Huamin Xu

Up-regulation of divalent metal transporter 1 in 6-hydroxydopamine intoxication is IRE/IRP dependent

Iron plays a key role in Parkinsons disease (PD). Increased iron content of the substantia nigra (SN) has been found in PD patients, and divalent metal transporter 1 (DMT1) has been shown to be up-regulated in the SN of both MPTP-induced PD models and PD patients. However, the mechanisms underlying DMT1 up-regulation are largely unknown. In the present study, we observed that in the SN of 6-hydroxydopamine (6-OHDA)-induced PD rats, DMT1 with the iron responsive element (IRE, DMT1+IRE), but not DMT1 without IRE (DMT1−IRE), was up-regulated, suggesting that increased DMT1+IRE expression might account for nigral iron accumulation in PD rats. This possibility was further assessed in an in vitro study using 6-OHDA-treated and DMT1+IRE-over-expressing MES23.5 cells. In 6-OHDA-treated MES23.5 cells, increased iron regulatory protein (IRP) 1 and IRP2 expression was observed, while silencing of IRPs dramatically diminished 6-OHDA-induced DMT1+IRE up-regulation. Pretreatment with N-acetyl-L-cysteine fully suppressed IRPs up-regulation by inhibition of 6-OHDA-induced oxidative stress. Increased DMT1+IRE expression resulted in increased iron influx by MES23.5 cells. Our data provide direct evidence that DMT1+IRE up-regulation can account for IRE/IRP-dependent 6-OHDA-induced iron accumulation initiated by 6-OHDA-induced intracellular oxidative stress and that increased levels of intracellular iron result in aggravated oxidative stress. The results of this study provide novel evidence supporting the use of anti-oxidants in the treatment of PD, with the goal of inhibiting iron accumulation by regulation of DMT1 expression.

Rg1 reduces nigral iron levels of MPTP-treated C57BL6 mice by regulating certain iron transport proteins

Elevated iron levels in the substantia nigra (SN) participate in neuronal death in Parkinsons disease, in which the misregulation of iron transporters such as divalent metal transporter (DMT1) and ferroportin1 (FP1) are involved. Our previous work observed that nigral iron levels were increased in MPTP-treated mice and Ginsenoside Rg1 which is one of the main components of ginseng, had neuroprotective effects against MPTP toxicity. Whether Rg1 could reduce nigral iron levels to protect the dopaminergic neurons? And whether its neuroprotective effect is achieved by regulating certain iron transporters? The present studies showed that Rg1 pre-treatment increased the dopamine and its metabolites contents in the striatum, as well as increased tyrosine hydroxylase expression in the SN. Further experiments observed that Rg1 pre-treatment substantially attenuated MPTP-elevated iron levels, decreased DMT1 expression and increased FP1 expression in the SN. These results suggest that the neuroprotective effect of Rg1 on dopaminergic neurons against MPTP is due to the ability to reduce nigral iron levels, which is achieved by regulating the expressions of DMT1 and FP1.

Increased iron levels correlate with the selective nigral dopaminergic neuron degeneration in Parkinson’s disease

The staging of Lewy-related pathology in sporadic Parkinson’s disease (PD) reveals that many brain nuclei are affected in PD during different stages, except the ventral tegmental area (VTA), which is close related to the substantia nigra (SN) and enriched in dopamine (DA) neurons. Why DA neurons are selectively degenerated in the SN of PD is far from known. In the present study, we observed that the number of tyrosine hydroxylase immunoreactive neurons decreased and iron-staining positive cells increased in the SN, but not in the VTA, in the chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated PD mice. Increased expression of divalent metal transporter 1 and decreased expression of ferroportin 1 might associate with this increased nigral iron levels. Lipofuscin granular aggregations and upregulation of alpha-synuclein (α-synuclein) were also observed only in the SN. These results suggest that increased iron levels associate with the selective degeneration of DA neurons in the SN. The intracellular regulation mechanisms for the iron transporters may be different in the SN and VTA under the same conditions. Moreover, the lipofuscin granular aggregations and upregulation of α-synuclein were also involved in the selective degeneration of dopaminergic neurons in the SN.

