ZBTB7A regulates MDD-specific chromatin signatures and astrocyte-mediated stress vulnerability in orbitofrontal cortex.

Hyperexcitability in the orbitofrontal cortex (OFC) is a key clinical feature of anhedonic domains of Major Depressive Disorder (MDD). However, the cellular and molecular substrates underlying this dysfunction remain unknown. Here, cell-population-specific chromatin accessibility profiling in human OFC unexpectedly mapped genetic risk for MDD exclusively to non-neuronal cells, and transcriptomic analyses revealed significant glial dysregulation in this region. Characterization of MDD-specific cis-regulatory elements identified ZBTB7A - a transcriptional regulator of astrocyte reactivity - as an important mediator of MDD-specific chromatin accessibility and gene expression. Genetic manipulations in mouse OFC demonstrated that astrocytic Zbtb7a is both necessary and sufficient to promote behavioral deficits, cell-type-specific transcriptional and chromatin profiles, and OFC neuronal hyperexcitability induced by chronic stress - a major risk factor for MDD. These data thus highlight a critical role for OFC astrocytes in stress vulnerability and pinpoint ZBTB7A as a key dysregulated factor in MDD that mediates maladaptive astrocytic functions driving OFC hyperexcitability.

Histone H3 serotonylation dynamics in dorsal raphe nucleus contribute to stress- and antidepressant-mediated gene expression and behavior.

Background: Major depressive disorder (MDD), along with related mood disorders, is a debilitating illness that affects millions of individuals worldwide. While chronic stress increases incidence levels of mood disorders, stress-mediated disruptions in brain function that precipitate these illnesses remain elusive. Serotonin-associated antidepressants (ADs) remain the first line of therapy for many with depressive symptoms, yet low remission rates and delays between treatment and symptomatic alleviation have prompted skepticism regarding precise roles for serotonin in the precipitation of mood disorders. Our group recently demonstrated that serotonin epigenetically modifies histone proteins (H3K4me3Q5ser) to regulate transcriptional permissiveness in brain. However, this phenomenon has not yet been explored following stress and/or AD exposures. Methods: We employed a combination of genome-wide and biochemical analyses in dorsal raphe nucleus (DRN) of male and female mice exposed to chronic social defeat stress to examine the impact of stress exposures on H3K4me3Q5ser dynamics, as well as associations between the mark and stress-induced gene expression. We additionally assessed stress-induced regulation of H3K4me3Q5ser following AD exposures, and employed viral-mediated gene therapy to reduce H3K4me3Q5ser levels in DRN and examine the impact on stress-associated gene expression and behavior. Results: We found that H3K4me3Q5ser plays important roles in stress-mediated transcriptional plasticity. Chronically stressed mice displayed dysregulated H3K4me3Q5ser dynamics in DRN, with both AD- and viral-mediated disruption of these dynamics proving sufficient to rescue stress-mediated gene expression and behavior. Conclusions: These findings establish a neurotransmission-independent role for serotonin in stress-/AD-associated transcriptional and behavioral plasticity in DRN.

Dissociable contributions of the amygdala and ventral hippocampus to stress-induced changes in defensive behavior.

Severe stress can produce multiple persistent changes in defensive behavior. While much is known about the circuits supporting stress-induced associative fear responses, how circuit plasticity supports the broader changes in defensive behavior observed after severe stress remains unclear. Here, we find that stress-induced plasticity in the ventral hippocampus (vHC) and basolateral amygdala (BLA) support doubly dissociable defensive behavioral changes. Stress-induced protein synthesis in the BLA was found to support lasting enhancements in stress sensitivity but not enhancements in exploratory anxiety-related behaviors, whereas protein synthesis in the vHC was found to support enhancements in anxiety-related behavior but not enhancements in stress sensitivity. Like protein synthesis, neuronal activity of the BLA and vHC were found to differentially support the expression of these same defensive behaviors. Lastly, blockade of associative fear had no impact on stress-induced changes in anxiety-related behavior. These findings highlight that multiple memory-systems support stress-induced defensive behavior changes.

