JPWO2014017462A1 - Pharmaceutical composition for suppressing abnormal spine formation - Google Patents

Abstract

In tuberous sclerosis, it was revealed that the amount of syntenin protein was increased. In addition, as the amount of the syntenin protein increases, it has also been clarified that spine formation abnormality occurs due to inhibition of the binding of syndecan-2 protein and CASK protein, which is known as a factor that induces spine formation. . In addition, by administering a syntenin gene-specific siRNA, it is found that abnormal spine formation in tuberous sclerosis can be suppressed, and further that abnormal behavior can be improved and epileptic seizures can be suppressed. Provided are a pharmaceutical composition and a vaccine composition, and a method for screening a compound having an activity to suppress abnormal spine formation.

Description

  The present invention relates to a pharmaceutical composition and a vaccine composition for suppressing abnormal spine formation. The present invention also relates to a method for screening a compound having an activity of suppressing abnormal spine formation.

  Tuberous sclerosis, also called Pringle's disease, is a nevus with multiple benign tumors called hamartomas throughout the body. Moreover, epilepsy, mental retardation, autism, etc. are combined as central nervous system symptoms. Although it has recently been reported that autism develops genetically, tuberous sclerosis is the disease that is said to cause hereditary autism from the earliest.

  In developmental disorders including autism, it has been reported that there are minute morphological changes in neurons in the brain. In particular, the spinous structure (spine) of dendrites forms the synapse side after the excitatory synapse, but spinal dysplasia and morphological changes are recognized in patients with developmental disorders. Abnormal formation of dendritic spines has been confirmed even in tuberous sclerosis (Non-patent Document 1). Spine dysplasia means that there is an abnormality in normal excitatory synaptic transmission, which is thought to be the cause of central symptoms of developmental disorders.

  It is known that the causative gene of tuberous sclerosis is the TSC1 gene or the TSC2 gene. TSC1 protein and TSC2 protein form a complex and activate GTPase, a small GTP-binding protein called Rheb. That is, it acts as a GTPase activator (GAP) for Rheb. However, if there is a mutation in the TSC1 protein or TSC2 protein, the Rheb protein becomes GTP type (active type) and activates serine threonine kinase called mTOR protein. And it has been thought that activation of mTOR protein promotes protein synthesis and develops autism and the like (Non-patent Document 2).

  However, as shown in Non-Patent Document 1 and as shown in Examples described later, even when rapamycin, an mTOR inhibitor, is administered, abnormal spine formation in tuberous sclerosis may not be improved. It has become clear.

  On the other hand, syntenin protein is a protein also called SDCBP (syndecan binding protein), MDA9 (melanoma differentiation-related gene 9) or TACIP18 (pro-TGF-α cytoplasmic domain binding protein 18), It is known to bind to various types of proteins via the two PDZ domains it has (Non-patent Document 3). Syntenin protein is known to be expressed in a large amount in neurons in the synapse formation / stable period. Furthermore, it has been clarified that overexpression of the syntenin protein in juvenile and mature neurons causes a marked change in neuronal morphology due to an increase in dendritic protrusion (non-dendritic growth) Patent Document 4). However, whether or not there is an association between the syntenin protein and tuberous sclerosis has not been clarified.

  Thus, since the mechanism which causes a spine formation abnormality is not yet elucidated, the effective therapeutic agent etc. with respect to the disease accompanied by a spine formation abnormality, such as tuberous sclerosis, are not yet developed.

Tabazoie SF. Nat Neurosci. 2005, Vol. 8, pp. 1727 to 1734 Inoki K.K. Et al., Nature Genetics, 2005, 37, 19-24. Beekman JM. Et al., J Cell Sci. 2008, 121 (Pt 9), pp. 1349-1355 Hirbec H.M. Et al., Mol Cell Neurosci. 2005, 28, 4, 737-746

  The present invention has been made in view of the above-described problems of the prior art, and includes a pharmaceutical composition and a vaccine composition for suppressing abnormal spine formation, and a method for screening a compound having an activity of suppressing abnormal spine formation. The purpose is to provide.

  In order to achieve the above object, the present inventors first focused on the Rheb protein located downstream of the TSC2 protein that causes tuberous sclerosis in the onset mechanism of tuberous sclerosis. We searched for proteins to bind. As a result, a syntenin protein was newly found as a protein that binds to the Rheb protein. Furthermore, the present inventors show that in tuberous sclerosis, the binding between the activated Rheb protein and the syntenin protein is weakened, the degradation of the syntenin protein by the ubiquitin-proteasome system is suppressed, and the amount of the syntenin protein is increased. Was revealed. In addition, as the amount of the syntenin protein increases, it has also been clarified that spine formation abnormality occurs due to inhibition of the binding of syndecan-2 protein and CASK protein, which is known as a factor that induces spine formation. (See FIGS. 1 and 2). Then, administration of a syntenin gene-specific siRNA to suppress the function of the syntenin gene can suppress spine formation abnormalities in tuberous sclerosis, and further improve behavioral abnormalities (context-dependent fear discrimination) and epilepsy It was found that seizures can be suppressed.

The present invention is based on the above findings, and more specifically, provides the following.
<1> A pharmaceutical composition for suppressing spine formation abnormality, comprising a molecule that suppresses the function of the syntenin gene as an active ingredient.
<2> The pharmaceutical composition according to <1>, wherein the molecule that suppresses the function of the syntenin gene is a molecule according to any one of the following (a) to (c): (a) a transcription product of the syntenin gene RNA to be bound or DNA encoding the RNA
(B) Anti-syntenin protein antibody (c) RNA or DNA that binds to syntenin protein.
<3> A vaccine composition for suppressing spine formation abnormality, comprising a syntenin protein or a partial peptide thereof as an active ingredient.
<4> A screening method of a candidate compound having an activity of suppressing spine formation abnormality including the following steps (a) to (c): (a) a step of contacting a test compound with a syntenin protein;
(B) detecting the binding between the test compound and the syntenin protein;
(C) selecting a compound that binds to the syntenin protein.
<5> A screening method for a compound having an activity to suppress abnormal spine formation, including the following steps (a) to (c): (a) contacting a syntenin protein with a syndecan-2 protein in the presence of a test compound The process of
(B) detecting the binding between the syntenin protein and the syndecan-2 protein;
(C) A step of selecting a compound that suppresses the binding.
<6> A screening method for a compound having an activity to suppress spine formation abnormality including the following steps (a) to (c): (a) a step of bringing a test compound into contact with a cell expressing the syntenin gene;
(B) detecting the expression of the syntenin gene in the cell,
(C) A step of selecting a compound that decreases the expression of the syntenin gene as compared with the case where the test compound is not contacted.
<7> A screening method for a compound having an activity to suppress abnormal spine formation, comprising the following steps (a) to (c): (a) a DNA having a reporter gene operably linked downstream of the promoter region of the syntenin gene; A step of contacting a test compound with cells having
(B) detecting the expression of the reporter gene in the cell;
(C) A step of selecting a compound that decreases the expression of the reporter gene as compared with the case where the test compound is not contacted.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the pharmaceutical composition and vaccine composition for suppressing a spine formation abnormality, and the screening method of the compound which has the activity which suppresses a spine formation abnormality, By extension, tuberous sclerosis Thus, it is possible to treat and prevent diseases accompanied by abnormal spine formation.

