Hannah M. Cates

Locus-specific epigenetic remodeling controls addiction- and depression-related behaviors

Chronic exposure to drugs of abuse or stress regulates transcription factors, chromatin-modifying enzymes and histone post-translational modifications in discrete brain regions. Given the promiscuity of the enzymes involved, it has not yet been possible to obtain direct causal evidence to implicate the regulation of transcription and consequent behavioral plasticity by chromatin remodeling that occurs at a single gene. We investigated the mechanism linking chromatin dynamics to neurobiological phenomena by applying engineered transcription factors to selectively modify chromatin at a specific mouse gene in vivo. We found that histone methylation or acetylation at the Fosb locus in nucleus accumbens, a brain reward region, was sufficient to control drug- and stress-evoked transcriptional and behavioral responses via interactions with the endogenous transcriptional machinery. This approach allowed us to relate the epigenetic landscape at a given gene directly to regulation of its expression and to its subsequent effects on reward behavior.

Incubation of Methamphetamine Craving Is Associated with Selective Increases in Expression of Bdnf and Trkb, Glutamate Receptors, and Epigenetic Enzymes in Cue-Activated Fos-Expressing Dorsal Striatal Neurons

Cue-induced methamphetamine seeking progressively increases after withdrawal (incubation of methamphetamine craving), but the underlying mechanisms are largely unknown. We determined whether this incubation is associated with alterations in candidate genes in dorsal striatum (DS), a brain area implicated in cue- and context-induced drug relapse. We first measured mRNA expression of 24 candidate genes in whole DS extracts after short (2 d) or prolonged (1 month) withdrawal in rats following extended-access methamphetamine or saline (control condition) self-administration (9 h/d, 10 d). We found minimal changes. Next, using fluorescence-activated cell sorting, we compared gene expression in Fos-positive dorsal striatal neurons, which were activated during “incubated” cue-induced drug-seeking tests after prolonged withdrawal, with nonactivated Fos-negative neurons. We found significant increases in mRNA expression of immediate early genes (Arc, Egr1), Bdnf and its receptor (Trkb), glutamate receptor subunits (Gria1, Gria3, Grm1), and epigenetic enzymes (Hdac3, Hdac4, Hdac5, GLP, Dnmt3a, Kdm1a) in the Fos-positive neurons only. Using RNAscope to determine striatal subregion and cell-type specificity of the activated neurons, we measured colabeling of Fos with Drd1 and Drd2 in three DS subregions. Fos expression was neither subregion nor cell-type specific (52.5 and 39.2% of Fos expression colabeled with Drd1 and Drd2, respectively). Finally, we found that DS injections of SCH23390 (C17H18ClNO), a D1-family receptor antagonist known to block cue-induced Fos induction, decreased incubated cue-induced methamphetamine seeking after prolonged withdrawal. Results demonstrate a critical role of DS in incubation of methamphetamine craving and that this incubation is associated with selective gene-expression alterations in cue-activated D1- and D2-expressing DS neurons.

Early life stress confers lifelong stress susceptibility in mice via ventral tegmental area OTX2

An early window of stress susceptibility defines a mouse’s response to stress in adulthood. Early life stress in depression susceptibility The linkage between stress early in life and behavioral depression in adulthood is complex. Peña et al. were able to define a time period in early development when mice are especially susceptible to stress. Mice subjected to stress during this time period were less resilient to stress in adulthood. Genes regulated by the transcription factor orthodenticle homeobox 2 (OTX2) primed the response toward depression in adulthood. Although early stress could establish the groundwork for later depression, that priming could be undone by intervention at the right moment. Science, this issue p. 1185 Early life stress increases risk for depression. Here we establish a “two-hit” stress model in mice wherein stress at a specific postnatal period increases susceptibility to adult social defeat stress and causes long-lasting transcriptional alterations that prime the ventral tegmental area (VTA)—a brain reward region—to be in a depression-like state. We identify a role for the developmental transcription factor orthodenticle homeobox 2 (Otx2) as an upstream mediator of these enduring effects. Transient juvenile—but not adult—knockdown of Otx2 in VTA mimics early life stress by increasing stress susceptibility, whereas its overexpression reverses the effects of early life stress. This work establishes a mechanism by which early life stress encodes lifelong susceptibility to stress via long-lasting transcriptional programming in VTA mediated by Otx2.

