Fig. 2
The formation of axonal spheroids in close proximity to axon terminals contributes to synaptic dysfunction due to anomalies in synaptic proteins involved in synaptic vesicle exocytosis. A To study the relationship between synaptic loss and axonal spheroid development, AAV1-hSyn-Cre was injected into the vDG of 6-month-old Ai6 control and 5xFAD; Ai6 transgenic mice. A schematic drawing depicts the Cre-Ai6 system used to record anterograde transsynaptic delivery events. B Whole-brain imaging of Ai6 and 5xFAD; Ai6 mice exhibited damaged synapses and reduced viral spreading across vDG connections in 5xFAD; Ai6 mice (bottom). Ai6 mice showed brain-wide spreading of Cre-mediated Ai6 expression (top). White arrow indicates Ai6 expression in the cortex, marked by transsynaptic transfer of AAV1-Cre. Scale bars = 2 mm (left), 1 mm (right). C Confocal images of coronal sections including the septum, hippocampus, and dentate gyrus of Ai6 and 5xFAD; Ai6 mouse brain immunostained for Aβ and Ctip2. In the septum, Ai6 mice showed transsynaptic labeling of a few neurons (yellow arrows) in contrast to the aggregation of Ai6 signals in the neuritic spheroids adjacent to Aβ plaques. In the cortex, Ai6 mice showed transsynaptic labeling of cortical neurons (yellow arrows), however, 5xFAD; Ai6 mice showed Ai6-positive neurites but did not show Ai6 labeled cortical neurons. In the dentate gyrus, Ai6-positive dentate granule neurons and hilus neurons were widely distributed, and a few Ai6-positive hilus neurons were visible in 5xFAD; Ai6 mice. Aggregation of Ai6 in the molecular layers depicts dystrophic axons near Aβ plaques. Scale bars = 100 μm. D Synaptosomes in the hippocampo-septal tracts of WT and 5xFAD mice were analyzed by LC–MS/MS to investigate the biological processes underlying axonal spheroid formation and synaptic dysfunction. E A total of 1,099 proteins, with 743 overlapping, 92 WT control-unique, and 264 5xFAD-unique proteins, were identified in the proteomic analysis of synaptosomes (Venn diagram). A scatter plot shows 337 up-regulated (log2 fold-change > 0.25, adjusted p-value < 0.1) and 109 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 (12-month-old, n = 4, 4 male) and WT (12-month-old, n = 4, 4 male) control synaptosomes and the weight value of this quantification. The position of the representative proteins selected for gene-set enrichment analysis is indicated in colors (blue = downregulated, red = upregulated). F Gene-set enrichment analysis based on GO Biological Process showed that both the downregulated and the upregulated proteins were linked to vesicle-related pathways, synaptic signaling pathways including chemical synaptic transmission and signal release from the synapse, and the neuron projection morphogenesis pathway. G PPI network analysis using the STRING database identified a network with 30 nodes and 50 edges from proteins in 10 upregulated pathways and a network with 20 nodes and 32 edges from proteins in 10 downregulated pathways, as shown in Fig. 2F