Retinal Pigment Epithelium Lipofuscin Proteomics

Abstract

Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein (∼2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E2 adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis. Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein (∼2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E2 adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis. Lipofuscin is a heterogeneous, fluorescent waste material that accumulates with age in active postmitotic cells such as cardiac myocytes, select neurons, and the retinal pigment epithelium (RPE) 1The abbreviations used are: RPE, retinal pigment epithelium; AMD, age-related macular degeneration; CEP, 2-(ω-carboxyethyl)pyrrole; A2E, 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6,-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium; mAb, monoclonal antibody; NEM, N-ethyl morpholine acetate; iso[4]LGE2, iso[4]levuglandin E2; TEM, transmission electron microscopy; DAB, diaminobenzidine; DHA, docosahexaenoyl. (for reviews, see Refs.1Sparrow J.R. Boulton M. RPE lipofuscin and its role in retinal pathobiology.Exp. Eye Res. 2005; 80: 595-606Crossref PubMed Scopus (498) Google Scholar, 2Brunk U.T. Terman A. Lipofuscin: mechanisms of age-related accumulation and influence on cell function.Free Radic. Biol. Med. 2002; 33: 611-619Crossref PubMed Scopus (696) Google Scholar, 3Rozanowska M. Rozanowski B. Visual transduction and age-related changes in lipofuscin.in: Tombran-Tink J. Barnstable C.J. Visual Transduction and Non-Visual Perception. Humana Press, Totowa, NJ2008: 405-446Crossref Google Scholar, 4Sparrow J.R. RPE lipofuscin: formation, properties, and relevance to retinal degeneration.in: Tombran-Tink J. Barnstable C.J. Retinal Degenerations: Biology, Diagnostics and Therapeutics. Human Press, Totowa, NJ2007: 213-236Crossref Google Scholar). RPE lipofuscin, monitored in vivo as fundus autofluorescence, may be associated with retinal degenerative diseases such as Best macular dystrophy, Stargardt disease, and age-related macular degeneration (AMD) (1Sparrow J.R. Boulton M. RPE lipofuscin and its role in retinal pathobiology.Exp. Eye Res. 2005; 80: 595-606Crossref PubMed Scopus (498) Google Scholar, 4Sparrow J.R. RPE lipofuscin: formation, properties, and relevance to retinal degeneration.in: Tombran-Tink J. Barnstable C.J. Retinal Degenerations: Biology, Diagnostics and Therapeutics. Human Press, Totowa, NJ2007: 213-236Crossref Google Scholar, 5Eldred G.E. Lipofuscin and other lysosomal storage deposits in the retinal pigment epithelium.in: Marmor M.F. Wolfensberger T.J. The Retinal Pigment Epithelium. Oxford University Press, New York1998: 651-668Google Scholar). Although studied intensely for over 30 years, the molecular composition and pathogenic mechanisms of RPE lipofuscin remain poorly defined. This is due in large part to the heterogeneous nature of most isolated lipofuscin preparations. RPE lipofuscin granules are considered membrane-bound residual bodies of the lysosomal compartment of the cell (1Sparrow J.R. Boulton M. RPE lipofuscin and its role in retinal pathobiology.Exp. Eye Res. 2005; 80: 595-606Crossref PubMed Scopus (498) Google Scholar, 4Sparrow J.R. RPE lipofuscin: formation, properties, and relevance to retinal degeneration.in: Tombran-Tink J. Barnstable C.J. Retinal Degenerations: Biology, Diagnostics and Therapeutics. Human Press, Totowa, NJ2007: 213-236Crossref Google Scholar). Estimates suggest that lipofuscin contains 30–70% protein depending upon tissue, species, and study (2Brunk U.T. Terman A. Lipofuscin: mechanisms of age-related accumulation and influence on cell function.Free Radic. Biol. Med. 2002; 33: 611-619Crossref PubMed Scopus (696) Google Scholar, 6Warburton S. Southwick K. Hardman R.M. Secrest A.M. Grow R.K. Xin H. Woolley A.T. Burton G.F. Thulin C.D. Examining the proteins of functional retinal lipofuscin using proteomic analysis as a guide for understanding its origin.Mol. Vis. 2005; 11: 1122-1134PubMed Google Scholar) and that this protein may contribute to pathogenesis. From 2002 to 2007 proteomics investigations have reported protein compositions for RPE lipofuscin (6Warburton S. Southwick K. Hardman R.M. Secrest A.M. Grow R.K. Xin