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Complement overactivation and the ubiquitine-proteasome pathway in age-related macular degeneration

Session Details

Session Title: AMD III

Session Date/Time: Sunday 20/09/2015 | 11:00-13:00

Paper Time: 11:24

Venue: Athena

First Author: : E.Ramos de Carvalho NETHERLANDS

Co Author(s): :    A. Bergen   C. van Noorden   E. Reits   I. Klaassen   T. Gorgels   R. Schlingemann

Abstract Details

PURPOSE:The ubiquitin-proteasome pathway (UPP) is a non-lysosomal protein-degradation nanomachinery present in all types of eukaryotic cells. Formation of drusen and choroidal neovascularization appears to be etiologically related to local inflammation, oxidative stress, complement overactivation, dysregulation of angiogenesis and accumulation of damaged or postsynthetically modified proteins. Removal of aberrant proteins by the ubiquitin-proteasome pathway is essential for normal cellular function. The purpose of our study was to investigate whether complement overactivation can influence UPP activity in human retinal pigment epithelial cell culture and in a mouse model of age-related macular degeneration.

Setting:

Departments of Ophthalmology and Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Methods:

In the retinal pigment epithelial cells (RPE) of monocyte chemoattractant protein-1-deficient CCL2(-/-) mice, a mouse model that may serve as a model for age-related atrophic degeneration of the RPE, proteasome function was investigated by immunohistochemistry of household (β5) and immuno (β5i) subunit expression. Subsequently, proteasome overall activity was determined using the BodipyFl-Ahx3L3VS probe in primary-cultured human retinal pigment epithelial cells (HRPE) cells that were exposed to different stimuli including C3a and C5a, using confocal laser scanning microscopy and flow cytometry. Gene expression and protein levels of proteasome subunits α7, PA28α, β5, and β5i were also studied in RPE cells after exposure to IFN-γ, C3a, and C5a by real-time PCR and Western blotting.

Results:

RPE cells of CCL2(-/-) mice showed immunoproteasome upregulation. C3a, but not C5a supplementation, induced a decreased proteasome overall activity in HRPE cells, whereas mRNA and protein levels of household proteasome and immunoproteasome subunits were unaffected.

Conclusions:

In HRPE cells, C3a induces decreased proteasome-mediated proteolytic activity, whereas in a mouse model of age-related RPE atrophy, the immunoproteasome was upregulated, indicating a possible role for complement-driven posttranslational alterations in proteasome activity in the cascade of pathologic events that result in AMD.

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