Conserved role for PCBP1 in altered RNA splicing in the hippocampus after chronic alcohol exposure.

We previously discovered using transcriptomics that rats undergoing withdrawal after chronic ethanol exposure had increased expression of several genes encoding RNA splicing factors in the hippocampus. Here, we examined RNA splicing in the rat hippocampus during withdrawal from chronic ethanol exposure and in postmortem hippocampus of human subjects diagnosed with alcohol use disorder (AUD). We found that expression of the gene encoding the splicing factor, poly r(C) binding protein 1 (PCBP1), was elevated in the hippocampus of rats during withdrawal after chronic ethanol exposure and AUD subjects. We next analyzed the rat RNA-Seq data for differentially expressed (DE) exon junctions. One gene, Hapln2, had increased usage of a novel 3' splice site in exon 4 during withdrawal. This splice site was conserved in human HAPLN2 and was used more frequently in the hippocampus of AUD compared to control subjects. To establish a functional role for PCBP1 in HAPLN2 splicing, we performed RNA immunoprecipitation (RIP) with a PCBP1 antibody in rat and human hippocampus, which showed enriched PCBP1 association near the HAPLN2 exon 4 3' splice site in the hippocampus of rats during ethanol withdrawal and AUD subjects. Our results indicate a conserved role for the splicing factor PCBP1 in aberrant splicing of HAPLN2 after chronic ethanol exposure. As the HAPLN2 gene encodes an extracellular matrix protein involved in nerve conduction velocity, use of this alternative splice site is predicted to result in loss of protein function that could negatively impact hippocampal function in AUD.

Unraveling the epigenomic and transcriptomic interplay during alcohol-induced anxiolysis.

Positive effects of alcohol drinking such as anxiolysis and euphoria appear to be a crucial factor in the initiation and maintenance of alcohol use disorder (AUD). However, the mechanisms that lead from chromatin reorganization to transcriptomic changes after acute ethanol exposure remain unknown. Here, we used Assay for Transposase-Accessible Chromatin followed by high throughput sequencing (ATAC-seq) and RNA-seq to investigate epigenomic and transcriptomic changes that underlie anxiolytic effects of acute ethanol using an animal model. Analysis of ATAC-seq data revealed an overall open or permissive chromatin state that was associated with transcriptomic changes in the amygdala after acute ethanol exposure. We identified a candidate gene, Hif3a (Hypoxia-inducible factor 3, alpha subunit), that had 'open' chromatin regions (ATAC-seq peaks), associated with significantly increased active epigenetic histone acetylation marks and decreased DNA methylation at these regions. The mRNA levels of Hif3a were increased by acute ethanol exposure, but decreased in the amygdala during withdrawal after chronic ethanol exposure. Knockdown of Hif3a expression in the central nucleus of amygdala attenuated acute ethanol-induced increases in Hif3a mRNA levels and blocked anxiolysis in rats. These data indicate that chromatin accessibility and transcriptomic signatures in the amygdala after acute ethanol exposure underlie anxiolysis and possibly prime the chromatin for the development of AUD.

Genome-wide methylation in alcohol use disorder subjects: implications for an epigenetic regulation of the cortico-limbic glucocorticoid receptors (NR3C1).

Environmental factors, including substance abuse and stress, cause long-lasting changes in the regulation of gene expression in the brain via epigenetic mechanisms, such as DNA methylation. We examined genome-wide DNA methylation patterns in the prefrontal cortex (PFC, BA10) of 25 pairs of control and individuals with alcohol use disorder (AUD), using the Infinium® MethylationEPIC BeadChip. We identified 5254 differentially methylated CpGs (pnominal < 0.005). Bioinformatic analyses highlighted biological processes containing genes related to stress adaptation, including the glucocorticoid receptor (encoded by NR3C1). Considering that alcohol is a stressor, we focused our attention on differentially methylated regions of the NR3C1 gene and validated the differential methylation of several genes in the NR3C1 network. Chronic alcohol drinking results in a significant increased methylation of the NR3C1 exon variant 1H, with a particular increase in the levels of 5-hydroxymethylcytosine over 5-methylcytosine. These changes in DNA methylation were associated with reduced NR3C1 mRNA and protein expression levels in PFC, as well as other cortico-limbic regions of AUD subjects when compared with controls. Furthermore, we show that the expression of several stress-responsive genes (e.g., CRF, POMC, and FKBP5) is altered in the PFC of AUD subjects. These stress-response genes were also changed in the hippocampus, a region that is highly susceptible to stress. These data suggest that alcohol-dependent aberrant DNA methylation of NR3C1 and consequent changes in other stress-related genes might be fundamental in the pathophysiology of AUD and lay the groundwork for treatments targeting the epigenetic mechanisms regulating NR3C1 in AUD.

