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.     

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.     

Iron-dependent functions of mitochondria—relation to neurodegeneration

A number of neurodegenerative diseases are associated with iron dyshomeostasis and mitochondrial dysfunction. However, the pathomechanistic interplay between iron and mitochondria varies. This review summarises the physiological role of iron in mitochondria and subsequently exemplifies two neurodegenerative diseases with disturbed iron function in mitochondria: inherited Friedreich ataxia (FRDA) and idiopathic Parkinson disease (PD). In eukaryotes, mitochondria are main consumers of iron. The respiratory chain relies on iron-containing redox systems in the form of complexes I–III with iron–sulphur clusters and cytochromes with haem as prosthetic groups. The bifunctional enzyme aconitase is not only important in the citric acid cycle, but also functions as a key regulator of cell iron metabolism. Haem biosynthesis occurs partially in mitochondria as well as the biogenesis of iron–sulphur clusters that are co-factors in numerous iron–sulphur proteins. FRDA is characterised by a mutation of the frataxin gene, the protein of which serves as an iron chaperone in iron–sulphur cluster assembly. The lack of frataxin expression leads to defective iron–sulphur cluster biogenesis with decreased respiratory and aconitase activity. The resulting mitochondrial iron overload might fuel reactive oxygen species formation and contribute to clinical signs of oxidative stress. PD is typically associated with an increased iron content of the substantia nigra, the causes of which are largely unknown. Recent research demonstrated raised iron levels in individual dopaminergic neurons of the substantia nigra. Moreover, transferrin/transferrin receptor 2 mediated transport of iron into the mitochondria of these neurons was identified together with increased transferrin immunoreactivity. Resulting accumulation of iron into mitochondria might lead to oxidative stress damaging iron–sulphur cluster-containing proteins.     

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.     

Friedreich ataxia: new pathways

Friedreich ataxia is a rare disorder characterized by an autosomal recessive pattern of inheritance. The disease is noted for a constellation of clinical symptoms, notably loss of coordination and a variety of neurologic and cardiac complications. More recently, scientists have focused their research on an array of general investigations of the underlying cellular basis for the disease, including mitochondrial biogenesis, iron-sulfur cluster synthesis, iron metabolism, antioxidant responses, and mitophagy. Combined with investigations that have explored the pathogenesis of the disease and the function of the protein frataxin, these studies have led to insights that will be key to identifying new therapeutic strategies for treating the disease.     

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.     

Review: Iron metabolism and the role of iron in neurodegenerative disorders

Iron plays a role for the biogenesis of two important redox‐reactive prosthetic groups of enzymes, iron sulphur clusters (ISC) and heme. A part of these biosynthetic pathways takes plays in the mitochondria. While several important proteins of cellular iron uptake and storage and of mitochondrial iron metabolism are well‐characterized, limited knowledge exists regarding the mitochondrial iron importers (mitoferrins). A disturbed distribution of iron, hampered Fe‐dependent biosynthetic pathways and eventually oxidative stress resulting from an increased labile iron pool are suggested to play a role in several neurodegenerative diseases. Friedreich's ataxia is associated with mitochondrial iron accumulation and hampered ISC/heme biogenesis due to reduced frataxin expression, thus representing a monogenic mitochondrial disorder, which is clearly elicited solely by a disturbed iron metabolism. Less clear are the controversially discussed impacts of iron dysregulation and iron‐dependent oxidative stress in the most common neurodegenerative disorders, i.e. Alzheimer's disease (AD) and Parkinson's disease (PD). Amyotrophic lateral sclerosis (ALS) may be viewed as a disease offering a better support for a direct link between iron, oxidative stress and regional neurodegeneration. Altogether, despite significant progress in molecular knowledge, the true impact of iron on the sporadic forms of AD, PD and ALS is still uncertain. Here we summarize the current knowledge of iron metabolism disturbances in neurodegenerative disorders.     

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.     

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.     

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.     

Mesenchymal Stem Cell-Derived Factors Restore Function to Human Frataxin-Deficient Cells

Friedreich’s ataxia is an inherited neurological disorder characterised by mitochondrial dysfunction and increased susceptibility to oxidative stress. At present, no therapy has been shown to reduce disease progression. Strategies being trialled to treat Friedreich’s ataxia include drugs that improve mitochondrial function and reduce oxidative injury. In addition, stem cells have been investigated as a potential therapeutic approach. We have used siRNA-induced knockdown of frataxin in SH-SY5Y cells as an in vitro cellular model for Friedreich’s ataxia. Knockdown of frataxin protein expression to levels detected in patients with the disorder was achieved, leading to decreased cellular viability, increased susceptibility to hydrogen peroxide-induced oxidative stress, dysregulation of key anti-oxidant molecules and deficiencies in both cell proliferation and differentiation. Bone marrow stem cells are being investigated extensively as potential treatments for a wide range of neurological disorders, including Friedreich’s ataxia. The potential neuroprotective effects of bone marrow-derived mesenchymal stem cells were therefore studied using our frataxin-deficient cell model. Soluble factors secreted by mesenchymal stem cells protected against cellular changes induced by frataxin deficiency, leading to restoration in frataxin levels and anti-oxidant defences, improved survival against oxidative stress and stimulated both cell proliferation and differentiation down the Schwann cell lineage. The demonstration that mesenchymal stem cell-derived factors can restore cellular homeostasis and function to frataxin-deficient cells further suggests that they may have potential therapeutic benefits for patients with Friedreich’s ataxia.     

