Lack of up-regulation of ferritin is associated with sustained iron

regulatory protein-1 binding activity in the substantia nigra

of patients with Parkinson’s disease

Baptiste A. Faucheux,*,� Marie-Elise Martin,� Carole Beaumont,� Stephane Hunot,*

Jean-Jacques Hauw,� Yves Agid* and Etienne C. Hirsch*

INSERM, *U289 and �U360 et Centre de Recherches de Neurologie Neuropathologie de l’Association Claude Bernard, Hopital de la

Salpetriere, Paris, France

�INSERM, U409, Faculte de Medecine X. Bichat, Paris, France

Abstract

Dopaminergic neurones degenerate during Parkinson’s dis-

ease and cell loss is most extensive in the subpopulation of

melanized neurones located in the substantia nigra pars

compacta. Iron accumulation, together with a lack of

up-regulation of the iron-storing protein, ferritin, has been

reported and may contribute to increased oxidative stress in

this region. We investigated the binding activity of iron regu-

latory protein-1 (IRP1) to the iron-responsive element that

precludes ferritin mRNA translation, in the substantia nigra of

a group of parkinsonian patients who presented a statistically

significant reduction in the number of nigral melanized-

neurones and an increased iron content, together with

unchanged H-ferritin and L-ferritin subunit levels as compared

to matched controls. The levels of ferritin mRNAs and the

binding activity of IRP1 to the iron-responsive element of

ferritin mRNA did not differ significantly between the two

groups. Moreover, there was no detectable contribution of the

iron regulatory protein-2 (IRP2) binding activity. No change in

IRP1 control of ferritin mRNA translation explains the lack of

up-regulation of ferritin expression in cytoplasmic extracts of

SNpc that would be normally expected with cytosolic iron

accumulation. The data of this study do not favor changes in

transcription and post-transcriptional regulation of ferritin

expression in Parkinson’s disease and suggest a �compart-

mentalized� iron accumulation.

Keywords: ferritin, iron regulatory proteins, iron, melanized

neurones, mesencephalon, Parkinson’s disease.

J. Neurochem. (2002) 83, 320–330.

Parkinson’s disease is associated with the death of dopam-

inergic neurones in the mesencephalon. The most severe

degeneration affects the subpopulation of melanized neu-

rones located in the substantia nigra pars compacta (SNpc)

where enhanced oxidative stress and damaged lipids,

proteins, and nucleic acids have been demonstrated, together

with iron accumulation (Alam et al. 1997a,b; Good et al.

1998; Hirsch and Faucheux 1998; Jenner and Olanow 1998;

Foley and Riederer 2000). Although no full demonstration

has been made of iron accumulation being a primary event in

the neurodegenerative process (Jenner and Olanow 1998),

the increased iron content in the SNpc, both in patients with

Parkinson’s disease and in experimental models reproducing

the human disease, raises the possibility that this metal

contributes to neuronal death in association with enhanced

oxidative stress. Iron, essential for all organisms, can be toxic

when present in excess, given the ability of its free Fe2+ form

to react with H2O2 (Fenton reaction) and to catalyse the

formation of the highly reactive hydroxyl radicals (Haber-

Weiss type of reaction). Intracellular free iron levels are

controlled by ferritin, the major iron storage protein, the

biosynthesis of which depends on both transcriptional control

Received March 4, 2002; revised manuscript received June 18, 2002;

accepted July 3, 2002.

Address correspondence and reprint requests to Dr Baptiste

A. Faucheux, INSERM U289, Batiment Pharmacie, Hopital de la

Salpetriere, 47 Boulevard de l’Hopital, F-75013 Paris, France.

E-mail: baptiste.faucheux@chups.jussieu.fr

Abbreviations used: A8, catecholaminergic cell group A8; CP, cerebral

peduncle; EMSA, electrophoretic mobility shift assay; Ft, ferritin; IRE,

iron-responsive element; IRP, iron regulatory protein; NS, not signifi-

cant; PAG, periaqueductal grey substance; SNpc, substantia nigra pars

compacta; RPA, ribonuclease protection assay; VTA, ventral tegmental

area.

Journal of Neurochemistry, 2002, 83, 320–330

320 � 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 320–330

and a post-transcriptional regulatory system (Harrison and

Arosio 1996; Hentze and Kuhn 1996). Two cytoplasmic

proteins, iron regulatory proteins 1 and 2 (IRP1 and IRP2),

control the synthesis of ferritin by binding to a stem–loop

structure located in the 5¢-untranslated region of the ferritin

mRNA and known as the �iron-responsive element� (IRE).

When the concentration of cellular iron is low, IRPs bind to

the IRE, which blocks translation, whereas when iron

concentration is high, IRPs do not bind to IRE, which

allows translation of ferritin mRNAs. The translation of

ferritin mRNAs involves the binding of IRP1 to the IRE in

the normal human brain (Hu and Connor 1996).

The levels of iron in the brain increase with age (Hallgren

and Sourander 1958) and there is also an elevation of ferritin

subunit levels in the SNpc of elderly subjects not affected by

any neurologic disorder (Connor et al. 1995). In contrast,

unchanged or low levels of ferritin subunits have been

reported in the SNpc of parkinsonian patients in spite of iron

accumulation (Dexter et al. 1991; Mann et al. 1994). Thus,

alterations in the molecular mechanisms that regulate ferritin

content and limit iron challenge may contribute to the

degeneration of dopaminergic neurones. This led us to

investigate the levels of ferritin mRNA transcripts and the

post-transcriptional regulation of ferritin synthesis in the

mesencephalon of patients with Parkinson’s disease.

First, using the ribonuclease protection assay, we exam-

ined whether mRNA levels of the H-Ft and L-Ft subunits of

ferritin were altered in parkinsonian patients in association

with an increased iron content in the SNpc. In addition, we

studied the expression of ferritin mRNAs by using in situ

hybridization. The mRNA levels in the SNpc were not

changed with Parkinson’s disease. Thus, we investigated the

post-transcriptional control of ferritin synthesis. We meas-

ured the binding activity of IRPs to an IRE–ferritin mRNA

probe, and did not find any significant change in IRP1

binding. We then investigated whether IRP2-binding activity

was present in cytoplasmic extracts of the SNpc from

controls and parkinsonian patients, and observed that

Parkinson’s disease was not associated with any detectable

binding activity. We conclude that ferritin expression is not

up-regulated in the SNpc, because there was no substantial

change at the transcriptional and post-transcriptional levels,

and we discuss possible explanations for this result.

Materials and methods

Human autopsy tissue

Brains were obtained postmortem from 10 control individuals, with

no known history of neurologic or psychiatric disorders, and from

six patients with clinically defined [stages II to IV on the rating scale

of Hoehn and Yahr (1967); levodopa response] and histologically

confirmed (nigral neuronal loss; presence of Lewy bodies in the SN

and locus ceruleus) Parkinson’s disease. Within 2 h after autopsy,

the brains were dissected and blocks of hemi-brainstem were frozen

in dry ice, reduced to powder and stored at )80�C. Serial, 20-lm-

thick sections were cut from the frozen blocks at )12�C using a

cryostat, thaw-mounted onto gelatin chrom-alun coated glass slides,

desiccated and stored at )80�C.

The specimens from parkinsonian patients and matched control

subjects did not differ significantly with respect to mean age at death

(range and mean ± SEM: 62–94 vs. 65–94 years; 74 ± 5 vs.

84 ± 3 years, respectively, in parkinsonian patients and control

subjects, p ¼ 0.11), time elapsed between the death of the patients

and the freezing of brain tissue (range and mean ± SEM: 10–31 vs.

10–27 h; 21 ± 4 vs. 20 ± 2 h, respectively, p ¼ 0.93) or gender

ratio (female/male: 0.50 vs. 0.60, respectively).

