Iron-induced Neurotoxicity Research Articles

Enhanced Phosphatidylinositol 3-kinase (PI3K)/Akt Signaling Has Pleiotropic Targets in Hippocampal Neurons Exposed to Iron-induced Oxidative Stress

The PI3K/Akt pathway is a key component in synaptic plasticity and neuronal survival. The aim of this work was to investigate the participation of the PI3K/Akt pathway and its outcome on different molecular targets such as glycogen synthase kinase 3β (GSK3β) and Forkhead box-O (FoxO) transcription factors during mild oxidative stress triggered by iron overload. The exposure of mouse hippocampal neurons (HT22) to different concentrations of Fe(2+) (25-200 μm) for 24 h led us to define a mild oxidative injury status (50 μm Fe(2+)) in which cell morphology showed changes typical of neuronal damage with increased lipid peroxidation and cellular oxidant levels but no alteration of cellular viability. There was a simultaneous increase in both Akt and GSK3β phosphorylation. Levels of phospho-FoxO3a (inactive form) increased in the cytosolic fraction of cells treated with iron in a PI3K-dependent manner. Moreover, PI3K and Akt translocated to the nucleus in response to oxidative stress. Iron-overloaded cells harboring a constitutively active form of Akt showed decreased oxidants levels. Indeed, GSH synthesis under oxidative stress conditions was regulated by activated Akt. Our results show that activation of the PI3K/Akt pathway during iron-induced neurotoxicity regulates multiple targets such as GSK3β, FoxO transcriptional activity, and glutathione metabolism, thus modulating the neuronal response to oxidative stress.

Pleiotropic effects of PI3K/Akt pathway in hippocampal neurons exposed to iron‐induced oxidative stress

PI3K/Akt pathway is a key component in synaptic plasticity and neuronal survival. The aim of this work was to investigate the participation of PI3K/Akt pathway and its outcome on different molecular targets such as GSK3β and FoxO transcription factors during mild oxidative stress triggered by iron overload. The exposure of mouse hippocampal neurons (HT22) to different concentrations of Fe2+ (25–200 μM) for 24 h led us to define a mild oxidative injury status (50 μM Fe2+) in which cell morphology showed typical changes of neuronal damage, with increased lipid peroxidation and cellular oxidant levels but no alterations of cellular viability. Under these experimental conditions, SOD1, SOD2 and Trx expression was diminished, catalase showed no changes, and HO1 was increased, with respect to the control. Simultaneously, both Akt and GSK3β phosphorylation was increased. Levels of phospho‐FoxO3a (inactive form) increased in the cytosolic fraction of cells treated with iron in a PI3K‐dependent manner. Moreover, PI3K and Akt translocated to the nucleus in response to oxidative stress. Iron‐overloaded cells harboring a contitutively active form of Akt showed decreased oxidants levels. Our results show that the activation of PI3K/Akt pathway during iron‐induced neurotoxicity regulates multiple targets such as GSK3β, FoxO transcriptional activity and the generation of reactive oxygen species.

Role of α-synuclein aggregation and the nuclear factor E2-related factor 2/heme oxygenase-1 pathway in iron-induced neurotoxicity

Abnormal aggregation of α-synuclein (α-syn) plays a critical role in the pathogenesis of Parkinson's disease (PD). Iron is also believed to serve as a major contributor by inducing oxidative stress and α-syn aggregation. Here, we report that down-regulation of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) may contribute to iron-induced α-syn aggregation. In this study, we show that ferrous iron down-regulates Nrf2 and HO-1 in a time-dependent manner in SK-N-SH neuroblastoma cells. Levels of both Nrf2 and HO-1 are decreased even more by ferrous iron in SK-N-SH cells that overexpress α-syn and results in greater cell toxicity. Consistent with these results, knockdown of α-syn expression prevents reduction of Nrf2 and HO-1 by ferrous iron, eliminates α-syn aggregates, and protects SK-N-SH cells against ferrous iron-induced cell damage. Furthermore, increased HO-1 expression exerts a protective role against ferrous iron. These results support a new hypothesis of synergistic α-syn/iron cytotoxicity, whereby ferrous iron induces α-syn aggregation and neurotoxicity by inhibiting Nrf2/HO-1. Inhibition of Nrf2/HO-1 leads to more α-syn aggregation and greater toxicity induced by iron, creating a vicious cycle of iron accumulation, α-syn aggregation and HO-1 disruption in PD.