It is the schematic which shows the molecular mechanism in the spine formation of a normal (wild type) neuron. In the figure, “synt” represents the syntenin protein, “sdc2” represents the syndecan-2 protein, and “Ub” represents ubiquitin (the same applies to FIG. 2). FIG. 2 is a schematic diagram showing the molecular mechanism in spine formation abnormality of tuberous sclerosis (TSC2 ^+/− ) neurons. It is a photograph which shows the result of having dye | stained the neuron derived from a wild type and TSC2 ^+/- with EGFP and an anti-vGlut1 (vesicular glutamate transporter) antibody, and observed with the fluorescence microscope. EGFP stains the entire neuron, and the anti-vGlut1 antibody specifically labels the excitatory synaptic marker molecule (vGlut1). In the figure, white arrows indicate that dendritic spines are formed (hereinafter the same in FIGS. 7, 14, 17, 24, 27, 31 and 43). Further, the scale bar in the figure indicates 5 μm (hereinafter the same in FIGS. 7, 14, 17, 24, 27, 31, 34, 43 and 46). FIG. 6 is a graph showing cumulative probabilities of protrusion length on dendrites in wild-type and TSC2 ^+/− derived neurons. It is a graph which shows the relative value of a spine synapse density in the neuron derived from TSC2 ^+/- . The relative value is a value when the spine synapse density in the wild-type neuron is 100%. It is a graph which shows the relative value of a shaft synapse density in the neuron derived from TSC2 ^+/- . The relative value is a value when the shaft synapse density in a wild-type neuron is 100%. It is a photograph which shows the result of having dye | stained the neuron derived from TSC2 ^{+/- which} culture | cultivated in the presence of 100 nM rapamycin or DMSO with EGFP and an anti-vGlut1 antibody, and observed with the fluorescence microscope. It is a graph which shows the cumulative probability of the length of the processus on the dendrite in the neuron derived from TSC2 ^+/− cultured in the presence of 100 nM rapamycin or DMSO. And 100nM rapamycin presence of ^{TSC2 +/-} derived cultured under neurons in the ^{TSC2 +/-} derived neurons cultured in the presence of DMSO, is a graph showing the spine synaptic density per each dendrite 10 [mu] m. It is the schematic which shows the structure of a syntenin protein and its variant. It is a photograph which shows the result of having analyzed the coupling ^| bonding of syntenin (synt) protein, and active type ^| mold Rheb (Rheb ^Q64V ) protein or inactive type ^| mold Rheb (Rheb ^D60I ) protein by GST precipitation method. It is a photograph which shows the result of having analyzed the coupling | bonding of syntenin (synt) protein and Rheb ^D60I protein or its effector domain variant ("T38M / D60I" or "I39K / D60I" in a figure) by GST precipitation method. It is a photograph which shows the result of having analyzed syntenin protein (in the figure, "synt") or the syntenin protein variant shown in FIG. 10, and the Rheb protein by GST precipitation method. It is the photograph which shows the result of having dye | stained the neuron derived from the wild type which overexpressed the syntenin gene ("synt" in a figure) with EGFP and anti-vGlut1 antibody, and observed with the fluorescence microscope. Moreover, the observation result of the wild-type neuron ("control" in the figure) that does not overexpress the syntenin gene is also shown. It is a graph which shows the accumulation probability of the length of the processus | protrusion on a dendrite in the neuron derived from the wild type which overexpressed the syntenin gene ("synt" in a figure). The results are also shown for wild-type neurons that do not overexpress the syntenin gene (“control” in the figure). For each 10 μm of dendrites in a wild-type neuron overexpressing the syntenin gene (“synt” in the figure) and a wild-type neuron not overexpressing the syntenin gene (“control” in the figure) It is a graph which shows the number of spine synapses. Neurons derived from TSC2 ^+/− introduced with syntenin siRNA (“synt siRNA” in the figure), neurons derived from TSC2 ^+/− introduced with non-specific siRNA (“non-specific siRNA” in the figure), and syntenin siRNA And ECFP-syntenin-introduced TSC2 ^+/- derived neurons ("synt siRNA + ECFP-synt ^* " in the figure) stained with EGFP and anti-vGlut1 antibody, and a photograph showing the results of observation with a fluorescence microscope is there. In the figure, “*” attached to “ECFP-synt” is a syntenin protein having resistance to synt siRNA because degenerate mutations are introduced into the target sequence of synt siRNA. This represents a nucleic acid encoding the mutant (the same applies to FIGS. 19 and 24 to 26). Neurons derived from TSC2 ^+/− introduced with syntenin siRNA (“synt siRNA” in the figure), neurons derived from TSC2 ^+/− introduced with non-specific siRNA (“non-specific siRNA” in the figure), and syntenin siRNA And ECSC-syntenin-introduced TSC2 ^+/− derived neurons (“synt siRNA + synt ^* ” in the figure) are graphs showing the cumulative probabilities of the length of protrusions on dendrites. Neurons derived from TSC2 ^+/− introduced with syntenin siRNA (“synt siRNA” in the figure), neurons derived from TSC2 ^+/− introduced with non-specific siRNA (“non-specific siRNA” in the figure), and syntenin siRNA 5 is a graph showing the number of spine synapses per 10 μm of each dendrite in neurons derived from TSC2 ^+/− introduced with ECFP-syntenin (“synt siRNA + ECFP-synt ^* ” in the figure). In the figure, “***” indicates p <0.001. It is a photograph which shows the result of having analyzed the quantity (in the figure, "synt") of the syntenin protein in the hippocampus of the wild type rat and the hippocampus of TSC2 ^+/- rat by the Western blot method. In the figure, “1” indicates the results for the hippocampus of wild-type rats, and “2” indicates the results for the hippocampus of TSC2 ^+/− rats. It is a graph which shows the relative value of the syntenin protein amount in the hippocampus of TSC2 ^+/- rat. The relative value is a value when the amount of the syntenin protein in the hippocampus of the wild type rat is 100%. In the figure, “**” indicates that p <0.01. It is a photograph showing the result of analyzing the amount of ubiquitinated syntenin protein (Ub-synt) in the presence of inactive Rheb (Rheb ^D60I ) protein or active Rheb (Rheb ^Q64V ) protein. The syntenin protein used in this ubiquitination assay is fused with a FLAG tag. In the figure, “1” and “2” are subjected to immunoprecipitation (IP) with an anti-FLAG antibody (anti-FLAG), and the precipitated protein is subjected to Western blotting using an anti-FLAG antibody (anti-FLAG) (IB ) Shows the result of analysis. “3” and “4” were subjected to immunoprecipitation (IP) with an anti-FLAG antibody, and the precipitated protein was analyzed by Western blotting (IB) using an anti-polyubiquitin antibody (anti-poly-Ub). Results are shown. It is a photograph which shows the result of having analyzed the quantity of ubiquitinated Rheb protein (Ub-Rheb) regarding inactive type Rheb (Rheb ^D60I ) protein and active type Rheb (Rheb ^Q64V ) protein. The Rheb protein used in this ubiquitination assay is fused with an EGFP tag. In the figure, “1” and “2” are subjected to immunoprecipitation (IP) with an anti-GFP antibody (anti-GFP), and the precipitated protein is subjected to Western blotting using an anti-GFP antibody (anti-GFP) (IB) ) Shows the result of analysis. “3” and “4” were subjected to immunoprecipitation (IP) with an anti-GFP antibody, and the precipitated protein was analyzed by Western blotting (IB) using an anti-polyubiquitin antibody (anti-poly-Ub). Results are shown. The result of observing a neuron derived from a wild type introduced with a syntenin gene or a mutant thereof (see FIG. 10) and a syntenin siRNA (synt siRNA) with an EGFP and an anti-vGlut1 antibody using a fluorescence microscope. It is a photograph shown. It is a graph which shows the cumulative probability of the length of the processus | protrusion on a dendrite in the neuron derived from a wild type which introduce | transduced the syntenin gene or its variant (refer FIG. 10), and syntenin siRNA. It is a graph which shows the number of spine synapses per 10 micrometers of each dendrite in the neuron derived from a wild type which introduce | transduced the syntenin gene or its variant (refer FIG. 10), and syntenin siRNA. In the figure, “***” indicates p <0.001. Syntenin siRNA (synt siRNA) and syndecan-2 siRNA (sdc2 siRNA) introduced TSC2 ^+/- derived neurons, syntenin siRNA and non-specific siRNA introduced TSC2 ^+/- derived neurons, EGFP and anti-neurons It is a photograph which shows the result of having dye | stained with vGlut1 antibody and observed with the fluorescence microscope. Neurons derived from TSC2 ^+/− introduced with syntenin siRNA and syndecan-2 siRNA (“sdc2 siRNA” in the figure) and neurons derived from TSC2 ^+/− introduced with syntenin siRNA and non-specific siRNA (in the figure, “ It is a graph which shows the accumulation probability of the length of the processus | protrusion on a dendrite in "non-specific siRNA"). Neurons derived from TSC2 ^+/− introduced with syntenin siRNA and syndecan-2 siRNA (“sdc2 siRNA” in the figure) and neurons derived from TSC2 ^+/− introduced with syntenin siRNA and non-specific siRNA (in the figure, “ It is a graph showing the number of spine synapses per 10 μm of each dendrite in “non-specific siRNA”). In the figure, “***” indicates p <0.001. When the amount of the syntenin protein (“FLAG-synt” in the figure) is increased by the GST precipitation method, a syndecan-2 protein (in the figure, which binds to a certain amount of CASK protein (“FLAG-CASK” in the figure)) , “GST-sdc2 IC”). It is the photograph which shows the result of having dye | stained the neuron derived from the wild type which made the quantity of CASTEN protein constant and the quantity of the syntenin protein increased with EGFP and anti-vGlut1 antibody, and observed with the fluorescence microscope. The figures in the figure are the plasmids encoding the EGFP-CASK fusion protein introduced into the primary cultured neurons (11 days after culture) derived from wild type rats by the lipofection method (using Lipofectamine 2000 manufactured by Invitrogen). In the figure, “CASK”) and the mass (μg) of the plasmid encoding the ECFP-syntenin fusion protein (“synt” in the figure) are shown. It is a graph which shows the number of spine synapses per 10 micrometers of each dendrite in the neuron derived from the wild type which made the quantity of CASTEN protein constant and increased the quantity of syntenin protein. In the figure, “***” indicates p <0.001. In addition, “synt0” indicates the result of a wild-type neuron in which only 1 μg of the CASK protein is introduced, and “synt0.5” indicates that 0.5 μg of syntenin protein is introduced with respect to 1 μg of the CASK protein. The results for wild-type neurons are shown, and “syntl” indicates the results for wild-type neurons into which 1 μg of syntenin protein is introduced per 1 μg of CASK protein. It is a photograph which shows the method of visualization of the in vivo dendritic spine using the diolisic method. It is a microscope picture which shows the result of having carried out the DiI labeling of the hippocampus of a wild type rat and a TSC2 ^+/- rat, and observing the form of the neuron. FIG. 5 is a graph showing cumulative probabilities of protrusion widths on dendrites in wild type and TSC2 ^+/− rat hippocampus. FIG. 6 is a graph showing cumulative probabilities of protrusion lengths on dendrites in wild type and TSC2 ^+/− rat hippocampus. syntenin siRNA-introduced TSC2 ^+/− rat hippocampus (in the figure, “syntenin siRNA”), non-specific siRNA-introduced TSC2 ^+/− rat hippocampus (in the figure, “non-specific siRNA”), It is a microscope picture which shows the result of having labeled the hippocampus of the TSC2 ^+/- rat which has not introduce | transduced siRNA (in the figure, "operation only") with DiI by the diolisic method, and having observed the form of the neuron. syntenin siRNA-introduced TSC2 ^+/− rat hippocampus (in the figure, “syntenin siRNA”), non-specific siRNA-introduced TSC2 ^+/− rat hippocampus (in the figure, “non-specific siRNA”), It is a graph which shows the cumulative probability of the width | variety of the processus | protrusion on a dendrite in the hippocampus (in the figure, "operation only") of TSC2 < ^+/- > rat which has not introduce | transduced siRNA. syntenin siRNA-introduced TSC2 ^+/− rat hippocampus (in the figure, “syntenin siRNA”), non-specific siRNA-introduced TSC2 ^+/− rat hippocampus (in the figure, “non-specific siRNA”), It is a graph which shows the cumulative probability of the length of the processus | protrusion on a dendrite in the hippocampus (in the figure, "operation only") of TSC2 < ⁺ > /-rat which has not introduce | transduced siRNA. It is a figure which shows the outline of the context-dependent fear discrimination test performed with respect to the TSC2 ^+/- rat which inject | poured syntenin siRNA continuously into the ventricle. The Syntenin siRNA is a graph showing the results of contextual fear discrimination test performed against ^{TSC2 +/-} rats continuous infusion intraventricularly ^(TSC2 +/- syntenin siRNA). The results of context-dependent fear discrimination tests for wild-type rats (WT) and TSC2 ^+/− rats not injected with syntenin siRNA (TSC2 ^+/− ) are also shown as controls. It is the schematic which shows the preparation scheme of syntenin ^+/- mouse. Hippocampal neurons from wild-type mice (WT), TSC2 ^+/− mice (Tsc2 ^+/− ) and TSC2 ^+/− ; syntenin ^+/− mice (Tsc2 ^+/− ; synt ^+/− ), EGFP and anti-vGlut1 (Vesicular glutamate transporter) It is a photograph showing the result of staining with an antibody and observing with a fluorescence microscope. Spinal synaptic density in hippocampal neurons from wild type mice (WT), TSC2 ^+/− mice (Tsc2 ^+/− ) and TSC2 ^+/− ; syntenin ^+/− mice (Tsc2 ^+/− ; synt ^+/− ) It is a graph which shows a relative value. The relative value is a value when the spine synapse density in a neuron derived from a wild type mouse is 100%. In the figure, “***” indicates p <0.001. Shaft synaptic density in hippocampal neurons from wild-type mice (WT), TSC2 ^+/− mice (Tsc2 ^+/− ) and TSC2 ^+/− ; syntenin ^+/− mice (Tsc2 ^+/− ; synt ^+/− ) It is a graph which shows a relative value. The relative value is a value when the shaft synapse density in a wild-type neuron is 100%. In the figure, “***” indicates p <0.001. Hippocampus derived from wild-type mice (WT), TSC2 ^+/− mice (Tsc2 ^+/− ) and TSC2 ^+/− ; syntenin ^+/− mice (Tsc2 ^+/− ; synt ^+/− ) were labeled with DiI by the diolitic method. It is a micrograph showing the result of observing the morphology of neurons. Each dendrite in hippocampal neurons from wild-type mice (WT), TSC2 ^+/− mice (Tsc2 ^+/− ) and TSC2 ^+/− ; syntenin ^+/− mice (Tsc2 ^+/− ; synt ^+/− ) It is a graph which shows the number of protrusions per micrometer. On dendrites in the hippocampus from wild type mice (WT), TSC2 ^+/− mice (Tsc2 ^+/− ) and TSC2 ^+/− ; syntenin ^+/− mice (Tsc2 ^+/− ; synt ^+/− ) It is a graph which shows the accumulation probability of the width | variety of a processus | protrusion. On dendrites in the hippocampus from wild type mice (WT), TSC2 ^+/− mice (Tsc2 ^+/− ) and TSC2 ^+/− ; syntenin ^+/− mice (Tsc2 ^+/− ; synt ^+/− ) It is a graph which shows the accumulation probability of the length of a projection. Context-dependent fear discrimination performed on wild-type mice (WT), TSC2 ^+/− mice (Tsc2 ^+/− ) and TSC2 ^+/− ; syntenin ^+/− mice (Tsc2 ^+/− ; synt ^+/− ) It is a graph which shows the result of a test. A graph showing the intensity of induced epileptic seizures by injecting pentylenetetrazole into wild type mice (WT), TSC2 ^+/− mice (tsc) and TSC2 ^+/− ; syntenin ^+/− mice (tsc; syn). is there. In the figure, the horizontal axis represents the number of administrations of pentylenetetrazole, and the vertical axis represents the seizure score (epileptic seizure intensity).