H. Woolley A.T. Burton G.F. Thulin C.D. Examining the proteins of functional retinal lipofuscin using proteomic analysis as a guide for understanding its origin.Mol. Vis. 2005; 11: 1122-1134PubMed Google Scholar, 7Schutt F. Ueberle B. Schnolzer M. Holz F.G. Kopitz J. Proteome analysis of lipofuscin in human retinal pigment epithelial cells.FEBS Lett. 2002; 528: 217-221Crossref PubMed Scopus (87) Google Scholar) and RPE melanolipofuscin (8Azarian S.M. McLeod I. Lillo C. Gibbs D. Yates J.R. Williams D.S. Proteomic analysis of mature melanosomes from the retinal pigmented epithelium.J. Proteome Res. 2006; 5: 521-529Crossref PubMed Scopus (33) Google Scholar, 9Warburton S. Davis W.E. Southwick K. Xin H. Woolley A.T. Burton G.F. Thulin C.D. Proteomic and phototoxic characterization of melanolipofuscin: correlation to disease and model for its origin.Mol. Vis. 2007; 13: 318-329PubMed Google Scholar). Oxidative protein modifications have also been associated with lipofuscin (6Warburton S. Southwick K. Hardman R.M. Secrest A.M. Grow R.K. Xin H. Woolley A.T. Burton G.F. Thulin C.D. Examining the proteins of functional retinal lipofuscin using proteomic analysis as a guide for understanding its origin.Mol. Vis. 2005; 11: 1122-1134PubMed Google Scholar, 7Schutt F. Ueberle B. Schnolzer M. Holz F.G. Kopitz J. Proteome analysis of lipofuscin in human retinal pigment epithelial cells.FEBS Lett. 2002; 528: 217-221Crossref PubMed Scopus (87) Google Scholar, 10Schutt F. Bergmann M. Holz F.G. Kopitz J. Proteins modified by malondialdehyde, 4-hydroxynonenal, or advanced glycation end products in lipofuscin of human retinal pigment epithelium.Investig. Ophthalmol. Vis. Sci. 2003; 44: 3663-3668Crossref PubMed Scopus (213) Google Scholar), but studies to date have not demonstrated that the identified proteins and oxidative modifications are from the granules rather than from the contaminating material that co-purifies with the granules. Because in vitro bioactivity studies of lipofuscin have also utilized preparations that contain extragranular contaminants, a clear understanding of the source of the bioactivities demonstrated in these studies remains elusive. In the RPE, lipofuscin is derived primarily from phagocytosis of shed photoreceptor outer segments and is associated with a functioning retinoid visual cycle. For example, RPE lipofuscin is significantly reduced in mice lacking the isomerohydrolase (RPE65) that generates 11-cis-retinol (11Katz M.L. Redmond T.M. Effect of Rpe65 knockout on accumulation of lipofuscin fluorophores in the retinal pigment epithelium.Investig. Ophthalmol. Vis. Sci. 2001; Google Scholar) and also by that the visual or that M. M. D. T.J. of the visual in vivo by acid from and a for in Sci. S. A. 2001; PubMed Scopus Google Scholar, S. with lipofuscin accumulation in a model of macular Sci. S. A. 2003; PubMed Scopus Google Scholar, J. J.R. the visual and lipofuscin 2006; PubMed Scopus Google Scholar, S. D. J. K. in accumulation of retinal a for treatment of retinal Ophthalmol. Vis. Sci. 2005; PubMed Scopus Google Scholar). fluorophores have been in RPE lipofuscin such as the and G.E. Retinal age generated by PubMed Scopus Google Scholar, J. K. G.E. age pigment Scopus Google Scholar, M. K. J. J. and of and fluorophores from human retinal pigment Sci. S. A. PubMed Scopus Google Scholar), of A2E, and such as all-trans-retinal J.R. RPE lipofuscin: formation, properties, and relevance to retinal degeneration.in: Tombran-Tink J. Barnstable C.J. Retinal Degenerations: Biology, Diagnostics and Therapeutics. Human Press, Totowa, NJ2007: 213-236Crossref Google Scholar, S. J.R. The all-trans-retinal of lipofuscin in retinal pigment epithelial cells in a Stargardt disease Sci. S. A. 2007; PubMed Scopus Google Scholar). in vitro studies have that of RPE cells to lipofuscin or in cell S. M. Boulton of lipofuscin in human retinal pigment epithelial Radic. Biol. Med. 2001; PubMed Scopus Google Scholar, F. S. Kopitz J. Holz F.G. Boulton to human RPE cells by a retinoid of Ophthalmol. Vis. Sci. Google Scholar, J.R. K. The lipofuscin to retinal pigmented epithelial Ophthalmol. Vis. Sci. Google Scholar, J.R. B. of of and by Ophthalmol. Vis. Sci. 2001; Google Scholar). The phototoxicity