Mechanisms Of DAPK1-ERK Signal In Ischemia Reperfusion Induced Neuronal Apoptosis

Part?Activation and combination of DAPK1 and ERK during neuronal ischemia reperfusion injury Objective: To investigate the activation pattern and apoptotic process of death-associated protein kinase 1(DAPK1)and extracellular signal-regulated kinase(ERK),as well as explore whether DAPK1 binds to ERK during neuronal ischemia reperfusion injury.Methods: In this part,mouse N2 a cell line was used as main object of study and primary cultured neurons along with male C57BL/6 mice were adopted as supplements,on which oxygen glucose deprivation(OGD),excitotoxicity insult and middle cerebral artery occlusion(MCAO)were performed separately as ischemia models.The experiment was parted as A,B and C.Part A was to investigate whether neuronal ischemia reperfusion could activate DAPK1-ERK,as well as the activation pattern.The experiment was divided into Non-OGD group,and OGD/reperfusion(OGD/R)1h,2h,6h,12 h,18h,24 h group,7 groups in total.Whole cell protein,cytoplasmic protein and nuclear protein were extracted from each group separately.Expression level of DAPK1,P-DAPK1,P-ERK,P-CREB,and P-MLC was measured by western blotting.Part B was to explore the neuronal apoptotic process induced by ischemia reperfusion.The experiment operation and group division were the same with part A.Total cell lysate was extracted from each group.The expression of apoptosis associated proteins including Bcl-2,Bax,cleaved-caspase3(Casp.3),and C-PARP was measured by western blotting.Cell immunofluorescence assay and quantitative analysis were performed to see the Casp.3 expression of Non-OGD group,OGD/R 12 h and 24 h group.Supernatant of cell culture media from Non-OGD group,OGD/R 1h,12 h and 24 h group was collected after centrifugation and tested for LDH content.Part C was to determine whether DAPK1-ERK combination exists during ischemia reperfusion.DAPK1-ERK co-localization of mouse N2 a cells was confirmed by carrying out double immunofluorescent staining on Non-OGD group and OGD/R 24 h group.Co-immunoprecipitation(Co-IP)was performed on mouse N2 a cells from Non-OGD group,OGD/R 1h and 24 h group to see the binding of DAPK1 and ERK.DAPK1-ERK co-localization of cortical primary neurons was confirmed by carrying out double immunofluorescent staining on solvent control group(Non)as well as Gly and NMDA administration group(Gly+NMDA).Cortices of the male C57 mice from sham-operated group(Sham),MCAO/reperfusion 0.5h,1h and 2h group were homogenized for whole cell extracts for Co-IP to determine the combination of DAPK1 and ERK.Results: The results of part A show that compared with Non-OGD group,DAPK1 activation and ERK nuclear translocation increased gradually in OGD/R group in a time-dependent manner and peaked at 24 h of ischemia reperfusion.P-MLC and P-CREB,as the substrate of DAPK1 and ERK respectively,were up-regulated accordingly.The results of part B demonstrate that compared with Non-OGD group,the ratio of Bcl-2 and Bax decreased gradually,while as the apoptotic executors,Casp.3 and its substrate,C-PARP increased gradually.Besides,LDH content of cell culture media supernatant increased gradually.Apoptotic effects induced by ischemia reperfusion peaked at 24 h.The results of part C manifest that DAPK1-ERK combination exists in the reperfusion of three different ischemia models including OGD on mouse N2 a cells,excitotoxicity insult on cortical primary neurons and MCAO on the male C57 mice.Conclusions: Neuronal ischemia reperfusion results in enhanced DAPK1 activation,promoted ERK nuclear translocation and increased cell apoptosis in a time-dependent manner.Combination of DAPK1 and ERK exists in the reperfusion of three different ischemia models including OGD,excitotoxicity insult and MCAO.Part?Effect of downregulation of DAPK1 or inhibition of its activity on ERK nuclear translocation and apoptosis of neurons subjected to ischemia reperfusionObjective: To investigate whether downregulation of DAPK1 has an impact on the apoptosis of neurons cultured under normoxia condition,as well as on the ERK nuclear translocation and apoptosis of neurons cultured under hypoxia condition.To explore the effect that DAPK1 inhibitor has on neuronal apoptosis induced by ischemia reperfusion and whether it's related to