MXPA01002752A - Ly6h gene - Google Patents

Abstract

A brain-specific gene useful in treating Alzheimer's disease, etc. which contains a base sequence encoding the amino acid sequence represented by SEQ ID NO:1;a fragment thereof;an expression vector containing the gene;a host cell containing the expression vector;an expression product of the gene;an antibody against the same;and remedies and preventives for nerve degeneration diseases, etc.

Description

GEN LY6H TECHNICAL FIELD The invention relates to a gene expressed with high specificity at a high level in the brain, more particularly to a gene coding for a novel protein belonging to the Ly6 family (see literature cited below), which has been used in the purification of stem cells of the blood, in studies on the differentiation of blood cells, in the activation of immune cells, in the inhibition of the production of active immune cells, in the treatment of tumors, and the like. The invention further relates to a novel protein encoded by said gene, and to its specific antibody. In addition, the invention relates to a therapeutic and prophylactic composition for a neurodegenerative disease such as Alzheimer's disease.

BACKGROUND OF THE INVENTION The proteins of the Ly6 family have a structure anchored to GPI of low molecular weight, and have been identified as a class of cell surface glycoproteins that form a cluster of genes on mouse chromosome 15 [Proc. Nati Acad. Sci., USA, 84, 1638-1643 (1987)].

The Ly6 family is specifically expressed at high levels in cells of the bone marrow and lymphoid cells, and therefore has been used as a marker for differentiation of T cells and for hematopoietic stem cells [Immunol. Cell Biol., 73, 277-296 (1995)]. Although much remains to be known about its in vivo functions, the finding that its expression is highly modulated in the lymphocyte system suggests that these proteins have important functions in the immune system, particularly in the differentiation and function of T cells. For example, Ly6c is reported to intervene in the direction of CD8 + T cells towards the lymphatic node by integrin-dependent adhesion [Proc. Nati Acad. Sci., USA, 94. 6898-6903 (1997)]. In addition, it is known that many GPI-anchored proteins interact with protein kinases [Science, 254, 1016-1019 (1991)]. For example, the interaction of Ly6 with p561ck and p59fyn suggests the likelihood of its involvement in the translation of T cell signal [Eur. J. Immunol., 23, 825-831 (1993)]. It has also been reported that T cells derived from mice defective in Ly6a, increased their ability to proliferate in response to antigenic stimulation [J. Exp. Med., 186, 705-717 (1997)]. It has also been suggested that they regulate the activation, not only of T cells, but also of B cells [J. Immunol., 144, 2197-2204 (1990)]. In addition, it is known that several GPI-anchored proteins are expressed and function both in the lymphocyte system and in the nervous system [Nature, 37_9, 826-829 (1996); Curr. Biol., 7, 705-708 (1997)]. In the Ly6 family, it is reported that Ly6a.2 and Ly6E are present and work in both systems [Proc. Nati Acad. Sci., USA, 85, 2255-2259 (1996); J. Immunol., 157, 969-973 (1996)]. It is considered that the elucidation of the physiological roles performed by said proteins of the Ly6 family and the genes coding for such proteins, and the resulting information, are useful in the field of fundamental scientific research, as well as in the pharmaceutical field with regarding the purification of blood stem cells, studies on the differentiation of blood cells, activation of immune cells, inhibition of immune cell activation, tumor therapy and the like. Recently, excessive atrophy of the brain's temporal lobe has been reported in patients with Alzheimer's disease compared with age-related brain atrophy [Jobst et al., Lancet, 343, 829-830 (1994)], suggesting that some gene or genes that have a relationship with the cerebral temporal lobe are associated in some way with the onset and progression of Alzheimer's disease. It is logical to assume that if such a gene could be identified or characterized, it could provide useful information for the therapy and prophylaxis of Alzheimer's disease. Therefore, an objective of the present invention is to provide the above information, necessary for those interested, particularly a f - "-» »jífi» lm¿iH- »- gi-«. ^^^^ g ^^ fe ^^^^^^^^^ i ^^^^^^?, a novel human protein that belongs to the Ly6 family, and a gene that codes for the protein.An additional objective of the invention is to provide a pharmaceutical composition for the therapy and prophylaxis of various neurodegenerative diseases, represented by Alzheimer's disease. the genes derived from various human tissues and succeeded in isolating and characterizing a novel brain-specific gene that meets the above objectives.The inventor also found that the level of expression of this newly isolated gene is depressed notably in the temporal lobe, including in the hippocampus and the entorinal cortex, of a patient with Alzheimer's disease; that this is a factor that causes the onset and progression of Alzheimer's disease and dementia and other disorders; and that this gene and its expression product can be used to advantage in the therapy and prophylaxis of the disease Alzheimer's. The present invention has been made based on the above findings.

DESCRIPTION OF THE INVENTION The present invention provides a gene comprising a nucleotide sequence encoding the following protein (a) or (b). (a) A protein having the amino acid sequence shown in SEQ ID NO: 1. (b) a protein having an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 1, by deletion, substitution or addition of one or a plurality of amino acids, and having at least one physiological activity selected from the group which consists of neuronal survival support activity, nerve lengthening activity, nerve regeneration activity, neuroglia activation activity, and mnemonic activity (of brain memory formation). The invention also provides the above gene, wherein the nucleotide sequence is shown in SEQ ID NO: 2, in particular, which is a human gene. In addition, the invention provides a gene comprising the following polynucleotides (a) or (b), particularly the corresponding human gene. (a) a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 3. (b) a polynucleotide that hybridizes under severe conditions to a DNA having the nucleotide sequence shown in SEQ ID NO: 3. The invention also provides a gene expression vector that stores said gene; a host cell that stores said gene expression vector; an expression product that is expressed by said host cell; a protein encoded by the gene of the invention; and an antibody that binds to said expression product or said protein.

The invention also provides a therapeutic and prophylactic composition for neurodegenerative disease, comprising said protein or an equivalent thereof, or said expression product, as an active ingredient in combination with a pharmaceutical carrier. More particularly, the invention provides the therapeutic and prophylactic composition for neurodegenerative disease, wherein said active ingredient is a protein having the amino acid sequence shown in SEQ ID NO: 1, or an equivalent thereof, or a product of gene that can be obtained by expressing a part or all of a gene comprising a The nucleotide sequence shown in SEQ ID NO: 2, and having at least one physiological action selected from the group consisting of supporting action of neuronal survival, nerve lengthening action, nerve regeneration action, action of neuroglia activation and brain memory formation action (mnemonic, coding). Especially, the invention provides the therapeutic and prophylactic composition for Alzheimer's disease, Alzheimer's dementia, cerebral ischemia and Parkinson's disease. In addition, the invention provides a sense chain oligonucleotide comprising at least 20 constituent nucleotides consecutive of the nucleotide sequence shown in SEQ ID NO: 2; a gene therapy composition comprising said sense chain oligonucleotide as an active ingredient in combination with a pharmaceutical carrier; and a gene-specific probe comprising a oligonucleotide sequence of at least 10 consecutive constituent nucleotides of the nucleotide sequence shown in SEQ ID NO: 2. In addition, the invention provides a method of selecting candidate compounds, capable of binding said protein, to equivalents thereof, or to expression products, or affecting their activity, which comprises using said protein, equivalent or expression product; a team for such selection; and said compounds thus selected. In the specification, the representation of amino acids, peptides, nucleotide sequences, nucleotides, etc., by means of abbreviations, is in accordance with the rules recommended by the IUPAC-IUB [Communication of the IUPAC-IUB on biological nomenclature, Eur. J. Biochem., 138. 9 (1984)], "Guideline for drafting patent specifications relative to nucleotide sequences and / or amino acid sequences" [Guidelines for drafting patent specifications related to nucleotide sequences and / or amino acid sequences] ( edited by the Patent Office of Japan), and the conventions of the art relating to the use of codes or symbols. A specific example of the gene of the invention is the gene deduced from the DNA sequence of the PCR product designated "LY6H", as described in the example hereinafter. Its nucleotide sequence is shown in SEQ ID NO: 3. The LY6H gene is a cDNA containing an open reading frame (ORF) of 420 codons, which codes for a novel brain-specific protein (LY6H protein) having a sequence of 140 amino acid residues as shown in SEQ ID NO: 1, and has a full-length sequence of 854 nucleotides. Through a search in the GenBank / EMBL databases, using the FASTA program (Person WR et al., Proc. Nati. Acad. Sci., USA, 85, 2444-2448 (1988)), it was found that the protein LY6H, which is the expression product of the gene of the invention, has high homology with the proteins of the Ly6 family of mice [Immunol. Cell Biol., 73, 277-296 (1995)]. In addition, high homology from gene to gene was recognized. Therefore, it is considered that the gene of the invention is a novel human Ly6 gene. By sequencing more than 28,000 cDNA clones randomly selected from a human fetal brain cDNA library, the LY6 gene of the invention was identified as a gene that is specifically expressed in the brain. By means of chromosome mapping RH [Hum. Mol. Genet, 5, 339-346 (1996)], it was found that the locus of the gene on the chromosome was 8q24.3. In this way, the gene and the expression product of the invention thus provided contribute to the detection of the expression of the gene in various tissues, to the production of human LY6H protein by genetic engineering techniques, and to the construction of an antibody for the same, thus allowing the purification of hematopoietic stem cells, the study of blood cell differentiation, the activation or suppression of immune cells, the therapy of tumors, and the like.

In addition, the expression product (polypeptide) of the invention thus provided allows the provision of a drug for prophylaxis and therapy of neurodegenerative diseases such as Alzheimer's disease, Alzheimer's dementia, Parkinson's disease and cerebral ischemia. Additionally, the sense chain of the gene according to the invention can be used as a pharmaceutical composition for gene therapy, with which the onset and progression of the aforementioned neurodegenerative diseases can be inhibited or stopped. The invention also provides a method of selecting compounds that bind to the expression product (polypeptide) of the invention, or that affect its activity, and a team relevant to the selection, and therefore also the compounds thus selected. For the identification of such compounds, an antibody that binds to the expression product of the gene of the invention can be used. In the specification, the term "gene" is used to refer to a double-stranded DNA and its constituent DNA of a single chain, either sense or antisense, without considering its length. Therefore, unless otherwise indicated, the gene (DNA) of the invention includes a double-stranded DNA that contains a human genomic DNA, a single-stranded DNA (sense chain) that includes the cDNA, a single chain DNA (antisense chain) having a sequence complementary to said sense chain, and fragments of said DNAs.

The gene (DNA) of the invention can contain a leader sequence, a coding region, exons and introns. The polynucleotide includes both RNA and DNA. The DNA includes cDNA, genomic DNA and synthetic DNA. The polypeptide includes its fragments, homologs, derivatives and mutants. Mutants include naturally occurring alleles, non-naturally occurring mutants, mutants that have mutated amino acid sequences by deletion, substitution, addition and / or insertion, and mutants that have functionally equivalent modified amino acid sequences. These modifications of amino acid sequences (for example mutations) can occur, for example, by spontaneous mutation or post-transcriptional modification, but can be induced artificially using a native gene (for example, the specific genes of the invention). The homology of such mutants with the non-mutated polypeptide can be at least 70%, preferably 80%, preferably 95%, and most preferably 97%. The above polypeptide and its mutants and homologues have a structural feature conserved in common and may have the biological activities of the expression product of the gene of the invention, such as neuronal survival support action, neuronal growth stimulating action, generation action of nerve and action of neuroglia stimulation. The homology of the polypeptides can be analyzed by searching through a database such as the SWISSPLOTS database, using a sequence analysis software such as FASTA [Clustal V., Methods Mol. Biol., 25, 307-318 (1994)].

The gene that codes for such a mutant is silent or conserved in amino acid substitution. In this manner, the amino acid residues encoded by the nucleotide sequence are not altered. The conservatively substitutable amino acid residues, i.e., the amino acid residues that can be substituted with other amino acid residues without losing the activities of the polypeptide having the original amino acid residues, and the corresponding original amino acid residues, are as follows. Amino acid residue Residue of amino acid original substituent conservative Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn or GIn lie Leu or Val Leu ile or VaL Lys Arg, Aln or Glu Met Leu or lie Phe Met, Leu or Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp or Phe Val lie or Leu In addition, Cys can substitute a different type of amino acid residue, for example Ser, Ala or Val. The gene and the expression product according to the invention provide information and means of great use for the elucidation, diffusion, diagnosis, prophylaxis and therapy of neurodegenerative diseases such as Alzheimer's disease, cerebral ischemia and Parkinson's disease. Also, the gene of the invention can be used with advantage for the development of new drugs capable of inducing the expression of the gene, for use in the treatment of said neurodegenerative diseases. Furthermore, the detection of the expression of the gene of the invention and of the resulting expression product in individuals or tissues, and the detection of mutation (deletion or point mutation) of the gene or of the abnormal expression thereof, can be used to advantage for elucidate and diagnose these neurodegenerative diseases. The gene of the invention includes, without limitation, the gene having the nucleotide sequence shown in SEQ ID NO: 2, which codes for a protein having the amino acid sequence shown in SEQ ID NO: 1, for example a gene (gene LY6H) having the nucleotide sequence shown in SEQ ID NO: 3. For example, the gene of the invention may be a gene encoding an amino acid sequence derived from the amino acid sequence defined above by a given modification, a gene encoding an amino acid sequence having a given degree of homology with the amino acid sequence defined above, or a gene having a nucleotide sequence that has a given degree of homology with any of the above genes. The given degree of homology mentioned above, with a A sequence of amino acids or a defined nucleotide sequence means a homology of at least not less than 70%, preferably not less than 90%, preferably not less than 95%, most preferably not less than 97%. The present invention encompasses homologs (gene homologs and protein homologs) having said homology. The gene of the invention includes "a gene encoding a polypeptide having an amino acid sequence (modified amino acid sequence) derived from the amino acid sequence shown in SEQ ID NO: 1 by deletion, substitution or addition of one or a plurality of amino acids ". The extension and position, or positions, of "Deletion, substitution or addition" are not particularly restricted as long as the resulting polypeptide having a modified amino acid sequence is equivalent in biological function to the polypeptide (LY6H protein) having the amino acid sequence shown in SEQ ID NO: 1 .

