JP2006213621A - Adoplin protein and utilization of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicament for inhibiting formation of a β-amyloid protein, to provide a remedy for Alzheimer's disease containing the medicament, and to provide a method for screening the remedy for the Alzheimer's disease. <P>SOLUTION: A substance which inhibits expression or functionality of the β-amyloid protein is developed based on success of identification of an Adoplin protein having interactive activity with PS1 participating in the formation of the β-amyloid protein. The substance inhibits the formation of the β-amyloid protein, and consequently it is found that the substance has an effect of treating the Alzheimer's disease. Further, it is possible that screening of the remedy for Alzheimer's disease is conducted by using expression of Adoplin as an indicator. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a β amyloid protein production inhibitor comprising an Adoplin protein expression inhibitor or a substance inhibitor.

  Alzheimer's disease is a progressive intellectual dysfunction that usually presents in late middle age and dies in about 5 to 10 years, presenting memory impairment, computational impairment, visual spatial disorientation, confusion, disorientation .

  The pathogenic factor of Alzheimer's disease, amyloid β protein (Aβ), is produced by undergoing a two-step cleavage from amyloid precursor protein (APP). Proteases that cleave the N-terminal side and the C-terminal side are called β-secretase and γ-secretase, respectively, and inhibitors of these enzymes may be applicable to the treatment of Alzheimer's disease. A recent study suggests that a complex containing Alzheimer's disease gene product Presenilin 1 (PS1) has a γ-secretase function.

  PS1 is a multifunctional protein that is essential for γ-secretase activity that cleaves transmembrane proteins such as APP and Notch in the membrane (see Non-Patent Documents 1 and 2), interacts with β-catenin. It is known to be involved in Wnt signaling. PS1 mutations affect Aβ production and relatively increase Aβ42 production. This Aβ42 is more likely to aggregate than normal Aβ (Aβ40) and deposits in the brain, suggesting that it causes senile plaque formation. An increase in Aβ is thought to promote senile plaque formation and cause Alzheimer's disease.

  However, there are still many unclear points about the onset mechanism of Alzheimer's disease, and an effective treatment method has not been established.

De Strooper B et al. Nature 1999, 398, 518 De Strooper B et al. Nature 1998, 391, 387

  An object of the present invention is to provide a therapeutic agent for Alzheimer's disease. More specifically, it is an object to provide a drug that inhibits the production of β-amyloid protein, a therapeutic agent for Alzheimer's disease containing the drug, and a screening method for a therapeutic drug for Alzheimer's disease.

  The present inventors have conducted intensive research to develop a therapeutic agent for Alzheimer's disease. The present inventors have conceived that PS1 involved in the production of β-amyloid protein may cause some change in gene expression, and searched for a gene whose expression changes specifically with PS1 mutation. As a result, we succeeded in identifying a gene whose expression is specifically decreased in the mutant PS1. We named this gene Adoplin-1. Two homologs having high homology with Adoplin-1 were named Adoplin-2 and Adoplin-3. Furthermore, the analysis results by the present inventors suggested that Adoplin-1 forms a gene family together with highly homologous homologues Adoplin-2 and 3.

  In addition, through coupled immunoprecipitation experiments, it was newly found that Adoplin-1 interacts with PS1. The above findings suggest that Adoplin is involved in γ-secretase activity through interaction with PS1.

  Furthermore, the present inventors have found by Western blot analysis that the Adoplin protein is expressed as a 17 kDa membrane protein in cultured cells or human brain extract. Since PS1 cleaves transmembrane proteins such as APP within the membrane, the above findings can be said to support that Adoplin is involved in PS1's γ-secretase activity as a membrane protein.

  The present inventors have further analyzed and discovered that inhibition of Adoplin expression by RNA interference decreases the γ-secretase activity of PS1. The above findings indicate that by suppressing the expression or function of Adoplin, the γ-secretase activity of PS1 can be inhibited, and as a result, it is possible to suppress the production of β-amyloid that is deeply involved in Alzheimer's disease. . That is, it was suggested that Alzheimer's disease can be treated by suppressing the expression or function of Adoplin.

In addition, as a result of the expression analysis of Adoplin, the present inventors have found that it is expressed in various organs including the brain.
Furthermore, as a result of immunostaining of brain sections of normal human cerebral cortex with Adoplin antibody, neurons were stained positively, and it was revealed that Adoplin was mainly expressed in neurons. In addition, in the brain of Alzheimer's disease, in addition to neurons, Adoplin immunoreactivity was also observed in degenerative neurites surrounding senile plaques.

  The above results indicate that Adoplin coexists with PS1 in cells and is mainly expressed in neurons in the brain, and Adpolin is involved in the pathogenesis of Alzheimer's disease, particularly β amyloid protein accumulation mechanism. It is shown.

  That is, the present inventors have shown for the first time that it is possible to develop a therapeutic agent for Alzheimer's disease having an action mechanism that targets Adoplin. A compound that inhibits the expression or function of Adoplin is considered to be a therapeutic agent for Alzheimer's disease. Further, by using the expression level or activity of Adoplin as an index, it is possible to efficiently screen for a therapeutic agent for Alzheimer's disease.

  As described above, the present inventors have found that Alzheimer's disease can be treated by inhibiting the production or production of β-amyloid by inhibiting the expression or function of Adoplin, and completed the present invention. .

