Fig. 3

Astrocyte-mediated synaptotoxicity in response to Aβ is accompanied by tau mislocalisation. A 14DIV neurons treated for 24 h with WTCM astro, TGCM astro or TGCM astro-ID were fixed and immunolabelled with antibodies against MAP2 (green) and tau (far red). Exposure to TGCM astro and TGCM astro-ID induced increased tau localisation in neurites with white arrows indicating beading of these neurites. Scale bar is 100 μm (20 μm in inset). B The number of MAP2 labelled neurites per cell containing missorted tau was quantified, showing increased tau mislocalisation upon exposure to conditioned medium from astrocytes exposed to Aβ (TGCM astro and TGCM astro-ID) (n = 4, four independent experiments each performed in triplicate). Data was analysed by ordinary one-way ANOVA and Tukey’s multiple comparison post-hoc test, and is shown relative to control (WTCM astro). C To confirm that Aβ induces tau mislocalisation in the presence of other neural cell types, 14DIV organotypic brain slice cultures prepared from CD1 mice were treated with WTCM or TGCM for 24 h. The cytosolic fraction (Cyt) and synaptoneurosomes (SNS) were extracted and immunoblotted with antibodies against PSD-95, total tau and tau phosphorylated at Ser396/404 (PHF1). GAPDH was used as a loading control. PSD-95 accumulates in the synaptic fraction showing successful SNS extraction. The abundance of D tau and E PHF1 in synaptoneurosomes relative to the amount of tau or PHF1 in the cytosolic fraction of the same sample was determined. Phosphorylated tau showed increased synaptic localisation following exposure to TGCM (n = 3, three independent slice culture preparations from which three wells of each were pooled). Data was analysed using unpaired t tests. Data on graphs is mean ± SEM and is shown relative to vehicle control. *p < 0.05