JPWO2007139120A1 - Amyloid β clearance promoter - Google Patents

Abstract

The present invention relates to a scavenger receptor protein, a protein having substantially the same activity as a scavenger receptor protein or a salt thereof, a DNA encoding a scavenger receptor protein, or a DNA comprising a base sequence complementary to the DNA, It is an amyloid β clearance promoter characterized by containing, as an active ingredient, DNA that contains a DNA that encodes a protein that hybridizes under stringent conditions and has substantially the same activity as the scavenger receptor protein. . The promoter is useful for clearance of amyloid β accumulated in the brain in Alzheimer's disease and the like.

Description

  The present invention relates to an amyloid β clearance promoter, and more particularly to an amyloid β clearance promoter comprising as an active ingredient a DNA containing a scavenger receptor protein or a DNA encoding the same.

Alzheimer's disease is a progressive neurodegenerative disease characterized by the accumulation of amyloid β in the brain, and its accumulation is considered to play an important role in the pathogenesis of Alzheimer's disease (Non-patent Document 1). In addition, it is considered that the cause of amyloid β-related diseases may be due to insufficient clearance of amyloid β by microglia, astrocytes, or the vascular system (Non-Patent Documents 2 to 4). Microglia and astrocytes are known to have the ability to clear amyloid β (Non-patent Documents 5 to 8). For example, cultured astrocytes of wild-type mice are amyloids of transgenic mouse models of Alzheimer's disease. It is known to contribute to the binding and clearance of amyloid β in β-accumulated fresh brain (Non-patent Document 8).
In addition, clearance of amyloid β by microglia, astrocytes and vascular system includes antibodies against amyloid β, TGF (transforming growth factor) β1, heat shock protein, apolipoprotein E (apoE), monoclonal antibody against amyloid β, and It is regulated by various substances including GM1 (ganglioside) (Non-Patent Documents 9 to 14).
Antibodies against amyloid β induce amyloid β plaque clearance by microglia through Fc (immunoglobulin) receptor-mediated phagocytosis and subsequent peptide degradation. (Non-patent document 9). These data demonstrate that amyloid β can be removed from the brain directly by glial cells, directly by blood vessel cells, or indirectly by the drainage channels of the perivascular interstitial fluid. These data also demonstrate that the effects of amyloid β clearance can be modulated. These results suggest that increased amyloid β clearance may be a promising therapeutic approach for reducing the pathogenesis mediated by amyloid β in Alzheimer's disease. Cell surface receptors such as scavenger receptors and related molecules may play a critical role in amyloid beta binding and clearance. However, the receptor assumed to be involved in the clearance of amyloid β has not yet been clearly identified (Non-Patent Documents 15 to 17).

Scavenger receptors with C-type lectins (SRCL; Non-Patent Documents 18, 19) are one of the family of scavenger receptors that include a coiled coil, collagen-like domain, and C-type lectin / sugar recognition domain (CRD). (Non-patent document 20). The major SRCL in humans is named human SRCL type I (hereinafter abbreviated as hSRCL-I). On the other hand, SRCL including a coiled coil excluding CRD and a collagen-like domain is referred to as human SRCL type II (hereinafter abbreviated as hSRCL-II) (Non-patent Document 18). Due to structural similarities, SRCL is considered to be closely related to the class A scavenger receptor (SR-A), which includes a coiled coil, collagen-like domain, and cysteine-rich domain (21). The ability of SRCL to bind to Gram-negative and Gram-positive bacteria as well as yeast strongly suggests a role for SRCL in host defense (Non-Patent Documents 18 and 19).
Tanzi RE et al., Cell, 2005, Volume 120 (No. 4), p. 545-555 Nicoll JA et al., Trends Mol. Med., 2003, Volume 9 (No. 7), p. 281-282 Tanzi RE et al., Neuron, 2004, Vol. 43 (No. 5), p. 605-608. Zlokovic BV et al., Journal of Neurochem., 2004, Vol. 89 (No. 4), p. 807-811 Guenette SY et al., Trends Mol. Med., 2003, Volume 9 (No. 7), p. 279-280 Guenette SY, Neuromolecular Med., 2003, Volume 4 (No. 3), p. 147-160 Paresce DM et al., Neuron, 1996, Vol. 17 (No. 3), p. 553-565 Wyss-Coray T et al., Nature Medicine, 2003, Volume 9 (No. 4), p. 453-457 Bard F et al., Nature Medicine (Nat. Med.), 2000, Vol. 6 (No. 8), p. 916-919 DeMattos RB et al., Science, 2002, 295 (No. 5563), p. 2264-2267 Kakimura J, et al., The FSB Journal, 2002, Volume 16 (No. 6), p. 601-603 Koistinaho M et al., Nature Medicine, 2004, Volume 10 (No. 7), p. 719-726 Matsuoka Y, et al., The Journal of Neuroscience (J. Neurosci.), 2003, Volume 23 (No. 1), p. 29-33 Wyss-Coray T et al., Nature Medicine, 2001, Volume 7 (No. 5), p. 612-618 Alarcon R et al., The Journal of Biological Chemistry (J. Biol. Chem.), 2005, 280 (No. 34), p. 30406-30415 El Khoury JB et al., The Journal of Experimental Medicine (J. Exp. Med.), 2003, Vol. 197 (No. 12), p. 1657-1666 Shibata M et al., The Journal of Clinical Investigation (J. Clin. Invest.), 2000, 106 (No. 12), p. 1489-1499 Nakamura K et al., Biochemical and Biophysical Research Communication (2001), Vol. 280 (No. 4), p. 1028-1035 Nakamura K et al., Biochim. Biophys. Acta., 2001, Volume 1522 (No. 1), p. 53-58 Murphy JE et al., Atherosclerosis, 2005, Volume 182 (No. 1), p. 1-15 Kodama T et al., Nature, 1990, 343 (No. 6258), p. 531-535

  The present invention provides a drug that clears amyloid β accumulated in the brain in Alzheimer's disease, senile dementia, presenile dementia and the like.

We examined the involvement of SRCL in amyloid β binding and clearance in Alzheimer's disease brains. We have determined that SRCL is amyloid in astrocytes, microglia and vascular / perivascular cells, both in Alzheimer's disease double transgenic mice (Tg-APP / PS1 mice) and samples from cadaver of Alzheimer's disease patients. It was found to be expressed depending on β. The inventors have further studied and found that SRCL can bind to fibrillar amyloid β _1-42 and that the binding is elevated in the brain of Alzheimer's disease patients. In addition, the present inventors have also confirmed that SRCL acts in a dose-dependent manner on amyloid β and binds to amyloid β from glia cells and blood vessels / perivascular cells of the brain affected by Alzheimer's disease. It has been found that it plays an important role in the clearance. Based on these findings, the inventors of the present invention have further studied and have completed the present invention.

That is, the present invention
[1] A scavenger receptor protein, a protein having substantially the same activity as a scavenger receptor protein or a salt thereof, a DNA encoding a scavenger receptor protein, or a DNA comprising a base sequence complementary to the DNA and a string An amyloid β clearance promoter characterized by containing, as an active ingredient, DNA containing a DNA that encodes a protein that hybridizes under gentle conditions and has substantially the same activity as the scavenger receptor protein,
[2] Whether the active ingredient is (a) a protein containing the same amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or (b) a protein containing the amino acid sequence substantially identical to the amino acid sequence or a salt thereof Or (c) hybridizes under stringent conditions with DNA consisting of the base sequence represented by SEQ ID NO: 1, 3 or 5 or (d) DNA consisting of a base sequence complementary to the base sequence of (c) above And the promoter according to the above [1], wherein the promoter comprises a DNA encoding a protein having substantially the same activity as the scavenger receptor protein.
[3] The active ingredient is a protein comprising (a) an amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or (b) a protein comprising substantially the same amino acid sequence as the amino acid sequence. The accelerator according to [1] or [2],
[4] The active ingredient is stringent with (c) DNA consisting of the base sequence represented by SEQ ID NO: 1, 3 or 5 or (d) DNA consisting of a base sequence complementary to the base sequence of (c) above. The promoter according to the above [1] or [2], which is a DNA comprising a DNA that hybridizes under conditions and encodes a protein having substantially the same activity as the scavenger receptor protein,
[5] DNA is a retrovirus vector, adenovirus vector, adeno-associated virus vector, herpes virus vector, vaccinia virus vector, poxvirus vector, poliovirus vector, shinbis virus vector, Sendai virus vector, SV40 vector and immunodeficiency The promoter according to any one of [1], [2] or [4], wherein the promoter is incorporated into at least one vector selected from viral vectors,
[6] The promoter according to any one of [1] to [5], wherein amyloid β is brain amyloid β,
[7] The promoter according to any one of [1] to [6], wherein the promoter is locally administered to an accumulation site of amyloid β,
[8] The promoter according to [4] or [5] above, which is administered intrathecally,
[9] An injection comprising cells transfected with the promoter according to [5] above,
[10] A scavenger receptor protein, a protein having substantially the same activity as a scavenger receptor protein or a salt thereof, a DNA encoding a scavenger receptor protein, or a DNA comprising a base sequence complementary to the DNA and a string A disease caused by accumulation of amyloid β, characterized by containing as an active ingredient a DNA containing a DNA encoding a protein that hybridizes under a gentle condition and has substantially the same activity as a scavenger receptor protein Preventive or therapeutic agent for
[11] Whether the active ingredient is (a) a protein comprising the same amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or (b) a protein comprising the amino acid sequence substantially identical to the amino acid sequence or a salt thereof Or (c) hybridizes under stringent conditions with DNA consisting of the base sequence represented by SEQ ID NO: 1, 3 or 5 or (d) DNA consisting of a base sequence complementary to the base sequence of (c) above And a prophylactic or therapeutic agent according to [10] above, which comprises DNA encoding a protein having substantially the same activity as the scavenger receptor protein.
[12] The preventive or therapeutic agent according to [10] or [11] above, wherein the disease is Alzheimer's disease,
[13] A therapeutically effective amount of a scavenger receptor protein, a protein having substantially the same activity as the scavenger receptor protein, or a salt thereof, or a scavenger receptor protein is given to a patient who needs to promote amyloid β clearance. Effective DNA that encodes a DNA that encodes a protein that is hybridized under stringent conditions with a DNA that encodes the DNA or a complementary base sequence thereof, and that has substantially the same activity as the scavenger receptor protein A method for promoting amyloid β clearance, characterized by being administered as a component,
[14] Whether the active ingredient is (a) a protein comprising the same amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or (b) a protein comprising the amino acid sequence substantially identical to the amino acid sequence or a salt thereof Or (c) hybridizes under stringent conditions with DNA consisting of the base sequence represented by SEQ ID NO: 1, 3 or 5 or (d) DNA consisting of a base sequence complementary to the base sequence of (c) above And the method for promoting amyloid β clearance according to [13] above, wherein the DNA comprises a DNA encoding a protein having substantially the same activity as the scavenger receptor protein.
[15] A scavenger receptor protein, a protein having substantially the same activity as the scavenger receptor protein, or a salt thereof, or a DNA encoding the scavenger receptor protein or the DNA for producing an amyloid β clearance promoter Use as an active ingredient of a DNA comprising a DNA encoding a protein that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to and having substantially the same activity as the scavenger receptor protein; and [16] Whether the active ingredient is (a) a protein containing the same amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or (b) a protein containing the amino acid sequence substantially identical to the amino acid sequence or a salt thereof Or (c) DNA comprising the base sequence represented by SEQ ID NO: 1, 3, or 5 or (D) DNA encoding a protein that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence of (c) and has substantially the same activity as the scavenger receptor protein. The use according to [15] above, characterized in that the DNA comprises DNA;
About.

