Hepatic mitochondrial dysfunction in Friedreich Ataxia

Background  

Mitochondrial dysfunction due to respiratory chain impairment is a key feature in pathogenesis of Friedreich ataxia. Friedreich ataxia affects the nervous system, heart and pancreas.     

Phylogenetic Analysis of the Friedreich Ataxia GAA Trinucleotide Repeat

Friedreich ataxia is an autosomal recessive neurodegenerative disorder associated with a GAA repeat expansion in the first intron of the gene (FRDA) encoding a novel, highly conserved, 210 amino acid protein known as frataxin. Normal variation in repeat size was determined by analysis of more than 600 DNA samples from seven human populations. This analysis showed that the most frequent allele had nine GAA repeats, and no alleles with fewer than five GAA repeats were found. The European and Syrian populations had the highest percentage of alleles with 10 or more GAA repeats, while the Papua New Guinea population did not have any alleles carrying more than 10 GAA repeats. The distributions of repeat sizes in the European, Syrian, and African American populations were significantly different from those in the Asian and Papua New Guinea populations (p < 0.001). The GAA repeat size was also determined in five nonhuman primates. Samples from 10 chimpanzees, 3 orangutans, 1 gorilla, 1 rhesus macaque, 1 mangabey, and 1 tamarin were analyzed. Among those primates belonging to the Pongidae family, the chimpanzees were found to carry three or four GAA repeats, the orangutans had four or five GAA repeats, and the gorilla carried three GAA repeats. In primates belonging to the Cercopithecidae family, three GAA repeats were found in the mangabey and two in the rhesus macaque. However, an AluY subfamily member inserted in the poly(A) tract preceding the GAA repeat region in the rhesus macaque, making the amplified sequence approximately 300 bp longer. The GAA repeat was also found in the tamarin, suggesting that it arose at least 40 million years ago and remained relatively small throughout the majority of primate evolution, with a punctuated expansion in the human genome. Received: 18 August 2000 / Accepted: 10 November 2000     

Novel Frataxin Isoforms May Contribute to the Pathological Mechanism of Friedreich Ataxia

Friedreich ataxia (FRDA) is an inherited neurodegenerative disease caused by frataxin (FXN) deficiency. The nervous system and heart are the most severely affected tissues. However, highly mitochondria-dependent tissues, such as kidney and liver, are not obviously affected, although the abundance of FXN is normally high in these tissues. In this study we have revealed two novel FXN isoforms (II and III), which are specifically expressed in affected cerebellum and heart tissues, respectively, and are functional in vitro and in vivo. Increasing the abundance of the heart-specific isoform III significantly increased the mitochondrial aconitase activity, while over-expression of the cerebellum-specific isoform II protected against oxidative damage of Fe-S cluster-containing aconitase. Further, we observed that the protein level of isoform III decreased in FRDA patient heart, while the mRNA level of isoform II decreased more in FRDA patient cerebellum compared to total FXN mRNA. Our novel findings are highly relevant to understanding the mechanism of tissue-specific pathology in FRDA.     

Regular insulin secretory oscillations despite impaired ATP synthesis in Friedreich Ataxia patients.

Friedreich Ataxia is an inherited disorder caused by decreased expression of a mitochondrial protein called frataxin. Deficiency of this protein causes reduced biogenesis of iron-sulfur clusters, and subsequently impaired synthesis and replenishment of ATP IN VIVO. Basal secretion of insulin occurs in an oscillating manner presumably triggered by ATP-dependent feedback inhibition of glycolytic flux. Hence, individuals with reduced ATP synthesis rates should possibly exhibit impaired insulin secretory oscillations if these were solely dependent on ATP. In the present study Friedreich Ataxia patients with a presumptive impairment of ATP synthesis in pancreatic beta-cells were evaluated for regularity of basal secretory oscillations of insulin. Healthy siblings were employed as controls. In conflict with the initial hypothesis, no differences in regards to oscillation patterns were observed between patients and controls. Supported by EX VIVO evidence, these findings tentatively suggest that pulsatile insulin secretion might not be exclusively dependent on ATP feedback inhibition in humans.     

