Mice harboring the FXN I151F pathological point mutation present decreased frataxin levels, a Friedreich ataxia-like phenotype, and mitochondrial alterations.

Friedreich Ataxia (FA) is a rare neuro-cardiodegenerative disease caused by mutations in the frataxin (FXN) gene. The most prevalent mutation is a GAA expansion in the first intron of the gene causing decreased frataxin expression. Some patients present the GAA expansion in one allele and a missense mutation in the other allele. One of these mutations, FXNI154F, was reported to result in decreased content of mature frataxin and increased presence of an insoluble intermediate proteoform in cellular models. By introducing this mutation into the murine Fxn gene (I151F, equivalent to human I154F) we have now analyzed the consequences of this pathological point mutation in vivo. We have observed that FXNI151F homozygous mice present low frataxin levels in all tissues, with no evidence of insoluble proteoforms. Moreover, they display neurological deficits resembling those observed in FA patients. Biochemical analysis of heart, cerebrum and cerebellum have revealed decreased content of components from OXPHOS complexes I and II, decreased aconitase activity, and alterations in antioxidant defenses. These mitochondrial alterations are more marked in the nervous system than in heart, precede the appearance of neurological symptoms, and are similar to those observed in other FA models. We conclude that the primary pathological mechanism underlying the I151F mutation is frataxin deficiency, like in patients carrying GAA expansions. Therefore, patients carrying the I154F mutation would benefit from frataxin replacement therapies. Furthermore, our results also show that the FXNI151F mouse is an excellent tool for analyzing tissue-specific consequences of frataxin deficiency and for testing new therapies.

The displacement of frataxin from the mitochondrial cristae correlates with abnormal respiratory supercomplexes formation and bioenergetic defects in cells of Friedreich ataxia patients.

Friedreich ataxia (FRDA) is a neurodegenerative disease resulting from a severe decrease of frataxin (FXN). Most patients carry a GAA repeat expansion in both alleles of the FXN gene, whereas a small fraction of them are compound heterozygous for the expansion and a point mutation in the other allele. FXN is involved in the mitochondrial biogenesis of the FeS-clusters. Distinctive feature of FRDA patient cells is an impaired cellular respiration, likely due to a deficit of key redox cofactors working as electrons shuttles through the respiratory chain. However, a definite relationship between FXN levels, FeS-clusters assembly dysregulation and bioenergetics failure has not been established. In this work, we performed a comparative analysis of the mitochondrial phenotype of cell lines from FRDA patients, either homozygous for the expansion or compound heterozygotes for the G130V mutation. We found that, in healthy cells, FXN and two key proteins of the FeS-cluster assembly machinery are enriched in mitochondrial cristae, the dynamic subcompartment housing the respiratory chain. On the contrary, FXN widely redistributes to the matrix in FRDA cells with defects in respiratory supercomplexes assembly and altered respiratory function. We propose that this could be relevant for the early mitochondrial defects afflicting FRDA cells and that perturbation of mitochondrial morphodynamics could in turn be critical in terms of disease mechanisms.

Increased airway iron parameters and risk for exacerbation in COPD: an analysis from SPIROMICS.

Levels of iron and iron-related proteins including ferritin are higher in the lung tissue and lavage fluid of individuals with chronic obstructive pulmonary disease (COPD), when compared to healthy controls. Whether more iron in the extracellular milieu of the lung associates with distinct clinical phenotypes of COPD, including increased exacerbation susceptibility, is unknown. We measured iron and ferritin levels in the bronchoalveolar lavage fluid (BALF) of participants enrolled in the SubPopulations and InteRmediate Outcome Measures In COPD (SPIROMICS) bronchoscopy sub-study (n = 195). BALF Iron parameters were compared to systemic markers of iron availability and tested for association with FEV1 % predicted and exacerbation frequency. Exacerbations were modelled using a zero-inflated negative binomial model using age, sex, smoking, and FEV1 % predicted as clinical covariates. BALF iron and ferritin were higher in participants with COPD and in smokers without COPD when compared to non-smoker control participants but did not correlate with systemic iron markers. BALF ferritin and iron were elevated in participants who had COPD exacerbations, with a 2-fold increase in BALF ferritin and iron conveying a 24% and 2-fold increase in exacerbation risk, respectively. Similar associations were not observed with plasma ferritin. Increased airway iron levels may be representative of a distinct pathobiological phenomenon that results in more frequent COPD exacerbation events, contributing to disease progression in these individuals.

