Quantitative determination method for mental state based on DRD1 and / or DRD5 and application of quantitative determination method

Abstract

A method of treating a psychiatric disorder in a subject comprises the step of normalizing the expression of DRD1 and DRD5 belonging to DRD1 class receptors in the subject to a preset standard level. By targeting an upstream promoter sequence of DRD1 / 5, the level and expression of DRD1 / 5 can be bidirectionally increased or decreased. The use of antipsychotic agents that directly target the DRD1 / 5 promoter can attenuate the DRD1 / 5 promoter activity. The antipsychotic agents used are typical and / or atypical antipsychotic agents, such as haloperidol, risperidone, clozapine and cariprazine. The treatment is also useful for subjects in which constitutive expression of the DRD1 / 5 gene is impaired. The methods can treat schizophrenia, autism, depression, mania, and bipolar affective disorders by normalizing DRD1 / 5 expression levels. Promoter gene sequencing of DRD1 and / or DRD5 can also be used for quantitative mental health screening by comparison to a standard sequence database.

Description

Technical Field

[0001] The present invention relates to dopamine receptor D1 (also known as DRD1) and dopamine receptor D5 (also known as D1BR or DRD5), and more particularly, to a quantitative method for determining the mental state of a human subject such as a psychiatric patient based on DRD1 and / or DRD5 and its application. Background Art

[0002] Currently, there is no quantitative method to determine the mental state of human subjects. For example, psychosis, which interferes with functional ability, is determined by symptoms such as delusions and hallucinations. The cause of mental disorders is still unclear, and commercially available antipsychotic drugs do not have a common mode of action.

[0003] The diagnosis of mental disorders relies heavily on signs and symptoms in human subjects. Examples include feeling sad or depressed, confusion or difficulty concentrating, excessive fear or worry, or extreme guilt, high and low mood swings, avoiding friends and activities, severe tiredness, sleeping problems, delusions, paranoia or hallucinations, excessive anger, hostility or violence. Sometimes, symptoms may include stomach pain, back pain, headaches or other unexplained pain. Determining a human subject's mental state is difficult because there are a large number of possible symptoms, most of which are based on the human subject's behavior and thinking.

[0004] There is currently some evidence of a link between schizophrenia and genetics. However, no single gene is thought to cause the disorder, and people who have family members with the disorder are simply at higher risk. The cause or link remains unknown.

[0005] Existing antipsychotic drugs are usually DRD2 antagonists. However, the current understanding of whether DRD2 is involved in the action of antipsychotic drugs is still controversial. Serotonin and dopamine are two common neurotransmitters that are believed to have a direct impact on mental disorders. Summary of the invention

[0006] One object of the present invention is to provide a method for quantitatively determining the mental state of a human subject based on DRD1 and / or DRD5 and its application and use.

[0007] Another object of the present invention is to provide antipsychotic drugs to chemically normalize DRD1 expression.

[0008] Another object of the present invention is to provide an antipsychotic drug that similarly affects the expression of DRD1 and DRD5, and is capable of normalizing or stabilizing up-regulated and down-regulated DRD1 and DRD5 expressions to normal expression levels.

[0009] Another object of the present invention is to provide antipsychotic drugs that directly target the DRD1 and / or DRD5 promoter sequence.

[0010] Another object of the present invention is to provide antipsychotic drugs involving a novel mode of action compared to conventional antipsychotic drugs, thereby enabling the development of new drugs based on maintaining the normalized expression levels of DRD1 and DRD5.

[0011] Another object of the present invention is to provide antipsychotic drugs that normalize DRD1 expression by targeting the 5'-regulatory element.

[0012] Another object of the present invention is to provide a method for treating depression and mania.

[0013] Another object of the present invention is to provide a therapeutic method for restoring positive and negative symptoms in psychiatric disorders by neutralizing the key processes involved in the bidirectional regulation of DRD1 and DRD5 expression.

[0014] Additional advantages and features of the invention will become apparent from the following description, and may be realized by means of the instrumentalities and combinations particularly pointed out in the appended claims.

[0015] According to the present invention, the above objects and advantages are achieved by a method for treating a mental disorder in a subject, the method comprising the steps of: The DRD1 levels and DRD1 expression of the subjects were normalized to the pre-set standard levels.

[0016] Preferably, DRD1 level and DRD1 expression can be bidirectionally increased or decreased by targeting the upstream promoter sequence of DRD1. Antipsychotic drugs that directly target the DRD1 promoter can weaken DRD1 promoter activity. The antipsychotic drugs used can be one or more typical and atypical antipsychotic drugs, such as haloperidol, risperidone, clozapine and cariprazine. This treatment is also useful for subjects with impaired DRD1 constitutive gene expression. This method can treat schizophrenia, autism, depression, mania and bipolar disorder by normalizing DRD1 expression levels.

[0017] According to another aspect of the invention, a method for treating a psychiatric disorder in a subject comprises the following steps: The overall expression levels of DRD1 and DRD5 of the subjects were bidirectionally normalized to the pre-set standard levels.

[0018] Preferably, the method further comprises the step of attenuating the promoter activity of DRD1 / DRD5 using an antipsychotic drug that directly targets the promoters of DRD1 and DRD5.

[0019] Preferably, the method further comprises the following steps: providing a sequence database related to polymorphisms of DRD1 and DRD5 promoter sequences and drug efficacy; and performing personalized drug screening on the efficacy of antipsychotic drugs based on the sequence database.

[0020] According to another aspect of the present invention, a method for screening a subject for mental disorder comprises the following steps: sequencing the DRD1 and DRD5 promoter genes of the subject to obtain a sample sequence, and comparing the sample sequence with a standard sequence database of human DRD1 / DR5 promoters to determine whether the subject suffers from a mental disorder.

