. 2018 Mar 7;97(5):1032-1048.e5.

doi: 10.1016/j.neuron.2018.02.002.

Elevated TREM2 Gene Dosage Reprograms Microglia Responsivity and Ameliorates Pathological Phenotypes in Alzheimer's Disease Models

Affiliations

¹ Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience & Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA; Brain Research Institute, University of California, Los Angeles, Los Angeles, CA, USA.
² Neuroscience and Aging Research Center, Sanford-Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.
³ Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience & Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA.
⁴ Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA.
⁵ Brain Research Institute, University of California, Los Angeles, Los Angeles, CA, USA; Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA; Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA.
⁶ Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience & Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA; Brain Research Institute, University of California, Los Angeles, Los Angeles, CA, USA; Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA.
⁷ Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience & Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA; Brain Research Institute, University of California, Los Angeles, Los Angeles, CA, USA. Electronic address: xwyang@mednet.ucla.edu.

PMID: 29518357
PMCID: PMC5927822
DOI: 10.1016/j.neuron.2018.02.002

Elevated TREM2 Gene Dosage Reprograms Microglia Responsivity and Ameliorates Pathological Phenotypes in Alzheimer's Disease Models

C Y Daniel Lee et al. Neuron. 2018.

. 2018 Mar 7;97(5):1032-1048.e5.

doi: 10.1016/j.neuron.2018.02.002.

Authors

Affiliations

¹ Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience & Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA; Brain Research Institute, University of California, Los Angeles, Los Angeles, CA, USA.
² Neuroscience and Aging Research Center, Sanford-Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.
³ Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience & Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA.
⁴ Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA.
⁵ Brain Research Institute, University of California, Los Angeles, Los Angeles, CA, USA; Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA; Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA.
⁶ Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience & Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA; Brain Research Institute, University of California, Los Angeles, Los Angeles, CA, USA; Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA.
⁷ Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience & Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA; Brain Research Institute, University of California, Los Angeles, Los Angeles, CA, USA. Electronic address: xwyang@mednet.ucla.edu.

PMID: 29518357
PMCID: PMC5927822
DOI: 10.1016/j.neuron.2018.02.002

Abstract

Variants of TREM2 are associated with Alzheimer's disease (AD). To study whether increasing TREM2 gene dosage could modify the disease pathogenesis, we developed BAC transgenic mice expressing human TREM2 (BAC-TREM2) in microglia. We found that elevated TREM2 expression reduced amyloid burden in the 5xFAD mouse model. Transcriptomic profiling demonstrated that increasing TREM2 levels conferred a rescuing effect, which includes dampening the expression of multiple disease-associated microglial genes and augmenting downregulated neuronal genes. Interestingly, 5xFAD/BAC-TREM2 mice showed further upregulation of several reactive microglial genes linked to phagocytosis and negative regulation of immune cell activation. Moreover, these mice showed enhanced process ramification and phagocytic marker expression in plaque-associated microglia and reduced neuritic dystrophy. Finally, elevated TREM2 gene dosage led to improved memory performance in AD models. In summary, our study shows that a genomic transgene-driven increase in TREM2 expression reprograms microglia responsivity and ameliorates neuropathological and behavioral deficits in AD mouse models.

Keywords: Alzheimer’s disease; BAC; RNA-sequencing; TREM2; amyloid plaque; gene dosage; microglia; mouse; neuroinflammation; reprogramming.

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Figures

**Figure 1. Generation and characterization of BAC-TREM2 mice**
(A) Schematic representation of the modification of TREM2-BAC. Red crosses indicate the deleted exons in TREM-like genes in the BAC construct. (B) UCSC genome browser track showing read coverage at the human TREM2 locus in TREM2 transgenic and wildtype animals. (C) Western blot was performed using the hippocampal lysates from 1.5 month-old WT and BAC-TREM2 mice with human TREM2 and mouse Trem2-specific antibodies. GAPDH served as a loading control. (D) The band intensity of Western blots was quantified using Image J and shown as ratio of mTrem2/GAPD (n = 4). (E–N) Brain sections from 1.5–2 month-old BAC-TREM2-GFP mice were double stained with GFP and cell-specific markers for microglia (Iba⁺, E–G), astrocytes (GFAP⁺, H–J), or neurons (NeuN⁺, K–M). Representative cortical images showed that BAC-TREM2-GFP colocalized with Iba1 (E–G) but not with GFAP (H–J) not NeuN (K–M). Bar = 100μm. (N) GFP⁺ cells were examined for the colocalization with cell-specific markers and presented as percent double labeled cells over GFP⁺ cells. The numbers below X-axis indicate the number of cell counted.

