Fig. 4

The accumulation of EVs in the axonal spheroids by dysfunctional vesicle exocytosis. A Tetraspanin proteins (CD9, CD63, and CD81), synaptic markers (Synapsin-1, PSD95: Postsynaptic density protein 95, and Synaptophysin), and Aβ protein were found in SDS-soluble lysates from hippocampal tissues and synaptosomes from 5xFAD (12-months-old, n = 4, four male) and WT control mice (12-months-old, n = 4, four male). 15 μg proteins from each sample were loaded. The plots are presented as the mean ± S.E.M. of four independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 as determined by the unpaired t-test. B Annexin-based biosensor called pSIVA, which detects exosome-like vesicles by binding to externalized phosphatidylserine was used to observe the accumulation of exosome-like vesicles at the axon terminals in the 5xFAD; Thy1-YFP mouse septum. pSIVA signals were detected in the DNs at 2-month-old mice but more localized in the Amylo-Glo-positive plaque core at 6-month-old mice. Scale bars = 10 μm. C Axonal spheroids surrounding the Aβ plaque of the 5xFAD; Thy1-YFP mice were fluorescently labeled for Aβ and CD63 to offer direct evidence of the accumulation of exosome-like vesicles at the axon terminals. Scale bars = 10 μm. D LC–MS/MS was utilized to identify CD63-immunoprecipitated proteins that were enriched in the synaptosomes collected from the hippocampo-septal tracts (hippocampo-septal synaptosome) of WT (6-months-old, n = 11, one female and ten male mice) and 5xFAD mice (6-months-old, n = 10, four female and six male mice) in order to explore the proteomic changes of EV states in the presence of Aβ plaques. The quality of the CD63-immunoprecipitated synaptosomal fractions was checked by western blot analysis of the pull-down, flow-through, and whole synaptosomes for CD63 and Aβ. E A total of 1,780 proteins were identified, including 1605 overlapping, 44 WT-unique, and 131 5xFAD-unique proteins. A scatter plot showed 211 up-regulated (log2 fold-change > 0.25, adjusted p-value < 0.1) and 65 down-regulated proteins (log2 fold-change < -0.25, adjusted p-value < 0.1) as a result of the differentially expressed protein analysis. Shown are the fold changes in protein abundance between 5xFAD and WT control CD63-IPed synaptosome samples and the weight value of this quantification. The positions of the significantly increased and decreased proteins are indicated. F The PPI network analysis using the STRING database identified a network with 19 nodes and 53 edges from the proteins in 11 upregulated pathways and a network with 7 nodes and 2 edges from the proteins in 5 downregulated pathways selected for gene-set enrichment analysis. G Gene-set enrichment analysis based on GO Biological Process showed 5 downregulated pathways related to vesicle-mediated transport and exocytosis and 11 upregulated pathways related to vesicle transport, endocytosis, and exocytosis