[0021] Preferably, polymorphisms in the DRD1 / DRD5 promoter sequence associated with clinically diagnosed psychiatric disorders are contained in standard sequence databases.

[0022] Further objects and advantages will become apparent by consideration of the ensuing description and accompanying drawings.

[0023] These and other objects, features and advantages of the present invention will become apparent from the following detailed description, the accompanying drawings and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A Shown is the relative expression of DRD1 gene mRNA on SH-SY5Y cells under the treatment of haloperidol (10 μM), SCH-23390 (10 μM), and SKF-81297 (10 μM).

[0025] Figure 1B Shown are the results of DRD1 protein expression on SH-SY5Y cells under haloperidol treatment.

[0026] Figure 1C The corresponding protein expression of DRD1 on SH-SY5Y cells under haloperidol treatment is shown.

[0027] Figure 2A The corresponding DRD1 mRNA expression of SCH-23390 treatment on SH-SY5Y cells is shown.

[0028] Figure 2B The results of DRD1 protein expression in SH-SY5Y cells treated with SCH-23390 are shown.

[0029] Figure 2C It shows the relative protein expression of DRD1 on SH-SY5Y cells.

[0030] Figure 2D ICC staining of SHSY5Y (36 h) treated with SCH under different conditions.

[0031] Figure 2E The percentages of positive H-DRD1 cells in SHSY-5Y cells under different treatments are shown.

[0032] Figure 3A Shown is the relative expression of DRD1 mRNA on SKF-81297 treated SH-SY5Y cells.

[0033] Figure 3B The results of protein expression of DRD1 under SKF-81297 treatment in SH-SY5Y cells are shown.

[0034] Figure 3C Shown is the relative protein expression of DRD1 upon SKF-81297 treatment on SH-SY5Y cells.

[0035] Figure 3D Shown is ICC staining of SKF-treated SHSY5Y under different treatments.

[0036] Figure 3E The percentages of H-DRD1-positive cells in SHSY-5Y cells under different treatments are shown.

[0037] Figure 4A Shown are the relative DRD1 promoter activities under SCH-23390 treatment on SH-SY5Y cells.

[0038] Figure 4B Shown is the relative activity of the DRD1 promoter under SKF-81297 treatment on SH-SY5Y cells.

[0039] Figure 5A and 5B Shown is a study of the regulation of DRD1 promoter activity by risperidone. Figure 5A The relative luciferase activity of DRD1 under SCH and risperidone treatment is shown (risperidone has a partial DRD1 antagonist effect when co-treated with SCH). Figure 5B The relative luciferase activity of DRD1 under SKF and risperidone treatment is shown (the combination of risperidone and SKF has a partial DRD1 antagonistic effect).

[0040] Fig. 6A and 6B A study of the regulation of DRD1 promoter activity by clozapine is described. Fig. 6A The relative luciferase activity of DRD1 under SCH and clozapine treatment is shown (clozapine has a partial DRD1 antagonistic effect when co-treated with SCH). Figure 6B The relative luciferase activity of DRD1 under SKF and clozapine treatment is shown (clozapine has a partial DRD1 antagonistic effect when co-treated with SKF).

[0041] Fig. 7A and 7B A study of the regulation of DRD5 mRNA expression is described. Fig. 7A Shown is the relative expression of DRD5 mRNA on SCH-23390 treated SH-SY5Y cells. Figure 7B The relative expression of DRD5 mRNA on SH-SY5Y cells treated with SKF-81297 and H is shown.

[0042] Figure 8 The relative mRNA expression of DRD1 and DRD5 in SH-SY5Y cells is shown. DETAILED DESCRIPTION

[0043] According to the present invention, DRD1 and DRD5, particularly the protein expression levels thereof, play a key role in maintaining the activity state of each individual functional cell such as neurons, muscle cells and endothelial cells.

[0044] Dopamine receptor D1 (DRD1) is encoded by the DRD1 gene and is one of the two members of the DRD1-like receptor family, which includes DRD1 and DRD5. DRD1 is mainly enriched in the central nervous system. DRD1 plays an important role in memory, learning, addiction, reward system, behavioral appearance and neuronal development. DRD1 is one of the most abundant dopamine receptors in the human brain and plays an important role in the etiology of psychiatric diseases, including schizophrenia, autism, depression, mania and bipolar disorder.

[0045] DRD1 and DRD5 belong to the dopamine D1 receptor family. Functionally, DRD1 and DRD5 are compensatory. Therefore, DRD1 and DRD5 can maintain the mental state compensatorily and coordinately. Preferably, both DRD1 and DRD5 are involved in the maintenance of the mental state, which helps maintain genetic and functional stability and can further explain the diversity of mental disorders.

[0046] According to the present invention, DRD1 dysfunction is associated with psychological disorders. Psychiatric disorders, including bipolar disorder, depression, mania and schizophrenia, are essentially promoter disorders. Psychosis is mainly due to promoter-related gene expression disorders, especially dopamine receptors. Therefore, antipsychotic drugs may work by normalizing chemically altered DRD1 expression back to constitutive levels. The action of antipsychotic drugs is bidirectional and can normalize up-regulated and down-regulated DRD1 expression. Antipsychotic drugs exert a "hormetic effect" by normalizing DRD1 expression.

[0047] According to the present invention, there are two possible mechanisms for regulating DRD1 expression: (a) There are two separate elements in the DRD1 promoter sequence: one is responsible for attenuating upregulated DRD1 expression; the other contributes to the reversal effect of antipsychotic drugs on downregulated DRD1 expression.

