Mechanism Of Amyloid Precursor Protein N-terminal Regulation In Phosphorylated Tau Aggregation And Synaptic Damage In Alzheimer’s-like Disease

Objective:At the molecular,cellular,and animal levels,it has been established that APPmediated tau protein uptake and APP overexpression exacerbate pathological manifestations of tau protein.At the molecular,cellular,human brain organoid,and animal levels,it has been definitively shown that the APPV225 A mutation enhances APP’s affinity for tau protein and increases neuronal cells’ efficiency in engulfing tau protein.This is achieved by promoting tau protein’s liquid-liquid phase separation(LLPS),which in turn leads to increased aggregation of phosphorylated tau protein,causing synaptic damage in neuronal cells,human brain organoid neurons,and animal neurons.This elucidates the molecular mechanism whereby the N-terminus of APP promotes the accumulation of intracellular phosphorylated tau protein,leading to synaptic damage in Alzheimer’s Disease(AD).Methods:At the molecular level,the stable interaction between APP and tau protein was verified by using Alpha Fold2 to simulate the docking of the APP N-terminus with tau protein,conducting Localized Surface Plasmon Resonance analysis(LSPR),and performing co-immunoprecipitation(Co-IP)experiments.Additionally,the affinity of the APPV225 A mutation with tau protein was confirmed.At the cellular level,we employed a combination of CRISPR/cas9-RNP point mutation gene editing,fluorescence labeling of tau protein,3D cell culture,p Hrodo-labeled tau protein endocytosis,and Signaling reporter islands(Si RIs)experiments to verify the APP Nterminus specifically mediates cell uptake of tau protein,and tau protein promotes APP endocytosis into host cells.Furthermore,through in vitro liquid-liquid phase separation,fluorescence recovery after photobleaching,cell surface biotinylation,and immunoelectron microscopy experiments,we verified that the APPV225 A mutation influences SH-SY5Y’s endocytosis of tau protein,the rate of tau-mediated APP endocytosis in SH-SY5 Y cells,and the synaptic dysfunction caused by tau protein’s liquid-liquid phase separation.At the level of human brain organoids and animals,we successfully induced the culture of APPV225 A mutated human brain organoids and built APPwt and APPV225 A mouse models using brain-localized infection of AAV adenoviruses.We validated that APP overexpression influences the propagation of htau protein in the brains of APP/PS1 mice,that the APPV225 A mutation affects tau phase separation in the neurons of human brain organoids,and in mouse brain neurons,and that it impacts synaptic damage in neurons and the aggregation of phosphorylated tau in mouse brain neurons.Results:This study confirmed at the molecular and cellular levels that there is a stable interaction between the N-terminus of APP and tau protein,and that the endocytosis system and APP overexpression promote the propagation of htau protein in the brains of APP/PS1 mice.We also confirmed that the APPV225 A mutation increases the affinity between the N-terminus of APP and tau protein,and that the APPV225 A mutation enhances the endocytosis of tau protein by SH-SY5 Y cells,increasing the rate of tau-mediated APP endocytosis in SH-SY5 Y cells.Furthermore,the APPV225 A mutation promotes liquid-liquid phase separation of tau protein,aggregation of phosphorylated tau231,and phase separation of tau protein in neuronal cells.In the neurons of mouse brain tissue,the APPV225 A mutation promotes phase separation of tau and synaptic damage in neurons,and it enhances the aggregation of phosphorylated tau in neurons of mouse brain tissue.In conclusion,this study reveals the interaction between the N-terminus of APP and tau protein,as well as the enhanced interaction caused by the APPV225 A mutation,providing important theoretical support for a deeper understanding of the regulatory mechanism of the N-terminus of APP on phosphorylated tau protein in neurodegenerative diseases,and its potential therapeutic targets.Conclusions:APP-mediated tau protein uptake and its overexpression lead to an exacerbation of tau protein pathology.The APPV225 A mutation promotes liquid-liquid phase separation of tau,resulting in the aggregation of phosphorylated tau and synaptic damage in neurons in Alzheimer’s disease(AD).The completion of this study theoretically refines the important molecular mechanism of the N-terminus of APP directly participating in the aggregation of phosphorylated tau protein in AD.At the same time,it provides important theoretical support for the clinical application of drugs targeting the N-terminus of APP to improve the onset and progression of AD.