TREM2-independent microgliosis promotes tau-mediated neurodegeneration in the presence of ApoE4

Abstract

In addition to tau and Aβ pathologies, inflammation plays an important role in Alzheimer's disease (AD). Variants in APOE and TREM2 increase AD risk. ApoE4 exacerbates tau-linked neurodegeneration and inflammation in P301S tau mice and removal of microglia blocks tau-dependent neurodegeneration. Microglia adopt a heterogeneous population of transcriptomic states in response to pathology, at least some of which are dependent on TREM2. Previously, we reported that knockout (KO) of TREM2 attenuated neurodegeneration in P301S mice that express mouse Apoe. Because of the possible common pathway of ApoE and TREM2 in AD, we tested whether TREM2 KO (T2KO) would block neurodegeneration in P301S Tau mice expressing ApoE4 (TE4), similar to that observed with microglial depletion. Surprisingly, we observed exacerbated neurodegeneration and tau pathology in TE4-T2KO versus TE4 mice, despite decreased TREM2-dependent microgliosis. Our results suggest that tau pathology-dependent microgliosis, that is, TREM2-independent microgliosis, facilitates tau-mediated neurodegeneration in the presence of ApoE4.

Keywords: Alzheimer’s disease; ApoE4; TREM2; microgliosis; tau pathology; tau-mediated neurodegeneration.

Figure 1.. TREM2 deletion increases the effect of ApoE4 on hippocampal atrophy and does not counteract the detrimental effect of ApoE4 on tau-mediated synaptic loss in 9-month-old TE4 mice.

(A) Representative images of Cresyl violet staining from brains of 9-month-old mice. Scale bars: 1 mm. (B–E) Quantification of the average volume of the hippocampus (B), entorhinal and piriform cortices (C), granule cell layer of the dentate gyrus (DG) (D), and protein levels of neurofilament light chain (NfL) (E). (F) Representative images and (G) Quantification of postsynaptic PSD-95 (red) puncta numbers per image (region of interest – ROI) in the HC. Scale bars: 10 μm. Data are presented as mean ± SEM. Significance was determined using a one-way ANOVA followed by a Tukey’s post hoc test for (C) and with a Kruskal–Wallis test followed by a Dunn’s post hoc test for (D, E, G) due to the nonparametric data set. Welch’s and Brown–Forsythe ANOVA test was used for (B) due to significantly different variances. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (n=20–27 mice/group).

Figure 2.. TREM2 deletion does not reduce tau pathology in 9-month-old TE4 mice.

(A-C) Representative images of pTau AT8 (A), AT180 (B) and tau confirmational dependent MC1 (C) staining in brain sections. Scale bars: 1 mm. Quantification of the percentage area covered by AT8, AT180, and MC1 staining in the hippocampus (C, D, and E respectively) and piriform cortex (G, H, and I respectively). (n=20–30 mice/group). (J) Western blot detection of AT8, AT180, total tau and actin protein levels in RIPA fraction extracted from hippocampal tissue (3 representative lanes/group). (K) Western blot detection of AT8, AT180 and total tau protein levels in Sarkosyl-insoluble fraction extracted from hippocampal tissue (3 representative lanes/group). (L- Q) Quantitative analysis of AT8, AT180 and total tau relative levels in RIPA (L, M, and N respectively) and Sarkosyl-insoluble (O, P, and Q respectively) fractions (n=7 mice/group). (R–U) Total hippocampal pTau and total tau concentrations in Sarkosyl-soluble (R and S respectively), and Sarkosyl-insoluble (T and U respectively) fractions assessed by ELISA. (n=15 mice/group). (V) Distribution of the four pTau staining types in hippocampus from AT8 staining. Type 1 has intense mossy fiber staining as well as diffuse cell body staining in the dentate gyrus granule cell layer and CA1 pyramidal cell layer; type 2 has compact and dense tangle-like cell body staining primarily in the dentate gyrus granule cells and CA3 pyramidal cells, but also has sparse staining in the CA1 region; type 3 has staining primarily in the neuropil of the stratum radiatum of the CA region with clear staining of dendrites from pyramidal neurons and only some staining in the neuronal cell bodies; type 4 has dense staining over the entire hippocampus, unlike other staining patterns; type 4 staining is fragmented, dotted, and grainy. Data are presented as the mean ± SEM. Significance was determined using a one-way ANOVA followed by a Tukey’s post hoc test for (E, G, H, L, M, N, R, S) and with a Kruskal–Wallis test followed by a Dunn’s post hoc test for (D, F, I, O, P, Q, U) due to the nonparametric data set. Welch’s and Brown–Forsythe ANOVA test was used for (T) due to significantly different variances. For (V), Fisher’s exact test two-sided was used with adjusted p value based on false-discovery rate. *, P < 0.05; **, P < 0.01.

