Human frataxin maintains mitochondrial iron homeostasis in Saccharomyces cerevisiae

Frataxin is a nuclear-encoded mitochondrial protein widely conserved among eukaryotes. Human frataxin (fxn) is severely reduced in Friedreich ataxia (FRDA), a frequent autosomal recessive neuro- and cardio-degenerative disease. Whereas the function of fxn is unknown, the yeast frataxin homolog (Yfh1p) has been shown to be involved in mitochondrial iron homeostasis and protection from free radical toxicity. Evidence of iron accumulation and oxidative damage in cardiac tissue from FRDA patients suggests that fxn may have a similar function, but whether yeast and human frataxin actually have interchangeable roles in mitochondrial iron homeostasis is unknown. We show that a wild-type FRDA cDNA can complement Yfh1p-deficient yeast (yfh1 delta) by preventing the mitochondrial iron accumulation and oxidative damage associated with loss of Yfh1p. We analyze the functional effects of two FRDA point mutations, G130V and W173G, associated with a mild and a severe clinical presentation, respectively. The G130V mutation affects protein stability and results in low levels of mature (m) fxn, which are nevertheless sufficient to rescue yfh1 delta yeast. The W173G mutation affects protein processing and stability and results in severe m-fxn deficiency. Expression of the FRDA (W173G) cDNA in yfh1 delta yeast leads to increased levels of mitochondrial iron which are not as elevated as in Yfh1p-deficient cells but are above the threshold for oxidative damage of mitochondrial DNA and iron-sulfur centers, causing a typical yfh1 delta phenotype. These results demonstrate that fxn functions like Yfh1p, providing experimental support to the hypothesis that FRDA is a disorder of mitochondrial iron homeostasis.     

Mitochondrial intermediate peptidase and the yeast frataxin homolog together maintain mitochondrial iron homeostasis in Saccharomyces cerevisiae.

Friedreich's ataxia (FRDA) is a neurodegenerative disease typically caused by a deficiency of frataxin, a mitochondrial protein of unknown function. In Saccharomyces cerevisiae, lack of the yeast frataxin homolog ( YFH1 gene, Yfh1p polypeptide) results in mitochondrial iron accumulation, suggesting that frataxin is required for mitochondrial iron homeostasis and that FRDA results from oxidative damage secondary to mitochondrial iron overload. This hypothesis implies that the effects of frataxin deficiency could be influenced by other proteins involved in mitochondrial iron usage. We show that Yfh1p interacts functionally with yeast mitochondrial intermediate peptidase ( OCT1 gene, YMIP polypeptide), a metalloprotease required for maturation of ferrochelatase and other iron-utilizing proteins. YMIP is activated by ferrous iron in vitro and loss of YMIP activity leads to mitochondrial iron depletion, suggesting that YMIP is part of a feedback loop in which iron stimulates maturation of YMIP substrates and this in turn promotes mitochondrial iron uptake. Accordingly, YMIP is active and promotes mitochondrial iron accumulation in a mutant lacking Yfh1p ( yfh1 [Delta]), while genetic inactivation of YMIP in this mutant ( yfh1 [Delta] oct1 [Delta]) leads to a 2-fold reduction in mitochondrial iron levels. Moreover, overexpression of Yfh1p restores mitochondrial iron homeostasis and YMIP activity in a conditional oct1 ts mutant, but does not affect iron levels in a mutant completely lacking YMIP ( oct1 [Delta]). Thus, we propose that Yfh1p maintains mitochondrial iron homeostasis both directly, by promoting iron export, and indirectly, by regulating iron levels and therefore YMIP activity, which promotes mitochondrial iron uptake. This suggests that human MIP may contribute to the functional effects of frataxin deficiency and the clinical manifestations of FRDA.     

