Skeletal Muscle Involvement in Friedreich Ataxia.

Friedreich Ataxia (FRDA) is an inherited neuromuscular disorder triggered by a deficit of the mitochondrial protein frataxin. At a cellular level, frataxin deficiency results in insufficient iron-sulfur cluster biosynthesis and impaired mitochondrial function and adenosine triphosphate production. The main clinical manifestation is a progressive balance and coordination disorder which depends on the involvement of peripheral and central sensory pathways as well as of the cerebellum. Besides the neurological involvement, FRDA affects also the striated muscles. The most prominent manifestation is a hypertrophic cardiomyopathy, which also represents the major determinant of premature mortality. Moreover, FRDA displays skeletal muscle involvement, which contributes to the weakness and marked fatigue evident throughout the course of the disease. Herein, we review skeletal muscle findings in FRDA generated by functional imaging, histology, as well as multiomics techniques in both disease models and in patients. Altogether, these findings corroborate a disease phenotype in skeletal muscle and support the notion of progressive mitochondrial damage as a driver of disease progression in FRDA. Furthermore, we highlight the relevance of skeletal muscle investigations in the development of biomarkers for early-phase trials and future therapeutic strategies in FRDA.

Deciphering the ferroptosis pathways in dorsal root ganglia of Friedreich ataxia models. The role of LKB1/AMPK, KEAP1, and GSK3ß in the impairment of the NRF2 response.

Friedreich ataxia (FA) is a rare neurodegenerative disease caused by decreased levels of the mitochondrial protein frataxin. Frataxin has been related in iron homeostasis, energy metabolism, and oxidative stress. Ferroptosis has recently been shown to be involved in FA cellular degeneration; however, its role in dorsal root ganglion (DRG) sensory neurons, the cells that are affected the most and the earliest, is mostly unknown. In this study, we used primary cultures of frataxin-deficient DRG neurons as well as DRG from the FXNI151F mouse model to study ferroptosis and its regulatory pathways. A lack of frataxin induced upregulation of transferrin receptor 1 and decreased ferritin and mitochondrial iron accumulation, a source of oxidative stress. However, there was impaired activation of NRF2, a key transcription factor involved in the antioxidant response pathway. Decreased total and nuclear NRF2 explains the downregulation of both SLC7A11 (a member of the system Xc, which transports cystine required for glutathione synthesis) and glutathione peroxidase 4, responsible for increased lipid peroxidation, the main markers of ferroptosis. Such dysregulation could be due to the increase in KEAP1 and the activation of GSK3ß, which promote cytosolic localization and degradation of NRF2. Moreover, there was a deficiency in the LKB1/AMPK pathway, which would also impair NRF2 activity. AMPK acts as a positive regulator of NRF2 and it is activated by the upstream kinase LKB1. The levels of LKB1 were reduced when frataxin decreased, in agreement with reduced pAMPK (Thr172), the active form of AMPK. SIRT1, a known activator of LKB1, was also reduced when frataxin decreased. MT-6378, an AMPK activator, restored NRF2 levels, increased GPX4 levels and reduced lipid peroxidation. In conclusion, this study demonstrated that frataxin deficiency in DRG neurons disrupts iron homeostasis and the intricate regulation of molecular pathways affecting NRF2 activation and the cellular response to oxidative stress, leading to ferroptosis.

Tissue Iron in Friedreich Ataxia.

Heart, dentate nucleus, and dorsal root ganglia (DRG) are targets of tissue damage in Friedreich ataxia (FA). This report summarizes the histology and histopathology of iron in the main tissues affected by FA. None of the affected anatomical sites reveals an elevation of total iron levels. In the myocardium, a small percentage of fibers shows iron-reactive granular inclusions. The accumulation of larger iron aggregates and fiber invasion cause necrosis and damage to the contractile apparatus. In the dentate nucleus, the principal FA-caused tissue injury is neuronal atrophy and grumose reaction. X-ray fluorescence mapping of iron in the dentate nucleus in FA shows retention of the metal in the center of the collapsed structure. Immunohistochemistry of ferritin, a surrogate marker of tissue iron, confirms strong expression in oligodendrocytes of the efferent white matter of the dentate nucleus and abundance of ferritin-positive microglia in the atrophic gray matter. Iron dysmetabolism in DRG is complex and consists of prominent expression of ferritin in hyperplastic satellite cells and residual nodules, also a loss of the iron export protein ferroportin from the cytoplasm of the remaining DRG nerve cells.