Figure 3.. snRNA-seq of microglia reveals changes regulated by TREM2 removal, ApoE4 and tau pathology in PU.1+ nuclei.

(A) Experimental design for isolation of PU.1⁺ nuclei from hippocampus. (B) Flow plots from the PU.1 sort gate showing expression of the nuclei markers NeuN and PU.1 for neurons and microglia, respectively. (C) UMAP for 25,166 nuclei from n=3 per group and annotated by cluster. (D) UMAP of canonical microglia markers Cx3cr1, Csf1r, and Hexb. (E) Expression of canonical marker genes delineates 9 microglial states and one macrophage (Mac) population (Homeo = homeostatic microglia; IRM = Interferon responsive microglia; Inflam = inflammatory microglia; Prol = proliferative microglia; TERM = Tau/ApoE4 reactive microglia, Mac = perivascular macrophages). (F) Donut plot for proportion of different PU.1+ clusters. (G) Stacked barplots showing the proportion of microglia states in the six experimental groups. (H) UMAP of IRM marker genes Oasl2 and Mx1. (I) Dot plot of IRM genes. (J) GO term analysis of IRM marker genes compared to homeostatic microglia.

Figure 4.. Microglial reactivity profile in 9-month-old TE4 and TEKO mice deleted for TREM2.

(A) UMAP showing the cluster distribution of all six experimental groups. (B) UMAP of selected up-regulated (Lpl, Spp1) and down-regulated (P2ry12, Sall1) Tau/ApoE4 reactive microglia marker genes. (C) Volcano plot showing the fold changes (log2(FC)) between Tau/ApoE4 reactive microglia to homeostatic microglia and their significance (-log(10) FDR). Significant up-regulated and down-regulated genes are depicted in red and blue, respectively. (D) GO analysis of 440 up-regulated genes in Tau/ApoE4 reactive microglia compared to homeostatic microglia. (E-F) Dot plot of selected genes showing fraction of nuclei and level of expression of homeostatic microglia genes from the Tau/ApoE4 reactive microglia cluster. (G) Representative images and quantification (H) of the average volume covered by P2ry12 in the HC. Scale bars: 40 μm. (I-J) Dot plot showing fraction of nuclei and level of expression of TREM2-dependent (I) and TREM2-independent (J) genes from the Tau/ApoE4 reactive microglia cluster. (G) Representative images and quantification (H) of the average volume covered by Clec7a in the HC. Scale bars: 40 μm. Data are presented as mean ± SEM. Significance was determined using a Kruskal–Wallis test followed by a Dunn’s post hoc test for (H) due to the nonparametric data set. Welch’s and Brown–Forsythe ANOVA test was used for (L) due to significantly different variances. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (n=20–27 mice/group).

Figure 5.. Lysosomal burden and synaptic engulfment by microglia are increased in TE4 mice despite TREM2 deletion.