[0048] (b) There is a specific element in the DRD1 promoter. When antipsychotic drugs target this sequence, they block the chemically altered DRD1 expression machinery, thereby stabilizing DRD1 expression at a constitutive level.

[0049] It is worth mentioning that clinically available antipsychotic drugs are more effective in treating bipolar disorder than schizophrenia. According to the principles of the present invention, the structural gene expression of DRD1 is significantly impaired, even after receiving antipsychotic drug treatment. After schizophrenia patients receive it, the expression of DRD1 cannot be restored to the normal DRD1 expression range. Therefore, the existing antipsychotic drugs are not effective in treating schizophrenia.

[0050] DRD1 and DRD5 work together to coordinate the mental state. The overall expression levels of DRD1 and DRD5 are controlled within the normal range to maintain a normal mental state.

[0051] In patients with psychosis, the overall expression levels of DRD1 and DRD5 are outside the normal range. Specifically, when the overall expression levels of DRD1 and DRD5 are very high, patients will show depression and negative symptoms. Conversely, when the overall expression levels of DRD1 and DRD5 are very low, patients will show mania and positive symptoms. In addition, when the promoters of DRD1 and DRD5 are susceptible to internal or external factors, causing their expression to drift easily, patients will develop bipolar disorder.

[0052] Promoter sequencing of human DRD1 and DRD5 can be used for diagnosis and screening of psychiatric disorders.

[0053] Our traditional understanding of the mode of action of antipsychotic drugs is largely based on DRD2. For example, all antipsychotic drugs reduce dopaminergic neurotransmission. The primary mode of action of most first- and second-generation antipsychotics appears to be postsynaptic blockade of DRD2-like receptors in the brain. In addition, most second-generation antipsychotics also inhibit serotonin receptors 5HT, such as 5HT1A / 2A.

[0054] Since the present invention provides that the overall expression level of DRD1 and DRD5 can be used as an objective factor of mental state, the expression level of DRD1 and / or DRD5 can now be directly related to mental health. Therefore, the effects of antipsychotic drugs on DRD1 and DRD5 can be further studied for drug development.

[0055] For example, DRD1 expression is significantly impaired in patients with schizophrenia. The importance of certain DRD1 promoter gene sequences can be used to modulate human psychiatric disorders by regulating the levels of DRD1 and DRD5.

[0056] According to a first preferred embodiment of the present invention, a method for treating mental disorders comprises the following steps: normalizing human DRD1 levels and expression to restore to normal levels; and / or normalizing human DRD5 levels and expression to restore to normal levels.

[0057] Preferably, the method may further comprise the following steps: increasing or decreasing the subject's constitutive expression of DRD1 by targeting the upstream promoter sequence of DRD1; and / or increasing or decreasing the subject's constitutive expression of DRD5 by targeting the upstream promoter sequence of DRD5.

[0058] Preferably, the method may further comprise the step of attenuating the promoter activity of DRD1 or DRD5 by using an antipsychotic drug that directly targets the promoter of DRD1 or DRD5.

[0059] The human DRD1 and DRD5 genes essentially contribute to the maintenance of mental states. Genetic and / or epigenetic alterations in the human DRD1 and DRD5 promoter sequences result in changes in their expression. Antipsychotic drugs directly target the human DRD1 and DRD5 promoters, normalizing exogenously or endogenously altered human DRD1 and DRD5 expression, thereby exerting a therapeutic effect.

[0060] Preferably, the method for treating a mental disorder comprises a pharmacological method or a non-pharmaceutical method to normalize DRD1 expression to return to within a predetermined normal level.

[0061] Preferably, antipsychotics are used to normalize the expression levels of DRD1 and / or DRD5. Antipsychotics can be selected from antipsychotics of different classes. Typical antipsychotics haloperidol and atypical antipsychotics risperidone, clozapine and cariprazine are selected. Therefore, these drugs can represent most antipsychotics in terms of mechanism.

[0062] According to a second preferred embodiment of the present invention, a method for quantitative screening and detection of mental illness comprises the following steps: using a genomic DNA sample extracted from a human subject to perform promoter sequencing of the DRD1 and / or DRD5 gene; then screening the sequence and comparing it with a human DRD1 and / or DRD5 promoter sequence database to determine whether the human subject suffers from a mental illness.

[0063] If at least one variant is found in DRD1 or DRD5, the human subject is confirmed to have a mental disorder. If at least two variants are found in DRD1 or DRD5, the human subject is confirmed to have an aggravated degree of mental disorder (a more severe mental disorder).

[0064] The sequencing project established a human DRD1 / DRD5 promoter sequence database. The polymorphism of the DRD1 / DRD5 promoter sequence in the sequence database is associated with clinically diagnosed mental disorders. In addition, the sequence database will also be used for prenatal and parent screening to predict the incidence of mental disorders in newborns. Variations in the DRD1 / DRD5 promoter sequence are associated with mental disorders.

[0065] In addition, the method further comprises the following steps: based on a database related to polymorphisms of DRD1 and DRD5 promoter sequences and medication efficacy, personalized medication screening is performed on the efficacy of antipsychotic drugs.

[0066] According to the present invention, the overall expression levels of DRD1 and DRD5 are maintained within a preset normal range for the treatment of mental disorders. Therefore, by screening known chemical libraries, potential candidate drugs that can normalize altered DRD1 and DRD5 expression can be selected for new drug development.

[0067] According to another preferred embodiment of the present invention, a quantitative screening method for mental disorders in a subject comprises the following three steps: Step 1: Perform genome sequencing on the subjects' DRD1 and DRD5 genes (including 7kb 5'-upstream sequence, protein coding sequence and 2kb 3'-downstream sequence) to establish a database of mental disorders and variation distribution in DRD1 and DRD5 genes (including the type, location and frequency of each gene variation).

