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, 170 (4), 649-663.e13

TREM2 Maintains Microglial Metabolic Fitness in Alzheimer's Disease

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TREM2 Maintains Microglial Metabolic Fitness in Alzheimer's Disease

Tyler K Ulland et al. Cell.

Abstract

Elevated risk of developing Alzheimer's disease (AD) is associated with hypomorphic variants of TREM2, a surface receptor required for microglial responses to neurodegeneration, including proliferation, survival, clustering, and phagocytosis. How TREM2 promotes such diverse responses is unknown. Here, we find that microglia in AD patients carrying TREM2 risk variants and TREM2-deficient mice with AD-like pathology have abundant autophagic vesicles, as do TREM2-deficient macrophages under growth-factor limitation or endoplasmic reticulum (ER) stress. Combined metabolomics and RNA sequencing (RNA-seq) linked this anomalous autophagy to defective mammalian target of rapamycin (mTOR) signaling, which affects ATP levels and biosynthetic pathways. Metabolic derailment and autophagy were offset in vitro through Dectin-1, a receptor that elicits TREM2-like intracellular signals, and cyclocreatine, a creatine analog that can supply ATP. Dietary cyclocreatine tempered autophagy, restored microglial clustering around plaques, and decreased plaque-adjacent neuronal dystrophy in TREM2-deficient mice with amyloid-β pathology. Thus, TREM2 enables microglial responses during AD by sustaining cellular energetic and biosynthetic metabolism.

Keywords: Alzheimer’s disease; TREM2; immunity; metabolism; microglia.

