6-Hydroxydopamine promotes iron traffic in primary cultured astrocytes

It is well known that disrupted brain iron homeostasis was involved in Parkinson’s disease. We previously reported 6-hydroxydopamine (6-OHDA) could enhance iron influx and attenuate iron efflux process, thus promote iron accumulation in neurons. Astrocytes, the major glial cell type in the central nervous system, are largely responsible for iron distribution in the brain. However, how iron metabolism changes in astrocytes with 6-OHDA treatment are not fully elucidated. In the present study, we first observed that both iron influx and efflux were enhanced with 10 μM 6-OHDA treatment for 24 h in primary cultured astrocytes. In accordance with these iron traffic modulations, both mRNA and protein levels of iron importer divalent metal transporter 1 with iron responsive element (DMT1+IRE) and exporter ferroportin 1 (FPN1) were up-regulated in these cells. L-ferritin mRNA levels were increased. Iron regulatory protein 1 (IRP1) showed a dynamic regulation with 6-OHDA treatment, as indicated by a moderate up-regulation at 12 h, however, down-regulation at 24 h. We further demonstrated that 6-OHDA treatment could induce activation of nuclear factor-kappaB (NF-κB) p65. IκBα activation inhibitor BAY11-7082 fully blocked 6-OHDA induced NF-κB p65 phosphorylation and DMT1 + IRE up-regulation. These results suggest that 6-OHDA might promote iron transport rate in astrocytes by regulating iron transporters, IRP1 expression and NF-κB p65 activation, indicating a different response between neurons and astrocytes.     

Iron and glutathione at the crossroad of redox metabolism in neurons

Neurons, as non-dividing cells, encounter a myriad of stressful conditions throughout their lifespan. In particular, there is increasing evidence that iron progressively accumulates in the brain with age and that iron induced oxidative stress is the cause of several forms of neurodegeneration. Here, we review recent evidence that gives support to the following notions: 1) neuronal iron accumulation leads to oxidative stress and cell death; 2) neuronal survival to iron accumulation associates with decreased expression of the iron import transporter DMT1 and increased expression of the efflux transporter IREG1; and 3) the adaptive process of neurons towards iron-induced oxidative stress includes a marked increase in both the expression of the catalytic subunit of gamma glutamate-cysteine ligase and glutathione. These findings may help to understand aging-related neurodegeneration hallmarks: oxidative damage, functional impairment and cell death.     

The Ferroportin Metal Efflux Proteins Function in Iron and Cobalt Homeostasis in Arabidopsis

Relatively little is known about how metals such as iron are effluxed from cells, a necessary step for transport from the root to the shoot. Ferroportin (FPN) is the sole iron efflux transporter identified to date in animals, and there are two closely related orthologs in Arabidopsis thaliana, IRON REGULATED1 (IREG1/FPN1) and IREG2/FPN2. FPN1 localizes to the plasma membrane and is expressed in the stele, suggesting a role in vascular loading; FPN2 localizes to the vacuole and is expressed in the two outermost layers of the root in response to iron deficiency, suggesting a role in buffering metal influx. Consistent with these roles, fpn2 has a diminished iron deficiency response, whereas fpn1 fpn2 has an elevated iron deficiency response. Ferroportins also play a role in cobalt homeostasis; a survey of Arabidopsis accessions for ionomic phenotypes showed that truncation of FPN2 results in elevated shoot cobalt levels and leads to increased sensitivity to the metal. Conversely, loss of FPN1 abolishes shoot cobalt accumulation, even in the cobalt accumulating mutant frd3. Consequently, in the fpn1 fpn2 double mutant, cobalt cannot move to the shoot via FPN1 and is not sequestered in the root vacuoles via FPN2; instead, cobalt likely accumulates in the root cytoplasm causing fpn1 fpn2 to be even more sensitive to cobalt than fpn2 mutants.     

