Caroline Dias

Epigenetic regulation of RAC1 induces synaptic remodeling in stress disorders and depression

Depression induces structural and functional synaptic plasticity in brain reward circuits, although the mechanisms promoting these changes and their relevance to behavioral outcomes are unknown. Transcriptional profiling of the nucleus accumbens (NAc) for Rho GTPase–related genes, which are known regulators of synaptic structure, revealed a sustained reduction in RAS-related C3 botulinum toxin substrate 1 (Rac1) expression after chronic social defeat stress. This was associated with a repressive chromatin state surrounding the proximal promoter of Rac1. Inhibition of class 1 histone deacetylases (HDACs) with MS-275 rescued both the decrease in Rac1 transcription after social defeat stress and depression-related behavior, such as social avoidance. We found a similar repressive chromatin state surrounding the RAC1 promoter in the NAc of subjects with depression, which corresponded with reduced RAC1 transcription. Viral-mediated reduction of Rac1 expression or inhibition of Rac1 activity in the NAc increases social defeat–induced social avoidance and anhedonia in mice. Chronic social defeat stress induces the formation of stubby excitatory spines through a Rac1-dependent mechanism involving the redistribution of synaptic cofilin, an actin-severing protein downstream of Rac1. Overexpression of constitutively active Rac1 in the NAc of mice after chronic social defeat stress reverses depression-related behaviors and prunes stubby spines. Taken together, our data identify epigenetic regulation of RAC1 in the NAc as a disease mechanism in depression and reveal a functional role for Rac1 in rodents in regulating stress-related behaviors.Depression involves plasticity of brain reward neurons, although the mechanisms and behavioral relevance are unknown. Transcriptional profiling of nucleus accumbens (NAc) for RhoGTPase related genes, known regulators of synaptic structure, following chronic social defeat stress, revealed a long-term reduction in Rac1 transcription. This was marked by a repressive chromatin state surrounding its proximal promoter. Inhibition of class 1 HDACs with MS-275 rescued both decreased Rac1 transcription and social avoidance behavior. A similar repressive chromatin state was found surrounding the Rac1 promoter in human postmortem NAc from depressed subjects, which corresponded with reduced Rac1 transcription. We show Rac1 is necessary and sufficient for social avoidance and anhedonia, and the formation of stubby excitatory spines by redistributing synaptic cofilin, an actin severing protein downstream of Rac1. Our data identifies epigenetic regulation of Rac1 in NAc as a bona fide disease mechanism in depression and reveals a functional role in regulating stress-related behaviors.

β-catenin mediates stress resilience through Dicer1/microRNA regulation

β-catenin is a multi-functional protein that has an important role in the mature central nervous system; its dysfunction has been implicated in several neuropsychiatric disorders, including depression. Here we show that in mice β-catenin mediates pro-resilient and anxiolytic effects in the nucleus accumbens, a key brain reward region, an effect mediated by D2-type medium spiny neurons. Using genome-wide β-catenin enrichment mapping, we identify Dicer1—important in small RNA (for example, microRNA) biogenesis—as a β-catenin target gene that mediates resilience. Small RNA profiling after excising β-catenin from nucleus accumbens in the context of chronic stress reveals β-catenin-dependent microRNA regulation associated with resilience. Together, these findings establish β-catenin as a critical regulator in the development of behavioural resilience, activating a network that includes Dicer1 and downstream microRNAs. We thus present a foundation for the development of novel therapeutic targets to promote stress resilience.

Rac1 is essential in cocaine-induced structural plasticity of nucleus accumbens neurons.

Repeated cocaine administration increases the dendritic arborization of nucleus accumbens neurons, but the underlying signaling events remain unknown. Here we show that repeated exposure to cocaine negatively regulates the active form of Rac1, a small GTPase that controls actin remodeling in other systems. Further, we show, using viral-mediated gene transfer, that overexpression of a dominant negative mutant of Rac1 or local knockout of Rac1 is sufficient to increase the density of immature dendritic spines on nucleus accumbens neurons, whereas overexpression of a constitutively active Rac1 or light activation of a photoactivatable form of Rac1 blocks the ability of repeated cocaine exposure to produce this effect. Downregulation of Rac1 activity likewise promotes behavioral responses to cocaine exposure, with activation of Rac1 producing the opposite effect. These findings establish that Rac1 signaling mediates structural and behavioral plasticity in response to cocaine exposure.

