Molecular Vision 2004; 10:598-607 Received 2 April 2004 | Accepted 19 August 2004 | Published 26 August 2004

Iron is essential for survival but is also highly toxic dueto its ability to generate free radicals via the Fenton reaction.Homeostatic mechanisms must thus delicately balance ironlevels to prevent the deleterious consequences of either ironoverload or deficiency. Cells regulate iron homeostasis in partby regulating the levels of iron import, storage, and exportproteins in response to iron levels. The major pathway for ironimport occurs when transferrin, the extracellular iron carrierprotein, binds transferrin receptor on the cell surface and isendocytosed. Iron is then released from transferrin by the acid-ity of the low pH endosome [1] and is exported from the en-dosome by divalent metal transporter-1 (DMT-1) for use, stor-age, or export by the cell [2]. DMT-1 is also responsible forabsorption of iron from the intestinal lumen into the intestinalendothelial cell [3].

Export of iron is achieved by iron transporters, such asferroportin (also known as MTP-1 and Ireg-1). Ferroportinexports ferrous (2+) iron, which must be oxidized to its ferric(3+) form to be accepted by circulating transferrin [4,5].Ferroportin is thus believed to cooperate with ferroxidases,

ceruloplasmin (Cp) and hephaestin (Heph); exogenous Cp hasbeen shown to augment iron export via ferroportin from oo-cytes [5], and co-immunoprecipitation studies on CNS astro-cytes have demonstrated interaction between ferroportin andceruloplasmin [6].

Storage of iron is achieved through sequestration by cy-tosolic ferritin, a multimeric heteropolymer comprised of 24subunits of both heavy (H) and light (L) ferritin [7]. H-ferritinhas ferroxidase activity essential for iron incorporation intothe macromolecule. A single ferritin complex can accommo-date up to 4500 iron atoms, making ferritin storage an effi-cient means of iron sequestration and detoxification.

Both ferroportin and H-/L-ferritin are post-transcription-ally regulated to increase with increasing cellular iron in or-der to maintain intracellular iron homeostasis. Iron regulatoryproteins -1 and -2 (IRP-1 and -2) can bind to iron-responsiveelements (IREs) in the 5' region of the mRNAs of ferroportinand H-/L-ferritin. As intracellular iron increases, IRPs losethe ability to bind IREs. IRP1 undergoes a conformationalchange upon insertion of an iron-sulfur cluster at the IRE bind-ing site, resulting in occlusion of the IRE binding site. IRP2undergoes iron-dependent degradation. Neither IRP binds toIREs in iron-replete cells, removing a steric obstruction totranslation and resulting in an increase in ferritin levels [8,9].

©2004 Molecular Vision

Immunolocalization and regulation of iron handling proteinsferritin and ferroportin in the retina

Paul Hahn,1 Tzvete Dentchev,1 Ying Qian,1 Tracey Rouault,2 Z. Leah Harris,3 Joshua L. Dunaief1

1F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, Philadelphia, PA; 2Cell Biology and Metabolism Branch,National Institute of Child Health and Human Development, Bethesda, MD; 3Department of Anesthesiology and Critical CareMedicine, Division of Pediatric Anesthesiology and Critical Care Medicine, The Johns Hopkins University, Baltimore, MD

Purpose: CNS iron accumulation is associated with several neurodegenerative diseases, including age-related maculardegeneration. Intracellular overload of free iron is prevented, in part, by the iron export protein, ferroportin, and the ironstorage protein, ferritin. The purpose of this study was to assess retinal localization and regulation of ferroportin andferritin.Methods: Normal murine retinas were analyzed by immunohistochemistry to localize ferroportin, cytosolic ferritin, andmitochondrial ferritin, with double-labeling using cell-specific markers to identify cell types. Retinas deficient in theferroxidases, ceruloplasmin and hephaestin, accumulate iron in their retinas and RPE, while retinas deficient in ironregulatory proteins (IRPs) lack the ability to regulate several proteins involved in iron metabolism; retinas from theseknockout mice along with their age matched wild type littermates were also examined to study regulation of ferritin andferroportin. To enable visualization of label in the retinal pigment epithelial cells, sections from pigmented mice werebleached with H

2O

2 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. Ferroportinwas found in RPE and Müller cells. Iron accumulation in mice deficient in ceruloplasmin and hephaestin was associatedwith upregulation of ferritin and ferroportin. Mice deficient in IRPs showed upregulation of ferritin and ferroportin, likelybecause of their inability to repress translation.Conclusions: Normal retinas contain ferritin and ferroportin, whose levels are regulated by iron-responsive, iron regula-tory proteins. Ferroportin colocalizes with ceruloplasmin and hephaestin to RPE and Müller cells, supporting a potentialcooperation between these ferroxidases and the iron exporter. Cytosolic ferritin accumulates in rod bipolar synaptic termi-nals, suggesting that ferritin may be involved in axonal iron transport. Mitochondrial ferritin increases with iron accumu-lation, suggesting a role in iron storage.

