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Rosario Fernandez-Godino, Eric Pierce, Donita Garland; Role of complement in altered RPE function and deposit formation in Efemp1 mutant mice: A primary cell culture model. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5007.
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© ARVO (1962-2015); The Authors (2016-present)
The R345W mutation in the Efemp1 gene causes the inherited macular degeneration Doyne Honeycomb Retinal Dystrophy/ Malattia Leventinese (DHRD/ML). Gene targeted Efemp1-R345W mice develop sub-RPE basal deposits and vacuoles in the RPE. In previous studies using Efemp1-R345W : complement C3 null mice we found that the complement system had a critical role in the formation of basal laminar deposits in the mutant mice. The purpose of this study was to use a primary RPE cell culture system to investigate the mechanisms involved in the pathogenesis of basal deposit formation in the Efemp1-R345W mutant mice and the role of the complement system in this process.
Primary RPE cells were cultured from Efemp1-R345W knockin, C3 null, and homozygous Efemp1-R345W:C3 null mice. The RPE cells, from 2.5 months old mice, were grown on a permeable support under polarizing conditions. mRNA expression levels of complement genes and Efemp1 were quantified by qRT-PCR. Protein expression in the RPE was determined by Western blot analysis and immunofluorescence using confocal microscopy. Proteins secreted from both the apical and basal surfaces were quantified by Western blot analyses.
Results of qRT-PCR analysis reproducibly showed that RPE cells from each of the mouse strains express complement C3 mRNA and protein. The C3 null mice used for these studies express an inactive form of C3. Moreover, the major complement components of the classical pathway, C1q, C2, C4 as well as Cfh of the alternative pathway were also expressed in the RPE of each mouse strain. The expression of Efemp1 mRNA was significantly higher in Efemp1-R345W knockin mice compared to wild type (Wt) (ANOVA,p=0.0005). However, this overexpression was repressed in the Efemp1-R345W:C3 null mice (p=0.0005).
The primary RPE cell culture provides an excellent approach to investigate the early effects of the Efemp1 mutation on RPE protein expression and also on the effects of alternations in the complement system on RPE function. The results suggested a critical role of C3 in the pathogenesis of the R345W mutation in DHRD/ML. They also suggest that the mutant EFEMP1 protein might cause local complement activation in the RPE, which in turn leads to RPE cell dysfunction and an altered pattern of proteins secreted to Bruch’s membrane.
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