Fig 2: TREM2-independent formation of interferon-responsive oligodendrocytes and microglia in the aged white matter.a, Immunofluorescence staining and b, quantification of MHC1+IBA1+ microglia in the white matter of 3-month old and 24-month old mice. Arrow marks MHC1+ nodules (n = 3 mice per group, **P = 0.0012; data are means±s.e.m; P value represents a two-sided Student’s t-test). Arrows mark double positive microglia nodulces. c, Immunofluorescence combined with RNAscope of MBP mRNA (green) within IBA1+ microglia nodule (red). Clipped 3D images show MBP mRNA in a microglia nodule. Arrow mark MBP mRNA inside microglia. Scale bar, 20 µm, 3D rendering 10 µm. d, Quantification of percentage of IBA1+ single microglia and IBA1+ nodule with MBP mRNA inside (n = 3 mice per group, *P = 0.0160; data are means±s.e.m; P values represent a two-sided Student’s t-test). Quantification of percentage of IBA1+ volume occupied by MBP mRNA (single microglia, n = 26 cells, IBA1+ nodules, n = 30; ***P = 0.0004; data are means±s.e.m.; P value represents two-tailed Mann-Whitney test). e, Immunofluorescence staining and quantification of CD8+ T (red) cells in the white matter of 18-month old wild type and Trem2-/- (n = 4 mice per group, *P = 0.0252; data are means±s.e.m; P value represents a two-sided Student’s t-test). Arrows indicate CD8+ T cells. Scale bar 20 µm. f, Immunofluorescence staining and quantification of STAT1+CC1+ oligodendrocytes in the white matter of 18-month old wild type and 18-month old Trem2-/- mice (n = 4 mice per group; data are means±s.e.m; P value represents a two-sided Student’s t-test). Scale bar, 20 µm. g, Immunofluorescence staining and quantification of STAT1+IBA1+ microglia in the white matter of 18-month old wild type and 18-month old Trem2-/- mice (n = 3 mice per group; data are means±s.e.m; P value represents a two-sided Student’s t-test). Scale bar, 20 µm. h, Immunofluorescence staining of CD8+ T cells and STAT1+IBA1+ microglia in the white matter of 18-month old wild type and 18-month old Trem2-/- mice. Scale bars, 20 µm. Quantification of STAT1+IBA1+ microglia and CD8+ T cells proximity (<20 µm) in 18-month old wild type and Trem2-/- mice (WT, n = 3; Trem2-/-, n = 4 mice; data are means ± s.e.m. P value represents a two-sided Student’s t-test). Source data

Fig 3: IFN-γ injections induce myelin and oligodendrocyte loss in aged mice.a, Diagram showing the model of IFN-γ injection in the corpus callosum (CC). The lesion area was identified by the positivity for monastral blue. b, Representative pictures of the CC after injection of 1 µl of a solution of 10 ng µl−1 of IFN-γ, 48 h postinjection. The intensity of the staining for MBP (green) was used to quantify the extent of demyelination (young, n = 4; old, n = 3; ***P = 0.0006; data are mean ± s.e.m.; P value represents a two-sided Student’s t-test). a.u., arbitrary units. c–e, Representative confocal images and quantifications of IFN-γ-mediated lesions in young and old mice, showing CC1+ oligodendrocytes (c), MAC2+ microglia (d) and NF200+ axons (e). The number of oligodendrocytes was expressed as number of cells per mm2; the amount of phagocytes was expressed as percentage area of the lesion occupied by MAC2+ cells; the extent of axonal damage was expressed as staining intensity of NF200 (young, n = 4; old, n = 3; CC1+, **P = 0.0031; MAC2+, *P = 0.0291; NF200+, *P = 0.0451; data are mean ± s.e.m.; P values represent a two-sided Student’s t-test). Scale bar, 20 µm. f–h, Representative confocal images and quantifications of IFN-γ-mediated lesions in old mice and of the vehicle control showing CC1+ oligodendrocytes (f), MAC2+ cells (g) and NF200+ axons (h) (n = 3 mice per group, CC1+, **P = 0.0025; MAC2+, *P = 0.0451; data are mean ± s.e.m.; P values represent a two-sided Student’s t-test). i, Representative images of cultured oligodendrocytes (labeled for O1 in red and STAT1 in white) after treatment with IFN-γ for 24 h compared with control. Quantification of the number of oligodendrocytes after IFN-γ or vehicle treatment, expressed as number of O1+ cells per mm2 (n = 4 biological replicates per group, **P = 0.0022; data are mean ± s.e.m.; P values represent a two-sided Student’s t-test). j, Representative images of oligodendrocytes cultured with microglia alone (MG) or microglia together with IFN-γ (MG+IFN-γ) and stained for O1 (oligodendrocytes) and IBA1 (microglia). Quantification of the number of oligodendrocytes, expressed as number of O1+ cells per mm2 (n = 4 biological replicates per group, **P = 0.0022; data are mean ± s.e.m.; P values represent a two-sided Student’s t-test).Source data

Fig 4: Detection of RABV P in non-neuronal Schwann cells of hind leg tissue after i.m. inoculation (9 days post-inoculation). a, b Maximum z-projection of high-resolution confocal z-stacks of RABV-infected nerve fiber (see also Fig. 1d) in hind leg sections [z = 45 µm; Scale bar: 100 µm] (a) and detail [z = 15 µm; Scale bar: 10 µm] (b). Red: RABV P; green: NEFM; blue: cell nuclei. c, d Volumetric 3D projections of (b) with different viewing angles. The light blue box indicates the clipping plane. Arrows: RABV P surrounding axonal NEFM. e, f Maximum z-projection of a hind leg section (e) [z = 134 µm; Scale bar: 200 µm] and detail (f) [z = 20 µm; Scale bar: 10 µm] stained for RABV P (red), MBP (green) and nuclei (blue). g, h Volumetric 3D projections of different viewing angles of (f). Arrowhead: RABV-infected Schwann cell i–k Single slices with RABV P (red), MBP (green) and nuclei (blue). Arrowheads indicate myelin sheath/nucleus surrounded by RABV P. Scale bar: 5 µm

Fig 5: RABV P in axons and infected Schwann cells in coronal head sections. a Maximum z-projection of high-resolution confocal z-stacks of mouse head sections after i.c. inoculation and staining for RABV P (red), NEFM (green) and nuclei (blue) [z = 38 µm]. Arrows indicate RABV P surrounding NEFM. Scale bar: 100 µm. b–e Maximum z-projections (b, d) and corresponding 3D projections (c, e) of details of (a) (white boxes). f Maximum z-projection of confocal z-stacks of mouse head sections after i.c. inoculation stained for RABV P (red), MBP (green) and nuclei (blue) [z = 29 µm]. Scale bar: 25 µm. g–i Single slices of (f). Arrowhead: RABV P surrounding MBP-positive myelin sheath. Star: RABV P surrounded by MBP-positive myelin sheath. Scale bar: 5 µm. j 3D projection of (g–h)