EP4646596A2 - Verfahren zur diagnose von depression und identifizierung von antidepressiver aktivität - Google Patents

Verfahren zur diagnose von depression und identifizierung von antidepressiver aktivität

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Publication number
EP4646596A2
EP4646596A2 EP24739008.1A EP24739008A EP4646596A2 EP 4646596 A2 EP4646596 A2 EP 4646596A2 EP 24739008 A EP24739008 A EP 24739008A EP 4646596 A2 EP4646596 A2 EP 4646596A2
Authority
EP
European Patent Office
Prior art keywords
gas
sample
subject
palmitoylation
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24739008.1A
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English (en)
French (fr)
Inventor
Mark M. Rasenick
Jason HICKOK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Illinois
US Department of Veterans Affairs
University of Illinois at Urbana Champaign
University of Illinois System
Original Assignee
University of Illinois
US Department of Veterans Affairs
University of Illinois at Urbana Champaign
University of Illinois System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Illinois, US Department of Veterans Affairs, University of Illinois at Urbana Champaign, University of Illinois System filed Critical University of Illinois
Publication of EP4646596A2 publication Critical patent/EP4646596A2/de
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • 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).
  • MDD major depressive disorder
  • 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.
  • the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample.
  • the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.
  • the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample.
  • 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 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.
  • the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample.
  • the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.
  • 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 activity 7 .
  • the cells can be cultured cells.
  • the cells can be blood cells.
  • identifying an agent having antidepressant activity in a depressed subject 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.
  • the cells can be cultured cells.
  • the cells can be blood cells.
  • 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.
  • the cells can be cultured cells.
  • the cells can be blood cells.
  • 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.
  • the cells can be cultured cells.
  • the cells can be blood cells.
  • 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.
  • the cells can be cultured cells.
  • the cells can be blood cells.
  • the cells can be obtained from saliva.
  • 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 determining the severity of depression in a subject 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.
  • 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.
  • MGC(p aim )LGNSK SEQ ID NO: 1
  • MGC(NEM)LGNSK SEQ ID NO: 1
  • SSRI escitalopram
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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).
  • 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.
  • 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. 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 his
  • 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.
  • FIGS. 8A-B show that other NMDA antagonist do not mediate Ga translocation from lipid rafts.
  • 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.
  • 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.
  • 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 recovery 7 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 activity 7 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.
  • MDD major depressive disorder
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the term “subject” refers to the target of administration, e.g., a human.
  • 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.).
  • a subject is a mammal.
  • 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.
  • the term “patient” refers to a subject afflicted with a disease or disorder.
  • the term “patient” includes human and veterinary subjects.
  • 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.
  • a treatment may include administration of a pharmaceutically effective amount of an antidepressant agent.
  • 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.
  • the disease, disorder, and/or condition can be depression or major depressive disorder.
  • preventing means preventing in whole or in part, or ameliorating or controlling.
  • “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.
  • 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.
  • 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.
  • the inhibition or reduction is 0-25, 25-50, 50-75, or 75- 100% as compared to native or control levels.
  • 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.
  • 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.
  • 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.
  • Gas As used herein. “Gas”. “Gas” and “Gsa” are used interchangeably and refer to the G- protein Gas.
  • 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.
  • 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.
  • G proteins/cAMP/CREB/'neurotrophin in depression and antidepressant action are G proteins/cAMP/CREB/'neurotrophin in depression and antidepressant action.
  • O’Donnell and Zhang 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).
  • increasing cAMP with inhibitors of phosphodiesterase showed promising antidepressantadjuvant properties (El-Haggar SM, et al. Psychother Psychosom. 2018; 87:331-9).
  • 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.
  • Gs cell surface G protein-coupled receptors
  • 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.
  • Gf> and Gy When not stimulated by a receptor, Ga is bound to GDP and to GPy to form the inactive G protein trimer.
  • the activated receptor 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • anti-depressant drugs are assumed to inhibit the proteins responsible for monoamine reuptake (transporters) or catabolism (monoamine oxidase).
  • STAR*D Sequenced Treatment Alternatives to Relieve Depression
  • 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.
  • 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.
  • 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).
  • adenylyl cyclase the enzyme activated by Gas
  • the measurements of Gas -activated adenylyl cyclase in platelets tracks with both depression severity' and antidepressant response.
  • 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.
  • 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.
  • the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample.
  • the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.
  • the method can be conducted without contacting the sample with a sulfhydryl reducing agent or otherwise in the absence of a sulfhydryl reducing agent.
