Loss of TREM2 function increases amyloid seeding but reduces plaque-associated ApoE

Abstract

Coding variants in the triggering receptor expressed on myeloid cells 2 (TREM2) are associated with late-onset Alzheimer's disease (AD). We demonstrate that amyloid plaque seeding is increased in the absence of functional Trem2. Increased seeding is accompanied by decreased microglial clustering around newly seeded plaques and reduced plaque-associated apolipoprotein E (ApoE). Reduced ApoE deposition in plaques is also observed in brains of AD patients carrying TREM2 coding variants. Proteomic analyses and microglia depletion experiments revealed microglia as one origin of plaque-associated ApoE. Longitudinal amyloid small animal positron emission tomography demonstrates accelerated amyloidogenesis in Trem2 loss-of-function mutants at early stages, which progressed at a lower rate with aging. These findings suggest that in the absence of functional Trem2, early amyloidogenesis is accelerated due to reduced phagocytic clearance of amyloid seeds despite reduced plaque-associated ApoE.

Figure 1.. Increased amyloidogenesis upon loss of Trem2 function.

(a) Schematic illustration of the study design and timeline. (b) In contrast to APPPS1/Trem2^+/+, Trem2 loss of function mice (APPPS1/Trem2^−/− and APPPS1/Trem2^p.T66M) show increased amyloid seeding in the dentate gyrus at four months when injected with APPPS1 brain homogenates. Insets show seeding pathology at higher magnification as indicated by the area in dotted white box. (c) Mice were analyzed for 4G8-positive immunoreactivity (n^+/+=8 mice, n^−/−=7 mice, n^p.T66M=7 mice; F_2,19=13.13, p=0.0003) and (d) ThioS-positive Aβ deposits (n^+/+=7 mice, n^−/−=6 mice, n^p.T66M=6 mice; F_2,16=16.73, p=0.0001). (e) Aβ₄₂ levels quantified in formic acid fractions using Meso-Scale discovery electrochemiluminescence assay confirm increased amyloid seeding in APPPS1/Trem2^−/− and APPPS1/Trem2p^.T66M mice compared to APPPS1/Trem2^+/+ mice (n=8 mice/genotype; F_2,21=9.016, p=0.0015). (f) Immunoblotting with the anti-Aβ antibody 6E10 demonstrates increased Aβ in the formic acid fraction of APPPS1/Trem2^−/− and APPPS1/Trem2^p.T66M as compared to APPPS1/Trem2^+/+ mice. Western blots were independently repeated five times to analyze n=10 mice/genotype. Full image of immunoblots are shown in Supplementary Fig. 7. Data represent mean ± SEM. One-way ANOVA, Dunnetts’s post hoc analysis; *p<0.05, **p<0.005, ***p<0.0005.

Figure 2.. Reduced microglial clustering, CD68 and Trem2 expression in microglia around newly seeded plaques in Trem2 loss of function mice.

(a) First panel: Random distribution of IBA1-positive microglia in C57BL6 mice. Second panel: Clustering of IBA1 and Trem2-positive microglia around seeded plaques in the hippocampus of APPPS1/Trem2^+/+. Third and fourth panel: Loss of Trem2 function reduces microglia clustering around seeded plaques. (b) IBA1-positive microglia clustering around seeded amyloid plaques (n^+/+=9 mice, n^−/−=7 mice, n^p.T66M=7 mice, F_2,20=38.87, p=1.5E-7) normalized to seeded amyloid pathology shown in Fig. 1c. (c) IBA1 immunoreactivity quantified in the hippocampus excluding seeded dentate gyrus (n^+/+=10 mice, n^−/−=9 mice, n^p.T66M=9 mice, F_2,25=1.657, p=0.2109) (d) First panel: CD68 expression is at the detection limit in C57BL6 mice. Second panel: Increased CD68 staining in IBA1-positive microglia around seeded plaques in the presence of functional Trem2. Third and fourth panel: reduced CD68 expression in microglia around seeded plaques in the absence of functional Trem2. (e) Quantification of CD68-positive microglia in the seeded dentate gyrus (n=6 mice/genotype, F_2,15=44.87, p=4.6E-7) normalized to seeded amyloid pathology shown in Fig. 1a. Dotted white boxes indicate the area in each staining that is merged and shown at higher magnification. Data represent mean ± SEM. One-way ANOVA, Dunnett’s post hoc analysis; n.s. p>0.05; ***p<0.0005.

Figure 3.. Increased amyloidogenesis in young Trem2 deficient mice is followed by lower amyloid accumulation rates during aging.

