Extended Data Fig. 8: The increase in tau secretion caused by CA inhibition is abrogated by ARL8BB and VAMP7 depletion and secreted tau is unable to seed.
From: Carbonic anhydrase inhibition ameliorates tau toxicity via enhanced tau secretion
a & b) Graphs showing the intracellular levels of GFP-tau_P301L upon transfection with siRNA-targeted genes Arl8B, VAMP7 or control scramble after carbonic anhydrase inhibition with methocarbamol (a) or methazolamide (b). Levels of intracellular tau were only significantly reduced in control cells, whereas abrogation of lysosomal secretion by silencing of Arl8B or VAMP7 blocked this effect. Data shown as mean corrected values of GFP vs total number of cells ± SEM. Values were normalised to mean of DMSO-treated of each independent experiment at each time point (n = 3 independent experiments; *p≤0.05, **p≤ 0.01, ***p≤ 0.001 vs DSMO analysed by Dunnet’s multiple comparisons two-way ANOVA, ##p≤ 0.01, ####p≤ 0.0001). c & d) Images and quantification of western blots for the knockdown efficiency of siRNA-targeted genes Arl8B (c) and VAMP7 (d) in GFP-tau_P301L cells, showing reduced Arl8B and VAMP7 levels after transfection. GAPDH was used as loading control for lysates. Values were normalized to control sample (siRNA ctr + DMSO) (mean of n = 3 ± SEM; *p≤0.05 and **p≤0.01 analysed by two-tailed one-sample t-test). e & f) Graphs showing the seeding ability of secreted tau in the media from inducible SH- Tau P301L cells treated with methocarbamol (MC) and methazolamide (MTZ) in comparison with recombinant tau seeding capacity in HEK293 cells expressing tau P301S Venus. Lipofectamine only (LF only) and SH-SY5Y cells transfected with empty GFP vector (GFP) were used as negative control. No seeding of secreted tau was observed, despite 0.1–0.2 nM recombinant tau being able to seed (e). As a negative control of the seeding, the entire experiment each time was run in parallel in the parental cell line, HEK293 cells not expressing tau P301S Venus, resulting in no seeding (f). Data shown as mean ± SEM of 3 independent experiments. ****p≤ 0.0001 vs DMSO, analysed by one-way ANOVA followed by Dunnet’s comparison test. g & h) Representative images of the secreted tau (media) and cellular tau (cell lysates), immune blotted with Tau5, PHF1 and AT8 antibodies, from 3 above seeding experiments in e and f, to show detection by Tau5, but not phospho-tau AT8 and PHF1 in secreted tau. This experiment was repeated 3 times. Only one representative blot for each antibody is shown.
