Dataset Information

C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway.

ABSTRACT: The mechanisms that connect complement system activation and basal deposit formation in early stages of age-related macular degeneration (AMD) are insufficiently understood, which complicates the design of efficient therapies to prevent disease progression. Using human fetal (hf) retinal pigment epithelial (RPE) cells, we have established an in vitro model to investigate the effect of complement C3a on RPE cells and its role in the formation of sub-RPE deposits. The results of these studies revealed that C3a produced after C3 activation is sufficient to induce the formation of sub-RPE deposits via complement-driven proteasome inhibition. C3a binds the C3a receptor (C3aR), stimulates deposition of collagens IV and VI underneath the RPE, and impairs the extracellular matrix (ECM) turnover by increased MMP-2 activity, all mediated by downregulation of the ubiquitin proteasome pathway (UPP). The formation of basal deposits can be prevented by the addition of a C3aR antagonist, which restores the UPP activity and ECM turnover. These findings indicate that the cell-based model can be used to test potential therapeutic agents in vitro. The data suggest that modulation of C3aR-mediated events could be a therapeutic approach for treatment of early AMD.

SUBMITTER: Fernandez-Godino R

PROVIDER: S-EPMC6018664 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway.

Fernandez-Godino Rosario R Pierce Eric A EA

Scientific reports 20180626 1

The mechanisms that connect complement system activation and basal deposit formation in early stages of age-related macular degeneration (AMD) are insufficiently understood, which complicates the design of efficient therapies to prevent disease progression. Using human fetal (hf) retinal pigment epithelial (RPE) cells, we have established an in vitro model to investigate the effect of complement C3a on RPE cells and its role in the formation of sub-RPE deposits. The results of these studies reve ...[more]

PMID: 29946065

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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.

Dataset Information

C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway.

Publications

C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway.

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