[0068] Step 2: We teased out variants that were highly associated with each mental disorder and developed target gene sequences for the human DRD1 and DRD5 genes that could predict the risk of developing mental disorders.

[0069] Step 3: Perform target gene sequencing of the DRD1 and DRD5 genes on new subjects to determine the risk of the subjects developing mental disorders.

[0070] In other words, a method for quantitative screening and testing the expression of human DRD1 and DRD5 in human subject cells (such as mucosal cells, skin cells, blood cells, etc.) can also be provided to establish the normal range of human DRD1 and DRD5. If the expression of DRD1 / 5 in a new subject exceeds the normal range in the cell, it can be used as an indicator of the subject's mental state.

[0071] Haloperidol is a typical antipsychotic drug commonly used to treat schizophrenia. The therapeutic effect of haloperidol is thought to be due to its inhibition of DRD2 activity in conventional treatment. By studying the relationship between haloperidol treatment and DRD1 and DRD5, rather than DRD2, a direct and effective correlation can be established for effective quantitative treatment.

[0072] Experiment 1: Haloperidol-DRD1 Project In general, the effect of a drug receptor depends on its binding affinity (Km) to its ligand and its total expression (Vmax). Binding affinity is mainly determined by its protein configuration. In contrast, receptor expression is affected by genetics and epigenetics.

[0073] To elucidate the role of DRD1 in the mode of action of antipsychotic drugs, in vitro studies determined drug modulation of DRD1 expression by treatment with typical and atypical antipsychotic drugs, including haloperidol, clozapine, risperidone, and cariprazine, in the presence of DRD1 agonists or antagonists.

[0074] The present study aimed to determine whether the widely used antipsychotic drugs haloperidol, risperidone, or clozapine could normalize chemically altered human DRD1 expression in human SH-SY5Y neuroblastoma cells.

[0075] refer to Figure 1A , shows the relative expression of DRD1 gene mRNA in SH-SY5Y cells under the treatment of haloperidol (10 μM), SCH-23390 (10 μM), and SKF-81297 (10 μM).

[0076] SCH-23390, also known as halogenated benzazepine, is a synthetic compound that acts as a DRD1 receptor antagonist. SKF-81297 is a synthetic compound of the benzazepine chemical class that acts as a selective dopamine D1 / D5 receptor full agonist. SH-SY5Y cells are a clonal subline of the human neuroblastoma cell line.

[0077] Experimental data show that haloperidol can normalize the expression of chemically altered human DRD1 in human SH-SY5Y neuroblastoma cells.

[0078] Materials and methods Haloperidol was purchased from Sigma-Aldrich (Catalog #H1512-10G, St. Louis, MO). SCH-23390 hydrochloride (Catalog #09-251-0) and SKF-81297 hydrobromide (Catalog #14-471-0) were purchased from FISHERSCIENTIFIC (Hanover Park, IL). Lipofectamine reagent was purchased from FISHER SCIENTIFIC (Catalog #NC0904455, Hanover Park, IL). mem-PER Plus Membrane Protein Extraction Kit (Catalog #89842) and NE-PER Nuclear and Cytoplasmic Extraction Kit (Catalog #78833) were purchased from Fisher Scientific (Hanover Park, IL). Zymo Plasmid Miniprep Kit (Catalog #D4036) and DNAClean & Concentrator Kit (Catalog #D4013) were purchased from Zymo Research Corporation (Irvine, CA). All other chemicals were purchased from ThermoFisher Scientific Inc. (Waltham, MA) unless otherwise stated.

[0079] Dopamine receptor D1 (DRD1) antibody was obtained from Abcam (Cat. No. ab216644, Cambridge, MA). Pierce goat anti-rabbit IgG (H+L) biotin-conjugated secondary antibody (1:5,000) and Pierce high sensitivity streptavidin-HRP (1:5,000; Cat. No. 21130) were purchased from Thermo Fisher Scientific (Waltham, MA).

[0080] Cell culture studies: Human SH-SY5Y neuroblastoma cell line was purchased from ATCC (Catalog #CRL-2266, Manassas, VA) and cultured in Eagle's Minimum Essential Medium (EMEM) (Catalog #ATCC® 30-2003, ATCC, Manassas, VA) containing 10% heat-inactivated fetal bovine serum (Atlanta Biologicals; Norcross, GA). 2 The cell culture incubator was maintained at 37°C and 5% CO 2 Treated cells in six-well cell culture plates were harvested for mRNA analysis by real-time quantitative PCR assay. When drug treatment exceeded 24 h, cells were plated at 3 × 10 5 The cells were seeded at a density of 4 × 10 / well in 6-well plates, and when drug treatment was less than 24 hours, the cells were plated at a density of 4 × 105 The cells were seeded into 6-well plates at a density of 10 / well. The treated cells in T-75 flasks were harvested and protein analysis was performed by Western blotting. 6 The cells were seeded at a density of 2.5 x 10 / flask (65% confluence) in T-75 flasks. 4 The cells were seeded at a density of 100 μg / well into 96-well plates and used for promoter analysis by performing a dual-luciferase reporter assay.

[0081] Time response study. We selected 10 μM haloperidol, 10 μM SCH-23390 or 10 μM SKF-81297 as the starting dose in the time response study. Cultured SH-SY5Y cells were treated with complete EMEM medium containing 10 μM haloperidol, 10 μM SCH-23390 or 10 μM SKF-81297 for 12, 18, 24, 30 or 32 hours, respectively. The control received DMSO treatment only. After each time period, the cells were harvested and processed for mRNA analysis of the human DRD1 gene.

