Fig. 6

Single-cell transcriptome profiling of the hippocampal tissues identified the specific neuron type associated with vesicle accumulation in the septum. A A UMAP plot shows the subtypes of neurons produced from WT (3-month-old, n = 3, 3 male; 6-month-old, n = 1, female) and 5xFAD (3-month-old, n = 4, male; 6-month-old, n = 2, female) hippocampus tissues. Using independent component analysis using canonical markers, nine different neuronal subclusters, including 1 undefined (u) were found from 5468 neurons, characterized by strong expression of Meg3 and Atp1b1. Each cluster is color-coded according to cell type. B A dot plot illustrated the expression of selected marker genes in each neuron subtype (u: undefined). C A permutation plot to calculate the average proportion of each neuron subtype between WT and 5xFAD mouse hippocampal tissues demonstrated that the number of neurons with Sst, Chgb, and Calb2 markers was significantly decreased in 5xFAD mice (ASC: astrocyte, Endo: endothelial cells, EPC: ependymal cells, NEUT: neutrophils, OPC: oligodendrocyte precursor cells, OLG: oligodendrocytes). D Differential gene expression analysis revealed 620 up-regulated (log₂ fold-change > 0.25, p-value < 0.01) and 234 down-regulated genes (log₂ fold-change < − 0.25, p-value < 0.01) in the SOM, and 738 up-regulated (log₂ fold-change > 0.25, p-value < 0.01) and 186 down-regulated genes (log₂ fold-change < -0.25, p-value < 0.01) in the Chgb-expressing neuron. Shown are the fold changes of gene expression between 5xFAD and WT control mouse hippocampus in SOM and the fold changes of gene expression between 5xFAD and WT control mouse hippocampus in Chgb-expressing neurons. E Gene-set enrichment analysis in GO biological process signatures showed that SOM and Chgb-expressing neurons commonly turned down pathways related to secretion, cell secretion, and the regulation of cell secretion, as well as the non-motile ciliary assembly pathway. F Gene network connections and associated functions were visualized based on GO biological process signatures derived from differential gene expression analysis of SOM. Gene lists for gene ontology terms are derived from SOM, as shown in Fig. 6E. The genes related to secretion, non-motile cilium assembly, phagocytosis, and synaptic plasticity were enclosed by black boxes. The gene symbols were matched with the STRING database, and we identified a network with 39 nodes and 132 edges. G Immunofluorescence analysis of hippocampal sections from WT and 5xFAD; Thy1-YFP mice showed that the number of SOM in the CA1 area of the hippocampus (left), and the dentate gyrus hilus (middle) was lower in 5xFAD mice than in WT. Plots represent the number of SOMs in each area. Higher magnification images of SST expression showed a bright aggregation of SST in the perinucleus of the SOM in 5xFAD. The data are presented as the mean ± S.E.M. **p < 0.01; ****p < 0.001 as determined by the unpaired t-test. Scale bars = 5 μm. H Axonal spheroids were observed in the vicinity of septal Aβ plaques for SOM by immunofluorescence staining for SST (white dashed line) in the 5xFAD; Thy1-YFP mouse septum. I Immunofluorescence analysis of the septum area in 5xFAD; Thy1-YFP mice showed that the axonal spheroids of SOM are greatly induced in the vicinity of Aβ plaques (Amylo-Glo) as early as 2-months old when Thy1-YFP-positive projection neurons initiate their axonal dystrophy. Scale bars = 5 μm. J Images of SOM in WT and 5xFAD mice were immunostained for Hoechst, CD63, ACIII, and SST. CD63 aggregates formed in the brightly stained perinuclear SST and primary cilia were short and weakly labeled in the SOMs of 5xFAD mice. Scale bar = 2 μm