That is, the present invention relates to an agent that inhibits the production of β-amyloid protein, an Alzheimer's disease therapeutic agent containing the agent, and a method for screening an Alzheimer's disease therapeutic agent.
[1] β-amyloid protein production inhibitor comprising an Adoplin protein expression inhibitor or function inhibitor,
[2] The β amyloid protein production inhibitor according to [1], wherein the Adoplin protein expression inhibitor is a compound selected from the group consisting of the following (a) to (c):
(A) an antisense nucleic acid for the transcription product of the Adoplin gene or a part thereof (b) a nucleic acid having ribozyme activity that specifically cleaves the transcription product of the Adoplin gene (c) has an inhibitory action by the RNAi effect on the expression of the Adoplin gene The β-amyloid protein production inhibitor according to [1], wherein the function inhibitor of nucleic acid [3] Adoplin protein is the following compound (a) or (b):
(A) an antibody that binds to Adoplin protein (b) a low molecular compound that binds to Adoplin protein [4] Alzheimer's disease comprising the β-amyloid protein production inhibitor according to any one of [1] to [3] as an active ingredient Therapeutic drugs,
[5] A screening method for a therapeutic agent for Alzheimer's disease, comprising selecting a compound that decreases the expression level or activity of Adoplin protein,
[6] A screening method for a therapeutic agent for Alzheimer's disease comprising the following steps (a) to (c):
(A) a step of bringing a test compound into contact with a cell expressing Adoplin protein (b) a step of measuring the expression level of Adoplin protein in the cell (c) compared with a case where measurement is performed in the absence of the test compound And [7] a method for screening a therapeutic agent for Alzheimer's disease comprising the following steps (a) to (c):
(A) a step of bringing a test compound into contact with a cell or cell extract containing DNA having a structure in which a transcriptional regulatory region of the Adoplin gene and a reporter gene are functionally linked, and (b) measuring the expression level of the reporter gene (C) a step of selecting a compound that reduces the expression level as compared with the case where it is measured in the absence of the test compound [8] Alzheimer, comprising the following steps (a) to (c): Screening method for therapeutic agents for diseases,
(A) a step of contacting Adoplin protein, or a cell or cell extract expressing the protein, with a test compound (b) a step of measuring the activity of the protein (c) measurement in the absence of the test compound A step of selecting a compound that reduces the activity of the protein as compared with the case [9] a method for screening an Alzheimer's disease therapeutic agent comprising the following steps (a) to (c):
(A) the step of bringing Adoplin protein and PS1 protein into contact with a test compound (b) the step of measuring the interaction activity between Adoplin protein and PS1 protein (c) the case of measurement in the absence of the test compound In comparison, the present invention provides a process for selecting a compound that decreases the interaction activity.

  The inventors have succeeded in finding a completely new target molecule for the treatment of Alzheimer's disease. According to the present invention, treatment of Alzheimer's disease targeting Adoplin is possible. For example, a compound that inhibits the expression or function of Adoplin is expected to be a therapeutic agent for Alzheimer's disease whose action mechanism is inhibition of β-amyloid protein production.

  The therapeutic agent for Alzheimer's disease of the present invention is very useful as a therapeutic agent having a completely new mechanism of action.

  In addition, by using the screening method provided by the present invention, it is possible to efficiently obtain a compound having a therapeutic effect against Alzheimer's disease.

  The present invention provides a β amyloid (Aβ) protein production inhibitor comprising an Adoplin protein expression inhibitor or a function inhibitor.

  Adoplin-1 (Adp-1) forms a gene family together with two highly homologous homologs Adoplin-2 (Adp-2) and Adoplin-3 (Adp-3). In the present invention, the Adoplin protein (gene) usually refers to any protein (gene) of Adoplin-1, Adoplin-2, or Adoplin-3. For example, 2 selected from these proteins (genes). It also includes the case of a seed protein (gene) or all these proteins (genes). The most preferred example of the Adoplin protein (gene) of the present invention is Adoplin-1 protein (gene).

  That is, in a preferred embodiment of the present invention, a substance that suppresses the expression of one or more proteins selected from the group consisting of Adoplin-1, Adoplin-2, and Adoplin-3, or a substance that suppresses the function of the protein. A β-amyloid protein production inhibitor, which is contained as an active ingredient, is provided.

  The Adoplin protein of the present invention is preferably a human Adoplin protein, but the biological species from which the Adoplin protein is derived is not particularly limited, and is a homologue of a human Adoplin protein in a non-human biological species (eg, a mammal such as a mouse). There may be. (Hjelmqvist L et al Genome Biol. 2002, 3, research0027.1-0027.16)

  As an example, the amino acid sequence of the human Adoplin-1 protein of the present invention is SEQ ID NO: 2, the amino acid sequence of the human Adoplin-2 protein is SEQ ID NO: 4, and the amino acid sequence of the human Adoplin-3 protein is SEQ ID NO: 6. Show.

  The Aβ protein production inhibitor of the present invention is a human Adoplin protein expression-suppressing substance or function-suppressing substance, and examples thereof include a human Adoplin-1 protein expression-suppressing substance or function-suppressing substance. Specifically, a protein expression inhibitor or function inhibitor consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4 or 6 can be shown.

  The “Adoplin protein” of the present invention can be prepared not only as a natural protein but also as a recombinant protein using gene recombination technology. Natural proteins can be prepared, for example, by a method using affinity chromatography using an antibody against Adoplin protein to an extract of cells (tissue) considered to be expressing Adoplin protein. On the other hand, recombinant protein can be prepared by culturing cells transformed with DNA encoding Adoplin protein.

  The “protein expression inhibitor” in the present invention is a substance that significantly suppresses (decreases) protein expression. The “expression suppression” of the present invention includes suppression of transcription of a gene encoding the protein and / or suppression of translation from a transcription product of the gene.

  Examples of the substance that suppresses the expression of the Adoplin protein of the present invention include substances that bind to the transcriptional regulatory region (eg, promoter region) of the Adoplin gene and suppress the transcription of the gene (such as a transcriptional repressing factor). .

  In the present invention, the measurement of the expression activity of Adoplin-1 protein can be easily carried out by those skilled in the art by methods well known to those skilled in the art, such as Northern blotting and Western blotting.

