Extended Data Fig. 8: snRNA-seq analysis of the transcriptomic perturbation signature of bumetanide in the hippocampus of J20/E4-KI mice.

a, Transcripts in 47,619 single nuclei from the hippocampus of bumetanide- and vehicle-treated J20/E4-KI mice (n = 3 mice per group) were sequenced. b, Clustering and visualization by t-SNE identifies 25 distinct cell clusters which are color-coded according to cell-type. c, Cell clusters color-coded by treatment groups. d, Histogram of the rank of FC of the human apoE4/4-specific transcriptomic signature of AD genes that were also detected by snRNA-seq in J20/E4-KI mouse hippocampi (calculated via FindMarkers with default parameters, Wilcoxon rank sum test, Seurat v_3.1.5.9005) in four representative cell clusters. The rank of the FC of these genes in a dentate gyrus granule cell cluster (1), a subiculum neuronal cluster (5), a microglial cluster (10), and an astrocyte cluster (17) in J20/E4-KI mouse hippocampi following bumetanide treatment as compared to vehicle treatment were plotted. The mean rank of all genes in this gene set is denoted by the black line, the average mean FC rank of up-regulated genes (colored red in histogram) is denoted by the red dashed line and the mean FC rank of down-regulated genes (colored blue in histogram) is denoted by the blue dashed line. P-value of significance of the “flip” of up- and down-regulated FC rank means away from the rank mean of all genes as calculated by Monte-Carlo simulation is shown (P < 0.05 considered significant. e, Heatmap of genes from apoE4/4-specific transcriptomic signature of AD, rank ordered and color coded (red for up, blue for down) by estimated FC in human apoE4/4 AD (top), then re-ordered by FC rank after bumetanide treatment in four representative cell types (clusters 1, 5, 10 and 17) in the J20/E4-KI mouse hippocampus. Bumetanide treatment flips the expression rank of both up- and down-regulated genes of the human apoE4/4-specific transcriptomic signature of AD in these four cell types. f, P-value of the “flip” of the apoE4/4-specific transcriptomic signature of AD, as calculated by Monte-Carlo simulation, is plotted on the y-axis versus the number of DE genes in each cell cluster on the x-axis. The red dashed line denotes P = 0.05. Dentate gyrus granule cells, subiculum neurons, OPCs, microglia, and astrocytes exhibit a significant “flip” of the apoE4/4-specific transcriptomic signature of genes in AD despite varying number of DE genes. g, Heatmap of the p-values of enriched ontological pathways in all cell clusters exhibiting the “flip” behavior of the apoE4/4-specific transcriptomic signatures of AD reveals 37 pathways that are affected in at least one of these cell types (unadjusted P < 0.005 by bespoke enrichment method, kegga function (limma v 3.36.5)). Pathway names highlighted in red (n = 7) are those shared with the apoE4/4-specific signature pathways of AD (see Fig. 1g and Supplementary Table 6 for human pathways).