Frataxin

Objective: Friedreich ataxia (FRDA) is an inherited neurodegenerative disease characterized by ataxia and cardiomy-opathy. Homozygous GAA trinucleotide repeat expansions in the first intron of FXN occur in 96% of affected individuals and reduce frataxin expression. Remaining individuals are compound heterozygous for a GAA expansion and a FXN point/insertion/deletion mutation. We examined disease-causing mutations and the impact on frataxin struc-ture/function and clinical outcome in FRDA. Methods: We compared clinical information from 111 compound heterozygotes and 131 individuals with homozy-gous expansions. Frataxin mutations were examined using structural modeling, stability analyses and systematic literature review, and categorized into four groups: (1) homozygous expansions, and three compound heterozygote groups; (2) null (no frataxin produced); (3) moderate/strong impact; and (4) minimal impact. Mean age of onset and the presence of cardiomyopathy and diabetes mellitus were compared using regression analyses. Results: Mutations in the hydrophobic core of frataxin affected stability whereas surface residue mutations affected interactions with iron sulfur cluster assembly and heme biosynthetic proteins. The null group of compound heterozy-gotes had significantly earlier age of onset and increased diabetes mellitus, compared to the homozygous expansion group. There were no significant differences in mean age of onset between homozygotes and the minimal and mod-erate/strong impact groups. Interpretation: In compound heterozygotes, expression of partially functional mutant frataxin delays age of onset and reduces diabetes mellitus, compared to those with no frataxin expression from the non-expanded allele. This integrated analysis of categorized frataxin mutations and their correlation with clinical outcome provide a definitive resource for investigating disease pathogenesis in FRDA.

Friedreich's ataxia (FRDA) is a rare inherited recessive disorder affecting the central and peripheral nervous systems and other extraneural organs such as the heart and pancreas. This incapacitating condition usually manifests in childhood or adolescence, exhibits an irreversible progression that confines the patient to a wheelchair, and leads to early death. FRDA is caused by a reduced level of the nuclear-encoded mitochondrial protein frataxin due to an abnormal GAA triplet repeat expansion in the first intron of the human FXN gene. FXN is evolutionarily conserved, with orthologs in essentially all eukaryotes and some prokaryotes, leading to the development of experimental models of this disease in different organisms. These FRDA models have contributed substantially to our current knowledge of frataxin function and the pathogenesis of the disease, as well as to explorations of suitable treatments. Drosophila melanogaster, an organism that is easy to manipulate genetically, has also become important in FRDA research. This review describes the substantial contribution of Drosophila to FRDA research since the characterization of the fly frataxin ortholog more than 15 years ago. Fly models have provided a comprehensive characterization of the defects associated with frataxin deficiency and have revealed genetic modifiers of disease phenotypes. In addition, these models are now being used in the search for potential therapeutic compounds for the treatment of this severe and still incurable disease.

abstract: Friedreich ataxia (FRDA), the most common of the hereditary ataxias, is an autosomal recessive, multisystem disorder characterised by progressive ataxia, sensory symptoms, weakness, scoliosis and cardiomyopathy. FRDA is caused by a GAA expansion in intron one of the FXN gene, leading to reduced levels of the encoded protein frataxin, which is thought to regulate cellular iron homeostasis. The cerebellar and spinocerebellar dysfunction seen in FRDA has known effects on motor function; however until recently slowed information processing has been the main feature consistently reported by the limited studies addressing cognitive function in FRDA. This chapter will systematically review the current literature regarding the neuropathological and neurobehavioural phenotype associated with FRDA. It will evaluate more recent evidence adopting systematic experimental methodologies that postulate that the neurobehavioural phenotype associated with FRDA is likely to involve impairment in cerebello‑cortico connectivity. Tandem Repeat Polymorphisms: Genetic Plasticity, Neural Diversity and Disease, edited by Anthony Hannan

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease most commonly caused by a GAA trinucleotide repeat expansion in the first intron of FXN, which reduces expression of the mitochondrial protein frataxin. Approximately 98% of individuals with FRDA are homozygous for GAA expansions, with the remaining 2% compound hetero-zygotes for a GAA expansion and a point mutation within FXN. Two siblings with early onset of symptoms experienced rapid loss of ambulation by 8 and 10 years. Diagnostic testing for FRDA demonstrated one GAA repeat expansion of 1010 repeats and one non-expanded allele. Sequencing all five exons of FXN identified a novel deletion-insertion mutation in exon 3 (c.371_376del6ins15), which results in a modified frataxin protein sequence at amino acid positions 124–127. Specifically, the amino acid sequence changes from DVSF to VHLEDT, increasing frataxin from 211 residues to 214. Using the known structure of human frataxin, a theoretical 3D model of the mutant protein was developed. In the event that the modified protein is expressed and stable, it is predicted that the acidic interface of frataxin, known to be involved in iron binding and interactions with the iron–sulphur cluster assembly factor IscU, would be impaired.

Objective: Friedreich ataxia (FA) is the most common ataxia and results from an expanded GAA repeat in the first intron of FXN. This leads to epigenetic modifications and reduced frataxin. We investigated the relationships between genetic, epigenetic, and clinical parameters in a large case–control study of FA. Methods: Clinical data and samples were obtained from individuals with FA during annual visits to our dedicated FA clinic. GAA expansions were evaluated by polymerase chain reaction (PCR) and restriction endonuclease digest. DNA methylation was measured using bisulfite-based EpiTYPER MassARRAY (Sequenom, San Diego, CA). FXN expression was determined using real-time reverse transcriptase PCR. Significant correlations between the different parameters were examined using the nonparametric Spearman rank correlation coefficient, as well as univariate and multivariate regression modeling. Results: Characteristic DNA methylation was identified upstream and downstream of the expansion, and validated in an independent FA cohort. Univariate and multivariate analyses showed significant inverse correlations between upstream methylation and FXN expression, and variation in downstream methylation and age of onset. FXN expression also inversely correlated with the Friedreich Ataxia Rating Scale score, an indicator of disease severity.

