Dual mechanisms for the regulation of brain-derived neurotrophic factor by valproic acid in neural progenitor cells

Abstract

Autism spectrum disorders (ASDs) are neurodevelopmental disorders that share behavioral features, the results of numerous studies have suggested that the underlying causes of ASDs are multifactorial. Behavioral and/or neurobiological analyses of ASDs have been performed extensively using a valid model of prenatal exposure to valproic acid (VPA). Abnormal synapse formation resulting from altered neurite outgrowth in neural progenitor cells (NPCs) during embryonic brain development has been observed in both the VPA model and ASD subjects. Although several mechanisms have been suggested, the actual mechanism underlying enhanced neurite outgrowth remains unclear. In this study, we found that VPA enhanced the expression of brain-derived neurotrophic factor (BDNF), particularly mature BDNF (mBDNF), through dual mechanisms. VPA increased the mRNA and protein expression of BDNF by suppressing the nuclear expression of methyl-CpG-binding protein 2 (MeCP2), which is a transcriptional repressor of BDNF. In addition, VPA promoted the expression and activity of the tissue plasminogen activator (tPA), which induces BDNF maturation through proteolytic cleavage. Trichostatin A and sodium butyrate also enhanced tPA activity, but tPA activity was not induced by valpromide, which is a VPA analog that does not induce histone acetylation, indicating that histone acetylation activity was required for tPA regulation. VPA-mediated regulation of BDNF, MeCP2, and tPA was not observed in astrocytes or neurons. Therefore, these results suggested that VPA-induced mBDNF upregulation was associated with the dysregulation of MeCP2 and tPA in developing cortical NPCs.

Keywords: Brain-derived neurotrophic factor; Methyl-CpG-binding protein 2; Tissue plasminogen activator; Valproic acid.

Fig. 1. Valproic acid (VPA)-induced upregulation of brain-derived neurotrophic factor (BDNF) in cultured neural progenitor cells (NPCs).

Neurospheres were cultured from the cortical region of embryonic-day-14.5-old brains and dissociated into single NPCs. The NPCs were exposed to 0.2 or 0.5 mM VPA for 3 or 6 h and then analyzed. (A) The expression of BDNF mRNA was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Gapdh was used as a loading control. All data are expressed as mean±standard error of the mean (SEM, n=3). ^*p<0.05 vs. Vehicle 3 h, and ^##p<0.01 vs. Vehicle 6 h [one-way analysis of variance (ANOVA) followed by post hoc Tukey's comparisons test]. (B) The expression levels of pro- and mature BDNF protein were analyzed by western blots. Acetylated histone 3 (AcH3) levels were increased by VPA treatment, and β-actin was used as a loading control. All data are expressed as mean±SEM (n=3). ^*p<0.05 vs. Vehicle 3 h, and ^#p<0.05 and ^###p<0.001 vs. Vehicle 6 h (one-way ANOVA followed by post hoc Tukey's comparisons test). ^&&p<0.01 vs. VPA 0.2 mM 3 h, and ^†p<0.05 vs. VPA 0.5 mM 3 h (Student's t-test).

Fig. 2. VPA reduced the levels of methyl-CpG-binding protein 2 (MeCP2) expression in NPCs.

Cultured NPCs were treated with 0.2 or 0.5 mM VPA for 3 h. (A, B) MeCP2 mRNA levels were analyzed with RT-PCR (A), and protein levels were analyzed with western blots (B). All data are expressed as mean±SEM (n=3). ^**p<0.01 and ^***p<0.001 vs. Vehicle 3 h (one-way ANOVA followed by post hoc Tukey's comparisons test). (C) Immunostaining of MeCP2 and diamidino-2-phenylindole (DAPI) in VPA-treated NPCs. The arrow indicates the coexpression of MeCP2 and DAPI, while the arrowhead indicates the region in which MeCP2 was expressed but DAPI staining cannot be observed. The magnified images are from the top row. All data are expressed as mean±SEM (n=3). ^**p<0.01 and ^***p<0.001 vs. Vehicle 3 h (one-way ANOVA followed by post hoc Tukey's comparisons test). Scale bar=10 µm.

Fig. 3. VPA enhanced the levels of tissue plasminogen activator (tPA) expression in NPCs.

Cultured NPCs were treated with VPA (0.2 and 0.5 mM) for 3 h or 6 h. (A-C) The levels of tPA mRNA (A), protein (B), and activity (C) were analyzed with RT-PCR, western blot, and casein zymography, respectively. ^**p<0.01 and ^***p<0.001 vs. Vehicle 3 h, and ^#p<0.05 and ^##p<0.01 vs. Vehicle 6 h (one-way ANOVA followed by post hoc Tukey's comparisons test). (D) The NPCs were treated with histone acetylation inhibitors (HDAC) inhibitors or valpromide (VPM) for 6 h then tPA activity was analyzed; VPA (0.5 mM), trTSA (trichostatin A, 20 nM), SB (sodium butyrate, 0.1 mM), and VPM (0.5 mM). All data are expressed as mean±SEM (n=3). *p<0.05 and **p<0.01 vs. Vehicle (Student's t-test).

Fig. 4. VPA did not affect the levels of MeCP2 or tPA expression in neurons or astrocytes.

Cortical neurons (A) or astrocytes (B) were treated with VPA for 6 h, then mRNA of BDNF, MeCP2, and tPA, and tPA activity were analyzed. GAPDH was used as a loading control.