of to RPE cells is in vitro studies have also associated J. J.R. by products of A2E, a lipofuscin of the retinal pigment Sci. S. A. 2006; PubMed Scopus Google Scholar) with in RPE cells to and that in RPE cells A. The lipofuscin in retinal pigment epithelial Sci. S. A. 2007; PubMed Scopus Google Scholar). bioactivities a role for lipofuscin in but the molecular mechanisms remain to be a understanding of lipofuscin pathogenic mechanisms, we studies to the RPE lipofuscin were by the significant heterogeneity of lipofuscin preparations. is the most part of proteomics we purified lipofuscin granules free of extragranular The purified granules were on a for amino oxidative modifications, bisretinoids, and Our that the purified lipofuscin organelles contain oxidative modifications and bisretinoids and are phototoxic to RPE cells but contain a minimal of protein. Human were from the Eye with for in to RPE cells were isolated from human by in and Lipofuscin isolated from RPE using in a as J. S. Boulton M. The of the retinal age pigment Biol. PubMed Scopus Google Scholar) and lipofuscin. crude lipofuscin preparations were from age from 30 age and from age Crude lipofuscin purified by (i) in N-ethyl morpholine SDS or (ii) with proteinase K in NEM, of crude lipofuscin, lipofuscin, and proteinase lipofuscin were by with a were also by with a and For analysis, lipofuscin preparations were with and the material and in SDS lipofuscin material to using a and with of the mAb, and E2 M. K. H. M. A. to the of age-related macular Sci. S. A. 2002; PubMed Scopus Google Scholar). the material by to and with as M. K. H. M. A. to the of age-related macular Sci. S. A. 2002; PubMed Scopus Google Scholar). Crude and proteinase lipofuscin were for in to on and with were from the to the of the proteins were reduced with with and in with and were for LC MS/MS M. K. H. M. A. to the of age-related macular Sci. S. A. 2002; PubMed Scopus Google Scholar). LC MS/MS with a using a LC a LC a and a of M. K. H. M. A. to the of age-related macular Sci. S. A. 2002; PubMed Scopus Google Scholar). LC with The in MS/MS with the most in to MS/MS of crude or lipofuscin preparations were to in 30 NEM, SDS of and were by using a a of and a of in with were by LC MS/MS on a as from utilized the and the The human a of a of and protein modifications for and of used for MS/MS of protein for protein of lipofuscin preparations of were to for and amino acid analysis as acid in New Scholar) using HPLC with a and crude and purified lipofuscin granules were in and in in a of to in and for M. K. H. M. A. to the of age-related macular Sci. S. A. 2002; PubMed Scopus Google Scholar). For were with a and with were with a with a For transmission electron microscopy the of on with and and in a electron with a For crude lipofuscin or purified granules were in of and for The with and and with for to and which the and for as For crude and purified lipofuscin granules were with a using a were in the and the using a of lipofuscin granules including and diameter were from using a generated in considered a of by the In this for is than for other the circularity of the is and by and are and not have Crude and lipofuscin were with and a in and The on HPLC with a a and and using with a of as M. K. J. J. and of and fluorophores from human retinal pigment Sci. S. A. PubMed Scopus Google Scholar). The lipofuscin A2E, isoA2E, and all-trans-retinal were identified and on and that to J.R. RPE lipofuscin: formation, properties, and relevance to retinal degeneration.in: Tombran-Tink J. Barnstable C.J. Retinal Degenerations: Biology, Diagnostics and Therapeutics. Human Press, Totowa, NJ2007: 213-236Crossref Google Scholar). cells were of crude or lipofuscin and in as F. S. Kopitz J. Holz F.G. Boulton to human RPE cells by a retinoid of Ophthalmol. Vis. Sci. Google Scholar). cells lipofuscin. the with and cells were in the or to for using the F. S. Kopitz J. Holz F.G. Boulton to human RPE cells by a retinoid of Ophthalmol. Vis. Sci. Google Scholar). RPE lipofuscin granules are isolated by J. S. Boulton M. The of the retinal age pigment Biol. PubMed Scopus Google Scholar) and contain significant of extragranular as and by light, confocal, and transmission electron