Iron uptake in quiescent and inflammation-activated astrocytes: A potentially neuroprotective control of iron burden

Astrocytes play a crucial role in proper iron handling within the central nervous system. This competence can be fundamental, particularly during neuroinflammation, and neurodegenerative processes, where an increase in iron content can favor oxidative stress, thereby worsening disease progression. Under these pathological conditions, astrocytes undergo a process of activation that confers them either a beneficial or a detrimental role on neuronal survival. Our work investigates the mechanisms of iron entry in cultures of quiescent and activated hippocampal astrocytes. Our data confirm that the main source of iron is the non-transferrin-bound iron (NTBI) and show the involvement of two different routes for its entry: the resident transient receptor potential (TRP) channels in quiescent astrocytes and the de novo expressed divalent metal transporter 1 (DMT1) in activated astrocytes, which accounts for a potentiation of iron entry. Overall, our data suggest that at rest, but even more after activation, astrocytes have the potential to buffer the excess of iron, thereby protecting neurons from iron overload. These findings further extend our understanding of the protective role of astrocytes under the conditions of iron-mediated oxidative stress observed in several neurodegenerative conditions.     

Alternative RNA splicing generates a glycosylphosphatidylinositol-anchored form of ceruloplasmin in mammalian brain

Ceruloplasmin is a copper-containing ferroxidase that is essential for normal iron homeostasis. Whereas ceruloplasmin in plasma is produced and secreted by hepatocytes, in the brain a glycosylphosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed on the surface of astrocytes. By using a cDNA cloning approach, we have now determined that the GPI-anchored form of ceruloplasmin is generated by alternative RNA splicing. The splicing occurs downstream of exon 18 and replaces the C-terminal 5 amino acids of the secreted form with an alternative 30 amino acids that signal GPI anchor addition. RNase protection analysis demonstrates that the GPI-anchored form is the major form in the brain, whereas the secreted form predominates in the liver. Individuals with aceruloplasminemia, a hereditary deficiency of ceruloplasmin, have severe iron deposition in a number of organs, including the brain where it results in neurodegeneration. Therefore, this novel GPI-anchored form of ceruloplasmin is likely to play an important role in iron metabolism in the central nervous system.     

A novel mammalian iron-regulated protein involved in intracellular iron metabolism

We have isolated and characterized a novel iron-regulated gene that is homologous to the divalent metal transporter 1 family of metal transporters. This gene, termed metal transporter protein (mtp1), is expressed in tissues involved in body iron homeostasis including the developing and mature reticuloendothelial system, the duodenum, and the pregnant uterus. MTP1 is also expressed in muscle and central nervous system cells in the embryo. At the subcellular level, MTP1 is localized to the basolateral membrane of the duodenal epithelial cell and a cytoplasmic compartment of reticuloendothelial system cells. Overexpression of MTP1 in tissue culture cells results in intracellular iron depletion. In the adult mouse, MTP1 expression in the liver and duodenum are reciprocally regulated. Iron deficiency induces MTP1 expression in the duodenum but down-regulates expression in the liver. These data indicate that MTP1 is an iron-regulated membrane-spanning protein that is involved in intracellular iron metabolism.     

Inflammation alters the expression of DMT1, FPN1 and hepcidin,and it causes iron accumulation in central nervous system cells

Inflammation and iron accumulation are present in a variety of neurodegenerative diseases that include Alzheimer's disease and Parkinson's disease. The study of the putative association between inflammation and iron accumulation in central nervous system cells is relevant to understand the contribution of these processes to the progression of neuronal death. In this study, we analyzed the effects of the inflammatory cytokines tumor necrosis factor alpha (TNF‐α) and interleukin 6 (IL‐6) and of lipopolysaccharide on total cell iron content and on the expression and abundance of the iron transporters divalent metal transporter 1 (DMT1) and Ferroportin 1 (FPN1) in neurons, astrocytes and microglia obtained from rat brain. Considering previous reports indicating that inflammatory stimuli induce the systemic synthesis of the master iron regulator hepcidin, we identified brain cells that produce hepcidin in response to inflammatory stimuli, as well as hepcidin‐target cells. We found that inflammatory stimuli increased the expression of DMT1 in neurons, astrocytes, and microglia. Inflammatory stimuli also induced the expression of hepcidin in astrocytes and microglia, but not in neurons. Incubation with hepcidin decreased the expression of FPN1 in the three cell types. The net result of these changes was increased iron accumulation in neurons and microglia but not in astrocytes. The data presented here establish for the first time a causal association between inflammation and iron accumulation in brain cells, probably promoted by changes in DMT1 and FPN1 expression and mediated in part by hepcidin. This connection may potentially contribute to the progression of neurodegenerative diseases by enhancing iron‐induced oxidative damage.     

