Joanne E. Levy

Transferrin receptor is necessary for development of erythrocytes and the nervous system

Plasma iron circulates bound to transferrin (Trf), which solubilizes the ferric ion and attenuates its reactivity. Diferric Trf interacts with cell-surface Trf receptor (Trfr) to undergo receptor-mediated endocytosis into specialized endosomes. Endosomal acidification leads to iron release, and iron is transported out of the endosome through the activity of divalent metal transporter 1 (DMT1, formerly Nramp2), a transmembrane iron transporter that functions only at low pH (ref. 1). Trf and Trfr then return to the cell surface for reuse, completing a highly efficient cycle. Although the Trf cycle is assumed to be the general mechanism for cellular iron uptake, this has not been validated experimentally. Mice with hypotransferrinaemia (hpx) have little or no plasma Trf (Refs 2,3). They have severe anaemia, indicating that the Trf cycle is essential for iron uptake by erythroid cells. Other hpx tissues, however, are generally normal, and there is a paradoxical increase in intestinal iron absorption and iron storage. To test the hypothesis that the Trf cycle has unique importance for erythropoiesis, we disrupted the Trfr gene in mice. This results in elimination of the Trf cycle, but leaves other Trf functions intact. Mice lacking Trfr have a more severe phenotype than hpx mice, affecting both erythropoiesis and neurologic development. Furthermore, haploinsufficiency for Trfr results in impaired erythroid development and abnormal iron homeostasis.

Genes that modify the hemochromatosis phenotype in mice

Hereditary hemochromatosis (HH) is a prevalent human disease caused by a mutation in HFE, which encodes an atypical HLA class I protein involved in regulation of intestinal iron absorption. To gain insight into the pathogenesis of hemochromatosis, we have bred Hfe knockout mice to strains carrying other mutations that impair normal iron metabolism. Compound mutant mice lacking both Hfe and its interacting protein, beta-2 microglobulin (B2m), deposit more tissue iron than mice lacking Hfe only, suggesting that another B2m-interacting protein may be involved in iron regulation. Hfe knockout mice carrying mutations in the iron transporter DMT1 fail to load iron, indicating that hemochromatosis involves iron flux through DMT1. Similarly, compound mutants deficient in both Hfe and hephaestin (Heph) show less iron loading than do Hfe knockout mice, indicating that iron absorption in hemochromatosis involves the function of Heph as well. Finally, compound mutants lacking Hfe and the transferrin receptor accumulate more tissue iron than do mice lacking Hfe alone, consistent with the idea that interaction between these two proteins contributes to the control of normal iron absorption. In addition to providing insight into the pathogenesis of HH, our results suggest that each of these genes might be a candidate modifier of the human hemochromatosis phenotype.

Molecular insights into mechanisms of iron transport.

The past 3 years have witnessed extraordinary progress in our understanding of mammalian iron transport and homeostasis. The first transmembrane iron transporter has been found. Mutations in this protein, in two animal models with iron-transport defects, have helped to define the roles of this protein in vivo. The gene defective in patients with hereditary hemochromatosis has been identified, and much has been learned about the structure and function of its gene product. Finally, our ability to make a molecular diagnosis of hereditary hemochromatosis has called attention to new iron-loading disorders, including African iron overload and juvenile hemochromatosis.

Iron metabolism in mice with partial frataxin deficiency

Friedreich ataxia (FRDA), the most common autosomal recessive inherited ataxic disorder, is the consequence of deficiency of the mitochondrial protein frataxin, typically caused by homozygous intronic GAA expansions in the corresponding gene. The yeast frataxin homologue (yfh1p) is required for cellular respiration. Yfh1p appears to regulate mitochondrial iron homeostasis and protect from free radical toxicity. Complete loss of frataxin in knockout mice leads to early embryonic lethality, indicating an important role for frataxin during development. Heterozygous littermates with partial frataxin deficiency are apparently healthy and have no obvious phenotype. Here we evaluate iron metabolism and sensitivity to dietary and parenteral iron loading in heterozygote frataxin knockout mice (Fx+-). Iron concentrations in the liver, heart, pancreas and spleen, and cellular iron distribution patterns were compared between wild type and Fx+- mice. Response to parenteral iron challenge was not different between Fx+- mice and wild type littermates, while sporadic iron deposits were observed in the hearts of dietary iron-loaded Fx+/- mice. Finally, we evaluated the effect of partial frataxin deficiency on susceptibility to cardiac damage in the mouse model of hereditary hemochromatosis (HH), the Hfe knockout mice. HH, an iron overload disease, is one of the most frequent genetic diseases in populations of European origin. By breeding Hfe-- with Fx+- mice, we obtained compound mutant mice lacking both Hfe and one frataxin allele. Sparse iron deposits in areas of mild to moderate cardiac fibrosis were found in the majority of these mice. However, they did not develop any neurological symptoms. Our studies indicate an association between frataxin deficiency, iron deposits and cardiac fibrosis, but no obvious association between iron accumulation and neurodegeneration similar to FRDA could be detected in our model. In addition, these results suggest that frataxin mutations may have a modifier role in HH, that predisposes to cardiomyopathy.