Acute and Chronic Electroconvulsive Seizures (ECS) Differentially Regulate the Expression of Epigenetic Machinery in the Adult Rat Hippocampus

Abstract

Background: Electroconvulsive seizure treatment is a fast-acting antidepressant therapy that evokes rapid transcriptional, neurogenic, and behavioral changes. Epigenetic mechanisms contribute to altered gene regulation, which underlies the neurogenic and behavioral effects of electroconvulsive seizure. We hypothesized that electroconvulsive seizure may modulate the expression of epigenetic machinery, thus establishing potential alterations in the epigenetic landscape.

Methods: We examined the influence of acute and chronic electroconvulsive seizure on the gene expression of histone modifiers, namely histone acetyltransferases, histone deacetylases, histone methyltransferases, and histone (lysine) demethylases as well as DNA modifying enzymes, including DNA methyltransferases, DNA demethylases, and methyl-CpG-binding proteins in the hippocampi of adult male Wistar rats using quantitative real time-PCR analysis. Further, we examined the influence of acute and chronic electroconvulsive seizure on global and residue-specific histone acetylation and methylation levels within the hippocampus, a brain region implicated in the cellular and behavioral effects of electroconvulsive seizure.

Results: Acute and chronic electroconvulsive seizure induced a primarily unique, and in certain cases bidirectional, regulation of histone and DNA modifiers, and methyl-CpG-binding proteins, with an overlapping pattern of gene regulation restricted to Sirt4, Mll3, Jmjd3, Gadd45b, Tet2, and Tet3. Global histone acetylation and methylation levels were predominantly unchanged, with the exception of a significant decline in H3K9 acetylation in the hippocampus following chronic electroconvulsive seizure.

Conclusions: Electroconvulsive seizure treatment evokes the transcriptional regulation of several histone and DNA modifiers, and methyl-CpG-binding proteins within the hippocampus, with a predominantly distinct pattern of regulation induced by acute and chronic electroconvulsive seizure.

Keywords: DNA demethylase; DNA methyltransferase; histone acetyltransferase; histone deacetylase; histone demethylase; histone methyltransferase; methyl-CpG-binding proteins.

Figure 1.

Influence of acute and chronic electroconvulsive seizure (ECS) on the expression of histone acetyl transferases (HATs) and histone deacetylases (HDACs) in the rat hippocampus. Shown is the experimental design for acute ECS (Ac. ECS, A) and chronic ECS (Chr. ECS, C) treatment. Represented are normalized gene expression levels in the hippocampus for specific HATs and HDACs following acute ECS (B) and chronic ECS (D) treatment compared with their respective sham groups. Heat maps indicate the magnitude of regulation, with upregulated genes shown in red and downregulated genes shown in green (key, E). Quantitative real-time PCR (qPCR) analysis results indicate differential regulation of several of the HATs and HDACs profiled following acute or chronic ECS treatment. The pattern of gene regulation shows a predominantly distinct, and in specific cases opposing, regulation following acute vs chronic ECS. The data are represented as fold change ± SEM with significance determined at *P<.05, Student’s t test (acute ECS: n = 6/group, chronic ECS: n = 10/group).

Figure 2.

Influence of acute and chronic electroconvulsive seizure (ECS) on the expression of histone methyltransferases (HMTs) and histone (lysine) demethylases (KDMs) in the rat hippocampus. Shown is the experimental design for acute ECS (Ac. ECS, A) and chronic ECS (Chr. ECS, C) treatment. Shown are normalized gene expression levels in the hippocampus for specific HMTs and KDMs relative to sham following acute ECS (B) and chronic ECS (D) treatment. Heat maps indicate the magnitude of regulation, with upregulated genes shown in red and downregulated genes shown in green (key, E). qPCR analysis results revealed differential regulation of several HMTs and KDMs in response to acute or chronic ECS. The data are represented as fold change ± SEM with significance determined at * P<.05, Student’s t test (acute ECS: n = 6/group, chronic ECS: n = 10/group).

Figure 3.

Influence of acute and chronic electroconvulsive seizure (ECS) on global and residue-specific levels of histone methylation and acetylation in the rat hippocampus. Hippocampal levels of residue-specific histone methylation (H3K9me2, H3K9me3, H3K27Me3, H3K4me3), and global, as well as residue-specific, histone acetylation (H3ac, H3K9ac) were determined by western-blot analysis in sham and ECS-treated animals following acute (A-G) and chronic ECS (H-N). The levels of the H3K9me2, H3K9me3, H3K27me3, H3K4me3, H3ac, and H3K9ac were normalized to H3 levels. Shown are representative images from acute (A) and chronic (H) ECS animals with their respective sham controls. Quantitative densitometric analysis is represented as percent of the sham controls. The results revealed no significant influence of acute or chronic ECS on levels of H3K9me2 (acute ECS: A, B; and chronic ECS: H, I), H3K9me3 (acute ECS: A, C; and chronic ECS: H, J), H3K27me3 (acute ECS: A, D; and chronic ECS: H, K), H3K4me3 (acute ECS: A, E; and chronic ECS: H, L) and H3ac (acute ECS: A, F; and chronic ECS: H, M). While acute ECS did not alter levels of H3K9ac (A, G), a significant decline in hippocampal H3K9ac levels was observed following chronic ECS treatment (H, N). Significance was determined at *P<.05, Student’s t test, acute ECS experiment: (sham, n = 6, acute ECS, n = 7) chronic ECS experiment: (sham, n = 4, chronic ECS, n = 3).

Figure 4.

Influence of acute and chronic electroconvulsive seizure (ECS) on the expression of DNA methylation machinery in the rat hippocampus. Shown is the experimental design for acute ECS (Ac. ECS, A) and chronic ECS (Chr. ECS, C) treatment. Shown are normalized gene expression levels in the hippocampus for specific DNMTs, DNA demethylases, and methyl-CpG-binding proteins relative to sham groups following acute ECS (B) and chronic ECS (D) treatment. Heat maps indicate the magnitude of regulation, with upregulated genes shown in red and downregulated genes shown in green (key, E). Quantitative real-time PCR (qPCR) analysis revealed differential regulation of the hippocampal expression of several DNMTs, DNA demethylases, and methyl-CpG-binding proteins in response to acute or chronic ECS. The pattern of regulation includes distinct, overlapping as well as opposing changes in specific DNMTs, DNA demethylases, and methyl-CpG-binding proteins. The data are represented as fold change ± SEM with significance determined at * P<.05, Student’s t test (acute ECS: n = 6/group, chronic ECS: n = 10/group).

Figure 5.

Transcriptional regulation of histone and DNA modifiers and methyl-CpG-binding proteins following acute and chronic electroconvulsive seizure (ECS). Shown is a summary of the transcriptional changes evoked in the hippocampal expression of both histone and DNA modifiers, as well as methyl-CpG-binding proteins, following acute or chronic ECS. We extensively profiled the effects of acute and chronic ECS on several histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMTs), histone (lysine) demethylases (KDMs), DNA methyltransferases (DNMTs), DNA demethylases, and methyl-CpG-binding proteins. This figure highlights the predominantly distinct pattern of gene regulation of histone and DNA modifying enzymes evoked by acute vs chronic ECS. We also observed specific cases of opposing patterns of regulation (Hdac3, Hdac4, Ezh2, Gadd45a, Mbd3) following acute and chronic ECS. The genes that showed an overlapping pattern of regulation with both acute and chronic ECS were Sirt4, Mll3, Jmjd3, Gadd45b, Tet2, and Tet3. Red indicates upregulation and green indicates downregulation of mRNA levels.