Hsa-miR223-3p circulating level is upregulated in Friedreich's ataxia and inversely associated with HCLS1 associated protein X-1, HAX-1.

Frataxin (FXN) deficiency is responsible for Friedreich's ataxia (FRDA) in which, besides the characteristic features of spinocerebellar ataxia, two thirds of patients develop hypertrophic cardiomyopathy that often progresses to heart failure and premature death. Different mechanisms might underlie FRDA pathogenesis. Among them, the role of miRNAs deserves investigations. We carried out an miRNA PCR-array analysis of plasma samples of early-, intermediate- and late-onset FRDA groups, defining a set of 30 differentially expressed miRNAs. Hsa-miR223-3p is the only miRNA shared between the three patient groups and appears upregulated in all of them. The up-regulation of hsa-miR223-3p was further validated in all enrolled patients (n = 37, Fc = +2.3; P < 0.0001). Using a receiver operating characteristic curve analysis, we quantified the predictive value of circulating hsa-miR223-3p for FRDA, obtaining an area under the ROC curve value of 0.835 (P < 0.0001) for all patients. Interestingly, we found a significant positive correlation between hsa-miR223-3p expression and cardiac parameters in typical FRDA patients (onset < 25 years). Moreover, a significant negative correlation between hsa-miR223-3p expression and HAX-1 (HCLS1-associated protein X-1) at mRNA and protein level was observed in all FRDA patients. In silico analyses suggested HAX-1 as a target gene of hsa-miR223-3p. Accordingly, we report that HAX-1 is negatively regulated by hsa-miR223-3p in cardiomyocytes (AC16) and neurons (SH-SY5Y), which are critically affected cell types in FRDA. This study describes for the first time the association between hsa-miR223-3p and HAX-1 expression in FRDA, thus supporting a potential role of this microRNA as non-invasive epigenetic biomarker for FRDA.

Defective palmitoylation of transferrin receptor triggers iron overload in Friedreich ataxia fibroblasts.

Friedreich ataxia (FRDA) is a frequent autosomal recessive disease caused by a GAA repeat expansion in the FXN gene encoding frataxin, a mitochondrial protein involved in iron-sulfur cluster (ISC) biogenesis. Resulting frataxin deficiency affects ISC-containing proteins and causes iron to accumulate in the brain and heart of FRDA patients. Here we report on abnormal cellular iron homeostasis in FRDA fibroblasts inducing a massive iron overload in cytosol and mitochondria. We observe membrane transferrin receptor 1 (TfR1) accumulation, increased TfR1 endocytosis, and delayed Tf recycling, ascribing this to impaired TfR1 palmitoylation. Frataxin deficiency is shown to reduce coenzyme A (CoA) availability for TfR1 palmitoylation. Finally, we demonstrate that artesunate, CoA, and dichloroacetate improve TfR1 palmitoylation and decrease iron overload, paving the road for evidence-based therapeutic strategies at the actionable level of TfR1 palmitoylation in FRDA.

A Drosophila model of Friedreich ataxia with CRISPR/Cas9 insertion of GAA repeats in the frataxin gene reveals in vivo protection by N-acetyl cysteine.

Friedreich ataxia (FA) is caused by GAA repeat expansions in the first intron of FXN, the gene encoding frataxin, which results in decreased gene expression. Thanks to the high degree of frataxin conservation, the Drosophila melanogaster fruitfly appears as an adequate animal model to study this disease and to evaluate therapeutic interventions. Here, we generated a Drosophila model of FA with CRISPR/Cas9 insertion of approximately 200 GAA in the intron of the fly frataxin gene fh. These flies exhibit a developmental delay and lethality associated with decreased frataxin expression. We were able to bypass preadult lethality using genetic tools to overexpress frataxin only during the developmental period. These frataxin-deficient adults are short-lived and present strong locomotor defects. RNA-Seq analysis identified deregulation of genes involved in amino-acid metabolism and transcriptomic signatures of oxidative stress. In particular, we observed a progressive increase of Tspo expression, fully rescued by adult frataxin expression. Thus, Tspo expression constitutes a molecular marker of the disease progression in our fly model and might be of interest in other animal models or in patients. Finally, in a candidate drug screening, we observed that N-acetyl cysteine improved the survival, locomotor function, resistance to oxidative stress and aconitase activity of frataxin-deficient flies. Therefore, our model provides the opportunity to elucidate in vivo, the protective mechanisms of this molecule of therapeutic potential. This study also highlights the strength of the CRISPR/Cas9 technology to introduce human mutations in endogenous orthologous genes, leading to Drosophila models of human diseases with improved physiological relevance.

