JP2008189662A - Release promoting function of neurotransmitter by sphingosine 1 phosphoric acid and its application - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To identify the action of sphingosine 1 phosphoric acid (S1P) specific to nervous system and apply the result to the treatment of diseases of the nervous system and the like. <P>SOLUTION: The invention is based on a neurospecific new action of the S1P identified this time such that the S1P promotes the release of a neurotransmitter from the neurocytes, and provides a method of regulating the release of the neurotransmitter from the neurocytes by administering an agonist or an antagonist of the S1P or its accepter to the neurocytes, or by controlling the production of the S1P in the neurocytes, also provides a release control agent of the neurotransmitter, a nervous disease treating medicine and a screening method of the release control agent of the neurotransmitter and the nervous disease treating medicine like this. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to a neurotransmitter release promoting function by sphingosine monophosphate and its application. For example, it is possible to promote or suppress the release of glutamate, a neurotransmitter, in hippocampal neurons in the central nervous system, and various neurological diseases such as the development of new antiepileptic drugs (especially hippocampal diseases and glutamatergic nervous system It can be applied to the treatment of diseases).
In addition, in this specification, when only the number is described in the parenthesis, the number indicates the corresponding number in the reference list listed at the end of the specification unless otherwise specified.

One of the major features of the central nervous system is the ability of a huge number of neurons (neurons) to communicate with each other to integrate and store countless information. Information transmission between neurons is performed through neurotransmitters at chemical synapses, and when neurotransmitters are released from presynaptic cells (hereinafter referred to as “presynaptics”) into the synaptic gap, Information is promptly transmitted from the pre-synapse to the post-synapse in the form that "the post-synapse" captures this information by its receptors.
Neurotransmitter release from presynapses includes phenomena such as movement of synaptic vesicles filled with neurotransmitters, fusion to cell membranes, and release of neurotransmitters into synaptic clefts (3, 17). In addition, the complex functions of the central nervous system, which has plasticity while accumulating a huge amount of information, and its research results suggest that there may be regulatory factors that control the release of neurotransmitters. By identifying such regulatory factors, the regulatory functions of these factors are used to control neurotransmitter release and, in turn, to control the transmission of information between neurons, thereby treating, diagnosing, and elucidating the pathology of various neurological diseases. It is expected to be applied to.

Sphingolipid metabolites such as sphingosine monophosphate (hereinafter sometimes abbreviated as “S1P”) are one of the substances that have recently attracted attention for their role in the nervous system (7). Sphingosine monophosphate is produced by phosphorylation of sphingosine by sphingosine kinase, and is considered to be an important lipid mediator that acts inside and outside the cell (22, 30). Extracellular sphingosine monophosphate binds to a member of the G protein-coupled S1P receptor family (S1P _1-5 ) to induce various cellular activities, causing angiogenesis, cardiac development, immunity, cell motility, nerves It has been reported that it is involved in various life phenomena such as elongation (31, 33, 38). On the other hand, sphingosine monophosphate has been reported to be involved in intracellular calcium migration, cell proliferation, and suppression of apoptosis in cells (14, 35). As yet another report, S12P accumulation occurs when PC12 cell depolarization is induced by high concentrations of potassium chloride (KCl) (1), and intracellularly generated S1P is released from cerebellar granule cells and astrocytes (2), sphingosine kinase 1 which is a synthase of S1P, and a receptor for S1P are abundantly present in the central nervous system (12, 13, 36). However, the physiological activity of S1P in the activity of nerve cells, its nerve-specific actions and roles have not been fully elucidated.
Therefore, the present invention aims to advance research on the action specific to the nervous system of sphingosine monophosphate (S1P), identify its action / function, and apply it to the treatment of neurological diseases and the like. .

  The lipid metabolizing enzyme sphingosine kinase 1 (hereinafter sometimes abbreviated as “SK1”) that phosphorylates sphingosine to produce S1P is abundant in the central nervous system as described above. The present inventor has found that, in hippocampal neurons, this SK1 is localized in the axon projection together with the vesicles. In addition, when the expression and activity of SK1 are suppressed, the release of depolarization-dependent neurotransmitter (glutamic acid) is suppressed, and at the time of depolarization, SK1 moves to the cell membrane and phosphorylates the substrate, Increasing the amount of S1P, and the generated S1P activates the S1P receptor in an autocrine or paracrine manner to promote the release of neurotransmitters from neurons, and this release by S1P. There are two different mechanisms of promoting action, S1P itself acts as an inducer to induce neurotransmitter release, and S1P as an enhancer to enhance and enhance depolarization-dependent neurotransmitter release As a result, it was found that these new findings can be applied to the treatment of neurological diseases, and the present invention has been completed.

That is, the present invention includes the following inventions (1) to (20) that are industrially useful.
(1) Regulating the release of neurotransmitters from neurons by administering sphingosine 1-phosphate or an agonist or antagonist of its receptor to neurons, or by controlling the production of sphingosine 1-phosphate in neurons how to.
(2) A method of suppressing the release of a neurotransmitter from a nerve cell by administering an antagonist of sphingosine 1-phosphate receptor to the nerve cell or suppressing production of sphingosine 1-phosphate in the nerve cell.
(3) A method for promoting the release of a neurotransmitter from a neuron by administering a sphingosine 1-phosphate or an agonist of its receptor to the neuron, or by promoting the production of sphingosine 1-phosphate in the neuron .
(4) The method according to any one of (1) to (3), wherein the neurotransmitter is glutamic acid or γ-aminobutyric acid (GABA).
(5) The method according to any one of (1) to (4) above, wherein the production of sphingosine 1-phosphate in the nerve cell is controlled by regulating the expression or activity of sphingosine kinase 1 in the nerve cell.
(6) The method according to any one of (1) to (5), wherein the nerve cell is a central nerve cell or derived from a central nerve cell.
(7) The method according to (6) above, wherein the nerve cells are derived from hippocampal neurons or hippocampal neurons.
(8) Release of a neurotransmitter containing as an active ingredient a substance selected from the group consisting of sphingosine 1-phosphate, an agonist or antagonist of sphingosine 1-phosphate receptor, and a substance that regulates the expression or activity of sphingosine kinase 1 Control agent.
(9) A neurotransmitter release inhibitor comprising as an active ingredient a substance selected from the group consisting of a sphingosine 1-phosphate receptor antagonist and a substance that suppresses the expression or activity of sphingosine kinase 1.
(10) A neurotransmitter release promoter comprising as an active ingredient a substance selected from the group consisting of sphingosine 1-phosphate, a sphingosine 1-phosphate receptor agonist, and a substance that promotes the expression or activity of sphingosine kinase 1 .
(11) A therapeutic agent for neurological diseases comprising as an active ingredient a substance selected from the group consisting of sphingosine 1-phosphate, an agonist or antagonist of sphingosine 1-phosphate receptor, and a substance that regulates the expression or activity of sphingosine kinase 1.
(12) The therapeutic agent for neurological disease according to (11), wherein the neurological disease is a central nervous disease.
(13) The therapeutic agent for neurological diseases according to (11), wherein the neurological disease is a hippocampal disease or a glutamatergic nervous system disease.
(14) The therapeutic agent for neurological diseases according to the above (11), wherein the neurological disease is selected from the group consisting of epilepsy, hippocampal sclerosis, Alzheimer's disease, schizophrenia and other psychoneurological diseases.
(15) A screening method for a neurotransmitter release-controlling agent or a therapeutic agent for neurological diseases, which comprises searching for an agonist or antagonist of sphingosine 1-phosphate receptor expressed in nerve cells.
(16) The screening method according to (15) above, wherein an agonist or antagonist of S1P ₁ or S1P ₃ which is a subtype of sphingosine 1-phosphate receptor is searched.
(17) It can be used in the screening method described in (15) or (16) above, and the presence or absence of activation of sphingosine 1-phosphate receptor expressed in the cells upon administration of a candidate substance to neurons A vector that expresses a fusion protein of a fluorescent protein and a sphingosine 1-phosphate receptor, prepared so that it can be detected by the FRET method.
(18) searching for a substance that regulates the expression or activity of sphingosine kinase 1 using the step of administering the candidate substance to a nerve cell or nerve tissue, and the change in expression or activity of sphingosine kinase 1 before and after administration of the candidate substance as an index A method for screening a neurotransmitter release-controlling agent or a neurological disease therapeutic agent.
(19) A method of regulating synapse formation between neurons by administering sphingosine 1-phosphate or an agonist or antagonist of a receptor thereof to neurons, or controlling production of sphingosine 1-phosphate in neurons.
(20) A method of suppressing synapse formation between nerve cells by inhibiting production of sphingosine 1-phosphate in nerve cells.

The present invention can be applied to the treatment, diagnosis, and pathological elucidation of various neurological diseases. For example, it can be used to develop new pharmacotherapy for intractable epilepsy such as temporal lobe epilepsy. Currently, pharmacotherapy such as valve acid (VPA) is the main treatment for temporal lobe epilepsy. However, there are many types in which the drug does not work, and teratogenicity due to this drug has been reported, and development of a safer and more effective drug is expected. Since this disease has a lesion in the hippocampus, the present invention can be applied to the treatment, diagnosis, pathological elucidation, etc. of temporal lobe epilepsy by specifically suppressing the release of glutamate from hippocampal neurons. it can.
Particularly in children with refractory temporal lobe epilepsy, causing intellectual disability due to repeated seizures is a major challenge in today's epilepsy treatment. The new trial of epilepsy treatment according to the present invention is based on the present finding that S1P promotes the release of neurotransmitter (glutamic acid) from hippocampal neurons and inhibits the action of S1P-producing enzyme SK1. One of them is to use a sphingosine kinase inhibitor for the treatment of epilepsy. Sphingosine kinase inhibitors are being developed with immunosuppressive drugs and the safety of the human body is being investigated. Therefore, a safer and more effective therapeutic effect can be expected by applying a sphingosine kinase inhibitor to a pharmacological treatment such as intractable temporal lobe epilepsy.
Of course, the present invention is applicable not only to the treatment of epilepsy but also to the treatment, diagnosis, and pathological elucidation of various neurological diseases. In particular, diseases caused by abnormal nerve activity in the center (disease caused by excessive nerve activity or conversely reduced nerve activity), especially hippocampal diseases caused by abnormal nerve activity in the hippocampal region, It is thought to be useful for the treatment, diagnosis, and pathological elucidation of diseases of the glutamatergic nervous system that transmits information between neurons using glutamate as a neurotransmitter. For example, in addition to epilepsy, clinical application to hippocampal sclerosis, Alzheimer's disease, schizophrenia, other neuropsychiatric disorders with memory and sensory impairment, and neurodegenerative disorders with tissue degeneration such as hippocampal atrophy can be considered.
Moreover, it has been found from the analysis results by immunohistochemistry that the S1P-producing enzyme SK1 is abundant not only in the hippocampal region but also in the olfactory bulb and cerebellar cortex. Therefore, the neurotransmitter release-promoting action of S1P identified this time is not limited to the hippocampal area, and S1P may have the same action in the brain area rich in SK1, and this is abnormal in areas other than the hippocampal area. This shows the possibility that the present invention can be clinically applied to the resulting brain diseases.
Furthermore, since S1P plays an important role in the formation and development of the nervous system, the present invention may be clinically applicable to diseases caused by abnormal formation of the nervous system. So far, the double knockout mouse SK1 / SK2 is S1P-producing enzyme, in both S1P ₁ deficient mice, a S1P receptor, it is reported to cause serious dysplasia of the nervous system, such as regurgitation of neural tube (26). In addition, since high expression of SK1 is observed in hippocampal mossy fibers where neurogenesis occurs frequently, S1P's neurotransmitter release-promoting action identified this time is the nervous system including synapse formation and thus circuit formation. It may be greatly involved in the formation and neoplasia. The present invention is also useful for the study of this molecular mechanism.
Recently, “spontaneous” release of neurotransmitters independent of action potential has been reported (19), but the molecular mechanism is not well understood. From this analysis, S1P enhances the action as a secretagogue that induces the release of neurotransmitters on its own without depolarization and the release of depolarization-dependent neurotransmitters. It has been clarified that it has an action as an enhancer. Of these two actions of S1P, the former action may be involved in the mechanism of spontaneous release of neurotransmitters. The present invention is useful for the study of this molecular mechanism, and can be widely used for other cranial nerve studies.