Regulation of Chromatin States by Drugs of Abuse

Drug addiction involves long-term behavioral abnormalities and gene expression changes throughout the mesolimbic dopamine system. Epigenetic mechanisms establish/maintain alterations in gene expression in the brain, providing the impetus for investigations characterizing how epigenetic processes mediate the effects of drugs of abuse. This review focuses on evidence that epigenetic events, specifically histone modifications, regulate gene expression changes throughout the reward circuitry. Drugs of abuse induce changes in histone modifications throughout the reward circuitry by altering histone-modifying enzymes, manipulation of which reveals a role for histone modification in addiction-related behaviors. There is a complex interplay between these enzymes, resulting in a histone signature of the addicted phenotype. Insights gained from these studies are key to identifying novel targets for diagnosis and therapy.

ACF chromatin-remodeling complex mediates stress-induced depressive-like behavior

Improved treatment for major depressive disorder (MDD) remains elusive because of the limited understanding of its underlying biological mechanisms. It is likely that stress-induced maladaptive transcriptional regulation in limbic neural circuits contributes to the development of MDD, possibly through epigenetic factors that regulate chromatin structure. We establish that persistent upregulation of the ACF (ATP-utilizing chromatin assembly and remodeling factor) ATP-dependent chromatin-remodeling complex, occurring in the nucleus accumbens of stress-susceptible mice and depressed humans, is necessary for stress-induced depressive-like behaviors. We found that altered ACF binding after chronic stress was correlated with altered nucleosome positioning, particularly around the transcription start sites of affected genes. These alterations in ACF binding and nucleosome positioning were associated with repressed expression of genes implicated in susceptibility to stress. Together, our findings identify the ACF chromatin-remodeling complex as a critical component in the development of susceptibility to depression and in regulating stress-related behaviors.

Ketamine and Imipramine Reverse Transcriptional Signatures of Susceptibility and Induce Resilience-Specific Gene Expression Profiles

BACKGROUND Examining transcriptional regulation by antidepressants in key neural circuits implicated in depression and understanding the relation to transcriptional mechanisms of susceptibility and natural resilience may help in the search for new therapeutic agents. Given the heterogeneity of treatment response in human populations, examining both treatment response and nonresponse is critical. METHODS We compared the effects of a conventional monoamine-based tricyclic antidepressant, imipramine, and a rapidly acting, non-monoamine-based antidepressant, ketamine, in mice subjected to chronic social defeat stress, a validated depression model, and used RNA sequencing to analyze transcriptional profiles associated with susceptibility, resilience, and antidepressant response and nonresponse in the prefrontal cortex (PFC), nucleus accumbens, hippocampus, and amygdala. RESULTS We identified similar numbers of responders and nonresponders after ketamine or imipramine treatment. Ketamine induced more expression changes in the hippocampus; imipramine induced more expression changes in the nucleus accumbens and amygdala. Transcriptional profiles in treatment responders were most similar in the PFC. Nonresponse reflected both the lack of response-associated gene expression changes and unique gene regulation. In responders, both drugs reversed susceptibility-associated transcriptional changes and induced resilience-associated transcription in the PFC. CONCLUSIONS We generated a uniquely large resource of gene expression data in four interconnected limbic brain regions implicated in depression and its treatment with imipramine or ketamine. Our analyses highlight the PFC as a key site of common transcriptional regulation by antidepressant drugs and in both reversing susceptibility- and inducing resilience-associated molecular adaptations. In addition, we found region-specific effects of each drug, suggesting both common and unique effects of imipramine versus ketamine.

MicroRNAs 146a/b-5 and 425-3p and 24-3p are markers of antidepressant response and regulate MAPK/Wnt-system genes

Antidepressants (ADs) are the most common treatment for major depressive disorder (MDD). However, only ∼30% of patients experience adequate response after a single AD trial, and this variability remains poorly understood. Here, we investigated microRNAs (miRNAs) as biomarkers of AD response using small RNA-sequencing in paired samples from MDD patients enrolled in a large, randomized placebo-controlled trial of duloxetine collected before and 8 weeks after treatment. Our results revealed differential expression of miR-146a-5p, miR-146b-5p, miR-425-3p and miR-24-3p according to treatment response. These results were replicated in two independent clinical trials of MDD, a well-characterized animal model of depression, and post-mortem human brains. Furthermore, using a combination of bioinformatics, mRNA studies and functional in vitro experiments, we showed significant dysregulation of genes involved in MAPK/Wnt signalling pathways. Together, our results indicate that these miRNAs are consistent markers of treatment response and regulators of the MAPK/Wnt systems.

Histone arginine methylation in cocaine action in the nucleus accumbens.

