GAA repeat expansion mutation mouse models of Friedreich ataxia exhibit oxidative stress leading to progressive neuronal and cardiac pathology

Friedreich ataxia (FRDA) is a neurodegenerative disorder caused by an unstable GAA repeat expansion mutation within intron 1 of the FXN gene. However, the origins of the GAA repeat expansion, its unstable dynamics within different cells and tissues, and its effects on frataxin expression are not yet completely understood. Therefore, we have chosen to generate representative FRDA mouse models by using the human FXN GAA repeat expansion itself as the genetically modified mutation. We have previously reported the establishment of two lines of human FXN YAC transgenic mice that contain unstable GAA repeat expansions within the appropriate genomic context. We now describe the generation of FRDA mouse models by crossbreeding of both lines of human FXN YAC transgenic mice with heterozygous Fxn knockout mice. The resultant FRDA mice that express only human-derived frataxin show comparatively reduced levels of frataxin mRNA and protein expression, decreased aconitase activity, and oxidative stress, leading to progressive neurodegenerative and cardiac pathological phenotypes. Coordination deficits are present, as measured by accelerating rotarod analysis, together with a progressive decrease in locomotor activity and increase in weight. Large vacuoles are detected within neurons of the dorsal root ganglia (DRG), predominantly within the lumbar regions in 6-month-old mice, but spreading to the cervical regions after 1 year of age. Secondary demyelination of large axons is also detected within the lumbar roots of older mice. Lipofuscin deposition is increased in both DRG neurons and cardiomyocytes, and iron deposition is detected in cardiomyocytes after 1 year of age. These mice represent the first GAA repeat expansion-based FRDA mouse models that exhibit progressive FRDA-like pathology and thus will be of use in testing potential therapeutic strategies, particularly GAA repeat-based strategies. © 2006 Elsevier Inc. All rights reserved

Generation and characterisation of Friedreich ataxia YG8R mouse fibroblast and neural stem cell models

This article has been made available through the Brunel Open Access Publishing Fund.Background: Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by GAA repeat expansion in the first intron of the FXN gene, which encodes frataxin, an essential mitochondrial protein. To further characterise the molecular abnormalities associated with FRDA pathogenesis and to hasten drug screening, the development and use of animal and cellular models is considered essential. Studies of lower organisms have already contributed to understanding FRDA disease pathology, but mammalian cells are more related to FRDA patient cells in physiological terms. Methodology/Principal Findings: We have generated fibroblast cells and neural stem cells (NSCs) from control Y47R mice (9 GAA repeats) and GAA repeat expansion YG8R mice (190+120 GAA repeats). We then differentiated the NSCs in to neurons, oligodendrocytes and astrocytes as confirmed by immunocytochemical analysis of cell specific markers. The three YG8R mouse cell types (fibroblasts, NSCs and differentiated NSCs) exhibit GAA repeat stability, together with reduced expression of frataxin and reduced aconitase activity compared to control Y47R cells. Furthermore, YG8R cells also show increased sensitivity to oxidative stress and downregulation of Pgc-1α and antioxidant gene expression levels, especially Sod2. We also analysed various DNA mismatch repair (MMR) gene expression levels and found that YG8R cells displayed significant reduction in expression of several MMR genes, which may contribute to the GAA repeat stability. Conclusions/Significance: We describe the first fibroblast and NSC models from YG8R FRDA mice and we confirm that the NSCs can be differentiated into neurons and glia. These novel FRDA mouse cell models, which exhibit a FRDA-like cellular and molecular phenotype, will be valuable resources to further study FRDA molecular pathogenesis. They will also provide very useful tools for preclinical testing of frataxin-increasing compounds for FRDA drug therapy, for gene therapy, and as a source of cells for cell therapy testing in FRDA mice. © 2014 Sandi et al

Severe reversible cardiac failure after bortezomib treatment combined with chemotherapy in a non-small cell lung cancer patient: a case report

BACKGROUND: Bortezomib (Velcade(®)), a dipeptide boronate proteasome inhibitor, is a novel anti-cancer agent registered for multiple myeloma (MM). It has also shown promising clinical activity in non-small cell lung cancer (NSCLC). Clinical experience with bortezomib so far indicates that overall incidence of cardiac failure associated with bortezomib therapy remains incidental. Nevertheless, acute development or exacerbation of congestive cardiac failure has been associated with bortezomib treatment. CASE PRESENTATION: We present here a case of severe, but reversible, congestive cardiac failure in a lung cancer patient who had no prior cardiac history, after receiving an experimental treatment of bortezomib combined with chemotherapy. Elevated levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP), as retrospectively measured in archived serum samples, were suggestive of pre-existent (sub-clinical) left ventricular dysfunction. CONCLUSION: Based on literature, we hypothesize that baseline presence of sub clinical cardiomyopathy, characterized by a dysregulation of the ubiquitin-proteasome system, could have predisposed this patient for a cardiac side effect induced by systemic proteasome inhibition. Patients with heart disease or risk factors for it should be closely monitored when being submitted to treatment with proteasome inhibition therapy such as bortezomib. Caution is therefore warranted in lung cancer patients who often present with cardiac comorbidities

