A modified mouse model of Friedreich's ataxia with conditional allele homozygosity delays onset of cardiomyopathy.

Friedreich's ataxia (FA) is an autosomal recessive disorder caused by a deficiency in frataxin (FXN), a mitochondrial protein that plays a critical role in the synthesis of iron-sulfur clusters (Fe-S), vital inorganic cofactors necessary for numerous cellular processes. FA is characterized by progressive ataxia and hypertrophic cardiomyopathy, with cardiac dysfunction as the most common cause of mortality in patients. Commonly used cardiac-specific mouse models of FA use the muscle creatine kinase (MCK) promoter to express Cre recombinase in cardiomyocytes and striated muscle cells in mice with one conditional allele and one floxed-out/null allele.

overexpression of frataxin causes toxicity mediated by iron-sulfur cluster deficiency.

Friedreich's ataxia is a rare disorder resulting from deficiency of frataxin, a mitochondrial protein implicated in the synthesis of iron-sulfur clusters. Preclinical studies in mice have shown that gene therapy is a promising approach to treat individuals with Friedreich's ataxia. However, a recent report provided evidence that AAVrh10-mediated overexpression of frataxin could lead to cardiotoxicity associated with mitochondrial dysfunction.

Correlation between frataxin expression and contractility revealed by in vitro Friedreich's ataxia cardiac tissue models engineered from human pluripotent stem cells.

Background: Friedreich's ataxia (FRDA) is an autosomal recessive disease caused by a non-coding mutation in the first intron of the frataxin (FXN) gene that suppresses its expression. Compensatory hypertrophic cardiomyopathy, dilated cardiomyopathy, and conduction system abnormalities in FRDA lead to cardiomyocyte (CM) death and fibrosis, consequently resulting in heart failure and arrhythmias. Murine models have been developed to study disease pathology in the past two decades; however, differences between human and mouse physiology and metabolism have limited the relevance of animal studies in cardiac disease conditions.

Structure of the human frataxin-bound iron-sulfur cluster assembly complex provides insight into its activation mechanism.

The core machinery for de novo biosynthesis of iron-sulfur clusters (ISC), located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreich's ataxia (FRDA). Here the 3.

4'-Phosphopantetheine and long acyl chain-dependent interactions are integral to human mitochondrial acyl carrier protein function.

The mitochondrial acyl carrier protein (human ACPM, yeast Acp1) is an essential mitochondrial protein. Through binding of nascent acyl chains on the serine (S112)-bound 4'-phosphopantetheine (4'-PP) cofactor, ACPM is involved in mitochondrial fatty acid synthesis and lipoic acid biogenesis. Recently, yeast Acp1 was found to interact with several mitochondrial complexes, including the iron-sulfur (Fe-S) cluster biosynthesis and respiratory complexes, the binding to LYRM proteins, a family of proteins involved in assembly/stability of complexes.

GLRX5 mutations impair heme biosynthetic enzymes ALA synthase 2 and ferrochelatase in Human congenital sideroblastic anemia.

Non-syndromic microcytic congenital sideroblastic anemia (cSA) is predominantly caused by defective genes encoding for either ALAS2, the first enzyme of heme biosynthesis pathway or SLC25A38, the mitochondrial importer of glycine, an ALAS2 substrate. Herein we explored a new case of cSA with two mutations in GLRX5, a gene for which only two patients have been reported so far. The patient was a young female with biallelic compound heterozygous mutations in GLRX5 (p.

Zinc(II) binding on human wild-type ISCU and Met140 variants modulates NFS1 desulfurase activity.

Human de novo iron-sulfur (Fe-S) assembly complex consists of cysteine desulfurase NFS1, accessory protein ISD11, acyl carrier protein ACP, scaffold protein ISCU, and allosteric activator frataxin (FXN). FXN binds the NFS1-ISD11-ACP-ISCU complex (SDAU), to activate the desulfurase activity and Fe-S cluster biosynthesis. In the absence of FXN, the NFS1-ISD11-ACP (SDA) complex was reportedly inhibited by binding of recombinant ISCU.

