Brigitte Sturm

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

To determine the role of recombinant human erythropoietin as a possible treatment option in Friedreichs ataxia, we performed an open‐label clinical pilot study. Primary outcome measure was the change of frataxin levels at week 8 versus baseline. Twelve Friedreichs 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. Ann Neurol 2007

Recombinant human erythropoietin: effects on frataxin expression in vitro.

Background  Friedreichs ataxia (FRDA) is a neurodegenerative disorder caused by decreased expression of the protein frataxin, recently described to be an iron chaperone for the assembly of iron‐sulphur clusters in the mitochondria, causing iron accumulation in mitochondria, oxidative stress and cell damage. Searching for compounds that could possibly influence frataxin expression, we found that the cytokine recombinant human erythropoietin (rhuEPO) significantly increases frataxin expression by a still unknown mechanism.

Hydrogen sulphide: A novel physiological inhibitor of LDL atherogenic modification by HOCl

Hypochlorite (HOCl), the product of the activated myeloperoxidase/H2O2/chloride (MPO/H2O2/Cl− ) system is favored as a trigger of LDL modifications, which may play a pivotal role in early atherogenesis. As HOCl has been shown to react with thiol-containing compounds like glutathione and N-acetylcysteine protecting LDL from HOCl modification, we have tested the ability of hydrogen sulfide (H2S)—which has recently been identified as an endogenous vasorelaxant—to counteract the action of HOCl on LDL. The results show that H2S could inhibit the atherogenic modification of LDL induced by HOCl, as measured by apolipoprotein alterations. Beside its HOCl scavenging potential, H2S was found to inhibit MPO (one may speculate that this occurs via H2S/heme interaction) and destroy H2O2. Thus, H2S may interfere with the reactants and reaction products of the activated MPO/H2O2/Cl− system. Our data add to the evidence of an anti-atherosclerotic action of this gasotransmitter taking the role of HOCl in the atherogenic modification of LDL into account.

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 Friedreichs 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.

Friedreich's Ataxia, No Changes in Mitochondrial Labile Iron in Human Lymphoblasts and Fibroblasts A DECREASE IN ANTIOXIDATIVE CAPACITY?

Friedreichs ataxia (FRDA) is caused by low expression of frataxin, a small mitochondrial protein. Studies with both yeast and mammals have suggested that decreased frataxin levels lead to elevated intramitochondrial concentrations of labile (chelatable) iron, and consequently to oxidative mitochondrial damage. Here, we used the mitochondrion-selective fluorescent iron indicator/chelator rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzylester (RPA) to determine the mitochondrial chelatable iron of FRDA patient lymphoblast and fibroblast cell lines, in comparison with age- and sex-matched control cells. No alteration in the concentration of mitochondrial chelatable iron could be observed in patient cells, despite strongly decreased frataxin levels. Uptake studies with 55Fe-transferrin and iron loading with ferric ammonium citrate revealed no significant differences in transferrin receptor density and iron responsive protein/iron regulatory element binding activity between patients and controls. However, sensitivity to H2O2 was significantly increased in patient cells, and H2O2 toxicity could be completely inhibited by the ubiquitously distributing iron chelator 2,2′-dipyridyl, but not by the mitochondrion-selective chelator RPA. Our data strongly suggest that frataxin deficiency does not affect the mitochondrial labile iron pool or other parameters of cellular iron metabolism and suggest a decreased antioxidative defense against extramitochondrial iron-derived radicals in patient cells. These results challenge current concepts favoring the use of mitochondrion-specific iron chelators and antioxidants to treat FRDA.

Carbamylated erythropoietin increases frataxin independent from the erythropoietin receptor.

Eur J Clin Invest 2010; 40 (6): 561–565

A high throughput electrochemiluminescence assay for the quantification of frataxin protein levels.

Friedreichs ataxia (FRDA) is an autosomal recessive neurodegenerative disease affecting 1 in 50,000 people and is caused by a GAA-trinucleotide expansion in the frataxin gene located on chromosome locus 9q13 which results in a markedly reduced expression of frataxin, a small mitochondrial protein. The exact function of frataxin is still unknown and currently there is no approved treatment available. In the near future there will be a high demand for measuring frataxin protein levels due to the development of therapeutic strategies for FRDA based on manipulating frataxin expression levels in vivo. In this paper we describe the development of an electrochemiluminescence assay (ECLIA) to measure frataxin protein levels in a 96-well plate format. The ECLIA for frataxin is able to measure human and mouse samples and is highly quantitative, accurate and reproducible, with low intra- and inter-assay error throughout a wide working range. The assay has an excellent precision and provides a new tool for the set up of high-throughput screening for basic research and for clinical studies with FRDA patients.

Correlation of frataxin content in blood and skeletal muscle endorses frataxin as a biomarker in Friedreich ataxia.

Friedreich ataxia is an autosomal recessive disorder caused by mutations in the frataxin gene, leading to reduced levels of the mitochondrial protein frataxin. Assays to quantitatively measure frataxin in peripheral blood have been established. To determine the validity of frataxin as a biomarker for clinical trials, we assessed frataxin in clinically affected tissue.

Sulfite facilitates LDL lipid oxidation by transition metal ions: A pro‐oxidant in wine?

Lipid oxidation in LDL may play a role in atherogenesis. It has been shown that sulfite – a compound in the aqueous fraction of wine – could inhibit free radical (AAPH) mediated oxidation of plasma. Thus, sulfite has been proposed as an antioxidant. In contrast, the aqueous phase of wine has recently been shown to contain not fully identified compounds promoting transition metal ion (Cu2+) initiated LDL oxidation. As transition metal ions can catalyse the auto‐oxidation of sulfite, we studied the influence of sulfite on Cu2+ initiated LDL oxidation. The results show that sulfite at concentrations found in vivo strongly facilitated LDL oxidation by Cu2+. The LDL‐oxidase activity of ceruloplasmin was also stimulated by sulfite. ROS formation by Cu 2 + / SO 3 2 ‐ was not inhibited by SOD but by catalase. We propose that formation of Cu+, sulfite radicals ( SO 3 − ) and hydroxyl radicals (OH) is a mechanism by which sulfite could act as a pro‐atherogenic agent in presence of transition metal ions.

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.