We examined ferritin expression and regulation in sections of the

caudal part of the SNpc, where a marked neuronal loss is observed.

For comparison, three other mesencephalic regions characterized by

lower rates of dopaminergic neuronal loss were also studied – the

ventral tegmental area (VTA), the catecholaminergic cell group A8

(A8) and the periaqueductal grey substance (PAG) – as well as the

cerebral peduncle (CP) as a control region with no dopaminergic

neurones. To determine accurately the boundaries of the SNpc and

the other anatomical subregions, adjacent sections were stained for

acetylcholinesterase activity (Geneser-Jensen and Blackstad method,

as modified by Graybiel and Ragsdale 1978). The boundaries of

subregions were delineated according to Hirsch et al. (1988).

To check the comparability of the tissue analysed from parkinso-

nian patients and controls, sections adjacent to those analysed for

iron and ferritin expression were treated with Nissl stain in order to

count the number of cell nuclei present in the studied subregions.

Stained cell nuclei and melanized neurones were counted in 20

circular fields (125 lm in diameter) randomly distributed through-

out each subregion (Faucheux et al. 1999).

To estimate the iron content in the SNpc of the individuals

studied, iron histochemistry was performed on adjacent sections

using Perls’ prussian blue reaction (non-heme iron), as described by

Smith et al. (1997). The surface area and number of stained deposits

were measured using a semiautomatic computer-assisted image

analysis system (Histoscan; Biocom, Les Ulis, France) within

10 fields of 360 lm · 510 lm. Staining and quantification were

carried out in duplicate in independent experiments. The segmen-

tation threshold of optical density signal (gray level) was determined

in order to detect deposits with an area of at least 1 lm2; this

threshold was identical for all the field images quantified in each

series of sections. Brown deposits of neuromelanin and artefacts

were examined by using microscopy and subtracted from the

number of detected objects that were colored blue by Perls’ reaction.

Preparation of tissue samples for assays

Tissue (3–12 mg) from each of the five midbrain subregions was

collected on serial sections (five sections for mRNAs and five for

proteins, in alternate order). Anatomic limits of the SNpc, VTA-Mv,

A8, PAG and CP were drawn in frozen tissue sections using the cold

straight tip of a metal probe, with reference to a section stained for

acetylcholinesterase. The frozen tissue of subregions was scraped

with a cold blade onto the glass slide, which was placed on dry ice.

The collected material was transferred to an Eppendorf tube (also

placed in dry ice) and stored at )80�C. For protein analyses [ELISA

and electrophoretic mobility shift assay (EMSA)], frozen specimens

Iron regulatory protein-1 in Parkinson’s disease 321

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 320–330

were suspended and homogenized in chilled 25 mM Tris–HCl,

pH 7.4, with 400 mM KCl, 5% (vol/vol) glycerol, 0.2% (vol/vol)

Nonidet P-40 (NP-40), 1 mM phenylmethylsulfonyl fluoride

(PMSF) and 5 lg/mL leupeptin. The resulting lysate was then

centrifuged at 3500 g for 15 min at 4�C. The supernatant (soluble

subcellular fraction) was removed, aliquoted and stored at )80�C.

The protein content of cytoplasmic extracts was evaluated in

triplicate using the Bio-Rad (Hercules, CA, USA) protein assay

calibrated with bovine serum albumin as standard, and optical

density was read at k¼ 595 nm (Hitachi, U-2000 spectrophoto-

meter; Hitachi Ltd, Tokyo, Japan). For mRNA quantification using

the ribonuclease protection assay, frozen specimens from adjacent

sections were homogenized in 5 M guanidium thiocyanate with

100 mM EDTA (pH 7.4) for 30 min at room temperature. The

resulting tissue lysates were centrifuged, aliquoted and stored at

)20�C. The DNA concentration of tissue extracts was determined in

duplicate according to the method of Labarca and Paigen (1980),

using bis-benzimide trihydrochloride (Sigma, St Louis, MO, USA)

with calf thymus DNA as standard (Sigma) and emitted fluorescence

was read at kem ¼ 458 nm with kexc ¼ 356 nm (Hitachi F2000

spectrofluorometer).

Determination of H-Ft and L-Ft proteins

The ferritin content of tissue lysates was measured using ELISA

based on monoclonal antibodies specific for the H-Ft or L-Ft

subunits, according to the method reported by Luzzago et al.

(1986), with minor modifications. Polystyrene 96-well ELISA plates

(Greiner Labortechnik, ELISA plate KO 96K; Greiner Bio-One

GmbH, Frickenhausen, Germany) were coated with 100 lL/well of

mouse monoclonal anti-H-Ft or anti-L-Ft subunit-specific antibodies

at 9 lg/mL (respectively, rH02 and LF03, a generous gift from

P. Santambrogio, Dibit, Institute H. San Raffaele, Milan, Italy), in

50 mM carbonate buffer (pH 9.6), by overnight incubation at 4�C.

The plates were then washed with 50 mM carbonate buffer and

100 lL of standards (30–500 ng/mL of recombinant H-Ft subunit or

L-Ft subunit) or samples, diluted in Dulbecco’s phosphate-buffered

saline containing 0.05% (vol/vol) Tween-20 and 2% (wt/vol)

defatted milk, were loaded in triplicate onto coated wells and

incubated at 37�C (2 h for H-Ft; 1.5 h for L-Ft). After washing with

50 mM carbonate buffer, the plates were loaded with a secondary

antibody coupled to horseradish peroxidase (rH02–HRP diluted

1 : 1500 for H-Ft, and LF03–HRP diluted 1 : 500 for L-Ft, a

generous gift from P. Arosio, Milan), and orthophenylene diamine

hydrochloride (Sigma) was added as a substrate to peroxidase with

hydrogen peroxide, before the absorbance was read at k¼ 492 nm

(Labsystems iEMS Reader MF; Thermo Labsystems, Vantaa,

Finland) after a 20-min incubation at room temperature in the dark.

Concentrations of H-Ft and L-Ft are expressed as ng of recombinant

subunit/mg of protein.

Electrophoretic mobility shift assay

Gel electrophoresis of complexes between IRP proteins and an IRE

mRNA probe was performed as described by Martin et al. (1998). A

[32P]-labeled IRE–H-Ft mRNA probe was prepared by in vitro

transcription of 4 lg of Xba-linearized pIL2CAT (IL2-CAT,

composed of 55 nucleotides including the IRE H-Ft sequence,

kindly provided by Dr M. Hentze, European Molecular Biology

Laboratory, Heidelberg, Germany) in the presence of 20 U of Rnase

inhibitor, 1 lL of 10 mM unlabeled ATP, CTP and GTP, 60 lCi of

[32P]-aUTP (400 Ci/mmol, Amersham Pharmacia Biotech, Piscat-

away, NJ, USA), and 60 U of T7 RNA polymerase. After

electrophoresis in a 15% (wt/vol) denaturing polyacrylamide gel

(acrylamide : bisacrylamide ¼ 19 : 1), the transcript (55 nt) was

eluted from the gel slice by incubation overnight at 25�C in 0.5 M

ammonium acetate and 1 mM EDTA, precipitated in ethanol and

resuspended in 50 lL H2O to a specific activity of 104 dpm/ng. The

binding reactions between tissue lysates and the probe were carried

out as described by Leibold and Munro (1988). Brain cytoplasmic

extracts (4 lg) were incubated with [32P]-labeled IRE–HFt mRNA

probe (3 · 104 dpm) in 20 lL of 10 mM HEPES buffer, pH 7.6

[containing 3 mM MgCl2, 1 mM dithiothreitol (DTT) and 5%

(vol/vol) glycerol] and 20 U of Rnase inhibitor, for 15 min at room

temperature, and then treated with 1.5 mg/mL heparin for 10 min at

room temperature. The IRP–IRE complexes were separated in a

non-denaturing 6% (wt/vol) native acrylamide/bisacrylamide gel

(acrylamide : bisacrylamide ¼ 19 : 1) for 2 h at 150 V, visualized

by autoradiography and quantified using an Instant Imager (Packard

Instrument Company, Meriden, CT, USA). To allow the full

expression of IRP–IRE-binding activity in vitro, additional samples

were treated in parallel with addition of b-mercaptoethanol [2%

(vol/vol) final concentration] before incubation with the [32P]-

labeled IRE–HFt mRNA probe. When supershift analyses were

performed, cytoplasmic extracts were incubated (at 0�C for 1 h)

with 3 lL of a rabbit anti-rat IRP2 serum (kindly provided by Dr E.