<Pharmaceutical composition for suppressing abnormal spine formation>
The present invention provides a pharmaceutical composition for suppressing spine formation abnormality, which contains, as an active ingredient, a molecule that suppresses the function of the syntenin gene.

  In the present invention, “spine” means a spinous structure on a dendrite that forms the post-synaptic side of an excitatory synapse. “Spine dysplasia” means a state in which the shape, size, and number of spines are different from those of normal (wild-type) neurons. For example, spine dysplasia (for example, before spine formation) Stop the transition from filopodia to spine).

  In the present invention, “suppression of gene function” includes both suppression of gene expression (suppression of transcription, suppression of translation) and suppression of activity of a gene translation product (protein).

  In the present invention, the “syntenin gene” that is the target of function suppression is typically a gene that encodes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 (SEQ ID NO: 1) as long as it is human. A gene comprising the base sequence described in 1). Typically, a mouse-derived gene encodes a protein consisting of an amino acid sequence specified by RefSeq ID: NP_05887.1 (a gene consisting of a base sequence specified by RefSeq ID: NM_0166807.1). . If it is derived from a rat, it is typically a gene encoding a protein consisting of an amino acid sequence specified by RefSeq ID: NP — 114192.1 (a gene consisting of a base sequence specified by RefSeq ID: NM — 031986.1). is there. However, the DNA sequence of a gene can be mutated in nature (ie, non-artificially) due to such mutations. Therefore, in the present invention, such a natural mutant can also be a target of function suppression.

  According to the homology analysis at the amino acid sequence level using BLASTP, the homology between the syntenin protein derived from rat and the syntenin protein derived from human is as high as 89%, and in particular, PDZ1 and 2 domains and the C-terminal side are It is highly preserved. In addition, as shown in the examples described later, the Rheb protein that was found to bind to the syntenin protein in the present invention is also a rat-derived Rheb protein (a protein consisting of an amino acid sequence specified by RefSeq ID: NP — 0334488.1). ) And a human-derived Rheb protein (a protein consisting of an amino acid sequence specified by RefSeq ID: NP_005605.1) is extremely high at 99%. Furthermore, regarding syndecan-2 protein that binds to syntenin protein, rat-derived syndecan-2 protein (refSeq ID: protein consisting of an amino acid sequence specified by NP — 03724.1) and human-derived syndecan-2 protein (SEQ ID NO: : A protein having the amino acid sequence described in 4 :) is as high as 81%. Thus, the signaling substances involved in spine formation from TSC protein to syndecan-2 protein shown in FIGS. 1 and 2 are highly conserved in rats and humans.

  Examples of the “molecule that suppresses the function of the syntenin gene” include RNA that binds to the transcription product of the syntenin gene or DNA encoding the RNA, anti-syntenin protein antibody, and RNA or DNA that binds to the syntenin protein.

  In addition, as shown in the below-mentioned Example, it became clear that a spine formation abnormality is suppressed by siRNA with respect to a syntenin gene, and a behavioral abnormality (context-dependent fear discrimination) is also improved, and suppression of the function of a syntenin gene It was also clarified that there is a causal relationship between suppression of abnormal spine formation and the like. Therefore, not only siRNA but also an anti-syntenin protein antibody or the like can be used to suppress abnormal spine formation by using a molecule that suppresses the function of the gene.

-RNA that binds to the transcript of the syntenin gene-
The RNA that binds to the transcription product of the syntenin gene contained as an active ingredient of the pharmaceutical composition for suppressing spine formation abnormality or the DNA encoding the RNA is complementary to the transcription product of the gene encoding the syntenin protein. Examples include dsRNA (double-stranded RNA) or DNA encoding the dsRNA.

  DNA encoding dsRNA includes antisense DNA encoding antisense RNA for any region of the transcript (mRNA) of the target gene, and sense DNA encoding sense RNA for any region of the mRNA, Antisense RNA and sense RNA can be expressed from the antisense DNA and the sense DNA, respectively. Moreover, dsRNA can be produced from these antisense RNA and sense RNA.

  As a configuration for holding a dsRNA expression system in a vector or the like, there are a case where an antisense RNA and a sense RNA are expressed from the same vector, and a case where an antisense RNA and a sense RNA are expressed from different vectors, respectively. As a configuration for expressing antisense RNA and sense RNA from the same vector, for example, an antisense RNA expression cassette in which a promoter capable of expressing a short RNA such as a polIII system is linked upstream of the antisense DNA and the sense DNA. And sense RNA expression cassettes are constructed, and these cassettes are inserted into the vector in the same direction or in the opposite direction.

  It is also possible to construct an expression system in which antisense DNA and sense DNA are arranged in opposite directions so as to face each other on different strands. In this configuration, one double-stranded DNA (siRNA-encoding DNA) in which an antisense RNA coding strand and a sense RNA coding strand are paired is provided, and antisense RNA and sense RNA from each strand are provided on both sides thereof. A promoter is provided oppositely so that it can be expressed. In this case, in order to avoid adding extra sequences downstream of the sense RNA and the antisense RNA, a terminator is added to the 3 ′ end of each strand (antisense RNA coding strand, sense RNA coding strand). It is preferable to provide. As this terminator, a sequence in which four or more A (adenine) bases are continued can be used. Further, in this palindromic style expression system, the two promoter types are preferably different.

  In addition, as a configuration for expressing antisense RNA and sense RNA from different vectors, for example, antisense RNA expression in which a promoter capable of expressing a short RNA such as a polIII system is linked upstream of the antisense DNA and the sense DNA. A cassette and a sense RNA expression cassette are constructed, and these cassettes are held in different vectors. In addition, those skilled in the art can prepare the dsRNA by chemically synthesizing each strand.

  The dsRNA used in the present invention is preferably siRNA or shRNA (short hairpin RNA). siRNA means double-stranded RNA consisting of short strands in a range that does not show toxicity in cells. In addition, shRNA is a single-stranded RNA in which a sense RNA and an antisense RNA are arranged with a spacer sequence, and hydrogen bonding occurs between the sense RNA and the antisense RNA in a cell or the like, resulting in a spacer. It means that the sequence becomes a hairpin structure and can be an siRNA by cleaving the hairpin structure in a cell.

  Furthermore, the chain length is not particularly limited as long as the expression of the target gene can be suppressed and it does not show toxicity. The chain length of dsRNA is, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30 base pairs.

  The dsRNA or the DNA encoding the dsRNA need not be completely identical to the base sequence of the target gene, but is at least 70% or more, preferably 80% or more, more preferably 90% or more (for example, 95%, 96 %, 97%, 98%, 99% or more). Sequence identity can be determined by the BLAST program.

  The dsRNA complementary to the transcription product of the gene encoding the syntenin protein used in the present invention has a high inhibitory effect on the expression of the syntenin gene. From the viewpoint of the human-derived syntenin gene, 894 to 894 described in SEQ ID NO: 1 A dsRNA complementary to DNA consisting of the nucleotide sequence at position 918 is preferred.

  Other embodiments of the “RNA that binds to the transcription product of the syntenin gene” in the present invention include antisense RNA complementary to the transcription product of the syntenin gene or DNA encoding the RNA (antisense DNA).

  Antisense RNA or antisense DNA suppresses target gene expression by inhibiting transcription initiation by triplex formation, transcription suppression by hybridization with a site where an open loop structure is locally created by RNA polymerase, Inhibition of transcription by hybridization with RNA, which is progressing synthesis, suppression of splicing by hybridization at the junction of intron and exon, suppression of splicing by hybridization with spliceosome formation site, nuclear to cytoplasm by hybridization with mRNA Suppression of translocation, suppression of splicing by hybridization with capping sites and poly (A) addition sites, suppression of translation initiation by hybridization with translation initiation factor binding sites, hybridization with ribosome binding sites in the vicinity of the start codon Translational repression by de formation, outgrowth inhibitory peptide chains by the formation of a hybrid with a coding region or polysome binding sites of mRNA, and the gene silencing due hybridization and interaction site between a nucleic acid and protein. These inhibit the transcription, splicing, or translation process and suppress the expression of the target gene (Hirashima and Inoue, "New Chemistry Laboratory Lecture 2 Nucleic Acid IV Gene Replication and Expression", Japan Biochemical Society, Tokyo Chemical) Doujin, 1993, pp. 319-347). The antisense RNA or antisense DNA used in the present invention may suppress the expression of the target gene by any of the actions described above. In one embodiment, if an antisense sequence complementary to the untranslated region near the 5 'end of the mRNA of the target gene is designed, it will be effective for inhibiting translation of the gene. However, sequences complementary to the coding region or 3 'untranslated region can also be used. Thus, RNA or DNA containing an antisense sequence of not only the translation region of a gene but also the sequence of an untranslated region is also included in the antisense RNA or antisense DNA used in the present invention. The antisense DNA to be used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 'side.

  Antisense RNA or antisense DNA can be prepared on the basis of the description of the phosphorothioate method (Stein, Nucleic Acids Res., 1988, Vol. 16, pages 3209-3221) based on the sequence information of the syntenin gene. .

