Experimental and real-world evidence supporting the computational repurposing of bumetanide for APOE4-related Alzheimer's disease

Abstract

The evident genetic, pathological, and clinical heterogeneity of Alzheimer's disease (AD) poses challenges for traditional drug development. We conducted a computational drug repurposing screen for drugs to treat apolipoprotein (apo) E4-related AD. We first established apoE-genotype-dependent transcriptomic signatures of AD by analyzing publicly-available human brain database. We then queried these signatures against the Connectivity Map database containing transcriptomic perturbations of >1300 drugs to identify those that best reverse apoE-genotype-specific AD signatures. Bumetanide was identified as a top drug for apoE4 AD. Bumetanide treatment of apoE4 mice without or with Aβ accumulation rescued electrophysiological, pathological, or cognitive deficits. Single-nucleus RNA-sequencing revealed transcriptomic reversal of AD signatures in specific cell types in these mice, a finding confirmed in apoE4-iPSC-derived neurons. In humans, bumetanide exposure was associated with a significantly lower AD prevalence in individuals over the age of 65 in two electronic health record databases, suggesting effectiveness of bumetanide in preventing AD.

Extended Data Fig. 1.. Bumetanide is also predicted to rescue the transcriptomic signature of aging in apoE4-KI mouse hippocampus.

a, PCA plot of top 500 variable genes in apoE4-KI mouse brain shows a distinct effect of age, with 3 month-old brains grouping separately from 12 and 24 month-old brains. b, Venn diagrams of the overlaping DE genes (defined as logFC > 2 and P < 0.05) and DE pathways (defined as P < 0.05) between 12 vs 3 month-old apoE4-KI brains and 24 vs 3 month-old apoE4-KI brains. c, Graphs of compounds ordered by CMap score against DE genes (defined as logFC > 2, P < 0.05) in 12 vs 3 month-old apoE4-KI brains (see Methods for details). Bumetanide has a negative CMap score in the 7^th percentile of all drugs in the CMap. d, Graphs of compounds ordered by CMap score against DE genes (defined as logFC > 2, P < 0.05) in 24 vs 3 month-old apoE4-KI brains. Bumetanide has a negative CMap score in the 8^th percentile of all drugs in the CMap. e, Histogram of the rank of FC of the DE genes (defined as logFC > 2, P < 0.05) in 12 vs 3 month-old apoE4-KI brains, which were also measured in the CMap database after bumetanide treatment. 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 dashed red line and the mean FC rank of the 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 = 0.056). This p-value does not reach significance even while the magnitude of the “flip” is quite large. f, Histogram of the rank of FC of the DE genes (defined as logFC > 2, p-value < 0.05) in 24 vs 3 month-old apoE4-KI brains which were also measured in the CMap database after bumetanide treatment. 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 dashed red line and the mean FC rank of the 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 = 0.064). This p-value does not reach significance even while the magnitude of the “flip” is quite large.

Extended Data Fig. 2.. Bumetanide treatment does not affect swim speed or visible trial performance in aged apoE4-KI mice and does not affect behavioral performance in wildtype (WT) mice.

a, Bumetanide did not significantly affect swim speed during hidden platform trials of apoE4-KI and apoE3-KI mice (n = 11 for each group) at 24 month of age over learning days 1–5. b, There was no significant difference between any groups in visible trials (measured by 2-way ANOVA) of apoE4-KI and apoE3-KI mice (n = 11 for each group), indicating there were no motor or vision impairment in any of the groups. c, Escape latency of vehicle (n = 16) and bumetanide (n = 15) treated WT mice during learning days 1–5 did not differ. d, Bumetanide did not significantly affect swim speed during hidden platform trials in WT mice (n = 15) as compared to vehicle treated WT controls (n = 16). e, In the 24-hour probe trial, both vehicle (n = 16, two way ANOVA with Bonferroni’s multiple comparisons test P < 0.0001) and bumetanide (n = 15, two way ANOVA with Bonferroni’s multiple comparisons test P = 0.0001) treated WT mice spent significantly more time in the target quadrant versus average percent time in the other quadrants. f, In the 72-hour probe trial, both vehicle (n = 16, two way ANOVA with Bonferroni’s multiple comparisons test P = 0.0054) and bumetanide (n = 15, two way ANOVA with Bonferroni’s multiple comparisons test P < 0.0001) treated WT mice spent more time in the target quadrant than the other quadrants. Values are mean ± SEM in a-f.

