Method for diagnosing methamphetamine dependence

TECHNICAL FIELD

The present invention relates to use of a novel mental disorder-related gene in the medical field and the field or research. In detail, the present invention provides a screening method, an antipsychotic drug, and the like, using the novel mental disorder-related gene.

BACKGROUND ART

At present, aging is progressing, and it is thought that the number of patients with a mental disorder will securely increase in the future. Furthermore, at present, the social structure has been changed and it is said that we live in “times of stress.” People including children and adults are exposed to various stresses every day. Abnormality in the mental condition due to stress is a problem for people of all ages. Meanwhile, dependence on psychostimulant drugs and the like, that is to say, drug dependence has been a serious social problem.

DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention

Under such circumstances, development of treatment methods for mental disorders such as schizophrenic disorder, drug dependence, and the like, is a very urgent problem. However, no radical treatment method has been established. By the way, in order to carry out an appropriate treatment, it is important to determine and understand the presence of morbidity, pathological conditions, and the like, accurately and objectively. However, under present circumstances, a diagnosis of mental disorders is much dependent upon the statement of a patient himself/herself. Therefore, correct and appropriate diagnosis may not be carried out in not a few cases. Furthermore, empirical facts show that appropriate treatment sometimes cannot be selected because there are various pathological conditions. On the other hand, if the morbidity risk is assessed in advance, preventive measures can be taken, so that the number of patients can be significantly reduced. It would bring measureless contribution to the medical field. Furthermore, many of onset and progression mechanisms of mental disorders have not been clarified, and study results are expected to be accumulated in the future.

The present invention addresses the problems discussed above, and aims to provide useful means for treatment or diagnosis of mental disorders. As to the treatment application, in particular, it is an object to provide a method of selecting drug candidates for mental disorders so as to contribute the establishment of the treatment method. As to diagnostic application, it is an object to provide a means capable of determining the presence of morbidity, understanding of pathological conditions, furthermore, assessment of morbidity risk, and the like. Furthermore, another object of the present invention is to provide a means that is directly effective in treating mental disorders. Furthermore, a further object of the present invention is to provide research tool effective for the purpose of study, for example, investigation of the causes of mental disorders.

Means to Solve the Problems

In order to achieve the above-mentioned objects, the present inventors have attempted to identify a gene that is related to mental disorders. Firstly, the present inventors have prepared a mouse with a mental disorder accompanying drug dependence so as to select a gene that is related to the expression of the mental disorder. Herein, when methamphetamine is administered to a mouse, the mouse shows hyperactivity, and when methamphetamine is administered to the mouse every day, the degree of hyperactivity is increased. This can be thought to be a mental disorder accompanying drug dependence. The present inventors have searched a gene in which the expression is extremely increased in the nucleus accumbens of this mouse. As a result, the present inventors have found three novel candidate genes. As a result of a further study, the present inventors have found the clear correlation between the expression amount of the gene and symptoms peculiar to drug dependence. It has been confirmed that the three genes are genes relating to a mental disorder. In other words, the present inventors have succeeded in identifying novel genes related to a mental disorder. This makes it possible to develop a drug for a mental disorder whose target molecule is this gene. On the other hand, this makes it possible to study for investigating the onset and progression mechanism of the mental disorder.

The present inventors further carried out homology search by using a public database under the prediction that there would be human homologous genes. As a result, the presence of a homologous gene with respect to each gene has been confirmed. That is to say, the present inventors have succeeded in identifying human genes related to a mental disorder. These genes are intended to be used in the treatment or diagnosis (including onset risk diagnosis) of a mental disorder or development of drugs for a mental disorder and in the investigation of the onset or progression mechanism of a mental disorder. Thus, these genes are very useful.

The present invention is mainly based on the above-mentioned results and findings, and has the following configuration. The first aspect of the present invention relates to a method for screening a compound that is effective for a mental disorder. The method includes: (1) preparing a cell capable of expressing a gene (target gene) selected from the group consisting of a gene having a base sequence of SEQ ID NO.: 1, a gene having a base sequence of SEQ ID NO.: 2, a gene having a base sequence of SEQ ID NO.: 3 and genes homologous to these genes; (2) exposing the cell to a test compound; (3) determining the expression level of the target gene in the cell after the exposure to the test compound; and (4) determining the change in expression level of the target gene due to exposure to the test compound.

According to one embodiment of the present invention, a screening method using an animal individual is provided. This screening method includes the steps of: (i) preparing a non-human animal; (ii) administering a test compound to the non-human animal; (iii) after administering the test compound, in the central nervous system tissue of the non-human animal, determining the expression level of the gene (target gene) selected from the group consisting of a gene having a base sequence of SEQ ID NO.: 1, a gene having a base sequence of SEQ ID NO.: 2, a gene having a base sequence of SEQ ID NO.: 3, and homologous genes thereof; and (iv) determining the change in expression level of the target cell due to the administration of the test compound.

In accordance with one embodiment of the present invention, as the non-human animal, a non-human animal with pathological condition of a mental disorder is used.

In the screening method of the present invention, as the non-human animal, for example, a mouse is used. In this case, a gene having a base sequence of SEQ ID NO.: 1, a gene having a base sequence of SEQ ID NO.: 2, or a gene having a base sequence of SEQ ID NO.: 3 are to be the target genes.

In the preferable embodiment, the screening method of the present invention has an object to select and identify a compound that is effective for drug dependence.

Another aspect of the present invention relates to use of a mental disorder-related gene to the medial application. In this aspect, a method for obtaining information for diagnosing a mental disorder is provided. The method includes the steps of a) preparing a biological sample collected from a subject; and b) determining the expression level in the biological sample of a gene selected from the group consisting of a gene having a base sequence of SEQ ID NO.: 4, a gene having a base sequence of SEQ ID NO.: 5, a gene having a base sequence of SEQ ID NO.: 6 and natural mutants of these genes.

A further aspect of the present invention relates to use of mental disorder-related gene for treatment application. In this aspect, an antipsychotic drug including a compound for increasing the expression level in the target tissue of a gene selected from the group consisting of a gene having a base sequence of SEQ ID NO.: 4, a gene having a base sequence of SEQ ID NO.: 5, a gene having a base sequence of SEQ ID NO.: 6 and natural mutants of these genes is provided.

In one embodiment of the present invention, an active ingredient of the antipsychotic drug includes a compound selected from the group consisting of an isolated protein having an amino acid sequence of SEQ ID NO.: 10, an isolated protein having an amino acid sequence of SEQ ID NO.: 11, an isolated protein having an amino acid sequence of SEQ ID NO.: 12, natural mutants of these proteins, an isolated nucleic acid encoding the amino acid sequence of SEQ ID NO.: 10, an isolated nucleic acid encoding the amino acid sequence of SEQ ID NO.: 11, an isolated nucleic acid encoding the amino acid sequence of SEQ ID NO.: 12, and isolated nucleic acids encoding the mutants.

A further aspect of the present invention relates to use of mental disorder-related gene for research purposes. This aspect provides a reagent for studying a mental disorder, including an isolated nucleic acid having any one of the base sequences of SEQ ID NOs.: 1 to 6, or a kit including thereof.

The present invention further provides an expression vector for treating or studying a mental disorder, which holds nucleic acid encoding any one of the amino acid sequences of SEQ ID NOs.: 7 to 12. The present invention further provides an antibody to protein including any one of the amino acid sequences of SEQ ID NOs.: 7 to 12 for treating, diagnosing or studying mental disorders. In one preferable embodiment, the antibody of the present invention shows a specific binding property to peptide including an amino acid sequence of SEQ ID NO.: 19 or 20.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing that the locomotor activity is enhanced when methamphetamine is administered to mice continuously. Methamphetamine (2 mg/kg, s.c.) was administered to mice for six days. The results are shown in a mean value±standard error (n=20-30). *P<0.05 compared with the physiological saline solution administered group. In a repetitive two-way layout analysis of variance, a significant difference was observed.

FIG. 2 is a list of genes in which the expression is largely increased in the mouse nucleus accumbens as a result of the screening.

FIG. 3 shows a result of the BLAST search based on the base sequence of Gene 1. In the lower column, primers for specifically amplifying only a partial region are shown.

FIG. 4 shows a result of the BLAST search based on the base sequence of Gene 2. In the lower column, primers for specifically amplifying only a partial region are shown.

FIG. 5 is a view showing the change of the expression of Gene 1 mRNA after continuous administration of methamphetamine. Methamphetamine (2 mg/kg, s.c.) was administered to mice for six days, and two hours after the final administration, the mice were subjected to decapitation (n=4-5).

FIG. 6 shows the size of Gene 1 and the change in the expression of Gene 1 mRNA after continuous administration of methamphetamine in the nucleus accumbens. Methamphetamine (2 mg/kg, s.c.) was administered to mice for six days, and two hours after the final administration, the mice were subjected to decapitation (n=4-5). The change in the expression of Gene 1 mRNA was examined by northern hybridization. The data were corrected by using GAPDH mRNA.

FIG. 7 shows the change of expression of Gene 2 mRNA after continuous administration of methamphetamine. After the final administration, the mice were subjected to decapitation (n=4-5).

FIG. 8 shows the size of Gene 2 and the change in the expression of Gene 2 mRNA after continuous administration of methamphetamine in the nucleus accumbens. Methamphetamine (2 mg/kg, s.c.) was administered to mice for six days, and two hours after the final administration, the mice were subjected to decapitation (n=4-5). The change in the expression of Gene 2 mRNA was examined by northern hybridization. The data were corrected by using GAPDH mRNA.

FIG. 9 shows the change in the expression of Piccolo mRNA in the nucleus accumbens after administration of methamphetamine. Methamphetamine (2 mg/kg, subcutaneous administration) was administered to a mouse for six days, and the nucleus accumbens extracted from the brain 24 hours after the final administration was used as a sample.

FIG. 10 shows the change in the expression of Gene 1 mRNA after single time administration and repeated administration of methamphetamines. Methamphetamine (2 mg/kg, s.c.) was administered to mice for six days, and two hours after the final administration, the mice were subjected to decapitation. The results are shown in a mean value±standard error (n=8). * P<0.05 compared with the physiological saline solution administered group.

FIG. 11 shows the change in the expression of Gene 2 mRNA after single time administration and repeated administration of methamphetamines. Methamphetamine (2 mg/kg, s.c.) was administered to mice for six days, and two hours after the final administration, the mice were subjected to decapitation. The results are shown in a mean value±standard error (n=8). * P<0.05 compared with the physiological saline solution administered group.

FIG. 12 shows the change in the expression of piccolo (Gene 3) in a mouse after treatment with methamphetamine. Single time administration and continuous administration of methamphetamine (1 mg/kg, subcutaneous administration) was carried out to mice for six days, and two hours after the final administration, the mice were subjected to decapitation and used as samples. The number of mice in each group was made to be 5 to 8. * P<0.05 compared with the physiological saline solution administered group.

FIG. 13 shows the expression level of Gene 1 in each tissue of a living body of a mouse.

FIG. 14 is a view showing an effect of Gene 1 antisense oligonucleotide in the methamphetamine-induced enhancement of locomotor activity. Methamphetamine (1 mg/kg, s.c.) was administered to mice for five days. Locomotor activity was measured for two hours. In the right cerebral ventricle (AP-0.5 mm, ML +1.0 mm from bregma, DV-2.0 mm from the skull), Gene 1 antisense oligonucleotide (Gene 1-AS, 1.8 nmol/6 μl/day), scramble control oligonucleotide (Gene 1-SC) and artificial cerebrospinal fluid (CSF) were infused continuously by using an osmotic pump. The results are shown in a mean value±standard error (n=5-7). In a repetitive two-way layout analysis of variance, a significant difference was observed. * P<0.05 compared with the physiological saline solution+CSF-treated group. # P<0.05 compared with the physiological saline solution+Gene 1-SC-treated group.

FIG. 15 is a view showing an effect of Gene 2 antisense oligonucleotide in the methamphetamine-induced enhancement of locomotor activity. Methamphetamine (1 mg/kg, s.c.) was administered to mice for five days. Locomotor activity was measured for two hours. In the right cerebral ventricle (AP-0.5 mm, ML +1.0 mm from bregma, DV-2.0 mm from the skull), Gene 2 antisense oligonucleotide (Gene 2-AS, 1.8 nmol/6 μl/day), scramble control oligonucleotide (Gene 2-SC) and artificial cerebrospinal fluid (CSF) were infused continuously by using an osmotic pump. The results are shown in a mean value±standard error (n=3-5).

