Fig. 3
The enrichment and direct interaction of CD81, a tetraspanin protein of EVs, with APP in the synaptosomes of the 5xFAD mouse brain. A A schematic drawing depicts the localization of axonal spheroids and pSer8-Aβ (Aβ with phosphorylation at the Ser8 residue) in the periphery of an Amylo-Glo-stained plaque core. Plaque-associated Iba1-positive microglia create barriers in their processes. A representative three-dimensional image of axonal spheroids with Aβ plaques consisting of the core (Amylo-Glo), the diffuse area (pSer8-Aβ), and a microglial barrier (Iba1). Scale bar = 5 μm. B Confocal mages of the septum from 5xFAD; Thy1-YFP mice at two different ages (2 and 6 months) were immunostained for Iba1 and Aβ. Higher magnification images of the white box are presented in (c). Aggregation of YFP-positive axons and clustering of microglia are visible at 6-months of age. Scale bars = 100 μm. C Higher magnification images of the white box in (b) show that the axonal spheroids became larger as the amount of pSer8-Aβ increased. Scale bar = 5 μm for 2-month-old mice, 10 μm for 6-month-old mice. D Primary hippocampal neurons were harvested at 48 h after treatment with 1 μM Aβ1-42 synthetic oligomer for 6 h. Proteins isolated from the whole lysate of the three independent experiments (n = 3) were analyzed by LC–MS/MS. E A scatter plot shows 907 up-regulated (log2 fold-change > 0.25, adjusted p-value < 0.1) and 233 downregulated 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 Aβ-treated and control primary hippocampal neurons and the weight value of this quantification. PPI network analysis using the STRING database identified a network of CD81, APP, and 65 other proteins with 310 interactions from the upregulated proteins of the differentially expressed protein analysis. F Gene-set enrichment analysis based on GO Biological Process showed 4 downregulated pathways related to cilia assembly and 18 upregulated pathways related to vesicle transport, endocytosis, and exocytosis. G The proteomic analysis of the APP C-terminal fragment-immunoprecipitated synaptosomes profiled the proteins involved in the initial formation of axonal spheroids during APP processing in the synaptosomes. A total of 1,070 proteins were identified with, 983 overlapping, 71 WT-unique, and 16 5xFAD-unique proteins. A volcano plot shows 519 up-regulated (log2 fold-change > 0.25, adjusted p-value < 0.1) and 307 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 (6-months-old, n = 4, one female and three male) and WT (6-months-old, n = 4, two female and two male) synaptosome samples and the adjusted p-value. The positions of the representative proteins selected for further PPI analysis are annotated. H 29 PPIs were linked to the 38 proteins from synaptosomes that were immunoprecipitated with APP. Several proteins, such as MIF (red) and CD81 (green), interacted directly with APP (blue)