Mouse Brains Deficient in H-Ferritin HaveNormal Iron Concentration but a ProteinProfile of Iron Deficiency and IncreasedEvidence of Oxidative Stress.

Khristy Thompson,1 Sharon Menzies,1 Martina Muckenthaler,2 Frank M. Torti,3,5

Teresa Wood,1 Suzy V. Torti,4,5 Matthias W. Hentze,2 John Beard,6 andJames Connor1*1Department of Neuroscience and Anatomy, The Pennsylvania State University College of Medicine,Hershey, Pennsylvania2European Molecular Biology Laboratory, Heidelberg, Germany3Department of Cancer Biology, Wake Forest University School of Medicine, Wake Forest University,Winston-Salem, North Carolina4Department of Biochemistry, Wake Forest University School of Medicine, Wake Forest University,Winston-Salem, North Carolina5Comprehensive Cancer Center, Wake Forest University School of Medicine, Wake Forest University,Winston-Salem, North Carolina6Department of Nutrition, The Pennsylvania State University, State College, Pennsylvania

Several neurodegenerative disorders such as Parkin-son’s Disease (PD) and Alzheimer’s Disease (AD) areassociated with elevated brain iron accumulation relativeto the amount of ferritin, the intracellular iron storageprotein. The accumulation of more iron than can be ad-equately stored in ferritin creates an environment of ox-idative stress. We developed a heavy chain (H) ferritin nullmutant in an attempt to mimic the iron milieu of the brainin AD and PD. Animals homozygous for the mutation diein utero but the heterozygotes (�/�) are viable. We ex-amined heterozygous and wild-type (wt) mice between 6and 8 months of age. Macroscopically, the brains of �/�mice were well formed and did not differ from controlbrains. There was no evidence of histopathology in thebrains of the heterozygous mice. Iron levels in the brainof the �/� and wild-type (�/�) mice were similar, but�/� mice had less than half the levels of H-ferritin. Theother iron management proteins transferrin, transferrinreceptor, light chain ferritin, Divalent Metal Transporter 1,ceruloplasmin, were increased in the �/� mice com-pared to �/� mice. The relative amounts of these pro-teins in relation to the iron concentration are similar tothat found in AD and PD. Thus, we hypothesized that thebrains of the heterozygote mice should have an increasein indices of oxidative stress. In support of this hypoth-esis, there was a decrease in total superoxide dismutase(SOD) activity in the heterozygotes coupled with an in-crease in oxidatively modified proteins. In addition, apo-ptotic markers Bax and caspase-3 were detected in neu-rons of the �/� mice but not in the wt. Thus, we havedeveloped a mouse model that mimics the protein profilefor iron management seen in AD and PD that also shows

evidence of oxidative stress. These results suggest thatthis mouse may be a model to determine the role of ironmismanagement in neurodegenerative disorders and fortesting antioxidant therapeutic strategies.© 2002 Wiley-Liss, Inc.

Key words: ferritin; iron; oxidative stress; neurodegen-eration; null mutant; Alzheimer’s disease; Parkinson’sdisease; iron proteins; mice

The central nervous system (CNS) appears to beparticularly vulnerable to damage by reactive oxygen spe-cies (ROS). A number of factors contribute to the rela-tively high vulnerability of the CNS to oxidative damageincluding low levels of the natural antioxidant glutathionein neurons (Cooper, 1997), membranes containing a highproportion of polyunsaturated fatty acids (Hazel and Wil-liams, 1990), and a relatively increased requirement foroxygen because of the high metabolic activities of thebrain (Benzi and Moretti, 1995). Multiple lines of evi-

Contract grant sponsor: Jane B. Barsumian Trust Fund; Contract grantsponsor: NIH; Contract grant number: NS34280.

*Correspondence to: James R. Connor, Ph.D., George M. Leader FamilyLaboratory, Department of Neuroscience and Anatomy, Pennsylvania StateUniversity College of Medicine, Hershey Medical Center, Hershey, PA17033. E-mail: jrc3@psu.edu

Received 19 June 2002; Revised 5 August 2002; Accepted 6 August 2002

Journal of Neuroscience Research 71:46–63 (2003)

© 2002 Wiley-Liss, Inc.

dence implicate redox-active transition metals, such asiron and copper, as mediators of oxidative stress and ROSproduction in neurodegenerative diseases (Sayre et al.,1999). Data are now rapidly accumulating to show thatmetallochemical reactions that result in formation of ROSmight be the common denominator underlying Alzhei-mer’s disease (AD), amyotrophic lateral sclerosis, priondisease, cataracts, mitochondrial disorders (Friedreich’sAtaxia) and Parkinson’s disease (PD); (Connor, 1997;Hirsch and Faucheux, 1998; Jellinger, 1999; Bush, 2000;Floor, 2000). Because iron is the most abundant transitionmetal in the brain, and in biological systems in general, itis considered the most potent potential toxin to cells.Histological and quantitative changes in iron and in pro-teins responsible for iron homeostasis have been reportedin most neurodegenerative diseases (Pinero and Connor,2000; Thompson et al., 2001).

Iron that is not utilized immediately in a cell is storedin ferritin, and ferritin accounts for one-third to three-fourths of brain iron (Hallgren, 1958; Octave et al., 1983).Apoferritin is a hollow protein shell (outside diameter12–13 nm, inside 7–8 nm) capable of storing up to 4,500Fe3� atoms as an inorganic complex (Ford et al., 1984;Harrison et al., 1991). In vivo, the ferritin molecule isactually composed of 24 subunits with a molecular weight(MW) about 500,000. Two functionally and geneticallydistinct subunits, known as H (heavy; 21 kDa) and L(light; 19 kDa), make up the 24-mer complex. Heavy (H)and right (L) chains of ferritin are present in varying ratiosin different tissues and within the brain, occur in differentratios in different cell types (Connor et al., 1994; Harrisonand Arosio, 1996). Functionally, subunits of type L con-tribute to the nucleation of the iron core, but lack theferroxidase activity necessary for uptake of ferrous (Fe2�)iron. Subunits of type H possess ferroxidase activity andpromote rapid uptake and oxidation of ferrous iron (Law-son et al., 1989).

In the brain, Connor et al. (1995) observed an in-crease in H and L ferritin that coincided with a normalage-related increase in iron. A similar pattern is seen inaging rats (Focht et al., 1997). In PD and AD, however,there is not a concomitant increase in ferritin despite anincrease in iron concentration (Connor et al., 1995). Fur-thermore, ferritin isolated from the brains of AD patientshave a higher iron content than does ferritin isolated fromcontrol human brains (Fleming and Joshi, 1987). Togetherthese data indicate that iron levels are increased in thebrains of AD and PD patients relative to the ferritin levels.The rise in levels of iron without a concomitant change inferritin provides a source of “free” iron for free radicalgeneration (Halliwell, 1996, 2001).

We developed a mouse genetically deficient inH-ferritin in an attempt to mimic the milieu found in ADand PD with respect to iron and proteins of iron metab-olism. The long-term goal for this model will be toascertain whether iron imbalance in the brain is a contrib-utor to neurodegenerative diseases, a causative agent, or issimply associated with the disease process. In this first

paper, we characterize the status of the brain following thegenetic manipulation of H-ferritin and test the hypothesisthat this mouse will be vulnerable to oxidative stress as aresult of the disruption of iron homeostasis.

MATERIALS AND METHODS

Disruption of HFRT Gene

A 129/ReJ mouse genomic library was screened with analkaline phosphatase labeled DNA probe corresponding to a498-bp PstI fragment from the human H-ferritin cDNA (J.Drysdale, Tufts University). Identified phage clones were fur-ther screened with an alkaline phosphatase-labeled 30-baseprobe corresponding to intron 1, 5�-AATGACATGCCTGAT-CTATGCAGGAGCCAC-3� followed by an addition screen-ing with an alkaline phosphatase labeled 30-base probe corre-sponding to a segment just upstream of the ATG site in exon 1,5�-TTCCTGCTTCAACAGTGCTTGAACGGAACC-3�. Aphage clone containing a 12.5-kb insert was identified. AHindIII-BamHI 2.5-kb 5� fragment was subcloned into thetargeting vector (a modified PBS[sk]II cassette, T. Wood, PennState College of Medicine; Wood et al., 2000) 5� of the neo-myosin resistance gene (NeoR). An AsnI 5-kb fragment wasligated into the vector flanked by NeoR and the thymidinekinase (TK) gene. The resulting targeting vector was linearizedwith NotI (Fig. 1) and 20 �g of plasmid DNA was introducedinto 129SvEv CCE embryonic stem (ES) cells by electropora-tion. G418 selection was started after 48 hours and gancyclovirselection after an additional 72 hours. Surviving colonies werepicked, expanded, and screened for homologous recombinan-tion by Southern blotting using a 5�-external probe that was a770-bp AvaII-AseI fragment excised from the HFRT promoterregion (Kwak et al., 1990). Five positive ES clones were iden-tified and used for injection into C57BL6/J blastocysts. Chi-meric males were obtained from two of the positive clones andbred to C57BL6/J females to produce F1 heterozygous micewhich were then interbred.

