A novel GAA-repeat-expansion-based mouse model of Friedreich’s ataxia

Friedreich’s 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 levels of frataxin protein. We have previously reported the generation of human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing 90–190 GAA repeats, but the presence of multiple GAA repeats within these mice is considered suboptimal. We now describe the cellular, molecular and behavioural characterisation of a newly developed YAC transgenic FRDA mouse model, designated YG8sR, which we have shown by DNA sequencing to contain a single pure GAA repeat expansion. The founder YG8sR mouse contained 120 GAA repeats but, due to intergenerational expansion, we have now established a colony of YG8sR mice that contain ~200 GAA repeats. We show that YG8sR mice have a single copy of the FXN transgene, which is integrated at a single site as confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We have identified significant behavioural deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8sR FRDA mice compared with control Y47R and wild-type (WT) mice. We have also detected increased somatic GAA repeat instability in the brain and cerebellum of YG8sR mice, together with significantly reduced expression of FXN, FAST-1 and frataxin, and reduced aconitase activity, compared with Y47R mice. Furthermore, we have confirmed the presence of pathological vacuoles within neurons of the dorsal root ganglia (DRG) of YG8sR mice. These novel GAA-repeat-expansion-based YAC transgenic FRDA mice, which exhibit progressive FRDA-like pathology, represent an excellent model for the investigation of FRDA disease mechanisms and therapy.KEY WORDS: GAA repeat, Friedreich’s ataxia, FRDA, YG8sR, Mouse model     

Inhibition of the SUV4-20 H1 histone methyltransferase increases frataxin expression in Friedreich's ataxia patient cells

The molecular mechanisms of reduced frataxin (FXN) expression in Friedreich''s ataxia (FRDA) are linked to epigenetic modification of the FXN locus caused by the disease-associated GAA expansion. Here, we identify that SUV4-20 histone methyltransferases, specifically SUV4-20 H1, play an important role in the regulation of FXN expression and represent a novel therapeutic target. Using a human FXN–GAA–Luciferase repeat expansion genomic DNA reporter model of FRDA, we screened the Structural Genomics Consortium epigenetic probe collection. We found that pharmacological inhibition of the SUV4-20 methyltransferases by the tool compound A-196 increased the expression of FXN by ∼1.5-fold in the reporter cell line. In several FRDA cell lines and patient-derived primary peripheral blood mononuclear cells, A-196 increased FXN expression by up to 2-fold, an effect not seen in WT cells. SUV4-20 inhibition was accompanied by a reduction in H4K20me2 and H4K20me3 and an increase in H4K20me1, but only modest (1.4–7.8%) perturbation in genome-wide expression was observed. Finally, based on the structural activity relationship and crystal structure of A-196, novel small molecule A-196 analogs were synthesized and shown to give a 20-fold increase in potency for increasing FXN expression. Overall, our results suggest that histone methylation is important in the regulation of FXN expression and highlight SUV4-20 H1 as a potential novel therapeutic target for FRDA.     

Two New Pimelic Diphenylamide HDAC Inhibitors Induce Sustained Frataxin Upregulation in Cells from Friedreich's Ataxia Patients and in a Mouse Model

Background

Friedreich''s ataxia (FRDA), the most common recessive ataxia in Caucasians, is due to severely reduced levels of frataxin, a highly conserved protein, that result from a large GAA triplet repeat expansion within the first intron of the frataxin gene (FXN). Typical marks of heterochromatin are found near the expanded GAA repeat in FRDA patient cells and mouse models. Histone deacetylase inhibitors (HDACIs) with a pimelic diphenylamide structure and HDAC3 specificity can decondense the chromatin structure at the FXN gene and restore frataxin levels in cells from FRDA patients and in a GAA repeat based FRDA mouse model, KIKI, providing an appealing approach for FRDA therapeutics.

Methodology/Principal Findings

In an effort to further improve the pharmacological profile of pimelic diphenylamide HDACIs as potential therapeutics for FRDA, we synthesized additional compounds with this basic structure and screened them for HDAC3 specificity. We characterized two of these compounds, 136 and 109, in FRDA patients'' peripheral blood lymphocytes and in the KIKI mouse model. We tested their ability to upregulate frataxin at a range of concentrations in order to determine a minimal effective dose. We then determined in both systems the duration of effect of these drugs on frataxin mRNA and protein, and on total and local histone acetylation. The effects of these compounds exceeded the time of direct exposure in both systems.

Conclusions/Significance

Our results support the pre-clinical development of a therapeutic approach based on pimelic diphenylamide HDACIs for FRDA and provide information for the design of future human trials of these drugs, suggesting an intermittent administration of the drug.     

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.     