In the crude preparations the lipofuscin granules showed and were in the In preparations with microscopy large extragranular were that were not associated with lipofuscin and We used to the extragranular to and functional characterization of the granules. of crude lipofuscin preparations with proteinase K the extragranular material the of the granules and we found that crude lipofuscin preparations with the SDS but no proteinase K also the extragranular and analyses that most granules in the crude were by purified granules as in analyses of granules in a crude lipofuscin and purified granules no statistically significant in diameter or circularity for differences granules from purified and crude preparations we not these differences to be The utilized that or granules with the that for the purified granules because of and granules than in the crude analyses of lipofuscin preparations were of crude and proteinase lipofuscin were by were in and proteomics analyses were pursued by LC From crude lipofuscin proteins were identified no proteins were identified from proteinase lipofuscin proteins and of the proteins in crude lipofuscin were in and significantly than the of modified and proteins analysis of the of crude lipofuscin proteins the of modified proteins For proteomics analyses of lipofuscin of crude or lipofuscin were in a were by and LC MS/MS the of proteins of 186 proteins were identified from the crude lipofuscin but no proteins were identified from the purified granules. LC MS/MS analysis of from the purified granules that exhibited a that were of very to from and Crude lipofuscin and lipofuscin granules were by analysis for nitrotyrosine and modifications derived from oxidative of or crude lipofuscin and the purified granules exhibited to adducts from M. K. H. M. A. to the of age-related macular Sci. S. A. 2002; PubMed Scopus Google Scholar), to adducts from lipids C. K. J. adducts of iso[4]levuglandin a of the in Biol. PubMed Scopus Google Scholar), and to nitrotyrosine and analysis used because of the purified granules to or analysis of crude lipofuscin demonstrated and the of analyses also demonstrated on the of the lipofuscin granules in purified and crude preparations and in the extragranular and lacking showed no in crude or purified lipofuscin preparations by and or by the from proteomics analysis of crude and purified lipofuscin amino acid analysis used to protein on a in proteinase and lipofuscin. amino acid analyses showed that the crude lipofuscin protein than the purified granules that (w/w) protein significant in the of amino in proteinase and lipofuscin granules. differences in amino acid composition were for preparations from and of amino acid content of lipofuscin of amino acid amino acid in a of crude or lipofuscin were with and and by HPLC for A2E, isoA2E, and all-trans-retinal significant in the of these bisretinoids from the crude and lipofuscin consistent with of the fluorophores to the granules. of crude or lipofuscin were to cultured cells with or to for and the cells were for from previous no significant in the of cells lacking lipofuscin and to or of cells lipofuscin but in the cells that were with membrane-bound lipofuscin granules or granules and to exhibited in This supports of the phototoxic activity of lipofuscin to the purified granules. a understanding of RPE lipofuscin composition and we purified RPE lipofuscin granules by treatment with proteinase K or SDS and demonstrated by light, confocal, and transmission electron analyses showed little in the and of the granules from purified or crude preparations with the diameter with the reported from microscopy C. and studies of human retinal lipofuscin Sci. S. A. 2003; PubMed Scopus Google Scholar). of the extragranular by of but this in the of the fluorescent granules. Our proteomics analyses showed that purified lipofuscin granules contain little protein (∼2% (w/w) on amino acid and no protein identifiable by with a of significant of the purified granules exhibited no in or and analyses the of oxidative protein modifications in the purified lipofuscin granules. In contrast, crude lipofuscin preparations were found to contain protein (w/w) and many identifiable We the bioactivity of the purified