A Novel Role of the WNT-Dishevelled-GSK3β Signaling Cascade in the Mouse Nucleus Accumbens in a Social Defeat Model of Depression

Based on earlier gene expression and chromatin array data, we identified the protein, dishevelled (DVL)-2, as being regulated in the nucleus accumbens (NAc), a key brain reward region, in the mouse social defeat model of depression. Here, we validate these findings by showing that DVL2 mRNA and protein levels are downregulated in NAc of mice susceptible to social defeat stress, effects not seen in resilient mice. Other DVL isoforms, DVL1 and DVL3, show similar patterns of regulation. Downregulation of DVL was also demonstrated in the NAc of depressed humans examined postmortem. Interestingly, several members of the WNT (Wingless)-DVL signaling cascade, including phospho-GSK3β (glycogen synthase kinase-3β), also show significant downregulation in the NAc of susceptible, but not resilient, mice, demonstrating concerted regulation of this pathway in the NAc due to social defeat stress. By using viral-mediated gene transfer to overexpress a dominant-negative mutant of DVL in NAc, or by using a pharmacological inhibitor of DVL administered into this brain region, we show that blockade of DVL function renders mice more susceptible to social defeat stress and promotes depression-like behavior in other assays. Similar prodepression-like effects were induced upon overexpressing GSK3β in the NAc, while overexpressing a dominant-negative mutant of GSK3β promoted resilience to social defeat stress. These findings are consistent with the knowledge that downregulation of DVL and phospho-GSK3β reflects an increase in GSK3β activity. These studies reveal a novel role for the DVL-GSK3β signaling pathway, acting within the brains reward circuitry, in regulating susceptibility to chronic stress.

Chronic cocaine-regulated epigenomic changes in mouse nucleus accumbens.

BackgroundIncreasing evidence supports a role for altered gene expression in mediating the lasting effects of cocaine on the brain, and recent work has demonstrated the involvement of chromatin modifications in these alterations. However, all such studies to date have been restricted by their reliance on microarray technologies that have intrinsic limitations.ResultsWe use next generation sequencing methods, RNA-seq and ChIP-seq for RNA polymerase II and several histone methylation marks, to obtain a more complete view of cocaine-induced changes in gene expression and associated adaptations in numerous modes of chromatin regulation in the mouse nucleus accumbens, a key brain reward region. We demonstrate an unexpectedly large number of pre-mRNA splicing alterations in response to repeated cocaine treatment. In addition, we identify combinations of chromatin changes, or signatures, that correlate with cocaine-dependent regulation of gene expression, including those involving pre-mRNA alternative splicing. Through bioinformatic prediction and biological validation, we identify one particular splicing factor, A2BP1(Rbfox1/Fox-1), which is enriched at genes that display certain chromatin signatures and contributes to drug-induced behavioral abnormalities. Together, this delineation of the cocaine-induced epigenome in the nucleus accumbens reveals several novel modes of regulation by which cocaine alters the brain.ConclusionsWe establish combinatorial chromatin and transcriptional profiles in mouse nucleus accumbens after repeated cocaine treatment. These results serve as an important resource for the field and provide a template for the analysis of other systems to reveal new transcriptional and epigenetic mechanisms of neuronal regulation.

Subregional, dendritic compartment, and spine subtype specificity in cocaine-regulation of dendritic spines in the nucleus accumbens

Numerous studies have found that chronic cocaine increases dendritic spine density of medium spiny neurons in the nucleus accumbens (NAc). Here, we used single-cell microinjections and advanced 3D imaging and analysis techniques to extend these findings in several important ways: by assessing cocaine regulation of dendritic spines in the core versus shell subregions of NAc in the mouse, over a broad time course (4 h, 24 h, or 28 d) of withdrawal from chronic cocaine, and with a particular focus on proximal versus distal dendrites. Our data demonstrate subregion-specific, and in some cases opposite, regulation of spines by cocaine on proximal but not distal dendrites. Notably, all observed density changes were attributable to selective regulation of thin spines. At 4 h after injection, the proximal spine density is unchanged in the core but significantly increased in the shell. At 24 h, the density of proximal dendritic spines is reduced in the core but increased in the shell. Such downregulation of thin spines in the core persists through 28 d of withdrawal, whereas the spine density in the shell returns to baseline levels. Consistent with previous results, dendritic tips exhibited upregulation of dendritic spines after 24 h of withdrawal, an effect localized to the shell. The divergence in regulation of proximal spine density in NAc core versus shell by cocaine correlates with recently reported electrophysiological data from a similar drug administration regimen and might represent a key mediator of changes in the reward circuit that drive aspects of addiction.

Regulator of G protein signaling 4 [corrected] is a crucial modulator of antidepressant drug action in depression and neuropathic pain models.

