Extended Data Fig. 6: Carbonic anhydrase inhibition increases tau clearance by promoting tau secretion.
From: Carbonic anhydrase inhibition ameliorates tau toxicity via enhanced tau secretion
a & b) Concentration-dependent decrease of GFP-tau_P301L accumulation when inducible SH-SY5Y cells expressing tau_P301L were treated with methocarbamol (MC) (1, 3, 10, 30 μM) (a), or methazolamide (MTZ) (0.1, 1, 3, 10, 30 μM) (b); positive controls (0.4 μM rapamycin, 1 μM torin, 100 mM trehalose). No tau detected in uninduced condition. Data are mean± SEM GFP values/cell numbers (Cell Tracker Red). Values were normalised to mean of DMSO-treatment in each independent experiment at each time point (n = 3 independent experiments for MC and n = 4 independent experiments for MTZ; ***p≤ 0.001 and ****p≤ 0.001 vs. DMSO; two-way ANOVA followed by Dunnett’s multiple comparisons test, ####p≤ 0.0001). To simplify graph, significance against DMSO were only annotated for final time point. c) GFP-trap pull-down experiment showing increased extracellular tau in cell media after treatment with 100 μM Bafilomycin A1 (BAF) versus DMSO-treated SH-SY5Y cells expressing GFP-tau_P301L after 24-hour induction with 0.2 µg/ml doxycycline. (i) Immunoblots showing tau levels in cell lysates and immunoprecipitated tau from media collected over 12 hours at 3-hour intervals. ß-actin was loading control for lysates and control for potential differential cell death/lysis in cell medium (ii) Quantification of tau secretion rate. Values normalized to DMSO at t = 3 h (mean of n = 3 experiments ± SD; *p≤0.05 and **p≤0.01 vs. DMSO; by two-way ANOVA followed by Dunnett’s multiple comparisons test, ###p≤ 0.001). (iii) Quantification of LDH release in media at each time point (mean of n = 3 experiments ± SD; ns for p>0.05 vs. DMSO; two-tailed Student’s t test). d) Split luciferase assay in stable SH-SY5Y cells expressing HiBit-tagged tau incubated in complete medium with DMSO or 100 μM Bafilomycin A1 (BAF) over 12 hours and collected at 4-hourly intervals. BAF treatment (i) without affecting LDH release (ii). Values normalized to DMSO at t = 4 h (mean of n = 3 experiments ± SD; *p≤0.05 and **p≤0.01 vs. DMSO; two-way ANOVA followed by Dunnett’s multiple comparisons test, ###p≤ 0.001). Bars indicate LDH release (mean of n = 3 experiments ± SD; ns for p>0.05 vs. DMSO; two-tailed Student’s t test).