the ERK nuclear translocation.Methods: In this part,mouse N2 a cell line was used as main object of study and OGD was performed as ischemia model.The experiment was divided as part A,B and C.Part A was to investigate the impact that neurons cultured under normoxia condition have on apoptosis.Lentiviral particles containing no sequence(empty),nonspecific sequence(sc DAPK1),and DAPK1 sh RNA sequence(sh DAPK1)were used to establish stable transfected cell lines respectively.This part was to explore whether there are any significant difference among wild type N2a(WT),empty,sc DAPK1 and sh DAPK1 cell line cultured under normoxia condition.Whole cell protein was extracted from these four cell lines mentioned above for western blotting to see the downregulation effect of DAPK1 and the expression level of apoptosis related proteins including Bcl-2,Bax,Casp.3 and C-PARP.Flow cytometry was conducted to determine the apoptosis of different cell lines.Part B was to investigate the differences that these four cell lines might have on cell apoptosis and ERK nuclear translocation on 24 h of reperfusion following OGD insult.Total cell lysate,cytoplasmic protein and nuclear protein were extracted separately for western blotting to determine the ERK nuclear translocation and its substrate P-CREB,as well as the expression level of apoptosis related proteins including Bcl-2,Bax,Casp.3 and C-PARP.Cell immunofluorescence assay was performed to see the intracellular location of P-ERK in WT and sh DAPK1 cell line for further analysis on nuclear translocation rate.Flow cytometry and LDH leakage assay were carried out to determine the differences between WT and sh DAPK1 cell line on apoptosis.Part C was to determine whether administration of ATP-competitive,highly selective DAPK1 inhibitor,TC-DAPK6 could inhibit the apoptosis of neurons subjected to OGD/R for 24 hrs and its relation with ERK nuclear translocation.Following 3hrs of OGD insult,WT cell line was administrated with solvent(Veh)or TC-DAPK6 and reperfused for 24 hrs.Then total cell lysate,cytoplasmic protein and nuclear protein were extracted separately for western blotting to determine the ERK nuclear translocation and its substrate P-CREB,as well as the expression level of apoptosis related proteins including Bcl-2,Bax,Casp.3 and C-PARP.Cell immunofluorescence assay was performed to see the intracellular location of P-ERK in Veh and TC-DAPK6 group for further analysis on nuclear translocation rate.Casp.3 immunofluorescent staining and LDH leakage assay were carried out to determine the differences between Veh and TC-DAPK6 group on apoptosis.Results: The results of part A show that compared with WT,empty and sc DAPK1 cell line group,DAPK1 was significantly downregulated in sh DAPK1 group with no effect on neuronal apoptosis.No significant differences could be detected among the groups on the expression level of apoptosis related proteins and results of flow cytometry apoptotic analysis.The results of part B demonstrate that compared with WT,empty and sc DAPK1 cell line group,ERK nuclear translocation increased dramatically with higher expression of P-CREB,and Bcl-2/Bax ratio increased while apoptotic executor Casp.3 and C-PARP decreased significantly in sh DAPK1 cell line group.Cell immunofluorescence assay demonstrated that more P-ERK was located in the nucleus in sh DAPK1 group than in WT group.Flow cytometry and LDH leakage assay showed alleviated apoptosis in sh DAPK1 group than in WT group.The results of part C manifest that compared with Veh group,DAPK1 activity was significantly inhibited and Bcl-2/Bax ratio was increased,and expression of apoptotic executor Casp.3 and C-PARP as well as LDH leakage were reduced dramatically in TC-DAPK6 group.However,no significant difference could be detected between two groups on DAPK1 expression and ERK nuclear translocation.Conclusions: When cultured under normoxia condition,downregulation of DAPK1 has no impact on neuronal apoptosis.During ischemia reperfusion,downregulation of DAPK1 could promote ERK nuclear translocation significantly and thus alleviate neuronal apoptosis.Inhibiting DAPK1 activity by administrating TC-DAPK6 could attenuate neuronal apoptosis induced by ischemia reperfusion and this anti-apoptotic mechanism is independent of ERK nuclear translocation.