Extended Data Fig. 3.. Violin plots of marker genes for 18 cell clusters and their properties identified by snRNA-seq in the hippocampus of aged apoE4-KI mice.

a, Violin plots of expression of marker genes for each of the 18 cell clusters identified by snRNA-seq in the hippocampus of aged apoE4-KI mice with and without bumetanide treatment. Y-axis is average imputed expression of a marker gene across all cells in a cluster (see Methods for details), x-axis denotes each cell cluster. b, snRNA-seq analysis of the hippocampus of aged apoE4-KI mice with and without bumetanide treatment identifies 18 unique cell clusters. c, Number of cells per cluster. d, Average number of genes identified per cell in each cluster (± SEM). Number of cells (n) for each cell cluster can be found in c (> 58 cells in any cluster). e, Average nUMI per cell for each cluster (± SEM). Number of cells (n) for each cell cluster can be found in c (> 58 cells in any cluster). f, Average % mitochondrial genes per cell in each cluster (± SEM). Number of cells (n) for each cell cluster can be found in c (> 58 cells in any cluster).

Extended Data Fig. 4.. Histograms of FC rank changes of human apoE4/4-specific AD signature genes in cell clusters 1, 4, 6, 7, 9–18 in aged apoE4-KI mice.

a–n, Histograms of the human apoE4/4-specific transcriptomic signature of AD geneset that was also detected by DE analysis of snRNA-seq in the apoE4-KI mouse hippocampus after bumetanide treatment as compared to controls in cell clusters 1, 4, 6, 7, 9–18. The rank of the FC of these genes in each cluster following bumetanide treatment, as compared to vehicle treatment, is plotted. The mean rank of all genes in this geneset 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 the down-regulated genes (colored blue in histogram) is denoted by the blue dashed line. P-value of the 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). Cell clusters 1, 4, 6, 9, 10, 13, 15, and 16, which include all excitatory neurons, mixed neurons, and endothelial/fibroblast-like cells have a significant “flip” of human apoE4/4-specific AD signiture genes, whereas cell clusters 7, 11, 12, 14, 17 and 18, which include oligodendrocytes, VIP-interneurons, OPC’s, RELN-interneurons, astrocytes, choroid plexus, are not significant. o, Histogram of the human apoE4/4-specific transcriptomic signature of AD geneset that was also detected by DE analysis of snRNA-seq in the apoE4-KI mouse hippocampus after bumetanide treatment as compared to controls in combined data from all neuronal clusters that exhibited a significant “flip” of human apoE4/4 AD genes (Clusters 1, 2, 3, 4, 5, 6, 8, 9, 10, 13, and 16 combined). The rank of the FC of these genes in the combined neuronal cells following bumetanide treatment, as compared to vehicle treatment, is plotted. The mean rank of all genes in this geneset 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 the down-regulated genes (colored blue in histogram) is denoted by the blue dashed line. P-value of the 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).

Extended Data Fig. 5.. The fold change size and directionality of all DE genes after bumetanide treatment in the five large excitatory neuronal cell types in aged apoE4-KI mouse hippocampus mimicked the fold change size and directionality after bumetanide treatment in PC3 cells in the CMap database.

