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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要酒精使用障碍 (AUD) 的一个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，对急性中毒剂量的反应水平存在差异酒精的产生主要是由于对酒精的急性耐受性的差异，耐受性对酒精的影响很大。消费；对酒精的奖励作用的容忍会鼓励更多的饮酒以达到预期的效果，而对酒精的厌恶特性的容忍会减少饮酒的抑制作用。我们的工作中最近发现磷酸二酯酶 4 (PDE4) 抑制剂可减少啮齿类动物的饮酒量。与 PDE4 抑制剂阿普斯特相比，我们对它减少酒精的能力之间的关系感到震惊 PDE4 抑制剂会降低 cAMP 的代谢，从而导致激活。众所周知，酒精对 GABAA 受体有显着的影响。多年来，PKA 通过 β1 和 β3 受体亚基的磷酸化改变 GABAA 受体的功能。我们的初步实验得出了总体假设：抑制 PDE4 会降低酒精耐受性通过 PKA 介导增加大脑中的 GABAA 受体功能来饮酒 GABAA 受体 β3 亚基的磷酸化，以及 PKA 介导的 β1 亚基的磷酸化计划通过确定是否会产生较小的相反效应来检验这一假设。 apremilast 增加野生海马切片中 PKA 介导的 b3 和 β1 亚基的磷酸化研究将使用 b3- 检验 PKA 磷酸化对酒精相关行为的作用。 S408A/S409A 和 b1-S409A 敲入小鼠，缺乏感兴趣的 PKA 磷酸化位点。使用 PDE4 同工酶检查特定大脑 PDE4 同工酶在这些酒精相关行为中的作用我们还将确定 b3 和 β1 亚基是否与选择性抑制剂和基因敲除小鼠相关。使用 Blue Native PAGE 分离 GABAA 受体复合物并对其进行分析，从而确定特定的 PDE4 亚型最后，我们将检验 b1 选择性、正变构的假设。调节剂（PAM）单独或与阿普斯特联合使用会降低酒精耐受性和酒精含量这些实验将表征一系列新的 10 种针对含 b1 的新型化合物。 GABAA 受体使用非洲爪蟾卵母细胞中表达的受体来识别 b1 选择性 PAM 适合在小鼠身上进行测试的特性。