Leibold, Utah State University, Salt Lake City, UT, USA) in the

presence of Rnase inhibitor, before addition of the labeled probe.

After incubation, the IRP1–IRE and IRP2–IRE complexes were

separated by electrophoresis in a 4% (wt/vol) non-denaturing

acrylamide gel (acrylamide : bisacrylamide ¼ 19 : 1) for 2 h, at

200 V and 4�C. After drying of the gels, autoradiographic images

were obtained by exposure of Kodak BioMax films (Eastman Kodak

Company, Rochester, NY, USA), and the amount of specific [32P]-

labeled IRP–mRNA complexes (band shift or �supershift�) was

determined by cpm counting using an Instant Imager (Packard

Instrument Company).

Rat brain tissue

To check the amplitude of the degradation of IRP1 and IRP2

proteins associated with postmortem delay, we measured the

changes in IRP–IRE-binding activity in cytosolic protein extracts

from animals that were stored for different durations at +4�Cbefore dissection. Male Wistar rats (eight animals, 225–250 g in

weight; Centre d’Elevage R. Janvier, Le Genest St Isle, France)

were killed by lethal anaesthesia of sodium pentobarbital (Sanofi,

Libourne, France) after intraperitoneal injection. Animal care was

in accordance with our University guidelines. At four time-points

(immediately after death, and 24, 48 and 72 h afterwards), the

brains were removed and dissected to separate the two hemi-

spheres, the cerebellum and the brainstem. Tissue blocks were

frozen and stored at )80�C until extraction of cytoplasmic

proteins, which was performed as described above for human

brain tissue.

Riboprobe preparation

The riboprobes for H-Ft mRNA quantification were obtained by

PCR amplification of nts 233–515 of H-Ft cDNA subcloned in

322 B. A. Faucheux et al.

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 320–330

reverse orientation for the antisense probe and of nts 233–453 for

the sense probe. Amplification products were cloned in the plasmid

pGEMT (Promega, Madison, WI, USA). Linearization of the

template at the SalI site and transcription by T7 polymerase

generated an antisense riboprobe of 356 nts, leading to an expected

protected H-Ft mRNA fragment of 282 nts. For the sense probe,

linearization at the SalI site and transcription by T7 polymerase gave

a 286-nt probe leading to an expected fragment of 220 nts. The

antisense riboprobe for L-Ft mRNA was obtained by cloning a PstI

restriction fragment containing nts 52–275 of the human L-Ft cDNA

in pSP65 vector (Promega). After linearization of the template at the

PpuMI site, a full-length probe of 173 nts, leading to a protected

fragment of 123 nts, was transcribed by SP6 polymerase.

The labeled antisense riboprobes were obtained using 1 lg of

linearized plasmids, transcribed by 20 U of RNA polymerase in the

presence of 50 lCi of [32P]-aUTP (400 Ci/mmol; Amersham

Pharmacia Biotech), unlabeled ATP, CTP and GTP each at

7.5 mM final concentration, 7 mM DTT and 20 U of Rnase inhibitor,

in a reaction volume of 15 lL. After 1 h, 15 U of Rnase-free DnaseI

was added and the transcripts purified in a Probe-Quant G50

microcolumn (Amersham) before ethanol precipitation. The pellets

were dissolved in 50 lL of 5 M GuSCN and 0.1 M EDTA, pH 7.0.

The specific activities were 7.5 · 108 dpm/lg of RNA. The

unlabeled H-Ft mRNA sense probe was synthesized using 20 U

of T7 polymerase in the presence of 4 lg of linearized plasmid,

5 lL of each ribonucleotide at 10 mM in 100 lL of reaction volume,

for 2 h at 37�C, and precipitated by ethanol. After centrifugation, the

pellet was solubilized in 50 lL of 5 M GuSCN containing 0.1 M

EDTA, and quantified at k¼ 260 nm. The concentrations were

adjusted to 100 pg/mL and aliquots stored at )80�C.

Quantification of ferritin mRNAs using the ribonuclease

protection assay

Total RNAs contained in 10 lL of tissue lysates at 150 ng of DNA/

lL were incubated overnight at 37�C with labeled H-Ft mRNA and

L-Ft mRNA antisense probes (2 · 105 dpm each) and 25 pg of H-Ft

mRNA sense probe as an internal standard. Single-strand, non-

hybridized RNAs were digested for 45 min at room temperature

with 380 lL of a solution containing 40 lg/mL of Rnase A and

625 U/mL of Rnase T1 in 1 mM EDTA, 300 mM NaCl, 30 mM Tris–

HCl pH 7.4. Rnases were inactivated by incubation for 30 min at

37�C with 60 lL of proteinase K (2 mg/mL) in 3.3% (vol/vol)

sodium dodecyl sulfate. Hybridized mRNAs were extracted in

phenol–chloroform–isoamyl alcohol (15 : 14 : 1), before ethanol

precipitation with 40 lg of yeast tRNA as a carrier. After

centrifugation at 4�C, the pellets were resuspended in 5 lL of

80% (vol/vol) formamide loading buffer. After denaturation by

heating for 2 min at 95�C, the duplexes of protected transcripts were

separated by electrophoresis in a 6% (wt/vol) denaturing acrylamide

gel (acrylamide : bisacrylamide ¼ 19 : 1) with 8 M urea. MspI-

digested, end-[32P]-labeled fragments of pBluescriptIISK+ were

used as molecular-weight markers. A control of the specificity was

obtained using tRNA instead of midbrain mRNA. After migration

for 2 h at 35 watts, the gel was dried and exposed for autoradiog-

raphy using Kodak BioMax film. Radioactive [32P]-labeled bands

were quantified using an Instant Imager (Packard). Concentrations

of H-Ft mRNA and L-Ft mRNA are expressed as pg of RNA/lg of

DNA, after correction for hybridization efficiency by the use of the

internal standard and normalization for DNA concentration in

lysates.

Investigation of ferritin mRNAs expression

by in situ hybridization35S-radiolabeled probes were synthesized by in vitro transcription

from linearized templates in the presence of 50 lCi of [35S]-aUTP

(> 1000 Ci/mmol, 10 lCi/lL; Amersham). In situ hybridization

was performed as described by Vila et al. (1997), and sections were

coated with NTB-2 emulsion (Kodak) diluted 1 : 1, air-dried, and

stored in lightproof boxes for 4 weeks at 4�C. Emulsions were

developed in Kodak D-19 and counterstained with 0.1% (wt/vol)

hematoxylin to localize cell nuclei.

Statistics

All values are expressed as the mean ± SEM. For statistical

comparisons between two groups, the Student’s t-test or Mann–

Whitney U-test were used, according to normality and variance of

data distributions (SIGMASTAT statistical program, version 2.0,

Jandel, SPSS Inc., Chicago, MI, USA). A p-value of less than

0.05 was considered to be statistically significant.

