The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases

Abstract

Microglia play a pivotal role in the maintenance of brain homeostasis but lose homeostatic function during neurodegenerative disorders. We identified a specific apolipoprotein E (APOE)-dependent molecular signature in microglia from models of amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and Alzheimer's disease (AD) and in microglia surrounding neuritic β-amyloid (Aβ)-plaques in the brains of people with AD. The APOE pathway mediated a switch from a homeostatic to a neurodegenerative microglia phenotype after phagocytosis of apoptotic neurons. TREM2 (triggering receptor expressed on myeloid cells 2) induced APOE signaling, and targeting the TREM2-APOE pathway restored the homeostatic signature of microglia in ALS and AD mouse models and prevented neuronal loss in an acute model of neurodegeneration. APOE-mediated neurodegenerative microglia had lost their tolerogenic function. Our work identifies the TREM2-APOE pathway as a major regulator of microglial functional phenotype in neurodegenerative diseases and serves as a novel target that could aid in the restoration of homeostatic microglia.

Keywords: APOE; Alzheimer’s disease; TREM2; amyotrophic lateral sclerosis; microglia; multiple sclerosis; neurodegeneration; transcriptional regulation.

Figure 1. Reciprocal induction of APOE and suppression of TGFβ signaling in disease-associated MGnD-microglia

(A) K-means clustering of 95 significantly affected common genes in FCRLS⁺ microglia during aging and disease by Nanostring). Vertical lanes are biological replicates per disease stage/condition in WT-aging (n = 12), EAE (n = 18), SOD1 (n = 11) and APP-PS1 (n = 12) mice. Cluster 1: suppressed homeostatic genes; cluster 2: upregulated genes. (B) Linear regression curve of Mef2a, Sall1, Tgfbr1 and Apoe in EAE (n = 4–6 mice/disease score) and SOD1 (n = 2–4 mice/disease score) spinal cord microglia and APP-PS1 (n = 3 mice/age) brain microglia. Thick line: 95% confidence interval of the regression line. (C) Selected homeostatic and disease-associated microglial genes during EAE (n = 3). Data are presented as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA followed by Dunnett’s multiple-comparison post-hoc test. (D) IPA shows common nodes significantly affected in microglia in all three-mouse models in disease. For each molecule, the expression fold change compared to normal, homeostatic microglia is presented. (E) Heatmap of significantly affected genes dysregulated in all 3 diseases (n = 3–4) determined by Nanostring. Vertical lanes: mean of biological replicates per disease stage/condition as indicated in (A). See also Figure S1 and Table S1.

Figure 2. MGnD-microglia are associated with neuritic Aβ-plaques

(A) Staining for P2ry12⁺ and Clec7a⁺ in Aβ-plaque microglia in 24-month-old APP-PS1 mice. MGnD around plaque (red arrows). MGnD transitioning to plaque (yellow arrows). M0-homeostatic microglia (white arrows). Scale bar, 20 μm. (B) Staining for P2ry12⁺ and Clec7a⁺ in microglia associated with neuritic plaque (pNF) in APP-PS1 mice (24 month). Scale bar, 20 μm. (C) Heatmap of significantly affected genes in Clec7a⁺ vs. Clec7a^int vs. Clec7a⁻ FCRLS⁺ microglia in APP-PS1 vs. WT mice (n = 5; 24 month). Selected homeostatic (blue) and inflammatory (red) genes. (D) Selected genes shown in (C). Dot plots: mRNAs transcripts (mean ± s.e.m). *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 by one-way ANOVA followed by Tukey’s multiple-comparison post-hoc test. (E) Heatmap of significantly affected genes determined by RNAseq in Clec7a⁺ vs. Clec7a⁻ FCRLS⁺ microglia in APP-PS1 vs. WT mice (n = 5; 9 month). (F) Selected genes shown in (E). Dot plots: FPKM (mean ± s.e.m). *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001 by one-way ANOVA followed by Tukey’s multiple-comparison post-hoc test. (G) Staining for P2RY12 with Aβ or pNF in diffuse vs. neuritic plaques in human AD brain. Scale bar, 100 μm. (H) Linear regression curve of P2RY12⁺ microglia with dystrophic axons in control temporal cortex and AD defined by CERAD criteria and Braak stages (n = 14) and age-matched controls (n = 10). See also Figure S2 and Table S2.

