|
Methocarbamol ameliorates tau-induced toxicity and reduces hyperphosphorylated and insoluble tau levels in zebrafish models. a, Representative images of retinal tissue from rho:eGFP–tau-WT and rho:eGFP–tau-P301L fish showing increased rod survival after treatment with 3 μM methocarbamol (MC). Scale bar, 50 μm. b, Quantification of fluorescent photoreceptor area (as shown in a) following treatment with 3 μM methocarbamol. Drug treatment increased the pixel area of eGFP–tau+ photoreceptors (n = minimum of 38 eyes per condition; data are shown as mean ± s.d.). Data were analyzed by one-way ANOVA, ####P ≤ 0.0001, followed by Tukey’s multiple comparisons test with the following significance values: **P ≤ 0.01 versus DMSO (–), *P ≤ 0.01 for tau-WT (–) versus tau-P301L (–). c, Proportion of morphological abnormalities in fish with pan-neuronal expression of Dendra–tau-A152T after treatment with 3 μM methocarbamol. Phenotypes were ranked as normal (green), mild (yellow), moderate (Mod.; orange) and severe (Sev.; red) according to the severity of deformities (representative images are shown above the graph). Methocarbamol reduced the proportion of fish in the severe category while increasing the proportion of normal fish compared with DMSO-treated siblings (–); n = 13 clutches with a minimum of 30 fish per group; ****P ≤ 0.0001 versus DMSO (–). Data were analyzed by χ2 test. d,e, Images (d) and quantification (e) of western blots showing levels of total (Tau5) and hyperphosphorylated tau (PHF1) in fish with pan-neuronal expression of Dendra–tau-A152T after treatment with 3 μM methocarbamol. Methocarbamol reduces the levels of total tau (Tau5/tubulin), total phospho-tau (p-tau; PHF1/tubulin) and relative amounts of phosphorylated tau (that is, the normalized ratio of (PHF1/tubulin)/(Tau5/tubulin) represented as PHF1/tau); n = 7 independent clutches with 10 fish each ± s.d.; *P ≤ 0.05 versus DMSO; ***P ≤ 0.001 versus DMSO. Data were analyzed by two-tailed one-sample t-test; Tub, tubulin. f,g, Images (f) and quantification (g) of western blots for the sarkosyl-soluble and sarkosyl-insoluble fractions of tau in fish 6 d.p.f. with pan-neuronal expression of Dendra–tau-A152T treated with either DMSO or 3 μM methocarbamol. Graphs show mean ratios (±s.d.) of soluble tau versus tubulin and insoluble tau versus soluble tau normalized to the mean DMSO value. Three independent clutches per condition were used; n = 50 fish per group; ****P ≤ 0.0001 versus DMSO; NS, not significant. Data were analyzed by two-tailed one-sample t-test. Source data
|