[0082] Haloperidol and SCH-23390 combination therapy study. Cultured SH-SY5Y cells were pretreated with complete EMEM medium containing 10 μM SCH-23390 for 6 hours or 12 hours. Then, the cells were treated with complete EMEM medium containing 10 μM haloperidol and 10 μM SCH-23390 for 12 hours or 24 hours. After treatment, the cells were harvested and processed for mRNA and protein analysis of the human DRD1 gene.

[0083] Haloperidol and SKF-81297 Combination Therapy Study. Cultured SH-SY5Y cells were pretreated with complete EMEM medium containing 10 μM SKF-81297 for 6 h or 12 h. Then, the cells were treated with complete EMEM medium containing 10 μM haloperidol and 10 μM SKF-81297 for 12 h or 24 h. After treatment, the cells were harvested and processed for mRNA and protein analysis of the human DRD1 gene.

[0084] Total RNA extraction and complementary DNA (cDNA) preparation RNA extraction was performed using TRIzol™ RNA extraction reagent (Catalog #15596026; Life Technologies; Carlsbad, CA). The concentration and purity of total RNA were determined by a biospectrometer (Eppendorf, Hauppauge, NY) at 260 nm. Only RNA samples with an A260 / A280 ratio between 1.8 and 2.0 were used for cDNA synthesis. Briefly, 3 pg of total RNA was reverse transcribed into cDNA using random primers (Catalog #PR-C1181, FisherScientific, Hanover Park, IL, polythymidine (Oligo 9dT)) primers (Catalog #FERSO131, FisherScientific, Hanover Park, IL) and SuperScript II reverse transcriptase (LifeTechnologies, Carlsbad, CA) according to the manufacturer's instructions. The synthesized cDNA samples were stored at -20°C.

[0085] Real-time quantitative PCR (qRT-PCR) detection Each cDNA sample was placed in duplicate in a 96-well plate and mixed with SYBR Select Master Mix (Life Technologies, Carlsbad, CA) and gene-specific primer sets. In general, the SYBR reaction in each well contained 5 ml SYBR Select Master Mix, 0.2 μl 10 μM gene-specific primer set, 1.8 μl deionized distilled water, and 3 μl cDNA sample. The AriaMx qRT-PCR system (Agilent Technologies, Santa Clara, CA) was used for real-time fluorescence quantitative PCR assays. The relative mRNA expression of each individual gene was calculated using the comparative delta-delta CT method. The results are expressed as relative multiples and normalized to the expression of 18s rDNA. All quantitative RT-PCR primers were designed using PubMed Primer-BLAST or Primer3 software (NIH) and synthesized by Eurofins (Eurofins MWG OperonUSA, Louisville, KY).

[0086] The real-time fluorescence quantitative PCR primer sequences for human DRD1 are as follows: Forward primer of human DRD1 gene: [Sequence ID: 1] 5'-CCATCACACAAAACGGTCAG-3', reverse primer: [Sequence ID: 2] 5'-GTGTGTTGGAAAGCAGC AGA-3'.

[0087] 18S rDNA gene forward primer: [SEQ ID: 3] 5'-GCAATTATTCCCCATGAACG-3'; reverse primer [SEQ ID: 4] 5'- GCCTC ACTAAACC ATCCAA-3'.

[0088] Western blotting Human SH-SY5Y neuroblastoma cells were grown to 65% confluence in T-75 flasks and then treated with chemicals as described above. Membrane proteins were extracted using the mem-PER Plus membrane protein extraction kit (Thermo Fisher Scientific, Waltham, Massachusetts). Protein concentration was semi-quantitated by spectrophotometry at 280 nm. Protein samples were prepared at a density of 30 mg / well, mixed with loading buffer, heated at 95°C for 5 minutes, and separated on a 12% SDS-polyacrylamide gel. The proteins were then electrotransferred to a polyvinyl difluoride (PVDF) membrane. The membrane was then blocked in Tris-buffered saline (TBS) supplemented with 2.5% skim milk. After blocking, the membrane was incubated overnight at 4°C with a primary antibody (human DRD1 antibody, 1:1000). On the second day, the membrane was washed 3 times with fresh TBS and then incubated for 2.5 hours at room temperature with secondary antibody [goat anti-rabbit biotin conjugated antibody (Abcam; Cambridge, MA) (1:5000)] in TBS supplemented with 2.5% BSA. The membrane was simply washed 3 times with fresh TBS and then placed at room temperature for 30 minutes in TBS supplemented with 0.5% BSA with secondary antibody (1:5000) linked with Pierce high sensitivity streptavidin HRP. The membrane was then rinsed 3 times with fresh TBS. Immunoreactive protein bands in the membrane were visualized using Immobilon chemiluminescent reagent (Millipore, Billerica, MA) by UVP Biospectrum imaging system (Upland, CA). Protein band analysis was performed using ImageJ (NIH, Bethesda, MD). In addition, the protein expression of β-actin as a loading control was determined using primary antibody (β-actin monoclonal antibody, 1:2000) and corresponding secondary antibody [goat anti-mouse IgG (H+L) secondary antibody, HRP 1:5000 (Catalog #31430)].

