Application of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine in treatment of depression

Abstract

The invention provides an application of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine in treatment of depression. Experiments prove the relationship between HPA / corticosterone / glucocorticoid receptor / GNMT / glycine and sarcosine, the HPA / corticosterone and sarcosine are in negative correlation regulation, and the glucocorticoid receptor / GNMT / glycine and sarcosine are in positive correlation regulation. The invention also provides a biomarker capable of effectively diagnosing depression. The biomarker comprises sarcosine, methyl stearate and LysoPE (16: 1 (9Z) / 0: 0) in the exosome. Besides, the depression animal model is constructed by reducing GNMT, and the depression animal model can be used for depression drug screening, efficacy evaluation or diagnosis, and can also be used for researching and revealing the pathogenesis of depression. The invention has important significance on the diagnosis and treatment of depression, and has wide application prospects in clinic.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine in the treatment of depression. Background Art

[0002] Major depressive disorder (MDD) is a common mental illness characterized by depressive mood and loss of interest, affecting approximately 6% of adults globally each year.

[0003] Recent studies have shown that the occurrence of depression is closely related to metabolic abnormalities. Certain metabolic diseases, such as obesity and diabetes, increase the risk of depression. In addition, maternal obesity and maternal diabetes have been reported to increase the risk of neuropsychiatric diseases in offspring, such as depression. In recent years, researchers have increasingly focused on the metabolites of MDD patients. The glial inflammatory metabolic pathway, plasma metabolomics, and hippocampal glucose metabolism in MDD patients have been explored, and many differential metabolites have been found compared with the HC population. However, the specific roles of these differential metabolites are still unclear, and these metabolites cannot yet be used for diagnosis or prediction of treatment prognosis. The research on biomarkers and antidepressant biologics for depression is limited due to the instability of samples. The reason may be that plasma biological indicators are vulnerable to contamination, and the biological activities of different batches of sera are inconsistent. In addition, the exploration of antidepressant drugs has been carried out for many years. However, current antidepressant drugs are not effective for all patients, and some antidepressant drugs even have side effects.

[0004] Therefore, there is an urgent need to find biomarkers that can effectively diagnose depression and antidepressant drugs that are effective and have no side effects. Summary of the Invention

[0005] In view of this, in order to make up for the deficiencies of the prior art, the present invention is specifically proposed.

[0006] The first aspect of the present invention provides the application of a promoter of glycine N-methyltransferase (GNMT) in the preparation of a drug for treating depression.

[0007] In the present invention, "treatment" refers to the improvement, prevention, or reversal of a disease or disorder or at least one distinguishable symptom thereof. Further, the "treatment" refers to the improvement, prevention, or reversal of at least one measurable physiological parameter related to the disease or disorder to be treated, and the parameter is not necessarily recognizable in or by a mammal. Further, the "treatment" refers to inhibiting or slowing down a disease or its course, and such inhibition or slowing down can be physical, such as certain distinguishable adverse symptoms. The "treatment" used in the present invention covers diseases of mammals, especially humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not been diagnosed with the disease. (b) Inhibiting the disease, such as blocking the development of the disease. Or (c) alleviating the disease, such as reducing the symptoms related to the disease.

[0008] Furthermore, the promoter includes, but is not limited to, nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, polypeptides, proteins, and reagents used in gene editing.

[0009] Furthermore, the promoter is selected from nucleic acid molecules, reagents used in gene editing, small molecule chemical drugs, and antibody drugs.

[0010] Furthermore, the nucleic acid molecule includes, but is not limited to, nucleic acids encoding glucocorticoid receptors, nucleic acids encoding GNMT, nucleic acids encoding corticosterone antibodies, and nucleic acids encoding cortisol antibodies.

[0011] Furthermore, the nucleic acid molecule is selected from nucleic acids encoding GNMT.

[0012] Furthermore, the reagent used in gene editing includes reagents used in gene knock-in.

[0013] Furthermore, the reagents used in gene knock-in include, but are not limited to, interfering lentiviruses, adeno-associated virus vectors, CRISPR / Cas9, and Cre-LoxP.

[0014] Furthermore, the adeno-associated virus vectors include AAV1, AAV2, AAV3, AAV5, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0015] Furthermore, the adeno-associated virus vector is selected from AAV9.

[0016] Furthermore, the small molecule chemical drugs include, but are not limited to, drugs that inhibit corticosterone or cortisol, and drugs that inhibit the HPA axis.

[0017] Furthermore, the drugs that inhibit corticosterone or cortisol include, but are not limited to, metyrapone tablets, mitotane tablets, phentolamine mesylate tablets, aminoglutethimide tablets, and ketoconazole tablets.

[0018] Furthermore, the antibody drugs include corticosterone antibodies and cortisol antibodies.

[0019] In the present invention, the depression includes, but is not limited to, major depressive disorder, unipolar depression, treatment-resistant depression, resistant depression, anxious depression, bipolar depression, and dysthymia. Furthermore, the depression is selected from major depressive disorder and anxious depression.

[0020] Furthermore, the drug includes its pharmaceutically acceptable carrier and / or excipient.

[0021] Further, the pharmaceutically acceptable carrier and / or excipient include diluents, binders, surfactants, wetting agents, adsorbent carriers, lubricants and / or disintegrants. Among them, diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, water; binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and its salts, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose; surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid esters, sodium dodecyl sulfate, monoglyceride stearate, cetyl alcohol; wetting agents include, but are not limited to, glycerol, starch; adsorbent carriers include, but are not limited to, starch, lactose, bentonite, silica gel, kaolin, saponite; lubricants include, but are not limited to, zinc stearate, glycerol monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, sucrose monolaurate, sodium lauryl sulfate, magnesium lauryl sulfate, sodium dodecyl magnesium sulfate.

[0022] Further, the drug can be administered by any applicable administration route.

[0023] Further, the administration routes include enteral administration and parenteral administration.

[0024] Further, the parenteral administration includes, but is not limited to, injection administration, respiratory administration, cavity administration, mucosal administration, skin administration.

[0025] Further, the sites for injection administration include, but are not limited to, vein, muscle, subcutaneous, intradermal, cavity, prefrontal cortex.

[0026] Further, the site for injection administration is selected from the prefrontal cortex.

[0027] The second aspect of the present invention provides any one of the following methods:

[0028] (1) A method for regulating sarcosine, the method regulates sarcosine by administering an HPA / corticosterone / glucocorticoid receptor / GNMT / glycine regulator.

[0029] (2) A method for regulating the AKT / mTOR signaling pathway, the method regulates the AKT / mTOR signaling pathway by administering a sarcosine regulator.

[0030] In the present invention, sarcosine has various uses. For example, sarcosine can be used as a dye stabilizer, participate in the synthesis of anti-enzyme agents, and can also be used as a biological reagent for laboratory research. In the field of daily chemicals, sarcosine can be used in amino acid-based surfactants, which helps to supplement the energy of skin cells and promote the repair of the self-fatigue state. Sarcosine is also beneficial for preventing hair damage and promoting the self-repair of damaged hair, enhancing the luster and elasticity of hair, and can be used for skin care and hair care.

[0031] In the present invention, the term "regulation" includes modulation and control, and the modulation includes increasing the expression level or activity of sarcosine, or decreasing the expression level or activity of sarcosine.

[0032] Furthermore, the regulator includes a promoter and an inhibitor.

[0033] In the present invention, the term "promoter" refers to any substance that can promote the activity of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine, promote the stability of the HPA / corticosterone / glucocorticoid receptor / GNMT / glycine gene or protein, promote the expression level of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine, and promote the effective action time of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine.

