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Friedreich ataxia is a hereditary neurodegenerative disorder resulting from reduced levels of the protein frataxin due to an expanded GAA repeat in the FXN gene. This deficiency causes progressive degeneration of specific neuronal populations in the cerebellum and the consequent loss of movement coordination and equilibrium, which are some of the main symptoms observed in affected individuals. Like in other neurodegenerative diseases, previous studies suggest that glial cells could be involved in the neurodegenerative process and disease progression in patients with Friedreich ataxia. In this work, we followed and characterized the progression of changes in the cerebellar cortex in the latest version of Friedreich ataxia humanized mouse model, YG8-800 (Fxn null :YG8s(GAA) >800 ), which carries a human FXN transgene containing >800 GAA repeats. Comparative analyses of behavioral, histopathological, and biochemical parameters were conducted between the control strain Y47R and YG8-800 mice at different time points. Our findings revealed that YG8-800 mice exhibit an ataxic phenotype characterized by poor motor coordination, decreased body weight, cerebellar atrophy, neuronal loss, and changes in synaptic proteins. Additionally, early activation of glial cells, predominantly astrocytes and microglia, was observed preceding neuronal degeneration, as was increased expression of key proinflammatory cytokines and downregulation of neurotrophic factors. Together, our results show that the YG8-800 mouse model exhibits a stronger phenotype than previous experimental murine models, reliably recapitulating some of the features observed in humans. Accordingly, this humanized model could represent a valuable tool for studying Friedreich ataxia molecular disease mechanisms and for preclinical evaluation of possible therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Friedreich's ataxia is a spinocerebellar degenerative disease caused by microsatellite (GAA.TTC)n repeat expansion in the first intron of FXN gene. Here, we developed iPSC lines from an FRDA patient (IGIBi016-A) and non-FRDA healthy control (IGIBi017-A). Both iPSC lines displayed typical iPSC morphology, expression of pluripotency markers, regular karyotypes (46, XY; 46, XX), capacity to grow into three germ layers, and FRDA hallmark -GAA repeat expansion and decreased FXN mRNA. Through these iPSC lines, FRDA phenotypes may be replicated in the in vitro assays, by creating neuron subtypes, cardiomyocytes and 3D organoids, for molecular and cellular biomarkers and therapeutic applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The funneled energy landscape theory suggests that the folding pathway of homologous proteins should converge at the late stages of folding. In this respect, proteins displaying a broad energy landscape for folding are particularly instructive, allowing inferring both the early, intermediate and late stages of folding. In this paper we explore the folding mechanisms of human frataxin, an essential mitochondrial protein linked to the neurodegenerative disorder Friedreich's ataxia. Building upon previous studies on the yeast homologue, the folding pathway of human frataxin is thoroughly examined, revealing a mechanism implying the presence of a broad energy barrier, reminiscent of the yeast counterpart. Through an extensive site-directed mutagenesis, we employed a Φ -value analysis to map native-like contacts in the folding transition state. The presence of a broad energy barrier facilitated the exploration of such contacts in both early and late folding events. We compared results from yeast and human frataxin providing insights into the impact of native topology on the folding mechanism and elucidating the properties of the underlying free energy landscape. The findings are discussed in the context of the funneled energy landscape theory of protein folding., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In this paper, we describe the development of a Dictyostelium discoideum strain deficient in frataxin protein (FXN). We investigated the conservation of function between humans and D. discoideum and showed that DdFXN can substitute the human version in the interaction and activation of the Fe-S assembly supercomplex. We edited the D. discoideum fxn locus and isolated a defective mutant, clone 8, which presents landmarks of frataxin deficiency, such as a decrease in Fe-S cluster-dependent enzymatic functions, growth rate reduction, and increased sensitivity to oxidative stress. In addition, the multicellular development is affected as well as growing on bacterial lawn. We also assessed the rescuing capacity of DdFXN-G122V, a version that mimics a human variant present in some FA patients. While the expression of DdFXN-G122V rescues growth and enzymatic activity defects, as DdFXN does, multicellular development defects were only partially rescued. The results of the study suggest that this new D. discoideum strain offers a wide range of possibilities to easily explore diverse FA FXN variants. This can facilitate the development of straightforward drug screenings to look for new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Computational techniques for analyzing biological images offer a great potential to enhance our knowledge of the biological processes underlying disorders of the nervous system. Friedreich's Ataxia (FRDA) is a rare progressive neurodegenerative inherited disorder caused by the low expression of frataxin, which is a small mitochondrial protein. In FRDA cells, the lack of frataxin promotes primarily mitochondrial dysfunction, an alteration of calcium (Ca2+) homeostasis and the destabilization of the actin cytoskeleton in the neurites and growth cones of sensory neurons. In this paper, a computational multilinear algebra approach was used to analyze the dynamics of the growth cone and its function in control and FRDA neurons. Computational approach, which includes principal component analysis and a multilinear algebra method, is used to quantify the dynamics of the growth cone (GC) morphology of sensory neurons from the dorsal root ganglia (DRG) of the YG8sR humanized murine model for FRDA. It was confirmed that the dynamics and patterns of turning were aberrant in the FRDA growth cones. In addition, our data suggest that other cellular processes dependent on functional GCs such as axonal regeneration might also be affected. Semiautomated computational approaches are presented to quantify differences in GC behaviors in neurodegenerative disease. In summary, the deficiency of frataxin has an adverse effect on the formation and, most importantly, the growth cones' function in adult DRG neurons. As a result, frataxin deficient DRG neurons might lose the intrinsic capability to grow and regenerate axons properly due to the dysfunctional GCs they build. [ABSTRACT FROM AUTHOR]