FIG. 16 is a view showing an effect of Gene 1 antisense oligonucleotide in the formation of the place preference by methamphetamine. During conditioning, methamphetamine (0.3 mg/kg, s.c.) or a physiological saline solution was administered to mice. In the right cerebral ventricle (AP-0.5 mm, ML +1.0 mm from bregma, DV-2.0 mm from the skull), Gene 1 antisense oligonucleotide (Gene 1-AS, 1.8 nmol/6 μl/day), scramble control oligonucleotide (Gene 1-SC) and artificial cerebrospinal fluid (CSF) are infused continuously by using an osmotic pump. The results are shown in a mean value±standard error (n=5-12). * P<0.05 compared with the physiological saline solution-treated group. # P<0.05 compared with the methamphetamine+CSF-treated group and a methamphetamine+Gene 1-SC-treated group.

FIG. 17 is a view showing an effect of Gene 2 antisense oligonucleotide in the formation of the place preference by methamphetamine. During conditioning, methamphetamine (0.3 mg/kg, s.c.) or a physiological saline solution was administered to mice. In the right cerebral ventricle (AP-0.5 mm, ML +1.0 mm from bregma, DV-2.0 mm from the skull), Gene 2 antisense oligonucleotide (Gene 1-AS, 1.8 nmol/6 μl/day), scramble control oligonucleotide (Gene 2-SC) and artificial cerebrospinal fluid (CSF) are infused continuously by using an osmotic pump. The results are shown in a mean value±standard error (n=4-8). * P<0.05 compared with the physiological saline solution-treated group. # P<0.05 compared with the methamphetamine+CSF-treated group.

FIG. 18 shows an influence of endogenous Piccolo (Gene 3) on the reverse tolerance of methamphetamine inducing property. Piccolo antisense oligonucleotide (antisense concentration: 0.6 nmol/6 μl/day), sense oligonucleotide (antisense concentration: 0.6 nmol/6 μl/day) and artificial cerebrospinal fluid were continuously infused in the cerebral ventricle by using an osmotic mini-pump. The site to be administered is a site that is 0.5 mm posterior and 1 mm left from the bregma, depth was made to be 2 mm. Methamphetamine was administered for six days. The number of individuals was 4 to 5.

FIG. 19 shows the change in the expression of Gene 1 mRNA by the continuous administration of methamphetamine in a mouse to which Gene 1 antisense oligonucleotide was infused. * P<0.05 compared with the physiological saline solution+CSF-treated group. # P<0.05 compared with the physiological saline solution+Gene 1-SC-treated group. $ P<0.05 compared with the physiological saline solution+Gene 1-AC-treated group. + P<0.05 compared with the methamphetamine+Gene 1-SC-treated group.

FIG. 20 shows an effect of dopamine D1 receptor antagonist R(+)-SCH₂₃₃₉₀and dopamine D2 receptor antagonist raclopride in the increase in the expression of Gene 1 mRNA in the nucleus accumbens induced by methamphetamine. * P<0.05 compared with the vehicle/physiological saline solution-administered group. # P<0.05 compared with the vehicle/methamphetamine-administered group.

FIG. 21 shows the change in the expression of TNF-α mRNA by the continuous administration of methamphetamine in a mouse to which Gene 1 antisense oligonucleotide was infused. * P<0.05 compared with the physiological saline solution+CSF-treated group. # P<0.05 compared with the physiological saline solution+Gene 1-SC-treated group. * P<0.05 compared with the physiological saline solution+CSF-treated group. # P<0.05 compared with the methamphetamine+Gene 1-SC-treated group.

FIG. 22 shows an in vivo effect of Gene 1 antisense oligonucleotide in the increase of the amount of extracellular dopamine induced by methamphetamine. * P<0.05 compared with the Gene 1-SC-infused group.

FIG. 23 shows an effect of Gene 1 in the reduction of the uptake of synaptosomal [³H]DA induced by methamphetamine. * P<0.05 compared with the physiological saline solution+CSF-infused group. # P<0.05 compared with the methamphetamine+Gene 1-SC-infused group.

FIG. 24 shows an effect of Gene 1 in the reduction of the uptake of synaptovesicle [³H]DA induced by methamphetamine. * P<0.05 compared with the physiological saline solution+CSF-infused group and physiological saline solution+Gene 1-SC-treated group. # P<0.05 compared with the methamphetamine+Gene 1-SC-infused group.

FIG. 25 shows a configuration of an expression vector (pcDNA-DEST53) used in expression experiment of the full length or a fragment of Gene 1. Regions of the base numbers 1650 and 3312 are replaced by Gene 1 DNA (full length or fragment). Into the site of attR1, any one of base numbers 1643 to 1767 and base numbers 3202 to 3326.

FIG. 26 shows an effect of transfected Gene 1 in the reduction of the uptake of [³H]DA induced by methamphetamine. * P<0.05 compared with pcDNA-DEST53 vector introduced cell. # P<0.05 compared with full length Gene 1-introduced cell. $ P<0.05 compared with Gene 1 fragment introduced cell. + P<0.05 compared with methamphetamine+pcDNA-DEST53 vector introduced cell.

FIG. 27 shows the change in the expression of Gene 1 mRNA after methamphetamine is acted in the PC 12 cell into which the full-length Gene 1 expression vector has been introduced. Into the PC12 cell, by using Lipofectamine, an expression vector for expressing the full length Gene 1, an expression vector for expressing the Gene 1 fragment or a pcDNA-DEST vector (empty vector) is transfected. In a 24-well plate, cells were cultured for 2 to 3 days, and then pre-treated with methamphetamine (1.0 μM) for 30 minutes. Thereafter, the expression level of Gene 1 mRNA in the cell was measured by a RT-PCR method. The results are shown in a mean value±standard error (n=8). * P<0.05 compared with pcDNA-DEST53 vector treated cells, # P<0.05 compared with full-length Gene 1 treated cells, $ P<0.05 compared with Gene 1 fragment treated cells, and + P<0.05 compared with methamphetamine+pcDNA-DEST53 vector treated cells.

FIG. 28 shows the localization of Gene 1 in the nucleus accumbens after the continuous administration of methamphetamine. Methamphetamine (2 mg/kg, s.c.) was administered to mice for six days, and 24 hours after the final administration, the mice were subjected to decapitation. The nucleus accumbens was immuno-stained with a polyclonal antibody to a partial peptide encoded by Gene 1. Methamphetamine/Gene 1 is a result of staining by using a polyclonal antibody to Gene 1 partial peptide. Furthermore, methamphetamine/NeuN is a result of staining with an anti-NeuN (nerve cell marker) antibody; methamphetamine/MAP2 is a result of staining with an anti-MAP2 (nerve cell marker) antibody, and methamphetamine/GFAP is a result of staining with an anti-GFAP (glia cell marker). Pictures of methamphetamine/merge in the right column show a merge of two pictures showing the results of staining, respectively. Scale bar: 20 μm.

FIG. 29 shows the change in expression of Gene 1 mRNA in nicotine, alcohol and phencyclidine. * P<0.05 compared with the physiological saline solution-administered group.

FIG. 30 shows the expression of Piccolo protein in a nerve cell. TH: immunostaining image using an anti-TH (tyrosine hydroxylase) antibody, Piccolo: immunostaining image using an anti-Piccolo antibody, and Merge: merge of two images at the left side.

FIG. 31 shows the expression of Piccolo protein in the anterior ganglion of midbrain dopamine neuron. TH: immunostaining image using an anti-TH (tyrosine hydroxylase) antibody, Piccolo: immunostaining image using an anti-Piccolo antibody, and Merge: merge of two images shown in the left side.

FIG. 32 shows an effect of a Piccolo C2A domain on the suppression of uptake of dopamine in the PC12 cell in which a human dopamine transporter has been forcedly expressed.

FIG. 33 shows an effect of the suppression of the expression of Piccolo in the PC12 cell in which a human dopamine transporter has been forcedly expressed.

FIG. 34 shows an effect of Piccolo on the intracellular movement of the dopamine transporter by methamphetamine.

FIG. 35 shows a localization of piccolo in the dopaminergic nerve cell.

FIG. 36 is a view summarizing the relation between the dopamine transporter between Piccolo protein in the PC12 cell. When methamphetamine is acted, the internalization of the dopamine transporter occurs (FIG. 36(a)). Furthermore, the C2A domain of the dopamine transporter and Piccolo are expressed in the same location (FIG. 36(b)).

BEST MODE FOR CARRYING OUT THE INVENTION

A mental disorder is generally classified into endogenous mental disorders (schizophrenic disorder and the like), exogenous mental disorders, organic mental disorders (dementia and the like), symptomatic mental disorders (mood swing and the like), toxic mental disorders (alcohol dependence, drug dependence, and the like), and psychogenic mental disorders (neurosis, psychosomatic disease, and the like) according to causes of disease. Preferably, the present invention is directed to endogenous mental disorders and toxic mental disorders. Further preferably, the present invention is directed to schizophrenic disorder that is one example of the endogenous mental disorders or drug dependence that is one example of the toxic mental disorder. Most preferably, the present invention is directed to preference drug dependence among the drug dependence. Depending upon the cause drugs, the drug dependence is characterized by psychic dependence that is dependence on a psychic action (effect) of drug, physical dependence for avoiding the biological response to drug withdrawal, or obtaining of tolerance (or combination thereof). An example of the cause drug of the drug dependence include methamphetamine, morphine, cocaine, nicotine, alcohol, phencyclidine, benzodiazepine, and the like (including each kind of salt). The drug dependence of the present invention is not limited to the drug dependence relating to these drugs.

In the present specification, “antipsychotic drug” refers to as drugs used for suppressing the onset of a mental disorder, or drugs used for reducing the symptoms of a mental disorder (including partial or complete healing). Therefore, the antipsychotic drugs of the present invention include drugs that can be used for preventive treatment of mental disorders and drugs that can be used for treating the mental disorders.

When the present invention refers to a gene, the natural mutant thereof may be contemplated.

(Screening Method)

One aspect of the present invention provides a method for screening a compound effective for a mental disorder. A compound selected by the screening method of the present invention is expected to be effective for medical measurement relating to mental disorders. That is to say, the compound selected by the screening method of the present invention becomes a promising candidate for drugs for mental disorders or a material useful in developing such drugs. When the selected compound has a sufficient drug efficacy with respect to mental disorders, an intact compound can be used as an active ingredient of drugs. On the other hand, when the selected compound does not have a sufficient drug efficacy, the compound can be used after the drug efficacy thereof is enhanced by subjecting the compound to modification such as chemical modification. Needless to say, even when the compound has a sufficient drug efficacy, it may be modified for the purpose of increasing the drug efficacy.

The screening method of the present invention can be carried out by using certain cells or animal individuals.

(Cell-Based Screening Method)

One embodiment of the present invention provides a method for cell-based screening. In this embodiment, whether or not the expression level of a certain gene in the cell is changed is examined by exposing (administering and adding) a test compound to the cell. Specifically, the screening method of the present invention carries out the following steps.

Step 1: preparing a cell capable of expressing a gene (target gene) selected from the group consisting of a gene having a base sequence of SEQ ID NO.: 1, a gene having a base sequence of SEQ ID NO.: 2, a gene having a base sequence of SEQ ID NO.: 3 and homologous genes thereof.

Step 2: exposing the cell to a test compound.

Step 3: determining the expression level of the target gene in the cell after the exposure to the test compound.

Step 4: determining the change in expression level of the target gene due to the exposure to the test compound.

Hereinafter, the detail for each step is described.

1. Step 1

In the step 1, a cell expressing a gene to be detected (which is referred to as “target gene” in the specification) is prepared. The target gene can be selected from a gene having a base sequence of SEQ ID NO.: 1 (which is also referred to as “target mouse gene 1” in the specification), a gene having a base sequence of SEQ ID NO.: 2 (which is also referred to as “target mouse gene 2” in the specification), a gene having a base sequence of SEQ ID NO.: 3 (which is also referred to as “target mouse gene 3” in the specification), and homologous genes thereof. Two kinds or more of genes may be a detection target. Note here that all of the target mouse genes 1 to 3 are mouse genes in which the relationship with respect to drug dependence has been observed (see the below-mentioned Examples).

