Biomarker for diagnosing depression and application thereof

Abstract

The invention provides a biomarker for diagnosing depression and application thereof. 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

[0001] This application is a divisional application of application number 202510477804.9, entitled "Application of GNMT promoter in the preparation of drugs for treating depression", filed on April 16, 2025. Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to biomarkers for diagnosing depression and their applications. Background Technology

[0003] Major depressive disorder (MDD) is a common mental illness characterized by depressed mood and loss of interest, affecting approximately 6% of adults worldwide each year. MDD can lead to serious self-harm and even suicidal behavior, placing a significant burden on individuals and society.

[0004] Recent studies have shown a close link between the development of depression and metabolic abnormalities. Certain metabolic diseases, such as obesity and diabetes, increase the risk of depression. Furthermore, maternal obesity and maternal diabetes have been reported to increase the risk of neuropsychiatric disorders, such as depression, in offspring. In recent years, researchers have increasingly focused on the metabolites of patients with MDD (Melanocytic Depression). Glial inflammatory metabolic pathways, plasma metabolomics, and hippocampal glucose metabolism in MDD patients have been explored, revealing many differentially expressed metabolites compared to the HC (Hypercholesterolaryngology) population. However, the specific roles of these differentially expressed metabolites remain unclear, and they cannot yet be used for diagnosis or predicting treatment outcomes. Research on biomarkers and antidepressant biologics for depression is limited by sample instability. This may be due to the susceptibility of plasma biomarkers to contamination and inconsistent bioactivity between different batches of serum. Additionally, while the exploration of antidepressants has been ongoing for many years, current antidepressants are not effective for all patients, and some even have side effects.

[0005] Therefore, there is an urgent need to find biomarkers that can effectively diagnose depression and effective antidepressants without side effects. Summary of the Invention

[0006] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0007] The first aspect of this invention provides the use of a promoter of glycosaminomethyltransferase (GNMT) in the preparation of a drug for treating depression.

[0008] In this invention, treatment refers to the improvement, prevention, or reversal of a disease or symptom or at least one identifiable symptom thereof. Further, treatment refers to the improvement, prevention, or reversal of at least one measurable physiological parameter associated with the disease or symptom to be treated, said parameter not necessarily being identifiable in or recognized by mammals. Further, treatment refers to the suppression or alleviation of a disease or its course, which may be physical, such as certain identifiable severe symptoms. The term "treatment" as used in this invention encompasses diseases in mammals, particularly humans, including: (a) preventing the occurrence of a disease or symptom in individuals susceptible to the disease but not yet diagnosed with it; (b) suppressing a disease, such as halting its progression; or (c) alleviating a disease, such as reducing symptoms associated with the disease.

[0009] Furthermore, the promoters include, but are not limited to, nucleic acid molecules, carbohydrates, liposomes, small molecule chemicals, antibody drugs, peptides, proteins, and reagents used in gene editing.

[0010] Furthermore, the promoter is selected from nucleic acid molecules, reagents used in gene editing, small molecule chemicals, and antibody drugs.

[0011] Furthermore, the nucleic acid molecules include, but are not limited to, nucleic acids encoding glucocorticoid receptors, nucleic acids encoding GNMT, nucleic acids encoding corticosterone antibodies, and nucleic acids encoding cortisol antibodies.

[0012] Furthermore, the nucleic acid molecule is selected from nucleic acids encoding GNMT.

[0013] Furthermore, the reagents used in the gene editing include those used in gene knock-in.

[0014] Furthermore, the reagents used for gene knock-in include, but are not limited to, interfering lentiviruses, adeno-associated virus vectors, CRISPR / Cas9, and Cre-LoxP.

[0015] Furthermore, the adeno-associated virus vector includes AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0016] Furthermore, the adeno-associated virus vector is selected from AAV9.

[0017] 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.

[0018] Furthermore, the drugs that inhibit corticosterone or cortisol include, but are not limited to, metheprone tablets, mitotane tablets, phentolamine mesylate tablets, aminoglutethimide tablets, and ketoconazole tablets.

[0019] Furthermore, the antibody drug includes corticosterone antibodies and cortisol antibodies.

[0020] In this invention, the depression includes, but is not limited to, major depressive disorder, unipolar depression, treatment-resistant depression, resistant depression, anxiety depression, bipolar depression, and dysphoric mood. Further, the depression is selected from major depressive disorder and anxiety depression.

[0021] Furthermore, the drug includes its pharmaceutically acceptable carrier and / or excipients.

[0022] Furthermore, the pharmaceutically acceptable carriers and / or excipients include diluents, binders, surfactants, humectants, adsorbents, lubricants, and / or disintegrants. The diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, and water; the binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginates, xanthan gum, hydroxypropylcellulose, and hydroxypropyl methylcellulose; the surfactants include, but are not limited to, polyethylene oxide sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol; the humectants include, but are not limited to, glycerol and starch; the adsorbents include, but are not limited to, starch, lactose, bentonite, silica gel, kaolin, and soap clay; and the lubricants include, but are not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium lauryl sulfate.

[0023] Furthermore, the drug can be administered in any applicable manner.

[0024] Furthermore, the administration method includes administration via the gastrointestinal tract and administration outside the gastrointestinal tract.

[0025] Furthermore, the non-gastrointestinal administration includes, but is not limited to, injection administration, respiratory administration, cavity administration, mucosal administration, and skin administration.

[0026] Furthermore, the injection sites include, but are not limited to, veins, intramuscular sites, subcutaneous sites, intradermal sites, intracavitary sites, and prefrontal cortex sites.

[0027] Furthermore, the injection site is selected from the prefrontal cortex.

[0028] The second aspect of the present invention provides any of the following methods:

[0029] (1) A method for regulating sarcosine, said method by administering an HPA / corticosterone / glucocorticoid receptor / GNMT / glycine regulator;

[0030] (2) A method for regulating the AKT / mTOR signaling pathway, wherein the method regulates the AKT / mTOR signaling pathway by applying a sarcosine regulator.

