Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologynumbers were similar to each other in the H vs J cybrids(1.34±0.0228 vs 1.36±0.017, p=0.59).Conclusions: The OCR/ECAR ratio confirms that the H cybrids haveincreased OXPHOS while the J cybrids use glycolysis. This findingis supported by lower expression levels of the mtDNA encoded genesin complex I (MT-ND1, MT-ND2, MT-ND3, MT-ND4/ND4L), IV(MT-CO2, MT-CO3) and V (MT-ATP6). Our findings show thatmtDNA variants can mediate energy production pathways andmtRNA expression which in turn may alter the signaling pathwaysand stress-response patterns of the cells.Commercial Relationships: Cristina M. Kenney, None; MarilynChwa, None; Shari Atilano, None; Payam Falatoonzadeh, None;Deepika Malik, None; Claudio A. Ramirez, None; S MichalJazwinski, None; Miceli V. Michael, None; Baruch D.Kuppermann, Alimera (C), Allegro (C), Allergan (C), Genentech(C), Glaukos (C), GSK (F), Novagali (C), Novartis (C), Ophthotech(C), Pfizer (C), Regeneron (C), Santen (C), SecondSight (C), Teva(C), ThromboGenics (C)Support: Supported by Discovery Eye Foundation, GuentherFoundation, Beckman Macular Research Initiative, Polly andMichael Smith Foundation, Max Factor Family Foundation, SkirballFoundation, Lincy Foundation, Iris and B. Gerald Cantor Foundation,Unrestricted grant from Research to Prevent Blindness, grant fromNational Institute on AgingProgram Number: 5006 Poster Board Number: A0135Presentation Time: 2:45 PM - 4:30 PMThe serine protease HTRA1 is a potential regulator of theinflammatory cytokine GDF15Chloe M. Stanton 1 , Elod Kortvely 2 , Caroline Hayward 1 , MariusUeffing 2, 3 , Alan F. Wright 1 . 1 MRC Human Genetics Unit, MRCIGMM, University of Edinburgh, Edinburgh, United Kingdom;2 Centre for Ophthalmology, Institute for Opthalmic ResearchTubingen, Tubingen, Germany; 3 Research Unit of Protein Science,Helmholtz Zentrum Munchen, Munich, Germany.Purpose: An AMD risk locus on chr10q26 spans two genes -ARMS2 and HTRA1 - and controversy exists as to whether one orboth genes contribute to AMD. This study was performed toinvestigate the protein interaction network of the serine proteaseHTRA1 (high-temperature requirement A-1) and to elucidate apossible role in AMD pathogenesis.Methods: Potential interacting partners of HTRA1 were identified ina yeast two-hybrid screen performed using a placental cDNA library.Protein interactions were subsequently verified using recombinantproteins in pull-down assays. Proteins found to interact directly withHTRA1 were subjected to protease activity assays to identifysubstrates of the protease. Plasma GDF15 was measured by ELISA,and gene expression determined by RT-PCR.Results: Growth differentiation factor 15 (GDF15), a divergentmember of the TGFβ superfamily, with a proposed role in mediatinginflammation, interacts directly with HTRA1 in vitro. ProGDF15 is asubstrate for the HTRA1 protease in vitro. GDF15 and HTRA1 areexpressed at very low levels by monocyte-like cells, and theirexpression is highly upregulated in activated macrophages. PlasmaGDF15 is significantly elevated in AMD cases carrying thechromosome 10 risk haplotype, relative to disease-free controls, or toAMD cases not carrying the chromosome 10 risk haplotype.Conclusions: ProGDF15 is a substrate for the HTRA1 protease invitro, suggesting a regulatory role for the protease on thisinflammatory cytokine. Given the previously described role ofGDF15, the substantial genetic risk for AMD associated with thechromosome 10 risk haplotype, and the involvement of macrophagesin AMD, GDF15 may contribute towards pathological inflammationobserved in the disease. Further work to understand the geneticcontribution to regulating GDF15 expression in the generalpopulation, and in disease, and to determine the biologicalsignificance of the interaction between HTRA1 and GDF15 is ongoing.Commercial Relationships: Chloe M. Stanton, None; ElodKortvely, None; Caroline Hayward, None; Marius Ueffing, None;Alan F. Wright, NoneSupport: UK Medical Research CouncilProgram Number: 5007 Poster Board Number: A0136Presentation Time: 2:45 PM - 4:30 PMRole of complement in altered RPE function and depositformation in Efemp1 mutant mice: A primary cell culture modelRosario Fernandez-Godino, Eric A. Pierce, Donita Garland.Ophthalmology, Ocular Genomics Institute. Mass Eye and EarInfirmary. Harvard Medical School, Boston, MA.Purpose: The R345W mutation in the Efemp1 gene causes theinherited macular degeneration Doyne Honeycomb RetinalDystrophy/ Malattia Leventinese (DHRD/ML). Gene targetedEfemp1-R345W mice develop sub-RPE basal deposits and vacuolesin the RPE. In previous studies using Efemp1-R345W : complementC3 null mice we found that the complement system had a critical rolein the formation of basal laminar deposits in the mutant mice. Thepurpose of this study was to use a primary RPE cell culture system toinvestigate the mechanisms involved in the pathogenesis of basaldeposit formation in the Efemp1-R345W mutant mice and the role ofthe complement system in this process.Methods: Primary RPE cells were cultured from Efemp1-R345Wknockin, C3 null, and homozygous Efemp1-R345W:C3 null mice.The RPE cells, from 2.5 months old mice, were grown on apermeable support under polarizing conditions. mRNA expressionlevels of complement genes and Efemp1 were quantified by qRT-PCR. Protein expression in the RPE was determined by Western blotanalysis and immunofluorescence using confocal microscopy.Proteins secreted from both the apical and basal surfaces werequantified by Western blot analyses.Results: Results of qRT-PCR analysis reproducibly showed that RPEcells from each of the mouse strains express complement C3 mRNAand protein. The C3 null mice used for these studies express aninactive form of C3. Moreover, the major complement components ofthe classical pathway, C1q, C2, C4 as well as Cfh of the alternativepathway were also expressed in the RPE of each mouse strain. Theexpression of Efemp1 mRNA was significantly higher in Efemp1-R345W knockin mice compared to wild type (Wt)(ANOVA,p=0.0005). However, this overexpression was repressed inthe Efemp1-R345W:C3 null mice (p=0.0005).Conclusions: The primary RPE cell culture provides an excellentapproach to investigate the early effects of the Efemp1 mutation onRPE protein expression and also on the effects of alternations in thecomplement system on RPE function. The results suggested a criticalrole of C3 in the pathogenesis of the R345W mutation in DHRD/ML.They also suggest that the mutant EFEMP1 protein might cause localcomplement activation in the RPE, which in turn leads to RPE celldysfunction and an altered pattern of proteins secreted to Bruch’smembrane.Commercial Relationships: Rosario Fernandez-Godino, None;Eric A. Pierce, None; Donita Garland, NoneSupport: Ocular Genomics InstituteProgram Number: 5008 Poster Board Number: A0137Presentation Time: 2:45 PM - 4:30 PM©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. 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