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Friedreich ataxia: a paradigm for mitochondrial diseases
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Friedreich ataxia: a paradigm for mitochondrial diseases Hélène Puccio* and Michel Kœnig† Friedreich ataxia (FRDA), a progressive neurodegenerative disease, is due to the partial loss of function of frataxin, a mitochondrial protein of unknown function. Loss of frataxin causes mitochondrial iron accumulation, deficiency in the activities of iron-sulfur (Fe–S) proteins, and increased oxidative stress. Mouse models for FRDA demonstrate that the Fe–S deficit precedes iron accumulation, suggesting that iron accumulation is a secondary event. Furthermore, increased oxidative stress in FRDA patients has been demonstrated, and in vitro experiments imply that the frataxin defect impairs early antioxidant defenses. These results taken together suggest that frataxin may function either in mitochondrial iron homeostasis, in Fe–S cluster biogenesis, or directly in the response to oxidative stress. It is clear, however, that the pathogenic mechanism in FRDA involves free-radical production and oxidative stress, a process that appears to be sensitive to antioxidant therapies. Addresses Institut de Génétique et de Biologie Moléculaire et Cellulaire (CNRS/INSERM/ULP), 1 rue Laurent Fries BP163, 67404 Illkirch, CU de Strasbourg, France *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Genetics & Development 2002, 12:272–277 0959-437X/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations 8OH2′′dG 8-hydroxy-2′-deoxyguanosine Fe–S iron–sulfur FRDA Friedreich ataxia SOD superoxide dismutase YAC yeast artificial chromosome YFH1 yeast frataxin homologue 1
Introduction Friedreich ataxia (FRDA) — the most common autosomal recessive ataxia (1 individual in 30,000) — is a neurodegenerative disease characterized by degeneration of the large sensory neurons and spinocerebellar tracts, cardiomyopathy and increased incidence in diabetes [1,2,3•]. FRDA is caused by severely reduced levels of frataxin as a result of a large GAA triplet repeat expansion within the first intron of the frataxin gene [4], leading to a reduction of frataxin expression by inhibiting transcription [5–7]. Frataxin is a ubiquitously expressed mitochondrial protein, highly conserved through evolution (from γ-purple bacteria to human). However, frataxin shows no similarity with protein domains of known function, and therefore its biochemical function cannot be deduced from its sequence. Both the physiological function of frataxin, and the pathophysiological process of the disease are controversial. In the present review, we discuss the recent advances that have
been made in deciphering the function of frataxin and the predominant hypotheses in the field. The generation of mouse models in order to help understand the pathophysiology and test pharmacological therapy are described. Finally, we conclude with promising prospect for therapy. The recent increased understanding of the effect of the GAA expansion on transcription and replication, as well as insight into the biogenesis of frataxin will not be discussed as it has recently been reviewed thoroughly [8].
The function of frataxin Although the exact physiological function of frataxin is not known, there are at least four predominant hypotheses for its role (Figure 1): mitochondrial iron transport, iron–sulfur (Fe–S) cluster biogenesis, iron binding/sequestration, and response to oxidative stress. In this review, we discuss the experimental data leading to these four different hypotheses. Frataxin and iron homeostasis
Yeast as a model organism proved to be an invaluable system for unraveling frataxin mitochondrial function. Deletion of the frataxin yeast homologue 1 (YFH1) results in mutant strains that show a growth defect on fermentable carbon source, accumulate mitochondrial iron and exhibit a high sensitivity to oxidative stress induced by oxidant agents such as hydrogen peroxide or iron, as well as a reduction in oxidative phosphorylation [9–13]. In parallel, not only is the high-affinity iron import system constitutively turned on in the ∆YFH1 mutants [9,12] but, using DNA microarray hybridization analysis, Foury and Talibi [14] found that the expression of all known members of the ‘iron regulon’ was increased in a frataxin-deficient strain. The implication being that cells are starved for cytosolic iron. Together, these observations lead to the hypothesis that frataxin is directly involved in iron homeostasis, possibly by mediating iron efflux. Therefore, the direct consequence of frataxin deficiency would be iron accumulation which likely catalyses the formation of hydroxyl radicals via the Fenton reaction, leading to an iron-induced oxidative stress provoking the loss of mitochondrial DNA and Fe–S proteins characteristic of the ∆YFH1 yeast [9,10]. There are multiple evidences that mammalian and yeast frataxins are true orthologues and that human frataxin might also be involved in iron homeostasis. Human frataxin can complement the defect in ∆YFH1 cells [13,15] and this complementation is abolished by the introduction of disease-causing missense mutations within the gene [15]. Moreover, pathological and clinical studies show that consequences of frataxin reduction are indeed similar in human cells. Iron deposits are consistently observed on autopsy in hearts of FRDA patients [16,17]. Magnetic resonance imaging data indicate that iron also accumulates
Friedreich ataxia: a paradigm for mitochondrial diseases Puccio and Kœnig
in the dentate nucleus [18], an affected cerebellar structure. Mitochondrial iron content of FRDA fibroblasts, an unaffected cell type in this disease, is minimally increased [19]. Moreover, an increased serum level of transferrin receptor, the major mediator of iron uptake, has been found in FRDA patients [20], which may indicate an abnormal intracellular distribution of iron. However, a similar increase in patients with other degenerative ataxia was observed [20], suggesting that the elevated transferrin levels is a feature of several degenerative ataxias, rather than specific to FRDA.
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Figure 1 Fe–S Biosynthesis
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Frataxin ?
