A quantitative method for determining a mental state based on DRD1 and / or DRD5, and its application

Abstract

A method for treating a mental disorder in a subject, comprising the step of normalizing the expression of both DRD1 and DRD5 belonging to the DRD1-like family of the subject to a preset standard level. The DRD1 / 5 level and expression can be increased or decreased bidirectionally by targeting the upstream promoter sequence of DRD1 / 5. The promoter activity of DRD1 / 5 can be attenuated by using antipsychotic drugs that directly target the DRD1 / 5 promoter. The antipsychotic drugs used are typical and / or atypical antipsychotic drugs such as haloperidol, risperidone, clozapine, and cariprazine. This treatment is also useful for subjects in whom the constitutive expression of the DRD1 / 5 gene is impaired. This method can be used to treat schizophrenia, autism, depression, mania, and bipolar disorder by normalizing the DRD1 / 5 expression level. The promoter gene sequencing of DRD1 and / or DRD5 can also be used for quantitative mental health screening by comparing with a standard sequence database.

Description

Technical Field

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

Background Art

[0002] <Description of Related Art> Currently, there is no quantitative method for determining the mental state of a human subject. For example, psychoses that interfere with the ability to function are determined by symptoms such as delusions and hallucinations. The etiology of psychotic disorders is unknown, and commercially available antipsychotics do not have common modes of action.

[0003] The diagnosis of mental disorders depends largely on the signs and symptoms of the human subject. Examples include feelings of sadness or depression, confused thinking or decreased concentration, excessive fear or worry, or extreme feelings of guilt, high and low mood swings, withdrawal from friends and activities, severe fatigue, sleep problems, delusions, paranoia or hallucinations, excessive anger, hostility or violence. Sometimes, the symptoms may include stomach aches, back aches, headaches, or other unexplained pains and aches. Since there are numerous possible symptoms mainly based on the behavior and thinking of the human subject, it is very difficult to determine the mental state of the human subject.

[0004] Currently, schizophrenia is associated with genetics. However, it is not thought to be caused by a single gene, and people with family members with psychotic disorders have only been determined to be at high risk. The cause or association remains unknown.

[0005] Existing antipsychotics are typically DRD2 antagonists. However, the current understanding of whether DRD2 is involved in the action of antipsychotics still remains controversial. Serotonin and dopamine are two common neurotransmitters that are thought to directly affect mental disorders. Disclosure of the Invention Problems to be Solved by the Invention

[0006] <Summary of the Invention> An 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, as well as its application and use.

[0007] Another object of the present invention is to provide an antipsychotic for normalizing chemically altered DRD1 expression.

[0008] Another object of the present invention is to provide an antipsychotic that similarly affects DRD1 and DRD5 expression, which can normalize or stabilize both up-regulated and down-regulated DRD1 and DRD5 expression to normal expression levels.

[0009] Another object of the present invention is to provide an antipsychotic that directly targets the DRD1 and / or DRD5 promoter sequences.

[0010] Another object of the present invention is to provide an antipsychotic with a new mechanism of action when compared to conventional antipsychotics, and thus it is possible to develop new drugs based on maintaining the normalized expression levels of DRD1 and DRD5.

[0011] Another object of the present invention is to provide an antipsychotic for normalizing DRD1 expression by targeting the 5'-control 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 treatment method for restoring both positive and negative symptoms in psychotic disorders by neutralizing an important process involving bidirectional regulation of DRD1 and DRD5 expression.

Means for Solving the Problems

[0014] Additional effects and features of the present invention will become apparent from the following description and can be realized by the means and combinations particularly shown in the appended claims.

[0015] According to the present invention, the above and other objects and advantages are achieved by a method for treating a mental disorder in a subject, the method comprising the following steps:

[0016] Normalizing the DRD1 level and DRD1 expression in the subject to a preset standard level.

[0017] Preferably, the DRD1 level and DRD1 expression can be increased or decreased bidirectionally by targeting the upstream promoter sequence of DRD1. The promoter activity of DRD1 can be attenuated by using an antipsychotic drug that directly targets the DRD1 promoter. The antipsychotic drug used can be one or more of typical and atypical antipsychotics such as haloperidol, risperidone, clozapine, and cariprazine. This treatment is also useful for subjects in whom the constitutive genetic expression of DRD1 is impaired. This method can be used to treat schizophrenia, autism, depression, mania, and bipolar disorder by normalizing the DRD1 expression level.

[0018] According to another aspect of the present invention, a method for treating a psychiatric disorder in a subject comprises the steps of:

[0019] Bidirectionally normalizing the subject's overall expression levels of DRD1 and DRD5 to a pre-established standard level.

[0020] Preferably, the method further comprises the step of attenuating promoter activity of DRD1 / DRD5 by using an antipsychotic drug that directly targets the DRD1 and DRD5 promoters.

[0021] Preferably, the method further comprises the steps of providing a sequence database in which polymorphisms in the DRD1 and DRD5 promoter sequences are associated with drug efficacy, and personalizing medication screening of antipsychotic drugs for efficacy based on the sequence database.

