Application of synaptic binding protein 11 as marker in preparation of products for diagnosis, auxiliary diagnosis, screening or auxiliary screening of schizophrenia

Abstract

The invention relates to the technical field of biology, in particular to application of synaptic binding protein 11 serving as a marker to preparation of products for diagnosis, auxiliary diagnosis, screening or auxiliary screening of schizophrenia and application of the synaptic binding protein 11 and / or a dopamine D2 receptor serving as a core target to treatment of schizophrenia. The application of the synaptic binding protein 11 or the transcriptional product mRNA or the mRNA fragment of the synaptic binding protein 11 as the marker in any one of the following items: 1) application in preparation of products for diagnosis or auxiliary diagnosis of schizophrenia of human or non-human mammals; 2) application in preparation of products for screening or auxiliary screening of schizophrenia of human or non-human mammals; 3) application in diagnosis or auxiliary diagnosis of schizophrenia of human or non-human mammals to be detected; and 4) application in screening or auxiliary screening of schizophrenia of human or non-human mammals to be detected. Meanwhile, the invention also provides application of treating three symptoms of schizophrenia through a brain region specific targeting dopamine D2 receptor. Researches find that synaptic binding protein 11 or a transcription product mRNA or mRNA fragment thereof can be used as a brand new functional schizophrenia diagnosis marker, and a chemical molecule or gene therapy drug targeting synaptic binding protein 11 or a dopamine D2 receptor can effectively improve three symptoms of schizophrenia.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to the use of synaptotagmin 11 as a marker in the preparation of products for diagnosis, auxiliary diagnosis, screening, and auxiliary screening of schizophrenia, and the use of synaptotagmin 11 and / or dopamine D2 receptors as core targets in the treatment of schizophrenia. Background Art

[0002] Schizophrenia (SCZ) is a serious mental illness, and about 1% of the world's population is affected by SCZ in their lifetime. The clinical manifestations of SCZ patients are divided into three categories: positive symptoms (auditory and visual hallucinations, delusions); negative symptoms (lack of will, emotional indifference, social disorders, and anhedonia) and cognitive dysfunction. Late adolescence and early adulthood are the peak periods of SCZ incidence, and patients' negative symptoms and cognitive impairment appear before positive symptoms. At present, there are no effective diagnostic markers and detection methods for SCZ. Therefore, the diagnosis of SCZ is a huge challenge for clinicians, which leads to the inability to carry out corresponding treatment in a timely manner.

[0003] There are currently many neurotransmitter hypotheses about the pathogenesis of SCZ, such as the glutamate hypothesis, the GABA hypothesis, the dopamine (DA) hypothesis, etc., but these neurotransmitter hypotheses fail to fully explain all the symptom phenotypes of schizophrenia. Among them, the DA hypothesis originated from the increase in the content of dopamine and its metabolites in the brain of patients with early schizophrenia. At the same time, studies have found that excessive use of amphetamine synthetic drugs can cause increased dopamine secretion and SCZ-like positive symptoms such as hallucinations and auditory hallucinations. At present, most of the clinical treatment drugs for SCZ target dopamine D2 receptors (D2R). However, these drugs targeting D2R are only effective in treating positive symptoms, and have almost no therapeutic effect on negative symptoms and cognitive impairment, and may even further aggravate negative symptoms and cognitive impairment. Therefore, the consensus reached in the field is that increased dopamine secretion in the striatum leads to positive symptoms of SCZ, while decreased dopamine secretion in the limbic system is the main cause of negative symptoms of SCZ. However, it is still unclear whether and how abnormal dopamine secretion is involved in the pathological process of schizophrenia. The pathological mechanism of SCZ negative symptoms and its clinical treatment strategy remain the most critical scientific issues and major medical problems in this field.

[0004] Large-scale genetic studies have revealed a large number of SCZ-related genes, however, few of these genes have been proven to cause SCZ alone. Currently, there are no biochemical markers that have been successfully applied to the clinical diagnosis of SCZ in the existing technology. The clinical diagnosis of SCZ and its differential diagnosis from other mental illnesses remain the most important and critical medical problems in this field. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a use of synaptotagmin 11 as a marker in the preparation of products for diagnosis, auxiliary diagnosis, screening, and auxiliary screening of schizophrenia, and the use of synaptotagmin 11 and / or dopamine D2 receptor as core targets for the treatment of schizophrenia.

[0006] The present study found that synaptotagmin-11 (Syt11) inhibits vesicle recycling and sustainable secretion of neurotransmitters by inhibiting endocytosis; at the same time, it was found that the abnormal reduction of synaptotagmin-11 in dopaminergic neurons leads to abnormal increase in dopamine secretion, which is an important mechanism leading to the pathological process of negative symptoms of schizophrenia. The transcription data of mRNA in the dorsolateral prefrontal cortex of schizophrenia patients in the three human brain banks of the Lieber Institute for Brain Development (LIBD), the CommonMindConsortium (CMC), and the Human Brain Collection Core (HBCC) were analyzed, and it was found that the transcription level of synaptotagmin 11 in schizophrenia patients was significantly reduced; and in the blood of schizophrenia patients, the level of synaptotagmin 11 protein was significantly reduced; after anti-schizophrenia treatment, the synaptotagmin 11 protein in the blood was significantly increased; knocking down synaptotagmin 11 in the midbrain dopamine neurons of mice, the mice showed all the behavioral manifestations related to the three major clinical symptoms of schizophrenia, positive symptoms, negative symptoms and cognitive impairment, indicating that the downregulation of synaptotagmin 11 expression is the core mechanism mediating the pathological process of SCZ. Therefore, the present invention not only clarifies that synaptotagmin 11 will be a single clinical diagnostic marker that effectively indicates the pathological process of SCZ, but also a key drug target for the clinical treatment of SCZ.

[0007] Therefore, the present invention proposes the following technical solution:

[0008] The embodiments of the present invention disclose the use of synaptotagmin 11 or its transcription product mRNA or mRNA fragment as a marker in any of the following:

[0009] 1) Use in the preparation of products for diagnosing or assisting in the diagnosis of schizophrenia in humans or non-human mammals;

[0010] 2) Application in the preparation of products for screening or assisting screening of schizophrenia in humans or non-human mammals;

[0011] 3) Use in the diagnosis or auxiliary diagnosis of schizophrenia in humans or non-human mammals;

[0012] 4) Application in screening or assisting screening of schizophrenia in humans or non-human mammals.

[0013] The embodiments of the present invention disclose the use of a product for detecting synaptotagmin 11 or its transcription product mRNA or mRNA fragment in any of the following aspects:

[0014] 1) Use in the preparation of products for diagnosing or assisting in the diagnosis of schizophrenia in humans or non-human mammals;

[0015] 2) Application in the preparation of products for screening or assisting screening of schizophrenia in humans or non-human mammals;

[0016] 3) Use in the diagnosis or auxiliary diagnosis of schizophrenia in humans or non-human mammals;

[0017] 4) Application in screening or assisting screening of schizophrenia in humans or non-human mammals.

[0018] In some embodiments, the product for detecting synaptotagmin 11 or its transcription product mRNA or mRNA fragment includes a product for detecting the expression level of synaptotagmin 11 or its transcription product mRNA or mRNA fragment in a test sample.

[0019] In some embodiments, the sample to be tested is brain tissue or body fluid of a human or non-human mammal; in some embodiments, the body fluid is selected from at least one of blood, cerebrospinal fluid and urine.

[0020] In some embodiments, the product for detecting synaptotagmin 11 or its transcription product mRNA or mRNA fragment comprises a kit, a reagent, a test strip, a primer, a probe or a chip;

[0021] In some embodiments, the reagent includes: an antibody, a protein and / or a polypeptide that specifically binds to synaptotagmin 11; or a reagent that detects mRNA or mRNA fragments of synaptotagmin 11 transcription products.

[0022] In some embodiments, the antibody, protein and / or polypeptide is modified with isotopes, horseradish peroxidase, colloidal gold, fluorescent probes, etc., or is unmodified.

[0023] In some embodiments, the reagent for detecting the mRNA or mRNA fragment of the synaptotagmin 11 transcription product includes a PCR reaction buffer or a nucleic acid molecule hybridization buffer; in some embodiments, it includes a synaptotagmin 11 gene fragment or a specific sequence fragment of synaptotagmin 11 mRNA, or a molecular probe containing these specific sequence fragments.

[0024] In some embodiments, the schizophrenia symptoms can be positive symptoms, negative symptoms, and / or cognitive impairment of schizophrenia;

[0025] In some embodiments, the non-human mammal comprises an animal model of schizophrenia;

[0026] In some embodiments, the human is a schizophrenia patient, a suspected schizophrenia patient, a person at high risk of schizophrenia, or a direct relative of a schizophrenia patient.

[0027] The embodiments of the present invention disclose a product for diagnosing, assisting in the diagnosis, screening or assisting in the screening of schizophrenia in humans or non-human mammals, including a product for detecting the expression level of synaptotagmin 11 or its transcription product mRNA or mRNA fragment in a sample to be tested.

[0028] In some embodiments, the product for detecting synaptotagmin 11 or its transcription product mRNA or mRNA fragment includes a kit, a reagent, a test strip, a primer, a probe or a chip.

[0029] In some embodiments, the sample to be tested is brain tissue or body fluid of a human or non-human mammal; optionally, the body fluid is selected from at least one of blood, cerebrospinal fluid and urine.

[0030] In some embodiments, the reagents include: antibodies, proteins and / or polypeptides that specifically bind to synaptotagmin 11; or reagents for detecting mRNA or mRNA fragments of synaptotagmin 11 transcription products; optionally, the antibodies, proteins and / or polypeptides are modified with isotopes, horseradish peroxidase, colloidal gold, fluorescent probes, etc., or are unmodified; optionally, the reagents for detecting mRNA or mRNA fragments of synaptotagmin 11 transcription products include PCR reaction buffer or nucleic acid molecule hybridization buffer; optionally, include synaptotagmin 11 gene fragments or specific sequence fragments of synaptotagmin 11 mRNA, or molecular probes containing these specific sequence fragments;

[0031] In some embodiments, the schizophrenia symptoms are manifested as positive symptoms, negative symptoms, and / or cognitive impairment of schizophrenia;

[0032] In some embodiments, the non-human mammal comprises an animal model of schizophrenia;

[0033] In some embodiments, the human is a schizophrenia patient, a suspected schizophrenia patient, a person at high risk of schizophrenia, or a direct relative of a schizophrenia patient.

[0034] Embodiments of the present invention disclose a system for diagnosing, assisting in diagnosis, screening or assisting in screening schizophrenia in humans or non-human mammals, comprising:

[0035] An obtaining device, wherein the obtaining device is used to obtain the expression level of synaptotagmin 11 or its transcription product mRNA or mRNA fragment in the sample to be tested;

[0036] A judgment device, the judgment device is connected to the acquisition device or is wirelessly connected to the judgment device, and the judgment device is used to diagnose or assist in diagnosis, screening or assist in screening whether a human or non-human mammal suffers from schizophrenia based on the expression level of synaptotagmin 11 or its transcription product mRNA or mRNA fragment in the sample to be tested.

[0037] In some embodiments, the system for diagnosing, assisting in diagnosis, screening or assisting in screening schizophrenia in humans or non-human mammals, compared with the expression level of synaptotagmin 11 or its transcription product mRNA or mRNA fragment in the sample of a normal human or normal non-human mammal, is lower than the expression level of synaptotagmin 11 in the sample of the human or non-human mammal to be tested, thereby judging that the human or non-human mammal to be tested is suffering from schizophrenia.

[0038] In some embodiments, a non-human mammal model of schizophrenia has a reduced expression level of synaptotagmin 11 in a sample of the non-human mammal model of schizophrenia compared to the expression level of synaptotagmin 11 or its transcript mRNA or mRNA fragment in a sample of a normal non-human mammal.