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Frataxin and the biogenesis of iron–sulfur clusters
A selective deficiency of the respiratory chain complexes I–III and of both mitochondrial and cytosolic aconitases activities in the heart biopsy and autopsy material of patients has been reported [16,21]. All the deficient enzymes and complexes contain Fe–S clusters in their active sites. Fe–S proteins are remarkably sensitive to free radicals, and their inactivation further suggests oxidative stress in FRDA-affected tissues which could be a consequence of iron accumulation. However, data from FRDA mouse models (see below) demonstrate that the Fe–S protein deficiency occurs prior to iron accumulation [22••], suggesting that iron accumulation might not be the primary consequence of frataxin deficiency. We cannot exclude, though, that deregulation of iron homeostasis without significant iron deposit or accumulation leads to ironinduced oxidative stress. Is the Fe–S protein deficiency either a cause or a consequence of mitochondrial iron accumulation? Yeast studies demonstrate that the presence of iron chelator in the culture media restores normal intramitochondrial iron levels and normal oxidative respiration in ∆YFH1 yeast cells, whereas the activity of aconitase is not fully restored [23]. These results suggest that the reduction in the activity of the respiratory chain complexes is a consequence of mitochondrial iron accumulation, whereas the reduced aconitase activity is directly linked, in part, to frataxin deficiency. Mitochondrial aconitase deficiency, therefore, does not seem to be a mere consequence of mitochondrial dysfunction. In addition, the inactivation of cytosolic aconitase in FRDA patients (yeast do not have cytosolic aconitase), suggests that a general Fe–S defect is the underlying mechanism. However, it is possible that the loss of cytosolic aconitase activity observed in FRDA might also reflect a decrease of cytosolic iron content, as cytosolic aconitase loses its Fe–S cluster to acquire iron-responsive element binding properties in response to low iron concentration [24].
Iron homeostasis
Oxidative stress
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Several concordant metabolic alterations have been observed in FRDA patient samplings and in animal and microorganism models. They can, however, result from each other in a vicious cycle: oxidative stress leading to oxidation of iron in Fe–S cluster, which is then released, causing itself oxidative stress through Fenton chemistry. Frataxin may prevent any one of these steps, including by iron storage as a buffer for free iron, and its deficiency will result in the full metabolic phenotype.
functional relation between frataxin and Atm1 have failed so far. Interestingly, mutations in ABC7, the human homologue of the Atm1 gene, is responsible for a rare X-linked recessive disease, associating spinocerebellar ataxia and anaemia [27]. The Ssq1 gene, encoding a low-abundance mitochondrial heat-shock 70 protein (mtHsp70) [28], was demonstrated recently to be required for Fe–S cluster assembly in mitochondria, together with the co-chaperone Jac1, a mitochondrial DnaJ homolog [29••]. Furthermore, the Fe–S cluster assembly into ferredoxin (itself involved in Fe–S assembly) requires frataxin [29••]. In concordance with these later results, the phylogenetic distribution of frataxin in 56 available genomes identified an identical phylogenetic distributions with hscA, a paralog of Ssq1 and hscB, an ortholog of Jac1 [30••]. Therefore, combining phylogenetic data with experimental and predicted cellular localization data supports the hypothesis that frataxin is involved directly in Fe–S cluster assembly. Again, however, this hypothesis is challenged by a study demonstrating that a lack of frataxin does not result in decreased Fe–S activities if iron accumulation is prevented [31], suggesting that the Fe–S deficiency is caused by oxidative damage, whether induced by iron or not. Frataxin as an iron-storage protein
It is interesting to note that two other yeast mutants (∆Atm1 and ∆Ssq1) that specifically accumulate iron in the mitochondria are involved in Fe–S cluster biogenesis. The Atm1 gene encodes a seven transmembrane ABC (ATPbinding cassette) ATPase (a putative ABC transporter) that appears to be involved in export of Fe–S clusters from the mitochondria [25,26]. Attempts to identify a direct
An attractive hypothesis recently put forward by Isaya and co-workers suggests that the function of frataxin is to bind iron and keep it in a soluble and bioavailable form [32]. By gel-filtration experiments and analytical ultracentrifugation, the authors demonstrated that recombinant purified yeast frataxin is a soluble monomer that contains no iron, which assembles into a high molecular weight regular spherical
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multimer sequestering >3000 atoms of iron upon titration with increasing concentration of ferrous iron. This function of frataxin is consistent with the role played by frataxin in iron export and would also account for the proposed role in Fe–S cluster assembly. Furthermore, storing uncomplexed iron should prevent iron toxicity, and (therefore) oxidative damage. To date, no other group has been able to reproduce the iron-binding results for human frataxin. Analysis of the solution [33] and crystal [34,35] structures of human frataxin and its bacterial Cyay homologue has revealed a compact and globular protein with a novel fold consisting of a five-stranded anti-parallel β sheet packing against a pair of parallel α helices. Frataxin does not have any features resembling known iron-binding sites, and the structure rather suggests that it interacts with a large ligand, probably a protein. However, attempts by different groups to identify the interacting protein have been unsuccessful, possibly because the interaction might be transient. Furthermore, both the crystal and NMR study suggest that human frataxin is not an iron-binding protein, under any concentration of iron tested [33,34]. Frataxin and oxidative stress
Whether frataxin is involved directly in iron homeostasis or Fe–S cluster biogenesis, the generation of reactive oxygen species plays an important role in the pathogenesis of FRDA. Consistent with this hypothesis, vitamin E deficiency produces a disease very similar to FRDA. Although the speculation of oxidative stress involvement in FRDA has long been accepted, it was not until very recently that an increased oxidative stress has been demonstrated in individuals with FRDA. Schulz et al. [36•] reported elevated urinary concentrations of 8-hydroxy2′-deoxyguanosine (8OH2′dG), a marker of oxidative damage to DNA, in patients with FRDA [36•]. Similarly, Edmond et al. [37•] found increased levels of plasma malondialdehyde, a product of lipid peroxidation. More recently, Piemonte et al. [38•] demonstrated decreased free glutathione concentration in blood of patients with FRDA. Glutathione, the most abundant antioxidant in the cell, can act either as a free radical scavenger or a co-substrate for important enzymes such as glutathione peroxidase and glutathione transferases [39]. These studies thus further imply a role of free radical cytotoxicity in the pathophysiology of the disease. In addition, these studies are important because they used readily available fluids, and thus the assays could provide a way to monitor disease status or treatment efficacy. It is unquestionable, therefore, that oxidative stress plays an important role in the pathogenesis of FRDA. Could frataxin be directly involved in oxidative stress response or is this oxidative stress iron-induced? To address this question, the Rustin group devised experimental conditions to endogenously produce superoxides in cells deficient in frataxin (FRDA fibroblasts) by inhibiting the ATP
synthase with oligomycin [40••]. This inhibition normally results in an important superoxide dismutase (SOD) induction, however FRDA cells failed to normally increase SOD activity and iron import and to decrease iron storage capacity, despite parallel loss of membrane-bound ironcontaining enzyme activity. Similarly, the Pandolfo group showed that FRDA fibroblasts exposed to iron, and therefore to iron-induced oxidative stress, failed to normally upregulate the mitochondrial SOD [41•]. Finally, measurements of SOD enzyme activity in the heart of the conditional mouse models of FRDA (see below) show lower SOD activity than in age-match control mice [40••]. These observations are consistent with expression data from DNA chip experiments that show no increase in SOD transcripts in ∆YFH1 yeast [14]. Therefore, together, these results suggest that either decreased or absent frataxin impairs early antioxidant defenses with the absence of SOD induction resulting in higher cell lethality in response to oxidative stress. The mechanism by which frataxin could be involved remains to be established. However, the primary involvement of superoxide injury is consistent with the pathology of the disease (neurons and cardiomyocytes have limited antioxidant defenses), and the increase in oxidative stress found in FRDA.
