 , Carlos Rodriguez-Galindo 2 , Bryan F. Shaw 4 .1 Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, MA;2 Pediatric Oncology, Dana Farber Cancer Institute, Boston, MA;3 Computer Science, Baylor University, Waco, TX; 4 Chemistry andBiochemistry, Baylor University, Waco, TX.Purpose: Early diagnosis and treatment of retinoblastoma areextremely important but remain challenging. Screening programs thatuse “red reflex” testing to detect leukocoria have improved the timingof diagnosis and treatment outcomes. Early detection of leukocoriavia amateur photography may facilitate earlier diagnosis. Thepurpose of this study is to determine: (i) whether leukocoria can bereliably detected with recreational flash photography during earlyretinoblastoma, and (ii) whether its detection correlates with diseaseprogression and treatment.Methods: 7377 consecutive, recreational digital photographs of onepatient with bilateral retinoblastoma were collected and analyzedover three years (1068 days) from birth, to retinoblastoma diagnosis,through treatment (systemic chemotherapy, enucleation of one eyeand laser, cryotherapy and proton radiation therapy in the fellow eye),and remission. For control, 305 pupils of 19 healthy children presentin the same photographs were analyzed. Images were converted toJPEG format, each pupil cropped, total pixel count determined usingAdobe Photoshop, and retrospectively analyzed for the presence ofleukocoria, typically defined by a pupil Value (brightness) ≥ 0.5 inHSV color space (Hue, Saturation, Value).Results: Leukocoria occurred in a total of 237 photographs andbegan at 12 days old seen in 0.5% of pictures taken per month. Thefrequency of occurrence increased from birth by up to 5% per month,reaching as high as 25% per month before decreasing exponentially(t1/2 = 1.1 month) to an average of 1.5% per month after successfultreatment.Conclusions: Leukocoria can be detected in the early stages ofretinoblastoma using recreational digital photography. Frequency ofleukocoria occurrence in digital photographs correlated with diseaseprogression and treatment. Development of leukocoria detection toolsbased on digital photography (e.g., leukocoria detection software)might improve the timeliness and accuracy of retinoblastomadiagnoses.Commercial Relationships: Katherine E. Talcott, None; ElizabethShaw, None; Rebecca Holden, None; Brandon W. Taylor, None;Erich J. Baker, None; Greg Hamerly, None; Alex Kentsis, None;Shizuo Mukai, None; Carlos Rodriguez-Galindo, None; Bryan F.Shaw, NoneProgram Number: 1585 Poster Board Number: D0012Presentation Time: 8:30 AM - 10:15 AMInitial proteomic analysis of structural changes in the aginghuman vitreousQianru Zhang 3, 1 , Ying-Bo Shui 1 , Anne Kettler 2 , James P. Malone 2 ,Robert R. Townsend 2 , David C. Beebe 1 . 1 Dept of Ophthal & Vis Sci,Washington Univ Sch of Med, Saint Louis, MO; 2 Proteomics CoreLab, Washington Univ Sch of Med, Saint Louis, MO;3 Ophthalmology, Zhejiang University, Hangzhou, China.Purpose: A substantial proportion of vision-threatening retinaldamage in older age is caused by degeneration of the vitreous body,including rhegmatogenous retinal detachment, macular hole,epiretinal membrane formation and macular traction syndrome. Toprevent these conditions we need to understand the changes invitreous structure that occur with age. We identify the structuralproteins of the human vitreous, setting the stage for identification ofchanges in these proteins during aging.Methods: Vitreous bodies were carefully dissected from donor eyes.Each vitreous body was divided into two portions. One was washedovernight in a mesh screen suspended in 1 L of PBS, while the othersample was analyzed in its native state. Samples were subjected toproteomic analysis by HPLC/high resolution mass spectroscopy orwestern blotting to identify and quantify the structural proteins of thevitreous.Results: A total of 407 proteins were identified in the vitreoussamples. Levels of most proteins decreased dramatically afterwashing, including alphaA- and alphaB-crystallins, Ig gamma-3chain C region, serum albumin, and glucose-6-phosphate isomerase.These proteins were considered as soluble components of thevitreous. Proteins that increased in relative abundance in washed,compared to whole vitreous were considered to be structuralcomponents of the vitreous. In addition to well-known vitreousstructural proteins, such as collagen alpha-1(II), collagen alpha-2(IX),opticin and chondromodulin-1, we identified fibrillin-1, collagenalpha-1 (XXII) and latent-transforming growth factor beta-bindingprotein 2. Using western blotting, we are measuring age-relatedchanges in the levels of collagen II, opticin and chondromodulin-1and will extend this analysis to each of the structural components ofthe vitreous.Conclusions: Using high-throughput proteomic analysis, weobtained the first comprehensive list of vitreous structural proteins.Quantification of changes in protein abundance and post-translationalmodifications, like proteolysis or glycation, should provide insightinto the cause and prevention of vitreous liquefaction and its impacton age-related retinal damage.Commercial Relationships: Qianru Zhang, None; Ying-Bo Shui,None; Anne Kettler, None; James P. Malone, None; Robert R.Townsend, None; David C. Beebe, FivePrime (C), Panoptica (C),Vistakon (Johnson and Johnson) (C)Support: China State‐Sponsored Postgraduate Study AbroadProgram, unrestricted grant from RPB, Core grant EY02687Program Number: 1586 Poster Board Number: D0013Presentation Time: 8:30 AM - 10:15 AMManipulation of the MicroRNA Content of EndothelialProgenitor Cell-derived Extracellular VesiclesDavid A. Simpson, Eoin Brown, Christina L. O'Neill, Reinhold J.Medina, Jasenka Guduric-Fuchs. Centre for Vision & VascularScience, Queens University Belfast, Belfast, United Kingdom.Purpose: The transfer of microRNAs between cells withinmembrane-bound extracellular vesicles has recently been confirmedas a novel mode of intercellular communication. We hypothesizedthat endothelial progenitor cells (EPCs) may employ this mechanismto communicate with endothelial cells during angiogenesis.Manipulation of the specific microRNAs transferred could potentiallybe used as an approach to modulate neovascularisation.Methods: Vesicles released from EPCs isolated from humanperipheral blood were collected by ultracentrifugation. ThemicroRNA content of EPCs and their vesicles was analyzed by deep©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologysequencing. Changes in mRNA expression in human microvascularendothelial cells (HMECs) following direct transfection withmicroRNA over-expression vectors were analysed by microarray.Expression of specific genes was measured by RT-qPCR.Results: The global profile of microRNAs within vesicles releasedfrom EPCs is distinct from that inside the cells. MicroRNAs highlyenriched in extracellular vesicles included miR-486, miR-4792, miR-216a and miR-143. Vesicles from EPCs transfected with severalvectors driving expression of microRNAs, including miR-146a andmiR-451, were highly enriched in the over-expressed microRNA.When EPC-derived vesicles enriched with miR-146a were incubatedwith human microvascular endothelial cells they causeddownregulation of miR-146a target genes.Conclusions: Endogenous microRNAs are selectively packaged intoextracellular vesicles. EPC vesicles can be manipulated to deliverspecific microRNAs to endothelial cells. Such vesicles carryingcandidate anti- or pro-angiogenic microRNAs may have therapeuticapplications in multiple ocular diseases.Commercial Relationships: David A. Simpson, None; EoinBrown, None; Christina L. O'Neill, None; Reinhold J. Medina,None; Jasenka Guduric-Fuchs, NoneSupport: Biotechnology and Biological Sciences Research Council(BBSRC), grant no. BB/H005498/1Program Number: 1587 Poster Board Number: D0014Presentation Time: 8:30 AM - 10:15 AMP2X7 Expression in Vitrectomy SamplesCheryl Chi 1 , Deeba Husain 1 , Celeste B. Rich 2 , Nicole H. Siegel 1 ,Vickery E. Trinkaus-Randall 1, 2 . 1 Ophthalmology, Boston UniversitySchool of Medicine, Boston, MA; 2 Biochemistry, Boston UniversitySchool of Medicine, boston, MA.Purpose: P2X7 is an ionotropic receptor activated by ATP releasedin stress conditions. The full-length form of P2X7 (P2X7A) isassociated with the induction of apoptosis in many cell types, but atruncated variant, P2X7j, has also recently been found in transitionalstates that require cell proliferation. Altered regulation of these formshas been observed in diabetic patients, but specific relationshipsbetween the disease and receptor have yet to be elucidated. Our goalis to demonstrate the expression of P2X7A and P2X7j and tocorrelate this with sulfated glycosaminoglycan (GAG).Methods: Vitreous samples from 46 patients were centrifuged andsupernatant collected. Real-time PCR and delta delta Ct analysis wereperformed to determine expression of P2X7 mRNA. Each form wasnormalized to 1 using a control sample. Sulfated GAG in thesupernatant were measured by Dimethylmethylene Blue.Measurements were averaged and standard statistical tests wereperformed.Results: We observed an increased trend in the ratio of P2X7j toP2X7A in diabetic patients over control. The level of P2X7j did notchange between diabetic or control patients, but the level of P2X7Adecreased in diabetics, indicative of a less apoptotic phenotype. Thiswas supported by lower expression of P2X7A in patients withproliferative diabetic retinopathy. Hypertensive control patientsshowed a large increase in ratio over non-hypertensive controlpatients, but no such trend was seen in diabetic patients. Conversely,diabetic patients with retained lens fragments showed larger ratiosthan those without retained lens fragments, but this trend was notseen in the control population. Diabetic patients overall showdecreased sulfated GAG compared to control. Diabetic patients withretained lens fragments show a decrease, but those with hypertensionshow an increase, when compared to the corresponding control.Conclusions: Expression of P2X7j or pro-proliferative form, alongwith a decrease in P2X7A, were reflected in the phenotypicexpectations of pathological states. The inflammation associated withhypertensive states is suggested by the increased ratio, and thedecreased pro-apoptotic form illustrates cell proliferation in PDR.Changes in P2X7 receptor expression and sulfated GAG reflect theinfluence of multiple underlying pathologies.Commercial Relationships: Cheryl Chi, None; Deeba Husain,None; Celeste B. Rich, None; Nicole H. Siegel, None; Vickery E.Trinkaus-Randall, NoneSupport: Boston University Department of Ophthalmology ResearchGrant, Massachusetts Lions Eye Research Fund, Inc.Program Number: 1588 Poster Board Number: D0015Presentation Time: 8:30 AM - 10:15 AMPro-inflammatory cytokines induce apoptosis of human retinalendothelial cells by downregulating Hsp27Ram H. Nagaraj, Allison Palmer, Rooban B. Nahomi.Ophthalmology and Visual Sciences, Case Western ReserveUniversity, Cleveland, OH.Purpose: The biochemical mechanisms for retinal capillary celldeath in diabetes are not clear. In the diabetic retina of humans androdents, pro-inflammatory cytokines are upregulated. In this study,we have investigated the effect if pro-inflammatory cytokines on asmall heat shock protein, Hsp27 in primary human retinal endothelialcells (HREC).Methods: HREC were cultured in the presence of pro-inflammatorycytokines, interferon -γ (IFN-γ, 50 and 100 units/ml), interleukin-1β(IL-1β, 10 and 20 ng/ml) and tumor necrosis factor-α (TNF-α, 10 and20 ng/ml) for 48 hrs and in the presence or absence of high glucose(25 mM, HG). The roles of the kynurenine pathway and NOS weredetermined by adding 20 μM 1-methyl tryptophan (MT) and 500 μML-Nω-nitroarginine methyl ester hydrochloride (L-NAME),respectively to the culture medium. The mRNA and protein levels ofHsp27 and heat shock factor-1 (HSF-1) were determined by PCR andWestern blotting. Apoptosis was assessed by Hoechst staining.Results: HREC cultured in the presence of mixed cytokines showeda significant downregulation of Hsp27 at the protein and mRNAlevels. This downregulation was not due to downregulation of thetranscription factor, HSF-1. The presence of high glucose (25 mM)further increased the effect of mixed cytokines. Mixed cytokinesactivated indoleamine 2,3-dioxygenase (IDO), enhanced kynurenineproduction and increased the ROS content in HREC. An inhibitor ofIDO (MT) inhibited the effects of mixed cytokines on Hsp27. Mixedcytokines upregulated NOS2 and consequently increased levels ofnitric oxide. A peroxynitrite donor and exogenous peroxynitritedrastically reduced Hsp27 in HREC. The cytokine and high glucosemediateddownregulation of Hsp27 was accompanied by increasedapoptosis of HREC, which was largely inhibited by treatment withMT or a NOS inhibitor, L-NAME. Downregulation of Hsp27 by asiRNA promoted apoptosis in HREC.Conclusions: Together our data suggest that pro-inflammatorycytokines induce formation of ROS through the IDO-mediatedkynurenine pathway, which through peroxynitrite production reducesHsp27 and brings about apoptosis of capillary endothelial cells. Ourresults suggest a novel mechanism for capillary cell death in diabeticretinopathy.Commercial Relationships: Ram H. Nagaraj, None; AllisonPalmer, None; Rooban B. Nahomi, NoneSupport: International Retina Research Foundation, RPB, OhioLions Eye Research Foundation, P30EY-11373Program Number: 1589 Poster Board Number: D0016Presentation Time: 8:30 AM - 10:15 AM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyProbing the sensitivity of rhodopsin expression to chromophorelevels in the retinaLauren L. Daniele 1 , Edward N. Pugh 2, 1 . 1 Cell Biology & HumanAnatomy, University of California Davis, Davis, CA; 2 Physiology &Molecular Biology, Univ of California Davis, Davis, CA.Purpose: To test the hypothesis that the decrease in rhodopsinexpression in young RPE65 -/- mice is due to enhanced ER associateddegradation (ERAD). Mice with deletions of proteins required for 11-cis retinal synthesis undergo retinal degeneration. While cones arewell established to have disrupted opsin expression and trafficking,and degenerate rapidly, rods degenerate more slowly. Nevertheless,rhodopsin levels are reduced early, suggesting 11-cis retinal can alsoaffect rhodopsin expression (Rohrer et al., 2003, IOVS; Feathers etal., 2008, IOVS; Sato et al., 2010, Exp Eye Res)Methods: Western blotting was used to estimate the relative amountsof rhodopsin (Rho) in total retina lysates of WT and Rpe65 -/- miceaged 2-3 months. To test for increased association of rhodopsin withproteins involved in ERAD, retina lysates were subject toimmunoprecipitation (IP). IP’s were performed with antibodies toVCP/p97 and EDEM1, key proteins known to be involved in ERADof mutant opsins. IP’s were analyzed by PAGE followed by WesternblottingResults: Western blots reveal Rho expression to be reduced to 71%of WT in 2-3 month old Rpe65 -/- mice (n=6 littermate pairs). Theseresults confirm previously published observations (Rohrer et al.,2003, IOVS; Feathers et al., 2008, IOVS; Sato et al., 2010, Exp EyeRes). When a Rho antibody was used for IPs of retina lysate, theamount of VCP detected relative to rhodopsin in theimmunoprecipitated fractions was constant for both genotypes.However, when a VCP antibody was used for IP’s, over 10-fold moreRho was detected relative to VCP in WT compared with Rpe65 -/-mice. IPs with EDEM1 antisera were unsuccessfulConclusions: Based on co-immunoprecipitation, a small fraction oftotal Rho is associated with VCP in WT and in Rpe65 -/- retinas,suggestive of active ERAD quality control operative in bothgenotypes. It is unclear why the Rho/VCP ratio in WT is so muchhigher relative to Rpe65 -/- in VCP pulldowns. The results suggest thatthe reduction in rhodopsin expression in RPE65 -/- is not due toincreased ERAD. However it is possible that rhodopsin undergoingERAD in the RPE65 -/- is degraded more rapidly and has a morefleeting interaction with VCP. Alternatively, as supported byevidence that Rho mRNA is reduced to ~ 70% of WT at 3-6 weeks ofage in Lrat -/- mice (Sato et al., Exp Eye Res, 2010), transcriptionalloss may underlie reduced Rho in the Rpe65 -/- miceCommercial Relationships: Lauren L. Daniele, None; Edward N.Pugh, NoneSupport: EY02660Program Number: 1590 Poster Board Number: D0017Presentation Time: 8:30 AM - 10:15 AMVitreoretinal interface abnormalities and viscoelastic behavior ofthe vitreousSanket U. Shah 1 , David C. Reed 1 , Sam Abbassi 1 , Ryan Freeman 2 ,Pooria Sharif-Kashani 2 , Pirouz Kavehpour 2 , Jean-PierreHubschman 1 . 1 Department of Ophthalmology - Retina Division, JulesStein Eye Institute and David Geffen School of Medicine, UCLA,Los Angeles, CA; 2 Mechanical and Aerospace Engineering, UCLA,Los Angeles, CA.Purpose: The study hypothesis is that the human vitreous hasviscoelastic properties that are related to tangential andanteroposterior tractional forces acting at the vitreoretinal interfacecausing vitreoretinal abnormalities. We evaluated the differences inthe viscoelastic properties of human vitreous samples followingvitrectomy in different vitreoretinal interface abnormalities.Methods: Undiluted chopped vitreous samples were collected duringvitrectomy using predetermined machine settings from patient eyeswith different vitreoretinal interface abnormalities. The viscosity andcreep compliance of these samples were measured using a parallelplate shear rheometer. Statistical analysis was performed toinvestigate association of different vitreoretinal diseases with theviscoelastic properties.Results: Of 81 patient eyes undergoing vitrectomy, the choppedvitreous was suitable for rheological tests to obtain creep compliancein 65 (80%) and viscosity in 30 (37%) patients. The vitreoretinalinterface abnormalities in 65 patients were grouped into four groupsbased on the type of vitreoretinal interface forces. These includedtangential vitreo-retinal (VR) traction (group A), anteroposterior VRtraction (group B), combined tangential and anteroposterior VRtraction (group C), and no VR traction (group D). The creepcompliances (mean ± SD) in these groups were 111.18 ± 47.99/Pa,104.50 ± 68.87/Pa, 49.38 ± 55.68/Pa, and 117.08 ± 61.67/Parespectively. The creep compliance of group C was significantlylower than groups A (p=0.007), B (p=0.011), and D (p=0.012).Viscosity data were available for 30 patients and did not showsignificant differences.Conclusions: The vitreous from eyes with vitreoretinal interfaceforces in both anteroposterior and tangential directions has lowercompliance (higher elasticity) than all other groups. Themacromolecular structural components of the vitreous that contributeto this low compliance may play a role in producing the prevalentvitreoretinal interface forces.Commercial Relationships: Sanket U. Shah, None; David C.Reed, None; Sam Abbassi, None; Ryan Freeman, None; PooriaSharif-Kashani, None; Pirouz Kavehpour, None; Jean-PierreHubschman, NoneProgram Number: 1591 Poster Board Number: D0018Presentation Time: 8:30 AM - 10:15 AMGene expression profiling of the human maculaKristis Vevis 1 , Michael B. Powner 1 , Jenny Mckenzie 1 , Meidong Zhu 2 ,Mark C. Gillies 2 , Marcus Fruttiger 1 . 1 UCL INSTITUTE OFOPHTHALMOLOGY, London, United Kingdom; 2 University ofSydney, Save Sight Institute, Sydney, ACT, Australia.Purpose: The human macula, which is located at the centre of theretina and has a diameter of approximately 3mm, is responsible forthe central vision and any damage in that area is immediatelyobvious. Macular telangiectasia type 2 (MacTel) is an eye diseaseaffecting specifically an area of 2mm by 3mm within the maculawhich is consistent from patient to patient and throughout the disease.This suggests some specific molecular and/or cellular properties existin that area making susceptible to the disease. Therefore we aimed toidentify differentially expressed genes in MacTel area, which mightreveal mechanisms of the disease and unique properties of the maculain general.Methods: Human retinas were isolated from fresh healthy humaneyes (enucleated 3-7 hours after death) and stored in RNAlater. Fromthe tissue five different areas were dissected; nasal to the optic nerve(1), in the macula (2), temporal to macula (3), inferior to the macula(4) and superior to macula (5, Figure 1). RNA isolation was followedusing Trizol and cDNA was then used for qPCR analysis anddifferential display, aiming to identify differentially expressed genesin the macula and the other four areas.Results: Initially, primers for genes with known distributions wereused in qPCR experiments to test the suitability of cDNA extractedfrom post-mortem tissue for gene expression analysis. As expectedmlOpsin (cones) was highest in the MacTel area, while rhodopsin©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biology(rods) and synuclein gamma (ganglion cells) were reduced. We thentested some candidate genes implicated in biochemical pathwaystaking place in the retina such as visual cycle and retinoic acidmetabolism. ALDH1A1, ALDH1A3, RDH5 and RDH10 were foundto be reduced within the MacTel area while PPARG was increased.From differential display, after testing ‘’macula’’ compared to‘’temporal to macula’’ areas, ADARB1 was found to be reduced inthe macula.Conclusions: Human retina up to 7 hours post mortem can be usedfor gene expression analysis experiments. Using qPCR anddifferential display techniques, genes differentially expressedbetween the macula and the peripheral retina were found.Confirmation of the differentially expressed genes needs to be doneby immunohistochemistry.Figure 1: Schematic representation of the location of the fivedifferent dissected retinal regions.Commercial Relationships: Kristis Vevis, None; Michael B.Powner, Novartis (R); Jenny Mckenzie, None; Meidong Zhu,None; Mark C. Gillies, Novartis (R), Pfizer (R), Allergan (F), Bayer(F); Marcus Fruttiger, AstraZeneca (F), Novartis (F), Novartis (C),Amakem (F)Support: Lowy Medical Research Institute LTDProgram Number: 1592 Poster Board Number: D0019Presentation Time: 8:30 AM - 10:15 AMIdentification of piRNAs in the RetinaYingfeng Zheng 1, 2 , Qilin Wang 1 , Xinyang Lin 1 , Mingguang He 1 .1 Zhongshan Ophthalmic Center, Guangzhou, China; 2 Singapore EyeResearch Institute, Singapore, Singapore.Purpose: Piwi-interacting RNAs (piRNAs), a class of small noncodingRNAs associated with Piwi, maintain genomic integrity bytransposon silencing. Outside of the germline, the roles of piRNAs insomatic tissues are not fully understood. We examine if piRNAs andPiwi-like molecules are expressed in retina.Methods: Mice aged 8 weeks were sacrificed and the retina, liver,and testis tissues were obtained. piRNA level was assessed byquantitative reverse transcription PCR (qPCR) in mouse retina.Mouse liver and testis served as controls. In another experiment, weassessed the expression of Piwi-like genes (Piwi-like-1 to -4) inhuman retinal pigment epithelial (RPE) cells in vitro using qPCR.Results: Expression of several piRNAs was observed in mouse retinatissue. These included DQ541777, DQ555094, DQ689086,DQ705026, and DQ719597 that have previously been reported toexpress in somatic tissues such as liver, heart and brain. We alsodemonstrated mRNA expression of Piwi-like genes, particularlyPiwi-like-2 and -4 genes, in cultured RPE cells.Conclusions: These findings indicate that piRNAs and Piwi-familyproteins exist in retina. They might play important roles in the properfunction of the eye.Commercial Relationships: Yingfeng Zheng, None; Qilin Wang,None; Xinyang Lin, None; Mingguang He, NoneSupport: This work is supported by National Natural ScienceFoundation of China 81100686Program Number: 1593 Poster Board Number: D0020Presentation Time: 8:30 AM - 10:15 AMUnpredictable Consequences of Systemic Valproic AcidTreatment on the Rate of Photoreceptor Loss in Different Pde6bRd-mutationsKenneth P. Mitton 1, 2 , David W. Byrd 1 , Eduardo Guzman 1, 2 , AdrianneWallace 1, 2 , Trung Tran 1 , Jason Sotzen 1 . 1 Control of Gene ExpressionLab, Eye Research Institute of Oakland University, Rochester, MI;2 Pediatric Retinal Research Laboratory, Eye Research Institute ofOakland University, Rochester, MI.Purpose: VPA is an FDA approved anticonvulsant whose activity asa histone deacetylase inhibitor was discovered only recently,generating intense interest for its potential use in epigenetic therapy.Clinical trials are recruiting patients to evaluate VPA’s therapeuticpotential for AD-RP. Given that VPA’s effects on gene expressionare non-specific, could VPA reduce or accelerate photoreceptor lossin RP-patients harboring different mutations? We compared systemicVPA treatment on retinal neurotrophic gene expression andphotoreceptor loss in rd-mice with different mutations of Pde6b.Methods: Expression of Bdnf, Gdnf, Cntf, and Fgf2 were measuredby real-time PCR after single and multiple VPA doses in wild-typeand Pde6b rd1/rd1 mice. Pde6b rd10/rd10 mice were also treated toevaluate a partial loss-of-function model. Retinal morphology wasassessed with virtual microscopy.Results: In post-natal mice, a single systemic dose of VPA causedsubstantial increases in the expression of Bdnf (3x) and Gdnf (10x) inthe neural retina within 18 hours. Cntf and Fgf2 expression decreased70%. These large gene expression changes did not persist aftermultiple days of dosing. Daily injections with VPA (P9-P19) reducedphotoreceptor loss in Pde6b rd1/rd1 mice. By P20, VPA treated micehad several rows of rod nuclei, compared to a single row remainingin PBS injected littermates. Starting treatment later, or dosing everysecond day, also rescued photoreceptors. VPA treatment did notreduce rod loss in Pde6b rd10/rd10 mice. Some PBS-treatedPde6b rd10/rd10 littermates may have had slightly more rod-nuclei thantheir VPA treated counterparts, although this difference was notstatistically significant with the numbers tested. VPA disrupted thelens bow region morphology in some mice.Conclusions: A single systemic dose of VPA caused increases anddecreases the retinal expression of different neurotrophic factorgenes. While systemic VPA treatment slowed the rate ofphotoreceptor loss in Pde6b rd1/rd1 mice, it did not benefit degeneratingphotoreceptors in Pde6b rd10/rd10 mice. We conclude that systemicVPA was not universally beneficial for degenerations involvingdifferent functional mutations of the same gene. These results suggestthat human clinical trials with VPA should best be limited to AD-RPpatients with identified molecular mutations.Commercial Relationships: Kenneth P. Mitton, None; David W.Byrd, None; Eduardo Guzman, None; Adrianne Wallace, None;Trung Tran, None; Jason Sotzen, NoneSupport: 1. ROPARD 2. OU Center for Biomedical Research 3.NIH/NEIProgram Number: 1594 Poster Board Number: D0021Presentation Time: 8:30 AM - 10:15 AMMicroRNA-21 (miR-21) Induces Down-regulation Of PEDF InRetinal Pigmented Epithelial Cells By Suppression Of PPARAlphaManuela Bartoli 1 , Chaunte E. Stampley 2 , Sean Shaw 1 , Pamela M.Martin 3 , Folami Lamoke 1 . 1 Ophthalmology, Georgia Health Sciences©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyUniversity, Augusta, GA; 2 Rollins School of Public Health, EmoryUniversity, Atlanta, GA; 3 Department of Biochemistry and MolecularBiology, Georgia Health Sciences University, Augusta, GA.Purpose: Micro-RNA 21 (miR-21) is up-regulated in the ischemicretina and hypoxia-induced miR-21 expression contributes toincreased matrix metalloproteinases activity (MMP2 and MMP9) inretinal endothelial cells. Increased MMP2 and MMP9 activity in theischemic retina has been linked to the proteolytic degradation ofpigmented epithelial derived factor (PEDF), a key retinal angiostaticfactor. Here we have investigated the effects of miR-21 on PEDFexpression and protein stability in retinal pigmented epithelial cells(ARPE19) and in the ischemic retina.Methods: Immunoblotting and quantitative RT-PCR were conductedto determine PEDF and peroxisome proliferator activated receptoralpha (PPARalpha) expression at protein and mRNA levels,respectively, in ARPE19 after transfection with miR-21 or a scramblemicro RNA (miR-s). The same assays were also used to determinethe expression of PEDF and PPAR alpha in a model of ischemicretinopathy. Mice were exposed from postnatal day 7 to 12 (P7 andOIR12 respectively) to 73% oxygen tension. At OIR12 the mice werereturned to normoxic conditions and analyzed at postnatal day 14 and17 (OIR14 and OIR17, respectively). In situ hybridization wasconducted using locked nucleic acid-based probes to determine miR-21 sites of production in the ischemic murine retina.Results: Transfection of ARPE19 with miR-21 significantlydecreased protein and mRNA expression of PEDF. We haveidentified a PPAR alpha responsive element on the PEDF promoterregion. PPAR alpha has shown to be a potential target for miR-21.Transfection of ARPE19 with miR-21 down-regulated PPARalphaexpression at protein and mRNA levels. In the ischemic retina,maximal expression of miR-21 (OIR14 and 17) correlated withmaximal downregulation of PEDF and PPAR-alpha protein levels. Insitu hybridization further confirmed production of miR-21 by theretinal pigmented epithelial cells in the ischemic retina.Conclusions: Expression and activity of small non-coding RNAs isemerging as critical regulation of retinal gene expression in normaland pathological conditions. Our results demonstrate a novel role formiR-21 in down-regulating PEDF expression by its ability to blockPPAR alpha expression, thus halting PPAR alpha mediated-PEDFexpression.Commercial Relationships: Manuela Bartoli, None; Chaunte E.Stampley, None; Sean Shaw, None; Pamela M. Martin, None;Folami Lamoke, NoneProgram Number: 1595 Poster Board Number: D0022Presentation Time: 8:30 AM - 10:15 AMSuppression of microglial inflammatory response by sigmareceptor 1 ligand (+)-PentazocineJing Zhao 1, 3 , Sylvia B. Smith 2, 3 , Kathryn Bollinger 1, 3 .1 Ophthalmology, Georgia Health Sciences University, Augusta, GA;2 Cell Biology and Anatomy, Georgia Health Sciences University,Augusta, GA; 3 Vision Discovery Institute, Georgia Health SciencesUniversity, Augusta, GA.Purpose: Sigma Receptor 1 (σR1) is a member of a family ofmembrane-associated proteins that are expressed throughout themammalian nervous system and visceral organs. The endogenousfunction of σR1is not known. However, ligands for this receptor haveshown robust neuroprotective effects in brain and retina. In addition,σR1 activation suppresses aspects of brain-derived microglialinflammatory response. The goal of this work is to determine whether(+)-PTZ can suppress inflammatory responses of retina-derivedmicroglia.Methods: Primary cultures of microglia were prepared frompostnatal (1-5 days) of Sprague-Dawley rat pups. Purity of culturedmicroglia was confirmed by specific expression of microglia markerIba-1 using immunocytochemistry. Purified microglia were subjectedto LPS (1μg/ml) in the presence/absence of (+)-PTZ (3μM) for 1, 3, 6or 24 hours. Supernatants were collected and TNF-α, IL-10 andMCP-1 release level were detected using ELISA assay. Nitric oxidelevel in the supernatant was determined using Griess assay.Intracellular ROS was detected by incubation cells with 5μMCellROX green dye followed by observation under fluorescencemicroscope. Immunocytochemistry was performed for detection ofany morphology change. Westerns were performed to detect MAPKand NF-κB expression after the cells were treated with LPS in thepresence/absence of (+)-PTZ.Results: Our data showed that σR1 ligand (+)-PTZ suppressedmicroglia morphology change in response to LPS stimulation at the 6hours time point. LPS stimulated primary retinal microglia to secretTNF-α, IL-10, MCP-1 and nitric oxide, and secretion levels weresignificantly decreased when the cells were pre-treated with (+)-PTZfor 30 min. LPS also stimulated intracellular ROS generation, andROS generation was significantly inhibited by (+)-PTZ. Our westernresults showed that ERK, JNK and p38 MAPKs were activated whenmicroglia were treated with LPS. (+)-PTZ inhibited MAPKphosphorylation.Conclusions: σR1 ligand (+)-PTZ suppresses inflammatoryresponses of retina-derived microglia and decreases MAPKactivation.Commercial Relationships: Jing Zhao, None; Sylvia B. Smith,None; Kathryn Bollinger, NoneSupport: National Eye Institute, K08 EY021758-02 and AmericanGlaucoma Society236 Glaucoma Biochemistry and MechanismsMonday, May 06, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 1596-1610/D0023-D0037Organizing Section: Biochemistry/Molecular BiologyProgram Number: 1596 Poster Board Number: D0023Presentation Time: 8:30 AM - 10:15 AMAssociation of LOXL1 Gene Polymorphisim in TurkishPopulation with Pseudoexfoliation Syndrome and GlaucomaNilgun Yildirim, Yetkin Yaz, Oguz Cilingir, Fez an Sahin, ZaferYüksel. Department of Ophthalmology, Eskisehir Osmangazi UnivMed Sch, Eskisehir, Turkey.Purpose: To investigate the three single nucleotid polymorphisms(SNPs) (rs3825942, rs1048661 and rs2165241) of LOXL1 gene in aTurkish population with pseudoexfoliation syndrome (PES) andpseudoexfoliation glucoma (PEG) cases.Methods: DNA was obtained from blood samples of 48 PES, 58PEG, and 171 control cases, after then three SNPs (rs3825942,rs1048661, rs2165241) of LOXL1 gene was investigated with RT-PCR; a probe-based genotyping method.Results: Results: All three SNPs of LOXL1 gene in PES and PEGcases were more prevalent than healty individuals (For PESrs3825942 p= 3.54x10-6 OR=∞, rs1048661 p= 0.008 OR= 2.18andrs2165241 p= 8.69x10-9 OR= 4.30. For PEG rs3825942 p=3.41x10-7 OR= ∞ , rs1048661 p= 1.75x10-5 OR= 3.78 andrs2165241 p=3.85x10-11 OR= 4.90). No significant association wasfound between none of these SNPs with PES and PEG. rs3825942SNP had 3-fold increased risk in men (p= 6.78x10-5 OR= 3.202).rs3825942 SNP was more valuable for distinguishingpseudoexfoliative cases from healthy individuals.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyConclusions: In a Turkish subpopulation, the LOXL1 genepolymorphism of PES and PEG were different from healtyindividuals, and genetic variation was similar to European andAmerican populations.Commercial Relationships: Nilgun Yildirim, None; Yetkin Yaz,None; Oguz Cilingir, None; Fez an Sahin, None; Zafer Yüksel,NoneSupport: Eskisehir Osmangaz UnversityProgram Number: 1597 Poster Board Number: D0024Presentation Time: 8:30 AM - 10:15 AMElevated TGFβ1 concentration in the plasma of glaucomapatientsJohn Kuchtey, L. Goodwin Burgess, Megan B. Parks, Jessica Kunkel,Milam A. Brantley, Rachel W. Kuchtey. Vanderbilt Eye Institute,Vanderbilt University, Nashville, TN.Purpose: Previously, we formed the hypothesis that microfibrildefects may contribute to glaucoma pathogenesis, based on ouridentification of the microfibril-associated gene, ADAMTS10, as thedisease gene in a colony of dogs with inherited primary open angleglaucoma (POAG). Since microfibrils are a major reservoir forTGFβ, and because other diseases caused by defective microfibrilsare associated with elevated plasma TGFβ, a corollary to themicrofibril hypothesis is that glaucoma patients may have a systemicelevation of TGFβ. The purpose of this study was to test thehypothesis that patients with POAG have elevated plasmaconcentrations of TGFβ.Methods: Blood samples from POAG patients and controls werecollected in lithium heparin tubes, centrifuged at 4 o C for 10 minutesat 1,500 x g and plasma aliquoted and stored at -80 o C.Concentrations of total TGFβ1 and total TGFβ2 were determined byELISA assay of acid-activated plasma samples from POAG patientsand controls matched for age, sex and ancestry.Results: In an initial set of 55 POAG patients and 55 controls, totalTGFβ1 was higher in the plasma of POAG patients compared tocontrols (median concentrations 3.12 vs. 2.54 ng/ml, respectively,p2SD above the mean of controls.Plasma total TGFβ2 concentrations were below limits of detection.Conclusions: Total TGFβ1 is higher in the plasma of POAG patientsas compared to controls, with some patients having pronouncedelevation. This finding is consistent with our hypothesis thatdefective microfibrils contribute to glaucoma pathogenesis andsuggest a mechanism to explain elevated TGFβ in the aqueous humorof glaucoma patients. Elevated plasma TGFβ1 may be used as abiomarker for glaucoma associated with defective microfibrils.Commercial Relationships: John Kuchtey, None; L. GoodwinBurgess, None; Megan B. Parks, None; Jessica Kunkel, None;Milam A. Brantley, None; Rachel W. Kuchtey, NoneSupport: NIH Grant EY020894Program Number: 1598 Poster Board Number: D0025Presentation Time: 8:30 AM - 10:15 AMAssociation of the T allele of rs41435250 of LOXL1 with elevatedrisk of exfoliation syndrome and exfoliation glaucoma in aMexican populationDalia C. Guadarrama 1 , Juan C. Zenteno 1, 2 , Antonio Miranda 3 , CeliaElizondo 1 . 1 GENETICS, INSTITUTE OF OPHTHALMOLOGYCONDE DE VALENCIANA, MEXICO, Mexico;2 BIOCHEMISTRY FACULTAD DE MEDICINA, NATIONALAUTONOMOUS UNIVERSITY OF MEXICO (UNAM), MEXICO,Mexico; 3 GENETICS, NATIONAL REHABILITATIONINSTITUTE, MEXICO, Mexico.Purpose: Pseudoexfoliation syndrome (XFS) is a major risk factorfor developing pseudoexfoliation glaucoma (XFG). The disease hasbeen associated with 3 high risk SNPs (rs3825942, rs1048661 andrs2165241) in the lysil oxidasa-like1 (LOXL1) gene. Particularly, theG allele of rs3825942 is considered the “universal” risk allele as it isconsistently associated to XFS/XFG in distinct ethnic groups.Previous studies in Mexican population showed that the T allele ofrs2165241 SNP confers a greater risk for XFS/XFG than the G alleleof rs3825942 in that population. The purpose of this study was toinvestigate the association of an additional LOXL1 SNP withXFS/XFG risk in Mexican patients.Methods: A total 102 subjects with PEX/PEG and 130 healthy adultcontrols were included. Genomic DNA was extracted fromleukocytes, and the LOXL1 exon 1 coding sequence of LOXL1 wasamplified by PCR and directly sequenced for genotyping the allelesof the rs41435250 SNP. STATA ver.10.0 statistical software packagewas utilized for calculations. Allele frequencies, HardyeWeinbergequilibrium (HWE) and haplotype association analyses were assessedwith Haplo View 4.0 software.Results: The mean age in the patient group was 75 years and of 72years in the control group. The rs41435250 TT genotype wasassociated with an elevated risk as it was observed in 3.92% ofpatients and in 1.54% controls (OR[95% CI]= 2.56 [0.44-14.58]; p=0.004500); accordingly, the GG genotype for this SNP had afrequency of 85.38% in controls and 70.59% in patients and wasassociated with a protective effect: OR[95% CI]= 0.43 [0.22-0.83];p= 0.013. Allele frequencies showed the same tendencies asmentioned for genotypes.Conclusions: Our results indicate that the T allele of rs41435250confers an elevated risk for the development of XFS/XFG in Mexicanpopulation.Commercial Relationships: Dalia C. Guadarrama, None; Juan C.Zenteno, None; Antonio Miranda, None; Celia Elizondo, NoneSupport: YesProgram Number: 1599 Poster Board Number: D0026Presentation Time: 8:30 AM - 10:15 AMRegulation of rhodopsin gene expression by DNA methylationJin Song, Tomohiro Masuda, Donald J. Zack, Shannath L. Merbs.Department of Ophthalmology, Johns Hopkins University, Baltimore,MD.Purpose: We have previously shown that Rhodopsin (RHO) has acell-specific DNA methylation pattern within the retina and ismethylated in non-expressing cells. We aimed to determine if DNAmethylation contributes directly to the suppression of RHOexpression.Methods: Cells expressing low levels of RHO (HEK 293, WERI andY79) were treated with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dc; 72 h treatment) to inducehypomethylation. Global DNA methylation was measured by LUMA(luminometric methylation assay). RHO methylation was measuredby bisulfite pyrosequencing, and the expression level determined byquantitative real-time PCR (qPCR). To explore whether the additionof transcription factors could further increase endogenous RHOexpression in 5-aza-dc-treated HEK 293 cells, a bovine Rho promoterreporter (Gluc luciferase) was co-transfected with CRX and NRLexpression vectors. Endogenous RHO methylation and expressionwas examined.Results: Treatment of HEK 293, WERI, and Y79 cells with 5-aza-dcinduced a drug-dependent global DNA hypomethylation in all threecell lines. At 17 CpG sites of RHO, DNA methylation was reduced in©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biology5-aza-dc -treated cells compared to controls and qPCR demonstrateda concomitant increase in RHO expression. In 5-aza-dc-treated HEK293 cells, addition of CRX and NRL greatly increased Gluc activityfrom the reporter plasmid and increased expression of endogenousRHO. Additional chromatin remodeling drugs, including HDACinhibitors, are currently being examined for possible synergisticeffects in the regulation of RHO gene expression.Conclusions: Our results show that DNA hypomethylation by 5-azadccan induce RHO expression from low-expressing cells.Endogenous RHO expression can be further increased by the additionof transcription factors required during normal gene expression. Ourresults suggest that DNA methylation plays an important role in thesuppression of RHO gene expression and is not a passive marker ofRHO silencing. The ability to manipulate the expression of retinaspecificgenes using epigenetic modifiers currently in therapeutic useenables further study into the role that these mechanisms may play inboth normal eye development and disease.Commercial Relationships: Jin Song, None; Tomohiro Masuda,None; Donald J. Zack, Alcon (C), Merck (F), Allergan (C);Shannath L. Merbs, NoneSupport: RPB unrestricted fundsProgram Number: 1600 Poster Board Number: D0027Presentation Time: 8:30 AM - 10:15 AMMicroRNA 184 regulates Ezrin expression and potentiallyimpacts Ezrin-dependent functions in human Retinal PigmentEpitheliumNishantha Gunawardena, Nady Golestaneh, Maria Kokkinaki, MiaGunawan. Georgetown University, Washington, DC.Purpose: The purpose of this study is to investigate the role ofmicroRNA 184 (miR-184) in regulating RPE functions. It is knownthat miR-184 plays a key role in neurological development and ishighly expressed in mice corneal epithelium and in human fetal RPE,however, its role in RPE is largely unknown.Methods: Proteomic analysis of RPE transfected with hairpins formiR-184 was performed and the data were analyzed to identify thegenes that were highly affected by the down-regulation of miR-184.Since RPE express miR-184, Hela cells were used to analyze thebinding of miR-184 to the 3’UTR of the targeted genes. Luciferaseassays were performed after transfection of Hela cells with bothplasmids for EZR 3’UTR in pEZX-MT01 vector and miR-184 codingDNA sequence in pEZX-MR04. Real Time PCR was used torelatively quantify the mRNA expression levels of the targeted gene.Western blot was used to measure the protein levels of ezrin in miR-184 transfected cells. RNAi technology was used to verify the effectof EZR down-regulation in RPE function. Phagocytosis assay andultrastructural analysis using electron microscopy were performed.Results: Our proteomic data showed that EZR is a potential target formiR-184.Ezrin is an actin-binding cytoplasmic peripheral membrane proteinthat functions as a protein-tyrosine kinase substrate in the apicalmicrovilli and as an intermediate between the plasma membrane andthe actin cytoskeleton. Luciferase assay revealed that miR-184 bindsto the 3’UTR of EZR and directly down-regulates EZR geneexpression. In addition, real time PCR revealed that EZR geneexpression is inhibited by miR-184. Western blot confirmed thedown-regulation of ezrin protein by miR-184. Using EZR siRNA, weshowed that RPE morphology and phagocytosis function wereaffected by ezrin down-regulation.Conclusions: Our results suggest that miR-184 affects actindepolymerization and in turn impacts membrane trafficking and RPEfunctions through down-regulation of EZR. In addition, since ezrin isexpressed in the apical region of polarized RPE, its regulation bymiR-184 might be crucial for RPE cell polarity and related biologicalfunctions. Further research is required to delineate the posttranslationalregulation of EZR mRNA by miR-184 and its possiblerole in RPE related diseases such as Age-related MacularDegeneration.Commercial Relationships: Nishantha Gunawardena, None;Nady Golestaneh, None; Maria Kokkinaki, None; Mia Gunawan,NoneSupport: Not applicable.Program Number: 1601 Poster Board Number: D0028Presentation Time: 8:30 AM - 10:15 AMThe role of Rgcs1 gene Spink2 in Autophagy and ganglion cellsusceptibility to optic nerve damageMargaret Maes 1, 2 , Joel Dietz 1 , Cassandra Schlamp 1 , Robert W.Nickells 1 . 1 Ophthalmology and Visual Sciences, University ofWisconsin-Madison, Madison, WI; 2 Cellular and MolecularPathology Graduate Program, University of Wisconsin-Madison,Madison, WI.Purpose: Rgcs1 is a QTL that influences ganglion cell death afteroptic nerve crush. Spink2, a gene of interest in this locus, isupregulated in mouse retina after crush. SPINK 2 is a serine proteaseinhibitor Kazal type 2. Previous studies suggest that these inhibitorsfunction as suppressors of autophagic flux (AF). High levels ofSPINK 2 may therefore alter the AF in RGCs, a process that has beenshown to increase their susceptibility to optic nerve damage.Methods: Resistant (DBA/2J) and susceptible (Balb/c) mice wereused to perform optic nerve crush. All crush studies compare thecrushed retina to the control (non-crushed) retina. Changes intranscript abundance of autophagy markers Beclin 1, Atg5, Atg7 andPik3c3 were examined 7 days post-crush using qPCR. Activatedcaspase 3/7 was measured using a cell death assay with D407 cells inthe presence or absence of SPINK 2 and/or staurosporine. Spinningdisc microscopy was used to image GFP-LC3 labeledautophagosomes in rapamycin-treated D407 RPE cells in thepresence or absence of SPINK 2. Fusion of the autophagosome andlysosome was analyzed using tfLC3 (GFP-RFP-LC3) in the presenceor absence of Rapamycin and/or SPINK 2. Quantification andkinetics of live imaging LC3 experiments were analyzed using Imarissoftware.Results: Transcript levels of autophagy markers were upregulatedpost-crush, and were significantly higher in resistant DBA/2J mice.D407 cells expressing Spink2 exhibited an increase in caspaseactivity in response to staurosporine, relative to control GFPtransfected cells. Rapamycin caused a significant increase in LC3containing vesicles within 30 minutes in cells with and withoutSPINK 2. D407 cells expressing Spink2 accumulated significantlymore vesicles. These remained small even 4 hours after rapamycintreatment, suggesting impaired fusion between autophagosomes andlysosomes. Further experiments directly examining this fusion eventare underway and results will be reported.Conclusions: SPINK 2 increases cell susceptibility to apoptoticstimuli, possibly by impairing the AF response. This provides arationale for the difference in ganglion cell susceptibility associatedwith the Rgcs1 locus.Commercial Relationships: Margaret Maes, None; Joel Dietz,None; Cassandra Schlamp, None; Robert W. Nickells, NoneSupport: NEI R01EY018869, P30 EY016665, and Research toPrevent BlindnessProgram Number: 1602 Poster Board Number: D0029Presentation Time: 8:30 AM - 10:15 AM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologymTor is Involved in the Dexamethasone Induced Expression ofβ3 Integrin in Human Trabecular Meshwork CellsJennifer A. Faralli 1 , Debjani Gagen 1 , Donna M. Peters 1, 2 .1 Pathology and Laboratory Medicine, University of Wisconsin,Madison, WI; 2 Ophthalmology and Visual Sciences, University ofWisconsin, Madison, WI.Purpose: αvβ3 integrin signaling is involved in the formation ofcross-linked actin networks (CLANs), a structure proposed to play arole in steroid induced glaucoma. The purpose of this study was todetermine the effect of dexamethasone (DEX) treatment on theexpression of β3 integrin in human trabecular meshwork (HTM)cells.Methods: Post-confluent HTM cells were treated with either 500nMDEX or 0.1% EtOH for 0-6 days. Western blot analysis and FACSwere used to determine changes in protein expression. Changes inmRNA expression were determined using qPCR in the absence orpresence of 5μg/ml actinomycin D or 25μg/ml cycloheximide. Theglucocorticoid inhibitor RU486 was used to determine if changes inmRNA levels were due to direct glucocorticoid receptor activation.The immunosuppressant rapamycin was used to determine if mTORwas involved. FKBP5 (positive control), and β1integrin subunit(housekeeping gene) were used as controls.Results: Protein expression of the β3 integrin subunit increasedstarting after 2 days of DEX treatment and remained high as long asDEX was present and for at least 14 days following the removal ofDEX. FACS analysis showed that DEX increased β3 integrinactivation at the cell surface and it remained activated for at least 7days after removal of DEX. By qPCR, DEX treatment of HTM cellsinduced a 6.2-fold increase (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyremoval of monocytes, from anticoagulated peripheral blood andmaintained under unstimulated conditions. Rotenone(0.5µM 24hrs), acomplex I inhibitor, was added to assess the susceptibility oflymphocytes to an insult causing mitochondrial dysfunction. Cellswere stained with the fluorescent red dye TMRM(25nM), incubatedat room temperature for 30min and analysed by flowcytometry(FACS Calibur, CellQuest software). A humanlymphocytic cell line(Raji) served as internal control acrossexperiments. Wilcoxon signed rank test was used to comparegroups(SPSS 20).Results: The TMRM staining expressed as the mean red fluorescence% difference from Raji (mean±SD) for the control, NTG and OHTgroups was 168.5±39.9, 166.2±39.2 and 187.2±46.2, respectively.The OHT lymphocyte ΔΨm was significantly higher than that in theNTG (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyeyes. A single antibody (Cell Signaling Technology) was able tospecifically detect recombinant p15INK4B by Western blot and inpositive skin squamous cells. This antibody labeled cell nuclei in theretinal inner nuclear and ganglion cell layers and glial columns in theoptic nerve.Conclusions: Analysis of the distribution of gene products from ornear the 9p21 gene region in the human eye indicated that restinglevels of P16INK4A/P14ARF were too low to detect but that bothMTAP and p15INK4B were present in different cells. This workprovides a framework for future studies that will seek to explore howthese gene products may influence the pathogenesis of POAG.Commercial Relationships: John P. Wood, None; Glyn Chidlow,None; Robert J. Casson, None; Shiwani Sharma, None; KathrynP. Burdon, None; Jamie E. Craig, NoneSupport: NH&MRC Grant ID APP1031347Program Number: 1607 Poster Board Number: D0034Presentation Time: 8:30 AM - 10:15 AMAnalysis of the covalent high molecular weight protein complexcontaining optineurin and its relationship with glaucomaJie Gao 1, 2 , Masafumi Ohtsubo 1 , Yoshihiro Hotta 2 , ShinseiMinoshima 1 . 1 Department of Photomedical Genomics, Basic MedicalPhotonics Laboratory, Medical Photonics Research Center,Hamamatsu University School of Medicine, Hamamatsu, Japan;2 Department of Ophthalmology, Hamamatsu University School ofMedicine, Hamamatsu, Japan.Purpose: We are analyzing the function of optineurin (OPTN) toelucidate the onset mechanism of glaucoma. It was reported that thephysiologically existing form of OPTN in cultured cells is oligomer,which is dissociated into monomers when detected by westernblotting (WB) using denatured gels under reducing condition. Werecently found an OPTN-containing high molecular weight complex(HMC) induced by H 2 O 2 treatment of cells using the same detectionmethod and named it covalent HMC. The aim of this study is toanalyze the covalent HMC and its relationship with glaucomapathogenesis.Methods: Plasmids to express wild-type (Wt) and a series of mutantOPTNs were constructed and transfected into HeLaS3 or NIH3T3cells. The mutants include the glaucoma causative ones, H26D,E50K, M98K, H486R, and R545Q, as well as a designed mutationD474N which was reported to abolish the binding with ubiquitin (Ub)and a truncated mutant LcUBD (lacking Ub-Binding Domain). TheHMC formed in the cells transfected with these constructswith/without stimuli was detected by WB using denatured gels underreducing condition. Dual-luciferase reporter assay was applied formeasuring TNFα-induced NF-κB activity (TINA) to examine if theinhibitory effect of OPTN on TINA is abolished by LcUBD andD474N.Results: 1. The covalent HMC induced by H 2 O 2 treatment of cellswas probably the covalent trimer of OPTN (cOPTN 3 ) according tothe estimated molecular weight from the WB results; 2. Theformation of cOPTN 3 was detected in not only Wt but also all of thepoint mutants including D474N when cells were stimulated withH 2 O 2 ; 3. Only in the case of E50K, the formation of cOPTN 3 wasinduced without H 2 O 2 addition; 4. cOPTN 3 was not detected inLcUBD with/without H 2 O 2 treatment; 5. TNFα did not induce theformation of cOPTN 3 .Conclusions: 1. Our results suggested that physiologically existingnon-covalent OPTN oligomer is cross-linked each other with somecovalent bond under oxidative environment with unknownmechanism. 2. E50K is a distinct mutation which possibly producesthe similar intracellular condition as H 2 O 2 treatment, suggesting apossible relationship of E50K with the severity of glaucomaphenotype. 3. The UBD is necessary for cOPTN 3 formation, but thesite essential for Ub binding seems unnecessary. 4. cOPTN 3 is notinvolved in TNFα-induced NF-κB signaling pathway.Commercial Relationships: Jie Gao, None; Masafumi Ohtsubo,None; Yoshihiro Hotta, None; Shinsei Minoshima, NoneProgram Number: 1608 Poster Board Number: D0035Presentation Time: 8:30 AM - 10:15 AMPost-transcriptional regulation of γ-synuclein expression and itsrole in glaucomatous alterationsAndrei Surguchov 1, 2 , Irina G. Surgucheva 1, 2 . 1 Research, RetinalBiology Lab, VAMCKC, Kansas City, MO; 2 Neurology, KansasUniversity, Kansas City, KS.Purpose: Glaucoma is related to the accumulation of aggregated andmisfolded proteins. γ-Synuclein (γ-Syn) accumulates in the retina andin the optic nerve astrocytes in large spheroidal structures in patientswith glaucoma and in animal models forming protease resistantintracellular inclusions. Defects in the regulation of γ-Syn expressionare responsible for the accumulation of such pathological deposits.microRNAs (miRNAs) are small, non-coding RNAs, that regulategene expression through their targets in the 3'untranslated regions ofmRNAs. Here we investigate the mechanism of post-transcriptionalregulation of γ-Syn expression by miRNAs.Methods: A 275 bp fragment was generated by PCR using human γ-Syn clone (long form, LF). LF and modified forms of this fragmentwith deleted putative miRNAs targets were inserted in a Luc reportervector and Luc activity was tested after transient transfection.Expression of miRNAs was carried out using “BLOCK-iT Pol II miRRNAi Expression Vector”. miRNAs were quantified using qRT-PCRwith TaqMan probes.Results: First we analyzed γ-Syn 3’-UTR by bioinformatics approachand identified putative targets for the following miRNAs: miR-103,107, 424, 497, 4437 and 4674 and 4722. The insertion of LFdownstream of the LUC gene caused a 51% reduction of LUCactivity, confirming the presence of effective targets for miRNAs inthis fragment. Deletion of targets for miR-103 and miR-107 from LFdoes not change the activity, although according to qRT-PCR datathese miRNAs are present in tested cell cultures. The effect oftruncated form (TF) of the 3’-UTR in which we have modifiedtargets for miR-4437 and miR-4674 on LUC activity was different: itdid not change LUC activity, while the deletion of miR-103 targetsfrom this TF increases the activity. To further investigate the effect ofmiR4437 and miR4674 on γ-Syn expression we performedexpression of these two miRNAs in SK-BR3 cells. According toWestern blotting data, expression of miR4437 caused a 69% andmiR4674 a 76% reduction of endogenous γ-Syn expression. On theother hand, in stable clones overexpressing γ-Syn no significanteffect of miRNAs on γ-Syn expression was found.Conclusions: 3’-UTR contains targets for miRNAs which are usedfor the regulation of γ-Syn expression. This mechanism of regulationis cell-specific and depends on the level of γ-Syn and miRNAsexpression.Commercial Relationships: Andrei Surguchov, None; Irina G.Surgucheva, NoneSupport: VA Merit Review, The Glaucoma FoundationProgram Number: 1609 Poster Board Number: D0036Presentation Time: 8:30 AM - 10:15 AMSoluble guanylate cyclase: an emerging therapeutic target inopen angle glaucomaEmmanuel S. Buys 1 , Yu-Chieh Ko 2 , Clemens Alt 3 , Haiyan Gong 4 ,Peter Brouckaert 5 , Janey L. Wiggs 6 , Meredith S. Gregory-Ksander 2, 6 ,Louis R. Pasquale 6, 7 , Kenneth D. Bloch 1, 8 , Bruce R. Ksander 2, 6 .