Biochemistry/Molecular Biology - ARVO

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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.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologypigment during dark adaptation. We also examined the role ofprocessing of visual chromophore (retinal) in cone outer segments(COS) by RDH8 and ABCA4 in cone dark adaptation.Methods: To test cone dark adaptation following >90% pigmentbleach, we performed transretinal ERG recordings (a-wave) fromcones in isolated Gnat1-/- mouse retinas lacking rod signaling in thepresence of synaptic blockers or standard ERG (b-wave) in liveanimals. To separate effects of the two visual cycles, we appliedintravitreal injections of light-inducible toxin Killer Red to destroyglial Müller cells that can regenerate 11-cis-retinol intraretinally or IPinjections of retinylamine, a potent inhibitor of the RPE visual cycle.The role of retinal processing in COS was tested in Rdh8-/-Abca4-/-Gnat1-/- triple knockout (TKO) mice. Photopic visual acuity inGnat1-/- and TKO mice was determined from optomotor responses.Results: M-cone dark adaptation was 4-6-fold slower in live Gnat1-/-mice treated with retinylamine indicating substantial contribution ofthe RPE visual pathway. In addition, cones in retinylamine-treatedmice were unable to maintain their sensitivity in steady bright light.On the other hand, retinylamine only slightly reduced the final levelof cone sensitivity recovery in retinas detached from the RPE. Miceinjected with Killer Red exhibited up to 2-fold lower postbleachsensitivity in their isolated retinas showing the importance of theretina visual cycle. Simultaneous deletion of RDH8 and ABCA4(whose expression in COS of Gnat1-/- mice was confirmed by IHC)retarded cone dark adaptation by 2-fold in both live animals andisolated retinas. Surprisingly, fully dark-adapted TKO cones were notchromophore-deficient, as evidenced by treatment with exogenous11-cis-retinal, but had accelerated responses compared with controlcones. Finally, the photopic visual acuity was reduced in adult TKOanimals.Conclusions: Dark adaptation of mouse cones requires the combinedaction of both RPE and retina visual cycles. Efficient processing ofretinal by RDH8 and ABCA4 in COS is essential for rapid andcomplete dark adaptation of cones.Commercial Relationships: Alexander V. Kolesnikov, None;Peter H. Tang, None; Akiko Maeda, None; Leah C. Byrne, None;John G. Flannery, None; Krzysztof Palczewski, QLT Inc (F),Polgenix Inc (E), Visum Inc (P), Amegen Inc (F); Vladimir J.Kefalov, NoneSupport: NIH Grants EY019312 and EY21126 and Research toPrevent Blindness (VJK), NIH Grants EY008061 and EY021126(KP), the Foundation Fighting Blindness (JGF).Program Number: 1700Presentation Time: 11:15 AM - 11:30 AMIdentification and Study of Two New 11-cis-Retinyl EsterSynthases in the RetinaJoanna J. Kaylor 1 , Jacob Makshanoff 1 , Jennifer Yong 1 , Tran N.Nguyen 1 , Roxana A. Radu 1 , Gabriel H. Travis 1, 2 . 1 Ophthalmology,UCLA-Jules Stein Eye Institute, Los Angeles, CA; 2 BiologicalChemistry, UCLA, Los Angeles, CA.Purpose: Biochemical and electrophysiological evidence show thatcones regenerate visual pigments by an enzymatic pathway thatoccurs in the Müller cells of the retina. Prior work from ourlaboratory indicates the existence of a retinol isomerase activity incone-dominant chicken and ground squirrel retinas. When all-transretinoland palmitoyl coenzyme A are added to microsomes preparedfrom these retinas predominantly 11-cis-retinyl esters are produced.We now have preliminarily identified the retinol isomerase asDesaturase 1 (DES1). This enzyme produces an equilibrium mixtureof cis-retinol products from all-trans-retinol, including 11-cis-retinol.In order for DES1’s retinol isomerase activity to be utilized forpigment