Histone H3 dopaminylation in nucleus accumbens, but not medial prefrontal cortex, contributes to cocaine-seeking following prolonged abstinence.

Enduring patterns of epigenomic and transcriptional plasticity within the mesolimbic dopamine system contribute importantly to persistent behavioral adaptations that characterize substance use disorders (SUD). While drug addiction has long been thought of as a disorder of dopamine (DA) neurotransmission, therapeutic interventions targeting receptor mediated DA-signaling have not yet resulted in efficacious treatments. Our laboratory recently identified a non-canonical, neurotransmission-independent signaling moiety for DA in brain, termed dopaminylation, whereby DA itself acts as a donor source for the establishment of post-translational modifications (PTM) on substrate proteins (e.g., histone H3 at glutamine 5; H3Q5dop). In our previous studies, we demonstrated that H3Q5dop plays a critical role in the regulation of neuronal transcription and, when perturbed within monoaminergic neurons of the ventral tegmental area (VTA), critically contributes to pathological states, including relapse vulnerability to both psychostimulants (e.g., cocaine) and opiates (e.g., heroin). Importantly, H3Q5dop is also observed throughout the mesolimbic DA reward pathway (e.g., in nucleus accumbens/NAc and medial prefrontal cortex/mPFC, which receive DA input from VTA). As such, we investigated whether H3Q5dop may similarly be altered in its expression in response to drugs of abuse in these non-dopamine-producing regions. In rats undergoing extended abstinence from cocaine self-administration (SA), we observed both acute and prolonged accumulation of H3Q5dop in NAc, but not mPFC. Attenuation of H3Q5dop in NAc during drug abstinence reduced cocaine-seeking and affected cocaine-induced gene expression programs associated with altered dopamine signaling and neuronal function. These findings thus establish H3Q5dop in NAc, but not mPFC, as an important mediator of cocaine-induced behavioral and transcriptional plasticity during extended cocaine abstinence.

Rescue of deficits by Brwd1 copy number restoration in the Ts65Dn mouse model of Down syndrome.

With an incidence of ~1 in 800 births, Down syndrome (DS) is the most common chromosomal condition linked to intellectual disability worldwide. While the genetic basis of DS has been identified as a triplication of chromosome 21 (HSA21), the genes encoded from HSA21 that directly contribute to cognitive deficits remain incompletely understood. Here, we found that the HSA21-encoded chromatin effector, BRWD1, was upregulated in neurons derived from iPS cells from an individual with Down syndrome and brain of trisomic mice. We showed that selective copy number restoration of Brwd1 in trisomic animals rescued deficits in hippocampal LTP, cognition and gene expression. We demonstrated that Brwd1 tightly binds the BAF chromatin remodeling complex, and that increased Brwd1 expression promotes BAF genomic mistargeting. Importantly, Brwd1 renormalization rescued aberrant BAF localization, along with associated changes in chromatin accessibility and gene expression. These findings establish BRWD1 as a key epigenomic mediator of normal neurodevelopment and an important contributor to DS-related phenotypes.

Histone H3 dopaminylation in ventral tegmental area underlies heroin-induced transcriptional and behavioral plasticity in male rats.

Persistent transcriptional events in ventral tegmental area (VTA) and other reward relevant brain regions contribute to enduring behavioral adaptations that characterize substance use disorder. Recent data from our laboratory indicate that aberrant accumulation of the newly discovered histone post-translational modification (PTM), H3 dopaminylation at glutamine 5 (H3Q5dop), contributes significantly to cocaine-seeking behavior following prolonged periods of abstinence. It remained unclear, however, whether this modification is important for relapse vulnerability in the context of other drugs of abuse, such as opioids. Here, we showed that H3Q5dop plays a critical role in heroin-mediated transcriptional plasticity in midbrain regions, particularly the VTA. In rats undergoing abstinence from heroin self-administration (SA), we found acute and persistent accumulation of H3Q5dop in VTA. Attenuation of H3Q5dop during abstinence induced persistent changes in gene expression programs associated with neuronal signaling and dopaminergic function in heroin abstinence and led to reduced heroin-seeking behavior. Interestingly, the observed changes in molecular pathways after heroin SA showed significant yet reversed overlap with the same genes altered in cocaine SA. These findings establish an essential role for H3Q5dop, and its downstream transcriptional consequences, in heroin-induced functional plasticity in VTA.