Akt/Nrf2 Activated Upregulation of Heme Oxygenase-1 Involves in the Role of Rg1 Against Ferrous Iron-Induced Neurotoxicity in SK-N-SH Cells

Iron accumulation is considered to be involved in the pathogenesis of Parkinson's disease (PD). Our previous studies have observed that Rg1, a major pharmacologically active ingredient from Ginseng, could protect dopaminergic neurons by reducing nigral iron levels through regulating the expression of iron transporters in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mice. The aim of this study is to investigate other mechanism involved in the cytoprotection of Rg1 against iron-induced neurotoxicity in human neuroblastoma SK-N-SH cells. Significant rescue of Rg1 on cell viability against 100μM ferrous iron-induced neurotoxicity was observed. Upregulation of heme oxygenase-1 (HO-1) and Cu-Zn superoxide dismutase (Cu/Zn SOD) were observed in Rg1 pretreated group. Moreover, Rg1 pretreatment induces Nrf2 nuclear translocation, which is upstream of HO-1 expression, and activated PI3K/Akt pathway was also observed in Rg1 pretreated group. This could antagonize iron-induced increase in intracellular reactive oxygen species and decrease in mitochondrial transmembrane potential. These results suggest that the neuroprotective effects of Rg1 against iron toxicity are attributed to the anti-oxidative properties by activating Akt/Nrf2 pathway and increasing Nrf2-induced expression of HO-1 and Cu/Zn SOD.

Alpha-tocopherol decreases iron-induced hippocampal and nigral neuron loss.

There are many studies about iron-induced neuronal hyperactivity and oxidative stress. Some reports also showed that iron levels rise in the brain in some neurodegenerative diseases such as Parkinson's (PD) and Alzheimer's disease (AD). It has been suggested that excessive iron level increases oxidative stress and causes neuronal death. Tocopherols act as a free radical scavenger when phenoxylic head group encounters a free radical. We have aimed to identify the effect of alpha-tocopherol (Vitamin E) on iron-induced neurotoxicity. For this reason, rats were divided into three groups as control, iron, and iron + alpha-tocopherol groups. Iron chloride (200 mM in 2.5 microl volume) was injected into brain ventricle of iron and iron + alpha-tocopherol group rats. Same volume of saline (2.5 microl) was given to the rats belonging to control group. Rats of iron + alpha-tocopherol group received intraperitoneally (i.p.) alpha-tocopherol (100 mg/kg/day) for 10 days. After 10 days, rats were perfused intracardially under deep urethane anesthesia. Removed brains were processed using standard histological techniques. The numbers of neurons in hippocampus and substantia nigra of all rats were estimated by stereological techniques. Results of present study show that alpha-tocopherol decreased hippocampal and nigral neuron loss from 51.7 to 12.1% and 41.6 to 17.8%, respectively. Findings of the present study suggest that alpha-tocopherol may have neuroprotective effects against iron-induced hippocampal and nigral neurotoxicity and it may have a therapeutic significance for neurodegenerative diseases involved iron.

Iron-Induced Oxidative Injury Differentially Regulates PI3K/Akt/GSK3β Pathway in Synaptic Endings from Adult and Aged Rats

In this work we study the state of phosphoinositide-3-kinase/Akt/glycogen synthase kinase 3 beta (PI3K/Akt/GSK3beta) signaling during oxidative injury triggered by free iron using cerebral cortex synaptic endings isolated from adult (4-month-old) and aged (28-month-old) rats. Synaptosomes were exposed to FeSO4 (50 microM) for different periods of time and synaptosomal viability and the state of the PI3K/Akt/GSK3beta pathway were evaluated in adult and aged animals. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and lactate dehydrogenase leakage were significantly affected in both age groups. However, aged animals showed a greater susceptibility to oxidative stress. In adults, Akt was activated after a brief exposure time (5 min), whereas in aged animals activation occurred after 5 and 30 min of incubation with the metal ion. GSK3beta phosphorylation showed the same activation pattern as that observed for Akt. Both Akt and GSK3beta phosphorylation were dependent on PI3K activation. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation was temporally coincident with Akt activation and was PI3K dependent in adults, whereas ERK1/2 activation in aged rats was higher than that observed in adults and showed no dependence on PI3K activity. We demonstrate here that synaptic endings from adult and aged animals subjected to iron-induced neurotoxicity show a differential profile in the activation of PI3K/Akt/GSK3beta. Our results strongly suggest that the increased susceptibility of aged animals to oxidative injury provokes a differential modulation of key signaling pathways involved in synaptic plasticity and neuronal survival.