Mouse models for FRDA To study further the disease pathology and to test pharmocological therapy, several mouse models have been generated. Our group generated a classical mouse model by constitutive inactivation of frataxin by homologous recombination [42]. Homozygous deletion of frataxin causes embryonic lethality a few days after implantation, demonstrating an important role for frataxin during early development. These results suggest that the milder phenotype in humans is caused by residual frataxin expression associated with the expansion mutations. No iron accumulation was observed during embryonic resorption, suggesting that cell death might be the result of a mechanism independent of iron accumulation. To circumvent embryonic lethality, we recently generated, in parallel, two different conditional knock-out models, based on the Cre-lox system, in which frataxin was deleted either specifically in skeletal and cardiac muscle (using transgenic mice expressing Cre under the Muscle Creatinine Kinase promoter) or predominantly in neuronal tissues (Neuron Specific Enolase promoter) [22••]. Both models are viable and reproduce some morphological and biochemical features observed in FRDA patients including cardiac hypertrophy without skeletal muscle involvement, large sensory neuron dysfunction without alteration of the small sensory and motor neurons, and deficient activities of complexes I–III of the respiratory chain and of the aconitases. Time-course experiments reveal that the Fe–S enzyme deficiencies begin in the initial phase of the pathology, and that intramitochondrial iron accumulation occurs later. In addition, as mentioned above, the SODs are abnormally low in the diseased mouse [40••]. These
Friedreich ataxia: a paradigm for mitochondrial diseases Puccio and Kœnig
mutant mice therefore represent the first mammalian models to evaluate treatment strategies (see below) for the human disease. More recently, Pandolfo’s group have attempted to generate a mouse model by introducing a (GAA)230 repeat within the mouse frataxin gene to mirror the chronically reduced levels of frataxin expression found in the human disease [43]. Bred with the Frda knockout, the authors obtained animals expressing 25–36% of wild-type frataxin levels, an expression level associated to mildly affected FRDA patients. Unfortunately, these mice did not develop abnormalities of motor coordination, cardiomyopathy, or iron metabolism. The Chamberlain group were able to overcome the embryonic lethality of the Frda knockout by generating transgenic mice that contain the entire frataxin gene within a human YAC clone onto the null mouse background [44]. Human frataxin was expressed in the appropriate tissues at levels comparable to the endogenous mouse frataxin, and was processed correctly and localized to mitochondria. Biochemical analysis of heart tissue demonstrated preservation of mitochondrial respiratory chain function. Therefore, it is possible to envision the generation of a mouse model expressing low levels of frataxin by using a human YAC derived from a patient, thereby having a repeat within the first intron inhibiting the expression of frataxin. It is clearly important to generate a mouse model that molecularly mirrors the human disease in addition to the pre-existing models.
Conclusion and prospect for therapy On the basis of the recent discoveries of the potential function of frataxin, and more specifically on the consequences of frataxin reduction, therapeutic advances can be envisioned. Whether the mitochondrial iron accumulation is either a primary or a secondary effect of frataxin deficiency, all data suggest that intracellular iron imbalance and oxidative stress are involved in the pathogenesis of FRDA. This led to initial enthusiasm for the use of iron chelators, such as desferrioxamine, as therapeutic reagents for treating the disease. However, desferrioxamine has a significant side effect profile and its effect on individuals who do not have a generalized iron overload is not well studied. FRDA patients have normal serum iron and ferritin levels [45]. Moreover, Rustin et al. have experimental evidences suggesting that caution should be taken when using iron chelators as a therapeutic agent as it could displace rather than protect against iron-mediated toxicity [46]. As it has been proposed that reducing the load of free radicals will slow the progression of the disease, antioxidants that are usually devoid of side-effects can be considered as potential therapeutic reagents. Rustin et al. have shown by in vitro experiments that the antioxidant idebenone, a short chain analogue of coenzyme Q10, protects the membrane respiratory chain enzymes against iron-induced
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injury without causing a reduction in soluble aconitase activity [46,47]. In addition, the short side chain of idebenone allows it to cross membranes readily (including the blood–brain barrier). Preliminary results on the cardiomyopathy of 40 FRDA patients [46,48••] are promising and warrant the establishment of a larger clinical trial using idebenone. Furthermore, Schulz et al. have found that eight weeks of treatment with idebenone decreased the urine levels of 8OH2′dG by 20% [36•]. Finally, our unpublished results of treatment of the conditional cardiac mouse model shows a statistically significant increase in the life span of the animals (H Puccio, M Kœnig, unpublished data). Therefore, idebenone appears to have some therapeutic effect, although the neurological consequences need to be evaluated in the long term. On the other hand, treatment of FRDA patients for six months with the antioxidants coenzyme Q10 and vitamin E resulted in an increase in the cardiac phosphocreatine to ATP ratios and improved muscle mitochondrial ATP production [49•]. Whether FRDA patients will benefit clinically from this bioenergetic improvement, however, must be evaluated by appropriate trials in the future. In conclusion, these findings contribute to the growing body of evidence that treatments aimed to enhance mitochondrial function and reduce toxic radical production are rational and may be a realistic hope for the patients and their families. The next challenge is to resolve the controversial issues as to the function of frataxin to provide solid bases for designing eagerly awaited therapeutic approaches for FRDA.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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29. Lutz T, Westermann B, Neupert W, Herrmann JM: The mitochondrial •• proteins Ssq1 and Jac1 are required for the assembly of iron sulfur clusters in mitochondria. J Mol Biol 2001, 307:815-825. See annotation [30••]. 