a, Scatterplot of average number of cells per cell cluster (of clusters with > 50 cells) in apoE4-KI hippocampi versus the percentile of CMap score against the DE genes in those clusters after bumetanide treatment (see Methods for details). The top 300 DE genes by p-value of the first five cell clusters (all excitatory neuronal cells) have a CMap score above the top 90 percentile of all drugs in the CMap database. b, Graphs of compounds ordered by CMap score against DE genes in Dentate Gyrus Granule Cells (see Methods for details). Bumetanide has one of the highest positive scores, suggesting that the signature in these cells in vivo is similar to the signature in the CMap database. c, Correlation analysis plot of rank of FC of genes in apoE4-KI Dentate Gyrus Granule Cells after bumetanide treatment versus rank of FC of genes in the CMap database after bumetanide treatment. There is a positive correlation with an R² = 0.08829 and a P-value = 3.6 × 10⁻¹², indicating that the FC of genes in these two signatures of DE genes after bumetanide treatment mimic each other. The shaded region represents the 95% confidence interval for predictions from the linear model. d, Graphs of compounds ordered by CMap score against DE genes in apoE4-KI CA1 neurons (see Methods for details). Bumetanide has one of the highest positive scores, suggesting that the signature in these cells in vivo is similar to the signature in the CMap database. e, Correlation analysis plot of rank of FC of genes in apoE4-KI CA1 neurons after bumetanide treatment versus rank of FC of genes in the CMap database after bumetanide treatment. There is a positive correlation with an R² = 0.08091 and a P-value = 1.9 x 10⁻¹⁰, indicating that the FC of genes in these two signatures of DE genes after bumetanide treatment mimic each other. The shaded region represents the 95% confidence interval for predictions from the linear model. f, Graphs of compounds ordered by CMap score against DE genes in apoE4-KI CA 2/3 Neurons (see Methods for details). Bumetanide has one of the highest positive scores, suggesting that the signature in these cells in vivo is similar to the signature in the CMap database. g, Correlation analysis plot of rank of FC of genes in apoE4-KI CA2/3 neurons after bumetanide treatment versus rank of FC of genes in the CMap database after bumetanide treatment. There is a positive correlation with an R² = 0.06221 and a P-value = 9.9 x 10⁻⁷, suggesting that the FC of genes in these two signatures of DE genes after bumetanide treatment mimic each other. The shaded region represents the 95% confidence interval for predictions from the linear model.

Extended Data Fig. 6.. Bumetanide treatment flips apoE4-mediated murine transcriptomic signature of aging in specific neuron subtypes in the hippocampus of aged apoE4-KI mice.

a, Number of upregulated (119) and downregulated (3) genes (defined as logFC > 2, P < 0.05) in 24 vs 3 month-old apoE4-KI cortex. b, Number of overlapping genes that were detected in clusters 1–18 in apoE4-KI hippocampi with and without bumetanide treatment. Due to sequencing depth and gene drop out, none of the 3 downregulated genes were detected in any cell cluster in aged apoE4-KI hippocampi. c, P-value of the “flip” of apoE4/4 specific transcriptomic signatures of AD in humans is plotted on the x-axis versus the P-value of the “flip” of aging signature of upregulated DE genes in 24 vs 3 month-old apoE4-KI hippocampus for each of the 18 cell clusters after bumetanide treatment in apoE4-KI mouse hippocampus. The black dashed lines denotes P = 0.05. Cell clusters 1, 2 and 4 have a significant P-value in each analysis, while Clusters 1–6 and cluster 13 have P-values either trending towards or reaching significance in both analyses, suggesting that most exitatory neuronal clusters experience a “flip” of both aging and apoE4/4 AD signatures when exposed to bumetanide in apoE4-KI hippocampi. d, Histogram of the rank of FC after bumetanide treatment in apoE4-KI Dentate Gyrus Granule Cell (Cluster 1) of the aging DE genes (defined as logFC > 2, P-value < 0.05) in 24 vs 3 month-old apoE4-KI hippocampus. 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 dashed red line. P-value of significance of the “flip” of up-regulated FC rank means away from the rank mean of all genes as calculated by Monte-Carlo simulation is shown (P = 0.015). e, Histogram of the rank of FC after bumetanide treatment in apoE4-KI Dentate Gyrus Granule Cell (Cluster 2) of the aging DE genes (defined as logFC > 2, P-value < 0.05) in 24 vs 3 month-old apoE4-KI hippocampus. 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 dashed red line. P-value of significance of the “flip” of up-regulated FC rank means away from the rank mean of all genes as calculated by Monte-Carlo simulation is shown (P = 0.031). f, Histogram of the rank of FC after bumetanide treatment in apoE4-KI CA 2/3 Neurons (Cluster 4) of the aging DE genes (defined as logFC > 2, P-value < 0.05) in 24 vs 3 month-old apoE4-KI hippocampus. 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 dashed red line. P-value of significance of the “flip” of up-regulated FC rank means away from the rank mean of all genes as calculated by Monte-Carlo simulation is shown (P = 0.007).