  Further, a substance that suppresses the function of Adoplin protein usually refers to a substance that can significantly suppress (inhibit) the function of Adoplin protein.

  Examples of the function (activity) possessed by the Adoplin protein include binding activity with the PS1 protein.

In a preferred embodiment of the present invention, the present invention relates to a β amyloid protein production inhibitor, wherein the Adoplin protein expression inhibitor is a compound selected from the group consisting of the following (a) to (c).
(A) an antisense nucleic acid for the transcription product of the Adoplin gene or a part thereof (b) a nucleic acid having a ribozyme activity that specifically cleaves the transcription product of the Adoplin gene (c) has an inhibitory action by the RNAi effect on the expression of the Adoplin gene Nucleic acid

  The “nucleic acid” in the present invention means RNA or DNA. As a method for inhibiting the expression of a specific endogenous gene, a method using an antisense technique is well known to those skilled in the art. There are several factors as described below for the action of the antisense nucleic acid to inhibit the expression of the target gene. Inhibition of transcription initiation by triplex formation, inhibition of transcription by hybridization with a site where an open loop structure was locally created by RNA polymerase, inhibition of transcription by hybridization with RNA undergoing synthesis, intron and exon Splicing inhibition by hybridization at splice point, splicing inhibition by hybridization with spliceosome formation site, inhibition of translocation from nucleus to cytoplasm by hybridization with mRNA, hybridization with capping site and poly (A) addition site Inhibition of splicing, translation initiation inhibition by hybridization with the translation initiation factor binding site, translation inhibition by hybridization with the ribosome binding site in the vicinity of the initiation codon, peptation by hybridization with the mRNA translation region and polysome binding site Elongation inhibition of de chain, and gene expression inhibition by hybrid formation at sites of interaction between nucleic acids and proteins, and the like. In this way, antisense nucleic acids inhibit the expression of target genes by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Shinsei Kagaku Kenkyu 2 Lecture and Expression of Nucleic Acid IV Gene, Japan Biochemical) (Academic Society, Tokyo Chemical Doujin, 1993, 319-347).

  The antisense nucleic acid used in the present invention may inhibit the expression of the Adoplin gene by any of the actions described above. As one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the Adoplin gene mRNA is designed, it is considered effective for inhibiting gene translation. In addition, a sequence complementary to the coding region or the 3 ′ untranslated region can also be used. Thus, the nucleic acid containing the antisense sequence of the sequence of the non-translated region as well as the translated region of the Adoplin gene is also included in the antisense nucleic acid used in the present invention. The antisense nucleic acid used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The nucleic acid thus prepared can be transformed into a desired animal by using a known method. The sequence of the antisense nucleic acid is preferably a sequence complementary to the endogenous gene or a part thereof possessed by the animal to be transformed, but it is not completely complementary as long as the gene expression can be effectively suppressed. May be. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcript of the target gene. In order to effectively suppress the expression of a target gene using an antisense nucleic acid, the length of the antisense nucleic acid is at least 15 bases, preferably 100 bases or more, more preferably 500 bases or more.

  Inhibition of the expression of the Adoplin gene can also be carried out using a ribozyme or a DNA encoding a ribozyme. A ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities, but research focusing on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes that cleave RNA in a site-specific manner. Some ribozymes have a size of 400 nucleotides or more, such as group I intron type and M1 RNA contained in RNase P, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type. (Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191.)

  For example, the self-cleaving domain of the hammerhead ribozyme cleaves 3 ′ of C15 in the sequence G13U14C15, but base pairing between U14 and A9 is important for its activity, and instead of C15, A15 or U15 However, it has been shown that it can be cleaved (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.). By designing a ribozyme whose substrate binding site is complementary to the RNA sequence in the vicinity of the target site, it is possible to create a restriction RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA (Koizumi, M. et al., FEBS Lett, 1988, 239, 285., Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191., Koizumi, M. et al., Nucl Acids Res, 1989, 17, 7059 .).

  Hairpin ribozymes are also useful for the purposes of the present invention. This ribozyme is found, for example, in the minus strand of tobacco ring spot virus satellite RNA (Buzayan, JM., Nature, 1986, 323, 349.). It has been shown that hairpin ribozymes can also produce target-specific RNA-cleaving ribozymes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.). Thus, the expression of the gene can be inhibited by specifically cleaving the transcript of the Adoplin gene in the present invention using a ribozyme.

  Inhibition of endogenous gene expression can also be carried out by RNA interference (RNAi) using double-stranded RNA having the same or similar sequence as the target gene sequence. RNAi refers to a phenomenon in which, when a double-stranded RNA having the same or similar sequence as a target gene sequence is introduced into a cell, the expression of the introduced foreign gene and target endogenous gene are both inhibited. Although the details of the RNAi mechanism are not clear, it is thought that the first introduced double-stranded RNA is decomposed into small pieces, and becomes an indicator of the target gene in some form, so that the target gene is decomposed. The RNA used for RNAi need not be completely identical to the Adoplin gene or a partial region of the gene, but preferably has complete homology.

  In the present invention, the sequence of the polynucleotide constituting the antisense nucleic acid or RNAi nucleic acid for the Adoplin gene can be appropriately designed by those skilled in the art based on the DNA sequence of the Adoplin gene listed in the sequence listing of the present specification. Is possible.

  Furthermore, a DNA (vector) capable of expressing the double-stranded RNA of the present invention is also included in a preferred embodiment of the compound capable of suppressing the expression of the Adoplin gene of the present invention. The DNA (vector) capable of expressing the double-stranded RNA of the present invention is usually a DNA encoding one strand of the double-stranded RNA and a DNA encoding the other strand of the double-stranded RNA, Each DNA has a structure linked to a promoter so that it can be expressed. Those skilled in the art can easily prepare the above-described DNA of the present invention by general genetic engineering techniques. More specifically, the expression vector of the present invention can be prepared by appropriately inserting DNA encoding the RNA of the present invention into various known expression vectors.

