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, 37 (7), 1772-1784

TREM2 Promotes Microglial Survival by Activating Wnt/β-Catenin Pathway

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TREM2 Promotes Microglial Survival by Activating Wnt/β-Catenin Pathway

Honghua Zheng et al. J Neurosci.

Abstract

Triggering Receptor Expressed on Myeloid cells 2 (TREM2), which is expressed on myeloid cells including microglia in the CNS, has recently been identified as a risk factor for Alzheimer's disease (AD). TREM2 transmits intracellular signals through its transmembrane binding partner DNAX-activating protein 12 (DAP12). Homozygous mutations inactivating TREM2 or DAP12 lead to Nasu-Hakola disease; however, how AD risk-conferring variants increase AD risk is not clear. To elucidate the signaling pathways underlying reduced TREM2 expression or loss of function in microglia, we respectively knocked down and knocked out the expression of TREM2 in in vitro and in vivo models. We found that TREM2 deficiency reduced the viability and proliferation of primary microglia, reduced microgliosis in Trem2-/- mouse brains, induced cell cycle arrest at the G1/S checkpoint, and decreased the stability of β-catenin, a key component of the canonical Wnt signaling pathway responsible for maintaining many biological processes, including cell survival. TREM2 stabilized β-catenin by inhibiting its degradation via the Akt/GSK3β signaling pathway. More importantly, treatment with Wnt3a, LiCl, or TDZD-8, which activates the β-catenin-mediated Wnt signaling pathway, rescued microglia survival and microgliosis in Trem2-/- microglia and/or in Trem2-/- mouse brain. Together, our studies demonstrate a critical role of TREM2-mediated Wnt/β-catenin pathway in microglial viability and suggest that modulating this pathway therapeutically may help to combat the impaired microglial survival and microgliosis associated with AD.SIGNIFICANCE STATEMENT Mutations in the TREM2 (Triggering Receptor Expressed on Myeloid cells 2) gene are associated with increased risk for Alzheimer's disease (AD) with effective sizes comparable to that of the apolipoprotein E (APOE) ε4 allele, making it imperative to understand the molecular pathway(s) underlying TREM2 function in microglia. Our findings shed new light on the relationship between TREM2/DNAX-activating protein 12 (DAP12) signaling and Wnt/β-catenin signaling and provide clues as to how reduced TREM2 function might impair microglial survival in AD pathogenesis. We demonstrate that TREM2 promotes microglial survival by activating the Wnt/β-catenin signaling pathway and that it is possible to restore Wnt/β-catenin signaling when TREM2 activity is disrupted or reduced. Therefore, we demonstrate the potential for manipulating the TREM2/β-catenin signaling pathway for the treatment of AD.

Keywords: Akt/GSK3β signaling pathway; Alzheimer's disease; TREM2; Wnt/β-catenin signaling pathway; cell survival; microglia.

