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.

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.

CB1 receptor neutral antagonist treatment epigenetically increases neuropeptide Y expression and decreases alcohol drinking.

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.

Epigenetic mechanisms underlying pathobiology of alcohol use disorder.

PURPOSE OF REVIEW: Chronic alcohol use is a worldwide problem with multifaceted consequences including multiplying medical costs and sequelae, societal effects like drunk driving and assault, and lost economic productivity. These large-scale outcomes are driven by the consumption of ethanol, a small permeable molecule that has myriad effects in the human body, particularly in the liver and brain. In this review, we have summarized effects of acute and chronic alcohol consumption on epigenetic mechanisms that may drive pathobiology of Alcohol Use Disorder (AUD) while identifying areas of need for future research. RECENT FINDINGS: Epigenetics has emerged as an interesting field of biology at the intersection of genetics and the environment, and ethanol in particular has been identified as a potent modulator of the epigenome with various effects on DNA methylation, histone modifications, and non-coding RNAs. These changes alter chromatin dynamics and regulate gene expression that contribute to behavioral and physiological changes leading to the development of AUD psychopathology and cancer pathology. SUMMARY: Evidence and discussion presented here from preclinical results and available translational studies have increased our knowledge of the epigenetic effects of alcohol consumption. These studies have identified targets that can be used to develop better therapies to reduce chronic alcohol abuse and mitigate its societal burden and pathophysiology.

A molecular mechanism for choosing alcohol over an alternative reward.

Alcohol addiction leads to increased choice of alcohol over healthy rewards. We established an exclusive choice procedure in which ~15% of outbred rats chose alcohol over a high-value reward. These animals displayed addiction-like traits, including high motivation to obtain alcohol and pursuit of this drug despite adverse consequences. Expression of the γ-aminobutyric acid (GABA) transporter GAT-3 was selectively decreased within the amygdala of alcohol-choosing rats, whereas a knockdown of this transcript reversed choice preference of rats that originally chose a sweet solution over alcohol. GAT-3 expression was selectively decreased in the central amygdala of alcohol-dependent people compared to those who died of unrelated causes. Impaired GABA clearance within the amygdala contributes to alcohol addiction, appears to translate between species, and may offer targets for new pharmacotherapies for treating this disorder.

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

The GABAB Positive Allosteric Modulator ADX71441 Attenuates Alcohol Self-Administration and Relapse to Alcohol Seeking in Rats.

GABAergic signaling is involved in modulating the reinforcing properties of alcohol, and GABAB receptors have been proposed as a potential target for clinical treatment of alcoholism. The orthosteric GABAB receptor agonist baclofen has been shown to suppress operant self-administration of alcohol in animals and alcohol use in alcohol-dependent patients, but its utility is limited by a narrow therapeutic index. We tested the effects of ADX71441, a novel GABAB receptor positive allosteric modulator, on alcohol-related behaviors in rats. We first assessed the effects of ADX71441 (1, 3, 10 and 30 mg/kg, I.P.) on both non-dependent and dependent male Wistar rats trained to self-administer 20% alcohol. We then determined the effects of ADX71441 on stress-induced as well as cue-induced relapse-like behavior. Finally, we sought to identify the brain regions through which ADX71441 may act to prevent relapse-like behavior by mapping the neuronal activation induced by stress-induced reinstatement of alcohol-seeking using c-Fos immunohistochemistry. ADX71441 dose-dependently decreased alcohol self-administration of both dependent and non-dependent animals, but its potency was higher in alcohol-dependent rats. Furthermore, both cue- and stress-induced alcohol seeking were blocked by the GABAB receptor positive allosteric modulator. Finally, pretreatment with 3 mg/kg of ADX71441 before stress-induced reinstatement significantly decreased c-Fos expression in a network of brain regions implicated in stress-induced relapse, comprising the nucleus accumbens shell, the dorsal raphe nucleus and the medial prefrontal cortex. Our findings support a causal role of GABAB receptors in alcohol reinforcement and relapse to alcohol seeking. These effects are observed in the absence of significant sedative side effects. Jointly, these observations indicate that GABAB receptor positive allosteric modulators merit being tested clinically for the treatment of alcoholism. Our data also point to a potential biomarker of target engagement for early clinical studies.

The mGluR2 Positive Allosteric Modulator, AZD8529, and Cue-Induced Relapse to Alcohol Seeking in Rats.

Group II metabotropic glutamate receptors (mGluR2 and mGluR3) may control relapse of alcohol seeking, but previously available Group II agonists were unable to discriminate between mGluR2 and mGluR3. Here we use AZD8529, a novel positive allosteric mGluR2 modulator, to determine the role of this receptor for alcohol-related behaviors in rats. We assessed the effects of AZD8529 (20 and 40 mg/kg s.c.) on male Wistar rats trained to self-administer 20% alcohol and determined the effects of AZD8529 on self-administration, as well as stress-induced and cue-induced reinstatement of alcohol seeking. The on-target nature of findings was evaluated in Indiana P-rats, a line recently shown to carry a mutation that disrupts the gene encoding mGluR2. The behavioral specificity of AZD8529 was assessed using self-administration of 0.2% saccharin and locomotor activity tests. AZD8529 marginally decreased alcohol self-administration at doses that neither affected 0.2% saccharin self-administration nor locomotor activity. More importantly, cue- but not stress-induced alcohol seeking was blocked by the mGluR2 positive allosteric modulator. This effect of AZD8529 was completely absent in P rats lacking functional mGluR2s, demonstrating the receptor specificity of this effect. Our findings provide evidence for a causal role of mGluR2 in cue-induced relapse to alcohol seeking. They contribute support for the notion that positive allosteric modulators of mGluR2 block relapse-like behavior across different drug categories.

Effects of ß-Arrestin-Biased Dopamine D2 Receptor Ligands on Schizophrenia-Like Behavior in Hypoglutamatergic Mice.

Current antipsychotic drugs (APDs) show efficacy with positive symptoms, but are limited in treating negative or cognitive features of schizophrenia. Whereas all currently FDA-approved medications target primarily the dopamine D2 receptor (D2R) to inhibit G(i/o)-mediated adenylyl cyclase, a recent study has shown that many APDs affect not only G(i/o)- but they can also influence ß-arrestin- (ßArr)-mediated signaling. The ability of ligands to differentially affect signaling through these pathways is termed functional selectivity. We have developed ligands that are devoid of D2R-mediated G(i/o) protein signaling, but are simultaneously partial agonists for D2R/ßArr interactions. The purpose of this study was to test the effectiveness of UNC9975 or UNC9994 on schizophrenia-like behaviors in phencyclidine-treated or NR1-knockdown hypoglutamatergic mice. We have found the UNC compounds reduce hyperlocomotion in the open field, restore PPI, improve novel object recognition memory, partially normalize social behavior, decrease conditioned avoidance responding, and elicit a much lower level of catalepsy than haloperidol. These preclinical results suggest that exploitation of functional selectivity may provide unique opportunities to develop drugs with fewer side effects, greater therapeutic selectivity, and enhanced efficacy for treating schizophrenia and related conditions than medications that are currently available.