  • 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.
  • the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non- palmitoylated Gas in the sample.
  • the amount of Gas palmitoylation in a sample can be determined as a percentage of total Gas in the sample.
  • 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.
  • the method can be conducted without contacting the sample with a sulfhydryl reducing agent or otherwise in the absence of a sulfhydryl reducing agent.
  • 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.
  • the amount of Gas palmitoylation in the sample can be indicative of the severity of depression in the subject.
  • 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.
  • 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.
  • 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).
  • the sample from the subject in step e) can be obtained one week after the sample obtained from the subject after step d).
  • the time the second sample is obtained can be dependent on the antidepressant that was administered to the subject.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • the therapeutic agent can be a therapeutic known to treat or ameliorate a symptom of depression.
  • the therapeutic agent can be, but is not limited to, any suitable pharmaceutical agent or other antidepressant agent.
  • 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).
  • the anti-pan palmitoylated antibody can bind a cysteine residue of the Gas palmitoylated protein.
  • the anti-pan palmitoylated antibody can be CBL-PTM-pal.
  • the anti-Gas antibody can bind the carboxy terminus of the Gas protein.
  • the anti-Gas antibody can be N192/12.
  • 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.
  • the anti-pan palmitoylated antibody and the anti-Gas antibody can be immobilized on a solid support.
  • the methods can utilize oligonucleotide-labeled secondary antibodies.
  • the oligonucleotides on the oligonucleotide-labeled secondary antibodies can hybridize to a connector plasmid that can be amplified by PCR or some variation thereof.
  • 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.
  • the proximity ligation assay can be performed using immunofluorescence or immunohistochemistry.
  • the detecting in step b) by the proximity ligation assay can be performed by flow cytometry.
  • the detecting in step b) by the proximity ligation assay can be performed using fluorescent detection methods (e.g., using secondary antibodies).
  • the fluorescent detection methods can be adapted for high throughput.
  • 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.
  • 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.
  • 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.
  • antibodies recognizing all Gas protein can be used in combination with and antibodies recognizing Gas cysteine palmitoylated proteins.
  • 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.
  • 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.
  • 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).
  • 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).
  • methods of assessing the efficacy of an anti-depressant agent in a subject 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.
  • 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.
  • the time the second sample can be obtained can be dependent on the antidepressant that was administered to the subject.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the anti-Gas antibody can be any commercially available anti-Gas antibody.
  • the anti-Gs protein, alpha subunit antibody (N 192/12; NeuroMab) can be used.
  • the cells can be cultured cells.
  • the cells can be blood cells.
  • the one or more cells can be any cultured cell line of neural or glial origin.
  • the one or more cells can be C6 glioma cells or SK N SH neuroblastoma cells.
  • the one or more cells can be patient- derived neural stem cells.
  • the cells can be hybrid cells.
  • the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cells can be cultured cells.
  • the cells can be blood cells.
  • 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.
  • the cells can be any cultured cell line of neural or glial origin.
  • 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.
  • cAMP cyclic adenosine monophosphate
  • 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.
  • an increase in the association of Gas with adenylyl cyclase is indicative of antidepressant activity.
  • 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.
  • cAMP cyclic adenosine monophosphate
  • 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 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.
  • 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.
  • 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 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.
  • the increase in cAMP generation is relative to control cells that have been exposed to a second agent having antidepressant activity.
  • the cultured cells comprise blood cells.
  • the blood cells can be erythrocy tes.
  • the blood cells can be leukocytes.
  • the blood cells can be platelets.
  • 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.
  • the cells can be cultured cells.
  • the cells can be blood cells.
  • the measured levels of Gas -activated adenylyl cyclase correlate negatively with the extent to which a subject is depressed.
  • 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.
  • the amount of the Gas palmitoylation in the sample can be indicative of the severity of depression in the subject.
  • the sample can comprise a blood cell.
  • the blood cell can be a leukocyte, an erythrocyte or a platelet.
  • 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.
  • the cells can be cultured cells.
  • the cells can be blood cells.
  • the extent of the subject's recovery correlates positively with the measured levels of Gas-activated adenylyl cyclase.
  • kits for treating a depressed patient or subject can comprise administering to the depressed patient or subject an antidepressant agent identified using any of the methods disclosed herein.
  • the sample can comprise one or more cells.
  • the sample can be whole cells.
  • the cells can be isolated from a bodily fluid.
  • the sample can be fresh cells or cultured cells.
  • the sample can be blood or comprise blood cells.
  • the blood cells can be leukocytes.