[0039] In some embodiments, a method of screening for a drug for treating schizophrenia comprises:

[0040] The candidate drugs are used in the non-human mammalian model of schizophrenia; the drugs that up-regulate the expression of synaptotagmin 11 or its transcription product mRNA or mRNA fragment or enhance its activity, or the drugs that up-regulate the expression of dopamine D2 receptor or its transcription product mRNA or mRNA fragment or enhance its activity, or the candidate drugs that alleviate schizophrenia are screened as the drugs for treating schizophrenia.

[0041] The embodiment of the present invention discloses a method for preparing a drug for preventing and / or treating schizophrenia, comprising: preparing the desired drug using protein and / or expressed cDNA or cDNA fragments.

[0042] In some embodiments, the protein is an expression product of synaptotagmin 11 gene cDNA or cDNA fragment, or an expression product of dopamine D2 receptor gene cDNA or cDNA fragment;

[0043] In some embodiments, the cDNA or cDNA fragment is derived from the synaptotagmin 11 gene, or the dopamine D2 receptor gene.

[0044] In some embodiments, the cDNA or cDNA fragment is unmodified, or contains at least one modified nucleotide or nucleotide analog; wherein the modification site of the modified nucleotide or nucleotide analog may be on the sugar group, main chain or base of the ribonucleotide;

[0045] In some embodiments, the nucleotide analogs include backbone-modified ribonucleotides with phosphorothioate groups, inosine, or tritylated bases.

[0046] In some embodiments, a method for preparing a drug for preventing and / or treating schizophrenia comprises the steps of preparing the desired drug using a virus that can promote the expression of synaptotagmin 11 and / or dopamine D2 receptor cDNA or cDNA fragments;

[0047] In some embodiments, the virus promoting the expression of synaptotagmin 11 and / or dopamine D2 receptor cDNA or cDNA fragments includes at least one of lentivirus, adenovirus, and adeno-associated virus.

[0048] In some embodiments, the cDNA or cDNA fragment expression virus is prepared by the following method: inserting synaptotagmin 11 and / or dopamine D2 receptor cDNA or cDNA fragment into the multiple cloning site region of an expression vector to construct a cDNA or cDNA fragment expression plasmid; co-transfecting the cDNA or cDNA fragment expression plasmid with an auxiliary plasmid into cells to produce virus particles, which are then recovered and concentrated to obtain the virus.

[0049] The embodiments of the present invention disclose a drug for preventing and / or treating schizophrenia, including synaptotagmin 11, synaptotagmin 11 cDNA or cDNA fragment; and / or, dopamine D2 receptor, dopamine D2 receptor cDNA or cDNA fragment; and / or, an expression virus containing synaptotagmin 11, synaptotagmin 11 cDNA or cDNA fragment gene; and / or, an expression virus containing dopamine D2 receptor, dopamine D2 receptor cDNA or cDNA fragment gene;

[0050] In some embodiments, the active ingredient of the drug includes a substance that can promote the expression of synaptotagmin 11 and / or dopamine D2 receptor, and / or a substance that increases the physiological activity of synaptotagmin 11 and / or dopamine D2 receptor.

[0051] In some embodiments, the virus comprises at least one of an adenovirus, a lentivirus, and an adeno-associated virus vector.

[0052] In some embodiments, a substance that can promote the expression of synaptotagmin 11 and / or dopamine D2 receptor genes or increase the content or activity of synaptotagmin 11 and / or dopamine D2 receptor, or synaptotagmin 11, synaptotagmin 11 cDNA or cDNA fragment, dopamine D2 receptor, dopamine D2 receptor cDNA or cDNA fragment, is used to prepare a drug or product having at least one of the following effects (1)-(5):

[0053] (1) Treatment of schizophrenia;

[0054] (2) Treat or alleviate the positive symptoms of schizophrenia;

[0055] (3) Treat or alleviate negative symptoms of schizophrenia;

[0056] (4) Treat or alleviate cognitive impairment in schizophrenia;

[0057] (5) Products that inhibit the excitation of dopamine neurons or the secretion of dopamine.

[0058] In some embodiments, the method of promoting the expression of synaptotagmin 11 and / or dopamine D2 receptor genes or increasing the content or activity of synaptotagmin 11 and / or dopamine D2 receptors is: enhancing the transcription and translation levels of synaptotagmin 11 and / or dopamine D2 receptor genes by gene editing, or increasing the stability of synaptotagmin 11 and / or dopamine D2 receptor mRNA, or increasing the stability of synaptotagmin 11 and / or dopamine D2 receptor proteins, or reducing the degradation level of synaptotagmin 11 and / or dopamine D2 receptor proteins.

[0059] In some embodiments, the synaptotagmin 11 protein and / or dopamine D2 receptor, or 11 cDNA or cDNA fragment, and / or dopamine D2 receptor cDNA or cDNA fragment, are delivered to the brain regions affected by schizophrenia in the following manners:

[0060] (1) injecting virus particles of synaptotagmin 11 protein or 11 cDNA or cDNA fragment into the brain area affected by schizophrenia lesions by stereotaxic injection;

[0061] (2) injecting a plasmid containing synaptotagmin 11 cDNA or cDNA fragment into the brain area affected by schizophrenia by stereotaxic injection, and transferring the plasmid into cells by electroporation;

[0062] (3) stereotaxic injection of plasmids and liposomes containing synaptotagmin 11 cDNA or cDNA fragments into the brain area affected by schizophrenia;

[0063] (4) Treatment of schizophrenia by injecting viral particles containing synaptotagmin 11 cDNA or cDNA fragments through the subarachnoid space;

[0064] In some embodiments, the affected brain regions of the schizophrenia patient include but are not limited to the ventral tegmental area of ​​the midbrain;

[0065] In some embodiments, the target cells for treating schizophrenia patients include but are not limited to dopamine neurons in the ventral tegmental area of ​​the midbrain.

[0066] In some embodiments, the functions of the drug or product include:

[0067] (1) The drug increases the expression level of synaptotagmin 11 and / or dopamine D2 receptor, or synaptotagmin 11 and / or dopamine D2 receptor mRNA;

[0068] (2) the drug improves the stability of synaptotagmin 11 and / or dopamine D2 receptor, or synaptotagmin 11 and / or dopamine D2 receptor mRNA;

[0069] (3) The drug improves the physiological function of synaptotagmin 11 and / or dopamine D2 receptor, or synaptotagmin 11 and / or dopamine D2 receptor mRNA;

[0070] (4) the drug increases the translation level of synaptotagmin 11 and / or dopamine D2 receptor, or synaptotagmin 11 and / or dopamine D2 receptor mRNA on the ribosome;

[0071] (5) Used to treat schizophrenia.

[0072] In some embodiments, the method of increasing the expression level of synaptotagmin 11 and / or dopamine D2 receptor is any one or more of the following:

[0073] (1) increasing the transcription level of synaptotagmin 11 and / or dopamine D2 receptor encoding genes;

[0074] (2) increasing the translation level of synaptotagmin 11 and / or dopamine D2 receptor protein;

[0075] (3) reduce the degradation rate of synaptotagmin 11 and / or dopamine D2 receptor;

[0076] In some embodiments, the drug improves the physiological function of synaptotagmin 11 and / or dopamine D2 receptor by inhibiting dopamine secretion by inhibiting exocytosis, inhibiting endocytosis, inhibiting vesicle uncoating, inhibiting vesicle recycling, etc.

[0077] In some embodiments, the schizophrenia patient has a family history of schizophrenia, or is a sporadic schizophrenia patient.

[0078] The embodiment of the present invention discloses a use of a dopamine receptor agonist or inhibitor in preparing a drug for treating schizophrenia, characterized in that it comprises:

[0079] In some embodiments, the use of an agonist of a dopamine neuron D2 receptor in the preparation of a drug for treating the three major symptoms of schizophrenia, optionally, the agonist of a dopamine neuron D2 receptor is one or more of quinpirole, bromocriptine, cabergoline, pergolide, apo, pramipexole and ropinirole; optionally, the agonist of a dopamine neuron D2 receptor is used in the preparation of a drug for local injection into the ventral tegmental area of ​​the midbrain;

[0080] In some embodiments, the inhibitor / antagonist of dopamine neuron D2 receptor is used in the preparation of a drug for treating negative symptoms of schizophrenia; optionally, the inhibitor / antagonist of dopamine neuron D2 receptor is one or more of haloperidol, risperidone, olanzapine, chlorpromazine, trifluoperazine hydrochloride, metoclopramide, doxilone or itopride; optionally, the inhibitor / antagonist of dopamine neuron D2 receptor is used in the preparation of a drug for local injection of the prefrontal cortex.

[0081] The technical solution of the present invention has the following advantages:

[0082] 1. Use of the synaptotagmin 1 or its transcription product mRNA or mRNA fragment provided by the present invention as a marker in any of the following items: 1) use in the preparation of products for diagnosing or assisting in the diagnosis of schizophrenia in humans or non-human mammals; 2) use in the preparation of products for screening or assisting in the screening of schizophrenia in humans or non-human mammals; 3) use in the diagnosis or assisting in the diagnosis of schizophrenia in humans or non-human mammals to be tested; 4) use in screening or assisting in the screening of schizophrenia in humans or non-human mammals to be tested; The present invention has found that synaptotagmin 11 or its transcription product mRNA or mRNA fragment can be used as a new functional early diagnostic marker for schizophrenia. The present invention finds that DA neuron-specific synaptotagmin 11 knockout mice (Syt11 cKO) show significant social disorders in three-box social tests, social novelty, open field social and home-cage social behaviors, and also have three typical clinical manifestations of SCZ, such as obvious increased activity, abnormal startle reflex, and cognitive function defects; the present invention also finds that knocking out synaptotagmin 11 in DA neurons in the early developmental stage will lead to SCZ-related behavioral changes in mice, and this behavioral change can persist into adulthood; the present invention also proves that excessive secretion of dopamine in the VTA to mPFC pathway during adolescence will lead to the phenotype of social disorder associated with schizophrenia in mice, and this social disorder is mediated by D2R. Therefore, it can be concluded that the decreased expression of synaptotagmin 11 or its transcript mRNA or mRNA fragments is the most important sign of schizophrenia, especially the early negative symptoms of SCZ. The clinical verification of the present invention shows that the expression level of synaptotagmin 11 or its transcript mRNA or mRNA fragments is the most ideal clinical diagnostic marker for SCZ so far, especially an important basis for the clinical diagnosis of early SCZ, with AUC, sensitivity and specificity of 0.91, 81% and 92%, respectively, indicating that the expression level of synaptotagmin 11 or its transcript mRNA or mRNA fragments has extremely high accuracy and reliability in diagnosing schizophrenia, especially negative schizophrenia.

[0083] In addition, the present invention also reveals the key role of synaptotagmin 11 in the pathogenesis of schizophrenia, providing a theoretical basis for the development of new treatment strategies. Through further research, we found that the loss or dysfunction of synaptotagmin 11 is closely related to the abnormal activity of the dopamine system, which provides a new target for the treatment of schizophrenia.

[0084] In terms of clinical application, the diagnostic markers provided by the present invention not only help to detect schizophrenia patients at an early stage, but also provide patients with personalized treatment plans. For example, by monitoring the expression level of synaptotagmin 11 or its transcripts, doctors can more accurately assess the patient's disease progression and treatment effect, thereby adjusting the treatment strategy and improving the pertinence and effectiveness of the treatment.

[0085] In addition, the present invention also provides a new idea for the prevention of schizophrenia. By early screening and monitoring the expression level of synaptotagmin 11 in high-risk populations, potential risks of disease can be discovered in time, so that preventive measures can be taken to reduce the incidence of schizophrenia. This not only helps to improve the quality of life of patients, but also reduces the burden on social medical resources.

