Nathan H. Wray and Mark M. Rasenick
Neuropsychopharmacology (2018) 0:1–2
No abstract available
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.
Harinder Singh et al. Neuropsychopharmacology volume 43, pages 1481–1491 (2018)
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 Gαs from lipid rafts toward increased association with adenylyl cyclase (AC). Concomitant with this is a sustained increase in cAMP production. While Gαs modifies microtubule dynamics, tubulin also acts as an anchor for Gαs in lipid-rafts. Since HDAC-6 inhibitors potentiate α-tubulin acetylation, we hypothesize that acetylation of α-tubulin disrupts tubulin-Gαs raft-anchoring, rendering Gαs 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 Gαs from lipid-rafts, without changing total Gαs. Reciprocally, depletion of α-tubulin acetyl-transferase-1 ablated this phenomenon. While escitalopram and imipramine also disrupt Gαs/tubulin complexes and translocate Gαs 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 Gαs translocation from lipid-rafts as a biosignature for antidepressant response that may be useful in the development of new antidepressant compounds.
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 (Gαs) is increasingly localized to lipid rafts in depressed subjects and that chronic antidepressant treatment translocates Gαs from lipid rafts. Translocation of Gαs, 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 Gαs 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 Gαs 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.
The heterotrimeric, G protein-coupled receptor-associated G protein, Gαs, binds tubulin with nanomolar affinity and disrupts microtubules in cells and in vitro. Here we determine that the activated form of Gαs binds tubulin with a KD of 100 nM, stimulates tubulin GTPase, and promotes microtubule dynamic instability. Moreover, the data reveal that the _3–_5 region of Gαs is a functionally important motif in the Gαs mediated microtubule destabilization. Indeed, peptides corresponding to that region of Gαs mimic Gαs protein in activating tubulin GTPase and increase microtubule dynamic instability.
We have identified specific mutations in peptides or proteins that interfere with this process. The data allow for a model of the Gαs/tubulin interface in which Gαs binds to the microtubule plus-end and activates the intrinsic tubulin GTPase. This model illuminates both the role of tubulin as an “effector” (e.g. adenylyl cyclase) for Gαs and the role of Gαs as a GTPase activator for tubulin. Given the ability of Gαs to translocate intracellularly in response to agonist activation, Gαs may play a role in hormone- or neurotransmitter-induced regulation of cellular morphology.
The biomolecular approach to major depression disorder is explained by the different steps that involve cell membrane viscosity, Gsα protein and tubulin. For the first time it is hypothesized that a biomolecular pathway exists, moving from cell membrane viscosity through Gsα protein and Tubulin, which can condition the conscious state and is measurable by electroencephalogram study of the brain’s γ wave synchrony.
Chronic antidepressant treatment has been shown to increase adenylyl cyclase activity, in part, due to translocation of Gsα from lipid rafts to a nonraft fraction of the plasma membrane where they engage in a more facile stimulation of adenylyl cyclase. This effect holds for multiple classes of antidepressants, and for serotonin uptake inhibitors, it occurs in the absence of the serotonin transporter. In the present study, we examined the change in the amount of Gsα in lipid raft and whole cell lysate after exposing C6 cells to escitalopram. The results showed that chronic (but not acute) escitalopram decreased the content of Gsα in lipid rafts, whereas there was no change in overall Gsα content. These effects were drug dose and exposure time-dependent. Although R-citalopram has been reported to antagonize some effects of escitalopram, this compound was without effect on Gsα localization in lipid rafts, and R-citalopram did not inhibit these actions of escitalopram. Escitalopram treatment increased cAMP accumulation, and this seemed due to increased coupling between Gsα and adenyly cyclase. Thus, escitalopram is potent, rapid and efficacious in translocating Gsα from lipid rafts, and this effect seems to occur independently of 5-hydroxytryptamine transporters. Our results suggest that, although antidepressants display distinct affinities for well identified targets (e.g., monoamine transporters), several presynaptic and postsynaptic molecules are probably modified during chronic antidepressant treatment, and these additional targets may be required for clinical efficacy of these drugs.
Donati RJ, et.al The Journal of Neuroscience, March 19, 2008 28(12):3042–3050
Recent in vivo and in vitro studies have demonstrated that Gsα migrates from a Triton X-100 (TX-100)-insoluble membrane domain (lipid raft) to a TX-100-soluble nonraft membrane domain in response to chronic, but not acute, treatment with tricyclic or selective serotonin reuptake inhibitor antidepressants. This migration resulted in a more facile association with adenylyl cyclase. Our hypothesis is that Gsα maybe ensconced, to a greater extent, in lipid rafts during depression, and that one action of chronic antidepressant treatment is to reverse this. In this postmortem study, we examined Gsα membrane localization in the cerebellum and prefrontal cortex of brains from nonpsychiatric control subjects and suicide cases with confirmed unipolar depression. Sequential TX-100 and TX-114 detergent extractions were performed on the brain tissue. In the cerebellum, the ratio of TX-100/TX-114-soluble Gsα is ~2:1 for control versus depressed suicides. Results with prefrontal cortex samples from each group demonstrate a similar trend. These data suggest that depression localizes Gsα to a membrane domain (lipid rafts) where it is less likely to couple to adenylyl cyclase and that antidepressants may upregulate Gsα signaling via disruption of membrane microenvironments. Raft localization of Gsα in human peripheral tissue may thus serve as a biomarker for depression and as a harbinger of antidepressant responsiveness.
Donati RJ, Rasenick MM. Future Neurology (2008) 3(5), 511-514
No abstract available.
Lipid rafts are specialized structures on the plasma membrane that have an altered lipid composition as well as links to the cytoskeleton. It has been proposed that these structures are membrane domains in which neurotransmitter signalling might occur through a clustering of receptors and components of receptor-activated signalling cascades. The localization of these proteins in lipid rafts, which is affected by the cytoskeleton, also influences the potency and efficacy of neurotransmitter receptors and transporters. The effect of lipid rafts on neurotransmitter signaling has also been implicated in neurological and psychiatric diseases.
Donati RJ, Rasenick MM. Neuropsychopharmacology (2005) 30, 1238–1245
Previous studies demonstrated that Gsa migrates from a Triton X-100 (TTX-100) insoluble membrane domain to a TTX-100 soluble membrane domain in response to chronic treatment with the antidepressants desipramine and fluoxetine. Antidepressant treatment also causes a Gsa redistribution in cells as seen by confocal microscopy. The current studies have focused on examining the possibility that the association between Gsa and the plasma membrane and/or cytoskeleton is altered in response to antidepressant treatment, and that this is relevant to both Gsa redistribution and the increased coupling between Gsa and adenylyl cyclase seen after chronic antidepressant treatment. Chronic treatment of C6 cells with two functionally and structurally distinct antidepressants, desipramine and fluoxetine, decreased the Gsa content of TTX-100 insoluble membrane domains by as much as 60%, while the inactive fluoxetine analog LY368514 had no effect. Disruption of these membrane domains with the cholesterol chelator methyl-b-cyclodextrin altered the localization of many proteins involved in the cAMP signaling cascade, but only Gsa localization was altered by antidepressant treatment. In addition, microtubule disruption with colchicine elicited the movement of Gsa out of detergent-resistant membrane domains in a manner identical to that seen with antidepressant treatment. The data presented here further substantiate the role of Gsa as a major player in antidepressant-induced modification of neuronal signaling and also raise the possibility that an interaction between Gsa and the cytoskeleton is involved in this process.