Cell-autonomous effects of APOE4 in restricting microglial response in brain homeostasis and Alzheimer's disease

Abstract

Microglial involvement in Alzheimer's disease (AD) pathology has emerged as a risk-determining pathogenic event. While apolipoprotein E (APOE) is known to modify AD risk, it remains unclear how microglial apoE impacts brain cognition and AD pathology. Here, using conditional mouse models expressing apoE isoforms in microglia and central nervous system-associated macrophages (CAMs), we demonstrate a cell-autonomous effect of apoE3-mediated microglial activation and function, which are negated by apoE4. Expression of apoE3 in microglia/CAMs improves cognitive function, increases microglia surrounding amyloid plaque and reduces amyloid pathology and associated toxicity, whereas apoE4 expression either compromises or has no effects on these outcomes by impairing lipid metabolism. Single-cell transcriptomic profiling reveals increased antigen presentation and interferon pathways upon apoE3 expression. In contrast, apoE4 expression downregulates complement and lysosomal pathways, and promotes stress-related responses. Moreover, in the presence of mouse endogenous apoE, microglial apoE4 exacerbates amyloid pathology. Finally, we observed a reduction in Lgals3-positive responsive microglia surrounding amyloid plaque and an increased accumulation of lipid droplets in APOE4 human brains and induced pluripotent stem cell-derived microglia. Our findings establish critical isoform-dependent effects of microglia/CAM-expressed apoE in brain function and the development of amyloid pathology, providing new insight into how apoE4 vastly increases AD risk.

Extended Data Fig. 1.. Induction of apoE isoforms in microglia/CAMs in iE3/Cre^ER/+ and iE4/Cre^ER/+ mice.

a, b, Upon TAM administration, GFP fluorescence which represents apoE distribution was exclusively detected in the Iba1⁺ microglia (a, Scale bar, 40 μm) and TMEM119⁺ microglia (b, Scale bar, 20 μm) in the iE3/Cre^ER/+ and iE4/Cre^ER/+ mice but not in control mice. The percentage of GFP⁺ cells in Iba1⁺microglia or TMEM119⁺ microglia in experimental mice (iE3/Cre^ER/+: Ctrl: n=5, TAM-induced: n=5; iE4/Cre^ER/+: Ctrl: n=5, TAM-induced: n=5) was quantified. **, P<0.0001. Two-tailed t-test. c, Brain tissues from TAM-induced mice were co-immunostained for GFP which represents apoE distribution (green), and astrocyte-specific (anti-GFAP; pink) or neuron-specific (anti-NeuN; red) markers. Experiments were repeated two times with similar results. Scale bar, 20 μm. d, The GFP fluorescence was colocalized with apoE signal (Red) detected by apoE antibody. Scale bar, 20 μm. e, The percentage of apoE⁺ cells in GFP⁺ microglia in iE3/Cre^ER/+ (Ctrl: n=5, TAM-induced: n=8) and iE4/Cre^ER/+ (Ctrl: n=5, TAM-induced: n=8) mice was quantified. **, P<0.0001. f, ApoE levels in the cortex of the iE3/Cre^ER/+ and iE4/Cre^ER/+ mice at 6 months of age upon vehicle or TAM administration (iE3/Cre^ER/+: Ctrl: n=7, TAM-induced: n=5; iE4/Cre^ER/+: Ctrl: n=7, TAM-induced: n=5) were analyzed by ELISA. **, P<0.0001. g, ApoE levels in the cortex of the iE3/Cre^ER/+ (Ctrl: n=8, TAM-induced: n=8) and iE4/Cre^ER/+ (Ctrl: n=9, TAM-induced: n=7) mice were examined by Western blotting and quantified. **, P<0.0001. h, ApoE levels in the plasma of the iE3/Cre^ER/+ (Ctrl: n=5, TAM-induced: n=5) and iE4/Cre^ER/+ (Ctrl: n=5, TAM-induced: n=3) mice upon vehicle or TAM administration were analyzed by ELISA. Plasma from APOE3-TR and APOE4-TR mice (n=4/group) was included as positive controls. **, P<0.0001, one-way ANOVA. a, b, g-h, Data represent mean ± s.e.m. N.S., not significant. e-g, two-way ANOVA with a Tukey’s post-hoc test.

