Fig. 4

Astrocytic inflammatory phenotypes are induced by Aβ. To explore the effects of physiological Aβ concentrations on astrocyte phenotypes, a number of inflammatory markers were examined. A Representative images from mouse astrocytes exposed to TGCM for 24 h showed increased GFAP immunoreactivity (red) relative to those treated with WTCM. Scale bar = 100 μm. Lysates from treated astrocytes were immunoblotted with antibodies against B GFAP, C phospho-NFkB (p65) and D Lcn2. GAPDH was used as a loading control. Quantification of band intensities showed increases in E GFAP, F pNFkB and G Lcn2 in TGCM-treated cultures relative to astrocytes treated with WTCM. Levels of the protein of interest were normalised to GAPDH in each case (n = 5, five independent cultures each containing three technical repeats). Data was quantified using unpaired t tests. Antibody-based cytokine arrays were used to provide unbiased analysis of cytokine content in medium from WTCM and TGCM exposed mouse and human astrocytes and in tissue homogenates from postmortem AD and control brain. Representative membranes showing increased CXCL1 in TGCM conditions are shown for H mouse astrocytes (n = 4, four independent experiments each performed in triplicate) and J human iNPC-astrocytes (n = 3, three independent experiments each performed in triplicate). L Total protein normalised cytosolic fractions of postmortem control and BA9 prefrontal cortex of AD brain at different Braak stages (n = 4 control, n = 7 stage I–II, n = 10 stage III–IV, n = 6 stage V–VI). Quantification of CXCL1 amounts in these samples revealed significant increases in CXCL1 in TGCM-treated I) mouse and K human astrocytes, and M in Braak stage V–VI human AD brain. Data was analysed using t test or one-way ANOVA with Dunnett’s post-hoc tests. Data on graphs is mean ± SEM and is relative to control (control brain or WTCM). *p < 0.05