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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.
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Biochemistry/Molecular Biology - ARVO

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