Biochemistry/Molecular Biology - ARVO

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.