[0089] Immunocytochemistry (ICC) staining. Human SH-SY5Y neuroblastoma cells were seeded on chamber slides (1×10 5 / well) and then treated with chemicals as described above. Fixation and blocking were performed as described previously (Bu et al., 2016). Briefly, cells were incubated with antibodies against human DRD1 protein (1:1000) at 4°C overnight. The next day, chamber slides were incubated with 1:300 Pierce goat anti-rabbit IgG (H+L) biotin-conjugated secondary antibody (Catalog #31820; Thermo Fisher Scientific Inc., where) for 4 hours at room temperature. Cells were then washed with 1× PBS and incubated with 1:400 Pierce High Sensitivity Streptavidin-HRP (Catalog #21130; Thermo Fisher Scientific Inc., where) for one hour at room temperature. After the final wash, color development was achieved using ImmPACTNovaRED Peroxidase (HRP) Substrate (Catalog #SK4805; Vector Laboratories, Inc.; Burlingame, CA) according to the manufacturer's instructions.

[0090] Cloning of human DRD1 gene promoter. Human DRD1 gene promoter sequences of different lengths (172 bp, 312 bp, 473 bp, 961 bp, and 2772 bp upstream of the transcription start site) were synthesized by GenScript (Piscataway, NJ) and inserted into the pGL3 basic vector (cat. # El 751; Promega, Fitchburg, WI) between the multiple cloning sites Nhel and Xhol. The sequences of the pGL3 recombinant constructs were verified by GenScript (Piscataway, NJ) or Eurofins (Eurofins MWG Operon USA, Louisville, KY).

[0091] DNA transfection and dual luciferase reporter gene detection. Following the manufacturer's instructions, pGL3 basic vector (catalog number #E1 75; Promega, Fitchburg, Wisconsin) and recombinant pGL3 vectors containing different lengths of human DRD1 gene promoter were transfected into human SH-SY5Y cells using Lipofectamine2000 transfection reagent (catalog number #11668027; Life Technologies, Inc.; Carlsbad, California). In brief, cells were inoculated into 96-well plates. Then, 180-200ng pGL3 plasmid DNA (pGL3-empty basic vector or recombinant pGL3 vector containing human DRD1 gene promoter) and 10ng pRL-CMV sea renilla luciferase control reporter vector (catalog number #E2261, Promega, Madison, Wisconsin) were used and mixed with Lipofectamine 2000 (1:1, v / v). Gently rotate to evenly mix the plasmids in each well. Then, the plate was placed in an incubator at a preset temperature for 24 hours. Transfection was stopped by replacing freshly prepared DMEM culture medium. After treatment, the culture medium was removed and the wells were rinsed once with PBS. The cells were then lysed. Dual luciferase reporter gene assay was performed according to the manufacturer's instructions (Catalog #E1910; Promega, Fitchburg, Wisconsin). All luminescent data of firefly and Renilla luciferase were read by Promega luminescence detector. The relative firefly / Renilla luciferase activity value of each sample was calculated by multiple by comparing the mean values ​​of the experimental group and the control group.

[0092] Statistical analysis. All data are expressed as mean ± standard error (n = 5-6 / treatment). Comparison of data from two treatment groups was analyzed by Student's t-test. Data from more than two treatment groups were compared by one-way analysis of variance (ANOVA) followed by Duncan's multiple comparison test (Sigmaplo; Systat Software, Inc.; San Jose, CA). p Data <0.05 were considered statistically significant.

[0093] Results: Temporal responses of haloperidol, SCH-23390, and SKF-81297 to DRD1 mRNA and protein expression in SH-SY5Y cells. Figure 1AAs shown, 10 μM haloperidol did not alter DRD1 mRNA expression at any time after treatment. In contrast, 10 μM SCH-23390 reduced DRD1 mRNA expression after 18 and 24 hours, but not at other time points. 10 μM SKF-81297 increased DRD1 mRNA expression in a time-dependent manner, reaching 6-fold after 24 hours. Then, DRD1 mRNA expression decreased to basal levels after 30 hours and thereafter. DRD1 protein was not detectable in the cytoplasm. As Figure 1B As shown, haloperidol treatment did not significantly change the DRD1 membrane protein level.

[0094] refer to Figure 2A-2E As shown, the results showed that haloperidol reversed SCH-23390-reduced DRD1 mRNA and protein expressions in SH-SY5Y cells.

[0095] like Figure 2A As shown in Figure 2, haloperidol did not alter DRD1 mRNA expression. SCH-23390 treatment for 18 hours reduced DRD1 mRNA expression, which was completely reversed by co-treatment with haloperidol for 12 hours. In contrast, SCH-23390 treatment for 24 hours still significantly reduced DRD1 mRNA expression, and co-treatment with haloperidol for 12 hours could not reverse this. This was due to insufficient co-treatment time.

[0096] After 36 hours of SCH23390 treatment, DRD1 membrane protein decreased by more than 70%. Co-treatment with haloperidol for 24 hours significantly restored the SCH-reduced DRD1 membrane protein to nearly 80% (Figure 2C). DRD1-positive cells appeared dark brown. Figure 2D As shown, haloperidol did not significantly change the counts of DRD1-positive cells. However, SCH significantly reduced the counts, which was reversed by haloperidol co-treatment ( Figure 2D and Figure 2E ).

[0097] refer to Figures 3A-3E As shown, the results showed that haloperidol attenuated SKF-81297-increased DRD1 mRNA and protein expressions in SH-SY5Y cells.

[0098] SKF-81297 treatment for 18 and 24 hours increased human DRD1 mRNA expression ( Figure 3A Haloperidol did not alter DRD1 mRNA expression after 18 or 24 hours. Haloperidol treatment for 12 hours completely reduced SKF-81297-induced DRD1 mRNA expression ( Figure 3ASKF-81297 treatment for 36 hours increased DRD1 membrane protein levels by more than 2-fold ( Figure 3B and 3C ). Haloperidol co-treatment for 24 hours significantly attenuated the SKF-81297-increased DRD1 membrane protein levels. Immunocytological staining showed that SKF-81297 increased the human DRD1-positive cell count by nearly 5 times compared with the control group ( Figure 3D ). This increase was completely abolished after co-treatment with haloperidol.