Hypoxia Rescues Frataxin Loss by Restoring Iron Sulfur Cluster Biogenesis.

Friedreich's ataxia (FRDA) is a devastating, multisystemic disorder caused by recessive mutations in the mitochondrial protein frataxin (FXN). FXN participates in the biosynthesis of Fe-S clusters and is considered to be essential for viability. Here we report that when grown in 1% ambient O2, FXN null yeast, human cells, and nematodes are fully viable. In human cells, hypoxia restores steady-state levels of Fe-S clusters and normalizes ATF4, NRF2, and IRP2 signaling events associated with FRDA. Cellular studies and in vitro reconstitution indicate that hypoxia acts through HIF-independent mechanisms that increase bioavailable iron as well as directly activate Fe-S synthesis. In a mouse model of FRDA, breathing 11% O2 attenuates the progression of ataxia, whereas breathing 55% O2 hastens it. Our work identifies oxygen as a key environmental variable in the pathogenesis associated with FXN depletion, with important mechanistic and therapeutic implications.

Correction of half the cardiomyocytes fully rescue Friedreich ataxia mitochondrial cardiomyopathy through cell-autonomous mechanisms.

Friedreich ataxia (FA) is currently an incurable inherited mitochondrial neurodegenerative disease caused by reduced levels of frataxin. Cardiac failure constitutes the main cause of premature death in FA. While adeno-associated virus-mediated cardiac gene therapy was shown to fully reverse the cardiac and mitochondrial phenotype in mouse models, this was achieved at high dose of vector resulting in the transduction of almost all cardiomyocytes, a dose and biodistribution that is unlikely to be replicated in clinic. The purpose of this study was to define the minimum vector biodistribution corresponding to the therapeutic threshold, at different stages of the disease progression. Correlative analysis of vector cardiac biodistribution, survival, cardiac function and biochemical hallmarks of the disease revealed that full rescue of the cardiac function was achieved when only half of the cardiomyocytes were transduced. In addition, meaningful therapeutic effect was achieved with as little as 30% transduction coverage. This therapeutic effect was mediated through cell-autonomous mechanisms for mitochondria homeostasis, although a significant increase in survival of uncorrected neighboring cells was observed. Overall, this study identifies the biodistribution thresholds and the underlying mechanisms conditioning the success of cardiac gene therapy in Friedreich ataxia and provides guidelines for the development of the clinical administration paradigm.

Frataxin-Mediated PINK1-Parkin-Dependent Mitophagy in Hepatic Steatosis: The Protective Effects of Quercetin.

SCOPE: Naturally occurring quercetin has been found to induce mitophagy and prevent nonalcoholic fatty liver disease (NAFLD). However, it still remains elusive whether frataxin upregulation by quercetin contributes to the beneficial effect through mitophagy or not. METHODS AND RESULTS: Adult male C57BL/J mice were fed a high-fat diet (HFD, 60% of energy from fat) with quercetin (100 mg kg-1 body weight) or not for 10 weeks. Quercetin alleviated HFD-induced histopathological changes, disorders of lipid metabolism, and mitochondrial damage. Moreover, quercetin blocked mitophagy suppression by HFD based on the increased LC3II, PTEN-induced putative kinase 1 (PINK1) and Beclin1 expressions, as well as decreased p62 levels. Quercetin also improved the Parkin translocation to mitochondria confirmed by immunofluorescence. Specifically, frataxin was lowered in the liver of HFD-fed mice or HepG2 cell incubated with oleate/palmitate but restored by quercetin, and quercetin's regulation of frataxin may depend on p53. Furthermore, lentivirus-mediated stable knockdown of frataxin in HepG2 inhibited PINK1-Parkin-associated mitophagy and resulted in lipid accumulation. Frataxin was further decreased by free fatty acids in knockdown cells concomitantly with depressed PINK1-Parkin-associated mitophagy, which was partially normalized by quercetin. CONCLUSION: Quercetin alleviated hepatic steatosis by enhancing frataxin-mediated PINK1/Parkin-dependent mitophagy, highlighting a promising preventive strategy and mechanism for NAFLD by quercetin.