[0022] According to another aspect of the present invention, a method for screening a subject for a psychiatric disorder comprises the steps of performing promoter gene sequencing of DRD1 and DRD5 of the subject to obtain a sample sequence, and comparing the sample sequence with a standard sequence database of human DRD1 / DR5 promoter to determine whether the subject is suffering from a psychiatric disorder.

[0023] Preferably, polymorphisms in the DRD1 / DRD5 promoter sequence associated with clinically diagnosed psychiatric disorders are included in the standard sequence database.

[0024] Still further objects and advantages will become apparent from a consideration of the following description and drawings.

[0025] 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 explanation of the drawings]

[0026] Figure 1A shows the relative DRD1 gene mRNA expression under haloperidol (10 μM), SCH-23390 (10 μM), and SKF-81297 (10 μM) treatment in SH-SY5Y cells.

[0027] Figure 1B shows the results of DRD1 protein expression under haloperidol treatment in SH-SY5Y cells.

[0028] Figure 1C shows the relative protein expression of DRD1 under haloperidol treatment in SH-SY5Y cells.

[0029] Figure 2A shows the relative DRD1 mRNA expression of SCH-23390 treatment in SH-SY5Y cells.

[0030] Figure 2B shows the results of DRD1 protein expression under SCH-23390 treatment in SH-SY5Y cells.

[0031] Figure 2C shows the relative protein expression of DRD1 in SH-SY5Y cells.

[0032] Figure 2D is a diagram showing ICC staining of SHSY5Y (36 hours) treated with SCH under different treatments.

[0033] Figure 2E shows the percentage of positive H-DRD1 cells on SH-SY5Y cells under different treatments.

[0034] Figure 3A shows the relative DRD1 mRNA expression of SKF-81297 treatment in SH-SY5Y cells.

[0035] Figure 3B shows the results of DRD1 protein expression under SKF-81297 treatment in SH-SY5Y cells.

[0036] Figure 3C shows the relative protein expression of DRD1 in SKF-81297 treatment in SH-SY5Y cells.

[0037] Figure 3D shows the ICC staining of SHSY5Y treated with SKF under different treatments.

[0038] Figure 3E shows the percentage of positive H-DRD1 cells on SHSY-5Y cells under different treatments.

[0039] Figure 4A shows the relative DRD1 promoter activity under SCH-23390 treatment in SH-SY5Y cells.

[0040] Figure 4B shows the relative DRD1 promoter activity under SKF-81297 treatment in SH-SY5Y cells.

[0041] Figures 5A and 5B show the study of the control of DRD1 promoter activity by risperidone. Figure 5A shows the relative luciferase activity of DRD1 under the treatment of SCH and risperidone. (Risperidone has a partial DRD1 antagonist effect using co-treatment with SCH) Figure 5B shows the relative luciferase activity of DRD1 under the treatment of SKF and risperidone. (Risperidone has a partial DRD1 antagonist effect using co-treatment with SKF)

[0042] Figures 6A and 6B show the study of the control of DRD1 promoter activity by clozapine. Figure 6A shows the relative luciferase activity of DRD1 under the treatment of SCH and clozapine (clozapine has a partial DRD1 antagonist effect using co-treatment with SCH), Figure 6B shows the relative luciferase activity of DRD1 under the treatment of SKF and clozapine (clozapine has a partial DRD1 antagonist effect using co-treatment with SKF)

[0043] Figures 7A and 7B show the study of the control of DRD5 mRNA expression. Figure 7A shows the relative DRD5 mRNA expression by the treatment of SCH-23390 on SHSY-5Y cells. Figure 7B shows the relative DRD5 mRNA expression by the treatment of SKF-81297 and H on SHSY-5Y cells.

[0044] Figure 8 shows the relative mRNA expression of DRD1 and DRD5 in SHSY5Y cells.

Mode for Carrying Out the Invention

[0045] <Detailed Description of Preferred Embodiments> According to the present invention, DRD1 and DRD5, particularly their protein expression levels, play important roles in maintaining the active state of individual functional cells such as neurons, muscles, and endothelial cells.

[0046] Dopamine receptor D1 (DRD1) encoded by the DRD1 gene is one of two members of the DRD1-like receptor family that includes DRD1 and DRD5. DRD1 is usually abundant in the central nervous system. DRD1 plays important roles in memory, learning, addiction, reward system, behavior patterns, and neuron development. DRD1 is one of the most abundant dopamine receptors in the human brain and plays an important role in the etiology of mental disorders including schizophrenia, autism, depression, mania, and bipolar disorder.

[0047] DRD1 and DRD5 belong to the D1-like dopamine 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 maintaining the mental state, which can help maintain genetic and functional stability and further explain the diversity of mental disorders.

[0048] According to the present invention, DRD1 dysfunction is associated with mental disorders. Psychoses including bipolar disorder, depression, mania, and schizophrenia are essentially promoter disorders. Psychoses are mainly due to promoter-related gene expression disorders, particularly by DRD. Therefore, antipsychotics can function by normalizing chemically altered DRD1 expression back to constitutive levels. The action of antipsychotics is two-ways and can normalize both upregulated and downregulated DRD1 expression. Antipsychotics exert a "hormesis" effect by normalizing DRD1 expression.

