Discovery and validation of novel targets for pharmacotherapy of alcoholism

酒精中毒药物治疗新靶点的发现和验证

• 批准号: 8941398
• 负责人: Markus Heilig
• 金额: $ 512.47万
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8941398
• 关键词: 3' Untranslated Regions; Agonist; Agreement; Alcohol consumption; Alcohol dependence; Alcoholism; Alcohols; Alleles; Amygdaloid structure; Animal Model; Animals; Anxiety; Attenuated; Automobile Driving; Behavior; Behavioral; Biological; Biological Assay; Brain; Brain-Derived Neurotrophic Factor; Breeding; C57BL/6 Mouse; Calcium; Chemicals; Clinical Trials; Cocaine; Collaborations; Consumption; Cues; Cyclic AMP; DNA Methylation; DNA Methyltransferase Inhibitor; Data; Dependence; Desire for food; Development; Dopamine; Dose; Down-Regulation; Emotional; Epigenetic Process; Ethanol Metabolism; Exposure to; Functional disorder; Genes; Genetic; Genetic Engineering; Genomics; Genotype; Goals; Heavy Drinking; Human; Hypermethylation; Hypothalamic structure; In Vitro; Infusion procedures; Intake; Interleukin-1; Intervention; Knock-out; Knockout Mice; Lead; Locomotion; Maps; Measures; Medial; Mediating; MicroRNAs; Microarray Analysis; Microinjections; Modeling; Molecular; Motivation; Mus; Naltrexone; Narcotic Antagonists; Natural Immunity; Neurons; Neuropeptide Receptor; Neuropeptides; Nucleus Accumbens; Nutrient; Pathway interactions; Peptides; Peripheral; Pharmaceutical Preparations; Phenotype; Phosphorylation; Play; Positron-Emission Tomography; Prefrontal Cortex; Property; Proteins; Rattus; Recording of previous events; Reflex action; Regulation; Relapse; Reporter; Resistance; Rewards; Rodent; Role; Self Administration; Signal Transduction; Sleep; Stimulus; Stress; Structure of terminal stria nuclei of preoptic region; Synapses; System; TACR1 gene; TNF gene; Target Populations; Taste Perception; Testing; Therapeutic; Time; Time Study; Transcript; Transgenic Organisms; Tumor Necrosis Factor-Beta; United States National Institutes of Health; Up-Regulation; Validation; Wistar Rats; Work; Yohimbine; addiction; alcohol behavior; alcohol exposure; alcohol relapse; alcohol response; alcohol reward; alcohol seeking behavior; alcoholism pharmacotherapy; alcoholism therapy; biological adaptation to stress; dorsal raphe nucleus; drinking; drug candidate; follow-up; in vivo; interest; melanin-concentrating hormone; mouse model; mu opioid receptors; neurotransmitter release; novel; overexpression; pre-clinical; preference; prevent; receptor; research study; response; sedative; social; synaptotagmin II; therapeutic target; vapor

