FIG. 5.

Proposed molecular mechanism behind abnormal neurogenesis in FXS. (A) Relative transcript levels of SOX2 and SOX9 following manipulation of FMR1 expression. FMR1 knockdown (“αFMR1-siRNA,” white) was carried out in HUES-13 and HUES-64 hNPCs, in three different experiments (20 nM, 48–72 h). Overexpression of FMR1 (“pcDNA-FMR1,” horizontal strips) was carried out in a FX3-hNPC line subclone, in which stable transfection was achieved by antibiotic selection (two different samples obtained from different passages were included in the analysis). Values were normalized to GAPDH. All values are mean ± SEM (n = 2–3/line, *P < 0.05; **P < 0.01; t-test). (B) Representative images of western blot analysis of SOX9 levels in hNPCs, corresponding to manipulation of FMR1 expression as described in (B). (C) Relative transcript levels of MAP2 and GFAP expression in FX-hNPC lines following siRNA-mediated knockdown of SOX2 (“αSOX2-siRNA”) or transiently induced overexpression of SOX9 (“pcDNA-SOX9”). Values were normalized to GAPDH. All values are mean ± SEM (n = 2–3/line, **P < 0.01; ANOVA, t-test). (D) Proposed molecular mechanism regulating abnormal human in vitro neurogenesis in FXS. FMRP inhibits SOX2 and enhances SOX9 to promote neural development. FMR1 downregulation in FXS leads to a decrease in FMRP, an increase in SOX2, and a decrease in SOX9, which result in reduced and delayed neural differentiation.