[0049] According to the present invention, there are two possible mechanisms for the control of DRD1 expression:

[0050] (a) There are two individual elements in the DRD1 promoter sequence: one is involved in the attenuation of upregulated DRD1 expression; the other is involved in the reversal effect (or, reversal effect) of antipsychotics on downregulated DRD1 expression.

[0051] (b) There are specific elements in the DRD1 promoter. When these target this sequence, antipsychotics block the chemically altered DRD1 expression mechanism and thus stabilize DRD1 expression to constitutive expression levels.

[0052] It is worth mentioning that clinically existing antipsychotics are more effective in bipolar disorder than in schizophrenia. According to the principle of the present invention, the constitutive gene expression of DRD1 is significantly impaired even after antipsychotic treatment. The expression of DRD1 after antipsychotic treatment in schizophrenia patients cannot be restored to the normal DRD1 expression range. As a result, existing antipsychotics are not effective in the treatment of schizophrenia.

[0053] DRD1 and DRD5 together regulate the mental state. The overall expression levels of DRD1 and DRD5 are regulated within a normal range to maintain a normal mental state.

[0054] In psychotic patients, 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, the patient exhibits depression and negative symptoms. In contrast, when the overall expression levels of DRD1 and DRD5 are very low, the patient exhibits mania and positive symptoms. Furthermore, when the promoters of DRD1 and DRD5 are prone to internal or external factors that cause easy drift in their expression, the patient exhibits bipolar disorder.

[0055] The promoter sequencing of human DRD1 and DRD5 can be used for diagnosing and screening mental disorders.

[0056] Our conventional understanding of the mechanism of action of antipsychotics is mainly based on DRD2. For example, all antipsychotics reduce dopaminergic neurotransmission. The main mechanism of action of most first-generation and second-generation antipsychotics is thought to be the postsynaptic blockade of DRD2-like receptors in the brain. Furthermore, most second-generation antipsychotics also inhibit serotonin receptors such as 5HT1A / 2A.

[0057] Since the present invention provides that the overall expression levels of DRD1 and DRD5 can serve as objective factors of mental state, it is now possible to directly correlate the expression levels of DRD1 and / or DRD5 with mental health. Therefore, the effects of antipsychotics on DRD1 and DRD5 can be further studied for drug development.

[0058] For example, DRD1 expression is severely impaired in patients with schizophrenia. At this time, by using the importance of specific DRD1 promoter gene sequences, human mental disorders can be regulated by controlling the levels of DRD1 and DRD5.

[0059] According to a first preferred embodiment of the present invention, a method for treating mental disorders includes the step of normalizing the human DRD1 level and expression to restore them to a normal level; and / or the step of normalizing the human DRD5 level and expression to restore them to a normal level.

[0060] Preferably, the method can further include the step of increasing or decreasing the constitutive expression of DRD1 in a subject by targeting the upstream promoter sequence of DRD1; and / or the step of increasing or decreasing the constitutive expression of DRD5 in a subject by targeting the upstream promoter sequence of DRD5.

[0061] Preferably, the method can further include the step of attenuating the promoter activity of DRD1 or DRD5 by using an antipsychotic drug that directly targets the DRD1 or DRD5 promoter.

[0062] The human DRD1 and DRD5 genes essentially contribute to the maintenance of mental state. Genetic and / or epigenetic changes in the human DRD1 and DRD5 promoter sequences result in mutations (or variations / change) in their expression. Antipsychotic drugs directly target the human DRD1 and DRD5 promoters to normalize the exogenously or endogenously altered human DRD1 and DRD5 expression, resulting in their therapeutic effects.

[0063] Preferably, the method for treating mental disorders includes both a drug therapy (or medications) approach or a non-drug therapy (or non-medications) approach for normalizing human DRD1 expression to restore it within a preset normal level.

[0064] Preferably, the antipsychotic drug is used to normalize the expression levels of DRD1 and / or DRD5. The antipsychotic drug can be selected from different categories of antipsychotic drugs. Haloperidol, a typical antipsychotic drug, and risperidone, clozapine, and cariprazine, atypical antipsychotic drugs, are selected. Thus, these drug therapies can mechanistically represent most antipsychotic drugs.

[0065] According to a second preferred embodiment of the present invention, a method for quantitative screening and testing for mental disorders comprises performing promoter sequencing of the DRD1 and / or DRD5 genes by applying an extracted genomic DNA sample of a human subject, and then screening this sequence and comparing it with a human DRD1 and / or DRD5 promoter sequence database to determine whether the human subject is suffering from a mental disorder.

[0066] If at least one mutation is found in DRD1 or DRD5, the human subject is confirmed to have a mental disorder. If at least two mutations are found in DRD1 or DRD5, the human subject is confirmed to have a mental disorder with increased levels (more severe mental disorder).

[0067] The human DRD1 / DRD5 promoter sequence database is established in a sequencing project. Polymorphisms in the DRD1 / DRD5 promoter sequence are clinically diagnosed in mental disorders in the sequence database. Furthermore, this sequence database is applied to prenatal screening and parental screening to predict the odds of mental disorders in newborns. Mutations in the DRD1 / DRD5 promoter sequence are associated with mental disorders.

[0068] In addition, the method further comprises the step of individualizing medication screening of antipsychotic drugs for efficacy based on a database in which polymorphisms in the DRD1 and DRD5 promoter sequences are related to dosing efficacy.