(A) UMAP of Grn gene from Tau/ApoE4 reactive microglia. (B) Dot plot of selected genes showing fraction of nuclei and level of expression of lysosomal-associated genes from the Tau/ApoE4 reactive microglia cluster. (C) Representative images and (D) Quantification of the CD68 (green) volume per microglia (Iba1 (red)) in the HC. Scale bars: 40 μm. (E) Representative images and (F) Quantification of engulfed PSD-95 (red) puncta within CD68⁺ (green) vesicles per microglia (Iba1 (white)) in the HC. Scale bars: 10 μm. (G) Representative images of Lamp1 (green), GFAP (white) and Iba1 (red) in the HC. Scale bars: 50 μm. (H) Quantification of ratios Lamp1 volume in Iba1+ cells/Iba1 volume (B) and LAMP1 volume in GFAP+ cells/GFAP volume (I). Data are presented as mean ± SEM. Significance was determined using a one-way ANOVA followed by a Tukey’s post hoc test for (H, I) and using a Kruskal–Wallis test followed by a Dunn’s post hoc test due to the nonparametric data set for (D, F). *, P < 0.05; **, P < 0.01; ****, P < 0.0001. (n=20–27 mice/group).

Figure 6.. Lysosomal damage and aberrant lysosomal lipid accumulation in TE4 mice despite TREM2 deletion.

(A) Representative images of Galectin-3 (red) and Iba1 (green) in the HC. Scale bars: 20 μm. (B) Quantification of the ratio of Galectin-3 colocalized with Iba1 volume per Iba1 microglia volume. (C) Representative images of LipidTox (green), CD68 (red) and Iba1 (purple) in the HC. Scale bars: 15 μm. (D) Quantification of the ratio of LipidTox volume within CD68⁺ vesicles per Iba1 microglia volume. (E) Representative proportion of the percent of genes from the CLEAR network within the upregulated genes in TE4-T2KO vs. TE4 microglia. Data are presented as mean ± SEM. Significance was determined using a Kruskal–Wallis test followed by a Dunn’s post hoc test due to the nonparametric data set for (B, D). For B, one outlier has been detected and excluded from the analysis in the TE4-T2KO group using the ROUT method based on the False Discovery Rate with Q=1%. For E, we used a Chi squared test = 53.948 with 1 degrees of freedom; two-tailed P value < 0.0000001. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. (n=20–27 mice/group).

Figure 7.. Human single-nuclei RNA sequencing from AD-E4 patients display similar microglia profiles in TREM2 variant carriers (p.R47H, p.R62H) vs. TREM2 common variant carriers) as in TE4 mice.

(A) UMAP representation of human microglia snRNA-seq data. Micro.0 corresponds to resting-state microglia, and Micro.1 corresponds to activated microglia. (B) Distribution of donors and genotypes for Micro.0 and Micro.1. (C) Gene signature score comparisons between Micro.0 and Micro.1. (D) Reactive microglia (Micro.1) gene expression patterns for the signatures of interest across TREM2 genotypes. (E) Gene signature comparisons for the signatures of interest across TREM2 genotypes. Gene signature comparisons p-values calculated using Wilcoxon rank-sum test and adjusted for multiple testing using a Bonferroni correction.

Figure 8.. TREM2 deletion increases GFAP+ astrocytes and ApoE levels in TE4 mice.

(A) Representative images of ApoE (green), Iba1 (red) and GFAP (white) in the HC. Scale bars: 30 μm. (B, C) Quantification of the average volume covered by Iba1 (B) and GFAP (C)/ROI. (D) Quantification of the ratio of ApoE volume within Iba1+ cells divided by Iba1 volume. (E) Quantification of the ratio of ApoE volume within GFAP+ cells divided by GFAP volume. (F) Representative images of ApoE (white), LAMP1 (cyan), Iba1 (red) and GFAP (green) in the HC. Scale bars: 15 μm. (G-H) Quantification of the colocalized ApoE-LAMP1 volume within GFAP+ (G) and Iba1+ (H) cells. Data are presented as mean ± SEM. Significance was determined using an unpaired, 2-tailed Mann-Whitney test due to the nonparametric data set, except for (C) where an unpaired, 2-tailed t-test with Welch’s correction was used due to significantly different variances. *, P < 0.05 P < 0.01; ****, P < 0.0001. (n=20–22 mice/group).