Impaired Nuclear Nrf2 Translocation Undermines the Oxidative Stress Response in Friedreich Ataxia

Background

Friedreich ataxia originates from a decrease in mitochondrial frataxin, which causes the death of a subset of neurons. The biochemical hallmarks of the disease include low activity of the iron sulfur cluster-containing proteins (ISP) and impairment of antioxidant defense mechanisms that may play a major role in disease progression.

Methodology/Principal Findings

We thus investigated signaling pathways involved in antioxidant defense mechanisms. We showed that cultured fibroblasts from patients with Friedreich ataxia exhibited hypersensitivity to oxidative insults because of an impairment in the Nrf2 signaling pathway, which led to faulty induction of antioxidant enzymes. This impairment originated from previously reported actin remodeling by hydrogen peroxide.

Conclusions/Significance

Thus, the defective machinery for ISP synthesis by causing mitochondrial iron dysmetabolism increases hydrogen peroxide production that accounts for the increased susceptibility to oxidative stress.     

The Friedreich Ataxia Critical Region Spans A 150-kb Interval on Chromosome 9q13

By analysis of crossovers in key recombinant families and by homozygosity analysis of inbred families, the Friedreich ataxia (FRDA) locus was localized in a 300-kb interval between the X104 gene and the microsatellite marker FR8 (D9S888). By homology searches of the sequence databases, we identified X104 as the human tight junction protein ZO-2 gene. We generated a largescale physical map of the FRDA region by pulsed-field gel electrophoresis analysis of genomic DNA and of three YAC clones derived from different libraries, and we constructed an uninterrupted cosmid contig spanning the FRDA locus. The cAMP-dependent protein kinase γ-catalytic subunit gene was identified within the critical FRDA interval, but it was excluded as candidate because of its biological properties and because of lack of mutations in FRDA patients. Six new polymorphic markers were isolated between FR2 (D9S886) and FR8 (D9S888), which were used for homozygosity analysis in a family in which parents of an affected child are distantly related. An ancient recombination involving the centromeric FRDA flanking markers had been previously demonstrated in this family. Homozygosity analysis indicated that the FRDA gene is localized in the telomeric 150 kb of the FR2-FR8 interval.     

化学药物导致的与Friedreich共济失调相关的GAA重复序列不稳定性的研究

Friedreich共济失调是由位于FRDA基因上的GAA重复序列动态突变扩增所引起的,目前其不稳定性机制还不清楚。为了探索化学药物对GAA重复序列的作用,本实验构建了含有(GAA)42的重组质粒,转入大肠杆菌中,分别经过丝裂霉素C和甲基磺酸乙酯连续多次处理后,检测其长度变化发现,两种化学药物在不同程度上都可以促使(GAA)42发生删除,且删除后长度多数处于体内的正常范围。试验结果表明,化学药物可以促进与Friedreich共济失调相关的GAA重复序列发生删除。本工作为研究Friedreich共济失调的致病机理及治疗方案奠定了一定的基础。     

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.     

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.     

Pharmacological Screening Using an FXN-EGFP Cellular Genomic Reporter Assay for the Therapy of Friedreich Ataxia

Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol.     

Mitochondrial Dysfunction in Friedreich's Ataxia: From Pathogenesis to Treatment Perspectives

Friedreich's ataxia (FRDA), the most common inherited ataxia, is an autosomal recessive degenerative disorder caused by a GAA triplet expansion or point mutations in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein, which is severely reduced in FRDA patients. The demonstration that deficit of frataxin in FRDA is associated with mitochondrial iron accumulation, increased sensitivity to oxidative stress, deficit of respiratory chain complex activities and in vivo impairment of cardiac and skeletal muscle tissue energy metabolism, has established FRDA as a "new" nuclear encoded mitochondrial disease. Pilot studies have shown the potential effect of antioxidant therapy based on idebenone or coenzyme Q 10 plus Vitamin E administration in this condition and provide a strong rationale for designing larger randomized clinical trials.     

