Deferiprone targets aconitase: implication for Friedreich's ataxia treatment.
ABSTRACT Friedreich ataxia is a neurological disease originating from an iron-sulfur cluster enzyme deficiency due to impaired iron handling in the mitochondrion, aconitase being particularly affected. As a mean to counteract disease progression, it has been suggested to chelate free mitochondrial iron. Recent years have witnessed a renewed interest in this strategy because of availability of deferiprone, a chelator preferentially targeting mitochondrial iron.
Control and Friedreich's ataxia patient cultured skin fibroblasts, frataxin-depleted neuroblastoma-derived cells (SK-N-AS) were studied for their response to iron chelation, with a particular attention paid to iron-sensitive aconitase activity.
We found that a direct consequence of chelating mitochondrial free iron in various cell systems is a concentration and time dependent loss of aconitase activity. Impairing aconitase activity was shown to precede decreased cell proliferation.
We conclude that, if chelating excessive mitochondrial iron may be beneficial at some stage of the disease, great attention should be paid to not fully deplete mitochondrial iron store in order to avoid undesirable consequences.
- SourceAvailable from: Dominique Chretien[show abstract] [hide abstract]
ABSTRACT: Friedreich ataxia (FRDA) is a common autosomal recessive degenerative disease (1/50,000 live births) characterized by a progressive-gait and limb ataxia with lack of tendon reflexes in the legs, dysarthria and pyramidal weakness of the inferior limbs. Hypertrophic cardiomyopathy is observed in most FRDA patients. The gene associated with the disease has been mapped to chromosome 9q13 (ref. 3) and encodes a 210-amino-acid protein, frataxin. FRDA is caused primarily by a GAA repeat expansion within the first intron of the frataxin gene, which accounts for 98% of mutant alleles. The function of the protein is unknown, but an increased iron content has been reported in hearts of FRDA patients and in mitochondria of yeast strains carrying a deleted frataxin gene counterpart (YFH1), suggesting that frataxin plays a major role in regulating mitochondrial iron transport. Here, we report a deficient activity of the iron-sulphur (Fe-S) cluster-containing subunits of mitochondrial respiratory complexes I, II and III in the endomyocardial biopsy of two unrelated FRDA patients. Aconitase, an iron-sulphur protein involved in iron homeostasis, was found to be deficient as well. Moreover, disruption of the YFH1 gene resulted in multiple Fe-S-dependent enzyme deficiencies in yeast. The deficiency of Fe-S-dependent enzyme activities in both FRDA patients and yeast should be related to mitochondrial iron accumulation, especially as Fe-S proteins are remarkably sensitive to free radicals. Mutated frataxin triggers aconitase and mitochondrial Fe-S respiratory enzyme deficiency in FRDA, which should therefore be regarded as a mitochondrial disorder.Nature Genetics 11/1997; 17(2):215-7. · 35.21 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: As an essential nutrient and a potential toxin, iron poses an exquisite regulatory problem in biology and medicine. At the cellular level, the basic molecular framework for the regulation of iron uptake, storage, and utilization has been defined. Two cytoplasmic RNA-binding proteins, iron-regulatory protein-1 (IRP-1) and IRP-2, respond to changes in cellular iron availability and coordinate the expression of mRNAs that harbor IRP-binding sites, iron-responsive elements (IREs). Nitric oxide (NO) and oxidative stress in the form of H2O2 also signal to IRPs and thereby influence cellular iron metabolism. The recent discovery of two IRE-regulated mRNAs encoding enzymes of the mitochondrial citric acid cycle may represent the beginnings of elucidating regulatory coupling between iron and energy metabolism. In addition to providing insights into the regulation of iron metabolism and its connections with other cellular pathways, the IRE/IRP system has emerged as a prime example for the understanding of translational regulation and mRNA stability control. Finally, IRP-1 has highlighted an unexpected role for iron sulfur clusters as post-translational regulatory switches.Proceedings of the National Academy of Sciences 09/1996; 93(16):8175-82. · 9.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The use of doxorubicin (Dox) and its derivatives as chemotherapeutic drugs to treat patients with cancer causes dilated cardiomyopathy and congestive heart failure due to Dox-induced cardiotoxicity. In this work, using heat shock factor-1 wild-type (HSF-1(+/+)) and HSF-1 knockout (HSF-1(-/-)) mouse fibroblasts and embryonic rat heart-derived cardiac H9c2 cells, we show that the magnitude of protection from Dox-induced toxicity directly correlates with the level of the heat shock protein 27 (HSP27). Western blot analysis of normal and heat-shocked cells showed the maximum expression of HSP27 in heat-shocked cardiac H9c2 cells and no HSP27 in HSF-1(-/-) cells (normal or heat-shocked). Correspondingly, the cell viability, measured [with (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay] after treatment with various concentrations of Dox, was the highest in heat-shocked H9c2 cells and the lowest in HSF-1(-/-) cells. Depleting HSP27 in cardiac H9c2 cells by small interfering (si)RNA also reduced the viability against Dox, confirming that HSP27 does protect cardiac cells against the Dox-induced toxicity. The cells that have lower HSP27 levels such as HSF-1(-/-), were found to be more susceptible for aconitase inactivation. Based on these results we propose a novel mechanism that HSP27 plays an important role in protecting aconitase from Dox-generated O(2)*(-), by increasing SOD activity. Such a protection of aconitase by HSP27 eliminates the catalytic recycling of aconitase released Fe(II) and its deleterious effects in cardiac cells.AJP Heart and Circulatory Physiology 12/2007; 293(5):H3111-21. · 3.63 Impact Factor
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Deferiprone targets aconitase: Implication for Friedreich's ataxia
Sergio Goncalves, Vincent Paupe, Emmanuel P Dassa and Pierre Rustin*
Address: Inserm, U676, Hôpital Robert Debré, Paris, F-75019 France and Université Paris 7, Faculté de Médecine Denis Diderot, IFR02, Paris,
Email: Sergio Goncalves - firstname.lastname@example.org; Vincent Paupe - email@example.com; Emmanuel P Dassa - firstname.lastname@example.org;
Pierre Rustin* - email@example.com
* Corresponding author
Background: Friedreich ataxia is a neurological disease originating from an iron-sulfur cluster
enzyme deficiency due to impaired iron handling in the mitochondrion, aconitase being particularly
affected. As a mean to counteract disease progression, it has been suggested to chelate free
mitochondrial iron. Recent years have witnessed a renewed interest in this strategy because of
availability of deferiprone, a chelator preferentially targeting mitochondrial iron.
Method: Control and Friedreich's ataxia patient cultured skin fibroblasts, frataxin-depleted
neuroblastoma-derived cells (SK-N-AS) were studied for their response to iron chelation, with a
particular attention paid to iron-sensitive aconitase activity.
Results: We found that a direct consequence of chelating mitochondrial free iron in various cell
systems is a concentration and time dependent loss of aconitase activity. Impairing aconitase activity
was shown to precede decreased cell proliferation.
Conclusion: We conclude that, if chelating excessive mitochondrial iron may be beneficial at some
stage of the disease, great attention should be paid to not fully deplete mitochondrial iron store in
order to avoid undesirable consequences.
Friedreich ataxia (FRDA) is a severe neurological disease
with progressive cerebellar ataxia associating cardiomyop-
athy. It originates from a triplet expansion in the first
intron of the gene coding for frataxin and the resulting
impaired transcription causes depletion of this mitochon-
drial protein . The actual consensus states that frataxin
function, through the handling of mitochondrial iron, is
intimately related with the synthesis of iron-sulfur clusters
(ISC) subsequently distributed to the various cell com-
partments . A number of ISC containing enzymes play
a crucial role in cell metabolism. In keeping with this, aco-
nitase was found severely affected in Friedreich ataxia and
its residual activity might be a crucial issue to determine
the course of the disease . Indeed, while the activity of
the mitochondrial enzyme is determinant for the meta-
bolic flux through the tricarboxylic acid in the mitochon-
dria , the cytosolic counterpart of the aconitase is
known to tightly regulate the overall iron metabolism of
mammal cells . Accordingly, loss of aconitase activity
has been previously shown to trigger cell death of cardiac
fibroblasts . Beside the impaired ISC assembly, an
increased susceptibility to oxidative insult  and a late
mitochondrial iron accumulation  have been reported
Published: 16 June 2008
BMC Neurology 2008, 8:20 doi:10.1186/1471-2377-8-20
Received: 5 February 2008
Accepted: 16 June 2008
This article is available from: http://www.biomedcentral.com/1471-2377/8/20
© 2008 Goncalves et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
BMC Neurology 2008, 8:20 http://www.biomedcentral.com/1471-2377/8/20
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as a result of frataxin depletion. Therefore, both antioxi-
dant- and mitochondrial iron chelator-based therapies
have been initially considered . However, while anti-
oxidants were readily trialled with some promising results
, iron-chelator therapy could not be initially assessed
because desferrioxamine, the only chelator in widespread
clinical use at that time, did not target mitochondrial iron
. Recently the interest in using an iron chelation
approach was renewed due to the availability of
deferiprone, a chelator specifically targeting mitochon-
drial iron [12,13]. In this context, we studied the in vitro
effect of deferiprone in control and FRDA patient cultured
skin fibroblasts and in a shRNA frataxin-depleted neurob-
lastoma-derived cell line (SK-N-AS cells).