Results

Validation of specimens analysed

The mean cell numbers were similar in both groups of

subjects, except for the number of melanized neurones,

which was decreased in the VTA-Mv ()19%) and SNpc

()68%) of the patients with Parkinson’s disease, thus

confirming that the SNpc of these patients was severely

affected by the neurodegenerative process.

To confirm whether an iron accumulation was present in

the SNpc of the parkinsonian patients, the histochemical

reaction of Perls was performed. A strong staining was

observed macroscopically in the SNpc, while there was no

staining in PAG, A8 or CP. Measurement of the surface area

and number of non-heme iron-stained deposits in the SNpc

showed that the iron content was significantly increased

in the group of parkinsonian patients, as compared to

control subjects (29 000 ± 9513 vs. 2732 ± 1251 lm2 of

deposits/mm2 of tissue section, p < 0.005). The sensitivity of

the method provided quantitative data for every subject,

ranging from a lowest mean value of 593 lm2 of deposits/

mm2 to a highest mean value of 63 063 lm2 of deposits/mm2

of tissue section. Quantification of the number of melanized

neurones and the iron content in the SNpc indicated that a

low cell count (high neuronal loss) was associated with a

high iron accumulation.

Expression of H-Ft and L-Ft subunits in the substantia

nigra pars compacta

H-Ft and L-Ft levels were determined in SNpc tissue

homogenates by using ELISA. H-Ft levels were higher than

L-Ft levels, with an H-Ft/L-Ft ratio slightly higher than 3.0 in

Iron regulatory protein-1 in Parkinson’s disease 323

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 320–330

control subjects (Table 1). The levels of H-Ft and L-Ft and

the H-Ft/L-Ft ratio did not differ significantly between

patients with PD and controls (H-Ft/L-Ft ratio: 2.2 ± 0.1 in

parkinsonian patients, vs. 3.3 ± 0.5 in control subjects,

p ¼ 0.09, NS). There was no significant correlation between

the subunit levels and the postmortem delay (H-Ft: r ¼ 0.01,

NS; L-Ft: r ¼ )0.33, NS).

Expression of H-Ft mRNA and L-Ft mRNA transcripts

in the substantia nigra pars compacta

The concentrations of H-Ft mRNA and L-Ft mRNA were

investigated and quantified by ribonuclease protection assay

(RPA) in SNpc tissue homogenates. In control subjects, the

mean levels of H-Ft mRNA and L-Ft mRNA were within the

same range, with a ratio of H-Ft mRNA to L-Ft mRNA of

less than 1 (0.8 ± 0.1). In patients with Parkinson’s disease,

the mean level of H-Ft mRNA and L-Ft mRNA did not vary

significantly in comparison to controls (H-Ft mRNA: 8 ± 2

vs. 11 ± 2 pg mRNA/lg DNA; L-Ft mRNA: 14 ± 2 vs.

13 ± 2 pg mRNA/lg DNA, in parkinsonian patients vs.

controls, respectively). There was no significant correlation

between the mRNA levels and the postmortem delay (H-Ft

mRNA: r ¼ )0.31, NS; L-Ft mRNA: r ¼ )0.04, NS).

To investigate at the cellular level whether melanized

neurones of the SNpc presented changes in H-Ft mRNA and

L-Ft mRNA transcript expression, some sections were

treated for in situ hybridization histochemistry. The emulsion

autoradiography showed that H-Ft mRNA labeling was not

increased over the cytoplasm of nigral-melanized neurones in

parkinsonian patients with a high iron content, as compared

to control subjects with a low iron content, and there was no

non-specific labeling over neuromelanin (Fig. 1). Among

melanized neurones of both control subjects and parkinso-

nian patients, the density of silver grains for L-Ft mRNA

labeling was similar to that of the environment surrounding

the neurones, indicating no detectable expression of L-Ft-

subunit messenger RNA in melanized neurones. An increase

in the level of H-Ft mRNA and L-Ft mRNA labeling was

observed over a subpopulation of small-cell nuclei in the

SNpc of parkinsonian patients (Fig. 1), possibly correspond-

ing to glial cells (astrocytes or microglia).

RNA band shift analysis of iron regulatory protein

activity in the substantia nigra pars compacta

To determine whether the absence of a significant increase in

the H-Ft and L-Ft levels observed in the SNpc of patients with

Parkinson’s disease could be attributed to an abnormal control

of Ft mRNA translation by IRPs, a series of RNA band-shift

analyses was performed. There was a considerable overlap in

individual values for binding activity in the groups of control

subjects and parkinsonian patients (Fig. 2), and the mean

level of binding activity of IRPs to the IRE–Ft mRNA probe

was not decreased in parkinsonian patients (52 ± 7 vs. 49 ± 4

dpm/lg of protein, respectively, in parkinsonian patients and

controls). There was no significant correlation between the

IRP–IRE-binding activity and the postmortem delay (r ¼)0.33, NS). Because a single band was observed after

migration in gel electrophoresis of IRP–IRE Ft mRNA

complexes (Fig. 2), additional experiments were performed

to investigate the possible contributions of both IRP1 and

IRP2 in IRP–IRE complexes in SNpc extracts from control

subjects and parkinsonian patients. The specificity of the

method was verified using animal brain tissues or cell lines

that express both IRP1 and IRP2. Separate IRP1–IRE Ft

mRNA and IRP2–IRE Ft mRNA complexes were detected in

homogenates of rat cerebellum (data not shown) and brain-

Table 1 Concentration of ferritin subunits in various regions of the human mesencephalon

H-Ft subunit

(ng/mg of proteins)

L-Ft subunit

(ng/mg of proteins)

Region C (n ¼ 10) PD (n ¼ 6) C (n ¼ 10) PD (n ¼ 6)

SNpc 11.5 ± 1.6a 9.6 ± 0.7 3.7 ± 0.3a 4.5 ± 0.4

VTA-Mv 4.6 ± 0.3 5.9 ± 0.8 2.1 ± 0.4b,c 3.1 ± 0.6

A8 5.0 ± 0.5 5.2 ± 0.5 0.6 ± 0.1 0.6 ± 0.03

PAG 3.5 ± 0.4 3.9 ± 0.6 0.6 ± 0.1 0.7 ± 0.1

CP 4.0 ± 0.3 4.6 ± 0.4 1.3 ± 0.2 1.6 ± 0.2

Data are expressed as mean ± SEM values of protein levels measured using ELISA in tissue homogenates from control subjects (C) and

parkinsonian patients (PD). No statistically significant differences were observed between controls and parkinsonian patients. Highly significant

differences were observed between anatomical subregions (H-Ft: F460 ¼ 27.53, p < 0.001; L-Ft: F4

60 ¼ 63.75, p < 0.001) by two-way analysis of

variance; comparison between means by Scheffe’s protected multiple t-test showed that levels of H-Ft and L-Ft in SNpc were significantly higher

than in the four other subregions, and levels of L-Ft in VTA-Mv were significantly higher than in A8, PAG and CP. ap < 0.001, for differences

between SNpc and other subregions. bp < 0.001, for differences between VTA-Mv and A8 or PAG. cp < 0.01, for differences between VTA-Mv and

CP. A8, catecholaminergic cell group A8; CP, pedonculus cerebri, or cerebral peduncle; PAG, griseum centrale mesencephali, or periaqueductal

grey substance; SNpc, substantia nigra pars compacta, or catecholaminergic cell group A9; VTA-Mv, medioventral part of the ventral tegmental

area, or catecholaminergic cell group A10.

