Effects Of Tau Acetylation And T217-phosphorylation On Its Properties And Cognitive Functions

Background: Alzheimer’s disease(AD)is the most prevalent neurodegenerative disease among aging population.One of pathological hallmarks of AD is the abnormal aggregation of intracellular pathological tau.The brain area where tau first showed pathological changes is the entorhinal cortex(EC).Recent studies have shown that the intracellular abnormal aggregation of tau can not only be deposited to form neurofibrillary tangles,but also propagate through various forms with the entorhinal cortex as the starting brain area to spread outward,and lead to adjacent brain areas or other brain areas with synaptic connections into pathological changes,the specific mechanism of transmission is elusive.Tau acetylation is an important modification after tau translation.Increased tau acetylation at K174,K274,K280,and K281 has been observed in the brains of Alzheimer’s disease(AD)patients or the transgenic mice.Many studies have shown that increased tau acetylation will lead to increased tau aggregation and obstruction of degradation.The specific mechanism is still unclear.It is unknown whether the spread of abnormally aggregated tau is affected by tau acetylation.Objective: To study the effect of tau acetylation in the entorhinal cortex and hippocampus on tau transmission and learning and memory.Methods: In this study,healthy and mature wild-type male mice with no significant difference in body weight were used as experimental subjects.AAV-Tau-4Q,which mimics tau acetylation,and AAV-Tau-4R,which mimics non-acetylated tau,as well as unmutated tau virus AAV-Tau-WT and AAV-Vector was used as controls.all virused have GFP tag which is not fused with tau.Brain stereotaxic injection was used to overexpress tau virus in mouse EC,or DG subset,and the control group was injected with the vector virus.Using novel object recognition(NOR),Morris water maze(MWM)and fear condition(FC)behaviors to test the learning and memory.Using open field(OF)and elevated plus maze(EPM)to detect mood changes in mice.Using MED64 isolated microelectrode array recording system to detect a long-term potentiation(LTP).Using immunohistochemistry and immunofluorescence to detect the glial activation and the transmission of tau in brain slices.Results:1.The acetylation level of tau in EC region is increased in AD model mice Compared with WT mice,the acetylation levels of tau K174 and K280 were significantly increased in EC subset of AD model mice.2.Overexpressing Tau-4Q in EC for 3 months and 6 months neither induce tau transmission to hippocampus nor learning and memory deficitOverexpressing different AAV-tau constructs in the EC subset of 2-month-old C57 mice after 3 months,except for microglial activation in the EC subset,there was no significant glial cell activation in the dentate gyrus(DG)subset of hippocampus,no obvious tau protein transmission to the hippocampus and no obvious learning and memory deficit in mice.Overexpressing different AAV-tau constructs in the EC subset after 6 months,microglia and astrocytes were obviously activated in the EC subset,but there was no significant tau protein transmission to the hippocampus and no obvious learning and memory deficit in mice.3.Overexpressing Tau-4Q in EC for 13.5 months promotes tau propagation to DG subset with memory deficitOverexpressing different AAV-tau constructs in the EC subset of 2-month-old C57 mice after 13.5 months,microglia and astrocyte in all three tau expression groups were significantly activated in the EC subset,the more significant effect was shown in Tau-4Q group;expressing Tau-WT induced tau propagation to the granular cell layer of DG,while expressing Tau-4Q promoted tau propagation to the inner hilus layer of DG;microglia and astrocyte in all three tau expression groups were activated in the DG subset,the more significant effect was shown in Tau-4Q group,the mice suffered memory deficit.4.Overexpressing Tau-4Q in hippocampal DG promotes its contralateral transmission and deteriorate Tau-WT induced impairment of synaptic function and cognitive deficitsOverexpressing different AAV-tau constructs in the unilateral hippocampus of 2-month-old C57 mice after 8 weeks,the HT7-positive cell number was much higher in Tau-4Q mice than the Tau-WT mice.Overexpressing Tau-4R abolished tau propagation to the contralateral hippocampus;overexpressing Tau-4Q induced very significant activation of microglial and astrocyte in both ipsilateral and the contralateral sites of the hippocampal DG,though fewer microglial activation was also shown in the ipsilateral hippocampus of Tau-WT group;the EPSP slope was decreased in all three groups with tau proteins overexpression,and the decrease was most significant in Tau-4Q group than the Tau-WT group,and no difference was shown between Tau-WT and the Tau-4R groups;significant cognitive impairments were detected in Tau-4Q and Tau-WT,Tau-4R restores learning and memory,significant anxiety-depression were detected in Tau-WT,Tau-4Q and Tau-4R.Conclusion: Tau protein acetylation promotes a time-dependent propagation from EC to DG,and only hippocampus but not EC tau accumulation induces cognitive deficits.Background: Alzheimer’s disease(AD)is the most prevalent neurodegenerative disease among aging population.One of pathological hallmarks of AD is the abnormal aggregation of intracellular pathological tau.A recent study found that the phosphorylation of tau T217 in the plasma of early mild