JP2007284351A - Substance inhibiting aggregation of amyloid protein and action thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To inhibit the aggregation of an amyloid protein and to inhibit/ameliorate symptoms of Alzheimer's diseases. <P>SOLUTION: A drug comprising a polypeptide selected from the group consisting of (1) an L-PGDS (lipocalin-type prostaglandin D synthase), (2) a mutant of the L-PGDS in which at least one cysteine residue of the L-PGDS is substituted with alanine or serine, (3) β-lactoglobulin and (4) a substance selected from the group consisting of estrogen, glucocorticoid, interleukin-1β and thyroid hormone and enhancing expression of the L-PGDS protein as an active ingredient is administered. The polypeptide binds to the β-amyloid protein and inhibits the aggregation thereof. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a drug that suppresses / ameliorates symptoms of Alzheimer's disease. More specifically, the present invention relates to a drug that suppresses aggregation of amyloid protein and suppresses / ameliorates symptoms of Alzheimer's disease.

With the advent of an aging society, the number of dementia patients is increasing, and it is said that about 5% of those over 65 years old are dementia patients. Alzheimer's disease, a neurodegenerative disease, is an important disease that accounts for most dementia in old age. It is. It is thought that the first stage of the pathogenesis is that Aβ protein has a sheet structure and aggregates and deposits in the brain parenchyma, and subsequently causes senile plaque formation and neurogenic changes in neurons. In recent years, administration of Aβ vaccine, anti-Aβ antibody, and β sheet breaker has been considered in order to inhibit the aggregation / deposition of Aβ protein (Hock C et al. Antibodies against beta-amyloid slow cognitive decline in Alzheimer's disease.
Neuron. 2003 38: 547-54., Janus C et al. A beta peptide immunization reduces
behavioral impairment and plaques in a model of Alzheimer's disease.Nature.
2000 408: 979-82., Soto C et al. Beta-sheet breaker peptides inhibit
fibrillogenesis in a rat brain model of amyloidosis: implications for Alzheimer's therapy. Nat Med. 1998 4: 822-6.). However, both are non-physiological substances and may have side effects.
Neuron. 2003 38: 547-554 Nature. 2000 408: 979-982 Nat Med. 1998 4: 822-826

An object of the present invention is to provide a method for suppressing aggregation and amelioration of Alzheimer's disease symptoms by suppressing aggregation of amyloid protein.

The inventor who achieves the above object has conducted extensive research and has completed the present invention based on the following findings.
1) L-PGDS is deposited on amyloid plaques in an autopsy brain of an Alzheimer's disease patient and an Alzheimer's disease model mouse.
2) Lipocalin-type plastaglandin D synthase (L-PGDS) exhibits a strong affinity for human amyloid protein, and further exhibits a strong aggregation inhibitory action in an aggregation test by thioflavin T (ThT) assay.
3) The effect of 2) is caused not only by the lipocalin-type plastaglandin D synthase but also by its mutants. Such mutants include those in which the cysteine residue in the active site of plastaglandin D synthase is substituted.

That is, the present invention is a drug used for suppressing / ameliorating symptoms of Alzheimer's disease,
1) Lipocalin-type prostaglandin D synthase (L-PGDS);
2) A variant of L-PGDS in which at least one cysteine residue of L-PGDS is replaced by alanine or serine;
3) β-Lactoglobulin 4) The gist of the present invention is a drug containing, as an active ingredient, a compound selected from the group consisting of substances that enhance the expression of L-PGDS protein in vivo, particularly in the brain.

  According to the drug of the present invention, the symptoms of Alzheimer's disease can be suppressed and improved.

  The first embodiment of the drug used for suppressing or improving the symptoms of Alzheimer's disease of the present invention comprises L-PGDS as an active ingredient. L-PGDS is derived from a mammal, derived from human (SEQ ID NO: 1, the first to 21st amino acids are signal peptides) and from mouse (SEQ ID NO: 2, the first to 24th amino acids are signal peptides). And those derived from humans are particularly preferred.