[0031] In this invention, sarcosine has multiple uses. For example, sarcosine can be used as a dye stabilizer, participate in the synthesis of anti-enzyme agents, and also as a biological reagent for laboratory research. In the field of daily chemical products, sarcosine can be used as an amino acid-based surfactant, which helps to replenish skin cell energy and promote the repair of fatigue. Sarcosine is also beneficial in preventing hair damage and promoting the self-repair of damaged hair, enhancing the shine and elasticity of hair, and can be used for skin and hair care.

[0032] In this invention, the term "regulation" includes regulation and control, wherein regulation includes increasing or decreasing sarcosine expression levels or activity.

[0033] Furthermore, the regulator includes promoters and inhibitors.

[0034] In this invention, the term "promoter" refers to any substance that can promote the activity of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine, promote the stability of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine genes or proteins, promote the expression level of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine, or promote the effective duration of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine.

[0035] In this invention, the effects of promoters can also be achieved through knock-in. Knock-in refers to the targeted insertion of a transgene into the host cell genome, resulting in transgene expression and / or altered expression of the endogenous target gene (e.g., increased (including ectopic) or decreased expression), for example, by introducing an additional copy of the target gene or by operatively inserting a regulatory sequence that provides an endogenous copy of the target gene to enhance expression. Knock-in transgenes can include heterozygous knock-in or homozygous knock-in. Knock-in also encompasses conditional knock-in, wherein transgene expression and / or altered expression of the endogenous target gene can occur, for example, by exposing an 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.

[0036] Furthermore, the promoters include nucleic acid molecules, carbohydrates, liposomes, small molecule chemicals, antibody drugs, peptides, proteins, or reagents used in gene editing. In this invention, the term nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). As equivalents, nucleic acids also include DNA or RNA analogs derived from nucleotide analogs and, where applicable, single-stranded (sense or antisense) and double-stranded polynucleotides. Liposomes refer to small vesicles composed of various types of lipids, phospholipids, and / or surfactants, which can be used for drug delivery to mammals. Antibodies cover complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two complete antibodies, and antibody fragments, provided they exhibit the desired antigen-binding activity and fall within the scope of this invention.

[0037] Furthermore, the promoter is selected from nucleic acid molecules.

[0038] In this 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 duration of action of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine.

[0039] Furthermore, the inhibitors include, but are not limited to, reagents used in gene editing, nucleic acid inhibitors, protein inhibitors, or compounds.

[0040] Furthermore, the reagents used in the gene editing include, but are not limited to, the 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.

[0041] Furthermore, the nucleic acid inhibitors include, but are not limited to, siRNA, shRNA, or miRNA.

[0042] In this invention, siRNA may include partially purified RNA, substantially pure RNA, synthetic RNA, or recombinant RNA, as well as RNA modified to differ from native RNA by adding, deleting, substituting, and / or altering one or more nucleotides. These modifications may include adding non-nucleotide substances, such as adding to the ends of the siRNA or to one or more internal nucleotides of the siRNA; modifications that make the siRNA resistant to ribozyme digestion (e.g., using 2'-substituted ribonucleotides or modifying the sugar phosphate backbone); or replacing one or more nucleotides in the siRNA with deoxyribonucleotides.

[0043] In this invention, shRNA is a non-coding small RNA molecule capable of forming a hairpin structure. shRNA can suppress gene expression through the RNA interference pathway. When shRNA is introduced into a cell, it is recognized by the intracellular ribozyme Dicer and cleaved into small interfering RNA (siRNA) of approximately 21 nucleotides. siRNA binds to a protein complex called the RNA-induced silencing complex (RISC). The Argonaute protein in RISC uses the antisense strand of siRNA to recognize and bind to the target mRNA, and then degrades the target mRNA through cleavage or inhibition of translation, thereby suppressing the expression of specific genes.

[0044] In this invention, the protein inhibitor is selected from substances capable of inhibiting HPA / corticosterone / glucocorticoid receptor / GNMT / glycine protein. The protein inhibitor includes proteolytic enzymes and protein-binding molecules. The proteolytic enzyme is selected from enzymes capable of catalyzing the hydrolysis of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine protein. The protein-binding molecule is 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.

[0045] Furthermore, the HPA axis is negatively correlated with creatine; when the HPA axis is overactive, creatine levels decrease. Furthermore, creatine production can be increased by inhibiting the HPA axis.

[0046] Furthermore, the corticosterone and creatine are negatively correlated; when corticosterone levels increase, creatine levels decrease, and vice versa. Furthermore, creatine production can be increased by adding a corticosterone inhibitor.

[0047] Furthermore, the glucocorticoid receptor and creatine are positively correlated in regulation; when the glucocorticoid receptor level increases, the creatine level increases, and when the glucocorticoid receptor level decreases, the creatine level decreases. Furthermore, creatine production can be increased by adding the glucocorticoid receptor itself or its promoters.

[0048] Furthermore, GNMT and creatine are positively correlated; when GNMT levels increase, creatine levels increase, and when GNMT levels decrease, creatine levels decrease. Furthermore, creatine production can be increased by adding GNMT itself or its promoters.

[0049] Furthermore, the glycine and sarcosine are positively correlated; when glycine levels increase, sarcosine levels increase, and when glycine levels decrease, sarcosine levels decrease. Furthermore, sarcosine production can be increased by adding glycine itself or its promoter.

[0050] The third aspect of the present invention provides any of the following applications:

[0051] (1) Application of reagents for detecting sarcosine in exosomes in the preparation of products for diagnosing depression;

[0052] (2) Application of reagents for detecting methyl stearate in exosomes in the preparation of products for diagnosing depression;

[0053] (3) Application of reagent for detecting LysoPE (16:1(9Z) / 0:0) in exosomes in the preparation of products for diagnosing depression;

[0054] (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.

[0055] Furthermore, the exosomes are derived from one or more of the following samples: cells, tissues, blood, urine, saliva, or mucus.

[0056] Furthermore, the cells include microglia, prefrontal cortex cells, and neurons.

[0057] Furthermore, the cells are selected from prefrontal cortex cells.