Mitochondrial frataxin interacts with ISD11 of the NFS1/ISCU complex and multiple mitochondrial chaperones

The neurodegenerative disorder Friedreich's ataxia (FRDA) is caused by mutations in frataxin, a mitochondrial protein whose function remains controversial. Using co-immunoprecipitation and mass spectrometry we identified multiple interactors of mitochondrial frataxin in mammalian cells. One interactor was mortalin/GRP75, a homolog of the yeast ssq1 chaperone that integrates iron-sulfur clusters into imported mitochondrial proteins. Another interactor was ISD11, recently identified as a component of the eukaryotic complex Nfs1/ISCU, an essential component of iron-sulfur cluster biogenesis. Interactions between frataxin and ISD11, and frataxin and GRP75 were confirmed by co-immunoprecipitation experiments in both directions. Immunofluorescence analysis demonstrated that ISD11 co-localized with both frataxin and with mitochondria. The point mutations I154F and W155R in frataxin cause FRDA and are clustered to one surface of the protein, and these mutations decrease the interaction of frataxin with ISD11. The frataxin/ISD11 interaction was also decreased by the chelator EDTA, and was increased by supplementation with nickel but not other metal ions. Nickel supplementation rescued the defective interaction of mutant frataxin I154F and W155R with ISD11. Upon ISD11 depletion by siRNA in HEK293T cells, the amount of the Nfs1/ISCU protein complex declined, as did the activity of the iron-sulfur cluster enzyme aconitase, while the cellular iron content was increased, as seen in tissues from FRDA patients. Furthermore, ISD11 mRNA levels were decreased in FRDA patient cells. These data suggest that frataxin binds the iron-sulfur biogenesis Nfs1/ISCU complex through ISD11, that the interaction is nickel-dependent, and that multiple consequences of frataxin deficiency are duplicated by ISD11 deficiency.     

Iron-sulfur protein maturation in human cells: evidence for a function of frataxin

The maturation of iron-sulfur (Fe/S) proteins in eukaryotes has been intensively studied in yeast. Hardly anything is known so far about the process in higher eukaryotes, even though the high conservation of the yeast maturation components in most Eukarya suggests similar mechanisms. Here, we developed a cell culture model in which the RNA interference (RNAi) technology was used to deplete a potential component of Fe/S protein maturation, frataxin, in human HeLa cells. This protein is lowered in humans with the neuromuscular disorder Friedreich's ataxia (FRDA). Upon frataxin depletion by RNAi, the enzyme activities of the mitochondrial Fe/S proteins, aconitase and succinate dehydrogenase, were decreased, while the activities of non-Fe/S proteins remained constant. Moreover, Fe/S cluster association with the cytosolic iron-regulatory protein 1 was diminished. In contrast, no alterations in cellular iron uptake, iron content and heme formation were found, and no mitochondrial iron deposits were observed upon frataxin depletion. Hence, iron accumulation in FRDA mitochondria appears to be a late consequence of frataxin deficiency. These results demonstrate (i) that frataxin is a component of the human Fe/S cluster assembly machinery and (ii) that it plays a role in the maturation of both mitochondrial and cytosolic Fe/S proteins.     

Friedreich ataxia: the oxidative stress paradox

Friedreich ataxia (FRDA) results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur protein activity initially ascribed to mitochondrial iron overload. Recent in vitro data suggest that frataxin is necessary for iron incorporation in Fe-S cluster (ISC) and heme biosynthesis. In addition, several reports suggest that continuous oxidative damage resulting from hampered superoxide dismutases (SODs) signaling participates in the mitochondrial deficiency and ultimately the neuronal and cardiac cell death. This has led to the use of antioxidants such as idebenone for FRDA therapy. To further discern the role of oxidative stress in FRDA pathophysiology, we have tested the potential effect of increased antioxidant defense using an MnSOD mimetic (MnTBAP) and Cu,ZnSOD overexpression on the murine FRDA cardiomyopathy. Surprisingly, no positive effect was observed, suggesting that increased superoxide production could not explain by itself the FRDA cardiac pathophysiology. Moreover, we demonstrate that complete frataxin-deficiency neither induces oxidative stress in neuronal tissues nor alters the MnSOD expression and induction in the early step of the pathology (neuronal and cardiac) as previously suggested. We show that cytosolic ISC aconitase activity of iron regulatory protein-1 progressively decreases, whereas its apo-RNA binding form increases despite the absence of oxidative stress, suggesting that in a mammalian system the mitochondrial ISC assembly machinery is essential for cytosolic ISC biogenesis. In conclusion, our data demonstrate that in FRDA, mitochondrial iron accumulation does not induce oxidative stress and we propose that, contrary to the general assumption, FRDA is a neurodegenerative disease not associated with oxidative damage.     

