Sex-Specific Role for the Long Non-coding RNA LINC00473 in Depression.

Depression is a common disorder that affects women at twice the rate of men. Here, we report that long non-coding RNAs (lncRNAs), a recently discovered class of regulatory transcripts, represent about one-third of the differentially expressed genes in the brains of depressed humans and display complex region- and sex-specific patterns of regulation. We identified the primate-specific, neuronal-enriched gene LINC00473 as downregulated in prefrontal cortex (PFC) of depressed females but not males. Using viral-mediated gene transfer to express LINC00473 in adult mouse PFC neurons, we mirrored the human sex-specific phenotype by inducing stress resilience solely in female mice. This sex-specific phenotype was accompanied by changes in synaptic function and gene expression selectively in female mice and, along with studies of human neuron-like cells in culture, implicates LINC00473 as a CREB effector. Together, our studies identify LINC00473 as a female-specific driver of stress resilience that is aberrant in female depression.

Early life stress alters transcriptomic patterning across reward circuitry in male and female mice.

Abuse, neglect, and other forms of early life stress (ELS) significantly increase risk for psychiatric disorders including depression. In this study, we show that ELS in a postnatal sensitive period increases sensitivity to adult stress in female mice, consistent with our earlier findings in male mice. We used RNA-sequencing in the ventral tegmental area, nucleus accumbens, and prefrontal cortex of male and female mice to show that adult stress is distinctly represented in the brain's transcriptome depending on ELS history. We identify: 1) biological pathways disrupted after ELS and associated with increased behavioral stress sensitivity, 2) putative transcriptional regulators of the effect of ELS on adult stress response, and 3) subsets of primed genes specifically associated with latent behavioral changes. We also provide transcriptomic evidence that ELS increases sensitivity to future stress through enhancement of known programs of cortical plasticity.

Multidimensional Predictors of Susceptibility and Resilience to Social Defeat Stress.

BACKGROUND: Previous studies identified several separate risk factors for stress-induced disorders. However, an integrative model of susceptibility versus resilience to stress including measures from brain-body domains is likely to yield a range of multiple phenotypic information to promote successful adaptation to stress. METHODS: We used computational and molecular approaches to test whether 1) integrative brain-body behavioral, immunological, and structural domains characterized and predicted susceptibility or resilience to social defeat stress (SDS) in mice and 2) administration of acetyl-L-carnitine promoted resilience at the SDS paradigm. RESULTS: Our findings identified multidimensional brain-body predictors of susceptibility versus resilience to SDS. The copresence of anxiety, decreased hippocampal volume, and elevated systemic interleukin-6 characterized a susceptible phenotype that developed behavioral and neurobiological deficits after exposure to SDS. The susceptible phenotype showed social withdrawal and impaired transcriptomic-wide changes in the ventral dentate gyrus after SDS. At the individual level, a computational approach predicted whether a given animal developed SDS-induced social withdrawal, or remained resilient, based on the integrative in vivo measures of anxiety and immune system function. Finally, we provide initial evidence that administration of acetyl-L-carnitine promoted behavioral resilience at the SDS paradigm. CONCLUSIONS: The current findings of multidimensional brain-body predictors of susceptibility versus resilience to stress provide a starting point for in vivo models of mechanisms predisposing apparently healthy individuals to develop the neurobiological and behavioral deficits resulting from stress exposure. This framework can lead to novel therapeutic strategies to promote resilience in susceptible phenotypes.

Adolescent exposure to Δ9-tetrahydrocannabinol alters the transcriptional trajectory and dendritic architecture of prefrontal pyramidal neurons.

Neuronal circuits within the prefrontal cortex (PFC) mediate higher cognitive functions and emotional regulation that are disrupted in psychiatric disorders. The PFC undergoes significant maturation during adolescence, a period when cannabis use in humans has been linked to subsequent vulnerability to psychiatric disorders such as addiction and schizophrenia. Here, we investigated in a rat model the effects of adolescent exposure to Δ9-tetrahydrocannabinol (THC), a psychoactive component of cannabis, on the morphological architecture and transcriptional profile of layer III pyramidal neurons-using cell type- and layer-specific high-resolution microscopy, laser capture microdissection and next-generation RNA-sequencing. The results confirmed known normal expansions in basal dendritic arborization and dendritic spine pruning during the transition from late adolescence to early adulthood that were accompanied by differential expression of gene networks associated with neurodevelopment in control animals. In contrast, THC exposure disrupted the normal developmental process by inducing premature pruning of dendritic spines and allostatic atrophy of dendritic arborization in early adulthood. Surprisingly, there was minimal overlap of the developmental transcriptomes between THC- and vehicle-exposed rats. THC altered functional gene networks related to cell morphogenesis, dendritic development, and cytoskeleton organization. Marked developmental network disturbances were evident for epigenetic regulators with enhanced co-expression of chromatin- and dendrite-related genes in THC-treated animals. Dysregulated PFC co-expression networks common to both the THC-treated animals and patients with schizophrenia were enriched for cytoskeletal and neurite development. Overall, adolescent THC exposure altered the morphological and transcriptional trajectory of PFC pyramidal neurons, which could enhance vulnerability to psychiatric disorders.

