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 Gα 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 Gα from lipid raft domains to non-raft domains. Other NMDA antagonist did not translocate Gα from lipid raft to non-raft domains. The ketamine-induced Gα plasma membrane redistribution allows increased functional coupling of Gα 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 Gα 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 Gα from lipid rafts is a reliable hallmark of antidepressant action that might be exploited for diagnosis or drug development.

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 βγ 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.

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.

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A single base mutation in the Gα 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 Gα (RGSi Gα) 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 Gα 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 Gα 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 Gα 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 Gα knock-in mice and that RGS proteins highly expressed in the hippocampus should be investigated as targets for novel antidepressant therapies.

Regulator of G‐protein signaling (RGS) proteins bind to the active GTP‐bound Gα subunit of heterotrimeric G‐proteins to accelerate hydrolysis of GTP and limit signaling downstream of G‐protein coupled receptors (GPCRs). Studies have shown that mice expressing Gαo protein that is insensitive to modulation by RGS proteins (RGSi Gαo) are less hyperalgesic due to enhanced signaling at the mu‐opioid receptor (MOPR). In contrast, data suggest these mice have a hyperalgesic response to mechanical stimulation. The goal of the present study was to determine the mechanism behind this apparent contradiction. Baseline mechanical hypersensitivity in naive RGSi Gαo mice was reversed by pretreatment with systemic or central administration of the nociceptin/orphanin FQ (N/OFQ) receptor (NOPR) selective antagonist J‐113397, while the opioid antagonist naltrexone further intensified the hypersensitivity. Intraplantar injection of λ‐carrageenan produced mechanical hypersensitivity, which was reversed by J‐113397 in wild‐type mice and exacerbated by naltrexone in mutant mice. Whole brain homogenates from RGSi Gαo mice showed no change from wild‐type littermates in levels of NOPR as determined by [3H]N/OFQ saturation binding and no change in the affinity of [3H]N/OFQ for NOPR, while N/OFQ had unaltered potency to activate G‐protein as measured by [35S]GTPγS binding. N/OFQ‐induced inhibition of presynaptic GABA release in the periaqueductal gray was reduced in the mutant mice compared to their wild‐type controls. Together, these results indicate that increased signaling downstream of NOPR leads to hyperalgesia in the RGSi Gαo knock‐in mice, while increased signaling downstream of MOPR reverses NOPR‐mediated hyperalgesia. Similar opposing actions of the systems are present after inflammatory pain in wild‐type mice, signifying that in both cases the balance between MOPR and NOPR signaling is disturbed. These data highlight a delicate homeostatic balance between two receptor systems that is dependent on pain state. Support or Funding Information Funded by R01 DA035316 and T32 DA007268. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Serotonergic hallucinogens psilocybin, and its active metabolite psilocin, have shown promise as rapid‐acting antidepressants. The FDA granted breakthrough therapy designation for psilocybin's use as an antidepressant in 2019, and phase II clinical trials are currently ongoing. Psilocybin is converted to psilocin following oral administration, and this metabolite is the primary active compound found in the central nervous system. As such, it is critically important to understand the cellular and molecular processes contributing to psilocin's antidepressant action, as well as the similarities and differences between the cellular effects of psilocin and those of more traditional antidepressants. Both traditional and rapid‐acting antidepressants cause translocation of Gαs from lipid‐rafts to non‐raft membranes. The sequelae of this translocation event include increased overall Gαs activity, leading to increased cellular cAMP. Antidepressants also accumulate in lipid‐raft membranes, even in cellular models lacking the presumed binding target for these drugs. The lipid‐raft binding target(s) are currently unknown, and the processes which contribute to Gαs translocation have not been fully elucidated. In this study we utilize a proteomic approach in a cellular model to investigate psilocin's effects on both the lipid‐raft and non‐raft proteome. We expect that additional proteins besides Gαs exhibit raft to non‐raft translocation following antidepressant treatment. Proteins exhibiting this translocation pattern may represent as‐of‐yet unrecognized contributors to antidepressant efficacy, or may serve as antidepressant biomarkers. We also measure signaling downstream of Gαs and Gαq in intact cells, as well as purified lipid‐raft membranes, using modified cAMP (AlphaScreen, cADDis) and inositol monophosphate (IP‐One) detection assays. We hypothesize that both psilocin and desipramine attenuate lipid‐raft Gαs (but notGαq) downstream signaling due to the loss of Gαs fromthese membranes. Membranes treated with psilocin or the tricyclic antidepressant desipramine are compared in order to identify candidate proteins that may be generally affected following antidepressant treatment. Both drugs cause raft to non‐raft translocation of hundreds of proteins. Several of these translocated proteins are common to both drug treatments, and we expect to identify novel contributors to the antidepressant response from this subset of proteins. In particular, observed effects on specific integrins, G‐proteins, and cholesterol biosynthesis pathways will be followed up with more in‐depth analyses in future studies. We also show that antidepressant treatments reduce lipid‐raft Gαs signaling, while simultaneously increasing whole cell Gαs signaling. This effect can persist for over 24 hr after drug withdrawal. These results represent one of the first direct measurements of lipid‐raft signaling following antidepressant treatment, and may provide a more complete understanding of both antidepressant action, and the molecular mechanisms by which ha...

Many patients experience a constellation of side effects following termination of antidepressant therapy. Collectively these effects are known as the antidepressant discontinuation syndrome. All currently prescribed antidepressant medications can cause this syndrome, regardless of primary mechanism of action. This suggests that some shared mechanism, independent of these drugs’ known binding targets, may contribute to the discontinuation syndrome. Lipid rafts are membrane microdomains with distinct signaling characteristics and protein expression compared to other membrane regions. Previous work has shown that chronic, but not acute, treatment with traditional antidepressants induces translocation of Gs out of lipid raft microdomains. In contrast, rapid acting antidepressants such as ketamine cause Gs translocation following acute exposure. This promotes Gs signaling in general, likely due to increased coupling to downstream effectors such as adenylyl cyclase. Using a lipid raft restricted cAMP biosensor we show that perturbations of Gs signaling persist for over 24 hr after antidepressant withdrawal in an in vitro model system. These signaling changes are variable between different subcellular microdomains, and between different cell types. In general, signaling changes in lipid rafts are more persistent than changes observed in non‐raft membrane regions, or in the cytoplasm. Effects are compared between c6 glioma, SK‐N‐SH neuronal, and HEK‐293 kidney derived cell lines. We also show that changes in lipid raft cAMP signaling are reflected by persistent reductions in lipid raft Gs expression following drug withdrawal. Protein expression is quantified following membrane isolation and sequential detergent extraction to separate lipid raft from non‐raft membranes. The observed antidepressant effects occur in model systems lacking the primary target for these drugs (e.g. SERT and escitalopram), and as such may represent a novel mechanism by which antidepressants regulate cellular activity. Understanding these residual antidepressant effects may aid in the development of novel antidepressants without an associated discontinuation syndrome, and in the discovery of new strategies to mitigate these persistent effects following drug withdrawal. Support or Funding Information Supported by: VA Merit BX001149, NIH T32 MH067631 and NIH R01 AT009169