Disabled early recruitment of antioxidant defenses in Friedreich's ataxia

Friedreich's ataxia (FRDA) results from a generalized deficiency of mitochondrial iron-sulfur protein activity ascribed to mitochondrial iron overload. However, iron overload appears to be a late event in the disease. Here we show that neither superoxide dismutases nor the import iron machinery was induced by an endogenous oxidative stress in FRDA patients' fibroblasts in contrast to control cells. Superoxide dismutase activity was not induced in the heart of conditional frataxin-KO mice either. This suggests that continuous oxidative damage to iron-sulfur clusters, resulting from hampered superoxide dismutase signaling, is causative of the mitochondrial deficiency and long term mitochondrial iron overload occurring in FRDA.     

The expression of human mitochondrial ferritin rescues respiratory function in frataxin-deficient yeast

Mitochondrial ferritin (MtF) is structurally and functionally similar to the cytosolic ferritins, molecules designed to store and detoxify cellular iron. MtF expression in human and mouse is restricted to the testis and few tissues, and it is abundant in the erythroblasts of patients with sideroblastic anemia, where it is thought to protect the mitochondria from the damage caused by iron loading. Mitochondria iron overload occurs also in cells deficient in frataxin, a mitochondrial protein involved in iron handling and implicated in Friedreich ataxia. We expressed human MtF in frataxin-deficient yeast cells, a well-characterized model of mitochondrial iron overload and oxidative damage. The human MtF precursor was efficiently imported by yeast mitochondria and processed to functional ferritin that actively sequestered iron in the organelle. MtF expression rescued the respiratory deficiency caused by the loss of frataxin protecting the activity of iron-sulfur enzymes and enabling frataxin-deficient cells to grow on non-fermentable carbon sources. Furthermore, MtF expression prevented the development of mitochondrial iron overload, preserved mitochondrial DNA integrity and increased cell resistance to H2O2. The data show that MtF can substitute for most frataxin functions in yeast, suggesting that frataxin is directly involved in mitochondrial iron-binding and detoxification.     

A structural approach to understanding the iron-binding properties of phylogenetically different frataxins

Friedreich's ataxia (FRDA), an autosomal recessive cardio- and neurodegenerative disease, is caused by low expression of frataxin, a small mitochondrial protein, encoded in the nucleus. At the biochemical level, the lack of frataxin leads to dysregulation of mitochondrial iron homeostasis and oxidative damage, which eventually causes neuronal death. It is, however, still unclear whether frataxin is directly involved in iron binding, since the yeast orthologue, but not the human protein, has been shown to form large aggregates in the presence of large iron excess. We have compared the properties of three proteins from the frataxin family--the bacterial CyaY from Escherichia coli, the yeast Yfh1 and human frataxin--as representative of organisms of increasing complexity. We show that the three proteins have the same fold but different thermal stabilities and iron-binding properties. While human frataxin has no tendency to bind iron, CyaY forms iron-promoted aggregates with a behaviour similar to that of yeast frataxin. However, aggregation can be competed by chelator agents or by ionic strength. At physiological salt conditions, almost no aggregation is observed. The design of mutants produced to identify the protein surface involved in iron-promoted aggregation allows us to demonstrate that the process is mediated by a negatively charged surface ridge. Mutation of three of these residues is sufficient to convert CyaY in a protein with properties similar to those of human frataxin. On the other hand, mutation of the exposed surface of the beta sheet, which contains most of the conserved residues, does not affect aggregation, suggesting that iron binding is a non-conserved part of a more complex cellular function of frataxins.     

