Study On The Significance And Mechanism Of Overexpression α-synuclein Induced Ferroptosis On PD Models

Parkinson’s disease（PD）is a common neurodegenerative disease which is characterized by movement disorders.Its pathological features include degeneration of dopaminergic neurons in the substantia nigra pars compacta（SNpc）,abnormal aggregation ofα-synuclein and Lewy bodies formation,and iron deposition.There are more than 2 million PD patients in China.According to the report of World Health Organization,the number of PD patients will reach 5 million by 2030 with the aging of population,which accounts for more than half of the total number of PD patients in the world.Although age,environment and genetic factors are all involved in the pathogenesis of PD,the exact mechanism has not been fully elucidated.Ferroptosis is a new cell death form which was firstly reported in Cell in 2012.Its major biochemical features include iron accumulation,depletion of reduced glutathione,and accumulation of lipid peroxidation on cell membrane,and all these features have been identified in the autopsy report of PD patients.Ferroptosis has been identified in both of the1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP）induced PD mouse model and overexpressionα-synuclein（A53T）transgenic mouse model.Moreover,the ferroptosis inhibitor Ferrostatin-1（Fer-1）can significantly inhibit the toxic effect of MPTP on the dopaminergic neurons,suggesting that ferroptosis may be involved in the degeneration of dopaminergic neurons in SNpc of PD.However,the role ofα-synuclein,which is the core pathogen of PD,in the ferroptosis is still unclear.In this study,three different PD animal models were employed:（1）transgenic mice model with overexpressed A53T mitantα-synucelin（hereinafter referred to as A53T mouse）,（2）brain stereotactic injection of adeno-associated virus（AAV）that carrying the SNCA^A53Tgene into SNpc and estabilished AAV^{SNCA（A53T）}rat model(hereinafter referred to as AAV^{SNCA（A53T）}rat),（3）rotenone-induced PD mouse model（hereinafter referred to as rotenone mouse）,and the PD cell model with overexpression of SNCA^A53T（inducible PC12,i PC12）.We investigated the induction of ferroptosis by overexpressedα-synuclein in the above-mentioned PD animal and cell models.Furthermore,the potential protein kinase that may be involved in the process ofα-synuclein induced ferroptosis were screened and identified on the AAV^{SNCA（A53T）}rat by the comnination of multi-omics（proteomics,phosphoproteomics,and transcriptomics）,bioinformatics and molecular biology.The experimental results are as follows:1、In the SNpc of A53T mice,Western Blotting results showed that the level of GPX4was significantly decreased（P<0.01,n≥6）which was accompanied with the overexpression ofα-synuclein（P<0.01,n≥6）.In the AAV ^{SNCA（A53T）}rat model,four weeks after the brain stereotactic injection with AAV,immunofluorescence results showed that the expression ofα-synuclein in the SNpc was significantly increased,and Western Blotting results revealed that the level of GPX4 in the left side of SNpc is significantly lower than that in the the right side of SNpc（P<0.01,n≥6）.In rotenone mice,Perls’staining showed significant iron deposition in the SNpc and Western blot results showed that the level of GPX4 is significantly decreased in the SNpc（P<0.05）.2、IPC12 cells were treated with doxycycline（DOX,2μg/m L）for 24 hours to induce the overexpression ofα-synuclein,Western Blotting results showed that the level of GPX4was significantly decreased（P<0.05）which was accompanied with the overexpression ofα-synuclein（P<0.01）.3、In order to explore the mechanism ofα-synuclein regulating ferroptosis,the SNpc brain tissue of the AAV ^{SNCA（A53T）}rat were subjected to analyzed by the comnination of multi-omics（proteomics,phosphoproteomics,and transcriptomics）,bioinformatics and molecular biology.And 7 kinase families,including PKC,MAPK,CK2,CK1,AGC,DYRK,and CAMKL,totally 30 protein kinases with increased activity were screened,which may be involved in the process ofα-synuclein regulating ferroptosis.4、In order to verify the function of these protein kinase,overexpression ofα-synuclein were induced by DOX（2μg/m L）treatment for 24 h in the i PC12 cells,and then knockdown the expression of these 30 kinases by siRNA interference technique respectively,and finally the protein levels of GPX4 were detected by Western Blotting.The results are as follows:（1）After knockdown the expression of kinases in the DYRK family respectively,including Hipk2,Dyrk1a or Dyrk1b,the level of GPX4 was significantly decreased（P<0.05,P<0.01）;while the level of GPX4 was significantly increased（P<0.05,P<0.01）after knockdown Hipk1 and Hipk4.And the level of GPX4 had no significant change after knockdown of Dyrk3.（2）After knockdown the expression of kinases in the PKC family respectively,including Prkcz,Prkcb or Prkcd of the PKC family respectively,the level of GPX4 was significantly decreased（P<0.05,P<0.01）,while the protein level of GPX4 was significantly increased（P<0.05,P<0.01）after knockdown of Prkch or Prkci.And there was no significant change in the protein level of GPX4 after knockdown of kinases Prkca,Prkce,Prkcg or Prkcq respectively.（3）After knockdown the expression of kinases in the CK1 family respectively,including Csnk1g2 and Ttkk2 of the CK1 family respectively,the level of GPX4 was significantly decreased（P<0.05）,while the level of GPX4 was significantly increased（P<0.05）after knockdown of Csnk1g1.There was no significant change in the protein level of GPX4 after knockdown of Csnk1e,Csnk1d,Ttbk1,Vrk2 or Vrk3 respectively.（4）After knockdown the expression of Mapk15 in the MAPK family,the level of GPX4was significantly decreased（P<0.05）,while there was no significant change in the protein level of GPX4 after knockdown of Mapk4.（5）There was no significant change in the protein level of GPX4 after knockdown the expression of Csnk2a1 and Csnk2a2 in the CK2 family.（6）There was no significant change in the protein level of GPX4 after knockdown the expression of Brsk2 and Mark2 in the CAMKL family.（7）There was no significant change in the protein level of GPX4 after knockdown the expression of Stk32a in the AGC family.In the above-mentioned siRNA interfering experiments,knockdown Hipk2 revealed the most significant change in the GPX4 level.Also,the result of LDH showed that compared with the control group,the release of LDH was significantly increased after knockdown the expression of Hipk2（P<0.01,P<0.001）.In summary,significant decrease of GPX4 were observed in in the SNpc of the three PD animal models,including genetic factors(A53T mice and AAV^{SNCA（A53T）}rats)and environmental factors（rotenone mice）models,which was also observed in the PD cell model with overexpression ofα-synuclein,suggesting that overexpression ofα-synuclein can induce ferroptosis.The results of multiple omics,bioinformatics and molecular biology showed that the DYRK family（Hipk2,Dyrk1a,Dyrk1b,Hipk1and Hipk4）and PKC family（Prkcz,Prkcb,Prkcd,Prkci and Prkch）may be involved inα-synuclein-induced ferroptosis.