  The present invention also provides a scavenger receptor protein, a protein having substantially the same activity as a scavenger receptor protein, or a salt thereof, a DNA encoding a scavenger receptor protein, or a DNA comprising a base sequence complementary to the DNA. Promotes the clearance of amyloid β, which comprises administering to a mammal DNA containing a DNA encoding a protein that hybridizes with a scavenger receptor protein and has substantially the same activity as a scavenger receptor protein Or a method for preventing or treating a disease caused by accumulation of amyloid β such as Alzheimer's disease. Further, the present invention substantially comprises a scavenger receptor protein or a scavenger receptor protein for producing a medicament for promoting clearance of amyloid β, or for preventing or treating a disease caused by accumulation of amyloid β such as Alzheimer's disease. It hybridizes under stringent conditions with a DNA having the same activity or a salt thereof, or a DNA encoding a scavenger receptor protein or a DNA comprising a base sequence complementary to the DNA, and substantially the same as a scavenger receptor protein. In particular, the present invention relates to the use of DNA comprising DNA encoding a protein having the same activity.

  The scavenger receptor protein used in the present invention has an effect of clearance of amyloid β. The amyloid β clearance promoter of the present invention is capable of degrading and metabolizing amyloid fibrils formed by the accumulation and aggregation of so-called senile plaques in so-called senile plaques in Alzheimer's disease and the like. It is useful for the prevention or treatment of).

FIG. 1 shows the results of Northern blot analysis of rat SRCL mRNA in rat tissue. Above: shows that rat SRCL was expressed as a single 3.0 kb transcript in various tissues.
The lower panel shows the results of densitometric analysis of SRCL mRNA relative to GAPDH mRNA.
FIG. 2 shows a representative photomicrograph (high magnification inset) image of A: rat neonatal microglia after 15 minutes of culture.
B: A representative photomicrograph (high magnification inset) image after 2 days of primary culture is shown.
C: shows RT-PCR of cultured neurons. In the figure, ASTRO represents mouse neonatal astrocytes, MG represents mouse neonatal microglia, 2d represents 2 days after culture, C6 represents human C6 glioma, and PC12 represents rat pheochromocytoma cells.
D: shows the results of RT-PCR analysis of rat neonatal microglia rat SRCL mRNA at different time points after the start of culture. P7 represents a 7-day-old mouse, MG represents a mouse newborn microglia, Br represents the brain, 1d represents 1 day after the start of culture, and 2d represents 2 days after the start of culture.
E: The upper figure shows the result of semi-quantitative RT-PCR analysis of astrocytes after treating cultured astrocytes with f amyloid β _1-42 for different times. The figure below shows the ratio of SRCL mRNA to control astrocytes to astrocytes after treatment of cultured astrocytes with f amyloid β _1-42 for different times. cont indicates control, Aβ indicates amyloid β treatment.
FIG. 3 is a diagram showing an immunostained image of CHO-K1 cells transfected with mSR-AI-Myc or hSRCL-I-Myc treated with A: f amyloid β _1-42 . fAβ1-42 shows a stained image of f amyloid β _1-42 ; merge shows a double-stained image of mSR-AI or hSRCL-I and f amyloid β _1-42 ; An enlarged view is shown. fAβ1-42 + Fucoidan shows a stained image when cultured in the presence of fucoidan.
B: CHO-K1 cells transfected with mSR-AI-Myc, hSRCL-I-Myc, hSRCL-II-Myc or Mock were treated with famyloid β _1-42 and excess poly (C) or poly (G) It is a figure which shows the immuno-staining image when it incubates with PBS containing.
FIG. 4 is a diagram showing an outline of the domain structure of A: hSRCL-I, hSRCL-II, and mSRCL.
B: Western blot results of CHO-K1 cells transfected with hSRCL-I-Myc / EGFP, hSRCL-II-Myc / EGFP, mSRCL-Myc / EGFP and Mock / EGFP expression vectors.
C: Immunostaining images of CHO-K1 cells transfected with hSRCL-I-Myc / EGFP, hSRCL-II-Myc / EGFP, mSRCL-Myc / EGFP and Mock / EGFP expression vectors. In the upper part, EGFP shows a green fluorescent color, and in the lower part, Myc is stained red.
FIG. 5 shows immunostained images of paraffin sections of A: 9-month-old Tg-APP / PS1 mice and wild-type littermates of cerebral cortex and hippocampus. Insert shows high magnification, GFAP indicates astrocyte marker, arrow indicates cells positive only for GFAP, arrowhead indicates cells positive for both SRCL and GFAP, merge overlaps SRCL and GFAP images CA1 and CA3 indicate hippocampal regions.
B: shows immunostained images of tissues of frozen cortical sections of 9-month-old Tg-APP / PS1 mice and wild-type littermates. merge represents an SRCL image and a Mac-1 image superimposed, the insertion represents a high-magnification view, the asterisk represents amyloid β plaques, the arrow represents immunopositive cells only in Mac-1, and the arrowheads represent SRCL and Mac-1 -1 shows immunopositive cells.
FIG. 6 shows double immunostaining images of SRCL and amyloid β in meningeal blood vessels. Arrowheads indicate amyloid β-immune positive and SRCL-immune positive meningeal blood vessels, arrows indicate amyloid β / SRCL-double positive blood vessels, insertion shows a high-magnification view, and bottom row shows anti-SRCL absence. Shows immunostaining control, m. v. Indicates meningeal blood vessels, and merge indicates an image in which an amyloid β-stained image and an SRCL-stained image are superimposed.
FIG. 7 shows a schematic representation of blood vessels / perivascular cells of A: 9 month old Tg-APP / PS1 mouse meningeal blood vessels (mv). MATO indicates MATO cells, SMC indicates smooth muscle cells, EC indicates vascular endothelial cells, Mφ indicates macrophages, Aβ indicates amyloid β aggregation in the extracellular matrix and amyloid β particles incorporated into the cells. Show.
B: Representative images of SRCL and CD31, αSMA or GFAP double staining in 9 month old Tg-APP / PS1 mouse meningeal vessels. m. v. Indicates meningeal blood vessels, arrows indicate CD31-positive ECs, and merge indicates an image obtained by superimposing the left two images.
C: A high-magnification image of double immunostaining of SRCL and CD31 or αSMA in meningeal blood vessels of 9 months old Tg-APP / PS1 mice.
D: shows a double-stained image of SRCL and GFAP in cerebral cortex tissue associated with amyloid plaques (asterisks) and cortical amyloid angiopathy in 9 month old Tg-APP / PS1 mice. The expression level of hSRCL mRNA is shown. mRNA levels are expressed as a ratio to GAPDH mRNA, bars indicate standard error.
FIG. 9 shows images obtained by immunostaining the brain tissue of A: formalin-fixed control and Alzheimer's disease patients with an anti-SRCL antibody that recognizes hSRCL.
B: shows a double-stained image of SRCL and GFAP of brain tissue of a Alzheimer's disease patient fixed with formalin. Arrowheads indicate amyloid β deposits (black asterisks) surrounding the hSRCL-immunopositive astrocyte.
C: An image of double staining of SRCL and Iba1 of brain tissue of a Alzheimer's disease patient fixed with formalin is shown. Arrows indicate hSRCL-immunopositive microglia cells, and arrowheads indicate hSRCL-immunopositive-MATO cells.
FIG. 10 shows images of A: formalin-fixed control and Alzheimer's disease brain tissue double-stained with vascular SRCL and amyloid β _1-42 associated with CAA. Asterisks indicate blood vessels, and arrowheads indicate SRCL / amyloid β double positive granules.
B: A highly enlarged view of the rectangular area shown in FIG. 2 of “FIG. 10A”. Arrowheads indicate SRCL / amyloid β double-stained particles, and asterisks indicate the intravascular space of meningeal blood vessels.
C: shows the result of the absorption test. Arrows indicate cells with hSRCL-immunity positivity, and arrowheads indicate that hSRCL-immunity positivity has disappeared due to preabsorption with the immunogen.
FIG. 11 shows an image obtained by immunostaining microglia cells treated with _famyloid β _1-42 for 1 hour. fAβ1-42 shows cells treated with f amyloid beta _1-42, merge represents a double stained image of the Mac-1 stained with fAβ1-42 stained, Magnified shows a high enlargement.
FIG. 12 shows CHO-K1 cells stably expressing mSRCL in the presence or absence of fucoidan.

The scavenger receptor (scavenger receptor; hereinafter abbreviated as SR) protein used in the present invention is involved in a mechanism for recognizing foreign substances such as oxidized modified low density lipoprotein (oxidized LDL) and taking in the foreign substances. As long as it is a membrane protein purified to such an extent that it can be used as a medicine, those prepared by various methods can be used. Examples of SR proteins include SR-AI / II (scavenger receptor-A I / II), SR-B1 (scavenger receptor class B type 1), SRCL (scavenger receptor with C-type lectin) -I, SRCL-II, Examples include LOX-1 (lectin-like oxidized LDL), CD36, SR-PSOX / XXCL16, CL-P1, and the like, but they are not limited to these as long as they have SR protein activity. In addition, as long as the SR protein has substantially the same action as the SR protein, one or more of the amino acid sequences of the SR protein (for example, about 2 to 20, preferably about 2 to 10; the same shall apply hereinafter) The amino acid may be substituted, deleted or added, and the sugar chain may be similarly substituted, deleted or added. Here, with respect to the amino acid sequence, “one or a plurality of amino acids are deleted, substituted or added” refers to genetic engineering techniques, well-known technical means such as site-directed mutagenesis, or the like. It means that a sufficient number (one to plural) is deleted, substituted or added. Examples of SR proteins in which sugar chains are substituted, deleted, or added include, for example, SR proteins in which sugar chains added to natural SR proteins are treated with enzymes or the like to delete sugar chains, and sugar chains are not added. Examples include those in which the amino acid sequence at the sugar chain addition site is mutated, or those in which the amino acid sequence is mutated so that the sugar chain is added to a site different from the natural sugar chain addition site.
Furthermore, a protein having at least about 80% homology with the amino acid sequence of SR protein, preferably a protein having about 90% homology, more preferably a protein having about 95% homology, A protein having substantially the same activity as that of the SR protein is also included in the SR protein used in the present invention. “Homology” in the above amino acid sequences means the degree of coincidence of amino acid residues constituting each sequence by comparing the primary structures of proteins.

  In the present invention, “activity of SR protein” includes, for example, activity of binding to oxidized LDL on the cell membrane and taking it into the cell.