The aforementioned biological "function" includes physiological functions such as neuronal survival support action, nerve lengthening action, nerve regeneration action, neuroglia activation action and mnemonic action, and the "equivalent" is a polypeptide that has such 5 functions. Therefore, the protein having said modified amino acid sequence includes a protein (equivalent) having a fragment (a fraction of consecutive residues) of the amino acid sequence shown in SEQ ID NO: 1, and has physiological activities similar to those mentioned above for the full length of said sequence of amino acids. In addition, the gene encoding a polypeptide having the modified amino acid sequence mentioned above can be a gene with which the gene of the invention encoding a polypeptide having the amino acid sequence prior to modification can be detected. The term plurality, as used in connection with said modification, usually means not less than 2, but up to several, although the range is not restrictive. The homolog of the LY6H gene (and the homolog of the gene expression product) according to the invention, means any of a series of related genes (and their expression products) that are homologs in sequence and are recognized as a family of genes based on their structural characteristics, common genetic expression pattern, and similarities in said biological function. This, of course, includes alleles of the genes of the invention. or . ¡& ?? . ~ t V ^ toi ^ &L Modification (mutation) of an amino acid sequence can occur naturally, for example by spontaneous mutation and post-translational modification, but can be induced artificially at the base of the native gene (for example the specific gene of the invention). The invention covers each and every one of the modified genes having the aforementioned characteristics, without considering the cause or the means of modification or mutation. Artificial means for such modification (mutation) of the amino acid sequence include genetic engineering techniques such as site-specific mutagenesis [Methods in Enzymology, 154. 350, 367-382 (1987); ibid, 100, 468 (1983); Nucleic Acids Res., 12, 9441 (1984); "Zoku Seikagaku Koza" [Experiments in Biochemistry, second series), 1: Idenshi Kenkyuho (Methods of genetic research) II, Biochemical Society of Japan (editors), page 105 (1986), etc.], chemical synthesis methods such as the phosphotriester method and the phosphoamidite method [J. Am. Chem. Soc, 89, 4801 (1967); ibid, 91, 3350 (1969); Science, 150, 178 (1968); Tetrahedron Lett., 22, 1859 (1981); ibid, 24, 245 (1983)], and combinations of such methods. More particularly, the DNA can be synthesized by means of a chemical method such as the phosphoamidite method or the phosphotriester method, and this synthesis can be carried out in a commercially available automatic oligonucleotide synthesizer. The double-stranded fragment can be obtained from the single-stranded fragment synthesized and "i¡r., and > Chemically, synthesizing a complementary strand and matching it under suitable conditions or adding the complementary strand using a suitable primer sequence and a DNA polymerase. A specific example of the gene according to the invention is the gene having the nucleotide sequence shown in SEQ ID NO: 3. The coding region (the sequence shown in SEQ ID NO: 2) of this nucleotide sequence is an example of codon combination specifying the respective amino acid residues of the amino acid sequence shown under SEQ ID NO: 1. The gene of the invention is not limited to the gene having said defined nucleotide sequence, but includes any gene having a nucleotide sequence that can be obtained by selecting any arbitrary combination of codons for each amino acid residue. The selection of codons can be done routinely, with reference to the use of the codon in the host to be used [Nucleic Acids Res., 9, 43 (1981)]. In addition, the gene of the invention includes one having a nucleotide sequence that shows a certain level of homology with the nucleotide sequence shown in SEQ ID NO: 3. Inferred by said level of homology, are polynucleotides and complementary polynucleotides having at least 70% homology, preferably at least 90% homology, most preferably at least 95% homology, with the nucleotide sequence shown in FIG. SEQ ID NO: 3 The gene having said level of homology, for example, can be characterized as a polynucleotide that hybridizes with a DNA having the nucleotide sequence shown in SEQ ID NO: 3 under severe conditions. More particularly, the gene having a nucleotide sequence that hybridizes to the DNA having the nucleotide sequence shown in SEQ ID NO: 3 under the condition of 6x SSC at 65 ° C overnight, or 50% formamide - SSC 4x at 37 ° C during the night, is included in the concept of the gene that has said level of homology. Here, SSC represents standard saline citrate (SSC 1x = 0.15 M NaCl, 0.015 M sodium citrate). The gene of the invention can be easily produced and isolated by means of general genetic engineering technology, based on the sequence information on any specific example of the gene of the invention as described in this specification [eg, "Molecular Cloning", 2nd. Edition, Cold Spring Harbor Lab. Press (1989); Zoku Seikagaku Jikken Koza (Biochemistry Experiments, second series): "Idenshi Kenkyuho" (Methods of genetic research) I, II, III, Biochemical Society of Japan (editors), (1986)]. More particularly, this can be done by preparing a cDNA library from a suitable source, in which the gene of the invention can be expressed, by a routine procedure and selecting a desired clone of this library using a suitable probe or antibody specific for the gene of the invention [Proc. Nati Acad. Sci., USA, 78, 6613 (1981); Science, 222, 778 (1983)].

The cDNA source that can be used in the above-mentioned process includes several cells and tissues expressing the gene of the invention, as well as cultured cells derived therefrom, particularly brain tissues. The isolation of total RNA from such a source, the isolation and purification of mRNA, and the acquisition and cloning of cDNA, can also be carried out in the conventional manner. In addition, cDNA libraries are commercially available, and the present invention can be practiced using such cDNA libraries, for example the cDNA libraries available from CLONTECH Lab. Inc. The method of selecting the gene of the invention from A cDNA library is not particularly restricted but the conventional method can be employed. Examples of screening methods include an immunoselection method using an antibody specific for the protein produced by a cDNA to select the corresponding cDNA clone, a method using a probe that selectively binds to the target DNA sequence, such as a plaque hybridization method, and a colony hybridization method, and a combination of such methods. Generally, the DNA synthesized chemically according to the nucleotide sequence information of the gene of the invention can be used as the probe for the above-mentioned method. It can also be advantageously used as the probe, the gene of the invention that has already been obtained, or a fragment thereof. The sense initiator and the antisense initiator established according to the information on the -jffe, J ^ á ^ fe ^ iBw-aá. { fei ^^ l #, nucleotide sequence of the gene of the invention, can be used as selection probes. For use as the probe, the nucleotide sequence can be a partial nucleotide sequence corresponding to SEQ ID NO: 2 and 5 comprising at least 10 consecutive nucleotides, preferably 20 consecutive nucleotides, preferably 30 consecutive nucleotides, most preferably 50 consecutive nucleotides. In addition, the positive clone having the oligonucleotide sequence shown in SEQ ID NO: 2, can be used as such as the probe. In obtaining the gene of the invention, DNA / RNA amplification can be advantageously used by PCR [Science, 230, 1350 (1985)]. Particularly, when it is difficult to obtain a full-length cDNA from a library, the RACE method [Rapid extension of cDNA ends; Jikken Igaku (Medicine Experimental), 12 (6), 35 (1994)], especially the 5'-RACE method [M.A. Frohman et al., Proc. Nati Acad. Sci., USA, 8, 8998 (1988)]. For use in such PCR methods, the primers can be conveniently established with reference to the sequence information of the gene of the invention as described herein, and can be synthesize by the routine procedure. The isolation and purification of the amplified DNA / RNA fragment can be carried out in the routine manner as mentioned above, for example with the gel electrophoresis method.

The sequencing of the gene of the invention obtained in the aforementioned manner, or several DNA fragments, can be done according to the dideoxy method [Proc. Nati Acad. Sci., USA, 74, 5463 (1977)] or the method of Maxam and Gilbert [Methods in Enzymology, 65, 499 (1980)] or more conveniently using a commercial sequencing equipment. For example, with the gene of the invention thus obtained, the expression or non-expression of the gene of the invention can be detected specifically in an individual or in a given tissue, using a portion or the entire nucleotide sequence of the gene of the invention. The above detection can be done with conventional procedures such as RNA amplification by RT-PCR [reverse transcription polymerase chain reaction; E. S. Kawasaki et al., Extension of RNA, in "PCR Protocol, A Guide to Methods and Applications", [PCR Protocol, Methods and Applications Guide], Academic Press Inc., San Diego, 21-27 (1991)], Northern blot analysis ["Molecular Cloning", Cold Spring Harbor Lab. (1989)], determination at the cellular level by in situ RT-PCR [Nucí. Acids Res., 21, 3159-3166 (1993)] or in situ hybridization, NASBA [amplification based on nucleic acid sequence, Nature, 350, 91-92 (1991)], and similar conventional techniques. The RT-PCR detection method is preferred. The initiator to be used when the PCR method is chosen for the above purpose is not particularly restricted, so long as it is characteristic of the gene of the invention and capable of selective amplification of the particular gene only, and can be established properly based on information on the sequence of the gene of the invention. Usually, one which has a partial sequence of the gene of the invention, which is about 10-35 nucleotides long, preferably about 15-30 nucleotides long, can be used as the primer. Thus, the gene of the invention includes the DNA fragment that can be used as a specific primer and / or probe specific for the detection of the LY6H gene of the invention. The DNA fragment mentioned above can be defined as a polynucleotide that hybridizes with the polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2 under severe conditions. The severe conditions mentioned above may be the ordinary conditions for initiators or probes and as such, are not particularly restricted. For example, we can mention the previous conditions of 15 SSC 6x, 65 ° C during the night or the formamide condition 4% -SSC 4 x, 37 ° C during the night. By applying the gene of the invention to normal genetic engineering technology, the expression product (polypeptide) of the gene or a protein containing it can be easily produced in large quantities and with good reproducibility. Therefore, the invention also provides a polypeptide having the amino acid sequence encoded by the gene of the invention (the expression product of the invention), a vector that stores the gene of the invention for the production of the polypeptide, a host cell transfected with the vector, and a method of producing the polypeptide of the invention comprising the development of the host cell. The polypeptide (LY6H protein) having the amino acid sequence shown under SEQ ID NO: 1, is a specific embodiment of the polypeptide of the invention. The polypeptide of the invention is not limited to this LY6H protein, but includes its homolog. The homolog can be a polypeptide having an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 1 by the deletion, substitution or addition of one or more amino acids and retaining the same function as the LY6H protein. A specific example of the homologue is the expression product of a homologue of said gene LY6H (the equivalent gene of LY6H, including the allele). In addition, the homologue of the LY6H protein of the invention includes proteins having the same activity or function as the polypeptide having the amino acid sequence shown in SEQ ID NO: 1, derived from any mammal such as equine, sheep, bovine, canine , monkey, cat, bear, etc., and rodents such as rat, mouse and rabbit. The polypeptide of the invention can be produced by conventional recombinant DNA technology [e.g., Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm., 130, 692 (1985); Proc. Nati Acad. Sci., USA, 80, 5990 (1983)] based on the sequence information of the gene provided by the present invention.

More particularly, the production of said polypeptide is carried out by the method comprising constructing a recombinant DNA (expression vector) that allows the expression of the gene coding for the desired protein in a host cell, transform the host cell with the vector, develop the resulting transformant, and harvest the polypeptide from the culture broth. The above-mentioned host cell can be any of a prokaryotic cell and a eukaryotic cell. As the prokaryotic host we can mention Escherichia coli, Bacillus subtilis and others common bacteria, and preferably Escherichia coli cells, particularly cells of Escherichia coli K12 can be used. The eukaryotic host cell includes vertebrate and yeast cells, and the former include the monkey COS cell line [Cell, 23: 175 (1981)], Chinese hamster ovary cells and the defective cells in dihydrofolate reductase thereof [Proc. Nati Acad. Sci., USA, TT, 4216 (1980)]. As the latter, yeast cells of the genus Saccharomyces can be advantageously used, but these are not exclusive choices. When prokaryotic cells such as host cells are used, a plasmid construct can be advantageously used.

Expression that is prepared using a vector that is replicable in the particular host cell, and adding a promoter and SD sequence (Shine and Dalgarno) at the 5 'end of the gene of the invention, such that the gene can be expressed in the same, as well as an initiation codon (for example ATG), necessary to initiate protein synthesis. As the vector mentioned above, it is usual to use plasmids derived from Escherichia coli such as pBR322, pBR325, pUC12, pUC13, etc. However, these are not exclusive choices, and several known vectors can be used. Examples of commercial vectors for use in expression systems employing E. coli include pGEX-4T (Amersham Pharmacia Biotech), pMAL-C2, pMA1-P2 (New England Biolabs), pET21, pET21 / lacq (Invitrogen) and pBAD / His (Invitrogen). To be used as the expression vector when cells of a vertebrate are used as host cells, the vector having a promoter at the 5 'end of the gene of the invention to be expressed, RNA splice sites, polyadenylation site and a transcription termination sequence, and this vector may also have an origin of replication when necessary. A specific example of the expression vector is pSV2dhfr, which stores an SV40 early promoter [Mol. Cell. Biol., 1: 854 (1981)]. Apart from the above, several commercially available known vectors can be employed. Examples of commercial vectors that are used in expression systems using animal cells include vectors for animal cells such as pEGFP-N, pEGFP-C (CLONTECH), pIND (Invitrogen), pcDNA3.1 / His (Invitrogen), etc. , and vectors for insect cells such as pFastBac HT (Gibci BRL), pAcGHLT ^ (PharMingen), pAc5 / V5-His, pMT V5-His and pMT / Bip / V5-his (all from Invitrogen).

PAM82 having a promoter for the acid phosphatase gene [Proc. Nati Acad. Sci., USA, 80: 1 (1983)] is a specific example of the expression vector to be used when yeast cells are used as host cells. Commercial expression vectors for yeast cells include pPICZ (Invitrogen) and pPICZ (Invitrogen). The promoter is not particularly restricted. When a strain of the genus Escherichia is used as the host, tryptophan (trp) promoter, Ipp promoter, lac promoter, recA promoter, PL / PR promoter can be advantageously used. When the host is a strain of the genus Bacillus, the SP01 promoter, the SP02 promoter, the penP promoter, etc. are preferably used. When a yeast is used as the host, the pH05 promoter, the PGK promoter, the GAP promoter, the ADH promoter, etc. can be advantageously used. The preferred promoter for use when the host cells are animal cells, includes promoters derived from SV40, retrovirus promoters, metalloprotein promoter, heat shock promoter, cytomegalovirus promoter and SR promoter. As the expression vector for the gene of the invention, the conventional fusion protein expression vector can be advantageously used. PGEX (Promega) for the expression of proteins fused with glutathione-S-transferase (GST) is a specific example of the vector. The polynucleotide sequence in which the coding sequence for a mature polypeptide aids the expression and secretion of a polypeptide from the host cells, includes the secretory sequence, the guide sequence and the marker sequence (hexahistidine tag, histidine tag) used in the purification of a mature fusion polypeptide in the case of bacterial cells, and the hemagglutinin tag (HA) in the case of mammalian cells. The method of introducing the recombinant DNA (expression vector) into the host cell and the associated transformation method are not particularly restricted but several standardized methods can be used. The transformant obtained can be cultured in the routine manner, by means of which the target protein encoded by the deliberately designed gene according to the invention is expressed and produced (accumulated and secreted) intracellularly, extracellularly or on the cell membrane. . The culture medium to be used can be selectively selected from various routine media according to the type of host cell adopted, and the culture is also carried out under conditions that favor the growth of the host cell. Optionally, the resulting recombinant protein (LY6H protein) according to the invention, can be isolated and purified by several separation techniques, taking advantage of for example its physical and / or chemical properties ["Seikagaku Data Book" (Book of biochemical data) II ", 1175-1259, first edition, 1st printing, June 23, 1980, Tokyo Kagaku Dojin K.K .; Biochemistry, 25 (25), 8274 (1986); Eur. J. Biochem., 163. 313 (1987), etc.]. Examples of such techniques are the conventional reconstitution method, treatment with a protein precipitating agent (salt separation method), centrifugation, osmotic shock method, sonic breakdown, ultrafiltration, various types of chromatography such as molecular sieve chromatography ( gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography and high performance liquid chromatography (HPLC), dialysis, and combinations of these techniques. The particularly preferred technique includes affinity chromatography using a column to which an antibody specific for the protein of the invention has been coupled. By designing the target gene encoding the polypeptide of the invention, the nucleotide sequence of the invention can be advantageously used. gene Y6H shown in SEQ ID NO: 2. If desired, this gene can be used after having correctly altered the codons that specify the respective amino acid residues. In addition, when any amino acid residue or partial sequence of the amino acid sequence encoded by the LY6H gene is to be modified by substitution, deletion or addition, such Modifications can be made by the different methods described above, for example by site-specific mutagenesis. The polypeptide of the invention can be produced by the standard protocol for chemical synthesis according to the sequence of amino acids shown in SEQ ID NO: 1. The method includes the liquid phase method and the solid phase method, conventional for the synthesis of peptides. More particularly, the method for the synthesis of peptides includes the so-called method of gradual elongation, in which the constituent amino acids are coupled one by one to extend the chain; and the fragment condensation method, which comprises pre-synthesizing fragments consisting of each of several amino acids, and coupling the fragments together. The synthesis of the protein of the invention can be carried out by any of the two previous methods. The condensation method for use in the peptide synthesis above can also be a conventional one, including the azide process, the mixed acid anhydride method, the DCC process, the active ester process, the redox process, the DPPA process ( diphenylphosphorylazide), the additive DCC + procedure (1-hydroxybenzotriazole, N-hydroxysuccinamide, N-hydroxy-5-norbomeno-2,3-dicarboximide) and the Woodward reagent procedure. The solvent to be used in these methods can also be selected from among several common solvents well known in the art for use in such condensation reactions for peptide formation. Examples of the solvents include dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate, etc., and mixtures thereof.

