Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyassociated vision loss. Our purpose is to investigate the structural andbiophysical effects of stabilizing compounds on the myocilin-OLFdomain, and evaluate their ability to prevent myocilin-OLFaggregation.Methods: Expression and purification of the OLF domain ofmyocilin, development of high throughput assay to discover ligands,development of assay to monitor rate and extent of aggregation,surface plasmon resonance to evaluate binding mode, molecularbiophysical characterization of formed aggregates.Results: Disease-associated OLF variants access a partially unfoldedstate under physiological conditions that increases their propensity togrow amyloid fibrils. Compounds that thermally stabilize the OLFdomain, such as osmolytes and calcium ions, reduce aggregation in acompound-specific manner. Ligands discovered by high throughputscreening also affect aggregation.Conclusions: Different categories of OLF-stabilizing smallmolecules, general and tailored, may prevent the accumulation ofmyocilin in TM cells, and thereby provide a potential new therapy formyocilin glaucoma.Commercial Relationships: Raquel L. Lieberman, None;Shannon E. Hill, None; Rebecca K. Donegan, None; Katherine C.Turnage, NoneSupport: NIH Grant R01EY021205Program Number: 4005Presentation Time: 9:15 AM - 9:30 AMPGC-1alpha Signaling Regulates Retinal Ganglion Cell InjuryResponses And Atrocyte ReactivityJeremy M. Sivak 1, 2 , Xiaoxin Guo 1 , Shirin Dason 2 , Adrian Nahirny 1 .1 Vision Sciences, Toronto Western Hospital, University HealthNetwork, Toronto, ON, Canada; 2 Ophthalmology and VisionSciences, University of Toronto, Toronto, ON, Canada.Purpose: Glaucomatous optic neuropathy is associated with acomplex combination of risk factors, including age, increasedintraocular pressure, and vascular disregulation. These stressesconverge to damage the sensitive nerve fiber layer (NFL), and opticnerve head, resulting in the death of retinal ganglion cells (RGCs).However, a molecular mechanism has not yet been identified thatintegrates local tissue responses to these varied insults. We havegenerated data identifying the transcriptional co-activator, PGC-1α(peroxisome proliferator-activated receptor gamma co-activator 1alpha), as a potential key mediator of this process. When activated,PGC-1α interacts with transcription factors regulating metabolic,oxidative, and ischemic stress. Through these interactions PGC-1αacts as a master cellular regulator of adaptive energy metabolism.Methods: Expression of PGC-1α was analyzed in acute and chronicmouse retinal injury models by quantitative rt-PCR, and in-situhybridization. Putative PGC-1α targets, markers of metabolic andoxidative stress, glial reactivity, and RGC death were assessed by rt-PCR, immunofluorescence microscopy, and TUNEL assay, incombination with loss- and gain-of-function techniques.Results: PGC-1α expression was increased in RGCs and the NFLwith age, and in a model of chronic ocular hypertension, as well asfollowing acute excitotoxic retinal injury. PGC-1α loss-of-functionresulted in significantly increased retinal astrocyte reactivity, andsensitivity of RGCs to damage, while increased activity wasprotective against oxidative stress.Conclusions: We have identified an important pathway that canintegrate retinal damage responses common to a variety ofpathologically relevant insults.Commercial Relationships: Jeremy M. Sivak, None; Xiaoxin Guo,None; Shirin Dason, None; Adrian Nahirny, NoneSupport: CNIB, Glaucoma Research Society of Canada, CanadianInstitutes for Health Research (CIHR), TGH and TWH FoundationGlaucoma Res ChairProgram Number: 4006Presentation Time: 9:30 AM - 9:45 AMSubcellular mislocalization and loss of function of mutantGpnmb proteinsAlexander C. Theos. Human Science, Georgetown University,Washington, DC.Purpose: Pigmentary glaucoma involves optic nerve degenerationafter accumulation of debris originating from pigmented cells withinthe eye, a frequent endpoint for Pigment Dispersion Syndrome (PDS)patients . The molecular events within pigmented ocular cells thatprecipitate this initial pigment dispersion remain uncharacterized.The DBA/2J mouse is an established model for human PDS andharbors a truncated mutant Gpnmb R150X allele which has beenproposed to be a functional null. To date the impact of C-terminaltruncation on the intracellular sorting and biology of Gpnmb has yetto be determined.Methods: We have characterized the intracellular transport pathwayfollowed by wild-type Gpnmb and compared this to both Gpnmb R150Xand cytoplasmic domain mutants, including C-terminal truncationmutants that lack candidate sorting signals within the cytoplasmicdomain of this integral membrane protein. We have assessed theintracellular transport of these mutant proteins by ectopic expressionin transfected tissue culture cells followed by immunofluorescencemicroscopy. Early secretory pathway localization of protein atsteady-state has been determined by endoglycosidase-H(endoH)-sensitivity in Western blotting experiments. Defects in endocyticsorting have been further analyzed by antibody uptake experiments.Results: The severe truncation mutant Gpnmb R150X is unable to leavethe endoplasmic reticulum after synthesis as shown by intracellularsteady-state localization to calnexin-positive membranes andcomplete endoH sensitivity. Cytoplasmic truncation mutants thatretain the single transmembrane domain are able to progress throughthe secretory pathway but fail to be internalized upon delivery to thecell surface. Efficient endocytosis is dependent upon a functionalcytoplasmic dileucine sorting signal, as shown by steady-stateindirect immunofluorescence microscopy as well as antibody uptakeendocytosis assays.Conclusions: The wild-type function of Gpnmb in healthy pigmentedcells within the eye requires the efficient intracellular targeting ofGpnmb to endosome-derived organelles. Loss of critical sortingsignals, including a conserved cytoplasmic dileucine, results inmissorting and so loss of function leading to aberrant melanosomemorpohology in DBA/2J animals. Our data support a model for aprotective function of Gpnmb within the internal environment of themelanosome.Commercial Relationships: Alexander C. Theos, NoneSupport: AHAF National Glaucoma Research, Standard AwardG2012025Program Number: 4007Presentation Time: 9:45 AM - 10:00 AMCochlin and phosphatidylcholines interact with SLC44A2channel on the trabecular meshwork cellsSanjoy K. Bhattacharya, Mitchell Martinez, Ayman Aljohani, RichardK. Lee. Bascom Palmer Eye Institute, Univ of Miami Miller Sch ofMed, Miami, FL.Purpose: To demonstrate that cochlin and phosphatidylcholines (PC)interact with choline transporter-like channel SLC44A2. Our overallhypothesis is that cochlin and cholines both interact and modulate©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.