[0069] 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 a known chemical library, potential drug candidates that can normalize the altered DRD1 and DRD5 expressions can be selected for new drug development.

[0070] According to another preferred embodiment of the present invention, a quantitative screening method for mental disorders in a subject includes the following three steps:

[0071] Step 1: Perform genomic sequencing of the DRD1 and DRD5 genes (including the 7-kb 5′-upstream sequence, protein-coding sequence, and 2-kb 3′-downstream sequence) of a human subject to establish the relationship between mental disorders and a variant distribution database (including the type, position, and frequency of each gene variant) in the DRD1 and DRD5 genes.

[0072] Step 2: Select variants highly associated with each mental disorder and develop target gene sequences of the human DRD1 and DRD5 genes that can predict the risk of developing mental disorders.

[0073] Step 3: Perform the target gene sequences of the DRD1 and DRD5 genes in a new subject to determine the risk of developing mental disorders in the subject.

[0074] In other words, a quantitative method for screening tests of human DRD1 and DRD5 expressions in cells (such as mucosal cells, skin cells, blood cells, etc.) of a human subject can also be provided to establish the normal range of human DRD1 and DRD5 expressions in the cells. If the DRD1 / 5 expression of a new subject is outside the normal range in the cells, it serves as an indicator of the mental state of the subject.

[0075] The typical antipsychotic haloperidol is commonly prescribed 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, a direct and effective correlation for effective quantitative treatment can be established instead of DRD2.

[0076] Experiment 1: Haloperidol-DRD1 Project

[0077] Generally, the action of a drug receptor depends on its binding affinity (Km) for its ligand and its total expression (Vmax). The binding affinity is determined by its protein configuration. In contrast, the expression of the receptor is affected genetically and epigenetically.

[0078] To explain the role of DRD1 in the mechanism of action of antipsychotics, an in vitro study is conducted to determine the regulation of DRD1 expression by several typical and atypical antipsychotics including haloperidol, clozapine, risperidone, and cariprazine in the presence of a DRD1 agonist or antagonist.

[0079] This study is designed to determine whether the widely prescribed antipsychotics haloperidol, risperidone, or clozapine can normalize chemically altered human DRD1 expression in cultured human SH-SY5Y neuroblastoma cells.

[0080] Referring to Figure 1A, the relative DRD1 gene mRNA expression under treatment with haloperidol (Halo) (10 μM), SCH-23390 (10 μM), and SKF-81297 (10 μM) in SH-SY5Y cells is shown.

[0081] SCH-23390, also known as halobenazepine, is a synthetic compound that acts as a D1 receptor antagonist. SKF-81297, a synthetic compound in the benzazepine chemical class, acts as a selective dopamine D1 / D5 receptor full agonist. SH-SY5Y cells are a cloned subline (or subline) of the human neuroblastoma cell line.

[0082] According to this experiment, haloperidol can normalize chemically altered human DRD1 expression in cultured human SH-SY5Y neuroblastoma cells.

[0083] Materials and Methods

[0084] Haloperidol was purchased from Sigma-Aldrich (Catalog No. H1512-10G, Saint Louis, MO). SCH23390 hydrochloride (Catalog No. 09-251-0) and SKF81297 hydrobromide (Catalog No. 14-471-0) were purchased from FISHER SCIENTIFIC (Hanover Park, IL). Lipofectamine reagent was purchased from FISHER SCIENTIFIC (Catalog No. NC0904455, HANOVER PARK, IL). Both the mem-PER Plus Membrane Protein Extraction Kit (Catalog No. 89842) and the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Catalog No. 78833) were purchased from Fisher Scientific (Hanover Park, IL). The Zymo Plasmid Miniprep Kit (Catalog No. D4036) and the DNA Clean&Concentrator Kit (Catalog No. D4013) were purchased from Zymo Research Corporation (Irvine, CA). All other chemicals were purchased from Thermo Fisher Scientific Inc. (Waltham, MA) unless otherwise specified.

[0085] The anti-dopamine receptor D1 (DRD1) antibody was obtained from Abcam (catalog number ab216644, Cambridge, MA). Both Pierce Goat anti-Rabbit IgG (H+L) biotin-conjugated secondary antibody (1:5,000) and Pierce High Sensitivity Streptavidin-HRP 1:5,000 (catalog number 21130) were purchased from Thermo Fisher Scientific (Waltham, MA).

[0086] Cell culture experiments: The human SH-SY5Y neuroblastoma cell line, originally purchased from ATCC (catalog number CRL-2266, Manassas, VA), was cultured in Eagle's Minimum Essential Medium (EMEM) (catalog number ATCC® 30-2003, ATCC, Manassas, VA) supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals; Norcross, GA). The CO2 cell incubator was maintained at 37 °C with 5% CO2 in a humidified atmosphere. Cells treated in 6-well plates were harvested for mRNA analysis by performing quantitative real-time PCR assays. When the drug treatment exceeded 24 hours, cells were seeded in 6-well plates at a concentration of 3×10 5 / well, and when the drug treatment was less than 24 hours, cells were seeded in 6-well plates at a concentration of 4×10 5 / well. Cells treated in T-75 flasks were harvested for protein analysis by performing Western blot assays. Cells were seeded in T-75 flasks at a concentration of 3×10 6 / flask (65% confluency). Cells seeded in 96-well plates at a concentration of 2.5×10 4 / well were used for promoter analysis by performing a Dual-Luciferase reporter assay.