Herein, the “homologous gene” denotes a homologous gene of human, rat, or the like, corresponding to any one of the target mouse genes 1 to 3 that are genes of a mouse. Human homologous genes corresponding to the target mouse gene 1, the target mouse gene 2 and the target mouse gene 3 are a gene having a base sequence of SEQ ID NO.: 4 (which is also referred to as “target human gene 1” in the specification), a gene having a base sequence of SEQ ID NO.: 5 (which is also referred to as “target human gene 2” in the specification), and a gene having a base sequence of SEQ ID NO.: 6 (which is also referred to as “target human gene 3” in the specification), respectively. Note here that other homologous genes can be found by homology search using public database (for example, BLAST search).

Herein, as the “cells,” mammalian cells may be used. Example of the mammalian cells include cells of rodents such as a mouse, a rat, a guinea pig, and cells of primates such as a human, a monkey, a chimpanzee. The origin of cells are not particularly limited. However, it is preferable to use cells derived from the central nervous system tissue. Among the central nervous system tissue, it is preferable to use cells derived from the prefrontal cortex of the forebrain, the nucleus accumbens, the striatum, the midbrain, or the hippocampus.

On the condition that cells of non-human animals (for example, mouse, rat, rabbit, chicken, and the like) are used, the cells may be used in a state in which it is not separated from the living body (that is to say, a state in which the cells constitute the living body).

In screening, it is possible to use a group of cells (for example, cells forming a specific tissue) in which a network is formed between cells instead of cells which are dispersing. Furthermore, by using two kinds or more of cells together, the screening method of the present invention may be carried out.

In addition to cells capable of inherently expressing a target gene, cells that can express a target gene after the cells are subjected to artificial manipulation can be used. For example, transformants obtained by introducing a target gene in a state in which it can express may be used. An example of the cell that can be used for transformation may include a HeLa cell, a COS cell, and a CHO cell. These cells are readly available from a cell bank such as ATCC.

The number of cells to be used in not particularly limited, and it can be determined while considering the detection sensitivity, experiment facility, and the like. For example, 1 to 10⁵cells, preferably, 10 to 10⁴cells, and further preferably, 10²to 10³cells can be used.

2. Step 2

In the step 2, the prepared cells are exposed to a test compound. The exposure to the test compound can be carried out by, for example, culturing cells in the condition in which the test compound is contained in a culture medium. Alternatively, the test compound, a solution containing the test compound, or the like, may be brought into direct contact with the cells.

The amount to be exposed can be set arbitrarily. For example, the maximum exposure amount can be employed as long as the exposure does not bring a lethal effect on the cells.

The exposure time is not particularly limited. For example, the exposure time can be set in the range from one minute to ten days. The exposure may be carried out continuously with any intervals.

The test compound used for the screening method of the present invention can include organic compounds with various molecular sizes (nucleic acid, peptide, protein, lipid (simple lipid, complex lipid (phosphoglyceride, sphingolipid, glycosyl glyceride, cerebroside, and the like), prostaglandin, isoprenoid, terpene, steroid, and the like)), or an inorganic compound. The test compound may be a naturally occurring compound or may be a synthesized compound. In the latter case, for example, it is possible to construct an effective screening system by using a means of the combinatorial synthesis. Note here that a cell extract, culture supernatant, and the like, may be used as the test compound.

3. Step 3

In the step 3, the cells exposed to the test compound are used so as to determine the expression level of target genes. In one embodiment of the present invention, as the expression level of the target gene, the amount of mRNA that is a transcriptional product of the target gene is measured. For the detection of mRNA, routine procedures such as an RT-PCR method, various hybridization methods using specific probes (for example, Southern hybridization, in situ hybridization), and the like can be used.

In another embodiment of the present invention, the amount of protein that is an expression product of the target gene is measured. For example, the detection (measurement) can be carried out by using a compound that specifically binds to a target protein. The detection method (or measurement) is not particularly limited to this alone. However, the detection (measurement) is preferably carried out by an immunological technique. In the immunological technique, an antibody against the specific protein is used, and the protein is detected by using a binding property (binding amount) of the antibody as an indicator. The term used herein “antibody” includes a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single strand antibody, a CDR graft antibody, a humanized antibody, or the fragment thereof, and the like. The antibody of the present invention can be prepared by using an immunological technique, a phage display method, a ribosome display method, and the like.

According to the immunological detection method, rapid and highly sensitive detection can be carried out and the operation is easy and simple. An example of the detection method includes an ELISA method, radioimmunoassay, FACS, an immunoprecipitation method, immunoblotting, and the like.

By using a labeled antibody, the above-mentioned detection can be carried out easily. For labeling of the antibody, for example, fluorescent dye such as fluorescein, rhodamine, Texas Red, Oregon Green, and the like, an enzyme such as horseradish peroxidase, micro peroxidase, alkaline phosphatase, β-D-galactosidase, and the like, chemiluminescence or bioluminescence compound such as luminal, acridine dye, and the like, radioisotope such as, ³²P, ¹³¹I, ¹²⁵I, and the like, and biotin, can be used.

4. Step 4

In the step 4, by using the measurement results of the above-mentioned steps, the change of the expression level of the target gene is determined. For example, cells that are exposed to the test compound (an exposure group) and cells that are not exposed to the test compound (a not exposure group, i.e., a control group) are prepared. The expression levels of the exposure group and the not exposure group are measured respectively, and compared with each other. Note here that the expression level of the target gene when exposure to the test compound is not carried out is known in advance, this expression amount can be used as the expression level of the not exposure group. From the comparison results, as a result of the exposure to the test compound, the degree of changing of the expression of the target gene (the change of the expression level) can be determined. Thus, the effect of the test compound on the expression level of the target gene is evaluated.

When the expression amount of the exposure group is larger as compared with the not exposure group, that is to say, when it is observed that the test compound has an increasing effect of the expression level, it can be determined that the test compound is a compound that is effective for a mental disorder. When the significant increase in the expression level is observed in the exposure group, it can be determined that the test compound is a compound that is particularly effective for a mental disorder.

As mentioned above, by using the results of this step, the effectiveness of the test compound to the mental disorder can be evaluated.

In a cell (or a group of cells), also by comparing the expression level of the gene before and after the exposure, the effect of the test compound can be evaluated. For example, (1) cells into which a target gene together with a reporter gene (for example, a luciferase gene) have been introduced are prepared; (2) the expression level of the introduced target gene is measured before and after the exposure to the test compound by using the expression level of the reporter gene as an indicator (reporter assay), and then, the measurement results are compared.

(Animal Individual Based Screening Method)

The screening of the compound effective for a mental disorder can be carried out by using an animal individual. That is to say, the present invention also provides an animal individual based screening method including the following steps.

Step i: preparing a non-human animal.

Step ii: administering a test compound to the non-human animal.

Step iii: determining the expression level of the gene (target gene) selected from the group consisting of a gene having a base sequence of SEQ ID NO.: 1, a gene having a base sequence of SEQ ID NO.: 2, a gene having a base sequence of SEQ ID NO.: 3 and homologous genes thereof in the central nervous system tissue of the non-human animal after administering the test compound.

Step iv: determining the change in expression level of the target cell due to the administration of the test compound.

Hereinafter, each step is described in detail. As the matters that are not specifically mentioned, the corresponding description of those for the cell-based screening method is applied to.

1. Step i

In the step i, a non-human animal is prepared. An example of the non-human animal includes non-human primates (a monkey, a chimpanzee, and the like), a mouse, a rat, a rabbit, a cow, a horse, a sheep, a dog, a cat, and the like. Among them, a mouse or a rat can be used preferably.

The non-human animal generally expresses homologous genes peculiar to the species with respect to the target mouse genes 1 to 3. Except for using genetically modified animals as mentioned below, in general, the expression level of this homologous gene is to be detected in this screening method.

It is preferable to use a non-human animal with pathological condition of a mental disorder. If such a disease model animal is used, in the mental disorder state, the effect of the test compound on the expression of the target gene can be examined. In other words, this screening system corresponds to an actual treatment. According to such a screening system, it is possible to select a compound that actually acts on the mental disorder and has an excellent effectiveness. On the other hand, when observation of the change in the pathological condition of the non-human animal is carried out in parallel with the measurement of the expression amount of the target gene, it is possible to examine the correlation between the change of the expression level of the target gene and the change of the pathological condition. Thus, it is possible to obtain useful information in evaluating the effect of the test compound.

As the non-human animal with pathological condition of a mental disorder, animals provided with a specific pathological condition by genetic modification and/or breeding under certain conditions can be used. For example, it has been proposed that a genetically modified mouse lacking NMDA receptors ε1 and ε4 is effective as a model animal of schizophrenic disorders (Miyamoto, Y., Yamada, K., Noda, Y., Mori, H., Mishina, M. and Nabeshima, T.: Hyperfunction of dopaminergic and serotonergic neuronal systems in mice lacking the NMDA receptor ε1 subunit.: FASEB J., 15, 1407-1409 (2001), Miyamoto, Y., Yamada, K., Noda, Y., Mori., H., Mishina, M. and Nabeshima, T., Lower sensitivity to stress and altered monoaminergic neuronal function in mice lacking the NDMA receptor ε4 subunit.: J. Neurosci., 21, 750-757 (2001)). These non-human animals can be used in the present invention. Furthermore, a mouse representing a symptom of schizophrenic disorder by administration of phencyclidine (PCP) (Noda Y. et al., Repeated phencyclidine treatment induces negative symptom-like behavior in forced swimming test in mice: imbalance of prefrontal serotonergic and dopaminergic functions. Neuropsychopharmacology. 2000 Oct. 23(4):375-87), and the like, can be used as a model animal of schizophrenic disorders of the present invention. On the other hand, as shown in the below-mentioned Examples, a mouse continuously administered with methamphetamine and the like presents pathological conditions of drug dependence. Therefore, a mouse that has been treated under such conditions can be preferably used as a model animal of drug dependence in the present invention.

2. Step ii

In the step ii, a test compound is administered to a non-human animal. The route of administration is not particularly limited and can be appropriately selected from oral administration, intravenous administration (injection), intradermal administration (injection), subcutaneous administration (injection), transdermal administration, intraoral administration, direct administration to the target tissue (injection), and the like. Herein, the target tissue is a tissue involved in mental disorder. A typical example thereof includes the central nervous system tissue (forebrain, in particular, prefrontal cortex, nucleus accumbens, striatum, midbrain, hippocampus, and the like).

The administration amount can be set arbitrarily. For example, a maximum amount can be employed as long as the test compound does not bring a lethal effect on the non-human animals.

The number of administration times may be arbitrarily set. For example, the number of administration may be in the range from once to 20 times.

3. Step iii

In the step iii, by using the non-human animal to which a test compound has been administered, the expression level of the target gene in the central nervous system tissue (forebrain, in particular, prefrontal cortex, nucleus accumbens, striatum, midbrain, hippocampus, and the like) is measured. In other words, the amount of mRNA and/or expression product (protein) of the target gene in the certain central nervous tissue is measured. Note here that any one of the measurement using a tissue extract and measurement using a tissue section (for example, immune structure dyeing) may be carried out.

If the non-human animal used herein is a mouse, the target to be detected is any of the target mouse genes 1 to 3 (or any combination thereof). If the non-human animal used herein is a species other than a mouse, the target to be detected is homologous genes of the species corresponding to the target mouse genes 1 to 3. For example, if a rat is used, the target to be detected is a rat homologous gene. When non-human animals (genetically modified animals) expressing homologous genes of other species are used, these gene are detected. Thus, by using a technique of genetic engineering, it may be possible to construct a screening method in which a subject to be detected may be a gene which the animal species does not inherently express.

4. Step iv

In the step iv, from the measurement results of the above-mentioned steps, the change of the expression level of the target gene is determined. For example, firstly, animal individuals to which the test compound is administered (an administered group) and animal individuals to which the test compound is not administered (a not-administered group) are prepared. The expression levels of the administered group and the not-administered group are measured, respectively. Then, the expression amount of the administered group and the expression amount of the not-administered group are compared with each other. From the comparison results, the degree of changing of the expression of the target gene (the change of the expression amount) as a result of the administration of the test compound can be determined. Thus, the effect of the test compound on the expression amount of the target gene is evaluated.