Extended Data Fig. 2.. Differential effects of microglia/CAM-derived apoE isoforms on amyloid pathology.

a, Brain sections from 9-month-old APP/iE3/Cre^ER/+ mice (Ctrl, n=16; TAM-induced, n=14) or APP/iE4/Cre^ER/+ mice (Ctrl, n=19; TAM-induced, n=21) were immunostained with a pan-Aβ antibody. The Aβ plaque burden in the hippocampus was quantified (*, P=0.048). Scale bar, 200 μm. b, c, Insoluble Aβ levels in the cortical tissues of APP/iE3/Cre^ER/+ mice (Ctrl, n=17; TAM, n=15) and APP/iE4/Cre^ER/+ mice (Ctrl, n=16; TAM, n=16) were examined by specific Aβ ELISA. (Aβ40:*, P=0.045; Aβ42:*, P=0.043) d, TBS- and TBSX-soluble Aβ levels in the cortex of the APP/iE3/Cre^ER/+ mice (Ctrl, n=17; TAM-induced, n=16) or APP/iE4/Cre^ER/+ mice (Ctrl, n=16; TAM-induced, n=16) were examined. *, P=0.010. e, TBS soluble, detergent-soluble (TBSX fraction) and insoluble (GDN fraction) apoE levels in the cortex of the APP/iE3/Cre^ER/+ (Ctrl, n=17; TAM-induced, n=16) or APP/iE4/Cre^ER/+ (Ctrl, n=16; TAM-induced, n=16) mice were examined. **, P<0.0001. f, Thioflavin S-positive plaques in the cortex of APP/iE3/Cre^ER/+ mice (Ctrl, n=10; TAM, n=10) and APP/iE4/Cre^ER/+ mice (Ctrl, n=13; TAM, n=17) were shown and quantified. Scale bar, 50 μm. In f-h, Images from APP/PS1 mice bred to apoE3-targeted replacement (TR) mice (APP/ApoE3) or apoE4-TR mice (APP/ApoE4) were included for a visualization of plaque morphology. g, Representative images of Aβ staining in the cortex of 9-month-old APP/iE3/Cre^ER/+ or APP/iE4/Cre^ER/+ mice (murine Apoe−/− background) are shown. Staining was performed on more than 10 animals/group with similar results. Scale bar, 200 μm. h, Representative images of plaque compactness examined by Thio S staining in APP/iE3/Cre^ER/+ mice (Ctrl, n=11; TAM, n=10) and APP/iE4/Cre^ER/+ mice (Ctrl, n=12; TAM, n=16) with vehicle treatment (Ctrl) or TAM administration. Scale bars, 10 μm. The shape compactness of amyloid plaque in the brain of experimental mice was quantified. *, P=0.048. a-f, h. Data represent mean ± s.e.m. N.S., not significant. Two-tailed, t-test.

Extended Data Fig. 3.. Expression of apoE4 compromises microglial barrier and phagocytic functions.

a, b, Brain sections from APP/iE3/Cre^ER/+ (Ctrl, n=18; TAM-induced, n=16) or APP/iE4/Cre^ER/+ mice (Ctrl, n=19; TAM-induced, n=21) at 9 months of age were immunostained with Iba1 antibody. The immunoreactivity of Iba1-positive microglia in cortex and hippocampus was quantified. Scale bar, 100 μm. c, d, TAM-induced APP/iE3/Cre^ER/+ mice (n=3) and APP/iE4/Cre^ER/+ male mice (n=4) at 9 months of age were subjected to 2-photon imaging. c, Representative images of GFP-positive microglia (green) and methoxy-X04-stained amyloid plaques (blue) monitored by two-photon in vivo imaging. Plaque associated microglia (number of microglia per 100 μm2; 1-3 fields/mouse) were quantified. Scale bar, 20 μm. *, P=0.018. d, Representative images of microglial phagocytosis in APP/iE3/Cre^ER/+ (n=3) and APP/iE4/Cre^ER/+ mice (n=4) examined by two-photon imaging. The colocalization (pink) of microglia and Aβ are pseudo colored to illustrate the phagocytosed Aβ in microglia which was quantified. Scale bar, 50 μm. *, P=0.032. a-d. Data represent mean ± s.e.m. N.S., not significant. Two-tailed, t-test.

