Effect Of Iron On Synaptic Function Of Dopamine Neurons And Its Underlying Mechanisms

Parkinson’s disease(PD)is the second most common neurodegenerative disorder.Its main clinical manifestations are bradykinesia,static tremor,rigidity,postural instability and so on.The main pathological feature of PD is the progressive degeneration of dopaminergic neurons in the substantia nigra(SN).Up to now,the etiology of PD has not been fully elucidated,but there is increasing evidence showing that excessive iron deposition in the SN might be one of the key factors in the pathogenesis of PD.The increased iron in the SN could produce reactive oxygen species and active nitrogen,promote the formation of intracellular α-synuclein,and induce the degeneration of dopamine neurons.Elevated iron might also mediate the death of dopamine neurons through ferroptosis.Growing evidence suggests that changes in synaptic function may be associated with pathophysiological processes in a variety of neuropsychiatric diseases.In autopsy studies of PD patients,reduced dendritic branches and density of dendritic spines were observed in neurons of Striatum(Str).In PD state,reduced dopamine(DA)in Str reduces the activity of the direct pathway of spinous projection neurons and cortical excitability.Moreover,the depletion of DA in PD will lead to the loss of a large number of dendrites.These results suggest that changes in synaptic function might be involved in the pathogenesis of PD.Glutamate is one of the most important excitatory neurotransmitters in the central nervous system of mammalian animals,which plays an important role in memory,synaptic plasticity and neuronal development.The changes of synaptic transmission efficiency are mainly related to the expression of N-methyl-D-aspartate receptor(NMDAR),α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid receptor(AMPAR)and the changes of excitatory postsynaptic potential or current mediated by NMDAR and AMPAR.Studies have shown that there are a large number of glutamate projection fibers in the SN and Str,and iron deposition were also detected in these areas,suggesting that there might be an interaction between them..Previous studies in our lab have shown that activation of NMDAR can decrease the expression of ferroportin1(FPN1)and increase the expression of iron importer divalent metal transport 1(DMT1),which indicates that activation of NMDAR could lead to a decrease in iron export and an increase in iron import,thus increasing intracellular iron.However,the effect of iron overload on the expression of NMDAR and related synaptic function are unclear.Therefore,in this study,iron dextran-induced iron overloaded Wistar rat model was prepared.The expression changes of synaptic function-related proteins in the SN and Str were detected by Western Blots.In addition,fluorescence microscopy,flow cytometry,Western blotting,Elisa and other techniques were used in this study to investigate the effects of ferric ammonium citrate(FAC)on expression changes of synaptic function-related proteins and the possible mechanisms in the primary cultured ventral midbrain(VM)neurons.The results are as follows:1.Wistar rats were intraperitoneally injected with iron dextran or normal saline for 4 hours,and open field test was used to test the changes of behavior.The results showed that,compared with the control group,the total movement distance of the rats(P<0.01),frequency in the center(P<0.05)and percent time spent in the center(P<0.05)in the iron dextran group were significantly decreased,and the difference was statistically significant.2.Western blotting was used to detect the expressions of dopamine transporter(DAT)and Vesicular transporter-2(VMAT-2)in the SN and Str.The results showed that compared with the control group,the expression of VMAT-2 in the SN of rats was not significantly changed in iron dextran group,and the expression of DAT was decreased(P<0.05),the difference was statistically significant.The expression of VMAT-2 and DAT in the Str did not change significantly in iron dextran group compared with the control group.3.The expressions of NMDAR1,NMDAR2 A and NMDAR2 B in the SN and Str of Wistar rats were detected by Western blotting.The results showed that compared with the control group,the expression of NMDAR1(P<0.05),NMDAR2A(P<0.05)and NMDAR2B(P<0.05)in the SN decreased in iron dextran-induced iron overloaded Wistar rat model,and the difference was statistically significant.Compared with the control group,the expression of NMDAR1(P<0.05),NMDAR2A(P<0.01)and NMDAR2B(P<0.05)in the Str of iron dextran-treated rats decreased,and the difference