Trem2 Deletion Reduces Late-Stage Amyloid Plaque Accumulation, Elevates the Aβ42:Aβ40 Ratio, and Exacerbates Axonal Dystrophy and Dendritic Spine Loss in the PS2APP Alzheimer's Mouse Model

Induction of Neurodegeneration-related and Proliferation gene modules and Wnt-related signaling components is impaired in Trem2^ko microglia. A, Heat map of differentially expressed genes (DEGs) between Trem2^wt and Trem2^ko microglia from 14 month PS2APP mouse brains (fold change ≥ 2, adjusted p ≤ 0.05), cross-referenced against previously published datasets from the PS2APP (GSE89482), APPswe/PS1de9 (GSE74615), and 5xFAD (GSE65067) models. Each row is a DEG, and each column is a microglial sort from a different mouse. z-score coloring represents a sample's distance in SDs from the mean expression value for a given gene across samples within a dataset. Most of the downregulated DEGs in Trem2^ko microglia were typically induced in models of β-amyloid pathology, and the majority also showed impaired microglial induction in Trem2^ko 5xFAD mice. For a complete list of genome-wide expression values in PS2APP;Trem2^wt and PS2APP;Trem2^ko microglia, see Figure 2-1). B, Induction of the Wnt/proliferation regulators Dkk2, Wif1, Ctnna3, and Asb11 was completely impaired in Trem2^ko microglia of PS2APP mice. C, DEGs identified by GO analysis as “positive regulators of Wnt signaling” with reduced expression in PS2APP;Trem2^ko microglia included Fzd9, Sulf2, Bambi, Ptk7, Aspm, and Dkk2. B, C, Bars and lines represent mean ± SEM, with each data point representing microglial gene expression level from a given mouse. For genes in B and C, see also Figure 2-2, showing microglial expression in the 5xFAD model with or without Trem2 (data from independent investigators). D, Top, Analysis of previously published expression profiles from the PS2APP model (GSE89482) indicated that six gene expression modules for brain myeloid cells (defined by Friedman et al., 2018) showed altered expression in microglia from brains with β-amyloid pathology. Bottom, Analysis of Trem2^ko and Trem2^wt microglia expression profiles from PS2APP mice showed that the Neurodegeneration-related and Proliferation gene sets showed significant Trem2 dependence. For data from independent investigators showing enriched expression of Proliferation module in 5xFAD model microglia and its dependence on Trem2 for full induction, see also Figure 2-2). For heat map displays of all individual genes in each module, see also Figure 2-3. Each data point represents a gene set expression score for microglia isolated from a given mouse. Lines indicate mean ± SEM. Two-tailed t tests assuming unequal variance between groups: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. E, Four-way plot of Neurodegeneration-related gene set, with each point representing a gene's fold change in expression between PS2APP versus nontransgenic microglia on the x axis (red and blue points, adjusted p < 0.05) and between PS2APP;Trem2^ko versus PS2APP;Trem2^wt microglia on the y axis (green and blue points, adjusted p < 0.05). Blue points represent significant differential expression in both datasets. Tiny black points represent genes not differentially expressed in either dataset.

Trem2 deletion shows age- and sex-dependent effects on amyloid plaque pathology, with reduced plaque accumulation at later ages. A, B, Amyloid plaque was visualized using the Campbell-Switzer silver stain method in nontransgenic (Ntg) controls or transgenic PS2APP mice with WT (wt), heterozygous (het), or homozygous KO (ko) Trem2 alleles. Quantification of the percent area covered by amyloid plaque was measured from coronal sections of indicated sex and age. Representative low (left, scale bar, 400 μm) and high (right, scale bar, 100 μm) magnification images of amyloid stains are shown. In the absence of Trem2, plaque loads were elevated in 6–7 month females but reduced in 12 month females and in 19–22 month males. For two-photon imaging of methoxy-X04-labeled plaque in somatosensory cortex of 6 month females and 8 month males, see also Figure 3-1. C, The Campbell-Switzer silver stain turns highly mature plaque cores amber (red arrowheads). Quantification plot (left) and representative images (right, scale bar, 20 μm) of amber core frequency in 19–22 month male mice. Error bars indicate mean ± SEM. Unpaired t test or ANOVA followed by Tukey's multiple-comparison test: *p < 0.05, **p < 0.01, ****p < 0.0001 versus PS2APP;Trem2^wt or as indicated.