Maturation of wild-type and mutated frataxin by the mitochondrial processing peptidase

Frataxin is a mitochondrial protein deficient in Friedreich ataxia (FRDA) and which is associated with abnormal intramitochondrial iron handling. We identified the mitochondrial processing peptidase beta (MPPbeta) as a frataxin protein partner using the yeast two-hybrid assay. In in vitro assays, MPPbeta binds frataxin which is cleaved by the reconstituted MPP heterodimer. MPP cleavage of frataxin results in an intermediate form (amino acids 41-210) that is processed further to the mature form. In vitro and in vivo experiments suggest that two C- terminal missense mutations found in FRDA patients modulate interaction with MPPbeta, resulting in a slower maturation process at the normal cleavage site. The slower processing rate of frataxin carrying such missense mutations may therefore contribute to frataxin deficiency, in addition to an impairment of its function.      

Reduction in frataxin causes progressive accumulation of mitochondrial damage

Frataxin protein controls iron availability in mitochondria and reduced levels lead to the human disease, Friedreich's ataxia (FRDA). The molecular aspects of disease progression are not well understood. We developed a highly regulatable promoter system for expressing frataxin in yeast to address the consequences of chronically reduced amounts of this protein. Shutting off the promoter resulted in changes normally associated with loss of frataxin including iron accumulation within the mitochondria and the induction of mitochondrial petite mutants. While there was considerable oxidative damage to mitochondrial proteins, the petites were likely due to accumulation of mitochondrial DNA lesions and subsequent DNA loss. Chronically reduced frataxin levels resulted in similar response patterns. Furthermore, nuclear DNA damage was detected in a rad52 mutant, deficient in double-strand break repair. We conclude that reduced frataxin levels, which is more representative of the disease state, results in considerable oxidative damage in both mitochondrial and nuclear DNA.     

The Friedreich's ataxia mutation confers cellular sensitivity to oxidant stress which is rescued by chelators of iron and calcium and inhibitors of apoptosis

Expansions of an intronic GAA repeat reduce the expression of frataxin and cause Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disease. Frataxin is a mitochondrial protein, and disruption of a frataxin homolog in yeast results in increased sensitivity to oxidant stress, increased mitochondrial iron and respiration deficiency. These previous data support the hypothesis that FRDA is a disease of mitochondrial oxidative stress, a hypothesis we have tested in cultured cells from FRDA patients. FRDA fibroblasts were hypersensitive to iron stress and significantly more sensitive to hydrogen peroxide than controls. The iron chelator deferoxamine rescued FRDA fibroblasts more than controls from oxidant-induced death, consistent with a role for iron in the differential kinetics of death; however, mean mitochondrial iron content in FRDA fibroblasts was increased by only 40%. Treatment of cells with the intracellular Ca2+chelator BAPTA-AM rescued both FRDA fibroblasts and controls from oxidant-induced death. Treatment with apoptosis inhibitors rescued FRDA but not control fibroblasts from oxidant stress, and staurosporine- induced caspase 3 activity was higher in FRDA fibroblasts, consistent with the possibility that an apoptotic step upstream of caspase 3 is activated in FRDA fibroblasts. These results demonstrate that FRDA fibroblasts are sensitive to oxidant stress, and may be a useful model in which to elucidate the FRDA mechanism and therapeutic strategies.      

