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.     

Quantitative profiling and identification of differentially expressed plasma proteins in friedreich's ataxia

Friedreich's ataxia (FRDA) is an autosomal recessive ataxia, characterized by progressive gait ataxia, limb ataxia, dysarthria, and areflexia associated with diabetes and hypertrophic cardiomyopathy. The primary cause of FRDA is the presence of expanded DNA triplet (GAA) repeats in the first intron of the fxn gene on chromosome 9q13. The expanded DNA repeats in fxn inhibit expression of the protein frataxin, which leads to neuronal degeneration. The aim of the study was to identify differentially expressed plasma proteins in FRDA patients for their diagnostic/prognostic applications. Clinically suspected FRDA patients (n = 42) were assessed on the International Co‐Operative Ataxia Rating Scale (ICARS), and genetic confirmation was performed by analyzing (GAA) repeats via PCR. Eighteen patients were confirmed to be homozygous for FRDA, with ICARS scores of 40 ± 8. Plasma proteomics of homozygous FRDA patients and age‐ and gender‐matched healthy controls was done using two‐dimensional difference in‐gel electrophoresis and LC‐MS/MS. Quantitative proteomic analysis (fold change ≥1.5; P < 0.05) revealed 13 differentially expressed protein spots. These proteins were found to be associated with neuropathy (α1‐antitrypsin), ataxia (apolipoprotein A‐I), oxidative stress (albumin), altered lipid metabolism (apolipoprotein C‐II, C‐III), etc. Further investigations of these differentially expressed proteins can aid in identifying prognostic/diagnostic markers for FRDA. © 2013 Wiley Periodicals, Inc.     

Comparison of three clinical rating scales in Friedreich ataxia (FRDA)

To test the validity and reliability of the scale for the assessment and rating of ataxia (SARA) in Friedreich ataxia (FRDA). SARA is limited to eight items and can be performed rapidly. Ninety‐six patients with a molecular genetic diagnosis of FRDA were rated using three different clinical scales, the FRDA Rating Scale (FARS), the International Cooperative Ataxia Rating Scale (ICARS), and SARA. Despite considerable discrepancies in scale size and subscale structure, SARA total scores were significantly correlated with ICARS (r = 0.953, P < 0.0001) and FARS (r = 0.938, P < 0.0001) total scores. SARA total scores also correlated with the activities of daily living (ADL, r = 0.929, P < 0.0001). Although originally developed for the use in dominantly inherited ataxias, which are primarily ataxias of the cerebellar type, SARA can also be used successfully to assess afferent ataxia, which is the predominant form in FRDA. Because SARA is characterized by high interrater reliability and practicability, SARA is applicable and well suited forclinical trials of FRDA. © 2009 Movement Disorder Society     

Coenzyme Q10 and vitamin E deficiency in Friedreich’s ataxia: predictor of efficacy of vitamin E and coenzyme Q10 therapy

Background and purpose: A pilot study of high dose coenzyme Q₁₀ (CoQ₁₀)/vitamin E therapy in Friedreich’s ataxia (FRDA) patients resulted in significant clinical improvements in most patients. This study investigated the potential for this treatment to modify clinical progression in FRDA in a randomized double blind trial. Methods: Fifty FRDA patients were randomly divided into high or low dose CoQ₁₀/ vitamin E groups. The change in International Co‐operative Ataxia Ratings Scale (ICARS) was assessed over 2 years as the primary end‐point. A post hoc analysis was made using cross‐sectional data. Results: At baseline serum CoQ₁₀ and vitamin E levels were significantly decreased in the FRDA patients (P < 0.001). During the trial CoQ₁₀ and vitamin E levels significantly increased in both groups (P < 0.01). The primary and secondary end‐points were not significantly different between the therapy groups. When compared to cross‐sectional data 49% of all patients demonstrated improved ICARS scores. This responder group had significantly lower baseline serum CoQ₁₀ levels. Conclusions: A high proportion of FRDA patients have a decreased serum CoQ₁₀ level which was the best predictor of a positive clinical response to CoQ₁₀/vitamin E therapy. Low and high dose CoQ₁₀/vitamin E therapies were equally effective in improving ICARS scores.     

Friedreich's ataxia: clinical pilot trial with recombinant human erythropoietin

To determine the role of recombinant human erythropoietin as a possible treatment option in Friedreich's ataxia, we performed an open-label clinical pilot study. Primary outcome measure was the change of frataxin levels at week 8 versus baseline. Twelve Friedreich's ataxia patients received 5,000 units recombinant human erythropoietin three times weekly subcutaneously. Frataxin levels were measured in isolated lymphocytes by enzyme-linked immunosorbent assay. In addition, urinary 8-hydroxydeoxyguanosine and serum peroxides, were measured. Treatment with recombinant human erythropoietin showed a persistent and significant increase in frataxin levels after 8 weeks (p < 0.01). All patients showed a reduction of oxidative stress markers.     