30. Huynen MA, Snel B, Bork P, Gibson TJ: The phylogenetic •• distribution of frataxin indicates a role in iron-sulfur cluster protein assembly. Hum Mol Genet 2001, 10:2463-2468. Lutz et al. [29••] developed an assay with which to assess directly the acquisition of Fe–S clusters by the apoform of mitochondrial ferredoxin in isolated yeast mitochondria. Using this assay, they have identified Ssq1, Jac1 and frataxin as the component involved in the process. Huynen et al. [30••] investigated by computational analysis whether the frataxin gene specifically cooccurs with any other genes in 56 available sequenced genomes in order to predict functional interaction of frataxin with other protein. They have found two genes with identical phylogenic distribution to the frataxin gene: hscA/Ssq1 and hscB/Jac1. Together, these two reports [29••,30••] suggest through experimental and phylogenetic data that frataxin is directly involved in the assembly of Fe–S cluster along with Ssq1 and Jac1. 31. Chen OS, Kaplan J: CCC1 suppresses mitochondrial damage in the yeast model of Friedreich’s ataxia by limiting mitochondrial iron accumulation. J Biol Chem 2000, 275:7626-7632. 32. Adamec J, Rusnak F, Owen WG, Naylor S, Benson LM, Gacy AM, Isaya G: Iron-dependent self-assembly of recombinant yeast frataxin: implications for Friedreich ataxia. Am J Hum Genet 2000, 67:549-562. 33. Musco G, Stier G, Kolmerer B, Adinolfi S, Martin S, Frenkiel T, Gibson T, Pastore A: Towards a structural understanding of Friedreich’s ataxia: the solution structure of frataxin. Structure Fold Des 2000, 8:695-707. 34. Dhe-Paganon S, Shigeta R, Chi YI, Ristow M, Shoelson SE: Crystal structure of human frataxin. J Biol Chem 2000, 275:30753-30756. 35. Cho SJ, Lee MG, Yang JK, Lee JY, Song HK, Suh SW: Crystal structure of Escherichia coli CyaY protein reveals a previously unidentified fold for the evolutionarily conserved frataxin family. Proc Natl Acad Sci USA 2000, 97:8932-8937. 36. Schulz JB, Dehmer T, Schols L, Mende H, Hardt C, Vorgerd M, • Burk K, Matson W, Dichgans J, Beal MF et al.: Oxidative stress in patients with Friedreich ataxia. Neurology 2000, 55:1719-1721. See annotation [38•]. 37. •
Edmond M, Lepage G, Vanasse M, Pandolfo M: Increased levels of plasma malondialdehyde in Friedreich ataxia. Neurology 2000, 55:1752-1753. See annotation [38•].
22. Puccio H, Simon D, Cossee M, Criqui-Filipe P, Tiziano F, Melki J, •• Hindelang C, Matyas R, Rustin P, Koenig M: Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits. Nat Genet 2001, 27:181-186. We have generated of conditional mouse models for FRDA that exhibit important pathophysiological and biochemical features of the human disease. The models demonstrate time-dependent intramitochondrial iron accumulation that does not represent the causative pathological mechanism. These mutant mice represent the first mammalian models to evaluate treatment strategies for the human disease.
38. Piemonte F, Pastore A, Tozzi G, Tagliacozzi D, Santorelli FM, • Carrozzo R, Casali C, Damiano M, Federici G, Bertini E: Glutathione in blood of patients with Friedreich’s ataxia. Eur J Clin Invest 2001, 31:1007-1011. These three reports [36•–38•] demonstrate increased oxidative stress in FRDA patients and provide the first relatively easy bioassays on readily available fluids to monitor disease status and treatment efficacy.
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40. 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, 10:2061-2067. Neither superoxide dismustases nor the import iron machinery was induced by an endogeneous oxidative stress in FRDA patients’ fibroblasts. This paper therefore suggests that continuous oxidative damage to Fe–S clusters, resulting from hampered superoxide dismutase signaling, is causative of the mitochondrial
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deficiency and long term mitochondrial iron overload. This is the first report that implies that frataxin is directly involved in the response to oxidative stress. 41. Jiralerspong S, Ge B, Hudson TJ, Pandolfo M: Manganese • superoxide dismutase induction by iron is impaired in Friedreich ataxia cells. FEBS Lett 2001, 509:101-105. This paper also reports an impaired regulation of mitochondrial superoxide dismutase by exposing patient fibroblast to iron, which normally results in upregulation of MnSOD (see [40••]). 42. Cossee M, Puccio H, Gansmuller A, Koutnikova H, Dierich A, LeMeur M, Fischbeck K, Dolle P, Koenig M: Inactivation of the Friedreich ataxia mouse gene leads to early embryonic lethality without iron accumulation. Hum Mol Genet 2000, 9:1219-1226. 43. Miranda C, Santos M, Ohshima K, Smith J, Cossee M, Koenig M, Sequeiros J, Kaplan J, Pandolfo M: Frataxin knock-in mouse. FEBS Lett 2002, 512:291-297. 44. Pook MA, Al-Mahdawi S, Carroll CJ, Cossee M, Puccio H, Lawrence L, Clark P, Lowrie MB, Bradley JL, Cooper JM et al.: Rescue of the Friedreich’s ataxia knockout mouse by human YAC transgenesis. Neurogenetics 2001, 3:185-193. 45. Wilson RB, Lynch DR, Fischbeck KH: Normal serum iron and ferritin concentrations in patients with Friedreich’s ataxia. Ann Neurol 1998, 44:132-134.
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46. Rustin P, von Kleist-Retzow JC, Chantrel-Groussard K, Sidi D, Munnich A, Rotig A: Effect of idebenone on cardiomyopathy in Friedreich’s ataxia: a preliminary study. Lancet 1999, 354:477-479. 47.
Rustin P, Munnich A, Rotig A: Quinone analogs prevent enzymes targeted in Friedreich ataxia from iron-induced injury in vitro. Biofactors 1999, 9:247-251.
48. Rustin P, Rötig A, Munnich A, Sidi D: Heart hypertrophy and •• function are improved by idebenone in Friedreich’s ataxia. Free Radic Research 2002, in press. Idebenone treatment is associated with improvement of hypertrophic cardiomyopathy in patients with FRDA. This work has great potential clinical significance for treatment of the disease and provides realistic hope for the patients and their families. The neurological benefit remains to be established. 49. Lodi R, Hart PE, Rajagopalan B, Taylor DJ, Crilley JG, Bradley JL, • Blamire AM, Manners D, Styles P, Schapira AH et al.: Antioxidant treatment improves in vivo cardiac and skeletal muscle bioenergetics in patients with Friedreich’s ataxia. Ann Neurol 2001, 49:590-596. Coenzyme Q10 and vitamin E resulted in a bioenergetic increase in heart and muscle, but whether the FRDA patients will benefit clinically needs to be evaluated by appropriate trials.