The present invention will be described in more detail below.
[1] Neurotransmitter release control method of the present invention In the neurotransmitter release control method of the present invention, S1P or an agonist or antagonist of its receptor is administered to a neuron, or S1P is produced in a neuron. It is characterized by regulating the release of neurotransmitters from nerve cells by controlling.
As described above, S1P produced by SK1 in nerve cells activates S1P receptors in an autocrine or paracrine manner to promote neurotransmitter release from nerve cells. Indeed, stimulation of hippocampal neurons with S1P induced the release of synaptic vesicles and the neurotransmitter glutamate in the vesicles (FIG. 6AB). This release inducing action was observed in a very small amount (picomolar concentration), and the maximum effect was observed at 10 nM. Also, administration of picomolar concentrations of S1P markedly enhanced depolarization-dependent glutamate release from neurons (FIG. 6D).
Therefore, administration of S1P or an agonist of its receptor to nerve cells can facilitate the release of neurotransmitters from the cells. In addition, among the members of the S1P receptor family, when the expression of S1P ₁ or S1P ₃ was suppressed, the release of neurotransmitter was suppressed, and when the expression of both was suppressed, the release of neurotransmitter was almost completely suppressed. (FIG. 6F). This result, as agonists of S1P receptors to be administered for the neurotransmitter release promoting, agonists of S1P ₁ or S1P ₃ is a subtype of S1P receptors (i.e., bind to S1P receptor, with S1P Similarly, it is preferable to use a substance that activates the receptor.
In addition, as can be seen from the results of FIG. 6F, by administering an antagonist of S1P receptor (that is, a substance that binds to S1P receptor and inhibits activation of receptor by S1P), conversely, neuronal cells The release of neurotransmitters from can be suppressed. As such agonists and antagonists used in the method of the present invention, known S1P receptor agonists and antagonists may be used. However, according to a method for detecting activation of S1P receptor by FRET method or the like, It is possible to search for agonists and antagonists for use in the invention. As an example of this method, a FRET method as shown in FIG. 5A can be adopted. In this method, first, a vector that expresses a fusion protein of an S1P receptor and a fluorescent protein (fluorophore) is prepared and introduced into a nerve cell. Then, various candidate substances are administered to nerve cells expressing this fusion protein, and the presence or absence of activation of the S1P receptor is detected by the FRET method. As a result, the substance that activates the S1P receptor has applicability as an agonist of the present invention, and as described later, it has applicability as a neurotransmitter release promoter or a therapeutic agent for neurological diseases.
On the other hand, various candidate substances were administered together with S1P to nerve cells expressing a fusion protein of S1P receptor and fluorescent protein (fluorophore), and activation of the receptor by S1P was administered together. Whether it is inhibited by a candidate substance may be detected by the FRET method. As a result, a substance that inhibits receptor activation by S1P can be used as an antagonist of the present invention, and can be used as a neurotransmitter release inhibitor or a therapeutic agent for neurological diseases as described later. Have
In addition, when neurons were treated with SK1 inhibitor dimethyl sphingosine (DMS) or SK1 expression was suppressed by RNA interference (siRNA), activation of S1P receptor was suppressed in either case (Fig. 5C). As can be seen from this result, it is possible to control the release of neurotransmitters from nerve cells by regulating the expression or activity of SK1 in neurons and regulating the production of S1P in the cells. is there. For example, it is possible to suppress the release of neurotransmitters from nerve cells by administering an SK1 inhibitor to suppress the production of S1P. As the SK1 inhibitor to be used, a known sphingosine kinase inhibitor (see, for example, JP-A No. 2001-122846) or a new SK1 inhibitor by screening may be used.
As described above, the neurotransmitter release control method of the present invention pays attention to the neurotransmitter release promoting function by S1P clarified by the present inventor, and by suppressing or promoting the function, A method for controlling the release of neurotransmitters. This method can be used for cultured neurons in vitro and for animals other than humans in vivo.

The above-described action of promoting the release of the neurotransmitter glutamate by S1P was confirmed not only by cultured hippocampal neurons but also by analysis using hippocampal tissue sections (FIG. 10). As a result of electrophysiological analysis, S1P promotes spontaneous glutamate release independent of electrical stimulation in hippocampal CA3 pyramidal cells (FIG. 11), and as an enhancement factor that enhances neurotransmitter release by electrical stimulation. An effect was also observed (FIG. 13).
Furthermore, as a neurotransmitter other than glutamic acid, S1P also has an action of promoting the release of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) (FIG. 14), CA3 pyramidal cells of the hippocampus and CA1 cones. Spontaneous GABA release was promoted in somatic cells (FIG. 15).
In addition, the present invention regulates synapse formation between neurons by administering sphingosine 1-phosphate or an agonist or antagonist of its receptor to neurons, or by controlling the production of sphingosine 1-phosphate in neurons. Provide a way to do it. For example, as described later, when the expression of sphingosine kinase 1 was suppressed in neurons, synapse formation between neurons was strongly suppressed (FIG. 16). Thus, it is possible to suppress the synapse formation between nerve cells by suppressing the production of sphingosine monophosphate in the nerve cells.

[2] Neurotransmitter release controlling agent and neurological disease therapeutic agent of the present invention As described above, the neurotransmitter release controlling agent and neurological disease therapeutic agent of the present invention are S1P, an agonist or antagonist of S1P receptor, Alternatively, a substance that regulates the expression or activity of SK1 is used as an active ingredient. As described above, the S1P receptor agonists and antagonists include, as described above, known S1P receptor agonists and antagonists, as well as novel S1P receptor agonists or antagonists by the method of detecting S1P receptor activation by the FRET method or the like. You may search for and use the resulting material.
When used as a therapeutic agent for neurological diseases, it is desirable to select an effective and appropriate substance from the candidate substance group according to the disease to be treated. For example, when an SK1 inhibitor that inhibits the expression or activity of SK1 is used as a therapeutic agent for temporal lobe epilepsy, a temporal lobe epilepsy model is selected from existing SK1 inhibitors or SK1 inhibitors newly obtained by screening. It is desirable to investigate the safety, translocation into the brain, effectiveness and specificity for epileptic seizures, and select more effective and appropriate substances using mice.

[3] Screening method of the present invention The screening method of the present invention focuses on the neurotransmitter release promoting action through activation of the S1P receptor, and screens for neurotransmitter release control agents or therapeutic agents for neurological diseases. The present invention is characterized by searching for an agonist or antagonist of the S1P receptor. For example, as described above, there is exemplified a screening method in which S1P ₁ or S1P ₃ which is a subtype of S1P receptor is expressed in nerve cells and an agonist or antagonist thereof is searched for by FRET method.
Another screening method of the present invention comprises a step of administering a candidate substance to nerve cells or nerve tissue in screening for a neurotransmitter release controlling agent or a neurological disease therapeutic agent, and the SK1 before and after administration of the candidate substance. And a step of searching for a substance that regulates the expression or activity of SK1 using the expression change or activity change as an index. The investigation of the change in the expression and activity of SK1 can be carried out by various known methods for examining the expression level of the gene / protein, the change in the phosphorylation activity of the kinase, etc., and is not particularly limited. Moreover, any of in vitro and in vivo screening systems may be used, and screening may be performed with a cell-free system. Further, the SK1 gene / protein may be derived from rats, mice and other animals in addition to those derived from humans. Of course, in silico screening may be performed using information on the higher order structure of the SK1 protein.

Next, examples of the present invention will be described, but the present invention is not limited to the following examples.
[Example 1]
[1] Materials and Methods [1-1] Preparation of Plasmids HA-mSK1 and HA-rSK2 plasmids were prepared in the same manner as in Reference 16, and cloned into the vector pEGFP-C1 (Clontech, Palo Alto, CA). .
The cDNAs of hSK1 (GenBank accession number, AF266756) and rSK1 (GenBank accession number, NM_133386) were amplified by PCR from a human brain cDNA library (Invitrogen) and a rat brain cDNA library (Clontech), respectively. In PCR, in order to attach an HA tag to the N-terminal, the following primers [1] and [2] were used for hSK1, and the primers [3] and [4] were used for rSK1 (described later). Each sequence of [1] to [31] corresponds to the sequence shown in SEQ ID NO: 1 to 31 in the sequence listing).
[1] sense primer, 5'-GCCGGTACCCCGCGGGTCGAGGTTATGG-3 '
[2] antisense primer, 5'-GCCGGTACCTCATAAGGGCTCTTCTGGC-3 '
[3] sense primer, 5'-CGGGGTACCATGCAACCAGCAGACTGTCCC-3 '
[4] antisense primer, 5'-CGGGGTACCTCATATTGGTTCTTCTGGAGGTGG-3 '
Stratagene (La Jolla, CA) A mutant hSK1G82D with inactive kinase activity was prepared by site-directed mutagenesis using the Quick Change site-directed mutagenesis kit. The resulting construct was sequenced over the entire coding sequence to confirm that the mutation was introduced.
A construct encoding rat synaptophysin fused with GFP at the C-terminus was reconstructed from pECFP incorporating synaptophysin-CFP.
The mouse S1P receptor, mS1P ₁ (GenBank accession number, NM_007901) cDNA was amplified from mouse brain cDNA. More specifically, after reverse transcription from mouse fetal brain mRNA (Invitrogen), PCR using the primers [5] and [6] below was performed to express pECFP-N1 expressing a construct in which CFP was fused to the C-terminus. Produced.
[5] sense primer, 5'-CGGAATTCGCCACCATGGTGTCCACTAGCATCC-3 '
[6] antisense primer, 5'-CGGAATTCGGGAAGAAGAATTGACGTTTCCAG-3 '
Mouse β-arrestin 2 (GenBank accession number, NM — 004313) cDNA was amplified from mouse brain cDNA. More specifically, after reverse transcription from mouse fetal brain mRNA (Invitrogen), PCR using the primers [7] and [8] below was performed, and pEYFP-C1 expressing a construct in which YFP was fused to the N-terminus was expressed. Produced.
[7] sense primer, 5'-GCCGGTACCCCGCGGGTCGAGGTTATGG-3 '
[8] antisense primer 5'-GCCGGTACCTCATAAGGGCTCTTCTGGC-3 '

[1-2] Cell culture All animal experiments were conducted in accordance with animal experiment guidelines of Kobe University.
Hippocampal neurons were prepared from embryonic day 18 (E18) rats (Wistar). Hippocampal neurons are glass bottom culture dishes (Matsunami Glass, Osaka, Japan) coated with 300 μg / ml poly-D-lysine and 25 μg / ml laminin in a 5% CO ₂ atmosphere in Neurobasal medium (B-27 (Invitrogen)). ), 1 mM L-glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin were added).
Various cDNA (0.53 μg) was introduced into cultured neurons (5 × 10 ⁴ cells, density 38,000 / cm ² ) using Lipofectamine 2000 (Invitrogen) 2-5 days before the assay. All experiments were performed on days 10-14 days of in vitro culture (DIV).