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.     

Variations of frataxin protein levels in normal individuals

Friedreich’s ataxia (FRDA) is the most common of the inherited ataxias and is associated with GAA trinucleotide repeat expansions within the first intron of the frataxin (FXN) gene. There are expanded FXN alleles from 66 to 1,700 GAA·TTC repeats in FRDA patients and correlations between number of GAA repeats and frataxin protein levels are assumed. Here, we present for the first time frataxin protein levels as well as analysis of GAA triplet repeats in the FXN gene in a population of 50 healthy Austrian people. Frataxin protein levels were measured in lymphocytes from blood samples by ELISA and GAA repeats were analyzed by capillary electrophoresis. Rather unexpectedly, we found a high variation of frataxin protein levels among the individuals. In addition, there was no correlation between frataxin levels, GAA repeats, age and sex in this group. However, these findings are of great importance for better characterization of the disease.     

Skeletal muscle involvement in friedreich ataxia and potential effects of recombinant human erythropoietin administration on muscle regeneration and neovascularization

Friedreich ataxia (FRDA) is caused by reduced expression of the mitochondrial protein frataxin. Cardiac muscle involvement has been attributed to mitochondrial dysfunction, but involvement of skeletal muscle has not been fully investigated. Improved motor skills in FRDA patients after administration of recombinant human erythropoietin (rhuEPO) have been reported. To elucidate the characteristics of skeletal muscle in FRDA and assess the potential effects of rhuEPO on skeletal muscle neovascularization and regeneration, 7 genetically confirmed FRDA patients underwent biopsy of the gastrocnemius muscle before and after administration of 3,000 international units of rhuEPO 3 times per week for 2 months. Muscle tissue was investigated using standard histologic methods, immunohistochemistry, and biochemical assays of mitochondrial enzymes. In pretreatment FRDA samples, there were neurogenic and myopathic changes and reduced capillary density versus that in healthy control biopsies (n = 4). Satellite cells were increased, but markers of satellite cell activation and differentiation did not differ from controls. Respiratory chain complex and citrate synthase activities were reduced in FRDA and remained unchanged after treatment. Administration of rhuEPO resulted in increases in muscle capillary densities and in endothelial progenitor cells in peripheral blood. These data indicate that there are morphological and biochemical abnormalities of skeletal muscle in FRDA. The rhuEPO-induced changes were subtle, but increased capillary density might result in improved oxygen supply and myofiber function.     

In children with Friedreich ataxia, muscle and ataxia parameters are associated

Aim In children with Friedreich ataxia (FRDA), ataxia is assessed using the surrogate marker the International Cooperative Ataxia Rating Scale (ICARS). We aimed to determine whether ICARS scores in children with FRDA are confounded by muscle weakness. Method In 12 children with FRDA (10 males, two females; mean age 13y 6mo, SD 2y 6mo) and 12 age‐matched children without FRDA (nine males; three females), we determined the association between muscle and ataxia parameters (i.e. muscle ultrasound density (MUD), muscle force, sensory evoked potentials, and ICARS scores). Children with FRDA were included on the basis of FXN gene analysis. Children in the comparison group were included on basis of uneventful pregnancy and normal cognitive and neurological development. Results In children with FRDA, muscle ultrasound density was homogeneously increased in the biceps, quadriceps, and tibialis anterior muscles (median 4SD). FRDA muscle weakness was significantly more pronounced in proximal than in distal muscles (−2SD vs −0.5SD respectively; p=0.004), with a stronger impairment of leg muscles than of arm muscles (−2SD vs −0. SD respectively; p=0.001). Comparing MUD between children with FRDA and an age‐matched comparison group revealed a relatively strong increase in MUD in the proximal leg muscles in the FRDA group. Under the condition of persistently absent sensory evoked potentials, leg ICARS subscores in the FRDA group appeared to be positively associated with leg muscle force until a maximal plateau level of ICARS subscores was reached. Interpretation In children with FRDA, ataxia scales based on ICARS are confounded by muscle weakness. Longitudinal ICARS evaluations in children with FRDA do not necessarily indicate altered ataxia.     

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’.     

