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Cited by 144Neuroinflammation and Neurodegeneration MechanismsAlzheimer's disease research and treatmentsPhagocytosis and Immune Regulation

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TREM2 promotes Aβ phagocytosis by upregulating C/EBPα-dependent CD36 expression in microglia

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Kim, Su-Man, Mun, Bo-Ram, Lee, Sun-Jun, Joh, Yechan, Lee, Hwa-Youn, Ji, Kon-Young, Choi, Ha-Rim, Lee, Eun-Hee, Kim, Eun-Mi, Jang, Ji-Hye, Song, Hyeong-Woo, Mook-Jung, Inhee, Choi, Won-Seok, Kang, Hyung-Sik·2017·Scientific Reports
DOI 10.1038/s41598-017-11634-xPMC PMC5593901
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Abstract

TREM2 plays a critical role in the alleviation of Alzheimer’s disease by promoting Aβ phagocytosis by microglia, but the detailed molecular mechanism underlying TREM2-induced direct phagocytic activity of Aβ remains to be revealed. We found that learning and memory functions were improved in aged TREM2 TG mice, with the opposite effects in KO mice. The amount of phagocytosed Aβ was significantly reduced in the primary microglia of KO mice. CD36 expression in primary microglia was greater in TG than in WT mice but was substantially decreased in KO mice. The expression of C/EBPα, an upstream transcriptional activator of CD36, was also elevated in primary microglia of TG mice but decreased in KO mice. The transcription of CD36 was markedly increased by TREM2 overexpression, and this effect was suppressed by a mutation of the C/EBPα binding site on the CD36 promoter. The TREM2-induced expression of CD36 and C/EBPα was inhibited by treatment with PI3K/AKT signaling blockers, and phosphorylation of AKT was elevated in TREM2-overexpressing BV2 cells. The present study provides evidence that TREM2 is required for preventing loss of memory and learning in Alzheimer’s disease by regulating C/EBPα-dependent CD36 expression and the consequent Aβ phagocytosis.

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Figure 1 Learning and memory was enhanced in TREM2 TG mice, but TREM2 KO mice showed a tendency toward defects. (A) Motor behavior was analyzed using the rotarod test in TREM2 TG mice (n = 8) and WT littermates (n = 8) as described in the Methods section. (B) Representative traces of swimming plots in the Morris water maze test of TREM2 TG and WT littermates. (C) Bar graphs calculated from data obtained from the Morris water maze test. Visible trial 4 is the last visible platform trial on day 1 before testing, representing the baseline. During testing on day 2, the time spent by each mouse before arriving at the hidden platform during trial 1 (hidden trial 1) was measured as the escape latency. (D) Rotarod test in TREM2 KO (n = 6) and WT littermates (n = 7). (E) Plots of swimming traces in TREM2 KO and WT littermates. (F) Bar graphs calculated from data obtained from the Morris water maze test. Data are presented as the mean ± SEM. One-way analysis of variance with Tukey’s post hoc multiple comparisons test was performed for all statistical analyses (*p < 0.05; other comparisons were not significant).
Figure 1 Learning and memory was enhanced in TREM2 TG mice, but TREM2 KO mice showed a tendency toward defects. (A) Motor behavior was analyzed using the rotarod test in TREM2 TG mice (n = 8) and WT littermates (n = 8) as described in the Methods section. (B) Representative traces of swimming plots in the Morris water maze test of TREM2 TG and WT littermates. (C) Bar graphs calculated from data obtained from the Morris water maze test. Visible trial 4 is the last visible platform trial on day 1 before testing, representing the baseline. During testing on day 2, the time spent by each mouse before arriving at the hidden platform during trial 1 (hidden trial 1) was measured as the escape latency. (D) Rotarod test in TREM2 KO (n = 6) and WT littermates (n = 7). (E) Plots of swimming traces in TREM2 KO and WT littermates. (F) Bar graphs calculated from data obtained from the Morris water maze test. Data are presented as the mean ± SEM. One-way analysis of variance with Tukey’s post hoc multiple comparisons test was performed for all statistical analyses (*p < 0.05; other comparisons were not significant).CC BY · PubMed Central
Figure 2 Aβ protein levels were increased in KO mice. (A) Immunohistochemistry for the neuronal marker NeuN and for Aβ was performed in hippocampal slices from WT and TREM2 KO mice (24 month-old). Scale Bar: 50 μm (B) Relative Aβ load in the hippocampus, DG was represented as a bar graph. (C) Immunostaining shows that the TREM2 expression level could affect neuronal cell number in WT, TREM2 TG and TREM2 KO mice. Scale Bar: 10 μm (D) Number of NeuN-positive cells in hippocampus, DG. Data are presented as the mean ± SEM (*p < 0.05, Student’s t test).