Figure 3. Phagocytosis of apoptotic neurons suppresses homeostatic microglia

(A) Staining for P2ry12⁺ microglia at the site of apoptotic neuron injection 16 h post-injection in WT mice (n = 6). Phagocytic (MG-dNΦ) and non-phagocytic (MG-nΦ) microglia. Scale bar, 80 μm. (B) Orthogonal projections of confocal z-stacks show intracellular Alexa488⁺ apoptotic neurons co-stained with DAPI in P2ry12⁺ microglia (n = 6). Scale bar, 5 μm. One of two individual experiments. (C) Staining for Apoe in P2ry12⁺ MG-dNΦ. White arrows: Apoe⁻ MG-nΦ; yellow arrows: Apoe⁺ MG-dNΦ (n = 3). Scale bar, 15 μm. (D) FACS-sorted CD11b⁺FCRLS⁺ MG-dNΦ containing Alexa488⁺ apoptotic neurons and MG-nΦ from the same injection site 16 h later. (E) Heatmap of affected genes in MG-dNΦ (n = 4) vs. MG-nΦ (n = 4) 16 h post-injection with apoptotic neurons. One of two individual experiments. (F) Top-20 affected genes shown in (E). Bars: mRNA Nanostring (nCounts) (mean ± s.e.m) detected in 100 ug total RNA. *p < 0.05 by Student t test, 2-tailed. (G) qPCR validation of selected genes. Gene expression level normalized against Gapdh using ΔCt (n = 3–4/group). Results: mean normalized expression ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA followed by Tukey’s multiple-comparison post hoc test. (H) Volcano plot of 552 TMT-mass spectrometry-identified proteins highlighting changes in MG-dNΦ vs. MG-nΦ. p < 0.05 by Student t test, 2-tailed. (I) Circos plot: connectivity map derived from the pairwise comparison of transcriptome data from mouse AD models, ALS and Mfp2^−/−, aging and irradiation shown in orange. Each line represents a pairwise data set overlap, determined using GSEA analysis and filtered by p < 0.001. See also Figure S3, S4, S5 and Tables S3, S4.

Figure 4. APOE regulates transcriptional and post-transcriptional program of MGnD phenotype

(A) K-means clustering (k = 4) of 2,234 affected genes in bulk microglia (1,000 cells per animal) from WT and Apoe^−/− mice. Vertical lane: mean of biological replicates of 1,000 MG-nΦ vs. MG-dNΦ cells from WT (n = 10) vs. Apoe^−/− mice for MG-nΦ (n = 10) and MG-dNΦ (n = 9). Microglia isolated 16 h post-injection of apoptotic neurons. (B–E) Selected Apoe-induced genes (B), Apoe-induced Transcription Factors (TFs) (C), Apoe-repressed genes (D) and Apoe- repressed TFs (E) from WT and Apoe^−/− mice 16 h post-injection of apoptotic neurons. Data: mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA followed by Tukey’s multiple-comparison post-hoc test. (F) qPCR validation of selected genes in MG-dNΦ from Cx3cr1^CreERT2:Apoe^fl/fl vs. Cx3cr1^wt:Apoe^fl/fl controls. Expression levels normalized against Gapdh using ΔCt (n = 6–10/group). Data: mean normalized expression ± s.e.m. *p < 0.05, **p < 0.01, by Student t test, 2-tailed. (G) qPCR validation of genes in MG-nΦ from Cx3cr1^CreERT2:Apoe^fl/fl vs. Cx3cr1^wt:Apoe^fl/fl controls. Expression level normalized against Gapdh using ΔCt (n = 6–10/group). Data: mean normalized expression ± s.e.m. *p < 0.05, **p < 0.01, by Student t test, 2-tailed. (H) Staining for NeuN⁺ and P2ry12⁺ in contralateral vs. axotomized facial motor nucleus 7 d post-surgery in WT (n = 5) vs. Apoe^−/− (n = 5) vs. Cx3cr1^CreERT2:Apoe^fl/fl (n = 4) vs. Cx3cr1^wt:Apoe^fl/fl (n = 4) control mice. Scale bar, 80 μm. (I) Quantification of NeuN⁺ per facial motor nucleus in contralateral vs. axotomized nucleus 7 d post-surgery (n = 5). Data: mean ± s.e.m. *p < 0.05, **p < 0.01, ****p < 0.0001 by one-way ANOVA followed by Tukey’s multiple-comparison post-hoc test. See also Figure S6 and Table S5.

