Frataxin: its role in iron metabolism and the pathogenesis of Friedreich's ataxia

Friedreich's ataxia (FA) is a severe neurodegenerative condition with an incidence of 1:50000 in the European population. In 97% of patients this disease is due to an intronic GAA triplet repeat expansion in the FRDA gene resulting in a marked decrease in its expression. The protein encoded by this gene is known as frataxin which is found within the mitochondrion. Upon deletion of the homologous gene (YFH1) in the yeast, there was an accumulation of iron (Fe) within the mitochondrion. When the YFH1 gene was reintroduced back into the yeast cell Fe was exported out of the mitochondrion and into the cytosol. Evidence that human frataxin is also involved in mitochondrial Fe-overload comes from studies in FA patients that have shown an accumulation of Fe within the heart. While the precise role of human frataxin remains to be determined, the molecule appears to be involved indirectly in regulating the export and/or import of mitochondrial Fe. The finding of mitochondrial Fe-overload suggests that the use of specific Fe chelators which can permeate the mitochondrion may have potential in the treatment of this disease.     

Mitochondrial dysfunction in Friedreich's ataxia: from pathogenesis to treatment perspectives

Friedreich's ataxia (FRDA), the most common inherited ataxia, is an autosomal recessive degenerative disorder caused by a GAA triplet expansion or point mutations in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein, which is severely reduced in FRDA patients. The demonstration that deficit of frataxin in FRDA is associated with mitochondrial iron accumulation, increased sensitivity to oxidative stress, deficit of respiratory chain complex activities and in vivo impairment of cardiac and skeletal muscle tissue energy metabolism, has established FRDA as a "new" nuclear encoded mitochondrial disease. Pilot studies have shown the potential effect of antioxidant therapy based on idebenone or coenzyme Q 10 plus Vitamin E administration in this condition and provide a strong rationale for designing larger randomized clinical trials.     

Reduced expression of frataxin extends the lifespan of Caenorhabditis elegans

Defects in the expression of the mitochondrial protein frataxin cause Friedreich's ataxia, an hereditary neurodegenerative syndrome characterized by progressive ataxia and associated with reduced life expectancy in humans. Homozygous inactivation of the frataxin gene results in embryonic lethality in mice, suggesting that frataxin is required for organismic survival. Intriguingly, the inactivation of many mitochondrial genes in the nematode Caenorhabditis elegans by RNAi extends lifespan. We therefore investigated whether inactivation of frataxin by RNAi-mediated suppression of the frataxin homolog gene (frh-1) would also prolong lifespan in the nematode. Frataxin-deficient animals have a small body size, reduced fertility and altered responses to oxidative stress. Importantly, frataxin suppression by RNAi significantly extends lifespan in C. elegans.     

Mitochondrial Dysfunction in Friedreich's Ataxia: From Pathogenesis to Treatment Perspectives

Friedreich's ataxia (FRDA), the most common inherited ataxia, is an autosomal recessive degenerative disorder caused by a GAA triplet expansion or point mutations in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein, which is severely reduced in FRDA patients. The demonstration that deficit of frataxin in FRDA is associated with mitochondrial iron accumulation, increased sensitivity to oxidative stress, deficit of respiratory chain complex activities and in vivo impairment of cardiac and skeletal muscle tissue energy metabolism, has established FRDA as a "new" nuclear encoded mitochondrial disease. Pilot studies have shown the potential effect of antioxidant therapy based on idebenone or coenzyme Q 10 plus Vitamin E administration in this condition and provide a strong rationale for designing larger randomized clinical trials.     

Friedreich ataxia: a paradigm for mitochondrial diseases

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.     

Mitochondrial dysfunction in friedreich's ataxia

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disorder caused in the vast majority of cases by a GAA triplet expansion in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein which is severely reduced in FRDA patients. Loss of the homologue of frataxin in yeast is associated with mitochondrial iron overload, increased sensitivity to oxidative stress and profound deficit of oxidative phosphorylation. The demonstration that the human pathology of FRDA is also characterised by mitochondrial iron accumulation, deficit of respiratory chain complex activities and in vivo deficit of tissue energy metabolism establishes FRDA as a 'new' nuclear encoded mitochondrial disease.     

Frataxin knockin mouse

Friedreich ataxia is the consequence of frataxin deficiency, most often caused by a GAA repeat expansion in intron 1 of the corresponding gene. Frataxin is a mitochondrial protein involved in iron homeostasis. As an attempt to generate a mouse model of the disease, we introduced a (GAA)(230) repeat within the mouse frataxin gene by homologous recombination. GAA repeat knockin mice were crossed with frataxin knockout mice to obtain double heterozygous mice expressing 25-36% of wild-type frataxin levels. These mice were viable and did not develop anomalies of motor coordination, iron metabolism or response to iron loading. Repeats were meiotically and mitotically stable.     

