Figure 4

A p110β-selective antagonist rescues increased protein synthesis in synaptic fractions from Fmr1 KO mice and in LCLs from a patient with FXS. (A–C) Treatment of synaptoneurosomes with TGX-221 (1 µmol/L, 30 min) reduces p110β-specific PI3K activity and phosphorylation of the downstream target AKT in both WT and Fmr1 KO SNS, shown by a radioactive PI3K assay and phosphoAkt-specific western blotting (A). (B) Quantification of PI3K activity using a competitive ELISA showed a significant reduction in PI3K activity in both genotypes after treatment (n = 4, 2-way ANOVA, *P (genotype) = 0.0086, *P (treatment) = 0.0087, P (interaction) = 0.7154]. (C) Densitometric quantification of phosphoAkt-specific western blots showed a significant effect of treatment (C, n = 4, 2-way ANOVA, *P (treatment) = 0.0005, P (genotype) = 0.372, P (interaction) = 0.4894). (D) The same dose of TGX-221 (1 µmol/L, 10 min) rescues excess protein synthesis in Fmr1 KO SNS to WT levels as measured by metabolic labeling using radioactive methionine (n = 7, 2-way ANOVA, P (treatment) = 0.611, *P (genotype) = 0.004, *P (interaction) = 0.024; LSD post hoc analyses: *P = 0.001, ^#P = 0.049). (E) TGX-221 treatment (0.5 µmol/L 30 min) of control and FXS LCLs reduces PI3K activity as shown by a radioactive PI3K assay of p110β-specific immunoprecipitates from LCL lysates, and decreases PI3K/mTOR downstream signaling as shown by strongly reduced phosphorylation of S6 independently of the genotype. (F, G) Quantification demonstrates a significant, dose-dependent effect of TGX-221 treatment on PI3K activity (F, competitive ELISA, n = 4, 2-way ANOVA, *P (genotype) = 0.0079, *P (treatment) = 0.0258, P (interaction) = 0.9418), and on S6 phosphorylation [G, ELISA, n = 4, 2-way ANOVA, *P (genotype) = 0.013), *P (treatment) < 0.0001, P (interaction) = 0.195]. (H) 30-min pretreatment of LCLs with 0.5 µmol/L TGX-221 significantly reduces protein synthesis in FXS patient cells (n = 40, 4 independent experiments, 2-way ANOVA: *P (genotype) < 0.001, *P (treatment) = 0.005, *P (interaction) < 0.001, Bonferroni post hoc analyses, *P = 0.001, ^#P = 0.002, ^≠P = 0.02]. Example images are shown on the left: upper panel: signal for newly synthesized proteins (red), lower panel: overlay with tubulin staining (green). Scale bar is 20 µm. (I) Increasing concentrations of TGX-221 (5 µmol/L and 10 µmol/L) further reduce protein synthesis rates in FXS LCLs to healthy control levels. (n (Ctr Untr) = 57, n (Ctr 5 µmol/L) = 57, n (Ctr 10 µmol/L) = 55, n (FXS Untr) = 59, n (FXS 5 µmol/L) = 55, n (FXS 10 µmol/L) = 58; 3 independent experiments, 2-way ANOVA, *P (genotype) = 0.001, *P (treatment) < 0.001, *P (interaction genotype-treatment) < 0.001; Games-Howell post hoc analyses, *P = 0.001, ^ns(1)P= 0.966, ^ns(2)P= 0.999). Protein synthesis rates in FXS cells were significantly reduced after treatment with either 5 µmol/L or 10 µmol/L TGX-221 (P(Ctr 5/10 µmol/L) < 0.001], whereas there was no significant difference between FXS cells treated with 5 and 10 µmol/L TGX-221 [P(5–10 µmol/L) = 0.992], or between healthy control cells after treatment [P(Ctr 5 µmol/L) = 0.271; P(Ctr 10 µmol/L) = 0.638]. a.u., Arbitrary units; Untr, untreated; ns(1), not significant (comparison 1); ns(2), not significant (comparison 2). B-D: □, WT; ■, KO; F-I: □, Ctr; ■, FXS.