Figure 5. Genetic targeting of Trem2 suppresses APOE pathway and restores the homeostatic microglia in APP-PS1 and SOD1 mice

(A) qPCR analysis of homeostatic genes in MG-dNΦ from Trem2^−/− vs. WT mice (n = 4). Data: mean ± s.e.m. *p < 0.05, **p < 0.01 by one way ANOVA. (B) qPCR analysis of Apoe and miR-155 expression in MG-dNΦ from Trem2^−/− vs. WT mice (n = 3–4/group). Data: mean ± s.e.m normalized against Gapdh and U6 expression using ΔCt. *p < 0.05, by Student t test, 2-tailed. (C) Quantification of NeuN⁺ per facial motor nucleus in contralateral vs. axotomized nucleus in WT vs. Trem2^−/− (n = 4–5/group), 7 d post-surgery. Data: mean ± s.e.m. *p < 0.05, **p < 0.01, ****p < 0.0001 by one-way ANOVA followed by Tukey’s multiple-comparison post-hoc test. (D) Volcano plot based on differentially expressed genes in microglia from APP-PS1 (n = 4) vs. APP-PS1:Trem2^−/− (n = 5) mice at 120 d. p < 0.05 by Student t test, 2-tailed. Selected restored homeostatic (red) and suppressed inflammatory (blue) genes. (E) Staining for P2ry12, Clec7a and Aβ plaque in APP-PS1 vs. APP-PS1:Trem2^−/− mice (120 d). Scale bar, 20 μm. (F and G) Quantification of P2ry12 and Clec7a intensity per Aβ plaque in APP-PS1 vs. APP-PS1:Trem2^−/− mice (n = 5; 120 d). ***p < 0.001, by Student t test, 2-tailed. (H) Quantification of Aβ plaque load per animal in APP-PS1 vs. APP-PS1:Trem2^−/− mice (n = 5; 120 d). **p < 0.01, by Student t test, 2-tailed. (I) Volcano plot based on differentially expressed genes in microglia from SOD1:Trem2^+/− (n = 9) vs. SOD1:Trem2^−/− (n = 13) mice at 115 d (neurological score 1). p < 0.05 by Student t test, 2-tailed. (J) Staining for P2ry12, Clec7a and NeuN in SOD1:Trem2^+/− vs. SOD1:Trem2^−/− mice (lumbar section, 115 d). M0-homeostatic microglia (white arrows). M0 microglia transitioning to MGnD (yellow arrows). MGnD at the epicenter of the ventral horn (red arrows). Scale bar, 50 μm. (K and L) Quantification of P2ry12 and Clec7a intensity in ventral horn of SOD1:Trem2^+/− (n = 4, n = 5 females) vs. SOD1:Trem2^−/− (n = 7 males, 5 females) mice (lumbar section, 115 d). **p < 0.001, ***p < 0.001 by Student t test, 2-tailed. (M) qPCR analysis of miR-155 expression in microglia isolated from ventral horn of SOD1:Trem2^+/− (n = 4) vs. SOD1:Trem2^−/− (n = 5) mice (lumbar section, 115 d). Data: mean ± s.e.m normalized against U6 expression using ΔCt. *p < 0.05, by Student t test, 2-tailed. (N) Volcano plot: differentially expressed genes in spinal cord microglia from male and female SOD1:Trem2^+/− (n = 4 males, 4 females) vs. SOD1:Trem2^−/− (n = 7 males, 5 females) mice at 115 d (neurological score 1). p < 0.05 by Student t test, 2-tailed. Cluster 1: female-specific differentially expressed genes; Cluster 2: differentially expressed genes in both genders; cluster 3: male-specific differentially expressed genes. (O) Heatmap of affected genes in male and female SOD1:Trem2^+/− (n = 4 males, 4 females) vs. SOD1:Trem2^−/− mice (n = 7 males, 5 females) at 115 d (cluster 2). Vertical lanes: biological replicates per genotype and gender. (P) Gender comparison of homeostatic (lower panel) and MGnD genes (upper panel) from SOD1:Trem2^+/− and SOD1:Trem2^−/− mice (n = 4–7/group). Data: mean ± s.e.m. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA, followed by Tukey’s multiple-comparison post-hoc test. See also Table S6.

