The Mechanism Of DAPK1-mediated BRCC3 In The Regulation Of Inflammasome Activation In Parkinson’s Disease

Objective:(1)To investigate the protein expression levels of DAPK1 and BRCC3 in Parkinson’s Disease(PD)cell model;(2)To discuss the relationship between DAPK1 and BRCC3 in primary neurons and HEK293 cells;(3)To explore the effect of DAPK1 on the expression level of NLRP3 inflammasome complex in PD cell model;(4)In PD cell model,DAPK1 regulates the expression of NLRP3 through BRCC3.Methods:(1)In the primary cultured neurons,the PD cell model was induced by 1-methyl-4-phenylpyridine ion(MPP~+)(100m M),and the protein expression level of TH(Tyrosine Hydroxylase)was detected by Western blot.(2)PD cell model was induced by MPP~+(100m M)in primary cultured neuron cells,and the protein expressions of DAPK1 and BRCC3 were detected Western blot and cellular immunofluorescence.(3)DAPK1-Flag and BRCC3-His plasmids were co-transfected with lipofectamine in HEK293 cells,the interaction between DAPK1 and BRCC3 were detected by co-immuneprecipitation.The colocalization of DAPK1 and BRCC3 was observed by Cellular immunofluorescence in primary cultured neurons.In HEK293 cells,DAPK1 different domains plasmids(DAPK1–Flag,DAPK1-K42A–Flag,DAPK1-△KD–Flag,DAPK1-△DD–Flag,DAPK1-△Ca M–Flag)were transfected,and the interaction between DAPK1different domains and BRCC3 were detected by Western blot.(4)PD cell model was induced by MPP~+(100m M)in the primary cultured neurons,and DAPK inhibitor(100m M)was given.The influence of DAPK1 on the expression of NLRP3 inflammasome complex was detected by Western blot and cellular immunofluorescence.(5)In the PD cell model,DAPK inhibitor(100m M)was given,and the expression of BRCC3 protein was detected by Western blot and cellular immunofluorescence.SH-SY5Y cells were infected with sh BRCC3lentivirus,and the protein expression of NLRP3 was detected by Western blot.SH-SY5Y cells were infected with Sh BRCC3 lentivirus and transfected with DAPK1-Flag plasmid.The protein expression level of NLRP3 was detected by Western blot.Results:(1)Western blot results showed that the TH protein level of MPP~+group was significantly lower than that of control group,indicating that the PD cell model was successfully established.(2)In the primary cultured neurons,the expression of DAPK1 protein was the highest at 8h after MPP~+treatment,then gradually decreased;In the PD cell model,the results of Western blot and cellular immunofluorescence manifested that DAPK1 and BRCC3 expression were increased.(3)In HEK293 cells,DAPK1-Flag and BRCC3-His plasmids were co-transfected,and the results indicated that DAPK1 interacted with BRCC3.In the primary cultured neurons,cell immunofluorescence results showed that DAPK1(green)and BRCC3(red)co-localized in the cytoplasm.In HEK293 cells,different domains of DAPK1 plasmid were transfected,and the results showed that the Ca M domain of DAPK1might be the domain that interacting with BRCC3.(4)In MPP~+induced PD cell model,Western blot results indicated the expression of NLRP3,Pro-Caspase-1,ASC and IL-1βwere higher than those in the control group.The expression of NLRP3,Pro-Caspase-1,ASC and IL-1βwere decreased after the application of DAPK inhibitor.The results of cell immunofluorescence were consistent with Western blot.These results indicated that DAPK1 regulates the expression of NLRP3 inflammasome complex in PD cell model.(5)In the PD cell model,after the application of DAPK inhibitor,the expression of BRCC3 in the inhibitor group was lower than that in the MPP~+group;The expression of BRCC3 protein was consistent with Western blot.These results demonstated that DAPK1regulated the expression of BRCC3 in PD model.SH-SY5Y cells were infected with sh BRCC3 lentivirus,and the expression of NLRP3 was significantly decreased by Western blot.SH-SY5Y cell line was infected with sh BRCC3 lentivirus and transfected with DAPK1-flag plasmid 48h later,the expression level of NLRP3inflammasome was detected.Western blot results showed that BRCC3was inhibited and the expression of DAPK1 was overexpressed,but the expression of NLRP3 was increased,indicating that DAPK1 could regulate the expression of NLRP3 through BRCC3.Conclusion:Death-related protein kinase DAPK1 is correlated with deubiquitination enzyme BRCC3,and DAPK1 positively regulates the expression of NLRP3 in PD cell model through BRCC3.