  • the blood cells can be erythrocytes.
  • the blood cells can be platelets.
  • the blood cells can be leukocytes, erythrocytes, platelets or a combination thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an antidepressant agent e.g., monoamine oxidase inhibitors, tricyclic antidepressants, SSRIs (selective serotonin reup
  • the amount of Gas palmitoylation can be expressed as a percent.
  • 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.
  • 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.
  • the methods can further comprise comparing the amount of Gas palmitoylation in a sample or subject to a predetermined reference value.
  • 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.
  • 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.
  • 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.
  • the reference value can be taken a different time point than to which it is being compared.
  • 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.
  • 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.
  • 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 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,.
  • 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.
  • 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.
  • 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 severity 7 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.
  • 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.
  • a change e.g., a decrease or a lower amount
  • 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.
  • “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.
  • 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).
  • 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).
  • 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.
  • the methods disclosed herein can further comprise calculating a ratio of Gas palmitoylation to nonpalmitoylated Gas in the sample.
  • the ratio of Gas palmitoylation to nonpalmitoylated Gas in the sample can be greater than 1 indicating depression in the subject.
  • 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.
  • the ratio of Gas palmitoylation to nonpalmitoylated Gas in the sample can be less than 1 indicating the absence of depression in the subject.
  • 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.
  • the sample will contain more palmitoylated Gas than nonpalmitoylated Gas.
  • 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.
  • 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.
  • 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).
  • 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).
  • MAOIs examples include but are not limited to ranylcypromine (Parnate), phenelzine (Nardil), isocarboxazid (Marplan), and Selegiline (Emsam).
  • rapid-acting antidepressants examples include but are not limited to ketamine or esketamine.
  • 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).
  • 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.
  • 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.
  • 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.
  • the difference in the amount of Gas palmitoylation measured between two or more samples can be significantly different.
  • the sulfhydryl reducing agent can N-ethylmaleimide (NEM), dithiothreitol (DTT), iodoacetamide, or N-methyl Maleimide (NMM).
  • NEM N-ethylmaleimide
  • DTT dithiothreitol
  • NMM N-methyl Maleimide
  • the methods can further comprise contacting the sample with a proteolytic enzyme after the step of contacting the sample with a sulfhydryl reducing agent.
  • Methods of determining the amount of Gas palmitoylation using an anti-Gas antibody 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.
  • the ratio of Gas palmitoylation to Gas in the sample can be greater than 1 indicates depression.
  • the amount of Gas palmitoylation in the sample can be determined using mass spectrometry.
  • the amount of Gas in the sample can be determined using mass spectrometry’.
  • the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.
  • the anti-Gas antibody can be any commercially available anti-Gas antibody.
  • 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.
  • the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.
  • the anti-Gas antibody can be any commercially available anti-Gas antibody.
  • the anti-Gs protein, alpha subunit antibody (N192/12; NeuroMab) can be used.
  • the amount of Gas palmitoylation in the sample can be determined as a percentage of total Gas.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the methods can comprise obtaining or having obtained a sample from the subject.
  • the method an include obtaining a blood sample from the subject.
  • the blood sample can comprise blood cells.
  • the blood cell can be a leukocyte, an erythrocyte, or a platelet.
  • the methods can comprise contacting the sample of a sulfhydryl reducing agent or an anti-Gas antibody.
  • the sulfhydryl reducing agent can be N-ethylmaleimide, dithiothreitol, iodoacetamide, or N- methyl maleimide.
  • the anti-Gas antibody can be any commercially available anti-Gas antibody.
  • the anti-Gs protein, alpha subunit antibody (N192/12; NeuroMab) can be used.
  • 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.
  • 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.
  • the amount of Gas palmitoylation in the sample is determined by determining the amount of palmitoylated Gas to non-palmitoylated Gas in the sample.
  • the method of diagnosing can further comprise a method of treating depression or major depressive disorder in a subject.
  • the subject or patient can be a human.
  • the subject can be an adult.
  • the subject can be an adolescent.
  • the subject can be a child.
  • 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.
  • the methods disclosed herein can comprise identifying a patient in need of treatment before the administration step.
  • the subject or patient can be identified of being in need of treatment using any of the methods disclosed herein.
  • the subject can be depressed.
  • the subject can have major depressive disorder.
  • the method an include obtaining a blood sample from the subject.
  • the method an include obtaining a blood sample from the subject in need of treatment.
  • the methods can include the step of administering a therapeutically effective amount of an antidepressant to the subject.
  • 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.