The present invention also provides a β-amyloid protein production inhibitor, wherein the function inhibitor of Adoplin protein is the following compound (a) or (b).
(A) Antibody that binds to Adoplin protein (b) Low molecular weight compound that binds to Adoplin protein

  Antibodies that bind to Adoplin protein (anti-Adoplin antibodies) can be prepared by methods known to those skilled in the art. If it is a polyclonal antibody, it can obtain as follows, for example. A serum is obtained by immunizing a small animal such as a rabbit with a recombinant Adoplin protein expressed in a microorganism such as Escherichia coli or a partial peptide thereof as a fusion protein with natural Adoplin protein or GST. This is prepared by, for example, purification by ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column coupled with Adoplin protein or synthetic peptide, or the like. In the case of a monoclonal antibody, for example, an Adoplin protein or a partial peptide thereof is immunized to a small animal such as a mouse, and the spleen is removed from the mouse, and this is ground to separate the cells. Are fused using a reagent such as polyethylene glycol, and a clone producing an antibody that binds to the Adoplin protein is selected from the fused cells (hybridomas) formed thereby. Subsequently, the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, Adoplin protein, It can be prepared by purification with an affinity column coupled with a synthetic peptide.

  The form of the antibody of the present invention is not particularly limited, so long as it binds to the Adoplin protein of the present invention, in addition to the above polyclonal antibody and monoclonal antibody, human antibodies, humanized antibodies by genetic recombination, antibody fragments thereof, Antibody modifications are also included.

  The Adoplin protein of the present invention used as a sensitizing antigen for obtaining an antibody is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammal such as a mouse or a human, and particularly preferably a protein derived from a human. Human-derived proteins can be obtained using the gene sequences or amino acid sequences disclosed herein.

  In the present invention, the protein used as the sensitizing antigen may be a complete protein or a partial peptide of the protein. Examples of the partial peptide of protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of protein. As used herein, “antibody” means an antibody that reacts with the full length or fragment of a protein.

  In addition to immunizing non-human animals with antigens to obtain the above hybridomas, human lymphocytes such as human lymphocytes infected with EB virus are sensitized with proteins, protein-expressing cells or lysates thereof in vitro. Lymphocytes can be fused with human-derived myeloma cells having permanent mitotic activity, such as U266, to obtain hybridomas that produce desired human antibodies having binding activity to proteins.

  The antibody that binds to the Adoplin protein of the present invention may be used for the purpose of treating Alzheimer's disease, for example. When the obtained antibody is used for the purpose of administering it to the human body (antibody treatment), a human antibody or a human-type antibody is preferable in order to reduce immunogenicity.

  Furthermore, the present invention also includes a low molecular weight substance that binds to the Adoplin protein as a substance that can inhibit the function of the Adoplin protein. The low molecular weight substance that binds to the Adoplin protein of the present invention may be a natural or artificial compound. Usually, it is a compound that can be produced or obtained by using methods known to those skilled in the art. The compound of the present invention can be obtained by the screening method described later.

  In addition, the β amyloid (Aβ) protein production inhibitor provided by the present invention inhibits (suppresses) the Aβ protein production reaction using amyloid precursor protein (APP) as a substrate by γ-secretase, and thus has a therapeutic effect on Alzheimer's disease. Is expected to have

  Therefore, the present invention relates to a therapeutic agent (pharmaceutical composition) for Alzheimer's disease containing the β amyloid protein production inhibitor of the present invention as an active ingredient.

  The present invention also provides a screening method for a therapeutic agent for Alzheimer's disease (a screening method for candidate compounds for a therapeutic agent for Alzheimer's disease).

  In a preferred embodiment of the screening method of the present invention, the method uses the expression level or activity of Adoplin protein as an index. That is, a compound that reduces the expression level or activity of Adoplin protein is expected to suppress the production of Aβ protein and, as a result, have a therapeutic effect on Alzheimer's disease.

  A preferred embodiment of the screening method of the present invention is a method characterized by selecting a compound that decreases the expression level or activity of the Adoplin protein from a test sample.

Moreover, in the preferable aspect of this invention, it is a screening method including the following processes (a)-(c).
(A) A step of contacting a test compound with a cell expressing Adoplin protein (b) A step of measuring the expression level of Adoplin protein in the cell (c) When measured in the absence of the test compound (test The step of selecting a compound that reduces the expression level compared to the case of not contacting the compound)

  In the above method of the present invention, first, a test compound is brought into contact with a cell expressing the Adoplin protein. The “cell” used in the present method is not particularly limited, but is preferably a human-derived cell. “Adoplin protein-expressing cell” refers to a cell expressing an endogenous Adoplin protein or a cell into which a gene encoding an exogenous Adoplin protein has been introduced and expressing the gene. it can. A cell in which a gene encoding an exogenous Adoplin protein is expressed can be usually prepared by introducing an expression vector into which a gene encoding an Adoplin protein is inserted into a host cell. The expression vector can be prepared by general genetic engineering techniques.

  There is no restriction | limiting in particular as a test compound used for the screening method of this invention. For example, natural compounds, organic compounds, inorganic compounds, proteins, peptides and other single compounds, as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts Products, plant extracts and the like, but are not limited thereto.

  In addition, these test compounds can be appropriately labeled as necessary. Examples of the label include a radiolabel and a fluorescent label.

