Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyβ shRNAs were overexpressed in purified P4 RGCs or E18hippocampal neurons, which were then immunostained and imagedafter 1-3 days in vitro. Neurite length was quantified using the ImageJ Neurite Tracer.Results: We found that in RGCs, full-length 39 kDa Set-β ispredominantly nuclear whereas a shorter 25 kDa isoform ispredominately cytoplasmic. Set-β overexpressed in postnatal RGCsor embryonic hippocampal neurons localized to the nucleus andsuppressed axon growth. In contrast, experimentally increasedmyristoylated-Set-β cytoplasmic localization, or shRNA knockdownof Set-β, promoted axon growth. Serine-9 phosphorylation of Set-βblocks Set-β’s ability to suppress axon growth. Finally, we showedthat in the CNS, Set-β binds selectively to PP2A-A-β but not αisoform; PP2A was recently shown to modulate axon growth in CNS.Conclusions: Full-length Set-β is developmentally upregulated inCNS primary neurons’ nuclei whereas a shorter isoform is found inthe cytoplasm. Set-β inhibits or promotes axon growth in the CNSneurons depending on its subcellular localization and serine-9phosphorylation, and Set-β binds selectively to PP2A-A-β but not αisoform. Manipulation of Set-β in vivo may provide a strategy toenhance regeneration.Commercial Relationships: Melina I. Morkin, None; Ephraim F.Trakhtenberg, None; Yan Wang, None; Stephanie Fernandez,None; Gregory M. Mlacker, None; Jeffrey L. Goldberg, NoneSupport: AHA (11PRE7310069, EFT), NEI (EY020913, JLG),Center grant P30 EY014801 (JLG), Research to Prevent Blindness,NIH (T32 NS007492, EFT), Lois Pope LIFE Fellowship (EFT)Program Number: 2474 Poster Board Number: D0079Presentation Time: 2:45 PM - 4:30 PMEarly Ischemia-reperfusion Injury Induces Retinal VascularPermeability in a VEGF Receptor 2 Dependent Manner Followedby Occludin Phosphorylation and UbiquitinationArivalagan Muthusamy, Cheng-mao Lin, Heather Lindner, SumathiShanmugam, Steven F. Abcouwer, David A. Antonetti.Ophthalmology & Vis Sciences, Univ of Michigan Kellogg Eye Ctr,Ann Arbor, MI.Purpose: Ischemia-reperfusion (IR) injury rapidly induces retinalvascular permeability by an unknown mechanism. Multiple tightjunction (TJ) proteins including the MARVEL family (occludin,tricellulin, and marvel D3 proteins) and the claudin family arerequired for the formation and maintenance of the blood-retinalbarrier (BRB). Vascular endothelial growth factor- (VEGF)-inducedpermeability of retinal endothelial cell cultures requiresphosphorylation of occludin on serine 490 (Ser-490), which leads toits ubiquitination and endocytosis. The present study tests thehypotheses that retinal IR injury stimulates vascular permeabilitythrough VEGF receptor 2 (VEGFR2) activation and alterations intight junction protein expression, modification and complexformation.Methods: Rats were subjected to ischemia for 45 minutes followedby reperfusion up to 4 h. BRB permeability was quantified by retinalaccumulation of Evans blue dye measured 2 h after its intravitrealinjection. Changes in retinal TJ protein content, occludinphosphorylation and ubiquitination were assessed by Westernblotting. To observe TJ organization, localization of TJ proteins wasexamined by immunohistochemistry (IHC) of flat-mounted retinas.Results: IR caused a rapid significant increase in permeabilityassessed during 0.25-2.25 h and 4-6 h following reperfusion. IR hadno effects on total protein content of any TJ protein examined, butrapidly diminished TJ protein localization at endothelial celljunctions within 15 min of reperfusion. IR caused phosphorylation ofVEGFR2 on tyrosine 1175 (Tyr-1175) at 15 min after reperfusion,coinciding with occludin phosphorylation at Ser-490 and polyubiquitination.Intravitreal injection of a tyrosine kinase inhibitorspecifically targeting VEGFR2 efficiently blocks both IR-inducedVEGFR2 Tyr-1175 phosphorylation and occludin Ser-490phosphorylation.Conclusions: These data are consistent with retinal IR injury causingvascular permeability through VEGFR2 activation andphosphorylation and ubiquitination of occludin leading to rapiddisassembly of tight junction complexes. This model suggests thatoccludin phosphorylation may represent a valid target for therapeuticprevention of VEGF-induced BRB breakdown and subsequentedema.Commercial Relationships: Arivalagan Muthusamy, None;Cheng-mao Lin, None; Heather Lindner, None; SumathiShanmugam, None; Steven F. Abcouwer, None; David A.Antonetti, NoneSupport: JDRFProgram Number: 2475 Poster Board Number: D0080Presentation Time: 2:45 PM - 4:30 PMOligonucleotide-mediated gene correction in human primaryfibroblastsMagdalena M. Staniszewska, Mark B. Consugar, Michael H. Farkas,Eric A. Pierce. Ocular Genomics Institute, Department ofOphthalmology, Massachusetts Eye and Ear Infirmary, HarvardMedical School, Boston, MA.Purpose: The use of stem cell transplantation has great potential forthe treatment of patients with inherited and other retinal degenerativedisorders. One approach to therapy currently being evaluated is theuse of gene-corrected induced pluripotent stem (iPS) cells. To testefficacy of gene editing induced by single-strandedoligodeoxynucleotides (ODNs), we evaluated the ODN-mediatedcorrection of the pathogenic mutation in fibroblasts from a patientwith gyrate atrophy. Next-generation sequencing (NGS) was utilizedto address the challenge of identifying cells with the corrected genein the absence of a selection system.Methods: Human skin fibroblasts isolated from patient with gyrateatrophy due to a p.Ala226Val (c.C677T) mutation in OAT gene werelipofected with correcting (WT) or control (mutant) 59-nucleotideODNs. The transfected cells were plated in a 96-well plate at adensity of 100 cells/well. The targeted OAT region was PCRamplifiedfrom each well of the 96-well plate with barcoded primers,and the purified amplicons were pooled. Illumina NGS was used toidentify wells containing corrected cells, which where enriched bysub-cloning. Diagnostic digestion of the PCR-amplified targetedOAT region was used to verify gene editing events in the sub-clonedfibroblasts.Results: NGS of the pooled PCR products generated an average of363,000 sequence reads per well. A total of 25 wells from the plate ofcells treated with the correcting ODN had the C677 nucleotidedetected above the background rate. Twenty one wells from thecontrol plate were also identified using this approach. Sub-clonalenrichment and diagnostic digestion of the amplified targeted OATregion showed that 22% (7/31) of viable sub-clones transfected withcorrecting ODN contained WT sequence. In contrast, none of theviable sub-clones from the cells receiving control ODN showed a WTdigestion pattern. We were not able to obtain clonal populations ofODN-corrected cells due to senescence of the fibroblasts.Conclusions: These results suggest that ODNs can be used to correctsingle point mutations in primary cells, and that corrected cells can bedetected using NGS without introduction of a selection cassette. Theexperiments also demonstrate that fibroblasts are not the optimal celltype for therapeutic gene correction, since they have a limited growth©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.