When the expression level of the administered group is larger than that of the not-administered group, that is to say, when it is observed that the test compound has an increasing effect of the expression level, it can be determined that the test compound is an effective compound for a mental disorder. When the significant increase in the expression level is observed in the administered group, it can be determined that the test compound is a particularly effective compound for a mental disorder.

As mentioned above, by using the results of the step, the effectiveness of the test compound for the mental disorder can be evaluated.

Similar to the cell-based screening method, in a certain animal individual (animal group), by comparing the gene expression level before and after administration of the test compound, the effect of the test compound may be evaluated.

(Diagnostic Application)

Another aspect of the present invention relates to a diagnostic application of genes which the present inventors have succeeded in identification. One embodiment of this aspect relates to a method for obtaining information for diagnosing a mental disorder and includes the following steps.

Step a: step of preparing a biological sample collected from a subject.

Step b: step of determining the expression level in the biological sample of a gene selected from the group consisting of a gene having a base sequence of SEQ ID NO.: 4, a gene having a base sequence of SEQ ID NO.: 5, a gene having a base sequence of SEQ ID NO.: 6, and natural mutants of these genes.

The information obtained by this method is used for diagnosis of mental disorders. For example, the information obtained by carrying out the above-mentioned method to patient with a mental disorder can be used for evaluation and understanding of the pathological conditions of the patient and evaluation of the treatment effect. For example, if the treatment for a mental disorder is carried out in parallel with the method of the present invention, the treatment effect can be evaluated based on the resultant information. Specifically, by carrying out the method of the present invention after administering drugs, it is possible to examine the change of the expression level of a certain gene and to determine the treatment effect from the increase and decrease of the expression level. Thus, the method of the present invention may be used for monitoring the treatment effect.

On the other hand, when the subject of the present invention is persons other than patient, that is to say, persons who have not recognized to have a mental disorder, the obtained information can be used for determination of the presence of the morbidity of mental disorders and the evaluation of the morbidity risk, and the like.

The method of the present invention can carry out a diagnosis of mental disorders based on the expression level of genes that is an objective indicator, it is extremely valuable in the filed of diagnosis of a mental disorder whose objective diagnosis has been difficult conventionally.

1. Step a

In the step a, a biological sample collected from a subject (tested subject) is prepared. The subject herein may include not only patients with mental disorders but also healthy persons (including persons who may have a mental disorder). For example, a tissue piece collected from the subject, a cell extract, cerebrospinal fluid, and the like, may be used as a biological sample. As the biological sample, a nucleic acid sample is preferably used. The nucleic acid sample can be prepared from blood, skin cells, mucosal cells, hair of a subject (a tested subject) by the conventional extract methods and purification methods.

2. Step b

In the step b, the expression level of a certain gene is determined by using a biological sample. The gene to be determined is a gene having a base sequence of SEQ ID NO.: 4 (target human gene 1), a gene having a base sequence of SEQ ID NO.: 5 (target human gene 2), or a gene having a base sequence of SEQ ID NO.: 6 (target human gene 3). The natural mutants of these genes may be a subject to be determined. When a plurality of the natural mutants are present, one or an arbitrary combination may be a subject to be determined.

Both the standard gene and the mutated gene may be a subject to be determined. For example, both the standard gene and the mutated gene are determined at the same time and the total expression level can be used as information for diagnosis. Alternatively, the expression level of the standard gene and the expression level of the mutated gene are determined respectively. Then, comparison data of the both levels may be used as information for diagnosis.

By measuring the corresponding the amount of mRNA or the amount of protein, the expression level of a certain gene can be obtained (as to the specific determination method, see the column of the above-mentioned screening method).

The present invention further relates to the application of the target human genes 1 to 3 to the risk diagnosis of mental disorders. In other words, this aspect relates to a method for obtaining information on the risk diagnosis of mental disorders and includes the following steps.

Step A: step of preparing a nucleic acid sample collected from a subject.

Step B: step of analyzing the genotype of a gene selected from the group consisting of a gene having a base sequence of SEQ ID NO.: 4, a gene having a base sequence of SEQ ID NO.: 5, and a gene having a base sequence of SEQ ID NO.: 6 in the nucleic acid sample.

The step A can be carried out by the same way as in the above-mentioned step a. The nucleic acid sample in the step A can be prepared from blood, skin cells, mucosal cells, hair, and the like, of a subject (a tested subject) by the conventional extract methods and purification methods. Genome DNA having any length can be used as a nucleic acid sample as long as it includes a gene to be analyzed. Furthermore, when a plurality of genes are to be analyzed, it is not always necessary that all genes to be analyzed should be present on the same nucleic acid. In other words, as the nucleic acid sample of the present invention, all genes to be analyzed may be present on one nucleic acid, or all the genes to be analyzed may be present on a plurality of nucleic acid. As the nucleic sample, a gene to be analyzed may not be present in a complete state (that is to say, the full length gene) but it may be a fragment gene or a partial gene as long as it includes a site necessary for analysis of the genotype (that is to say, polymorphic site).

In the step B, the genotype is analyzed. In other words, a specific genetic polymorphism is analyzed. In general, in a gene, there is an individual difference of the DNA sequence constituting the gene. This individual difference is referred to as genetic polymorphism. As the genetic polymorphism, a polymorphism in which one base is substituted by another base (SNP (single nucleotide polymorphism)), a polymorphism in which one to several tens of bases are deleted or inserted (insertion/deletion type polymorphism), a polymorphism in which the number of repetition of the repetitive sequence including two to several bases is different (VNTR (variable number of tandem repeat), microsatellite polymorphism, and the like, are well known. These genetic polymorphisms may specify the expression state of the gene or may change the amino acid in a protein encoded by the gene and affect the function thereof. Therefore, by analyzing a certain genetic polymorphism, it is possible to evaluate the potential function of the protein encoded by the gene.

The information on the resultant genotype in the step B can be used for risk diagnosis of mental disorders. For example, based on the genotype (combination of specific alleles), the degree of genetic risk of a mental disorder can be determined.

The analyzing method of polymorphism is not particularly limited and may employ well-known methods. Example of the method include a method for analyzing the polymorphism of the amplification by a PCR method by using allele specific primer (and probe), a method for analyzing the polymorphism of the amplified product by fluorescence or emission, a PCR-RFLP (restriction fragment length polymorphism) method using a PCR (polymerase chain reaction) method, a PCR-SSCP (single strand conformation polymorphism) method (Orita, M. et al., Proc. Natl. Acad. Sci., U.S.A., 86, 2766-2770 (1989), etc.), a PCR-SSO (specific sequence oligonucleotide) method, an ASO (allele specific oligonucleotide) hybridization method combining the PCR-SSO method and a dot hybridization method (Saiki, Nature, 324, 163-166 (1986), etc.), or a TaqMan-PCR method (Livak, K J, Genet Anal, 14, 143 (1999), Morris, T. et al., J. Clin. Microbiol., 34, 2933 (1996)), an Invader method (Lyamichev V et al., Nat Biotechnol, 17, 292 (1999)), MALDI-TOF/MS (matrix) method using a primer elongation process (Haff L A, Smimov I P, Genome Res 7, 378 (1997)), a RCA (rolling cycle amplification) method (Lizardi P M et al., Nat Genet 19, 225 (1998)), a method a using DNA chip or a microarray (Wang D G et al., Science 280, 1077 (1998), etc.), a primer elongation process, a Southern blotting hybridization method, a dot hybridization method (Southern, E., J. Mol. Biol. 98, 503-517 (1975)), and the like. Furthermore, the analysis may be carried out by directly sequencing a portion of the polymorphism to be analyzed. Note here that combination of these methods may be used for analyzing the polymorphism. Furthermore, after the nucleic acid sample is amplified in advance (including amplification of a partial region of the nucleic acid sample) by a nucleic acid amplification method such as a PCR method or a method applying the PCR method, and the like, any of the above-mentioned analyzed methods may be employed.

When a large number of nucleic acid samples are analyzed, it is preferable to use an analyzing method capable of analyzing a large number of specimens for a relatively short time. Such analyzing method includes an allele specific PCR method, an allele specific hybridization method, a TaqMan-PCR method, an Invader method, a MALDI-TOF/MS (matrix) method using primer elongation process, an RCA (rolling cycle amplification) method, a method a using DNA chip or a microarray, and the like.

The polymorphisms of each gene can be analyzed by using mRNA that is a transcriptional product of gene to be analyzed. After mRNA of gene to be analyzed is extracted/purified from blood, urine, etc. from a subject, a northern blotting method (Molecular Cloning, Third Edition, 7.42, Cold Spring Harbor Laboratory Press, New York), a dot blot method, (Molecular Cloning, Third Edition, 7. 46, Cold Spring Harbor Laboratory Press, New York), an RT-PCR method (Molecular Cloning, Third Edition, 8.46, Cold Spring Harbor Laboratory Press, New York), a method using a DNA chip (DNA array), and the like, are executed. Thereby, polymorphism analysis using mRNA as a starting material can be analyzed.

The polymorphism can be analyzed by using the expression product of gene to be analyzed. In this case, a partial protein or a partial peptide can be used as a sample for analysis as long as it contains amino acid corresponding to a polymorphism site.

As the analysis method using the expression product of a gene, a method for directly analyzing amino acid at the polymorphism site, or a method of immunological analysis by using the change of the three-dimensional structure and the like, can be carried out. For the former example, a well-known amino acid sequence analyzing method (a method using an Edman's method) can be used. As the latter method, by using a monoclonal antibody or a polyclonal antibody having a binding property specific to an expression product of gene having any genotype constituting a polymorphism, an ELISA method (enzyme-linked immunosorbent assay), radioimmunoassay, an immunoprecipitation method, an immunodiffusion technique, and the like, can be used.

(Treatment Application)

The further aspect of the present invention relates to treatment application of genes which the present inventors have successfully identified. In one embodiment of this aspect, an antipsychotic drug is provided. The antipsychotic drug of the present invention includes a compound for increasing the expression level in the target tissue of a gene selected from the group consisting of a gene having a base sequence of SEQ ID NO.: 4 (target human gene 1), a gene having a base sequence of SEQ ID NO.: 5 (target human gene 2), a gene having a base sequence of SEQ ID NO.: 6 (target human gene 3), and the natural mutant thereof. Thus, the antipsychotic drug of the present invention has an effect of increasing the expression level in the target tissue of gene.

In general, herein, a central nervous system tissue is “target tissue.” When the expression level of any of the above genes is increased in the central nervous system tissue, the direct effect on mental disorders can be expected.

A specific example of compounds (active ingredients) contained in the drug of the present invention may include an isolated protein having an amino acid sequence of SEQ ID NO.: 10, an isolated protein having an amino acid sequence of SEQ ID NO.: 11, or an isolated protein having an amino acid sequence of SEQ ID NO.: 12. The proteins are expression products of the target human genes 1 to 3, respectively. Therefore, according to the drugs containing the proteins, when the drugs are delivered to a target tissue, the amount of the expression product of the target human gene in the target tissue is increased. Note here that the natural mutant (corresponding to the natural mutant of the target human gene) of the any of the above-mentioned proteins can be used as an active ingredient.

The term “isolated” used herein about protein refers to a state in which the protein is extracted from the original environment (for example, natural environment in the case of the natural material), that is, a state in which the protein existing in a state that is different from the state by the artificial manipulation.

When the protein of the present invention is derived from the natural material, the “isolated” state is generally a state in which the natural material does not substantially contain a component other than the protein (in particular, contaminated protein is not substantially contained). Specifically, in the isolated protein of the present invention, the content of the contaminated protein on a weight basis is, for example, less than about 20%, preferably less than about 10%, further preferably, less than about 5%, and still further preferably, less than about 1% with respect to total weight.

On the other hand, when a protein is produced by recombinant DNA technology, the “isolated” state generally refers to a state that is free from other components derived from the used host cell, a culture medium, or the like. Specifically, in the isolated protein, the content of the contaminated protein on a weight basis is, for example, less than about 20%, preferably less than about 10%, further preferably, less than about 5%, and still further preferably, less than about 1%, with respect to total weight.