Pharmacological screening using an FXN-EGFP cellular genomic reporter assay for the therapy of Friedreich ataxia

Copyright @ 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol.Muscular Dystrophy Association (USA), the National Health and Medical Research Council (Australia), the Friedreich’s Ataxia Research Alliance (USA), the Brockhoff Foundation (Australia), the Friedreich Ataxia Research Association (Australasia), Seek A Miracle (USA) and the Victorian Government’s Operational Infrastructure Support Program

Targeting lipid peroxidation and mitochondrial imbalance in Friedreich's ataxia

Friedreich's ataxia (FRDA) is an autosomal recessive disorder, caused by reduced levels of the protein frataxin. This protein is located in the mitochondria, where it functions in the biogenesis of iron-sulphur clusters (ISCs), which are important for the function of the mitochondrial respiratory chain complexes. Moreover, disruption in iron biogenesis may lead to oxidative stress. Oxidative stress can be the cause and/or the consequence of mitochondrial energy imbalance, leading to cell death. Fibroblasts from two FRDA mouse models, YG8R and KIKO, were used to analyse two different categories of protective compounds: deuterised poly-unsaturated fatty acids (dPUFAs) and Nrf2-inducers. The former have been shown to protect the cell from damage induced by lipid peroxidation and the latter trigger the well-known Nrf2 antioxidant pathway. Our results show that the sensitivity to oxidative stress of YG8R and KIKO mouse fibroblasts, resulting in cell death and lipid peroxidation, can be prevented by d4-PUFA and Nrf2-inducers (SFN and TBE-31). The mitochondrial membrane potential (ΔΨm) of YG8R and KIKO fibroblasts revealed a difference in their mitochondrial pathophysiology, which may be due to the different genetic basis of the two models. This suggests that variable levels of reduced frataxin may act differently on mitochondrial pathophysiology and that these two cell models could be useful in recapitulating the observed differences in the FRDA phenotype. This may reflect a different modulatory effect towards cell death that will need to be investigated further.RA is supported by FARA and GoFar, A&C Simeone Foundation.PG has received funding from FARA and GoFar, A&C Simeone Foundation and the European Union Seventh Framework Programme(FP7/2007-2013) under grant agreement number 242193/EFACTS.PG is supported by the National Institute for Health Research, University College London Hospitals, Biomedical Research Centre. EU and IR were supported by MSc scholarships

'Mitochondrial energy imbalance and lipid peroxidation cause cell death in Friedreich's ataxia'

Friedreich's ataxia (FRDA) is an inherited neurodegenerative disease. The mutation consists of a GAA repeat expansion within the FXN gene, which downregulates frataxin, leading to abnormal mitochondrial iron accumulation, which may in turn cause changes in mitochondrial function. Although, many studies of FRDA patients and mouse models have been conducted in the past two decades, the role of frataxin in mitochondrial pathophysiology remains elusive. Are the mitochondrial abnormalities only a side effect of the increased accumulation of reactive iron, generating oxidative stress? Or does the progressive lack of iron-sulphur clusters (ISCs), induced by reduced frataxin, cause an inhibition of the electron transport chain complexes (CI, II and III) leading to reactive oxygen species escaping from oxidative phosphorylation reactions? To answer these crucial questions, we have characterised the mitochondrial pathophysiology of a group of disease-relevant and readily accessible neurons, cerebellar granule cells, from a validated FRDA mouse model. By using live cell imaging and biochemical techniques we were able to demonstrate that mitochondria are deregulated in neurons from the YG8R FRDA mouse model, causing a decrease in mitochondrial membrane potential (▵Ψm) due to an inhibition of Complex I, which is partially compensated by an overactivation of Complex II. This complex activity imbalance leads to ROS generation in both mitochondrial matrix and cytosol, which results in glutathione depletion and increased lipid peroxidation. Preventing this increase in lipid peroxidation, in neurons, protects against in cell death. This work describes the pathophysiological properties of the mitochondria in neurons from a FRDA mouse model and shows that lipid peroxidation could be an important target for novel therapeutic strategies in FRDA, which still lacks a cure

Endothelial Cell and Platelet Bioenergetics: Effect of Glucose and Nutrient Composition

