Human Mesenchymal Stem Cells Increase Anti-oxidant Defences in Cells Derived from Patients with Friedreich’s Ataxia

Friedreich??s ataxia (FRDA) is a progressive neurodegenerative disorder which is, at present, incurable. Oxidative damage and inhibition of mitochondrial function are key determinants of cellular damage in FRDA, since there is greater sensitivity to oxidative stress in cells with frataxin deficiency. In addition, frataxin-deficient cells have an impaired ability to recruit antioxidant defences against endogenous oxidative stress. We have recently shown that factors derived from bone marrow-derived mesenchymal stem cells (MSCs) increase hydrogen peroxide scavenging enzymes and offer protection against hydrogen peroxide-mediated injury in cells derived from patients with FRDA. Here we extend these studies and have performed a series of experiments showing that expression of superoxide dismutase (1 and 2) enzymes is reduced in FRDA cells but can be restored by treatment with conditioned medium from human MSCs. Furthermore, we have demonstrated that exposure to factors secreted by MSCs increases resistance to nitric oxide-induced oxidative stress in FRDA fibroblasts through, at least in part, restoring the expression of the superoxide dismuting enzymes and via modulation of PI₃ kinase/Akt pathways. These findings suggest that MSCs secrete factors that improve the cellular homeostasis of cells derived from FRDA patients and provide suitable support for their enhanced survival. This study further suggests the potential therapeutic use of MSCs in patients with FRDA.     

Therapeutic developments in friedreich ataxia

Friedreich ataxia is an inherited, severe, progressive neuro- and cardiodegenerative disorder for which there currently is no approved therapy. Friedreich ataxia is caused by the decreased expression and/or function of frataxin, a mitochondrial matrix protein that binds iron and is involved in the formation of iron-sulfur clusters. Decreased frataxin function leads to decreased iron-sulfur cluster formation, mitochondrial iron accumulation, cytosolic iron depletion, oxidative stress, and mitochondrial dysfunction. Cloning of the disease gene for Friedreich ataxia and elucidation of many aspects of the biochemical defects underlying the disorder have led to several major therapeutic initiatives aimed at increasing frataxin expression, reversing mitochondrial iron accumulation, and alleviating oxidative stress. These initiatives are in preclinical and clinical development and are reviewed herein.     

Progress in the treatment of Friedreich ataxia

Friedreich ataxia (FRDA) is a progressive neurological disorder affecting approximately 1 in 29,000 individuals of European descent. At present, there is no approved pharmacological treatment for this condition however research into treatment of FRDA has advanced considerably over the last two decades since the genetic cause was identified. Current proposed treatment strategies include decreasing oxidative stress, increasing cellular frataxin, improving mitochondrial function as well as modulating frataxin controlled metabolic pathways. Genetic and cell based therapies also hold great promise. Finally, physical therapies are being explored as a means of maximising function in those affected by FRDA.     

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.     

The molecular pathogenesis of Friedreich ataxia

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

Iron dysregulation in Friedreich ataxia

Friedreich ataxia is the most common hereditary ataxia. The signs and symptoms of the disorder derive from decreased expression of the protein frataxin, which is involved in iron metabolism. Frataxin chaperones iron for iron-sulfur cluster biogenesis and detoxifies iron in the mitochondrial matrix. Decreased expression of frataxin is associated with impairments of iron-sulfur cluster biogenesis and heme synthesis, as well as with mitochondrial dysfunction and oxidative stress. Compounds currently in clinical trials are directed toward improving mitochondrial function and lessening oxidative stress. Iron chelators and compounds that increase frataxin expression are under evaluation. Further elucidation of frataxin's function should lead to additional therapeutic approaches.     

Unanswered questions in friedreich ataxia

During the past 15 years, the pace of research advancement in Friedreich ataxia has been rapid. The abnormal gene has been discovered and its gene product characterized, leading to the development of new evidence-based therapies. Still, various unsettled issues remain that affect clinical trials. These include the level of frataxin deficiency needed to cause disease, the mechanism by which frataxin-deficient mitochondrial dysfunction leads to symptomatology, and the reason selected cells are most affected in Friedreich ataxia. In this review, we summarize these questions and propose testable hypotheses for their resolution.     