Figure 6. TREM2 haplodeficiency in human AD lead to preservation of microglia morphology and TMEM119 immunoreactivity

(A) Staining for Aβ (red), IBA1 (blue) in AD control subjects (AD-TREM2^WT, n = 6) and TREM2 variant carriers (AD-TREM2^R47H, n = 2 and AD-TREM2^R62H, n = 2). Lower panels: single distribution of IBA1⁺ microglia in the same section areas. Scale bar, 100 μm. (B) Higher magnification of the plaques indicated by * and + in (A). Scale bar, 50 μm. (C) Quantification of IBA1⁺ microglia associated with Aβ-plaque in AD-TREM2^WT and AD-TREM2^Mut cortical tissue. Student’s t test, 2-tailed. (D) Cortical tissue from AD-TREM2^WT and AD-TREM2^R62H cases stained with TMEM119. Panels show basis for scoring microglia staining, described in (E). Scores: − no cells stained; +/− single cells stained; + moderate number of microglia with TMEM119 reactivity; ++ most microglia are TMEM119 positive. (E) Quantification of TMEM119 in AD-TREM2^WT and AD-TREM2^Mut cortical tissue. TMEM119 expression in microglia rated as in (D); the ratings are shown in numbers: − (0), +/− (1), + (2), ++ (3). Student’s t test, 2-tailed. See also Table S7.

Figure 7. Activation of APOE pathway suppresses tolerogenic function in microglia

(A) CFSE⁺ CD3⁺ T cells cultured with spinal cord microglia of WT-naïve mice. Cultures treated with soluble rApoe (100 ng ml⁻¹) for 72 h. (B) T cell proliferation: mean ± s.e.m ***p < 0.001, Student’s t test, 2-tailed (n = 3). (C) CFSE⁺ CD3⁺ T cells cultured for 72 h with spinal cord microglia from naive or EAE mice at disease peak. (D) T cell proliferation: mean ± s.e.m **p < 0.01, Student’s t test, 2-tailed (n = 3). (E) CFSE⁺ CD3⁺ T cells cultured for 72 h with spinal cord microglia from EAE WT vs. Apoe^−/− mice disease peak. (F) T cell proliferation: mean ± s.e.m ***p < 0.001, Student’s t test, 2-tailed (n = 3). (G) CFSE⁺ CD3⁺ T cells cultured for 72 h with spinal cord microglia from EAE Apoe^−/− mice at disease peak. Cultures treated with soluble rApoe (100 ng ml⁻¹) for 72 h. (H) T cell proliferation: as mean ± s.e.m ***p < 0.001, Student’s t test, 2-tailed (n = 3). (I) CFSE⁺ CD3⁺ T cells cultured for 72 h with spinal cord microglia from WT vs. SOD1 mice at disease peak (neurologic score 4). (J) T cell proliferation: mean ± s.e.m **p < 0.01, Student’s t test, 2-tailed (n = 3). Black line: isotype control. Numbers on FACS plots: representative T cell proliferation (%). Data are representative of 3 independent experiments (A–J).