Friedreich ataxia: a paradigm for mitochondrial diseases Hélène Puccio* and Michel Kœnig† Friedreich ataxia (FRDA), a progressive neurodegenerative disease, is due to the partial loss of function of frataxin, a mitochondrial protein of unknown function. Loss of frataxin causes mitochondrial iron accumulation, deficiency in the activities of iron-sulfur (Fe–S) proteins, and increased oxidative stress. Mouse models for FRDA demonstrate that the Fe–S deficit precedes iron accumulation, suggesting that iron accumulation is a secondary event. Furthermore, increased oxidative stress in FRDA patients has been demonstrated, and in vitro experiments imply that the frataxin defect impairs early antioxidant defenses. These results taken together suggest that frataxin may function either in mitochondrial iron homeostasis, in Fe–S cluster biogenesis, or directly in the response to oxidative stress. It is clear, however, that the pathogenic mechanism in FRDA involves free-radical production and oxidative stress, a process that appears to be sensitive to antioxidant therapies. Addresses Institut de Génétique et de Biologie Moléculaire et Cellulaire (CNRS/INSERM/ULP), 1 rue Laurent Fries BP163, 67404 Illkirch, CU de Strasbourg, France *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Genetics & Development 2002, 12:272–277 0959-437X/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations 8OH2′′dG 8-hydroxy-2′-deoxyguanosine Fe–S iron–sulfur FRDA Friedreich ataxia SOD superoxide dismutase YAC yeast artificial chromosome YFH1 yeast frataxin homologue 1
Introduction Friedreich ataxia (FRDA) — the most common autosomal recessive ataxia (1 individual in 30,000) — is a neurodegenerative disease characterized by degeneration of the large sensory neurons and spinocerebellar tracts, cardiomyopathy and increased incidence in diabetes [1,2,3•]. FRDA is caused by severely reduced levels of frataxin as a result of a large GAA triplet repeat expansion within the first intron of the frataxin gene [4], leading to a reduction of frataxin expression by inhibiting transcription [5–7]. Frataxin is a ubiquitously expressed mitochondrial protein, highly conserved through evolution (from γ-purple bacteria to human). However, frataxin shows no similarity with protein domains of known function, and therefore its biochemical function cannot be deduced from its sequence. Both the physiological function of frataxin, and the pathophysiological process of the disease are controversial. In the present review, we discuss the recent advances that have
been made in deciphering the function of frataxin and the predominant hypotheses in the field. The generation of mouse models in order to help understand the pathophysiology and test pharmacological therapy are described. Finally, we conclude with promising prospect for therapy. The recent increased understanding of the effect of the GAA expansion on transcription and replication, as well as insight into the biogenesis of frataxin will not be discussed as it has recently been reviewed thoroughly [8].
The function of frataxin Although the exact physiological function of frataxin is not known, there are at least four predominant hypotheses for its role (Figure 1): mitochondrial iron transport, iron–sulfur (Fe–S) cluster biogenesis, iron binding/sequestration, and response to oxidative stress. In this review, we discuss the experimental data leading to these four different hypotheses. Frataxin and iron homeostasis
Yeast as a model organism proved to be an invaluable system for unraveling frataxin mitochondrial function. Deletion of the frataxin yeast homologue 1 (YFH1) results in mutant strains that show a growth defect on fermentable carbon source, accumulate mitochondrial iron and exhibit a high sensitivity to oxidative stress induced by oxidant agents such as hydrogen peroxide or iron, as well as a reduction in oxidative phosphorylation [9–13]. In parallel, not only is the high-affinity iron import system constitutively turned on in the ∆YFH1 mutants [9,12] but, using DNA microarray hybridization analysis, Foury and Talibi [14] found that the expression of all known members of the ‘iron regulon’ was increased in a frataxin-deficient strain. The implication being that cells are starved for cytosolic iron. Together, these observations lead to the hypothesis that frataxin is directly involved in iron homeostasis, possibly by mediating iron efflux. Therefore, the direct consequence of frataxin deficiency would be iron accumulation which likely catalyses the formation of hydroxyl radicals via the Fenton reaction, leading to an iron-induced oxidative stress provoking the loss of mitochondrial DNA and Fe–S proteins characteristic of the ∆YFH1 yeast [9,10]. There are multiple evidences that mammalian and yeast frataxins are true orthologues and that human frataxin might also be involved in iron homeostasis. Human frataxin can complement the defect in ∆YFH1 cells [13,15] and this complementation is abolished by the introduction of disease-causing missense mutations within the gene [15]. Moreover, pathological and clinical studies show that consequences of frataxin reduction are indeed similar in human cells. Iron deposits are consistently observed on autopsy in hearts of FRDA patients [16,17]. Magnetic resonance imaging data indicate that iron also accumulates
Friedreich ataxia: a paradigm for mitochondrial diseases Puccio and Kœnig
in the dentate nucleus [18], an affected cerebellar structure. Mitochondrial iron content of FRDA fibroblasts, an unaffected cell type in this disease, is minimally increased [19]. Moreover, an increased serum level of transferrin receptor, the major mediator of iron uptake, has been found in FRDA patients [20], which may indicate an abnormal intracellular distribution of iron. However, a similar increase in patients with other degenerative ataxia was observed [20], suggesting that the elevated transferrin levels is a feature of several degenerative ataxias, rather than specific to FRDA.
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Figure 1 Fe–S Biosynthesis
?
Frataxin ?
?
