TREM2 Acts Downstream of CD33 in Modulating Microglial Pathology in Alzheimer's Disease

Abstract

The microglial receptors CD33 and TREM2 have been associated with risk for Alzheimer's disease (AD). Here, we investigated crosstalk between CD33 and TREM2. We showed that knockout of CD33 attenuated amyloid beta (Aβ) pathology and improved cognition in 5xFAD mice, both of which were abrogated by additional TREM2 knockout. Knocking out TREM2 in 5xFAD mice exacerbated Aβ pathology and neurodegeneration but reduced Iba1⁺ cell numbers, all of which could not be rescued by additional CD33 knockout. RNA-seq profiling of microglia revealed that genes related to phagocytosis and signaling (IL-6, IL-8, acute phase response) are upregulated in 5xFAD;CD33^-/- and downregulated in 5xFAD;TREM2^-/- mice. Differential gene expression in 5xFAD;CD33^-/- microglia depended on the presence of TREM2, suggesting TREM2 acts downstream of CD33. Crosstalk between CD33 and TREM2 includes regulation of the IL-1β/IL-1RN axis and a gene set in the "receptor activity chemokine" cluster. Our results should facilitate AD therapeutics targeting these receptors.

Keywords: Alzheimer's; CD33; IL-1beta; RNA-seq; TREM2; amyloid beta; microglia; neuroinflammation; pathway analysis; transcriptomics.

Figure 1.. CD33 knock-out restores retention memory in 5xFAD mice, which is abrogated by additional knock-out of TREM2.

7-month-old WT (n=5M/4F), CD33^−/− (n=4M/4F), TREM2^−/− (n=4M/4F), CD33^−/−;TREM2^−/− (n=4M/4F), 5xFAD (n=5M/4F), 5xFAD;CD33^−/− (n=4M/4F), 5xFAD;TREM2^−/− (n=4M/4F) and 5xFAD;CD33^−/−;TREM2^−/− (n=5M/5F) mice were evaluated in the open field (A and B) and Morris water maze (C-G) tests. (A and B) All mouse genotypes were characterized by similar total distance moved (A, Kruskal-Wallis ANOVA, Dunn’s test) and time spent in the center area (B, one-way ANOVA, Tukey’s test). (C) Time needed to reach the hidden platform was plotted across training days. A two-way repeated measures ANOVA revealed significant effects for days (F_(6,360)=71.24, p<0.0001) and for groups (F_(7,60)=5.781, p<0.0001), but not for interaction (F_(42,360)=1.407, p=0.0539). Two-way ANOVA, Tukey’s test revealed a difference in 5xFAD;CD33^−/− mice versus 5xFAD;CD33^−/−;TREM2^−/− on days 6 (p<0.01) and 7 (p<0.05). (D and E) Time spent by the mice in the area surrounding the platform location (platform plus, D) and the number of platform plus crossovers (E) were recorded during the probe test (day 8). For (D), *p<0.05, **p<0.01, Kruskal-Wallis ANOVA (p<0.0001), Dunn’s test. For (E), **p<0.01, one-way ANOVA (F_(7,60)=9.134, p<0.0001), Tukey’s test. (F) All mouse groups showed similar swim speed during the test (one-way ANOVA, Tukey’s test). (G) On days 9 and 10, no differences in latencies to the visible platform were found among mouse groups (one-way ANOVA, Tukey’s test). Data are represented as mean ± SEM. See also Figure S1.

Figure 2.. CD33 knock-out leads to decreased levels of formic acid-soluble Aβ42 and Aβ plaque burden, abolished by additional knock-out of TREM2.

(A–D) ELISA analysis of Aβ40 (A and C) and Aβ42 (B and D) in TBS-soluble (A and B) and formic acid (FA)-soluble (C and D) fractions isolated from the cortex of 8-month-old mice. (E) Images of cortical and hippocampal fields from mice of indicated genotypes, labeled with the anti-Aβ antibody 3D6. Scale bar represents 100 μm. (F and G) Quantification of amyloid plaque burden in cortex (F) and hippocampus (G) of 8-month-old mice. For (A)-(G), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA, Tukey’s test, 5xFAD (n=7M/7F), 5xFAD;CD33^−/− (n=7M/7F), 5xFAD;TREM2^−/− (n=4M/4F) and 5xFAD;CD33^−/−;TREM2^−/− (n=5M/6F) mice. Data are represented as mean ± SEM. See also Figure S2.

Figure 3.. TREM2 knock-out leads to neuronal cell loss in 5xFAD mice, which is not rescued by additional knock-out of CD33.

