Discovery and validation of novel targets for pharmacotherapy of alcoholism

编码mu-阿片受体的人类OPRM1基因中的非同义SNP A118G可能调节酒精奖励和对纳曲酮（NTX）的治疗反应。为了确定人类OPRM1 A118G变异的机制作用，并从连锁不平衡的等位基因中分离出这种多态性的影响，我们创建了两个分别携带人类等位基因（118AA和118GG）的人源化小鼠系。我们观察到酒精对118GG小鼠伏隔核（NAc）多巴胺（DA）释放的显著增强。这一发现与在人类中平行进行的正电子发射断层扫描研究一致（Ramchandani et al., 2011）。118GG小鼠系通过酒精诱导的颅内脑刺激（ICSS）阈值降低显示酒精奖励增强。ICSS阈值抑制被NTX选择性阻断（与Robinson和Malanga合作，UNC）。因此，与118AA系相比，118GG小鼠的自愿酒精摄入量增加，并且118GG小鼠对NTX优先敏感（Thorsell et al., in prep）。

A non-synonymous SNP, A118G in the human OPRM1 gene that encodes the mu-opioid receptor may modulate alcohol reward and therapeutic response to naltrexone (NTX). In an attempt to establish the mechanistic role of human OPRM1 A118G variation, and to isolate the effects of this polymorphism from those of alleles in linkage disequilibrium, we created two lines of humanized mice carrying the respective human allele (118AA and 118GG). We observed a markedly enhanced dopamine (DA) release in the nucleus accumbens (NAc) of 118GG mice in response to alcohol. This finding was consistent with a positron emission tomography study conducted in parallel in humans (Ramchandani et al., 2011). The 118GG mouse line revealed an enhanced alcohol reward as measured by alcohol-induced decrease in intracranial brain stimulation (ICSS) threshold. The ICSS threshold suppression is selectively blocked by NTX (collaboration with Robinson and Malanga, UNC). Accordingly, voluntary alcohol consumption in 118GG mice is increased compared to the 118AA line, and the 118GG mice are preferentially sensitive to NTX (Thorsell et al., in prep). Pharmacological blockade of neurokinin 1 receptors (NK1R) using the NK1R antagonist L-703,606 suppresses alcohol consumption (Thorsell et al., 2010). L822429, a rat-specific NK1 antagonist dose-dependently suppressed stress-induced reinstatement of alcohol seeking in Wistar rats, but had no effect on cue-induced reinstatement (Schank et al., 2011). In follow-up work using genetically selected alcohol preferring P-rats, L-822429 dose-dependently suppressed self-administration in P-rats but was ineffective in non-dependent Wistar rats. We found an up regulation of NK1R expression in the central amygdata (CeA) of P-rats; microinjections of the antagonist into this region mimicked systemic effects (Schank et al., 2013). Fos-mapping showed that stress-induced reinstatement of alcohol seeking is associated with neuronal activation in the amygdala (amg), NAc, dorsal raphe nucleus (DR), medial prefrontal cortex (mPFC) and bed nucleus of the stria terminalis. NK1R antagonism suppressed the stress-induced increase in Fos in a subset of these regions, particularly the DR and NAc shell, suggesting that these regions mediate the effect of NK1R antagonism to block stress-induced relapse. Neuropeptide S (NPS) suppresses anxiety and appetite in experimental animals, and ourinitial work in collaboration with the Ciccocioppo laboratory indicated that NPS signaling may play a role in relapse to alcohol seeking (Canella et al., 2009). We then developed a screenable assay for the NPS receptor, and in collaboration with the NIH Chemical Genomics Center (NCGC) identified a lead molecule, NCG001865684, that was determined to be brain penetrant upon peripheral administration in rats. In vitro, NCGC00185684 shows biased antagonist properties, and preferentially blocks ERK-phosphorylation over intracellular cAMP- or calcium-responses to NPS. In vivo, pretreatment with NCG001865684 was found to block alcohol-induced ERK-phosphorylation in the rat CeA, a region involved in regulation of alcohol intake. NCGC00185684 also decreases operant alcohol self-administration, and lowers alcohol reward as measured using progressive ratio responding. These effects are behaviorally specific, in that they are observed at doses that do not influence locomotor activity or reinstatement responding following extinction. Taken together, these data provide an initial validation of the NPSR as a therapeutic target in alcoholism (Thorsell et al., 