Friedreich ataxia is an autosomal recessive, neurodegenerative disease characterized by the deficiency of the iron‐sulfur cluster assembly protein frataxin. Loss of this protein impairs mitochondrial function. Mitochondria alter their morphology in response to various stresses; however, such alterations to morphology may be homeostatic or maladaptive depending upon the tissue and disease state. Numerous neurodegenerative diseases exhibit excessive mitochondrial fragmentation, and reversing this phenotype improves bioenergetics for diseases in which mitochondrial dysfunction is a secondary feature of the disease. This paper demonstrates that frataxin deficiency causes excessive mitochondrial fragmentation that is dependent upon Drp1 activity in Friedreich ataxia cellular models. Drp1 inhibition by the small peptide TAT‐P110 reverses mitochondrial fragmentation but also decreases ATP levels in frataxin‐knockdown fibroblasts and FRDA patient fibroblasts, suggesting that fragmentation may provide a homeostatic pathway for maintaining cellular ATP levels. The cardiolipin‐stabilizing compound SS‐31 similarly reverses fragmentation through a Drp1‐dependent mechanism, but it does not affect ATP levels. The combination of TAT‐P110 and SS‐31 does not affect FRDA patient fibroblasts differently from SS‐31 alone, suggesting that the two drugs act through the same pathway but differ in their ability to alter mitochondrial homeostasis. In approaching potential therapeutic strategies for FRDA, an important criterion for compounds that improve bioenergetics should be to do so without impairing the homeostatic response of mitochondrial fragmentation. [ABSTRACT FROM AUTHOR]

In this work, we have designed and generated a Fe(III)-binding protein with thiol oxidoreductase activity. The consensus iron-binding motif EExxED from the frataxin protein family was grafted on a model peptide and on the surface of thioredoxin (TRX) from E. coli. We investigated metal interactions with a family of peptides containing the motif EExxED or altered versions obtained by removing negatively charged residues: EExxEx, xExxED, and xExxEx. The interaction of the metal ion with the peptides was studied by circular dichroism, and our results indicated that the motif EExxED retained its functional properties and also that this motif is able to bind Ga(III) and Al(III). The interaction of the grafted TRX with iron(III) was investigated by NMR, showing that the motif was functional in the context of the protein structure, and also the binding of two equivalents of Fe(III) per TRX molecule was stable in a non-chelating neutral buffer. Protein conformation, stability, and enzymatic activity were studied by applying experimental and computational approaches. Interestingly, the thiol oxidoreductase activity was modulated by interaction with Ga(III), a Fe(III) mimetic ion. Furthermore, the design of functional proteins with both functions, oxidoreductase activity and metal-ion binding ability, should consider the reorganisation of the electrostatic network. Similarly, studying the crosstalk and electrostatic balance among different metal-binding sites may be critical. [ABSTRACT FROM AUTHOR]

Genetic disorders are due to mutation of some gene. For example, Friedreich's Ataxia is due to mutation of frataxin gene. The mutation causes transmission error (noise) in the transmission channel. In this paper we show how this transmission error can be calculated using information theory approach. [ABSTRACT FROM AUTHOR]

Iron-sulfur clusters are evolutionarily conserved biological structures which play an important role as cofactor for multiple enzymes in eukaryotic cells. The biosynthesis pathways of the iron-sulfur clusters are located in the mitochondria and in the cytosol. The mitochondrial iron-sulfur cluster biosynthesis pathway (ISC) can be divided into at least twenty enzymatic steps. Since the description of frataxin deficiency as the cause of Friedreich’s ataxia, multiple other deficiencies in ISC biosynthesis pathway have been reported. In this paper, an overview is given of the clinical, biochemical and genetic aspects reported in humans affected by a defect in iron-sulfur cluster biosynthesis. [ABSTRACT FROM AUTHOR]