Correspondence to: Joshua L. Dunaief, 305 Stellar Chance Labs, 422Curie Boulevard, Philadelphia, PA, 19104; Phone: (215) 898-5235;FAX: (215) 573-3918; email: jdunaief@mail.med.upenn.edu

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Iron-dependent changes in ferroportin levels can act in both asimilar and opposite manner as ferritin. In liver, iron deple-tion results in ferroportin downregulation, while iron deple-tion in duodenum results in increased ferroportin [10].

Another form of ferritin, mitochondrial ferritin (MtF), hasrecently been identified [11]. Most similar to H-ferritin, MtFhas ferroxidase activity and high iron affinity suggesting thatit may store iron in the mitochondria. Increased MtF has beendetected in the mitochondria of iron-loaded sideroblasts frompatients with sideroblastic anemia [12], but MtF does not havea recognizable IRE and has not been shown to be increased inresponse to other pathological conditions of iron overload.

Iron accumulation has been demonstrated in a variety ofneurodegenerative diseases, including Alzheimer’s disease,Parkinson’s disease, and age-related macular degeneration [13-15], implicating iron in their pathogenesis and indicating aneed to understand normal mechanisms of iron detoxificationin the CNS. The retina is a particularly suitable model for CNSiron studies. The retina is constantly exposed to photo-oxida-tive stress and is thus especially vulnerable to damaging freeradicals generated with iron excess. Diseases such asaceruloplasminemia and age-related macular degeneration aswell as conditions such as siderosis bulbi and subretinal hem-orrhage are associated with increased intraocular iron, whichmay contribute to the ensuing retinal degenerations [15-20].Additionally, the eye is particularly iron-dependent. The ex-tensive membrane biogenesis necessary to replenish continu-ally shed photoreceptor outer segments requires iron as anessential cofactor [21]. These outer segments are phagocy-tosed by the retinal pigment epithelium (RPE), which mustprocess the associated iron; further, as part of the blood-reti-nal barrier, the RPE functions to regulate flow of iron andother nutrients between the outer retina and the choroidal vas-culature. Iron in a normal adult rat retina has been detected athighest levels in the choroid, RPE, and photoreceptor innerand outer segments [22].

The roles of iron handling proteins in the retina have be-gun to be investigated. Import of iron into the retina can occurby transferrin-receptor-mediated endocytosis in both retinalvascular endothelial cells and RPE, whose tight junctions formthe basis of the blood-retinal barrier in the retinal and choroi-dal vasculature, respectively [23,24]. Iron in the retina maybe bound to transferrin, which is expressed by RPE and neu-ral retina, and can be endocytosed by cells throughout eachretinal layer, which contain transferrin receptor [22]. Iron stor-age and export in the retina has been less extensively studied.Ferritin immunolocalized in the rat retina to the choroid, RPE,and inner segments [22]. Strong expression of murine MtFmRNA has been observed by RT-PCR analysis in testis and inlower amounts in brain, thymus, kidney, heart, and retina.Immunohistochemical localization of MtF has been success-ful in erythroid cells, in sperm, and in mitochondria-denseendocrine cells of the testis and pancreas [25]; retinal local-ization of MtF protein has not yet been published. Ferroportin,originally detected in the basolateral membrane of enterocytes,is also found in CNS neurons and cultured astrocytes [6,26]but has not been immunolocalized within retina.

The purpose of the current study was to systematicallyinvestigate the distribution of some of the major proteins in-volved in iron detoxification (ferroportin, H- and L-ferritin,and MtF) in the murine retina. We used an immunohistochemi-cal approach in normal retinas, in iron overloaded retinas offerroxidase-deficient, Cp-/-Heph-/Y mice, and in IRP deficientretinas with defective IRP-mediated iron regulation. The lo-calization of these proteins sheds light on potential functions,both systemically and in the retina, and provides a baselinepattern from which to compare their levels and distribution inpathology.

METHODSGeneration of mice and fixation of eyes: Retinas from wildtype C57BL/6 and BALB/c mice (Jackson Laboratories, Bar

©2004 Molecular VisionMolecular Vision 2004; 10:598-607

Figure 1. Ferroportin is present in normal retina at high levels in the Müller cell endfeet and RPE. Normal BALB/c retina immunolabeled withomission of primary antibody (A) has undetectable autofluorescence or background immunolabel even at longer exposure times than all otherdisplayed panels. Nuclei are labeled with DAPI (blue). Ferroportin label (red) in normal BALB/c retina (B) localizes to Müller endfeet nearthe ILM, in photoreceptor inner segments (IS), and in the RPE, as demonstrated by the co-label with CRALBP (green), a marker for Müllercells and RPE (C,D). Ferroportin in the RPE excludes its apical microvilli, the green only label indicated with an asterisk (“*”) in D and in theinset of D, which shows a high power image of RPE co-labeled with ferroportin and CRALBP. Ferroportin is also present in a punctate patternthroughout the inner retina.

