The Role Of Ferroptosis In The Degeneration Of Dopaminergic Neurons And Its Underlying Mechanism In Parkinson’s Disease

Parkinson’s disease（PD）is a neurodegenerative disease with a second highest incidence of Alzheimer’s disease（AD）.The main pathological change is the selective deletion of dopaminergic（DAergic）neurons in the substantia nigra pars compacta（SNpc）,and alpha-synuclein（α-Syn）in the remaining neurons abnormal aggregation to form Lewy body（LB）,which leads to clinical manifestations such as resting tremor,muscle rigidity,bradykinesia and abnormal posture reflex.At present,the pathogenesis of PD remains unclear,genetic,environmental,aging,inflammation,oxidative stress and abnormal accumulation of iron are involved in the development of PD.A variety of cell death modes such as apoptosis,autophagy and necrosis are involved in the degenerative process of DA neurons in the PD substantia nigra.Ferroptosis is a cell death mode caused by iron-dependent oxidative damage,which is quite different from apoptosis,necrosis and autophagy in morphology,biochemistry and genetics.As an iron-dependent cell death mode,iron plays an important role in ferroptosis.Neuroimaging and postmortem studies reported that iron selective aggregation in the SNpc led to the increased iron content in residual DA neurons,suggesting that the misregulation of iron metabolism played a key factor in neuronal injury in PD.Therefore,iron selective aggregation of PD patients in the SN suggests that iron can be used as a biomarker and imaging index to reflect the progression of PD.Recent studies have found that ferroptosis is involved in the development of PD,but the role of ferroptosis in PD SN degeneration is unknown,and the relationship with other death patterns is unclear.The clarification of this issue will provide new ideas for understanding the iron aggregation mechanism and pathogenesis of PD.In the present study,MES23.5 cells with DA neurons characteristics were selected,high-iron cell models of MES23.5 cells were damaged by different concentrations of ferric ammonium citrate（FAC）,and 200μmol/L H₂O₂ was used as cell death induced by oxidative stress and 10μmol/L erastin was used as a control for ferroptosis.Transmission electron microscopy,flow cytometry and western blots were used to observe the morphology,intracellular ROS level,expression of glutathione peroxidase（GPX4）protein and lipid peroxidation malondialdehyde（MDA）content after FAC injury,to explore the phenomenon of ferroptosis.The expression of Bcl2 and Bax protein levels and the change of caspase-3 activity were detected to explore the apoptosis phenomenon in FAC injury.Pretreatment of FAC-injured MES23.5 cells with ferroptosis specific inhibitor Ferrostatin-1（Fer-1）,iron chelator deferoxamine mesylate salt（DFO）and apoptosis inhibitor ABT-263,to detecte the changes in protein levels of GPX4,Bcl2 and Bax.To further investigate the changes of MAPK and p53 signaling pathways induced by different concentrations of FAC,and to explore the possible molecular mechanism of FAC-induced ferroptosis.On the PD transgenic mice carrying the human A53T mutantα-Syn(α-SynA53T^+/+),weatern blots were used to detect changes in GPX4,Bcl2 and Bax proteins in the SN,to analyze the relationship between ferroptosis and apoptosis in FAC induced cell death.The experimental results were as follows:1.MES23.5 cells treated with different concentrations FAC（10 mg/L,50 mg/L and 100mg/L）for 24 hours,the morphological changes in the nucleus of 10 mg/L FAC and 50 mg/L FAC were not observed,however,the nucleus of the cells showed chromatin condensation and marginalization and the membrane of mitochondria appears autolysis after 100 mg/L FAC treatment.There was no change in the nucleus after treatment with the erastin,while nucleus pyknosis was observed in the H₂O₂ group.Measurements of the long-axis of mitochondria showed that mitochondrial length of