Fig 2: AAV-mediated sTREM2 expression reduces amyloid plaque deposition and increases the number of plaque-associated microglia. a, b AAV-mediated sTREM2 expression in 5×FAD mice. Neonatal 5×FAD mice were injected with either AAV2/8-CAG-EGFP (Ctrl) or AAV2/8-CAG-EGFP-2A-sTREM2-3×FLAG (human TREM2 1–171 aa) (sTREM2) in the cerebral ventricles and analyzed after 7 months for sTREM2 expression at the mRNA (a, n = 8 mice per group, unpaired Student’s t test) or protein (b, n = 8 mice for Ctrl, n = 9 mice for sTREM2, unpaired Student’s t test) levels in the cortex and hippcampus using RT-qPCR or ELISA, respectively. c The 5×FAD mice injected with control AAV or AAV-sTREM2 were analyzed for amyloid plaque pathology using an anti-Aβ antibody MOAB-2. Representative images of the hippocampus and the cortex of those mice are shown. Original magnification ×10; scale bar, 200 μm. Blue, DAPI; red, Aβ antibody MOAB-2. d Quantitation of Aβ plaque area in the hippocampus and cortex of 5×FAD mice injected with either control AAV or AAV-sTREM2 (n = 5 mice per group for the hippocampus or the cortex, 12 fields of the hippo and 28 fields of the cortex for analysis, unpaired Student’s t test). e Coronal sections from the 5×FAD mice injected with control AAV or AAV-sTREM2 were stained with DAPI (blue) for nuclei, MOAB-2 (green) for an amyloid plaque, and Iba1 (red) for microglia. Representative z-stack images of the hippocampus and cortex region are shown. Scale bar, 25 μm. f Quantitation of the number of plaque-associated microglia in e (n = 5 mice, 35 plaques of ctrl and 37 plaques of sTREM2 in the hippocampus or 64 plaques of ctrl and 67 plaques of sTREM2 in the cortex for analysis, unpaired Student’s t test). Plaques with 50 μm in diameter were selected for analysis. All data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001

Fig 3: C3G attenuates neuroinflammation in the cortex of APPswe/PS1ΔE9 model mice. Mice were divided into three groups: non-transgenic mice (VC), AD mice administered PBS (APPswe), and AD mice administered 30 mg/kg/day C3G (APPswe + C3G). RNA was extracted from the cortex region of mouse brains and analyzed for mRNA expression of a–c inflammatory cytokines (IL-1β, TNF-α, and IL-6), d M1-specific marker (CD86), e–f M2-specific markers (Arg1 and CD206), and g-h PPARγ and TREM2 using RT-PCR. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig 4: C3G mediates Aβ42-phagocytosis in Aβ42-treated HMC3 cells via triggering receptor expressed on myeloid cells (TREM2). HMC3 cells were treated with 1 μM Aβ42 and co-treated with 1 μM Aβ42 and 50 μM C3G for 24 h. a Cell surface protein expression of TREM2 was determined using flow cytometry. b Intercellular uptake of Aβ42 was determined using flow cytometry. c–d HMC3 cells were pretreated with 1 μM TREM2-blocking antibody for 1 h, followed by co-treatment with 1 μM Aβ42 and 50 μM C3G for 24 h. Intercellular uptake of Aβ42 was determine using flow cytometry and fluorescence microscopy (d). **p < 0.01, ***p < 0.001, and ****p < 0.0001. Scar bar is 50 μm

Fig 5: Plaque-associated microglia from DSCR1 KO/5xFAD mice exhibited increased Iba1 expression, and microglia from DSCR1 KO mice accelerated the degradation of fAβ compared with WT mice.(A) Representative IHC image of Aβ (MOAB2) and microglia (Iba1) with similar plaque sizes in 5xFAD mice and DSCR1 KO/5xFAD mice. Scale bar = 10 µm. (B, C, D) Number of Iba1-positive microglia associated with Aβ plaques (B), microgliosis (C), and Iba1 intensity (D) (5xFAD, n = 78 plaques; DSCR1 KO/5xFAD, n = 50 plaques). (E) Representative images of Aβ (MOAB2), TREM2, and microglia (Iba1) in 5xFAD mice and DSCR1 KO/5xFAD mice at 5 mo. Scale bar = 10 µm. (F) Quantification of microglial TREM2 intensity (5xFAD, n = 499 Iba1-positive (Iba1+) cells; DSCR1 KO/5xFAD, n = 399 Iba1+ cells). (G) Representative images from the engulfment assay. Scale bar = 20 µm. (H) Quantification of internalized fluorescence fAβ intensity in both cell types at different time points after fAβ treatment (WT, n = 71–124 cells; DSCR1 KO, n = 105–144 cells). (I) Representative images from the degradation assay. Scale bar = 20 µm. (J) Quantification of degraded fAβ intensity at different time points after fAβ washing (WT, n = 153–333 cells; DSCR1 KO, n = 135–351 cells). (B, C, D, F, I, J) Values are shown as the mean ± SEM and were tested for statistical significance using a two-tailed unpaired t test. N.S., not significant, **P < 0.01, and ****P < 0.0001.