©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biology1 Anesthesia, Critical Care, and Pain Medicine, MassachusettsGeneral Hospital, Boston, MA; 2 Ophthalmology, Schepens EyeResearch Institute, Boston, MA; 3 Wellman Center for Photomedicineand Center for Systems Biology, Massachusetts General Hospital,Boston, MA; 4 Ophthalmology, Boston University School ofMedicine, Boston, MA; 5 VIB Department of Molecular BiomedicalResearch, Ghent University, Ghent, Belgium; 6 Ophthalmology,Massachusetts Eye and Ear Infirmary, Boston, MA; 7 ChanningDivision of Network Medicine, Brigham and Women’s Hospital,Boston, MA; 8 Cardiology Division, Department of Medicine,Massachusetts General Hospital, Boston, MA.Purpose: Primary open angle glaucoma (POAG) is a leading causeof blindness. Available therapies offer incomplete protection. Themolecular signaling involved in the pathogenesis of POAG remainsunknown. Here, we identify the nitric oxide (NO) receptor solubleguanylate cyclase (sGC) as a key enzyme in the etiology of POAG,using a novel murine gene knockout model and genetic data from asubgroup of POAG patients.Methods: Female wild-type (WT) mice and mice lacking the α1subunit of sGC (sGCα1-/-) were studied. Thickness of the retinalnerve fiber layer (RNFL) was measured via spectral domain opticalcoherence tomography (SD-OCT). Retinal ganglion cells (RGCs) andoptic nerve axons were detected histochemically and counted.Morphology of the iridocorneal angle was examined by lightmicroscopy, ultrasound biomicroscopy, and SD-OCT. Intraocularpressure (IOP) was measured serially with a TonoLab-Tonometer.Retinal arterial function was assessed via in vivo laserophthalmoscopy. POAG cases and controls from the GlaucomaGenes and Environment (GLAUGEN) study were studied for theirassociation with genetic variants in the sGC locus.Results: Age-dependent thinning of the RNFL and loss of RGC’sand optic nerve axons in the context of an open iridocorneal anglewas observed in sGCα1-/- but not age-matched WT mice. The opticneuropathy associated with sGCα1-deficiency was accompanied by amodest (~2mmHg) and age-dependent increase in IOP and by retinalartery dysfunction. A candidate gene association study of POAG withparacentral vision loss, a POAG subtype thought to be associatedwith vascular dysregulation, identified a variant in the locuscontaining the genes encoding the α1 and β1 subunits of sGC.Together, these results highlight the relevance of sGC (and ouranimal model) in the pathogenesis of POAG.Conclusions: This study provides new insights into the pathogenesisand genetics of POAG and represents a paradigm shift in POAGresearch. Our findings suggest that therapies that do not focus onlowering IOP (e.g. targeting retinal vascular dysfunction) constitute acomplementary approach to treating POAG. In addition, ourobservations unequivocally identify perturbation of a wellcharacterizedsignaling pathway (NO-cGMP signaling) as a keymechanism in the etiology of POAG. Identifying sGC as a potentialtherapeutic target for POAG may inform the clinical development ofexisting cGMP-elevating therapeutic compounds.Commercial Relationships: Emmanuel S. Buys, None; Yu-ChiehKo, None; Clemens Alt, None; Haiyan Gong, None; PeterBrouckaert, None; Janey L. Wiggs, None; Meredith S. Gregory-Ksander, None; Louis R. Pasquale, None; Kenneth D. Bloch,None; Bruce R. Ksander, NoneSupport: NEI R21 EY020987, NEI R01 EY022746-01Program Number: 1610 Poster Board Number: D0037Presentation Time: 8:30 AM - 10:15 AMMammalian Expression and Biophysical Examination of HumanWild-Type Optineurin ProteinHongyu Ying, Xiang Shen, Minhua Wang, Beatrice Yue. Ophthal &Visual Sciences, Univ of Illinois at Chicago, Chicago, IL.Purpose: To express optineurin in mammalian cells and attain highlypurified optineurin protein for biophysical examinations. Optineurinis a gene linked to normal tension glaucoma and amyotrophic lateralsclerosis.Methods: Tetracycline inducible (Tet-on) neuronal RGC5 cell linesthat express Halo tagged wild-type optineurin (OPTN WT -Halo oroptineurin-Halo) were created by transfecting RGC5 cells withplasmid vector pTRE-OPTN WT -Halo-IRES-GFP-INS-rtTA-IREShyg-pcDNA3.1z.Cells were selected in hygromycin (100 μg/ml)-containing medium and subsequently screened for optineurinexpression by Western blotting or based on GFP expression afterdoxycycline (Dox) induction by fluorescence microscopy.Optineurin-Halo collected from the cell lysate was purified usingPromega’s HaloTag based affinity purification technique undernative conditions. The purity of the isolated protein was assessed bySDS-PAGE and Western blotting. The secondary structure ofpurified optineurin was examined by Jasco 710 circular dichroism(CD) spectropolarimeter.Results: Tet-on inducible RGC5 cell lines were established toexpress optineurin-Halo upon Dox induction. Following affinitypurification, Western blotting with anti-optineurin antibody detecteda single band at approximately 74 kDa. This highly purified tag-freeoptineurin protein was isolated with a yield of 23 µg/500 cm 2 (22 cmsquare dish). A circular dichroism (CD) spectrum indicated thatoptineurin is folded, containing both α-helical and β-sheet secondarystructures.Conclusions: Human optineurin protein expressed in stable induciblecell lines was purified and isolated. The inducible cell lines wouldallow production of highly purified protein in a scaled-up and costeffective manner, facilitating biophysical characterization ofoptineurin including its secondary structure, thermal stability andaggregation.Commercial Relationships: Hongyu Ying, None; Xiang Shen,None; Minhua Wang, None; Beatrice Yue, NoneSupport: Grants EY018828 and EY005628 (to B.Y.J.T.Y.) and coregrant EY001792 from the National Eye Institute, Bethesda,Maryland.240 Visual Cycle, Retinoids and CarotenoidsMonday, May 06, 2013 11:00 AM-12:45 PM6A Paper SessionProgram #/Board # Range: 1699-1705Organizing Section: Biochemistry/Molecular BiologyProgram Number: 1699Presentation Time: 11:00 AM - 11:15 AMMouse Cone Dark Adaptation Relies on Two Visual Cycles andIs Substantially Retarded in Mice Lacking RDH8 and ABCA4Alexander V. Kolesnikov 1 , Peter H. Tang 2 , Akiko Maeda 3 , Leah C.Byrne 4 , John G. Flannery 4 , Krzysztof Palczewski 3 , Vladimir J.Kefalov 1 . 1 Ophthalmology and Visual Sciences, WashingtonUniversity School of Medicine, Saint Louis, MO; 2 Ophthalmology,Medical University of South Carolina, Charleston, SC;3 Pharmacology, Case Western Reserve University, Cleveland, OH;4 Molecular and Cell Biology, The University of California, Berkeley,CA.Purpose: Rapid regeneration of the visual pigment following itsphotoactivation is critical for the function of cone photoreceptors. Weinvestigated the contributions of classic RPE visual cycle and novelcone-specific retina visual cycle to regeneration of mouse M-cone©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologypigment during dark adaptation. We also examined the role ofprocessing of visual chromophore (retinal) in cone outer segments(COS) by RDH8 and ABCA4 in cone dark adaptation.Methods: To test cone dark adaptation following >90% pigmentbleach, we performed transretinal ERG recordings (a-wave) fromcones in isolated Gnat1-/- mouse retinas lacking rod signaling in thepresence of synaptic blockers or standard ERG (b-wave) in liveanimals. To separate effects of the two visual cycles, we appliedintravitreal injections of light-inducible toxin Killer Red to destroyglial Müller cells that can regenerate 11-cis-retinol intraretinally or IPinjections of retinylamine, a potent inhibitor of the RPE visual cycle.The role of retinal processing in COS was tested in Rdh8-/-Abca4-/-Gnat1-/- triple knockout (TKO) mice. Photopic visual acuity inGnat1-/- and TKO mice was determined from optomotor responses.Results: M-cone dark adaptation was 4-6-fold slower in live Gnat1-/-mice treated with retinylamine indicating substantial contribution ofthe RPE visual pathway. In addition, cones in retinylamine-treatedmice were unable to maintain their sensitivity in steady bright light.On the other hand, retinylamine only slightly reduced the final levelof cone sensitivity recovery in retinas detached from the RPE. Miceinjected with Killer Red exhibited up to 2-fold lower postbleachsensitivity in their isolated retinas showing the importance of theretina visual cycle. Simultaneous deletion of RDH8 and ABCA4(whose expression in COS of Gnat1-/- mice was confirmed by IHC)retarded cone dark adaptation by 2-fold in both live animals andisolated retinas. Surprisingly, fully dark-adapted TKO cones were notchromophore-deficient, as evidenced by treatment with exogenous11-cis-retinal, but had accelerated responses compared with controlcones. Finally, the photopic visual acuity was reduced in adult TKOanimals.Conclusions: Dark adaptation of mouse cones requires the combinedaction of both RPE and retina visual cycles. Efficient processing ofretinal by RDH8 and ABCA4 in COS is essential for rapid andcomplete dark adaptation of cones.Commercial Relationships: Alexander V. Kolesnikov, None;Peter H. Tang, None; Akiko Maeda, None; Leah C. Byrne, None;John G. Flannery, None; Krzysztof Palczewski, QLT Inc (F),Polgenix Inc (E), Visum Inc (P), Amegen Inc (F); Vladimir J.Kefalov, NoneSupport: NIH Grants EY019312 and EY21126 and Research toPrevent Blindness (VJK), NIH Grants EY008061 and EY021126(KP), the Foundation Fighting Blindness (JGF).Program Number: 1700Presentation Time: 11:15 AM - 11:30 AMIdentification and Study of Two New 11-cis-Retinyl EsterSynthases in the RetinaJoanna J. Kaylor 1 , Jacob Makshanoff 1 , Jennifer Yong 1 , Tran N.Nguyen 1 , Roxana A. Radu 1 , Gabriel H. Travis 1, 2 . 1 Ophthalmology,UCLA-Jules Stein Eye Institute, Los Angeles, CA; 2 BiologicalChemistry, UCLA, Los Angeles, CA.Purpose: Biochemical and electrophysiological evidence show thatcones regenerate visual pigments by an enzymatic pathway thatoccurs in the Müller cells of the retina. Prior work from ourlaboratory indicates the existence of a retinol isomerase activity incone-dominant chicken and ground squirrel retinas. When all-transretinoland palmitoyl coenzyme A are added to microsomes preparedfrom these retinas predominantly 11-cis-retinyl esters are produced.We now have preliminarily identified the retinol isomerase asDesaturase 1 (DES1). This enzyme produces an equilibrium mixtureof cis-retinol products from all-trans-retinol, including 11-cis-retinol.In order for DES1’s retinol isomerase activity to be utilized forpigment regeneration the 11-cis-retinol production must be driven bymass action due to other 11-cis-specific enzymes downstream.Identification of the enzyme(s) that are functionally coupled to DES1that produce 11-cis-retinyl esters is the goal of this project.Methods: DES1, DGAT1 and MFAT cDNA were subcloned intomammalian expression vector pcDNA3.1. dgat1-/- mice wereobtained from Dr. Robert Farese at UCSF. Enzyme activity assayswere conducted by transfection of clones into HEK 293T cells(Polyfect, Qiagen). In vitro homogenate assays using different retinolisomers as substrate were carried out in pH 7.2 40 mM Tris buffer.Retinoids were extracted with hexane and high performance liquidchromotography (HPLC) was used to identify and quantify retinoidsformed. Numerous experimental techniques were undertaken tounderstand these proteins including gene expression (qRT-PCR andWestern), Michaelis-Menten kinetic analysis, and co-expressionstudies with DES1.Results: DGAT1 and MFAT catalyze the synthesis of 11-cis-retinylesters. MFAT preferentially esterifies 11-cis-retinol better thanDGAT1 based on kinetic analysis. Gene expression studies confirmthey are both located in the retina of the eye. dgat1-/- mice havereduced retinyl esters in the eye and assays on homogenates of eyetissues from these mice have greatly reduced 11-cis-retinyl estersynthesis. DGAT1 or MFAT coexpressed with DES1 showproduction of 11-cis-retinyl esters.Conclusions: MFAT and DGAT1 are robust 11-cis-retinyl estersynthases that may be functionally coupled to DES1 and act asdownstream regulators of 11-cis-specificity in the alternate visualcycle of the retina.Commercial Relationships: Joanna J. Kaylor, None; JacobMakshanoff, None; Jennifer Yong, None; Tran N. Nguyen, None;Roxana A. Radu, None; Gabriel H. Travis, NoneSupport: US National Eye Institute R01-EY11713Program Number: 1701Presentation Time: 11:30 AM - 11:45 AMSubstrate Specificity and Localization of AL-OL CouplingReaction in Carp ConesSatoru Kawamura 1, 2 , Shinya Sato 2 , Shuji Tachibanaki 1, 2 , TakashiFukagawa 1 . 1 Grad Sch of Frontier Biosci, Osaka Univ, Suita, Japan;2 Dept of Biol Sci, Faculty of Sci, Osaka Univ, Toyonaka, Japan.Purpose: Cones are known to regenerate visual pigments from 11-cisretinol. Our previous study showed that carp cones specifically havean enzyme activity to oxidize 11-cis retinol (alcohol) to 11-cis retinal(aldehyde) with concomitant reduction of all-trans retinal to all-transretinol (AL-OL coupling reaction, Miyazono et al., 2008, PNAS 105:16051). In this reaction, it was not necessary to add NADP + , acofactor required for a conventional retinol dehydrogenase activity.In the present study, we tried to examine the substrate specificity andlocalization of this AL-OL coupling reaction.Methods: Cones were isolated from carp retinas using a stepwisePercoll density gradient (Tachibanaki et al., PNAS, 102: 9329).Substrate specificity was examined by a pair of alcohol and aldehydeof various retinoids and related compounds. Reaction products wereanalyzed and quantified with HPLC. To examine the localization ofthe reaction, the activity was first measured in the membrane fractionand in the soluble fraction of our cone preparation, and then in a coneouter segment (COS)-rich and cone inner segment (CIS)-richmembrane fractions prepared by centrifugation after mechanicaltreatment of purified cones.Results: In the presence of all-trans retinal, 11-cis retinol and 9-cisretinol were oxidized, but other alcohols including all-trans retinoland 13-cis retinol were not oxidized. Besides all-trans retinal,hydrophobic aldehydes such as benzaldehyde and dodecanal wereeffective in the oxidation of 11-cis retinol. AL-OL coupling activity©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologywas found in the membrane fraction, and was not co-localized withvisual pigment. The activity was extracted by a detergent.Conclusions: Substrate specificity for oxidation of alcohol was high,but that for reduction of aldehyde was low. Presumably, the bindingsite for alcohol is specific only for 11-cis and 9-cis form of retinol,but the site for aldehyde accepts a wide range of hydrophobicaldehydes. This AL-OL coupling activity seems to be localized in theCIS membrane. These findings suggest that in the AL-OL couplingreaction, not only all-trans retinal produced by visual pigment bleachbut also other hydrophobic aldehydes produced in the CIS could bethe substrate to oxidize 11-cis retinol.Commercial Relationships: Satoru Kawamura, None; ShinyaSato, None; Shuji Tachibanaki, None; Takashi Fukagawa, NoneSupport: JSPS KAKENHI Grant Number 23227002Program Number: 1702Presentation Time: 11:45 AM - 12:00 PMTargeting the STRA6/RBP4 Binding interaction to TreatMacular DegenerationKrysten M. Farjo, Gennadiy P. Moiseyev, Jian-Xing Ma. Physiology,Univ of Oklahoma Hlth Sciences, Oklahoma City, OK.Purpose: Macular degeneration is initiated and perpetuated by theaccumulation of toxic Vitamin A derivatives in the retinal pigmentepithelium (RPE). Pharmacological inhibition of Vitamin A deliveryor metabolism in the RPE can significantly slow and reduce visionloss in animal models of macular degeneration. Serum retinol bindingprotein (RBP4) transports Vitamin A, and delivers Vitamin A to RPEthrough binding to its receptor, STRA6. We sought to develop andcharacterize novel inhibitory peptides that target the STRA6/RBP4interaction to reduce Vitamin A delivery to RPE.Methods: We synthesized candidate inhibitory peptides based on theprotein domains of STRA6 that are implicated in binding to RBP4(including C43, C194-Q196, Y335-G341, and R461-N462). HEK-293 cells were used to generate a STRA6-stable cell line, whichserved as the basis for the development of a high throughputSTRA6/RBP4 competition binding assay in which alkalinephosphatase-tagged RBP4 (AP-RBP4) is incubated with STRA6-stable cells alone or in combination with increasing concentrations ofcandidate inhibitory peptides. Quantification of AP activity isproportional to the amount of AP-RBP4 bound to STRA6-stablecells. In addition, we have established a cellular Vitamin A uptakeassay to evaluate the efficacy of inhibitory peptides to reduceSTRA6-mediated uptake of Vitamin A in STRA6-stable cells.Results: We have identified four inhibitory peptides that significantlyreduce the STRA6/RBP4 binding interaction and/or inhibit STRA6-mediated Vitamin A uptake.Conclusions: Peptides derived from the STRA6 protein domainsimplicated in binding to RBP4 have inhibitory activity tosignificantly reduce STRA6/RBP4 binding and STRA6-mediatedVitamin A uptake in cell culture. Ongoing studies are evaluating theability of these peptides to inhibit Vitamin A uptake and visual cycleactivity in mouse eyecups. These inhibitory peptides could serve asthe basis for the development of a novel therapeutic to treat maculardegeneration.Commercial Relationships: Krysten M. Farjo, None; Gennadiy P.Moiseyev, Charlesson LLC (E); Jian-Xing Ma, NoneSupport: AHAF M2012057, OCAST HR10-150, FFS-GIA-10-015Program Number: 1703Presentation Time: 12:00 PM - 12:15 PMEvaluation of different classes of RBP4 antagonists as potentialtreatments for AMDNicoleta Dobri 1 , Qiong Qin 1 , Jian Kong 1 , Rando Allikmets 1, 2 , JanetR. Sparrow 1, 2 , Konstantin Petrukhin 1 . 1 OPHTHALMOLOGY,COLUMBIA UNIVERSITY, NEW YORK, NY; 2 PATHOLOGY,COLUMBIA UNIVERSITY, NEW YORK, NY.Purpose: Pharmacological inhibition of the retinol-inducedinteraction of Retinol-Binding Protein 4 (RBP4) with transthyretin(TTR) in the serum may decrease the uptake of serum retinol to theretina and reduce formation of lipofuscin bisretinoids. A1120 is nonretinoidRBP4 ligand capable of antagonizing retinol-induced RBP4-TTR interaction. Here we present characterization of A1120 andother classes of RBP4 agonists in a battery of in vivo and in vitroassays.Methods: RBP4 binding potency, ability to antagonize RBP4-TTRinteraction and compound specificity were compared for therepresentatives of different classes of RBP4 antagonists. Specificityof compounds was confirmed in in vitro assays probing thecompound effect on the activity of protein targets capable of bindingdifferent types of retinoids. The in vivo effect of compoundadministration on levels of serum RBP4, visual cycle retinoids,lipofuscin bisretinoids, and retinal visual function was evaluatedusing a combination of biochemical and electrophysiologicaltechniquesResults: We documented significant reduction of serum RBP4 inresponse to administration of RBP4 antagonists representing differentstructural classes. Compound administration induced partial depletionof visual cycle retinoids and significant inhibition of bisretinoidaccumulation in the mouse model of enhanced lipofuscinogenesiswhile no significant changes in kinetics of dark adaptation after thephotobleach were evident following long-term compound dosing.Conclusions: Medicinal chemistry optimization in selected structuralseries may yield drug candidates with optimized potency andimproved pharmacokinetic characteristics which could allow theiruse in treatment of dry AMD, Stargardt disease and other conditionscharacterized by excessive lipofuscin accumulation.Commercial Relationships: Nicoleta Dobri, None; Qiong Qin,None; Jian Kong, None; Rando Allikmets, None; Janet R.Sparrow, None; Konstantin Petrukhin, NoneSupport: NIH Grants R21 NS067594 (KP), U01 NS074476 (KP),R24 EY019861 (RA, JRS, KP), R01 EY012951 (JRS), P30EY019007 (Core Support for Vision Research), and unrestrictedfunds from Research to Prevent Blindness (New York, NY) to theDepartment of Ophthalmology, Columbia University, and gifts fromThe Burch Family Foundation, the Mary Jaharis-John CatsimatidisScholarship Fund, the Kaplen Foundation, and the Eye Surgery FundProgram Number: 1704Presentation Time: 12:15 PM - 12:30 PMBestrophinopathy Large Animal Model Shows AbnormalAccumulation of Lipofuscin in the Retinal Pigment EpitheliumNestor Mas Gomez 1 , Emily V. Dutrow 1 , Simone Iwabe 1 , Frank P.Stefano 2 , Kathleen Boesze-Battaglia 2 , Gustavo D. Aguirre 1 , KarinaE. Guziewicz 1 . 1 Department of Clinical Studies, University ofPennsylvania, Philadelphia, PA; 2 Department of Biochemistry,University of Pennsylvania, Philadelphia, PA.Purpose: BEST1 gene encodes for bestrophin-1, an integralmembrane protein localized basolaterally in the RPE. BEST1mutations are responsible for a group of inherited retinal disordersknown as bestrophinopathies. A major pathological hallmark of thesediseases is lipofuscin overload within the RPE cell monolayer. Themain lipofuscin component, fluorophore A2E, has been shown tocause accumulation of free and esterified cholesterol in RPE cells.The aim of this study was to investigate whether canine multifocalretinopathy 1 (cmr1), a large animal model for human©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologybestrophinopathies, demonstrates this extensive buildup of lipofuscin,and whether these accumulations correlate with increased levels ofcholesterol in the cmr1-affected RPE.Methods: cmr1 disease progression was monitored in vivo bycSLO/SD-OCT using NIR and FAF modes. Retinal cryosectionswere obtained from control and dogs with advanced disease selectedfor autofluorescence spectra study, and lipofuscin granules werequantified in comparison to WT control. Sections were excited at406nm and emission spectra were recorded from 406-731nm.Cholesterol was localized and quantified by filipin binding. Forcholesterol and A2E correlation studies, sections were excited at406nm and images were collected at 420-440nm and 470-570nm,respectively. The percentage of co-localization was determined asPearson coefficient. Data analysis was performed with the 4.0Elements software of the Nikon A1R laser scanning confocalmicroscope.Results: Both the cmr1-affected and the WT RPE accumulatedautofluorescencent debris with age; however, cmr1 tissue showed anabnormal buildup and 2.5 fold increase (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyResults: The results show that only the fraction corresponding to2.5% BSA contained most cells displaying PROX-1 and Islet-1positive immunoreactivities with a typical HC morphology. Based onan accurate morphological analysis, a number of cells in this fractionresembled H1- and H3-types of HCs. Strikingly, Opn4ximmunoreactivitywas observed in cultures from both the 2.5 and 3 %BSA gradient phases. It is noteworthy that the 3% phase containscells that express the neuronal filament of 200 KDa (NF200) anddisplay longer processes that morphologically resemble RGCs. Byflow cytometry we found a 30% of cells from the wholedisaggregated retina and 80% of cells from the 2.5% of BSA gradientthat were positively labeled for Prox-1 respectively. Afterimmunopurification, cultures contained only OPN4x (+) HCs whichwere also found to express the Gq mRNA.Conclusions: In conclusion, by means of this method we selectivelyobtained primary cultures highly enriched in HCs expressing nonvisualphotoreceptor components.Commercial Relationships: Mario E. Guido, None; Luis P.Morera, None; Nicolás Díaz, NoneSupport: ANPCyT-FONCyT PICT Bicentenario 2010 Nr. 647,PICT, CONICET, SeCyT-UNC, and MinCyT of Córdoba.Program Number: 2008 Poster Board Number: D0039Presentation Time: 11:00 AM - 12:45 PMEffect of prostaglandin E2 on collagen gel contraction in mouseretinal pigment epithelium cellsTomoko Orita, Kazuhiro Kimura, Koh-hei Sonoda. Ophthalmology,Yamaguchi University, Ube, Japan.Purpose: An epithelial to mesenchymal transition of retinal pimentepithelium (RPE) cells plays an important role in the formation ofmembrane in proliferative vitreoretinopathy (PVR) or scar tissue inage-related macular degeneration. Growth factors and inflammatorycytokines are involved in this process. Meanwhile prostaglandin E2(PGE2) has a variety of strong physiological effects and is concernedwith inflammation. Some articles reported that the level of PGE2 invitreous body increases in PVR patients. In this study, weinvestigated that effect of PGE2 on fibrillary twitch of RPE cells byusing collagen gel contraction model.Methods: Mouse RPE cells were prepared and incubated in threedimensional type 1 collagen gel culture system with transforminggrowth factor (TGF)-β2 (1ng/ml) in the absence or presence of PGE2(0-10μM). The expression of α-smooth muscle actin and matrixmetalloproteinase (MMP)-3 as well as the phosphorylation of Smad2and paxillin were examined by immunoblot analysis.Results: TGF-β2 significantly caused a gel contraction in RPE cells,oherwise PGE2 inhibited this effect of TGF-β2 in does dependentmanner. The expression of α-smooth muscle actin as well as thephosphorylation of Smad2 and paxillin induced by TGF-β2 was alsoreduced by PGE2. In contrast, TGF-β2 have no effect on theexpression of MMP-3.Conclusions: PGE2 inhibited the RPE cell mediated collagen gelcontraction. This indicates that PGE2 may inhibit the contraction ofproliferative membrane in progressive vitreoretinal diseases.Commercial Relationships: Tomoko Orita, None; KazuhiroKimura, None; Koh-hei Sonoda, NoneProgram Number: 2009 Poster Board Number: D0040Presentation Time: 11:00 AM - 12:45 PMAge-dependent Biochemical Changes in the Retina ofGlutaredoxin 2 (Grx2) Knockout MiceHongli Wu 1 , Marjorie F. Lou 1, 2 . 1 VBS, University of Nebraska-Lincoln, Lincoln, NE; 2 Redox Biology Center, University ofNebraska-Lincoln, Lincoln, NE.Purpose: Glutaredoxin 2 (Grx2) is a mitochondrial isozyme ofthioltransferase in the oxidoreductase family that is a key regulator ofredox homeostasis in the cells through dethiolation of proteinglutathionemixed disulfides (PSSG). Previously, we have found thatGrx2 gene knockout (KO) mouse developed cataract faster duringaging, and that the lens epithelial cells were more sensitive tooxidative stress-induced apoptosis. In this study, we investigated thepresence of Grx2 in the mouse retina, and the potential biochemicalchanges in the mouse retina with Grx2 KO during aging.Methods: Grx2 gene KO mouse model was used to study the agedependentchanges in the retina. Neruo-retina tissues from 1, 7, and16 months old Grx2 KO mice were surgically removed. Five pairs ofthe retina tissues were pooled, homogenized with a glass-to-glasshomogenizer. The mitochondrial fraction was isolated followingpublished procedure. Retina from age-matched wild type (WT)littermates were processed in parallel and used as the control. Thelevels of glutathione (GSH) and protein thiols (PSH) were eachquantified by DTNB colorimetric method. PSSG were analyzed usingWestern blot with GSH-specific antibody. Mitochondrial complex I,complex IV activity and ATP levels was each measured usingmicroplate assay kit.Results: Grx2 was found in the neuro-retinal tissues with higherprotein level and enzyme activity than that of the lens from the sameanimal. Grx2 was absent in the retina of the Grx2 KO model. Withincreasing age, both WT and Grx2 KO groups showed a gradualdecrease in GSH and PSH levels, but this alteration was moreexaggerated in the Grx2 KO group. Glutathionylated protein or PSSGaccumulation in the retina was more prominent in the Grx2 KO micethan that of the WT controls. Remarkably, in comparison with theWT control, the retina in Grx2 gene KO mice showed extensive loss(50%) in complex I and complex IV enzyme activities. The ATP poolin the same Grx2 null retina was correspondingly suppressed to lessthan 30% as that of the WT control.Conclusions: Grx2 was found in the retina and deletion of its geneaffected the redox homeostasis and compromised the mitochondrialfunction. It is likely that mitochondrial dethiolase Grx2 may play animportant role in regulating retinal function.Commercial Relationships: Hongli Wu, None; Marjorie F. Lou,NoneSupport: NIH Grant EY010595Program Number: 2010 Poster Board Number: D0041Presentation Time: 11:00 AM - 12:45 PMRole of Hypoxia-inducible Factor 1 Alpha in Hydrogen PeroxideinducedHuman Retinal Pigment Epithelial Cell DeathPiyush C. Kothary, Patrick Lee, Neil B. Parikh, Sara Abraham,Monte A. Del Monte. Ophthalmology, Univ of Michigan-Kellogg EyeCtr, Ann Arbor, MI.Purpose: Molecular biological investigations led the development ofanti-vascular endothelial growth factor (VEGF) therapy for choroidalneovascularization (CNV). Since anti-VEGF therapy has not beeneffective in all patients, we investigated the mechanism of action ofVEGF: whether there is a role for hypoxia-inducible Factor 1 Alphain the VEGF pathway in our cultured human retinal pigmentepithelial (hRPE) cell model.Methods: Human RPE cells were cultured from normal eyesobtained from the Michigan Eye Bank. Cellular proliferation in thepresence of increasing concentrations of FBS was measured by 3(H)thymidine incorporation (3H-thy) and cell viability by the trypan blueexclusion method. Intracellular synthesis of caspase 3, an apoptosiscell death marker, HIF 1 alpha, a regulatory subunit of hypoxiainduciblefactor 1 and VEGF were quantitated and localized byimmunoprecipitation and immunohistochemistry. Statistical analysis©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologywas done by Student t-test with p≤0.05 considered to be significant.Results: Fetal bovine serum stimulated hRPE cell proliferation andH2O2 exposure inhibited proliferation of viable hRPE cells also in ain a dose dependent manner. H2O2 (0.5 mM) exposure decreasedhRPE cell number by 209.6% (5.70±0.73 vs 2.72±0.35, (Viable cellsx 10,000±SEM, n=4, p≤0.05)) and increased 14C-caspase-3 and 14C-HIF1 alpha synthesis in a dose dependent manner. H2O2 (0.5 mM)exposure increased 14C-caspase synthesis by 209.4% (1022.44±432vs. 2141.26± 666.31, n=4, p≤0.05)) and HIF1 alpha by 211.1%(197.62±43.44 vs. 417.20± 21.34, n=6, p≤0.05)). H2O2 (0.5 mM)exposure also increased 14C-VEGF synthesis by 130.0% (613.43.26±143.49 vs.793.68 ±124.02, n=4, p≤0.05)). Phase contrast microscopyconfirmed oxidative damage hRPE cell morphology following H2O2exposure. Immunohistochemical analysis confirmed and localizedincreased expression of HIF1 alpha and VEGF expression inpresence of H2O2 when compared with controls.Conclusions: Our data suggest that H2O2 may stimulateangiogenesis in CNV by stimulating HIF1 alpha as well as VEGF,suggesting anti-HIF1 alpha targeted therapy may provide a novelapproach to treating CNV.Commercial Relationships: Piyush C. Kothary, None; PatrickLee, None; Neil B. Parikh, None; Sara Abraham, None; Monte A.Del Monte, NoneSupport: Supported by the Skillman Foundation EndowmentMADM.Program Number: 2011 Poster Board Number: D0042Presentation Time: 11:00 AM - 12:45 PMExtremely brief light-preconditioning in pigmented mouse modelof light-induced retinal degeneration (LIRD)Priscila P. Cunha, Micah A. Chrenek, Jana T. Sellers, Jeffrey H.Boatright. Ophthalmology, Emory University School of Medicine,Atlanta, GA.Purpose: Preconditioning of albino mice using moderate intensitycyclic light may be confounded due to shortened photoreceptor outersegments and reduced rhodopsin expression such that the retinas areless sensitive to light. We hypothesize that preconditioning can beinduced in pigmented mice with brief, sub-toxic light exposure. Herewe describe the parameters used to induce such preconditioning.Methods: Sixty-four male 129sv mice were obtained from CharlesRiver (100 days old) and kept in a 12:12 light: dark cycle. Lightpreconditioningwas done using the following procedure: mice weretreated with 1% atropine eye drops at 9:00AM and maintained in thedark for 1 hour to allow the pupils to dilate. Non-toxic light exposureconsisted of 5k lux of white light for four hours using a LED lamp.Light-preconditioning was done either 1 or 3 days before LIRD.LIRD was done according to the following procedure: at 9:00AMmice were treated with 1% atropine eye drops and maintained in thedark for 1 hour to allow the pupils to dilate. Toxic light exposureconsisted of 40-50k lux of white light for four hours using a LEDlamp. Visual acuity was measured using OptoMetry optokinetictracking system (OKT). Retinal function was assessed using ERGs:scotopic a- and b-waves were measured using 5 flash intensities(0.0039-24.9 cd s/m^2).Results: We found that intense white light causes light damage in129sv mice. This can reliably be measured with either OKT (82% ofdim controls, P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyMicah A. Chrenek, Jana T. Sellers, Stephanie L. Foster, Dustin R.Zuelke, Tiffany L. Liao, John M. Nickerson, Jeffrey H. Boatright.Ophthalmology, Emory University, Atlanta, GA.Purpose: We have developed a pigmented mouse model for lightinduced retinal degeneration (LIRD). Transgenic mice are commonlyavailable on the 129sv background and these mice can be used foroptokinetic tracking (OKT). Here we test our hypothesis that it ispossible to light damage retinas in a pigmented strain of mice usinghigh intensity white-light LED boxes and that the retinal damagewould be similar to LIRD in BALB/c albino mice.Methods: Light damage: 129sv and BALB/c mice were obtainedfrom Charles River. 129sv mice were pretreated with 1% atropineeye drops to dilate pupils prior to light exposure. BALB/c mice weretreated with 5-10k lux white light for 4 hours starting at ZT2. 129svmice were treated with 50-70k lux for 4 hours starting at ZT2. OKT:visual acuity in 129sv mice was measured using an OptoMotry OKTsystem 6 days after light damage. ERG: retinal function wasmeasured 7-10 days following light exposure using scotopic ERGs.Histology: eyes were embedded in epon and toluidine blue sectionswere imaged using brightfield microscopy.Results: Exposure to toxic light induces LIRD in 129sv mice. OKTresults showed a dose dependent reduction in visual acuity with 50klux for 4 hours (0.292+/-0.021) significantly (P=0.029) reducingOKT response compared to dim controls (0.446+/-0.081) and nodetectable response with 70k lux (0+/-0) for 4 hours (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologymacula compared to the periphery (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologybe associated to the abnormal pigmentation observed in the RPE ofthe Oa1-/- mouse model of ocular albinism.Commercial Relationships: Sonia Guha, None; Alejandra Young,None; Debora B. Farber, NoneSupport: The Vision of Children GrantProgram Number: 2018 Poster Board Number: D0049Presentation Time: 11:00 AM - 12:45 PMRosemary extract and its effects on retinal gene expression withand without photic challengeAlison Ziesel 1 , Daniel T. Organisciak 3 , Ruth Darrow 3 , ChristineRapp 3 , John C. Lang 2 , Paul Wong 1 . 1 Emory University, Atlanta, GA;2 University of Texas at Arlington, Cedar Hill, TX; 3 Wright StateUniversity, Dayton, OH.Purpose: Components of the herb rosemary (Rosemarinusofficinalis) are known to possess antioxidant properties. We haveundertaken a study of these effects and their efficacy in reducing orpreventing oxidative damage done to the retina during acute lightdamage by examining the effects of rosemary treatment on retinalgene expression in the presence and absence of intense light mediateddamage.Methods: Dark-reared male Sprague-Dawley rats were either (a)injected with rosemary extract or a vehicle solution, treated with 24hours of green light challenge followed by a 24 hour light recovery,or (b) injected with a rosemary extract and maintained under normalconditions for zero, 1 or 5 hours post-injection. Subjects were theneuthanized and retinas excised and snap frozen. RNA was extractedusing a Trizol/Qiagen RNEeasy extraction method and subjected toAgilent NanoDrop analysis to ensure high RNA integrity. RNA wasthen assayed on Affymetrix Rat Gene 1.0 ST microarrays, and theresulting data were analyzed using R and BioConductor.Results: Comparing rosemary treated, non-light damaged retina tountreated controls, we see a modest decrease in expression of Hspafamily genes, homologous to HSP70 in humans. A decreased level ofHspa family expression in this study may suggest a lower turnover ofHspa proteins. Similarly, many members of the crystallin family sawa slight decrease in expression level, possibly implicating a similaractivity by rosemary's antioxidant components on crystallin proteins.In our light damage rosemary treated versus vehicle treated studies,most Hspa family genes do not exhibit a marked change in geneexpression, tending towards slightly higher in the rosemary treatedretinas. Conversely most crystallin family genes show a steepincrease in expression in vehicle treated light damaged retinas, withcrystallin expression levels remaining comparatively lower in therosemary treated retinas.Conclusions: We believe that our initial findings support the notionthat rosemary's antioxidant components may confer a resistance tooxidative damage by protecting oxidation response proteins frominitial damage. We believe this produces a protective context thateither slows or mitigates initial oxidative damage, and that moredetailed investigation of rosemary's mode of action on retinal geneexpression warrants consideration.Commercial Relationships: Alison Ziesel, None; Daniel T.Organisciak, Alcon Research, Ltd (F); Ruth Darrow, None;Christine Rapp, None; John C. Lang, None; Paul Wong, NoneSupport: NIH P30EY006360, Ohio Lions Eye Research Foundation,Alcon Research Ltd.Program Number: 2019 Poster Board Number: D0050Presentation Time: 11:00 AM - 12:45 PMDopamine Entrains The Circadaian Rhythm of PER2::LUCBioluminescence in The Mouse Retinal Pigment EpitheliumKenkichi Baba, Susana Contreras-Alcantara, Gianluca Tosini.Morehouse School of Medicine, Atlanta, GA.Purpose: The retinal pigment epithelium (RPE) plays an importantrole in the maintenance of the health and function of photoreceptors.Previous studies have shown that the RPE is also involved in theregulation of disc shedding, a process that is vital for photoreceptorhealth. This process has been shown to be under circadian control.We have recently reported that Per2 mRNA levels in the RPEchoroidshow a clear circadian rhythm in vivo, and the culturing ofRPE-choroid tissue obtained from PERIOD 2::LUCIFERASE(PER2::LUC) mice showed robust circadian rhythm inbioluminescence. Additional studies have indicated that thePER2::LUC rhythm was not phase-shifted by light, suggesting thatother signals are used by the retina to entrain this circadian rhythm.In this study we investigated whether dopamine (DA) and/ormelatonin (MLT) are capable to phase-shift this circadian rhythm.Methods: Eyes were obtained from PER2::LUC mice, the eye-cupscontaining RPE were dissected, flattened and cultured at 37 oC with199 medium containing D-Luciferin K salt. The bioluminescenceemitted by the tissue was recorded by a luminometer (Lumicycle).After 3-4 days of culture, DA (100uM), MLT (100nM) and DAreceptor agonists (SKF38393: 50uM, Quinpirole: 50uM) were addedto the culture at different circadian times, and the recording wascontinued another 5 days.Results: Administration of DA was able to phase-shift PER2::LUCbioluminescence rhythm in the RPE-choroid during the earlysubjective day CT0-CT6, the action of DA appeared to be mediatedby D2-like receptors, since Quinpirole (D2-like agonist) induced asignificant phase-shift during the early subjective day, whereasSKF38393 (D1-like agonist) was not able to produce a significantphase-shift of the PER2::LUC bioluminescence rhythm. Consistentlywith these results, we observed that Period1 mRNA level was upregulatedsignificantly up-regulated 1 hour after the addition of DA tothe culturing medium. MLT did not produce any significant phaseshifton the circadian rhythm in PER2::LUC bioluminescence.Conclusions: Our data indicate that DA via D2-like receptors canphase-shift the circadian rhythm in PER2::LUC bioluminescencerhythms in the mouse RPE. Our study also indicates that this newpreparation may be useful for elucidating the cellular and molecularmechanisms responsible for the regulation of the physiologicalrhythmic event in the RPE.Commercial Relationships: Kenkichi Baba, None; SusanaContreras-Alcantara, None; Gianluca Tosini, NoneSupport: NIH grants EY0208821 and EY 022216Program Number: 2020 Poster Board Number: D0051Presentation Time: 11:00 AM - 12:45 PMThe transcription factor neural retina leucine zipper controlsphotoreceptor-specific expression of Reep6Hong Hao 1 , Shobi Veleri 1 , Bo Sun 1 , Raman Sood 2 , Paul Liu 2 , AnandSwaroop 1 . 1 Neurobiol, Neurodegnrtn & Rpr Lab, National EyeInstitute, Bethesda, MD; 2 National Human Genome ResearchInstitute, Bethesda, MD.Purpose: The transcription factor neural retina leucine zipper (NRL)is essential to specify rod photoreceptor cell fate during retinadevelopment, as targeted deletion of Nrl (-/-) in mice led to acomplete absence of rod photoreceptors. Nrl -/- rod precursors fail toturn on rod genes and differentiate into functional conephotoreceptors. Previous ChIP-seq analysis of mouse retina revealedNRL binding to the intron of the receptor accessory protein 6(Reep6). Our goal is to elucidate the mechanism of transcriptionalregulation of the Reep6 gene and to understand the molecularmechanism of NRL action.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyMethods: We performed morpholino experiments in zebra fish totest the functional relevance of Reep6 in retina development. Tostudy the transcriptional regulation of Reep6 gene, we used acombined approach including rapid analysis of cDNA ends (5’-RACE), chromatin immunoprecipitation, luciferase reporter assay,promoter mapping in retina explant and in vivo shRNA analysis.Results: Knockdown of Reep6 in zebrafish by morpholinos led tomicrophthalmia. NRL binding to the Reep6 intron enhanced itspromoter activity in a luciferase reporter assay and also in a GFPreporter assay in retina explant.Conclusions: Reep6 expression is controlled by NRL through anenhancer located within its intron. NRL direct target genes serve asexcellent candidates for retinal disease gene discovery.Commercial Relationships: Hong Hao, None; Shobi Veleri, None;Bo Sun, None; Raman Sood, None; Paul Liu, None; AnandSwaroop, NoneSupport: NEI intramural supportProgram Number: 2021 Poster Board Number: D0052Presentation Time: 11:00 AM - 12:45 PMElucidating the mechanism behind enhanced retinal transductionof an AAV2 variantKenton T. Woodard, Richard Samulski. Gene Therapy, Neurobiology,Univ of NC at Chapel Hill, Chapel Hill, NC.Purpose: Photoreceptor dystrophies lead to reduced vision as thephotoreceptors degenerate. Gene therapy using Adeno-associatedvirus (AAV) has shown to be safe and effective at preservingphotoreceptors. This preservation is robust when AAV is deliveredsubretinally, but inefficient when delivered intravitreally (IVit) eventhough IVit delivery of AAV offers practical advantages. Of thenaturally occurring AAV serotypes, most don't transduce the retinaIVit, and of the serotypes that can, transduction is limited to theganglion cell layer. Efforts are now underway to develop mutantAAVs that are capable of photoreceptor transduction via IVitdelivery.Methods: We developed mutations on the AAV2 capsid whichdisplayed enhanced transduction of multiple retinal cell layers acrossseveral animal models. This mutant was dubbed AAV2.5. Ourinterest was to determine the mechanism of action for thisenhancement. We assessed difference in receptor usage and receptorbinding affinity of AAV2 and AAV2.5 in vitro using several celllines and affinity chromatography. To identify key amino acidsinvolved in the enhancement, we generated chimeric capsid carryingeach of the 5 amino acids modified in AAV2.5 separately andproduced viral preps carrying reporter gene for testing in vivo.Finally, we tried to promote greater transduction in the retina bymixing soluble heparin with AAV2 and AAV2.5.Results: We saw no difference in receptor usage or affinity betweenAAV2 and AAV2.5, indicating a still unknown mechanism for theenhancement in the retina. Similar to published results in muscle, thethreonine insertion found in AA2.5 but absent from AAV2 seemed tobe a key amino acid necessary for increased retinal transduction.While soluble heparin mixed with AAV2 showed a greater number ofretinal cells being transduced, soluble heparin mixed with AAV2.5showed no increase in transduction.Conclusions: The lack of difference in primary heparin sulfatereceptor usage and affinity may indicate that AAV2.5 has asecondary receptor usage or downstream advantages in the viralendocytic pathway. Current work is aimed at determining whatmechanisms are at play for enhanced retinal transduction, notably byreal time imaging of capsid particle critical steps. Future work willinvestigate newly identified amino acid insertions in combinationwith chimeric AAV2.5 to fully exploit maximum robust transductionprofile in ocular target tissue.Commercial Relationships: Kenton T. Woodard, None; RichardSamulski, Asklepios BioPharmaceutical, Inc. (I)Support: NEI Grant EY0199555-01Program Number: 2022 Poster Board Number: D0053Presentation Time: 11:00 AM - 12:45 PMCharacterization of Age Related Maculopathy Susceptibility 2(ARMS2) transcripts in human and Cynomolgus macaque retinaPeng Yu, Darius Donohue, Kristina L. Rhoades, Carl Romano,Rajkumar V. Patil. Opthalmology, Novartis Institutes for BiomedicalResearch, Alcon Research Ltd., Fort Worth, TX.Purpose: ARMS2 gene is the second major risk allele for AMD,contributing independently of complement factor H to disease risk.The rs10490924 (A69S) SNP in ARMS2 accounts for approximately35% of the population-attributable risk for the development of AMD.However, there are controversies regarding its function, localization,expression and whether it is a real gene or pseudo gene. The presentstudy was undertaken to gain an understanding of ARMS2 transcriptsin human and macaque retina.Methods: Total RNA from human and macaque retinas was reversetranscribed and PCR and qPCR were performed using customdesigned primers and probes. For macaque, primers were designedbased on homologous human sequence. The cDNAs of varioushuman tissues including placenta were purchased from Zyagen. ThePCR products of interest were purified and sequenced to confirm theauthenticity of the amplified products.Results: PCR and qPCR results showed that two splice variants ofARMS2 mRNAs are expressed in human retina. DNA sequencingresults of PCR products revealed that a larger splice variant ofARMS2 (designated as variant A) was detected in 5 human retinaswith more common alleles at R38 and A69, whereas, a shorter splicevariant of ARMS2 (designated as variant B) was detected in 7 humanretinas and all of them showed “C to T” SNP at rs2736911.Quantitative RT-PCR results suggested that expression level ofvariant B was much higher than the variant A (the Δcycle is about 4-5). In macaque retina, only variant A was detected. In human,ARMS2 expression was detected only in placenta besides retina.Conclusions: Two ARMS2 splice variants are expressed in humanretina, whereas, only variant A is expressed in macaque retina.Variant B is the major transcript of ARMS2 in human retina. Ourresults suggest that variant A carried more common alleles at both,R38 and A69 positions, whereas, variant B always carried R38X SNPand common allele at A69 suggesting that R38X variation may be aprerequisite for variant B splicing. Further studies are required tounderstand the function, cellular localization, and the pathogenic roleof ARMS2 splice variants in the development of AMD.Commercial Relationships: Peng Yu, Alcon/Novartis (E); DariusDonohue, Alcon/Novartis (E); Kristina L. Rhoades, Alcon/Novartis(E); Carl Romano, Alcon/Novartis (E); Rajkumar V. Patil,Alcon/Novartis (E)Program Number: 2023 Poster Board Number: D0054Presentation Time: 11:00 AM - 12:45 PMGeneration of GARP2-Specific Knockout Mice Using Zinc FingerNuclease TechnologySteven J. Pittler 1 , Delores Davis 1 , Larry W. Johnson 2 , Robert A.Kesterson 2 . 1 Vision Sciences, Univ of Alabama at Birmingham,Birmingham, AL; 2 Genetics Research Division, Univ of Alabama atBirmingham, Birmingham, AL.Purpose: The Cngb1 locus encodes the rod photoreceptor cGMPgatedcation channel β-subunit and two soluble glutamic acid rich©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyproteins, GARP1 and GARP2. GARPs are generated by alternativesplicing towards the 5’-end of the gene. A knockout null mousedeleted for all three proteins in rods exhibits disorganized rod outersegment (ROS) structure and an attenuated photoresponse. To furtherunderstand the role of GARPs in ROS we have used zinc fingernuclease (ZFN) genome editing technology to selectively remove aGARP2 unique exon from the mouse genome.Methods: A FokI restriction nuclease site adjacent to suitable zincfinger DNA recognition sequences was identified downstream ofGARP2-specific exon 12a. Two ZFN mRNAs encoding zinc fingerDNA recognition domains and half of FokI restriction endonucleasewere generated by T7 transcription using Sigma CompoZrtechnology and tested for nuclease activity in cultured mouseNeuro2A cells. ZFN RNAs were co-injected into male pronuclei ofC57Bl/6 fertilized embryos and founder mice were screened fordeletions by PCR and DNA sequencing.Results: Zinc finger nuclease RNA introduced into cultured cellsyielded significant target site cleavage of up to 11% of targetedplasmid. Microinjection of 235 embryos produced 14 founder mice ofwhich one, by PCR indicated different deletions on both alleles. DNAsequence analysis of the PCR products demonstrates 826 and 514 bpdeletions. The 826 bp deletion covers all of GARP2 specific exon 12aand the 514 bp deletion retains the acceptor splice site and the 8amino acid protein coding region and stop codon within this exon.This founder mouse was crossed to wild type mice yielding expectedMendelian transmission of each deletion allele establishing germlinetransmission.Conclusions: Two germline transmitting GARP2-specific exonknockout mice have been generated using ZFN technology. One KOallele is a GARP2-specific null that will allow assessment of therequirement for GARP2 function in rods, and the partial KO may bea GARP2 hypomorph with reduced expression of GARP2 that willcomplement our GARP2 overexpression studies. ZFN technology is arapid and efficient method of targeted genome editing.Program #/Board # Range: 2131-2134Organizing Section: Biochemistry/Molecular BiologyProgram Number: 2131Presentation Time: 2:50 PM - 3:15 PMTherapeutics and Diagnostics for the Elimination ofOnchocerciasisKim D. Janda. Chemistry, Immunology and Microbial Sciences, TheScripps Research Institute, La Jolla, CA.Commercial Relationships: Kim D. Janda, NoneProgram Number: 2132Presentation Time: 3:15 PM - 3:40 PMInterrogating chemical libraries for therapeutic insightsJames Inglese. Pre-Clinical Innovation, National Center forAdvancing Translational Sciences (NCATS), Rockville, MD.Commercial Relationships: James Inglese, NoneProgram Number: 2133Presentation Time: 3:40 PM - 4:05 PMDietary Omega 3 Fatty Acids and RetinopathyLois E. Smith. Ophthalmology, Harvard Univ/Childrens Hospital,Boston, MA.Commercial Relationships: Lois E. Smith, NoneProgram Number: 2134Presentation Time: 4:05 PM - 4:30 PMA Kinetic Brake to Prevent Vitamin A DimerizationIyas Washington. CUMC, New York, NY.Commercial Relationships: Iyas Washington, None292 Phototransduction, Retinal Development, Molecular Biologyand BiochemistryMonday, May 06, 2013 2:45 PM-4:30 PMExhibit Hall Poster SessionProgram #/Board # Range: 2450-2478/D0055-D0083Organizing Section: Biochemistry/Molecular BiologyZFN-mediated genome editing PCR results. WT mice yield a 2.15 kbamplicon. Crossing of the founder mouse (F0) to WT mice producedoffspring that were heterozygous for the WT allele and one of thedeletion alleles (Δ826 or Δ514).Commercial Relationships: Steven J. Pittler, None; Delores Davis,None; Larry W. Johnson, None; Robert A. Kesterson, NoneSupport: R01 EY018143, P30 EY03039 to SJP; Services providedthrough the UAB Transgenic Mouse Facility supported by NIH P30CA13148, P30 AR048311, and P30 DK074038RAK to RAK273 Application of Pharmacologic / Chemical BiologicApproaches to Eye Disease - MinisymposiumMonday, May 06, 2013 2:45 PM-4:30 PM6A MinisymposiumProgram Number: 2450 Poster Board Number: D0055Presentation Time: 2:45 PM - 4:30 PMRetinal Horizontal cell numbers are modulated by neuronspecific methyl transferase, PRMT8Ratnesh Singh 1 , Yasutake Mori 2 , Shingo Miyata 2 , Masaya Tohyama 2 ,Visvanathan Ramamurthy 1 . 1 Ophthalmology, West VirginiaUniversity, Morgantown, WV; 2 Anatomy and Neuroscience, OsakaUniversity, Osaka, Japan.Purpose: Arginine methylation is a common posttranslational proteinmodification found in eukaryotic cells. This modification is catalyzedby family of enzymes called protein arginine methyltransferases(PRMTs). These enzymes are thought to regulate gene expressionthrough methylation of histone and non histone proteins. PRMTs aredistributed ubiquitously throughout the body including neurons.Among PRMTs, PRMT8 is unique as it is selectively present in theneurons. PRMT8 shares 80% homology with PRMT1 and isproposed to function as heterodimer. In this study, we focused on therole for arginine methylation by PRMT8 in development and functionof retinal neurons.Methods: We checked subcellular distribution of PRMT8 andPRMT1 in mouse retina by immunohistochemistry. To understandthe function of Prmt8 in retina we generated a complete knockout ofPrmt8 in mice. Electroretinographic recordings were used to assessthe physiological role of Prmt8 in retina. We examined horizontal©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologycell number by whole mount staining with calbindin antibody andcone cell numbers by a combination of opsin and peanut agglutininstaining.Results: PRMT8 staining was confined to cone photoreceptors,horizontal cell nuclei and processes, subset of amacrine cells andinner plexiform layer. Co-labeling study with cell type specificmarkers and absence of PRMT8 signal in knockout animalsconfirmed our PRMT8 localization in retina. PRMT1, a closehomologue of PRMT8 was found mainly in all inner nuclei andganglion cells indicating a distribution pattern that is different thanPRMT8. Interestingly, our preliminary study showed an increase(40%) in horizontal cell density in central part of retina in animallacking PRMT8 compared to littermate controls. Increase inhorizontal cell density was supported by increased levels of calbindinby western blotting.Conclusions: Our results demonstrate that PRMT8 and PRMT1localization differs in the retina suggesting that each enzyme has aunique functional role independent of other. Additionally our resultsshowing differences in the horizontal cell number in animals lackingPRMT8 suggest that intercellular spacing and density of horizontalcells may be controlled by epigenetic mechanisms or posttranslational protein modification by arginine methylation.Commercial Relationships: Ratnesh Singh, None; Yasutake Mori,None; Shingo Miyata, None; Masaya Tohyama, None;Visvanathan Ramamurthy, NoneProgram Number: 2451 Poster Board Number: D0056Presentation Time: 2:45 PM - 4:30 PMCharacterization of recombinant intra-melanosomal domain ofhuman tyrosinaseMonika B. Dolinska 1 , Peter S. Backlund 2 , Brian P. Brooks 1 , Yuri V.Sergeev 1 . 