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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.
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