Aberrant H3.3 dynamics in NAc promote vulnerability to depressive-like behavior

Abstract

Human major depressive disorder (MDD), along with related mood disorders, is among the world's greatest public health concerns; however, its pathophysiology remains poorly understood. Persistent changes in gene expression are known to promote physiological aberrations implicated in MDD. More recently, histone mechanisms affecting cell type- and regional-specific chromatin structures have also been shown to contribute to transcriptional programs related to depressive behaviors, as well as responses to antidepressants. Although much emphasis has been placed in recent years on roles for histone posttranslational modifications and chromatin-remodeling events in the etiology of MDD, it has become increasingly clear that replication-independent histone variants (e.g., H3.3), which differ in primary amino acid sequence from their canonical counterparts, similarly play critical roles in the regulation of activity-dependent neuronal transcription, synaptic connectivity, and behavioral plasticity. Here, we demonstrate a role for increased H3.3 dynamics in the nucleus accumbens (NAc)-a key limbic brain reward region-in the regulation of aberrant social stress-mediated gene expression and the precipitation of depressive-like behaviors in mice. We find that molecular blockade of these dynamics promotes resilience to chronic social stress and results in a partial renormalization of stress-associated transcriptional patterns in the NAc. In sum, our findings establish H3.3 dynamics as a critical, and previously undocumented, regulator of mood and suggest that future therapies aimed at modulating striatal histone dynamics may potentiate beneficial behavioral adaptations to negative emotional stimuli.

Keywords: H3.3; chronic social defeat stress; depression; histone dynamics; nucleus accumbens.

Fig. 1.

H3.3 dynamics in NAc are increased in human MDD and in response to chronic stress in rodents. (A) H3F3B, but not H3F3A, expression is increased in NAc of nonmedicated subjects with MDD vs. controls, an effect that is reversed with ADs (*P < 0.05, ***P < 0.0001 by one-way ANOVA followed by Dunnett’s post hoc test; n = 4–11 per group). (B) Mice were segregated into susceptible vs. resilient populations following CSDS using SI testing for subsequent molecular analyses (***P < 0.0001 by one-way ANOVA followed by Dunnett’s post hoc test; n = 8 per group). (C) H3f3b, but not H3f3a, is significantly increased in expression in NAc of susceptible, but not resilient, mice following CSDS. This effect is reversed by chronic imipramine treatments in behaviorally responsive susceptible animals (*P < 0.05 by one-way ANOVA followed by Dunnett’s post hoc test; n = 3–13 per group; in some cases, tissues from multiple animals were pooled/biological replicate). (D) ELS (maternal separation) in mice results in faster rates of H3.3 chromatin accumulation in NAc with age in comparison with SR animals. LC-MS/MS analysis [*P < 0.05, **P < 0.01 by unpaired Student’s t test; n = 3 (pooled/biological replicate) per group]. (E) EE during adolescence promotes reduced expression of H3f3b, but not H3f3a, in NAc in comparison with normally housed mice (*P < 0.05 by unpaired Student’s t test; n = 7 per group) and (F) renders animals resilient to subsequent bouts of CSDS (**P < 0.01 by unpaired Student’s t test; n = 5 per group). Data are represented as means ± SEM.

Fig. 2.

Blockade of H3.3 dynamics in NAc promotes resilience to CSDS. (A) Viral targeting of AAV-miR (H3f3a/b)-IRES-GFP in NAc. (Magnification: 20×.) (B) Confirmation of H3f3a and H3f3b mRNA expression in NAc following viral knockdown (**P < 0.01, ***P < 0.001 by unpaired Student’s t test; n = 3 per group). (C) Reducing H3.3 dynamics in NAc promotes increased SI following CSDS (**P < 0.01 by two-way ANOVA followed by Bonferroni post hoc test; n = 7–10 per group; (^#P < 0.05 by planned Student’s t test postdetermination of main-effect significance). (D) Reducing H3.3 dynamics in NAc is anxiolytic post-CSDS (*P < 0.05 by two-way ANOVA followed by Bonferroni post hoc test; n = 7–9 per group). (E) Stalling H3.3 dynamics in NAc does not affect locomotor behavior. Data are represented as means ± SEM.

Fig. 3.

Chronic stress-mediated H3.3 dynamics promote transcriptional dysregulation of synaptic-related genes in NAc. (A) Genomic distribution (and numbers) of H3.3 differential enrichment events in NAc in response to either an acute stress (control vs. acute) or following re-exposure to stress in susceptible mice (control vs. susceptible + acute). (B) Venn diagrams showing minimal overlap between protein-coding genes displaying differential H3.3 enrichment in control vs. acute and control vs. susceptible + acute stress groups. (C) IPA-based pathway analysis of unique protein-coding genes displaying differential H3.3 enrichment in control vs. acute and control vs. susceptible + acute stress groups. (D) IPA-based network prediction diagram showing assumed relationships between unique genes displaying differential H3.3 enrichment in control vs. susceptible + acute NAc. Focus on most heavily enriched molecular and cellular function category of cellular morphology indicates a role for these genes in the inhibition of morphological/dendritic plasticity. (E) Odds ratio analyses of differential enrichment events for H3.3 (both control vs. acute and control vs. susceptible + acute stress groups) vs. stress-mediated gene expression in NAc indicates a significant relationship between H3.3 dynamics in susceptible mice only and reduced gene expression, effects that are predicted to disinhibit cellular morphology-associated gene pathways. (F) qPCR validation of candidate genes (predicted a priori from sequencing data) displaying H3.3 dynamics and reduced gene expression in NAc following CSDS. Knockdown of H3f3a and H3f3b results in a partial renormalization of transcriptional deficits post-CSDS (*P < 0.05 by unpaired Student’s t test vs. miR (–) control; n = 5 per group). Data are represented as means ± SEM. See Materials and Methods, H3.3 ChIP-seq Analysis and Correlations with Gene Expression for statistical comparisons, FC cutoffs, and numbers.