  Specific examples of the SR protein include, but are not limited to, proteins having SR protein activity registered with an accession number (Accession No.) by GeneBank / EMBL / DDBJ, for example. The SR protein is preferably a human-derived SR protein. Examples of human-derived SR proteins include SR-AI / II (eg, Accession No. NP — 619729, NP — 002436), SR-B1 (eg, Accession No. AAI12038, NP — 005496, etc.), SRCL-I [eg, Accession No. BAB39147 (SEQ ID NO: 2) etc.], SRCL-II [eg Accession No. BAB39148 (SEQ ID NO: 4) etc.], LOX-1 (eg Accession No. NP — 002534 etc.), CD36, etc. are exemplified, but not limited thereto. SRCL-I or SRCL-II is preferred.

  As a protein containing an amino acid sequence substantially identical to the SR protein, for example, a protein having an amino acid sequence highly homologous to the amino acid sequence of the SR protein by homology search by DDBJ (DNA Data Bank of Japan) or the like. And a protein having SR protein activity. For example, the protein comprising the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 is at least about 80% or more, preferably about 90% or more, more preferably, the amino acid sequence represented by SEQ ID NO: 2. Is a protein comprising an amino acid sequence having an identity of about 95% or more, and a protein having substantially the same activity as the SR protein, for example, one to a plurality of amino acids from the amino acid sequence represented by SEQ ID NO: 2 Includes amino acid sequences in which residues are inserted or deleted, amino acid sequences in which one or more amino acid residues are replaced with other amino acid residues, or amino acid sequences in which one or more amino acid residues are modified Examples of the protein include proteins having substantially the same activity as the SR protein. Specific examples of the protein having substantially the same activity as SEQ ID NO: 2 include a protein comprising the amino acid sequence represented by SEQ ID NO: 4, or Accession No. Human-derived protein registered as BAB83592, or mouse SRCL (Accession No. BAB82497; SEQ ID NO: 6), human SRCL (Accession No. AAH60879), mouse SRCL (Accession No. NP569716), mouse SRCL (Accession No. BAC05523) ), Rat SRCL (Accession No. XP341575), chicken SRCL (UD.GG.Contig 23617), and the like. The amino acid to be inserted or substituted may be an unnatural amino acid other than the 20 types of amino acids encoded by the gene. The non-natural amino acid may be any compound as long as it has an amino group and a carboxyl group, and examples thereof include γ-aminobutyric acid. These proteins may be used alone or as a mixed protein thereof.

In the SR protein used in the present invention, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO—), an amide (—CONH ₂ ), or an ester (—COOR). Here, R in the ester is, for example, a C1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, for example, phenyl, α -C6-12 aryl groups such as naphthyl, for example, C7-14 aralkyl groups such as phenyl-C1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C1-2 alkyl groups such as α-naphthylmethyl, A pivaloyloxymethyl group or the like is used. When the SR protein used in the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the SR protein of the present invention. As the ester in this case, for example, the above C-terminal ester or the like is used. Furthermore, in the SR protein used in the present invention, the amino group of the N-terminal methionine residue is a protecting group (for example, a C1-6 acyl group such as a C2-6 alkanoyl group such as formyl group and acetyl). Etc.), N-terminal side cleaved in vivo, glutamyl group produced by pyroglutamine oxidation, substituent on the side chain of amino acid in the molecule (for example, —OH, —SH, amino group) , An imidazole group, an indole group, a guanidino group, etc.) are protected with an appropriate protecting group (for example, a C1-6 acyl group such as a C2-6 alkanoyl group such as formyl group or acetyl), or a sugar chain Also included are complex proteins such as so-called glycoproteins.

  Examples of the salt of the SR protein used in the present invention include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  The SR protein is transformed, for example, by inserting a gene encoding the SR protein into an appropriate vector by genetic engineering techniques, inserting the gene into an appropriate host cell, and transforming it from the cultured cells of the transformant. It can be obtained by separating the SR protein or the like (see, for example, Biochem. Biophys. Res. Commun. 2001, Vol. 280, p. 1028-1035). The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, yeast or animal cells can be used.

  In the present invention, “DNA encoding SR protein” refers to DNA capable of expressing SR protein. Examples of DNA containing DNA encoding SR protein include Proc. Natl. Acad. Sci. USA. 1990, vol. 87, p. 9133-9137; Biol. Chem. 1997, 272, p. 17551-17557; Nature, 1997, 386, p. 73-77; Biochem. Biophys. Res. Commun. 2001, Vol. 280, p. 1028-1035 etc., for example, Accession No. in GeneBank / EMBL / DDBJ. NM_138715 (DNA encoding SR-A1), NM_002445 (DNA encoding SR-A2), BC112037, NM_005505 (DNA encoding SR-B1), ABO38518 (SEQ ID NO: 1; DNA encoding SRCL-I), Preferred examples include DNA containing DNA encoding a human-derived SR protein registered as ABO52103 (SEQ ID NO: 3; DNA encoding SRCL-II, etc.) or NM_002543 (DNA encoding LOX-1). However, it is not limited to these. The DNA containing the DNA encoding the SR protein of the present invention is a DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the DNA, and is substantially the same as the SR protein. For example, DNA encoding a protein that binds to, for example, oxidized LDL on the cell membrane and takes up into the cell.

  Examples of the DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the DNA include, for example, a colony hybridization method, a plaque hybridization method, or a Southern blot hybridization using the partial sequence of the DNA as a probe. Examples thereof include DNA obtained by using a hybridization method or the like. Specifically, hybridization was performed at about 65 ° C. in the presence of about 0.7 to 1.0 M sodium chloride using a filter on which DNA derived from colonies or plaques was immobilized, and then about 0.1 to DNA that can be identified by washing the filter under a condition of about 65 ° C. using a SSC solution of a double concentration (the composition of the SSC solution of a single concentration consists of 150 mM sodium chloride and 15 mM sodium citrate). Can be mentioned.

  DNA that hybridizes under stringent conditions with DNA comprising a base sequence complementary to the DNA is, for example, about 80% or more, preferably about 90% or more, more than the DNA by homology search using DDBJ or the like. Preferably, it includes DNA having a homology of about 95% or more and containing DNA encoding a protein having SR protein activity. Specifically, for example, a DNA having a base sequence having a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more with the base sequence represented by SEQ ID NO: 1 (Accession No. AB038518) For example, Accession No. AB052103 (SEQ ID NO: 3), Accession No. AB038519 (SEQ ID NO: 5) or Accession No. Examples include DNA registered as AB034251. Hybridization is performed by a known method such as molecular cloning (Molecular Cloning, A laboratory Manual, Third Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001: hereinafter abbreviated as Molecular Cloning 3rd Edition). )), Etc. When a commercially available library is used, it can be carried out according to the method described in the attached instruction manual.

  Furthermore, the DNA containing the DNA encoding the SR protein of the present invention is not limited to the above, and as long as the expressed protein is a DNA containing a DNA encoding a protein having substantially the same activity as the SR protein, It can be used as DNA containing DNA encoding SR protein.

  The DNA containing the DNA encoding the SR protein can be easily obtained by, for example, the usual hybridization method or PCR (Polymerase Chain Reaction) method. Specifically, the DNA can be obtained by, for example, the molecular cloning method 3 described above. This can be done with reference to basic documents such as editions.

Preferred examples of DNA containing the DNA encoding the SR protein used in the present invention include genomic DNA, genomic DNA library, cell / tissue-derived cDNA, cell / tissue-derived cDNA library, or synthetic DNA. . Examples of the vector into which the genomic DNA fragment is cloned in the library include bacteriophage, plasmid, cosmid, or phagemid.
The RNA encoding the SR protein used in the present invention can also be used in the present invention as long as the SR protein or the partial peptide can be expressed by reverse transcriptase. Examples of the RNA include RNA obtained by preparing an mRNA fraction from a cell or tissue and amplified by RT-PCR, and are within the scope of the present invention. The RNA can also be obtained by known means.

  In addition, as for the DNA containing SR protein used in the present invention or DNA encoding the same, those described above derived from humans are preferably used when applied to humans, but mammals other than humans (for example, monkeys, cows, SR protein derived from horses, pigs, sheep, dogs, cats, rats, mice, birds, etc.) or DNA encoding the same may be used. For example, mouse-derived SR protein includes, for example, mouse SRCL (Accession No. BAB82497; SEQ ID NO: 6; hereinafter abbreviated as mSRCL), and the DNA containing the DNA encoding mSRCL is SEQ ID NO: 5. Examples thereof include DNA (Accession No. AB038519; SEQ ID NO: 5) comprising the represented base sequence.

In the present invention, “amyloid β clearance” means removal of amyloid β, and includes amyloid β degradation, phagocytosis (including endocytosis or phagocytosis) that incorporates and degrades amyloid β into cells. . Amyloid β is toxic to nerve cells and causes cell death. For example, amyloid β _1-42 and amyloid β _1-40 are included, and they are further modified by apo E or presenilin 1, 2 or the like. The received protein is also included. Amyloid β _1-42 or amyloid β _1-40 has an amino acid from about 40 to 42 that can be produced from a precursor protein (β-amyloid protein precursor; hereinafter abbreviated as APP) by the action of β- and γ-secretase. It is a peptide. γ-secretase can cleave APP at the 40th or 42nd amino acid to produce amyloid β _1-42 or amyloid β _1-40 . Amyloid β is highly hydrophobic and can form aggregates or modifications to form fibrils.
In the present invention, “accumulation of amyloid β” means that amyloid β is not decomposed or suppressed, and amyloid β accumulates in the body, and amyloid β aggregates to form insoluble fibers. Is included.
In addition, amyloid β includes amyloid β present in any part of the body, but is preferably cerebral amyloid β.

  Examples of the diseases caused by accumulation of amyloid β include Alzheimer's disease in which amyloid β accumulates and aggregates in the brain to form insoluble fibers. The Alzheimer's disease includes Alzheimer type senile dementia. The major pathological changes of Alzheimer's disease include senile plaques and neurofibrillary tangles. The senile plaques are recognized from the early stage of onset, and the main component is amyloid β.

  The amyloid β clearance promoter or the preventive or therapeutic agent for diseases caused by accumulation of amyloid β according to the present invention is not limited to humans and mammals other than humans (for example, monkeys, cows, horses, pigs, sheep, dogs, cats, rats). , Mouse, etc.).

