164,624 research outputs found
GAA repeat expansion mutation mouse models of Friedreich ataxia exhibit oxidative stress leading to progressive neuronal and cardiac pathology
No full textFriedreich 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
Somatic instability of the expanded GAA triplet-repeat sequence in Friedreich ataxia progresses throughout life
No full textFriedreich ataxia (FRDA) patients are homozygous for expanded GAA triplet-repeat
alleles in the FXN gene. Primary neurodegeneration involving the dorsal root ganglia
(DRG) results in progressive ataxia. While it is known that DRG are inherently sensitive
to frataxin deficiency, recent observations also indicate that they show age-dependent,
further expansion of the GAA triplet-repeat mutation. Whether somatic instability is
progressive has not been systematically investigated in FRDA patients. Small pool PCR
analysis of ~2300 individual molecules from tissues of an 18-week fetus, homozygous
for expanded alleles, revealed very low levels of instability compared with adult-derived
tissues (4.2% versus 30.6%, P<0.0001). Mutation load in blood samples from multiple
patients and carriers increased significantly with age, ranging from 7.5% at 18-weeks
gestation to 78.7% at 49y (R=0.91; P=0.0001). Therefore, somatic instability in FRDA
occurs mostly after early embryonic development and progresses throughout life,
lending further support to the role of postnatal somatic instability in disease
pathogenesis
The Friedreich ataxia GAA repeat expansion mutation induces comparable epigenetic changes in human and transgenic mouse brain and heart tissues
No full textFriedreich ataxia (FRDA) is caused by a homozygous GAA repeat expansion mutation
within intron 1 of the FXN gene, leading to reduced expression of frataxin protein.
Evidence suggests that the mutation may induce epigenetic changes and heterochromatin
formation, thereby impeding gene transcription. In particular, studies using FRDA patient
blood and lymphoblastoid cell lines have detected increased DNA methylation of specific
CpG sites upstream of the GAA repeat and histone modifications in regions flanking the
GAA repeat. In this report we show that such epigenetic changes are also present in
FRDA patient brain, cerebellum and heart tissues, the primary affected systems of the
disorder. Bisulfite sequence analysis of the FXN flanking GAA regions reveals a shift in
the FRDA DNA methylation profile, with upstream CpG sites becoming consistently
hypermethylated and downstream CpG sites becoming consistently hypomethylated. We
also identify differential DNA methylation at three specific CpG sites within the FXN
promoter and one CpG site within exon 1. Furthermore, we show by chromatin
immunoprecipitation (ChIP) analysis that there is overall decreased histone H3K9
acetylation together with increased H3K9 methylation of FRDA brain tissue. Further
studies of brain, cerebellum and heart tissues from our GAA repeat expansion-containing
FRDA YAC transgenic mice reveal comparable epigenetic changes to those detected in
FRDA patient tissue. We have thus developed a mouse model that will be a valuable
resource for future therapeutic studies targeting epigenetic modifications of the FXN gene
to increase frataxin expression
Generation and characterisation of Friedreich ataxia YG8R mouse fibroblast and neural stem cell models
No full textThis 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
Cellular, molecular and functional characterisation of YAC transgenic mouse models of Friedreich Ataxia
No full textCopyright © 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
Genotype and phenotype characterisation of Friedreich ataxia mouse models and cells
No full textThis thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel UniversityFriedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by a GAA repeat expansion mutation within intron 1 of the FXN gene, resulting in reduced level of frataxin protein. Normal individuals have 5 to 40 GAA repeat sequences, whereas affected individuals have approximately 70 to more than 1000 GAA triplets. Frataxin is a mitochondrial protein involved in iron-sulphur cluster and heme biosynthesis. The reduction in frataxin expression leads to oxidative stress, mitochondrial iron accumulation and consequential cell death with the primary sites of neurons of the dorsal root ganglia and the dentate nucleus of the cerebellum. FRDA, which is the most common inherited ataxia, affecting 1:50,000 Caucasians, is characterised by neurodegeneration, cardiomyopathy, diabetes mellitus and skeletal deformities. To investigate FRDA molecular disease mechanisms and therapy, several human FXN YAC transgenic mouse models have been established: Y47R, containing normal-sized (GAA)9 repeats; YG8R and YG22R, which initially contained expanded GAA repeats of 90-190 units and 190 units, respectively, but which have subsequently been bred to now contain expanded GAA repeats of 120-220 units and 170-260 units, respectively, and YG8sR (YG8R with a small GAA band) that was recently generated from YG8R breeding. To determine the FXN transgene copy number in the enhanced GAA repeat expansion-based FRDA mouse lines, a TaqMan qPCR assay was developed. The results demonstrated that the YG22R and Y47R lines had a single copy of the FXN transgene while the YG8R line had two copies. The YG8s lines showed less than one copy of the target gene, suggesting potential deletion of the FXN gene. Single integration sites of all transgenes were confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. However, in the YG8s line, at least 25% of the YG8s cells had no signals, while the remaining cells showed one signal corresponding to the transgenic FXN gene. In addition, the analysis of FXN exons in YG8s rescue mice by PCR confirmed the presence of all FXN exons in these lines, suggesting the incidence of somatic mosaicism in these lines. Extended functional analysis was carried out on these mice from 4 to 12 months of age. Coordination ability of YG8R, YG8sR and YG22R ‘FRDA-like’ mice, together with Y47R and C57BL6/J wild-type control mice, was assessed using accelerating rotarod analysis. The results indicated a progressive decrease in the motor coordination of YG8R, YG22R and YG8sR mice compared to Y47R or C57BL6/J controls. Locomotor activity was also assessed using an open field beam-breaker apparatus followed by four additional functional analyses including beam-walk, hang wire, grip strength and foot print tests. The results indicated significant functional deficits in the FRDA mouse models. Glucose and insulin tolerance tests were also conducted in the FRDA mouse models, indicating glucose intolerance and insulin hypersensitivity in the aforementioned lines.
