Antidepressants Produce Persistent Gα s-Associated Signaling Changes in Lipid Rafts after Drug Withdrawal.

Termination of antidepressant therapy often has negative consequences. Although symptoms of antidepressant withdrawal are widely recognized, the molecular processes that underlie them are not well characterized. We show that certain aspects of Gα s signaling remain suppressed after antidepressant withdrawal, even after others have reverted to baseline. Antidepressant treatment causes translocation of Gα s protein from lipid rafts to nonraft membrane regions. This results in augmented Gα s signaling, including facilitated activation of adenylyl cyclase and increased cAMP accumulation. Using CC6 or SK-N-SH cells and a lipid raft-localized cAMP sensor, we show that Gα s signaling is reduced in lipid rafts, even while signaling is enhanced elsewhere in the cell. These signaling changes mirror the changes in Gα s localization observed after antidepressant treatment. Furthermore, we show that suppression of Gα s signaling in lipid rafts persists at least 24 hours after cessation of antidepressant treatment. Gα s localization was quantified after membrane isolation and sequential detergent extraction. We show that suppression of lipid raft Gα s signaling persists for an extended time period after antidepressant withdrawal, whereas increased nonraft membrane Gα s signaling reverts partially or fully upon cessation of antidepressant treatment. Translocation of Gα s out of lipid rafts is also persistent. These events may reflect cellular adaptations to antidepressant treatment that contribute to antidepressant discontinuation syndromes and may aid in the discovery of new treatments and strategies to mitigate the symptoms of depression and antidepressant withdrawal. SIGNIFICANCE STATEMENT: This work explores, for the first time, the effects of antidepressants on Gα s signaling after drug withdrawal. This provides novel insight into the cellular and molecular processes affected by antidepressant drugs and their persistence after discontinuation of treatment.

NMDAR-independent, cAMP-dependent antidepressant actions of ketamine.

Ketamine produces rapid and robust antidepressant effects in depressed patients within hours of administration, often when traditional antidepressant compounds have failed to alleviate symptoms. We hypothesized that ketamine would translocate Gαs from lipid rafts to non-raft microdomains, similarly to other antidepressants but with a distinct, abbreviated treatment duration. C6 glioma cells were treated with 10 µM ketamine for 15 min, which translocated Gαs from lipid raft domains to non-raft domains. Other NMDA antagonist did not translocate Gαs from lipid raft to non-raft domains. The ketamine-induced Gαs plasma membrane redistribution allows increased functional coupling of Gαs and adenylyl cyclase to increase intracellular cyclic adenosine monophosphate (cAMP). Moreover, increased intracellular cAMP increased phosphorylation of cAMP response element-binding protein (CREB), which, in turn, increased BDNF expression. The ketamine-induced increase in intracellular cAMP persisted after knocking out the NMDA receptor indicating an NMDA receptor-independent effect. Furthermore, 10 µM of the ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) also induced Gαs redistribution and increased cAMP. These results reveal a novel antidepressant mechanism mediated by acute ketamine treatment that may contribute to ketamine's powerful antidepressant effect. They also suggest that the translocation of Gαs from lipid rafts is a reliable hallmark of antidepressant action that might be exploited for diagnosis or drug development.

Regulator of G-Protein Signaling (RGS) Protein Modulation of Opioid Receptor Signaling as a Potential Target for Pain Management.

Opioid drugs are the gold standard for the management of pain, but their use is severely limited by dangerous and unpleasant side effects. All clinically available opioid analgesics bind to and activate the mu-opioid receptor (MOR), a heterotrimeric G-protein-coupled receptor, to produce analgesia. The activity of these receptors is modulated by a family of intracellular RGS proteins or regulators of G-protein signaling proteins, characterized by the presence of a conserved RGS Homology (RH) domain. These proteins act as negative regulators of G-protein signaling by serving as GTPase accelerating proteins or GAPS to switch off signaling by both the Gα and ßγ subunits of heterotrimeric G-proteins. Consequently, knockdown or knockout of RGS protein activity enhances signaling downstream of MOR. In this review we discuss current knowledge of how this activity, across the different families of RGS proteins, modulates MOR activity, as well as activity of other members of the opioid receptor family, and so pain and analgesia in animal models, with particular emphasis on RGS4 and RGS9 families. We discuss inhibition of RGS proteins with small molecule inhibitors that bind to sensitive cysteine moieties in the RH domain and the potential for targeting this family of intracellular proteins as adjuncts to provide an opioid sparing effect or as standalone analgesics by promoting the activity of endogenous opioid peptides. Overall, we conclude that RGS proteins may be a novel drug target to provide analgesia with reduced opioid-like side effects, but that much basic work is needed to define the roles for specific RGS proteins, particularly in chronic pain, as well as a need to develop newer inhibitors.

The Role of G-proteins and G-protein Regulating Proteins in Depressive Disorders.

Progress toward new antidepressant therapies has been relatively slow over the past few decades, with the result that individuals suffering from depression often struggle to find an effective treatment - a process often requiring months. Furthermore, the neural factors that contribute to depression remain poorly understood, and there are many open questions regarding the mechanism of action of existing antidepressants. A better understanding of the molecular processes that underlie depression and contribute to antidepressant efficacy is therefore badly needed. In this review we highlight research investigating the role of G-proteins and the regulators of G-protein signaling (RGS) proteins, two protein families that are intimately involved in both the genesis of depressive states and the action of antidepressant drugs. Many antidepressants are known to indirectly affect the function of these proteins. Conversely, dysfunction of the G-protein and RGS systems can affect antidepressant efficacy. However, a great deal remains unknown about how these proteins interact with antidepressants. Findings pertinent to each individual G-protein and RGS protein are summarized from in vitro, in vivo, and clinical studies.

Role of hippocampal 5-HT1A receptors in the antidepressant-like phenotype of mice expressing RGS-insensitive Gαi2 protein.

A single base mutation in the Gαi2 protein (G184S) renders this Gα subunit insensitive to the negative modulatory effects of Regulator of G-protein Signaling (RGS) proteins. Mice expressing this RGS insensitive (RGSi) variant of Gαi2 (RGSi Gαi2) display a spontaneous antidepressant-like phenotype that is reversible by treatment with the 5-HT1A receptor (5-HT1AR) antagonist WAY100635. Here we test the hypothesis that increased activity of 5-HT1ARs in the hippocampus of RGSi Gαi2 knock-in mice is responsible for the expression of the observed antidepressant-like behavior. We administered the 5-HT1AR antagonist WAY100635 or the agonist 8-OH-DPAT via bilateral intra-hippocampal infusion cannulae and evaluated antidepressant-like behavior using the tail suspension test (TST). WAY100635 reversed the antidepressant-like phenotype of the RGSi Gαi2 knock-in mice and 8-OH-DPAT produced an antidepressant-like response in wild type mice that was blocked by systemic WAY100635. Furthermore, intra-hippocampal infusion of the RGS19/4 inhibitor CCG-203769 produced an antidepressant-like effect in female mice. Ex-vivo slice recording confirmed the 5-HT1AR-mediated decrease in hippocampal CA1 pyramidal neuron excitability was enhanced in the RGSi Gαi2 knock-in mice. There was no change in hippocampal 5-HT1AR expression as measured by ligand binding but there was a compensatory reduction in Gαi proteins. The findings demonstrate that RGS protein control of hippocampal 5-HT1AR signaling is necessary and sufficient to account for the antidepressant-like phenotype in the RGSi Gαi2 knock-in mice and that RGS proteins highly expressed in the hippocampus should be investigated as targets for novel antidepressant therapies.