granules and found no significant differences in phototoxicity or content crude lipofuscin granules and the purified granules. analyses that the bioactivity of lipofuscin is associated with the granules (1Sparrow J.R. Boulton M. RPE lipofuscin and its role in retinal pathobiology.Exp. Eye Res. 2005; 80: 595-606Crossref PubMed Scopus (498) Google Scholar, 2Brunk U.T. Terman A. Lipofuscin: mechanisms of age-related accumulation and influence on cell function.Free Radic. Biol. Med. 2002; 33: 611-619Crossref PubMed Scopus (696) Google Scholar, 4Sparrow J.R. RPE lipofuscin: formation, properties, and relevance to retinal degeneration.in: Tombran-Tink J. Barnstable C.J. Retinal Degenerations: Biology, Diagnostics and Therapeutics. Human Press, Totowa, NJ2007: 213-236Crossref Google Scholar) in to previous a role for lipofuscin protein in Although that lipofuscin granules contain a of of protein compositions for human RPE lipofuscin have to a that lipofuscin contains protein (6Warburton S. Southwick K. Hardman R.M. Secrest A.M. Grow R.K. Xin H. Woolley A.T. Burton G.F. Thulin C.D. Examining the proteins of functional retinal lipofuscin using proteomic analysis as a guide for understanding its origin.Mol. Vis. 2005; 11: 1122-1134PubMed Google Scholar, 7Schutt F. Ueberle B. Schnolzer M. Holz F.G. Kopitz J. Proteome analysis of lipofuscin in human retinal pigment epithelial cells.FEBS Lett. 2002; 528: 217-221Crossref PubMed Scopus (87) Google Scholar). The the of for proteomics Although we no proteins in purified granules, we identified 186 proteins in crude lipofuscin, including lysosomal protein the proteins identified by in RPE lipofuscin, over were in crude lipofuscin that the previous the protein composition of extragranular and not of lipofuscin granules. of the proteins identified in RPE melanosomes (8Azarian S.M. McLeod I. Lillo C. Gibbs D. Yates J.R. Williams D.S. Proteomic analysis of mature melanosomes from the retinal pigmented epithelium.J. Proteome Res. 2006; 5: 521-529Crossref PubMed Scopus (33) Google Scholar) and of the proteins reported in RPE melanolipofuscin S. Davis W.E. Southwick K. Xin H. Woolley A.T. Burton G.F. Thulin C.D. Proteomic and phototoxic characterization of melanolipofuscin: correlation to disease and model for its origin.Mol. Vis. 2007; 13: 318-329PubMed Google Scholar) were also identified in analyses of crude lipofuscin. extragranular is in the from these studies (6Warburton S. Southwick K. Hardman R.M. Secrest A.M. Grow R.K. Xin H. Woolley A.T. Burton G.F. Thulin C.D. Examining the proteins of functional retinal lipofuscin using proteomic analysis as a guide for understanding its origin.Mol. Vis. 2005; 11: 1122-1134PubMed Google Scholar, S.M. McLeod I. Lillo C. Gibbs D. Yates J.R. Williams D.S. Proteomic analysis of mature melanosomes from the retinal pigmented epithelium.J. Proteome Res. 2006; 5: 521-529Crossref PubMed Scopus (33) Google Scholar, 9Warburton S. Davis W.E. Southwick K. Xin H. Woolley A.T. Burton G.F. Thulin C.D. Proteomic and phototoxic characterization of melanolipofuscin: correlation to disease and model for its origin.Mol. Vis. 2007; 13: 318-329PubMed Google Scholar). RPE melanolipofuscin is poorly granular waste material isolated by with lipofuscin. The most protein identified the study and the other proteomics lipofuscin and melanolipofuscin the proteins are in of the proteins we in crude lipofuscin visual proteins from the RPE such as and 11-cis-retinol and photoreceptor proteins such as and is the protein composition we for crude lipofuscin and that reported for J. S. S. C. M. The Biol. 2001; PubMed Scopus Google Scholar) and A. A. S. F. J. Proteomic analysis of human to and 2002; PubMed Scopus Google Scholar) from Oxidative been associated with a of age-related and been of to Our of and in purified granules are consistent with a role for in lipofuscin B. of of fluorescent PubMed Scopus Google Scholar). adducts of protein amino are generated from a derived from lipids M. K. H. M. A. to the of age-related macular Sci. S. A. 2002; PubMed Scopus Google Scholar) that are in photoreceptor outer In AMD, adducts are in M. K. H. M. A. to the of age-related macular Sci. S. A. 2002; PubMed Scopus Google Scholar), the the RPE from the