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Harbour, ME) 3-4 months old (n=2) and 6-7 months old (n=2)were studied for normal immunolocalization of proteins. Tostudy the effects of iron accumulation, retinas from C57BL/6mice with a mutation in Cp and/or Heph (Cp-/- and Cp-/-Heph-/Y) were studied along with their age-, strain-, and fixationmatched, wild type littermates (n=4 for each genotype). Addi-tionally, retinas from 9-12 month old C57BL/6 mice withmutations in IRP-1 and IRP-2 (IRP1+/-IRP2-/-, as IRP1-/-IRP2-/- mice die during embryogenesis) were studied alongwith their age-, strain-, and fixation matched, wild type litter-mates (n=2 for each genotype). Heph, an X-linked gene, isalso called sla, as it was originally identified in sex-linkedanemia mice [27].

Mice were reared with a 12 h light-dark cycle in a dedi-cated animal facility throughout their lives until sacrifice dur-ing daylight hours. Eyes from knockout mice and their ageand strain matched wild type littermate controls were enucle-ated immediately after sacrifice and fixed overnight in 4%paraformaldehyde. In addition, wild type C57BL/6 and BALB/c eyes were enucleated and lightly fixed in 4% paraformalde-hyde for 2 h to increase the sensitivity of immunohistochem-istry for those sections.

Within each figure, all retinas were from age and strainmatched mice and were fixed and immunostained simulta-neously and identically. All results were verified by repeatingthe staining on retinas from independent sets of genotype

matched mice. All procedures conformed to the ARVO State-ment for the Use of Animals in Ophthalmic and Vision Re-search, and the procedures were approved by the InstitutionalAnimal Care and Use Committee of the University of Penn-sylvania.

Immunohistochemistry: Fixed globes were rinsed in PBSand prepared as eyecups, cryoprotected in 30% sucrose, andembedded in Tissue-Tek OCT (Sakura Finetek, USA, Inc.,Torrance, CA). Immunohistochemistry was performed oncryosections 10 µm thick as published [28]. When compari-sons were made among panels of individual figures, retinas ineach panel were processed for immunohistochemistry identi-cally and simultaneously. Primary antibodies were selectedbased on previous demonstrations of their specificity by West-ern analysis and immunohistochemistry. Rabbit anti-ferroportin [10], a gift from D. Haile (University of TexasHealth Science Center, San Antonio, Texas), was diluted 1:20.Mouse anti-CRALBP [29], a gift from J. Saari (University ofWashington, Seattle, Washington), was diluted 1:250. Mouseanti-PKC-alpha, which labels rod bipolar cells [30], was pur-chased from Pharmingen (San Diego, CA) and was diluted1:500. Mouse anti-mitochondrial ATPase complex V (clone7H10) was purchased from Molecular Probes (Eugene, OR)and was diluted 1:200. P. Santambrogio, S. Levy, and P. Arosio(IRCCS, Milan, Italy) generously provided the following an-tibodies: rabbit anti-light ferritin (F17, 1:2500, 6.2 µg/ml),

©2004 Molecular VisionMolecular Vision 2004; 10:598-607

Figure 2. Cp-/-Heph-/Y retinas have increased ferroportin. Fluorescence photomicrographs of age matched wild type, Cp-/-, and Cp-/-Heph-/Y retinas immunolabeled for ferroportin (red) and imaged under identical exposure parameters. Nuclei were counterstained with DAPI (blue).Differences between wild type (A) and Cp-/- (B) were subtle, but there is a clear increase in ferroportin label in the Müller endfeet of the Cp-/-Heph-/Y retina (C). The punctate ferroportin label throughout the IPL is also increased in the Cp-/-Heph-/Y retina. In order to optimally detectdifferences in ferroportin in the pigmented RPE, it was necessary to pre-bleach sections (D-F). Equivalently bleached retinas immunolabeledwith ferroportin and imaged with equivalent exposure parameters reveals a robust increase in the Cp-/-Heph-/Y RPE of ferroportin, whichlocalized to both the apical and basolateral surfaces of the RPE (demarcated with brackets). Scale bars represent 50 µm.

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rabbit anti-heavy ferritin [31] (Y17; 1:2500, 4.2 µg/ml), rab-bit anti-mitochondrial ferritin (1:1000) [32]. Control sectionswere treated identically but with omission of primary anti-body.