Frataxin deficiency and mitochondrial dysfunction

Friedreich ataxia (FA) is an inherited recessive disorder characterized by progressive neurological disability and heart abnormalities. The Friedreich ataxia gene (FRDA) encodes a small mitochondrial protein, frataxin, which is produced in insufficient amounts in the disease as a consequence of a GAA triplet repeat expansion in the first intron of the gene. Frataxin deficiency leads to excessive free radical production, dysfunction of Fe-S center containing enzymes (in particular respiratory complexes I, II and III, and aconitase), and progressive iron accumulation in mitochondria. Frataxin may be a mitochondrial iron-binding protein that prevents this metal from participating in Fenton chemistry to generate toxic hydroxyl radicals. We investigated whether frataxin deficiency may in addition interfere with signaling pathways. First, we showed that exposure of FA fibroblasts to iron fails to produce the normally observed increase in expression of the stress defense protein manganese superoxide dismutase. This impaired induction involves a nuclear factor-kappaB-independent pathway that does not require free radical signaling intermediates. We also examined the role of frataxin in neuronal differentiation by using stably transfected clones of P19 embryonic carcinoma cells with antisense or sense frataxin constructs. We found that during retinoic acid-induced neurogenesis frataxin deficiency enhances apoptosis and reduces the number of terminally differentiated neuronal-like cells. The addition of the antioxidant N-acetyl-cysteine only rescues cells non-committed to the neuronal lineage, indicating that frataxin deficiency impairs differentiation mechanisms and survival responses through different mechanisms. Both studies suggest that some abnormalities in frataxin-deficient cells are related to free radical independent signaling pathways.     

Rapamycin reduces oxidative stress in frataxin-deficient yeast cells

Friedreich ataxia (FRDA) is a common form of ataxia caused by decreased expression of the mitochondrial protein frataxin. Oxidative damage of mitochondria is thought to play a key role in the pathogenesis of the disease. Therefore, a possible therapeutic strategy should be directed to an antioxidant protection against mitochondrial damage. Indeed, treatment of FRDA patients with the antioxidant idebenone has been shown to improve neurological functions. The yeast frataxin knock-out model of the disease shows mitochondrial iron accumulation, iron-sulfur cluster defects and high sensitivity to oxidative stress. By flow cytometry analysis we studied reactive oxygen species (ROS) production of yeast frataxin mutant cells treated with two antioxidants, N-acetyl-L-cysteine and a mitochondrially-targeted analog of vitamin E, confirming that mitochondria are the main site of ROS production in this model. Furthermore we found a significant reduction of ROS production and a decrease in the mitochondrial mass in mutant cells treated with rapamycin, an inhibitor of TOR kinases, most likely due to autophagy of damaged mitochondria.     

Lipophilic methylene blue analogues enhance mitochondrial function and increase frataxin levels in a cellular model of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder resulting from reduced expression of the protein frataxin (FXN). Although its function is not fully understood, frataxin appears to help assemble iron sulfur clusters; these are critical for the function of many proteins, including those needed for mitochondrial energy production. Finding ways to increase FXN levels has been a major therapeutic strategy for this disease. Previously, we described a novel series of methylene violet analogues and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Presently, a series of methylene blue analogues has been synthesized and characterized for their in vitro biochemical and biological properties in cultured Friedreich’s ataxia lymphocytes. Favorable methylene blue analogues were shown to increase frataxin levels and mitochondrial biogenesis, and to improve aconitase activity. The analogues were found to be good ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I and from glutathione depletion. The analogues also preserved mitochondrial membrane potential and augmented ATP production. Our results suggest that analogue 5, emerging from the initial structure of the parent compound methylene blue (MB), represents a promising lead structure and lacks the cytotoxicity associated with the parent compound MB.     