Friedreich ataxia (FRDA) is an autosomal reces-sive disorder characterised by neurodegeneration and cardiomyopathy. It is caused by a trinucleotide (GAA) repeat expansion in the first intron of the FXN gene that results in reduced synthesis of FXN mRNA and its protein product, frataxin. We report the generation of induced pluripotent stem (iPS) cell lines derived from skin fibro-blasts from two FRDA patients. Each of the patient-derived iPS (FA-iPS) cell lines maintain the GAA repeat expansion and the reduced FXN mRNA expression that are characteristic of the patient. The FA-iPS cells are pluripotent and form teratomas when injected into nude mice. We demonstrate that following in vitro differentiation the FA-iPS cells give rise to the two cell types primarily affected in FRDA, peripheral neurons and cardiomyocytes. The FA-iPS cell lines have the potential to provide valuable models to study the cellular pathology of FRDA and to develop high-throughput drug screening assays. We have previously demonstrated that stable insertion of a functional human BAC containing the intact FXN gene into stem cells results

BACKGROUND:

Friedreich's ataxia (FA), the most frequent form of inherited ataxias in the Caucasian population, is caused by a reduced expression of frataxin, a highly conserved protein. Model organisms have contributed greatly in the efforts to decipher the function of frataxin; however, the precise function of this protein remains elusive. Overexpression studies are a useful approach to investigate the mechanistic actions of frataxin; however, the existing literature reports contradictory results. To further investigate the effect of frataxin overexpression, we analyzed the consequences of overexpressing human (FXN) and fly (FH) frataxins in Drosophila.
METHODOLOGY/PRINCIPAL FINDINGS:

We obtained transgenic flies that overexpressed human or fly frataxins in a general pattern and in different tissues using the UAS-GAL4 system. For both frataxins, we observed deleterious effects at the biochemical, histological and behavioral levels. Oxidative stress is a relevant factor in the frataxin overexpression phenotypes. Systemic frataxin overexpression reduces Drosophila viability and impairs the normal embryonic development of muscle and the peripheral nervous system. A reduction in the level of aconitase activity and a decrease in the level of NDUF3 were also observed in the transgenic flies that overexpressed frataxin. Frataxin overexpression in the nervous system reduces life span, impairs locomotor ability and causes brain degeneration. Frataxin aggregation and a misfolding of this protein have been shown not to be the mechanism that is responsible for the phenotypes that have been observed. Nevertheless, the expression of human frataxin rescues the aconitase activity in the fh knockdown mutant.
CONCLUSION/SIGNIFICANCE:

Our results provide in vivo evidence of a functional equivalence for human and fly frataxins and indicate that the control of frataxin expression is important for treatments that aim to increase frataxin levels.

Friedreich’s ataxia (FRDA) is a rare inherited recessive disorder affecting the central and peripheral nervous systems and other extraneural organs such as the heart and pancreas. This incapacitating condition usually manifests in childhood or adolescence, exhibits an irreversible progression that confines the patient to a wheelchair, and leads to early death. FRDA is caused by a reduced level of the nuclear-encoded mitochondrial protein frataxin due to an abnormal GAA triplet repeat expansion in the first intron of the human FXN gene. FXN is evolutionarily conserved, with orthologs in essentially all eukaryotes and some prokaryotes, leading to the development of experimental models of this disease in different organisms. These FRDA models have contributed substantially to our current knowledge of frataxin function and the pathogenesis of the disease, as well as to explorations of suitable treatments. Drosophila melanogaster, an organism that is easy to manipulate genetically, has a...

The relationships between conformational dynamics, stability and protein function are
not obvious. Frataxin (FXN) is an essential protein that forms part of a supercomplex
dedicated to the iron-sulfur (Fe-S) cluster assembly within the mitochondrial matrix. In
humans, the loss of FXN expression or a decrease in its functionality results in
Friedreich’s Ataxia, a cardio-neurodegenerative disease. Recently, the way in which
FXN interacts with the rest of the subunits of the supercomplex was uncovered. This
opens a window to explore relationships between structural dynamics and function. In
this study, we prepared a set of FXN variants spanning a broad range of conformational
stabilities. Variants S160I, S160M and A204R were more stable than the wild-type and
showed similar biological activity. Additionally, we prepared SILCAR, a variant that
combines S160I, L203C and A204R mutations. SILCAR was 2.4 kcal mol-1 more stable
and equally active. Some of the variants were significantly more resistant to proteolysis
than the wild-type FXN. SILCAR showed the highest resistance, suggesting a more
rigid structure. It was corroborated by means of molecular dynamics simulations.
Relaxation dispersion NMR experiments comparing SILCAR and wild-type variants
suggested similar internal motions in the microsecond to millisecond timescale. Instead,
variant S157I showed higher denaturation resistance but a significant lower function,
similarly to that observed for the FRDA variant N146K. We concluded that the
contribution of particular side chains to the conformational stability of FXN might be
highly subordinated to their impact on both the protein function and the stability of the
functional supercomplex.