[1-3] Production of siRNA As the rat SK1 siRNA, the following [9] and [10] sequences were synthesized and used.
[9] 5'-GGGCAAGGCUCUGAAGCUCdTdT-3 '
[10] 5'-GAGCUUCAGAGCCUUGCCCdTdT-3 '
Further, those of the following sequences [11] [12] As a control siRNA, those of the following sequences [13] [14] as siRNA of SlP _1, the following sequences [15] [16] as siRNA of S1P ₃ Each was synthesized and used.
[11] 5'-UUCUCCGAACGUGUCACGUdTdT-3 '
[12] 5'-ACGUGACACGUUCGGAGAAdTdT-3 '
[13] 5'-CUGACUUCAGUGGUGUUCAdTdT-3 '
[14] 5'-UGAACACCACUGAAGUCAGdTdT-3 '
[15] 5'-CCCUCUACUCCAAGAAAUACA-3 '
[16] 5'-UAUUUCUUGGAGUAGAGGGGC-3 '
In the experiment, the generated siRNA and empty vector or various expression vector constructs were transfected into neurons 2-5 days prior to the assay.

[1-4] Preparation of anti-SK1 antibody A rabbit polyclonal anti-mouse SK1 antibody is a fusion protein of a synthetic peptide (corresponding to amino acid residues 362-381) having the amino acid sequence of [17] below and glutathione S-transferase. Was used as an antigen and prepared by a conventional method. The obtained anti-SK1 antibody was affinity purified using antigen-immobilized Sepharose 4B.
[17] GSRDAPSGRDSRRGPPPEEP

[1-5] Immunoprecipitation Cultured hippocampal neurons were treated with cell lysis buffer (20 mM Tris-HCl, pH 7.4, 1 mM EGTA, 1 mM EDTA, 150 mM NaCl, 1% (w / v) Triton X-100, protease inhibitors (Roche Applied Science)), and the resulting lysate was sonicated and then centrifuged at 10,000 xg. The supernatant was incubated with anti-SK1 antibody (1: 100 dilution) for 1 hour at 4 ° C., followed by addition of protein A-sepharose and further incubation for 1 hour. Thereafter, the sample was centrifuged at 2,000 × g for 5 minutes at 4 ° C., and the resulting pellet was washed three times with a cell lysis buffer not containing a surfactant. The pellet was dissolved in a cell lysis buffer not containing a surfactant and subjected to immunoblot analysis and sphingosine kinase (SK) assay.

[1-6] Immunofluorescence staining Rat hippocampal neurons were immunostained on the 10th to 14th day of culture in the same manner as in the method of Reference 11.
Polyclonal antibodies were used as the anti-SK1 antibody (1: 100 dilution) and the anti-SK2 antibody (1: 1000 dilution). Anti-Tau antibody (Sigma Aldrich; 1: 100), anti-MAP2 antibody (Sigma Aldrich; 1: 100), and anti-synaptophysin antibody (CHEMICON International, Temecula, CA; 1:50) used monoclonal antibodies.
For the fluorescence observation, a confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Jena, Germany) was used. More specifically, Alexa488 fluorescence was excited at 488 nm using a 505-530 nm band-pass barrier filter, and Alexa594 fluorescence was excited at 543 nm using a 560 nm long-pass barrier filter. Each was observed.
In addition, an experiment was conducted in which a neuron was transfected with a plasmid DNA encoding GFP-SK1 and labeled with FM4-64 dye, and then an active protrusion (functional puncta) was detected.

[1-7] Monitoring with FM4-64 Presynaptic synaptic vesicles were analyzed using a styryl fluorescent dye probe FM4-64 (Molecular Probes, Eugene, OR) for membrane staining in substantially the same manner as in Reference 34. Labeled. In the experiment, synaptic vesicles were labeled with 15 μM FM4-64 in 50 mM KCl for 1 minute, and then washed with a dye-free solution for 10 minutes. Then, extracellular release of vesicles induced by high-concentration potassium (50 mM KCl) or glutamic acid was applied to buffers (135 mM NaCl, 5.4 mM KCl, 1 mM MgCl ₂ , 1.8 mM CaCl ₂ , 10 mM glucose, 5 mM HEPES). , pH 7.3).
FM4-64 fluorescence was measured with a confocal laser scanning microscope (Zeiss LSM 510 META) at an excitation wavelength of 488 nm using a 640 nm long-pass barrier filter. The fluorescence at each protrusion (at least 100 different regions) was analyzed using Zeiss LSM 510 software. The average of the fluorescence values from the experiment performed three times independently was taken, and the change with time (decay) of FM4-64 fluorescence was plotted. Moreover, in siRNA experiment etc., FM4-64 fluorescence was measured in the GFP positive projection part expressing synaptophysin-GFP.

[1-8] Measurement of glutamic acid Glutamic acid released from cultured hippocampal neurons was measured using Amplex ^™ Red Glutamic Acid / Glutamate Oxidase Assay Kit (Invitrogen). Primary hippocampal neurons were treated for 1 minute in the presence or absence of 50 mM KCl or 10 nM S1P. Thereafter, the medium was collected, and the amount of glutamic acid was measured using the kit. Changes in fluorescence over time were measured using a fluorescence spectrometer (F-2500, Hitachi, Tokyo) at an excitation wavelength of 540 nm and an emission wavelength of 590 nm.

[1-9] Measurement of S1P amount in hippocampal neurons The amount of S1P in hippocampal neurons was measured by slightly modifying the method of Reference 10. In the experiment, KCl-treated (or control) hippocampal neurons (2.5 × 10 ⁶ cells / sample) were collected with methanol and S1P was extracted. The extracted S1P was dephosphorylated with alkaline phosphatase (Sigma) derived from bovine small intestinal mucosa, and phosphorylated again using [γ- ³² P] ATP-labeled recombinant mSK1. Then, radioactive S1P was quantified by thin layer chromatography using standard S1P.

[1-10] Real-time quantitative RT-PCR
Total RNA was extracted from rat hippocampal neurons (4 × 10 ⁵ cells) using ISOGEN (Nippon gene, Toyama, Japan). cDNA synthesis and quantitative PCR were performed in the same manner as in Reference 27. The used primer sequences for gene amplification are as follows.
The sense primer and antisense primer of rat _{S1P 1 [18] 5'-TTG} TTG CAA ATG CCC CAA CG-3 '
[19] 5'-TTT GCT GCG GCT AAA TTC CAT G-3 '
Rat S1P ₂ sense primer and antisense primer [20] 5'-TCG CCA AGG TCA AGC TCT ACG-3 '
[21] 5'-AGA CAA TTC CAG CCC AGG ATG G-3 '
The sense primer and antisense primer of rat _{S1P 3 [22] 5'-CAC} CTG ACC ATG ATC AAG ATG AG-3 '
[23] 5'-ACC CAG CGA GAA GGC AAT TAG C-3 '
Sense primer and antisense primer of rat _{S1P 4 [24] 5'-TGT} GTA TGG CTG CAT CGG TCT G-3 '
[25] 5'-GAG CAC ATA GCC CTT GGA GTA G-3 '
Sense primer and antisense primer of rat _{S1P 5 [26] 5'-GCT} CTA CGC CAA GGC CTA TGT G-3 '
[27] 5'-GCA CCT GAC AGT AAA TCC TTG C-3 '
Rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sense and antisense primer [28] 5'-TGC CCC CAT GTT TGT GAT G-3 '
[29] 5'-TGT GGT CAT GAG CCC TTC C-3 '
Rat SK1 sense primer and antisense primer [30] 5'-CTGGAGGAGGCTGAGGTATC-3 '
[31] 5'-CCAGTGACCCAGTTCTTCTGC-3 '
The expression of each mRNA was normalized by GAPDH mRNA expression.

[1-11] FRAP (fluorescence recovery after photobleaching)
FRAP analysis was performed using a Zeiss LSM 510 META confocal laser scanning microscope, as in the method of Reference 6. In experiments, GFP-SK1, GFP-mutated SK1, synaptophysin-GFP or GFP alone were transfected into primary hippocampal neurons and treated for 1 minute in the presence or absence of 50 mM KCl or 100 μM glutamate. Thereafter, the annular region of each selected projection was scanned for 8 seconds with the maximum output argon laser, and photobleached. After photobleaching, the recovery of fluorescence in each selected region was analyzed over time with a confocal fluorescence microscope with low laser output. Line scan averaging reduced the noise level of all images obtained.
FRAP in each selected area was calculated by the following formula (32).
[F (t)-Fp] / [Fi-Fp] x 100
("Fi" is fluorescence in the selected area before photo bleaching, "Fp" is fluorescence in the selected area immediately after photo bleaching, and "F (t)" is in the selected area at the time t (seconds) after photo bleaching. Fluorescent).