糖尿病心肌病发病机制的研究进展

糖尿病心肌病（diabetic cardiomyopathy, DCM）是一种特殊类型的心脏疾病,在一定程度上增加了糖尿病患者发生心力衰竭的风险,也是糖尿病患者死亡的主要原因之一。DCM的发病机制涉及多个方面,心肌细胞代谢紊乱（如高血糖和胰岛素抵抗）、心肌炎症和纤维化是DCM发病的基础,这些因素单独或联合作用于DCM的发生和发展。目前临床上尚无根治DCM的有效药物,研究疾病的发病机制在开发靶向治疗药物中具有重要意义。主要对当前DCM发病机制的研究进展展开综述,以期为DCM的早期预防和治疗提供理论基础。     

Recombinations in Individuals Homozygous by Descent Localize the Friedreich Ataxia Locus in a Cloned 450-kb Interval

The locus for Friedreich ataxia (FRDA), a severe neurodegenerative disease, is tightly linked to markers D9S5 and D9S15, and analysis of rare recombination events has suggested the order cen–FRDA–D9S5–D9S15–qter. We report here the construction of a YAC contig extending 800 kb centromeric to D9S5 and the isolation of five new microsatellite markers from this region. In order to map these markers with respect to the FRDA locus, all within a 1-cM confidence interval, we sought to increase the genetic information of available FRDA families by considering homozygosity by descent and association with founder haplotypes in isolated populations. This approach allowed us to identify one phase-known recombination and one probable historic recombination on haplotypes from Réunion Island patients, both of which place three of the five markers proximal to FRDA. This represents the first identification of close FRDA flanking markers on the centromeric side. The two other markers allowed us to narrow the breakpoint of a previously identified distal recombination that is >180 kb from D9S5 (26P). Taken together, the results place the FRDA locus in a 450-kb interval, which is small enough for direct search of candidate genes. A detailed rare cutter restriction map and a cosmid contig covering this interval were constructed and should facilitate the search of genes in this region.     

Bioenergetics of the Calf Muscle in Friedreich Ataxia Patients Measured by 31P-MRS Before and After Treatment with Recombinant Human Erythropoietin

Friedreich ataxia (FRDA) is caused by a GAA repeat expansion in the FXN gene leading to reduced expression of the mitochondrial protein frataxin. Recombinant human erythropoietin (rhuEPO) is suggested to increase frataxin levels, alter mitochondrial function and improve clinical scores in FRDA patients. Aim of the present pilot study was to investigate mitochondrial metabolism of skeletal muscle tissue in FRDA patients and examine effects of rhuEPO administration by phosphorus 31 magnetic resonance spectroscopy (31P MRS). Seven genetically confirmed FRDA patients underwent 31P MRS of the calf muscles using a rest-exercise-recovery protocol before and after receiving 3000 IU of rhuEPO for eight weeks. FRDA patients showed more rapid phosphocreatine (PCr) depletion and increased accumulation of inorganic phosphate (Pi) during incremental exercise as compared to controls. After maximal exhaustive exercise prolonged regeneration of PCR and slowed decline in Pi can be seen in FRDA. PCr regeneration as hallmark of mitochondrial ATP production revealed correlation to activity of complex II/III of the respiratory chain and to demographic values. PCr and Pi kinetics were not influenced by rhuEPO administration. Our results confirm mitochondrial dysfunction and exercise intolerance due to impaired oxidative phosphorylation in skeletal muscle tissue of FRDA patients. MRS did not show improved mitochondrial bioenergetics after eight weeks of rhuEPO exposition in skeletal muscle tissue of FRDA patients.

Trial Registration

EU Clinical Trials Register 2008-000040-13