[0034] In the present invention, the effects achieved by the promoter can also be realized by knock-in. Knock-in refers to the targeted insertion of a transgene into the genome of a host cell, resulting in the expression of the transgene and / or altered expression of the endogenous target gene (such as elevated (including ectopic) or decreased expression), for example, by introducing additional copies of the target gene or by operatively inserting regulatory sequences that enhance the expression of the endogenous copy of the target gene. The knock-in transgene can include a heterozygous knock-in or a homozygous knock-in of the transgene. Knock-in also encompasses conditional knock-in, in which, for example, by exposing the animal to a substance that promotes such expression, by introducing an enzyme that promotes recombination at the targeted insertion site, or by some other method for altering the targeted insertion site, the expression of the transgene and / or the altered expression of the endogenous target gene can occur.

[0035] Furthermore, the promoter includes nucleic acid molecules, carbohydrates, liposomes, small molecule chemicals, antibody drugs, polypeptides, proteins, or reagents used in gene editing. In the present invention, the term nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Equivalently, nucleic acids also include DNA or RNA analogs generated from nucleotide analogs and, when applicable, single-stranded (sense or antisense strands) and double-stranded polynucleotides. Liposomes refer to small vesicles composed of various types of lipids, phospholipids, and / or surfactants that can be used to deliver drugs to mammals. Antibodies cover intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed by at least two intact antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity, all fall within the scope of protection of the present invention.

[0036] Furthermore, the promoter is selected from nucleic acid molecules.

[0037] In the present invention, the term "inhibitor" refers to any substance that can inhibit the activity of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine, inhibit the stability of the HPA / corticosterone / glucocorticoid receptor / GNMT / glycine gene or protein, inhibit the expression level of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine, or inhibit the effective action time of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine.

[0038] Furthermore, the inhibitor includes, but is not limited to, reagents used in gene editing, nucleic acid inhibitors, protein inhibitors, or compounds.

[0039] Furthermore, the reagents used in gene editing include, but are not limited to, reagents used in adeno-associated virus vectors, zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS, or phiC31.

[0040] Furthermore, the nucleic acid inhibitors include, but are not limited to, siRNA, shRNA, or miRNA.

[0041] In the present invention, the siRNA may include partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as modified RNA that differs from natural RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The alterations may include the addition of non-nucleotide substances, such as addition to the ends of the siRNA or to one or more internal nucleotides of the siRNA. Modifications that render the siRNA resistant to ribonuclease digestion (e.g., using 2'-substituted ribonucleotides or modifying the sugar-phosphate backbone), or replacement of one or more nucleotides in the siRNA with deoxyribonucleotides.

[0042] In the present invention, shRNA is a non-coding small RNA molecule capable of forming a hairpin structure, and shRNA can inhibit gene expression through the RNA interference pathway. When shRNA is introduced into cells, it is recognized and cleaved by the ribonuclease Dicer in the cells into small interfering RNAs (siRNAs) of approximately 21 nucleotides. The siRNAs bind to a protein complex called the RNA-induced silencing complex (RISC). The Argonaute protein in RISC uses the antisense strand of the siRNA to recognize and bind to the target mRNA, and then degrades the target mRNA by cleavage or inhibition of translation, etc., thereby achieving the inhibition of the expression of a specific gene.

[0043] In the present invention, the protein inhibitor is selected from substances capable of inhibiting HPA / corticosterone / glucocorticoid receptor / GNMT / glycine protein, and the protein inhibitor includes proteolytic enzymes and protein-binding molecules. The proteolytic enzymes are selected from enzymes capable of catalyzing the hydrolysis of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine protein. The protein-binding molecules are selected from substances that specifically bind to HPA / corticosterone / glucocorticoid receptor / GNMT / glycine protein, such as antibodies or ligands capable of inhibiting the activity of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine protein.

[0044] Furthermore, the HPA is negatively correlated with the regulation of sarcosine. When the HPA axis is hyperactive, the sarcosine level decreases. Further, the production of sarcosine can be increased by inhibiting the HPA axis.

[0045] Furthermore, the corticosterone is negatively correlated with the regulation of sarcosine. When the corticosterone level increases, the sarcosine level decreases, and when the corticosterone level decreases, the sarcosine level increases. Further, the production of sarcosine can be increased by adding a corticosterone inhibitor.

[0046] Furthermore, the glucocorticoid receptor is positively correlated with sarcosine regulation. When the level of the glucocorticoid receptor increases, the level of sarcosine will increase; when the level of the glucocorticoid receptor decreases, the level of sarcosine will decrease. Further, the production of sarcosine can be increased by adding the glucocorticoid receptor itself or its promoter.

[0047] Furthermore, the GNMT is positively correlated with sarcosine regulation. When the level of GNMT increases, the level of sarcosine will increase; when the level of GNMT decreases, the level of sarcosine will decrease. Further, the production of sarcosine can be increased by adding GNMT itself or its promoter.

[0048] Furthermore, glycine is positively correlated with sarcosine regulation. When the level of glycine increases, the level of sarcosine will increase; when the level of glycine decreases, the level of sarcosine will decrease. Further, the production of sarcosine can be increased by adding glycine itself or its promoter.

[0049] The third aspect of the present invention provides any one of the following applications:

[0050] (1) The application of a reagent for detecting sarcosine in exosomes in the preparation of a product for diagnosing depression;

[0051] (2) The application of a reagent for detecting methyl stearate in exosomes in the preparation of a product for diagnosing depression;

[0052] (3) The application of a reagent for detecting LysoPE(16:1(9Z) / 0:0) in exosomes in the preparation of a product for diagnosing depression;

[0053] (4) The application of a reagent for detecting sarcosine, methyl stearate and LysoPE(16:1(9Z) / 0:0) in exosomes in the preparation of a product for diagnosing depression.

[0054] Furthermore, the reagent includes an oligonucleotide probe that specifically recognizes sarcosine in exosomes or methyl stearate in exosomes or LysoPE(16:1(9Z) / 0:0) in exosomes, a primer that specifically amplifies sarcosine in exosomes or methyl stearate in exosomes or LysoPE(16:1(9Z) / 0:0) in exosomes, a binder that specifically binds to the protein encoded by sarcosine in exosomes or methyl stearate in exosomes or LysoPE(16:1(9Z) / 0:0) in exosomes, or a chip that specifically analyzes sarcosine in exosomes or methyl stearate in exosomes or LysoPE(16:1(9Z) / 0:0) in exosomes.

[0055] Further, the reagent includes an oligonucleotide probe that specifically recognizes sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes, a primer that specifically amplifies sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes, a binder that specifically binds to the proteins encoded by sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes, or a chip that specifically analyzes sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes.

[0056] Further, the product includes a chip, a kit, or a nucleic acid membrane strip.

[0057] Further, the chip includes a gene chip and a protein chip.

[0058] Further, the gene chip includes an oligonucleotide probe for detecting the transcriptional level of sarcosine or methyl stearate or LysoPE(16:1(9Z) / 0:0) in exosomes, which is specific for sarcosine or methyl stearate or LysoPE(16:1(9Z) / 0:0) in exosomes.

[0059] Further, the gene chip includes an oligonucleotide probe for detecting the transcriptional levels of sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes, which is specific for sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes.

[0060] Further, the protein chip includes a specific binder for the sarcosine or methyl stearate or LysoPE(16:1(9Z) / 0:0) protein in exosomes.

[0061] Further, the protein chip includes a specific binder for the sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) proteins in exosomes.

[0062] Further, the kit includes reagents for detecting the gene or protein expression levels of sarcosine or methyl stearate or LysoPE(16:1(9Z) / 0:0) in exosomes by RT-PCR, qRT-PCR, biochip detection, DNA blotting, in situ hybridization, immunoblotting, or mass spectrometry.

[0063] Further, the kit includes reagents for detecting the gene or protein expression levels of sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes by RT-PCR, qRT-PCR, biochip detection, DNA blotting, in situ hybridization, immunoblotting, or mass spectrometry.

[0064] Further, the exosomes are derived from one or more of the following samples: cells, tissues, blood, urine, saliva, or mucus.

[0065] Further, the cells include microglia, prefrontal cortex cells, and neurons.

[0066] Further, the cells are selected from prefrontal cortex cells.

[0067] Further, the tissue includes prefrontal cortex tissue.

[0068] Further, the kit includes instruments or reagents for processing samples.