In carrying out the peptide synthesis reactions, the carboxyl group of any amino acid or peptide fragment that should not take part in the reaction can be protected in advance, usually by esterification in the form of a lower alkyl ester such as ester. methyl, ethyl ester, tert-butyl ester, etc., or an aralkyl ester such as benzyl ester, p-methoxybenzyl ester, p-nitrobenzyl ester, etc. With respect to any amino acid having a functional group in its side chain, for example, the hydroxyl group of a tyrosine residue can be protected in advance with an acetyl group, benzyl, benzyloxycarbonyl, tert-butyl or other, although such protection is not necessarily indispensable. In addition, the guanidino group of an arginine residue can be protected with a suitable protecting group such as nitro, tosyl, p-methoxybenzenesulfonyl, methylene-2-sulfonyl, benzyloxycarbonyl, isobomyloxycarbonyl, adamantyloxycarbonyl, or the like. The reactions for removing such protecting groups from the protected amino acids, peptides or the final protein product of the invention can also be carried out in the routine manner, for example by catalytic reduction, or by a method employing ammonia. liquid / sodium, hydrogen fluoride, hydrogen bromide, chloride of hydrogen, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid or other reagent. The polypeptide of the invention thus produced can be purified as necessary by the various techniques mentioned above such as ion exchange resin chromatography, partition chromatography, gel chromatography, countercurrent distribution, and similar methods routinely used in the field of peptide chemistry. The polypeptide of the invention can be advantageously used as an immunogen for the preparation of its specific antibody. Using this immunogen, the antiserum (polyclonal antibody) and the monoclonal antibody can be provided. The technology for producing antibodies is well known to the person skilled in the art and the known methods can be employed in the art. present invention [for example, Zoku Seikagaku Jikken Koza (Biochemistry experiments, second series), "Men-eki Seikagaku Kenkyuho (Methods of immunobiochemistry)", edited by the Biochemical Society of Japan (1986)]. For example, as the immune animal to harvest the desired antiserum therefrom, the animals can be arbitrarily selected. ordinary such as rabbit, guinea pig, rat, mouse, chicken, etc., and immunization with said immunogen and blood extraction can be carried out by conventional procedures. The preparation of a monoclonal antibody can also be carried out by the conventional technique, which comprises constructing a hybridoma between the plasma cell (immune cell) of an animal immunized with said immunogen, and a plasmacytoma cell; select the clones that produce the desired antibody, and grow the clones. Generally, the immune animal is selected considering its compatibility with the cell of Plasmacytoma to be used for cell fusion, and usually the mouse or rat are advantageously used. The immunization procedure can be the same used for the preparation of said antiserum and, if desired, the immunization can be done using a conventional adjuvant in combination. The plasmacytoma cell for use in such hybridization is not particularly restricted, and includes several myeloma cells such as p3 (p3 / x63-Ag8) [Nature, 256: 495-497 (1975)], p3-U1 [Current Topics in Microbiology and Immunology, 81: 1-7 (1978)], NS-1 [Eur. J. Immunol., 6: 511-519 (1976)], MPC-11 [Cell, 8: 405-415 (1976)], SP2 / 0 [Nature, 276: 269-271 (1978)], etc., R210 [Nature, 277: 131-133 (1979)], and others in rats, and cells derived therefrom. Hybridization between said immune cell and said plasmacytoma cell can be performed by known technology, in the presence of a conventional hybridization promoter such as polyethylene glycol (PEG) or Sendai virus (HVJ), and the separation of the target hybridoma can also be carried out in the known way [Meth. in Enzymol., 73: 3 (1981); Zoku Seikagaku Jikken Koza (idem)]. The search for the clone of cells that produce the target antibody, and the preparation of the monoclonal antibody, can also be carried out in the routine manner. For example, the search for the antibody-producing hybridoma can be done by any of the various techniques of routine use for the detection of antibodies, such as ELISA.

A. ^^ ---.- rf --'- ^ - ^^^^ -.'- ¿? ..

[Meth. in Enzymol., 70: 419-439 (1980)], the plaque method, the spotting method, the agglutination reaction method, the Ouchterlony method, radioimmunoassay and the like, using the protein of the invention as an antigen. Harvesting the antibody of the invention from the resulting hybridoma can be performed by culturing the hybridoma in the routine manner and recovering the antibody as a culture supernatant, or administering the hybridoma to a compatible mammal and recovering the antibody in the form of ascites. The first method is suitable for the production of the high purity antibody, while the latter method is suitable for high production of the antibody. The antibody thus produced can be further purified with conventional means such as salting out, gel filtration, affinity chromatography and the like. The antibody thus obtained is characterized by its binding affinity to the LY6H protein of the invention, and can be used advantageously for the purification of the LY6H protein and determination or differentiation of the protein by immunological techniques. In addition, since a diminished expression of the gene of the invention has been confirmed in the temporal lobe of the brain of a patient with Alzheimer's disease, which is a neurodegenerative disease, this antibody can be used in the selection of protein agonists or antagonists. LY6H. The present invention also provides the novel antibody described above.

The polypeptide of the invention is useful in the field of medicine as a pharmaceutical product containing it as an active ingredient. Therefore, the invention provides a pharmaceutical composition comprising the polypeptide of the invention as an active ingredient. The utility of the polypeptide of the invention in, or as, said pharmaceutical composition, is attributable to the action of supporting neuronal survival, nerve lengthening action, nerve regeneration action, neuroglia activation action and inherent mnemonic action of this brain-specific polypeptide. Examples of 0 methods to confirm these actions include the following methods for each action. (1) Neural survival support action The following method can be used to quantify the action of neuronal survival support of the polypeptide of the invention. For example, the hippocampus of the entire brain of a fetal rat SD is aseptically isolated and treated with an enzyme; it is seeded in a 96-well plate precoated with poly-L-lysine (Sigma) containing 10% fetal calf serum-DMEM, at a final concentration of 2 x 10 5 cells / cm 2. 0 The cells are developed for 24 hours, after which the culture medium is changed to DMEM containing 1% N2 supplement (Gibco). Then, the polypeptide of the invention is added as an active ingredient (group of the invention). As a control, the The present invention relates to a polypeptide of the invention that has been heat-treated in a boiling water bath for 5 minutes (boiling protein group). The cells (culture) in each group, prepared in the above manner, are cultured for 72 hours. Then, the effect of supporting the neuronal survival of the polypeptide of the invention can be evaluated on the neurons of the hippocampus by performing an MTT test [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide. ] using the 96-well Promega cell-title test system. Similarly, aseptically isolating the average brain of the same complete brain of a fetal rat SD as above, and by carrying out an MTT test in the same manner as above, the support effect of the neuronal survival of the polypeptide of the invention on midbrain neurons can be investigated. (2) Dopaminergic neuronal survival support action As a method for evaluating the neuronal survival support action of the polypeptide of the invention, the following method of quantifying the dopaminergic neuronal survival support activity can be mentioned. In this way, the cells (the culture) in each group, prepared as in (1) above, are cultured for 72 hours and then fixed with 4% paraformaldehyde -PBS for 15 minutes, resting at room temperature. Then, the culture is passed through a membrane using Triton 1% x 100 / PBS.

To prevent non-specific binding of the antibody, the cells are incubated in 10% goat serum-PBS for 1 hour, and then, using a polyclonal anti-tyrosine hydroxylase antibody (Chemicon, diluted 1000-fold in PBS), incubated additionally at 4 ° C for 16 hours. After removing the antibody fluid, the cells are washed with PBS and, after adding goat anti-rabbit immunoglobulin, coupled with dextran polymer and labeled with peroxidase (Dako), they are incubated at room temperature for 1 hour. Tyrosine hydroxylase-positive cells can be detected by color reaction using diaminobenzidine as the substrate. In this way, the dopaminergic neuronal survival support activity of the polypeptide of the invention can be tested, using the number of tyrosine-hydroxylase-positive cells as the indicator. (3) Nerve lengthening action The determination of the nerve elongation action (axonal elongation promotion action) of the polypeptide of the invention can be carried out using PC12 cells [Accession number ATCC CRL1721; Science, 229, 393-395 (1985)], as follows. Thus, subcultured PC12 cells in modified Dalbecco MEM (D-MEM) containing 5% heat-inactivated horse serum (56 ° C, 30 minutes) and 10% fetal calf serum (FCS), are transplanted in a plastic Petri dish coated with collagen 35 mm in diameter, at a concentration of 6 x 104 cells / 3 ml.

On day 2 after transplantation, the medium is replaced with D-MEM containing a variable concentration of the polypeptide of the invention, as well as nerve growth factor (NGF; Wako Puré Chemical Ind.) And FCS, and culture is continued. in each case. On day 3, the morphological changes of the cells are examined with a phase contrast microscope. By determining whether neurite formation or neurite prolongation promotion is observed compared to the control, the potential for promotion of axonal elongation of the polypeptide of the invention can be evaluated. (4) Neuroglia activation action The action of neuroglia activation can be evaluated for example by determining the effect of the polypeptide of the invention on the activation of neuroglia by FGF according to the method of Kniss et al., Or the Bogler method and others [Kniss, DA and Burry R.W., Brain Res., 439, 281-288 (1988); Bogler O., and others, Proc. Nati Acad. Sci., USA, 87 816), 6368-6372 (1990)]. (5) Mnemonic action The mnemonic action can be evaluated, for example, according to the Morris water maze protocol [Morris R.G.M., J. Neurosci. Meth., 11, 47-60 (1984)].

Another evaluation method comprises administering the LY6H protein or an agonist or antagonist of the LY6H protein selected by a selection technique, to an animal model of Alzheimer's disease such as a transgenic mouse of the mutant precursor-amyloid protein gene or the mutant gene of presenilin 1 [eg, Nature, 373, 523-527 (1995); Nature Med., 5, 560-564 (1999)] and evaluating the degree of disease progression or the degree of nerve degeneration compared to an untreated control group. Furthermore, to have the gene expressed in the human temporal lobe (gene therapy), for example, an adenovirus vector is used [Straus E.S., Plenum Press New York, 451-496 (1984); Setoguchi Y. and others, Blood, 84. 2953-2964 (1994)]. In this way, a possible method comprises cloning the gene of the invention into an adenovirus vector, culturing it in the stem cell, administering it directly in the temporal lobe or intravenously through a peripheral blood vessel, and checking to see if it has improved the Alzheimer's dementia or Alzheimer's disease, or if its progress has been inhibited. As the active ingredient of the pharmaceutical composition of the invention, the polypeptide includes its pharmaceutically acceptable salt. Said salt includes non-toxic salts with alkali metals, alkaline earth metals or ammonium, such as the sodium, potassium, lithium, calcium, magnesium, barium and ammonium salts. These salts can be prepared by conventional methods in the art. In addition, said salts include non-toxic acid addition salts which can be prepared by reacting the polypeptide active ingredient of the invention with suitable organic or inorganic acids. Representative non-toxic acid addition salts include hydrochloride, bromohydrate, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, phosphate, p-toluenesulfonate (tosylate), citrate, maleate, fumarate, succinate , tartrate, sulfonate, glycolate, maleate, ascorbate, benzenesulfonate, naphthalenesulfonate and the like. The pharmaceutical composition of the invention includes a composition comprising a pharmacologically effective amount of the polypeptide of the invention, and a suitable non-toxic pharmaceutical carrier or diluent. The pharmaceutical carrier that can be used for said pharmaceutical composition (pharmaceutical preparation) includes diluents or excipients that are conventionally used in accordance with the dosage forms, such as fillers, volume fillers, binders, humectants, disintegrants, surfactants and lubricants. These can be selected correctly and can be used according to the unit dosage form of the composition. The particularly preferred composition of the invention can be prepared using various additives that can be formulated into ordinary protein preparations, such as stabilizer, biocide, buffer, isotonicity agent, chelating agent, pH controlling agent and surfactant.

* Sü¡ & - The stabilizer includes human serum albumin, an L-amino acid, a sugar, and a cellulose derivative, which may be mentioned for example. These may be used individually or in combination with a surfactant or the like when necessary. The use in combination with a surfactant can lead to a more effective stabilization of the active ingredient in particular. The L-amino acid is not particularly restricted, but can be for example any of glycine, cysteine and glutamic acid. The sugar is not particularly restricted but includes monosaccharides such as glucose, mannose, galactose, fructose, etc .; sugar alcohols such as mannitol, inositol, xylitol, etc .; disaccharides such as sucrose, maltose, lactose, etc .; polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate, hyaluronic acid, etc .; and its derivatives. The surfactant is not particularly restricted but both ionic and nonionic surfactants can be used. Examples of surfactants are alkyl esters of polyoxyethylene glycol sorbitan, polyoxyethylene alkyl ethers, sorbitan monoacrylic esters and fatty acid glycerides. The cellulose derivative that can be used is not particularly restricted, but includes methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and sodium carboxymethylcellulose.

The level of addition of any of said sugar additives and other additives can be selected accurately with reference to the amount commonly used. In general, the sugar is used in a proportion of not less than about 0.0001 mg, preferably within the range of about 0.01 to about 10 mg, per g of active ingredient. The surfactant is generally used in a proportion of not less than about 0.00001 mg, preferably within the range of about 0.0001 to about 0.01 mg per g of the active ingredient. Human serum albumin, an example of the stabilizer, may be used in a proportion of not less than about 0.0001 mg, preferably within the range of about 0.001 to about 0.1 mg per g of the active ingredient. The amount of the amino acid, another example of stabilizer, can be selected from the scale of about 0.001 to about 10 mg per g of the ingredient active. The level of addition of the cellulose derivative is not less than about 0.00001 mg, and is preferably selected from the scale of about 0.001 to about 0.1 mg. The amount of the active ingredient in the pharmaceutical composition of the invention can be selected freely from a wide scale, but it is usually selected from the scale of about 0.00001 to about 70% by weight, preferably about 0.0001 to about 5% by weight.