  The “contact” of the test compound to the cell expressing the Adoplin protein gene is usually performed by adding the test compound to the culture medium of the cell expressing the Adoplin protein, but is not limited to this method. When the test compound is a protein or the like, “contact” can be performed by introducing a DNA vector expressing the protein into the cell.

  In this method, the expression level of Adoplin protein in cells expressing Adoplin protein is then measured. The expression level can be easily measured by those skilled in the art and can be appropriately performed by a general method such as Northern blotting or Western blotting. For example, it is also possible to measure using a label attached to a test compound brought into contact with a cell expressing Adoplin protein as an index.

  In the present invention, “expression” means any of transcription from a gene encoding a protein (generation of mRNA) or translation from a transcription product of the gene.

  In this method, a compound that decreases the expression level is then selected as compared to the case where the test compound is not contacted (control). The compound thus selected becomes a candidate compound for the treatment of Alzheimer's disease.

Moreover, in the preferable aspect of this invention, the screening method of the Alzheimer's disease therapeutic agent containing the process of the following (a)-(c) is provided.
(A) a step of bringing a test compound into contact with a cell or cell extract containing DNA having a structure in which a transcriptional regulatory region of the Adoplin gene and a reporter gene are functionally linked, and (b) measuring the expression level of the reporter gene (C) a step of selecting a compound that reduces the expression level as compared with the case where measurement is performed in the absence of the test compound (when the test compound is not contacted).

  In this method, first, a test compound is brought into contact with a cell or cell extract containing DNA having a structure in which a transcriptional regulatory region of an Adoplin gene and a reporter gene are functionally linked. Here, “functionally linked” means that the transcriptional regulatory region of the gene is bound to the reporter gene so that expression of the reporter gene is induced by binding of the transcription factor to the transcriptional regulatory region of the Adoplin gene. It means doing. Therefore, even when the reporter gene is bound to another gene and forms a fusion protein with another gene product, the transcription factor binds to the transcriptional regulatory region of the gene, Any expression that is induced is included in the meaning of “functionally linked”. Based on the cDNA base sequence of the Adoplin gene, those skilled in the art can obtain the transcriptional regulatory region of the gene present in the genome by a well-known method.

  The reporter gene used in the present method is not particularly limited as long as its expression can be detected, and examples thereof include a CAT gene, a lacZ gene, a luciferase gene, and a GFP gene. Examples of the “cell containing DNA having a structure in which the transcriptional regulatory region of the Adoplin gene and the reporter gene are functionally linked” include, for example, a vector having a structure in which the transcriptional regulatory region of the Adoplin gene and the reporter gene are functionally linked. Examples include introduced cells. Those skilled in the art can produce the above-mentioned vector by a general genetic engineering technique. Introduction of the vector into the cells can be carried out by a general method such as calcium phosphate precipitation, electric pulse perforation, lipophetamine, microinjection and the like. “Cells containing DNA having a structure in which a transcriptional regulatory region of an Adoplin gene and a reporter gene are functionally linked” also include cells in which the structure is inserted into a chromosome. Insertion of a DNA structure into a chromosome can be performed by methods generally used by those skilled in the art, for example, random integration or gene transfer methods utilizing homologous recombination.

  “A cell extract containing DNA having a structure in which a transcriptional regulatory region of an Adoplin gene and a reporter gene are functionally linked” refers to, for example, a cell extract contained in a commercially available in vitro transcription translation kit, Examples include those to which DNA having a structure in which a transcriptional regulatory region and a reporter gene are functionally linked is added.

  The “contact” in the present method includes adding a test compound to a culture solution of “a cell containing DNA having a structure in which a transcriptional regulatory region of an Adoplin gene and a reporter gene are functionally linked” or containing the DNA. Although it can carry out by adding a test compound to the commercially available cell extract of this, it is not limited to these methods. When the test compound is a protein, for example, contact can be performed by introducing a DNA vector expressing the protein into the cell.

  In this method, the expression level of the reporter gene is then measured. The expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of the reporter gene. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting acetylation of chloramphenicol by the gene product. When the reporter gene is a lacZ gene, by detecting the color development of the dye compound catalyzed by the gene expression product, and when the reporter gene is a luciferase gene, the fluorescent compound catalyzed by the gene expression product By detecting fluorescence, and in the case of a GFP gene, the expression level of the reporter gene can be measured by detecting fluorescence due to the GFP protein.

  In this method, a compound that lowers the measured expression level of the reporter gene is then selected as compared to the case where it is measured in the absence of the test compound. The compound thus selected is expected to have a therapeutic effect on Alzheimer's disease.

  Another aspect of the present invention is a method for screening a therapeutic agent for Alzheimer's disease using the activity of a protein encoded by the Adoplin gene as an index.

The present invention provides a method for screening an Alzheimer's disease therapeutic drug, comprising the following steps (a) to (c).
(A) a step of contacting Adoplin protein, or a cell or cell extract expressing the protein, with a test compound (b) a step of measuring the activity of the protein (c) measurement in the absence of the test compound A step of selecting a compound that reduces the activity of the protein compared to the case (when the test compound is not contacted)

  Specific examples of the activity of the protein include binding activity (interaction activity) with PS1. The activity can be appropriately measured by those skilled in the art by a known method such as a conjugate immunoprecipitation experiment. Adoplin protein is considered to be involved in PS1 γ-secretase activity through interaction with PS1. Therefore, the activity in the above method can be appropriately evaluated (measured) using, for example, the γ-secretase activity of PS1 as an index. The γ-secretase activity can be evaluated, for example, by measuring the amount of Aβ produced using APP as a substrate.

  Furthermore, the screening method of the present invention can be carried out using the interaction between Adoplin protein and PS1 as an index. Compounds that reduce the interaction activity between Adoplin and PS1 are expected to inhibit Aβ production and become candidate compounds for Alzheimer's disease therapeutics.

