RESUMEN
Alcohol use and anxiety disorders occur in both males and females, but despite sharing similar presentation and classical symptoms, the prevalence of alcohol use disorder (AUD) is lower in females. While anxiety is a symptom and comorbidity shared by both sexes, the common underlying mechanism that leads to AUD and the subsequent development of anxiety is still understudied. Using a rodent model of adolescent intermittent ethanol (AIE) exposure in both sexes, we investigated the epigenetic mechanism mediated by enhancer of zeste 2 (EZH2), a histone methyltransferase, in regulating both the expression of activity-regulated cytoskeleton-associated protein (Arc) and an anxiety-like phenotype in adulthood. Here, we report that EZH2 protein levels were significantly higher in PKC-δ positive GABAergic neurons in the central nucleus of amygdala (CeA) of adult male and female rats after AIE. Reducing protein and mRNA levels of EZH2 using siRNA infusion in the CeA prevented AIE-induced anxiety-like behavior, increased H3K27me3, decreased H3K27ac at the Arc synaptic activity response element (SARE) site, and restored deficits in Arc mRNA and protein expression in both male and female adult rats. Our data indicate that an EZH2-mediated epigenetic mechanism in the CeA plays an important role in regulating anxiety-like behavior and Arc expression after AIE in both male and female rats in adulthood. This study suggests that EZH2 may serve as a tractable drug target for the treatment of adult psychopathology after adolescent alcohol exposure.
Asunto(s)
Ansiedad , Núcleo Amigdalino Central , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética , Etanol , Animales , Masculino , Femenino , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Núcleo Amigdalino Central/metabolismo , Núcleo Amigdalino Central/efectos de los fármacos , Ratas , Ansiedad/metabolismo , Ansiedad/genética , Etanol/farmacología , Modelos Animales de Enfermedad , Alcoholismo/genética , Alcoholismo/metabolismo , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/efectos de los fármacos , Ratas Sprague-Dawley , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
BACKGROUND: The ventral tegmental area (VTA) is a dopaminergic brain area that is critical in the development and maintenance of addiction. During withdrawal from chronic ethanol exposure, the response of VTA neurons to GABA (gamma-aminobutyric acid) is reduced through an epigenetically regulated mechanism. In the current study, a whole-genome transcriptomic approach was used to investigate the underlying molecular mechanism of GABA hyposensitivity in the VTA during withdrawal after chronic ethanol exposure. METHODS: We performed RNA sequencing of the VTA of Sprague Dawley male rats withdrawn for 24 hours from a chronic ethanol diet as well as sequencing of the VTA of control rats fed the Lieber-DeCarli diet. RNA sequencing data were analyzed using weighted gene coexpression network analysis to identify modules that contained coexpressed genes. Validation was performed with quantitative polymerase chain reaction, gas chromatography-mass spectrometry, and electrophysiological assays. RESULTS: Pathway and network analysis of weighted gene coexpression network analysis module 1 revealed a significant downregulation of genes associated with the cholesterol synthesis pathway. Consistent with this association, VTA cholesterol levels were significantly decreased during withdrawal. Chromatin immunoprecipitation indicated a decrease in levels of acetylated H3K27 at the transcriptional control regions of these genes. Electrophysiological studies in VTA slices demonstrated that GABA hyposensitivity during withdrawal was normalized by addition of exogenous cholesterol. In addition, inhibition of cholesterol synthesis produced GABA hyposensitivity, which was reversed by adding exogenous cholesterol to VTA slices. CONCLUSIONS: These results suggest that decreased expression of cholesterol synthesis genes may regulate GABA hyposensitivity of VTA neurons during alcohol withdrawal. Increasing cholesterol levels in the brain may be a novel avenue for therapeutic intervention to reverse detrimental effects of chronic alcohol exposure.