Blood cells from Friedreich ataxia patients harbor frataxin deficiency without a loss of mitochondrial function

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by GAA triplet expansions or point mutations in the FXN gene on chromosome 9q13. The gene product called frataxin, a mitochondrial protein that is severely reduced in FRDA patients, leads to mitochondrial iron accumulation, Fe-S cluster deficiency and oxidative damage. The tissue specificity of this mitochondrial disease is complex and poorly understood. While frataxin is ubiquitously expressed, the cellular phenotype is most severe in neurons and cardiomyocytes. Here, we conducted comprehensive proteomic, metabolic and functional studies to determine whether subclinical abnormalities exist in mitochondria of blood cells from FRDA patients. Frataxin protein levels were significantly decreased in platelets and peripheral blood mononuclear cells from FRDA patients. Furthermore, the most significant differences associated with frataxin deficiency in FRDA blood cell mitochondria were the decrease of two mitochondrial heat shock proteins. We did not observe profound changes in frataxin-targeted mitochondrial proteins or mitochondrial functions or an increase of apoptosis in peripheral blood cells, suggesting that functional defects in these mitochondria are not readily apparent under resting conditions in these cells.     

A pool of extramitochondrial frataxin that promotes cell survival

Frataxin is a mitochondrial protein involved in iron metabolism. Defective expression of frataxin causes Friedreich ataxia (FA), an inherited degenerative syndrome characterized by ataxia, cardiomyopathy, and high incidence of diabetes. Here we report that frataxin-deficient cells are more prone to undergo stress-induced mitochondrial damage and apoptosis, while the overexpression of frataxin confers protection to a variety of cell types. Moreover, we reveal the existence of an extramitochondrial pool of frataxin, which can efficiently prevent mitochondrial damage and apoptosis in different cellular systems. Remarkably, extramitochondrial frataxin can fully replace mitochondrial frataxin in promoting survival of FA cells.     

The First Cellular Models Based on Frataxin Missense Mutations That Reproduce Spontaneously the Defects Associated with Friedreich Ataxia

Background

Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)_n expansion within the first intron of the frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation.

Methodology

We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN^G130V and hFXN^I154F) frataxin. Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN^I154F mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients.

Conclusions

These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating frataxin deficiency.     

Towards a structural understanding of Friedreich's ataxia: the solution structure of frataxin

BACKGROUND: Lesions in the gene for frataxin, a nuclear-encoded mitochondrial protein, cause the recessively inherited condition Friedreich's ataxia. It is thought that the condition arises from disregulation of mitochondrial iron homeostasis, with concomitant oxidative damage leading to neuronal death. Very little is, as yet, known about the biochemical function of frataxin. RESULTS: Here, we show that the mature form of recombinant frataxin behaves in solution as a monodisperse species that is composed of a 15-residue-long unstructured N terminus and an evolutionarily conserved C-terminal region that is able to fold independently. The structure of the C-terminal domain consists of a stable seven-stranded antiparallel beta sheet packing against a pair of parallel helices. The structure is compact with neither grooves nor cavities, features that are typical of iron-binding modules. Exposed evolutionarily conserved residues cover a broad area and all cluster on the beta-sheet face of the structure, suggesting that this is a functionally important surface. The effect of two clinically occurring mutations on the fold was checked experimentally. When the mature protein was titrated with iron, no tendency to iron-binding or to aggregation was observed. CONCLUSIONS: Knowledge of the frataxin structure provides important guidelines as to the nature of the frataxin binding partner. The absence of all the features expected for an iron-binding activity, the large conserved area on its surface and lack of evidence for iron-binding activity strongly support an indirect involvement of frataxin in iron metabolism. The effects of point mutations associated with Friedreich's ataxia can be rationalised by knowledge of the structure and suggest possible models for the occurrence of the disease in compound heterozygous patients.     

Yeast and human frataxin are processed to mature form in two sequential steps by the mitochondrial processing peptidase.

Frataxin is a nuclear-encoded mitochondrial protein which is deficient in Friedreich's ataxia, a hereditary neurodegenerative disease. Yeast mutants lacking the yeast frataxin homologue (Yfh1p) show iron accumulation in mitochondria and increased sensitivity to oxidative stress, suggesting that frataxin plays a critical role in mitochondrial iron homeostasis and free radical toxicity. Both Yfh1p and frataxin are synthesized as larger precursor molecules that, upon import into mitochondria, are subject to two proteolytic cleavages, yielding an intermediate and a mature size form. A recent study found that recombinant rat mitochondrial processing peptidase (MPP) cleaves the mouse frataxin precursor to the intermediate but not the mature form (Koutnikova, H., Campuzano, V., and Koenig, M. (1998) Hum. Mol. Gen. 7, 1485-1489), suggesting that a different peptidase might be required for production of mature size frataxin. However, in the present study we show that MPP is solely responsible for maturation of yeast and human frataxin. MPP first cleaves the precursor to intermediate form and subsequently converts the intermediate to mature size protein. In this way, MPP could influence frataxin function and indirectly affect mitochondrial iron homeostasis.