The Role Of NLRP3 Regulation In The Occurrence And Development Of Alzheimer’s Disease

Objective:Alzheimer’s disease(AD)is the most common neurodegenerative disease in the elderly.The continuous and excessive inflammatory response is one of the main factors leading to the damage of neurons in AD.Aβ plaque induces the activation of NLRP3 inflammasome in microglia which promotes microglia in proinflammatory role and reduces capacity for phagocytosis and clearing of Aβ plaque.NLRP3 protein is a rate-limiting protein in assembling NLRP3 inflammasome.However,the interacting proteins involved in the regulation of NLRP3 expression are still unclear.We screened out the target protein p62 may interacts with NLRP3 through immunoprecipitation combined with mass spectrometry method.As p62 participates in the degradation of ubiquitinized proteins through autophagy-lysosome pathway,this study aims to investigate whether p62 interacts with NLRP3 and participates in the degradation of NLRP3 protein through the autophagosomal pathway,as well as its effects on microglial polarization status and pathological changes in AD.Methods:1)In vivo experiment,in order to explore the effect of NLRP3 knockout on the development of cognitive function and pathological changes in 5XFAD mice,NLRP3 knockout 5XFAD mouse model was made to explore cognition function using open field experiment,new object recognition experiment,Y maze experiment,and Morris water maze experiment.We alos observed the morphology of microglia,neurons,deposition of Aβ plaque and ultrastructure of brain tissue.In order to explore the effect of NLRP3 expression on the microglia in AD,we stimulated the BV2 cells with LPS+Aβ1-42 oligomer as AD inflammatory model in vitro.Western Blot and ELISA were used to verify the NLRP3 inflammasome synthesized and activated in the AD inflammatory model,and flow cytometry was used to detect the polarization status of BV2 cells in the AD inflammatory model.Over/down-expression NLRP3 by lentivirus transfection in BV2 cells were made to investigate the effect of NLRP3 expression on the polarization state of microglia.2)In order to screen out the protein that regulate the expression of NLRP3 and has interaction relationship with NLRP3,immunoprecipitation(co-IP)combined mass spectrometry was used in BV2 cells of AD model in vitro.Co-IP and co-localization method were used to verify the protein interactions between NLRP3 and p62.Schematic diagram of combination interaction was made by computer.In order to investigate the effect of p62 on the regulation of NLRP3 expression,the target gene was transfected into BV2 cells by lentiviral vector,and the BV2 cells were stimulated by 3-MA to construct the autophagy lysosome inhibition model.Western blot,qRT-PCR,flow cytometry and other methods were used to verify the experimental results.3)Western blot,immunofluorescence,RNA-seq and other methods were used to explore the protein expression of apoptosis-related pathways related to p62.Results:1)In phenotypic validation,reduced NLRP3 expression decreased the M1 polarization status of microglia and improved cognitive impairment of AD by reducing Aβ plaque deposition in 5XFAD mouse model.2)Co-IP combined with mass spectrometry analysis showed that p62 might interact with NLRP3.P62 can recognize K63-ubiquitin modified NLRP3 which was verified by the results of co-IP and immunofluorescence co-localization.The possible protein interaction sites between NLRP3 and p62 and the combination mode stimulated by computer 3D model.The expression of p62 promoted the degradation of NLRP3 through the autophagy-lysosomal pathway,and reduced the formation of NLRP3 inflammasome,and slowed down the polarization of microglia to M1 type.3)The expression of autophagy pathway related proteins LC3A/B,LAMP-1 and Beclin-1 was increased in AD model in vivo and in vitro.The expression levels of p-Akt and p-mTOR were decreased in the Akt-mTOR pathway.Conclusion:P62 can recognize K63-ubiquitin modified NLRP3,degrade it through autophagy lysosomal pathway,inhibit the assembly and synthesis of NLRP3 inflammasome which reduce the polarization of microglia M1,maintain the recognition and phagocytosis ability of microglia to Aβ plaque and improve the cognitive function of AD.