FIG 8.
The GSK3β-CUGBP1 pathway is abnormal in DMSXL brain. (A) TG corrects the levels of GSK3β in the brains of DMSXL mice. Western blotting of protein extracts from the whole brains of 2-month-old WT and untreated and treated with TG hom DMSXL mice with antibodies to GSK3β and actin as a control. (B) Correction of GSK3β restores CUGBP1 activity in the brains of DMSXL mice. (Top) Interactions of CUGBP1 with inactive p-S51-eIF2α in the cytoplasmic extracts from the whole brains of 2-month-old (male) WT, untreated, and TG-treated (0.1 μg/g, 2 times) hom DMSXL mice were examined by the IP-Western blot analysis. (Bottom) Input of p-S51-eIF2α prior to precipitation. (C) The list of mRNAs encoding RNA-binding proteins altered in the whole brain of neonatal hom Celf1 KO mice, determined by the global microarray analysis of gene expression (13). Superscript letter “a” indicates genes examined in this study. (D) Confirmation of the altered expression of mRNAs, downstream of CUGBP1, in 0- to 5-day-old Celf1 KO brains by qRT-PCR. Rbm45 and Smn1 were examined in hom Celf1 KO brains, whereas Mbnl3 and Fgf-2 were analyzed in het Celf1 KO mice. (E) Rbm45, Mbnl3, Smn1, and Fgf-2 show similar patterns of expression in the brains of 1-month-old CUGBP1 S302A-KI mice (males) that contain inactive CUGBP1REP due to a mutation of the GSK3β-cyclin D3-CDK4 site. Rbm45 was measured by qRT-PCR in het CUGBP1-S302A-KI brains, whereas Mbnl3, Smn1, and Fgf-2 were tested in the brains of hom CUGBP1-S302A-KI mice. (F) Correction of GSK3β normalizes the downstream targets of CUGBP1 in DMSXL brains. Shown is the qRT-PCR analysis of Mbnl3, Fgf-2, and Smn1 in the whole-brain extracts from neonatal WT mice, untreated hom DMSXL mice, and hom DMSXL mice produced by females treated 1 to 2 times with TG during gestation. Rbm45 was measured in the whole brains of 6-week-old WT mice, untreated hom DMSXL mice, and hom DMSXL mice produced by TG-treated females during gestation. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.