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-阿片受体：人类 OPRM1 基因中编码 mu-阿片受体的非同义 A118G SNP 可能调节酒精奖赏和对纳曲酮 (NTX) 的治疗反应。我们之前创建了两个携带各自人类等位基因（118AA 和 118GG）的人源化小鼠品系，并观察到 ​​118GG 小鼠的伏核（NAc）中对酒精的反应显着增强了多巴胺（DA）的释放。这一发现与在人体中并行进行的正电子发射断层扫描研究一致（Ramchandani 等，2011）。最近的研究表明，通过酒精引起的颅内脑刺激（ICSS）阈值降低来测量 118GG 小鼠的酒精奖赏增强。 ICSS 阈值抑制可被阿片类拮抗剂纳曲酮和批准的酗酒药物选择性阻断（与北卡罗来纳大学 Robinson 和 Malanga 合作）。因此，与 118AA 系相比，118GG 小鼠的自愿饮酒量增加，并且 118GG 小鼠优先对纳曲酮敏感（Bilbao 等人，Biol Psychiat，出版中）。 NK1 受体：我们之前发现 L822429（一种大鼠特异性 NK1 拮抗剂）剂量依赖性地抑制 Wistar 大鼠应激诱导的酒精寻求恢复，但对提示诱导的恢复没有影响（Schank 等，2011）。在后续工作中，L-822429 在偏好酒精的 P 大鼠中剂量依赖性地抑制自我给药，但在非依赖性 Wistar 大鼠中无效。我们发现 P 大鼠中央杏仁核 (CeA) 中 NK1R 表达上调；将拮抗剂显微注射到该区域模拟了全身效应（Schank 等，2013）。 Fos 映射显示，压力诱导的酒精寻求恢复与杏仁核 (amg)、NAc、中缝背核 (DR)、内侧前额叶皮层 (mPFC) 和终纹床核的神经元激活有关。 NK1R 拮抗作用抑制了这些区域的一部分（特别是 DR 和 NAc 壳）中应激诱导的 Fos 增加，表明这些区域介导了 NK1R 拮抗作用以阻止应激诱导的复发。此外，我们发现 NK-1 拮抗作用会减弱育亨宾诱导的酒精寻求和可卡因寻求的恢复（Shank 等，2014）。 神经肽 S (NPS) 受体：NPS 信号传导可能在酗酒复发中发挥作用（Canella 等，2009）。我们与 NIH 化学基因组中心 (NCGC) 合作，鉴定了一种先导分子 NCG001865684，该分子在大鼠外周给药后具有脑渗透性。在体外，NCGC00185684 显示出偏向的拮抗剂特性，并且优先阻断 ERK 磷酸化，而不是细胞内 cAMP 或钙对 NPS 的反应。在体内，NCG001865684 预处理可阻断大鼠 CeA（参与调节酒精摄入的区域）中酒精诱导的 ERK 磷酸化。 NCGC00185684 还减少了操作性酒精自我管理，并降低了使用渐进比率响应测量的酒精奖励。这些影响是行为特定的。总而言之，这些数据初步验证了 NPSR 作为酒精中毒治疗靶点的作用（Thorsell 等，2013）。 黑色素浓缩激素 (MCH)：MCH 是一种下丘脑食欲调节肽，还参与啮齿动物中由 MCH-1 受体 (MCH-1R) 介导的奖励、动机和焦虑样行为。 MCH1-R 阻断可抑制酒精自我给药（Cippitelli 等人，2010）。由于酒精也是一种热量营养素，因此自我给药结果可能会受到食欲影响的影响。因此，我们在 MCH-1R 敲除 (KO) 小鼠中使用条件位置偏好 (CPP) 评估了 MCH 系统对酒精奖励特性的作用。与野生型C57BL/6小鼠相比，MCH-1R KO:s在2g/kg剂量下没有产生位置偏好。这种效应是行为特异性的，因为在运动、翻正反射丧失 (LORR) 或酒精代谢和消除方面没有观察到差异（Karlsson 等人，准备中）。 促炎活性：先天免疫可能参与应激反应和与成瘾相关的行为。我们开始研究 IL-1 受体 (IL-1R) 在酒精相关行为中的作用。在两瓶选择测试中，缺乏 IL-1R 的小鼠饮酒量较少，但 CPP 实验表明，IL-1R 小鼠的饮酒量减少并不是由于酒精奖励的影响。在评估 LORR 时，IL1-R KO 小鼠的睡眠时间显着更长，表明与对照组相比，对酒精的镇静/共济失调特性具有更高的敏感性。这与经常观察到的酒精镇静/共济失调效应敏感性与消费之间的负相关一致。在压力诱导饮酒、社交挫败压力（SDS）模型中，两种基因型都显着增加了饮酒量，但没有发现基因型差异。由于 IL-1β 和 TNF-α 具有重叠的途径，因此有人认为它们可以并行作用，并且可以相互替代。因此，我们接下来使用缺乏 IL-1 和 TNF-1 受体的双 KO 小鼠模型。双 KO 消耗的酒精较少，并且不会随着酒精浓度的增加而增加。这不太可能是由于酒精的奖励特性不同造成的。双 KO 小鼠表现出酒精诱导的 CPP 的强烈表达。进一步对 Double KO 小鼠的 LORR、酒精代谢和味觉偏好进行评估。没有观察到差异表明行为特定的影响。 有趣的是，双敲除小鼠在暴露于 SDS 后对压力诱导的饮酒不太敏感，并且相对于基线摄入量没有增加其摄入量。 （Karlsson 等人，准备中）。 DNA 甲基化：大量证据支持表观遗传机制在调节酒精相关行为中的作用。我们发现 i.c.v.输注 RG-108（一种特定的 DNA 甲基转移酶抑制剂）可减少依赖后大鼠的操作性酒精自我给药，并逆转依赖史诱导的 mPFC 中编码参与神经递质释放的突触蛋白的长期表达变化。 RG-108 的效果通过局部输注至 mPFC 7-10 天来重现，并且具有行为选择性。输注 RG108 可以防止 PD 大鼠中酒精消耗的增加和依赖性诱导的 7 个转录本中 4 个转录本的下调。具体来说，RG108 治疗直接逆转了突触结合蛋白 2 (syt2) 的下调，这是由酒精诱导的高甲基化引起的。 慢病毒抑制 mPFC 中 Syt2 的表达会增加厌恶性饮酒，支持 Syt2 在酒精寻求中的机制作用。 （Barbier 等人提交）。 microRNA 调节：根据最近的微阵列筛选（Tapocik 等，2012），我们的 qPCR 证实酒精依赖会导致 mPFC 中 miR-206 的持续上调。非依赖性大鼠 mPFC 中 miR-206 的过度表达再现了自我饮酒的升级。因此，在微阵列分析中，PD 大鼠中 BDNF 下调，这一点通过 qPCR 得到了证实。在体外，BDNF 表达在 3'UTR 报告基因测定中被 miR-206 抑制，证实 BDNF 是 miR-206 的功能靶点。抑制分化大鼠皮质原代神经元中 miR-206 的表达可显着增加 BDNF 的分泌水平。因此，mPFC 中 miR-206 的募集有助于增加酒精消耗和 BDNF 调节（Tapocik 等，2014）。我们最近在酒精依赖小鼠模型中证实了这一发现，其中 BDNF 表达下降，而 mir-206 表达上调。 此外，miR-206 KO 小鼠对酒精蒸汽的乙醇反应减弱（Tapocik 等人，准备中）。