Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyof this study was to determine in human samples whether a lipidomicapproach based on red blood cells could reveal associations betweencirculating and ocular lipid profiles.Methods: Red blood cells, retinas and optic nerves were collectedfrom 9 human donors. The lipidomic analyses on these tissues weredone by both, gas chromatography and liquid chromatographycoupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS/MS).Results: Gas chromatographic approach did not show any relevantassociation between circulating and ocular lipids except forarachidonic acid whose circulating amounts were positivelyassociated with its retinal and optic nerve levels. However,significant associations emerged from LC-ESI-MS/MS analyses.Indeed, phospholipid species in red blood cells were positively ornegatively associated with representative pools of retinal DHA(docosahexaenoic acid), retinal VLC-PUFA (very-long chainpolyunsaturated fatty acids) or optic nerve plasmalogens.Conclusions: First, our results show that LC-ESI-MS/MSmethodology is more appropriate than gas chromatography forlipidomics on red blood cells, and further extrapolation to ocularlipids. Second, this study has identified several individual lipidspecies that can be good candidates to represent circulatingbiomarkers of ocular lipids.Commercial Relationships: Niyazi Acar, None; Olivier Berdeaux,None; Zhiguo He, None; Stéphanie Cabaret, None; Philippe Gain,None; Gilles Thuret, None; Catherine P. Garcher, Alcon (C),Allergan (C), Baush and Lomb (C), Bayer Pharma (C), Novartis (C),Laboratoire Théa (C); Alain M. Bron, Allergan (C), Bausch Lomb(C), Horus (F), Théa (C); Lionel Bretillon, NoneProgram Number: 3758 Poster Board Number: A0097Presentation Time: 2:45 PM - 4:30 PMInflammatory cytokines decrease cell viability and alterganglioside profile in retinal pigment epithelium cells (ARPE19)Elodie A. Masson 1 , Olivier Berdeaux 1, 2 , Stéphanie Cabaret 1, 2 , LionelBretillon 1 . 1 Eye and Nutrition Research Group, UMR CSGA-1324INRA-6265 CNRS-University of Burgundy, Dijon, France;2 ChemoSens Platform, UMR CSGA-1324 INRA-6265 CNRS-University of Burgundy, Dijon, France.Purpose: Early stages of Age related Macular Degeneration (AMD)are characterized by dysfunction and degeneration of the retinalpigment epithelium (RPE) cells. These alterations participate in thedeath of the overlying photoreceptors ultimately leading to loss ofvision. It is therefore crucial to understand this initial event in orderto prevent the pathology development. Gangliosides (GG) make awide family of sialic acid-containing glycosphingolipids. Both theoligosaccharidic chain and the ceramide moiety (sphingoïd base andfatty acid) exhibit a huge heterogeneity in their structure. Thebiological role of this heterogeneity is largely unknown. GG areparticularly abundant in the central nervous system, including theretina. While their developmental and neuroprotective actions havebeen demonstrated, their precise role in retina’s function and itspathologies is still poorly understood.In the present study, we aimed to investigate the role of GG in theresponse of RPE cells to inflammation, which is known as one of thepathophysiological features of AMD.Methods: Cultured human RPE cells (ARPE19) were exposed to aninflammatory cytokine mix (ICM): TNF-α, IL-1β and IFN-γ for 72hours. Cell viability was assessed by direct cell counting andmeasurement of total DNA content. GG were analyzed by ReversedPhase High Pressure Liquid Chromatography, coupled with tandemmass spectrometry (RPHPLC-ESI-MSMS).Results: ICM had deleterious effects on ARPE19 viability. Indeed,the cell number decreased by half between control and treatedconditions based both on cell counting and measurement of DNAcontent. GM3 appeared to be the main GG class present in ARPE19cells. Interestingly, ICM exposure was associated with modificationsin the GM3 profile. Especially, the relative amounts of d16:1/18:0and/or d18:1/16:0 species increased whereas those of the d18:1/24:1and the d18:1/24:0 and/or d20:1/22:0 species decreased. Thesechanges reflected an increase in short chain fatty acids to thedetriment of long chain fatty acids.Conclusions: Our observations suggest that GG might be implicatedin ARPE19 cell response to inflammatory cytokines. Furtherinvestigations are required to understand the precise biological role ofthis change in fatty acid profile of GM3 in the deleterious effects ofinflammation on RPE cells.Commercial Relationships: Elodie A. Masson, None; OlivierBerdeaux, None; Stéphanie Cabaret, None; Lionel Bretillon, NoneProgram Number: 3759 Poster Board Number: A0098Presentation Time: 2:45 PM - 4:30 PMN-Terminal Region of Fatty Acid Transport Protein 4 (FATP4) isImportant for Interacting with and Inhibiting RPE65 IsomeraseSonghua Li 1 , John F. Green 2 , Jean T. Jacob 2 , Minghao Jin 1, 2 .1 Neuroscience, LSU Health Sciences Center, New Orleans, LA;2 Ophthalmology, LSU Health Sciences Center, New Orleans, LA.Purpose: In a previous study, we identified FATP4 as negativeregulator of RPE65 isomerase, which catalyzes isomerization of alltransretinyl fatty acid esters to 11-cis retinol (11cROL) for the visualcycle in the retinal pigment epithelium (RPE). The purpose of thisstudy is to identify the region(s) of FATP4 that interacts with RPE65and inhibits synthesis of 11-cis retinol.Methods: A mammalian expression vector encoding the bovineFATP4 (pRK-FATP4) was used to construct various mutant FATP4swith a Flag tag. A series of N-terminal or C-terminal deletionmutations and the Ichthyosis Prematurity Syndrome (IPS)-associatedmissense or non-sense mutations (A92T, S247P, Q300R, and C168X)were individually introduced into pRK-FATP4. These mutantFATP4s and RPE65 were expressed in 293T cells stably expressingLRAT and/or CRALBP (293T-LC or 293T-C cells). Interaction ofwild-type and mutant FATP4s with RPE65 in the cells wasdetermined by immunoprecipitation. The inhibitory effects of themutant FATPs on RPE65 activity were compared by measuringsynthesis of 11cROL from all-trans retinyl palmitate or all-transretinol added into the cell homogenates or medium of the culturecells.Results: Synthesis of 11-cis retinol in the cells transfected with pRK-RPE65 and pRK-FATP4 (wild-type FATP4) was 60% lower thanthat of control cells transfected with pRK-RPE65 and pRK5 mockvector. Under the same experimental conditions, C-terminal regionsof FATP4 had no inhibitory effect on the synthesis of 11-cis retinol.These regions were not able to co-precipitate RPE65 inimmunoprecipitation. In contrast, N-terminal region of FATP4significantly inhibited synthesis of 11cROL. The inhibitory effect ofthe N-terminal region was appromaximitely 70% of wild-typeFATP4. The N-terminal region of FATP4 was able to co-precipitateRPE65 in immunoprecipitation. The IPS-associated FATP4s, whichlost very long-chain fatty acid-CoA synthetase activity, also inhibitedsynthesis of 11cROL. However, their inhibitory efficiencies werereduced 20-30% compared to the inhibitory efficiency of wild-typeFATP4.Conclusions: Our results indicate that the very long-chain fatty acid-CoA synthetase activity of FATP4 partially contributes to itsinhibitory effect on synthesis of 11cROL and the N-terminal region©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.