Furthermore, when a protein is produced by chemical synthesis, the “isolated” state generally refers to a state that is free from precursors (raw materials), chemical materials used in the synthesizing process, or the like. Specifically, in the isolated protein, the content of a precursor on a weight basis is, for example, less than about 20%, preferably less than about 10%, further preferably, less than about 5%, and still further preferably, less than about 1%, with respect to total weight.

The protein of the present invention is preferably prepared by using genetic engineering techniques. For example, a target protein can be obtained by, for example, introducing nucleic acid encoding the target protein into an appropriate host cell and recovering the protein expressed in the transformant. The recovered protein is purified if necessary. The target protein can be obtained by using a cell-free protein synthesis system. The cell-free protein synthesis system means that from nucleic acid (DNA and mRNA) as a template, mRNA or protein encoded by the nucleic acid is synthesized in vitro by using ribosome derived from living cells (or obtained by genetic engineering techniques) or a transcription—translation factor instead of using living cells. The cell-free synthesis system generally uses a cell extract obtained by purifying a cell homogenized solution if necessary. The cell extract generally includes ribosome that is necessary for protein synthesis, various factors such as an initiation factor, and various enzymes such as tRNA. When protein is synthesized, various amino acids, energy source such as ATP, GTP, and the like, creatine phosphate, and other materials necessary for synthesizing protein are added to this cell extract. Needless to say, when protein is synthesized, ribosome prepared separately, various factors, and/or various enzymes may be supplemented if necessary. The development of a transcription/translation system for reconstructing various molecules (factors) necessary for protein synthesis has been reported (Shimizu, Y. et al.: Nature Biotech., 19, 751-755, 2001). In this synthesis system, genes of three kinds of initiation factors constituting the protein synthesis system of bacteria, three kinds of elongation factors, four kinds of factors involved in termination, 20 kinds of aminoacyl tRNA synthesis synthase for binding various amino acid to tRNA, and 31 kinds of factors including methionyl tRNA formyl transferase are amplified from Escherichia coli genome. By using them, a protein synthesis system is reconstructed in vitro. In the present invention, such a reconstructed synthesis system may be used.

The term “cell-free transcription/translation system” can be used compatibly with a cell-free protein synthesis system, an in vitro translation system or an in vitro transcription/translation system. In the in vitro translation system, protein is synthesized by using RNA as a template. An example of the template RNA includes total RNA, mRNA, in vitro transcriptional product, and the like. On the other hand, in the in vitro transcription/translation system, DNA is used as a template. The template DNA should include a ribosome binding region and preferably include an appropriate terminator sequence. The in vitro transcription/translation system sets conditions in which factors necessary to each reaction are added so that the transcription reaction and the translation reaction proceed continuously.

The cell-free synthesis systems widely used at present include the following systems: an Escherichia coli S30 fraction extract system (prokaryotic cell system), a wheat germ extract system (eukaryotic cell system), and a rabbit reticulocyte solubilizer system (eukaryotic cell system). These systems are commercially available as a kit and can be used easily.

The target protein can be prepared by separation and purification of a natural source (obtaining source). An example of the source of protein in the present invention includes animal cells (including human cells), plant cells, body fluid (blood, urine, etc.), and the like.

Other specific examples of the compound (active ingredient) contained in the antipsychotic drug of the present invention can include an isolated nucleic acid encoding any of the above-mentioned proteins. That is to say, an example of the active ingredient can include an isolated nucleic acid encoding an amino acid sequence of SEQ ID NO.: 10, an isolated nucleic acid encoding an amino acid sequence of SEQ ID NO.: 11, and an isolated nucleic acid encoding an amino acid sequence of SEQ ID NO.: 12. These nucleic acids are contained in a state in which they can be expressed in the target tissue when the antipsychotic drug of the present invention is administered. For example, a nucleic acid in a state in which it is inserted in an appropriate expression vector is used. That is to say, the present invention provides an expression vector comprising a nucleic acid (specifically, for example, DNA having a base sequence of any of SEQ ID NOs.: 1 to 6) encoding an amino acid sequence of any of SEQ ID NOs.: 7 to 12. The nucleic acid is inserted into the expression vector in a state in which it is linked operatively to an appropriate regulatory sequence working in the target tissue.

The term “nucleic acid” used herein includes DNA (including cDNA and genome DNA), RNA (including mRNA), a DNA analogue and an RNA analogue. The form of nucleic acid of the present invention is not particularly limited. That is to say, it may be any of a single stranded DNA and a double stranded DNA. Preferably, it is a double stranded DNA. Furthermore, codon degeneracy is contemplated. That is to say, in the case of nucleic acid encoding protein, the nucleic acid may include any base sequences as long as the protein can be obtained as an expression product.

In this specification, “nucleic acid encoding protein” denotes nucleic acid from which the protein is obtained when it is expressed. The nucleic acid includes not only nucleic acid having a base sequence corresponding to the amino acid sequence of the protein but also nucleic acid obtained by adding a sequence that does not encode the amino acid sequence to the above-mentioned nucleic acid (for example, DNA including one or a plurality of introns).

Furthermore, in this specification, the term “isolated nucleic acid” is typically one which is separated from other nucleic acids co-existing in the natural state when the nucleic acid is a naturally occurring nucleic acid (for example, nucleic acid in a living human body). However, the isolated nucleic acid may include a part of other nucleic acid components, for example, nucleic acid sequences flanking in the natural state. Preferably, the “isolated nucleic acid” is substantially free of other DNA components (including DNA sequences which naturally flank the nucleic acid) co-existing in a natural state in, for example, the genomic DNA.

Preferably, the “isolated nucleic acid” such as a cDNA molecule can be substantially free of other cellular components or culture medium when produced by recombination techniques. Similarly, preferably, the “isolated nucleic acid” such as a ddNTP can be substantially free of precursors (raw materials), other chemicals used in chemical synthesis, or the like, when chemically synthesized.

When nucleic acid is present as a part of a vector or a composition, or a nucleic acid is present in a cell as an exogenous molecule, the nucleic acid may be an “isolated nucleic acid” as long as it is present as a result of an artificial manipulation.

The nucleic acid used in the present invention can be prepared into an “isolated state” by referring to the sequence information disclosed in the present specification or attached sequence list and by using standard genetic engineering technique, molecular biological technique, biochemical technique, and the like. For example, the nucleic acid can be isolated by a hybridization method using the base sequence of the target nucleic acid or the entire or part of the complementary sequence as a probe. Furthermore, the nucleic acid can be isolated by using a nucleic acid amplification reaction (for example, PCR) using a synthesized oligonucleotide primer that has been designed to specifically hybridize to a part of the base sequence. Note here that the oligonucleotide primer can be easily synthesized by using a commercially available automated DNA synthesizer.

The preferable embodiment of the nucleic acid to be used in the present invention has a base sequence of any of SEQ ID NOs.: 4 to 6. In the other embodiment of the present invention, a DNA molecule (for example, nucleic acid including only a coding region) in which any one or more of the 5′ non-translation region or a part thereof and the 3′ non-translation region or a part thereof is deleted from the base sequence of any of SEQ ID NOs.: 4 to 6. Note here that a nucleic acid combining a non-translation region that is different from the original non-translation region may be used as long as it does not adversely affect the translation in the coding region.

Drugs of the present invention can be formulated according to the conventional method. In formulation, other ingredients acceptable for formulation (for example, carrier, vehicle, disintegrating agents, buffer agent, emulsifying agent, suspending agent, soothing agent, stabilizer, preservative, antiseptic agent, physiological saline, and the like) can be contained. An example of the vehicle may include lactose, starch, sorbitol, D-mannitol, and sucrose. An example of the disintegrating agents may include starch, carboxymethyl cellulose, calcium carbonate, and the like. An example of the buffer agent may include phosphate, citrate, acetate, and the like. An example of the emulsifying agent may include gum Arabic, alginate sodium, tragacanth, and the like. An example of the suspending agent may include glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate, and the like. An example of the soothing agent may include benzyl alcohol, chlorobutanol, sorbitol, and the like. An example of the stabilizer may include propylene glycol, diethylene sulfite, ascorbic acid, and the like. An example of the preservative may include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like. An example of the antiseptic agent may include benzalkonium chloride, parahydroxybenzoate, chlorobutanol, and the like.

The dosage form in the formulation is not particularly limited. An example of the dosage form may include tablet, powdered drug, fine subtilae, granule, capsules, syrup, injectable drug, external preparation, and suppository.

The thus formulated drug of the present invention can be administered to a patient by oral administration or parenteral administration (intravenous, intra-arterial, subcutaneous, intramuscular, intraperitoneal injection, and the like) depending upon the dosage form.

The content of the active ingredient (compound) in the drug of the present invention is, for example, in the range from about 0.001 wt. % to about 90 wt. % generally depending upon the dosage form. Thus, a predetermined dosage amount can be achieved.

Another aspect of the present invention provides a prevention method or a treatment method (hereinafter, these two methods are together referred to as “treatment method and the like”) of mental disorders by using the above-mentioned drug. The treatment method and the like of the present invention includes a step of administering the antipsychotic drug of the present invention to a living body. The route of administration is not particularly limited and can includes, for example, oral, intravenous, subcutaneous, intramuscular, intraperitoneal, transdermal, transmucosal administrations, and the like. The dosage amount of the drug will vary depending on the symptoms, age, sex, body weight, and the like, of the patient, but the person skilled in the art can set an appropriate dosage amount. For example, the dosage amount can be set so that the dosage amount of active ingredient for adult (body weight: about 60 kg) per day is about 0.001 mg to about 100 mg. The administration regimen can include, for example, once to several times a day, once per two days, or once per three days. For setting administration schedule, conditions of a patient, efficacy duration time of the drug, and the like, can be considered.

(Application for Study)

The present invention further relates to an application for study of genes that have been successfully identified. One embodiment of this aspect provides a reagent for application for study of mental disorders, which includes the isolated nucleic acid having any of the base sequences of SEQ ID NOs.: 1 to 6 or the homologous nucleic acid thereof. Another embodiment of this aspect provides a reagent for application for study of mental disorders, which includes isolated protein having an amino acid sequence of SEQ ID NOs.: 7 to 12 or the homologous protein thereof.

The reagent of the present invention can be used as a research tool for studying the onset mechanism or developing mechanism of symptom of mental disorders. Furthermore, it can be used as a reagent for carrying out the method of the present invention (a screening method, a method of obtaining information for diagnosis, and the like), or can be used as a component constituting the drug of the present invention. The reagent of the present invention can be used for producing a chimera mouse or a transgenic mouse as a model animal of mental disorders.

The reagent of the present invention contains nucleic acid in a state in which, for example, it is inserted into an appropriate vector (for example, an expression vector). In other words, the present invention provides an expression vector comprising a nucleic acid encoding an amino acid sequence of any of SEQ ID NOs.: 7 to 12. Specific embodiment of the expression vector of the present invention comprises a DNA having a base sequence of any of SEQ ID NOs.: 1 to 6.

The “homologous nucleic acid” herein is referred to as nucleic acid in which, as compared with the reference nucleic acid (nucleic acid having a base sequence of any of SEQ ID NOs.: 1 to 6), the function of protein encoded thereby is equal to that of the reference nucleic acid but a part of the base sequence is different from that of the reference base sequence. An example of the homologous nucleic acid includes DNA encoding a protein having a feature that it has a base sequence including substitution, deletion, insertion, addition or inversion in one or a plurality of base when the base sequence of any of SEQ ID NOs.: 1 to 6 is made to be a reference base sequence, and that the expression amount is increased relating to mental disorder. The substitution, deletion, or the like, of the base may occur in a plurality of sites. Herein, “plurality” denotes, for example, 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 bases, although it depends upon the positions or kinds of the amino acid residue in the three-dimensional structure of the protein encoded by the nucleic acid. The above-mentioned homologous nucleic acid can be obtained by genetically modifying nucleic acid having a sequence of SEQ ID NOs.: 1 to 6 so that a certain site may include substitution, deletion, insertion, addition or inversion of base by using a site specific mutation method. Furthermore, homologous nucleic acid can be obtained by other method such as irradiation with ultraviolet ray.