  The sequence of the antisense RNA or antisense DNA is preferably a sequence complementary to the transcriptin of syntenin, but may not be completely complementary as long as it can effectively inhibit gene expression. Antisense RNA or antisense DNA preferably has a complementarity of 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more) to the transcript of the target gene. In order to effectively inhibit the expression of the target gene, the length of the antisense RNA or antisense DNA is at least 15 bases or more, preferably 100 bases or more, more preferably 500 bases or more. Usually, the length of antisense RNA or antisense DNA used is shorter than 5 kb, preferably shorter than 2.5 kb.

  Other embodiments of the “RNA that binds to the transcriptin of the syntenin gene” in the present invention include RNA having a ribozyme activity that specifically cleaves the transcript of the syntenin gene, or DNA encoding the RNA. Some ribozymes have a group I intron type and a size of 400 nucleotides or more like M1RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 1990, 35, 2191).

  For example, the self-cleaving domain of hammerhead ribozyme cleaves on the 3 ′ side of C15 of G13U14C15, but it is important for activity that U14 forms a base pair with A at position 9, and the base at position 15 is In addition to C, A or U is also shown to be cut. If the ribozyme substrate binding site is designed to be complementary to the RNA sequence in the vicinity of the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA. (Koizumi et al., FEBS Lett. 1988, 239, 285-288, Koizumi Makoto and Otsuka Eiko, Protein Nucleic Acid, 1990, 35, 2191, Koizumi et al., Nucleic. Acids. Res. 1989. 17 pp. 7059).

  Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzyan, Nature, 1986, 323, 349). It has been shown that this ribozyme can also be designed to cause target-specific RNA cleavage (Kikuchi and Sasaki et al., Nucleic Acids Res, 1992, 19, 6751, Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112 pages). A ribozyme designed to cleave a target is linked to a promoter and a transcription termination sequence so as to be transcribed in a nerve cell or the like. Such a structural unit can be arranged in tandem so that a plurality of sites in the target gene can be cleaved to further increase the effect (Yuyama et al., Biochem. Biophys. Res. Commun., 1992, 186, 1271).

-Anti-syntenin protein antibody-
The anti-syntenin protein antibody may be a polyclonal antibody, a monoclonal antibody, or a functional fragment of the antibody. “Antibody” also includes all classes and subclasses of immunoglobulins. The “functional fragment” of an antibody means a part (partial fragment) of an antibody that specifically recognizes a syntenin protein. Specifically, Fab, Fab ′, F (ab ′) 2, variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc (Fv) 2, diabody, multispecific antibody, And polymers thereof.

  Anti-syntenin protein antibodies include chimeric antibodies, humanized antibodies, human antibodies, and functional fragments of these antibodies. When the antibody of the present invention is administered to a human as a therapeutic agent, a chimeric antibody, a humanized antibody or a human antibody is desirable from the viewpoint of reducing side effects.

  In the present invention, the “chimeric antibody” is an antibody in which the variable region of a certain antibody is linked to the constant region of a heterologous antibody. A chimeric antibody, for example, immunizes a mouse with an antigen, cuts out an antibody variable region (variable region) that binds to the antigen from the mouse monoclonal antibody gene, and binds to a human bone marrow-derived antibody constant region (constant region) gene. Can be obtained by incorporating it into an expression vector and introducing it into a host for production (for example, JP-A-8-280387, U.S. Pat. No. 4,816,397, U.S. Pat. No. 4,816,567, U.S. Pat. 5807715). Further, in the present invention, the “humanized antibody” is an antibody obtained by grafting (CDR grafting) the gene sequence of the antigen binding site (CDR) of a non-human-derived antibody to a human antibody gene, and its production method is publicly known. (See, for example, EP239400, EP125503, WO90 / 07861, WO96 / 02576). In the present invention, a “human antibody” is an antibody derived from all regions. In the production of human antibodies, it is possible to use a transgenic animal (for example, a mouse) that can produce a repertoire of human antibodies by immunization. Techniques for producing human antibodies are known (for example, Nature, 1993, 362, 255-258, Inter. Rev. Immunol, 1995, 13, 65-93, J. Mol. Biol, 1991). Year, 222, 581-597, Nature Genetics, 1997, 15, 146-156, Proc. Natl. Acad. Sci. USA, 2000, 97, 722-727, JP-A-10-146194 No. 10-155492, No. 2938569, No. 11-206387, No. 8-509612, No. 11-505107.

  In addition, the amino acid sequence of an anti-syntenin protein antibody is modified without reducing the desired activity (binding activity to syntenin protein, activity to suppress spine formation abnormality, and / or other biological properties, etc.). Antibodies. Amino acid sequence variants can be made by introducing mutations into DNA encoding the antibody chain or by peptide synthesis. The site where the amino acid sequence of the antibody is modified may be the constant region of the heavy chain or light chain of the antibody as long as it has an activity equivalent to that of the antibody before modification, and further, the variable region (framework region and CDR). Modification of amino acids other than CDR is considered to have a relatively small effect on the binding affinity with the antigen, but at present, the amino acid of the CDR is modified to screen for an antibody having an increased affinity for the antigen. Methods are known (PNAS, 2005, 102, 8466-8471, Protein Engineering, Design & Selection, 2008, 21, 485-493, WO2002 / 051870, J. Biol. Chem., 2005, 280 Volume, 24880-24887, Protein Engineering, Design & Selection, 2008, 21, 345-351).

  The number of amino acids to be modified is preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids (eg, within 2 amino acids, 1 amino acid). The amino acid modification is preferably a conservative substitution. In the present invention, “conservative substitution” means substitution with another amino acid residue having a chemically similar side chain. Groups of amino acid residues having chemically similar amino acid side chains are well known in the technical field to which the present invention belongs. For example, acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), neutral amino acids, amino acids having a hydrocarbon chain (glycine, alanine, valine, leucine, isoleucine, proline), hydroxy group Amino acids with amino acids (serine / threonine), amino acids with sulfur (cysteine / methionine), amino acids with amide groups (asparagine / glutamine), amino acids with imino groups (proline), amino acids with aromatic groups (phenylalanine / tyrosine / (Tryptophan). The amino acid sequence variant preferably has the same binding activity to the antigen as the target antibody. The binding activity to the antigen can be evaluated by analysis using, for example, a flow cytometer, ELISA, Western blotting, immunoprecipitation method or the like.

  Furthermore, in the present invention, deamidation is suppressed by substituting an amino acid adjacent to the amino acid deamidated or deamidated with another amino acid for the purpose of increasing the stability of the antibody. May be. Alternatively, glutamic acid can be substituted with other amino acids to increase antibody stability. The present invention also provides the antibody thus stabilized.

  In addition, as described above, the antibody of the present invention may be any antibody that specifically recognizes syntenin protein, but inhibits the binding between syntenin protein and syndecan-2 protein, and syndecan-2 protein and CASK protein. An antibody that specifically recognizes the PDZ2 domain of the syntenin protein is preferable from the viewpoint that the spine formation abnormality can be suppressed by maintaining the interaction with. The PDZ2 domain is a polypeptide consisting of the 196th to 269th amino acid sequences in the human-derived syntenin protein (protein consisting of the amino acid sequence described in SEQ ID NO: 2).

-RNA or DNA binding to syntenin protein-
The RNA or DNA that binds to the syntenin protein contained as an active ingredient of a pharmaceutical composition for suppressing spine formation abnormality is typically a nucleic acid aptamer.

  The nucleic acid aptamer of the present invention is preferably DNA from the viewpoint of high in vivo stability. Furthermore, from the same viewpoint, the nucleic acid aptamer of the present invention may include a nucleic acid molecule in which the phosphate skeleton in the nucleic acid molecule structure is modified to increase the in vivo stability. Examples of the phosphate skeleton include a phosphate skeleton including a phosphorothioate bond, a phosphorodithioate bond, a phosphoramidothioate bond, a phosphoramidate bond, a phosphordiamidate bond, and a methylphosphonate bond.

  The nucleic acid aptamer of the present invention has, as structural features, at least one loop structure, a primary structure containing many deoxyguanosines (including guanosine and guanosine analogs), or a deuteroguanosine tetramer cluster structure (so-called “G quartet”). Structure "). The nucleic acid aptamer of the present invention may be either a double-stranded nucleic acid or a single-stranded nucleic acid, but a single-stranded nucleic acid is preferred.

  The length of the nucleic acid aptamer of the present invention is not particularly limited as long as it has the number of bases that can specifically bind to the syntenin protein. For example, the length is 10 to 200 bases, preferably 20 to 150 bases. More preferably, it is 30 to 150 bases, more preferably 50 to 150 bases, and specific examples include 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 and 150 base aptamers. .

  In addition, the nucleic acid aptamer of the present invention may be any one that specifically binds to the syntenin protein, but inhibits the binding between the syntenin protein and the syndecan-2 protein so that the syndecan-2 protein and the CASK protein interact with each other. From the viewpoint that spine formation abnormality can be suppressed by maintaining the action, a nucleic acid that specifically binds to the PDZ2 domain of the syntenin protein is preferable.

  The nucleic acid aptamer of the present invention can be produced by a person skilled in the art by appropriately selecting a known technique. Known methods include, for example, in vitro selection method (SELEX method) (Tuerk, C. et al., Science, 1990, 249, pp. 505-510, Green, L. et al., Meths. Enzymol., 1991, 2 Vol. 75-86, Gold, L. et al., Annu. Rev. Biochem., 1995, 64, 763-797, Uphoff, KW et al., Curr. Opin. Struct. , Vol. 6, pages 281 to 288).

  The in vitro selection method is a method of selecting a nucleic acid having affinity for a desired protein from a pool of nucleic acids containing a random sequence and excluding nucleic acids having no affinity.

  Specifically, when the nucleic acid aptamer of the present invention is produced by the in vitro selection method, first, a random base sequence of about 20 to 300 bases, preferably 30 to 150 bases, more preferably about 30 to 100 bases is included. Prepare the nucleic acid. The nucleic acid may be synthesized directly, or in the case of RNA molecules, the DNA molecules may be synthesized first and then prepared by a transcription reaction. When the nucleic acid is DNA, it is preferable to have a base sequence serving as a primer for enabling PCR amplification at both ends. The primer binding sequence portion may be prepared so as to have an appropriate restriction enzyme recognition sequence for excising the primer portion with a restriction enzyme after PCR amplification. The length of the primer binding sequence portion is not particularly limited, but is usually 20 to 50, preferably 20 to 30 bases. Further, in order to separate the DNA after PCR amplification at the 5 'end of the primer by electrophoresis or the like, labeling may be performed using a radioactive substance, a fluorescent substance or the like. Furthermore, when RNA is prepared, it may be prepared by transcription from a DNA molecule to an RNA molecule using an appropriate promoter as a 5'-end primer, such as DNA having a T7 promoter sequence.