[0086] At the same time, the present invention also provides a new therapeutic target and therapeutic strategy for the clinical treatment of schizophrenia. By specifically intervening in the expression of synaptotagmin 11 or dopamine D2 receptors in the ventral tegmental area of ​​the midbrain, or using dopamine D2 receptor agonists to inhibit dopamine neuron excitation and dopamine secretion levels, the behavioral manifestations related to schizophrenia can be improved comprehensively and long-term, providing an effective way for the clinical cure of schizophrenia, especially its negative symptoms and cognitive impairment.

[0087] In summary, the present invention has important application value in the diagnosis, treatment and prevention of schizophrenia. Through in-depth research on synaptotagmin 11 and its mechanism of action in schizophrenia, we are expected to develop more effective diagnostic tools and therapeutic drugs, bringing new hope to schizophrenia patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0089] Figure 1 The expression levels of synaptotagmin 11 in brain tissue and peripheral blood of SCZ patients and healthy people in Example 1, as well as the expression changes of synaptotagmin 11 after treatment. Figures A and B show the expression levels of synaptotagmin 11 mRNA in brain tissue of healthy people (HC) and SCZ patients in three brain banks, LIBD, CMC and HBCC; Figures C and D are representative Western blot graphs and statistical graphs of the expression levels of synaptotagmin 11 in peripheral blood of healthy people (HC) and SCZ patients; Figure 1E is the expression level of synaptotagmin 11 in the peripheral blood of healthy people (HC) and SCZ patients before and after treatment; Figure FH is the symptom improvement score, synaptotagmin 11 expression level score of SCZ before and after treatment with three drugs, and the correlation analysis between the two scores.

[0090] Figure 2 The immunofluorescence images of dopaminergic neurons in the VTA brain region of synaptotagmin 11 cKO mice and the recording images of DA release in the nucleus accumbens (NAc) in Example 2. A in the figure is a schematic diagram of the construction of synaptotagmin 11 cKO transgenic mice; B and C in the figure are immunofluorescence images of dopaminergic neurons and tyrosine hydroxylase (TH) and synaptotagmin 11 in the VTA brain region of the control group and Syt11 cKO mice; D in the figure is a schematic diagram and principle diagram of carbon fiber electrode recording in NAc; E in the figure is a representative image and statistical analysis of dopamine release in control mice and Syt11 cKO mice.

[0091] Figure 3 The method for evaluating the social ability of 6-8 week old Syt11 cKO mice in Example 3 is as follows. Figure 3 AC are heat maps and statistical graphs of social preference of control mice and Syt11 cKO mice in three-box experiments; Figure 3 Middle (DE) are schematic diagrams and statistical graphs of the social contact experiment of control mice and Syt11 cKO mice; Figure 3 Middle FH are heat maps and statistical graphs of social novelty in the three-box experiment for control mice and Syt11 cKO mice; Figure 3 Center I is a schematic diagram and statistical graph of the home cage social behavior experiment of control mice and Syt11 cKO mice.

[0092] Figure 4 The social ability of 3-month-old adult Syt11 cKO mice was evaluated in Example 4. Figure 4 Center AC is the heat map and statistical graph of social preference of control mice and Syt11 cKO mice in the three-box experiment; Figure 4 Middle D is the heat map and statistical graph of the social contact experiment of control mice and Syt11 cKO mice; Figure 4 Middle EG is the heat map and statistical graph of social novelty in the three-box experiment for control mice and Syt11 cKO mice; Figure 4 (H) is a statistical graph of the social behavior experiment in the home cage of control mice and Syt11 cKO mice.

[0093] Figure 5 The behavior analysis of Syt11 cKO mice in Example 5 shows SCZ-like behavior instead of autism. Figure 5 Middle A is the movement trajectory diagram of the open field test for 6-8 week old control mice and Syt11 cKO mice; Figure 5Middle B is the statistical analysis of the total movement distance and maximum speed of control mice and Syt11cKO mice; Figure 5 Middle C is a schematic diagram of the T-maze for testing the short-term memory ability of mice; Figure 5 Middle D is a statistical analysis chart of short-term memory in experimental mice aged 6-8 weeks and 3-4 months; Figure 5 Middle E is a statistical analysis chart of the startle reflex experiment of experimental mice in the PPI test for 6-8 weeks; Figure 5 Middle F is a statistical analysis chart of the startle reflex test of 3-4 month old experimental mice; Figure 5 Middle G is the bead-burying experiment to test the repetitive stereotyped behavior of 6-8 week old experimental mice; Figure 5 Figure H in the figure is the repetitive stereotyped behavior detected by grooming behavior in 6-8 week old experimental mice.

[0094] Figure 6 This is a statistical diagram for analyzing the effect of synaptotagmin 11 on vesicle circulation and dopamine secretion in Example 6. Figure 6 A in the middle is a schematic diagram, representative graphs and statistical graphs of dopamine release recorded by carbon fiber electrodes in the NAc of control mice and Syt11 cKO mice; Figure 6 Middle B is a schematic diagram of the injection of optogenetics and TH-Cre mixed viruses into the VTA of 3-week-old Syt11 flox / flox mice, as well as a representative diagram of dopamine signals generated by light stimulation and paired pulse ratio statistics recorded on NAc brain slices; Figure 6 Middle C is a schematic diagram of VTA injection of chemical genetics and TH-Cre mixed virus in 3-week-old Syt11 flox / flox mice; Figure 6 Middle D is the immunofluorescence image after injection of mixed virus in VTA; Figure 6 E in the middle and F in the 6th middle are Figure 6 (C) Schematic diagram, representative graphs and statistical analysis graphs of dopamine secretion recorded in mouse mPFC after injection of mixed virus.

[0095] Figure 7 The mechanism of enhanced excitability of dopamine neurons in Syt11-cKO mice in Example 7. Figure 7 Middle A is a schematic diagram of the VTA brain region of Syt11-cKO mice injected with TH-Cre-EGFP virus at day 0 after birth; Figure 7 Middle B is the action potential discharge diagram and statistical diagram of dopamine neurons in control mice and cKO mice; Figure 7 C in the middle and D in the middle are representative images and statistical graphs of action potential discharges of dopamine neurons in control mice and cKO mice in response to Haloperidol; Figure 7 Figure 7E and Figure 7F are representative and statistical graphs of action potential discharges of dopamine neurons in control mice and cKO mice in response to Quinpirole drug; Figure 7Middle G: Immunofluorescence images and statistical graphs of D2R expression after knockdown of synaptotagmin 11 in SH-SY5Y cell line; Figure 7 H in the middle is a schematic diagram of the relationship between D2R, DA secretion and vesicle circulation after synaptotagmin 11 knockout.

[0096] Figure 8 This is Example 8 to verify that enhanced DA transmission leads to negative symptoms of SCZ. Figure 8 Middle A and Figure 8 Middle B is a schematic diagram and immunofluorescence image of dopamine neurons specifically infected by injection of TH-Cre and Cre-dependent chemically activated mixed virus into the VTA of 3-week-old Syt11 flox / flox mice; Figure 8 Middle C and Figure 8 D in the middle is a carbon fiber recording photo of mPFC slices of control mice and Syt11-cKO mice under CNO stimulation, a typical dopamine secretion graph, and a dopamine secretion amplitude statistical graph; Figure 8 Middle E is a schematic diagram of the injection of TH-Cre and Cre-dependent chemical activation mixed virus into the VTA of 3-week-old C57 mice to specifically infect dopamine neurons, and a statistical graph of the social behavior of mice detected after administration of CNO; Figure 8 Middle F is a schematic diagram of the VTA injection of TH-Cre and Cre-dependent chemically activated mixed viruses to specifically infect dopamine neurons in 3-week-old C57 mice, as well as a schematic diagram of the long-term activation of dopamine neurons in childhood (intraperitoneal injection of CNO at p7, once every other day, a total of 4 injections, and behavioral testing starting at 6 weeks of age) and a statistical graph of social behavior analysis.

[0097] Fig. 9 Example 9 is that excessive secretion of dopamine in the VTA-mPFC circuit leads to social deficits in mice. Fig. 9 A in the middle is a schematic diagram of the VTA brain region of 3-week-old C57 mice injected with TH-Cre and Cre-dependent light-activated mixed virus to specifically infect dopamine neurons and their main projections to downstream brain regions NAc and mPFC; Fig. 9 Middle B is a representative picture of action potential recording of dopamine neurons under light stimulation (blue laser with a wavelength of 473nm and a frequency of 20Hz) in the VTA brain region after virus infection, as well as carbon fiber recording under light stimulation in the mPFC and NAc brain regions; Fig. 9 Middle C and Fig. 9 Middle D shows a schematic diagram of the implantation of optical fibers in the mPFC and NAc at 4 weeks of age after injection of mixed viruses into the VTA and NAc brain regions of 3-week-old C57 mice, as well as the experimental flow chart of social behavior testing of adolescent mice (6-8 weeks of age) and social behavior statistics.

[0098] Fig.10The figure shows the changes in the cell structure of the mPFC of Syt11 cKO mice in Example 10. Fig.10 Middle A (upper) and Fig.10 Middle B is a representative microscopic image and statistical graph of MAP2-positive neurons in the mPFC of adult Syt11 cKO mice and control mice; Fig.10 Middle A (lower) and Fig.10 (C) Representative microscopic images and statistical graphs of TH-positive neurons in the mPFC of adult Syt11 cKO mice and control mice.

[0099] Fig.11 The figure shows the changes in electrophysiological function of mPFC neurons in Syt11 cKO mice in Example 10. Fig.11 Middle A: Representative electrophysiological recordings of sEPSCs in mPFC neurons of adult Syt11-cKO mice and statistical graphs of amplitude and frequency; Fig.11 The left figure in middle B is a schematic diagram of injecting TH-Cre-EGFP or TH-EGFP virus into the VTA of newborn Syt11-flox / flox mice (P0) to obtain Syt11-cKO or control mice. The middle and right figures are representative electrophysiological recordings and statistical graphs of spontaneous action potentials (APs) of mPFC neurons in adult (3-month-old) Syt11-cKO mice and control mice; Fig.11 The left figure in center C is a schematic diagram and experimental flow chart of co-injection of TH-Cre and DIO-hM3Dq-mCherry / DIO-mCherry viruses into the VTA of newborn C57 mice (P0). The middle and right figures are representative electrophysiological recordings and statistical graphs of spontaneous APs in mPFC neurons of adult (3-month-old) hM3Dq-expressing mice after continuous CNO treatment and control mice.

[0100] Fig.12 1 shows the changes in transcriptome expression in mPFC of Syt11 cKO mice in Example 10. Fig.12 Middle A heatmap shows the gene expression profiles determined by whole-genome RNA sequencing (RNA-seq) of the mPFC in Syt11 cKO mice (n=4) and controls (n=3), with rows representing differentially expressed genes (DEGs) and columns representing transcriptome animal individual maps; Fig.12 The volcano plot in middle B shows the gene expression profiles in the mPFC of Syt11 cKO mice and control mice. The x-axis represents the log2 fold change (FC) between the two groups.

[0101] Fig.13 It is a scatter plot of the expression of synaptotagmin 11 protein in the peripheral blood of the schizophrenia patient group and the normal healthy population group in Example 11.

[0102] Fig.14This is the operating characteristic curve fitted based on the expression level of synaptotagmin 11 protein of the subjects in Example 11.

[0103] Fig.15 In Example 12, local injection of D2 receptor antagonist haloperidol into the VTA of the midbrain of adolescent schizophrenia model mice and local injection of D2 receptor agonist quinpirol into the mPFC can treat social disorders in schizophrenia;

[0104] Fig.16 As described in Example 12, local injection of D2 receptor antagonist haloperidol into the VTA of the midbrain of adult schizophrenia model mice and local injection of D2 receptor agonist quinpirol into the mPFC can treat social disorders in schizophrenia. DETAILED DESCRIPTION

[0105] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0106] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0107] Experimental animals and experimental methods involved in the embodiments of the present invention:

[0108] 1. Experimental Animals

[0109] The following mouse strains are mainly used in the present invention: transgenic mice (The Jackson Laboratory, USA) include floxedSyt11-null mice (B6.129-Syt11tm1Sud / J) and DAT-Cre mice (B6.SJL-Slc6a3tm1.1(cre)Bkmn / J). In order to obtain mice with dopaminergic neuron-specific knockout of synaptotagmin 11, floxed Syt11-null mice are hybridized with DAT-Cre mice, and hybrid mice Syt11 cKO mice with homozygous dopaminergic neuron-specific knockout of synaptotagmin 11 are selected.

