Fig 2: TREM2 deficiency locks microglia into a homeostatic state in TDP-43-induced neurodegeneration.In adult local injection model, hTDP-43 or GFP-hTDP-43 was expressed in the primary motor cortex of 2-month-old mice via stereotactic intracerebral injection of AAV9.CAG.hTDP-43 or AAV9.CAG.hTDP-43.GFP (AAV9.CAG.Empty or AAV9.CAG.GFP as control). a, Timeline for local injection model. b, Representative images (upper panels. Scale bar, 100 μm) and skeletal images (lower panels. Scale bar, 10 μm) of GFP-expressing microglia in the primary motor cortex of indicated groups at 14 dpi. c-e, Quantification of microglia number of branches (c), soma size (d), and process length (e) in b. f,g, Representative images of P2Y12 expression (f) and TMEM119 expression (g) in the primary motor cortex of indicated groups at 7 dpi. Scale bar, 100 μm. h, i, Quantification of relative P2Y12 (h) and TMEM119 (i) positive area. j, Representative images of CD11c (purple), hTDP-43 (green), and DAPI staining (blue) in the primary motor cortex of indicated groups at 28 dpi. Scale bar, 100 μm. High magnification images as indicated by area in dotted white box showing on the right. Scale bar, 10 μm. k, Analysis of co-localization of CD11c with GFP-hTDP-43 in the primary motor cortex of indicated groups at 28 dpi. Fluorescence intensity profiles of CD11c and GFP-hTDP-43 show the distribution of fluorescence across the yellow dotted arrows in j. l, Quantification of the CD11c+ microglia in i. Significances were calculated using either two-way ANOVA, Tukey’s post-hoc analysis (c-e, h and i) or two-tailed unpaired Student’s t-test (l). Data represented as mean ± SEM. n.s., not significant, *P < 0.05, **P < 0.01, *** P < 0.001. c, n = 10 per group, P < 0.0001, F 3,27 = 80.39; d, n = 15 per group, P < 0.0001, F 3,42 = 38.44; e, n = 10 per group, P < 0.0001, F 3,27 = 89.88; h, n = 10 per group, P < 0.0001, F 3,27 = 24.12; i, n = 10 per group, P = 0.0146, F 3,27 = 14.90; l, n = 7 per group, P < 0.0001, t = 20.47, d.f. = 12.

Fig 3: Differentially expressed (DE) and microglia-specific genes in PLOSL. (A) Volcano plots displaying DE genes in PLOSL against a baseline of gene expression in controls. (B) Venn diagram and list of shared DE genes (FDR < 0.1) between TREM2 R47H AD and PLOSL. (C) Enriched ontology clusters in DE TREM2 R47H AD and PLOSL. Metascape cluster analysis of GO term enrichment among DE genes (FDR < 0.1) between TREM2 R47H AD and PLOSL. Eight hundred genes on the NanoString panel were used as a background for enrichment calculations. GO ID, Description: Representative GO term; Log10(P) – p-value for enrichment. (D) Microglia-specific genes (MG) expression is highly perturbed in PLOSL as compared with AD groups. Distribution of fold changes in MG (N = 134); X-axis displays log2 of fold changes between gene expression in cases vs. controls (∗p-value < 0.05; ****p-value < 0.0001, one-way ANOVA, Tukey’s multiple comparison test); (E) Protein–protein association network formed by differentially expressed MG in PLOSL, as visualized by STRING database (http:/string-db.org).

Fig 4: Phagocytosis of aggregated pHrodo-labeled amyloid beta-peptide. THP-1, unmodified cells; THP-Cas9, THP1 stably expressing Cas9 nuclease; KO, TREM2 knockout; KO + CV, KO + R47H, KO expressing common variant (CV) or R47H TREM2, respectively. All cells were treated with doxycycline, then incubated with pHrodo-labeled Aβ. Data are shown as mean of biological triplicates ± SD (NS, non-significant; ∗∗p < 0.01; ∗∗∗p < 0.001; and ****p < 0.0001, one-way ANOVA, Tukey’s multiple comparison).

Fig 5: (a) ELISA of IL-1beta protein secretion in supernatant from iPS-Mg from control, TREM2 R47Hhet and R47Hhom lines following treatment overnight with PS+ SH-SY5Y cells or overnight L PS+ 30 min ATP; data shown is mean ± SEM, n = 3–9 cell lines from 5 individual experiments, *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA with Tukey’s correction). (b–f) qPCR analysis in control and R47Hhet iPS-Mg following overnight treatment with PS+ SH-SY5Y cells or LPS of (b) IL1B, (c) NLRP3, (d) NLRP1, (f) CASP1 and (f) NFKB1; data are mean ± SEM, n = 2–3, *P < 0.05, ***P < 0.001 (two-way ANOVA with Tukey’s correction), ns, non-significant.