A novel role for E2F3b in regulating cocaine action in the prefrontal cortex.

Drug abuse is a multifaceted disorder that involves maladaptive decision making. Long-lasting changes in the addicted brain are mediated by a complex circuit of brain reward regions. The prefrontal cortex (PFC) is one region in which chronic drug exposure changes expression and function of upstream transcriptional regulators to alter drug responses and aspects of the addicted phenotype. We reported recently that the transcription factor E2F3a is a critical mediator of cocaine responses in the nucleus accumbens. E2F3a is one of two splice variants of the E2f3 gene; the other is E2F3b. Another recent study predicted E2F3 as an upstream regulator of the transcriptional response to cocaine self-administration (SA) in PFC. Based on previous findings that E2F3a and E2F3b have divergent regulatory roles, we set out to study the putative transcriptional role of these transcripts in PFC in the context of repeated I.P. cocaine exposure. We implemented viral-mediated isoform-specific gene manipulation, RNA-sequencing, advanced bioinformatics analyses, and animal behavior to determine how E2F3a and E2F3b contribute to persistent cocaine-induced transcriptional changes in PFC. We show that E2F3b, but not E2F3a, in PFC is critical for cocaine locomotor and place preference behaviors. Interestingly, RNA-seq of PFC following E2f3b overexpression or I.P. cocaine exposure showed very different effects on expression levels of differentially expressed genes. However, we found that E2F3b drives a similar transcriptomic pattern to that of cocaine SA with overlapping upstream regulators and downstream pathways predicted. These findings reveal a novel transcriptional mechanism in PFC that controls behavioral and molecular responses to cocaine.

Genome-wide transcriptional profiling of central amygdala and orbitofrontal cortex during incubation of methamphetamine craving.

Methamphetamine (Meth) seeking progressively increases after withdrawal (incubation of Meth craving), but the transcriptional mechanisms that contribute to this incubation are unknown. Here we used RNA-sequencing to analyze transcriptional profiles associated with incubation of Meth craving in central amygdala (CeA) and orbitofrontal cortex (OFC), two brain areas previously implicated in relapse to drug seeking. We trained rats to self-administer either saline (control condition) or Meth (10 days; 9 h/day, 0.1 mg/kg/infusion). Next, we collected brain tissue from CeA and OFC on withdrawal day 2 (when Meth seeking is low and non-incubated) and on day 35 (when Meth seeking is high and incubated), for subsequent RNA-sequencing. In CeA, we identified 10-fold more differentially expressed genes (DEGs) on withdrawal day 35 than day 2. These genes were enriched for several biological processes, including protein ubiquitination and histone methylation. In OFC, we identified much fewer expression changes than in CeA, with more DEGs on withdrawal day 2 than on day 35. There was a significant overlap between upregulated genes on withdrawal day 2 and downregulated genes on withdrawal day 35 in OFC. Our analyses highlight the CeA as a key region of transcriptional regulation associated with incubation of Meth seeking. In contrast, transcriptional regulation in OFC may contribute to Meth seeking during early withdrawal. Overall, these findings provide a unique resource of gene expression data for future studies examining transcriptional mechanisms in CeA that mediate Meth seeking after prolonged withdrawal.

Transcriptional and physiological adaptations in nucleus accumbens somatostatin interneurons that regulate behavioral responses to cocaine.

The role of somatostatin interneurons in nucleus accumbens (NAc), a key brain reward region, remains poorly understood due to the fact that these cells account for < 1% of NAc neurons. Here, we use optogenetics, electrophysiology, and RNA-sequencing to characterize the transcriptome and functioning of NAc somatostatin interneurons after repeated exposure to cocaine. We find that the activity of somatostatin interneurons regulates behavioral responses to cocaine, with repeated cocaine reducing the excitability of these neurons. Repeated cocaine also induces transcriptome-wide changes in gene expression within NAc somatostatin interneurons. We identify the JUND transcription factor as a key regulator of cocaine action and confirmed, by use of viral-mediated gene transfer, that JUND activity in somatostatin interneurons influences behavioral responses to cocaine. Our results identify alterations in NAc induced by cocaine in a sparse population of somatostatin interneurons, and illustrate the value of studying brain diseases using cell type-specific whole transcriptome RNA-sequencing.