Asunto(s)
Alcoholismo , Síndrome de Abstinencia a Sustancias , Ratas , Masculino , Animales , Ácido gamma-Aminobutírico/metabolismo , Síndrome de Abstinencia a Sustancias/genética , Síndrome de Abstinencia a Sustancias/metabolismo , Área Tegmental Ventral , Alcoholismo/metabolismo , Ratas Sprague-Dawley , Etanol/farmacologíaRESUMEN
SUN, a multi-targeted tyrosine kinase inhibitor, exerts cardiotoxicity which hinders its clinical use. It is necessary to elucidate molecular mechanism of SUN-induced cardiotoxicity. To elucidate molecular mechanism of SUN-induced cardiotoxicity and whether it is related to Nrf2-dependent ferroptosis, in vitro model with H9c2 cells derived from rat heart tissue and in vivo model (C57BL/6J male mouse) were used in the present study. In vivo model was established by oral treatment of SUN at dose of 10, 20, 40 mg/kg for 14 days. Body weight, ECG, plasma enzyme activities, histology staining were performed to evaluate heart function. Western-blot was performed to analyze the level of ferroptosis-related proteins. In vitro results indicated that SUN markedly induced ferroptosis embodied as collapsed MMP, accumulated iron and elevated ROS. In vivo results showed that SUN significantly impaired cardiac function. Abnormal electrocardiogram, increased serum CK and lactate LDH levels were significantly observed in SUN groups. Histology staining showed that SUN caused structural injuries and fibrosis deposition. Moreover, SUN increased the level of MDA and Fe2+ content, decreased the level of GSH. Both in vitro and in vivo experiments indicated that SUN reduced the expression of Nrf2, HO-1, NQO1, GPX4 and FTH1, enhanced the TfR expression. This study suggested that oxidative stress and Nrf2-dependent ferroptosis played a vital role in SUN-induced cardiotoxicity.
Asunto(s)
Ferroptosis , Factor 2 Relacionado con NF-E2 , Masculino , Ratones , Animales , Ratas , Ratones Endogámicos C57BL , Sunitinib , Cardiotoxicidad , Estrés OxidativoRESUMEN
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.
Asunto(s)
Alcoholismo , Epigénesis Genética , Animales , Ratas , Epigénesis Genética/genética , Etanol/farmacología , Cromatina , Perfilación de la Expresión Génica , Alcoholismo/genética , ARN Mensajero/metabolismo , Factores de Transcripción/genéticaRESUMEN
Background: Alcohol use disorder (AUD) is a complex and chronic relapsing brain disease, which is often co-morbid with psychiatric disorders such as anxiety and depression. AUD phenotypes differ in men and women. Although genetic factors play an important role in its pathophysiology, epidemiologic evidence suggests that during prenatal development, individuals are more vulnerable to the negative effects of environmental factors that may predispose them to AUD later in life. We explored the effects of prenatal stress on the development of AUD phenotypes as well as anxiety- and depression-like behaviors using rat model. Methods: In this study, timed-pregnant Sprague Dawley dams were used. Dams in the control group were left undisturbed throughout gestation, whereas dams in stress groups were either subjected to protracted or acute restraint stress under bright light. At adulthood, the anxiety-like, ethanol drinking, and sucrose drinking behaviors were measured using the Light/Dark Box test and two-bottle free-choice procedure. Results: Compared to the control group, both the male and female offspring in the stress groups exhibited anxiety-like behavior and consumed significantly higher amounts of ethanol in which the acute stress group demonstrated the higher ethanol preference. Moreover, male but not female offspring from the stress groups had decreased sucrose preferences. Conclusion: These findings suggest that protracted and acute prenatal stress in late pregnancy can induce in anxiety-, depressive-like behaviors, and excessive ethanol intake in adult offspring.
RESUMEN
Adolescent binge drinking is a major risk factor for psychiatric disorders later in life including alcohol use disorder. Adolescent alcohol exposure induces epigenetic reprogramming at the enhancer region of the activity-regulated cytoskeleton-associated protein (Arc) immediate-early gene, known as synaptic activity response element (SARE), and decreases Arc expression in the amygdala of both rodents and humans. The causal role of amygdalar epigenomic regulation at Arc SARE in adult anxiety and drinking after adolescent alcohol exposure is unknown. Here, we show that dCas9-P300 increases histone acetylation at the Arc SARE and normalizes deficits in Arc expression, leading to attenuation of adult anxiety and excessive alcohol drinking in a rat model of adolescent alcohol exposure. Conversely, dCas9-KRAB increases repressive histone methylation at the Arc SARE, decreases Arc expression, and produces anxiety and alcohol drinking in control rats. These results demonstrate that epigenomic editing in the amygdala can ameliorate adult psychopathology after adolescent alcohol exposure.
Asunto(s)
Alcoholismo , Epigenómica , Adolescente , Alcoholismo/genética , Animales , Ansiedad/genética , Etanol/efectos adversos , Histonas/metabolismo , Humanos , RatasRESUMEN
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.
