Identification of nonferritin mitochondrial iron deposits in a mouse model of Friedreich ataxia.

There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreich ataxia (FA). This disease is due to decreased expression of the mitochondrial protein, frataxin, which leads to alterations in mitochondrial iron (Fe) metabolism. The identification of potentially toxic mitochondrial Fe deposits in FA suggests Fe plays a role in its pathogenesis. Studies using the muscle creatine kinase (MCK) conditional frataxin knockout mouse that mirrors the disease have demonstrated frataxin deletion alters cardiac Fe metabolism. Indeed, there are pronounced changes in Fe trafficking away from the cytosol to the mitochondrion, leading to a cytosolic Fe deficiency. Considering Fe deficiency can induce apoptosis and cell death, we examined the effect of dietary Fe supplementation, which led to body Fe loading and limited the cardiac hypertrophy in MCK mutants. Furthermore, this study indicates a unique effect of heart and skeletal muscle-specific frataxin deletion on systemic Fe metabolism. Namely, frataxin deletion induces a signaling mechanism to increase systemic Fe levels and Fe loading in tissues where frataxin expression is intact (i.e., liver, kidney, and spleen). Examining the mutant heart, native size-exclusion chromatography, transmission electron microscopy, Mössbauer spectroscopy, and magnetic susceptibility measurements demonstrated that in the absence of frataxin, mitochondria contained biomineral Fe aggregates, which were distinctly different from isolated mammalian ferritin molecules. These mitochondrial aggregates of Fe, phosphorus, and sulfur, probably contribute to the oxidative stress and pathology observed in the absence of frataxin.

Mitochondrial iron trafficking and the integration of iron metabolism between the mitochondrion and cytosol.

The mitochondrion is well known for its key role in energy transduction. However, it is less well appreciated that it is also a focal point of iron metabolism. Iron is needed not only for heme and iron sulfur cluster (ISC)-containing proteins involved in electron transport and oxidative phosphorylation, but also for a wide variety of cytoplasmic and nuclear functions, including DNA synthesis. The mitochondrial pathways involved in the generation of both heme and ISCs have been characterized to some extent. However, little is known concerning the regulation of iron uptake by the mitochondrion and how this is coordinated with iron metabolism in the cytosol and other organelles (e.g., lysosomes). In this article, we discuss the burgeoning field of mitochondrial iron metabolism and trafficking that has recently been stimulated by the discovery of proteins involved in mitochondrial iron storage (mitochondrial ferritin) and transport (mitoferrin-1 and -2). In addition, recent work examining mitochondrial diseases (e.g., Friedreich's ataxia) has established that communication exists between iron metabolism in the mitochondrion and the cytosol. This finding has revealed the ability of the mitochondrion to modulate whole-cell iron-processing to satisfy its own requirements for the crucial processes of heme and ISC synthesis. Knowledge of mitochondrial iron-processing pathways and the interaction between organelles and the cytosol could revolutionize the investigation of iron metabolism.

The ins and outs of mitochondrial iron-loading: the metabolic defect in Friedreich's ataxia.

Friedreich's ataxia is a cardio- and neurodegenerative disease due to decreased expression of the mitochondrial protein, frataxin. This defect results in mitochondrial iron-overload, and in this review, we discuss the mechanisms that lead to this iron accumulation. Using a conditional knockout mouse model where frataxin is deleted in the heart, it has been shown that this mutation leads to transferrin receptor-1 upregulation, resulting in increased iron uptake from transferrin. There is also marked downregulation of ferritin that is required for iron storage and decreased expression of the iron exporter, ferroportin 1, leading to decreased cellular iron efflux. The increased mitochondrial iron uptake is facilitated by upregulation of the mitochondrial iron transporter, mitoferrin 2. This stimulation of iron uptake probably attempts to rescue the deficit in mitochondrial iron metabolism that is due to downregulation of mitochondrial iron utilization, namely, heme and iron-sulfur cluster (ISC) synthesis and also iron storage (mitochondrial ferritin). The resultant decrease in heme and ISC synthesis means heme and ISCs are not exiting the mitochondrion for cytosolic use. Hence, increased mitochondrial iron uptake coupled with decreased utilization and release leads to mitochondrial iron-loading. More generally, disturbance of mitochondrial iron utilization in other diseases probably also results in similar compensatory alterations.

Elucidation of the mechanism of mitochondrial iron loading in Friedreich's ataxia by analysis of a mouse mutant.

We used the muscle creatine kinase (MCK) conditional frataxin knockout mouse to elucidate how frataxin deficiency alters iron metabolism. This is of significance because frataxin deficiency leads to Friedreich's ataxia, a disease marked by neurologic and cardiologic degeneration. Using cardiac tissues, we demonstrate that frataxin deficiency leads to down-regulation of key molecules involved in 3 mitochondrial utilization pathways: iron-sulfur cluster (ISC) synthesis (iron-sulfur cluster scaffold protein1/2 and the cysteine desulferase Nfs1), mitochondrial iron storage (mitochondrial ferritin), and heme synthesis (5-aminolevulinate dehydratase, coproporphyrinogen oxidase, hydroxymethylbilane synthase, uroporphyrinogen III synthase, and ferrochelatase). This marked decrease in mitochondrial iron utilization and resultant reduced release of heme and ISC from the mitochondrion could contribute to the excessive mitochondrial iron observed. This effect is compounded by increased iron availability for mitochondrial uptake through (i) transferrin receptor1 up-regulation, increasing iron uptake from transferrin; (ii) decreased ferroportin1 expression, limiting iron export; (iii) increased expression of the heme catabolism enzyme heme oxygenase1 and down-regulation of ferritin-H and -L, both likely leading to increased "free iron" for mitochondrial uptake; and (iv) increased expression of the mammalian exocyst protein Sec15l1 and the mitochondrial iron importer mitoferrin-2 (Mfrn2), which facilitate cellular iron uptake and mitochondrial iron influx, respectively. Our results enable the construction of a model explaining the cytosolic iron deficiency and mitochondrial iron loading in the absence of frataxin, which is important for understanding the pathogenesis of Friedreich's ataxia.