When administering the amyloid β clearance promoter of the present invention or a prophylactic or therapeutic agent for diseases caused by accumulation of amyloid β to a patient, the administration form, administration method, dosage and the like are such that the active ingredient is SR protein or SR protein. In the case of a protein having substantially the same quality of activity or a salt thereof (hereinafter referred to as SR protein) and a DNA encoding the SR protein or a DNA comprising a base sequence complementary to the DNA under stringent conditions It may be slightly different from the case of DNA containing a DNA that encodes a protein that hybridizes with and has substantially the same activity as the SR protein (hereinafter referred to as SR gene).
For example, when the active ingredient is an SR protein or the like, various preparation forms such as liquids and solids can be used, but in general, only an SR protein or the like and a conventional carrier and injection, propellant, liposome Alternatively, it is preferable to use a sustained-release preparation (for example, a depot). The injection may be either an aqueous injection or an oily injection. In the case of an aqueous injection, according to a known method, for example, an aqueous solvent (water for injection, purified water, etc.) is added to a pharmaceutically acceptable additive such as an isotonic agent (sodium chloride, potassium chloride, glycerin, mannitol, Sorbitol, boric acid, borax, glucose, propylene glycol, etc.), buffer (phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, epsilon) Aminocaproic acid buffer, etc.), preservative (methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boro Acid, borax, etc.), thickener (hydroxyethylcellulose) Hydroxypropyl cellulose, polyvinyl alcohol, polyethylene glycol, etc.), stabilizers (sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, dibutylhydroxytoluene, etc.) or pH adjusters (hydrochloric acid, sodium hydroxide) , Phosphoric acid, acetic acid, etc.) can be prepared by dissolving SR protein, etc. in a solution, filtered and sterilized with a filter, etc., and then filled into an aseptic container. Further, an appropriate solubilizing agent such as alcohol (ethanol etc.), polyalcohol (propylene glycol, polyethylene glycol etc.) or nonionic surfactant (polysorbate 80, polyoxyethylene hydrogenated castor oil 50 etc.) may be further blended. Good. In the case of an oily injection, for example, sesame oil or soybean oil is used as the oily solvent, and benzyl benzoate or benzyl alcohol may be blended as a solubilizing agent. The prepared injection solution is usually filled in an appropriate ampoule or vial. The SR protein content in the injection can be adjusted to about 0.0002 to 0.2 w / v%, preferably about 0.001 to 0.1 w / v%. In addition, it is desirable to store liquid preparations such as injections after removing moisture by freezing or lyophilization. The freeze-dried preparation is used by adding distilled water for injection at the time of use and re-dissolving it.

  Propellants can also be prepared by routine pharmaceutical methods. In the case of producing as a propellant, the additive added to the propellant may be any additive as long as it is generally used for inhalation preparations. A solvent, a preservative, a stabilizer, an isotonic agent, a pH adjuster and the like can be blended. Examples of the propellant include a liquefied gas propellant or a compressed gas. Examples of the liquefied gas propellant include fluorinated hydrocarbons (alternative chlorofluorocarbons such as HCFC22, HCFC-123, HCFC-134a, and HCFC142), liquefied petroleum, dimethyl ether, and the like. Examples of the compressed gas include soluble gas (carbon dioxide gas, nitrous oxide gas, etc.) or insoluble gas (nitrogen gas, etc.).

  The liposome is not particularly limited as long as it is a vesicle having a capsule-like structure having an artificial lipid membrane mainly composed of phospholipid, but a lipid bilayer membrane vesicle comprising a lipid having a positive charge as a constituent ( Cationic liposomes), temperature sensitive liposomes, pH sensitive liposomes and the like are preferred. Of these, cationic liposomes are preferred. Since liposomes can encapsulate various compounds, they are also used as food carriers and pharmaceutical materials as chemical carriers and microcapsules. By encapsulating the SR protein or the like according to the present invention in a liposome and injecting it into the body, it becomes possible to maintain the effect of the SR protein or the like, or to bring the effect of the SR protein or the like only to a specific organ. Liposomes vary in size depending on the preparation method and materials, but are preferably about 50 nm to 10 μm in diameter. Examples of cationic liposomes include liposomes having cell fusion proteins on their surfaces (HVJ-liposomes), HVJ-E (Sendai virus envelope) vectors (Neuroscience Letters 378 (2005) 18-21), and the like. As a method for encapsulating or binding the SR protein or the like according to the present invention in a cationic liposome, for example, about 1 to 500 μL, preferably about 10 to 100 μL of HVJ-E vector (HVJ-liposome: GenomONE, manufactured by Ishihara Sangyo Co., Ltd.) ) Is added at about 0.1 to 100 μL, allowed to stand at about 1 to 5 ° C. for about 2 to 30 minutes, and then about 0.1 to 100 μL of SR protein solution (0.1 to 10 mg / mL, preferably 0.2 to 2 mg / mL) and about 0.1 to 100 μL of enclosing factor, and after mixing, centrifuge at about 2000 to 10,000 × g for about 1 to 30 minutes at about 1 to 5 ° C., Collect the precipitate. An SRCL protein-HVJ-E liposome solution can be prepared by dissolving this in about 10 to 1000 μL of phosphate buffered saline (PBS; pH 7.4).

  In addition, the SR protein and the like used in the present invention can be used as a sustained-release preparation (for example, a depot) together with a biodegradable polymer. By using SR protein or the like as a depot, effects such as reduction in the number of doses, sustained action and reduction in side effects can be expected. The sustained-release preparation can be produced according to a known method. The biodegradable polymer used in the sustained-release preparation can be appropriately selected from known biodegradable polymers. For example, polysaccharides such as starch, dextran or chitosan, proteins such as collagen or gelatin, etc. , Polyglutamic acid, polylysine, polyleucine, polyalanine or polymethionine polyamino acid, polylactic acid, polyglycolic acid, lactic acid / glycolic acid copolymer, polycaprolactone, poly-β-hydroxybutyric acid, polymalic acid, polyanhydride Or polyester such as fumaric acid / polyethylene glycol / vinyl pyrrolidone copolymer, polyorthoester or polyalkyl cyanoacrylic acid such as polymethyl-α-cyanoacrylic acid, polycarbonate such as polyethylene carbonate or polypropylene carbonate, and the like. Polyester is preferable, and polylactic acid or lactic acid / glycolic acid copolymer is more preferable. When a lactic acid-glycolic acid copolymer is used, the composition ratio (lactic acid / glycolic acid) (mol%) varies depending on the sustained release period. For example, the sustained release period is about 2 to 3 months, preferably about 2 weeks. In the case of 1 month, about 100/0 to 50/50 is preferable. The weight average molecular weight of the lactic acid-glycolic acid copolymer is generally preferably about 5,000 to 20,000. Polylactic acid and lactic acid-glycolic acid copolymer can be produced according to a known production method, for example, the production method described in JP-A No. 61-28521. The blending ratio of the biodegradable polymer and SR protein is not particularly limited. For example, the SR protein is preferably about 0.01 to 30% by mass with respect to the biodegradable polymer.

  As an administration method, an injection or a spray is directly injected into the amyloid β accumulation site (intrathecal administration or spinal parenchymal administration, intrathecal continuous administration by a sustained release pump, etc.) or sprayed, or sustained release It is preferable to implant the preparation (depot agent) in a site close to the tissue of the accumulation site of amyloid β. The dosage is appropriately selected according to the dosage form, the degree of disease or age, but is usually 1 μg to 500 mg, preferably 10 μg to 50 mg, more preferably 1 to 25 mg per dose. In addition, the number of administrations is appropriately selected according to the dosage form, the degree of disease, age, or the like, and can be administered once or continuously at a certain interval. In the case of continuous administration, the administration interval may be from once a day to once every several months. For example, in the case of administration by a sustained release preparation (depot) or intrathecal continuous administration by a sustained release pump, several months It may be once.

On the other hand, in order to administer the SR gene to a patient, conventional methods, for example, separate experimental medicine, basic technology of gene therapy, Yodosha, 1996, separate experimental medicine, gene transfer & expression analysis experimental method, Yodosha, 1997 It is preferable to carry out according to the method described in the Gene Therapy Society of Japan Handbook of Gene Therapy Development, NTS, 1999, etc.
As a specific administration method, for example, a method of locally injecting a recombinant expression vector or the like in which an SR gene is incorporated into a tissue (for example, brain) where amyloid β is accumulated, or a tissue or spinal cord of a patient's lesion site After removing cells from the body, blood, bone marrow, etc., and introducing and transfecting the cells with a recombinant expression vector incorporating the SR gene, the transfected cells are transferred to the patient, for example, by intravenous injection or the like. A method of transplantation, a method of transplanting into a lesion site or spinal cord of a patient, or SR genes such as SRCL genes (SRCL-I, SRCL-II, etc.) are introduced into phagocytic cells such as microglia ex vivo, and the cells are introduced into blood. The method of supplying SR protein expression phagocytic cells, such as srcl, etc. in the brain etc. is mentioned. Preferred examples of the cells include glial cells such as astrocytes and microglia, and blood myeloid cells such as macrophages.

Expression vectors include plasmid DNA, detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus (such as type I herpes simplex virus), vaccinia virus, pox virus, poliovirus, symbis virus, Sendai virus, SV40 or Examples include, but are not limited to, DNA viruses such as immunodeficiency virus (HIV) or RNA viruses. By introducing a target gene into the expression vector and infecting the cell with a recombinant virus, the SR gene can be introduced into the cell. Among them, type I herpes simplex virus (HSV-1) vector, adenovirus vector, adeno-associated virus (AAV) vector, and the like are preferable.
The HSV-1 vector is a neurotropic vector. The HSV-1 vector has a large (152 kb) genome that incorporates multiple genes (up to 30 kb) and has the ability to potentially establish infection in the cerebral cortex and hippocampus of the brain throughout life. Those are preferred. A specific HSV-1 vector is a non-replicating HSV-1 that is in a severely impaired state due to the deletion of three genes encoding ICR4, ICP34.5 and VP16 (vmw65) for viral replication. (HSV1764 / 4 / pR19) vector (Coffin RS, et al., J. Gen. Virol. 1998, 79, p. 3019-3026; Palmer JA, et al., J. Virol., 2000) 74, p. 5604-5618; Lilley CE, et al., J. Virol., 2001, vol. 75, p. 4343-4356) or the above-described liposomes (for example, HVJ-liposomes, HVJ-). E vectors) and the like, but is not limited thereto. AAV vectors belong to non-pathogenic viruses, are highly safe, and can efficiently introduce genes into non-dividing cells such as nerve cells. AAV-2, AAV-4, AAV-5 etc. are mentioned as an AAV vector. These HSV-1 vectors and AAV vectors can express the target gene in the cerebral cortex and hippocampus of the brain for a long time. Since the disease caused by accumulation of amyloid β such as Alzheimer's disease completes its pathological condition after a long period of time, the vector used in the present invention is not particularly limited, but an HSV-1 vector enabling long-term expression, AAV vectors, or HVJ-liposomes or HVJ-E vectors are particularly preferred.

As the dosage form, various known dosage forms [for example, injections, sprays, sustained-release preparations (depots), microcapsules, liposomes, etc.] suitable for each of the above administration forms can be taken. Injections, sprays, sustained-release preparations (depots) or liposomes can be prepared in the same manner as for SR proteins and the like. Further, for example, a host cell into which an expression plasmid containing an SR gene is introduced is used as a core substance, and this is covered with a coating substance according to a method known per se (eg, coacervation method, interfacial polymerization method or double nozzle method). Microcapsules containing fine particles of about 1 to 500 μm, preferably about 100 to 400 μm can be produced. Coating materials include carboxymethyl cellulose, cellulose acetate phthalate, ethyl cellulose, alginic acid or salts thereof, gelatin, gelatin gum arabic, nitrocellulose, polyvinyl alcohol or hydroxypropyl cellulose, polylactic acid, polyglycolic acid, chitosan-alginate, cellulose sulfate -Film-forming polymers such as poly (dimethyldiallyl) ammonium chloride, hydroxyethyl methacrylate-methyl methacrylate, chitosan-carboxymethylcellulose, alginate-polylysine-alginate and the like.
The content and dosage of DNA in the preparation can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient. The dose varies depending on the type of SR gene transfer vector, but is usually 1 × 10 ⁶ pfu to 1 × 10 ¹² pfu, preferably 1 × 10 ⁷ pfu to 2 × 10 ¹¹ pfu in terms of SR gene transfer vector. More preferably, 1.5 × 10 ⁷ pfu to 1.5 × 10 ¹¹ pfu are preferably administered once, or once every several days to several months or years.

