Fig. 7
Silencing of TIA1 expression protects against oligomeric Tau toxicity. a Representative images showing the co-localization of phosphorylated tau inclusions CP13 (red) with TIA1 granules (green) in hippocampal neurons over-expressing human P301L tau, at 1 h after S1p or vehicle treatment. Co-labeled markers are MAP-2 (violet) for neurons and DAPI (blue) for nuclear. Scale bar 20 µm. b To show the translocation of TIA1 from nuclear to cytosol and co-localization with tau inclusions in neuronal soma, high-magnification images were enlarged fivefold from the boxed neurons in a. c CP13 staining representative images show that TIA1 knock down disperses S1p induced bead-like tau granules in synapse and dendrites, creating a more continuous, less consolidated distribution of CP13 positive tau. CP13 staining of hippocampal cultures over-expressing human 4R0 N WT or P301L tau fixed at 24 h after treatment. d TIA1 knockdown prevents the elevation of CP13 phospho-tau from S1p or P3 treatment. Immunoblots of primary neuron lysates expressing either 4R0N WT or P301L tau, knocked down with either shCtrl or shTIA1 and treated for 24 h with S1p or P3 fractions. Phosphorylated tau detected by CP13 and MAP-2 internal control. e Quantification of CP13 immunoblot in cell lysate of hippocampal cultures over-expressing human 4R0 N WT tau, transduced with shTIA1 or shCtrl AAV on day- 4, treated with vehicle control, S1p or P3 on day 14 and harvested at 24 h after treatment. N = 3, in shCtrl, S1p compared to vehicle **p < 0.001, P3 to vehicle **p < 0.001. For S1p treatment, shTIA1 compared to shCtrl, ##p = 0.001. For P3 treatment, shTIA1 compared to shCtrl, ##p = 0.0016. f Quantification of CP13 immunoblot in cell lysate of hippocampal cultures over-expressing human P301L tau, transduced with shTIA1 or shCtrl AAV on day 4, treated with vehicle control, S1p or P3 on day 14 and harvested at 24 h after treatment. N = 3, in shctrl, S1p compared to vehicle **p = 0.007, P3 to vehicle **p = 0.006. For S1p treatment, shTIA1 compared to shCtrl, ##p = 0.006. For P3 treatment, shTIA1 compared to shCtrl, ##p = 0.006. Two-way ANOVA multiple comparison test by Fisher’s LSD. g NeuN-positive neurons in shctrl and shTIA1 cultures after saline or S1p treatment. **p < 0.0001 compared to corresponding control, ##p = 0.0055 compared to S1p group in shctrl cultures. h LDH assay with conditioned medium at 24 h after vehicle or S1p treatment in hippocampal neurons over-expressed with human WT or P301L Tau. The S1p fractions are from three genotypes of 9-month-old brains, including PS19 Tia1+/+, PS19 Tia1+/− and C57BL/6 wild-type mice. N = 4 separate times of experiment (triplicate wells each time). **p < 0.001 compared to corresponsive saline control. In WT tau over-expressed neurons, Tia1+/+ S1p compared to Tia1+/− S1p, #p = 0.0402. In P301L tau over-expressed neurons, Tia1+/+ S1p compared to Tia1+/− S1p, #p = 0.0394. Two-way ANOVA, post hoc multiple comparison test by Fisher’s LSD. Data are represented as mean ± SEM