Extended Data Fig. 7.. Violin plots of marker genes for 25 cell clusters and their properties identified by snRNA-seq in the hippocampus of J20/E4-KI mice.

a, Violin plots of expression of marker genes for each of the 25 cell clusters identified by snRNA-Seq in the hippocampus of J20/E4-KI mice with and without bumetanide treatment. Y-axis is average imputed expression of a marker gene across all cells in a cluster (see Methods for details), x-axis denotes each cell cluster. b, snRNA-seq analysis of the hippocampus of aged apoE4-KI mice with and without bumetanide treatment identifies 25 unique cell clusters. c, Number of cells per cluster. d, Average number of genes identified per cell in each cluster (± SEM). Number of cells (n) for each cell cluster can be found in c (> 177 cells in any cluster). e, Average nUMI per cell for each cluster (± SEM). Number of cells (n) for each cell cluster can be found in c (> 177 cells in any cluster). f, Average % mitochondrial genes per cell in each cluster (± SEM). Number of cells (n) for each cell cluster can be found in c (> 177 cells in any cluster).

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 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 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 (P < 0.005). Pathway names highlighted in red (n = 7) are those shared with the apoE4/4-specific signature pathways of AD (see Figure 1g and Supplementary Table 6 for human pathways).

Extended Data Fig. 9.. Analyses of overlapping enriched ontological pathways among bumetanide-treated apoE4-KI mice, J20/E4-KI mice, human iPSC-derived neurons, and human apoE4/4-specific transcriptomic signature of AD.

a, 22 overlapping enriched ontological pathways (Supplementary Table 17) in bumetanide-flipped cell clusters in apoE4-KI mice vs J20/E4-KI mice. b, Six overlapping enriched ontological pathways (Supplementary Table 18) in bumetanide-flipped cell clusters in apoE4-KI mice vs J20/E4-KI mice vs E4/4-hiPSC neurons. c, Three overlapping enriched ontological pathways in bumetanide-flipped cell clusters in apoE4-KI mice vs J20/E4-KI mice vs E4/4-hiPSC neurons vs human E4/4 signature of AD, which include GABAergic Synapse, Circadian Entrainment, Morphine Addiction pathways.

Extended Data Fig. 10.. Bumetanide exposure is associated with a significantly lower AD prevalence in individuals over the age of 65 in two independent EHR databases.

We evaluated two large-scale EHR databases (UCSF EHR and Mt. Sinai EHR) in a cross-sectional manner to test the association of bumetanide exposure with AD prevalence in individuals with the age of 65 or above using a propensity score matching approach to control cohort creation. a, AD prevalence in bumetanide-exposed cohort is significantly lower than those in all 10 randomly selected non-bumetanide-exposed cohorts in the UCSF EHR database. All 10 randomly selected 1:2 control cohorts were matched on propensity score which included age, sex, race, and hypertension and edema diagnosis. χ-squared test, all P < 0.05. b, AD prevalence in bumetanide-exposed cohort is significantly lower than those in all 10 randomly selected non-bumetanide-exposed cohorts in the Mt. Sinai EHR database. All 10 randomly selected 1:2 control cohorts were matched on propensity score which included age, sex, race, and hypertension and edema diagnosis. χ-squared test, all P < 0.0001. c, AD prevalence in bumetanide-exposed cohort is significantly lower than those in 8 out of 10 randomly selected non-bumetanide-exposed cohorts controlled for non-bumetanide diuretic drug use for hypertension and edema treatment in the UCSF EHR database. χ-squared test, 8 out of 10 P < 0.05. d, AD prevalence in bumetanide-exposed cohort is significantly lower than those in all 10 randomly selected non-bumetanide-exposed cohorts controlled for non-bumetanide diuretic drug use for hypertension and edema treatment in the Mt. Sinai EHR database. χ-squared test, all P < 0.0001.

Fig. 1.. ApoE-genotype-dependent transcriptomic signatures of AD.