[1-12] FRET (fluorescence resonance energy transfer)
Primary hippocampal neurons cultured in a glass-bottom culture dish were co-transfected with S1P ₁ -CFP (donor) and YFP-β-arrestin (acceptor) expression plasmid at a donor / acceptor ratio of 1: 1 (both cells Introduced). Following excitation wavelength 458 nm, using a lambda mode and analysis software Zeiss LSM 510 META confocal laser scanning microscope, eight channels of 20nm intervals from 473nm to 633nm, (S1P ₁ -CFP or YFP-beta-arrestin The emission spectra of CFP and YFP were obtained (from cells expressing singly). Two days after co-transfection, neurons were treated with various agonists and each protrusion was FRET analyzed. The FRET efficiency was measured after the acceptor photobleaching as in the method of Reference 4. After obtaining a mixed spectrum image of co-transfected cells, the target protrusion or dendrite region was selected for YFP photobleaching. Then, while the selected region is photobleached (acceptor photobleaching) for 2 seconds with a 100% 514 nm laser output, images before and after the photobleaching are recorded using a 10% laser output for limiting photobleaching and an excitation wavelength of 458 nm. The method was adopted. FRET is understood as an increase in the CFP (donor) signal that comes close after the photobleaching of YFP (acceptor). The FRET efficiency (E) is calculated from the relative fluorescence intensity between the fluorescence intensity (Ipost) of the energy donor (CFP) after photobleaching and the fluorescence intensity (Ipre) of the energy acceptor (YFP) before photobleaching by the following formula. Determined (25).
E = 1- (Ipre / Ipost)

[1-13] Immunoblot analysis and the like Immunoblot analysis was performed in the same manner as in Reference Document 20. The activity of sphingosine kinase 1 was measured in substantially the same manner as in Reference Document 16, but 0.5% Triton x-100 was added to the reaction solution to suppress sphingosine kinase 2 (SK2) activity (24).

[2] Results [2-1] Expression of SK1 in the functional puncta of hippocampal neuron axons To investigate the role of S1P in neuronal activity and its neuron-specific function, first, sphingosine kinase (SK) in hippocampal neurons. ) Was examined.
First, an anti-SK1 antibody produced using mouse SK1 (mSK1) as an antigen specifically recognized rat SK1 (rSK1), but did not recognize rat SK2 (rSK2) (FIG. 1A). As a result of immunoblot analysis on rat brain lysate using this anti-SK1 antibody, a single band was observed at around 40 kDa corresponding to the molecular weight of recombinant rat SK1 (rSK1) (FIG. 1B left). This band disappeared when the antigen peptide was added to the reaction with the anti-SK1 antibody (right in the figure).
In addition, immunoprecipitation was performed from rat brain lysate using anti-SK1 antibody, and sphingosine kinase (SK) activity of the immunoprecipitate was analyzed. As a result, when an antigen-antibody reaction was performed in the absence of the antigen peptide, SK activity was observed, whereas no SK activity was observed in the presence of the antigen peptide (FIG. 1C).
In addition, isolated primary cultured rat hippocampal neurons were stained with anti-SK1 antibodies and antibodies against various marker proteins. As a result of immunohistochemistry on the primary hippocampal neurons, SK1 was stained in a punctate pattern along neurites. Almost all SK1 colocalized with the axon marker Tau, but did not colocalize with the dendritic marker microtubule-associated protein 2 (MAP2) (FIG. 1D). .
The SK1 punctate pattern was also co-labeled with synaptophysin, a marker of synaptic vesicles released extracellularly from presynapses. In addition, when the membrane staining substance FM4-64 that labels active presynaptic vesicles was used, almost all punctate patterns containing GFP-SK1 were co-labeled with FM4-64 (FIG. 1D).
From the above results, it is considered that SK1 is localized in the functional puncta of the presynapse in hippocampal neurons. In contrast, SK2, an isozyme of SK1, was mainly localized in the nuclei of hippocampal neurons, and was not present in axon projections or dendritic spines. This observation is consistent with previous reports (16, 27).

[2-2] Involvement of SK1 in neurotransmitter release by depolarization in hippocampal neurons From the observation that SK1 is localized in the presynaptic functional puncta, SK1 is We examined whether it is involved in neuron-specific effects such as release.
First, the influence of dimethyl sphingosine (DMS), a competitive enzyme inhibitor of SK1, on neurotransmitter release was examined. The neurotransmitter glutamate released from hippocampal neurons can be quantified by an enzyme fluorescence assay. Glutamate released from neurons by depolarization induced by high concentrations of potassium was strongly suppressed by the addition of DMS (FIG. 2A), suggesting the involvement of sphingosine kinase in the release of neurotransmitters.
Furthermore, the involvement of sphingosine kinase in the release of neurotransmitters induced by depolarization was investigated in detail by the FM dye method. In experiments, after pretreatment of hippocampal neurons in the presence or absence of DMS, presynaptic active synaptic vesicles were briefly treated with FM4-64 (1) under depolarizing conditions using a buffer containing 50 mM KCl. Min) Labeled. Then, after various depolarization stimuli, the release (secretion) of synaptic vesicles was evaluated by examining the fluorescence change (decay) of FM4-64 dye (FIG. 2B). As a result, DMS remarkably suppressed release (secretion) induced by depolarization by high-concentration potassium (FIG. 2C). This result supports the involvement of sphingosine kinase in vesicle release (secretion). Since there was no change in the emission spectrum of the FM dye before and after depolarization, the observed fluorescence change is considered not a result of changes in the membrane lipid environment after depolarization. Further, DMS itself had no substantial effect on the fluorescent labeling efficiency.
Furthermore, also in the release of synaptic vesicles (and neurotransmitters in the vesicles) from the presynaptic induced by glutamic acid, the release suppressing effect by DMS was observed (FIG. 2D). In addition, the release of synaptic vesicles was observed earlier in time when induced by glutamic acid than when induced by high-concentration potassium.
Next, the involvement of endogenous SK1 in neurotransmitter release was examined by a SK1 expression suppression experiment using siRNA. When cultured hippocampal neurons were treated with rSK1-siRNA, the expression of recombinant rSK1 was strongly suppressed, but the expression of human SK1 (hSK1) and hSK1G82D was not suppressed (FIG. 3A). When hippocampal neurons were treated with rSK1-siRNA, SK1 protein expression was suppressed by about 60% compared to control siRNA-treated neurons (FIG. 3B).
When hippocampal neurons were treated with rSK1-siRNA, the release of synaptic vesicles (and neurotransmitters in the vesicles) induced by high concentrations of potassium (50 mM) was markedly suppressed (FIG. 3C). This suppression of release was almost completely rescued when wild-type hSK1 was co-expressed, but was not rescued even when the inactive mutant hSK1G82D was co-expressed. This result indicates that SK1 kinase activity is very important in the release mechanism induced by depolarization.
Even with rSK2-siRNA treatment, no significant difference was observed in the release induced by high concentrations of potassium. In addition, when release was induced using glutamic acid, release in rSK1-siRNA-treated cells was suppressed particularly at an early stage (FIG. 3D). This release suppression was rescued when wild-type hSK1 was co-expressed, but was not rescued by co-expression of the inactive mutant hSK1G82D.
From the above results, it is considered that endogenous SK1 has an action and a role of up-regulating neurotransmitter release from hippocampal neurons induced by depolarization through production of S1P.

[2-3] Movement of SK1 from cytoplasm to membrane in depolarization It has been reported that when cells are treated with various agonists, intracellular SK1 is stimulated to move to cell membranes and organelle membranes. (8, 18, 29, 37). Therefore, by FRAP (fluorescence recovery after photobleaching) analysis, it was examined whether or not the position movement of SK1 is induced by depolarization in the axon protrusion (FIG. 1D) where SK1 is abundant. Plasmids encoding various GFP fusion proteins were transfected into hippocampal neurons and the movement of the fusion protein after photobleaching at the presynaptic protrusion was observed.
Before induction by depolarization, GFP protein seemed to freely diffuse and migrate in the cytoplasm in cells transfected with GFP alone, but synaptophysin, a presynaptic membrane protein, diffuses freely. The situation was not recognized. On the other hand, SK1 was observed to move in two ways, and the majority of SK1 seemed to diffuse and move freely, but part of it was localized in the membrane and not freely diffused. (FIG. 4A).
When depolarization was induced by KCl, the proportion of SK1 that did not diffuse freely increased, and SK1 in the protrusions moved from the cytoplasm to the membrane by depolarization. In contrast, the distribution pattern of GFP protein and synaptophysin did not change even when depolarization was induced by KCl (FIG. 4A). Furthermore, even when depolarization was induced by glutamic acid, the proportion of SK1 that did not diffuse freely increased, and the movement of SK1 from the cytoplasm to the membrane was observed, but this rate induced depolarization by KCl. There were few compared with the case (FIG. 4B).

[2-4] SK1 activity-dependent S1P receptor activation in depolarization Depolarization induces SK1 movement from the cytoplasm to the membrane in the axonal projection (FIG. 4), and SK1 activity is In view of the above results that are very important for the release of transmitter (FIG. 3CD), we next examined whether S1P newly produced by SK1 activates the S1P receptor (S1P ₁ ). In the experiment, FRET (fluorescence resonance energy transfer) analysis was performed to examine the activation of the S1P receptor (S1P ₁ ). β-arrestin binds rapidly to the receptor when the G protein-coupled receptor is activated (5, 21). In view of this, vectors expressing S1P ₁ -CFP and YFP-β-arrestin fusion proteins were prepared. If β-arrestin binds to S1P ₁ and CFP and YFP are in close proximity, excitation of CFP will stimulate emission from the YFP fluorophore (FIG. 5A). By this method, it is possible to capture the activation of S1P receptor in cells spatially and temporally. FRET produced by two fluorophore-conjugated proteins using the two expression vectors co-transfected into hippocampal neurons and specific photobleaching of the acceptor fluorophore YFP followed by FRET efficiency values based on deenchenching of the CFP signal The signal was confirmed.
FRET efficiency was increased to the same extent as stimulation by the receptor ligand S1P by depolarization induced by KCl or glutamate (FIG. 5B). This indicates that the S1P receptor is stimulated upon depolarization. When neurons were treated with DMS or rSK1-siRNA, the increase in FRET efficiency disappeared when depolarization was induced by KCl or glutamate, except when stimulated by S1P (FIG. 5C). This result indicates that the S1P ₁ receptor is activated in the vicinity of the region where S1P is produced by SK1 during depolarization. This result is also consistent with the previous results that release induced by depolarization is suppressed by DMS (FIG. 2) or rSK1-siRNA (FIG. 3CD) treatment.
Furthermore, the increase in FRET efficiency induced by depolarization was detected only in axon protrusions (FIG. 1D) in which SK1 is abundant, and not in dendritic regions with low SK1 content (FIG. 5C). ) Confirmed the importance of S1P production by SK1 during depolarization.