The pharmaceutical composition of the invention can be further supplemented with a buffer, an isotonicity agent and a chelating agent. The buffer includes boric acid, phosphoric acid, acetic acid, citric acid, aminocaproic acid, glutamic acid, and the corresponding salts (the alkali metal or alkaline earth metal salts thereof, such as the sodium, potassium, calcium and magnesium). The isotonicity agent includes sodium chloride, potassium chloride, sugars and glycerol. The chelating agent includes sodium edetate and citric acid. The level of addition of any of these additives may be within the conventional range. The pharmaceutical preparation of the invention can be provided in the form of a solution and in a lyophilized form that can be stored. Such lyophilized preparations can be dissolved extemporaneously for example in a buffer, including water, saline or the like, at a suitable concentration. With respect to the unit dosage form of the pharmaceutical composition of the invention, several forms can be selected according to the therapeutic objective. The representative form includes solid dosage forms such as tablets, pills, powders, pure powders, granules, capsules, etc., and liquid dosage forms such as solutions, suspensions, emulsions, syrups, elixirs, etc. These dosage forms are generally classified by their route of administration in oral preparations, parenteral preparations, nasal preparations, vaginal suppositories, rectal suppositories, sublingual tablets, ointments and others. Each of these dosage forms can be formulated and molded, or prepared in some other way, by the established pharmaceutical procedure. For example, the tablets can be manufactured using as said pharmaceutical carrier, any of several excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc.; binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, polyvinyl pyrrolidone, etc .; disintegrants such as sodium carboxymethylcellulose, calcium carboxymethylcellulose, low-substitution hydroxypropylcellulose, dried starch, sodium alginate, powdered agar, powder laminate, sodium acid carbonate, calcium carbonate, etc .; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglycerol stearate, etc .; disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, etc .; absorption promoters such as quaternary ammonium bases, sodium lauryl sulfate, etc .; humectants such as glycerol, starch, etc .; adsorbents such as starch, lactose, kaolin, bentonite, colloidal silica, etc .; and lubricants such as purified talc, salts of stearic acid, boric acid powder, polyethylene glycol, etc. • 'acjtí. AiA -. - M ^? ? á & ß ¿6Vk¡ & kM & * l * * .- ^ * AJüma? »* a & Y.

When necessary, such tablets can be coated with conventional coatings to provide sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, and film-coated tablets. It is also possible to use double-layer or multi-layer tablets. The pharmaceutical carrier that can be used for the production of pills includes various excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, taco, etc .; binders such as gum arabic powder, powdered tragacanth, gelatin, ethanol, etc .; and disintegrants such as laminating and agar. Generally, the capsules can be manufactured in the conventional manner by mixing the active ingredient of the invention with the pharmaceutical carrier (s) and filling the resulting composition into hard gelatin capsules, soft capsules or the like. The liquid preparation for oral administration includes pharmaceutically acceptable solutions, emulsions, suspensions, syrups, elixirs, etc., formulated with routine inert diluents such as water, and these dosage forms can contain a wetting agent, an emulsifier, a suspending agent and / or other auxiliary additives. These can be manufactured by established pharmaceutical procedures. The liquid preparation for parenteral administration, including aqueous and non-aqueous solutions, sterile emulsions and suspensions, is "* £ _-» -J? »* T <l> faith» afcri «gt». »« H »» «» «tt *». »-. *» «- - L * Má * ß & a ? H? &Stototr y *. ».A» fey-i «« j. A¿ ^^? / T can be prepared using diluents such as water, ethyl alcohol, propylene glycol, polyethylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol , polyoxyethylene sorbitan fatty acid esters and vegetable oils such as olive oil In addition, 5 injectable organic esters such as ethyl oleate can be formulated In addition, any of the solubilizers, buffers, wetting agents, emulsifiers, conventional suspension agents, preservatives, dispersants, etc. The different dosage forms given above are sterilize in the routine manner. This sterilization can be achieved by filtration through a bacterial filter, formulation of a biocide, irradiation, or a heat treatment. In addition, they can be provided in the form of sterile solid compositions that can be dissolved extemporaneously in sterile water or in a suitable sterilizable medium. For the manufacture of dosed forms for rectal or vaginal administration, pharmaceutical carriers such as polyethylene glycol, cocoa butter, higher alcohols, higher alcohol esters, gelatin, semi-synthetic glycerides, etc. can be prepared. Ointments such as pastes can be prepared, creams and gels using a diluent such as white petrolatum, paraffin, glycerol, cellulose derivatives, propylene glycol, polyethylene glycol, silicone, bentonite and vegetable oils such as olive oil.

Compositions for transnasal or sublingual administration can be prepared in the routine manner, using a well known standard excipient. When necessary, the pharmaceutical compositions of the invention can be supplemented with coloring agents, preservatives, perfumes, flavoring agents, sweeteners and other drugs. The method of administration of such pharmaceutical preparations is not particularly restricted, but can be selected according to the specific dosage form, the age of the patient, sex and other factors, the severity of the disease and other variables. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are administered orally, while parenteral products are administered intravenously, either alone or in a mixture with the glucose, amino acid or other conventional infusion, or when necessary , are administered alone intramuscularly, intradermally, subcutaneously or intraperitoneally. Rectal suppositories are administered in the rectum; vaginal suppositories are administered in the vagina; nasal preparations are administered in the nostril, sublingual preparations are administered buccally, and ointments are administered topically for transdermal release of the drug. The dosage of any of the above pharmaceutical preparations is not particularly restricted, but can be conveniently selected on a wide scale according to the therapeutic effect expected, the method of administration, the duration of treatment, patient history such as age and sex, and other factors. Generally, the usual recommended dosage of the active ingredient is about 0.01 g - 10 mg / day, preferably about 0.1 5 g - 1 mg / day, per kg of the patient's body weight. The previous dose can be administered once a day or in 2 or more divided doses. Furthermore, as noted in the working example presented hereinafter, the expression of the gene of the invention is canceled or reduced in the temporal lobe of patients with Alzheimer's disease. Therefore, by constructing an arbitrary expression vector that hosts all or part of the gene of the invention, and introducing the expression vector into the tissue of the temporal lobe for the forced expression of the gene in the tissue, neurodegenerative changes can be inhibited. excessive atrophy of neurons in the temporal lobe, and therefore, can be stop Alzheimer's disease. Therefore, the present invention also provides a pharmaceutical composition for gene therapy (gene therapeutic agent) which possesses said neurodegeneration inhibitory action. The present invention further provides the above vector or expression vectors for gene therapy, cells transfected with the gene for the invention by introducing said vector, and a pharmaceutical composition for gene therapy comprising any of the foregoing as the active component.

The gene therapy using said gene therapeutic agent is carried out by administering at least one member selected from the group consisting of the vector for introduction and expression of the gene of the invention, and cells transfected with the gene of the invention, by introducing said gene. vector in the neurons of the brain or tissue of the temporal lobe of a patient with neurodegenerative disease. By means of said method, neurodegenerative changes in said tissue can be inhibited, and the symptoms of Alzheimer's disease, Alzheimer's dementia, Parkinson's disease, cerebral ischemia, etc. can be alleviated. Gene therapy is now described in greater detail. In the following form of gene therapy, routine techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, and immunology may be employed, unless otherwise specified. These techniques are described, among others, by Maniatis T. et al., "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Laboratory), Cold Spring Harbor, New York (1982); Sambrook J. et al., "Molecular Cloning: A laboratory manual" [Molecular Cloning, Laboratory Manual], 2a. edition (Cold Spring Harbor Laboratory), Cold Spring Harbor, New York (1981); Ausbel F.M. and others, "Current Protocols in Molecular Biology", John Wiley and Sons, New York (1992), Glover D., "DNA Cloning, I and II" [DNA Cloning, I and II ] (Oxford Press (1985)); Anand "Techniques for the Analysis of Complex Genomes" (Techniques for the analysis of complex genomes) (Academic Press - * ¿* & & amp; a »a« -. i (1992)), Guthrie G. and others, "Guide to Yeast Genetics and Molecular Biology" (Academic Press (1991)), and Fink and others, Hum. Gene Ther., 3, 11-19 (1992). Gene therapy can be carried out using a gene therapy vector that stores all or part of the gene of the invention, or cells transfected with the gene of the invention by introducing said vector. This gene therapy for example may be a method of delivery of the LY6H gene or its function to cells in which said gene has not been expressed. By such gene therapy, neurodegeneration around the recipient cell / target cell is inhibited. The gene of the invention or a fragment of said gene, they can be introduced into the cells by means of a vector adapted to maintain the gene extrachromosomally. In such cases, the particular gene can be made to be expressed by the cells from an extrachromosomal position. Furthermore, when the LY6H gene is to be expressed by introducing a fragment of the gene into the site of the temporal lobe of the nervous system of the brain where expression of the gene is not found, the particular fragment of the gene may be a fragment encoding a part of the gene. LY6H protein, which is necessary for the survival or non-tumorigenic growth of cells. The gene transfer vector can be any of several known vectors in which the gene of the invention has been subcloned as will be described hereinafter. - * £ & The introduction of the gene transfer vector into the target cell can be easily effected by the established technology of introducing DNA into several cells, which is already known to the person skilled in the art, such as electroporation, transfection with calcium phosphate 5 ( coprecipitation), virus transduction and other techniques. Cells transfected with the gene of the invention can be used as a drug for neurodegenerative disorders of the brain, including an inhibitor of premature atrophy of the nervous system of the brain, or as models for therapeutic research. As mentioned above, the gene or gene fragment of the invention, introduced by means of gene therapy according to the invention, increases the expression of the corresponding product of the gene in the nerve of the surrounding brain or tissue to thereby inhibit the atrophy of the cerebral nerve in the tissue that expresses the gene. Such gene therapy can be advantageously applied to the neuronal tissue of the brain where the expression of the LY6H gene or the LY6H protein is canceled, as well as to the neuronal tissue of the brain where the level of expression of said gene is depressed. The gene therapy according to the present invention is carried out as follows. In the first place, a selection of candidate patients for gene therapy recording a computer tomogram (CT) with the position of fixed sweep in the temporal lobe of the patient with Alzheimer-type dementia or Alzheimer's disease to review the temporal lobe atrophy or progression of atrophy. yt? d * 4 ^ ». . ^^^^^^^^^^^^^^^^^^^^^^^^^^^ Next, to achieve the expression of the gene of invention, creates the intracellular LY6H mRNA in the target cell, and its translation is promoted to accelerate the expression of the LY6H gene. For this purpose, a sense oligonucleotide corresponding to the mRNA of the gene is preferably produced and delivered to the target cell. By providing the cell with the activity to promote the expression of the LY6H gene by the previous gene therapy, the neurodegenerative change in the recipient cell of the brain / target cell can be inhibited. Based on previous gene therapy using said In the sense oligonucleotide, objective inhibition of the neurodegenerative change of the brain and the consequent alleviation or arrest of advancing neurodegenerative symptoms can be successfully obtained by subcloning the LY6H gene into a retrovirus, adenovirus or AAV-derived vector, and infecting the nerve cells. objective of the brain with the vector, to cause This is the expression of the sense oligonucleotide. When a sense oligonucleotide of the invention gene is introduced into neurons or brain tissue to increase the expression of the LY6H protein, the sense oligonucleotide is not required to be a full-length nucleotide of the LY6H gene, but may be the product of Modification as long as it retains a function substantially identical to the function of the parent gene and promotes the expression of the LY6H gene, or a fragment of the gene comprising a partial sequence that retains said function.

The vectors that can be used to introduce a target gene, both for DNA recombination and for extrachromosomal maintenance of the gene, are already known in the art and any of these known vectors can be used in the practice of the invention. For example, a virus or a plasmid vector including a copy of the sense oligonucleotide of the LY6H gene linked to the expression control element can be used as such vectors, and is capable of expressing the sense oligonucleotide product in the target cell. Usually any of the aforementioned vectors may be employed, but vectors constructed using any of the vectors described in USP 5252479 and WO 93/07282 (specifically pWP-7A) are preferred., pWP-19, pWU-1, pWP-8A, pWP-21 and / or pRSVL) or pRC / CMV (Invitrogen) as the vector source. Most preferred are the various virus vectors described hereinafter. As a promoter for use in the vector for gene therapy, those promoters that are intrinsic to the affected tissues to be treated in various diseases are preferably employed. Examples of the promoters are albumin, -fetoprotein, 1-antitrypsin, transferrin and transthyretin for the liver, and carbonic anhydrase I and carcinoembryonic antigen for the colon. When the affected tissues are the uterus and the placenta, it can be exemplified by estrogen, aromatase, cytochrome P450, enzyme P450 for breaking the cholesterol side chain and 17 -hydroxylase P450.

For the prostate, prostate-specific antigens, gp91-fox gene and prostate-specific kalikrein can be exemplified. For the breast, erb-B2, erb-B3, -casein, - lactoglobin and whey protein can be exemplified. For the lung, surfactant protein C and uroglobin can be used. For the skin, one can put for example K-14-keratin, human keratin 1 or 6 and leucine. For the brain, glial fibrillar acid, mature astrocyte-specific protein, myelin, pancreatic tyrosine hydroxylase villin and amyloid polypeptide of the Langerhans is exemplary. For the thyroid, can be exemplified by thyroglobulin and calcitonin. For bone, collagen 1, osteocalcin and bone sialoglycoprotein can be exemplified. For the kidney, one can put for example renin, liver / bone / kidney alkaline phosphatase, and erythropoietin; and for the pancreas, amylase and PAPI can be exemplified. In addition, in the production of a vector for the introduction of a sense oligonucleotide, the sense oligonucleotide to be introduced (one having a full-length or partial sequence corresponding to the sequence of the gene of the invention), can be prepared and easily acquired by normal genetic engineering techniques based on the information on the nucleotide sequence of the gene of the invention as described hereinabove. The transfer of said vector for the introduction of a sense oligonucleotide into the cells can be carried out by means of y.í »sg? '.A», ^ aafc * ^ .- ^ s3- > aa3? > b »- j. . jt faisafe.,., - gÉ * Ífea¿. .- .-. ^^^^ several techniques already known in the field, such as electroporation, transfection with calcium phosphate (coprecipitation), transduction of viruses and the like. Cells transfected with said sense oligonucleotide, as such and in isolated form, have an inhibitory action of brain neurodegeneration, in such a way that they can be used as a drug, or a therapeutic research model for inhibition or also the stopping the progression of neurodegenerative lesions. In gene therapy, the vector above for the introduction of a sense oligonucleotide can be injected topically into the lobe temporal or surrounding region of the patient, or systemically. In addition, stem cells can be cultured together and then administered by local or systemic injection. By such administration, the vector can be introduced into the nerve cells of the patient's brain. In case the transduced gene is not permanently taken on the chromosome of each target cell, the administration may be repeated periodically. The method of gene therapy according to the invention includes the technique both in vivo which comprises administering a construct for the introduction of said sense oligonucleotide (a sense oligonucleotide transfer vector) directly into the body, and the ex vivo technique that comprises transferring the gene into cultured stem cells and then culturing, transplanting or otherwise introducing the cells into the patient's body. A gene therapy comprising the introduction of said sense oligonucleotide directly into the cell is also feasible.

