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.     

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.     

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.     

Normal serum iron and ferritin concentrations in patients with Friedreich's ataxia

Friedreich's ataxia (FRDA) is caused by point mutations or trinucleotide repeat expansions in both alleles of the gene encoding frataxin. Studies of frataxin homologues in lower eukaryotes suggest that mitochondrial iron accumulation may underlie the pathophysiology of FRDA. To evaluate the possible role of iron-chelation therapy for FRDA, we measured serum iron and ferritin concentration in 10 FRDA patients. The measurements were within normal limits, suggesting that iron-chelation therapy for FRDA may be problematic.     

Friedreich's ataxia with chorea and myoclonus caused by a compound heterozygosity for a novel deletion and the trinucleotide GAA expansion.

Friedreich's ataxia (FRDA) is the most common hereditary ataxia, affecting about 1 in 50,000 individuals. It is caused by mutations in the frataxin gene; 98% of cases have homozygous expansions of a GAA trinucleotide in intron 1 of the frataxin gene. The remaining 2% of patients are compound heterozygotes, who have a GAA repeat expansion in one allele and a point mutation in the other allele. FRDA patients with point mutation have been suggested to have atypical clinical features. We present a case of compound heterozygotes in a FRDA patient who has a deletion of one T in the start codon (ATG) of the frataxin gene and a GAA repeat expansion in the other allele. The patient presented with chorea and subsequently developed FRDA symptoms. The disease in this case is the result of both a failure of initiation of translation and the effect of the expansion. This novel mutation extends the range of point mutations seen in FRDA patients, and also broadens the spectrum of FRDA genotype associated with chorea.     

Rescue of the Friedreich's ataxia knockout mouse by human YAC transgenesis

We have generated and characterised transgenic mice that contain the entire Friedreich's ataxia gene (FRDA) within a human YAC clone of 370 kb. In an effort to overcome the embryonic lethality of homozygous Frda knockout mice and to study the behaviour of human frataxin in a mouse cellular environment, we bred the FRDA YAC transgene onto the null mouse background. Phenotypically normal offspring that express only YAC-derived human frataxin were identified. The human frataxin was expressed in the appropriate tissues at levels comparable to the endogenous mouse frataxin, and it was correctly processed and localised to mitochondria. Biochemical analysis of heart tissue demonstrated preservation of mitochondrial respiratory chain function, together with some increase in citrate synthase and aconitase activities. Thus, we have demonstrated that human frataxin can effectively substitute for endogenous murine frataxin in the null mutant. Our studies are of immediate consequence for the generation of Friedreich's ataxia transgenic mouse models, and further contribute to the accumulating knowledge of human-mouse functional gene replacement systems. Electronic Publication     

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’.     

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.     

Frataxin point mutations in two patients with Friedreich's ataxia and unusual clinical features

Two patients with a progressive ataxia are presented with clinical features consistent with classic Friedreich's ataxia (FRDA), but also with features unusual for FRDA. Analysis of DNA showed that each patient is heterozygous for the expanded GAA repeat of FRDA, but carries a base change on his other frataxin allele. For one patient a non-conservative arginine to cysteine amino acid change is predicted at amino acid 165 whereas the other mutation is found at the junction of exon one and intron one. Muscle biopsy showed an absence of frataxin immunoreactivity in the patient harbouring the intronic mutation, confirming the pathological nature of the base change. These mutations extend the range of point mutations seen in FRDA, and agree with recent reports suggesting phenotypic variation in patients with FRDA harbouring point mutations in conjunction with an expanded GAA repeat.     

Mapping of the second Friedreich's ataxia (FRDA2) locus to chromosome 9p23-p11: evidence for further locus heterogeneity

Friedreich's ataxia (FRDA), the most-common form of autosomal recessive ataxia, is inherited in most cases by a large expansion of a GAA triplet repeat in the first intron of the frataxin (X25) gene. Genetic heterogeneity in FRDA has been previously reported in typical FRDA families that do not link to the FRDA locus on chromosome 9q13. We report localization of a second FRDA locus (FRDA2) to chromosome 9p23-9p11, and we provide evidence for further genetic heterogeneity of the disease, in a family with the classic FRDA phenotype.     

Multimodal Analysis of the Visual Pathways in Friedreich's Ataxia Reveals Novel Biomarkers

Background

Optic neuropathy is a near ubiquitous feature of Friedreich's ataxia (FRDA). Previous studies have examined varying aspects of the anterior and posterior visual pathways but none so far have comprehensively evaluated the heterogeneity of degeneration across different areas of the retina, changes to the macula layers and combined these with volumetric MRI studies of the visual cortex and frataxin level.

