Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biology1 Anesthesia, Critical Care, and Pain Medicine, MassachusettsGeneral Hospital, Boston, MA; 2 Ophthalmology, Schepens EyeResearch Institute, Boston, MA; 3 Wellman Center for Photomedicineand Center for Systems Biology, Massachusetts General Hospital,Boston, MA; 4 Ophthalmology, Boston University School ofMedicine, Boston, MA; 5 VIB Department of Molecular BiomedicalResearch, Ghent University, Ghent, Belgium; 6 Ophthalmology,Massachusetts Eye and Ear Infirmary, Boston, MA; 7 ChanningDivision of Network Medicine, Brigham and Women’s Hospital,Boston, MA; 8 Cardiology Division, Department of Medicine,Massachusetts General Hospital, Boston, MA.Purpose: Primary open angle glaucoma (POAG) is a leading causeof blindness. Available therapies offer incomplete protection. Themolecular signaling involved in the pathogenesis of POAG remainsunknown. Here, we identify the nitric oxide (NO) receptor solubleguanylate cyclase (sGC) as a key enzyme in the etiology of POAG,using a novel murine gene knockout model and genetic data from asubgroup of POAG patients.Methods: Female wild-type (WT) mice and mice lacking the α1subunit of sGC (sGCα1-/-) were studied. Thickness of the retinalnerve fiber layer (RNFL) was measured via spectral domain opticalcoherence tomography (SD-OCT). Retinal ganglion cells (RGCs) andoptic nerve axons were detected histochemically and counted.Morphology of the iridocorneal angle was examined by lightmicroscopy, ultrasound biomicroscopy, and SD-OCT. Intraocularpressure (IOP) was measured serially with a TonoLab-Tonometer.Retinal arterial function was assessed via in vivo laserophthalmoscopy. POAG cases and controls from the GlaucomaGenes and Environment (GLAUGEN) study were studied for theirassociation with genetic variants in the sGC locus.Results: Age-dependent thinning of the RNFL and loss of RGC’sand optic nerve axons in the context of an open iridocorneal anglewas observed in sGCα1-/- but not age-matched WT mice. The opticneuropathy associated with sGCα1-deficiency was accompanied by amodest (~2mmHg) and age-dependent increase in IOP and by retinalartery dysfunction. A candidate gene association study of POAG withparacentral vision loss, a POAG subtype thought to be associatedwith vascular dysregulation, identified a variant in the locuscontaining the genes encoding the α1 and β1 subunits of sGC.Together, these results highlight the relevance of sGC (and ouranimal model) in the pathogenesis of POAG.Conclusions: This study provides new insights into the pathogenesisand genetics of POAG and represents a paradigm shift in POAGresearch. Our findings suggest that therapies that do not focus onlowering IOP (e.g. targeting retinal vascular dysfunction) constitute acomplementary approach to treating POAG. In addition, ourobservations unequivocally identify perturbation of a wellcharacterizedsignaling pathway (NO-cGMP signaling) as a keymechanism in the etiology of POAG. Identifying sGC as a potentialtherapeutic target for POAG may inform the clinical development ofexisting cGMP-elevating therapeutic compounds.Commercial Relationships: Emmanuel S. Buys, None; Yu-ChiehKo, None; Clemens Alt, None; Haiyan Gong, None; PeterBrouckaert, None; Janey L. Wiggs, None; Meredith S. Gregory-Ksander, None; Louis R. Pasquale, None; Kenneth D. Bloch,None; Bruce R. Ksander, NoneSupport: NEI R21 EY020987, NEI R01 EY022746-01Program Number: 1610 Poster Board Number: D0037Presentation Time: 8:30 AM - 10:15 AMMammalian Expression and Biophysical Examination of HumanWild-Type Optineurin ProteinHongyu Ying, Xiang Shen, Minhua Wang, Beatrice Yue. Ophthal &Visual Sciences, Univ of Illinois at Chicago, Chicago, IL.Purpose: To express optineurin in mammalian cells and attain highlypurified optineurin protein for biophysical examinations. Optineurinis a gene linked to normal tension glaucoma and amyotrophic lateralsclerosis.Methods: Tetracycline inducible (Tet-on) neuronal RGC5 cell linesthat express Halo tagged wild-type optineurin (OPTN WT -Halo oroptineurin-Halo) were created by transfecting RGC5 cells withplasmid vector pTRE-OPTN WT -Halo-IRES-GFP-INS-rtTA-IREShyg-pcDNA3.1z.Cells were selected in hygromycin (100 μg/ml)-containing medium and subsequently screened for optineurinexpression by Western blotting or based on GFP expression afterdoxycycline (Dox) induction by fluorescence microscopy.Optineurin-Halo collected from the cell lysate was purified usingPromega’s HaloTag based affinity purification technique undernative conditions. The purity of the isolated protein was assessed bySDS-PAGE and Western blotting. The secondary structure ofpurified optineurin was examined by Jasco 710 circular dichroism(CD) spectropolarimeter.Results: Tet-on inducible RGC5 cell lines were established toexpress optineurin-Halo upon Dox induction. Following affinitypurification, Western blotting with anti-optineurin antibody detecteda single band at approximately 74 kDa. This highly purified tag-freeoptineurin protein was isolated with a yield of 23 µg/500 cm 2 (22 cmsquare dish). A circular dichroism (CD) spectrum indicated thatoptineurin is folded, containing both α-helical and β-sheet secondarystructures.Conclusions: Human optineurin protein expressed in stable induciblecell lines was purified and isolated. The inducible cell lines wouldallow production of highly purified protein in a scaled-up and costeffective manner, facilitating biophysical characterization ofoptineurin including its secondary structure, thermal stability andaggregation.Commercial Relationships: Hongyu Ying, None; Xiang Shen,None; Minhua Wang, None; Beatrice Yue, NoneSupport: Grants EY018828 and EY005628 (to B.Y.J.T.Y.) and coregrant EY001792 from the National Eye Institute, Bethesda,Maryland.240 Visual Cycle, Retinoids and CarotenoidsMonday, May 06, 2013 11:00 AM-12:45 PM6A Paper SessionProgram #/Board # Range: 1699-1705Organizing Section: Biochemistry/Molecular BiologyProgram Number: 1699Presentation Time: 11:00 AM - 11:15 AMMouse Cone Dark Adaptation Relies on Two Visual Cycles andIs Substantially Retarded in Mice Lacking RDH8 and ABCA4Alexander V. Kolesnikov 1 , Peter H. Tang 2 , Akiko Maeda 3 , Leah C.Byrne 4 , John G. Flannery 4 , Krzysztof Palczewski 3 , Vladimir J.Kefalov 1 . 1 Ophthalmology and Visual Sciences, WashingtonUniversity School of Medicine, Saint Louis, MO; 2 Ophthalmology,Medical University of South Carolina, Charleston, SC;3 Pharmacology, Case Western Reserve University, Cleveland, OH;4 Molecular and Cell Biology, The University of California, Berkeley,CA.Purpose: Rapid regeneration of the visual pigment following itsphotoactivation is critical for the function of cone photoreceptors. Weinvestigated the contributions of classic RPE visual cycle and novelcone-specific retina visual cycle to regeneration of mouse M-cone©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.