Introduction: Friedreich's ataxia is the most common inherited ataxia. Sources of data: Literature search using PubMed with keywords Friedreich's ataxia together with published papers known to the authors. Areas of agreement: The last decade has seen important advances in our understanding of the pathogenesis of disease. In particular, the genetic and epigenetic mechanisms underlying the disease now offer promising novel therapeutic targets. Areas of controversy: The search for effective disease-modifying agents continues. It remains to be determined whether the most effective approach to treatment lies with increasing frataxin protein levels or addressing the metabolic consequences of the disease, for example with antioxidants. Areas timely for developing research: Management of Freidreich's ataxia is currently focussed on symptomatic management, delivered by the multidisciplinary team. Phase II clinical trials in agents that address the abberrant silencing of the frataxin gene need to be translated into large placebocontrolled Phase III trials to help establish their therapeutic potential. [ABSTRACT FROM AUTHOR]

Friedreich ataxia (FRDA) is a life-threatening hereditary ataxia; its incidence is 1:50,000 individuals in the Caucasian population. A unique therapeutic drug for FRDA, the antioxidant Omaveloxolone, has been recently approved by the US Food and Drug Administration (FDA). FRDA is a multi-systemic neurodegenerative disease; in addition to a progressive neurodegeneration, FRDA is characterized by hypertrophic cardiomyopathy, diabetes mellitus and musculoskeletal deformities. Cardiomyopathy is the predominant cause of premature death. The onset of FRDA typically occurs between the ages of 5 and 15. Given the complexity and heterogeneity of clinical features and the variability of their onset, the identification of biomarkers capable of assessing disease progression and monitoring the efficacy of treatments is essential to facilitate decision making in clinical practice. We conducted an RNA-seq analysis in peripheral blood mononuclear cells from FRDA patients and healthy donors, identifying a signature of small non-coding RNAs (sncRNAs) capable of distinguishing healthy individuals from the majority of FRDA patients. Among the differentially expressed sncRNAs, microRNAs are a class of small non-coding endogenous RNAs that regulate posttranscriptional silencing of target genes. In FRDA plasma samples, hsa-miR-148a-3p resulted significantly upregulated. The analysis of the Receiver Operating Characteristic (ROC) curve, combining the circulating expression levels of hsa-miR-148a-3p and hsa-miR-223-3p (previously identified by our group), revealed an Area Under the Curve (AUC) of 0.86 (95%, Confidence Interval 0.77–0.95; p-value < 0.0001). An in silico prediction analysis indicated that the IL6ST gene, an interesting marker of neuroinflammation in FRDA, is a common target gene of both miRNAs. Our findings support the evaluation of combined expression levels of different circulating miRNAs as potent epi-biomarkers in FRDA. Moreover, we found hsa-miR-148a-3p significantly over-expressed in Intermediate and Late-Onset Friedreich Ataxia patients' group (IOG and LOG, respectively) compared to healthy individuals, indicating it as a putative prognostic biomarker in this pathology. [ABSTRACT FROM AUTHOR]

Friedreich's ataxia (FRDA) is a rare childhood neurologic disorder, affecting 1 in 50,000 Caucasians. The disease is caused by the abnormal expansion of the GAA repeat sequence in intron 1 of the FXN gene, leading to the reduced expression of the mitochondrial protein frataxin. The disease is characterised by progressive neurodegeneration, hypertrophic cardiomyopathy, diabetes mellitus and musculoskeletal deformities. The reduced expression of frataxin has been suggested to result in the downregulation of endogenous antioxidant defence mechanisms and mitochondrial bioenergetics, and the increase in mitochondrial iron accumulation thereby leading to oxidative stress. The confirmation of oxidative stress as one of the pathological signatures of FRDA led to the search for antioxidants which can be used as therapeutic modality. Based on this observation, antioxidants with different mechanisms of action have been explored for FRDA therapy since the last two decades. In this review, we bring forth all antioxidants which have been investigated for FRDA therapy and have been signed off for clinical trials. We summarise their various target points in FRDA disease pathway, their performances during clinical trials and possible factors which might have accounted for their failure or otherwise during clinical trials. We also discuss the limitation of the studies completed and propose possible strategies for combinatorial therapy of antioxidants to generate synergistic effect in FRDA patients. [ABSTRACT FROM AUTHOR]