324 B. A. Faucheux et al.

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 320–330

Fig. 1 Emulsion autoradiography after in situ hybridization of H-Ft

mRNA (a–c, a¢–c¢) and L-Ft mRNA (d–f, d¢–f¢) with 35S-labeled

riboprobes in substantia nigra pars compacta (SNpc) sections. Silver

grains indicate probe binding. In a representative control subject they

show a weak but clearly visible expression of H-Ft mRNA (a and a¢)and an expression of L-Ft mRNA at the level of the background (d and

d¢) in melanized neurones; in addition, a low level expression of L-Ft

mRNA was detected in some cells surrounding melanized neurones (d

and d¢). The labeling of H-Ft mRNA (b and b¢) and L-Ft mRNA

(e and e¢) was not increased in melanized neurones of parkinsonian

patients with a high nigral iron expression, whereas expression was

increased (arrows) in a subpopulation of scattered small cells that

were probably glial (note the small size of their nuclei) and often in the

proximity of neuromelanin aggregates, both for H-Ft mRNA (b and b¢;c and c¢) and L-Ft mRNA (e and e¢; f and f¢). (a)–(f) Bright field

transmitted light; (a¢)–(f¢) polarized-light epi-illumination with dark field.

Counterstain: haematoxylin. Scale bar, 25 lm.

Iron regulatory protein-1 in Parkinson’s disease 325

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 320–330

stem, and mouse RR4 microglial cells (Fig. 2). The specificity

of the binding was demonstrated by competition with a 100-

fold molar excess of unlabeled probe, which abolished the

formation of both complexes (Fig. 2). To separate the binding

activity of IRP2 from that of IRP1, incubations were

performed in the presence of specific antibodies directed

against IRP2 to slow its migration during electrophoresis. As

expected, the serum was able to supershift the IRP2–IRE Ft

mRNA complex of rat brain stem homogenates and mouse

RR4 microglial cells without altering the IRP1–IRE Ft mRNA

complex (Fig. 2). However, it did not reveal any IRP2–IRE Ft

mRNA complex in human SNpc homogenates, either from

controls or parkinsonian patients, although a supershifted

complex was generated with extracts from human intestinal

CaCo2 cells (data not shown). As no supershifted band could

be detected in the human SNpc homogenates, in contrast to

human intestinal cells, these results suggest a very low level of

expression of IRP2 in the SNpc of control subjects and

patients with Parkinson’s disease.

To control the validity of our assay, we incubated

the cytoplasmic extracts in the presence of 2% (vol/vol)

b-mercaptoethanol, a reductant that promotes the IRP1

conformation and recruits all IRP1/aconitase molecules

present in tissue homogenates. As expected, this treatment

increased the IRP1–IRE Ft mRNA-binding activity in

extracts from rat cerebellum and brainstem, mouse RR4

microglial cells, and in the human SNpc (Fig. 2).

As the absence of IRP2 detection could be caused by a

constitutive low expression and/or protein alteration, IRP1-

and IRP2-binding activities were studied in the rat brainstem

for postmortem delays of 0, 24, 48 and 72 h to estimate the

amplitude of IRP2 loss of binding activity during the time

elapsed between the death of the patients and the freezing of

brain tissue. As expected, the IRP-binding activity was

decreased as a function of postmortem delay (Fig. 3), but

IRP–mRNA complexes remained detectable, even after 24,

48 and 72 h (IRP1: )7%, )15%, )20%; IRP2: )23%,

)33%, )40%). The level of binding activity for all IRP1

Fig. 2 Iron regulatory protein (IRP)-binding activity in cytosolic protein

extracts from the substantia nigra pars compacta (SNpc) of control

subjects (C) and parkinsonian patients (P). Electrophoretic mobility

shift assays (EMSA) were performed using a [32P]-labeled iron-

responsive element (IRE)–Ft mRNA probe of 55 nucleotides, and

IRP–IRE complexes were resolved by electrophoresis in non-dena-

turing acrylamide : bisacrylamide gels either at 6% (wt/vol) (panels a,

b and c) or 4% (wt/vol) (panels d and e). Panel (a), cytoplasmic

extracts from the SNpc of control subjects (lanes C1 to C10) and

parkinsonian patients (lanes P1 to P6) were assayed for IRP activity

(4 lg of proteins and 30 000 dpm per lane) with binding reactions in

control conditions; radioactivity in the bands was determined using an

Instant Imager (Packard) and mean levels of duplicate experiments

are presented in the text. Panel (b), EMSA was performed on rat

brainstem extracts (20 lg of proteins and 70 000 dpm per lane) with

binding reaction either in control conditions (lane 1), or in the presence

of specific antibodies directed against the IRP2 protein (lane 2), or a

100-fold molar excess of the unlabeled 55-oligonucleotide probe (lane

3), or 2% (vol/vol) b-mercaptoethanol, a reductant that allows the full

expression of IRP1–IRE-binding activity (lane 4). Panel (c), EMSA was

performed on human SNpc extracts pooled for controls (C) and pooled

for patients with Parkinson’s disease (P) (10 lg of proteins and 70 000

dpm per lane) with binding reactions either under control conditions or

in the presence of 2% (vol/vol) b-mercaptoethanol. Panels (d) and (e),

EMSA was performed on mouse RR4-immortalized microglial

cells and human SNpc extracts pooled for controls (C) and pooled for

patients with Parkinson’s disease (P) (20 lg of proteins and 70 000

dpm per lane) with binding reactions either under control conditions

(lanes 1, C, P), or in the presence of specific antibodies directed

against the IRP2 protein (lanes 2, C, P) or 2% (vol/vol) b-mercapto-

ethanol (lane 3). The arrows indicate well separated IRP1–IRE Ft

mRNA, IRP2–IRE Ft mRNA and supershifted IRP2*–IRE Ft mRNA

complexes in the rat brainstem and mouse RR4 microglial cells, and

an IRP1–IRE Ft mRNA complex with possible – but not detectable –

IRP2 contribution in the human SNpc.

326 B. A. Faucheux et al.

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 320–330

molecules that could be recruited in the extracts in the

presence of 2% (vol/vol) b-mercaptoethanol was decreased

by 13% after 24 h and 26% after 72 h. These results show

that our method allowed the detection of IRP2–IRE Ft

mRNA-binding activity in rat brainstem, even for tissues

stored for 72 h at +4�C, a much longer storage time than the

postmortem delay of the studied human brains.

Expression of H-Ft and L-Ft in the subregions

of the mesencephalon

For control comparison, the expression of H-Ft and L-Ft

subunits was also assessed in those regions of the mesen-

cephalon where the vulnerability of dopaminergic neurone

subpopulations is lower than in the SNpc and where

iron accumulation is low or undetectable: the mean concen-

trations of the two subunits were highest in the SNpc

(Table 1).

Discussion

In this study of the human brain, no up-regulation was

observed of ferritin subunits in the SNpc of parkinsonian

patients in parallel with increased iron content. To understand

this paradoxical result, the ribonuclease protection assay and

in situ hybridization were used to investigate ferritin mRNA

expression levels, and RNA gel-shift assays were used to

assess IRP–IRE-Ft mRNA-binding activity. The levels of Ft

mRNAs and the control of Ft mRNA translation by the IRP–

IRE system were not modified in parkinsonian patients as

compared to control subjects.

Expression of ferritin subunits

Many studies of parkinsonian patients have shown iron

accumulation in the SNpc and within nigral melanized

neurones (Earle 1968; Dexter et al. 1989; Good et al. 1992;

Riederer et al. 1992; Gorell et al. 1995), even though some

other studies found no change (Loeffler et al. 1995). Nigral

iron content is also increased in animals that develop

parkinsonian syndromes after intoxication by 1-methyl-4-

phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 6-hydroxydop-

amine (Mochizuki et al. 1994; He et al. 1996; Foley and

Riederer 2000). The iron accumulation accompanying neuro-

nal loss in parkinsonian patients was not associated with a

parallel increase of Ft-subunit concentrations, in contrast to the

data of controls in which there were higher levels of Ft subunits

in the SNpc than in the other mesencephalic subregions, in

parallel with iron content. This finding is consistent with the

results of studies performed both in parkinsonian patients and

in MPTP-intoxicated monkeys (Dexter et al. 1991; Mann

et al. 1994; Connor et al. 1995; Goto et al. 1996).