To investigate the correlation between the FRDA-like pathological phenotype and frataxin deficiency in the FRDA mouse models, frataxin mRNA and protein levels as well as somatic GAA repeat instability were examined. The results indicated that somatic GAA repeats increased in the cerebellum and brain of YG22R, YG8R and YG8sR mice, together with significantly reduced levels of FXN mRNA and protein in the liver of YG8R and YG22R compared to Y47R. However, YG8sR lines showed a significant decrease in FXN mRNA in all of the examined tissues compared to Y47R human FXN and C57BL6/J mouse Fxn mRNA. Protein expression levels were also considerably reduced in all the tissues of YG8sR mice compared to Y47R. Subsequently, the telomere length of human and mouse FRDA and control fibroblasts was assessed using qPCR and Q-FISH. The results indicated that the FRDA cells had chromosomes with relatively longer telomeric repeats in comparison to the controls. FRDA cells were screened for expression of telomerase activity using the TRAP assay and a quantitative assay for hTERT mRNA expression using TaqMan qRT-PCR. The results indicated that telomerase activity was not present in the FRDA cells. To investigate whether FRDA cells maintained their telomeres by ALT associated PML bodies (APBs), co-localisation of PML bodies with telomeres was assessed in these cells using combined immunofluorescence to PML and Q-FISH for telomere detection. The results demonstrated that the FRDA cells had significantly higher co-localised PML foci with telomeric DNA compared to the normal cells. Moreover, telomere sister chromatid exchange (T-SCE) frequencies were analysed in the human FRDA cell lines using chromosome orientation FISH (CO-FISH). The results indicated a significant increase in T-SCE levels of the FRDA cell lines relative to the controls. Furthermore, growth curve and population doubling analysis of the human FRDA and control fibroblasts was carried out. The results showed that the FRDA fibroblast cell cultures underwent growth arrest with higher cumulative population doubling compared to the controls. Though, further analysis of telomere length at different passage numbers revealed that the FRDA cells lost telomeres faster than the controls. Finally, the telomere dysfunction-induced foci (TIF) assay was performed to detect DNA damage in the human FRDA fibroblast cells using an antibody against DNA damage marker γ-H2AX and a synthetic PNA probe for telomeres. The frequency of γ-H2AX foci was significantly higher in the FRDA cells compared to the controls. Similarly, the FRDA cells had greater frequencies of TIFs in comparison to the controls, suggesting induced telomere dysfunction in the FRDA cells
Pharmacological screening using an FXN-EGFP cellular genomic reporter assay for the therapy of Friedreich ataxia
No full textCopyright @ 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
Quantitative investigations of FXN transcription and epigenetic modifications, including histone acetylation and methylation, in FRDA human and mouse tissues
No full textThis thesis was submitted for the degree of Master of Philosophy and awarded by Brunel University.Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative
disorder characterized by progressive gait and, limb ataxia, cardiomayopathy,
diabetes mellitus, optic atrophy and hearing loss. It is most often caused by
homozygous expanded (GAA)∙(TTC)n repeats within intron 1 of the FXN gene,
resulting in severely reduced levels of frataxin protein. The exact mechanisms of
how the expanded (GAA)∙(TTC)n repeats reduce FXN transcription are not fully
understood. However, many studies have suggested that the expanded repeat may
induce epigenetic modifications that cause the FXN transcription inhibition to occur.