a, Experimental workflow, including dataset selection, apoE genotype stratification, analysis of DE genes, drug repurposing analysis, efficacy validation in apoE4-KI and J20/E4-KI mice, and transcriptomic validation in apoE4-KI and J20/E4-KI mice and apoE4/4-iPSC-derived human neurons. b, ApoE genotype composition of AD (n = 97 individuals) and control (n = 116 individuals) dataset of GSE15222 (total n = 213 individuals). ApoE4 carrier representation (apoE3/4 and apoE4/4) was greater in AD groups (χ² test of apoE4 carriers versus non-carriers in AD versus control populations, two-sided χ² = 52.236, df = 2, unadjusted p-value = 4.541e⁻¹²). c, PCA of the temporal lobe transcriptomic data showed the first principal component (PC1) is correlated with the diagnosis covariate (two sided Pearson’s correlation, Pearson’s r = −0.4127, unadjusted P = 3.28x10⁻¹⁰). d, PCA of the transcriptomic data showed the first principal component (PC1) is correlated with the apoE genotype covariate (two sided Pearson’s correlation, Pearson’s r = −0.215, unadjusted P = 0.0016). e, f, Venn diagram of overlapping and uniquely upregulated (e) and downregulated (f) DE genes (estimated absolute logFC > 0.4 across 10 sex-matched permutations, P-value < 0.05 in all 10 sex matched permutations) from apoE-genotype-specific DE analysis. 93 genes were uniquely significantly upregulated in apoE4/4 AD, 11 in apoE3/4 AD, and 324 in apoE3/3 AD. Only 76 DE genes were shared across these groups. 142 genes were uniquely significantly downregulated in apoE4/4 AD, 39 in apoE3/4 AD, and 318 in apoE3/3 AD. Only 32 DE genes were shared across all three AD groups. g, Venn diagram of shared and uniquely significantly enriched ontological pathways across apoE-genotype-specific up and down regulated gene groups. Eight pathways were uniquely significantly enriched in apoE4/4 AD, 2 in apoE3/4 AD, and 58 in apoE3/3 AD. Only 7 pathway were shared across all three AD groups.

Fig. 2.. ApoE-genotype-dependent drug repurposing analysis identifes bumetanide as a top predicted drug candidate for apoE4 AD.

a–d, Graphs of compounds ordered by CMap score against (a) apoE4/4 AD, (b) apoE3/4 AD, (c) apoE3/3 AD, and (d) all AD transcriptomic signatures (see Methods for details). The transcriptomic effects of bumetanide showed apoE4/4 genotype preference (ranked number 4), with a stepwise weaker CMap score (still a negative number toward therapeutic direction) against apoE3/4 AD, apoE3/3 AD, and AD status not controlling for apoE genotype. e, Histogram of the rank of FC of the human apoE4/4-specific transcriptomic signature of AD genes which were also measured in the CMap database after bumetanide treatment. 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 dashed red line and the mean FC rank of the 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 < 0.001). f, 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) and then re-color-coded by FC rank after bumetanide treatment in the CMap database. Bumetanide flips the expression rank of both up- and down-regulated genes in the human apoE4/4-specific transcriptomic signature of AD.

Fig. 3.. Bumetanide treatment rescues AD-like neuronal excitability and plasticity deficits as well as learning impairment in aged apoE4-KI mice.

a, Female apoE3-KI and apoE4-KI mice were treated with bumetanide (0.2 mg/kg, daily intraperitoneal injection) for 8 weeks. Input-output relationships in Schaeffer collaterals-CA1 network (inset: example traces) were measured on ex-vivo hippocampal slices from vehicle-treated apoE3-KI (n = 23 slices from 7 mice), bumetanide-treated apoE3-KI (n = 10 slices from 3 mice), vehicle-treated apoE4-KI (n = 11 slices from 3 mice), and bumetanide-treated apoE4-KI (n = 12 slices from 3 mice) mice at 17 months of age. The average fPSP slope values were compared among different groups (Kruskal-Wallis test, P = 0.0011). b, c, LTP was measured on hippocampal slices from vehicle-treated apoE3-KI (n = 14 slices from 6 mice), bumetanide-treated apoE3-KI (n = 8 slices from 3 mice), vehicle-treated apoE4-KI (n = 14 slices from 6 mice), and bumetanide-treated apoE4-KI (n = 12 slices from 3 mice) mice at 17 months of age. Average fPSP slope values were binned to one-minute intervals and normalized to control (b). LTP gain outcomes were summarized as compared to pre-TBS baseline, paired two-sided t test (c). Vehicle-treated (P = 0.0007) and bumetanide-treated (P = 0.0125) apoE3-KI slices and bumetanide-treated apoE4-KI slices (P < 0.0001) showed significant gain while vehicle-treated apoE4-KI slices did not. One way ANOVA, P = 0.0008. d, Escape latency of bumetanide- and vehicle-treated apoE3-KI and apoE4-KI mice (n = 11 for each group) at 24 months of age. One way repeated-measures ANOVA, P = 0.0037 between treatment groups; Tukey’s multiple conparisons test, P = 0.0345 bumetanide-treated apoE4-KI versus vehicle-treated apoE4-KI groups. e, In the 120-hour probe trial, both vehicle-treated (n = 11) and bumetanide-treated (n = 10) apoE4-KI mice as well as vehicle-treated apoE3-KI mice (n = 11), but not bumetanide-treated apoE3-KI mice (n = 11), demonstrated a significant preference for the target quadrant (one way ANOVA p < 0.0001, with Tukey’s multiple comparisons test). Values are mean ± SEM in a-e. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 in a-c.