Physiological and pathological aspects of Aβ in iron homeostasis via 5'UTR in the APP mRNA and the therapeutic use of iron-chelators

Many studies have highlighted the pathological involvement of iron accumulation and iron-related oxidative stress (OS) in Alzheimer's disease (AD). Iron was further demonstrated to modulate expression of the Alzheimer's amyloid precursor holo-protein (APP) by a mechanism similar to that of regulation of ferritin-L and -H mRNA translation through an iron-responsive element (IRE) in their 5' untranslated regions (UTRs). Here, we discuss two aspects of the link between iron and AD, in relation to the recently discovered IRE in the 5'UTR of APP mRNA. The first is the physiological aspect: a compensatory neuroprotective response of amyloid-β protein (Aβ) in reducing iron-induced neurotoxicity. Thus, given that Aβ possesses iron chelation sites, it is hypothesized that OS-induced intracellular iron may stimulate APP holo-protein translation (via the APP 5'UTR) and subsequently the generation of its cleavage product, Aβ, as a compensatory response that eventually reduces OS. The second is the pathological aspect: iron chelating compounds target the APP 5'UTR and possess the capacity to reduce APP translation, and subsequently Aβ levels, and thus represent molecules with high potential in the development of drugs for the treatment of AD.

Role of Nitric Oxide Synthesis Inhibitors in Iron-Induced Nigral Neurotoxicity: A Mechanistic Exploration

ABSTRACTIn the central nervous system, nitric oxide (NO) has been suggested to be a cell-to-cell signaling molecule that regulates guanylyl cyclase, aconitase, and iron regulatory protein. NO is also one of the substances that is involved in neuronal death. On the other hand, iron overload and enhanced hydroxyl radical formation have been implicated as the causative factors of some neurodegenerative disorders. The present study was performed to clarify whether nitric oxide is involved in iron-induced neuron death. Neurotoxicity was produced by microinjection of iron chloride (200 mM, 2.5 μL) into the left cerebral ventricle. After the intracerebroventricular (ICV) injection, all animals were kept alive for 10 days. During this period, animals in the iron + L-NAME (N-nitro-L-arginine methyl ester) and iron + aminoguanidine groups received intraperitoneal (IP) L-NAME (30 mg/kg) and aminoguanidine (100 mg/kg) injections once a day, respectively. Rats belonging to the control group also received intraperitoneally the same amount of saline. After 10 days, the rats were perfused intracardially under deep urethane anesthesia. Removed brains were processed using the standard histological techniques. The total numbers of neurons in substantia nigra of all rats were estimated with stereological techniques. It was found that L-NAME significantly decreased nigral cell loss from 43.2% to 14.0%, while aminoguanidine did not affect cell loss. Results of the present study suggest that NOS inhibition by L-NAME seems to have neuroprotective effects on iron-induced nigral neurotoxicity.

Neuroprotective effect of aminoguanidine on iron-induced neurotoxicity

Iron is a commonly used metal to induce neuronal hyperactivity and oxidative stress. Iron levels rise in the brain in some neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. A body of evidence indicates a link between neuronal death and nitric oxide. The present study was performed to investigate whether nitric oxide produced by inducible nitric oxide synthase is involved in iron-induced neuron death. For this purpose rats were divided into four groups: control, iron, aminoguanidine and iron + aminoguanidine. Animals in iron and iron + aminoguanidine groups received intracerebroventricular FeCl 3 injection (200 mM, 2.5 μl). Rats belonging to control and aminoguanidine groups received the same amount of saline into the cerebral ventricles. All animals were kept alive for 10 days following the operation and animals in aminoguanidine and iron + aminoguanidine groups received intraperitoneal aminoguanidine injections once a day (100 mg/kg day) during this period. After 10 days, rats were perfused intracardially under deep urethane anesthesia. Removed brains were processed using the standard histological techniques. The total numbers of neurons in hippocampus of all rats were estimated with the unbiased stereological techniques. It was found that aminoguanidine decreased mean neuron loss from 43.4% to 20.3%. Results of the present study suggest that aminoguanidine may attenuate the neurotoxic effects of iron by inhibiting inducible nitric oxide synthase.

