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.     

Iron status and lipopolysaccharide regulate Ndfip1 by activation of nuclear factor-kappa B

Nedd4 family interacting protein 1 (Ndfip1) is an adaptor protein for the Nedd4 family of ubiquitin ligases that target proteins for degradation. Recent studies confirmed the role of Ndfip1 as a regulator of iron metabolism and pointed out that Ndfip1 was involved in iron homeostasis by regulating the degradation of iron importer divalent metal transporter 1 (DMT1). However, little is known about how Ndfip1 is regulated. The aim of this article was to investigate the regulation of Ndfip1 levels and the possible mechanisms. In this study, we investigated the effect of various stimuli, including iron status and lipopolysaccharide (LPS) on Ndfip1 expression in MES23.5 dopaminergic cell lines. Results showed that Ndfip1 expression in these cells was enhanced by ferrous iron overload, but not ferric iron overload, and decreased after iron deprivation by deferoxamine. In addition, LPS could significantly increase the expression of Ndfip1. Furthermore, we demonstrated that the regulation of Ndfip1 expression by these various stimuli was achieving by activation of nuclear factor-kappa B. We speculate that iron status and LPS may contribute to the changes of Ndfip1 expression by activation of nuclear factor-kappa B.     

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.     

Intranasal administration of α-synuclein preformed fibrils triggers microglial iron deposition in the substantia nigra of Macaca fascicularis

Iron deposition is present in main lesion areas in the brains of patients with Parkinson’s disease (PD) and an abnormal iron content may be associated with dopaminergic neuronal cytotoxicity and degeneration in the substantia nigra of the midbrain. However, the cause of iron deposition and its role in the pathological process of PD are unclear. In the present study, we investigated the effects of the nasal mucosal delivery of synthetic human α-synuclein (α-syn) preformed fibrils (PFFs) on the pathogenesis of PD in Macaca fascicularis. We detected that iron deposition was clearly increased in a time-dependent manner from 1 to 17 months in the substantia nigra and globus pallidus, highly contrasting to other brain regions after treatments with α-syn PFFs. At the cellular level, the iron deposits were specifically localized in microglia but not in dopaminergic neurons, nor in other types of glial cells in the substantia nigra, whereas the expression of transferrin (TF), TF receptor 1 (TFR1), TF receptor 2 (TFR2), and ferroportin (FPn) was increased in dopaminergic neurons. Furthermore, no clear dopaminergic neuron loss was observed in the substantia nigra, but with decreased immunoreactivity of tyrosine hydroxylase (TH) and appearance of axonal swelling in the putamen. The brain region-enriched and cell-type-dependent iron localizations indicate that the intranasal α-syn PFFs treatment-induced iron depositions in microglia in the substantia nigra may appear as an early cellular response that may initiate neuroinflammation in the dopaminergic system before cell death occurs. Our data suggest that the inhibition of iron deposition may be a potential approach for the early prevention and treatment of PD.Subject terms: Parkinson''s disease, Parkinson''s disease     

Differential regulation of estrogen in iron metabolism in astrocytes and neurons

Previous studies have demonstrated an effect of estrogen on iron metabolism in peripheral tissues. The role of estrogen on brain iron metabolism is currently unknown. In this study, we investigated the effect and mechanism of estrogen on iron transport proteins. We demonstrated that the iron exporter ferroportin 1 (FPN1) and iron importer divalent metal transporter 1 (DMT1) were upregulated and iron content was decreased after estrogen treatment for 12 hr in primary cultured astrocytes. Hypoxia-inducible factor-1 alpha (HIF-1α) was upregulated, but HIF-2α remained unchanged after estrogen treatment for 12 hr in primary cultured astrocytes. In primary cultured neurons, DMT1 was downregulated, FPN1 was upregulated, iron content decreased, iron regulatory protein (IRP1) was downregulated, but HIF-1α and HIF-2α remained unchanged after estrogen treatment for 12 hr. These results suggest that the regulation of iron metabolism by estrogen in astrocytes and neurons is different. Estrogen increases FPN1 and DMT1 expression by inducing HIF-1α in astrocytes, whereas decreased expression of IRP1 may account for the decreased DMT1 and increased FPN1 expression in neurons.     