Asunto(s)
Depresores del Sistema Nervioso Central/efectos adversos , Ataxia Cerebelosa/inducido químicamente , Cerebelo/efectos de los fármacos , Etanol/efectos adversos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Intoxicación Alcohólica/etiología , Intoxicación Alcohólica/metabolismo , Animales , Depresores del Sistema Nervioso Central/administración & dosificación , Ataxia Cerebelosa/metabolismo , Cerebelo/metabolismo , Epigénesis Genética/efectos de los fármacos , Etanol/administración & dosificación , Código de Histonas/efectos de los fármacos , Masculino , Ratas Sprague-DawleyRESUMEN
Alcohol use disorder (AUD) is highly comorbid with depression. Withdrawal from chronic alcohol drinking results in depression and understanding brain molecular mechanisms that drive withdrawal-related depression is important for finding new drug targets to treat these comorbid conditions. Here, we performed RNA sequencing of the rat hippocampus during withdrawal from chronic alcohol drinking to discover key signaling pathways involved in alcohol withdrawal-related depressive-like behavior. Data were analyzed by weighted gene co-expression network analysis to identify several modules of co-expressed genes that could have a common underlying regulatory mechanism. One of the hub, or highly interconnected, genes in module 1 that increased during alcohol withdrawal was the transcription factor, signal transducer and activator of transcription 3 (Stat3), a known regulator of immune gene expression. Total and phosphorylated (p)STAT3 protein levels were also increased in the hippocampus during withdrawal after chronic alcohol exposure. Further, pSTAT3 binding was enriched at the module 1 genes Gfap, Tnfrsf1a, and Socs3 during alcohol withdrawal. Notably, pSTAT3 and its target genes were elevated in the postmortem hippocampus of human subjects with AUD when compared with control subjects. To determine the behavioral relevance of STAT3 activation during alcohol withdrawal, we treated rats with the STAT3 inhibitor stattic and tested for sucrose preference as a measure of anhedonia. STAT3 inhibition alleviated alcohol withdrawal-induced anhedonia. These results demonstrate activation of STAT3 signaling in the hippocampus during alcohol withdrawal in rats and in human AUD subjects, and suggest that STAT3 could be a therapeutic target for reducing comorbid AUD and depression.
Asunto(s)
Factor de Transcripción STAT3 , Transcriptoma , Anhedonia , Animales , Etanol , Hipocampo/metabolismo , Ratas , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
Alcohol consumption is mediated by several important neuromodulatory systems, including the endocannabinoid and neuropeptide Y (NPY) systems in the limbic brain circuitry. However, molecular mechanisms through which cannabinoid-1 (CB1) receptors regulate alcohol consumption are still unclear. Here, we investigated the role of the CB1 receptor-mediated downstream regulation of NPY via epigenetic mechanisms in the amygdala. Alcohol drinking behavior was measured in adult male C57BL/6J mice treated with a CB1 receptor neutral antagonist AM4113 using a two-bottle choice paradigm while anxiety-like behavior was assessed in the light-dark box (LDB) test. The CB1 receptor-mediated changes in the protein levels of phosphorylated cAMP-responsive element binding protein (pCREB), CREB binding protein (CBP), H3K9ac, H3K14ac and NPY, and the mRNA levels of Creb1, Cbp, and Npy were measured in amygdaloid brain structures. Npy-specific changes in the levels of acetylated histone (H3K9/14ac) and CBP in the amygdala were also measured. We found that the pharmacological blockade of CB1 receptors with AM4113 reduced alcohol consumption and, in an ethanol-naïve cohort, reduced anxiety-like behavior in the LDB test. Treatment with AM4113 also increased the mRNA levels of Creb1 and Cbp in the amygdala as well as the protein levels of pCREB, CBP, H3K9ac and H3K14ac in the central and medial nucleus of amygdala, but not in the basolateral amygdala. Additionally, AM4113 treatment increased occupancy of CBP and H3K9/14ac at the Npy gene promoter, leading to an increase in both mRNA and protein levels of NPY in the amygdala. These novel findings suggest that CB1 receptor-mediated CREB signaling plays an important role in the modulation of NPY function through an epigenetic mechanism and further support the potential use of CB1 receptor neutral antagonists for the treatment of alcohol use disorder.