[0087] <Timeless response research.> As the starting dose in the timeless response research, we selected 10 μM haloperidol, 10 μM SCH23390, or 10 μM SKF81297. Cultured SH-SY5Y cells were treated with complete EMEM medium containing 10 μM haloperidol, 10 μM SCH23390, or 10 μM SKF81297 for 12, 18, 24, 30, or 32 hours respectively. The control received only DMSO. After each period, the cells were harvested and processed for mRNA analysis of the human DRD1 gene.

[0088] Co-treatment test of haloperidol and SCH23390. Cultured SH-SY5Y cells were pretreated with complete EMEM medium containing 10 μM SCH23390 for 6 hours or 12 hours. Then, the cells were treated with complete EMEM medium containing 10 μM haloperidol and 10 μM SCH23390 for 12 hours or 24 hours. After the treatment, the cells were harvested and processed for mRNA and protein analysis of the human DRD1 gene.

[0089] Co-treatment test of haloperidol and SKF81297. Cultured SH-SY5Y cells were pretreated with complete EMEM medium containing 10 μM SKF81297 for 6 hours or 12 hours. Then, the cells were treated with complete EMEM medium containing 10 μM haloperidol and 10 μM SKF81297 for 12 hours or 24 hours. After the treatment, the cells were harvested and processed for mRNA and protein analysis of the human DRD1 gene.

[0090] Total RNA extraction and complementary DNA (cDNA) preparation

[0091] RNA extraction was performed using TRIzol (trademark) RNA extraction reagent (catalog number 15596026; Life Technologies; Carlsbad, CA). The concentration and purity of total RNA were measured at 260 nm using a Biospectrometer (Eppendorf, Hauppauge, NY). Only RNA samples with an A260 / A280 ratio between 1.8 and 2.0 were used for cDNA synthesis. Briefly, 3 μg of total RNA was reverse transcribed into cDNA using Random and Oligo 9dT) primers (catalog number PR-C1181, Fisher Scientific, Hanover Park, IL), Oilgo dT (catalog number FERSO131, Fisher Scientific, Hanover Park, IL), and SuperScript II reverse transcriptase (Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. The synthesized cDNA samples were stored at -20°C.

[0092] Quantitative real-time PCR (qRT-PCR) assay

[0093] 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. Generally, each SYBR reaction in each well contained 5 μl of SYBR Select Master Mix, 0.2 μl of 10 μM gene-specific primer set, 1.8 μl of deionized distilled water, and 3 μl of cDNA sample. The qRT-PCR assay was performed on an AriaMx qRT-PCR system (Agilent Technologies; Santa Clara, CA). The relative mRNA expression of each individual gene was calculated according to the comparative delta-delta CT method. The results were expressed as relative fold (or relative multiple) 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 MWG Operon USA (Louisville, KY).

[0094] The sequences of the RT-PCR primers for human DRD1 are as follows:

[0095] Gene human DRD1, forward primer: [SEQ ID: 1] 5′-CCATCACACAAAACGGTCAG-3′, reverse primer: [SEQ ID: 2] 5′-GTGTGTTGGAAAGCAGCAGA-3?

[0096] Gene 18s rDNA, NM number NR_003278.3, forward primer [SEQ ID: 3] 5′-GCAATTATTCCCCATGAACG-3′; reverse primer [SEQ ID: 4] 5′-GCCTCACTAAACCATCCAA-3′

[0097] Western Blotting

[0098] Human SH-SY5Y neuroblastoma cells were grown to 65% confluence in a T-75 flask and then treated with the chemical substances as described above. Membrane proteins were extracted using the mem-PER Plus Membrane Protein Extraction Kit (ThermoFisher Scientific, Waltham, MA). Protein concentration was semi-quantified spectrophotometrically 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 onto a polyvinyl difluoride (PVDF) membrane. The membrane was then blocked in Tris-buffered saline (TBS) supplemented with 2.5% non-fat milk. After blocking, the membrane was incubated overnight at 4 °C with a primary antibody (human DRD1 antibody, 1:1000). The next day, the membrane was washed three times with fresh TBS and then incubated for 2.5 hours at room temperature with a secondary antibody [goat anti-rabbit biotin-conjugated antibody (Abcam; Cambridge, MA) (1:5000)] in TBS supplemented with 2.5% BSA. The membrane was briefly washed three times with fresh TBS and then incubated for 30 minutes at room temperature with Pierce™ High Sensitivity Streptavidin HRP-linked secondary antibody (1:5000) in TBS supplemented with 0.5% BSA. The membrane was rinsed three times with fresh TBS. Immunoreactive protein bands in the membrane were visualized using Immobilon Chemiluminescence reagents (Millipore, Billerica, MA) via a UVP Biospectrum Imaging system (Upland, CA). Protein bands analysis was performed using ImageJ (NIH, Bethesda, MD).Furthermore, the protein expression of beta-actin, which is a loading control, was determined using a primary antibody (beta-actin monoclonal antibody, 1:2000) and the corresponding secondary antibody [goat anti-mouse IgG (H+L) secondary antibody, HRP 1:5000 (catalog number 31430)].