[0058] Furthermore, the tissue includes prefrontal cortex tissue.

[0059] Furthermore, the kit includes instruments or reagents for processing samples.

[0060] The fourth aspect of the present invention provides any of the following products:

[0061] (1) A product for diagnosing depression, said product comprising a reagent capable of detecting sarcosine expression levels in exosomes;

[0062] (2) A product for diagnosing depression, said product comprising a reagent capable of detecting the expression level of methyl stearate in exosomes;

[0063] (3) A product for diagnosing depression, said product comprising a reagent capable of detecting the expression level of LysoPE (16:1(9Z) / 0:0) in exosomes;

[0064] (4) A product for diagnosing depression, the product comprising reagents capable of detecting the expression levels of sarcosine, methyl stearate and LysoPE (16:1(9Z) / 0:0) in exosomes.

[0065] Furthermore, the products include chips, reagent kits, or nucleic acid membrane strips.

[0066] The fifth aspect of the present invention provides a method for constructing a non-human animal model of depression, the method comprising administering a GNMT inhibitor to a non-human animal.

[0067] Furthermore, the inhibitors include reagents used in gene editing, nucleic acid inhibitors, protein inhibitors, or compounds.

[0068] Furthermore, the reagents used in the 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.

[0069] Furthermore, the nucleic acid inhibitor includes siRNA, shRNA, or miRNA.

[0070] Furthermore, the reagents used in the gene editing are selected from adeno-associated virus vectors.

[0071] Furthermore, the adeno-associated virus vector includes AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0072] Furthermore, the adeno-associated virus vector is selected from AAV9.

[0073] Furthermore, the non-human animal model refers to a non-human animal that has or displays characteristics of disease or symptom.

[0074] Furthermore, the non-human animal in question is a mammal.

[0075] Furthermore, the mammals include mice, rats, rabbits, dogs, pigs, monkeys, and sheep.

[0076] Furthermore, the mammal is selected from mice.

[0077] Furthermore, the method includes injecting mice with an AAV9 vector to express GNMT recombinase and GFAP, thereby specifically reducing GNMT expression driven by the human synaptophysin 1 promoter (AAV9--ITR-hsyn-GNMT-GFAP).

[0078] Furthermore, the injection site is the prefrontal cortex.

[0079] Furthermore, the injection method is bilateral injection.

[0080] Furthermore, the method can specifically reduce the expression of GNMT in neurons.

[0081] Furthermore, the method includes injecting an AAV9 vector into the prefrontal cortex of mice to express GNMT recombinase and GFAP, which specifically reduces GNMT expression in neurons by being driven by the human synaptophysin 1 promoter (AAV9--ITR-hsyn-GNMT-GFAP).

[0082] The sixth aspect of the present invention provides any of the following applications:

[0083] (1) The application of the non-human animal model of depression prepared by the method of the fifth aspect of the present invention in screening drug candidates for the treatment of depression;

[0084] (2) The application of the 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;

[0085] (3) The application of the non-human animal model of depression prepared by the method in the fifth aspect of the present invention in the study of the pathogenesis of depression;

[0086] (4) Application of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine in the regulation of sarcosine.

[0087] The seventh aspect of the present invention provides any of the following methods:

[0088] (1) A method for screening drug candidates for the treatment of depression, the method comprising:

[0089] a) Applying the screening reagent to non-human animals with depression prepared by the method described in the fifth aspect of the present invention;

[0090] b) Analyze and evaluate the therapeutic effects of the reagents to be screened, and select the reagents that can significantly improve depressive-like behavior in mouse models;

[0091] Furthermore, the drug candidates include selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, tricyclic drugs, tetracyclic drugs, serotonin modulators, and stimulants;

[0092] (2) A method for evaluating the efficacy of a drug for treating depression, the method comprising:

[0093] a) Applying the drug to a non-human animal with depression prepared by the method described in the fifth aspect of the present invention;

[0094] b) Evaluate the therapeutic effect of the drug on the depression;

[0095] (3) A method for studying the pathogenesis of depression, wherein the method is to use non-human animals with depression prepared by the method described in the fifth aspect of the present invention to study the pathogenesis of depression.

[0096] Furthermore, the evaluation indicators include, but are not limited to, animal behavioral tests, neurogenic performance, and microglial cell status.

[0097] Furthermore, animal behavioral tests include, but are not limited to, the Open Field Test (OFT), the Novel Environment Diet Inhibition Test (NSFT), the Forced Swimming Test (FST), and the Suspended Tail Test (TST).

[0098] The advantages and beneficial effects of this invention are as follows:

[0099] This invention provides the application of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine in the treatment of depression. The invention experimentally demonstrates the relationship between HPA / corticosterone / glucocorticoid receptor / GNMT / glycine and sarcosine, wherein HPA / corticosterone is negatively correlated with sarcosine, and glucocorticoid receptor / GNMT / glycine is positively correlated with sarcosine. This invention also provides biomarkers for the effective diagnosis of depression, including sarcosine, methyl stearate, and LysoPE (16:1(9Z) / 0:0) in exosomes. Furthermore, this invention constructs an animal model of depression by reducing GNMT. This animal model can be used for drug screening, efficacy evaluation, or diagnosis of depression, and can also be used to study and elucidate the pathogenesis of depression. This invention is of great significance for the diagnosis and treatment of depression and has broad clinical application prospects. Attached Figure Description

[0100] Figure 1Figure 1 shows the bioinformatics analysis results of exosomes from serum samples and exosomes from prefrontal cortex tissue. Figure 2 shows the shared metabolites of exosomes from serum samples and exosomes from prefrontal cortex tissue of adult mice; Figure 3 is a heatmap; Figure 4 shows the quantified results of 447 metabolites from exosomes from serum samples of patients with depression; Figure 5 shows the quantified results of 480 differentially expressed metabolites from exosomes from prefrontal cortex tissue of mice; Figure 6 shows the relative levels of 30 named metabolites that differed between patients with major depressive disorder (MDD) and healthy controls (HC); Figure 7 shows the levels of 63 named metabolites that were altered in prefrontal cortex samples of mice; Figure 8 is a flowchart of exosome collection; Figure 9 shows the structural diagram of sarcosine; Figure 10 shows the molecular fragmentation diagram of sarcosine; Figure 11 shows the results of reduced sarcosine in serum exosomes of patients with depression; and Figure 12 shows the results of reduced sarcosine in exosomes from prefrontal cortex of depressed mice.