[0069] The fourth aspect of the present invention provides any one of the following products:

[0070] (1) A product for diagnosing depression, the product including a reagent capable of detecting the expression level of sarcosine in exosomes;

[0071] (2) A product for diagnosing depression, the product including a reagent capable of detecting the expression level of methyl stearate in exosomes;

[0072] (3) A product for diagnosing depression, the product including a reagent capable of detecting the expression level of LysoPE(16:1(9Z) / 0:0) in exosomes;

[0073] (4) A product for diagnosing depression, the product including reagents capable of detecting the expression levels of sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes.

[0074] Further, the product includes a chip, a kit, or a nucleic acid membrane strip.

[0075] The fifth aspect of the present invention provides a method for constructing a non-human animal model of depression, the method including administering a GNMT inhibitor to a non-human animal.

[0076] Further, the inhibitor includes reagents used in gene editing, nucleic acid inhibitors, protein inhibitors, or compounds.

[0077] Further, the reagents used for gene editing include those used in adeno-associated virus vectors, zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS or phiC31.

[0078] Further, the nucleic acid inhibitors include siRNA, shRNA or miRNA.

[0079] Further, the reagents used for gene editing are selected from adeno-associated virus vectors.

[0080] Further, the adeno-associated virus vectors include AAV1, AAV2, AAV3, AAV5, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12.

[0081] Further, the adeno-associated virus vector is selected from AAV9.

[0082] Further, the non-human animal model refers to a non-human animal having or showing characteristics of a disease or disorder.

[0083] Further, the non-human animal is a mammal.

[0084] Further, the mammals include mice, rats, rabbits, dogs, pigs, monkeys, sheep.

[0085] Further, the mammal is selected from mice.

[0086] Further, the method includes injecting an AAV9 vector expressing GNMT recombinase and GFAP driven by the human synapsin 1 promoter (AAV9--ITR-hsyn-GNMT-GFAP) into a mouse to specifically reduce the expression of GNMT.

[0087] Further, the injection site is the prefrontal cortex.

[0088] Further, the injection method is bilateral injection.

[0089] Further, the method can specifically reduce the expression of GNMT in neurons.

[0090] Further, the method includes injecting an AAV9 vector expressing GNMT recombinase and GFAP driven by the human synapsin 1 promoter (AAV9--ITR-hsyn-GNMT-GFAP) into the prefrontal cortex of a mouse to specifically reduce the expression of GNMT in neurons.

[0091] The sixth aspect of the present invention provides any one of the following applications:

[0092] (1) Use of a non-human animal model of depression prepared by the method described in the fifth aspect of the present invention in screening drug candidates for treating depression;

[0093] (2) Use of a non-human animal model of depression prepared by the method described in the fifth aspect of the present invention in evaluating the therapeutic effect of drugs for treating depression;

[0094] (3) Use of a non-human animal model of depression prepared by the method described in the fifth aspect of the present invention in studying the pathogenesis of depression;

[0095] (4) Use of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine in regulating sarcosine.

[0096] The seventh aspect of the present invention provides any one of the following methods:

[0097] (1) A method for screening drug candidates for treating depression, the method comprising:

[0098] a) Administering a reagent to be screened to a non-human animal with depression prepared by the method described in the fifth aspect of the present invention;

[0099] b) Analyzing and evaluating the therapeutic effect of the reagent to be screened, and selecting a reagent that can significantly improve the depressive-like behavior of the mouse model;

[0100] Furthermore, the drug candidates include selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, tricyclic drugs, tetracyclic drugs, serotonin regulators and stimulants;

[0101] (2) A method for evaluating the efficacy of a drug for treating depression, the method comprising:

[0102] a) Administering the drug to a non-human animal with depression prepared by the method described in the fifth aspect of the present invention;

[0103] b) Evaluating the therapeutic effect of the drug on the depression;

[0104] (3) A method for studying the pathogenesis of depression, which is to use a non-human animal with depression prepared by the method described in the fifth aspect of the present invention to study the pathogenesis of depression.

[0105] Furthermore, the evaluation indicators include but are not limited to animal behavior tests, neurogenesis performance, and microglial cell status.

[0106] Furthermore, the ethological tests include, but are not limited to, open field test (OFT), novel environment feeding suppression test (NSFT), forced swimming test (FST), and tail suspension test (TST).

[0107] Advantages and beneficial effects of the present invention:

[0108] The present invention provides the application of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine in the treatment of depression. The present invention proves the relationship between HPA / corticosterone / glucocorticoid receptor / GNMT / glycine and sarcosine through experiments, wherein HPA / corticosterone is negatively correlated with sarcosine, and glucocorticoid receptor / GNMT / glycine is positively correlated with sarcosine. The present invention also provides biomarkers capable of effectively diagnosing depression, including sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes. In addition, the present invention constructs a depression animal model by reducing GNMT, and the depression animal model can be used for screening of antidepressant drugs, evaluation of drug efficacy or diagnosis, and can also be used to study and reveal the pathogenesis of depression. The present invention is of great significance for the diagnosis and treatment of depression and has broad application prospects in clinical practice. Brief Description of the Drawings

[0109] Figure 1 It is a result diagram of bioinformatics analysis of exosomes in serum samples and exosomes in prefrontal cortex tissues. Among them, Figure A is the shared metabolites of exosomes in serum samples and exosomes in prefrontal cortex tissues of adult mice; Figure B is a heat map; Figure C is a result diagram of 447 quantified metabolites of exosomes in serum samples of depression patients; Figure D is a result diagram of 480 differential metabolites quantified in exosomes of mouse prefrontal cortex tissues; Figure E is a relative level diagram of 30 named metabolites different between the major depressive disorder (MDD) patients and healthy control (HC) groups; Figure F is a level diagram of 63 named metabolites changed in mouse prefrontal cortex samples; Figure G is a flowchart for exosome collection; Figure H is a structural diagram of sarcosine; Figure I is a molecular fragmentation diagram of sarcosine; Figure J is a result diagram of reduced sarcosine in serum exosomes of depression patients; Figure K is a result diagram of reduced sarcosine in prefrontal cortex exosomes of depressed mice.

[0110] Figure 2It is the result diagram of the functional role of sarcosine in MDD. Among them, Figure A is the experimental protocol diagram of the intravenous injection experiment for studying the functional role of sarcosine in MDD; Figure B is the experimental result diagram of the open field test (OFT); Figure C is the experimental result diagram of the novel environment feeding suppression test (NSFT); Figure D is the experimental result diagram of the tail suspension test (TST); Figure E is the experimental result diagram of the forced swimming test (FST); Figures F - I and R - U are the result diagrams of the significant increase in the protein levels of p - AKT / AKT and p - mTOR / mTOR after sarcosine treatment; Figure M is the experimental protocol diagram of the prefrontal cortex targeted injection experiment for studying the functional role of sarcosine in MDD; Figures J - L and V - X are the expression result diagrams of the neuronal synaptic proteins PSD95, Syn, and SYP.

[0111] Figure 3 It is the result diagram of corticosterone directly regulating GNMT expression through glucocorticoid receptor and affecting sarcosine content. Among them, Figure A is the schematic diagram showing that glycine is the precursor of sarcosine; Figure B is the result diagram of the increased corticosterone level in depressive mice; Figure C is the PC12 cell culture diagram; Figures D and E are the result diagrams showing that corticosterone can reduce GNMT expression; Figure F is the result diagram of the significantly reduced GNMT expression level in the prefrontal cortex of depressive mice; Figures G - L are the result diagrams showing that GR directly binds to the promoter of the GNMT gene to promote GNMT expression.

[0112] Figure 4 It is the result diagram of the role of GNMT in the development of depression. Among them, Figures A and B are the result diagrams of specifically reducing GNMT expression; Figures C, D, E, and F are the result diagrams of specifically reducing GNMT in neurons can induce depressive - like behaviors.