  Next, the random nucleic acid obtained by PCR amplification and the syntenin protein or a partial peptide thereof are mixed and incubated. After incubation, the mixture is electrophoresed to separate the nucleic acid-syntenin protein complex and free nucleic acid. Then, the nucleic acid forming a complex with the syntenin protein is extracted according to a known method. When the recovered nucleic acid is DNA, PCR amplification is further performed, and the amplified DNA is recovered by heat denaturation or the like, and recovered as single-stranded DNA. When the recovered nucleic acid is RNA, the RNA is reverse transcribed into cDNA, PCR amplified, RNA is prepared by transferring the amplified DNA. In addition, the step of mixing the above-mentioned nucleic acid and the syntenin protein, the step of separating the nucleic acid bound to the syntenin protein, the step of PCR amplification (in the case of RNA, reverse transcription and amplification), the amplified nucleic acid again in the syntenin protein The operation consisting of the steps used for binding to is repeated several times. Sequencing of the obtained nucleic acid can be performed according to a known method.

  The nucleic acid aptamer of the present invention is produced by chemical synthesis, in vitro transcription using DNA-dependent RNA polymerase or DNA-dependent DNA polymerase, and amplification by PCR based on the nucleic acid sequence obtained by the above-described operation. May be.

  Since the pharmaceutical composition of the present invention can suppress abnormal spine formation by suppressing the function of the syntenin gene, diseases associated with abnormal spine formation such as tuberous sclerosis (Pringle disease), epilepsy (nodule) Refractory epilepsy etc. in multiple sclerosis), mental retardation, autism, Alzheimer's disease and the like can be suitably used as a pharmaceutical composition to be administered for the treatment or prevention.

  The pharmaceutical composition of the present invention can be formulated by a known pharmaceutical method. For example, capsule, tablet, pill, liquid, powder, granule, fine granule, film coating, pellet, troche, sublingual, chewing agent, buccal, paste, syrup, suspension, Use orally or parenterally as elixirs, emulsions, coatings, ointments, plasters, poultices, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc. Can do.

  In these preparations, a pharmacologically acceptable carrier, specifically, sterilized water or physiological saline, vegetable oil, solvent, base, emulsifier, suspension, surfactant, stabilizer, pH adjuster, Flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, tonicity agent, soothing agent, extender, disintegrant, buffering agent, coating agent, lubricant, colorant, sweetness Can be suitably combined with an agent, a thickening agent, a flavoring agent, a solubilizing agent or other additives.

  You may use the pharmaceutical composition of this invention together with the well-known pharmaceutical composition used for the treatment or prevention of the said autism etc. Moreover, the pharmaceutical composition of the present invention can be used for animals including humans. There is no restriction | limiting in particular as an animal other than a human, Various livestock, a poultry, a pet, a laboratory animal, etc. can be made into object.

  The dosage form of the pharmaceutical composition of the present invention is not particularly limited. For example, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration and intramuscular administration, by infusion Administration and administration directly into the brain (local administration) can be mentioned.

  As a method for directly administering the pharmaceutical composition of the present invention into the brain, for example, as shown in the examples described later, a sustained-release drug delivery system containing the pharmaceutical composition of the present invention (for example, permeation manufactured by ALZET) A pressure pump) is embedded in the brain, the head is opened, the pharmaceutical composition of the present invention is introduced by electroporation, or a cannula is inserted by stereotactic brain surgery, and administered through the cannula into the brain. A method is mentioned.

  In addition, when the pharmaceutical composition of the present invention is not directly administered into the brain, a method in which a brain barrier permeating substance is bound to a molecule that suppresses the function of the syntenin gene may be used. Examples of the brain barrier permeating substance include a glycoprotein consisting of 29 amino acids derived from rabies virus (see Kumar et al., Nature, July 5, 2007, 448, pages 39-43). Not limited.

  When administering the pharmaceutical composition of the present invention, the dosage is appropriately selected according to the age, weight, symptom, health condition, etc. of the subject. For example, the dosage of the pharmaceutical composition of the present invention (effective (Equivalent component amount) varies depending on the type of active ingredient. For example, when the active ingredient is RNA or DNA, it is usually 0.1 μg to 10 mg / kg body weight, and the active ingredient is an antibody. Is usually 1 to 10 mg / kg body weight. Also, the number of administrations or intakes per day is not particularly limited and can be appropriately selected in consideration of the various factors as described above. When the active ingredient is DNA encoding RNA that binds to the transcription product of the syntenin gene, an expression construct may be prepared and administered so that an appropriate amount of RNA is expressed.

  Thus, the present invention also provides a method for treating or preventing a disease associated with spine formation in a subject, characterized by causing the subject (such as an autistic patient) to administer the pharmaceutical composition of the present invention. .

  The pharmaceutical composition or product (pharmaceutical product) of the present invention or instructions thereof may be labeled with an indication that it is used for the treatment or prevention of diseases associated with spine formation. Here, “labeled product or instructions” means that the product body, container, packaging, etc. are marked, or instructions, package inserts, promotional materials, or other printed materials that disclose product information. It means that the display is attached to. In the indication that it is used for the treatment or prevention of diseases associated with spine formation, it contains information on the mechanism by which abnormal spine formation is suppressed by administering a molecule that suppresses the function of the syntenin gene of the present invention. Can do.

<Vaccine composition>
In the present invention, since a molecule that suppresses the function of the syntenin gene, such as an anti-syntenin protein antibody, has been found to have an activity to suppress abnormal spine formation, the syntenin protein or a partial peptide thereof can be used as a vaccine for mammals including humans. It is also possible to administer to animals (for example, JP 2007-277251 A, JP 2006-052216 A). The present invention also provides a vaccine composition for suppressing abnormal spine formation, comprising a syntenin protein or a partial peptide thereof as an active ingredient, which is used for such vaccine applications. As a “partial peptide” of a syntenin protein, it is possible to suppress spine formation abnormality by inhibiting the binding between the syntenin protein and the syndecan-2 protein and maintaining the interaction between the syndecan-2 protein and the CASK protein. The PDZ2 domain of the syntenin protein is preferred. In the case of formulation, like the pharmaceutical composition of the present invention, pharmacologically acceptable carriers such as stabilizers, preservatives, tonicity agents and the like can be appropriately combined.

  Thus, the present invention also provides a method for treating or preventing a disease associated with spine formation in a subject, characterized by causing the subject (such as an autistic patient) to administer the vaccine composition of the present invention. be able to.

  The product (vaccine) of the vaccine composition of the present invention or the instructions thereof may be labeled with an indication that it is used for the treatment or prevention of diseases associated with spine formation. Here, “labeled product or instructions” means that the product body, container, packaging, etc. are marked, or instructions, package inserts, promotional materials, or other printed materials that disclose product information. It means that the display is attached to. In the indication of being used for treatment or prevention of a disease associated with spine formation, information on the mechanism by which spine formation abnormality is suppressed by administering a syntenin protein or a partial peptide thereof can be included.

<Screening method>
As shown in the below-mentioned Example, it discovered that spine formation abnormality was suppressed by suppressing the function of a syntenin gene. Therefore, in screening for compounds having an activity to suppress abnormal spine formation, it is effective to select a compound that binds to the syntenin protein as a candidate for a compound that suppresses the function of the syntenin gene. That is, this invention provides the screening method of the candidate compound which has the activity which suppresses spine formation abnormality including the process of following (a)-(c) as a 1st aspect of the screening method of this invention.
(A) contacting the test compound with the syntenin protein;
(B) detecting the binding between the test compound and the syntenin protein;
(C) selecting a compound that binds to the syntenin protein.

  The “test compound” used in the screening method of the present invention is not particularly limited, and examples thereof include expression products of gene libraries, synthetic low molecular compound libraries, peptide libraries, polynucleotide libraries, antibodies, and bacterial release substances. Cell (microbe, plant cell, animal cell) extract and culture supernatant, purified or partially purified polypeptide, marine organism, plant or animal extract, soil, random phage peptide display library. The test compound may be synthesized based on an in silico design based on the three-dimensional structure of the syntenin protein.

  Examples of the “syntenin protein” used in the screening method of the present invention include a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. In addition, the syntenin protein used in the screening method of the present invention may be a partial peptide thereof, and the partial peptide inhibits the binding between the syntenin protein and the syndecan-2 protein, and the syndecan-2 protein and the CASK protein. The PDZ2 domain of the syntenin protein (for example, a polypeptide comprising the 196th to 269th amino acid sequences described in SEQ ID NO: 2) is preferable from the viewpoint that spine formation abnormality can be suppressed by maintaining the interaction with.

  Moreover, as the syntenin protein used in the screening method of the present invention, in addition to the natural type protein and its partial peptide, a modified form, a modified form, and the like can be used. For example, expressed as a fusion protein with a marker protein for easy detection, an enzyme such as alkaline phosphatase (SEAP), β-galactosidase, a fluorescent protein such as glutathione-S-transferase (GST), or a green fluorescent protein (GFP) What is obtained can be used.

In the step (b), the binding between the test compound and the syntenin protein is detected, and a known technique can be appropriately employed to detect the binding. For example, the test compound is a synthetic low molecular weight compound, a natural product (bacterial release material, cell (microorganism, plant cell, animal cell) extract and culture supernatant, purified or partially purified polypeptide, marine organism, plant or animal. In the case of an extract derived from soil, soil) or a random phage peptide display library, a method of screening a molecule that binds to the syntenin protein by allowing these compounds to act on the immobilized syntenin protein can be mentioned. In addition, when the test compound is a synthetic low molecular weight compound, a screening method using high throughput by combinatorial chemistry technology (Wrightton
NC et al., Science, 1996, 273, 458-464, Verdine GL. Nature 1996, 384, 11-13, Hogan JC.
Jr. Nature, 1996, 384, 17-19). Furthermore, when the test compound is a polynucleotide library, the above-described in vitro selection method can be mentioned. In addition, a synthetic low molecular weight compound, the natural product, and a peptide library are provided on an affinity column to which a syntenin protein is immobilized, and a compound that binds to the syntenin protein is purified and identified. Furthermore, when the test compound is a gene library, a yeast two-hybrid system in which the syntenin protein is expressed and used as a bait protein can be preferably used. When the test compound is a peptide library or an antibody, GST precipitation or immunoprecipitation can be used. Furthermore, in the present invention, a biosensor using the surface plasmon resonance phenomenon can also be used as means for detecting the binding between the test compound and the syntenin protein.

  The method of “contact between test compound and syntenin protein” in step (a) and “selection of compound that binds to syntenin protein” in step (c) can be used by those skilled in the art to detect the binding. It can be implemented depending on the type of method.