the 10 mg/L,50 mg/L and 100 mg/L FAC were reduced by 5%,11% and 23%,respectively,with a statistically significant difference（P<0.05,P<0.01）.The mitochondrial length of cells in the H₂O₂ group increased by 22.6%（P<0.001）.The mitochondrial length of the erastin treated group was reduced by 10%（P<0.05）.2.MES23.5 cells were treated with different concentrations of FAC,the intracellular ROS content of 10 mg/L,50 mg/L and 100 mg/L FAC groups increased by 83.5%,126.6%and 128.2%,respectively,with a statistically significant difference（P<0.05,P<0.001）.In the erastin and H₂O₂ groups,the intracellular ROS content increased by 46.3%and 48.8%,respectively,with a statistically significant difference（P<0.01）.3.MES23.5 cells were treated with different factors for 24 hours,there was no significant change in GPX4 protein expression in the 10 mg/L FAC group（P>0.05）.The levels of GPX4 protein in the cells after 50 mg/L and 100 mg/L FAC treatment were down-regulated by 27%and 45.6%,respectively,with a statistically significant difference（P<0.01,P<0.001）.The level of GPX4 protein in the erastin treatment group was down-regulated by 43.5%（P<0.001）.There was no significant change in the level of GPX4 protein in the H₂O₂ treatment group（P>0.05）.The MDA content in the cells treated with 10 mg/L,50 mg/L and 100 mg/L FAC increased by 66.9%,102.3%and 166.8%,respectively,with a statistically significant difference （P<0.001）.The MDA content in the cells was up-regulated by 144.6%after erastin treatment（P<0.001）.The MDA content in the cells was up-regulated by 70.4% after H₂O₂ treatment.4.After treatment of MES23.5 cells with different concentrations of FAC for 24 hours,the results showed that 10 mg/L FAC had no significant effect on the intracellular Bcl2/Bax protein ratio（P>0.05）.The protein ratio of Bcl2/Bax in the cells treated with 50 mg/L and 100 mg/L FAC were down-regulated by 11%and 74%,respectively,with a statistically significant difference（P<0.05,P<0.001）.There was no significant change in the protein level of Bcl2/Bax in the erastin treatment group （P>0.05）.The intracellular Bcl2/Bax protein ratio was down-regulated by 33% after H₂O₂ treatment.Tthere was no significant effect on the activity of caspase-3 after treatment with 10 mg/L FAC（P>0.05）.After 50 mg/L and 100 mg/L FAC treatment,the activation of caspase-3 were significantly up-regulated by 172.5%and 307%,respectively,with a statistically significant difference（P<0.01,P<0.001）.Erastin treatment had no significant effect on caspase-3 activity（P>0.05）.H₂O₂ treatment increased the activity of caspase-3 by 284%（P<0.001）.5.After pretreatment of MES23.5 cells with Fer-1 for 30 min,the GPX4 protein levels in the 50 mg/L FAC and 100 mg/L FAC cells after Fer-1 pretreatment were 156% and 180%of the single FAC group,respectively,the difference was statistically significant compared to the single FAC group（P<0.001）.The Bcl2/Bax protein ratio of the 50 mg/L FAC and 100 mg/L FAC cells after Fer-1 pretreatment were 141.6% and 145.9%of the the single FAC group,respectively,the difference was statistically significant compared to the single FAC group（P<0.001）.6.After pretreatment of MES23.5 cells with DFO for 30 min,the GPX4 protein levels in the 50 mg/L FAC and 100 mg/L FAC cells after DFO pretreatment were 151% and 161%of the single FAC group,respectively,the difference was statistically significant compared to the single FAC group（P<0.01,P<0.001）.The Bcl2/Bax protein ratio of the 50 mg/L FAC and 100 mg/L FAC cells after DFO pretreatment were 141%and 250%of the the single FAC group,respectively,the difference was statistically significant compared to the single FAC group（P<0.05,P<0.001）.7.After pretreatment of MES23.5 cells with ABT-263 for 30 min,the GPX4 protein levels in the 50 mg/L FAC and 100 mg/L FAC cells after DFO pretreatment were 97%and 97.5%of the single FAC group,respectively,the difference is not statistically significant