[0113] Figure 5 It is the result diagram of the relationship between depressive - like behaviors and neuronal synapses. Among them, Figures A - F are the result diagrams of overexpressing GNMT; Figure G is the experimental result diagram of the novel environment feeding suppression test (NSFT); Figure H is the experimental result diagram of the open field test (OFT); Figure I is the experimental result diagram of the tail suspension test (TST); Figure J is the experimental result diagram of the forced swimming test (FST); Figures K - O are the result diagrams showing that the antidepressant - like behavior of the GNMT gene is closely related to the AKT / mTOR signaling pathway.

[0114] Figure 6Results graph of the association between exosomes and antidepressant effects. Among them, Figure A is a graph of bone marrow mesenchymal stem cells; Figures B and C are graphs of the results of flow cytometry detecting that the BMSC marker CD90 of bone marrow mesenchymal stem cells is positive; Figure D is a graph of the results of flow cytometry detecting that the BMSC marker CD105 of bone marrow mesenchymal stem cells is positive; Figures E - G are graphs of the results of exosome extraction and identification of the third-generation BMSC; Figure H is a graph of the result that exosomes can enter the prefrontal cortex through the blood-brain barrier; Figure I is an animal experiment procedure; Figure J is a graph of the result that the sarcosine content in exosomes of mesenchymal stem cells with downregulated GNMT is significantly reduced; Figure K is a graph of the result of downregulating GNMT; Figures L - O are graphs of the results of behavioral tests (OFT, NSFT, TST, and FST); Figures P - S are graphs of the results related to the antidepressant effect of GNMT and the AKT / mTOR signaling pathway and the regulation of neuronal synapse membranes.

[0115] Figure 7 Graph of the potential mechanism of depression.

[0116] Figure 8 ROC curves for diagnosing depression with sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes in the training set. Among them, Figure A is the ROC curve for diagnosing depression with methyl stearate in exosomes; Figure B is the ROC curve for diagnosing depression with LysoPE(16:1(9Z) / 0:0) in exosomes; Figure C is the ROC curve for diagnosing depression with sarcosine in exosomes; Figure D is the ROC curve for diagnosing depression by combining sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes.

[0117] Figure 9 ROC curves for diagnosing depression with sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes in the validation set. Among them, Figure A is the ROC curve for diagnosing depression with methyl stearate in exosomes; Figure B is the ROC curve for diagnosing depression with LysoPE(16:1(9Z) / 0:0) in exosomes; Figure C is the ROC curve for diagnosing depression with sarcosine in exosomes; Figure D is the ROC curve for diagnosing depression by combining sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes. Detailed implementation

[0118] The above disclosure provides a general description of the present invention. A more complete understanding can be obtained by referring to the following specific embodiments. The purpose of describing these embodiments is merely illustrative and is not intended to limit the scope of the present invention. Formal transformations and equivalent substitutions are considered as circumstances may suggest or afford convenience. Although specific terms are used herein, these terms are used for descriptive purposes and not for limitation.

[0119] Example

[0120] I. Materials and Methods

[0121] Ethical Statement and Participants

[0122] All participants (if they were 18 years of age or older) or their guardians (if under 18 years of age) signed a written informed consent form prior to study inclusion. The experimental protocol was reviewed and approved by the Ethics Committees of the Third People's Hospital of Foshan and the Fourth People's Hospital of Urumqi. The experiment was conducted in accordance with the Declaration of Helsinki. From June 2019 to August 2019, we recruited 37 MDD patients and 33 healthy control (HC) participants from the Third People's Hospital of Foshan (Guangdong, China). We recruited 32 MDD patients and 41 HC participants from the Fourth People's Hospital of Urumqi (Xinjiang, China). All HCs (n = 74), including doctors and nurses, were from local hospitals. All MDD patients (n = 69) were diagnosed by experienced psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) and the International Classification of Diseases, Tenth Revision (ICD-10). Two standardized and validated scales, namely the Montgomery-Åsberg Depression Rating Scale (MADRS) and the Hamilton Depression Rating Scale (HAMD), were used to quantify the severity of depression. Patients included in the study were patients with moderate to severe depression (i.e., MADRS ≥22 or HAMD ≥17) and had no co-existing somatic, neurological, or psychiatric diseases.

[0123] Exosome Isolation

[0124] Serum exosome isolation and tissue exosome isolation were performed as previously described. Briefly, exosomes (serum and tissue) were isolated using a qEV column according to the manufacturer's protocol (Izon, Oxford, UK). Exosomes were centrifuged at 10,000×g using a 30K MWCO PES protein concentrator Vivaspin (Sartorius, Göttingen, Germany).

[0125] Nanoparticle Tracking Analysis (NTA)

[0126] A NanoSight NS300 (NanoSight NTA 2.3 nanoparticle tracking and analysis release version build 0033) (Malvern Instruments) was used to measure the concentration and size of the particles. Samples were diluted 1:1000 in PBS, and the camera was set to capture 3 videos for each sample, each video lasting 30 seconds. The videos were then analyzed to determine the particle size distribution and approximate the number of particles.

[0127] Sample Collection and Preparation

[0128] Fasting blood samples of patients and HC were collected and allowed to clot at room temperature for 1 hour, then the samples were centrifuged for 10 minutes (centrifugation speed: 3000×g, temperature: 4 °C). Serum (about 1 mL of supernatant) was carefully aspirated and stored at -80 °C until exosomes were isolated. Briefly, according to the manufacturer's protocol (Izon, Oxford, UK), serum exosomes were isolated on a qEV column and then concentrated in a protein concentrator Vivaspin® (Sartorius, Göttingen, Germany) for 60 minutes (centrifugation speed: 10000×g, temperature: 4 °C). The concentrated exosomes were resuspended in 200 µL of cold phosphate-buffered saline and stored frozen at -80 °C for subsequent UPLC-MS / MS analysis. As described previously, negative staining electron microscopy and nanoparticle tracking analysis were applied to verify the isolated exosomes.

[0129] Animals and Treatments

[0130] Male C57BL / 6 mice were obtained from Vital River Laboratories (Beijing, China). The animals were housed at 24 ± 1 °C and 50 ± 1% humidity with a 14 / 10-hour light / dark cycle and had free access to food and water. The animal procedures were approved by the Animal Care and Use Committee of Minzu University of China. The mice were randomly divided into four groups according to the experimental requirements. Isolation of mouse brain exosomes was performed according to the method of Vella et al.

[0131] Stereotaxic Injection

[0132] In this experiment, the mice were placed in an anesthesia induction chamber and anesthetized with 3% isoflurane. Then we fixed the mice on a brain stereotaxic apparatus and measured the target nucleus mPFC using the nuclear localization coordinates obtained from the mouse brain atlas. Next, we drilled a hole with a dental drill and inserted a special cannula. Finally, we injected the RWD catheter with a microinjection pump.

[0133] UPLC-MS / MS Analysis

[0134] Wide-targeted metabolomics analysis of exosomes from participants' serum samples and mouse brain tissue samples was performed using UPLC (Shim-pack UFLC SHIMADZU CBM30A system; Shimadzu Corporation, Kyoto, Japan) and tandem mass spectrometer (MS / MS) (4500 QTRAP; Applied Biosystems, Foster City, CA, USA) equipment at Wuhan Metware Biotechnology Co., Ltd. Briefly, the exosomes extracted from participants' serum were freeze-dried and vortexed for 30 seconds by adding 1 mL of 70% MeOH aqueous mixture. Then, the samples were placed in liquid nitrogen for 5 minutes and then on ice for 3 minutes. After repeating three times, they were sonicated at 30 HZ for 3 minutes, vortexed for about 30 seconds, and centrifuged for 10 minutes (centrifugation speed: 12000 rpm, temperature: 4°C). Then, the supernatant was concentrated and 150 μL of 70% MeOH aqueous solution was added. Finally, it was centrifuged for 10 minutes (centrifugation speed: 12000 rpm, temperature: 4°C), and the supernatant was mixed with 100 μL of mobile phase B containing 10% acetonitrile, 90% isopropanol, 0.04% acetic acid, and 5 mmol / L ammonium formate for UPLC-MS / MS analysis.