Lifelong reductions of PKMζ in ventral hippocampus of nonhuman primates exposed to early-life adversity due to unpredictable maternal care.

Protein kinase Mζ (PKMζ) maintains long-term potentiation (LTP) and long-term memory through persistent increases in kinase expression. Early-life adversity is a precursor to adult mood and anxiety disorders, in part, through persistent disruption of emotional memory throughout life. Here we subjected 10- to 16-wk-old male bonnet macaques to adversity by a maternal variable-foraging demand paradigm. We then examined PKMζ expression in their ventral hippocampi as 7- to 12-yr-old adults. Quantitative immunohistochemistry reveals decreased PKMζ in dentate gyrus, CA1, and subiculum of subjects who had experienced early-life adversity due to the unpredictability of maternal care. Adult animals with persistent decrements of PKMζ in ventral hippocampus express timid rather than confrontational responses to a human intruder. Persistent down-regulation of PKMζ in the ventral hippocampus might reduce the capacity for emotional memory maintenance and contribute to the long-lasting emotional effects of early-life adversity.

Early Life Stress and the Fate of Kynurenine Pathway Metabolites.

Early life stress (ELS) precedes alterations to neuro-immune activation, which may mediate an increased risk for stress-related psychiatric disorders, potentially through alterations of central kynurenine pathway (KP) metabolites, the latter being relatively unexplored. We hypothesized that ELS in a non-human primate model would lead to a reduction of neuroprotective and increases of neurotoxic KP metabolites. Twelve adult female bonnet macaques reared under conditions of maternal variable foraging demand (VFD) were compared to 27 age- and weight-matched non-VFD-exposed female controls. Baseline behavioral observations of social affiliation were taken over a 12-week period followed by the first cerebrospinal fluid (CSF) sample. Subjects were then either exposed to a 12-week repeated separation paradigm (RSP) or assigned to a "no-RSP" condition followed by a second CSF. We used high-performance liquid chromatography for kynurenine (KYN), tryptophan, 5-hydroxyindoleacetic acid, kynurenic acid (KYNA), and anthranilic acid (ANTH) as a proxy for quinolinic acid determination. At baseline, social affiliation scores were reduced in VFD-reared versus control subjects. CSF log KYNA and log KYNA/KYN ratio were lower in VFD-reared versus control subjects. CSF log KYNA/KYN was positively correlated with CSF log ANTH in VFD only (r = 0.82). Controlling for log KYNA/KYN, log ANTH was elevated in VFD-reared subjects versus controls. CSF log KYNA/KYN obtained post-RSP was positively correlated with mean social affiliation scores during RSP, specifically in VFD. ELS is associated with a reduced neuroprotective and increased neurotoxic pathway products. That the two contrasting processes are paradoxically correlated following ELS suggests a cross-talk between two opposing KP enzymatic systems.

Developmental Antecedents of Adult Macaque Neurogenesis: Early-Life Adversity, 5-HTTLPR Polymorphisms, and Adolescent Hippocampal Volume.