Expression of ferritin mRNA transcripts

This study found no change in H-Ft mRNA and L-Ft mRNA

concentrations in patients with Parkinson’s disease, as shown

by quantification of SNpc tissue homogenates and melanized

neurones after in situ hybridization histochemistry. The very

low H-Ft mRNA and undetectable L-Ft mRNA signals

observed after in situ hybridization for melanized neurones

of control subjects are consistent with the data of immuno-

histochemical detection of the corresponding proteins

(Connor and Menzies 1995). Our data are also in agreement

with experiments performed in the rat model of parkinsonian

syndrome induced by 6-hydroxydopamine intoxication,

where the H-Ft mRNA level of expression has also been

reported to remain low in dopaminergic neurones of the

SNpc (Foster et al. 1991). Moreover, we observed, in the

SNpc of parkinsonian patients, a few small cells, sometimes

Fig. 3 Detection of iron-responsive protein-1–iron-responsive ele-

ment (IRP1–IRE) ferritin (Ft) mRNA and IRP2–IRE Ft mRNA-binding

activity in rat brainstem cytoplasmic extracts, as a function of post-

mortem delay. Cytosolic protein extracts were isolated from

homogenates of control brainstems (C) and from brainstems obtained

after 24 h (24), 48 h (48) and 72 h (72) of storage at +4�C. Panel (a),

electrophoretic mobility shift assays (EMSA) were performed using a

[32P]-labeled IRE Ft mRNA probe and complexes were electropho-

retically separated in a 4% (wt/vol) acrylamide : bisacrylamide gel

(20 lg of protein per lane; 150 V) under control conditions (–) or in the

presence (+) of 2% (vol/vol) b-mercaptoethanol. Panel (b), graphic

representation of the relative changes in the levels of the binding

activity (in the absence of b-mercaptoethanol treatment) after 24 h,

48 h and 72 h of storage at +4�C. Values are expressed as

the mean ± SEM of three experiments performed with samples in

duplicate (two animals) at each time-point.

Iron regulatory protein-1 in Parkinson’s disease 327

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 320–330

located in the vicinity of melanized neurones, which showed

an increased expression of the two transcripts. This probably

reflects an increased iron storage in a subset of glial cells, and

agrees with the reported high level of L-Ft expression

detected by immunohistochemical staining and observed in

some reactive microglial cells often adjacent to remaining

nigral melanized neurones of patients with Parkinson’s

disease (Jellinger et al. 1990).

Control of ferritin mRNA translation, assessed by the

binding activity of iron regulatory protein-1

Ferritin synthesis depends on both a transcriptional regula-

tion (Ponka et al. 1998) and a post-transcriptional control

mechanism through IRP1 and IRP2 proteins that bind to 5¢-untranslated regions and prevent recruitment of the 40S small

ribosomal subunit to the Ft mRNAs (Muckenthaler et al.

1998). We observed no change in the binding activity of

IRPs to an IRE–Ft mRNA probe, which indicates no

substantial change in post-transcriptional regulation in the

SNpc of parkinsonian patients.

The IRP1–IRE and IRP2–IRE complexes were not

separated in RNA band-shift assays of human brainstem,

and IRP2 was not detectable in supershift experiments. These

results suggest a very low expression of IRP2 in the

brainstem of 65- to 94-year-old human subjects and no

detectable expression associated with Parkinson’s disease, in

contrast to the findings in Alzheimer’s disease (Smith et al.

1998). Our results are in agreement with those of Pinero and

Connor (2000), who reported no supershift of IRP2–IRE

complexes for cytoplasmic extracts from the temporal cortex

after addition of IRP2 antiserum. A postmortem complete

degradation of IRP2 in the studied brains was not supported

by the control experiments we performed with rat brainstem

extracts: 77% of the binding activity remained detectable

after 24 h of body storage at 4�C. We conclude that the level

of IRP2 in the active conformation is low in the human SNpc

of controls and not increased to detectable levels in

parkinsonian patients. This result is also compatible with

the rapid degradation of IRP2 reported in iron-replete cells

(see Butt et al. 1996).

The IRP1–IRE-binding activity was increased (1.1- to 2.0-

fold) in control subjects and parkinsonian patients when

protein extracts were treated with 2% (vol/vol) b-mercapto-

ethanol. As this reductant recruits all IRP1 molecules

becoming competent for IRE binding (Drapier and Bouton

1996), these data suggest that a small fraction of IRP1/

aconitase molecules were in their aconitase form without

affinity for mRNA and could be recruited in the human

SNpc. Therefore, our study indicates that IRP1 is predom-

inant in the human mesencephalon, as in most human cell

lines and tissues examined to date (Henderson et al. 1993;

Guo et al. 1995; Hu and Connor 1996), with no substantial

change in the IRP1–IRE regulatory system in the SNpc of

parkinsonian patients.

Several factors may explain the absence

of ferritin up-regulation

Taken together, our combined morphological and molecular

investigation shows that both transcriptional and post-

transcriptional regulatory mechanisms of ferritin expression

were not substantially modified despite an iron accumulation

in the SNpc of patients with Parkinson’s disease. According

to the hypothesis that iron accumulation is associated with an

increase of the intracellular free-iron pool, iron homeostasis

would be expected to induce an up-regulation of buffering

systems. A lack of ferritin up-regulation may reflect either

some pathological effects interacting with ferritin synthesis

or some �normal� mechanisms that need further investigation.

Some astroglial and microglial cells localized in the

vicinity of vulnerable dopaminergic neurones of the SNpc

produce nitric oxide and proinflammatory cytokines in

parkinsonian patients and in experimental models of the

disease (Hunot et al. 1996, 1999; Good et al. 1998; Hirsch

et al. 1998; Liberatore et al. 1999). As nitric oxide increases

the binding activity of IRP1 to IRE Ft mRNAs and blocks Ft

mRNA translation (Mulero and Brock 1999), the production

of nitric oxide could interfere with the binding of IRP1 to

IRE Ft mRNAs and may contribute to a deficiency in ferritin

subunit synthesis in the SNpc of patients with Parkinson’s

disease.

However, the absence of ferritin up-regulation may also

reflect a non-significant increase in the cytosolic iron pool.

Indeed, methods with a subcellular resolution, such as laser

microprobe mass analysis and dispersive X-ray microanal-

ysis, have conduced to the measurement of iron accumula-

tion within melanized neurones in neuromelanin granules

(Good et al. 1992; Jellinger et al. 1992) and in Lewy bodies

(Hirsch et al. 1991). Neuromelanin may be involved in

intraneuronal iron homeostasis as a result of its strong

chelating ability for iron (Zecca et al. 2001) and therefore

may limit increases in the labile free-iron pool in patients

with Parkinson’s disease. Some iron sequestration may also

be attributable to a-synuclein, one of the main components of

Lewy bodies (Jenner and Olanow 1998; Ostrerova-Golts

et al. 2000). The relative importance of this �compartmen-

talized� iron accumulation remains to be established, but

these data support the speculation that intracellular location

of iron in Parkinson’s disease may be different to that of

control subjects. In that case, iron accumulation in Parkin-

son’s disease would not induce a change in the IRP1 control

of ferritin mRNA translation. The IRP1–IRE-binding activity

that regulates the levels of ferritin subunits in the cytoplasm

may therefore appear to be essentially normal in the SNpc of

patients with Parkinson’s disease.

