The Mechanism Of CDK5-mediated BRCC3 In The Regulation Of Inflammasome Activation In Parkinson's Disease

Objective:(1)To investigate the expression of BRCC3 in PD animal model and PD cell model.(2)To investigate the effect of BRCC3 on expression of NLRP3 inflammasome and IL-1?in PD cell model.(3)The interaction between CDK5 and BRCC3 was investigated in HEK293 cells and in PD cell model.(4)To determine whether CDK5 mediates BRCC3 to regulate the expression of inflammasome in PD model.Methods:(1)C57BL/6J mice were randomly divided into the control group and the model group.MPTP was injected intraperitoneally in the model group at the dose of 25mg/kg*7 days,and normal saline was injected in the control group at the same dose.As a marker of DA,tyrosine hydroxylase(TH)in brain tissue was used to establish the animal model of PD through Western blot and neurobehavioral changes(including balance bar and climbing bar).Western blot was used to detect the BRCC3 protein level in PD animal models and PD cell models.Cell immunofluorescence was used to observe the expression of BRCC3 and intracellular localization in PD cell model.PCR was employed to detect the m RNA level of BRCC3 in PD animal model and PD cell model.(2)Studies have shown that BRCC3 activates NLRP3 inflammasome through ubiquitination in macrophages,but the mechanisn of BRCC3 in PD model has not been determined.Deubiquitination inhibitors(G5)and infection with BRCC3 sh RNA were used to knock down the expression of BRCC3 in PD cell models,Western blot was used to detect the expression of NLRP3inflammasome(NLRP3,ASC,pro-caspase-1)and IL-1?protein when the expression of BRCC3 was down-regulated.Cellular immunofluorescence was used to detect the intracellular localization and expression of NLPR3 and BRCC3 proteins in PD cells infected with BRCC3-sh RNA.(3)CDK5 inhibitor(Roscovitine)was treated in the PD cell model,and the level of BRCC3 protein was detected by Western blot.The interaction between CDK5 and BRCC3 was detected by immunoprecipitation in vivo.(4)NLRP3,pro-caspase-1,ASC,and IL-1?were detected by Western blot using Roscovitine,a CDK5 inhibitor,in a PD cell model.Roscovitine,a CDK5 inhibitor,was used to measure the NLRP3,pro-caspase-1,ASC and IL-1?proteins.CDK5/P35 and BRCC3 plasmids were overexpressed in HEK293 cells by transfection of target plasmids with liposomes.Western blot was used to detect the expression of NLRP3,pro-caspase-1 and ASC without LPS+ATP treatment or LPS+ATP treatment,respectively.ELISA was used to detect the exocytosis of IL-1?from PD model cells that decreased the expression of BRCC3 through inhibitor G5 and infection with BRCC3-sh RNA in normal neurons,PD cell models.Results:(1)Western blot results showed that TH expression was decreased and BRCC3 protein expression was up-regulated compared with the control group in the PD animal model group.The mice injected with MPTP showed significant behavioral impairment,and the experiment duration was longer than that of the control group.When MPP~+intervention was given to neurons at different times,we found that the level of BRCC3 protein increased significantly after 8h of MPP~+treatment.RT-PCR results indicate whether it is in the use of MPP~+PD cell model of building or PD animal model by intraperitoneal injection of MPTP BRCC3 m RNA level were no significant change,but then we in the MPP~+PD cell model of building detection in the different time BRCC3m RNA level,found that the MPP~+with 1 hour of neurons BRCC3 m RNA expression increased significantly,reduce after 4 hours.(2)Western blot results showed that the expression of BRCC3 was inhibited and the expression levels of NLRP3,pro-caspase-1,ASC and IL-1?proteins were decreased in the PD model.Immunofluorescence results showed that NLRP3 was located in the cell membrane and part of the cytoplasm.And when the expression of BRCC3 was knocked down,the fluorescence intensity expressed by NLRP3 was also decreased.(3)Western blot results showed that the level of BRCC3 protein was significantly increased after transfection with plasmid CDK5-HA and plasmid P35-Myc,and the expression of BRCC3 was also decreased when the CDK5was inhibited by Roscovitine in PD cell model.The co-immunoprecipitation results showed that the interaction between CDK5 and BRCC3 was found in HEK293 cells transfected with BRCC3-His plasmid and CDK5-HA plasmid.(4)The expression of NLRP3,pro-caspase-1,ASC and IL-1?were down-regulated by the application of CDK5 inhibitor Roscovitine in PD cell model.In the inflammatory model constructed by LPS+ATP in HEK293 cells,the co-transfected plasmids CDK5-HA plasmid,P35-Myc plasmid and BRCC3-His plasmids were treated with Roscovitine and the results were the same as those obtained by MPP~+.Western blot results showed that BRCC3increased the expression of NLRP3 inflammasome in the LPS+ATP-constructed inflammatory model of HEK293 cells,and inhibition of CDK5 significantly reduced the expression of NLRP3 inflammasome significantly.ELISA data showed that BRCC3 knocked down by G5 inhibitor and infection with BRCC3-sh RNA reduced inflammation in PD cell model.Conclusions:(1)The expression of BRCC3 was upregulated in PD models,MPTP Parkinson's mice model and MPP~+Parkinson's cell model was established succesfully.(2)BRCC3 regulates the expression of NLRP3 inflammasome and IL-1?protein positively in MPP~+Parkinson's cell model.(3)CDK5 can regulate the expression of NLRP3 inflammasome positively.(4)The regulatory effect of BRCC3 on NLRP3 inflammasome depends on the involvement of CDK5.(5)Inhibition of BRCC3 can reduce the secretion of IL-1?.