  The agent of the present invention can be used for diseases caused by accumulation of amyloid β, such as Alzheimer's disease and Alzheimer type senile dementia.

  The clearance of amyloid β by SR protein or the like is described in, for example, a method of transfecting the SR gene into an accumulation site of amyloid β using an expression vector, for example, the cerebral cortex or hippocampus, and a test example described later. It can be measured and evaluated using a method or the like.

  Hereinafter, the present invention will be described using test examples, but the present invention is not limited thereto. In the following test examples,% indicates mass% unless otherwise specified.

[Test Example 1]
I. Test method Experimental Animals SD (Sprague-Dawley) rats and C57 / BL6 mice whose gestation dates were confirmed were purchased from SLC (Shizuoka, Japan). Double transgenic mice (Tg-APP / PS1 mice) and wild-type littermates (WT) are PS1 'knock-in' mice [Nakano Y et al., Eur. J. Neurosci. 1999, Volume 11 (No. 7): p. 2577-2581] and a transgenic mouse (Tg2576; Hsiao K et al., Science, 1996, Vol. 274 (No. 5284): p. 99-102) that overexpresses the Swedish mutation of human amyloid precursor protein. Made. Animals were placed in individual cages and housed in a temperature controlled room under a 12 hour light / 12 hour dark cycle. All experiments were conducted according to the guidelines of the Osaka University Animal Ethics Committee. All efforts made the animal burden as small as possible and used as few animals as possible.

2. Human samples Human samples were obtained from the University of Maryland brain and tissue bank. Using temporal cortical tissue (mean age of death: 84.2 ± 2.8) from 4 cadaver of Alzheimer's disease patients and 3 cortical samples of non-Alzheimer's disease (mean age of death 56.7 ± 26.1) did. All samples were handled according to procedures approved by the Institutional Review Board for Human Subject Research.

3. CDNA cloning of rat SRCL coiled-coil region The sequence of rat SRCL coiled-coil region was cloned by PCR using the primers of Table 1 and hexaoligonucleotide-primed cDNA of rat microglia total RNA as a template.

The forward primer corresponds to an assumed residue of the mSRCL ORF (Open Reading Frame) (base sequence at positions 493-510 of SEQ ID NO: 6), and the reverse primer corresponds to an assumed residue of the mSRCL (SEQ ID NO: SEQ ID NO: 6 base sequence at positions 1129-1146).
The obtained PCR product was subcloned into a pGEM-T vector (manufactured by Promega). The reliability of the vector was confirmed by transforming competent cells (E. coli) and purifying and sequencing the expressed DNA.

4). Northern blot, RT-PCR and quantitative real-time RT-PCR analysis (1) Total RNA
Total RNA is obtained from cells and tissues of adult rats and 7-day-old rats (P7) using ISOGEN Kit (Nippon Gene) or RNeasy Micro Kit (QIAGEN) according to the instructions for the kit. Prepared. Tissue from the temporal cortex of human pathological anatomical samples was similarly prepared.

(2) Northern blotting For Northern blotting, poly A + RNA was prepared from total RNA of adult rats. 2 μg of the prepared poly A + RNA was subjected to electrophoresis with 1% agarose / 0.7% formamide gel, and blotted on a high bond N + nylon membrane (hereinafter also referred to as filter membrane). Subsequently, the RNA transcribed to the filter membrane was hybridized with a ³² P-labeled cDNA fragment (nucleotide at positions 493 to 1146 of hSRCL). Thereafter, the filter membrane was washed, and the method of Nakamura et al. (Biochem. Biophys. Res. Commun. 2001, Vol. 280 (No. 4), p. 1028-1035; Biochim. Biophys. Acta. 2001, 1522. Volume (No. 1), p.53-58). Rat GAPDH probe was used as a loading control.

(3) RT-PCR (reverse transcription-PCR)
The total RNA prepared in (1) above is pretreated with RNase-free DNase (Quiagen), and 1 μg of RNA is subjected to reverse transcription reaction using SuperScript (registered trademark) II (Invitrogen) according to the instruction manual. went.
Rat RT-PCR was carried out using a GeneAmp Gold PCR Reagent Kit (manufactured by Perkin-Elmer), followed by a denaturation reaction at 95 ° C for 5 minutes, then 94 ° C for 1 minute, 58 ° C for 1 minute, 72 ° C. 1 minute was carried out for 32 cycles, and then an extension reaction was carried out at 72 ° C. for 7 minutes. The number of amplification cycles and the amount of cDNA used for each reaction were optimized in advance in preliminary tests for RNA quantification. PCR products were separated by 1.5% agarose gel electrophoresis. The separated gel was stained with ethidium bromide. As the rat SRCL primer, the primers shown in Table 2 were used.

GAPDH mRNA was used for normalization of experimental data.

  Mouse RT-PCR was performed using the GeneAmp Gold PCR Reagent Kit. The PCR reaction was performed at 94 ° C for 5 minutes, followed by 32 cycles of 94 ° C for 30 seconds, 63 ° C for 30 seconds, and 72 ° C for 3 minutes, followed by extension reaction at 72 ° C for 5 minutes. Went. The following Table 3 was used for the mSRCL primer and mouse GAPDH.

(4) Quantitative real-time RT-PCR
For quantification by real-time RT-PCR, 2.5 μg of each RNA was reverse-transcribed using SuperScript ^™ II (manufactured by Invitrogen) according to the instruction manual of the product. Quantitative real-time RT-PCR using the ABI PRISM 7900 Sequence Detection System (Applied Biosystems) using the High-capacity cDNA Archive Kit (Applied Biosystems) for quantitative evaluation of mRNA levels in human samples (TaqMan method). A universal PCR master mixture (Applied Biosystems) was used for the reaction, and TaqMan MGB (fluorescent label (FAM) added) was used for the probe. Exon 5-6, Hs00560477m1 (Applied Biosystems) was used as a probe for hSRCL. TaqMan VICTM Probe; 4326317E (Applied Biosystems) was used as the human GAPDH probe. The PCR reaction was first denatured at 50 ° C. for 2 minutes, followed by denaturation at 95 ° C. for 10 minutes, followed by 40 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute.

(5) Statistical processing In order to quantify the PCR reaction three times for each sample without variation among the samples, the SRCL mRNA value was shown as a relative value to the GAPDH mRNA value. The notation was expressed as mean ± standard error (SE), and statistical significance was evaluated by Student's t test. A level of P <0.05 was used as a criterion for significant difference. Significant differences were assessed by at least two separate experiments.

5. Culture of Cell Lines CHO-K1 cell line, C6 glioma cell (hereinafter abbreviated as C6), mouse macrophage J774A.1 cell line and rat pheochromocytoma PC12 cell (hereinafter abbreviated as PC12) are Funakoshi. H et al. Biol. Chem. 1991, 266, p. 15614-15620; Nakamura K et al., Biochem. Biophys. Res. Commun. 2001, Vol. 280, p. 1028-1035; Nakamura K et al., Biochim. Biophys. Acta. 2001, vol. 1522, p. 53-58; or Naveilhan P et al., Brain Res. Mol. Brain Res. 1996, 41, p. The cells were cultured by the method described in 259-268.

6). Culture of microglia Mixed glia cells isolated from C57 / BL6 mice of 1 to 3 days of age and SD rats were precultured, and microglia were isolated by the method of Funakoshi et al. (J. Neurosci. Res., 2002, 68th). Volume, p. 150-160). In other words, the cerebral cortex was a tissue from which the meninges were completely removed. The tissue was then minced and the fine sections were suspended in PBS containing 0.05% trypsin (GIBCO), and the cells were trypsinized at 37 ° C. for 10 minutes. The trypsinized cells were incubated for 5 minutes at room temperature in PBS containing 0.01% DNase I (Sigma). After incubation, the cells were centrifuged to precipitate the cells. The cell pellet was washed 3 times. The cells were filtered through a 75 μm nylon mesh and centrifuged to precipitate the cells. The centrifuged cells were used as mixed glial cells. Low-glucose Dulbecco's modified Eagle's medium containing 10% (v / v) FBS (fetal bovine serum; JRH Bioscience), 100 IU / mL penicillin and 100 μg / mL streptomycin (low concentration DMEM; manufactured by Nacalai Tesque) ) And transferred to a culture dish. When the cells became confluent, the mixed glial cells were washed with PBS and suspended in PBS containing trypsin. Suspended mixed glial cells were placed in a 6-well plate and subcultured in DMEM containing 1% (v / v) FBS. After 3 days, the culture plate was rocked vigorously for 2 hours at room temperature to separate microglia. Cells detached from the culture plate and floating in the culture medium were collected and used as microglia. Mouse microglia were passaged with DMEM (Nacalai Tesque) containing 10% (v / v) FBS (JRH Bioscience). Rat microglia were passaged in serum-free modified N3 medium consisting of high-concentration glucose DMEM / ham F12 medium containing transferrin, insulin, progesterone and sodium selenite (J. Neurosci. Res., 2002, Vol. 68, p. 150-160). The purity of microglia was about 95% as analyzed (assayed) by Mac-1 (CD11b) immunostaining.

7). Preparation of mouse astrocytes And prepared from mixed glial cells isolated from 1-day-old C57 / BL6 mice [Funakoshi H et al., J Neurosci Res, 2002, 68 (2): 150-160]. Mixed glial cells were isolated and cultured as in 6 above. After 3 days of culture, the culture plate was vigorously shaken at room temperature for 2 hours to remove microglia and the floating cells were removed. The purity of astrocytes after 3 days of culture is determined by anti-GFAP (glial fibrillary acidic protein; manufactured by Chemicon International) and ionized calcium-binding adapter molecule-1 (hereinafter abbreviated as IbaI). Y) analysis (assay) by double staining using an antibody (manufactured by Wako Pure Chemical Industries, Ltd.), it was about 95% or more.

8). Fibrous amyloid β binding and competition assay Human fibrillar synthetic amyloid β _1-42 peptide (hereinafter abbreviated as f amyloid β _1-42 ; manufactured by Seikagaku Corporation) was dissolved in dimethyl sulfoxide (DMSO) and 1 mg. The solution was diluted with sterilized water according to the instruction manual of the product so as to be / mL, and fibrillated immediately before use. Mouse microglia isolated from mixed glial cell cultures were isolated for 20 hours at 37 ° C. in DMEM supplemented with 10% (v / v) FBS with 2.5 μg / mL famyloid β _1-42 20 hours after isolation. Processed. The treated mouse microglia was washed thoroughly with ice-cold PBS and fixed with PBS containing 4% (v / v) paraformaldehyde (PFA). The immobilized mouse microglia were then incubated with primary antibodies anti-CD11b (Mac1) and anti-amyloid β (6E10) followed by anti-IgG conjugated with Alexa546 (stained red) or Alexa488 (stained green) Incubated with antibody (Molecular Probes; secondary antibody). Nuclei were counterstained with Hoechst 33342 (Molecular Probes; Invitrogen). Stained mouse microglia were observed under an LSM510 confocal microscope.