Fibroblasts derived from forearm biopsies taken with
informed consent from healthy controls and two FRDA
patients (10–20% residual frataxin mRNA in their fibrob-
lasts) were grown under standard conditions in Dul-
becco's modified Eagle's medium (DMEM; Gibco
Invitrogen, Cergy Pontoise, France) supplemented with
10% foetal calf serum, 10 mg/ml penicillin/streptomycin
and 2 mM L-Glutamine (as Glutamax™; Gibco Invitro-
gen). Final iron content in culture medium amounted to
2–3 μM. The medium (4 ml/25 cm2 flask; 3 ml/10 cm2
well) was changed each three days. Fibroblasts were
seeded at 18 × 103 cells/cm2. SK-N-AS cells, seeded at 150
× 103 cells/cm2, were grown in the same culture medium
added with 100 μM non essential amino acid mixture
(Gibco Invitrogen) and 200 μM uridine (Sigma-Aldrich,
St Quentin, Falavier, France). Fibroblasts were treated
with 25, 75 or 150 μM deferiprone for 7 days. SK-N-AS
cells were treated for a variable duration (0, 1, 7 days)
with 150 μM deferiprone final. The deferiprone effect on
these cells was in addition tested for 7 days using 10, 50,
150 μM of the drug. Cell counting was done after trypsi-
nation using the Quick Read Precision Cell (Globe Scien-
tific Inc., NJ, USA).
For the sake of comparison between cell types, SK-N-AS
cells derived from a neuroblastoma were also used after
frataxin silencing using shRNA. In brief, frataxin-depleted
SK-N-AS were obtained by transducing lentiviral particles
carrying a gene encoding for frataxin-directed shRNA and
a puromycin resistance cassette (MISSION™ TRC shRNA;
Sigma-Aldrich, St Louis, Missouri USA). Frataxin-targeting
sequence of the
GCTAAAGAGTCCAGCTTTTT. SK-N-AS cells were seeded
in 24-well plates. After 24 h, 2 μg/ml hexadimethrine bro-
mide was added just before infection with lentiviral parti-
cles. After 18 h, cells were washed 3 times with PBS.
Deferiprone targets aconitase enzyme
Deferiprone targets aconitase enzyme. Aconitase inhibition triggered by deferiprone in (A) cultured skin fibroblasts from
control (open square) and two Friedreich's ataxia patients after 7 d of culture. Time-dependent aconitase inhibition by 150 μM
deferiprone in (B) control (open square) and frataxin-depleted (shRNA treated) SK-N-AS cells. Error bars correspond to 1 SE
(n = 3); *** denotes p < 0.001; n.s. non significant. Experimental conditions as described under Methods.
0 1 7 0 1 7
(nmol/min/mg prot) 9
0 150 0 150 0 150
25 25 25
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Successfully infected cells were selected with 10 μg/ml
puromycin (15 d).
Aconitase (EC 22.214.171.124) measurement was spectrophoto-
metrically carried out by following aconitate production
from citrate at 240 nm  on the supernatant (800 g × 5
min) of detergent-treated cells (0.2% lauryl maltoside).
All chemicals were of the purest grade available from
Sigma-Aldrich (St Quentin; Falavier, France). Protein con-
centration was measured according to Bradford method.
Data are shown as mean ± 1 SE. Measurements were ana-
lyzed by t test. All statistical tests were calculated using Sig-
maStat software (Sigma, St Louis, USA); p < 0.001 was
considered to indicate statistical significance and denoted
by *** for each value; n.s. non significant.