The MCK mouse heart model of Friedreich's ataxia: Alterations in iron-regulated proteins and cardiac hypertrophy are limited by iron chelation.

There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreich's ataxia (FA). The identification of potentially toxic mitochondrial (MIT) iron (Fe) deposits in FA suggests that Fe plays a role in its pathogenesis. This study used the muscle creatine kinase conditional frataxin (Fxn) knockout (mutant) mouse model that reproduces the classical traits associated with cardiomyopathy in FA. We examined the mechanisms responsible for the increased cardiac MIT Fe loading in mutants. Moreover, we explored the effect of Fe chelation on the pathogenesis of the cardiomyopathy. Our investigation showed that increased MIT Fe in the myocardium of mutants was due to marked transferrin Fe uptake, which was the result of enhanced transferrin receptor 1 expression. In contrast to the mitochondrion, cytosolic ferritin expression and the proportion of cytosolic Fe were decreased in mutant mice, indicating cytosolic Fe deprivation and markedly increased MIT Fe targeting. These studies demonstrated that loss of Fxn alters cardiac Fe metabolism due to pronounced changes in Fe trafficking away from the cytosol to the mitochondrion. Further work showed that combining the MIT-permeable ligand pyridoxal isonicotinoyl hydrazone with the hydrophilic chelator desferrioxamine prevented cardiac Fe loading and limited cardiac hypertrophy in mutants but did not lead to overt cardiac Fe depletion or toxicity. Fe chelation did not prevent decreased succinate dehydrogenase expression in the mutants or loss of cardiac function. In summary, we show that loss of Fxn markedly alters cellular Fe trafficking and that Fe chelation limits myocardial hypertrophy in the mutant.

Proteomic analysis of hearts from frataxin knockout mice: marked rearrangement of energy metabolism, a response to cellular stress and altered expression of proteins involved in cell structure, motility and metabolism.

A frequent cause of death in Friedreich's ataxia patients is cardiomyopathy, but the molecular alterations underlying this condition are unknown. We performed 2-DE to characterize the changes in protein expression of hearts using the muscle creatine kinase frataxin conditional knockout (KO) mouse. Pronounced changes in protein expression profile were observed in 9 week-old KO mice with severe cardiomyopathy. In contrast, only several proteins showed altered expression in asymptomatic 4 week-old KO mice. In hearts from frataxin KO mice, components of the iron-dependent complex-I and -II of the mitochondrial electron transport chain and enzymes involved in ATP homeostasis (creatine kinase, adenylate kinase) displayed decreased expression. Interestingly, the KO hearts exhibited increased expression of enzymes involved in the citric acid cycle, catabolism of branched-chain amino acids, ketone body utilization and pyruvate decarboxylation. This constitutes evidence of metabolic compensation due to decreased expression of electron transport proteins. There was also pronounced up-regulation of proteins involved in stress protection, such as a variety of chaperones, as well as altered expression of proteins involved in cellular structure, motility and general metabolism. This is the first report of the molecular changes at the protein level which could be involved in the cardiomyopathy of the frataxin KO mouse.

Iron: a new target for pharmacological intervention in neurodegenerative diseases.

Iron (Fe) is an essential element that is imperative for the redox-driven processes of oxygen transport, electron transport, and DNA synthesis. However, in the absence of appropriate storage or chelation, excess-free Fe readily participates in the formation of toxic-free radicals, inducing oxidative stress and apoptosis. A growing body of evidence suggests that Fe may play some role in neurodegenerative diseases such as Huntington disease, Alzheimer's disease, Parkinson's disease, and particularly Friedreich's ataxia. This review examines the role of Fe in the pathology of these conditions and the potential use of Fe chelators as therapeutic agents for the treatment of neurodegenerative disorders. Consideration is given to the features that comprise a clinically successful Fe chelator, with focus on the development of ligands such as desferrioxamine, clioquinol, pyridoxal isonicotinoyl hydrazone, and other novel aroylhydrazones.

A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics.

Novel chemotherapeutics with marked and selective antitumor activity are essential to develop, particularly those that can overcome resistance to established therapies. Iron (Fe) is critical for cell-cycle progression and DNA synthesis and potentially represents a novel molecular target for the design of new anticancer agents. The aim of this study was to evaluate the antitumor activity and Fe chelation efficacy of a new class of Fe chelators using human tumors. In this investigation, the ligands showed broad antitumor activity and could overcome resistance to established antitumor agents. The in vivo efficacy of the most effective chelator identified, di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT), was assessed by using a panel of human xenografts in nude mice. After 7 weeks, net growth of a melanoma xenograft in Dp44mT-treated mice was only 8% of that in mice treated with vehicle. In addition, no differences in these latter animals were found in hematological indices between Dp44mT-treated mice and controls. No marked systemic Fe depletion was observed comparing Dp44mT- and vehicle-treated mice, probably because of the very low doses required to induce anticancer activity. Dp44mT caused up-regulation of the Fe-responsive tumor growth and metastasis suppressor Ndrg1 in the tumor but not in the liver, indicating a potential mechanism of selective anticancer activity. These results indicate that the novel Fe chelators have potent and broad antitumor activity and can overcome resistance to established chemotherapeutics because of their unique mechanism of action.