[0135] Qualitative analysis was based on a self-made target database MWDB (MetWare database) and a public metabolite information database, and was carried out according to the retention time RT (Retention time), precursor ion pair information, and secondary spectrum data. Quality control (QC) samples were prepared by equally mixing all exosome samples before sample analysis, and were injected every ten samples to monitor the repeatability during the instrument analysis process.

[0136] Behavioral tests

[0137] The open field test (OFT), novel environment feeding suppression test (NSFT), tail suspension test (TST), and forced swimming test (FST) were selected to evaluate the depressive behavior of mice. Behavioral tests were performed after modeling and treatment. Open field test (OFT): The mice were placed in a corner of an open field device (50×50×45 cm). Each mouse was allowed to move freely for six minutes, and the total moving distance in the first five minutes was recorded. Novel environment feeding suppression test (NSFT): The open field device was used to evaluate the behavioral test. Food pellets were placed in the center of the device. The mice were placed separately on the grids at the floor corners and allowed to explore freely for five minutes. The latency to start eating was recorded. Tail suspension test (TST): In this test, the tip of the mouse's tail was suspended by a rope. The mice were separated, and the head of each mouse was 15 cm from the ground. The total duration was six minutes, and the immobile time of the mice in the last five minutes was recorded. Forced swimming test (FST): In this behavioral test, each mouse was placed in a cylindrical container at a temperature of 23±2°C. Each mouse was forced to swim for 6 minutes, and the immobile time in the last 5 minutes was recorded.

[0138] Quantitative RT-PCR

[0139] Total RNA was extracted from 15 mg of cryopreserved prefrontal tissue and cell samples using Trizol reagent. 1 μg of RNA was used for the synthesis of the first-strand cDNA, and quantitative real-time PCR was performed using LightCycler 96 RT-PCR. The β-actin gene was used as a reference gene for normalization. The relative changes in gene expression were analyzed using the 2−ΔΔCT method. The forward primer for GNMT was 5′CTTCAGCGTGATGAGCGTGGAC-3′; the reverse primer for GNMT was 5′ AGGGTTACCATGTGGGCTTTGTTG-3′.

[0140] Western blot analysis

[0141] Frontal tissue and cell samples were lysed using RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentrations were measured using the BCA assay and adjusted to the same final concentration. After heat denaturation at 95 °C for 10 minutes, 30 - 50 μg of total protein was separated by SDS-PAGE electrophoresis and then transferred to a nitrocellulose membrane. The nitrocellulose membrane was incubated with the following primary antibodies: anti-GNMT (#224753, Abcam, 1:1000), anti-AKT (AG-20B-0014, Adipogen, 1:1000), anti-p-AKT (#11948S, Cell signaling Technology, 1:1000), anti-mTOR (AG-20B-0014, Adipogen, 1:1000), anti-p-mTOR (#11948S, Cell signaling Technology, 1:1000), anti-PSD95 (ab12093, Abcam, 1:1000), anti-SYP (ab267272, Abcam, 1:1000), anti-Syn (ab32127, Abcam, 1:1000), anti-CD63 (#5275, Senta, 1:300), anti-TSG101 (#133586, Abcam, 1:1000), anti-GM130 (#55590, Senta, 1:300), anti-HSP70 (#5439, Abcam, 1:1000), anti-β-Actin (#4967S, Cell signaling Technology, 1:1000).

[0142] Immunostaining

[0143] After anesthesia, the mice were sacrificed and perfused with saline. The mouse brains were isolated and fixed with 4% paraformaldehyde for 48 hours, then dehydrated once in 20% sucrose solution and twice in 30% sucrose solution. The whole brain tissues were embedded in optimal cutting temperature compound (OCT) and cut into 35 μm thickness. Primary antibodies for immunofluorescence staining were used. Images were captured using a laser scanning confocal microscope (Leica Microsystems, Germany). The results were analyzed using Image J software.

[0144] Luciferase assay

[0145] The promoter region was cloned into a luciferase reporter vector. The plasmid sequence was verified by sequencing. The GNMT luciferase reporter plasmid and the glucocorticoid receptor luciferase reporter plasmid had been constructed and used in our previous study. According to the manufacturer's protocol (Promega), the dual-luciferase reporter assay system was used to measure the luciferase assay.

[0146] Diagnostic efficacy verification

[0147] Twenty-seven clinical samples of depression and 29 healthy control samples were collected as the training set. The expression levels of sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes were detected, and the ROC curves were plotted. The AUC values, sensitivity, and specificity of sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) were analyzed respectively to judge the diagnostic efficacy of sarcosine, methyl stearate, LysoPE(16:1(9Z) / 0:0), and sarcosine + methyl stearate + LysoPE(16:1(9Z) / 0:0) for depression.

[0148] Twenty-eight clinical samples of depression and 35 healthy control samples were collected as the validation set. The expression levels of sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) in exosomes were detected, and the ROC curves were plotted. The AUC values, sensitivity, and specificity of sarcosine, methyl stearate, and LysoPE(16:1(9Z) / 0:0) were analyzed respectively to judge the diagnostic efficacy of sarcosine, methyl stearate, LysoPE(16:1(9Z) / 0:0), and sarcosine + methyl stearate + LysoPE(16:1(9Z) / 0:0) for depression.

[0149] Statistical analysis

[0150] For clinical characteristics, all data are presented as mean ± standard deviation. Statistical analysis was performed by one-way analysis of variance (ANOVA) and the Mann-Whitney U test when applicable. For animals, data are presented as mean ± standard error. A p-value less than 0.05 was considered statistically significant. The ROC curve was generated by SPSS 25.0 statistical analysis software.

[0151] II. Results

[0152] Metabolomic analysis of exosomes from serum of patients with depression and from cortical tissues of mice with depression

[0153] Metabolic disorders can drive neurophysiological dysfunction and subsequently lead to neurodegenerative diseases. To characterize the molecular mechanisms underlying the behavioral phenotypes of depressed mice and patients with depression, we performed metabolomic analysis of exosomes from serum samples of participants (72 patients with MDD and 73 HC individuals) and from prefrontal cortical tissues of adult mice (5 depressed model mice and 5 control mice). Bioinformatics analysis showed that a total of 447 metabolites were quantified in exosomes from serum samples of participants ( Figure 1 C), and a total of 480 differential metabolites were quantified in exosomes from prefrontal cortical tissues of adult mice ( Figure 1 D). Among these metabolites, 229 were shared between exosomes from serum samples of participants and exosomes from prefrontal cortical tissues of adult mice ( Figure 1 A). These features are shown as heatmaps ( Figure 1 B). These data indicate disordered exosomal metabolic features. Notably, only a few metabolites changed significantly in the depressed group compared with the control group. The bilateral Wilcoxon rank-sum test was used to evaluate different exosomal metabolites in the two groups (patients with MDD, n = 73; HC participants, n = 72; CUMS mice, n = 5; control mice, n = 5). Figure 1 E shows the relative levels of 30 named metabolites that differed between patients with MDD and the HC group. Similarly, Figure 1 F shows the levels of 63 named metabolites that were altered in samples of the prefrontal cortex of mice. Notably, a few metabolites (sarcosine, LysoPE(16:1(9Z) / 0:0)(16:1(9Z) / 0:0), methyl stearate) were common among the altered metabolites in these different types of biological samples. Methyl stearate increased, while sarcosine and LysoPE(16:1(9Z) / 0:0)(16:1(9Z) / 0:0) decreased compared with the control group. Among them, sarcosine showed the most significant decrease. The results showed that sarcosine was in the prefrontal cortical exosomes of depressed mice ( Figure 1 K) and in the serum exosomes of patients with depression ( Figure 1J) Significantly reduced. To confirm the decreased sarcosine levels in depressive mice, we first collected cortical tissue exosomes from adult mice ( Figure 1 G). Then, we used a highly sensitive and specific method (LC / MS / MS) to accurately quantify the sarcosine content. The structure of sarcosine ( Figure 1 H) and the molecular fragmentation of sarcosine ( Figure 1 I) showed that the quantitative results confirmed a significant reduction in the sarcosine content in exosomes of the prefrontal cortex.