(A) Representative pictures from CA1 of 8-month-old mice of indicated genotypes, labeled with anti-NeuN antibody. Scale bar represents 50 μm. (B) Summary of NeuN⁺ cell numbers in CA1 of 8-month-old mice. (C) Representative images from the cortex and hippocampus of 8-month-old mice of indicated genotypes, labeled with an antibody against activated Caspase-3. Scale bar represents 50 μm. (D and E) Quantification of Caspase-3⁺ cells in cortex (D) and hippocampus (E) of 8-month-old mice. For (B), (D) and (E), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA, Tukey’s test, WT (n=4M/4F), CD33^−/− (n=4M/4F), TREM2^−/− (n=4M/4F), CD33^−/−;TREM2^−/− (n=4M/4F), 5xFAD (n=7M/7F), 5xFAD;CD33^−/− (n=7M/7F), 5xFAD;TREM2^−/− (n=4M/4F) and 5xFAD;CD33^−/−;TREM2^−/− (n=5M/6F) mice. Data are represented as mean ± SEM. See also Figure S3.

Figure 4.. TREM2 knock-out leads to reduced Iba1⁺ cell numbers and clustering of Iba1⁺ cells around Aβ plaques in 5xFAD mice, both of which are not rescued by additional knock-out of CD33.

(A) Brain sections were labeled with Iba1 (green) and 3D6 antibody (red) for Aβ plaques. Representative images of cortex and hippocampus from of 8-month-old mice of indicated genotypes. Scale bar represents 50 μm. (B and C) Quantification of Iba1⁺ cells in cortex (B) and hippocampus (C). (D and E) Quantification of Iba1⁺ cells associated with plaques of similar sizes in cortex (D) and hippocampus (E). (F and G) Plaque-associated Iba1⁺ cells were analyzed for their distance from the center of adjacent plaque in cortex (F) and hippocampus (G). For (B)-(G), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA, Tukey’s test, 8-month-old WT (n=4M/4F), CD33^−/− (n=4M/4F), TREM2^−/− (n=4M/4F), CD33^−/−;TREM2^−/− (n=4M/4F), 5xFAD (n=7M/7F), 5xFAD;CD33^−/− (n=7M/7F), 5xFAD;TREM2^−/− (n=4M/4F) and 5xFAD;CD33^−/−;TREM2^−/− (n=5M/6F) mice. (H) Images from the cortex of 8-month-old mice of indicated genotypes, stained with P2ry12 (red) and Iba1-specific antibody (green). Scale bar represents 50 μm. (I and J) Quantification of P2ry12⁺Iba1⁺ cells in cortex (I) and hippocampus (J) of 8-month-old mice. (K and L) Quantification of % P2ry12⁺Iba1⁺/Iba1⁺ cells in cortex (K) and hippocampus (L) of 8-month-old mice. For (I)-(L), **p<0.01, ***p<0.001, one-way ANOVA, Tukey’s test, 5xFAD (n=4M/4F), 5xFAD;CD33^−/− (n=4M/4F), 5xFAD;TREM2^−/− (n=4M/4F) and 5xFAD;CD33^−/−;TREM2^−/− (n=4M/4F) mice. Data are represented as mean ± SEM. See also Figure S4.

Figure 5.. The microglial transcriptome changes in a progressive, age-dependent manner in 5xFAD mice.

(A) Expression heatmap of top 40 DE genes in 4-month-old 5xFAD microglia compared to WT by RNA-seq. Genes were ranked by FC; samples were grouped by genotype (WT: n=13M/14F, 5xFAD: n=14M/14F mice). (B) Corresponding volcano plot of 5xFAD microglia versus WT showed 481 upregulated (log₂FC>1, FDR<0.05, red) and 5 downregulated (log₂FC<−1, FDR<0.05, blue) genes. (C) Expression heatmap of top 40 DE genes in 8-month-old 5xFAD microglia compared to WT, ranked by FC, and grouped by genotype (WT: n=8M/8F, 5xFAD: n=10M/9F mice). (D) Corresponding volcano plot of 5xFAD microglia relative to WT. (E) Heatmap of top 40 DE genes in 8 and 4-month-old 5xFAD microglia compared to WT, ranked by FC of 8-month-old 5xFAD versus WT, grouped by genotype and time point. (F) Anti-inflammatory, pro-inflammatory and inflammasome-associated genes were concurrently upregulated in 8-month-old 5xFAD microglia versus WT. (G) Genes related to pathogen sensing and host defense (Clec7a➔C5ar1, blue and navy bars) were upregulated in 8-month-old 5xFAD mice compared to WT. Genes related to sensing endogenous ligands (Siglech➔Clec4a2, red bars) were downregulated. (H) Upregulated (left) or downregulated (right) genes in 5xFAD microglia versus WT at each time point, displayed as Venn diagrams. See also Tables S1 and S2.

