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.

G proteins mediate signals from membrane G protein coupled receptors to the cell interior, evoking significant regulation of cell physiology. The cytoskeleton contributes to cell morphology, motility, division, and transport functions. This review will discuss the interplay between heterotrimeric G protein signaling and elements of the cytoskeleton. Also described and discussed will be the interplay between tubulin and G proteins that results in atypical modulation of signaling pathways and cytoskeletal dynamics. This will be extended to describe how tubulin and G proteins act in concert to influence various aspects of cellular behavior.

GPCR signaling is modified both in major depressive disorder and by chronic antidepressant treatment. Endogenous Ga s redistributes from raft-to nonraft-membrane fractions after chronic antidepressant treatment. Modification of G protein anchoring may participate in this process. Regulation of Ga s signaling by antidepressants was studied using fluorescence recovery after photobleaching (FRAP) of GFP-Ga s . Here we find that extended antidepressant treatment both increases the half-time of maximum recovery of GFP-Ga s and decreases the extent of recovery. Furthermore, this effect parallels the movement of Ga s out of lipid rafts as determined by cold detergent membrane extraction with respect to both dose and duration of drug treatment. This effect was observed for several classes of compounds with antidepressant activity, whereas closely related molecules lacking antidepressant activity (eg, R-citalopram) did not produce the effect. These results are consistent with previously observed antidepressant-induced translocation of Ga s , but also suggest an alternate membrane attachment site for this G protein. Furthermore, FRAP analysis provides the possibility of a relatively high-throughput screening tool for compounds with putative antidepressant activity.

Depression is a significant public health problem for which currently available medications, if effective, require weeks to months of treatment before patients respond. Previous studies have shown that the G protein responsible for increasing cAMP (G␣ s ) is increasingly localized to lipid rafts in depressed subjects and that chronic antidepressant treatment translocates G␣ s from lipid rafts. Translocation of G␣ s , which shows delayed onset after chronic antidepressant treatment of rats or of C6 glioma cells, tracks with the delayed onset of therapeutic action of antidepressants. Because antidepressants appear to specifically modify G␣ s localized to lipid rafts, we sought to determine whether structurally diverse antidepressants accumulate in lipid rafts. Sustained treatment of C6 glioma cells, which lack 5-hydroxytryptamine transporters, showed marked concentration of several antidepressants in raft fractions, as revealed by increased absorbance and by mass fingerprint. Closely related molecules without antidepressant activity did not concentrate in raft fractions. Thus, at least two classes of antidepressants accumulate in lipid rafts and effect translocation of G␣ s to the non-raft membrane fraction, where it activates the cAMP-signaling cascade. Analysis of the structural determinants of raft localization may both help to explain the hysteresis of antidepressant action and lead to design and development of novel substrates for depression therapeutics.Depression is the leading cause of long term disability in the industrialized world (1). Although depression is a significant health problem in the United States and antidepressants are heavily prescribed (2), the mechanism of action for these drugs is not understood. Further, nearly a third of those treated with these drugs do not achieve remission of their depression (3). Although most of these drugs do interfere with monoamine uptake or catabolism, they exert this effect within hours, even though most of the compounds require weeks before alleviation of symptoms is observed (4). Thus, other targets for antidepressant drugs may exist (4).Chronic antidepressant treatment engages signaling pathways apart from increasing monoamine density in the synaptic cleft. One of these is an increased accumulation of cellular cAMP and sequelae thereof, such as increased cAMP-response element-binding protein (CREB) phosphorylation and increased transcription of cAMP-regulated genes (e.g. BDNF) (5). Moreover, positron emission tomography (PET) evidence suggests that cAMP is diminished throughout the brain of depressed human subjects (6). Thus, it is possible that some antidepressant effects are mediated through induction of the cAMP-generating system, including G␣ s and adenylyl cyclase.Previous studies demonstrated that chronic antidepressant treatment translocates G␣ s from lipid rafts, whereupon it engages in a more facile activation of adenylyl cyclase (7,8). Lipid rafts are regions of the plasma membrane rich in caveolin, cholesterol, sphingolipids, and cytoskeletal an...