Extended Data Fig. 4.. Differential effects of microglia/CAM-derived apoE isoforms on astrogliosis.

a, b, Brain sections from the APP/iE3/Cre^ER/+ mice (Ctrl, n=16; TAM-induced, n=16) or APP/iE4/Cre^ER/+ mice (Ctrl, n=19; TAM-induced, n=22) at 9 months of age were immunostained with GFAP antibody for astrogliosis, and quantified. The immunoreactivities of GFAP-positive astrocyte in cortex and hippocampus were quantified. Scale bar, 100 μm. iE3:*, P=0.028; iE4:*, P=0.016. c, The correlations between the levels of GFAP-positive astrocytes and amyloid plaque in the APP/iE3/Cre^ER/+ mice and APP/iE4/Cre^ER/+ mice. Correlation coefficient (r) and P-value were acquired by Pearson's correlation analysis. d, The levels of APP, PSD-95, (iE3: Ctrl, n=9; TAM, n=8; iE4: Ctrl, n=9; TAM, n=8), GFAP (iE3: Ctrl, n=9; TAM, n=8; iE4: Ctrl, n=9; TAM, n=7), Synaptophysin (Syp) and aquaporin 4 (Aqp4) (iE3: Ctrl, n=7; TAM, n=7; iE4: Ctrl, n=7; TAM, n=7) in the cortex of the APP/iE3/Cre^ER/+ mice or APP/iE4/Cre^ER/+ mice were examined by Western blotting and quantified. *, P=0.015 (GFAP); *, P=0.013 (Aqp4). a, b, d. Data represent mean ± s.e.m. N.S., not significant. Two-tailed, t-test.

Extended Data Fig. 5.. Single cell transcriptomic analysis of mice with apoE3 or apoE4 expression in microglia/CAMs.

Brain cortical tissues from the APP/iE3/Cre^ER/+ mice (Ctrl, n=6; TAM-induced, n=6) or APP/iE4/Cre^ER/+ mice (Ctrl, n=6; TAM-induced, n=6) at 9 months of age were subjected to single cell RNA-sequencing (scRNA-seq). a, Uniform manifold approximation and projection (UMAP) plot showing the clusters of single cell events captured in scRNA-seq analysis. MG, microglia; EC, endothelial cell; SMC, smooth muscle cell; PC, pericyte; MФ, microphage; CP, choroid plexus. b, Split dot plot depicting marker genes for each cell population in the APP/iE3/Cre^ER/+ mice and APP/iE4/Cre^ER/+ mice. The expression level (color intensity) and the percentage of cells in a cluster expressing a given gene (size of the dot) are reflected in circles. c, Feature plots of canonical markers defining major cell types identified from scRNA-seq analysis. d, The relative proportion of each cell cluster identified in the APP/iE3/Cre^ER/+ mice and APP/iE4/Cre^ER/+ mice with or without TAM administration. e, Proportions of cell types identified from the APP/iE3/Cre^ER/+ mice and APP/iE4/Cre^ER/+ mice with or without TAM administration. MG, microglia; AS, astrocyte; OL, oligodendrocyte; VC, vascular cell; MФ, microphage; CP, choroid plexus.

Extended Data Fig. 6.. Single cell transcriptomic profiling reveals microglial apoE isoform-dependent pathways responding to amyloid pathology.

a, Feature plots showing the expression of selected markers in various microglial subtypes. Legend shows a color gradient of normalized expression. b, UMAP revealed an increased ARM4 (Cluster 9) cell population in mice with apoE3 expression in microglia. c, Percentage of cells from the APP/iE3/Cre^ER/+ and APP/iE4/Cre^ER/+ groups (Ctrl, n=6; TAM-induced, n=6) for each microglial cluster identified. Data represent mean ± s.e.m. **, P<0.0001; N.S., not significant, two-tailed, t-test. The fold changes of cell numbers for each microglial cluster in TAM-induced mice compared with control mice (set as 1) are shown. d, Gene ontology (GO) enrichment analysis for genes in the Cluster 7 (ARM3) of the APP/iE3/Cre^ER/+ mice. e, Gene ontology (GO) enrichment analysis for genes in the Cluster 7 (ARM3) of the APP/iE4/Cre^ER/+ mice.