(a) Cortical microglial activity measured by in vivo TSPO μPET at ages of three (p=0.418), six (p=0.426) and twelve months (p=0.002) for Trem2 deficient APPPS1 mice compared to APPPS1/Trem2^+/+. Age matched results for C57BL6 mice with and without Trem2 deficiency are implemented for comparison with mice lacking amyloid pathology. (b) Corresponding results for cortical fibrillar amyloidogensis assessed by in vivo Amyloid μPET at three (p=0.026), six (p=0.007) and twelve months (p=0.895). (APPPS1: three months Aβ PET n^+/+=4 mice, TSPO PET n^+/+=5 mice, Aβ PET n^−/−=7 mice, TSPO PET n^−/−=9 mice; six months Aβ PET n^+/+=12 mice, TSPO PET n^+/+=13 mice, Aβ PET n^−/−=9 mice, TSPO PET n^−/−=11 mice; twelve months Aβ PET n^+/+=11 mice, TSPO PET n^+/+=11 mice, Aβ PET n^−/−=7 mice, TSPO PET n^−/−=7 mice. C57BL6: three months Aβ PET n^+/+=6 mice, TSPO PET n^+/+=6 mice, Aβ PET n^−/−=5 mice, TSPO PET n^−/−=6 mice; six months Aβ PET n^+/+=25 mice, TSPO PET n^+/+=20 mice, Aβ PET n^−/−=6 mice, TSPO PET n^−/−=8 mice; twelve months Aβ PET n^+/+=21 mice, TSPO PET n^+/+=20 mice, Aβ PET n^−/−=7 mice, TSPO PET n^−/−=7 mice). Error bars represent mean ± SD. Two-sided t-test. (c) Coronal and axial slices show Z-scores of increased TSPO μPET against age matched C57BL6 (n=18 mice) to compare APPPS1/Trem2^−/− and APPPS1/Trem2^+/+ at three, six and twelve months of age using an MRI template. (d) Coronal and axial slices show Z-scores of increased Amyloid μPET against age matched C57BL6 (n=18 mice) to compare APPPS1/Trem2^−/− and APPPS1/Trem2^+/+ at three, six and twelve months. (e) Serial imaging shows distinctly lower monthly increases for TSPO μPET during aging (6–12 months) in APPPS1/Trem2^−/− mice compared to APPPS1/Trem2^+/+ mice (n^+/+=11 mice, n^−/−=11 mice, 3–5 months p=0.756, 6–8 months p=8.03E-7, 9–11 months p=0.003). (f) Serial Amyloid μPET indicates an increased accumulation rate of fibrillar amyloidogenesis in young APPPS1/Trem2^−/− mice (3–5 months) compared to age matched APPPS1/Trem2^+/+ mice. Importantly, the accumulation rate of fibrillar amyloid in APPPS1/Trem2^−/− mice declines below those of APPPS1/Trem2^+/+ during aging (6–12 months) n^+/+=10 mice, n^−/−=9 mice, 3–5 months p=1.52E-4, 6–8 months p=0.003, 9–11 months p=1.48E-4). Thick lines in e and f represent polynomic functions of longitudinal changes, whereas dotted lines represent functions of SEM. Two-sided t-test. *p<0.05; **p<0.01; ***p<0.001.

Figure 4.. Relative protein quantification of ApoE in microglia-enriched and microglia-depleted lysates using mass spectrometry and label free quantification (LFQ).

(a) Immunoblotting of microglia-enriched (MG+) and microglia-depleted (MG-) lysates from twelve months old APPPS1/Trem2^+/+ and APPPS1/Trem2^−/− mice. Brain cell types in each fraction were identified by detection of IBA1 for microglia, GFAP for astrocytes, Tuj1 for neurons and CNPase for oligodendrocytes. Full image of each immunoblot is shown in Supplementary Fig. 7. (b) ApoE LFQ intensities of microglia-enriched lysates APPPS1/Trem2^+/+ mice compared to age matched C57BL6 controls. Two-sided Student’s t-test comparing log² transformed LFQ intensities of APPPS1 (n=3 mice) and C57BL6 (n=3 mice) separately for three (p=0.061), six (p=0.00372) and twelve months (p=1.78E-5) of age (**p<0.01; ***p<0.001). (c) ApoE LFQ intensities of microglia-enriched lysates from twelve months old APPPS1/Trem2^−/− mice show significantly reduced ApoE compared to age matched APPPS1/Trem2^−/− controls (n=3 mice/genotype). APPPS1 mice show significant reduction in ApoE after loss of Trem2 (p=0.00105). (d) ApoE and IBA1 costained in twelve months old APPPS1/Trem2^+/+ and APPPS1/Trem2^−/− mice. White boxes indicate the area in each staining that is magnified as inset. (e) Microglia-depleted lysates from twelve months old APPPS1 mice show no statistically significant changes regardless of Trem2 expression (n=3 mice/genotype; p=0.979). Data represent mean ± SD. (f) ApoE and GFAP costained in twelve months old APPPS1/Trem2^+/+ and APPPS1/Trem2^−/− mice. White boxes indicate the area in each staining that is magnified as inset.

