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

项目总结/摘要 发生酒精使用障碍（AUD）的一个主要风险因素是对酒精的反应水平降低。在 酒精代谢正常的人，对急性中毒剂量的 酒精的产生主要是由于对酒精的急性耐受性的差异。耐受性对酒精有重大影响 消费;对酒精的奖励效应的耐受性鼓励更多的饮酒以达到预期的效果， 而对酒精的厌恶性的耐受性减少了对饮酒的抑制。该提案基于 最近发现磷酸二酯酶4（PDE 4）抑制剂可减少啮齿动物的饮酒量。在我们的工作中 对于PDE 4抑制剂阿普斯特，我们被其减少酒精和 耐受性和乙醇消耗。PDE 4抑制剂降低cAMP代谢，导致活化 蛋白激酶A（PKA）。酒精对GABAA受体有显着影响，这一点众所周知 PKA通过磷酸化β1和β3受体亚基改变GABAA受体的功能。 我们的初步实验导致我们的总体假设，即PDE 4抑制降低酒精耐受性 通过PKA介导的增加大脑中GABAA受体的功能， GABAA受体β3亚基磷酸化，PKA介导的β1亚基磷酸化 产生较小的相反效果。研究计划通过确定是否 阿普斯特增加PKA介导的野生型海马脑片b3和β1亚基磷酸化 型小鼠。研究将使用b3-磷酸化蛋白激酶A（PKA）对酒精相关行为的作用进行检测。 S408 A/S409 A和b1-S409 A敲入小鼠，其缺乏感兴趣的PKA磷酸化位点。我们将 使用PDE 4同工酶检查特定脑PDE 4同工酶在这些酒精相关行为中的作用- 选择性抑制剂和基因敲除小鼠。我们还将确定b3和β1亚基是否与 通过使用蓝色非变性PAGE分离GABAA受体复合物并分析它们， 质谱法（LS-MS/MS）。最后，我们将检验一个假设，即一个b1选择性的，积极的变构 单独或与阿普斯特组合的PAM调节剂将降低酒精耐受性和酒精浓度。 消费这些实验将表征一系列新的10种新型化合物， GABAA受体，使用爪蟾卵母细胞中表达的受体，鉴定具有b1选择性的PAM， 适合在小鼠中测试的性质。

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.