Figure 5
From: BIN1 favors the spreading of Tau via extracellular vesicles
Deletion of Bin1 in microglia reduces the release of Tau via EVs in vitro as well as decreases pTau spreading in male mice in vivo. (A) Experimental timeline for in vitro microglia-derived Tau-containing EVs analysis. (B) Western blot of Tau and BIN1 in cell lysates (top) and EVs (bottom) from Bin1 wt and cKO primary microglia cells after administration of Tau fibrils and stimulation with LPS and ATP, as reported in A. β-actin: cell lysate loading control. Exosome marker Alix: EVs loading control. Each experiment performed by pooling together microglia from more than 10 pups of the same genotype. One representative experiment shown out of 4 independent replicates. WB images shown represent cropped sections of full blots shown in Supplementary Fig. 13. (C) Plot representing densitometric analysis of Tau band intensity normalized to Alix band intensity, as obtained by WB from EVs purified from CMs of microglia Bin1 wt and cKO, respectively. Plot represents mean and SD of 4 independent replicates. Paired parametric Student’s t-test two tailed, 95% confidence: p-value: *p < 0.05; **<0.01; ***p < 0.001. (D) Experimental scheme. Artwork by Channa Bao. (E) Representative images of immunohistochemical staining of pTau (40E8) in contralateral hippocampi of Bin1flox/flox:: Cx3Cr1-Cre+::PS19Hemi and Bin1wt:: Cx3Cr1-Cre−::PS19Hemi littermate male mice injected with Tau fibrils. Scale: 700 μm. (F) Plot representing the percentage of pTau staining area normalized to the size of the hippocampus for each animal. Each symbol represents one animal. (G) Representative images of immunohistochemical staining of pTau (40E8) in contralateral hippocampi of Bin1flox/flox:: Cx3Cr1-Cre+:: PS19Hemi and Bin1wt:: Cx3Cr1-Cre−:: PS19Hemi littermate female mice injected with Tau fibrils. Scale: 700 μm. (H) Plot representing the percentage of pTau staining area normalized to over the size of the hippocampus for each animal. Each symbol represents one single animal. Two-way ANOVA with Holm’s p-value adjustment (two-sided) (see Supplementary Fig. 9): p-value: *p < 0.05; **<0.01; ***p < 0.001.
