Patent Application
20080038198
When central nervous system stimulants, other dopamine agonists and the like are given to nematodes, which have outstanding characteristics as molecular neurobiological laboratory animals, behavioral sensitization characteristic to psychiatric disorders such as schizophrenia is established. The screening method of the present invention is a method for screening a gene relating to a psychiatric disorder or a method for screening a substance useful for prophylactic and/or therapeutic treatment of a psychiatric disorder, and is characterized by the use of nematodes behaviorally sensitized as described above.
Various information on nematodes (C. elegans) is provided by the Japanese Nematological Society. As for experimental methods using nematodes, detailed information on methods for morphological observation, culture of nematodes and the like can be obtained by referring to “The Nematode Caenorhabditis elegans” (Wood W. B., Cold Spring Harbor Laboratory Press). Therefore, those skilled in the art can easily obtain and utilize nematodes for the present invention.
The behavior to be sensitized in nematodes is not particularly limited. An example includes locomotion of nematodes or the like. However, the behavior is not limited to the above example, and may be any of behaviors that can be observed under microscope or the like, for example, pharynx pumping.
Types of agents used for behavioral sensitization in nematodes are not particularly limited. For example, nematodes can be behaviorally sensitized by using central nervous system stimulants or other dopamine agonists. More specifically, it is preferable to use nematodes behaviorally sensitized by any of a psychostimulant, an apomorphine, and a narcotic analgesic, and most preferably, nematodes behaviorally sensitized by any of amphetamine, methamphetamine, apomorphine and cocaine can be used. However, agents used for the behavioral sensitization are not limited to these examples.
According to the screening method of the-present invention, for example, a nematode with inhibited gene expression is constructed beforehand by a method for inhibiting expression of a specific gene, for example, by means of RNAi or the like, and the resulting nematode is treated with an agent that induces behavioral sensitization characteristic to a psychiatric disorder. Then, after a washout period, whether or not the gene associates with the establishment of behavioral sensitization characteristic to the psychiatric disorder can be judged based on the presence or absence of establishment of behavioral sensitization in response to a stress loaded with the agent. For the method for constructing a nematode with inhibited expression of a specific gene, for example, “The Nematode Caenorhabditis elegans” (Wood W. B., Cold Spring Harbor Laboratory Press) can be referred to. If behavioral sensitization is not established, the gene is determined to be a gene relating to the psychiatric disorder. In this case, a substance that inhibits expression of the gene is useful as an active ingredient of a medicament for prophylactic and/or therapeutic treatment of the psychiatric disorder. If behavioral sensitization is promoted, the gene is determined to be a gene relating to inhibition of onset of the psychiatric disorder. In this case, a compound that increases expression of the gene is useful as a therapeutic agent for the psychiatric disorder.
According to the present invention, the following two methods can be used as typical examples of the methods for screening target genes to be treated which relate to vulnerability to the onset of psychiatric disorders such as schizophrenia, manic depression, and drug dependence.
A mutation can be randomly introduced into the genome of C. elegans by applying a reported method (Dev. Biol., 221, pp. 295-307, 2000). An individual having the mutation in the gene relating to MAP-induced behavioral sensitization can be found by comparing MAP-induced behavioral sensitization in mutants with that in a wild type. According to a method of published report, the gene into which the mutation is introduced can be identified in the mutant found (Dev,Biol., 221, pp. 295-307, 2000).
A knockout library of C. elegans in which deletions of a predetermined level are introduced over the entire genome of C. elegans is constructed according to a reported method, and the library can be screened by PCR for a mutant in which deletion occurs in a specific gene (Nat. Genet., 17, pp. 119-121, 1997). A gene relating to behavioral sensitization can be identified by analyzing MAP-induced behavioral sensitization by using animals in which the specific isolated and identified gene is knocked out and comparing the result with that obtained by using a wild type.