[0099] <Immunocytochemistry (ICC) staining.> Human SH-SY5Y neuroblastoma cells were seeded in chamber slides (1×10 5 / well), and then treated with the chemical substances as described above. Fixation and blocking were performed as previously described (Bu et al., 2016). Briefly, the cells were incubated overnight at 4°C with an antibody (1:100) against human DRD1 protein (1;1000). The next day, the chamber slides were incubated for 4 hours at room temperature with a 1:300 Pierce goat anti-rabbit IgG (H+L) biotin-conjugated secondary antibody (Pierce Goat Anti-Rabbit IgG(H+L) Biotin Conjugated secondary antibody) (catalog number 31820; Thermo Fisher Scientific Inc.). Then, the cells were washed with 1×PBS and incubated for 1 hour at room temperature with 1:400 Pierce High Sensitivity Streptavidin-HRP (Pierce High Sensitivity Streptavidin-HRP) (catalog number 21130; Thermo Fisher Scientific Inc.). After the final wash, color development was achieved using ImmPACT NovaRED Peroxidase (HRP) substrate (catalog number SK4805; Vector Laboratories, Inc.; Burlingame, CA) according to the manufacturer's instructions.

[0100] <Cloning of the human DRD1 gene promoter.> The human DRD1 gene promoter sequences (172 bp, 312 bp, 473 bp, 961 bp, and 2772 bp upstream of the transcription start site) were synthesized and engineered into the pGL3-basic vector (catalog number E1751; Promega; Fitchburg, WI) at the polylinker site between NheI and XhoI by GenScript (Piscataway, NJ). The sequences of the recombinant pGL3 constructs were verified by GenScript (Piscataway, NJ) or Eurofins (Eurofins MWG Operon USA, Louisville, KY).

[0101] <DNA transfection and Dual-Luciferase Reporter Assay.> The pGL3-basic vector (Catalog No. E1751; Promega; Fitchburg, WI) and recombinant pGL3 vectors containing human DRD1 gene promoters of various lengths were transfected into human SH-SY5Y cells according to the manufacturer's instructions using Lipofectamine 2000 transfection reagent (Catalog No. 11668027; Life Technologies, Inc.; Carlsbad, CA). Briefly, cells were seeded in 96-well plates. Then, 180 - 200 ng of pGL3 plasmid DNA (either the empty pGL3-basic vector or a recombinant pGL3 vector containing the human DRD1 gene promoter) and 10 ng of pRL-CMV Renilla luciferase control reporter vector (Catalog No. E2261; Promega; Madison, Wisconsin) were transfected into each well and mixed with Lipofectamine 2000 (1:1, v / v). Gently vortex to evenly mix the plasmids in each well. Then, leave the plate in the incubator at the preset temperature for 24 hours. Transfection was stopped by replacing with freshly prepared DMEM medium. After treatment, the medium was removed and the wells were rinsed with PBS. Then, the cells were lysed. The Dual-Luciferase Reporter Assay was performed according to the manufacturer's instructions (Catalog No. E1910; Promega; Fitchburg, WI). All luminescence data for both firefly and Renilla luciferase were read with a luminometer (Promega). The relative firefly / Renilla luciferase activity value for each sample was calculated in fold by comparing the mean value of the experimental group with the control group.

[0102] <Statistical analysis> All data are presented as mean ± standard error (n = 5 - 6 / treatment). Data comparison between two treatment groups was analyzed by Student’s t-test. Data comparison from three or more treatment groups was analyzed by one-way ANOVA, followed by Duncan’s post-hoc (Sigmaplot; Systat Software, Inc.; San Jose, CA), and data with p < 0.05 were considered statistically significant.

[0103] Results:

[0104] <Time course of DRD1 mRNA and protein expression by haloperidol, SCH23390, and SKF81297 in SH-SY5Y cells.> As shown in Figure 1A, 10 μM haloperidol did not change DRD1 mRNA expression at any time point after treatment. In contrast, 10 μM SCH23390 decreased DRD1 mRNA expression at 18 and 24 h, but not at other time points. 10 μM SKF81297 increased DRD1 mRNA expression in a time-dependent manner, reaching 6-fold after 24 h. Subsequently, the expression of DRD1 mRNA decreased to basal levels after 30 h. DRD1 protein was not detectable in the cytosolic fraction. As shown in Figure 1B, haloperidol treatment did not change DRD1 membrane protein levels.

[0105] Referring to Figures 2A - 2E of the drawings, the results show that haloperidol (Halo) reverses the decrease in DRD1 mRNA and protein expression induced by SCH23390 in SH-SY5Y cells.

[0106] As shown in Fig. 2A, haloperidol did not change DRD1 mRNA expression. 18-hour SCH23390 treatment decreased DRD1 mRNA expression, which was completely reversed by 12-hour co-treatment with haloperidol. In contrast, 24-hour SCH23390 treatment still significantly decreased DRD1 mRNA expression, which was not reversed by 12-hour co-treatment with haloperidol. This is due to insufficient co-treatment time.