Epigenetic Regulation of GABAergic Neurotransmission and Neurosteroid Biosynthesis in Alcohol Use Disorder.

BACKGROUND: Alcohol use disorder (AUD) is a chronic relapsing brain disorder. GABAA receptor (GABAAR) subunits are a target for the pharmacological effects of alcohol. Neurosteroids play an important role in the fine-tuning of GABAAR function in the brain. Recently, we have shown that AUD is associated with changes in DNA methylation mechanisms. However, the role of DNA methylation in the regulation of neurosteroid biosynthesis and GABAergic neurotransmission in AUD patients remains under-investigated. METHODS: In a cohort of postmortem brains from 20 male controls and AUD patients, we investigated the expression of GABAAR subunits and neurosteroid biosynthetic enzymes and their regulation by DNA methylation mechanisms. Neurosteroid levels were quantified by gas chromatography-mass spectrometry. RESULTS: The α 2 subunit expression was reduced due to increased DNA methylation at the gene promoter region in the cerebellum of AUD patients, a brain area particularly sensitive to the effects of alcohol. Alcohol-induced alteration in GABAAR subunits was also observed in the prefrontal cortex. Neurosteroid biosynthesis was also affected with reduced cerebellar expression of the 18kDa translocator protein and 3α-hydroxysteroid dehydrogenase mRNAs. Notably, increased DNA methylation levels were observed at the promoter region of 3α-hydroxysteroid dehydrogenase. These changes were associated with markedly reduced levels of allopregnanolone and pregnanolone in the cerebellum. CONCLUSION: Given the key role of neurosteroids in modulating the strength of GABAAR-mediated inhibition, our data suggest that alcohol-induced impairments in GABAergic neurotransmission might be profoundly impacted by reduced neurosteroid biosynthesis most likely via DNA hypermethylation.

Persistence of cerebellar ataxia during chronic ethanol exposure is associated with epigenetic up-regulation of Fmr1 gene expression in rat cerebellum.

BACKGROUND: Alcohol intoxication produces ataxia by affecting the cerebellum, which coordinates movements. Fragile X mental retardation (FMR) protein is a complex regulator of RNA and synaptic plasticity implicated in fragile X-associated tremor/ataxia syndrome, which features ataxia and increased Fmr1 mRNA expression resulting from epigenetic dysregulation of FMRP. We recently demonstrated that acute ethanol-induced ataxia is associated with increased cerebellar Fmr1 gene expression via histone modifications in rats, but it is unknown whether similar behavioral and molecular changes occur following chronic ethanol exposure. Here, we investigated the effects of chronic ethanol exposure on ataxia and epigenetically regulated changes in Fmr1 expression in the cerebellum. METHODS: Male adult Sprague-Dawley rats were trained on the accelerating rotarod and then fed with chronic ethanol or a control Lieber-DeCarli diet while undergoing periodic behavioral testing for ataxia during ethanol exposure and withdrawal. Cerebellar tissues were analyzed for expression of the Fmr1 gene and its targets using a real-time quantitative polymerase chain reaction assay. The epigenetic regulation of Fmr1 was also investigated using a chromatin immunoprecipitation assay. RESULTS: Ataxic behavior measured by the accelerating rotarod behavioral test developed during chronic ethanol treatment and persisted at both the 8-h and 24-h withdrawal time points compared to control diet-fed rats. In addition, chronic ethanol treatment resulted in up-regulated expression of Fmr1 mRNA and increased activating epigenetic marks H3K27 acetylation and H3K4 trimethylation at 2 sites within the Fmr1 promoter. Finally, measurement of the expression of relevant FMRP mRNA targets in the cerebellum showed that chronic ethanol up-regulated cAMP response element binding (CREB) Creb1, Psd95, Grm5, and Grin2b mRNA expression without altering Grin2a, Eaa1, or histone acetyltransferases CREB binding protein (Cbp) or p300 mRNA transcripts. CONCLUSIONS: These results suggest that epigenetic regulation of Fmr1 and subsequent FMRP regulation of target mRNA transcripts constitute neuroadaptations in the cerebellum that may underlie the persistence of ataxic behavior during chronic ethanol exposure and withdrawal.

Human Plasma BDNF Is Associated With Amygdala-Prefrontal Cortex Functional Connectivity and Problem Drinking Behaviors.

