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.     

Oxidative stress induces mitochondrial fragmentation in frataxin-deficient cells

Friedreich ataxia (FA) is the most common recessive neurodegenerative disease. It is caused by deficiency in mitochondrial frataxin, which participates in iron-sulfur cluster assembly. Yeast cells lacking frataxin (Δyfh1 mutant) showed an increased proportion of fragmented mitochondria compared to wild-type. In addition, oxidative stress induced complete fragmentation of mitochondria in Δyfh1 cells. Genetically controlled inhibition of mitochondrial fission in these cells led to increased resistance to oxidative stress. Here we present evidence that in yeast frataxin-deficiency interferes with mitochondrial dynamics, which might therefore be relevant for the pathophysiology of FA.     

Friedreich's ataxia, no changes in mitochondrial labile iron in human lymphoblasts and fibroblasts: a decrease in antioxidative capacity?

Friedreich's ataxia (FRDA) is caused by low expression of frataxin, a small mitochondrial protein. Studies with both yeast and mammals have suggested that decreased frataxin levels lead to elevated intramitochondrial concentrations of labile (chelatable) iron, and consequently to oxidative mitochondrial damage. Here, we used the mitochondrion-selective fluorescent iron indicator/chelator rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzylester (RPA) to determine the mitochondrial chelatable iron of FRDA patient lymphoblast and fibroblast cell lines, in comparison with age- and sex-matched control cells. No alteration in the concentration of mitochondrial chelatable iron could be observed in patient cells, despite strongly decreased frataxin levels. Uptake studies with (55)Fe-transferrin and iron loading with ferric ammonium citrate revealed no significant differences in transferrin receptor density and iron responsive protein/iron regulatory element binding activity between patients and controls. However, sensitivity to H(2)O(2) was significantly increased in patient cells, and H(2)O(2) toxicity could be completely inhibited by the ubiquitously distributing iron chelator 2,2'-dipyridyl, but not by the mitochondrion-selective chelator RPA. Our data strongly suggest that frataxin deficiency does not affect the mitochondrial labile iron pool or other parameters of cellular iron metabolism and suggest a decreased antioxidative defense against extramitochondrial iron-derived radicals in patient cells. These results challenge current concepts favoring the use of mitochondrion-specific iron chelators and antioxidants to treat FRDA.     

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.     

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.     

Frataxin Deficiency Promotes Excess Microglial DNA Damage and Inflammation that Is Rescued by PJ34

An inherited deficiency in the frataxin protein causes neurodegeneration of the dorsal root ganglia and Friedreich''s ataxia (FA). Frataxin deficiency leads to oxidative stress and inflammatory changes in cell and animal models; however, the cause of the inflammatory changes, and especially what causes brain microglial activation is unclear. Here we investigated: 1) the mechanism by which frataxin deficiency activates microglia, 2) whether a brain-localized inflammatory stimulus provokes a greater microglial response in FA animal models, and 3) whether an anti-inflammatory treatment improves their condition. Intracerebroventricular administration of LPS induced higher amounts of microglial activation in the FA mouse model vs controls. We also observed an increase in oxidative damage in the form of 8-oxoguanine (8-oxo-G) and the DNA repair proteins MUTYH and PARP-1 in cerebellar microglia of FA mutant mice. We hypothesized that frataxin deficiency increases DNA damage and DNA repair genes specifically in microglia, activating them. siRNA-mediated frataxin knockdown in microglial BV2 cells clearly elevated DNA damage and the expression of DNA repair genes MUTYH and PARP-1. Frataxin knockdown also induced a higher level of PARP-1 in MEF cells, and this was suppressed in MUTYH-/- knockout cells. Administration of the PARP-1 inhibitor PJ34 attenuated the microglial activation induced by intracerebroventricular injection of LPS. The combined administration of LPS and angiotensin II provoke an even stronger activation of microglia and neurobehavioral impairment. PJ34 treatment attenuated the neurobehavioral impairments in FA mice. These results suggest that the DNA repair proteins MUTYH and PARP-1 may form a pathway regulating microglial activation initiated by DNA damage, and inhibition of microglial PARP-1 induction could be an important therapeutic target in Friedreich''s ataxia.     

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.     

The chemical form of mitochondrial iron in Friedreich's ataxia

Friedreich's ataxia (FRDA) results from cellular damage caused by a deficiency in the mitochondrial matrix protein frataxin. To address the effect of frataxin deficiency on mitochondrial iron chemistry, the heavy mitochondrial fraction (HMF) was isolated from primary fibroblasts from FRDA affected and unaffected individuals. X-ray absorption spectroscopy was used to characterize the chemical form of iron. Near K-edge spectra were fitted with a series of model iron compounds to determine the proportion of each iron species. Most of the iron in both affected and unaffected fibroblasts was ferrihydrite. The iron K-edge from unaffected HMFs were best fitted with poorly organized ferrihydrite modeled by frataxin whereas HMFs from affected cells were best fitted with highly organized ferrihydrite modeled by ferritin. Both had several minor iron species but these did not differ consistently with disease. Since the iron K-edge spectra of ferritin and frataxin are very similar, we present additional evidence for the presence of ferritin-bound iron in HMF. The predominant ferritin subunit in HMFs from affected cells resembled mitochondrial ferritin (MtFt) in size and antigenicity. Western blotting of native gels showed that HMF from affected cells had 3-fold more holoferritin containing stainable iron. We conclude that most of the iron in fibroblast HMF from both affected and unaffected cells is ferrihydrite but only FRDA affected cells mineralize significant iron in mitochondrial ferritin.     

