Genotype and phenotype characterisation of Friedreich ataxia mouse models and cells

This 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

Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids.

Oncogenic transformation is associated with profound changes in cellular metabolism, but whether tracking these can improve disease stratification or influence therapy decision-making is largely unknown. Using the iKnife to sample the aerosol of cauterized specimens, we demonstrate a new mode of real-time diagnosis, coupling metabolic phenotype to mutant PIK3CA genotype. Oncogenic PIK3CA results in an increase in arachidonic acid and a concomitant overproduction of eicosanoids, acting to promote cell proliferation beyond a cell-autonomous manner. Mechanistically, mutant PIK3CA drives a multimodal signaling network involving mTORC2-PKCζ-mediated activation of the calcium-dependent phospholipase A2 (cPLA2). Notably, inhibiting cPLA2 synergizes with fatty acid-free diet to restore immunogenicity and selectively reduce mutant PIK3CA-induced tumorigenicity. Besides highlighting the potential for metabolic phenotyping in stratified medicine, this study reveals an important role for activated PI3K signaling in regulating arachidonic acid metabolism, uncovering a targetable metabolic vulnerability that largely depends on dietary fat restriction. VIDEO ABSTRACT

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

MutLα heterodimers modify the molecular phenotype of Friedreich ataxia

This article has been made available through the Brunel Open Access Publishing Fund.Background: Friedreich ataxia (FRDA), the most common autosomal recessive ataxia disorder, is caused by a dynamic GAA repeat expansion mutation within intron 1 of FXN gene, resulting in down-regulation of frataxin expression. Studies of cell and mouse models have revealed a role for the mismatch repair (MMR) MutS-heterodimer complexes and the PMS2 component of the MutLα complex in the dynamics of intergenerational and somatic GAA repeat expansions: MSH2, MSH3 and MSH6 promote GAA repeat expansions, while PMS2 inhibits GAA repeat expansions. Methodology/Principal Findings: To determine the potential role of the other component of the MutLα complex, MLH1, in GAA repeat instability in FRDA, we have analyzed intergenerational and somatic GAA repeat expansions from FXN transgenic mice that have been crossed with Mlh1 deficient mice. We find that loss of Mlh1 activity reduces both intergenerational and somatic GAA repeat expansions. However, we also find that loss of either Mlh1 or Pms2 reduces FXN transcription, suggesting different mechanisms of action for Mlh1 and Pms2 on GAA repeat expansion dynamics and regulation of FXN transcription. Conclusions/Significance: Both MutLα components, PMS2 and MLH1, have now been shown to modify the molecular phenotype of FRDA. We propose that upregulation of MLH1 or PMS2 could be potential FRDA therapeutic approaches to increase FXN transcription. © 2014 Ezzatizadeh et al.This article has been made available through the Brunel Open Access Publishing Fund

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

Proteomics profiling of interactome dynamics by colocalisation analysis (COLA)

Localisation and protein function are intimately linked in eukaryotes, as proteins are localised to specific compartments where they come into proximity of other functionally relevant proteins. Significant co-localisation of two proteins can therefore be indicative of their functional association. We here present COLA, a proteomics based strategy coupled with a bioinformatics framework to detect protein–protein co-localisations on a global scale. COLA reveals functional interactions by matching proteins with significant similarity in their subcellular localisation signatures. The rapid nature of COLA allows mapping of interactome dynamics across different conditions or treatments with high precision.Cancer Research UK; BBSRC

PARK2 Depletion Connects Energy and Oxidative Stress to PI3K/Akt Activation via PTEN S-Nitrosylation

© 2017 The Authors. PARK2 is a gene implicated in disease states with opposing responses in cell fate determination, yet its contribution in pro-survival signaling is largely un-known. Here we show that PARK2is altered in over a third of all human cancers, and its depletion results in enhanced phosphatidylinositol 3-kinase/Akt (PI3K/Akt) activation and increased vulnerability to PI3K/Akt/mTOR inhibitors. PARK2 depletion contributes to AMPK-mediated activation of endothelial nitricoxide synthase (eNOS), enhanced levels of reactiveoxygen species, and a concomitant increase inoxidized nitric oxide levels, thereby promoting theinhibition of PTEN by S-nitrosylation and ubiquitination. Notably, AMPK activation alone is sufficient to induce PTEN S-nitrosylation in the absence of PARK2 depletion. Park2 loss and Pten loss also display striking cooperativity to promote tumorigenesis in vivo. Together, our findings reveal an important missing mechanism that might account for PTEN suppression in PARK2-deficient tumors, and they highlight the importance of PTEN S-nitrosylationin supporting cell survival and proliferation under conditions of energy deprivation.NIH P01-CA120964 (J.M.A. and L.C.C.) and R01-GM041890; Ministry of Education, Culture and Sport under the Program for Promoting and Hiring of Talent and its Employability (Subprogram for Mobility) of the Spanish Government; ICR; MRC grant MC_UP_1202/1

Transgene copy number.

<p>(<b>a</b>) Two TaqMan copy number assays were applied; Hs05092416-cn assay, represented in black, was designed to amplify a 106 bp fragment of <i>FXN</i> within intron 3 and Hs02407730-cn assay, represented in grey, was designed to amplify an 80 bp fragment of <i>FXN</i> within intron 1 and exon 2. Wild type (WT) served as a negative control with no copy number. Error bars = SD. <i>n</i> = 2. (<b>b</b> and <b>c</b>) Determination of the integration site of the transgenic <i>FXN</i> gene by FISH. Biotin-labelled RP11-265B8 and digoxigenin- labelled RP11-876N18 probes were hybridised onto interphase and metaphase chromosomes (DAPI stained) of YG8R, YG22R and Y47R cells. (<b>b</b>) All three cell lines showed a single integration site of the <i>FXN</i> transgene by metaphase analysis. (<b>c</b>) YG8R showed three hybridisation signals corresponded to the transgenic <i>FXN</i>, whereas YG22R and Y47R showed one signal indicating one copy of the <i>FXN</i> transgene. Scale bare = 10 µm.</p

Grip strength analysis.

<p>(<b>a</b>) Analysis of YG8R and YG22R mice revealed significantly reduced grip strength compared to B6 and Y47R controls when both males and females were analysed together (<i>n</i> = 10 mice per genotype). Analysis of (<b>b</b>) males and (<b>c</b>) females separately (<i>n</i> = 5 mice per genotype) revealed a significant decrease in grip strength of all mutant mice compared to the controls Values represent mean ± SEM. ^**P<0.01 and ^***P<0.001. Statistical differences between mutant and B6 control mice are indicated by the top bar while the bottom barindicates the differences between mutant and Y47R control mice.</p