Robert J. Donati

G protein signaling and the molecular basis of antidepressant action.

Over the past four decades, a variety of interventions have been used for the treatment of clinical depression and other affective disorders. Several distinct pharmacological compounds show therapeutic efficacy. There are three major classes of antidepressant drugs: monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs), and tricyclic compounds. There are also a variety of atypical antidepressant drugs, which defy ready classification. Finally, there is electroconvulsive therapy, ECT. All require chronic (2-3 weeks) treatment to achieve a clinical response. To date, no truly inclusive hypothesis concerning a mechanism of action for these diverse therapies has been formed. This review is intended to give an overview of research concerning G protein signaling and the molecular basis of antidepressant action. In it, the authors attempt to discuss progress that has been made in this arena as well as the possibility that some point (or points) along a G protein signaling cascade represent a molecular target for antidepressant therapy that might lead toward a unifying hypothesis for depression. This review is not designed to address the clinical studies. Furthermore, as it is a relatively short paper, citations to the literature are necessarily selective. The authors apologize in advance to authors whose work we have failed to cite.

Postmortem Brain Tissue of Depressed Suicides Reveals Increased Gsα Localization in Lipid Raft Domains Where It Is Less Likely to Activate Adenylyl Cyclase

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 may be 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.

Chronic antidepressant treatment prevents accumulation of Gsα in cholesterol-rich, cytoskeletal-associated, plasma membrane domains (lipid rafts)

Previous studies demonstrated that Gsα 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 Gsα redistribution in cells as seen by confocal microscopy. The current studies have focused on examining the possibility that the association between Gsα and the plasma membrane and/or cytoskeleton is altered in response to antidepressant treatment, and that this is relevant to both Gsα redistribution and the increased coupling between Gsα and adenylyl cyclase seen after chronic antidepressant treatment. Chronic treatment of C6 cells with two fuctionally and structurally distinct antidepressants, desipramine and fluoxetine, decreased the Gsα 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-β-cyclodextrin altered the localization of many proteins involved in the cAMP signaling cascade, but only Gsα localization was altered by antidepressant treatment. In addition, microtubule disruption with colchicine elicited the movement of Gsα 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 Gsα as a major player in antidepressant-induced modification of neuronal signaling and also raise the possibility that an interaction between Gsα and the cytoskeleton is involved in this process.

Treatment of C6 Glioma Cells and Rats with Antidepressant Drugs Increases the Detergent Extraction of Gsα from Plasma Membrane

Abstract : Results from previous studies suggested that chronic treatment of rats or C6 glioma cells with antidepressants augments the coupling between Gs and adenylyl cyclase. As these effects on C6 glioma cells are seen in the absence of presynaptic input, several antidepressant drugs may have a direct “postsynaptic” effect on their target cells. It was hypothesized that the target of antidepressant action was some membrane protein that may regulate coupling between G proteins and adenylyl cyclase. To test this, C6 glioma cells were treated with amitriptyline, desipramine, iprindole, or fluoxetine for 3 days. Chlorpromazine served as a control for these treatments. Membrane proteins were extracted sequentially with Triton X‐100 and Triton X‐114 from C6 glioma cells. Triton X‐100 extracted more Gsα in membranes prepared from antidepressant‐treated C6 glioma cells than from control groups. In addition, cell fractionation studies revealed that the amount of Gsα in caveolin‐enriched domains was reduced after antidepressant treatment and that adenylyl cyclase comigrated with Gsα in the gradients. These data suggest that some postsynaptic component that increases availability of Gs to activate effector molecules, such as adenylyl cyclase, might be a target of antidepressant treatment.

Heterotrimeric G-Proteins Interact Directly with Cytoskeletal Components to Modify Microtubule-Dependent Cellular Processes

A large percentage of current drugs target G-protein-coupled receptors, which couple to well-known signaling pathways involving cAMP or calcium. G-proteins themselves may subserve a second messenger function. Here, we review the role of tubulin and microtubules in directly mediating effects of heterotrimeric G-proteins on neuronal outgrowth, shape and differentiation. G-protein-tubulin interactions appear to be regulated by neurotransmitter activity, and, in turn, regulate the location of Gα in membrane microdomains (such as lipid rafts) or the cytosol. Tubulin binds with nanomolar affinity to Gsα, Giα1 and Gqα (but not other Gα subunits) as well as Gβ1γ2 subunits. Gα subunits destabilize microtubules by stimulating tubulin’s GTPase, while Gβγ subunits promote microtubule stability. The same region on Gsα that binds adenylyl cyclase and Gβγ also interacts with tubulin, suggesting that cytoskeletal proteins are novel Gα effectors. Additionally, intracellular Giα-GDP, in concert with other GTPase proteins and Gβγ, regulates the position of the mitotic spindle in mitosis. Thus, G-protein activation modulates cell growth and differentiation by directly altering microtubule stability. Further studies are needed to fully establish a structural mechanism of this interaction and its role in synaptic plasticity.

