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The lipofuscin fluorophore A2E perturbs cholesterol metabolism in retinal pigment epithelial cells.

ABSTRACT: Proteins involved in cholesterol trafficking are known to contribute to the pathogenesis of atherosclerosis and Alzheimer's disease. Allelic variants in the cholesterol transporters apolipoprotein E and ATP-binding cassette protein A1 (ABCA1) have recently been associated with susceptibility to age-related macular degeneration (AMD). Histopathological analyses of eyes with AMD demonstrate the presence of cholesterol and cholesteryl ester deposits beneath the retinal pigment epithelium (RPE), implicating abnormal cholesterol trafficking in disease progression. Here, we show that A2E, a quaternary amine and retinoid by-product of the visual cycle, causes the accumulation of free and esterified cholesterol in RPE cells. The mechanism involves neither generalized alterations in late endosomal/lysosomal pH nor a direct inhibition of acid lipase activity. Rather, A2E prevents cholesterol efflux from these organelles, which in turn indirectly inhibits acid lipase, leading to a subsequent accumulation of cholesteryl esters. Transcriptional activation of the ABCA1 cholesterol transporter by agonists of the liver X receptor/peroxisome proliferator-activated receptor pathway relieves the A2E-induced block on cholesterol efflux and restores cholesterol homeostasis in RPE cells. Our data also demonstrate that A2E, which is a cone-shaped lipid, increases the chemical activity and displacement of cholesterol from model membranes, providing a biophysical mechanism for cholesterol sequestration in A2E-loaded cells. Although endogenously produced A2E in the RPE has been associated with macular degeneration, the precise mechanisms are unclear. Our results provide direct evidence that A2E causes aberrant cholesterol metabolism in RPE cells which could likely contribute to AMD progression.

SUBMITTER: Lakkaraju A

PROVIDER: S-EPMC1904145 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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The lipofuscin fluorophore A2E perturbs cholesterol metabolism in retinal pigment epithelial cells.

Lakkaraju Aparna A Finnemann Silvia C SC Rodriguez-Boulan Enrique E

Proceedings of the National Academy of Sciences of the United States of America 20070619 26

Proteins involved in cholesterol trafficking are known to contribute to the pathogenesis of atherosclerosis and Alzheimer's disease. Allelic variants in the cholesterol transporters apolipoprotein E and ATP-binding cassette protein A1 (ABCA1) have recently been associated with susceptibility to age-related macular degeneration (AMD). Histopathological analyses of eyes with AMD demonstrate the presence of cholesterol and cholesteryl ester deposits beneath the retinal pigment epithelium (RPE), imp ...[more]

PMID: 17578916

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Project description:Age-related macular degeneration (AMD) is the leading cause of central vision loss in the elderly. Degeneration of retinal pigment epithelial (RPE) cells is a crucial causative factor responsible for the onset and progression of AMD. A2E, a major component of toxic lipofuscin implicated in AMD, is deposited in RPE cells with age. However, the mechanism whereby A2E may contribute to the pathogenesis of AMD remains unclear. We demonstrated that A2E was a danger signal of RPE cells, which induced autophagy and decreased cell viability in a concentration- and time-dependent manner. Within 15 min after the treatment of RPE with 25 μM A2E, the induction of autophagosome was detected by transmission electron microscopy. After continuous incubating RPE cells with A2E, intense punctate staining of LC3 and increased expression of LC3-II and Beclin-1 were identified. Meanwhile, the levels of intercellular adhesion molecule (ICAM), interleukin (IL)1β, IL2, IL-6, IL-8, IL-17A, IL-22, macrophage cationic peptide (MCP)-1, stromal cell-derived factor (SDF)-1, and vascular endothelial growth factor A (VEGFA) were elevated. The autophagic inhibitor 3-methyladenine (3-MA) and activator rapamycin were also used to verify the effect of autophagy on RPE cells against A2E. Our results revealed that 3-MA decreased the autophagosomes and LC3 puncta induced by A2E, increased inflammation-associated protein expression including ICAM, IL1β, IL2, IL-6, IL-8, IL-17A, IL-22, and SDF-1, and upregulated VEGFA expression. Whereas rapamycin augmented the A2E-mediated autophagy, attenuated protein expression of inflammation-associated and angiogenic factors, and blocked the Akt/mTOR pathway. Taken together, A2E induces autophagy in RPE cells at the early stage of incubation, and this autophagic response can be inhibited by 3-MA or augmented by rapamycin via the mTOR pathway. The enhancement of autophagy has a protective role in RPE cells against the adverse effects of A2E by reducing the secretion of inflammatory cytokines and VEGFA.

Project description:With ageing extracellular material is deposited in Bruch's membrane, as drusen. Lipofuscin is deposited in retinal pigment epithelial cells. Both of these changes are associated with age related macular degeneration, a disease now believed to involve chronic inflammation at the retinal-choroidal interface. We hypothesise that these molecules may act as danger signals, causing the production of inflammatory chemokines and cytokines by the retinal pigment epithelium, via activation of pattern recognition receptors.ARPE-19 cells were stimulated in vitro with the following reported components of drusen: amyloid-ß (1-42), Carboxyethylpyrrole (CEP) modified proteins (CEP-HSA), N?-(Carboxymethyl)lysine (CML) modified proteins and aggregated vitronectin. The cells were also stimulated with the major fluorophore of lipofuscin: N-retinylidene-N-retinylethanolamine (A2E). Inflammatory chemokine and cytokine production was assessed using Multiplex assays and ELISA. The mechanistic evaluation of the NLRP3 inflammasome pathway was assessed in a stepwise fashion.Of all the molecules tested only A2E induced inflammatory chemokine and cytokine production. 25 µM A2E induced the production of significantly increased levels of the chemokines IL-8, MCP-1, MCG and MIP-1?, the cytokines IL-1ß, IL-2, IL-6, and TNF-?, and the protein VEGF-A. The release of IL-1ß was studied further, and was determined to be due to NLRP3 inflammasome activation. The pathway of activation involved endocytosis of A2E, and the three inflammasome components NLRP3, ASC and activated caspase-1. Immunohistochemical staining of ABCA4 knockout mice, which show progressive accumulation of A2E levels with age, showed increased amounts of IL-1ß proximal to the retinal pigment epithelium.A2E has the ability to stimulate inflammatory chemokine and cytokine production by RPE cells. The pattern recognition receptor NLRP3 is involved in this process. This provides further evidence for the link between A2E, inflammation, and the pathogenesis of AMD. It also supports the recent discovery of NLRP3 inflammasome activation in AMD.

Project description: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 ( approximately 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 E(2) 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.

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The lipofuscin fluorophore A2E perturbs cholesterol metabolism in retinal pigment epithelial cells.

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The lipofuscin fluorophore A2E perturbs cholesterol metabolism in retinal pigment epithelial cells.

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