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.

Identification of the di-pyridyl ketone isonicotinoyl hydrazone (PKIH) analogues as potent iron chelators and anti-tumour agents.

(1) In an attempt to develop chelators as potent anti-tumour agents, we synthesized two series of novel ligands based on the very active 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) group. Since lipophilicity and membrane permeability play a critical role in Fe chelation efficacy, the aldehyde moiety of the PCIH series, namely 2-pyridylcarboxaldehyde, was replaced with the more lipophilic 2-quinolinecarboxaldehyde or di-2-pyridylketone moieties. These compounds were then systematically condensed with the same group of acid hydrazides to yield ligands based on 2-quinolinecarboxaldehyde isonicotinoyl hydrazone (QCIH) and di-2-pyridylketone isonicotinoyl hydrazone (PKIH). To examine chelator efficacy, we assessed their effects on proliferation, Fe uptake, Fe efflux, the expression of cell cycle control molecules, iron-regulatory protein-RNA-binding activity, and (3)H-thymidine, (3)H-uridine and (3)H-leucine incorporation. (2) Despite the high lipophilicity of the QCIH ligands and the fact that they have the same Fe-binding site as the PCIH series, surprisingly none of these compounds were effective. In contrast, the PKIH analogues showed marked anti-proliferative activity and Fe chelation efficacy. Indeed, the ability of these ligands to inhibit proliferation and DNA synthesis was similar or exceeded that found for the highly cytotoxic chelator, 311. In contrast to the PCIH and QCIH analogues, most of the PKIH group markedly increased the mRNA levels of molecules vital for cell cycle arrest. (3) In conclusion, our studies identify structural features useful in the design of chelators with high anti-proliferative activity. We have identified a novel class of ligands that are potent Fe chelators and inhibitors of DNA synthesis, and which deserve further investigation.

Hepcidin expression inversely correlates with the expression of duodenal iron transporters and iron absorption in rats.

BACKGROUND & AIMS: Hepcidin is an antimicrobial peptide thought to be involved in the regulation of intestinal iron absorption. To further investigate its role in this process, we examined hepatic and duodenal gene expression in rats after the switch from a control diet to an iron-deficient diet. METHODS: Adult rats on an iron-replete diet were switched to an iron-deficient diet and the expression of iron homeostasis molecules in duodenal and liver tissue was studied over 14 days. Intestinal iron absorption was determined at these same time-points by measuring the retention of an oral dose of (59)Fe. RESULTS: Iron absorption increased 2.7-fold within 6 days of switching to an iron-deficient diet and was accompanied by an increase in the duodenal expression of Dcytb, divalent metal transporter 1, and Ireg1. These changes precisely correlated with decreases in hepatic hepcidin expression and transferrin saturation. No change in iron stores or hematologic parameters was detected. CONCLUSIONS: This study showed a close relationship between the expression of hepcidin, duodenal iron transporters, and iron absorption. Both hepcidin expression and iron absorption can be regulated before iron stores and erythropoiesis are affected, and transferrin saturation may signal such changes.

Erythroid differentiation and protoporphyrin IX down-regulate frataxin expression in Friend cells: characterization of frataxin expression compared to molecules involved in iron metabolism and hemoglobinization.

Friedreich ataxia (FA) is caused by decreased frataxin expression that results in mitochondrial iron (Fe) overload. However, the role of frataxin in mammalian Fe metabolism remains unclear. In this investigation we examined the function of frataxin in Fe metabolism by implementing a well-characterized model of erythroid differentiation, namely, Friend cells induced using dimethyl sulfoxide (DMSO). We have characterized the changes in frataxin expression compared to molecules that play key roles in Fe metabolism (the transferrin receptor [TfR] and the Fe transporter Nramp2) and hemoglobinization (beta-globin). DMSO induction of hemoglobinization results in a marked decrease in frataxin gene (Frda) expression and protein levels. To a lesser extent, Nramp2 messenger RNA (mRNA) levels were also decreased on erythroid differentiation, whereas TfR and beta-globin mRNA levels increased. Intracellular Fe depletion using desferrioxamine or pyridoxal isonicotinoyl hydrazone, which chelate cytoplasmic or cytoplasmic and mitochondrial Fe pools, respectively, have no effect on frataxin expression. Furthermore, cytoplasmic or mitochondrial Fe loading of induced Friend cells with ferric ammonium citrate, or the heme synthesis inhibitor, succinylacetone, respectively, also had no effect on frataxin expression. Although frataxin has been suggested by others to be a mitochondrial ferritin, the lack of effect of intracellular Fe levels on frataxin expression is not consistent with an Fe storage role. Significantly, protoporphyrin IX down-regulates frataxin protein levels, suggesting a regulatory role of frataxin in Fe or heme metabolism. Because decreased frataxin expression leads to mitochondrial Fe loading in FA, our data suggest that reduced frataxin expression during erythroid differentiation results in mitochondrial Fe sequestration for heme biosynthesis.