Atypical Friedreich ataxia in patients with FXN p.R165P point mutation or comorbid hemochromatosis

BackgroundCompound heterozygosity for a trinucleotide repeat expansion and a point mutation in the FXN gene is a rare cause of Friedreich ataxia (FRDA).MethodsWe identified three Swedish FRDA patients with an FXN p.R165P missense mutation and compared their clinical features with six homozygote trinucleotide repeat expansion carriers. Patients were assessed clinically. Trinucleotide expansion length was determined and lymphocyte frataxin levels measured.Resultsp.R165P mutation carriers became wheelchair bound early, but had retained reflexes, better arm function, milder dysarthria, and were more independent in activities of daily living. One p.R165P mutation carrier developed psychosis. Frataxin levels were higher than in homozygous trinucleotide expansion patients. One patient with homozygous trinucleotide repeat expansions and comorbid hemochromatosis had more severe FRDA symptoms than his sibling without hemochromatosis.Conclusionp.R165P patients progress to a less disabling disease state than typical FRDA. Comorbid hemochromatosis may worsen FRDA symptoms through additive effects on iron metabolism.     

Radial diffusivity in the cerebellar peduncles correlates with clinical severity in Friedreich ataxia

Friedreich ataxia (FRDA) is a common inherited ataxia, caused by an expanded GAA repeat sequence in the Frataxin (FXN) gene. The proprioceptive system, which enters the cerebellum through the cerebellar peduncles, is a primary focus of pathology. In this study, we investigate the relationship of clinical and genetic data with diffusion-tensor imaging (DTI) indices reflecting white matter integrity of the cerebellar peduncles. Nine FRDA patients underwent DTI. After between-subject registration using tract-based spatial statistics, a white matter atlas was used for computing average values of DTI indices in the regions of interest. These were the inferior, middle and superior cerebellar peduncles (ICP, MCP, SCP). For Bonferroni correction, significance threshold was set to p < 0.0056. We found that radial diffusivity (D^⊥) within the ICP significantly correlated with scores on the Friedreich Ataxia Rating Scale (FARS, Spearman’s ρ = 0.883, p = 0.0016, all two-sided) and, at trend level, with number of trinucleotide repeats (ρ = 0.812, p = 0.008). D^⊥ in the SCP correlated with scores on the Scale for the Assessment and Rating of Ataxia (SARA, ρ = 0.867, p = 0.0025). These findings support the role of DTI, and especially D^⊥, as an informative biomarker in FRDA.     

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.     

Combined Cerebellar Proton MR Spectroscopy and DWI Study of Patients with Friedreich’s Ataxia

Friedreich’s ataxia (FRDA) is the commonest autosomal recessive ataxia, caused by GAA triplet expansion in the frataxin gene. Neuropathological studies in FRDA demonstrate that besides the primary neurodegeneration of the dorsal root ganglia, there is a progressive atrophy of the cerebellar dentate nucleus. Diffusion-weighted imaging (DWI) detected microstructural alterations in the cerebellum of FRDA patients. To investigate the biochemical basis of these alterations, we used both DWI and proton MR spectroscopy (¹H-MRS) to study the same cerebellar volume of interest (VOI) including the dentate nucleus. DWI and ¹H-MRS study of the left cerebellar hemisphere was performed in 28 genetically proven FRDA patients and 35 healthy controls. In FRDA mean diffusivity (MD) values were calculated for the same ¹H-MRS VOI. Clinical severity was evaluated using the International Cooperative Ataxia Rating Scale (ICARS). FRDA patients showed a significant reduction of N-acetyl-aspartate (NAA), a neuroaxonal marker, and choline (Cho), a membrane marker, both expressed relatively to creatine (Cr), and increased MD values. In FRDA patients NAA/Cr negatively correlated with MD values (r?=??0.396, p?=?0.037) and with ICARS score (r?=??0.669, p?<?0.001). Age-normalized NAA/Cr loss correlated with the GAA expansion (r?=??0.492, p?=?0.008). The reduced cerebellar NAA/Cr in FRDA suggests that neuroaxonal loss is related to the microstructural changes determining higher MD values. The correlation between NAA/Cr and the severity of disability suggests that this biochemical in vivo MR parameter might be a useful biomarker to evaluate therapeutic interventions.     

Multicellular models of Friedreich ataxia

Patients with Friedreich ataxia (FRDA) have severely reduced levels of the mitochondrial protein frataxin, which results from a large GAA triplet-repeat expansion within the frataxin gene (FXN). High evolutionary conservation of frataxin across species has enabled the development of disease models of FRDA in various unicellular and multicellular organisms. Mouse models include classical knockout models, in which the Fxn gene is constitutively inactivated, and knock-in models, in which a GAA repeat mutation or the conditional allele is inserted into the genome. Recently, “humanised” GAA repeat expansion mouse models were obtained by combining the constitutive knockout with the transgenic expression of a yeast artificial chromosome carrying the human FRDA locus. In lower organisms such as Caenorhabditis elegans and Drosophila, straight-forward and conditional RNA interference technology has provided an easy way to knock down frataxin expression. Conditional mouse models have been used for pre-clinical trials of potential therapeutic agents, including idebenone, MnTBAP (a superoxide dismutase mimetic), and iron chelators. Various models of FRDA have shown that different, even opposite, phenotypes can be observed, depending on the level of frataxin expression. Additional studies with animal models will be essential for an enhanced understanding of the disease pathophysiology and for the development of better therapies.     