Figure 2 Aβ protein levels were increased in KO mice. (A) Immunohistochemistry for the neuronal marker NeuN and for Aβ was performed in hippocampal slices from WT and TREM2 KO mice (24 month-old). Scale Bar: 50 μm (B) Relative Aβ load in the hippocampus, DG was represented as a bar graph. (C) Immunostaining shows that the TREM2 expression level could affect neuronal cell number in WT, TREM2 TG and TREM2 KO mice. Scale Bar: 10 μm (D) Number of NeuN-positive cells in hippocampus, DG. Data are presented as the mean ± SEM (*p < 0.05, Student’s t test).CC BY · PubMed Central
Figure 3 TREM2 increased the viability of neuronal cells in the presence of Aβ. Neonatal and embryonic day 14 (E14) neuronal cells isolated from the brains of mice treated with Aβ for 48 h. (A) Neuronal cells were stained with anti-NeuN and Alexa 488-conjugated secondary antibody. The number of NeuN-positive cells was quantified and calculated as the percentage of viable cells. (B) The percentage of NeuN-positive cells. (C) Amount of released LDH in the supernatant of neonatal cells treated with Aβ. (D) The percentage of released LDH. (E) The percentage of released LDH in KO and WT E14 cells treated with Aβ. (F) Amount of released LDH in the supernatant of TG or WT microglia co-cultured with E14 cells in the presence of Aβ. Data shown are representative of five independent experiments, and the error bars represent the mean ± SEM of five independent experiments. (*p < 0.05; **p < 0.01; ***p < 0.005; N.S.: not significant).
Figure 3 TREM2 increased the viability of neuronal cells in the presence of Aβ. Neonatal and embryonic day 14 (E14) neuronal cells isolated from the brains of mice treated with Aβ for 48 h. (A) Neuronal cells were stained with anti-NeuN and Alexa 488-conjugated secondary antibody. The number of NeuN-positive cells was quantified and calculated as the percentage of viable cells. (B) The percentage of NeuN-positive cells. (C) Amount of released LDH in the supernatant of neonatal cells treated with Aβ. (D) The percentage of released LDH. (E) The percentage of released LDH in KO and WT E14 cells treated with Aβ. (F) Amount of released LDH in the supernatant of TG or WT microglia co-cultured with E14 cells in the presence of Aβ. Data shown are representative of five independent experiments, and the error bars represent the mean ± SEM of five independent experiments. (*p < 0.05; **p < 0.01; ***p < 0.005; N.S.: not significant).CC BY · PubMed Central
Figure 4 Deficiency of TREM2 signaling impairs the phagocytic activity of microglia. (A) Primary microglia of WT or KO mice were stained with PE-conjugated anti-CD11b or isotype control antibody, and the purity of primary microglia was determined using flow cytometric analysis. (B) Primary microglia were treated with 8 μM Aβ in the presence of 10 μM cytochalasin D (CD) as an inhibitor of actin-mediated phagocytosis, and the percentage of Aβ phagocytosed was determined using the phagocytosis assay. (C) The percentage of Aβ phagocytosed by BV2 cells after treatment with anti-TREM2 (1 μg/ml). (D) The percentage of Aβ phagocytosed by BV2 cells after treatment of soluble TREM2 (sTREM2, 2 μg/ml). (E) Representative fluorescence microscopic views (20×) from brain slices of 5XFAD mice stained with Thioflavin T after co-culture with primary microglia. Scale bar: 200 μm (F) The numbers of Aβ plaques quantified from the brain slice assay in (E). Data shown are representative of three independent experiments, and the error bars indicate the mean ± SEM of three independent experiments. (*p < 0.05; **p < 0.01).