Zinc regulates the function and expression of the iron transporters DMT1 and IREG1 in human intestinal Caco-2 cells.

Trace metals influence the absorption of each other from the diet and it has been suggested that the divalent metal transporter (DMT1) represents a common uptake pathway for these important micronutrients. However, compelling evidence from our laboratory suggests that DMT1 is predominantly an iron transporter, with lower affinity for other metals. Several studies have shown that increasing dietary iron downregulates DMT1. Interestingly, our current data indicate that zinc upregulates DMT1 protein and mRNA expression and also pH-dependent iron uptake. Transepithelial flux of iron was also increased and was associated with a rise in IREG1 mRNA expression.     

Intestinal expression of genes involved in iron absorption in humans

Hereditary hemochromatosis (HHC) is one of the most frequent genetic disorders in humans. In healthy individuals, absorption of iron in the intestine is tightly regulated by cells with the highest iron demand, in particular erythroid precursors. Cloning of intestinal iron transporter proteins provided new insight into mechanisms and regulation of intestinal iron absorption. The aim of this study was to assess whether, in humans, the two transporters are regulated in an iron-dependent manner and whether this regulation is disturbed in HHC. Using quantitative PCR, we measured mRNA expression of divalent cation transporter 1 (DCT1), iron-regulated gene 1 (IREG1), and hephaestin in duodenal biopsy samples of individuals with normal iron levels, iron-deficiency anemia, or iron overload. In controls, we found inverse relationships between the DCT1 splice form containing an iron-responsive element (IRE) and blood hemoglobin, serum transferrin saturation, or ferritin. Subjects with iron-deficiency anemia showed a significant increase in expression of the spliced form, DCT1(IRE) mRNA. Similarly, in subjects homozygous for the C282Y HFE mutation, DCT1(IRE) expression levels remained high despite high serum iron saturation. Furthermore, a significantly increased IREG1 expression was observed. Hephaestin did not exhibit a similar iron-dependent regulation. Our data show that expression levels of human DCT1 mRNA, and to a lesser extent IREG1 mRNA, are regulated in an iron-dependent manner, whereas mRNA of hephaestin is not affected. The lack of appropriate downregulation of apical and basolateral iron transporters in duodenum likely leads to excessive iron absorption in persons with HHC.     

Tumour necrosis factor alpha causes hypoferraemia and reduced intestinal iron absorption in mice

Cytokines are implicated in the anaemia of chronic disease by reducing erythropoiesis and increasing iron sequestration in the reticuloendotheial system. However, the effect of cytokines, in particular TNFalpha (tumour necrosis factor alpha), on small bowel iron uptake and iron-transporter expression remains unclear. In the present study, we subjected CD1 male mice to intraperitoneal injection with TNFalpha (10 ng/mouse) and then examined the expression and localization of DMT1 (divalent metal transporter 1), IREG1 (iron-regulated protein 1) and ferritin in duodenum. Liver and spleen samples were used to determine hepcidin mRNA expression. Changes in serum iron and iron loading of duodenum, spleen and liver were also determined. We found a significant (P<0.05) fall in serum iron 3 h post-TNFalpha exposure. This was coincident with increased iron deposition in the spleen. After 24 h of exposure, there was a significant decrease in duodenal iron transfer (P<0.05) coincident with increased enterocyte ferritin expression (P<0.05) and re-localization of IREG1 from the basolateral enterocyte membrane. Hepatic hepcidin mRNA levels remained unchanged, whereas splenic hepcidin mRNA expression was reduced at 24 h. In conclusion, we provide evidence that TNFalpha may contribute to anaemia of chronic disease by iron sequestration in the spleen and by reduced duodenal iron transfer, which seems to be due to increased enterocyte iron binding by ferritin and a loss of IREG1 function. These observations were independent of hepcidin mRNA levels.     

