APOE4 Regulates Synaptic Function by Directly Modulating the SNARE Complex Assembly

Abstract Background: The ε4 allele of the Apolipoprotein E ( APOE ) gene is a major genetic risk determinant of sporadic Alzheimer's disease (AD). Its protein product APOE4 has been demonstrated to coffers deleterious effects for various neurodegenerative disorders related to cognitive impairment, including AD. A line of evidence implied that APOE4 affects these diseases partly through its synaptic damage. However, the mechanisms underlying this have not been fully interpreted. Methods: Proteomics analysis, Co-immunoprecipitation assay (Co-IP), Bimolecular fluorescence complementation (BIFC), and Proximity ligation assay (PLA) assays were used to screen and verify the interactome of APOE, which in an APOE4-priority manner. The molecular docking and molecular dynamic analysis were conducted to elucidate the molecular mechanisms that APOE3 differs from APOE4 in the binding ability of VAMP2. Adeno-associated virus expressing APOE3 and APOE4 was stereotaxically injected into the Hippocampus of Apoe-/- mice, and in vitro recombinant proteins experiments were conducted to verify the AOPE on soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assemble. FM4-64 fluorescent dye labeling assay was explored in hippocampus from APOE3-TR and APOE4-TR mice to study the APOE genotype effect on synaptic vesicle release . Results: Using proteomics analysis, we screened interactomes of APOE3 and APOE4 in neurons, respectively. Then, VAMP2 protein was selected for further analysis through related bioinformatics analysis. Via Co-IP, BIFC, and PLA assays,we demonstrated that APOE directly interacts with VAMP2 in an E4 > E3 manner in vitro and in vivo . The molecular docking and molecular dynamic analysis suggested that the APOE4-VAMP2 complex was more stable and had higher affinity than APOE3-VAMP2, may due to the increased contribution of hydrogen bonding, hamper VAMP2 to form the SNARE complex. The further in vitro and in vivo results suggest that APOE4 blocks the SNARE complex assembly, negatively regulating synaptic vesicle release, finally contributing to the synaptic damage and cognitive impairment. Conclusions :Our findings identify SNARE protein as an APOE interactor, and APOE4 isoform effects on SNARE complex formation, mediates APOE4-induced synaptic dysfunction. Our results provide insights into APOE4-mediated synapse toxicity, and suggested new avenues for specifically targeting early presynaptic dysfunction in AD.