Myriam Rai

HDAC inhibitors correct frataxin deficiency in a Friedreich ataxia mouse model.

Background Friedreich ataxia, an autosomal recessive neurodegenerative and cardiac disease, is caused by abnormally low levels of frataxin, an essential mitochondrial protein. All Friedreich ataxia patients carry a GAA⋅TTC repeat expansion in the first intron of the frataxin gene, either in the homozygous state or in compound heterozygosity with other loss-of-function mutations. The GAA expansion inhibits frataxin expression through a heterochromatin-mediated repression mechanism. Histone modifications that are characteristic of silenced genes in heterochromatic regions occur at expanded alleles in cells from Friedreich ataxia patients, including increased trimethylation of histone H3 at lysine 9 and hypoacetylation of histones H3 and H4. Methodology/Principal Findings By chromatin immunoprecipitation, we detected the same heterochromatin marks in homozygous mice carrying a (GAA)230 repeat in the first intron of the mouse frataxin gene (KIKI mice). These animals have decreased frataxin levels and, by microarray analysis, show significant gene expression changes in several tissues. We treated KIKI mice with a novel histone deacetylase inhibitor, compound 106, which substantially increases frataxin mRNA levels in cells from Friedreich ataxia individuals. Treatment increased histone H3 and H4 acetylation in chromatin near the GAA repeat and restored wild-type frataxin levels in the nervous system and heart, as determined by quantitative RT-PCR and semiquantitative western blot analysis. No toxicity was observed. Furthermore, most of the differentially expressed genes in KIKI mice reverted towards wild-type levels. Conclusions/Significance Lack of acute toxicity, normalization of frataxin levels and of the transcription profile changes resulting from frataxin deficiency provide strong support to a possible efficacy of this or related compounds in reverting the pathological process in Friedreich ataxia, a so far incurable neurodegenerative disease.

Neurons and cardiomyocytes derived from induced pluripotent stem cells as a model for mitochondrial defects in Friedreich's ataxia

SUMMARY Friedreich’s ataxia (FRDA) is a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy. FRDA is due to expanded GAA repeats within the first intron of the gene encoding frataxin, a conserved mitochondrial protein involved in iron-sulphur cluster biosynthesis. This mutation leads to partial gene silencing and substantial reduction of the frataxin level. To overcome limitations of current cellular models of FRDA, we derived induced pluripotent stem cells (iPSCs) from two FRDA patients and successfully differentiated them into neurons and cardiomyocytes, two affected cell types in FRDA. All FRDA iPSC lines displayed expanded GAA alleles prone to high instability and decreased levels of frataxin, but no biochemical phenotype was observed. Interestingly, both FRDA iPSC-derived neurons and cardiomyocytes exhibited signs of impaired mitochondrial function, with decreased mitochondrial membrane potential and progressive mitochondrial degeneration, respectively. Our data show for the first time that FRDA iPSCs and their neuronal and cardiac derivatives represent promising models for the study of mitochondrial damage and GAA expansion instability in FRDA.

Functional genomic analysis of frataxin deficiency reveals tissue-specific alterations and identifies the PPARγ pathway as a therapeutic target in Friedreich’s ataxia

Friedreich’s ataxia (FRDA), the most common inherited ataxia, is characterized by focal neurodegeneration, diabetes mellitus and life-threatening cardiomyopathy. Frataxin, which is significantly reduced in patients with this recessive disorder, is a mitochondrial iron-binding protein, but how its deficiency leads to neurodegeneration and metabolic derangements is not known. We performed microarray analysis of heart and skeletal muscle in a mouse model of frataxin deficiency, and found molecular evidence of increased lipogenesis in skeletal muscle, and alteration of fiber-type composition in heart, consistent with insulin resistance and cardiomyopathy, respectively. Since the peroxisome proliferator-activated receptor gamma (PPARγ) pathway is known to regulate both processes, we hypothesized that dysregulation of this pathway could play a key role in frataxin deficiency. We confirmed this by showing a coordinate dysregulation of the PPARγ coactivator Pgc1a and transcription factor Srebp1 in cellular and animal models of frataxin deficiency, and in cells from FRDA patients, who have marked insulin resistance. Finally, we show that genetic modulation of the PPARγ pathway affects frataxin levels in vitro, supporting PPARγ as a novel therapeutic target in FRDA.

Epigenetic therapy for Friedreich ataxia.

To investigate whether a histone deacetylase inhibitor (HDACi) would be effective in an in vitro model for the neurodegenerative disease Friedreich ataxia (FRDA) and to evaluate safety and surrogate markers of efficacy in a phase I clinical trial in patients.

Grafting Neural Precursor Cells Promotes Functional Recovery in an SCA1 Mouse Model

The B05 transgenic SCA1 mice, expressing human ataxin-1 with an expanded polyglutamine tract in cerebellar Purkinje cells (PCs), recapitulate many pathological and behavioral characteristics of the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1), including progressive ataxia and PC loss. We transplanted neural precursor cells (NPCs) derived from the subventricular zone of GFP-expressing adult mice into the cerebellar white matter of SCA1 mice when they showed absent (5 weeks), initial (13 weeks), and significant (24 weeks) PC loss. Only in mice with significant cell loss, grafted NPCs migrated into the cerebellar cortex. These animals showed improved motor skills compared with sham-treated controls. No grafted cell adopted the morphological and immunohistochemical characteristics of PCs, but the cerebellar cortex in NPC-grafted SCA1 mice had a significantly thicker molecular layer and more surviving PCs. Perforated patch-clamp recordings revealed a normalization of the PC basal membrane potential, which was abnormally depolarized in sham-treated animals. No significant increase in levels of several neurotrophic factors was observed, suggesting, along with morphological observation, that the neuroprotective effect of grafted NPCs was mediated by direct contact with the host PCs. We postulate that a similar neuroprotective effect of NPCs may be applicable to other cerebellar degenerative diseases.