Mice Genotyping

DNA from mice tails were prepared by overnight lysis ina proteinase K-containing buffer followed by phenol extractionsand ethanol precipitation. The DNA was digested with NcoI andmice were genotyped by Southern blot as described above.

Animals

H-ferritin heterozygote mice of mixed C57BL6/J X129SvEv genetic background were maintained under normalhousing conditions and regularly intercrossed. All mice weremaintained in accordance with the NIH Guide for the Care andUse of Laboratory Animals. Mice were anesthetized with CO2and organs removed following perfusion for homogenization orprocessed for paraffin embedding and immunohistochemicalanalysis. Male mice from the F2 and F3 generations were ex-amined at 6 to 8 months of age.

Tissue Homogenization

Mouse brain tissue was collected following saline perfu-sion, weighed and homogenized in homogenization buffer(0.32 M sucrose, 0.1 M phosphate-buffered saline [PBS] and

Hypoferritinemic Mice as an Oxidative Stress Model 47

protease-inhibitor mixture) at 4°C using an Ultra-Turrax T25tissue homogenizer (IKA Works, Inc.).

Measurement of Brain Iron

Brain iron was measured in triplicate by graphite furnaceatomic absorption spectrophotometry (model 5100 AA, Perkin-Elmer, Norwalk, CT) according to standard methods (Eriksonet al., 1997).

Hematological Analyses

Blood samples were obtained prior to perfusions by car-diac puncture. Hematocrit was determined by centrifugation ofblood collected into heparinized microcapillary tubes. Hemo-globin concentration was measured by the cyanmethemoglobinmethod (procedure no. 525, Sigma Chemical, St. Louis, MO).

RNA Preparation

Brains were removed and immediately stored in RNA-later (Ambion, Austin, TX) until processed. The tissue wasremoved later from the RNA. One hemisphere was submergedin 5 ml Trizol (Invitrogen, Carlsbad, CA) and homogenized for3 � 20 seconds using a Polytron PT2100 (Novodirect) at setting19. The RNA was extracted according to the manufacturer’sinstructions (Trizol/Invitrogen).

Selection of cDNA Clones

We established a cDNA based microarray that representsa selection of 300 genes that either encode proteins that aredirectly involved in iron metabolism or in one of the interlinkedpathways, such as Cu-metabolism, NO-metabolism, or the ox-idative stress pathway. For the list of genes and correspondinggene accession number, see Mouse chip version http://www.embl-heidelberg.de/ExternalInfo/hentze/suppinfo.html.The genes that are immobilized for the microarray platformwere selected from: (1) the literature, (2) microarray experi-ments performed on filters that contain approximately 20,000human nonredundant “expressed sequence tags” (ESTs) com-paring hemin and desferrioxamine treated CaCo2 cells, and (3)gene lists from published microarray studies that address meta-bolic pathways of interest. Three hundred EST clones frommouse, which were sequence-verified from both ends, werechosen for the microarray platform. The ESTs were selected tocontain the 3�end of a cDNA (i.e., the polyadenylation signal)and to extend for at least 300 bp towards the 5�end. The cloneswere purchased from the German Resource Center (RZPD).

Preparation of the Microarray Platform

The preparation of the microarray platform, which in-cludes amplification, spotting, and attachment of the cDNAs, isdescribed elsewhere (Richter et al., 2002). The same referencedescribes the use of negative hybridization controls to determinethe background noise of a microarray experiment. Briefly, weselected sequences from Arabidopsis and bacterial genomes forspotting. We consider a gene detectable and expressed if thesignal intensity exceeds two standard deviations of the averagesignal intensity of all negative controls.

Synthesis of Fluorescent cDNA Probes

Total RNAs derived from three control litter mates �/�or three HFRT �/� mice were pooled. Fluorescent cDNA

probes were synthesized from 5 �g total RNA using a linearmRNA amplification protocol, exactly as described in (http://cmgm.stanford.edu/pbrown/protocols/ampprotocol_3.html).Three �g of the T7 RNA polymerase amplified antisense RNAwas subsequently subjected to a direct labeling reaction byincorporation of Cy3 and Cy5 fluorescent dyes (Cy3 or Cy5)(http://cmgm.stanford.edu/pbrown/protocols/4_human_RNA.html). Two experiments were performed. Cy3 fluorescent dyeswere either incorporated into the cDNA synthesized from thewild-type mice and Cy5 fluorescent dyes into cDNA synthe-sized from the HFRT �/� mice or vice versa. This switch canidentify artifacts that could derive from different dye usage in theexperimental and control sample. Genes were only scored aschanging expression if they were identified in both experiments.

Prehybridization and Denaturation of Microarrays

The microarrays were immersed at 42°C in 6 � standardsodium citrate (SSC)/0.5% sodium dodcylsulfate (SDS)/1% bo-vine serum albumin (BSA) for 40 minutes and subsequentlywashed briefly with ddH2O at room temperature. Prior tohybridization, the spotted polymerase chain reaction (PCR)products were denatured by immersing the slides at 95°C inddH2O for 2 minutes. Excess liquid was removed from theslides by centrifuging them briefly at 715 � g in a microtiterplate centrifuge (Z320, Hermle, Wehingen, Germany).

Hybridization of Microarrays

Prior to hybridization, the purified Cy3- and Cy5-labeledcDNAs were mixed, 5 �g poly(dA) and 1 �g human Cot1DNA were added and subsequently evaporated in a speedvac at60°C. The resulting pellet was dissolved in 12 �l hybridizationbuffer (50% formamide /6 � SSC /0.5% SDS/5 � Denhardt)and denatured by incubating at 95°C for 2 minutes. The probewas then transferred onto the array under a 24 � 24 mmcoverslip and incubated in a humid chamber (GeneMachines)containing 2 � SSC drops for providing humidity. Hybridiza-tion was performed for 12 hours to 16 hours in a 42°C water-bath. After hybridization, the microarrays were washed in 0.1 �SSC/0.1% SDS for 10 minutes and twice with 0.1 � SSC for5 minutes (on an orbital shaker), followed by a brief immersionof the slides in ddH2O. All washing steps were performed atroom temperature.

Scanning and Data Analysis

All microarrays were scanned on a GenePix 4000B Microar-ray Scanner (Axon Instruments, Union City, CA). For each mi-croarray, individual laser power and photomultiplier settings wereused, allowing all signals to remain in the linear range of thescanner. Separate scan images for Cy3 and Cy5 were produced andanalyzed using the ChipSkipper microarray data evaluation soft-ware (http://pc-ansorge11.embl-heidelberg.de/chipskipper). In-tensity values for each spot were calculated by subtraction of thelocal background surrounding the spot. All spots were used for thecalculation of a linear regression line. The parameters of the regres-sion line (offset, slope) were used for normalization. The resultingdata were analyzed in Excel (Microsoft Corp., Redmond, WA).For each individual chip, all triplicate spots representing one cDNAwere averaged. For those genes that are represented by multiple

48 Thompson et al.

cDNA clones on the microarry platform, the average of ratios wascalculated and the standard deviation determined.

Northern Blot Analysis

Ten �g of total brain RNA from three HFRT �/� andthree wild-type control littermates were separated on a 1%formaldehyde agarose gel and blotted onto a Nylon membrane(Nytran N, Schleicher and Schuell, Dassel, Germany). Themembrane was subsequently hybridized to radioactively labeledprobes that are specific for H-ferritin, transferrin, transferrinreceptor, light chain ferritin, ceruloplasmin, divalent metaltransporter 1 (DMT1), and beta-actin mRNA. Church bufferwas used for the hybridizations (Sambrook et al., 1989). Thesignals obtained were quantified on a fluoroimager (MolecularDynamics, Sunnvale, CA, Amersham, Arlington Heights, IL).