Mu-opioid receptors: A non-synonymous A118G SNP in the human OPRM1 gene that encodes the mu-opioid receptor may modulate alcohol reward and therapeutic response to naltrexone (NTX). We previously created two lines of humanized mice carrying the respective human allele (118AA and 118GG), and 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). More recent work revealed that the enhanced alcohol reward in the 118GG mouse as measured by alcohol-induced decrease in intracranial brain stimulation (ICSS) threshold. The ICSS threshold suppression is selectively blocked by the opioid antagonist naltrexone, and approved alcoholism medication (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 naltrexone (Bilbao et al., Biol Psychiat, in press). NK1 receptors: We previously found that 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,, L-822429 dose-dependently suppressed self-administration in alcohol preferring 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. Furthermore, we identified that NK-1 antagonism attenuates yohimbine-induced reinstatement of alcohol seeking and cocaine seeking (Shank et al., 2014). Neuropeptide S (NPS) receptors: NPS signaling may play a role in relapse to alcohol seeking (Canella et al., 2009). In collaboration with the NIH Chemical Genomics Center (NCGC), we identified a lead molecule, NCG001865684, that is 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 blocks 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. 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): 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 evaluated the role of the MCH system for the rewarding properties of alcohol using conditioned place preference (CPP) in MCH-1R knockout (KO) mice. In contrast to wildtype C57BL/6 mice, MCH-1R KO:s did not develop place preference for 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: Innate immunity 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. This is not likely to be due to differences in the rewarding properties of alcohol. Double KO mice show a robust expression of alcohol induced CPP. Further Double KO mice were assessed on LORR, alcohol metabolism and taste preference. No differences were observed suggesting behaviorally specific effects. Interestingly, double-KO mice are less sensitive to stress induced drinking after SDS exposure and did not increase their consumption from baseline intake. (Karlsson et al., in prep). DNA methylation: Substantial evidence supports a role of epigenetic mechanisms in the regulation of alcohol-related behaviors. We found that i.c.v. infusion of RG-108, a specific DNA methyltransferase inhibitor, decreased operant alcohol self-administration in post-dependent rats, and reversed long-term expression changes encoding synaptic proteins involved in neurotransmitter release in the mPFC 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. Infusion of RG108 prevented both escalation of alcohol consumption and dependence-induced down-regulation of 4 out of the 7 transcripts modified in PD rats. Specifically, RG108 treatment directly reversed the down-regulation of synaptotagmin 2 (syt2), which was caused by alcohol-induced hypermethylation. Lentiviral inhibition of Syt2 expression in the mPFC increased aversion-resistant alcohol drinking, supporting a mechanistic role of Syt2 in alcohol seeking. (Barbier et al., in submission). microRNA regulation: 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, 2014). We have recently confirmed this finding in a mouse model of alcohol dependence where BDNF expression is down and mir-206 expression is upregulated. Furthermore, miR-206 KO mice have a blunted ethanol response to alcohol vapor (Tapocik et al.,in prep).

