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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.
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