1 OGVFB, National Eye Institute, Bethesda, MD;2 NICHD/NIH, Bethesda, MD.Purpose: Mutations in tyrosinase gene cause an autosomal recessivedisorder, oculocutaneous albinism Type 1 (OCA1). OCA1 isclassified into a disorder with complete absence of tyrosinase activity(OCA1A) and with residual tyrosinase activity (OCA1B). Tyrosinaseis an enzyme which catalyzes the rate-limiting conversions oftyrosine to L-DOPA and dopachrome in melanin production in theskin and eye. Here we characterized intra-melanosomal domain ofhuman tyrosinase, hTyrCtr (residues 19 - 469 of the native protein).Methods: Recombinant hTyrCtr was produced in larvae (C-PERL,MD) and purified from the soluble fraction by immobilized metalaffinity and size-exclusion chromatography. The effect of variousinhibitors and activators, kinetics parameters and optimumtemperature and pH were examined by using L-DOPA and L-tyrosineenzymatic reactions. N-glycosylation sites and a composition ofsugar components in hTyrCtr were determined using MS/MS, Maldi-Tof MS, and Q-Tof MS/MS mass spectroscopy combined withtrypsin, asp-proteinase (peptide sequencing) and PNGase-Fproteinase digests (sugar composition).Results: Recombinant hTyrCtr show maximum enzymatic activity atoptimal temperature of 37°C and neutral pH. The hTyrCtr is N-glycosylated as shown by the PNGase de-glycosylation. Five N-glycosylation sites are either modified (N86, N337) or partiallymodified (N111, N230, N290) by carbohydrates. Expected asparaginsN161 and N371 are not glycosylated. Molecular modeling of humantyrosinase suggests that all observed N-glycosylation sites are locatedat the surface of intra-melanosomal domain with sugar chainsaccessible for inter-molecular interactions within the melanosome.Conclusions: Recombinant hTyrCtr is a functional N-glycosylatedenzyme. In contrast to current observation that genetic mutationsaffect just a few N-glycosylation sites our work suggests that a widerspectrum of sequence positions associated with N- glycosylationsequons, 84-91, 109-116, 228-235, 288-295, and 335-342, and mightbe associated with genetic defects. The recombinant hTyrCtr could beused to search for small molecule activators of tyrosinase mutantvariants as a potential treatment of OCA1 type of albinism.Commercial Relationships: Monika B. Dolinska, None; Peter S.Backlund, None; Brian P. Brooks, None; Yuri V. Sergeev, NoneProgram Number: 2452 Poster Board Number: D0057Presentation Time: 2:45 PM - 4:30 PMInteractions between Dopamine Receptor D4 and VisualArrestinsJanise D. Deming 1, 2 , Kayleen Lim 1, 2 , Bruce M. Brown 1, 2 , JosephPak 1, 2 , Kathleen Van Craenenbroeck 3 , Cheryl M. Craft 1, 2 . 1 Mary D.Allen Lab for Vision Research, Doheny Eye Institute, Los Angeles,CA; 2 Ophthalmology & Cell & Neurobiology, Keck School ofMedicine of Univ. Southern Calif., Los Angeles, CA; 3 Physiology,University of Gent, Gent, Belgium.Purpose: Dopamine (DA) and its G-protein coupled receptors(GPCR) contribute to normal photopic vision, including lightadaptation and contrast sensitivity, but the specific cellular pathwaysby which they contribute are unclear. Similar defective visualphenotypes are observed when either the DA receptor D4 (DRD4) orcone arrestin (ARR4) is not expressed. The purpose of this study wasto determine the contribution of ARR4 in the desensitization ofDRD4 in vitro and in photopic vision.Methods: Immunohistochemistry (IHC) was performed usingspecific antibodies on frozen sections of mouse retinas or fixedtransfected cells. C57Bl/6J mice were used, along with Drd4 -/- andthe four ARR knockout mice (J. Chen, USC; R. Lefkowitz, Duke) asnegative controls. Co-immunoprecipitation (co-IP) followed byimmunoblot analysis was used for protein-protein interactions. DRD4desensitization was measured using in vitro internalizationexperiments with tagged plasmid constructs of human DRD4 andfour ARRs in transfected HEK 293T cells incubated with or without10 µM DA.Results: IHC of control compared to Drd4 -/- retinas revealed the coexpressionof DRD4 and ARR4 in cone pedicles, with higher DRD4expression in dark vs. light adapted mice. Both βARR1 and βARR2were also expressed in photoreceptors. DRD4 interaction withβARR2 is DA independent, but our co-IP data demonstrated a DAdependent interaction between DRD4 and ARR4 but not ARR1. Invitro studies showed DRD4 internalization only when a βARR and avisual ARR were co-transfected with DRD4. Internalization washighest with βARR2 and ARR4, but other combinations revealedlimited internalization; however, none was detectable with twoβARRs or two visual ARRs.Conclusions: DRD4 and ARR4 are required for normal photopicvision and are co-expressed with ARR1 and βARRs in conephotoreceptors, particularly in the pre-synaptic terminals. Our studiesdemonstrate that for complete desensitization of the DRD4, indicatedby internalization after DA stimulation, at least one visual ARR mustbe co-expressed with a βARR. To our knowledge, no GPCR haspreviously been shown to require the expression of two differentARRs for desensitization. Our in vitro experiments indicate that notonly is the interaction of DRD4, a visual arrestin, and a βARRpossible in the cone photoreceptors, but their interaction may play anessential role in maintaining normal high acuity vision.Commercial Relationships: Janise D. Deming, None; KayleenLim, None; Bruce M. Brown, None; Joseph Pak, None; KathleenVan Craenenbroeck, None; Cheryl M. Craft, NoneSupport: NIH Grant EY015851(CMC), P30EY03040(DEI),Research to Prevent Blindness (DEI), Mary D. Allen Endowment,©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyDorie Miller, William Hansen Sandberg Found.(JDD, KL), USCProvost Undergrad. Research(KL)Program Number: 2453 Poster Board Number: D0058Presentation Time: 2:45 PM - 4:30 PMCharacterization and Post-Translational Modifications ofProline-Rich Protein 4 (PRR4) in Basal and Reflex TearProteome of HumanNatarajan Perumal, Sebastian Funke, Norbert Pfeiffer, Franz H.Grus. Experimental Ophthalmology, University Medical CenterMainz, Mainz, Germany.Purpose: It was previously reported that PRR4 is down-regulated inthe tears of dry eye patients. This study was initiated considering thelack of comprehensive characteristics profile of PRR4 in tears ofhealthy subjects and, to understand the physiological roles of PRR4in tears. Post-translational modifications and characterization ofPRR4 in basal and reflex tears were determined in the studypresented herein.Methods: Tear samples were collected employing capillary methodfor basal and reflex tears. Proteomics approaches based on 1-DE and2-DE combined with LC-ESI-LTQ-Orbitrap MS system wereemployed for characterization and determination of post-translationalmodifications of PRR4.Results: Identification and characterization of PRR4 in 2-DEutilizing LC-ESI-LTQ-Orbitrap MS yielded 10 PRR4 spots with vastpolymorphisms (molecular weight: 17 - 30 kDa, pI: 3.5 - 6.6). A newisoform designated as PRR4γ was also identified in addition to thetwo existing isoforms of PRR4. Further characterization of the PRR4isoforms identified multiple post-translational modifications, whichare methylation, oxidation and pyroglutamate formation. Label-freequantification analysis of basal and reflex tear proteome employing1-DE and LC-ESI-LTQ-Orbitrap MS demonstrated significantincrement of PRR4 for the first time in reflex tears. Correspondingly,the result also showed that more methylation, oxidation andpyroglutamate formation were found in reflex tears than in basaltears. Besides, reflex tear sample also showed significant incrementof prolactin, serum albumin, zinc-α-2-glycoprotein, haptoglobin,mesothelin and β-2-microglobulin and, decrement of Ig α-1 chain C,cystatin-S, clusterin, lipid transfer protein II and phospholipase A2compared to basal tears.Conclusions: The results of this study provide fundamental insightinto the complex characteristics of PRR4 in basal and reflex tears.These findings highlight that the multiple post-translationalmodifications of PRR4 isoforms may play crucial roles in the tearsystem and ocular surface of healthy people as well as in dry eyepatients.Commercial Relationships: Natarajan Perumal, None; SebastianFunke, None; Norbert Pfeiffer, Sensimed AG (F), Sensimed AG(R), MSD (F), MSD (R), Alcon (F), Allergan (F), Novartis (F),Novartis (R), Bayer (F), Heidelberg Engineering (F), Bausch&Lomb(F), Boehringer-Ingelheim (F), Carl Zeiss Meditech (F), Chibret (F),Nidek (F), Pfizer (F), Santen (F), Santen (R), Topcon (F), Ivantis Inc(F), Ivantis Inc (R); Franz H. Grus, NoneProgram Number: 2454 Poster Board Number: D0059Presentation Time: 2:45 PM - 4:30 PMSteric Volume Exclusion Leads to Size-dependent ProteinDistribution in Mouse Ciliary PhotoreceptorsMehdi Najafi 1, 2 , Peter D. Calvert 1, 2 . 1 Department of Ophthalmology,SUNY Upstate Medical University, Syracuse, NY; 2 SUNY EyeInstitute, Syracuse, NY.Purpose: We recently demonstrated that ‘steric volume exclusion’mediates size-dependent soluble protein distribution in Xenopuslaevis rod photoreceptors 1 . However, the large variation in themorphology of photoreceptors among species raises the question ofwhether the steric volume exclusion effect is significant for allphotoreceptors. We therefore examined the subcellular distributionsof soluble molecules of different sizes in mouse photoreceptors usinglive cell confocal imaging.Methods: Microslices of retina from dark-adapted mice expressingEGFP in rod cells under the NRL promoter were prepared underinfrared illumination, placed in an imaging chamber containingmouse ringer solution, and imaged using our custom confocalmicroscope. Distribution of EGFP in rods was compared to that ofthe small fluorescent probe Calcein introduced into WT retinas thatwere similarly prepared. Corrections were made to account for thefact that mouse rods are below the resolution limit of confocalmicrocopy.Results: The distribution of Calcein significantly differed from thatof EGFP in mouse photoreceptors. The ratio of the meanfluorescence in the outer segment to that in the myoid region(F OS /F MY ) was 0.91±0.03 in photoreceptors containing Calcein, anddropped to 0.44 ±0.06 in EGFP expressing rods. A size-dependentdistribution was evident in other areas of the cell as well. WhileCalcein uniformly filled the nucleus, EGFP was excluded from thecentral areas of this compartment where heterochromatin is located.Conclusions: Our results indicate that steric volume exclusiongoverns protein distribution in mammalian photoreceptors in muchthe same manner as in Xenopus. The lower F OS /F MY ratio in EGFPexpressing rods compared to Calcein demonstrates a size-dependentexclusion from the outer segment due to the higher packing densityof disk membranes in this compartment. We project a molecular sizecut-off of ~4.0nm for protein access to the outer segment in mousewhich is consistent with our previous estimation in Xenopusphotoreceptors (4.2 nm). Our experiments indicate that despitesignificant variation in photoreceptor morphology among species,steric volume exclusion remains a general mechanism for regulationof protein access to the ciliary outersegment compartment.1. Najafi et al. PNAS, 012 Jan 3;109(1):203-8Commercial Relationships: Mehdi Najafi, None; Peter D. Calvert,NoneSupport: NIH Grant EY018421, Research to Prevent BlindnessProgram Number: 2455 Poster Board Number: D0060Presentation Time: 2:45 PM - 4:30 PMRefined estimates of rhodopsin deactivation kinetics from mouserods lacking rhodopsin phosphorylation or Arr1 bindingOwen P. Gross 1, 2 , Edward N. Pugh 2, 4 , Marie E. Burns 2, 3 . 1 VollumInstitute, Oregon Health & Science University, Portland, OR; 2 Centerfor Neuroscience, UC Davis, Davis, CA; 3 Ophthalmology & VisionScience, UC Davis, Davis, CA; 4 Cell Biology and Human Anatomy,UC Davis, Davis, CA.Purpose: Deactivation of rhodopsin (R*) requires multiplephosphorylation of its COOH-terminal serine and threonine residuesby Grk1, followed by the binding of arrestin-1 (Arr1). Single cellrecordings from rods that under-express Arr1 have suggested that R*lifetime is normally brief and dictated largely by phosphorylation(Gross and Burns, 2010). Here, we apply a recently developed modelof phototransduction to single photon responses (SPRs) of rodslacking either phosphorylation or Arr1 binding, providing newquantitative estimates of the in situ kinetics of R* deactivation.Methods: Suction electrode recordings from intact rod outersegments were performed as previously described. Measured SPRswere compared to simulations produced by a spatiotemporalphototransduction model (Gross et al 2012a). The model input was atime series representing R* activity, generated assuming a Markov©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologydeactivation process in which Arr1 binds with high affinity after threephosphorylations.Results: The Grk1-/- SPR is approximately a step function (Chen etal., 1999), whose ~2-fold larger amplitude constrains the maximal R*activity in the context of the downstream model, and whose 3-4 smean duration sets the upper limit for the rate of Arr1 binding tounphosphorylated R*. In contrast, Arr1-/- SPRs exhibit peakamplitudes similar to wild-type SPRs and have long-lasting tailcurrents with amplitudes ~50% of the peak (e.g. Xu et al., 1997).Simulated SPRs revealed that this tail current can be explained by arelatively small fraction (~5%) of maximal R* activity that persistsafter extensive phosphorylation of R*. In order to achieve this lowlevel of R* activity on the time scale of the SPR, phosphorylation ofR* must proceed at a maximal, initial rate of 100/s.Conclusions: The deduced R* deactivation kinetics predict that 3-4G protein/PDE (G*-E*) complexes are active during the plateau tailphase of the Arr1-/- SPR, a number that corresponds to ~50% of theG*-E* level at the peak of the wild-type SPR. In contrast, ~ 60 G*-E* complexes are predicted for the Grk1-/- SPR. The grossdisproportionality between the number of G*-E* complexes and theresponse plateau amplitudes reveals the consequences of cascadesaturation and the disproportionate role of calcium feedback to cGMPsynthesis on larger responses (Gross et al., 2012b).Commercial Relationships: Owen P. Gross, None; Edward N.Pugh, None; Marie E. Burns, NoneSupport: NIH Grant R01-EY014047 (MEB)Program Number: 2456 Poster Board Number: D0061Presentation Time: 2:45 PM - 4:30 PMRobust self-association of arrestin-1 is a neuroprotectivemechanismEugenia V. Gurevich 1 , Xiufeng Song 1 , Jungwon Seo 1 , FaizaBaameur 1 , Sergey A. Vishnivetskiy 1 , Qiuyan Chen 1 , Miyeon Kim 2 ,Jeannie Chen 3 , Wayne L. Hubbell 2 , Vsevolod V. Gurevich 1 .1 Pharmacology, Vanderbilt University, Nashville, TN; 2 University ofCalifornia Los Angeles, Los Angeles, CA; 3 University of SouthernCalifornia, Los Angeles, CA.Purpose: To determine the biological role of arrestin-1 selfassociation.Methods: Retinal morphology and the health of rod photoreceptorsin mice of different ages expressing wild type (WT) arrestin-1 andmutant with impaired self-association was compared.Results: Arrestin-1 binds light-activated phosphorhodopsin andensures timely signal shutoff. Arrestin-1 self-association is conservedin at least three mammalian species, but its biological role remainsobscure. We found that high expression of arrestin-1 mutant withimpaired oligomerization, but not of WT arrestin-1 that robustlyoligomerizes, results in progressive rod degeneration via apoptosis.Synaptic terminals of rods are adversely affected earlier than othercell compartments. Dark rearing does not prevent mutant-inducedcell death, ruling out the role of arrestin-rhodopsin complexes. WTarrestin-1 co-expressed with the mutant delays retinal degeneration,likely by forming mixed oligomers.Conclusions: Monomeric arrestin-1 is cytotoxic, and it likelyacquired propensity to oligomerize to reduce the concentration of themonomer in the cell. This finding suggests that cones express thebulk of their arrestin complement (~98%) in the form of arrestin-1because cone-specific arrestin-4 is naturally oligomerization-deficientand therefore likely cytotoxic. The results also suggest that arrestin-1binding to microtubules largely serves to minimize free monomer inthe cell body, rather than to localize arrestin-1 away from the outersegment in the dark.Commercial Relationships: Eugenia V. Gurevich, None; XiufengSong, None; Jungwon Seo, None; Faiza Baameur, None; Sergey A.Vishnivetskiy, None; Qiuyan Chen, None; Miyeon Kim, None;Jeannie Chen, None; Wayne L. Hubbell, None; Vsevolod V.Gurevich, NoneSupport: NIH grants, EY011500, GM077561, GM081756 (VVG),NS45117 and NS065868 (EVG), EY05216 and the Jules SteinProfessorship Endowment (WLH), EY012155 (JC)Program Number: 2457 Poster Board Number: D0062Presentation Time: 2:45 PM - 4:30 PMAdaptive acceleration in mouse rods is mediated by slowfeedback via guanylate cyclase activating proteinsMarie E. Burns 1, 2 , Owen P. Gross 1 , Claudia M. Krispel 3, 4 . 1 Centerfor Neuroscience, Univ of California-Davis, Davis, CA; 2 CellBiology and Human Anatomy, Univ of California-Davis, Davis, CA;3 Ophthalmology & Vision Science, Univ of California-Davis, Davis,CA; 4 Wilmer Eye Institute, Johns Hopkins Hospital, Baltimore, MD.Purpose: In retinal photoreceptors, adaptation to steady backgroundlight produces several specific changes in phototransductionbiochemistry and the incremental flash response. A long-lasting formof adaptation termed “adaptive acceleration” (AA) has been found toshorten responses for many tens of seconds following the offset ofsaturating light, but the mechanism and functional significance of thisform of adaptation has been unknown.Methods: Suction electrodes were used to record the outer segmentmembrane current of individual rods of mice before, during, and afteran adapting stimulus (500 nm light) that varied both in intensity andduration. Adaptive acceleration was probed using brief (10 ms)flashes of light of fixed intensity. The dominant time constant ofrecovery was probed using saturating flashes whose intensity varied2-8 fold in the period immediately before and after the adapting light,and by measuring the time that these flash responses remained insaturation.Results: As described previously, AA was easily induced usingsteady light that suppressed the rod’s circulating current. Theinduction of AA required extended periods of response saturation,with an onset time constant of 35 s, and faded with a time constant of~80 s. Analysis of a sequence of saturating flash responsesimmediately after the adapting light offset revealed that, contrary toan earlier report, AA was not associated with the shortening of thedominant time constant of recovery, indicating that it is not caused byacceleration of GTPase activity. Indeed, rods that overexpressed theRGS9 complex or which lacked several other targets for adaptation,including recoverin, showed the normal induction and offset featuresof AA. However, AA was completely abolished in the rods of micelacking guanylate cyclase activating proteins, or GCAPs.Furthermore, AA improved the flicker response properties of wildtyperods, but not in rods lacking GCAPs.Conclusions: GCAPs have previously been described to play a rolein setting the single photon response amplitude and in preventingsaturation in steady light (Mendez et al., 2001; Burns et al., 2002;Gross et al 2012). Our findings in the current study suggest thatGCAPs also mediate a longer-lasting change in guanylate cyclaseactivity that improves the temporal resolution of the rods following abright steady light.Commercial Relationships: Marie E. Burns, None; Owen P.Gross, None; Claudia M. Krispel, NoneSupport: NIH Grant R01-EY014047 (MEB)Program Number: 2458 Poster Board Number: D0063Presentation Time: 2:45 PM - 4:30 PM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyCritical role of central 139 loop in stability and binding selectivityof arrestin-1Sergey A. Vishnivetskiy, Faiza Baameur, Kristen R. Findley,Vsevolod V. Gurevich. Pharmacology, Vanderbilt University,Nashville, TN.Purpose: To elucidate the functional role of central 139-loop on thereceptor-binding surface of arrestin-1Methods: Deletions of 139-loop and mutations that disrupt itsinteractions with other elements of the molecule were introduced inwild type arrestin-1 and its enhanced 3A mutant with high binding tounphosphorylated light-activated rhodopsin (Rh*). We tested bindingselectivity and thermal stability of these mutantsResults: Using intra-molecular distance measurements by DEER wedetected large movement of arrestin-1 139-loop to the side, awayfrom the incoming receptor, upon its binding to activephosphorhodopsin (P-Rh*). Therefore, we hypothesized thatelimination of this loop would promote rhodopsin binding. Deletionsof increasing length in 139-loop progressively enhanced arrestin-1binding to non-preferred forms of rhodopsin: Rh*, dark P-Rh, andphospho-opsin. The interactions of 139-loop with the neighboringfinger loop involved in receptor binding were disrupted by chargeeliminations and reversals on both sides. These milder perturbationsincreased arrestin-1 binding to non-preferred forms of rhodopsin, butto a lesser extent. In all cases an increase in binding to Rh*, dark P-Rh, and phospho-opsin correlated with a decrease in thermal stabilityof the mutants. Similar effects of deletions and mutations in 139-loopon both binding selectivity and thermal stability were observed in thecontext of WT arrestin-1 and an enhanced 3A mutant. whichdemonstrates higher binding to non-preferred forms of rhodopsinConclusions: Central 139 loop is an earlier unappreciated elementstabilizing basal arrestin-1 conformation and precluding its binding tonon-preferred forms of rhodopsin. Thermal stability of the basal stateof arrestin-1 directly correlates with its selectivity for P-Rh*. NIHgrants EY011500, GM077561, and GM081756 (VVG).Commercial Relationships: Sergey A. Vishnivetskiy, None; FaizaBaameur, None; Kristen R. Findley, None; Vsevolod V. Gurevich,NoneSupport: NIH grants EY011500, GM077561, and GM081756(VVG).Program Number: 2459 Poster Board Number: D0064Presentation Time: 2:45 PM - 4:30 PMModulation of Mouse Rod cGMP-Gated Channels by Grb14Raju V. Rajala 1 , Michael L. Woodruff 2 , Gordon L. Fain 2, 3 .1 Ophthal/Dean McGee Eye Inst, Univ of Oklahoma Hlth Sci Ctr,Oklahoma City, OK; 2 Integrative Biology and Physiology, UCLA,Los Angeles, CA; 3 Jules Stein Eye Inst, UCLA, Los Angeles, CA.Purpose: Previous experiments indicated that growth factor receptorboundprotein 14 (Grb14) may modulate rod cyclic guanosinenucleotide (cGMP) gated channels by decreasing channel affinity forcGMP. We tested this hypothesis by recording electrical responsesfrom rods in which the gene for the Grb14 protein had been deleted.Methods: Grb14 -/- mice were obtained from Dr. Roger Daly, GarvanInstitute of Medical Research, Australia. Suction-electrode recordingswere made from single mouse rods by methods previously described(e.g. Chen et al., J. Neurosci. 30:16232-40, 2010).Results: Rod responses of dark-adapted Grb14 -/- mice decayed morerapidly than strain-controlled wild-type (WT) rods (Figure 1) withdecreased values of τ REC at all light intensities; integration timedecreased from 292 ± 20 ms (SE, n=15) in WT to 215 ± 12 ms (17)in Grb14 -/- mice. This result is consistent with an increase in channelaffinity for cGMP produced by deletion of Grb14. Grb14 -/- mouserods also showed a large and significant decrease in the limiting timeconstant τ D from 171 ± 15 ms (15) in WT to 118 ± 5 ms (17) inGrb14 -/- rods. Similar though smaller decreases in both τ REC and τ Dwere previously reported from mice whose channels lack thecalmodulin binding site (Chen et al., 2010), which should also resultin increased channel affinity for cGMP. Although Grb14 wasreported to translocate from inner to outer segment in the light(Rajala et al. Biochemistry 48:5563-5572, 2009), we saw no apparentdifference in the effect of Grb14 gene deletion on response decay indark-adapted and bleached or background adapted rods, and lightadaption in Grb14 -/- rods was essentially the same as in WT.Conclusions: Our results confirm a role of Grb14 as a modulator ofmouse rod cGMP-gated channels in both dark-adapted and lightadaptedrods. The physiological role of this modulation remains to beelucidated.Figure 1. Responses of WT and Grb14 -/- rods to 10 ms flashes ofintensities from 2.4 to 2600 photons μm -2 .Commercial Relationships: Raju V. Rajala, None; Michael L.Woodruff, None; Gordon L. Fain, NoneSupport: NIH/NEI grant (EY01844, EY016507, EY00871)Program Number: 2460 Poster Board Number: D0065Presentation Time: 2:45 PM - 4:30 PMIndependent manipulation of binding selectivity and selfassociationof arrestin-1Vsevolod V. Gurevich 1 , Sergey A. Vishnivetskiy 1 , Qiuyan Chen 1 ,Maria C. Palazzo 1 , Evan K. Brooks 2 , Christian Altenbach 2 , Tina M.Iverson 1 , Wayne L. Hubbell 2 . 1 Pharmacology, Vanderbilt University,Nashville, TN; 2 JSEI, UCLA, Los Angeles, TN.Purpose: To test whether binding selectivity and self-association ofarrestin-1 can be independently manipulated for research andtherapeutic useMethods: Versions of arrestin-1 with activating mutations enhancingthe binding to unphosphorylated light-activated rhodopsin (Rh*) andsubstitutions suppressing self-association were constructed,expressed, and their binding selectivity, stability, and oligomerizationwere testedResults: Arrestin-1 preferentially binds active phosphorylatedrhodopsin (P-Rh*). An enhanced mutant with increased binding toRh* partially compensates for the lack of rhodopsin phosphorylationin vivo. We show that reengineering of the receptor-binding surfaceof arrestin-1 further improves the binding to Rh* while preservingprotein stability. Mammalian arrestin-1 readily self-associates atphysiological concentrations. To elucidate the biological role of thisphenomenon, wild type arrestin-1 in living animals must be replacedwith a non-oligomerizing mutant retaining all other functions.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyConstitutively monomeric forms of arrestin-1 with wild typeselectivity for P-Rh* are sufficiently stable for in vivo expression.The same mutations eliminate self-association of enhanced forms,while preserving high Rh* binding and stabilityConclusions: Individual functions of arrestin-1 can be independentlymanipulated to generate mutants with the desired combinations offunctional characteristics. Stable forms of arrestin-1 with high Rh*binding can be constructed with or without the ability to selfassociate.These results pave the way for testing of the biological roleof arrestin-1 self-association and elucidation of full potential ofcompensational gene therapy of gain-of-function GPCR mutations.NIH grants EY011500, GM077561, GM081756 (VVG), EY05216and the Jules Stein Professorship Endowment (WLH), GM079419,GM095633 (TII)Commercial Relationships: Vsevolod V. Gurevich, None; SergeyA. Vishnivetskiy, None; Qiuyan Chen, None; Maria C. Palazzo,None; Evan K. Brooks, None; Christian Altenbach, None; TinaM. Iverson, None; Wayne L. Hubbell, NoneSupport: NIH grants EY011500, GM077561, GM081756 (VVG),EY05216 and the Jules Stein Professorship Endowment (WLH),GM079419, GM095633 (TII)Program Number: 2461 Poster Board Number: D0066Presentation Time: 2:45 PM - 4:30 PMA New Form of Photoreceptor Light Adaptation: EnhancedCurrent Following Sustained IlluminationAlex S. McKeown, Timothy W. Kraft. Vision Sciences, Univ ofAlabama at Birmingham, Birmingham, AL.Purpose: Photoreceptors have been extensively studied for the pasthalf century, but important details remain undiscovered in the realmof biochemical adaptation. Certain stimulus conditions can elicitprotein translocation, but these occur well outside the working rangeof rods. More relevant to fast adaptation are the biochemical changesthat occur in response to changing calcium levels, which result inaltered phototransduction response kinetics. Here we present a uniqueform of photoreceptor adaptation that results in increased totalcirculating dark current following sustained illumination. We alsoexamine the idea that the adaptation is caused by the dynamicinteraction of calmodulin with the cyclic nucleotide gated (CNG)channels of the rod outer segment.Methods: Using single cell suction electrode recordings of mousephotoreceptors, we have discovered a form of adaptation that occursin response to a steady illumination capable of bleaching only 1-4%of the photopigment in mouse rod cells. Following illumination of 1-3 minutes (5000 photons µm-2 s-1) there is a transient 35% increasein response amplitude that returns to baseline with a time constant of10 seconds. We also investigated this unique form of adaptation in amutant mouse lacking the calmodulin binding site on the beta subunitof the rod outer segment CNG channel (CaM-del mouse).Results: Following exposures of sustained illumination, rods exhibita 35% increase in circulating current upon return to darkness (n=11WT). Following identical duration and intensity of sustainedillumination, calmodulin mutant rods exhibit a 65% (vs. 35% in WT)increase in circulating current upon returning to darkness (p = 0.006)(n=14 CaM-del).Conclusions: These findings demonstrate both a unique form of lightadaptation in photoreceptors that we call dark potentiation, andsuggest a role for calmodulin in attenuating this form of adaptation.The excess channel opening following the light step is likely due tofluctuations in the concentration of cGMP, as determined byguanylate cyclase and phosphodiesterase. The role of calcium andGCAPs in this form of adaptation is being investigated. Additionalpsychophysical studies may reveal how such photoreceptor changescan impact human perception. The adaptation may enhancesensitivity in dim environments like those found in a deep forest, orduring specific times of day, such as dawn or dusk.Commercial Relationships: Alex S. McKeown, None; Timothy W.Kraft, NoneProgram Number: 2462 Poster Board Number: D0067Presentation Time: 2:45 PM - 4:30 PMEM-structure and functional properties of the rhodopsintransducincomplexBeata Jastrzebska, Tivadar Orban, Marcin Golczak, Andreas Engel,Krzysztof Palczewski. Pharmacology, Case Western ReserveUniversity, Cleveland, OH.Purpose: To determine the molecular assembly, structure andfunctional properties of the visual G protein-coupled receptor(GPCR), rhodopsin (Rho), in the complex with its cognitiveheterotrimeric G protein, transducin (Gt).Methods: By photoactivation of Rho (Meta II or Rho*) andnucleotide depletion, we trapped and purified the nucleotide-freeRho*-Gt complex and calculated its molecular envelope fromprojections of negatively stained Rho*-Gt particles. Using sConA asa probe, we identified the quaternary organization of Rho moleculesin this complex. Functional properties of Rho molecules bound to Gtwithin the stable Rho*-Gt complex was investigated by UV-visiblespectroscopy and HPLC retinoid analysis.Results: The 3-D envelope determined for the Rho*-Gt complexaccommodated two Rho molecules and one Gt heterotrimer(pentameric assembly). Within the complex, the photoactivated Rhodimer serves as a platform for binding the Gt heterotimer. We foundthat binding of Gt stabilizes one Rho in its active Meta II state,whereas the second progresses toward the opsin conformation.Therefore, each monomer contributes unevenly to the pentamericcomplex, indicating an asymmetry of the Rho dimer.Conclusions: This study unequivocally demonstrates dimericassembly of Rho* in the active complex with transducin and providesa clear functional distinction between each monomer, indicating thatoligomeric assembly and activation of family A GPCRs areintimately associated.Commercial Relationships: Beata Jastrzebska, None; TivadarOrban, None; Marcin Golczak, None; Andreas Engel, None;Krzysztof Palczewski, QLT Inc (F), Polgenix Inc (E), Visum Inc(P), Amegen Inc (F)Support: RES506768Program Number: 2463 Poster Board Number: D0068Presentation Time: 2:45 PM - 4:30 PMMechanisms of Retinal Vascular Alteration inHyperhomocysteinemiaAmany M. Tawfik 1, 2 , Shanu Markand 1, 2 , Sylvia Magyerdi 2, 3 ,Mohamed A. Al-Shabrawey 2, 3 , Sylvia B. Smith 1, 2 . 1 Cellular Biology& Anatomy, Georgia Health Sciences Univ, Augusta, GA; 2 VisionDiscovery Institute, GHSU, Augusta, GA; 3 Oral Biology/Anatomy,GHSU, Augusta, GA.Purpose: Excess homocysteine (Hcy), a sulfur containing nonproteinogenicamino acid, is implicated in human vascular disorders.Recently, we described the retinal phenotype inhyperhomocysteinemic mice lacking/deficient in the gene encodingcystathionine β−synthase (CBS). These mice have marked disruptionof retinal neuronal layers, decreased ERG function & altered retinalvasculature suggestive of ischemic retinopathy. In this study weexplored the mechanism of Hcy-induced retinopathy specificallyscreening markers of ER stress, oxidative stress, inflammation &NMDA receptor (NMDAr) activation.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyMethods: Retinas of cbs+/+ (n=21) & cbs-/- (n=22) mice wereharvested at ~3 wks. RNA & protein were isolated & subjected toqPCR & western blotting, respectively to analyze expression of ERstress genes & the proteins they encode including BiP, PERK,pPERK, CHOP, ATF6 & RE1α. Retinal cryosections were subjectedto immunofluorescence to evaluate BiP, CHOP, NMDAr, TNFα andthe oxidative stress marker DACF-2DA. To investigate whether Hcyaltered vascular permeability, human retinal endothelial cells (HREC)were exposed to Hcy-thiolactone (20µM, 50µM, 100µM) in thepresence/absence of the NMDAr inhibitor MK801 (25µM) or the ERstress inhibitor phenylbutyric acid, PBA (10mM) followed byincubation with FITC-dextran as an indicator of leakage.Results: Gene & protein analysis revealed ~40% increase in BiP &PERK expression in cbs-/- retinas compared to cbs+/+; there was nosignificant change in expression of ATF4, ATF6, CHOP, IRE1α .Immunofluorescence studies in retinal cryosections of cbs-/- miceshowed a dramatic increase in BiP & CHOP as well as NMDAr,TNFα & DACF-2A. In vitro studies of Hcy-treated HREC showed a25% increase in FITC-dextran leakage through the HREC monolayer,which was reduced significantly by MK801 and PBA.Conclusions: Retinal neurovascular alterations observed in severelyhyperhomocysteinemic cbs mice are accompanied by increased levelsof ER & oxidative stress as well as activation of NMDAr.Importantly, increased vascular permeability may be associated withHcy-induced leakiness, which can be attenuated by inhibiting ERstress or NMDAr.Commercial Relationships: Amany M. Tawfik, None; ShanuMarkand, None; Sylvia Magyerdi, None; Mohamed A. Al-Shabrawey, None; Sylvia B. Smith, NoneSupport: NIH Grant EYO12830 and RO1 EYO14560Program Number: 2464 Poster Board Number: D0069Presentation Time: 2:45 PM - 4:30 PMA Prospective Study of In-Office Diagnostic Vitreous Sampling inPatients with Vitreoretinal Pathology, 2007-2011Bert M. Glaser, Stephanie M. Ecker, Joshua C. Hines, Ann O. Igbre.Ocular Proteomics, National Retina Institute, Towson, MD.Purpose: This prospective study has been compiled to demonstratethat in-office vitreous sampling can be done safely and efficiently inpatients with a variety of retinal pathologies.Methods: Briefly, a vitreous aspiration was performed before aninjection of Anti-VEGF or of corticosteroid for treatment of retinaldisease. Each sample was taken using a standard technique, whichincludes 1) application of 2% topical liodcaine gel followed byplacement of a soaked cotton-tip applicator with 4% Lidocainesolution (in the inferior temporal quadrant; 2) a Sterile Lid Speculumwas placed followed by a drop of betadine 5%; 3) next, a 25 gauge,5/8 inch Terumo Needle was placed via pars plana approach into themid-vitreous cavity and 0.05 mL to 0.10mL of vitreous fluid wasaspirated into a 1mL syringe. After removal of the needle theintravitreal injection is directed via or close to the same spot. Afterthe injection the patient is discharged with post-injection warningstypical of patients following intra-vitreal injections.Results: As of December 31, 2011, a total of 830 patients wereenrolled in the Western IRB approved Vitreous Proteomics study atour institute. A total of 3,741 attempts were made to acquire avitreous sample, resulting in 3,245 samples, yielding an 86.7% rate ofsuccess over a 4 year period. The majority of the vitreoretinaldiagnoses were AMD, DR and RVO, but there were 93 patients withother retinal diseases that were also sampled in this study. Out of the830 patients in the study, there have been 24 (2.89%) patients thathad a complication. The majority of the complications (79.16%,n=19) were mild vitreous hemorrhages, present at the one monthfollow-up visit. All vitreous hemorrhages resolved without sequelae.The other 5 adverse events consisted of a corneal abrasion (n=1),endophthalmitis (n=1), pseudo-endophthalmitis (n=2) and scleritis(n=1). There were no cases identified of acute posterior vitreousdetachment, retinal tears or retinal detachments.Conclusions: This proves that in-office vitreous sampling of 50-100µl is a safe and reproducible procedure. The low rate of adverseevents and the 4 year duration of this study give solid evidence thatvitreous sampling is in fact a safe in-office procedure.Commercial Relationships: Bert M. Glaser, Ocular Proteomics,LLC (E); Stephanie M. Ecker, Ocular Proteomics LLC (E); JoshuaC. Hines, Ocular Proteomics (E); Ann O. Igbre, NoneProgram Number: 2465 Poster Board Number: D0070Presentation Time: 2:45 PM - 4:30 PMMouse Vitreoretinal ProteomeJessica M. Skeie 1, 2 , Stephen H. Tsang 3 , Vinit B. Mahajan 1, 2 .1 Ophthalmology and Visual Sciences, University of Iowa, Iowa City,IA; 2 Omics Laboratory, University of Iowa, Iowa City, IA; 3 Bernardand Shirlee Brown Glaucoma Laboratory, Department ofOphthalmology, Columbia University, New York, NY.Purpose: To identify the protein profiles of mouse retina andvitreous and determine unique protein pathways and interactionnetworks in these tissues.Methods: Vitreous and retina samples from normal mice werecollected by an evisceration technique developed in our laboratoryand pooled. Samples were analyzed in technical triplicate by LC-MS/MS. The proteins discovered were further analyzed usingbioinformatic software to determine significant pathways present,statistically significant protein expression differences betweenvitreous and retina, gene ontologies, and protein interaction networks.Results: In the retina, we identified 5,729 unique spectra with106,734 peptide hits, corresponding to 1,682 unique proteins and1,085 unique spectra with 45,507 peptide hits, corresponding to 677unique proteins in the vitreous. Unbiased clustering of the proteinsverified that the vitreous and retina were significantly different withdifferent gene ontology distributions. The most highly representedpathways that distinguished the retina from the vitreous (p < 0.05)were neuronal cell synapses (LRRK2), pyruvate metabolism,neurophysiological processes, apoptosis/survival, cytoskeletalremodeling, and G-protein signaling (CFTR). Pathways thatdistinguished the vitreous from the retina (p < 0.05) werephenylalanine metabolism and nitrogen metabolism. Some clusters ofinteracting proteins were identified in both the retina and vitreous.These included crystallin cell regulation and oxidative stress proteins.Conclusions: We identified several unique proteins, pathways, andinteraction networks that distinguish the normal mouse retina andvitreous. This methodology can be applied to mouse models ofhuman vitreoretinal disease.Commercial Relationships: Jessica M. Skeie, None; Stephen H.Tsang, None; Vinit B. Mahajan, NoneSupport: NIH 1F32EY022280-01A1Program Number: 2466 Poster Board Number: D0071Presentation Time: 2:45 PM - 4:30 PMThe novelty of Toll like receptor-4 ligand and RGC degenerationYasunari Munemasa, Kazuhide Takada, Kaori Kojima, Satoki Ueno,Yasushi Kitaoka. Ophthalmology, St Marianna University, Kawasaki,Japan.Purpose: Toll like receptor-4 (TLR-4) has been indicated in responseto various ligands related with cell death signaling, in addition toautoimmune response. The purpose of this study is to investigate thenovelty of TLR-4 in RGC degeneration.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyMethods: Eight week male C3H/HeJ and C3H/HeN mice were usedin this study. C3H/HeJ mice show dysfunction of TLR-4. In contrast,C3H/HeN mice show normal TLR-4 function. To generate opticnerve crush (ONC), the nerve of one eye was exposed and clampedapproximately 0.5 mm from the globe with self-closing forceps for10s, contralateral eye was used as a control. RGC was labeled withfluoro-gold at superior colliculus 5 days before ONC andmorphological analysis was performed with whole mount retina at 2weeks after ONC. Matrix-assisted laser desorption/ionisation-time offlight mass spectrometry (MALDI-TOFMS) was used to detect TLR-4 ligands after immunoprecipitation with TLR-4 antibody andisolation of retinal membrane proteins at 5 days after ONC. Inductionof RGC degeneration with TLR-4 ligand was studied with cellviability assay using RGC-5 cells and intravitreal injection of TLR-4ligand on C3H/HeJ and C3H/HeN mice.Results: A significant decrease in RGC was observed in the retina ofC3H/HeJ mice compared to C3H/HeN mice 2 weeks after ONC(C3H/HeJ 0.24 fold / control vs C3H/HeN 0.40 fold / control, P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyto ganglion cell and optic nerve development. Gradients of ephrinreceptors (Eph) create retinotopic maps that pattern RGC axonprojections to the brain, but little is known about the regulation ofthese gradients. In order to test the hypothesis that nasal/temporaldifferences in transcription factor expressions regulate EphA5 andEphA6 mRNA gradient pattern in RGCs, this initial study focuses onoptimizing laser capture microdissection (LCM) to yield high-qualityRNA for transcriptome sequencing.Methods: C57Bl/6 mice (postnatal day 2) eyes were enucleated andfrozen in molds with OCT compound with or without prior sucrosecryoprotection. Eyes were cryosectioned at 7µm and mounted ontoPEN-Membrane slides held at 4°C or room temperature. Sectionswere dehydrated prior to LCM of nasal and temporal thirds of theretinal ganglion cell layer (GCL). RNA quality was assessed usingRNA Nano and Pico chips (Agilent Bioanalyzer). Nasal/temporalsamples were tested in triplicate with quantitative reversetranscriptase PCR (qRT-PCR) and analyzed with Relative ExpressionSoftware Tool-MCSv2.Results: RNA quality was highest with flash frozen vs. sucrosecryoprotection of the eyes, with RNA Integrity Numbers (RINs) of7.6 and 5.9 respectively (scale 0 low-10 high). Mounting sections on4°C slides yielded better RNA quality than on room temperatureslides, with RINs of 8.8 and 7.8 respectively. Minimal RNAdegradation was detected within 90 mins following dehydration, withRINs declining from 8.8 to 8.5. qRT-PCR showed about a 3.5-foldenrichment of Pou4f2 mRNA in LCM samples vs. whole retina.There was about a 2-fold enrichment of EphA5 mRNA in temporalGCL vs. nasal GCL.Conclusions: LCM provides a powerful technique to extract highqualityRNA with sufficient yields for downstream, high-contentsequencing. Histological sections and RT-qPCR support anenrichment of RGC mRNA. Nasal/temporal differences in EphA5were detectable. Additional samples will be collected underoptimized conditions and analyzed prior to whole transcriptomeanalysis of the nasal and temporal GCL.Commercial Relationships: Steve Huynh, None; Deborah C.Otteson, NoneSupport: NIH R01 EY021792; NIH/NEI P30 EY007551Program Number: 2470 Poster Board Number: D0075Presentation Time: 2:45 PM - 4:30 PMA Non-Radioactive Assay for Measuring Retinal Base ExcisionRepair CapabilityVincent T. Ciavatta 1, 2 , Priscila P. Cunha 2 , Jeffrey H. Boatright 2 ,Sophia M. Tang 2 . 1 Rehabilitation R & D, Center of Excellence, USDept of Veterans Affairs, Decatur, GA; 2 Ophthalmology, EmoryUniversity School of Medicine, Atlanta, GA.Purpose: We are attempting to use endogenous retinal DNA repaircapability as part of a novel gene therapy approach. We aim toenhance DNA repair capability through physiological andpharmacological means. Oxoguanine glycosylase (OGG1) is anenzyme needed for repairing oxidized guanine residues, and it isexpressed in several mammalian retina cell types. To assess theimpact of our efforts, we developed a relatively inexpensive,fluorescence-based, non-radioactive assay to quantify OGG1 enzymeactivity.Methods: Methods - For substrate, a 50-base oligonucleotide with an8-oxodG residue at nucleotide (nt) position 26 and a fluorescentmoiety at the 5’ end was annealed to its complementary strand.Annealing (100 fmol/µL each oligonucleotide) was done according toLan et al., 2003. Protein extracts were prepared from fresh frozenC57BL6 mouse cortex and retina from C57BL6 and RD10 miceaccording to Bigot et al., 2009 and stored at -80°C. Cut reactionswere performed at 32°C for 1 h in 50 or 100 µL using 250 fmoldouble-stranded target, 5 to 100 µg protein extract, 50 mM HEPES(pH 7.6), 2 mM EDTA, 50 mM NaCl, 5% glycerol, and 0.1 mg/mLBSA. Reactions were stopped with 0.1 M NaOH and 37°C for 15min. DNA was recovered by ethanol precipitation, dissolved in 90%formamide, heat denatured, resolved by 7M urea, 15% PAGE, andDNA bands were photographed, digitized, and quantified.Results: Intensity of a 24 nt band showed a dose dependentrelationship with amount of retinal protein added up to 100 µg. Thediagnostic 24 nt band was detected in all retina and brain sampleswhen using the lowest amount of protein (5 µg). Omitting proteinproduced no detectable 24 nt band. Product was nearly eliminated if0.1 M NaOH was not used to stop the reaction. In weanling-agedmice, OGG1 activity was greater in C57BL6 than rd10 retina.Conclusions: This non-radioactive OGG1 assay is a uniquerefinement of one established OGG1 activity assay that usesradiolabeled substrates and another fluorescence-based method usinga hairpin, single-stranded oligonucleotide. Assay sensitivityapproximates that from the established methods. The assay isamenable to high throughput, fluorescence-based detection systemsand is useful for measuring effects of various independent variableson retinal OGG1 activity. Retinas undergoing degeneration may haveless DNA repair capability than wildtype retinas.Commercial Relationships: Vincent T. Ciavatta, None; Priscila P.Cunha, None; Jeffrey H. Boatright, None; Sophia M. Tang, NoneSupport: NEI R01 EY014026, NEI P30 EY06360, Research toPrevent Blindness (RPB), The Abraham & Phyllis Katz FoundationProgram Number: 2471 Poster Board Number: D0076Presentation Time: 2:45 PM - 4:30 PMEvolutionarily Conserved Minor Spliceosome is Required forDifferentiating Mouse Retinal NeuronsRahul N. Kanadia, Ashley M. Kilcollins. Physiology andNeurobiology, University of Connecticut, Storrs, CT.Purpose: Splicing removes introns and fuses exons, which isessential for eukaryotic gene expression and is carried out by themajor spliceosome. The major spliceosome consists of a core set ofsnRNAs including, U1, U2, U4, U5 and U6 that are required forsplicing. Interestingly, in eukaryotes, there exists anotherspliceosome called the minor spliceosome that is evolutionarilyconserved and consists of snRNAs including, U11, U12, U4atac andU6atac. Named thusly, for it removes introns in only 3% of thegenes. Given this small subset of introns that it regulates, we wantedto address the following questions. 1) Are the minor spliceosomeassociated snRNAs expressed in the developing retina? 2) Is theminor spliceosome function required for retinal development?Methods: We determined the expression of the minor spliceosomeassociated snRNAs by RT-PCR and in situ. We also employed P0 invivo retinal electroporation to inactivate U12 snRNA.Results: To test the presence of a functioning spliceosome in thedeveloping retina we examined the expression of U11 and U12snRNA. Surprisingly, U11 and U12 snRNAs were enriched in newlydifferentiating neurons, but absent in progenitor cells. Expression ofU11 and U12 was observed as distinct puncta in the nuclei of retinalganglion cells at P0, followed by amacrines and P4 and later in theONL at P10 and P14. Also, within the nuclei of the same cell, U11and U12 snRNAs do not overlap, which is surprising since they arethought to work as a di-snRNP. This might suggest that in the retinae,they function independently. Finally, inactivation of U12 snRNA didnot perturb the progenitor cell function, but it led to death ofterminally differentiating neurons. Specifically, the neuronal deathprogressed in the order in which they were differentiating. Forexample, amacrine cell death preceded the rod photoreceptor death.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyConclusions: The minor spliceosome components such as U11 andU12 are expressed in terminally differentiating retinal neurons. Also,U12 snRNA is required for terminally differentiating retinal neuronspostnatally, but is not expressed or required for progenitor cellsurvival.Double Fluorescent in situ for U11 (red) and U12 (green).processes, including cell-cycle arrest, differentiation, morphogenesis,and apoptosis. TGF-β promotes extracellular matrix production andsuppresses cell proliferation. Morphogenetic responses to TGF-βmembers include cell migration and epithelial-mesenchymaltransitions (EMTs), which are critical during embryogenesis,development of fibrotic diseases, and advanced carcinoma spreading.The purpose of this study is to clarify how can survive human retinalpigment epithelial cells during TGF-β induced EMT.Methods: Serum-starved ARPE-19 cells were incubated with vehiclealone or 10ng/ml TGF-β1. Flow cytometric analysis of ARPE-19cells treated for 24 h with 10 ng/ml TGF-β1, followed by incubationwith Annexin V-FITC and propidium iodide (PI), showed theapoptotic fraction. Using siRNA targeting for Survivin, we show thatthese proteins are critical to TGF-β1 induced EMT. To determine thekey signaling mediator responsible for the up-regulation of survivinin response to TGF-β1, we used kinase inhibitors to individuallyblock each signaling pathway in ARPE-19 cells treated with TGF-β1,and then examined the level of survivin expression.Results: Apoptosis analysis showed that the apoptosis was notinduced in ARPE-19 cells treated with TGF-β1. Using siRNAtargeting for Survivin, we show that cell apoptosis increased intreated ARPE-19 cells lacking survivin compared to control cellstreated with TGF-β1 only. Inhibition of MEK or PI3K blocked theup-regulation of survivin following TGF-β1 treatment, whileinhibition of Rho did not.Conclusions: In conclusion, we showed that induction of EMT inhuman RPE cells led to up-regulation of Survivin expression, andinhibition of Survivin iduced apoptosis. We demonstrate thatSurvivin involves in the Transforming Growth Factor β1-mediatedEpithelial-Mesenchymal Transition of retinal pigment epithelial cellsby evading cell apoptosis.Commercial Relationships: Jungeun Lee, None; Jung-Ha Choi,None; Choun-Ki Joo, NoneSupport: National Research Foundation of Korea (NRF) 2011-0013562In situ (purple) for U12 snRNA and TUNEL+ (red) cells in P10 andP14 retina after knockdown of U12 snRNA. Cells are marked withGFP (green)Commercial Relationships: Rahul N. Kanadia, None; Ashley M.Kilcollins, NoneSupport: This project was supported by award numberR00EY019547 (to RK) from the National Eye Institute and by awardnumber 5P30NS069266 from National Institute for NeurologicalDisorders and Stroke.Program Number: 2472 Poster Board Number: D0077Presentation Time: 2:45 PM - 4:30 PMRetinal pigment epithelial cells evade apoptosis during TGF-β1-induced epithelial-mesenchymal transition adopting survivinJungeun Lee, Jung-Ha Choi, Choun-Ki Joo. Catholic Institutes ofVisual Science, College of Medicine, Catholic University of Korea,Seoul, Republic of Korea.Purpose: Members of the transforming growth factorβ(TGF-β)superfamily are multifunctional cytokines that regulate cellularProgram Number: 2473 Poster Board Number: D0078Presentation Time: 2:45 PM - 4:30 PMRegulation of Set-β’s subcellular localization andposttranslational modifications affect axon growth andregenerationMelina I. Morkin 1 , Ephraim F. Trakhtenberg 1, 2 , Yan Wang 1 ,Stephanie Fernandez 3 , Gregory M. Mlacker 4 , Jeffrey L. Goldberg 1, 2 .1 Ophthalmology, Bascom Palmer Eye Institute, Miami, FL;2 Neuroscience Program, University of Miami, Miami, FL;3 University of Miami, Miami, FL; 4 Miller School of Medicine,University of Miami, Miami, FL.Purpose: Adult mammalian central nervous system (CNS) neuronsare unable to regrow axons after injury, but immature CNS axons canregenerate. Manipulation of various cell-autonomous factors alongwith overcoming the inhibitory adult CNS environment only partiallyrestores regeneration. However, regenerative capacity of CNSneurons themselves declines after birth. We found that an epigeneticfactor Set-β, previously reported to be upregulated in Alzheimer’sdisease patients’ neurons, is also postnatally upregulated in retinalganglion cells’ nuclei. Here we investigated the role of Set-β in axongrowth of RGCs.Methods: Embryonic and postnatal rat retinal sections were costainedagainst RGC marker Brn3b and Set-β; immunofluorescenceintensity was analyzed with AxioVision. Cytoplasmic and nuclearfractions of P5 RGCs were separated and immunoblotted for Set-β.Wild-type Set-β, myristoylated-Set-β, Set-β serine-9 phosphomutantsand nuclear localization signal deletion mutants, or anti-Set-©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyβ shRNAs were overexpressed in purified P4 RGCs or E18hippocampal neurons, which were then immunostained and imagedafter 1-3 days in vitro. Neurite length was quantified using the ImageJ Neurite Tracer.Results: We found that in RGCs, full-length 39 kDa Set-β ispredominantly nuclear whereas a shorter 25 kDa isoform ispredominately cytoplasmic. Set-β overexpressed in postnatal RGCsor embryonic hippocampal neurons localized to the nucleus andsuppressed axon growth. In contrast, experimentally increasedmyristoylated-Set-β cytoplasmic localization, or shRNA knockdownof Set-β, promoted axon growth. Serine-9 phosphorylation of Set-βblocks Set-β’s ability to suppress axon growth. Finally, we showedthat in the CNS, Set-β binds selectively to PP2A-A-β but not αisoform; PP2A was recently shown to modulate axon growth in CNS.Conclusions: Full-length Set-β is developmentally upregulated inCNS primary neurons’ nuclei whereas a shorter isoform is found inthe cytoplasm. Set-β inhibits or promotes axon growth in the CNSneurons depending on its subcellular localization and serine-9phosphorylation, and Set-β binds selectively to PP2A-A-β but not αisoform. Manipulation of Set-β in vivo may provide a strategy toenhance regeneration.Commercial Relationships: Melina I. Morkin, None; Ephraim F.Trakhtenberg, None; Yan Wang, None; Stephanie Fernandez,None; Gregory M. Mlacker, None; Jeffrey L. Goldberg, NoneSupport: AHA (11PRE7310069, EFT), NEI (EY020913, JLG),Center grant P30 EY014801 (JLG), Research to Prevent Blindness,NIH (T32 NS007492, EFT), Lois Pope LIFE Fellowship (EFT)Program Number: 2474 Poster Board Number: D0079Presentation Time: 2:45 PM - 4:30 PMEarly Ischemia-reperfusion Injury Induces Retinal VascularPermeability in a VEGF Receptor 2 Dependent Manner Followedby Occludin Phosphorylation and UbiquitinationArivalagan Muthusamy, Cheng-mao Lin, Heather Lindner, SumathiShanmugam, Steven F. Abcouwer, David A. Antonetti.Ophthalmology & Vis Sciences, Univ of Michigan Kellogg Eye Ctr,Ann Arbor, MI.Purpose: Ischemia-reperfusion (IR) injury rapidly induces retinalvascular permeability by an unknown mechanism. Multiple tightjunction (TJ) proteins including the MARVEL family (occludin,tricellulin, and marvel D3 proteins) and the claudin family arerequired for the formation and maintenance of the blood-retinalbarrier (BRB). Vascular endothelial growth factor- (VEGF)-inducedpermeability of retinal endothelial cell cultures requiresphosphorylation of occludin on serine 490 (Ser-490), which leads toits ubiquitination and endocytosis. The present study tests thehypotheses that retinal IR injury stimulates vascular permeabilitythrough VEGF receptor 2 (VEGFR2) activation and alterations intight junction protein expression, modification and complexformation.Methods: Rats were subjected to ischemia for 45 minutes followedby reperfusion up to 4 h. BRB permeability was quantified by retinalaccumulation of Evans blue dye measured 2 h after its intravitrealinjection. Changes in retinal TJ protein content, occludinphosphorylation and ubiquitination were assessed by Westernblotting. To observe TJ organization, localization of TJ proteins wasexamined by immunohistochemistry (IHC) of flat-mounted retinas.Results: IR caused a rapid significant increase in permeabilityassessed during 0.25-2.25 h and 4-6 h following reperfusion. IR hadno effects on total protein content of any TJ protein examined, butrapidly diminished TJ protein localization at endothelial celljunctions within 15 min of reperfusion. IR caused phosphorylation ofVEGFR2 on tyrosine 1175 (Tyr-1175) at 15 min after reperfusion,coinciding with occludin phosphorylation at Ser-490 and polyubiquitination.Intravitreal injection of a tyrosine kinase inhibitorspecifically targeting VEGFR2 efficiently blocks both IR-inducedVEGFR2 Tyr-1175 phosphorylation and occludin Ser-490phosphorylation.Conclusions: These data are consistent with retinal IR injury causingvascular permeability through VEGFR2 activation andphosphorylation and ubiquitination of occludin leading to rapiddisassembly of tight junction complexes. This model suggests thatoccludin phosphorylation may represent a valid target for therapeuticprevention of VEGF-induced BRB breakdown and subsequentedema.Commercial Relationships: Arivalagan Muthusamy, None;Cheng-mao Lin, None; Heather Lindner, None; SumathiShanmugam, None; Steven F. Abcouwer, None; David A.Antonetti, NoneSupport: JDRFProgram Number: 2475 Poster Board Number: D0080Presentation Time: 2:45 PM - 4:30 PMOligonucleotide-mediated gene correction in human primaryfibroblastsMagdalena M. Staniszewska, Mark B. Consugar, Michael H. Farkas,Eric A. Pierce. Ocular Genomics Institute, Department ofOphthalmology, Massachusetts Eye and Ear Infirmary, HarvardMedical School, Boston, MA.Purpose: The use of stem cell transplantation has great potential forthe treatment of patients with inherited and other retinal degenerativedisorders. One approach to therapy currently being evaluated is theuse of gene-corrected induced pluripotent stem (iPS) cells. To testefficacy of gene editing induced by single-strandedoligodeoxynucleotides (ODNs), we evaluated the ODN-mediatedcorrection of the pathogenic mutation in fibroblasts from a patientwith gyrate atrophy. Next-generation sequencing (NGS) was utilizedto address the challenge of identifying cells with the corrected genein the absence of a selection system.Methods: Human skin fibroblasts isolated from patient with gyrateatrophy due to a p.Ala226Val (c.C677T) mutation in OAT gene werelipofected with correcting (WT) or control (mutant) 59-nucleotideODNs. The transfected cells were plated in a 96-well plate at adensity of 100 cells/well. The targeted OAT region was PCRamplifiedfrom each well of the 96-well plate with barcoded primers,and the purified amplicons were pooled. Illumina NGS was used toidentify wells containing corrected cells, which where enriched bysub-cloning. Diagnostic digestion of the PCR-amplified targetedOAT region was used to verify gene editing events in the sub-clonedfibroblasts.Results: NGS of the pooled PCR products generated an average of363,000 sequence reads per well. A total of 25 wells from the plate ofcells treated with the correcting ODN had the C677 nucleotidedetected above the background rate. Twenty one wells from thecontrol plate were also identified using this approach. Sub-clonalenrichment and diagnostic digestion of the amplified targeted OATregion showed that 22% (7/31) of viable sub-clones transfected withcorrecting ODN contained WT sequence. In contrast, none of theviable sub-clones from the cells receiving control ODN showed a WTdigestion pattern. We were not able to obtain clonal populations ofODN-corrected cells due to senescence of the fibroblasts.Conclusions: These results suggest that ODNs can be used to correctsingle point mutations in primary cells, and that corrected cells can bedetected using NGS without introduction of a selection cassette. Theexperiments also demonstrate that fibroblasts are not the optimal celltype for therapeutic gene correction, since they have a limited growth©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologycapacity. Testing of similar approaches in iPS cells may bewarranted.Commercial Relationships: Magdalena M. Staniszewska, None;Mark B. Consugar, Agilent Technologies, Inc. (R); Michael H.Farkas, None; Eric A. Pierce, NoneSupport: Foundation Fighting Blindness, Ocular Genomics InstituteProgram Number: 2476 Poster Board Number: D0081Presentation Time: 2:45 PM - 4:30 PMCompensation for the loss of the alpha7 nAChR in the retina ofthe alpha7 nAChR knockout mouseMarci L. Smith 1 , Fred Souza 2 , Christianne E. Strang 2 , Barbara J.Morley 3 , Kent T. Keyser 2 . 