[0110] 2. Genotyping of hybrid mice

[0111] DNA extraction: Cut about 0.5 cm from the tail of the mouse, put the tail into a numbered 1.5ml centrifuge tube and cover it. Add 0.5ml lysis solution and 50μl proteinase K storage solution to each tube and seal the lid tightly. Place the centrifuge tube in a water bath at 55℃ overnight (more than 12 hours). The next day, centrifuge the sample at 13000rpm for 10 minutes. Pipette 200ul supernatant into a 1.5ml centrifuge tube, add 1ml anhydrous ethanol, cover the lid and shake gently, and flocculent precipitation can be seen. 13000rpm, 15min, discard the supernatant. Let it air dry at room temperature for 10-15min. Add 30-50μl sterile water to each tube, store at -20℃, or directly perform PCR or hybridization experiments.

[0112] Lysis solution formula: 100mmol Tris-Hcl (PH8.0); 25mmol EDTA (PH 8.0); 500mmol NaCl; 1% SDS.

[0113] PCR hybridization experiment:

[0114] PCR reaction system 25ul system: Tiangen Biotechnology Co., Ltd. 2×Taq PCR Mix (KT201) 2*MIX (12.5ul); Primer 1 (0.5ul) / Primer 2 (0.5ul); DNA template (3ul); dd H2O2 (8.5ul);

[0115] DAT-Cre primers: 1, 5-GTT GAT GAG GGT GGAGTT GGT C-3, SEQ ID NO. 1;

[0116] 2. 5-GCC GCATAACCAGTG AAA CAG C-3, SEQ ID NO.2;

[0117] 3. 5-TCC ATA GCC AAT CTC TCC AGT C-3, SEQ ID NO.3;

[0118] PCR cycle: 94°C for 5 min; 94°C for 1 min, 60°C for 30 s, 72°C for 1 min and 10 s (repeat steps 2-4 for 35 cycles); 72°C for 10 min; maintain at 4°C.

[0119] floxed Syt11-null primer: 1. 5-AAT CTC AGC ACT CAG GAG TCA G-3, SEQ ID NO.4; 2. 5-CTC TTG CTT ACT GAT TGG CAG C-3, SEQ ID NO.5;

[0120] PCR cycle: 94°C for 3 min; 94°C for 30 s, 68°C for 1 min, 72°C for 1 min (repeat steps 2-4 for 35 cycles); 2°C for 2 min; maintain at 10°C.

[0121] Electrophoresis

[0122] Prepare gel: Prepare 1-3% agarose gel according to the target band, dissolve 1-3g agarose in 100ml TAE buffer, heat in a microwave until the agarose melts, add 10ul nucleic acid dye (EB substitute; Bio-Time D0143), pour into the electrophoresis template, insert the comb, and use after condensation.

[0123] Sample addition: Add an appropriate amount of DNA to the agarose gel. It is recommended to add 10ul to the small wells and 20ul to the large wells. Select a suitable DNA marker according to the target band size and add it to the gel.

[0124] Gel running: Set the electrophoresis instrument to 120V and run for 20-30 minutes.

[0125] Imaging: Use gel imaging system to observe and take pictures. (DAT-Cre: wild-type band is 400bp; transgenic homozygous mouse band is 700bp; floxed Syt11-null: wild-type band is 361bp; transgenic homozygous mouse band is 500bp); edit gel image and generate genotype identification result report.

[0126] 3. Peripheral Blood Protein Extraction

[0127] Take 5 μl of human blood sample and add it to 95 μL IP cell lysate. After homogenization, centrifuge at 12000g and 4℃ for 15 min. Take the supernatant, add 1 / 4 volume of supernatant 5X SDS-PAGE loading buffer, boil at 95-100℃ for 10 min to obtain protein samples for Western blot analysis.

[0128] 4. Western blot

[0129] The obtained protein solution was subjected to electrophoresis and transferred to a PVDF membrane, blocked with a TBST solution containing 5% skim milk powder at room temperature for 1-1.5 h, washed three times, incubated in a 2% BSA TBST solution containing Syt11 primary antibody (SYSY, 270003) at 4°C overnight, washed five times with TBST and then incubated with the corresponding secondary antibody (111-035-003 rabbit antibody, 115-035-003 mouse antibody, Jackson ImmunoResearch) at room temperature for 1.5-2 h, washed five times with TBTS and scanned in a Clinx Chemicapture imaging system.

[0130] 5. Immunohistochemistry

[0131] After intraperitoneal anesthesia, the animals were perfused with normal saline and 4% paraformaldehyde respectively. The brain was removed and immersed in the same 4% paraformaldehyde for 24 hours and then precipitated for 3 days (4°C). 30 μm thick sections were cut from each tissue and routinely stained using a two-step method. The tissue sections were incubated with the corresponding receptor antibody serum and the secondary antibody overnight. The images were visualized using a laser confocal microscope and the immunohistochemical staining results were observed.

[0132] 6. Carbon fiber electrochemical recording

[0133] Electrochemical recordings were performed using a 200 μm long, 7 μm diameter carbon fiber microelectrode (CFE). The exposed tip of the carbon fiber electrode was inserted into the brain slice at a 30° angle, 300 μm away from the stimulating electrode. The voltage of the recording electrode was stabilized at 780 mV using an EPC9 / 2 amplifier and Pulse software (HEKA Electronic, Lambrecht / Pfalz, Germany). The stimulating electrode was a bipolar platinum wire electrode (diameter ∼150 μm, Plastics One Inc., USA), which was controlled by a Master-8 stimulator (AMPI, Israel). The duration of each pulse was 0.2 ms, the amplitude was 0.6 mA, and the time interval between each two stimulations was 3 min. The amperometric signal (Iamp) was collected at a frequency of 3.13 kHz and low-frequency filtered at 100 Hz. Off-line analysis was performed using Igor software (WaveMetrix).

[0134] 7. In vivo and in vitro optogenetic and chemogenetic experiments

[0135] For in vivo optogenetics experiments, after the virus is injected into the corresponding brain area, the mouse virus is expressed for one week before the fiber is buried in the nucleus accumbens (NAc: front and back +1.25, left and right + / -0.75, up and down -4.3) or medial prefrontal cortex (mPFC: front and back +2.8, left and right + / -0.5, up and down -1.7), and the mouse recovers for one week and is stroked and adapted for five minutes every day. This operation will be performed for three consecutive days. After the mouse is docked with the optical fiber and the ceramic ferrule of the head, a behavioral detection device is placed, and a laser is used to activate the photosensitive protein that has been integrated and detect behavior. The laser is preferably a yellow light stimulator (596nm laser) and a blue light (488nm laser) laser purchased from Qian Aoxingke, using a laser with a wavelength of 488nm / 596nm. For in vitro optogenetics, the inventor also uses a laser with a wavelength of 488nm / 596nm in the corresponding brain area to activate the photosensitive protein that has been integrated, and record the response of its neurons. Before the experiment, a power meter (Thorlabs) was used to adjust the laser intensity. In this experiment, the laser intensity was approximately 10-15 milliwatts (mW).

[0136] For in vivo chemical genetics experiments, the experimental methods and operations are basically the same as those for optogenetics. Clozapine (CNO) is administered intraperitoneally before the experiment and behavioral tests are performed. For in vitro chemical genetics experiments, after the virus is injected into the corresponding brain area, in vitro brain slices are prepared after the mouse virus expression is appropriate, CNO is administered in the recording area, and the response to activated nerves is recorded.

[0137] 8. Virus Injection

[0138] After the mice were anesthetized with 3% isoflurane, the hair on the top of the skull was removed and fixed on a stereotaxic injection instrument. The skin on the top of the skull was cut to expose the cross seam. The three-dimensional parameters were adjusted and a small animal skull drill was used to drill a hole to expose the choroid plexus. 500 nl of virus (titer: 2-5×10 12 vg / ml), slowly lower the glass electrode to the ventral tegmentalarea (VTA), the coordinates of VTA are: Anterior-posterior (AP) -3.4mm, Middle-lateral (ML) + / -0.5mm, Dorsal-ventral (DV) -4.4mm, inject the virus (100nl / min) through the microelectrode injection pump, stay for 15min after the injection, then slowly turn the regulator of the positioning instrument to pull out the electrode (to prevent the virus from being taken out with the electrode), use a cotton swab dipped in saline to keep the skull moist, suture the mouse scalp, and apply erythromycin ointment on the suture. The whole process needs to use a heating blanket (KEL-2000, Nanjing) to maintain the body temperature of the mouse at 37℃. After the operation, it is still placed on the heating blanket and returned to the mouse cage after it wakes up.

[0139] The virus stereotaxic injection of P0 (newborn mice) mice was performed according to the mouse brain atlas. P0 mice were placed in ice for 5 minutes for ice anesthesia. The anesthetized P0 mice were fixed to the brain stereotaxic instrument (Narishige, Japan) with ear bars glued with plastic foam. A box was placed under the mouse, which was filled with the bedding of the mouse cage. 150nl of virus suspension (titer: 2-5×10 12 vg / ml), slowly inserted into VTA (AP+0.1mm, DV-3.7mm relative to Lambda), injected virus with a microelectrode injection pump (100nl / min), stayed for 3min after injection and then pulled out, wiped excess blood from the wound with alcohol, placed the bedding in the cage on the heating blanket, put the mouse on the heating blanket, and put it back into the cage after the mouse woke up and returned to normal body temperature.

[0140] 9. Three-Box Social Behavior Experiment

[0141] The three boxes consist of three rectangular boxes (20cm×40cm×23cm) with a passage in the middle connecting the three boxes. The experiment includes three stages. The first stage is the adaptation stage. Two empty restraint cages are placed in the boxes on both sides of the three boxes. The experimental mice can freely explore the three boxes. The whole process lasts for 10 minutes. The second stage is the social stage. Stranger mouse (Stranger Mouse1, M1) or fake mouse (Fake mouse, F) are randomly placed in the restraint cages on both sides of the three boxes. Then the contact time of the experimental mice with M1 and fake mouse during the 10 minutes of free exploration is recorded to detect the social behavior of mice; the third stage is the social novelty stage. The model mouse in the restraint cage of the second stage is replaced with the second stranger mouse (Stranger Mouse 2, M2). Then the sniffing contact time of the experimental mice with M1 and M2 during the 10 minutes of free exploration of the three boxes is recorded. The sniffing contact time (Sniffering time) of the mice with the stranger mice, the social index (Social index), and the total social time (Total social time) of social novelty are recorded throughout the process.

[0142] 10. Open field exercise test

[0143] The mouse motor ability test was conducted in an open field (40×40×40 cm). The mice were placed in the open field and the Anymaze recording and tracking system was used to record the mice's free exploration of the open field for 15 minutes. The movement distance and maximum speed of the mice were used as indicators of the mouse's motor ability.

[0144] 11. Social Approach Behavior Experiment

[0145] A restraint cage was placed in the center of the open field, and a strange mouse was placed in the restraint cage. Then the experimental mouse was placed in the open field, and the Anymaze recording and tracking system was used to record the experimental mouse's approach behavior to the strange mouse in real time. The total duration was recorded for 30 minutes, and the contact time of the experimental mouse with the strange mouse in the restraint cage was counted.