A misregulation of the labile iron pool may participate in

the degenerative process of melanized neurones. Further

investigation is needed to determine possible defects in iron

regulation at the cellular and molecular levels under patho-

logical conditions in the human brain. The inhibition of

328 B. A. Faucheux et al.

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 320–330

complex I of the mitochondrial respiratory chain has been

proposed as a primary cause of nigral cell death (Jenner and

Olanow 1998), but it has been demonstrated that the 75-kDa

subunit of mitochondrial complex I is regulated by a novel

IRP, thus supporting the argument that iron misregulation

may be the basis of idiopathic Parkinson’s disease (Lin et al.

2001). Our results stress the complexity of iron homeostasis

in the SNpc, which probably involves intricately related

mechanisms. The control process of ferritin synthesis at the

transcriptional level and at the post-transcriptional level by

the binding activity of IRP1, both in melanized neurones and

glial cells in pathological conditions, remains unresolved.

Identification of other mechanisms modulated by levels of

iron or altering iron homeostasis and cell survival will

contribute to the understanding of the pathological process

leading to neurodegeneration in Parkinson’s disease.

Acknowledgements

We are grateful to Drs H Beck, JY Beinis, AM Bonnet, JP Bouchon,

C Duyckaerts, J Hogenhuis, M Laurent, R Moulias, F Piette, A

Sachet, D Seilhean, O Saint-Jean, V Sazdovitch and M Verny for

their clinical investigations and co-operation in providing the brain

specimens, P Arosio and P Santambrogio (Milan) and E Leibold

(Salt Lake City) for their generous gifts of antibodies, and F Darios,

S Haık, C Hanus, N Lambeng, P Michel, A Mouatt-Prigent, MP

Muriel, G Orieux, K Parain, C Perier and JD Troadec for their

technical advice and contribution. This work was supported by the

INSERM, the Association Claude Bernard pour le developpement

des recherches biologiques et medicales dans les hopitaux de

l’Assistance Publique a Paris (to BAF and JJH), the CNRS, and the

National Parkinson Foundation (Miami, Florida).

References

Alam Z. I., Daniel S. E., Lees A. J., Marsden D. C., Jenner P. and

Halliwell B. (1997a) A generalized increase in protein carbonyls

in the brain in Parkinson’s but not incidental Lewy body disease.

J. Neurochem. 69, 1326–1329.

Alam Z. I., Jenner A., Daniel S. E., Lees A. J., Cairns N., Marsden C. D.,

Jenner P. and Halliwell B. (1997b) Oxidative DNA damage in the

parkinsonian brain: an apparent selective increase in 8-hydroxy-

guanine levels in substantia nigra. J. Neurochem. 69, 1196–1203.

Butt J., Kim H. Y., Basilion J. P., Cohen S., Iwai K., Philpott C. C.,

Altschul S., Klausner R. D. and Rouault T. A. (1996) Differences

in the RNA binding sites of iron regulatory proteins and potential

target diversity. Proc. Natl Acad. Sci. USA 93, 4345–4349.

Connor J. R. and Menzies S. L. (1995) Cellular management of iron in

the brain. J. Neurol. Sci. 134, 33–44.

Connor J. R., Snyder B. S., Arosio P., Loeffler D. A. and LeWitt P.

(1995) A quantitative analysis of isoferritins in select regions of

aged, parkinsonian, and Alzheimer’s diseased brains. J. Neuro-

chem. 65, 717–724.

Dexter D. T., Wells F. R., Lees A. J., Agid F., Agid Y., Jenner P. and

Marsden C. D. (1989) Increased nigral iron content and alterations

in other metal ions occurring in brain in Parkinson’s disease.

J. Neurochem. 52, 1830–1836.

Dexter D. T., Carayon A., Javoy-Agid F., Agid Y., Wells F. R., Daniel

S. E., Lees A. J., Jenner P. and Marsden D. (1991) Alterations in

the levels of iron, ferritin and other trace metals in Parkinson’s

disease and other neurodegenerative diseases affecting the basal

ganglia. Brain 114, 1953–1975.

Drapier J. C. and Bouton C. (1996) Modulation by nitric oxide of

metalloprotein regulatory activities. Bioessays 18, 549–556.

Earle K. M. (1968) Studies on Parkinson’s disease including x-ray

fluorescent spectroscopy of formalin fixed brain tissue. J. Neuro-

pathol. Exp. Neurol. 27, 1–14.

Faucheux B. A., Bonnet A. M., Agid Y. and Hirsch E. C. (1999) Blood

vessels change in the mesencephalon of patients with Parkinson’s

disease. Lancet 353, 981–982.

Foley P. and Riederer P. (2000) Influence of neurotoxins and oxidative

stress on the onset and progression of Parkinson’s disease.

J. Neurol. 247, II82–II94.

Foster O. J. F., Kingsbury A., Dexter D. T., Blunt S., Lightman S. L.,

Marsden C. D. and Jenner P. (1991) Ferritin mRNA expression in

rat brain: effects of 6-hydroxydopamine lesions and levodopa

administration. Soc. Neurosci. Abstracts 17, 1288.

Good P. F., Olanow C. W. and Perl D. P. (1992) Neuromelanin-

containing neurons of the substantia nigra accumulate iron and

aluminium in Parkinson’s disease: a LAMMA study. Brain Res.

593, 343–346.

Good P. F., Hsu A., Werner P., Perl D. P. and Olanow C. W. (1998)

Protein nitration in Parkinson’s disease. J. Neuropathol. Exp.

Neurol. 57, 338–342.

Gorell J. M., Ordidge R. J., Brown G. G., Deniau J. C., Buderer N. M.

and Helpern J. A. (1995) Increased iron-related MRI contrast in

the substantia nigra in Parkinson’s disease. Neurology 45, 1138–

1143.

Goto K., Mochizuki H., Imai H., Akiyama H. and Mizuno Y. (1996) An

immuno-histochemical study of ferritin in 1-methyl-4-phenyl-

1,2,3,6-tetrahydropyridine (MPTP)-induced hemiparkinsonian

monkeys. Brain Res. 724, 125–128.

Graybiel A. M. and Ragsdale C. W. (1978) Histochemically distinct

compartments in the striatum of human, monkey and cat demon-

strated by acetylcholinesterase stain. Proc. Natl Acad. Sci. USA

75, 1723–1726.

Guo B., Brown F. M., Phillips J. D., Yu Y. and Leibold E. A. (1995)

Characterization and expression of iron regulatory protein 2

(IRP2). Presence of multiple IRP2 transcripts regulated by intra-

cellular iron levels. J. Biol. Chem. 270, 16529–16535.

Hallgren B. and Sourander P. (1958) The effect of age on the non-haemin

iron in the human brain. J. Neurochem. 3, 41–51.

Harrison P. M. and Arosio P. (1996) The ferritins: molecular properties,

iron storage function and cellular regulation. Biochim. Biophys.

Acta 1275, 161–203.

He Y., Thong P. S. P., Lee T., Leong S. K., Shi C. Y., Wong P. T. H.,

Yuan S. Y. and Watt F. (1996) Increased iron in the substantia nigra

of 6-OHDA induced parkinsonian rats: a nuclear microscopy study.

Brain Res. 735, 149–153.

Henderson B. R., Seiser C. and Kuhn L. C. (1993) Characterization of a

second RNA-binding protein in rodents with specificity for iron-

responsive elements. J. Biol. Chem. 268, 27327–27334.

Hentze M. W. and Kuhn L. C. (1996) Molecular control of vertebrate

iron metabolism: mRNA-based regulatory circuits operated by

iron, nitric oxide, and oxidative stress. Proc. Natl Acad. Sci. USA

93, 8175–8182.

