EP4646596A2 - Methods for diagnosing depression and identifying antidepressant activity - Google Patents

Abstract

Disclosed herein, are methods of determining the amount of Gas palmitoylation in a blood sample using mass spectrometry. Also disclosed herein, are methods of determining the severity of depression in a subject as well as determining the whether a subject will respond to antidepressant therapy by determining the amount of Gas palmitoylation in a blood sample using mass spectrometry. Further disclosed herein are methods of determining the effectiveness of antidepressant agent in a depressed subject by measuring the association of Gas with adenylyl cyclase by fluorescence or by methods for measuring cAMP.

Description

METHODS FOR DIAGNOSING DEPRESSION AND IDENTIFYING ANTIDEPRESSANT ACTIVITY

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos. 63/478,610, filed January 5, 2023 and 63/520,158, filed August 17, 2023. The content of these earlier filed applications is hereby incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant number BX001149 awarded by the United States Department of Veterans Affairs and grant numbers AT009169 and R41MH113398 awarded by the National Institute of Health. The government has certain rights in the invention.

INCORPORATION OF THE SEQUENCE LISTING

The present application contains a sequence listing that is submitted concurrent with the filing of this application, containing the file name “37759_0451Pl_SL” which is 4.096 bytes in size, created on December 22, 2023, and is herein incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND

The World Health Organization (WHO) states that major depressive disorder (MDD) is the most common cause of disability worldwide, and has been exacerbated during the COVID pandemic. Although approximately 1 in 6 Americans will suffer from MDD during their lifetime, roughly half of the patients with MDD go undiagnosed in primary care settings, and another fifth are incorrectly diagnosed with MDD, while suffering from another psychiatric illness. Currently, no biologic test can diagnose major depressive disorder (MDD) or distinguish it from other psychiatric conditions. The above factors create a tremendous need for a simple, objective, fast and accurate test for MDD diagnosis and prediction of treatment response. Moreover, the desperate need for antidepressants is stymied by high development costs and the inability' to employ screening methods for potential drugs in a manner that accounts for hysteresis of therapeutic onset. SUMMARY

Disclosed herein are methods comprising: a) contacting a sample with a sulfhydryl reducing agent; and b) determining the amount of Gas palmitoylation in the sample by mass spectrometry. In some aspects, the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample. In some aspects, the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.

Disclosed herein are methods of determining the amount of Gas palmitoylation in a subject, the methods comprising: a) contacting a sample obtained from the subject with a sulfhydryl reducing agent; and b) detemiining the amount of Gas palmitoylation in the sample by mass spectrometry. In some aspects, the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample. In some aspects, the amount of Gas palmitoylation in the sample can be determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.

Disclosed herein are methods of determining the severity of depression in a subject, the methods comprising: a) contacting a sample obtained from the subject with a sulfhydryl reducing agent; and b) determining the amount of Gas palmitoylation in the sample by mass spectrometry, wherein the amount of Gas palmitoylation in the sample is indicative of the severity of depression in the subject. In some aspects, the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample. In some aspects, the amount of Gas palmitoylation in the sample can be determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.

Disclosed herein are methods comprising: a) obtaining a sample from a subject; b) contacting the sample with a sulfhydryl reducing agent; c) determining the amount of Gas palmitoylation in the sample in b) by mass spectrometry'; d) administering an anti-depressant agent to the subject; e) obtaining a sample from the subject after step d); f) contacting the sample with a sulfhydryl reducing agent; and g) determining the amount of Gas palmitoylation in the sample in step f) by mass spectrometry; and h) comparing the amount of Gas palmitoylation from step c) with the amount of Gas palmitoylation from step g). In some aspects, the amount of Gas palmitoylation can be decreased in step g) compared to the amount Gas palmitoylation in step c) indicating the efficacy of the anti-depressant agent. In some aspects, the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample. In some aspects, the amount of Gas palmitoylation in the sample can be determined by determining the amount of palmitoylated Gas to non- palmitoylated Gas in the sample.

Disclosed herein are methods comprising: a) contacting a sample obtained from a subject with a sulfhydryl reducing agent; b) determining the amount of Gas palmitoylation in the sample in by mass spectrometry; c) administering an anti-depressant agent to the subject;; d) contacting a second sample obtained from a subject after step c) with a sulfhydryl reducing agent; and e) determining the amount of Gas palmitoylation in the sample in step d) by mass spectrometry; and e) comparing the amount of Gas palmitoylation from step b) with the amount of Gas palmitoylation from step e). In some aspects, the amount of Gas palmitoylation can be decreased in step e) compared to the amount Gas palmitoylation in step b) indicating the efficacy of the anti-depressant agent. In some aspects, the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample. In some aspects, the amount of Gas palmitoylation in the sample can be determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.

Disclosed herein are methods comprising: a) administering an anti-depressant agent to a subject; b) obtaining a sample from the subject after step a) at a first time point; c) obtaining a sample at from the subject after step a) at a second time point, wherein the second time point is after the first time point; d) contacting the samples from step b) and step c) with a sulfhydryl reducing agent; e) determining the amount of Gas palmitoylation in the samples from b) and c) by mass spectrometry; and f) comparing the amount of Gas palmitoylation from the first time point with the amount of Gas palmitoylation from the second time point. In some aspects, the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample. In some aspects, the amount of Gas palmitoylation in the sample can be determined by determining the amount of palmitoylated Gas to non- palmitoylated Gas in the sample.

Disclosed herein are methods comprising: a) administering an anti-depressant agent to a subject; b) contacting a first and second sample obtained from the subject with a sulfhydryl reducing agent, wherein the first sample was obtained at a first time point after step a) and the second sample w as obtained at a second time point after step a), wherein the second time point is after the first time point; c) determining the amount of Gas palmitoylation in the first and second samples by mass spectrometry; and d) comparing the amount of Gas palmitoylation from the first sample with the amount of Gas palmitoylation from the second sample. In some aspects, the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample. In some aspects, the amount of Gas palmitoylation in the sample can be determined by determining the amount of palmitoylated Gas to non- palmitoylated Gas in the sample.

Disclosed herein are methods of screening a candidate compound for antidepressant activity, the methods comprising: a) contacting the candidate compound with one or more cells; b) incubating the one or more cells in step a) with an anti-Gas antibody; c) detecting the amount of Gas palmitoylation in the one or more cells after step b) by mass spectrometry: d) comparing the amount of Gas palmitoylation in the one or more cells in step a) with the amount of Gas palmitoylation in control cells that have not been exposed to the candidate compound, and wherein when the amount of Gas palmitoylation is decreased compared to the Gas palmitoylation of control cells that have not been exposed to the candidate compound is indicative of antidepressant activity. In some aspects, the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample. In some aspects, the amount of Gas palmitoylation in the sample can be determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.

Disclosed herein are methods of determining the severity of depression in a subject, the methods comprising: a) contacting a sample with an anti-Gas antibody; and b) determining the amount of Gas palmitoylation in the sample in a) by mass spectrometry; wherein the amount of the Gas palmitoylation in the sample is indicative of the severity of depression in the subject. In some aspects, the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample. In some aspects, the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.

Disclosed herein are methods for identifying an agent having antidepressant activity, the methods comprising: a) contacting the agent with cells; b) incubating the cells for a period of up to 72 hours; and c) measuring the association of Gas with adenylyl cyclase by cAMP fluorescence, wherein an increase in the association of Gas with adenylyl cyclase relative to control cells that have not been exposed to the agent is indicative of antidepressant activity⁷. In some aspects, the cells can be cultured cells. In some aspects, the cells can be blood cells. Disclosed herein are methods of identifying an agent having antidepressant activity in a depressed subject, the methods comprising: a) contacting the agent with cells, wherein the cells are obtained from the depressed subject and maintained in culture; b) incubating the cells for a period of up to 72 hours with putative antidepressant agents; and c) measuring the association of Gas with adenylyl cyclase by fluorescence, wherein an increase in the association of Gas with adenylyl cyclase is indicative of antidepressant activity. In some aspects, the cells can be cultured cells. In some aspects, the cells can be blood cells.

Disclosed herein are methods of determining the effectiveness of antidepressant agent in a depressed subject, the method comprising: a) contacting the agent with cells obtained from the depressed subject, wherein the depressed subject is being treated with the antidepressant agent at a first time point and a second time point; b) incubating the cells for a period of up to 72 hours; and c) measuring the association of Gas with adenylyl cyclase by fluorescence, wherein an increase in the association of Gas with adenylyl cyclase measured in the cells obtained at the second time point relative to the association of Gas with adenylyl cyclase measured in the cells obtained at the first time point indicates that the antidepressant therapy is effective. In some aspects, the cells can be cultured cells. In some aspects, the cells can be blood cells.

Disclosed herein are methods of determining the severity of depression in a subject, the methods comprising measuring Gas-activated adenylyl cyclase in cells obtained from the subject wherein the measured levels of Gas-activated adenylyl cyclase correlate negatively with the extent to which a subject is depressed. In some aspects, the cells can be cultured cells. In some aspects, the cells can be blood cells.

Disclosed herein are methods of monitoring a subject’s recovery from depression after antidepressant treatment, the methods comprising measuring levels of Gas-activated adenylyl cyclase in cells obtained from the subject, wherein the extent of the subject’s recovery correlates positively with the measured levels of Gas-activated adenylyl cyclase. In some aspects, the cells can be cultured cells. In some aspects, the cells can be blood cells. In some aspects, the cells can be obtained from saliva.

Disclosed herein are methods of identifying a subject that will respond to a therapeutic agent by comparing the association of Gas with adenylyl cyclase or comparing the levels of Gas-activated adenylyl cyclase before and after administering the therapeutic agent to the subject. Disclosed herein are methods comprising: a) contacting a sample with an anti-pan palmitoylated antibody and an anti-Gas antibody; b) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity ligation assay; and c) determining the amount of Gas palmitoylation in the sample.

Disclosed herein are methods comprising: a) obtaining a sample from a subject; b) contacting the sample with an anti -pan palmitoylated antibody and an anti-Gas antibody; c) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity ligation assay; d) determining the amount of Gas palmitoylation in the sample; e) administering an anti-depressant agent to the subject; f) obtaining a sample from the subject after step e); g) contacting the sample with an anti -pan palmitoylated antibody and an anti-Gas antibody; h) detecting Gas palmitoylated protein and Gas protein in the sample in step g) by a proximity ligation assay; i) determining the amount of Gas palmitoylation in the sample in step h); and j) comparing the amount of Gas palmitoylation from step d) with the amount of Gas palmitoylation from step i).

Disclosed herein are methods comprising: a) contacting a sample obtained from a subject with an anti-pan palmitoylated antibody and an anti-Gas antibody; b) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity ligation assay; c) determining the amount of Gas palmitoylation in the sample; d) administering an antidepressant agent to the subject; e) contacting a sample obtained from the subject after step d) with an anti-pan palmitoylated antibody and an anti-Gas antibody; f) detecting Gas palmitoylated protein and Gas protein in the sample in step e) by a proximity ligation assay; f) determining the amount of Gas palmitoylation in the sample in step f); and g) comparing the amount of Gas palmitoylation from step c) with the amount of Gas palmitoylation from step f).

Disclosed herein are methods of screening a candidate compound for antidepressant activity⁷, the methods comprising: a) contacting the candidate compound with one or more cells; b) incubating the one or more cells in step a) with an anti-pan palmitoylated antibody and an anti-Gas antibody; c) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity ligation assay; d) determining the amount of Gas palmitoylation in the one or more cells after step c); e) comparing the amount of Gas palmitoylation in the one or more cells in step a) with the amount of Gas palmitoylation in control cells that have not been exposed to the candidate compound, and wherein when the amount of Gas palmitoylation is decreased compared to the Gas palmitoylation of control cells that have not been exposed to the candidate compound is indicative of antidepressant activity.

Disclosed herein are methods of determining the severity of depression in a subject, the methods comprising: a) contacting a sample obtained from the subject with an anti -pan palmitoylated antibody and an anti -Gas antibody; b) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity ligation assay; and c) determining the amount of Gas palmitoylation in the sample; wherein the amount of the Gas palmitoylation in the sample is indicative of the severity of depression in the subject.

Other features and advantages of the present compositions and methods are illustrated in the description below, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A-B show that Ga_s is nascently palmitoylated and is depalmitoylated in response to escitalopram treatment. Gas is natively palmitoylated and antidepressant stimulation mediates its depalmitoylation. Immunoprecipitations of Gas from C6 plasma membrane preparations were reacted with N-ethylmaleimide (NEM); depalmitoylated peptides are NEM conjugated. Subsequent treatment with hydroxylamine was used to control for false positive identification. LC-MS/MS experiments were performed as described in the Methods. FIG. 1 A shows representative MSi spectra of the palmitoylated N- terminal Ga_s peptide (MGC(Paim)LGNSK; SEQ ID NO: 1) immunoprecipitated Ga_s from naive C6 cell plasma membrane as well as following 72h treatment with escitalopram (SSRI); with and without subsequent hydroxylamine treatment is included. FIG. IB shows representative MS2 fragmentation spectra for the palmitoylated Gas peptide (naive membrane) and NEM conjugated Ga_s peptide (10 pM escitalopram, 3-day treated membrane) respectively. Detection of MGC(p_aim)LGNSK (SEQ ID NO: 1) as compared with MGC(NEM)LGNSK (SEQ ID NO: 1) revealed escitalopram (SSRI) mediates depalmitoylation of Gas.

FIGS. 2A-B show chronic treatment with antidepressant drugs but not the antipsychotic, olanzapine, or the inactive citalopram enantiomer, R-citalopram. increases depalmitoylation of Gas. Representative MSi spectrum antidepressant treated (72 h) C6 cells with subsequent hydroxylamine treatment. LC-MS/MS experiments were performed as described in the Methods. FIG. 2A shows the detection of the NEM conjugated peptide (MGC(NEM)LGNSK; SEQ ID NO: 1, green peak) revealed phenelzine (MAOI), desipratnine (TCA), fluoxetine (SSRI), and escitalopram (SSRI). but not the inactive stereoisomer R- citalopram. nor the antipsychotic olanzapine. The classes of antidepressant treatments tested yielded NEM-conjugated Gets peptides (depalmitoylation of Gets), although the magnitude of this effect varied among drugs. FIG. 2B shows peak integrations for MGC(NEM)LGNSK (SEQ ID NO: 1) are normalized to the aggregate total integration for each immunoprecipitation. Normalized means are presented as fold change from control with SEM and analyzed by one-way ANOVA followed by a post-hoc Dunnetf s t-test (n=3: *, p<0 05; **, p<0.01; ***, p<0.001).

FIGS. 3A-D show that the depalmitoylation inhibitor, palmostatin-B, blocks escitalopram-induced translocation of Gets out of lipid rafts. C6 cells were treated with either vehicle (FIG. 3A), escitalopram (FIG. 3B), palmostatin-B (FIG. 3C) or escitalopram plus palmostatin-B (FIG. 3D) for 3 days. Treated cells were fractionated by sucrose density centrifugation and fractions were probed for Ga_s and caveolin-1 by immunoblotting. Translocation out of caveolin-1 rich fractions (lipid rafts) was observed in escitalopram treated cells and this was blocked by the addition of palmostatin-B, which blocks antidepressant-induced depalmitoylation of Gets.

FIGS. 4A-C show GFP-Ga_s palmitoylation mutants are not translocated after antidepressant treatment with ketamine. C6 cells were transfected with wild type (Ga_s-GFP) and modified Gas-GFP (NoPalm and MyrPalm) constructs, respectively. FIG. 4A shows that confocal imaging of N-terminal mutants of Ga_s-GFP reveals their differential membrane localization. Ga_s-GFP is natively palmitoylated and localizes to lipid rafts. Mutating the palmitoylation site (NoPalm) results in an entirely cytosolic distribution whereas modification of the N-terminus to resemble Go, (MyrPalm) results in both membrane and particulate distribution within the cell interior. After 24 hours, cells were treated with lOpM ketamine for 30 min. and harvested in TME buffer. Cell pellets are then homogenized, cell membrane, TX100 (non-raft) and TX114 (raft) soluble fractions are obtained by sequential centrifugation. FIG. 4B show s representative western blots of endogenous and the three GFP- Gas constructs are shown with control and ketamine treatments. The upper band is the GFP- Gas construct and the lower band, native, endogenously expressed Ga_s. About one third of cells are transfected, so net cellular expression of the constructs is about 3x that of the native Ga_s. FIG. 4C shows the ratio of Ga_s and Gas-GFP in TTX100 and TTX114 fractions are plotted. The higher the TX-100/TX-114. the less Ga_s in lipid rafts. NEE terminal sequences for the constructs are Ga_s(WT): MGCLGNSK (SEQ ID NO: 1): Gas(no palm) MGSLGNSK (SEQ ID NO: 2); Ga_s (myr-palm) MGCTLSSK (SEQ ID NO: 3).

FIGS. 5 A-B show that N-terminal acylation of Gets is a major determinant of subcellular localization and response to antidepressant treatment. Confocal imaging of N- terminal mutants of Gas-GFP reveals their differential membrane localization and indicates the importance of acylation in the response to antidepressants. FIG. 5A shows Gas-GFP is natively palmitoylated and localizes to lipid rafts (Gets and Gas-GFP). Modification of the N- terminus to resemble Gai (Myr/Palm: MGCTSLSK; SEQ ID NO: 3), results in both membrane and particulate distribution within the cell interior, whereas mutating the palmitoylation site results in an entirely cytosolic distribution palmitoylation deficient Ga_s mutant (C3S) (No palm: MGSLGNSK; SEQ ID NO: 2). Gcts(WT): MGCLGNSK (SEQ ID NO: 1). FIG. 5B shows that modification of acylation impairs the antidepressant response where doubly acylated Gets no longer responds to escitalopram by translocating from the lipid raft. Chronic antidepressant treatment does not appreciably affect expression of Go or Gas- GFP. Sample means are presented with SEM and analyzed by one-way ANOVA followed by a post-hoc Dunnetfs t-test (n=3; *, p<0.05; **, p<0.01).

FIGS. 6A-D show that ketamine decreases Ga lipid raft localization. FIG. 6 A shows C6 cells that were treated with 10 pm ketamine for 15 min or 24 h and lipid raft fractions isolated and probed for Gas. Both groups show a statistical decrease of Gas in lipid raft fractions indicating ketamine-mediated Ga translocation from lipid rafts into non-raft regions of the plasma membrane, blots were re-probed for caveolin-1 to confirm lipid raft fractions. The histogram on the left represents the average lipid raft localization of 4 experiments. FIG. 6B shows Ga-GFP C6 cells that were treated for 15 min or 24 h and Ga lateral mobility⁷ analyzed by fluorescence recovery after photobleaching (FRAP), which revealed a statistical increase of recovery half-time consistent with augmented association of Ga and adenylyl cyclase. FIGS. 6C-6D show the dose-response of ketamine-mediated Ga translocation analyzed by TX100 lipid raft isolation (FIG. 6C) and FRAP (FIG. 6D), both methods reveal a dose dependency of ketamine for Ga translocation, which occurred at clinically relevant levels of drug (n < 4) *p < 0.05; **p < 0.01; ***p < 0.001;****p < 0.0001.

FIG. 7 shows that Ga lipid raft localization returns 24 h after ketamine withdrawal. C6 cells were treated with ketamine for 15 min and drug was washed off. Cells were collected at 15 min, 1, 6, 12 and 24 h afterward, lipid rafts were isolated and probed for Ga. Data reveal the localization of Ga in non-raft regions is maintained for 12 h after 15-minute treatment (n = 4). *p < 0.05; **p < 0.01. Ketamine effects were no longer evident by 24 h after washout. This in-vitro method mirrors the clinical effects of ketamine which last for a few days after infusion.

FIGS. 8A-B show that other NMDA antagonist do not mediate Ga translocation from lipid rafts. C6 cells were treated with NMD AR antagonists AP-V. memantine, and MK-801 for 15 min and Ga translocation was analyzed by (FIG. 8A) lipid raft isolation (n = 3) and (FIG. 8B) FRAP (n > 48). Both methods reveal no effect upon Ga lipid raft localization, indicating ketamine-mediated Ga translocation may result from an NMDAR-independent target of ketamine.

FIGS. 9A-C show that ketamine increases phosphorylation of PKA-associated proteins and BDNF in primary astrocytes. Western blot analysis of cAMP/PKA-associated proteins, C6 cells were treated for 15 min with 10 pM ketamine and collected at the indicated time point and probed for (FIG. 9 A) phosphory lation of CREB at Ser- 133 which showed elevated levels for 24 h and statistically significant increases at 15 min and 2 h. The histogram represents the average of CREB phosphorylation from 5 determinations. The blot is a representative determination. FIG. 9B shows phosphorylation of eEF2 at Thr-56 showed increased levels for 24 h reaching after reaching statistical significance at Ih (n = 4). FIG. 9C shows primary astrocytes that were treated with ketamine or ketamine plus 1 pM cAMPS-Rp (a cAMP antagonist) and cells collected 24 h later, the ketamine-treated group showed an increase in BDNF, which was abolished by the cAMP antagonist. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIG. 10 shows that (2 R,6 R)-Hydroxynorketamine, a ketamine analog that does not inhibit NMDA receptors, mediates attenuated Ga lipid raft localization and cAMP accumulation similarly to ketamine. Ga -GFP monoclonal C6 cells were treated for 15 mm with 10 pM (2 R,6 R)-HNK and Ga lateral mobility analyzed by FRAP (n > 75), which revealed an increase of recovery⁷ half-time, suggesting augmented association of Ga and adenylyl cyclase. ***p < 0.001; **** > < 0.0001.

