PDE4 regulation of GABA-A receptors in alcohol tolerance and consumption

PDE4 对酒精耐受和消费中 GABA-A 受体的调节

• 批准号: 10706954
• 负责人: ROBERT O. MESSING
• 金额: $ 42.96万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10706954
• 关键词: Acute; Alcohol consumption; Alcohols; Animals; Antibodies; Ataxia; Behavior; Behavioral; Blood alcohol level measurement; Brain; Complex; Consumption; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Development; Disincentive; Dose; Enzymes; Ethanol; Ethanol Metabolism; GABA-A Receptor; Goals; Hippocampus; Immunoprecipitation; Impairment; Intoxication; Isoenzymes; Knock-in Mouse; Knockout Mice; Mass Spectrum Analysis; Mediating; Metabolism; Minor; Mus; Mutate; Mutation; Oocytes; PDE4B; Persons; Pharmaceutical Preparations; Pharmacodynamics; Phosphodiesterase Inhibitors; Phosphorylation; Phosphorylation Site; Property; Protein Isoforms; Proteins; Regulation; Risk Factors; Rodent; Role; Series; Slice; Testing; Wild Type Mouse; Work; Xenopus laevis; Xenopus oocyte; alcohol behavior; alcohol response; alcohol reward; alcohol use disorder; drinking; experimental study; improved; inhibitor; interest; novel; phosphodiesterase IV; positive allosteric modulator; prevent; protein activation; receptor; receptor function; response; side effect; targeted treatment

PROJECT SUMMARY/ABSTRACT A major risk factor for developing alcohol use disorder (AUD) is a reduced level of response to alcohol. In persons with normal alcohol metabolism, differences in level of response to an acute intoxicating dose of alcohol result mainly from differences in acute tolerance to alcohol. Tolerance has a major influence on alcohol consumption; tolerance to alcohol’s rewarding effects encourages more drinking to achieve a desired effect, whereas tolerance to alcohol’s aversive properties reduces a disincentive to drink. This proposal is based on recent findings that inhibitors of phosphodiesterase 4 (PDE4) reduce alcohol drinking in rodents. In our work with the PDE4 inhibitor apremilast, we were struck by the relationship between its ability to reduce both alcohol tolerance and ethanol consumption. Inhibitors of PDE4 reduce metabolism of cAMP which leads to activation of protein kinase A (PKA). Alcohol has prominent effects on GABAA receptors, and it has been known for many years that PKA alters the function of GABAA receptors through phosphorylation of β1 and β3 receptor subunits. Our preliminary experiments led us to our overall hypothesis that PDE4 inhibition reduces alcohol tolerance and alcohol consumption by increasing GABAA receptor function in the brain through PKA-mediated phosphorylation of GABAA receptor β3 subunits, with PKA-mediated phosphorylation of β1 subunits contributing a minor opposite effect. Studies are planned to test this hypothesis by determining whether apremilast increases PKA-mediated phosphorylation of b3 and β1 subunits in hippocampal slices from wild type mice. Studies will examine the role of PKA phosphorylation on alcohol-related behaviors using b3- S408A/S409A and b1-S409A knock-in mice, which lack the PKA phosphorylation sites of interest. We will examine the role of specific brain PDE4 isozymes in these alcohol-related behaviors using PDE4 isozyme- selective inhibitors and knockout mice. We will also determine whether b3 and β1 subunits associate with specific PDE4 isoforms by isolating GABAA receptor complexes using Blue Native PAGE and analyzing them with mass spectrometry (LS-MS/MS). Finally, we will test the hypothesis that a b1-selective, positive allosteric modulator (PAM) alone or in combination with apremilast will reduce alcohol tolerance and alcohol consumption. These experiments will characterize a new series of 10 novel compounds targeting b1-containing GABAA receptors using receptors expressed in Xenopus oocytes, to identify a b1-selective PAM with properties suitable for testing in mice.

项目摘要/摘要 发展饮酒障碍（AUD）的主要危险因素是对酒精的反应水平降低。 酒精代谢正常的人，对急性醉酒剂量的反应水平的差异 酒精主要是由于急性耐受性对酒精的差异而产生的。耐受对酒精有重大影响 消耗;对酒精的奖励效果的容忍会鼓励更多的饮酒实现预期的效果， 而对酒精厌恶性能的耐受性会减少饮酒的抑制作用。该提议基于 最近的发现是磷酸二酯酶4（PDE4）抑制剂减少啮齿动物中的饮酒。在我们的工作中 使用PDE4抑制剂APREMILAST，我们对降低两种酒精的能力之间的关系感到震惊 耐受性和乙醇消耗。 PDE4的抑制剂减少了CAMP的代谢，从而导致激活 蛋白激酶A（PKA）的。酒精对GABAA受体具有显着影响，并且以许多人闻名 PKA通过β1和β3受体亚基的磷酸化来改变GABAA受体的功能。 我们的初步实验使我们提出了PDE4抑制作用降低酒精耐受性的总体假设 通过PKA介导的大脑中的GABAA受体功能来增加饮酒 GABAA受体β3亚基的磷酸化，PKA介导的β1亚基的磷酸化 造成较小的相反效果。计划通过确定是否是否 Apremilast增加了来自野外海马切片中B3和β1亚基的PKA介导的磷酸化 类型的鼠标。研究将研究使用B3-的PKA磷酸化对酒精相关行为的作用 S408A/S409A和B1-S409A敲入小鼠，它们缺乏感兴趣的PKA磷酸化位点。我们将 使用PDE4同工酶 - 检查特异性脑PDE4同工酶在这些与酒精相关的行为中的作用 选择性抑制剂和敲除小鼠。我们还将确定B3和β1亚基是否与 特定的PDE4同工型通过使用蓝色本地页面隔离GABAA接收器配合物并分析它们 具有质谱（LS-MS/MS）。最后，我们将检验以下假设，即B1选择性阳性变构性 单独或与Apremilast结合使用调节器（PAM）将降低酒精耐受性和酒精 消耗。这些实验将表征一个新的10种针对B1的新型化合物 使用在异爪蟾卵母细胞中表达的受体的GABAA受体，以鉴定B1选择性PAM与 适用于小鼠测试的特性。