Regulator of G protein signaling 4 (Rgs4) is a signal transduction protein that controls the function of monoamine, opiate, muscarinic, and other G protein-coupled receptors via interactions with Gα subunits. Rgs4 is expressed in several brain regions involved in mood, movement, cognition, and addiction and is regulated by psychotropic drugs, stress, and corticosteroids. In this study, we use genetic mouse models and viral-mediated gene transfer to examine the role of Rgs4 in the actions of antidepressant medications. We first analyzed human postmortem brain tissue and found robust up-regulation of RGS4 expression in the nucleus accumbens (NAc) of subjects receiving standard antidepressant medications that target monoamine systems. Behavioral studies of mice lacking Rgs4, including specific knockdowns in NAc, demonstrate that Rgs4 in this brain region acts as a positive modulator of the antidepressant-like and antiallodynic-like actions of several monoamine-directed antidepressant drugs, including tricyclic antidepressants, selective serotonin reuptake inhibitors, and norepinephrine reuptake inhibitors. Studies using viral-mediated increases in Rgs4 activity in NAc further support this hypothesis. Interestingly, in prefrontal cortex, Rgs4 acts as a negative modulator of the actions of nonmonoamine-directed drugs that are purported to act as antidepressants: the N-methyl-D-aspartate glutamate receptor antagonist ketamine and the delta opioid agonist (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide. Together, these data reveal a unique modulatory role of Rgs4 in the brain region-specific actions of a wide range of antidepressant drugs and indicate that pharmacological interventions at the level of RGS4 activity may enhance the actions of such drugs used for the treatment of depression and neuropathic pain.

Fluoxetine epigenetically alters the CaMKIIα promoter in nucleus accumbens to regulate ΔFosB binding and antidepressant effects.

Chronic social defeat stress in mice produces a susceptible phenotype characterized by several behavioral abnormalities consistent with human depression that are reversed by chronic but not acute exposure to antidepressant medications. Recent work in addiction models demonstrates that the transcription factor ΔFosB and protein kinase calmodulin-dependent protein kinase II (CaMKII) are co-regulated in nucleus accumbens (NAc), a brain reward region implicated in both addiction and depression models including social defeat. Previous work has also demonstrated that ΔFosB is induced in NAc after chronic social defeat stress or after chronic antidepressant treatment, wherein it mediates a pro-resilience or antidepressant-like phenotype. Here, using chromatin immunoprecipitation assays, we found that ΔFosB binds the CaMKIIα gene promoter in NAc and that this binding increases after mice are exposed to chronic social defeat stress. Paradoxically, chronic exposure to the antidepressant fluoxetine reduces binding of ΔFosB to the CaMKIIα promoter and reduces CaMKII expression in NAc, despite the fact that ΔFosB is induced under these conditions. These data suggest a novel epigenetic mechanism of antidepressant action, whereby fluoxetine induces some chromatin change at the CaMKIIα promoter, which blocks the ΔFosB binding. Indeed, chronic fluoxetine reduces acetylation and increases lysine-9 dimethylation of histone H3 at the CaMKIIα promoter in NAc, effects also seen in depressed humans exposed to antidepressants. Overexpression of CaMKII in NAc blocks fluoxetine’s antidepressant effects in the chronic social defeat paradigm, whereas inhibition of CaMKII activity in NAc mimics fluoxetine exposure. These findings suggest that epigenetic suppression of CaMKIIα expression in NAc is behaviorally relevant and offer a novel pathway for possible therapeutic intervention in depression and related syndromes.

Epigenetic basis of opiate suppression of Bdnf gene expression in the ventral tegmental area

Brain-derived neurotrophic factor (BDNF) has a crucial role in modulating neural and behavioral plasticity to drugs of abuse. We found a persistent downregulation of exon-specific Bdnf expression in the ventral tegmental area (VTA) in response to chronic opiate exposure, which was mediated by specific epigenetic modifications at the corresponding Bdnf gene promoters. Exposure to chronic morphine increased stalling of RNA polymerase II at these Bdnf promoters in VTA and altered permissive and repressive histone modifications and occupancy of their regulatory proteins at the specific promoters. Furthermore, we found that morphine suppressed binding of phospho-CREB (cAMP response element binding protein) to Bdnf promoters in VTA, which resulted from enrichment of trimethylated H3K27 at the promoters, and that decreased NURR1 (nuclear receptor related-1) expression also contributed to Bdnf repression and associated behavioral plasticity to morphine. Our findings suggest previously unknown epigenetic mechanisms of morphine-induced molecular and behavioral neuroadaptations.

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.