After observing that pigment within the RPE quenchesimmunofluorescence, some sections were pre-treated with 3%H

2O

2 for 13 h in order to bleach endogenous melanin. In the

past, bleaching with H2O

2 has been used prior to

immunostaining of melanotic neoplasms [33,34]. We haveapplied a similar bleaching technique, which, to our knowl-edge, is its first reported use in the RPE. In the past, we haveused H

2O

2 bleaching prior to Perl’s staining for iron [15], with

preservation of retinal morphology. In the current paper, com-parison of anti-ferroportin labeling in bleached sections frompigmented mice to unbleached sections from albino mice sug-gests that H

2O

2 bleaching prior to retinal IHC is useful and

valid. Like H2O

2 bleached sections from pigmented, iron over-

loaded cp-/-heph-/Y mice, the RPE in sections from albinomice labels with anti-ferroportin (Figure 1).

Secondary antibodies (donkey anti-rabbit and anti-mouse)were labeled with Cy-3 (red; Jackson ImmunoResearch Labo-ratories, Inc., West Grove, PA) and/or Cy-2 (green). Nucleiwere counterstained with DAPI (1.5 µg/ml)-supplementedVectashield mounting medium (Vector Laboratories,Burlingame, CA). Sections were analyzed by fluorescencemicroscopy using identical exposure parameters across geno-types when comparisons were made. Confocal microscopy wasperformed with a Zeiss LSM 510 confocal microscope (CarlZeiss, Inc., Oberkochen, Germany), and epifluorescence mi-croscopy was performed with a Nikon TE-300 microscope(Nikon Inc., Kanagawa, Japan) and SpotRT Slider camera(Diagnostic Instruments, Inc., Sterling Heights, MI) withImagePro Plus software, version 4.1 (Media Cybernetics, Sil-

ver Spring, MD). All images were photographed at 1000 µmfrom the optic nerve head.

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis: C57BL/6 mice were sacrificed and eyespromptly enucleated. Anterior segments were removed, andretinas were completely dissected away from the underlyingRPE. Retinas were then flash frozen and stored at -80 °C. RPEwas enzymatically detached from the remaining posterior seg-ment by incubation in 0.25% trypsin at 37 °C, 5% CO

2 for 20

min. Detached RPE was collected in an eppendorf tube,pelleted, flash frozen and stored at -80 °C.

RNA was extracted from primary murine retina or RPEusing TRIZOL (Gibco, BRL Life Technologies, Rockville,MD) and used to generate first strand cDNA with a T7-(dT)

24

oligomer primer and the SuperScript II reverse transcriptase(InVitrogen, Carlsbad, CA). Specific sequences were thenamplified by PCR using a MJ research PTC-0200 thermocycler(MJ Research, Inc., Waltham, MA).

Primers specific for murine H-ferritin or L-ferritin weredesigned to span intron-exon boundaries within genomic DNAin order to amplify cDNA and minimize amplification of po-tentially contaminating genomic DNA. The H-ferritin forwardprimer was 5'-TTT GAG CCT GAG CCC TTT G-3', and thereverse primer was 5'-TCA AAG AGA TAT TCT GCC ATGC-3'. The L-ferritin forward primer was 5'-AGC GTC TCCTCG AGT TTC AG-3', and the reverse primer was 5'-AGGTTG GTC AGA TGG TTG C-3'.

RESULTSFerroportin is present in the RPE and Müller cell endfeet:Normal lightly fixed retinas (n=4) labeled for the iron trans-porter ferroportin (Figure 1) had positive Müller endfeet atthe inner surface of the retina (Figure 1B), as confirmed by

©2004 Molecular VisionMolecular Vision 2004; 10:598-607

Figure 3. IRP1+/-IRP2-/-retinas have increasedferroportin. Fluorescencephotomicrographs of agematched wild type andIRP1+/-IRP2-/- retinasimmunolabeled forferroportin (red), counter-stained for nuclei with DAPI(blue), and imaged underidentical exposure param-eters. Ferroportin label is in-creased in the IRP1+/-IRP2-/- inner retina includingMüller endfeet. Scale barsrepresent 50 µm.

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double labeling with cellular retinaldehyde binding protein(CRALBP; Figure 1C-D), a marker of Müller cells and RPE.Ferroportin was occasionally detectable within Müller pro-cesses, but levels were highest near the Müller endfeet.Ferroportin was also present in photoreceptor inner segments,in the outer plexiform layer (OPL), and in a punctate patternthroughout the inner plexiform layer (IPL). BALB/c (Figure1) and C57BL/6 (not shown) retinas had an identical patternof localization within Müller endfeet and occasionally withinMüller processes. Ferroportin was present in normal RPE (Fig-ure 1D; yellow label in RPE), with exclusion from the RPE’sapical microvilli (Figure 1D; green only CRALBP label inRPE). Because the pigment in C57BL/6 eyes quenches fluo-rescent label in the RPE, albino BALB/c mice are shown infigures of normal mice to best represent retinal and RPE pat-terns of immunolocalization. In figures of knockout mice (be-low), C57BL/6 mice are shown, to match the genetic back-ground of the knockout mice.

