Generation and Characterisation of Friedreich Ataxia YG8R Mouse Fibroblast and Neural Stem Cell Models

Chiranjeevi Sandi,Sara Anjomani-Virmouni,Harvinder Jassal,Sahar Al-Mahdawi,Mark A Pook,Madhavi Sandi,Vahid Ezzatizadeh,Annalisa Pastore

doi:10.1371/journal.pone.0089488

Abstract

BackgroundFriedreich 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 FindingsWe 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/SignificanceWe 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.

Highlights

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative mitochondrial disorder caused primarily by a homozygous GAA repeat expansion mutation within intron 1 of the frataxin (FXN) gene, located on chromosome 9q21.1 [1]
Fibroblasts were obtained from freshly isolated kidneys, while neural stem cells (NSCs) were isolated from the sub-ventricular zone (SVZ) of the brain and dissociated mechanically followed by enzymatic digestion of the tissue
The results clearly indicated the presence of neurons, oligodendrocytes and astrocytes in the differentiated NSCs

Summary

Introduction

FRDA is an autosomal recessive neurodegenerative mitochondrial disorder caused primarily by a homozygous GAA repeat expansion mutation within intron 1 of the frataxin (FXN) gene, located on chromosome 9q21.1 [1]. The effect of the GAA repeat expansion is to decrease expression of the essential and ubiquitously expressed mitochondrial protein frataxin, with levels in FRDA patients ranging from 4% to 29% that of normal [15]. Asymptomatic carriers produce about 50% frataxin levels compared to unaffected individuals [16]. 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. 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

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Results

Conclusion