Frataxin and the biogenesis of iron–sulfur clusters
A selective deficiency of the respiratory chain complexes I–III and of both mitochondrial and cytosolic aconitases activities in the heart biopsy and autopsy material of patients has been reported [16,21]. All the deficient enzymes and complexes contain Fe–S clusters in their active sites. Fe–S proteins are remarkably sensitive to free radicals, and their inactivation further suggests oxidative stress in FRDA-affected tissues which could be a consequence of iron accumulation. However, data from FRDA mouse models (see below) demonstrate that the Fe–S protein deficiency occurs prior to iron accumulation [22••], suggesting that iron accumulation might not be the primary consequence of frataxin deficiency. We cannot exclude, though, that deregulation of iron homeostasis without significant iron deposit or accumulation leads to ironinduced oxidative stress. Is the Fe–S protein deficiency either a cause or a consequence of mitochondrial iron accumulation? Yeast studies demonstrate that the presence of iron chelator in the culture media restores normal intramitochondrial iron levels and normal oxidative respiration in ∆YFH1 yeast cells, whereas the activity of aconitase is not fully restored [23]. These results suggest that the reduction in the activity of the respiratory chain complexes is a consequence of mitochondrial iron accumulation, whereas the reduced aconitase activity is directly linked, in part, to frataxin deficiency. Mitochondrial aconitase deficiency, therefore, does not seem to be a mere consequence of mitochondrial dysfunction. In addition, the inactivation of cytosolic aconitase in FRDA patients (yeast do not have cytosolic aconitase), suggests that a general Fe–S defect is the underlying mechanism. However, it is possible that the loss of cytosolic aconitase activity observed in FRDA might also reflect a decrease of cytosolic iron content, as cytosolic aconitase loses its Fe–S cluster to acquire iron-responsive element binding properties in response to low iron concentration [24].
Iron homeostasis
Oxidative stress
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Several concordant metabolic alterations have been observed in FRDA patient samplings and in animal and microorganism models. They can, however, result from each other in a vicious cycle: oxidative stress leading to oxidation of iron in Fe–S cluster, which is then released, causing itself oxidative stress through Fenton chemistry. Frataxin may prevent any one of these steps, including by iron storage as a buffer for free iron, and its deficiency will result in the full metabolic phenotype.
functional relation between frataxin and Atm1 have failed so far. Interestingly, mutations in ABC7, the human homologue of the Atm1 gene, is responsible for a rare X-linked recessive disease, associating spinocerebellar ataxia and anaemia [27]. The Ssq1 gene, encoding a low-abundance mitochondrial heat-shock 70 protein (mtHsp70) [28], was demonstrated recently to be required for Fe–S cluster assembly in mitochondria, together with the co-chaperone Jac1, a mitochondrial DnaJ homolog [29••]. Furthermore, the Fe–S cluster assembly into ferredoxin (itself involved in Fe–S assembly) requires frataxin [29••]. In concordance with these later results, the phylogenetic distribution of frataxin in 56 available genomes identified an identical phylogenetic distributions with hscA, a paralog of Ssq1 and hscB, an ortholog of Jac1 [30••]. Therefore, combining phylogenetic data with experimental and predicted cellular localization data supports the hypothesis that frataxin is involved directly in Fe–S cluster assembly. Again, however, this hypothesis is challenged by a study demonstrating that a lack of frataxin does not result in decreased Fe–S activities if iron accumulation is prevented [31], suggesting that the Fe–S deficiency is caused by oxidative damage, whether induced by iron or not. Frataxin as an iron-storage protein
It is interesting to note that two other yeast mutants (∆Atm1 and ∆Ssq1) that specifically accumulate iron in the mitochondria are involved in Fe–S cluster biogenesis. The Atm1 gene encodes a seven transmembrane ABC (ATPbinding cassette) ATPase (a putative ABC transporter) that appears to be involved in export of Fe–S clusters from the mitochondria [25,26]. Attempts to identify a direct
An attractive hypothesis recently put forward by Isaya and co-workers suggests that the function of frataxin is to bind iron and keep it in a soluble and bioavailable form [32]. By gel-filtration experiments and analytical ultracentrifugation, the authors demonstrated that recombinant purified yeast frataxin is a soluble monomer that contains no iron, which assembles into a high molecular weight regular spherical
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multimer sequestering >3000 atoms of iron upon titration with increasing concentration of ferrous iron. This function of frataxin is consistent with the role played by frataxin in iron export and would also account for the proposed role in Fe–S cluster assembly. Furthermore, storing uncomplexed iron should prevent iron toxicity, and (therefore) oxidative damage. To date, no other group has been able to reproduce the iron-binding results for human frataxin. Analysis of the solution [33] and crystal [34,35] structures of human frataxin and its bacterial Cyay homologue has revealed a compact and globular protein with a novel fold consisting of a five-stranded anti-parallel β sheet packing against a pair of parallel α helices. Frataxin does not have any features resembling known iron-binding sites, and the structure rather suggests that it interacts with a large ligand, probably a protein. However, attempts by different groups to identify the interacting protein have been unsuccessful, possibly because the interaction might be transient. Furthermore, both the crystal and NMR study suggest that human frataxin is not an iron-binding protein, under any concentration of iron tested [33,34]. Frataxin and oxidative stress
Whether frataxin is involved directly in iron homeostasis or Fe–S cluster biogenesis, the generation of reactive oxygen species plays an important role in the pathogenesis of FRDA. Consistent with this hypothesis, vitamin E deficiency produces a disease very similar to FRDA. Although the speculation of oxidative stress involvement in FRDA has long been accepted, it was not until very recently that an increased oxidative stress has been demonstrated in individuals with FRDA. Schulz et al. [36•] reported elevated urinary concentrations of 8-hydroxy2′-deoxyguanosine (8OH2′dG), a marker of oxidative damage to DNA, in patients with FRDA [36•]. Similarly, Edmond et al. [37•] found increased levels of plasma malondialdehyde, a product of lipid peroxidation. More recently, Piemonte et al. [38•] demonstrated decreased free glutathione concentration in blood of patients with FRDA. Glutathione, the most abundant antioxidant in the cell, can act either as a free radical scavenger or a co-substrate for important enzymes such as glutathione peroxidase and glutathione transferases [39]. These studies thus further imply a role of free radical cytotoxicity in the pathophysiology of the disease. In addition, these studies are important because they used readily available fluids, and thus the assays could provide a way to monitor disease status or treatment efficacy. It is unquestionable, therefore, that oxidative stress plays an important role in the pathogenesis of FRDA. Could frataxin be directly involved in oxidative stress response or is this oxidative stress iron-induced? To address this question, the Rustin group devised experimental conditions to endogenously produce superoxides in cells deficient in frataxin (FRDA fibroblasts) by inhibiting the ATP
synthase with oligomycin [40••]. This inhibition normally results in an important superoxide dismutase (SOD) induction, however FRDA cells failed to normally increase SOD activity and iron import and to decrease iron storage capacity, despite parallel loss of membrane-bound ironcontaining enzyme activity. Similarly, the Pandolfo group showed that FRDA fibroblasts exposed to iron, and therefore to iron-induced oxidative stress, failed to normally upregulate the mitochondrial SOD [41•]. Finally, measurements of SOD enzyme activity in the heart of the conditional mouse models of FRDA (see below) show lower SOD activity than in age-match control mice [40••]. These observations are consistent with expression data from DNA chip experiments that show no increase in SOD transcripts in ∆YFH1 yeast [14]. Therefore, together, these results suggest that either decreased or absent frataxin impairs early antioxidant defenses with the absence of SOD induction resulting in higher cell lethality in response to oxidative stress. The mechanism by which frataxin could be involved remains to be established. However, the primary involvement of superoxide injury is consistent with the pathology of the disease (neurons and cardiomyocytes have limited antioxidant defenses), and the increase in oxidative stress found in FRDA.