FIG. 11 shows that PGE1 -activated adenylyl cyclase is significantly lower in platelets from depressed subjects, while the innate adenylyl cyclase activity⁷ does not differ between groups. Platelets collected from 41 subjects with major depressive disorder (MDD) and 44 healthy controls at the screen visit were isolated and assayed for cAMP. Values displayed are mean +/- SEM. Basal (intrinsic) cAMP accumulation at the screen visit was 24.3 ± 13.6 (SD) nM cAMP/well in the MDD subjects and 27.1 ± 14. 1 nM cAMP/well in the healthy controls: t= -0.87; p=ns.

FIG. 12 shows a change from screen values of PGE-1 stimulated cAMP signaling response in platelets from major depressive disorder (MDD) treatment responders and nonresponders. Platelets were collected at pre-treatment (visit 1) and post-treatment after 6 weeks of antidepressant treatment (visit 3) from 19 MDD subjects. PGE1 change from screen to visit 3 (6 weeks) was 1.35 ± 2.3 (SD) in treatment responders compared to 0.32 ± 1.0 nm cAMP/well in nonresponders: t=2. 14; p-0.050. nPGEl stimulation of adenylyl cyclase activity normalized over basal activity at that visit was assessed and the difference between the screen and 6 weeks scores was calculated. Antidepressant responders were defined as MDD subjects who had total HamD17 score improvement >50% between the screen visit 1 and visit 3.

FIG. 13 shows that for individual subjects responding to antidepressant therapy, decrease in depression severity (HAM-D) correlates with Gas-activated adenylyl cyclase increase. Platelets were collected at pre-treatment (visit 1) and post-treatment after 6 weeks of antidepressant treatment (visit 3) from 19 subjects with major depressive disorder (MDD). PGE1 change from screen to visit 3 (6 weeks) was 1.35 ± 2.3 (SD) in treatment responders compared to -0.32 ± 1.0 nm cAMP/well in nonresponders: t=2. 14; p=0.050. PGE1 stimulation of adenylyl cyclase activity normalized over basal activity at that visit was assessed and the difference between the screen and 6 weeks scores was calculated. Antidepressant responders were defined as MDD subjects who had total HamD17 score improvement >/= 50% betw een the screen visit 1 and visit 3.

FIG. 14 shows blood that was collected and then separated into components and stored at -80°C. Adenylyl cyclase (AC) assays were carried out as described in Targum et.al. Mol. Psy chiatty, 2022, 27(3): 1640-1646) and AC activity in the presence of PGE1 was compared to that in the absence of activator. The net activation by PGE1 represents the Gas-activated-AC score. Platelets from MDD subjects exhibited lower levels of Gas- activated AC. which correlated with the severity of depression as measured by FIAM-D. (p=0.02, r=-0.54).

FIG. 15 show s Quick Inventory of Depressive Symptomatology -Self Report (QIDS- SRie) patient self-ratings suggest that PGE-1 -activated adenylyl cyclase in platelets acts as a biomarker for mild depression. ANOVA: mildly depressed subjects (QIDS-SRis >6) vs. healthy controls: F=11.6; p=0.002; and mildly depressed subjects (QIDS-SRis >6) vs. asymptomatic depressed subjects (QIDS-SR16 <6): F=12.7; p= 0.001,

FIG. 16 shows the stability of measuring Gas -activated- AC in platelets.

FIG. 17 shows that the Gas-activated-AC is stable in a subject with stable QIDS, and varies with changes in depression severity.

DETAILED DESCRIPTION

The present disclosure can be understood more readily by reference to the following detailed description of the invention, the figures and the examples included herein.

Before the present compositions and methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

Moreover, it is to be understood that unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, and the number or type of aspects described in the specification.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

Ranges can be expressed herein as from “about” or “approximately” one particular value, and/or to “about” or “approximately” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” or “approximately,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value " 10" is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not.

As used herein, the term “subject” refers to the target of administration, e.g., a human. Thus, the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). In some aspects, a subject is a mammal. In some aspects, a subject is a human. The term does not denote a particular age or sex. Thus, adult, child, adolescent and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

As used herein, the term “patient” refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects. In some aspects of the disclosed methods, the “patient” has been diagnosed with a need for treatment for depression, such as, for example, prior to an administering step. “Treatment” and “treating” refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, a treatment may include administration of a pharmaceutically effective amount of an antidepressant agent.

As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting or slow ing progression of. reducing severity of. and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. For example, the disease, disorder, and/or condition can be depression or major depressive disorder.

The terms “preventing,” “blocking,” “antagonizing,” or “reversing” mean preventing in whole or in part, or ameliorating or controlling.

The terms “diminishing,” “reducing,” or “preventing,” “inhibiting,” and variations of these terms, as used herein include any measurable decrease, including complete or substantially complete inhibition. The terms “enhance” or “enhanced” as used herein include any measurable increase or intensification.

“Inhibit,” “inhibiting” and “inhibition” mean to diminish or decrease an activity, level, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% inhibition or reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, in some aspects, the inhibition or reduction can be a 10, 20, 30, 40, 50, 60, 70, 80. 90. 100%, or any amount of reduction in between as compared to native or control levels. In some aspects, the inhibition or reduction is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% as compared to native or control levels. In some aspects, the inhibition or reduction is 0-25, 25-50, 50-75, or 75- 100% as compared to native or control levels.

As used herein, the term “sample” is meant a tissue or organ from a subject; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), which is assayed as described herein. A sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells or cell components.

As used herein, the term '‘determining” can refer to measuring or ascertaining a quantity or an amount or a change in activity'. For example, determining the amount of a disclosed polypeptide, protein, gene or antibody in a sample as used herein can refer to the steps that the skilled person would take to measure or ascertain some quantifiable value of the polypeptide protein, gene or antibody in the sample. The art is familiar with the ways to measure an amount of the disclosed polypeptide, proteins, genes or antibodies in a sample.

As used herein, the terms “disease” or “disorder” or “condition” are used interchangeably referring to any alternation in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with a person. A disease or disorder or condition can also related to a distemper, ailing, ailment, malady, disorder, sickness, illness, complaint, affection.

As used herein. “Gas”. “Gas” and “Gsa” are used interchangeably and refer to the G- protein Gas.

Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of’), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.

The World Health Organization currently lists depression as the leading cause of disability', yet successful treatment is hindered in part by the limited understanding of how antidepressants function to treat the disease. It has recently been shown that antidepressants accumulate over time in lipid rafts, independent of the serotonin transporter, whereby they function to mediate the redistribution of Gas to non-raft membrane regions, but previously it was unclear whether this targeting was affected by antidepressants. It is demonstrated herein that Ga_s is depalmitoylated following sustained exposure to several classes of antidepressants. Moreover, mutant forms of Gas, in which palmitoyl ati on is altered or blocked, are not displaced from rafts by antidepressant treatment. Thus, several classes of antidepressants associate with lipid rafts and effect depalmitoylation of Gets, translocating it to the non-raft membrane fraction where it potentiates the cAMP-signaling cascade. Described herein are methods useful diagnosing and treating depression wherein the amount of palmitoylated Gets is determined in a sample from a subject.

G proteins/cAMP/CREB/'neurotrophin in depression and antidepressant action.

Several studies (vide infra) indicate that chronic antidepressant treatment increases physical coupling between Gsalpha (Gsa) and adenylyl cyclase, resulting in increased cAMP generation. This finding is consistent with the observation that chronic treatment with antidepressants results in long-term increases in cellular cAMP (Malberg JE and Blendy JA. Trends Pharm Sci. 2005; 26:631-8). Consistent with this, depressed subjects show decreased ⁿC rolipram binding that recovers with successful antidepressant therapy (Fujita M, et al. Mol Psychiatry. 2017; 22:754-9). Evidence also exists that cAMP signaling may be involved in antidepressant action. O’Donnell and Zhang (O’Donnell JM and Zhang HT. Trends Pharm Sci. 2004; 25: 158-63) discuss antidepressant properties of PDE4 inhibitors (and PDE4 knockout in mice). The long-term sequelae of antidepressant treatment include sustained cAMP increases as well as cAMP-induced transcription of growth factor genes (Racagni G and Popoli M. Dialogues Clin Neurosci. 2008; 10:385-400). In a recent clinical study, increasing cAMP with inhibitors of phosphodiesterase showed promising antidepressantadjuvant properties (El-Haggar SM, et al. Psychother Psychosom. 2018; 87:331-9). The initial studies showing that CREB knockout blocks the behavioral response to antidepressants date back 20 years and more recent papers target serotonergic and noradrenergic neurons in achieving this effect (Blendy JA. Biol Psychiatry⁷. 2006; 59:1144-50; and Rafa-Zablocka K, et al. Sci Rep. 2017; 7: 13515). BDNF and TrkB knockout also ablated antidepressant effects in mice (Bjorkholm C and Monteggia LM. Neuropharmacology. 2016; 102:72-79). Both humans and mice with the BDNF val66met allele are more vulnerable to stress-induced anxiety⁷ and depression, but this is variable with age and sex (Hwang JP, et al. Neurobiol Aging. 2006; 27:1834-7; and Verhagen M, et al. Mol Psychiatry. 2010; 15:260-71). A polymorphism in the regulatory region of the human BDNF gene, which reduces BDNF expression and release, is also associated with depression (Bjorkholm C and Monteggia LM. Neuropharmacology. 2016;l 02:72-79). The Gs alpha subunit (Gas, Gsa) is a subunit of the hetero trimeric G protein Gs that stimulates the cAMP-dependent pathway by activating adenylyl cyclase. Gsa is a GTPase that functions as a cellular signaling protein. Gsa is the founding member of one of the four families of heterotrimeric G proteins, defined by the alpha subunits they contain: the Gas family, Gai/Gao family, Gaq family, and Gal2/Gal3 family. The Gs-family has two members: the other member is Golf, named for its predominant expression in the olfactory system. In humans, Gsa is encoded by the GNAS complex locus, while Golfa is encoded by the GNAL gene.

The general function of Gs is to activate intracellular signaling pathways in response to activation of cell surface G protein-coupled receptors (GPCRs). GPCRs function as part of a three-component system of receptor-transducer-effector. The transducer in this system is a heterotrimeric G protein, composed of three subunits: a Ga protein such as Gsa, and a complex of two tightly linked proteins called Gf> and Gy in a G0y complex. When not stimulated by a receptor, Ga is bound to GDP and to GPy to form the inactive G protein trimer. When the receptor binds an activating ligand outside the cell (such as a hormone or neurotransmitter), the activated receptor acts as a guanine nucleotide exchange factor to promote GDP release from and GTP binding to Ga, which drives dissociation of GTP-bound Ga from GPy. In particular, GTP-bound, activated Gsa binds to adenylyl cyclase to produce the second messenger cAMP, which in turn activates the cAMP-dependent protein kinase (also called Protein Kinase A or PKA).

Although each GTP-bound Gsa can activate one adenylyl cyclase enzyme, amplification of the signal occurs because one receptor can activate multiple copies of Gs while that receptor remains bound to its activating agonist, and each Gsa-bound adenylyl cyclase enzyme can generate substantial cAMP to activate many copies of PKA.

G protein signaling and lipid rafts. The localization of G proteins to specific membrane domains such as caveolae and lipid rafts has generated interest in these cholesterol and sphingolipid-rich detergent-resistant membrane domains and how they affect G protein targeting and function (Li S, Okamoto T, et al. J Biol Chem. 1995; 270: 15693-701; and Allen JA, et al. Nat Rev Neurosci. 2007; 8: 128-40). Lipid rafts have variable effects on signaling, as they promote Gqalpha (Gqa) signaling and inhibit Gsa signaling (Allen JA, et al. Lipid raft microdomains and neurotransmitter signaling. Nat Rev Neurosci. 2007; 8: 128- 40; Bhatnagar A, et al. J Biol Chem. 2004; 279:34614-23; Allen JA, et al. Mol Pharm. 2005; 67: 1493-504; and Allen JA, et al. Mol Pharmacol. 2009; 76: 1082-108). A long experimental history of agents that increase “membrane fluidity'’ increasing agonist- and Gsa-mediated adenylyl cyclase exist (Rimon G, et al. Nature. 1978; 276:394-6; and Rasenick MM, et al. Nature. 1981; 294:560-2). Gsa stimulates adenylyl cyclase more efficiently outside of lipid rafts and chronic treatment with antidepressants facilitates G protein exodus from those rafts (Allen JA, et al. Mol Pharm. 2005;67: 1493-504; Allen JA, et al. Mol Pharmacol. 2009;76: 1082-108; Donati RJ and Rasenick MM. Neuropsychopharmacology.

2005;30: 1238-45; Zhang L and Rasenick MM. J Pharm Exp Ther. 2010;332:977-84; Czysz AH, et al. Neuropsychopharmacology. 2015;40:766-73; Singh H, et al.

Neuropsychopharmacology. 2018;43: 1481-91; and Liu JJ, et al. Neuropsychopharmacology. 2018;43:2165-79). Sublette et al. recently reviewed lipid rafts and the possible roles of these structures in the biology of depression and Kapoor and colleagues have demonstrated direct effects of antidepressants on lipid bilayers (Liu JJ, et al. Neuropsychopharmacology. 2018;43:2165-79; and Kapoor R, et al. J Gen Physiol. 2019;151 :342-56).

The accumulating evidence as disclosed herein suggests that antidepressants may translocate Gsa from lipid rafts and. in so doing, facilitate the stimulation of adenylyl cyclase by Gsa in the non-raft membrane fraction. These findings suggest that lipid rafts from depressed subjects would show enriched Gsa consistent with diminished cAMP signaling. Donati and colleagues observed enriched Gsa in lipid rafts in both the prefrontal cortex and cerebellum (Donati RJ, et al. J Neurosci. 2008; 28:3042-50). No major change in the total Gsa, and no other G proteins were altered in lipid raft localization in MDD subjects versus controls. More recent post-mortem data showed that the anchors (non-acetylated tubulin) for Gsa in lipid rafts were increased significantly in samples from MDD subjects (Singh H, et al. J Neurosci. 2020;40:4033-41), providing a cellular rationale for the enrichment of Gsa in rafts from depressed subjects. Antidepressant treatment compromised the association between Gsa and tubulin in lipid rafts (Singh H, et al. Neuropsychopharmacology. 2018; 43: 1481-91).

Disclosed herein are methods, including, for example, a adenylyl cyclase assay using blood cells that can be used to diagnosis of MDD and antidepressant response that can also be adaptable to high throughput screening.

Depression is the leading cause of long-term disability’ in the world (Chen, G, et al. (2010) Science translational medicine 2, 54ps51). Canonically, anti-depressant drugs are assumed to inhibit the proteins responsible for monoamine reuptake (transporters) or catabolism (monoamine oxidase). However, the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) trial revealed that roughly two thirds of subjects with major depressive disorder (MDD) fail to remit following their initial drug (citalopram) trial and 40% of those relapse within ayear (Rush, A. J., et al. (2006) Am J Psychiat 163, 1905-1917). Furthermore, monoamine-centric antidepressants exhibit several weeks delay before therapeutic onset (hysteresis) (Maes. M., et al. (2009) Metab Brain Dis 24, 27-53), and recent positron emission tomography (PET) evidence suggests a global reduction in cAMP in depressed humans that is normalized effective antidepressant treatment (Fujita, M., et al. (2017) Molecular psychiatry 22, 754-759). Taken together, this information suggests different mechanistic targets apart from/complimentary to the monoamine system.

Disclosed herein are methods, for example, a high-throughput blood biomarker-based test to evaluate, preemptively, antidepressant-efficacy and improve current MDD treatment strategies.

The effects of lipid rafts and sustained antidepressant treatments on the membrane redistribution of the G protein can affect the accumulation of cAMP (Ga_s). Lipid rafts are regions of the plasma membrane rich in caveohn and cholesterol.

Palmitoylation of native Ga_s occurs on the third cysteine residue, and enzymatic digestion results in a doubly charged try ptic peptide of sequence: MGCLGNSK (SEQ ID NO: 1). Results disclosed herein that examine this peptide reveal a mechanistic explanation consistent with delayed therapeutic response for antidepressants. These results also are consistent with the translocation of Gets from lipid rafts as a reproducible biomarker for antidepressant efficacy.

Disclosed herein are methods for determining the effectiveness of antidepressant therapy, methods for screening for agents having antidepressant activity’, and methods of treating a patient suffering from depression. Disclosed herein are methods for determining the effectiveness of antidepressant therapy, methods for screening for agents having antidepressant activity, and methods of treating a patient suffering from depression wherein the amount of palmitoylated Ga_s is determined in a sample from a patient.

Described herein are cellular assays that can be used to confirm the depressive state of a sample from a subject, and that can also be used to predict the efficacy of a compound in the treatment of depression. The assay enriches a protein, Gas in cholesterol-rich membrane structures (lipid rafts) in depression and the translocation of Gas from those lipid rafts during effective antidepressant treatment.

Translocation of Gas from lipid rafts is coincides with an increase in the activity of adenylyl cyclase (the enzyme activated by Gas). As described herein, the measurements of Gas -activated adenylyl cyclase in platelets tracks with both depression severity' and antidepressant response. These data demonstrate that an assay that measures cAMP (the product of adenylyl cyclase) can form the basis of a high throughput screen for antidepressant activity that could also be used in antidepressant drug development.

In addition, the extent of Gas -activated adenylyl cyclase measures correlates negatively with the extent to which a subject is depressed, and the extent to which a subject recovers from depression as a result of antidepressant treatment is correlated positively with Gas -activated adenylyl cyclase and the effectiveness of an antidepressant corresponds to the loss of lipid-raft specific adenylyl cyclase.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

METHODS

As described herein, are methods of determining the amount of Gas palmitoylation in a sample by mass spectrometry.

Disclosed herein are methods comprising: contacting a sample with a sulfhydryl reducing agent; and determining the amount of Gas palmitoylation in the sample by mass spectrometry. In some aspects, the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample. In some aspects, the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample. In some aspects, the method can be conducted without contacting the sample with a sulfhydryl reducing agent or otherwise in the absence of a sulfhydryl reducing agent.

Disclosed herein are methods of determining the amount of Gas palmitoylation in a subject. In some aspects, the methods can comprise: contacting a sample obtained from the subject with a sulfhydryl reducing agent; and determining the amount of Gas palmitoylation in the sample by mass spectrometry. In some aspects, the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non- palmitoylated Gas in the sample. In some aspects, the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample. In some aspects, the amount of Gas palmitoylation in the sample can be determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample. In some aspects, the method can be conducted without contacting the sample with a sulfhydryl reducing agent or otherwise in the absence of a sulfhydryl reducing agent.

Disclosed herein are methods of determining the severity of depression in a subject. In some aspects, the methods can comprise: contacting a sample obtained from the subject with a sulfhydryl reducing agent; and determining the amount of Gas palmitoylation in the sample by mass spectrometry. In some aspects, the amount of Gas palmitoylation in the sample can be indicative of the severity of depression in the subject. In some aspects, the amount of Gas palmitoylation in the sample can be determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample. In some aspects, the method can be conducted without contacting the sample with a sulfhydryl reducing agent or otherwise in the absence of a sulfhydry l reducing agent.

As also described herein, are methods of predicting drug (or therapeutic agent) responsiveness in samples from depressed subjects or from subjects with major depressive disorder.

Disclosed herein are methods comprising: a) obtaining a sample from a subject; b) contacting the sample with a sulfhydry l reducing agent; c) determining the amount of Gas palmitoylation in the sample in b) by mass spectrometry’; d) administering an anti-depressant agent to the subject; e) obtaining a sample from the subject after step d); I) contacting the sample with a sulfhydryl reducing agent; and g) determining the amount of Gas palmitoylation in the sample in step f) by mass spectrometry’; and h) comparing the amount of Gas palmitoylation from step c) with the amount of Gas palmitoy lation from step g). In some aspects, the sample from the subject in step e) can be obtained one week after the sample obtained from the subject after step d). In some aspects, the time the second sample is obtained can be dependent on the antidepressant that was administered to the subject. For example, the sample obtained from the subject at the second time point can be one day after the sample obtained from the subject at the first time point when the antidepressant administered was ketamine. In some aspects, the sample obtained from the subject at the second time point can be between 1 and 24 hours, 1 days and 7 days, 1 week and 4 weeks or any time in between after the sample obtained from the subject at the first time point. In some aspects, the method can be conducted without steps b) and f) (e.g., without contacting the sample with a sulfhydryl reducing agent or otherwise in the absence of a sulfhydryl reducing agent.

Disclosed herein are methods comprising: a) contacting a sample obtained from a subject with a sulfhydryl reducing agent; b) determining the amount of Gas palmitoylation in the sample in by mass spectrometry; c) administering an anti-depressant agent to the subject: d) contacting a second sample obtained from a subject after step c) with a sulfhydryl reducing agent; and e) determining the amount of Gas palmitoylation in the sample in step d) by mass spectrometry; and e) comparing the amount of Gas palmitoylation from step b) with the amount of Gas palmitoylation from step e). In some aspects, the amount of Gas palmitoylation can be decreased in step e) compared to the amount Gas palmitoylation in step b) indicating the efficacy of the anti-depressant agent. In some aspects, the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample. In some aspects, the amount of Gas palmitoylation in the sample can be determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.