2013). Melanin Concentrating Hormone (MCH) is a hypothalamic appetite regulating peptide that is also involved in reward, motivation and anxiety-like behaviors mediated by the MCH-1 receptor (MCH-1R) in rodents. MCH1-R blockade suppresses alcohol self-administration (Cippitelli et al., 2010). Because alcohol is also a caloric nutrient, self-administration results could be confounded by appetite effects. We have therefore directly evaluated the role of the MCH system for the rewarding properties of alcohol using conditioned place preference (CPP) in MCH-1R knockout (KO) mice. MCH-1R KO mice did not develop alcohol preference compared to WT controls at a 2g/kg dose. This effect was behaviorally specific, as no differences were observed on locomotion, loss of righting reflex (LORR), or alcohol metabolism and elimination (Karlsson et al., in prep). Proinflammatory activity might be involved in stress responses and behaviors related to addiction. We began investigating the role of the IL-1 receptor (IL-1R) in alcohol related behaviors. Mice lacking IL-1Rs consumed less alcohol in a two bottle choice test, but CPP experiments showed that the decreased alcohol consumption in the IL-1R mice is not due to effects on alcohol reward. IL1-R KO mice had significantly longer sleep time when evaluated for LORR, indicating higher sensitivity to sedative / ataxic properties of alcohol than controls. This is in agreement with a frequently observed inverse correlation between sensitivity to sedative / ataxic effects of alcohol and consumption. In a model of stress-induced drinking, social defeat stress (SDS), both genotypes potently increased their consumption acutely, but no genotype differences were seen. Because IL-1 beta and TNF-a share overlapping pathways, it has been suggested that they can act in parallel, and can substitute for each other. We next therefore used a double KO mouse model lacking both IL-1 and TNF-1 receptors. The double KOs consumed less alcohol, and did not escalate their consumption with increasing alcohol concentrations. Preliminary data also suggest that the double-KO mice are less sensitive to stress induced drinking after SDS exposure (Karlsson et al., in prep). We found that intracerebroventricular infusion of RG-108, a specific DNA methyltransferase inhibitor, decreased operant alcohol self-administration in post-dependent rats, and reversed long-term expression changes induced by a history of dependence. The effects of RG-108 were reproduced with local infusion into the mPFC over 7-10 days and were behaviorally selective. Since inhibition of DNA methylation was able to reverse escalation of alcohol self-administration, we asked whether inducing increased central DNA methylation would be sufficient to reproduce escalated self-administration in rats without a history of dependence. Subchronic (10 days) systemic treatment with the methyl group donor L- methionine significantly increased DNA methylation in the PFC. This treatment, as well as direct delivery of L-methionine into the mPFC delivery increased operant alcohol self-administration as well as increased motivation to consume alcohol. Once again, the effects of L-methionine on alcohol-related behavior were specific (Barbier et al., in prep). Following up on a recent microarray screen (Tapocik et al., 2012), we qPCR confirmed that alcohol dependence results in persistent up-regulation of miR-206 in the mPFC. Overexpression of miR-206 in the mPFC of non-dependent rats reproduced the escalation of alcohol self-administration. Accordingly, BDNF was downregulated in PD rats on microarray analysis, and this was confirmed by qPCR. In vitro, BDNF expression was repressed by miR-206 in a 3'UTR reporter assay, confirming BDNF as a functional target of miR-206. Inhibition of miR-206 expression in differentiated rat cortical primary neurons significantly increased secreted levels of BDNF. Therefore, recruitment of miR-206 in the mPFC contributes to escalated alcohol consumption and BDNF regulation (Tapocik et al, in prep).