Pathogenesis Of AEP-dependent Tau Cleavage And Truncated Tau-induced Neurodegeneration Under NGF Deprivation

Background:Alzheimer’s disease(AD)is the most common dementia and neurofibrillary tangles(NFTs)consisting of hyperphosphorylated Tau are one of the characteristics of AD.Nerve growth factor(NGF)plays a vital role in neuronal survival and function.NGF-deprivation mouse model shows NFTs,but the mechanism behind it is still unknown.Asparaginyl endopeptidase(AEP)is involved in the pathologic processes of AD and cleavage of Tau by AEP mediates the neurofibrillary pathology in AD.Objective:To explore whether Src mediates NGF-deprevation-induced AEP activation and Tau truncation and whether AEP-cleaved Tau-induced Tau pathology and neurodegeneration depend on its phosphorylation.Methods:Deprivation of NGF in primary rat neurons and detecting Src,AEP,Tau368 by Western-Blot.Induce Src activation in HEK293t cells by EGF and measure the changes of related molecules by Western-Blot.Overexpressing or knock-down of Src/Traf6 to study their influence on AEP.Using mass spectrum to detect the phosphorylation sites in Traf6 by Src.Mutation of these sites to study its influence on AEP activation by Western-Blot.Using Src inhibitor to treat the neurons derived from IPSC,constructing Traf6 conditional knockout mice,injection of AAV-Src virus in the brain,and using the Western-Blot to detect the changes of Src,AEP,Tau368,and Tau phosphorylation.According to the cleavage sites of Tau by AEP,different Tau fragments are constructed,with either phosphorylation or non-phosphorylation mimic mutations.In vitro,these phosphorylated or non-phosphorylated truncated Tau are overexpressed in cells respectively.Detergent-insoluble fraction is separated and the aggregation of these truncated Tau is detected by Western-Blot.Flow cytometry was used to measure the transmission of truncated Tau between neurons.Behavior test was used to test cognitive function and neuronal apoptosis,synapse plasticity,and neuroinflammation were measured by immunofluorescence.Inflammatory factors were measured by ELISA.Results:Deprivation of NGF in primary hippocampal neurons induces AEP activation and Tau truncation which depend on Src activation.Src also mediates EGF-induced AEP activation and polyubiquitination.Knockdown of Traf6 suppresses Src-induced AEP activation.Biochemical studies and mass spectrum show that Src induces AEP activation through phosphorylating Traf6.Inhibition of Src activity in neurons derived from the induced pluripotent stem cell(IPSC)suppresses Traf6 phosphorylation and AEP activation.Conditional knock-out Traf6 in neurons reduces AEP level and inhibits Src-induced AEP activation and Tau truncation in vivo but does not affect Src-induced Tau phosphorylation.The self-aggregation of phospho-truncated Tau is significantly influenced by the domain it contains.N-terminal inhibits,proline-rich domain promotes and C-terminus has no impacts on phospho-truncated Tau aggregation.Phosphorylation of truncated Tau 1-368 induces endogenous Tau phosphorylation and aggregation.Flow cytometry results demonstrate that Tau1-368 transmission is not influenced by phosphorylation.In vivo,Tau1-368-induced anxiety behavior in C57BL/6 mice is phosphorylation-independent.Recognition memory of mice is impaired by phospho-Tau1-368,but not by non-phospho-Tau 1-368.Immunofluorescence(IF)staining shows that overexpressing phospho-Tau1-368 results in neuronal loss and gliosis in the hippocampus.Conclusion:NGF deprivation promotes AEP-mediated Tau truncation by activating Src/Traf6 pathway,and phosphorylation of AEP-cleaved Tau promotes endogenous Tau pathology and cognitive impairments.