It has been suggested that cells that are independent of insulin for glucose uptake, when exposed to high glucose or other nutrient concentrations, manifest enhanced mitochondrial substrate oxidation with consequent enhanced potential and generation of reactive oxygen species (ROS); a paradigm that could predispose to vascular complications of diabetes. Here we exposed bovine aortic endothelial (BAE) cells and human platelets to variable glucose and fatty acid concentrations. We then examined oxygen consumption and acidification rates using recently available technology in the form of an extracellular oxygen and proton flux analyzer. Acute or overnight exposure of confluent BAE cells to glucose concentrations from 5.5 to 25 mM did not enhance or change the rate of oxygen consumption (OCR) under basal conditions, during ATP synthesis, or under uncoupled conditions. Glucose also did not alter OCR in sub-confluent cells, in cells exposed to low serum, or in cells treated with added pyruvate. Likewise, overnight exposure to fatty acids of varying saturation had no such effects. Overnight exposure of BAE cells to low glucose concentration decreased maximal uncoupled respiration, but not basal or ATP related oxygen consumption. Labeled glucose oxidation to CO2 increased, but only marginally after high glucose exposure while oleate oxidation to CO2 decreased. Overnight exposure to linolenic acid, but not oleic or linoleic acid increased extracellular acidification consistent with enhanced glycolytic metabolism. We were unable to detect an increase in production of reactive oxygen species (ROS) from BAE cells exposed to high medium glucose. Like BAE cells, exposure of human platelets to glucose did not increase oxygen consumption. As opposed to BAE cells, platelet mitochondria demonstrate less respiratory reserve capacity (beyond that needed for basal metabolism). Our data do not support the concept that exposure to high glucose or fatty acids accelerates mitochondrial oxidative metabolism in endothelial cells or platelets

A Combined Nucleic Acid and Protein Analysis in Friedreich Ataxia: Implications for Diagnosis, Pathogenesis and Clinical Trial Design

BACKGROUND: Friedreich's ataxia (FRDA) is the most common hereditary ataxia among caucasians. The molecular defect in FRDA is the trinucleotide GAA expansion in the first intron of the FXN gene, which encodes frataxin. No studies have yet reported frataxin protein and mRNA levels in a large cohort of FRDA patients, carriers and controls. METHODOLOGY/PRINCIPAL FINDINGS: We enrolled 24 patients with classic FRDA phenotype (cFA), 6 late onset FRDA (LOFA), all homozygous for GAA expansion, 5 pFA cases who harbored the GAA expansion in compound heterozygosis with FXN point mutations (namely, p.I154F, c.482+3delA, p.R165P), 33 healthy expansion carriers, and 29 healthy controls. DNA was genotyped for GAA expansion, mRNA/FXN was quantified in real-time, and frataxin protein was measured using lateral-flow immunoassay in peripheral blood mononuclear cells (PBMCs). Mean residual levels of frataxin, compared to controls, were 35.8%, 65.6%, 33%, and 68.7% in cFA, LOFA, pFA and healthy carriers, respectively. Comparison of both cFA and pFA with controls resulted in 100% sensitivity and specificity, but there was overlap between LOFA, carriers and controls. Frataxin levels correlated inversely with GAA1 and GAA2 expansions, and directly with age at onset. Messenger RNA expression was reduced to 19.4% in cFA, 50.4% in LOFA, 52.7% in pFA, 53.0% in carriers, as compared to controls (p<0.0001). mRNA levels proved to be diagnostic when comparing cFA with controls resulting in 100% sensitivity and specificity. In cFA and LOFA patients mRNA levels correlated directly with protein levels and age at onset, and inversely with GAA1 and GAA2. CONCLUSION/SIGNIFICANCE: We report the first explorative study on combined frataxin and mRNA levels in PBMCs from a cohort of FRDA patients, carriers and healthy individuals. Lateral-flow immunoassay differentiated cFA and pFA patients from controls, whereas determination of mRNA in q-PCR was sensitive and specific only in cFA

Essential role of proteasomes in maintaining self-renewal in neural progenitor cells

Protein turnover and homeostasis are regulated by the proteasomal system, which is critical for cell function and viability. Pluripotency of stem cells also relies on normal proteasomal activity that mitigates senescent phenotypes induced by intensive cell replications, as previously demonstrated in human bone marrow stromal cells. In this study, we investigated the role of proteasomes in self-renewal of neural progenitor cells (NPCs). Through both in vivo and in vitro analyses, we found that the expression of proteasomes was progressively decreased during aging. Likewise, proliferation and self-renewal of NPCs were also impaired in aged mice, suggesting that the down-regulation of proteasomes might be responsible for this senescent phenotype. Lowering proteasomal activity by loss-of-function manipulations mimicked the senescence of NPCs both in vitro and in vivo; conversely, enhancing proteasomal activity restored and improved self-renewal in aged NPCs. These results collectively indicate that proteasomes work as a key regulator in promoting self-renewal of NPCs. This potentially provides a promising therapeutic target for age-dependent neurodegenerative diseases