Methods

We investigated 62 genetically confirmed FRDA patients using an integrated approach as part of an observational cohort study. We included measurement of frataxin protein levels, clinical evaluation of visual and neurological function, optical coherence tomography to determine retinal nerve fibre layer thickness and macular layer volume and volumetric brain MRI.

Results

We demonstrate that frataxin level correlates with peripapillary retinal nerve fibre layer thickness and that retinal sectors differ in their degree of degeneration. We also shown that retinal nerve fibre layer is thinner in FRDA patients than controls and that this thinning is influenced by the AAO and GAA1. Furthermore we show that the ganglion cell and inner plexiform layers are affected in FRDA. Our MRI data indicate that there are borderline correlations between retinal layers and areas of the cortex involved in visual processing.

Conclusion

Our study demonstrates the uneven distribution of the axonopathy in the retinal nerve fibre layer and highlight the relative sparing of the papillomacular bundle and temporal sectors. We show that thinning of the retinal nerve fibre layer is associated with frataxin levels, supporting the use the two biomarkers in future clinical trials design. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.     

Neurological effects of recombinant human erythropoietin in Friedreich's ataxia: A clinical pilot trial

In a “proof‐of‐concept” study, we demonstrated that recombinant human erythropoietin (rhuEPO) increases frataxin levels in Friedreich's ataxia (FRDA) patients. We now report a 6‐month open‐label clinical pilot study of safety and efficacy of rhuEPO treatment in FRDA. Eight adult FRDA patients received 2.000 IU rhuEPO thrice a week subcutaneously. Clinical outcome measures included Ataxia Rating Scales. Frataxin levels and indicators for oxidative stress were assessed. Hematological parameters were monitored biweekly. Scores in Ataxia Rating Scales such as FARS (P = 0.0063) and SARA (P = 0.0045) improved significantly. Frataxin levels increased (P = 0.017) while indicators of oxidative stress such as urine 8‐OHdG (P = 0.012) and peroxide levels decreased (P = 0.028). Increases in hematocrit requiring phlebotomies occurred in 4 of 8 patients. In this explorative open‐label clinical pilot study, we found an evidence for clinical improvement together with a persistent increase of frataxin levels and a reduction of oxidative stress parameters in patients with FRDA receiving chronic treatment with rhuEPO. Safety monitoring with regular blood cell counts and parameters of iron metabolism is a potential limitation of this approach. © 2008 Movement Disorder Society     

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.     

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.     

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.     

A novel deletion–insertion mutation identified in exon 3 of <Emphasis Type="Italic">FXN</Emphasis> in two siblings with a severe Friedreich ataxia phenotype

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease most commonly caused by a GAA trinucleotide repeat expansion in the first intron of FXN, which reduces expression of the mitochondrial protein frataxin. Approximately 98% of individuals with FRDA are homozygous for GAA expansions, with the remaining 2% compound heterozygotes for a GAA expansion and a point mutation within FXN. Two siblings with early onset of symptoms experienced rapid loss of ambulation by 8 and 10 years. Diagnostic testing for FRDA demonstrated one GAA repeat expansion of 1010 repeats and one non-expanded allele. Sequencing all five exons of FXN identified a novel deletion-insertion mutation in exon 3 (c.371_376del6ins15), which results in a modified frataxin protein sequence at amino acid positions 124–127. Specifically, the amino acid sequence changes from DVSF to VHLEDT, increasing frataxin from 211 residues to 214. Using the known structure of human frataxin, a theoretical 3D model of the mutant protein was developed. In the event that the modified protein is expressed and stable, it is predicted that the acidic interface of frataxin, known to be involved in iron binding and interactions with the iron–sulphur cluster assembly factor IscU, would be impaired.     

Friedreich's ataxia: a clinical and genetic analysis

We report a patient with genetically confirmed Friedreich's ataxia (FRDA) who developed a previously unreported feature of a mixed sleep apnea. Initial mutation analysis, by PCR, of the parental frataxin alleles showed an apparent de novo mutation in the maternal germline. Further investigation using Southern blot analysis showed that the mother did carry an expanded mutant frataxin allele. Based upon published data, FRDA resulting from at least one allelic spontaneous expansion mutation is rare with a frequency of less than 1/1,000,000. The presence of such a mutation should be confirmed by Southern blot analysis. Our patient expands the neurological features of FRDA to include sleep apnea. The genetic analysis of the family demonstrates the importance of Southern blot analysis for accurate genotyping which, in turn, has implications for genetic counseling.