**Figure 2. Increased TREM2 gene dosage ameliorates amyloid pathology and remodels amyloid plaque types**
(A–C) Matched brain sections from 7-months-old 5xFAD (A) and 5xFAD/TREM2 mice (B) were stained with ThioS (green) and NeuN (red) to visualize the amyloid plaques in the cortex. Z-stack confocal images (20X) were utilized to measure total plaque area in the field using ImageJ. The results are presented as ThioS⁺ plaque area (μm²) per mm² of the cortical area (C). n = 7 per genotype, **p < 0.01. Bar = 50μm. (D–G) The levels of soluble and insoluble Aβ₄₂ (D and E) and Aβ₄₀ (F and G) in the cortex of 4 and 7-month-old mice were measured by ELISA. n = 6 per genotype, *p < 0.05. (H–J) Matched brain sections from 7-month-old 5xFAD (H) and 5xFAD/TREM2 mice (I) were stained with ThioS and anti-Aβ antibody (6E10). Z-stack confocal images (40X) were utilized to quantify 3 different forms of plaques using ImageJ (J). A total of 502 plaques were analyzed and are presented as mean ± SEM. n = 4 per genotype, **p < 0.01, ***p < 0.001. Bar = 50μm.

**Figure 3. Transcriptomic and coexpression network analyses reveal partial rescue of transcriptomic changes in 5xFAD mice with increased TREM2 gene dosage**
(A) Numbers of DE genes (FDR < 0.1) in genotype contrasts. Blue/red bars represent significantly down–/up-regulated genes. n=6 per genotype and time point except n=5 for 5xFAD at 7 m. (B–C) Transcriptome-wide “rescue plots” at 4 and 7 months. The plots show Z statistics for DE in 5xFAD vs. 5xFAD/TREM2 (y-axis) and 5xFAD vs. WT (x-axis) for all genes (each gene corresponds to one point). Rescued (concordant in this plot) and exacerbated (discordant) genes that pass the FDR threshold of 0.1 in both comparisons are shown in blue and red colors, respectively. Genome-wide correlations of Z statistics and the corresponding correlation p-values (for which the n=15809 genes are considered independent) are indicated on the top of each panel. (D) DE analysis of module eigengenes. Rows correspond to modules and columns to selected genotype contrasts. Numbers in the heatmap show the Z statistics and the corresponding p-values of module eigengene association with the genotype. n=6 per genotype and time point except n=5 for 5xFAD at 7m. (E) Expression variation of module eigengene expression with age in WT (black), 5xFAD (blue) and 5xFAD/TREM2 (Orange) samples. Points represent means of eigengene values across samples at the same age. Error bars give SEM. Network of top 10 hub genes are presented on the right.

**Figure 4. Increased TREM2 gene dosage reprogrammed disease-associated microglia gene expression**
(A) Heatmap representation of differential expression Z statistics for TD1-3 genes. Genes are divided into TD1 (blue), TD2 (green) and TD3 (red). (B–C) Fold changes of individual TD1 (B) and TD2 (C) genes in 5xFAD (red) and 5xFAD/TREM2 (blue) vs. wildtype mice at 7m. Stars indicate FDR-corrected significance (***FDR<0.001, **FDR<0.01, *FDR<0.1). n=6 per genotype, except n=5 for 5xFAD. (D–F) Venn diagrams of overlaps of TD1 (blue) and TD2 (green) genes with published genesets (orange).