In the second embodiment of the drug used for suppressing or improving the symptoms of Alzheimer's disease of the present invention, an L-PGDS mutant is used as an active ingredient. Preferably, the L-PGDS variant in the present invention is one in which at least one of the cysteine residues contained in L-PGDS is substituted with another amino acid residue, preferably alanine or serine.
1) A protein in which cysteines at positions 65 and 167 of human L-PGDS (SEQ ID NO: 1) are substituted with alanine or serine (hereinafter, may be abbreviated as “Ala1.3” and “Ser1.3”, respectively);
2) Proteins of human L-PGDS (SEQ ID NO: 1) in which cysteines at positions 89, 167 and 186 are substituted with alanine or serine (hereinafter abbreviated as “Ala2.3.4” and “Ser2.3.4”, respectively) There is)
3) Protein obtained by substituting the 65th cysteine of mouse L-PGDS (SEQ ID NO: 2) with alanine or serine (hereinafter sometimes abbreviated as “Ala1” and “Ser1”);
4) Proteins obtained by substituting the cysteines at positions 89 and 186 of mouse L-PGDS (SEQ ID NO: 2) with alanine or serine (hereinafter sometimes abbreviated as “Ala2.3” and “Ser2.3”, respectively)
It is.

  Mammalian-derived L-PGDS can be obtained from mammalian tissue, but it is convenient to produce it by genetic recombination techniques. The amino acid sequence of human or mouse-derived lipocalin-type prostaglandin D synthase (L-PGDS) is known. The amino acid sequence of human-derived L-PGDS is shown in SEQ ID NO: 1 (the amino acid residues 1 to 21 of SEQ ID NO: 1 are signal peptides). The amino acid sequence of L-PGDS derived from mouse is shown in SEQ ID NO: 2 (the amino acid residues 1 to 24 of SEQ ID NO: 1 are signal peptides). The nucleotide sequence of cDNA encoding L-PGDS is also known. The nucleotide sequence of cDNA encoding human-derived L-PGDS is shown in SEQ ID NO: 3 (nucleotide residues 1 to 63 of SEQ ID NO: 3 encode a signal peptide). The nucleotide sequence of cDNA encoding mouse-derived L-PGDS is shown in SEQ ID NO: 4 (nucleotide residues 1 to 72 of SEQ ID NO: 4 encode a signal peptide). Therefore, an expression vector that expresses recombinant L-PGDS in an appropriate host system can be constructed. Recombinant L-PGDS can be produced by transforming host cells with the obtained expression vector and culturing the transformant under conditions suitable for the expression of DNA encoding L-PGDS.

  An expression vector containing DNA encoding L-PGDS of the present invention can be constructed by methods known to those skilled in the art. A vector suitable for L-PGDS expression would have a promoter for transcription initiation immediately upstream of the DNA insertion site. Appropriate promoters are also known in the art and can be selected according to functional properties in the host cell. For example, an SV40 virus early gene promoter, a peptide chain elongation factor EF-1α promoter, a metallothionein gene promoter, a β-actin promoter, a CMV virus promoter, etc. are expressed in an animal cell system, and a T7 polymerase promoter or beta A galactosidase gene promoter or the like can be used for expression in bacteria and Escherichia coli. It is desirable that there is a transcription termination signal downstream of the insertion site of human hematopoietic PGD synthase DNA.

  Desirably, a selectable marker such as a drug resistance marker is present in the vector. Or you may transform simultaneously using the plasmid which codes drug resistance, such as an expression vector containing the polynucleotide which codes L-PGDS, and a separate antibiotic.

  In order to construct an expression vector, DNA encoding L-PGDS is inserted into an appropriate vector. Appropriate vectors are selected from those known in the art in view of promoters, transcription termination signals, selectable markers and other conditions. DNA vectors that can be used for the purpose of inserting L-PGDS cDNA, introducing it into cultured cells and expressing this cDNA, such as pKCR, pEF-BOS, CDM8, pCEV4 bovine papillomavirus DNA, etc. In Escherichia coli, pGEMEX, pUC and the like can be mentioned.