Structural model of a complex between the heterotrimeric G protein, Gsα, and tubulin

A number of studies have demonstrated interplay between the cytoskeleton and G protein signaling. Many of these studies have determined a specific interaction between tubulin, the building block of microtubules, and G proteins. The alpha subunits of some heterotrimeric G proteins, including Gsalpha, have been shown to interact strongly with tubulin. Binding of Galpha to tubulin results in increased dynamicity of microtubules due to activation of GTPase of tubulin. Tubulin also activates Gsalpha via a direct transfer of GTP between these molecules. Structural insight into the interaction between tubulin and Gsalpha was required, and was determined, in this report, through biochemical and molecular docking techniques. Solid phase peptide arrays suggested that a portion of the amino terminus, alpha2-beta4 (the region between switch II and switch III) and alpha3-beta5 (just distal to the switch III region) domains of Gsalpha are important for interaction with tubulin. Molecular docking studies revealed the best-fit models based on the biochemical data, showing an interface between the two molecules that includes the adenylyl cyclase/Gbetagamma interaction regions of Gsalpha and the exchangeable nucleotide-binding site of tubulin. These structural models explain the ability of tubulin to facilitate GTP exchange on Galpha and the ability of Galpha to activate tubulin GTPase.

Tubulin as a regulator of G-protein signaling.

Tubulin is known to form high-affinity complexes with certain G proteins. The formation of such complexes allows tubulin to activate Galpha and fosters a system whereby elements of the cytoskeleton can influence G-protein signaling. This article describes the interaction between tubulin and G proteins and discusses methods for examining this interaction.

Differential effects of antidepressants escitalopram versus lithium on Gs alpha membrane relocalization

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.

Polypharmacy and the lack of oculo-visual complaints from those with mental illness and dual diagnosis

BACKGROUND Individuals with mental illness (MI) and intellectual disability (ID) are characterized as dually diagnosed (DD). These individuals are known to have numerous systemic and oculo-visual anomalies. This comorbidity of conditions should elicit frequent oculo-visual complaints from these patients during the initial review of systems. A search of MedLine yielded one article that was published on oculo-visual symptomology/pain associated with MI and DD. This report appears to be the first to assess the frequency of these symptoms within these 2 unique populations. METHODS A retrospective analysis of all medical records for patients (N = 202) evaluated at the Neumann Association (NA) Developmental Disabilities Service of the Illinois Eye Institute was completed. Only the records of patients who had either MI or DD and who were prescribed antipsychotics, antidepressants, anticonvulsants, or tranquilizers/anxiolytics were used for our analysis. Upon record review, 89.9% of MI and 59.4% of DD individuals met the above subject criteria. We determined the frequency of ocular anomalies, drugs taken, and complaints reported by patients during the initial review of systems. RESULTS The most common documented side effects for the targeted drug types were decreased or blurred vision (near or far), visual hallucinations, decreased accommodation, and eyelid/conjunctiva irregularities. In our sample, the most frequent ocular anomalies encountered were astigmatism (50% MI and 37.84% DD), myopia (60.71% MI and 62.16% DD), presbyopia (35.71% MI and 37.84%DD), and blepharitis (32.14% MI and 32.43%DD). Additionally, the most frequently encountered complaints were no complaints (45.16% MI and 46.84% DD), blurry vision (17.74% MI and 17.72% DD), and need new glasses (11.29% MI and 17.72% DD). CONCLUSIONS It has been established that MI and DD populations exhibit a higher incidence of oculo-visual anomalies (uncorrected refractive error, binocular vision anomalies, ocular pathology) than noted in the general population. They are also typically taking 1 or more neuropsychotropic medications that are frequently associated with undesirable visual side effects. Individuals with MI and DD should report numerous complaints associated with the medications they take and the oculo-visual anomalies they exhibit during the initial case history and the review of systems. The data from this study suggest that this is not the case and that only about 50% of those who should have complaints actually report them.

Lipid rafts, G proteins and the etiology of and treatment for depression: progress toward a depression biomarker

Despite decades of research, the cellular target(s)of chronic antidepressant treatment remainunknown. For the most part, antidepressant treat-ments, whether drug or direct stimulation,require repeated administration for approximatelya month before achieving clinical benefit, whilecognitive therapy typically requires 12–16 weeksnot including follow-up visits. Many antidepres-sant drugs are suggested to increase synapticmonoamine content as they prevent the reuptakeor breakdown of monoamines by presynapticnerve terminals. These actions are contemporane-ous with acute drug exposure and it is difficult tocorrelate these actions with the required chronictreatment. We hypothesize that chronic anti-depressant drug treatment reorganizes the synap-tic membrane and modifies neurotransmittersignaling and that these effects are relevant to thebiology and treatment of depression.Receptors for monoamine neurotransmittersare often G protein-coupled, and those that arecoupled to G proteins, activate adenylyl cyclaseand generate cAMP. Extensive studies on the roleof antidepressant treatment and the G