Figure 4 Deficiency of TREM2 signaling impairs the phagocytic activity of microglia. (A) Primary microglia of WT or KO mice were stained with PE-conjugated anti-CD11b or isotype control antibody, and the purity of primary microglia was determined using flow cytometric analysis. (B) Primary microglia were treated with 8 μM Aβ in the presence of 10 μM cytochalasin D (CD) as an inhibitor of actin-mediated phagocytosis, and the percentage of Aβ phagocytosed was determined using the phagocytosis assay. (C) The percentage of Aβ phagocytosed by BV2 cells after treatment with anti-TREM2 (1 μg/ml). (D) The percentage of Aβ phagocytosed by BV2 cells after treatment of soluble TREM2 (sTREM2, 2 μg/ml). (E) Representative fluorescence microscopic views (20×) from brain slices of 5XFAD mice stained with Thioflavin T after co-culture with primary microglia. Scale bar: 200 μm (F) The numbers of Aβ plaques quantified from the brain slice assay in (E). Data shown are representative of three independent experiments, and the error bars indicate the mean ± SEM of three independent experiments. (*p < 0.05; **p < 0.01).CC BY · PubMed Central
Figure 5 TREM2 promotes Aβ phagocytosis by upregulating CD36 in microglia. (A,B) CD36 mRNA expression was determined by semi-quantitative RT-PCR from primary microglia (left), and the relative mRNA expression levels were quantitatively analyzed by normalization against GAPDH (right panels) (full-length blots/gels are presented in Supplementary Fig. S8). (C) Immunofluorescent staining of primary microglia. (D) Surface expression of CD36 on primary microglia analyzed using flow cytometry. (E) BV2 cells were treated with recombinant CD36 (rCD36, 1 μg/ml) in the presence of Aβ (8 μM) at 24 h after transfection with a control vector or a plasmid encoding TREM2, and the phagocytosis assay was performed. (F) The percentage of phagocytosed Aβ in primary microglia infected with lentivirus encoding shRNA targeting CD36. Data shown are representative of three independent experiments, and the error bars indicate the mean ± SEM of three independent experiments. (*p < 0.05; **p < 0.01).
Figure 5 TREM2 promotes Aβ phagocytosis by upregulating CD36 in microglia. (A,B) CD36 mRNA expression was determined by semi-quantitative RT-PCR from primary microglia (left), and the relative mRNA expression levels were quantitatively analyzed by normalization against GAPDH (right panels) (full-length blots/gels are presented in Supplementary Fig. S8). (C) Immunofluorescent staining of primary microglia. (D) Surface expression of CD36 on primary microglia analyzed using flow cytometry. (E) BV2 cells were treated with recombinant CD36 (rCD36, 1 μg/ml) in the presence of Aβ (8 μM) at 24 h after transfection with a control vector or a plasmid encoding TREM2, and the phagocytosis assay was performed. (F) The percentage of phagocytosed Aβ in primary microglia infected with lentivirus encoding shRNA targeting CD36. Data shown are representative of three independent experiments, and the error bars indicate the mean ± SEM of three independent experiments. (*p < 0.05; **p < 0.01).CC BY · PubMed Central
Figure 6 TREM2-induced C/EBPα promotes the transcription of CD36 in microglia. (A) The expression levels of C/EBPα and CD36 in primary mouse microglia were analyzed by western blot (full-length blots/gels are presented in Supplementary Fig. S9). (B) After transfection of BV2 cells with an empty vector or a plasmid encoding TREM2, the cells were treated for 24 h with signaling blockers, such as PD (PD98059), KN (KN62), LY (LY294002) and MK (MK2206), and cell surface expression of CD36 was analyzed using flow cytometry. The CD36-positive populations from data obtained from flow cytometry are represented as percentages in bar graphs. (C) Western blot analysis in BV2 cells transfected with vector or TREM2 (full-length blots/gels are presented in Supplementary Fig. S10). (D) Western blot analysis in BV2 cells treated with PBS or soluble TREM2 (sTREM2) (full-length blots/gels are presented in Supplementary Fig. S11). (E) After transfection of BV2 cells with vector or TREM2 and with scramble or C/EBPα siRNA, cell surface expression of CD36 was plotted as the mean fluorescence intensity (MFI) on a histogram of flow cytometric analysis. (F) Western blot analysis in BV2 cells transfected with the indicated expression or siRNA plasmids (full-length blots/gels are presented in Supplementary Fig. S12). The western blot and flow cytometry were quantified by using ImageJ or WinMDI software and are presented as bar graphs. (G) BV2 cells were co-transfected with the expression vector pGL3 basic-Luc, CD36-Luc or ΔCE-Luc and TREM2. After 48 h, luciferase activity was determined by a luminometer, and the data were normalized against β-galactosidase. All data shown are representative of three independent experiments, and the error bars indicate the mean ± SEM of three independent experiments. (*p < 0.05**p < 0.01; ***p < 0.001).