[0146] 12. Home cage social behavior experiment

[0147] Before the social behavior experiment in the home cage, the experimental mice need to be kept in a single cage 5 days in advance. The bedding will not be changed during the single cage. During the experiment, the water and food in the home cage will be removed and the home cage will be placed in the open field. A stranger mouse of the same age / sex is placed in the home cage, and the Anymaze recording and tracking system is used to record in real time for 30 minutes, and the time and number of active contact between the experimental mouse and the stranger mouse are counted.

[0148] 13. Buried Bead Test

[0149] Before the bead burying test, the bedding in the mouse cage needs to be replaced with corn cob bedding 4 days in advance. During the experiment, a clean mouse cage filled with corn cob bedding (about 5 cm deep) is used. After the corn cob bedding is compacted and flattened, clean glass marbles are neatly arranged in a 3×6 grid on the corn cob bedding. The experimental mouse is placed in the cage with glass marbles and allowed to explore freely for 15 minutes. The marbles buried in the corn cob bedding are counted. Marbles that are buried are considered buried if 2 / 3 of the volume of the entire marble is buried by the corn cob bedding.

[0150] 14. Grooming test

[0151] The mice were placed in an open field and recorded for 30 minutes. The number of times or duration of grooming behaviors were recorded to assess the stereotyped repetitive behaviors of the mice. The number of times the mice rubbed their faces, scratched / rubbed their heads, ears, or combed their entire bodies was counted.

[0152] 15. Food-induced T-maze

[0153] Before the T-maze, mice need to be restricted in advance, and the restriction standard is no more than 10% of body weight. The T-maze consists of a starting arm and two target arms (each arm is 30 cm long, 10 cm wide, and 20 cm high), and the maze is T-shaped. During the training phase, one side arm of the T-maze is randomly defined as the target arm, and 2g of ordinary feed particles are placed in the target arm. The mice are allowed to adapt to the starting arm for 90 seconds, and the baffle is removed after 90 seconds. After the test mice enter the food target arm, the food target arm is closed. After the mice start eating, wait for 30 seconds, take the mice out and put them back into the cage. Repeat the test after 10 minutes. During the test, no food is put into the previous food target arm. The time when the mouse enters the food target arm from the starting arm is recorded to evaluate the short-term memory of the mouse. In order to prevent mice from identifying the target arm by smell, the entire maze needs to be thoroughly wiped with 75% ethanol before and after each experiment.

[0154] 16. Hearing test

[0155] The auditory test uses the acoustic startle response (ASR) to test. The entire sound stimulation-induced startle reflex test is conducted in a soundproof box. The mouse is placed in a transparent rectangular organic glass test box with a gap. The box is inserted into the center of the gravity sensing base in the startle reflex box. There is a piezoelectric accelerometer under the base, which can convert the pressure changes caused by the animal's movement into electrical signals, and then the amplifier amplifies the signal. In order to ensure the stability of the experiment, the mouse is placed in the test box and then placed in the startle reflex box for adaptation two days in advance. Each adaptation lasts for 30 minutes for two days. The background noise (65dB) is turned on throughout the adaptation process. During the test, the background noise (65dB) is turned on throughout the process. After the mouse has adapted for 5 minutes, it is given sound stimulation of different decibels. One sound stimulation is a trial, and the interval time is 10 to 30s (random). The sound stimulation changes gradually from 70 to 125dB, with a gradient of 5dB, and each stimulation time is 20ms. Each decibel is given 10 times of stimulation, and different decibels appear in a random manner.

[0156] 17. Prepulse Inhibition (PPI)

[0157] Before the PPI test, the mice need to be placed in the test box and then in the startle reflex test box for two days of adaptation, 30 minutes each time, and the background white noise (65dB) is turned on throughout the adaptation process. On the third day of the formal experiment, the 65dB background white noise is turned on throughout the experiment. The test mice are placed in the test box and then in the startle reflex test box to adapt for 5 minutes (there is only 65dB background noise during the whole process). This stage is the adaptation period, and then enter the Block I stage, that is, the basic startle reflex of the mice is tested with 120dB sound stimulation (120dB, 40ms). One stimulus is a trial, and the setting of Block I is: 120dB 40ms, 5 trials, and an interval of 15s (inter-trial interval) between each trial. After Block I, we enter Block II. There are 5 stimulation modes in Block II (no sound stimulation; 72, 76 or 80dB prepulse stimulation + 120dB startle stimulation; 120dB startle stimulation), a total of 50 trials, 5 kinds of stimulation each appeared 10 times, the number of appearances was random, and the interval time was 10 to 30s (random). The startle response was the maximum response displayed within the 65ms sampling window after each group of stimulation. PPI% = (1-prepulse startle reflex / 120dB startle reflex) × 100%.

[0158] 18. Implanted catheter medication

[0159] Schizophrenia was treated by local injection of D2 receptor antagonist haloperidol into the VTA and D2 receptor agonist quinpirol into the mPFC. Drugs were administered through bilateral stainless steel inner cannulas (RWD, China) controlled by a microinjection pump. D2 agonist quinpirol (QP, 1 μg / μl, 0.2 μl / side), D2 antagonist haloperidol (Halo, 50 μM, 0.2 μl / side) or saline was infused into the desired local brain area at a rate of 100 nl / min. All drugs were prepared in buffered saline. The inner cannulas were removed 2 minutes after infusion, and behavioral tests were performed 10 minutes after drug administration.

[0160] 19. Whole-cell patch clamp

[0161] Spontaneous excitatory postsynaptic currents (sEPSCs and mEPSCs) of the mPFC were recorded by whole-cell voltage clamp recording using an EPC10 / 2 amplifier controlled by Patchmaster software (HEKA Elektronik, Germany). VTA DA neurons and mPFC cortical neurons were identified by cells labeled with EGFP / mCherry, and their AP discharges were recorded by current clamp in the whole-cell configuration. The seal during patch clamp recording was monitored by series conductance and membrane conductance. The signal was sampled at 20 kHz, low-pass filtered at 2.9 kHz, and the electrode resistance was controlled at 3-5 MΩ. CNO (5 μM, 500 nl), D2R agonist quinpirole (50 nM, 500 nl), and D2R antagonist haloperidol (50 nM, 500 nl) were administered by gravity perfusion system (MPS-2, Yibo Inc., Wuhan).

[0162] 20. Cell Culture and Transfection

[0163] Human neuroblastoma SH-SY5Y cells ( CRL-2266TM) were cultured in Dulbecco's modified Eagle's medium-F12 supplemented with 10% fetal bovine serum at 37°C and 5% CO2. When the cells reached ~75% confluency, SH-SY5Y cells were infected with Syt11-shRNA or control AAV virus (Shanghai Obio Technology Co., Ltd.) to knock down the expression of synaptotagmin 11. Immunoblotting experiments were performed three days after transfection.

[0164] 21. Statistical analysis

[0165] Data are presented as mean ± SEM. Data were analyzed using Prism (GraphPad). Pairwise comparisons were performed using a two-tailed Student's T test. Other data were analyzed using one-way or two-way ANOVA with correction for multiple comparisons. In all cases, p < 0.05 was considered significant. *p < 0.05, **p < 0.01, ***p < 0.001.

[0166] Example 1. Synaptotagmin 11 is a novel risk factor for schizophrenia

[0167] Refer to the above experimental operations 1-20 to implement this embodiment

[0168] By searching the expression of synaptotagmin 11 in the dorsolateral prefrontal cortex (dlPFC) of healthy people and SCZ people in the LIBD brain bank, it was found that the expression of synaptotagmin 11 in SCZ people was significantly lower than that in normal people ( Figure 1 A), and also showed significant differences in the other two brain banks (CMC and HBCC) ( Figure 1 Plasma samples from normal people and SCZ patients were collected and Western blot analysis revealed that the expression of synaptotagmin 11 in the plasma of SCZ patients was also significantly lower than that in normal people ( Figure 1 C and D). After SCZ patients were treated with haloperidol, risperidone or olanzapine, the changes in the content of synaptotagmin 11 in plasma samples were detected again. At this time, the expression of synaptotagmin 11 was significantly increased ( Figure 1 At the same time, the symptom change scores and the change scores of synaptotagmin 11 content before and after the three different drugs for treating SCZ were evaluated. The results showed that the score of haloperidol was significantly higher than that of the other two drugs ( Figure 1 F, G), and the synaptotagmin 11 change score after haloperidol treatment was positively correlated with the symptom change score ( Figure 1 Middle H).

[0169] The above results indicate that synaptotagmin 11 is associated with SCZ.

[0170] Example 2. Increased dopamine release in the NAc brain region of Syt11 cKO mice

[0171] By immunohistochemistry, control mice and Syt11 cKO mice ( Figure 2In center A, immunofluorescence staining of the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) of floxed Syt11-null mice hybridized with DAT-Cre mice to obtain homozygous synaptotagmin 11 knockout mice was performed, and synaptotagmin 11 and tyrosine hydroxylase (TH) were co-stained. The results showed that synaptotagmin 11 was completely knocked out in the midbrain dopamine neurons of Syt11 cKO mice ( Figure 2 (B, C) Carbon fiber electrodes were used to record dopamine release in the nucleus accumbens (NAc) slices of Syt11 cKO mice ( Figure 2 The results showed that the dopamine secretion in the NAc brain region of Syt11 cKO mice under electrical stimulation was significantly higher than that in the control group (DAT-Cre mice) ( Figure 2 Middle E), indicating that knocking out synaptotagmin 11 increases DA release.

[0172] Example 3. Syt11 cKO mice show social impairment

[0173] Refer to the above experimental operations 1-20 to implement this embodiment

[0174] 1. A three-box social experiment was used to observe the social behavior of 6-8 week-old adolescent Syt11 cKO mice, divided into 19 control mice (DAT-Cre mice) and 21 Syt11 cKO mice. In the social phase, a stranger mouse (Mouse 1, M1) was placed in the restraint cage of one side of the three boxes, and a fake mouse (F) was placed in the other side ( Figure 3 The results showed that the control mice (DAT-Cre) showed obvious social preference, and the contact time with the stranger mouse was much longer than that with the mold mouse, while the active sniffing contact time of Syt11 cKO mice to M1 was significantly reduced compared with the control mice ( Figure 3 Middle B), by analyzing the social preference index of the two groups of mice, it was found that the social preference index of Syt11 cKO mice was significantly lower than that of the control group ( Figure 3 Then the mold mouse was replaced with another new stranger mouse (Mouse 2, M2) ( Figure 3 F), this stage of the experiment is called the three-box social novelty test. In this stage, the control mice actively contacted M2 for a much longer time than M1, while the Syt11 cKO mice actively contacted M1 and M2 for a significantly lower time than the control mice ( Figure 3 Middle G), the total social preference index of Syt11 cKO mice was significantly lower than that of the control group ( Figure 3 Middle H).

[0175] 2. A social contact experiment was used to detect the social behavior of 6-8 week-old adolescent Syt11 cKO mice in an unfamiliar environment. The mice were divided into control mice (DAT-Cre mice) and Syt11 cKO mice, with 18 mice in each group. The unfamiliar mice were placed in a restraint cage in the middle of the open field, and the social preference of the two groups of mice towards the unfamiliar mice was tested ( Figure 3 (D) Consistent with the results of the three-box social preference test, Syt11 cKO mice showed significantly reduced active contact and sniffing time toward their own kind compared to control mice ( Figure 3 Middle E).

[0176] 3. Social Behavior in Living Cages The social behavior of 6-8 week old adolescent mice was tested. They were divided into 10 control mice (DAT-Cre mice) and 13 Syt11 cKO mice. Each mouse was housed in a single cage for 5 days. On the 6th day, the food and water boxes in the living cage were removed, and the unfamiliar mice were placed in the living cage of the experimental mice. The time and frequency of active socialization of the experimental mice with their peers in a familiar environment were observed. The time and frequency of contact between Syt11 cKO mice and their peers were significantly lower than those of control mice ( Figure 3 Middle I).

