Study Of Aberrant P53 SUMOylation In Senescence And Alzheimer's Disease

Background: At present,China is stepping into an aging society,and the number of disabled or semi-disabled elderly with neurodegenerative changes will increase sharply,which will bring great burden to the society and family.Therefore,the research on how to delay aging and prevent neurodegenerative diseases caused by aging has become a top priority.Alzheimer's disease(AD)is the most common type of neurodegenerative disease,accounting for about 60-70% of all dementia cases.AD is clinically characterized by memory loss and progressive neurocognitive impairment,which can subsequently lead to speech and visual impairment,often accompanied by behavioral disorders,resulting in loss of the ability to live independently.The etiology of AD involves the combined action of individual genetics and living environment,and the pathogenesis of AD is not fully understood.Exploration of the new pathogenesis of AD is of great significance for drugs development and its treatment.Cell senescence is a complex process regulated by multi-factor stress signals.As an important tumor suppressor gene,p53 is also a key molecule involved in the regulation of aging and senescence.It can mediate cell cycle arrest and lead to the cell senescence by regulating the downstream pro-senescence molecule p21.In addition,activation of p53 affects the aging of organs and participates in the pathological process of AD by regulating reactive oxygen species,m TOR signal,mitochondria and autophagy,but its molecular mechanism has not been fully elucidated.SUMOylation is an important type of posttranslational modifications(PTMs).It can regulate the function of the protein by changing the subcellular localization,stability,transcriptional regulation and proteinprotein interaction.Current studies have shown that SUMOylation is involved in physiological processes such as neuronal differentiation,synaptic formation,transmitter release,vesicle circulation and cell survival,and it also plays an important role in the pathological process of AD by mediating the SUMOylation of key pathogenic molecules of AD.Some studies have reported that p53 can be SUMOylated,but whether the SUMOylation of p53 in the nervous system mediates the senescence of nerve cells and the occurrence of neurodegeneration has not been studied.Therefore,research on the molecular mechanism of p53 SUMOylation involved in aging and the pathogenesis of AD can help further study the occurrence and development mechanism of neurodegenerative diseases.Objective: Study the correlation between aberrant p53 SUMOylation in senescence and AD,and the mechanism by which p53 SUMOylation promotes the dissociation of the p53/SET complex,resulting in abnormal translocation of SET and its retention in cytoplasm,leading to abnormal hyperphosphorylation of Tau and neurodegeneration.It provides new clues for the pathogenesis of AD in the aging process and provides new molecular targets for clinical drug screening.Methods: The expression levels of p53 in brain slices of young 2-month-old mice and aged 18-month-old mice were analyzed.Immunohistochemistry was used to observe the expression distribution of p53 in brain slices.The expression levels of p53 protein,SUMO1 and SUMO2/3 in hippocampal tissues were detected by Western blot.The immunoprecipitation was used to compare the SUMOylation level of p53 in the hippocampus of young and aged mice.The primary neurons of rats were isolated and treated with A?.Cell survival was detected by CCK8.The expression levels of SUMO1 and SUMO2/3 were detected by Western blot,and the expression levels of p21 were detected.?-gal staining and Western blot were used to detect the expression of p16 and analyze the senescence of cells.N2 a cells were treated with A?,and the senescence of cells was analyzed by Western blot.The level of p53 SUMOylation was detected by immunoprecipitation.In HEK293 cells and N2 a cells,Flag-p53 plasmid and MycSUMO1 plasmid were transfected simultaneously.The SUMOylation level of p53 was detected by immunoprecipitation,and the phosphorylation level of Tau and cell senescence level were detected by western blot.The plasmid Flag-p53 K386 R was constructed,N2 a cells was transfected with Flag-p53 or Flag-p53 K386 R plasmid,and then treated with A?.The effect of down-regulating p53 SUMOylation on A?-induced Tau hyperphosphorylation and cell senescence was detected.The binding level of p53 with SET was detected by immunoprecipitation.The cytoplasmic and nucleus part of cells was isolated,and the effect of p53 SUMOylation on the SET cytoplasmic retention was detected.Cells were treated with cycloheximide(CHX)to detect the effect of p53 SUMOylation on its degradation.APP/PS1 mice were stereotaxically injected with p53 or SUMOylation defective p53 K386 R virus in hippocampal CA1 region,or injected with Vector virus as control.After 4 weeks of virus expression,the mice behavior was detected by Open field test,Elevated plus maze test,Novel object recognition test and Morris water maze test.Western blot assay was used to detect the expression of presynaptic and postsynaptic proteins in the hippocampus of mice.Golgi staining was used to detect the changes in the number of synapses in the CA1 region of the hippocampus of mice.q PCR was used to detect the expressions of inflammatory cytokines in the hippocampus of mice.RNA-seq was used to detect the effect of aberrant p53 SUMOylation on gene expression in the hippocampus.Results: Compared with young mice,the expression level of p53 in the hippocampus of aged mice was significantly up-regulated,and the SUMOylation level of p53 was increased.In the primary neurons treated with A?,the expression level of SUMO1 was up-regulated,the expression levels of p53 and SUMO2/3 were not changed,the modification level of p53 SUMOylation was significantly increased,and the cells showed senescence phenotype.In A? treated N2 a cells,the phosphorylation level of p53 at Ser15 was upregulated,the modification level of p53 SUMOylation was significantly increased,as well as the expression levels of p21 and p16,and the cells showed senescent phenotype.The co-transfection of p53 and SUMO1 in N2 a cells increased the phosphorylation level of Tau and cell senescence.Transfection of p53 but not p53 SUMOylation defective Flag-p53 K386 R plasmid promoted A?