[0099] refer to Figure 4A-4B , the results showed that haloperidol reversed the DRD1 promoter activity altered by SCH23390 or SKF21897.

[0100] The engineered recombinant pGL3 reporter construct containing the 2.8 kb human DRD1 gene promoter was transfected into SH-SY5Y cells. Luciferase reporter gene assay showed that haloperidol did not change the human DRD1 gene promoter activity ( Figure 4A In contrast, SCH-23390 treatment for 36 h trans-inhibited DRD1 promoter activity, whereas co-treatment with haloperidol for 24 or 36 h reversed this activity ( Figure 4A In addition, SKF-81297 treatment for 36 h transactivated DRD1 promoter activity ( Figure 4B ), and co-treatment with haloperidol for 24 and 36 h significantly attenuated this activity.

[0101] Similarly, the regulation of DRD5 mRNA by SCH+haloperidol and SKF+haloperidol was also studied. Fig. 7A and 7B The results showed that the effects of SCH+haloperidol and SKF+haloperidol on DRD5 were similar to those on DRD1.

[0102] The effects of several antipsychotic drugs on DRD1 promoter activity have also been investigated, including risperidone, clozapine, and cariprazine.

[0103] 5A and 5B of the accompanying drawings, the results show that risperidone trans-stabilizes the DRD1 promoter activity altered by SCH-2339 or SKF-81297.

[0104] See also Fig. 6A and 6B , the results showed that clozapine trans-stabilized the DRD1 promoter activity altered by SCH-23390 or SKF-81297.

[0105] The results also indicated that cariprazine trans-stabilized the DRD1 promoter activity altered by SCH-23390 or SKF-81297.

[0106] In summary, our results show that: 1) 10 μM haloperidol did not alter DRD1 expression. In contrast, 10 μM SCH-23390 decreased, while 10 μM SKF-81297 increased DRD1 mRNA and protein expression in a time-dependent manner.

[0107] 2) Haloperidol co-treatment reversed the SCH-23390-reduced DRD1 mRNA and protein expressions.

[0108] 3) The up-regulated DRD1 mRNA and protein expressions by SKF-81297 were attenuated by co-treatment with haloperidol.

[0109] 4) Dual luciferase reporter gene assays showed that SCH-23390 transactivated, while SKF-81297 inhibited the activity of the 2.8 kb human DRD1 gene promoter. In addition, haloperidol reversed the human DRD1 promoter activity altered by SCH-23390 or SKF-81297.

[0110] 5) Similar to haloperidol, risperidone and clozapine reversed and normalized the human DRD1 promoter activity altered by SCH-23390 or SKF-81297. These data suggest that antipsychotics can bidirectionally normalize chemical alterations in human DRD1 expression by targeting its upstream promoter sequence. These findings support why most antipsychotics are effective in treating bipolar disorder and substance-induced psychotic disorders, in which DRD1 expression is genetically normal but chemically altered.

[0111] With reference to the attached drawings Figure 8 , shows the relative mRNA expression of DRD1 and DRD5 in SH-SY5Y cells. SHSY5Y cells were cultured in T-25 flasks (1x10 6 cells / T-25 flask) and treated as follows: (a) Haloperidol: Haloperidol (10 μM) for 20 hours; (b) SCH: SCH-23390 (10 μM) for 20 hours: (c) SKF: SKF-21987 (10 μM) for 20 hours; (d) Haloperidol + SCH: SCH23390 (10 μM) and haloperidol (10 μM), in which SCH-23390 was pretreated for 6 hours, and then SCH-23390 and haloperidol were co-treated for another 14 hours; (e) Haloperidol + SKF: SKF81297 (10 μM) and haloperidol (10 μM), in which SKF-81297 was pretreated for 6 hours, and then SKF81297 and haloperidol were co-treated for another 14 hours; (f) DMSO treatment was used as a control treatment.

[0112] After treatment, SH-SY5Y cells were harvested and subjected to real-time quantitative PCR analysis. Data are presented as the mean of three individual samples for each treatment.

[0113] Clinical studies of DRD1 and DRD5 genomic sequence variations in human subjects: The genomic sequences of human DRD1 and DRD5 were analyzed using an Illumina MiniSeq instrument. Genomic DNA was extracted from human blood using the Qiagen Blood and Cell Culture DNA Kit according to the manufacturer's instructions (Qiagen, Beverly, MA). Genomic DNA sequence data were analyzed using the Illumina Apps DNA Amplicon and DRAGEN Amplicon.

[0114] Three typical human subjects were selected to demonstrate the relationship between variations in human DRD1 genomic DNA and mental disorders.

[0115] The results are summarized in Table 1: Variation of human DRD1 genomic DNA (including 7 kb 5'-regulatory sequence, gene coding sequence and 1 kb 3'-regulatory sequence) in three human subjects with psychiatric disorders.

[0116] In our clinical studies, at least one variant of DRD1 or DRD5 was found in human subjects with psychiatric disorders. The results also showed that when the number of variants increased, the severity of the psychiatric disorder increased.

[0117] In other words, mental disorders can be quantitatively identified by locating at least one variation in DRD1 or at least one variation in DRD5. More severe types of mental disorders can be identified by two or more variations in DRD1 and DRD5.

[0118] Those skilled in the art will appreciate that the embodiments of the present invention shown in the drawings and described above are intended to be illustrative only and not restrictive.