In addition, as shown in Examples described later, it was revealed that the syntenin protein binds with the syndecan-2 protein, thereby suppressing the interaction between the syndecan-2 protein and the CASK protein and causing abnormal spine formation. . Therefore, it is effective to select a compound that suppresses the binding between the syntenin protein and the syndecan-2 protein in screening for compounds having an activity to suppress abnormal spine formation. That is, this invention provides the screening method of the compound which has the activity which suppresses spine formation abnormality including the process of following (a)-(c) as a 2nd aspect of the screening method of this invention.
(A) contacting a syntenin protein and a syndecan-2 protein in the presence of a test compound;
(B) detecting the binding between the syntenin protein and the syndecan-2 protein;
(C) A step of selecting a compound that suppresses the binding.

  The “test compound” and “syntenin protein” used in the second embodiment are as described above. Examples of the “syndecan-2 protein” used in the screening method of the present invention include a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4. Further, the syndecan-2 protein used in the screening method of the present invention may be a partial peptide thereof, and as the partial peptide, the binding between the syntenin protein and the syndecan-2 protein is inhibited, and the syndecan-2 protein and From the viewpoint that spine formation abnormality can be suppressed by maintaining the binding to the CASK protein, the intracellular domain of the syndecan-2 protein is preferable.

  In the step (a) of the second embodiment, the syntenin protein and the syndecan-2 protein are contacted in the presence of the test compound, and such contact is performed in the absence of the test compound with the syntenin protein and the syndecan-2 protein. What is necessary is just to carry out on the conditions which are not suppressed.

  In addition, in the step (b) of the second aspect, the binding between the syntenin protein and the syndecan-2 protein is detected using an immunoprecipitation method, a GST precipitation method, an ELISA method, or an affinity chromatography in which the syntenin protein is immobilized. Known methods such as a method, a method using a surface plasmon resonance phenomenon, a method using FRET (fluorescence resonance energy transfer), and the like can be appropriately employed.

  Further, in the step (c) of the second aspect, a compound that suppresses the binding is selected. For example, when immunoprecipitation is used, the syntenin protein is specifically expressed in the presence of the test compound. Evaluation can be made by comparing the amount of syndecan-2 protein co-precipitated when precipitated with an antibody with the amount of syndecan-2 protein in the absence of the test compound (control value). That is, when the amount of syndecan-2 protein in the presence of the test compound is lower than the amount in the absence of the test compound (for example, 80% or less, 50% or less, 30% or less of the control value). Can evaluate the test compound as a compound having an activity of suppressing abnormal spine formation. Similarly, when a method other than the immunoprecipitation method is used in the detection of the binding, the degree of binding in the absence of the test compound can be similarly evaluated as a control value.

In addition, as shown in Examples described later, the amount of syntenin protein is higher in tuberous sclerosis neurons than in normal (wild-type) neurons, and the large amount of syntenin protein contributes to spine formation abnormality. It became clear that Therefore, it is effective to select a compound that decreases the expression level of the syntenin gene or the transcription product (mRNA) of the syntenin gene in screening for a compound having an activity to suppress abnormal spine formation. That is, this invention provides the screening method of the compound which has the activity which suppresses spine formation abnormality including the process of following (a)-(c) as a 3rd aspect of the screening method of this invention.
(A) contacting a test compound with a cell expressing the syntenin gene;
(B) detecting the expression of the syntenin gene in the cell,
(C) A step of selecting a compound that decreases the expression of the syntenin gene as compared with the case where the test compound is not contacted.

  The “test compound” used in the third embodiment is as described above. Examples of the “syntenin gene” to be detected in the screening method of the present invention include a gene consisting of the base sequence described in SEQ ID NO: 1.

Examples of “cells expressing the syntenin gene” used in the screening method of the present invention include nerve cells derived from tuberous sclerosis model animals and cells derived from patients such as autism. Examples of “tuberous sclerosis model animals” include TSC2 ^+/− rats, TSC2 ^+/− mice (Dan Ehinger et al., Nat Med., 2008, 14, No. 8, pp. 843-848, Kobayashi et al. Cancer Research, 1999, Vol. 59, see pages 1206-1211).

  In the step (a) of the third aspect, the “contact” can be performed, for example, by adding a test compound to the cell culture medium.

  A known technique can be used for “detection of expression of the syntenin gene” in the step (b) of the third embodiment. “Detection of gene expression” may be detected at the transcription level (mRNA level) or at the translation level (protein level). Examples of the detection method at the transcription level include RT-PCR, Northern blotting, in situ hybridization, dot blot, and RNase protection assay. Moreover, as a method of detecting at the translation level, for example, a method of detecting using antibodies such as immunohistochemical staining, imaging cytometry, flow cytometry, ELISA, radioimmunoassay, immunoprecipitation, Western blot (Immunological technique).

  In step (c) of the third embodiment, a compound that decreases the expression of the syntenin gene is selected. For example, in the case of using Western blotting, syntenin detected in the method in the presence of the test compound. It can be evaluated by comparing the amount of the protein (for example, the intensity of the band derived from the syntenin protein) with the amount of the syntenin protein in the absence of the test compound (control value). That is, when the amount of the syntenin protein in the presence of the test compound is low compared to the amount in the absence of the test compound (for example, 80% or less, 50% or less, 30% or less of the control value), The test compound can be evaluated as a compound having an activity of suppressing abnormal spine formation. Similarly, when a method other than Western blotting is used in the detection of the expression, the expression level in the absence of the test compound can be used as a control value for evaluation.

Screening for a compound that decreases the expression level of the syntenin gene can also be performed in a system using a reporter gene. That is, this invention provides the screening method of the compound which has the activity which suppresses spine formation abnormality including the process of following (a)-(c) as a 4th aspect of the screening method of this invention.
(A) contacting a test compound with a cell having DNA to which a reporter gene is operably linked downstream of the promoter region of the syntenin gene;
(B) detecting the expression of the reporter gene in the cell;
(C) A step of selecting a compound that decreases the expression of the reporter gene as compared with the case where the test compound is not contacted.

  The “syntenin gene promoter region” is a region that controls transcription of the syntenin gene and means an upstream region of the coding region of the syntenin gene to which a transcription factor can bind. The “syntenin gene promoter region” can be prepared by those skilled in the art by cloning, for example, by screening a genomic DNA library using the nucleotide sequence of SEQ ID NO: 1 or a part thereof as a probe. .

  The “reporter gene” is not particularly limited as long as its expression is detectable. For example, a CAT gene, a lacZ gene, a luciferase gene, a β-glucuronidase gene (GUS) commonly used by those skilled in the art, and A GFP gene can be mentioned.

  In the present invention, the reporter gene “functionally bound” downstream of the promoter region of the syntenin gene means that the transcription factor is bound to the promoter region of the syntenin gene so that expression of the reporter gene is induced. It means that the promoter region of the syntenin gene is linked to the reporter gene.

  The “syntenin gene”, “test compound” and “contact” in the fourth embodiment are the same as those in the third embodiment. The “cell” used in the fourth embodiment may be any cell in which the expression of the reporter gene is induced by binding of a transcription factor to the promoter region of the syntenin gene. For example, HEK293T cells, COS cells, nodules Examples include neuronal cells derived from a model animal of sclerosis and cells derived from patients such as autism.

  In the step (b) of the fourth embodiment, the expression of the reporter gene detected can be detected by a method known to those skilled in the art depending on the type of reporter gene used. For example, when the reporter gene is a CAT gene, the expression of the reporter gene can be detected by detecting acetylation of chloramphenicol by the gene product. When the reporter gene is a lacZ gene, the color development of the dye compound is detected by the catalytic action of the gene expression product. When the reporter gene is a luciferase gene, chemiluminescence by the catalytic action of the gene expression product is detected. When GUS is detected, glucuron luminescence or 5-bromo-4-chloro-3-indolyl-β-glucuronide (X-Gluc) coloration is detected by the catalytic action of the gene expression product. Thus, in the case of the GFP gene, the expression of the reporter gene can be detected by detecting the fluorescence of the GFP protein.

  In the step (c) of the fourth embodiment, a compound that decreases the expression of the reporter gene is selected. For example, when a luciferase gene is used as the reporter gene, chemiluminescence detected in the presence of the test compound And the intensity of chemiluminescence in the absence of the test compound (control value) can be evaluated. That is, when the luminescence intensity in the presence of the test compound is lower than the luminescence intensity in the absence of the test compound (for example, 80% or less, 50% or less, 30% or less of the control value), The test compound can be evaluated as a compound having an activity of suppressing abnormal spine formation. In the detection of the expression, the expression level in the absence of the test compound can be similarly evaluated in the case other than the case of using the luciferase gene as a control value.

  As mentioned above, although the screening method of this invention was demonstrated, it is preferable to verify the activity in vivo further about the compound selected by these methods. For example, as shown in the Examples described later, the compound is brought into contact with a neuron (neuron) derived from a tuberous sclerosis model animal, and the degree of suppression of spine formation abnormality in the neuron is evaluated, thereby enabling nodular sclerosis. It can be selected as an effective compound by prevention or treatment of diseases such as infectious diseases. In addition, in vivo activity verification is carried out by administering a compound selected by the screening method of the present invention into the brain of a tuberous sclerosis model animal, as shown in the examples described later. It can also be done by evaluating the degree.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to a following example.

<Primary culture of hippocampal neurons>
On day 20 of embryonic day, the hippocampus was excised from TSC2 ^+/− (Eker) rat or wild type rat, and treated in Hanks buffer containing 0.25% trypsin at 37 ° C. for 10 minutes. After washing, the cells were suspended, and cultured under a condition of 37 ° C. and 5% CO ₂ using a Neurobasal culture solution (Gibco) containing B-27 additive. Lipofectamine 2000 (manufactured by Invitrogen) was used for gene forced expression.

<SiRNA>
In order to suppress the expression of the syntenin gene, siRNA comprising the following base sequence
(DsRNA) was used.
syntenin siRNA
5′-GAAGGAUGCUCAGAUUGCAGAUAUA-3 ′ (SEQ ID NO: 5)
5′-UAUAUUCUGCAAUUCAGAGCAUCCUUC-3 ′ (SEQ ID NO: 6)
As a negative control for the syntenin siRNA, siRNA (dsRNA) having the following base sequence was also used.
Non-specific siRNA (syntenin)
5′-GAACGUAGACUGUUAAGACUGGAUA-3 ′ (SEQ ID NO: 7)
5′-UAUCCAGUCUUACAGUCUCUACGUUC-3 ′ (SEQ ID NO: 8)
In order to suppress the expression of the syndecan-2 gene, siRNA (dsRNA) having the following base sequence was used.
syndecan-2 siRNA
5′-CAACUGAUUCCAAGGAUCUCCUCUCA-3 ′ (SEQ ID NO: 9)
5′-UGAGGGGAGAUCCUUGGAAUCAGUGUG-3 ′ (SEQ ID NO: 10)
As a negative control for the syndecan-2 siRNA, siRNA (dsRNA) having the following base sequence was also used.
Non-specific siRNA (syndecan-2)
5′-CAAUUAGAACCUAGGCCUCCUCUCA-3 ′ (SEQ ID NO: 11)
5′-UGAGAGGAGGCCUAGGGUUCUAAUG-3 ′ (SEQ ID NO: 12).

