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Research Report

Chronic ferritin expression within murine dopaminergicmidbrain neurons results in a progressiveage-related neurodegeneration

Deepinder Kaura, Subramanian Rajagopalana, Shankar Chintaa, Jyothi Kumara,Donato Di Monteb, Robert A. Chernyc, Julie K. Andersena,⁎aBuck Institute for Research in Aging, 8001 Redwood Blvd., Novato, CA 94945, USAbParkinson's Institute, Sunnyvale, CA 94089, USAcDepartment of Pathology, University of Melbourne, and the Mental Health Research Institute of Victoria, Parkville 3052, Australia

A R T I C L E I N F O

⁎ Corresponding author.E-mail address: jandersen@buckinstitute.

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.03.006

A B S T R A C T

Article history:Accepted 7 March 2006Available online 2 May 2006

Ferritin elevation has been reported by some laboratories to occur within the substantianigra (SN), the area of the brain affected in Parkinson's disease (PD), but whether such anincrease could be causatively involved in neurodegeneration associated with the disorder isunknown. Here, we report that chronic ferritin elevation in midbrain dopamine-containingneurons results in a progressive age-related neurodegeneration of these cells. This providesstrong evidence that chronic ferritin overload could be directly involved in age-relatedneurodegeneration such as occurs in Parkinson's and other related diseases.

© 2006 Elsevier B.V. All rights reserved.

Keywords:Iron chelationFerritinAgingDopamineNeurodegenerationParkinson's disease

1. Introduction

Increased ferritin levels have been reported by somelaboratories to occur in the substantia nigra (SN) ofParkinsonian patients, the midbrain region preferentiallyimpacted by the disorder (Bartzokis et al., 2004; Griffiths etal., 1999). Targeted disruption of the gene encoding ironregulatory protein 2 (IRP2) in mice is also associated with,among several other changes, an increase in ferritin levelsalong with a substantial accumulation of iron in the centralnervous system proceeding onset of a progressive age-related ataxic movement disorder (LaVaute et al., 2001).Neuroferritinopathy, a recently recognized human genetic

org (J.K. Andersen).

er B.V. All rights reserved

disease resulting in an age-dependent progressive move-ment disorder, also appears to involve abnormal ferritinaccumulation in the brain (Crompton et al., 2002; Curtis etal., 2001). While ferritin increase is correlated with neurode-generation in both human disease and the IRP2 knockoutmouse model, many other alterations also occur whichcould conceivably be responsible for ensuing age-relatedneurodegeneration. Previously, we created transgenic mouselines in which ferritin levels were selectively elevated withindopaminergic SN neurons resulting in an increase in ferritin-bound iron within these cells (Kaur et al., 2003). In younganimals, this was found to afford protection againstneurodegeneration associated with two widely used animal

.

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models of the disease, systemic 1-methyl-4-phenyl 2,3,6-tetrahydropyridine (MPTP) (Kaur et al., 2003) and paraquatadministration (McCormack et al., 2005). Here, we examinedthe effects of chronic ferritin elevation in these neurons toexplore whether its persistent elevation could be a directcause of age-related neurodegeneration.

2. Results

As previously reported by our laboratory, ferritin transgenicsdisplay an elevation in dopaminergic SN iron levels by an earlyage (Kaur et al., 2003) which is found to bemaintained into lateadulthood. This represents an increase in both total iron levelsas assessed by inductively coupled plasmamass spectroscopy(ICP-MS, Fig. 1A) and ferritin-bound iron as measured bymagnetic resonance imaging (MRI, Fig. 1B). Although noneuropathology was previously observed in the younger (2–4 months) animals (Kaur et al., 2003), by 8–12 months of age,the ferritin transgenics began to display a progressiveneurodegeneration initially characterized by axonal degener-ation within the striatum as measured by fluorojade staining(Fig. 2A) accompanied by ameasurable decline in spontaneouslocomotor activity. Fluorojade, an anionic fluorescein deriva-tive, specifically binds to degenerating neuronal elementswithin neurons including distal dendrites, axons, and axonterminals, making them fluoresce brightly (Schmued andHopkins, 2000). Open field analyses demonstrated both asignificant decrease in movement episodes (Fig. 3A) andincreased rest time (Fig. 3B). This is consistent with retrogradedegeneration of the dopaminergic SN neurons as demonstrat-ed by increased silver staining in this brain region (Fig. 2B). Atlater ages (16–24 months), significant declines in both striataldopamine content (Fig. 4A) and tyrosine-hydroxylase-positiveSN cell numbers (Fig. 5) were observed, hallmarks of dopami-nergic SN cell loss as occurs in both other animalmodels of thedisease and in PD itself. In addition, in contrast to what was

