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Esther Clark, Delu Song, Jacob Sterling, Steven Grieco, Joshua L Dunaief; AAV gene transfer of the iron-regulatory hormone hepcidin increases Muller cell iron levels. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5834.
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© ARVO (1962-2015); The Authors (2016-present)
Hepcidin, a peptide hormone, plays a key role in iron regulation within the gut; it triggers degradation of the only cellular iron exporter, ferroportin. Yet, little is known about how this hormone regulates iron transport in the retina. Iron is implicated in retinal diseases, such as age-related macular degeneration (AMD); therefore, it is important to understand hepcidin’s role within the retina. To facilitate these studies, we also assessed spatial viral transduction patterns when two AAV-viruses were co-injected, to determine whether AAV-GFP can serve as a marker for expression of a gene encoded by a co-injected virus.
AAV2/8-CMV-Hepcidin was co-injected with AAV2/8-CMV-GFP into the left vitreous of C57BL/6J male hepcidin knockout mice (N=10). The right eye received only AAV-GFP as a control (N=10). The retinas were analyzed with immunofluorescence to assess the levels and localization of iron, ferritin, hepcidin, the iron transporter ferroportin, and transferrin receptor. C57BL/6J wild type mice were co-injected with AAV-GFP and red fluorescent virus (AAV-RFP) to determine the extent of transduction co-localization when the two viruses are co-injected at equal titers (N=4).
Ferritin immunoreactivity (which is directly correlated with iron levels) in Muller cells was increased in areas that received co-injection of AAV-GFP and AAV-Hepcidin when compared to control retinas that only received AAV-GFP. Moreover, AAV-Hepcidin caused an increase in retinal pigment epithelium (RPE) ferritin levels and a decrease in RPE ferroportin and transferrin receptor levels when compared to control retinas. AAV-GFP co-injected with AAV-RFP showed co-localized expression.
These findings indicate that hepcidin produced within the retina can regulate iron transport by causing the internalization and degradation of ferroportin. Thus, iron is trapped within cells, particularly Mullers. Since hepcidin can be produced by the retina, and is upregulated by elevated retinal iron levels or inflammation, endogenous hepcidin production may influence patterns of retinal iron accumulation. Moreover, AAV-GFP provides a spatial label for co-injected AAV-Hepcidin. This co-injection approach should be generally applicable for AAV gene therapy experiments studying other genes.
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