[0101] Figure 2 The diagrams show the results of the functional role of sarcosine in MDD. Figure A shows the intravenous injection protocol for studying the functional role of sarcosine in MDD; Figure B shows the results of the Open Field Test (OFT); Figure C shows the results of the Novel Environment Diet Suppression Test (NSFT); Figure D shows the results of the Suspended Tail Test (TST); Figure E shows the results of the Forced Swimming Test (FST); Figures F-I and R-U show the significant increase in p-AKT / AKT and p-mTOR / mTOR protein levels after sarcosine treatment; Figure M shows the prefrontal cortex targeted injection protocol for studying the functional role of sarcosine in MDD; and Figures J-L and V-X show the expression results of neuronal synaptic proteins PSD95, Syn, and SYP.

[0102] Figure 3 The diagrams show the results of corticosterone directly regulating GNMT expression and affecting sarcosine levels through glucocorticoid receptors. Figure A illustrates glycine as a precursor to sarcosine; Figure B shows increased corticosterone levels in depressed mice; Figure C shows PC12 cell culture; Figures D and E show that corticosterone can reduce GNMT expression; Figure F shows a significant decrease in GNMT expression levels in the prefrontal cortex of depressed mice; and Figures G-L show that GR directly binds to the GNMT gene promoter to promote GNMT expression.

[0103] Figure 4 This is a diagram showing the role of GNMT in the development of depression. Figures A and B show the results of specifically reducing GNMT expression; Figures C, D, E, and F show the results of specifically reducing GNMT in neurons inducing depressive-like behaviors.

[0104] Figure 5These are diagrams showing the relationship between depressive-like behaviors and neuronal synapses. Figures A-F show the results of GNMT overexpression; Figure G shows the results of the Novel Environment Food Suppression Test (NSFT); Figure H shows the results of the Open Field Test (OFT); Figure I shows the results of the Suspended Tail Test (TST); Figure J shows the results of the Forced Swimming Test (FST); and Figures K-O show the close relationship between the antidepressant-like behaviors of the GNMT gene and the AKT / mTOR signaling pathway.

[0105] Figure 6 This is a graph showing the association between exosomes and antidepressant effects. Figure A shows bone marrow mesenchymal stem cells (BMSCs); Figures B and C show the positive results of flow cytometry detection of the BMSC marker CD90 in BMSCs; Figure D shows the positive results of flow cytometry detection of the BMSC marker CD105 in BMSCs; Figures E-G show the results of exosome extraction and identification from third-generation BMSCs; Figure H shows the results of exosomes crossing the blood-brain barrier and entering the prefrontal cortex; Figure I shows the animal experimental procedure; Figure J shows the results of downregulating GNMT, which significantly reduces sarcosine content in exosomes from mesenchymal stem cells; Figure K shows the results of downregulating GNMT; Figures L-O show the results of behavioral tests (OFT, NSFT, TST, and FST); and Figures P-S show the results of the antidepressant effect of GNMT and its relationship with the AKT / mTOR signaling pathway and regulation of neuronal synaptic membranes.

[0106] Figure 7 This is a diagram illustrating the potential mechanisms of depression.

[0107] Figure 8 The ROC curves for diagnosing depression using sarcosine, methyl stearate, and LysoPE (16:1(9Z) / 0:0) in exosomes from the training set are shown in Figure A. Figure B shows the ROC curve for diagnosing depression using methyl stearate in exosomes; Figure C shows the ROC curve for diagnosing depression using methyl stearate in exosomes; and Figure D shows the ROC curve for diagnosing depression using the combined combination of sarcosine, methyl stearate, and LysoPE (16:1(9Z) / 0:0) in exosomes.

[0108] Figure 9The ROC curves of sarcosine, methyl stearate, and LysoPE (16:1(9Z) / 0:0) in exosomes for diagnosing depression are shown in Figure A. Figure B shows the ROC curve of methyl stearate in exosomes for diagnosing depression. Figure C shows the ROC curve of sarcosine in exosomes for diagnosing depression. Figure D shows the ROC curve of the combined use of sarcosine, methyl stearate, and LysoPE (16:1(9Z) / 0:0) in exosomes for diagnosing depression. Detailed Implementation

[0109] The foregoing disclosure provides a general description of the invention. A more complete understanding can be obtained by referring to the specific embodiments below. These embodiments are described for illustrative purposes only and are not intended to limit the scope of the invention. Formal variations and equivalent substitutions are considered as may be suggested or provided by the circumstances. Although specific terminology is used herein, it is descriptive and not limiting.

[0110] Example

[0111] I. Materials and Methods

[0112] Ethical statements and participants

[0113] All participants (if they were 18 years of age or older) or their guardians (if under 18 years of age) signed written informed consent prior to study inclusion. The study 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, and the study was conducted in accordance with the Declaration of Helsinki. From June to August 2019, we recruited 37 MDD patients and 33 healthy controls (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 Uygur Autonomous Region, China). All HCs (n=74), including physicians 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, the Montgomery-Asperger's Depression Rating Scale (MADRS) and the Hamilton Depression Rating Scale (HAMD), were used to quantify the severity of depression. Patients included in the study had moderate to severe depression (i.e., MADRS ≥22 or HAMD ≥17) and no comorbid physical, neurological, or psychiatric illnesses.

[0114] Exosome isolation

[0115] Serum and tissue exosome isolation were performed according to the previously described method. Briefly, exosomes (serum and tissue) were isolated using qEV columns 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).