[0119] It can be seen that the purpose of the present invention has been fully and effectively achieved. The embodiments of the present invention have been shown and described for the purpose of illustrating the functional and structural principles of the present invention, and changes can be made without departing from these principles. Therefore, the present invention includes all modifications included in the spirit and scope of the appended claims.

Claims

1. A method for treating a mental disorder in a subject, comprising the steps of normalizing the DRD1 level and DRD1 expression of the subject to a preset standard level.

2. The method for treating a mental disorder in a subject according to claim 1, characterized in that Further comprising the step of increasing or decreasing the subject's constitutive expression of DRD1 by targeting an upstream promoter sequence of DRD1.

3. The method for treating a mental disorder in a subject according to claim 1, characterized in that: The method further comprises the step of normalizing the subject's DRD5 level and DRD5 expression to a preset standard level.

4. The method for treating a mental disorder in a subject according to claim 3, characterized in that: Further comprising the steps of: increasing or decreasing the subject's constitutive expression of DRD1 or DRD5 by targeting the upstream promoter sequence of DRD1 or DRD5; and attenuating the promoter activity of DRD1 or DRD5 by using an antipsychotic drug that directly targets the DRD1 or DRD5 promoter.

5. The method for treating a mental disorder in a subject according to claim 3, characterized in that: The antipsychotic is selected from the group consisting of haloperidol, risperidone, clozapine and cariprazine.

6. The method for treating a mental disorder in a subject according to claim 4, wherein: The antipsychotic is selected from the group consisting of haloperidol, risperidone, clozapine and cariprazine.

7. The method for treating a mental disorder in a subject according to claim 1, wherein: The subject has impaired constitutive expression of the DRD1 gene.

8. The method for treating a mental disorder in a subject according to claim 2, wherein: The subject has impaired constitutive expression of the DRD1 gene.

9. The method for treating a mental disorder in a subject according to claim 4, wherein: The subject has impaired constitutive expression of the DRD1 or DRD5 gene.

10. The method for treating a mental disorder in a subject according to claim 2, wherein: The psychiatric disorders include schizophrenia, autism, depression, mania and bipolar disorder.

11. The method for treating a mental disorder in a subject according to claim 4, wherein: Mental disorders include schizophrenia, autism, depression, mania, and bipolar disorder.

12. A method for treating a mental disorder in a subject, comprising the step of: bidirectionally normalizing the overall expression level of DRD1, DRD5, or DRD1 and DRD5 of the subject to a preset standard level.

13. The method for treating a mental disorder in a subject according to claim 12, characterized in that: Further comprising the step of attenuating the promoter activity of DRD1 / DRD5 by using an antipsychotic drug that directly targets the DRD1 and / or DRD5 promoter.

14. The method for treating a mental disorder in a subject according to claim 13, wherein: The antipsychotic is selected from the group consisting of haloperidol, risperidone, clozapine and cariprazine.

15. The method for treating a mental disorder in a subject according to claim 13, characterized in that: The psychiatric disorders include schizophrenia, autism, depression, mania and bipolar disorder.

16. The method for treating a mental disorder in a subject according to claim 14, wherein: The psychiatric disorders include schizophrenia, autism, depression, mania and bipolar disorder.

17. The method for treating a mental disorder in a subject according to claim 13, characterized in that: The method further comprises the steps of: providing a sequence database, wherein the polymorphism of the DRD1 and / or DRD5 promoter sequence is associated with the drug efficacy; and performing personalized drug screening on the efficacy of antipsychotic drugs based on the sequence database.

18. A quantitative screening method for mental disorders in subjects, comprising the steps of: performing genome sequencing on the DRD1 and DRD5 genes of multiple subjects to establish a database of mental disorders and variation distribution in the DRD1 and DRD5 genes; screening out one or more variants that are highly correlated with a mental disorder to form target gene sequences of the DRD1 and DRD5 genes for determining the risk of mental disorders; performing target gene sequencing on the DRD1 and DRD5 genes in the target subjects to determine the risk of mental disorders in the target subjects; the DRD1 and DRD5 genes of multiple subjects include a 7kb 5'-upstream sequence, a protein coding sequence, and a 2kb 3'-downstream sequence; the database of mental disorders and variation distribution includes the type, location, and frequency of each gene variation.

19. A quantitative screening method for psychiatric disorders in subjects, wherein two or more variants are associated with a psychiatric disorder.

20. The method for quantitative screening of mental disorders in a subject according to claim 19, characterized in that: Polymorphisms in the DRD1 and / or DRD5 promoter sequences that are associated with clinically diagnosed psychiatric disorders are included in the Variant Distribution Database.

21. The method for quantitative screening of mental disorders in a subject according to claim 19, characterized in that: The method is a prenatal screening approach in which polymorphisms in the DRD1 and / or DRD5 promoter sequences that are associated with the risk of clinically diagnosed mental disorders are included in a variant distribution database.

22. A quantitative screening assay for expression of human DRD1 and DRD5 in cells from a human subject, comprising the steps of: Establish normal ranges for human DRD1 and DRD5 expression in cells; Determine human DRD1 and DRD5 expression in cells from human subjects; The expression of human DRD1 and DRD5 in cells of the human subject is compared to a normal range to determine the mental status of the human subject, wherein the cells are selected from the group consisting of mucosal cells, skin cells, and blood cells.

23. A quantitative method for screening new drugs for mental disorders, comprising the steps of: bidirectionally normalizing the overall expression level of DRD1 and / or making the subject's DRD5 reach a preset standard level, wherein the mental disorders include schizophrenia, autism, depression, mania and bipolar disorder, and wherein the sample sequence includes at least one variation compared to a standard sequence database.

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