Extended Data Fig. 10: C1q and C3b produced by Grn^−/− microglia promote TDP-43 granule formation and cell death in Grn^−/− neurons.

a, Immunohistochemical images of Grn^+/+, Grn^−/− and Grn^−/−;C1qa^−/−;C3^−/− mice at 7 months show the upregulation of C1q and C3b in the ventral thalamus of Grn^−/− mice. No C1q or C3b staining is detected in Grn^−/−;C1q^−/−;C3^−/− mouse brain, confirming the specificity of these antibodies. Insets in Grn^−/− panels represent higher magnification of the boxed regions in the ventral thalamus. Results were analysed in 3 mice per genotype. b, ELISA assays for C1q and C3b show increases of both proteins in Grn^−/− MCM, but no C1q or C3b is detected in Grn^−/−;C1qa^−/−;C3^−/− MCM. Data represent mean ± s.e.m. from 8 independent microglial cultures for Grn^+/+ and Grn^−/− MCM, and 3 independent cultures from Grn^−/−;C1qa^−/−;C3^−/− MCM. Statistics use two-tailed, unpaired Student’s t-test. c, Confocal images of cultured Grn^+/+ and Grn^−/− cortical neurons treated with purified human C1q (1 μg/ml) or C1q+C3b (1 μg/ml, each) indicate that complements are sufficient to promote the formation of TDP-43 granules in Grn^+/+ and Grn^−/− cortical neurons, whereas Grn^−/−;C1qa^−/−;C3^−/− MCM fail to induce TDP-43 granule formation. d, Quantification of cytoplasmic TDP-43 intensity (upper panel) and cell death (lower panel) in Grn^+/+ and Grn^−/− neurons treated with C1q, C1q+C3b and C4. N in the upper panel and the lower panel indicates the number of independent cultures analysed. On average, 6-8 images were obtained from each culture. Statistics use two-tailed, unpaired Student’s t-test. e, Quantification of cytoplasmic TDP-43 intensity (upper panel) and cell death (lower panel) in Grn^+/+ and Grn^−/− neurons treated with control media, Grn^−/− MCM, Grn^−/−;C1qa^−/− MCM or Grn^−/−;C1qa^−/−;C3^−/− MCM. Data represent mean ± s.e.m. Statistics use two-tailed, unpaired Student’s t-test. ns, not significant. N in the upper panel and the lower panel indicates the number of independent cultures analysed. On average, 6-8 images were obtained from each culture. f, Quantification of cell death of Grn^+/+ and Grn^−/− neurons treated with Grn^−/− MCM (250 μg/ml) and two different concentrations of vitronectin (50 or 500 ng/ml), an inhibitor of the complement membrane attack complex. Data represent mean ± s.e.m. Statistics uses two-tailed, unpaired Student’s t-test. ns, not significant. Data are obtained from 3 independent cultures.