Extended Data Fig. 7.. eIF2 pathway, oxidative stress and components of complement system are differentially modulated by apoE3 or apoE4 expression in microglia/CAMs.

a, Feature plots of Cluster 7 (ARM3) and Cluster 9 (ARM4) microglial populations. b, c, Heatmap and Violin plots for selected genes in cluster 7 (ARM3) identified from scRNA-seq of the APP/iE4/Cre^ER/+ mice. d, e, Heatmap and Violin plots for selected genes in cluster 9 (ARM4) identified from scRNA-seq of the APP/iE4/Cre^ER/+ mice. f, Venn diagrams indicating up-regulated and down-regulated DEGs in microglia cluster 7 (ARM3) between the APP/iE3/Cre^ER/+ mice and APP/iE4/Cre^ER/+ mice. g, h, Heatmap and Violin plots for selected genes in cluster 9 (ARM4) identified from scRNA-seq of the APP/iE3/Cre^ER/+ mice and APP/iE4/Cre^ER/+ mice. i, Gene ontology (GO) enrichment analysis for genes in the Cluster 9 (ARM4) of the APP/iE3/Cre^ER/+ mice or APP/iE4/Cre^ER/+ mice.

Extended Data Fig. 8.. ApoE isoforms differentially impact transcriptional signatures for ARM1 and ARM2 in amyloid pathogenesis.

a, b, Volcano plots illustrating up- or down-regulated DEGs in activated microglia Cluster 3 (ARM1) or Cluster 4 (ARM2) from the APP/iE3/Cre^ER/+ mice (a) or APP/iE4/Cre^ER/+ mice (b) identified from scRNA-seq analysis. Genes significant at the P value ≤ 0.05 and fold change ≥ 1.2 are denoted red in color for apoE3 mice, and blue in color for apoE4 mice. c, Violin plots showing the mean and variance differences for selected DEGs from ARM1 or ARM2 identified from scRNA-seq analysis in the experimental mice. d, Venn diagrams indicating up-regulated and down-regulated DEGs in microglia cluster 3 (ARM1) between the APP/iE3/Cre^ER/+ mice and APP/iE4/Cre^ER/+ mice. e, Pseudotime analyses of scRNA-seq data showing the transition and trajectory of microglia from homeostatic status to activated signature.

Extended Data Fig. 9.. The effects of apoE expression in microglia/CAMs on anxiety-related behaviors and synaptic transmission.

a, The general locomotor activity, and anxiety-like behaviors in the iE3/Cre^ER/+ mice (Ctrl, n=19; TAM-induced, n=18) or iE4/Cre^ER/+ mice (Ctrl, n=19; TAM-induced, n=20) at 9 months of age were assessed by open field tests. The total distance traveled, average speed, time spent mobile, and the center vs. total distance travelled are shown. b, The anxiety-like behaviors in the iE3/Cre^ER/+ (Ctrl, n=19; TAM-induced, n=18) or iE4/Cre^ER/+ mice (Ctrl, n=19; TAM-induced, n=20) were assessed by elevated plus maze test. Total distance traveled, average speed, total time spent mobile, and ratio of open vs. closed arms are shown. In a, b, Data represent mean ± s.e.m. N.S., not significant. Two-tailed, t-test. c, d, Input-output curve in Schaeffer collaterals-CA1 network showing fEPSPs in CA1 stratum radiatum in response to increased stimulus in the iE3/Cre^ER/+ mice (c; Ctrl, n=12; TAM, n=10 brain slices from 5-6 mice/group) or iE4/Cre^ER/+ mice (d; Ctrl, n=16; TAM, n=12 brain slices brain slices from 5-6 mice/group). The linear regression of fEPSP slope is shown. e, f, Paired-pulse facilitation (PPF) achieved with increased inter-pulse intervals in the iE3/Cre^ER/+ mice (e; Ctrl, n=10; TAM, n=8 brain slices from 5-6 mice/group) or iE4/Cre^ER/+ mice (f; Ctrl, n=11; TAM, n=7 brain slices from 5-6 mice/group) are shown. *, P=0.044; 0.011; 0.015; 0.004; 0.0027. Wilcoxon Rank-sum tests.