The screening method of another embodiment typically comprises at least (1) the step of introducing a test gene into a nematode, and (2) the step of measuring behavioral changes in the nematode due to a subsequent drug treatment or stress to the nematode. As for the method for introducing a gene into a nematode in an expressible state, for example, “The Nematode Caenorhabditis elegans” (Wood, W. B., Cold Spring Harbor Laboratory Press) can be referred to. If behavioral sensitization is not established, the gene can be determined to be a gene relating to inhibition of onset of a psychiatric disorder. In this case, a substance that promotes expression of the gene is useful as an active ingredient of a medicament for prophylactic and/or therapeutic treatment of the psychiatric disorder. If behavioral sensitization is promoted, the gene can be determined to be a gene relating to onset of a psychiatric disorder. In this case, a compound that inhibits expression of the gene is useful as a therapeutic agent of the psychiatric disorder. A novel or known arbitrary gene can be used as the gene.
Further, according to the screening method of the present invention, a nematode is treated beforehand with an agent that induces behavioral sensitization characteristic to a psychiatric disorder, then the treatment with the agent is discontinued and a treatment with a test substance is performed, followed by observation of whether behavioral sensitization is inhibited by subsequent stress load, for example, a treatment with amphetamine or the like. By means of the aforementioned procedure, it can be determined whether the test substance is useful as an active ingredient of a medicament for prophylactic and/or therapeutic treatment of the psychiatric disorder, more specifically, whether the substance has a pharmacological effect of regulating progression of behavioral sensitization characteristic to the psychiatric disorder. The type of the test substance is not particularly limited, and may be antibodies, antisense nucleic acids, RNAi and the like, as well as low molecular weight compounds, natural substances and the like.
The present invention will be explained more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples.
Breeding of Nematode:
Nematodes were bred on a lawn of E. coli OP50 coated on a 60-mm NGM plate (Genetics, 77, pp. 71-94, 1974) in an incubator at 20° C.
Drug Treatment and Behavior Observation:
MAP hydrochloride was dissolved in sterilized pure water at a 100-fold concentration, and 50 μL of the solution was applied to a 35-mm NGM plate (5 mL). As a control group, sterilized water used as a solvent was similarly applied to prepare an assay plate. Apomorphine (hereinafter also abbreviated as “APO”) dissolved in 0.1% aqueous ascorbic acid as a solvent was applied as a solution of 100-fold concentration in a similar manner to prepare an assay plate. For a control group, 0.1% aqueous ascorbic acid was used. The periphery of each assay plate was applied with a 3.5 mol/L sucrose solution to prevent nematodes from invading the back of the plate. Adult-stage nematodes bred on the E. coli lawn were washed with S-basal buffer (Genetics, 77, pp. 71-94, 1974), then transferred to each of the aforementioned assay plates, and left at room temperature with light shielding for one hour in the MAP treatment or 30 minutes in the APO treatment, and the number of body bends over 20 seconds was counted under a stereoscopic microscope. In the behavioral sensitization analysis, nematodes treated on the MAP-containing NGM plate for one hour were returned to the E. coli lawn, bred at 20° C. for a predetermined time and then treated with various drugs, and their behavior was observed.
Imipramine Treatment:
Nematodes washed with S-basal buffer were transferred onto a 35-mm assay plate prepared by applying imipramine (hereinafter abbreviated as “IMI”) in the same manner as that used for MAP, and the number of eggs laid during 90 minutes was counted. Five nematodes were transferred to each plate, and the count was obtained for each plate.
It is known that the DA signal plays a major role in real-time recognition of the presence or absence of bacteria as feed (Escherichia coli), as one kind of important environmental information. Specifically, locomotion on a plate without feed is more suppressed than that under a condition with feed, and nematodes behave to stay longer in that environment. This slowing response is regulated by the DA system in the nerves (Neuron, 26, pp. 619-631, 2000; J. Neurosci., 21, pp. 5871-5884, 2001). MAP functions as an indirect DA agonist in mammals by acting on DAT of presynapse to elevate the DA concentration between synapses (Eur. J. Pharmacol., 361, pp. 269-275, 1998), and accordingly, it was inferred that MAP induced the slowing responses in nematodes.
As shown in
From the above results, it was confirmed for the first time that MAP-induced behavioral sensitization showing characteristics common to that in mammals was also established in nematodes. Specifically, it was found that susceptibility to MAP or other DA agonists used in the subsequent treatment was continuously increased for a prolonged time by experiencing MAP beforehand.
