Epigenetics in Friedreich's ataxia: Challenges and opportunities for therapy

Copyright © 2013 Chiranjeevi Sandi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by homozygous expansion of a GAA·TTC trinucleotide repeat within the first intron of the FXN gene, leading to reduced FXN transcription and decreased levels of frataxin protein. Recent advances in FRDA research have revealed the presence of several epigenetic modifications that are either directly or indirectly involved in this FXN gene silencing. Although epigenetic marks may be inherited from one generation to the next, modifications of DNA and histones can be reversed, indicating that they are suitable targets for epigenetic-based therapy. Unlike other trinucleotide repeat disorders, such as Huntington disease, the large expansions of GAA·TTC repeats in FRDA do not produce a change in the frataxin amino acid sequence, but they produce reduced levels of normal frataxin. Therefore, transcriptional reactivation of the FXN gene provides a good therapeutic option. The present paper will initially focus on the epigenetic changes seen in FRDA patients and their role in the silencing of FXN gene and will be concluded by considering the potential epigenetic therapies.This study is supported by funding from the European Union Seventh Framework Programme (FP7/2007–2013) under Grant agreement no. 242193/EFACTS; and by funding from theWellcome Trust (089757)

The emerging role of 5-hydroxymethylcytosine in neurodegenerative diseases

Copyright © 2014 Al-Mahdawi, Anjomani Virmouni and Pook. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.This article has been made available through the Brunel Open Access Publishing Fund.DNA methylation primarily occurs within human cells as a 5-methylcytosine (5mC) modification of the cytosine bases in CpG dinucleotides. 5mC has proven to be an important epigenetic mark that is involved in the control of gene transcription for processes such as development and differentiation. However, recent studies have identified an alternative modification, 5-hydroxymethylcytosine (5hmC), which is formed by oxidation of 5mC by ten-eleven translocation (TET) enzymes. The overall levels of 5hmC in the mammalian genome are approximately 10% of 5mC levels, although higher levels have been detected in tissues of the central nervous system (CNS). The functions of 5hmC are not yet fully known, but evidence suggests that 5hmC may be both an intermediate product during the removal of 5mC by passive or active demethylation processes and also an epigenetic modification in its own right, regulating chromatin or transcriptional factors involved in processes such as neurodevelopment or environmental stress response. This review highlights our current understanding of the role that 5hmC plays in neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), fragile X-associated tremor/ataxia syndrome (FXTAS), Friedreich ataxia (FRDA), Huntington's disease (HD), and Parkinson's disease (PD).Sara Anjomani Virmouni was supported by funding to Mark A. Pook from the Friedreich’s Ataxia Research Alliance(FARA)

Comparative (computational) analysis of the DNA methylation status of trinucleotide repeat expansion diseases

Copyright © 2013 Mohammadmersad Ghorbani et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.This article has been made available through the Brunel Open Access Publishing Fund.Previous studies have examined DNA methylation in different trinucleotide repeat diseases. We have combined this data and used a pattern searching algorithm to identify motifs in the DNA surrounding aberrantly methylated CpGs found in the DNA of patients with one of the three trinucleotide repeat (TNR) expansion diseases: fragile X syndrome (FRAXA), myotonic dystrophy type I (DM1), or Friedreich’s ataxia (FRDA). We examined sequences surrounding both the variably methylated (VM) CpGs, which are hypermethylated in patients compared with unaffected controls, and the nonvariably methylated CpGs which remain either always methylated (AM) or never methylated (NM) in both patients and controls. Using the J48 algorithm of WEKA analysis, we identified that two patterns are all that is necessary to classify our three regions CCGG* which is found in VM and not in AM regions and AATT* which distinguished between NM and VM + AM using proportional frequency. Furthermore, comparing our software with MEME software, we have demonstrated that our software identifies more patterns than MEME in these short DNA sequences. Thus, we present evidence that the DNA sequence surrounding CpG can influence its susceptibility to be de novo methylated in a disease state associated with a trinucleotide repeat.European Union Seventh Framework Programme and The Brunel University Graduate Program

The mismatch repair system protects against intergenerational GAA repeat instability in a Friedreich ataxia mouse model