[0154] Sarcosine treatment alleviated depressive-like behaviors in mice, and the antidepressant effect of sarcosine was related to the AKT / mTOR signaling pathway and the regulation of neuronal synaptic membranes.

[0155] Based on the finding of a significant reduction in sarcosine content in prefrontal cortex exosomes, we explored the possible role of sarcosine in CUMS-induced depressive-like behaviors. To study the functional role of sarcosine in MDD, we used two administration methods (intravenous injection and prefrontal cortex targeted injection). For intravenous sarcosine supplementation, the experimental protocol was as Figure 2 shown in A. After sarcosine supplementation, depressive-like behaviors were alleviated in several established behavioral tests. Specifically, in the open field test (OFT) ( Figure 2 B), the total moving distance significantly increased after sarcosine supplementation; in the novelty-suppressed feeding test (NSFT) ( Figure 2 C), the feeding latency significantly shortened. In addition, in the forced swimming test (FST) ( Figure 2 E), the immobility time significantly decreased; in the tail suspension test (TST) ( Figure 2 D), the immobility time significantly decreased (p<0.001, p<0.05, p<0.01). For prefrontal cortex targeted sarcosine injection, the experimental protocol was as Figure 2 shown in M. As expected, after sarcosine supplementation, depressive-like behaviors were also alleviated (p<0.001, p<0.01, p<0.05, p<0.01, Figure 2 N- Figure 2Q). These results confirmed that the administration of sarcosine could improve the behavioral impairments in CUMS-induced mice, and prefrontal cortex-targeted injection of sarcosine was a more effective drug delivery method. Sarcosine is an endogenous amino acid and a competitive inhibitor of the co-agonist of type I glycine transporter (GlyT1) and N-methyl-D-aspartic acid receptor (NMDAR). Studies have shown that rapid activation of the mammalian target of rapamycin (mTOR) signaling pathway by stimulating α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) is the main mechanism by which the NMDAR antagonist ketamine exerts rapid antidepressant effects. Therefore, we investigated the role of the mTOR signaling pathway in the antidepressant effect of sarcosine. The results showed that after sarcosine treatment, the protein levels of p-AKT / AKT and p-mTOR / mTOR increased significantly (p < 0.05, p < 0.01, Figure 2 F- Figure 2 I、 Figure 2 R- Figure 2 U). In addition, we detected the expression of neuronal synaptic proteins PSD95, Syn, and SYP and found that the expression in the synaptic membrane decreased ( Figure 2 J- Figure 2 L, Figure 2 V-2X). These results together indicated that the antidepressant-like behavior of sarcosine was closely related to the AKT / mTOR signaling pathway.

[0156] Corticosterone reduces GNMT gene expression and sarcosine content through glucocorticoid receptors

[0157] Since sarcosine is reduced in depressed mice and sarcosine supplementation has been found to be effective against depression, we speculated that the pathway affecting sarcosine synthesis might play an important role in the occurrence and development of depression. It is well known that sarcosine can be produced in some metabolic processes in the body, and glycine is the precursor of sarcosine ( Figure 3 A). In addition, GNMT is a multifunctional protein that can promote the conversion of glycine to sarcosine. Therefore, we inferred that GNMT expression was abnormal in depressed mice. Therefore, we specifically detected the expression level of GNMT by RT-PCR test and western blot detection. In addition, the results of RT-PCR and western blot showed that the expression level of GNMT in the prefrontal cortex of depressed mice was significantly reduced ( Figure 3 F). Next, we determined whether GNMT was involved in the development of depression by affecting sarcosine content. To verify our hypothesis, we cultured mesenchymal stem cells derived from bone marrow and then transfected the GNMT overexpression plasmid into the mesenchymal stem cells. The results showed that after overexpressing GNMT, the sarcosine content in the exosomes secreted by the mesenchymal stem cells increased. In addition, previous studies have shown that the corticosterone level increases in depressed mice. Our experimental results confirmed this (Figure 3 B). To investigate whether increased corticosterone levels affect GNMT expression, we cultured PC12 cells ( Figure 3 C). Our study showed that a small amount of corticosterone could reduce GNMT expression ( Figure 3 D, Figure 3 E). Studies have shown that the androgen receptor (AR) can directly regulate muscle amino acid levels by regulating GNMT transcription, while corticosterone acts by affecting the glucocorticoid receptor (GR). Next, we will study whether corticosterone can directly regulate GNMT expression levels by affecting the glucocorticoid receptor. To address this question, we cultured 293T cells and constructed a dual-luciferase reporter gene system. The results confirmed that GR directly binds to the GNMT gene promoter to promote GNMT expression ( Figure 3 G-L). These results together indicate that corticosterone directly regulates GNMT expression through the glucocorticoid receptor and affects sarcosine content.

[0158] GNMT plays an important role in the development of depression

[0159] Given that overexpression of GNMT can increase sarcosine content, we hypothesized that reducing GNMT expression could induce the occurrence of depression, and overexpressing GNMT could relieve depressive-like behaviors by altering sarcosine content. To further determine the functional role of GNMT in CUMS-induced depressive-like behaviors, we specifically reduced GNMT by bilateral injection of an AAV9 vector expressing GNMT recombinase and GFAP, driven by the human synapsin 1 promoter (AAV9--ITR-hsyn-GNMT-GFAP). An AAV9 vector expressing GFAP was used as a control in the cortex of depressive mice (AAV9--ITR-hsyn-GFAP) ( Figure 4 A, Figure 4 B). We found that specifically reducing GNMT in neurons could induce depressive-like behaviors ( Figure 4 C- Figure 4 F). We further demonstrated that depressive-like behaviors were related to neuronal synapses. In addition, GNMT was specifically overexpressed by bilateral injection of an AAV9 vector expressing GNMT recombinase and GFAP, driven by the human synapsin 1 promoter (AAV9--ITR-hsyn-GNMT-GFAP). An AAV9 vector expressing GFAP was used as a control in the cortex of depressive mice (AAV9--ITR-hsyn-GFAP) ( Figure 5 A). We found that GNMT expression in the prefrontal cortex of mice increased significantly after injection ( Figure 5B). Behavioral results showed that specific overexpression of GNMT in the prefrontal cortex significantly alleviated depressive-like behaviors in mice. Specifically, compared with the model group, the total moving distance in the open field test (OFT) was significantly increased ( Figure 5 H), the feeding latency in the novelty-suppressed feeding test (NSFT) was significantly shortened ( Figure 5 G), and the immobility time in the forced swimming test (FST) ( Figure 5 J) and the tail suspension test (TST) ( Figure 5 I) was significantly reduced (p < 0.01).

[0160] GNMT is mainly highly expressed in neurons and is involved in the occurrence and development of depression

[0161] In addition, we further detected that GNMT was mainly expressed in neurons and hardly expressed in microglia. To further prove the role of GNMT in prefrontal cortex neurons. We injected the overexpressing adenovirus AAV9-GNMT-CMV-FLEX-MCS-EGFP into the prefrontal cortex of camkIIa-cre mice to specifically overexpress GNMT in neurons. In addition, we detected the expression of GNMT in cortical tissues and found that after expressing GNMT, the expression of GNMT in cortical tissues increased (p < 0.01, Figure 5 C- Figure 5 F). After administration, the depressive-like behaviors of camkIIa-cre mice subjected to chronic unpredictable mild stress were alleviated. Specifically, in the open field test (OFT), the total moving distance was significantly increased ( Figure 5 H), in the novelty-suppressed swimming test (NSFT), the feeding latency was significantly shortened ( Figure 5 G), and the immobility time in the forced swimming test (FST) ( Figure 5 J) and the tail suspension test (TST) ( Figure 5 I) was significantly reduced (p < 0.05). These results indicate that specific overexpression of GNMT in prefrontal cortex neurons can significantly alleviate depressive-like behaviors. We investigated the role of the AKT / mTOR signaling pathway and synaptic proteins in the antidepressant effect of GNMT. The results showed that the antidepressant-like behaviors of the GNMT gene were closely related to the AKT / mTOR signaling pathway ( Figure 5 K- Figure 5 O).

