Molecular and functional alterations in a mouse cardiac model of Friedreich ataxia: activation of the integrated stress response, eIF2α phosphorylation, and the induction of downstream targets.

Friedreich ataxia (FA) is a neurodegenerative and cardiodegenerative disease resulting from marked frataxin deficiency. The condition is characterized by ataxia with fatal cardiomyopathy, but the pathogenic mechanisms are unclear. We investigated the association between gene expression and progressive histopathological and functional changes using the muscle creatine kinase conditional frataxin knockout (KO) mouse; this mouse develops a severe cardiac phenotype that resembles that of FA patients. We examined KO mice from 3 weeks of age, when they are asymptomatic, to 10 weeks of age, when they die of the disease. Positive iron staining was identified in KO mice from 5 weeks of age, with markedly reduced cardiac function from 6 weeks. We identified an early and marked up-regulation of a gene cohort responsible for stress-induced amino acid biosynthesis and observed markedly increased phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α), an activator of the integrated stress response, in KO mice at 3 weeks of age, relative to wild-type mice. Importantly, the eIF2α-mediated integrated stress response has been previously implicated in heart failure via downstream processes such as autophagy and apoptosis. Indeed, expression of a panel of autophagy and apoptosis markers was enhanced in KO mice. Thus, the pathogenesis of cardiomyopathy in FA correlates with the early and persistent eIF2α phosphorylation, which precedes activation of autophagy and apoptosis.

Biochemistry of cardiomyopathy in the mitochondrial disease Friedreich's ataxia.

FRDA (Friedreich's ataxia) is a debilitating mitochondrial disorder leading to neural and cardiac degeneration, which is caused by a mutation in the frataxin gene that leads to decreased frataxin expression. The most common cause of death in FRDA patients is heart failure, although it is not known how the deficiency in frataxin potentiates the observed cardiomyopathy. The major proposed biochemical mechanisms for disease pathogenesis and the origins of heart failure in FRDA involve metabolic perturbations caused by decreased frataxin expression. Additionally, recent data suggest that low frataxin expression in heart muscle of conditional frataxin knockout mice activates an integrated stress response that contributes to and/or exacerbates cardiac hypertrophy and the loss of cardiomyocytes. The elucidation of these potential mechanisms will lead to a more comprehensive understanding of the pathogenesis of FRDA, and will contribute to the development of better treatments and therapeutics.

Hepcidin bound to α2-macroglobulin reduces ferroportin-1 expression and enhances its activity at reducing serum iron levels.

Hepcidin regulates iron metabolism by down-regulating ferroportin-1 (Fpn1). We demonstrated that hepcidin is complexed to the blood transport protein, α2-macroglobulin (α2M) (Peslova, G., Petrak, J., Kuzelova, K., Hrdy, I., Halada, P., Kuchel, P. W., Soe-Lin, S., Ponka, P., Sutak, R., Becker, E., Huang, M. L., Suryo Rahmanto, Y., Richardson, D. R., and Vyoral, D. (2009) Blood 113, 6225-6236). However, nothing is known about the mechanism of hepcidin binding to α2M or the effects of the α2M·hepcidin complex in vivo. We show that decreased Fpn1 expression can be mediated by hepcidin bound to native α2M and also, for the first time, hepcidin bound to methylamine-activated α2M (α2M-MA). Passage of high molecular weight α2M·hepcidin or α2M-MA·hepcidin complexes (≈725 kDa) through a Sephadex G-25 size exclusion column retained their ability to decrease Fpn1 expression. Further studies using ultrafiltration indicated that hepcidin binding to α2M and α2M-MA was labile, resulting in some release from the protein, and this may explain its urinary excretion. To determine whether α2M-MA·hepcidin is delivered to cells via the α2M receptor (Lrp1), we assessed α2M uptake and Fpn1 expression in Lrp1(-/-) and Lrp1(+/+) cells. Interestingly, α2M·hepcidin or α2M-MA·hepcidin demonstrated similar activities at decreasing Fpn1 expression in Lrp1(-/-) and Lrp1(+/+) cells, indicating that Lrp1 is not essential for Fpn1 regulation. In vivo, hepcidin bound to α2M or α2M-MA did not affect plasma clearance of α2M/α2M-MA. However, serum iron levels were reduced to a significantly greater extent in mice treated with α2M·hepcidin or α2M-MA·hepcidin relative to unbound hepcidin. This effect could be mediated by the ability of α2M or α2M-MA to retard kidney filtration of bound hepcidin, increasing its half-life. A model is proposed that suggests that unlike proteases, which are irreversibly bound to activated α2M, hepcidin remains labile and available to down-regulate Fpn1.

Mitochondrial mayhem: the mitochondrion as a modulator of iron metabolism and its role in disease.

The mitochondrion plays vital roles in various aspects of cellular metabolism, ranging from energy transduction and apoptosis to the synthesis of important molecules such as heme. Mitochondria are also centrally involved in iron metabolism, as exemplified by disruptions in mitochondrial proteins that lead to perturbations in whole-cell iron processing. Recent investigations have identified a host of mitochondrial proteins (e.g., mitochondrial ferritin; mitoferrins 1 and 2; ABCBs 6, 7, and 10; and frataxin) that may play roles in the homeostasis of mitochondrial iron. These mitochondrial proteins appear to participate in one or more processes of iron storage, iron uptake, and heme and iron-sulfur cluster synthesis. In this review, we present and critically discuss the evidence suggesting that the mitochondrion may contribute to the regulation of whole-cell iron metabolism. Further, human diseases that arise from a dysregulation of these mitochondrial molecules reveal the ability of the mitochondrion to communicate with cytosolic iron metabolism to coordinate whole-cell iron processing and to fulfill the high demands of this organelle for iron. This review highlights new advances in understanding iron metabolism in terms of novel molecular players and diseases associated with its dysregulation.

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.

Hepcidin, the hormone of iron metabolism, is bound specifically to alpha-2-macroglobulin in blood.

Hepcidin is a major regulator of iron metabolism. Hepcidin-based therapeutics/diagnostics could play roles in hematology in the future, and thus, hepcidin transport is crucial to understand. In this study, we identify alpha2-macroglobulin (alpha2-M) as the specific hepcidin-binding molecule in blood. Interaction of 125I-hepcidin with alpha2-M was identified using fractionation of plasma proteins followed by native gradient polyacrylamide gel electrophoresis and mass spectrometry. Hepcidin binding to nonactivated alpha2-M displays high affinity (Kd 177 +/- 27 nM), whereas hepcidin binding to albumin was nonspecific and displayed nonsaturable kinetics. Surprisingly, the interaction of hepcidin with activated alpha2-M exhibited a classical sigmoidal binding curve demonstrating cooperative binding of 4 high-affinity (Kd 0.3 microM) hepcidin-binding sites. This property probably enables efficient sequestration of hepcidin and its subsequent release or inactivation that may be important for its effector functions. Because alpha2-M rapidly targets ligands to cells via receptor-mediated endocytosis, the binding of hepcidin to alpha2-M may influence its functions. In fact, the alpha2-M-hepcidin complex decreased ferroportin expression in J774 cells more effectively than hepcidin alone. The demonstration that alpha2-M is the hepcidin transporter could lead to better understanding of hepcidin physiology, methods for its sensitive measurement and the development of novel drugs for the treatment of iron-related diseases.