Ferroportin increases with iron accumulation: To exam-ine the effects of iron accumulation on ferroportin levels, weexamined Cp-/-Heph-/Y retinas, which have RPE and neuralretinal iron overload by 6 months [35]. Six month old Cp-/-Heph-/Y retinas were compared with retinas from age, strain,and fixation matched wild type and Cp-/- littermates (n=4 foreach genotype) immunolabeled for ferroportin. Cp-/- retinas(Figure 2B) had a modest increase in ferroportin label com-pared to wild type retinas (Figure 2A), and Cp-/-Heph-/Y reti-nas had substantially increased ferroportin (Figure 2C).Ferroportin immunolabel in the RPE was obscured by endog-enous melanin of the pigmented mice, and bleaching of theRPE was required to optimally observe ferroportin levelswithin the RPE. In Cp-/-Heph-/Y bleached RPE (Figure 2F)ferroportin localized to basolateral and apical regions, andferroportin levels were strikingly increased compared with wildtype (Figure 2D) and Cp-/- (Figure 2E) mice. The ferroportininner segment label was highly fixation dependent and was

©2004 Molecular VisionMolecular Vision 2004; 10:598-607

Figure 4. H- and L-ferritin are present in normal retina in rod bipolarcells. Normal retina labeled for H-ferritin (red; A) and L-ferritin(red; D). Nuclei are counterstained with DAPI (blue). Both ferritinsubunits have punctate label in the inner IPL, the region adjacent tothe ganglion cell layer (GCL). This punctate label is characteristic ofthe synaptic terminals of rod bipolar cells, labeled with anti-PKCα(green), which colocalizes (gold) with both H-ferritin (B-C) and L-ferritin (E-F). This colocalization is best seen in high power imagesof these synaptic terminals which are positive for both PKCα and L-ferritin (arrowheads, inset F). H- and L-ferritin mRNAs are also ex-pressed in retina and RPE, as indicated by bands from ethidium bro-mide-stained agarose gels corresponding to RT-PCR amplificationproducts of the indicated mRNAs and of the expected size from dis-sected C57BL/6 murine RPE and retina (G).

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visible in lightly fixed sections (Figure 1) but not in these moreheavily fixed sections (Figure 2). Sections within each panelof Figure 2 were fixed identically to allow semi-quantitativecomparisons among panels in Figure 2 but not between themore heavily fixed retinas of Figure 2 and the lightly fixedretina in Figure 1.

Ferroportin increases with IRP-deficiency: To determinewhether IRPs mediated the iron overload-induced increase inretinal ferroportin, we immunolabeled ferroportin in retinasfrom mice with IRP deficiency (n=2) and their wild type, agematched littermates (n=2). These mice had the genotypeIRP1+/- IRP2-/- which results in neurodegenerative diseaseby 9-12 months that is much more severe than that of IRP2-/-animals [36,37]. IRP1 contributes to baseline iron homeosta-sis [38], and since loss of one IRP1 allele significantly wors-ens the neurodegenerative disease of IRP2-/- animals [36],we chose to examine ferritin and ferroportin expression in theseseverely affected animals. These IRP deficient retinas (Figure3B) had increased ferroportin in the inner segments, Müllerendfeet, and inner retina compared to their age, strain, andfixation matched wild type retinas (Figure 3A), suggestingthat in the retina, ferroportin levels are regulated by IRPs.

H- and L-ferritin are present in rod bipolar cells: Nor-mal lightly fixed BALB/c (Figure 4) and C57BL/6 (not shown)retinas (n=3) labeled for H- or L-ferritin both had positiveprocesses in the photoreceptor inner segments, outer plexi-form layer (OPL), inner nuclear layer (INL) cell bodies, andsmall spheres in the innermost inner plexiform layer (IPL)adjacent to the ganglion cell layer (GCL; Figure 4A). Speci-ficity of ferritin label was confirmed by pre-incubation of an-