Trem2 deletion impairs the consolidation of β-amyloid into dense plaque. A, β-amyloid plaques were visualized by X-34 staining and confocal z-stack imaging. Representative maximum projection images of X-34⁺ plaques from 12 month PS2APP females of different Trem2 genotypes are shown (top). High-magnification images show a representative plaque from each genotype with the outlined masks used to delineate the compact core (green) and compact+diffuse (blue) areas of the plaque (bottom). Scale bars, 20 μm. B, The diffuseness index (see Materials and Methods) of the X-34⁺ plaques in cohorts of the indicated age and sex was quantified. Each data point represents 1 animal's plaque diffuseness index averaged from 3 to 4 FOV. Error bars indicate mean ± SEM. Unpaired t test or ANOVA followed by Tukey's multiple-comparison test: **p < 0.01, ***p < 0.001, ****p < 0.0001 versus PS2APP;Trem2^wt.

Trem2 deletion increases the Aβ42:Aβ40 ratio and soluble fibrillar Aβ oligomers in PS2APP brains. A, B, Frozen hippocampal tissues were homogenized and processed for measuring the abundance of Aβ40 and Aβ42 peptides by ELISA in the soluble (TBS) and insoluble (GuHCl) fractions. The ratio of Aβ42:Aβ40 in the TBS (A) and GuHCl (B) fractions is shown. Each data point represents 1 animal's Aβ42:Aβ40 ratio. For individual Aβ40 and Aβ42 peptide measurements, see Figure 5-1. C, Image of nondenaturing dot blot of hippocampal soluble TBS homogenates from 12 month female animals immunostained with the fibrillar oligomeric Aβ antibody OC (green) and control β-actin antibody (red). Squares outline PS2APP;Trem2^wt samples. Circles outline PS2APP;Trem2^ko samples. #, Control sample from a PS2APP;Bace1^ko mouse. D, Signal intensity ratios of OC antibody to actin antibody are plotted for the dot blot shown in C. Dotted line indicates the OC:actin ratio for a control sample from a PS2APP;Bace1^ko mouse. E, The Aβ42:Aβ40 ratio (from A) and normalized OC⁺ dot blot signal (from D) in the TBS soluble fraction showed a significant positive correlation (linear regression; df(1,28), F = 8.63, p < 0.01). F, Same as in C, except immunostained with pan-Aβ antibody 6E10 (green) and control GAPDH antibody (red). G, Signal intensity ratios of Aβ 6E10 antibody to control GAPDH antibody are plotted for the dot blot shown in F. The PS2APP;Bace1^ko control sample still has substantial 6E10 signal (see dotted line) since the N-terminal Aβ residues recognized by 6E10 are present in soluble APP when α-secretase is the responsible enzyme. H, The Aβ42:Aβ40 ratio (from A) and normalized Aβ 6E10 dot blot signal (from G) in the TBS soluble fraction showed a significant negative correlation (linear regression; df(1,28), F = 4.98, p < 0.05). Error bars indicate mean ± SEM. Unpaired t test or ANOVA followed by Tukey's multiple-comparison test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus PS2APP;Trem2^wt or as indicated.