Lactoferrin Protects Human Mesenchymal Stem Cells from Oxidative Stress-Induced Senescence and Apoptosis.

Mesenchymal stem cells (MSCs) have been suggested as a primary candidate for cell therapy applications because they have self-renewal and differentiation capabilities. Although they can be expanded in ex vivo system, clinical application of these cells is still limited because they survive poorly and undergo senescence or apoptosis when transplanted and exposed to environmental factors such as oxidative stress. Thus, reducing oxidative stress is expected to improve the efficacy of MSC therapy. The milk protein lactoferrin is a multifunctional iron-binding glycoprotein that plays various roles, including reduction of oxidative stress. Thus, we explored the effect of lactoferrin on oxidative stress-induced senescence and apoptosis of human MSCs (hMSCs). Measurement of reactive oxygen species (ROS) revealed that lactoferrin inhibited the production of hydrogen peroxide-induced intracellular ROS, suggesting lactoferrin as a good candidate as an antioxidant in hMSCs. Pretreatment of lactoferrin suppressed hydrogen peroxide-induced senescence of hMSCs. In addition, lactoferrin reduced hydrogen peroxide-induced apoptosis via inhibition of caspase-3 and Akt activation. These results demonstrate that lactoferrin can be a promising factor to protect hMSCs from oxidative stress-induced senescence and apoptosis, thus increasing the efficacy of MSC therapy.

Biogenesis and functions of mammalian iron-sulfur proteins in the regulation of iron homeostasis and pivotal metabolic pathways.

Fe-S cofactors are composed of iron and inorganic sulfur in various stoichiometries. A complex assembly pathway conducts their initial synthesis and subsequent binding to recipient proteins. In this minireview, we discuss how discovery of the role of the mammalian cytosolic aconitase, known as iron regulatory protein 1 (IRP1), led to the characterization of the function of its Fe-S cluster in sensing and regulating cellular iron homeostasis. Moreover, we present an overview of recent studies that have provided insights into the mechanism of Fe-S cluster transfer to recipient Fe-S proteins.

Frataxin Is Localized to Both the Chloroplast and Mitochondrion and Is Involved in Chloroplast Fe-S Protein Function in Arabidopsis.

Frataxin plays a key role in eukaryotic cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (Fe-S) cluster biosynthesis. However, its precise role has yet to be elucidated. In this work, we studied the subcellular localization of Arabidopsis frataxin, AtFH, using confocal microscopy, and found a novel dual localization for this protein. We demonstrate that plant frataxin is targeted to both the mitochondria and the chloroplast, where it may play a role in Fe-S cluster metabolism as suggested by functional studies on nitrite reductase (NIR) and ferredoxin (Fd), two Fe-S containing chloroplast proteins, in AtFH deficient plants. Our results indicate that frataxin deficiency alters the normal functioning of chloroplasts by affecting the levels of Fe, chlorophyll, and the photosynthetic electron transport chain in this organelle.

Molecular insights into frataxin-mediated iron supply for heme biosynthesis in Bacillus subtilis.

Iron is required as an element to sustain life in all eukaryotes and most bacteria. Although several bacterial iron acquisition strategies have been well explored, little is known about the intracellular trafficking pathways of iron and its entry into the systems for co-factor biogenesis. In this study, we investigated the iron-dependent process of heme maturation in Bacillus subtilis and present, for the first time, structural evidence for the physical interaction of a frataxin homologue (Fra), which is suggested to act as a regulatory component as well as an iron chaperone in different cellular pathways, and a ferrochelatase (HemH), which catalyses the final step of heme b biogenesis. Specific interaction between Fra and HemH was observed upon co-purification from crude cell lysates and, further, by using the recombinant proteins for analytical size-exclusion chromatography. Hydrogen-deuterium exchange experiments identified the landscape of the Fra/HemH interaction interface and revealed Fra as a specific ferrous iron donor for the ferrochelatase HemH. The functional utilisation of the in vitro-generated heme b co-factor upon Fra-mediated iron transfer was confirmed by using the B. subtilis nitric oxide synthase bsNos as a metabolic target enzyme. Complementary mutational analyses confirmed that Fra acts as an essential component for maturation and subsequent targeting of the heme b co-factor, hence representing a key player in the iron-dependent physiology of B. subtilis.