tibody with a 5 M excess of purified H or L-ferritin (gift fromP. Arosio), each of which eliminated label by the correspond-ing antibody but did not affect label with the unmatched anti-body (not shown). Double labeling with PKCα, a marker forrod bipolar cells, and H-ferritin (Figure 4A-C) or L-ferritin(Figure 4D-F) demonstrated that many of the ferritin positiveINL cells were rod bipolars, with ferritin accumulation in theiraxon terminals (punctate label in the IPL, Figure 4C,F). In theRPE, both ferritin subtypes had minimal immunofluorescenceeven in non-pigmented BALB/c RPE (Figure 4A,D). To con-firm ferritin expression in retina and to determine whether fer-ritin is expressed in the RPE, RT-PCR analysis was performedon cDNA from freshly harvested murine retina and RPE cellsusing primers specific for either H- or L-ferritin. In two dif-ferent experiments, one from a single 6 month old C57BL/6mouse (Figure 4G) and another from 3 mice aged 3, 5, and 7months (not shown), both H- and L-ferritin mRNAs were de-tected in both retina, consistent with immunohistochemicalresults, and RPE. The purity of isolated RPE was demonstratedby amplification of RPE65, an RPE specific gene, but notopsin, a retina specific gene.

Ferritin increases with IRP-deficiency: We have previ-ously demonstrated that iron accumulation in Cp-/-Heph-/Yretinas results in an increase in both H- and L-ferritin levels(unpublished data). To determine whether these changes inferritin levels might result from IRP regulation, weimmunolabeled retinas from IRP deficient animals (n=2) andtheir age matched, wild type littermates (n=2) for L-ferritin. Ifretinal ferritin is IRP-regulated, these deficient retinas shouldhave increased ferritin levels from relief of IRP-mediated in-

©2004 Molecular VisionMolecular Vision 2004; 10:598-607

Figure 5. IRP1+/-IRP2-/- retinas haveincreased L-ferritin.Fluorescence pho-tomicrographs ofage matched wildtype and IRP1+/-IRP2-/- retinasimmunolabeled forL-ferritin (red),counterstained fornuclei with DAPI(blue), and imagedunder identical ex-posure parameters.L-ferritin is in-creased in theIRP1+/-IRP2-/- in-ner retina, includingbipolar cell syn-apses in the IPL, aswell as in the OPLand inner segments.Scale bars represent50 µm.

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hibition of ferritin translation. Retinas from IRP deficient micehad increased L-ferritin label compared to their age, strain,and fixation matched wild type retinas. The L-ferritin labelwas increased in rod bipolar axon termini, outer plexiformprocesses, and photoreceptor inner segments of IRP deficientmice, suggesting that retinal ferritin levels are regulated byIRPs. As with ferroportin, the ferritin inner segment label wasfixation dependent and was visible in lightly fixed sections(Figure 4) but difficult to visualize in more heavily fixed sec-tions (Figure 5). Sections within each panel of Figure 5 werefixed identically to each other to allow semi-quantitative com-parisons between the panels of Figure 5 only.

Mitochondrial ferritin is present in inner segments andincreases with iron accumulation: Normal retinas had subtlemitochondrial ferritin (MtF) label in the mitochondria-richinner segments of photoreceptors (Figure 6A) and diffuselythroughout the inner retina. To confirm that this label corre-sponded to MtF and to examine the effect of iron accumula-tion on MtF levels, retinas with iron accumulation from defi-ciency of Cp and Cp/Heph (n=3 for each genotype) were alsolabeled for MtF. All genotypes had a diffuse pattern of MtFlabel throughout the inner retina (not shown). MtF label in thephotoreceptor inner segments was increased in Cp-/- retinas(Figure 6B) compared with their age, strain, and fixationmatched wild type retinas (Figure 6A), and MtF was furtherincreased in Cp-/-Heph-/Y retinas (Figure 6C). To verify thatanti-MtF labels mitochondria, retinas were co-labeled withanti-MtF and a mitochondria-specific antibody recognizingan ATPase in Complex V of the electron transport chain. Mi-tochondria are prominent in the inner segment ellipsoids, ex-cluding the inner segment myoid, which lies between the el-lipsoid and the photoreceptor nuclei [39]. As shown in a Cp-/- retina (Figure 6D-F), the mitochondria-specific ATPase andMtF co-localize specifically to ellipsoids and exclude themyoid (arrowheads, Figure 6D-F).

DISCUSSION Iron toxicity has been implicated in the pathogenesis of sev-eral neurodegenerative diseases, but mechanisms which regu-late normal iron homeostasis are incompletely understood.Cells can handle excess iron by decreasing import of iron orby increasing iron export by ferroportin, or by increasing ironstorage by cytosolic ferritin. Another type of ferritin, mito-chondrial ferritin, can bind iron and protect mitochondria fromiron toxicity [32]. In this study, we used an immunohistochemi-cal approach to shed light on the retinal functions and regula-tion of ferroportin, cytosolic ferritin, and mitochondrial fer-ritin.