BACKGROUND: Preclinical studies suggest that decreased levels of brain-derived neurotrophic factor in the amygdala play a role in anxiety and alcohol use disorder. The association between brain-derived neurotrophic factor levels and amygdala function in humans with alcohol use disorder is still unclear, although neuroimaging studies have also implicated the amygdala in alcohol use disorder and suggest that alcohol use disorder is associated with disrupted functional connectivity between the amygdala and prefrontal cortex during aversive states. METHODS: The current study investigated whether plasma brain-derived neurotrophic factor levels in individuals with and without alcohol use disorder (n = 57) were associated with individual differences in amygdala reactivity and amygdala-prefrontal cortex functional connectivity during 2 forms of aversive responding captured via functional magnetic resonance imaging: anxiety elicited by unpredictable threat of shock and fear elicited by predictable threat of shock. We also examined whether brain-derived neurotrophic factor and brain function were associated with binge drinking episodes and alcohol use disorder age of onset. RESULTS: During anxiety, but not fear, lower levels of plasma brain-derived neurotrophic factor were associated with less connectivity between the left amygdala and the medial prefrontal cortex and the inferior frontal gyrus. In addition, within individuals with alcohol use disorder (only), lower levels of brain-derived neurotrophic factor and amygdala-medial prefrontal cortex functional connectivity during anxiety were associated with more binge episodes within the past 60 days and a lower age of alcohol use disorder onset. There were no associations between brain-derived neurotrophic factor levels and focal amygdala task reactivity. CONCLUSIONS: Together, the results indicate that plasma brain-derived neurotrophic factor levels are related to amygdala circuit functioning in humans, particularly during anxiety, and these individual differences may relate to drinking behaviors.

Essential role for neuronal nitric oxide synthase in acute ethanol-induced motor impairment.

The cerebellum is widely known as a motor structure because it regulates and controls motor learning, coordination, and balance. However, it is also critical for non-motor functions such as cognitive processing, sensory discrimination, addictive behaviors and mental disorders. The cerebellum has the highest relative abundance of neuronal nitric oxide synthase (nNos) and is sensitive to ethanol. Although it has been demonstrated that the interaction of γ-aminobutyric acid (GABA) and nitric oxide (NO) might play an important role in the regulation of ethanol-induced cerebellar ataxia, the molecular mechanisms through which ethanol regulates nNos function to elicit this behavioral effect have not been studied extensively. Here, we investigated the dose-dependent effects of acute ethanol treatment on motor impairment using the rotarod behavioral paradigm and the alterations of nNos mRNA expression in cerebellum, frontal cortex (FC), hippocampus and striatum. We also examined the link between acute ethanol-induced motor impairment and nNos by pharmacological manipulation of nNos function. We found that acute ethanol induced a dose-dependent elevation of ethanol blood levels which was associated with the impairment of motor coordination performance and decreased expression of cerebellar nNos. In contrast, acute ethanol increased nNos expression in FC but did not to change the expression for this enzyme in striatum and hippocampus. The effects of acute ethanol were attenuated by l-arginine, a precursor for NO and potentiated by 7-nitroindazole (7-NI), a selective inhibitor of nNos. Our data suggests that differential regulation of nNos mRNA expression in cerebellum and frontal cortex might be involved in acute ethanol-induced motor impairment.

Neuroimmune and epigenetic involvement in adolescent binge ethanol-induced loss of basal forebrain cholinergic neurons: Restoration with voluntary exercise.

Binge drinking and alcohol abuse are common during adolescence and cause lasting pathology. Preclinical rodent studies using the adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2-day on/2-day off from postnatal day [P]25 to P55) model of human adolescent binge drinking report decreased basal forebrain cholinergic (ie, ChAT+) neurons that persist into adulthood (ie, P56-P220). Recent studies link AIE-induced neuroimmune activation to cholinergic pathology, but the underlying molecular mechanisms contributing to the persistent loss of basal forebrain ChAT+ neurons are unknown. We report here that the AIE-induced loss of cholinergic neuron markers (ie, ChAT, TrkA, and p75NTR ), cholinergic neuron shrinkage, and increased expression of the neuroimmune marker pNF-κB p65 are restored by exercise exposure from P56 to P95 after AIE. Our data reveal that persistently reduced expression of cholinergic neuron markers following AIE is because of the loss of the cholinergic neuron phenotype most likely through an epigenetic mechanism involving DNA methylation and histone 3 lysine 9 dimethylation (H3K9me2). Adolescent intermittent ethanol caused a persistent increase in adult H3K9me2 and DNA methylation at promoter regions of Chat and H3K9me2 of Trka, which was restored by wheel running. Exercise also restored the AIE-induced reversal learning deficits on the Morris water maze. Together, these data suggest that AIE-induced adult neuroimmune signaling and cognitive deficits are linked to suppression of Chat and Trka gene expression through epigenetic mechanisms that can be restored by exercise. Exercise restoration of the persistent AIE-induced phenotypic loss of cholinergic neurons via epigenetic modifications is novel mechanism of neuroplasticity.