Iron-binding E3 ligase mediates iron response in plants by targeting basic helix-loop-helix transcription factors.

Iron uptake and metabolism are tightly regulated in both plants and animals. In Arabidopsis (Arabidopsis thaliana), BRUTUS (BTS), which contains three hemerythrin (HHE) domains and a Really Interesting New Gene (RING) domain, interacts with basic helix-loop-helix transcription factors that are capable of forming heterodimers with POPEYE (PYE), a positive regulator of the iron deficiency response. BTS has been shown to have E3 ligase capacity and to play a role in root growth, rhizosphere acidification, and iron reductase activity in response to iron deprivation. To further characterize the function of this protein, we examined the expression pattern of recombinant ProBTS::ß-GLUCURONIDASE and found that it is expressed in developing embryos and other reproductive tissues, corresponding with its apparent role in reproductive growth and development. Our findings also indicate that the interactions between BTS and PYE-like (PYEL) basic helix-loop-helix transcription factors occur within the nucleus and are dependent on the presence of the RING domain. We provide evidence that BTS facilitates 26S proteasome-mediated degradation of PYEL proteins in the absence of iron. We also determined that, upon binding iron at the HHE domains, BTS is destabilized and that this destabilization relies on specific residues within the HHE domains. This study reveals an important and unique mechanism for plant iron homeostasis whereby an E3 ubiquitin ligase may posttranslationally control components of the transcriptional regulatory network involved in the iron deficiency response.

Human frataxin activates Fe-S cluster biosynthesis by facilitating sulfur transfer chemistry.

Iron-sulfur clusters are ubiquitous protein cofactors with critical cellular functions. The mitochondrial Fe-S assembly complex, which consists of the cysteine desulfurase NFS1 and its accessory protein (ISD11), the Fe-S assembly protein (ISCU2), and frataxin (FXN), converts substrates l-cysteine, ferrous iron, and electrons into Fe-S clusters. The physiological function of FXN has received a tremendous amount of attention since the discovery that its loss is directly linked to the neurodegenerative disease Friedreich's ataxia. Previous in vitro results revealed a role for human FXN in activating the cysteine desulfurase and Fe-S cluster biosynthesis activities of the Fe-S assembly complex. Here we present radiolabeling experiments that indicate FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting persulfide species is viable in the subsequent synthesis of Fe-S clusters. Additional mutagenesis, enzyme kinetic, UV-visible, and circular dichroism spectroscopic studies suggest conserved ISCU2 residue C104 is critical for FXN activation, whereas C35, C61, and C104 are all essential for Fe-S cluster formation on the assembly complex. These results cannot be fully explained by the hypothesis that FXN functions as an iron donor for Fe-S cluster biosynthesis, and further support an allosteric regulator role for FXN. Together, these results lead to an activation model in which FXN accelerates persulfide formation on NFS1 and favors a helix-to-coil interconversion on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2 as an initial step in Fe-S cluster biosynthesis.

Pathophysiogical and therapeutic progress in Friedreich ataxia.

Friedreich ataxia (FRDA) is the most common hereditary autosomal recessive ataxia, but is also a multisystemic condition with frequent presence of cardiomyopathy or diabetes. It has been linked to expansion of a GAA-triplet repeat in the first intron of the FXN gene, leading to a reduced level of frataxin, a mitochondrial protein which, by controlling both iron entry and/or sulfide production, is essential to properly assemble and protect the Fe-S cluster during the initial stage of biogenesis. Several data emphasize the role of oxidative damage in FRDA, but better understanding of pathophysiological consequences of FXN mutations has led to develop animal models. Conditional knockout models recapitulate important features of the human disease but lack the genetic context, GAA repeat expansion-based knock-in and transgenic models carry a GAA repeat expansion but they only show a very mild phenotype. Cells derived from FRDA patients constitute the most relevant frataxin-deficient cell model as they carry the complete frataxin locus together with GAA repeat expansions and regulatory sequences. Induced pluripotent stem cell (iPSC)-derived neurons present a maturation delay and lower mitochondrial membrane potential, while cardiomyocytes exhibit progressive mitochondrial degeneration, with frequent dark mitochondria and proliferation/accumulation of normal mitochondria. Efforts in developing therapeutic strategies can be divided into three categories: iron chelators, antioxidants and/or stimulants of mitochondrial biogenesis, and frataxin level modifiers. A promising therapeutic strategy that is currently the subject of intense research is to directly target the heterochromatin state of the GAA repeat expansion with histone deacytelase inhibitors (HDACi) to restore frataxin levels.