Therefore, in a preferred embodiment of the present invention, the present invention relates to a method for screening a therapeutic agent for Alzheimer's disease comprising the following steps (a) to (c).
(A) A step of bringing Adoplin protein and PS1 protein into contact with a test compound (b) A step of measuring interaction activity (binding activity) between Adoplin protein and PS1 protein (c) In the absence of the test compound Step of selecting a compound that reduces the interaction activity compared to the case of measurement (when the test compound is not contacted)

  In the above method, the “contact” in step (a) can be carried out, for example, by adding a test compound to a culture solution of cells co-expressing Adoplin and PS1.

  Usually, detection of the interaction activity between Adoplin protein and PS1 protein can be easily performed by those skilled in the art, and the measurement of the interaction activity in the above step (b) is performed by a general method, for example, immune co-operation. It is possible to implement appropriately using a sedimentation method or the like. Specifically, the interaction activity can be evaluated (measured) by the method described in Examples described later.

  In addition, the Adoplin protein and PS1 protein used in the above method are preferably wild-type proteins that do not contain mutations. However, as long as they have interaction activity, some amino acid sequences of these proteins are substituted or missing. It may be a lost protein (polypeptide).

  The Aβ protein production inhibitor or Alzheimer's disease therapeutic agent of the present invention may be mixed with a physiologically acceptable carrier, excipient, diluent or the like and administered orally or parenterally as a pharmaceutical composition. it can. As oral preparations, dosage forms such as granules, powders, tablets, capsules, solvents, emulsions or suspensions can be used. As the parenteral preparation, a dosage form such as an injection, an infusion, an external medicine, or a suppository can be selected. Examples of injections include subcutaneous injections, intramuscular injections, intraperitoneal injections, and the like. Examples of the medicine for external use include a nasal administration agent or an ointment. The preparation technique of the above dosage form so as to include the differentiation-inducing agent of the present invention which is the main component is known.

  For example, a tablet for oral administration can be produced by adding an excipient, a disintegrant, a binder, a lubricant and the like to the differentiation-inducing agent of the present invention, and mixing and compression-molding. As the excipient, lactose, starch, mannitol or the like is generally used. As the disintegrant, calcium carbonate, carboxymethyl cellulose calcium and the like are generally used. As the binder, gum arabic, carboxymethylcellulose, or polyvinylpyrrolidone is used. As the lubricant, talc, magnesium stearate and the like are known.

  The tablet containing the differentiation-inducing agent according to the present invention can be coated with a known coating for masking or enteric preparation. As the coating agent, ethyl cellulose, polyoxyethylene glycol, or the like can be used.

  The injection can be obtained by dissolving the differentiation inducer of the present invention, which is the main component, together with an appropriate dispersant, or dissolving or dispersing in a dispersion medium. Depending on the selection of the dispersion medium, either a water-based solvent or an oil-based solvent can be used. In order to use an aqueous solvent, distilled water, physiological saline, Ringer's solution, or the like is used as a dispersion medium. In the oil solvent, various vegetable oils and propylene glycol are used as a dispersion medium. At this time, a preservative such as paraben may be added as necessary. In addition, known isotonic agents such as sodium chloride and glucose can be added to the injection. Further, a soothing agent such as benzalkonium chloride or procaine hydrochloride can be added.

  Further, the Aβ protein production inhibitor or the Alzheimer's disease therapeutic agent of the present invention can be used as an external preparation by making it into a solid, liquid, or semisolid composition. About a solid or liquid composition, it can be set as an external preparation by setting it as the composition similar to what was described previously. A semi-solid composition can be prepared by adding a thickener to an appropriate solvent as required. As the solvent, water, ethyl alcohol, polyethylene glycol, or the like can be used. As the thickener, bentonite, polyvinyl alcohol, acrylic acid, methacrylic acid, or polyvinylpyrrolidone is generally used. A preservative such as benzalkonium chloride can be added to the composition. Also, a suppository can be obtained by combining an oily base material such as cacao butter or an aqueous gel base material such as a cellulose derivative as a carrier.

  The necessary amount of the Aβ protein production inhibitor or the Alzheimer's disease therapeutic agent of the present invention is administered to mammals including humans within the safe dosage range. The dosage of the drug of the present invention can be appropriately determined finally based on the judgment of a doctor or veterinarian in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, and the like.

  The “therapeutic agent” in the present invention includes a drug having a preventive effect in addition to a drug having a therapeutic effect. That is, in the present invention, a drug containing the β-amyloid protein production inhibitor of the present invention as an active ingredient can also be called an Alzheimer's disease onset inhibitor.

  Furthermore, the present invention relates to a method for preventing or treating Alzheimer's disease, which comprises the step of administering the Aβ protein production inhibitor or the Alzheimer's disease therapeutic agent of the present invention to an individual (for example, a patient). The administration method is not particularly limited, and examples thereof include intravenous administration, arterial administration, subcutaneous administration, and oral administration.

  Furthermore, the present invention relates to the use of a compound that suppresses the expression of the Adoplin gene or a compound that inhibits the function of the protein encoded by the gene for the production of an Aβ protein production inhibitor or a therapeutic agent for Alzheimer's disease.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not restrict | limited by these Examples.