The target cells in which the sense oligonucleotide of the gene of the invention is introduced can be selected according to the purpose of the gene therapy (treatment). For example, target cells include neurons of the brain and nerve tissues of the brain, as well as lymphocytes, fibroblasts, hepatocytes, and hematopoietic cells. The method of introducing the sense oligonucleotide in the previous gene therapy includes a viral introduction technique and a non-viral introduction technique. With respect to the viral introduction technique, considering the fact that the sense oligonucleotide to be transferred is a foreign substance that is expressed especially in normal brain cells, the method using a retrovirus vector can be exemplified. Other viral vectors that can be used include the adenovirus vector, the HIV vector (human immunodeficiency virus), adeno-associated virus vector (AAV), herpes virus vector, herpes simplex virus vector (HSV) and vector of the Epstein-Barr virus (EBV). The method of constructing a viral vector to transfer a sense oligonucleotide, and the method for transferring the sense oligonucleotide to the target cell or target tissue are now specifically described. The retroviral vector system consists of a viral vector and an auxiliary cell (packaging cell). The auxiliary cell means a cell that has expressed genes encoding the structural protein gag (structural protein within the viral particle), pol (reverse transcriptase), env (envelope protein), etc., of a retrovirus, but which has not formed viral particles. On the other hand, the viral vector has the signal of packaging and LTR (long terminal repeats), but lacks structural genes such as gag, pol, env, etc., which are necessary for viral replication. The packaging signal is a sequence that functions as a mark in the assembly of a viral particle. Instead of the viral genes, selective genes (neo, hyg) and the oligonucleotide are inserted of objective sense linked in the cloning site. To obtain a high titer of viral particles, it is important to use an insert as short as possible, provide a broad packaging signal including a part of the gag gene, and be careful not to leave ATG of the gag gene. As the vector DNA that guards the sense oligonucleotide When the target is transferred to the helper cell, the genomic RNA of the vector is packaged by the structural protein of the virus formed by the helper cell, whereby the viral particles are formed and secreted. The viral particle as a recombinant virus, infects the target cell and, as a result, the reverse transcribed DNA sequence of the genomic RNA of the virus, integrates into the nucleus of the cell in such a way that the sense gene inserted in the vector can be expressed. A technique can be employed using a fragment of fibronectin that contains the cell adhesion domain, the binding site of heparin and a conjugation segment [Hanenberg H. et al., Exp. Hemat, 23, 747 (1995)], to increase the transfer efficiency of the target gene. An example of the retroviral vector for use in the aforementioned retroviral vector system 5 is the retrovirus derived from the mouse leukemia virus [McLachlin, J.R. and others, Proc. Nati Acad. Res. Molec. Biol., 38, 91-135 (1990)]. The method using an adenoviral vector is now described in detail. The adenoviral vector can be constructed according to the methods that describe Berkner K.L., Curr. Microbiol. Immunol., 158. 39-66 (1992), Setoguchi Y. and others, Blood, 84, 2946-2953 (1994), Kanegae H. and others [Jikken Igaku (Experimental Medicine), 12, 28-34 (1984) ] and Ketner G. et al., Proc. Nati Acad. Sci. USA, 91, 6186-6190 (1994). For example, to build a non-proliferative adenoviral vector, First, the early E1 and / or E3 region of the adenovirus is excised. Then, a plasmid vector containing the desired expression unit of the foreign gene (consisting of the sense oligonucleotide to be transferred, the promoter for the transcription of said sense oligonucleotide, poly-A to ensure the stability of the transcribed gene) is used. and a part of the genomic DNA of the adenovirus, and a plasmid containing the adenovirus genome, to cotransfect the 293 cell, for example. As a homologous recombination between them is thus caused for the replacement of the gene expression unit by E1, a non-proliferative adenovial vector is obtained i. ^ »» * • -? «, Woa« & 3. - I know.-. as a vector that stores the oligonucleotide of objective sense. A 3 'end adenoviral vector with an added terminal protein can also be constructed by ligating the adenoviral genomic DNA in a cosmid vector. In addition, the YAC vector can also be used for the construction of an adenoviral vector. The production of an adeno-associated virus (AAV) vector is now briefly described. The AAV was discovered as a small virus contaminant of adenovirus culture systems. With respect to this virus, the existence of the genus Parvovirus, capable of autonomous proliferation within the host cell without requiring an auxiliary virus for viral replication, and of the genus Dependovirus has been identified., which requires an auxiliary virus. This AAV has a wide range of hosts and is one of the common viruses that infect several types of cells. The viral genome is a single-stranded linear DNA consisting of 4680 nucleotides, the 145 nucleotides at both ends having a characteristic sequence known as ITR (inverted terminal repeat). This ITR region functions as the origin of replication and has the function of an initiator. This ITR is also essential for packaging viral particles and integrating AAV into the chromosomal DNA of the host cell. With respect to the viral protein, the left half of the genome encodes the non-structural protein, which is the regulatory protein Rep that controls replication and transcription. The construction of the recombinant AAV can be carried out using the property of AAV to integrate into the chromosomal DNA, by means from which the desired gene transfer vector can be prepared. This method can be described in detail as follows. First, a plasmid (AAV vector plasmid) is constructed that retains the ITRs at the 5 'and 3' ends of a wild-type AW, and stores the sense oligonucleotide to be transferred, interposed between them. The viral protein necessary for viral replication and virus particle formation is supplied from a separate auxiliary plasmid. It is necessary to ensure that there is no common nucleotide sequence between the two plasmids so that a wild-type virus does not appear by DNA recombination. Then, the two plasmids are transferred into the 293 cell by transfection, for example, and subsequently the cells are infected with an adenovirus such as the helper virus (when the 293 cell is used, this adenovirus can be a non-proliferative one), by means of from which the non-proliferative recombinant AAV is produced. Since this recombinant AAV is present in the nucleus, the cells are subjected to freeze-thawing and recovered, and the contaminating adenovirus is inactivated by heating at 56 ° C. Then, if necessary, the recombinant AAV is separated and concentrated by ultracentrifugation using cesium chloride. In this way, the desired recombinant AAV is obtained for the gene transfer. The production of an EBV vector can be carried out by the method of Shimidzu et al. [Shimidzu N., SAIBÓ KOUGAKU (Cell Technology), 14 (3), 280-287 (1995)].

The production of the EBV vector for the transfer of the sense oligonucleotide according to the invention is now briefly described. The EB virus (Epstein-Barr virus) is a virus of the family Herpes viridae, which was first isolated by Epstein and collaborators of cultured cells 5 derived from Burkitt's lymphoma [Kieff E. and Liebowitz D., "Virology" [Virology] ], 2nd. Edition, Raven Press, New York, 1990, pages 1889-1920]. This EBV has cell transforming activity, and to use it as a vector for the transfer of genes, it is necessary to prepare a defective virus of this transforming activity. This can be done as follows. First, the EBV genome is cloned in the vicinity of the target DNA in which the desired foreign gene is to be inserted. Next, a DNA fragment of the foreign gene and a drug resistance gene are inserted to construct a vector for the preparation of a recombinant virus. Then, the vector for the preparation of a virus Recombinant, cut with a suitable restriction enzyme, is transfected to EBV-positive Akata cells. Recombinant virus formed by homologous recombination is recovered, together with the wild-type Akata EBV, by stimulation of viral production by treatment with surface anti-immunoglobulin. The recombinant virus becomes infected in cells Akata EBV-negative and, in the presence of a drug, resistant clones are selected, whereby Akata cells infected exclusively with the recombinant virus, free of wild-type EBV, can be obtained. In addition, the target recombinant virus vector can be produced in large quantities inducing viral activity in Akata cells infected with the recombinant virus. The method of introducing the target gene into the target cell or target tissue in the gene therapy of the invention includes the following two representative methods. The first method comprises harvesting the target cells of a patient to be treated, developing the cells ex vivo, for example under the addition of interleukin-2 (IL-2) or the like, to transfer the sense oligonucleotide housed in the retroviral vector, and retransplantar the resulting cells (ex vivo method). This method is suitable for the therapy of genetic diseases caused by defective genes and cancer, for example. The second method is a method for direct gene transfer, which comprises directly injecting the sense oligonucleotide into the body of the patient or the target site, such as brain tissue (direct method). More particularly, the first method can be carried out in the following manner, for example. Mononuclear cells, such as stem cells, harvested from the patient, are fractionally separated from monocytes using a blood classifier, and cultured in the presence of IL-2 in a suitable medium such as AIM-V medium for approximately 72 hours, followed by by adding the vector that stores the sense oligonucleotide to be introduced. To increase the transfer efficiency of the sense oligonucleotide, the cells can be developed in the presence of protamine at 32 ° C for 1 hour, centrifuge at 2500 rpm, and then cultivate under 10% carbon dioxide gas at 37 ° C for 24 hours. After repeating this procedure a few times, the cells are further cultured in the presence of IL-2, for example in AIM-V medium for 48 hours, and then washed with saline. Viable cells are counted and the efficiency of the introduction of the sense oligonucleotide is evaluated by said PCR in situ or, when the target is the enzymatic activity, the degree of enzymatic activity is determined. To confirm safety, safety checks such as bacterial and fungal culture, review of the presence or absence of mycoplasma infection, search for endotoxins, etc., are carried out in the cultured cells. Subsequently, the cultured cells transformed with the predicted effective dose of the sense oligonucleotide are returned to the patient by intravenous drip injection. He The above procedure is repeated at intervals of several weeks or a few months to complete gene therapy. The dosage of the viral vector is selected according to the target cell. The usually preferred dose may be, for example, 1 x 103 cfu-1 x 108 cfu, in terms of virus titer per 1 x 108. target cells. An alternative version of the first mentioned method can be adopted, which comprises co-culturing the virus-producing cells that have the retrovirus vector that stores the sense oligonucleotide. target, and the cells of the patient, to thereby introduce the sense oligonucleotide into the target cells. In carrying out the second method (direct method) for gene therapy, it is particularly preferred to develop a preliminary ex vivo experiment to see if the target sense oligonucleotide can actually be introduced by means of the gene transfer method, performing PCR of the CDNA of the vector gene or PCR in situ, or see if the desired therapeutic effect can actually be achieved, for example elevation of a specific activity or growth or inhibition of cell growth objective, by introducing the objective sense oligonucleotide. Furthermore, when a viral vector is used, it is of course of great importance to confirm the safety of introducing the sense oligonucleotide into gene therapy, performing a PCR search of proliferative retroviruses and the like, determining the reverse transcriptase activity, or monitoring the envelope protein gene (env) by the RCR technique. The gene therapy of the invention in Alzheimer's disease, Alzheimer's dementia or Parkinson's disease, may for example be a neurodegenerative disease therapy comprising harvesting stem cells or nerve cells from the brain of the patient. patient, establish a cell line cultured by enzymatic treatment or the like, introduce the oligonucleotide of objective direction into the nerve cells of the brain that are targeted, using AAV or the like, carry out a selection with G418 cells, measure the amount of expression of IL-12 or similar in vivo, give a radiation treatment and inoculate the cells in the brain tissue of the patient or the site of the temporal lobe. The present invention also provides a pharmaceutical composition or preparation (a gene therapeutic agent) comprising a sense oligonucleotide transfer vector of the invention, or a cell line transformed with the sense oligonucleotide as an active ingredient, in a pharmacologically amount effective, in combination with a suitable non-toxic pharmaceutical vehicle or diluent. The pharmaceutical carrier that can be used in the pharmaceutical composition (pharmaceutical preparation) of the invention, includes diluents or excipients, for example fillers, volume fillers, binders, humectants, disintegrants, surfactants, lubricants, etc., which are usually employed depending on the mode of use of said composition, and can be used selectively according to the unit dosage form contemplated in the preparation. The unit dosage form of the pharmaceutical preparation of the invention can be the same as mentioned for the preparation of the polypeptide of the invention, and one suitable one can be selected appropriately according to the therapeutic objective. The therapeutic and prophylactic method for neurodegenerative diseases according to the invention is now described in detail. The present invention provides a method for therapy of neurodegenerative diseases, such as Alzheimer's disease, dementia of Alzheimer's type, cerebral ischemia, Parkinson's disease and similar diseases in which an excess or deficit of the LY6H polypeptide is involved. When the activity of LY6H is excessive, several approaches can be taken. The first method comprises administering an inhibitory compound (antagonist) in an amount effective to inhibit the function of the LY6H polypeptide by blocking its binding to a ligand, substrate, receptor, enzyme, or the like, or inhibiting a secondary signal in combination with a pharmaceutically carrier. acceptable to improve with it an abnormal state. An alternative method comprises administering a LY6H polypeptide in a soluble form, capable of binding to a ligand, substrate, enzyme, receptor or the like, in competition with endogenous LY6H. A typical example of such a competitive substance includes a fragment of the LY6H polypeptide. In another method, a soluble form of the LY6H polypeptide capable of binding to a ligand in competition with endogenous LY6H can be administered. An example Typical of said competitive substance includes a fragment of the LY6H polypeptide. In another method, the expression of the gene encoding the endogenous LY6H polypeptide can be inhibited by applying a technique of inhibiting the expression of the gene to the LY6H gene product. The technique known of this type includes the use of an internally generated or separately administered antisense sequence [eg, "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression" - Oligodeoxynucleotides as antisense inhibitors of expression genetic- CRC Press, Boca Raton, Florida (1988), O'Connor, J. Neurochem. 56: 560 (1991)]. As an alternative method, an oligonucleotide capable of forming a triple helix with the gene may be provided [eg, Lee et al., Nucleic Acids Res., 6: 3073 (1979); Cooney et al., Science, 24: 456 (1988); Dervan et al., Science 251: 1360 (1991)]. These oligomers can be administered as such or related oligomers can be made to be expressed in vivo. For the therapy of abnormal symptoms related to a low expression of LY6H and its activity, several methods can be used. The first method comprises administering a compound capable of activating LY6H (agonist) in a therapeutically effective amount, together with a pharmaceutically acceptable carrier to a subject, to thereby improve the abnormal symptoms. In another method, endogenous production of LY6H by related cells in the subject can be promoted by gene therapy. For example, the polynucleotide of the present invention can be manipulated in such a way that it is expressed with a defective retroviral vector as mentioned hereinabove. Then, this retroviral expression construct is isolated and introduced into packaging cells transduced with a retroviral plasmid vector which stores the RNA encoding the polypeptide of the invention, so that the packaging cells form infectious viral particles containing the target gene. These producer cells are administered to the subject for in vivo manipulation of the cells, so that the polypeptide can be expressed in vivo. For a review of gene therapy, reference can be made to "Human Molecular Genetics" - Human Molecular Genetics -, T.

Strachan and A.P. Read, BIOS Scientific Publishers Ltd. (1996), chapter 20, Gene therapy and other therapeutic approaches based on molecular genetics, including the specific references cited therein. An alternative method comprises administering a therapeutic dose of LY6H polypeptide in combination with a suitable pharmaceutical carrier. For example, the cells can be formulated in phosphate buffered saline (pH 7.4), Ringer's solution or in an intracellular composition injection, or in such a dosage form can be administered in combination with a conductive substance for increased transfer efficiency. of gene, such as protamine. The method of administration of the above pharmaceutical preparation is not particularly restricted, but an appropriate regimen can be established according to the particular dosage form, the age of the patient, sex and other factors, the severity of the disease and the like. The amount of the active ingredient to be incorporated in the pharmaceutical preparation and the dosage is not particularly restricted, but each can be freely selected from a wide range according to the expected therapeutic benefit, the method of administration, the duration of treatment, background of the patient including age and sex, and other variables.