Hirsch E. C. and Faucheux B. A. (1998) Iron metabolism and Parkin-

son’s disease. Mov. Disord. 13, 39–45.

Hirsch E., Graybiel A. M. and Agid Y. A. (1988) Melanized dopamin-

ergic neurons are differentially susceptible to degeneration in

Parkinson’s disease. Nature 334, 345–348.

Hirsch E. C., Brandel J. P., Galle P., Javoy-Agid F. and Agid Y. (1991)

Iron and aluminum increase in the substantia nigra of patients with

Iron regulatory protein-1 in Parkinson’s disease 329

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 320–330

Parkinson’s disease: an X-ray microanalysis. J. Neurochem. 56,

446–451.

Hirsch E. C., Hunot S., Damier P. and Faucheux B. (1998) Glial cells

and inflammation in Parkinson’s disease: a role in neurodegener-

ation? Ann. Neurol. 44, S115–S120.

Hoehn M. M. and Yahr M. D. (1967) Parkinsonism: onset, progression

and mortality. Neurology 17, 427–442.

Hu J. and Connor J. R. (1996) Demonstration and characterization of the

iron regulatory protein in human brain. J. Neurochem. 67, 838–

844.

Hunot S., Boissiere F., Faucheux B., Brugg B., Mouatt-Prigent A., Agid

Y. and Hirsch E. C. (1996) Nitric oxide synthase and neuronal

vulnerability in Parkinson’s disease. Neuroscience 72, 355–363.

Hunot S., Dugas N., Faucheux B., Hartmann A., Tardieu M., Debre P.,

Agid Y., Dugas B. and Hirsch E. C. (1999) FceRII/CD23 is

expressed in Parkinson’s disease and induces, in vitro, produc-

tion of nitric oxide and tumor necrosis factor-a in glial cells.

J. Neurosci. 19, 3340–3347.

Jellinger K., Paulus W., Grundke-Iqbal I., Riederer P. and Youdim M. B.

H. (1990) Brain iron and ferritin in Parkinson’s and Alzheimer’s

diseases. J. Neural Transm. (Park. Dis. Dementia Section) 2, 327–

340.

Jellinger K., Kienzl E., Rumpelmair G., Riederer P., Stachelberger H.,

Ben-Shachar D. and Youdim M. B. H. (1992) Iron–melanin

complex in substantia nigra of parkinsonian brains: an X-ray

microanalysis. J. Neurochem. 59, 1168–1171.

Jenner P. and Olanow C. W. (1998) Understanding cell death in Par-

kinson’s disease. Ann. Neurol. 44, S72–S84.

Labarca C. and Paigen K. (1980) A simple, rapid, and sensitive DNA

assay procedure. Anal. Biochem. 102, 344–352.

Leibold E. A. and Munro H. N. (1988) Cytoplasmic protein binds in vitro

to a highly conserved sequence in the 5¢ untranslated region of

ferritin heavy- and light-subunit mRNAs. Proc. Natl Acad. Sci.

USA 85, 2171–2175.

Liberatore G. T., Jackson-Lewis V., Vukosavic S., Mandir A. S., Vila M.,

McAuliffe W. G., Dawson V. L., Dawson T. M. and Przedborski S.

(1999) Inducible nitric oxide synthase stimulates dopaminergic

neurodegeneration in the MPTP model of Parkinson disease. Nat.

Med. 5, 1403–1409.

Lin E., Graziano J. H. and Freyers G. A. (2001) Regulation of the

75-kDa subunit of mitochondrial Complex I by iron. J. Biol. Chem.

276, 27685–27692.

Loeffler D. A., Connor J. R., Juneau P. L., Snyder B. S., Kanaley L.,

DeMaggio A. J., Nguyen H., Brickman C. M. and LeWitt P. A.

(1995) Transferrin and iron in normal, Alzheimer’s disease, and

Parkinson’s disease brain regions. J. Neurochem. 65, 710–716.

Luzzago A., Arosio P., Iacobello C., Ruggeri G., Capucci L., Brocchi E.,

De Simone F., Gamba D., Gabri E., Levi S. and Albertinin A.

(1986) Immunochemical characterization of human liver and heart

ferritins with monoclonal antibodies. Biochim. Biophys. Acta 872,

61–71.

Mann V. M., Cooper J. M., Daniel S. E., Srai K., Jenner P., Marsden

C. D. and Schapira A. H. V. (1994) Complex I, iron, and ferritin in

Parkinson’s disease substantia nigra. Ann. Neurol. 36, 876–881.

Martin M. E., Fargion S., Brissot P., Pellat B. and Beaumont C. (1998) A

point mutation in the bulge of the iron-responsive element of the L

ferritin gene in two families with the hereditary hyperferritinemia–

cataract syndrome. Blood 91, 319–323.

Mochizuki H., Imai H., Endo K., Yokomizo K., Murata Y., Hattori N.

and Mizuno Y. (1994) Iron accumulation in the substantia nigra of

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced

hemiparkinsonian monkeys. Neurosci. Lett. 168, 251–253.

Muckenthaler M., Gray N. K. and Hentze M. W. (1998) IRP-1 binding to

ferritin mRNA prevents the recruitment of the small ribosomal

subunit by the cap-binding complex eIF4F. Mol. Cell 2, 383–388.

Mulero V. and Brock J. H. (1999) Regulation of iron metabolism

in murine J774 macrophages: role of nitric oxide-dependent and

-independent pathways following activation with gamma interferon

and lipopolysaccharide. Blood 94, 2383–2389.

Ostrerova-Golts N., Petrucelli L., Hardy J., Lee J. M., Farer M. and

Wolozin B. (2000) The A53T a-synuclein mutation increases iron-

dependent aggregation and toxicity. J. Neurosci. 20, 6048–6054.

Pinero D. J. and Connor J. R. (2000) Alterations in the interaction

between iron regulatory proteins and their iron responsive element

in normal and Alzeimer’s diseased brains. Cell. Mol. Biol. 46, 761–

776.

Ponka P., Beaumont C. and Richardson D. R. (1998) Function and

regulation of transferrin and ferritin. Semin. Hematol. 35, 35–54.

Riederer P., Dirr A., Goetz M., Sofic E., Jellinger K. and Youdim M. B.

H. (1992) Distribution of iron in different brain regions and sub-

cellular compartments in Parkinson’s disease. Ann. Neurol. 32,

S101–S104.

Smith M. A., Harris P. L. R., Sayre L. M. and Perry G. (1997) Iron

accumulation in Alzheimer disease is a source of redox-generated

free radicals. Proc. Natl Acad. Sci. USA 94, 9866–9868.

Smith M. A., Wehr K., Harris P. L. R., Siedlak S. L., Connor J. R. and

Perry G. (1998) Abnormal localization of iron regulatory protein in

Alzheimer’s disease. Brain Res. 788, 232–236.

Vila M., Levy R., Herrero M. T., Ruberg M., Faucheux B., Obeso J. A.,

Agid Y. and Hirsch E. C. (1997) Consequences of nigrostriatal

denervation on the functioning of the basal ganglia in human and

nonhuman primates: an in situ hybridization study of cytochrome

oxidase subunit I mRNA. J. Neurosci. 17, 765–773.

Zecca L., Gallorini M., Schunemann V., Trautwein A. X., Gerlach M.,

Riederer P., Vezzoni P. and Tampellini D. (2001) Iron, neuromel-

anin and ferritin content in the substantia nigra of normal subjects

at different ages: consequences for iron storage and neurodegen-

erative processes. J. Neurochem. 76, 1766–1773.

330 B. A. Faucheux et al.

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 320–330