Fig. 1 – Increased iron levels in the SN of older (12 months) ferriICP-mass spectrometry as previously described (Kaur et al., 2003Ferritin-bound iron as measured by magnetic resonance imaginassess hypo-intensity of T2-weighted SN samples in the coronaldensitometric units normalized to cortical white matter (Scanalyanimals per measurement. Abbreviations, Wt = wildtype litterm

previously observed in younger animals (Kaur et al., 2003),chronic elevations in dopaminergic ferritin levels rendered theolder animals more rather than less susceptible to theneurotoxic effects of acute MPTP administration (Fig. 4B).Our data demonstrate that not only does chronic ironchelation via prolonged dopaminergic ferritin expressionresult in age-related neurodegeneration but actually appearsto worsen acutely induced Parkinson degeneration in olderanimals.

3. Discussion

We report here that prolonged elevation of ferritin levelswithin dopaminergic midbrain neurons results in theirprogressive age-related neurodegeneration. This is initiallymanifested as a striatal axonopathy accompanied by loss inlocomotor function and is followed by a significant loss instriatal dopamine content and subsequent SN dopaminergiccell loss. This transgenic line was originally created in ourlaboratory in order to assess the impact of alterations inintradopaminergic nigral iron content on susceptibility toMPTP. We found that iron chelation was efficacious inpreventing selective dopaminergic midbrain neurodegenera-tion associated with the neurotoxin in young (3–4 months old)animals (Kaur et al., 2003). Our intent in this current studywasto assess the impact of chronic iron chelation during aging asthis has been suggested as a possible therapeutic for thedisease. We found that continued chronic up-regulation offerritin in the affected cells rather than being protectiveactually resulted in a progressive age-related neurodegenera-tion. We presume that, in young brains, when iron levels arelower, increased ferritin levels are sufficient to keep iron in anon-reactive form due to a relatively high ferritin to iron ratio.As the brain ages and accumulates iron, ferritin could becomeoverloaded (and at some point saturated) with iron. At thetime of ferritin turnover, the amount of iron releasedwould be

tin transgenics. (A) Total SN iron content as measured by). Data are presented as μg iron/g wet SN tissue weight. (B)g (MRI) as previously described using a 7.0 T MRI system toplane (Kaur et al., 2003). Data are presented in representativetics, Inc). *P < 0.05 compared to wildtype littermates, n = 5–6ates; Tg = ferritin transgenics.

Fig. 2 – Degenerating neurites in ST and neurons in SN of ferritin Tg vs.WT. (A) Representative striatal (ST) axonal degenerationas observed in transgenics expressing ferritin in a dopamine cell-specific fashion by 8–12 months of age via fluorojadestaining of neurites; no comparable degeneration is noted in the wildtype controls. Data are presented at both 40× and 100×magnification for better visualization. (B) Representative silver staining of degenerating SN neurons of 16months wildtype andferritin transgenic mice. Data presented at 10× magnification show more silver impregnation in the dopaminergic neuronsof transgenic mice.

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presumed to be much higher in older compared to youngermice. This could be compounded by an age-related increase inoxidative stress via elevations in such enzymes as mono-amine oxidase B (MAO-B) which can react with this momen-tarily unprotected iron, making oxidative damage more likely.Evidence exists suggesting that within dopaminergic neuronsferric iron stored in the ferritin core can be easily reduced bycytotoxic by-products of dopamine oxidation such as super-oxide and 6-hydroxydopamine (6-OHDA), allowing its releasefrom ferritin as ferrous iron (Double et al., 1998; Kienzl et al.,1995; Linert et al., 1996; Monteiro and Winterbourn, 1989;Thomas and Aust, 1986). In this case, increased storage of iron

in ferritin could create a pool of iron which could be easilymobilized via conversion to its reduced form and therebymade available to participate in deleterious oxidative events.Conversely, iron maintained primarily bound to ferritin couldresult in an iron deficit which could impact on cellularfunction by reducing iron for the synthesis of importantiron–sulfur containing enzymes particularly those of themitochondria.