Generally, the dosage of the retroviral vector which stores the sense oligonucleotide as a pharmaceutical preparation can be, for example, from about 1 x 10 3 pfu to 1 x 10 15 pfu, in terms of retrovirus titer per kilogram of body weight per day. In the case of cells carrying the sense oligonucleotide for introduction, the dosage can appropriately be selected from the scale of about 1 x 10 4 cells / body to 1 x 10 15 cells / body. The above preparation can be administered once a day or in a few divided doses per day, or even intermittently at intervals of 1 or several weeks. Preferably, a substance conducive to increased efficiency of gene transfer, such as protamine, or a preparation containing the same, can be administered in combination. When the gene therapy according to the invention is applied to the therapy of a neurodegenerative disease, it can be performed in a suitable combination with other gene therapies (joint gene therapy), or in combination with pharmacotherapy using an acetylcholinesterase inhibitor or the like, and / or a rehabilitation therapy. The gene therapy of the invention can be performed with reference to the NIH guidelines, including its security aspect [Recombinant DNA Advisory Committee, Human Gene Therapy, 4, 365-389 (1993)]. Furthermore, according to the invention, in order to detect the presence of the LY6H gene, it is possible to prepare a biological sample .- * mJs £ k «> - * y * S ¡_ _- > .V. , -, - ^ J ^^^ ft8 ^ iW ^^ feAta-M ^ Ééatej & ^^ hfca ^! ^^ - such as blood or serum, optionally extract the nucleic acid and analyze it with respect to the LY6H gene. The method of detecting the gene can comprise the preparation of a DNA fragment of the gene of the invention and its design, in such a way that it can be used in the selection of the LY6H gene and / or its amplification. More specifically, it is possible to construct a DNA fragment having the properties of a probe for plaque hybridization, colony hybridization, Southern blotting, Northern blotting, etc., or a probe for the preparation of a full-length or partial DNA of the DNA. gene of the invention expanded by a polymerase chain reaction (PCR) that extends a nucleotide sequence with a polymerase. For this purpose, an initiator having the same sequence as the LY6H gene is first prepared. Then, this primer is reacted, as a probe for selection, with a biological sample (nucleic acid sample) to see if there is the presence of the particular sequence of the LY6H gene. The nucleic acid sample can be prepared by any of several techniques that facilitate the detection of the target sequence, such as denaturation, digestion with restriction enzymes, electrophoresis or dot blotting. As the method for such selection, the use of a PCR technique from the point of view of sensitivity is particularly preferred, and this technique is not particularly restricted as long as a fragment of the gene of the invention is used as an initiator. In this way, any of the techniques known up to now [Science, 230. 1350-1354 (1985)] and the modified versions of PCR that have been developed recently or will be developed in the future [Sakaki, Yoshiyuki and others. (ed.), Jikken Igaku (Experimental Medicine), supplement 8 (9) (1990), Yodosha; Protein, Nucleic Acid, Enzyme: special supplement, Kyoritsu Shuppan, 35 (7) (1990)]. The DNA fragment to be used as the primer is a chemically synthesized oligo-DNA, and said oligo-DNA can be synthesized using an automatic DNA synthesizer or the like, for example the gene setter Pharmacia LKB Gene Assembler Plus (Pharmacia). The preferred length of the primer to be synthesized (sense primer or antisense primer) can be, for example, about 10-30 nucleotides. The probe for the present in said selection is usually a labeled probe, but may be an unmarked one, or the detection may be done according to the specific binding to a directly or indirectly labeled ligand. The proper label and the labeling method for the probe or ligand belong to the prior art. Thus, the prior art mark includes radioisotopes, biotin, fluorescent groups, chemiluminescent groups, enzymes, antibodies, etc., which can be taken by known methods such as nick translation, random initiation and kinase treatment. The PCR technique to be used for detection can be for example RT-PCR, but several modifications of the technique that are of routine use can be used.

In addition, the above test method can be conveniently carried out using a reagent kit to detect a LY6H gene in the samples. Therefore, the present invention provides a kit of LY6H gene detection reagent 5 comprising a DNA fragment of the gene of the invention. This reagent kit comprises at least one DNA fragment that hybridizes with part or all of the nucleotide sequence shown in SEQ ID NO: 2 or its complementary nucleotide sequence, as an essential component, and optionally may contain other components such as a labeling agent and PCR reagents (eg, Taq DNA polymerase, deoxynucleotide triphosphates, initiators, etc.). The labeling agent can be a radioisotope or a chemical modifier such as a fluorescent substance, but the DNA fragment 15 as such can be conjugated to said labeling agent. This reagent kit may additionally contain a suitable reaction solvent or diluent, standard antibody, buffer, wash solution, reaction stop solution, etc., which make it easier to conduct a test. In a further aspect, the present invention provides a method of diagnosing neurodegenerative diseases, comprising using the above test method, and a diagnostic agent or a diagnostic reagent kit, for use in the practice of said method.

By direct or indirect sequencing of the LY6H genes obtained from test samples using the above method, it is possible to find new genes related to LY6H that have high homology with the wild-type LY6H gene. Therefore, the present invention also provides a method of selecting genes related to the human LY6H gene in samples, which comprises performing said test and sequencing the LY6H genes contained in test samples. The wild-type LY6H and / or the LY6H mutant can be determined using the protein encoded by the human LY6H gene of the invention (a polypeptide having the amino acid sequence shown in SEQ ID NO: 1), a polypeptide having a sequence of amino acids derived from the sequence shown in SEQ ID NO: 1 by deletion, substitution or addition of 1 or a plurality of amino acids, a fragment of any of them, or an antibody to any such protein. Therefore, the invention provides a method of determining a wild-type anti-LY6H antibody and / or anti-LY6H mutant, or a method of antigen determination. By this method, the degree of brain nerve deterioration can be detected from a change in wild-type LY6H (polypeptide). Such changes can be detected by sequencing LY6H by means of the well established technology described here above, preferably by detecting the differences in the LY6H polypeptide or the presence or absence of LY6H polypeptide, using said antibody (polyclonal or monoclonal antibody). The following is a specific example of determination of said wild-type and / or mutant LY6H. The anti-LY6H antibody can be used to immunoprecipitate LY6H polypeptide from a solution containing a biological sample obtained from a human body, such as blood or serum, or it can be reacted with the LY6H polypeptide on polyacrylamide gel of Western blot or immunoblot. The LY6H polypeptide can be detected in a paraffin section or in a specimen of frozen tissue by means of a immunohistochemical technique using the anti-LY6H antibody. Antibody production and purification technology is well known in the art and suitable techniques can be employed selectively. The preferred preferred technology for the detection of a wild type LY6H or a mutant thereof, includes the immunosorbent test linked to enzyme (ELISA), radioimmunoassay (RIA), immunoradiometric test (IRMA) and immunoenzymometric test (IEMA), with a sandwich technique using a monoclonal antibody and / or a polyclonal antibody. The invention also provides a ligand of LY6H or a receptor of LY6H that exists in a cell membrane fraction or on a cell surface, and has binding affinity for the LY6H polypeptide. The LY6H receptor can be obtained by conjugating a labeled LY6H polypeptide in a biological sample containing a fraction of the cell membrane, extracting, isolating and purifying the conjugation product and identifying the amino acid sequence of the isolated product. The procedure for the preparation and sequencing method of this LY6H receptor polypeptide are obvious to the person skilled in the art. In addition, by applying the LY6H receptor or a fragment thereof to a drug selection technique, the invention allows the selection of several compounds (which react with the LY6H receptor, including low molecular weight compounds, high molecular weight compounds, proteins, protein fragments, antigens, antibodies, etc.). Preferably, the LY6H receptor is used in its entirety. The LY6H receptor polypeptide or a fragment thereof, for use in such selection, can be immobilized in a solid matrix or can be a free substance in a solution to be transported to the surface of the cell. An example of the aforementioned drug selection is a selection system in which prokaryotic or eukaryotic host cells stably transformed with a recombinant DNA encoding a LY6H polypeptide, or a fragment thereof, are preferably used in a binding assay competitive Alternatively, said host cells, either in the free form or immobilized, are used in the standard binding test. More particularly, the aforementioned drug screening may comprise the reaction of the LY6H receptor polypeptide, or a fragment thereof, with the LY6H polypeptide, or a fragment thereof, in the presence of a candidate drug, to cause the formation of a complex and detecting the degree of inhibition of complex formation of said candidate drug. Thus, in accordance with the invention, a method of drug selection is provided which comprises contacting a candidate drug with the LY6H receptor polypeptide or a fragment thereof, and then detecting the presence of the resulting complex or the presence of a complex of the LY6H receptor polypeptide, or a fragment thereof, with a ligand, by means of a technique known per se. In addition, by determining the activity of the LY6H receptor, it is possible to evaluate whether a candidate drug is capable of antagonizing the LY6H receptor and consequently can modify the activity of LY6H defined above, that is, it can be able to modulate the growth of neurons, or modulate the protein-protein conjugation or the complexing activity of the complex. In said competitive binding test, the LY6H receptor polypeptide, or a fragment thereof, is labeled. When the free LY6H receptor polypeptide or the fragment thereof is separated from the protein-protein complex and the amount of labeling of the free substance (non-complex-forming) is measured, the measured value serves as a measurement standard of the binding of the test factor for the LY6H receptor. The measured value also serves as a measure of the inhibition of LY6H receptor binding to the LY6H polypeptide. By analyzing in this manner a small peptide (pseudopeptide) of the LY6H polypeptide, the candidate drug can be determined as a substance having LY6H receptor antagonistic activity.

Another protocol for drug selection according to the invention is to select a compound that has an adequate binding affinity for the LY6H receptor polypeptide. Briefly, this method comprises synthesizing a large number of different test peptide compounds on a solid support such as the surface of a plastic spike or other material, by reacting the test peptide compounds with the LY6H receptor polypeptide and after washing, detect the products of the binding reaction of the LY6H receptor polypeptide by a known method [eg, PCT patent publication No. WO 84-03564]. The purified LY6H receptor can be coated directly on the plate to be used in said drug selection process. The antibody can be captured with a non-neutralizing antibody against the polypeptide, and the LY6H receptor polypeptide can be immobilized on a solid phase. The invention is also directed to the use of a competitive drug selection test. For binding to the LY6H receptor polypeptide, or a fragment thereof, a neutralizing antibody capable of binding specifically to the LY6H receptor polypeptide with the candidate compound is competed. By such a competitive reaction with the neutralizing antibody, the presence of any peptide having one or more antigenic determinants of the LY6H receptor polypeptide can be detected.

As an additional method of drug selection, the LY6H polypeptide of the invention, or the product of the LY6H gene of the invention, can be used in the selection of compounds that activate (agonists) or inhibit (antagonists or inhibitors) the activity of the polypeptide LY6H or the product of the LY6H gene. Using the LY6H polypeptide or the LY6H gene product of the invention, cell agonists or antagonists, cell-free preparations, chemical libraries and compositions of natural origin can be identified. These agonists or antagonists can be natural or modified substrates, ligands, enzymes or receptors of the LY6H polypeptide of the invention or structural or functional copies of the polypeptide of the invention [Coligan et al., Current Protocols in Immunology, 1 (2), chapter 5 ( 1991)]. In situ hybridization studies revealed the expression of the LY6H gene of the invention in various tissues of the normal human brain, at particularly high levels in the hippocampus and entorhinal cortex, which are usually severely impaired in Alzheimer's patients, and it was found that their level of expression was considerably depressed in the temporal lobe, including the hippocampus and the entorinal cortex, of patients with Alzheimer's disease. It is, therefore, very likely that this gene is associated with the onset and progression of said disease. Therefore, it is expected that an agonist or antagonist of this LY6H protein, or a product of the LY6H gene, have application as a therapeutic or prophylactic drug for neurodegenerative diseases such as Alzheimer's disease, Alzheimer's dementia, cerebral ischemia and Parkinson. The compounds obtained from the selection of candidate drugs for said diseases related to the gene LY6H, have the functions of the protein of the invention (the expression product of the gene of the invention), such as action of support of neuronal survival , nerve lengthening action, nerve regeneration action, neuroglia activation action, etc., in the central nervous system and other nervous systems, and mnemonic action of the brain (memory formation), among other physiological actions, and they can therefore be used as a therapeutic or prophylactic drug for various neurodegenerative diseases such as Alzheimer's disease, Alzheimer's dementia, cerebral ischemia and Parkinson's disease. In this way, the proteins of the invention (including the gene expression products, partial peptides thereof and their salts), can be used as reagents for the selection of compounds that promote the functions of the protein of the invention. The invention provides a method of selecting compounds that promote the functions of the protein of the invention (hereinafter referred to each time as a functional enhancer of the protein of the invention). More particularly, the invention provides (a) a method of selecting a functional enhancer of the protein of the invention, comprising contacting (1) the protein of the invention with cells -i ^ k yy-i ^^ tít ^ nervous or a nervous tissue on the one hand, and (2) on the other hand the protein and a test compound with said cells or nervous tissue, and compare the results, and (b) a method of selecting a functional enhancer of the protein of the invention, comprising administering (1) the protein of the invention to a vertebrate, on the one hand, and (2) on the other hand, the protein of the invention and a compound test to the vertebrate; and compare the results. More particularly, in the selection method (a) above, a physiological activity is measured in the central nervous system or other nervous system, such as neuronal survival support activity, nerve elongation activity, nerve regeneration activity or activity of activation of neuroglia, under conditions (1) and (2) above, and the results are compared. In the selection method (b), the mnemonic activity (of memory formation) in the brain is measured, for example, under said two conditions (1) and (2), and the results are compared. Nerve cells (neurons and glia) for use in the above selection, include neuroblastoma cells, glioma cells, and their hybridoma cells (e.g. N18TG-2, IMR-32, GOTO (e.g., GOTO-P3) cells) , NB1, C6BU-1, U251, KNS42, KNS81 and NG108-15, and PC12 cells that have a differentiation power for nerve cells). The nerve tissue that can be used includes the mouse neuroepithelial cell, rat hippocampal primary cultured cell, Prukinje cell from fetal mouse culture, and mouse dorsal root ganglia. The test compound includes peptides, proteins, non-peptide compounds, ~ As such, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma can be novel compounds or known compounds by carrying out said selection method. (a), the protein of the invention (including a partial peptide thereof or a salt thereof), is dissolved or suspended in a selection buffer to prepare a sample of the protein of the invention. any buffer solution that does not interfere with the contact between the protein of the invention and the nerve cell or tissue (eg, phosphate buffer, Tris-HCl buffer, etc., at a pH of about 4-10, preferably at a pH of about 6-8) The duration of the contact is usually about 1-10 days, preferably about 7-10 days.The contact temperature is usually about 37 ° C. of the protein of the invention in the central nervous system or other nervous systems, such as support for neuronal survival, nerve lengthening activity, nerve regeneration activity, and neuroglia activation activity, can be determined by the methods Routines such as visual determination of axonal elongation, measurement of intracellular Ca 2+ concentration, and the like. Any test compound that promotes any of said physiological activities, such as neuronal survival support activity, nerve elongation activity, nerve regeneration activity, neuroglia activation activity, at least For this purpose, about 20%, preferably not less than about 30%, preferably not less than about 50%, most preferably not less than about 70%, under the condition (2) mentioned above, compared to condition (1), can be selected as a functional enhancer of the protein of the invention. In carrying out selection method (b) above, the protein of the invention, alone or in combination with the test compound, is administered to test animals by intravenous, subcutaneous or intramuscular injection, or orally. The dosage of the protein of the invention for oral administration is generally about 0.1-100 mg / day, preferably about 1.0-50 mg / day, very preferably about 1.0-20 mg / day, per mammal (based on 50 kg body weight). The parenteral dose should be selected according to the recipient and method of administration, but it is preferable to administer about 0.01-30 mg / day, preferably about 0.1-20 mg / day, very preferably about 0.1-10 mg / day, per mammal (50 kg body weight) by the intravenous route. Test animals include mammals such as man, monkey, chimpanzee, mouse, rat, rabbit, sheep, pig, bovine, horse, cat and dog, and fish (eg, carp, salmon, herring, trout, gold carp, etc.). The mnemonic activity (of memory formation) in the brain of the protein of the invention can be determined for example in accordance with a water maze test protocol [Morris R.G.M., J. Neurosci. Meth., 11, 47-60 (1984)]. Any test compound that promotes the mentioned mnemonic effect, in not less than about 20%, preferably not less than 50%, preferably not less than 70%, under said condition (2), in comparison with said condition (1), it is useful as a functional enhancer of the protein of the invention. The screening equipment, as a further embodiment of the invention, contains the protein of the invention (including the gene expression product, a partial peptide thereof, and any salt of any of them) as an essential component. A kit consisting of the following components 1-4 is an example of the selection equipment of the invention. Component 1: Hanks solution as a test buffer. Component 2: standard protein (protein of the invention or a salt thereof). Component 3: nerve cells or a nervous tissue (a culture of said nerve cells or nerve tissue in a 24-well plate), 104 cells / well, developed using Eagle's MEM, Hanks solution under 5% C02 at 37 ° C). Component 4: An inverted microscope for observation. The selection with the previous selection team can be carried out in the following way.