INTRODUCTION: Attenuated adult hippocampal neurogenesis may manifest in affective symptomatology and/or resistance to antidepressant treatment. While early-life adversity and the short variant ('s') of the serotonin transporter gene's long polymorphic region (5-HTTLPR) are suggested as interacting risk factors for affective disorders, no studies have examined whether their superposed risk effectuates neurogenic changes into adulthood. Similarly, it is not established whether reduced hippocampal volume in adolescence, variously identified as a marker and antecedent of affective disorders, anticipates diminished adult neurogenesis. We investigate these potential developmental precursors of neurogenic alterations using a bonnet macaque model. METHODS: Twenty-five male infant bonnet macaques were randomized to stressed [variable foraging demand (VFD)] or normative [low foraging demand (LFD)] rearing protocols and genotyped for 5-HTTLPR polymorphisms. Adolescent MRI brain scans (mean age 4.2y) were available for 14 subjects. Adult-born neurons were detected post-mortem (mean age 8.6y) via immunohistochemistry targeting the microtubule protein doublecortin (DCX). Models were adjusted for age and weight. RESULTS: A putative vulnerability group (VG) of VFD-reared 's'-carriers (all 's/l') exhibited reduced neurogenesis compared to non-VG subjects. Neurogenesis levels were positively predicted by ipsilateral hippocampal volume normalized for total brain volume, but not by contralateral or raw hippocampal volume. LIMITATIONS: No 's'-carriers were identified in LFD-reared subjects, precluding a 2×2 factorial analysis. CONCLUSION: The 's' allele (with adverse rearing) and low adolescent hippocampal volume portend a neurogenic deficit in adult macaques, suggesting persistent alterations in hippocampal plasticity may contribute to these developmental factors' affective risk in humans.

Epigenetics of Drug Addiction.

Substance use disorders (SUDs) are chronic brain diseases characterized by transitions from recreational to compulsive drug use and aberrant drug craving that persists for months to years after abstinence is achieved. The transition to compulsive drug use implies that plasticity is occurring, altering the physiology of the brain to precipitate addicted states. Epigenetic phenomena represent a varied orchestra of transcriptional tuning mechanisms that, in response to environmental stimuli, create and maintain gene expression-mediated physiological outcomes. Therefore, epigenetic mechanisms represent a convergent regulatory framework through which the plasticity required to achieve an addicted state can arise and then persist long after drug use has ended. In the first section, we will introduce basic concepts in epigenetics, such as chromatin architecture, histones and their posttranslational modifications, DNA methylation, noncoding RNAs, and transcription factors, along with methods for their investigation. We will then examine the implications of these mechanisms in SUDs, with a particular focus on cocaine-mediated neuroepigenetic plasticity across multiple behavioral models of addiction.

Dopaminylation of histone H3 in ventral tegmental area regulates cocaine seeking.

Vulnerability to relapse during periods of attempted abstinence from cocaine use is hypothesized to result from the rewiring of brain reward circuitries, particularly ventral tegmental area (VTA) dopamine neurons. How cocaine exposures act on midbrain dopamine neurons to precipitate addiction-relevant changes in gene expression is unclear. We found that histone H3 glutamine 5 dopaminylation (H3Q5dop) plays a critical role in cocaine-induced transcriptional plasticity in the midbrain. Rats undergoing withdrawal from cocaine showed an accumulation of H3Q5dop in the VTA. By reducing H3Q5dop in the VTA during withdrawal, we reversed cocaine-mediated gene expression changes, attenuated dopamine release in the nucleus accumbens, and reduced cocaine-seeking behavior. These findings establish a neurotransmission-independent role for nuclear dopamine in relapse-related transcriptional plasticity in the VTA.

Translational Molecular Approaches in Substance Abuse Research.

Excessive abuse of psychoactive substances is one of the leading contributors to morbidity and mortality worldwide. In this book chapter, we review translational research strategies that are applied in the pursuit of new and more effective therapeutics for substance use disorder (SUD). The complex, multidimensional nature of psychiatric disorders like SUD presents difficult challenges to investigators. While animal models are critical for outlining the mechanistic relationships between defined behaviors and genetic and/or molecular changes, the heterogeneous pathophysiology of brain diseases is uniquely human, necessitating the use of human studies and translational research schemes. Translational research describes a cross-species approach in which findings from human patient-based data can be used to guide molecular genetic investigations in preclinical animal models in order to delineate the mechanisms of reward circuitry changes in the addicted state. Results from animal studies can then inform clinical investigations toward the development of novel treatments for SUD. Here we describe the strategies that are used to identify and functionally validate genetic variants in the human genome which may contribute to increased risk for SUD, starting from early candidate gene approaches to more recent genome-wide association studies. We will next examine studies aimed at understanding how transcriptional and epigenetic dysregulation in SUD can persistently alter cellular function in the disease state. In our discussion, we then focus on examples from the literature illustrating molecular genetic methodologies that have been applied to studies of different substances of abuse - from alcohol and nicotine to stimulants and opioids - in order to exemplify how these approaches can both delineate the underlying molecular systems driving drug addiction and provide insights into the genetic basis of SUD.