Figures

Figure 1
Figure 1. Defect in TREM2 enhances autophagy in vivo in the 5XFAD mouse model and in AD patients
(A) TEM images of microglia (CD45+, CD11b+, F4/80+ cells) sorted from 8-month-old WT, Trem2−/−, 5XFAD, and Trem2−/− 5XFAD mice. (B) Average number of multivesicular and multilamellar structures/cell (30 cells analyzed/genotype). (C) Confocal images of plaque bearing regions of the cortex (1.1 mm Bregma to 0.8 mm Bregma) of 8-month-old WT, Trem2−/−, 5XFAD, and Trem2−/− 5XFAD mice show Iba-1+ microglia (red), methoxy X04+ plaques (blue), and LC3 (green). Z-stacks composed of ~30 images taken at 1.2 μm intervals were analyzed. Results are reported as an average of 2 regions of interest (ROI) analyzed. (D) Quantification of the % of microglia that are positive for LC3 puncta. ~150–400 microglia/HPF were analyzed depending on the genotype of the animal. (E) Confocal images of sections from post-mortem brains of R47H+ AD patients and case-matched controls (CV, common variant of TREM2) show Iba-1+ microglia (red), methoxy X04+ plaques (blue), and LC3 (green). 3 ROIs/donor were analyzed and between 400 and 700 microglia/individual were analyzed. (F) Percentages of LC3+ microglia in post mortem specimens of AD patients with different genotypes. *** p<0.005, ****p<0.001 by One-way ANOVA with Holm-Sidak’s multiple comparisons test. 15 cells from 2 separate mice were visualized for TEM (A, B). Confocal images are representative of 3 female mice per group (C, I) or 7 R47H, 4 R62H, and 8 case matched AD patients for post-mortem specimens (E). Immunoblots are representative of 3 independent experiments from microglia from 3 separate mice per group (G). Arrowheads indicate multilamellar and multivesicular structures (A) or LC3+ vesicles (C, E, I). See also Figure S1 and Table S1.
Figure 2
Figure 2. Defect in TREM2 impairs mTOR activation and elicits AMPK activation, autophagy and cell death in microglia from 5XFAD mice
(A) Microglia were sorted as in Figure 1A. Immunoblots for LC3I/II, p62, phospho-Akt (serine 473), phospho-AMPK, phospho-NDRG1, phospho-4EBP1, phospho-ULK1 (serine 757), and β-actin were performed on cell lysates. (B) Quantification of the LC3II/I ratio in microglia from different genotypes. (C) Single cell suspensions of brain tissue were incubated with MitoTraker Green and stained for CD45+, CD11b+, F4/80+. Representative histograms comparing unstained 5XFAD microglia and stained 5XFAD and Trem2−/− 5XFAD microglia are shown. (D) Quantification of the geometric mean fluorescence intensity (gMFI) of microglia from 3 mice of each genotype is shown. (E) Confocal images of brain sections of 8 month-old WT, Trem2−/−, 5XFAD, and Trem2−/− 5XFAD mice were taken as in Fig. 1C. Images depict Iba-1+ microglia (red), methoxy X04+ plaques (blue), and cleaved caspase-3 (green). (F) Quantification of the % of LC3+ microglia that are positive for cleaved caspase-3. ****p<0.001 by One-way ANOVA with Holm-Sidak’s multiple comparisons test (B and F). ** p<0.01 by Student’s T test (D). Immunoblots are representative of 3 independent experiments from microglia from 3 separate mice per group (A). Confocal images are representative of 3 female mice per group (E). See also Figure S2.
Figure 3
Figure 3. TREM2 deficiency affects mTOR signaling and induces autophagy in BMDM
(A) TEM images of WT and Trem2−/− BMDM cultured overnight in either in 10% or 0.5% LCCM as source of CSF1. (B) Number of multivesicular structures/cell observed in the TEM images. 30 cells/genotype and condition were analyzed. (C) Quantification of the LC3II/LC3I ratio in BMDMs from WT and Trem2−/− mice cultured in 10% or 0.5% LCCM overnight or starved in HBSS for 4 hours prior to lysis. (D) Immunoblots for LC3 and actin performed on lysates from WT and Trem2−/− BMDMs cultured in 10% or 0.5% LCCM overnight. Cell were treated with bafilomycin for 5 hours prior to harvest at a final concentration of 0.5 μg/ml (E) Quantification of LC3II/LC3I ratio in BMDMs from WT and Trem2−/− mice treated as indicated. (F–H) Immunoblots for phosphorylated Akt (serine 473), NDRG1, S6K, 4EBP1, (F), AMPK, (G), Ulk1 (serine 317 and serine 757) (H) and relative controls. Lysates were from WT and Trem2−/− BMDM cultured overnight in 10% or 0.5% LCCM (I) Immunoblots for phosphorylated Akt (serine 473), NDRG1, S6K, 4EBP1, mTOR, total S6K, Akt, and actin performed on lysates from WT and Trem2−/− BMDMs cultured overnight in 10% or 0.5% LCCM followed by the addition of wortmannin for 3 hours prior to harvest. (J, K) Immunoblots for LC3 and phosphoserine 473 Akt in WT and Trem2−/− BMDM cultured in 10% LCCM with the indicated concentration of tunicamycin. Bar graph shows LC3II/LC3I ratios. Error bar represents mean ± SEM. *p<0.05, **p<0.01, or ****p<0.001 by One-way ANOVA with Holm-Sidak’s multiple comparisons test (B, C, E, K). Data are representative of at least 3 independent experiments. Arrowheads indicate multilamellar and multivesicular structures (A). See also Figure S3.
Figure 4