[0116] Nanoparticle tracking analysis (NTA)

[0117] The NanoSight NS300 (NanoSight NTA 2.3 Nanoparticle Tracking and Analysis Release Version 0033) (Malvern Instruments) was used to measure particle concentration and size. Samples were diluted 1:1000 in PBS, and the camera was set to capture 3 videos per sample, each lasting 30 seconds. The videos were then analyzed to determine the particle size distribution and approximate particle number.

[0118] Sample collection and preparation

[0119] Fasting blood samples were collected from patients and HC and allowed to coagulate at room temperature for 1 hour. The samples were then centrifuged for 10 minutes (3000×g, 4°C), and serum (approximately 1 mL of supernatant) was carefully aspirated and stored at -80°C until exosomes were isolated. In summary, serum exosomes were isolated on a qEV column according to the manufacturer's protocol (Izon, Oxford, UK) and then concentrated in a Vivaspin® protein concentrator (Sartorius, Göttingen, Germany) for 60 minutes (10000×g, 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. The isolated exosomes were validated using negative staining electron microscopy and nanoparticle tracking analysis as previously described.

[0120] Animals and processing

[0121] Male C57BL / 6 mice were obtained from Viton Lever Laboratories (Beijing, China). Animals were housed at 24±1°C and 50±1% humidity with a 14 / 10 hour light / dark cycle and free access to food and water. Animal procedures were approved by the Animal Care and Use Committee of Minzu University of China. Mice were randomly assigned to four groups according to experimental requirements. Isolation of mouse brain exosomes was performed according to the method of Vella et al.

[0122] Stereotactic injection

[0123] In this experiment, mice were placed in an anesthesia induction chamber and anesthetized using 3% isoflurane. The mice were then fixed to a stereotactic brain apparatus, and the target nucleus mPFC was measured using nuclear localization coordinates obtained from mouse brain atlases. Next, a hole was drilled with a dental drill and a special cannula was inserted. Finally, the RWD catheter was injected using a microinjection pump.

[0124] UPLC-MS / MS analysis

[0125] Exosome-based targeted metabolomics analysis of participant serum samples and mouse brain tissue samples was performed at Wuhan Metawell Biotechnology Co., Ltd. using UPLC (Shim-pack UFLC SHIMADZU CBM30A system; Shimadzu Corporation, Kyoto, Japan) and tandem mass spectrometry (MS / MS) (4500 QTRAP; Applied Biosystems, Foster City, CA, USA). Briefly, exosomes extracted from participant serum were freeze-dried and vortexed for 30 seconds with 1 mL of 70% MeOH-water mixture. The samples were then placed in liquid nitrogen for 5 minutes, followed by 3 minutes on ice. This process was repeated three times. Afterward, the samples were sonicated at 30 Hz for 3 minutes, vortexed for approximately 30 seconds, and centrifuged for 10 minutes (centrifugation speed: 12000 rpm, temperature: 4℃). The supernatant was then concentrated, and 150 μL of 70% MeOH-water was added. Finally, centrifuge for 10 minutes (centrifugation speed: 12000 rpm, temperature: 4℃), and mix the supernatant 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.

[0126] Qualitative analysis was performed based on a self-developed target database MWDB (MetWare database) and a public metabolite information database, according to retention time (RT), precursor ion pair information, and secondary spectrum data. Quality control (QC) samples were prepared by mixing all exosome samples in equal volumes before sample analysis, and injected every ten samples to monitor the repeatability of the instrument analysis process.

[0127] Behavioral testing

[0128] The Open Field Test (OFT), Novel Environment Diet Suppression Test (NSFT), Suspended Tail Test (TST), and Forced Swimming Test (FST) were used to assess depressive behavior in mice. Behavioral tests were conducted after modeling and treatment. Open Field Test (OFT): Mice were placed in a corner of an open field apparatus (50×50×45 cm). Each mouse was allowed to move freely for six minutes, and the total distance moved in the first five minutes was recorded. Novel Environment Diet Suppression Test (NSFT): An open field apparatus was used to assess the behavior. Food particles were placed in the center of the apparatus. Mice were placed on individual grids in the corners of the floor and allowed to explore freely for five minutes. The latency period at which the mice began eating was recorded. Suspended Tail Test (TST): In this test, the tips of the mice's tails were suspended by a rope. The mice were separated, with each mouse's head 15 cm above the ground. The total duration was six minutes, and the immobility time 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℃. Each mouse was forced to swim for 6 minutes, and the immobility time in the last 5 minutes was recorded.

[0129] Quantitative RT-PCR

[0130] Total RNA was extracted from 15 mg of deep-frozen prefrontal lobe tissue and cell samples using Trizol reagent. 1 μg of RNA was used to synthesize first-strand cDNA, and quantitative real-time PCR was performed using a LightCycler 96 RT-PCR instrument. Normalization was performed using the β-actin gene as a reference gene. 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′.

[0131] Western blot analysis

[0132] Frontal lobe tissue and cell samples were lysed using RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentration was measured using the BCA assay and adjusted to the same final concentration. After heat denaturation at 95°C for 10 min, 30–50 μg of total protein was separated, electrophoresed by SDS-PAGE, and then transferred to a nitrocellulose membrane. The nitrocellulose membrane was incubated with the following major 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), and anti-p-mTOR (#11948S, Cell Signaling Technology). 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).

[0133] Immunostaining

[0134] After anesthesia, mice were euthanized and perfused with physiological saline. Mouse brains were isolated and fixed with 4% paraformaldehyde for 48 hours, followed by dehydration once in 20% sucrose solution and twice in 30% sucrose solution. Whole brain tissue was embedded with an optimal sectioning temperature compound (OCT) and cut into 35 μm thicknesses. The main antibody used for immunofluorescence staining was employed. Images were captured using a laser scanning confocal microscope (Leica Microsystems, Germany). Results were analyzed using ImageJ software.

[0135] Luciferase analysis

[0136] The promoter region was cloned into the luciferase reporter vector. The plasmid sequence was validated by sequencing. The GNMT luciferase reporter plasmid and the glucocorticoid receptor luciferase reporter plasmid have been constructed and used in our previous studies. Luciferase assays were performed using a dual-luciferase reporter analysis system according to the manufacturer's protocol (Promega).

