Murine models have made valuable contributions to our understanding of iron metabolism. Investigation of mice with inherited forms of anemia has led to the discovery of novel proteins involved in iron homeostasis. A growing number of murine models are being developed to investigate mitochondrial iron metabolism. Mouse strains are available for the major forms of hereditary hemochromatosis. Findings in murine models support the concept that the pathogenesis of nearly all forms of hereditary hemochromatosis involves inappropriately low expression of hepcidin. The availability of mice with floxed iron-related genes allows the study of the in vivo consequences of cell-selective deletion of these genes.

BACKGROUND & AIMS:The liver, being the major site of iron storage, is particularly exposed to the toxic effects of iron. Transcription factor NRF2 is critical for protecting the liver against disease by activating the transcription of genes encoding detoxification/antioxidant enzymes. We aimed to determine if the NRF2 pathway plays a significant role in the protection against hepatic iron overload. METHODS:Wild-type and Nrf2(-/-) mouse primary hepatocytes were incubated with ferric ammonium citrate. Wild-type and Nrf2(-/-) mice were fed standard rodent chow or iron-rich diet for 2weeks, with or without daily injection of the antioxidant mito-TEMPOL. RESULTS:In mouse hepatocytes, iron induced the nuclear translocation of NRF2 and the expression of cytoprotective genes in an NRF2-dependent manner. Moreover, Nrf2(-/-) hepatocytes were highly susceptible to iron-induced cell death. Wild-type and Nrf2(-/-) mice fed iron-rich diet accumulated similar amounts of iron in the liver and were equally able to increase the expression of hepatic hepcidin and ferritin. Nevertheless, in Nrf2-null mice the iron loading resulted in progressive liver injury, ranging from mild confluent necrosis to severe necroinflammatory lesions. Hepatocytic cell death was associated with gross ultrastructural damage to the mitochondria. Notably, liver injury was prevented in iron-fed animals that received mito-TEMPOL. CONCLUSIONS:NRF2 protects the mouse liver against the toxicity of dietary iron overload by preventing hepatocytic cell death. We identify NRF2 as a potential modifier of liver disease in iron overload pathology and show the beneficial effect of the antioxidant mito-TEMPOL in a mouse model of dietary iron-induced liver injury.

Iron is an abundant transition metal that is essential for life, being associated with many enzyme and oxygen carrier proteins involved in a variety of fundamental cellular processes. At the same time, the metal is potentially toxic due to its capacity to engage in the catalytic production of noxious reactive oxygen species. The control of iron availability in the cells is largely dependent on ferritins, ubiquitous proteins with storage and detoxification capacity. In mammals, cytosolic ferritins are composed of two types of subunits, the H and the L chain, assembled to form a 24-mer spherical cage. Ferritin is present also in mitochondria, in the form of a complex with 24 identical chains. Even though the proteins have been known for a long time, their study is a very active and interesting field yet. In this review, we will focus our attention to mammalian cytosolic and mitochondrial ferritins, describing the most recent advancement regarding their storage and antioxidant function, the effects of their genetic mutations in human pathology, and also the possible involvement in non-iron-related activities. We will also discuss recent evidence connecting ferritins and the toxicity of iron in a set of neurodegenerative disorder characterized by focal cerebral siderosis.

Pathological overload of iron in the mitochondrial matrix has been observed in numerous diseases, including sideroblastic anemias, which have many causes, and in genetic diseases that affect iron-sulfur cluster biogenesis, heme synthesis, and mitochondrial protein translation and its products. Although high expression of the mitochondrial iron importer, mitoferrin, appears to be an underlying common feature, it is unclear what drives high mitoferrin expression and what other proteins are involved in trapping excess toxic iron in the mitochondrial matrix. Numerous examples of human diseases and model systems suggest that mitochondrial iron homeostasis is coordinated through transcriptional remodeling. A cytosolic/nuclear molecule may affect a transcriptional factor to coordinate the events that lead to iron accumulation, but no candidates for this role have yet been identified.

AIM:Iron dyshomeostasis is one of the primary causes of neuronal death in Alzheimer's disease (AD). Huperzine A (HupA), a natural inhibitor of acetylcholinesterase (AChE), is a licensed anti-AD drug in China and a nutraceutical in the United Sates. Here, we investigated the protective effects of HupA against iron overload-induced injury in neurons. METHODS:Rat cortical neurons were treated with ferric ammonium citrate (FAC), and cell viability was assessed with MTT assays. Reactive oxygen species (ROS) assays and adenosine triphosphate (ATP) assays were performed to assess mitochondrial function. The labile iron pool (LIP) level, cytosolic-aconitase (c-aconitase) activity and iron uptake protein expression were measured to determine iron metabolism changes. The modified Ellman's method was used to evaluate AChE activity. RESULTS:HupA significantly attenuated the iron overload-induced decrease in neuronal cell viability. This neuroprotective effect of HupA occurred concurrently with a decrease in ROS and an increase in ATP. Moreover, HupA treatment significantly blocked the upregulation of the LIP level and other aberrant iron metabolism changes induced by iron overload. Additionally, another specific AChE inhibitor, donepezil (Don), at a concentration that caused AChE inhibition equivalent to that of HupA negatively, influenced the aberrant changes in ROS, ATP or LIP that were induced by excessive iron. CONCLUSION:We provide the first demonstration of the protective effects of HupA against iron overload-induced neuronal damage. This beneficial role of HupA may be attributed to its attenuation of oxidative stress and mitochondrial dysfunction and elevation of LIP, and these effects are not associated with its AChE-inhibiting effect.

