Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyquantified via retinal whole mounts and caspase 3+ cells wereevaluated on retina cross-sections at 8, 12, and 22 days. IgM and IgGantibody deposits and GFAP reactivity were detected at the samepoints in time.Results: We detected no difference in caspase 3+ cells 8 days afterimmunization (p=0.23). At 14 days distinct IgM deposits wereobserved epiretinally in ONA animals and these animals had highercaspase 3+ rates (2.0±0.3) than controls (0.4±0.2; p=0.004). But bothgroups showed no difference in RGC density (p=0.62) at this time. At22 days we detected a significantly higher number of apoptotic RGCsin ONA animals (ONA: 2.0±0; CO: 0.4±0.2; p=0.0007) andconcurrent a lower RGC density (p=0.04). Regarding Ig deposits, 14and 22 days after immunization we observed Igs in the ganglion celllayer (GCL) of ONA retinas. Especially 22 days after immunizationwe noted the IgG deposits are often in close vicinity to caspase+ cellsin the GCL.The GFAP+ area in ONA retinas continuously increased. Already at8 days ONA animals had a significantly higher mean GFAP+ area(7.2±0.5%) than CO (5.0±0.8%; p=0.02). Similar differences werenoted at 14 days (CO=6.3±0.5%; ONA=7.1±1.0%; p=0.04). Weobserved the largest percentage of GFAP+ area in the ONA group(8.5±0.7%) at 22 days (CO: 6.1±0.6%; p=0.02).Conclusions: Our data suggest that immunization with ocularantigens leads to apoptotic RGC death. Antibody deposits aredetectable in the retina when apoptosis occurs. Therefore, weconclude that antibodies are engaged in eliciting RGC apoptosis inthis animal model.Commercial Relationships: Stephanie C. Joachim, None;Christine Mondon, None; Sandra Kuehn, None; Sabrina Reinehr,None; Franz H. Grus, None; Burkhard H. Dick, NoneSupport: German Research Foundation grant JO 886/1-1; FoRUMFoundation (Ruhr University)Program Number: 6111 Poster Board Number: B0182Presentation Time: 10:30 AM - 12:15 PMEpigenetic Regulation of Rod Photoreceptor DevelopmentSarah Cheng, Hyun-Jin Yang, Anand Swaroop. N-NRL, Bldg 6,National Eye Institute, Bethesda, MD.Purpose: Stringent control of gene expression is critical forphotoreceptor development and function. A handful of transcriptionfactors regulate photoreceptor-specific gene expression. Interestingly,a group of their downstream target genes, includingphototransduction genes, exhibit delayed onset of expression indeveloping rod photoreceptors, suggesting additional regulatorymechanisms. In this study, we have explored epigenetic regulation ofphotoreceptor gene expression, with focus on gene-silencing histonemodification H3K27Me3 and gene-activating histone modificationH3K4Me3.Methods: Rod photoreceptors were purified from Nrl-GFP mice byfluorescence activated cell sorting. We performed chromatinimmunoprecipitation using the flow-sorted mouse rod photoreceptorsto characterize changes in H3K4Me3 and H3K27Me3 profiles duringrod development. We then knock-down specific components ofhistone modifying complexes using in vivo electroporation of P0mouse retina with shRNA. Expression of rod genes is analyzed at P6-10 by immunohistochemistry.Results: We found that rod genes were marked with H3K27Me3 innewborn rod photoreceptors. There was then subsequent change inhistone modification profile towards H3K4Me3, which accompaniedgene activation as rods further differentiate. The knock-downexperiments are in progress. We expect that changes in methylationcomponents would bias retinal gene expression profile toward a moreimmature state.Conclusions: This is the first study to directly examine epigeneticregulation of gene expression during development of a single retinalcell type in vivo. Our results affirm that histone modifications areimportant contributors to gene regulation in developing rodphotoreceptors.Commercial Relationships: Sarah Cheng, None; Hyun-Jin Yang,None; Anand Swaroop, NoneSupport: NIH intramural research programProgram Number: 6112 Poster Board Number: B0183Presentation Time: 10:30 AM - 12:15 PMHistone Deacetylase 3 (HDAC3) plays an important role inretinal ganglion cell death after acute optic nerve injuryHeather Schmitt 1, 2 , Cassandra Schlamp 1 , Robert W. Nickells 1 .1 Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI; 2 Cellular and Molecular Pathology,University of Wisconsin-Madison, Madison, WI.Purpose: HDAC activity mediates gene silencing and apoptosis indying retinal ganglion cells. An important role in these processes maybe played by HDAC3, which exhibits nuclear translocation inganglion cells shortly after optic nerve injury. The purpose of thisstudy was to test the hypothesis that knock out of Hdac3 transcriptionwill lead to protection of retinal ganglion cells from histonedeacetylation-related gene silencing and death following acute injury.Methods: Hdac3 fl/fl mice were given 1μL intravitreal injections ofAAV2-CRE virus (10 12 genome copies/mL) into the OS eye to exciseHdac3. Rosa26-LacZ fl/fl or Rosa26-LacZ fl/fl mice were treatedidentically to serve as controls. After eight weeks, injected eyesunderwent optic nerve crush. Five days post-optic nerve crush,retinas were harvested and analyzed using fluorescent microscopy orqPCR quantification of ganglion cell mRNAs. Analyses includedHDAC2, HDAC3, activated caspase 3, and acetylated histone 4(AcH4) labeling. Eyes were also harvested 14 days post-optic nervecrush and retinas analyzed using fluorescent microscopy for totalretinal ganglion cell counts.Results: : Rosa26-LacZ fl/fl mouse eyes expressed HDAC2 andHDAC3 at 5 days following optic nerve crush, but exhibited widespreadhistone deacetylation in the ganglion cell layer characteristicof optic nerve crush. Deletion of Hdac3 in ganglion cells of Hdac3 fl/flmice led to loss of HDAC3 expression and retained expression ofHDAC2 and AcH4. Ganglion cells of Rosa26-LacZ fl/fl mice alsolabeled positive for active caspase 3, an indicator of cell apoptosis,more frequently than those of Hdac3 fl/fl mice (P