Deferiprone gradually abolishes aconitase activity in
human cultured cells
The activity of cytosolic and mitochondrial aconitase, two
ISC containing proteins, is known to be highly dependent
on iron availability . We thus first investigated in
human cultured skin fibroblasts the capacity of the
enzymes to resist the iron depletion resulting from a
deferiprone treatment (Fig. 1A). Mitochondrial aconitase
activity represents up to 80% of the total aconitase activity
measurable in these cells. We observed that 150 μM
deferiprone treatments resulted in a significant and pro-
gressive loss of aconitase activity (up to 60% loss after 7
days) in control and patient's fibroblasts. An even more
pronounced loss of aconitase activity was induced by 150
μM deferiprone in frataxin-depleted and non-depleted
SK-N-AS cells (Fig. 1B). Deferiprone-induced loss of aco-
nitase activity was both dose- and time-dependent (Fig.
1). Noticeably, 150 μM deferiprone had no effect on the
activity of aconitase when added during enzyme assay
(not shown), indicative that the loss of aconitase activity
observed in cells should be ascribed to the chelation of
available iron rather than to a direct effect of the chelator
on the ISC of the enzyme.
Deferiprone inhibits growth of human cultured cells
We next investigated the consequences of the loss of aco-
nitase activity induced by 150 μM deferiprone (7 d) on
cell proliferation (Fig. 2). Not surprisingly in view of the
critical role of theses enzymes in cell metabolism , we
observed that the severe loss of aconitase activity was con-
comitant with a major impairment of cell growth of con-
trol and patient's fibroblasts (Fig. 2A, B) or of control and
frataxin-depleted SK-N-AS cells (Fig. 2C).
The results reported in this study confirm that deferiprone
is a potent chelator of mitochondrial matrix iron . As
such, it also potently impairs aconitase activity, presuma-
bly through reduced synthesis of the iron-sulfur cluster
machinery which makes use of available iron in the mito-
chondrial matrix with the help of frataxin . Conflict-
ing results have been reported concerning mitochondrial
iron content of FRDA patient's fibroblasts which may or
not accumulate iron . We show here that the abnor-
Deferiprone decreases cell proliferation
Deferiprone decreases cell proliferation. Effect of 7 d-treatment with deferiprone (0–150 μM) on (A) control (open sym-
bol) and patient fibroblasts and on (C) control (open symbol) and frataxin-depleted SK-N-AS cells. A light microscope view (B)
(×4) of control (a, b, c) and patient (d, e, f) fibroblasts, before treatment (18 h after seeding; a, d), or 7 d in the absence (b, e)
or presence (c, f) of 150 μM deferiprone. Error bars correspond to 1 SE (n = 3); *** denotes p < 0.001; n.s. non significant.
Experimental conditions as described under Methods.
Cell number (/105)
0 50 100 1500 50 100 150
Cell number (/106)
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mal status of mitochondrial iron in Friedreich ataxia pos-
sibly at the origin of the hypersensitivity of these cells to
oxidative insults – associated or not with an increased
mitochondrial iron content – does not protect frataxin
depleted cells from the deleterious effect of iron chelation.
So far we have no idea of the concentration of deferiprone
which might be reached in brain tissues of treated
patients. Nevertheless, a mean peak plasma concentration
of deferiprone has been estimated to be about 150–200
μM when deferiprone is provided in one daily dose of 25
mg/kg of body weight ; a value similar to the 150 μM
of deferiprone used in this in vitro study. A lower concen-
tration is probably maintained in tissues, however if
deferiprone concentration is efficient to empty mitochon-
drial iron it should also gradually impair aconitase activity
in the mitochondrial matrix. Obviously, the activity of
additional iron-requiring enzymes might be affected as
well due to excessive lowering of mitochondrial iron, e.g.
the mitochondrial ribonucleotide reductase . Assay-
ing the iron-sensitive activity of these mitochondrial
enzymes might provide useful maker(s) for iron chelator
toxicity in future clinical studies.
Consequently, while iron chelation might help the mito-
chondrion to cope with excessive iron at some particular
step of the disease, i.e. when iron accumulation is instru-
mental, its putative efficacy on a long term will predicta-
bly be associated with a detrimental mitochondrial iron
deprivation. Ultimately, this might further impair the
mitochondrial synthesis of ISC, already a critical issue in
The authors declare that they have no competing interests.
SG conceived and performed the experiments. VP gener-
ated and studied the frataxin-depleted SK-N-AS cells. ED
characterized the chelator-treated cells and interpreted the
results. PR conceived the study and wrote the manuscript.
