Study On The Role And Mechanism Of Anti-ASC Antibody In Alzheimer’s Disease

Background and ObjectiveAlzheimer’s disease(AD)is an aging-related neurodegenerative disease characterized by progressive dementia.The excessive production and abnormal deposition of amyloid-beta(Aβ)in the brain is the key pathological process of AD.Apoptosis-associated speck-like protein containing a CARD(ASC)is expressed in microglia,dendritic cells,macrophages and other immune cells.ASC plays an important role in the regulation of Aβ aggregation and deposition.In AD,Aβ deposition is accompanied by autoimmune reaction.After microglia activation,ASC transfers from the nucleus to extracellular and aggregates to form ASC specks.ASC specks quickly bind to Aβ,promote the spread of Aβ lesions,increase the formation of Aβoligomers and Aβ deposition,and enhance their neurotoxicity.In addition,Aβ can promote the expression of ASC,which forms a positive feedforward cycle that promotes each other.Therefore,blocking the co-deposition of ASC specks and Aβ is of great significance to reduce the accumulation and deposition of Aβ in AD.In this study,we used the N-terminal and Cterminal antibodies of ASC to interfere with APP/PS1 mice to explore the behavioral and pathological changes,which is of certain significance to reveal the new pathogenesis of AD and provide a new target for the prevention and treatment of AD.Materials and methods1.Effect of anti-ASC antibodies on cognitive function of APP/PS1 miceWe used brain stereotaxic technique to inject anti-ASC antibody and control antibody into the lateral ventricle of 8-month-old APP/PS1 mice.The groups are as follows: Tg Control group injected with homotype control antibody rabbit-derived Ig G,Tg ASC-N group injected with ASC N-terminal antibody SAB4501315,Tg ASC-C group injected with ASC Cterminal antibody ab155970,5μl every time,once a week,one month later,we added monthold sex-matched WT mice for Y-maze and field experiments.2.Effect of anti-ASC antibodies on Aβ deposition in the brain of APP/PS1 mice(1)Immunohistochemical staining: the area and number of total Aβ plaques in the brains of Tg ASC-Control,Tg ASC-N and Tg ASC-C mice were detected by 6E10 antibody against human Aβ,and the positive area and number of compact Aβ plaques in the brains of the three groups were further detected by thioflavine staining.(2)Western blotting: the levels of Aβ and APP metabolites in mouse brain were detected with 6E10,171610 and 22C11 antibodies,β-shearing enzyme and α-shearing enzyme were detected with BACE1 and ADAM1 antibody,Aβ-degrading enzyme was detected with antiIDE and NEP,and Aβ-transporting enzyme level was detected with LRP1 and RAGE antibody.3.Effect of anti-ASC antibodies on phosphorylated tau in the brain of APP/PS1mice(1)Immunohistochemical staining: PT231 antibody was used to detect the area of p Tau231 positive neurons in the brain of three groups of mice.(2)Western blotting: pS396,pT231,pS199 and pT181 antibodies were used to further detect the content of Tau protein at multiple phosphorylation sites in the mouse brain,.4.Effects of anti-ASC antibodies on astrocyte and microglia in the brain of APP/PS1miceImmunohistochemical staining and analysis: We used astrocytes specific antibody GFAP and microglial specific antibody IBA1 to detect the proportion of positive area of activated astrocytes and microglia in mouse brain.5.Effects of anti-ASC antibodies on the levels of neuron and synaptic associated protein in APP/PS1 mice(1)Immunofluorescence co-staining: the proportion of positive area of neurons in the brain of mice was analyzed by fluorescence co-staining with Map-2 and Neu N antibodies.(2)Western blotting: PSD95,SYN1,SNAP25 and VAMP1 antibodies were used to further detect the level of synaptic-related proteins in mouse brain homogenate.Results1.Anti-ASC antibodies improve cognitive impairment in APP/PS1 mice1.1 Y maze test: In the alternation experiment,the percentage of alternation in the Tg ASC-N and Tg ASC-C mice was higher than that in the Tg Control group,which suggested that the APP/PS1 mice treated with ASC antibody had better spatial memory ability.1.2 Open filed test: Compared with Tg Control mice,Tg ASC-N and Tg ASC-C mice showed more attachment times and central area/peripheral area time ratio.This indicated that the exploratory behavior of APP/PS1 mice treated with ASC antibody increased while the anxiety behavior decreased.2.Anti-ASC antibodies attenuate Aβ deposition in the brain of APP/PS1 miceCompared with Tg control mice,Tg ASC-N and Tg ASC-C mice displayed a lower 6E10 positive area fraction and total number of plaques in the hippocampus and neocortex,respectively.The Thioflavine staining showed that Tg ASC-N and Tg ASC-C mice had a lower Aβ positive area fraction than Tg control mice in the neocortex,the number of hippocampus plaques in Tg ASC-C group was lower than that in Tg control group,and there was no significant difference in the number of neocortex plaques among the three groups.Western blotting analysis showed that the level of Aβ in the brain of Tg ASC-N and Tg ASCC mice was lower than that of Tg Control mice,but there was no significant difference in the expression of full-length APP,s APPα,s APPβ,CTFα and CTFβ among the three groups.Taken together,our findings showed that Anti-ASC antibodies attenuate the brain Aβ burden of APP/PS1 mice.3.Anti-ASC antibodies had no significant effect on the levels of APP splicing enzymes and Aβ metabolism-related enzymes in the brain of APP/PS1 mice.Western blotting analysis showed that there was no significant difference in the levels of ADAM10,BACE1,IDE,NEP,LRP1 and RAGE in the brain of Tg Control,Tg ASC-N and Tg ASC-C groups.4.Anti-ASC antibodies reduce the level of pT231 in the brain of APP/PS1 mice(1)pTau231 immunohistochemistry: compared with Tg Control mice,the percentage of p Tau231 positive neurons in hippocampal CA1 and locus coeruleus decreased in Tg ASC-N and Tg ASC-C groups.(2)Western blotting: compared with Tg Control mice,the content of p Tau231 protein in Tg ASC-N and Tg ASC-C groups decreased,but there was no significant difference in p S396,p S199 and p T181.5.Anti-ASC antibodies inhibit the activation of astrocytes and microglia in the brain of APP/PS1 mice.(1)GFAP immunohistochemical staining: compared with Tg Control mice,Tg ASC-N and Tg ASC-C mice showed lower proportion of astrocyte positive area in cortex and hippocampus.(2)IBA1 immunohistochemical staining: compared with Tg Control mice,the cortex of Tg ASC-N and Tg ASC-C groups showed lower proportion of microglia positive area,and the hippocampus of Tg ASC-C showed lower proportion of microglia positive area.6.Anti-ASC antibodies reduce neuronal loss in the brain of APP/PS1 mice.Compared with Tg Control mice,the proportion of Map-2 and Neu N positive area in hippocampus of Tg ASC-N and Tg ASC-C mice increased,the level of SYN1 in the brain of Tg ASC-C mice increased,and the brain SYN1 of Tg ASC-N mice showed an upward trend.Therefore,it was suggested that anti-ASC antibody might reduce the loss of neurons in the brain of APP/PS1 mice.ConclusionOur study found that both N-terminal antibody SAB4501315 and C-terminal antibody ab155970 of ASC could reduce the deposition of Aβ and abnormal phosphorylation of tau protein in the brain of APP/PS1 mice,inhibit the activation of microglia and astrocytes,and may reduce the loss of neurons in the brain of APP/PS1 mice and save the cognitive impairment of APP/PS1 mice.Therefore,immunotherapy for ASC may be a potential treatment strategy for AD.