Alterations in ferritin in the Parkinsonian substantia nigra(SN) have been reported by some laboratories to be elevated(Bartzokis et al., 2004; Dexter et al., 1991; Faucheux et al., 2002),although this is controversial (Chen et al., 1993; Connor et al.,

Fig. 3 – (A, B) Decreased spontaneous locomotor activity isdetectable in ferritin transgenics as early as 8 months ofage. (A) Decrease in total movement episodes and (B) anincrease in rest time were measured via open field analysesusing an automated Truscan photobeam apparatus(Colbourne Instruments, Allentown, PA) under illuminationas previously described (Peng et al., 2002).

Fig. 4 – Reduction in ST dopamine (DA) content in older(16months) ferritin transgenics as previously described (Kauret al., 2003) in the absence and presence of 2 × 20 mg/kgbody weight MPTP vs. saline. Values represent ng of DA permg wet weight tissue as measured via reverse phase HPLC,*P < 0.01 vs. Wt, n = 5–6 animals per condition. (A) STdopamine content in untreated animals. (B) Percentagedecrease in ST dopamine content following MPTPadministration.

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1995; Faucheux et al., 2002; Mann et al., 1994; Riederer et al.,1989). It is not clear whether this is due to increases in neuronsversus, for example, oligodendrocytes which are the site ofiron-mediated myelin synthesis and are where most ironincreases occur during normal aging. Due to the controversyregarding the status of ferritin in the human disease stateitself, it is impossible to definitely verify that our modelexactly emulates what occurs in Parkinson's disease. Howev-er, what we observe is in line with the hypothesis that the age-related neurodegeneration such as that observed in RoaualtIRP2−/− mouse line is likely due to increased ferritin levelsrather than the multiple other alterations elicited by thisgenetic manipulation in vivo. IRP1+/−,IRP2−/− mice generatedhave been reported to have more severe neurodegenerationthan in the IRP−/− lines which was accompanied by evenhigher levels of ferritin accumulation (Smith et al., 2004). Itshould be noted however that IRP2−/− mice developed byanother laboratory which displayed altered body iron distri-bution showed no apparent neurodegeneration (Galy et al.,2005). Neuroferritinopathy or hereditary ferritinopathy, a

dominantly inherited movement disorder, is also character-ized by deposition of iron and ferritin in the brain. The disease,also named dominant adult-onset basal ganglia disease, isassociated with mutations in the ferritin L-chain. The twoseparate L ferritin variants studied revealed that, whenmutated L ferritins co-assemble with normal H and L ferritins,they form abnormal ferritin shells. These shells are unstableand appear to undergo either expediated degradation orabnormal aggregation. In either case, they significantly affectcellular iron homeostasis resulting in an increase in intracel-lular iron availability and oxidative damage (Levi et al., 2005;Mancuso et al., 2005; Vidal et al., 2004).

Our data suggest that prolonged ferritin elevation indopaminergic SN neurons could contribute to selective age-related neurodegeneration of this brain region. The fact thatchronic ferritin elevation in addition acts to exacerbate MPTPneurotoxicity in older animals also suggests that the use ofiron chelation as a therapy for the disease should be carefully

Fig. 5 – Stereological cell counts of SN TH+ cell numbersdemonstrate a significant loss of DA SN neurons in 24-monthfer Tgs (30 ± 4.3%) vs. age-matched Wt littermate controls;this loss is progressive (vs. 22 ± 2.3% by 16 months, data notshown). Cells were counted from a total of 15–20 sections ineach field per brain at a magnification of 100× using theoptical fractionator approach as previously described (Kaur etal., 2003). *P < 0.05 vs. Wt, n = 5–6 animals per condition.

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considered in elderly patients with the disorder especiallyover prolonged periods of time.

4. Experimental procedures

4.1. Animals

Construction and characterization of the ferritin transgenicsand wildtype littermate controls used in this study werepreviously described (Kaur et al., 2003). Transgenics wereconstructed in the hybrid B6D2 background strain. Thetyrosine hydroxylase (TH) promoter was used to drivehuman H ferritin expression in these mice. Both male andfemale mice were used in this study as no sex-dependentphenotypic differences were noted. Animals were bred in-house and housed according to standard animal care proto-cols, fed Harlan Teklad 7912 irradiated chow ad libitum, kepton a 12-h light/dark cycle, and maintained in a pathogen-freeenvironment in the Buck Institute Vivarium. All animalexperiments were approved by local IACUC review andconducted according to current NIH policies on the use ofanimals in research. Cohorts of transgenic and wildtypelittermates were aged 2, 12, 16 and 24 months for further sub-sequent analyses. Animals received 2 injections of 20 mg/kgMPTP or saline vehicle intraperitoneally (ip), 12 h apart.