Maternal Cerebrospinal Fluid Glutamate in Response to Variable Foraging Demand: Relationship to Cerebrospinal Fluid Serotonin Metabolites in Grown Offspring.

BACKGROUND: Maternal response to allostatic overload during infant rearing may alter neurobiological measures in grown offspring, potentially increasing susceptibility to mood and anxiety disorders. We examined maternal cerebrospinal fluid (CSF) glutamate response during exposure to variable foraging demand (VFD), a bonnet macaque model of allostatic overload, testing whether activation relative to baseline predicted concomitant CSF elevations of the stress neuropeptide, corticotropin-releasing factor. We investigated whether VFD-induced activation of maternal CSF glutamate affects maternal-infant attachment patterns and offspring CSF 5-hydroxyindoleacetic acid concentrations. METHODS: Mother-infant dyads were exposed to the "VFD stressor," a paradigm in which mothers experience 16 weeks of foraging uncertainty while rearing their infant offspring. Through staggering the infant age of VFD onset, both a cross-sectional design and a longitudinal design were used. Maternal CSF glutamate and glutamine concentrations post-VFD exposure were cross-sectionally compared to maternal VFD naive controls. Proportional change in concentrations of maternal glutamate (and glutamine), a longitudinal measure, was evaluated in relation to VFD-induced elevations of CSF corticotropin-releasing factor. The former measure was related to maternal-infant proximity scores obtained during the final phases of VFD exposure. Maternal glutamatergic response to VFD exposure was used as a predictor variable for young adolescent offspring CSF metabolites of serotonin, dopamine, and norepinephrine. RESULTS: Following VFD exposure, maternal CSF glutamate concentrations correlated positively with maternal CSF CRF concentrations. Activation relative to baseline of maternal CSF glutamate concentrations following VFD exposure correlated directly with a) increased maternal-infant proximity during the final phases of VFD and b) offspring CSF concentrations of monoamine metabolites including 5-hydroxyindoleacetic acid, which was elevated relative to controls. CONCLUSIONS: Activation of maternal CSF glutamate in response to VFD-induced allostasis is directly associated with elevations of maternal CSF corticotropin-releasing factor. Maternal CSF glutamate alterations induced by VFD potentially compromise serotonin neurotransmission in grown offspring, conceivably modeling human vulnerability to treatment-resistant mood and anxiety disorders.

Reduced left ventricular dimension and function following early life stress: A thrifty phenotype hypothesis engendering risk for mood and anxiety disorders.

BACKGROUND: Early life stress (ELS) in macaques in the form of insecure maternal attachment putatively induces epigenetic adaptations resulting in a "thrifty phenotype" throughout the life cycle. For instance, ELS induces persistent increases in insulin resistance, hippocampal and corpus callosum atrophy and reduced "behavioral plasticity", which, taken together, engenders an increased risk for mood and anxiety disorders in humans but also a putative sparing of calories. Herein, we test the hypothesis whether a thrifty phenotype induced by ELS is peripherally evident as hypotrophy of cardiac structure and function, raising the possibility that certain mood disorders may represent maladaptive physiological and central thrift adaptations. METHODS: 14 adult bonnet macaques (6 males) exposed to the maternal variable foraging demand (VFD) model of ELS were compared to 20 non-VFD adult subjects (6 males). Left ventricle end-diastolic dimension (LVEDD), Left ventricle end-systolic dimension (LVESD) and stroke volume (SV) were calculated using echocardiography. Blood pressure and heart rate were measured only in females. Previously obtained neurobehavioral correlates available only in males were analyzed in the context of cardiac parameters. RESULTS: Reduced LVESD (p < 0.05) was observed when controlled for age, sex, body weight and crown-rump length whereas ejection fraction (EF) (p = 0.037) was greater in VFD-reared versus non-VFD subjects. Pulse pressure was lower in VFD versus non-VFD females (p < 0.05). Male timidity in response to a human intruder was associated with reduced LVEDD (p < 0.05). CONCLUSIONS: ELS is associated with both structural and functional reductions of left ventricular measures, potentially implying a body-wide thrifty phenotype. Parallel "thrift" adaptations may occur in key brain areas following ELS and may play an unexplored role in mood and anxiety disorder susceptibility.