Disclosed herein are methods comprising: a) obtaining a sample from a subject; b) contacting the sample with a sulfhydryl reducing agent; c) determining the amount of Gas palmitoylation in the sample in b) by mass spectrometry; d) administering a therapeutic agent to the subject; e) obtaining a sample from the subject after step d); f) contacting the sample with a sulfhydryl reducing agent; and g) determining the amount of Gas palmitoylation in the sample in step f) by mass spectrometry; and h) comparing the amount of Gas palmitoylation from step c) with the amount of Gas palmitoylation from step g). In some aspects, a decrease in the amount of Gas palmitoylation from step g) as compared to the amount of Gas palmitoylation from step c) indicates the therapeutic agent has antidepressant activity. In some aspects, the therapeutic agent can be a therapeutic known to treat or ameliorate a symptom of depression. For example, the therapeutic agent can be, but is not limited to, any suitable pharmaceutical agent or other antidepressant agent. In some aspects, the method can be conducted without step b) (e.g., without contacting the sample with a sulfhydryl reducing agent) or otherwise in the absence of a sulfhydryl reducing agent).

Disclosed herein are methods comprising: a) contacting a sample with an anti-pan palmitoylated antibody and an anti-Gas antibody; b) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity' ligation assay; and c) determining the amount of Gas palmitoylation in the sample. In some aspects, the anti-pan palmitoylated antibody can bind a cysteine residue of the Gas palmitoylated protein. In some aspects, the anti-pan palmitoylated antibody can be CBL-PTM-pal. In some aspects, the anti-Gas antibody can bind the carboxy terminus of the Gas protein. In some aspects, the anti-Gas antibody can be N192/12. In some aspects, the proximity ligation assay can comprise contacting the sample with pair of oligonucleotide-labeled secondary’ antibodies (also referred to herein as “proximity ligation assay probes”) that bind to the anti-pan palmitoylated antibody and an anti-Gas antibody, respectively. In some aspects, the anti-pan palmitoylated antibody and the anti-Gas antibody can be immobilized on a solid support. In an aspect, in the proximity' ligation assays disclosed herein, for example, the methods can utilize oligonucleotide-labeled secondary antibodies. In some aspects, the oligonucleotides on the oligonucleotide-labeled secondary antibodies can hybridize to a connector plasmid that can be amplified by PCR or some variation thereof. In some aspects, the methods can further comprise contacting the sample with hybridizing connector oligonucleotides capable of joining the oligonucleotide- labeled secondary antibodies. Hybridizing connector oligonucleotides are commercially available. In some aspects, the proximity ligation assay can be performed using immunofluorescence or immunohistochemistry. In some aspects, the detecting in step b) by the proximity ligation assay can be performed by flow cytometry. In some aspects, the detecting in step b) by the proximity ligation assay can be performed using fluorescent detection methods (e.g., using secondary antibodies). In some aspects, the fluorescent detection methods can be adapted for high throughput. For example, fluorescent detection methods that utilize multiple fluorophores of different wavelengths that can be quantified in a fluorescence plate reader or flow-through instrument can be used. Another example of a fluorescent detection method that can be used includes methods that implement FRET (Fluorescence (or Fourier) Resonance Energy Transfer. In this case, the two secondary antibodies can each have fluorescence probes wherein one fluorescence probe can be activated by the other fluorescence probe, provided that the molecules are in close proximity. Yet, another example of a fluorescent detection method that can be used in the disclosed methods can be bimolecular fluorescence complementation wherein each secondary antibody has a portion of a fluorescent protein, and when brought into proximity, a fluorescent probe can be generated.

As described herein, antibodies recognizing all Gas protein can be used in combination with and antibodies recognizing Gas cysteine palmitoylated proteins. In some aspects, the methods described herein can detect a single Gas protein that is bound to both of these antibodies. As such, specificity can be derived from the two antibodies binding to the same molecule. In some aspects of, an antibody can be developed in a species other than mouse to the palmitoylation sequence (including palmitate) on Gas. Such a method can be adapted for either fluorescence or colorimetric detection, in which a field test can be developed to screen for depression using a drop of blood. In some aspects, Gsa can be present on a plate, a bead, or a radial flow chamber and the methods can include the step of contacting the Gsa with a palmitoylated Gs antibody (or alternatively, an antipalmitoylated cysteine). Such methods can be used to generate a point of care test or an automated immunodetection assay.

Also disclosed herein are methods comprising: a) obtaining a sample from a subject; b) contacting the sample with an anti-pan palmitoylated antibody and an anti-Gas antibody ; c) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity ligation assay; d) determining the amount of Gas palmitoylation in the sample; e) administering an anti-depressant agent to the subject; f) obtaining a sample from the subject after step e); g) contacting the sample with an anti -pan palmitoylated antibody and an anti-Gas antibody; h) detecting Gas palmitoylated protein and Gas protein in the sample in step g) by a proximity ligation assay; i) determining the amount of Gas palmitoylation in the sample in step h); and j) comparing the amount of Gas palmitoylation from step d) with the amount of Gas palmitoylation from step i). In some aspects, when the amount of Gas palmitoy lation is decreased in step g) compared to the amount Gas palmitoylation in step c) can indicate the efficacy of the anti-depressant agent. IN some aspects, the blood cell can be a leukocyte, an erythrocyte or a platelet.

Disclosed herein are methods comprising: a) contacting a sample obtained from a subject with an anti -pan palmitoylated antibody and an anti-Gas antibody ; b) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity ligation assay; c) determining the amount of Gas palmitoylation in the sample; d) administering an antidepressant agent to the subject; e) contacting a sample obtained from the subject after step d) with an anti-pan palmitoylated antibody and an anti-Gas antibody; f) detecting Gas palmitoylated protein and Gas protein in the sample in step e) by a proximity ligation assay; f) determining the amount of Gas palmitoylation in the sample in step f); and g) comparing the amount of Gas palmitoylation from step c) with the amount of Gas palmitoylation from step f).

Also, disclosed herein are methods of assessing the efficacy of an anti-depressant agent in a subject. For example, disclosed herein are methods comprising: a) administering an anti-depressant agent to a subject; b) obtaining a sample at from the subject after step a) at a first time point; c) obtaining a sample at from the subject after step a) at a second time point, wherein the second time point is after the first time point; d) contacting the samples from step b) and step c) with a sulfhydryl reducing agent; e) determining the amount of Gas palmitoylation in the samples from b) and c) by mass spectrometry; and f) comparing the amount of Gas palmitoylation from the first time point with the amount of Gas palmitoylation from the second time point. In some aspects, the sample obtained from the subject at the second time point can be one week after the sample obtained from the subject at the first time point. In some aspects, the time the second sample can be obtained can be dependent on the antidepressant that was administered to the subject. For example, the sample obtained from the subject at the second time point can be one day after the sample obtained from the subject at the first time point when the antidepressant administered was ketamine. In some aspects, , the sample obtained from the subj ect at the second time point can be between 1 and 24 hours, 1 days and 7 days, 1 week and 4 weeks or any time in between after the sample obtained from the subject at the first time point. In some aspects, the method can be conducted without contacting the sample with a sulfhydryl reducing agent or otherwise in the absence of a sulfhydryl reducing agent.

Disclosed herein are methods comprising: a) administering an anti-depressant agent to a subject; b) contacting a first and second sample obtained from the subject with a sulfhydryl reducing agent, wherein the first sample was obtained at a first time point after step a) and the second sample w as obtained at a second time point after step a), wherein the second time point is after the first time point; c) determining the amount of Gas palmitoylation in the first and second samples by mass spectrometry; and d) comparing the amount of Gas palmitoylation from the first sample with the amount of Gas palmitoylation from the second sample. In some aspects, the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample. In some aspects, the amount of Gas palmitoylation in the sample can be determined by determining the amount of palmitoylated Gas to non- palmitoylated Gas in the sample. In some aspects, the sample obtained from the subject at the second time point can be one week after the sample obtained from the subj ect at the first time point. In some aspects, the time the second sample can be obtained can be dependent on the antidepressant that was administered to the subject. For example, the sample obtained from the subject at the second time point can be one day after the sample obtained from the subject at the first time point when the antidepressant administered was ketamine. In some aspects, the sample obtained from the subject at the second time point can be between 1 and 24 hours, 1 days and 7 days, 1 week and 4 weeks or any time in between after the sample obtained from the subj ect at the first time point. In some aspects, the method can be conducted without step b) (e.g., without contacting the sample with a sulfhydryl reducing agent) or otherwise in the absence of a sulfhydryl reducing agent.

Disclosed herein are methods of screening a candidate compound for antidepressant activity⁷. In some aspects, the methods can comprise: a) contacting the candidate compound with one or more cells; b) incubating the one or more cells in step a) with an anti-Gas antibody; c) determining the amount of Gas palmitoylation in the one or more cells after step b) by mass spectrometry; d) comparing the amount of Gas palmitoylation in the one or more cells in step a) with the amount of Gas palmitoylation in control cells that have not been exposed to the candidate compound. In some aspects, the amount of Gas palmitoylation can decrease compared to the Gas palmitoylation of control cells that have not been exposed to the candidate compound is indicative of antidepressant activity. In some aspects, the anti-Gas antibody can be any commercially available anti-Gas antibody. For example, the anti-Gs protein, alpha subunit antibody (N 192/12; NeuroMab) can be used. In some aspects, the cells can be cultured cells. In some aspects, the cells can be blood cells. In some aspects, the one or more cells can be any cultured cell line of neural or glial origin. In some aspects, the one or more cells can be C6 glioma cells or SK N SH neuroblastoma cells. In some aspects, other neural or glial cell lines can be used. In some aspects, the one or more cells can be patient- derived neural stem cells. In some aspects, the cells can be hybrid cells. In some aspects, the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.

Also disclosed herein are methods of screening a candidate compound for antidepressant activity. In some aspects, the methods can comprise: a) contacting the candidate compound with one or more cells; b) incubating the one or more cells in step a) with an anti-pan palmitoylated antibody and an anti-Gas antibody; c) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity ligation assay; d) determining the amount of Gas palmitoylation in the one or more cells after step c); e) comparing the amount of Gas palmitoylation in the one or more cells in step a) w ith the amount of Gas palmitoylation in control cells that have not been exposed to the candidate compound. In some aspects, when the amount of Gas palmitoylation is decreased compared to the Gas palmitoylation of control cells that have not been exposed to the candidate compound can be indicative of antidepressant activity. In some aspects, the one or more cells can be C6 glioma cells, SK-N-SH neuroblastoma cells, or patient-derived neural stem cells. In some aspects, other cells or cell lines known to one of ordinary skill in the art can be used. Also, disclosed herein are methods of treating depression in a subject. In some aspects, the methods can comprise administering to a subject with depression an effective amount of the candidate compound identified using the methods of screening a candidate compound for antidepressant activity disclosed herein. In some aspects, the methods can comprise: a) contacting the candidate compound with one or more cells obtained from a subject; b) incubating the one or more cells in step a) with an anti-pan palmitoylated antibody and an anti-Gas antibody; c) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity ligation assay; d) determining the amount of Gas palmitoylation in the one or more cells after step c); e) comparing the amount of Gas palmitoylation in the one or more cells in step a) with the amount of Gas palmitoylation in control cells that have not been exposed to the candidate compound, wherein when the amount of Gas palmitoylation is decreased compared to the Gas palmitoylation of control cells that have not been exposed to the candidate compound can be indicative of antidepressant activity and further comprising administering the candidate compound to the subject.

Disclosed herein are methods for identifying an agent having antidepressant activity. In some aspects, the methods can comprise a) contacting the agent with cells; b) incubating the cells for a period of up to 72 hours; and c) measuring the association of Gas w ith adenylyl cyclase by cAMP fluorescence. In some aspects, the cells can be cultured cells. In some aspects, the cells can be blood cells. In some aspects, an increase in the association of Gas with adenylyl cyclase relative to control cells that have not been exposed to the agent is indicative of antidepressant activity. In some aspects, the cells can be any cultured cell line of neural or glial origin. In some aspects, the cells can be C6 glioma cells or SKN SH neuroblastoma cells. In some aspects, the cells can be patient-derived neural stem cells. In some aspects, the cells can be hybrid cells. In some aspects, the association of Gas with adenylyl cyclase can be determined by measuring cyclic adenosine monophosphate (cAMP) levels in the cells using a fluorescent cAMP reporter or fluorescent or luminescence-based cAMP assays. In some aspects, the association of Gas with adenylyl cyclase can be measured by fluorescence recovery' after photobleaching of GFP-Gas.

Disclosed herein are methods of identifying an agent having antidepressant activity in depressed subject, the methods comprising: a) contacting the agent with cells obtained from the depressed subject and maintained in culture; b) incubating the cells for a period of up to 72 hours w ith putative antidepressant agents; and c) measuring the association of Gas w ith adenylyl cyclase by fluorescence. In some aspects, an increase in the association of Gas with adenylyl cyclase is indicative of antidepressant activity. In some aspects, the association of Gas with adenylyl cyclase can be determined by measuring cyclic adenosine monophosphate (cAMP) levels in the cultured cells using a fluorescent cAMP reporter. In some aspects, the association of Gas with adenylyl cyclase can be measured by fluorescence recovery after photobleaching of GFP- of Gas. In some aspects, the increase in cAMP generation is relative to control cells that have not been exposed to the agent. In some aspects, the increase in cAMP generation is relative to control cells that have been exposed to a second agent having antidepressant activity. In some aspects, the cells can be cultured cells. In some aspects, the cells can be blood cells. In some aspects, the cultured cells can comprise blood cells. In some aspects, the blood cells can be erythrocytes. In some aspects, the blood cells can be leukocytes. In some aspects, the blood cells can be platelets. In some aspects, the methods further comprise administering the agent having antidepressant activity to the subject. Disclosed herein are methods of identifying an agent having antidepressant activity in depressed subject, the methods comprising: a) contacting the agent with cells obtained from the depressed subject and maintained in culture; b) incubating the cells for a period of up to 72 hours with putative antidepressant agents; c) measuring the association of Gas with adenylyl cyclase by fluorescence, wherein an increase in the association of Gas with adenylyl cyclase is indicative of antidepressant activity; and (d) administering the agent having antidepressant activity to the subject.

Disclosed herein are methods of determining the effectiveness of an antidepressant agent in a depressed subject, the methods comprising: a) contacting the agent with cells obtained from the depressed subject being treated with the antidepressant agent at a first time point and a second time point; b) incubating the cells for a period of up to 72 hours; and c) measuring the association of Gas with adenylyl cyclase by fluorescence. In some aspects, an increase in the association of Gas with adenylyl cyclase measured in the cells obtained at the second time point relative to the association of Gas with adenylyl cyclase measured in the cells obtained at the first time point indicates that the antidepressant therapy can be effective. In some aspects, the association of Gas with adenylyl cyclase can be determined by measuring cyclic adenosine monophosphate (cAMP) levels in the cultured cells using a fluorescent cAMP reporter. In some aspects, the association of Gas with adenylyl cyclase can be measured by fluorescence recovery after photobleaching of GFP- Gas. In some aspects, the increase in cAMP generation can be relative to control cells that have not been exposed to the agent. In some aspects, the cells can be cultured cells. In some aspects, the cells can be blood cells. Disclosed herein are methods of determining the effectiveness of an antidepressant agent in a depressed subject, the methods comprising: a) contacting the agent with cells obtained from the depressed subject being treated with the antidepressant agent at a first time point and a second time point; b) incubating the cells for a period of up to 72 hours; c) measuring the association of Gas with adenylyl cyclase byfluorescence, wherein an increase in the association of Gas with adenylyl cyclase measured in the cells obtained at the second time point relative to the association of Gas with adenylyl cyclase measured in the cells obtained at the first time point indicates that the antidepressant therapy is effective; and (d) administering the antidepressant agent to the subject.

In some aspects, the increase in cAMP generation is relative to control cells that have been exposed to a second agent having antidepressant activity. In some aspects, the cultured cells comprise blood cells. In some aspects, the blood cells can be erythrocy tes. In some aspects, the blood cells can be leukocytes. In some aspects, the blood cells can be platelets. In some aspects, the blood cells can be leukocytes. Disclosed herein are methods of determining the severity of depression in a subject comprising measuring Gas-activated adenylyl cyclase in cells obtained from the subject. In some aspects, the cells can be cultured cells. In some aspects, the cells can be blood cells. In some aspects, the measured levels of Gas -activated adenylyl cyclase correlate negatively with the extent to which a subject is depressed.

Further disclosed herein are methods of determining the severity of depression in a subject. In some aspects, the methods can comprise: a) contacting a sample obtained from the subject with an anti -pan palmitoylated antibody and an anti-Gas antibody; b) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity ligation assay; and c) determining the amount of Gas palmitoylation in the sample. In some aspects, the amount of the Gas palmitoylation in the sample can be indicative of the severity of depression in the subject. In some aspects, the sample can comprise a blood cell. In some aspects, the blood cell can be a leukocyte, an erythrocyte or a platelet.

Disclosed herein are methods of monitoring a subject’s recover}’ from depression as a result of antidepressant treatment comprising measuring levels of Gsa-activated adenylyl cyclase in cells obtained from the subject. In some aspects, the cells can be cultured cells. In some aspects, the cells can be blood cells. In some aspects, the extent of the subject's recovery correlates positively with the measured levels of Gas-activated adenylyl cyclase.

Also disclosed herein are methods of treating a depressed patient or subject. In some aspects, the methods can comprise administering to the depressed patient or subject an antidepressant agent identified using any of the methods disclosed herein.

Procedures for collecting or obtaining a sample from a subject can be done by methods known in the art. In some aspects, the sample can comprise one or more cells. In some aspects, the sample can be whole cells. In some aspects, the cells can be isolated from a bodily fluid. In some aspects, the sample can be fresh cells or cultured cells. In some aspects, the sample can be blood or comprise blood cells. In some aspects, the blood cells can be leukocytes. In some aspects, the blood cells can be erythrocytes. In some aspects, the blood cells can be platelets. In some aspects, the blood cells can be leukocytes, erythrocytes, platelets or a combination thereof. In some aspects, the one or more cells can be obtained from the subject after antidepressant therapy can be obtained one week after the start of antidepressant therapy. In some aspects, the one or more cells obtained from the subject at a second time point can be after one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, ten hours, eleven hours, twelve hours, thirteen hours, fourteen hours, fifteen hours, sixteen hours, seventeen hours, eighteen hours, nineteen hours, twenty hours, twenty-one hours, twenty-two hours, twenty-three hours, twenty-four hours, one day, two days, three days, four days, five days, six days, or seven days after administration of an antidepressant agent. In some aspects, the one or more cells obtained from the subject at a second time point can be after one week, two weeks, three weeks, or four weeks after administration of an antidepressant agent. In some aspects, the one or more cells obtained from the subject at a second time point can be after one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months or twelve months weeks after administration of an antidepressant agent.

In some aspects, the amount of Gas palmitoyl ati on in a sample can be determined as a percentage of total Gas in the sample. In some aspects, the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non- palmitoylated Gas in the sample. In some aspects, the sample can be from the same subject at the same time point. In some aspects, the sample can be from the same subject but at different time points. In some aspects, samples from a subject can be compared with samples contacted with a therapeutic agent or from subjects administered a therapeutic agent (e g., an antidepressant agent) to determine the percent change to identify a change in one or more symptoms of depression or major depressive disorder or to determine the severity of depression or major depressive disorder in a subject or a sample that will be (or will not be) responsive to, for example, an antidepressant agent (e.g., monoamine oxidase inhibitors, tricyclic antidepressants, SSRIs (selective serotonin reuptake inhibitors), SNRIs (serotonin, norepinephrine uptake inhibitors), ketamine and esketamine, an atypical antidepressant, and any other compounds showing antidepressant activity) or another treatment or therapeutic agent. In other w ords, the amount of Gas palmitoylation can be expressed as a percent. For example, the percent change in the amount of Gas palmitoylation can be decreased (or is lower) by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to the amount of Gas palmitoylation in a sample not contacted, administered or exposed to an anti-depressant agent or to the amount of Gas palmitoylation in a sample contacted, administered or exposed to an anti-depressant agent. Alternatively, the percent change in the amount of Gas palmitoylation can be increased (or higher) by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% when to the amount of Gas palmitoylation in a sample not contacted, administered or exposed to an anti-depressant agent or to the amount of Gas palmitoylation in a sample contacted, administered or exposed to an antidepressant agent.

In some aspects, the methods can further comprise comparing the amount of Gas palmitoylation in a sample or subject to a predetermined reference value.

As used herein, the term '‘reference,” “reference expression,” '‘reference sample,” “reference value,” “control,” “control sample” and the like, when used in the context of a sample or amount of Gas palmitoylation in a sample refers to a reference standard wherein the reference is measured at a constant level among a particular sample type, and is unaffected by the experimental conditions, and is indicative of the amount of Gas palmitoylation in a sample of a predetermined disease status (e.g., not suffering from depression) or whether a given amount of Gas palmitoylation in a sample (or subject) will respond to a therapeutic agent or treatment. The reference value can be a predetermined standard value or a range of predetermined standard values, representing no illness, or a predetermined type or severity of illness or representing the likelihood the disease (e.g., depression) will be responsive to a particular type of therapeutic agent or treatment.

