Ferrodifferentiation regulates neurodevelopment via ROS generation.

Iron is important for life, and iron deficiency impairs development, but whether the iron level regulates neural differentiation remains elusive. In this study, with iron-regulatory proteins (IRPs) knockout embryonic stem cells (ESCs) that showed severe iron deficiency, we found that the Pax6- and Sox2-positive neuronal precursor cells and Tuj1 fibers in IRP1-/-IRP2-/- ESCs were significantly decreased after inducing neural differentiation. Consistently, in vivo study showed that the knockdown of IRP1 in IRP2-/- fetal mice remarkably affected the differentiation of neuronal precursors and the migration of neurons. These findings suggest that low intracellular iron status significantly inhibits neurodifferentiation. When supplementing IRP1-/-IRP2-/- ESCs with iron, these ESCs could differentiate normally. Further investigations revealed that the underlying mechanism was associated with an increase in reactive oxygen species (ROS) production caused by the substantially low level of iron and the down-regulation of iron-sulfur cluster protein ISCU, which, in turn, affected the proliferation and differentiation of stem cells. Thus, the appropriate amount of iron is crucial for maintaining normal neural differentiation that is termed ferrodifferentiation.

A novel treatment strategy to prevent Parkinson's disease: focus on iron regulatory protein 1 (IRP1).

We propose that neural damage in Parkinson's disease (PD) is due to dysregulation of iron utilization rather than to high iron levels per se. Iron deposits are associated with neuronal cell death in substantia nigra (SN) resulting in PD where high levels of iron in SNs are due to dysregulation of iron utilization. Cytosolic aconitase (ACO1) upon losing an iron-sulfur cluster becomes iron regulatory protein 1 (IRP1). Rotenone increases levels of IRP1 and induces PD in rats. An increase in iron leads to inactivation of IRP1. We propose a novel treatment strategy to prevent PD. Specifically in rats given rotenone by subcutaneous injections, iron, from iron carbonyl from which iron is slowly absorbed, given three times a day by gavage will keep iron levels constant in the gut whereby iron levels and iron utilization systematically can be tightly regulated. Rotenone adversely affects complex 1 iron-sulfur proteins. Iron supplementation will increase iron-sulfur cluster formation switching IRP1 to ACO1. With IRP1 levels kept constantly low, iron utilization will systematically be tightly regulated stopping dysregulation of complex 1 and the neural damage done by rotenone preventing PD.

Translational repression of HIF2α expression in mice with Chuvash polycythemia reverses polycythemia.

Chuvash polycythemia is an inherited disease caused by a homozygous germline VHLR200W mutation, which leads to impaired degradation of HIF2α, elevated levels of serum erythropoietin, and erythrocytosis/polycythemia. This phenotype is recapitulated by a mouse model bearing a homozygous VhlR200W mutation. We previously showed that iron-regulatory protein 1-knockout (Irp1-knockout) mice developed erythrocytosis/polycythemia through translational derepression of Hif2α, suggesting that IRP1 could be a therapeutic target to treat Chuvash polycythemia. Here, we fed VhlR200W mice supplemented with Tempol, a small, stable nitroxide molecule and observed that Tempol decreased erythropoietin production, corrected splenomegaly, normalized hematocrit levels, and increased the lifespans of these mice. We attribute the reversal of erythrocytosis/polycythemia to translational repression of Hif2α expression by Tempol-mediated increases in the IRE-binding activity of Irp1, as reversal of polycythemia was abrogated in VhlR200W mice in which Irp1 was genetically ablated. Thus, a new approach to the treatment of patients with Chuvash polycythemia may include dietary supplementation of Tempol, which decreased Hif2α expression and markedly reduced life-threatening erythrocytosis/polycythemia in the VhlR200W mice.

A synergistic role of IRP1 and FBXL5 proteins in coordinating iron metabolism during cell proliferation.