Figure 4. TREM2 deficiency reduces anabolic and energetic metabolism in BMDM
(A) Top most changed metabolites between WT and Trem2−/− BMDM cultured overnight in 10% LCCM. Defined as p≤0.01 and identified in the mouse metabolic network analysis in B. (B) Shiny-genes and metabolites (GAM) output for network analysis combining mass spectrometry and RNA-seq data highlights differences between WT and Trem2−/− BMDM cultured in 10% LCCM. Enzyme-encoding mRNAs and metabolites downregulated or upregulated in Trem2−/− cells vs WT cells are indicated with green or red nodes and connectors, respectively. (C) Top most changed metabolites between WT and Trem2−/− BMDM cultured in 0.5% LCCM. Defined as p≤0.01 and identified in the mouse metabolic network analysis in Fig. S4C. (D) ATP content of WT and Trem2−/− BMDM cultured in the indicated concentration of LCCM overnight. (E) Extracellular acidification rate (ECAR) and baseline oxygen consumption rate (OCR) by WT and Trem2−/− BMDM cultured overnight in the indicated concentration of LCCM. (F, G) Mitochondrial mass of WT and Trem2−/− BMDM assessed by Mito Tracker Green incorporation (F) and by the ratio of mitochondrial-to nuclear DNA (G). Error bar represents mean ± SEM. *p<0.05, ** p<0.01, or ****p<0.001 by One-way ANOVA with Holm-Sidak’s multiple comparisons test (C) or Student’s T test (F, G). Data are representative of at least 3 independent experiments. See also Figure S4.
Figure 5
Figure 5. Enhanced energy storage or dectin-1 signaling can compensate for TREM2 deficiency
(A) ECAR of WT and Trem2−/− BMDM incubated overnight in 0.5% LCCM ± 10 mM cyclocreatine. (B) Viability of WT and Trem2−/− BMDM incubated for 40 hours in 0.5% LCCM ± cyclocreatine. (C) Immunoblots of LC3, phosphorylated mTOR, phosphorylated Akt (serine 473), and actin in WT and Trem2−/− BMDM incubated overnight in 0.5% LCCM ± 5 mM cyclocreatine. (D, F) LC3, phosphoserine 473 Akt, p62, and actin immunoblots from WT and Trem2−/− BMDM incubated overnight in the indicated concentration of LCCM ± depleted zymosan. (E) Quantification of the LC3II/LC3I ratio derived from immunoblots of LC3 as shown in D. (G) ATP content of WT and Trem2−/− BMDM cultured in the indicated concentration of LCCM ± zymosan overnight. *p<0.05 or ****p<0.001 by One-way ANOVA with Holm-Sidak’s multiple comparisons test (B, E, G). Data are representative of results from at least 3 independent experiments.
Figure 6
Figure 6. Enhanced Energy Storage Can Correct Microglial Defects in TREM2-deficent 5XFAD Mice
(A) TEM images of microglia sorted from 8-month-old 5XFAD, and Trem2−/− 5XFAD mice ± cyclocreatine. (B) Quantification of the number of multivesicular and multilamellar structures/cell from A. (C) Confocal images of brain sections of 8-month-old 5XFAD, and Trem2−/− 5XFAD mice ± cyclocreatine show Iba-1+ microglia (red), methoxy X04+ plaques (blue), and LC3 (green). (D) Quantification of the number of LC3 puncta per HPF in the cortexes of the indicated mice. (E) Quantification of the percentage of microglia that were cleaved caspase-3 positive from the indicated mice. (F) Clustering analysis quantifying the number of microglia per mm3 within 15 μm of the surface of plaques. *p<0.05, ***p<0.005 and ****p<0.001 by One-way ANOVA with Holm-Sidak’s multiple comparisons test (B, D–F) results pooled from 2 independent experiments representing a total of 5–8 male and female mice per treatment group. Arrowheads indicate multilamellar and multivesicular structures (A) or LC3+ vesicles (C). See also Figure S5.
Figure 7
Figure 7. Energy Supplementation Can Offset Neuronal Damage in TREM2 Deficient 5XFAD mice
(A) Representative images depicting plaques (X04 in blue), nuclei (To-Pro3 in white), microglia (Iba-1 in red), and Spp1 (in green) staining in cortexes of mice from the indicated genotypes. (B) Quantification of the percentage of microglia that were Spp1+ in the indicated genotypes of mice. Confocal images were taken as in Figure 1C. (C) Immunoblots performed on lysates of microglia sorted from the indicated genotype and treatment group of mice. Immunoblots for phosphorylated Akt (serine 473), NDRG1, total LC3, Akt, and actin. (D) Quantification of the LC3II/LC3I ratio observed in immunoblots from 3 mice of each of the indicated genotypes and treatment groups. (E) Average intensity of the plaques observed in the cortexes of mice from the indicated genotypes and treatment groups. (F) Representative images depicting plaques (X04 in blue), nuclei (To-Pro3 in white), and N-terminus APP (green) from the indicated mice and treatment groups. Confocal images were taken as in Figure 1C. (G) Quantification of the number of dystrophic neurites/plaque in the indicated mice and treatment group. N.S. indicates not significant, *p<0.05, and ****p<0.001 by One-way ANOVA with Holm-Sidak’s multiple comparisons test (A, C, D, F) results pooled from 2 independent experiments representing a total of 5–8 male and female mice per treatment group. See also Figure S5.

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