Deficiency of ABCA1 impairs apolipoprotein E metabolism in brain

ABCA1 is a cholesterol transporter that is widely expressed throughout the body. Outside the central nervous system (CNS), ABCA1 functions in the biogenesis of high-density lipoprotein (HDL), where it mediates the efflux of cholesterol and phospholipids to apolipoprotein (apo) A-I. Deficiency of ABCA1 results in lack of circulating HDL and greatly reduced levels of apoA-I. ABCA1 is also expressed in cells within the CNS, but its roles in brain lipid metabolism are not yet fully understood. In the brain, glia synthesize the apolipoproteins involved in CNS lipid metabolism. Here we demonstrate that glial ABCA1 is required for cholesterol efflux to apoA-I and plays a key role in facilitating cholesterol efflux to apoE, which is the major apolipoprotein in the brain. In both astrocytes and microglia, ABCA1 deficiency reduces lipid efflux to exogenous apoE. The impaired ability to efflux lipids in ABCA1-/- glia results in lipid accumulation in both astrocytes and microglia under normal culture conditions. Additionally, apoE secretion is compromised in ABCA1-/- astrocytes and microglia. In vivo, deficiency of ABCA1 results in a 65% decrease in apoE levels in whole brain, and a 75-80% decrease in apoE levels in hippocampus and striatum. Additionally, the effect of ABCA1 on apoE is selective, as apoJ levels are unchanged in brains of ABCA1-/- mice. Taken together, these results show that glial ABCA1 is a key influence on apoE metabolism in the CNS.     

A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation

Iron absorption by the duodenal mucosa is initiated by uptake of ferrous Fe(II) iron across the brush border membrane and culminates in transfer of the metal across the basolateral membrane to the portal vein circulation by an unknown mechanism. We describe here the isolation and characterization of a novel cDNA (Ireg1) encoding a duodenal protein that is localized to the basolateral membrane of polarized epithelial cells. Ireg1 mRNA and protein expression are increased under conditions of increased iron absorption, and the 5' UTR of the Ireg1 mRNA contains a functional iron-responsive element (IRE). IREG1 stimulates iron efflux following expression in Xenopus oocytes. We conclude that IREG1 represents the long-sought duodenal iron export protein and is upregulated in the iron overload disease, hereditary hemochromatosis.     

Glial Fibrillary Acidic Protein is Greatly Modified by Oxidative Stress in Aceruloplasminemia Brain

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by mutations in the ceruloplasmin (Cp) gene. The neuropathological hallmark of this disease is intracellular iron overload, which is thought to lead to neuronal cell death through increased oxidative stress. We evaluated and characterized protein oxidation in the brain of a patient with this disease. The protein carbonyl content in the cerebral cortex of the patient was elevated compared to controls. Furthermore, peptide mass fingerprinting and partial amino acid sequencing identified glial fibrillary acidic protein (GFAP) as the major carbonylated protein in the cerebral cortex of the patient. In conjunction with the facts that Cp mainly localizes to astrocytes in the central nervous system and that astrocytes are loaded with much more iron than neurons in the cerebral cortex, our findings indicate that Cp deficiency may primarily damage astrocytes. We speculate that the dysfunction of astrocytes may be causatively related to neuronal cell loss in aceruloplasminemia.     