cognitive impairment(MCI)and AD patients,with higher accuracy than established plasma-and MRI-based biomarkers,can be better distinguished from other neurodegenerative diseases compared with the cerebral spinal fluid(CSF)phosphorylation tau T181,the earlier prediction of AD.However,the effect of early phosphorylation of tau T217 on the pathological process of AD is unclear.Objective: To study the potential toxic role of tau T217-phosphorylation.Methods: In this study,healthy and mature wild-type male mice with no significant difference in body weight were used as experimental subjects.AAV-tau-T217 E,which mimics tau phosphorylation,and AAV-tau-T217 A,which mimics non-phosphorylation tau,and AAV-tau-P301L-T217 E,AAV-tau-P301L-T217 A,which mimics tau phosphorylation and non-phosphorylation on the basis of tau P301 L,as well as unmutated tau virus AAV-tau-WT and vector was used as controls.all virused have GFP tag which is not fused with tau.Brain stereotaxic injection was used to overexpress tau virus in mouse CA3 subset,and the control group was injected with the vector virus.Using novel object recognition(NOR),Morris water maze(MWM)and fear condition(FC)behaviors to test the learning and memory ability of mice.Using open field(OF)and elevated plus maze(EPM)to detect mood changes in mice.Using immunohistochemistry and immunofluorescence to detect the glial activation and the transmission of tau in brain slices.Using Western blotting to detect proteins expression.Using Golgi staining to detect spine.Using in vitro recombinant tau to detect polymerization.Results:1.Level of T217-phosphorylated tau is increased in the hippocampus of 3x Tg AD miceBy Western blotting,we observed that the level of tau-p T217 in the total hippocampal extracts was significantly increased when compared with the age-and sex-matched controls.2.T217-phosphorylation on wild-type tau enhances tau phosphorylation at other sites with inhibited tau degradation and increased fibrillization in vitroHEK293 cells were transfected with plasmids after 48 hours,tau-T217 E significantly increased tau phosphorylation level at other sites,inhibited tau degradation compared with wild-type tau and tau-T217 A groups.By Thioflavin T staining,a significantly enhanced aggregation of tau-T217 E was shown compared with wild-type tau and tauT217 A groups.3.T217-phosphorylation on wild-type tau exacerbates tau-induced cognitive damages in miceOverexpressing different AAV-tau constructs in the CA3 subset of 2-month-old C57 mice after 1 month,different types tau induce cognitive deficit,the changes were more significant in tau-T217 E group than wild-type tau and tau-T217 A group,and the T 217 A restored the behavioral performance of the mice.4.T217-phosphorylation on wild-type tau exacerbates tau pathologies with aggravated spine in miceOverexpressing different AAV-tau constructs in the CA3 subset of 2-month-old C57 mice after 1 month,tau-T217 E significantly increased the phosphorylation level of tau at T181,T205,S214,T231,S262,and S422 compared with wild-type tau and remarkably increased the levels of truncated tau-N368(AEP-cleaved tau)and tau-C3(caspase-cleaved tau at Asp421)compared with tau-T217.By Golgi staining,all tauexpressing groups showed a significantly reduced number of neuronal dendritic spine in hippocampal CA3 subset.Overexpressing tau-T217 A was restored to the level of the wild-type tau group.5.T217-phosphorylation on wild-type tau exacerbates tau-induced glial activation without affecting tau propagationOverexpressing different AAV-tau constructs in the CA3 subset of 2-month-old C57 mice after 1 month,all tau-overexpressing groups showed microglial activation in both ipsilateral and contralateral hippocampal CA3,and the activation was most significant in tau-T217 E group,and T217 A restored IBA1 to normal control level.For astrocytes,overexpressing tau only induced astrocytes activation in ipsilateral side with similar feature as IBA1,but without affecting the contralateral side.6.T217-phosphorylation on tau-P301 L does not enhance tau phosphorylation at other sitesHEK293 cells were transfected with plasmids after 48 hours,all tau-overexpressing groups did not induce significant elevation of tau phosphorylation at S202,T205,S214,T231,S262,S396.7.T217-phosphorylation on tau-P301 L does not exacerbate cognitive impairment though it promotes tau cleavageOverexpressing different AAV-tau constructs in the CA3 subset of 2-month-old C57 mice after 1 month,tau-P301L-T217 E remarkably increased the level of cleaved tauN368 without affecting its phosphorylation at T181 and S214 in mice and all three types of tau induced similar level of behavioral abnormalities measured by open field test,novel object recognition test,and Morris water maze test.8.T217-phosphorylation on tau-P301 L promotes propagation and glial activationOverexpressing different AAV-tau constructs in the CA3 subset of 2-month-old C57 mice after 1 month,significant HT7-positive signal was detected on the contralateral hippocampal CA1 in tau-LE group.By immunofluorescence co-staining with IBA1 and GFAP,we also observed that T217 phosphorylation exacerbated tau-P301L-induced glial activation.Conclusion: T217-phosphorylation exacerbates wild-type tau hyperphosphorylation with aggravated tau cleavage/fibrillization and cognitive impairments,while overexpressing T217 E on the basis P301 L does not exacerbate tau phosphorylation or the P301L-induced cognitive deficits although it aggravates tau cleavage and propagation.