Asunto(s)
Consumo de Bebidas Alcohólicas/metabolismo , Amígdala del Cerebelo/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Etanol/farmacología , Neuropéptido Y/metabolismo , Receptor Cannabinoide CB1/antagonistas & inhibidores , Amígdala del Cerebelo/metabolismo , Animales , Ansiedad/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Masculino , Ratones , Fosforilación/efectos de los fármacos , Pirazoles/farmacologíaRESUMEN
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.
Asunto(s)
Trastornos del Sistema Nervioso Inducidos por Alcohol/metabolismo , Ataxia Cerebelosa/metabolismo , Etanol/efectos adversos , Óxido Nítrico Sintasa de Tipo I/metabolismo , Trastornos Psicomotores/metabolismo , Trastornos del Sistema Nervioso Inducidos por Alcohol/inducido químicamente , Animales , Arginina/farmacología , Ataxia Cerebelosa/inducido químicamente , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Inhibidores Enzimáticos/farmacología , Lóbulo Frontal/efectos de los fármacos , Lóbulo Frontal/metabolismo , Indazoles/farmacología , Masculino , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Trastornos Psicomotores/inducido químicamente , Ratas Sprague-DawleyRESUMEN
Adolescent binge drinking is a serious public health concern and a risk factor for alcohol use disorder (AUD) and comorbid anxiety in adulthood. Chromatin remodeling mediated by epigenetic enzymes including lysine-specific demethylase 1 (LSD1) due to adolescent alcohol exposure may play a role in adult psychopathology. The mechanism by which adolescent alcohol exposure mechanistically regulates epigenetic reprogramming and behavioral changes in adulthood is unknown. We investigated the role of microRNA-137 (miR-137), which is crucial for normal neurodevelopment and targets LSD1, in adolescent intermittent ethanol (AIE) exposure-induced anxiety-like and alcohol-drinking behaviors and related epigenetic reprogramming in the amygdala in adulthood. Adolescent rats were exposed to 2 g/kg ethanol (2 d on/off; AIE) or adolescent intermittent saline (AIS) during postnatal days (PND)28-PND41 and allowed to grow to adulthood for analysis of behavior, miRNA expression, and epigenetic measures in the amygdala. Interestingly, miR-137 was increased and its target genes Lsd1 and Lsd1 + 8a were decreased in the AIE adult amygdala. Infusion of miR-137 antagomir directly into the central nucleus of the amygdala (CeA) rescues AIE-induced alcohol-drinking and anxiety-like behaviors via normalization of decreased Lsd1 expression, decreased LSD1 occupancy, and decreased Bdnf IV expression due to increased H3K9 dimethylation in AIE adult rats. Further, concomitant Lsd1 small interfering RNA (siRNA) infusion into the CeA prevents the miR-137-mediated reversal of AIE-induced adult anxiety and chromatin remodeling at the Bdnf IV promoter. These novel results highlight miR-137 as a potential therapeutic target for anxiety and AUD susceptibility after adolescent alcohol exposure in adulthood.
Asunto(s)
Consumo de Bebidas Alcohólicas/metabolismo , Amígdala del Cerebelo/metabolismo , Conducta Animal/fisiología , Etanol/administración & dosificación , MicroARNs/metabolismo , Consumo de Bebidas Alcohólicas/genética , Amígdala del Cerebelo/efectos de los fármacos , Animales , Antagomirs/farmacología , Ansiedad/genética , Ansiedad/metabolismo , Conducta Animal/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Epigénesis Genética , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , ARN Interferente Pequeño , RatasRESUMEN
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.