The cells that can be used for L-PGDS expression may be any cells that can replicate and can express DNA encoding L-PGDS. For example, prokaryotic microorganisms such as E. coli; Eukaryotic microorganisms such as cerevisiae and even mammalian cells are used. Tissue culture cells include avian or mammalian cells such as murine, rat and monkey cells. Selection and use methods of an appropriate host cell-vector system are known to those skilled in the art, and a system suitable for expression of cDNA encoding L-PGDS can be arbitrarily selected from them.
A desired protein can be obtained by culturing the transformed cells according to a conventional method. The medium used for the culture can be appropriately selected according to the properties of the host. For example, when the host is Escherichia coli, the LB medium or TB medium is appropriately selected. When the host is a mammalian cell, RPMI 1640 medium or the like is appropriately selected. Can be used.
Isolation and purification of the protein used in the present invention from the culture obtained by this culture can be performed by conventional methods. For example, the culture is subjected to various treatment operations utilizing physical and chemical properties of the protein. Can be used. Specifically, treatment with a protein precipitating agent, ultrafiltration, high performance liquid chromatography, centrifugation, electrophoresis, affinity chromatography and the like can be used alone or in combination. The obtained protein solution can be powdered by lyophilization if necessary. In lyophilization, stabilizers such as sorbitol, mannitol, dextrose, maltose, glycerol can be added.

In order to produce a mutant of L-PGDS, a site-directed mutagenesis method is preferably used. This method is well known, and kits for implementation (for example, QuickChange (registered trademark) Site-Directed Mutagenesis Kit manufactured by STRATAGENE) are commercially available.
In order to introduce a mutation by the site-directed mutagenesis method, a primer pair of about 30 to 40 bases containing a mismatch codon at a target site is synthesized, and Pfu polymerase is used with these primers as a template using a wild-type plasmid as a template. PCR amplify the entire plasmid. After completion of the PCR, the template plasmid and the synthesized mutant plasmid coexist in the reaction solution. Therefore, the DpnI endonuclease enzyme treatment is performed, and the template plasmid is decomposed and purified to purify the mutant plasmid.

  In the third aspect of the drug used for suppressing or improving the symptoms of Alzheimer's disease of the present invention, β-lactoglobulin is used as an active ingredient. β-lactoglobulin is a protein that is contained in milk and belongs to the lipocalin family. Commercially available β-lactoglobulin (for example, Sigma) can be used.

  In the fourth aspect of the drug used for suppressing or improving the symptoms of Alzheimer's disease of the present invention, a substance that enhances the expression of L-PGDS is used as an active ingredient. As a substance that enhances the expression of L-PGDS, estrogen (Otsuki M et al. Specific regulation of lipocalin-type prostaglandin D synthase in mouse heart by estrogen receptor beta. Mol Endocrinol. 2003 Sep; 17 (9): 1844-55 Epub 2003 Jun 26.), glucocorticoid (Garcia-Fernandez LF et al. Dexamethasone induces lipocalin-type prostaglandin D synthase gene expression in mouse neuronal cells. J Neurochem. 2000 Aug; 75 (2): 460-70.), Interleukin-1β (Fujimori K et al. Regulation of lipocalin-type prostaglandin D synthase gene expression by Hes-1 through E-box and interleukin-1 beta via two NF-kappa B elements in rat leptomeningeal cells.J Biol Chem. 2003 Feb 21; 278 (8): 6018-26. Epub 2002 Dec 17.), Thyroid hormone (Leone MG et al. Lipocalin type prostaglandin D-synthase: which role in male fertility Contraception. 2002 Apr; 65 (4): 293-5.). These substances are known to enhance the expression of L-PGDS in vivo, particularly in the brain.