was statistically significant.4.Western blotting was used to detect other proteins related to synaptic plasticity,such as AMPAR1,the activity-regulated cytoskeleton associated protein(Arc)and the post-synaptic density 95(PSD-95)in the SN and Str.The results showed that,compared with the control group,the expression of AMPAR1(P<0.05),Arc(P<0.05)and PSD-95(P<0.01)in the SN decreased in iron dextran-induced iron overloaded Wistar rat model,and the difference was statistically significant.The expression of AMPAR1(P<0.05),Arc(P<0.05)and PSD-95(P<0.01)also decreased in the Str of iron dextran-treated rats,compared with the control group,and the difference was statistically significant.5.The expression of neuronsal nitric oxide synthase(nNOS)in the SN and Str of Wistar rats was also detected by Western blotting.The results showed that compared with the control group,the expression of nNOS in the SN was significantly increased in the iron dextran group(P<0.05),the difference was statistically significant,while there was no significant change in the expression of nNOS in the Str.6.Labile iron pool(LIP),reactive oxygen species(ROS)and mitochondrial transmembrane potential were detected in primary cultured VM neurons after 24 hours of FAC treatment.The results showed that the intracellular LIP was significantly increased(P<0.01),the mitochondrial transmembrane potential decreased(P<0.01),the level of ROS increased(P<0.001)significantly in the FAC group compared with the control group.7.The expression of VMAT-2 and DAT in primary cultured VM neurons were detected by Western blotting.Results showed that the expression of VMAT-2 decreased in the FAC group compared to the control group(P<0.05),the difference was statistically significant,while the expression of DAT was not significantly changed.8.The content of glutamate in the supernatant of the primary cultured VM neurons was detected by Elisa,and the expressions of NMDAR1,NMDAR2 A and NMDAR2 B in the VM neurons were detected by Western blotting.The results showed that,compared with the control group,the level of glutamate in the supernatant of FAC-treated VM neurons was significantly increased,the difference was statistically significant(P<0.05).The expression of NMDAR1(P<0.05),NMDAR2A(P<0.01)and NMDAR2B(P<0.05)in FAC-treated VM neurons decreased compared to the control,and the difference was statistically significant.9.The expressions of AMPAR1,PSD-95 and Arc in the primary cultured VM neurons were detected by Western blotting.The results showed that the expression of AMPAR1 was significantly reduced in the FAC group compared with the control group(P<0.05),the expression of Arc was also significantly decreased(P<0.05),the expression of PSD-95 was significantly increased(P<0.01),the difference was statistically significant.10.The protein expression of NMDAR(NMDAR1,NMDAR2 A,NMDAR2B)in the membrane of VM neurons was detected by Western blotting.The results showed that the expression of NMDAR1,NMDAR2 A and NMDAR2 B in the membrane of VM neurons increased in the FAC group compared with the control group(P<0.05,P<0.05,P<0.05),the difference was statistically significant.11.The expression of nNOS in primary cultured VM neurons was detected by Western blotting.The results showed that the expression of nNOS was significantly increased in the FAC group compared to the control group(P<0.05),the difference was statistically significant.These results showed that DA transportation was disturbe,the expression of NMDAR,AMPAR and the proteins related to synaptic plasticity in iron dextran-induced iron overloaded Wistar rat model.This indicated that iron overload may play a role in the pathogenesis of PD by affecting DA homeostasis,excitatory synaptic transmission and synaptic function.Further in vitro experiments confirmed that high level of iron can increase intracellular free iron and ROS production and reduce mitochondrial transmembrane potentia of primary cultured VM neurons.Moreover,the expression of NMDAR,especially NMDAR2 B on the cell membranes was increased by increased expression of PSD-95.This might lead to the excitotoxicity and the damage of VM neurons.In addition,iron overload can also reduce the expression of synaptic plasticity related proteins,suggesting that high iron may be involved in the functional regulation of dopamine neurons by affecting synaptic plasticity.The results of this study will help to further understand the regulatory effect of iron on synaptic function and the possible mechanism.This will provide an experimental basis for the study of the relationship between iron deposition and synaptic function.