Curcumin protects nigral dopaminergic neurons by iron-chelation in the 6-hydroxydopamine rat model of Parkinson’s disease

ObjectiveCurcumin is a plant polyphenolic compound and a major component of spice turmeric (Curcuma longa). It has been reported to possess free radical-scavenging, iron-chelating, and anti-inflammatory properties in different tissues. Our previous study showed that curcumin protects MES23.5 dopaminergic cells from 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro. The present study aimed to explore this neuroprotective effect in the 6-OHDAlesioned rat model of Parkinson’s disease in vivo.MethodsRats were given intragastric curcumin for 24 days. 6-OHDA lesioning was conducted on day 4 of curcumin treatment. Dopamine content was assessed by high-performance liquid chromatography with electrochemical detection, tyrosine hydroxylase (TH)-containing neurons by immunohistochemistry, and iron-containing cells by Perls’ iron staining.ResultsThe dopamine content in the striatum and the number of TH-immunoreactive neurons decreased after 6-OHDA treatment. Curcumin pretreatment reversed these changes. Further studies demonstrated that 6-OHDA treatment increased the number of iron-staining cells, which was dramatically decreased by curcumin pretreatment.ConclusionThe protective effects of curcumin against 6-OHDA may be attributable to the ironchelating activity of curcumin to suppress the iron-induced degeneration of nigral dopaminergic 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 Parkinsons 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.

Rg1 protects the MPP+-treated MES23.5 cells via attenuating DMT1 up-regulation and cellular iron uptake.

Ginsenoside-Rg1 is one of the pharmacologically active component isolated from ginseng. Our previous study observed the protective effect of Rg1 on iron accumulation in the substantia nigra (SN) in 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-treated Parkinsons disease (PD) mice. However, the mechanisms of this neuroprotective effect of Rg1 are unknown. In this study, we elucidated possible mechanisms for this effect using 1-methyl-4-phenylpyridinium (MPP(+))-treated MES23.5 cells. Previous study showed MPP+ treatment induced up-regulation of divalent metal transporter 1 without iron responsive element (DMT1-IRE) in MES23.5 cells. In the present study, we observed that pretreatment with Rg1 could inhibit MPP+-induced up-regulation of DMT1-IRE in MES23.5 cells. Up-regulation of DMT1-IRE by MPP+ treatment was associated with ROS production and translocation of nuclear factor-kappaB (NF-kappaB) to nuclei, both of which were significantly inhibited by Rg1 pretreatment. The role of ROS and NF-kappaB in the up-regulation of DMT1-IRE was supported by application of an antioxidant NAC and BAY 11-7082, an inhibitor of IkappaBalpha phosphorylation. Furthermore, we also showed Rg1 could decrease DMT1-mediated ferrous iron uptake and iron-induced cell damage by inhibiting the up-regulation of DMT1-IRE. These results indicate that Rg1 protected the MPP+-treated MES23.5 cells via attenuating DMT1-IRE up-regulation likely through inhibition of ROS-NF-kappaB pathway; Attenuation of DMT1-IRE expression decreased the iron influx and iron-induced oxidative stress.

Neurorescue Effect of Rosmarinic Acid on 6-Hydroxydopamine-Lesioned Nigral Dopamine Neurons in Rat Model of Parkinson's Disease

Rosmarinic acid (RA) is a naturally occurring polyphenolic compound. It has been reported that RA possessed antioxidant and anti-inflammatory properties. Our previous study showed that RA could protect MES23.5 dopaminergic cells against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro. The purpose of this study was to explore the neuroreparative (neurorescue) effect of RA on 6-OHDA-lesioned rat model of Parkinsons disease (PD) in vivo. In this study, the rats were given RA orally after intrastriatal 6-OHDA lesion. Results showed that the dopamine content in the striatum decreased and the numbers of tyrosine hydroxylase-immunoreactive neurons reduced after 6-OHDA treatment. RA treatment after 6-OHDA administration could restore these changes. Further studies demonstrated that 6-OHDA treatment increased the iron-staining positive cells, which were markedly decreased by RA treatment. Moreover, RA suppressed the increased ratio of Bax/Bcl-2 at gene level induced by 6-OHDA. This indicates that the neurorescue effects of RA against 6-ODHA-induced degeneration of the nigrostriatal dopaminergic system were achieved by decreasing nigral iron levels and regulating the ratio of Bcl-2/Bax gene expression.

