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

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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.