Excess phosphoinositide 3-kinase subunit synthesis and activity as a novel therapeutic target in fragile X syndrome - PubMed (original) (raw)
PI3K antagonists rescue dysregulated basal and stimulus-induced synaptic translation rates in Fmr1 KO SNS. A, Basal translation in cortical _Fmr_1 KO SNS is increased ∼30% compared with WT. Translation rates were analyzed by metabolic labeling of SNS with [35S]methionine, and radioactivity incorporation during a 5 min time period was quantified (n = 6, *p = 0.03, paired t test). B, C, Pretreatment of SNS with an mGluR5 antagonist (B: MPEP, 10 μ
m
) or an mGluR1 antagonist (C: LY367385, 10 μ
m
) significantly decreased translation rates in Fmr1 KO but not in WT SNS (MPEP: n = 8, *p wt–ko untreated = 0.043, *p ko untreated–treated = 0.002; LY367385: n = 5, *p wt–ko untreated = 0.04, *p ko untreated–treated = 0.007; Tukey's HSD post hoc tests; two-way ANOVAs detect significant interaction between genotype and treatment; MPEP: *p < 0.001; LY367385: *p < 0.001). D, Treatment with a specific NMDAR antagonist (APV, 50 μ
m
) affected translation similarly across genotypes (n = 3; two-way ANOVA: no significant interaction between genotype and treatment; p = 0.782) (also see supplemental Fig. S5_A_,B, available at
as supplemental material). E, F, Treatment of SNS with two different PI3K antagonists, LY294002 (E; 50 μ
m
) and wortmannin (F; 100 n
m
), significantly reduced amino acid incorporation rates in Fmr1 KO but not in WT (n = 6; E, LY294002: *p wt–ko untreated = 0.001, *p ko treated–untreated = 0.026; F, wortmannin: *p wt–ko untreated < 0.001, *p ko treated–untreated = 0.002; Tukey's HSD post hoc tests; two-way ANOVA: significant interaction between genotype and treatment; LY294002: *p < 0.001; wortmannin: *p < 0.001). LY303511, an inactive analog of LY294002, did not alter translation rates (supplemental Fig. S5_C_, available at
as supplemental material). G, An ERK1/2 antagonist (U0126, 20 μ
m
) did not show a genotype-specific effect on translation rates [n = 5, two-way ANOVA: significant effects of treatment (*p = 0.001) and genotype (*p = 0.001) but no significant interaction between genotype and treatment (p = 0.68)] (also see supplemental Fig. S5_D_,E, available at
as supplemental material). H, DHPG-induced translational activation in Fmr1 KO SNS is occluded and can be rescued by antagonizing PI3K signaling (wortmannin) but not by ERK1/2 inhibition (U0126) (wortmannin: n = 4, one-way ANOVA, *p = 0.012, Tukey's HSD post hoc tests: *p untrctr–wortctr = 0.045, p wortctr–wortDHPG = 0.010; U0126: n = 4, one-way ANOVA, *p = 0.001, Tukey's HSD post hoc tests: *p untrctr–untrDHPG = 0.029, *p untrctr–U0126ctr/DHPG = 0.006/0.001, *p untrDHPG–U0126ctr/DHPG = 0.019/0.004). I, Inhibitors of PI3K signaling (Wort) and ERK1/2 signaling (U0126) abolish DHPG-induced (15 min, 100 μ
m
) translational activation in WT SNS (wortmannin: n = 4, one-way ANOVA, *p = 0.002, Tukey's HSD post hoc tests: *p untrctr–untrDHPG = 0.006, *p wortctr–wortDHPG = 0.004; U0126: n = 3, one-way ANOVA, *p < 0.001, Tukey's HSD post hoc tests: *p untrctr–untrDHPG = 0.029, *p untrctr/DHPG–U0126DHPG = 0.022/0.001). Results were normalized to control. All error bars represent SEM.