In normal adult retinas, ferroportin was present in photo-receptor inner segments, the outer plexiform layer, Müllerendfeet, and RPE. Iron overload in Cp-/-Heph-/Y retinas re-sulted in increased ferroportin levels in Müller endfeet and inthe apical and basolateral surfaces of the RPE. This increasedferroportin is likely a result of iron-mediated inhibition of IRPfunction, as IRP deficient retinas also have increasedferroportin levels relative to their age matched wild type lit-termates. As ferroportin functions as an iron exporter [4,10],IRPs respond to iron overload in the retina by increasing lev-els of ferroportin, presumably to increase iron export.Ferroportin also immunolocalized to the outer plexiform layer,which contains synapses of photoreceptor axons and innernuclear layer neuronal dendrites. Since ferroportin has recentlybeen identified in association with brain synaptic vesicles [40],immuno-electron microscopy studies will be performed todetermine whether ferroportin is present in synaptic vesiclesin the retina.

Ferroportin transports ferrous iron [5], which must be oxi-dized to its ferric form to bind transferrin. Analogously, inyeast, the transporter protein Ftr1 acts in concert withferroxidase Fet3 to efficiently transport iron across the yeastmembrane [41]. A critical question in mammalian iron biol-

©2004 Molecular VisionMolecular Vision 2004; 10:598-607

Figure 6. Mitochondrial ferritin is present in normal inner segments and is increased in Cp-/- and Cp-/-Heph-/Y retinas. Fluorescence photo-micrographs of age matched wild type, Cp-/-, and Cp-/-Heph-/Y retinas immunolabeled for mitochondrial ferritin (A-C) and imaged underidentical exposure parameters. Cp-/- retinas (B) have increased mitochondrial ferritin (red) in the inner segments (IS) compared to wild type(A), and Cp-/-Heph-/Y retinas (C) have further increased mitochondrial ferritin. The label in the Cp-/- inner segments (E) excludes the innersegment myoid (arrowhead, D-F) and colocalizes with a mitochondria-specific antibody (green) to the inner segment ellipsoid (D,F), suggest-ing mitochondrial localization of MtF. Nuclei are labeled with DAPI (blue). Scale bars represent 50 µm.

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ogy is whether ferroportin activity is similarly coupled toferroxidases, Heph and/or Cp. Previous reports have demon-strated that addition of exogenous Cp can increase iron exportfrom oocytes [4] and from Cp-/- mouse brain astrocytes inculture [6]. Further, ferroportin and Cp have been shown toco-immunoprecipitate in cultured astrocytes [6]. We have pre-viously shown by immunohistochemistry, western analysis,and RT-PCR that Cp and Heph are present in both RPE andMüller cells, particularly their endfeet [35]. Our finding inthis report that ferroportin is also present in Müller endfeetand RPE further supports a cooperative role for ferroportinand both ferroxidases in RPE iron export or in Müller celliron export into the vitreous; Cp is also present in the vitreous[42] and in plasma of the choriocapillaris and may further fa-cilitate iron export through Müller endfeet and RPE, respec-tively.

Prominent ferritin label was present in murine rod bipo-lar cells, particularly their cell bodies in the INL and their syn-aptic terminals in the innermost IPL. Retinal ferritin appearsto be IRP-regulated, as IRP-deficient retinas have increasedferritin label relative to their age matched wild type controls.H- and L-ferritin label was present in inner segments of nor-mal murine photoreceptors, consistent with a previous reportstudying rat retinas [22]. This report also detected high levelsof iron in photoreceptor inner segments, and inner segmentlevels of ferritin appear sensitive to IRPs, suggesting that theinner segment may be an important site for iron sequestration.In contrast, neither H- nor L-ferritin was detected in photore-ceptor outer segments, which are shed daily and phagocytosedby RPE. The inner segment label for both ferritin andferroportin was present in retinas from both BALB/c (Figure1 and Figure 4) and C57BL/6 (not shown) mice, but only whenthe retinas were lightly fixed. Perhaps surprisingly, little fer-ritin protein was detected in RPE, although we did detect H-and L-ferritin expression in RPE by RT-PCR. As the RPE com-prises the outer blood-retinal barrier and thus may be subjectto high iron flux, normal, non-iron overloaded RPE, whichhas robust ferroportin label, may have little need to store ironin ferritin, explaining low protein levels.

