Changes in mitochondrial glutathione levels and protein thiol oxidation in ∆yfh1 yeast cells and the lymphoblasts of patients with Friedreich's ataxia

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by low levels of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulfur cluster defects and high sensitivity to oxidative stress. Frataxin deficiency is also associated with severe impairment of glutathione homeostasis and changes in glutathione-dependent antioxidant defenses. The potential biological consequences of oxidative stress and changes in glutathione levels associated with frataxin deficiency include the oxidation of susceptible protein thiols and reversible binding of glutathione to the SH of proteins by S-glutathionylation. In this study, we isolated mitochondria from frataxin-deficient ?yfh1 yeast cells and lymphoblasts of FRDA patients, and show evidence for a severe mitochondrial glutathione-dependent oxidative stress, with a low GSH/GSSG ratio, and thiol modifications of key mitochondrial enzymes. Both yeast and human frataxin-deficient cells had abnormally high levels of mitochondrial proteins binding an anti-glutathione antibody. Moreover, proteomics and immunodetection experiments provided evidence of thiol oxidation in α-ketoglutarate dehydrogenase (KGDH) or subunits of respiratory chain complexes III and IV. We also found dramatic changes in GSH/GSSG ratio and thiol modifications on aconitase and KGDH in the lymphoblasts of FRDA patients. Our data for yeast cells also confirm the existence of a signaling and/or regulatory process involving both iron and glutathione.     

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.     

Changes in mitochondrial glutathione levels and protein thiol oxidation in ?yfh1 yeast cells and the lymphoblasts of patients with Friedreich's ataxia

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by low levels of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulfur cluster defects and high sensitivity to oxidative stress. Frataxin deficiency is also associated with severe impairment of glutathione homeostasis and changes in glutathione-dependent antioxidant defenses. The potential biological consequences of oxidative stress and changes in glutathione levels associated with frataxin deficiency include the oxidation of susceptible protein thiols and reversible binding of glutathione to the SH of proteins by S-glutathionylation. In this study, we isolated mitochondria from frataxin-deficient ?yfh1 yeast cells and lymphoblasts of FRDA patients, and show evidence for a severe mitochondrial glutathione-dependent oxidative stress, with a low GSH/GSSG ratio, and thiol modifications of key mitochondrial enzymes. Both yeast and human frataxin-deficient cells had abnormally high levels of mitochondrial proteins binding an anti-glutathione antibody. Moreover, proteomics and immunodetection experiments provided evidence of thiol oxidation in α-ketoglutarate dehydrogenase (KGDH) or subunits of respiratory chain complexes III and IV. We also found dramatic changes in GSH/GSSG ratio and thiol modifications on aconitase and KGDH in the lymphoblasts of FRDA patients. Our data for yeast cells also confirm the existence of a signaling and/or regulatory process involving both iron and glutathione.     

Deferiprone and idebenone rescue frataxin depletion phenotypes in a Drosophila model of Friedreich's ataxia

Friedreich's ataxia (FRDA), the most common inherited ataxia, is a neurodegenerative disease caused by a reduction in the levels of the mitochondrial protein frataxin, the function of which remains a controversial matter. Several therapeutic approaches are being developed to increase frataxin expression and reduce the intramitochondrial iron aggregates and oxidative damage found in this disease. In this study, we tested separately the response of a Drosophila RNAi model of FRDA ( Llorens et al., 2007) to treatment with the iron chelator deferiprone (DFP) and the antioxidant idebenone (IDE), which are both in clinical trials. The FRDA flies have a shortened life span and impaired motor coordination, and these phenotypes are more pronounced in oxidative stress conditions. In addition, under hyperoxia, the activity of the mitochondrial enzyme aconitase is strongly reduced in the FRDA flies. This study reports that DFP and IDE improve the life span and motor ability of frataxin-depleted flies. We show that DFP eliminates the excess of labile iron in the mitochondria and thus prevents the toxicity induced by iron accumulation. IDE treatment rescues aconitase activity in hyperoxic conditions. These results validate the use of our Drosophila model of FRDA to screen for therapeutic molecules to treat this disease.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Primary and secondary drug screening assays for Friedreich ataxia

Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardiodegenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix for the assembly of iron-sulfur clusters (ISCs), which are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain (ETC). Decreased expression of frataxin or the yeast frataxin orthologue, Yfh1p, is associated with decreased ISC assembly, mitochondrial iron accumulation, and increased oxidative stress, all of which contribute to mitochondrial dysfunction. Using yeast depleted of Yfh1p, a high-throughput screening (HTS) assay was developed in which mitochondrial function was monitored by reduction of the tetrazolium dye WST-1 in a growth medium with a respiration-only carbon source. Of 101 200 compounds screened, 302 were identified that effectively rescue mitochondrial function. To confirm activities in mammalian cells and begin understanding mechanisms of action, secondary screening assays were developed using murine C2C12 cells and yeast mutants lacking specific complexes of the ETC, respectively. The compounds identified in this study have potential relevance for other neurodegenerative disorders associated with mitochondrial dysfunction, such as Parkinson disease.     

Frataxin deficiency in neonatal rat ventricular myocytes targets mitochondria and lipid metabolism

Friedreich ataxia (FRDA) is a hereditary disease caused by deficient frataxin expression. This mitochondrial protein has been related to iron homeostasis, energy metabolism, and oxidative stress. Patients with FRDA experience neurologic alterations and cardiomyopathy, which is the leading cause of death. The specific effects of frataxin depletion on cardiomyocytes are poorly understood because no appropriate cardiac cellular model is available to researchers. To address this research need, we present a model based on primary cultures of neonatal rat ventricular myocytes (NRVMs) and short-hairpin RNA interference. Using this approach, frataxin was reduced down to 5 to 30% of control protein levels after 7 days of transduction. At this stage the activity and amount of the iron–sulfur protein aconitase, in vitro activities of several OXPHOS components, levels of iron-regulated mRNAs, and the ATP/ADP ratio were comparable to controls. However, NRVMs exhibited markers of oxidative stress and a disorganized mitochondrial network with enlarged mitochondria. Lipids, the main energy source of heart cells, also underwent a clear metabolic change, indicated by the increased presence of lipid droplets and induction of medium-chain acyl-CoA dehydrogenase. These results indicate that mitochondria and lipid metabolism are primary targets of frataxin deficiency in NRVMs. Therefore, they contribute to the understanding of cardiac-specific mechanisms occurring in FRDA and give clues for the design of cardiac-specific treatment strategies for FRDA.     

Role of Oxidative Damage in Friedreich's Ataxia

Plasma malondialdehyde (MDA) levels were raised in Friedreich's ataxia (FRDA) patients. These levels correlated with increasing age and disease duration, suggesting lipid peroxidation increased with disease progression. Using fibroblasts from FRDA patients we observed that GSH levels and aconitase activities were normal, suggesting their antioxidant status was unchanged. When exposed to various agents to increase free radical generation we observed that intracellular superoxide generation induced by paraquat caused enhanced oxidative damage. This correlated with the size of the GAA1 expansion, suggesting decreased frataxin levels may render the cells more vulnerable to mild oxidative stress. More severe oxidative stress induced by hydrogen peroxide caused increased cell death in FRDA fibroblasts but was not significantly different from control cells. We propose that abnormal respiratory chain function and iron accumulation may lead to a progressive increase in oxidative damage, but increased sensitivity to free radicals may not require detectable respiratory chain dysfunction.