Fig. 3: Impairment of TGFβ signaling in microglia results in their activation and subsequent demyelination in the DC.

a, A schematic illustration of Tgfbr2 deletion blocking TGFβ ligand binding in microglia of Cx3cr1^CreER:Tgfbr2^fl/fl mice. b–d, Immunofluorescent staining of IBA1 (green), MHC-II (magenta) and nuclei (Hoechst, blue) in cervical spinal cord sections of Tgfbr2^fl/fl (WT) and Cx3cr1^CreER:Tgfbr2^fl/fl (KO) mice (~12 months old) at 7, 20, 30 and 60 days post-tamoxifen injection (b), with the quantification of microglia number (c) and mean fluorescent intensity (MFI) of IBA1 and MHC-II (d). Scale bar, 200 μm; n = 4, 3, 3, 5 and 5 mice for each time point. e,f, Flow cytometry analyses comparing the expression of microglial surface markers MHC-II and CD45 in CD11b⁺CD45⁺Ly6C⁻Ly6G⁻ cells microdissected from the DC, VC and GM regions of adult WT and Cx3cr1^CreER:Tgfbr2^fl/fl mice at 18 or 28 days post-tamoxifen administration (e) and quantification comparing the frequency of MHC-II^high and CD45^high microglia across the different spinal cord regions (f); n = 3, 5, and 4 mice for each group. g,h, Staining of fluoromyelin (green), IBA1 (red) and nuclei (Hoechst, blue) in cervical spinal cord sections of ~12-month-old WT and KO mice at 20, 30 or 60 days post-tamoxifen administration (g) and quantification comparing the percentages of demyelinated area between DC and VC (D30) (h). Scale bar, 200 μm, n = 4 mice. i, Representative immunofluorescent staining of IBA1⁺ microglia (green), PLP1⁺ myelin (magenta) and TUJ1⁺ axons (cyan) in the DC of KO mice 25 days post-tamoxifen administration, with an orthogonal display illustrating the spatial arrangement of axons surrounded by microglia. A 3D reconstruction of microglia encompassing the axon–myelin units is shown below. Scale bar, 3 μm. j, Representative TEM images in the DC and VC of ~12-month-old WT and KO mice (25 days post-tamoxifen administration). Scale bar, 20 μm. The insets below indicate degenerated axons (orange arrows) and redundant myelin (red arrows). Scale bar, 2 μm. k,l, Quantification of myelinated axons (k) and myelin abnormalities (l) in the DC and VC of WT and KO mice; n = 4 and 3 mice for WT and KO_D25, respectively. m,n, Spontaneous behavioral deficits in KO mice were assessed using the disease score (m) and four-paw hanging wire behavioral test (n); n = 18 mice for the young female (YF) and older female (OF) groups, and n = 11 and 12 mice for the young male (YM) and older male (OM) groups, respectively. Young mice: 2–3 months; older mice: 8–12 months. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001. A two-way ANOVA with Sidak’s post hoc test was used for c and d. Cross-region comparisons within each group in f were analyzed using repeated-measures one-way ANOVA with Geisser–Greenhouse correction, followed by Dunnett’s post hoc test. A two-tailed paired or unpaired t-test was used for h and i, respectively. Multiple unpaired t-tests with false discovery rate (FDR) correction were used for k. The cumulative disease score in m was analyzed using the Kruskal–Wallis with Dunnett’s post hoc test, and the mean time in the four-paw hanging wire test in n was analyzed using one-way ANOVA with Tukey’s post hoc test. p.t., post-tamoxifen administration. Panel a created with BioRender.com.

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