PROJECT SUMMARY Iron plays a critical role in both the healthy and diseased retina. The long term goals of the proposed studies are to understand regulation of retinal iron flux, determine why iron accumulates in retinal disease, and discover how to protect against retina iron toxicity. Iron is necessary in the retina for oxidative phosphorylation, membrane biogenesis and retinol isomerization, but becomes a central producer of oxidative stress when improperly regulated. Iron toxicity is evident in retinal disease: it causes rapid retinal degeneration following entry into the eye carried by an intraocular foreign body. Iron accumulation has also been noted in retinal diseases including AMD, where it may exacerbate oxidative stress. Further, patients with the inherited disease aceruloplasminemia, caused by mutation of the ferroxidase ceruloplasmin (Cp), have retinal iron accumulation with RPE pigment abnormalities, and occasionally early onset macular degeneration. Mice with knockout for Cp and its homolog hephaestin (Heph) have age-dependent retinal iron overload and degeneration of photoreceptors and RPE. Evidence from other organs suggests that Cp or Heph can cooperate with the sole plasma membrane iron exporter, ferroportin (Fpn), to export iron from cells. Yet, results from the previous funding period indicate that retina-specific knockout of Fpn has no impact on retinal iron levels while retina-specific knockout of Heph leads to retinal iron accumulation. These data point to the importance of ferroxidases Cp and Heph for keeping intraocular iron in its ferric (Fe3+) state. We will test this hypothesis using AAV-Cp gene therapy in the absence of Fpn, as well as an oxidation resistant form of the lipid DHA, which will be tested for retinal protection against reactive oxygen species produced by Fe2+.