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.     

The role of the mitochondrion in cellular iron homeostasis

The yeast ATM1 protein is essential for normal mitochondrial iron homeostasis. Deletion of ATM1 results in mitochondrial iron accumulation and oxidative mitochondrial damage. Mutations in ABC7, the human homolog of ATM1, result in X-linked sideroblastic anemia and ataxia. Here we show that a deletion of ATM1 also has effects on extra-mitochondrial iron metabolism. ATM1-deficient cells have an increased iron requirement for growth. When grown in iron-rich medium, mutant cells accumulate excess mitochondrial iron and have increased expression of the genes required for both high and low affinity iron uptake. Thus, ATM1 mutant cells simultaneously demonstrate features of both iron overload and iron starvation. Yfh1p is the yeast homolog of the human frataxin protein, which is deficient in Friedreich's ataxia. As in atm1 cells, a yfh1 deletion results in both mitochondrial iron accumulation and cytosolic iron starvation. In spite of their apparent roles in cellular iron homeostasis, we find that the expression of neither ATM1 nor YFH1 is responsive to cellular iron status. Based on these observations, we propose a model in which cellular iron is prioritized for use by the mitochondrion, and available to the remainder of the cell only after mitochondrial needs have been fulfilled.     

Lymphoblast Oxidative Stress Genes as Potential Biomarkers of Disease Severity and Drug Effect in Friedreich's Ataxia

There is no current approved therapy for the ultimately lethal neuro- and cardio-degenerative disease Friedreich''s ataxia (FA). Finding minimally-invasive molecular biomarkers of disease progression and drug effect could support smaller, shorter clinical trials. Since we and others have noted a deficient oxidative stress response in FA, we investigated the expression of 84 genes involved in oxidative stress, signaling, and protection in control and FA lymphoblasts ranging from 460 to 1122 GAA repeats. Several antioxidant genes responded in a dose-dependent manner to frataxin expression at the mRNA and protein levels, which is inversely correlated with disease progression and severity. We tested the effect of experimental Friedreich’s ataxia therapies dimethyl fumarate (DMF) and type 1 histone deacetylase inhibitor (HDACi) on biomarker mRNA expression. We observed that exposure of lymphoblasts to DMF and HDACi dose-dependently unsilenced frataxin expression and restored the potential biomarkers NCF2 and PDLIM1 expression to control levels. We suggest that in addition to frataxin expression, blood lymphoblast levels of NCF2 and PDLIM1 could be useful biomarkers for disease progression and drug effect in future clinical trials of Friedreich’s ataxia.     

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.     

Assembly of the iron-binding protein frataxin in Saccharomyces cerevisiae responds to dynamic changes in mitochondrial iron influx and stress level

Defects in frataxin result in Friedreich ataxia, a genetic disease characterized by early onset of neurodegeneration, cardiomyopathy, and diabetes. Frataxin is a conserved mitochondrial protein that controls iron needed for iron-sulfur cluster assembly and heme synthesis and also detoxifies excess iron. Studies in vitro have shown that either monomeric or oligomeric frataxin delivers iron to other proteins, whereas ferritin-like frataxin particles convert redox-active iron to an inert mineral. We have investigated how these different forms of frataxin are regulated in vivo. In Saccharomyces cerevisiae, only monomeric yeast frataxin (Yfh1) was detected in unstressed cells when mitochondrial iron uptake was maintained at a steady, low nanomolar level. Increments in mitochondrial iron uptake induced stepwise assembly of Yfh1 species ranging from trimer to > or = 24-mer, independent of interactions between Yfh1 and its major iron-binding partners, Isu1/Nfs1 or aconitase. The rate-limiting step in Yfh1 assembly was a structural transition that preceded conversion of monomer to trimer. This step was induced, independently or synergistically, by mitochondrial iron increments, overexpression of wild type Yfh1 monomer, mutations that stabilize Yfh1 trimer, or heat stress. Faster assembly kinetics correlated with reduced oxidative damage and higher levels of aconitase activity, respiratory capacity, and cell survival. However, deregulation of Yfh1 assembly resulted in Yfh1 aggregation, aconitase sequestration, and mitochondrial DNA depletion. The data suggest that Yfh1 assembly responds to dynamic changes in mitochondrial iron uptake or stress exposure in a highly controlled fashion and that this may enable frataxin to simultaneously promote respiratory function and stress tolerance.     

Yeast frataxin mutants display decreased superoxide dismutase activity crucial to promote protein oxidative damage

Iron overload is involved in several pathological conditions, including Friedreich ataxia, a disease caused by decreased expression of the mitochondrial protein frataxin. In a previous study, we identified 14 proteins selectively oxidized in yeast cells lacking Yfh1, the yeast frataxin homolog. Most of these were magnesium-binding proteins. Decreased Mn-SOD activity, oxidative damage to CuZn-SOD, and increased levels of chelatable iron were also observed in this model. This study explores the relationship between low SOD activity, the presence of chelatable iron, and protein damage. We observed that addition of copper and manganese to the culture medium restored SOD activity and prevented both oxidative damage and inactivation of magnesium-binding proteins. This protection was compartment specific: recovery of mitochondrial enzymes required the addition of manganese, whereas cytosolic enzymes were recovered by adding copper. Copper treatment also decreased Δyfh1 sensitivity to menadione. Finally, a Δsod1 mutant showed high levels of chelatable iron and inactivation of magnesium-binding enzymes. These results suggest that reduced superoxide dismutase activity contributes to the toxic effects of iron overloading. This would also apply to pathologies involving iron accumulation.