Asunto(s)
Analgésicos/farmacología , Conducta Animal/efectos de los fármacos , Etanol , Inhibidores de Histona Desacetilasas/farmacología , Hiperalgesia/tratamiento farmacológico , Umbral del Dolor/efectos de los fármacos , Síndrome de Abstinencia a Sustancias/tratamiento farmacológico , Vorinostat/farmacología , Animales , Hiperalgesia/inducido químicamente , Hiperalgesia/fisiopatología , Hiperalgesia/psicología , Masculino , Ratas Sprague-Dawley , Síndrome de Abstinencia a Sustancias/etiología , Síndrome de Abstinencia a Sustancias/fisiopatología , Síndrome de Abstinencia a Sustancias/psicologíaRESUMEN
Binge drinking during adolescence increases the risk for neuropsychiatric disorders including alcoholism in adulthood. DNA methylation in post-mitotic neurons is an important epigenetic modification that plays a crucial role in neurodevelopment. We examined the effects of intermittent ethanol exposure during adolescence on adult behavior and whether DNA methylation changes provide a plausible explanation for the lasting effects of this developmental insult. One hour after last adolescent intermittent ethanol (AIE), growth arrest and DNA damage inducible protein 45 (Gadd45a, Gadd45b, and Gadd45g) mRNA expression was increased and DNA methyltransferase (DNMT) activity and Dnmt3b expression was decreased in the amygdala as compared to adolescent intermittent saline (AIS) rats. However, AIE rats 24â¯h after last exposure displayed increased DNMT activity but normalized Gadd45 and Dnmt3b mRNA expression compared to AIS rats. In adulthood, rats exposed to AIE show increased Dnmt3b mRNA expression and DNMT activity, along with decreased Gadd45g mRNA expression in the amygdala. DNA methylation of neuropeptide Y (Npy) and brain-derived neurotrophic factor (Bdnf) exon IV is increased in the AIE adult amygdala compared to AIS adult rats. Treatment with the DNMT inhibitor 5-azacytidine (5-azaC) at adulthood normalizes the AIE-induced DNA hypermethylation of Npy and Bdnf exon IV with concomitant reversal of AIE-induced anxiety-like and alcohol-drinking behaviors. These results suggest that binge-like ethanol exposure during adolescence leads to dysregulation in DNA methylation mechanisms in the amygdala which may contribute to behavioral phenotypes of anxiety and alcohol use in adulthood.
Asunto(s)
Consumo de Bebidas Alcohólicas/fisiopatología , Amígdala del Cerebelo/metabolismo , Ansiedad/fisiopatología , Metilación de ADN/fisiología , Etanol/farmacología , Factores de Edad , Animales , Antígenos de Diferenciación/biosíntesis , Ansiedad/inducido químicamente , Azacitidina/farmacología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteínas de Ciclo Celular/biosíntesis , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasas/biosíntesis , Etanol/antagonistas & inhibidores , Exones/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Masculino , Neuropéptido Y/metabolismo , Ratas , ADN Metiltransferasa 3B , Proteinas GADD45RESUMEN
Withdrawal from chronic alcohol drinking can cause depression, leading to an inability to function in daily life and an increased risk for relapse to harmful drinking. Understanding the causes of alcohol withdrawal-related depression may lead to new therapeutic targets for treatment. Epigenetic factors have recently emerged as important contributors to both depression and alcohol use disorder (AUD). Specifically, acetylation of the N-terminal tails of histone proteins that package DNA into nucleosomes is altered in stress-induced models of depression and during alcohol withdrawal. The goal of this study was to examine depression-like behavior during alcohol withdrawal and associated changes in histone acetylation and expression of histone deacetylase 2 (HDAC2) in the hippocampus, a brain region critical for mood regulation and depression. Male Sprague-Dawley rats were treated with the Lieber-DeCarli ethanol liquid diet for 15 days and then underwent withdrawal. Rats were treated with the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), during withdrawal and were tested for depression-like behavior. In a separate group of rats, the hippocampus was analyzed for mRNA and protein expression of HDAC2 and levels of histone H3 lysine 9 acetylation (H3K9ac) during chronic ethanol exposure and withdrawal. Rats undergoing ethanol withdrawal exhibited depression-like behavior and had increased HDAC2 and decreased H3K9ac levels in specific structures of the hippocampus. Treatment with SAHA during withdrawal ameliorated depression-like behavior and normalized changes in hippocampal HDAC2 and H3K9ac levels. These results demonstrate that ethanol withdrawal causes an altered epigenetic state in the hippocampus. Treatment with an HDAC inhibitor can correct this state and alleviate depression-like symptoms developed during withdrawal. Targeting histone acetylation may be a novel strategy to reduce ethanol withdrawal-induced depression.