Copyright @ 2012 Elsevier. The article can be accessed from the link below.This article has been made available through the Brunel Open Access Publishing Fund.Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a dynamic GAA repeat expansion mutation within intron 1 of the FXN gene. Studies of mouse models for other trinucleotide repeat (TNR) disorders have revealed an important role of mismatch repair (MMR) proteins in TNR instability. To explore the potential role of MMR proteins on intergenerational GAA repeat instability in FRDA, we have analyzed the transmission of unstable GAA repeat expansions from FXN transgenic mice which have been crossed with mice that are deficient for Msh2, Msh3, Msh6 or Pms2. We find in all cases that absence of parental MMR protein not only maintains transmission of GAA expansions and contractions, but also increases GAA repeat mutability (expansions and/or contractions) in the offspring. This indicates that Msh2, Msh3, Msh6 and Pms2 proteins are not the cause of intergenerational GAA expansions or contractions, but act in their canonical MMR capacity to protect against GAA repeat instability. We further identified differential modes of action for the four MMR proteins. Thus, Msh2 and Msh3 protect against GAA repeat contractions, while Msh6 protects against both GAA repeat expansions and contractions, and Pms2 protects against GAA repeat expansions and also promotes contractions. Furthermore, we detected enhanced occupancy of Msh2 and Msh3 proteins downstream of the FXN expanded GAA repeat, suggesting a model in which Msh2/3 dimers are recruited to this region to repair mismatches that would otherwise produce intergenerational GAA contractions. These findings reveal substantial differences in the intergenerational dynamics of expanded GAA repeat sequences compared with expanded CAG/CTG repeats, where Msh2 and Msh3 are thought to actively promote repeat expansions.This study is funded under European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 242193/EFACTS. This article is made available through the Brunel Open Access Publishing Fund

FXN promoter silencing in the humanized mouse model of Friedreich Ataxia

Background - Friedreich ataxia is caused by an expanded GAA triplet-repeat sequence in intron 1 of the FXN gene that results in epigenetic silencing of the FXN promoter. This silencing mechanism is seen in patient-derived lymphoblastoid cells but it remains unknown if it is a widespread phenomenon affecting multiple cell types and tissues. Methodology / Principal Findings - The humanized mouse model of Friedreich ataxia (YG8sR), which carries a single transgenic insert of the human FXN gene with an expanded GAA triplet-repeat in intron 1, is deficient for FXN transcript when compared to an isogenic transgenic mouse lacking the expanded repeat (Y47R). We found that in YG8sR the deficiency of FXN transcript extended both upstream and downstream of the expanded GAA triplet-repeat, suggestive of deficient transcriptional initiation. This pattern of deficiency was seen in all tissues tested, irrespective of whether they are known to be affected or spared in disease pathogenesis, in both neuronal and non-neuronal tissues, and in cultured primary fibroblasts. FXN promoter function was directly measured via metabolic labeling of newly synthesized transcripts in fibroblasts, which revealed that the YG8sR mouse was significantly deficient in transcriptional initiation compared to the Y47R mouse. Conclusions / Significance- Deficient transcriptional initiation accounts for FXN transcriptional deficiency in the humanized mouse model of Friedreich ataxia, similar to patient-derived cells, and the mechanism underlying promoter silencing in Friedreich ataxia is widespread across multiple cell types and tissues.This research was supported by grants from the National Institutes of Health (R01 NS072418), and the Muscular Dystrophy Association to S.I.B. Y.K.C. is supported by a postdoctoral research fellowship from the Million Dollar Bike Ride Grant Program of the Orphan Disease Center at University of Pennsylvania. T.T.H. was supported by the American College of Medical Genetics Foundation. A.C.P. and M.G.M. were supported by the SURE and OSCTR programs at OUHSC, respectively

Cellular, molecular and functional characterisation of YAC transgenic mouse models of Friedreich Ataxia

Copyright © 2014 Anjomani Virmouni 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.This article has been made available through the Brunel Open Access Publishing Fund.Background - Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by a GAA repeat expansion mutation within intron 1 of the FXN gene. We have previously established and performed preliminary characterisation of several human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing GAA repeat expansions, Y47R (9 GAA repeats), YG8R (90 and 190 GAA repeats) and YG22R (190 GAA repeats). Methodology/Principal Findings - We now report extended cellular, molecular and functional characterisation of these FXN YAC transgenic mouse models. FXN transgene copy number analysis of the FRDA mice demonstrated that the YG22R and Y47R lines each have a single copy of the FXN transgene while the YG8R line has two copies. Single integration sites of all transgenes were confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We identified significant functional deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8R and YG22R FRDA mice compared to Y47R and wild-type control mice. We also confirmed increased somatic GAA repeat instability in the cerebellum and brain of YG22R and YG8R mice, together with significantly reduced levels of FXN mRNA and protein in the brain and liver of YG8R and YG22R compared to Y47R. Conclusions/Significance - Together these studies provide a detailed characterisation of our GAA repeat expansion-based YAC transgenic FRDA mouse models that will help investigations of FRDA disease mechanisms and therapy.European Union, Ataxia UK and FARA