Extended Data Fig. 10.. Analyses on behaviors, synaptic plasticity, and microglial responses to focal injury in mice expressing apoE3 or apoE4 in microglia in the absence of Cx3cr1.

a, b, The associative memory of iE3/Cre^ER/ER mice (Ctrl, n=13; TAM, n=14) or iE4/Cre^ER/ER mice (Ctrl, n=15; TAM, n=14) at 6 months of age was assessed by contextual and cued fear conditioning tests. **, P=0.007. c, The general locomotor activity, and anxiety-related behaviors in the iE3/Cre^ER/ER mice (Ctrl, n=13; TAM, n=14) or iE4/Cre^ER/ER mice (Ctrl, n=15; TAM, n=14) at 6 months of age were examined by open field tests. The total distance traveled, average speed, and center: distance are shown. d, The anxiety-like behaviors in the iE3/Cre^ER/ER mice (Ctrl, n=13; TAM, n=14) or iE4/Cre^ER/ER mice (Ctrl, n=15; TAM, n=13) were examined by elevated plus maze test. Total distance traveled, average speed, and open: closed arm are shown. Data represent mean ± SEM. N.S., not significant, Student's t-test. e, f, The time courses of normalized initial slope measurements of fEPSP in hippocampal CA1 neurons in brain slices from the iE3/Cre^ER/ER mice (Ctrl, n=12; TAM, n=13 brain slices from 5-6 mice/group) or iE4/Cre^ER/ER mice (Ctrl, n=7; TAM, n=9 brain slices from 4-5 mice/group). Averages of the last 5 min of fEPSP recording were quantified. **, P=0.001. g, Microglial processes responded and extended toward the ablation (15-20 μm in diameter). h, The kinetics of microglial responses over 30 min; microglial process movement towards a laser-induced focal lesion (circle) in the iE3/Cre^ER/ER mice or iE4/Cre^ER/ER mice (7-9 microglia from 4 mice/group). a-f. Data represent mean ± s.e.m. N.S., not significant. Two-tailed, t-test.

Fig. 1 ∣. ApoE3 expression in microglia/CAMs protects against the development of amyloid pathology and associated neurotoxicity, whereas apoE4 expression is associated with lipid accumulation.

a, The schematic structure of the ROSA-targeting vector for APOE and eGFP in apoE inducible mice. Under murine Apoe null background, apoE inducible mice were bred to Cx3cr1-Cre^ER mice to remove loxP-flanked Neo^r gene, generating tamoxifen (TAM)-inducible, microglia-specific apoE3 or apoE4 mice. b, Illustration of TAM administration paradigms in amyloid model mice. c, d, Brain sections from 9-month-old APP/iE3/Cx3cr1-Cre^ER/+ mice (Ctrl, n=17; TAM, n=15;) (c) and APP/iE4/Cx3cr1-Cre^ER/+ mice (Ctrl, n=19; TAM, n=21) (d) were immunostained with a pan-Aβ antibody. Representative images of immunostaining for Aβ pathology, and quantification of amyloid burden in the cortex of mice with vehicle treatment (Ctrl) or TAM administration (*P=0.024). Scale bar, 1 mm. Closed circles are males; open circles are females. e, Representative images from brains of APP/iE3/Cre^ER/+ mice (Ctrl, n=10; TAM, n=10) and APP/iE4/Cre^ER/+ mice (Ctrl, n=12; TAM, n=16) stained with Iba1 (for labeling microglia) and Thio S (for labeling fibrillary amyloid plaques). The plaque-associated microglia (<30 μm radius) in experimental mice were quantified (*P=0.034). Scale bars, 50 μm. f, Plaque-associated neuritic dystrophy in the cortex was examined by co-immunofluorescence staining of LAMP1 (green) and Aβ plaques (red). The LAMP1 immunoreactivity in the brains of APP/iE3/Cre^ER/+ mice (Ctrl, n=15; TAM, n=16) and APP/iE4/Cre^ER/+ mice (Ctrl, n=13; TAM, n=16) was quantified (*, P=0.037). Scale bar, 50 μm. g, h, Representative images from the brains of APP/iE3/Cre^ER/+ mice (Ctrl, n=10; TAM, n=9) and APP/iE4/Cre^ER/+ mice (Ctrl, n=9; TAM, n=9) stained with Iba1 (green), Plin2 (red), and Aβ plaque (blue). Plaque-associated Plin2 immunoreactivity (<50 μm) in experimental mice was quantified (*P= 0.023; **P=0.002). Scale bars, 25 μm. c-h, Data represent mean ± s.e.m. N.S., not significant. Two-tailed t-test.