Deficiency in human mature frataxin (hFXN-M) protein is responsible for the devastating neurodegenerative and cardiodegenerative disease of Friedreich's ataxia (FRDA). It results primarily through epigenetic silencing of the FXN gene by GAA triplet repeats on intron 1 of both alleles. GAA repeat lengths are most commonly between 600 and 1200 but can reach 1700. A subset of approximately 3% of FRDA patients have GAA repeats on one allele and a mutation on the other. FRDA patients die most commonly in their 30s from heart disease. Therefore, increasing expression of heart hFXN-M using gene therapy offers a way to prevent early mortality in FRDA. We used rhesus macaque monkeys to test the pharmacology of an adeno-associated virus (AAV)hu68.CB7.hFXN therapy. The advantage of using non-human primates for hFXN-M gene therapy studies is that hFXN-M and monkey FXN-M (mFXN-M) are 98.5% identical, which limits potential immunologic side-effects. However, this presented a formidable bioanalytical challenge in quantification of proteins with almost identical sequences. This could be overcome by the development of a species-specific quantitative mass spectrometry-based method, which has revealed for the first time, robust transgene-specific human protein expression in monkey heart tissue. The dose response is non-linear resulting in a ten-fold increase in monkey heart hFXN-M protein expression with only a three-fold increase in dose of the vector. Species-specific quantitative mass spectrometry revealed robust human frataxin expression in monkey heart tissue after dosing with an AAV vector expressing frataxin and a non-linear increase in protein expression with increasing amounts of vector (248 characters). [ABSTRACT FROM AUTHOR]

Frataxin (FH) plays a crucial role in the biogenesis of mitochondria and the regulation of iron in the cells of various organisms. However, there has been very little research on FH in plants. In this study, the potato FH gene (StFH) was identified and characterized using a genome-wide approach, and its sequence was compared to those of FH genes from Arabidopsis, rice, and maize. The FH genes were found to have a lineage-specific distribution and were more conserved in monocots than in dicots. While multiple copies of FH genes have been reported in some species, including plants, only one isoform of FH was found in potato. The expression of StFH in leaves and roots was analyzed under two different abiotic stress conditions, and the results showed that StFH was upregulated more in leaves and that its expression levels increased with the severity of the stress. This is the first study to examine the expression of an FH gene under abiotic stress conditions. [ABSTRACT FROM AUTHOR]

Iron–sulfur (Fe–S) clusters are protein cofactors of a multitude of enzymes performing essential biological functions. Specialized multi-protein machineries present in all types of organisms support their biosynthesis. These machineries encompass a scaffold protein on which Fe–S clusters are assembled and a cysteine desulfurase that provides sulfur in the form of a persulfide. The sulfide ions are produced by reductive cleavage of the persulfide, which involves specific reductase systems. Several other components are required for Fe–S biosynthesis, including frataxin, a key protein of controversial function and accessory components for insertion of Fe–S clusters in client proteins. Fe–S cluster biosynthesis is thought to rely on concerted and carefully orchestrated processes. However, the elucidation of the mechanisms of their assembly has remained a challenging task due to the biochemical versatility of iron and sulfur and the relative instability of Fe–S clusters. Nonetheless, significant progresses have been achieved in the past years, using biochemical, spectroscopic and structural approaches with reconstituted system in vitro. In this paper, we review the most recent advances on the mechanism of assembly for the founding member of the Fe–S cluster family, the [2Fe2S] cluster that is the building block of all other Fe–S clusters. The aim is to provide a survey of the mechanisms of iron and sulfur insertion in the scaffold proteins by examining how these processes are coordinated, how sulfide is produced and how the dinuclear [2Fe2S] cluster is formed, keeping in mind the question of the physiological relevance of the reconstituted systems. We also cover the latest outcomes on the functional role of the controversial frataxin protein in Fe–S cluster biosynthesis. [ABSTRACT FROM AUTHOR]

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Iron-sulfur (Fe-S) clusters function as protein cofactors for a wide variety of critical cellular reactions. In human mitochondria, a core Fe–S assembly complex [called SDUF and composed of NFS1, ISD11, ISCU2, and frataxin (FXN) proteins] synthesizes Fe–S clusters from iron, cysteine sulfur, and reducing equivalents and then transfers these intact clusters to target proteins. In vitro assays have relied on reducing the complexity of this complicated Fe–S assembly process by using surrogate electron donor molecules and monitoring simplified reactions. Recent studies have concluded that FXN promotes the synthesis of [4Fe-4S] clusters on the mammalian Fe–S assembly complex. Here the kinetics of Fe–S synthesis reactions were determined using different electron donation systems and by monitoring the products with circular dichroism and absorbance spectroscopies. We discovered that common surrogate electron donor molecules intercepted Fe–S cluster intermediates and formed high-molecular weight species (HMWS). The FIMWS are associated with iron, sulfide, and thiol-containing proteins and have properties of a heterogeneous solubilized mineral with spectroscopic properties remarkably reminiscent of those of [4Fe-4S] clusters. In contrast, reactions using physiological reagents revealed that FXN accelerates the formation of [2Fe-2S] clusters rather than [4Fe-4S] clusters as previously reported. In the preceding paper [Fox, N. G., et al. (2015) Biochemistry 54, DOI: 10.1021/bi5014485], [2Fe-2S] intermediates on the SDUF complex were shown to readily transfer to uncomplexed ISCU2 or apo acceptor proteins, depending on the reaction conditions. Our results indicate that FXN accelerates a rate-limiting sulfur transfer step in the synthesis of [2Fe-2S] clusters on the human Fe-S assembly complex. [ABSTRACT FROM AUTHOR]

