Junxia Xie

Ghrelin antagonizes MPTP-induced neurotoxicity to the dopaminergic neurons in mouse substantia nigra

Ghrelin, a stomach-derived hormone which induces growth hormone release and promotes positive energy balance, has been reported to inhibit cell apoptosis in endotheliocytes, osteoblasts and cardiocytes. Recent evidence has shown that ghrelin can also inhibit neuronal apoptosis of the hypothalamus and the hippocampus. However, little is known about the effects of ghrelin on the substantia nigra pars compacta (SNpc) neurons in which ghrelins receptor, growth hormone secretagogue receptor (GHSR)-1a, is highly expressed. In the present study, we investigated whether ghrelin could protect nigral dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in mice. We observed that ghrelin, acting through GHS-R 1a, inhibited MPTP-induced dopaminergic neuronal loss in the SNpc as well as dopamine depletion in the striatum. Ghrelin could also reverse the down-regulated the expression of Bcl-2, up-regulated the expression of Bax, and caspase-3 activation caused by MPTP. This study demonstrated that ghrelin might be a potential protector of dopaminergic neurons in a therapeutic strategy for Parkinsons disease.

Curcumin attenuates 6-hydroxydopamine-induced cytotoxicity by anti-oxidation and nuclear factor-kappaB modulation in MES23.5 cells

Oxidative stress has been implicated in the degeneration of dopaminergic neurons in the substantia nigra of Parkinsons disease patients, and several anti-oxidants have been shown to be effective on the treatment of Parkinsons disease. Curcumin has been previously reported to possess radical scavenger, iron chelating, anti-inflammatory properties in different tissues. The aim of present study is to explore the cytoprotection of curcumin against 6-hydroxydopamine (6-OHDA)-induced neuronal death, as well as the underlying mechanisms in MES23.5 cells. Our results showed that 6-OHDA significantly reduced the cell viability of MES23.5 cells. Curcumin protected MES23.5 cells against 6-OHDA neurotoxicity by partially restoring the mitochondrial membrane potential, increasing the level of Cu-Zn superoxide dismutase and suppressing an increase in intracellular reactive oxygen species. Furthermore, curcumin pretreatment significantly inhibited 6-OHDA induced nuclear factor-kappaB translocation. These results suggest that the neuroprotective effects of curcumin are attributed to the antioxidative properties and the modulation of nuclear factor-kappaB translocation.

Up-regulation of divalent metal transporter 1 is involved in 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis in MES23.5 cells

Apoptosis has been identified as one of the important mechanisms involved in the degeneration of dopaminergic neurons in Parkinsons disease (PD). Our previous study showed increased iron levels in the substantia nigra as well as loss of dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mouse models. 1-Methyl-4-phenylpyridinium (MPP(+)) is commonly used to establish a cellular model of PD. Although intracellular iron plays a crucial role in MPP(+)-induced apoptosis, the molecular mechanism linking increased iron and MPP(+)-induced neurodegeneration is largely unknown. In the present study, we investigate the involvement of divalent metal transporter 1 (DMT1) that accounts for the ferrous iron transport in MPP(+)-treated MES23.5 cells. In the treated cells, a significant influx of ferrous iron was observed. This resulted in a decreased mitochondrial membrane potential. Additionally, an elevated level of ROS production and activation of caspase-3 were also detected, as well as the subsequent cell apoptosis. These effects could be fully abolished by iron chelator desferal (DFO). Increased DMT1 (-IRE) expression but not DMT1 (+IRE) accounted for the increased iron influx. However, there were no changes for iron regulatory protein 1 (IRP1), despite decreased expression of IRP2. Iron itself had no effect on IRP1 and IRP2 expression. Our data suggest that although DMT1 mRNA contains an iron responsive element, its expression is not totally controlled by this. MPP(+) could up-regulate the expression of DMT1 (-IRE) in an IRE/IRP-independent manner. Our findings also show that MPP(+)-induced apoptosis in MES23.5 cells involves DMT1-dependent iron influx and mitochondria dysfunction.

Ferroportin1 and hephaestin are involved in the nigral iron accumulation of 6-OHDA-lesioned rats

Elevated iron levels in the substantia nigra (SN) participate in neuronal death in Parkinsons disease (PD). While the mechanisms underlying the increased iron are still unknown, some iron transport proteins may be involved. The nigral iron accumulation could be a result of either increased import or decreased export. The mechanisms of iron import have received considerable attention, but little is known about iron export mechanisms. Ferroportin1 (FP1) and hephaestin (HP), two newly discovered iron export proteins, cooperate in the iron export in the gut. Here, we investigated their expression in the SN of rats lesioned by 6‐hydroxydopamine (6‐OHDA). Using immunofluorescence, we showed that FP1 and HP were both expressed on astrocytes, microglia, oligodendrocytes and neurons in the SN. By immunohistochemistry, we showed that 1 day after 6‐OHDA lesion, the expression of the two proteins decreased compared with the control. When rats began showing rotation behaviour induced by apomorphine, usually 6 weeks after 6‐OHDA lesion, they are considered PD models. In these PD models, a further decrease in the two proteins was observed. Reverse transcriptase‐polymerase chain reaction showed that the mRNA levels of FP1 and HP decreased 1 day after 6‐OHDA lesion compared with the control, and further decrease was also observed in the PD model rats. These results show for the first time that FP1 and HP co‐localize in the rat brain, and suggest that decreased expression of these transporters in the SN can account for the increased iron levels.