<Analysis of protein-protein interaction>
By the GST precipitation method or the immunoprecipitation method, the binding site of Rheb and syntenin and the binding of CASK and syndecan-2 were analyzed according to a conventional method.

<Ubiquitination assay>
In the Rheb and syntenin ubiquitination assay, first, EGFP-Rheb fusion protein and FLAG-syntenin fusion protein were forcibly expressed in HEK293T cells. Next, a protein extract of the cells was prepared and immunoprecipitated with a GFP antibody. The obtained sample was analyzed by Western blotting using an anti-ubiquitin antibody.

<Immune cell staining>
Cultured neurons were fixed with 4% paraformaldehyde. The fixed neurons were blocked with 10% goat serum-containing buffer and reacted with the primary antibody. Furthermore, the target protein was labeled using a fluorescent secondary antibody.

<Diolisitic labeling method>
Normal rats and TSC2 mutant rats were opened, and siRNA was introduced into each brain by electroporation. Then, the brain into which siRNA was introduced was removed and fixed with 2% paraformaldehyde. Next, the fixed brain was removed and sliced with a vibratome to a thickness of 250 μm with a forehead. Neurons were fluorescently labeled by introducing gold particles with DiI dye attached to sliced brain slices using a gene gun (see FIG. 33). The labeled neurons were observed with a laser microscope (product name: LSM780, manufactured by Carl Zeiss).

<Administration of siRNA>
150 μl of syntenin siRNA 250 μM / saline solution is placed in an osmotic pump (manufactured by Alzet, mini pump # 1002) and placed in the lateral ventricle, so that 6 μl of syntenin siRNA per day is maintained in the lateral ventricle for 14 days. Administered.

<Context-dependent fear discrimination test>
The context-dependent fear discrimination test was performed according to the method described in “Osterweil EK. Et al., Nature, 2011, 480, 7375, pages 63-68” (see FIG. 40). That is, normal rats or TSC2 ^+/− rats (all adult rats aged 6 to 12 weeks) were subjected to an operation in which an osmotic pump was implanted as described above. Two weeks later, 1-second electrical stimulation (foot shock) was given three times in a bright room. Then, 24 hours later, the rat was transferred to a bright room, which is the same environment as before, or a dark room, which is a different environment, and the time for freezing (non-moving state) of the rat was measured.

Example 1
<Verification of presence / absence of association between mTOR protein and spine formation>
Hippocampal neurons were isolated from a rat having a mutation in the causative gene TSC2 of tuberous sclerosis (also referred to as “Eker rat” or “TSC2 ^+/− rat”) and cultured in primary culture. The morphology of hippocampal neurons after 21 days in culture was compared with that of wild type rats. The obtained results are shown in FIGS.

As shown in FIGS. 3 and 4, spine was formed in wild type rats, whereas spine was not formed in TSC2 ^+/− rats, and filopodia, an immature form before becoming spine, was observed. . In addition, whether or not excitatory synapses were formed was examined by immunocytochemical staining using an antibody against vGlut1, and TSC2 ^+/− rat filopodia did not form synapses, but synapses directly on dendrites. (See FIGS. 3, 5 and 6).

It is speculated that the activation of mTOR protein contributes to the abnormal spine formation in such TSC2 ^+/− rats. That is, when a mutation occurs in TSC2, the Rheb protein becomes active (Rheb-GTP), and the local translation of the protein in the spine is enhanced through activation of the downstream mTOR protein. And it is speculated that abnormally synthesized proteins inhibit spine formation.

Therefore, in order to examine whether this spine formation abnormality is due to activation of mTOR protein, rapamycin, an mTOR inhibitor, was added to the medium, and the morphology of hippocampal neurons of TSC2 ^+/− rats was observed in the same manner as described above. The obtained results are shown in FIGS.

As is apparent from the results shown in FIGS. 7 to 9, between the hippocampal neurons cultured in the medium supplemented with rapamycin and those cultured in the medium supplemented with DMSO as a negative control, the morphology of the neurons, the dendritic state There was no significant difference regarding the length of the protrusion on the protrusion and the spine synapse density. Thus, rapamycin did not restore spine formation abnormalities in TSC2 ^+/− rats, suggesting that mTOR protein does not contribute to spine formation.

(Example 2)
<Analysis of binding between Rheb protein and syntenin protein>
Based on the results described in Example 1, assuming a spine formation mechanism not mediated by the mTOR protein, proteins that bind to the Rheb protein upstream of the mTOR protein were searched using the yeast two-hybrid method. As a result, it was revealed that the syntenin protein and the Rheb protein bind to each other. As shown in FIG. 10, the syntenin protein has two PDZ domains on the C-terminal side and is known to bind to various proteins (see Non-Patent Document 3). However, the fact that syntenin protein and Rheb protein bind to each other is a finding that has been revealed for the first time.

As described above, in TSC2 ^+/− rats, the Rheb protein is in the active form (Rheb-GTP) due to the mutation of TSC2. Therefore, it was analyzed whether there was a difference between the active Rheb protein and the inactive Rheb protein with respect to the binding to the syntenin protein. That is, the Rheb (Rheb ^Q64V ) protein in which the 64th amino acid is replaced with glutamine to valine, which is known as an active mutant, and the 60th amino acid, which is known as an inactive mutant, is asparagine. Using Rheb (Rheb ^D60I ) protein in which acid is replaced with isoleucine, the binding to syntenin protein was analyzed by GST precipitation method. The obtained results are shown in FIG.

As is clear from the results shown in FIG. 11, the amount of Rheb ^Q64V protein bound to the syntenin protein was significantly lower than those of the Rheb protein and the Rheb ^D60I protein. Therefore, it was revealed that when the Rheb protein is activated, the binding to the syntenin protein is weakened.

Next, identification of the binding site of the syntenin protein in the Rheb protein was attempted. That is, first, as the effector domain variant, 38 th amino acid with ^{Rheb D60I} protein has been replaced with a methionine threonine (T38M / D60I), 39 th ^{Rheb D60I} protein amino acids have been substituted with a lysine isoleucine ( I39K / D60I). And the coupling | bonding of these Rheb protein variants and a syntenin protein was analyzed by GST precipitation method. The obtained result is shown in FIG.

  The Rheb protein is a kind of low molecular weight GTP binding protein, and like other low molecular weight GTP binding proteins, it is known to transmit a signal downstream by binding to an effector molecule. It has also been clarified that the region to which this effector molecule binds is an effector domain, and in the Rheb protein, a region consisting of amino acids 35 to 43.

As is clear from the results shown in FIG. 12, the amount of the effector domain mutant that binds to the syntenin protein is low compared to those of the Rheb protein and the Rheb ^D60I protein (D60I), particularly in the T38M / D60I mutant. , Almost no binding to the syntenin protein was detected. Therefore, it was revealed that the Rheb protein is bound to the syntenin protein via the effector domain.

  Next, an attempt was made to identify the binding site of the Rheb protein in the syntenin protein. That is, first, a mutant of the syntenin protein shown in FIG. 10 was prepared. And the coupling | bonding of these syntenin protein variants and Rheb protein was analyzed by GST precipitation method. The obtained result is shown in FIG.

  As is clear from the results shown in FIG. 13, the amount of syntenin protein (ΔC (278-300) and ΔC (289-300))) in which mutation was introduced into the C-terminal region of the PDZ2 domain, was bound to the Rheb protein. Was significantly lower compared to wild-type syntenin protein (synt) and other mutants. Therefore, it was revealed that the syntenin protein is bound to the Rheb protein via the C-terminal region.

Example 3
<Verification of the existence of the relationship between syntenin and spine formation>
Next, in order to investigate the presence or absence of the relationship between the syntenin protein and spine formation, the syntenin gene was overexpressed by introducing a syntenin gene expression vector into cultured hippocampal neurons derived from wild type rats. In addition, the expression of the syntenin gene in cultured hippocampal neurons derived from TSC2 ^+/− rats was knocked down by RNAi. The obtained results are shown in FIGS.

As is clear from the results shown in FIGS. 14 to 16, filopodia increased in cultured hippocampal neurons derived from wild-type rats due to overexpression of the syntenin gene. On the other hand, as shown in FIGS. 17 to 19, it was revealed that spine was formed in cultured hippocampal neurons derived from TSC2 ^+/− rats by suppressing the expression of the syntenin gene.

Furthermore, when the amount of syntenin protein in cultured hippocampal neurons derived from TSC2 ^+/- rats was analyzed by Western blotting, it was found that the amount was increased compared to that of the wild type (see FIGS. 20 and 21). . Therefore, the degree of ubiquitination in the syntenin protein and the Rheb protein was evaluated in order to elucidate the mechanism of the increase in the amount of the syntenin protein in the TSC2 ^+/− rat. The obtained results are shown in FIGS.

As shown in FIG. 22, there was no difference in the degree of ubiquitination of the ^syntenin protein between the presence of the active Rheb (Rheb ^Q64V ) protein and the presence of the inactive Rheb (Rheb ^D60I ) protein.

Meanwhile, as shown in FIG. 23, in the ubiquitination of Rheb protein Trip inactive ^{Rheb (Rheb D60I)} protein than active ^{Rheb (Rheb Q64V)} protein, the apparent that likely to be more ubiquitinated became.

  From the above results, it was revealed that in the neurons (normal neurons) of wild type rats, the Rheb protein exists in an inactive form and forms a complex with the syntenin protein. Furthermore, since the inactive Rheb protein is easily ubiquitinated, it was also revealed that the amount of the syntenin protein is kept low by degrading the syntenin protein together with the complex by the ubiquitin-proteasome system. (See Figure 1).

On the other hand, in TSC2 ^+/- rat neurons (neurons of tuberous sclerosis), the Rheb protein exists in an active form due to mutations in the TSC protein. The binding between the active Rheb protein and the syntenin protein is weak, and the active Rheb protein is less likely to be ubiquitinated than the inactive type. Therefore, in the neurons of TSC2 ^+/− rats, syntenin protein degradation did not occur so much, and the amount of syntenin protein was found to be higher than that of wild type rats (see FIG. 2). .

Example 4
<Analysis of spine formation inhibition by syntenin protein>
As shown in Example 3, it was revealed that spine formation was suppressed by increasing the amount of syntenin protein. Therefore, in order to clarify the mechanism of action of the syntenin protein to suppress spine formation, an attempt was made to identify a region necessary for the suppression of spine formation in the syntenin protein. That is, by introducing an endogenous syntenin gene-specific siRNA and a plasmid capable of expressing an RNAi-resistant syntenin gene into a cultured hippocampal neuron derived from a wild type rat, exogenously instead of the endogenous syntenin protein. Various introduced syntenin protein mutants (see FIG. 10) and the like were overexpressed in cultured hippocampal neurons derived from wild-type rats, and the morphology of the neurons, the length of protrusions on dendrites, and the spine synapse density were analyzed. The obtained results are shown in FIGS.

