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Selected missense mutations impair frataxin processing in Friedreich ataxia.

ABSTRACT: Frataxin (FXN) is a highly conserved mitochondrial protein. Reduced FXN levels cause Friedreich ataxia, a recessive neurodegenerative disease. Typical patients carry GAA repeat expansions on both alleles, while a subgroup of patients carry a missense mutation on one allele and a GAA repeat expansion on the other. Here, we report that selected disease-related FXN missense mutations impair FXN localization, interaction with mitochondria processing peptidase, and processing.Immunocytochemical studies and subcellular fractionation were performed to study FXN import into the mitochondria and examine the mechanism by which mutations impair FXN processing. Coimmunoprecipitation was performed to study the interaction between FXN and mitochondrial processing peptidase. A proteasome inhibitor was used to model traditional therapeutic strategies. In addition, clinical profiles of subjects with and without point mutations were compared in a large natural history study.FXNI154F and FXNG130V missense mutations decrease FXN 81-210 levels compared with FXNWT, FXNR165C, and FXNW155R, but do not block its association with mitochondria. FXNI154F and FXNG130V also impair FXN maturation and enhance the binding between FXN 42-210 and mitochondria processing peptidase. Furthermore, blocking proteosomal degradation does not increase FXN 81-210 levels. Additionally, impaired FXN processing also occurs in fibroblasts from patients with FXNG130V. Finally, clinical data from patients with FXNG130V and FXNI154F mutations demonstrates a lower severity compared with other individuals with Friedreich ataxia.These data suggest that the effects on processing associated with FXNG130V and FXNI154F mutations lead to higher levels of partially processed FXN, which may contribute to the milder clinical phenotypes in these patients.

SUBMITTER: Clark E

PROVIDER: S-EPMC5553228 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Project description: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 GAATTC 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.

Project description: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. Keywords: drug response

Project description:Frataxin deficiency is the primary cause of Friedreich ataxia (FRDA), an autosomal recessive cardiodegenerative and neurodegenerative disease. Frataxin is a nuclear-encoded mitochondrial protein that is widely conserved among eukaryotes. Genetic inactivation of the yeast frataxin homologue (Yfh1p) results in mitochondrial iron accumulation and hypersensitivity to oxidative stress. Increased iron deposition and evidence of oxidative damage have also been observed in cardiac tissue and cultured fibroblasts from patients with FRDA. These findings indicate that frataxin is essential for mitochondrial iron homeostasis and protection from iron-induced formation of free radicals. The functional mechanism of frataxin, however, is still unknown. We have expressed the mature form of Yfh1p (mYfh1p) in Escherichia coli and have analyzed its function in vitro. Isolated mYfh1p is a soluble monomer (13,783 Da) that contains no iron and shows no significant tendency to self-associate. Aerobic addition of ferrous iron to mYfh1p results in assembly of regular spherical multimers with a molecular mass of approximately 1. 1 MDa (megadaltons) and a diameter of 13+/-2 nm. Each multimer consists of approximately 60 subunits and can sequester >3,000 atoms of iron. Titration of mYfh1p with increasing iron concentrations supports a stepwise mechanism of multimer assembly. Sequential addition of an iron chelator and a reducing agent results in quantitative iron release with concomitant disassembly of the multimer, indicating that mYfh1p sequesters iron in an available form. In yeast mitochondria, native mYfh1p exists as monomer and a higher-order species with a molecular weight >600,000. After addition of (55)Fe to the medium, immunoprecipitates of this species contain >16 atoms of (55)Fe per molecule of mYfh1p. We propose that iron-dependent self-assembly of recombinant mYfh1p reflects a physiological role for frataxin in mitochondrial iron sequestration and bioavailability.

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Selected missense mutations impair frataxin processing in Friedreich ataxia.

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