Rg1 protects iron‐induced neurotoxicity through antioxidant and iron regulatory proteins in 6‐OHDA‐treated MES23.5 cells

Ginsenoside‐Rg1 is one of the pharmacologically active components isolated from ginseng. It was reported that Rg1 protected dopamine (DA) neurons in 6‐hydroxydopamine (6‐OHDA)‐induced Parkinson's disease (PD) models in vivo and in vitro. Our previous study also demonstrated that iron accumulation was involved in the toxicity of 6‐OHDA. However, whether Rg1 could protect DA neurons against 6‐OHDA toxicity by modulating iron accumulation and iron‐induced oxidative stress is not clear. Therefore, the present study was carried out to elucidate this effect in 6‐OHDA‐treated MES23.5 cells and the possible mechanisms were also conducted. Findings showed Rg1 restored iron‐induced decrease in mitochondrial transmembrane potential in MES23.5 cells, and increased ferrous iron influx was found in 6‐OHDA‐treated cells. Rg1 pretreatment could decrease this iron influx by inhibiting 6‐OHDA‐induced up‐regulation of an iron importer protein divalent metal transporter 1 with iron responsive element (DMT1 + IRE). Furthermore, findings also showed that the effect of Rg1 on DMT1 + IRE expression was due to its inhibition of iron regulatory proteins (IRPs) by its antioxidant effect. These results suggested that the neuroprotective effect of Rg1 against iron toxicity in 6‐OHDA‐treated cells was to decrease the cellular iron accumulation and attenuate the improper up‐regulation of DMT1 + IRE via IRE/IRP system. This provides new insight to understand the pharmacological effects of Rg1 on iron‐induced degeneration of DA neurons. J. Cell. Biochem. 111: 1537–1545, 2010. © 2010 Wiley‐Liss, Inc.     

Brain iron transport

Brain iron is a crucial participant and regulator of normal physiological activity. However, excess iron is involved in the formation of free radicals, and has been associated with oxidative damage to neuronal and other brain cells. Abnormally high brain iron levels have been observed in various neurodegenerative diseases, including neurodegeneration with brain iron accumulation, Alzheimer's disease, Parkinson's disease and Huntington's disease. However, the key question of why iron levels increase in the relevant regions of the brain remains to be answered. A full understanding of the homeostatic mechanisms involved in brain iron transport and metabolism is therefore critical not only for elucidating the pathophysiological mechanisms responsible for excess iron accumulation in the brain but also for developing pharmacological interventions to disrupt the chain of pathological events occurring in these neurodegenerative diseases. Numerous studies have been conducted, but to date no effort to synthesize these studies and ideas into a systematic and coherent summary has been made, especially concerning iron transport across the luminal (apical) membrane of the capillary endothelium and the membranes of different brain cell types. Herein, we review key findings on brain iron transport, highlighting the mechanisms involved in iron transport across the luminal (apical) as well as the abluminal (basal) membrane of the blood–brain barrier, the blood–cerebrospinal fluid barrier, and iron uptake and release in neurons, oligodendrocytes, astrocytes and microglia within the brain. We offer suggestions for addressing the many important gaps in our understanding of this important topic, and provide new insights into the potential causes of abnormally increased iron levels in regions of the brain in neurodegenerative disorders.     

Increased isoprostane and prostaglandin are prominent in neurons in Alzheimer disease

Parkinson's disease is the most common movement disorder characterized by dopaminergic dysfunction and degeneration. Loss-of-function mutations in the DJ-1 gene have been linked to autosomal recessive forms of early-onset familial Parkinson's disease. DJ-1 is thought to play roles in protection of cells against oxidative stress and in maintenance of the normal dopaminergic function in the nigrostriatal pathway. Here we investigate the consequence of both DJ-1 inactivation and aging in mice. We found that DJ-1-/- mice at the age of 24–27 months have normal numbers of dopaminergic neurons in the substantia nigra and normal levels of dopamine and its major metabolites in the striatum. The number of noradrenergic neurons in the locus coeruleus is also unchanged in DJ-1-/- mice. Moreover, there is no accumulation of oxidative damage or inclusion bodies in aged DJ-1-/- brains. Together, these results indicate that loss of DJ-1 function alone is insufficient to cause nigral degeneration and oxidative damage in the life span of mice.     

Parkin-Dependent Degradation of the F-Box Protein Fbw7β Promotes Neuronal Survival in Response to Oxidative Stress by Stabilizing Mcl-1

Parkinson''s disease (PD) is characterized by progressive loss of midbrain dopaminergic neurons resulting in motor dysfunction. While most PD is sporadic in nature, a significant subset can be linked to either dominant or recessive germ line mutations. PARK2, encoding the ubiquitin ligase parkin, is the most frequently mutated gene in hereditary Parkinson''s disease. Here, we present evidence for a neuronal ubiquitin ligase cascade involving parkin and the multisubunit ubiquitin ligase SCF^Fbw7β. Specifically, parkin targets the SCF substrate adapter Fbw7β for proteasomal degradation. Furthermore, we show that the physiological role of parkin-mediated regulation of Fbw7β levels is the stabilization of the mitochondrial prosurvival factor Mcl-1, an SCF^Fbw7β target in neurons. We show that neurons depleted of parkin become acutely sensitive to oxidative stress due to an inability to maintain adequate levels of Mcl-1. Therefore, loss of parkin function through biallelic mutation of PARK2 may lead to death of dopaminergic neurons through unregulated SCF^Fbw7β-mediated ubiquitylation-dependent proteolysis of Mcl-1.     

