Epigenetic Mechanism of 5-HT/NE/DA Triple Reuptake Inhibitor on Adult Depression Susceptibility in Early Stress Mice

Abstract

Major depressive disorder (MDD) is a chronic, remitting and debilitating disease and the etiology of MDD is highly complicated that involves genetic and environmental interactions. Despite many pharmacotherapeutic options, many patients remain poorly treated and the development of effective treatments remains a high priority in the field. LPM570065 is a potent 5-hydroxytryptamine (5-HT), norepinephrine (NE) and dopamine (DA) triple reuptake inhibitor and both preclinical and clinical results demonstrate significant efficacy against MDD. This study extends previous findings to examine the effects and underlying mechanisms of LPM570065 on stress vulnerability using a "two-hit" stress mouse model. The "two-hit" stress model used adult mice that had experienced early life maternal separation (MS) stress for social defeat stress (SDS) and then they were evaluated in three behavioral assays: sucrose preference test, tail suspension test and forced swimming test. For the mechanistic studies, methylation-specific differentially expressed genes in mouse hippocampal tissue and ventral tegmental area (VTA) were analyzed by whole-genome transcriptome analysis along with next-generation bisulfite sequencing analysis, followed by RT-PCR and pyrophosphate sequencing to confirm gene expression and methylation. LPM570065 significantly reversed depressive-like behaviors in the mice in the sucrose preference test, the tail suspension test, and the forced swimming test. Morphologically, LPM570065 increased the density of dendritic spines in hippocampal CA1 neurons. Hypermethylation and downregulation of oxytocin receptor (Oxtr) in the hippocampal tissues along with increased protein expression of Dnmt1 and Dnmt3a in mice that experienced the "two-hit" stress compared to those that only experienced adulthood social defeat stress, and LPM570065 could reverse these changes. Combined, these results suggest that methylation specificity of the gene Oxtr in the hippocampus may play an important role in early life stress-induced susceptibility to depression and that the5-HT/NE/DA triple reuptake inhibitor LPM570065 may reduce depression susceptibility via the reversal of the methylation of the gene Oxtr.

Keywords: DNA methylation; depression susceptibility; epigenetics; second stress; triple reuptake inhibitors.

FIGURE 1

Chemical structure of LPM570065.

FIGURE 2

LPM570065 reversed the increased susceptibility to depression in mice experiencing “two-hit” stress. (A) A schematic of mother-infant separation. (B) A schematic of repeated social defeat stress. (C) Experimental timeline. (D) Results of sucrose preference test. (E) Results of tail suspension test. (F) Results of forced swimming test. One-way ANOVA with Bonferroni multiple comparison correction and Student‘s t test. Data are mean ± SEM (n = 24 mouse per group), and statistical analysis is shown in Supplementary Data. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Double stress group.

FIGURE 3

LPM570065 increased the density of dendritic spines in the hippocampus. Dendritic spine density in the hippocampus of mice that experienced early life stress further decreased after a second stressful event in adulthood (p < 0.001). The density of dendritic spines in the hippocampus of mice treated with LPM570065 increased (p < 0.001). Histograms showed number of dendritic spines per 10 µm of dendrite length for hippocampal pyramidal neuron. One-way ANOVA with Bonferroni multiple comparison correction. Data are mean ± SEM (n = 9 neurons). ***p < 0.001 vs. Double stress group.

FIGURE 4

Transcriptome sequencing analysis of the mouse hippocampus and cluster analysis of mouse hippocampal DEGs. For the overall analysis of each group of genes, (A) Venn diagram of the clustering analysis of the two compared groups, and gene enrichment GO analysis. (B) Clustering heat map of each sample illustrated good inter-group homogeneity and clustering repeatability between samples. (C) go function enrichment. (D,E) KEGG analysis of differential gene enrichment pathways. (n = 3/group, two mice were mixed in each sample, |Fold-change| > 1.5, p < 0.05).

FIGURE 5

RRBS sequencing for further screening of differential genes for methylation. (A,B) Representation in bar graphs of DMRs hypermethylation or hypomethylation in the hippocampus of double stress vs. single stress and LPM570065 vs. double stress mice. (n = 2/group, each two mice mixed as one sample, |Fold-change| > 1.5, p < 0.05). (C) Venn diagram of cluster analysis of the two compared groups identified DMRs between the two compared groups. (D) KEGG enrichment analysis, 17% of the genes were found to be enriched in the signaling pathway.

FIGURE 6

Methylation and validation of DEGs. (A) Venn diagram of joint analysis of differential genes screened by RNA-seq and clustered with genes that underwent methylation in RRBS. (B) Methylation of gene-specific hippocampal Oxtr was assessed by pyrophosphate sequencing in each group of hippocampal tissues. (C) Validation of Oxtr expression in each group of hippocampal tissues by RT-qPCR. One-way ANOVA with Bonferroni multiple comparison correction. Data expressed as mean ± SEM, n = 5–6. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Double stress group.

FIGURE 7

DNMT1 and DNMT3a protein expression in hippocampus. One-way ANOVA with Bonferroni multiple comparison correction. Data are normalized to controls and are expressed as means ± SEM (n = 8/group) ***p < 0.001 vs. Double stress group.