CHO-K1 cells were transfected with an expression vector or a mock expression vector (control vector) introduced with hSRCL-I-Myc, hSRCL-II-Myc, or mSR-AI-Myc with a Myc tag added to each gene. Twenty-four hours after transfection with the expression vector, transfected CHO-K1 cells were mixed with famyloid β _1-42 2.5 μg / mL, fucoidan 500 μg / mL, polyguanine (hereinafter abbreviated as poly (G)). 200 μg / mL or polycytosine (hereinafter abbreviated as “poly (C)”) 10% (v / v) FBS-added Ham F12 (F12 Nutrient Mixture) medium with or without 200 μg / mL (manufactured by GIBCO) And treated at 37 ° C. for 30 minutes. Cells were visualized using anti-Myc (12A5) and anti-β _1-42 -amyloid antibody described below and detected with Alexa546 (stained red) and Alexa488 (stained green) conjugated with the antibody, respectively. did.

9. The process f amyloid beta _1-42 in primary cultured mouse astrocytes for f amyloid beta _1-42 was dissolved in DMSO, diluted with PBS so as to be 300 [mu] M (equivalent to 1.35mg / mL), 3 days of culture at 37 ° C. did. The prepared f amyloid β _1-42 was decomposed by ultrasonic waves when necessary.

10. Preparation of antibody Synthetic peptide QPD (KAGQPDNWGHGHGPGEDC; SEQ ID NO: 15 (positions 691-708 of SEQ ID NO: 2)); equivalent to the CRD region of hSRCL; Biochem. Biophys. Res. Commun., 2001, 280 Volume (No. 4), p. 1028-1035; Biochim. Biophys. Acta., 2001, Volume 1522 (No. 1), pages 53-58) were obtained from MBL (FIG. 4A). Rabbits were immunized with SRCL / QPD peptide conjugated with keyhole limpet hemocyanin (KLH) by m-maleimidobenzol-N-hydroxysuccinimide ester. After immunization, blood was collected from rabbits, and serum was separated according to a standard method. The antiserum was purified with a CNBr activated Sepharose 4B (Amersham) affinity column conjugated with antigen only (excluding the KLH sequence) to obtain an antibody (the obtained antibody is hereinafter referred to as an anti-SRCL antibody). . Serum anti-SRCL antibody titers were raised against peptides containing QPD sequences in hSRCL CRD.

11. Western blot CHO-K1 cells and mouse macrophage J774A.1 cells were lysed 24 hours after transfection with hSRCL-I-Myc, hSRCL-II-Myc, mSRCL-Myc or Mock. Then, an extract with an equal protein mass of the lysate was subjected to SDS-PAGE (SDS-polyacrylamide gel electrophoresis). Immunoblotting was then performed with anti-SRCL antibody. An ECL system was used to visualize the resulting immunoblot.

12 Immunohistochemistry Mice were deeply anesthetized, cold PBS was injected from the heart to perfuse the whole body of the mice, and then the mice were perfused and fixed with cold 4% (v / v) PFA-containing PBS. The brain was removed, fixed with PBS containing 4% (v / v) PFA cold at 4 ° C. for 1.5 hours, and then divided into two on the midline in order to halve the brain. One half was placed in a series of PBS solutions containing saccharose (10%, 20%) and frozen in CO ₂ gas to prepare frozen sections.
The other half was dehydrated by a known general operation technique and embedded in paraffin to obtain a section. Regarding human material, brain tissue fixed with 10% (v / v) formalin was embedded in paraffin to prepare a section. The prepared paraffin sections were deparaffinized by sequentially incubating in 100% (v / v), 95% (v / v), and 75% (v / v) ethanol for 5 minutes each for rehydration. The deparaffinized sections were then washed twice with water and twice with PBS. To quench endogenous peroxidase activity, it was incubated for 5 minutes in PBS containing 3% (v / v) H ₂ O ₂ . In order to activate the antigen, the sections were immersed in 50 mM Tris-HCl (pH 8.0) containing 0.4 mg / mL protease K for 10 minutes at room temperature and washed with PBS. The washed sections were blocked for 30 minutes at room temperature with blocking buffer supplemented with 5% (v / v) normal goat serum and 0.3% Triton X-100. For immunostaining of amyloid peptide (amyloid β), sections were pretreated with 70% formic acid for 1 minute, blocked with blocking buffer and incubated with primary antibody for 1.5 hours at room temperature.
The following antibodies were used as antibodies.
Affinity-purified rabbit anti-SRCL / QPD (OT667, 3 μg / mL), mouse anti-GFAP (1: 400; MAB3402, Chemicon; for astrocytes), rat anti-Mac-1 (1: 100; Chemicon; MATO cells For microglia and macrophages), rabbit anti-Iba-1 (1: 1,000; manufactured by Wako Pure Chemical Industries, Ltd .; for microglia and macrophages containing MATO cells), anti-CD-31 (1: 200; PECAM-1, Antibody produced by Pharmingen; activated Lewis rat microglia as an immunogen; activated microglia / macrophages, for MATO cells and endothelial cells), anti-αSMA (1: 500; manufactured by DAKO, for smooth muscle cells), rabbit anti-amyloid _{β 40 (1: 1,500; FCA3340} ) and rabbit anti-amyloid β ₄₂ (1: 1 500; FCA3542) antibody (FCA3340 and FCA3542 was kindly provided to Dr. F. Checler)..

Sections were washed 3 times with PBS and then incubated for 20 minutes at room temperature with a secondary antibody conjugated with Alexa488 (stained green) or Alexa546 (stained red). Thereafter, it was washed with PBS to prepare a specimen. Human tissue sections were incubated with EnVision + system (K4001 mouse antibody / K4003 rabbit antibody; manufactured by Dako) and 3,3′-diaminobenzidine tetrahydrochloride (DAB; manufactured by Wako Pure Chemical Industries, Ltd.) as a chromogen. To visualize as a brown signal. For double-labeled immunohistochemistry, DAB was used to visualize with the primary antibody, and then the sections were placed in citrate buffer (pH 6.0) and heated in a microwave for 5 minutes. Sections were washed and incubated with secondary antibody. The sample was visualized as a red signal using alkaline phosphatase-conjugated anti-rabbit IgG (Vector) and Vector Red Alkaline Phosphatase Substrate Kit (Vector) according to the instruction manual, and Mayer's hematoxylin (Wako Pure Chemical Industries, Ltd.). ) Counterstained. Preabsorption was performed by incubating the sections with rabbit anti-SRCL / QPD antibody (OT667) pretreated with excess SRCL / QPD immunogen.
For double immunostaining of amyloid β _1-42 / amyloid β _1-40 and SRCL, rabbit anti-amyloid β _1-42 / amyloid β _1-40 antibody and affinity purified rabbit anti-SRCL antibody are Zenon Alexa Fluor Rabbit IgG Using Labeling Kit (Molecular Probes), each was directly labeled with Alexa546 and Alexa488 according to the instruction manual and used for immunofluorescence staining.

II. Test results 1. CDNA cloning of rat SRCL coiled-coil region The sequence of the rat SRCL cDNA region encoding the coiled-coil region was first compared with the sequence of human and mouse. For comparison, RT-PCR was performed using cDNA generated from rat microglia amplified with primers based on the mSRCL cDNA. The cloned cDNA corresponding to the rat SRCL coiled-coil region is 88% and 95% identical at the nucleotide level to that of hSRCL-I and mSRCL, respectively, and 91.7% and 95.9% at the amino acid level, respectively. And are very similar and contain eight possible N-glycosylation sites. Since the N-glycosylation site is conserved, the N-glycosylation site is thought to be important for SRCL protein folding, maturation, and / or function.

2. Expression of rat SRCL mRNA Human SRCL is transcribed as two different isoforms (isoforms), hSRCL-I and hSRCL-II. HSRCL-I containing C-type CRD (carbohydrate recognition domain) is a major transcript found in human tissues. hSRCL-I is separated into 3.0 kilobase (kb) bands by Northern blot. On the other hand, hSRCL-II lacking C-type CRD shows a 4.5 kb band by Northern blot. mSRCL was separated into one 3.0 kb transcript corresponding to hSRCL-I and no other transcripts were observed. This result indicates that only one isoform is expressed in mouse SRCL. In rats, it was found that rat SRCL mRNA was isolated as a single 3.0 kb band using a probe targeting the coiled-coil region of SRCL. Rat SRCL mRNA was expressed in a wide range (brain, lung, heart, spleen, stomach, small intestine, large intestine, kidney, muscle) (FIG. 1). SRCL mRNA expression was highest in the lung, followed by the spleen, small intestine, large intestine, stomach, and brain. On the other hand, the expression of rat SRCL mRNA in the liver, adrenal gland and thymus was below the detection limit. Thus, in the same rodent mouse and rat, only the SRCL transcript corresponding to hSRCL-I was expressed.
GAPDH serves as a loading control.

3. Expression of SRCL mRNA in rodent microglia and astrocytes In the brains of Alzheimer's patients, microglia and astrocytes are associated with senile plaques and recognize fibrillar amyloid β (f amyloid β) from the senile plaques. It is assumed that it contributes to capture and deletion. This can be easily done with cell surface scavenger receptors. To determine whether these cells express SRCL, we examined SRCL mRNA expression in rodent microglia (FIG. 2A, B) and astrocytes cultured using semi-quantitative RT-PCR. did. After 24 hours of culture (1 day in vitro, 1 DIV), SRCL mRNA was detected in astrocytes, and after 48 hours of culture (2 DIV), it was detected in microglia (MG) (FIG. 2C). On the other hand, SRCL mRNA expression in rat pheochromocytoma cells (PC12) and human C6 glioma cells (C6) was below the detection limit (FIG. 2C). These results indicate that SRCL mRNA is first expressed in glial cells among neural cells but not in neurons in vitro. Furthermore, it was found that the level of SRCL mRNA increases in a culture time-dependent manner (MG) or is regulated by the use of amyloid β-peptide (ASTRO). In microglia, the expression level of SRCL mRNA increased according to the culture time, and increased after 2 days compared to 1 day after the start of culture. And its level at 2 days in culture was higher than that in 7 days old brain, indicating that it was activated (FIG. 2D). From this result, it was considered that the level of SRCL mRNA in microglia correlated with the activity state of microglia. To determine whether f amyloid β _1-42 can alter SRCL mRNA expression, cultured mouse neonatal astrocytes are treated with f amyloid β _1-42 for the time indicated in FIG. 2E to reduce SRCL mRNA expression. analyzed. In analysis using semi-quantitative RT-PCR, SRCL mRNA levels increased proportionally with time following amyloid β _1-42 treatment compared to that of the corresponding control astrocytes (FIG. 2E; GAPDH is a loading control) To serve as.) Results analyzed using other primer sets of SRCL mRNA were similar. The induction level of SRCL mRNA in microglia was almost the same as that of astrocytes in vitro (1.9 vs. 2.4). A report by Nakamura et al. (Biochem. Biophys. Res. Commun., 2001, Vol. 280 (No. 4), p. 1028-1035; Biochim. Biophys. Acta., 2001) that SRCL acts as a scavenger receptor. 1522 (No. 1), pp. 53-58), and these results collectively, activated glial cells expressing SRCL are neurodegenerative in the brain affected by Alzheimer's disease. It is thought that it recognizes various substances, such as f amyloid β, which are present in the tissues.