Mechanisms of Mitochondrial Iron-Sulfur Protein Biogenesis.

Mitochondria are essential in most eukaryotes and are involved in numerous biological functions including ATP production, cofactor biosyntheses, apoptosis, lipid synthesis, and steroid metabolism. Work over the past two decades has uncovered the biogenesis of cellular iron-sulfur (Fe/S) proteins as the essential and minimal function of mitochondria. This process is catalyzed by the bacteria-derived iron-sulfur cluster assembly (ISC) machinery and has been dissected into three major steps: de novo synthesis of a [2Fe-2S] cluster on a scaffold protein; Hsp70 chaperone-mediated trafficking of the cluster and insertion into [2Fe-2S] target apoproteins; and catalytic conversion of the [2Fe-2S] into a [4Fe-4S] cluster and subsequent insertion into recipient apoproteins. ISC components of the first two steps are also required for biogenesis of numerous essential cytosolic and nuclear Fe/S proteins, explaining the essentiality of mitochondria. This review summarizes the molecular mechanisms underlying the ISC protein-mediated maturation of mitochondrial Fe/S proteins and the importance for human disease.

Nanoscopic X-ray imaging and quantification of the iron cellular architecture within single fibroblasts of Friedreich's ataxia patients.

Friedreich's ataxia (FRDA) is a neurodegenerative disease characterized by an increase in intracytoplasmic iron concentration. Here the nanoscale iron distribution within single fibroblasts from FRDA patients was investigated using synchrotron-radiation-based nanoscopic X-ray fluorescence and X-ray in-line holography at the ID16A nano-imaging beamline of the ESRF. This unique probe was deployed to uncover the iron cellular two-dimensional architecture of freeze-dried FRDA fibroblasts. An unsurpassed absolute detection capability of 180 iron atoms within a 30 nm × 50 nm nanoscopic X-ray beam footprint was obtained using state-of-the-art X-ray focusing optics and a large-solid-angle detection system. Various micrometre-sized iron-rich organelles could be revealed for the first time, tentatively identified as endoplasmic reticulum, mitochondria and lysosomes. Also a multitude of nanoscopic iron hot-spots were observed in the cytosol, interpreted as chaperoned iron within the fibroblast's labile iron pool. These observations enable new hypotheses on the storage and trafficking of iron in the cell and ultimately to a better understanding of iron-storage diseases such as Friedreich's ataxia.

Targeting NRF2 for the Treatment of Friedreich's Ataxia: A Comparison among Drugs.

NRF2 (Nuclear factor Erythroid 2-related Factor 2) signaling is impaired in Friedreich's Ataxia (FRDA), an autosomal recessive disease characterized by progressive nervous system damage and degeneration of nerve fibers in the spinal cord and peripheral nerves. The loss of frataxin in patients results in iron sulfur cluster deficiency and iron accumulation in the mitochondria, making FRDA a fatal and debilitating condition. There are no currently approved therapies for the treatment of FRDA and molecules able to activate NRF2 have the potential to induce clinical benefits in patients. In this study, we compared the efficacy of six redox-active drugs, some already adopted in clinical trials, targeting NRF2 activation and frataxin expression in fibroblasts obtained from skin biopsies of FRDA patients. All of these drugs consistently increased NRF2 expression, but differential profiles of NRF2 downstream genes were activated. The Sulforaphane and N-acetylcysteine were particularly effective on genes involved in preventing inflammation and maintaining glutathione homeostasis, the dimethyl fumarate, omaxevolone, and EPI-743 in counteracting toxic products accumulation, the idebenone in mitochondrial protection. This study may contribute to develop synergic therapies, based on a combination of treatment molecules.

Physiologically relevant reconstitution of iron-sulfur cluster biosynthesis uncovers persulfide-processing functions of ferredoxin-2 and frataxin.

Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich's ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich's ataxia therapies.