Yap1 mediates tolerance to cobalt toxicity in the yeast Saccharomyces cerevisiae.

BACKGROUND: Cobalt has a rare occurrence in nature, but may accumulate in cells to toxic levels. In the present study, we have investigated how the transcription factor Yap1 mediates tolerance to cobalt toxicity. METHODS: Fluorescence microscopy was used to address how cobalt activates Yap1. Using microarray analysis, we compared the transcriptional profile of a strain lacking Yap1 to that of its parental strain. To evaluate the extent of the oxidative damage caused by cobalt, GSH was quantified by HPLC and protein carbonylation levels were assessed. RESULTS: Cobalt activates Yap1 under aerobiosis and anaerobiosis growth conditions. This metal generates a severe oxidative damage in the absence of Yap1. However, when challenged with high concentrations of cobalt, yap1 mutant cells accumulate lower levels of this metal. Accordingly, microarray analysis revealed that the expression of the high affinity phosphate transporter, PHO84, a well-known cobalt transporter, is compromised in the yap1 mutant. Moreover, we show that Yap1 is a repressor of the low affinity iron transporter, FET4, which is also known to transport cobalt. CONCLUSIONS: Cobalt activates Yap1 that alleviates the oxidative damage caused by this metal. Yap1 partially controls cobalt cellular uptake via the regulation of PHO84. Although FET4 repression by Yap1 has no effect on cobalt uptake, it may be its first line of defense against other toxic metals. GENERAL SIGNIFICANCE: Our results emphasize the important role of Yap1 in mediating cobalt-induced oxidative damages and reveal new routes for cell protection provided by this regulator.

Fixing frataxin: 'ironing out' the metabolic defect in Friedreich's ataxia.

The metabolically active and redox-active mitochondrion appears to play a major role in the cellular metabolism of the transition metal, iron. Frataxin, a mitochondrial matrix protein, has been identified as playing a key role in the iron metabolism of this organelle due to its iron-binding properties and is known to be essential for iron-sulphur cluster formation. However, the precise function of frataxin remains elusive. The decrease in frataxin expression, as seen in the inherited disorder Friedreich's ataxia, markedly alters cellular and mitochondrial iron metabolism in both the mitochondrion and the cell. The resulting dysregulation of iron trafficking damages affects tissues leading to neuro- and cardiodegeneration. This disease underscores the importance of iron homeostasis in the redox-active environment of the mitochondrion and the molecular players involved. Unravelling the mechanisms of altered iron metabolism in Friedreich's ataxia will help elucidate a biochemical function for frataxin. Consequently, this will enable the development of more effective and rationally designed treatments. This review will focus on the emerging function of frataxin in relation to the observed alterations in mitochondrial iron metabolism in Friedreich's ataxia. Tissue-specific alterations due to frataxin loss will also be discussed, as well as current and emerging therapeutic strategies.

Mitoneet mediates TNFα-induced necroptosis promoted by exposure to fructose and ethanol.

Fructose and ethanol are metabolized principally in the liver and are both known to contribute to the development of hepatic steatosis that can progress to hepatic steatohepatitis. The present study indentifies a synergistic interaction between fructose and ethanol in promoting hepatocyte sensitivity to TNFα-induced necroptosis. Concurrent exposure to fructose and ethanol induces the overexpression of the CDGSH iron-sulfur domain-containing protein 1 (CISD1 or mitoneet), which is localized to the outer mitochondrial membrane. The increased expression of mitoneet primes the hepatocyte for TNFα-induced cytotoxicity. Treatment with TNFα induces the translocation of a Stat3-Grim-19 complex to the mitochondria, which binds to mitoneet and promotes the rapid release of its 2Fe-2S cluster, causing an accumulation of mitochondrial iron. The dramatic increase of mitochondrial iron provokes a surge in formation of reactive oxygen species, resulting in mitochondrial injury and cell death. Additionally, mitoneet is constitutively expressed at high levels in L929 fibrosarcoma cells and is required for L929 cells to undergo TNFα-induced necroptosis in the presence of caspase inhibition, indicating the importance of mitoneet to the necroptotic form of cell death.

