Fig. 8
From: BIN1 is a key regulator of proinflammatory and neurodegeneration-related activation in microglia
LPS-induced up-regulation of IFITM3 in microglia is dependent on BIN1. A qRT-PCR analysis of whole-brain cDNA found inflammation-induced upregulation of key homeostatic and DAM genes are dependent on BIN1. Upregulation of a crucial myeloid transcription factor (Sfpi1), as well as an interferon-induced innate immune gene (Ifitm3), were also BIN1-dependent. Raw dataset is provided in Fig. S8. B NanoString analysis of transcripts in FACS-isolated microglia demonstrates an up-regulation of Ifitm3 following in vivo LPS injections. This effect is augmented in Cx3cr1CreER microglia and is dependent on BIN1. Main effects were found for genotype (F2,16 = 26.538, p < 0.001) and LPS treatment (F1,16 = 66.105, p < 0.001), and a significant genotype*LPS interaction was found (F2,16 = 20.609, p < 0.001) (by two-way ANOVA). Post-hoc pairwise comparisons found Cx3cr1CreER to be different from both Bin1fl/fl (p < 0.001) and Cx3cr1CreER-Bin1 cKO (p < 0.001) (with Fisher’s LSD applied). C qRT-PCR analysis of whole-brain transcripts validated the pattern of microglial expression. A main effect for LPS treatment was found (F1,18 = 17.497, p < 0.001), but the effect for genotype (F2,18 = 3.189, p = 0.065) and the genotype*LPS interaction (F2,18 = 2.734, p = 0.092) failed to reach significance (by two-way ANOVA). Despite this, post-hoc pairwise comparisons found Cx3cr1CreER to be different from Cx3cr1CreER-Bin1 cKO (p = 0.036). However, the comparison with Bin1fl/fl genotype failed to reach significance (p = 0.051) (with Fisher’s LSD applied). D Immunoblot analysis of whole-brain lysates confirmed the transcriptional regulation results in similar IFITM3 protein level changes. Whilst a main effect for LPS treatment was found (F1,8 = 6.156, p = 0.038), genotype (F1,8 = 4.788, p = 0.06) and the genotype*LPS interaction (F1,8 = 4.126, p = 0.077) failed to reach significance in our data (by two-way ANOVA). E NanoString analysis of transcripts in primary cultured microglia shows Ifitm3 expression is blunted in Bin1 KD cells. Main effects for siRNA treatment (F1,8 = 53.326, p < 0.001) and LPS (F1,8 = 43.226, p < 0.001) were found. There was no siRNA*LPS interaction (F1,8 = 3.137, p = 0.115). F CRISPR-edited BIN1 BV2 KO microglia validate that Ifitm3 upregulation in response to LPS stimulation is impaired in Bin1 KO cells, with main effects for Bin1 (F1,20 = 44.503, p < 0.001) and LPS (F1,20 = 23.945, p < 0.001), and a significant Bin1*LPS interaction (F1,20 = 16.023, p < 0.001). G Immunofluorescence detection of IFITM3 in mouse brain demonstrates that IFITM3 expression throughout the LPS-treated Cx3cr1CreER microglia but not in Cx3cr1CreER-Bin1 cKO. Note that microglia are indicated by white arrows, and IFITM3 labelling of blood vessels is indicated by small yellow arrowheads. *, p < 0.05; **, p < 0.01; ***, p < 0.001; by post-hoc t-test with Bonferroni correction for multiple comparisons. All data plotted as mean ± SEM