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M. Samardzija, S. Joly, M. Thiersch, C. Grimm; Differential Regulation of Iron Metabolism-Related Genes in Rpe65 Mutant Mice. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3056.
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RPE65 is an iron-containing enzyme strongly expressed in the retinal pigment epithelium (RPE). A change in the abundance of RPE65 protein in Rpe65 mutant or deficient mice may affect the iron metabolism in the retina and the RPE. To address this possibility, we analyzed expression and localization of various proteins involved in iron homeostasis in wild-type and Rpe65 mutant mice.
Retinas and eyecups of wild-type and Rpe65 mutant mice (Rpe65-/- and Rpe65R91W) were collected at different ages (4 to 24 weeks and up to 1.5 years). Gene expression was analyzed by RT-PCR and Western blotting. Tissue localization of specific proteins was studied using immunohistochemistry. The genes/proteins analyzed included: ceruloplasmin (Cp), hemochromatosis (Hfe), ferroportin (Slc40a1), ferritin (Ft), transferrin (Tf), transferrin receptor (Tfrc), hephaestin (Heph).
Retinal mRNA levels of Tfrc and Tf were elevated in old mutant mice (24 wks) relative to wild-type controls. Expression of Cp, Heph, Hfe and Slc40a1 remained constant in retinas of mutant mice and was comparable to wild-types. In the eyecup, however, Tfrc was elevated in the young Rpe65-/- and remained high until 24 wks of age while Rpe65R91W and wild-type animals had basal levels throughout the period of analysis. In addition, Hfe was also strongly upregulated in eyecups of Rpe65-/- but not in those of Rpe65R91W or wild-type mice. Ft protein showed an age-dependent increase in the eyecup of Rpe65R91W mutant but not of wild-type mice.
These initial results demonstrate that the status of the RPE65 protein in the retinal pigment epithelium differentially influences expression of several genes involved in iron metabolism in the RPE as well as in the neuronal retina. Ongoing experiments aim at the identification of the regulatory differences between the Rpe65 knock-out and the Rpe65R91W knock-in mouse lines with respect to iron metabolism.
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