A new FRDA mouse model [Fxnnull:YG8s(GAA) > 800] with more than 800 GAA repeats

Introduction: Friedreich’s ataxia (FRDA) is an inherited recessive neurodegenerative disorder caused by a homozygous guanine-adenine-adenine (GAA) repeat expansion within intron 1 of the FXN gene, which encodes the essential mitochondrial protein frataxin. There is still no effective therapy for FRDA, therefore the development of optimal cell and animal models of the disease is one of the priorities for preclinical therapeutic testing. Methods: We obtained the latest FRDA humanized mouse model that was generated on the basis of our previous YG8sR, by Jackson laboratory [YG8JR, Fxnnull:YG8s(GAA) > 800]. We characterized the behavioral, cellular, molecular and epigenetics properties of the YG8JR model, which has the largest GAA repeat sizes compared to all the current FRDA mouse models. Results: We found statistically significant behavioral deficits, together with reduced levels of frataxin mRNA and protein, and aconitase activity in YG8JR mice compared with control Y47JR mice. YG8JR mice exhibit intergenerational GAA repeat instability by the analysis of parent and offspring tissue samples. Somatic GAA repeat instability was also detected in individual brain and cerebellum tissue samples. In addition, increased DNA methylation of CpG U13 was identified in FXN GAA repeat region in the brain, cerebellum, and heart tissues. Furthermore, we show decreased histone H3K9 acetylation and increased H3K9 methylation of YG8JR cerebellum tissues within the FXN gene, upstream and downstream of the GAA repeat region compared to Y47JR controls. Discussion: These studies provide a detailed characterization of the GAA repeat expansion-based YG8JR transgenic mouse models that will help investigations of FRDA disease mechanisms and therapy.Friedreich’s Ataxia Research Alliance (FARA) (2018) and Brunel University London (BRIEF Award; 2019)

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. (c) 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

Interruptions of the FXN GAA Repeat Tract Delay the Age at Onset of Friedreich’s Ataxia in a Location Dependent Manner

Copyright: © 2021 by the authors. Friedreich’s ataxia (FRDA) is a comparatively rare autosomal recessive neurological disorder primarily caused by the homozygous expansion of a GAA trinucleotide repeat in intron 1 of the FXN gene. The repeat expansion causes gene silencing that results in deficiency of the frataxin protein leading to mitochondrial dysfunction, oxidative stress and cell death. The GAA repeat tract in some cases may be impure with sequence variations called interruptions. It has previously been observed that large interruptions of the GAA repeat tract, determined by abnormal MboII digestion, are very rare. Here we have used triplet repeat primed PCR (TP PCR) assays to identify small interruptions at the 5′ and 3′ ends of the GAA repeat tract through alterations in the electropherogram trace signal. We found that contrary to large interruptions, small interruptions are more common, with 3′ interruptions being most frequent. Based on detection of interruptions by TP PCR assay, the patient cohort (n = 101) was stratified into four groups: 5′ interruption, 3′ interruption, both 5′ and 3′ interruptions or lacking interruption. Those patients with 3′ interruptions were associated with shorter GAA1 repeat tracts and later ages at disease onset. The age at disease onset was modelled by a group-specific exponential decay model. Based on this modelling, a 3′ interruption is predicted to delay disease onset by approximately 9 years relative to those lacking 5′ and 3′ interruptions. This highlights the key role of interruptions at the 3′ end of the GAA repeat tract in modulating the disease phenotype and its impact on prognosis for the patient.European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement number 242193/EFACTS; National Brain Appeal—Small Acorns Fund; The Wellcome Centre for Human Neuroimaging is supported by core funding from the Wellcome (203147/Z/16/Z); Department of Health’s National Institute for Health Research Biomedical Research Centres funding scheme; CRN; North Thames, National Institute for Health Research (NIHR). Medical Research Council (MR/N028767/1)