Effect Of Iron Overload On The Expression Of DAT And VMAT-2

Parkinson's disease?PD?is a common neurodegenerative disease,one of its main pathological features is the selective degeneration of dopaminergic neurons.The products of dopamine?DA?metabolic process could damage cells,such as hydrogen peroxide,hydroxyl radicals and some quinone compounds.Studies have shown that the abnormal deposition of iron may cause the death of dopaminergic neurons,iron and DA are a toxic couple.H₂O₂ produced by DA metabolism can react with Fe?II?to generate hydroxyl radicals through Fenton reaction,induced the synthesis of DNA adducts,lipid peroxidation,loss of plasma membrane integrity,and apoptosis.Dopamine transporter?DAT?and vesicular monoamine transporter-2?VMAT-2?are two important proteins responsible for DA transport.VMAT-2 uptake DA into synaptic vesicles,and then releases them into the synaptic cleft,acting on the corresponding receptors;DA is taken up by DAT from synaptic cleft into presynaptic neurons,DAT and VMAT-2 together maintain the DA levels homeostasis in the presynaptic neurons and synaptic cleft.Studies have shown that the expression of VMAT-2 and DAT proteins was significantly decreased in the substantia striatum system,and DA uptake capacity of isolated synaptic vesicles from PD patients was decreased about 87%.Iron deposition in substantia nigra pars compacta?SNpc?were found in PD patients and PD animal models,while the expression of DAT and VMAT-2 has also decreased significantly.Deferoxamine?DFO?has been shown to cause decreased stability of DAT mRNA and promote membrane endocytosis,reducing DAT expression on the cell membrane in N2a cells,indicating that intracellular iron content may affect DAT protein expression.However,the effect of intracellular iron overload on the expression of DAT and VMAT-2 has not been reported.In this study,we observed the protein expression of DAT and VMAT-2 in olfactory bulbs?OB?,substantia nigra?SN?,and striatum?Str?in Wistar rat with intraperitoneal injection of dextran iron,as well as DAT and VMAT-2 proteins expression in SN and Str in C57BL/6 mouse with high-iron diet.We also explore the effects of intracellular iron levels on the expression of DAT and VMAT-2 mRNA and protein in a PC12 dopaminergic cell,and observed the effects of intracellular iron overload and DA on cell activity and production of reactive oxygen species?ROS?.The results were shown as follows:1.Wistar rats were intraperitoneally injected with dextran iron or normal saline for 1-4weeks,and the changes of DAT and VMAT-2 proteins expression in the OB,SN and Str were detected by Western blot.The results showed that compared with the control group,the expression levels of DAT and VMAT-2 proteins in the OB were decreased by 29.05%and 26.42%,respectively,at 1-week?P<0.001,P<0.001?.The ratio of VMAT-2 to DAT protein expression was decreased by 21.78%at the second week?P<0.05?.The protein expression in other time groups did not change significantly.The DAT and VMAT-2proteins,as well as the ratio between the two proteins did not change in the SN.The expression level of VMAT-2 protein was decreased by 32.44%?P<0.01?,the ratio of VMAT-2 and DAT proteins expression was decreased by 28.22%in the Str at the second week?P<0.01?.The ratio of VMAT-2 to DAT proteins expression was decreased by 18.78%at the 4-week?P<0.05?;the protein in other time groups did not change significantly.2.Wistar rats were intraperitoneally injected with dextran iron or normal saline for 1-4weeks.Compared with the control group,dihydroxyphenylacetic acid?DOPAC?content was decreased by 38.89%?P<0.05?in the Str of rats at 2-week.No significant change in DOPAC content at other times,and DA and homo vanillic acid?HVA?content did not change significantly within 1-4 weeks.3.C57BL/6 mice were fed with 3%carbonyl iron formulated food for 2 weeks to 1 month.Compared with the control group,the DAT protein expression in SN were increased by34.66%?P<0.001?,VMAT-2 protein expression did not change significantly in mice with high-iron diet for 2 weeks;VMAT-2 protein expression level in the Str was decreased by23.56%?P<0.001?in mice with high-iron diet for 1 month,and the DAT protein expression did not change significantly.4.PC12 cells were treated with ferric ammonium citrate?FAC?,DFO and DA for 24 h.Compared with the control group,the expression of Ferritin protein in the FAC treated group and DA treated group were increased by 4.33 times?P<0.001?and 79.84%?P<0.01?,respectively,the ferritin protein in the DFO treated group was decreased by 36.66%?P<0.05?.5.PC12 cells were treated with FAC,DFO and DA for 24 h.Compared with the control group,the DAT mRNA expression level was decreased by 21.62%?P<0.05?in the DFO treated group.There was no significant change in FAC and DA group.No significant change in VMAT-2 mRNA expression levels in FAC,DFO,and DA group.The DAT protein expression was increased by 32.66%?P<0.001?,and the VMAT-2 protein expression was decreased by 31.92%?P<0.01?in the FAC treatment group;the DAT protein expression in the DFO treatment group did not change significantly,and the VMAT-2 protein expression was increased by 22.72%?P<0.01?.The DAT protein expression in the DA treatment group was decreased by 11.72%?P<0.01?,and there was no significant change in VMAT-2 protein expression.6.CCK-8 results showed that the cell viability was decreased by 38.17%?P<0.001?and45.71%?P<0.001?respectively,in PC12 cells treated with FAC for 4 h or 24 h.When PC12cells were treated with DA for 24 h,the cell viability was increased by 30.09%?P<0.001?.The cell viability of the FAC?4 h?+DA?24 h?group had no significant change compared with the control group.The intracellular ROS levels were measured by flow cytometry,and the results showed that compared with the control group,intracellular ROS levels were increased by 80.37%?P<0.05?and 72.96%?P<0.01?in the FAC?4 h?and FAC?24 h?groups,respectively,and the intracellular ROS levels were decreased by 50.73%?P<0.01?in the DA?24 h?group.The intracellular ROS level in the FAC?4 h?+DA?24 h?group was not significantly different from the control group.Intracellular ROS levels were unchanged in VMAT-2 reversible inhibitor TBZ?24 h?treated group,and TBZ have no effect on the increased ROS levels in the FAC group and the decreased ROS levels in the DA group.The above results showed that the expression of DAT and VMAT-2 protein were decreased significantly in the OB of dextran iron treated rats.The expression of VMAT-2protein in the Str was decreased significantly at 2 weeks,however,DAT and VMAT-2protein expression did not change in the SN.There was no significant change in DA content in the Str of iron dextran treated rats.In the high-iron diet mouse model,the DAT protein expression in the SN increased significantly at 2 weeks,and the VMAT-2 protein expression in the Str decreased significantly at 1 month.These results suggest that the regulation of DAT and VMAT-2 protein expression by peripheral iron overload in vivo needs to be further explored.In PC12 dopaminergic cell lines,iron overload can induce increased DAT expression and reduced VMAT-2 expression,and iron chelator?DFO?can induce increased VMAT-2 protein expression,suggesting that intracellular iron levels can regulate DAT and VMAT-2 protein expression.FAC-induced decreased cell activity and increased ROS levels can be completely blocked by co-treatment of DA,indicating that the cooperative actions of FAC and DA exert protective effects,which might be due to interactions between iron and DA.These data explored the effects of iron overload on the expression of DAT and VMAT-2 in animal models and cell models,as well as the effects of iron overload and dopamine on intracellular ROS levels.The results will help to elucidate the role of increased intracellular iron levels in regulating dopamine metabolism,and provide an experimental basis for the relationship between iron deposition and DA metabolism in PD.