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Depresión/tratamiento farmacológico , Hipocampo/metabolismo , Histona Desacetilasa 2/química , Inhibidores de Histona Desacetilasas/uso terapéutico , Síndrome de Abstinencia a Sustancias/tratamiento farmacológico , Vorinostat/uso terapéutico , Acetilación , Animales , Epigénesis Genética , Histonas/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Adolescent intermittent ethanol (AIE) exposure is an emerging risk factor for adult psychopathology, such as anxiety disorders. Enhancer RNAs (eRNAs) are short noncoding RNAs transcribed from enhancer regions that regulate synaptic plasticity-associated gene expression, including Arc, but their role in AIE-induced susceptibility to anxiety in adulthood is unknown. METHODS: Rats were exposed to AIE (ethanol exposure 2 days on/off) or intermittent normal saline during postnatal days 28 to 41 and allowed to grow to adulthood for analysis of behavior and biochemical measures. Some AIE rats and rats with intermittent normal saline exposure were exposed to an acute challenge with ethanol in adulthood. Cohorts of alcohol-naïve adult rats were cannulated in the central nucleus of amygdala and infused with either Kdm6b small interfering RNA or an antisense locked nucleic acid oligonucleotide specific to Arc eRNA before behavioral and biochemical analysis. RESULTS: AIE adult rats displayed heightened anxiety and decreased Arc eRNA expression, which is regulated epigenetically through decreased Kdm6b expression. This triggered condensed chromatin at the synaptic activity response element site and promoter of the Arc gene, facilitating increased negative elongation factor binding to the Arc promoter and decreasing Arc expression in the amygdala. Knockdown of Kdm6b or Arc eRNA expression in the central nucleus of amygdala provoked anxiety in alcohol-naïve adult rats and recapitulated the molecular and epigenetic phenotypes of AIE. CONCLUSIONS: These data suggest that eRNA regulation via epigenetic reprogramming in the amygdala, particularly at the Arc synaptic activity response element site, contributes to adult anxiety after adolescent alcohol exposure.
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Ansiedad/genética , Proteínas del Citoesqueleto/genética , Elementos de Facilitación Genéticos/genética , Proteínas del Tejido Nervioso/genética , ARN Pequeño no Traducido/biosíntesis , Factores de Edad , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Animales , Proteínas del Citoesqueleto/biosíntesis , Epigénesis Genética/efectos de los fármacos , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Masculino , Microinyecciones , Proteínas del Tejido Nervioso/biosíntesis , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacología , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/farmacología , ARN Pequeño no Traducido/genética , RatasRESUMEN
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.
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Amígdala del Cerebelo , Ansiolíticos/farmacología , Ansiedad/tratamiento farmacológico , Tolerancia a Medicamentos , Etanol/farmacología , N-Metiltransferasa de Histona-Lisina , Neuropéptido Y , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Animales , Ansiolíticos/administración & dosificación , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Epigénesis Genética/efectos de los fármacos , Etanol/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/metabolismo , Masculino , Neuropéptido Y/efectos de los fármacos , Neuropéptido Y/metabolismo , Quinazolinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
GABAérgicos/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Síndrome de Abstinencia a Sustancias/tratamiento farmacológico , Área Tegmental Ventral/efectos de los fármacos , Vorinostat/uso terapéutico , Ácido gamma-Aminobutírico/farmacología , Animales , Dieta , Histona Desacetilasa 2/metabolismo , Histonas/metabolismo , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/biosíntesis , Receptores de GABA-A/efectos de los fármacos , Área Tegmental Ventral/metabolismoRESUMEN
Binge alcohol drinking in adolescence leads to increased risk for alcohol use and other psychiatric disorders in adulthood. The transcription factor cAMP-response element binding (CREB) protein is involved in the neuronal response to adult ethanol exposure, but its role in the enduring effects of adolescent alcohol exposure in adulthood is unknown. We exposed male rats to adolescent intermittent ethanol (AIE) or saline (AIS) during post-natal days 28-41 and evaluated the epigenetic regulation of CREB dynamics in the adult amygdala. A subset of these adult rats was exposed to an acute ethanol challenge. AIE decreased CREB, phosphorylated CREB, CREB-binding protein (CBP) and p300 protein levels in adult amygdaloid brain structures. AIE exposure also causes deficits in Creb1, Cbp, and p300 mRNA expression in the amygdala of AIE adult rats which are normalized after acute ethanol exposure. Interestingly, occupancy of acetylated histone H3K9/14 proteins at specific locations in the Creb1, Cbp, and p300 gene promoter regions was decreased in the amygdala of AIE adult rats and was normalized by acute ethanol exposure. These results suggest that AIE exposure epigenetically reduces CREB and other related transcriptional activators in the amygdala in adulthood that may be associated with the behavioral effects of adolescent alcohol exposure.