Reference amount of Gas palmitoylation in a sample or total amount of Gas palmitoylation in a sample described herein in a reference sample from a subject, or a pool of subjects, not suffering from depression or with a known response (or lack thereof) to a particular treatment. In some aspects, the reference value can be the amount of Gas palmitoylation in a sample of a subject, or subjects, wherein the subject or subjects known to be a responder to a particular therapeutic agent or is known to be not be responsive to a particular therapeutic agent. In some aspects, the reference value can be the amount of Gas palmitoylation in a sample of the same subject before or after administration of or exposure to a particular therapeutic agent. In some aspects, the reference value can be taken a different time point than to which it is being compared.

As used herein, a “reference value” can be an absolute value; a relative value; a value that has an upper and/or lower limit; a range of values; an average value; a median value, a mean value, or a value as compared to a particular control or baseline value. A reference value can be based on an individual sample value, such as for example, a value obtained from a sample from the individual before administration of or exposure to a particular therapeutic agent, but at an earlier point in time, or a value obtained from a sample from depressive patient other than the individual being tested, or a '‘normal” individual, that is an individual not diagnosed with depression or major depressive disorder. The reference value can be based on a large number of samples, such as from depressive patients or normal individuals or based on a pool of samples including or excluding the sample to be tested. The reference value can also be based on a sample from a depressive patient other than the individual being tested, or a “normal” individual that is an individual not diagnosed with depression or major depressive disorder that has not or has been administered or exposed to a particular therapeutic agent.

The reference level used for comparison with the amount of Gas palmitoylation in a sample can vary, depending on the method begin practiced, as will be understood by one of ordinary skill in the art. For methods for determining the likelihood depression, a subject or a sample will be responsive to a particular ty pe of therapeutic agent or treatment, the “reference level” is typically a predetermined reference level, such as an average of levels obtained from a population that has either been exposed or has not been exposed to particular type of therapeutic agent or treatment, but in some instances, the reference level can be a mean or median level from a group of individuals that are responders or non-responders. In some instances, the predetermined reference level can be derived from (e g., is the mean or median of) levels obtained from an age-matched population.

Age-matched populations (from which reference values may be obtained) can be populations that are the same age as the individual being tested, but approximately age- matched populations are also acceptable. Approximately age-matched populations may be within 1, 2. 3, 4, or 5 years of the age of the individual tested, or may be groups of different ages which encompass the age of the individual being tested. Approximately age-matched populations may be in 2, 3, 4, 5, 6, 7, 8, 9, or 10 year increments (e.g. a “5 year increment” group which serves as the source for reference values for a 62 year old individual might include 58-62 year old individuals, 59-63 year old individuals, 60-64 year old individuals, 61- 65 year old individuals, or 62-66 year old individuals).

Determining the amount of Gas palmitoylation in a sample can include determining whether the amount of Gas palmitoylation in a sample is increased as compared to a control or reference sample or a sample that has been contacted, administered or exposed to a particular therapeutic agent or treatment, decreased compared to a control or reference sample or a sample that has been contacted, administered or exposed to a particular therapeutic agent or treatment, or unchanged compared to a control or reference sample or a sample that has been contacted, administered or exposed to a particular therapeutic agent or treatment,. As used herein, the terms, “increased” or “increased amount” or “increased Gas palmitoylation” or “increased amount of Gas palmitoylation in a sample” or “high” or “higher amount” or “higher amount of Gas palmitoylation in a sample” refers to an amount of Gas palmitoylation in a sample, that is measured wherein the quantity of Gas palmitoylation in a sample exhibits an increased level when compared to a reference sample or “normal” control or a sample that has been contacted, administered or exposed to a particular therapeutic agent or treatment. An “increased amount” or “higher amount” refers to an increase in expression of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or more, or greater than 1-fold, up to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more. As used herein, the terms “decreased,” “decreased amount,” or “decreased amount of Gas palmitoylation” or “low” or "lower amount” or “lower amount of Gas palmitoylation” refers to an amount of Gas palmitoylation in a sample that is expressed wherein the measure of the Gas palmitoylation in a sample exhibits a decreased level when compared to a reference sample or “normal” control or a sample that has been contacted, administered or exposed to a particular therapeutic agent or treatment. A “decreased amount” or “lower amount” refers to a decrease in expression of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or more, or greater than 1-fold, up to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more.

In some aspects, samples from a subject can be compared with reference samples or samples that have been contacted, administered or exposed to a particular therapeutic agent or treatment to determine the ratio of the biological sample amount of Gas palmitoylation in a sample to identify the severity⁷ of a depression or general status of the depression in a subject or a sample that will be (or will not be) responsive to, for example, an antidepressant agent, or another treatment or therapeutic agent. By comparing the amount, for example, in a sample of Gas palmitoylation in a sample with the amount of Gas palmitoylation in a sample or total amount of Gas in a sample that was also contacted with, for example, an antidepressant agent applying the methods disclosed herein, it is possible to identify⁷ the severity of the depression or depression or the sample from a subject with depression that will be responsive (or will not be responsive) to the antidepressant agent. Suitable statistical and other analysis can be carried out to confirm a change (e.g., a decrease or a lower amount) of Gas palmitoylation in a sample disclosed herein when compared with the amount of Gas palmitoylation or total amount of Gas in a sample that was also contacted with a therapeutic agent, wherein a ratio of the sample amount of Gas palmitoylation in a sample disclosed herein to the amount of the Gas palmitoylation in a sample that was also contacted with a therapeutic agent.

In some aspects, the amount of Gas palmitoylation in a sample can be compared to the total amount of Gas in a sample to determine the disease state of the sample (e.g., whether the disease is present or not or the severity of the disease).

The amount of the Gas palmitoylation in a sample or total amount of Gas in a sample can be a measure, for example, per unit weight or volume. In some aspects, the amount can be a ratio (e.g., the amount of Gas palmitoylation in a sample relative to the amount of the Gas palmitoylation in a sample of a reference value or in a sample that was also contacted with a therapeutic agent or the amount of Gas palmitoylation in a sample relative to the total amount of the Gas in a sample).

The method of comparing a measured value and a reference value or a measured value before and after contact with a therapeutic agent can be carried out in any convenient manner appropriate to the type of measured value. For example, "measuring’ can be performed using quantitative or qualitative measurement techniques, and the mode of comparing a measured value and a reference value can van depending on the measurement technology employed. For example, the measured values used in the methods described herein can be quantitative values (e.g.. quantitative measurements of concentration, such as nanograms per milliliter of sample, or absolute amount). As with qualitative measurements, the comparison can be made by inspecting the numerical data, by inspecting representations of the data (e.g., inspecting graphical representations such as bar or line graphs).

In some aspects, the sample not contacted, administered or exposed to an antidepressant agent or to the amount of Gas palmitoylation in a sample contacted, administered or exposed to an anti -depressant agent.

In some aspects, the methods disclosed herein can further comprise calculating a ratio of Gas palmitoylation to nonpalmitoylated Gas in the sample. In some aspects, the ratio of Gas palmitoylation to nonpalmitoylated Gas in the sample can be greater than 1 indicating depression in the subject. In some aspects, the ratio of Gas palmitoylation to nonpalmitoylated Gas in the sample can be 1.1: 1, 1.2: 1, 1.3: 1, 1.4: 1, 1.5: 1, 1.6: 1, 1.7: 1, 1.8: 1, 1.9: 1, 2: 1, 2.2: 1, 2.3: 1, 2.4: 1, 2.5: 1, 2.6: 1, 2.7: 1, 2.8: 1, 2.9: 1, 3: 1, 3.2:1, 3.3: 1, 3.4: 1, 3.5: 1, 3.6: 1, 3.7: 1, 3.8: 1, 3.9: 1, 4.0, 5:0, 6:0, etc. and any value in between. In some aspects, the ratio of Gas palmitoylation to nonpalmitoylated Gas in the sample can be less than 1 indicating the absence of depression in the subject. In some aspects, the ratio of Gas palmitoylation to nonpalmitoylated Gas in the sample can be 0.9: 1, 0.8: 1, 0.7: 1, 0.6: 1, 0.5: 1, 0.4: 1, 0.3: 1, 0.2: 1, 0. 1 : 1, or less, and any value in between. In some aspects, the sample will contain more palmitoylated Gas than nonpalmitoylated Gas. In some aspects, the amount or relative amount of palmitoylated Gas can be less in non-depressed or subjects with major depressive disorder or subjects with depression or major depression disorder treated successfully with antidepressants.

In some aspects, the one or more symptoms of depression or maj or depressive disorder can be anxiety, apathy, general discontent, guilt, hopelessness, loss of interest or pleasure in activities, mood swings, sadness, agitation, excessive crying, irritability, restlessness, social isolation, early awakening, excess sleepiness, insomnia, restless sleep, excessive hunger, fatigue, loss of appetite, lack of concentration, slowness in activity', weight gain, weight loss, poor appetite, repeatedly going over thoughts, or thoughts of suicide.

In some aspects, the antidepressant agent can be a selective serotonin reuptake inhibitor (SSRI), a serotonin and norepinephrine reuptake inhibitor (SNRI), an atypical antidepressant, a tricyclic antidepressant or a monoamine oxidase inhibitor (MAOI). Examples of SSRIs include but are not limited to fluoxetine (Prozac), paroxetine (Paxil, Pexeva), sertraline (Zoloft), citalopram (Celexa), vilazodone (Viibryd), and escitalopram (Lexapro). Examples of SNRIs include but are not limited to venlafaxine (Effexor) and duloxetine (Cymbalta). Examples of antidepressants include but are not limited to trazodone, mirtazapine (Remeron), vortioxetine (Trintellix), and bupropion (Forfivo XL, Wellbutrin SR). Examples of tricyclic antidepressants include but are not limited to imipramine, nortriptyline (Pamelor), amitriptyline, doxepin and desipramine (Norpramin). Examples of MAOIs include but are not limited to ranylcypromine (Parnate), phenelzine (Nardil), isocarboxazid (Marplan), and Selegiline (Emsam). Examples of rapid-acting antidepressants include but are not limited to ketamine or esketamine. Other examples of antidepressants include but are not limited to psilocybin, LSD and MDMA as well as other agents that act as antidepressants, including but not limited to biologies and devices (e.g., rTMS. ECT). In some aspects, one or more antidepressant agents can be combined.

The methods and assays described herein can be performed over time, and the change in the amount of Gas palmitoylation assessed. For example, the assays can be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter carried out as needed. Assays can also be completed prior to. during, or after a treatment protocol. Together, the information provided using any of the methods disclosed herein can be used to profile an individual's likelihood or responding to a particular therapeutic agent or treatment as well as determine that the subject has depression or major depressive disorder or determine the severity of the subject’s depression. In some aspects, the difference in the amount of Gas palmitoylation measured between two or more samples can be significantly different.

In some aspects, the sulfhydryl reducing agent can N-ethylmaleimide (NEM), dithiothreitol (DTT), iodoacetamide, or N-methyl Maleimide (NMM).

In some aspects, the methods can further comprise contacting the sample with a proteolytic enzyme after the step of contacting the sample with a sulfhydryl reducing agent.

Further disclosed herein are methods of determining the amount of Gas palmitoylation using an anti-Gas antibody. Disclosed herein are methods comprising: a) contacting a sample with an anti-Gas antibody; b) determining the amount of Gas palmitoylation and the amount of Gas in the sample; and c) determining the ratio of Gas palmitoylation to Gas in the sample. In some aspects, wherein the ratio of Gas palmitoylation to Gas in the sample can be greater than 1 indicates depression. In some aspects, the amount of Gas palmitoylation in the sample can be determined using mass spectrometry. In some aspects, the amount of Gas in the sample can be determined using mass spectrometry’. In some aspects, the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample. In some aspects, the anti-Gas antibody can be any commercially available anti-Gas antibody. For example, the anti-Gs protein, alpha subunit antibody (N192/12; NeuroMab) can be used in the methods disclosed herein.

Disclosed herein are methods comprising: a) administering an anti -depressant agent to a subject; b) obtaining a sample at from the subject after step a) at a first time point; c) obtaining a sample at from the subject after step a) at a second time point, wherein the second time point is after the first time point; d) contacting the samples from step b) and step c) with an anti-Gas antibody; e) determining the amount of Gas palmitoylation in the samples from b) and c) by mass spectrometry; and f) comparing the amount of Gas palmitoylation from the first time point with the amount of Gas palmitoylation from the second time point. In some aspects, the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample. In some aspects, the anti-Gas antibody can be any commercially available anti-Gas antibody. For example, the anti-Gs protein, alpha subunit antibody (N192/12; NeuroMab) can be used. In some aspects, the amount of Gas palmitoylation in the sample can be determined as a percentage of total Gas. In some aspects, the sample obtained from the subject at the second time point can be one week after the sample obtained from the subject at the first time point. In some aspects, the time the second sample can be obtained can be dependent on the antidepressant that was administered to the subject. For example, the sample obtained from the subject at the second time point can be one day after the sample obtained from the subject at the first time point when the antidepressant administered was ketamine. In some aspects, the sample obtained from the subject at the second time point can be between 1 and 24 hours. 1 days and 7 days, 1 week and 4 weeks or any time in between after the sample obtained from the subject at the first time point.

In some aspects, the subject can be a human subject. In some aspects, the subject can be an adult. In some aspects, the subject can be an adolescent. In some aspects, the subject can be a child. In some aspects, the subject can be depressed. In some aspects, the subject can have major depressive disorder. In some aspects, the subject has one or more symptoms of depression or major depressive disorder. In some aspects, the subject can maintained on the antidepressant therapy. In some aspects, the subject can have depression and anxiety.

METHODS OF TREATMENT

Disclosed herein are methods of treating a subject diagnosed using the methods disclosed herein.

Disclosed herein are methods of treating a subject in need thereof comprising: administering to the subject an antidepressant agent, wherein the subject in need thereof was identified using one or more of the methods disclosed herein. For example, disclosed herein are methods of treating a subject in need thereof comprising: administering to the subject an antidepressant agent, wherein the subject in need thereof was identified by: a) contacting a sample from the subject with a sulfhydry l reducing agent; and b) determining the amount of Gas palmitoylation in the sample by mass spectrometry, wherein the amount of Gas palmitoylation relative to total Gas in the sample is indicative of a subject in need of treatment with an antidepressant agent. In some aspects, the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non- palmitoylated Gas in the sample.

Disclosed herein are methods of treating a subject in need thereof comprising: administering to the subject an antidepressant agent, wherein the subject in need thereof was identified by measuring Gas-activated adenylyl cyclase in cells obtained from the subject wherein the measured levels of Gas-activated adenylyl cyclase identify a subject in in need of treatment with an antidepressant agent.

Disclosed herein, are methods of diagnosing a subject or a patient. Disclosed herein, are methods of diagnosing depression or major depressive disorder in a subject. In some aspects, the methods can comprise obtaining or having obtained a sample from the subject. In some aspects, the method an include obtaining a blood sample from the subject. In some aspects, the blood sample can comprise blood cells. In some aspects, the blood cell can be a leukocyte, an erythrocyte, or a platelet. In some aspects, the methods can comprise contacting the sample of a sulfhydryl reducing agent or an anti-Gas antibody. In some aspects, the sulfhydryl reducing agent can be N-ethylmaleimide, dithiothreitol, iodoacetamide, or N- methyl maleimide. In some aspects, the anti-Gas antibody can be any commercially available anti-Gas antibody. For example, the anti-Gs protein, alpha subunit antibody (N192/12; NeuroMab) can be used. In some aspects, the method can comprise determining the amount of Gas palmitoylation in the sample by mass spectrometry'. In some aspects, the amount of Gas palmitoylation in the sample can be determined as a percentage of total Gas. In some aspects, the comparative amount of Gas palmitoylation in the sample can be in an amount that indicates that the subject has depression or major depressive disorder. In some aspects, the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample. In some aspects, the method of diagnosing can further comprise a method of treating depression or major depressive disorder in a subject. In some aspects, the subject or patient can be a human. In some aspects, the subject can be an adult. In some aspects, the subject can be an adolescent. In some aspects, the subject can be a child. In some aspects, the method can be conducted without contacting the sample with a sulfhydry l reducing agent or otherwise in the absence of a sulfhydryl reducing agent.

Disclosed herein, are methods of treating a subject or a patient. In some aspects, the methods disclosed herein can comprise identifying a patient in need of treatment before the administration step. In some aspects, the subject or patient can be identified of being in need of treatment using any of the methods disclosed herein. In some aspects, the subject can be depressed. In some aspects, the subject can have major depressive disorder. In some aspects, the method an include obtaining a blood sample from the subject. In some aspects, the method an include obtaining a blood sample from the subject in need of treatment. In some aspects, the methods can include the step of administering a therapeutically effective amount of an antidepressant to the subject. In some aspects, the antidepressant agent can be a selective serotonin reuptake inhibitor (SSRI), a serotonin and norepinephrine reuptake inhibitor (SNRI), an atypical antidepressant, a tricyclic antidepressant, ketamine and esketamine, a monoamine oxidase inhibitor (MAOI) or any other compounds showing antidepressant activity. In some aspects, the methods can include the step of administering a therapeutically effective amount of a candidate compound identified as having antidepressant activity⁷ using any of the methods disclosed herein to the subject. In some aspects, the methods can include the step of administering a therapeutically effective amount of an antidepressant agent to the subject when it was determined that the subject will respond to the antidepressant agent by applying the methods disclosed herein. In some aspects of the methods disclosed herein, the antidepressant agent or therapeutic agent can be a selective serotonin reuptake inhibitor, a serotonin and norepinephrine reuptake inhibitor, an atypical antidepressant, a tricyclic antidepressant, a monoamine oxidase inhibitor, ketamine and esketamine, an atypical antidepressant, and any other compounds showing antidepressant activity.

Therapeutic administration encompasses prophylactic applications. Based on genetic testing and other prognostic methods, a physician in consultation with their patient can choose a prophylactic administration where the patient has a clinically determined predisposition or increased susceptibility (in some cases, a greatly increased susceptibility) to a type of condition disorder or disease.

In some aspects, the subject can be at risk for developing depression or major depressive disorder. The therapeutic agent, antidepressant agent or treatment described herein can be administered to the subject (e.g.. a human patient) in an amount sufficient to delay, reduce, or preferably prevent the onset of clinical disease. Accordingly, in some aspects, the patient can be a human patient. In therapeutic applications, compositions are administered to a subject (e.g., a human patient) already with or diagnosed with a condition, disorder or disease in an amount sufficient to at least partially improve a sign or symptom or to inhibit the progression of (and preferably arrest) the symptoms of the condition, its complications, and consequences. An amount adequate to accomplish this is defined as a ‘"therapeutically effective amount.” A therapeutically effective amount of the cells described herein can be an amount that achieves a cure, but that outcome is only one among several that can be achieved. One or more of the symptoms can be less severe. Recovery’ can be accelerated in an individual who has been treated.

The therapeutically effective amount of the therapeutic agent, antidepressant agent or treatment described herein and used in the methods as disclosed herein applied to mammals (e.g., humans) can be determined by one of ordinary’ skill in the art with consideration of individual differences in age, yveight, and other general conditions (as mentioned herein).

Amounts effective for this use can depend on the severity of the disease and the yveight and general state and health of the subject. Suitable regimes for initial administration and booster administrations are typified by an initial administration followed by repeated doses at one or more hourly, daily, weekly, or monthly intervals by a subsequent administration. For therapeutic uses, the peptides and compositions can include a pharmaceutically acceptable excipient. Such compositions can be formulated yvithout undue experimentation for administration to a mammal, including humans, as appropriate for the particular application. Additionally, proper dosages of the compositions can be determined without undue experimentation using standard dose-response protocols. For example, a subject can receive any of the peptides or compositions disclosed herein one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 or more times per week).

The therapeutic agent, agent or treatment as described herein can be prepared for parenteral administration. The therapeutic agent, agent or treatment prepared for parenteral administration include those prepared for intravenous (or intra-arterial), intramuscular, subcutaneous, intraperitoneal, transmucosal (e.g., intranasal, intravaginal, or rectal), or transdermal (e.g., topical) administration. PHARMACEUTICAL COMPOSITIONS

As disclosed herein, are pharmaceutical compositions, comprising any of the antidepressant agents described herein or any of the antidepressant agents identified using any of the methods disclosed herein and a pharmaceutical acceptable carrier. Examples of antidepressant agents include but are not limited to selective serotonin reuptake inhibitor (SSRI). a serotonin and norepinephrine reuptake inhibitor (SNRI), an atypical antidepressant, a tricyclic antidepressant, a monoamine oxidase inhibitor (MAOI), ketamine and esketamine, an atypical antidepressant, and any other compounds showing antidepressant activity.

As used herein, the term “pharmaceutically acceptable carrier” refers to solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants that can be used as media for a pharmaceutically acceptable substance. The pharmaceutically acceptable carriers can be lipid-based or a polymer-based colloid. Examples of colloids include liposomes, hydrogels, microparticles, nanoparticles and micelles. The compositions can be formulated for administration by any of a variety of routes of administration and can include one or more physiologically acceptable excipients, which can vary depending on the route of administration.

As used herein, the term “excipient” means any compound or substance, including those that can also be referred to as “carriers” or “diluents.” Preparing pharmaceutical and physiologically acceptable compositions is considered routine in the art, and thus, one of ordinary skill in the art can consult numerous authorities for guidance if needed. The compositions can also include additional agents (e.g., preservatives).

