Fig. 2: Soluble tau aggregates destabilize microtubules, reduce microtubule density, and block eNOS activation in human brain microvascular endothelial cells.
A, B Soluble tau aggregates destabilize microtubules in primary human brain microvascular endothelial cells (HBEC). A Representative electropherograms from capillary electrophoresis immunoassays of acetyl-α-tubulin and α-tubulin in lysates of cells treated with recombinant human cytokeratin-8 (KRT8), monomeric tau protein (M. Tau), soluble tau aggregates (O.Tau), or vehicle (control). B Quantitative analyses of data represented in (A). (F(3,19) = 9.871, ANOVA, P = 0.0004, O. Tau different from Control by Tukey’s test, ***p < 0.001. Control, n = 11; KRT8, n = 4; M. Tau, n = 4, O.Tau, n = 5 biologically independent samples examined over 6 independent experiments. Data are means ± SEM. C, D Soluble tau aggregates reduce microtubule density in HBEC. C Representative images of β-tubulin immunoreactivity in HBEC exposed to KRT8, M. Tau, O.Tau, or vehicle (control). Scale bar is 200 µm. D Quantitative analyses of microtubule density distributions from data in C (H = 49.37, P < 0.0001, Kruskal–Wallis. M. Tau and O. Tau different from Control by Dunn’s ****P < 0.0001. Control, n = 31; KRT8, n = 8; M. Tau, n = 8, O. Tau, n = 27 biologically independent samples examined over three independent experiments. Data are representative images and means ± SEM. E, F Soluble tau aggregates inhibit eNOS translocation to the cell membrane. E Representative images of eNOS and β-tubulin immunoreactivity in HBEC exposed to KRT8, M. Tau, O.Tau and induced with acetylcholine (ACh) or vehicle (control). The scale bar is 200 µm. F Quantitation of edge-associated eNOS (H = 12.04, p = 0.0002, Kruskal-Wallis, O.Tau different from Control by Dunn’s, **p = 0.002), n = 6 (all experimental groups) biologically independent samples examined over 2 independent experiments and G perinuclear-localized eNOS from images in E in ACh-induced cells normalized to the vehicle for each treatment group (H = 1.719, P = 0.4527, Kruskal–Wallis, n.s., n = 6 (all experimental groups) biologically independent samples examined over 2 independent experiments. Soluble tau aggregates decrease nitric oxide (NO) production in HBEC. H Quantification of the relative fluorescence of DAF-FM, a nitric oxide indicator, in HBEC treated with M. Tau, O. Tau, or vehicle (Control) (F(2,45)=22.15, ANOVA, p < 0.0001; Control vs M. Tau, ** p = 0.0048; Control vs O.Tau, ****p < 0.0001, M. Tau vs O.Tau, #p = 0.0473 by Tukey’s. Control, n = 30; M. Tau, n = 9, O. Tau, n = 9 biologically independent samples examined over three independent experiments. Data are means ± SEM. I, J Soluble tau aggregates decrease eNOS activation by phosphorylation at S1177. I Representative electropherograms from capillary electrophoresis immunoassays of phosphorylated eNOS (S1177) in HBEC treated with KRT8, M. Tau, O.Tau, or vehicle (control). J Quantitative analyses of data in G (F(3,18) = 3.903, ANOVA, p = 0.027, Control vs. O. Tau, *p < 0.05; M. Tau vs. O. Tau, #, p < 0.05; Control vs. M. Tau, n.s., from Tukey’s. Control, n = 11; KRT8, n = 3; M. Tau, n = 3, O.Tau, n = 5 biologically independent samples examined over 6 independent experiments. Data are means ± SEM. K, L Soluble tau aggregates promote eNOS inhibition by phosphorylation at Thr495. K Representative electropherograms from capillary electrophoresis immunoassays of phosphorylated eNOS (Thr495) in HBEC treated with KRT8, M. Tau, O.Tau, or vehicle (Control). L Quantitative analyses of data in I (F(3,16)=5.079, ANOVA, p = 0.012, Control vs O.Tau, *p = 0.03; M. Tau vs O.Tau, #p = 0.014, Control vs. M. Tau, n.s., p = 0.79. Control, n = 8; KRT8, n = 4; M. Tau, n = 4, O.Tau, n = 4 biologically independent samples examined over four independent experiments. Data are representative electropherograms, and means ± SEM. For post-hoc analyses, lack of a specific P value in the legend reflects the information reported by GraphPad Prism Version 9.4.0.
