The Effect Of Alpha-synuclein Knockout On Iron Accumulation In The Substantia Nigra And Its Mechanism

Parkinson’s disease（PD）is a common neurodegenerative disease of the elderly.The major pathological feature of PD is the selective loss of dopaminergic neurons in substantia nigra（SN）,eventually leading to depletion of DA in the striatum.The pathogenesis of PD mainly includes oxidative stress,abnormal accumulation of iron in SN,inflammation,abnormal protein aggregation,apoptosis and mitochondrial dysfunction.With the further development of social aging,PD has become the second killer of nervous system diseases after Alzheimer’s disease（AD）.Although PD is a chronic disease,which has no acute lethal effect,it seriously affects the quality of life of patients and places a heavy burden on families and society.Alpha-synuclein（α-syn）is a soluble protein abundantly expressed in the central nervous system.It is closely related to the pathogenesis and the dysfunction of PD and is the main component of lewy bodies（LBs）.Recent studies have found thatα-syn is a ferrireductase that can reduce Fe³⁺to Fe²⁺.The cytotoxicity of iron plays a key role in the paththogenesis and progression of PD.Iron accumulation in the SN is involved in PD.Previous studies have shown that（divalent metal transporter 1,DMT1）,a key iron importer,is upregulating,which can account for iron accumulate in PD.So,we speculate thatα-syn and DMT1 may synergistically participate in cellular iron uptake in the SN.To investigate the changes of iron ferrireductase activity inα-syn knockout（KO）mice and its effect on iron metabolism in SN,we measured the changes of ferrireductase activity using Microplate reader.We detected changes of iron content as well as the Fe²⁺/Fe³⁺ratio in the midbrain of KO mice using ICP-MS and iron staining techniques.We detected the expressions of DMT1 and TH in SN by Western blots.In addition,we silenced thea-syn gene in SH-SY5Y cells and observed the iron uptake of SH-SY5Y using a laser confocal microscope.The results are as follows:1.No matter the mice were fed with 3%carbonyl iron powder or normal diet,the expression of DMT1 decreased significantly in the KO group compared with the WT group in the SN（P<0.01,P<0.01,n=6）.No matter in KO or WT mice,there was no difference of DMT1 expression between normal diet and iron-supplied diet group（P>0.05,n=6）.2.ICP-MS was used to detect the iron content in the SN of mice.Iron content of WT mice in the iron-supplied diet group was significantly higher than that in the normal diet group（P<0.001,n=6）,and there was no differences between normal and iron-supplied diet of KO mice（P>0.05,n=6）.Iron content was increased in WT mice compared with the KO mice of iron-supplied diet group（P<0.01,n=6）,and there was no differences between WT and KO of normal diet of group（P>0.05,n=6）.3.The Fe²⁺and Fe³⁺were significantly increased in the iron-supplied diet compared with the normal diet of WT mice（P<0.01,P<0.001,n=6）,and there was no differences between the iron-supplied diet and the normal diet in the KO mice（P>0.05,n=6）.The Fe²⁺and Fe³⁺were increased in WT mice compared with the KO mice of iron-supplied diet group（P<0.001,P<0.001,n=6）,and there was no differences between WT and KO of normal diet of group（P>0.05,n=6）.The Fe²⁺/Fe³⁺was significantly increased in the iron-supplied diet compared with the normal diet of WT mice（P<0.001,n=6）,and there was no differences between the iron-supplied diet and the normal diet in the KO mice（P>0.05,n=6）.The Fe²⁺/Fe³⁺was increased in WT mice compared with the KO mice of iron-supplied diet group（P<0.001,n=6）,and there was no differences between WT and KO of normal diet of group（P>0.05,n=6）.4.The ferrireductase activity of SN in mice was significantly decreased in the KO group compared with the WT group（P<0.001,n=6）.Ferrireductase activity was different at different time points（P<0.05,P<0.001,n=6）.5.The expression of TH in iron-supplied diet group significantly decreased compared with the normal diet group of WT mice（P<0.01,n=6）,and there was no differences between normal and iron-supplied diet of KO mice（P>0.05,n=6）;The expression of TH was decreased in KO mice compared with the WT mice of normal diet group（P<0.01,n=6）,the expression of TH was decreased in KO mice compared with the WT mice of iron-supplied diet group（P<0.05,n=6）.6.The ferrireductase activity significantly decreased inα-syn gene silencing group compared with the control group（P（27）0.001,n=6）.Ferrireductase activity was different at different time points within group comparison（P（27）0.01,n=6）.7.The expression of DMT1 significantly decreased inα-syn gene silencing group compared with the control group（P<0.05,n=6）.8.There was a time-dependent intracellular fluorescence quenching with 1 mmol/L ferrous iron perfusion,indicating the increased intracellular iron level.Fluorescence quenching was slower inα-syn gene silencing group compared with control group detected at 72 h after infection with lentivirus,indicating that the cells can reduce the influx of iron after silencing theα-syn gene（P<0.05,n=6）.The fluorescence intensities were different at different time points within group comparison（P<0.05,P<0.01,n=6）.The results suggest that:Because of the lack ofα-syn,KO mice exhibited a decrease in the activity of ferrireductase.The iron content and Fe²⁺/Fe³⁺ratio in the SN decreased,and the expression level of the DMT1 decreased.The above factors led to a decrease in the level of oxidative stress in SN,which reduced the toxic effect of iron on DA neurons.We confirmed the above results by silencing thea-syn gene expression in SH-SY5Y cells.α-syn silencing decreased iron uptake in the cells.The experimental results provide experimental basis for the involvement ofα-syn protein in the pathogenesis of PD,and provide new idea for the prevention and treatment of PD.