Fig. 2 ∣. Transcriptomic analysis reveals apoE4-associated microglial dysfunction in activation and inflammatory responses to amyloid pathology.

a, Cortical brain tissues from 9-month-old control mice and mice expressing apoE3 or apoE4 in microglia/CAMs (n=6/genotype) were subjected to scRNA-seq. Schematic of the droplet-based scRNA-seq workflow is shown. b, Violin plots of selected marker genes for each identified microglial cluster: HM (Clusters 0, 1, 2, 6, 8); ARM (Clusters 3, 4, 7, 9); CPM (Cluster 16). c, Feature plots showing the expression of selected markers in various microglial subtypes. Legend shows a color gradient of normalized expression. d, Microglial cell clusters from scRNA-seq analysis visualized by UMAP. Microglial cells are colored according to identified subpopulations. e, The numbers of DEGs in ARM and CPM clusters identified in the APP/iE3/Cre^ER/+ and APP/iE4/Cre^ER/+ mice. f, g, Volcano plots illustrating up- or down-regulated DEGs in activated microglia Cluster 7 (ARM3) or Cluster 9 (ARM4) from the APP/iE3/Cre^ER/+ mice (f) or APP/iE4/Cre^ER/+ mice (g) (+TAM vs. −TAM). Genes significant at the p value ≤ 0.05 and fold change ≥ 1.2 are denoted red in color for apoE3 mice, and blue in color for apoE4 mice. h, Heatmap revealing the expression of DEGs in activated microglia Cluster 7 (ARM3) from the APP/iE3/Cre^ER/+ mice or APP/iE4/Cre^ER/+ mice with or without TAM administration. i, Violin plots showing the mean and variance differences for selected DEGs in the experimental mice. j, GSEA for the biological pathways and processes that are up-regulated (red) or down-regulated (blue) in each activated microglial cluster in the APP/iE3/Cre^ER/+ and APP/iE4/Cre^ER/+ mice. ARM1 (Cluster 3); ARM2 (Cluster 4); ARM3 (Cluster 7); ARM4 (Cluster 9); CPM (Cluster 16). The CPM cluster was not included because no downregulated pathway was identified in Cluster 16 in the APP/iE3/Cre^ER/+ or APP/iE4/Cre^ER/+ mice.

Fig. 3 ∣. ApoE4 microglia display compromised microglial dynamics and responses to injury with reduced ability to support synaptic function compared to apoE3 microglia.

The iE3/Cre^ER/+ (n=3) and iE4/Cre^ER/+ (n=4) mice (in the absence of amyloid pathology) at 6 months of age were used in this study. a, Time-lapse imaging of the microglial branches in the experimental mice displayed rapid extension and retraction of fine microglial processes over time. Microglial image at 0 min (red) was overlayed with that at 20 min (green). Scale bar, 20 μm. Representative processes that extend and retract over time are shown. b, The experimental paradigm of the two-photon imaging studies. c, The retraction and extension of microglial processes were quantified (**P=002). d, Microglial responses to focal brain injury. Following localized ablation inside the cortex (20 μm in diameter) with a two-photon laser, microglial processes moved rapidly towards the site of injury over time. Scale bar, 50 μm. The time course of microglial processes moved towards a laser-induced focal lesion (circle) in iE3/Cre^ER/+ or iE4/Cre^ER/+ mice over 30 min period of time (9-13 microglia from 4-5 mice/group). e, The morphology of microglia in the cortex of the iE3/Cre^ER/+ or iE4/Cre^ER/+ mice (n=5/group) was examined and skeletonized for analysis. The branch length for each microglia and number of branch points were quantified (**P<0.0001). f, Memory performance of the iE3/Cre^ER/+ mice (Ctrl, n=19; TAM, n=18) and iE4/Cre^ER/+ mice (Ctrl, n=19; TAM, n=20) at 6 months of age examined by contextual (*P=0.044) and cued fear conditioning tests. Closed circle: male; Open circle: female. g, h, Long-term potentiation of the iE3/Cre^ER/+ mice (Ctrl, n=16; TAM, n=11 brain slices from 5-6 mice/group) and iE4/Cre^ER/+ mice (Ctrl, n=17; TAM, n=14 brain slices brain slices from 5-6 mice/group) was examined. Normalized field excitatory postsynaptic potential fEPSP responses to field stimulation were summarized during recordings from the CA1 region of hippocampal slices. Averages of the last 5 min of fEPSP recording were quantified. (g: *P=0.044; h: *P=0.048). In c and e-h, data represent mean ± s.e.m. Two-tailed t-test.