[2-5] Role of exogenous S1P in neurotransmitter release In order to examine the effect of S1P receptor activation in depolarization, we next examined the release of neurotransmitters from hippocampal neurons. Thus, the influence of exogenous S1P and dihydro S1P (DHS1P), which is a phosphorylated product of SK1, was examined.
As measured by the FM dye method, S1P itself has the effect of promoting the release of vesicles (and neurotransmitters in the vesicles) with EC ₅₀ = ˜20 pM in a dose-dependent manner, with a maximum effect observed at 10 nM. (FIG. 6A). DHS1P also promoted release similar to S1P. This indicates that the S1P release promoting action acts via a receptor.
Similar results were obtained by directly measuring glutamate release from hippocampal neurons. Exogenous S1P promoted glutamate release at S1P concentrations comparable to the results measured using the FM dye method (FIG. 6B). The effect of S1P was rapid and maximum extracellular release (exocytosis) occurred in about 2 seconds (FIG. 6C).
Next, the effect of S1P in combination with glutamic acid was analyzed in order to examine the effect of S1P via the receptor on neurotransmitter release induced by glutamic acid. Release induced by glutamic acid is strongly suppressed (75%) by DMS treatment (FIG. 2D, FIG. 6C). This indicates the importance of sphingosine kinase (SK) activity in glutamate-induced release. Significantly, this suppression by DMS was alleviated by co-administration of suboptimal concentrations (1 pM) of S1P (FIG. 6C). However, sufficient exocytosis could not be obtained by administration of S1P alone at this level (black triangles in FIGS. 6A and C). This result shows that, in addition to S1P itself acting as an inducer that induces release, neurotransmitter release induced by glutamate is enhanced by S1P, that is, S1P acts as an enhancer. It shows that it has. Indeed, the dose-dependent curve of glutamate-induced neurotransmitter release (exocytosis) was shifted to the left by administration of 1 pM exogenous S1P (FIG. 6D).
In order to examine whether the above-mentioned action of exogenous S1P is acting through activation of S1P receptor, S1P-induced neurotransmitter release was measured in S1P receptor knockdown cells.
First, when the expression of S1P receptor in hippocampal neurons is evaluated by real-time quantitative PCR, among the _five S1P receptor subtypes (S1P _{1 to} S1P ₅ ), mRNAs of S1P ₁ and S1P ₃ are mainly in hippocampal neurons. It was expressed (FIG. 6E). Therefore, the siRNA of the S1P ₁ or S1P ₃ Transfection in hippocampal neurons, as compared to control siRNA transfection neurons, SlP stimulated emission was either clearly suppressed (FIG. 6F). Furthermore, when co-transfected with the siRNA of S1P ₁ and SlP _3, SlP stimulated emission was almost completely suppressed. This indicates that both S1P ₁ receptor and S1P ₃ receptor activation are involved in S1P induced release.

[2-6] Autocrine or paracrine action of S1P in the induction and enhancement of glutamate release The action of S1P as a secretagogue mediated by the receptor (FIG. 6AB) is achieved with a Na ⁺ -channel blocker. Further examination was performed using a certain tetrodotoxin (TTX).
When hippocampal neurons were treated with TTX, S1P-induced release was moderately (approximately 50%) suppressed (FIG. 7A). In contrast, when the receptor was induced mainly by NMDA (N-methyl-D-aspartic acid) present in the post-synaptic membrane, the release induced by NMDA was almost completely lost by TTX treatment. (FIG. 7B).
This indicates that S1P promotes neurotransmitter release by two different mechanisms. The first is a mechanism that induces the release of a neurotransmitter (glutamic acid) by directly acting on the protuberance of TTX-insensitive presynapse, and the second is deactivation through activation of a TTX-sensitive NMDA-type glutamate receptor. A mechanism that enhances neurotransmitter release induced by polarization. Actually, the glutamate release promoting action of S1P was partially suppressed by TTX treatment (FIG. 7C). This indicates that two different mechanisms (ie, release induction mechanism and enhancement mechanism) are involved in the glutamate release promoting action by S1P.
Glutamate release induced by KCl was strongly suppressed by DMS (FIG. 2A), whereas glutamate release induced by S1P was insensitive to DMS (FIG. 7C). From this result, S1P generated in the local region where SK1 moved during depolarization then activates nearby S1P receptors and induces the release of glutamate from the presynapse, and the nerve induced by glutamate It is conceivable to exert the effect of enhancing the release of the transmitter substance. As supporting data, co-localization of active presynaptic vesicles labeled with FM4-64 and S1P ₁ or S1P ₃ receptors was observed (FIG. 7E).
Furthermore, the amount of intracellular S1P before and after depolarization was measured. When neurons were treated with high concentrations of potassium, the amount of intracellular S1P increased by approximately 50% compared to untreated neurons (FIG. 7D). This indicates that S1P produced during depolarization plays an important role in both induction and enhancement of glutamate release.

[Example 2]
[1] Materials and Methods [1-1] Cell Culture All animal experiments were conducted in accordance with Kobe University animal experiment guidelines. As hippocampal neurons, embryonic day 18 Wister rats or embryonic day 16 C57BL mice were used. Nerve cells were coated with 300 μg / ml polylysine and 25 μg / ml laminin on a glass-bottom culture dish, B27 was added to Neurobasal medium, 1 mM glutamine, 100 units of penicillin, and 100 μg / ml streptomycin were added. Culturing was performed under 5% atmospheric pressure carbon dioxide. Cultured neurons (cell density of 5 × 10 ⁴ cells per 38,000 / cm ² ) were transfected with various cDNAs (0.53 μg) using Lipofectamine 2000 (Invitrogen) and used for experiments after 4-5 days. All experiments were performed 10-14 days after neuronal culture.

[1-2] Glutamic acid and GABA release experiment Hippocampal slices (400 μm thick) were prepared from 6-week-old male Wister rats, and standard cranial nerve artificial fluid (ACSF) (117 mM NaCl, 3.6 mM KCl, 1.2 mM) NaH ₂ PO ₄ , 1.2 mM MgCl ₂ , 2.5 mM CaCl ₂ , 25 mM NaHCO ₃ , 11.5 mM glucose) are cultured for 1 hour at room temperature while passing 95% oxygen and 5% carbon dioxide, and further cultured at 34 ° C. for 50 minutes. did. Thereafter, the slice was transferred to a culture dish filled with ACSF medium in the presence or absence of tetrodotoxin (TTX, 0.5 μM) and S1P, and cultured at 34 ° C. for 20 minutes while passing 95% oxygen and 5% carbon dioxide gas. . After the operation, the culture broth was collected, and glutamic acid and GABA were labeled with 4-fluoro-7-nitrobenzofurazan (NBD-F). 24 μl of sample was injected into a column connected to an HPLC system (Shimadzu, Kyoto, Japan). NBD-F was excited at a wavelength of 350 nm, and the emitted light at 450 nm was measured using a fluorescence measuring instrument (Shimadzu, Kyoto, Japan). The total protein amount was measured using a protein quantification kit (Dojindo, Osaka, Japan). Statistical analysis was performed using an unpaired t test.

[1-3] Recording of EPSCs and IPSCs Slice patches were performed using the CA3 or CA1 region of hippocampal slices prepared from 6-week-old male Wister rats. Spontaneous excitatory postsynaptic currents (AMPA-mEPSCs) mediated by spontaneous AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptors are specific to standard ACSF fluid and to NMDA receptors Added the inhibitory inhibitor DL-2-amino-5-phosphonovaleric acid (DL-AP5, 100 μM), the GABA-A receptor specific inhibitors bicuculline (20 μM) and TTX (0.5 μM) at 34 ° C. The cells were cultured while passing through% oxygen and 5% carbon dioxide gas, and measured using an Axopatch-200A amplifier (Axon Instruments, USA).
GABA receptor-dependent microinhibitory post-synaptic potentials (GABA-mIPSCs) are expressed in standard ASCF fluid, AMPA receptor specific inhibitor 6,7-dinitro-2,3-dihydroxyquinoxaline (DNQX) (20 μM), DL-AP5 (100 μM) and TTX (0.5 μM) were added, and measurement was performed while a slice was cultured at 34 ° C. with aeration of 95% oxygen and 5% carbon dioxide gas. The experiment was conducted while the recording chamber was constantly refluxing the ACSF solution at a flow rate of 2 ml / min. When S1P (1 μM), DMS (10 μM, Sigma) or HACPT (3 μM) was added, it was added so that the slices were immersed using a three-way stopcock. The patch electrode solution is Cs ₂ SO ₄ (110 mM), tetraethylammonium chloride (5 mM), MgCl ₂ (2 mM), CaCl ₂ (0.5 mM), ethylene glycol-bis (β-aminoethyl ether) -N, N, N ', N'-tetraacetate (EGTA) (5 mM), Hepes (5 mM), MgATP (5 mM), CsCl (in place of CsSO ₄ (110 mM) for mIPSC measurement 135 mM) was used. The Kolmogorov-Smirnov test was used for statistical analysis.

[1-4] In situ hybridization histochemical staining Ten days old male C57BL mice were transcardially refluxed with 0.1 M phosphate buffer containing 4% paraformaldehyde, and the brain was excised from the head. Fixed overnight. Next, it was dehydrated using special grade ethanol, washed with toluene, embedded in paraffin, and serial sections were prepared with a thickness of 4 μm. CDNAs for all genes of the mouse S1P _1-5 receptor were prepared using the PCR method. DIG-labeled antisense and sense RNA probes were prepared in vitro in the presence of DIG-labeled dUTP (Roche, Sydney, Australia) using the cDNA fragment as a template. In situ hybridization was performed with reference to a manual prepared by the manufacturer.

[1-5] Preparation of siRNA of sphingosine kinase 1 (SK1), etc. siRNA of rat sphingosine kinase 1 (rSK1) (5′-GGGCAAGGCUCUGAAGCUCdTdT-3 ′ (SEQ ID NO: 9), 5′-GAGCUUCAGAGCCUUGCCCdTdT-3 ′ (SEQ ID NO: 10) )) And control siRNA (5′-UUCUCCGAACGUGUCACGUdTdT-3 ′ (SEQ ID NO: 11), 5′-ACGUGACACGUUCGGAGAAdTdT-3 ′ (SEQ ID NO: 12)) were synthesized at Japan Bio Services (Saitama, Japan). Production of HA-hSK1 and its activity-deficient mutant was performed in the same manner as in Example 1. Neurons were transfected with the above siRNA in the presence of an empty vector or a plasmid vector containing various hSK1, and used for experiments after 2-5 days.

[1-6] Anti-SK1 Antibody A rabbit anti-mouse SK1 polyclonal antibody prepared against a synthetic peptide was prepared in the same manner as in Example 1.

[1-7] Immunohistochemical staining The affinity-purified anti-mouse SK1 polyclonal antibody was diluted 500 times and used. Horizontal sections of adult C57BL / 6 mouse brains were immunostained according to the method described in Sakai et al., Genes Cells 9: 945-957 (2004).

[1-8] Analysis of synapse formation 50,000 cultured hippocampal neurons were transfected with siRNA for synaptophysin-GFP and rat SK1, or in some experiments, further with a plasmid containing human HA-SK1 cDNA, It was used for the experiment after 5 days. On day 14 after culturing, nerve cells were fixed, and immunostaining was performed using an anti-PSD95 antibody (Bioreagents, Golden, CO; used at 500-fold dilution). Fluorescence images were captured and analyzed in a continuous image acquisition mode using a confocal microscope. In each analysis, a granular structure (puncta) labeled with at least 500 synaptophysin-GFP in each series was arbitrarily selected and analyzed, and three series were analyzed for statistical analysis. In the measurement of PSD-95 accumulation size, a single puncta that expresses both synaptophysin-GFP-derived GFP and PSD-95-derived fluorescence is arbitrarily selected, and the size of the synaptic region after PSD-95 positive is selected with Zeiss LSM 510 software. Analysis and cumulative frequency distribution analysis.