A gene expression phenotype in lymphocytes from Friedreich ataxia patients.

Gene expression studies in peripheral tissues from patients with neurodegenerative disorders can provide insights into disease pathogenesis, and identify potential biomarkers, an important goal of translational research in neurodegeneration. Friedreich Ataxia (FRDA) is a chronic neurodegenerative disease caused by reduced transcription of frataxin, a ubiquitously expressed protein. We studied in vitro lymphocytes from FRDA patients and carriers to identify a peripheral gene expression phenotype. Peripheral biomarkers related to disease status would be extremely valuable for assessing drug efficacy and could provide new pathophysiological insights.

Central role and mechanisms of β-cell dysfunction and death in friedreich ataxia-associated diabetes.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused in almost all cases by homozygosity for a GAA trinucleotide repeat expansion in the frataxin gene. Frataxin is a mitochondrial protein involved in iron homeostasis. FRDA patients have a high prevalence of diabetes, the pathogenesis of which is not known. We aimed to evaluate the relative contribution of insulin resistance and β‐cell failure and the pathogenic mechanisms involved in FRDA diabetes.

Progression characteristics of the European Friedreich’s Ataxia Consortium for Translational Studies (EFACTS): a 2 year cohort study

BACKGROUND The European Friedreichs Ataxia Consortium for Translational Studies (EFACTS) is a prospective international registry investigating the natural history of Friedreichs ataxia. We used data from EFACTS to assess disease progression and the predictive value of disease-related factors on progression, and estimated sample sizes for interventional randomised clinical trials. METHODS We enrolled patients with genetically confirmed Friedreichs ataxia from 11 European study sites in Austria, Belgium, France, Germany, Italy, Spain, and the UK. Patients were seen at three visits-baseline, 1 year, and 2 years. Our primary endpoint was the Scale for the Assessment and Rating of Ataxia (SARA). Secondary outcomes were the Inventory of Non-Ataxia Signs (INAS), the Spinocerebellar Ataxia Functional Index (SCAFI), phonemic verbal fluency (PVF), and the quality of life measures activities of daily living (ADL) and EQ-5D-3L index. We estimated the yearly progression for each outcome with linear mixed-effect modelling. This study is registered with ClinicalTrials.gov, number NCT02069509, and follow-up assessments and recruitment of new patients are ongoing. FINDINGS Between Sept 15, 2010, and Nov 21, 2013, we enrolled 605 patients with Friedreichs ataxia. 546 patients (90%) contributed data with at least one follow-up visit. The progression rate on SARA was 0·77 points per year (SE 0·06) in the overall cohort. Deterioration in SARA was associated with younger age of onset (-0·02 points per year [0·01] per year of age) and lower SARA baseline scores (-0·07 points per year [0·01] per baseline point). Patients with more than 353 GAA repeats on the shorter allele of the FXN locus had a higher SARA progression rate (0·09 points per year [0·02] per additional 100 repeats) than did patients with fewer than 353 repeats. Annual worsening was 0·10 points per year (0·03) for INAS, -0·04 points per year (0·01) for SCAFI, 0·93 points per year (0·06) for ADL, and -0·02 points per year (0·004) for EQ-5D-3L. PVF performance improved by 0·99 words per year (0·14). To detect a 50% reduction in SARA progression at 80% power, 548 patients would be needed in a 1 year clinical trial and 184 would be needed for a 2 year trial. INTERPRETATION Our results show that SARA is a suitable clinical rating scale to detect deterioration of ataxia symptoms over time; ADL is an appropriate measure to monitor changes in daily self-care activities; and younger age at disease onset is a major predictor for faster disease progression. The results of the EFACTS longitudinal analysis provide suitable outcome measures and sample size calculations for the design of upcoming clinical trials of Friedreichs ataxia. FUNDING European Commission.

Autosomal recessive cerebellar ataxia of adult onset due to STUB1 Mutations

Autosomal recessive ataxias affect about 1 person in 20,000. Friedreich ataxia accounts for one-third of the cases in Caucasians; the others are due to a growing list of very rare molecular defects, including mild forms of metabolic diseases. In nearly 50%, the genetic cause remains undetermined.1

MME mutation in dominant spinocerebellar ataxia with neuropathy (SCA43)

Objective: To identify the causative gene mutation in a 5-generation Belgian family with dominantly inherited spinocerebellar ataxia and polyneuropathy, in which known genetic etiologies had been excluded. Methods: We collected DNA samples of 28 family members, including 7 living affected individuals, whose clinical records were reviewed by a neurologist experienced in ataxia. We combined linkage data of 21 family members with whole exome sequencing in 2 affected individuals to identify shared heterozygous variants mapping to potentially linked regions. Variants were screened for rarity and for predicted damaging effect. A candidate mutation was confirmed by Sanger sequencing and tested for cosegregation with the disease. Results: Affected individuals presented with late-onset sensorimotor axonal polyneuropathy; all but one also had cerebellar ataxia. We identified a variant in the MME gene, p.C143Y, that was absent from control databases, cosegregated with the phenotype, and was predicted to have a strong damaging effect on the encoded protein by all algorithms we used. Conclusions: MME encodes neprilysin (NEP), a zinc-dependent metalloprotease expressed in most tissues, including the central and peripheral nervous systems. The mutated cysteine 143 forms a disulfide bridge, which is 100% conserved in NEP and in similar enzymes. The recent identification of recessive MME mutations in 10 unrelated individuals from Japan with axonal polyneuropathy further supports the causality of the mutation, despite the dominant mode of inheritance and the presence of cerebellar involvement in our study family. Functional studies are needed to identify the mechanisms underlying these differences.