Cellular,Molecular and Functional Characterisation of YAC Transgenic Mouse Models of Friedreich Ataxia

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.     

Understanding the genetic and molecular pathogenesis of Friedreich’s ataxia through animal and cellular models

In 1996, a link was identified between Friedreich’s ataxia (FRDA), the most common inherited ataxia in men, and alterations in the gene encoding frataxin (FXN). Initial studies revealed that the disease is caused by a unique, most frequently biallelic, expansion of the GAA sequence in intron 1 of FXN. Since the identification of this link, there has been tremendous progress in understanding frataxin function and the mechanism of FRDA pathology, as well as in developing diagnostics and therapeutic approaches for the disease. These advances were the subject of the 4th International Friedreich’s Ataxia Conference held on 5th–7th May in the Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France. More than 200 scientists gathered from all over the world to present the results of research spanning all areas of investigation into FRDA (including clinical aspects, FRDA pathogenesis, genetics and epigenetics of the disease, development of new models of FRDA, and drug discovery). This review provides an update on the understanding of frataxin function, developments of animal and cellular models of the disease, and recent advances in trying to uncover potential molecules for therapy.     

Activating frataxin expression by single-stranded siRNAs targeting the GAA repeat expansion

Friedreich's ataxia (FRDA) is an incurable neurodegenerative disorder caused by reduced expression of the mitochondrial protein frataxin (FXN). The genetic cause of the disease is an expanded GAA repeat within the FXN gene. Agents that increase expression of FXN protein are a potential approach to therapy. We previously described anti-trinucleotide GAA duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) that activate FXN protein expression in multiple patient derived cell lines. Here we test two distinct series of compounds for their ability to increase FXN expression. ASOs with butane linkers showed low potency, which is consistent with the low T_m values and suggesting that flexible conformation impairs activity. By contrast, single-stranded siRNAs (ss-siRNAs) that combine the strengths of dsRNA and ASO approaches had nanomolar potencies. ss-siRNAs provide an additional option for developing nucleic acid therapeutics to treat FRDA.     

Progress towards drug discovery for Friedreich’s Ataxia: Identifying synthetic oligonucleotides that more potently activate expression of human frataxin protein

Friedreich’s Ataxia (FRDA) is an incurable genetic disease caused by an expanded trinucleotide AAG repeat within intronic RNA of the frataxin (FXN) gene. We have previously demonstrated that synthetic antisense oligonucleotides or duplex RNAs that are complementary to the expanded repeat can activate expression of FXN and return levels of FXN protein to near normal. The potency of these compounds, however, was too low to encourage vigorous pre-clinical development. We now report testing of “gapmer” oligonucleotides consisting of a central DNA portion flanked by chemically modified RNA that increases binding affinity. We find that gapmer antisense oligonucleotides are several fold more potent activators of FXN expression relative to previously tested compounds. The potency of FXN activation is similar to a potent benchmark gapmer targeting the nuclear noncoding RNA MALAT-1, suggesting that our approach has potential for developing more effective compounds to regulate FXN expression in vivo.     

The First Cellular Models Based on Frataxin Missense Mutations That Reproduce Spontaneously the Defects Associated with Friedreich Ataxia

Background

Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)_n expansion within the first intron of the frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation.

Methodology

We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN^G130V and hFXN^I154F) frataxin. Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN^I154F mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients.

Conclusions

These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating frataxin deficiency.     

DNA triplex structures in neurodegenerative disorder, Friedreich’s ataxia

It is now established that a small fraction of genomic DNA does adopt the non-canonical B-DNA structure or 'unusual' DNA structure. The unusual DNA structures like DNA-hairpin, cruciform, Z-DNA, triplex and tetraplex are represented as hotspots of chromosomal breaks, homologous recombination and gross chromosomal rearrangements since they are prone to the structural alterations. Friedreich's ataxia (FRDA), the autosomal recessive degenerative disorder of nervous and muscles tissue, is caused by the massive expansion of (GAA) repeats that occur in the first intron of Frataxin gene X25 on chromosome 9q13-q21.1. The purine strand of the DNA in the expanded (GAA) repeat region folds back to form the (R.R*Y) type of triplex, which further inhibits the frataxin gene expression, and this clearly suggests that the shape of DNA is the determining factor in the cellular function. FRDA is the only disease known so far to be associated with DNA triplex. Structural characterization of GAA-containing DNA triplexes using some simple biophysical methods like UV melting, UV absorption, circular dichroic spectroscopy and electrophoretic mobility shift assay are discussed. Further, the clinical aspects and genetic analysis of FRDA patients who carry (GAA) repeat expansions are presented. The potential of some small molecules that do not favour the DNA triplex formation as therapeutics for FRDA are also briefly discussed.