  When the active ingredient of the drug of the present invention is a protein, the protein can be mixed and diluted with a carrier known per se and formulated as a liquid preparation, for example. The liquid preparation can be prepared, for example, using purified water, physiological saline, alcohols such as ethanol / propylene glycol / glycerin / polyethylene glycol, and solvents such as triacetin. The liquid thus prepared can be used by diluting, for example, a lactate Ringer's solution, a maintenance solution composed of an electrolyte solution for infusion, a postoperative recovery solution, a dehydration replenisher, a drip saline solution, or the like. Additives appropriately selected and used as a normal solution, for example, pH adjusting buffer (for example, phosphate buffer, borate buffer, citrate buffer, tartrate buffer, acetate buffer, etc.), isotonic Agents (eg, sorbitol, glycerin, polyethylene glycol, propylene glycol, glucose, sodium chloride, etc.) Such formulations further include pharmaceutically acceptable benzalkonium chloride, paraoxybenzoates, benzyl alcohol, parachlorometa Suitable antiseptic fungicides such as xinol, chlorcresol, phenethyl alcohol, sorbic acid or salts thereof, thimerosal, chlorobutanol, wetting agents such as talc, emulsifiers such as polyethylene glycol monooleate, dispersants, sulfites, hydrogen sulfites Add adjuvants such as stabilizers such as salt and metabisulfite It may be. It can also be said that one of the preferable dosage forms is to administer as a suspending agent using gum arabic, kaolin, methylcellulose, sodium carboxymethylcellulose or the like as a suspending agent.

  The administration route is preferably non-caliber administration, and includes, for example, intravenous administration, transcerebral spinal fluid administration, transarterial local administration by catheter, or surgical local administration. The dosage of the active ingredient of the present invention is 0.01 to 20.0 mg / kg / day, preferably 0.5 to 5 mg / kg / day.

  Even when the active ingredient of the drug of the present invention is a low molecular compound such as estrogen or glucocorticoid, the drug can be produced by mixing with a pharmaceutically acceptable carrier. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. As the administration route, either parenteral administration or oral administration can be used. Powders, pills, capsules and tablets for oral administration are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl It can be produced using a binder such as alcohol, hydroxypropylcellulose, gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin. The dosage of the active ingredient of the present invention is 0.001 to 10 mg / kg / day, preferably 0.01 to 1 mg / kg / day.

Furthermore, the present invention uses L-PGDS and mutant genes for gene therapy, and suppresses the aggregation / deposition of Aβ protein by locally expressing L-PGDS or mutants in the brain, thereby causing Alzheimer's disease. The method used for the prevention, suppression, and improvement of symptoms is provided.
The gene of L-PGDS of the present invention or a mutant thereof is incorporated into a vector and introduced into the brain. Examples of vectors used for gene therapy include retrovirus vectors, all virus vectors including adenovirus vectors, and liposome vectors. More specifically, a viral vector is a gene that uses a portion of the virus shell and part of the gene, cuts out the gene responsible for pathogenicity, and instead contains an L-PGDS gene that has been modified to be expressed in brain membranes or oligodendrocytes. is there. Liposome vectors are those in which L-PGDS genes modified to be expressed in brain membranes or oligodendrocytes are incorporated into microspheres made mainly of artificial lipid bilayer membranes (lipofection method). Usually, liposomes composed of phosphatidylserine are used. Instead of this negatively charged phospholipid, a positive cation called DOTMA (N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride), which makes it easier to produce more stable liposomes Ionic lipids have become commercially available. Positively charged liposomes adsorb negatively charged cells to the surface and fuse with the cell membrane to introduce DNA into the cells. This vector is administered by intravenous injection, arterial injection, intramuscular injection, intrathecal administration, or local administration.
EXAMPLES The present invention will be further described below with reference to examples, but it is obvious that the present invention is not limited to the examples.

Example 1
Production of human-derived lipocalin-type prostaglandin D synthase The human L-PGDS gene from Ala21 to C-terminus was obtained by PCR amplification from a cDNA library, the signal peptide was deleted, and the 67th to 3rd of the DNA sequence of SEQ ID NO: 3 'Up to the end was inserted into an expression vector pGEX-2T (Amersham Pharmacia Biotech), and Escherichia coli DH5α was transformed with the expression vector. pGEX-2T is an expression vector for inserting the target gene downstream of the glutathione-S-tospherase (GST) gene and expressing the target protein as a GST fusion protein. Subsequently, the vector was transformed into Escherichia coli BL21 strain. The E. coli strain transformed with the expression plasmid was cultured at 37 ° C. in LB medium (containing 50 μg / mL ampicillin), and then IPTG (isopropyl-1-thio-β-D-galactoside) was added at a final concentration of 0.4. To 0.6 mM to induce expression of a fusion protein of GST and L-PGDS, and further cultured for 4-6 hours.
After completion of the culture, E. coli was recovered from the culture solution, and proteins were extracted from the cells by ultrasonic disruption. Unnecessary substances were removed by centrifugation and then retained on a glutathione sepharose 4B column. The fusion protein retained on the column was digested with thrombin, and the L-PGDS fraction was eluted. Subsequently, purification was performed by gel filtration column chromatography to obtain a human-derived lipocalin type prostaglandin D synthase.

