Gadd45b and N-methyl-D-aspartate induced DNA demethylation in postmitotic neurons

Abstract

Aim: In nondividing neurons examine the role of Gadd45b in active 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) removal at a gene promoter highly implicated in mental illnesses and cognition, Bdnf.

Materials & methods: Mouse primary cortical neuronal cultures with and without Gadd45b siRNA transfection were treated with N-methyl-d-aspartate (NMDA). Expression changes of genes reportedly involved in DNA demethylation, Bdnf mRNA and protein and 5MC and 5HMC at Bdnf promoters were measured.

Results: Gadd45b siRNA transfection in neurons abolishes the NMDA-induced increase in Bdnf IXa mRNA and reductions in 5MC and 5HMC at the Bdnf IXa promoter.

Conclusion: These results contribute to our understanding of DNA demethylation mechanisms in neurons, and its role in regulating NMDA responsive genes implicated in mental illnesses.

Keywords: CpG; alcoholism; epigenetic; histone; schizophrenia.

Figure 1. Hypothetical pathway of activity-dependent Bdnf DNA demethylation

(A) Effects of neuronal depolarization on DNA demethylation pathway. (1) Neuronal activity decreases DNA methyltransferase (DNMT) expression leading to less production of 5-methylcytosine (5MC). This decreases the amount of substrate used to produce 5-hydroxymethylcytosine (5HMC). (2) Tet1-catalyzed conversion of 5MC to 5HMC at Bdnf IXa may maintain the promoter in a state primed for demethylation. However, once the neuron is depolarized Tet1 expression decreases lead to reduced 5HMC production. (3) Neuronal activity increases Gadd45b expression. Gadd45b participates in Bdnf IXa promoter demethylation by facilitating the removal of 5HMC directly, and as a direct or indirect negative regulator on Tet1 expression. Both these actions serve to decrease 5HMC. (B) Effects of Gadd45b knockdown on activity-dependent Bdnf demethylation. (1) In the current study, we knocked down Gadd45b expression using siRNA. This may prevent the Gadd45b-coordinated N-methyl-D-aspartate (NMDA)- induced removal of 5HMC. (2) Gadd45b knockdown may also prevent it from suppressing Tet1 expression following NMDA treatment. Inhibition of both actions of Gadd45b would lead to an accumulation of 5HMC at Bdnf promoters. This may explain why Gadd45b knockdown did not merely prevent the removal 5HMC from Bdnf promoters, but it actually led to an increase following NMDA treatment. (3) By preventing the removal of 5HMC, the pathway is blocked, leading to a failure in NMDA-dependent 5MC removal. 5HMC: 5-hydroxymethylcytosine; 5MC: 5-methylcytosine.

Figure 2. N-methyl-D-aspartate induced changes in DNA methylating and demethylating factors in mouse primary cortical neuronal cultures

(A) NMDA 45 μM at 2 h significantly reduces Dnmt3a and 3b mRNA expression with a trend for a decrease in Dnmt1. (B) NMDA induces significant reductions in the expression of Tet1 mRNA. (C) Gadd45b mRNA is significantly induced by NMDA, but expression of no other base excision repair pathway genes measured are affected. (D) NMDA significantly induces Gadd45b and g, which is prevented by co-treatment with NMDA antagonist MK-801 50 μM. MK-801 50 μM alone has no effect on Gadd45 expression. Error bars represent standard error of mean. *p < 0.05, **p < 0.01, ***p < 0.001. CTL: Control; NMDA: N-methyl-D-aspartate.