Iron–protein interactions are involved in electron transfer reactions. Alterations of these processes are present in a number of human pathologies; among them, in Friedreich's ataxia, in which a deficiency of functional frataxin, an iron-binding protein, leads to progressive neuromuscular degenerative disease. The putative iron-binding motif of acidic residues EExxED was selected from the first α-helical stretch of the frataxin protein family and grafted onto a foreign peptide scaffold corresponding to the C-terminal α-helix from E. coli thioredoxin. The resulting grafted peptide named GRAP was studied by applying experimental (circular dichroism, isothermal titration calorimetry, capillary zone electrophoresis, thermal denaturation, NMR) and computational approaches (docking, molecular dynamics simulations). Although isolated GRAP lacks a stable secondary structure in solution, when iron is added, the peptide acquires an α-helical structure. Here we have shown that the designed peptide is able to specifically bind Fe3+ with a moderate affinity (KD = 1.9 ± 0.2 μM) and a 1 : 1 stoichiometry. Remarkably, the GRAP/Fe3+ interaction is entropically driven (ΔH° = −1.53 ± 0.03 kcal mol−1 and TΔS° = 6.26 kcal mol−1). Experiments and simulations indicate that Fe3+ interacts with the peptide through three acidic side chains, inducing an α-helical conformation of the grafted motif. In addition, the acidic side chains involved undergo significant conformational rearrangements upon binding, as judged by the analysis of MDs. Altogether, these results contribute to an understanding of the iron-binding mechanisms in proteins and, in particular, in the case of human frataxin. [ABSTRACT FROM AUTHOR]

Frataxin (FXN) is a protein involved in storage and delivery of iron in the mitochondria. Single-point mutations in the FXN gene lead to reduced production of functional frataxin, with the consequent dyshomeostasis of iron. FXN variants are at the basis of neurological impairment (the Friedreich's ataxia) and several types of cancer. By using altruistic metadynamics in conjunction with the maximal constrained entropy principle, we estimate the change of free energy in the protein unfolding of frataxin and of some of its pathological mutants. The sampled configurations highlight differences between the wild-type and mutated sequences in the stability of the folded state. In partial agreement with thermodynamic experiments, where most of the analyzed variants are characterized by lower thermal stability compared to wild type, the D104G variant is found with a stability comparable to the wild-type sequence and a lower water-accessible surface area. These observations, obtained with the new approach we propose in our work, point to a functional switch, affected by single-point mutations, of frataxin from iron storage to iron release. The method is suitable to investigate wide structural changes in proteins in general, after a proper tuning of the chosen collective variable used to perform the transition. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Frataxin, a small nuclear-encoded protein targeted to mitochondria, is known to play an important role in both the mitochondrial respiratory chain and iron homeostasis. The protein is highly conserved in most eukaryotic organisms with no major structural changes, suggesting that it serves a crucial function in all organisms. Recently, purified frataxin was used as a therapeutic treatment of Friedreich's ataxia, a common degenerative disorder that results from a frataxin protein deficiency, by directly applying the protein to the diseased cells. In this report, we describe a novel and rapid method of synthesizing genes encoding frataxin proteins for the purpose of efficient protein production. The artificial yeast and human frataxin genes were synthesized by direct assembly of serial deoxyoligonucleotide primers designed based on the optimal nucleotide sequences. When we tested the expression of these synthetic genes in two E. coli host strains, the yeast frataxin gene was expressed 20 folds higher in Rosetta (DE3) cells than in BL21 (DE3) cells, whereas the expression levels of human frataxin were similar in both E. coli strains. Attenuation of the Fenton reactions by the purified yeast and human frataxin proteins was observed under the defined conditions, which suggests that the recombinant frataxin proteins are active and functional. The procedure described here could be applied to many known genes or to generate novel synthetic genes that can be redesigned by arranging functional domains from previously identified genes and to study the structure and function of synthetic recombinant proteins and potential usage. [ABSTRACT FROM AUTHOR]