[2] Results [2-1: SK1 and S1P ₁ receptors are both abundantly expressed in dentate gyrus granule cells hippocampal]
Previous Example 1 showed that S1P is involved in neurotransmitter release in the hippocampal primary neurons. Furthermore, in order to analyze the function of neurotransmitter release of S1P, first, immunohistochemistry of the enzyme SK1 involved in S1P production was analyzed using the adult mouse brain. As a result, as shown in FIG. 8AB, SK1 was widely distributed in the olfactory bulb, caudate nucleus-palmoid bulb, substantia nigra, hippocampus and cerebellar cortex. Interestingly, SK1 is preferentially found in the clear layer of the CA3 region of the hippocampus, where mossy fibers from dentate granule cells synapse to the proximal dendrites of CA3 pyramidal cells, CA3 pyramidal cells were not found in the Schaffer collateral path projecting onto CA1 pyramidal cells (FIGS. 8C-F). This suggests that S1P is present in a high concentration in mossy fibers.
S1P transmits signals through the S1P receptor. When analyzed using in situ hybridization histochemistry, S1P ₁ receptor mRNA positive cells were distributed in large numbers in the dentate granule cells and weakly distributed from CA3 to CA1 (arrows in FIG. 9). Similar results were obtained using S1P ₃ receptor mRNA. In Example 1, it is revealed that these S1P ₁ and S1P ₃ receptors are both involved in the release of glutamate from hippocampal neurons. From the above, it was found that the distribution of S1P ₁ receptor is consistent with that of SK1 in the hippocampus.

[2-2: S1P causes AMPA-mEPSC in CA3 pyramidal cells but not CA1 pyramidal cells in rat hippocampal slices]
To further investigate the effect of S1P on neurotransmitter release, rat hippocampal slices were used to examine the effect of S1P on glutamate release in the presence of voltage-dependent sodium channel blocker TTX. As a result, S1P caused glutamate release from the hippocampal slice in a concentration-dependent manner (0.01-1 μM) (FIG. 10). This indicates that S1P causes the release of glutamate from synapses in a state where no static depolarization occurs.
To gain further insight into the release of glutamate in S1P, AMPA-mEPSC was measured using rat hippocampal slices. As a result, S1P (1 μM) significantly increased the frequency without changing the intensity of AMPA-mEPSC recorded from CA3 pyramidal cells (P <0.01, Kolmogorov-Simirnov test, FIG. 11A). This indicates that S1P promotes spontaneous glutamate release at synapses that project from mossy fibers to the proximal dendrites of CA3 pyramidal cells. On the other hand, such a phenomenon by S1P was not observed in CA1 pyramidal cells with low expression levels of SK1 (FIG. 8C) and S1P ₁ receptor (FIG. 9) (FIG. 11B). From these results, sphingosine kinase (SK) / S1P signal is an important factor that causes spontaneous glutamate release at least in the hippocampus, and this signal is not CA1 in which Schaffer side branch exists but CA3 region in which mossy fibers are present. It was strongly suggested that it plays an important role in spontaneous excitatory signaling.

[2-3: Spontaneous release of glutamate is no longer observed with inhibitors of sphingosine kinase (SK)]
In Example 1, it was shown using neuronal cultured cells of primary hippocampus that so-called “automatic activation” in which endogenously produced S1P activates nearby S1P ₁ receptors causes glutamate release. In this connection, the SK inhibitor DMS significantly reduced the frequency, not the intensity of spontaneous AMPA-mEPSC from CA3 pyramidal cells (P <0.01, Kolmogorov-Simirnov test, FIG. 12A). Similar suppression was also observed using another more specific SK inhibitor 2- (p-Hydroxyanilino) -4- (p-chlorophenyl) thiazole (HACPT) (P <0.01, Kolmogorov-Simirnov test, FIG. 12B). Taken together, this confirms from a specific phenomenon that endogenous S1P produced by the action of SK regulates the spontaneous release of glutamate through the automatic activation of the S1P receptor.

[2-4: S1P enhances EPSC caused by electrical stimulation]
Next, the effects of post-excitatory synaptic potential (eEPSC) and S1P in CA3 pyramidal cells caused by electrical stimulation were examined using hippocampal slices. Consistent with the results of Example 1, S1P (1 μM) enhanced eEPSC (FIG. 13A), and this enhancing effect was not suppressed by DMS and did not affect the strength of eEPSC (FIG. 13B). Thus, exogenously added S1P promotes depolarization-dependent glutamate release via the S1P receptor, whereas endogenous S1P produced through the action of sphingosine kinase does not appear to affect eEPSCs.

[2-5: S1P induces GABA-mIPSCs from both CA3 and CA1 pyramidal cells in rat hippocampal slices]
To investigate whether S1P causes only excitatory neurotransmitter release, we investigated the relationship between the release of GABA, an inhibitory neurotransmitter, from rat hippocampal slices in the presence of TTX (0.5 μM) and S1P. It was. As a result, S1P caused GABA release in a concentration-dependent manner (0.01-1 μM) as in glutamate release (FIG. 14).
In addition, GABA receptor-dependent microinhibitory post-synaptic potentials (GABA-mIPSCs) in CA3 or CA1 pyramidal cells were examined using rat hippocampal slices. As a result, S1P (1 μM) increased the frequency predominantly without changing the intensity of GABA-mIPSCs in CA3 pyramidal cells (P <0.01, Kolmogorov-Simirnov test, FIG. 15A). These facts indicate that S1P causes spontaneous release of both glutamic acid and GABA. Furthermore, unlike AMPA-mEPSCs, S1P (1 μM) also increased the frequency of GABA-mIPSCs in CA1 pyramidal cells (P <0.01, Kolmogorov-Simirnov test, FIG. 15B). CA1 and CA3 pyramidal cells are known to be innervated by GABA interneurons, and it is likely that sphingosine kinase and S1P receptors are expressed in these interneurons.

[2-6: Sphingosine kinase (SK) / S1P signal promotes synapse formation in neuronal cell culture experiments in rat primary hippocampus]
Previous reports have shown that a causal relationship exists between neurotransmitter release and synaptogenesis (Han et al., Nature 349: 697-700 (1991); Kabayama et al., Neurosci. 88 : 999-1003 (1999)). Therefore, whether or not S1P is involved in synapse formation was examined using a rat primary hippocampal neuron culture. When SK1-specific siRNA was used, endogenous SK1 expression was suppressed (FIG. 16B). Next, the frequency of co-staining of synaptophysin, a presynaptic marker, and PSD-95, a post-synaptic marker, was reduced by about 60% in neurons treated with SK1-specific siRNA (FIG. 16AC). ). The assembly size of PSD-95 was clearly reduced compared to the control (FIG. 16D). Furthermore, the phenomenon caused by the SK1-specific siRNA was recovered by expressing human SK1, but it could not be recovered by expressing an activity-deficient mutant (FIG. 16ACD). This suggests that the S1P signal is necessary for synapse formation.