Western Blots

Total brain homogenates (50 �g; determined with Brad-ford reagent (Bio-Rad, Richmond, CA) were analyzed persample by sodium dodecylsulfate-polyacrylamide gel electro-phoresis (SDS-PAGE). Five heterozygote (HFRT�/�) andfive wild-type (HFRT�/�) animals were analyzed per gel.Samples were diluted in SDS sample buffer and boiled for5 minutes. Samples were loaded on either 10%, 12%, or 15%SDS-polyacrylamide minigels followed by electroporesis andtransfer to nitrocellulose membranes (OPTITRAN, Schleicher& Schuell). Membranes were stained with ponceau-S to verifyequal loading of protein samples followed by rinses in ddH2O.Membranes were then blocked with 5% nonfat dry milk andproteins detected with antibodies to H-ferritin (1:1,500; rabbitanti-human recombinant H-ferritin (Cheepsunthorn et al.,1998), L-ferritin (1:1,500; mouse anti-horse ferritin; JacksonLabs, West Grove, PA), transferrin (1:1,500; goat anti-mousetransferrin; ICN, Irvine, CA), transferrin receptor (1:1,500; rab-bit anti-human placenta transferrin receptor; P. Seligman, Col-orado), Divalent Metal Transporter 1 (1:300; rabbit anti-ratDMT1), recognizes both iron responsive elements (IRE) andnon-IRE containing forms; (QCB) (Burdo et al., 2001), MetalTransport Protein 11 (1:500; Abboud and Haile, 2000b), ceru-loplasmin (1:3,500; rabbit anti-human Cp; DAKO, Carpinteria,CA) and heme oxygenase-1 (1:2,000; rabbit anti-rat heme-oxygenase 1; StressGen Biotechnologies Corp., Vancouver,B.C., Canada). Secondary anti-mouse or anti-rabbit horseradishperoxidase-linked antibodies, enhanced chemiluminescent de-tection (KPL) and x-ray films were used to visualize signals.LabWorks Software was used to determine the maximal inten-sity of each band. The mean value was determined for eachgroup and S.E.M. calculated. Student’s two-tailed t-test wasused to determine statistical significance between heterozygotesand wild-type mice.

Immunohistochemistry

Mice were perfused pericardially first with ice-cold salinefollowed by 4% paraformaldehyde. Brains were removed andplaced in 4% paraformaldehyde overnight, paraffin-embeddedand 6-�m-thick sections were prepared. The sections were

immunoreacted for H-ferritin, L-ferritin, Bax, and caspase-3using the avidin biotin complex (ABC) protocol and the reac-tion visualized using 3-amino-9-ethylcarbazole (AEC; Scytek,Logen, UT). As a control for nonspecific antibody immunore-actions, sections were incubated in the absence of the primaryantibody and taken through the immunoreaction process. Noimmunoreaction product was seen in any of the negative con-trols. In addition to the antibodies mentioned in the descriptionof the Western blot analysis, Bax (1:100; Trevigen, Gaithers-berg, MD) and caspase-3 (active form, 1:100; R&D Systems,Minneapolis, MN) cellular distributions were also determinedimmunohistochemically.

SOD Activity

Total superoxide dismutase (SOD) activity was measuredin homogenized brain tissue extracts by autoxidation of 5,6,6a,11b-tetrahydro-3, 9, 10-trihydroxybenzo[c]fluorene using acommercially available assay (Oxis Research; Bioxtech SOD-525). The assay was performed in duplicate on each sample. Theduplicate samples generated one mean. The mean value wasthen determined for each group and S.E.M. calculated. Stu-dent’s two-tailed t-test was used to determine statistical signifi-cance between heterozygotes and wild-type mice. A total of fiveanimals were used in each group.

Detection of Oxidatively Modified Proteins

Metal-catalyzed oxidation of proteins introduces carbonylgroups at specific amino acid residues in a site-specific manner(Levine, 1983). These carbonyl groups were detected in proteinlysates from homogenized brain tissue by reacting with 2,4-dinitrophenylhydrazine using a commercially available assay(Intergen Company, Purchase, NY; OxyBlot). KPL was used tovisualize signal intensity. LabWorks Software was used to de-termine the maximal intensity of each band. The mean valuewas determined for each group and S.E.M. calculated. Student’stwo-tailed t-test was used to determine statistical significancebetween heterozygotes and wild-type mice. A total of five ofanimals were used in each group.

RESULTSTargeted Deletion of the H-Ferritin Gene

A null ferritin allele was created by replacing exon 1to intron 1 with the neomyocin resistance gene (Fig. 1A).This mutation resulted in the deletion of the ATG startsite. SvEv129 ES cells were electroporated, selected inG418/gancyclovir containing media and surviving cellscarrying the H ferritin recombination were identified bySouthern blotting. Selected cells were used to produceheterozygous mice on a mixed 129SvEv � C57BL/6genetic background. Southern blot analyses of the littersrevealed only heterozygote and wild-type animals (Fig.1B). Western blot analyses of brain homogenates frommice genotyped in Figure 1B show that the heterozygotemice have decreased levels of H-ferritin protein in thebrain (Fig. 1C).

Phenotype of H Ferritin-Deficient MiceIn general, the HFRT �/� mice were indistin-

guishable at birth and during development from wild-type1This protein is also known as ferroportin IREG1

Hypoferritinemic Mice as an Oxidative Stress Model 49

littermate controls. The litter size of HFRT �/� matingpairs was decreased compared to wild-type control matingpairs (four to six pups versus eight to 10 pups, respective-ly). The smaller littersize for the HFRT �/� mice mostlikely reflects the in utero death of the homozygote ani-mals. No gross tissue abnormalities were observed duringnecropsy on the 6-month-old mice. HFRT �/� micewere fertile. The weights of various body organs (brain,heart, spleen, and kidney) as a percent of body weightwere analyzed with no statistically significant differences(Table I). There were no differences in the hematocrit orhemoglobin measurements (Table I). There were no mi-croscopic differences or evidence of histopathology in thebrains for the HFRT �/� mice compared to controls.

IronTotal brain iron measured by atomic absorption

spectrophotometry was the same in the HFRT�/� andwild-type mice (Fig. 2). Perl’s reaction product showedsimilar cell staining for iron between the two groups andsimilar regional distributions (Fig. 3). The iron stainingwas strongest in substantia nigra and in white matter tractsin both groups. The cell type that stains most prominentlyfor iron is oligodendrocytes. Although the distribution andcell pattern seen for iron staining was similar between thetwo groups, many of the iron-positive oligodendrocytes in

the white matter tracts had more processes in the HFRT�/� mice compared to littermate controls (Fig. 3Cand D).

Proteins of Iron Homeostasis: H-Ferritin,L-Ferritin, Transferrin, Transferrin Receptor,DMT1, MTP1, HO-1, and Ceruloplasmin

Western blot analyses of brain homogenates revealeddifferences in the levels of proteins involved in iron ho-meostasis in the HFRT�/� mice compared to littermatecontrols. H-ferritin levels are decreased by 83% in theHFRT�/� mice (Fig. 4A; P � 0.05). Levels of L-ferritin

Fig. 1. Generation of the H-ferritn null mutant. A: Structure of thenative locus, targeting construct and HFRT mutant allele are shown.Boxes represent HFRT exons. Restriction enzymes sites are marked asA (AsnI), B (BamHI), H (HindIII), and N (NcoI). NeoR and hsv-tkrepresent the neomycin resistance gene and the thymidine kinase generespectively. B: Southern analysis of Nco1 digest of genomic DNAisolated from mouse tail DNA preparations. The probe used for thegenomic Southern blot was the 5� AvaII/AseI fragment of the HFRTgenomic clone. Genomic DNA from HFRT �/� mice in lanes 1 and2 show the wild-type band of 10 kb and the 4.5-kb mutant band;genomic DNA from wild-type mice in lanes 3 and 4 show a band of10 kb. C: Western blot analysis of brain homogenates of HFRT �/�and littermate wild-type controls from B. Lanes 1 and 2 are HFRT�/� mice and lanes 3 and 4 are wild-type mice.