Friedreich ataxia: effects of genetic understanding on clinical evaluation and therapy

The discovery of the genetic cause of Friedreich ataxia has significantly affected our understanding of the disorder at both the clinical and basic science levels. Friedreich ataxia results from a deficiency of functional frataxin, a protein that appears to be involved in mitochondrial iron homeostasis. This leads to iron accumulation and mitochondrial abnormalities with consequent oxidant damage. The clinical spectrum of Friedreich ataxia has also expanded with the recognition of broader phenotypic features, including the absence of classical Friedreich ataxia features, later age at onset, and spasticity instead of ataxia. Although no proven therapy is yet available, antioxidants are a potential method for therapeutic intervention.     

Molecular pathogenesis of Friedreich ataxia.

Friedreich ataxia, the most common type of inherited ataxia, is itself caused in most cases by a large expansion of an intronic GAA repeat, resulting in decreased expression of the target frataxin gene. The autosomal recessive inheritance of the disease gives this triplet repeat mutation some unique features of natural history and evolution. Frataxin is a mitochondrial protein that has homologues in yeast and even in gram-negative bacteria. Yeast organisms deficient in the frataxin homologue accumulate iron in mitochondria and show increased sensitivity to oxidative stress. This suggests that Friedreich ataxia is caused by mitochondrial dysfunction and free radical toxicity.     

The pathogenesis of Friedreich ataxia and the structure and function of frataxin

Understanding the role of frataxin in mitochondria is key to an understanding of the pathogenesis of Friedreich ataxia. Frataxins are small essential proteins whose deficiency causes a range of metabolic disturbances, which include oxidative stress, deficit of iron-sulphur clusters, and defects in heme synthesis, sulfur amino acid and energy metabolism, stress response, and mitochondrial function. Structural studies carried out on different orthologues have shown that the frataxin fold consists of a flexible N-terminal region present only in eukaryotes and in a highly conserved C-terminal globular domain. Frataxins bind iron directly but with very unusual properties: iron coordination is achieved solely by glutamates and aspartates exposed on the protein surface. It has been suggested that frataxin function is that of a ferritin-like protein, an iron chaperone of the ironsulphur cluster machinery and heme metabolism and/or a controller of cellular oxidative stress. To understand FRDA pathogenesis and to design novel therapeutic strategies, we must first precisely identify the cellular role of frataxin.     

Cardiomyopathy in friedreich ataxia: clinical findings and research

Friedreich ataxia is the most common human ataxia and results from inadequate production of the frataxin protein, most often the result of a triplet expansion in the nuclear FXN gene. The gene cannot be transcribed to generate the messenger ribonucleic acid for frataxin. Frataxin is an iron-binding protein targeted to the mitochondrial matrix. In its absence, multiple iron-sulfur-dependent proteins in mitochondria and the cytosol lack proper assembly, destroying mitochondrial and nuclear function. Mitochondrial oxidant stress may also participate in ongoing cellular injury. Although progressive and debilitative ataxia is the most prominent clinical finding, hypertrophic cardiomyopathy with heart failure is the most common cause of early death in this disease. There is no cure. In this review the authors cover recent basic and clinical findings regarding the heart in Friedreich ataxia, offer recommendations for clinical management of the cardiomyopathy in this disease, and point out new research directions to advance the field.     

Clinical monitoring in a patient with friedreich ataxia and osteogenic sarcoma

Friedreich ataxia is an autosomal recessive neurodegenerative disorder caused by mutations in the FXN gene that result in abnormally low levels of the mitochondrial protein frataxin. The authors recently used a lateral flow immunoassay to measure frataxin levels in a large cohort of controls, carriers, and patients with the condition. The findings show that frataxin levels do not appreciably change over time and correlate well with GAA(1) repeat length and age of onset; thus, frataxin is a reliable and stable marker for severity of disease. In this article, the authors present a patient diagnosed as having Friedreich ataxia and osteosarcoma who received combined methotrexate, doxorubicin (Adriamycin), and cisplatin (MAP) chemotherapy over 8 months. The authors assessed the effect of treatment on frataxin levels, blood cell counts, and clinical markers of cardiomyopathy. Results of the regimen and the use of MAP chemotherapy for treatment of neoplasms in individuals with Friedreich ataxia are discussed.     

