{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T11:24:13Z","timestamp":1789125853840,"version":"build-2803163510"},"reference-count":36,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,10,20]],"date-time":"2023-10-20T00:00:00Z","timestamp":1697760000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Mol. Neurosci."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>The vertebrate retinal pigment epithelium (RPE) lies adjacent to the photoreceptors and is responsible for the engulfment and degradation of shed photoreceptor outer segment fragments (POS) through receptor-mediated phagocytosis. Phagocytosis of POS is critical for maintaining photoreceptor function and is a key indicator of RPE functionality. Popular established methods to assess RPE phagocytosis rely mainly on quantifying POS proteins, especially their most abundant protein rhodopsin, or on fluorescent dye conjugation of bulk, unspecified POS components. While these approaches are practical and quantitative, they fail to assess the fate of POS lipids, which make up about 50% of POS by dry weight and whose processing is essential for life-long functionality of RPE and retina.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>We have developed a novel very-long-chain polyunsaturated fatty acids (VLC-PUFA)-based approach for evaluating RPE phagocytic activity by primary bovine and rat RPE and the human ARPE-19 cell line and validated its results using traditional methods.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results and discussion<\/jats:title><jats:p>This new approach can be used to detect <jats:italic>in vitro<\/jats:italic> the dynamic process of phagocytosis at varying POS concentrations and incubation times and offers a robust, unbiased, and reproducible assay that will have utility in studies of POS lipid processing.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fnmol.2023.1279457","type":"journal-article","created":{"date-parts":[[2023,10,20]],"date-time":"2023-10-20T06:14:06Z","timestamp":1697782446000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":8,"title":["A novel quantification method for retinal pigment epithelium phagocytosis using a very-long-chain polyunsaturated fatty acids-based strategy"],"prefix":"10.3389","volume":"16","author":[{"given":"Fangyuan","family":"Gao","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emily","family":"Tom","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephanie A.","family":"Lieffrig","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Silvia C.","family":"Finnemann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dorota","family":"Skowronska-Krawczyk","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2023,10,20]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1016\/0042-6989(91)90069-H","article-title":"Anti-rhodopsin monoclonal antibodies of defined specificity: characterization and application","volume":"31","author":"Adamus","year":"1991","journal-title":"Vis. Res."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"12843","DOI":"10.1073\/pnas.0802607105","article-title":"Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids","volume":"105","author":"Agbaga","year":"2008","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"1613","DOI":"10.1007\/s00417-017-3679-9","article-title":"A novel MERTK mutation causing retinitis pigmentosa","volume":"255","author":"Al-khersan","year":"2017","journal-title":"Graefes Arch. Clin. Exp. Ophthalmol."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"1172","DOI":"10.1016\/S0021-9258(19)75767-6","article-title":"A novel group of very long chain polyenoic fatty acids in dipolyunsaturated phosphatidylcholines from vertebrate retina","volume":"262","author":"Avelda\u00f1o","year":"1987","journal-title":"J. Biol. Chem."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"175","DOI":"10.1002\/jps.2600770216","article-title":"Estimation of the rate constants in a data-sparse environment: comparison of a mathematical method and least squares analysis","volume":"77","author":"Barcia","year":"1988","journal-title":"J. Pharm. Sci."