[0162] Exosomes of bone marrow mesenchymal stem cells carry the sarcosine gene and alleviate depressive-like symptoms in mice

[0163] To further determine the functional role of GNMT in CUMS-induced depressive mice, we specifically investigated the association between bone marrow mesenchymal stem cell-derived exosomes and antidepressant effects. The animal experimental procedures are as Figure 6 shown in Figure 6 I. We isolated and cultured bone marrow mesenchymal stem cells ( Figure 6 A). The specific surface antigens of bone marrow mesenchymal stem cells were detected by flow cytometry. The results showed that BMSC markers CD90 and CD105 were positive, while CD34 was negative (Figure Figure 6 B- Figure 6 D). This indicated that we had isolated mesenchymal stem cells. We extracted and identified the exosomes of the third generation of BMSC ( Figure 6 E- Figure 6 G). We injected the exosomes into the tail vein of depressive mice and found that the exosomes could enter the prefrontal cortex through the blood-brain barrier ( Figure 6 H). In addition, the results of behavioral tests showed that exosomes derived from bone marrow mesenchymal stem cells could relieve depressive-like behaviors. Specifically, after injecting exosomes derived from bone marrow mesenchymal stem cells, the total moving distance increased, and the time to explore food in the NSFT test was shortened ( Figure 6 L). Compared with the model group, the immobility time in the forced swimming test (FST) ( Figure 6 N) and the tail suspension test (TST) ( Figure 6 M) was significantly reduced after administration (p < 0.05). The glycine-N-methyltransferase (GNMT) gene encodes a protein that promotes the conversion of glycine to sarcosine. To verify this hypothesis, we cultured mesenchymal stem cells and downregulated GNMT ( Figure 6 K). We found that the sarcosine content in the exosomes of mesenchymal stem cells with downregulated GNMT was significantly reduced ( Figure 6 J). Further studies showed that exosomes derived from mesenchymal stem cells with downregulated GNMT were ineffective in relieving depressive-like behaviors. Behavioral tests (OFT, NSFT, TST, and FST) did not show significant improvement (p < 0.01, p < 0.05, p < 0.01, p < 0.01, Figure 6 L- Figure 6 O). These data further indicated that GNMT plays an important role in the antidepressant process. Further experiments showed that the antidepressant effect of GNMT is related to the AKT / mTOR signaling pathway and the regulation of neuronal synapse membranes ( Figure 6 P- Figure 6 S).

[0164] Hyperfunction of the HPA axis affects the expression of sarcosine in the glycine metabolic pathway

[0165] Hyperactivity of the HPA axis is one of the common neurobiological manifestations of depression and a significant feature of depression. Hyperactivity of the HPA axis leads to increased cortisol secretion. Our results also showed that the cortisol expression in the prefrontal cortex tissue and serum of depressive mice was higher than that of healthy control mice. Cortisol affects neuronal function by binding to glucocorticoid receptors and membrane receptors, and its function is not yet clear. Metabolomics studies have shown that the occurrence of depression is closely related to the glycine metabolic pathway. The sarcosine expression in the prefrontal cortex tissue and serum of depressive mice was lower than that of healthy control mice. We speculate that hyperactivity of the HPA axis leads to an increase in cortisol content, affects the glycine metabolic pathway by binding to membrane receptor proteins, reduces creatine content, and exacerbates depression. GNMT is an important enzyme in the glycine metabolic pathway. In cell experiments, we found that downregulating GNMT reduced sarcosine content, while overexpressing GNMT increased the amount of creatine secreted by cells. In addition, our results showed that the expression level of GNMT in the prefrontal cortex tissue of depressive mice was low. Dual-luciferase reporter assays showed that the binding of cortisol to glucocorticoid receptors reduced GNMT expression. Taken together, our results indicate that hyperactivity of the HPA axis leads to an increase in cortisol content, which is caused by GNMT, interferes with the glycine metabolic pathway, promotes depression by reducing GNMT expression and reducing creatine content. The potential mechanism revealed in this paper is as Figure 7 shown.

[0166] Diagnostic efficacy verification

[0167] As Figure 8 shown, in the training set, the AUC of methyl stearate was 0.692 ( Figure 8 A), the AUC of LysoPE(16:1(9Z) / 0:0) was 0.558 ( Figure 8 B), the AUC of sarcosine was 0.697 ( Figure 8 C), and the combined AUC of sarcosine, methyl stearate and LysoPE(16:1(9Z) / 0:0) was 0.824 ( Figure 8 D).

[0168] As Figure 9 shown, in the validation set, the AUC of methyl stearate was 0.659 ( Figure 9 A), the AUC of LysoPE(16:1(9Z) / 0:0) was 0.661 ( Figure 9 B), the AUC of sarcosine was 0.804 ( Figure 9 C), and the combined AUC of sarcosine, methyl stearate and LysoPE(16:1(9Z) / 0:0) was 0.804 ( Figure 9 D).

[0169] The combination of sarcosine, methyl stearate and LysoPE(16:1(9Z) / 0:0) has better diagnostic efficacy than sarcosine, methyl stearate and LysoPE(16:1(9Z) / 0:0) alone.

[0170] The description of the above embodiments is only for understanding the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of GNMT promoters in the preparation of drugs for treating depression; Preferably, the promoter includes nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, peptides, proteins, and reagents used for gene editing; Preferably, the promoter is selected from nucleic acid molecules, reagents used in gene editing, small molecule chemical drugs, and antibody drugs; Preferably, the reagents used for gene editing include reagents used for gene knock-in; Preferably, the reagents used for gene knock-in include interfering lentivirus, adeno-associated virus vector, CRISPR / Cas9, Cre-LoxP; Preferably, the adeno-associated virus vector includes AAV1, AAV2, AAV3, AAV5, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12; Preferably, the adeno-associated viral vector is selected from AAV9; Preferably, the small molecule chemical drugs include drugs that inhibit corticosterone or cortisol, and drugs that inhibit the HPA axis; Preferably, the antibody drug includes corticosterone antibody and cortisol antibody; Preferably, the depression includes major depressive disorder, unipolar depression, treatment-resistant depression, resistant depression, anxious depression, bipolar depression, and dysthymia; Preferably, the depression is selected from major depression and anxious depression; Preferably, the drug includes a pharmaceutically acceptable carrier and / or excipient.

2. Any of the following methods: (1) A method for regulating sarcosine, characterized in that: The method modulates sarcosine by administering an HPA / corticosterone / glucocorticoid receptor / GNMT / glycine modulator; (2) A method for regulating the AKT / mTOR signaling pathway, characterized in that the method regulates the AKT / mTOR signaling pathway by administering a sarcosine regulator.

3. The method according to claim 2, characterized in that The regulators include promoters and inhibitors; Preferably, the promoter includes nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, peptides, proteins, and reagents used for gene editing; Preferably, the promoter is selected from nucleic acid molecules; Preferably, the inhibitor includes reagents, nucleic acid inhibitors, protein inhibitors or compounds used in gene editing; Preferably, the reagents used for gene editing include adeno-associated virus vectors, zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS or reagents used for phiC31; Preferably, the nucleic acid inhibitor comprises siRNA, shRNA or miRNA; Preferably, the HPA is negatively correlated with the creatine; Preferably, the corticosterone is negatively correlated with the creatine; Preferably, the glucocorticoid receptor is positively correlated with the sarcosine; Preferably, the GNMT is positively correlated with the sarcosine; Preferably, the glycine and the sarcosine are regulated in a positive correlation.