TABLE I. Biological Variables of Six- to Eight-Month-Old MaleHFRT �/� and Littermate Wild-Type Control Mice*

�/� �/�

Body wt (g) 38.6 � 5.97 35.9 � 1.07Brain (%) 1.21 � 0.18 1.29 � 0.09Heart (%) 0.52 � 0.18 0.45 � 0.11Spleen (%) 0.33 � 0.14 0.29 � 0.06Kidney (%) 1.28 � 0.46 1.46 � 0.05Hematocrit 0.54 � 0.04 0.47 � .05Hemoglobin (g/L) 162 � 11.03 141 � 18.32

*The weights of brain, heart, spleen, and kidney are expressed as per centof body weight (n � 5 �/� and 5 �/�). Hematocrit and hemoglobinvalue were determined by standard methods (see Materials and Methods; n� 10 �/� and 10 �/�). The results are shown as the mean and standarderror. None of the differences in the means are statistically significant.

Fig. 2. Total brain iron concentration in HFRT �/� and wild-typemice. Total brain iron concentrations were determined by atomicabsorption spectrophotometry (see Materials and Methods) in 6- to8-month-old mice (n � 5 �/� and 5 �/�). To minimize contam-ination from blood, the brains were perfused with saline. Once thebrains were removed, they were processed through additional salinerinses. The data are reported as nmol of iron (Fe) per gram of tissue (wetweight). There are no statistically significant differences (t-test).

50 Thompson et al.

Fig. 3. Cellular brain iron distribution in HFRT �/� and wild-typelittermate controls. Brains were stained for iron using the Perl’s reaction(see Materials and Methods). Stainable iron is present in oligodendro-cytes of both HFRT �/� mice (B,D,F) and wild-type littermatecontrols (A,C,E). In A and B, the distribution of iron positive oligo-dendrocytes is primarily in the subcortical white matter (area betweenthe arrows). The white matter tract in the �/� mice appears thinnerwhich is under further investigation. In C, the iron-positive oligoden-drocytes appear in rows (arrowhead) which is the classical pattern forinterfascicular oligodendrocytes. Oligodendrocytes that are process

bearing are rare even if the cell is not in a row (arrow). In contrast inthe HFRT �/� mice, (D), it is difficult to find oligodendrocytes inrows and many of the oligodendrocytes are processing bearing (arrows).Numerous iron-positive processes are visible throughout the whitematter. The presence of process bearing oligodendrocytes are consistentwith a disruption in myelin. bv, blood vessel. E and F are from thesubstantia nigra. The intensity of staining and the number of cellsstained are similar in both animals. There is a suggestion of increasediron positive processes in the substantia nigra. Magnification � 50� inA,B,E,F; 200� in C,D.

increased by a similar percentage (71%) in the HFRT�/� mice (Fig. 4B; P � 0.05). Transferrin and transferrinreceptor levels were increased in the HFRT �/� mice(Fig. 5A,B; P � 0.001). The levels of DMT1 were alsoincreased in the HFRT �/� mouse (Fig. 5C; P � 0.05).Metal transport protein 1 (MTP1) levels were not statis-tically significantly different (Fig. 5D). The amount ofheme oxygenase-1 (HO-1) did not vary between the twogroups (Fig. 5E). Ceruloplasmin levels were increased inthe HFRT �/� mice compared to the wild-type (wt)controls (Fig. 5F; P � 0.05).

Gene Array AnalysisMessenger RNA expression levels of genes that play

a role in iron metabolism and related pathways weredetermined by DNA microarray analysis. The microarrayis referred to as the “iron chip” in the rest of this manu-script. To define the set of genes that is detectable in thebrains of the HFRT �/� mice and the wild-type littermates, we utilized a set of negative controls that arespotted on the iron chip (Richter et al., 2002). We con-sider a gene detectable and expressed if the signal intensityexceeds two standard deviations of the average signalintensity of all negative controls. The approximate posi-tion of the background cut-off is indicated in Figure 6.

The result of the “iron chip” experiment is summa-rized in a representative scatter blot (Fig.6). The mRNAsrepresenting the heavy- and light chain ferritin, the trans-ferrin receptor, transferrin, and ceruloplasmin are clearlydetectable. In contrast, MTP1/ferroportin/IREG1, andDCT1/DMT1/Nramp2 lie closer to the cut-off for back-ground noise (Fig. 6). H-ferritin mRNA levels are re-duced 1.8 fold in the HFRT �/� mice. The DNAmicroarray data were confirmed by Northern blotting,analyzing total RNA prepared from individual mice.H-ferritin mRNA levels were normalized to actin mRNA

levels. An average reduction to 54% was calculated with astandard deviation of 4% (Fig. 7).An approximately 2-foldreduction in H-ferritin mRNA levels is consistent withthe expected phenotype in the heterozygotic mice. Inaddition, DNA microarray analysis showed a 1.4-fold re-duction of transferrin receptor mRNA levels in theH-ferritin �/� mice (Fig. 6). This was independent ofwhether the Cy3 dye was incorporated into cDNA pre-pared from the HFRT�/� brain and the Cy5 dye intocDNA derived from the littermate control, or vice versa(see Material and Methods). Taken in isolation, a 1.4-foldchange on the microarray would be considered borderlinein spite of its consistency in the “dye switch” experiment.However, Northern blotting demonstrated a similar de-crease in TfR mRNA levels in each of the HFRT�/�mice analyzed. The average reduction is 69% with astandard deviation of 11% (data not shown). It is interest-ing to note that the iron chip revealed this minor change.For the other approximately 300 genes tested, the generesponses recorded in the total brain of HFRT �/� miceis very similar to that of the wild-type control (data notshown). We did not observe a change in mRNA levels inthe HFRT �/� mice for transferrin, light chain ferritin,Divalent Metal Transporter 1, and ceruloplasmin in com-parison to �/� mice. The lack of change in expressionlevels of the transferrin and light chain ferritin genes wasconfirmed by Northern blotting (data not shown).

At the cellular level, the type of cell staining forH-ferritin protein was similar between the HFRT �/�mice and wild-type littermate controls. However, theintensity of staining was dramatically decreased in theneurons of the �/� mice (Fig. 8) throughout the brain,consistent with the decrease in H-ferritin determined byimmunoblotting. Oligodendrocytes in white matter tractsand in the striatum stained with a similar intensity for

Fig. 4. Quantitative analysis of heavy (H) and light (L)-ferritin in brainsof HFRT �/� and �/� mice. Brain homogenates from five HFRT�/� and five littermate control (�/�) mice were examined for thepresence of H-ferritin using a polyclonal rabbit anti-human recombi-nant H-ferritin antibody (A). The presence of L-ferritin was deter-mined using a polyclonal rabbit anti-horse spleen ferritin antibody (B).

Densitometric analysis revealed a statistically significant decrease inH-ferritin and increase in L-ferritin in the heterozygotes for the ferritinmutation (�/�) compared to littermate controls (�/�). The resultswere analyzed densitometrically and the data are reported as meanoptical density with S.E.M.

52 Thompson et al.

Fig. 5. Reduction of H-ferritin in mice results in alterations in the levels of proteins involved in ironhomeostasis in the brain. Brain homogenates from five littermate control mice (�/�) and five HFRT�/� were examined for the presence of transferrin (A), transferrin receptor (B), divalent metaltransporter 1 (DMT1; C), Metal transport protein 1 (MTP1); D), heme oxygenase-1 (HO-1; E), andceruloplasmin (F). A mean optical density was determined and the results reported as mean � S.E.M.

Hypoferritinemic Mice as an Oxidative Stress Model 53

H-ferritin in both groups. Immunohistochemical analysisof L-ferritin revealed a similar pattern of cell stainingbetween the two groups of mice except in the striatumwhere an increase in L-ferritin processes on the cells wasclearly present (Fig. 9).

Indices of Cell StressSOD activity was measured as an index of oxidative

stress. Total SOD activity was decreased by approximately50% in heterozygote mice compared to wild-type controls(Fig. 10; P � 0.05 ). Oxidatively modified proteins asdetermined by the carbonyl assay were increased 2-fold inthe heterozygote mice (Fig. 11; P � 0.05).