Significance We demonstrate that protein-R (arginine)-methyltransferase-6 (PRMT6) and its associated histone mark, asymmetric dimethylation of R2 on histone H3 (H3R2me2a), are decreased in the nucleus accumbens (NAc) of mice and rats after repeated cocaine exposure, as well as in the NAc of cocaine-addicted humans. We show that cocaine-induced PRMT6 down-regulation occurs selectively in NAc medium spiny neurons expressing dopamine D2 receptors (D2-MSNs) and serves to protect against cocaine-induced behavioral abnormalities. Furthermore, we provide the first, to our knowledge, genome-wide characterization of H3R2me2a within a specific brain region in vivo, and identify Src kinase signaling inhibitor 1 (Srcin1 or p140Cap) as a key target for this chromatin modification. Srcin1 induction in the NAc after cocaine exposure, which is associated with reduced Src signaling, decreases cocaine reward. Repeated cocaine exposure regulates transcriptional regulation within the nucleus accumbens (NAc), and epigenetic mechanisms—such as histone acetylation and methylation on Lys residues—have been linked to these lasting actions of cocaine. In contrast to Lys methylation, the role of histone Arg (R) methylation remains underexplored in addiction models. Here we show that protein-R-methyltransferase-6 (PRMT6) and its associated histone mark, asymmetric dimethylation of R2 on histone H3 (H3R2me2a), are decreased in the NAc of mice and rats after repeated cocaine exposure, including self-administration, and in the NAc of cocaine-addicted humans. Such PRMT6 down-regulation occurs selectively in NAc medium spiny neurons (MSNs) expressing dopamine D2 receptors (D2-MSNs), with opposite regulation occurring in D1-MSNs, and serves to protect against cocaine-induced addictive-like behavioral abnormalities. Using ChIP-seq, we identified Src kinase signaling inhibitor 1 (Srcin1; also referred to as p140Cap) as a key gene target for reduced H3R2me2a binding, and found that consequent Srcin1 induction in the NAc decreases Src signaling, cocaine reward, and the motivation to self-administer cocaine. Taken together, these findings suggest that suppression of Src signaling in NAc D2-MSNs, via PRMT6 and H3R2me2a down-regulation, functions as a homeostatic brake to restrain cocaine action, and provide novel candidates for the development of treatments for cocaine addiction.

Threonine 149 Phosphorylation Enhances ΔFosB Transcriptional Activity to Control Psychomotor Responses to Cocaine

Stable changes in neuronal gene expression have been studied as mediators of addicted states. Of particular interest is the transcription factor ΔFosB, a truncated and stable FosB gene product whose expression in nucleus accumbens (NAc), a key reward region, is induced by chronic exposure to virtually all drugs of abuse and regulates their psychomotor and rewarding effects. Phosphorylation at Ser27 contributes to ΔFosBs stability and accumulation following repeated exposure to drugs, and our recent work demonstrates that the protein kinase CaMKIIα phosphorylates ΔFosB at Ser27 and regulates its stability in vivo. Here, we identify two additional sites on ΔFosB that are phosphorylated in vitro by CaMKIIα, Thr149 and Thr180, and demonstrate their regulation in vivo by chronic cocaine. We show that phosphomimetic mutation of Thr149 (T149D) dramatically increases AP-1 transcriptional activity while alanine mutation does not affect transcriptional activity when compared with wild-type (WT) ΔFosB. Using in vivo viral-mediated gene transfer of ΔFosB-T149D or ΔFosB-T149A in mouse NAc, we determined that overexpression of ΔFosB-T149D in NAc leads to greater locomotor activity in response to an initial low dose of cocaine than does WT ΔFosB, while overexpression of ΔFosB-T149A does not produce the psychomotor sensitization to chronic low-dose cocaine seen after overexpression of WT ΔFosB and abrogates the sensitization seen in control animals at higher cocaine doses. We further demonstrate that mutation of Thr149 does not affect the stability of ΔFosB overexpressed in mouse NAc, suggesting that the behavioral effects of these mutations are driven by their altered transcriptional properties.

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

Significance Human major depressive disorder is a chronic remitting syndrome that affects millions of individuals worldwide; however, the molecular mechanisms mediating this syndrome remain elusive. Here, using a unique combination of epigenome-wide and behavioral analyses, we demonstrate a role for histone variant dynamics in the nucleus accumbens (NAc)—a critical brain center of reward and mood—contributing to stress susceptibility in mice. These studies, which also demonstrate that molecular blockade of aberrant dynamics in the NAc promotes resilience to chronic stress, promise to aid in the identification of novel molecular targets (i.e., downstream genes displaying altered expression as the result of stress-induced histone dynamics) that may be exploited in the development of more effective pharmacotherapeutics. 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.