Another example of the homologous nucleic acid can include nucleic acid that hybridizes to the complementary strand of the nucleic acid of any of base sequences of SEQ ID NOs.: 1 to 6 under stringent conditions. Herein, “stringent conditions” are referred to as conditions in which a so-called specific hybrid can be formed and a nonspecific hybrid cannot be formed. Such stringent conditions are known to person skilled in the art and can be set with reference to, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) and Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). An example of the stringent conditions can include a condition in which a hybridization solution (50% formamide, 10×SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 5×Denhardt solution, 1% SDS, 10% dextran sulfate, and 10 μg/ml modified salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)) is used and incubated at about 42° C. to about 50° C., thereafter, 0.1×SSC and 0.1% SDS are used and washed at about 65° C. to about 70° C. Further preferable stringent conditions can include conditions of using, for example, a hybridization solution (50% formamide, 5×SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 1×Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg/ml modified salmon sperm DNA and 50 mM phosphate buffer (pH 7.5)).

Further example of the homologous nucleic acid can include nucleic acid in which the above-mentioned difference in the base is recognized due to the polymorphism represented by SNP.

(Kit Used in the Present Invention)

Each method of the present invention (screening method, a method for obtaining information for diagnosis, and the like) may be carried out by using a kit of reagent and the like. Another aspect of the present invention provides a kit used for such a purpose. For example, nucleic acid (probe and primer), reaction reagent, dilution, a reactor vessel, and the like, that are used for the method of the present invention can be contained in the kit. Note here that the kit of the present invention is generally includes instruction.

(Antibody to the Protein of the Present Invention)

The present invention further provides an isolated antibody having a specific binding property to the protein (target protein) having amino acid sequences of any of SEQ ID NOs.: 7 to 12. The antibody of the present invention is useful as a tool for studying mental disorders. For example, the antibody of the present invention is used for, for example, detecting, measuring and determining the target protein and expected to be contributed to elucidation of the action mechanism of mental disorders. The antibody of the present invention is expected to be applied to treatment or diagnosis of mental disorders.

The antibody of the present invention may be any one of a polyclonal antibody, an oligoclonal antibody (mixture of several kinds to several tens kinds of antibodies), and a monoclonal antibody. As the polyclonal antibody or the oligoclonal antibody, in addition to an IgG fraction derived from antiserum obtained by immunizing an animal, it is possible to use an antibody obtained by affinity-purifying with antigen. The antibody may be any antibody fragments such as Fab, Fab′, F(ab′)₂, scFv, and dsFv antibodies, and the like.

The antibody of the present invention may be prepared by using an immunological technique, a phage display method, a ribosome display method, and the like. A polyclonal antibody by an immunological technique can be prepared by the following procedures. Antigen (target protein or a part thereof) is prepared and an animal such as a rabbit, mouse, rat, or the like, is immunized with this prepared antigen. The antigen can be obtained by purifying a biological sample. Furthermore, recombinant protein (or peptide) can be used as an antigen. As mentioned below, the present inventors have obtained an antibody having a specific binding property to the protein having a amino acid sequence of SEQ ID NO.: 7 by using two kinds of partial peptides (CNTAFRGLRQHPRTQLL: SEQ ID NO.: 19, and CMSVDSRFRGKGIAKALG: SEQ ID NO.: 20) as an antigen.

Recombinant protein (or peptide) as an antigen can be prepared by, for example, introducing a gene (a part of a gene may be included) encoding an amino acid sequence of any of SEQ ID NOs.: 7 to 12 into an appropriate host by using a vector and expressing the gene in the obtained recombinant cell.

In order to enhance an immune provocation effect, an antigen to which a carrier protein is bonded may be used. As the carrier protein, KLH (Keyhole Limpet Hemocyanin), BSA (Bovine Serum Albumin), OVA (Ovalbumin), and the like, can be used. For the binding of the carrier protein, a carbodiimide method, a glutaraldehyde method, a diazo condensation method, an MBS (maleimide benzoyloxysuccinimide) method, and the like, can be used. On the other hand, it is also possible to use an antigen in which the target protein (or a part thereof) is expressed as a fused protein with GST, β galactosidase, maltose binding protein, histidine (His) tag, or the like, can be used. Such a fused protein can be purified by a general method in a simple way.

Immunization is repeated if necessary. At the time when the antibody titer is sufficiently increased, blood is collected and centrifuged so as to obtain serum. The obtained antiserum is affinity-purified so as to obtain a polyclonal antibody.

On the other hand, the monoclonal antibody can be prepared by the following procedures. Firstly, by the same procedure as mentioned above, immunization operation is carried out. If necessary, immunization is repeated. At the time when the antibody titer is sufficiently increased, antibody production cells are extracted from the immunized animal. Next, the obtained antibody production cells and myeloma cells are fused, so that hybridoma is obtained. Subsequently, this hybridoma is made into monoclones, followed by selecting a clone capable of producing antibody having a high specificity against an objective protein. By purifying the culture medium of the selected clone, the objective antibody can be obtained. On the other hand, after the hybridoma is proliferated into a predetermined number or more, this is transplanted into the peritoneal cavity of an animal (for example, a mouse) and proliferated in the ascites, followed by purifying the ascites. Thereby, the objective antibody can be obtained. For the purification of the above-mentioned culture medium or the purification of the ascites, affinity chromatography using the protein G, protein A, and the like, is preferably used. Furthermore, an affinity chromatography in which antigen is made into a solid phase can be used. Furthermore, an ion exchange chromatography, a gel filtration chromatography, ammonium sulfate fractionation, and centrifugation, and the like, can be used. These methods can be used singly or in a combination of any of them.

EXAMPLES
1. Experimental Materials and Methods

1-1. Search of Genes Related to Drug Dependence

1-1-1 Extract of mRNA

By using RNeasy mini kit (QIAGEN), mRNA was prepared by the following procedure. Mouse brain was taken out and the tissue was divided so as to be 30 mg or less. The brain tissue was dissolved in a solution containing a protein denaturizing agent and centrifuged with a spin column. Ethanol precipitation was repeated. Finally, mRNA was purified with silica gel.

1-1-2 cDNA Subtraction

The cDNA subtraction was carried out by using CLONTECH PCR-Select™ cDNA Subtraction Kit (CLONTECH) according to the attached instruction manual.

1-2. Place Preference Test

1-2-1. Animals

In the test, c57/black6 male mice (Japan SLC, Hamamatsu) that were 8-week old when the test was started. The mice were bred under conditions of light and dark cycle of 12 hours (light up at A.M 8:00), room temperature of 23±1° C., and humidity of 50±5%. The mice were freely fed with food (CE2: CLEA Japan, Tokyo) and water. This research program was approved by Animal Research Committee, Nagoya University School of Medicine and carried out in accordance with Guideline of Animal Research of Nagoya University School of Medicine and Principles of Laboratory Animal Care (National Institutes of Health Publication 85-23, 1985).

1-2-2. Drug

In the test, methamphetamine hydrochloride (philopon, Dainihon Seiyaku, Osaka) dissolved in saline was used.

1-2-3 Device for Test

In the test, a device having two chambers (light and dark chambers) and being provided with a partition between two chambers so that a mouse cannot come and go between two chambers (15 cm in length, 15 cm in width, and 15 cm in height). Staying time in each box was measured by using SCANET (Neuroscience, Tokyo).

1-2-4 Conditioned Place Preference Test

Conditioned place preference test was carried out in accordance with the method by Noda et al. (Noda Y, Miyamoto Y, Mamiya T, Kamei H, Furukawa H, Nabeshima T: Involvement of dopaminergic system in phencyclidine-induced place preference in mice pretreated with phencyclidine repeatedly. J Pharmacol Exp Ther 286: 44-51 (1998)). In the 2-day pre-conditioning test, mice were allowed to go and come between both chambers for 15 minutes a day for three days so that the mice became accustomed to the device. On day 3, the time staying in each chamber was measured (pre-value). After the pre-conditioning test, the 6-day conditioning test was carried out. In the conditioning test, a partition was put between chambers and mice were made to stay in only one of the chambers. On day 4, 6 and 8, right after methamphetamine or morphine was administered to the mice by subcutaneous administration, the mice were placed in the chamber with lower preference (the chamber with shorter staying time was shown in the pre-conditioning test). On day 5, 7 and 9, right after a physiological saline solution was administered to the mice by subcutaneous administration, the mice were placed in the opposite chambers. In the post conditioning test, the mice were allowed to go and come between the chambers and the staying time of each chamber, that is, the post-value was measured. The place preference was calculated from “(post-value)−(pre-value).” Note here that during all days of test, Leu-Ile was administered by intraperitoneal injection to the mice one hour before they were placed in the chamber.

1-2-5. Statistical Analysis

All the results are shown in a mean value and standard error. For the statistical analysis, one-way layout analysis of variance was carried out. When the significant difference was observed, multiple comparison test by Bonferroni was further carried out. Note here that, the difference with significance level of 5% or less was defined as the significant difference.

1-3. Measurement of Locomotor Activity

1-3-1. Animal

In the test, ICR male mice (Japan SLC, Hamamatsu) that were 8-week old when the test was started. The mice were bred under conditions of light and dark cycle of 12 hours (light up: A.M 8:00), room temperature 23±1° C., and humidity 50±5%. The mice were freely fed with food (CE2: CLEA Japan, Tokyo) and water. Note here that this research program was approved by Animal Research Committee, Nagoya University School of Medicine and carried out in accordance with Guideline of Animal Research of Nagoya University School of Medicine and Principles of Laboratory Animal Care (National Institutes of Health Publication 85-23, 1985).

1-3-2. Drug

In the test, methamphetamine hydrochloride (philopon, Dainihon Seiyaku, Osaka) dissolved in physiological saline solution (saline) was used.

1-3-3 Device for Test

In the test, an acrylic cage (28 cm in length, 17 cm in width and 13 cm in height) that is the same kind as the home case was used. On the floor, a small amount of breeding chip was placed. From the starting day to the completion date, cage and breeding chip at the measurement time were same for each mouse. For measuring the locomotor activity, an infrared detector (Neuroscience, Tokyo) was used. Analysis was carried out by using AB305 Measure and ABTEXT (Neuroscience, Tokyo).

1-3-4. Locomotor Activity Test

On days 1 to 3, mice were placed in a measurement cage for two hours so that the mice became accustomed to the measurement environment. Later than the fourth day, methamphetamine was administered to the mice by subcutaneous administration once a day. Right after the administration, the total locomotor activity for two hours was measured.

1-3-5. Statistical Analysis

All the results are shown in a mean value and standard error. For the statistical analysis, two-way layout analysis of variance was carried out. When the significant difference was observed, multiple comparison test by Bonferroni was further carried out. Note here that, the difference with significance level of 5% or less was defined as the significant difference.

2. Search of Novel Gene Related to Drug Dependence

2-1. Search of Novel Gene by Using Model Animal

Methamphetamine-administered mice show hyperactivity, and when methamphetamine is administered to mice every day, the degree of hyperactivity is increased. This can be thought to be a mental disorder accompanying drug dependence (FIG. 1). Then, mRNA was taken out from the mouse (C57black6) nucleus accumbens to which methamphetamine had been administered for 10 consecutive days. Based on this, cDNA subtraction (CLONTECH) was carried out. In mice to which methamphetamine had been administered, a gene in which mRNA expression amount increased by 20 times or more than that of a control group was searched. As a result, five genes satisfy the requirement (FIG. 2). In the genes, three genes except for CREB3 and Odz4 were used for the later research. For convenience of description, each gene is referred to as Gene 1, Gene 2 and Gene 3 (Piccolo).

When Gene 1, 2 and Gene 3 were retrieved by BLAST search, they were completely matched to partial sequences of the genes registered as BC034068 (GenBank Accession No. NM_—001001985 XM_—194205, Mus musculus cDNA sequence BC034068 (BC034068), mRNA: SEQ ID NO.: 1), 8430437G11Rik (GenBank Accession No. NM_—028990, Mus musculus RIKEN cDNA 8430437G11 gene (8430437G11Rik), mRNA.: SEQ ID NO.: 2), and Piccolo or Aczonin (GenBank Accession No. NM_—011995, Mus musculus piccolo (presynaptic cytomatrix protein) (Pclo), mRNA.: SEQ ID NO.: 3), respectively (FIGS. 3 and 4). The amino acid sequences encoded by Gene 1 (BC034068), 2 (8430437G11Rik) and 3 (Piccolo or Aczonin) are shown in SEQ ID NO.: 7, SEQ ID NO.: 8, and SEQ ID NO.: 9, respectively. Note here that Gene 1 was registered under the designation of mouse Shati in GeneBank (Accession No. DQ174094).