Mouse models for FRDA To study further the disease pathology and to test pharmocological therapy, several mouse models have been generated. Our group generated a classical mouse model by constitutive inactivation of frataxin by homologous recombination [42]. Homozygous deletion of frataxin causes embryonic lethality a few days after implantation, demonstrating an important role for frataxin during early development. These results suggest that the milder phenotype in humans is caused by residual frataxin expression associated with the expansion mutations. No iron accumulation was observed during embryonic resorption, suggesting that cell death might be the result of a mechanism independent of iron accumulation. To circumvent embryonic lethality, we recently generated, in parallel, two different conditional knock-out models, based on the Cre-lox system, in which frataxin was deleted either specifically in skeletal and cardiac muscle (using transgenic mice expressing Cre under the Muscle Creatinine Kinase promoter) or predominantly in neuronal tissues (Neuron Specific Enolase promoter) [22••]. Both models are viable and reproduce some morphological and biochemical features observed in FRDA patients including cardiac hypertrophy without skeletal muscle involvement, large sensory neuron dysfunction without alteration of the small sensory and motor neurons, and deficient activities of complexes I–III of the respiratory chain and of the aconitases. Time-course experiments reveal that the Fe–S enzyme deficiencies begin in the initial phase of the pathology, and that intramitochondrial iron accumulation occurs later. In addition, as mentioned above, the SODs are abnormally low in the diseased mouse [40••]. These
Friedreich ataxia: a paradigm for mitochondrial diseases Puccio and Kœnig
mutant mice therefore represent the first mammalian models to evaluate treatment strategies (see below) for the human disease. More recently, Pandolfo’s group have attempted to generate a mouse model by introducing a (GAA)230 repeat within the mouse frataxin gene to mirror the chronically reduced levels of frataxin expression found in the human disease [43]. Bred with the Frda knockout, the authors obtained animals expressing 25–36% of wild-type frataxin levels, an expression level associated to mildly affected FRDA patients. Unfortunately, these mice did not develop abnormalities of motor coordination, cardiomyopathy, or iron metabolism. The Chamberlain group were able to overcome the embryonic lethality of the Frda knockout by generating transgenic mice that contain the entire frataxin gene within a human YAC clone onto the null mouse background [44]. Human frataxin was expressed in the appropriate tissues at levels comparable to the endogenous mouse frataxin, and was processed correctly and localized to mitochondria. Biochemical analysis of heart tissue demonstrated preservation of mitochondrial respiratory chain function. Therefore, it is possible to envision the generation of a mouse model expressing low levels of frataxin by using a human YAC derived from a patient, thereby having a repeat within the first intron inhibiting the expression of frataxin. It is clearly important to generate a mouse model that molecularly mirrors the human disease in addition to the pre-existing models.
Conclusion and prospect for therapy On the basis of the recent discoveries of the potential function of frataxin, and more specifically on the consequences of frataxin reduction, therapeutic advances can be envisioned. Whether the mitochondrial iron accumulation is either a primary or a secondary effect of frataxin deficiency, all data suggest that intracellular iron imbalance and oxidative stress are involved in the pathogenesis of FRDA. This led to initial enthusiasm for the use of iron chelators, such as desferrioxamine, as therapeutic reagents for treating the disease. However, desferrioxamine has a significant side effect profile and its effect on individuals who do not have a generalized iron overload is not well studied. FRDA patients have normal serum iron and ferritin levels [45]. Moreover, Rustin et al. have experimental evidences suggesting that caution should be taken when using iron chelators as a therapeutic agent as it could displace rather than protect against iron-mediated toxicity [46]. As it has been proposed that reducing the load of free radicals will slow the progression of the disease, antioxidants that are usually devoid of side-effects can be considered as potential therapeutic reagents. Rustin et al. have shown by in vitro experiments that the antioxidant idebenone, a short chain analogue of coenzyme Q10, protects the membrane respiratory chain enzymes against iron-induced
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injury without causing a reduction in soluble aconitase activity [46,47]. In addition, the short side chain of idebenone allows it to cross membranes readily (including the blood–brain barrier). Preliminary results on the cardiomyopathy of 40 FRDA patients [46,48••] are promising and warrant the establishment of a larger clinical trial using idebenone. Furthermore, Schulz et al. have found that eight weeks of treatment with idebenone decreased the urine levels of 8OH2′dG by 20% [36•]. Finally, our unpublished results of treatment of the conditional cardiac mouse model shows a statistically significant increase in the life span of the animals (H Puccio, M Kœnig, unpublished data). Therefore, idebenone appears to have some therapeutic effect, although the neurological consequences need to be evaluated in the long term. On the other hand, treatment of FRDA patients for six months with the antioxidants coenzyme Q10 and vitamin E resulted in an increase in the cardiac phosphocreatine to ATP ratios and improved muscle mitochondrial ATP production [49•]. Whether FRDA patients will benefit clinically from this bioenergetic improvement, however, must be evaluated by appropriate trials in the future. In conclusion, these findings contribute to the growing body of evidence that treatments aimed to enhance mitochondrial function and reduce toxic radical production are rational and may be a realistic hope for the patients and their families. The next challenge is to resolve the controversial issues as to the function of frataxin to provide solid bases for designing eagerly awaited therapeutic approaches for FRDA.