[Reference list]
1. Alemany, R., B. Kleuser, L. Ruwisch, K. Danneberg, H. Lass, R. Hashemi, S. Spiegel, KH Jakobs, and D. Meyer zu Heringdorf 2001. Depolarisation induces rapid and transient formation of intracellular sphingosine-1-phosphate. FEBS Lett. 509: 239-244.
2. Anelli, V., R. Bassi, G. Tettamanti, P. Viani, and L. Riboni 2005. Extracellular release of newly synthesized sphingosine-1-phosphate by cerebellar granule cells and astrocytes. J. Neurochem. 92: 1204- 1215.
3. Bajjalieh, SM 1999. Synaptic vesicle docking and fusion. Curr. Opin. Neurobiol. 9: 321-328.
4. Bastiaens, PI, IV Majoul, PJ Verveer, HD Soling, and TM Jovin 1996. Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin. EMBO J. 15: 4246-4253.
5. Benovic, JL, H. Kuhn, I. Weyand, J. Codina, MG Caron, and RJ Lefkowitz 1987. Functional desensitization of the isolated betaadrenergic receptor by the beta-adrenergic receptor kinase: potential role of an analog of the retinal protein arrestin (48-kDa protein). Proc. Natl. Acad. Sci. USA. 84: 8879-8882.
6. Carrero, G., E. Crawford, J. Th'ng, G. de Vries, and MJ Hendzel 2004. Quantification of protein-protein and protein-DNA interactions in vivo, using fluorescence recovery after photobleaching. Methods Enzymol. 375 : 415-442.
7. Colombaioni, L., and M. Garcia-Gil 2004. Sphingolipid metabolites in neural signaling and function.Brain Res. Rev. 46: 328-355.
8. Delon, C., M. Manifava, E. Wood, D. Thompson, S. Krugmann, S. Pyne, and NT Ktistakis 2004. Sphingosine kinase 1 is an intracellular effector of phosphatidic acid. J. Biol. Chem. 279 : 44763-44774.
9. Doussau, F., S. Gasman, Y. Humeau, F. Vitiello, M. Popoff, P. Boquet, MF Bader, and B. Poulain 2000. A Rho-related GTPase is involved in Ca ²⁺ -dependent neurotransmitter exocytosis. J. Biol. Chem. 275: 7764-7770.
10. Edsall, LC, and S. Spiegel 1999. Enzymatic measurement of sphingosine 1-phosphate. Anal. Biochem. 272: 80-86.
11. Fujita, T., T. Okada, S. Hayashi, S. Jahangeer, N. Miwa, and S. Nakamura 2004. Delta-catenin / NPRAP (neural plakophilin-related armadillo repeat protein) interacts with and activates sphingosine kinase 1 Biochem. J. 382: 717-723.
12. Fukuda, Y., A. Kihara, and Y. Igarashi 2003. Distribution of sphingosine kinase activity in mouse tissues: contribution of SPHK1. Biochem. Biophys. Res. Commun. 309: 155-160.
13. Harada, J., M. Foley, MA Moskowitz, and C. Waeber 2004. Sphingosine-1-phosphate induces proliferation and morphological changes of neural progenitor cells. J. Neurochem. 88: 1026-1039.
14. Hla, T., MJ Lee, N. Ancellin, CH Liu, S. Thangada, BD Thompson, and M. Kluk 1999. Sphingosine-1-phosphate: extracellular mediator or intracellular second messenger? Biochem. Pharmacol. 58: 201 -207.
15. Humeau, Y., MR Popoff, H. Kojima, F. Doussau, and B. Poulain 2002. Rac GTPase plays an essential role in exocytosis by controlling the fusion competence of release sites. J. Neurosci. 22: 7968-7981 .
16. Igarashi, N., T. Okada, S. Hayashi, T. Fujita, S. Jahangeer, and S. Nakamura 2003. Sphingosine kinase 2 is a nuclear protein and inhibits DNA synthesis. J. Biol. Chem. 278: 46832 -46839.
17. Jahn, R., and TC Sudhof 1999. Membrane fusion and exocytosis. Annu. Rev. Biochem. 68: 863-911.
18. Johnson, KR, KP Becker, MM Facchinetti, YA Hannun, and LM Obeid 2002. PKC-dependent activation of sphingosine kinase 1 and translocation to the plasma membrane. Extracellular release of sphingosine-1-phosphate induced by phorbol 12-myristate 13 -acetate (PMA). J. Biol. Chem. 277: 35257-35262.
19. Katz, B. 1969. The release of neural transmitter substances.Liverpool University Press, Liverpool, UK
20. Kosaka, Y., K. Ogita, K. Ase, H. Nomura, U. Kikkawa, and Y. Nishizuka 1988. The heterogeneity of protein kinase C in various rat tissues. Biochem. Biophys. Res. Commun. 151: 973-981.
21. Krasel, C., M. Bunemann, K. Lorenz, and MJ Lohse 2005. Betaarrestin binding to the β2-adrenergic receptor requires both receptor phosphorylation and receptor activation. J. Biol. Chem. 280: 9528-9535.
22. Le Stunff, H., S. Milstien, and S. Spiegel 2004. Generation and metabolism of bioactive sphingosine-1-phosphate. J. Cell Biochem. 92: 882-899.
23. Lee, MJ, S. Thangada, KP Claffey, N. Ancellin, CH Liu, M. Kluk, M. Volpi, RI Sha'afi, and T. Hla 1999. Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine -1-phosphate. Cell. 99: 301-312.
24. Liu, H., M. Sugiura, VE Nava, LC Edsall, K. Kono, S. Poulton, S. Milstien, T. Kohama, and S. Spiegel 2000. Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform. J. Biol. Chem. 275: 19513-19520.
25. Liu, J., SA Ernst, SE Gladycheva, YY Lee, SI Lentz, CS Ho, Q. Li, and EL Stuenkel 2004. Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo. J. Biol Chem. 279: 55924-55936.
26. Mizugishi, K., T. Yamashita, A. Olivera, GF Miller, S. Spiegel, and RL Proia 2005. Essential role for sphingosine kinases in neural and vascular development. Mol. Cell. Biol. 25: 11113-11121.
27. Okada, T., G. Ding, H. Sonoda, T. Kajimoto, Y. Haga, A. Khosrowbeygi, S. Gao, N. Miwa, S. Jahangeer, and S. Nakamura 2005. Involvement of N-terminal -extended form of sphingosine kinase 2 in serum-dependent regulation of cell proliferation and apoptosis.J. Biol. Chem. 280: 36318-36325.
28. Okamoto, H., N. Takuwa, T. Yokomizo, N. Sugimoto, S. Sakurada, H. Shigematsu, and Y. Takuwa 2000. Inhibitory regulation of Rac activation, membrane ruffling, and cell migration by the G protein coupled sphingosine-1-phosphate receptor EDG5 but not EDG1 or EDG3. Mol. Cell. Biol. 20: 9247-9261.
29. Pitson, SM, PA Moretti, JR Zebol, HE Lynn, P. Xia, MA Vadas, and BW Wattenberg 2003. Activation of sphingosine kinase 1 by ERK1 / 2-mediated phosphorylation. EMBO J. 22: 5491-5500.
30. Pyne, S., JS Long, NT Ktistakis, and NJ Pyne 2005. Lipid phosphate phosphatases and lipid phosphate signaling Biochem. Soc. Trans. 33: 1370-1374.
31. Rosen, H., and EJ Goetzl 2005. Sphingosine 1-phosphate and its receptors: an autocrine and paracrine network. Nat. Rev. Immunol. 5: 560-570.
32. Samuel, AD, RA Silva, and VN Murthy 2003. Synaptic activity of the AFD neuron in Caenorhabditis elegans correlates with thermotactic memory. J. Neurosci. 23: 373-376.
33. Sanchez, T., and T. Hla 2004. Structural and functional characteristics of S1P receptors. J. Cell Biochem. 92: 913-922.
34. Sankaranarayanan, S., PP Atluri, and TA Ryan 2003. Actin has a molecular scaffolding, not propulsive, role in presynaptic function. Nat. Neurosci. 6: 127-135.
35. Spiegel, S., and S. Milstien 2003. Exogenous and intracellularly generated sphingosine 1-phosphate can regulate cellular processes by divergent pathways. Biochem. Soc. Trans. 31: 1216-1219.
36. Terada, N., Y. Banno, N. Ohno, Y. Fujii, T. Murate, JR Sarna, R. Hawkes, Z. Zea, T. Baba, and S. Ohno 2004. Compartmentation of the mouse cerebellar cortex by sphingosine kinase. J. Comp. Neurol. 469: 119-127.
37. Thompson, CR, SS Iyer, N. Melrose, R. VanOosten, K. Johnson, SM Pitson, LM Obeid, and DJ Kusner 2005. Sphingosine kinase 1 (SK1) is recruited to nascent phagosomes in human macrophages: inhibition of SK1 translocation by Mycobacterium tuberculosis. J. Immunol. 174: 3551-3561.
38. Yatomi, Y. 2006. Sphingosine 1-phosphate in vascular biology: possible therapeutic strategies to control vascular diseases. Curr. Pharm. Des. 12: 575-587.
39. Zhang, YH, MR Vasko, and GD Nicol 2006. Intracellular sphingosine 1-phosphate mediates the increased excitability produced by nerve growth factor in rat sensory neurons. J. Physiol. In press.