also contains of adducts and M. protein adducts and for age-related macular Biol. 2003; PubMed Scopus Google Scholar). adducts in vivo K. J. A. B. oxidative protein modifications for age-related macular Sci. S. A. 2006; PubMed Scopus Google Scholar) and a in a model of Oxidative age-related macular Med. PubMed Scopus Google Scholar). reactive to the is generated by free of and of modifications, including C. K. J. adducts of iso[4]levuglandin a of the in Biol. PubMed Scopus Google Scholar, S. E2 PubMed Scopus Google Scholar). adducts have been in from with and end disease K. A.M. adducts in products of free lipid the PubMed Scopus Google Scholar). In of in cells by PubMed Scopus Google Scholar) and and H. E2 and PubMed Scopus Google Scholar). We the that and adducts with amino in and but not yet been such We nitrotyrosine in crude lipofuscin and the purified granules that is from the of free or with nitrogen oxide species such as free nitrogen and of and have been associated with including and diseases B. K. M. oxide and The the and the not a of Radic. Res. PubMed Scopus Google Scholar). oxidative protein modifications reported in RPE lipofuscin and adducts and advanced glycation end products F. Bergmann M. Holz F.G. Kopitz J. Proteins modified by malondialdehyde, 4-hydroxynonenal, or advanced glycation end products in lipofuscin of human retinal pigment epithelium.Investig. Ophthalmol. Vis. Sci. 2003; 44: 3663-3668Crossref PubMed Scopus (213) Google Scholar). remains to be these other modifications are associated with the granules because the analyses were with crude lipofuscin preparations F. Bergmann M. Holz F.G. Kopitz J. Proteins modified by malondialdehyde, 4-hydroxynonenal, or advanced glycation end products in lipofuscin of human retinal pigment epithelium.Investig. Ophthalmol. Vis. Sci. 2003; 44: 3663-3668Crossref PubMed Scopus (213) Google Scholar). protein modifications have the to J. M. K. by adducts with active Sci. 2002; 11: PubMed Scopus Google Scholar), and contribute to the accumulation of The of protein in lipofuscin granules by amino acid analysis modified and proteins that are to and protein by modifications but not be to CEP, and This is the study to date that that RPE lipofuscin granules contain minimal protein. This suggests that lipofuscin protein minimal impact on lipofuscin pathogenesis. We have demonstrated that the of lipofuscin are associated with the granules, which contain phototoxic bisretinoids and many lipids in human of age with outer segments and Ophthalmol. Vis. Sci. Google Scholar). lipofuscin in these remains to be studies have demonstrated that RPE lipofuscin is a potent generator of reactive oxygen species including and lipid that have the to the and other such as and the as demonstrated in cell J.R. RPE lipofuscin: formation, properties, and relevance to retinal degeneration.in: Tombran-Tink J. Barnstable C.J. Retinal Degenerations: Biology, Diagnostics and Therapeutics. Human Press, Totowa, NJ2007: 213-236Crossref Google Scholar, M. A. J. M. D. Lipofuscin is a free Biol. PubMed Scopus Google Scholar, M. J. Boulton of retinal age In vitro of Biol. PubMed Scopus Google Scholar, J. studies on human retinal PubMed Scopus Google Scholar, M. M. Rozanowski B. The of Sci. PubMed Google Scholar, S. Boulton M. and free in epithelial Biol. 2005; PubMed Scopus Google Scholar). the of lipids and and to by free and lipofuscin granules be considered a source of reactive oxidative reactive fragments amino and the and contribute to in the cell adducts and and protein been to upon and been to be in RPE the that reactive fragments may from RPE cells J. J.R. by products of A2E, a lipofuscin of the retinal pigment Sci. S. A. 2006; PubMed Scopus Google Scholar). For example, a of the oxidative acid may from the RPE and contribute to the of adducts and in M. protein adducts and for age-related macular Biol. 2003; PubMed Scopus Google Scholar). a pathogenic for RPE lipofuscin, we that oxidative of lipid and retinoid the generates a of reactive of which and and with The and of and retinoid and protein or upon the of the RPE and This hypothesis not for other in lipofuscin and is to the molecular of the mechanisms of lipofuscin the we of for the of the and and for