Epigenetic regulation of brain region-specific microglia clearance activity.

The rapid elimination of dying neurons and nonfunctional synapses in the brain is carried out by microglia, the resident myeloid cells of the brain. Here we show that microglia clearance activity in the adult brain is regionally regulated and depends on the rate of neuronal attrition. Cerebellar, but not striatal or cortical, microglia exhibited high levels of basal clearance activity, which correlated with an elevated degree of cerebellar neuronal attrition. Exposing forebrain microglia to apoptotic cells activated gene-expression programs supporting clearance activity. We provide evidence that the polycomb repressive complex 2 (PRC2) epigenetically restricts the expression of genes that support clearance activity in striatal and cortical microglia. Loss of PRC2 leads to aberrant activation of a microglia clearance phenotype, which triggers changes in neuronal morphology and behavior. Our data highlight a key role of epigenetic mechanisms in preventing microglia-induced neuronal alterations that are frequently associated with neurodegenerative and psychiatric diseases.

Cocaine Self-administration Alters Transcriptome-wide Responses in the Brain's Reward Circuitry.

BACKGROUND: Global changes in gene expression underlying circuit and behavioral dysregulation associated with cocaine addiction remain incompletely understood. Here, we show how a history of cocaine self-administration (SA) reprograms transcriptome-wide responses throughout the brain's reward circuitry at baseline and in response to context and/or cocaine re-exposure after prolonged withdrawal (WD). METHODS: We assigned male mice to one of six groups: saline/cocaine SA + 24-hour WD or saline/cocaine SA + 30-day WD + an acute saline/cocaine challenge within the previous drug-paired context. RNA sequencing was conducted on six interconnected brain reward regions. Using pattern analysis of gene expression and factor analysis of behavior, we identified genes that are strongly associated with addiction-related behaviors and uniquely altered by a history of cocaine SA. We then identified potential upstream regulators of these genes. RESULTS: We focused on three patterns of gene expression that reflect responses to 1) acute cocaine, 2) context re-exposure, and 3) drug + context re-exposure. These patterns revealed region-specific regulation of gene expression. Further analysis revealed that each of these gene expression patterns correlated with an addiction index-a composite score of several addiction-like behaviors during cocaine SA-in a region-specific manner. Cyclic adenosine monophosphate response element binding protein and nuclear receptor families were identified as key upstream regulators of genes associated with such behaviors. CONCLUSIONS: This comprehensive picture of transcriptome-wide regulation in the brain's reward circuitry by cocaine SA and prolonged WD provides new insight into the molecular basis of cocaine addiction, which will guide future studies of the key molecular pathways involved.

Corrigendum: Sex-specific transcriptional signatures in human depression.

This corrects the article DOI: 10.1038/nm.4386.

Estrogen receptor α drives pro-resilient transcription in mouse models of depression.

Most people exposed to stress do not develop depression. Animal models have shown that stress resilience is an active state that requires broad transcriptional adaptations, but how this homeostatic process is regulated remains poorly understood. In this study, we analyze upstream regulators of genes differentially expressed after chronic social defeat stress. We identify estrogen receptor α (ERα) as the top regulator of pro-resilient transcriptional changes in the nucleus accumbens (NAc), a key brain reward region implicated in depression. In accordance with these findings, nuclear ERα protein levels are altered by stress in male and female mice. Further, overexpression of ERα in the NAc promotes stress resilience in both sexes. Subsequent RNA-sequencing reveals that ERα overexpression in NAc reproduces the transcriptional signature of resilience in male, but not female, mice. These results indicate that NAc ERα is an important regulator of pro-resilient transcriptional changes, but with sex-specific downstream targets.

Suppression of RGSz1 function optimizes the actions of opioid analgesics by mechanisms that involve the Wnt/ß-catenin pathway.