Immunohistochemical analysis for the presence ofBax and the active form of caspase-3 was performed todetermine whether any specific cell type may be differen-tially expressing one of these indicators of apoptosis re-

sulting from oxidative stress. Bax staining was detected inneurons in the cortex, lateral hypothalamic nucleus, andstriatum in the HFRT�/� mice but not in the wt (Fig.12). Caspase-3 staining was also present in cortical andstriatal neurons as well as neurons of the hippocampus ofthe HFRT �/� mice but not in the wt (Fig. 13). How-ever, the Purkinje cells in the cerebellum were intenselystained in both groups (Fig. 13G and H).

DISCUSSIONThere were no live births of mice that were ho-

mozygous for the H-ferritin null mutation. This obser-vation is consistent with a previous report thatH-ferritin-deficient mice die between embryonic days3.5 and 9.5 (Ferreira et al., 2000). The heterozygotes forthe null mutation are viable and have significant alter-ations in amounts of brain iron management proteins

Fig. 6. Gene expression profile from total brain of HFRT �/� mice.A scatter plot analysis of a DNA microarray experiment performed onthe iron chip (version 2.0) is shown. The y-axis represents the expres-sion levels of genes in three pooled HFRT �/� mice (logarithmicscale). The x-axis represents the expression levels of genes in threepooled wt control mice (logarithmic scale). Each gene selected for the

array is represented by multiple spots derived either from a single ormultiple independent cDNA clones. A selection of spots is enlarged andshown in color. The identity of the genes represented by these spots isindicated in the legend. Spots above the background cut-off value areconsidered detectably expressed in the mouse brain.

54 Thompson et al.

but not total iron. In the brain, the relatively low levelof expression of H-ferritin in the presence of normallevels of iron is similar to that seen in brains of indi-viduals with Alzheimer’s or Parkinson’s disease (Con-nor et al., 1995). This relationship of ferritin to iron isexpected to promote oxidative stress. The CNS of theheterozygote mouse is under oxidative stress as evi-denced by the significant increase detected in oxida-tively modified proteins and the decrease in SOD ac-tivity. More specifically, the oxidative stress appearslocalized to the neurons because they alone expressimmunodetectable levels of activated caspase-3 andBax. The increased detection of caspase-3 and Bax isconsistent with observation in AD brains (Paradis et al.,1996; MacGibbon et al., 1997; Kitamura et al., 1998;Tortosa et al., 1998; Giannakopoulos et al., 1999; Sta-delmann et al., 1999, 2001; Su et al., 2000; Rohn et al.,2001). These results indicate that H-ferritin heterozy-gote mutant mice are a good model in which to studythe contribution of loss of iron homeostasis to oxidativestress in the brain.

Changes in Iron Management Proteins in theBrain

H-ferritin protein expression in the HFRT�/�mice was les than 20% of normal. At the cellular level,H-ferritin staining was decreased in neurons throughoutthe brain. However, the staining intensity in oligodendro-

cytes for both iron and H-ferritin throughout the brainwas similar to wt. This observation suggests selective tar-geting of iron to oligodendrocytes and that oligodendro-cytic function may not be impaired. However, the in-creased branching seen in many of the oligodendrocytessuggests these cells may be more immature than normal.The decrease in H-ferritin staining in the neurons suggeststhat these cells may be iron-deficient. This notion isconsistent with the observation that transferrin receptorprotein levels are increased 2.5� in the HFRT �/� micecompared to wt. Transferrin receptors are predominantlyexpressed on neurons in the brain (Octave et al., 1983;Jefferies et al., 1984; Giometto et al., 1990; Mash et al.,1990; Moos, 1996; Dickinson and Connor, 1998; Kissel etal., 1998). An increase in transferrin receptor is a consistentresponse to iron deficiency including within the brain(Pinero et al., 2000).

No change in cellular staining pattern was seen in theHFRT �/� mice compared to wt. Thus the change in Tfreceptor protein levels should have come mostly fromneurons. The proposed mechanism by which oligoden-drocytes acquire iron is via a ferritin binding protein(Hulet et al., 2000). The notion that neurons are irondeficient is further supported by the lack of L-ferritindetection in these cells despite a 71% increase in L-ferritinprotein in the brain. Neurons have the capacity to expressL-ferritin when challenged with iron (Malecki et al., 2002)and L-ferritin expression could have been induced ifH-ferritin levels were not sufficient to handle the incom-ing iron. There was no difference in the cell types thatexpress the L ferritin subunit between HFRT �/� miceand wt.

Additional indications that the HFRT �/�mouse brain is phenotypically iron deficient comesfrom the finding of a 5� increase in transferrin. Trans-ferrin is an iron mobilization protein and is synthesizedin oligodendrocytes and choroid plexus within thebrain (Bloch et al., 1985; Aldred et al., 1987). Anincrease in brain transferrin has been reported in brainsthat are iron-deficient (Pinero et al., 2000) and in thefrontal cortex of Alzheimer’s disease brains (Loeffler etal., 1995). DMT1 is present in endocytic vesicles and isresponsible for the transport of iron from the vesicle tothe cytosol. In the brain, DMT1 is found in the micro-vasculature and in the astrocytes associated with theblood vessels, suggesting it is involved in brain ironuptake (Burdo et al., 2001). Belgrade rats, which have adefect in DMT1, have less iron in the brain than normal(Burdo et al., 1999). DMT1 levels are increased 65% inthe heterozygotes compared to control. This observa-tion is also consistent with the idea that the HFRT�/� brain is iron-deficient. MTP1/ferroportin. IREG1is thought to be involved in iron egress from the cell(Abboud and Haile, 2000a; Donovan et al., 2000;McKie et al., 2000) and is found predominantly inneurons but is also present in oligodendrocytes (Burdoet al., 2001). The concentration of this protein is notsignificantly altered in the heterozygote mice. The re-

Fig. 7. H-ferritin Northern blot analysis. A Northern blot analysis wasperformed to confirm the observations with the gene array. In thisfigure, the results of the H-ferritin Northen blots are shown. The dataare presented as the mean of three animals in each group. The amountof H-ferritin mRNA was compared to Actin mRNA and the ratio isplotted. The source of mRNA was total brain homogenate. OD,optical density.

Hypoferritinemic Mice as an Oxidative Stress Model 55

Fig. 8. Immunohistochemical localization of H-ferritin. Neurons ex-press H-ferritin in the cortex (arrows in A,B) in both �/� (A) andHFRT �/� mice (B). However the staining intensity is much less inthe HFRT �/� mice. A similar pattern is seen in the hippocampus(C,D), where strong neuronal staining is observed in the pyramidal

layer (arrow), of the �/� (C) mice but only light staining is observedin the HFRT �/� mice (arrow in D). In the striatum (E,F), H-ferritinstaining is found in oligodendrocytes (e.g., at arrow) in both groups ata similar intensity. Magnification in all panels � 100�.

56 Thompson et al.

sponse of this protein to iron status is an organ-specificresponse. MTP1 is not detected in liver in the iron-deficient state, but is increased in the gut (Abboud andHaile, 2000). Thus, it is not possible at this time tointerpret the lack of response of MTP1 to the loss ofH-ferritin in the brain.

Taken together, the proteins involved in iron ho-meostasis in the brain in the H-ferritin null mutant het-erozygote mice reflect a state of iron deficiency despite thenormal levels of iron and an increase in L-ferritin. Apossible explanation for the protein profile of iron defi-ciency despite the normal concentration of iron is thatmuch of the iron could be bound in L-ferritin and thus lessbioavailable than normal (Lawson et al., 1989; Harrisonand Arosio, 1996).