},{"key":"ref6","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1007\/978-1-4614-3209-8_11","article-title":"In vivo and in vitro monitoring of phagosome maturation in retinal pigment epithelium cells","volume-title":"Retinal degenerative diseases","author":"Esteve-Rudd","year":"2014"},{"key":"ref7","doi-asserted-by":"publisher","first-page":"12932","DOI":"10.1073\/pnas.94.24.12932","article-title":"Phagocytosis of rod outer segments by retinal pigment epithelial cells requires \u03b1v\u03b25 integrin for binding but not for internalization","volume":"94","author":"Finnemann","year":"1997","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"3842","DOI":"10.1073\/pnas.052025899","article-title":"The lipofuscin component A2E selectively inhibits phagolysosomal degradation of photoreceptor phospholipid by the retinal pigment epithelium","volume":"99","author":"Finnemann","year":"2002","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref9","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1007\/0-387-32442-9_69","article-title":"Mertk activation during RPE phagocytosis in vivo requires \u03b1V\u03b25 integrin","volume-title":"Retinal degenerative diseases","author":"Finnemann","year":"2006"},{"key":"ref10","doi-asserted-by":"publisher","first-page":"270","DOI":"10.1038\/81555","article-title":"Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa","volume":"26","author":"Gal","year":"2000","journal-title":"Nat. Genet."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"118","DOI":"10.1073\/pnas.2017739118","article-title":"Retinal bioavailability and functional effects of a synthetic very-long-chain polyunsaturated fatty acid in mice","volume":"118","author":"Gorusupudi","year":"2021","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"509","DOI":"10.1006\/exer.2001.1062","article-title":"Outer segment phagocytosis by cultured retinal pigment epithelial cells requires Gas6","volume":"73","author":"Hall","year":"2001","journal-title":"Exp. Eye Res."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"277","DOI":"10.1002\/jlb.45.4.277","article-title":"Confocal scanning fluorescence microscopy: a new method for phagocytosis research","volume":"45","author":"Hook","year":"1989","journal-title":"J. Leukoc. Biol."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/s12967-018-1434-6","article-title":"RPE phagocytic function declines in age-related macular degeneration and is rescued by human umbilical tissue derived cells","volume":"16","author":"Inana","year":"2018","journal-title":"J. Transl. Med."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"1071","DOI":"10.1126\/science.982063","article-title":"Rod outer segment disk shedding in rat retina: relationship to cyclic lighting","volume":"194","author":"LaVail","year":"1976","journal-title":"Science"},{"key":"ref16","doi-asserted-by":"publisher","first-page":"4941","DOI":"10.1074\/jbc.M114.628297","article-title":"Cleavage of Mer tyrosine kinase (MerTK) from the cell surface contributes to the regulation of retinal phagocytosis","volume":"290","author":"Law","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"101037","DOI":"10.1016\/j.preteyeres.2021.101037","article-title":"Dynamic lipid turnover in photoreceptors and retinal pigment epithelium throughout life","volume":"89","author":"Lewandowski","year":"2021","journal-title":"Prog. Retin. Eye Res."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/s12860-018-0177-9","article-title":"Kif17 phosphorylation regulates photoreceptor outer segment turnover","volume":"19","author":"Lewis","year":"2018","journal-title":"BMC Cell Biol."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"191","DOI":"10.1016\/j.chroma.2013.07.103","article-title":"Comprehensive and sensitive quantification of long-chain and very long-chain polyunsaturated fatty acids in small samples of human and mouse retina","volume":"1307","author":"Liu","year":"2013","journal-title":"J. Chromatogr. A"},{"key":"ref20","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1007\/978-1-62703-080-9_20","article-title":"Analysis of photoreceptor outer segment phagocytosis by RPE cells in culture","volume-title":"Retinal degeneration","author":"Mao","year":"2012"},{"key":"ref21","doi-asserted-by":"publisher","first-page":"1104","DOI":"10.1091\/mbc.e11-10-0840","article-title":"Essential diurnal Rac1 activation during retinal phagocytosis requires \u03b1v\u03b25 integrin but not tyrosine kinases focal adhesion kinase or Mer tyrosine kinase","volume":"23","author":"Mao","year":"2012","journal-title":"Mol. Biol. Cell"},{"key":"ref22","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1007\/978-1-4939-8669-9_7","article-title":"Advanced analysis of photoreceptor outer segment phagocytosis by RPE cells in culture","volume-title":"Retinal degeneration: methods and protocols","author":"Mazzoni","year":"2019"},{"key":"ref23","doi-asserted-by":"publisher","first-page":"12005","DOI":"10.1073\/pnas.0704756104","article-title":"Essential role for MFG-E8 as ligand for \u03b1v\u03b25 integrin in diurnal retinal phagocytosis","volume":"104","author":"Nandrot","year":"2007","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"81","DOI":"10.1016\/0165-5728(90)90150-L","article-title":"Adult thymus expresses an embryonic nicotinic acetylcholine receptor-like protein","volume":"29","author":"Nelson","year":"1990","journal-title":"J. Neuroimmunol."