Structure of the human frataxin-bound iron-sulfur cluster assembly complex provides insight into its activation mechanism.

The core machinery for de novo biosynthesis of iron-sulfur clusters (ISC), located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreich's ataxia (FRDA). Here the 3.2 Å resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates the interactions of FXN with other component proteins of the complex. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure reveals how FXN facilitates ISC production through stabilizing key loop conformations of NFS1 and ISCU at the protein-protein interfaces, and suggests how FRDA clinical mutations affect complex formation and FXN activation.

New Insights into the Hepcidin-Ferroportin Axis and Iron Homeostasis in iPSC-Derived Cardiomyocytes from Friedreich's Ataxia Patient.

Iron homeostasis in the cardiac tissue as well as the involvement of the hepcidin-ferroportin (HAMP-FPN) axis in this process and in cardiac functionality are not fully understood. Imbalance of iron homeostasis occurs in several cardiac diseases, including iron-overload cardiomyopathies such as Friedreich's ataxia (FRDA, OMIM no. 229300), a hereditary neurodegenerative disorder. Exploiting the induced pluripotent stem cells (iPSCs) technology and the iPSC capacity to differentiate into specific cell types, we derived cardiomyocytes of a FRDA patient and of a healthy control subject in order to study the cardiac iron homeostasis and the HAMP-FPN axis. Both CTR and FRDA iPSCs-derived cardiomyocytes express cardiac differentiation markers; in addition, FRDA cardiomyocytes maintain the FRDA-like phenotype. We found that FRDA cardiomyocytes show an increase in the protein expression of HAMP and FPN. Moreover, immunofluorescence analysis revealed for the first time an unexpected nuclear localization of FPN in both CTR and FRDA cardiomyocytes. However, the amount of the nuclear FPN was less in FRDA cardiomyocytes than in controls. These and other data suggest that iron handling and the HAMP-FPN axis regulation in FRDA cardiac cells are hampered and that FPN may have new, still not fully understood, functions. These findings underline the complexity of the cardiac iron homeostasis.

Mechanism of frataxin "bypass" in human iron-sulfur cluster biosynthesis with implications for Friedreich's ataxia.

In humans, mitochondrial iron-sulfur cluster biosynthesis is an essential biochemical process mediated by the assembly complex consisting of cysteine desulfurase (NFS1), LYR protein (ISD11), acyl-carrier protein (ACP), and the iron-sulfur cluster assembly scaffold protein (ISCU2). The protein frataxin (FXN) is an allosteric activator that binds the assembly complex and stimulates the cysteine desulfurase and iron-sulfur cluster assembly activities. FXN depletion causes loss of activity of iron-sulfur-dependent enzymes and the development of the neurodegenerative disease Friedreich's ataxia. Recently, a mutation that suppressed the loss of the FXN homolog in Saccharomyces cerevisiae was identified that encodes an amino acid substitution equivalent to the human variant ISCU2 M140I. Here, we developed iron-sulfur cluster synthesis and transfer functional assays and determined that the human ISCU2 M140I variant can substitute for FXN in accelerating the rate of iron-sulfur cluster formation on the monothiol glutaredoxin (GRX5) acceptor protein. Incorporation of both FXN and the M140I substitution had an additive effect, suggesting an acceleration of distinct steps in iron-sulfur cluster biogenesis. In contrast to the canonical role of FXN in stimulating the formation of [2Fe-2S]-ISCU2 intermediates, we found here that the M140I substitution in ISCU2 promotes the transfer of iron-sulfur clusters to GRX5. Together, these results reveal an unexpected mechanism that replaces FXN-based stimulation of the iron-sulfur cluster biosynthetic pathway and suggest new strategies to overcome the loss of cellular FXN that may be relevant to the development of therapeutics for Friedreich's ataxia.

Assessment of cell-free levels of iron and copper in patients with Friedreich's ataxia.

