Phosphorylation Of POP1 By CDK5 Mediates The Activation Of Inflammasome In Parkinson’s Disease

Objective:The inflammasome,the major immune response pathway,promotes the maturation of the inflammatory cytokines Interleukin 1β(IL-1β)and Interleukin 18(IL-18).It has been shown to participate in regulating the process of programmed cell death in many immune related diseases.Parkinson’s disease was also suggested that associated with chronic inflammation over the recent decades.However,the precise mechanism under the relationship between inflammasome and Parkinson’s disease was still not clear.Here,we tried to demonstrate the inflammasome whether exist in neurons and the detailed mechanism under the activation of the inflammasome in Parkinson’s disease.Methods:(1)To identify the key proteins of inflammasome whether expression in neurons by RT-PCR,western blot and immunofluorescence.(2)The inflammasome was activated in neurons under the treatment of classical inflammation stimulation or rotenone-induced Parkinson’s disease cell model by ELISA and western blot.(3)Human cytokines IL-1β and IL-18 in cerebrospinal fluid and serum were measure by ELISA.(4)Detection the expression of IL-1β and IL-18 by RT-PCR and western blot in substantia nigra(SN)region with MPTP-induced and transgenic animal model of Parkinson’s disease.(5)Detection with multiple levels to illustrate CDK5 participates in regulating the activation of inflammasome under the treatment with inhibitor of CDK5 or conditional knock out CDK5 in Parkinson’s disease.(6)Combination with bioinformatical prediction,western blot and mass spectrometry to confirm POP1 was the phosphorylated substrate of CDK5.(7)By using GST pull down,Bimolecular Fluorescence Complementation(BiFC)and Luciferase reporter gene assay to verify the phosphorylation of POP1 whether involved in altering the activity of NF-κB pathway and the ability of association between POP1 and ASC.Results:(1)The core molecules of the inflammasome,such as NALP3,ASC,caspase-1,and IL-1β were expressing in rat primary cultured cortical neurons.Additionally,LPS-primed ATP could robustly increase the secretion of mature IL-1βand IL-18.(2)Activated caspase-1,mature and secreted IL-1β were enhanced in a rotenone-induced PD cell model.(3)Mature IL-1β was elevated in the MPTP-induced mouse model of PD.(4)Mature IL-1β was elevated in the SNCA*A30P*A53T transgenic mouse model of PD.(5)Elevated IL-1β and IL-18 levels in the CSF of PD patients,but the serum not.The PD patients and healthy individuals were the similar age-and sex-distribution.(6)Ros,a chemical CDK5 inhibitor,powerfully blocked the acti-vation of caspase-1,the maturation and secretion of IL-1β.(7)Cdk5 deletion blocked the rotenone-induced IL-1β in neurons.(8)Knockout CDK5.strongly inhibited the activation of NF-κB pathway,but the pathway such as C/EBP.cAMP-PKA、Glucocorticoid、PKC-Ca2+、TGF-β not.(9)Scansite 3beta and GPS 2.0 showed the Thr16 site of POP1 was the substrate of CDK5.3D protein model displayed the Thr16 site located on the surface of whole protein structure and this site was evolutionarily conserved.(10)Kinase assay in vitro and MS/MS also indicated CDK5 could phosphorylated Thr16 of POP1,but T16A mutation not.(11)The phosphorylation of POP1 is involved in decreasing the ability of association between POP1 and ASC through GST pull down and BiFC.(11)TE could significantly abolished the activation of NF-κB pathway,but the TA not.Conclusion:(1)The key proteins of inflammasome were expression in neurons and it was activated in Parkinson’s disease cell model.(2)The inflammasome was also activated in Parkinson’s disease patients,MPTP-induced and transgenic animal model of Parkinson’s disease.(3)CDK5 mediates the activation of inflammasome in Parkinson’s disease.(4)CDK5 phosphorylates the Thr16 site of POP1.(5)The phosphorylation of POP1 is involved in decreasing the ability of association between POP1 and ASC,and also regulating the activity of inflammasome related cell signaling pathway of NF-κB.