regeneration the 11-cis-retinol production must be driven bymass action due to other 11-cis-specific enzymes downstream.Identification of the enzyme(s) that are functionally coupled to DES1that produce 11-cis-retinyl esters is the goal of this project.Methods: DES1, DGAT1 and MFAT cDNA were subcloned intomammalian expression vector pcDNA3.1. dgat1-/- mice wereobtained from Dr. Robert Farese at UCSF. Enzyme activity assayswere conducted by transfection of clones into HEK 293T cells(Polyfect, Qiagen). In vitro homogenate assays using different retinolisomers as substrate were carried out in pH 7.2 40 mM Tris buffer.Retinoids were extracted with hexane and high performance liquidchromotography (HPLC) was used to identify and quantify retinoidsformed. Numerous experimental techniques were undertaken tounderstand these proteins including gene expression (qRT-PCR andWestern), Michaelis-Menten kinetic analysis, and co-expressionstudies with DES1.Results: DGAT1 and MFAT catalyze the synthesis of 11-cis-retinylesters. MFAT preferentially esterifies 11-cis-retinol better thanDGAT1 based on kinetic analysis. Gene expression studies confirmthey are both located in the retina of the eye. dgat1-/- mice havereduced retinyl esters in the eye and assays on homogenates of eyetissues from these mice have greatly reduced 11-cis-retinyl estersynthesis. DGAT1 or MFAT coexpressed with DES1 showproduction of 11-cis-retinyl esters.Conclusions: MFAT and DGAT1 are robust 11-cis-retinyl estersynthases that may be functionally coupled to DES1 and act asdownstream regulators of 11-cis-specificity in the alternate visualcycle of the retina.Commercial Relationships: Joanna J. Kaylor, None; JacobMakshanoff, None; Jennifer Yong, None; Tran N. Nguyen, None;Roxana A. Radu, None; Gabriel H. Travis, NoneSupport: US National Eye Institute R01-EY11713Program Number: 1701Presentation Time: 11:30 AM - 11:45 AMSubstrate Specificity and Localization of AL-OL CouplingReaction in Carp ConesSatoru Kawamura 1, 2 , Shinya Sato 2 , Shuji Tachibanaki 1, 2 , TakashiFukagawa 1 . 1 Grad Sch of Frontier Biosci, Osaka Univ, Suita, Japan;2 Dept of Biol Sci, Faculty of Sci, Osaka Univ, Toyonaka, Japan.Purpose: Cones are known to regenerate visual pigments from 11-cisretinol. Our previous study showed that carp cones specifically havean enzyme activity to oxidize 11-cis retinol (alcohol) to 11-cis retinal(aldehyde) with concomitant reduction of all-trans retinal to all-transretinol (AL-OL coupling reaction, Miyazono et al., 2008, PNAS 105:16051). In this reaction, it was not necessary to add NADP + , acofactor required for a conventional retinol dehydrogenase activity.In the present study, we tried to examine the substrate specificity andlocalization of this AL-OL coupling reaction.Methods: Cones were isolated from carp retinas using a stepwisePercoll density gradient (Tachibanaki et al., PNAS, 102: 9329).Substrate specificity was examined by a pair of alcohol and aldehydeof various retinoids and related compounds. Reaction products wereanalyzed and quantified with HPLC. To examine the localization ofthe reaction, the activity was first measured in the membrane fractionand in the soluble fraction of our cone preparation, and then in a coneouter segment (COS)-rich and cone inner segment (CIS)-richmembrane fractions prepared by centrifugation after mechanicaltreatment of purified cones.Results: In the presence of all-trans retinal, 11-cis retinol and 9-cisretinol were oxidized, but other alcohols including all-trans retinoland 13-cis retinol were not oxidized. Besides all-trans retinal,hydrophobic aldehydes such as benzaldehyde and dodecanal wereeffective in the oxidation of 11-cis retinol. AL-OL coupling activity©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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