Essential Role of Histone Methyltransferase G9a in Rapid Tolerance to the Anxiolytic Effects of Ethanol.

BACKGROUND: Tolerance to ethanol-induced anxiolysis promotes alcohol intake, thus contributing to alcohol use disorder development. Recent studies implicate histone deacetylase-mediated histone H3K9 deacetylation in regulating neuropeptide Y expression during rapid ethanol tolerance to the anxiolytic effects of ethanol. Furthermore, the histone methyltransferase, G9a, and G9a-mediated H3K9 dimethylation (H3K9me2) have recently emerged as regulators of addiction and anxiety; however, their role in rapid ethanol tolerance is unknown. Therefore, we investigated the role of G9a-mediated H3K9me2 in neuropeptide Y expression during rapid ethanol tolerance. METHODS: Adult male rats were administered one injection of n-saline followed by single acute ethanol injection (1 g/kg) 24 hours later (ethanol group) or 2 injections (24 hours apart) of either n-saline (saline group) or ethanol (tolerance group). Anxiety-like behaviors and global and Npy-specific G9a and H3K9me2 levels in the amygdala were measured. Effects of G9a inhibitor (UNC0642) treatment on behavioral and epigenetic measures were also examined. RESULTS: Acute ethanol produced anxiolysis and decreased global H3K9me2 and G9a protein levels in the central and medial nucleus of the amygdala as well as decreased occupancy levels of H3K9me2 and G9a near a putative binding site for cAMP-response element binding protein on the Npy gene. Two identical doses of ethanol produced no behavioral or epigenetic changes relative to controls, indicating development of rapid ethanol tolerance. Interestingly, treatment with UNC0642, before the second ethanol dose reversed rapid ethanol tolerance, decreased global H3K9me2 and increased neuropeptide Y levels in the central and medial nucleus of the amygdala. CONCLUSIONS: These results implicate amygdaloid G9a-mediated H3K9me2 mechanisms in regulating rapid tolerance to the anxiolytic effects of ethanol via neuropeptide Y expression regulation.

Effect of Histone Deacetylase Inhibitor on Ethanol Withdrawal-Induced Hyperalgesia in Rats.

BACKGROUND: Increased pain sensitivity is observed following alcohol withdrawal, and attempts to alleviate this hyperalgesia can contribute to the cycle of addiction. The aim of this study was to determine if alcohol withdrawal-induced hyperalgesia was observed in a chronic ethanol exposure model and if this pain was affected by histone deacetylase inhibitors, thus revealing an epigenetic mechanism. METHODS: Adult male Sprague Dawley rats received Lieber-DeCarli liquid control or ethanol (9% v/v) diet for 15 days. Mechanical sensitivity was measured with von Frey hair stimulation of the hindpaw during ethanol administration and 24- and 72-hour withdrawal. RESULTS: Ethanol withdrawal produced severe and sustained mechanical hyperalgesia, an effect not observed in the control or ethanol-maintained groups. Furthermore, this hyperalgesia was attenuated by the histone deacetylase inhibitor, suberoylanilide hydroxamic acid treatment. CONCLUSIONS: Heightened pain sensitivity was observed following withdrawal from chronic ethanol exposure, and histone deacetylase inhibitors could be novel treatments for this alcohol withdrawal-induced hyperalgesia.

The development of a mouse model of mTBI-induced post-traumatic migraine, and identification of the delta opioid receptor as a novel therapeutic target.

BACKGROUND: Post-traumatic headache is the most common and long-lasting impairment observed following mild traumatic brain injury, and frequently has migraine-like characteristics. The mechanisms underlying progression from mild traumatic brain injury to post-traumatic headache are not fully understood. The aim of this study was to develop a mouse model of post-traumatic headache and identify mechanisms and novel targets associated with this disorder. METHODS: We combined the closed head weight-drop method and the nitroglycerin chronic migraine model. To induce mild traumatic brain injury, a weight was dropped onto intact crania of mildly anesthetized mice, and mechanical responses to chronic-intermittent administration of nitroglycerin, a human migraine trigger, were determined at multiple time points post-injury. RESULTS: Low dose nitroglycerin (0.1 mg/kg) evoked acute periorbital and hind paw allodynia in both mild traumatic brain injury and sham animals. However, only mild traumatic brain injury mice developed chronic hypersensitivity to low dose nitroglycerin. Migraine medications, sumatriptan and topiramate, inhibited post-traumatic headache-associated allodynia. In addition, the delta opioid receptor agonist, SNC80, also blocked post-traumatic headache-associated allodynia. Finally, we examined the expression of calcitonin gene-related peptide within this model and found that it was increased in trigeminal ganglia two weeks post-mild traumatic brain injury. CONCLUSIONS: Overall, we have established a mouse model of post-traumatic headache and identified the delta opioid receptor as a novel therapeutic target for this disorder.