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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29. Lutz T, Westermann B, Neupert W, Herrmann JM: The mitochondrial •• proteins Ssq1 and Jac1 are required for the assembly of iron sulfur clusters in mitochondria. J Mol Biol 2001, 307:815-825. See annotation [30••]. 30. Huynen MA, Snel B, Bork P, Gibson TJ: The phylogenetic •• distribution of frataxin indicates a role in iron-sulfur cluster protein assembly. Hum Mol Genet 2001, 10:2463-2468. Lutz et al. [29••] developed an assay with which to assess directly the acquisition of Fe–S clusters by the apoform of mitochondrial ferredoxin in isolated yeast mitochondria. Using this assay, they have identified Ssq1, Jac1 and frataxin as the component involved in the process. Huynen et al. [30••] investigated by computational analysis whether the frataxin gene specifically cooccurs with any other genes in 56 available sequenced genomes in order to predict functional interaction of frataxin with other protein. They have found two genes with identical phylogenic distribution to the frataxin gene: hscA/Ssq1 and hscB/Jac1. Together, these two reports [29••,30••] suggest through experimental and phylogenetic data that frataxin is directly involved in the assembly of Fe–S cluster along with Ssq1 and Jac1. 31. Chen OS, Kaplan J: CCC1 suppresses mitochondrial damage in the yeast model of Friedreich’s ataxia by limiting mitochondrial iron accumulation. J Biol Chem 2000, 275:7626-7632. 32. Adamec J, Rusnak F, Owen WG, Naylor S, Benson LM, Gacy AM, Isaya G: Iron-dependent self-assembly of recombinant yeast frataxin: implications for Friedreich ataxia. Am J Hum Genet 2000, 67:549-562. 33. Musco G, Stier G, Kolmerer B, Adinolfi S, Martin S, Frenkiel T, Gibson T, Pastore A: Towards a structural understanding of Friedreich’s ataxia: the solution structure of frataxin. Structure Fold Des 2000, 8:695-707. 34. Dhe-Paganon S, Shigeta R, Chi YI, Ristow M, Shoelson SE: Crystal structure of human frataxin. J Biol Chem 2000, 275:30753-30756. 35. Cho SJ, Lee MG, Yang JK, Lee JY, Song HK, Suh SW: Crystal structure of Escherichia coli CyaY protein reveals a previously unidentified fold for the evolutionarily conserved frataxin family. Proc Natl Acad Sci USA 2000, 97:8932-8937. 36. Schulz JB, Dehmer T, Schols L, Mende H, Hardt C, Vorgerd M, • Burk K, Matson W, Dichgans J, Beal MF et al.: Oxidative stress in patients with Friedreich ataxia. Neurology 2000, 55:1719-1721. See annotation [38•]. 37. •
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22. Puccio H, Simon D, Cossee M, Criqui-Filipe P, Tiziano F, Melki J, •• Hindelang C, Matyas R, Rustin P, Koenig M: Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits. Nat Genet 2001, 27:181-186. We have generated of conditional mouse models for FRDA that exhibit important pathophysiological and biochemical features of the human disease. The models demonstrate time-dependent intramitochondrial iron accumulation that does not represent the causative pathological mechanism. These mutant mice represent the first mammalian models to evaluate treatment strategies for the human disease.
38. Piemonte F, Pastore A, Tozzi G, Tagliacozzi D, Santorelli FM, • Carrozzo R, Casali C, Damiano M, Federici G, Bertini E: Glutathione in blood of patients with Friedreich’s ataxia. Eur J Clin Invest 2001, 31:1007-1011. These three reports [36•–38•] demonstrate increased oxidative stress in FRDA patients and provide the first relatively easy bioassays on readily available fluids to monitor disease status and treatment efficacy.
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deficiency and long term mitochondrial iron overload. This is the first report that implies that frataxin is directly involved in the response to oxidative stress. 41. Jiralerspong S, Ge B, Hudson TJ, Pandolfo M: Manganese • superoxide dismutase induction by iron is impaired in Friedreich ataxia cells. FEBS Lett 2001, 509:101-105. This paper also reports an impaired regulation of mitochondrial superoxide dismutase by exposing patient fibroblast to iron, which normally results in upregulation of MnSOD (see [40••]). 42. Cossee M, Puccio H, Gansmuller A, Koutnikova H, Dierich A, LeMeur M, Fischbeck K, Dolle P, Koenig M: Inactivation of the Friedreich ataxia mouse gene leads to early embryonic lethality without iron accumulation. Hum Mol Genet 2000, 9:1219-1226. 43. Miranda C, Santos M, Ohshima K, Smith J, Cossee M, Koenig M, Sequeiros J, Kaplan J, Pandolfo M: Frataxin knock-in mouse. FEBS Lett 2002, 512:291-297. 44. Pook MA, Al-Mahdawi S, Carroll CJ, Cossee M, Puccio H, Lawrence L, Clark P, Lowrie MB, Bradley JL, Cooper JM et al.: Rescue of the Friedreich’s ataxia knockout mouse by human YAC transgenesis. Neurogenetics 2001, 3:185-193. 45. Wilson RB, Lynch DR, Fischbeck KH: Normal serum iron and ferritin concentrations in patients with Friedreich’s ataxia. Ann Neurol 1998, 44:132-134.
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46. Rustin P, von Kleist-Retzow JC, Chantrel-Groussard K, Sidi D, Munnich A, Rotig A: Effect of idebenone on cardiomyopathy in Friedreich’s ataxia: a preliminary study. Lancet 1999, 354:477-479. 47.
Rustin P, Munnich A, Rotig A: Quinone analogs prevent enzymes targeted in Friedreich ataxia from iron-induced injury in vitro. Biofactors 1999, 9:247-251.
48. Rustin P, Rötig A, Munnich A, Sidi D: Heart hypertrophy and •• function are improved by idebenone in Friedreich’s ataxia. Free Radic Research 2002, in press. Idebenone treatment is associated with improvement of hypertrophic cardiomyopathy in patients with FRDA. This work has great potential clinical significance for treatment of the disease and provides realistic hope for the patients and their families. The neurological benefit remains to be established. 49. Lodi R, Hart PE, Rajagopalan B, Taylor DJ, Crilley JG, Bradley JL, • Blamire AM, Manners D, Styles P, Schapira AH et al.: Antioxidant treatment improves in vivo cardiac and skeletal muscle bioenergetics in patients with Friedreich’s ataxia. Ann Neurol 2001, 49:590-596. Coenzyme Q10 and vitamin E resulted in a bioenergetic increase in heart and muscle, but whether the FRDA patients will benefit clinically needs to be evaluated by appropriate trials.