4). Mouse microglia binds and takes up f amyloid β _1-42 microaggregates. To investigate the role of SRCL expressed in activated glial cells in the evaluation of f amyloid β _1-42 , fibrous β _1-42 − Conditions for positive control in which amyloid was recognized and taken up were established using microglia. When f amyloid β _1-42 prepared from synthetic amyloid β _1-42 -peptide and microglia were incubated at 37 ° C. for 3 days, f amyloid β _1-42 microaggregates (stained in green) In particular, it was found that the microglia (stained red) was incorporated and incorporated (FIG. 11).

5. For hSRCL-I and hSRCL-II expressed in CHO-K1 cells to investigate whether it has an activity of SRCL that bind f amyloid beta _1-42 binds the f amyloid beta _1-42, measure for the scavenger receptor In the presence or absence of one of the ligands, fucoidan, famyloid β _1-42 that binds to CHO-K1 cells expressing hSRCL-I was examined. CHO-K1 cells expressing the mouse class A type I scavenger receptor (mSR-AI) serve as a positive control. CHO-K1 cells were washed with PBS, fixed, and immunostained with antibodies against famyloid β _1-42 and Myc to visualize mSR-AI-Myc or hSRCL-I-Myc. fAmyloid β _1-42 binding (stained in green) was clearly seen in CHO-K1 cells expressing mSR-AI-Myc or hSRCL-I-Myc (stained in red). On the other hand, the f amyloid β _1-42 binding significantly competed in the presence of excessive amounts of fucoidan (FIG. 3A).
In order to further confirm the activity of SRCL that binds famyloid β _1-42 , CHO-K1 that stably expresses mSRCL-Fla in which a flag tag was fused to the cytoplasmic end of mSRCL was prepared. Similar results test f-amyloid β _1-42 (Cy3-f amyloid β _1-42 ) fluorescently labeled with cyanine 3 (Cy3) bound to CHO-K1 cells stably expressing mSRCL. (Fig. 12).
Compared to the omission of parental CHO-K1 cells that bind to Cy3-f amyloid β _1-42 , CHO-K1 cells stably expressing mSRCL are Cy3-f amyloid β _1-42 (in red) Strong binding ability). This binding was competed by fucoidan (FIG. 12). These results demonstrate that SRCL is specifically bound to _famyloid β _1-42 by using the same residues that interact with other scavenger receptor ligands such as fucoidan. In order to further investigate whether the CRD region of SRCL is essential for the binding of famyloid β _1-42 to SRCL, mSR-AI-Myc-, hSRCL-I-Myc-, hSRCL-II-Myc- or Mock- The binding between CHO-K1 cells transformed with the expression vector and f-amyloid β _1-42 was examined. Cells were detected and visualized with antibodies conjugated with anti-Myc and anti-amyloid β antibodies and Alexa546 (stained red) and Alexa488 (stained green). f Amyloid β _1-42 microaggregates (stained in green) are specific to mSR-AI-Myc-, hSRCL-I-Myc- and hSRCL-II-Myc-expressing cells (stained in red) Although bound, it did not bind in cells transfected with Mock. In addition to the CHO-K1 cells expressing mSR-AI, those cells expressing each isoform of hSRCL binds efficiently f amyloid beta _1-42, mock transfected cells f amyloid beta _{1- 42} was not bound. If there is a difference in the domain organization between hSRCL-I and -II, amyloid β _1-42 binding is initially mediated by the SRCL collagen-like domain, and the CRD of hSRCL-I contributes relatively little to binding. It is obvious.
Finally, famyloid β _1-42 that binds to these cells in the presence of poly (G) was examined. The scavenger receptor ligand inhibits _famyloid β _1-42 which binds to the polycationic region of the collagen-like domain of SR-AI. Poly (C) serves as a negative control. Binding of famyloid β _1-42 to CHO-K1 cells expressing mSR-AI was not inhibited by 200 μg / mL poly (C), but was inhibited by 200 μg / mL poly (G). To see if hSRCL binding to famyloid β _1-42 is mechanistically similar to binding by mSR-AI, the same analysis was performed on cells expressing hSRCL-I- or -II. In contrast to the mSR-AI results, binding to famyloid β _1-42 by cells expressing each isoform did not respond sensitively to 200 μg / mL poly (G) (FIG. 3C). Although the possibility that hSRCL-I and -II bind to f amyloid β _1-42 in a different way than mSR-AI could not be excluded, the most likely difference is in the single domain of SR-AI. In contrast, it appears to be in the large number of polycationic regions present in the collagen-like domain of SRCL (three domains). And it is thought that the binding affinity of SRCL for f amyloid β _1-42 is higher than that of SR-AI. In addition, the outline which shows the position of the coiled coil area | region, collagen-like domain, and CRD area | region in the domain structure of hSRCL-I, hSRCL-II, and mSRCL is shown to FIG. 4A.

6). Immunohistochemical localization of mSRCL in Tg-APP / PS1 mice For immunolocalization analysis of SRCL protein in brains that developed Alzheimer's disease, a rabbit anti-antibody specific for the QPD sequence in the CRD region of hSRCL -Created hSRCL polyclonal antiserum. The antiserum was purified using an affinity column carrying an immunizing peptide. The obtained purified antibody is hereinafter referred to as anti-SRCL antibody. Since hSRCL-I and mSRCL except for hSRCL-II have a CRD region containing a QPD sequence, anti-SRCL antibodies were expected to specifically recognize hSRCL-I and mSRCL. From Western blot analysis using anti-SRCL antibody, cells transformed with human SRCL-I-Myc / EGFP (hSRCL-I-Myc / EGFP), cells transformed with mouse SRCL-Myc / EGFP (MSRCL-Myc / EGFP) and mouse macrophages J774A.1 recognize SRCL protein with the expected molecular size, but transformed with Mock / EGFP and transformed with hSRCL-II-Myc / EGFP It was found that it was not recognized by the treated cells (FIG. 4B). Slight differences in the molecular weight of reactive proteins may reflect differences in glycosylation patterns. Furthermore, in order to confirm the specificity of this antibody, CHO-K1 cells transformed with hSRCL-I-Myc / EGFP, hSRCL-I-Myc / EGFP and SRCL-Myc / EGFP were immunostained with an anti-SRCL antibody. (FIG. 4C). By immunostaining of transformed cells, this antibody temporarily recognizes CHO-K1 cells expressing hSRCL-I-Myc / EGFP and mSRCL-Myc / EGFP, but Mock / EGFP and hSRCL-II-Myc It was found that CHO-K1 cells expressing / EGFP are not recognized. Moreover, pre-absorption of anti-SRCL antibody with the immunogen abolished the immunoreactivity of this antibody that recognizes CHO-K1 cells expressing hSRCL-I-Myc / EGFP.
These observations clearly show the specificity of the rabbit anti-SRCL antibody for hSRCL-I and mSRCL.

  Using this antibody, immunolocalization of SRCL in Tg-APP / PS1 mice (a dual transgenic mouse model of familial Alzheimer's disease co-expressing mutant amyloid β precursor protein and mutant human presenilin 1) It was investigated. In wild-type littermates, except for specific staining of SRCL present in perivascular macrophages (data not shown), no specific SRCL-immunity positivity of microglia and astrocytes located in the cerebral cortex was observed (FIG. 5A). , Upper figure, arrow). In contrast, massive SRCL-immunity positivity was visualized in astrocytes that react around GFAP-positive plaques in both cerebral cortex and hippocampus of 9-month-old Tg-APP / PS1 mice (FIG. 5A). The intensity of SRCL immunoreactivity was strong in GFAP positive astrocytes near amyloid β plaques (FIG. 5A, arrowheads) but weak in astrocytes located distal to such amyloid β plaques (FIG. 5A). , Arrow). These data are consistent with amyloid β increasing astrocyte SRCL levels in vitro (FIG. 2E). On the other hand, SRCL-immunity positivity was not detected in wild type mice, but was only slightly detected in neurons in the hippocampal amyloid CA1 region in the context of amyloid β plaques in 9 month old Tg-APP / PS1 mice. (FIG. 5A, bottom diagram). In microglia, SRCL-immunity was below the detection limit in wild type mice (FIG. 5B, top panel). On the other hand, in Tg-APP / PS1, a slight up-regulation (increase) of SRCL-immunity of microglia was observed in vivo with amyloid β plaque. These results indicate that SRCL plays an important role in glial cells such as astrocytes and microglia in Alzheimer patients.