Acute Ethanol Produces Ataxia and Induces Fmr1 Expression via Histone Modifications in the Rat Cerebellum.

BACKGROUND: The cerebellum is fundamental for motor coordination and therefore crucial in ethanol (EtOH)-induced ataxia. EtOH contributes to cerebellar pathophysiology. Fragile-X mental retardation protein (FMRP) is a complex regulator of RNA and synaptic plasticity implicated in fragile-X tremor and ataxia syndrome, a phenotype featuring increased Fmr1 mRNA expression. Recent studies have implicated glutamatergic targets of FMRP in hereditary cerebellar ataxias including the main cerebellar excitatory amino acid (Eaa1) transporter and a subtype of metabotropic glutamate receptor (Grm5). However, EtOH-induced changes in cerebellar Fmr1 expression and its epigenetic regulation have not been investigated. Here, we examined the effects of acute EtOH exposure on ataxic behavior, gene expression, and epigenetic regulation of the Fmr1 gene and its glutamatergic targets in the rat cerebellum. METHODS: Male adult Sprague Dawley rats received acute EtOH (2 g/kg) intraperitoneally 1 hour prior to ataxic behavioral testing on the accelerating rotarod and were sacrificed immediately thereafter. Cerebellar tissues were analyzed for gene expression and epigenetic regulation of the Fmr1 gene and its glutamatergic targets in the rat cerebellum using real-time quantitative polymerase chain reaction (PCR) and chromatin immunoprecipitation. RESULTS: Acute EtOH exposure caused marked ataxia on the accelerating rotarod test compared with saline-treated controls. This ataxic response was associated with increases in mRNA levels of Fmr1, postsynaptic density 95 (Psd95), Eaa1, and Grm5 in the cerebellum. In addition, we found increased H3K27 acetylation both at the promoter region of Fmr1 and at a proposed cyclic adenosine monophosphate (cAMP) response-element binding (CREB) site downstream of the Fmr1 transcription start site. Furthermore, acute EtOH exposure significantly increased Creb1 and the histone acetyltransferases (HAT) CREB binding protein (Cbp), and p300 mRNA transcripts. CONCLUSIONS: Overall, EtOH regulates cerebellar Fmr1 expression most likely via HAT-mediated increase in histone acetylation. We propose that FMRP regulation of glutamatergic transcripts plays an important role in disrupting the excitatory-inhibitory balance in the cerebellum underlying EtOH-induced ataxia.

Mechanisms of Persistent Neurobiological Changes Following Adolescent Alcohol Exposure: NADIA Consortium Findings.

The Neurobiology of Adolescent Drinking in Adulthood (NADIA) Consortium has focused on the impact of adolescent binge drinking on brain development, particularly on effects that persist into adulthood. Adolescent binge drinking is common, and while many factors contribute to human brain development and alcohol use during adolescence, animal models are critical for understanding the specific consequences of alcohol exposure during this developmental period and the underlying mechanisms. Using adolescent intermittent ethanol (AIE) exposure models, NADIA investigators identified long-lasting AIE-induced changes in adult behavior that are consistent with observations in humans, such as increased alcohol drinking, increased anxiety (particularly social anxiety), increased impulsivity, reduced behavioral flexibility, impaired memory, disrupted sleep, and altered responses to alcohol. These behavioral changes are associated with multiple molecular, cellular, and physiological alterations in the brain that persist long after AIE exposure. At the molecular level, AIE results in long-lasting changes in neuroimmune/trophic factor balance and epigenetic-microRNA (miRNA) signaling across glia and neurons. At the cellular level, AIE history is associated in adulthood with reduced expression of cholinergic, serotonergic, and dopaminergic neuron markers, attenuated cortical thickness, decreased neurogenesis, and altered dendritic spine and glial morphology. This constellation of molecular and cellular adaptations to AIE likely contributes to observed alterations in neurophysiology, measured by synaptic physiology, EEG patterns, and functional connectivity. Many of these AIE-induced brain changes replicate findings seen in postmortem brains of humans with alcohol use disorder (AUD). NADIA researchers are now elucidating mechanisms of these adaptations. Emerging data demonstrate that exercise, antiinflammatory drugs, anticholinesterases, histone deacetylase inhibitors, and other pharmacological compounds are able to prevent (administered during AIE) and/or reverse (given after AIE) AIE-induced pathology in adulthood. These studies support hypotheses that adolescent binge drinking increases risk of adult hazardous drinking and influences brain development, and may provide insight into novel therapeutic targets for AIE-induced neuropathology and AUDs.