compared to the single FAC group（P>0.05）.The Bcl2/Bax protein ratio of the 50 mg/L FAC and 100 mg/L FAC cells after ABT-263 pretreatment were 131%and 146%of the the single FAC group,respectively,the difference was statistically significant compared to the single FAC group（P<0.05,P<0.001）.8.MES23.5 cells were treated with different treatment factors for 24 hours.The results showed that the phosphorylation level of Erk in the 10 mg/L FAC treatment group did not change compared with the control group（P>0.05）.Erk phosphorylation levels were down-regulated 22.8%and 42.6%in 50 mg/L and 100 mg/L FAC treated groups（P<0.05,P<0.01）.The phosphorylation level of Erk in erastin treatment group was increased by 27%（P<0.05）.The phosphorylation level of Erk in the H₂O₂ treatment group was down-regulated by 21.2%（P<0.05）.The phosphorylation level of JNK in the 10 mg/L FAC treatment group did not change significantly compared with the control group（P>0.05）.The phosphorylation levels of JNK in the 50 mg/L and 100 mg/L FAC treatment groups were up-regulated by 33.4%and 50.4%,respectively,with a statistically significant difference（P<0.05）.The phosphorylation level of JNK in the erastin treatment group was up-regulated by 62%（P<0.001）.The phosphorylation level of JNK in the H₂O₂ treatment group did not change significantly compared with the control group（P>0.05）.Different treatment factors had no significant effect on the phosphorylation level of p38（P>0.05）.9.After treatment with MES23.5 cells for 24 hours,western blots were used to detect the change in intracellular p53 proteinphosphorylation levels.The results showed that the phosphorylation levels of p53 in cells of 10 mg/L,50 mg/L and 100 mg/L FAC groups were up-regulated by 56.2%,45.7%and 43.7%,respectively,with a statistically significant difference（P<0.01,P<0.001）.The phosphorylation level of p53 in the erastin treatment group was up-regulated by 60.2%（P<0.001）.The phosphorylation level of p53 in the H₂O₂ treatment group was up-regulated by 42.1%（P<0.01）.10.On the 3 months oldα-Syn A53T^+/+mice,the level of GPX4 protein in the SN was decreased by 25.8%,compared with the control group,the difference was statistically significant（P<0.05）.There was no significant change in the protein levels of Bcl2 and Bax（P>0.05）.On the 6 months oldα-Syn A53T^+/+mice,the level of GPX4 protein in the SN was down-regulated by 33%,compared with the control group,the difference was statistically significant（P<0.05）.The protein level of Bcl2 in the SN was down-regulated by 20%,the protein level of Bax was up-regulated by 30%,and the ratio of Bcl2/Bax was decreased by 41.5%,compared with the control group,the difference was statistically significant（P<0.05）.The above results indicate that in the process of cell death caused by iron overload,low concentration of FAC can induce ferroptosis,and as the concentration of FAC increases,apoptosis occurs.Therefore,during the iron selective deposition of PD,the ferroptosis of DA neurons may exist in the early stage of PD and induces apoptosis with the increase of iron overload.Ferroptosis inhibitors can antagonize ferroptosis and apoptosis caused by iron overload,and inhibitors of apoptosis can not inhibit the occurrence of ferroptosis,indicating that the activation of ferroptosis can promotes the occurrence of apoptosis during iron treatment.This process may involved the p53signaling pathway and is not dependent on the MAPK signaling pathway.In the PD transgenic animal model,changes in ferroptosis-related proteins were first observed in the SN,and changes in apoptosis-related proteins occurred as the disease progressed,indicating that ferroptosis occurred early in PD.This study has important scientific significance for elucidating the role and mechanism of ferroptosis in DA degenerative neuropathy caused by PD SN iron accumulation,and provides a new potential drug target for the treatment of PD.