Glial fibrillary acidic protein is greatly modified by oxidative stress in aceruloplasminemia brain

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by mutations in the ceruloplasmin (Cp) gene. The neuropathological hallmark of this disease is intracellular iron overload, which is thought to lead to neuronal cell death through increased oxidative stress. We evaluated and characterized protein oxidation in the brain of a patient with this disease. The protein carbonyl content in the cerebral cortex of the patient was elevated compared to controls. Furthermore, peptide mass fingerprinting and partial amino acid sequencing identified glial fibrillary acidic protein (GFAP) as the major carbonylated protein in the cerebral cortex of the patient. In conjunction with the facts that Cp mainly localizes to astrocytes in the central nervous system and that astrocytes are loaded with much more iron than neurons in the cerebral cortex, our findings indicate that Cp deficiency may primarily damage astrocytes. We speculate that the dysfunction of astrocytes may be causatively related to neuronal cell loss in aceruloplasminemia.     

Corticosterone Induces Dysregulation of Iron Metabolism in Hippocampal Neurons In Vitro

Iron is required for neuronal function but in excess generates neurodegeneration. Although the iron homeostasis machinery in neurons has been described extensively, little is known about the influence of corticosterone on the iron homeostasis in neurons. In this study, we characterized the response of hippocampal neurons to a model of progressive corticosterone condition. We found that increasing extracellular corticosterone-induced iron accumulation killed a large proportion of neurons. Iron concentrations were significantly increased in the corticosterone-treated cells. In the hippocampal neurons, corticosterone decreased expression of ferritin and increased expression of transferrin receptor1 (TfR1), iron regulatory protein1 (IRP1), and divalent metal transporter 1. Corticosterone-induced elevation of IRP1 expression can cause increase of TfR1 and decrease of ferritin expression, which further leads to iron accumulation in hippocampal neurons and subsequently increases the oxidative damage of the neurons; it is indicated that corticosterone might be an important reason for iron deposition-caused neurodegenerative diseases.     

Erratum to: Corticosterone Induces Dysregulation of Iron Metabolism in Hippocampal Neurons In Vitro

Iron is required for neuronal function but in excess generates neurodegeneration. Although the iron homeostasis machinery in neurons has been described extensively, little is known about the influence of corticosterone on the iron homeostasis in neurons. In this study, we characterized the response of hippocampal neurons to a model of progressive corticosterone condition. We found that increasing extracellular corticosterone-induced iron accumulation killed a large proportion of neurons. Iron concentrations were significantly increased in the corticosterone-treated cells. In the hippocampal neurons, corticosterone decreased expression of ferritin and increased expression of transferrin receptor1 (TfR1), iron regulatory protein1 (IRP1), and divalent metal transporter 1. Corticosterone-induced elevation of IRP1 expression can cause increase of TfR1 and decrease of ferritin expression, which further leads to iron accumulation in hippocampal neurons and subsequently increases the oxidative damage of the neurons; it is indicated that corticosterone might be an important reason for iron deposition-caused neurodegenerative diseases.     

Hypoxic preconditioning increases iron transport rate in astrocytes

The mechanisms involved in the neuroprotection induced by hypoxic preconditioning (HP) have not been fully elucidated. The involvement of hypoxia-inducible factor-1 alpha (HIF-1alpha) in such neuroprotection has been confirmed. There is also evidence showing that a series of genes with important functions in iron metabolism, including transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1), are regulated by HIF-1alpha in response to hypoxia in extra-neural organs or cells. We therefore hypothesized that HP is able to affect the expression of iron metabolism proteins in the brain and that changes in these proteins induced by HP might be associated with the HP-induced neuroprotection. We herein demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (ferroportin1) proteins, and thus increase tansferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release in astrocytes. Moreover, HP could lead to a progressive increase in cellular iron content. We concluded that HP has the ability to increase iron transport speed in astrocytes. Based on our findings and the importance of astrocytes in neuronal survival in hypoxic/ischemic preconditioning, we proposed that the increase in iron transport rate and cellular iron in astocytes might be one of the mechanisms associated with the HP-induced neuroprotection. We also demonstrated that ferroportin1 expression was significantly affected by HIF-1alpha in astrocytes, implying that the gene encoding this iron efflux protein might be a hypoxia-inducible one.     