[0137] Diagnostic efficacy verification

[0138] Twenty-seven clinical samples of depression and 29 healthy control samples were collected as training sets. The expression levels of sarcosine, methyl stearate, and LysoPE (16:1(9Z) / 0:0) in exosomes were detected, and ROC curves were plotted. The AUC values, sensitivity, and specificity of sarcosine, methyl stearate, and LysoPE (16:1(9Z) / 0:0) were analyzed to determine 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.

[0139] Twenty-eight clinical samples of depression and 35 healthy control samples were collected as a validation set. The expression levels of sarcosine, methyl stearate, and LysoPE (16:1(9Z) / 0:0) in exosomes were detected, and ROC curves were plotted. The AUC values, sensitivity, and specificity of sarcosine, methyl stearate, and LysoPE (16:1(9Z) / 0:0) were analyzed to determine 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.

[0140] Statistical analysis

[0141] For clinical characteristics, all data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA and, where applicable, the Mann-Whitney U test. For animals, data are expressed as mean ± standard error. A p-value less than 0.05 was considered statistically significant. ROC curves were generated using SPSS 25.0 statistical analysis software.

[0142] II. Results

[0143] Metabolomics analysis of serum exosomes from patients with depression and cortical tissue exosomes from mice with depression

[0144] Metabolic disorders can drive neurophysiological dysfunction, leading to neurodegenerative diseases. To characterize the molecular mechanisms underlying the behavioral phenotypes in depressed mice and patients with depression, we performed metabolomics analysis on exosomes from serum samples of participants (72 MDD patients and 73 HC individuals) and exosomes from prefrontal cortex tissue of adult mice (5 depression model mice and 5 control mice). Bioinformatics analysis revealed that a total of 447 metabolites were quantified in the exosomes from the participants' serum samples. Figure 1 C), 480 differentially metabolites were quantified in exosomes from the prefrontal cortex of adult mice. Figure 1D). Of these metabolites, 229 were shared in exosomes from participant serum samples and exosomes from the prefrontal cortex of adult mice. Figure 1 A). These features are displayed as a heatmap ( Figure 1 B). These data indicate a disordered exosome metabolic profile. Notably, only a few metabolites were significantly different in the depression group compared to the control group. Different exosome metabolites in the two groups were assessed using a two-sided Wilcoxon rank-sum test (MDD patients, 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 the MDD and HC groups. Similarly, Figure 1 F shows the altered levels of 63 named metabolites in mouse prefrontal cortex samples. Notably, a few metabolites (sarcosine, LysoPE (16:1(9Z) / 0:0), methyl stearate) were co-present in the altered metabolites across these different types of biological samples. Compared to the control group, methyl stearate was increased, while sarcosine and LysoPE (16:1(9Z) / 0:0) were decreased. Sarcosine showed the most significant decrease. The results indicate that sarcosine levels in the exosomes of the prefrontal cortex of depressed mice ( Figure 1 K) and serum exosomes of patients with depression ( Figure 1 J) was significantly reduced. To confirm the decreased creatine levels in depressed mice, we first collected cortical exosomes (J) from adult mice. Figure 1 G). Then, we used a highly sensitive and specific method (LC / MS / MS) to accurately quantify the creatine content. The structure of creatine ( Figure 1 H) and molecular fragmentation of sarcosine ( Figure 1 I) showed quantitative results confirming a significant reduction in creatine content in exosomes of the prefrontal cortex.

[0145] Creatine treatment alleviates depressive-like behavior in mice. The antidepressant effect of creatine is related to the AKT / mTOR signaling pathway and regulation of neuronal synaptic membranes.

[0146] Based on the finding of significantly reduced sarcosine levels in prefrontal cortex exosomes, we explored the potential role of sarcosine in CUMS-induced depressive-like behaviors. To investigate the functional role of sarcosine in MDD, we employed two administration methods (intravenous injection and prefrontal cortex-targeted injection). For intravenous sarcosine supplementation, the experimental protocol was as follows: Figure 2 As shown in Figure A. Creatine supplementation alleviated depressive-like behaviors in several established behavioral tests. Specifically, in the open field (OFT) experiment ( Figure 2B), after creatine supplementation, the total distance traveled increased significantly; in the novel environment dietary inhibition experiment (NSFT) ( Figure 2 C), the feeding latency was significantly shortened. Furthermore, in the forced swimming test (FST) ( Figure 2 E), the immobility time was significantly reduced; in the tail suspension test (TST) ( Figure 2 D), immobility time was significantly reduced (p<0.001, p<0.05, p<0.01). For prefrontal cortex targeted sarcosine injection, the experimental protocol is as follows: Figure 2 As shown in M. It was expected that creatine supplementation would also alleviate depressive-like behaviors (p<0.001, p<0.01, p<0.05, p<0.01). Figure 2 N- Figure 2 Q). These results confirm that administration of sarcosine can improve CUMS-induced behavioral impairment in mice, and that prefrontal cortical-targeted injection of sarcosine is a more effective drug delivery method. Sarcosine is an endogenous amino acid and a competitive inhibitor of type I glycine transporter (GlyT1) and N-methyl-D-aspartate receptor (NMDAR) co-agonists. Studies have shown that rapid activation of the mammalian target of rapamycin (mTOR) signaling pathway through stimulation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) is the main mechanism by which the NMDAR antagonist ketamine exerts its rapid antidepressant effect. Therefore, we investigated the role of the mTOR signaling pathway in the antidepressant effect of sarcosine. The results showed that after sarcosine treatment, the levels of p-AKT / AKT and p-mTOR / mTOR proteins significantly increased (p<0.05, p<0.01, ...). Figure 2 F- Figure 2 I, Figure 2 R- Figure 2 U). Furthermore, we examined the expression of neuronal synaptic proteins PSD95, Syn, and SYP, and found that their expression at the synaptic membrane was reduced (U). Figure 2 J- Figure 2 L, Figure 2 These results collectively indicate that the antidepressant-like behavior of creatine is closely related to the AKT / mTOR signaling pathway.

