Figure 2.

α-Syn-HDO activates BDNF transcription

(A) Schematic illustration of the construction of α-Syn-HDO. (B) The internalization of FAM-α-Syn-HDO visualized by microscopy at 0 min, 30 min, and 1 h following transfection of FAM-α-Syn-HDO (200 nM). Scale bar, 50 μm. (C) Western blot analysis of α-Syn, p-CREB, CREB, MeCP2, and BDNF in SH-SY5Y cells treated with various dosages of α-Syn-HDO (mean ± SEM, n = 4 per group, one-way ANOVA, ∗p < 0.05 and ∗∗p < 0.01). (D) Luciferase assay for BDNF IV promoters. BDNF exon IV luciferase promoters and/or α-Syn-HDO were transfected into HEK293T cells (mean ± SEM, n = 4 per group, one-way ANOVA, ∗∗∗p < 0.001). (E) BDNF exon IV luciferase promoter and α-Syn-HDO, siRNA-CREB plasmids, or mutation (Mut) plasmids were cotransfected into HEK293T cells (mean ± SEM, n = 4 per group, one-way ANOVA, ∗∗p < 0.01 and ∗∗∗p < 0.001). (F) ChIP-PCR assays demonstrated that p-CREB specifically binds to genomic DNA of BDNF exon IV promoter binding motifs. p-CREB protein-DNA crosslinking samples were obtained from SH-SY5Y cells treated with α-Syn-HDO or vehicle via coimmunoprecipitation with an anti-p-CREB antibody. PCR was performed with primers targeting the BDNF exon IV promoter. An anti-histone H3 antibody coupled with GAPDH primers was used as the positive control (mean ± SEM, n = 4 per group, Student’s t test, ∗p < 0. 05). (G) qPCR assay for BDNF in SH-SY5Y cells treated with α-Syn-HDO (mean ± SEM, n = 5 per group, Student’s t test, ∗p < 0. 05).