Figure 6.

PTHS-associated missense mutations alter the transcriptional activity of TCF4 in rat cortical and hippocampal primary neurons.A–C, luciferase reporter assay with WT and mutant TCF4 in rat cortical and hippocampal primary neurons. The cells were cotransfected with WT or mutant TCF4 vectors alone or with ASCL1 vector, firefly luciferase reporter construct carrying 12 μE5 E-box regulatory sequences (CACCTG) in front of TK promoter and Renilla luciferase construct with PGK promoter for normalization. The transfected neurons were left untreated (CNTR) or treated with 25 mM KCl for 8 h (KCl) to induce membrane depolarization. Luciferase assays were performed with TCF4-B¯ carrying RTT-like syndrome, MMID, or PTHS-associated mutations (A), PTHS mutations containing TCF4-A¯ (B) or TCF4-B¯ (C). D, index of cooperation between ASCL1 and WT or mutant TCF4 calculated from data in (B) or (C). Three independent experiments were performed in duplicates. The luciferase data is presented as fold-induced levels above the signals measured from empty vector-transfected (vector) untreated cells. The error bars indicate SEM. For statistical analysis, one-way ANOVA (A: RTT-like syndrome mutant F (5, 10) = 88.62 p < 0.0001; MMID and SCZ mutants F (13, 26) = 70.52 p < 0.0001) (B: F (27, 54) = 79.12 p < 0.0001) (C: F (19, 38) = 48.36 p < 0.0001) followed by Holm–Sidak's multiple comparisons test (A–C) or one-way ANOVA (TCF4-A¯ F (5, 10) = 22.05 p < 0.0001; TCF4-B¯ F (6, 12) = 31.49 p < 0.0001) Dunnett's multiple comparisons test (D) was used. The individual data points are shown as white diamonds. Statistical significance shown with asterisks is relative to cells overexpressing WT TCF4-B¯ (A and C), WT TCF4-A¯ (B and C) or between the bars connected with lines; ∗p, < 0.05; ∗∗p, < 0.01; ∗∗∗p, < 0.001. MMID, Mild-to-moderate intellectual disability; PTHS, Pitt-Hopkins syndrome; RLU, relative luciferase units; RTT-like, Rett-like syndrome; SCZ, Schizophrenia; TCF4, transcription factor 4.