Ghrelin prevents 1-methyl-4-phenylpyridinium ion-induced cytotoxicity through antioxidation and NF-κB modulation in MES23.5 cells

Ghrelin, a 28-amino acid peptide, is an endogenous ligand for the growth hormone secretagogue (GHS) receptor. Our previous data showed that ghrelin could inhibit apoptosis in Parkinsons disease (PD) models both in vitro and in vivo. There is now growing evidence that oxidative stress has a critical role in the etiology of PD. And ghrelin was reported to possess anti-inflammatory, antioxidant effects. Dose ghrelin protect dopaminergic neurons by its antioxidant effect? In the present study, 1-methyl-4-phenylpyridinium (MPP(+)) was used to evaluate the possible antioxidant effects of ghrelin on MPP(+)-induced neurotoxicity in MES23.5 cells and the underlying mechanisms. Our results showed that MPP(+) significantly increased malonaldehyde (MDA) level and Bax/Bcl2 ratio, reduced the level of Cu-Zn superoxide dismutase (SOD) and catalase (CAT). Ghrelin protected MES23.5 cells against MPP(+)-induced neurotoxicity by reversing these changes. Furthermore, ghrelin pretreatment significantly inhibited MPP(+)-induced nuclear factor-kappaB translocation. These results suggest that the protective effects of ghrelin on MPP(+)-induced cytotoxicity may be ascribed to its antioxidative properties, and the modulation of nuclear factor-kappaB.

Alpha-synuclein aggregation is involved in the toxicity induced by ferric iron to SK-N-SH neuroblastoma cells

Increased nigral iron levels and intracytoplasmic Lewy bodies (LBs) in the degenerating neurons are found in Parkinson’s disease (PD). Whether LBs formation is involved in the toxicity of iron is largely unknown. In the present study, we observed that the toxicity of ferric iron was enhanced when SK-N-SH cells were overexpressed with wild-type alpha-synuclein. The mitochondrial transmembrane potential (Δψm) and cell viability were decreased in these cells, while the intracellular reactive oxygen species (ROS) were increased, compared with that of control. More aggregated alpha-synuclein was observed in these cells. However, while silencing the expression of alpha-synuclein in SK-N-SH cells with siRNA, iron-induced toxicity could be partially alleviated, indicated by the returned Δψm and cell viability and reduced ROS formation compared with that of control. No alpha-synuclein aggregation could be observed in these cells. The results suggest that alpha-synuclein aggregation was involved in the toxicity of iron to SK-N-SH neuroblastoma cells. Targeting the aggregation of alpha-synuclein might provide a therapeutic strategy for PD in the future.

Rosmarinic Acid Inhibits 6-OHDA-induced Neurotoxicity by Anti-oxidation in MES23.5 Cells

Rosmarinic acid (RA) is a naturally occurring polyphenolic compound. It is found in several herbs in the Lamiaceae family, such as Perilla frutescens. RA has been reported to exert anti-oxidative effects on rat erythrocyte, liver, and kidney cells. However, little is known about the effects of RA on dopaminergic cells. In the present study, we investigated whether RA could protect MES23.5 dopaminergic cells from 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. The results showed that RA pretreatment significantly prevented 6-OHDA-induced cell viability reduction. Further experiments demonstrated that 6-OHDA induced intracellular reactive oxygen species generation and decreased the mitochondria membrane potential (ΔΨm). These effects could be partially reversed by RA pretreatment. However, RA had no direct chemical reaction with 6-OHDA extracellularly in a cell-free system. Taken together, these results suggest that RA could exert its protective effects against 6-OHDA-induced neurotoxicity through its anti-oxidation properties. Thus, we propose that RA should be viewed as a potential chemotherapeutic in Parkinson’s disease patients.