[0147] Corticosterone reduces GNMT gene expression and sarcosine levels through glucocorticoid receptors.

[0148] Since creatine levels are reduced in depressed mice, and creatine supplementation has been found to be effective in combating depression, we hypothesize that pathways affecting creatine synthesis may play an important role in the occurrence and development of depression. It is well known that creatine can be produced in some metabolic processes in the body, and glycine is a precursor to creatine. Figure 3A). Furthermore, GNMT is a multifunctional protein that promotes the conversion of glycine to sarcosine. Therefore, we inferred that GNMT expression was abnormal in depressed mice. Therefore, we specifically detected GNMT expression levels using RT-PCR and Western blot analysis. Moreover, RT-PCR and Western blot results showed that GNMT expression levels were significantly reduced in the prefrontal cortex of depressed mice (A). Figure 3 F). Next, we determined whether GNMT participates in the development of depression by affecting creatine levels. To test our hypothesis, we cultured bone marrow-derived mesenchymal stem cells and then transfected them with a GNMT overexpression plasmid. The results showed that creatine levels in exosomes secreted by mesenchymal stem cells increased after GNMT overexpression. Furthermore, previous studies have shown increased corticosterone levels in depressed mice. Our experimental results confirm this. Figure 3 B). To investigate whether increased corticosterone levels affect GNMT expression, we cultured PC12 cells ( Figure 3 C). Our study showed that small amounts of corticosterone can reduce GNMT expression (C). Figure 3 D、 Figure 3 E). Studies have shown that the androgen receptor (AR) can directly regulate muscle amino acid levels by modulating GNMT transcription, while corticosterone exerts its effect by affecting the glucocorticoid receptor (GR). Next, we will investigate 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 (E). Figure 3 These results collectively indicate that corticosterone directly regulates GNMT expression and affects sarcosine levels through glucocorticoid receptors.

[0149] GNMT plays an important role in the development of depression.

[0150] Given that GNMT overexpression can increase creatine levels, we hypothesized that reducing GNMT expression could induce depression, while GNMT overexpression could alleviate depressive-like behaviors by altering creatine levels. To further determine the functional role of GNMT in CUMS-induced depressive-like behaviors, we bilaterally injected an AAV9 vector to express GNMT recombinase and GFAP, specifically reducing GNMT driven by the human synaptophysin 1 promoter (AAV9-ITR-hsyn-GNMT-GFAP). GFAP expression using the AAV9 vector in the cortex of depressed mice served as a control (AAV9-ITR-hsyn-GFAP). Figure 4 A, Figure 4B). We found that specifically reducing GNMT in neurons can induce depressive-like behavior (B). Figure 4 C- Figure 4 F). We further demonstrated that depressive-like behavior is associated with neuronal synapses. Furthermore, GNMT was specifically overexpressed by the human synaptophysin 1 promoter-driven expression (AAV9-ITR-hsyn-GNMT-GFAP) via bilateral injection of the AAV9 vector expressing GNMT recombinase and GFAP. GFAP expression using the AAV9 vector in the cortex of depressed mice served as a control (AAV9-ITR-hsyn-GFAP). Figure 5 A). We found that GNMT expression in the prefrontal cortex of mice was significantly increased after injection. Figure 5 B). Behavioral results showed that specific overexpression of GNMT in the prefrontal cortex significantly alleviated depressive-like behavior in mice. Specifically, compared with the model group, the total movement distance in the open field test (OFT) was significantly increased ( Figure 5 H), the food latency time in the novel environment diet suppression test (NSFT) was significantly shortened ( Figure 5 G), in the forced swimming test (FST) Figure 5 J) and the tail suspension test (TST) Figure 5 The immobility time in I) was significantly reduced (p<0.01).

[0151] GNMT is highly expressed primarily in neurons and is involved in the occurrence and development of depression.

[0152] Furthermore, we detected that GNMT was primarily expressed in neurons, but almost entirely absent in microglia. To further demonstrate the role of GNMT in prefrontal cortical neurons, we injected adenovirus AAV9-GNMT-CMV-FLEX-MCS-EGFP into the prefrontal cortex of CamkIIa-cre mice to specifically overexpress GNMT in neurons. We also examined GNMT expression in the cortical tissue, finding that GNMT expression increased significantly after expression (p<0.01). Figure 5 C- Figure 5 F). Following administration, depressive-like behaviors in camkIIa-cre mice subjected to chronic, unpredictable, mild stress were alleviated. Specifically, in the open field test (OFT), the total distance traveled was significantly increased (F). Figure 5 H), in the novelty inhibition swimming test (NSFT), the feeding latency was significantly shortened ( Figure 5 G), in the forced swimming test (FST) Figure 5 J) and the tail suspension test (TST) Figure 5Immobility time was significantly reduced in I) (p<0.05). These results indicate that specific overexpression of GNMT in prefrontal cortex neurons can significantly alleviate depressive-like behavior. We investigated the roles of the AKT / mTOR signaling pathway and synaptic proteins in the antidepressant effect of GNMT. The results showed that the antidepressant behavior of the GNMT gene is closely related to the AKT / mTOR signaling pathway ( Figure 5 K- Figure 5 O).

[0153] Exosomes from bone marrow mesenchymal stem cells carry the sarcosine gene and alleviate depressive-like symptoms in mice.