Mice deficient in Cp and Heph accumulate iron in theirRPE and retinas by 6 months, and mice deficient in IRPs be-come neurologically symptomatic by 9-12 months; we thusstudied 6 month old Cp/Heph-deficient mice and 9-12 monthold IRP-deficient mice along with their age matched litter-mate wild type controls. Because immunohistochemistry is asemi-quantitative technique, we have taken many measuresto standardize experimental conditions. Within each figure,retinas were age, strain, and fixation matched, and retinas fromthe individual panels in each figure were processed in paral-lel, beginning at the time of enucleation until the end ofimmunolabeling. While we did not observe any obvious dif-ferences in immunolabel of any studied proteins between nor-mal mice at 3-4 months and 6-7 months, there could be subtleage-related changes in iron-handling proteins. Therefore, com-parisons in label among different genotypes were always madewith simultaneously and identically processed age matchedlittermate wild type controls. Differences in label intensity

within each figure were striking and reproducible among rep-licate experiments. At the ages studied herein, despite evidenceof abnormal iron metabolism, electroretinography on IRP de-ficient mice was normal. These results indicate that mice ofthese ages have normal summated rod, cone, and bipolar cellfunction, but do not preclude the possibility that further dis-ruption of iron homeostasis in older mice might lead to abnor-mal ERGs.

In IRP2-deficient mice, axon bundles in the brain haveincreased ferritin label, and the first sign of pathology is ax-onal degeneration, suggesting that ferritin may be involved iniron trafficking from the cell body to the synaptic terminal[36]. In retina, the unmyelinated rod bipolar cell axons labelfor ferritin, particularly at their synaptic terminals, supportingthe hypothesis that ferritins may be involved in iron traffick-ing to synaptic terminals.

Mitochondrial ferritin (MtF) is a third, recently describedferritin subtype [11]. We provide immunohistochemical iden-tification of MtF in the mitochondria of photoreceptor innersegment ellipsoids. MtF is increased in conditions of iron ex-cess induced by deficiency of Cp and further increased bydeficiency of both Cp and Heph. While the MtF in Cp-/- innersegments colocalizes with ellipsoid mitochondria, the MtF inthe Cp-/-Heph-/Y inner segments appear less specifically lo-calized to ellipsoids and present more diffusely throughoutboth the myoid and ellipsoid. MtF is translated in the cytosolwithin the myoid, and a mitochondrial localization signal se-quence targets the newly translated protein to the mitochon-dria [11]. Perhaps the dramatic iron overload in the Cp-/-Heph-/Y retina upregulates MtF translation to the point at which itexceeds the capacity for translocation into mitochondria, re-sulting in a more diffuse localization to the Cp-/-Heph-/Y in-ner segment myoid and ellipsoid. The increases in MtF withinCp-/- and Cp-/-Heph-/Y iron-overloaded inner segments areconsistent with increases observed in sideroblastic anemia [12].MtF, however, does not have a known iron-responsive ele-ment, and further studies will be needed to understand the roleand regulation of MtF.

The retina has two blood supplies, each with a blood-brain barrier. Supplying the inner retina is the retinal vascula-ture, whose blood-brain barrier consists of tight junctions be-tween retinal endothelial cells. Supplying the photoreceptorsand RPE is the choroidal circulation, whose blood-brain bar-rier consists of the tight junctions between RPE cells. Importof iron into the retina can occur by typical transferrin receptormediated endocytosis in both RPE and retinal endothelial cells[23,24]. Transferrin is expressed by both RPE and neural retina,including photoreceptors, and may deliver iron to transferrinreceptor, which is present on cells within each retinal layer,including photoreceptor inner segments [22,43].

Export of iron in the retina has been less extensively stud-ied, although the data presented herein suggests that at leastone mechanism of iron export from the retina may be into thechoroidal circulation through the RPE or into the vitreousthrough Müller cell endfeet at the inner limiting membrane.Together with the exclusion of ferroportin from the apical sur-face of normal RPE, these data suggest that at least one path-

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way for iron flux through the retina may be to normally enterthe retina via the retinal vasculature and exit the retina throughthe basolateral RPE into the choroidal circulation or throughthe Müller cell endfeet and the ILM into the vitreous. Futurestudies will investigate the subcellular localization of iron han-dling proteins, including iron import, storage, and export pro-teins, and in vitro RPE culture systems will be used to betterunderstand the normal flux of iron and its regulatory mecha-nisms, ultimately to further understand the effects of ironmisregulation in neurodegenerative diseases including AMD.

ACKNOWLEDGEMENTS We gratefully acknowledge the generous antibody gifts fromP. Arosio, P. Santambrogio, S. Levy, D. Haile, and J. Saari.This work was supported by NIH: R01EY015240,K08EY00417, MSTP T32GM7170, DK02464 and DK58086,a Career Development Award from Research to Prevent Blind-ness, International Retina Research Foundation, the SteinbachFoundation, and the Paul and Evanina Bell Mackall Founda-tion Trust.

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The print version of this article was created on 26 Aug 2004. This reflects all typographical corrections and errata to the article through thatdate. Details of any changes may be found in the online version of the article.