Hypoxic preconditioning increases iron transport rate in astrocytes

The mechanisms involved in the neuroprotection induced by hypoxic preconditioning (HP) have not been fully elucidated. The involvement of hypoxia-inducible factor-1 alpha (HIF-1alpha) in such neuroprotection has been confirmed. There is also evidence showing that a series of genes with important functions in iron metabolism, including transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1), are regulated by HIF-1alpha in response to hypoxia in extra-neural organs or cells. We therefore hypothesized that HP is able to affect the expression of iron metabolism proteins in the brain and that changes in these proteins induced by HP might be associated with the HP-induced neuroprotection. We herein demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (ferroportin1) proteins, and thus increase tansferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release in astrocytes. Moreover, HP could lead to a progressive increase in cellular iron content. We concluded that HP has the ability to increase iron transport speed in astrocytes. Based on our findings and the importance of astrocytes in neuronal survival in hypoxic/ischemic preconditioning, we proposed that the increase in iron transport rate and cellular iron in astocytes might be one of the mechanisms associated with the HP-induced neuroprotection. We also demonstrated that ferroportin1 expression was significantly affected by HIF-1alpha in astrocytes, implying that the gene encoding this iron efflux protein might be a hypoxia-inducible one.     

MPTP诱导小鼠黑质区铁摄取和DMT1表达增加

铁在帕金森病(Parkinson‘s disease,PD)的发病机制中起着非常关键的作用,为了探讨PD中铁升高的机制,本实验观察了1-甲基4-苯基—1,2,3,6-四氢吡啶(MPTP)处理小鼠黑质(substantia nigra,SN)内铁摄取及新的铁转运蛋白二价金属离子转运蛋白1(DMT1)的表达变化。结果表明：(1)MPTP处理组小鼠SN内铁染色增高,注射MPTP7d组明显高于3d组。(2)MPTP处理组小鼠,酪氨酸羟化酶(TH)免疫阳性细胞数目显著减少。(3)MPTP处理组小鼠,“-IRE”型DMT1表达在各组中均增加,而“ IRE”型DMT1仅在MPTP处理后7d才出现变化。上述结果提示,这种新发现的哺乳动物跨膜铁转运蛋白表达增加可能是引起MPTP处理小鼠SN中铁升高的关键因素,铁的升高进一步导致DA神经元的死亡。     

Iron trafficking inside the brain

Iron, an essential element for all cells of the body, including those of the brain, is transported bound to transferrin in the blood and the general extracellular fluid of the body. The demonstration of transferrin receptors on brain capillary endothelial cells (BCECs) more than 20 years ago provided the evidence for the now accepted view that the first step in blood to brain transport of iron is receptor-mediated endocytosis of transferrin. Subsequent steps are less clear. However, recent investigations which form the basis of this review have shed some light on them and also indicate possible fruitful avenues for future research. They provide new evidence on how iron is released from transferrin on the abluminal surface of BCECs, including the role of astrocytes in this process, how iron is transported in brain extracellular fluid, and how iron is taken up by neurons and glial cells. We propose that the divalent metal transporter 1 is not involved in iron transport through the BCECs. Instead, iron is probably released from transferrin on the abluminal surface of these cells by the action of citrate and ATP that are released by astrocytes, which form a very close relationship with BCECs. Complexes of iron with citrate and ATP can then circulate in brain extracellular fluid and may be taken up in these low-molecular weight forms by all types of brain cells or be bound by transferrin and taken up by cells which express transferrin receptors. Some iron most likely also circulates bound to transferrin, as neurons contain both transferrin receptors and divalent metal transporter 1 and can take up transferrin-bound iron. The most likely source for transferrin in the brain interstitium derives from diffusion from the ventricles. Neurons express the iron exporting carrier, ferroportin, which probably allows them to excrete unneeded iron. Astrocytes lack transferrin receptors. Their source of iron is probably that released from transferrin on the abluminal surface of BCECs. They probably to export iron by a mechanism involving a membrane-bound form of the ferroxidase, ceruloplasmin. Oligodendrocytes also lack transferrin receptors. They probably take up non-transferrin bound iron that gets incorporated in newly synthesized transferrin, which may play an important role for intracellular iron transport.