It is known that behavioral sensitization induced by central nervous system stimulants in mammals is a prolonged phenomenon associated with morphological changes in synapses (J. Neurosci., 17, pp. 8491-8497, 1997; Eur. J. Neurosci., 11, pp. 1598-1604, 1999; Nat. Neurosci., 4, pp. 1217-1223, 2001; J. Neurochem., 85, pp. 14-22, 2003). Under the experimental conditions in the present study, it was confirmed that the MAP-induced behavioral sensitization in nematodes was a phenomenon prolonged for at least 2 days (
Another characteristic of the behavioral sensitization is that cross sensitization is observed over drugs that differ in the sites and modes of action. Accordingly, effect of a pretreatment with MAP, i.e., an indirect DA agonist that acts on DAT in presynapse, on susceptibility to APO which is a direct DA agonist that acts on DA receptor in postsynapse was analyzed. Acute treatment with APO inhibited the locomotion in a concentration-dependent manner as MAP and induced a slowing response (
To examine a possibility that the observed MAP-APO cross behavioral sensitization depends on non-specific mechanisms such as change in the absorption system for the agent applied on a plate, analysis was performed to know whether susceptibility to IMI, which affects egg laying behavior primarily via the serotonin system, was effected by an MAP pretreatment. It was confirmed that IMI applied on an NGM plate promoted egg laying in a concentration-dependent manner (
The experimental results suggested that the phenomena of cross sensitization between indirect and direct DA agonists and sustainability thereof are an aspect of the neuroplasticity as in mammals, and it was inferred that at least a part of the mechanism is common to that in mammals.
Breeding of Nematode:
In an incubator inside of which was maintained at 20° C., nematodes were bred on an Escherichia coli lawn prepared by applying the E. coli OP50 strain on a 60-mm NGM plate (Genetics, 77, pp. 71-94, 1974).
Drug Treatment and Behavior Observation:
MAP hydrochloride was dissolved in pure water to prepare a 30 mM solution, and 50 μL of the solution was applied to a 35-mm NGM plate (5 mL) to prepare a 300 μM MAP plate. This MAP plate was further applied with 50 μL of an ondansetron solution (0.1 or 1 mM) dissolved in 100 mM NaCl or 0.1 N hydrochloric acid and adjusted to neutral pH by using NaOH to complete an assay plate. Nematodes (adults) bred on the Escherichia coli lawn were washed with S-basal buffer (Genetics, 77, pp. 71-94, 1974) for a short period of time, and then incubated on each of the aforementioned assay plates at 20° C. for one hour with light shielding. For this procedure, the periphery of each assay plate was applied with a 3.5 M sucrose solution to prevent nematodes from invading the back of the assay plate. After completion of the incubation for one hour, the nematodes were transferred to a plate with an Escherichia coli lawn prepared on a 35-mm NGM plate (5 mL) coated with 50 μL of 100 mM NaCl or a neutral ondansetron solution (0.1 or 1 mM), and incubated overnight at 20° C. with light shielding. On the following day, the nematodes were washed with S-basal buffer for a short period of time and transferred onto a MAP challenge plate prepared by applying 50 μL of a 10 μM MAP solution on a 35-mm NGM plate (5 mL), and the number of body bends over 20 seconds was counted under a stereoscopic microscope.
Pharmacological actions of ondansetron, widely used as an antiemetic drug, on the central nerve system have been reported, including a therapeutic effect for mental conditions (elicitation of hallucination and delusion), which develop during dopamine replacement therapy for Parkinson's disease (Neurology, 45, pp. 1305-1308, 1995; Scand. J. Rheumatol. Suppl., 113, pp. 37-45, 2000), effectiveness in initial treatment of alcoholism (Psychopharmacology, 149, pp. 327-344, 2000), and the like. However, its detailed action mechanism remains unknown. As shown in
The present invention provides a method for efficiently and inexpensively screening a gene relating to a psychiatric disorder such as schizophrenia. The present invention also provides a method for screening a gene relating to a psychiatric disorder or a substance useful for prophylactic and/or therapeutic treatment of the psychiatric disorder.
| Number | Date | Country | Kind |
|---|---|---|---|
| 000179/2004 | Jan 2004 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP05/00009 | 1/5/2005 | WO | 00 | 5/23/2007 |