Cardiomyopathy in Friedreich's ataxia

Friedreich's ataxia (FRDA), an autosomal recessive disorder, is characterized by spinocerebellar degeneration and cardiomyopathy. Here we explore some of the putative mechanisms underlying the cardiomyopathy in FRDA that have been elucidated using different experimental models. FRDA is characterized by a deficiency in frataxin, a protein vital in iron handling. Iron accumulation, lack of functional iron-sulphur clusters, and oxidative stress seem to be among the most important consequences of frataxin deficiency explaining the cardiac abnormalities in FRDA.     

Neurochemical alterations in spinocerebellar ataxia type 1 and their correlations with clinical status

Robust biomarkers of neurodegeneration are critical for testing of neuroprotective therapies. The clinical applicability of such biomarkers requires sufficient sensitivity to detect disease in individuals. Here we tested the sensitivity of high field (4 tesla) proton magnetic resonance spectroscopy (¹H MRS) to neurochemical alterations in the cerebellum and brainstem in spinocerebellar ataxia type 1 (SCA1). We measured neurochemical profiles that consisted of 10 to 15 metabolite concentrations in the vermis, cerebellar hemispheres and pons of patients with SCA1 (N = 9) and healthy controls (N = 15). Total NAA (N‐acetylaspartate + N‐acetylaspartylglutamate, tNAA) and glutamate were lower and glutamine, myo‐inositol and total creatine (creatine + phosphocreatine, tCr) were higher in patients relative to controls, consistent with neuronal dysfunction/loss, gliotic activity, and alterations in glutamate–glutamine cycling and energy metabolism. Changes in tNAA, tCr, myo‐inositol, and glutamate levels were discernible in individual spectra and the tNAA/myo‐inositol ratio in the cerebellar hemipheres and pons differentiated the patients from controls with 100% specificity and sensitivity. In addition, tNAA, myo‐inositol, and glutamate levels in the cerebellar hemispheres and the tNAA and myo‐inositol levels in the pons correlated with ataxia scores (Scale for the Assessment and Rating of Ataxia, SARA). Two other biomarkers measured in the cerebrospinal fluid (CSF) of a subset of the volunteers (F₂‐isoprostanes asa marker of oxidative stress and glial fibrillary acidic protein (GFAP) as a marker of gliosis) were not different between patients and controls. These data demonstrate that ¹H MRS biomarkers can be utilized to noninvasively assess neuronal and glial status in individual ataxia patients. © 2010 Movement Disorder Society     

Frataxin gene point mutations in Italian Friedreich ataxia patients

Friedreich ataxia (FRDA) is associated with a GAA-trinucleotide-repeat expansion in the first intron of the FXN gene (9q13-21), which encodes a 210-amino-acid protein named frataxin. More than 95% of patients are homozygous for 90-1,300 repeat expansion on both alleles. The remaining patients have been shown to be compound heterozygous for a GAA expansion on one allele and a micromutation on the other. The reduction of both frataxin messenger RNA (mRNA) and protein was found to be proportional to the size of the smaller GAA repeat allele. We report a clinical and molecular study of 12 families in which classical FRDA patients were heterozygous for a GAA expansion on one allele. Sequence analysis of the FXN gene allowed the identification of the second disease-causing mutation in each heterozygous patient, which makes this the second largest series of FRDA compound heterozygotes reported thus far. We have identified seven mutations, four of which are novel. Five patients carried missense mutations, whereas eight patients carried null (frameshift or nonsense) mutations. Quantitation of frataxin levels in lymphoblastoid cell lines derived from six compound heterozygous patients showed a statistically significant correlation of residual protein levels with the age at onset (r = 0.82, p < 0.05) or the GAA expansion (r = -0.76, p < 0.1). In the group of patients heterozygous for a null allele, a strong (r = -0.94, p < 0.01) correlation was observed between the size of GAA expansion and the age at onset, thus lending support to the hypothesis that the residual function of frataxin in patients' cells derive exclusively from the expanded allele.     

Sudomotor dysfunction is frequent and correlates with disability in Friedreich ataxia

Objectives

To evaluate autonomic symptoms and function in Friedreich’s Ataxia (FRDA).