The pharmaceutical compositions as disclosed herein can be prepared for, for example, parenteral administration. Pharmaceutical compositions prepared for parenteral administration include those prepared for intravenous (or intra-arterial), intramuscular, intervertebral subcutaneous, or intraperitoneal. Paternal administration can be in the form of a single bolus dose, or may be, for example, by a continuous pump. Topical administration includes ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery. Aerosol inhalation can also be used to deliver any of the compositions described herein. Pulmonary administration includes inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal. In some aspects, the compositions can be prepared for parenteral administration that includes dissolving or suspending the compounds in an acceptable carrier, including but not limited to an aqueous carrier, such as water, buffered water, saline, buffered saline (e.g., PBS), and the like. One or more of the excipients included can help approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like. Where the compositions include a solid component (as they may for oral administration), one or more of the excipients can act as a binder or filler (e.g., for the formulation of a tablet, a capsule, and the like). Where the compositions are formulated for application to the skin or to a mucosal surface, one or more of the excipients can be a solvent or emulsifier for the formulation of a cream, an ointment, and the like.

The pharmaceutical compositions can be sterile and sterilized by conventional sterilization techniques or sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation, which is encompassed by the present disclosure, can be combined with a sterile aqueous carrier prior to administration. The pH of the pharmaceutical compositions ty pically will be between 3 and 11 (e.g., between about 5 and 9) or between 6 and 8 (e.g., between about 7 and 8). The resulting compositions in solid form can be packaged in multiple single dose units, each containing a fixed amount of the above- mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment. The compositions can also be formulated as powders, elixirs, suspensions, emulsions, solutions, syrups, aerosols, lotions, creams, ointments, gels, suppositories, sterile injectable solutions and sterile packaged powders. The active ingredient can be nucleic acids or vectors described herein in combination with one or more pharmaceutically acceptable carriers. As used herein “pharmaceutically acceptable" means molecules and compositions that do not produce or lead to an untoward reaction (i.e., adverse, negative or allergic reaction) when administered to a subject as intended (i.e., as appropriate).

The pharmaceutical compositions described herein can also be formulated so as to provide slow, prolonged, or controlled release. For example, a controlled-release preparation is a pharmaceutical composition capable of releasing the peptides or compositions disclosed herein at a desired or required rate to maintain constant activity for a desired or required period of time. KITS

In some aspects, kits are provided for measuring or determining the amount of Gas palmitoylation in a sample or subject. The kits can comprise materials and reagents that can be used for measuring or determining the amount of Gas palmitoylation. These kits can include the reagents needed to carry out the measurements of the determining the amount of Gas palmitoylation. Alternatively, the kits can further comprise additional materials and reagents.

EXAMPLES

Example 1: Acylation status of the G protein, Ga_s, directs membrane localization and the response to chronic antidepressant stimulation.

Materials and Methods. Chemicals. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, trypsin, and penicillin/streptomycin were purchased from Sigma- Aldrich, St Louis, MO. Cell culture flasks were from NUNC (VWR International, West Chester, PA). Escitalopram and //-citalopram were kindly provided from H. Lundbeck A/S, Copenhagen, Denmark. Desipramine hydrochloride and olanzapine were purchased from Tocris Bioscience, Ellisville, MO. Phenelzine sulfate, fluoxetine hydrochloride. N-ethylmaleimide. and Hydroxylamine were purchased from Sigma-Aldrich, St Louis, MO.

Drug Treatments. C6 cells were cultured in DMEM, 4.5 g of glucose/L, 10% newborn calf serum (Hyclone Laboratories, Logan, UT), 100 mg/mL bacteriostatic penicillinstreptomycin at 37 °C in humidified 5% CO2 atmosphere to a confluence of -40% before drug treatments were begun. Treatment with 10 pM for 72 hrs is a standard assay condition (Zhang, L., and Rasenick, M. M. (2010) The Journal of pharmacology arid experimental therapeutics 332, 977-984), however, these drugs show effects in this in-vitro system at concentrations as low as 50 nM (Czysz. A. H., et al. (2015) Neuropsychopharmacology 40, 766-773). Culture medium and drug were changed daily and no apparent change in cell morphology was observed during treatment. Before assay, cells were rinsed twice with prewarmed IX phosphate buffered saline (PBS) to remove debris and wash aw ay unbound drugs. Patients (defined as depressed through inventories such as HAM-D, MADRS, PHQ-9 or others) are treated with antidepressant drugs, alone or in combination. Blood is taken before initial treatment and after one week and assayed for Gas-activated adenylyl cyclase (Targum et al., Mol. Psychiatry, 2022, 27(3): 1640-1646). To verify the antidepressant response, blood can also be taken 8 weeks after the initiation of treatment. Lipid Raft Isolation. Cells were washed and harvested in ice-cold IX PBS and lipid raft fractions (Erb, S. J., et al. (2016) Antidepressants Accumulate in Lipid Rafts Independent of Monoamine Transporters to Modulate Redistribution of the G protein, Galphas. The Journal of biological chemistry).

Ga_s-GFP tagging and N-t er minal mutant generation. A GFP tagged Ga_s construct was used that behaves much the same as wild type Ga_s when treated with antidepressants (moves out of lipid rafts) (Y u, J. Z., and Rasenick, M. M. (2002) Mol. pharmacology 61. 352- 359). Based on this construct, acylation mutants of Gas-GFP at Cys3Ser to impair palmitoylation and Asn6Ser to provide the recognition sequence important for myristoylation were constructed (Hannoush, R. N., and Sun, J. (2010) Nature chemical biology' 6, 498-506; and Thiyagarajan, M. M., et al. (2002)). The Gots-GFP constructs were made monomeric (Zacharias, D. A., et al. (2002) Science (New York. N. Y.) 296, 913-916).

C6 glioma cells were cultured until 80% confluence and trypsinized into suspension for electroporation with the Invitrogen Neon Transfection System according to the manufacturer’s protocols. Approximately 15 pg of DNA was used per one million cells. After transfection, cells were plated in an appropriate dish and further selected with G418 for 24 h before further study.

Microscopy and Live-Cell Imaging. Cells were plated on glass microscopy dishes in DMEM with 10% FBS and 1 mM G418. One hour before live cell imaging, complete medium was replaced with serum-free DMEM supplemented with 20 mM HEPES. Fluorescent images were obtained using an inverted microscope equipped for fluorescent microscopy (Nikon Eclipse TE 300, excitation wavelength, 547 nm; emission wavelength, 579 nm; via high pressure Nikon Xenon XBO 100 W lamp; Nikon, Toky o, Japan); a digital camera [RTE/CCD-1300 Y/HS (Roper Scientific, Trenton, NJ), MicroMAX camera controller (Princeton Instruments Inc.. Scientific Instruments. Monmouth Junction. NJ), and Lambda 10-2 shutter (Sutter Instrument Company, Novato, CA)], and imageprocessing software (IPLab, Scanalytics, Fairfax, VA). The images shown were obtained using oil immersion with a 60x objective lens. Scale bars = 10 pm.

Western blotting. Westerns were conducted according to standard protocols with a mouse monoclonal anti-Ga_s (1 : 1 ,000; NeuroMab clone N 192/12, Davis, CA, USA, catalog # 75-211, RRID #AB_2315846), rabbit polyclonal anti-Caveolin 1 (1: 10,000; BD Biosciences, Franklin Lakes, NJ, USA catalog # 610059), and mouse monoclonal anti-P-actin (1 :5,000; Sigma-Aldrich, St. Louis, MO, USA catalog # A5441).

Ga_s Acylation Determination. Lipid rafts were extracted from purified membranes via sucrose density gradient and Gets immunoprecipitated with an anti-Ga_s monoclonal antibody. Immunoprecipitations of Ga_s were concentrated by chloroform: methanol precipitation and free sulfhydryls blocked with NEM, which covalently couples to free cysteine sulfhydryls (Wan. J., et al. (2007) Nature protocols 2, 1573-158) and allows for the differentiation between antidepressant-mediated versus loss of modification during LC- MS/MS analysis. To further control for false positive detection, sites of S-palmitoylation were cleaved with hydroxylamine and the extent of palmitoylation determined by LC- MS/MS. The immunoprecipitations were prepared (Drisdel, R. C., and Green, W. N. (2004) Biotechniques 36, 276-285) without radiolabeled N-ethylmaleimide (NEM). Peptide identification was accomplished via peak identification in the LC-MS/MS spectrum (Wan, J., Roth, et al. (2007) Nature protocols 2, 1573-1584). Briefly, protein samples were treated as follows:

1) Free sulfhydryls were covalently blocked with 100 mM NEM and unreacted NEM was removed via 10K molecular weight cutoff (MWCO) spin filter to prevent undesired side reactions;

2) Samples were reduced with IM hydroxylamine; Cys-palmitoyl is reduced whereas Cys-NEM is not reducible with hydroxylamine (Smyth. D. G., et al. (1964) The Biochemical Journal 91, 589-595). Importantly, a sample omitting hydroxylamine was kept to control for false positive detection. Unreacted hydroxylamine was removed with a 10K MWCO spin filter to prevent undesired effects on precipitating antibodies;

3) Samples were precleared with sepharose A resin and immunoprecipitated with two pg of an anti-Gas monoclonal antibody in IX PBS. Gets was immunoprecipitated after treatment of samples with NEM and hydroxyl amine in order to protect protein palmitoylation;

4) Immunoprecipitations were washed and digested overnight at 4 °C with one pg Trypsin and gentle rocking. Beads were pelleted and the peptide supernatant desalted with Cl 8 zip-tips (Millipore*); and

5) NEM conjugation (indicates drug mediated depalmitoylation of Cys3) of the N- terminal Ga_s peptide, MGCLGNSK (SEQ ID NO: 1), was analyzed by LC-MS/MS with peptide mass tolerance of ± 10 parts per million (ppm) and fragment mass tolerance of ± 0.6 Da.

Liquid Chromatography Tandem Mass Spectrometry (LC-MS MS). Chromatographic separation of peptides was accomplished by gradient elution on an Agilent 1200 binary HPLC coupled to an Orbitrap Velos Pro™ Hybrid Ion Trap-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany). Chromatographic separation was conducted using a ZORBAX 300SB-C18-microbore column (5 * 0.3 mm ID, 5 pm; Agilent Technologies, Santa Clara, CA) at 4 °C. Peptides were detected by full-scan mass analysis from m^zz 400 to 1800 at a resolving power of 30,000 (at m/z 400, full width at half maximum [FWHM]) and followed by data-dependent multiple stage mass analysis at a resolving power of 7500, which was triggered by the most abundant ions from a parent list of triply-, doubly-, and singly-charged peptides at a flow rate of 250 nL min'¹ into the ESI source. CID was conducted with an isolation width of three Da, normalized collision energy of 30%, and an activation time of 10 millisec. Data acquisition and reduction was carried out using Xcalibur version 2. 1 (Thermo Fisher Scientific, Bremen, Germany).

Statistical Analysis. The measurements are presented as the mean (a minimum of n=3) ± standard error of the mean (SEM). Calculation error was propagated throughout each calculation. Each data set was further subjected to statistical analyses using GraphPad Prism (version 5.0), using a one-way analysis of variance (ANOVA) followed by a post-hoc Dunnetf s t-test (multiple groups) (95% C.I.).

Results. Acylation state directs the subcellular localization of Ga_s as well as the response to antidepressants. Gets is singly palmitoylated and the Ga that internalizes when activated (Wedegaertner, P. B., et al. (1996) Molecular biology of the cell 7, 1225-1233)). By contrast, Ga; is both myristoylated and palmitoylated in the N-terminal region. To examine whether secondary myristoylation of Ga_s would prevent antidepressant mediated translocation of Gcu from lipid rafts, N-terminal acylation mutants of Ga were prepared (Wedegaertner, P. B. (1998) Lipid modifications and membrane targeting of G alpha. Biological signals and receptors 7, 125-135 (Wedegaertner, P. B. (1998) Biological signals and receptors 7, 125-135). Mutant constructs were ensured GFP was monomeric (Zacharias. D. A., et al. (2002) Science (New York. N. Y.) 296, 913-916), and stable C6 cell clones selected with G418. Mutation of Gas at an N-terminal glycine residue (N6S) provides the recognition sequence for myristoylation and makes Gas both palmitoylated and myristoylated (Gai like Ga_s). By contrast, mutation of the N-terminal cysteine residue (C3S) renders Gas acylation deficient. See, for example, FIGS. 4 and 5.

Imaging by confocal microscopy revealed that G s-GFP primarily localized to the plasma membrane (FIG. 1 A). For the Myr-Palm mutant (Gai like Gas), about 50% is localized to the plasma membrane, while the rest is bound to membrane structures in the cell interior (Wedegaertner, P. B., et al. (1993) The Journal of biological chemistry 268, 25001- 25008). Moreover, sucrose density gradient fractionation shows that neither mutant translocates from lipid rafts in response to chronic antidepressant treatment (FIGS. 4 and 5).

Note that the native Gas is translocated in each of these cells by antidepressant treatments even when these same treatments fail to translocate the mutant proteins. This further shows that the Myr-Palm Ga_s-GFP mutant (Gai like Gas) remains palmitoylated following treatment with escitalopram. When Ga, contains both fatty acid anchors, it preferentially localizes to lipid rafts, whereas myristoylated and depalmitoylated Gai prefers other ty pes of ordered lipid microdomains (Alvarez, R., et al. (2015) Biochimica et biophysica acta 1851, 1511-1520).

Chronic treatment with antidepressants mediates the depalmitoylation ofGa_s. Since antidepressant treatment mediates a translocation of Gas from lipid rafts, and altering the native palmitoylation state prevents this, it was tested whether antidepressant treatment evokes depalmitoylation of Ga_s. This was examined in response to stimulation with the antidepressant escitalopram and compared to a treatment naive control. Identified Gas peptides revealed, as expected, that Gas retains its acylation status in the absence of antidepressant treatment, but also that three-day treatment with escitalopram mediates the depalmitoy lation of Gas (FIG. 2A). This is evidenced by the lack of palmitoy lated Gas peptide in the drug treated spectrum and further by the disappearance in the treatment naive sample upon reaction with hydroxylamine, which cleaves sites of S-palmitoylation. Thus, escitalopram’s effects on Ga_s palmitoylation state provide a potential mechanistic explanation for its action on Gas shown in FIGS. 1 and 2.

The MSi peak identifications were within 10 ppm of expected (Table 1). Confirmation of peak identity was made identifying MS2 spectrum b and v ions. Fragmentation of the N-terminal Gas peptide MGCLGNSK (SEQ ID NO: 1), depicted in FIG. 2A, produces b andy ions (131.041, 188.062, 416.196, 529.281, 586.302, 701.326, 788.358; and 128.095, 215.127, 330.151, 387.172, 500.256, 728.391, 785.412) respectively, when NEM conjugated and b and .p ions (131.041, 188.062, 291.071, 404.155, 461.177, 576.201, 663.233; and 128.095, 215.127, 330.151, 387.172. 500.256. 603.266, 660.287) respectively, when palmitoylated. In order to verify MSi peak identifications and control for false positives, the MS2 ions were verified. Representative MS2 spectra for treatment naive control and escitalopram treated are presented (FIG. 2B).

Table 1. Peptide profiles for antidepressant mediated depalmitoylated Gets. LC- MS/MS experiments were performed. The classes of antidepressant treatments tested produced NEM-conjugated Ga_s peptides. The magnitude of this effect varied among drugs, rather than classes. Neither R-cital opram, nor the antipsychotic olanzapine had any effect on the palmitoylation state of Ga_s. Theoretical MSi and MS2m/z are provided along with observed. The MSi peptide mass to charge ratios (m/z) were within 10 parts per million (PPm).

Treatment Peptide Charge Observed Expected

Control MGCLGNSK M+2H⁺ 515.89 515.881

Phenelzine MGC*LGNSK M+2H⁺ 459.19 459.234

Desipramine MGC*LGNSK M+2H⁺ 459.27 459.234

Fluoxetine MGC*LGNSK M+2H⁺ 459.23 459.234

Escitalopram MGC*LGNSK M+2H' 459.29 459.234

R-Citalopram MGCLGNSK M+2H⁺ 515.51 515.881

Olanzapine MGCLGNSK M+2H⁺ 515.51 515.881

*- NEM conjugated, otherwise palmitoylated

In addition to escitalopram, its inactive isomer / -cital opram, and antidepressants phenelzine, desipramine. and fluoxetine, as well as the antipsychotic olanzapine were assessed for their capacity to mediate the depalmitoylation of Ga_s. Three-day treatment with phenelzine, desipramine, fluoxetine, and escitalopram each yielded a doubly charged NEM conjugated MGCLGNSK peptide (SEQ ID NO: 1) of mass to charge ratio (m^zz) 459.23 (FIG. 3 and Table 1). Three-day treatment with either R-citalopram or the antipsychotic olanzapine, which do not mediate translocation of Gets, and do not accumulate in lipid rafts (Erb, S. J., et al. (2016) The Journal of biological chemistry), did not result in NEM conjugation and thus do not evoke depalmitoylation of Ga_s. These results are thus consistent with a model of antidepressant-induced depalmitoylation of Ga_s in order to achieve efficacy. These results showing antidepressant action on Ga_s are label free. Specific molar quantification cannot be provided. However, relative quantification is accomplished through comparison of the NEM conjugated Gok peptide in each treatment with the naive control (Asara, J. M., et al. (2008) Proteomics 8, 994-999). Integration of the confirmed MSi peptide peaks for the treatments were normalized by the aggregate total integrations of the peaks in each spectrum. The resulting normalized means are reported as a fold change from the treatment naive control NEM conjugated Ga_s peptide (FIG. 3B). The antidepressants examined are thus able to significantly mediate a decrease in palmitoyl ati on of Gets.

Discussion. The antidepressants examined herein move Gou from lipid rafts (Czysz, A. H., et al. (2015) Neuropsychopharmacology 40, 766-773; and Singh, H., et al. (2018) Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology). The data indicate different/multiple mechanisms may exist for the actions of different antidepressants. It has been shown that antidepressants do not accumulate in lipid rafts (assays are complicated by the observation that some tricyclics disrupt lipid rafts) (Erb, S. J., et al. (2016) Antidepressants Accumulate in Lipid Rafts Independent of Monoamine Transporters to Modulate Redistribution of the G protein, Galphas. The Journal of biological chemistry), but the results described herein correlate accumulation with mediated depalmitoylation of Ga_s. Further support for distinct molecular drug targets, comes from the enantiomer-selective accumulation (Erb, S. J., Schappi, J. M., and Rasenick, M. M. (2016) Antidepressants Accumulate in Lipid Rafts Independent of Monoamine Transporters to Modulate Redistribution of the G protein, Galphas. The Journal of biological chemistry’) and depalmitoylation of Go mediated by escitalopram, but not its inactive stereoisomer 7?-ci talopram. Regardless, the results show that, independent of monoamine transporters, antidepressants have a similar molecular footprint to exploit for the purposes of diagnostics.

Monoamine-centric antidepressants require several weeks of treatment in order to achieve clinical efficacy. This is modeled (albeit accelerated to 3 days) in C6 glioma cells, where compounds with antidepressant properties translocate Go from lipid rafts (Zhang. L., and Rasenick, M. M. (2010) The Journal of pharmacology and experimental therapeutics 332, 977-984; Czysz, A. H., et al. (2015) Neuropsychopharmacology 40, 766-773; and Singh, H., et al. (2018) Disruption of lipid-raft localized Galphas/tubulin complexes by antidepressants: a unique feature of HDAC6 inhibitors. SSRI and tricyclic compounds. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology) . The extent of translocation is dependent upon both drug dose and duration and has been observed in both rats (Toki, S., et al. (1999) J. Neurochem 73, 1114-1120) and cell culture (Zhang, L., and Rasenick, M. M. (2010) The Journal of pharmacology⁷ and experimental therapeutics 332, 977-984; Donah, R. J., and Rasenick, M. M. (2005) Neuropsychopharmacology 30, 1238-1245; and Donati, R. J , et al. (2001) Molecular pharmacology 59, 1426-1432). Curiously, the molecular entities most commonly associated with antidepressants, serotonin and norepinephrine transporters, are absent in C6 cells, and are not required to observe this biological hallmark of antidepressant efficacy (Erb, S. J., et al. (2016) Antidepressants Accumulate in Lipid Rafts Independent of Monoamine Transporters to Modulate Redistribution of the G protein, Galphas. The Journal of biological chemistry, and Eshleman, A. J., et al. (1997) Journal of neurochemistry 69, 1459-1466). It is noteworthy that the time required for these drugs to inhibit serotonin or norepinephrine transport is relatively short. Thus, additional molecular mechanism may explain antidepressant efficacy.

Targeting of G protein alpha subunits to the plasma membrane, primarily lipid rafts, is mediated through N-terminal palmitoylation (Dunphy, J. T., and Linder, M. E. (1998) Biochimica et biophysica acta 1436, 245-261; and Michaelson, D., et al. (2002) Molecular biology of the cell 13, 3294-3302). Lipid raft disruption through cholesterol depletion or cytoskeletal disruption displaces many raft proteins. However, singly palmitoylated Gm is the Ga that internalizes when activated (Wedegaertner, P. B., et al. (1996) Molecular biology of the cell 7, 1225-1233), whereas Gm (myristoylated and palmitoylated) and Ga_q (doubly palmitoylated) do not (Wedegaertner, P. B., et al. (1995) The Journal of biological chemistry 270, 503-506). For example, see, FIGS. 4 and 5.