1 Psychology, University of Alabama atBirmingham, Birmingham, AL; 2 Vision Science, University ofAlabama at Birmingham, Birmingham, AL; 3 Boys Town NationalResearch Hospital, Omaha, NE.Purpose: Nicotinic acetylcholine receptors (nAChRs) are pentamericligand-gated cation channels expressed throughout the centralnervous system, including in the retina. The alpha7 nAChR subtypeis expressed by bipolar, amacrine, and ganglion cells in the rabbitretina (Dmitrieva et al., 2007). These receptors may play both a directand modulatory role in visual signaling. Alzheimer’s disease (AD)and schizophrenia are disease states which have visual dysfunctionand decreased alpha7 nAChR expression. In contrast, the alpha7nAChR knockout (KO) mouse has a relatively mild phenotype, withonly mild visual dysfunction (Morley & Rodriguez-Sierra, 2004;Origlia et al., 2012). Consequently, we predict that there iscompensatory up-regulation of other genes, including muscarinic andnicotinic AChRs, during development that is not present in AD andschizophrenia.Methods: We employed quantitative real-time polymerase chainreaction (qPCR), using RNA extracted from whole retina as well asRNA extracted from specific retinal layers obtained by laser capturemicrodissection, to quantify expression of AChR transcripts.Results: In whole retina alpha2, alpha9, alpha10, beta4, m1, and m4AChR transcripts were up-regulated. In the ganglion cell layer beta3and beta4 nAChR transcripts were significantly down-regulatedwhile m2 and m4 mAChR transcripts were significantly up-regulated.In the inner portion of the inner nuclear layer (INL) alpha2, alpha9,beta4, m3 and m4 AChR subunits were all significantly downregulated.Finally, in the outer portion of the INL beta2, beta4 andm4 AChR transcripts were all significantly up-regulated.Conclusions: Thus, rather than a simple up-regulation of a singleAChR subtype, compensation for the loss of the alpha7 nAChR in theKO mouse was differentially mediated by changes in mRNA levelsof a number of different AChR transcripts in different retinal layers.Compensatory changes in subunit transcription may provide insightinto potential receptor based pharmaceutical targets for treatment ofAD and schizophrenia symptoms, success of which may be testablethrough assessment of visual dysfunction.Commercial Relationships: Marci L. Smith, None; Fred Souza,None; Christianne E. Strang, None; Barbara J. Morley, None;Kent T. Keyser, NoneSupport: R01DC006907, P30EY03039, and the EyesightFoundation of AlabamaProgram Number: 2477 Poster Board Number: D0082Presentation Time: 2:45 PM - 4:30 PMThe role of extracellular histones in retinal detachmentHiroki Kawano 1 , Takashi Ito 2 , Ikuro Maruyama 2 , Taiji Sakamoto 1 .1 Opthalmology, Kagoshima University Graduate School of Medicaland Dental School, Kagoshima, Japan; 2 Systems Biology inThromboregulation, Kagoshima University Graduate School ofMedical and Dental Sciences, kagoshima, Japan.Purpose: Histones are DNA-binding proteins and are involved inchromatin remodeling and regulation of gene expression. Histonescan be released from dying cell and extracellular histones causecellular damage and organ dysfunction during sepsis, sterileinflammatory liver injury, and acute kidney injury. Regardless ofthese clinical significances, its role and relevance to ocular diseaseshave been mostly unknown. The purpose of this study was toinvestigate the role of histone on retinal cells and their pathologyfocusing on retinal detachment (RD).Methods: The oxidative stress was introduced with H2O2 in culturedrat retinal progenitor cells R28 and the expression of histone H3 wasevaluated by Western blot. RD model was produced by subretinalinjection of hyaluronan in rats and the expression of H3 wasexamined by immunohistochemistry. In addition, we assessed thevitreous concentrations of histone H3 by enzyme-linked immunesorbentassay and their relationships with cytokine levels a usingcytometric bead array in human eyes with various ocular pathologies.Furthermore, the cytokine profile analysis and cell viability assaywere performed to show the toxicity of histones to human retinalpigment epithelial (RPE) cell line, ARPE-19 using WST-1 assay.Results: Oxidative stress induced the release of histone H3 fromretinal R28 cells. In rat eyes, histone H3 was observed in the outerside of detached retina associating with photoreceptor necrosis butnot apoptosis. Furthermore, the vitreous levels of histone H3 inpatients with RD (30.9 ng/ml) were significantly higher than those ofmacular hole or epiretinal membrane (less than detection level,P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyWe describe a second mouse model lacking the TF, Prdm8, thatphenocopies the Bhlhb4 -/- model. Both TFs regulate the final stagesof RB development in the retina and have parallel functions in theregulation of interneuron development elsewhere in the CNS.Methods: Prdm8 and Bhlhb4 expression in wildtype (wt) retina andCNS was analysed by immunofluorescence using cell specificmarkers. Retinal transcriptomes of Prdm8 -/- , Bhlhb4 -/- and wt mice atPN6 were compared using Affymetrix Murine Genome 430 2.0arrays with a false discovery rate (FDR) of up to 10%. Differentialgene expression between mutant and littermatched wt retina wasquantified by rtPCR. Morphological differences between mutant andwt brains were analysed by MRI; and neurobehavioural phenotypeswith SHIRPA screening tests and the Morris water maze.Results: Adult Prdm8 -/- and Bhlhb4 -/- retinas lack RBs anddemonstrate b wave deficits on dark adapted ERGs, consistent withcompromised RB circuitry. In both models, RBs are born in normalnumbers but die apoptotically from PN8. Prdm8 and Bhlhb4 are coexpressedin RBs at PN5 to PN12 and from PN14 to adulthood. AtPN6, Bhlhb4 is downregulated in Prdm8 -/- retina (p=0.003) whilstPrdm8 expression is unchanged in Bhlhb4 -/- retina (p=0.5). Nonoverlappinggenes are differentially expressed in Prdm8 -/- andBhlhb4 -/- retina at PN6. In the latter, the most significantlydownregulated genes, c-Rel and Neu3 (FDR=0%), are implicated inapoptosis.In the CNS, Prdm8 and Bhlhb4 are coexpressed in adult cerebellumand hippocampus. Prdm8 -/- mice demonstrate neurobehaviouraldeficits and MRI changes consistent with impaired hippocampal andcerebellar development that overlap with deficits in Bhlhb4 -/- mice.Conclusions: We propose a model in which Prdm8 and Bhlhb4coordinately regulate the development of specific interneuronsubtypes in retina and brain.Commercial Relationships: Sasha Woods, None; AndrewStewart, None; Cynthia Jung, None; Sarah Ross, None; AndrewD. Dick, Novartis (C), Novartis (F), GSK (F), Abbott (F); CleaWarburton, None; Roderick R. McInnes, None; Denize Atan,NoneSupport: National Eye Research Centre, Bristol, UK301 Gene Regulation: Transcription and EpigeneticsTuesday, May 07, 2013 8:30 AM-10:15 AM6A Paper SessionProgram #/Board # Range: 2612-2618Organizing Section: Biochemistry/Molecular BiologyProgram Number: 2612Presentation Time: 8:30 AM - 8:45 AMTranscriptional and post-transcriptional regulation links RPE65,RLBP1, and RGR in the RPENoriko Esumi 1 , Tomohiro Masuda 1 , Jun Wan 1 , Karl J. Wahlin 1 , JaredIacovelli 2 , Natalie Wolkow 2 , Joshua L. Dunaief 2 , Donald J. Zack 1, 3 ,Jiang Qian 1 . 1 Wilmer Eye Institute, Johns Hopkins Univ Sch of Med,Baltimore, MD; 2 F.M. Kirby Center for Molecular Ophthalmology,Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA;3 Neuroscience, Molecular Biology and Genetics, and Institute ofGenetic Medicine, Johns Hopkins Univ Sch of Med, Baltimore, MD.Purpose: RPE cells perform many specialized functions that requirethe precisely regulated expression of a number of RPE-specificgenes. We have been studying the mechanisms that regulate geneexpression in the RPE, with particular interest in examining thehypothesis that genes involved in the same pathway are controlled bya common regulatory network. Here, we aim to test this hypothesisusing the visual cycle as a model system.Methods: In this study, we focused on three visual cycle genes,RPE65, RLBP1, and RGR. For transcriptional regulation, we usedRT-qPCR and immunohistochemistry for gene expression, DNase Ihypersensitivity assay for defining proximal promoters, celltransfection for promoter function, and chromatinimmunoprecipitation (ChIP) for binding of transcription factors. Sox9conditional knockout mice were used to study SOX9’s role in RPEgene expression. For post-transcriptional regulation, we usedbioinformatics to predict microRNA (miRNA) binding sites in the 3’-untranslated region (3’UTR), and cotransfection of miRNA mimicswith 3’UTR-luciferase constructs to test miRNA-target mRNAinteraction.Results: Conditional knockout of Sox9 in mature RPE resulted insignificantly reduced expression of several visual cycle genesincluding Rpe65, Rlbp1, and Rgr. Promoter analyses bycotransfection showed that SOX9 and OTX2 activate the RPE65 andRLBP1 promoters, whereas SOX9 and LHX2 activate the RGRpromoter. ChIP revealed that SOX9, OTX2, and LHX2 all bind to theproximal upstream region of RPE65, RLBP1, and RGR in bovineRPE cells. The expression of SOX9 and OTX2 in the RPE is wellknown; however, LHX2 has not been reported to be involved in RPEgene regulation. We found that LHX2 is highly expressed in matureRPE, with similar expression patterns to those of SOX9 in that bothgenes are expressed exclusively in Muller glia and RPE cells inadults. The 3’UTR of the three genes, particularly RPE65 andRLBP1, share multiple predicted miRNA sites, and cotransfection ofa miR-137 mimic significantly decreased luciferase activity of theconstructs containing the 3’UTR of RPE65 and RLBP1.Conclusions: These results indicate the important role of SOX9 as acentral link in the transcriptional regulation of RPE65, RLBP1, andRGR. In addition, RPE65 and RLBP1 are targets of the samemiRNA(s), suggesting that these genes share a common coreregulatory structure.Commercial Relationships: Noriko Esumi, None; TomohiroMasuda, None; Jun Wan, None; Karl J. Wahlin, None; JaredIacovelli, None; Natalie Wolkow, None; Joshua L. Dunaief,ApoPharma, Inc. (F), ApoPharma, Inc. (P); Donald J. Zack, Alcon(C), Merck (F), Allergan (C); Jiang Qian, NoneSupport: NIH Grant EY016398, AHAF Grant M2012102Program Number: 2613Presentation Time: 8:45 AM - 9:00 AMGTF2IRD1 is a Dual-FunctionTranscription Factor thatPromotes M Opsin Gene Expression and Suppresses S OpsinGene Expression in M ConesDonald J. Zack 1 , Anitha Yerrabelli 1 , Xiaodong Zhang 2 , Elizabeth A.Conner 3 , Snorri S. Thorgeirsson 3 , Anand Swaroop 4 , Shiming Chen 2 ,Tomohiro Masuda 1 . 1 Ophthalmology, Wilmer Eye Inst, JohnsHopkins Univ, Baltimore, MD; 2 Ophthalmology and Visual Sciences,Washington University School of Medicine, St. Louis, MO; 3 NationalCancer Institute, Bethesda, MD; 4 National Eye Institute, Bethesda,MD.Purpose: The mechanisms controlling cone photoreceptor-specificgene expression are not fully understood. Our aim was to identifytranscription factors (TFs) that regulate cone opsin gene expression.Methods: Yeast one hybrid (YOH) screening of a retinal library wasperformed to identify TFs that interact with the locus control region(LCR) of the mouse M opsin gene. Histological studies were appliedto examine the expression pattern of identified TFs in the developingmouse retina. Interaction of the identified TF with the upstreamregions of opsin genes was examined by ChIP. Transcriptionalactivity was characterized by siRNA-mediated gene knockdown inprimary murine retinal cells, and transient co-transfection assay in©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyHEK 293 cells with a reporter containing opsin gene upstreamregions. Finally, we analyzed the retina of a Gtf2ird1-null mouseusing molecular, histological, and electrophysiological approaches.Results: YOH screening identified several TFs that bind to the LCR.Among those was Gtf2ird1, whose role in the retina has not beenstudied. Gtf2ird1 expression in M cones is first detectable at P10 andis maintained through adulthood. ChIP assays showed that Gtf2ird1interacts with the LCR and promoter regions of the M and S opsingenes. siRNA-mediated Gtf2ird1 knockdown suppressed M opsinexpression; transient co-transfection assays showed that Gtf2ird1synergistically enhanced L opsin promoter activity in the presence ofCRX. On the other hand, Gtf2ird1 abolished S opsin promoteractivity enhanced by CRX with RORα or RORβ. Consistent withthese results, M opsin gene expression in the Gtf2ird1-null mouseretina was significantly suppressed, while S opsin gene expressionwas significantly increased. Histological studies of Gtf2ird1-nullmouse retina showed that S opsin immuno-positive cells wereincreased in the dorsal retina, where S cones are normally scarce.Interestingly, most of them were also M opsin immuno-positive. Onthe other hand, there was no significant change between Gtf2ird1-nulland wild-type mouse retinas in both the number of cells and thedorsal-ventral distribution pattern of M opsin immuno-positive cells.ERGs reflected these cone changes.Conclusions: Our results indicate that Gtf2ird1 is a dual-functiontranscription factor that maintains M cone function by promoting Mopsin expression and suppressing S opsin expression in M cones.Commercial Relationships: Donald J. Zack, Alcon (C), Merck (F),Allergan (C); Anitha Yerrabelli, None; Xiaodong Zhang, None;Elizabeth A. Conner, None; Snorri S. Thorgeirsson, None; AnandSwaroop, None; Shiming Chen, None; Tomohiro Masuda, NoneSupport: the National Eye Institute, Guerrieri Family Foundation,Foundation Fighting Blindness, and Research to Prevent Blindness,Inc.Program Number: 2614Presentation Time: 9:00 AM - 9:15 AMTranscription and splicing associated protein NonO/p54nrbregulates rod specific genes including rhodopsin and theirregulators required for rod differentiation and homeostasisSharda P. Yadav, Hong Hao, Marie-Audrey I. Kautzmann, MatthewBrooks, Jacob Nellissery, Anand Swaroop. National Eye Institute,National Institutes of Health, Bethesda, MD.Purpose: We have identified Non pou domain containing octamerbinding protein (NonO) from bovine retinal extracts using therhodopsin distal enhancer region. The purpose of current study is toelucidate the role of NonO in regulating the expression of rhodopsin.Methods: To understand role of NonO in regulation of rhodopsinexpression we performed shRNA knockdown of NonO using in vivoelectroporation. P0 CD1 pups were co-electroporated with Rhodopsinpromoter driving DsRed reporter along with either control or NonOshRNA. To visualize the electroporated cells, both shRNA constructscontained GFP reporter driven by signal recognition particle alphapromoter. GFP and DsRed positive cells in outer nuclear layer of thecontrol and NonO shRNA electroporated retinas were examined. Toexplore in vivo role of NonO in rod photoreceptors of P21 retinas,chromatin immunoprecipitation was performed followed by deepsequencing (Chip-Seq). We choose zeitgeber (ZT) 6 and ZT18 asgenes implicated in rod outer segment disc renewal exhibitdifferential expression at these times. Occupancy of NonO on genepromoters was analyzed using Genomatix software and UCSCgenome browser.Results: Knockdown of NonO by shRNA resulted in death ofdeveloping rod photoreceptors. The rod death could be partiallyrescued by a truncated NonO, which contained C-terminal 224 to 473amino acids. Luciferase reporter assays showed that C-terminalregion of NonO is sufficient to activate Rhodopsin promoterluciferase reporter. Chip-Seq data suggested that NonO occupiedgenes are mostly involved in phototransduction pathway. NonOoccupancy on the phototransduction genes is higher at ZT6 comparedto ZT18. These results suggest that NonO is involved in circadianregulation of rhodopsin as well as other phototransduction genes andtheir regulators in mouse rod photoreceptors.Conclusions: NonO is critical for rod development and itsknockdown leads to rod cell death. C-terminal 224 to 473 aminoacids of NonO are sufficient to activate rhodopsin promoterluciferasereporter expression in vitro. Chip-Seq results suggest thatNonO plays an important role in modulating the levels ofphototransduction genes during the circadian cycle. Further analysisof NonO function in mice retina should provide new insights into theregulation of rod outer segment biogenesis.Commercial Relationships: Sharda P. Yadav, None; Hong Hao,None; Marie-Audrey I. Kautzmann, None; Matthew Brooks,None; Jacob Nellissery, None; Anand Swaroop, NoneSupport: National Eye InstituteProgram Number: 2615Presentation Time: 9:15 AM - 9:30 AMCorrelating Whole Genome DNA Methylation Patterns withRetinal Expression and Alternative SplicingJiang Qian, Jun Wan, Verity F. Oliver, Donald J. Zack, Shannath L.Merbs. Department of Ophthalmology, Johns Hopkins School ofMedicine, Baltimore, MD.Purpose: DNA methylation is a major epigenetic modification thatplays an important role in multiple cellular processes. It is generallybelieved that DNA methylation inhibits gene expression. However,how exactly the DNA methylation regulates tissue-specific geneexpression and splicing remains unclear. In this study, we performedgenome-wide DNA methylation profiling in retina and brain toevaluate the correlation between DNA methylation and tissuespecificgene regulation.Methods: DNA methylation profiling on two adult mouse tissues,retina and brain, was conducted using the comprehensive highthroughputarray for relative methylation (CHARM) NimbleGentiling array. Affymetrix exon microarrays were also used to measurethe gene expression and splicing differences between retina andbrain. Bioinformatics methods were developed and employed toanalyze the data and correlate the DNA methylation, gene expressionand splicing.Results: Numerous tissue-specific differentially methylated regions(tDMRs) were identified. These tDMRs located in various genomicregions, including promoter, exon, intron and intergenic regions.They were enriched in CpG island shores, but depleted in CpGislands. We then integrated these tDMRs with the transcriptome datameasured in the corresponding tissues by exon microarray. DMRsthat negatively correlated with downstream gene expression tended tobe close to transcription start sites. Tissue-specific genes were morelikely to be regulated by differential methylation than expected. Alarge number of DMRs located in exons or introns correlated with theexpression of an adjacent exon, suggesting a potential role of DNAmethylation in tissue-specific splicing.Conclusions: Our work provides a large-scale survey of differentialDNA methylation of retina-specific genes and lays the foundation forfurther mechanistic studies of the biological role of DNA methylationin tissue-specific gene regulation.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyCommercial Relationships: Jiang Qian, None; Jun Wan, None;Verity F. Oliver, None; Donald J. Zack, Alcon (C), Merck (F),Allergan (C); Shannath L. Merbs, NoneSupport: NIH Grants EY021897 EY018703 and Unrestricted fundsfrom RPBProgram Number: 2616Presentation Time: 9:30 AM - 9:45 AMHistone marks predict cell plasticity of the adult human retinalpigment epitheliumTimothy A. Blenkinsop 1 , Alvaro Rada-Iglesias 2 , Joanna Wysocka 2 ,Sally Temple 1 . 1 Macular Degeneration Program, Neural Stem CellInstitute, Rensselaer, NY; 2 Developmental Biology, StanfordUniversity, Stanford, CA.Purpose: Retinal pigment epithelial (RPE) cells are one of the fewcell types well recognized to change fate in lower vertebrates. Werecently showed adult human RPE as old as 99 years differentiatedown mesenchymal and neural lineages using commercially availabledifferentiation media. Addressing whether this was due to themultipotency of the RPE cells or to a contaminating cell type, forexample MSCs, in the cultures was critical. Therefore, wedemonstrated that single, cloned RPE cells exhibit this ability,irrefutably demonstrating that they are multipotent cells. Wetherefore sought to understand the basis for this observed plasticity.Histone marks at promoter and enhancer sites can reveal both genesthat are actively being transcribed - i.e. active, and genes that arepoised - i.e. currently inactive, but can be activated by underappropriate conditions. Recently histone H3K27me3 mark has beenshown to be enriched in regions several kilobases upstream from thepromoter around enhancer regions that are poised in humanembryonic stem cells (hESCs). In contrast, histone mark H3K27acmarks similar locations when the downstream gene is active.Therefore, we hypothesized that these two marks can predict genechanges from being poised to active.Methods: We cultured human retinal pigment epithelium intopolarized layers and verified their terminal differentiation throughtransepithelial resistance (TER) in Ωcm2. These monolayersexpressed TER of > 200Ωcm2 similar to native RPE. We alsoconfirmed their identity immunohistochemically, by quantitativePCR. We then conducted H3K27ac and H3K27me3 Chromatinimmunoprecipitation-sequencing (ChIP-seq) on adult human RPEmonolayers.Results: We found RPE specific genes posses H3K27ac marksupstream of their promotor regions, whereas the genes we have foundto increase expression during differentiation, for example RUNX2during osteogenic conditions, possess the poised mark H3K27me3upstream of their promotor.Conclusions: These results suggest the histone marks can predict notonly the cell identity, but also the poised mark can predict RPEplasticity.Commercial Relationships: Timothy A. Blenkinsop, None; AlvaroRada-Iglesias, None; Joanna Wysocka, None; Sally Temple,Athghin Biotech (I)Support: Ey022079-01Program Number: 2617Presentation Time: 9:45 AM - 10:00 AMThe MLL1 Histone Methyltransferease is Essential forDevelopment of Photoreceptor FunctionDiana S. Brightman, Ray Suzuki, Shiming Chen. Ophthalmology andVisual Sciences, Washington University in St. Louis, St. Louis, MO.Purpose: Development and maintenance of retinal photoreceptorfunction requires precisely controlled gene expression. This isregulated by both photoreceptor-specific and general transcriptionregulators, including histone modification enzymes. Members of theMLL family of histone H3K4 methyltransferases are expressed inmouse photoreceptors. The most prominent of these is MLL1 whoseexpression increases between postnatal day 2 (P2) and P14, a criticalperiod for photoreceptor terminal differentiation. MLL1 expressiondepends on the key photoreceptor-specific transcription factor CRX,suggesting a potential role of MLL1 in the CRX regulatory pathway.We determined the role of MLL1 in photoreceptor gene expression,development and survival using a loss-of-function approach.Methods: To avoid embryonic lethality, Cre-loxP-mediatedconditional knockout (CKO) was used. Mice with Mll1 floxed alleles(Mll1 fl/fl ) were crossed with Crx-Cre mice to create Mll1 CKO in thedeveloping retina. Mll1 CKO mice show morphological changes inthe retina by H&E staining, immunohistochemistry (IHC) andelectron microscopy (EM), and visual function changes byelectroretinogram (ERG).Results: Cre activity was found in all cell layers of the developingMll1 CKO retina, in a superior-to-inferior gradient. As a result, Mll1expression was uniformly depleted in the superior region, where allretinal layers were significantly thinner by morphometry. IHC andEM analyses show abnormal superior outer plexiform layer (OPL)synapses. The intensity of the presynaptic markers V-GLUT1 andCTBP2 were markedly decreased at the OPL. Calbindin-positivehorizontal cells were also reduced, indicative of postsynaptic defects.Consistent with these defects, both dark and light-adapted ERGs of 1-month-old Mll1 CKO mice were significantly decreased, suggestingdefects in rod, cone, and inner retina functions. The morphologicaland functional changes are stable to 6 months of age, suggesting adevelopmental origin, not degeneration.Conclusions: MLL1 is required for the development of appropriateretinal structure and function. Additional Mll1 CKO usingphotoreceptor and INL cell-type-specific Cre lines are in progress todetermine cell autonomy and underlying molecular mechanisms.These studies will shed light on how general epigenetic modulatorscontribute to the regulation of cell-type specific gene expression anddevelopment.Commercial Relationships: Diana S. Brightman, None; RaySuzuki, None; Shiming Chen, NoneSupport: NIH Grant EY012543 (to SC), EY02687 (to WU-DOVS),RBP Lew R. Wasserman Award (to SC) and unrestricted funds (toWU-DOVS)Program Number: 2618Presentation Time: 10:00 AM - 10:15 AMRole of 5-hydroxymethylcytosine during postnatal retinaldevelopmentStylianos Michalakis 1 , Arshan Perera 1 , Susanne Koch 1 , MirkoWagner 2 , Lukas Windhager 3 , Kerstin Nagel-Wolfrum 4 , Tim M.Strom 5, 6 , Ralf Zimmer 3 , Thomas Carell 2 , Martin Biel 1 . 1 Center forIntegrated Protein Science at the Department of Pharmacy-Center forDrug Research, Ludwig-Maximilians-Universität München, Munich,Germany; 2 Center for Integrated Protein Science at the Department ofChemistry, Ludwig-Maximilians-Universität München, Munich,Germany; 3 Institute for Informatics, Ludwig-Maximilians-UniversitätMünchen, Munich, Germany; 4 Department of Cell and MatrixBiology, Institute of Zoology, Johannes Gutenberg University-Mainz,Mainz, Germany; 5 Institute of Human Genetics, TechnischeUniversität München, Munich, Germany; 6 Institute of HumanGenetics, Helmholtz Zentrum München, German Research Center forEnvironmental Health, Neuherberg, Germany.Purpose: 5-hydroxymethylcytosine (5hmC), also known as the sixthbase of the genome, is a recently discovered oxidative product of 5-©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologymethylcytosine (5mC) generated by the enzymatic action of TenEleven Translocation (TET) family members. The functional role of5hmC is largly unknown. However, studies in embryonic stem cellsand cancer tissues suggest that TET enzymes and 5hmC may beinvolved in gene regulation. Eye opening at postnatal week (pw) 2 isa key time point during mouse retinal development. Maturation ofretinal cells as well as formation of the retinal network is ongoingafter eye opening and is completed only one to two weeks later. Here,we analyzed the role of 5hmC during this phase of postnatal retinaldevelopment in the mouse.Methods: 5hmC was localized to specific retinal cell types usingimmunohistochemistry. Global 5hmC levels in retinal samples werequantified using ultra high pressure liquid chromatography-tandemmass spectrometry (UHPLC-MS/MS). 5hmC marks were mapped tothe retinal genome by hydroxymethylated DNA immunoprecipitation(hMeDIP) followed by next generation sequencing and weresubsequently correlated with retinal gene expression data obtainedfrom microarray experiments.Results: At pw 2 5hmC weakly localized to nuclei of cells within theganglion cell layer and the inner nuclear layer of the retina. Thelevels of 5hmC in these cells were increased at pw 3. In addition,5hmC was now detectable in retinal photoreceptors were it colocalizedwith histone marks of the euchromatin. Global levels of5hmC also increased from pw 2 to pw 3, whereas 5-methylcytosinewas unchanged. hMeDIP experiments revealed a developmentallyprogrammed acquisition of 5hmC during retinal maturation at generichregions and in genes containing activating histone marks.Conclusions: Our data suggest that 5hmC is dynamically regulatedduring postnatal retinal development and is capable to elevate geneexpression of retina-specific genes.Commercial Relationships: Stylianos Michalakis, None; ArshanPerera, None; Susanne Koch, None; Mirko Wagner, None; LukasWindhager, None; Kerstin Nagel-Wolfrum, None; Tim M. Strom,None; Ralf Zimmer, None; Thomas Carell, None; Martin Biel,NoneSupport: Deutsche Forschungsgemeinschaftbut also therapeutic targets and vehicles. EVs were previously shownto participate in angiogenesis, tissue remodeling and tissueregeneration. In this work we aimed at first time characterization ofprotein and RNA profiles of EVs isolated from human vitreoussamples.Methods: We analyzed EVs from vitreous fluid collected duringvitrectomy from patients with proliferative diabetic retinopathy andprimary rhegmatogenous retinal detachment. EVs were isolated usingdifferential centrifugation, and were visualized by transmissionelectron microscopy (TEM). Size histograms of EV preparationswere determined by a resistive pulse sensing approach (qNano).Cellular origin of EVs was determined by flow cytometry (FC).Protein and RNA profiles of EVs were analyzed by massspectrometry (MS) and bioanalyzer assay (Agilent).Results: TEM clearly shows various populations of EVs in thevitreous fluid (Figure 1). Peak EV size was around 150 nm indiameter. The presence of EVs in vitreous fluid was also confirmedusing FC based on annexin V binding. Most EVs in the vitreous fluidwere derived from platelets and endothelial cells. MS revealedclassical EV-associated proteins including actin, actin-bindingproteins (e.g. ankyrin), tubulin, clusterin, heat shock proteins andenzymes. However, we also identified eye-specific proteins in EVsincluding retinal dehydrogenase, retinol binding protein and lensspecific proteins (lensin, crystallin etc.). Most importantly RNAprofiling has revealed that miRNA molecules were present invitreous-fluid-derived EVs in very high amounts (Figure 2).Conclusions: In this work we successfully isolated and characterizedEVs from vitreous fluid, and demonstrated the presence of miRNAsin these structures. Demonstration of angiogenesis-inducing miRNAsin vitreous fluid EVs may lead to identification of new biomarkers ornovel therapeutic targets in eye disorders.325 Biochemical and Molecular Mechanisms of Diabetic andRetinal DiseaseTuesday, May 07, 2013 11:00 AM-12:45 PM6A Paper SessionProgram #/Board # Range: 3148-3153Organizing Section: Biochemistry/Molecular BiologyContributing Section(s): Biochemistry/Molecular BiologyFigure 1. EVs visualized by TEM. Magnification is 30,000xProgram Number: 3148Presentation Time: 11:00 AM - 11:15 AMAnalysis of extracellular vesicles in vitreous samplesBence Gyorgy 1, 2 , Zsuzsanna Récsán 2 , Ágnes Kittel 3 , KrisztinaPálóczi 1 , Lilla Turiák 4 , Károly Vékey 4 , Janos Nemeth 2 , Edit I. Buzas 1 ,Zoltán Zsolt Nagy 2 . 1 Department of Genetics, Cell- andImmunobiology, Semmelweis University, Budapest, Hungary;2 Department of Ophthalmology, Semmelweis University, Budapest,Hungary; 3 Institute of Experimental Medicine, Hungarian Academyof Sciences, Budapest, Hungary; 4 Chemical Research Center,Hungarian Academy of Sciences, Budapest, Hungary.Purpose: Extracellular vesicle (EV) secretion represents anevolutionally conserved feature of living cells. EVs are known totransfer protein and RNA cargos between cells placing EV analysisinto the mainstream of biomedical research. The assessment of EVsmay provide insight into the pathomechanism of various disorders.Furthermore, they may not only serve as potential novel biomarkers,Figure 2. RNA profile from EVs showing mostly small RNAmolecules.Commercial Relationships: Bence Gyorgy, None; ZsuzsannaRécsán, None; Ágnes Kittel, None; Krisztina Pálóczi, None; LillaTuriák, None; Károly Vékey, None; Janos Nemeth, None; Edit I.Buzas, None; Zoltán Zsolt Nagy, NoneSupport: This work was supported by OTKA K 73247, NK 84043and K77537, Kerpel-Fronius Ödön Fellowship, Baross Gábor (REG-KM-09-1-2009-0010) and FP7-PEOPLE-2011-ITN - PITN-GA-©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biology2011-289033 “DYNANO” and BM1202 European Network onMicrovesicles and Exosomes in Health and Disease (ME-HAD).Bence György is a Kerpel-Fronius Ödön Fellows.Program Number: 3149Presentation Time: 11:15 AM - 11:30 AMNephrocystin-5 knockout mice recapitulate retina and kidneypathologies of Senior-Løken SyndromeCecinio Ronquillo, Jeanne M. Frederick, Wolfgang Baehr.Ophthalmology and Visual Sciences, Moran Eye Center, Universityof Utah, Salt Lake City, UT.Purpose: Senior-Løken syndrome is an autosomal recessive diseasecharacterized by retinal degeneration (RP/LCA) andnephronophthisis (kidney fibrosis and presence of cysts). Mutationsin Nephrocystin-5 (NPHP5) have been shown to be the most commoncause of Senior-Løken syndrome in humans; however, the function ofNPHP5 is poorly understood. Here, we describe a novel mammalianmodel of Senior-Løken syndrome generated by deleting NPHP5 inthe mouse.Methods: A gene trap containing a reporter gene (β-gal) was insertedin intron 4 of the mouse Nphp5 gene causing premature terminationof Nephrocystin-5 protein expression. Retina and renal tissue werecharacterized by immunocystochemistry using various antibodies atdevelopmental time points. ERGs were recorded at different timepoints. Ultrastructure of the photoreceptor connecting cilium andbasal body was examined on postnatal days P6 and P10.Results: Global knockout mice are viable and fertile. Retinalfunction in knockout animals was undetectable by ERG at P12 (eyeopening). At P10, knockout animals already showed absence of outersegments and decreased number of cells in the outer nuclear layer.Rhodopsin transport is impaired as early as P6 and rhodopsinaccumulates in rod perinuclear regions (Fig. 1). Apoptosis, fibrosisand presence of cysts are observed in the kidneys of knockout mice(Fig. 2). Knockdown of NPHP5 in a kidney cell line shows decreasednumbers of primary cilia suggesting a role of NPHP5 in ciliogenesisor ciliary maintenance.Conclusions: NPHP5 global knockout mouse is a novel model ofSenior-Løken syndrome recapitulating the retina and renalpathologies observed in humans. This model will be an importanttool to provide insight into detailed molecular mechanisms of NPHP5function in ciliogenesis and primary cilium structure.Figure 1. Rhodopsin mislocalizes to rod perinuclear regions of P10NPHP5 KO retina (red arrow). Rhodopsin (green, 1D4), cone arrestin(red, mCAR), nuclei (blue, DAPI).Figure 2. NPHP5 knockout kidneys have increased cell death byTUNEL stain (green). Also shown is an example of a cyst present inknockout kidneys (red arrow).Commercial Relationships: Cecinio Ronquillo, None; Jeanne M.Frederick, None; Wolfgang Baehr, NoneSupport: National Eye Institute F31 NRSA - F31EY021972-01A1Program Number: 3150Presentation Time: 11:30 AM - 11:45 AMLong-term preservation of immature cone-like photoreceptors ina mouse model of human LCA caused by dominant CRXframeshift mutationJerome E. Roger 1 , Avinash H. Hiriyanna 1 , Debbie F. Cheng 1 ,Norimoto Gotoh 1 , Rinki Ratna Priya 1 , Matthew Brooks 1 , Harsha K.Rajasimha 1 , Bo Chang 2 , Anand Swaroop 1 . 1 Neurobiol-Neurodegnt'nRep Lab, NEI / National Institutes of Health, Bethesda, MD; 2 TheJackson Laboratory, Bar Harbor, ME.Purpose: Leber’s congenital amaurosis (LCA) is a severe form ofchildhood blindness representing 5% of all retinopathies. Mutationsin CRX, a key transcription factor for photoreceptor development, areassociated with retinal dystrophies including LCA. The pathogenicmechanisms of CRX disease have not been elucidated, and thereported Crx-/- mouse model does not reflect the human phenotype.Here, we describe a spontaneous mutant with autosomal dominantcongenital blindness named RIP (Retina with ImmaturePhotoreceptors), caused by a frameshift mutation in Crx leading tothe differentiation of retina with immature cone-like photoreceptors.Methods: Linkage analysis and exome capture sequencing were usedto identify the mutation. Fundus photography, ERG,immunohistochemistry, RNA-seq, luciferase and gel-shift assayswere performed to characterize the RIP mouse and the mutant CRXprotein. CRX and frameshift mutants were transfected in mouseretina by in vivo electroporation. Crxp::Nrl mice were used for someof the rescue experiments.Results: The RIP mutant revealed autosomal dominant pattern ofinheritance with congenital blindness and exhibited pigmentation andattenuated vasculature upon fundus examination at one-month of age.We identified a frameshift mutation in Crx resulting in the deletion ofthe Otx-like domain and an extended C-terminal region. Interestingly,several LCA-causing CRX mutations result in similar proteinmodifications. In the RIP mutant, the retinal photoreceptors hadcone-like nuclei, but no outer segments; however, unlike Crx-/- micethe photoreceptors did not degenerate. RNAseq analysis of P2 andP21 retina revealed a loss of Nrl and Nr2e3 expression and anabsence of most rod visual signaling components only at P21. Wealso showed the lack of transcriptional activity in CrxRIP was due toits inability to bind DNA. By mating RIP mutant with Crxp-Nrl micein which Nrl is expressed in all photoreceptor precursors, we partiallyrescued the phenotype due to the existence of only rods with shortouter segments. Electroporation in wild type mice of LCA-causingCRX frameshift mutations led to an arrest of photoreceptor©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologymaturation.Conclusions: Our findings show the dominant negative effect of agroup of CRX mutations preventing the establishment of rod generegulatory network and reveal the RIP mouse as a good model forLCA-causing CRX mutations.Commercial Relationships: Jerome E. Roger, None; Avinash H.Hiriyanna, None; Debbie F. Cheng, None; Norimoto Gotoh, None;Rinki Ratna Priya, None; Matthew Brooks, None; Harsha K.Rajasimha, Genome International Corporation (C), DovelTechnologies (E), George Mason University (S), Rare GenomicsInstitute (S), National Eye Institute (C); Bo Chang, None; AnandSwaroop, NoneSupport: NIH Intramural Research ProgramProgram Number: 3151Presentation Time: 11:45 AM - 12:00 PMNatriuretic peptides regulate MAP kinases via PKG to protectthe RPE from VEGF actionZsolt Ablonczy 1 , Mohammad Dahrouj 1 , Yueying Liu 1 , KumarSambamurti 2 , Craig E. Crosson 1 . 1 Ophthalmology, MedicalUniversity of South Carolina, Charleston, SC; 2 Ophthalmology,Medical University of South Carolina, Charleston, SC.Purpose: Diabetic retinopathy-associated vision loss is oftenattributed to edema within the layers of the retina. Diabetes is knownto alter the levels of natriuretic peptides (NP) expressed by the retinalpigment epithelium (RPE). However, the function of these peptidesand their contribution to diabetic retinopathy remains unclear. Wehave previously shown that NP receptor 2 (NPR-2) activation canblock the breakdown of the RPE barrier. In this project, we haveinvestigated the molecular mechanism of NP activity in the RPE.Methods: Barrier breakdown was induced in fetal human RPE cellsby 100 mg/L glycated albumin (glyc-alb) or VEGF (10 μg/L) andmeasured by transepithelial resistance (TER). NPs (ANP or CNP;nmol/L) were administered apically. Administration of the cGMPanalog, 8-Br-cGMP (100 μmol/L), or the PKG inhibitor, KT5823 (5μmol/L) was used to measure the involvement of signaling eventsdownstream of NPR-2. Enzyme-linked immunoassays determinedchanges in VEGF secretion. VEGF-R2 was inhibited by ZM323881(10 nmol/L). Map kinase activation was assessed by pretreatmentwith pathway inhibitors (U0126 for ERK1/2, SB203580 for p38,SP600125 for JNK, LY294002 for PI3K; all at 0.1-10 μM) andwestern blot analysis of ERK1/2.Results: Glycated albumin induced an apically-directed VEGFrelease paralleled with a rapid breakdown of RPE barrier function.The glyc-alb response was reversed by the administration of ANP orCNP in a concentration-dependent fashion and also by the VEGF-R2antagonist, ZM323881. However, VEGF-release was notsignificantly altered by NPs. Pretreatment with 8-Br-cGMPsubstituted NP-induced responses and pretreatment with KT5823abolished any NP-responses. VEGF-induced barrier breakdownrequired the activity of the ERK1/2 pathway but not that of the JNK,p38 and PI3K pathways. Similarly to U0126, NPs inhibited theVEGF-induced activation of ERK1/2.Conclusions: These studies provided evidence that in RPE cells, NPscan oppose the effects of advanced glycation end-products, one of themost important complicating factors in diabetes. NPR-2 activationinduced PKG, eventually targeting the ERK1/2 MAP kinasedownstream of VEGF-R2. These results are consistent with the ideathat NPs are part of an endogenous system necessary for maintainingthe barrier integrity of the RPE. Therefore, we conclude that NPsplay a critical role in protecting vision against diabetic retinopathyand retinal edema.Commercial Relationships: Zsolt Ablonczy, None; MohammadDahrouj, None; Yueying Liu, None; Kumar Sambamurti, None;Craig E. Crosson, Alimera Sciences (C), Lexicon Pharmaceuticals,Inc (R)Support: Supported by NIH/NEI EY019065 (ZA), NIH/NEIEY009741 (CEC), NIH/NIA AG022103 (KS); and an unrestrictedgrant fro RPB to the Department of Ophthalmology at MUSC.Program Number: 3152Presentation Time: 12:00 PM - 12:15 PMRestoration of vitreous structure after degradationYing-Bo Shui 1 , Benjamen Filas 1 , Qianru Zhang 1 , Shaili Sharma 3 ,Alyssa Panitch 3 , David C. Beebe 1, 2 . 1 Dept of Ophthalmology andVisual Sciences, Washington Univ Sch of Med, St Louis, MO; 2 Deptof Cell Biology and Physiology, Washington Univ Sch of Med, StLouis, MO; 3 Weldon School of Biomedical Engineering, PurdueUniversity, West Lafayette, IN.Purpose: Age-related vitreous degeneration leads to several sightthreateningdiseases, including retinal detachment, macular hole, andpre-retinal membranes. Delaying degeneration or restoring thestructure of the vitreous gel would prevent these diseases. Recently,synthetic proteoglycan mimics that bind to hyaluronic acid orcollagen have been shown to increase the compressive strength ofcartilage and protect other components of the extracellular matrixfrom proteolytic degradation, thereby replicating the functions of thecartilage proteoglycan, aggrecan. Since the vitreous is essentially“dilute cartilage,” we tested the ability of three proteoglycan mimicsto restore the physical properties and structure of trypsin-treatedbovine vitreous.Methods: A core of fresh bovine vitreous is carefully removed with atrephine and injected with PBS (sham), purified trypsin, trypsin(TRYP) plus mimics , or mimics alone. The mimics were designed tobind hyaluronan (GAH), collagen type II (WYR) or both hyaluronanand collagen type II (WYR/GAH). Samples are incubated at 4 degreeovernight and their viscoelastic properties measured using arheometer. Morphological analysis is performed by deep-etchelectron microscopy (DEEM).Results: Trypsin-treated vitreous had a significant, 50% decrease instorage modulus (stiffness) relative to untreated and PBS--shamsamples. The stiffness of vitreous samples pre-treated with trypsin iscompletely restored by addition of each of the three proteoglycanmimics. Vitreous treated with proteoglycan mimics alone also showsa tendency toward increased storage modulus but this was notstatistically significant (figure 1). In trypsin-treated vitreous DEEMshowed thinner, more discontinuous fibrils, decreased “knobby”projections and increased open space. Analysis by DEEM of trypsinandmimic-treated vitreous is in process.Conclusions: Synthetic proteoglycan mimics restore the physicalproperties of degraded vitreous and may have clinical applications forprevention of age-related retinal damage. In vivo examination of thesafety and stability the mimics in the vitreous is warranted.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologySupport: NIH R01 EY020861, RPB Challenge Grant, RPB Lew R.Wasserman Merit Award, HHMI Medical Research Fellows ProgramCommercial Relationships: Ying-Bo Shui, None; Benjamen Filas,None; Qianru Zhang, None; Shaili Sharma, None; Alyssa Panitch,Purdue Research Foundation (P); David C. Beebe, FivePrime (C),Panoptica (C), Vistakon (Johnson and Johnson) (C)Support: EY021515-01, Unrestricted grant from RPB, Core grantEY02687Program Number: 3153Presentation Time: 12:15 PM - 12:30 PMInsulin Stimulates Increased Production of Soluble Betacellulinin Retinal Pigment Epithelium CellsBailey Y. Shen, Mariya Ali, Alecia Cutler, Bela Anand-Apte.Ophthalmology, Cole Eye Institute, Cleveland Clinic, Cleveland, OH.Purpose: To determine the effects of high glucose and extracellularinsulin on insulin signaling, ADAM 10 (A Disintegrin andMetalloproteinase 10), and betacellulin (BTC) expression in retinalpigment epithelium (RPE) cells in vitro.Methods: ARPE-19 cells were incubated in low (5 mM) or high (25mM) glucose media containing varying doses of regular humaninsulin (0 pM - 100 nM). After 48 hours, cells were harvested bymechanical lysis, and western blots of cell lysates were performed tomeasure expression of insulin receptor, phosphorylated ERK-1 (amember of the insulin signaling pathway), ADAM 10, andbetacellulin. As ADAM 10 is known to cleave pro-BTC (MW ~46kDa) into soluble BTC (22 kDa) and BTC cytoplasmic remnant (~25kDa), two different types of betacellulin antibodies, one against its N-terminal ectodomain, and one against its cytoplasmic domain, wereused to study BTC cleavage.Results: Insulin concentrations greater than or equal to 10 nM causedincreased soluble BTC expression in RPE cells, while glucoseconcentrations did not have any effect on soluble BTC expression.Insulin also upregulated ADAM 10 expression, but, surprisingly, didnot increase levels of BTC cytoplasmic remnant, which would beexpected to accumulate if insulin enhanced ADAM 10-mediatedcleavage of pro-BTC into soluble BTC. Finally, insulin increasedexpression of phosphorylated ERK-1 and decreased expression ofinsulin receptor.Conclusions: Soluble betacellulin is a molecule known to increaseretinal vascular permeability, and a molecule thought to play a role inthe pathogenesis of diabetic macular edema. This study suggests thatextracellular insulin acting upon RPE cells may contribute to theincreased soluble betacellulin previously measured in retinas ofdiabetic animals and humans. Our results also raise the possibilitythat insulin-mediated increases in soluble BTC in RPE cells may beindependent of pro-BTC cleavage.Commercial Relationships: Bailey Y. Shen, None; Mariya Ali,None; Alecia Cutler, None; Bela Anand-Apte, 7 183 256 B2 (P)362 Biochemistry and Regulation of Proteins in AMDTuesday, May 07, 2013 2:45 PM-4:30 PM6A Paper SessionProgram #/Board # Range: 3651-3657Organizing Section: Biochemistry/Molecular BiologyProgram Number: 3651Presentation Time: 2:45 PM - 3:00 PMAge-Dependent Changes In Heparan Sulfate In Human Bruch’sMembrane: Implications For Age-Related Macular DegenerationTiarnan D. Keenan 1, 4 , Claire Pickford 2 , Rebecca Holley 2, 3 , Simon J.Clark 1, 4 , Catherine Merry 2 , Anthony J. Day 3 , Paul N. Bishop 1, 4 .1 Faculty of Medical and Human Sciences, University of Manchester,Manchester, United Kingdom; 2 Faculty of Engineering and PhysicalSciences, University of Manchester, Manchester, United Kingdom;3 Faculty of Life Sciences, University of Manchester, Manchester,United Kingdom; 4 Centre for Advanced Discovery and ExperimentalTherapeutics, Manchester Academic Health Sciences Centre,Manchester, United Kingdom.Purpose: Heparan sulfate (HS) plays an important role in retinalhomeostasis. It may be implicated in age-related maculardegeneration (AMD), as it is the major binding partner forcomplement factor H (CFH) in human macular Bruch’s membrane.The aim of this study was to investigate potential changes with age inthe quantity and composition of HS in the human retina.Methods: Postmortem human ocular tissue was obtained fromconsenting eye donors without known retinal disease (n=4 pairs ofyounger globes, age 26-35 years; n=6 pairs of older globes, age 71-88years). Retinal tissue was dissected, and HS was extracted andpartially purified by anion exchange chromatography. Followingheparinase digestion, HS disaccharides were labeled with AMAC andquantified according to sulfation pattern by HPLC alongsidereference standards. Separately, HS was detected byimmunohistochemistry in frozen human macular tissue sections (n=6from younger globes, age 18-34 years; n=5 from older globes, age79-89 years).Results: The quantity of HS in Bruch’s membrane was 44% lower inolder donors (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyProgram Number: 3652Presentation Time: 3:00 PM - 3:15 PMTREM2 (chr6p21.1) and CFH (chr1q32) regulation by NF-kBsensitivemiRNAs in age-related macular degeneration (AMD)and Alzheimer's disease (AD)Walter J. Lukiw 1, 2 , Brandon M. Jones 1 , Surjyadipta J.Bhattacharjee 1 , Peter N. Alexandrov 2 , Prerna Dua 3 , Yuahi Zhao 1, 4 .1 Neuroscience & Ophthalmology, Lousiana State Univ HealthSciences Center, New Orleans, LA; 2 Russian Academy of MedicalSciences, Moscow, Russian Federation; 3 Louisiana TechnicalUniversity, Ruston, LA; 4 University of Texas Health SciencesCenter, Houston, TX.Purpose: Age-related macular degeneration (AMD) and Alzheimer’sdisease (AD), progressive degenerations of layered multicellularassemblies in the retina and neocortex, are both associated withaltered innate-immune responses, amyloidogenesis and progressiveneurodegeneration. Very recently, loss-of-function for the triggeringreceptor expressed in myeloid and microglial cells 2 (TREM2;chr6p21.1), have been associated with inflammatoryneurodegeneration in AD. The pathological role of TREM2 appearsto be related to aberrant complement factor H (CFH; chr 1q32)signaling functions in both AMD and AD. The purpose of thesestudies was to examine the epigenetic regulation of TREM2 and CFHin AMD and AD, and the up-regulated micro RNAs (miRNAs) whichregulate their expression.Methods: 3’-untranslated region (3’-UTR) sequence and vectoranalysis; Aβ42-peptide+TNFα-induced and/or hydrogen peroxideinducedstress; bioinformatics; CAPE, CAY10512, DNA array; DNAsequencing; human brain postmortem tissue; human retinal tissue;human retinal and brain cells in primary culture; gel shift assay;LED-Northern micro-dot blot analysis; micro-RNA array; protectedanti-miRNAs; RT-PCR; TREM-2- and CFH-3’-UTR-luciferasereportertransfection assay; Western immuno-histochemistryResults: In AMD retina, in AD brain and in stressed primary humanbrain and retinal cells we identified small families of NF-kBregulatedmiRNAs that include miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a and miRNA-155. Each of the promoters thatregulate transcription of the precursors for these 5 miRNAs containfunctional NF-kB binding domains. Inducible combinations of thesemature miRNAs target the TREM2 mRNA-3’-UTR and/or the CFHmRNA-3’-UTR, resulting in significant decreases in TREM2 or CFHexpression (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologynovel biomarkers for AMD. We performed the whole proteomicprofiling of AH of patients, conditioned media (CM) from ARPE-19cells exposed to 400 µM paraquat 24 h and exosomes derived fromAH and CM by LC-MS/MS. Exosome pellets from AH and CM wereobtained with ExoQuickTM Exosome Precipitation Solution. Toconfirm exosome and exosomal proteins, transmission electronmicroscopy (TEM) and Western blot analysis for exosomal markerproteins (CD63 and Hsp70) were performed. Finally, liquidchromatography multiple reaction monitoring (LC-MRM) analysis ofAH of 15 treatment-naïve patients with neovascular AMD andcontrol subjects were performed to validate the target proteins.Results: TEM revealed that exosomes from the AH of patients andCM from ARPE-19 cells as round shaped membrane vesicles sized50-100 nm in diameter. CD63 and Hsp70 in exosomes from AH andCM were detected by Western blot analysis. Among 70 proteinsidentified both in AH and CM, six proteins including cathepsin D and26S proteasome non-ATPase were found in exosomes of AH. Sevenproteins including cytokeratin 8 and Hsp70 were found in exosomesof CM. Two proteins were detected in exosomes from both. In total,11 exosomal proteins were increased in AH of 15 patients withvarious levels of decrease after intravitreal injection of ranibizumabby LC-MRM.Conclusions: The present study has identified potential biomarkersand therapeutic target proteins of AMD and provided an effectiveapproach to identify AMD-associated proteins that are secreted byRPE cells in vivo. In addition, we provide new evidence that thesecretory mechanisms of these proteins might be closely related toexocytic activity as in the formation of drusens which are considereda risk factor for developing AMD.Commercial Relationships: Hyewon Chung, None; Ae Jin Choi,None; Eun Sook Lee, None; Gum-Yong Kang, None; SoyoungChoi, None; Hyunjung J. Lim, NoneSupport: National Research Foundation of Korea (NRF) funded bythe Ministry of Education, Science and Technology(2012M3A9B2028333)Program Number: 3655Presentation Time: 3:45 PM - 4:00 PMShort-term exposure to thrombin induces long lasting disruptionof barrier properties and proangiogenic signals in RPETami Livnat 1 , Omer Bialer 2 , Yael Nisgav 1 , Mor Dachbash 1 , RimaDardik 1, 3 , Dov Weinberger 2, 4 . 