-induced Tau hyperphosphorylation,p53 phosphorylation at Ser15,and cell senescence.When N2 a cells stimulated by A?,over expression of Flag-p53 decreased the binding level of p53 with SET,and the binding level of p53 with SET was increased after down-regulation of p53 SUMOylation.SUMOylation of p53 reduced the distribution of SET in the nucleus and increased its distribution in the cytoplasm.CHX results showed that p53 degradation was not affected by its SUMOylation.The behavioral results showed that compared with the control group,the anxiety level and cognitive impairment was significantly increased in mice injected with p53 virus,and the mice injected with SUMOylation defective p53 K386 R virus significantly ameliorated the anxiety level and cognitive ability.Western blot assay and Golgi staining assay showed that the number of synapses and the expression of synaptic protein was decreased in the hippocampus region of mice injected with p53 virus,while the number of synaptic protein and the expression of synaptic protein in hippocampus region of mice injected with SUMOylation defective p53 K386 R virus were not impaired.The q PCR results showed that the expression of inflammatory cytokines in the hippocampus of mice was inhibited by the down-regulation of p53 SUMOylation.RNA-seq showed that down-regulation of p53 SUMOylation alleviated the neurogenesis disorder.Conclusion: In the hippocampus of aged mice,p53 SUMOylation was increased.The primary neurons and N2 A cells were treated with A?,the key pathogenetic molecule of AD,which induced an increase of p53 SUMOylation,and the cells showed senescent phenotype.A?-induced hyperphosphorylation of Tau was promoted by the p53 SUMOylation.By promoting the dissociation of p53/SET complex,p53 SUMOylation induced the entry and retention of PP2 A inhibitor SET into the cytoplasm,promoting the hyperphosphorylation of Tau.p53 SUMOylation accelerated cognitive impairment and synaptic damage in APP/PS1 mice,and downregulated p53 SUMOylation alleviated cognitive dysfunction and neuroinflammation in APP/PS1 mice.The results of this study preliminarily confirmed that the p53 SUMOylation promoted senescence,induced Tau hyperphosphorylation and cognitive impairment,and provided new clues for the pathogenesis of AD.Background: Alzheimer's disease(AD)is a neurodegenerative disease characterized by memory loss and progressive cognitive impairment.Currently,the global prevalence of dementia is about 47 million,and the number is predicted to be more than tripled by 2050.Two important pathological features of AD are senile plaques(SP)composed mainly of amyloid-?(A?)and neurofibrillary tangles(NFTs)made of hyperphosphorylated Tau.The imbalance between the production and clearance of A?42 and its related A? peptides leads to abnormal accumulation of extracellular A?,which can lead to long-term synaptic and memory impairment in the hippocampus.Moreover,exposure to A?42 also trigger hyperphosphorylation of tau at AD-relevant epitopes in cultured neurons.Beta-site APP cleaving enzyme 1(BACE1),a single transmembrane aspartyl-protease,which is responsible for the cleavage of amyloid precursor protein(APP)to produce APP?,is the rate limiting enzyme in the amyloidogenic APP processing and thus plays an important role in A? generation.Therefore,inhibition of BACE1 has become a promising strategy to reduce the A? toxicity in the AD therapeutics.As the etiology of AD is multifactorial,natural products which usually contain numerous ingredients with synergic effect would be beneficial for the treatment of the condition,for their bioavailable peculiarity and relatively less side-effects.Codonopsis pilosula(CP)is a kind of Chinese herbal medicine with long history,which has complex component including polysaccharides,sesquiterpenes,saponins,polyphenolic glycosides,polyacetylenes,alkaloids,essential oils,and phytosterols.The CP polysaccharides(CPPs)are active compounds extracted from CP,which have been identified to possess multiple pharmacological functions such as antitumor,antimicrobial,antioxidant,and immunoenhancing properties.It has been reported that heparan sulfate polysaccharides interact with BACE1 and regulate its APP cleaving activity,mainly by blocking access of substrate to the active site.Previous studies have also provided evidence that natural polysaccharides mitigated cognitive deficits in animal models of AD.However,whether CPPs alleviate AD pathological process,especially as anti-A? accumulation is yet to be known.Objective: Our previous work showed that CPPs alleviated Tau hyperphosphorylation and cognitive impairment in over-expressing Tau mice.In this study,we investigated the effects of CPPs treatment on A? toxicity and cognitive impairment in APP/PS1 mice.Methods: APP/PS1 mice were given different concentrations of CPPs by intragastric administration for 1 month,and the anxiety-like behavior and cognitive function of the mice were detected by behavioral test.Western blot was used to detect the expression of synapse-related proteins and A? pathway related proteins in the hippocampus of treated mice,the enzyme activity of BACE1 was detected by fluorometric Assay.Cell model N2A-APP and APP transfected HEK293 cells were treated with different concentrations of CPPs in vitro to detect the expression level and activity of BACE1.The effect of CPPs on BACE1 activity was detected by co-incubation with recombinant BACE1 enzyme at different concentrations in vitro.Results: In this study,we found that CPPs significantly alleviated the cognitive impairment of APP/PS1 mice after 1 month of intragastric administration,recoverd the loss of synaptic plasticity in the hippocampus,and increased the expression of synaptic related proteins synaptotagmin and PSD95.In addition,the activity of BACE1 enzyme and the expression of A?40 and A?42 in the hippocampus of the CPPs treatment group was significantly decreased.In vitro cultured cells,CPPs co-incubations with cells inhibited intracellular BACE1 activity.In vitro,different concentrations of CPPs were co-incubated with recombinant BACE1 enzyme,and the results showed that CPPs had obvious inhibitory effect on the activity of BACE1.Conclusion: Taken together,these results suggest that down-regulating BACE1 might be the underlaying mechanism which could be a therapeutic target for alleviating cognitive defects in AD pathology.