Friedreich's ataxia (FRDA), a progressive neurodegenerative disorder caused by trinucleotide (GAA) repeat expansion in frataxin (fxn) gene which results in decreased levels of frataxin protein. Insufficient frataxin levels leads to iron and copper deposits in the brain and cardiac cells. A total of hundred and twenty patients, suspected of FRDA were screened for the (GAA) repeats in the fxn gene and only confirmed patients (n = 25) were recruited in the study. The total Iron and total copper concentrations were measured in blood plasma using Nitro PAPS and Dibrom PAESA method, respectively both in patients and age, sex matched healthy controls. The iron levels mean ± SD (6.2 ± 3.8) in plasma of FRDA patients were found to be significantly decreased as compared to healthy controls mean ± SD (15.2 ± 4.2). A similar trend was observed in case of plasma copper levels in FRDA patient (8.15 ± 4.6) as compared to controls (17.5 ± 3.40). Present results clearly prove abnormal distribution of extra-cellular iron in FRDA patients, which is in accordance with the well established fact of intracellular iron overload, which is the key feature of the pathogenesis of this disease. This can be of importance in understanding the pathophysiology of the disease in association with frataxin/iron. It appears that intracellular sequestration of trace metals in FRDA patients (due to low frataxin) results in their sub-optimal levels in blood plasma (extra-cellular) an observation that can find prognostic application in clinical trials.

Ferroptosis as a Novel Therapeutic Target for Friedreich's Ataxia.

Friedreich ataxia (FRDA) is a progressive neuro- and cardio-degenerative disorder characterized by ataxia, sensory loss, and hypertrophic cardiomyopathy. In most cases, the disorder is caused by GAA repeat expansions in the first introns of both alleles of the FXN gene, resulting in decreased expression of the encoded protein, frataxin. Frataxin localizes to the mitochondrial matrix and is required for iron-sulfur-cluster biosynthesis. Decreased expression of frataxin is associated with mitochondrial dysfunction, mitochondrial iron accumulation, and increased oxidative stress. Ferropotosis is a recently identified pathway of regulated, iron-dependent cell death, which is biochemically distinct from apoptosis. We evaluated whether there is evidence for ferroptotic pathway activation in cellular models of FRDA. We found that primary patient-derived fibroblasts, murine fibroblasts with FRDA-associated mutations, and murine fibroblasts in which a repeat expansion had been introduced (knockin/knockout) were more sensitive than normal control cells to erastin, a known ferroptosis inducer. We also found that the ferroptosis inhibitors ethyl 3-(benzylamino)-4-(cyclohexylamino)benzoate (SRS11-92) and ethyl 3-amino-4-(cyclohexylamino)benzoate, used at 500 nM, were efficacious in protecting human and mouse cellular models of FRDA treated with ferric ammonium citrate (FAC) and an inhibitor of glutathione synthesis [L-buthionine (S,R)-sulfoximine (BSO)], whereas caspase-3 inhibitors failed to show significant biologic activity. Cells treated with FAC and BSO consistently showed decreased glutathione-dependent peroxidase activity and increased lipid peroxidation, both hallmarks of ferroptosis. Finally, the ferroptosis inhibitor SRS11-92 decreased the cell death associated with frataxin knockdown in healthy human fibroblasts. Taken together, these data suggest that ferroptosis inhibitors may have therapeutic potential in FRDA.

Longitudinal evaluation of iron concentration and atrophy in the dentate nuclei in friedreich ataxia.

BACKGROUND: Friedreich ataxia is a recessively inherited, progressive neurological disease characterized by impaired mitochondrial iron metabolism. The dentate nuclei of the cerebellum are characteristic sites of neurodegeneration in the disease, but little is known of the longitudinal progression of abnormalities in these structures. METHODS: Using in vivo magnetic resonance imaging, including quantitative susceptibility mapping, we investigated changes in iron concentration and volume in the dentate nuclei in individuals with Friedreich ataxia (n = 20) and healthy controls (n = 18) over a 2-year period. RESULTS: The longitudinal rate of iron concentration was significantly elevated bilaterally in participants with Friedreich ataxia relative to healthy controls. Atrophy rates did not differ significantly between groups. Change in iron concentration and atrophy both correlated with baseline disease severity or duration, indicating sensitivity of these measures to disease stage. Specifically, atrophy was maximal in individuals early in the disease course, whereas the rate of iron concentration increased with disease progression. CONCLUSIONS: Progressive dentate nucleus abnormalities are evident in vivo in Friedreich ataxia, and the rates of change of iron concentration and atrophy in these structures are sensitive to the disease stage. The findings are consistent with an increased rate of iron concentration and atrophy early in the disease, followed by iron accumulation and stable volume in later stages. This pattern suggests that iron dysregulation persists after loss of the vulnerable neurons in the dentate. The significant changes observed over a 2-year period highlight the utility of quantitative susceptibility mapping as a longitudinal biomarker and staging tool. © 2019 International Parkinson and Movement Disorder Society.