[Example 1] Analysis of Adoplin gene family PS1 genes of human wild type and mutant type (I143T, G384A) are incorporated into expression vector pCEP4 and introduced into human neuroblastoma cell SH-SY5Y for stable expression Got the stock.
Differential display analysis was performed as follows using a Differential Display Kit (Takara).
Total RNA was extracted from the above cell line and purified by DNase treatment. Using this as a template, reverse transcription reaction was performed using 9 types of downstream primers (T11VV) to synthesize cDNA. These were combined with the above oligo (dT) primer and 24 types of upstream primers, and PCR was performed at a low annealing temperature of 40 ° C. ³⁵ S-dATP was used for labeling. The obtained PCR products were separated by polyacrylamide gel electrophoresis and detected by autoradiography.
As a result of analysis with attention to reproducibility, four types of bands in which expression was increased or decreased in two types of mutant PS1-expressing cells were obtained compared to wild-type PS1-expressing cells. These bands were excised from the gel, and DNA fragments obtained by PCR reamplification were subcloned into a plasmid (pUC18) to determine the sequence.
We examined changes in mRNA expression by Northern blot analysis using the DNA fragments obtained from the four genes obtained as a probe. Could not be confirmed. One of the two confirmed species was considered an unknown gene.

In order to obtain a full-length cDNA of this unknown gene, screening of a human brain cDNA library was performed by a plaque hybridization method. As a result, a gene (Adoplin-1) encoding a protein consisting of 153 amino acids could be isolated.
This sequence was registered in the database as Accession Nos. AB064959 and AB064960.
As a result of database search, the presence of a gene homologous to Adoplin-1 was suggested, and this gene was similarly isolated by cDNA library screening. This was named Adoplin-2 (Accession No AB064961).
It was also found that a third homologous gene was present (ORMDL-3, Accession No AF395708), and the present inventors decided to paraphrase this as Adoplin-3.
Adoplin-1 was shown to form a gene family together with two highly homologous homologs, Adoplin-2 and 3.

  These base sequences are shown in FIG. 1, and the amino acid sequences are shown in FIG. Further, the base sequence of Adoplin-1 is SEQ ID NO: 1, the amino acid sequence of the protein encoded by the base sequence is SEQ ID NO: 2, the base sequence of Adoplin-2 is SEQ ID NO: 3, and the base sequence The amino acid sequence of the encoded protein is described in SEQ ID NO: 4, the base sequence of Adoplin-3 is described in SEQ ID NO: 5, and the amino acid sequence of the protein encoded by the base sequence is described in SEQ ID NO: 6.

[Example 2] Expression of Adoplin in cells and human tissues Northern expression using mRNA extracted from each cell for expression of Adoplin-1, Adoplin-2, Adoplin-3 mRNA in wild-type and mutant PS1-expressing SH-SY5Y cells Investigated by blotting. Adoplin-1 mRNA was reduced to about 1/3 of wild type cells in mutant cells. Adoplin-2 and Adoplin-3 also showed a downward trend in mutant cells (FIG. 3).
The mRNA expression of Adoplin-1, Adoplin-2 and Adoplin-3 in human tissues was similarly analyzed by Northern blot. Specifically, according to a conventional method using each cDNA as a probe, a ³² P-labeled probe was hybridized with human multiple tissue Northern blots (Clontech) and detected with an image analyzer (BAS5000).
As a result, these three genes were found to be expressed in various organs including the brain (FIG. 4).

[Example 3] Adoplin expression in membrane fractions of cultured cells and brain tissue Homogenize cultured cells (SH-SY5Y cells, SH-SY5Y cells that stably express Adoplin-1, Adplin-2) and human brain tissue with Tris buffer The membrane fraction after ultracentrifugation was solubilized with RIPA buffer. The protein was immunoprecipitated with Adoplin C-terminal antibody, and the precipitate was analyzed by Western blot using the same antibody.
As a result, it was confirmed that Adoplin protein was expressed as a 17 kDa membrane protein in cultured cells and human brain extract (FIG. 5).

[Example 4] Expression analysis of Adoplin in brain tissue Expression of Adoplin-1 in cells and in human brain was analyzed by immunostaining.
First, SH-SY5Y cells that stably express Adoplin-1 were further transfected with wild-type PS1 to prepare a cell line that co-expresses Adoplin-1 and PS1. The cells were double-stained with Adoplin antibody and monoclonal PS1 antibody and observed with a confocal laser microscope. As a result, it was shown that the localization of both Adoplin-1 and PS1 proteins almost coincided (FIG. 6).
In addition, brain sections of human cerebral cortex (normal) were immunostained with Adoplin antibody. As a result, neurons were stained positively, and Adoplin was found to be expressed mainly in neurons. In the Alzheimer's disease brain, Adoplin immunoreactivity was also observed in degenerative neurites surrounding senile plaques in addition to nerve cells (FIG. 7).

[Example 5] Protein interaction analysis by conjugate immunoprecipitation
Using human SH-SY5Y cell line co-expressing Adoplin-1 and PS1, the interaction between Adoplin and PS1 was examined by coupled immunoprecipitation experiments.
The cell membrane fraction was solubilized with a buffer containing 1% CHAPSO, and fractionated according to the size of the molecular weight by glycerol density gradient centrifugation. Each fraction was immunoprecipitated with Adoplin antibody and the precipitate was Western blotted with PS1 antibody.
As a result, Western blot S1, C-terminal and N-terminal fragments were coprecipitated with Adoplin in the high molecular weight fraction. This indicates that PS1 (or a fragment thereof) interacts with Adoplin. At the same time, PEN-2, one of the components of β-catenin and PS1 complex, was found to coprecipitate with Adoplin (FIG. 8).

[Example 6] Effect of PS1 on γ-secretase activity by suppressing Adoplin expression
We investigated the effect of suppression of Adoplin expression on γ-secretase activity of PS1 by RNA interference experiments.
SiRNA (short interfering RNA) specific to Adoplin-1, Adoplin-2, and Adoplin-3 was simultaneously introduced into Hela cells using oligofectamine reagent (Invitorgen). Non-silencing siRNA and Nicastrin-specific siRNA were used as controls.