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.

Ferroportin 1 but not hephaestin contributes to iron accumulation in a cell model of Parkinson''s disease

Iron-induced oxidative stress is thought to play a crucial role in the pathogenesis of Parkinsons disease (PD). Based on our previous in vivo experiments showing that down-regulation of the iron transporters ferroportin 1 (FP1) and hephaestin (HP) might account for the nigral iron accumulation in 6-hydroxydopamine (6-OHDA)-lesioned animal models, in this study we investigated whether FP1 and HP were involved in cellular iron accumulation and the underlying mechanisms in a cell model of PD. The findings showed that 6-OHDA induced FP1 and HP down-regulation, followed by decreased iron efflux and iron accumulation in primary ventral mesencephalic neurons and MES23.5 dopaminergic cells. Silencing of FP1 but not HP led to increased iron levels and aggravated reactive oxygen species generation in MES23.5 cells. Under iron-overloaded conditions, FP1 showed dose-dependent up-regulation, whereas HP showed no response, indicating their down-regulation was not due to the increased intracellular iron content. In 6-OHDA-treated cells, both iron-regulatory protein (IRP) 1 and IRP2 were up-regulated, and silencing of IRPs in MES23.5 cells dramatically blocked 6-OHDA-induced FP1 down-regulation and reversed HP down-regulation. These results suggest that FP1 but not HP contributes to 6-OHDA-induced intracellular iron accumulation, and down-regulation of FP1 and HP by 6-OHDA is IRPs-dependent.

Divalent metal transporter 1 up-regulation is involved in the 6-hydroxydopamine-induced ferrous iron influx.

The reasons underlying the high iron content found in the substantia nigra (SN) of Parkinsons disease (PD) are largely unknown. We suppose, based on our previous studies, that the newly discovered iron transporter divalent metal transporter 1 (DMT1) might be involved in this SN iron accumulation process. To investigate this, we first observed the cellular expression of DMT1 in rat SN, both with the iron response element (+IRE) and without the IRE (−IRE) forms. The results showed that both forms of DMT1 were expressed on neurons, astrocytes, and microglia but not on oligodendrocytes. We further observed the relationship between the increased iron influx and DMT1 expression in 6‐hydroxydopamine (6‐OHDA)‐treated C6 cells. 6‐OHDA (10 μmol/liter) caused a significant increase in ferrous iron influx, with the increased expression of DMT1+IRE, both in protein and in mRNA levels, whereas no change was observed for DMT1−IRE. To clarify further that the increased expression of DMT1 was not due to the increased intracellular iron content, C6 cells were overloaded with ferric ammonium citrate (100 μg/ml). Decreased expression of both forms of DMT1 was observed. Our data suggest that DMT1 is highly expressed in rat SN in a cell‐specific manner. Increased DMT1+IRE expression is the mechanism behind ferrous iron influx induced by 6‐OHDA treatment in C6 cells. This may give some evidence for the involvement of DMT1 in the iron accumulation in PD.

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.

Peptide hormone ghrelin enhances neuronal excitability by inhibition of Kv7/KCNQ channels

The gut-derived orexigenic peptide hormone ghrelin enhances neuronal firing in the substantia nigra pars compacta, where dopaminergic neurons modulate the function of the nigrostriatal system for motor coordination. Here we describe a novel mechanism by which ghrelin enhances firing of nigral dopaminergic neurons by inhibiting voltage-gated potassium Kv7/KCNQ/M-channels through its receptor GHS-R1a and activation of the PLC-PKC pathway. Brain slice recordings of substantia nigra pars compacta neurons reveal that ghrelin inhibits native Kv7/KCNQ/M-currents. This effect is abolished by selective inhibitors of GHS-R1a, PLC and PKC. Transgenic suppression of native Kv7/KCNQ/M-channels in mice or channel blockade with XE991 abolishes ghrelin-induced hyperexcitability. In vivo, intracerebroventricular ghrelin administration causes increased dopamine release and turnover in the striatum. Microinjection of ghrelin or XE991 into substantia nigra pars compacta results in contralateral dystonic posturing, and attenuation of catalepsy elicited by systemic administration of the D2 receptor antagonist haloperidol. Our findings indicate that the ghrelin/KCNQ signalling is likely a common pathway utilized by the nervous system.

DMT1 polymorphism and risk of Parkinson's disease.

Growing evidence suggests that iron accumulation in the substantia nigra (SN) is involved in the pathology of Parkinsons diseases (PD). Divalent metal transporter 1 (DMT1) is an endogenous transporter for ferrous iron, the levels of which are significantly increased in the SN in postmortem PD brains. To study the possible association of DMT1 gene with PD occurrence, one mutation (1303C/A) and two single nucleotide polymorphisms (SNPs) (1254T/C and IVS4+44C/A) in DMT1 gene were investigated in 192 PD patients in a Han Chinese population and 193 healthy controls by method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Direct sequencing was performed in 10% of the samples to validate the genotyping results. Our results failed to find any significant association between the tested genotypes, alleles or mutation and PD, however, a haplotype (C alleles of 1254T and IVS4+44C/A polymorphisms) occurred at greater frequencies in PD subjects compared with that of control (18.2% versus 11.4%, OR=1.72, 95% CI=1.15-2.59, P=0.01). These results suggest that CC haplotype in DMT1 gene is a possible risk factor for PD in this Han Chinese population.