Regulator of G protein signaling z1 (RGSz1), a member of the RGS family of proteins, is present in several networks expressing mu opioid receptors (MOPRs). By using genetic mouse models for global or brain region-targeted manipulations of RGSz1 expression, we demonstrated that the suppression of RGSz1 function increases the analgesic efficacy of MOPR agonists in male and female mice and delays the development of morphine tolerance while decreasing the sensitivity to rewarding and locomotor activating effects. Using biochemical assays and next-generation RNA sequencing, we identified a key role of RGSz1 in the periaqueductal gray (PAG) in morphine tolerance. Chronic morphine administration promotes RGSz1 activity in the PAG, which in turn modulates transcription mediated by the Wnt/ß-catenin signaling pathway to promote analgesic tolerance to morphine. Conversely, the suppression of RGSz1 function stabilizes Axin2-Gαz complexes near the membrane and promotes ß-catenin activation, thereby delaying the development of analgesic tolerance. These data show that the regulation of RGS complexes, particularly those involving RGSz1-Gαz, represents a promising target for optimizing the analgesic actions of opioids without increasing the risk of dependence or addiction.

Transcription Factor E2F3a in Nucleus Accumbens Affects Cocaine Action via Transcription and Alternative Splicing.

BACKGROUND: Lasting changes in gene expression in brain reward regions, including nucleus accumbens (NAc), contribute to persistent functional changes in the addicted brain. We and others have demonstrated that altered expression of several candidate transcription factors in NAc regulates drug responses. A recent large-scale genome-wide study from our group predicted transcription factor E2F3 (E2F3) as a prominent upstream regulator of cocaine-induced changes in gene expression and alternative splicing. METHODS: We studied expression of two E2F3 isoforms-E2F3a and E2F3b-in mouse NAc after repeated cocaine administration and assayed the effects of overexpression or depletion of E2f3 isoforms in NAc on cocaine behavioral responses. We then performed RNA sequencing to investigate the effect of E2f3a overexpression in this region on gene expression and alternative splicing and performed quantitative chromatin immunoprecipitation at downstream targets in NAc following E2f3a overexpression or repeated cocaine exposure. Sample sizes varied between experiments and are noted in the text. RESULTS: We showed that E2f3a, but not E2f3b, overexpression or knockdown in mouse NAc regulates cocaine-induced locomotor and place conditioning behavior. Furthermore, we demonstrated that E2f3a overexpression substantially recapitulates genome-wide transcriptional profiles and alternative splicing induced by cocaine. We further validated direct binding of E2F3a at key target genes following cocaine exposure. CONCLUSIONS: This study establishes E2F3a as a novel transcriptional regulator of cocaine action in NAc. The findings reveal a crucial role for E2F3a in the regulation of cocaine-elicited behavioral states. Moreover, the importance of this role is bolstered by the extensive recapitulation of cocaine's transcriptional effects in NAc by overexpression of E2f3a.

Sex-specific transcriptional signatures in human depression.

Major depressive disorder (MDD) is a leading cause of disease burden worldwide. While the incidence, symptoms and treatment of MDD all point toward major sex differences, the molecular mechanisms underlying this sexual dimorphism remain largely unknown. Here, combining differential expression and gene coexpression network analyses, we provide a comprehensive characterization of male and female transcriptional profiles associated with MDD across six brain regions. We overlap our human profiles with those from a mouse model, chronic variable stress, and capitalize on converging pathways to define molecular and physiological mechanisms underlying the expression of stress susceptibility in males and females. Our results show a major rearrangement of transcriptional patterns in MDD, with limited overlap between males and females, an effect seen in both depressed humans and stressed mice. We identify key regulators of sex-specific gene networks underlying MDD and confirm their sex-specific impact as mediators of stress susceptibility. For example, downregulation of the female-specific hub gene Dusp6 in mouse prefrontal cortex mimicked stress susceptibility in females, but not males, by increasing ERK signaling and pyramidal neuron excitability. Such Dusp6 downregulation also recapitulated the transcriptional remodeling that occurs in prefrontal cortex of depressed females. Together our findings reveal marked sexual dimorphism at the transcriptional level in MDD and highlight the importance of studying sex-specific treatments for this disorder.

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

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.

Neuropathic pain promotes adaptive changes in gene expression in brain networks involved in stress and depression.