Example 2
Production of mouse-derived lipocalin-type prostaglandin D synthase In Example 1, the cDNA encoding human-derived L-PGDS was replaced with the cDNA encoding mouse-derived L-PGDS (from the 70th to the 3 'end of the DNA sequence of SEQ ID NO: 4). A mouse-derived lipocalin-type prostaglandin D synthase was obtained in the same manner as in Example 1 except that the above was changed.

Example 3
Manufacture of a mutant (Ala1.3) in which human L-PGDS 65 and 167th cysteines were substituted with alanine To obtain a mutant in which human L-PGDS Cys65 was substituted with alanine, A pair of synthetic oligomers containing a mismatch codon at the target site (Cys65) using the expression vector as a template:
5'-gcgttgtccatgGCCaagtctgtggtggcc-3 '(SEQ ID NO: 5)
5'-ggccaccacagacttGGCcatggacaacgc-3 '(SEQ ID NO: 6)
A Cys65Ala expression vector was prepared using the QuickChange Site-Directed Mutagenesis Kit manufactured by STRATAGENE.
Further, using the obtained expression vector as a template, a pair of synthetic oligomers containing a mismatch codon at the target site (Cys167):
5'-attcaccgccttcGCCaaggcccagggct-3 '(SEQ ID NO: 7)
5'-agccctgggccttGGCgaaggcggtgaat-3 '(SEQ ID NO: 8)
And a Cys65,167Ala expression vector was prepared using the QuickChange Site-Directed Mutagenesis Kit.

An expression vector in which Cys65 and 167 were substituted with alanine was transformed into Escherichia coli BL21 strain. The E. coli strain transformed with the expression plasmid was cultured at 37 ° C. in LB medium (containing 50 μg / mL ampicillin), and then IPTG (isopropyl-1-thio-β-D-galactoside) was added at a final concentration of 0.4. To 0.6 mM to induce expression of a fusion protein of GST and L-PGDS, and further cultured for 4-6 hours.
After completion of the culture, E. coli was recovered from the culture solution, and proteins were extracted from the cells by ultrasonic disruption. Unnecessary substances were removed by centrifugation and then retained on a glutathione sepharose 4B column. The fusion protein retained on the column was digested with thrombin, and the L-PGDS fraction was eluted. Subsequently, a human PGDS mutant (Ala1.3) was obtained by purification by gel filtration column chromatography and substituting cysteines of human L-PGDS 65 and 167 with alanine.

Example 4
Manufacture of a mutant (Ser1.3) in which 65th and 167th cysteines of human L-PGDS are replaced with serine In order to create a mutant in which Cys65 is replaced with serine, the expression vector obtained in Example 1 is used as a template. As a pair of synthetic oligomers containing a mismatch codon at the target site (Cys65) (a synthetic oligomer for Cys-65-Ser):
5'-gcgttgtccatgTCCaagtctgtggtggcc-3 '(SEQ ID NO: 9)
5'-ggccaccacagacttGGAcatggacaacgc-3 '(SEQ ID NO: 10)
A Cys65Ser expression vector was prepared using the QuickChange Site-Directed Mutagenesis Kit manufactured by STRATAGENE.
Further, using the obtained expression vector as a template, a pair of synthetic oligomers containing a mismatch codon at the target site:
5'-attcaccgccttcTCCaaggcccagggct-3 '(SEQ ID NO: 11)
5'-agccctgggccttGGAgaaggcggtgaat-3 '(SEQ ID NO: 12)
And the Cys65 and 167Ser expression vectors were prepared using the QuickChange Site-Directed Mutagenesis Kit.