[0177] The above results show that Syt11 cKO mice showed significantly reduced contact time with stranger mice compared with control mice, indicating that Syt11 cKO mice showed social disorders in adolescence.

[0178] Example 4. Social deficits in Syt11 cKO mice persist into adulthood

[0179] Corresponding behavioral tests were performed on adult (3-4 months old) Syt11 cKO and control mice (DAT-Cre) using the same method as in Example 3. The results showed that in the three-box social behavior ( Figure 4 A), divided into 13 control mice and 8 Syt11 cKO mice. The adult control mice still showed obvious social tendencies, as shown by the significantly higher contact time with stranger mice than with mold mice ( Figure 4 In contrast, the time of contact with stranger mice by adult Syt11 cKO mice was not significantly different from that with model mice ( Figure 4 In the social novelty stage ( Figure 4 Middle E), divided into 13 control mice and 8 Syt11 cKO mice, adult Syt11 cKO mice spent less time with new and old mice than control mice, thus showing a significant decrease in the total time of social contact ( Figure 4In the social contact experiment, there were 8 control mice and 7 Syt11 cKO mice. The adult Syt11 cKO mice showed a significant decrease in social contact time compared with the control mice, and the difference became larger as time went by ( Figure 4 In the social experiment of the home cage, there were 12 control mice and 11 Syt11 cKO mice. The active social contact time of adult Syt11 cKO mice with stranger mice was still significantly lower than that of control mice with stranger mice ( Figure 4 Middle H).

[0180] The above results indicate that Syt11 cKO mice not only show obvious social defects during adolescence, but also persist into adulthood.

[0181] Example 5. Other SCZ behavioral phenotypes of Syt11 cKO mice

[0182] Refer to the above experimental operations 1-20 to implement this embodiment

[0183] 1. The open field test was used to detect the motor ability of 6-8 week old Syt11 cKO mice ( Figure 5 A), divided into 13 control mice (DAT-Cre mice) and 12 Syt11 cKO mice. The results showed that Syt11 cKO mice showed an overexcited state during adolescence, which was manifested by a significant increase in movement distance ( Figure 5 B), and their maximum speed was no different from that of the control group mice ( Figure 5 Middle B), showing that Syt11 cKO mice have increased early motor ability.

[0184] 2. The short-term memory ability of Syt11 cKO mice aged 6-8 weeks and 3-4 months was tested by T-maze. There were 10 control mice (DAT-Cre mice) and 12 Syt11 cKO mice aged 6-8 weeks; there were 11 control mice (DAT-Cre mice) and 12 Syt11 cKO mice aged 3-4 months. In the food-induced T-maze, food was placed on one side of the T-maze, and the experimental mice whose weight was controlled were placed in the starting arm of the T-maze. After the mice adapted, the barrier between the starting arm and the target arm was removed, and the experimental mice were allowed to explore freely. When the experimental mice found the food arm, the food arm was closed, and it was taken out 30 seconds after the experimental mice had eaten. This was to detect the effect of synaptotagmin 11 knockout in dopamine neurons on the spatial memory of mice ( Figure 5 During the test phase, it was found that the time taken by Syt11cKO mice in adolescence (6-8 weeks) to find the food arm did not change significantly compared with control mice ( Figure 5The time taken by adult Syt11 cKO mice to find the food arm was significantly increased compared with that of control mice ( Figure 5 Middle D, right), indicating a significant decline in short-term memory ability.

[0185] 3. Auditory startle reflex detection and PPI test were performed on adolescent Syt11 cKO mice. There were 13 control mice (DAT-Cre mice) and 13 Syt11 cKO mice aged 6-8 weeks; 23 control mice (DAT-Cre mice) and 16 Syt11 cKO mice aged 3-4 months. There was no difference in startle reflex between 6-8 week old control mice and Syt11 cKO mice to 70-90dB sound stimulation, indicating that there was no abnormality in the auditory system of the two mice ( Figure 5 In the prepulse inhibition experiment, 72dB, 76dB and 80dB prepulse stimulations were given respectively. Under the prompt of three prepulse stimulations, the control mice showed a large decrease in the amplitude of the startle reflex caused by the prepulse stimulation in the 120dB startle reflex, while the adolescent Syt11 cKO mice showed a smaller decrease in the amplitude of the startle reflex caused by the prepulse stimulation than the control mice, showing a prepulse inhibition defect ( Figure 5 Similarly, the auditory startle reflex (120 dB, 40 ms) was tested in 3-4 month old adult Syt11 cKO mice. There was no significant difference in the startle reflex between control mice and Syt11 cKO mice, indicating that the mice had normal hearing ( Figure 5 In contrast, in 3-4 month old adult Syt11 cKO mice, the magnitude of the startle reflex reduction under prepulse cueing was reduced compared with the control group, and they also showed a prepulse inhibition defect ( Figure 5 center F, right).

[0186] 4. In the bead-burying test, there were 9 control mice (DAT-Cre mice) and 9 Syt11 cKO mice aged 6-8 weeks. In the bead-burying test, the glass beads placed at fixed intervals are unfamiliar and potentially threatening objects to mice. The mice's cleaning behavior of these glass beads reflects the mice's anxiety-like phenotype and stereotyped repetitive behavior. In the bead-burying test, there was almost no difference in the buried glass beads between Syt11 cKO mice and control mice ( Figure 5 In the grooming experiment, there were 12 3-4 month old control mice (DAT-Cre mice) and 10 Syt11 cKO mice. The grooming experiment results showed that there was no difference in the grooming time of Syt11 cKO mice compared with control mice ( Figure 5 Middle (H), the above two experiments showed that Syt11 cKO mice had no obvious stereotyped repetitive behaviors.

[0187] Example 6. Deletion of synaptotagmin 11 leads to increased dopamine release

[0188] Refer to the above experimental operations 1-20 to implement this embodiment

[0189] Micro carbon fiber electrodes were used to record dopamine secretion in the NAc, a brain area downstream of the VTA. The results showed that the amount of dopamine released by adult Syt11 cKO mice aged 3-4 months was significantly higher than that of the control group (DAT-Cre mice) of the same age ( Figure 6 (A) A mixed virus of rAAV-TH-Cre-WPRE-hGH pA and pAAV-EF1a-DIO-ChR2(H134R)-mCherry purchased from Privy Technology was injected into the VTA of 3-week-old Syt11 flox / flox mice and C57 mice (titer of each was 2-5×10 12 vg / ml, injection volume 500nl) After 3 weeks of viral expression, paired pulses were given to continuously stimulate neurons in NAc slices. The results showed that the paired pulse ratio of Syt11 cKO mice was significantly higher than that of the control group ( Figure 6 At the same time, the mixed viruses of rAAV-TH-Cre-WPRE-hGH pA and rAAV-EF1α-DIO-hM3D(Gq)-mCherry-WPREs purchased from Shumi Technology were injected into the VTA brain region of 3-week-old Syt11 flox / flox mice and C57 mice (the titers of the two were 2-5×10 12 vg / ml, injection volume 500nl)( Figure 6 C), immunofluorescence was used to detect the infection efficiency of the mixed virus in the VTA, and it was found that most dopamine neurons were infected with Gq virus ( Figure 6 Middle D), a carbon fiber electrode was placed in the projection area of ​​fluorescent dopamine neurons in the mPFC, and CNO was used to activate downstream dopamine secretion ( Figure 6 Middle E), the results showed that the amplitude of dopamine secretion in the mPFC of mice with synaptotagmin 11 knockout in dopamine neurons increased significantly compared with control mice under CNO activation ( Figure 6 Middle F). The above results indicate that knockout of synaptotagmin 11 leads to increased secretion of the dopamine system.

[0190] Example 7. Synaptotagmin 11 deficiency leads to D2R-mediated DA neuron hyperexcitability

[0191] The VTA brain regions of Syt11 loxp mice at day 0 were injected with TH-Cre-EGFP / TH-EGFP virus (purchased from Shumi Technology Co., Ltd., virus titer 2-5×10 12vg / ml, injection volume 200nl), and the dopamine neurons infected by virus on midbrain slices of Syt11-p0-cKO mice (Syt11-cKO mice p0) were electrophysiologically detected by patch clamp technique ( Figure 7 The results showed that the action potential firing frequency of dopamine neurons in Syt11-p0-cKO mice with synaptotagmin 11 knocked out was significantly higher than that in control mice (Syt11 loxp mice injected with TH-EGFP virus) ( Figure 7 Middle B). Using SCZ treatment drugs Haloperidol and Quinpirole on mPFC brain slices can significantly reduce the firing frequency of dopamine neuron action potentials ( Figure 7 In SH-SY5Y cell line (transfected with AAV-shSyt11 virus, purchased from Heyuan Biotechnology, titer: 2-5×10 12 vg / ml, volume: 2ul / well), and immunofluorescence was used to detect the expression of D2R on the surface of SH-SY5Y cells. It was found that the fluorescence value of D2R on the surface of SH-SY5Y cells with synaptotagmin 11 knockdown was significantly lower than that of the control cells without transfection ( Figure 7 Middle G). The results showed that the loss of synaptotagmin 11 led to increased vesicle recycling capacity and increased dopamine release, while also leading to a decrease in the number of D2Rs, further promoting dopamine release ( Figure 7 Middle H).

[0192] Example 8. Excessive release of dopamine is key to mediating negative symptoms in SCZ

[0193] The VTA of 3-week-old Syt11 flox / flox mice and C57 mice were injected with a mixed virus of rAAV-TH-Cre-WPRE-hGH pA and rAAV-EF1α-DIO-hM3D(Gq)-mCherry-WPREs purchased from Primi Technology (the titers of both were 2-5×10 12 vg / ml, injection volume 500nl)( Figure 8 Middle A), immunofluorescence was used to detect the infection efficiency of the mixed virus in the VTA, and it was found that most dopamine neurons were infected with Gq virus ( Figure 8 Middle B). In the mPFC, a carbon fiber electrode was placed in the projection area of ​​fluorescent dopamine neurons, and CNO was used to activate the secretion of downstream dopamine. The mPFC-specific dopamine secretion was recorded in combination with the carbon fiber electrode ( Figure 8 Middle C). The dopamine secretion amplitude of the mPFC brain region of mice with synaptotagmin 11 knockout in dopamine neurons was significantly increased under CNO activation compared with that of the mPFC brain region of control mice ( Figure 8 (middle D).

[0194] Using chemical genetics, the VTA of 3-week-old C57 mice was injected with a mixture of rAAV-TH-Cre-WPRE-hGH pA (TH-Cre) and rAAV-EF1α-DIO-hM3D(Gq)-mCherry-WPREs or rAAV-EF1α-DIO-mCherry-WPREs (DIO-hM3D-mCherry / DIO-mCherry) purchased from Shumi Technology (the titers of the two were 2-5×10 12 vg / ml, the injection volume of the mixed virus was 500nl). When the mice were 6 weeks old, behavioral experiments were started. Before each experiment, CNO (0.5mg / kg) was injected intraperitoneally to activate dopamine neurons, and behavioral tests were started half an hour later ( Figure 8 The results showed that after intraperitoneal injection of CNO, the control mice spent much more time with the stranger mice than with the mold mice in social behavior ( Figure 8 E), while the time of contact with stranger mice (hM3D) after injection of CNO to activate dopamine neurons in real time was no different from that of contact with mold mice. In addition, the time of contact with stranger mice M1 by chemically activated mice (hM3D) was significantly shorter than that of control mice, and their social tendency was significantly lower than that of control mice ( Figure 8 Middle E).