First, it was confirmed by Western blot that Adoplin protein was significantly reduced by Adoplin siRNA. At that time, the expression of endogenous PS1 and Nicastrin was examined. A slight decrease in endogenous PS1 was observed with a decrease in Adoplin. In addition, a significant decrease in mature Nicastrin was also observed (FIG. 9 left). This result suggests that Adoplin is necessary for maturation and stabilization of PS1 and Nicastrin as proteins.
Next, when APP CTFβ, the precursor protein of amyloid β protein, was transiently transfected into cells treated with Adoplin siRNA, and the amount of APP CTF was examined by Western blot, the amount of APP CTF was found in cells treated with Adoplin siRNA. Was significantly increased (FIG. 9 right). This suggests that APP CTF accumulated due to a decrease in γ-secretase activity.
In addition, when Notch receptor partial protein NotchΔE was expressed in similar cells, accumulation of NotchΔE was observed in cells treated with Adoplin siRNA (right in FIG. 9). This is also data supporting a decrease in γ-secretase activity.

  From the above results, it was shown that when Adoplin expression was inhibited by RNA interference, γ-secretase activity was significantly reduced.

FIG. 1 is a diagram showing the base sequences of Adoplin-1, 2, and 3 side by side. FIG. 2 is a diagram showing the amino acid sequences of Adoplin-1, 2, and 3 side by side. FIG. 3 is a photograph showing the results of analysis of the expression of Adoplin-1, 2, and 3 mRNA in wild-type and mutant PS1-expressing SH-SY5Y cells by Northern blotting using mRNA extracted from various cells. FIG. 4 is a photograph showing the results of analyzing the expression of Adoplin-1, 2, 3 mRNA in human tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas) by Northern blotting. . FIG. 5 is a photograph showing the results of Western blot analysis of Adoplin protein expression in cultured cells (SH-SY5Y cells, SH-SY5Y cells stably expressing Adoplin-1, Adoplin-2) and human brain tissue. . FIG. 6 is a photograph showing the results of immunohistochemical analysis of the localization of Adoplin-1 protein and PS1 protein in SH-SY5Y cells. The photograph on the left is stained with Adoplin-1 antibody, the photograph on the center is stained with PS1 antibody, and the photograph superimposed on the right is the photograph on the right. FIG. 7 is a photograph showing the results of immunohistochemical analysis of the localization of Adoplin-1 protein and PS1 protein in human cerebral cortex. The upper photo is the normal brain, and the lower photo is the Alzheimer's brain. The photograph on the left is stained with an anti-Adoplin-1 antibody, the photograph on the center is stained with an anti-amyloid β antibody, and these are superimposed on the right. FIG. 8 shows that the membrane fraction of human SH-SY5Y cells co-expressing Adoplin-1 and PS1 was fractionated by glycerol density gradient centrifugation, immunoprecipitated with Adoplin antibody, and then the precipitate was PS1 antibody, β-catenin antibody, It is a photograph showing the results of Western blotting with PEN-2 antibody or Adoplin antibody. FIG. 9 is a photograph showing the results of confirming the effect of Adoplin RNAi by Western blot. The left shows HeLa cells treated with Non-silencning siRNA, Nicastrin siRNA or Adoplin siRNA. The right shows HeLa cells that were treated with Non-silencning siRNA or Adoplin siRNA and then transfected with APP CTF or NotchΔE.

Claims (9)

  A β-amyloid protein production inhibitor comprising an expression inhibitor or function inhibitor of Adoplin protein. The β amyloid protein production inhibitor according to claim 1, wherein the expression inhibitor of Adoplin protein is a compound selected from the group consisting of the following (a) to (c).
(A) an antisense nucleic acid for the transcription product of the Adoplin gene or a part thereof (b) a nucleic acid having ribozyme activity that specifically cleaves the transcription product of the Adoplin gene (c) has an inhibitory action by the RNAi effect on the expression of the Adoplin gene Nucleic acid The β amyloid protein production inhibitor according to claim 1, wherein the function inhibitor of Adoplin protein is the following compound (a) or (b):
(A) Antibody that binds to Adoplin protein (b) Low molecular weight compound that binds to Adoplin protein   A therapeutic agent for Alzheimer's disease comprising the β-amyloid protein production inhibitor according to any one of claims 1 to 3 as an active ingredient.   A method for screening a therapeutic agent for Alzheimer's disease, comprising selecting a compound that decreases the expression level or activity of Adoplin protein. A screening method for an Alzheimer's disease therapeutic drug, comprising the following steps (a) to (c).
(A) a step of bringing a test compound into contact with a cell expressing Adoplin protein (b) a step of measuring the expression level of Adoplin protein in the cell (c) compared with a case where measurement is performed in the absence of the test compound And selecting a compound that reduces the expression level A screening method for a therapeutic agent for Alzheimer's disease, comprising the following steps (a) to (c):
(A) a step of bringing a test compound into contact with a cell or cell extract containing DNA having a structure in which a transcriptional regulatory region of the Adoplin gene and a reporter gene are functionally linked, and (b) measuring the expression level of the reporter gene (C) a step of selecting a compound that reduces the expression level as compared with the case of measuring in the absence of the test compound A screening method for a therapeutic agent for Alzheimer's disease, comprising the following steps (a) to (c):
(A) a step of contacting Adoplin protein, or a cell or cell extract expressing the protein, with a test compound (b) a step of measuring the activity of the protein (c) measurement in the absence of the test compound Selecting a compound that reduces the activity of the protein compared to the case A screening method for an Alzheimer's disease therapeutic drug, comprising the following steps (a) to (c).
(A) a step of bringing Adoplin protein and PS1 protein into contact with a test compound (b) a step of measuring an interaction activity between Adoplin protein and PS1 protein (c) a case of measurement in the absence of the test compound A step of selecting a compound that reduces the interaction activity in comparison