This work was supported by AFAF (Association Française contre l'Ataxie
de Friedreich) to SG, VP and PR, ACHAF (Association Suisse contre
l'Ataxie de Friedreich) to PR, F.A.R.A. (Friedreich's Ataxia Research Alli-
ance) to EPD and PR, AFM (Association Française contre les Myopathies)
1. Campuzano V, Montermini L, Molto MD, Pianese L, Cossee M, Cav-
alcanti F, Monros E, Rodius F, Duclos F, Monticelli A, Zara F, Caniza-
res J, Koutnikova H, Bidichandani SI, Gellera C, Brice A, Trouillas P,
De Michele G, Filla A, De Frutos R, Palau F, Patel PI, Di Donato S,
Mandel JL, Cocozza S, Koenig M, Pandolfo M: Friedreich's ataxia:
autosomal recessive disease caused by an intronic GAA tri-
plet repeat expansion. Science 1996, 271(5254):1423-1427.
2. Wilson RB: Iron dysregulation in Friedreich ataxia. Semin Pedi-
atr Neurol 2006, 13(3):166-175.
Rotig A, de Lonlay P, Chretien D, Foury F, Koenig M, Sidi D, Munnich
A, Rustin P: Aconitase and mitochondrial iron-sulphur protein
deficiency in Friedreich ataxia. Nat Genet 1997, 17(2):215-217.
Tzagoloff A: Mitochondria. In Cellular Organelles Edited by: Siekevitz
P. New York , Plenum Press; 1982:1-342.
Hentze MW, Kuhn LC: Molecular control of vertebrate iron
metabolism: mRNA-based regulatory circuits operated by
iron, nitric oxide, and oxidative stress. Proc Natl Acad Sci U S A
Turakhia S, Venkatakrishnan CD, Dunsmore K, Wong H, Kuppusamy
P, Zweier JL, Ilangovan G: Doxorubicin-induced cardiotoxicity:
direct correlation of cardiac fibroblast and H9c2 cell survival
and aconitase activity with heat shock protein 27. Am J Physiol
Heart Circ Physiol 2007, 293(5):H3111-21.
Chantrel-Groussard K, Geromel V, Puccio H, Koenig M, Munnich A,
Rotig A, Rustin P: Disabled early recruitment of antioxidant
defenses in Friedreich's ataxia. Hum Mol Genet 2001,
Waldvogel D, van Gelderen P, Hallett M: Increased iron in the
dentate nucleus of patients with Friedrich's ataxia. Ann Neurol
Rustin P: The use of antioxidants in Friedreich's ataxia treat-
ment. Expert Opin Investig Drugs 2003, 12(4):569-575.
Di Prospero NA, Baker A, Jeffries N, Fischbeck KH: Neurological
effects of high-dose idebenone in patients with Friedreich's
ataxia: a randomised, placebo-controlled trial. Lancet Neurol
Richardson DR, Mouralian C, Ponka P, Becker E: Development of
potential iron chelators for the treatment of Friedreich's
ataxia: ligands that mobilize mitochondrial iron. Biochim Bio-
phys Acta 2001, 1536(2-3):133-140.
Glickstein H, El RB, Shvartsman M, Cabantchik ZI: Intracellular
labile iron pools as direct targets of iron chelators: a fluores-
cence study of chelator action in living cells. Blood 2005,
Boddaert N, Le Quan Sang KH, Rotig A, Leroy-Willig A, Gallet S,
Brunelle F, Sidi D, Thalabard JC, Munnich A, Cabantchik ZI: Selective
iron chelation in Friedreich ataxia: biologic and clinical impli-
cations. Blood 2007, 110(1):401-408.
Tong WH, Rouault TA: Metabolic regulation of citrate and iron
by aconitases: role of iron-sulfur cluster biogenesis. Biometals
Muhlenhoff U, Richhardt N, Gerber J, Lill R: Characterization of
iron-sulfur protein assembly in isolated mitochondria. A
requirement for ATP, NADH, and reduced iron. J Biol Chem
Wong A, Yang J, Cavadini P, Gellera C, Lonnerdal B, Taroni F, Cor-
topassi G: The Friedreich's ataxia mutation confers cellular
sensitivity to oxidant stress which is rescued by chelators of
iron and calcium and inhibitors of apoptosis. Hum Mol Genet
Limenta LM, Jirasomprasert T, Tankanitlert J, Svasti S, Wilairat P,
Chantharaksri U, Fucharoen S, Morales NP: UGT1A6 genotype-
related pharmacokinetics of deferiprone (L1) in healthy vol-
unteers. Br J Clin Pharmacol 2008.
Young P, Leeds JM, Slabaugh MB, Mathews CK: Ribonucleotide
reductase: evidence for specific association with HeLa cell
mitochondria. Biochem Biophys Res Commun 1994, 203(1):46-52.
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