[0195] Using chemical genetics, the VTA of newborn (p0) C57 mice was injected with a mixture of rAAV-TH-Cre-WPRE-hGH pA (TH-Cre) and rAAV-EF1α-DIO-hM3D(Gq)-mCherry-WPREs or rAAV-EF1α-DIO-mCherry-WPREs (DIO-hM3D-mCherry / DIO-mCherry) purchased from Shumi Technology (the titers of the two were 2-5×10 12 vg / ml, the mixed virus injection volume is 200nl) at P7 (the seventh day after birth), the virus-injected mice were intraperitoneally injected with CNO (0.5mg / kg) to activate dopamine neurons, and then injected once every other day for 4 consecutive times. When the mice were 6 weeks old, the corresponding social behavior tests were performed ( Figure 8 Middle F). It was found that mice with chronic dopamine neuron activation in childhood (hM3D) had significantly less contact time with stranger mice in a three-box social behavior experiment in adolescence than control mice, but no difference in contact time with mold mice compared to control mice, and showed a significant decrease in social tendency ( Figure 8 Middle F).

[0196] Example 9. Excessive dopamine secretion in the VTA-mPFC circuit mediates negative symptoms of SCZ

[0197] The VTA of 3-week-old C57 mice was injected with a mixed virus of rAAV-TH-Cre-WPRE-hGH pA and pAAV-EF1a-DIO-hChR2(H134R)-mCherry or pAAV-EF1a-DIO-mCherry purchased from Primi Technology (the titers of both were 2-5×10 12 vg / ml, the injection volume of the mixed virus was 500nl). After 3 weeks of virus expression, patch clamp recording was used in the VTA brain region to detect the effectiveness of light-activated virus in the virus-transfected neurons ( Fig. 9 The results showed that dopamine neurons in the VTA brain region can accurately respond to pulses of light stimulation. In addition, micro-carbon fiber electrodes were used to record dopamine release signals induced by light activation in two downstream brain regions, mPFC and NAc ( Fig. 9 Therefore, the above results verify the effectiveness of light-activated viruses.

[0198] When the mice were 4 weeks old, optical fibers were implanted in the mPFC and NAc brain regions of the mice injected with the virus, and behavioral tests were performed on the mice at 6 weeks old ( Fig. 9 C). During the social behavior test in the home cage, 10mW, 20Hz, 473nm wavelength blue pulse light stimulation was given. The experimental results showed that real-time stimulation of excessive release of dopamine in the mPFC brain region led to a significant decrease in the social frequency and time of mice in the social behavior test in the home cage compared with control mice. This indicates that excessive increase in dopamine secretion in the VTA to mPFC projection circuit can cause social disorders in mice ( Fig. 9 Middle C).

[0199] Similarly, social behavior experiments were conducted on mice with optical fibers embedded in the NAc brain region. It was found that the same 10mW, 20Hz, 473nm wavelength blue pulse light stimulation directly and in real time over-activated the release of dopamine in the NAc brain region ( Fig. 9 Middle D), the number and duration of contact with stranger mice in the social behavior of the mice in the home cage did not change significantly compared with the control mice ( Fig. 9 (middle D).

[0200] These results suggest that schizophrenia-related social impairments are mediated by the projection circuit from VTA to mPFC dopamine neurons.

[0201] Example 10. Multidimensional changes in mPFC in SCZ

[0202] Immunofluorescence detection was performed in the mPFC of 3-month-old Syt11 cKO mice and control mice (DAT-Cre mice). Fig.10 The results showed that the number of MAP2-positive neurons in the mPFC layer I, layer II / III, and layer VI of Syt11 cKO mice was significantly less than that of the control group ( Fig.10 Middle A, Fig.10 Middle B), TH-positive neurites were significantly lower than those in the control group ( Fig.10 Middle A, Fig.10 (C) The results suggest that the over-secretion of DA in the early stage in the mPFC leads to changes in the neural structure of the mPFC.

[0203] The patch clamp technique was used to perform electrophysiological testing on the mPFC of 3-month-old Syt11 cKO mice and control mice (DAT-Cre mice). The results showed that the amplitude of the excitatory postsynaptic potential of Syt11 cKO mice was significantly lower than that of the control group, but there was no significant difference in the firing frequency between the two ( Fig.11 TH-Cre-EGFP or TH-EGFP virus (titer of 2-5×10 12 vg / ml, injection volume was 200nl). When the mice were 3 months old, the patch clamp technique was used to perform electrophysiological detection of neurons in the mPFC brain slices of Syt11-p0-cKO mice ( Fig.11 The results showed that the action potential firing frequency of neurons in the mPFC of Syt11-p0-cKO mice was significantly lower than that of control mice ( Fig.11 (middle B).

[0204] Using chemical genetics, the VTA of newly born (p0) C57 mice was injected with a mixed virus of rAAV-TH-Cre-WPRE-hGH and rAAV-EF1α-DIO-hM3D(Gq)-mCherry-WPREs (titer of 2-5×10 12 vg / ml, injection volume was 200 nl) or rAAV-TH-Cre-WPRE-hGH and rAAV-EF1α-DIO-mCherry-WPREs mixed virus as control (titer was 2-5×10 12 vg / ml, injection volume was 200nl). At P7 (the seventh day after birth), the virus-injected mice were intraperitoneally injected with CNO (0.5mg / kg) to activate dopamine neurons, followed by injections every other day for 4 consecutive times. When the mice were 3 months old, electrophysiological testing was performed ( Fig.11The results showed that the firing frequency of the mPFC neurons of mice (hM3D) after long-term activation of dopamine neurons in childhood was significantly reduced compared with the control group (Ctrl) ( Fig.11 C).

[0205] By performing genome-wide RNA sequencing of the mPFC of Syt11 cKO mice ( Fig.12 Figure 2 (A) shows that compared with the gene expression profile of the control group (DAT-Cre) mice, 170 differentially expressed genes (DEGs) were identified in Syt11 cKO mice, including 97 upregulated genes and 73 downregulated genes. Among these DEGs, typical risk genes for schizophrenia such as Nrg1, Arc, Ddc, and Icam were found. Fig.12 Middle B). The results showed that transcriptional changes occurred in the mPFC of Syt11 cKO mice.

[0206] Example 11 Verification of the diagnostic effect of synaptotagmin 11 as a biomarker for schizophrenia

[0207] Schizophrenia group (84 people) and normal healthy group (87 people), both groups age range 16 to 60, no age difference. Inclusion criteria: 1) confirmed by experienced clinical attending physicians, meet the diagnostic criteria of schizophrenia in the Diagnostic and Statistical Manual of Mental Disorders-Fifth Edition (DSM-5); 2) aged between 16 and 60 years old, primary school education or above; 3) symptom severity using the Positive and Negative Composite Scale (PANSS), the baseline total score is 60 points or more; 4) no electrotherapy in the past 6 months. Exclusion criteria: including organic mental illness, any neurological abnormality, dementia, other serious physical illness, brain injury, drug abuse or addiction, and inability to give informed consent. Diagnostic criteria: Both inclusion criteria and exclusion criteria are indispensable.

[0208] Blood was drawn from confirmed schizophrenia patients and healthy controls of the same age range, and the expression of synaptotagmin 11 protein in peripheral blood was detected by Western Blots. The two groups of data were statistically plotted as follows: Fig.13 As shown in Figure 1, the lower 95% quantile of the control group is used as the detection standard, and the sensitivity of synaptotagmin 11 as a biomarker for schizophrenia is 81% and the specificity is 92%. The two sets of values ​​are mixed and arranged from low to high. The sensitivity and specificity of the subject can be obtained according to whether the subject is a schizophrenia patient. The receiver operating characteristic curve (ROC curve) is made with sensitivity and 1-specificity, as shown in Figure 1. Fig.14As shown in the figure, the AUC value is 0.91 obtained by calculating the area under the curve. The above shows that using synaptotagmin 11 or its transcription product mRNA or mRNA fragment as a diagnostic marker for schizophrenia has a very accurate predictive ability and precise diagnostic efficacy, and can objectively diagnose patients with schizophrenia, providing a reference for the clinical diagnosis and targeted treatment of patients with schizophrenia.

[0209] Example 12 Dopamine receptor agonists or inhibitors for the treatment of schizophrenia-like social deficits

[0210] Syt11 cKO schizophrenia model mice were constructed by injecting TH-Cre-EGFP virus into the midbrain of P0 Syt11-loxp mice (i.e., floxed Syt11-null mice). At 4 weeks of age, cannulas were implanted in the VTA / mPFC, and at 6 weeks of age, D2 receptor agonist Quinpirol (1 μg / μl, 0.2 μl / side, single injection) was injected locally into the VTA. A three-box social test ( Fig.15 The D2 receptor antagonist haloperidol was injected locally into the mid-AC and mPFC (injection dose was 50 μM, 0.2 μl / side, single injection), and a three-box social test was performed 5 minutes after injection ( Fig.15 DF). The results show that both methods can reverse the social deficit phenotype of schizophrenia mice. The control mice in the figure are Syt11 loxp injected TH-EGFP mice (the virus titer is 2-5×10 12 vg / ml, injection volume was 200nl), and the same volume of normal saline was given during the experiment.

[0211] Similarly, the above mice were tested again in adulthood and it was found that regardless of whether the D2 receptor antagonist haloperidol was injected multiple times locally in the VTA (injection started at 6 weeks, injection dose was 50μM, 0.2μl / side, injection three times, injection once every other day) Fig.16 Multiple local injections of D2 receptor agonist Quinpirol (1 μg / μl, 0.2 μl / side, three times, once every other day) were performed in the AD and mPFC (starting at 6 weeks, injection dose was 1 μg / μl, 0.2 μl / side, injection once every other day) Fig.16 In the EH, after treatment, the social time of Syt11 cKO mice in the three-box test remained unchanged compared with that of control mice, indicating that the treatment effect can last into adulthood. The control mice in the figure are Syt11 loxp injected TH-EGFP mice (virus titer is 2-5×10 12 vg / ml, injection volume was 200nl), and the same volume of normal saline was given during the experiment.

[0212] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of synaptotagmin 11 or its transcription product mRNA or mRNA fragment as a marker in any of the following: 1) Use in the preparation of products for diagnosing or assisting in the diagnosis of schizophrenia in humans or non-human mammals; 2) Application in the preparation of products for screening or assisting screening of schizophrenia in humans or non-human mammals; 3) Use in the diagnosis or auxiliary diagnosis of schizophrenia in humans or non-human mammals; 4) Application in screening or assisting screening of schizophrenia in humans or non-human mammals.

2. A product for detecting synaptotagmin 11 or its transcription product mRNA or mRNA fragment for any of the following purposes: 1) Use in the preparation of products for diagnosing or assisting in the diagnosis of schizophrenia in humans or non-human mammals; 2) Application in the preparation of products for screening or assisting screening of schizophrenia in humans or non-human mammals; 3) Use in the diagnosis or auxiliary diagnosis of schizophrenia in humans or non-human mammals; 4) Application in screening or assisting screening of schizophrenia in humans or non-human mammals.

3. The use according to claim 2, characterized in that: The product for detecting synaptotagmin 11 or its transcription product mRNA or mRNA fragments includes a product for detecting the expression level of synaptotagmin 11 or its transcription product mRNA or mRNA fragments in a sample to be tested; optionally, the sample to be tested is brain tissue or body fluid of a human or non-human mammal; optionally, the body fluid is selected from at least one of blood, cerebrospinal fluid and urine; And / or, the product for detecting synaptotagmin 11 or its transcription product mRNA or mRNA fragment comprises a kit, a reagent, a test strip, a primer, a probe or a chip; Optionally, the reagent includes: an antibody, protein and / or polypeptide that specifically binds to synaptotagmin 11; or a reagent that detects mRNA or mRNA fragments of synaptotagmin 11 transcription products; Optionally, the antibody, protein and / or polypeptide is modified with an isotope, horseradish peroxidase, colloidal gold or a fluorescent probe, or is unmodified; Optionally, the reagent for detecting the synaptotagmin 11 transcription product mRNA or mRNA fragment includes a PCR reaction buffer or a nucleic acid molecule hybridization buffer; optionally, it includes a synaptotagmin 11 gene fragment or a specific sequence fragment of its mRNA, or a molecular probe containing these specific sequence fragments.