Epigenetic modulation of intestinal Na+/H+ exchanger-3 expression.

Na+/H+ exchanger-3 (NHE3) is crucial for intestinal Na+ absorption, and its reduction has been implicated in infectious and inflammatory bowel diseases (IBD)-associated diarrhea. Epigenetic mechanisms such as DNA methylation are involved in the pathophysiology of IBD. Whether changes in DNA methylation are involved in modulating intestinal NHE3 gene expression is not known. Caco-2 and HuTu 80 cells were used as models of human intestinal epithelial cells. Normal C57/BL6, wild-type, or growth arrest and DNA damage-inducible 45b (GADD45b) knockout (KO) mice were used as in vivo models. NHE3 gene DNA methylation levels were assessed by MBDCap (MethyMiner) assays. Results demonstrated that in vitro methylation of NHE3 promoter construct (p-1509/+127) cloned into a cytosine guanine dinucleotide-free lucia vector decreased the promoter activity in Caco-2 cells. DNA methyltransferase inhibitor 5-azacytidine (10 µM, 24 h) caused a significant decrease in DNA methylation of the NHE3 gene and concomitantly increased NHE3 expression in Caco-2 cells. Similarly, 5-azacytidine treatment increased NHE3 mRNA levels in HuTu 80 cells. 5-Azacytidine treatment for 3 wk (10 mg/kg body wt ip, 3 times/wk) also resulted in an increase in NHE3 expression in the mouse ileum and colon. Small-interfering RNA knockdown of GADD45b (protein involved in DNA demethylation) in Caco-2 cells decreased NHE3 mRNA expression. Furthermore, there was a significant decrease in NHE3 mRNA and protein expression in the ileum and colon of GADD45b KO mice. Our findings demonstrate that NHE3 gene expression is regulated by changes in its DNA methylation. NEW & NOTEWORTHY Our studies for the first time demonstrate that Na+/H+ exchanger-3 gene expression is regulated by an epigenetic mechanism involving DNA methylation.

Transcriptome analysis of alcohol-treated microglia reveals downregulation of beta amyloid phagocytosis.

BACKGROUND: Microglial activation contributes to the neuropathology associated with chronic alcohol exposure and withdrawal, including the expression of inflammatory and anti-inflammatory genes. In the current study, we examined the transcriptome of primary rat microglial cells following incubation with alcohol alone, or alcohol together with a robust inflammatory stimulus. METHODS: Primary microglia were prepared from mixed rat glial cultures. Cells were incubated with 75 mM ethanol alone or with proinflammatory cytokines ("TII": IL1ß, IFNγ, and TNFα). Isolated mRNA was used for RNAseq analysis and qPCR. Effects of alcohol on phagocytosis were determined by uptake of oligomeric amyloid beta. RESULTS: Alcohol induced nitrite production in control cells and increased nitrite production in cells co-treated with TII. RNAseq analysis of microglia exposed for 24 h to alcohol identified 312 differentially expressed mRNAs ("Alc-DEs"), with changes confirmed by qPCR analysis. Gene ontology analysis identified phagosome as one of the highest-ranking KEGG pathways including transcripts regulating phagocytosis. Alcohol also increased several complement-related mRNAs that have roles in phagocytosis, including C1qa, b, and c; C3; and C3aR1. RNAseq analysis identified over 3000 differentially expressed mRNAs in microglia following overnight incubation with TII; and comparison to the group of Alc-DEs revealed 87 mRNAs modulated by alcohol but not by TII, including C1qa, b, and c. Consistent with observed changes in phagocytosis-related mRNAs, the uptake of amyloid beta1-42, by primary microglia, was reduced by alcohol. CONCLUSIONS: Our results define alterations that occur to microglial gene expression following alcohol exposure and suggest that alcohol effects on phagocytosis could contribute to the development of Alzheimer's disease.

Histone Deacetylase Inhibitor Suberanilohydroxamic Acid Treatment Reverses Hyposensitivity to γ-Aminobutyric Acid in the Ventral Tegmental Area During Ethanol Withdrawal.