Figure 5.. Decreased ApoE in newly seeded hippocampal plaques in the absence of functional Trem2.

(a) Left: ApoE (gray) staining in seeded hippocampus of APPPS1/Trem2^+/+ mice. Inset shows a higher magnification of ApoE (green) colocalised with x34-positive seeded plaques (blue) in the same area shown in yellow dotted box. Middle: 3D reconstructed images of x34/ApoE/IBA1 stainings of seeded area in APPPS1/Trem2^+/+ dentate gyrus. Enlarged areas of each staining (top - ApoE in green, middle – IBA1 in magenta, bottom - merged in white) are shown adjacently. Right: 3D reconstructed images of x34/ApoE/Aβ (3552) stainings of seeded area in APPPS1/Trem2^+/+ dentate gyrus. (b) Left: Reduced ApoE staining (gray) in seeded APPPS1/Trem2^−/− mice and (c, left) APPPS1/Trem2^p.T66M mice compared to APPPS1/Trem2^+/+ mice. Insets show a higher magnification of ApoE (green) with seeded amyloid pathology (x34; blue) in the same area indicated in yellow dotted box. Middle: 3D reconstructed images of x34/ApoE/IBA1 show reduced IBA1 and ApoE colocalisation in APPPS1/Trem2^−/− mice and (c, middle) APPPS1/Trem2^p.T66M mice. Right: 3D reconstructed images of x34/ApoE/Aβ (3552) stainings of seeded area in APPPS1/Trem2^−/− and APPPS1/Trem2^p.T66M dentate gyrus show increased amyloid pathology despite reduced ApoE levels. Note increased staining of non-fibrillar (x34 negative) Aβ upon Trem2 loss of function in (b) and (c) as compared to (a). (d) Quantification of mean ApoE density over seeded amyloid pathology area (n^+/+=7 mice, n^−/−=6 mice, n^p.T66M=6 mice; F_2,16=10.35, p=0.0013). (e) Immunoblotting with the anti-ApoE antibody HJ6.3 demonstrates decreased hippocampal ApoE in the formic acid fraction of seeded APPPS1/Trem2^−/− and APPPS1/Trem2^p.T66M as compared to APPPS1/Trem2^+/+ mice. Western blots were independently repeated three times to analyze n=6 mice/genotype. Full image of immunoblots are shown in Supplementary Fig. 7. Data represent mean ± SEM. One-way ANOVA, Dunnett’s post hoc analysis; **p<0.005.

Figure 6.. Decreased ApoE in non-experimentally seeded cortical Aβ plaques after loss of Trem2 function.

(a) Left: ApoE (gray) staining of non-experimentally seeded cortical Aβ plaques of APPPS1/Trem2^+/+ mice. Inset demonstrates a higher magnification of ApoE (green) colocalised with x34-positive cortical plaque (blue) in the same area shown in yellow dotted box. Middle: High-resolution confocal images of x34/ApoE/IBA1 stained cortical amyloid plaque in APPPS1/Trem2^+/+ were 3D reconstructed. Selected areas of each staining (top - ApoE in green, middle - IBA1 in magenta, bottom - merged in white) are shown adjacently. Right: 3D reconstructed high-resolution confocal images of x34/ApoE/Aβ (3552) stained amyloid plaque in APPPS1/Trem2^+/+ cortex. Immunopositive Aβ (red) and ApoE (green) are strongly colocalised (yellow). (b) Left: Reduced ApoE staining (gray) in cortical amyloid plaques of APPPS1/Trem2^−/− mice and (C, left) APPPS1/Trem2^p.T66M mice. Dotted cyan box indicates the area that is magnified as inset (ApoE – green, x34 – blue). Middle: 3D reconstructed images of x34/ApoE/IBA1 show reduced IBA1 and ApoE colocalisation in APPPS1/Trem2^−/− mice and (c, middle) APPPS1/Trem2^p.T66M mice. Right: 3D reconstructed images of x34/ApoE/Aβ (3552) stained amyloid plaque in APPPS1/Trem2^−/− and APPPS1/Trem2^p.T66M cortex show very little to no colocalisation between ApoE (green) and immunopositive-Aβ (red). (d) Immunoblotting with the anti-ApoE antibody HJ6.3 shows decreased cortical ApoE in the formic acid fraction of seeded APPPS1/Trem2^−/− and APPPS1/Trem2^p.T66M as compared to APPPS1/Trem2^+/+ mice. Western blots were independently repeated four times to analyze at least n=8 mice/genotype. Full image of immunoblots are shown in Supplementary Fig. 7. (e) Quantification of ApoE density in cortex (n=5 mice/genotype; F_2,12=14.31, p=0.0007). (f) Quantification of ApoE colocalisation in IBA1-positive microglia in APPPS1/Trem2^+/+, APPPS1/Trem2^−/− and APPPS1/Trem2^p.T66M mice (n=5 mice/genotype; F_2,12=18.82, p=0.0002). Data represent mean ± SEM. One-way ANOVA, Dunnett’s post hoc analysis; **p<0.005.