Abstract: Severe mitochondria deficiency leads to a number of devastating degenerative disorders, yet, mild mitochondrial dysfunction in different species, including the nematode Caenorhabditis elegans, can have pro-longevity effects. This apparent paradox indicates that cellular adaptation to partial mitochondrial stress can induce beneficial responses, but how this is achieved is largely unknown. Complete absence of frataxin, the mitochondrial protein defective in patients with Friedreich''s ataxia, is lethal in C. elegans, while its partial deficiency extends animal lifespan in a p53 dependent manner. In this paper we provide further insight into frataxin control of C. elegans longevity by showing that a substantial reduction of frataxin protein expression is required to extend lifespan, affect sensory neurons functionality, remodel lipid metabolism and trigger autophagy. We find that Beclin and p53 genes are required to induce autophagy and concurrently reduce lipid storages and extend animal lifespan in response to frataxin suppression. Reciprocally, frataxin expression modulates autophagy in the absence of p53. Human Friedreich ataxia-derived lymphoblasts also display increased autophagy, indicating an evolutionarily conserved response to reduced frataxin expression. In sum, we demonstrate a causal connection between induction of autophagy and lifespan extension following reduced frataxin expression, thus providing the rationale for investigating autophagy in the pathogenesis and treatment of Friedreich''s ataxia and possibly other human mitochondria-associated disorders. [Copyright &y& Elsevier]

Frataxin is a conserved mitochondrial protein deficient in patients with Friedreich's ataxia. Frataxin has been implicated in control of iron homoeostasis and Fe–S cluster assembly. In yeast or human mitochondria, frataxin interacts with components of the Fe–S cluster synthesis machinery, including the cysteine desulfurase Nfs1, accessory protein Isd11 and scaffold protein Isu. In the present paper, we report that a single amino acid substitution (methionine to isoleucine) at position 107 in the mature form of Isu1 restored many deficient functions in Δyfh1 or frataxin-depleted yeast cells. Iron homoeostasis was improved such that soluble/usable mitochondrial iron was increased and accumulation of insoluble/non-usable iron within mitochondria was largely prevented. Cytochromes were returned to normal and haem synthesis was restored. In mitochondria carrying the mutant Isu1 and no frataxin, Fe–S cluster enzyme activities were improved. The efficiency of new Fe–S cluster synthesis in isolated mitochondria was markedly increased compared with frataxin-negative cells, although the response to added iron was minimal. The M107I substitution in the highly conserved Isu scaffold protein is typically found in bacterial orthologues, suggesting that a unique feature of the bacterial Fe–S cluster machinery may be involved. The mechanism by which the mutant Isu bypasses the absence of frataxin remains to be determined, but could be related to direct effects on Fe–S cluster assembly and/or indirect effects on mitochondrial iron availability. [ABSTRACT FROM AUTHOR]

Abstract: As in the case of aging, many degenerative disorders also result from progressive mitochondrial deterioration and cellular damage accumulation. Therefore, preventing damage accumulation may delay aging and help to prevent degenerative disorders, especially those associated with mitochondrial dysfunction. In the nematode Caenorhabditis elegans a mild mitochondrial dysfunction prolongs the lifespan. We previously proposed that, following a mild mitochondrial dysfunction, protective stress responses are activated in a hormetic-like fashion, and ultimately account for extended animal’s lifespan. We recently showed that in C. elegans, lifespan extension induced by reduced expression of different mitochondrial proteins involved in electron transport chain functionality requires p53/cep-1. In this paper we find that reducing the expression of frataxin, the protein defective in patients with Friedreich’s ataxia, triggers a complex stress response, and that the associated induction of the antioxidant glutathione-S-transferase is regulated by cep-1. Given the high percentage of homology between human and nematode genes and the conservation of fundamental intracellular pathways between the two species, identification of molecular mechanisms activated in response to frataxin suppression in C. elegans may suggest novel therapeutic approaches to prevent the accumulation of irreversible damage and the consequent appearance of symptoms in Friedreich’s ataxia and possibly other human mitochondrial-associated diseases. The same pathways could be exploitable for delaying the aging process ascribed to mitochondrial degeneration. [Copyright &y& Elsevier]

Abstract: Friedreich''s ataxia (FRDA) is an autosomal recessive neurodegenerative disease affecting 1 in 50,000 people and is caused by a GAA-trinucleotide expansion in the frataxin gene located on chromosome locus 9q13 which results in a markedly reduced expression of frataxin, a small mitochondrial protein. The exact function of frataxin is still unknown and currently there is no approved treatment available. In the near future there will be a high demand for measuring frataxin protein levels due to the development of therapeutic strategies for FRDA based on manipulating frataxin expression levels in vivo. In this paper we describe the development of an electrochemiluminescence assay (ECLIA) to measure frataxin protein levels in a 96-well plate format. The ECLIA for frataxin is able to measure human and mouse samples and is highly quantitative, accurate and reproducible, with low intra- and inter-assay error throughout a wide working range. The assay has an excellent precision and provides a new tool for the set up of high-throughput screening for basic research and for clinical studies with FRDA patients. [Copyright &y& Elsevier]