MU-Apoid受体：编码Mu-Apioid受体的人OPRM1基因中的非同义A118G SNP可能会调节对纳曲酮（NTX）的酒精奖励和治疗反应。我们以前创建了两条携带各个人类等位基因（118AA和118GG）的人源化小鼠，并观察到118GG小鼠的伏隔核（NAC）在对酒精响应的118GG小鼠中明显增强的多巴胺（DA）释放。这一发现与人类并行进行的正电子发射断层扫描研究一致（Ramchandani等，2011）。最近的工作表明，通过酒精引起的颅内脑刺激（ICS）阈值减少的118GG小鼠的酒精奖励增强。 ICSS阈值抑制被阿片类拮抗剂Naltrexone选择性阻止，并批准了酒精中毒药物（与Robinson和Malanga，UNC的合作）。因此，与118AA线相比，118GG小鼠的自愿性饮酒量增加了，而118GG小鼠优先对纳曲酮敏感（Bilbao等人，Biol Psychiat，印刷中）。 NK1受体：我们先前发现L822429是一种大鼠特异性的NK1拮抗剂剂量依赖性地抑制了wistar大鼠中寻求酒精的应激诱导的恢复，但对提示诱导的恢复没有影响（Schank等，2011）。在后续工作中，L-822429剂量依赖性地抑制了酒精中的自我给药，但在非依赖性Wistar大鼠中无效。我们发现在p鼠的中央杏仁核（CEA）中，NK1R表达的调节。拮抗剂对该区域的微分注射模仿了全身效应（Schank等，2013）。 FOS映射表明，应力诱导的酒精寻求恢复与杏仁核（AMG），NAC，NAC，背侧Raphe核（DR），内侧前额叶皮层（MPFC）和Stria端的床核的神经元激活有关。 NK1R拮抗作用抑制了这些区域（尤其是DR和NAC壳）中FOS的应力诱导的增加，这表明这些区域介导了NK1R拮抗作用，以阻止应力诱导的复发。此外，我们确定NK-1拮抗作用减弱了Yohimbine引起的恢复饮酒和可卡因寻求的恢复（Shank等，2014）。 神经肽S（NP）受体：NPS信号传导可能在复发中发挥作用（Canella等，2009）。与NIH化学基因组学中心（NCGC）合作，我们确定了铅分子NCG001865684，即大脑对大鼠外周施用的脑渗透。在体外，NCGC00185684显示出有偏见的拮抗剂特性，优先将ERK-磷酸化在NPS上的细胞内cAMP或钙回答上。在体内，使用NCG001865684预处理可阻止酒精诱导的大鼠CEA中的ERK-磷酸化，该区域参与了酒精摄入的调节。 NCGC00185684还减少了饮酒的自我给药，并降低了使用进行性比率响应来衡量的酒精奖励。这些效果在行为上是特定的。综上所述，这些数据提供了NPSR作为酒精中毒的治疗靶标的初始验证（Thorsell等，2013）。 黑色素浓缩激素（MCH）：MCH是调节肽的下丘脑食欲，还参与了啮齿动物中MCH-1受体（MCH-1R）介导的奖励，动机和焦虑行为。 MCH1-R封锁抑制了酒精自我给药（Cippitelli等，2010）。由于酒精也是一种热量营养素，因此食欲效应可能会混淆自我管理结果。因此，我们已经评估了MCH系统在MCH-1R基因敲除（KO）小鼠中使用条件位置偏好（CPP）奖励酒精特性的作用。与WildType C57BL/6小鼠相反，MCH-1R KO：S并未以2g/kg剂量形成偏好。这种效果在行为上是特定的，因为没有观察到对运动的差异，矫正反射（LORR）或酒精代谢和消除的差异（Karlsson等人，在PREP中）。 促进活动：与成瘾有关的压力反应和行为可能涉及先天免疫。我们开始研究IL-1受体（IL-1R）在酒精相关行为中的作用。在两次瓶装测试中，缺乏IL-1R的小鼠消耗的酒精少量，但是CPP实验表明，IL-1R小鼠的酒精消耗量降低并不是由于对酒精奖励的影响。评估LORR时，IL1-R KO小鼠的睡眠时间明显更长，表明对酒精的镇静 /共同特性的敏感性高于对照。这与对酒精和消费的镇静 /共同作用的敏感性之间经常观察到的相关性一致。在压力引起的饮酒模型中，社会失败压力（SDS），两种基因型都有效地增加了消耗量，但没有看到基因型差异。因为IL-1β和TNF-A共享重叠的途径，因此有人建议它们可以并行起作用，并且可以互相代替。因此，我们接下来使用了缺乏IL-1和TNF-1受体的双KO小鼠模型。双Kos消耗的酒精含量较少，并且没有随着酒精浓度的增加而升级其消费量。这不太可能是由于酒精的奖励性质差异。双KO小鼠表现出强大的酒精诱导CPP表达。对LORR，酒精代谢和味道偏好进行了进一步的双重KO小鼠。没有观察到差异提示在行为方面的效果。 有趣的是，双KO小鼠对SDS暴露后的压力引起的饮酒不太敏感，并且不会增加基线摄入量的消耗。 （Karlsson等人，在准备中）。 DNA甲基化：大量证据支持表观遗传机制在与酒精相关行为的调节中的作用。我们发现I.C.V.输注RG-108是一种特定的DNA甲基转移酶抑制剂，降低了依赖后大鼠中的作战醇自我给药，并逆转了长期表达变化，编码了由依赖病史引起的MPFC中涉及神经递质释放的突触蛋白的长期表达变化。在7-10天内，在局部输注MPFC的情况下，RG-108的作用在行为上选择性。输注RG108可以防止饮酒的升级和依赖性引起的在PD大鼠中修饰的7个转录本中的4个下调。具体而言，RG108处理直接逆转了由酒精诱导的高甲基化引起的突触毒素2（SYT2）的下调。 MPFC中SYT2表达的慢病毒抑制增加了抗厌食的饮酒，从而支持SYT2在寻求酒精中的机械作用。 （Barbier等人，在提交中）。 MicroRNA调节：在最近的微阵列屏幕上进行后续（Tapocik等，2012），我们qpcr证实酒精依赖性导致MPFC中miR-206的持续上调。 miR-206在非依赖大鼠的MPFC中的过表达再现了酒精自我给药的升级。因此，在微阵列分析中，PD大鼠中BDNF被下调，这是由qPCR证实的。在体外，在3'UTR报告基因测定中，miR-206抑制了BDNF的表达，确认BDNF为miR-206的功能靶标。分化大鼠皮质原发性神经元中miR-206表达的抑制显着增加了BDNF的分泌水平。因此，MPFC中miR-206的募集促进了饮酒和BDNF调节的升级（Tapocik等，2014）。我们最近在酒精依赖性的小鼠模型中证实了这一发现，即BDNF表达降低并上调miR-206表达。 此外，miR-206 KO小鼠对乙醇对酒精蒸气的反应钝化（Tapocik等，在Prep中）。