BACKGROUND: The ventral tegmental area (VTA) is important for alcohol-related reward and reinforcement. Mouse VTA neurons are hyposensitive to γ-aminobutyric acid (GABA) during ethanol (EtOH) withdrawal, and GABA responsiveness is normalized by in vitro treatment with histone deacetylase inhibitors (HDACi). The present study examined the effect of a systemically administered HDACi, suberanilohydroxamic acid (SAHA) on GABA sensitivity, and related molecular changes in VTA neurons during withdrawal after chronic EtOH intake in rats. METHODS: Sprague Dawley male adult rats were fed with Lieber-DeCarli diet (9% EtOH or control diet) for 16 days. Experimental groups included control diet-fed and EtOH diet-fed (0- or 24-hour withdrawal) rats treated with either SAHA or vehicle injection. Single-unit recordings were used to measure the response of VTA neurons to GABA. Immunohistochemistry was performed to examine levels of HDAC2, acetylated histone H3 lysine 9 (acH3K9), and GABAA receptor α1 and α5 subunits in the VTA; quantitative polymerase chain reaction was performed to examine the mRNA levels of HDAC2 and GABAA receptor subunits. RESULTS: VTA neurons from the withdrawal group exhibited GABA hyposensitivity. In vivo SAHA treatment 2 hours before sacrifice normalized the sensitivity of VTA neurons to GABA. EtOH withdrawal was associated with increased HDAC2 and decreased acH3K9 protein levels; SAHA treatment normalized acH3K9 levels. Interestingly, no significant change was observed in the mRNA levels of HDAC2. The mRNA levels, but not protein levels, of GABAA receptor α1 and α5 subunits were increased during withdrawal. CONCLUSIONS: Withdrawal from chronic EtOH exposure results in a decrease in GABA-mediated inhibition, and this GABA hyposensitivity is normalized by in vivo SAHA treatment. Disruption of signaling in the VTA produced by alteration of GABA neurotransmission could be 1 neuroadaptive physiological process leading to craving and relapse. These results suggest that HDACi pharmacotherapy with agents like SAHA might be an effective treatment for alcoholism.

Donepezil Reverses Dendritic Spine Morphology Adaptations and Fmr1 Epigenetic Modifications in Hippocampus of Adult Rats After Adolescent Alcohol Exposure.

BACKGROUND: Adolescent intermittent ethanol (AIE) exposure produces persistent impairments in cholinergic and epigenetic signaling and alters markers of synapses in the hippocampal formation, effects that are thought to drive hippocampal dysfunction in adult rodents. Donepezil (Aricept), a cholinesterase inhibitor, is used clinically to ameliorate memory-related cognitive deficits. Given that donepezil also prevents morphological impairment in preclinical models of neuropsychiatric disorders, we investigated the ability of donepezil to reverse morphological and epigenetic adaptations in the hippocampus of adult rats exposed to AIE. Because of the known relationship between dendritic spine density and morphology with the fragile X mental retardation 1 (Fmr1) gene, we also assessed Fmr1 expression and its epigenetic regulation in hippocampus after AIE and donepezil pretreatment. METHODS: Adolescent rats were administered intermittent ethanol for 16 days starting on postnatal day 30. Rats were treated with donepezil (2.5 mg/kg) once a day for 4 days starting 20 days after the completion of AIE exposure. Brains were dissected out after the fourth donepezil dose, and spine analysis was completed in dentate gyrus granule neurons. A separate cohort of rats, treated identically, was used for molecular studies. RESULTS: AIE exposure significantly reduced dendritic spine density and altered morphological characteristics of subclasses of dendritic spines. AIE exposure also increased mRNA levels and H3-K27 acetylation occupancy of the Fmr1 gene in hippocampus. Treatment of AIE-exposed adult rats with donepezil reversed both the dendritic spine adaptations and epigenetic modifications and expression of Fmr1. CONCLUSIONS: These findings indicate that AIE produces long-lasting decreases in dendritic spine density and changes in Fmr1 gene expression in the hippocampal formation, suggesting morphological and epigenetic mechanisms underlying previously reported behavioral deficits after AIE. The reversal of these effects by subchronic, post-AIE donepezil treatment indicates that these AIE effects can be reversed by up-regulating cholinergic function.

Adolescent Alcohol Exposure-Induced Changes in Alpha-Melanocyte Stimulating Hormone and Neuropeptide Y Pathways via Histone Acetylation in the Brain During Adulthood.