Iron-regulatory protein 1 (IRP1) belongs to a family of RNA-binding proteins that modulate metazoan iron metabolism. Multiple mechanisms are employed to control the action of IRP1 in dictating changes in the uptake and metabolic fate of iron. Inactivation of IRP1 RNA binding by iron primarily involves insertion of a [4Fe-4S] cluster by the cytosolic iron-sulfur cluster assembly (CIA) system, converting it into cytosolic aconitase (c-acon), but can also involve iron-mediated degradation of IRP1 by the E3 ligase FBXL5 that also targets IRP2. How CIA and FBXL5 collaborate to maintain cellular iron homeostasis through IRP1 and other pathways is poorly understood. Because impaired Fe-S cluster biogenesis associates with human disease, we determined the importance of FBXL5 for regulating IRP1 when CIA is impaired. Suppression of FBXL5 expression coupled with induction of an IRP1 mutant (IRP13C>3S) that cannot insert the Fe-S cluster, or along with knockdown of the CIA factors NUBP2 or FAM96A, reduced cell viability. Iron supplementation reversed this growth defect and was associated with FBXL5-dependent polyubiquitination of IRP1. Phosphorylation of IRP1 at Ser-138 increased when CIA was inhibited and was required for iron rescue. Impaired CIA activity, as noted by reduced c-acon activity, was associated with enhanced FBXL5 expression and a concomitant reduction in IRP1 and IRP2 protein level and RNA-binding activity. Conversely, expression of either IRP induced FBXL5 protein level, demonstrating a negative feedback loop limiting excessive accumulation of iron-response element RNA-binding activity, whose disruption reduces cell growth. We conclude that a regulatory circuit involving FBXL5 and CIA acts through both IRPs to control iron metabolism and promote optimal cell growth.

Iron-induced epigenetic abnormalities of mouse bone marrow through aberrant activation of aconitase and isocitrate dehydrogenase.

Iron overload remains a concern in myelodysplastic syndrome (MDS) patients. Iron chelation therapy (ICT) thus plays an integral role in the management of these patients. Moreover, ICT has been shown to prolong leukemia-free survival in MDS patients; however, the mechanisms responsible for this effect are unclear. Iron is a key molecule for regulating cytosolic aconitase 1 (ACO1). Additionally, the mutation of isocitrate dehydrogenase (IDH), the enzyme downstream of ACO1 in the TCA cycle, is associated with epigenetic abnormalities secondary to 2-hydroxyglutarate (2-HG) and DNA methylation. However, epigenetic abnormalities observed in many MDS patients occur without IDH mutation. We hypothesized that iron itself activates the ACO1-IDH pathway, which may increase 2-HG and DNA methylation, and eventually contribute to leukemogenesis without IDH mutation. Using whole RNA sequencing of bone marrow cells in iron-overloaded mice, we observed that the enzymes, phosphoglucomutase 1, glycogen debranching enzyme, and isocitrate dehydrogenase 1 (Idh1), which are involved in glycogen and glucose metabolism, were increased. Digital PCR further showed that Idh1 and Aco1, enzymes involved in the TCA cycle, were also elevated. Additionally, enzymatic activities of TCA cycle and methylated DNA were increased. Iron chelation reversed these phenomena. In conclusion, iron activation of glucose metabolism causes an increase of 2-HG and DNA methylation.

Iron-dependent turnover of IRP-1/c-aconitase in kidney cells.

The kidney plays an important role in iron homeostasis and actively reabsorbs citrate. The bifunctional iron-regulatory protein IRP-1 potentially regulates iron trafficking and participates in citrate metabolism as a cytosolic (c-) aconitase. We investigated the role of cellular iron status in determining the expression and dynamics of IRP-1 in two renal cell types, with the aim of identifying a role of the protein in cellular ROS levels, citrate metabolism and glutamate production. The effects of iron supplementation and chelation on IRP-1 protein and mRNA levels and protein turnover were compared in cultured primary rat mesangial cells and a porcine renal tubule cell line (LLC-PK1). Levels of ROS were measured in both cell types, and c-aconitase activity, glutamate, and glutathione were measured in LLC-PK1 cells, with and without IRP-1 silencing and in glutamine-supplemented or nominally glutamine-free medium. Iron supplementation decreased IRP-1 levels (e.g., approx. 40% in mesangial cells treated with 10 µg ml(-1) iron for 16 h) and increased ubiquitinated IRP-1 levels in both cells types, with iron chelation having the opposite effect. Although iron increased ROS levels (three-fold with 20 µg ml(-1) iron in mesangial cells and more modestly by about 30% with 50 µg ml(-1) in LLC-PK1 cells, both after 24 h), protein degradation was not ROS-dependent. In LLC-PK1 cells, 10 µg ml(-1) iron (24 h) increased both aconitase activity (30%) and secreted glutamate levels (65%). Silencing did not remove the glutamate response to iron but decreased the c-aconitase activity of the residual protein independent of iron loading (37% and 46% of control levels, without and with iron treatment, respectively). However, in glutamine-free medium, glutamate was still increased by iron, even in IRP-1-silenced cells, and did not correspond to c-aconitase. Silencing decreased the amount of ferritin measured in response to iron loading, decreased the affect of iron on total glutathione by 48%, and increased the response of ROS to iron loading by 38%. We conclude that iron increases turnover of IRP-1 in kidney cells, while increasing aconitase activity, suggesting that the apoprotein (aconitase-inactive) form is not exclusively responsible for turnover. Iron increases glutamate levels in tubule epithelial cells, but this appears to be independent of c-aconitase activity or the availability of extracellular glutamine. IRP-1 protein levels are not regulated by ROS, but IRP-1-dependent ferritin expression may decrease ROS and increase total glutathione levels, suggesting that ferritin levels are more important than citrate metabolism in protecting renal cells against iron.