Clinical, biochemical and molecular genetic correlations in Friedreich's ataxia

Friedreich's ataxia (FRDA) is an autosomal recessive disorder with a frequency of 1 in 50 000 live births. In 97% of patients it is caused by the abnormal expansion of a GAA repeat in intron 1 of the FRDA gene on chromosome 9, which encodes a 210 amino acid protein called frataxin. Frataxin is widely expressed and has been localized to mitochondria although its function is unknown. We have investigated mitochondrial function, mitochondrial DNA levels, aconitase activity and iron content in tissues from FRDA patients. There were significant reductions in the activities of complex I, complex II/III and aconitase in FRDA heart. Respiratory chain and aconitase activities were decreased although not significantly in skeletal muscle, but were normal in FRDA cerebellum and dorsal root ganglia, although there was a mild decrease in aconitase activity in the latter. Mitochondrial DNA levels were reduced in FRDA heart and skeletal muscle, although in skeletal muscle this was paralleled by a decline in citrate synthase activity. Increased iron deposition was seen in FRDA heart, liver and spleen in a pattern consistent with a mitochondrial location. The iron accumulation, mitochondrial respiratory chain and aconitase dysfunction and mitochondrial DNA depletion in FRDA heart samples largely paralleled those in the yeast YFH1 knockout model, suggesting that frataxin may be involved in mitochondrial iron regulation or iron sulphur centre synthesis. However, the severe deficiency in aconitase activity also suggests that oxidant stress may induce a self-amplifying cycle of oxidative damage and mitochondrial dysfunction, which may contribute to cellular toxicity.     

Inactivation of the Friedreich ataxia mouse gene leads to early embryonic lethality without iron accumulation

Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is caused in almost all cases by homozygous intronic expansions resulting in the loss of frataxin, a mitochondrial protein conserved through evolution, and involved in mitochondrial iron homeostasis. Yeast knockout models, and histological and biochemical data from patient heart biopsies or autopsies indicate that the frataxin defect causes a specific iron-sulfur protein deficiency and mitochondrial iron accumulation leading to the pathological changes. Affected human tissues are rarely available to further examine this hypothesis. To study the mechanism of the disease, we generated a mouse model by deletion of exon 4 leading to inactivation of the Frda gene product. We show that homozygous deletions cause 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 due to residual frataxin expression associated with the expansion mutations. Surprisingly, in the frataxin knockout mouse, no iron accumulation was observed during embryonic resorption, suggesting that cell death could be due to a mechanism independent of iron accumulation.     

The in vivo mitochondrial two-step maturation of human frataxin

Deficiency in the nuclear-encoded mitochondrial protein frataxin causes Friedreich ataxia (FRDA), a progressive neurodegenerative disorder associating spinocerebellar ataxia and cardiomyopathy. Although the exact function of frataxin is still a matter of debate, it is widely accepted that frataxin is a mitochondrial iron chaperone involved in iron-sulfur cluster and heme biosynthesis. Frataxin is synthesized as a precursor polypeptide, directed to the mitochondrial matrix where it is proteolytically cleaved by the mitochondrial processing peptidase to the mature form via a processing intermediate. The mature form was initially reported to be encoded by amino acids 56-210 (m(56)-FXN). However, two independent reports have challenged these studies describing two different forms encoded by amino acids 78-210 (m(78)-FXN) and 81-210 (m(81)-FXN). Here, we provide evidence that mature human frataxin corresponds to m(81)-FXN, and can rescue the lethal phenotype of fibroblasts completely deleted for frataxin. Furthermore, our data demonstrate that the migration profile of frataxin depends on the experimental conditions, a behavior which most likely contributed to the confusion concerning the endogenous mature frataxin. Interestingly, we show that m(56)-FXN and m(78)-FXN can be generated when the normal maturation process of frataxin is impaired, although the physiological relevance is not clear. Furthermore, we determine that the d-FXN form, previously reported to be a degradation product, corresponds to m(78)-FXN. Finally, we demonstrate that all frataxin isoforms are generated and localized within the mitochondria. The clear identification of the N-terminus of mature FXN is an important step for designing therapeutic approaches for FRDA based on frataxin replacement.     

Friedreich's ataxia: from disease mechanisms to therapeutic interventions

Friedreich's ataxia (FRDA) is the most common inherited ataxia. FRDA 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 that is severely reduced in FRDApatients. The function of frataxin has not been established yet. Studies of the yeast and animal model of the disease as well as of tissues from FRDA patients have demonstrated that deficit of frataxin is associated with mitochondrial iron accumulation, increased sensitivity to oxidative stress, deficit of respiratory chain complex activities and in vivo impairment of tissue energy metabolism. Pilot studies have shown the potential effect of antioxidant therapy in this condition and provide a strong rationale for designing larger clinical randomized trials.     