Bone marrow mesenchymal stem cells (BMSCs) are an expandable population of stem cells which can differentiate into osteoblasts, chondrocytes and adipocytes. Dysfunction of BMSCs in response to pathological stimuli contributes to bone diseases. Melatonin, a hormone secreted from pineal gland, has been proved to be an important mediator in bone formation and mineralization. The aim of this study was to investigate whether melatonin protected against iron overload-induced dysfunction of BMSCs and its underlying mechanisms. Here, we found that iron overload induced by ferric ammonium citrate (FAC) caused irregularly morphological changes and markedly reduced the viability in BMSCs. Consistently, osteogenic differentiation of BMSCs was significantly inhibited by iron overload, but melatonin treatment rescued osteogenic differentiation of BMSCs. Furthermore, exposure to FAC led to the senescence in BMSCs, which was attenuated by melatonin as well. Meanwhile, melatonin was able to counter the reduction in cell proliferation by iron overload in BMSCs. In addition, protective effects of melatonin on iron overload-induced dysfunction of BMSCs were abolished by its inhibitor luzindole. Also, melatonin protected BMSCs against iron overload-induced ROS accumulation and membrane potential depolarization. Further study uncovered that melatonin inhibited the upregulation of p53, ERK and p38 protein expressions in BMSCs with iron overload. Collectively, melatonin plays a protective role in iron overload-induced osteogenic differentiation dysfunction and senescence through blocking ROS accumulation and p53/ERK/p38 activation.

Beta-propeller protein-associated neurodegeneration is a subtype of monogenic neurodegeneration with brain iron accumulation caused by de novo mutations in WDR45. The WDR45 protein functions as a beta-propeller scaffold and plays a putative role in autophagy through its interaction with phospholipids and autophagy-related proteins. Loss of WDR45 function due to disease-causing mutations has been linked to defects in autophagic flux in patient and animal cells. However, the role of WDR45 in iron homeostasis remains elusive. Here we studied patient-specific WDR45 mutant fibroblasts and induced pluripotent stem cell-derived midbrain neurons. Our data demonstrated that loss of WDR45 increased cellular iron levels and oxidative stress, accompanied by mitochondrial abnormalities, autophagic defects, and diminished lysosomal function. Restoring WDR45 levels partially rescued oxidative stress and the susceptibility to iron treatment, and activation of autophagy reduced the observed iron overload in WDR45 mutant cells. Our data suggest that iron-containing macromolecules and organelles cannot effectively be degraded through the lysosomal pathway due to loss of WDR45 function.

Excess iron accumulation and cardiac oxidative stress have been shown as important mediators of cardiac hypertrophy, whereas it remains largely elusive about the occurrence of mitochondrial iron overload and its significance during cardiac hypertrophy. In the present study, we aim to investigate the role of NCOA4-mediated ferritinophagy and SFXN1-dependent mitochondria iron overload in apelin-13-induced cardiomyocytes hypertrophy. Apelin-13 significantly promotes ferric citrate (FAC)-induced total cellular and mitochondria ion production, as well as mitochondria ROS contents. Mechanistically, apelin-13 effectively induces the expression of SFXN1, a mitochondria iron transporting protein and NCOA4, a cargo receptor of ferritinophagy in dose and time-dependent manner. Conversely, blockade of APJ by F13A abolishes these stimulatory effects. In addition, apelin-13-triggered mitochondria iron overload is reversed by the genetic inhibition of SFXN1 and NCOA4. NCOA4 deficiency via its silencing also interferes with the enhanced expression of SFXN1 evoked by apelin-13. In apelin-13-treated H9c2 cells, the promotion in cell diameter, volume as well as protein contents are obviously suppressed by the knockdown of NCOA4 and SFXN1 with their corresponding siRNAs. Remarkably, the human and murine hypertrophic hearts models, as well as apelin-13-injected mice models, present evident cardiac mitochondrial iron deposition and raised expressions of NCOA4 and SFXN1. Taken together, these results provide experimental evidences that NCOA4-mediated ferritinophagy might be defined as an essential mechanism leading to apelin-13-cardiomyocytes hypertrophy in SFXN1-dependent mitochondria iron overload manners.

Loss-of-function mutations in the ATP-binding cassette (ABC) transporter of the inner mitochondrial membrane, ABCB7, cause X-linked sideroblastic anemia with ataxia, a phenotype that remains largely unexplained by the proposed role of ABCB7 in exporting a special sulfur species for use in cytosolic iron-sulfur (Fe-S) cluster biogenesis. Here, we generated inducible ABCB7-knockdown cell lines to examine the time-dependent consequences of loss of ABCB7. We found that knockdown of ABCB7 led to significant loss of mitochondrial Fe-S proteins, which preceded the development of milder defects in cytosolic Fe-S enzymes. In erythroid cells, loss of ABCB7 altered cellular iron distribution and caused mitochondrial iron overload due to activation of iron regulatory proteins 1 and 2 in the cytosol and to upregulation of the mitochondrial iron importer, mitoferrin-1. Despite the exceptionally large amount of iron imported into mitochondria, erythroid cells lacking ABCB7 showed a profound hemoglobinization defect and underwent apoptosis triggered by oxidative stress. In ABCB7-depleted cells, defective heme biosynthesis resulted from translational repression of ALAS2 by iron regulatory proteins and from decreased stability of the terminal enzyme ferrochelatase. By combining chemical crosslinking, tandem mass spectrometry and mutational analyses, we characterized a complex formed of ferrochelatase, ABCB7 and ABCB10, and mapped the interfaces of interactions of its components. A dimeric ferrochelatase physically bridged ABCB7 and ABCB10 homodimers by binding near the nucleotide-binding domains of each ABC transporter. Our studies not only underscore the importance of ABCB7 for mitochondrial Fe-S biogenesis and iron homeostasis, but also provide the biochemical characterization of a multiprotein complex required for heme biosynthesis.