Ga_s is a membrane-associated protein that inhabits cholesterol rich lipid raft microdomains (Donati, R. J., et al. (2008) J. Neuroscience 28, 3042-3050; and Donati, R. J., and Rasenick, M. M. (2005) Neuropsychopharmacology 30, 1238-1245)). Lipid rafts contain many of the anchoring cytoskeletal-associated membrane structures and facilitate molecular association(s) of a vast array of different membrane-embedded and associated proteins to initiate intracellular signaling. While lipid rafts can facilitate this clustering of signaling molecules, the rigid structure afforded by increased cholesterol content appears to have a globally dampening effect on Ga_s signaling by inhibiting association(s) between raft and non- raft based molecules (Allen, J. A., Halverson-Tamboli, R. A., and Rasenick, M. M. (2007) Nat Rev Neurosci 8, 128-140).

Dampened signaling, through Ga_s and associated receptors, is consistent with the observed increase in Ga_s association with rafts seen in postmortem MDD brain (Donati, R. J., et al. (2008) J. Neuroscience 28, 3042-3050). Attenuated coupling between Ga_s and AC is also observed in peripheral tissue from subjects with MDD (Hines, L. M., and Tabakoff, B. (2005) Biological psychiatry 58, 955-962; and Mooney, J. J., et al. (2013) Journal of Psychiatric Research 47, 706-711). Spatial sequestration in lipid rafts is thus a barrier to functional activity, which can be achieved through lipid raft disruption or removal of the anchoring palmitoylation. It is noteworthy, that internalized Gcu is maintained in the active conformation, where it continues to signal in the intracellular milieu (Vilardaga, J. P., et al. (2012) Trends in pharmacological sciences 33, 423-431; and Eichel, K., and von Zastrow, M. (2018) Trends in pharmacological sciences 39, 200-208). However, neither the intrinsic GTPase activity of Ga_s nor the intrinsic activity of AC is altered in response to chronic antidepressant treatment (Ozawa, H., and Rasenick. M. M. (1991) Journal of neurochemistry 56, 330-338). Note also that activated Ga_s is internalized (Allen, J. A., et al. (2005) Molecular pharmacology 67, 1493-1504)), depalmitoylated (Wedegaertner, P. B., and Bourne, H. R. (1994) Cell 77, 1063-1070), associates with microtubules, increasing their dynamicity, and neurite outgrowth (Y u, J. Z., et al. (2009) The Journal of biological chemistry 284, 10462-10472; and Sarma, T., et al. (2015) The Journal of biological chemistry 290, 10045-10056). However, increased physical coupling between Gou and AC following chronic, but not acute antidepressant treatment (Chen, J., and Rasenick, M. M. (1995) 77?e Journal of pharmacology and experimental therapeutics 275, 509-517), suggests that Ga_s remains membrane associated following stimulation.

Among the heterotri meric G proteins, the antidepressant response is exclusive to Ga_s (Toki, S., et al. (1999) J. Neurochem 73, 1114-1120). As neither Gai nor Gaq are displaced following antidepressant stimulation the secondary' anchoring modification in Gai and Ga_q either prevents depalmitoylation or abrogates the membrane mobility effects thereof. Supporting evidence is found in the observation that depalmitoylation of Gai allows for its redistribution from lipid rafts (Alvarez, R., et al. (2015) Biochimica et biophysica acta 1851, 1511-1520). Taken together, these findings demonstrates that antidepressant treatment mediates the depalmitoylation of Ga_s and not Ga; or Ga_q. Moreover, this is bome out by the results showing the failure of acylation-modified Ga_s-GFP to be displaced by antidepressant treatment.

Ga; is secondarily myristoylated. which was replicated using aN-terminal mutant Ga_s-GFP construct. Mutation of the N-terminus of Ga_s affects acylation status and prevents translocation from lipid rafts following chronic escitalopram treatment. Myristoylated and palmitoylated (G ilike) Gas is not translocated following antidepressant treatment (FIGS. 3- 5) and palmitoylati on-deficient Ga« does not appreciably localize to lipid rafts (FIGS. 4 and 5), effectively making it antidepressant-insensitive. This is consistent with reports that palmitoylation of the G alpha subunit is important for plasma membrane localization irrespective of secondary lipid modification (Michaelson, D., et al. (2002) Molecular biology of the cell 13, 3294-3302). Furthermore, the data (e.g., FIGS. 1 and 2) show that antidepressants decrease Ga_s palmitoylation; thereby mechanistically demonstrating how these compounds potentiate cAMP signaling.

The drugs that mediate the depalmitoylation of Gas are also those that accumulate in lipid rafts (Erb, S. J., et al. (2016) The Journal of biological chemistry , and are those that increase the fluorescence recovery after photobleaching (FRAP) recovery half-time of Gos- GFP (Czysz, A. H., et al. (2015) Neuropsychopharmacology 40, 766-773). By contrast, those drugs that do not increase the FRAP recovery half time of Ga_s, nor accumulate in lipid rafts, did not mediate the depalmitoylation of Ga_s. In addition, others have shown that G ; maintains its raft localization while both palmitoylated and myristoylated (Alvarez, R.. et al. (2015) Biochimica et biophysica acta 1851, 1511-1520). Thus, depalmitoylation of Gas appears to be mechanistically relevant to the antidepressant response through Ga_s.

The notion that one action of antidepressants is to move Ga_s out of lipid rafts may represent a consistent biochemical hallmark for antidepressant action — especially subsequent to the recent finding that ketamine also evokes this response. Identification of the antidepressant-sensitive molecular anchor for Gas in lipid rafts may lead to the development of more targeted therapies for depression, including compounds that may have a more rapid course of action. Moreover, the antidepressant-induced biochemical alterations of Gas maybe exploitable for creating more targeted pharmacological therapies. Finally, the findings in this study show that G protein translocation from lipid rafts is a biosignature for effective antidepressant action that can be useful in predicting efficacy of therapeutic agents. The results described herein demonstrate that antidepressants mediate Gets depalmitoylation, in order to induce translocation of Gets from lipid rafts, providing a biochemical explanation for their therapeutic delay. Further elucidation of the proteins integral to the translocation and depalmitoylation of Gets can be used for the development of therapeutic compounds for depression.

Example 2: Measurement of Gsa-activated adenylyl cyclase in platelets.

Methods. Fluorescence recovery after photobleaching. C6 cells were transfected or infected with GFP-Ga and cells expressing GFP-Ga were analyzed (Czysz AH, et al. Neuropsychopharmacology. 2014; 40: 1-8). For infection protocols, GFP-Ga was inserted into a baculoviral vector by Montana Molecular - Bozeman MT and used according to the manufacturer's directions. One hundred fifty data points, -300 ms apart (including ten prebleach values) were measured for each cell. Zeiss Zen software was used to calculate fluorescence recovery after photobleaching (FRAP) recovery half-time utilizing a one-phase association fit, correcting for total photobleaching of the analyzed regions.

Viral Infection and cAMP quantification. Cells are grown on glass bottom microscope dishes and infected with (1.09 x 10 VG/mL) cADDlS BacMam virus encoding the green upward cAMP sensor (Montana Molecular, Bozeman, MT, USA), supplemented with sodium butyrate at a final concentration of 2 mM, and grown for 24-26 h before live imaging under a x40 objective on a Zeiss 880 microscope. Cells were serum starved with 1% serum for 2-3 h before drug treatments. Images were taken every 30 s. Average responses from 4 to 10 cells were selected randomly from the visual field and fluorescence was normalized to baseline fluorescence for each experiment (Senese and Rasenick, 2021). cAMP assay. Cells are plated in black-sided clear-bottom plates (96, 384 or 1536 well format). Twenty -four hours before measurement at a density of 48,000 (or 12,000 or 4.000 for 384 and 1536 well format) cells per well. At time of plating, each well is infected with (2.18 x 10 VG/mL) c ADDIS BacMam virus Green cADD is upward cAMP sensor and supplemented with sodium butyrate at a final concentration of 2 mM. Final volume of each well was brought up to a volume of 140 pL with culture media (DMEM supplemented with 10% newborn calf serum). Twenty-four hours after plating, media was replaced with fresh culture media, with or without Ketamine at a final concentration of 10 pM. After 15 min, the culture media was replaced with 200 pL DPBS. GFP signal intensity was determined on a Biotek Synergy H4 plate reader (Biotek Instruments Inc.) using in-built monochromator with excitation set to 488 nM and emission set to 525 nM. Wells were read from the bottom with detector set to 90% sensitivity. Each condition was measured in duplicate wells for each experiment. Blank subtracted baseline measurements were obtained for each well before isoproterenol challenge. Wells containing 200 pL DPBS were used for blank subtraction. Following baseline measurement, either isoproterenol or vehicle (ddH O) was added to each well and GFP signal intensity was measured. In some aspects, for monoaminergic antidepressants (MAOI, SSRI. SNRI, Tricyclic), cells can be incubated with an antidepressant agent for two days prior to cADDIS virus (or other fluorescent cAMP reporter) infection and an additional day after infection. In some aspects, for a candidate compound suspected to have antidepressant activity, two or more incubation time points will be carried out.

Measurement of Gsa-activated adenyfyl cyclase in platelets. Platelets from depressed subjects at week 0 (pre-treatment) and week 8, were assayed for adenylyl cyclase in the presence of buffer (basal), PGE1 and Forskolin. Change in HAMD and change in adenylyl cyclase stimulation were calculated. Both forskolin and PGE1 -stimulated adenylyl cyclase values were normalized to the unstimulated/basal value. The results are plotted in FIG. 10 and FIG. 11.

Statistical analysis. Western blot bands were quantified using Biorad image lab software or Wes (protein simple) internal software. Control values were set to one and compared to treatment values. The graphs are represented with either fold change or percent change with p values. Data are represented from at least three biological replicate experiments. Statistical significant differences (p < 0.05) were determined by unpaired t-test or two-way ANOVA, or if variances were unequal a two-way Kruskal-Wallis test was performed followed by Dunn’s post hoc test for multiple comparisons. Unpaired t-test for control vs treatment conditions were performed followed by Welch’s correction. In cAMP dose-response experiments F was defined as the mean signal intensity of the vehicle + vehicle condition.

Isoproterenol EC and maximal efficacy were calculated by fitting the data to a standard agonist concentration versus response curve (Hill slope = 1). For time course experiments, signal intensity was measured with a 20 s read interval for the duration of the experiment. Results are represented as mean ± S.E.M. Statistical analysis was performed with the Prism version 5.0 software package for statistical analysis (GraphPad Software Inc., San Diego, CA).

Results. Brief ketamine treatment or sustained treatment with traditional antidepressants redistributes plasma membrane Got into non-raft regions of the plasma membrane of C6 glioma cells Ga localization in lipid rafts is decreased after 3-day treatment with 10 pM antidepressants in C6 cells and 3-week treatment in several regions of rat brain (Toki S, et al. J Neurochem. 1999; 73: 1114-20; and Czysz AH. et al. Neuropsychopharmacology. 2014; 40: 1-8). Likewise, ketamine concentration rises to ~10 pM in the brains of rats after they have been treated with an antidepressant dose (Zanos P, et al. Nature. 2016; 533:481-6). To test if ketamine induced a similar redistribution, C6 cells were treated for 15 min or 24 h with lOpM ketamine and lipid raft fractions were isolated using a detergent-free sucrose density gradient method. The results show ketamine liberated Ga from lipid rafts after as little as 15 min treatment (FIG. 6A). Additional experiments were restricted to 15-min treatment as this is sufficient time to observe a phenoty pic antidepressant response in cells after ketamine administration. This time course represents an appropriate juxtaposition of the rapid onset of antidepressant effects seen in humans compared with the delayed therapeutic onset of traditional antidepressant compounds (Berman, RM, et al. Biol Psychiatry. 2000;47:351-4; and Zarate CA, et al. Arch Gen Psychiatry. 2006;63:856-64)

Translocation of Ga from lipid rafts was also determined using fluorescence recover}' after photobleaching (FRAP) (Czysz et.al. 2015). FRAP of GFP-Ga from antidepressant treated C6 cells results in decreased lateral membrane diffusion due to its increased association between GFP-Ga and adenylyl cyclase. Treatment (3 days) of C6 cells with all tested antidepressants translocate GFP- Ga from lipid rafts as indicated by FRAP (Malberg JE, Blendy JA. Trends Pharmacol Sci. 2005; 26:631-8). Data presented here record an “antidepressant signature” for ketamine after 15 mm treatment (FIG.6B). comparable to 3- day treatment with “classic” antidepressants. These data corroborate the data derived from lipid-raft isolation (FIG. 6A). Note that compounds not possessing antidepressant properties (e.g., antipsychotics, mood stabilizers or anxiolytics) do not displace Gas from lipid rafts.

Ketamine translocates Ga from lipid rafts at clinically relevant concentrations .

Patients are commonly treated with one 0.5 mg/kg infusion of ketamine over 40 min, where ketamine serum levels after 10 min and 30 min increase to 1.3 pM and 1.0 pM, respectively (Sos P, et al. Neuro Endocrinol Lett. 2013;34:287-93). To determine if ketamine translocation of Ga from lipid rafts occurs at clinically relevant concentrations, C6 cells were exposed to 1. 3, and 10 pM ketamine for 15 min and lipid rafts were isolated using TX100 and sucrose density protocol to allow equal protein and the groups to be loaded on one gel. There was a dose-dependent Ga exodus from lipid rafts at concentrations achieved in human subjects (FIG. 6C). These biochemical data for Ga translocation are corroborated by FRAP (FIG. 6D). Similar results are seen for several other antidepressants (SSRI. SNRI, tricyclic and MAO inhibitors), but require longer (72 hr) treatment time to see maximal effects. This reflects the delayed clinical response of these drugs vs. the rapid response of ketamine.

Ketamine-induced translocation of Ga returns to baseline after 24 h. The duration of ketamine's antidepressant effects are highly variable, lasting from 24 h to 2 weeks, with an average remission of depressive symptoms for one week (Berman, RM, et al. Biol Psychiatry. 2000;47:351-4; and Zarate CA, et al. Arch Gen Psychiatry. 2006;63:856-64). The duration of Ga translocation to non-raft microdomains after one 15-minute treatment with ketamine was determined. C6 cells were treated with 10 pM ketamine and collected 15 min, 1. 6, 12, and 24 h afterwards, lipid rafts were isolated and Ga quantified by immunoblotting.

Translocation of Ga from lipid rafts w as sustained significantly for 12 h after transient 15-min treatment, returning to baseline after 24 h (FIG. 7), showing rapid, transient effects, similar to those seen in human subjects, in the cellular model system. This is also reflective of the clinical situation, as ketamine’s antidepressant effects are short lived.

Other NMDA antagonists, MOR agonists, or KOR antagonist do not mediate Ga translocation from lipid rafts C6 cells lack monoamine transporters yet show delayed effects of antidepressant treatment (Erb. SJ, et al. J Biol Chem. 2016; 291: 19725-33). Given that C6 cells express NMDARs, the canonical target of ketamine’s anesthetic action, it was tested whether Ga lipid raft exodus was a result of NMD AR antagonism. To test this. C6 cells were treated w ith 10 pM of the NMDA antagonists MK-801, memantine or AP-V for 15 min and Ga lipid raft localization w as evaluated via TX100 and sucrose density⁷ gradient lipid raft isolation. No other NMD AR antagonist had a significant effect on Ga lipid raft localization suggesting that ketamine may be acting on a target other than the NMDA receptors (FIG. 8A).

Again, these data w ere corroborated with FRAP after a 15-min treatment and no other NMD AR antagonist altered Ga rate of lateral membrane diffusion (FIG. 3B) These data agree with reports suggesting targets other than NMDA receptors are responsible for ketamine's antidepressant action (Newport DJ, et al. Am J Psychiatry.2015; 172:950-66).

Ketamine enhances isoproterenol elicited cAMP accumulation in NR1 knockdown C6 cells. As antidepressant treatment translocates Ga from lipid rafts, it increases association of Ga with adenylyl cyclase, increasing cellular cAMP production (Zhang, L, & Rasenick, MM. J Pharmacol Exp Ther. 2010; 332: 977-84). To test if ketamine had a similar effect, cAMP was measured in live C6 cells using exchange factor directly activated by cAMP (EPAC)- based fluorescent biosensors (Tewson PH, et al. J Biomol Screen. 2016; 21:298-305). For these experiments C6 cells were infected with (1.09 x 10 VG/mL) cADDIS virus and imaged every 30 s and 10 pM ketamine was added after the fourth frame (n = 2). No difference in basal fluorescence between control and ketamine-treated cells was observed. However, when cells were treated with ketamine for 15 min and then stimulated by the Ga -coupled agonist isoproterenol (C6 cells have endogenous |3 adrenergic receptors; n=4) after drug washout, ketamine-treated cells show both a quicker and more robust increase in fluorescence, consistent with an enhanced coupling of Ga and adenylyl cyclase. These data agree with previous reports, which show the requirement of an activator of Gas to detect changes in cAMP production after antidepressant treatment (Zhang, L, & Rasenick, MM J Pharmacol Exp Ther. 2010; 332: 977-84). To investigate ketamine-mediated cAMP production independent of its canonical target, the NMD AR was ablated. The NR1 subunit was knocked down with siRNA, preventing proper assembly of the NMDA receptor complex (Ulbrich MH, Isacoff EY. Proc Natl Acad Sci USA. 2008;105: 14163-8). The ketamine-treated NR 1 knockdown group shows an increase in cAMP similar to ketamine-treated groups in which NMD AR is present. These data further support an NMDAR-independent antidepressant target of ketamine. To determine if ketamine influences Ga -coupled GPCR potency and efficacy, the isoproterenol mediated cAMP accumulation C6 cells was examined.

C6 cells in a 96-well plate were infected with (2.18 x 10 VG/mL) cADDIS virus and treated with 10 pM ketamine or vehicle for 15 min. Vehicle or isoproterenol was added to each well and GFP signal intensity was measured. Dose-response reveals ketamine treatment increases, significantly, percent stimulation (efficacy) over baseline (180 ± 10) compared to control (130 ± 11) while EC50 (potency) of ketamine (-9.0 ± 0.23) compared to (-8.2 ± 0.28) was unaffected. These data reveal a statistically significant increase in efficacy of adenylyl cyclase activation but not agonist potency, (n = 3) *p < 0.05; ****p < 0.0001. Transient ketamine treatment promotes sustained phosphorylation of CREB.

Activation of protein kinase A (PKA) by cAMP results in the phosphorylation of cAMP response element-binding protein (CREB) at serine- 133 (Ser- 133). Phosphorylated CREB (pCREB) then translocates to the nucleus where it acts as a transcription factor for genes involved in growth and survival, neuroprotection, and synaptic plasticity⁷ (Dwivedi Y, Pandey GN. Neuropsychiatr Dis Treat. 2008;4 (1 A): 161-76; and Duman RS, et al. Biol Psychiatry⁷. 1999; 46: 1181—91). CREB is both upregulated and phosphorylated at Ser- 133 after chronic antidepressant treatment in animals and cultured cells (Dwivedi Y, Pandey GN.

Neuropsychiatr Dis Treat. 2008; 4 (1 A): 161-76). The effect of ketamine-mediated cAMP elevation on CREB phosphorylation was evaluated. To test this, C6 cells were treated with 10 pM ketamine for 15 min, drug was washed off and cells harvested 15 min, 1. 2, 6, and 24 h after treatment. The data are consistent with a sustained increase of pCREB after a transient exposure to ketamine with significant increases shown at 15 min and again at 2 h (FIG. 9A). Total cellular CREB content remains constant. Increased pCREB is aligned, temporally, with the observed increase of cAMP production. Interestingly, increase in cAMP-dependent CREB phosphorylation was achieved without the addition of a Got -coupled agonist. This may be the result from activation of endogenous GPCRs by⁷ agonists present in serum or an increase of cAMP below the range of the sensor (Tewson PH, et al. J Biomol Screen. 2016; 21 :298-305).

Ketamine increases BDNF in a cAMP-dependent manner in primary astrocytes.

Numerous reports indicate that chronic treatment with antidepressants increase BDNF mRNA and protein levels in the rodent brain and in cell cultures (Nibuya M, et al. J Neurosci. 1995; 15:7539- 47; Dwivedi Y, Pandey GN. Neuropsychiatr Dis Treat. 2008; 4 (1 A): 161-76; and Coppell AL, et al. Neuropharmacology⁷. 2003; 44:903-10). Emerging evidence also indicates that acute ketamine treatment results in increased levels of BDNF (Li, N. mTOR-dependent synapse formation. Science. 2010; 329:959-65; Autry AE, et al. Nature. 2011; 475:91-95; and Zanos P, et al. Nature. 2016; 533:481-6) and, ketamine treatment does not elicit an antidepressant behavioral response in mice lacking forebrain BDNF (Autry AE, et al. Nature. 2011; 475:91-95). Given PKA phosphorylation of CREB has been shown to increase the transcription of BDNF, the effects of these cellular events was determined. C6 cells were treated with 10 pM ketamine for 15 min and were collected after either 1 h or 24 h. At 24 h after ketamine treatment, a significant increase in BDNF was observed indicating the possibility of a cAMP/PKA/pCREB-mediated transcriptional-dependent increase.

