Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologya major mechanism for oxidative stress-induced RPE cell death.Moreover, we have screened a collection of 2,000 FDA-approvedcompounds for molecules that inhibit oxidative stress-induced RPEnecrosis.Methods: ARPE-19 cells were treated with different concentration ofH2O2 and harvested at different time points followed by cell deathmarker staining, as well as RNA, DNA and protein analyses.Compound screening was performed using MTT assay.Results: Morphology and molecular hallmarks of dying ARPE-19cells show strong evidence of necrotic cell death induced byoxidative stress. We observed partial chromosomal condensation(pyknosis) and permeability of cell membrane by propidium iodine(PI). ARPE-19 cells treated with H2O2 did not exhibit apoptoticDNA fragmentation, consistent with our finding that ARPE-19 cellshave very low level of DFF45 protein, inhibitory subunit of DFF40main apoptotic endonuclease. RPE cell death is accompanied byincreased levels of PGAM5 and RIP3 both critical for undergoingprogrammed necrosis. Consistently, necrostatin-1, but not caspaseinhibitor zVAD, rescued oxidative stress-induced RPE cell death.Additionally our results showed that autophagic cell death is not aresult of H2O2 treatment in RPE cells. Furthermore, we screenedFDA-approved compounds and identified candidates thatsuccessfully rescued RPE cells from oxidative stress-induced celldeath.Conclusions: We provide unequivocal evidence that necrosis is amajor mechanism for oxidative stress-induced RPE cell death. Wealso identified natural compounds that can rescue oxidative stressinducedRPE cell death. Taken together, these findings provide novelinsight into the mechanism of RPE cell death, and will beinstrumental for developing novel therapeutics for dry AMD,especially GA.Commercial Relationships: Jakub Hanus, None; William C.Anderson, None; Peng Jin, None; Qinghua Liu, None; ShushengWang, UT Southwestern Medical Center (P)Support: Startup fund of Tulane University (S.W.), President’sResearch Council New Investigator Award from UT SouthwesternMedical Center (S.W.), NIH Grants EY021862 (S.W.), CareerDevelopment Award from the Research to Prevent BlindnessFoundation (S.W.).Program Number: 5002 Poster Board Number: A0131Presentation Time: 2:45 PM - 4:30 PMPhosphorylation Networks in Age-Related Macular DegenerationSrinivas R. Sripathi 1 , O'Donnell Sylvester 2 , Trevor Moser 1 , Paul S.Bernstein 3 , Folami Lamoke 4 , Manuela Bartoli 4 , Wan Jin Jahng 2 .1 Biological Sciences, Michigan Tech University, Houghton, MI;2 Retina Proteomics Laboratory, Petroleum Chemistry, AmericanUniversity of Nigeria, Yola, Nigeria; 3 Ophthalmology and VisualSciences, Moran Eye Center, University of Utah, Salt Lake City, UT;4 Ophthalmology, Georgia Health Sciences University, Augusta, GA.Purpose: Age-related macular degeneration (AMD) results from thechronic atrophy of retina and retinal pigment epithelial (RPE) cells(dry AMD) or choroidal neovascularization (wet AMD). However,the molecular mechanism underlying the progression of AMD is notwell defined yet. Light-induced oxidative stress, lipid oxidation,accumulation of soft drusen, and crystalline aggregation maycontribute to AMD pathogenesis. The aim of our phosphoproteomestudy is to identify the early biochemical events and specificphosphorylated sites in the macular, peripheral retina and RPE. Ourdata demonstrated that specific kinase/phosphatase regulatedapoptotic phosphorylation signaling in AMD progression.Methods: The senescence-associated molecular events during AMDprogression were identified by comparing the phosphoproteome ofmacular and peripheral retina, RPE from different stages of AMDcompared to age-matching control donor eyes. Phosphoproteins wereenriched by charge-based spin column chromatography and resolvedby 2D gel electrophoresis. Tryptic peptides were enriched usingGa3+/TiO2 immobilized metal ion chromatography. Elutedphosphopeptides were analyzed and confirmed by MALDI-TOF orESI/MS/MS mass spectrometry and phospho-Western blotrespectively.Results: Our preliminary data shows tyrosine phosphorylation ofbeta crystallin A3 and A4 increased but serine phosphoryaltion ofbeta crystallin decreased in RPE under stress. Our phospho-Westernanalysis reveals phosphorylation of intermediate filament vimentin(Ser) and mitochondrial heat shock protein mt Hsp70.Serine/threonine phosphatase PP2A-Cα/β (Tyr) and tubulin α1B/β2were dephosphorylated in the RPE. Further, our data shows thatprohibitin is upregulated in the retina, but down regulated in the RPEfrom AMD donor eyes. Our study suggests that altered lipidcomposition may determine phosphorylation-dependent interactionsof target proteins that contain PH, PX, and SH domains.Conclusions: The region specific proteome changes incentral/peripheral retina and RPE from AMD donor eyes and agematchingcontrols were analyzed. Phosphorylation changes anddownstream activation may leads to the early biochemical events inAMD. Our phosphoproetomic strategy provides the detailedphosphorylation signaling,phospholipid changes, altered proteinaggregation,binding affinity and expression that include depletedlevels of anti-apoptotic, mitochondrial chaperone prohibitin in theRPE of AMD eyes.Commercial Relationships: Srinivas R. Sripathi, None; O'DonnellSylvester, None; Trevor Moser, None; Paul S. Bernstein, Kalsec(C), Kemin Health (R), Science Based Health (C), Abbott Nutrition(F), Genentech (C), DSM (R), Sequenom (R), NuSkin/Pharmanex(P), Aciont (C), Thrombogenics (C); Folami Lamoke, None;Manuela Bartoli, None; Wan Jin Jahng, NoneProgram Number: 5003 Poster Board Number: A0132Presentation Time: 2:45 PM - 4:30 PMEvaluation of RPE cell senescence as a mechanism for agerelatedmacular degeneration (AMD),Michael R. Kozlowski. Arizona College of Optometry, MidwesternUniversity, Glendale, AZ.Purpose: The purpose of this work is to assess whether or not thechanges seen in retinal pigment epithelial (RPE) cells as theyapproach cell senescence are consistent with the pathologicalprocesses that occur in age-related macular degeneration (AMD).Methods: In this study, ARPE-19 cells grown on membranecontainingmulti-well plate inserts or in cell culture flasks were usedto model the natural RPE cell layer. The cells were cultured to highpopulation doubling levels (PDL’s) and several measures of cellgrowth and function were assessed including doubling rate, viability,morphology, terminal restriction fragment (TRF) length (as ameasure of telomere erosion), trans-epithelial electrical resistance(TEER, as a measure of the quality of their tight junctions), andsenescence associated beta-galactosidase activity (SABG).Results: As ARPE-19 cells reached high population doubling levels(PDL), which is one trigger for cell senescence, they experiencedsignificant telomere erosion and displayed several senescenceassociatedchanges. First, their staining for SABG, a marker forsenescence which is related to lysosomal dysfunction, was elevated.Second, their shape became more rounded, possibly indicating achange in their adhesion characteristics. Finally, the depression oftheir TEER values by VEGF was significantly enhanced.Conclusions: The changes seen in ARPE-19 cells that had reached©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.