Figure 3. N-methyl-D-aspartate induces Bdnf DNA demethylation

(A) Map of the Bdnf promoter regions examined in this study. The top figure represents the promoter region of Bdnf IV. Each CpG site is represented by a red vertical line. MeDIP and hydroxyl-MeDIP primers encompass a region from −156 to +31 relative to the transcription start site (black horizontal line). This region includes nine CpG sites. For Bdnf IXa, MeDIP and hMeDIP primers encompass a region from −171 to +32 (blue line) relative to the transcription start site. This region includes seven CpG sites. (B) NMDA significantly induces the expression of Bdnf IV and IXa mRNA. (C) NMDA increases Bdnf protein expression after 2 h. Below bar graph is a representative western blot with control (C) and NMDA (N) conditions. (D) NMDA treatment induces a reduction in 5HMC at Bdnf IV and IXa promoters. (E) NMDA treatment leads to a reduction in at Bdnf IXa, while having no effect on Bdnf IV 5MC levels. Error bars represent standard error of mean. *p < 0.05, ***p < 0.001. 5HMC: 5-hydroxymethylcytosine; 5MC: 5-methylcytosine; CTL: Control; MeDIP: Methylated DNA immunoprecipitation; NMDA: N-methyl-D-aspartate. For color images please see online at: www.futuremedicine.com/doi/full/10.2217/EPI.15.12

Figure 4. Effect of Gadd45b knockdown via siRNA in mouse primary cortical neuron culture

(A) Neurons were transfected with either a scrambled nontargeting siRNA (siScr) or a Gadd45b specific siRNA (siGadd45b) at the sixth day in vitro (DIV). Successful knockdown of Gadd45b was assessed based on reduced mRNA expression. siGadd45b produces no significant changes to, Gadd45a or g, the other two Gadd45 isoforms. (B) siGadd45b transfection also has no effect on Tet1 mRNA expression. Error bars represent standard error of mean. ***p < 0.001.

Figure 5. Gadd45b is necessary for N-methyl-D-aspartate induction of Bdnf expression

(A) Neurons were transfected with a scrambled nontargeting siRNA (siScr) or a Gadd45b specific siRNA (siGadd45b) at the sixth day in vitro, then treated with NMDA 45 μM for 2 h on day in vitro 8. Results and significance levels are expressed as fold change in NMDA treated cells relative to no NMDA control condition (dotted line). NMDA induces the expression of Bdnf IV and Bdnf IXa in cells transfected with siScr compared with control condition. siGadd45b transfection does not affect NMDA-induced increase in Bdnf IV, but does prevent the induction of Bdnf IXa mRNA expression relative to no NMDA control. (B) Similarly, NMDA induces Bdnf protein expression based on western blot in siScr transfected cells, but fails to in siGadd45b transfected cells. (C) NMDA induces a reduction in Tet1 mRNA expression in siScr neurons relative to control, but siGadd45b transfected cells showed no decrease in expression. Error bars represent standard error of mean. **p < 0.01, ***p < 0.001. NMDA: N-methyl-D-aspartate; NS: Non significant.

Figure 6. Gadd45b is necessary for N-methyl-D-aspartate induced demethylation of Bdnf

(A) Neurons were transfected with a scrambled nontargeting siRNA (siScr) or a Gadd45b-specific siRNA (siGadd45b) at day 6 in vitro (DIV), then treated with NMDA 45 μM for 2 h on day 8 in vitro. Results and significance levels are expressed as fold change in NMDA treated cells relative to no NMDA control condition (dotted line). In siScr transfected cells, NMDA treatment significantly reduces Bdnf IXa promoter 5MC, but does not alter 5MC at the Bdnf IV promoter. siGadd45b eliminates the NMDA-induced reduction in 5MC at the Bdnf IXa promoter. (B) In siScr transfected cells, NMDA treatment significantly reduces Bdnf IV and IXa promoter 5HMC. siGadd45b does not merely prevent the reduction in 5HMC, but in fact causes increases in 5HMC at Bdnf IV and IXa promoters. Error bars represent SEMs. *p < 0.05, **p < 0.01. 5HMC: 5-hydroxymethylcytosine; 5MC: 5-methylcytosine; NMDA: N-methyl-D-aspartate.