Next, whether SRCL can be induced in cells following the onset of cerebral amyloid angiopathy (CAA), a typical pathological feature of Alzheimer's disease, SRCL and 9 month old Tg-APP / PS1 mice And SRCL and amyloid β _1-42 or β _1-40 double immunostaining in the cerebral cortex of their wild-type littermates. Paraffin sections of cerebral cortex of 9 month old Tg-APP / PS1 mice and wild-type littermates were analyzed using Alexon 546 (stained in red) and Alexa 488 (stained in green) using Zenon rabbit IgG Labeling Kit (manufactured by Invitrogen). Incubated with rabbit anti-amyloid β _{1-40 / 1-42} and rabbit anti-SRCL antibody, respectively, previously labeled. In order to visualize cell nuclei, counterstaining was performed with Hoechst33342 (stained in blue). Except for specific SRCL staining of perivascular macrophages (mv; arrowheads in the upper part of FIG. 6) of wild-type littermates of the same age, specific staining for amyloid β-immunity positive or SRCL-immunity positive is the meninges Was not seen inside the blood vessels. In contrast, SRCL-immunopositive and amyloid β _{1-42 / 1-40} colocalization was prominent, especially in meningeal blood vessels showing the characteristics of CAA in Tg-APP / PS1 mice ( Fig. 6, arrows). At high magnification, SRCL-immunopositive and amyloid β _1-40 -immunopositive appeared as particles. Many of them co-localized inside the blood vessel wall of blood vessels / perivascular cells (FIG. 6). Sections incubated without the first antibody were unstained (FIG. 6, bottom panel), confirming the specificity of SRCL-immunity positivity.
FIG. 7A shows a schematic representation of cells in the blood vessels / perivascular of wild-type mice and meningeal blood vessels (mv) of Tg-APP / PS1. In wild type mice, m. v. Contains vascular endothelial cells (ECs: stained bright green) surrounded by smooth muscle cells (SMCs: stained orange), and the vessel wall is thin. Near the blood vessels are perivascular macrophages (MATO cells: stained yellow). Tg-APP / PS1 m. v. Then, a large amount of amyloid β (stained red) is localized in the blood vessel wall. As many macrophages (Mφ: stained yellow) adhere to and penetrate the vessel wall, the number of SMCs increases and the vessel wall becomes thinner during disease progression. Blood vessels / perivascular cells contain a large number of amyloid β particles (stained red) inside the cells, presumably as a result of phagocytosis. These amyloid β-containing cells include not only macrophages (Mφ) and MATO cells, but also SMCs and ECs. Even if the localization is recognized by amyloid β, the molecule involved in the binding and ingestion of amyloid β has not been identified.
Since SRCL-immunopositive co-localized with Tg-APP / PS1-intravascular amyloid β _{1-40 / 1-42} -immunopositive (FIG. 6), vascular / perivascular cell specific antibody [anti-rat CD31 (Active Lewis rat microglia as antigen, markers as microglia / macrophages containing MATO cells and ECs), anti-αSMA (marker as MCs), and anti-GFAP antibody (marker as astrocytes)] The blood vessels / perivascular cells were examined whether the blood vessels / perivascular cells express SRCL. In wild-type littermates, except for MATO cells (FIG. 7B, arrowheads), SR31-immunity positive (stained in green) coexisting with CD31-immunity positive (stained in red; FIG. 7B, upper panel) is easy Could not be observed. Wild type litter m. v. In SRCL-immunopositive, only a few were detected in αSMA-positive MCs, but a significant amount was detected in CD31-positive ECs (FIG. 7B, center and top, respectively). In contrast to wild-type littermates, SRg-immunopositive (stained green) was strongly detected in a small population of blood vessels / perivascular cells in Tg-APP / PS1 mice (FIGS. 7C and 7D). The strength of SRCL-immunity positive (stained in green) is the same as that of MATO cells, in addition to CD31-immunity positivity in Tg-APP / PS1, and SRCL-immunity positivity is an invasive macrophage. Was very strong in the inner wall of the meningeal vascular wall, as well as in ECs (FIG. 7B, top view, arrow; stained red). This suggests the induction of SRCL in macrophages. In a highly magnified view (FIG. 7C, top view), SRCL-immunopositive (stained in green) appears as a number of particles inside CD31-positive macrophages (stained in red) that have accumulated in the vessel wall. It was. This indicates that SRCL is taken up into cells by macrophages. The strength of SRCL-immunopositive (stained in green) was significantly increased in αSMA-positive SMCs from Tg-APP / PS mice (FIG. 7B, middle panel). At high magnification (FIG. 7C, bottom panel), SRCL-immunostaining was co-stained intracellularly as a number of particles in αSMA-positive cells. This indicates that SRCL was taken up into cells in SMCs of Tg-APP / PS1. In Tg-APP / PS1, GFAP positive astrocyte cell bodies (stained in red) and limbs surrounding brain microvessels (cv) associated with cerebral amyloid angiitis (CAA) (stained in red) ) A strong induction of SRCL-immunopositive (stained green) was observed within both. Astrocytes associated with amyloid plaques (asterisks) were markedly intracellularly expressed SRCL-immunopositive. This indicates that amyloid β is taken up into cells by SRCL in astrocytes. Occasional astrocytes were observed that stretched toward both amyloid plaques (asterisks) and brain microvessels (cv). This finding is derived from amyloid plaques c. v. It shows that SRCL of astrocytes plays a role in causing amyloid β clearance due to movement into the systemic circulation via.

From the above immunostaining results, in wild-type littermates, SRCL-immunostaining ability was slight in SMCs but was present in MATO cells. On the other hand, in Tg-APP / PS1 mice, it was found that SRCL-immunity positivity was remarkably induced inside the blood vessel wall and colocalized with amyloid β _{1-40 / 1-42} -immunity positivity as particles. Within blood vessels / perivascular cells, SRCL was induced in invasive macrophages, MATO cells, SMCs, ECs and Tg-APP / PS1 astrocytes. This indicates that SRCL up-regulated (induced) in a sub-population of blood vessels / perivascular cells plays an important role in amyloid β binding and clearance in the Tg-APP / PS1 brain.
7). Quantification of SRCL expression in the brains of Alzheimer's disease patients by real-time RT-PCR Pathological anatomy from Alzheimer's disease patients and non-Alzheimer's disease (control) listed in Table 4 with respect to the expression and localization of SRCL in the brains of Alzheimer's disease patients The expression of SRCL mRNA in the sample was examined.

  Although the number of pathological anatomical samples is limited and insufficient for statistical analysis, quantitative real-time RT-PCR showed a trend towards increased levels of SRCL mRNA in the cortical tissue of Alzheimer's disease patients compared to control subjects (Figure 8).

8). Expression of hSRCL in the brain of Alzheimer's disease patients The immunolocalization of SRCL in the temporal cortex of Alzheimer's disease patients was examined in detail. Immune activity was visualized using DAB (stained brown). As a result, it was confirmed that many SRCL immunoreactive cells were present in the brain tissue of Alzheimer's disease, but there were few SRCL immunoreactive cells in the control subject samples (FIG. 9A). Using SRCL / GFAP-double immunostaining, a strong SRCL-immunopositive (pink) is found in the reactive astrocytes surrounding the plaque (asterisk) and the end of the perivascular astrocytes (brown) , Colocalization with GFAP- (brown) was observed (FIG. 9B). At high magnification, SRCL-immunostaining was observed as particles in the cells (FIG. 9B, right panel). Furthermore, when SRCL and Iba-1 (microglia / macrophages containing MATO cells) were co-stained, SRCL-immunostaining (stained in pink) is an Iba1-positive reaction in the brain affected by Alzheimer's disease. Type microglia (arrows) or vascular macrophages and MATO cells (arrowheads) were detected (FIG. 9C). Moreover, double staining of SRCL (stained pink) and amyloid β _{1-40 / 1-42} (stained brown) indicates that SRCL-immunopositive vascular cells of Alzheimer patients, such as invasive macrophages, It was clarified that co-localization with amyloid β was performed as multiple particles in the intracellular compartments of SMCs and certain ECs (FIGS. 10A and 10B, arrowheads). This result is consistent with the result with Tg-APP / PS1 (FIGS. 6 and 7C). The specificity of SRCL-immunity was confirmed by preabsorbing the antibody with its antigen. In the brain of Alzheimer's disease, SRCL-immunity positivity is associated with plaque-associated reactive astrocytes, activated microglia and numerous particles inside blood vessels / perivascular cells, such as invasive macrophages within the vessel wall and CAA It appeared as SMCs and ECs etc. in agreement with the results in the Tg-APP / PS1 mice. The results indicate that reactive astrocyte up-regulated SRCL, activated microglia and vascular / perivascular cells are involved in amyloid β binding and clearance in Alzheimer patients.

  The therapeutic agent or disease-suppressing agent of the present invention is useful as a drug for preventing or treating Alzheimer's disease.

Claims (16)

  A scavenger receptor protein or a protein having substantially the same activity as a scavenger receptor protein or a salt thereof, a DNA encoding a scavenger receptor protein, or a DNA comprising a base sequence complementary to the DNA, and stringent conditions An amyloid β clearance promoter comprising, as an active ingredient, DNA containing DNA that encodes a protein that hybridizes below and has substantially the same activity as the scavenger receptor protein.   The active ingredient is (a) a protein containing the same amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or (b) a protein containing the amino acid sequence substantially identical to the amino acid sequence or a salt thereof, or ( c) hybridizes under stringent conditions with DNA consisting of the base sequence represented by SEQ ID NO: 1, 3 or 5 or (d) DNA consisting of a base sequence complementary to the base sequence of (c) above; and The promoter according to claim 1, wherein the promoter comprises DNA encoding a protein having substantially the same activity as that of a scavenger receptor protein.   The active ingredient is a protein comprising (a) the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 or (b) a protein comprising substantially the same amino acid sequence as the amino acid sequence. 3. The accelerator according to item 1 or 2.   Under active conditions, the active ingredient is (c) DNA consisting of a base sequence represented by SEQ ID NO: 1, 3 or 5 or (d) DNA consisting of a base sequence complementary to the base sequence of (c) above. The promoter according to claim 1 or 2, which is a DNA containing a DNA that encodes a protein that hybridizes and has substantially the same activity as that of a scavenger receptor protein.   DNA from retrovirus vector, adenovirus vector, adeno-associated virus vector, herpes virus vector, vaccinia virus vector, poxvirus vector, poliovirus vector, shinbis virus vector, Sendai virus vector, SV40 vector and immunodeficiency virus vector The promoter according to any one of claims 1, 2 or 4, which is incorporated into at least one selected vector.   The promoter according to any one of claims 1 to 5, wherein amyloid β is cerebral amyloid β.   The promoter according to any one of claims 1 to 6, wherein the promoter is locally administered to an accumulation site of amyloid β.   6. The promoter according to claim 4 or 5, which is administered intrathecally.   An injection comprising the cell transfected with the promoter according to claim 5.   A scavenger receptor protein or a protein having substantially the same activity as a scavenger receptor protein or a salt thereof, a DNA encoding a scavenger receptor protein, or a DNA comprising a base sequence complementary to the DNA, and stringent conditions Prevention of a disease caused by accumulation of amyloid β, comprising as an active ingredient a DNA comprising a DNA that encodes a protein that hybridizes under and has substantially the same quality of activity as a scavenger receptor protein Therapeutic agent.   The active ingredient is (a) a protein containing the same amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or (b) a protein containing the amino acid sequence substantially identical to the amino acid sequence or a salt thereof, or ( c) hybridizes under stringent conditions with DNA consisting of the base sequence represented by SEQ ID NO: 1, 3 or 5 or (d) DNA consisting of a base sequence complementary to the base sequence of (c) above; and 11. The preventive or therapeutic agent according to claim 10, which is a DNA comprising a DNA encoding a protein having substantially the same activity as a scavenger receptor protein.   12. The preventive or therapeutic agent according to claim 10 or 11, wherein the disease is Alzheimer's disease.   DNA that encodes a therapeutically effective amount of a scavenger receptor protein, a protein having substantially the same activity as a scavenger receptor protein, or a salt thereof, or a scavenger receptor protein in patients who need to promote amyloid β clearance Alternatively, a DNA containing a DNA encoding a protein that hybridizes under stringent conditions with a DNA having a base sequence complementary to the DNA and has substantially the same activity as the scavenger receptor protein is administered as an active ingredient. A method for promoting amyloid β clearance, characterized by comprising:   The active ingredient is (a) a protein containing the same amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or (b) a protein containing the amino acid sequence substantially identical to the amino acid sequence or a salt thereof, or ( c) hybridizes under stringent conditions with DNA consisting of the base sequence represented by SEQ ID NO: 1, 3 or 5 or (d) DNA consisting of a base sequence complementary to the base sequence of (c) above; and The method for promoting amyloid β clearance according to claim 13, which is a DNA comprising a DNA encoding a protein having substantially the same activity as that of a scavenger receptor protein.   A scavenger receptor protein, a protein having substantially the same activity as that of a scavenger receptor protein, or a salt thereof, or a DNA encoding a scavenger receptor protein or a DNA complementary thereto, for producing an amyloid β clearance promoter Use as an active ingredient of DNA containing DNA encoding a protein that hybridizes with stringent DNA under stringent conditions and that has substantially the same quality of activity as a scavenger receptor protein.   The active ingredient is (a) a protein containing the same amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or (b) a protein containing the amino acid sequence substantially identical to the amino acid sequence or a salt thereof, or ( c) hybridizes under stringent conditions with DNA consisting of the base sequence represented by SEQ ID NO: 1, 3 or 5 or (d) DNA consisting of a base sequence complementary to the base sequence of (c) above; and The use according to claim 15, which is a DNA comprising a DNA encoding a protein having substantially the same activity as that of a scavenger receptor protein.