Glucagon-like peptide-1 as predictor of body mass index and dentate gyrus neurogenesis: neuroplasticity and the metabolic milieu.

Glucagon-like peptide-1 (GLP-1) regulates carbohydrate metabolism and promotes neurogenesis. We reported an inverse correlation between adult body mass and neurogenesis in nonhuman primates. Here we examine relationships between physiological levels of the neurotrophic incretin, plasma GLP-1 (pGLP-1), and body mass index (BMI) in adolescence to adult neurogenesis and associations with a diabesity diathesis and infant stress. Morphometry, fasting pGLP-1, insulin resistance, and lipid profiles were measured in early adolescence in 10 stressed and 4 unstressed male bonnet macaques. As adults, dentate gyrus neurogenesis was assessed by doublecortin staining. High pGLP-1, low body weight, and low central adiposity, yet peripheral insulin resistance and high plasma lipids, during adolescence were associated with relatively high adult neurogenesis rates. High pGLP-1 also predicted low body weight with, paradoxically, insulin resistance and high plasma lipids. No rearing effects for neurogenesis rates were observed. We replicated an inverse relationship between BMI and neurogenesis. Adolescent pGLP-1 directly predicted adult neurogenesis. Two divergent processes relevant to human diabesity emerge-high BMI, low pGLP-1, and low neurogenesis and low BMI, high pGLP-1, high neurogenesis, insulin resistance, and lipid elevations. Diabesity markers putatively reflect high nutrient levels necessary for neurogenesis at the expense of peripheral tissues.

Elevated cerebrospinal fluid 5-hydroxyindoleacetic acid in macaques following early life stress and inverse association with hippocampal volume: preliminary implications for serotonin-related function in mood and anxiety disorders.

BACKGROUND: Early life stress (ELS) is cited as a risk for mood and anxiety disorders, potentially through altered serotonin neurotransmission. We examined the effects of ELS, utilizing the variable foraging demand (VFD) macaque model, on adolescent monoamine metabolites. We sought to replicate an increase in cerebrospinal fluid (CSF) 5-hydroxyindoleacetic acid (5-HIAA) observed in two previous VFD cohorts. We hypothesized that elevated cisternal 5-HIAA was associated with reduced neurotrophic effects, conceivably due to excessive negative feedback at somatodendritic 5-HT1A autoreceptors. A putatively decreased serotonin neurotransmission would be reflected by reductions in hippocampal volume and white matter (WM) fractional anisotropy (FA). METHODS: When infants were 2-6 months of age, bonnet macaque mothers were exposed to VFD. We employed cisternal CSF taps to measure monoamine metabolites in VFD (N = 22) and non-VFD (N = 14) offspring (mean age = 2.61 years). Metabolites were correlated with hippocampal volume obtained by MRI and WM FA by diffusion tensor imaging in young adulthood in 17 males [10 VFD (mean age = 4.57 years)]. RESULTS: VFD subjects exhibited increased CSF 5-HIAA compared to non-VFD controls. An inverse correlation between right hippocampal volume and 5-HIAA was noted in VFD- but not controls. CSF HVA and MHPG correlated inversely with hippocampal volume only in VFD. CSF 5-HIAA correlated inversely with FA of the WM tracts of the anterior limb of the internal capsule (ALIC) only in VFD. CONCLUSIONS: Elevated cisternal 5-HIAA in VFD may reflect increased dorsal raphe serotonin, potentially inducing excessive autoreceptor activation, inducing a putative serotonin deficit in terminal fields. Resultant reductions in neurotrophic activity are reflected by smaller right hippocampal volume. Convergent evidence of reduced neurotrophic activity in association with high CSF 5-HIAA in VFD was reflected by reduced FA of the ALIC.