[0154] To further determine the functional role of GNMT in CUMS-induced depressed mice, we specifically investigated the association between exosomes derived from bone marrow mesenchymal stem cells and their antidepressant effect. The animal experimental procedure is as follows: Figure 6 As shown in Figure I, we isolated and cultured bone marrow mesenchymal stem cells (BMSCs). Figure 6 A). Specific surface antigens of bone marrow mesenchymal stem cells (BMSCs) were detected by flow cytometry. Results showed that BMSCs were positive for the markers CD90 and CD105, but negative for CD34 (Figure 1). Figure 6 B- Figure 6 D). This indicates that we have isolated mesenchymal stem cells. We extracted and identified exosomes of third-generation BMSCs ( Figure 6 E- Figure 6 G). We injected exosomes into the tail vein of depressed mice and found that the exosomes could cross the blood-brain barrier and enter the prefrontal cortex (G). Figure 6 H). Furthermore, behavioral testing results showed that exosomes derived from bone marrow mesenchymal stem cells could alleviate depressive-like behaviors. Specifically, after injection of exosomes derived from bone marrow mesenchymal stem cells, total movement distance increased, and the food exploration time in the NSFT test was shortened (H). Figure 6 L). Compared with the model group, the forced swimming test (FST) after drug administration ( Figure 6 N) and the tail suspension test (TST) Figure 6 The immobility time in mesenchymal stem cells (M) was significantly reduced (p<0.05). The glycosaminoglycine-N-methyltransferase (GNMT) gene encodes a protein that promotes the conversion of glycine to sarcosine. To test this hypothesis, we cultured mesenchymal stem cells and downregulated GNMT ( Figure 6 K). We found that downregulating GNMT significantly reduced sarcosine levels in mesenchymal stem cell exosomes (K). Figure 6 Further research showed that exosomes derived from mesenchymal stem cells with downregulated GNMT were ineffective in alleviating 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, p<0.01). Figure 6 L- Figure 6 These data further indicate 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 synaptic membranes (O). Figure 6 P- Figure 6 S).

[0155] HPA axis hyperfunction affects sarcosine expression in the glycine metabolic pathway.

[0156] Hyperfunction of the HPA axis is a common neurobiological manifestation of depression and a prominent feature of the condition. Hyperfunction of the HPA axis leads to increased cortisol secretion. Our results also show that cortisol expression in the prefrontal cortex and serum of depressed mice is higher than in healthy controls. Cortisol affects neuronal function by binding to glucocorticoid receptors and membrane receptors, but its function remains unclear. Metabolomics studies have shown that the occurrence of depression is closely related to the glycine metabolic pathway. Creatine expression in the prefrontal cortex and serum of depressed mice is lower than in healthy controls. We hypothesize that hyperfunction of the HPA axis leads to increased cortisol levels, which, through binding to membrane receptor proteins, affects the glycine metabolic pathway, reducing creatine levels and exacerbating depression. GNMT is an important enzyme in the glycine metabolic pathway. In cell experiments, we found that downregulation of GNMT reduces creatine levels, while overexpression of GNMT increases the amount of creatine secreted by cells. Furthermore, our results show that GNMT expression levels are low in the prefrontal cortex of depressed mice. Dual-luciferase reporter assays showed that cortisol and glucocorticoid receptor binding reduces GNMT expression. In summary, our results indicate that hyperfunction of the HPA axis leads to increased cortisol levels, induced by GNMT, which interferes with the glycine metabolic pathway, thereby promoting depression by reducing GNMT expression and creatine levels. The underlying mechanisms revealed in this study are as follows: Figure 7 As shown.

[0157] Diagnostic efficacy verification

[0158] like Figure 8 As shown, in the training set, the AUC of methyl stearate is 0.692 ( Figure 8 The AUC of LysoPE(16:1(9Z) / 0:0) is 0.558. Figure 8 B), the AUC of creatine is 0.697 ( Figure 8 C), the AUC of the combination of creatine, methyl stearate and LysoPE (16:1(9Z) / 0:0) was 0.824 ( Figure 8 D).

[0159] like Figure 9 As shown, in the validation set, the AUC of methyl stearate was 0.659 ( Figure 9 The AUC of LysoPE(16:1(9Z) / 0:0) is 0.661 (A). Figure 9 B), the AUC of creatine is 0.804 ( Figure 9 C), the AUC of the combination of creatine, methyl stearate and LysoPE (16:1(9Z) / 0:0) was 0.804 ( Figure 9 D).

[0160] 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.

[0161] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Biomarkers for diagnosing depression, characterized in that, The biomarkers include creatine, methyl stearate, and LysoPE (16:1(9Z) / 0:0).

2. The biomarker according to claim 1, characterized in that, The biomarker is derived from exosomes; 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 cortex cells, and neurons; Preferably, the cells are selected from prefrontal cortex cells; Preferably, the tissue comprises prefrontal cortex tissue.

3. The use of a reagent for detecting the biomarker according to any one of claims 1-2 in the preparation of products for diagnosing depression; Preferably, the depression includes major depressive disorder, unipolar depression, treatment-resistant depression, resistant depression, anxiety depression, bipolar depression, and dysphoric mood. Preferably, the depression is selected from major depressive disorder and anxiety depression.

4. The application according to claim 3, characterized in that, The product described is used in quantitative analysis methods. Preferably, the quantitative analysis method is selected from UPLC-MS / MS.

5. The application according to any one of claims 3-4, characterized in that, The product includes a reagent kit.

6. A product for diagnosing depression, characterized in that, The product contains a reagent for detecting the expression level of the biomarker according to any one of claims 1-2; Preferably, the product includes a reagent kit.

7. The product according to claim 6, characterized in that, The kit also contains standards for biomarkers; Preferably, the kit further includes reagents for processing samples.

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

9. The method according to claim 8, characterized in that, The regulators include promoters and inhibitors; Preferably, the promoter includes nucleic acid molecules, carbohydrates, liposomes, small molecule chemicals, antibody drugs, peptides, proteins, and reagents used in gene editing; Preferably, the promoter is selected from nucleic acid molecules; Preferably, the inhibitor includes reagents used in gene editing, nucleic acid inhibitors, protein inhibitors, or compounds; Preferably, the reagents used in the 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. Preferably, the nucleic acid inhibitor includes siRNA, shRNA, or miRNA; Preferably, the HPA is negatively correlated with the creatine level; Preferably, the corticosterone is negatively correlated with the sarcosine; Preferably, the glucocorticoid receptor is positively correlated with the sarcosine aminotransferase. Preferably, the GNMT is positively correlated with the creatine content; Preferably, the glycine is positively correlated with the sarcosine.

10. Application of HPA / corticosterone / glucocorticoid receptor / GNMT / glycine in the regulation of sarcosine.

Images