One protein that is capable of replacing the ferroxi-dase activity in the brain that would be diminished by aloss of H-ferritin is ceruloplasmin. Indeed, ceruloplasminhas been suggested to provide the ferroxidase activityresponsible for iron uptake into ferritin (Juan et al., 1997)and can accelerate the iron incorporation into apotrans-ferrin (Osaki and Johnson, 1969). Ceruloplasmin report-edly increases in iron-deficient states and increased by7-fold in the brains of the HFRT �/� mice, consistentwith the overall profile of brain iron deficiency (Shermanand Moran, 1984; Mukhopadhyay et al., 2000). The ele-vation of ceruloplasmin could be further indication ofoxidative stress within the HFRT �/� brain, althoughceruloplasmin can have both prooxidant and antioxidanteffects (Samokyszyn et al., 1988). In AD brains, cerulo-

Fig. 9. Immunohistochemical localization of L-ferritin. L-ferritin im-munostaining is relatively weak in the cerebral cortex in both thelittermate controls (A) and the �/� mice (B). In both animals, thestaining is confined to oligodendrocytes (e.g., at arrow). In the striatum,there are occasional L-ferritin-positive oligodendrocytes in the wild

type animals (arrow in C) with relatively little staining in the paren-chyma. In contrast the L-ferritin staining intensity is robust in the �/�mice (D) in oligodendrocytes (arrow) and in processes. Magnificationin all panels � 100�.

Hypoferritinemic Mice as an Oxidative Stress Model 57

plasmin has been reported to both increase and decrease(Connor et al., 1993; Loeffler et al., 1996). Thus, therelationship of the ceruloplasmin response to that seen inAD brain tissue cannot be established without furtherstudy.

Gene Expression ProfileFerritin and transferrin receptors levels are posttran-

scriptionally regulated through the interaction of iron reg-ulatory proteins (IRPs), which are cytoplasmic mRNAbinding proteins and IREs on their mRNAs (Eisensteinand Munro, 1990). The reduction in H-ferritin proteinlevels seem to be a result of reduced mRNA levels in theHFRT �/� mice. However, protein levels decrease fur-ther than the mRNA levels. In addition, there is a 1.4-folddownregulation of the transferrin receptor mRNA whenan increase might be expected. These observations mighthave important implications for the posttranscriptionalregulatory mechanism. The most parsimonious explana-tion is that the total brain homogenate has obscured theresponses of the individual cell types. It is known that theIRPs have different cell distributions in the brain (Leiboldet al., 2001) and a knockout of IRP2 has selective effectswithin the brain (LaVaute et al., 2001). Thus, furtherunderstanding of the expression of ferritin and transferrinreceptors and their regulation in this model must await cellculture analyses.

Changes in transferrin, light chain ferritin, DMT-1,MTP-1, and ceruloplasmin protein levels are not due to a

change in the amount of mRNA. L-ferritin and DMT1are posttranscriptionally regulated (Munro et al., 1993; Leeet al., 1998), which may account to the absence of changein mRNA levels. Tf mRNA is present in the brain inoligodendrocytes and choroid plexus. The iron stainingintensity in the oligodendrocytes appears normal; thus,these cells may not be expected to increase Tf mRNAexpression. The increase in Tf protein in the brain couldbe the result of increased transcytosis from the serum(Dickinson et al., 1996). Ceruloplasmin mRNA is foundin astrocytes in the brain (Klomp et al., 1996). The in-crease in ceruloplasmin protein in the HFRT �/� braincould also be due to an increase production by the choroidplexus. A caveat in these interpretations is that changes inmRNA levels in specific regions of the brain might bemasked by the use of total RNA derived from whole brainextracts.

Increased Indices of Oxidative StressWe hypothesized that the milieu of low ferritin

relative to normal iron should promote oxidative stress(Orino et al., 1999, 2001). The increase in oxidativelymodified proteins in the H-ferritin null mutant heterozy-gote mice is an indication that oxidative stress is occurringin the CNS of the mutant mice. The decrease in SODactivity also suggests the heterozygote animals are at in-creased risk for oxidative stress. SOD activity has been

Fig. 10. Superoxide dismutase (SOD) activity. Superoxide dismutaseactivity was determined in whole brain homogenates from HFRT�/� and �/� mice. There were five mice in each group. The meansand standard error are plotted as units of activity per total protein (mg).The results are statistically significant (P � 0.05).

Fig. 11. Carbonyl assay for oxidatively modified proteins. Oxidativelymodified proteins were detected using the carbonyl assay (see text). Theresults indicate that oxidatively modified proteins are elevated in theHFRT �/� mice compared to the control group (�/�). The levelsof oxidatively modified proteins were determined by densitometry.The mean and standard errors for five animals in each group are shownin this graph. The results are statistically significant (P � 0.05).

58 Thompson et al.

Fig. 12. Immunohistochemical analyses of Bax. Bax staining was minimal in the brains of �/� micecompared to the HFRT �/� mice. The brain areas represented in this figure are cortex (A,B), lateralhypothalamic nucleus (C,D), and striatum (E,F). In each area, the HFRT �/� mice (B,D,F) hadincreased Bax staining compared to the littermate controls (A,C,E). The Bax staining in each brainarea was found only in neurons (arrows). Magnification in all panels � 100�.

Hypoferritinemic Mice as an Oxidative Stress Model 59

Fig. 13. Immunohistochemical analyses of caspase-3 active form. Im-munostaining for the active form of caspase-3 was minimal in the brainsof �/� mice compared to the HFRT �/� mice. The brain areasrepresented in this figure are cortex (A,B), hippocampus (C,D), stria-tum (E,F) and cerebellum (G,H). In each area, the HFRT �/� mice(B,D,F,H) have increased staining for the active form of the caspase-3enzyme compared to the littermate controls (A,C,E,G).except for in

the Purkinje cells of the cerebellum (G,H) where staining was intensein both conditions. Staining for the active form of the caspase-3 enzymein each brain area was found primarily in neurons (arrows). However,in the cortex, an occasional glial cell (arrowhead) was visible and somewere associated with the vasculature (white arrowhead) Magnifica-tion � 100� in A–D; 50� in E,F; 200� in G,H.

related to cellular metal status. Studies by Isler et al. (2002)found decreased SOD activity in erythrocytes from pa-tients with iron-deficient anemia. Toxicity by mercuricchloride resulted in a decrease in SOD activity (Hussain etal., 1997). The decrease in SOD activity seen in theH-ferritin heterozygote mice may be a result of irondeficiency at the cellular level. The mechanism by whichSOD activity is decreased in this model will be a subjectfor future studies.

A particularly salient observation in this analysis wasthe loss of H-ferritin staining in neurons and the lack ofdetectable L-ferritin in neurons. This observation led toour hypothesis that neurons would be particularly vulner-able to oxidative stress in this animal model. The cellularanalysis of caspase and Bax supports our hypothesis.Caspase-3 and Bax were both detected in neurons in thecortex and striatum. Caspase-3 was also found in theneurons of the hippocampus and Bax in the neurons of thelateral hypothalamic nucleus. No other cell type was ob-served expressing Bax or the active caspase-3 enzyme.Increases in caspase-3 have been implicated in AD (Su etal., 2000) and p53 Bax activation has been found followingtreatment with beta-amyloid or presenilin in neuronalcultures (Alves da Costa et al., 2002; Zhang et al., 2002).For reasons discussed above, the presence of apoptoticmarkers in neurons could be due to an insufficient ironsupply in these cells.

In conclusion, the HFRT �/� mouse model rep-resents a unique opportunity to study the influence of ironhomeostatic mechanisms on oxidative stress. The futureplans are to use this animal model to determine whetherloss of iron homeostatic mechanisms is a contributingfactor, a causative factor, or is simply associated withneurodegenerative disorders. The first stage of the analysis,reported here, suggests that iron imbalance contributes tooxidative stress, suggesting that we have developed ananimal model in which efficacy of anti-oxidant strategiescan be evaluated. Ultimately this animal model will beuseful to determine the contributions of iron to neurode-generative processes.

ACKNOWLEDGMENTSThe CCE ES cells were provided by Dr. E. Rob-

ertson. This work was supported by the Jane B. BarsumianTrust Fund and NIH grant NS34280. The authors grate-fully acknowledge the technical support of Bonnie Del-linger. The MTP1 antibody was generously provided byDavid Haile. The authors thank Belen Minana and Jos deGraaf for outstanding technical help in the establishmentof the microarray platform and sample analysis. M.W.H.gratefully acknowledges support by the Deutsche For-schungsgemeinschaft (Gottfried-Wilhelm-Leibniz Prize).Supported in part by DK42412 (F.M.T.).

REFERENCESAbboud S, Haile DJ. 2000. A novelmammalian iron-regulated protein

involved in intracellular iron metabolism. J Biol Chem 275:19906–19912.

Aldred AR, Dickson PW, Marley PD, Schreiber G. 1987. Distribution of

transferrin synthesis in brain and other tissues in the rat. J Biol Chem262:5293–5297.