Neuroimaging, nutrition, and iron-related genes.

Several dietary factors and their genetic modifiers play a role in neurological disease and affect the human brain. The structural and functional integrity of the living brain can be assessed using neuroimaging, enabling large-scale epidemiological studies to identify factors that help or harm the brain. Iron is one nutritional factor that comes entirely from our diet, and its storage and transport in the body are under strong genetic control. In this review, we discuss how neuroimaging can help to identify associations between brain integrity, genetic variations, and dietary factors such as iron. We also review iron's essential role in cognition, and we note some challenges and confounds involved in interpreting links between diet and brain health. Finally, we outline some recent discoveries regarding the genetics of iron and its effects on the brain, suggesting the promise of neuroimaging in revealing how dietary factors affect the brain.

Leishmania donovani depletes labile iron pool to exploit iron uptake capacity of macrophage for its intracellular growth.

Intracellular pathogens employ several strategies for iron acquisition from host macrophages for survival and growth, whereas macrophage resists infection by actively sequestering iron. Here, we show that instead of allowing macrophage to sequester iron, protozoan parasite Leishmania donovani (LD) uses a novel strategy to manipulate iron uptake mechanisms of the host and utilizes the taken up iron for its intracellular growth. To do so, intracellular LD directly scavenges iron from labile iron pool of macrophages. Depleted labile iron pool activates iron sensors iron-regulatory proteins IRP1 and IRP2. IRPs then bind to iron-responsive elements present in the 3' UTR of iron uptake gene transferrin receptor 1 by a post-transcriptional mRNA stability mechanism. Increased iron-responsive element-IRP interaction and transferrin receptor 1 expressions in spleen-derived macrophages from LD-infected mice confirm that LD employs similar mechanism to acquire iron during infection into mammalian hosts. Increased intracellular LD growth by holo-transferrin supplementation and inhibited growth by iron chelator treatment confirm the significance of this modulated iron uptake pathway of host in favour of the parasite.

Tempol-mediated activation of latent iron regulatory protein activity prevents symptoms of neurodegenerative disease in IRP2 knockout mice.

In mammals, two homologous cytosolic regulatory proteins, iron regulatory protein 1 (also known as IRP1 and Aco1) and iron regulatory protein 2 (also known as IRP2 and Ireb2), sense cytosolic iron levels and posttranscriptionally regulate iron metabolism genes, including transferrin receptor 1 (TfR1) and ferritin H and L subunits, by binding to iron-responsive elements (IREs) within target transcripts. Mice that lack IRP2 develop microcytic anemia and neurodegeneration associated with functional cellular iron depletion caused by low TfR1 and high ferritin expression. IRP1 knockout (IRP1(-/-)) animals do not significantly misregulate iron metabolism, partly because IRP1 is an iron-sulfur protein that functions mainly as a cytosolic aconitase in mammalian tissues and IRP2 activity increases to compensate for loss of the IRE binding form of IRP1. The neurodegenerative disease of IRP2(-/-) animals progresses slowly as the animals age. In this study, we fed IRP2(-/-) mice a diet supplemented with a stable nitroxide, Tempol, and showed that the progression of neuromuscular impairment was markedly attenuated. In cell lines derived from IRP2(-/-) animals, and in the cerebellum, brainstem, and forebrain of animals maintained on the Tempol diet, IRP1 was converted from a cytosolic aconitase to an IRE binding protein that stabilized the TfR1 transcript and repressed ferritin synthesis. We suggest that Tempol protected IRP2(-/-) mice by disassembling the cytosolic iron-sulfur cluster of IRP1 and activating IRE binding activity, which stabilized the TfR1 transcript, repressed ferritin synthesis, and partially restored normal cellular iron homeostasis in the brain.