Friedreich ataxia

There has been rapid progress in the understanding of several aspects of Friedreich's ataxia (FA) since the gene mutation was identified in 1996. At the clinical level, now it is possible to confirm that the majority of patients fullfilling clinical criteria for classic FA have the FA gene mutation but some do not, indicating genetic heterogeneity. Also, the phenotype associated with the FA mutation is much wider than that defined by clinical criteria and includes ataxia with retained or brisk reflexes as well as late onset ataxia with or without retained reflexes. It is now clear that the unstable GAA expansion that underlies FA causes a deficiency of the mitochondrial protein frataxin, leading to potentially harmful oxidative injury associated with excessive iron deposits in mitochondria. In addition, pathogenesis may involve a primary defect in synthesis of iron-sulfur cluster containing enzymes. Therapeutic attempts are already using anti-oxidant strategies and such efforts are likely to be enhanced by the rapid availability of animal models of the disease.     

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’.     

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 and iron metabolism

The possible causes of abnormal iron metabolism in patients with Friedreich's ataxia are considered. Reduced expression of a frataxin homologue in yeast is associated with mitochondrial iron accumulation at the expense of cytosolic iron, and the same phenomenon can be demonstrated in these patients. A decrease in cytosolic iron causes the expression of a high-affinity iron-uptake protein, and therefore Friedreich's ataxia can be considered to be a disease of abnormal intracellular iron distribution. Friedreich's ataxia is of autosomal recessive inheritance, and the gene associated with it has been mapped to chromosome 9. This encodes the protein frataxin which regulates mitochondrial iron transport. The commonest mutation causing this disorder is an expanded GAA repeat in the gene for this protein. Different point mutations may account for some of the variations in the phenotypic features that are often found, and these variations are discussed. These findings have raised therapeutic possibilities in a condition for which previously there was no specific treatment. There are intracellular enzymes which are very sensitive to injury by oxygen-free radicals. Treatment has therefore been tried with ibebenone which acts as a free-radical scavenger, with some evidence of improvement. Iron chelating agents, such as deferoxamine, have also been given, but the finding of normal serum iron and ferritin casts doubt on the rationale of this. However the finding that the accumulation of iron in the mitochondria of the cells in patients with this form of ataxia will cause oxidative stress and cell death, gives hope for more effective treatment in the future, possibly with gene therapy.     

Iron and Friedreich ataxia

Friedreich ataxia is due to insufficient levels of frataxin, a mitochondrial iron chaperone that shields this metal from reactive oxygen species (ROS) and renders it bioavailable as Fe II. Frataxin participates in the synthesis of iron-sulfur clusters (ISCs), cofactors of several enzymes, including mitochondrial and cytosolic aconitase, complexes I, II and III of the respiratory chain, and ferrochelatase. It also plays a role in the maintenance of ISCs, in particular for mitochondrial aconitase. A role of frataxin in heme synthesis has been postulated, but is controversial. Insufficient frataxin leads to deficit of ISC enzymes and energy deficit. Iron levels increase in mitochondria. Oxidative stress may result from respiratory chain dysfunction and from direct reaction between iron and ROS. Stress pathways are activated that may lead to apoptosis or other forms of cell death. The basis for the selective vulnerability of specific neurons, like sensory neurons, is still unknown.     

Effects of Erythropoietin on Frataxin Levels and Mitochondrial Function in Friedreich Ataxia �C a Dose�CResponse Trial

Friedreich ataxia (FRDA) is an autosomal recessive inherited neurodegenerative disorder leading to reduced expression of the mitochondrial protein frataxin. Previous studies showed frataxin upregulation in FRDA following treatment with recombinant human erythropoietin (rhuEPO). Dose-response interactions between frataxin and rhuEPO have not been studied until to date. We administered escalating rhuEPO single doses (5,000, 10,000 and 30,000?IU) in monthly intervals to five adult FRDA patients. Measurements of frataxin, serum erythropoietin levels, iron metabolism and mitochondrial function were carried out. Clinical outcome was assessed using the "Scale for the assessment and rating of ataxia". We found maximal erythropoietin serum concentrations 24?h after rhuEPO application which is comparable to healthy subjects. Frataxin levels increased significantly over 3?months, while ataxia rating did not reveal clinical improvement. All FRDA patients had considerable ferritin decrease. NADH/NAD ratio, an indicator of mitochondrial function, increased following rhuEPO treatment. In addition to frataxin upregulation in response to continuous low-dose rhuEPO application shown in previous studies, our results indicate for a long-lasting frataxin increase after single high-dose rhuEPO administration. To detect frataxin-derived neuroprotective effects resulting in clinically relevant improvement, well-designed studies with extended time frame are required.     

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.