ISCA1 is essential for mitochondrial FeS biogenesis in vivo.

Mammalian A-type proteins, ISCA1 and ISCA2, are evolutionarily conserved proteins involved in iron-sulfur cluster (Fe-S) biogenesis. Recently, it was shown that ISCA1 and ISCA2 form a heterocomplex that is implicated in the maturation of mitochondrial FeS proteins. Here we report that mouse ISCA1 and ISCA2 are FeS-containing proteins that combine all features of Fe-S carrier proteins.

Iron regulatory protein 1 sustains mitochondrial iron loading and function in frataxin deficiency.

Mitochondrial iron accumulation is a hallmark of diseases associated with impaired iron-sulfur cluster (Fe-S) biogenesis, such as Friedreich ataxia linked to frataxin (FXN) deficiency. The pathophysiological relevance of the mitochondrial iron loading and the underlying mechanisms are unknown. Using a mouse model of hepatic FXN deficiency in combination with mice deficient for iron regulatory protein 1 (IRP1), a key regulator of cellular iron metabolism, we show that IRP1 activation in conditions of Fe-S deficiency increases the available cytosolic labile iron pool.

The diabetes drug target MitoNEET governs a novel trafficking pathway to rebuild an Fe-S cluster into cytosolic aconitase/iron regulatory protein 1.

In eukaryotes, mitochondrial iron-sulfur cluster (ISC), export and cytosolic iron-sulfur cluster assembly (CIA) machineries carry out biogenesis of iron-sulfur (Fe-S) clusters, which are critical for multiple essential cellular pathways. However, little is known about their export out of mitochondria. Here we show that Fe-S assembly of mitoNEET, the first identified Fe-S protein anchored in the mitochondrial outer membrane, strictly depends on ISC machineries and not on the CIA or CIAPIN1.

Dysregulation of cellular iron metabolism in Friedreich ataxia: from primary iron-sulfur cluster deficit to mitochondrial iron accumulation.

Friedreich ataxia (FRDA) is the most common recessive ataxia in the Caucasian population and is characterized by a mixed spinocerebellar and sensory ataxia frequently associating cardiomyopathy. The disease results from decreased expression of the FXN gene coding for the mitochondrial protein frataxin. Early histological and biochemical study of the pathophysiology in patient's samples revealed that dysregulation of iron metabolism is a key feature of the disease, mainly characterized by mitochondrial iron accumulation and by decreased activity of iron-sulfur cluster enzymes.

Mammalian frataxin controls sulfur production and iron entry during de novo Fe4S4 cluster assembly.

Iron-sulfur (Fe-S) cluster-containing proteins are essential components of cells. In eukaryotes, Fe-S clusters are synthesized by the mitochondrial iron-sulfur cluster (ISC) machinery and the cytosolic iron-sulfur assembly (CIA) system. In the mammalian ISC machinery, preassembly of the Fe-S cluster on the scaffold protein (ISCU) involves a cysteine desulfurase complex (NFS1/ISD11) and frataxin (FXN), the protein deficient in Friedreich's ataxia.

Clinical data and characterization of the liver conditional mouse model exclude neoplasia as a non-neurological manifestation associated with Friedreich's ataxia.

Friedreich's ataxia (FRDA) is the most common hereditary ataxia in the caucasian population and is characterized by a mixed spinocerebellar and sensory ataxia, hypertrophic cardiomyopathy and increased incidence of diabetes. FRDA is caused by impaired expression of the FXN gene coding for the mitochondrial protein frataxin. During the past ten years, the development of mouse models of FRDA has allowed better understanding of the pathophysiology of the disease.

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.

Mammalian frataxin: an essential function for cellular viability through an interaction with a preformed ISCU/NFS1/ISD11 iron-sulfur assembly complex.