An expression vector in which Cys65 and 167 were substituted with serine was transformed into Escherichia coli BL21 strain. The E. coli strain transformed with the expression plasmid was cultured at 37 ° C. in an LB medium (containing 50 μg / mL ampicillin), and then IPTG (isopropyl-1-thio-β-D-galactoside) was added at a final concentration of 0.4. To 0.6 mM to induce expression of a fusion protein of GST and L-PGDS, and further cultured for 4-6 hours.
After completion of the culture, E. coli was recovered from the culture solution, and proteins were extracted from the cells by ultrasonic disruption. Unnecessary substances were removed by centrifugation and then retained on a glutathione sepharose 4B column. The fusion protein retained on the column was digested with thrombin, and the L-PGDS fraction was eluted. Subsequently, purification was performed by gel filtration column chromatography to obtain a mutant (Ser1.3) in which the 65th and 167th human L-PGDS were substituted with serine.

Example 5
Production of mutant (Ala 2.3.4) in which cysteines at positions 89, 167 and 186 of human L-PGDS were substituted with alanine In Example 4, except that the design of the synthetic oligomer was changed, the same procedure as in Example 3 was performed. A mutant (Ala 2.3.4) in which cysteines at positions 89, 167 and 186 of human L-PGDS were substituted with alanine was produced.

Example 6
Production of a mutant (Ala1) in which the 65th cysteine of mouse L-PGDS is substituted with alanine
Using the mouse-derived L-PGDS expression plasmid obtained in Example 2 as a template, a mutant in which the 65th cysteine of mouse L-PGDS is substituted with alanine (Ala1) using the STRATAGENE QuickChange Site-Directed Mutagenesis Kit Was manufactured as follows.
A pair of synthetic oligomers containing a mismatch codon so that Cys65 is replaced with Ala
5'-gctgtattgtatatgGCAaagacagtggta-3 '(SEQ ID NO: 13)
5'-taccactgtcttTGCcatatacaatacagc-3 '(SEQ ID NO: 14)
Was used to PCR amplify the Cys65Ala mutant plasmid using the L-PGDS expression plasmid as a template. The obtained mutant expression plasmid was transformed into Escherichia coli BL21 strain, and a mutant (Ala1) in which the 65th cysteine of mouse L-PGDS was substituted with alanine was produced in the same manner as in Example 2.

Example 7
Production of a mutant (Ala2.3) in which cysteines at positions 89 and 186 of mouse L-PGDS were substituted with alanine In Example 6, except that the design of the synthetic oligomer was changed, mouse L-PGDS was prepared in the same manner as in Example 6. A mutant (Ala2.3) was prepared by substituting the 89th and 186th cysteines with alanine.

Example 8
Binding of lipocalin-type prostaglandin D synthase to amyloid plaques in Alzheimer's disease model mice. Autopsy brain of Alzheimer's disease patients and Alzheimer's disease model mice Tg2576. 274: 99-102.1996; Hsiao KK et al. Age-related CNS disorder and early death in transgenic FVB / N mice overexpressing Alzheimer amyloid precursor proteins. Neuron.15: 1203-18. 1995) -The relationship between plaque and L-PGDS was analyzed by immunohistochemical staining (see FIGS. 1A and 1B). L-PGDS was identified to bind to the amyloid plaque site in both the autopsy brain of Alzheimer's disease patients and the Alzheimer's disease model mice.

Example 9
Binding ability of L-PGDS to human amyloid beta protein Using the surface plasmon resonance method (BIAcore), the binding ability of L-PGDS recombinant protein to human amyloid beta 1-40 protein and 1-42 protein was analyzed ( (See FIG. 2). When human amyloid beta 1-40 and 1-42 proteins were previously bound to a sensor chip on which an L-PGDS recombinant protein had been immobilized, the dissociation constants were 88.4 nM and 65.2 nM, respectively. Indicated.