[Iron's ups and downs]. / Le fer dans tous ses états.

Iron is an essential trace metal whose extracellular concentration and stores are efficiently regulated. Systemic iron homeostasis assures a stable milieu in which each cell regulates its iron uptake to meet its own requirements. The system is challenged by variable availability of iron in the diet, by occasional iron losses through bleeding and by the fluctuations in the iron request by iron requiring processes such as erythropoiesis, growth, pregnancy and lactation; but also by pathologic processes involving aberrant iron retention leading to tissue iron overload and finally to end organ damage. A low serum ferritin is 100% specific for iron deficiency ; conversely hyperferritinemia is not a reliable sign of iron overload. Iron deficiency is a pan-ethnic disorder more prevalent in western and ageing people. Anemia represents the end stage of iron deficiency. During inflammatory states, iron becomes unavailable for erythropoiesis although adequate stores are present. This phenomenon is called functional iron deficiency and is characteristic of anemia of chronic disorders. Hyperferritinemia may exist in the presence or in the absence of iron overload. A cut off value of > 45% for transferrine saturation has been suggested to discriminate both settings. All the acquired conditions associated with hyperferritinemia must be excluded before performing genetic testing. Perfect understanding of iron homeostasis regulation as well as an adequate use of analyses exploring iron metabolism are mandatory for proper clinical management of iron deficiency and overload states.

[Therapeutic developments in chronic ataxias]. / Avances en el tratamiento de las ataxias crónicas.

Autosomal recessive cerebellar ataxias belong to a broader group of disorders known as inherited ataxias. In most cases onset occurs before the age of 20. These neurological disorders are characterized by degeneration or abnormal development of the cerebellum and spinal cord. Currently, specific treatment is only available for some of the chronic ataxias, more specifically those related to a known metabolic defect, such as abetalipoproteinemia, ataxia with vitamin E deficiency, and cerebrotendinous xanthomatosis. Treatment based on a diet with reduced intake of fat, supplementation of oral vitamins E and A, and the administration of chenodeoxycholic acid could modify the course of the disease. Although for most of autosomal recessive ataxias there is no definitive treatment, iron chelators and antioxidants have been proposed to reduce the mitochondrial iron overload in Friederich's ataxia patients. Corticosteroids have been used to reduce ataxia symptoms in ataxia telangiectasia. Coenzyme Q10 deficiency associated with ataxia may be responsive to Co Q10 or ubidecarenone supplementations. Early treatment of these disorders may be associated with a better drug response.

Frataxin: a protein in search for a function.

Reduced levels of the protein frataxin cause the neurodegenerative disease Friedreich's ataxia. Pathology is associated with disruption of iron-sulfur cluster biosynthesis, mitochondrial iron overload, and oxidative stress. Frataxin is a highly conserved iron-binding protein present in most organisms. Despite the intense interest generated since the determination of its pathology, identification of the cellular function of frataxin has so far remained elusive. In this review, we revisit the most significant milestones that have led us to our current understanding of frataxin and its functions. The picture that emerges is that frataxin is a crucial element of one of the most essential cellular machines specialized in iron-sulfur cluster biogenesis. Future developments, therefore, can be expected from further advancements in our comprehension of this machine.

Animal and cellular models of Friedreich ataxia.

The development and use of animal and cellular models of Friedreich ataxia (FRDA) are essential requirements for the understanding of FRDA disease mechanisms and the investigation of potential FRDA therapeutic strategies. Although animal and cellular models of lower organisms have provided valuable information on certain aspects of FRDA disease and therapy, it is intuitive that the most useful models are those of mammals and mammalian cells, which are the closest in physiological terms to FRDA patients. To date, there have been considerable efforts put into the development of several different FRDA mouse models and relevant FRDA mouse and human cell line systems. We summarize the principal mammalian FRDA models, discuss the pros and cons of each system, and describe the ways in which such models have been used to address two of the fundamental, as yet unanswered, questions regarding FRDA. Namely, what is the exact pathophysiology of FRDA and what is the detailed genetic and epigenetic basis of FRDA?