Fig. 4.. snRNA-seq analysis of the transcriptomic perturbation signature of bumetanide in the hippocampus of aged apoE4-KI mice.

a, Transcripts in ~27,000 single nuclei from the hippocampus of bumetanide- and vehicle-treated female apoE4-KI mice at 17 months (n = 3 per group, 8-week treatment) were sequenced. b, Clustering and visualization by t-SNE identifies 18 distinct cell clusters. c, Cell clusters color-coded by treatment groups. d, Histogram of the rank of FC of apoE4/4-specific transcriptomic signature of AD genes that were also detected by snRNA-seq in vehicle- and bumetanide-treated apoE4-KI mouse hippocampus in four representative cell clusters. The mean rank of all genes in this gene set is denoted by the black line. The average mean FC ranks of up-regulated (red histogram) and down-regulated (blue histogram) genes are denoted by the red and blue dashed lines, respectively. P-value 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 (unadjusted P < 0.05 considered significant). e, Heatmap of genes from apoE4/4-specific transcriptomic signature of AD were rank ordered and color coded by estimated FC in human apoE4/4 AD (top) and then re-color-coded by FC rank after bumetanide treatment in combined neuron types exhibiting a significant “flip” of apoE4/4 AD signature genes (clusters 1, 2, 3, 4, 5, 6, 8, 9, 10, 13, 16 combined) and in representative neuron types (clusters 2, 3, 5, 8) in apoE4-KI hippocampus. f, P-value of the “flip” of apoE4/4-specific transcriptomic signatures of AD (y-axis), as calculated by Monte-Carlo simulation, is plotted versus number of DE genes in each cell cluster (x-axis). The red dashed line denotes P = 0.05. g, Heatmap of P-values of enriched pathways for all DE genes from each cell cluster after bumetanide treatment revealed 135 pathways that were affected in at least one of the cell types that had a significant “flip” of human apoE4/4 AD signature genes (unadjusted P < 0.005 by the bespoke enrichment method employed by the kegga function (limma v 3.36.5)). 28 pathways highlighted in red are those shared with apoE4/4-specific signature pathways of AD (P = 0.001 by Monte Carlo Simulation, see Figure 1g and Supplementary Table 6 for human pathways).

Fig. 5.. Bumetanide treatment rescues AD-like neuronal excitability and plasticity deficits and reduces Aβ plaque loads in the hippocampus and cortex in J20/E4-KI mice.

a, Female J20/E-KI mice were treated with bumetanide (0.2 mg/kg, daily intraperitoneal injection) for 12 weeks. Input-output relationships in Schaeffer collaterals-CA1 network were measured on ex-vivo hippocampal slices from vehicle-treated J20/E3-KI (n = 15 slices from 5 mice), bumetanide-treated J20/E3-KI (n = 21 slices from 7 mice), vehicle-treated J20/E4-KI (n = 18 slices from 5 mice), and bumetanide-treated J20/E4-KI (n = 27 slices from 7 mice) mice at 13 months of age. The average fPSP slope values were compared by one way ANOVA (P = 0.0023), with Tukey’s multiple comparisons test P = 0.0205 for bumetanide-treated J20/E4-KI mice versus vehicle-treated J20/E4-KI mice and P = 0.0033 for bumetanide-treated J20/E4-KI mice versus vehicle-treated J20/E3-KI mice. b, c, LTP was measured on ex-vivo hippocampal slices from vehicle-treated J20/E3-KI (n = 12 slices from 5 mice), bumetanide-treated J20/E3-KI (n = 11 slices from 5 mice), vehicle-treated J20/E4-KI (n = 15 slices from 5 mice), and bumetanide-treated J20/E4-KI (n = 15 slices from 5 mice) mice at 13 months of age. Average fPSP slope values were binned to one-minute intervals and normalized to control (b). LTP gain outcomes were summarized as compared to pre-TBS baseline, paired two-sided t test (c). Bumetanide treatment resulted in a significant increase in LTP gain outcome for both treated J20/E3-KI (P = 0.041 versus vehicle-treated J20/E3-KI) and J20/E4-KI (P = 0.0446 versus vehicle-treated J20/E4-KI) mice. d, e, Representative images of Aβ immunostaining from vehicle-treated (d) and bumetanide-treated (e) J20/E4-KI mice at 13 months of age (n = 8 per group, 12-week treatment). Scale bars = 300 μm. f–i, Quantifications of Aβ plaque number (f, h) and area (g, i) in the hippocampus (f, g) and the cortex (h, i) from vehicle-treated or bumetanide-treated J20/E4-KI mice at 13 months (n = 8 per group, 12-week treatment). Unpaired and two-sided t test in f-i. Values are mean ± SEM in a-c and f-i. * P < 0.05; ** P < 0.01; *** P < 0.001 in a-c.

