Minimal effect of conditional ferroportin KO in the neural retina implicates ferrous iron in retinal iron overload and degeneration

Abstract

Purpose : Iron-induced oxidative stress has been implicated in retinal degenerative diseases. Mouse models with systemic, or neural retina-specific knockout (KO) of homologous ferroxidases ceruloplasmin (Cp) and hephaestin (Heph) have retinal iron overload. We previously reported age-dependent iron overload in the retina of Cp/Heph DKO mice by 6mo, and retinal degeneration with features of age-related macular degeneration by 9mo. Because Cp and Heph can facilitate cellular iron export by ferroportin (Fpn), we hypothesized that the impairment of ferroxidase-facilitated, Fpn-mediated iron export from retinal neurons and glia might account for these results.

Methods : To investigate the role of Fpn in retinal iron regulation, we developed mice with neural retina-specific Fpn KO (mRx-Cre+, Fpn^flox/flox). Mice were aged to 7-10 months of age, and retinal iron levels were analyzed using qPCR and immunolabeling for ferritin light chain (Ft-L). To further test the hypothesis that a combination of elevated ferrous iron plus deletion of Fpn might cause retinal iron accumulation, we developed mice with neural retina-specific Fpn KO and systemic Cp KO. To increase the serum iron level, mice were given an intraperitoneal injection of iron at 3 months of age. Retina iron levels were analyzed using qPCR and immunolabeling for Ft-L.

Results : There was no indication of retinal iron overload in neural retina-specific Fpn KO mice at 7-10 months of age: the mRNA levels of transferrin receptor (Tfrc) measured by qPCR were not altered in retina-specific Fpn KO mice and immunolabeling for Ft-L showed similar intensity in retina-specific Fpn KO mice and controls. Iron injected mice with systemic Cp KO and retina-specific Fpn KO had a significant but small decrease in retinal Tfrc mRNA. There was no effect on Dmt1 mRNA no change in Ft-L immunolabeling, which we have typically observed are less sensitive measures of retinal iron overload than levels of Tfrc mRNA.

Conclusions : These results indicate that impaired Fpn mediated cellular iron export is not sufficient to explain the retinal iron overload observed in Cp/Heph DKO mice. Elevated levels of ferrous iron are likely to contribute in the absence of ferroxidases. Supporting this, injection of ferrous sulfate into the vitreous increased retinal iron levels indicated by significantly decreased Tfrc mRNA followed by rapid retinal degeneration.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.