[0107] 36-hour SCH23390 treatment decreased DRD1 membrane protein by more than 70%. 24-hour co-treatment with haloperidol significantly restored the DRD1 membrane protein decreased by SCH to nearly 80% (Fig. 2C). DRD1-positive cells are shown in dark brown. As shown in Fig. 2D, haloperidol did not significantly change the number of DRD1-positive cells. SCH significantly decreased the number, which was reversed by co-treatment with haloperidol (Fig. 2D and Fig. 2E).

[0108] Referring to FIGS. 3A-3E of the drawings, the results show that haloperidol (Halo) attenuated the DRD1 mRNA and protein expression increased by SKF81297 in SH-SY5Y cells.

[0109] 18-hour and 24-hour SKF81297 treatment increased human DRD1 mRNA expression (Fig. 3A). On the other hand, haloperidol did not change DRD1 mRNA expression after 18 or 24 hours. 12-hour co-treatment with haloperidol completely decreased the DRD1 mRNA expression increased by SKF81297 (Fig. 3A). 36-hour SKF81297 treatment increased the membrane DRD1 protein level by more than two-fold (Fig. 3B and 3C). 24-hour co-treatment with haloperidol significantly attenuated the DRD1 membrane protein level increased by SKF81297. Immunocytostaining showed that SKF81297 increased the number of human DRD1-positive cells by nearly five-fold compared to the control (Fig. 3D). Such an increase completely disappeared after co-treatment with haloperidol.

[0110] Referring to FIGS. 4A - 4B of the drawings, the results show that haloperidol reversed the DRD1 promoter activity altered by SCH23390 / SKF21897.

[0111] An engineered recombinant pGL3 reporter construct containing the 2.8 kb human DRD1 gene promoter was transfected into SH - SY5Y cells. The luciferase reporter assay showed that haloperidol did not change the human DRD1 gene promoter activity (FIG. 4A). In contrast, 36 - hour SCH23390 treatment trans - repressed the DRD1 promoter activity, which was reversed by 24 - hour or 36 - hour co - treatment with haloperidol (FIG. 4A). Furthermore, 36 - hour SKF81297 treatment trans - activated the DRD1 promoter activity (FIG. 4B), which was significantly attenuated by co - treatment with haloperidol for both 24 hours and 36 hours.

[0112] Similarly, the regulation of DRD5 mRNA by SCH + haloperidol (Halop) and SKF + haloperidol has also been studied. The results are shown in FIGS. 7A and 7B. The results show that the effects of SCH + haloperidol and SKF + haloperidol on DRD5 are similar to those on DRD1.

[0113] The effects of several antipsychotics, including risperidone, clozapine, and cariprazine, on DRD1 promoter activity have also been studied.

[0114] Referring to FIGS. 5A and 5B of the drawings, the results show that risperidone trans - stabilizes the DRD1 promoter activity altered by SCH23390 / SKF81297.

[0115] Referring to FIGS. 6A and 6B of the drawings, the results show that clozapine trans - stabilizes the DRD1 promoter activity altered by SCH23390 / SKF81297.

[0116] The results also show that cariprazine trans-stabilizes the DRD1 promoter activity altered by SCH23390 / SKF81297.

[0117] In summary, our results show the following:

[0118] 1) 10 μM haloperidol does not change DRD1 expression. In contrast, 10 μM SCH23390 decreases it, while 10 μM SKF81297 increases DRD1 mRNA and protein expression in a time-dependent manner.

[0119] 2) The DRD1 mRNA and protein expression decreased by SCH23390 are reversed by co-treatment with haloperidol.

[0120] 3) The upregulated DRD1 mRNA and protein expression by SKF-81297 are attenuated by co-treatment with haloperidol.

[0121] 4) The dual luciferase reporter assay demonstrates that SKF81297 inhibits the activity of the 2.8 kb human DRD1 gene promoter, while SCH23390 trans-activates it. Furthermore, haloperidol reversed the human DRD1 promoter activity altered by either SCH23390 or SKF81297.

[0122] 5) Similar to haloperidol, risperidone and clozapine reverse and normalize the human DRD1 promoter activity altered by either SCH23390 or SKF81297. These data indicate that antipsychotics can bidirectionally normalize chemically altered human DRD1 expression by targeting its upstream promoter sequence. These findings support the reason why most antipsychotics are effective in the treatment of bipolar disorder and substance-induced psychotic disorder (where DRD1 expression is genetically normal but chemically altered).

[0123] Referring to FIG. 8 of the drawings, the relative mRNA expression of DRD1 and DRD5 in SHSY5Y cells is shown. SHSY5Y cells are cultured in T-25 flasks (1X106 cells / T-25 flask) and treated with the following:

[0124] (a) Halo: Haloperidol (10 μM) for 20 hours; (b) SCH: SCH23390 (10 μM) for 20 hours; (c) SKF: SKF21987 (10 μM) for 20 hours; (d) Halo+SCH: SCH23390 (10 μM) and haloperidol (10 μM), SCH is pretreated for 6 hours, and then SCH23390 and haloperidol are co-treated for an additional 14 hours; (e) Halo+SKF: SKF81297 (10 μM) and haloperidol (10 μM), SKF is pretreated for 6 hours, and then SKF81297 and haloperidol are co-treated for an additional 14 hours; and (f) CONT: DMSO only as a control treatment.