Figure 7.. Decreased ApoE in cortical Aβ plaques after microglia depletion.

(a) Schematic figure outlining study design and timeline of GCV application and subsequent repopulation phase. (b) An overview of fibrillar and immunopositive amyloid plaques costained with ApoE. Left: APPPS1/TK- mice show strong colocalisation of amyloid plaques and ApoE compared to APPPS1/TK+ mice (right). Right: Reduced plaque associated ApoE shown at larger magnification. (c) IMARIS 3D reconstructed high-resolution confocal images of costainings shown in b. White box indicates the area that is magnified for each immunostaining and placed adjacently. (d & e) 3D reconstructed images of x34/IBA1/ApoE/GFAP quadruple immunostaining in APPPS1/TK- and APPPS1/TK+ mice. Channels from the same confocal image were split to focus on costaining with IBA1 or GFAP individually. (d) Left: x34/ApoE/IBA1 costaining shows increased IBA1 and ApoE colocalisation with amyloid plaques in APPPS1/TK- mice compared to APPPS1/TK+ mice (right). Smaller images placed adjacently display each channel separately and merged channels (indicated by white box). (e) x34/ApoE/GFAP immunostaining demonstrates no qualitative differences in APPPS1/TK- mice (left) compared to APPPS1/TK+ mice (right). White box indicates the area that is magnified for each immunostaining and placed adjacently. (f) Plaque associated ApoE staining quantified from immunostainings shown in b and c (n=3 mice/genotype, p=0.0034). (g) Quantification of percentage IBA1/ApoE (n^ALL=3, p=0.0004) and GFAP/ApoE colocalisation (n=3 mice/genotype, p=0.4464) from stainings displayed in d and e, respectively. Two-tailed unpaired T-test with Welch’s correction; n.s. p>0.05; **p<0.005; ***p<0.001. Data represent mean ± SD.

Figure 8.. Reduced ApoE levels in Aβ plaques and impaired microglial clustering in TREM2 coding variants.

(a) Temporal neocortex of human AD patients with and without the indicated TREM2 variants stained for Aβ by 4G8 immunohistochemistry. (b) In sections consecutive to those of the left column, AD patients with TREM2 variants show reduced ApoE immunoreactivity within amyloid plaques compared to an AD case with no TREM2 coding variants (no CV). Note that the ApoE reduction is most pronounced in cases with an amino acid exchange at position 62. Red boxes indicate the area in each staining that is magnified as inset. (c) More examples of ApoE stainings comparing the same region in consecutive Aβ stained sections. (d) IBA1 (brown) and 4G8 (red) costaining in temporal neocortex. Microglia cells association with amyloid plaques as seen in a no CV case (last row) is severely impaired in AD cases with different TREM2 coding variants (rows 1–4). Furthermore, the overall density of microglial cells is reduced in TREM2 variant AD cases when compared to no CV. Dotted black boxes indicate the area magnified as inset. (e) ApoE immunoblotted in plaque-enriched formic acid fractions from cases shown in a-d and additional cases shown in Supplementary Fig. 5. Full image of the immunoblot is shown in Supplementary Fig. 7. (f) ApoE/Aβ₄₂ ratio quantified from plaque-enriched formic acid fraction shows significantly decreased ApoE levels in AD cases with TREM2 coding variants (n^R47H=2 cases, n^R62H=4 cases; n^R62C=1 case) compared to no CV (n=4 cases; p=0.0121). Of note, frozen material from p.D87N cases was not available and therefore not included. One of the no CV cases was excluded due to diagnosed Hepatitis. (g) Number of IBA1-positive microglia per plaque quantified from images shown in d and supplementary Fig. 5d (n^R47H=2 cases, n^R62H=4 cases; n^R62C=1 case; n^D87N=2 cases; n^{no CV}=3 cases; p=3.9E-4). Noteworthy, sections from only three no CV cases were available in comparison to frozen material. No subjects were excluded in this analysis. Medial temporal cortex at the level of anterior hippocampus was used for all experiments. Data represent as mean ± SD. Unpaired Two-tailed T-test with Welch’s correction; *p<0.05; ***p<0.001.