| Parkinson’s disease(PD), a common neurodegenerative disease, is characterized by a pathologically selective loss of dopaminergic neurons and accumulation of intracellular inclusions known as Lewy bodies(LBs) in the substantia nigra(SN). The missense mutation of alpha-synuclein(α-Syn) is the main cause of familial PD. Neurological imaging and postmortem studies have demonstrated that iron selectively accumulates in the SN of PD patients. Although the cause of selective iron deposits in PD has not been elucidated, our lab had previously demonstrated that the abnormal expressions of iron transporters in the SN, such as divalent metal transporter 1(DMT1) and ferroportin1(FPN1) were involved in the nigral iron accumulation in PD animal models. Iron regulatory protein 1(IRP1), cytoplasmic RNA-binding protein, regulates the translation or stability of m RNAs encoding proteins for iron acquisition(DMT1) and export(FPN1) by binding to iron regulatory elements(IREs)in the untranslated regions(UTRs) of m RNA. As two main pathogenic factors of PD, little is known about the relationships between α-Syn and iron. So it is of great importance to investigate the potential relationships of α-Syn aggregation and iron deposit in dopaminergic cells, which will provide new insights into the pathogenesis of PD.Recent studies have displayed that iron can promote α-Syn aggregation mainly via three ways. Firstly, it directly binds to α-Syn, and induces the conformational changes of α-Syn. Secondly, hydroxyl free radical(·OH) generated by iron through the Fenton reaction accelerates α-Syn aggregation. Furthmore, iron could also cause α-Syn aggregation by adjusting the phosphorylation of α-Syn. Meanwhile, α-Syn was regarded as a reductase of iron, which could induce ferrous iron reduced to ferric iron in cells. Besides, the upregulation of α-Syn could also influence the iron levels. After treated with iron, an increased iron level and the iron inclusions containing rich α- Syn redistributed from cytoplasm to perinuclear were observed in PC12 cells overexpressed with α-Syn or primary midbrain neurons of rats. However, the mechanism underlying how α-Syn regulates intracellular iron levels is largely unknown. In our studies, cell viability was tested by MTT mehods, mitochondrial transmembrane potential(ΔΨm) and reactive oxide species(ROS) were tested by flow cytometry, the function for ferrous iron uptake was tested by confocal microscopy, and the protein expression levels were tested by western blots. For the purpose of our study, we investigated the changes of function for ferrous iron uptake and the ferroportinin protein expression levels in wild-type α-synuclein(WT α-Syn) and A53 T α-synuclein(A53T α-Syn) transfected SH-SY5 Y cell, and then we investigate the possible mechanisim underlying α-Syn-induced iron deposits. The results are as follows:1. WT α-Syn and A53 T α-Syn over-expression for 24 hours had no effects on SH-SY-5Y cells viability when compared with the control group.2. After overexpression of vector, WT α-Syn and A53 T α-Syn for 24 hours in SH-SY5 Y cells, the changes of the iron uptake function were detected. Iron uptake function was significantly increased in WT α-Syn and A53 T α-Syn transfected SH-SY-5Y cells when compared with the control group.3. After overexpression of vector, WT α-Syn and A53 T α-Syn for 24 hours in SH-SY5 Y cells, ferrous iron(100 μmol/L) incubation for 4 hours resulted in a significant reduction in ΔΨm and increase in ROS generation in WT α-Syn and A53 T α-Syn transfected SH-SY-5Y cells when compared with the control group. ΔΨm was shown a 11.77% and 11.52% reduction, respectively(P<0.001). ROS was shown a 77.89% and 76.2% increase, respectively(P<0.001).4. After overexpression of vector, WT α-Syn and A53 T α-Syn for 24 hours in SH-SY5 Y cells, we detected the protein levels of IRP1, DMT1 and FPN1. Increased DMT1 expression levels were observed in WT α-Syn and A53 T α-Syn transfected SH-SY-5Y cells, which were shown a 56.18% and 61.80% increase, respectively(P<0.05). However, no significant changes were observed in the protein levels of IRP1 and FPN1.5. After overexpression of vector, WT α-Syn and A53 T α-Syn for 24 hours in SH-SY5 Y cells, a significant decrease in proteasome activity was detected in WT α-Syn and A53 T α-Syn transfected SH-SY-5Y cells when compared with the control group, which was shown a 69.37% and 65.35% reduction, respectively(P<0.001). After treated with 30 μM chloroquine for 24 hours, no significant changes of proteasome activity were observed in WT α-Syn and A53 T α-Syn transfected SH-SY-5Y cells when compared with that of control.6. After overexpression of vector, WT α-Syn and A53 T α-Syn and treated with 30 μM chloroquine for 24 hours, no significant changes were observed in the protein levels of IRP1, DMT1 and FPN1 in WT α-Syn and A53 T α-Syn group when compared with the control group.These results indicated that overexpression of WT α-Syn and A53 T α-Syn was able to increase the levels of DMT1, which promoted the iron intake into the cells. Increased intracellular iron levels caused the imparied mitochondrial function, resulting in a decrease in ΔΨm and increase in ROS generation. However, protein levels of IRP1 and FPN1 were unchanged, proteasome activity was significantly decreased. It was inferred that the change of DMT1 protein level was attributed to inhibition of ubiquitination proteasome induced by WT α-Syn and A53 T α-Syn overexpression, rather than the IRP1 regulation. The study clarified the potential relation of α-Syn aggregation and iron deposit in dopaminergic cells. Overexpression of α-Syn caused an increae in the function of iron uptake, aggravated dopaminergic neurons damage. And it may provide potential targets for pathogenesis research of PD. |