Background: Frataxin, the mitochondrial protein deficient in Friedreich ataxia, a rare autosomal recessive neurodegenerative disorder, is thought to be involved in multiple iron-dependent mitochondrial pathways. In particular, frataxin plays an important role in the formation of iron-sulfur (Fe-S) clusters biogenesis.

Methodology/principal Findings: We present data providing new insights into the interactions of mammalian frataxin with the Fe-S assembly complex by combining in vitro and in vivo approaches.

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.

The in vivo mitochondrial two-step maturation of human frataxin.

Deficiency in the nuclear-encoded mitochondrial protein frataxin causes Friedreich ataxia (FRDA), a progressive neurodegenerative disorder associating spinocerebellar ataxia and cardiomyopathy. Although the exact function of frataxin is still a matter of debate, it is widely accepted that frataxin is a mitochondrial iron chaperone involved in iron-sulfur cluster and heme biosynthesis. Frataxin is synthesized as a precursor polypeptide, directed to the mitochondrial matrix where it is proteolytically cleaved by the mitochondrial processing peptidase to the mature form via a processing intermediate.

Zinc adaptation and resistance to cadmium toxicity in mammalian cells: molecular insight by proteomic analysis.

To identify proteins involved in cellular adaptive responses to zinc, a comparative proteome analysis between a previously developed high zinc- and cadmium-resistant human epithelial cell line (high zinc-resistant HeLa cells, HZR) and the parental HeLa cells has been carried out. Differentially produced proteins included cochaperones, proteins associated with oxido-reductase activities, and ubiquitin. Biochemical pathways to which these proteins belong were probed for their involvement in the resistance of both cell lines against cadmium toxicity.

Frataxin is essential for extramitochondrial Fe-S cluster proteins in mammalian tissues.

Friedreich ataxia, the most common recessive ataxia, is caused by the deficiency of the mitochondrial protein frataxin (Fxn), an iron chaperone involved in the assembly of Fe-S clusters (ISC). In yeast, mitochondria play a central role for all Fe-S proteins, independently of their subcellular localization. In mammalian cells, this central role of mitochondria remains controversial as an independent cytosolic ISC assembly machinery has been suggested.

Folding and turnover of human iron regulatory protein 1 depend on its subcellular localization.

Aconitases are iron-sulfur hydrolyases catalysing the interconversion of citrate and isocitrate in a wide variety of organisms. Eukaryotic aconitases have been assigned additional roles, as in the case of the metazoan dual activity cytosolic aconitase-iron regulatory protein 1 (IRP1). This human protein was produced in yeast mitochondria to probe IRP1 folding in this organelle where iron-sulfur synthesis originates.

Photoreactive threading agent that specifically binds to abasic sites in DNA.

We report the synthesis and study of a photoreactive nitrobenzamide containing acridine that specifically interacts at abasic site in DNA by threading intercalation and introduces under irradiation a lesion on the opposite strand at the unpaired pyrimidine.

IRP1 Ser-711 is a phosphorylation site, critical for regulation of RNA-binding and aconitase activities.

In iron-starved cells, IRP1 (iron regulatory protein 1) binds to mRNA iron-responsive elements and controls their translation or stability. In response to increased iron levels, RNA-binding is inhibited on assembly of a cubane [4Fe-4S] cluster, which renders IRP1 to a cytosolic aconitase. Phosphorylation at conserved serine residues may also regulate the activities of IRP1.

Zinc and cadmium specifically interfere with RNA-binding activity of human iron regulatory protein 1.

The cellular pro-oxidative stress induced by high zinc concentrations or cadmium is most likely mediated by disruption of redox (mainly thiol) homeostasis or by mishandling of redox-active transition metals. The impact of zinc and cadmium on the main regulators of iron homeostasis in metazoans, the iron regulatory proteins (IRP) 1 and 2, has been probed with the human recombinant proteins. Using purified proteins or extracts of yeast producing human IRP, zinc and cadmium were shown to interfere with the IRE-binding activity of IRP1, but not with that of IRP2 or the aconitase activity of IRP1.