Iron–sulfur cluster synthesis,iron homeostasis and oxidative stress in Friedreich ataxia

Friedreich ataxia (FRDA) is an autosomal recessive, multi-systemic degenerative disease that results from reduced synthesis of the mitochondrial protein frataxin. Frataxin has been intensely studied since its deficiency was linked to FRDA in 1996. The defining properties of frataxin – (i) the ability to bind iron, (ii) the ability to interact with, and donate iron to, other iron-binding proteins, and (iii) the ability to oligomerize, store iron and control iron redox chemistry – have been extensively characterized with different frataxin orthologs and their interacting protein partners. This very large body of biochemical and structural data [reviewed in (Bencze et al., 2006)] supports equally extensive biological evidence that frataxin is critical for mitochondrial iron metabolism and overall cellular iron homeostasis and antioxidant protection [reviewed in (Wilson, 2006)]. However, the precise biological role of frataxin remains a matter of debate. Here, we review seminal and recent data that strongly link frataxin to the synthesis of iron–sulfur cluster cofactors (ISC), as well as controversial data that nevertheless link frataxin to additional iron-related processes. Finally, we discuss how defects in ISC synthesis could be a major (although likely not unique) contributor to the pathophysiology of FRDA via (i) loss of ISC-dependent enzymes, (ii) mitochondrial and cellular iron dysregulation, and (iii) enhanced iron-mediated oxidative stress. This article is part of a Special Issue entitled ‘Mitochondrial function and dysfunction in neurodegeneration’.     

In vivo assessment of OXPHOS capacity using 3 T CrCEST MRI in Friedreich’s ataxia

Journal of Neurology - Friedreich’s ataxia (FRDA) is a neurodegenerative disease caused by decreased expression of frataxin, a protein involved in many cellular metabolic processes, including...     

Iron metabolism in mice with partial frataxin deficiency

Friedreich ataxia (FRDA), the most common autosomal recessive inherited ataxic disorder, is the consequence of deficiency of the mitochondrial protein frataxin, typically caused by homozygous intronic GAA expansions in the corresponding gene. The yeast frataxin homologue (yfh1p) is required for cellular respiration. Yfh1p appears to regulate mitochondrial iron homeostasis and protect from free radical toxicity. Complete loss of frataxin in knockout mice leads to early embryonic lethality, indicating an important role for frataxin during development. Heterozygous littermates with partial frataxin deficiency are apparently healthy and have no obvious phenotype. Here we evaluate iron metabolism and sensitivity to dietary and parenteral iron loading in heterozygote frataxin knockout mice (Fx(+/-)). Iron concentrations in the liver, heart, pancreas and spleen, and cellular iron distribution patterns were compared between wild type and Fx(+/-) mice. Response to parenteral iron challenge was not different between Fx(+/-) mice and wild type littermates, while sporadic iron deposits were observed in the hearts of dietary iron-loaded Fx(+/-) mice. Finally, we evaluated the effect of partial frataxin deficiency on susceptibility to cardiac damage in the mouse model of hereditary hemochromatosis (HH), the Hfe knockout mice. HH, an iron overload disease, is one of the most frequent genetic diseases in populations of European origin. By breeding Hfe(-/-) with Fx(+/-) mice, we obtained compound mutant mice lacking both Hfe and one frataxin allele. Sparse iron deposits in areas of mild to moderate cardiac fibrosis were found in the majority of these mice. However, they did not develop any neurological symptoms. Our studies indicate an association between frataxin deficiency, iron deposits and cardiac fibrosis, but no obvious association between iron accumulation and neurodegeneration similar to FRDA could be detected in our model. In addition, these results suggest that frataxin mutations may have a modifier role in HH, that predisposes to cardiomyopathy.     