1 Laboratory of Eye Research,Felsenstein Medical Research Center, PetachTikva, Israel;2 Department of Ophthalmology, Rabin Medical Center, Petah Tiqwa,Israel; 3 The Israeli National Hemophilia Center and Institute ofThrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer,Israel; 4 Sackler School of Medicine, Tel Aviv University, Tel Aviv,Israel.Purpose: Thrombin is a multifunctional protease playing a centralrole in coagulation. Apart from its major function in vein and arteryocclusion, thrombin was found to play a significant role ininflammatory diseases. Thrombin exerts cellular effects through itsmembrane receptor (PAR). In vivo testing of thrombin is difficultsince its existence in plasma is short-term and transient. The retinalpigment epithelium (RPE) forms the outer blood retina barrier (BRB)and its integrity is essential for normal function of the retina. Retinalpathologies involving BRB disruption, such as diabetic retinopathy,inflammation, vascular occlusions and tumors may be accompaniedby elevation in thrombin levels.In the present work, we aimed to explore the impact of thrombin onthe integrity and function of the RPE blood barrier. We induced invitroa short-term exposure of RPE to pathological levels of thrombinand studied the immediate and long lasting changes in the RPEpermeability and angiogenic balance.Methods: ARPE-19 cells were grown for a month to achieve definitepolarity properties. Following a short (10 minutes) exposure tothrombin, the cells were washed and covered with new medium untilmeasurements were performed. Permeability was evaluated based onspectrophotometric monitoring of the leakage of labeled dextranmolecules. The expression of pro- and anti- angiogenic genes wasevaluated using real time PCR. Protein levels were measured byELISA or by FlowCytomix. MMPs activity was examined byzymography.Results: Short-term exposure to thrombin induced a long lasting (forhours) increase in RPE permeability to 10, 40 and 70 KD dextran.Decrease in PEDF mRNA and increase in VEGF and HGF mRNAexpression and protein levels were detected even 24 hours after the10 minute exposure to thrombin. MMPs 2 and 9 activities were alsoincreased hours after the short-term exposure to thrombin.Conclusions: Short-term exposure to thrombin inducesproangiogenic signals in RPE and disruption of barrier properties.The data indicate that changes in permeability, gene expression,proteins levels and activity persisted for hours after short-termexposure to thrombin. Based on our findings, we suggest thataccumulation of short-term exposures to thrombin over yearscontributes to the pathological processes involved in CNVdevelopment in the elderly.Commercial Relationships: Tami Livnat, None; Omer Bialer,None; Yael Nisgav, None; Mor Dachbash, None; Rima Dardik,None; Dov Weinberger, NoneProgram Number: 3656Presentation Time: 4:00 PM - 4:15 PMDICER1 is regulated via cAMP in an EPAC/Rap1-dependentmanner in human retinal pigment epithelial cells - implicationsfor age-related macular degenerationThomas P. Sauer, Sabine Kurth, Frank G. Holz, Tim U. Krohne.Ophthalmology, University of Bonn, Bonn, Germany.Purpose: DICER1 deficiency has recently been implicated in thepathogenesis of geographic atrophy (GA) due to age-related maculardegeneration (AMD). However, little is known about DICER1 generegulation in RPE cells. Cyclic adenosine monophosphate (cAMP)regulates DICER1 in human melanocytes. Therefore, we investigatedthe effects of cAMP on DICER1 gene expression in human RPE cellsMethods: The human RPE cell line ARPE-19 was treated withadenylate cyclase agonist forskolin, protein kinase A (PKA) inhibitorH89, Rap1 inhibitor GGTI-298, and Epac agonist 8-pCPT-2-0-MecAMP,both as single and combination treatments. Subsequently,cells were lysed and total mRNA was isolated. To assess DICER1gene expression, real-time quantitative PCR was carried out usingDICER1- specific primers: forward 5-CCCGGCTGAGAGAACTTACG-, reverse 5-CTGTAACTTCGACCAACACCTTTAAA-3Results: In forskolin-treated cells, increased cAMP levels resulted indownregulation of DICER1 (0.04 ± 0.036 relative gene expression).Inhibition of PKA did not prevent cAMP-induced downregulation(0.47 ± 0.41) whereas inhibition of Rap1 did (5.67 ± 5.23). Evenwithout forskolin treatment, activation of Epac downregulatedDICER1 (0.21 ± 0.21) whereas inhibition of Rap1 resulted in anupregulation (4.37 ± 3.52)Conclusions: cAMP-mediated downregulation of DICER1 in humanRPE cells is not solely explained by PKA action but involves theEpac/Rap1 pathway. As DICER1 has been demonstrated to preventAlu RNA-mediated damage in RPE, Rap1 represents a noveltherapeutic target for advanced dry AMD©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyCommercial Relationships: Thomas P. Sauer, None; SabineKurth, None; Frank G. Holz, Acucela (C), Allergan (C), Genentech(F), Heidelberg Engineering (F), Zeiss (F), Novartis (F), Novartis(C), Optos (F), Merz (C), Bayer (F), Bayer (C), BoehringerIngelheim (C); Tim U. Krohne, Novartis Pharma GmbH,Nuremberg, Germany (F), Novartis Pharma GmbH, Nuremberg,Germany (C), Novartis Pharma GmbH, Nuremberg, Germany (R)Program Number: 3657Presentation Time: 4:15 PM - 4:30 PMUsing extremes to identify rare pathogenic variants in agerelated-maculardegenerationCodrut C. Paun 1, 2 , Dzenita Smailhodzic 1 , Camiel J. Boon 1 , Lies H.Hoefsloot 2 , Mohamed R. Daha 3 , Carel B. Hoyng 1 , Anneke I. DenHollander 1, 2 . 1 Ophthalmology, Radboud Univ Nijmegen Med Ctr,Nijmegen, Netherlands; 2 Human Genetics, Radboud UniversityNijmegen Medical Centre, Nijmegen, Netherlands; 3 Nephrology,Leiden University Medical Center, Leiden, Netherlands.Purpose: Age-related macular degeneration (AMD) has a stronggenetic component, and it has been estimated that 50% of theheritability of AMD is explained by common variants in thecomplement system. Despite the progress that has been made inidentifying common variants in AMD, few studies have assessed therole of rare variants in the pathogenesis of AMD. The purpose of thisstudy was to identify rare variants in AMD patients with high levelsof complement activation.Methods: Complement component C3 and the activation fragmentC3d were measured in serum samples of 197 AMD patients and 150unaffected age-matched controls. The C3d/C3 ratio was calculated asan indicator of C3 activation. DNA samples of patients with highC3d/C3 ratios (>3), were screened for mutations in the CFH gene bySanger sequencing. DNA samples of 8 patients with the highestC3d/C3 ratios (>4) were analyzed by exome sequencing.Results: AMD patients displayed an increased median C3d/C3 ratiocompared to controls (p3 were observed in37 AMD patients, but only in 4 control individuals. Sequenceanalysis of the CFH gene revealed a heterozygous frameshiftmutation (c.1769_1779del; p.Gly590fsX) in a patient with a C3d/C3ratio of 4.4. Exome sequencing in 8 patients with C3d/C3 ratios >4revealed rare variants in 5 patients in key components of thecomplement pathway.Conclusions: High levels of complement activation in AMD patientscan be caused by rare variants in components of the complementpathway, which have previously not been associated with AMD.High complement activation may therefore direct a genetic searchtoward complement gene abnormalities. This genotype-serotypecorrelation approach is able to identify new risk factors in AMD.Commercial Relationships: Codrut C. Paun, None; DzenitaSmailhodzic, None; Camiel J. Boon, None; Lies H. Hoefsloot,None; Mohamed R. Daha, None; Carel B. Hoyng, None; Anneke I.Den Hollander, NoneSupport: Foundation Fighting Blindness USA center grant C-GE-0811-0548-RAD04374 Lipids, Retinoids and Macular PigmentsTuesday, May 07, 2013 2:45 PM-4:30 PMExhibit Hall Poster SessionProgram #/Board # Range: 3756-3783/A0095-A0122Organizing Section: Biochemistry/Molecular BiologyProgram Number: 3756 Poster Board Number: A0095Presentation Time: 2:45 PM - 4:30 PMAnalysis of Retinal Gangliosides by Hydrophilic InteractionLiquid Chromatography - Electrospray Ionization Tandem MassSpectrometryOlivier Berdeaux 1, 2 , Elodie A. Masson 2 , Stéphanie Cabaret 1, 2 , AnneAthias 3 , Jean-Paul Pais De Barros 3 , Lionel Bretillon 2 . 1 ChemoSensPlatform, INRA, Dijon, France; 2 Eye & Nutrition Research Group,INRA, Dijon, France; 3 Lipidomic Analytical Platform, University ofBurgundy, Dijon, France.Purpose: Gangliosides (GG) are sialic acid-containingglycosphingolipids. A wide variety of GG have been described basedon differences in the oligosaccharidic chain, fatty acids and longchain sphingoid bases of the ceramide moiety. The GG profile isspecific to the organ, its differentiation and pathophysiological status.An efficient method of separation, identification and quantification istherefore crucial ifthe huge heterogeneity among these compounds isto be understood and changes highlighted. We report here a powerfulanalytical method based on hydrophilic interaction liquidchromatography-electrospray ionization tandem mass spectrometry(HILIC-ESI-MS/MS), further tested on rat retina. Indeed, GG areparticularly abundant in the central nervous system, including theretina. While their involvement in the development of the retina andtheir neuroprotective action have been demonstrated, their preciserole in the retina and its pathologies is still poorly understood.Methods: GG classes were separated under HILIC conditions. Usinga triple quadrupole MS instrument equipped with an ESI source,GM3 and GM2 molecular species were structurally characterized bycollision-induced dissociation (CID) of [M-H] - and GD3, GD1a,GD1b, GT1b and GQ1b by CID of [M-XH]X- in negative modes.The semi-quantitative analysis of the GG molecular species wasperformed in the negative mode by single reaction monitoring(SRM). An N-acetylneuraminic acid fragment at m/z 290 wasproduced in the collision cell from the GG molecular species andused in SRM for quantification.Results: This method enables the major and minor GG species to beeffectively separated in a highly heterogeneous GG mixture.Furthermore, it provides an accurate characterization of the widelyvariable ceramide moiety, whose role is not well understood. In ratretina, the main long chain bases are d18:1 and d20:1. Fatty acids aresaturated or monounsaturated, mainly 16:0, 18:0, 20:0 and 22:0. Thedistribution of the different ceramide species varies among the GGclasses, even though d18:1/18:0 and d18:1/20:0 (or d20:1/18:0)consistently appear to be the major ones.Conclusions: Applied to retinal samples, this glycolipidomicapproach offers an efficient, sensitive, straightforward, highlyaccurate and reliable tool to investigate the role of GG in the functionand pathologies of the retina.Commercial Relationships: Olivier Berdeaux, None; Elodie A.Masson, None; Stéphanie Cabaret, None; Anne Athias, None;Jean-Paul Pais De Barros, None; Lionel Bretillon, NoneProgram Number: 3757 Poster Board Number: A0096Presentation Time: 2:45 PM - 4:30 PMCirculating markers of retinal and optic nerve lipidsNiyazi Acar 1 , Olivier Berdeaux 1 , Zhiguo He 2 , Stéphanie Cabaret 1 ,Philippe Gain 2 , Gilles Thuret 2 , Catherine P. Garcher 3, 1 , Alain M.Bron 3, 1 , Lionel Bretillon 1 . 1 INRA, University of Burgundy, Eye &Nutrition Research Group, Dijon, France; 2 EA2521, University JeanMonnet, Laboratory "Biology, Imaging, and Engineering of CornealGrafts", Saint-Etienne, France; 3 University Hospital, Department ofOphthalmology, Dijon, France.Purpose: Blood lipids are frequently used as a surrogate of lipidcomposition of peripheral tissues. Even if it is well accepted, such arelationship has never been clearly demonstrated for the eye. The aim©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyof this study was to determine in human samples whether a lipidomicapproach based on red blood cells could reveal associations betweencirculating and ocular lipid profiles.Methods: Red blood cells, retinas and optic nerves were collectedfrom 9 human donors. The lipidomic analyses on these tissues weredone by both, gas chromatography and liquid chromatographycoupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS/MS).Results: Gas chromatographic approach did not show any relevantassociation between circulating and ocular lipids except forarachidonic acid whose circulating amounts were positivelyassociated with its retinal and optic nerve levels. However,significant associations emerged from LC-ESI-MS/MS analyses.Indeed, phospholipid species in red blood cells were positively ornegatively associated with representative pools of retinal DHA(docosahexaenoic acid), retinal VLC-PUFA (very-long chainpolyunsaturated fatty acids) or optic nerve plasmalogens.Conclusions: First, our results show that LC-ESI-MS/MSmethodology is more appropriate than gas chromatography forlipidomics on red blood cells, and further extrapolation to ocularlipids. Second, this study has identified several individual lipidspecies that can be good candidates to represent circulatingbiomarkers of ocular lipids.Commercial Relationships: Niyazi Acar, None; Olivier Berdeaux,None; Zhiguo He, None; Stéphanie Cabaret, None; Philippe Gain,None; Gilles Thuret, None; Catherine P. Garcher, Alcon (C),Allergan (C), Baush and Lomb (C), Bayer Pharma (C), Novartis (C),Laboratoire Théa (C); Alain M. Bron, Allergan (C), Bausch Lomb(C), Horus (F), Théa (C); Lionel Bretillon, NoneProgram Number: 3758 Poster Board Number: A0097Presentation Time: 2:45 PM - 4:30 PMInflammatory cytokines decrease cell viability and alterganglioside profile in retinal pigment epithelium cells (ARPE19)Elodie A. Masson 1 , Olivier Berdeaux 1, 2 , Stéphanie Cabaret 1, 2 , LionelBretillon 1 . 1 Eye and Nutrition Research Group, UMR CSGA-1324INRA-6265 CNRS-University of Burgundy, Dijon, France;2 ChemoSens Platform, UMR CSGA-1324 INRA-6265 CNRS-University of Burgundy, Dijon, France.Purpose: Early stages of Age related Macular Degeneration (AMD)are characterized by dysfunction and degeneration of the retinalpigment epithelium (RPE) cells. These alterations participate in thedeath of the overlying photoreceptors ultimately leading to loss ofvision. It is therefore crucial to understand this initial event in orderto prevent the pathology development. Gangliosides (GG) make awide family of sialic acid-containing glycosphingolipids. Both theoligosaccharidic chain and the ceramide moiety (sphingoïd base andfatty acid) exhibit a huge heterogeneity in their structure. Thebiological role of this heterogeneity is largely unknown. GG areparticularly abundant in the central nervous system, including theretina. While their developmental and neuroprotective actions havebeen demonstrated, their precise role in retina’s function and itspathologies is still poorly understood.In the present study, we aimed to investigate the role of GG in theresponse of RPE cells to inflammation, which is known as one of thepathophysiological features of AMD.Methods: Cultured human RPE cells (ARPE19) were exposed to aninflammatory cytokine mix (ICM): TNF-α, IL-1β and IFN-γ for 72hours. Cell viability was assessed by direct cell counting andmeasurement of total DNA content. GG were analyzed by ReversedPhase High Pressure Liquid Chromatography, coupled with tandemmass spectrometry (RPHPLC-ESI-MSMS).Results: ICM had deleterious effects on ARPE19 viability. Indeed,the cell number decreased by half between control and treatedconditions based both on cell counting and measurement of DNAcontent. GM3 appeared to be the main GG class present in ARPE19cells. Interestingly, ICM exposure was associated with modificationsin the GM3 profile. Especially, the relative amounts of d16:1/18:0and/or d18:1/16:0 species increased whereas those of the d18:1/24:1and the d18:1/24:0 and/or d20:1/22:0 species decreased. Thesechanges reflected an increase in short chain fatty acids to thedetriment of long chain fatty acids.Conclusions: Our observations suggest that GG might be implicatedin ARPE19 cell response to inflammatory cytokines. Furtherinvestigations are required to understand the precise biological role ofthis change in fatty acid profile of GM3 in the deleterious effects ofinflammation on RPE cells.Commercial Relationships: Elodie A. Masson, None; OlivierBerdeaux, None; Stéphanie Cabaret, None; Lionel Bretillon, NoneProgram Number: 3759 Poster Board Number: A0098Presentation Time: 2:45 PM - 4:30 PMN-Terminal Region of Fatty Acid Transport Protein 4 (FATP4) isImportant for Interacting with and Inhibiting RPE65 IsomeraseSonghua Li 1 , John F. Green 2 , Jean T. Jacob 2 , Minghao Jin 1, 2 .1 Neuroscience, LSU Health Sciences Center, New Orleans, LA;2 Ophthalmology, LSU Health Sciences Center, New Orleans, LA.Purpose: In a previous study, we identified FATP4 as negativeregulator of RPE65 isomerase, which catalyzes isomerization of alltransretinyl fatty acid esters to 11-cis retinol (11cROL) for the visualcycle in the retinal pigment epithelium (RPE). The purpose of thisstudy is to identify the region(s) of FATP4 that interacts with RPE65and inhibits synthesis of 11-cis retinol.Methods: A mammalian expression vector encoding the bovineFATP4 (pRK-FATP4) was used to construct various mutant FATP4swith a Flag tag. A series of N-terminal or C-terminal deletionmutations and the Ichthyosis Prematurity Syndrome (IPS)-associatedmissense or non-sense mutations (A92T, S247P, Q300R, and C168X)were individually introduced into pRK-FATP4. These mutantFATP4s and RPE65 were expressed in 293T cells stably expressingLRAT and/or CRALBP (293T-LC or 293T-C cells). Interaction ofwild-type and mutant FATP4s with RPE65 in the cells wasdetermined by immunoprecipitation. The inhibitory effects of themutant FATPs on RPE65 activity were compared by measuringsynthesis of 11cROL from all-trans retinyl palmitate or all-transretinol added into the cell homogenates or medium of the culturecells.Results: Synthesis of 11-cis retinol in the cells transfected with pRK-RPE65 and pRK-FATP4 (wild-type FATP4) was 60% lower thanthat of control cells transfected with pRK-RPE65 and pRK5 mockvector. Under the same experimental conditions, C-terminal regionsof FATP4 had no inhibitory effect on the synthesis of 11-cis retinol.These regions were not able to co-precipitate RPE65 inimmunoprecipitation. In contrast, N-terminal region of FATP4significantly inhibited synthesis of 11cROL. The inhibitory effect ofthe N-terminal region was appromaximitely 70% of wild-typeFATP4. The N-terminal region of FATP4 was able to co-precipitateRPE65 in immunoprecipitation. The IPS-associated FATP4s, whichlost very long-chain fatty acid-CoA synthetase activity, also inhibitedsynthesis of 11cROL. However, their inhibitory efficiencies werereduced 20-30% compared to the inhibitory efficiency of wild-typeFATP4.Conclusions: Our results indicate that the very long-chain fatty acid-CoA synthetase activity of FATP4 partially contributes to itsinhibitory effect on synthesis of 11cROL and the N-terminal region©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyof FATP4 is important for interacting with RPE65 and for inhibitingsynthesis of 11cROL.Commercial Relationships: Songhua Li, None; John F. Green,None; Jean T. Jacob, 4,865,601 (P), 5,282,851 (P); Minghao Jin,NoneSupport: EY021208Program Number: 3760 Poster Board Number: A0099Presentation Time: 2:45 PM - 4:30 PMCloning and Characterization of a Carboxylesterase from BovineRetinal Pigment EpitheliumGennadiy P. Moiseyev 1 , Bill X. Wu 2 , Yusuke Takahashi 5 , Ying Chen 1 ,Andrew T. Tsin 4 , Rosalie K. Crouch 3 , Jian-Xing Ma 1 . 1 Physiology,Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK; 2 Biochemistry& Molecular Biol, Medical Univ of South Carolina, Charleston, SC;3 Ophthalmology, Medical Univ of South Carolina, Charleston, SC;4 Biology, University of Texas San Antonio, San Antonio, TX;5 Medicine-Endocrinology, Univ of Oklahoma Hlth Sci Ctr,Oklahoma City, OK.Purpose: Retinyl esters are stored in lipid droplets in retinal pigmentepithelium (RPE) and can serve as a substrate for RPE65isomerohydrolase for generation of 11-cis retinol, the precursorrhodopsin chromophore. At present, RPE65 is the only known retinylester hydrolase in the eye. Some carboxylesterases of the liver andother tissues have been shown to hydrolyze retinyl esters.Carboxylesterases may also participate in xenobiotic, drug and lipidmetabolism. However, little is known about carboxylesterasesexpression and function in the eye. The aim of this study was toexamine whether RPE carboxylesterase has retinyl ester hydrolyzingactivity or demonstrates other substrate preference.Methods: Carboxylesterase (CES1) has been cloned from bovineRPE cDNA library by PCR methods. A 293A cell line stablyexpressing CES1 with His-tag using pcDNA6 vector was generatedand used to express CES1. CES1 was purified from the culturemedium using N-NTA resin. CES1 activity was determinedspectrophotometrically in the reaction with various esters. Retinylester hydrolyzing activity was assayed by HPLC.Results: The cloned bovine CES1 cDNA sequence contains 1695 bp(accession number AY369075 in GenBank). The deduced protein hasa calculated molecular weight 61,780 Da and isoelectric point 6.79.CES1 mRNA was detected as a single transcript in the bovine RPE,kidney, liver and lung. No signal was detected in other bovine tissuesanalyzed, including the retina, pancreas, skeletal muscles, spleen,heart and brain. Immunohistochemistry showed the specificlocalization of CES1 in Bruch’s membrane. CES1 was purified tohomogeneity from culture medium of 293A cell line that stablyexpressed this protein. CES1 cleaves various esters producing alcoholand acid. Kinetic parameters kcat and Km were determined forparanitrophenyl acetate, vinyl acetate, vinyl butyrate, vinyl lauratewhich were found to be substrates for CES1. Lipid esters triolein andretinyl palmitate were not hydrolyzed by CES1.Conclusions: Bovine CES1 showed significant activity towardsesters with a small alcohol group. However, CES1 did not cleaveretinyl palmitate suggesting that it is not a retinyl ester hydrolase. Wehypothesize that CES1 may have a physiological role as a detoxifyingenzyme serving as a protector of RPE from various xenobiotic esters.Commercial Relationships: Gennadiy P. Moiseyev, CharlessonLLC (E); Bill X. Wu, None; Yusuke Takahashi, None; Ying Chen,None; Andrew T. Tsin, None; Rosalie K. Crouch, None; Jian-XingMa, NoneSupport: NIH grants EY018659, EY012231, EY019309 andP20GM104934Program Number: 3761 Poster Board Number: A0100Presentation Time: 2:45 PM - 4:30 PMThe rd12 allele of Rpe65 exerts a dominant-negative effect onvision because it may exhibit altered intracytoplasmic traffickingCharles B. Wright 1 , Micah A. Chrenek 1 , Stephanie L. Foster 1 ,Machelle T. Pardue 2 , Jeffrey H. Boatright 1 , John M. Nickerson 1 .1 Ophthalmology, Emory University, Atlanta, GA; 2 EmoryUniv/Ophthal, Atlanta VA Medical Center, Decatur, GA.Purpose: The rd12 allele of mouse Rpe65 has a nonsense mutation inexon 3 of the gene. The rd12 allele exerts a dominant-negative effecton vision that extends beyond the loss of visual function caused by alack of Rpe65 enzymatic activity. The purpose of this study is toattempt to define a possible mechanism-of-action for the rd12 allele.Methods: Optokinetic tracking (OKT) and electroretinography(ERG) were used to assess the visual acuity and retinal function ofC57BL/6J (+/+), Rpe65 KO (KO/KO), tvrm148 (tvrm148/tvrm148),rd12 (rd12/rd12), rd12/+, KO/+, tvrm148/+, KO/rd12, andKO/tvrm148 mice. Immunoblotting with an antibody specific to theN-terminus of Rpe65 was performed to detect protein, and qRT-PCRwas used to detect steady-state mRNA levels of genes of interest.RNAfold was used to predict the centroid structures of the Rpe65mRNAs from the +, tvrm148, and rd12 alleles. RNAimmunoprecipitation (RIP) was used to detect Rpe65 mRNA bindingto eIF4E.Results: Mice heterozygous for the rd12 allele lost 21% of theirvisual acuity by P210 while mice heterozygous for other mutationsdid not. Mice homozygous for the rd12 allele lost 100% visual acuityby P120, while mice homozygous for KO or tvrm148 alleles did notlose 100% visual acuity until P210. KO/rd12 mice had visualfunction loss that resembled rd12 homozygote mice. qRT-PCR for allRpe65 exon boundaries showed the rd12 allele has no splicing defectsin the mutant mRNA. No other coding mutations were found.Structural prediction of the mRNA predicted the rd12 allele mRNAdid not adopt a different secondary structure than wild type. qRT-PCR showed the same level of wild type and mutant mRNA in thecytoplasm, but RIPs showed the mutant mRNA was not bound byeIF4E, a translation initiation factor found in polysomes.Conclusions: Loss of visual function in mice homozygous andheterozygous for the rd12 allele shows the allele acts in a dominantnegativemanner to other Rpe65 mutant alleles, and that one copy ofthe rd12 allele is sufficient to drive this dominant-negative visualfunction loss. The rd12 allele does not produce a detectable protein,but does produce an mRNA that is appropriately transcribed, spliced,folded, and exported from the nucleus to the cytoplasm but does nottraffic to polysomes, suggesting the mutant mRNA may induce harmto the visual system because of altered intracytoplasmic trafficking.Commercial Relationships: Charles B. Wright, None; Micah A.Chrenek, None; Stephanie L. Foster, None; Machelle T. Pardue,None; Jeffrey H. Boatright, None; John M. Nickerson, NoneSupport: NIH GrantP30EY06360,R01EY016470,R24EY017045,R01EY014026,T32EY007092,RPBProgram Number: 3762 Poster Board Number: A0101Presentation Time: 2:45 PM - 4:30 PMMechanism for a Dominant-Acting D477G Mutation in RPE65Leading to Vision ImpairmentOlga Nikolaeva 1 , Gennadiy P. Moiseyev 1 , Yusuke Takahashi 2 , Jian-Xing Ma 1 . 1 Department of Physiology, OUHSC, Oklahoma City, OK;2 Endocrinology, OUHSC, Oklahoma City, OK.Purpose: RPE65 is a key enzyme in the visual cycle. Multiplerecessive point mutations in the RPE65 gene have been shown to leadto impaired vision in patients with retinitis pigmentosa (RP) and©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyLeber congenital amaurosis (LCA). Recently, a dominant mutationD477G of RPE65 has been reported to cause RP through themechanism yet to be identified. The purpose of this study is to revealhow the mutation D477G of RPE65 affects its enzymatic activity.Methods: Recombinant human wt RPE65 (hRPE65) with a His-tagand its mutant D477G with a 1D4 tag were generated by site-directedmutagenesis. Wt hRPE65 and the mutant were expressed in 293-LRAT cells using either adenovirus or plasmid transfection and theirexpression was analyzed by Western blot analysis. Subcellularfractionation was performed using FractPrepTM kit. Catalyticactivities of wt hRPE65 and the mutant were quantified by HPLC.Velocity sedimentation through a sucrose gradient was used toidentify oligomer formation between the mutant and wt RPE65.Results: Expression level of the D477G mutant was found similar tothat of wt hRPE65. The wt hRPE65 showed a high stability, with ahalf-life more than 10 h whereas the mutant has a decreased half-life,suggesting that the mutation decreases the protein stability. Themutant showed a diminished catalytic activity in comparison to thatof wt hRPE65. The subcellular fractionation revealed thatconsiderable amount of wt and D477G were located in membranefractions. However, when wt and D477G were co-expressed, itcaused a decrease of wt RPE65 in the membrane fraction while asimultaneous increase in its inclusion body fraction. Moreover, coexpressionof the D477G mutant with wt RPE65 enhances the RPE65aggregation and causes oligomerization of the wt and the mutantRPE65.Conclusions: The presence of the D477G mutant affects membraneassociation of wt RPE65. The D477G mutation enhances theformation of RPE65 oligomers rather than dimers or monomers. Inturn, the oligomerization affects the isomerase activity of wt RPE65and leads to impaired vision in patients with the D477G mutation ofRPE65.Commercial Relationships: Olga Nikolaeva, None; Gennadiy P.Moiseyev, Charlesson LLC (E); Yusuke Takahashi, None; Jian-Xing Ma, NoneSupport: NIH EY018659, EY012231, EY019309 andP20GM104934Program Number: 3763 Poster Board Number: A0102Presentation Time: 2:45 PM - 4:30 PMHuman iPS-RPE Synthesize and Release 11-cis Retinaldehydefrom Exogenous All-trans RetinolAlberto Muniz 1, 2 , Mark L. Plamper 2 , Jae Hyek Choi 1, 2 , Whitney A.Greene 1, 2 , Anthony J. Johnson 1 , Andrew T. Tsin 3 , Heuy-Ching H.Wang 1 . 1 Ocular Trauma, United States Army Institute of SurgicalResearch, Fort Sam Houston, TX; 2 National Research Council,Washington, DC; 3 Biology, The University of Texas at San Antonio,San Antonio, TX.Purpose: Retinal pigment epithelium (RPE) has recently beenderived from human induced pluripotent stem (iPS) cells. Beforenewly derived iPS-RPE cells can be used for stem cell therapies, it isimportant to determine the functional ability of iPS-RPE. One crucialrole of the RPE is uptake and processing of retinoids to supplyphotoreceptors with visual chromophore. However, the ability of iPS-RPE to support visual pigment regeneration has not beendemonstrated. The purpose of this study is to investigate the retinoidprocessing ability of the visual cycle in cultured iPS-RPE.Methods: RPE derived from human iPS cells were analyzed by RT-PCR and western blot analyses to detect expression of RPE visualcycle genes and proteins LRAT, RPE65, CRALBP and the RPEspecific gene PEDF. To examine the retinoid processing ability of theiPS-RPE, all-trans retinol (all-trans ROL) was delivered to culturediPS-RPE. After a 24 hour incubation period, retinoids were extractedfrom cell homogenates and the culture media. The extracts wereanalyzed by HPLC on a 0.2% to 10% dioxane/hexane gradientsystem. Retinoids were identified by retention time and respectiveabsorbance spectrum.Results: Expression of the critical visual cycle components LRAT,RPE65, CRALBP and the RPE specific gene PEDF was confirmedby RT-PCR and western blot in iPS-RPE. In addition, followingincubation with all-trans ROL, all-trans retinyl palmitate wasdetected from cell homogenates of iPS-RPE by HPLC . Furthermore,HPLC analysis of the culture media revealed a peak corresponding tothe retention time and absorbance spectrum of 11-cis retinaldehyde(11-cis RAL). Retinoids were not detected in control samples.Conclusions: iPS-RPE maintains expression of visual cycle proteins.The detection of all-trans retinyl palmitate in iPS-RPE and 11-cisRAL in the culture media demonstrates that the iPS-RPE can uptake,process, and secrete retinoids, confirming that the visual cycleproteins expressed in iPS-RPE are functional and can support visualpigment regeneration. This is the first study to demonstrateexpression of visual cycle machinery and complete visual cycleactivity in stem cell-derived RPE.Commercial Relationships: Alberto Muniz, None; Mark L.Plamper, None; Jae Hyek Choi, None; Whitney A. Greene, None;Anthony J. Johnson, None; Andrew T. Tsin, None; Heuy-ChingH. Wang, NoneSupport: US Army Medical Research Command Clinical andRehabilitative Medicine Research Program.Program Number: 3764 Poster Board Number: A0103Presentation Time: 2:45 PM - 4:30 PMFunctional Characterization of Peropsin in the Retinal PigmentEpitheliumJeremy D. Cook, Roxana A. Radu, Hui Sun, Gabriel H. Travis.Ophthalmology, Jules Stein Eye Institute, UCLA School ofMedicine, Los Angeles, CA.Purpose: Peropsin (RRH) is a non-visual opsin of unknown functionlocated in apical processes of the retinal pigment epithelium (RPE).The current study is an attempt to determine the function of peropsin.We began with the hypothesis that peropsin is involved in theprocessing of visual retinoids. To this end, we compared levels ofvisual retinoids in wild-type and peropsin-knockout (rrh-/-) miceunder dark-adapted conditions, and at different times following adeep photobleach. We are employing a cell-culture system to studythe role of peropsin on retinoid processing by enzymes of the visualcycle in RPE cells.Methods: Mice homozygous for a null mutation in the rrh gene weregenerated using conventional gene-targeting methods. Both the rrh-/-and strain-129 control mice (WT) were homozygous for the L450(normal) allele of rpe65. Mice were dark-adapted overnight. A subsetof WT and rrh-/- mice were euthanized and their eyes collected,while the remainder were exposed to fluorescent light at 1,000 lux for5 minutes. Subsets of these mice were euthanized immediatelyfollowing light exposure, or returned to darkness for different timeperiods before euthanasia. Retinas and RPE-containing eyecupsminus retinas were dissected, homogenized, and extracted withhexane for HPLC analysis of retinoids. To assay peropsin expressionand function in cultured HEK-293T cells, a construct containing thegene for mouse peropsin was subcloned into pcDNA and used fortransient transfection. Expression was confirmed by immunoblotanalysis and confocal microscopy, retinoid uptake was examined byHPLC.Results: Levels of all-trans-retinyl esters were several-fold lower inRPE from rrh-/- versus WT mice under all light conditions. Fiveminutes following the photobleach, levels of 11-cis-retinaldehyde and©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biology11-cis-retinyl esters were higher in the RPE of rrh-/- versus WTmice. Levels of LRAT, Rpe65, RGR-opsin and LRAT were similarin RPE from the two strains. Peropsin immunoreactivity wasobserved in the plasma membranes of 293T cells following transienttransfection with the peropsin expression plasmid.Conclusions: The altered retinoid dynamics in rrh-/- mice suggestdelayed removal of 11-cis-retinaldehyde from RPE and delayeduptake of all-trans-retinol by RPE from bleached photoreceptors.Peropsin-transfected HEK-293T cells represent a suitable system tostudy the role of peropsin on the uptake and processing of visualretinoids.Commercial Relationships: Jeremy D. Cook, None; Roxana A.Radu, None; Hui Sun, None; Gabriel H. Travis, NoneSupport: 5R01 EY011713Program Number: 3765 Poster Board Number: A0104Presentation Time: 2:45 PM - 4:30 PMInterphotoreceptor Retinoid Binding Protein Protects andDelivers Retinoids in the Cone Visual CycleAndrew T. Tsin 1 , Joshua M. Mimun 1 , Federico Gonzalez-Fernandez 2 .1 Biology, University of Texas San Antonio, San Antonio, TX;2 Veterans Affairs and SUNY Eye Institute, State University of NewYork at Buffalo, Buffalo, NY.Purpose: In the cone cycle, the recycling of all-trans retinol (atROL)and 11-cis-retinol (11cROL), between Müller cells and conephotoreceptor cells occurs within the retina. Interphotoreceptorretinoid binding protein (IRBP), the most abundant soluble protein inthe interphotoreceptor matrix, can bind to these photosensitiveretinoids, suggesting a role for IRBP as a transfer protein in the conevisual cycle. Thus, we hypothesized that IRBP is able to protectatROL and 11cROL from light induced degradation anddeliver/retrieve the retinoids to/from Müller cells.Methods: Protection studies: atROL and 11cROL (3.0 µM each)were exposed to 2,000 Lux, in the presence and absence of 3.0 µMIRBP. Retinoids were extracted every 10 min for 30 min andanalyzed by HPLC. Delivery/release studies: Primary chicken Müllercells (CMCs) were incubated for 24 hrs in serum free media (SFM)with 2.0 µM atROL and 2.0 µM IRBP or 2.0 µM BSA. To test ifthese proteins facilitated the release of retinoids from CMCs after aninitial 24 hr incubation with atROL and BSA (2.0 µM each) in SFM,the CMCs were re-incubated with SFM, 2.0 µM IRBP in SFM, or 2.0µM BSA in SFM for an additional 24hrs. Cellular homogenate andmedia were extracted for retinoids and analyzed via HPLC.Results: 30 min of light exposure resulted in degradation of 11cROL(83%) and atROL (67%). IRBP reduced these levels to 21%(11cROL) and 18% (atROL). After incubation with atROL for 24 hrs,the CMCs contained 0.32 pm/mg cell protein(cp) of all-trans retinylpalmitate (atRP) when incubated without proteins, 6.3 pm/mg cp ofatRP when incubated with BSA, and 0.85 pm/mg cp of atRP whenincubated with IRBP. The addition of BSA or IRBP into culturemedia of CMCs pre-incubated with atROL resulted in a significantincrease of atROL when compared to SFM (BSA: 31.7 pm/mg cp;IRBP: 40.1 pm/mg cp; SFM: 0.56 pm/mg cp).Conclusions: IRBP protects retinoids in situ from light-induceddegradation. IRBP delivery and retrieval experiments suggest thatIRBP delivers retinoids to the CMCs as indicated by the increase ofatRP present in the cellular homogenate and retrieves retinoids fromthe CMCs as indicated by the increase of atROL present in theculture media. IRBP could act as a transport protein of the conevisual cycle by protecting and facilitating transport of retinols toMüller cells.Commercial Relationships: Andrew T. Tsin, None; Joshua M.Mimun, None; Federico Gonzalez-Fernandez, PathRD Inc. (S)Support: ), NIH SCORE Program Grant # GM08194; NationalCenter for Research Resources (5G12RR013646-12); NationalInstitutes of Minority Health and Health Disparities(G12MD007591); Merit Review Award I01BX007080; NIH RO1EY09412Program Number: 3766 Poster Board Number: A0105Presentation Time: 2:45 PM - 4:30 PMBiochemical Characterization of Retinoid Isomerases in the RPEand RetinaQuan Yuan, Joanna J. Kaylor, Shanta Sarfare, Tongzhou Xu, JacobMakshanoff, Pengfei Cheng, Gabriel H. Travis. Jules Stein EyeInstitute, UCLA, Los Angeles, CA.Purpose: Enzyme mediated conversion of all-trans-retinoids to 11-cis-retinoids plays a key role in the biochemical process thatregenerates visual pigment for photoreceptor cells. RPE65, a knownprotein in RPE cell, has been confirmed as the isomerase in retinalpigment epithelium cells. Recently we identified dihydroceramidedesaturase 1 (DES1) as the retinoid isomerase for regenerating coneopsins in daylight. However the biochemical mechanism of theenzyme still remains elusive. Here we characterize the biochemicalproperties of DES1 using a variety of biochemical and biophysicalmethods.Methods: GST fused DES1 and His-CRALBP protein were purifiedby chromatography. Pull-down assays were carried out with thepurified proteins. The isomerase activities of RPE65 and DES1 incorresponding animal tissues and cell expression in the presence of avariety of specific inhibitors have been examined.Results: The physiological and reciprocal pull-down assays confirmthe direct interaction between DES1 and CRALBP. DES1 isomeraseactivity is dependent on the cytochrome b5 electron transfer chain.Inhibition of DES1 isomerase activity by spin traps and probesdemonstrate a possible radical intermediate pathway. DES1 is aFe(III) dependent retinol isomerase.Conclusions: Similarity towards different inhibitors for chickenretina and HEK-DES1 suggests DES1 is the isomerase-2 responsiblefor the alternative visual cycle in retina. The reaction mechanism ofDES1 appears to involve a free-radical intermediate.Commercial Relationships: Quan Yuan, None; Joanna J. Kaylor,None; Shanta Sarfare, None; Tongzhou Xu, None; JacobMakshanoff, None; Pengfei Cheng, None; Gabriel H. Travis, NoneSupport: 5R01 EY011713Program Number: 3767 Poster Board Number: A0106Presentation Time: 2:45 PM - 4:30 PMIsomerase 2 Activity in Primary Müller Cells from conedominatedChicken Retinas is iron-dependentBrandi S. Betts-Obregon, Andrew S. Mendiola, Andrew T. Tsin.Biology, The University of Texas at San Antonio, San Antonio, TX.Purpose: Previous studies have shown that there are two parallelvisual cycles in the cone dominated chicken retina (Muniz et al2009). Takahashi et al 2011 showed that RPE65c is an irondependentIsomerase 2 in the retinal Müller cells of the conedominant zebrafish. Kaylor et al 2012 showed that DES1 is anIsomerase 2 in the Müller glial cells of the retina. This DES1 proteinhas ion-binding di-iron ligands. We hypothesize that Müller cellslocated in the chicken retina have an iron-dependent IsomeraseMethods: Primary Müller cells were harvested from chicken retinasand seeded into T75 flasks. Cells were grown until confluence(approx 10-14 days) and homogenates prepared. Homogenates werepre-incubated with or without 5mM of 2,2’-Bipyridine (a knownisomerase 2 inbhibitor) for 30 minutes at room temperature.Homogenates were then incubated with 20 µM all-trans retinol©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologydelivered in BSA along with 100 µM PalmCoA and 30 µM CRALBPfor one hour in a 37C shaking water bath. Retinoids were extractedand analyzed by HPLC in comparison to retention times andauthentic retinoid standards.Results: Bovine RPE homogenates (chelator control) yielded 80pmol of 11cisRE/mg of protein when incubated with all-trans retinol.In the presence of 2,2’-bipyridne, the amount of 11-cisRE’s droppedto 38 pmol/mg of protein. Primary Chicken Müller cell homogenatesincubated with all-trans retinol yielded 116 pmol of 11-cisRE/mg ofprotein. Primary Chicken Müller cell homogenates that wereincubated with the iron chelator, 2,2’-bipyridine, yielded no 11-cisRE’s. Levels of 11-cis retinol remained constant at 24 pmol/mg ofprotein in homogenates incubated both with and without the additionof 2,2’-bypridine iron chelator.Conclusions: Isomerase 2 activity in Müller cells of the conedominatedchicken retina is iron-dependent.Commercial Relationships: Brandi S. Betts-Obregon, None;Andrew S. Mendiola, None; Andrew T. Tsin, NoneSupport: NIH SCORE Program Grant # GM08194, National Centerfor Research Resources (5G12RR013646-12) and the NationalInstitutes of Minority Health and Health Disparities (G12MD007591)Program Number: 3768 Poster Board Number: A0107Presentation Time: 2:45 PM - 4:30 PMSerum and Macular Response to Lutein Supplementation: ASystematic Review and Meta-AnalysisEstelle S. Lowry 2 , Christopher Cardwell 1 , Jayne Woodside 1 , Ruth E.Hogg 2 . 1 Centre for Public Health, Queens University Belfast, Belfast,United Kingdom; 2 Centre for Vision and Vascular Science, QueensUniversity Belfast, Belfast, United Kingdom.Purpose: To systematically review the evidence currently availableon the effects of lutein supplementation on serum lutein and macularpigment and investigate the concept of retinal nonresponse.Methods: Three databases were searched systematically to identifyhuman studies in English language, published up to February 2012.Studies were selected using pre-specified criteria. Of 77 potentialarticles identified, 24 articles met the selection criteria. Meta-analysisand meta-regression techniques were used to quantitatively poolresults and investigate the association between study characteristicsand mean changes in serum lutein and macular pigment (MP).Results: Intervention studies using lutein only and combined luteinsupplements were included. Daily dosage ranged from 2.4mg to30mg lutein and duration of the supplementation period varied from35 to 365 days. The review included 19 open label studies, 12placebo-controlled and 10 randomised, placebo-controlled studies.All studies reported an increase in mean serum lutein and all but tworeported an increase in MP levels. There was strong heterogeneityamongst studies. Dosage was found to be a significant influence inthe increase of serum lutein levels in healthy volunteers. For eachincrease of 10mg of lutein, there was an increase of 0.02µmol-1 (95%CI 0.003, 0.04) P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologySheng 2 , Paul S. Bernstein 1 . 1 Moran Eye Center, University of UtahSchool of Medicine, Salt Lake City, UT; 2 Pediatrics, University ofUtah School of Medicine, Salt Lake City, UT; 3 Physics andAstronomy, University of Utah, Salt Lake City, UT.Purpose: Lutein and zeaxanthin are dietary carotenoids specificallyconcentrated in the human macula that may be important in infantmacular and visual development. Infants affected by intrauterinegrowth restriction (IUGR) may have low carotenoid levels. Weevaluated the correlation of carotenoid levels between mothers andtheir infants at birth.Methods: We conducted a prospective, controlled, clinical studycomparing carotenoids in mothers, 30 term infants, and 10 IUGRinfants. Skin carotenoids were measured with resonance Raman skindevices. Blood and breast milk carotenoids were examined viaHPLC. Macular pigment optical density (MPOD) was assessed on asubgroup of 14 infants using RetCam imaging.Results: Serum and breast milk carotenoids were lower in the IUGRmothers than controls (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biology0.5 eccentricity increased significantly in Group 1 (0.103±0.076[30%]; P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologySupport: European Research Council ref: 281096.Clinical Trial: ISRCTN13894787Program Number: 3774 Poster Board Number: A0113Presentation Time: 2:45 PM - 4:30 PMLens status and macular pigment optical density measured by thetwo wavelength autofluorescence method: the Alienor StudyMarie B. Rougier 1 , Cecile Delcourt 2, 3 , Marie-Noelle Delyfer 1, 2 ,Melanie Le Goff 2, 3 , Florence Malet 1 , Jean Francois Dartigues 2, 3 ,Joseph Colin 1, 3 , Jean-Francois Korobelnik 1, 2 . 1 Service dOphtalmologie, CHU-Bordeaux Univ de Bordeaux, Bordeaux,France; 2 INSERM U897, Bordeaux, France; 3 Université Bordeaux2,Bordeaux, France.Purpose: To determine whether the lens status (lens opacities, lensextraction with PC IOL) is associated with differences in macularpigment optical density measured with the two wavelengthautofluorescence method.Methods: 963 residents of Bordeaux (France), aged 73 years ormore, were included in 2006-2008. About 18 months later, acomplementary eye examination was proposed to all subjectsdiagnosed with early ARM at baseline, and an equal number ofsubjects without early ARM. It included a measurement of macularpigment optical density (MPOD) using two wavelengthautofluorescence method (Modified HRA I, Heidelberg Engineering,Heidelberg, Germany). Lens opacities were graded at slit lampaccording to the LOCSII methodology. Nuclear cataract was definedas NO grade greater than 3 and/or NC grade greater than 3. Corticalcataract was defined as C grade greater or equal to 0.5. Posteriorsubcapsular cataract was defined as P grade greater or equal to 0.3.Among 395 included subjects, we excluded 66 subjects with late agerelatedmacular degeneration and 93 subjects with other maculardiseases (macular hole, retinal occlusion, myopic maculopathy,retinitis pigmentosa, laser scars, epiretinal membranes …) and 30subjects using dietary supplements containing lutein and zeaxanthin.Among the remaining 206 subjects, 181 subjects (321 eyes) hadcomplete data for lens opacities and macular pigment measurements.Associations of MPOD with lens status were estimated usingmultivariate mixed linear regression models, taking into account datafrom both eyes and their intra-individual correlations.Results: After adjustment for age and gender, MPOD within 0.5°was higher in eyes having undergone lens extraction ( +0.071 opticaldensity units, p=0.002) and lower in eyes with nuclear cataract (-0.19optical density units, p=0.0001), but was not different according tocortical or posterior subcapsular opacities. Similarly, MPOD with 1°was higher in eyes having undergone lens extraction (+0.069 opticaldensity units, p=0.001) and lower in eye with nuclear cataract (-0.15optical density units, p=0.001).Conclusions: Lens status appears to affect macular pigment opticaldensity measured using the two wavelength autofluorescence method.Commercial Relationships: Marie B. Rougier, THEA (C),Bausch&Lomb (C), Allergan (C), Kemin (C); Cecile Delcourt,Laboratoires Théa (F), Novartis (C), Bausch+Lomb (C); Marie-Noelle Delyfer, Thea Laboratories (F); Melanie Le Goff, None;Florence Malet, None; Jean Francois Dartigues, Novartis (F),IPSEN (F); Joseph Colin, Alcon (C), Abbott (C),AdditionTechnology (C); Jean-Francois Korobelnik, Alcon (C),Allergan (C), Bayer (C), Carl Zeiss Meditec (C), Novartis (C), Thea(F)Support: Financial support: Laboratoires THEAProgram Number: 3775 Poster Board Number: A0114Presentation Time: 2:45 PM - 4:30 PMThe relationship between visual performance and AMD severityin subjects with early age-related macular degenerationJohn M. Nolan 1 , Sarah Sabour-Pickett 1 , Jim Stack 1 , JamesLoughman 2 , Alan Howard 3 , Stephen Beatty 1 . 1 Macular PigmentResearch Group, Waterford Institute of Technology, Waterford,Ireland; 2 Optometry, Dublin Institute of Technology, Dublin, Ireland;3 Howard Foundation, Cambridge University, Cambridge, UnitedKingdom.Purpose: To investigate the relationship between measures of visualperformance (VP) (using a range of psychophysical parameters) andAMD severity in subjects with early age-related maculardegeneration (AMD) (ARERDS grade 2 to 8).Methods: Data was collected from 66 subjects with early AMD.Corrected distance visual acuity (CDVA) was measured using alogMAR chart. Contrast sensitivity (CS) and glare disability (GD)were measured using the sine wave grating-based Functional VisionAnalyser (FVA) . Retinotopic ocular sensitivity (ROS) wasmeasured by microperimetry. Stereo fundus images were graded forAMD status at the accredited reading centre at the University ofWisconsin.Results: There was a statistically significant inverse relationshipbetween measures of ROS and AMD-severity grade (p ≤ 0.01). Noother parameters of VP were significantly related to AMD severity (p> 0.05, for all).Conclusions: AMD status is related to ROS measurements, but is notrelated to other psychophysical measurements of VP, includingCDVA, in subjects with early AMD. This is an important finding, asCDVA is typically the only outcome measure used in the clinical andresearch setting for this condition.Commercial Relationships: John M. Nolan, None; Sarah Sabour-Pickett, None; Jim Stack, None; James Loughman, None; AlanHoward, None; Stephen Beatty, NoneSupport: Howard Foundation UKClinical Trial: ISRCTN81595685Program Number: 3776 Poster Board Number: A0115Presentation Time: 2:45 PM - 4:30 PMConcordance of macular pigment measurements usingheterochromatic flicker photometry, autofluorescence, andreflectometryJessica L. Dennison, Jim Stack, Stephen Beatty, John M. Nolan.Chemical & Life Sciences, Waterford Institute of Technology,Waterford, Ireland.Purpose: This study compares in vivo measurements of MP obtainedusing customized heterochromatic flicker photometry (cHFP)(Densitometer), dual-wavelength autofluorescence (HeidelbergSpectralis) and single-wavelength reflectance (Zeiss Visucam).Methods: MP was measured in one eye of 49 subjects using each ofthe three devices. Agreement between the Densitometer andSpectralis devices was investigated at various eccentricities (circa0.25, 0.50, 1.00, and 1.75 degrees eccentricity), using a variety ofquantitative and graphical methods (including Pearson correlation tomeasure degree of scatter (precision [prcn]), accuracy coefficient(acc) to measure shifts in location or scale, concordance correlationcoefficient (ccc) calculated from the acc and prcn coefficients, pairedt-test, and Bland-Altman plot). Because data at different eccentricitiesis not yielded from the Visucam, agreement between central MPusing the Visucam and each of the other devices was investigatedusing regression methods.Results: Agreement was strong between the Densitometer andSpectralis at all central eccentricities (e.g. at circa 0.25 degreeseccentricity: acc= 0.97, prcn= 0.90, ccc= 0.87). Regression analysisshowed a very weak relationship between the Densitometer and the©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyVisucam data (e.g. multiple regression of max MP from the Visucamon Densitometer MP at eccentricities 0.25 and 0.5 degrees had anassociated R 2 value of just 0.008 and was not statistically significant,p=0.843). Regression analysis also showed a weak relationshipbetween MP measurements from the Spectralis and Visucam devices(e.g. multiple regression of max MP from the Visucam on SpectralisMP at eccentricities 0.23 and 0.27 degrees had an associated R 2 valueof just 0.047 and was not statistically significant, p=0.348).Regression results between readings obtained on the Visucam andthose obtained on each of the other two instruments remained poorwhen orthogonal regression was used in place of ordinary leastsquares.Conclusions: MP values obtained using the Heidelberg Spectralis arecomparable to MP values obtained using the Densitometer. Incontrast, MP values obtained using the Zeiss Visucam are notcomparable with either the Densitometer or the Spectralis MPmeasuring devices. Taking cHFP as the current gold standard, theSpectralis is suitable for use in a clinical and research setting,whereas the Visucam is not.Commercial Relationships: Jessica L. Dennison, None; Jim Stack,None; Stephen Beatty, None; John M. Nolan, NoneSupport: European Research Council: CREST 281096Program Number: 3777 Poster Board Number: A0116Presentation Time: 2:45 PM - 4:30 PMA Comparison of the MacuScope and QuantifEye MacularPigment Densitometers in Two Distinct Population TypesRobert J. Donati, Elizabeth Wyles. Illinois College of Optometry,Chicago, IL.Purpose: Studies have suggested that reduced levels of macularpigment (MP) may increase risk for developing age-related maculardegeneration (AMD). There are two compact commercially availableheterochromic flicker photometry instruments that measure MP in theUSA. Previous studies revealed significant variability betweeninstruments. Our aim was to determine if the same variability wouldbe found in a young, healthy, population compared to an olderpopulation for which these instruments have more significance.Methods: Twenty young healthy adults (21-29 years old) and 28older adults (50-70 years old) with and without early signs of AMDwere recruited from the Illinois Eye Institute patient base. Macularpigment optical density (MPOD) was measured using the MacuScopeand QuantifEye. Data was collected for each patient in one session. Asingle, but different operator collected data for each of the patientpopulations. Two measurements per eye were taken on eachinstrument and each eye was used as a separate data point. If thedifference was greater than 0.04 absorbance units between twomeasurements on a single instrument, a third measurement was taken.Invalid readings were excluded. Paired t-tests and ANOVA weredone to compare the statistical significance of the results from bothinstruments and age groups. Bland-Altman plots were done for anadded comparison.Results: Mean MPOD for the combined age groups was 0.335 ±0.137 for the MacuScope (n=83) and 0.350 ± 0.186 for theQuantifEye (n=83). There was no significant difference between theMPOD means. The mean standard deviation of each subject’s MPODreadings was 0.0943 ± 0.0822 for the MacuScope (n=87) and 0.0508± 0.0496 for the QuantifEye (n=86). There is a significant differencebetween the two instruments (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyNicole K. Scripsema 1, 3 , Nishit Shah 2 , Patricia Garcia 1 , DavidWarrow 1 , Karen Tobias 2 , Gennady Landa 1, 3 , Richard B. Rosen 1, 3 .1 The Retina Center, The New York Eye and Ear Infirmary, NewYork, NY; 2 The Einhorn Clinical Research Center, The New YorkEye and Ear Infirmary, New York, NY; 3 Ophthalmology, New YorkMedical College, Valhalla, NY.Purpose: Previously, our group reported objective evidence of lowermacular pigment density (MP) levels in patients with Type IIDiabetes (DM) with early retinopathy, which inversely correlatedwith their hemoglobin A1c. The purpose of this study was todetermine if lutein (L) or zeaxanthin (Z) supplementation couldincrease levels of MP in a similar population.Methods: Patients with DM with early retinopathy were enrolled inthe clinical trial. Each was assigned to one of four supplementationformulations containing different doses of L or Z (ZeaVision,Chesterfield, MO). Patients were examined at baseline, 1 month, 3months, 6 months, and 1 year. At each visit, patients were imagedtwo objective techniques for measuring MP. Total MPOD at 1.0degree from the fovea was measured with a modified scanning laserophthalmoscope (HRA Heidelberg Retinal Angiograph, Heidelberg,Germany). Individual LOD and ZOD at 1.0 degee from the foveawere measured with the Macular Pigment Reflectometer (MPR,Maastricht, Netherlands). Paired t-tests were used to compare meanoptical densities. We are reporting preliminary 6 month data.Results: 24 eyes of 13 patients were included. Two eyes wereexcluded due to inadequate image analysis. 