Figures

Figure 1.
Figure 1.
TREM2 deficiency in microglia leads to decreased cell viability and increased cell death. A, Knock-down of Trem2 with two independent siRNAs in mouse primary microglia was confirmed by qRT-PCR. B, Knock-down of Trem2 suppressed microglial viability compared with the control nontarget (NT) group as examined by MTT assay. C, Trem2 knock-down suppressed microglial proliferation as assessed by BrdU incorporation assay. D, Decreased cell viability in Trem2−/− microglia compared with WT cells. E, Proliferation was suppressed in Trem2−/− microglia compared with WT cells. F, Trem2−/− microglia were cultured for the indicated times and apoptosis and necrosis were assessed by TUNEL staining. TUNEL-positive, apoptotic cells (green) were quantified as a percentage of total DAPI-positive cells (blue). The number of TUNEL-positive cells increased in Trem2−/− microglia compared with WT cells. G, Cell lysates from WT or Trem2−/− microglia were analyzed for pro-Caspase3 (pro-Casp3), cleaved Caspase-3 (c-Casp3), and Bcl-2 levels by Western blotting. Protein levels were quantified by densitometry ratio to α-tubulin for comparison. The level of c-Casp3 was increased, whereas pro-Casp3 and Bcl-2 were suppressed in Trem2−/− microglia compared with WT cells. Data are plotted as mean ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 2.
Figure 2.
TREM2 deficiency leads to reduced microgliosis in response to KA treatment in mouse brain. Trem2−/− or WT mice were intraperitoneally injected with 25 mg/kg KA at 8 weeks of age and mouse brains were dissected 3 d later. A, Coronal sections from Trem2−/− or WT mouse brains were stained with Iba-1 (green) for microglia. Representative images are shown. B, D, Number of microglia double stained with DAPI (blue) and Iba1 (green) was quantified per HPF in Trem2−/− or WT hippocampi and cortices. C, E, Microglia number response to KA was assessed by quantifying the cell number ratio of KA group to control group in WT or Trem2−/− brains. F, G, Quantification of microglial cell body (area, in square micrometers) per HPF in the hippocampi and cortices of WT or Trem2−/− brains with or without KA treatment. H, I, Quantification of microglial process per HPF in the hippocampi and cortices of WT or Trem2−/− brains with or without KA treatment. Scale bar, 100 μm. Data are plotted as mean ± SEM (n = 5). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
Figure 3.
Figure 3.
TREM2 deletion in microglia leads to cell cycle arrest at the G1/S checkpoint. A, Trem2−/ or WT microglia were fixed, stained with DAPI, and analyzed by flow cytometry. B, Ratio of stage-specific (G1/G0, S, or G2/M) cell numbers to total cell numbers was quantified for comparison. The number of G1/G0 phase cells increased, whereas S-phase cells decreased in Trem2−/− microglia compared with WT microglia. C, Cyclin D1 and c-Myc mRNA levels were decreased in Trem2−/− microglia compared with WT cells as quantified by qRT-PCR. D, Cell lysates from Trem2−/− microglia were analyzed for Cyclin D1 and c-Myc by Western blotting. Protein levels were quantified by densitometry normalized to α-tubulin. Note that Cyclin D1 and c-Myc were downregulated in Trem2−/− microglia compared with WT cells. Data are plotted as mean ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4.
Figure 4.
β-catenin was downregulated in Trem2-depleted microglia. A, Trem2 was knocked down by two independent siRNAs. Protein levels of β-catenin, phosphorylated p38 (p-P38), phosphorylated ERK1/2 (p-ERK1/2), total p38, total ERK1/2 from nontarget (NT), or Trem2 knock-down microglia were analyzed by Western blotting. B, Protein levels were quantified by densitometry and presented as ratios to α-tubulin. β-catenin was suppressed in Trem2 knock-down microglia compared with NT microglia. The amount of p-P38 or p-ERK1/2 was not altered in Trem2 knock-down microglia compared with NT microglia. C, D, Cell lysates of WT or Trem2−/− microglia were analyzed by Western blotting. The amount of β-catenin was reduced in Trem2−/− microglia. Protein levels were quantified by densitometry and are presented as ratios to α-tubulin. p-P38 or p-ERK1/2 levels were not altered in Trem2−/− microglia compared with WT microglia. Data are plotted as mean ± SEM (n = 3). **p < 0.01; ns, not significant.
Figure 5.
Figure 5.