Self-association and ligand-induced conformational changes of iron regulatory proteins 1 and 2.

Iron regulatory proteins (IRPs) regulate iron metabolism in mammalian cells. We used biophysical techniques to examine the solution properties of apo-IRP1 and apo-IRP2 and the interaction with their RNA ligand, the iron regulatory element (IRE). Sedimentation velocity and equilibrium experiments have shown that apo-IRP1 exists as an equilibrium mixture of monomers and dimers in solution, with an equilibrium dissociation constant in the low micromolar range and slow kinetic exchange between the two forms. However, only monomeric IRP1 is observed in complex with IRE. In contrast, IRP2 exists as monomer in both the apo-IRP2 form and in the IRP2/IRE complex. For both IRPs, sedimentation velocity and dynamic light-scattering experiments show a decrease of the Stokes radius upon binding of IRE. This conformational change was also observed by circular dichroism. Studies with an RNA molecule complementary to IRE indicate that, although specific base interactions can increase the stability of the protein/RNA complex, they are not essential for inducing this conformational change. The dynamic change of the IRP between different oligomeric and conformational states induced by interaction with IRE may play a role in the iron regulatory functions of IRPs.

An insulin-response element-binding protein that ameliorates hyperglycemia in diabetes.

Insulin modulates glucose homeostasis, but the role of insulin-responsive transcription factors in such actions is not well understood. Recently, we have identified the insulin-response element-binding protein-1 (IRE-BP1) as a transcription factor that appears to mediate insulin action on multiple target genes. To examine the possibility that IRE-BP1 is an insulin-responsive glucoregulatory factor involved in the metabolic actions of insulin, we investigated the effect of adenoviral overexpression of hepatic IRE-BP1 on the glycemic control of insulin-resistant diabetic rats. Adenoviral IRE-BP1 lowered both fasting and postprandial glucose levels, and microarray of hepatic RNA revealed modulation of the expression of genes involved in gluconeogenesis, lipogenesis, and fatty acid oxidation. The insulin mimetic effects of IRE-BP1 were also confirmed in L6 myocytes; stable constitutive expressions of IRE-BP1 enhanced glucose transporter expression, glucose uptake, and glycogen accumulation in these cells. These findings showed physiologic sufficiency of IRE-BP1 as the transcriptional mediator of the metabolic action of insulin. Understanding IRE-BP1 action should constitute a useful probe into the mechanisms of metabolic regulation and an important target to develop therapeutic agents that mimic or enhance insulin action.

Compartment-specific protection of iron-sulfur proteins by superoxide dismutase.

Iron and oxygen are essential but potentially toxic constituents of most organisms, and their transport is meticulously regulated both at the cellular and systemic levels. Compartmentalization may be a homeostatic mechanism for isolating these biological reactants in cells. To investigate this hypothesis, we have undertaken a genetic analysis of the interaction between iron and oxygen metabolism in Drosophila. We show that Drosophila iron regulatory protein-1 (IRP1) registers cytosolic iron and oxidative stress through its labile iron sulfur cluster by switching between cytosolic aconitase and RNA-binding functions. IRP1 is strongly activated by silencing and genetic mutation of the cytosolic superoxide dismutase (Sod1), but is unaffected by silencing of mitochondrial Sod2. Conversely, mitochondrial aconitase activity is relatively insensitive to loss of Sod1 function, but drops dramatically if Sod2 activity is impaired. This strongly suggests that the mitochondrial boundary limits the range of superoxide reactivity in vivo. We also find that exposure of adults to paraquat converts cytosolic aconitase to IRP1 but has no affect on mitochondrial aconitase, indicating that paraquat generates superoxide in the cytosol but not in mitochondria. Accordingly, we find that transgene-mediated overexpression of Sod2 neither enhances paraquat resistance in Sod1+ flies nor compensates for lack of SOD1 activity in Sod1-null mutants. We conclude that in vivo, superoxide is confined to the subcellular compartment in which it is formed, and that the mitochondrial and cytosolic SODs provide independent protection to compartment-specific protein iron-sulfur clusters against attack by superoxide generated under oxidative stress within those compartments.