Mitochondrial impairment of human muscle in Friedreich ataxia in vivo

Friedreich ataxia occurs due to mutations in the gene encoding the mitochondrial protein frataxin. This (31)P magnetic resonance spectroscopy study on the calf muscle of Friedreich ataxia patients provides in vivo evidence of a severe impairment of mitochondrial function. Mitochondrial adenosine triphosphate resynthesis was studied by means of the post-exercise recovery of phosphocreatine. After ischemic exercise in calf muscles of all patients, phosphocreatine recovery was dramatically delayed. Time constants of recovery correlated with mutations of the frataxin gene, the age of the patients, and disease duration. (31)P magnetic resonance spectroscopy represents the first expedient tool for monitoring therapeutic trials in Friedreich ataxia non-invasively.     

Friedreich's ataxia

Friedreich’s ataxia, the most common hereditary ataxia, is caused by expansion of a GAA triplet located within the first intron of the frataxin gene on chromosome 9q13. There is a clear correlation between size of the expanded repeat and severity of the phenotype. Frataxin is a mitochondrial protein that plays a role in iron homeostasis. Deficiency of frataxin results in mitochondrial iron accumulation, defects in specific mitochondrial enzymes, enhanced sensitivity to oxidative stress, and eventually free-radical mediated cell death. Friedreich’s ataxia is considered a nuclear encoded mitochondrial disease.

This review discusses the major and rapid progress made in Friedreich’s ataxia from gene mapping and identification of the gene to pathogenesis and encouraging therapeutic implications.     

Friedreich's ataxia: past, present and future

Friedreich's ataxia (FRDA) is an autosomal recessive inherited disorder characterized by progressive gait and limb ataxia, dysarthria, areflexia, loss of vibratory and position sense, and a progressive motor weakness of central origin. Additional features include hypertrophic cardiomyopathy and diabetes. Large GAA repeat expansions in the first intron of the FXN gene are the most common mutation underlying FRDA. Patients show severely reduced levels of a FXN-encoded mitochondrial protein called frataxin. Frataxin deficiency is associated with abnormalities of iron metabolism: decreased iron-sulfur cluster (ISC) biogenesis, accumulation of iron in mitochondria and depletion in the cytosol, enhanced cellular iron uptake. Some models have also shown reduced heme synthesis. Evidence for oxidative stress has been reported. Respiratory chain dysfunction aggravates oxidative stress by increasing leakage of electrons and the formation of superoxide. In vitro studies have demonstrated that Frataxin deficient cells not only generate more free radicals, but also show a reduced capacity to mobilize antioxidant defenses. The search for experimental drugs increasing the amount of frataxin is a very active and timely area of investigation. In cellular and in animal model systems, the replacement of frataxin function seems to alleviate the symptoms or even completely reverse the phenotype. Therefore, drugs increasing the amount of frataxin are attractive candidates for novel therapies. This review will discuss recent findings on FRDA pathogenesis, frataxin function, new treatments, as well as recent animal and cellular models. Controversial aspects are also discussed.     

Therapeutic strategies in Friedreich's ataxia

Friedreich's ataxia is caused by a pronounced lack of frataxin, a mitochondrial protein of not fully understood function. Lack of frataxin homologues in yeast and mice leads to increased sensitivity to oxidative stress, depletion of proteins with iron-sulfur clusters like respiratory chain complexes I-III and aconitase, and to iron accumulation in mitochondria. Similar effects have been demonstrated in human disease with increased markers of oxidative DNA damage in urine and impaired oxidative phosphorylation in in vivo exercise studies using 31 Phosphorus magnetic resonance spectroscopy (31P-MRS). Therapeutical trials mainly focus on antioxidative treatment with coenzyme Q10 or its short-chain variant idebenone. Promising effects on cardiac hypertrophy in uncontrolled preliminary studies contrast with minor effects in controlled trials and no effect of antioxidants on neurological deficits has been established. Preliminary encouraging 31P-MRS data exist for the treatment with L-carnitine but not with creatine. However, all these interventions may take effect too late in the pathogenic process. Alternative strategies aiming at an enhancement of frataxin by stem cell transplantation, gene transfer or frataxin supplementation are desirable. Additionally, more efficient biomarkers are needed to monitor treatment effects.