2-2. Search of Human Homologous Gene

Human homologous genes of Genes 1, 2 and 3 were retrieved by BLAST search. As a result, human homologous genes of Genes 1, 2 and 3 (which were referred to as Gene 1 human homologous gene, Gene 2 human homologous gene and Gene 3 human homologous gene, respectively) were found as follows. Two of the human homologous genes were genes whose function was not known, and remaining one gene was a gene identified as Piccolo or Aczonin.

Gene 1 human homologous gene: Homo sapiens cDNA FLJ3478 fis, clone BRAWH2013219, weakly similar to Homo sapiens N-acetyltransferase Camello 2 (CML2) mRNA (Genbank Accession No. AK094797: SEQ ID NO.: 4)

Gene 2 human homologous gene: Homo sapiens cDNA FLJ13576 fis, clone PLACE1008715 (Genbank Accession No. AK023638: SEQ ID NO.: 5)

Gene 3 human homologous gene: Homo sapiens piccolo (presynaptic cytomatrix protein) (PCLO), mRNA (Genbank Accession No. NM_—033026, XM_—168530: SEQ ID NO.: 6)

Note here that amino acid sequences encoded by Gene 1 human homologous gene, Gene 2 human homologous gene and Gene 3 human homologous gene are shown in SEQ ID NO.: 10, SEQ ID NO.: 11 and SEQ ID NO.: 12, respectively.

3. Change of Expression of Novel Gene

As to Genes 1 to 3, a part of each gene was subjected to RT-PCR method, in all the combination of primers, the increase of mRNA was observed in the methamphetamine administered group as compared with the control group (FIGS. 5 to 9). Furthermore, when the increase of the expression amount was examined by a real time RT-PCR method, the expression amount was increased in the methamphetamine-administered cortex of frontal lobe, nucleus accumbens and striatum, respectively (FIGS. 10 to 12).

When the expression amount of Gene 1 in each tissue of mouse was examined by RT-PCR, it was confirmed that Gene 1 was highly expressed in the cerebrum and cerebellum (FIG. 13). Furthermore, also in the liver, kidney, and spleen, Gene 1 was highly expressed. Three kinds of primer sets and PCR conditions used in RT-PCR are shown below.

(1) Primer Set 1

Forward primer:

cttgcctccccagcccatca
(SEQ ID NO.: 13)

and

reverse primer:

ctgggggccagggttctgct
(SEQ ID NO.: 14)

Reaction conditions: reaction was carried out at 95° C. for 5 minutes, followed by repeating reaction cycles 35 times each cycle including reaction at 94° C. for 30 seconds, at 70° C. for 40 seconds and at 72° C. for one minute, then reaction was carried out at 72° C. for 5 minutes and left at 4° C.

(2) Primer Set 2

Forward primer:

gggtggccgggtaggtggaa
(SEQ ID NO.: 15)

and

reverse primer:

ggcagtgcccagcccttcct
(SEQ ID NO.: 16)

Reaction conditions: reaction was carried out at 95° C. for 5 minutes, followed by repeating reaction cycles 35 times each cycle including reaction at 94° C. for 30 seconds, at 71° C. for 40 seconds, and at 72° C. for one minute, then reaction was carried out at 72° C. for 5 minutes and left at 4° C.

(3) Primer Set 3

Forward primer:

tgtacattcctccctggtggtg
(SEQ ID NO.: 17)

and

reverse primer:

aaatctgagagctgcaagaaaataggg
(SEQ ID NO.: 18)

Reaction conditions: reaction was carried out at 95° C. for 5 minutes, followed by repeating reaction cycles 35 times each cycle including reaction at 94° C. for 30 seconds, at 65° C. for 40 seconds, and at 72° C. for one minute, then reaction was carried out at 72° C. for 5 minutes and left at 4° C.

4. Expression Suppression Experiment

4-1. Effect of Gene 1 Antisense Oligonucleotide and Gene 2 Antisense Oligonucleotide in Enhancement of Locomotor Activity Induced by Methamphetamine

Each of the antisense nucleotide of Gene 1 and 2 was infused into the mice cerebral ventricle continuously by using a mini osmotic pump and methamphetamine was administered to the animals every day. The locomotor activity on the first, third and fifth days was measured. Note here that methamphetamine (1 mg/kg, s.c.) was administered to the mice for five days. The locomotor activity was measured for two hours. In the right cerebral ventricle (AP −0.5 mm, ML +11.0 mm from bregma, DV −2.0 mm from the skull), antisense oligonucleotide (Gene 1-AS or Gene2-AS, 1.8 nmol/6 μl/day), scramble control oligonucleotide (Gene 1-SC or Gene2-SC), and artificial cerebrospinal fluid (CSF) were infused continuously by using an osmotic pump.

The measurement result of Gene 1 is shown in FIG. 14; and the measurement result of Gene 2 is shown in FIG. 15. The results are shown in a mean value±standard error (Gene 1: n=5-7, Gene 2: n=3-5). As to Gene 1, in the repetitive two-way layout analysis of variance, between Gene 1 antisense oligonucleotide treated group and the control group and scramble control oligonucleotide group, the significant difference was observed (* P<0.05 compared with the physiological saline solution+CSF-treated group. # P<0.05 compared with the physiological saline solution+Gene 1-SC-treated group).

4-2. Effect of Gene 1 Antisense Oligonucleotide and Gene 2 Antisense Oligonucleotide in Place Preference Formation by Methamphetamine

As to Gene 1 and Gene 2, by using antisense oligonucleotide, the place preference test was carried out. During the conditioning, methamphetamine (0.3 mg/kg, s.c.) or a physiological saline solution was administered to mice. In the right cerebral ventricle (AP-0.5 mm, ML +1.0 mm from bregma, DV −2.0 mm from the skull), antisense oligonucleotide (Gene 1-AS or Gene2-AS, 1.8 nmol/6 μl/day), scramble control oligonucleotide (Gene 1-SC or Gene2-SC) and artificial cerebrospinal fluid (CSF) were infused continuously by using an osmotic pump.

The measurement result of Gene 1 is shown in FIG. 16; and the measurement result of Gene 2 is shown in FIG. 17. The results are shown in a mean value±standard error (n=5-12). Between the oligonucleotide treated group and the control group and scramble control oligonucleotide group, the significant difference was observed (*P<0.05 compared with the physiological saline solution-treated group. # P<0.05 compared with the methamphetamine+CSF-treated group).

4-3. Effect of Gene 3 Antisense Oligonucleotide in Enhancement of Locomotor Activity Induced by Methamphetamine

As to Gene 3 (piccolo), the antisense nucleotide was infused into the mouse cerebral ventricle continuously by using a mini osmotic pump and methamphetamine was administered to the animals every day. The locomotor activity on the first, third and fifth days was measured. Methamphetamine (1 mg/kg, subcutaneous administration) was administered to the mice for six consecutive days. 24 hours after the final administration, the mice were subjected to decapitation so as to obtain a sample. The number of mice in each group was five.

The measurement results are shown in FIG. 18. The results are shown in a mean value±standard error (n=5). In the repetitive two-way layout analysis of variance, between the Gene 3 antisense oligonucleotide treated group and the control group and sense oligonucleotide group, the significant difference was observed (*** p<0.0005 compared with physiological saline solution).

4-4. Change of Expression of Gene 1 mRNA in Gene 1 Antisense Oligonucleotide Treated Mouse by Continuous Administration of Methamphetamine

Antisense nucleotide of Gene 1 was continuously infused into the mouse cerebral ventricle by using a mini osmotic pump and methamphetamine was administered to the animals every day. Three days after the start of experiment, the mice were subjected to decapitation and the Gene 1 expression amount in the nucleus accumbens was measured by a real time RT-PCR method. The measurement results are shown in FIG. 19. The results are shown in a mean value±standard error (n=8). In the repetitive two-way layout analysis of variance, between the Gene 1 antisense oligonucleotide treated group and the control group and scramble control oligonucleotide group, the significant difference was observed (* P<0.05 compared with the physiological saline solution+CSF-treated group. #P<0.05 compared with the physiological saline solution+Gene 1-SC-treated group. $ P<0.05 compared with the physiological saline solution+Gene 1-AC-treated group. + P<0.05 compared with the methamphetamine+Gene 1-SC-treated group).

5. Effect and Action Mechanism of Gene 1

5-1. Effect of Dopamine D1 Receptor Antagonist R(+)-SCH₂₃₃₉₀and Dopamine D2 Receptor Antagonist Raclopride in the Increase of Expression of Gene 1 mRNA in the Nucleus Accumbens Induced by Methamphetamine

Methamphetamine (2 mg/kg, s.c.) was administered to mice once a day for six days, and 30 minutes before methamphetamine was administered, dopamine D1 receptor antagonist R(+)-SCH23390 (0.1 mg/kg, i.p.) or dopamine D2 receptor antagonist raclopride (2 mg/kg, i.p.) were administered. Two hours after the final administration, the mice were subjected to decapitation and the Gene 1 expression amount in the nucleus accumbens was measured by a RT-PCR method. The measurement results are shown in FIG. 20. The results are shown in a mean value±standard error (n=6-8). In the repetitive two-way layout analysis of variance, in the vehicle administered group, R(+)-SCH23390 administered group and raclopride administered group, the significant difference was observed (* P<0.05 compared with the vehicle/physiological saline solution-administered group. # P<0.05 compared with the vehicle/methamphetamine administered group). The results suggest that the increase of mRNA expression amount of Gene 1 is controlled by a signal via a dopamine receptor.

5-2. Change of Expression of TNF-α mRNA in Gene 1 Antisense Oligonucleotide Treated Mouse by Continuous Administration of Methamphetamine

Methamphetamine (1 mg/kg, s.c.) was administered to mice once a day for five days. During this time, in the right cerebral ventricle (AP −0.5 mm, ML +1.0 mm from bregma, DV −2.0 mm from the skull), Gene 1 antisense oligonucleotide (Gene 1-AS, 1.8 nmol/6 μl/day), scramble control oligonucleotide (Gene 1-SC) and artificial cerebrospinal fluid (CSF) were continuously infused by using an osmotic pump. Two hours after the final administration of methamphetamine, the mice were subjected to decapitation and TNF-α mRNA expression amount in the nucleus accumbens was measured by a RT-PCR method. The measurement results are shown in FIG. 21. The results are shown in a mean value±standard error (n=8-10). In Gene 1 antisense oligonucleotide treated group, control (CSF treated) group and scramble control oligonucleotide group, the significant difference was observed (*P<0.05 compared with physiological saline solution-administered group. # P<0.05 compared with the physiological saline solution+CSF-treated group and a physiological saline solution+Gene 1-SC-treated group. $ P<0.05 compared with the methamphetamine+Gene 1-SC treated group). The results suggest that Gene 1 may act via TNF-α.

5-3. In Vivo Effect of Gene 1 Antisense Oligonucleotide in the Increase of the Amount of Extracellular Dopamine Induced by Methamphetamine

Methamphetamine (1 mg/kg, s.c.) was administered to mice for two days. During this time, in the right cerebral ventricle (AP −0.5 mm, ML +1.0 mm from bregma, DV −2.0 mm from the skull), Gene 1 antisense oligonucleotide (Gene 1-AS, 1.8 nmol/6 μl/day), scramble control oligonucleotide (Gene 1-SC), or artificial cerebrospinal fluid (CSF) was continuously infused by using an osmotic pump. On day 3, by in vivo microdialysis method, the amount of extracellular dopamine in the nucleus accumbens (AP +1.7 mm, ML −0.8 mm from bregma, DV −4.0 mm from the skull) was measured for 220 minutes after the administration of methamphetamine. The measurement results are shown in FIG. 22. The results are shown in a mean value±standard error (n=5-6). In the repetitive two-way layout analysis of variance, between the Gene 1 antisense oligonucleotide treated group and the control (CSF) group and scramble control oligonucleotide group, the significant difference was observed (* P<0.05 compared with the Gene 1-SC-treated group). The results suggest that Gene 1 suppresses the increase of the amount of extracellular dopamine induced by methamphetamine.

