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Natalie Wolkow, Ying Song, Delu Song, Jennifer Lee, Joshua L. Dunaief; RPE Degeneration and Iron Accumulation in Mice with Cre Recombinase Mediated RPE-specific Knockout of the Ferroxidase Hephaestin. Invest. Ophthalmol. Vis. Sci. 2011;52(14):973.
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© ARVO (1962-2015); The Authors (2016-present)
Ferroxidases Hephaestin (Heph) and Ceruloplasmin (Cp) are necessary for retinal iron homeostasis; when Heph is systemically mutated and Cp is systemically deleted in mice (Cp/sla), RPE cells become iron overloaded and hypertrophic, and a retinal degeneration results. Although it is clear that Cp and Heph are important for normal retinal function, they are expressed in several retinal cell types and the cell-autonomous functions of these proteins are unknown. The purpose of our study was to investigate the RPE-specific role of Heph.
We deleted Heph specifically from the RPE cells using the cre-lox system; Heph-floxed mice were crossed with BEST1-cre mice (Iacovelli, et al. IOVS 2010), which have cre expression limited to the RPE within the eye. Since Cp and Heph are highly homologous, and Cp might compensate for Heph’s absence, we utilized a Cp-/- background. To determine if Heph recombination occurs, we used PCR assays on RPE DNA and mRNA. Immunofluorescence was performed on cryosections. Plastic sections were used to assess retinal degeneration.
Heph recombination occurs specifically in the RPE cells, as determined by PCR detecting DNA recombination and qPCR for Heph mRNA levels (p=0.0050). When Heph is deleted in RPE cells on a systemic Cp-/- background, RPE cells accumulate iron, as indicated by a decrease in transferrin receptor mRNA levels by qPCR (p=0.0115) and increased L-ferritin levels by immunofluorescence (p=0.0202). This iron accumulation results in RPE pathology, including RPE hypertrophy and atrophy, but, unlike Cp/sla mice, not RPE autofluorescence.
RPE-specific Heph deletion, on a systemic Cp-/- background, results in RPE cell iron accumulation, hypertrophy and atrophy. These results indicate that Heph functions in an RPE-autonomous manner to regulate intracellular iron levels, most likely by facilitating cellular iron export. The RPE-iron accumulation, however, is less than what is seen in Cp/sla mice of the same age. The retinal pathology differs from the pathology of the Cp/sla mice, likely because Cp/sla mice also accumulate iron within photoreceptors.
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