Frataxin expression rescues mitochondrial dysfunctions in FRDA cells

Friedreich's ataxia (FRDA) is the result of mutations in the nuclear-encoded frataxin gene, which is expressed in mitochondria. Several lines of evidence have suggested that frataxin is involved in mitochondrial iron homeostasis. We have transfected the frataxin gene into lymphoblasts of FRDA compound heterozygotes (FRDA-CH) with deficient frataxin expression to produce FRDA-CH-t cells in which message and protein are rescued to near-physiological levels. FRDA-CH cells were more sensitive to oxidative stress by challenge with free iron, hydrogen peroxide and the combination, consistent with a Fenton chemical mechanism of pathophysiology, and this sensitivity was rescued to control levels in FRDA-CH-t cells. Iron challenge caused increased mitochondrial iron levels in FRDA-CH cells, and a decreased mitochondrial membrane potential (MMP), both of which were rescued in FRDA-CH-t cells. The rescue of the low MMP, and high mitochondrial iron concentration by frataxin overexpression suggests that these cellular phenotypes are relevant to the central pathophysiological process in FRDA which is aggravated by exposure to free iron. However, even at physiological iron concentrations, FRDA-CH cells had decreased MMP as well as lower activities of aconitase and ICDH (two enzymes supporting MMP), and twice the level of filtrable mitochondrial iron (but no increase in total mitochondrial iron), and the observed phenotypes were either fully or partially rescued in FRDA-CH-t cells. Free iron is known to be toxic. The observation that frataxin deficiency (either directly or indirectly) causes an increase in filtrable mitochondrial iron provides a new hypothesis for the mechanism of cell death in this disease, and could be a target for therapy.     

Recent advances in the molecular pathogenesis of Friedreich ataxia

Friedreich ataxia, the most frequent cause of recessive ataxia, is due in most cases to a homozygous intronic expansion resulting in the loss of function of frataxin. Frataxin is a mitochondrial protein conserved through evolution. Yeast knock-out models and histological data from patient heart autopsies have shown that frataxin defect causes mitochondrial iron accumulation. Biochemical data from patient heart biopsies or autopsies have revealed a specific deficiency in the activities of aconitases and of mitochondrial iron-sulfur proteins. These results suggest that frataxin may play a role either in mitochondrial iron transport or in iron-sulfur cluster assembly or transport. Iron abnormalities suggest a pathogenic mechanism involving free radical production and oxidative stress, a process that might be sensitive to antioxidant therapies.     

Mitochondrial iron detoxification is a primary function of frataxin that limits oxidative damage and preserves cell longevity

Friedreich ataxia is a severe autosomal-recessive disease characterized by neurodegeneration, cardiomyopathy and diabetes, resulting from reduced synthesis of the mitochondrial protein frataxin. Although frataxin is ubiquitously expressed, frataxin deficiency leads to a selective loss of dorsal root ganglia neurons, cardiomyocytes and pancreatic beta cells. How frataxin normally promotes survival of these particular cells is the subject of intense debate. The predominant view is that frataxin sustains mitochondrial energy production and other cellular functions by providing iron for heme synthesis and iron-sulfur cluster (ISC) assembly and repair. We have proposed that frataxin not only promotes the biogenesis of iron-containing enzymes, but also detoxifies surplus iron thereby affording a critical anti-oxidant mechanism. These two functions have been difficult to tease apart, however, and the physiologic role of iron detoxification by frataxin has not yet been demonstrated in vivo. Here, we describe mutations that specifically impair the ferroxidation or mineralization activity of yeast frataxin, which are necessary for iron detoxification but do not affect the iron chaperone function of the protein. These mutations increase the sensitivity of yeast cells to oxidative stress, shortening chronological life span and precluding survival in the absence of the anti-oxidant enzyme superoxide dismutase. Thus, the role of frataxin is not limited to promoting ISC assembly or heme synthesis. Iron detoxification is another function of frataxin relevant to anti-oxidant defense and cell longevity that could play a critical role in the metabolically demanding environment of non-dividing neuronal, cardiac and pancreatic beta cells.