5-4. Effect of Gene 1 in the Reduction of Uptake of Synaptosomal [³H]DA Induced by Methamphetamine

Methamphetamine (1 mg/kg, s.c.) was administered to mice for three days. During this time, in the right cerebral ventricle (AP −0.5 mm, ML +1.0 mm from bregma, DV −2.0 mm from the skull), Gene 1 antisense oligonucleotide (Gene 1-AS, 1.8 nmol/6 μl/day), scramble control oligonucleotide (Gene 1-SC), and artificial cerebrospinal fluid (CSF) were continuously infused by using an osmotic pump. The final concentration of [³H]DA was 5 nM. Two hours after the final administration of methamphetamine, the mice were subjected to decapitation and the amount of [³H]DA in the midbrain synaptosome was measured. The measurement results are shown in FIG. 23. The results are shown in a mean value±standard error (n=7-8). The amount of uptake of synaptosomal [³H]DA in the physiological saline solution+CSF-treated group, physiological saline solution+Gene 1-SC-treated group, physiological saline solution+Gene 1-AS-treated group, methamphetamine+CSF-treated group, methamphetamine+Gene 1-SC-treated group, methamphetamine+Gene 1-AS-treated group were 0.32±0.04, 0.29±0.03, 0.20±0.02, 0.18±0.01, 0.20±0.01, and 0.09±0.01 μmol/4 minutes/mg of proteins. This result suggest that Gene 1 suppresses the reduction of uptake of the dopamine into synaptosome induced by methamphetamine.

5-5. Effect of Gene 1 in the Reduction of Uptake Synaptovesicular [³H]DA Induced by Methamphetamine

Methamphetamine (1 mg/kg, s.c.) was administered to mice for three days. During this time, in the right cerebral ventricle (AP −0.5 mm, ML +1.0 mm from bregma, DV −2.0 mm from the skull), Gene 1 antisense oligonucleotide (Gene 1-AS, 1.8 nmol/6 μl/day), scramble control oligonucleotide (Gene 1-SC), and artificial cerebrospinal fluid (CSF) were continuously infused by using an osmotic pump. The final concentration of [³H]DA was 30 nM. Two hours after the final administration of methamphetamine, the mice were subjected to decapitation and the amount of [³H]DA in the midbrain synaptosome was measured. The measurement results are shown in FIG. 24. The results are shown in a mean value±standard error (n=8). The amount of uptake of synaptovesicular [³H]DA in the physiological saline solution+CSF-treated group, physiological saline solution+Gene 1-SC-treated group, physiological saline solution+Gene 1-AS-treated group, methamphetamine+CSF-treated group, methamphetamine+Gene 1-SC-treated group, methamphetamine+Gene 1-AS-treated group were 3.76±0.25, 4.05±0.29, 2.80±0.20, 1.74±0.21, 1.85±0.14, and 0.90±0.14 μmol/4 minutes/mg of proteins. These results suggest that Gene 1 suppresses the reduction of dopamine uptake induced by methamphetamine synaptovesicle.

5-6. Effect of Tranfection of Gene 1 in Reduction of Uptake of [³H]DA Induced by Methamphetamine

By using pcDNA-DEST53 (Invitrogen), an expression vector in which the full length Gene 1 or a fragment of Gene 1 had been incorporated was constructed (FIG. 25). Into the PC22 cell, an expression vector for expressing the full length Gene 1, an expression vector for expressing the fragment of Gene 1, or a pcDNA-DEST vector (empty vector) was transfected with Lipofectamine. After cells were cultured in a 24-well plate for 2 to 3 days, the cells were pre-treated with methamphetamine (1.0 μM) for 30 minutes. Then, by using Krebs-Ringer HEPES buffer containing 10 μM pagyline and 10 μM ascorbic acid, the amount of [³H]DA uptake in the cell was measured. The final concentration of [³H]DA was 20 nM. Furthermore, the cells were incubated at 22° C. for 10 minutes. The results are shown in a mean value±standard error (n=9-12). The measurement results are shown in FIG. 26. In the cell in which the expression vector for expressing the full length Gene 1 had been transfected, it was shown that the reduction of the uptake amount of [³H]DA induced by methamphetamine was suppressed (* P<0.05 compared with the pcDNA-DEST53 vector treated cell. #P<0.05 compared with the full length Gene 1 treated cell. $ P<0.05 compared with the Gene 1 fragment treated cell. + P<0.05 compared with the methamphetamine+pcDNA-DEST53 vector treated cell).

On the other hand, as a result of the measurement of the expression amount of intracellular Gene 1 mRNA by the RT-PCR method, in the cell in which the expression vector for expressing the full length Gene 1 was transfected, the significant increase in the expression amount of Gene 1 mRNA was confirmed (FIG. 27).

The above-mentioned results suggest that the Gene 1 suppresses the reduction of uptake of dopamine into the cell induced by methamphetamine.

5-7. Localization of Gene 1 in the Nucleus Accumbens after Continuous Administration of Methamphetamine

A rabbit was immunized with the partial peptide encoded by Gene 1 (S-3 antigen: CNTAFRGLRQHPRTQLL: SEQ ID NO.: 19 or S-4 antigen: CMSVDSRFRGKGIAKALG: SEQ ID NO.: 20) as an antigen so as to obtain two kinds of polyclonal antibodies (Gene 1 antibody S-3 and Gene 1 antibody S-4). On the other hand, methamphetamine (2 mg/kg, s.c.) was administered to mice for six days. 24 hours after the final administration of methamphetamine, the mice were subjected to decapitation. A specimen sample of the nucleus accumbens was produced. By using the above-mentioned polyclonal antibody (Gene 1 antibody S-3), immunostaining was carried out (FIG. 28). The staining results shown in FIG. 28 shows that Gene 1 is localized in the cytoplasm of the nerve cell. Also when Gene 1 antibody S-4 is used, the same stained image was obtained (not shown).

6. Change in Expression of Gene 1 mRNA in Nicotine, Alcohol and Phencyclidine

The responsibility of Gene 1 to nicotine, alcohol and phencyclidine was examined.

(1) Nicotine

Nicotine (0.05-1.0 mg/kg, s.c.) was administered to mice for 12 days. Two hours after the final administration, the mice were subjected to decapitation and the Gene 1 mRNA amount in the nucleus accumbens was measured by a RT-PCR method. The measurement results are shown in FIG. 29(a). The results are shown in a mean value±standard error (n=5). As shown in the figure, the correlation between the administration of nicotine and the expression amount of Gene 1 was observed (* P<0.05 compared with the physiological saline solution-administered group).

(2) Alcohol

A mouse was fed with 6% ethanol or water (control) for 12 days, and then was subjected to decapitation. The amount of Gene 1 mRNA in the nucleus accumbens was measured by a RT-PCR method. The measurement results are shown in FIG. 29 (b). The results are shown in a mean value±standard error (n=7-8). As shown in the figure, no significant correlation between the administration of ethanol and the expression amount of Gene 1 was observed.

(3) Phencyclidine

Phencyclidine (10 mg/kg, s.c.) was administered to mice for 14 days. Two hours after the final administration, the mice were subjected to decapitation and the Gene 1 mRNA amount in the cortex of frontal lobe was measured by a RT-PCR method. The measurement results are shown in FIG. 29(c). The results are shown in a mean value±standard error (n=8-9). As shown in the figure, no significant correlation between the administration of phencyclidine and the expression amount of Gene 1 was observed.

7. Expression of Gene 3 (Piccolo)

The expression state of Piccolo protein in the nerve cell was examined by the following procedure. By using cultured midbrain cells extracted from 13-day old rat, fluorescence double staining of tyrosine hydroxylase and Piccolo was carried out. The staining results are shown in FIGS. 30 and 31. This results show that the Piccolo protein is expressed in the anterior ganglion of the dopaminergic nerve cell and the midbrain dopamine neuron.

8. Effect of Piccolo on Suppression of Dopamine Uptake by Methamphetamine

8-1. Effect of Piccolo C2a Domain on the Suppression of Dopamine Uptake in PC12 Cell in which the Human Dopamine Transporter was Forcedly Expressed

An expression vector for expressing certain domains (CA2 and PDZ) of Piccolo was constructed (not shown). This expression vector was transfected into the PC12 cell in which the human dopamine transporter was forcedly expressed, then 1 μM methamphetamine was acted on the cell for 30 minutes. Then, the amount of uptake of dopamine into the cell was measured. Note here that dopamine labeled with ³H was acted on for 10 minutes so that the final concentration became 20 mM. The measurement result is shown in FIG. 32. As shown in FIG. 32, when Piccolo C2A domain was expressed in the PC12 cell in which a human dopamine transporter was forcedly expressed, the suppression effect of uptake of dopamine by methamphetamine was reduced.

8-2. Effect of Piccolo Expression Suppression on Uptake of Dopamine in PC12 Cell in Which Human Dopamine Transporter was Forcedly Expressed

Into the PC12 cell in which a human dopamine transporter was forcedly expressed, the oligonucleotide antisense or sense of the Piccolo protein was introduced, and then dopamine labeled with ³H was acted on for 10 minutes so that the final concentration became 20 nM and the radioactivity in the cell was measured. The measurement results are shown in FIG. 33. This results suggest that the Piccolo inhibits the suppression of uptake of dopamine by methamphetamine in the PC12 cell in which a human dopamine transporter was forcedly expressed.

8-3. Relation Between Dopamine Transporter and Piccolo Protein in PC12 Cell

(1) Effect of Piccolo on Intracellular Movement of Dopamine Transporter by Methamphetamine

In the PC12 cell, the human dopamine transporter was forcedly expressed and fluorescence immunostaining of a human dopamine transporter was carried out before and after methamphetamine was acted. When methamphetamine was acted on, the intracellular movement of the human dopamine transporter was observed. Furthermore, in the same cell, when the C2A domain of Piccolo was also expressed, the same intracellular localization was observed in both proteins.

From the above-mentioned results, it was determined that the Piccolo CA2 domain promoted the intracellular movement of the dopamine transporter by methamphetamine.

(2) Localization of Piccolo in Dopaminergic Nerve Cell

The expression state of the Piccolo protein in the dopaminergic nerve cell was examined by the following procedure. By using the dopaminergic nerve cell extracted from 13-day fetal rat midbrain, double staining of Piccolo and dopamine transporter was carried out. The staining results were sown in FIG. 35. FIG. 35 shows that the dopamine transporter and the Piccolo are localized in the same region in the dopaminergic nerve cell.

From the above-mentioned results (1) and (2), it was shown that when methamphetamine is acted on, internalization of the dopamine transporter occurs (FIG. 36(a)) and that the dopamine transporter and the C2A domain of the Piccolo were expressed in the same place (FIG. 36(b)).

9. Conclusion

From the experimental results, it was clarified that in Genes 1 to 3 that has been successfully identified, the expression level in the central nervous system tissue was increased in accordance with the formation of the drug dependence. Furthermore, it was clarified that Gene 1 was involved in the uptake of dopamine into the nerve cell. Furthermore, it was determined that Gene 1 had responsibility to nicotine. It was determined that Gene 3 (Piccolo) was involved in the internalization of the dopamine transporter. Thus, genes having the strong relationship with respect to the drug dependence were identified. Furthermore, the human homologous genes of Genes 1 to 3 were also identified. These genes are expected to be useful in the diagnosis and treatment of drug dependence.

INDUSTRIAL APPLICABILITY

The mental disorder-related genes provided by the present invention are used in screening of compound effective for a mental disorder or treatment, diagnosis and study of the mental disorder, and the like.

The present invention is not limited to the description of the above exemplary embodiments and Examples. A variety of modifications, which are within the scopes of the following claims and which are easily achieved by a person skilled in the art, are included in the present invention.

Contents of the theses, Publication of Patent Applications, Patent Publications, and other published documents referred to in this specification are herein incorporated by reference in its entity.

Method for diagnosing methamphetamine dependence

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