38% were male. Meanage was 61.0±8.2 yrs. 46% were Hispanic, 24% Asian, 15%Caucasian, 15% African American. All patients had Type II DM withearly retinopathy. Mean duration of DM was 21.3±11.2 yrs and meanHbA1c was 7.4±1.4 mg/dL. Changes in mean MPOD, ZOD, andLOD are shown in Table 1. Each parameter showed significantincreases from baseline after 1-3 months of supplementation.Conclusions: These results suggest that in Type II DM patients withearly retinopathy, supplementation of L or Z can increase MP levelsin the parafoveal area. This may prevent the onset of maculopathy bymaintaining or restoring the oxidant-antioxidant balance in conditionassociated with increased levels of oxidative stress in the retina.Commercial Relationships: Nicole K. Scripsema, None; NishitShah, None; Patricia Garcia, None; David Warrow, None; KarenTobias, None; Gennady Landa, None; Richard B. Rosen, Opko-OTI (C), Optos (C), Clarity (C), OD-OS (C), Topcon (R), Zeavision(F), Genetech (F), Optovue (C)Support: The Bendheim-Lowenstein Retina Fund of the New YorkEye and Ear InfirmaryClinical Trial: NYEE0936France; 4 Unité de Soutien Méthodologique à la Recherche (USMR),CHU de Bordeaux, Bordeaux, France; 5 Service d’Ophtalmologie,CHU de Dijon, Dijon, France.Purpose: The macular pigment may protect against age-relatedmacular degeneration (AMD). It is formed of lutein (L) andzeaxanthin (Z), two dietary carotenoids. Some studies have suggestedthat omega 3 fatty acids may help increasing macular pigmentdensity. We performed a randomized trial testing the efficacy of adietary supplement containing L, Z, omega 3 fatty acids andantioxidants for increasing macular pigment density in subjects athigh genetic risk for AMD.Methods: The Limpia Study is a double-blind, placebo controlled,prospective randomized clinical trial performed in 120 subjects withat least one parent affected by neovascular AMD. To be included,subjects had to be aged 40-70 years, have best-corrected visual acuity(BCVA) greater than 20/25, be free of late AMD and other major eyeconditions (severe glaucoma, high myopia, severe retinal disease,cataract surgery…). Included subjects were randomly assigned toreceive Nutrof® Total (2 gels/day, representing lutein 10 mg/day,zeaxanthin 2 mg/day, long chain omega 3 fatty acids 540 mg/day,vitamin C 180 mg/day, vitamin E 30 mg/day, zinc 15 mg/day, copper1 mg/day and resveratrol 1 mg/day ) or placebo for 6 months (Figure1). The primary outcome was the variation of macular pigmentoptical density (MPOD). Secondary outcomes included visualfunction, nutritional biomarkers and cognitive function. MPOD wasmeasured by two methods (modified Heidelberg Retinal Analyzer,Heidelberg, Germany and Visucam 200 MPD, Carl Zeiss Meditec,Germany) every 3 months during one year. This allows studying thevariation of macular pigment density during the 6 monthsupplementationphase, and during 6 months after the cessation of thesupplementation.Results: 120 subjects were included from December 2010 to January2012. Mean age was 56.7 years and 71.7 % were women. The motherof 97 subjects (80.8 %) was affected by AMD, while in only 25subjects (20.8 %) the father was affected. At baseline, mean MPODwithin 0.51°, measured with the modified HRA method, was 0.5(standard deviation 0.2). Maximal MPOD, measured with Visucam,was 0.4 (standard deviation 0.1).Conclusions: Middle-aged subjects with at least one parent affectedby AMD appear to have similar macular pigment density as observedin the general population.Commercial Relationships: Jean-Francois Korobelnik, Alcon (C),Allergan (C), Bayer (C), Carl Zeiss Meditec (C), Novartis (C), Thea(F); Marie-Noelle Delyfer, Thea Laboratories (F); Marie B.Rougier, THEA (C), Bausch&Lomb (C), Allergan (C), Kemin (C);Hélène Savel, None; Geneviève Chêne, None; Cecile Delcourt,Laboratoires Théa (F), Novartis (C), Bausch+Lomb (C); CatherineP. Garcher, Alcon (C), Allergan (C), Baush and Lomb (C), BayerPharma (C), Novartis (C), Laboratoire Théa (C)Support: Grants from Laboratoires Thea and Carl Zeiss MeditecClinical Trial: NCT01269697Program Number: 3780 Poster Board Number: A0119Presentation Time: 2:45 PM - 4:30 PMA randomized trial of supplementation with lutein, zeaxanthin,omega 3 fatty acids and antioxidants for increasing macularpigment optical density in high-risk subjects: the LIMPIA StudyJean-Francois Korobelnik 1, 2 , Marie-Noelle Delyfer 1, 2 , Marie B.Rougier 1 , Hélène Savel 4 , Geneviève Chêne 4 , Cecile Delcourt 2, 3 ,Catherine P. Garcher 5 . 1 Service d'Ophtalmologie, Hopital Pellegrin,Bordeaux, France; 2 Univ. Bordeaux, ISPED, Centre INSERM U897-Epidemiologie-Biostatistique, Bordeaux, France; 3 INSERM, ISPED,Centre INSERM U897-Epidemiologie-Biostatistique, Bordeaux,Program Number: 3781 Poster Board Number: A0120Presentation Time: 2:45 PM - 4:30 PMComparison of macular pigment optical density measured byautofluorescence and reflectometry: the LIMPIA StudyCatherine P. Garcher 1, 2 , Marie-Noelle Delyfer 3, 4 , Marie B. Rougier 3 ,Hélène Savel 6 , Geneviève Chêne 6 , Cecile Delcourt 5, 6 , Jean-FrancoisKorobelnik 3, 4 . 1 Ophthalmology, University Hospital, Dijon, France;2 Eye and Nutrition Research Group, UMR CSGA-1324 INRA-6265CNRS-Université de Bourgogne-AgroSup, Dijon, Dijon, France;3 Ophthalmology, CHU Bordeaux, Bordeaux, France; 4 Univ.Bordeaux, ISPED, Centre INSERM U897-Epidemiologie-©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyBiostatistique, Bordeaux, France; 5 INSERM, ISPED, CentreINSERM U897-Epidemiologie-Biostatistique, Bordeaux, France;6 Unité de soutien méthodologique à la recherche clinique etépidémiologie (USMR), Pôle de Santé Publique, CHU Bordeaux,Bordeaux, France.Purpose: To compare macular pigment optical density (MPOD)measured by two techniques (autofluorescence, reflectometry) inhealthy subjects at high risk for age-related macular degeneration(AMD).Methods: The Limpia Study is a double-blind, placebo controlled,prospective randomized clinical trial performed in 120 subjects withat least one parent affected by neovascular AMD. To be included,subjects had to be aged 40-70 years, have best-corrected visual acuity(BCVA) greater than 20/25, be free of late AMD and other major eyeconditions (severe glaucoma, high myopia, severe retinal disease,cataract surgery…). Subjects having used supplements containinglutein and/or zeaxanthin in the preceding year were not included.MPOD was measured using two methods: the two-wavelengthautofluorescence method with a modified scanning laserophthalmoscope (Heidelberg Retinal Analyzer (HRA), Heidelberg,Germany) and a method based on reflectometry (Visucam200 MPD,Carl ZeissMeditec, Germany).Results: At baseline, mean MPOD within 0.51°, measured with themodified HRA method, was 0.5 ± 0.2. Maximal MPOD, measuredwith Visucam, was 0.4 ± 0.1. Parameters from the modified HRA(optical density within 0.5°, 1°, 2° and 6°) and from the Visucammethod (volume, area, mean and maximum optical density) wereweakly correlated. The best correlations were observed for MPODwithin 2° and 6° with volume from Visucam (r=0.21 and r=0.22,respectively), and mean MPOD from Visucam (r=0.21 and r=0.18).Conclusions: The two methods propose different parameters toevaluate macular pigment, which overall correlated weakly in thispopulation of middle-aged healthy subjects at high risk for AMD.Further research is needed to characterize the differences between themethods and identify the best parameters and techniques to measuremacular pigment.Commercial Relationships: Catherine P. Garcher, Alcon (C),Allergan (C), Baush and Lomb (C), Bayer Pharma (C), Novartis (C),Laboratoire Théa (C); Marie-Noelle Delyfer, Thea Laboratories (F);Marie B. Rougier, THEA (C), Bausch&Lomb (C), Allergan (C),Kemin (C); Hélène Savel, None; Geneviève Chêne, None; CecileDelcourt, Laboratoires Théa (F), Novartis (C), Bausch+Lomb (C);Jean-Francois Korobelnik, Alcon (C), Allergan (C), Bayer (C), CarlZeiss Meditec (C), Novartis (C), Thea (F)Support: Théa, Carl Zeiss MeditecClinical Trial: NCT01269697Program Number: 3782 Poster Board Number: A0121Presentation Time: 2:45 PM - 4:30 PMSerial Assessment of Macular Pigment Distribution ProfilesObtained Using Minimum Motion Photometry: 10 to 15-YearFollow-UpJack D. Moreland 1 , Anthony G. Robson 2, 3 , Daniel Pauleikhoff 4 ,Frederik J. van Kuijk 5 . 1 Life Sciences, Keele University, Keele,United Kingdom; 2 Electrophysiology, Moorfields Eye Hospital,London, United Kingdom; 3 Institute of Ophthalmology, UniversityCollege London, London, United Kingdom; 4 St Franziskus Hospital,Muenster, Germany; 5 Life Sciences, Keele University, Keele, UnitedKingdom.Purpose: To monitor macular pigment (MP) distribution profilesover periods of more than a decade in order to assess the long-termstability of MP optical density values at different retinal locations.Methods: Multiple MP spatial distribution profiles were obtainedusing a motion photometer in 4 healthy subjects (A-D) monitoredover periods of 15.6, 14.7, 11.7 and 10.7 years respectively. A squarewave grating (460nm and 580nm) was moved at constant horizontalvelocity (26 deg/sec and/or 37 deg/sec) within 2 circular fields(radius 0.45° and 1.1°) and 11 annular segments (maximum radius7.5°). The radiance of the 580nm stimulus was adjusted to minimizethe perceived motion. Optical density (OD) was computed at eachlocation relative to the most eccentric area from log (Rref/R), whereRref is the mean radiance setting for the most eccentric locations andR is the radiance setting at any location. The shape of the meanspatial distribution profiles for each of the 4 subjects (average of 46,42, 12 and 10 profiles respectively) were characterised in detail.Results: Mean peak MPOD values obtained with a circular field(radius = 0.45°) were 0.89 (A), 0.76 (B), 0.45 (C) and 0.17 (D). Theshapes of the mean MP profiles varied in a way representative of anormal population and were best described by the sum of 2 Gaussianfunctions, accounting for at least 99% of the variance. Linearregression through serial data points (circular field; radius 1.1°) gavegradients of 0.0093 (A), 0.0012 (B), -0.0003 (C) and -0.0024 (D) peryear. Maximum gradients at 4 eccentricities at or between 0.82 and2.31 degrees were 0.0075 (A), -0.0070 (B), 0.0026 (C) and 0.0049(D) per year.Conclusions: Widely different MP distribution profiles in healthysubjects demonstrate a high degree of stability over periods of up to15 years. The findings provide an assessment of normal stability thatis pertinent to studies that aim to monitor MP in disease.Commercial Relationships: Jack D. Moreland, None; Anthony G.Robson, None; Daniel Pauleikhoff, None; Frederik J. van Kuijk,NoneSupport: Foundation Fighting Blindness and NIHR MoorfieldsBiomedical Research Centre (AGR). Research to Prevent Blindnessunrestricted grant to University of Minnesota (EVK)Program Number: 3783 Poster Board Number: A0122Presentation Time: 2:45 PM - 4:30 PMEffects of Zeaxanthin on Constitutive and TPA-InducedSecretion of VEGF by Human Retinal Pigment Epithelial CellsIn VitroDan-Ning Hu, Richard B. Rosen, Anthony Sclafani, Steven A.McCormick. Pathology & Ophthalmology, New York Eye & EarInfirmary, New York, NY.Purpose: TPA (12-0-tetradecanoyl phorbol 13-acetate) is a proteinkinase C activator, which can stimulate the secretion of VEGF byretinal pigment epithelial (RPE) cells. The purposes of this studywere to investigate the effects of zeaxanthin on the constitutive andTPA-induced secretion of VEGF by human RPE cells in vitro.Methods: Human RPE cells (ARPE19) were seeded into 12-wellplates and cultured with F12 medium with 10% serum. After 24hours, culture medium was replaced by F12 medium without serum.Zeaxanthine at various concentrations (1, 10 and 100 µM) was added.TPA (100 ng/ml) was added 1 hour later. After 24 hours, conditionedmedium was collected and the amount of VEGF was measured usinga sandwich enzyme-linked immunosorbent assay kit (R & DSystems). All tests were performed in triplicate. A primary culture ofRPE cells isolated from a donor eye was also tested.Results: VEGF could be detected in the conditioned medium fromcultured ARPE cells (70.7±5.5 pg/ml). TPA significantly stimulatedthe secretion of VEGF (149.3±12.1 pg/ml, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologylevels in conditioned medium from cells cultured with TPA andzeaxanthin (100 µM) was 93.3 ± 7.8 pg/ml, which was significantlylower than that in cells cultured with TPA alone (p < 0.01) but stillhigher than that in cells cultured without TPA (0.05 > p > 0.01).Study in primary culture of cell line obtained a similar result.Conclusions: Zeaxanthin only slightly inhibits constitutive secretionof VEGF by human RPE cells in vitro but significantly reduced TPAinducedsecretion of VEGF by RPE cells in a dose-dependentmanner.Commercial Relationships: Dan-Ning Hu, Zeovision (F); RichardB. Rosen, Opko-OTI (C), Optos (C), Clarity (C), OD-OS (C),Topcon (R), Zeavision (F), Genetech (F), Optovue (C); AnthonySclafani, None; Steven A. McCormick, NoneSupport: Supported by the Pathology Research Fund, New York Eyeand Ear Infirmary and the Research Fund from ZeaVision.401 Biochemistry of GlaucomaWednesday, May 08, 2013 8:30 AM-10:15 AM6A Paper SessionProgram #/Board # Range: 4002-4008Organizing Section: Biochemistry/Molecular BiologyContributing Section(s): Biochemistry/Molecular BiologyProgram Number: 4002Presentation Time: 8:30 AM - 8:45 AMMyocilin affects differentiation of oligodendrocytes and regulatesmyelination of the optic nerve via Lingo-1/RhoA signalingStanislav I. Tomarev 1 , Heung Sun Kwon 1 , Naoki Nakaya 1 , Mones S.Abu-Asab 2 . 1 SRGCB, LRCMB, National Eye Institute, Bethesda,MD; 2 Histopathology Core Facility, National Eye Institute, Bethesda,MD.Purpose: To investigate the functions of the glaucoma-associatedprotein, myocilin, in the optic nerve.Methods: Optic nerve astrocytes and oligodendrocytes were isolatedfrom postnatal day 6 (P6) or P7 mice. Optic nerves were examined byelectron microscopy. Binding of myocilin to the Lingo-1/NgR1complex was assessed by co-immunoprecipitation from the opticnerve and by binding assays using myocilin-alkaline phosphatasefusion proteins. Conductivity of the optic nerve was evaluated by avisual evoked potential test. Changes in gene expression weremeasured by RNA sequencing.Results: Myocilin was first detected in optic nerve astrocytes at P6and its level increased with age. Myocilin was also detected inastrocytes but not oligodendrocytes that were isolated and culturedfrom the optic nerve. Addition of purified myocilin to immatureoligodendrocytes stimulated their differentiation and increasedmRNAs encoding myelin-associated proteins (MBP, MAG andMOG) when compared with untreated oligodendrocytes. MBP levelsin the optic nerve of Myoc null mice were lower than in wild-typelittermates at early postnatal stages (P5-P21). Electron microscopyexamination of the optic nerve showed that myelin thickness wasreduced and the microvilli at the nodes of Ranvier were disorganizedin P13 Myoc null mice when compared with wild-type littermates.These defects in the myelination of the optic nerve were accompaniedby a reduction of RhoA activity. Myocilin regulates RhoA activitythrough interaction with the Lingo-1/NgR1 complex. Changes in themyelination of the optic nerve led to a delayed latency and reducedamplitude of the visually evoked potential in adult Myoc null micewhen compared to wild-type littermates. The levels of multiplemRNAs in the optic nerves of adult mice were different from wildtype,Myoc null and transgenic mice expressing mutated humanmyocilin (Tyr437His).Conclusions: Our data suggest that myocilin regulates myelination ofthe optic nerve via the Lingo-1/RhoA signaling pathway.Commercial Relationships: Stanislav I. Tomarev, None; HeungSun Kwon, None; Naoki Nakaya, None; Mones S. Abu-Asab,NoneProgram Number: 4003Presentation Time: 8:45 AM - 9:00 AMGenomewide DNA methylation analysis of human trabecularmeshwork cells with dexamethasone stimulationAkira Matsuda, Nobuyuki Ebihara, Akira Murakami. Department ofOphthalmology, Juntendo Univ School of Med, Tokyo, Japan.Purpose: To investigate the change of DNA methylation statusduring glucocorticoid exposure using cultured human trabecularmeshwork cells.Methods: Human trabecular meshwork (TM) cells established fromglaucoma patients (GTM) and normal control (NTM) were culturedwith and without 100nM dexamethasone (DEX) for 14 days. Genomewide methylation analysis was carried out using Illumina 450Kmethylation analysis. Gene expression analysis was also carried outwith Agilent 44K whole human genome array using cultured TMcells. The effect of DNA methylation in response to DEX treatmentsfor gene expression was verified by DNA methyltransferase inhibitor(5-aza-2’-deoxycytidine) treatment and subsequent realtime PCRanalysis.Results: We found 174/297 cytosine-phosphate-guanine (CpG) sitesshowing significant demethylation and 102/184 CpG sites showingsignificant methylation by 2 week-DEX treatment in GTM/NTM,respectively. We found 23 sets of demethylated-CpG sites after DEXexposure with significant changes of corresponding gene expression,and 15 sets methylated-CpG sites after DEX exposure withsignificant changes of corresponding gene expression. Among themwe further evaluated the expression of FKBP5, ZBTB16 and DUSP1.ZBTB16 gene-body methylation was observed after DEX treatment,accompanied with increased gene expression. 5-aza-2’-deoxycytidine-treatment of TM cells abolished the increase ofZBTB16 mRNA expression. DNA-demethylation of FKBP5promoter region was observed with increased FKBP5 geneexpression by DEX treatment. Promoter region of DUSP1 generegion is highly methylated in NTM cells. Significant DNAdemethylationand increase of DUSP1 gene expression was observedafter DEX treatment in NTM cells but not in GTM cells.Conclusions: DEX treatments induce the alteration of DNAmethylation status in human TM cells. Epigenetic modification couldaffect TM gene expression profile.Commercial Relationships: Akira Matsuda, None; NobuyukiEbihara, None; Akira Murakami, SEED(Japan) JP4855782 (P),SEED(Japan) JP5132958 (P)Support: Grants-in-Aid from MEXT Japan (No. 24592652).Program Number: 4004Presentation Time: 9:00 AM - 9:15 AMStabilization of Myocilin with Small Molecules Prevents ItsAggregationRaquel L. Lieberman, Shannon E. Hill, Rebecca K. Donegan,Katherine C. Turnage. School of Chemistry & Biochemistry, GeorgiaInstitute of Technology, Atlanta, GA.Purpose: The vast majority of mutations in the myocilin geneleading to glaucoma are located within the 30 kDa C-terminalolfactomedin (OLF) domain. Disease-causing myocilin variantsaggregate within human trabecular meshwork (TM) cells. Thisaccumulation taxes the cells and leads to TM cell death, resulting inincreased intraocular pressure, and a hastening of glaucoma-©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyassociated vision loss. Our purpose is to investigate the structural andbiophysical effects of stabilizing compounds on the myocilin-OLFdomain, and evaluate their ability to prevent myocilin-OLFaggregation.Methods: Expression and purification of the OLF domain ofmyocilin, development of high throughput assay to discover ligands,development of assay to monitor rate and extent of aggregation,surface plasmon resonance to evaluate binding mode, molecularbiophysical characterization of formed aggregates.Results: Disease-associated OLF variants access a partially unfoldedstate under physiological conditions that increases their propensity togrow amyloid fibrils. Compounds that thermally stabilize the OLFdomain, such as osmolytes and calcium ions, reduce aggregation in acompound-specific manner. Ligands discovered by high throughputscreening also affect aggregation.Conclusions: Different categories of OLF-stabilizing smallmolecules, general and tailored, may prevent the accumulation ofmyocilin in TM cells, and thereby provide a potential new therapy formyocilin glaucoma.Commercial Relationships: Raquel L. Lieberman, None;Shannon E. Hill, None; Rebecca K. Donegan, None; Katherine C.Turnage, NoneSupport: NIH Grant R01EY021205Program Number: 4005Presentation Time: 9:15 AM - 9:30 AMPGC-1alpha Signaling Regulates Retinal Ganglion Cell InjuryResponses And Atrocyte ReactivityJeremy M. Sivak 1, 2 , Xiaoxin Guo 1 , Shirin Dason 2 , Adrian Nahirny 1 .1 Vision Sciences, Toronto Western Hospital, University HealthNetwork, Toronto, ON, Canada; 2 Ophthalmology and VisionSciences, University of Toronto, Toronto, ON, Canada.Purpose: Glaucomatous optic neuropathy is associated with acomplex combination of risk factors, including age, increasedintraocular pressure, and vascular disregulation. These stressesconverge to damage the sensitive nerve fiber layer (NFL), and opticnerve head, resulting in the death of retinal ganglion cells (RGCs).However, a molecular mechanism has not yet been identified thatintegrates local tissue responses to these varied insults. We havegenerated data identifying the transcriptional co-activator, PGC-1α(peroxisome proliferator-activated receptor gamma co-activator 1alpha), as a potential key mediator of this process. When activated,PGC-1α interacts with transcription factors regulating metabolic,oxidative, and ischemic stress. Through these interactions PGC-1αacts as a master cellular regulator of adaptive energy metabolism.Methods: Expression of PGC-1α was analyzed in acute and chronicmouse retinal injury models by quantitative rt-PCR, and in-situhybridization. Putative PGC-1α targets, markers of metabolic andoxidative stress, glial reactivity, and RGC death were assessed by rt-PCR, immunofluorescence microscopy, and TUNEL assay, incombination with loss- and gain-of-function techniques.Results: PGC-1α expression was increased in RGCs and the NFLwith age, and in a model of chronic ocular hypertension, as well asfollowing acute excitotoxic retinal injury. PGC-1α loss-of-functionresulted in significantly increased retinal astrocyte reactivity, andsensitivity of RGCs to damage, while increased activity wasprotective against oxidative stress.Conclusions: We have identified an important pathway that canintegrate retinal damage responses common to a variety ofpathologically relevant insults.Commercial Relationships: Jeremy M. Sivak, None; Xiaoxin Guo,None; Shirin Dason, None; Adrian Nahirny, NoneSupport: CNIB, Glaucoma Research Society of Canada, CanadianInstitutes for Health Research (CIHR), TGH and TWH FoundationGlaucoma Res ChairProgram Number: 4006Presentation Time: 9:30 AM - 9:45 AMSubcellular mislocalization and loss of function of mutantGpnmb proteinsAlexander C. Theos. Human Science, Georgetown University,Washington, DC.Purpose: Pigmentary glaucoma involves optic nerve degenerationafter accumulation of debris originating from pigmented cells withinthe eye, a frequent endpoint for Pigment Dispersion Syndrome (PDS)patients . The molecular events within pigmented ocular cells thatprecipitate this initial pigment dispersion remain uncharacterized.The DBA/2J mouse is an established model for human PDS andharbors a truncated mutant Gpnmb R150X allele which has beenproposed to be a functional null. To date the impact of C-terminaltruncation on the intracellular sorting and biology of Gpnmb has yetto be determined.Methods: We have characterized the intracellular transport pathwayfollowed by wild-type Gpnmb and compared this to both Gpnmb R150Xand cytoplasmic domain mutants, including C-terminal truncationmutants that lack candidate sorting signals within the cytoplasmicdomain of this integral membrane protein. We have assessed theintracellular transport of these mutant proteins by ectopic expressionin transfected tissue culture cells followed by immunofluorescencemicroscopy. Early secretory pathway localization of protein atsteady-state has been determined by endoglycosidase-H(endoH)-sensitivity in Western blotting experiments. Defects in endocyticsorting have been further analyzed by antibody uptake experiments.Results: The severe truncation mutant Gpnmb R150X is unable to leavethe endoplasmic reticulum after synthesis as shown by intracellularsteady-state localization to calnexin-positive membranes andcomplete endoH sensitivity. Cytoplasmic truncation mutants thatretain the single transmembrane domain are able to progress throughthe secretory pathway but fail to be internalized upon delivery to thecell surface. Efficient endocytosis is dependent upon a functionalcytoplasmic dileucine sorting signal, as shown by steady-stateindirect immunofluorescence microscopy as well as antibody uptakeendocytosis assays.Conclusions: The wild-type function of Gpnmb in healthy pigmentedcells within the eye requires the efficient intracellular targeting ofGpnmb to endosome-derived organelles. Loss of critical sortingsignals, including a conserved cytoplasmic dileucine, results inmissorting and so loss of function leading to aberrant melanosomemorpohology in DBA/2J animals. Our data support a model for aprotective function of Gpnmb within the internal environment of themelanosome.Commercial Relationships: Alexander C. Theos, NoneSupport: AHAF National Glaucoma Research, Standard AwardG2012025Program Number: 4007Presentation Time: 9:45 AM - 10:00 AMCochlin and phosphatidylcholines interact with SLC44A2channel on the trabecular meshwork cellsSanjoy K. Bhattacharya, Mitchell Martinez, Ayman Aljohani, RichardK. Lee. Bascom Palmer Eye Institute, Univ of Miami Miller Sch ofMed, Miami, FL.Purpose: To demonstrate that cochlin and phosphatidylcholines (PC)interact with choline transporter-like channel SLC44A2. Our overallhypothesis is that cochlin and cholines both interact and modulate©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologySLC44A2 resulting in alteration of trabecular meshwork (TM) cellshape and behavior.Methods: The previously prepared human TM cells (two differentcell lines) and primary TM cells (derived from 5 different donorseach) were used for these studies. The protein extracts were preparedfrom approximately 1 million cells. In the suspension buffer, acombination of octylpyranoside (0.1% w/v), genapol (0.1%w/v),triton X-100 (0.5% v/v) and Tween-20 detergents (0.05% w/v) wasused to ensure suspension of SLC44A2 channel.Immunoprecipitation (IP) of 0.5-1 mg cell extracts was performedusing anti-cochlin (rabbit and chicken polyclonal antibodies) andanti-SLC44A2 (rabbit polyclonal and monoclonal) antibodies. The IPproducts were further extracted using Bligh and Dyer method. Lipidswere subjected to mass spectrometry for identification ofcholines/phosphatidylcholines and aqueous phase extracted proteinswere subjected to Western blot analyses and/or mass spectrometry forprotein identification. Fluorescent analogs of select PCs wereincubated with TM cells and subjected to UV cross-linking, proteinextraction, SDS-PAGE separation and mass spectrometry foridentification of cross-linked proteins.Results: The reciprocal IP showed precipitation of cochlin as well asphosphatidylcholines with anti-SLC44A2 antibodies. Cochlinantibodies precipitated SLC44A2. Fluorescent analogs performed forseveral PC species (not control PS species) showed cross-linkagewith SLC44A2.Conclusions: Several cholines and cochlin both interact withSLC44A2 channel. These results suggest that SLC44A2 channelactivity could be potentially modulated by interactions with bothphosphatidylcholines and cochlin.Commercial Relationships: Sanjoy K. Bhattacharya, None;Mitchell Martinez, None; Ayman Aljohani, None; Richard K.Lee, National Eye Institute (F)Support: Supported by an unrestricted grant from RPB to Universityof Miami, NIH grants P30EYEY014801 and R01EY016112,R01EY016112S1.Program Number: 4008Presentation Time: 10:00 AM - 10:15 AMDisruption of the homeostatic balance between TGF-β and BMPsin the outflow tissues of the CTGF-based Glaucoma mouse modelSabrina Kuespert, Ernst R. Tamm, Rudolf Fuchshofer. UniversityRegensburg, Institut of Human Anatomy and Embryology,Regensburg, Germany.Purpose: To investigate the homeostatic balance between TGF-β2and its endogenous antagonists, the bone morphogenetic proteins(BMPs), in CTGF-overexpressing mice (Junglas et al., Am J. Path.2012) with primary open-angle glaucoma (POAG).Methods: 2 month-old βB1-Crystallin-CTGF mice and wild-typelittermates were analyzed. The amounts of BMPs, TGF-β2 and theirmRNA expression, as well as that of other molecules involved intheir respective signaling pathways (pSmad 1/5/8; pSmad 2/3; Smad6/7, Gremlin) were analyzed by immunoblotting,immunohistochemistry and real-time RT-PCR. Cultured humantrabecular meshwork (HTM) cells were treated under serum freeconditions with different concentrations of CTGF (5 ng-100 ng) for24 h. Changes in the expression pattern of TGF-β2 were determinedby Western blot analysis and real-time RT-PCR. Blocking of theCTGF pathway was performed with an inhibitor against MEK1/2(UO126).Results: CTGF overexpression in βB1-Crystallin-CTGF with POAGleads to a significant decrease of BMP synthesis and signaling inaqueous humor outflow tissues when compared to wild-typelittermates. In contrast, the expression of TGF-β2 and activity of itssignaling were enhanced in 2-month-old transgenic mice. Theexamination of the inhibitory Smads showed that Smad6 wassignificantly enhanced in transgenic mice, whereas Smad7 wasunaffected. Gremlin, a specific BMP inhibitor, was also significantlyincreased in CTGF overexpressing mice with POAG. In vitro, CTGFtreatment led to an induction of TGF-β2 via the ERK1/2 pathway.Conclusions: CTGF is a key modulator in the pathogenesis of POAGleading to changes in the homeostatic balance between TGF-β andBMPs. The shift towards the TGF-β pathway is initiated by anincreased TGF-β expression versus a reduced BMP synthesis. Inaddition, an upstream blockage of BMPs by Gremlin and anenhanced downstream inhibition by Smad6 was observed in thetransgenic animals. The effects appear to involve the ERK1/2pathway. Modification of CTGF signaling seems to be a promisingstrategy to treat POAG.Commercial Relationships: Sabrina Kuespert, None; Ernst R.Tamm, None; Rudolf Fuchshofer, NoneSupport: DFG FOR1075/TP3436 AMD I, BIWednesday, May 08, 2013 11:00 AM-12:45 PMExhibit Hall Poster SessionProgram #/Board # Range: 4576-4594/A0095-A0113Organizing Section: Biochemistry/Molecular BiologyProgram Number: 4576 Poster Board Number: A0095Presentation Time: 11:00 AM - 12:45 PMAutophagy disregulation in the retina during aging andneurodegenerationEnrique J. de la Rosa 1 , Natalia Rodriguez-Muela 2 , Hiroshi Koga 3 ,Lucia Garcia-Ledo 2 , Pedro de la Villa 4 , Ana M. Cuervo 3 , PatriciaBoya 2 . 1 Cell & Molecular Medicine, Centro de InvestigacionesBiologicas, Madrid, Spain; 2 Cell & Molecular Biology, Centro deInvestigaciones Biologicas CSIC, Madrid, Spain; 3 Institute for AgingStudies, Albert Einstein College of Medicine, Bronx, NY;4 Physiology, School of Medicine, University of Alcala, Alcala deHenares, Spain.Purpose: Accumulation of undegraded proteinaceus materialrepresents a hallmark in many retinopathies including age-relatedmacular degeneration, the major cause of blindness in the elderlypopulation. Autophagy contributes to maintain cellular homeostasisthrough the removal of damaged cellular components in lysosomes.Although alterations in autophagic function underlie a growingnumber of human diseases, very little is known about the contributionof this quality control mechanism to the retinal pathophysiology.Methods: To determine how aging affects the functional state ofautophagy in the different cell types of the retina, we comparedyoung and old C57B6/J mice. In addition, we analyzed theconsequences of macroautophagic blockage in retinal quality controlby crossing the conditional Atg5 knockout animals (Atg5flox/flox)with the nestin-Cre mice to delete Atg5 in neural precursors. In bothmodels, we determined several parameters related with autophagy,the levels of apoptosis, as well as the visual function byelectroretinography.Results: We found that autophagic flux is less efficient in the agedretinas in both basal and under starvation conditions. In parallel, wefound increased levels of lipofuscin, ubiquitinated proteins and p62 inall retinal layers from old C57B6/J mice. Decreased expression of themRNA of the autophagy regulator Beclin1, a main component of theinitiation complex, and of Atg7, the limiting enzyme for theelongation of the autophagosome membrane, were also observed inthe aged mice. Further, we found TUNEL-positive nuclei in the retina©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyfrom Atg5flox/flox; nestin-Cre mice, as well as a clear reduction intheir visual function.Conclusions: We have identified a primary malfunction ofmacroautophagy in the aging retina that may contribute to the agerelatedretinopathies and reduction of visual function.Commercial Relationships: Enrique J. de la Rosa, ProRetinaTherapeutics, S.L. (F), ProRetina Therapeutics, S.L. (I), ProRetinaTherapeutics, S.L. (C), US2010/0042 A1 (P); Natalia Rodriguez-Muela, None; Hiroshi Koga, None; Lucia Garcia-Ledo, None;Pedro de la Villa, ProRetina Therapeutics, S.L. (I), US2010/0042 A1(P); Ana M. Cuervo, None; Patricia Boya, US2010/0042 A1 (P),ProRetina Therapeutics, S.L. (I)Support: SAF-2010-21879 to EJdlR and PdlV, CONSOLIDERCSD2010-000454 to EJdlR and PB, NIH-AG031782 and AG038072to AMC, SAF-2009-08086 to PBProgram Number: 4577 Poster Board Number: A0096Presentation Time: 11:00 AM - 12:45 PMHigh glucose activates ChREBP-mediated HIF-1α and VEGFexpression in human RPE cells under normoxiaMin-Lee Chang, Chung-Jung Chiu, Fu Shang, Allen Taylor. HNRCATufts University, Boston, MA.Purpose: Because retina-damaging angiogenesis is controlled byVEGF and people with higher glucose intakes are more susceptible toretinal complications that may be due to increased VEGF, it is crucialto elucidate relations between glucose exposure and VEGFexpression. We aimed to determine if a carbohydrate responseelement binding protein (ChREBP) plays a role in the transcriptionalup-regulation of hypoxia-inducible factor-1α (HIF-1α) anddownstream vascular endothelial growth factor (VEGF) expression inretinal pigment epithelial (RPE) cells exposed to normoxia and highglucose.Methods: ARPE19 cells were exposed to 5.6, 11, 17, 25 and 30 mMglucose for 48h in serum-free culture media under normoxic (21%O2) conditions. Protein and mRNA expression of indicated geneswere determined by immunoblot analyses and real-time RT-PCR,respectively. An enzyme-linked immunosorbent assay (ELISA) wasused to detect the concentrations of VEGF in the media.Immunofluorescence (IF) and chromatin immunoprecipitation (ChIP)for ChREBP were used to demonstrate nuclear translocation andHIF-1α gene promoter association, respectively.Results: Immunoblot analyses showed that HIF-1α levels werepositively related to levels of glucose exposure between 5.6-25 mMin the RPE cells, indicating the induction and stabilization of HIF-1αby elevated glucose under normoxic conditions. Human lensepithelial cells and HeLa cells did not respond to high glucose,implying that this phenomenon is cell type-specific. Real-time RT-PCR for HIF-1α and VEGF and ELISA for VEGF indicated that highglucose is associated with elevated production of HIF-1α-inducedVEGF, an established inducer of neovascularization, in the RPE cells.IF analyses showed that, although ChREBP was equally expressedunder both low (5.6 mM) and high (25 mM) glucose conditions, itappeared more in the nuclear region than in the cytosol of the RPEcells after the high glucose treatment. ChIP analyses suggested aHIF-1α gene promoter association with ChREBP under the highglucose condition. These results imply that RPE cells use cytosolicChREBP as a glucose sensor to up-regulate HIF-1α expression.Conclusions: These results suggest a high glucose-induced,ChREBP-mediated, and normoxic HIF activation that may bepartially responsible for the neovascularization in both diabetic andage-related retinopathy.Commercial Relationships: Min-Lee Chang, None; Chung-JungChiu, None; Fu Shang, None; Allen Taylor, NoneSupport: NEI R01 EY021826, EY021212, EY013250, Ross AgingInitiative, and USDA agreements 1950-5100-060-01A.Program Number: 4578 Poster Board Number: A0097Presentation Time: 11:00 AM - 12:45 PMAMD-associated silent polymorphisms in HtrA1 ameliorate IGF-1 antagonismSarah Melissa P. Jacobo 1, 2 , Margaret M. DeAngelis 3, 4 , AndriusKazlauskas 1, 2 . 1 Ophthalmology, Harvard Medical School, Boston,MA; 2 The Schepens Eye Research Institute, Boston, MA;3 Ophthalmology and Visual Sciences, University of Utah, Salt LakeCity, UT; 4 John A. Moran Eye Center, Salt Lake City, UT.Purpose: Single nucleotide polymorphisms (SNPs) within atranscript’s coding region that do not change the amino acid sequenceof the protein product are intuitively assumed to have no effect onprotein properties and function. The synonymous SNPs rs1049331and rs2293870 within the exon 1 of HTRA1 are associated withincreased risk of neovascular age-related macular degeneration(NvAMD), and convert the common codons for Ala34 and Gly36 toleast frequently used codons. The purpose of this project was toassess the impact of these SNPs on HtraA1’s properties andfunctions.Methods: To evaluate the effect of optimum-to-rare codonconversion on the rate of protein synthesis, synthetic mRNAtemplates encoding HtrA1+SNPs or WT HtrA1 were used in in vitrotranslation. We evaluated the effect of the synonymous SNPs onprotein structure by partial proteolysis and immunoprecipitation witha conformation-sensitive antibody. The expression level of HtrA1 inneovascular AMD (NvAMD) patients homozygotic for rs1049331and rs2293870 were compared to age-matched unaffected controls.Given that rs1049331 and rs2293870 lie within HtrA1’s Mac25domain, we evaluated the ability of HtrA1 to influence IGF-1signaling in endothelial cells.Results: We report that this common-to-rare codon conversionreduced mRNA translation rate, increased susceptibility to partialproteolysis, and reduced recognition by an antibody raised against theregion of HtrA1 that includes exon 1. Exon 1 encodes a Mac25domain, which is structurally very similar to proteins that bindinsulin-like growth factor-1 (IGF-1). The NvAMD-associated SNPsreduced HtrA1’s ability to antagonize IGF-1-stimulated signalingevents, and to suppress IGF-1-dependent tube formation of primaryhuman choroidal endothelial cells.Conclusions: These observations demonstrate the potential ofsynonymous SNPs within HTRA1 to impact its previouslyunappreciated ability to titrate IGF-1, and suggest that the loss of thiscapacity contributes to pathogenesis of NvAMD.Commercial Relationships: Sarah Melissa P. Jacobo, None;Margaret M. DeAngelis, None; Andrius Kazlauskas, NoneSupport: NIH Grant EY014458Program Number: 4579 Poster Board Number: A0098Presentation Time: 11:00 AM - 12:45 PMDifferences in Reactive Oxygen Species (ROS) Production andExpression of Genes from the Complement and InflammatoryPathways Between H and K Haplogroup CybridsJavier Cáceres del Carpio 1 , Mohamed Tarek 1 , Claudio A. Ramirez 1 ,Payam Falatoonzadeh 1 , Marilyn Chwa 1 , Deepika Malik 1 , S MichalJazwinski 2 , Miceli V. Michael 2 , Baruch D. Kuppermann 1 , Cristina M.Kenney 1 . 1 Gavin Herbert Eye Institute, University of CaliforniaIrvine, Irvine, CA; 2 Tulane Center for Aging, Tulane University,New Orleans, LA.Purpose: Age-related Macular Degeneration (AMD) is the leadingcause of blindness in people older than 50 in developed countries.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyMitochondrial DNA (mtDNA) polymorphisms, that are maternallyinheritedand are classified into haplogroups, can be associated withAMD. Individuals with haplogroup H, have less risk of developingAMD. Haplogroup K is related to AMD in some families. In thisstudy we hypothesize that different haplogroups can affect nucleargene expression and influence the production of ROS. We establisheda cybrid (cytoplasmic hybrid) model that has human RPE cells withidentical nuclei but different mitochondria (H vs K haplogroups) andcompared the differences in ROS production and expression of genesinvolved in the complement and inflammation pathways.Methods: Human ARPE-19 cells without mitochondria (Rho0) werefused with platelets from individuals that had either H or Khaplogroup genetic background. The haplogroup profiles weredetermined with PCR and restriction enzyme digestion. The ROSassay was performed with 2'7' dichlorodihydrofluorescein diacetate(H2DCFDA) dye. Gene expression profiles were determined usingQuantitative-PCR (Q-PCR) for selected genes of the complement andinflammatory pathways: Complement Component 3 (C3),Complement Factor H (CFH), CD59, CD55, Transforming GrowthFactor Alpha (TGFA), Transforming Growth Factor Beta 2 (TGFB2),Interleukin 6 (IL-6) and Retinoic Acid Receptor Alpha 1 (RARA1).Results: ROS production for haplogroup K cybrids was68.78±7.07% compared to H, which had been normalized to 100%(p=0.02). Expression levels for complement and inflammation geneswere significantly lower in K haplogroup cybrids compared to the Hcybrids: C3, 0.07 fold, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyAPNpII treated group was 38% less in 24 hours and 59% less in 48hours after injury compared to control. Expression of PCNA inwounded area was 20% more in APNpII treated ARPE-19 cellscompared to control group.Conclusions: RPE cells are potential target for anti-angiogenictherapy. APNpII may be used for inhibiting the VEGF and CCL2secretion by RPE cells. APNpII peptide may have therapeutic use forAMD treatment.Commercial Relationships: Puran S. Bora, None; Nalini S. Bora,None; Valeriy V. Lyzogubov, NoneSupport: Jones Eye Institute, University of Arkansas for MedicalSciencesProgram Number: 4582 Poster Board Number: A0101Presentation Time: 11:00 AM - 12:45 PMIsoform-specific interactions between human apolipoprotein Eand amyloid-beta in the retinal pigment epitheliumKimberly A. Toops, Jin Xu, Aparna Lakkaraju. Ophthalmology andVisual Sciences, University of Wisconsin - Madison, Madison, WI.Purpose: To investigate how different apolipoprotein E (ApoE)isoforms (a) influence the expression of amyloid-beta (Aβ) in theretinal pigment epithelium (RPE) and (b) preferentially interact withAβ under conditions of RPE stress. Human ApoE occurs in threeisoforms (E2, E3, E4) that vary in only two amino acids but havedifferent conformations, stabilities and interactions with lipids andproteins such as Aβ. The E4 isoform is strongly associated withincreased risk of Alzheimers’ disease (AD), but has a protectiveeffect in age-related macular degeneration (AMD). Drusen containApoE and Aβ and the RPE may be a source of both proteins.Increased intracellular cholesterol escalates neuronal Aβ productionand ApoE participates in clearing Aβ. Since RPE cells withlipofuscin store cholesterol, we investigated how lipofuscin andcholesterol storage affects Aβ expression and ApoE-Aβ interactions.Methods: Primary porcine RPE cells were transfected with GFPtaggedApoE2, E3 or E4 expressed from either a CMV or Best1promoter. Cells were treated with the lipofuscin component A2E orwith U18666A to induce cholesterol storage directly. Expression ofApoE and Aβ was measured by immunoblot in total cell lysates orafter immunoprecipitation with an ApoE antibody. ApoE and Aβcellular localization was examined by immunocytochemistry.Cholesterol was measured biochemically and by filipin staining. Liveimaging was performed using spinning disk confocal microscopy.Results: Expression of human ApoE isoforms in pig RPE induces Aβproduction. The amount of Aβ varies depending on the ApoEisoform. Pull-down assays show that the Aβ-ApoE interaction isresistant to reducing agents. Our data also suggest that lipofuscin andintracellular cholesterol affect numerous steps of the ApoE-Aβcascade including ApoE trafficking, the amount of Aβ generated inthe cell and the binding of ApoE to Aβ.Conclusions: ApoE trafficking and ApoE-Aβ interactions aremodulated by intracellular cholesterol and lipofuscin content in theRPE. In the retina, it is likely that ApoE isoforms participate inclearing Aβ from the RPE and direct it into the choroidal circulation.Thus, while there may be common pathological processes in AD andAMD, our data point to key functional differences at the cellular levelthat could help explain how the same ApoE isoform can confer riskin one disease and protection in another.Commercial Relationships: Kimberly A. Toops, None; Jin Xu,None; Aparna Lakkaraju, NoneSupport: American Health Assistance Foundation (M2009093); KarlKirchgessner Foundation; McPherson Eye Research Institute(Rebecca Meyer Brown Professorship); Research to PreventBlindness Career Development Award; Reeves Foundation forMacular Degeneration ResearchProgram Number: 4583 Poster Board Number: A0102Presentation Time: 11:00 AM - 12:45 PMEffects of the absence of both complement factor H andapolipoprotein E on VEGF expression, MMP2/9 activity andcaspase-1Laura Garcia-Garcia, Patricia Fernandez, Sergio Recalde, MaiteMoreno-Orduña, Vanessa Fernandez-Garcia, Alfredo Garcia-Layana. Ophthalmology Experimental Laboratory, ClinicaUniversidad de Navarra, Pamplona, Spain.Purpose: Apolipoprotein E deficient mouse (apoE-/-), experimentalmodel of genetic hypercholesterolemia, shows retinal proteinexpression patron alterations. Complement factor H (CFH) isinvolved in inflammatory process associated to retinal degenerations.We aimed to investigate the effect of the absence of CFH and apoEgenes in mouse protein expression in the retina.Methods: Wild type (WT), CFH-/-/apoE-/- (DKO) and CFH+/-/apoE+/- (DH) mice were used and divided in groups of age (youngeror older than 12 months) having 3-5 animals per group (all mice werein C57BL6/J background). Western blot analysis for vascularendothelial growth factor (VEGF) expression, zymography for matrixmetalloproteinase (MMP) activity and immunohistochemistrytechniques for caspase-1 detection were assessed. For comparisonsafter a significant ANOVA or Kruskal-Wallis among groups, posthoctests or U Mann-Whitney were applied (SPSS v.15.0).Results: VEGF expression was significantly lower in DH groupcompared with WT and DK (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologynegative regulator of alternative pathway complement activation),native properdin (P2 dimer, P3 trimer, and P4 tetramer; a positiveregulator of the same pathway), and complement C3b binding toCEP-BSA.Methods: SPR experiments were performed using a Biacore 3000.CEP-BSA with pyrrole to protein (P/P) ratio 7 (as measured by theEhrlich assay) and BSA were immobilized on CM5 chips by aminecoupling. The human CFH, C3b, P2, P3, or P4, or recombinant CFH(rCFH) fragments diluted in HBS-P buffer were injected over thechips, and the sensorgrams analyzed. rCFH fragments includedcomplement control protein (CCP) repeat domains 1-3, 5-7, 6-8, 7-9,17-19 (with 6XHis tag), as well as CCPs 18-20 and 19-20.Results: CFH, native properdin forms, and C3b bound to CEP-BSAwhile they didn’t bind to the reference surface where BSA wasimmobilized. Kinetic rate constants were determined usingBiaevaluation software (V4.0.1) by the global-fit method. The datashowed affinity values (KD) in the range 10 -6 M (C3b) to 10 -7 M(P3). CFH appears to have the highest association-rate constant (ka),while the native properdins have the slowest dissociation kinetics(kd). The rCFH fragments CCPs 5-7, 6-8, 18-20 and 19-20 bound toCEP-BSA P/P 7, with affinity values in the range of 1.4 X 10 -6 M to4.4 X 10 -6 M. The other recombinants did not bind.Conclusions: CFH likely binds to CEP moieties through its “heparinbinding”domains, CCPs 18-20 and 6-8. Properdin, a positivelychargedmultimer, avidly bound to the CEP moieties. Therefore, thenegative charges of CEP are probably responsible for attractionthrough electrostatic interactions. As the CEP-BSA was bound byboth positive and negative regulators of the complement pathway, abalance may be necessary for allowing complement activation(promoted by properdin), while regulating the extent of activation (byCFH) to a level that facilitates opsonization and removal of lipidoxidation products, but not tissue damage.Commercial Relationships: Lisa Kuttner-Kondo, None; VivianaP. Ferreira, None; Claudio Cortes, None; Satya P. Yadav, None;Joe G. Hollyfield, NoneSupport: Research to Prevent Blindness Unrestricted Grant & NIHR01EY014240-10 (JGH); NIH RR016789-01A1 (SPY);AHA12GRNT10240003 (VPF)Program Number: 4585 Poster Board Number: A0104Presentation Time: 11:00 AM - 12:45 PMThe aging retina: Macula-less rat and macula-bearing monkeyretinae exhibit common age-related changes in their retinalproteinsMichael R. Boehm 1, 2 , Sonja Mertsch 1 , Harutyun Melkonyan 1 , SolonThanos 1 . 1 Institute for Experimental Ophthalmology, School ofMedicine, WWU Muenster, Muenster, Germany; 2 CurrentAffiliation: Department of Ophthalmology, University of MuensterMedical Center, Muenster, Germany.Purpose: The visual consequences of age-related alterations in theneural retina are well documented, but little is known about theirmolecular bases. We performed a comparative proteomic analysis ofthe retinae in marmosets and rats, in order to identify proteins forwhich the expression profiles in retinas are altered with age.Methods: Protein profiles were compared in the newborn, juvenile,middle-aged, and aged retinae using two-dimensional gelelectrophoresis. Seven proteins exhibited changes in contentthroughout the life of rats, and 21 proteins that exhibited age-relatedcontent changes in marmosets were identified by matrix-assisteddesorption ionization-time-of-flight mass spectrometry. Western-blot(WB), quantitative reverse-transcriptase PCR (qRT-PCR), andimmunohistochemistry (IHC) analyses of selected proteins and theirmRNA were employed to determine whether changes identified byproteomics were verifiable at the cellular and molecular levels.Results: We found four proteins common to both species [Parkinsondisease (autosomal recessive, early onset) 7/DJ-1, stathmin,peroxiredoxin, and β-synuclein] whose contents were regulated withage. WB, IHC, and qRT-PCR analyses confirmed these findings. Theproteins were localized in certain layers and subsets of cells withinthe retinae of both species. Alterations in the expressions of theseproteins between the localization of the macular and retinal peripherywere analyzed with IHC. Detection of found proteins in the adulthuman retina were confirmed with IHC.Conclusions: This study is the first to provide evidence that theaging retina is physiologically characterized by specific changes in itsproteome. These changes occur in key functional pathways during theprocessing of visual signals and may be involved in the developmentof age-related pathologies, such as age-related macular degeneration.Commercial Relationships: Michael R. Boehm, None; SonjaMertsch, None; Harutyun Melkonyan, None; Solon Thanos, NoneSupport: Supported by Interdisciplinary Centre for Clinical Researchof Muenster (IZKF) grants to MRRB and ST (Tha3/002/09)Program Number: 4586 Poster Board Number: A0105Presentation Time: 11:00 AM - 12:45 PMGene expression profile of Retinal Pigment Epithelium derivedARPE-19 cells under serum starvationSanghamitra Mishra 1 , Katherine M. Peterson 1 , Alan E. Berger 2 ,Graeme J. Wistow 1 . 1 Section for Molecular Structure and FunctionalGenomics, National Eye Institute, Bethesda, MD; 2 JHBMC LoweFamily Genomics Core, Johns Hopkins University - School ofMedicine, Baltimore, MD.Purpose: Age Related Macular Degeneration (AMD) is associatedwith formation of subretinal deposits (drusen) and damage to Bruch’smembrane (BM) basal to the retinal pigment epithelium (RPE). Thiscould lead to serum deprivation of overlying RPE. Here we studiedgene expression in the human RPE derived cell line, ARPE-19,during a seven day time course of serum starvation.Methods: Gene expression levels in serum starved ARPE-19 cellswere measured for 0, 1, 3, 5, and 7 days using cDNA microarrays.Standard statistical tools and pathway analysis software were used toidentify regulated genes and altered pathways. Differentiallyexpressed genes and pathways, identified from microarray analysis,and the effect of supplements were confirmed by qPCR, western blotanalysis and confocal microscopy.Results: Differentially expressed genes at days 1, 3, 5, and 7 wereidentified. The most notable changes in gene expression were upregulationof cholesterol and lipid gene pathways and downregulationof methallothionein genes. This was confirmed by qPCRand western blotting. Supplementing LDL reversed the change incholesterol pathway genes. Uptake of LDL in the serum starved cellswas also tested. Cells at days 3, 5 and 7 of serum starvation showedincreasing uptake of LDL-Dy549, whereas no uptake was seen atDay 0. The labeled LDL uptake was abolished in the presence ofunlabeled LDL. Downregulation of metallothionein genes along withupregulation of zinc binding genes is consistent with a reduction inthe intracellular levels of bio-available zinc. Zinc supplementationreversed expression changes of the metallothionein and zinctransportergenes.Conclusions: Serum starved ARPE-19 cells showed significantchanges in expression patterns of cholesterol and zinc-relatedpathway genes. Cholesterol synthesis enzymes were upregulated andcells showed increased uptake of exogenous cholesterol. Thissuggests that serum deprivation initiates a cellular program requiringcholesterol in RPE cells. The cells also have an increasedrequirement of zinc. Zinc-binding proteins frequently function as©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyregulators of transcription, which have a profound effect on cellfunction when altered. These observations are interesting,considering that both cholesterol and zinc have been implicated inAMD. This raises the possibility that the response of RPE-derivedcells to serum-deprivation may model some processes related toAMD.Commercial Relationships: Sanghamitra Mishra, None;Katherine M. Peterson, None; Alan E. Berger, None; Graeme J.Wistow, NoneProgram Number: 4587 Poster Board Number: A0106Presentation Time: 11:00 AM - 12:45 PMHSPA5 - a possible new contributing gene in atrophic age-relatedmacular degenerationSumana Chintalapudi, Yin H. Chan Lau, Shwetapadma Sahu, MonicaM. Jablonski. Ophthalmology, The University of Tennessee HealthScience Center, Memphis, TN.Purpose: To investigate the contribution of extracellular HSPA5 (akaGRP78) in human atrophic age-related macular degeneration usingnovel systems genetic methods, mathematical modeling andimmunostaining techniques.Methods: To determine the cellular localization and contribution ofextracellular HSPA5, single and double immunostaining in humannormal and AMD retinas were performed using well-characterizedantibodies. Quantitative trait locus (QTL) mapping was used to mapthe genomic region(s) that regulate Hspa5 in a BXD geneticreference panel. Multiple partial correlation and heatmap analyseswere performed to define the relationship between the expression ofHspa5 and all genes within the mouse genome. The top candidategenes were identified and further evaluated using SNP screening.Results: In non-AMD retinas, extracellular HSPA5 was localizedintensely around cones within the macular area; however it wasscattered in the peripheral retina. In contrast, in AMD retinas, therewere very few HSPA5 immunopositive cones, especially in themacula. In addition, the immunolabeling profiles were aberrant. Ourmapping studies in BXD mice indicate a single highly significanttrans-eQTL with an LRS of 18.4 on Chr2:169-175Mb and asuggestive trans-eQTL on Chr15:75-100Mb, both of which suggestthat other genes control Hspa5 expression either directly orindirectly. Partial correlation analyses and heatmaps confirmed theQTLs on Chrs 2 and 15. The best candidate extracellular genes inthose intervals were B4galt5, Dpm1, Sulf2, Fbln1, Glt8d3 and Pmm1.Direct correlation analyses showed that expression of Sulf2, Pmm1and Fbln1 were significantly correlated with Hspa5. Only Sulf2 had anon-synonymous SNP, which makes it our top candidate forcontrolling Hspa5 expression.Conclusions: The presence of HSPA5 in the interphotoreceptormatrix around cones in healthy human retinas and its absence inatrophic AMD retinas supports our assertion that HSPA5 may play arole in cone structural integrity in the macula. Integrated approachesusing mouse and human analyses suggest that HSPA5 may be a locusfor AMD progression and/or susceptibility. Our studies furthersuggest that Sulf2 is the best candidate gene for controlling Hspa5expression in the retina.Commercial Relationships: Sumana Chintalapudi, None; Yin H.Chan Lau, None; Shwetapadma Sahu, None; Monica M.Jablonski, 8,092,825 (P)Support: Research to Prevent BlindnessProgram Number: 4588 Poster Board Number: A0107Presentation Time: 11:00 AM - 12:45 PMQuercetin Inhibits H2O2 Stimulated PEDF and FGF2 Synthesisin hRPE CellsNandita Anand, Piyush C. Kothary, Monte A. Del Monte. Universityof Michigan, Ann Arbor, MI.Purpose: Age related macular degeneration (AMD) is a major causeof blindness. Proliferation and damage of the human retinal pigmentepithelial (hRPE) cells is involved in the pathogenesis of AMD.Oxidative stress induces in hRPE cell death and stimulates thesynthesis of angiogenic factors in hRPE cells. We studied the effectof quercetin (Q), a flavonoid that protects cells from oxidative stress,on the synthesis of the angiogenic factor, fibroblast growth factor 2(FGF2), and the anti-angiogenic factor, pigment epithelial derivedfactor (PEDF) in hRPE cells.Methods: hRPE cells were cultured from eyes obtained from theMichigan Eye Bank.Cellular proliferation in the presence ofincreasing concentrations of fetal bovine serum (FBS) was measuredby 3[H] thymidine incorporation. hRPE cell viability was measuredin the presence of increasing concentrations of FBS and H2O2 by thetrypan blue exclusion method (T).The effect of (Q) on hRPE cellviability was also measured in presence of H2O2 by (T).The effect ofvarious concentrations of H2O2 on intracellular synthesis of PEDFand FGF2 in presence and absence of (Q) was measured byimmunoprecipitating 14C-methionine-PEDF (14C Met-PEDF) and14C-methionine-FGF2 (14C Met-FGF2) and immunocytochemical(IC) analysis.Results: FBS (0-10%) stimulated cell proliferation and cell viabilityin cultured hRPE cells. H2O2 (0-0.5mM) decreased cell viability (T)in a dose dependent manner. The addition of (Q) (50μM) to 0.5mMH2O2 treated cells increased hRPE cell viability(130,000 ± 48,989vs. 40,625± 28,252, n=8, cells±SEM, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyphenotype of Tg and age-matched wild-type (Wt) mice in 18- to 24-month-old mice. Real time PCR, immunohistology, transmissionelectron microscopy and fluorescein angiography (FA) were used toanalyze the phenotype of this transgenic mouse model and agematchedcontrols.Results: This BMP4 over-expressing mouse accumulated remarkableamounts of oxidized lipid in RPE and on Bruch’s membrane (BM).This suggests that over-expressed BMP4 induces oxidative stress ofRPE which might lead to subsequent AMD phenotypes, includingRPE atrophy and thickened BM. RPE in Tg mice showed decreasedheight, hypo- or hyper-pigmentation, disorganized basal infoldingsand cellular lipid vesicles. Some Tg mice eventually developedchoroidal neovascularization (CNV) with fluorescein leakage on FA,isolectin B4 positive staining on flat mounted posterior eye cups andCNV-like lesions by histology. These changes were not seen in aged-matched controls.Conclusions: The BMP4 over-expressing mice mimic thepathological process of AMD and may be a good model for studyingAMD. Our work suggests that a dysregulated growth factor, BMP4,may be involved in the pathogenesis of AMD.Commercial Relationships: Danhong Zhu, None; Jing Xu, None;Xuemei Deng, None; Jamie Hsiung, None; Stephen J. Ryan,Alergan (S); David R. Hinton, RPT (I), RPT (P)Support: EY03040Program Number: 4590 Poster Board Number: A0109Presentation Time: 11:00 AM - 12:45 PMVitamin D Status and Subretinal Fibrosis in Neovascular Age-Related Macular DegenerationAmardeep Singh 1, 2 , Mads K. Falk 1, 2 , Yousif Subhi 1, 2 , Torben L.Sørensen 1, 2 . 