  • the methods can include the step of administering a therapeutically effective amount of a candidate compound identified as having antidepressant activity 7 using any of the methods disclosed herein to the subject.
  • 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.
  • 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.
  • 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.
  • the patient can be a human patient.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • SSRI selective serotonin reuptake inhibitor
  • SNRI serotonin and norepinephrine reuptake inhibitor
  • MAOI monoamine oxidase inhibitor
  • ketamine and esketamine an atypical antidepressant, and any other compounds showing antidepressant activity.
  • 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.
  • 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).
  • 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.
  • 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.
  • an aqueous carrier such as water, buffered water, saline, buffered saline (e.g., PBS), and the like.
  • an aqueous carrier such as water, buffered water, saline, buffered saline (e.g., PBS), and the like.
  • the excipients included can help approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like.
  • 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
  • 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.
  • 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.
  • 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).
  • compositions described herein can also be formulated so as to provide slow, prolonged, or controlled release.
  • 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 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.
  • Example 1 Acylation status of the G protein, Ga s , directs membrane localization and the response to chronic antidepressant stimulation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • LC-MS MS Liquid Chromatography Tandem Mass Spectrometry
  • Peptides were detected by full-scan mass analysis from m z z 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).
  • G s-GFP primarily localized to the plasma membrane (FIG. 1 A).
  • Myr-Palm mutant Gai like Gas
  • sucrose density gradient fractionation shows that neither mutant translocates from lipid rafts in response to chronic antidepressant treatment (FIGS. 4 and 5).
  • the native Gas is translocated in each of these cells by antidepressant treatments even when these same treatments fail to translocate the mutant proteins.
  • Myr-Palm Ga s -GFP mutant (Gai like Gas) remains palmitoylated following treatment with escitalopram.
  • 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).
  • phenelzine desipramine
  • fluoxetine as well as the antipsychotic olanzapine were assessed for their capacity to mediate the depalmitoylation of Ga s .
  • 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 pharmacology 7 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).
  • 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.
  • 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.
  • the antidepressant response is exclusive to Ga s (Toki, S., et al. (1999) J. Neurochem 73, 1114-1120).
  • 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).
  • 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.
  • 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).
  • FRAP fluorescence recovery after photobleaching
  • Example 2 Measurement of Gsa-activated adenylyl cyclase in platelets.
  • 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).
  • 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.
  • cADDIS virus or other fluorescent cAMP reporter
  • 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).
  • FRAP fluorescence recover ⁇ ' after photobleaching
  • Ketamine translocates Ga from lipid rafts at clinically relevant concentrations .
  • 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.
  • 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).
  • 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 density 7 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).
  • Ketamine enhances isoproterenol elicited cAMP accumulation in NR1 knockdown C6 cells.
  • 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).
  • 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.
  • 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 then translocates to the nucleus where it acts as a transcription factor for genes involved in growth and survival, neuroprotection, and synaptic plasticity 7 (Dwivedi Y, Pandey GN. Neuropsychiatr Dis Treat. 2008;4 (1 A): 161-76; and Duman RS, et al. Biol Psychiatry 7 . 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.
  • Ketamine increases BDNF in a cAMP-dependent manner in primary astrocytes.
  • 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.
  • (2R,6R)-HNK robustly increased cAMP indicating a possible role of cAMP in the antidepressant effects of (2R,6R)-HNK.
  • 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 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).
  • NMD AR antagonists other than ketamine may act at both neurons and glia, and it is possible that NMD AR are relevant for the former.
  • 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).
  • 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).
  • 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).
  • Example 3 A biomarker for depression and antidepressant 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).
  • IDS-SR30 Inventory of Depressive Symptomatology Selfreport
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • EC50 for PGE1 was 0.19 x
  • 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.
  • results described herein include the demographic and treatment data and corresponding changes of the total HamD17 scores and PGE1 stimulation of adenyly l cyclase activity 7 (normalized over basal activity 7 ) 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).
  • 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 (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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • Example 4 Gsa is a stable biomarker for depression.
  • Subject recruitment Subjects were recruited and had been diagnosed with major depressive disorder (MDD).
  • MDD major depressive disorder
  • 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.
  • C-SSRS Columbia Suicide Severity Rating Scale
  • suicide attempt(s) in the last 3 months severe cognitive impairment secondary 7 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 Clinical assessment.
  • 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.
  • 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. 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.
  • 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.
  • 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. 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.
  • 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.
  • QIDS Quick Inventory of Depressive Symptomatology
  • 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.

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