Fig. 6

TAOK2 regulates spine function through RhoA signaling. a Western blot of RhoA-GTP (arrow) and total RhoA in cortical and hippocampal lysates from P21 WT, Het, and KO Taok2 mice. Arrowhead indicates background from Rhotekin-GST. b Increased levels of total RhoA and reduced levels of Rho-GTP (normalized to β-actin) in the Taok2 KO mice cortex. c Reduced levels of RhoA-GTP (normalized to β-actin) in the hippocampus compared with the cortex in WT mice. (n = 4 western blots; one-sample t-test; Hippocampus: t(3) = 46.03, p < 0.0001). (Eight separate mouse cortices per condition; total RhoA: one-sample t-test; WT vs. KO: t(7) = 2.905, p = 0.0228; RhoA-GTP: one-sample t-test; WT vs. KO: t(7) = 5.55, p = 0.0009). d Western blot of RhoA-GTP and total RhoA in LCLs of the A135P proband and the unaffected father. e Increased levels of total RhoA and reduced levels of Rho-GTP in LCLs of the A135P proband compared with the unaffected father (nine separate lysates per LCL; RhoA levels (normalized to tubulin): one-sample t-test; t(80) = 2.499, p = 0.0370; RhoA-GTP: one-sample t-test; t(8) = 2.399, p = 0.0433). f Western blots of the SHSY-5Y neuroblastoma cell lines showing increased RhoA-GTP levels after addition of the RhoA activator (CN01) (arrow). Arrowhead indicates background from Rhotekin-GST. g Snapshots from time-lapse analysis (0, 90, 189, and 302 s) of dendritic filopodia/spines from DIV14 cortical neurons labeled with Lifeact-GFP from WT and KO Taok2 mice and Taok2 KO treated with CN01 (1 U/ml). Red arrowheads indicate actin-rich protrusions selected for kymograph analysis. Bottom: kymographs reveal diffuse and unstable spine movement in Taok2 KO neurons that is rescued by CN01. Scale bars represent 3 µm. h CN01 reduces spine motility of Taok2 KO neurons during a 5-min period (KO = 105, KO + CN01 = 109 spines from 9 cells per condition; Wilcoxon-matched paired t-test, p = 0.0039). i CN01 increased percentage of stable spines and reduced the percentage of collapsed spines in Taok2 KO neurons during a 5-min period (WT = 418 spines from 8 cells, KO = 603 spines from 9 cells and KO + CN01 = 433 spines from 7 cells; Filopodia: one-way ANOVA, post hoc Tukey’s test; F_{2, 21}= 0.2249, p = 0.8005; Spines: one-way ANOVA, post hoc Tukey’s test; F_{2, 21}= 4.66, p = 0.0211; WT vs. KO + CN01 p = 0.4905, KO vs. KO + CN01 p = 0.0180; Collapsed: one-way ANOVA, post hoc Tukey’s test; F_{2, 21}= 12.5, p = 0.0003; WT vs. KO p = 0.0047, WT vs. KO + CN01 p = 0.4473, KO vs. KO + CN01 p = 0.0003). j Images of DIV18-19 Taok2 KO neurons with or without addition of CN01 for 30–60 min, stained with phalloidin and SynGAP. Scale bars represent 10 µm. k CN01 increases the number of SynGAP + spines in Taok2 KO neurons compared with DMSO control KO neurons (WT + DMSO = 6 cells, WT + CN01 (0.5U/ml/30′) = 8 cells, WT + CN01(0.5U/ml/60′) = 8 cells, KO + DMSO = 9 cells, KO + CN01 (0.5U/ml/30’) = 9 cells; KO + CN01 (0.5U/ml/60’) = 9 cells; one-way ANOVA, post hoc Bonferroni’s test; F_{5, 43}= 3.192, p = 0.0154; WT + DMSO vs. KO + DMSO p = 0.0156, WT + DMSO vs. WT + CN01(0.5U/ml/60’) p = 0.0611, KO + DMSO vs. KO + CN01(0.5U/ml/60′) p = 0.0235). *p < 0.05, **p < 0.01, and ***p < 0.001. Values are mean ± s.e.m.