1 Ophthalmology, Copenhagen University HospitalRoskilde, Roskilde, Denmark; 2 University of Copenhagen,Copenhagen, Denmark.Purpose: Vitamin D has been shown to have anti-fibrotic propertiesand deficient levels of circulating 25-hydroxyvitamin D have beenassociated with fibrogenesis in several extra-ocular tissues. Evidencelinking vitamin D deficiency to age-related macular degeneration(AMD) is inconsistent. Thus, we sought to investigate 25-hydroxyvitamin D levels across clinical subgroups of AMD andwhether the presence of subretinal fibrosis in neovascular AMD atdiagnosis is associated with lower levels of circulating vitamin D.Methods: Plasma samples were collected from 202 participantsacross 4 groups: controls (n=73), early AMD (n=22), geographicatrophy (n=12) and neovascular AMD (n=95). All participants weresubjected to a structured interview and retinal examination wasperformed using stereoscopic funduscopy on mydriatic eyes, digitalcolor fundoscopy, autofluorescence imaging, spectral-domain opticalcoherence tomography and fluorescein and indocyanin greenangiography in patients suspected of having neovascular age-relatedmacular degeneration. Clinical grading was performed using theClinical Age-Related Maculopathy Grading System. Venous bloodwas obtained for measurement of 25-hydroxyvitamin D2 and D3 inplasma using liquid chromatography-tandem mass spectrometry.Genomic DNA was extracted from leukocytes and genotyping wasperformed for single nucleotide polymorphisms in the vitamin Dmetabolism.Results: Patients with subretinal fibrosis in addition to neovascularAMD had significantly lower levels of circulating 25-hydroxyvitaminD compared to patients without subretinal fibrosis (47.2 versus 75.6nmol/L, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyresults demonstrate spontaneous degenerative changes in the RPEand the neuroretina of Cd46 -/- mice. These changes resemble thoseobserved in patients with dry-type AMD.Conclusions: Cd46 -/- mouse develops a phenotype similar to drytypeAMD. It represents a tractable model to facilitate ourunderstanding of AMD.Commercial Relationships: Valeriy V. Lyzogubov, None; XiaoboWu, None; Grant Kolar, None; Puran S. Bora, None; John P.Atkinson, None; Nalini S. Bora, NoneSupport: The Edward N. & Della L. Thome Memorial Foundation,Lions of Arkansas Foundation, Inc, and the Pat and Willard WalkerEye Research Center at the Jones Eye Institute, University ofArkansas for Medical Sciences, Little Rock, AR.Program Number: 4592 Poster Board Number: A0111Presentation Time: 11:00 AM - 12:45 PMTissue Inhibitor of Metalloproteinase-3 (TIMP3) InducesEndothelial Apoptosis via MMP- and Caspase-IndependentMechanismsJian H. Qi, Bela Anand-Apte. Ophthalmic Research-ColeEye Inst,Cole Eye Institute, Cleveland, OH.Purpose: TIMP3 has been shown to induces apoptosis in certain nonendothelialcell types. Mutations in TIMP3 gene causes Sorsbyfundus dystrophy (SFD, a rare, dominantly inherited, early onsetmacular degenerative disease. SFD has similar disease phenotypes toage-related macular degeneration (AMD) including increasedaccumulation of TIMP3 in Bruch’s membrane (BM) and an earlyphase of atrophy of the choriocapillaris, retinal pigment epithelium(RPE) and photoreceptor. To address the contribution of TIMP3 tothe disease process, the apoptotic activity and pathways of TIMP3were investigated in endothelial cells (ECs).Methods: human RPE cells, monkey choroidal ECs, PAE/KDR cellline expressing wild type or SFD-S156C-TIMP3 and mouse aorticECs derived from WT, heterozygous TIMP3+/S156 and homozygousTIMP3S156C/S156C mice were treated with recombinantadenoviruses carrying the wild-type (WT) TIMP3 or TIMP3 Cys1-Ser or serum deprivation. Apoptosis was analyzed by ApopTag directkit, FACS or CasPASETM apoptosis assays. Immunoprecipitationand/or immunbotting were used to analyze FAK cleavage, levels ofBax and Bcl2 and FAK tyrosine phosphorylation and associationwith paxillin. Focal adhesion formation was determined byimmunocytochemical staining.Results: Expression of TIMP3 promoted apoptosis in ECs, and to alesser extent, in RPE cells. It appears that TIMP3-induced apoptosisis independent of MMP inhibition. This is because 1) TIMP3 Cys1-Ser, devoid of inhibitory activity towards MMP2, promotedapoptosis, and 2) S156C-TIMP3, which has attenuated MMPinhibitory activity, promoted or even enhanced apoptosis. TIMP3-induced apoptosis was associated with activation of caspases, upregulationof bax/Bcl2 ratio and inhibition of tyrosinephosphorylation of FAK, association with paxillin and focal adhesionformation on matrix. However, cleavage of FAK, a prominentcaspase substrate, was not increased by TIMP3. Furthermore, either apolycaspase inhibitor or a specific inhibitor of caspase 3 or 8 wereineffective against TIMP3-mediated apoptosis and inhibition of focaladhesion formation.Conclusions: TIMP3 has a direct effect on ECs by triggeringapoptosis via MMP- and caspase-independent pathways, which likelyunderlies atrophy of the choriocapillaris and subsequent RPE orphotoreceptor in AMD and SFD disease processes.Commercial Relationships: Jian H. Qi, None; Bela Anand-Apte, 7183 256 B2 (P)Support: REY022768AProgram Number: 4593 Poster Board Number: A0112Presentation Time: 11:00 AM - 12:45 PMLocalization of mechanotransduction channels in the trabecularmeshwork of glaucomatous DBA/2J micePhi Ho, Vu T. Tran, Lais Cabrera, Sanjoy K. Bhattacharya. BascomPalmer Eye Institute, University of Miami Miller School ofMedicine, Miami, FL.Purpose: To determine whether selected mechanotransductionchannels (TREK-1, TRPM2, TRPA1 and TRPC1-6) localize in thetrabecular meshwork (TM) of glaucomatous DBA/2J mice. Todetermine whether exposure of selected lipids results in modulationof level of any of these channels on TM cells. Our hypothesis is thatselect lipids modulate mechanotransduction channels leading toalteration of TM cell shape and behavior.Methods: All studies were performed adhering to the ARVOstatement for use of animals in vision research under IACUCapproved protocols. DBA/2J mice of age 3-8 months (n=40 at eachpoint) was used for detection of mechanotransduction channels.Immunohistochemistry and Western Blot analyses were carried outusing commercial antibodies to mechanotransduction channels(TREK-1, TRPM2, TRPA1 and TRPC1-4 and 6) that were found inthe DBA/2J TM by genomic analyses (from GEO database confirmedwith RT-PCR analyses). Two human TM cell lines maintained in thelaboratory were subjected to treatment with select phospholipids anddetermination of changes in the level using polyclonal antibodies toTREK-1. The phospholipids for these experiments were selectedbased on their prior differential identification in normal but notglaucomatous TM using shotgun lipidomics approach in ourlaboratory.Results: We detected TREK-1, TRPM2, TRPC3 and TRPC6 in theTM of DBA/2J mice at 3-8 months of age. We detected somechanges in the level of these channels with aging in DBA/2J mice.We found level changes in TREK-1 channels (normalized withGAPDH or actin) determined by Western Blot analyses in human TMcells in response to exposure to select exogenous addedphosphatidylserines and sphingomyelins in cell culture.Conclusions: Our investigation confirms presence of TREK-1,TRPM2, TRPC1-4 and TRPC6 mechanotransduction channels atprotein levels in the DBA/2J mice TM. This is in parallel toobservation in the human TM. Levels of TREK-1 was modulated inhuman TM cells in response to exposure to select exogenously addedlipids.Commercial Relationships: Phi Ho, None; Vu T. Tran, None; LaisCabrera, None; Sanjoy K. Bhattacharya, NoneSupport: Supported by RPB Career award (SKB), an unrestrictedgrant from RPB to University of Miami, NIH grants R01EY016112and P30EYEY014801Program Number: 4594 Poster Board Number: A0113Presentation Time: 11:00 AM - 12:45 PMCTRP5 over-expression in RPE cells leads to loss of RPE celladhesion and retinal degeneration in wild-type miceAstra Dinculescu 1 , Seok-Hong Min 1 , Wen-Tao Deng 1 , Jie Li 1 , ReneeC. Ryals 1 , Rachel M. Stupay 1 , Ping Zhu 1 , Bhubanananda Sahu 2 ,Radha Ayyagari 2 , William W. Hauswirth 1 . 1 Ophthalmology,University of Florida, Gainesville, FL; 2 Shiely Eye Center,University of California, San Diego, La Jolla, CA.Purpose: The complement C1q tumor necrosis factor-related protein-5 (C1QTNF5/CTRP5) is highly expressed in the RPE and cilliaryepithelial layers in the eye. A pathogenic mutation, S163R, causesautosomal dominant late-onset retinal degeneration, with thickextracellular sub-RPE deposits, and choroidal neovascularization at©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologylater stages. Here, our goal was to test the effects of wild-type CTRP5protein overexpression in RPE cells on retinal structure and functionin normal mice.Methods: We generated AAV vectors as tools to overexpress eitherCTRP5 or its dicistronic partner, the membrane-type frizzled relatedprotein (MFRP), specifically in RPE cells of C57BL/6 mice. Onemicroliter (10 9 total vector genomes) of scAAV8 (Y733F) vectorscontaining an RPE-specific BEST1 promoter driving either a mouseCtrp5 or Mfrp cDNA was injected subretinally into one eye of adultC57BL/6 mice, while the contralateral eyes were untreated andserved as controls. Retinal function was assessed by full-fieldelectroretinography (ERG) under scotopic (dark-adapted) conditionsat 2 months post-injection. Pathological changes were investigated byoptical coherence tomography, digital fundus imaging, evaluation ofretinal morphology and immunohistochemistry.Results: RPE targeted overexpression of CTRP5 leads to severeretinal degeneration, with pathological effects on both photoreceptorsand RPE. The scAAV8 (Y733F)-CTRP5 treated eyes displayed adramatic loss of photoreceptors over the entire retina. In addition, theRPE cells appeared to have lost their intercellular junctional integrity,and separated from their lateral neighbor in the RPE monolayer.Some areas of CTRP5 treatment suffered complete loss of both RPEand photoreceptor cells by 3 months post-injection. In contrast,scAAV8 (Y733F)-MFRP treatment led to no detrimental effects onretinal structure or function, with the RPE appearing healthy evenwith robust overexpression of MFRP within the apical membrane andits microvilli.Conclusions: Abnormally increased levels of CTRP5 in RPE lead towidespread photoreceptor cell death, and loss of RPE intercellularadhesion. This suggests that CTRP5 may play a key role inmodulating the RPE intercellular tight junctions, and its excess maycontribute to the progression of choroidal neovascularization (CNV)in pathological conditions such as age related macular degeneration(AMD).Commercial Relationships: Astra Dinculescu, None; Seok-HongMin, None; Wen-Tao Deng, None; Jie Li, None; Renee C. Ryals,None; Rachel M. Stupay, None; Ping Zhu, None; BhubananandaSahu, None; Radha Ayyagari, None; William W. Hauswirth,AGTC (I), Bionic Sight (I), AGTC (C), Syncona (C), RetroSense (C)Support: EY013198, Foundation Fighting Blindness, and Researchto Prevent Blindness, Inc.466 AMD II, BIWednesday, May 08, 2013 2:45 PM-4:30 PMExhibit Hall Poster SessionProgram #/Board # Range: 4985-5016/A0114-A0145Organizing Section: Biochemistry/Molecular BiologyProgram Number: 4985 Poster Board Number: A0114Presentation Time: 2:45 PM - 4:30 PMCOMPARISON OF THE GENE EXPRESSION BYHAPLOGROUPS H AND J: IMPLICATIONS FOR AMD (AGERELATED MACULAR DEGENERATION)Claudio A. Ramirez 1 , Marilyn Chwa 1 , Shari Atilano 1 , DeepikaMalik 1 , Javier Cáceres del Carpio 1 , Mohamed Tarek 1 , S MichalJazwinski 2 , Miceli V. Michael 2 , Baruch D. Kuppermann 1 , Cristina M.Kenney 1 . 1 Gavin Herbert Eye Institute, University of California,Irvine, Irvine, CA; 2 Tulane Center for Aging, Tulane University,New Orleans, LA.Purpose: AMD is a leading cause of vision loss in the elderlypopulation. Mitochondrial genetics is an important area of researchthat may help us understand the predisposition for AMD between andwithin racial groups.Previous studies have shown that the mtDNAhaplogroup J is associated with AMD, while the H haplogroup isprotective. However, the functional consequences of this differenceare not understood. We have used cybrids (cytoplasmic hybrids) tostudy the characteristics and biochemical differences between varioushaplogroups. Our hypothesis is that cybrids, dissimilar only in theirmtDNA haplogroup will behave differently in vitro.Methods: Cybrids were created by introducing the mitochondriafrom human individuals platelets into a host cell line (ARPE-19) thatwas devoid of mitochondrial DNA (Rho0). Therefore, all cybridscarry the same nuclear genes but vary only in their mitochondrialcontent. Cybrid cultures H and J were pelleted, the RNA was isolatedand then quantified. RNA samples were reverse transcribed intocDNA. The RNAs from the three H and the three J haplogroupscybrid cultures were combined into a single sample each for analyseswith the Affymetrix Human U133 Plus 2.0 Array. The geneexpression results were analyzed with pathway analysis software(INGENUITY Systems). Q-PCR was performed using primers forgenes associated with inflammation (TGFA.TGFB2, and IL6) andapoptosis (RARA1, BBC-3 and BCL2L13).Results: The array analyses showed that H and J cybrids had alteredexpression of nuclear genes involved in inflammation and apoptoticpathways. Q-PCR analyses showed that J cybrids had decreasedexpression levels for TGFA (0.43 fold, p=0.03), RARA (0.57 fold,p=0.007), and BLC2L13 (0.56 fold, p=0.005). There were nosignificant changes in expression levels for TGFB2 (0.86 fold,p=0.41), IL-6 (0.38 fold, p=0.09), and BBC-3 (0.70 fold, p=0.11) inthe H versus J cybrids.Conclusions: This study demonstrates that cybrids may be a usefultool to study the effects of mtDNA variants on gene expression. Ourfindings suggest that mtDNA haplogroup differences have functionalconsequences. This data may have implications for understanding theassociation between haplogroup J and its increased risk for AMD.Commercial Relationships: Claudio A. Ramirez, None; MarilynChwa, None; Shari Atilano, None; Deepika Malik, None; JavierCáceres del Carpio, None; Mohamed Tarek, None; S MichalJazwinski, None; Miceli V. Michael, None; Baruch D.Kuppermann, Alimera (C), Allegro (C), Allergan (C), Genentech(C), Glaukos (C), GSK (F), Novagali (C), Novartis (C), Ophthotech(C), Pfizer (C), Regeneron (C), Santen (C), SecondSight (C), Teva(C), ThromboGenics (C); Cristina M. Kenney, NoneSupport: Supported by Discovery Eye Foundation, GuentherFoundation, Beckman Macular Research Initiative, Polly andMichael Smith Foundation, Max Factor Family Foundation, SkirballFoundation,Lincy Foundation, Iris and B. Gerald Cantor Foundation,Unrestricted grant from Research to Prevent Blindness, grant fromthe National Institute on AgingProgram Number: 4986 Poster Board Number: A0115Presentation Time: 2:45 PM - 4:30 PMHops Extract Xanthohumol Protects Visual Acuity and FunctionAfter Light DamageStephanie L. Foster, Nathaniel F. Henneman, Micah A. Chrenek,Charles B. Wright, Jeffrey H. Boatright. Ophthalmology, EmoryUniv School of Med, Atlanta, GA.Purpose: While hops is commonly recognized as an ingredient ofbeer, one of its lesser-known constituents, the flavonoid derivativexanthohumol (Xn), has been shown to scavenge reactive oxygenspecies. If xanthohumol can mitigate oxidative damage in the eye, itmay be a valuable treatment for retinal degenerations. In thisexperiment, we sought to test the ability of xanthohumol to preservevision in the light-induced retinal degeneration (LIRD) mouse, anenvironmental model of blindness.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyMethods: Twelve male 129SV mice (Charles River Laboratory)were given two intraperitoneal injections of Xn (0.8 mg/kg) orvehicle (4 PBS: 1 ethanol: 1 cremophor) prior to light damage.Atropine eye drops (1%) were administered twice before lightdamage to dilate pupils. Bright Xn and bright vehicle treatmentgroups were exposed to 50,000 lux of white LED light for 6 hours,while dim Xn and dim vehicle groups received 50 lux of normallight. After light damage, injections were administered five times perweek to maintain systemic drug levels. To assess visual function andacuity, optokinetic tracking (OKT) and electroretinograms (ERGs)were performed once weekly after light damage.Results: Mean ERG b-wave amplitudes were significantly higher forbright Xn mice as compared to bright vehicle, with representativeamplitudes (in microvolts ± SEM) of 445±62 and 263±29,respectively, at a flash intensity of 24.9 cd s/m 2 (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyResults: The Affymetrx array analyses showed that compared to Hcybrids, the L cybrids and J cybrids had 5.7 fold and 3.5 fold lowerexpression levels of IL-33 but the K cybrids were similar (1.7 folddifference). As determined by Q-PCR, the gene expression levels ofIL-33 for cybrids J, K, and L were measured relative to theexpression in H cybrid. An analysis of the data showed decreasedexpression of the IL-33 gene in J cybrids (0.46 fold, p=0.004) and Lcybrids (0.30 fold, p=0.002), but similar expression levels of IL-33 inthe K cybrids and H cybrids (1.35 fold, p=0.40).Conclusions: This study demonstrates that expression of the IL-33gene varies in cybrids that have identical nuclei but differenthaplogroups defined by specific mtDNA variants. This is significantbecause it suggests that the mtDNA variants may mediate IL-33, apro-inflammatory cytokine that may be important in the pathogenesisof AMD.Commercial Relationships: Thomas A. Vo, None; Marilyn Chwa,None; Shari Atilano, None; Deepika Malik, None; Claudio A.Ramirez, None; Javier Cáceres del Carpio, None; S MichalJazwinski, None; Miceli V. Michael, None; Baruch D.Kuppermann, Alimera (C), Allegro (C), Allergan (C), Genentech(C), Glaukos (C), GSK (F), Novagali (C), Novartis (C), Ophthotech(C), Pfizer (C), Regeneron (C), Santen (C), SecondSight (C), Teva(C), ThromboGenics (C); Cristina M. Kenney, NoneSupport: Discovery Eye Foundation, Guenther Foundation,Beckman Macular Research Initiative, Polly and Michael SmithFoundation, Max Factor Family Foundation, Skirball Foundation,Lincy Foundation, Iris and B. Gerald Cantor Foundation,Unrestricted grant from Research to Prevent Blindness, grant fromNational Institute on Aging (AG006168)Program Number: 4989 Poster Board Number: A0118Presentation Time: 2:45 PM - 4:30 PMResveratrol Protects Human Retinal Pigment Epithelial Cellsfrom Inflammatory InsultsR K. Kutty 1 , Chandra N. Nagineni 1 , William Samuel 1 , Todd Duncan 1 ,Camasamudram Vijayasarathy 2 , Cynthia Jaworski 1 , T. MichaelRedmond 1 . 1 National Eye Institute, National Institutes of Health,Bethesda, MD; 2 National Institute on Deafness and OtherCommunication Disorders, National Institutes of Health, Bethesda,MD.Purpose: Inflammatory responses of retinal pigment epithelium(RPE) are implicated in the pathogenesis of age-related maculardegeneration (AMD). RPE cells in culture respond to theproinflammatory cytokines IFN-γ, TNF-α and IL-1β by increasingthe expression of chemokines and cytokines. Resveratrol, a naturallyoccurring polyphenol, is a well characterized anti-inflammatoryantioxidant. The purpose of the present study is to investigatewhether resveratrol can modulate the effect of IFN-γ, TNF-α and IL-1β on RPE cells.Methods: Confluent cultures of ARPE-19 cells were treated with aproinflammatory cytokine mixture containing IFN-γ, TNF-α and IL-1β in a serum free medium for various time intervals. Total RNAfraction was then isolated and real-time PCR analysis of geneexpression was performed using TaqMan reagents (AppliedBiosystems) with GAPDH as an endogenous control. The activationof nuclear factor-κB (NFκB) was examined by western immunoblotanalysis of cell extracts using anti-phospho-NFκB p65 (Ser536)antibody.Results: Pretreatment of ARPE-19 cells with resveratrol (50 µM)effectively blocked the induction of CCL5, CXCL9, CSF2 andNOS2A (inducible form of nitric oxide synthase) by theproinflammatory cytokines (IFN-γ + TNF-α + IL-1β). Resveratrolalso prevented the decrease in the expression of HMOX1 (hemeoxygenase-1) in cells exposed to proinflammatory cytokines.Immunoblot analysis showed that the phospo-NFκB p65 increased incells in response to the treatment with proinflammatory cytokines.This increase in phosphorylation was considerably reduced in thepresence of resveratrol.Conclusions: Resveratrol attenuated the inflammatory response ofthe RPE cells in culture by its ability to block the activation of NFκBsignaling pathway by the proinflammatory cytokines. Thus,resveratrol, due to its ability to protect RPE from inflammatoryinsults, may have therapeutic potential as a nutraceutical in arrestingthe development of ocular degenerative diseases such as AMD.Commercial Relationships: R K. Kutty, None; Chandra N.Nagineni, None; William Samuel, None; Todd Duncan, None;Camasamudram Vijayasarathy, None; Cynthia Jaworski, None;T. Michael Redmond, NoneSupport: Intramural Research Program of the National Eye Institute,National Institutes of HealthProgram Number: 4990 Poster Board Number: A0119Presentation Time: 2:45 PM - 4:30 PMBone morphogenetic protein 4 induces ID1 and ID3 transcriptionfactors in trabecular meshwork cellsAvani A. Mody, Robert J. Wordinger, Abbot F. Clark. Visual sicence/NTERI, UNTHSC, Fort Worth, TX.Purpose: Bone morphogenetic protein4 (BMP4) attenuatestransforming growth factor β2 (TGFβ2) mediated responses intrabecular meshwork (TM) cells. However, The overall regulatorymechanisms of this inhibition remain unclear. BMP4 regulatesvarious cellular processes by induction of transcription factor knownas inhibitors of DNA binding protein (ID1, ID3) which bind andsuppress other transcription factors. The purpose of the current studyis to determine whether BMP4 will induce ID1 and ID3 expression incultured human TM cells. This study will aid in understandingwhether BMP4 transcriptionally suppresses TGFβ-2 responses in TMcells via induction of IDs.Methods: Transformed ( GTM-3) and primary TM cells were treatedwith different doses of recombinant BMP-4 for various time points(0-48 hr) to study ID1/ID3 induction. Quantitative RT- PCR andwestern immunoblotting were used to study mRNA and proteinexpression of ID1 and ID3.Results: BMP-4 significantly induces ID1 and ID3 m RNA levels inTM cells (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyPurpose: We recently reported the generation of the first PCV modelby transgenically expressing human HTRA1, a multi-functionalserine protease, in mouse retinal pigment epithelium (RPE). Weshowed that increased HTRA1 induced characteristic features ofPCV, including branching networks of choroidal vessels andpolypoidal lesions. Transgenic hHTRA1+ mice also developed occultCNV. To test the hypothesis that the proteolytic activity of HTRA1 isresponsible for its pathological role in PCV, we have generatedtransgenic mice expressing the protease inactive mutant of HTRA1(S328A) in mouse RPE. In addition, we generated an improvedtransgenic HTRA1 mouse model Tg44, with more consistentphenotypes.Methods: The expression of HTRA1 and HTRA1-S328A wasdetermined by western blotting and immunohistochemistry. Thephenotypes of various HTRA1 mice were examined by fluoresceinangiography (FA), indocyanine green angiography (ICGA), fundusimaging, spectral-domain optical coherence tomography (SD-OCT),histology and electron microscopy (EM).Results: We have generated two S328A mouse lines (Tg26 andTg33) and one HTRA1 mouse line Tg44. The protein level ofHTRA1-S328 is ~ 2-3 times that of HTRA1 in Tg44. The proteinlevel of HTRA1-S328 in Tg33 is similar to that of HTRA1 in Tg44.All transgenic proteins were expressed in mouse RPE. On ICGA,Tg44 mice exhibited characteristic features of PCV: 1) lategeographic hyperfluorescence and hyperfluorescent plaque; 2)polypoidal lesions (polyp and branching vascular network). SD-OCTdepicted RPE atrophy in lesion areas. Color fundus photographshowed hemorrhagic pigment epithelial detachment and scarformation at polypoidal lesions. Histology staining revealed serousexudation, thin-walled choroidal vessels, and regional loss ofchoriocapillaris. EM analysis revealed deteriorative changes in theelastic lamina of Bruch’s membrane, RPE, photoreceptors andchoroid vasculature. In contrast to Tg44 mice, neither Tg26 nor Tg33mice expressing HTRA1-S328A show any PCV phenotypes.Conclusions: Our results provide strong evidence that the proteolyticactivity of HTRA1 is responsible for its pathological role in PCV.The transgenic HTRA1 mice exhibited cardinal features of humanPCV, which will be invaluable for further studies on the mechanismsand treatment strategies of PCV.Commercial Relationships: Sandeep Kumar, None; Alex Jones,None; Zach Berriochoa, None; Shixian Wang Wang, None;Yingbin Fu, Methods of Diagnosing and Treating VascularAssociated Maculopathy and Symptoms Thereof (P)Support: Career Development Award from RPB; RPB departmentalunrestricted grant; Carl Marshall Reeves & Mildred Almen ReevesFoundation Equipment Grant; Moran Center for TranslationalMedicine Research GrantProgram Number: 4992 Poster Board Number: A0121Presentation Time: 2:45 PM - 4:30 PMAssociations between Posterior Vitreous Detachment andConcentrations of Various Cytokines in Eyes with Age-relatedMacular Degeneration and Normal Control EyesHidenori Takahashi 1 , Xue Tan 2 , Yoko Nomura 2 , Aya Iriyama 2 , YujiroFujino 3 , Yuko Okubo 1 , Aya Sato 1 , Mikiko Takezawa 1 , HidetoshiKawashima 1 , Yasuo Yanagi 2 . 1 Ophthalmology, Jichi MedicalUniversity, Shimotsuke-shi, Japan; 2 Ophthalmology, University ofTokyo, Tokyo, Japan; 3 Ophthalmology, Tokyo KoseiNenkinHospital, Tokyo, Japan.Purpose: We previously reported the prevalence of posterior vitreousdetachment (PVD) to be lower in eyes with age-related maculardegeneration (AMD) and that, eyes with PVD had a lowerconcentration of vascular endothelial growth factor (VEGF) in theaqueous humor. Herein, we investigated changes in theconcentrations of various cytokines in the aqueous humor and theircorrelation with PVD in eyes with AMD and normal control eyes.Methods: This is a prospective IRB-approved study. We studied 53eyes with exudative AMD initially treated with ranibizumab, and 79receiving cataract surgery without fundus lesions (control eyes) atTokyo Kosei Nenkin Hospital. Among the 5 eyes, 22 eyes were withtypical AMD and 31 were with polypoidal choroidal vasculopathy(PCV). Concentrations of the following cytokines were determinedusing a multiplex cytokine assay: IP-10, MCP-1, MMP-9, CXCL 12,IL-6, IL-10, CXCL 1, CXCL 13, and CCL 11. Explanatory variableswere age, sex, type of disease (typical AMD, PCV, and control),greatest linear diameter of the lesion (μm), disease duration (months),axial length of the eye (mm), PVD, and vitreomacular adhesion.Response variables were the concentrations of the various cytokines.For model selection in the general linear model, Akaike's InformationCriterion was used.Results: The cytokine level results were nearly log-normallydistributed. The correlation coefficient was very high (approximately0.9) for the levels of CXCL 13, IP-10, and MMP-9. Other correlationcoefficients did not exceed 0.5. PVD remained as an explanatoryvariable in a good fit model for IP-10 (p < 0.0001*), MCP-1 (p =0.0009*), MMP-9 (p = 0.0051*), CXCL 12 (p = 0.0182), IL-6 (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyimmunoprecipitation of protein complexes. Stable Isotope Labelingof Amino acids in Cell culture (SILAC) approach have been used toidentify binding partners of Htra1. Besides, the proteolytically activeHtra1 naturally expressed by a human ovarian adenocarcinoma cellline (SKOV3) was immunoprecipitated along with its interactingpartners and subjected for labeling (isotope-coded protein label,ICPL) and subsequent protein identification. Finally, this latter highthroughputquantitative proteome profiling approach was used tocompare the protein composition of retinas isolated from wild-typevs. Htra1 knock-out mice.Results: More than 25 proteins were identified as binding partnersfor Htra1, many of which are putative substrates or members of theserine protease inhibitor family (SERPIN). Importantly, using ARPE-19 cells, Htra1 was coprecipitated with components of thecomplement systems. Our data also revealed interactions betweenbasement membrane-specific heparan sulfate containing proteins andHtra1.Conclusions: These results suggest that the compromised regulationof Htra1 may contribute to AMD pathogenesis. The interaction withextracellular matrix proteins is of pivotal importance in spatiallyrestricting the activity of this enzyme. The targeted regulation ofHtra1 by using its specific inhibitors or activators might be animportant therapeutic strategy for AMD in the future.Commercial Relationships: Lili Feng, None; Elod Kortvely, None;Andreas Vogt, None; Karsten Boldt, None; Marius Ueffing, NoneProgram Number: 4994 Poster Board Number: A0123Presentation Time: 2:45 PM - 4:30 PMAmyloid Beta 1- 42 Promotes NLRP3-inflammasome ActivationIn Retinal Pigmented Epithelial CellsMatthew West 1 , Folami Lamoke 1 , AnnaLisa Montemari 2 , GiovanniParisi 3 , Guido Ripandelli 3 , Dennis M. Marcus 4 , Manuela Bartoli 1 .1 Department of Ophthalmology, Georgia Health Sciences University,Augusta, GA; 2 Department of Experimental Medicine and Pathology,University of Rome, Rome, Italy; 3 IRCCS Fondazione GB Bietti,Rome, Italy; 4 Southeast Retina Center, Augusta, GA.Purpose: Accumulation of amyloid beta peptides (A-beta) has beenshown to be a potential contributing factor for the development ofage-related macular degeneration (AMD). Recent work has shed lighton the role of the pro-inflammatory macromolecular complex of theNLRP3-inflammasome in the pathogenesis of geographical atrophy(GA). Our previous studies have shown that A-beta increases theexpression of the thioredoxin-interacting protein (TXNIP), aconstituent of the NLRP3-inflammasome complex. In this study wewanted to determine whether A-beta stimulation of retinal pigmentedepithelial cells (ARPE19) promoted the expression, activation andprotein-protein interaction of constituents of the NLRP3-inflammasome.Methods: Human retinal pigmented epithelial cells (ARPE19) werestimulated for 24 hours with 10μM of A-beta 1-42 or 10μM of thereverse peptide A-beta 42-1, which was used as control. Westernblotting and immunoprecipitation analyses were performed todetermine the expression and protein-protein interaction of theinflammasome constituent TLR4, NLRP3, ASC and TXNIP and todetermine the expression of A-beta receptor (RAGE). NLRP3-activation was determined by measuring caspase1 activity. RAGEinhibition was achieved by transfecting ARPE19 with specificsiRNA.Results: A-beta treatment of ARPE19 promoted the expression ofRAGE, TLR4, NLRP3 and TXNIP. Immunocytochemistry revealedthat A-beta stimulated NLRP3 interaction with ASC and TXNIP andthis effect was followed by increased caspase1 activity. Selectiveblockade of RAGE, by RNA silencing, blocked A-beta -inducedactivation of NLRP3-inflammasome in ARPE19, as assessed bymonitoring caspase 1 activity.Conclusions: The specific role of A-beta in the pathogenesis of wetAMD or GA is still controversial. Our data demonstrating that A-betacan promote RPE cells damage by inducing the NLRP3-inflammasome, further confirm a clear role of A-beta in the earlypathogenesis of AMD.Commercial Relationships: Matthew West, None; FolamiLamoke, None; AnnaLisa Montemari, None; Giovanni Parisi,None; Guido Ripandelli, None; Dennis M. Marcus, Genentech (C),Genentech (F), Regeneron (F), Regeneron (C), Thrombogenics (F),Thrombogenics (C), Allergan (F), Pfizer (F), Santen (F), Alimera (F),LPath (F), Acucela (F), Galaxo Smith Kline (F); Manuela Bartoli,NoneProgram Number: 4995 Poster Board Number: A0124Presentation Time: 2:45 PM - 4:30 PMConditional ablation of VEGFR-1 in photoreceptors inducesretinal angiomatous proliferation: A transgenic mouse model ofAMDLing Luo 1, 2 , Xiaohui Zhang 1 , Subrata K. Das 1 , Hironori Uehara 1 ,Tad Miya 1 , Thomas Olsen 1 , Bonnie Archer 1 , Yingbin Fu 1 , WolfgangBaehr 1 , Balamurali K. Ambati 1 . 1 Moran eye center, University ofUtah, Salt lake city, UT; 2 Department of Ophthalmology, The 306thhospital of PLA, China, Beijing, China.Purpose: To determine whether retinal angiomatous proliferation(RAP, a subtype of age related macular degeneration, or AMD) isdeveloped spontaneously without mechanical injury (e.g. subretinalinjection) by conditional ablation of VEGF receptor-1 (VEGFR-1, orFlt-1) in the murine photoreceptors.Methods: We interbred iCre-75+ mice (Cre recombinase expressedspecifically in phtotoreceptors) with floxed Flt-1 mice. Creexpression in above Cre lines was identified by interbreeding withmT/mG mice expressing tomato/EGFP in all tissues. At 21days to 3months after born, the fundi were observed in vivo by fluoresceinangiography (FA) and indocyanine green (ICG) angiography usingthe Heidelberg Retina Angiograph. Histology was performed byhematoxylin and eosin (H&E) stainining and Transmission electronmicroscope (TEM). Cre expression in the iCre-75+ lines wasidentified by interbreeding with mT/mG mice expressingtomato/EGFP in all tissues; The sFlt-1 expression was analyzed byimmunohisochemistry (IHC) and in situ hybridization.Results: Cre expression, as indicated by tomato deletion resulting inexclusive Egfp fluorescence, was restricted to photoreceptors infloxed mT/mG mice crossed with the iCre-75+ line. At 1-3 months ofage, both homozygous (iCre-75+ flt-1lox/lox, 8/14 eyes, 57%,P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyProgram Number: 4996 Poster Board Number: A0125Presentation Time: 2:45 PM - 4:30 PMIn-depth analysis of blood plasma proteome for discovery of agerelatedmacular degeneration biomarkersSe Joon Woo 1, 2 , Hye-Jung Kim 3 , Eui Jin Suh 3 , Ji Hyun Park 1, 2 ,Jeeyun Ahn 1, 2 , Duck Jin Hwang 1, 2 , Ji Eun Lee 3 , Kyu Hyung Park 1, 2 ,Cheolju Lee 3, 4 . 1 Ophthalmology, Seoul National University Collegeof Medicine, Seoul, Republic of Korea; 2 Ophthalmology, SeoulNational University Bundang Hospital, Seongnam, Republic ofKorea; 3 Theragnosis Research Center, Korea Institute of Science andTechnology, Seoul, Republic of Korea; 4 Biomolecular Science,University of Science and Technology, Daejeon, Republic of Korea.Purpose: To find plasma biomarkers for age-related maculardegeneration, we performed comprehensive proteomic analyses onblood plasma of AMD and age-matched healthy controlsMethods: We used a 4D protein profiling method on pooled plasmaof patient and control groups. (1) Abundant protein depletion coupledwith separation strategies - Gel-Eluted Liquid Fraction EntrapmentElectrophoresis (GELFrEE) for protein and (2) Isoelectrofocusing oftryptic peptides by OFFGEL (3) Liquid chromatography and tandemmass analsys (4) LC-MS/MS. Global functional analyses andcanonical pathway analyses were carried out using IngenuityPathway Analysis for proteins which were differentially expressed(more than 2- fold). Validation of candidate biomarkers wasperformed using ELISA and western blot on individual samples.Results: Using this profiling strategy, we identify 5042 uniquepeptides from 353 proteins, ranging over six orders of magnitude inabundance. Canonical pathways associated with AMD wereidentified for example, LXR/RXR activation, acute phase responsesignaling, complement cascades and actin cytoskeleton signaling. Todevelop potential biomarker proteins for AMD, we selected 7proteins for further verification.Conclusions: Our results implicate altered systemic pathways ofAMD and provide a new protein database for hypothesis-driven anddiscovery based studies of AMD.Commercial Relationships: Se Joon Woo, None; Hye-Jung Kim,None; Eui Jin Suh, None; Ji Hyun Park, None; Jeeyun Ahn, None;Duck Jin Hwang, None; Ji Eun Lee, None; Kyu Hyung Park,None; Cheolju Lee, NoneSupport: This study was partly supported by a grant from the KoreaHealth Technology R&D Project, Ministry of Health and Welfare,Republic of Korea (Grant No. A111161).Program Number: 4997 Poster Board Number: A0126Presentation Time: 2:45 PM - 4:30 PMPost-Transcriptional Gene Interactions in Retinal IschemiaKalina Andreeva, Maha Soliman, Nigel G. Cooper. AnatomicalSciences & Neurobiology, University of Louisville School ofMedicine, Louisville, KY.Purpose: Ischemia-Reperfusion (IR) injury has been implicated innumerous retinal disorders, the etiological expression of which can beassociated with various protein coding and micro RNA genes. Whilemuch progress has been made towards identification of genes andpathways associated with retinal disorders, the regulatorymechanisms for their coordinated expression are just beginning to beuncovered.Methods: Our laboratory has generated microarray mRNA andmiRNA data using a rat ischemic model for 3 post-ischemic timepoints (0h, 24h and 7d). We computed correlation coefficientsbetween miRNAs and their target genes in our datasets based onexpression values over the three post-ischemic time points. We thangrouped the genes and miRNAs based on their expressioncorrelations.Results: The graphic representations of changes in expression ofgenes and miRNAs showed inverse relationships, such that whengenes were elevated in expression miRNAs showed reducedexpression and vice versa. These findings support the hypothesis thatthe changes in expression of protein coding genes are in part afunction of changes in expression of their corresponding miRNAs.Conclusions: Based on preliminary data, we propose that regulatedgene expression may control different phases of the disorder.Commercial Relationships: Kalina Andreeva, None; MahaSoliman, None; Nigel G. Cooper, NoneSupport: Supported by EY017594Program Number: 4998 Poster Board Number: A0127Presentation Time: 2:45 PM - 4:30 PMAn In-Vitro Study of Wnt Pathway Modulation in Cybrid H andJ Mitochondrial Haplogroups and the Possible Implications forAge-related Macular DegenerationPayam Falatoonzadeh 1 , Grace B. Woo 1 , Nitin Udar 1 , Shari Atilano 1 ,Marilyn Chwa 1 , Miceli V. Michael 2 , S Michal Jazwinski 2 , Cristina M.Kenney 1 . 1 Ophthalmology, Gavin Herbert Eye Inst., UC Irvine,Irvine, CA; 2 Tulane Center for Aging, Tulane University, NewOrleans, LA.Purpose: The wet form of age-related macular degeneration (AMD)can be associated with increased VEGF, vascular cell proliferation,and induced choroidal neovascularization. Mitochondrial DNA(mtDNA) haplogroups are defined by accumulation of SNPs thatrepresent different geographic origins of populations. Individualswith haplogroup H mtDNA are lower risk for AMD, whilehaplogroup J individuals have higher predisposition. The Wntpathway and β-Catenin, one of its downstream proteins, arecommonly associated with cell proliferation, and studies havefocused on its role in angiogenesis related to cancer and possiblyAMD. Our present study uses a mitochondrial haplogroupcytoplasmic hybrid (cybrid) model to assess the effects of mtDNAvariations on the Wnt pathway.Methods: Mitochondria-deficient (Rho0) human ARPE-19 cellswere fused with platelets from individuals with mtDNA haplogroupsH (n=3) or J (n=3) to establish cybrid cell lines. RNA was extractedfrom each cybrid and cDNA synthesized. Q-PCR was performedusing primers for various genes associated with the Wnt pathway andanalyzed for changes in gene expression levels of J cybrids relative toH cybrids.Results: Gene expression levels were significantly decreased in Jcybrids relative to H cybrids for inhibitors of the Wnt pathway,including SFRP1 (0.29 fold, p=0.0001), DKK3 (0.46 fold, p=0.004),and RARA1 (0.57 fold, p=0.007). There was also a relative increasein expression of GSK3 (1.6 fold, p=0.005). Expression levels of othermembers of the Wnt pathway, including CSNK1E, WNT9A, LRP1,KREMEN1, MDM2, and TGFβ were not found to be significantlydifferent in the J versus the H cybrids.Conclusions: SFRP1 and DKK3 inhibit the binding and interactionof the Wnt ligand with cell surface receptors. Decreased expressionof these genes in J cybrids could potentially lead to more Wnt ligandbinding and increased signal transduction within the cell. This wouldinclude recruitment of increased GSK3A away from the β-catenindegradation complex. Also, decreased RARA1 levels, a nuclearinhibitor of β-catenin, would allow for more β-catenin driventranscription. These observations correlate with faster cybrid Jgrowth patterns in culture, and may also have implications for howdifferent haplogroups might modulate the Wnt pathway and possiblyinfluence AMD.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyCommercial Relationships: Payam Falatoonzadeh, None; GraceB. Woo, None; Nitin Udar, Illumina (E); Shari Atilano, None;Marilyn Chwa, None; Miceli V. Michael, None; S MichalJazwinski, None; Cristina M. Kenney, NoneSupport: Supported by Discovery Eye Foundation, GuentherFoundation, Beckman Macular Research Initiative, Polly andMichael Smith foundation, Max Factor Family Foundation, SkirballFoudnation, Lincy Foundation, Iris and B. Gerald Cantor Foundation,Unrestricted grant from Research to Prevent Blindness, grant fromNational Institute on Aging (AG006168)Program Number: 4999 Poster Board Number: A0128Presentation Time: 2:45 PM - 4:30 PMExpression of VEGF-C, VEGF-D and their Cognate Receptors inExperimental Choroidal Neovascularization and Clinical AMDKameran Lashkari 1 , Jie Ma 1 , Gianna Teague 1 , Jorge G. Arroyo 2 .1 Schepens Eye Research Institute/ Massachusetts Eye & Ear, HarvardMed School, Boston, MA; 2 Beth Israel Deaconess Medical Center,Harvard Med School, Boston, MA.Purpose: VEGF-C has been implicated in tumor-associated andcorneal angiogenesis through its effects on lymphangiogenesis andactivation of VEGFR3. However, recent evidence implicates VEGF-C and D in pathological angiogenesis in absence of lymphaticinvolvement, possibly through activation of VEGFR2. The goal ofthis study is to define the role of VEGF-C and its cognate receptors inretinal angiogenesis including neovascular AMD and relateddisorder.Methods: The levels of VEGF-C and D, and soluble VEGFreceptors, R1, R2 and R3 were measured in vitreous samples ofsubjects with various vitreoretinal conditions using combination ofLuminex and Elisa assays. The mouse model of laser-inducedchoroidal neovascularization (CNV) was used to detect theexpression of VEGF-C, D and receptors, R2 and R3 using imagingand immunohistochemistry. Expression of these factors was alsoexamined in donor eyes with various stages of AMD.Results: Both VEGF-C and D and soluble (s) R1, R2 and R3 weredetectable in the human vitreous. sR3 levels averaged 200 pg/ml incontrol subjects, and one log order higher than sR2 levels. Therespective concentrations of VEGF-C and D positively correlatedwith age and were affected by retinal diseases including AMD.VEGF-C and its cognate receptors were detected within cellularcomponents of laser-induced CNV membranes. A similar expressionpattern was seen in clinical specimens of AMD.Conclusions: Based on these examinations, VEGF-C, D andsVEGFRs are detectable in the human vitreous, positively associatedwith age, and affected by disease states such as AMD. VEGF-C andD were detected within experimental and clinical CNV membranes.VEGF-C and D may participate in pathological angiogenic processessuch as neovascular AMD in absence of any observedlymphangiogenesis.Commercial Relationships: Kameran Lashkari, CircadianTechnologies (F), Regeneron (R); Jie Ma, None; Gianna Teague,None; Jorge G. Arroyo, NoneSupport: Circadian TechnologiesProgram Number: 5000 Poster Board Number: A0129Presentation Time: 2:45 PM - 4:30 PMMitochondrial DNA Damage Increases with Age-related MacularDegeneration in the RPE but not Neural RetinaMARCIA R. TERLUK 1 , Lauren M. Soukup 2 , Sandra R. Montezuma 1 ,Deborah A. Ferrington 1 . 1 Ophthalmology and Visual Neurosciences,University of Minnesota, Minneapolis, MN; 2 Biology, University ofSt. Thomas, St. Paul, MN.Purpose: Reports of increased mitochondrial DNA (mtDNA)damage in the retinal pigment epithelium (RPE) from human donorswith age-related macular degeneration (AMD) provide compellingevidence supporting the hypothesis that mitochondrial dysfunctionplays a prominent role in AMD pathology (Karunadharma et al.,2010; Lin et al., 2011). However, it is still not clear whether thisdamage is limited to the RPE or if it also involves the neural retina.The purpose of this study was to determine if (1) mtDNA damageincreases in the neural retina with AMD, and (2) the extent ofmtDNA damage in the RPE correlates with the extent of damage inthe neural retina in individual donors.Methods: Human donor eyes were obtained from the MinnesotaLions Eye Bank, Minneapolis, MN. Genomic DNA was isolated froma 5 mm trephine punch of the macular region of the RPE and neuralretina from human donor eyes categorized into four progressivestages of AMD (MGS 1-4, Olsen and Feng, 2003). MitochondrialDNA damage was assessed in 32 donors (n=8/stage) using the longextension polymerase chain reaction. In this assay, decreasedamplification reflects increased DNA damage. The amplified productwas quantified using Pico Green fluorescent dye. Statistical analysisincluded one way ANOVA and Tukey’s post-hoc tests to determine ifthere was a significant increase in mtDNA damage with AMDprogression. Pearson Product-Moment Correlation analysis wasperformed comparing the extent of damage in the RPE and retina foreach donor to determine if there was an association between damagein these two ocular regions.Results: There was no significant change in mtDNA damage in themacula of the neural retina when comparing age-matched controlsand donors with AMD (p=0.29). Furthermore, comparison of mtDNAdamage in the RPE and retina from individual donors showed nocorrelation (Pearson’s r=0.03; p>0.10) between the extent of mtDNAdamage in these two macular tissues.Conclusions: Collectively our data indicates that mtDNA damagethat increases with AMD is limited to the RPE and not the retina.These results suggest that targeting treatments that protect themitochondria in the RPE may be the most efficacious therapeuticstrategy for AMD.Commercial Relationships: MARCIA R. TERLUK, None;Lauren M. Soukup, None; Sandra R. Montezuma, None; DeborahA. Ferrington, NoneSupport: Unrestricted grant to the Department of OVNS fromResearch to Prevent Blindness; Beckman Initiative for MacularResearch, Minnesota Lions Club.Program Number: 5001 Poster Board Number: A0130Presentation Time: 2:45 PM - 4:30 PMOxidative Stress-Induced Necrosis in RPE CellsJakub Hanus 1 , William C. Anderson 1 , Peng Jin 3 , Qinghua Liu 4 ,Shusheng Wang 1, 2 . 1 Cell and Molecular Biology, Tulane University,New Orleans, LA; 2 Ophthalmology, Tulane University, New Orleans,LA; 3 Human Genetics, Emory University, Atlanta, GA;4 Biochemistry, University of Texas Southwestern Medical Center,Dallas, TX.Purpose: Atrophy of retinal pigment epithelial (RPE) cells andphotoreceptors in the macula is characteristic of geographic atrophy(GA), an advanced state of dry age-related macular degeneration(AMD). RPE cells are subjected to chronic oxidative stress.Currently, the mechanism of oxidative stress-induced RPE cell deathis controversial, with most pointing to a dominant role for apoptosis.However, apoptosis does not induce inflammatory response, which isnot consistent with chronic inflammation observed in dry AMD. Weset out to clarify the mechanism of oxidative stress-induced RPE celldeath using cultured RPE cells. We test the hypothesis that necrosis is©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologya major mechanism for oxidative stress-induced RPE cell death.Moreover, we have screened a collection of 2,000 FDA-approvedcompounds for molecules that inhibit oxidative stress-induced RPEnecrosis.Methods: ARPE-19 cells were treated with different concentration ofH2O2 and harvested at different time points followed by cell deathmarker staining, as well as RNA, DNA and protein analyses.Compound screening was performed using MTT assay.Results: Morphology and molecular hallmarks of dying ARPE-19cells show strong evidence of necrotic cell death induced byoxidative stress. We observed partial chromosomal condensation(pyknosis) and permeability of cell membrane by propidium iodine(PI). ARPE-19 cells treated with H2O2 did not exhibit apoptoticDNA fragmentation, consistent with our finding that ARPE-19 cellshave very low level of DFF45 protein, inhibitory subunit of DFF40main apoptotic endonuclease. RPE cell death is accompanied byincreased levels of PGAM5 and RIP3 both critical for undergoingprogrammed necrosis. Consistently, necrostatin-1, but not caspaseinhibitor zVAD, rescued oxidative stress-induced RPE cell death.Additionally our results showed that autophagic cell death is not aresult of H2O2 treatment in RPE cells. Furthermore, we screenedFDA-approved compounds and identified candidates thatsuccessfully rescued RPE cells from oxidative stress-induced celldeath.Conclusions: We provide unequivocal evidence that necrosis is amajor mechanism for oxidative stress-induced RPE cell death. Wealso identified natural compounds that can rescue oxidative stressinducedRPE cell death. Taken together, these findings provide novelinsight into the mechanism of RPE cell death, and will beinstrumental for developing novel therapeutics for dry AMD,especially GA.Commercial Relationships: Jakub Hanus, None; William C.Anderson, None; Peng Jin, None; Qinghua Liu, None; ShushengWang, UT Southwestern Medical Center (P)Support: Startup fund of Tulane University (S.W.), President’sResearch Council New Investigator Award from UT SouthwesternMedical Center (S.W.), NIH Grants EY021862 (S.W.), CareerDevelopment Award from the Research to Prevent BlindnessFoundation (S.W.).Program Number: 5002 Poster Board Number: A0131Presentation Time: 2:45 PM - 4:30 PMPhosphorylation Networks in Age-Related Macular DegenerationSrinivas R. Sripathi 1 , O'Donnell Sylvester 2 , Trevor Moser 1 , Paul S.Bernstein 3 , Folami Lamoke 4 , Manuela Bartoli 4 , Wan Jin Jahng 2 .1 Biological Sciences, Michigan Tech University, Houghton, MI;2 Retina Proteomics Laboratory, Petroleum Chemistry, AmericanUniversity of Nigeria, Yola, Nigeria; 3 Ophthalmology and VisualSciences, Moran Eye Center, University of Utah, Salt Lake City, UT;4 Ophthalmology, Georgia Health Sciences University, Augusta, GA.Purpose: Age-related macular degeneration (AMD) results from thechronic atrophy of retina and retinal pigment epithelial (RPE) cells(dry AMD) or choroidal neovascularization (wet AMD). However,the molecular mechanism underlying the progression of AMD is notwell defined yet. Light-induced oxidative stress, lipid oxidation,accumulation of soft drusen, and crystalline aggregation maycontribute to AMD pathogenesis. The aim of our phosphoproteomestudy is to identify the early biochemical events and specificphosphorylated sites in the macular, peripheral retina and RPE. Ourdata demonstrated that specific kinase/phosphatase regulatedapoptotic phosphorylation signaling in AMD progression.Methods: The senescence-associated molecular events during AMDprogression were identified by comparing the phosphoproteome ofmacular and peripheral retina, RPE from different stages of AMDcompared to age-matching control donor eyes. Phosphoproteins wereenriched by charge-based spin column chromatography and resolvedby 2D gel electrophoresis. Tryptic peptides were enriched usingGa3+/TiO2 immobilized metal ion chromatography. Elutedphosphopeptides were analyzed and confirmed by MALDI-TOF orESI/MS/MS mass spectrometry and phospho-Western blotrespectively.Results: Our preliminary data shows tyrosine phosphorylation ofbeta crystallin A3 and A4 increased but serine phosphoryaltion ofbeta crystallin decreased in RPE under stress. Our phospho-Westernanalysis reveals phosphorylation of intermediate filament vimentin(Ser) and mitochondrial heat shock protein mt Hsp70.Serine/threonine phosphatase PP2A-Cα/β (Tyr) and tubulin α1B/β2were dephosphorylated in the RPE. Further, our data shows thatprohibitin is upregulated in the retina, but down regulated in the RPEfrom AMD donor eyes. Our study suggests that altered lipidcomposition may determine phosphorylation-dependent interactionsof target proteins that contain PH, PX, and SH domains.Conclusions: The region specific proteome changes incentral/peripheral retina and RPE from AMD donor eyes and agematchingcontrols were analyzed. Phosphorylation changes anddownstream activation may leads to the early biochemical events inAMD. Our phosphoproetomic strategy provides the detailedphosphorylation signaling,phospholipid changes, altered proteinaggregation,binding affinity and expression that include depletedlevels of anti-apoptotic, mitochondrial chaperone prohibitin in theRPE of AMD eyes.Commercial Relationships: Srinivas R. Sripathi, None; O'DonnellSylvester, None; Trevor Moser, None; Paul S. Bernstein, Kalsec(C), Kemin Health (R), Science Based Health (C), Abbott Nutrition(F), Genentech (C), DSM (R), Sequenom (R), NuSkin/Pharmanex(P), Aciont (C), Thrombogenics (C); Folami Lamoke, None;Manuela Bartoli, None; Wan Jin Jahng, NoneProgram Number: 5003 Poster Board Number: A0132Presentation Time: 2:45 PM - 4:30 PMEvaluation of RPE cell senescence as a mechanism for agerelatedmacular degeneration (AMD),Michael R. Kozlowski. Arizona College of Optometry, MidwesternUniversity, Glendale, AZ.Purpose: The purpose of this work is to assess whether or not thechanges seen in retinal pigment epithelial (RPE) cells as theyapproach cell senescence are consistent with the pathologicalprocesses that occur in age-related macular degeneration (AMD).Methods: In this study, ARPE-19 cells grown on membranecontainingmulti-well plate inserts or in cell culture flasks were usedto model the natural RPE cell layer. The cells were cultured to highpopulation doubling levels (PDL’s) and several measures of cellgrowth and function were assessed including doubling rate, viability,morphology, terminal restriction fragment (TRF) length (as ameasure of telomere erosion), trans-epithelial electrical resistance(TEER, as a measure of the quality of their tight junctions), andsenescence associated beta-galactosidase activity (SABG).Results: As ARPE-19 cells reached high population doubling levels(PDL), which is one trigger for cell senescence, they experiencedsignificant telomere erosion and displayed several senescenceassociatedchanges. First, their staining for SABG, a marker forsenescence which is related to lysosomal dysfunction, was elevated.Second, their shape became more rounded, possibly indicating achange in their adhesion characteristics. Finally, the depression oftheir TEER values by VEGF was significantly enhanced.Conclusions: The changes seen in ARPE-19 cells that had reached©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyhigh PDL’s are consistent with pathological processes that occur inAMD. For example, a change in RPE cell attachment characteristics,such as that seen in the present study, could lead to a compromise inthe transport of materials across Bruch’s membrane and, thereby, tothe development of drusen. Likewise, a change in lysosomalfunctioning, as was indicated in this study by the increased SABGstaining, could cause a buildup of improperly process outer segmentmaterials, which would also contribute to the development of drusen.In addition, increased responsiveness to VEGF in terms of looseningof tight junctions, as was indicated by the present TEER results,could be permissive to the development choroidal neovascularizationin wet AMD. These finding demonstrate that RPE cell senescencecould contribute to the occurrence of AMD.Commercial Relationships: Michael R. Kozlowski, NoneSupport: Intramural Research Stimulation GrantProgram Number: 5004 Poster Board Number: A0133Presentation Time: 2:45 PM - 4:30 PMAn In-Vitro Study of the Effect of Ultraviolet Radiation onGrowth Patterns and Gene Expression of Human ARPE-19Cybrid H and J, and their Implications with Age-related MacularDegeneration (AMD)Deepika Malik 1 , Payam Falatoonzadeh 1 , Tiffany Hsu 1, 2 , Claudio A.Ramirez 1 , Javier Cáceres del Carpio 1 , Mohamed Tarek MohamedMoustafa 1 , S Michal Jazwinski 3 , Miceli V. Michael 3 , Cristina M.Kenney 1 , Baruch D. Kuppermann 1 . 