Activation Of NMDA Receptor Is Involved In 6-OHDA-Induced Iron Accumulation In MES23.5 Cells

Parkinson’s disease(PD) is a neurodegenerative disease mainly affecting the motor system. The pathological characteristics of PD are selective loss of dopamine(DA) neurons in the substantia nigra pars compacta(SNpc) and degeneration of DA nerve terminals in the striatum(Str). This causes major physiological disruptions in the basal ganglia motor circuit. However, the etiology of PD has not been clarified until now. Increasing evidence has confirmed that iron accumulation in the substantia nigra(SN) was an important factor contributed to the damage of DA neurons in PD. Previous studies have confirmed that dysregulation of iron transporters including divalent metal transporter 1(DMT1) and ferroportin 1(Fpn1) was involved in this nigral iron accumulation in PD. Therefore, researches on the regulating mechanisms of iron transport proteins will provide theoretical basis to elucidate the mechanisms of iron accumulation in PD.N-methyl-D-asparate(NMDA) receptor is a glutamate receptor which mediates excitatory neurotransmission and participates in the regulation of many important physiological functions in nervous system. However, excessive activation of NMDA receptors has been implicated in the neuronal degeneration due to excitatory neurotoxicity. NMDA receptors activation leads to calcium influx via its coupled calcium-gated channels, and then activates neuronal nitric oxide synthase(n NOS) to produce NO, which causes excitability toxicity. In addition, it has been reported that the activation of NMDA receptors could increase the ability of iron uptake to enhance intracellular iron levels in PC12 cells. This suggests that the activation of NMDA receptors might be involved in the iron regulation of neurons. However, the role and mechanisms of the abnormal activation of NMDA receptors in iron accumulation of DA neurons in PD are not clear. Previous studies in our laboratory showed that abnormal expression of DMT1 and Fpn1 in PD might be associated with dysregulation of iron regulatory proteins(IRPs). Thus we hypothesize that the activation of NMDA receptors might up-regulate IRPs through activating n NOS to produce NO. The up-regulation of IRPs could increase the expression of DMT1 and decrease the expression of Fpn1 through IRPs/iron responsive element(IRE) mechanism, eventually leading to intracellular iron aggregation. To verify the hypothesis mentioned above, we chose MES23.5 dopaminergic cells as the experimental neuronal model. Using flow cytometry, Western blots, real-time PCR and other methods, we detected the role of activation of NMDA receptors in iron transport, and the levels of iron transporters including DMT1 and Fpn1. Furthermore, we detected the effect of NMDA receptor inhibitor dizocilpine(MK-801) on iron transport, m RNA and protein levels of IRP1, DMT1 and Fpn1 in 6-hydroxydopamine(6-OHDA)-treated MES23.5 dopaminergic cells and the possible mechanisms.The results are as follows:1. After treatment with 50 μM NMDA for 24 hours, iron influx in MES23.5 cells was increased by 18.1%, compared with Fe2+ group(P<0.05). The mitochondrial transmembrane potential(ΔΨm) of iron-treated cells was decreased by 9.62%, compared with the control group. Pretreatment with 50 μM NMDA for 24 hours enhanced iron-induced damage of ΔΨm compared with Fe2+ group(P<0.05). 2. After treatment with 50 μM NMDA for 24 hours, the m RNA levels of DMT1+IRE were increased by 99.9%(P<0.01), the m RNA levels of Fpn1 were decreased by 29%, compared with the control(P<0.05). 3. Iron influx was increased by 21% with 10 μM 6-OHDA treatment for 24 hours, compared with the control(P<0.001). After pretreatment with MK801, iron influx was decreased by 12%, compared with the 6-OHDA group(P<0.05). 4. Iron levels were increased by 24% with 10 μM 6-OHDA treatment for 24 hours, compared with control(P<0.01). After pretreatment with MK801, iron levels were increased by 43%, compared with the 6-OHDA group(P<0.001). 5. After 10 μM 6-OHDA treatment for 24 hours, the m RNA levels of DMT1+IRE were increased by 38% and the m RNA levels of Fpn1 were decreased by 30%, compared with the control(P<0.05). After pretreatment with MK801, DMT1+IRE m RNA levels were decreased by 35%(P<0.01), Fpn1 m RNA levels were increased by 82%, compared with the 6-OHDA group(P<0.001). 6. The protein levels of DMT1+IRE were increased by 52% with 10 μM 6-OHDA treatment for 24 hours, compared with control(P<0.01). After pretreatment with MK801, DMT1+IRE m RNA levels were decreased by 36%, compared with the 6-OHDA group(P<0.01). The protein levels of Fpn1 were decreased by 41% with 10 μM 6-OHDA treatment for 24 hours, compared with the control(P<0.01). After pretreatment with MK801, Fpn1 m RNA levels were increased by 47%, compared with the 6-OHDA group(P<0.05).7. The m RNA levels of IRP1 were increased by 29% with 10 μM 6-OHDA treatment for 24 hours, compared with the control(P<0.01). After pretreatment with MK801, IRP1 m RNA levels were decreased by 27%, compared with the 6-OHDA group(P<0.05). The protein levels of IRP1 were increased by 20% with 10 μM 6-OHDA treatment for 24 hours, compared with the control(P<0.05). After pretreatment with MK801, IRP1 m RNA levels were decreased by 27%, compared with the 6-OHDA group(P<0.01). 8. The protein levels of n NOS were increased by 58% with 10 μM 6-OHDA treatment for 24 hours, compared with the control(P<0.001). 9. The m RNA levels of IRP1 or DMT1+IRE were increased by 29% or 38% with 10 μM 6-OHDA treatment for 24 hours, compared with the control(P<0.01, 0.05). After pretreatment with spermidine, IRP1 or DMT1+IRE m RNA levels were decreased by 20% or 32%, compared with the 6-OHDA group(P<0.01, 0.05). The m RNA levels of Fpn1 were decreased by 30% with 10 μM 6-OHDA treatment for 24 hours, compared with the control(P<0.05). After pretreatment with spermidine, Fpn1 m RNA levels were increased by 62%, compared with the 6-OHDA group(P<0.001).These results indicate that activation of NMDA receptors can enhance the iron transport and intensify ΔΨm damage caused by high intracellular iron. Further experimental results show that NMDA receptor inhibitor MK801 could antagonize 6-OHDA-induced iron accumulation by up-regulation of IRP1, DMT1 and down-regulation of Fpn1. In addition, results also showed that MK801 reduced 6-OHDA induced expression of n NOS. In conclusion, the activation of NMDA receptors in PD may up-regulate IRP1 through activating n NOS to produce NO, which could increase IPP1. The up-regulation of IRP1 increased DMT1 and decreased Fpn1 expression, eventually leading to intracellular iron aggregation. This study provides a new experimental basis and new strategies for the prevention and treatment of iron accumulation in PD.