Fig. 9

Microglia promote the formation of a C-terminal lacking amyloid fragment in vitro. a 3D reconstruction by Imaris software of microglia cells (Iba1 positive, red) treated or not for 3 h with 1 μM Aβ42–488 (green). Scale bar: 4 μm. b Bright field images of microglia cells stained with 6E10 (red) or anti-Aβ42 (green) antibodies, before (left panel) or after (right panel) 24 h incubation with H-Aβ42. Arrows indicate 6E10 positive puncta not co-localizing with anti-Aβ42 positive domains. Scale bar: 10 μm. On top: schematic representation of H-Aβ42 sequence, showing the binding sites for the different antibodies. c Dot blot analysis of the extracellular medium collected 6 and 24 h after microglia exposure to H-Aβ42. Histograms represent the densitometry quantification upon staining with anti-Aβ42 C-terminal antibody (left histogram) or 6E10 N-terminal antibody (right histogram). Intensity values are shown. N = 5 independent experiments, statistical analysis was performed by One way Anova, Bonferroni multiple comparison test (**P < 0.01, *P < 0.05). d Quantification of Aβ42 extracellular levels in microglia cultures exposed to H-Aβ42 for the indicated time points. Two different ELISA kits based on a capture antibody against the C-terminal (left) or N-terminal (right) domains of the protein were used. N = 5 independent experiments. Statistical analysis was performed by One way Anova, Bonferroni’s post hoc test for multiple comparisons (*** P < 0.001). e ELISA of C-terminal-containing Aβ42 in the extracellular medium of microglia exposed to H-Aβ42 in both control conditions and in the presence of protease inhibitors. N = 3 independent experiments. Statistical analysis was performed by two way Anova, Bonferroni’s post hoc test for multiple comparisons. f ELISA of C-terminal-containing Aβ42 in the extracellular medium of N9 cells exposed to siRNAcontrol or MMP9siRNA. Statistical analysis was performed by two way Anova, Bonferroni’s post hoc test for multiple comparisons. g Representative dot blots of the extracellular medium from H-Aβ42-treated microglia in control conditions, in the presence of protease inhibitors, from siRNAcontrol or MMP9siRNA or from MMP9^−/− microglia. Blots are immunostained with anti-Aβ42 or 6E10 antibodies. h-j Dot blot quantification of the extracellular medium collected from microglia in control conditions or in the presence of protease inhibitors h), from siRNAcontrol or MMP9siRNA i) or from MMP9^−/− microglia j) after 6 and 24 h of Aβ42 treatment. k) Apical-to-basolateral exchange across the BBB of fluorescent Aβ42 over 120 min upon pre-incubation with Aβ42-treated microglia conditioned medium in the presence or absence of protease inhibitors. Quantification of unidirectional trans-endothelial Aβ42 transport by fluorescence spectrophotometry of N = 2 independent experiments; statistical analysis was performed by One way Anova, Bonferroni’s post hoc test for multiple comparisons (**P < 0.01)