  As is clear from the results shown in FIGS. 24 to 26, only the syntenin protein having a mutation inserted in PDZ2 did not show an inhibitory effect on spine formation. Therefore, it was revealed that the inhibitory effect on spine formation is exhibited through the PDZ2 domain of the syntenin protein.

Based on this finding, we focused on the syndecan-2 protein, which has been shown to interact with the PDZ2 domain and is known to be involved in spine formation. Then, the expression of the syntenin gene and the syndecan-2 gene in cultured hippocampal neurons derived from TSC2 ^+/− rats was suppressed by RNAi, and the morphology of the neurons, the length of projections on dendrites, and the spine synaptic density were analyzed. . The obtained results are shown in FIGS.

As is clear from the results shown in FIGS. 27 to 29, spine formation recovered by reducing the amount of syntenin protein suppresses the expression of the syndecan-2 gene in cultured hippocampal neurons derived from TSC2 ^+/− rats. It was solved by that.

  Next, we focused on the CASK protein that is known to control spine formation by interacting with the syndecan-2 protein (Chao HW. Et al., J Cell Biol., 2008, Vol. 182, No. 1, See pages 141-155). And, it is thought that the interaction between syndecan-2 protein and CASK protein necessary for spine formation is disrupted by syntenin protein, and whether syntenin protein and CASK protein can compete in the binding with syndecan-2 protein. Were examined by the GST precipitation method. The obtained result is shown in FIG. Furthermore, in cultured hippocampal neurons derived from wild type rats, the form of the neurons and the number of spine synapses when the amount of syntenin protein was increased relative to a certain amount of CASK protein were evaluated. The obtained results are shown in FIGS.

  As is apparent from the results shown in FIGS. 30 to 32, when the amount of syntenin protein is increased with respect to a certain amount of CASK protein, the binding between CASK protein and syndecan-2 protein is weakened, and spine formation by CASK protein is performed. Was also suppressed.

  From the above results, in the tuberous sclerosis neuron, increased syntenin protein binds with syndecan-2 protein, thereby competitively suppressing the binding between CASK and syndecan-2 protein and suppressing spine formation. (See Figure 2).

(Example 5)
<Verification of therapeutic effect on tuberous sclerosis by syntenin gene-specific siRNA>
As described above, it was revealed that the spine formation in cultured neurons of tuberous sclerosis was restored by siRNA against the syntenin gene. Then, next, it was examined whether there is a similar effect also in vivo. That is, syntenin siRNA was introduced into the hippocampus of TSC2 ^+/− rats by electroporation, and brain sections thereof were prepared. They were labeled with DiI by the diolisic method, and the morphology of individual neurons was examined (see FIG. 33). The obtained results are shown in FIGS.

As shown in FIGS. 34 to 36, spine formation is delayed in TSC2 ^+/− rats, as in cultured neurons, compared to wild type. On the other hand, it was revealed that spine formation was recovered in TSC2 ^+/− rats introduced with syntenin siRNA (see FIGS. 37 to 39).

Furthermore, it was investigated whether this syntenin siRNA was effective in behavioral abnormalities in tuberous sclerosis. It is known that there is an abnormality in context-dependent fear discrimination in tuberous sclerosis model mice. When a context-dependent fear discrimination test (see FIG. 40) was performed on TSC2 ^+/− rats, similar abnormalities were confirmed (see FIG. 41, particularly, results in “different environment” of “TSC2 ^+/− ”). ). Thus, it was revealed that context-dependent fear discrimination was improved by continuously injecting syntenin siRNA into the ventricle of TSC2 ^+/− rats and reducing the amount of syntenin protein in the entire brain (see FIG. 41). Therefore, it was revealed that central symptoms of tuberous sclerosis can be reduced by suppressing the amount of syntenin protein in the brain by using syntenin siRNA or the like.

(Example 6)
<Verification of spine formation and memory recovery in TSC2 ^+/- mice by syntenin hetero knockout>
In order to confirm the results obtained using TSC2 ^+/− rats in mice, TSC2 ^+/− mice and syntenin ^+/− mice were mated to produce TSC2 ^+/− ; syntenin ^+/− mice.

As the TSC2 ^+/− mouse, the mouse described in Kobayashi et al., Cancer Research, 1999, 59, 1206-1211, was used. Syntenin ^+/− mice were prepared according to a standard method using Cre / lox and FRT / FLP site-specific recombination systems. That is, first, by causing homologous recombination between the targeting vector shown in FIG. 42 and the wild type allele, a selection marker gene having FRT sequences at both ends in the mouse syntenin gene region (wild type allele in FIG. 42). (LacZ and neo etc.) were inserted between exons 2 and 3, and further, an attempt was made to insert loxp sequences on both sides of exon 3 (see target allele in FIG. 42). Next, using the expression of the selectable marker gene as an index, a mouse having the target allele was selected, and the mouse and FLP mouse were crossed to prepare a mouse from which the selectable marker gene was removed (in FIG. 42, See Phlox Allele). Then, a mouse having exon 3 of the syntenin gene was prepared by mating a mouse having a floxed allele with a Cre mouse (see Floxed allele in FIG. 42). In addition, by deleting exon 3 from the mouse syntenin gene region, a syntenin mutant consisting of amino acids 1 to 17 is transcribed and translated.

The dendrites of hippocampal neurons were observed in TSC2 ^+/− hetero knockout mice and TSC2 ^+/− ; syntenin ^+/− mice and wild type mice prepared as described above. The obtained results are shown in FIGS. In addition, these mice were used to conduct a context-dependent fear discrimination learning test in the same manner as the aforementioned rats. The obtained results are shown in FIG.

As shown in FIGS. 43 to 49, spine formation was remarkably suppressed in TSC2 ^+/− mice as in the above-mentioned TSC2 ^+/− rats, and it was confirmed that most were filopodia. However, it was revealed that spine formation was restored to the same level as that of the wild type in double hetero mice (TSC2 ^+/− ; syntenin ^+/− mice) with syntenin.

Further, as shown in FIG. 50, discrimination was observed in TSC2 ^+/− mice, but in TSC2 ^+/− ; syntenin ^+/− mice, it was recovered to the same level as in the wild type, as in the aforementioned rat.

  The above results are considered to be due to the recovery of spine formation in TSC mice and normalization of memory due to the decrease in syntenin. Therefore, it was revealed that molecules that suppress the function of the syntenin gene, such as syntenin siRNA, are effective in the treatment of mental retardation in tuberous sclerosis.

(Example 7)
<Epileptic effect of TSC mice due to syntenin deficiency>
Tuberous sclerosis is complicated by intractable epilepsy. Therefore, the effect of syntenin knockout on epilepsy was examined. In other words, wild-type mice, ^{TSC2 +/-} mice and ^TSC2 +/-; to Syntenin ^+/- mice, are each convulsant agent pentylenetetrazol (2 mg / ml, Sigma) and a 30mg per mouse body weight 1kg Thus, intraperitoneal injection was performed every other day to induce epileptic seizures. Then, the behavior of the mice is observed for 30 minutes after each injection, and the intensity of epileptic seizures is scored according to a modified index of Ito et al. (Neuroscience, 2004, 129, 757-766) (0-6) did.
Specifically, seizure intensity was scored based on the following criteria. The obtained results are shown in FIG.
0: normal state, exploratory behavior 1: weakness, immobility state 2: neck swing movement, involuntary movement from one limb to two limbs 3: involuntary movement from three limbs to four limbs, clonic seizure 4 in which body movement stops : Falls, tonic spasm, clonic cramps that strongly bend or warp the body 5: tonic cramps that occur repeatedly, intense jumping behavior 6: death.

As shown in FIG. 51, TSC2 ^+/− mice were found to cause severe epileptic seizure earlier than wild-type mice. However, in TSC2 ^+/− ; syntenin ^+/− mice, it was found that the progression of epileptic seizures was always milder than in TSC2 ^+/− mice, similar to the wild type.

Therefore, syntenin
It has been clarified that molecules that suppress the function of the syntenin gene of siRNA are also effective for the treatment or prevention of intractable epilepsy in tuberous sclerosis.

As described above, spine synapses are decreased in TSC2 ^+/− mice, and shaft synapses are increased at a cost. In spine synapses, the inflow of extracellular Ca ²⁺ ions and the like stays in the spine head, but in the shaft synapse, ions directly flow into dendrites, which is thought to enhance synaptic transmission. That is, in TSC2 ^+/− mice, the influx of ions into the dendrites is large, and thus it is considered that hyperexcitation tends to occur and the epileptogenicity is enhanced.

  As described above, according to the present invention, it is possible to suppress abnormal spine formation by suppressing the function of the syntenin gene. Therefore, the pharmaceutical composition of the present invention is useful in the treatment or prevention of abnormal spine formation diseases such as epilepsy, mental retardation, autism, tuberous sclerosis and the like.

SEQ ID NOs: 5-12
<223> Artificially synthesized siRNA sequences

Claims (7)

  A pharmaceutical composition for suppressing abnormal spine formation, comprising a molecule that suppresses the function of the syntenin gene as an active ingredient. The pharmaceutical composition according to claim 1, wherein the molecule that suppresses the function of the syntenin gene is a molecule according to any one of the following (a) to (c): RNA that binds to a transcription product of the syntenin gene Or DNA encoding the RNA
(B) Anti-syntenin protein antibody (c) RNA or DNA that binds to syntenin protein.   A vaccine composition for suppressing abnormal spine formation, comprising a syntenin protein or a partial peptide thereof as an active ingredient. A method for screening a candidate compound having an activity of suppressing abnormal spine formation, comprising the following steps (a) to (c): (a) a step of contacting a test compound with a syntenin protein;
(B) detecting the binding between the test compound and the syntenin protein;
(C) selecting a compound that binds to the syntenin protein. A method for screening a compound having an activity of suppressing abnormal spine formation, comprising the following steps (a) to (c): (a) a step of contacting a syntenin protein with a syndecan-2 protein in the presence of a test compound;
(B) detecting the binding between the syntenin protein and the syndecan-2 protein;
(C) A step of selecting a compound that suppresses the binding. A method for screening a compound having an activity of suppressing spine formation abnormality including the following steps (a) to (c): (a) a step of contacting a test compound with a cell expressing the syntenin gene;
(B) detecting the expression of the syntenin gene in the cell,
(C) A step of selecting a compound that decreases the expression of the syntenin gene as compared with the case where the test compound is not contacted. A method for screening a compound having an activity to suppress abnormal spine formation, comprising the following steps (a) to (c): (a) a cell having DNA functionally linked to a reporter gene downstream of the promoter region of the syntenin gene; A step of contacting a test compound,
(B) detecting the expression of the reporter gene in the cell;
(C) A step of selecting a compound that decreases the expression of the reporter gene as compared with the case where the test compound is not contacted.