Current antidepressant therapies meet with variable therapeutic success and there is increasing interest in therapeutic approaches not based on monoamine signaling. Histone deacetylase 6 (HDAC6), which also deacetylates α-tubulin shows altered expression in mood disorders and HDAC6 knockout mice mimic traditional antidepressant treatments. Nonetheless, a mechanistic understanding for HDAC6 inhibitors in the treatment of depression remains elusive. Previously, we have shown that sustained treatment of rats or glioma cells with several antidepressants translocates Gα from lipid rafts toward increased association with adenylyl cyclase (AC). Concomitant with this is a sustained increase in cAMP production. While Gα modifies microtubule dynamics, tubulin also acts as an anchor for Gα in lipid-rafts. Since HDAC-6 inhibitors potentiate α-tubulin acetylation, we hypothesize that acetylation of α-tubulin disrupts tubulin-Gα raft-anchoring, rendering Gα free to activate AC. To test this, C6 Glioma (C6) cells were treated with the HDAC-6 inhibitor, tubastatin-A. Chronic treatment with tubastatin-A not only increased α-tubulin acetylation but also translocated Gα from lipid-rafts, without changing total Gα. Reciprocally, depletion of α-tubulin acetyl-transferase-1 ablated this phenomenon. While escitalopram and imipramine also disrupt Gα/tubulin complexes and translocate Gα from rafts, they evoke no change in tubulin acetylation. Finally, two indicators of downstream cAMP signaling, cAMP response element binding protein phosphorylation (pCREB) and expression of brain-derived-neurotrophic-factor (BDNF) were both elevated by tubastatin-A. These findings suggest HDAC6 inhibitors show a cellular profile resembling traditional antidepressants, but have a distinct mode of action. They also reinforce the validity of antidepressant-induced Gα translocation from lipid-rafts as a biosignature for antidepressant response that may be useful in the development of new antidepressant compounds.

In contrast to healthy controls, the heterotrimeric G protein, Gsalpha (Gsα) is ensconced predominantly in lipid rafts in subjects with major depressive disorder (MDD) resulting in impaired stimulation of adenylyl cyclase. In this small proof-of-concept study, we examined the hypothesis that translocation of Gsα from lipid rafts toward a more facile activation of adenylyl cyclase is a biomarker for clinical response to antidepressants. There were 49 subjects with MDD (HamD 17 score ≥15) and 59 healthy controls at the screen visit. The AlphaScreen (PerkinElmer) assay measured both basal activity and prostaglandin E1 (PGE1) stimulation of Gsα-adenylyl cyclase to assess the extent of coupling of Gsα with adenylyl cyclase. At screen, platelet samples obtained from MDD subjects revealed significantly lower PGE1 activation of adenylyl cyclase activity than controls (p = 0.02). Subsequently, 19 consenting MDD subjects completed a 6-week open label antidepressant treatment trial. The 11 antidepressant responders (HamD 17 improvement ≥50% from screen) revealed significant increase in PGE1-stimulated adenylyl cyclase compared to non-responders (p = 0.05) with an effect size of 0.83 for the PGE1/Gsα lipid-raft biomarker. PGE1 stimulation increased by ≥30% from screen assessment in eight responders (72.7%) and two non-responders (25.0%) [Fisher exact = 0.07] with a positive predictive value for response of 80.0%. In this small, pilot study, increased PGE1 stimulated adenylyl cyclase was associated with antidepressant response in MDD subjects. These data suggest that a simple, high-throughput-capable assay for depression and antidepressant response can be developed. Future studies are needed to evaluate the utility of this biomarker for the treatment of MDD.

BackgroundPlasma membrane localization can play a significant role in the ultimate function of certain proteins. Specific membrane domains like lipid rafts have been shown to be inhibitory domains to a number of signaling proteins, including Gsα, and chronic antidepressant treatment facilitates Gs signaling by removing Gsα form lipid rafts. The intent of this study is to compare the effects of the selective serotnin reuptake inhibitor, escitalopram, with that of the mood stabilizing drug, lithium.ResultsThere are a number of mechanisms of action proposed for lithium as a mood stabilizing agent, but the interactions between G proteins (particularly Gs) and mood stabilizing drugs are not well explored. Of particular interest was the possibility that there was some effect of mood stabilizers on the association between Gsα and cholesterol-rich membrane microdomains (lipid rafts), similar to that seen with long-term antidepressant treatment. This was examined by biochemical and imaging (fluorescence recovery after photobleaching: FRAP) approaches. Results indicate that escitalopram was effective at liberating Gsα from lipid rafts while lithium was not.ConclusionsThere are a number of drug treatments for mood disorders and yet there is no unifying hypothesis for a cellular or molecular basis of action. It is evident that there may in fact not be a single mechanism, but rather a number of different mechanisms that converge at a common point. The results of this study indicate that the mood stabilizing agent, lithium, and the selective serotonin reuptake inhibitor, escitalopram, act on their cellular targets through mutually exclusive pathways. These results also validate the hypothesis that translocation of Gsα from lipid rafts could serve as a biosignature for antidepressant action.

The relationship between depression, its etiology and therapy, and the cAMP signaling system have been studies for decades. This review will focus on cAMP, G proteins and adenylyl cyclase and depression or antidepressant action. Both human and animal studies are compared and contrasted. It is concluded that there is some synteny in the findings that cAMP signaling is attenuated in depression and that this is reversed by successful antidepressant therapy. The G protein that activates adenylyl cyclase, Gαs, appears to have diminished access to adenylyl cyclase in depression, and this is rectified by successful antidepressant treatment. Unfortunately, attempts to link specific isoforms of adenylyl cyclase to depression or antidepressant action suffer from discontinuity between human and animal studies.