GRP75 overexpression rescues frataxin deficiency and mitochondrial phenotypes in Friedreich ataxia cellular models.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by the deficiency of frataxin, a mitochondrial protein crucial for iron-sulfur cluster biogenesis and adenosine triphosphate (ATP) production. Currently, there is no therapy to slow down the progression of FRDA. Recent evidence indicates that posttranslational regulation of residual frataxin levels can rescue some of the functional deficit of FRDA, raising the possibility of enhancing levels of residual frataxin as a treatment for FRDA. Here, we present evidence that mitochondrial molecular chaperone GRP75, also known as mortalin/mthsp70/PBP74, directly interacts with frataxin both in vivo in mouse cortex and in vitro in cortical neurons. Overexpressing GRP75 increases the levels of both wild-type frataxin and clinically relevant missense frataxin variants in human embryonic kidney 293 cells, while clinical GRP75 variants such as R126W, A476T and P509S impair the binding of GRP75 with frataxin and the effect of GRP75 on frataxin levels. In addition, GRP75 overexpression rescues frataxin deficiency and abnormal cellular phenotypes such as the abnormal mitochondrial network and decreased ATP levels in FRDA patient-derived cells. The effect of GRP75 on frataxin might be in part mediated by the physical interaction between GRP75 and mitochondrial processing peptidase (MPP), which makes frataxin more accessible to MPP. As GRP75 levels are decreased in multiple cell types of FRDA patients, restoring GRP75 might be effective in treating both typical FRDA patients with two guanine-adenine-adenine repeat expansions and compound heterozygous patients with point mutations.

Identification of cardioprotective drugs by medium-scale in vivo pharmacological screening on a Drosophila cardiac model of Friedreich's ataxia.

Friedreich's ataxia (FA) is caused by reduced levels of frataxin, a highly conserved mitochondrial protein. There is currently no effective treatment for this disease, which is characterized by progressive neurodegeneration and cardiomyopathy, the latter being the most common cause of death in patients. We previously developed a Drosophila melanogaster cardiac model of FA, in which the fly frataxin is inactivated specifically in the heart, leading to heart dilatation and impaired systolic function. Methylene Blue (MB) was highly efficient to prevent these cardiac dysfunctions. Here, we used this model to screen in vivo the Prestwick Chemical Library, comprising 1280 compounds. Eleven drugs significantly reduced the cardiac dilatation, some of which may possibly lead to therapeutic applications in the future. The one with the strongest protective effects was paclitaxel, a microtubule-stabilizing drug. In parallel, we characterized the histological defects induced by frataxin deficiency in cardiomyocytes and observed strong sarcomere alterations with loss of striation of actin fibers, along with full disruption of the microtubule network. Paclitaxel and MB both improved these structural defects. Therefore, we propose that frataxin inactivation induces cardiac dysfunction through impaired sarcomere assembly or renewal due to microtubule destabilization, without excluding additional mechanisms. This study is the first drug screening of this extent performed in vivo on a Drosophila model of cardiac disease. Thus, it also brings the proof of concept that cardiac functional imaging in adult Drosophila flies is usable for medium-scale in vivo pharmacological screening, with potent identification of cardioprotective drugs in various contexts of cardiac diseases.

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.

The role of oxidative stress in Friedreich's ataxia.

Oxidative stress and an increase in the levels of free radicals are important markers associated with several pathologies, including Alzheimer's disease, cancer and diabetes. Friedreich's ataxia (FRDA) is an excellent paradigmatic example of a disease in which oxidative stress plays an important, albeit incompletely understood, role. FRDA is a rare genetic neurodegenerative disease that involves the partial silencing of frataxin, a small mitochondrial protein that was completely overlooked before being linked to FRDA. More than 20 years later, we now know how important this protein is in terms of being an essential and vital part of the machinery that produces iron-sulfur clusters in the cell. In this review, we revisit the most important steps that have brought us to our current understanding of the function of frataxin and its role in disease. We discuss the current hypotheses on the role of oxidative stress in FRDA and review some of the existing animal and cellular models. We also evaluate new techniques that can assist in the study of the disease mechanisms, as well as in our understanding of the interplay between primary and secondary phenotypes.