Alves da Costa C, Paitel E, Mattson MP, Amson R, Telerman A, AncolioK, Checler F. 2002. Wild-type and mutated presenilins 2 trigger p53-dependent apoptosis and down-regulate presenilin 1 expression inHEK293 human cells and in murine neurons. Proc Natl Acad Sci U S A99:4043–4048.

Benzi G, Moretti A. 1995. Are reactive oxygen species involved in Alz-heimer’s disease? [see comments]. Neurobiol Aging 16:661–674.

Bloch B, Popovici T, Levin M, Tuil D, Kahn A. 1985. Transferrin geneexpression visualized in oligodendrocytes of the rat brain using in situhybridization and immunohistochemistry. Proc Natl Acad Sci USA 82:6706–6710.

Burdo JR, Martin J, Menzies SL, Dolan KG, Romano MA, Fletcher RJ,Garrick MD, Garrick LM, Connor JR. 1999. Cellular distribution of ironin the brain of the Belgrade rat. Neuroscience 93:1189–1196.

Burdo JR, Menzies SL, Simpson IA, Garrick LM, Garrick MD, Dolan KG,Haile DJ, Beard JL, Connor JR. 2001. Distribution of divalent metaltransporter 1 and metal transport protein in the normal and belgrade rat.J Neurosci Res 66:1198–1207.

Bush AI 2000. Metals and neuroscience. Curr Opin Chem Biol 4:184–191.Cheepsunthorn P, Palmer C, Connor JR. 1998. Cellular distribution of

ferritin subunits in postnatal rat brain. J Comp Neurol 400:73–86.Connor JR. 1997. Metals and oxidative damage in neurological disorders.

New York: Plenum Press.Connor JR, Tucker P, Johnson M, Snyder B. 1993. Ceruloplasmin levels

in the human superior temporal gyrus in aging and Alzheimer’s disease.Neurosci Lett 159:88–90.

Connor JR, Boeshore KL, Benkovic SA. 1994. Isoforms of ferritin have aspecific cellular distribution in the brain. J Neurosci Res 37: 461–465.

Connor JR, Snyder BS, Arosio P, Loeffler DA, LeWitt P. 1995. A quan-titative analysis of isoferritins in select regions of aged, parkinsonian, andAlzheimer’s diseased brains. J Neurochem 65:717–724.

Cooper AJL. 1997. Glutathione in the brain: disorders of glutathionemetabolism. In: Rosenberg RN, Prusiner SB, DiMauro S, Barch RL,Klunk LM, editors. The molecular and genetic basis of neurologicaldisease. Boston: Butterworth-Heinemann. p 1242–1245.

Dickinson TK, Connor JR. 1998. Immunohistochemical analysis of trans-ferrin receptor: regional and cellular distribution in the hypotransferrine-mic (hpx) mouse brain. Brain Res 801:171–181.

Dickinson TK, Devenyi AG, Connor JR. 1996. Distribution of injectediron 59 and manganese 54 in hypotransferrinemic mice. J Lab Clin Med128:270–278.

Donovan A, Brownlie A, Zhou Y, Shepard J, Pratt SJ, Moynihan J, PawBH, Drejer A, Barut B, Zapata A, Law TC, Brugnara C, Lux SE, PinkusGS, Pinkus JL, Kingsley PD, Palis J, Fleming MD, Andrews NC, Zon LI.2000. Positional cloning of zebrafish ferroportin1 identifies a conservedvertebrate iron exporter. Nature 403:776–781.

Eisenstein RS, Munro HN. 1990. Translational regulation of ferritin syn-thesis by iron. Enzyme 44:42–58.

Erikson KM, Pinero DJ, Connor JR, Beard JL. 1997. Regional brain iron,ferritin and transferrin concentrations during iron deficiency and ironrepletion in developing rats. J Nutr 127:2030–2038.

Ferreira C, Bucchini D, Martin ME, Levi S, Arosio P, Grandchamp B,Beaumont C. 2000. Early embryonic lethality of H ferritin gene deletionin mice. J Biol Chem 275:3021–3024.

Fleming J, Joshi JG. 1987. Isolation of aluminum-ferritin complex frombrain. Proc Natl Acad Sci 84:7866–7870.

Floor E. 2000. Iron as a vulnerability factor in nigrostrriatal degeneration inaging and Parkinson’s disease. Cell Mol Biol 46:709–720.

Focht SJ, Snyder BS, Beard JL, Van Gelder W, Williams LR, Connor JR.1997. Regional distribution of iron, transferrin, ferritin, and oxidatively-modified proteins in young and aged Fischer 344 rat brains. Neuroscience79:255–261.

Hypoferritinemic Mice as an Oxidative Stress Model 61

Ford GC, Harrison PM, Rice DW, Smith JM, Treffry A, White JL, YarivJ. 1984. Ferritin: design and formation of an iron-storage molecule. PhilosTrans R Soc Lond B Biol Sci 304:551–565.

Giannakopoulos P, Kovari E, Savioz A, de Bilbao F, Dubois Dauphin M,Hof PR, Bouras C. 1999. Differential distribution of presenilin-1, Bax,and Bcl-X(L) in Alzheimer’s disease and frontotemporal dementia. ActaNeuropathol (Berl) 98:141–149.

Giometto B, Bozza F, Argentiero V, Gallo P, Pagni S, Piccinno MG,Tavolato B. 1990. Transferrin receptors in rat central nervous system. Animmunocytochemical study. J Neurol Sci 98:81–90.

Hallgren B. 1958. The effect of age on the nonhaemin iron in the humanbrain. J Neurochem 3:41–51.

Halliwell B. 1996. Mechanisms involved in the generation of free radicals.Pathol Biol (Paris) 44:6–13.

Halliwell B. 2001. Role of free radicals in the neurodegenerative diseases:therapeutic implications for antioxidant treatment. Drugs Aging 18:685–716.

Harrison PM, Arosio P. 1996. The ferritins: molecular properties, ironstorage function and cellular regulation. Biochim Biophys Acta 1275:161–203.

Harrison PM, Ford GC, Smith JM, White JL. 1991. The location of exonboundaries in the multimeric iron-storage protein ferritin. Biol Met4:95–99.

Hazel JR, Williams EE. 1990. The role of alterations in membrane lipidcomposition in enabling physiological adaptation of organisms to theirphysical environment. Prog Lipid Res 29:167–227.

Hirsch EC, Faucheux BA. 1998. Iron metabolism and Parkinson’s disease.Mov Disord 13 Suppl 1:39–45.

Hulet SW, Heyliger SO, Powers S, Connor JR. 2000. Oligodendrocyteprogenitor cells internalize ferritin via clathrin-dependent receptor medi-ated endocytosis. J Neurosci Res 61:52–60.

Hussain S, Rodgers DA, Duhart HM, Ali SF. 1997. Mercuric chloride-induced reactive oxygen species and its effect on antioxidant enzymes indifferent regions of rat brain. J Environ Sci Health B 32:395–409.

Isler M, Delibas N, Guclu M, Gultekin F, Sutcu R, Bahceci M, Kosar A.2002. Superoxide dismutase and glutathione peroxidase in erythrocytes ofpatients with iron deficiency anemia: effects of different treatment mo-dalities. Croat Med J 43:16–19.

Jefferies WA, Brandon MR, Hunt SV, Williams AF, Gatter KC, MasonDY. 1984. Transferrin receptor on endothelium of brain capillaries.Nature 312:162–163.

Jellinger KA. 1999. The role of iron in neurodegeneration: prospects forpharmacotherapy of Parkinson’s disease. Drugs Aging 14:115–140.

Juan SH, Guo JH, Aust SD. 1997. Loading of iron into recombinant ratliver ferritin heteropolymers by ceruloplasmin. Arch Biochem Biophys341:280–286.

Kissel K, Hamm S, Schulz M, Vecchi A, Garlanda C, Engelhardt B. 1998.Immunohistochemical localization of the murine transferrin receptor(TfR) on blood-tissue barriers using a novel anti-TfR monoclonal anti-body. Histochem Cell Biol 110:63–72.