Friedreich's ataxia (FA) is an inherited neurodegenerative disorder caused by decreased expression of frataxin (FXN) protein. Previous studies have shown that antisense oligonucleotides (ASOs) and single-stranded silencing RNAs can be used to increase expression of frataxin in cultured patientderived cells. In this study, we investigate the potential for oligonucleotides to increase frataxin expression in a mouse model for FA. After confirming successful in vivo delivery of oligonucleotides using a benchmark gapmer targeting the nuclear noncoding RNA Malat1, we tested anti-FXN oligonucleotides designed to function by various mechanisms. None of these strategies yielded enhanced expression of FXN in the model mice. Our inability to translate activation of FXN expression from cell culture to mice may be due to inadequate potency of our compounds or differences in the molecular mechanisms governing FXN gene repression and activation in FA model mice. [ABSTRACT FROM AUTHOR]

Abstract Friedreich ataxia is an autosomal recessive, neurodegenerative disease characterized by the deficiency of the iron‐sulfur cluster assembly protein frataxin. Loss of this protein impairs mitochondrial function. Mitochondria alter their morphology in response to various stresses; however, such alterations to morphology may be homeostatic or maladaptive depending upon the tissue and disease state. Numerous neurodegenerative diseases exhibit excessive mitochondrial fragmentation, and reversing this phenotype improves bioenergetics for diseases in which mitochondrial dysfunction is a secondary feature of the disease. This paper demonstrates that frataxin deficiency causes excessive mitochondrial fragmentation that is dependent upon Drp1 activity in Friedreich ataxia cellular models. Drp1 inhibition by the small peptide TAT‐P110 reverses mitochondrial fragmentation but also decreases ATP levels in frataxin‐knockdown fibroblasts and FRDA patient fibroblasts, suggesting that fragmentation may provide a homeostatic pathway for maintaining cellular ATP levels. The cardiolipin‐stabilizing compound SS‐31 similarly reverses fragmentation through a Drp1‐dependent mechanism, but it does not affect ATP levels. The combination of TAT‐P110 and SS‐31 does not affect FRDA patient fibroblasts differently from SS‐31 alone, suggesting that the two drugs act through the same pathway but differ in their ability to alter mitochondrial homeostasis. In approaching potential therapeutic strategies for FRDA, an important criterion for compounds that improve bioenergetics should be to do so without impairing the homeostatic response of mitochondrial fragmentation.

Friedreich's ataxia (FRDA) is the most frequent autosomal recessive ataxia in western countries, with a mean age of onset at 10–15 years. Patients manifest progressive cerebellar and sensory ataxia, dysarthria, lower limb pyramidal weakness, and other systemic manifestations. Previously, we described a family displaying two expanded GAA alleles not only in the proband affected by late-onset FRDA but also in the two asymptomatic family members: the mother and the younger sister. Both of them showed a significant reduction of frataxin levels, without any disease manifestation. Here, we analyzed if a protective mechanism might contribute to modulate the phenotype in this family. We particularly focused on the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), the first line of antioxidant defense in cells, and on the glutathione (GSH) system, an index of reactive oxygen species (ROS) detoxification ability. Our findings show a great reactivity of the GSH system to the frataxin deficiency, particularly in the asymptomatic mother, where the genes of GSH synthesis [glutamate–cysteine ligase (GCL)] and GSSG detoxification [GSH S-reductase (GSR)] were highly responsive. The GSR was activated even in the asymptomatic sister and in the proband, reflecting the need of buffering the GSSG increase. Furthermore, and contrasting the NRF2 expression documented in FRDA tissues, NRF2 was highly activated in the mother and in the younger sister, while it was constitutively low in the proband. This suggests that, also under frataxin depletion, the endogenous stimulation of NRF2 in asymptomatic FRDA subjects may contribute to protect against the progressive oxidative damage, helping to prevent the onset of neurological symptoms and highlighting an "out-brain origin" of the disease. [ABSTRACT FROM AUTHOR]