Pro-inflammatory cytokines modulate iron regulatory protein 1 expression and iron transportation through reactive oxygen/nitrogen species production in ventral mesencephalic neurons

Both inflammatory processes associated with microglia activation and abnormal iron deposit in dopaminergic neurons are involved in the pathogenesis of Parkinson's disease (PD). However, the relationship between neuroinflammation and iron accumulation was not fully elucidated. In the present study, we aimed to investigate whether the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) released by microglia, could affect cellular iron transportation in primary cultured ventral mesencephalic (VM) neurons. The results showed that IL-1β or TNF-α treatment led to increased ferrous iron influx and decreased iron efflux in these cells, due to the upregulation of divalent metal transporter 1 with the iron response element (DMT1 + IRE) and downregulation of ferroportin1 (FPN1). Increased levels of iron regulatory protein 1 (IRP1), transferrin receptor 1 (TfR1) and hepcidin were also observed in IL-1β or TNF-α treated VM neurons. IRP1 upregulation could be fully abolished by co-administration of radical scavenger N-acetyl-l-cysteine and inducible NO synthetase inhibitor N_ω-nitro-l-arginine methyl ester hydrochloride. Further experiments demonstrated that IL-1β and TNF-α release was remarkably enhanced by iron load in activated microglia triggered by lipopolysaccharide or 1-methyl-4-phenylpyridinium (MPP⁺). In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice, salicylate application could not block DMT1 + IRE upregulation in dopaminergic neurons of substantia nigra. These results suggested that IL-1β and TNF-α released by microglia, especially under the condition of iron load, might contribute to iron accumulation in VM neurons by upregulating IRP1 and hepcidin levels through reactive oxygen/nitrogen species production. This might provide a new insight into unraveling that microglia might aggravate this iron mediated neuropathologies in PD.     

α-Synuclein and neuronal cell death

Mutations in the DJ-1 gene have been linked to autosomal recessive familial Parkinson's disease. To understand the function of DJ-1, we determined the DJ-1 expression in both zebrafish and post mortem human brains. We found that DJ-1 was expressed early during zebrafish development and throughout adulthood. Knock down (KD) of DJ-1 by injection of morpholino did not cause dramatic morphologic alterations during development, and no loss of dopaminergic neurons was observed in embryos lacking DJ-1. However, DJ-1 KD embryos were more susceptible to programmed cell death. While a slight reduction in staining for islet-1 positive neurons was observed in both DJ-1 KD and H₂O₂ treated embryos, the number of apoptotic cells was significantly increased in both KD and H₂O₂ treated embryos. Interestingly, DJ-1 expression was increased in brains of zebrafish under conditions of oxidative stress, indicating that DJ-1 is a part of stress-responsive machinery. Since oxidative stress is one of the major contributors to the development of Alzheimer's disease (AD), we also examined DJ-1 expression in AD brains. Using DJ-1 specific antibodies, we failed to detect a robust staining of DJ-1 in brain tissues from control subjects. However, DJ-1 immunoreactivity was detected in hippocampal pyramidal neurons and astrocytes of AD brains. Therefore, our results strongly suggest that DJ-1 expression is not necessary during zebrafish development but can be induced in zebrafish exposed to oxidative stress and is present in human AD brains.     

Pathogenic Mutation in VPS35 Impairs Its Protection against MPP+ Cytotoxicity

Parkinson''s disease primarily results from progressive degeneration of dopaminergic neurons in the substantia nigra. Both neuronal toxicants and genetic factors are suggested to be involved in the disease pathogenesis. The mitochondrial toxicant 1-methyl-4-phenylpyridinium (MPP⁺) shows a highly selective toxicity to dopaminergic neurons. Recent studies indicate that mutation in the vacuolar protein sorting 35 (vps35) gene segregates with Parkinson''s disease in some families, but how mutation in the vps35 gene causes dopaminergic cell death is not known. Here, we report that enhanced VPS35 expression protected dopaminergic cells against MPP⁺ toxicity and that this neuroprotection was compromised by pathogenic mutation in the gene. A loss of neuroprotective functions contributes to the pathogenesis of VPS35 mutation in Parkinson''s disease.     