Figure 6.. Differential gene expression in 5xFAD;CD33^−/− microglia is contingent upon the presence of TREM2, but this is not the case for CD33 in 5xFAD;TREM2^−/− microglia.

(A) Expression heatmap of top 40 DE genes in 4-month-old 5xFAD;CD33^−/− microglia compared to 5xFAD (left) and 5xFAD;CD33^−/−;TREM2^−/− relative to 5xFAD;TREM2^−/− (right). Genes were ranked by FC; samples were grouped by genotype (5xFAD: n=14M/14F, 5xFAD;CD33^−/−: n=6M/6F, 5xFAD;TREM2^−/−: n=11M/11F and 5xFAD;CD33^−/−;TREM2^−/−: n=5M/5F mice). (B) Volcano plot of 5xFAD;CD33^−/− microglia in the presence of TREM2 (left) showed 24 downregulated (log₂FC<−1, FDR<0.05, blue) and 275 upregulated (log₂FC>1, FDR<0.05, red) genes. Volcano plot of 5xFAD;CD33^−/− microglia in the absence of TREM2 (right) yielded only 2 downregulated and 8 upregulated genes. (C) Expression heatmap of top 40 DE genes in 4-month-old 5xFAD;TREM2^−/− microglia versus 5xFAD (left) and 5xFAD;CD33^−/−;TREM2^−/− compared to 5xFAD;CD33^−/− (right). (D) Volcano plot of 5xFAD;TREM2^−/− microglia in the presence of CD33 (left) showed 125 downregulated and 9 upregulated genes. Volcano plot of 5xFAD;TREM2^−/− in the absence of CD33 (right) yielded 238 downregulated and 30 upregulated genes. (E) Top 60 DE genes (p-value<0.001 and FDR<0.05) in 5xFAD versus WT were selected and corresponding log₂FC values were hierarchically clustered (euclidean distance, average linkage) across the datasets: 5xFAD;CD33^−/− versus 5xFAD, 5xFAD;CD33^−/−;TREM2^−/− versus 5xFAD;TREM2^−/−, 5xFAD;TREM2^−/− versus 5xFAD, and 5xFAD;CD33^−/−;TREM2^−/− versus 5xFAD;CD33^−/−. The heatmap is presented with row dendrograms and cluster membership (by row color). See also Figures S5, S6 and Tables S2 and S3.

Figure 7.. Crosstalk between CD33 and TREM2 in 5xFAD microglia includes regulation of gene sets related to the extracellular collagen matrix and receptor activity chemokine clusters.

(A) Expression heatmap of top 40 DE genes in 8-month-old 5xFAD;CD33^−/− microglia that were also DE in 4-month-old 5xFAD;CD33^−/− (compared to 5xFAD). Genes were ranked by FC of 8-month-old 5xFAD;CD33^−/− versus 5xFAD; samples were grouped by genotype and time point (5xFAD: n=14M/14F, 5xFAD;CD33^−/−: n=6M/6F at 4 months; and 5xFAD: n=12M/12F, 5xFAD;CD33^−/−: n=13M/13F mice at 8 months). (B) Heatmap of top 40 DE genes in 8-month-old 5xFAD;TREM2^−/− microglia that were also DE in 4-month-old 5xFAD;TREM2^−/− (compared to 5xFAD). Genes were ranked by FC of 8-month-old 5xFAD;TREM2^−/− versus 5xFAD; samples were grouped by genotype and time point (5xFAD: n=14M/14F, 5xFAD;TREM2^−/−: n=11M/11F at 4 months; and 5xFAD: n=10M/9F, 5xFAD;TREM2^−/−: n=9M/8F mice at 8 months). (C) Volcano plot of 5xFAD;CD33^−/− microglia versus 5xFAD at 8 months revealed 8 downregulated (log₂FC<−1, FDR<0.05, blue) and 274 upregulated (log₂FC>1, FDR<0.05, red) genes. (D) Volcano plot of 8-month-old 5xFAD;TREM2^−/− microglia versus 5xFAD. (E) Gene set enrichment analysis revealed 277 gene sets enriched for 5xFAD;CD33^−/− versus 5xFAD and 342 gene sets for 5xFAD;TREM2^−/− versus 5xFAD group (p-value<0.001, FDR<0.05) at 8 months. The top 30 significant gene sets are shown for each group. (F) Enrichment maps were generated for 8-month-old 5xFAD;CD33^−/− versus 5xFAD and 5xFAD;TREM2^−/− versus 5xFAD datasets. Four edges (connections) were found between the gene sets related to the extracellular collagen matrix and chemokine receptor activity cluster. See also Figures S7, S8 and Tables S2 and S5.