"A [Method] The number of positive axonal elongation cells per field of vision is counted in the well containing the test compound, and compared to the number of positive axonal elongation cells in the control well (compound free) test), and the difference is statistically tested The compound or salt obtained with the selection method or with the selection equipment according to the invention, is a member selected from the above-mentioned class consisting of peptides, proteins, compounds non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and is a compound capable of promoting the function of the protein of the invention.The compound that promotes the functions of the protein of the invention as such, show physiological activities such as neuronal survival support activity, nerve lengthening activity, activity d of nerve regeneration, neuroglia activation activity, etc., and thereby promote the function of the protein of the invention or the like, additively or synergistically or, although without showing said physiological activity on its own, it can promote the function of the protein of the invention. Examples of the salts of the compound include salts with physiologically acceptable bases (for example alkali metals) or acids (for example organic acids, inorganic acids). Particularly preferred are physiologically acceptable acid addition salts, such as 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, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid). The compound or salt that promotes the function of the protein of the invention is of value as a safe therapeutic-prophylactic drug, of low toxicity for several neurodegenerative diseases such as Alzheimer's disease, Alzheimer's dementia, cerebral ischemia and Parkinson's disease. . The above selection procedure includes the use of cells that express the LY6H polypeptide on the cell surface or that respond to the protein of the invention. Among such cells, there are cells derived from mammalian animals, yeasts, Drosophila and E. coli. Cells expressing the LY6H polypeptide (or the cell membrane having the expressed polypeptide) or responding to the LY6H polypeptide are contacted with the test compound to observe stimulation or inhibition of functional binding or response. The LY6H activity of the cells in contact with the candidate compound is then compared with that of similar cells that are not in contact. The above test can be carried out by detecting adhesion to cells that store the LY6H polypeptide, using a tag directly or indirectly coupled to a candidate compound, or in a test system that uses a competition with a substance that competes with the brand. In this way, the binding of the candidate compound can be easily tested. In addition, using a suitable detection system for cells carrying the LY6H polypeptide in such tests, it can be tested whether the candidate compound produces a signal attributable to the activation of the LY6H polypeptide. Generally, the activation inhibitor is tested in the presence of a known agonist and the effect of the candidate compound on activation due to the agonist is observed. The test may comprise a simple procedure comprising mixing the candidate compound with a solution containing the LY6H polypeptide to form a mixture, determining the activity of LY6H in the mixture, and comparing the activity of LY6H in the mixture with a standard. The low molecular weight compound (agonist or antagonist) which binds to the LY6H protein can be obtained for example by a selection with BIACORE 2000 [Markgren P.O. and others, Analytical Biochemistry, 265, 340-350 (1998)]. In accordance with the invention, for the purpose of developing a more active or stabilized LY6H-derived polypeptide, or a drug that increases or blocks the function of the LY6H polypeptide in vivo, it is possible to construct a biologically active polypeptide or a structural analogue thereof for interaction, such as a LY6H agonist, LY6H antagonist, LY6H inhibitor or the like. The aforementioned structural analog can be obtained for example by determining the three-dimensional structure of a complex of the LY6H polypeptide with another protein, by X-ray crystallography, computer modeling, or a combination of such techniques. Information on the structure of a structural analog can also be acquired by modeling the polypeptide based on the structures of homologous proteins. To obtain said polypeptide derived from more active or stabilized LY6H, analysis by sweeping alanine can be used. This method comprises replacing each amino acid residue with Ala to determine the influence of the substitution on the activity of the peptide. From In this manner, as each amino acid residue of a peptide is analyzed, the region of importance for the activity or stability of the peptide is determined. With this method it is possible to design a more active or stable LY6H-derived polypeptide. It is also possible to isolate the specific antibody for the target, selected by functional test, and analyze its crystal structure. As a general rule, through this approach we obtain the drug core that provides a basis for the subsequent design of the same. By producing an anti-idiotypic antibody for the pharmacologically active functional antibody, it is possible to identify and isolate a peptide from a peptide library generated chemically or biologically. Therefore, it is predictable that the selected peptide can also serve as a drug core.

»*» -JHíMB **. a »and develop drugs that have or act as inhibitors, agonists or antagonists of LY6H activity. The evaluation of said drug can be done by titrating its effect on neuronal survival using primary culture hippocampal neurons [Japan. J. Pharmacol., 53, 221-227 (1990)] or investigating their effect on neurodegenerative lesions in Alzheimer model animals such as transgenic mice of mutant precursor-amyloid protein gene or mutant presenilin 1 gene [Nature, 373, 523 -527 (1995); Nature Med., 5, 560-564 (1999)]. The compound thus obtained can be used not only as a drug for Alzheimer's disease, but also as a therapeutic drug for cerebral infarction and other neurodegenerative diseases. In addition, according to the invention, by constructing mice carrying the destroyed LY6H gene (transgenic mice with a history of destruction of LY6H), it is possible to determine which site or sites of the nucleotide sequence of the LY6H gene have influence on said multiple activities of LY6H in vivo, that is, what functions the expression products of the LY6H gene and a modified LY6H gene have in vivo. This method is a technique to intentionally modify the genetic information of a living being using homologous recombinant genes, and includes a method that uses embryonic stem cells from mouse (ES cells) as an example [Ca ^ cchi M.R., Science, 244, 1288-1292 (1989)]. The method of construction of said mutant mice is currently a routine technology for those skilled in the art, and mutant mice can be easily constructed by applying a human wild-type LY6H gene or a mutant LY6H gene to a modified version of the aforementioned technology [Noda, Testuo (ed.) Jikken Igaku (Experimental Medicine), supplement, 14 (20) (1996), Yodosha]. Therefore, using this technique, it is possible to design and develop drugs that have improved or stabilized LY6H activity, or inhibitors, agonists and antagonists of LY6H activity.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of Northern blots showing the pattern of expression of the LY6H gene in various brain sites of a patient with Alzheimer's disease.

PREFERRED MODALITY OF THE INVENTION The following examples are intended to illustrate the invention in greater detail. < .í * a & »3 * lifelá? b? £. * (1) Cloning and DNA sequencing of the human LY6H gene. Mice extracted from the human fetal brain, from CLONTECH Laboratories, were purchased and used as starting material. From this mRNA, a cDNA was synthesized and ligated into the ZAPII vector (Stratagene) to construct a cDNA library (Otsuka GEN Research Institute, Otsuka Pharmaceutical Co.). Using the in vivo excision method [excision in vivo: Short J.M. et al., Nucleic Acids Res., 16, 7583-7600 (1988)], colonies of Escherichia coli carrying the human gene were formed on agar medium and randomly collected in a 96 well microplate to record the E clones. Coli who carried the human gene. These clones were stored at -80 ° C. After, each registered clone was cultured overnight in 1.5 ml of LB medium, and the DNA was extracted and purified using an automatic plasmid extractor PI-100 (Kurabo). The contaminated E. coli RNA was decomposed with RNAse and removed. Finally, 30 I of a DNA solution was prepared and, using a portion of 2 I, the approximate size and amount of DNA was checked with the minigel method. A portion of 7 I was used for a sequencing reaction and the remaining 21 I were stored as plasmid DNA at 4 ° C. With this method, it was possible to extract a cosmid that can also be used as a probe for FISH (in situ hybridization with fluorescence), which is described below, with a minor modification of the program. A dideoxy terminator reaction of Sanger et al. Was then carried out using T3, T7 or a synthetic oligonucleotide primer [Sanger F. et al., Proc. Nati Acad. Sci., USA, 7_4, 5463-5467 (1977)] or a cyclic sequencing reaction [Carothers A.M. and others, Bio. Techniques, 7, 494-499 (1989)], which is the dideoxy termination reaction plus PCR. These are techniques for chain extension with specific termination for 4 types of bases, using a small amount (approximately 0.1-0.5 g) of plasmid DNA as a template. Using an initiator labeled with FITC (fluorescein isothiocyanate) as the starter sequence, approximately 25 reaction cycles were carried out using Taq polymerase. From the fluorescently labeled DNA fragment, the sequence of approximately 400 nucleotides was determined from the 5 'end of the cDNA with the automatic DNA sequencer ALF ™ DNA Sequencer (Pharmacia). The 3 'untranslated region is high in heterogeneity between genes, and is suitable for differentiation of individual genes. Therefore, sequencing of the 3 'end region was also performed in some cases. The huge nucleotide sequence information generated with the DNA sequencer was transmitted to the 64-bit computer DEC3400 for computerized homology analysis. This analysis of homology was carried out by means of a database search (GenBank, EMBL) according to the FASTA program of UWGCG [Pearson W.R. and Lipman D.J. Proc. Nati Acad. Sci. USA., 85, 2444-2448 (1988)]. Fujiwara et al. Describe in detail the above-mentioned method of analysis of a human fetal brain cDNA library [Fujiwara T. et al., DNA Res., 2, 107-11 1 (1991)]. The ESTs (expressed sequence tags: partial DNA sequences of the expressed gene fragment) were then sequenced randomly from the human fetal brain cDNA library constructed as mentioned above. It was found that the clone designated GEN-425D01 in the GenBank / EMBL sequence search according to the FASTA program was highly homologous with the gene encoding the mouse Ly6 family protein. Using a double-stranded DNA inserted into a vector (pBluescript vector, Stratagene) as a template and a synthetic oligonucleotide as an initiator, the nucleotide sequence of the cDNA was determined by including the entire coding region of the aforementioned clone by the terminating method. Sanger dideoxy chain. Sequencing with an ABIPRISMTM377 automatic DNA sequencer revealed that the cDNA sequence of the clone obtained above contained a deduced amino acid coding region of 420 bases, and the amino acid sequence encoded by it had 140 amino acid residues. The nucleic acid sequence of the full-length cDNA clone was composed of 854 nucleotides. The complete sequence is shown in SEQ ID NO: 3; the nucleotide sequence of the open reading frame is shown in SEQ ID NO: 2; and the deduced amino acid sequence encoded by said nucleotide sequence is shown in SEQ ID NO: 1. The amino acid sequence of the human LY6H protein was compared to the sequences of other proteins of the Ly6 family, and the nucleotide sequence conserved in the translation initiation region amino acids [Kozak M. J., Biol. Chem., 266, 19867-19879 (1990)] was compared to the 5 'region of the human LY6H gene. The initiation codon thus determined was located at position 99-101, which is the second triplet ATG of the nucleotide sequence shown in SEQ ID NO: 3. In addition, the polyadenylation signal (AATAAA) was located at position 832-837 of the same nucleotide sequence. (2) Northern blot analysis To define the expression profile of LY6H in tissues, a Northern blot analysis was performed using various human tissues. 20 In the Northern blot analysis Blot I and II human MTN (Northern of multiple tissues, CLONTECH) was used. The cDNA fragment was amplified by PCR using a series of T3 and T7 promoter sequence primers.

The amplified PCR product of said clone GEN-425D01cDNA was labeled with [3 P -dCTP (randomly initiated DNA labeling equipment, Boehringer Mannheim GmbH) for use as a probe. The blot containing the amplification product was prehybridized (under conditions according to the product protocol), and then subjected to hybridization according to the product protocol. Hybridization was performed at 65 ° C overnight in a solution composed of 1 M NaCl / 50 mM Tris-HCl (pH 7.5) / Denhardt 2x solution / 10% dextran sulfate / 1% SDS solution (containing 100 μg ml of DNA from denatured salmon sperm). It was then washed twice with 2x SSC / 0.1% SDS at room temperature and the product was washed once with 0.1x SSC / 0.1% SDS at 65 ° C for 40 minutes. The filter was exposed against X-ray film (Kodak) at -70 ° C for 18 hours. The previous test was performed using the following adult human tissues: brain, pancreas, testes, small intestine, colon, thymus, prostate, ovary, heart, placenta, lung, liver, skeletal muscle, kidney, spleen, testes, and leukocyte. peripherally blood. As a result, transcripts of approximately 1 kb showing homology with LY6H were observed in brain, pancreas, testes, small intestine, colon, thymus, prostate and ovary, particularly high in the brain. (3) Localization of the gene in the chromosome by FISH using cosmid clones FISH was carried out for chromosomal localization using 0.5 g of each cosmid DNA as a probe, according to the known method [Takahashi E. et al., Hum. Genet., 86, 14-16 (1990)]. FISH signals were taken using Provia 100 film (Fuji, ISO 100) or CCD camera system (Applied Imaging Cyto Vision). As a result, it was found that the human LY6H gene was located at q24.3 of chromosome 8. In this way, GEN425D01 was mapped on the band of chromosome 8q24.3. Antibodies against proteins belonging to the Ly6 family have been used in the purification of stem cells of the blood as a target of gene therapy [van de Rijn M. et al., Proc. Nati Acad. Sci. USA, 86, 4634-4638 (1989)], in studies on the differentiation of blood cells [van de Rijn M. et al., Proc. Nati Acad. Sci. USA, 86, 4634-4638 (1989)], Classon B.J. and Coverdale L., Proc. Nati Acad. Sci. USA, 91, 5296-5300 (1994)], in the activation of immune cells [Malek T.R. et al., J. Exp. Med., 164, 709-722 (1986)], on the inhibition of the production of active immune cells [Haque A. et al., Immunology 69, 558-563 (1990)], and the like , and they have also been found to have effects against tumors [Lu L. et al., J. Immunol., 142, 719-725 (1989)]. The human LY6H gene provided in the present example allows the detection of the expression of the gene in various tissues, the production of the human LY6H protein by means of genetic engineering and the construction of an antibody using the gene, thus allowing said purification of stem cells of the blood, research in the differentiation of blood cells, activation of immune cells, inhibition of the activation of immune cells and therapy of 5 tumors . In addition, the LY6H expressed at a high level in the brain, allows the investigation of the differentiation of nerve cells, the activation of neurons and the therapy of neural and mental diseases. It also became possible to select compounds with the human LY6H protein as a target, and the compounds thus obtained are as useful as the anti-human LY6H protein.

EXAMPLE 2 (1) Northern blot analysis in the brain tissues of a patient with Alzheimer's disease Northern blot analysis was performed according to example 1 (2). To investigate the expression of the LY6H gene in the brain tissues of patients with Alzheimer's disease, Northern blot analysis was performed using brain tissues from an Alzheimer's patient and normal human brain tissues.

JWjfcfc SMlIfilItt * - »fc fa? S3hH» Northern blotting was performed using human normal brain blot II and human Alzheimer's blott II (both Invltrogen), and expression of the LY6H gene was compared in various brain tissues, particularly the frontal lobe, the temporal lobe, the parietal lobe, the occipital lobe, the bridge, the thalamus and corpus callosum, between normal brains and with Alzheimer's. The results are shown in figure 1. As noted in example 1, the LY6H gene is expressed at a high level in the brain. The above analysis revealed that, while gene expression was confirmed in several tissues of the normal human brain, the gene was expressed at particularly high levels in the temporal lobe, including the hippocampus and the entorinal cortex, which is known to deteriorate Severely in patients with Alzheimer's disease, while significant reductions were found in the temporal lobe, including the hippocampus and the entorinal cortex, in the patient with Alzheimer's disease, indicating that it is very likely that the gene is involved in the beginning. and advance of this disease. Therefore, it is expected that the sense chain of the LY6H gene, the expression product of LY6H and the LY6H protein, have application as therapeutic drugs for Alzheimer's disease, Alzheimer's type dementia, cerebral ischemia and Parkinson's disease. In addition, agonists and antagonists of the LY6H protein are also expected to be useful as therapeutic drugs for Alzheimer's disease and other diseases.