Fig. 4 ∣. Expression of apoE isoforms in microglia/CAMs differentially impacts the development of amyloid pathology and gliosis in the presence of mouse endogenous apoE.

a, ApoE inducible mice in the presence of mouse endogenous Apoe were bred to Cx3cr1-Cre^ER mice. Mice were further bred with 5xFAD amyloid model mice, and the TAM administration paradigm is shown. b, c, Brain sections from 5-month-old 5xFAD; iE3/Cre^ER/+ (Ctrl, n=22; TAM-induced, n=21) or 5xFAD; iE4/Cre^ER/+ mice (Ctrl, n=20; TAM-induced, n=18) were immunostained with Aβ antibody, and quantified (b: *P=0.041; c: *P=0.046). (Top) Scale bar, 1 mm; (Bottom) Scale bar, 200 μm. d-g, Brain sections from 5-month-old 5xFAD; iE3/Cre^ER/+ (Ctrl, n=21; TAM-induced, n=21) or 5xFAD; iE4/Cre^ER/+ mice (Ctrl, n=20; TAM-induced, n=18) were immunostained with Iba1 or GFAP antibody. The immunoreactivities of Iba1 (d,e) and GFAP (f,g) were quantified (d: *P=0.046; g: *P=0.010). Scale bar, 1 mm (top); and 200 μm (Bottom). In b-g, data represent mean ± s.e.m. Two-tailed t-test.

Fig. 5 ∣. APOE4 reduces microglial responses to AD pathology in human brains and human microglia.

a, Volcano plot highlighting DEGs in the total human microglia population from human brain of APOE3/4 and APOE3/3 carriers. Significance at P value ≤ 0.05 and fold change ≥ 1.2 is denoted in red. b, Microglia clusters visualized by UMAP in the scRNA-seq dataset. Activated microglia (ARM)-like cell cluster are shown (right). c, Violin plots of microglial marker genes and selected genes for ARM-like cells. d, The proportion of ARM-like cluster (Cluster 5) among the total microglial population in human brains of APOE3/3 (n=5) or APOE4 (n=3). P= 0.01, Wilcoxon rank-sum test. e-h, Microglial cell clusters in a single-nucleus RNA-Seq dataset from human brain tissues. e, f, ARM-like cell cluster (e), microglial marker genes and selected genes for ARM-like cells (f) are shown. g, Violin plots of selected genes in MHC classes. h, The proportion of ARM-like cell cluster (Cluster 5) among the total microglia population in human brain tissues of APOE3 (n=11) or APOE4 (n=9). P= 0.008, Wilcoxon rank-sum test. i-k, Human AD brain tissues from APOE3/3 or APOE4 carriers were subjected to staining for Iba1 (green), Lgals3 (an activated and phagocytic marker for microglia; red), and Amylo-Glo (blue). Scale bar, 50 μm. The immunoreactivities of amyloid plaque associated Iba1 (j) and Lgals3 (k) in the human brain tissues (n=13/group) were quantified. Horizontal lines are medians and boxes are interquartile ranges (25^th - 75^th Percentile). *P=0.046; **P=0.006, two-tailed t-test. l-n, Schematic illustration for generating iMGL cells. The isogenic iPSCs carrying APOE3 or APOE4 were differentiated into iMGLs. The iPSCs, hematopoietic Progenitor Cells (iHPCs), and iMGLs expressing Tmem119 (red) are shown (l). Scale bars, 200 μm (iPSCs), 500 μm (iHPCs) and 50 μm (iMGL cells)). (m, n) The APOE3 or APOE4 iMGLs were treated with or without Aβ42 (1 μM); BODIPY signals (lipid droplets; green) in Iba1⁺ microglia (red) were quantified. Scale bar, 10 μm. Experiments (n=6) were performed three times from different batches of iMGL differentiation. Data represent mean ± s.e.m. Two-way ANOVA with a Tukey’s post-hoc test.