4. Any of the following applications: (1) Application of reagents for detecting sarcosine in exosomes in the preparation of products for diagnosing depression; (2) Application of reagents for detecting methyl stearate in exosomes in the preparation of products for diagnosing depression; (3) Application of reagents for detecting LysoPE (16:1(9Z) / 0:0) in exosomes in the preparation of products for diagnosing depression; (4) Application of reagents for detecting sarcosine, methyl stearate and LysoPE (16:1(9Z) / 0:0) in exosomes in the preparation of products for diagnosing depression; Preferably, the depression includes major depressive disorder, unipolar depression, treatment-resistant depression, resistant depression, anxious depression, bipolar depression, and dysthymia; Preferably, the depression is selected from major depressive disorder and anxious depression.

5. The use according to claim 4, characterized in that: The reagents include oligonucleotide probes that specifically recognize sarcosine in exosomes, methyl stearate in exosomes, or LysoPE (16:1 (9Z) / 0:0) in exosomes, primers that specifically amplify sarcosine in exosomes, methyl stearate in exosomes, or LysoPE (16:1 (9Z) / 0:0) in exosomes, binding agents that specifically bind to proteins encoded by sarcosine in exosomes, methyl stearate in exosomes, or LysoPE (16:1 (9Z) / 0:0) in exosomes, or chips that specifically analyze sarcosine in exosomes, methyl stearate in exosomes, or LysoPE (16:1 (9Z) / 0:0) in exosomes; Preferably, the reagents include oligonucleotide probes that specifically recognize sarcosine, methyl stearate and LysoPE (16:1 (9Z) / 0:0) in exosomes, primers that specifically amplify sarcosine, methyl stearate and LysoPE (16:1 (9Z) / 0:0) in exosomes, binders that specifically bind to proteins encoded by sarcosine, methyl stearate and LysoPE (16:1 (9Z) / 0:0) in exosomes, or chips that specifically analyze sarcosine, methyl stearate and LysoPE (16:1 (9Z) / 0:0) in exosomes.

6. The use according to claim 4, characterized in that: The product includes a chip, a test kit or a nucleic acid membrane strip; Preferably, the chip includes a gene chip and a protein chip; Preferably, the gene chip comprises oligonucleotide probes for sarcosine in exosomes or methyl stearate in exosomes or LysoPE (16:1 (9Z) / 0:0) in exosomes for detecting the transcription level of sarcosine in exosomes or methyl stearate in exosomes or LysoPE (16:1 (9Z) / 0:0) in exosomes; Preferably, the gene chip comprises oligonucleotide probes for sarcosine, methyl stearate and LysoPE (16:1 (9Z) / 0:0) in exosomes for detecting the transcription levels of sarcosine, methyl stearate and LysoPE (16:1 (9Z) / 0:0) in exosomes; Preferably, the protein chip comprises a specific binding agent for sarcosine in exosomes or methyl stearate in exosomes or LysoPE (16:1 (9Z) / 0:0) protein in exosomes; Preferably, the protein chip comprises specific binding agents for sarcosine, methyl stearate and LysoPE (16:1 (9Z) / 0:0) protein in exosomes; Preferably, the kit comprises a reagent for detecting the gene or protein expression level of sarcosine in exosomes or methyl stearate in exosomes or LysoPE (16:1 (9Z) / 0:0) in exosomes by RT-PCR, qRT-PCR, biochip detection, Southern blotting, in situ hybridization, immunoblotting, or mass spectrometry; Preferably, the kit comprises reagents for detecting the gene or protein expression levels of sarcosine, methyl stearate and LysoPE (16:1 (9Z) / 0:0) in exosomes by RT-PCR, qRT-PCR, biochip detection, Southern blotting, in situ hybridization, immunoblotting, and mass spectrometry; Preferably, the exosomes are derived from one or more of the following samples: cells, tissues, blood, urine, saliva or mucus; Preferably, the cells include microglia, prefrontal cortical cells, neurons; Preferably, the cells are selected from prefrontal cortex cells; Preferably, the tissue comprises prefrontal cortex tissue; Preferably, the kit includes instruments or reagents for processing samples.

7. Any of the following products: (1) A product for diagnosing depression, characterized in that: The product includes a reagent capable of detecting the expression level of sarcosine in exosomes; (2) A product for diagnosing depression, characterized in that the product comprises a reagent capable of detecting the expression level of methyl stearate in exosomes; (3) A product for diagnosing depression, characterized in that the product comprises a reagent capable of detecting the expression level of LysoPE (16:1(9Z) / 0:0) in exosomes; (4) A product for diagnosing depression, characterized in that the product comprises a reagent capable of detecting the expression levels of sarcosine, methyl stearate and LysoPE (16:1(9Z) / 0:0) in exosomes; Preferably, the depression includes major depressive disorder, unipolar depression, treatment-resistant depression, resistant depression, anxious depression, bipolar depression, and dysthymia; Preferably, the depression is selected from major depression and anxious depression; Preferably, the product comprises a chip, a kit or a nucleic acid membrane strip.

8. A method for constructing a non-human animal model of depression, characterized in that: The method comprises administering a GNMT inhibitor to a non-human animal; Preferably, the inhibitor includes reagents, nucleic acid inhibitors, protein inhibitors or compounds used in gene editing; Preferably, the reagents used for gene editing include adeno-associated virus vectors, zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR-Cas9, Cre-LoxP, FLP / FRT, R / RS, Gin / gix, Cin H / RS2, Par A / MRS or reagents used for phiC31; Preferably, the nucleic acid inhibitor comprises siRNA, shRNA or miRNA; Preferably, the reagent used for gene editing is selected from adeno-associated virus vectors; Preferably, the adeno-associated virus vector includes AAV1, AAV2, AAV3, AAV5, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12; Preferably, the adeno-associated viral vector is selected from AAV9; Preferably, the non-human animal model refers to a non-human animal that has or displays characteristics of a disease or condition; Preferably, the non-human animal is a mammal; Preferably, the mammals include mice, rats, rabbits, dogs, pigs, monkeys, and sheep; Preferably, the mammal is selected from mice; Preferably, the method comprises injecting mice with an AAV9 vector expressing GNMT recombinase and GFAP driven by the human synapsin 1 promoter (AAV9--ITR-hsyn-GNMT-GFAP) to specifically reduce the expression of GNMT; Preferably, the injection site is the prefrontal cortex; Preferably, the injection method is bilateral injection; Preferably, the method is capable of specifically reducing the expression of GNMT in neurons; Preferably, the method comprises injecting into the prefrontal cortex of mice an AAV9 vector expressing GNMT recombinase and GFAP driven by the human synapsin 1 promoter (AAV9--ITR-hsyn-GNMT-GFAP) to specifically reduce the expression of GNMT in neurons; Preferably, the depression includes major depressive disorder, unipolar depression, treatment-resistant depression, resistant depression, anxious depression, bipolar depression, and dysthymia; Preferably, the depression is selected from major depressive disorder and anxious depression.

9. Any of the following applications: (1) Use of the non-human animal model of depression prepared by the method of claim 8 in screening drug candidates for treating depression; (2) Use of the non-human animal model of depression prepared by the method of claim 8 in evaluating the therapeutic effect of drugs for treating depression; (3) Use of the non-human animal model of depression prepared by the method of claim 8 in studying the pathogenesis of depression; (4) Application of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine in regulating sarcosine.

10. Any of the following methods: (1) A method for screening drug candidates for treating depression, characterized in that: The method comprises: a) administering the agent to be screened to the non-human animal with depression prepared by the method of claim 8; b) Analyze and evaluate the therapeutic effects of the agents to be screened, and select agents that can significantly improve the depressive-like behavior of the mouse model; Preferably, the drug candidates include selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, tricyclic drugs, tetracyclic drugs, serotonin modulators and stimulants; (2) A method for evaluating the efficacy of a drug for treating depression, characterized in that the method comprises: a) administering a drug to a non-human animal with depression prepared by the method of claim 8; b) evaluating the therapeutic effect of the drug on the depression; (3) A method for studying the pathogenesis of depression, characterized in that the method comprises using a non-human animal with depression prepared by the method of claim 8 to study the pathogenesis of depression.

Images