4. The use according to any one of claims 1 to 4, characterized in that The symptoms of schizophrenia are positive symptoms, negative symptoms and cognitive impairment of schizophrenia; and / or, the non-human mammal comprises an animal model of schizophrenia; And / or, the person is a schizophrenia patient, a suspected schizophrenia patient, a schizophrenia high-risk person, or a direct relative of a schizophrenia patient.

5. A product for diagnosing, assisting in the diagnosis, screening or assisting in the screening of schizophrenia in humans or non-human mammals, characterized in that: The invention comprises products for detecting the expression level of synaptotagmin 11 or its transcription product mRNA or mRNA fragment in a sample to be tested.

6. The product for diagnosing, assisting in diagnosis, screening or assisting in screening schizophrenia in humans or non-human mammals according to claim 5, characterized in that: The product for detecting synaptotagmin 11 or its transcription product mRNA or mRNA fragment comprises a kit, a reagent, a test strip, a primer, a probe or a chip; And / or, the sample to be tested is human or non-human mammal brain tissue or body fluid; Optionally, the body fluid is selected from at least one of blood, cerebrospinal fluid and urine; and / or, the reagents include: antibodies, proteins and / or polypeptides that specifically bind to synaptotagmin 11; Or a reagent for detecting the mRNA or mRNA fragment of the synaptotagmin 11 transcription product; optionally, the antibody, protein and / or polypeptide is modified with an isotope, horseradish peroxidase, colloidal gold or a fluorescent probe, or is unmodified; optionally, the reagent for detecting the mRNA or mRNA fragment of the synaptotagmin 11 transcription product comprises a PCR reaction buffer or a nucleic acid molecule hybridization buffer; optionally, it comprises a synaptotagmin 11 gene fragment or a specific sequence fragment of its mRNA, or a molecular probe containing these specific sequence fragments; and / or, the schizophrenia symptoms are positive symptoms, negative symptoms and cognitive impairment of schizophrenia; and / or, the non-human mammal comprises an animal model of schizophrenia; And / or, the person is a schizophrenia patient, a suspected schizophrenia patient, a schizophrenia high-risk person, or a direct relative of a schizophrenia patient.

7. A system for diagnosing, assisting in diagnosis, screening or assisting in screening of schizophrenia in humans or non-human mammals, characterized in that: include: An obtaining device, wherein the obtaining device is used to obtain the expression level of synaptotagmin 11 or its transcription product mRNA or mRNA fragment in the sample to be tested; A judgment device, the judgment device is connected to the acquisition device or is wirelessly connected to the judgment device, and the judgment device is used to diagnose or assist in diagnosis, screening or assist in screening whether a human or non-human mammal suffers from schizophrenia based on the expression level of synaptotagmin 11 or its transcription product mRNA or mRNA fragment in the sample to be tested.

8. The system for diagnosing, assisting in diagnosis, screening or assisting in screening schizophrenia in humans or non-human mammals according to claim 7, characterized in that: Compared with the expression level of synaptotagmin 11 or its transcription product mRNA or mRNA fragment in the sample of a normal human or normal non-human mammal, the expression level of synaptotagmin 11 in the sample of the human or non-human mammal to be tested is reduced, and the human or non-human mammal to be tested is judged to suffer from schizophrenia.

9. A non-human mammal model of schizophrenia, characterized in that: Compared with the expression level of synaptotagmin 11 or its transcription product mRNA or mRNA fragment in the sample of a normal non-human mammal, the expression level of synaptotagmin 11 in the sample of the non-human mammal model of schizophrenia is reduced.

10. A method for screening drugs for treating schizophrenia, characterized in that: include: Using the candidate drug in the non-human mammal model of schizophrenia as described in claim 9; screening drugs that up-regulate the expression of synaptotagmin 11 or its transcription product mRNA or mRNA fragment or enhance its activity, or up-regulate the expression of dopamine D2 receptor or its transcription product mRNA or mRNA fragment or enhance its activity, or screening candidate drugs that alleviate schizophrenia as the drug for treating schizophrenia.

11. A method for preparing a drug for preventing and / or treating schizophrenia, characterized in that: The method comprises the steps of using the protein and / or expressed cDNA or cDNA fragment to prepare the desired drug; The protein is an expression product of synaptotagmin 11 gene cDNA or cDNA fragment, or an expression product of dopamine D2 receptor gene cDNA or cDNA fragment; The cDNA or cDNA fragment is derived from synaptotagmin 11 gene or dopamine D2 receptor gene.

12. The method for preparing the drug for preventing and / or treating schizophrenia according to claim 11, characterized in that: The cDNA or cDNA fragment is unmodified, or contains at least one modified nucleotide or nucleotide analog; wherein the modification site of the modified nucleotide or nucleotide analog may be on the sugar group, main chain or base of the ribonucleotide; The nucleotide analogs include backbone-modified ribonucleotides with phosphorothioate groups, inosine, or tritylated bases.

13. A method for preparing a drug for preventing and / or treating schizophrenia, characterized in that: The method comprises the steps of preparing the desired drug by using a virus that can promote the expression of synaptotagmin 11 and / or dopamine D2 receptor cDNA or cDNA fragment; The virus promoting the expression of synaptotagmin 11 and / or dopamine D2 receptor cDNA or cDNA fragment includes at least one of lentivirus, adenovirus and adeno-associated virus.

14. The method for preparing the drug for preventing and / or treating schizophrenia according to claim 13, characterized in that: The cDNA or cDNA fragment expression virus is prepared by the following method: inserting synaptotagmin 11 and / or dopamine D2 receptor cDNA or cDNA fragment into the multiple cloning site region of the expression vector to construct a cDNA or cDNA fragment expression plasmid; co-transfecting the cDNA or cDNA fragment expression plasmid with an auxiliary plasmid into cells to generate virus particles, and recovering and concentrating the virus to obtain the virus.

15. A drug for preventing and / or treating schizophrenia, characterized in that: The drug includes synaptotagmin 11, synaptotagmin 11 cDNA or cDNA fragment; and / or, dopamine D2 receptor, dopamine D2 receptor cDNA or cDNA fragment; and / or, an expression virus containing synaptotagmin 11, synaptotagmin 11 cDNA or cDNA fragment gene; and / or, an expression virus containing dopamine D2 receptor, dopamine D2 receptor cDNA or cDNA fragment gene; or, The active ingredients of the drug include substances that can promote the expression of synaptotagmin 11 and / or dopamine D2 receptors, and / or substances that increase the physiological activity of synaptotagmin 11 and / or dopamine D2 receptors; Preferably, the virus comprises at least one of an adenovirus, a lentivirus, and an adeno-associated virus vector.

16. A substance that can promote the gene expression of synaptotagmin 11 and / or dopamine D2 receptor or increase the content or activity of synaptotagmin 11 and / or dopamine D2 receptor, or use of synaptotagmin 11, synaptotagmin 11 cDNA or cDNA fragment, dopamine D2 receptor, dopamine D2 receptor cDNA or cDNA fragment for preparing a drug or product having at least one of the following effects (1) to (5): (1) Treatment of schizophrenia; (2) Treat or alleviate the positive symptoms of schizophrenia; (3) Treat or alleviate negative symptoms of schizophrenia; (4) Treat or alleviate cognitive impairment in schizophrenia; (5) Products that inhibit the excitation of dopamine neurons or the secretion of dopamine.

17. The use according to claim 16, characterized in that The method of promoting the expression of synaptotagmin 11 and / or dopamine D2 receptor genes or increasing the content or activity of synaptotagmin 11 and / or dopamine D2 receptors is: enhancing the transcription and translation levels of synaptotagmin 11 and / or dopamine D2 receptor genes by gene editing, or increasing the stability of synaptotagmin 11 and / or dopamine D2 receptor mRNA, or increasing the stability of synaptotagmin 11 and / or dopamine D2 receptor proteins, or reducing the degradation level of synaptotagmin 11 and / or dopamine D2 receptor proteins.

18. The use according to claim 16, characterized in that The synaptotagmin 11 protein and / or dopamine D2 receptor, or synaptotagmin 11 cDNA or cDNA fragment, and / or dopamine D2 receptor cDNA or cDNA fragment, are delivered to the brain regions affected by schizophrenia in the following ways: (1) injecting virus particles of synaptotagmin 11 protein or synaptotagmin 11 cDNA or cDNA fragment into the brain area affected by schizophrenia lesions by stereotaxic injection; (2) injecting a plasmid containing synaptotagmin 11 cDNA or cDNA fragment into the brain area affected by schizophrenia by stereotaxic injection, and transferring the plasmid into cells by electroporation; (3) stereotaxic injection of plasmids and liposomes containing synaptotagmin 11 cDNA or cDNA fragments into the brain area affected by schizophrenia; (4) Treatment of schizophrenia by injecting viral particles containing synaptotagmin 11 cDNA or cDNA fragments through the subarachnoid space; Optionally, the brain regions affected in the schizophrenia patient include but are not limited to the ventral tegmental area of ​​the midbrain; Optionally, the target cells for treating schizophrenia patients include but are not limited to dopamine neurons in the ventral tegmental area of ​​the midbrain.

19. The use according to claim 16, characterized in that The functions of the drug or product include: (1) The drug increases the expression level of synaptotagmin 11 and / or dopamine D2 receptor, or synaptotagmin 11 and / or dopamine D2 receptor mRNA; (2) the drug improves the stability of synaptotagmin 11 and / or dopamine D2 receptor, or synaptotagmin 11 and / or dopamine D2 receptor mRNA; (3) The drug improves the physiological function of synaptotagmin 11 and / or dopamine D2 receptor, or synaptotagmin 11 and / or dopamine D2 receptor mRNA; (4) the drug increases the translation level of synaptotagmin 11 and / or dopamine D2 receptor, or synaptotagmin 11 and / or dopamine D2 receptor mRNA on the ribosome; (5) Used to treat schizophrenia.

20. The use according to claim 19, characterized in that: The method of increasing the expression level of synaptotagmin 11 and / or dopamine D2 receptor is any one or more of the following: (1) increasing the transcription level of synaptotagmin 11 and / or dopamine D2 receptor encoding genes; (2) increasing the translation level of synaptotagmin 11 and / or dopamine D2 receptor protein; (3) reduce the degradation rate of synaptotagmin 11 and / or dopamine D2 receptor; The drug improves the physiological function of synaptotagmin 11 and / or dopamine D2 receptors, specifically, the drug inhibits dopamine secretion by inhibiting exocytosis, inhibiting endocytosis, inhibiting vesicle uncoating, inhibiting vesicle circulation, etc.; Optionally, the schizophrenia patient has a family history of schizophrenia, or is a sporadic schizophrenia patient.

21. Use of synaptotagmin 11 protein and / or dopamine D2 receptor, or synaptotagmin 11 and / or dopamine D2 receptor mRNA and fragments thereof as therapeutic targets for schizophrenia.

22. Use of a dopamine receptor agonist or inhibitor in the preparation of a drug for treating schizophrenia, characterized in that: include: Use of an agonist of a dopamine neuron D2 receptor in preparing a drug for treating schizophrenia, wherein the agonist of the dopamine neuron D2 receptor is one or more of quinpirole, bromocriptine, cabergoline, pergolide, apoxicam, pramipexole and ropinirole; and optionally, the agonist of the dopamine neuron D2 receptor is used in preparing a drug for local injection into the ventral tegmental area of ​​the midbrain; And / or, use of an inhibitor / antagonist of dopamine neuron D2 receptor in the preparation of a drug for treating negative symptoms of schizophrenia; optionally, the inhibitor / antagonist of dopamine neuron D2 receptor is one or more of haloperidol, risperidone, olanzapine, chlorpromazine, trifluoperazine hydrochloride, metoclopramide, doxiritone or itopride; optionally, use of the inhibitor / antagonist of dopamine neuron D2 receptor in the preparation of a drug for local injection of the prefrontal cortex.

Images