Neuropathic pain is a complex chronic condition characterized by various sensory, cognitive, and affective symptoms. A large percentage of patients with neuropathic pain are also afflicted with depression and anxiety disorders, a pattern that is also seen in animal models. Furthermore, clinical and preclinical studies indicate that chronic pain corresponds with adaptations in several brain networks involved in mood, motivation, and reward. Chronic stress is also a major risk factor for depression. We investigated whether chronic pain and stress affect similar molecular mechanisms and whether chronic pain can affect gene expression patterns that are involved in depression. Using two mouse models of neuropathic pain and depression [spared nerve injury (SNI) and chronic unpredictable stress (CUS)], we performed next-generation RNA sequencing and pathway analysis to monitor changes in gene expression in the nucleus accumbens (NAc), the medial prefrontal cortex (mPFC), and the periaqueductal gray (PAG). In addition to finding unique transcriptome profiles across these regions, we identified a substantial number of signaling pathway-associated genes with similar changes in expression in both SNI and CUS mice. Many of these genes have been implicated in depression, anxiety, and chronic pain in patients. Our study provides a resource of the changes in gene expression induced by long-term neuropathic pain in three distinct brain regions and reveals molecular connections between pain and chronic stress.

Tet1 in Nucleus Accumbens Opposes Depression- and Anxiety-Like Behaviors.

Depression is a leading cause of disease burden, yet current therapies fully treat <50% of affected individuals. Increasing evidence implicates epigenetic mechanisms in depression and antidepressant action. Here we examined a possible role for the DNA dioxygenase, ten-eleven translocation protein 1 (TET1), in depression-related behavioral abnormalities. We applied chronic social defeat stress, an ethologically validated mouse model of depression-like behaviors, and examined Tet1 expression changes in nucleus accumbens (NAc), a key brain reward region. We show decreased Tet1 expression in NAc in stress-susceptible mice only. Surprisingly, selective knockout of Tet1 in NAc neurons of adult mice produced antidepressant-like effects in several behavioral assays. To identify Tet1 targets that mediate these actions, we performed RNAseq on NAc after conditional deletion of Tet1 and found that immune-related genes are the most highly dysregulated. Moreover, many of these genes are also upregulated in the NAc of resilient mice after chronic social defeat stress. These findings reveal a novel role for TET1, an enzyme important for DNA hydroxymethylation, in the brain's reward circuitry in modulating stress responses in mice. We also identify a subset of genes that are regulated by TET1 in this circuitry. These findings provide new insight into the pathophysiology of depression, which can aid in future antidepressant drug discovery efforts.

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.

Circuit-wide Transcriptional Profiling Reveals Brain Region-Specific Gene Networks Regulating Depression Susceptibility.

Depression is a complex, heterogeneous disorder and a leading contributor to the global burden of disease. Most previous research has focused on individual brain regions and genes contributing to depression. However, emerging evidence in humans and animal models suggests that dysregulated circuit function and gene expression across multiple brain regions drive depressive phenotypes. Here, we performed RNA sequencing on four brain regions from control animals and those susceptible or resilient to chronic social defeat stress at multiple time points. We employed an integrative network biology approach to identify transcriptional networks and key driver genes that regulate susceptibility to depressive-like symptoms. Further, we validated in vivo several key drivers and their associated transcriptional networks that regulate depression susceptibility and confirmed their functional significance at the levels of gene transcription, synaptic regulation, and behavior. Our study reveals novel transcriptional networks that control stress susceptibility and offers fundamentally new leads for antidepressant drug discovery.

In vivo imaging identifies temporal signature of D1 and D2 medium spiny neurons in cocaine reward.

The reinforcing and rewarding properties of cocaine are attributed to its ability to increase dopaminergic transmission in nucleus accumbens (NAc). This action reinforces drug taking and seeking and leads to potent and long-lasting associations between the rewarding effects of the drug and the cues associated with its availability. The inability to extinguish these associations is a key factor contributing to relapse. Dopamine produces these effects by controlling the activity of two subpopulations of NAc medium spiny neurons (MSNs) that are defined by their predominant expression of either dopamine D1 or D2 receptors. Previous work has demonstrated that optogenetically stimulating D1 MSNs promotes reward, whereas stimulating D2 MSNs produces aversion. However, we still lack a clear understanding of how the endogenous activity of these cell types is affected by cocaine and encodes information that drives drug-associated behaviors. Using fiber photometry calcium imaging we define D1 MSNs as the specific population of cells in NAc that encodes information about drug associations and elucidate the temporal profile with which D1 activity is increased to drive drug seeking in response to contextual cues. Chronic cocaine exposure dysregulates these D1 signals to both prevent extinction and facilitate reinstatement of drug seeking to drive relapse. Directly manipulating these D1 signals using designer receptors exclusively activated by designer drugs prevents contextual associations. Together, these data elucidate the responses of D1- and D2-type MSNs in NAc to acute cocaine and during the formation of context-reward associations and define how prior cocaine exposure selectively dysregulates D1 signaling to drive relapse.