4.2. Measurement of SN iron levels by inductively coupledplasma mass spectrometry (ICP-MS)

Measurements of SN iron levels were assessed by ICP-MS aspreviously described by our laboratory (Kaur et al., 2003).Briefly, tissue was dissected, frozen, lyophilized, weighed,taken up in concentrated nitric acid and allowed to digestovernight. Samples were then heated to 70 °C for 15 min,cooled and diluted 1:40 into 1%HNO3 before being subjected to

ICP-MS using a Varian Ultramass 700 instrument in peak-hopping mode with 0.100 AMU spacing, 1 point per peak, 50scans per replicate, 3 replicates per sample.

4.3. Measurement of SN iron levels by magnetic resonanceimaging (MRI)

MRI studies were performed using a Biospec7T, a 7.0 T MRIsystem from Bruker as previously described by our laboratory(Kaur et al., 2003). Brains were fixed in 4% paraformaldehydeand MRI performed on 2-mm-thick slices in the coronal plane.Comparisons of SNhypo-intensity onT2-weightedMRI imagesencompassing the SN were performed by defining regions ofinterest using ChemiImager (Alpha Innotech). Intensity wasnormalized to cortical white matter (Kaur et al., 2003).

4.4. Fluorojade staining of ST neurite degeneration

Fluorojade staining for detection of degenerating striatalneurites was performed according to the method of Schmuedet al. (1997). Following cardiac perfusion with phosphate-buffered saline (PBS) then 4% paraformaldehyde, brains wereremoved, dehydrated in 30% sucrose and sectioned at 40 μm.Sections were then immersed in 100% ethyl alcohol for 3 min,70% alcohol for 1 min, and distilled H2O for 1 min before beingtransferred for 15 min to a solution of 0.06% potassiumpermanganate. Slides were next rinsed with distilled waterthen transferred to a 0.001% fluorojade solution (Histo-ChemInc.) for 30 min. Sections were rinsed 3 × 1 min in distilled H2Oair-dried, immersed in xylene and coverslipped for viewing onan epifluorescence microscope using fluorescein compatiblefilters.

4.5. Silver staining

Silver staining was used for detection of degenerating STneurites. FD NeuroSilver Kit (FD Neuro Technologies) wasemployed to do the staining on SN sections pre-immunos-tained with TH antibody as described in Section 4.8.

4.6. Open field analysis of locomotor behavior

Motor function was assessed using an automated Truscanphotobeamapparatus (Colbourne Instruments, Allentown, PA)under illumination as previously described (Peng et al., 2002).Animals were habituated to the apparatus for 10 min, thendata collected over a 10-min period followed by analysis usingTruscan 99 software. Spontaneous horizontal movement wasassessed in both young (2 months) and older (8 months) Wtand Tg mice via both the number of movement episodes in a10-min period (left panel) and rest time, i.e. the number ofperiods of inactivity over 2 s in length in a 10-min period.

4.7. Measurement of striatal dopamine content in theabsence and presence of MPTP administration

Dopamine from dissected striata was analyzed by HPLC usinga 5 μm C-18 reverse phase column and precolumn (BrownleeLabs) followed by electrochemical detection with a glassycarbon electrode (Kaur et al., 2003).

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4.8. Stereological SN TH+ cell counts

Sections from the SN were processed as described above.Immunocytochemistry was performed using antibody againsttyrosine hydroxylase (TH, Chemicon, 1:500) followed by biotin-labeled secondary antibody and development using DAB(Vector Laboratories) to immunostain dopaminergic neuronsthroughout the SN pars compacta. TH+ cells were countedstereologically using the optical fractionator method aspreviously described by our laboratory (Kaur et al., 2003).Sections were cut at a 40 μm thickness, and every 4th sectionwas counted using a grid of 100 × 100 μm. Dissector size usedwas 35 × 35 × 12 μm. TH staining was verified with Nissl.

4.9. Statistical analyses

All data are expressed as mean ± SD for the number (n) ofindependent experiments performed. Statistical analyses ofthe data were performed using analysis of variance and posthoc Students' t testing. Values of P < 0.05 were taken as beingstatistically significant.

Acknowledgments

This work was funded by the National Institutes of Healthgrant NS41264 (JKA), NHMRC (Australia) and the Percy BaxterTrust (RAC) (RC).

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