**Figure 5. Upregulation of TREM2 altered microglial response to the amyloid plaque**
(A–H) Representative images demonstrated the interaction between microglia (Iba1⁺, green) and the plaque (6E10⁺, red) in 7 months old 5xFAD (A–D) and 5xFAD/TREM2 mice (E–H). Bar = 25 μm. (I) Amyloid plaques in cortex were randomly selected and Z-stack confocal images were taken for counting plaque-associated microglia (Iba1⁺). n = 4 per genotype. ***p < 0.001. (J–L) The morphological properties of plaque-associated microglia were measured by Imaris using z-stack confocal images taken under a 63X objective lens. The results are presented as total process length (J), process volume (K) and branch numbers (L) per microglia. Images of more than 15 plaques with over 150 microglia per genotypes were analyzed and presented as mean ± SEM. n = 4 per genotypes, **p < 0.01, *p < 0.05.

**Figure 6. Increased TREM2 gene dosage upregulated expression of phagocytic markers and enhanced phagocytic activity in microglia**
(A–C) Matching cortical sections from 7 month-old 5xFAD (A) and 5xFAD/BAC-TREM2 mice (B) were stained with CD68 (green), Iba1 (red) and ThioS (blue). (C) Area of CD68⁺ labeling per plaque was measured on z-stack confocal images. n = 4 per genotypes, *p < 0.05. Bar = 25μm. (D–F) Matching cortical sections from 7 month-old 5xFAD (D) and 5xFAD/BAC-TREM2 mice (E) stained with Lgals3 (dark blue) and Congo Red (pink). (F) Number of Lgal3⁺ cells per plaque were counted under microscope by a blinded observer. n = 3 per genotypes, ***p < 0.001. Bar = 50μm. (G) Phagocytosis of Alexa488-conjugated microsphere by primary microglia were measured by flow cytometry. Phagocytic microglia were detected with strong fluorescent signal in the cells. The graph is pooled results from 4 independent experiments and presented as mean ± SEM. n = 3–6 per genotypes; **p < 0.01, *p < 0.05 compared to WT; ^#p < 0.05 compared to *Trem2^+/−*; ^§p < 0.05 compared to *Trem2^−/−*.

**Figure 7. Increase TREM2 gene dosage in microglia reduced plaque-associated neuritic pathology and ameliorate cognitive deficit**
(A–D) Representative images of APP-immunolabeled dystrophic neurites (red) surrounding the plaques co-labelled with 4G8 (purple) and thioflavin S (green) in the cortex of 5xFAD and 5xFAD/TREM2 mice. Bar = 25μm. (E) Total dystrophy neurites volume was quantified in the cortex of 5xFAD and 5xFAD/TREM2 mice. n = 3 per genotype. Data presented as mean ± SEM, **p < 0.01. (F) The contextual memory function of mice from the cohort of BAC-TREM2 �� 5xFAD (n = 8–14 per genotype) was evaluated by contextual fear conditioning and is presented as percent time freezing. Power analysis was performed to ensure >80% confidence levels with the number of animals used. **p < 0.01, *p < 0.05.

**Figure 8. Increased TREM2 gene dosage alters plaque-associated microglia morphology and ameliorates behavioral deficit in a second mouse model of AD**
(A–F) Representative confocal images from 11 month-old APP/PS1 (A–C) and APP/PS1;TREM2 (D–F) mice stained with anti-Iba1 (green) and 6E10 (red) antibodies. Bar = 25μm. (G) The contextual memory function was evaluated by contextual fear conditioning and is presented as percent time freezing. n = 14–18 per genotype, ***p < 0.001, **p < 0.01.

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Comment in

TREM2 and Amyloid Beta: A Love-Hate Relationship.
Udeochu J, Sayed FA, Gan L. Udeochu J, et al. Neuron. 2018 Mar 7;97(5):991-993. doi: 10.1016/j.neuron.2018.02.018. Neuron. 2018. PMID: 29518360

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Elevated TREM2 Gene Dosage Reprograms Microglia Responsivity and Ameliorates Pathological Phenotypes in Alzheimer's Disease Models

Affiliations

Elevated TREM2 Gene Dosage Reprograms Microglia Responsivity and Ameliorates Pathological Phenotypes in Alzheimer's Disease Models

Authors

Affiliations

Abstract

Figures

Comment in

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Molecular Biology Databases

Research Materials