Accelerated degradation of β-catenin in Trem2-depleted microglia. A, β-catenin mRNA levels were not altered in Trem2−/− microglia compared with WT cells as quantified by qRT-PCR. B, Trem2−/− or WT microglia were treated with cycloheximide (CHX; 100 μg/ml) or MG132 (10 μm) for the indicated times and cell lysates were analyzed by Western blotting. C, Protein levels were quantified by densitometry and normalized to GAPDH. Upon CHX treatment, β-catenin protein levels increased significantly in WT microglia at 4 and 5 h compared with Trem2−/− cells. D, When cells were treated with MG132, β-catenin protein level increased in both Trem2−/− and WT microglia in a time-dependent manner. Data are plotted as mean ± SEM (n = 3). *p < 0.05; **p < 0.01.
Figure 6.
Figure 6.
Decreased phosphorylation of GSK3β and AKT in Trem2-depleted microglia. A, Protein levels in cell lysates from Trem2 knock-down microglia were analyzed by Western blotting. Representative Western blots of phosphorylated GSK3βS9, phosphorylated AktS473, total GSK3β, and total Akt are shown. B, C, Protein levels were quantified by densitometry and normalized to total GSK3β/Akt and α-tubulin. The levels of phosphorylated GSK3βS9 and phosphorylated AktS473 were decreased in Trem2 knock-down microglia compared with nontarget (NT) cells. D, Cell lysates from Trem2−/− or WT microglia were analyzed to assess the levels of the indicated proteins by Western blotting. Representative Western blots of phosphorylated GSK3βS9, phosphorylated AktS473, total GSK3β, and total Akt are shown. E, F, Protein levels were quantified by densitometry and normalized to total GSK3β/Akt and α-tubulin. The levels of phosphorylated GSK3βS9 and phosphorylated AktS473 were decreased in Trem2−/− microglia compared with WT cells. Data are plotted as mean ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 7.
Figure 7.
Wnt3a restores β-catenin level and cell growth in Trem2−/− microglia. A, Trem2−/− or WT microglia were treated with LCM or Wnt3aCM for 24 h. The β-catenin level was analyzed by Western blotting. B, Amount of β-catenin was increased in Trem2−/− microglia treated with Wnt3aCM compared with the cells treated with LCM. Protein levels were quantified by densitometry and presented as ratios to GAPDH. C, Wnt3a promoted the cell viability of Trem2−/− microglia. D, Wnt3a partially rescued the cell proliferation of Trem2−/− microglia as examined by BrdU incorporation assay. Data are plotted as mean ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 8.
Figure 8.
LiCl and TDZD-8 rescue β-catenin signaling and cell survival in Trem2−/− microglia. A, Trem2−/− or WT microglia were treated with or without LiCl for 24 h. β-catenin level was analyzed by Western blotting. B, LiCl increased β-catenin in Trem2−/− microglia compared with control treated cells. C, LiCl enhanced the viability of Trem2−/− microglia. D, LiCl restored Trem2−/− microglial proliferation as examined by BrdU incorporation assay. E, Trem2−/− or WT microglia were treated with or without 5 μm TDZD-8 for 24 h. β-catenin level was analyzed by Western blotting. F, TDZD-8 increased β-catenin in Trem2−/− microglia compared with vehicle treated cells. Data are plotted as mean ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 9.
Figure 9.
LiCl rescues β-catenin signaling and cell survival in Trem2−/− mouse brain. A, Trem2−/− or WT mice at 8 weeks of age were intraperitoneally injected with LiCl (200 mg/kg) for 3 consecutive days and mouse brains were harvested and dissected. Brain sections from Trem2−/− or WT mice were double stained with Iba-1 (green) and DAPI (blue) for microglia. Representative images are shown. B, Number of microglia double stained with DAPI (blue) and Iba1 (green) was quantified per HPF in hippocampi and cortices. C, Protein levels in brain lysates were analyzed by Western blotting. Protein levels were quantified by densitometry and normalized to total GSK3β and/or α-tubulin. The levels of β-catenin, Cyclin D1, c-Myc, and phosphorylated GSK3βS9 were increased in Trem2−/− mice treated with LiCl compared with WT mice. Scale bar, 100 μm. Data are plotted as mean ± SEM (n = 5). *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 10.
Figure 10.
Schematic model of TREM2 in the regulation of β-catenin signaling and microglial survival. TREM2 interacts with DAP12 to activate PI3K/Akt signaling. Akt phosphorylates the serine 9 residue of GSK3β (GSK3βS9), leading to GSK3β inactivation and stabilization of β-catenin. β-catenin accumulates in the cytoplasm and then enters the nucleus, where it regulates the expression of target genes such as Cyclin D1, c-Myc, and Bcl-2.

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