EXAMPLE 3 (1) Construction of an LY6H expression vector The LY6H cDNA obtained by the in vivo excision method is cut with Mv1 l and Xhol to give a fragment of approximately 800 bases. This fragment containing the entire coding region of the LY6H gene shown in SEQ ID NO: 1 is ligated to pAc5.1 / V5-HisA cut with EcoRV / Xhol (Invitrogen) to construct an expression vector (expression vector pAC / LY6H). (2) Expression and purification of the active protein of the invention The DNA of the expression vector pAc / LY6H and DNA of the vector pCoHYGRO (Invitrogen) are mixed in a ratio of 19: 1, and introduced into fruit fly cells. (Schneider 2) by phosphate transfection of calcium. The cells are then cultured in 10% fetal calf serum - DES expression medium (Invitrogen) at 23 ° C for 48 hours, 300 μg / ml hygromycin (Hygromycin B, Boehringer Mannheim) is added to the culture and the selection of clones of drug resistant cells for 2 weeks. A stable transformant is subjected to stationary culture to a concentration of 5 x 106 cells / ml using Falcon 5000 culture flasks (Becton Dickinson) containing 20 ml of 10% fetal calf serum-DES expression medium (Invitrogen), and the cultured cells are harvested. After washing twice with phosphate buffered saline (PBS), the Tiftisf «aK« dfl- & - -. nf - »ffet« ¡s. * - > t, suasito, ryíüJSKX -ase cells are resuspended in PBS containing 2% bovine serum albumin and 0.5 U / ml of phosphatidylinositol-specific phospholipase C (PIPLC), and cultured at 37 ° C for 1 hour. The target protein can be purified from the culture supernatant by means of ion exchange chromatography or the like. (3) Isolation and culture of hippocampal neurons The entire brain is aseptically isolated from fetal rats SD on embryonic day 18 and the hippocampus is removed. The excised tissue is cut into thin slices with a surgical knife and incubated for enzymatic treatment in PBS containing 0.25% trypsin and 0.002% DNase I at 37 ° C for 20 minutes. After stopping the enzymatic reaction by adding fetal calf serum, the aspiration-expulsion of the cell digested product is repeated 3 times with a pipette having a plastic tip to disperse the cells. The cell dispersion is passed through a filter consisting of 2 stacked sheets of lens paper to remove undigested tissue and centrifuged at 1000 rpm for 5 minutes. The cells are washed with DMEM (Gibco) and seeded in a 96-well plate coated with poly-L-lysine (Sigma) containing 10% FCS-DMEM at a final concentration of 2 x 10 5 cells / cm 2. (4) Treatment with the active protein of the invention The cells above are finalized 24 hours and after changing the culture medium to DMEM supplemented with N2 1% (Gibco), the active protein of the invention prepared according to (2) is added. ) (group of the invention). For comparison, the active protein of the invention is treated with heat in a boiling water bath for 5 minutes and added (boiling protein group). (5) Evaluation of neuronal survival of the hippocampus The cells (culture) in each group prepared according to (4) are cultured for 72 hours. The hippocampal neuronal survival support effect of the active protein of the invention can then be evaluated by the MTT test [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide]. This MTT test can be performed using for example the "CelITiter 96" test system from Promega. (6) Isolation and culture of midbrain neurons The entire brain is aseptically isolated from fetal rats SD on embryonic day 14 and the ventral midbrain is removed. The tissue is cut into thin slices with a surgical knife and incubated for enzymatic treatment in phosphate buffer saline (PBS) containing 0.25% trypsin and 0.002% DNase., at 37 ° C for 20 minutes. After stopping the enzymatic reaction by adding fetal calf serum, the aspiration-expulsion of the cell digestion product is repeated 3 times with a pipette that has a plastic tip to disperse the cells. The cell dispersion is passed through a filter consisting of 2 stacked sheets of lens paper to remove undigested tissue and centrifuged at 1000 rpm for 5 minutes. The cells are washed with DMEM / F12 (Gibco) and seeded in a 96-well plate coated with poly-L-lysine (Sigma) containing 10% FCS-DMEM / F12 at a final concentration of 3 x 10 5 cells / cm 2. (7) Treatment with the active protein of the invention The cells prepared according to (6) are cultured 24 hours, and after changing the culture medium to DMEM supplemented with N2 1% -DMEM / F12 (Gibco), the protein is added. active ingredient of the invention prepared according to (2) (group of the invention). For comparison, the active protein of the invention is treated with heat in a boiling water bath for 5 minutes and added (boiling protein group). (8) Evaluation of the medium brain neuronal survival support effect The cells (culture) in each group prepared according to (7) are cultured for 72 hours. The effect of the medium brain neuronal survival support of the active protein of the invention can then be evaluated by the MTT test [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] . This MTT test can be performed using for example the "CelITiter 96" test system from Promega. (9) Evaluation of the support effect of dopaminergic neuronal survival The cells (culture) in each group prepared according to (7), are cultured for 72 hours and then fixed, allowing them to stand in paraformaldehyde 4% -PBS at room temperature for 15 minutes. . Then, they are passed through a membrane using Triton 1% x100 / PBS. To prevent non-specific binding of the antibody, the cells are incubated in 10% goat serum-PBS for 1 hour and then, using a polyclonal anti-tyrosine hydroxylase antibody (Chemicon, diluted 1000 times with PBS), the cells are incubate at 4 ° C for 16 hours. After removing the antibody solution, the cells are washed with PBS and, with goat anti-rabbit immunoglobulin, conjugated with peroxidase-labeled dextran polymer (Dako), the cells are incubated at room temperature for 1 hour. Tyrosine hydroxylase positive cells can be detected by the color reaction using diaminobenzidine as the substrate. Using the number of tyrosine hydroxylase positive cells as a marker, the dopaminergic neuronal survival support effect can be evaluated.

INDUSTRIAL APPLICABILITY The present invention provides a specific novel gene of the brain and a protein encoded by it, and using both can provide valuable technologies for the purification of stem cells of the blood, investigation of the differentiation of blood cells, activation of immune cells , inhibition of the production of active immune cells and tumor therapies. Also, the present invention provides novel genes that have physiological activities such as neuronal brain survival support activity, nerve elongation activity, nerve regeneration activity, neuroglia activation activity, and memory memory activity of the brain. In view of the remarkable depression of its level of expression in the temporal lobe of the brain of patients with Alzheimer's disease, it is considered that the gene of the invention inhibits neurodegenerative tissue changes, thus being useful as a gene therapy drug. In addition, the expression product of the gene of the invention has application as a prophylactic and therapeutic drug for said neurodegenerative diseases.

LIST OF SEQUENCES < 110 > Ostuka Pharmaceutical Co., Ltd. < 120 > GenLY6H < 130 > P99-45 < 160 > 3 < 170 > PatentlnVer.2.0 < 210 > 1 < 211 > 140 < 212 > PRT < 213 > Human embryonic brain < 400 > 1 Met Leu Pro Ala Ala Met Lys Gly Leu Gly Leu Ala Leu Leu Ala Val 1 5 10 15 Leu Leu Cys Ser Wing Pro Wing His Gly Leu Trp Cys Gln Asp Cys Thr 25 30 Leu Thr Thr Asn Ser Ser His Cys Thr Pro Lys Gln Cys Gln Pro Ser 40 45 Asp Thr Val Cys Wing Ser Val Arg He Thr Asp Pro Ser Ser Ser Arg 50 55 60 Lys Asp His Ser Val Asn Lys Met Cys Wing Ser Ser Cys Asp Phe Val 65 70 75 80 Lys Arg His Phe Phe Ser Asp Tyr Leu Met Gly Phe He Asn Ser Gly 85 90 95 .. At-c &S m¿t £ l '' - t ¿> .

He Leu Lys Val Asp Val Asp Cys Cys Glu Lys Asp Leu Cys Asn Gly 100 105 110 Ala Ala Gly Ala Gly His Ser Pro Trp Ala Leu Ala Gly Gly Leu Leu 115 120 125 Leu Ser Leu Gly Pro Ala Leu Leu Trp Ala Gly Pro 30 135 140 < 210 > 2 < 211 > 420 < 212 > DNA ío < 213 > human embryonic brain < 400 > 2 atgctgcctg cagccatgaa gggcctcggc ctggcgctgc tggccgtcct gctgtgctcg 60 gcgcccgctc atggcctgtg gtgccaggac tgcaccctga ccaccaactc cagccattgc 120 accccaaagc agtgccagcc gtccgacacg gtgtgtgcca gtgtccgaat caccgatccc 180 ggaaggatca agcagcagca ctcggtgaac aagatgtgtg cctcctcctg tgacttcgtt 240 aagcgacact ttttctcaga ctatctgatg gggtttatta actctgggat cttaaaggtc 300 gacgtggact gctgcgagaa ggatttgtgc aatggggcgg caggggcagg gcacagcccc 360 tgggccctgg ccggggggct cctgctcagc ctggggcctg ccctcctctg ggctgggccc 420 < 210 > 3 < 211 > 854 < 212 > DNA < 213 > human embryonic brain < 220 > < 221 > CDS < 222 > (99) .. (518) < 400 > 3 acgccgcccg agcccggagt gcggacaccc ccgggatgct tgcgccccag aggacccgcg 60 ccccaagccc ccgcgccgcc cccaggccca cccggagc atg ctg ect gca gcc atg 116 Met Leu Pro Ala Ala Met 1 5 aag ggc etc ggc ctg gcg ctg ctg gcc gtc ctg ctg tgc tcg gcg ccc 164 Lys Gly Leu Gly Leu Wing Leu Leu Wing Val Leu Leu Cys Ser Wing Pro 10 15 20 gct fell ggc ctg tgg tgc cag gac tgc ac ctg ac acc aac tcc age 212 Wing His Gly Leu Trp Cys Gln Asp Cys Thr Leu Thr Thr Asn Ser Ser 25 30 35 cat tgc acc cea aag cag tgc cag ceg tcc gac acg gtg tgt gcc agt 260 His Cys Thr Pro Lys Gln Cys Gln Pro Ser Asp Thr Val Cys Wing Ser 40 45 50 gtc cga ate acc gat ccc age age agg aag gat tcg gtg aac 308 Val Arg He Thr Asp Pro Ser Ser Ser Arg Lys Asp His Ser Val Asn 55 60 65 70 aag atg tgt gcc tcc tcc tgt gac ttc gtt aag cga cae ttt ttc tea 356 Lys Met Cys Wing Being Ser Cys Asp Phe Val Lys Arg His Phe Phe Ser 75 80 85 gac tat ctg atg ggg ttt att aac tct ggg ate tta aag gtc gac gtg 404 Asp Tyr Leu Met Gly Phe He Asn Ser Gly He Leu Lys Val Asp Val 90 95 100 gac tgc tgc gag aag gat ttg tgc aat ggg gcg gca ggg gca ggg drops 452 tS & amp; Sk¡y ?. ",, .-. »., 10 Asp Cys Cys Glu Lys Asp Leu Cys Asn Gly Wing Wing Gly Wing Gly His 105 110 115 age ccc tgg gcc clg gcc ggg ggg etc ctg etc age ctg ggg ect gcc 500 Ser Pro Trp Ala Leu Ala Gly Gly Leu Leu Leu Ser Leu Gly Pro Wing 120 125 130 etc etc tgg gct ggg ccc tgatgtctcc tccttcccac ggggcttctg 548 Leu Leu Trp Wing Gly Pro 135 140 agcttgctcc cctgagcctg tggctgccct ctccccagcc tggcgtggct ggggctgggg 608 gcagccttgg cccagctccg tggctgtggc ctgtggctct cactcctccc ccgacgtgaa 668 gcctccctgt ctctccgcca gctctgagtc ccaggcagct ggacatctcc aggaaaccag 728 gccatctggg caggaggcct ggggaígagg gtgggggggg acccccaggt cccggagggg 788 aagtgaagca acagcccagc tggaagggcg tcttctgcgg agaaataaag tcacttttga 848 gtcctg 854 ^^^ < ? & s ^^

Claims (19)

NOVELTY OF THE INVENTION CLAIMS

1. A gene comprising a nucleotide sequence encoding the following protein (a) or (b): (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; (b) a protein having an amino acid sequence derived from the amino acid sequence shown in SEQ ID NO: 1 by deletion, substitution or addition of one or a plurality of amino acids, and having at least one physiological activity selected from the group which consists of neuronal survival support activity, nerve elongation activity, nerve regeneration activity, neuroglia activation activity and brain memory formation activity.

2. The gene according to claim 1, further characterized in that the nucleotide sequence is that shown in SEQ ID NO: 2.

3. A gene comprising the following polynucleotide (a) or (b): (a) a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 3; (b) a polynucleotide that hybridizes under severe conditions with a DNA having the nucleotide sequence shown in SEQ ID NO: 3.

4. The gene according to any of claims 1-3, further characterized in that the gene is a human gene.

* > ** »- ~ ^. ^ .- ^^ ...,. ..., - ^ ^^^^^ _ ^^^. ^ ^ S ^ ñ * 5.- A gene expression vector that comprises the gene that is claimed in claim 2 or 3.

6.- A cell host comprising the gene expression vector claimed in claim 5.

7. An expression product that is expressed by the host cell claimed in claim 6.

8. A protein that is encoded by the gene which is claimed in claim 1.

9. An antibody having a binding affinity for the expression product that is claimed in claim 7 or the protein claimed in claim 8.

10. An expression product that comprises all or part of a gene of the nucleotide sequence shown in SEQ ID NO: 2, and having at least one physiological activity selected from the group consisting of neuronal survival support activity, nerve elongation activity, activity of nerve regeneration, neurogli activation activity a and brain memory training activity.

11. A therapeutic and prophylactic composition for neurodegenerative disease, comprising the protein claimed in claim 8, an equivalent protein having a partial amino acid sequence thereof, or the expression product claimed in claim 10 , as an active ingredient, in combination with a pharmaceutical vehicle.

12. The therapeutic and prophylactic composition for neurodegenerative disease according to claim 11, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Alzheimer's type dementia, cerebral ischemia and Parkinson's disease.

13. A sense chain oligonucleotide comprising at least 20 consecutive constituent nucleotides of the nucleotide sequence shown in SEQ ID NO: 2.

14. A composition for gene therapy comprising the sense chain oligonucleotide that is claimed in claim 13 as an active ingredient, in combination with a pharmaceutical carrier.

15. A gene-specific probe comprising an oligonucleotide sequence of at least 10 consecutive constituent nucleotides of the nucleotide sequence shown in SEQ ID NO: 2.

16. A method of selecting candidate compounds, comprising using the protein claimed in claim 8, an equivalent protein having a partial amino acid sequence thereof, or the expression product claimed in claim 10; said candidate compounds bind or affect the protein, the equivalent protein or the gene product.

17. The use of the protein claimed in claim 8, an equivalent protein having a partial amino acid sequence of

the same, or the expression product, for the manufacture of a medicament for therapy and prophylaxis of neurodegenerative disease in a patient.

18. The use according to claim 17, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Alzheimer-type dementia, cerebral ischemia and Parkinson's disease.

19. The use of the sense chain oligonucleotide claimed in claim 13, for the manufacture of a medicament for gene therapy in a patient.