Figure 6 TREM2-induced C/EBPα promotes the transcription of CD36 in microglia. (A) The expression levels of C/EBPα and CD36 in primary mouse microglia were analyzed by western blot (full-length blots/gels are presented in Supplementary Fig. S9). (B) After transfection of BV2 cells with an empty vector or a plasmid encoding TREM2, the cells were treated for 24 h with signaling blockers, such as PD (PD98059), KN (KN62), LY (LY294002) and MK (MK2206), and cell surface expression of CD36 was analyzed using flow cytometry. The CD36-positive populations from data obtained from flow cytometry are represented as percentages in bar graphs. (C) Western blot analysis in BV2 cells transfected with vector or TREM2 (full-length blots/gels are presented in Supplementary Fig. S10). (D) Western blot analysis in BV2 cells treated with PBS or soluble TREM2 (sTREM2) (full-length blots/gels are presented in Supplementary Fig. S11). (E) After transfection of BV2 cells with vector or TREM2 and with scramble or C/EBPα siRNA, cell surface expression of CD36 was plotted as the mean fluorescence intensity (MFI) on a histogram of flow cytometric analysis. (F) Western blot analysis in BV2 cells transfected with the indicated expression or siRNA plasmids (full-length blots/gels are presented in Supplementary Fig. S12). The western blot and flow cytometry were quantified by using ImageJ or WinMDI software and are presented as bar graphs. (G) BV2 cells were co-transfected with the expression vector pGL3 basic-Luc, CD36-Luc or ΔCE-Luc and TREM2. After 48 h, luciferase activity was determined by a luminometer, and the data were normalized against β-galactosidase. All data shown are representative of three independent experiments, and the error bars indicate the mean ± SEM of three independent experiments. (*p < 0.05**p < 0.01; ***p < 0.001).CC BY · PubMed Central

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Authors
Kim, S.-M., Mun, B.-R., Lee, S.-J., Joh, Y., Lee, H.-Y., Ji, K.-Y., Choi, H.-R., Lee, E.-H., Kim, E.-M., Jang, J.-H., Song, H.-W., Mook-Jung, I., Choi, W.-S., & Kang, H.-S.
Affiliations
  • Chonnam National University (KR)
  • Daegu-Gyeongbuk Medical Innovation Foundation (KR)
  • Nambu University (KR)
  • Korea Atomic Energy Research Institute (KR)
  • Korea Institute of Toxicology (KR)
Corresponding author
  • Choi, Won-Seok
  • Kang, Hyung-Sik
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2017-09-11
Volume
7
Issue
1
Pages
11118-11118
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-017-11634-x
PMC
PMC5593901
OpenAlex
W2751088224
Cited by
143
References
32
Citations by year
2026: 12 · 2025: 13 · 2024: 38 · 2023: 23 · 2022: 18 · 2021: 13 · 2020: 9 · 2019: 13 · 2018: 4
FWCI
3.54
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Research Foundation
  • National Research Foundation of Korea (2016R1D1A3B03931710)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Immunology and Microbiology › Immunology › Phagocytosis and Immune Regulation
Keywords
TREM2, amyloid-beta phagocytosis, microglia, CD36, C/EBPα, Alzheimer's disease, learning and memory, PI3K/Akt signaling, Akt phosphorylation, TREM2 knockout mice
MeSH terms
Animals, Immunohistochemistry, Membrane Glycoproteins, Mice, Transgenic, Neurons, Phagocytes, Phagocytosis, Receptors, Immunologic, Biomarkers, Gene Expression Regulation, Neoplastic, Amyloid beta-Peptides, Microglia, Mice, Knockout, CD36 Antigens, CCAAT-Enhancer-Binding Proteins, Mice

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