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

项目摘要/摘要 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……里面 酒精代谢正常的人，对急性醉酒剂量的反应水平不同 酒精主要是由于对酒精的急性耐受性不同造成的。耐受性对酒精有很大的影响 消费；对酒精的有益影响的容忍鼓励更多的饮酒以达到预期的效果， 然而，对酒精令人厌恶的特性的容忍会减少饮酒的抑制。这项建议是基于 最近发现，磷酸二酯酶4(PDE4)的抑制剂可以减少啮齿动物的饮酒。在工作中 对于PDE4抑制剂阿普米司特，我们被其减少酒精含量的能力之间的关系所震惊 耐受性和酒精消耗量。PDE4抑制剂降低cAMP的代谢，从而导致激活 蛋白激酶A(PKA)的活性。酒精对GABAA受体有显著的影响，它已经被很多人所知 多年来，PKA通过β1和β3受体亚基的磷酸化改变了GABA受体的功能。 我们的初步实验得出了我们的总体假设，即抑制PDE4会降低酒精耐受性 通过PKA介导增加大脑中GABAA受体的功能和饮酒 GABAA受体β3亚基的磷酸化，以及PKA介导的β1亚基的磷酸化 产生了轻微的相反效果。计划进行的研究将通过确定 阿普利司特增加pKA介导的野生型海马片b3和β-1亚单位的磷酸化 输入老鼠。研究将使用b3-来研究PKA磷酸化在酒精相关行为中的作用。 S408A/S409A和b1-S409A敲入小鼠，它们缺乏感兴趣的PKA磷酸化位点。我们会 使用PDE4同工酶检测特定的大脑PDE4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亚单位是否与 用Blue Native PAGE分离GABAA受体复合体并分析其特异性PDE4亚型 用质谱仪(LS-MS/MS)分析。最后，我们将检验b1选择性正变构的假设。 调节剂(PAM)单独或与阿波司特联用会降低酒精耐量和酒精含量 消费。这些实验将表征一系列新的10种针对b1的新型化合物。 利用非洲爪哇卵母细胞表达的GABAA受体鉴定具有b1选择性的PAM 适合在小鼠身上测试的特性。

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.