Example 10
L-PGDS and its variants against human amyloid beta 1-40 protein, and β-lactoglobilin aggregation inhibition ThT assay (Ban T et al., Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence. J Biol Chem 278: 16462-5. 2003), and L-PGDS (βTRACE) purified from human cerebrospinal fluid for aggregation of Aβ1-40 protein and a variant of human L-PGDS (Ala 2.3.4, Ala 1.3, The effects of Ala2.3, Ala1) and β-lactoglobulin were analyzed (see FIGS. 3A-C).
ThT is a fluorescent substance that specifically recognizes the cross-beta sheet structure, and it is known that the fluorescence intensity of ThT increases with the elongation of amyloid fibrils in vitro. Therefore, a mixed solution of phosphate buffer (50 mM, pH 7.5), NaCl (100 mM) and Aβ1-40 (50 μM) was prepared and kept at 37 ° C. At that time, a group in which similar concentrations of L-PGDS similar substances were mixed was also set. 5 μl of each was mixed with 1 ml of ThT (5 μM) and glycine buffer (50 mM, pH 8.5), and the fluorescence intensity was measured over time with a fluorometer (excitation wavelength 446 nm, fluorescence wavelength 490 nm).
Both L-BGDS (βTRACE) and its mutants, and β-lactoglobilin exhibited a strong aggregation inhibitory action.

It is a microscope picture which shows the coupling | bonding of L-PGDS to the elderly group of the autopsy brain of an Alzheimer's disease patient by an immunohistochemical staining method. It is a microscope picture which shows L-PGDS localized in the amyloid plaque of Alzheimer's disease model mouse | mouth Tg2576 by an immunohistochemical staining method. It is a graph which shows the binding ability of L-PGDS with respect to human amyloid beta 1-40 and 1-42 protein by the surface plasmon resonance method. It is a microscope picture which shows the inhibitory effect of Ala2,3,4 on the aggregation of A (beta) 1-40 protein by ThT assay. It is a graph which shows the dose-dependent inhibitory effect of Ala2.3.4 with respect to the aggregation of A (beta) 1-40 protein by ThT assay. It is a graph which shows the inhibitory effect of L-PGDS, Ala2.3.4, Ala1.3, Ala2.3, Ala1, and β-lactoglobulin on aggregation of Aβ1-40 protein by ThT assay.

Claims (8)

A drug used to suppress or improve the symptoms of Alzheimer's disease,
1) Lipocalin-type prostaglandin D synthase (L-PGDS);
2) A variant of L-PGDS in which at least one cysteine residue of L-PGDS is replaced by alanine or serine;
3) β-lactoglobulin; and 4) a substance that enhances the expression of L-PGDS protein selected from the group consisting of estrogen, glucocorticoid, interleukin-1β, thyroid hormone;
A drug comprising a polypeptide selected from the group consisting of as an active ingredient.   The drug according to claim 1, wherein the lipocalin-type prostaglandin D synthase (L-PGDS) is derived from human (SEQ ID NO: 1) or mouse (SEQ ID NO: 2). A variant of L-PGDS is
(1) a protein in which the 65th and 167th cysteines of human L-PGDS of SEQ ID NO: 1 are substituted with alanine or serine;
(2) a protein obtained by substituting the cysteines at positions 89, 167 and 186 of human L-PGDS of SEQ ID NO: 1 with alanine or serine;
(3) a protein in which the 65th cysteine of mouse L-PGDS of SEQ ID NO: 2 is substituted with alanine or serine; and (4) the 89th and 186th cysteines of mouse L-PGDS of SEQ ID NO: 2 are substituted with alanine or serine. Protein;
The drug according to claim 1 or 2, which is selected from the group consisting of:   The nucleic acid which codes L-PGDS and its variant of Claims 1-3 used for the gene therapy method with respect to suppression of the symptom of Alzheimer's disease, and improvement. A protein in which the 65th and 167th cysteines of human L-PGDS of SEQ ID NO: 1 are substituted with alanine or serine.   A protein obtained by substituting the cysteines at positions 89, 167 and 186 of human L-PGDS of SEQ ID NO: 1 with alanine or serine.   A protein in which the 65th cysteine of mouse L-PGDS of SEQ ID NO: 2 is substituted with alanine or serine.   A protein in which the 89th and 186th cysteines of mouse L-PGDS of SEQ ID NO: 2 are substituted with alanine or serine.

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