Fig. 6.. RNA-seq analysis of the transcriptomic perturbation signature of bumetanide in apoE4/4-iPSC-derived human neurons.

a, ApoE4/4-iPSC-derived human neurons were treated for 6 hours with 10 μM bumetanide or vehicle (n = 3 for each group) and transcriptomic changes were analyzed by RNA-seq. b, PCA of 500 most variale genes in bumetanide treated apoE4/4-iPSC-derived human neurons does not clearly separate bumetanide treated samples from vehicle treated samples. c, PCA of DE genes in bumetanide treated apoE4/4-iPSC-derived human neurons separates bumetanide treated samples from vehicle treated samples. d, Histogram of the human apoE4/4-specific transcriptomic signature of AD geneset that were also detected by RNA-seq in apoE4/4-iPSC-derived human neurons after bumetanide treatment (up and down-regulated gene signatures as denoted in Fig. 1e, f). The rank of the FC of these genes in apoE4/4-iPSC-derived human neurons following bumetanide treatment as compared to vehicle treatment is plotted on the x-axis versus number of genes at that rank on the y-axis. The mean rank of all genes in this geneset 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 the down-regulated genes (colored blue in histogram) is denoted by the blue dashed line. P-value of the 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 < 0.001). e, Heatmap of genes from human apoE4/4-specific transcriptomic signature of AD, rank ordered and color coded (red for up, blue for down) by estimated FC in apoE4/4 AD (top) and then re-color-coded by FC rank in apoE4/4-iPSC-derived human neurons after bumetanide treatment (bottom). f, Heatmap of the p-value of enriched ontological pathways (n = 19) in apoE4/4-iPSC-derived human neurons after bumetanide treatment and their corresponding enrichment p-values in human apoE4/4-specific AD. Pathways highlighted in red (n = 3 pathways) denote those that are shared between apoE4/4-iPSC-derived human neurons and human apoE4/4-specific AD signature pathways (see Figure 1g and Supplementary Table 6 for human pathway information).

Fig. 7.. Bumetanide exposure is associated with a significantly lower AD prevalence in individuals over the age of 65 in two independent EHR databases.

a, Workflow of evaluation of the UCSF EHR database and the Mt. Sinai EHR database for association of bumetanide exposure with AD prevalence. We evaluated two large-scale EHR databases in a cross-sectional manner to test the association of bumetanide exposure with AD prevalence in individuals with the age of 65 or above using a propensity score matching approach to control cohort creation. b, AD prevalence (the left y-axis) in bumetanide-exposed cohort is significantly lower than those in 10 randomly selected non-bumetanide-exposed cohorts controlled for non-bumetanide diuretic drug use for hypertension and edema treatment (Bootstrapped two-sided χ²(1) = 4.530, P = 0.0333, median difference = 0.0133 (95% CI: 0.0123 – 0.0142)) in the UCSF EHR database. The right y-axis represents the paired mean differences in the UCSF EHR database. c, AD prevalence (the left y-axis) in bumetanide-exposed cohort is significantly lower than those in 10 randomly selected non-bumetanide-exposed cohorts controlled for non-bumetanide diuretic drug use for hypertension and edema treatment (Bootstrapped two-sided χ²(1)= 32.846, P = 1x10⁻⁸, median difference = 0.0463 (95% CI: 0.0445–0.0477)) in the Mt. Sinai EHR database. The right y-axis represents the paired mean differences in the Mt. Sinai EHR database.