It is a figure which shows the result of having examined about the expression of SK1 in the active processus part (functional puncta) of a hippocampal neuron axon. (A) The result of immunoblot analysis using anti-SK1 antibody or anti-HA antibody on COS7 cells in which HA-rSK1 or HA-rSK2 was transiently expressed. (B) The results of analysis of endogenous SK1 expression by performing immunoblot analysis using an anti-SK1 antibody in the presence or absence of an antigen peptide on rat brain lysate. (C) Results of analysis of sphingosine kinase (SK) activity of immunoprecipitates by immunoprecipitation from rat brain lysate using anti-SK1 antibody in the presence or absence of antigenic peptide. (D) Results of double staining of rat primary hippocampal neurons with anti-SK1 antibody or anti-SK2 antibody and anti-Tau antibody, anti-MAP2 antibody or anti-synaptophysin (SynPhy) antibody. In addition, neurons that transiently express GFP-SK1 were labeled with a membrane staining material FM4-64, and fluorescence localization in living cells was observed. A differential interference (DIC) image and a double dye merge image are also shown. Scale bar 10μm It is a figure which shows that the release of glutamic acid by depolarization was suppressed by DMS. (A) Neurons cultured in a 6 cm culture dish were washed three times with PBS, pretreated for 30 minutes in the presence or absence of 10 μM DMS, and treated with a buffer containing or not containing 50 mM KCl (Buffer). It is a graph which shows the result of having analyzed glutamic acid release after 1 minute. (B) It is a figure explaining the protocol of the neurotransmitter release measurement using FM4-64 dye method. (C) Rat hippocampal neurons were treated for 30 minutes in the presence or absence (control) of 10 μM DMS. Neurons were washed, labeled with FM dye, and introduced into active presynaptic vesicles. Then, after depolarization induced by 50 mM KCl, the fluorescence change (Ft%) of the dye at each protrusion was measured over time. (D) Neurons treated in the presence or absence (control) of 10 μM DMS were labeled with FM dye as described above. Then, after the treatment with 100 μM glutamic acid, the fluorescence change (Ft%) of the dye at each protrusion was measured over time. The arrow indicates the point of addition of glutamic acid. Data are mean ± SEM of three independent experiments (C, D). It is a figure which shows the result of having examined the involvement of SK1 in the neurotransmitter release induced by depolarization. (A) Rat hippocampal neurons were transfected with control siRNA or rSK1-siRNA together with an expression vector encoding rSK1, hSK1, or hSK1G82D. Two days later, neuronal SK1 expression was analyzed by immunoblot analysis. (B) Endogenous SK1 protein was detected in neurons transfected with control siRNA or rSK1-siRNA by immunoblot analysis using anti-SK1 antibody. (C, D) Neurons transfected with control siRNA or rSK1-siRNA with an expression vector encoding hSK1 or hSK1G82D or an empty vector (mock) are pretreated with FM4-64 dye, 50 mM KCl (C) or 100 μM After the treatment with glutamic acid (D), the fluorescence change (Ft%) of the dye at each protrusion was measured over time. The arrow indicates the point of addition of glutamic acid. Data are mean ± SEM of three independent experiments (C, D). It is a graph which shows the result of having investigated whether the position shift of SK1 is induced by depolarization. Expression vectors encoding GFP-SK1, GFP alone or SynPhy-GFP were transfected into hippocampal neurons. Two days later, FRAP analysis of living cells was performed using a confocal laser scanning microscope. Depolarization was induced with 50 mM KCl (A) or 100 μM glutamic acid (B), or without induction, axon protrusions expressing GFP fusion proteins were photobleached and then imaged over time. The fluorescence intensity at each protrusion (minimum fluorescence intensity was 0 second) was measured, and the ratio of fluorescence recovery was calculated based on the initial value before photobleaching. Data are mean ± SEM of 3 independent experiments. That activation of S1P receptors (S1P ₁₎ by depolarization is induced illustrates by FRET analysis. (A) is a diagram illustrating a detecting binding by FRET between S1P ₁ after S1P ₁ activation β- arrestin (βArr). (B) Hippocampal neurons were transfected together with expression plasmids encoding S1P ₁ -CFP and YFP-β-arrestin, respectively, in the presence or absence (buffer) of 50 mM KCl, 100 μM glutamic acid, or 10 nM S1P. The treated cells were subjected to FRET analysis. At each protrusion, the detection light from the increase in donor fluorescence after acceptor photobleaching was measured and expressed as FRET efficiency. The FRET efficiency showed a substantial increase not only when depolarization was induced by KCl or glutamic acid but also when treated with S1P (n = 50, p <0.01, Student's paired t test). (C) Expression plasmids encoding S1P ₁ -CFP and YFP-β-arrestin, respectively, were transfected together into hippocampal neurons and stimulated with various agonists for 30 minutes in the presence or absence of 10 μM DMS ( 10 μM methanol). In addition, experiments were carried out in which neurons were transfected with control siRNA or rSK1-siRNA together with a plasmid encoding a fluorescent substance-coupled protein. Neurons were stimulated and fixed in the presence or absence (buffer) of 50 mM KCl, 100 μM glutamate, or 10 nM S1P, and then the FRET efficiency of axonal or dendritic regions after photobleaching was measured. . It is a figure which shows the experimental result which clarified the effect | action and role of exogenous S1P as an inducer of neurotransmitter release, and an enhancement factor. (A) After pre-labeling rat primary hippocampal neurons with FM4-64 dye, the labeled cells were stimulated with various concentrations of S1P or DHS1P for 6 seconds, and changes in fluorescence intensity were measured. In all cases, the concentration of vehicle (methanol) was kept constant (1 μM). (B) After washing the neurons 3 times with PBS, they were stimulated with various concentrations of S1P for 6 seconds. Thereafter, the released glutamic acid was measured by enzyme fluorescence assay. (C) Neurons were treated with or without 10 μM DMS for 30 minutes and labeled with FM dye. The neurons were then stimulated with various combinations of 1 pM S1P and 100 μM glutamate and changes in fluorescence intensity were measured over time. Arrows indicate the time point of agonist addition. (D) Neurons pre-labeled with FM dye were treated for 6 seconds in the presence or absence of various concentrations of glutamate and 1 pM S1P, and changes in fluorescence intensity were measured. (E) Each S1P receptor mRNA was quantified from rat primary hippocampal neurons by real-time quantitative reverse transcription PCR. mRNA values were normalized by GAPDH expression. (F) Hippocampal neurons were transfected with control siRNA, S1P ₁ -siRNA, S1P ₃ -siRNA in various combinations and pre-labeled with FM4-64. Thereafter, neurons were stimulated with 10 nM S1P, and changes in fluorescence intensity were measured. The arrow indicates the point of addition of S1P. Data are mean ± SEM of experiments performed 3 times (AD, F) and 6 times (E) independently. It is a figure which shows the analysis result of the effect | action of S1P in induction and enhancement of glutamate release. Hippocampal neurons pre-labeled with (A, B) FM dye were treated for 10 minutes in the presence or absence of 1 μM TTX. Thereafter, neurons were stimulated with 10 nM S1P (A) or 100 μM NMDA (B), and changes in fluorescence intensity were measured. Arrows indicate the time point of agonist addition. (C) Neurons cultured in 6 cm culture dishes were washed 3 times with PBS and then pretreated for 30 minutes with 10 μM DMS, 10 minutes with 1 μM TTX, or in the absence of these (control). The neurons were then treated with a buffer with or without 10 nM S1P and analyzed for glutamate release after 1 minute. (D) Neurons cultured in a 10 cm culture dish were washed three times with PBS and then treated for 1 minute in the presence (depolarization) or absence (control) of 50 mM KCl. Thereafter, lipids were extracted from neurons, and the amount of S1P was analyzed (n = 5, p <0.05, Student's paired test). (E) Rat primary hippocampal neurons expressing S1P ₁ -GFP or S1P ₃ -GFP were labeled with a membrane stain FM4-64, and the fluorescence localization of living cells was analyzed. A differential interference (DIC) image and a double dye merge image are also shown. Scale bar 10μm It is a figure which shows the result of having examined about the expression of sphingosine kinase 1 (SK1) in an adult mouse hippocampus. (A) cerebrum, (B) cerebellum, OB: olfactory bulb, CPu: caudate nucleus-palliary bulb, SN: substantia nigra, Hp: hippocampus. (C) is an enlarged view (x 100) of the hippocampal region, DG: dentate gyrus. (D) schematically shows mossy fibers from dentate granule cells, and (E) and (F) are views in which (C) is further partially enlarged (x 8). The In situ hybridization is a graph showing a result of examining expression of S1P ₁ receptor mRNA in the mouse hippocampus. S1P ₁ receptor mRNA was highly expressed in dentate gyrus (DG) granule cells, and was weakly distributed from CA3 to CA1 (arrow). The bar length is 500 μm, and the lower left is for low magnification (x 1/12). It is a graph which shows the result of having examined how release of glutamic acid from a rat hippocampal slice changes with S1P concentration in the presence of TTX by the method mentioned above. The average of eight experiments is shown, and * indicates that there is a significant difference (P <0.01) compared to the case where S1P is not present. Spontaneous AMPA-mEPSCs (via AMPA receptors) from rat hippocampal slices (A) CA3 pyramidal cells and (B) CA1 pyramidal cells in the presence or absence (Control) of S1P (1 μM) It is a figure which shows the measurement result of a microexcitatory post-synaptic current. The experiment was performed four times at a holding potential of -70 mV, each showing a typical current, and the cumulative probabilities for recorded AMPA-mEPSCs intensity (amplitude, pA) and frequency (inter-event interval, s) were graphed. Show. It is a figure which shows the result of having measured spontaneous AMPA-mEPSCs using the CA3 pyramidal cell of a rat hippocampal slice in the presence or absence (Control) of a sphingosine kinase inhibitor (A: DMS, B: HACPT). . The experiment was performed four times at a holding potential of -70 mV, each showing a typical current, and the cumulative probabilities for recorded AMPA-mEPSCs intensity (amplitude, pA) and frequency (inter-event interval, s) were graphed. Show. It is a graph which shows the result of having examined the influence of S1P with respect to the post-excitatory synaptic potential (eEPSC) in the CA3 pyramidal cell caused by electrical stimulation using the rat hippocampal slice. At the time of electrical stimulation, S1P (1 μM) was treated for 10 minutes in the absence (A) or presence (B) of DMS (10 μM), and eEPSC was measured. The experiment was performed 4 times, and each value represents the average percentage of eEPSC intensity at 0 minutes. It is a graph which shows the result of having examined how discharge | release of GABA from a rat hippocampal slice changes with S1P density | concentration in the presence of TTX by the method mentioned above. The average of 8 experiments is shown, and * indicates that there is a significant difference (P <0.01) compared to the case where S1P is not present. Spontaneous GABA-mIPSCs from rat hippocampal slices (A) CA3 pyramidal cells and (B) CA1 pyramidal cells in the presence or absence (Control) of S1P (1 μM). It is a figure which shows the measurement result of inhibitory post-synaptic potential). The experiment was performed four times at a holding potential of -70 mV, each showing a typical current, and the cumulative probabilities for recorded GABA-mIPSCs intensity (amplitude, pA) and frequency (inter-event interval, s) were graphed. Show. It is a figure which shows the result of having examined about involvement of S1P in synapse formation. (A) Rat primary hippocampal neurons with synaptophysin (SynPhy) -GFP, i) control siRNA, ii) rat sphingosine kinase 1 (rSK1) -specific siRNA, iii) rSK1-specific siRNA + human sphingosine kinase 1 (hSK1) This is a result of immunostaining using an anti-PSD-95 antibody after introducing an expression plasmid or iv) an rSK1-specific siRNA + activity-deficient mutant human sphingosine kinase 1 (hSK1G82D) expression plasmid. Also shown is a Merge image of SynPhy and PSD-95. Scale bar 5 μm. (B) The result of analyzing the expression of sphingosine kinase 1 (SK1) in rat hippocampal neurons under each condition by immunoblotting. (C) Of all puncta that was positive for synaptophysin (SynPhy), the percentage of puncta that was positive for both SynPhy and PSD-95 was calculated and plotted as a “Synaptic index”. (D) The cumulative frequency (%) of the aggregate size of PSD-95 for puncta that was positive for both SynPhy and PSD-95 is shown in the graph.

Claims (20)

  A method of regulating the release of a neurotransmitter from a nerve cell by administering to the nerve cell an agonist or antagonist of sphingosine 1-phosphate or its receptor, or by controlling the production of sphingosine 1-phosphate in the nerve cell.   A method for suppressing the release of a neurotransmitter from a neuron by administering a sphingosine 1-phosphate receptor antagonist to the neuron or suppressing the production of sphingosine 1-phosphate in the neuron.   A method of promoting the release of a neurotransmitter from a neuron by administering a sphingosine 1-phosphate or an agonist of its receptor to the neuron, or promoting the production of sphingosine 1-phosphate in the neuron.   The method according to claim 1, wherein the neurotransmitter is glutamic acid or γ-aminobutyric acid (GABA).   The method according to any one of claims 1 to 4, wherein the production of sphingosine 1-phosphate in the nerve cell is controlled by regulating the expression or activity of sphingosine kinase 1 in the nerve cell.   The method according to claim 1, wherein the nerve cell is a central nerve cell or derived from a central nerve cell.   The method according to claim 6, wherein the nerve cell is derived from a hippocampal nerve cell or a hippocampal nerve cell.   A neurotransmitter release regulator comprising as an active ingredient a substance selected from the group consisting of sphingosine 1-phosphate, a sphingosine 1-phosphate receptor agonist or antagonist, and a substance that regulates the expression or activity of sphingosine kinase 1.   A neurotransmitter release inhibitor comprising as an active ingredient a substance selected from the group consisting of a sphingosine 1-phosphate receptor antagonist and a substance that suppresses the expression or activity of sphingosine kinase 1.   A neurotransmitter release promoter comprising as an active ingredient a substance selected from the group consisting of sphingosine 1-phosphate, a sphingosine 1-phosphate receptor agonist, and a substance that promotes the expression or activity of sphingosine kinase 1.   A therapeutic agent for neurological diseases comprising as an active ingredient a substance selected from the group consisting of sphingosine 1-phosphate, a sphingosine 1-phosphate receptor agonist or antagonist, and a substance that regulates the expression or activity of sphingosine kinase 1.   The therapeutic agent for neurological disease according to claim 11, wherein the neurological disease is a central nervous disease.   The therapeutic agent for a neurological disease according to claim 11, wherein the neurological disease is a hippocampal disease or a glutamatergic nervous system disease.   The therapeutic agent for neurological disease according to claim 11, wherein the neurological disease is selected from the group consisting of epilepsy, hippocampal sclerosis, Alzheimer's disease, schizophrenia and other psychoneurological diseases.   A screening method for a neurotransmitter release-controlling agent or a therapeutic agent for a neurological disease, comprising searching for an agonist or antagonist of a sphingosine 1-phosphate receptor expressed in a nerve cell. The screening method according to claim 15, wherein an agonist or antagonist of S1P ₁ or S1P ₃ which is a subtype of sphingosine 1-phosphate receptor is searched.   It can be used for the screening method according to claim 15 or 16, and upon administration of a candidate substance to a nerve cell, the presence or absence of activation of sphingosine 1-phosphate receptor expressed in the cell can be detected by the FRET method. A vector for expressing a fusion protein of a fluorescent protein and a sphingosine 1-phosphate receptor prepared in (1).   A step of administering a candidate substance to a nerve cell or a nerve tissue, and a step of searching for a substance that regulates the expression or activity of sphingosine kinase 1 using, as an index, a change in expression or activity of sphingosine kinase 1 before and after administration of the candidate substance. A method for screening a neurotransmitter release-controlling agent or a therapeutic agent for neurological diseases.   A method of regulating synapse formation between neurons by administering sphingosine 1-phosphate or an agonist or antagonist of a receptor thereof to neurons, or by controlling the production of sphingosine 1-phosphate in neurons.   A method of suppressing synapse formation between nerve cells by inhibiting production of sphingosine monophosphate in the nerve cells.