Kitamura Y, Shimohama S, Kamoshima W, Ota T, Matsuoka Y, NomuraY, Smith MA, Perry G, Whitehouse PJ, Taniguchi T. 1998. Alteration ofproteins regulating apoptosis, Bcl-2, Bcl-x, Bax, Bak, Bad, ICH-1 andCPP32, in Alzheimer’s disease. Brain Res 780:260–269.

Klomp LW, Farhangrazi ZS, Dugan LL, Gitlin JD. 1996. Ceruloplasmingene expression in the murine central nervous system [see comments].J Clin Invest 98:207–215.

Kwak EL, Torti SV, Torti FM. 1990. Murine ferritin heavy chain: isolationand characterization of a functional gene. Gene 94:255–261.

LaVaute T, Smith S, Cooperman S, Iwai K, Land W, Meyron-Holtz E,Drake SK, Miller G, Abu-Asab M, Tsokos M, Switzer Rr, Grinberg A,Love P, Tresser N, Rouault TA. 2001. Targeted deletion of the geneencoding iron regulatory protein-2 causes misregulation of iron metabo-lism and neurodegenerative disease in mice. Nat Genet 27:209–214.

Lawson DM, Treffry A, Artymiuk PJ, Harrison PM, Yewdall SJ, LuzzagoA, Cesareni G, Levi S, Arosio P. 1989. Identification of the ferroxidasecentre in ferritin. FEBS Lett 254:207–210.

Lee PL, Gelbart T, West C, Halloran C, Beutler E. 1998. The humanNramp2 gene: characterization of the gene structure, alternative splicing,promoter region and polymorphisms. Blood Cells Mol Dis 24:199–215.

Leibold EA, Gahring LC, Rogers SW. 2001. Immunolocalization of ironregulatory protein expression in the murine central nervous system.Histochem Cell Biol 115:195–203.

Levine RL. 1983. Oxidative modification of glutamine synthetase. I. In-activation is due to loss of one histidine residue. J Biol Chem 258:11823–11827.

Loeffler DA, Connor JR, Juneau PL, Snyder BS, Kanaley L, DeMaggio AJ,Nguyen H, Brickman CM, LeWitt PA. 1995. Transferrin and iron innormal, Alzheimer’s disease, and Parkinson’s disease brain regions. J Neu-rochem 65:710–724.

Loeffler DA, LeWitt PA, Juneau PL, Sima AA, Nguyen HU, DeMaggioAJ, Brickman CM, Brewer GJ, Dick RD, Troyer MD, Kanaley L. 1996.Increased regional brain concentrations of ceruloplasmin in neurodegen-erative disorders. Brain Res 738:265–274.

MacGibbon GA, Lawlor PA, Sirimanne ES, Walton MR, Connor B,Young D, Williams C, Gluckman P, Faull RL, Hughes P, Dragunow M.1997. Bax expression in mammalian neurons undergoing apoptosis, and inAlzheimer’s disease hippocampus. Brain Res 750:223–234.

Malecki EA, Cable EE, Isom HC, Connor JR. 2002. The lipophilic ironcompound TMH-ferrocene [(3,5,5-trimethylhexanoyl)ferrocene] in-creases iron concentrations, neuronal L-ferritin, and heme oxygenase inbrains of BALB/c mice. Biol Trace Elem Res 86:73–84.

Mash DC, Pablo J, Flynn DD, Efange SM, Weiner WJ. 1990. Character-ization and distribution of transferrin receptors in the rat brain. J Neuro-chem 55:1972–1979.

McKie AT, Marciani P, Rolfs A, Brennan K, Wehr K, Barrow D, Miret S,Bomford A, Peters TJ, Farzaneh F, Hediger MA, Hentze MW, SimpsonRJ. 2000. A novel duodenal iron-regulated transporter, IREG1, impli-cated in the basolateral transfer of iron to the circulation. Mol Cell5:299–309.

Moos T. 1996. Immunohistochemical localization of intraneuronal trans-ferrin receptor immunoreactivity in the adult mouse central nervoussystem. J Comp Neurol 375:675–692.

Mukhopadhyay CK, Mazumder B, Fox PL. 2000. Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by irondeficiency. J Biol Chem 2750:21048–21054.

Munro HN, Kikinis Z, Eisenstein RS. 1993. Iron-dependent regulation offerritin synthesis. In: Berdanier C, Hargrove JL, editors. Nutrition andgene expression. Boca Raton: CRC Press. p 525–545.

Octave JN, Schneider YJ, Trouet A, Crichton RR. 1983. Iron uptake andutilization by mammalian cells. I. cellular uptake of transferrin and iron.Trends Biochem Sci 8:217–221.

Orino K, Tsuji Y, Torti FM, Torti SV. 1999. Adenovirus E1A blocksoxidant-dependent ferritin induction and sensitizes cells to pro-oxidantcytotoxicity. FEBS Lett 461:334–338.

Orino K, Lehman L, Tsuji Y, Ayaki H, Torti SV, Torti FM. 2001. Ferritinand the response to oxidative stress. Biochem J 357:241–247.

Osaki S, Johnson DA. 1969. Mobilization of liver iron by ferroxidase(ceruloplasmin). J Biol Chem 244:5757–5758.

Paradis E, Douillard H, Koutroumanis M, Goodyer C, LeBlanc A. 1996.Amyloid beta peptide of Alzheimer’s disease downregulates Bcl-2 andupregulates bax expression in human neurons. J Neurosci 16:7533–7539.

Pinero DJ, Connor JR. 2000. Iron in the brain: an important contributorin normal and diseased states. Neuroscientist 6:435–453.

Pinero DJ, Li NQ, Connor JR, Beard JL. 2000. Variations in dietary ironalter brain iron metabolism in developing rats. J Nutr 130:254–263.

62 Thompson et al.

Richter A, Schwager C, Ansorge W, Hentze MW, Muckenthal M. 2002.Comparison of fluorescent tag DNA labeling methods used for expressionanalysis by DNA microarrays. Biotechniques 33:620–630.

Rohn TT, Head E, Su JH, Anderson AJ, Bahr BA, Cotman CW, CribbsDH. 2001. Correlation between caspase activation and neurofibrillarytangle formation in Alzheimer’s disease. Am J Pathol 158:189–198.

Samokyszyn VM, Thomas CE, Reif DW, Saito M, Aust SD. 1988. Releaseof iron from ferritin and its role in oxygen radical toxicities. Drug MetabRev 19:283–303.

Sayre LM, Perry G, Smith MA. 1999. Redox metals and neurodegenerativedisease. Curr Opin Chem Biol 3:220–225.

Sherman AR, Moran PE. 1984. Copper metabolism in iron-deficientmaternal and neonatal rats. J Nutr 114:298–306.

Stadelmann C, Deckwerth TL, Srinivasan A, Bancher C, Bruck W, Jell-inger K, Lassmann H. 1999. Activation of caspase-3 in single neurons andautophagic granules of granulovacuolar degeneration in Alzheimer’s dis-ease. Evidence for apoptotic cell death. Am J Pathol 155:1459–1466.

Stadelman C, Mews I, Srinivasan A, Deckwerth TL, Lassmann H, Bruck

W. 2001. Expression of cell death-associated proteins in neuronal apo-ptosis associated with pontosubicular neuron necrosis. Brain Pathology11:273–281.

Su JH, Nichol KE, Sitch T, Sheu P, Chubb C, Miller BL, Tomaselli KJ,Kim RC, Cotman CW. 2000. DNA damage and activated caspase-3expression in neurons and astrocytes: evidence for apoptosis in fronto-temporal dementia. Exp Neurol 163:9–19.

Thompson KJ, Shoham S, Connor JR. 2001. Iron and neurodegenerativedisorders. Brain Res Bull 55:155–164.

Tortosa A, Lopez E, Ferrer I. 1998. Bcl-2 and Bax protein expression inAlzheimer’s disease. Acta Neuropathol (Berl) 95:407–412.

Wood TL, Rogler LE, Czick ME, Schuller AG, Pintar JE. 2000. Selectivealterations in organ sizes in mice with a targeted disruption of theinsulin-like growth factor binding protein-2 gene. Mol Endocrinol 14:1472–1482.

Zhang Y, McLaughlin R, Goodyer C, LeBlanc A. 2002. Selective cyto-toxicity of intracellular amyloid beta peptide1-42 through p53 and Bax incultured primary human neurons. J Cell Biol 156:519–529.

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