In the past few years, epigenetic modifications have been given considerable
attention as an important mechanism that is contributing to the aetiology of FRDA.
This thesis investigated histone acetylation and methylation in three different regions
of the FXN gene: FXN promoter, upstream GAA and downstream GAA, using
chromatin immunoprecipitation (ChIP) and quantitative reverse transcriptase PCR
(qRT-PCR) of the human and transgenic mouse brain tissues. Furthermore, the
frataxin mRNA levels were investigated in autopsied brain tissues from an FRDA
patient and FXN transgenic mouse brain, heart and liver tissues. In addition, a
preliminary study that investigated the effect of a histone deacetylase inhibitor
(HDACi) on FXN transcription and histone modifications (acetylation) of transgenic
mouse brain, heart and liver tissues was conducted.
Results showed an overall significant decrease in the acetylation pattern of
H3K9ac and H4K16ac residues in all three regions within FXN gene. Moreover, a
significant increase in the di- and trimethylation pattern of the H3K9me2 and the
H3K9me3 residues was identified in all three regions of the FXN gene. The results
were comparable between the FRDA patient and transgenic mouse (YG8, YG22)
brain tissues. The FXN mRNA levels showed a significant decrease in all transgenic
mouse brain, heart and liver tissues, which is comparable with the FXN mRNA level
of the FRDA patient brain and heart tissues.Results for the preliminary HDACi study showed an approximate 20-30 %
increase in the FXN mRNA level in different transgenic mouse tissues after 3 days
intake at 150mg/kg dose. In addition, there was an increase in the acetylation pattern
of the H3K9ac and the H4K12ac in the HDACi treated transgenic mouse brain
tissues.
These studies will aid the understanding of FXN epigenetic modifications and
their contribution to FRDA disease; this is an exciting challenge leading to a new
effective FRDA therapeutic pathway
Friedreich ataxia patient tissues exhibit increased 5-hydroxymethylcytosine modification and decreased CTCF binding at the FXN locus
No full text© 2013 Al-Mahdawi 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.Friedreich ataxia (FRDA) is caused by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene, which induces epigenetic changes and FXN gene silencing. Bisulfite sequencing studies have identified 5-methylcytosine (5 mC) DNA methylation as one of the epigenetic changes that may be involved in this process. However, analysis of samples by bisulfite sequencing is a time-consuming procedure. In addition, it has recently been shown that 5-hydroxymethylcytosine (5 hmC) is also present in mammalian DNA, and bisulfite sequencing cannot distinguish between 5 hmC and 5 mC.The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 242193/EFACTS (CS), the Wellcome Trust [089757] (SA) and Ataxia UK (RMP) to MAP
Prolonged treatment with pimelic o-aminobenzamide HDAC inhibitors ameliorates the disease phenotype of a Friedreich ataxia mouse model
No full textNOTICE: this is the author’s version of a work that was accepted for publication in Neurobiology of Disease. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication.Friedreich ataxia (FRDA) is an inherited neurodegenerative disorder caused by GAA repeat expansion within the FXN gene, leading to epigenetic changes and heterochromatin-mediated gene silencing that result in a frataxin protein deficit. Histone deacetylase (HDAC) inhibitors, including pimelic o-aminobenzamide compounds 106, 109 and 136, have previously been shown to reverse FXN gene silencing in short-term studies of FRDA patient cells and a knock-in mouse model, but the functional consequences of such therapeutic intervention have thus far not been described. We have now investigated the long-term therapeutic effects of 106, 109 and 136 in our GAA repeat expansion mutation-containing YG8R FRDA mouse model. We show that there is no overt toxicity up to 5 months of treatment and there is amelioration of the FRDA-like disease phenotype. Thus, while the neurological deficits of this model are mild, 109 and 106 both produced an improvement of motor coordination, whereas 109 and 136 produced increased locomotor activity. All three compounds increased global histone H3 and H4 acetylation of brain tissue, but only 109 significantly increased acetylation of specific histone residues at the FXN locus. Effects on FXN mRNA expression in CNS tissues were modest, but 109 significantly increased frataxin protein expression in brain tissue. 109 also produced significant increases in brain aconitase enzyme activity, together with reduction of neuronal pathology of the dorsal root ganglia (DRG). Overall, these results support further assessment of HDAC inhibitors for treatment of Friedreich ataxia.This work was supported by Repligen Corporation; Muscular Dystrophy Association
(MDA) USA; Ataxia UK; Friedreich's Ataxia Research Alliance (FARA); GoFAR; and the Wellcome Trust [089757]
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