Immunolocalization and regulation of iron handling proteins ferritin and ferroportin in the retina.
OBJECTIVE
CNS iron accumulation is associated with several neurodegenerative diseases, including age-related macular degeneration. Intracellular overload of free iron is prevented, in part, by the iron export protein, ferroportin, and the iron storage protein, ferritin. The purpose of this study was to assess retinal localization and regulation of ferroportin and ferritin.


METHODS
Normal murine retinas were analyzed by immunohistochemistry to localize ferroportin, cytosolic ferritin, and mitochondrial ferritin, with double-labeling using cell-specific markers to identify cell types. Retinas deficient in the ferroxidases, ceruloplasmin and hephaestin, accumulate iron in their retinas and RPE, while retinas deficient in iron regulatory proteins (IRPs) lack the ability to regulate several proteins involved in iron metabolism; retinas from these knockout mice along with their age matched wild type littermates were also examined to study regulation of ferritin and ferroportin. To enable visualization of label in the retinal pigment epithelial cells, sections from pigmented mice were bleached with H2O2 prior to IHC, a novel use of this technique for study of the RPE.


RESULTS
In normal retinas, cytosolic ferritins were found predominantly in rod bipolar cells and photoreceptors. Ferroportin was found in RPE and Müller cells. Iron accumulation in mice deficient in ceruloplasmin and hephaestin was associated with upregulation of ferritin and ferroportin. Mice deficient in IRPs showed upregulation of ferritin and ferroportin, likely because of their inability to repress translation.


CONCLUSIONS
Normal retinas contain ferritin and ferroportin, whose levels are regulated by iron-responsive, iron regulatory proteins. Ferroportin colocalizes with ceruloplasmin and hephaestin to RPE and Müller cells, supporting a potential cooperation between these ferroxidases and the iron exporter. Cytosolic ferritin accumulates in rod bipolar synaptic terminals, suggesting that ferritin may be involved in axonal iron transport. Mitochondrial ferritin increases with iron accumulation, suggesting a role in iron storage.
Hahn
Paul
P
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, Philadelphia, PA, USA.
Dentchev
Tzvete
T
Qian
Ying
Y
Rouault
Tracey
T
Harris
Z Leah
ZL
Dunaief
Joshua L
JL
D000818:Animals; D001052:Apoferritins; D027682:Cation Transport Proteins / Q000378:metabolism*; D005293:Ferritins / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D058851:GPI-Linked Proteins; D007501:Iron / Q000378:metabolism; D035925:Iron-Regulatory Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008667:Metalloproteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009363:Neoplasm Proteins / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000098742:Ferroportin
D027682:Cation Transport Proteins; D058851:GPI-Linked Proteins; D035925:Iron-Regulatory Proteins; C425068:Meltf protein, mouse; D008565:Membrane Proteins; D008667:Metalloproteins; D009363:Neoplasm Proteins; D000098742:Ferroportin; D005293:Ferritins; D001052:Apoferritins; D007501:Iron