ApoE markedly accumulates on plaques and in microglia in PS2APP;Trem2^ko females. A, Plaque-associated ApoE signal was quantified for each animal in each cohort using 2 or 3 sections per animal, with each section having a range of ∼50–800 plaques contributing to the analysis. Significant increases were observed in 6–7 month and 12 month female (f) PS2APP;Trem2^ko mice, but not in males (m). B, Representative low-magnification images of the hippocampus (top row) and high-magnification images of the subiculum (enlarged from the boxed regions) from 12 month female brain sections stained with methoxy-X04 to label plaque (blue), anti-Iba1 to label microglia (green), and anti-ApoE (red). Arrows point to examples of plaques with intense ApoE staining, which are atypical in Trem2^wt females but typical in Trem2^ko females at this age. C, Representative 20× maximum projection of confocal z-stacks imaged from cortex, highlighting ApoE (red) localization in microglia (Iba1, green) and plaque (blue). Microglia with small ApoE puncta (arrows) are typical in Trem2^wt, whereas microglia with enlarged ApoE structures (arrowheads) are frequent in Trem2^ko. Plaques with strong ApoE labeling are more common in Trem2^ko (asterisks). D, Analysis of the fraction of Iba1 signal that overlaps with ApoE staining (Mander's colocalization coefficient) in 12 month PS2APP females revealed increased colocalization in Trem2^ko microglia. E, RNA-Seq profiles of microglia FACS-purified from brains of 14 month PS2APP mice showed no difference in Apoe expression between Trem2^wt and Trem2^ko microglia (MG). F, Expression profiles of microglia FACS-purified from brains of 8 month nontransgenic or 5xFAD mice revealed strong Apoe induction by β-amyloid pathology in both Trem2^wt and Trem2^ko microglia, with induction in 5xFAD;Trem2^ko relative to 5xFAD;Trem2^wt microglia being twofold reduced in one dataset (GSE132508, RNA-Seq) and not significantly different in another (GSE65067, microarray). Error bars indicate mean ± SEM. Unpaired t test or ANOVA followed by Tukey's multiple-comparison test: *p < 0.05, **p < 0.01, ****p < 0.0001 versus PS2APP;Trem2^wt or as indicated. n.s. - not significant.

Trem2 deletion exacerbates plaque-proximal axonal dystrophy. A, Representative images from 12 month brain sections stained with methoxy-X04 to label plaque, anti-Iba1 to label microglia, and anti-Lamp1 to label dystrophic axons around plaque. B, Microglial clustering around plaque is impaired in Trem2-deficient mice. Plaque-associated Iba1 signal was quantified for each animal in each cohort using 2 or 3 sections per animal, with each section having hundreds or thousands of plaques contributing to the analysis. C, Axonal dystrophy per plaque is exacerbated in Trem2-deficient mice. Plaque-associated Lamp1 signal was quantified for each animal in a similar manner as Iba1 signal in B. D, Total axonal dystrophy is exacerbated in Trem2-deficient mice. Total Lamp1 signal in each section was quantified, and each data point represents the average score from 2 or 3 sections per animal. Error bars indicate mean ± SEM. Unpaired t test or ANOVA followed by Tukey's multiple-comparison test: **p < 0.01, ***p < 0.001, ****p < 0.0001 versus PS2APP;Trem2^wt or as indicated.

Disintegrative degeneration staining and CSF NfL measurements reveal exacerbated neuronal damage in Trem2-deficient mice at later ages. A, Quantification of sections from nontransgenic (Ntg) and PS2APP mice with indicated Trem2 genotypes stained using an amino cupric silver staining method that labels degenerative neuronal processes. Each data point represents the average percent area covered/section for ∼10 sections per animal. B, Representative low (left, scale bar, 400 μm) and high (right, scale bar, 100 μm) magnification images from the 12 month female and 19–22 month male cohorts are shown. Degenerative signal is apparent in plaque-accompanying foci (red arrowheads) throughout the cortex and hippocampus and in certain white matter tracts, including the corpus callosum (cc). C, Plasma (left) and CSF (right) NfL were measured from a separate, mixed sex cohort of 12 months PS2APP;Trem2^wt and PS2APP;Trem2^ko mice. Error bars indicate mean ± SEM. Unpaired t test or ANOVA followed by Tukey's multiple-comparison test: **p < 0.01, ****p < 0.0001 versus PS2APP;Trem2^wt or as indicated.