Friedreich ataxia (FA) is a neurodegenerative disease caused by the deficiency of frataxin, a mitochondrial protein. In primary cultures of dorsal root ganglia neurons, we showed that frataxin depletion resulted in decreased levels of the mitochondrial calcium exchanger NCLX, neurite degeneration and apoptotic cell death. Here, we describe that frataxindeficient dorsal root ganglia neurons display low levels of ferredoxin 1 (FDX1), a mitochondrial Fe/S cluster-containing protein that interacts with frataxin and, interestingly, is essential for the synthesis of calcitriol, the active form of vitamin D. We provide data that calcitriol supplementation, used at nanomolar concentrations, is able to reverse the molecular and cellular markers altered in DRG neurons. Calcitriol is able to recover both FDX1 and NCLX levels and restores mitochondrial membrane potential indicating an overall mitochondrial function improvement. Accordingly, reduction in apoptotic markers and neurite degeneration was observed and, as a result, cell survival was also recovered. All these beneficial effects would be explained by the finding that calcitriol is able to increase the mature frataxin levels in both, frataxin-deficient DRG neurons and cardiomyocytes; remarkably, this increase also occurs in lymphoblastoid cell lines derived from FA patients. In conclusion, these results provide molecular bases to consider calcitriol for an easy and affordable therapeutic approach for FA patients. [ABSTRACT FROM AUTHOR]

Ferroptosis is a newly discovered form of non-apoptotic regulated cell death and is characterized by iron-dependent and lipid peroxidation. Due to the enhanced dependence on iron in cancer cells, induction of ferroptosis is becoming a promising therapeutic strategy. However, the precise underlying molecular mechanism and regulation process of ferroptosis remains largely unknown. In the present study, we demonstrate that the protein Frataxin (FXN) is a key regulator of ferroptosis by modulating iron homeostasis and mitochondrial function. Suppression of FXN expression specifically repressed the proliferation, destroyed mitochondrial morphology, impeded Fe–S cluster assembly and activated iron starvation stress. Moreover, suppression of FXN expression significantly enhanced erastin-induced cell death through accelerating free iron accumulation, lipid peroxidation and resulted in dramatic mitochondria morphological damage including enhanced fragmentation and vanished cristae. In addition, this type of cell death was confirmed to be ferroptosis, since it could be pharmacologically restored by ferroptotic inhibitor Fer-1 or GSH, but not by inhibitors of apoptosis, necrosis. Vice versa, enforced expression of FXN blocked iron starvation response and erastin-induced ferroptosis. More importantly, pharmacological or genetic blocking the signal of iron starvation could completely restore the resistance to ferroptosis in FXN knockdown cells and xenograft graft in vivo. This paper suggests that FXN is a novel ferroptosis modulator, as well as a potential provided target to improve the antitumor activity based on ferroptosis.

The common clinical symptoms of Friedreich's ataxia (FRDA) include ataxia, muscle weakness, type 2 diabetes and heart failure, which are caused by impaired mitochondrial function due to the loss of frataxin (FXN) expression. Endurance exercise is the most powerful intervention for promoting mitochondrial function; however, its impact on FRDA has not been studied. Here we found that mice with genetic knockout and knock-in of the Fxn gene (KIKO mice) developed exercise intolerance, glucose intolerance and moderate cardiac dysfunction at 6 months of age. These abnormalities were associated with impaired mitochondrial respiratory function concurrent with reduced iron regulatory protein 1 (Irp1) expression as well as increased oxidative stress, which were not due to loss of mitochondrial content and antioxidant enzyme expression. Importantly, long-term (4 months) voluntary running in KIKO mice starting at a young age (2 months) completely prevented the functional abnormalities along with restored Irp1 expression, improved mitochondrial function and reduced oxidative stress in skeletal muscle without restoring Fxn expression. We conclude that endurance exercise training prevents symptomatic onset of FRDA in mice associated with improved mitochondrial function and reduced oxidative stress. These preclinical findings may pave the way for clinical studies of the impact of endurance exercise in FRDA patients. [ABSTRACT FROM AUTHOR]

Abnormalities in actin cytoskeleton have been linked to Friedreich's ataxia (FRDA), an inherited peripheral neuropathy characterised by an early loss of neurons in dorsal root ganglia (DRG) among other clinical symptoms. Despite all efforts to date, we still do not fully understand the molecular events that contribute to the lack of sensory neurons in FRDA. We studied the adult neuronal growth cone (GC) at the cellular and molecular level to decipher the connection between frataxin and actin cytoskeleton in DRG neurons of the well-characterised YG8R Friedreich's ataxia mouse model. Immunofluorescence studies in primary cultures of DRG from YG8R mice showed neurons with fewer and smaller GCs than controls, associated with an inhibition of neurite growth. In frataxin-deficient neurons, we also observed an increase in the filamentous (F)-actin/monomeric (G)-actin ratio (F/G-actin ratio) in axons and GCs linked to dysregulation of two crucial modulators of filamentous actin turnover, cofilin-1 and the actin-related protein (ARP) 2/3 complex. We show how the activation of cofilin is due to the increase in chronophin (CIN), a cofilin-activating phosphatase. Thus cofilin emerges, for the first time, as a link between frataxin deficiency and actin cytoskeleton alterations. [ABSTRACT FROM AUTHOR]