Background: Adolescent intermittent ethanol exposure causes long-lasting alterations in brain epigenetic mechanisms. Melanocortin and neuropeptide Y signaling interact and are affected by ethanol exposure in the brain. Here, the persistent effects of adolescent intermittent ethanol on alpha-melanocyte stimulating hormone, melanocortin 4 receptor, and neuropeptide Y expression and their regulation by histone acetylation mechanisms were investigated in adulthood. Methods: Male rats were exposed to adolescent intermittent ethanol (2 g/kg, i.p.) or volume-matched adolescent intermittent saline from postnatal days 28 to 41 and allowed to grow to postnatal day 92. Anxiety-like behaviors were measured by the elevated plus-maze test. Brain regions from adult rats were used to examine changes in alpha-melanocyte stimulating hormone, melanocortin 4 receptor, and neuropeptide Y expression and the histone acetylation status of their promoters. Results: Adolescent intermittent ethanol-exposed adult rats displayed anxiety-like behaviors and showed increased pro-opiomelanocortin mRNA levels in the hypothalamus and increased melanocortin 4 receptor mRNA levels in both the amygdala and hypothalamus compared with adolescent intermittent saline-exposed adult rats. The alpha-Melanocyte stimulating hormone and melanocortin 4 receptor protein levels were increased in the central and medial nucleus of the amygdala, paraventricular nucleus, and arcuate nucleus of the hypothalamus in adolescent intermittent ethanol-exposed compared with adolescent intermittent saline-exposed adult rats. Neuropeptide Y protein levels were decreased in the central and medial nucleus of the amygdala of adolescent intermittent ethanol-exposed compared with adolescent intermittent saline-exposed adult rats. Histone H3K9/14 acetylation was decreased in the neuropeptide Y promoter in the amygdala but increased in the melanocortin 4 receptor gene promoter in the amygdala and the melanocortin 4 receptor and pro-opiomelanocortin promoters in the hypothalamus of adolescent intermittent ethanol-exposed adult rats compared with controls. Conclusions: Increased melanocortin and decreased neuropeptide Y activity due to changes in histone acetylation in emotional brain circuitry may play a role in adolescent intermittent ethanol-induced anxiety phenotypes in adulthood.

Emerging Role of One-Carbon Metabolism and DNA Methylation Enrichment on δ-Containing GABAA Receptor Expression in the Cerebellum of Subjects with Alcohol Use Disorders (AUD).

Background: Cerebellum is an area of the brain particularly sensitive to the effects of acute and chronic alcohol consumption. Alcohol exposure decreases cerebellar Purkinje cell output by increasing GABA release from Golgi cells onto extrasynaptic α6/δ-containing GABAA receptors located on glutamatergic granule cells. Here, we studied whether chronic alcohol consumption induces changes in GABAA receptor subunit expression and whether these changes are associated with alterations in epigenetic mechanisms via DNA methylation. Methods: We used a cohort of postmortem cerebellum from control and chronic alcoholics, here defined as alcohol use disorders subjects (n=25/group). S-adenosyl-methionine/S-adenosyl-homocysteine were measured by high-performance liquid chromatography. mRNA levels of various genes were assessed by reverse transcriptase-quantitative polymerase chain reaction. Promoter methylation enrichment was assessed using methylated DNA immunoprecipitation and hydroxy-methylated DNA immunoprecipitation assays. Results: mRNAs encoding key enzymes of 1-carbon metabolism that determine the S-adenosyl-methionine/S-adenosyl-homocysteine ratio were increased, indicating higher "methylation index" in alcohol use disorder subjects. We found that increased methylation of the promoter of the δ subunit GABAA receptor was associated with reduced mRNA and protein levels in the cerebellum of alcohol use disorder subjects. No changes were observed in α1- or α6-containing GABAA receptor subunits. The expression of DNA-methyltransferases (1, 3A, and 3B) was unaltered, whereas the mRNA level of TET1, which participates in the DNA demethylation pathway, was decreased. Hence, increased methylation of the δ subunit GABAA receptor promoter may result from alcohol-induced reduction of DNA demethylation. Conclusion: Together, these results support the hypothesis that aberrant DNA methylation pathways may be involved in cerebellar pathophysiology of alcoholism. Furthermore, this work provides novel evidence for a central role of DNA methylation mechanisms in the alcohol-induced neuroadaptive changes of human cerebellar GABAA receptor function.

Emerging Role of Epigenetic Mechanisms in Alcohol Addiction.

Alcohol use disorder (AUD) is a complex brain disorder with an array of persistent behavioral and neurochemical manifestations. Both genetic and environmental factors are known to contribute to the development of AUD, and recent studies on alcohol exposure and subsequent changes in gene expression suggest the importance of epigenetic mechanisms. In particular, histone modifications and DNA methylation have emerged as important regulators of gene expression and associated phenotypes of AUD. Given the therapeutic potential of epigenetic targets, this review aims to summarize the role of epigenetic regulation in our current understanding of AUD by evaluating known epigenetic signatures of brain regions critical to addictive behaviors in both animal and human studies throughout various stages of AUD. More specifically, the effects of acute and chronic alcohol exposure, tolerance, and postexposure withdrawal on epigenetically induced changes to gene expression and synaptic plasticity within key brain regions and the associated behavioral phenotypes have been discussed. Understanding the contribution of epigenetic regulation to crucial signaling pathways may prove vital for future development of novel biomarkers and treatment agents in ameliorating or preventing AUD.