Next, the relationship of ketamine-induced cAMP increase with that of the ketamine- induced increase in BDNF was established. Mature primary rat astrocytes were treated (15 min) with ketamine alone or ketamine plus the cell permeable cAMP competitive antagonist cAMPs-Rp and collected after 24 h. Staining for GFAP confirmed presence of astrocytes. The data show a significant increase in BDNF 24 h after ketamine treatment, which was attenuated by 1 pM cAMPs-Rp (FIG. 9C). These data show a ketamine-induced. cAMP- dependent, production of BDNF.

The ketamine metabolite, (2R, 6R) -hydroxynor ketamine, increases cAMP accumulation. Recent reports indicate that the ketamine metabolite (2R,6R)- hydroxynorketamine (2R,6R)-HNK produces rapid and robust antidepressant effects through increased expression of BDNF, GluAl, GluA2, and de-phosphorylation of eEF2k in synaptosomes, through an unknown mechanism.

Extensive binding displacement studies have shown that these effects are independent of NMD AR antagonism as (2R,6R)-HNK lacks affinity for the NMD AR (Zanos P. et al. Nature. 2016;533:481-6). Suzuki et al. have suggested a possible role of NMD AR antagonism for (2R,6R)-HNK, yet this remains controversial (Suzuki K, et al. Nature. 2017;546:El-E3). Hence, it was tested whether (2R,6R)-HNK decreases translocation of Ga -GFP similarly to ketamine. A 15 -minute treatment with (2R,6R)-HNK had effects similar to ketamine (FIG. 10). To determine the functional consequences of augmented Ga and adenylyl cyclase coupling, C6 cells were infected with (1.09 x 10 VG/rnL) cADDIS virus, pretreated with 10 pM (2R,6R)-HNK, imaged every' 30 s and 10 pM isoproterenol was added after the fourth frame, (n = 4), for 15 min, drug washed off, stimulated with isoproterenol and fluorescence was measured. Indeed, (2R,6R)-HNK robustly increased cAMP indicating a possible role of cAMP in the antidepressant effects of (2R,6R)-HNK.

Data presented herein suggest astrocytic cAMP-dependent BDNF may be involved in antidepressant actions of ketamine. Some of the effects of ketamine (e.g., Ga translocation and cAMP production) were immediate and others (e.g., CREB phosphorylation and BDNF production) were delayed. Ketamine has a metabolic half-life of ~6 h, while remission of depressive symptoms on average persist for 1-2 weeks, suggesting deployment of both rapid and sustained cellular pathways for antidepressant effects (Berman, RM, et al. Biol Psychiatry. 2000;47:351-4; and Zarate CA, et al. Arch Gen Psychiatry. 2006;63:856-64). The results described herein show cAMP-dependent production of BDNF 24 h after ketamine treatment in astrocytes. Reports are conflicting on the timeframe of increased BDNF expression; some showing increased BDNF production after 24 h, while others observe this within 30 min (Li, N. Science. 2010;329:959-65; Autry AE, et al. Nature. 2011;475:91-95; Zanos P, et al. Nature. 2016;533:481-6; and Popp S, et al. FlOOOResearch. 2016;5:634). The method of tissue collection and data presented here may explain this controversy in the literature, given isolation of synaptosomes would limit glia derived BDNF. Moreover, the cAMP-dependent effect may be specific to glia, as reports on primary neurons and animals show antidepressant doses of ketamine decrease phosphorylation of eEF2 and show conflicting reports on the effect of phosphorylation of CREB (Autry AE, et al. Nature. 2011;475:91-95; Zanos P, et al. Nature. 2016;533:481-6; Xue W, et al. Sci Rep. 2016:6:26331; and Reus GZ, et al. Behav Brain Res. 2011;221: 166-71).

The inability of NMD AR antagonists other than ketamine to elicit the Gas biosignature, combined with these cellular antidepressant hallmarks persisting after reduced expression of NMDARs suggests that ketamine actions are NMDAR-independent, at least in astrocytes and C6 cells. This is further reinforced by the positive effect seen with (2R,6R)- HNK since it does not bind the NMD AR at 10 pM, yet still elicits antidepressant biochemical and behavioral responses (Zanos P, et al. Nature. 2016;533:481-6). This report also contrasts studies, where NMD AR antagonists other than ketamine elicit a similar biochemical antidepressant response to ketamine (Li. N. Science. 2010; 329:959-65; and Autry AE. et al. Nature. 2011; 475:91-95). However, ketamine may act at both neurons and glia, and it is possible that NMD AR are relevant for the former.

Note that accumulating evidence suggests glia contribute to the pathophysiology of MDD. Several post-mortem histological investigations of depressed subjects report reduced astrocyte numbers in brain regions implicated in depression, including the prefrontal cortex, dorsolateral prefrontal cortex, orbitofrontal cortex, and hippocampus. This is consistent with impaired astrocyte function in the pathophysiology of MDD (Wang Q, et al. Glia. 2017; 65: 1227-50). Furthermore, PET studies showing global reduction of cAMP in the brains of depressed subjects cannot differentiate between cell types (Fujita M, et al. Mol Psychiatry. 2017; 22:754-9). The role of cAMP in astrocytes or other glia in MDD and the antidepressant response has been largely unexplored. Given the delayed increase in BDNF expression in astrocytes compared to studies performed in neurons, the NMDAR-independent effects of ketamine in translocating Ga from lipid rafts may contribute to the maintenance of the antidepressant action of ketamine. BDNF is an important component of antidepressant action, and glia synthesize and secrete a variety of neurotrophic factors, including BDNF. Astrocyte-derived BDNF is involved in synaptogenesis, dendritic arborization, and increased dendritic spine density (Wang Q, et al. Glia. 2017; 65: 1227-50). One may speculate that a major loss of neurotrophic producing cells, or a loss of astrocytic neurotrophic production would result in depression. Parallel to this concept, increased astrocytic neurotrophic production may have antidepressant effects. Indeed, preclinical data suggests overexpressing BDNF in mouse hippocampal astrocytes produces antidepressant effects (Quesseveur G, et al. Transl Psychiatry. 2013; 3:e253). Data presented herein suggest cAMP-dependent BDNF production may play a role in the antidepressant actions of ketamine. Recently, it was reported that the translocation of Ga from lipid rafts was mediated by disruption of lipid raft tubulin/Ga complexes after sustained treatment with a variety of antidepressant compounds (Singh, H, et al. Neuropsychopharmacology. 2018; 1-11). Unlike previously tested compounds, ketamine's action is dramatically more rapid, suggesting a different portal to the translocation of Ga. Like many lipophilic anesthetic compounds, ketamine partitions into the plasma membrane (Jerabek H, et al. J Am Chem Soc. 2010; 132:7990-7) and may interact directly with lipids or proteins in the plasma membrane to influence the localization of Ga. Indeed, other anesthetics have been shown to alter plasma membrane localization of transmembrane proteins within minutes of treatment (Bademosi AT, et al. Cell Rep. 2018; 22:427-40). The translocation of Ga from lipid rafts may be considered a cellular hallmark of antidepressant action and provides a biosignature with which to identify compounds with antidepressant potential. The notion that ketamine displays this signature along a time course relevant for its antidepressant effects is intriguing. Thus, these results reveal a mechanism for ketamine action, but also suggest a cAMP-dependent mechanism of action of many classes of antidepressants.

Example 3: A biomarker for depression and antidepressant response.

It was tested whether antidepressant treatment changes Gsa such that it exits from the lipid raft, moves to the non-raft region, and completes the process of neurotransmitter action by stimulating the enzy me, adenylyl cyclase. Subjects with MDD were tested the effects of antidepressant treatment on the putative Gsa biomarker.

Materials/Subjects and Methods. The clinical data and platelets for this study came from a 6-week open-label, naturalistic antidepressant trial of participants experiencing an acute maj or depressive episode to assist the development of a test for quantitation of depression and prediction of therapeutic response.

Subject selection. Depressed subjects with non-psychotic MDD meeting DSM-IV TR criteria for MDD based upon the Structured Clinical Interview for DSM-IV (First MB, et al. Structured Clinical Interview for DSM-IV Axis I Disorders-Patient Edition (SCID-I/P, Version 2.0). Biometrics Research Department, New York State Psychiatric Institute, New York; American Psychiatric Association. Diagnostic and statistical manual of mental disorders, 4th edition (DSM-IV). Washington DC. 1994; and American Psychiatric Association. Diagnostic and statistical manual of mental disorders, 5th edition. Arlington VA: American Psychiatric Press. 2013), and mentally healthy controls were used for this study. The subjects were male and females between the ages of 30-65.

Eligible depressed subjects had a 17-item Hamilton rating scale for depression (HamD17) score that was >15 at the screen visit (Hamilton M. Br J Psychiatry. 1960; 134:382-9). Depressed subjects were excluded if they met criteria for bipolar disorder, a primary psychotic disorder, dementia, substance abuse or dependence, within 3 months of the screening visit or presented with a clinically significant suicide risk. The Columbia Suicide Severity Rating Scale was used as part of the assessment of suicidal risk (Posner K, et al. Am J Psychiatry. 2011; 168: 1266-77). Eligible depressed subjects had not been taking antidepressant or other psychotropic medications (except for sedatives) for at least 4 weeks prior to the initiation of treatment. Healthy controls had scores <7 on the HamD17 and had no history of MDD or dysthymia, and no current psychiatric diagnosis as assessed by the SCID- IV.

Study design. The study consisted of 2 required visits and an optional third visit for treated MDD subjects. At Visit 1, consenting participants underwent a diagnostic interview by a study psychiatrist who completed the SCID-IV to confirm the presence of MDD and absence of exclusionary diagnoses. In addition, clinical ratings scales were administered by a trained rater and participants completed the Inventory of Depressive Symptomatology Selfreport (IDS-SR30) to assess severity of depressive symptoms over the past week (Rush AJ, et al. Psychiatry' Res. 1986:18:65-87). Vital signs, medical history and physical examination were performed, followed by screening laboratory urine testing (urine drug screen, urinalysis, urine pregnancy test) and phlebotomy for complete metabolic profile, complete blood count, thyroid stimulating hormone test, and for collection of the Gsa blood draw (30 ml of whole blood). The sample was collected without regard for fasting status or time of day (usually between 10 A.M. and 3 P.M.), as initial volunteer blood draws for methods development showed that these vanables are unrelated to the Gsa content or distribution in platelets.

Visit 2 was scheduled 7 days after Visit 1 and consisted of vital signs, adverse events, and concomitant medications review, and repeat of the Gsa blood draw (30 ml) to assess the test-retest reliability of the Gsa measure over two separate time points. At this visit, MDD participants who desired pharmacotherapy were prescribed an antidepressant medication after consultation with a study psychiatrist. Participants who completed 6 weeks of pharmacotherapy were invited to return for a repeat assessment of their symptoms and to repeat the Gsa 30 ml blood draw to evaluate change in the Gsa measure after treatment.

Visit 3 was scheduled 6 weeks after the initiation of the open-label antidepressant medication. Visit 3 included clinical ratings, participant self-ratings, a blood draw of 30 ml, vital signs, and a review of concomitant medications and adverse events.

Visits 1 and 3 assessments included clinician ratings of the HamD17 (primary measure), HamD6 (derived from the HamD17), Montgomery Asberg depression rating scale (MADRS). Hamilton rating scale for anxiety (Ham-A), the Clinical Global Impression of Severity (CGI-S), and a self-report measure (the Inventory of Depressive Symptoms-self rating form, IDS-SR30) that was completed by the study participant (Hamilton M. Br J Psychiatry. 1960;134:382-9; Rush AJ, et al. Psychiatry Res. 1986;18:65-87; Hamilton M. Br J Med Psychol. 1959;32:50-55; Guy W. Early Clinical Drug Evaluation (ECDEU) assessment manual for psycho-pharmacology. Publication No. 76-338. Rockville, Md: National Institute of Mental Health. 1976; Montgomery S and Asberg M. Br J Psychiatry. 1979; 134:382-9; and Bech P. Dialogues Clin Neurosci. 2006; 8:207-15).

Preparation of Gsa samples. After each blood draw, blood was separated into red blood cells, white blood cells, and platelet-rich plasma. To ensure blinding, the blood samples were identified by the participant study ID number.

Each sample was divided into aliquots and frozen at -80 °C until further use. Prior to use, platelets were thawed, diluted 1: 10 in TEM buffer (10 mM Tris HC1. 1 mM MgC12, 150 mM NaCl 1 mM EDTA pH 7.5, protease inhibitor cocktail, Sigma # P2714) and adjusted to a concentration of 1 pg/pL for the adenylyl cyclase assay.

Adenylyl cyclase activity was assayed in a 384 well plate using PerkinElmer’s AlphaScreen cAMP assay kit following the manufacturer’s directions. Briefly, 2.5 ML of platelets was mixed with 1.5 ML of stimulation buffer (1 MM Hepes pH 7.5, 500 MM IBMX, 0. 1% BSA. 25 mM MgC12, 375 mM NaCl, 250 MM ATP, 2.5 MM GDP, 2.5 nM GTP in HBSS) in one well of a 384 well plate. Subsequently. 2.5 ML of acceptor beads in stimulation buffer were mixed with the platelets and 5 ML total volume of cells/beads was added to each well. Adenylyl cyclase activity was measured both without stimulating agent (basal, 5 ML stimulation buffer) and in the presence of 10 MM prostaglandin El (PGE1) in 5 ML of stimulation buffer. The 384 well plate was incubated for 30 min at RT to allow cAMP accumulation. The reaction was stopped by adding 15 ML of 1.67 x biotin-cAMP/ Streptavidin Donor Bead Detection Mix. The plate was sealed and kept in the dark overnight. Plates were read on a Molecular Devices SpectraMax i3x plate reader. cAMP produced was calculated from a standard curve run with each assay.

Data analyses. Basal adenylyl cyclase activity and the magnitude of PGE1 stimulated adenylyl cyclase activity w as compared in platelet samples obtained from healthy controls and MDD subjects at visit 1 (screen) and at visit 3 from the MDD subjects who participated in the 6-week open label treatment program. The changes from visit 1 (screen) to visit 3 (post-treatment) of PGE1 stimulation of adenylyl cyclase activity over basal activity in antidepressant treatment responders and non-responders were evaluated. Antidepressant treatment response w as defined as >50% improvement after 6-weeks of treatment from the total HamD17 score at the screen visit. Statistical analyses included Student’s t tests for independent samples and Spearman’s correlation coefficient as appropriate.

Results. Forty-nine study participants who met DSM-IV criteria for MDD, and 59 healthy controls seen at the screen visit ranging in age from 30 to 64 years old. Twelve men and 37 women were in the MDD group (mean age = 49. 1 ± 7.8 years) and 18 men and 41 women were in the healthy control group (mean age =43.6 ± 11.3 years). At the screen visit, the mean HamD17 was 20.5 ± 3.3 in the whole MDD group, the CGI-S was 4.6 ± 0.6, and the IDS-SR30 was 35.4 ± 10.9. The healthy controls had a mean CGI-S of 1 .0 ± 1.0 and IDS- SR30 of 2.9 ± 3.0 at the screen visit. Platelet samples were available from 41 MDD subjects and 44 healthy controls at the screen visit. No significant age, sex, or weight differences were observed between the MDD subjects and healthy controls. The mean basal cAMP activity was 24.3 ± 13.6 nM cAMP/well in the MDD subjects and 27.1 ± 14. 15 nM cAMP/well in the healthy controls (t = -0.87; p = ns). As shown in FIG. 11, PGE1 stimulation of adenylyl cyclase activity⁷ yielded a significantly lower stimulation response in the MDD subj ects than the healthy controls (t = -2.3; p = 0.02). EC50 for PGE1 was 0.19 x | ()’^s for healthy controls and 1.9 x 10 ⁹ for MDD subj ects.

Twenty -five MDD subjects began antidepressant treatment. As per protocol, treated subjects were not required to return for visit 3. Nineteen subjects completed 6 weeks of treatment and had reliable visit 1 and visit 3 clinical data and platelet samples available for analysis. Six men and 13 women were in the group of 19 treated MDD subjects who ranged in age from 35 to 60 years (mean age = 50.6 ± 6.2 years). The antidepressants prescribed were escitalopram [7], citalopram [4], fluoxetine [3], duloxetine [2], venlafaxine XR [2], and nortriptyline [1], Eleven treated subjects were antidepressant treatment responders at 6 weeks. No significant demographic differences were observed between the treatment responders and non-responders. The mean total HamD17 score at screen was 20.4 ± 2.5 (SD) in the treatment responders and 20. 1 ± 5.0 in the non- responders (t = 0. 19; p = ns). After 6 weeks of treatment, the HamD17 score improved by 14.6 ± 3.6 points in the responders and 6.0 ± 2.3 in the non-responders (t = 5.87; p < 0.0001). The other clinical metrics tracked with the HamD17 score changes. At 6 weeks, treatment responders had significantly greater improvement than non-responders on the HamD6 (t = 3.06; p = 0.007), MADRS (t = 3.70; p < 0.002), and the CGI-S (t = 3.87; p = 0.05) and revealed non- significant trends on the Ham- A (t = 1.45; p = 0. 16), and self-rated IDS-SR30 (t = 1.91; p = 0.08).

The results described herein include the demographic and treatment data and corresponding changes of the total HamD17 scores and PGE1 stimulation of adenyly l cyclase activity⁷ (normalized over basal activity⁷) from screen (visit 1) to visit 3 for each of the 19 treated subjects. Antidepressant treatment responders had a significant increase in PGE1 stimulated adenylyl cyclase activity at 6 weeks compared to non-responders (FIG. 12). After 6 weeks of antidepressant treatment, PGE1 stimulation of adenylyl cyclase over basal cAMP activity⁷ increased 1.35 ± 2.3 (SD) nM cAMP/well from the screen visit in the 11 treatment responders, whereas it decreased -0.32 ± 1.0 nM cAMP/well in the eight non-responders (t = 2.14; p = 0.050). FIG. 13 displays graphically, the change of PGE1 stimulation of adenylyl cyclase activity normalized over basal activity between visits 1 and 3. The calculated effect size (Cohen’s d) was 0.83 for the PGEl/Gsa lipid-raft biomarker. Antidepressant treatment responders revealed a 62.0% mean increase of PGE1 stimulation from the screen assessment in contrast to a -4. % decrement in the non-responder cohort. A modest correlation was observed between increased cAMP activity and improvement on the total HamD17 score between visits 1 and 3 in the 19 treated MDD subjects (Spearman’s rank-order correlation: rs = 0.339, t = 1.49, p = 0.15). PGE1 stimulation of adenylyl cyclase activity increased by at least 30% from the screen assessment in 8 of the 11 responders (72.7%) on contrast to 2 of the 8 non-responders (25.0%) in this small group of subjects (Fisher exact = 0.07), and the positive predictive value of PGE1 activation for treatment response was 80.0%.

The relationship of changes in Gsa-stimulated adenylyl cyclase activity in MDD subjects was evaluated in a 6-week open-label antidepressant treatment trial. The results show that MDD subjects had significantly lower PGE1 activation of adenylyl cyclase activity' in platelet samples than healthy controls at the screen visit (p = 0.02). The results also show that antidepressant treatment responders had a marked increase in PGE1 stimulated adenylyl cyclase after 6-weeks of treatment compared to non-responders (t = 2. 14; p = 0.050) with a calculated effect size of 0.83 for the PGE/Gsa lipid-raft biomarker.

PGE1 stimulation of adenylyl cyclase improved by at least 30% from the screen assessment in 8 of the 11 antidepressant treatment responders (72.7%) in contrast to 2 of the 8 non-responders (25.0%) in this study (Fisher exact = 0.07). A (five-fold) decrease in 5HT potency was observed for controls vs. MDD subjects, consistent with the notion that the relevant biology underlying this biomarker is the coupling between Gsa and adenylyl cyclase dictated by the extent of lipid raft localization of Gsa.

Post-mortem evidence demonstrates that lipid rafts from subjects with MDD are enriched in the heterotrimeric G protein, Gsa, consistent with diminished cAMP signaling (Donati RJ, et al. J Neurosci. 2008;28:3042-50; and Singh H, et al. J Neurosci. 2020;40:4033-41). Gsa located outside lipid rafts stimulates adenylyl cyclase more efficiently than when it is located within lipid rafts and chronic treatment with antidepressants facilitates G protein exodus from those rafts (Allen JA, et al. Mol Pharmacol. 2009;76: 1082-108; Donati RJ and Rasenick MM. Neuropsychopharmacology.

2005;30: 1238-45; Zhang L and Rasenick MM. J Pharm Exp Ther. 2010;332:977-84; Czysz AH, et al. Neuropsychopharmacology. 2015;40:766-73; Singh H, et al. Neuropsychopharmacology. 2018;43: 1481-91; and Senese NB and Rasenick MM. Mol Pharm. 2021;100:66-81).

Earlier studies that employed more cumbersome cAMP assays suggested that attenuated PGE1 in platelets (Mooney JJ, et al. Biol Psychiatry. 1988; 23:543-59) and impaired P-adrenergic signaling in white blood cells (Pandey GN, et al. Acta Pharm Toxicol. 1985; 56 (Suppl 1):S66-S79) could be associated with both depression and clinical improvement. It is noteworthy, however, that, in lymphoblasts from depressed subjects, increased cAMP signaling was observed which was reversed by antidepressant treatment (Chukaew P, et al. Mol Psychiatry. 2021; 26:2402-14). Unlike platelets, lymphocytes are negatively affected by cAMP, and attenuated cAMP is "‘favorable'’ for the lymphocyte. Caruncho et al. found altered membrane clustering of 5HT2A receptors and serotonin transporters in lymphocytes from depressed subj ects and attributed this finding to altered cytoskeletal association with these proteins (Caruncho HJ, et al. Front Pharm. 2019; 10: 19047). This finding could be attributed to altered association of these components with lipid rafts.