1 Ophthalmology, Gavin HerbertEye Institute, Irvine, CA; 2 Warren Alpert Medical School, BrownUniversity, Providence, RI; 3 Tulane Center for Aging, TulaneUniversity, New Orleans, LA.Purpose: Studies have shown that mitochondrial DNA (mtDNA)plays an important role in the aging process. Various haplogroups ofmtDNA have been classified based on variable combinations ofsingle nucleotide polymorphisms (SNPs). Cybrids (cytoplasmichybrids) have identical nuclei but mitochondria of differenthaplogroups. Although both haplogroups H and J representEuropean-Caucasian populations, individuals with haplogroup J havea higher susceptibility to develop AMD and H is protective. The aimof this study was to evaluate the effect of UV radiation (a knownenvironmental risk factor for AMD) on cell growth patterns and geneexpression in various cellular pathways in H and J cybrids.Methods: ARPE-19 cells were depleted of mitochondria (Rho0) andfused with platelets isolated from haplogroup H or J subjects to createindividual cybrids. Cybrids were treated with 10mJ of UV radiation.Growth patterns of cybrids H and J were studied over 14 days in bothUV-treated and untreated groups. RNA extraction and cDNAsynthesis were done at 0, 72 and 120 hours, after exposure. Variouspro-apoptotic genes RARA (Retinoic Acid Receptor Alpha), BBC3(BLC2 Binding Protein) and BLC2L13 (BCL2 like 13) (both BBC3and BCL2L13 induce mitochondrial outer membranepermeabilization and caspase activation); pro-inflammatory geneIL33 (Interleukin-33); and angiogenic gene TGF-A (TransformingGrowth Factor-Alpha) were studied using Q-PCR.Results: A stepladder growth pattern was seen in UV-treated groups,which was significantly different than control groups. J cybridsshowed more growth than H cybrids at all studied time points. At 120hours, both UV-treated cybrids H and J showed increase in geneexpression levels for pro-apoptotic genes RARA, BBC3 andBCL2L13, but cybrid H showed a greater increase. UV-treated cybridJ showed higher expression of pro-inflammatory gene IL-33 andangiogenic gene TGF-A as compared to UV-treated cybrid H.Table 1: Fold differences relative to untreated cybrid H (0 hours)Conclusions: Our study shows that SNP variations in mtDNA mayalter nuclear gene expression and cellular responses to stress, andcontribute to differential genetic risk factors for AMD.Fold differences relative to Untreated Cybrid H (0 Hours)Commercial Relationships: Deepika Malik, None; PayamFalatoonzadeh, None; Tiffany Hsu, None; Claudio A. Ramirez,None; Javier Cáceres del Carpio, None; Mohamed TarekMohamed Moustafa, None; S Michal Jazwinski, None; Miceli V.Michael, None; Cristina M. Kenney, None; Baruch D.Kuppermann, Alimera (C), Allegro (C), Allergan (C), Genentech(C), Glaukos (C), GSK (F), Novagali (C), Novartis (C), Ophthotech(C), Pfizer (C), Regeneron (C), Santen (C), SecondSight (C), Teva(C), ThromboGenics (C)Support: Discovery Eye Foundation, Guenther Foundation,Beckman Macular Research Initiative, Polly and Michael SmithFoundation, Max Factor Family Foundation, Skirball Foundation,Lincy Foundation, Iris and B. Gerald Cantor Foundation,Unrestricted grant from Research to Prevent Blindness, grant fromNational Institute on Aging (AG006168)Program Number: 5005 Poster Board Number: A0134Presentation Time: 2:45 PM - 4:30 PMCybrids with Different mtDNA Haplogroups Show DifferentialExpression of Respiratory Complex GenesCristina M. Kenney 1 , Marilyn Chwa 1 , Shari Atilano 1 , PayamFalatoonzadeh 1 , Deepika Malik 1 , Claudio A. Ramirez 1 , S MichalJazwinski 2 , Miceli V. Michael 2 , Baruch D. Kuppermann 1 .1 Ophthalmology, Gavin Herbert Eye Inst, UC Irvine, Irvine, CA;2 Tulane Center for Aging, Tulane University, New Orleans, LA.Purpose: Mitochondrial DNA (mtDNA) haplogroups representdifferent human populations and are defined by accumulation ofspecific SNPs. The J haplogroup is high risk for age-related maculardegeneration (AMD) while the H haplogroup is protective for AMD.Cybrids (cytoplasmic hybrids) are an excellent model to studymitochondrial functions because the cells have identical nuclei andvary only in the mtDNA content. This study was designed to analyzethe oxygen consumption rates (OCR representing OXPHOS) andextracellular acidification rates (ECAR representing glycolysis) andthe expression of mtDNA encoded genes that are associated with theelectron transport chain complexes I, III, IV, and V.Methods: Cybrids were created by fusing Rho0 ARPE-19 cells(depleted of mitochondria) with platelets isolated from haplogroup Hor J subjects (n=3 each). The Seahorse FX24 extracellular fluxanalyzer was used to measure OCR and ECAR in H vs J cybrids.These measurements allow for evaluation of cellular metabolicactivity, mitochondrial function, and energy expenditure. Q-PCR wasperformed on H cybrids vs J cybrids using primers for the mtDNAgenes of complexes I, III, IV, and V. mtDNA copy numbers weremeasured by comparing nDNA:mtDNA ratios (18S:MT-ND2).Results: The J cybrids had a significantly lower OCR/ECAR ratiothan the H cybrids (14±2.7 vs 24 ±2.5, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologynumbers were similar to each other in the H vs J cybrids(1.34±0.0228 vs 1.36±0.017, p=0.59).Conclusions: The OCR/ECAR ratio confirms that the H cybrids haveincreased OXPHOS while the J cybrids use glycolysis. This findingis supported by lower expression levels of the mtDNA encoded genesin complex I (MT-ND1, MT-ND2, MT-ND3, MT-ND4/ND4L), IV(MT-CO2, MT-CO3) and V (MT-ATP6). Our findings show thatmtDNA variants can mediate energy production pathways andmtRNA expression which in turn may alter the signaling pathwaysand stress-response patterns of the cells.Commercial Relationships: Cristina M. Kenney, None; MarilynChwa, None; Shari Atilano, None; Payam Falatoonzadeh, None;Deepika Malik, None; Claudio A. Ramirez, None; S MichalJazwinski, None; Miceli V. Michael, None; Baruch D.Kuppermann, Alimera (C), Allegro (C), Allergan (C), Genentech(C), Glaukos (C), GSK (F), Novagali (C), Novartis (C), Ophthotech(C), Pfizer (C), Regeneron (C), Santen (C), SecondSight (C), Teva(C), ThromboGenics (C)Support: Supported by Discovery Eye Foundation, GuentherFoundation, Beckman Macular Research Initiative, Polly andMichael Smith Foundation, Max Factor Family Foundation, SkirballFoundation, Lincy Foundation, Iris and B. Gerald Cantor Foundation,Unrestricted grant from Research to Prevent Blindness, grant fromNational Institute on AgingProgram Number: 5006 Poster Board Number: A0135Presentation Time: 2:45 PM - 4:30 PMThe serine protease HTRA1 is a potential regulator of theinflammatory cytokine GDF15Chloe M. Stanton 1 , Elod Kortvely 2 , Caroline Hayward 1 , MariusUeffing 2, 3 , Alan F. Wright 1 . 1 MRC Human Genetics Unit, MRCIGMM, University of Edinburgh, Edinburgh, United Kingdom;2 Centre for Ophthalmology, Institute for Opthalmic ResearchTubingen, Tubingen, Germany; 3 Research Unit of Protein Science,Helmholtz Zentrum Munchen, Munich, Germany.Purpose: An AMD risk locus on chr10q26 spans two genes -ARMS2 and HTRA1 - and controversy exists as to whether one orboth genes contribute to AMD. This study was performed toinvestigate the protein interaction network of the serine proteaseHTRA1 (high-temperature requirement A-1) and to elucidate apossible role in AMD pathogenesis.Methods: Potential interacting partners of HTRA1 were identified ina yeast two-hybrid screen performed using a placental cDNA library.Protein interactions were subsequently verified using recombinantproteins in pull-down assays. Proteins found to interact directly withHTRA1 were subjected to protease activity assays to identifysubstrates of the protease. Plasma GDF15 was measured by ELISA,and gene expression determined by RT-PCR.Results: Growth differentiation factor 15 (GDF15), a divergentmember of the TGFβ superfamily, with a proposed role in mediatinginflammation, interacts directly with HTRA1 in vitro. ProGDF15 is asubstrate for the HTRA1 protease in vitro. GDF15 and HTRA1 areexpressed at very low levels by monocyte-like cells, and theirexpression is highly upregulated in activated macrophages. PlasmaGDF15 is significantly elevated in AMD cases carrying thechromosome 10 risk haplotype, relative to disease-free controls, or toAMD cases not carrying the chromosome 10 risk haplotype.Conclusions: ProGDF15 is a substrate for the HTRA1 protease invitro, suggesting a regulatory role for the protease on thisinflammatory cytokine. Given the previously described role ofGDF15, the substantial genetic risk for AMD associated with thechromosome 10 risk haplotype, and the involvement of macrophagesin AMD, GDF15 may contribute towards pathological inflammationobserved in the disease. Further work to understand the geneticcontribution to regulating GDF15 expression in the generalpopulation, and in disease, and to determine the biologicalsignificance of the interaction between HTRA1 and GDF15 is ongoing.Commercial Relationships: Chloe M. Stanton, None; ElodKortvely, None; Caroline Hayward, None; Marius Ueffing, None;Alan F. Wright, NoneSupport: UK Medical Research CouncilProgram Number: 5007 Poster Board Number: A0136Presentation Time: 2:45 PM - 4:30 PMRole of complement in altered RPE function and depositformation in Efemp1 mutant mice: A primary cell culture modelRosario Fernandez-Godino, Eric A. Pierce, Donita Garland.Ophthalmology, Ocular Genomics Institute. Mass Eye and EarInfirmary. Harvard Medical School, Boston, MA.Purpose: The R345W mutation in the Efemp1 gene causes theinherited macular degeneration Doyne Honeycomb RetinalDystrophy/ Malattia Leventinese (DHRD/ML). Gene targetedEfemp1-R345W mice develop sub-RPE basal deposits and vacuolesin the RPE. In previous studies using Efemp1-R345W : complementC3 null mice we found that the complement system had a critical rolein the formation of basal laminar deposits in the mutant mice. Thepurpose of this study was to use a primary RPE cell culture system toinvestigate the mechanisms involved in the pathogenesis of basaldeposit formation in the Efemp1-R345W mutant mice and the role ofthe complement system in this process.Methods: Primary RPE cells were cultured from Efemp1-R345Wknockin, C3 null, and homozygous Efemp1-R345W:C3 null mice.The RPE cells, from 2.5 months old mice, were grown on apermeable support under polarizing conditions. mRNA expressionlevels of complement genes and Efemp1 were quantified by qRT-PCR. Protein expression in the RPE was determined by Western blotanalysis and immunofluorescence using confocal microscopy.Proteins secreted from both the apical and basal surfaces werequantified by Western blot analyses.Results: Results of qRT-PCR analysis reproducibly showed that RPEcells from each of the mouse strains express complement C3 mRNAand protein. The C3 null mice used for these studies express aninactive form of C3. Moreover, the major complement components ofthe classical pathway, C1q, C2, C4 as well as Cfh of the alternativepathway were also expressed in the RPE of each mouse strain. Theexpression of Efemp1 mRNA was significantly higher in Efemp1-R345W knockin mice compared to wild type (Wt)(ANOVA,p=0.0005). However, this overexpression was repressed inthe Efemp1-R345W:C3 null mice (p=0.0005).Conclusions: The primary RPE cell culture provides an excellentapproach to investigate the early effects of the Efemp1 mutation onRPE protein expression and also on the effects of alternations in thecomplement system on RPE function. The results suggested a criticalrole of C3 in the pathogenesis of the R345W mutation in DHRD/ML.They also suggest that the mutant EFEMP1 protein might cause localcomplement activation in the RPE, which in turn leads to RPE celldysfunction and an altered pattern of proteins secreted to Bruch’smembrane.Commercial Relationships: Rosario Fernandez-Godino, None;Eric A. Pierce, None; Donita Garland, NoneSupport: Ocular Genomics InstituteProgram Number: 5008 Poster Board Number: A0137Presentation Time: 2:45 PM - 4:30 PM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyQuantifying progression of disease in aging mouse and humanRPEJohn M. Nickerson 1 , Xin Qi 2 , Tingting Jiang 3 , Yuwei Cheng 3 , Jing M.Zhang 3 , Micah A. Chrenek 1 , Alia Rashid 1 , Shagun K. Arora 1 , Hans E.Grossniklaus 1 , Yi Jiang 2 . 1 Ophthalmology, Emory Univ, Atlanta, GA;2 Mathematics, Georgia State University, Atlanta, GA; 3 Statistics,Yale University, New Haven, CT.Purpose: RPE sheet patterns change with age and progression indiseased RPE. We developed quantitative predictors of age andgenotype from the RPE sheet, and built a model of how the RPEsheet changes in disease. Using functional principal componentanalysis (FPCA) and multidimensional scaling (MDS), we classifiedRPE sheets into major categories of genotypes and age groups.Methods: Over 130 mice of many different ages and genotypes wereused, including WT C57BL/6J and mutant rd10, rpe65 KO, rd12,IRBP KO, and tvrm148, which were congenic with C57BL/6J. Over16 human cadaveric eyes were tested. ~10,000 cells per eye wereanalyzed. Borders of RPE cells were identified with anti-ZO-1 orphalloidin. Thresholding was by Ridler-Calvard adaptive method.RPE segmentation and analysis with 23 metrics were done withCellProfiler. Four classification methods were used after FPCA andMDS.Results: We compared the morphology of RPE cells from eyesacross various mouse strains and normal human and AMD eyes ofdifferent ages. Disrupted RPE had distinctive characteristics withgreat variability in size and shape of cells. In old RPE, there weregaps, atrophy, and interruptions in the sheet with significant showthroughof the choroid. Joint aspect ratio and cell area providedprincipal components predictive of age (< or > 70 days) and genotype(WT or rd10) in mice with high accuracy (>98%). With furtherdivision of mice into four age groups and spatial locations, PCA andMDS showed that no single metric was significant as a signature. Butspatial information revealed regional changes. At >P180 days, therewas extensive deformation and a subpopulation of large cells. Amathematical model of RPE cell death recapitulated changes in vivoin AMD: Normal RPE progressed to abnormal only when multiplelocal clusters of RPE cells were killed. The death of single cells orsparse clusters of RPE cells were handled effectively by stretchingthe sheet. Preliminary analysis on human normal RPE showed noage-related trends or changes in morphology.Conclusions: Morphometrics readily discriminates young vs old,normal vs mutant RPE sheets. Classification accuracy was high anddepended on metric and location of the RPE cell. Tissue-stretchingrobustly maintains layer integrity. However, death of multiple localclusters of RPE cells is overwhelming and results in highly variableappearance and eventually frank holes.Commercial Relationships: John M. Nickerson, None; Xin Qi,None; Tingting Jiang, None; Yuwei Cheng, None; Jing M. Zhang,None; Micah A. Chrenek, None; Alia Rashid, None; Shagun K.Arora, None; Hans E. Grossniklaus, None; Yi Jiang, NoneSupport: RPB; NIH (R01EY016470, P30EY06360, TL1TR000456,UL1TR000454)Program Number: 5009 Poster Board Number: A0138Presentation Time: 2:45 PM - 4:30 PMZinc Staining of sub-RPE Deposits in Murine Models of RetinalDegenerationRebecca J. Kapphahn 1 , Heidi Roehrich 2 , Deborah A. Ferrington 1 ,Frederik J. van Kuijk 1 . 1 Ophthalmology, University of Minnesota,Minneapolis, MN; 2 Histology Core for Vision Research, Universtiyof Minnesota, Minneapolis, MN.Purpose: One of the hallmarks of age-related macular degeneration(AMD) is the accumulation of sub-retinal pigment epithelial (RPE)deposits, including drusen and basal laminar deposits, in Bruch’smembrane. We have previously shown that these sub-RPE depositscontain high concentrations of zinc in human donors with AMD andthat these zinc-enriched deposits were especially prevalent in themacula (Lengyel et al, 2007). Since sub-RPE deposits may contributeto AMD pathology, zinc-containing sub-RPE deposits may be animportant characteristic for animal models used to investigate AMDmechanisms or test therapeutic interventions. The purpose of thisstudy was to determine if zinc-enriched sub-RPE deposits are presentin animal models of retinal degeneration.Methods: The presence of zinc-enriched sub-RPE deposits wasassessed in multiple murine models that exhibit retinal degeneration(sod1-/-, mice carrying the rd8 mutation, old C57BL6). In order tovisualize sub-RPE zinc deposits, the anterior segment was removedfrom enucleated globes. The retina and RPE were then removedusing zinc-free PBS and the unfixed eye cup was flat mounted on aglass slide without a cover slip. A Leica DM4000B microscope wasused to visualize the autofluorescence associated with the optic nerve.Exposure time was then decreased to determine the exposureparameters where autoflourescence was no longer visible. Theseparameters were used to capture all subsequent images. Membranepermeable fluorescent zinc stain, Zinpyr-1 (Strem Chemicals,Newburyport, MA) (10uM diluted in PBS) was then applied to sub-RPE flat mounts and incubated for 5 min. Excess Zinpyr-1 wasremoved by rinsing with zinc-free PBS and the sections were coverslipped. The fluorescence associated with zinc deposits wasimmediately visualized.Results: High levels of zinc-containing sub-RPE deposits wereobserved in sod1-/- mice, mice carrying the rd8 mutation, and old (15months) but not young (3 months) C57BL6 mice. The presence ofhigh levels of zinc-enriched sub-RPE deposits in the sod1-/- mouse,proposed as a model for AMD, further validates its use to investigateAMD disease pathology and test interventions.Conclusions: Membrane permeable fluorescent zinc stainingprovides a quick assessment of sub-RPE deposits that can be easilyquantified. Staining of sub-RPE deposits helps to validate this AMDassociatedphenotype in animal models used in AMD research.Commercial Relationships: Rebecca J. Kapphahn, None; HeidiRoehrich, None; Deborah A. Ferrington, None; Frederik J. vanKuijk, NoneSupport: : Unrestricted grant from Research to Prevent Blindness tothe Department of Ophthalmology and Visual Neurosciences,Beckman Initiative for Macular Research, Minnesota Lions Club,NIH (P30-EY00374).Program Number: 5010 Poster Board Number: A0139Presentation Time: 2:45 PM - 4:30 PMVEGF, VEGF-C, and VEGF-D Increases in Aqueous HumorSamples in a LPS Induced Model of Uveitis in New ZealandWhite RabbitsDouglas Decker, Jeffery E. Burr, Kelly Lester, Vicki Zaworski, StevenJ. Weber. Biochemistry, PharmOptima, Portage, MI.Purpose: To determine the levels of VEGF, VEGF-C, and VEGF-Dfrom aqueous humor samples in an E.coli lipopolysaccharide (LPS)induced model of uveitis in rabbits.Methods: Uveitis was induced by intravitreal (IVT) injection of LPSinto the right eye(OD) of New Zealand Rabbits (NZW). The left eye(OS) received an IVT injection of vehicle. At 24 hours post injectionrabbits were evaluated by an ophthalmic exam and the aqueoushumor (AH) collected for cell counts and protein. VEGF, VEGF-C,and VEGF-D were quantitated in AH using a Meso Scale DrugDiscovery (MSD) electrochemiluminescent ELISA.Results: Ophthalmic exams of LPS treated rabbit eyes (OD)©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyindicated substantial inflammation compared to control eyes (OS).Cell counts from OS eyes were very low or undetectable andincreased to 2.1 x 106 cells/ml of AH in OD eyes. Protein from OSeyes averaged 3.2 mg/ml and increased to 32.9 mg/ml in OD eyes.VEGF and VEGF-C were below detection limits in OS eyes andincreased to 7.1 pg/ml and 75.5 pg/ml, respectively, in OD eyes.VEGF-D in OS eyes averaged 96.0 pg/ml and increased to 853 pg/mlin OD eyes.Conclusions: VEGF, VEGF-C, and VEGF-D levels increasedsignificantly in LPS treated eyes of NZW and are indicative of aninflammatory response. Ophthalmic exams, cell counts, and proteinincreases in LPS treated eyes support increased inflammation in thisrabbit model of uveitis. The significantly increased levels of VEGF-Cand VEGF-D in AH are new findings.Commercial Relationships: Douglas Decker, PharmOptima (I),PharmOptima (S); Jeffery E. Burr, PharmOptima LLC (E); KellyLester, PharmOptima (E); Vicki Zaworski, PharmOptima (E);Steven J. Weber, PharmOptima LLC (I), PharmOptima LLC (E)Program Number: 5011 Poster Board Number: A0140Presentation Time: 2:45 PM - 4:30 PMThe role of Cannabinoid receptors on light-inducedphotoreceptor degenerationTomoyo Imamura, Yuta Ohno, Kazuhiro Tsuruma, MasamitsuShimazawa, Hideaki Hara. Gifu Pharmaceutical University, Gifu,Japan.Purpose: Cannabinoid receptors are known as G protein-coupledreceptors and classified in CB1 receptor and CB2 receptor. CB1receptor is mainly localization in the presynaptic terminal of neurons,and regulates the release of neurotransmitters. In contrast, CB2receptors are expressed in the peripheral nervous system and organs,such as tonsils, spleen, these are involved in the regulation ofinflammation and immune response. It has been reported thatcannabinoid receptors are related to some neurodegenerativediseases, and expressed in retina. However, the role of cannabinoidreceptors in retina remains to be elucidated. Here, we investigated therole of cannabinoid receptors in light-induced photoreceptordegeneration, which is associated with retinitis pigmentosa and agerelatedmacular degeneration.Methods: An murine photoreceptor cell line, 661W, was irradiatedby white light at 2,500 lx. To examine the effect of CB1 and CB2receptors on light-induced retinal cell death, we used each receptorsagonist and antagonist, and evaluated cell viability by nuclearstaining with propidium iodine and Hoechst33342. In addition, 8weeks old male ddY mice were induced retinal damage by white lightirradiation at 8,000 lx illumination for 3 hours after the darkadaptation for 24 hours. To determine the expression of CB1 andCB2 receptor after light exposure, time-dependent changes ofexpression level and localization in retina and in 661W cells weremeasured by Western blotting and immunostaining.Results: CB1 and CB2 receptors existed in all layers of the retina.Furthermore, the expression level of CB1 receptor was increased bylight irradiation, whereas that of CB2 receptor was decreased. Inaddition, SR141617A, a CB1 receptor antagonist, showed asignificant protective effect. On the other hand, HU-308, a CB2receptor agonist, showed a significant protective effect, whereasSR144528, which is a CB2 antagonist, did not show a clear effect.Conclusions: CB1 receptor antagonist showed a significantprotective effect, whereas a CB2 receptor agonist showed asignificant protective effect. Furthermore, the expression level ofCB1 and CB2 receptor was changed in light retinal degenerationmodel. These results suggest that cannnabinoid receptors may beinvolved in the pathogenesis of retinal diseases such as retinitispigmentosa and age-related macular degeneration.Commercial Relationships: Tomoyo Imamura, None; Yuta Ohno,None; Kazuhiro Tsuruma, None; Masamitsu Shimazawa, None;Hideaki Hara, NoneProgram Number: 5012 Poster Board Number: A0141Presentation Time: 2:45 PM - 4:30 PMThe involvement of heparin-binding epidermal growth factor likegrowth factor on light-induced retinal degenerationYuki Inoue, Tomohiro Nakanishi, Astushi Oyagi, Yuta Ohno,Tomohiro Otsuka, Kazuhiro Tsuruma, Masamitsu Shimazawa,Hideaki Hara. Gifu Pharmaceutical University, Gifu, Japan.Purpose: Heparin-binding epidermal growth factor like growthfactor (HB-EGF) is a member of the EGF family of growth factor,and has been reported that it protects against various neuronal celldamages and expresses in the retina. Here, we investigated its role inlight-induced photoreceptor degeneration using 661W cells, atransformed mouse cone-cell line, and conditional Hb-egf knockout(KO) mice.Methods: 661W cells were used to investigate the effect of HB-EGFon light irradiation-induced cell-death using HB-EGF siRNA andHB-EGF treatments. Time-dependent changes in retinal HB-EGFwere measured using quantitative RT-PCR and Western blotting.Moreover, disruption of Hb-egf in the retinas from ventral forebrainspecific Hb-egf KO mice, was confirmed by LacZ staining and RT-PCR. Retinal damage was induced by exposure to whitefluorescentlight. Recombinant human HB-EGF was directly injectedinto vitreousbody in mice intravitreally. Electroretinogram (ERG)and histological analyses were performed.Results: The 661W cell death induced by light irradiation wasexacerbated by Hb-egf knockdown. Treatment with HB-EGFprotected against light-induced cell-death in 661W cells. Hb-egf andpro-HB-EGF levels were increased after light exposure in wild-type(WT) mice. LacZ-positive cells were observed, and Hb-egf deletionwas confirmed in the retinas of Hb-egf KO mice. Exposure to whitefluorescent light for 3 h reduced the a- and b-wave amplitudes of thedark-adapted ERG, and ONL thickness in Hb-egf KO mice versusWT mice. HB-EGF treatment improved both the a- and b-waveamplitudes, and the thickness of the ONL.Conclusions: These data suggest that HB-EGF plays a pivotal role inlight-induced retinal damage. It may become a potential therapeutictarget for age-related macular degeneration.Commercial Relationships: Yuki Inoue, None; TomohiroNakanishi, None; Astushi Oyagi, None; Yuta Ohno, None;Tomohiro Otsuka, None; Kazuhiro Tsuruma, None; MasamitsuShimazawa, None; Hideaki Hara, NoneSupport: None.Program Number: 5013 Poster Board Number: A0142Presentation Time: 2:45 PM - 4:30 PMComparison Study of EFEMP1 Gene Expression Levels inHuman ARPE-19 Cybrid Models Having Mitochondrial DNA ofHaplogroups H, J, K or LDanli Xing 1 , Deepika Malik 1 , Payam Falatoonzadeh 1 , Shari Atilano 1 ,Marilyn Chwa 1 , S Michal Jazwinski 2 , Miceli V. Michael 2 , Baruch D.Kuppermann 1 , Cristina M. Kenney 1 . 1 Ophthalmology, UC Irvine,Long Beach, CA; 2 Tulane Center for Aging, Tulane University, NewOrleans, LA.Purpose: EFEMP1 (Epidermal Growth Factor-containing fibulin-likeextracellular matrix protein) is a gene expressed in retinal pigmentepithelium (RPE) cells and is one of the high-risk genes associatedwith Age-related Macular Degeneration (AMD). EFEMP1 suppresses©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyVEGFA-induced angiogenesis, and mutations in this gene areassociated with drusen formation. Histology of AMD eyes shows thatEFEMP1 protein can be found between the RPE cell layer anddrusen. It has been suggested that misfolded EFEMP1 proteinaccumulates within RPE cells causing altered cellular function andinflammation. The aim of this study is to assess the baselineexpression of the EFEMP1 gene in a human ARPE-19 cybridcytoplasmic hybrid (cybrid) model where the cells have identicalnuclei, but contain mitochondrial DNA haplogroup H, J, K or L.Haplogroup H and J are of Northern-European descent, however, Jgenerally has more risk of developing AMD while H is protective.Haplogroup K, of Central-European descent, has some risk, andhaplogroup L, of African descent, has the least predisposition todeveloping AMD.Methods: Human ARPE-19 cells that were depleted of mitochondria(Rho0) were fused with platelets from individuals with known H, J,K or L haplogroups to create unique cybrid cell lines. RNA wasextracted from individual cybrids and cDNA was synthesized.EFEMP1 gene expression levels were measured using Q-PCR andanalyzed for changes in gene expression levels relative to cybrid H.Results: The expression levels of the EFEMP1 gene were found tobe significantly lower in Cybrids J, K and L when compared tocybrid H: cybrid J= 0.25 fold, p=0.0001; cybrid L= 0.4 fold, p=0.008;and cybrid K= 0.17 fold, p=0.003.Conclusions: This study demonstrates that human ARPE-19 cybridswith identical nuclei but variable mitochondrial DNA haplogroupshave differentially expressed levels of the EFEMP1 gene which isinvolved in VEGF-A induced angiogenesis. Cybrids J, K and Lshowed significantly lower levels of EFEMP1 gene expression ascompared to cybrid H in the following order H > L > J > K. Thisfinding may help us in further understanding the pathogenesis ofAMD with regards to differential risks amongst populationsCommercial Relationships: Danli Xing, None; Deepika Malik,None; Payam Falatoonzadeh, None; Shari Atilano, None; MarilynChwa, None; S Michal Jazwinski, None; Miceli V. Michael, None;Baruch D. Kuppermann, Alimera (C), Allegro (C), Allergan (C),Genentech (C), Glaukos (C), GSK (F), Novagali (C), Novartis (C),Ophthotech (C), Pfizer (C), Regeneron (C), Santen (C), SecondSight(C), Teva (C), ThromboGenics (C); Cristina M. Kenney, NoneSupport: Discovery Eye Foundation, Guenther Foundation,Beckman Macular Research Initiative, Polly and Michael SmithFoundation, Max Factor Family Foundation, Skirball Foundation,Lincy Foundation, Iris and B. Gerald Cantor Foundation,Unrestricted grant from Research to Prevent Blindness, grant fromNational Institute on Aging (AG006168)Program Number: 5014 Poster Board Number: A0143Presentation Time: 2:45 PM - 4:30 PMInositol phosphatase INPP5E in primary cilia and eyedevelopmentNa Luo 1 , Michael Conwell 1 , Akhilesh Kumar 1 , Ryan M. Anderson 2 ,Yang Sun 1 . 1 Ophthalmology, Indiana University, Indianapolis, IN;2 Pediatrics/Endocrinology, Indiana University, Indianapolis, IN.Purpose: To investigate the function of inositol 5-phosphataseINPP5E in cilia development in the retina. Mutations of INPP5Ecause Joubert Syndrome, which is characterized by retinaldegeneration, renal cysts, polydactyly, and mental retardation.Previous studies have implicated primary cilia defects in JoubertSyndrome, but the function and mechanisms of INPP5E in ciliaformation and retina development are not well understood.Methods: Using antisense morpholino oligonucleotides, knockdownof INPP5E was performed in zebrafish and rescue experiments weredone using wild type and mutant human mRNA. INPP5E mRNAsmutations of R378C and R435Q were generated using QuikChangeIIand Ambion mMessage mMachine®. Immunofluorescence wasperformed to study the functional aspect of cilia in zebrafishembryos. mTOR inhibitors treatments were performed in zebrafishand cell lines to examine the role of INPP5E in cilia signaling.Results: In zebrafish INPP5E morphants, embryos showed a dosedependentphenotype of retinal degeneration, microphthalmia, andbody axis asymmetry. The Inpp5e morphant zebrafish exhibitedshortened and decreased cilia formation in the Kupffer’s vesicle andpronephric ducts as compared to control zebrafish. Epinephrinestimulatedmelanosome trafficking was delayed in the Inpp5ezebrafish morphants. The phenotypes were rescued by co-injection ofhuman wide type INPP5E but not R378C or R435Q hINPP5EmRNA. Rapamycin was found to slow the development of the retinalphenotype in the zebrafish morphants.Conclusions: Our data supports an important role of INPP5E inciliary development and maintenance and provides a novel model toevaluate small molecules in the function of retinal development.Commercial Relationships: Na Luo, None; Michael Conwell,None; Akhilesh Kumar, None; Ryan M. Anderson, None; YangSun, NIH (F), American Glaucoma Society (R), Knights TemplarEye Foundation (R), Reeves Foundation (R)Support: NIH-EY-K08-022058; American Glaucoma society;Knights Templar Eye foundationProgram Number: 5015 Poster Board Number: A0144Presentation Time: 2:45 PM - 4:30 PMHuman Complement Factor H (CFH) Transgene ExpressionRescues the Visual Function and Retina Abnormalities in Agedcfh-/- MiceJindong Ding 1 , Una L. Kelly 1 , Marybeth Groelle 1 , Catherine BowesRickman 1, 2 . 1 Ophthalmology, Duke University Medical Center,Durham, NC; 2 Cell Biology, Duke University Medical Center,Durham, NC.Purpose: Complement factor H (CFH) polymorphisms are thestrongest genetic risk factors associated with age-related maculardegeneration. We have previously generated bacterial artificialchromosome (BAC) transgenic mouse lines expressing the humanY402 or H402 CFH variants. We have also shown that human CFHprotein can replace mouse cfh in the mouse complement alternativepathway when crossed to the cfh-/- mice (CFH-Tg,cfh-/-). In thepresent work, we evaluated the ocular phenotype of the CFH-Tg,cfh-/- mice. Specifically, we examined whether the retinal degenerationin the cfh-/- mice can be rescued by the human CFH protein.Methods: Experiments were performed on two-year-old cfh-/-, CFH-Tg,cfh-/- and C57 mice. The retina visual function was measured byelectroretinogram (ERG). Eyes were fixed and embedded in resin.Semi-thin sections were examined to evaluate the gross morphologyof the retina. Approximately 60 transmission electron microscopicimages per retina were taken at regular intervals across the superiorinferiorplane to measure the thickness of sub-RPE deposit.Results: The b-wave amplitude of the ERG was significantly reducedin the cfh-/- mice, compared to the C57 mice. The b-wave amplitudeof the CFH-Tg,cfh-/- mice was partially recovered. Initial evaluationdid not detect any differences in the gross retina structure. Contraryto previous studies, we found that aged cfh-/- mice have considerablymore sub-RPE deposit than age-matched C57 mice. However, thethickness of these basal deposits were reduced in the CFH-Tg,cfh-/-mice.Conclusions: Human CFH protein can partially rescue the retinalERG deficit and reduce the amount of sub-RPE basal deposit causedby cfh gene deletion.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyCommercial Relationships: Jindong Ding, None; Una L. Kelly,None; Marybeth Groelle, None; Catherine Bowes Rickman, NoneSupport: NIH Grants EY019038, P30 EY005722, Edward N. &Della L. Thome Memorial Foundation Award, and Research toPrevent Blindness, Inc.538 Apoptosis and Cell StressThursday, May 09, 2013 10:30 AM-12:15 PMExhibit Hall Poster SessionProgram #/Board # Range: 6109-6118/B0180-B0189Organizing Section: Biochemistry/Molecular BiologyProgram Number: 5016 Poster Board Number: A0145Presentation Time: 2:45 PM - 4:30 PMCasein Kinase 1 Epsilon Is Identified As A Potential TherapueticTarget For Dry AMD By A Multi-Pronged Gene ExpressionApproachGeorge Inana 1 , Christopher Murat 1 , Margaret J. McLaren 2 .1 Ophthalmology, Bascom Palmer Eye Institute, Miami, FL;2 Graymatter Research, Miami, FL.Purpose: To continue to analyze therapeutic targets for age-relatedmacular degeneration (AMD) identified by a multi-pronged geneexpression profiling approach combining gene expression in AMD,retinal pigment epithelium (RPE) phagocytosis, and cigarettesmoking.Methods: In vitro rod outer segment (ROS) phagocytosis assay ofRPE, collection and classification of post-mortem human eyes,cigarette smoke exposure of mice, RNA isolation, gene expressionprofiling (total genome and custom CHANGE), northernhybridization, qRT-PCR, immunofluorescence microscopy,transgenic mouse construction.Results: Casein kinase 1 Epsilon (Ck1e), a critical regulator ofcircadian oscillations, was identified among genes changed inexpression in AMD, ROS phagocytosis, and smoke exposure, afirmly established environmental factor for AMD. Ck1e significantlydecreased and increased in expression, 5 and 15 hours, respectively,after addition of ROS to RPE in the in vitro assay, consistent with itsinvolvement in phagocytosis. Consistent with a diurnal regulation,the expression of Ck1e peaked at 6pm and 3am in the retina andeyecup (EC), respectively. Expression of Ck1e was increased inAMD retina and EC in correlation to severity, peaking in grade 3corresponding to severe atrophic AMD. A monkey model of dryAMD also showed increase in Ck1e expression in the retina.Exposure of mice to cigarette smoke increased Ck1e expression after3 and 5 weeks in the EC and retina, respectively. The increase inCk1e was confirmed in the ROS by immunofluorescence.Significantly, the smoke exposure shifted the diurnal peaks of Ck1eexpression by 3 and 9 hours in the retina and EC, respectively.Conditional Ck1e over-expression transgenic mouse model wasconstructed, and preliminary result confirmed induced overexpressionof Ck1e, setting the stage for phenotypic analysis of themodel.Conclusions: A multi-pronged approach based on gene expression inAMD, RPE phagocytosis, and smoking identified a potentialcandidate gene for dry AMD, Ck1e, that shows a significantcorrelation to dry AMD in humans and a monkey model and phaseshiftsits diurnal peaks of expression after cigarette smoke exposure,possibly affecting critical circadian processes such as RPEphagocytosis of ROS. The conditional over-expression transgenicmodel should provide an excellent opportunity to investigate thiscandidate gene.Commercial Relationships: George Inana, iTherapeutics (I), US7,309,487 (P); Christopher Murat, None; Margaret J. McLaren,iTherapeutics (I), iTherapeutics (C), US 7,309,487 (P)Support: Flight Attendant Medical Research Institute, NIH P30EY014801, an unrestricted grant to the University of Miami fromResearch to Prevent Blindness, Inc.Program Number: 6109 Poster Board Number: B0180Presentation Time: 10:30 AM - 12:15 PMRag1 expression in RGCs is involved in programmed cell deathTakao Hirano 1 , Takuma Hayashi 2 , Toshinori Murata 1 . 1 Depart ofOphthalmology, Shinshu University, Matsumoto, Japan; 2 Departmentof Molecular and Cellular Immunology, Shinshu University,Matsumoto, Japan.Purpose: The recombination activating gene 1 (RAG1) plays animportant role in the rearrangement and recombination of genes inimmune cells. Although recent studies have focused on theexpression of Rag1 in the hippocampus, cerebellum, and elsewhere inthe central nervous system, its presence and function in retinalganglion cells (RGCs) remains unclear. The purpose of this study isto investigate the distribution of Rag1 in RGCs and examine itsinvolvement in programmed cell death.Methods: RGCs were purified from the retinas of 6-month-oldC57/BL/6 mice using microbeads-conjugated anti-mouse Thy-1antibodies by positive selection. RT-PCR of total RNA from thepurified-RGCs was then performed to demonstrate the geneexpression of Rag1. To examine the role of Rag1 in vivo, the totalnumber of RGCs was calculated in 0.3-milimeter-sections taken0.35mm from both the edge of the optic disk and the orra serata ineach of 4 groups: NFkBp50 knockout (p50KO) mice in which wehave reported increased age-related RGC death, Rag1KO mice,p50/Rag1 double KO (DKO) mice, and age-matched wild-type (WT)mice.Results: Rag1 gene expression was detected in the pan-purifiedRGCs. The numbers of RGCs in the WT, p50KO, Rag1KO, andDKO groups were 61.8±4.2, 54.7±4.5, 58.8±3.5 and 58.0±4.8(mean±SD, n=8-12), respectively. The number of RGCs in p50KOmice was significantly decreased compared with that in WT mice (p< 0.01). However, there was no significant difference in the numberof RGCs between DKO and WT mice.Conclusions: Our findings indicate that Rag1 is present in RGCs andplays a role in programmed cell death.Commercial Relationships: Takao Hirano, None; TakumaHayashi, None; Toshinori Murata, NoneProgram Number: 6110 Poster Board Number: B0181Presentation Time: 10:30 AM - 12:15 PMApoptotic retinal ganglion cell death in an autoimmune glaucomamodel is accompanied by antibody depositionsStephanie C. Joachim 1 , Christine Mondon 1 , Sandra Kuehn 1 , SabrinaReinehr 1 , Franz H. Grus 2 , Burkhard H. Dick 1 . 1 Experimental EyeResearch Institute, Ruhr University, Bochum, Germany;2 Experimental Ophthalmology, University Medical Center, Mainz,Germany.Purpose: Glaucoma is characterized by death of retinal ganglioncells (RGCs), but its cause is still unknown. Our studies indicate thatimmunization with ocular antigens leads to RGC loss in a model ofExperimental Autoimmune Glaucoma. To gain further knowledgeabout the mechanism and time-course of RGC cell death caspase 3levels and possible antibody appearances were evaluated in thismodel.Methods: Lewis rats were immunized with a optic nerve homogenatein Freund’s adjuvant and pertussis toxin (ONA), while the controlgroup was injected with sodium chloride (CO). RGC density was©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyquantified via retinal whole mounts and caspase 3+ cells wereevaluated on retina cross-sections at 8, 12, and 22 days. IgM and IgGantibody deposits and GFAP reactivity were detected at the samepoints in time.Results: We detected no difference in caspase 3+ cells 8 days afterimmunization (p=0.23). At 14 days distinct IgM deposits wereobserved epiretinally in ONA animals and these animals had highercaspase 3+ rates (2.0±0.3) than controls (0.4±0.2; p=0.004). But bothgroups showed no difference in RGC density (p=0.62) at this time. At22 days we detected a significantly higher number of apoptotic RGCsin ONA animals (ONA: 2.0±0; CO: 0.4±0.2; p=0.0007) andconcurrent a lower RGC density (p=0.04). Regarding Ig deposits, 14and 22 days after immunization we observed Igs in the ganglion celllayer (GCL) of ONA retinas. Especially 22 days after immunizationwe noted the IgG deposits are often in close vicinity to caspase+ cellsin the GCL.The GFAP+ area in ONA retinas continuously increased. Already at8 days ONA animals had a significantly higher mean GFAP+ area(7.2±0.5%) than CO (5.0±0.8%; p=0.02). Similar differences werenoted at 14 days (CO=6.3±0.5%; ONA=7.1±1.0%; p=0.04). Weobserved the largest percentage of GFAP+ area in the ONA group(8.5±0.7%) at 22 days (CO: 6.1±0.6%; p=0.02).Conclusions: Our data suggest that immunization with ocularantigens leads to apoptotic RGC death. Antibody deposits aredetectable in the retina when apoptosis occurs. Therefore, weconclude that antibodies are engaged in eliciting RGC apoptosis inthis animal model.Commercial Relationships: Stephanie C. Joachim, None;Christine Mondon, None; Sandra Kuehn, None; Sabrina Reinehr,None; Franz H. Grus, None; Burkhard H. Dick, NoneSupport: German Research Foundation grant JO 886/1-1; FoRUMFoundation (Ruhr University)Program Number: 6111 Poster Board Number: B0182Presentation Time: 10:30 AM - 12:15 PMEpigenetic Regulation of Rod Photoreceptor DevelopmentSarah Cheng, Hyun-Jin Yang, Anand Swaroop. N-NRL, Bldg 6,National Eye Institute, Bethesda, MD.Purpose: Stringent control of gene expression is critical forphotoreceptor development and function. A handful of transcriptionfactors regulate photoreceptor-specific gene expression. Interestingly,a group of their downstream target genes, includingphototransduction genes, exhibit delayed onset of expression indeveloping rod photoreceptors, suggesting additional regulatorymechanisms. In this study, we have explored epigenetic regulation ofphotoreceptor gene expression, with focus on gene-silencing histonemodification H3K27Me3 and gene-activating histone modificationH3K4Me3.Methods: Rod photoreceptors were purified from Nrl-GFP mice byfluorescence activated cell sorting. We performed chromatinimmunoprecipitation using the flow-sorted mouse rod photoreceptorsto characterize changes in H3K4Me3 and H3K27Me3 profiles duringrod development. We then knock-down specific components ofhistone modifying complexes using in vivo electroporation of P0mouse retina with shRNA. Expression of rod genes is analyzed at P6-10 by immunohistochemistry.Results: We found that rod genes were marked with H3K27Me3 innewborn rod photoreceptors. There was then subsequent change inhistone modification profile towards H3K4Me3, which accompaniedgene activation as rods further differentiate. The knock-downexperiments are in progress. We expect that changes in methylationcomponents would bias retinal gene expression profile toward a moreimmature state.Conclusions: This is the first study to directly examine epigeneticregulation of gene expression during development of a single retinalcell type in vivo. Our results affirm that histone modifications areimportant contributors to gene regulation in developing rodphotoreceptors.Commercial Relationships: Sarah Cheng, None; Hyun-Jin Yang,None; Anand Swaroop, NoneSupport: NIH intramural research programProgram Number: 6112 Poster Board Number: B0183Presentation Time: 10:30 AM - 12:15 PMHistone Deacetylase 3 (HDAC3) plays an important role inretinal ganglion cell death after acute optic nerve injuryHeather Schmitt 1, 2 , Cassandra Schlamp 1 , Robert W. Nickells 1 .1 Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI; 2 Cellular and Molecular Pathology,University of Wisconsin-Madison, Madison, WI.Purpose: HDAC activity mediates gene silencing and apoptosis indying retinal ganglion cells. An important role in these processes maybe played by HDAC3, which exhibits nuclear translocation inganglion cells shortly after optic nerve injury. The purpose of thisstudy was to test the hypothesis that knock out of Hdac3 transcriptionwill lead to protection of retinal ganglion cells from histonedeacetylation-related gene silencing and death following acute injury.Methods: Hdac3 fl/fl mice were given 1μL intravitreal injections ofAAV2-CRE virus (10 12 genome copies/mL) into the OS eye to exciseHdac3. Rosa26-LacZ fl/fl or Rosa26-LacZ fl/fl mice were treatedidentically to serve as controls. After eight weeks, injected eyesunderwent optic nerve crush. Five days post-optic nerve crush,retinas were harvested and analyzed using fluorescent microscopy orqPCR quantification of ganglion cell mRNAs. Analyses includedHDAC2, HDAC3, activated caspase 3, and acetylated histone 4(AcH4) labeling. Eyes were also harvested 14 days post-optic nervecrush and retinas analyzed using fluorescent microscopy for totalretinal ganglion cell counts.Results: : Rosa26-LacZ fl/fl mouse eyes expressed HDAC2 andHDAC3 at 5 days following optic nerve crush, but exhibited widespreadhistone deacetylation in the ganglion cell layer characteristicof optic nerve crush. Deletion of Hdac3 in ganglion cells of Hdac3 fl/flmice led to loss of HDAC3 expression and retained expression ofHDAC2 and AcH4. Ganglion cells of Rosa26-LacZ fl/fl mice alsolabeled positive for active caspase 3, an indicator of cell apoptosis,more frequently than those of Hdac3 fl/fl mice (P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyReserve University, Cleveland, OH; 2 Ophthalmology and VisualSciences, Case Western Reserve University, Cleveland, OH.Purpose: It is impossible to overstate the ubiquity of miRNAmediatedgene regulation in the biology of plants and animals.miRNAs have been implicated in virtually every physiologicalprocess and disease pathology in which they have been studied.Despite this widespread functional relevance, surprisingly little isknown about the physiological impact of miRNAs in the retina.Inactivation of the miRNA processing enzyme, Dicer, in thedeveloping retina leads to defects in cell fate determination andsurvival of progenitors, as well as progressive retinal degeneration.However, it’s unclear which retinal cell types require miRNA generegulation for survival and function or whether miRNAs are requiredfor survival or function of the mature retina. The aim of this study isto address the impact of loss of miRNA gene regulation in mature rodphotoreceptors.Methods: To address these questions we have developed a rodspecificconditional Dicer knockout mouse model. We have analyzedthe morphology of mutant mice using light microscopy,immunohistochemistry, and electron microscopy. We are alsoanalyzing the function of the visual system using electroretinogram,single cell electrophysiological approaches, and retinoid analysis.Results: These animals develop progressive retinal degeneration,beginning with disorganization of rod outer segments around andculminating with degeneration of nearly all photoreceptors. ERGanalysis shows progressive loss of rod function, followed by loss ofcone-driven visual responses. Electrophysiological and highresolution imaging studies are underway to gain insight into themechanism of photoreceptor death in these mice.Conclusions: The observed retinal degeneration demonstrates thatmiRNAs are essential for the survival and function of mature rodphotoreceptors. In addition, these results suggest that miRNAs otherthan the photoreceptor specific miR-183/96/182 cluster are essentialfor rod survival and vision.Commercial Relationships: Thomas Sundermeier, None; NingZhang, None; Debarshi Mustafi, None; Hideo Kohno, None;Krzysztof Palczewski, QLT Inc (F), Polgenix Inc (E), Visum Inc(P), Amegen Inc (F)Support: NIH Fellowship 1F32EY022830Program Number: 6114 Poster Board Number: B0185Presentation Time: 10:30 AM - 12:15 PMTransient Upregulation and then Downregulation of BACE 1 andPS-1 in ER Stress Induced Apoptosis of Retinal Ganglion Cells invitroBingqian Liu, Jian Ge. State Key Laboratory of Ophthalmology,Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou,China.Purpose: To investigate the level of beta-secretase 1 (BACE-1),presenilin-1 (PS-1) and full length amyloid precursor protein (APP)in Endoplasmic Reticulum (ER) stress induced retinal ganglion cellapoptosis in vitro.Methods: Tunicamycin (Tm, 2μg/ml) were added for 48 hours toinduce ER stress in retinal ganglion cells (RGC-5 cell line) culturedin Dulbecco’s modified Eagle’s medium supplemented with 10%fetal bovine serum. Protein levels of 78 kDa glucose-regulatedprotein (GRP-78), full length APP, BACE1, PS-1, and cleavedcaspase 3 were examined by western blot analysis.Results: ER stress marker GRP-78 was increased in RGC-5 cellstreated with Tm in a time-dependent manner. Protein level of fulllength APP was decreased steadily after Tm treatment. BACE-1, andPS-1 were both increased significantly at 2 hour time point in Tmgroup compared to vehicle control. Then, BACE-1 decreased rapidly,while PS-1 decreased slowly, to an undetectable level. Cleavedcaspase-3 was increased significantly under ER stress.Conclusions: BACE-1 and PS-1 were initially increased and thendecreased in ER stress induced apoptosis of retinal ganglion cells invitro. Full length APP might be cleaved by the transient upregulationof BACE-1 and PS-1. Further study is needed to determine whetherthere is a negative feedback between the production of amyloid betaand the BACE-1 and PS-1 activities in this process.Commercial Relationships: Bingqian Liu, None; Jian Ge, NoneSupport: National Natural Science Foundation of China 81000376.Program Number: 6115 Poster Board Number: B0186Presentation Time: 10:30 AM - 12:15 PMProgranulin Mutant Mice Exhibit Accumulation ofIntraneuronal Lipofuscin Aggregates in the RetinaBrian P. Hafler 1 , Zoe A. Klein 2 , Stephen Strittmatter 2 .1 Ophthalmology and Visual Science, Yale School of Medicine, NewHaven, CT; 2 Program in Cellular Neuroscience, Neurodegeneration,and Repair, Yale School of Medicine, New Haven, CT.Purpose: Patients lacking progranulin develop neuronal ceroidlipofuscinosis, a neurodegenerative lysosomal disorder characterizedby abnormal lipofuscin accumulation. Previous investigations haveshown that mice lacking progranulin, a secreted glycoproteinencoded by the granulin (GRN) gene, develop abnormalintraneuronal lipofuscin accumulation in the brain. To determine ifprogranulin mutant mice have a similar phenotype in the retina asobserved in humans with mutations in the progranulin gene, GRN-/-retinas were examined for lipofuscin accumulation.Methods: In order to address whether the age-accelerated lipofuscinaccumulation in the brains of GRN /- mice was present in the retina,we examined the retinas of progranulin deficient mice. Thegeneration of GRN-/- mice has been described previously (Kayasugaet al., 1997). GRN -/- genotype was detected using PCR with primersdirected against GRN and western blot for progranulin protein. GRN-/- mice were examined for lipofuscin using confocal microscopyautofluorescence.Results: As expected, GRN-/- mice lacked progranulin compared tocontrols using western blot. Given than humans deficient forprogranulin develop retinal dystrophy, GRN-/- mice were examinedfor lipofuscin in 12 month old mice. Retinas from GRN-/- mice wereexamined using autofluorescence, and interestingly, there wasintracellular accumulation of highly autofluorescent lipofuscinmaterial in neurons as compared to GRN+/+ mice.Conclusions: These data indicate that GRN-/- mice exhibit anaccumulation of intraneuronal autofluorescent lipofuscin aggregates.As humans with the neurodegenerative disease neuronal ceroidlipofuscinosis linked to progranulin gene mutations on chromosome17 have excessive accumulation of lipofuscin in the retina, this studyis, to our knowledge, is the first example demonstrating a similarretinal phenotype of GRN-/- mice providing a new model toinvestigate the retinal pathology found in this disorder.Commercial Relationships: Brian P. Hafler, None; Zoe A. Klein,None; Stephen Strittmatter, NoneSupport: Department of Ophthalmology and Visual Science, YaleSchool of Medicine, and Kavli Institute for Neuroscience at YaleUniversityProgram Number: 6116 Poster Board Number: B0187Presentation Time: 10:30 AM - 12:15 PMC1qa deficiency exacerbates RGC loss in a mouse glaucomamodel©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyQiong Ding 1 , Michael G. Anderson 2 , Amy Cook 1 , Markus H. Kuehn 1 .1 Ophthalmology and Visual Sciences, U of Iowa, Iowa City, IA;2 Molecular Physiology and Biophysics, U of Iowa, Iowa City, IA.Purpose: The development of glaucoma is associated with activationof the complement cascade and the marked accumulationcomplement components 1q (C1q) and 3 (C3) in association withretinal ganglion cells (RGC). In order to determine the functionalsignificance of C1q and C3 in the pathophysiology of glaucoma, weevaluated retinal ganglion cell (RGC) and axonal loss in B6-Sh3pxd2nee (nee) mice with and without targeted deletions in either the C1qaor C3 genes.Methods: Nee mice were bred either with those containing targeteddeletions in C1q or C3 to create double knockouts. At 8 weeks of agethe intraocular pressure (IOP) was determined using a reboundtonometer. The animals were then euthanized and the eyes and opticnerves were harvested. The number of surviving gamma synucleinimmunoreactive RGC was determined in flat mounted preparations ofretina and axonal damage in the optic nerve was evaluated using a 5-point grading scheme. Differences between strains were evaluatedusing either one way ANOVA or Kruskal-Wallis test.Results: The presence of the nee allele caused a marked elevation ofIOP above that observed in control mice (+6.0 mmHg, p=0.0002),but no difference was observed with respect to the absence orpresence of functional complement genes. The elevation in IOPcaused a decline in RGC numbers and an increase in optic nervedamage scores. Among mice homozygous for the nee mutation, theobserved damage was particularly marked in C1q-/- (-74.3%) miceand significantly higher than that observed in C3-/- mice or littermatecontrols (-33.6% and -32.3%, respectively, p160 nt). Smaller chimeras allow3D RNA algorithms for structural design and biophysicalinvestigation of hhRz structure/function performance.Methods: CELO-VAI scaffold, designed using RNAStructure, wasPCR constructed with partially overlapping DNA primers, thendirectionally ligated into pNEB193-T7. hhRz cDNA or controlsequence was cloned into an adapter placed at a unique CELO-VAIrestriction site. Target hRHO fragment cDNA (510 nt RNA)containing the hhRz cleavage site was cloned into pBlueScript.Transcription with T7 RNA polymerase generated chimeric CELO-VAI-hhRzs and target RNA to test in vitro cleavage. In HEK293 cellsfull length hRHO cDNA was expressed from pCDNA3.1, and CELO-VAI-hhRzs or controls (in pNEB193-T7) were expressed from strongintragenic Pol-III promoters. hRHO mRNA was assayed byquantitative RT/PCR with controls. ANOVA and t-tests were used tostatistically evaluate outcomes.Results: The hhRz that demonstrated cellular cleavage activitywithin a larger human VAI RNA scaffold had only pure antisensetarget suppression (64% knockdown, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyupstream tertiary accessory element (TAE) to drive formation of acatalytically active hhRz conformation state recovered both catalyticintracellular knockdown (41%, p