[0125] After treatment, SHSY5Y cells are harvested and processed for RT-PCR analysis. The data are presented as the mean of three individual samples per treatment.

[0126] Clinical trials of genomic sequence variations of DRD1 and DRD5 in human subjects

[0127] The genomic sequences of human DRD1 and DRD5 are analyzed using an Illumina MiniSeq instrument. Genomic DNA is analyzed using Qiagen Blood & Cell Culture DNA Kits according to the manufacturer's instructions (Qiagen, Beverly, MA). Genomic DNA sequence data are analyzed using Illumina apps DNA Amplicon and DRAGEN Amplicon.

[0128] To demonstrate the relationship between mutations in human DRD1 genomic DNA and mental disorders, three exemplary human subjects are selected.

[0129] The results are summarized in Table 1 below: Mutations in human DRD1 genomic DNA in three human subjects with mental disorders (including the 7-kb 5'-regulatory sequence, the gene coding sequence, and the 1-kb 3'-regulatory sequence). JPEG2025525131000001.jpg254169

[0130] In the clinical trials of the present inventors, at least one mutation in DRD1 or DRD5 is found in human subjects with mental disorders. The results also indicate that as the number of mutations increases, the level of severity of the mental disorder increases.

[0131] In other words, mental disorders can be quantitatively identified by detecting at least one mutation in DRD1 or at least one mutation in DRD5. More severe types of mental disorders can be identified by two or more mutations in DRD1 and DRD5.

[0132] Those skilled in the art will understand that the embodiments of the present invention as shown in the drawings and described above are merely illustrative and not intended to be limiting.

[0133] Therefore, it will be understood that the object of the present invention has been fully and effectively achieved. The embodiments have been shown and described for the purpose of illustrating the functional and structural principles of the present invention and may be changed without departing from such principles. Therefore, the present invention includes all modifications within the spirit and scope of the following claims.

Claims

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

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

3. The method for treating a mental disorder in a subject according to claim 1, further comprising the step of normalizing the DRD5 level and DRD5 expression in the subject to a preset standard level.

4. The method for treating a mental disorder in a subject according to claim 3, further comprising: increasing or decreasing the constitutive expression of DRD1 or DRD5 in the subject 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, wherein the antipsychotic drug 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 drug 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 constitutive expression of the DRD1 gene in the subject is impaired.

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

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

10. The method for treating a mental disorder in a subject according to claim 2, wherein the mental disorder includes schizophrenia, autism, depression, mania, and bipolar disorder.

11. The method for treating a mental disorder in a subject according to claim 4, wherein the mental disorder includes schizophrenia, autism, depression, mania, and bipolar disorder.

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

13. The method for treating mental disorders in a subject according to claim 12, 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 mental disorders in a subject according to claim 13, wherein the antipsychotic drug is selected from the group consisting of haloperidol, risperidone, clozapine, and cariprazine.

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

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

17. The method for treating mental disorders in a subject according to claim 13, further comprising the step of providing a sequence database in which polymorphisms of the DRD1 and / or DRD5 promoter sequence are related to the dosing effect, and the step of individualizing drug screening regarding the effectiveness of an antipsychotic drug based on the sequence database.

18. A quantitative screening method for mental disorders in a subject, comprising the steps of performing genomic sequencing of the DRD1 and DRD5 genes (including the 7-kb 5'-upstream sequence, protein-coding sequence, and 2-kb 3'-downstream sequence) of a plurality of subjects to establish a mental disorder and mutation distribution database (including the type, position, and frequency of each gene variant) in the DRD1 and DRD5 genes, selecting one or more variants highly related to one mental disorder for the target gene sequence of the DRD1 and DRD5 genes, which can be used to determine the risk of developing the mental disorder, and performing the target gene sequence of the DRD1 and DRD5 genes in the target subject to determine the risk of developing the mental disorder in the target subject.

19. The quantitative screening method for mental disorders in the subject, wherein three or more variants are related to one mental disorder.

20. The quantitative screening method for mental disorders in the subject according to claim 19, wherein polymorphisms of the DRD1 and / or DRD5 promoter sequence related to clinically diagnosed mental disorders are included in the variant distribution database.

21. The method according to claim 19, wherein the method is a prenatal screening method, and a polymorphism of the DRD1 and / or DRD5 promoter sequence associated with the risk of a clinically diagnosed mental disorder is included in the variant distribution database, for quantitatively screening for a mental disorder of a subject.

22. A quantitative screening test for human DRD1 and DRD5 expression in cells of a human subject, comprising: establishing a normal range of human DRD1 and DRD5 expression in the cells; determining the human DRD1 and DRD5 expression in the cells of the human subject; and comparing the human DRD1 and DRD5 expression in the cells of the human subject with the normal range to determine the mental state 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 for a novel drug for a mental disorder, comprising: bidirectionally normalizing the overall expression level of DRD1 and / or DRD5 of the subject to a preset standard level; wherein the mental disorder includes schizophrenia, autism, depression, mania, and bipolar disorder; the method, wherein the sample sequence contains at least one variant when compared with the standard sequence database.

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