In preclinical studies, selective serotonin reuptake inhibitors, serotoninnorepinephrine reuptake inhibitors, tricyclic antidepressants, and monoamine oxidase inhibitors each increased Gsa signaling and evoked translocation of Gsa from lipid rafts. Studies in rodents required three weeks of drug treatment while studies in neuronal and glial cells required three days. Extending treatment time in cellular models allowed drug concentrations used to mirror those used in the clinic. Rapid-acting antidepressant drugs such as ketamine had similar effects to traditional antidepressants in the cellular model systems, but on a much more rapid timescale (15 min rather than 3 days) (Wray N, et al. Mol Psychiatry. 2019; 24: 1833-43). Thus, antidepressants concentrate in lipid rafts contributing at least in part for their antidepressant effect (Eisensamer B, et al. J Neurosci. 2005; 25:10198- 206; and Erb SJ, et al. J Biol Chem. 2016; 291: 19725-33). While most antidepressants sort slowly into lipid rafts, ketamine appears do so rapidly (Wray N, et al. Mol Psychiatry. 2019; 24: 1833-43; and Casarotto PC, et al. Cell. 2021; 184: 1299-313). Taken together, a model for Gsa sequestration in rafts during depression and subsequent liberation by antidepressants, which as described herein detected as an increase in PGE1 -activated adenylyl cyclase, can be used to demonstrate the effects of depression and antidepressant treatment have on Gsa plasma membrane localization. Briefly, Gsa is distributed between non-raft regions of the membrane where it moves freely and promotes neurotransmitter-activated adenylyl cyclase activity and a specialized region of the membrane rich in cholesterol (lipid raft), where the movement/adenylyl cyclase activation of Gsa is impaired. During depression, Gsa is ensconced in the lipid raft region where it is anchored by the structural protein, tubulin (Ta/TB). Antidepressant treatment changes Gsa such that it exits from the raft and moves to the non-raft region where it completes the process of neurotransmitter action by activating the enzyme, adenylyl cyclase. Thus, the translocation of Gsa from lipid rafts, as reflected by an increase in PGE1 stimulated adenylyl cyclase, can serve as a biomarker for clinical response to antidepressants.

The methods described herein can be used as a quantifiable diagnostic and treatment paradigm.

As disclosed herein, the translocation of Gsa from lipid rafts, as reflected by an increase in PGE1 stimulated adenylyl cy clase, was used as a biomarker reflecting cAMP signaling/lipid-raft status of Gsa, and was associated with antidepressant treatment response in MDD subjects. These data demonstrate that a simple, high-throughput-capable assay for MDD and antidepressant response can be used for personalized medicine for subjects with MDD.

Example 4: Gsa is a stable biomarker for depression.

Subject recruitment. Subjects were recruited and had been diagnosed with major depressive disorder (MDD).

Inclusion criteria. The patients were between 25-70 years old; and met MDD criteria according to DSM-5 criteria for major depressive disorder. Patients with co-morbid anxiety were not excluded.

Exclusion criteria. Patients with active suicidal ideation as determined by the Columbia Suicide Severity Rating Scale (C-SSRS), suicide attempt(s) in the last 3 months; severe cognitive impairment secondary⁷ to a neurological disorder (e.g., mild cognitive impairment, neurocognitive disorders, traumatic brain injury, developmental delay) defined by a Montreal Cognitive Assessment (MoCA) Score < 26); active moderate or severe alcohol and/or substance use disorders; major medical or neurologic illness that would interfere with protocol adherence and/or interpretation of findings; and/or the presence of contraindications to MRI were excluded. Clinical assessment. Upon study entry, the participants received a physical exam, and the following clinical tests/instruments (Week 0): 1) Structural Clinical Interview for DSM-5 (SCID); 2) The Cumulative Illness Rating Scale (CIRS); 3); Hamilton Rating Scale for Depression (HAM-D); 4) Young Mania Rating Scale (YMRS); 5) The Inventory of Depressive Symptomatology (IDS-C/QIDS-C); 6) Frequency , Intensity , Burden of Side Effects Rating Scale (60); 7) UKU Side Effect Self Rating Scale (UKU-SERS-Pat); and 10) a brief neuropsychological protocol focused on cognitive domains of processing speed, executive function and attention using the following tests from the NIH Toolbox: Pattern Comparison Processing Speed Test, Dimensional Change Card Sort Test, List Sorting Working Memory Test and Flanker Inhibitory Control and Attention Test. These tests have been shown to have good test-retest reliability and standardized effect sizes for practice effects. Psychiatrically-healthy control subjects were recruited from UIC clinic patients enrolled in other studies at the Clinical Translational Science Center at UIC.

Blood collection. Blood was collected, and one sample (about 8 ml) was collected in an EDTA-containing (lavender top) vacutainer tube and separated into RBC, WBC and platelet-rich plasma fractions. Platelets were collected from platelet rich plasma by centrifugation and frozen at -80°C until assay.

Blood was assayed for Gas-activated adenylyl cyclase as described in Targum et al., Mol. Psychiatry', 2022, 27(3): 1640-1646. cAMP assay was performed on thawed platelets using Perkin-Elmer Alpha screen assay according to the manufacturer’s directions. cAMP values were obtained for samples in the presence and absence of prostaglandin (PGE1 or PGE2) and calculated as a percentage of the unstimulated (no PGE) level. This ratio is the number used in the calculations.

FIG. 14 plots HAM-D at visit one vs. Gas-activated-AC values. Subjects with MDD are included in these data, which show a correlation r= -0.54 between severity of depression and Gas-activated-AC values.

FIG. 15 compares Gas-activated-AC values for healthy control subjects with subjects diagnosed with MDD who have responded to treatment and are currently asymptomatic and subjects showing mild depression as indicated by their QIDS score. Subjects with MDD who are asymptomatic do not separate out from healthy controls, while the mildly depressed subjects separate from both groups at p<0.002. These results confirm results disclosed herein in a new set of subjects and demonstrated that Gas-activated- AC is a reliable biomarker for depression.

PGE1 and non-activated adenylyl cyclase were assayed in platelets according to the methods of figure 11 QIDS-SRie scores between 6 andlO reflect mild depression whereas scores >11 reflect moderate depression. Samples depicted in FIG. 15 were from mildly depressed subjects, subjects initially diagnosed with moderate depression and currently asymptomatic (n=19) and psychiatrically healthy controls (n=10). Gas-activated- AC values in asymptomatic MDD subjects were not different from values obtained with platelets from healthy controls, whereas Gas-activated-AC values from mildly depressed subjects were significantly lower than both groups.

In sum. QIDS-SR16 patient self-ratings show that PGE- 1 -activated adenylyl cyclase in platelets acts as a biomarker for mild depression and that subjects diagnosed with major depression, but not currently symptomatic, resemble healthy controls in their Gas- activatedAC activity.

FIG. 16 compares Gas-activated-AC values from MDD subjects in blood drawn at visits 2 weeks apart and demonstrates the remarkable stability for within-subject Gsa/AC biomarker values between two blood draws. Platelets were collected from MDD (n=20) and control (n=6) subjects at the screen visit; were isolated and assayed for cAMP. HAM-D score was measured the same day with platelet collection at 2 different time points in 2 weeks (Visit 1, Visit 2). Note equivalence of week 1 and week 2 Gas-activated-AC values. MDD was defined as HAM-D > 7.

FIG. 17 represents individual subject data from FIG. 16. The data demonstrates the stability for individuals across two blood draws and shows that minor variations in depression severi ty between the two visits are represented by corresponding changes in Gas-activated- AC values. For example. FIG. 17 provides evidence that the Gas-activated-AC is a stable biomarker in subjects with stable QIDS, but varies with change in depression severity.

PGE 1 -activated adenylyl cyclase as a percentage of unstimulated adenylyl cyclase was calculated for platelets from 2 patient visits, one week change in the Quick Inventory of Depressive Symptomatology (QIDS) score and a corresponding change in PGE1 stimulated cAMP response. The values between the two time points are consistent. For subjects with equal QIDS scored on each visit, the Gas-activated-AC values remain stable. For those with small changes in QIDS (there was little to no change in most subjects), the Gas-activated-AC score reflected the limited increase or decrease in depression severity.

Although not represented here, blood from a single volunteer was collected in five vacutainer tubes. Platelets from one of the tubes were prepared immediately, while one was stored at 4°C for 5 days and the others remained at room temperature for 3 and 5 days before platelets were prepared. The tubes were assayed for basal or PGE1 -stimulated adenylyl cyclase at the same time. Gas-activated-AC values varied by < 10% for the samples, indicating the stability of samples collected for the Gas-activated-AC assay.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other aspects of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method comprising: a) contacting a sample with a sulfhydryl reducing agent; and b) determining the amount of Gas palmitoylation in the sample by mass spectrometry.

2. A method of determining the amount of Gas palmitoylation in a subject, the method comprising: a) contacting a sample obtained from the subject with a sulfhydryl reducing agent; and b) determining the amount of Gas palmitoylation in the sample by mass spectrometry.

3. A method of determining the severity of depression in a subject, the method comprising: a) contacting a sample obtained from the subject with a sulfhydryl reducing agent; and b) determining the amount of Gas palmitoylation in the sample by mass spectrometry, wherein the amount of Gas palmitoylation in the sample is indicative of the severity of depression in the subject.

4. A method comprising: a) obtaining a sample from a subject; b) contacting the sample with a sulfhydryl reducing agent; c) determining the amount of Gas palmitoylation in the sample in b) by mass spectrometry; d) administering an anti-depressant agent to the subject; e) obtaining a sample from the subject after step d); f) contacting the sample with a sulfhydryl reducing agent; g) determining the amount of Gas palmitoylation in the sample in step f) by mass spectrometry; and h) comparing the amount of Gas palmitoylation from step c) with the amount of Gas palmitoylation from step g).

5. The method of claim 4, wherein when the amount of Gas palmitoylation is decreased in step g) compared to the amount Gas palmitoylation in step c) indicating the efficacy of the anti-depressant agent.

6. A method comprising: a) administering an antidepressant agent to a subject; b) obtaining a sample at from the subject after step a) at a first time point; c) obtaining a sample at from the subject after step a) at a second time point, wherein the second time point is after the first time point; d) contacting the samples from step b) and step c) with a sulfhydryl reducing agent; e) determining the amount of Gas palmitoylation in the samples from b) and c) by mass spectrometry; and f) comparing the amount of Gas palmitoylation from the first time point with the amount of Gas palmitoylation from the second time point.

7. The method of any of claims 1-6, wherein the amount of Gas palmitoylation in the sample is determined as a percentage of total Gas.

8. The method of claims 1-6, further comprising comparing the amount of Gas palmitoylation to a predetermined reference value.

9. The method of any of the preceding claims, wherein the sample comprises a blood cell.

10. The method of claim 9, wherein the blood cell is a leukocyte, an erythrocyte or a platelet.

11. The method of any of claims 1-6, further comprising calculating a ratio of Gas palmitoylation to nonpalmitoylated Gas in the sample.

12. The method of any of claims 1-6, wherein the sulfhydryl reducing agent is N- ethylmaleimide, dithiothreitol, iodoacetamide, or N-methyl maleimide.

13. The method of any of the preceding claims, wherein the sample comprises a blood cell.

14. The method of claim 13, wherein the blood cell is a leukocyte, an erythrocyte or a platelet.

15. The method of any of the preceding claims, wherein the subject is a human subject.

16. The method of any of the preceding claims, wherein the subject is an adult.

17. The method of any of the preceding claims, wherein the subject is an adolescent or a child.

18. The method of any of the preceding claims, wherein the subject is maintained on the antidepressant therapy.

19. A method of screening a candidate compound for antidepressant activity, the method comprising: a) contacting the candidate compound with one or more cells; b) incubating the one or more cells in step a) with an anti-Gas antibody; c) determining the amount of Gas palmitoylation in the one or more cells after step b) by mass spectrometry; d) comparing the amount of Gas palmitoylation in the one or more cells in step a) with the amount of Gas palmitoylation in control cells that have not been exposed to the candidate compound, and wherein when the amount of Gas palmitoylation is decreased compared to the Gas palmitoylation of control cells that have not been exposed to the candidate compound is indicative of antidepressant activity.

20. The method of claim 19, wherein the one or more cell are C6 glioma cells, SK-N-SH neuroblastoma cells, or patient-derived neural stem cells.

21. A method of treating depression in a subject, the method comprising administering to a subject with depression an effective amount of the candidate compound identified using the method of claim 19.

22. A method of determining the severity of depression in a subject, the method comprising: a) contacting a sample obtained from the subject with an anti-Gas antibody; and b) determining the amount of Gas palmitoylation in the sample in a) by mass spectrometry; and wherein the amount of the Gas palmitoylation in the sample is indicative of the severity of depression in the subject.

23. The method of claim 22, wherein the sample comprises a blood cell.

24. The method of claim 23, wherein the blood cell is a leukocyte, an erythrocyte or a platelet.

25. A method for identifying an agent having antidepressant activity, the method comprising: a) contacting the agent with cells; b) incubating the cells for a period of up to 72 hours; and c) measuring the association of Gas with adenylyl cyclase by cyclic adenosine monophosphate (cAMP) fluorescence, wherein an increase in the association of Gas with adenylyl cyclase relative to control cells that have not been exposed to the agent is indicative of antidepressant activity.

26. The method of claim 25, wherein the association of Gas with adenylyl cyclase is determined by measuring cAMP levels in the cells using a fluorescent cAMP reporter.

27. The method of claim 25, wherein the association of Gas with adenylyl cyclase is measured by fluorescence recovery after photobleaching of GFP-Gs.

28. The method of claim 25, wherein the cells comprise C6 glioma cells or SK N SH neuroblastoma cells.

29. A method of identifying an agent having antidepressant activity in a depressed subject, the method comprising: a) contacting the agent with cells, wherein the cells are obtained from the depressed subject and maintained in culture; b) incubating the cells for a period of up to 72 hours with putative antidepressant agents; and c) measuring the association of Gas with adenylyl cyclase by fluorescence, wherein an increase in the association of Gas with adenyl yl cyclase is indicative of antidepressant activity.

30. The method of claim 29, wherein the association of Gas with adenylyl cyclase is determined by measuring cyclic adenosine monophosphate (cAMP) levels in the cells using a fluorescent cAMP reporter, wherein the presence of the fluorescent cAMP reporter is indicative of total cellular cAMP or lipid raft cAMP.

31. The method of claim 29, wherein the association of Gas with adenylyl cyclase is measured by fluorescence recovery after photobleaching of GFP- Gas.

32. The method of claim 29, wherein the increase in cAMP levels is relative to control cells that have not been exposed to the agent.

33. The method of claim 29, wherein the increase in cAMP levels is relative to control cells that have been exposed to a second agent having antidepressant activity.

34. The method of claim 33, wherein the cells comprise blood cells.

35. The method of claim 34, wherein the blood cells are leukocytes.

36. A method of determining the effectiveness of antidepressant agent in a depressed subject, the method comprising: a) contacting the antidepressant agent with cells obtained from the depressed subject, wherein the depressed subject is being treated with the antidepressant agent at a first time point and a second time point; b) incubating the cells for a period of up to 72 hours; and c) measuring the association of Gas with adenylyl cyclase by fluorescence, wherein an increase in the association of Gas with adenylyl cyclase measured in the cells obtained at the second time point relative to the association of Gas with adenylyl cyclase measured in the cells obtained at the first time point indicates that the antidepressant therapy is effective.

37. The method of claim 36, wherein the association of Gas with adenylyl cyclase is determined by measuring cyclic adenosine monophosphate (cAMP) levels in the cells using a fluorescent cAMP reporter.

38. The method of claim 36, wherein the association of Gas with adenylyl cyclase is measured by fluorescence recovery after photobleaching of GFP- Gas.

39. The method of claim 36, wherein the increase in cAMP levels is relative to control cells that have not been exposed to the agent.

40. The method of claim 36, wherein the increase in cAMP levels is relative to control cells that have been exposed to a second agent having antidepressant activity.

41 . The method of claim 36, wherein the cells comprise blood cells.

42. The method of claim 41, wherein the blood cells are erythrocytes, leukocytes, platelets or a combination thereof.

43. The method of claim 42, wherein the blood cells comprise leukocytes.

44. The method of claim 36, wherein the effectiveness of the antidepressant agent corresponds to the loss of lipid-raft specific adenylyl cyclase.

45. A method of treating a depressed patient comprising administering to the patient an antidepressant identified using the method of claim 25.

46. A method of treating a depressed patient comprising administering to the patient an antidepressant identified using the method of claim 29.

47. A method of determining the severity of depression in a subject, the method comprising measuring Gas-activated adenylyl cyclase in cells obtained from the subject wherein the measured levels of Gas-activated adenylyl cyclase correlate negatively with the extent to which a subject is depressed.

48. A method of monitoring a subject’s recovery from depression as a result of antidepressant treatment, the method comprising measuring levels of Gas-activated adenylyl cyclase in cells obtained from the subject, wherein the extent of the subject’s recovery correlates positively with the measured levels of Gas-activated adenylyl cyclase.

49. A method comprising: a) contacting a sample with an anti-pan palmitoylated antibody and an anti-Gas antibody; b) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity ligation assay; and c) determining the amount of Gas palmitoylation in the sample.

50. The method of claim 49, wherein the anti-pan palmitoylated antibody binds a cysteine residue of the Gas palmitoylated protein.

51. The method of claim 50, wherein the anti-pan palmitoylated antibody is CBL-PTM-pal.

52. The method of claim 49, wherein the anti-Gas antibody binds the carboxy terminus of the Gas protein.

53. The method of claim 52, wherein the anti-Gas antibody is N192/12.

54. The method of any of claims 49-53, wherein the proximity ligation assay comprises contacting the sample with pair of oligonucleotide-labeled secondary antibodies (PLA probes) that bind to the anti-pan palmitoylated antibody and an anti-Gas antibody, respectively.

55. The method of claim 54, further comprising contacting the sample with hybridizing connector oligonucleotides capable of joining the PLA probes.

56. The method of claim 49, wherein the proximity ligation assay is performed using immunofluorescence or immunohistochemistry.

57. The method of claim 49, wherein the detecting in step b) by the proximity ligation assay is performed by flow cytometry.

58. The method of claim 49, wherein the anti-pan palmitoylated antibody and the anti-Gas antibody are immobilized on a solid support.

59. A method comprising: a) obtaining a sample from a subject; b) contacting the sample with an anti-pan palmitoylated antibody and an anti-Gas antibody; c) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity ligation assay; d) determining the amount of Gas palmitoylation in the sample; e) administering an anti-depressant agent to the subject; f) obtaining a sample from the subject after step e); g) contacting the sample with an anti-pan palmitoylated antibody and an anti-Gas antibody; h) detecting Gas palmitoylated protein and Gas protein in the sample in step g) by a proximity ligation assay; i) determining the amount of Gas palmitoylation in the sample in step h); and j) comparing the amount of Gas palmitoylation from step d) with the amount of Gas palmitoylation from step i).

60. The method of claim 59, wherein when the amount of Gas palmitoylation is decreased in step g) compared to the amount Gas palmitoylation in step c) indicating the efficacy of the anti -depressant agent.

61. The method of any of the preceding claims, wherein the sample comprises a blood cell.

62. The method of claim 61, wherein the blood cell is a leukocyte, an erythrocyte or a platelet.

63. The method of any of the preceding claims, wherein the subject is a human subject.

64. The method of any of the preceding claims, wherein the subject is an adult.

65. The method of any of the preceding claims, wherein the subject is an adolescent or a child.

66. A method of screening a candidate compound for antidepressant activity, the method comprising: a) contacting the candidate compound with one or more cells; b) incubating the one or more cells in step a) with an anti-pan palmitoylated antibody and an anti-Gas antibody; c) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity ligation assay; d) determining the amount of Gas palmitoylation in the one or more cells after step c); e) comparing the amount of Gas palmitoylation in the one or more cells in step a) with the amount of Gas palmitoylation in control cells that have not been exposed to the candidate compound, and wherein when the amount of Gas palmitoylation is decreased compared to the Gas palmitoylation of control cells that have not been exposed to the candidate compound is indicative of antidepressant activity.

67. The method of claim 66, wherein the one or more cell are C6 glioma cells, SK-N-SH neuroblastoma cells, or patient-derived neural stem cells.

68. A method of treating depression in a subject, the method comprising administering to a subject with depression an effective amount of the candidate compound identified using the method of claim 66.

69. A method of determining the severity of depression in a subject, the method comprising: a) contacting a sample with an anti-pan palmitoylated antibody and an anti-Gas antibody; b) detecting Gas palmitoylated protein and Gas protein in the sample by a proximity ligation assay; and c) determining the amount of Gas palmitoylation in the sample; wherein the amount of the Gas palmitoylation in the sample is indicative of the severity of depression in the subject.

70. The method of claim 69, wherein the sample comprises a blood cell.

71. The method of claim 70, wherein the blood cell is a leukocyte, an erythrocyte or a platelet.