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"3096","DOI":"10.3390\/nu15143096","article-title":"Understanding the roles of very-long-chain polyunsaturated fatty acids (VLC-PUFAs) in eye health","volume":"15","author":"Nwagbo","year":"2023","journal-title":"Nutrients"},{"key":"ref26","doi-asserted-by":"publisher","first-page":"2604","DOI":"10.1128\/IAI.69.4.2604-2611.2001","article-title":"Helicobacter pylori resists phagocytosis by macrophages: quantitative assessment by confocal microscopy and fluorescence-activated cell sorting","volume":"69","author":"Ramarao","year":"2001","journal-title":"Infect. Immun."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"6228","DOI":"10.1038\/ncomms7228","article-title":"Adiponectin receptor 1 conserves docosahexaenoic acid and promotes photoreceptor cell survival","volume":"6","author":"Rice","year":"2015","journal-title":"Nat. Commun."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"e202101063","DOI":"10.1083\/jcb.202101063","article-title":"Nano-scale resolution of native retinal rod disk membranes reveals differences in lipid composition","volume":"220","author":"Sander","year":"2021","journal-title":"J. Cell Biol."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"1347","DOI":"10.1016\/j.stemcr.2020.10.013","article-title":"A human retinal pigment epithelium-based screening platform reveals inducers of photoreceptor outer segments phagocytosis","volume":"15","author":"Schreiter","year":"2020","journal-title":"Stem Cell Rep."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"6494","DOI":"10.1074\/jbc.M115.687004","article-title":"Regulation of phagolysosomal digestion by caveolin-1 of the retinal pigment epithelium is essential for vision","volume":"291","author":"Sethna","year":"2016","journal-title":"J. Biol. Chem."},{"key":"ref31","first-page":"2310","article-title":"Bruch\u2019s membrane aging decreases phagocytosis of outer segments by retinal pigment epithelium","volume":"13","author":"Sun","year":"2007","journal-title":"Mol. Vis."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"108503","DOI":"10.1016\/j.exer.2021.108503","article-title":"Deterioration of phagocytosis in induced pluripotent stem cell-derived retinal pigment epithelial cells established from patients with retinitis pigmentosa carrying Mer tyrosine kinase mutations","volume":"205","author":"Tagawa","year":"2021","journal-title":"Exp. Eye Res."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41598-020-69833-y","article-title":"Positional determination of the carbon\u2013carbon double bonds in unsaturated fatty acids mediated by solvent plasmatization using LC-MS","volume":"10","author":"Takashima","year":"2020","journal-title":"Sci. Rep."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"3661","DOI":"10.3390\/ijms23073661","article-title":"Probing photoreceptor outer segment phagocytosis by the RPE in vivo: models and methodologies","volume":"23","author":"Vargas","year":"2022","journal-title":"Int. J. Mol. Sci."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"6282","DOI":"10.1167\/iovs.12-9721","article-title":"Using flow cytometry to compare the dynamics of photoreceptor outer segment phagocytosis in iPS-derived RPE cells","volume":"53","author":"Westenskow","year":"2012","journal-title":"Invest. Ophthalmol. Vis. Sci."},{"key":"ref36","first-page":"105","article-title":"The daily rhythm of shedding and degradation of rod and cone outer segment membranes in the chick retina","volume":"17","author":"Young","year":"1978","journal-title":"Invest. Ophthalmol. Vis. Sci."}],"container-title":["Frontiers in Molecular Neuroscience"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fnmol.2023.1279457\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,10,20]],"date-time":"2023-10-20T06:14:12Z","timestamp":1697782452000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fnmol.2023.1279457\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,10,20]]},"references-count":36,"alternative-id":["10.3389\/fnmol.2023.1279457"],"URL":"https:\/\/doi.org\/10.3389\/fnmol.2023.1279457","relation":{},"ISSN":["1662-5099"],"issn-type":[{"value":"1662-5099","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,10,20]]},"article-number":"1279457"}}