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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.
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Biochemistry/Molecular Biology - ARVO

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