Glutathione depletion in immortalized midbrain-derived dopaminergic neurons results in increases in the labile iron pool: Implications for Parkinson's disease

Glutathione depletion is one of the earliest detectable events in the Parkinsonian substantia nigra (SN), but whether it is causative for ensuing molecular events associated with the disease is unknown. Here we report that reduction in levels of glutathione in immortalized midbrain-derived dopaminergic neurons results in increases in the cellular labile iron pool (LIP). This increase is independent of either iron regulatory protein/iron regulatory element (IRP/IRE) or hypoxia inducible factor (HIF) induction but is both H₂0₂ and protein synthesis-dependent. Our findings suggest a novel mechanistic link between dopaminergic glutathione depletion and increased iron levels based on translational activation of TfR1. This may have important implications for neurodegeneration associated with Parkinson's disease in which both glutathione reduction and iron elevation have been implicated.     

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson''s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.     

Enhanced autophagy from chronic toxicity of iron and mutant A53T α-synuclein: implications for neuronal cell death in Parkinson disease

Parkinson disease (PD), a prevalent neurodegenerative motor disorder, is characterized by the rather selective loss of dopaminergic neurons and the presence of α-synuclein-enriched Lewy body inclusions in the substantia nigra of the midbrain. Although the etiology of PD remains incompletely understood, emerging evidence suggests that dysregulated iron homeostasis may be involved. Notably, nigral dopaminergic neurons are enriched in iron, the uptake of which is facilitated by the divalent metal ion transporter DMT1. To clarify the role of iron in PD, we generated SH-SY5Y cells stably expressing DMT1 either singly or in combination with wild type or mutant α-synuclein. We found that DMT1 overexpression dramatically enhances Fe(2+) uptake, which concomitantly promotes cell death. This Fe(2+)-mediated toxicity is aggravated by the presence of mutant α-synuclein expression, resulting in increased oxidative stress and DNA damage. Curiously, Fe(2+)-mediated cell death does not appear to involve apoptosis. Instead, the phenomenon seems to occur as a result of excessive autophagic activity. Accordingly, pharmacological inhibition of autophagy reverses cell death mediated by Fe(2+) overloading. Taken together, our results suggest a role for iron in PD pathogenesis and provide a mechanism underlying Fe(2+)-mediated cell death.     

Ferroportin1 and hephaestin overexpression attenuate iron‐induced oxidative stress in MES23.5 dopaminergic cells

Elevated iron was found in the substantia nigra (SN) of patients with Parkinson's disease (PD). Our previous in vivo experiments suggested that decreased ferroportin1 (FPN1) and hephaestin (HP) expression might account for the cellular iron accumulation and resulting dopaminergic neurons loss in the SN of PD animal models. In the present study, we investigated whether increased FPN1 and/or HP expression could attenuate iron‐induced oxidative stress in the dopaminergic MES23.5 cell line. We generated MES23.5 cells with stable overexpression of FPN1 and/or HP. Our study showed that overexpression of FPN1 and/or HP increased iron efflux, lowered cellular iron level, suppressed reactive oxygen species production, and restored mitochondrial transmembrane potential, similar to the effects seen for the iron chelator deferoxamine. These results suggest that FPN1 and/or HP might directly contribute to iron efflux process from neurons in conditions of overexpression, thus prevent cellular iron accumulation and eventually protect cells from iron‐induced oxidative stress. J. Cell. Biochem. 110: 1063–1072, 2010. Published 2010 Wiley‐Liss, Inc.     

Cellular distribution of ferric iron, ferritin, transferrin and divalent metal transporter 1 (DMT1) in substantia nigra and basal ganglia of normal and beta2-microglobulin deficient mouse brain.

We examined whether high levels of circulatory iron may cause iron accumulation in the brain. In particular, we focussed on the substantia nigra and basal ganglia as several papers have indicated that iron may accumulate here and cause death of dopaminergic neurons. Normal mice and a mouse model of hereditary haemochromatosis, the beta2-microglobulin (beta2m) knock out [beta2m (-/-)] mouse, which has high levels of circulating iron due to increased iron absorption, were examined. The iron concentration in livers were: 170+/-15 microg/g (mean +/- SD) in controls and 1010+/-50 microg/g in beta2m (-/-) mice (p<0.001), whereas in the brain the respective values were 47 +/-1 microg/g and 53+/-2 microg/g (p<0.02). Hence, the difference between cerebral iron levels of normal and beta2m (-/-) mice was small. Histological examination of the brains revealed an unequivocal distribution of ferric iron, ferritin, transferrin and divalent metal transporter 1 (DMT1), which were indistinguishable when normal and beta2m (-/-) mice were compared. In the substantia nigra and basal ganglia, ferric iron and the iron-binding proteins were present in identical cell types, which mainly comprised oligodendrocytes and microglia. Neurons were lightly labelled with transferrin and DMT1. The virtual lack of an increase in cerebral iron in beta2m (-/-) mice clearly shows that the blood-brain barrier (BBB) is capable of restricting the transport of excess plasma iron into the brain.