Nitric oxide synthesis inhibition attenuates iron-induced neurotoxicity: A stereological study

Iron overload and enhanced hydroxyl radical formation have been implicated as the causative factors of some neurodegenerative disorders. Therefore, iron is commonly used as a metal to induce neuronal hyperactivity and oxidative stress. A body of evidence indicates a relationship between iron-induced neuronal death and nitric oxide (NO). Data are, however, controversial because it is not clear whether NO has neuroprotective or neurotoxic effects on neurotoxicity. To determine the contribution of NO to iron-induced hippocampal cell loss, l-arginine, the NO synthesis precursor, and a nonselective nitric oxide synthase inhibitor N G-nitro- l-arginine methyl ester ( l-NAME) were used. Animals were divided into four groups as follows: control, iron, iron + l-NAME and iron + l-arginine. Neurotoxicity was produced by microinjection of iron chloride (200 mM, 2.5 μl) into the left cerebral ventricle in iron-treated groups while control group rats received same amount of saline. After the intracerebroventricular injection, all animals were kept alive for 10 days. During this period, animals in iron + l-NAME and iron + l-arginine groups received intraperitoneal (i.p.) l-NAME (30 mg/kg) and l-arginine (1000 mg/kg) injections once a day, respectively. Rats belonging to control group also received the same amount of saline intraperitoneally. After 10 days, rats were perfused intracardially under deep urethane anesthesia. Removed brains were processed using the standard histological techniques. The total numbers of neurons in hippocampus of all rats were estimated with stereological techniques. It was found that l-NAME decreased iron-induced cell loss from 44.7 to 13.7%, while l-arginine increased cell loss from 44.7 to 57.5%. Results of the present study suggest that inhibition of NO synthesis may attenuate the neurotoxic effects of iron.

Human Abeta1-42 reduces iron-induced toxicity in rat cerebral cortex.

Senile plaques, the major neuropathological lesions of Alzheimer's disease (AD), are composed primarily of amyloid-beta (Abeta) peptide and contain high concentrations of iron (1.0 mM). We have previously shown that intracortical injections of 1.0 mM iron to adult rats produce significantly more neuronal loss than control injections of saline vehicle, whereas injections of Abeta do not. Because iron has been shown to increase the in vitro toxicity of Abeta, the present study was undertaken to determine whether iron can make Abeta neurotoxic in vivo. Abeta and 1.0 mM iron (as ferric ammonium citrate) were coinjected into rat cerebral cortex, and the neuronal loss was compared with that produced by pure Abeta or pure iron. The human and rat variants of Abeta(1-42) were compared to determine whether they produce the same amount of neuronal loss when combined with iron. Coinjection of iron with either Abeta variant caused significantly more neuronal loss than Abeta peptide alone, suggesting that iron may contribute to the toxicity associated with senile plaques. Rat Abeta(1-42) combined with iron was as toxic as iron alone, whereas iron combined with human Abeta(1-42) was significantly less toxic. This latter finding indicates that fibrillar human Abeta is able to reduce iron-induced neurotoxicity in vivo and raises the interesting possibility that senile plaques in AD may represent a neuroprotective response to the presence of elevated metal ions.

Systemic vitamin D3 attenuated oxidative injuries in the locus coeruleus of rat brain.

Iron-induced oxidative injuries in locus coeruleus (LC), a major source of noradrenergic projections in the central nervous system (CNS), were investigated in chloral-hydrate anesthetized rats. Local infusion of iron dose-dependently elevated lipid peroxidation of iron-infused LC seven days after infusion. At the same time, norepinephrine content in the hippocampus ipsilateral to the iron-infused LC was decreased in a concentration-dependent manner. Our immunostaining study demonstrated reduced tyrosine hydroxylase-positive neurons in the iron-infused LC, indicating a reduction of neuron number by iron infusion. The involvement of apoptosis in iron-induced oxidative injuries was studied. An abrupt increase in cytosolic cytochrome c content was demonstrated in the infused LC 48 hours after iron infusion. TUNEL-positive cells, an indication of apoptosis, were detected in the iron-infused LC. In an attempt to prevent iron-induced neurotoxicity, vitamin D3, an active metabolite of vitamin D, was systemically administered. Iron-induced increases in cytosolic cytochrome c and TUNEL-positive cells were reduced by this treatment. Furthermore, systemic administration of vitamin D3 attenuated iron-induced oxidative injuries in the infused LC. Our data suggest that local infusion of iron in LC induced oxidative stress and resulted in programmed cell death in the LC-hippocampal noradrenergic system. Furthermore, vitamin D3 may be neuroprotective and therapeutic in attenuating iron-induced neurotoxicity in CNS.