Extended Data Fig. 7: Carbonic anhydrase inhibition causes redistribution of lysosomes and tau secretion by lysosomal exocytosis.
From: Carbonic anhydrase inhibition ameliorates tau toxicity via enhanced tau secretion
a) Images and quantification of lysosomal re-distribution from the perinuclear region to the periphery in SH-SY5H cells upon treatment with DMSO, 100 μM Bafilomycin A1 (BAF), 30 μM methazolamide (MTZ) or 30 μM methocarbamol (MC) for 6 h. (i) Representative confocal images showing immunofluorescence staining of lysosomes (LAMP1 in green) and nuclei stained with DAPI (magenta). Arrowheads point to the perinuclear localization of lysosomes in the DMSO group. Scale bar: 50 µm). (ii) Quantification of LAMP1 distribution using CellProfiler Analyst. (n = 3 independent experiments with a minimum of 40 cells/condition for each; graph represents mean ± SD; *p≤0.05, **p≤ 0.01, ***p≤ 0.001 vs. DMSO analysed by two-tailed Student’s t test). b) Knockdown efficiency of siRNA-targeted genes VAMP7 and Arl8B in SH-SY5Y cells expressing HiBit-tagged tau incubated in complete medium in presence of DMSO, 30 μM MTZ or 30 μM MC for 12 hours. (i) Western blots and their relative quantification (ii) showing reduced VAMP7 and Arl8B levels after transfection with indicated siRNAs. ß-actin was used as loading control for lysates. Values were normalized to control sample (siRNA ctr + DMSO) (mean of n = 3 experiments ± SD; ***p≤0.001 analysed by two-tailed Student’s t test). c) Quantification of tau secretion by the split luciferase complementation assay in SH-SY5Y cell line expressing HiBit-tagged tau transfected with siRNAs targeting VAMP7 and ARL8B. Abrogation of lysosomal exocytosis by siRNAs reduces drug-dependent tau secretion to the media without affecting cell integrity (LDH release). d) Quantification of cathepsin D release by ELISA in SH-SY5Y cells transfected with siRNAs targeting VAMP7 and ARL8B. Drug effect on cathepsin D secretion is lost after preventing lysosomal exocytosis. For c and d, cells were incubated in complete medium with DMSO, 30 μM MTZ or 30 μM MC for 12 hours. Values were normalized to DMSO (mean of n = 3 experiments ± SD; *p≤0.05, **p≤0.01 vs. DMSO analysed by two-tailed Student’s t test). Bars in the bottom graph indicate LDH release (mean of n = 3 biological replicates ± SD; ns for p>0.05 vs. DMSO analysed by two-tailed Student’s t test).
