WO2002102144A1 - Cell deah-inducible model nonhuman animal - Google Patents

Abstract

It is intended to provide a cell death-inducible model nonhuman animal wherein cell death can be induced specifically in nervous system cells or immune system cells and thus the cytokine signal transfer mechanism can be studied and analyzed; a method of screening a cell death promoter or inhibitor by using the model animal, etc. The cell death-inducible model nonhuman animal is constructed by replacing a fragment containing the whole AMSH gene or a part thereof by a neomycin tolerance cassette, inserting loxP into the side thereof, constructing a target vector by inserting a polyA domain-free diphtheria toxin A chain gene cassette as a negative selection marker, transferring the targeting vector into embryonic stem cells, microinjecting an ES clone deficient in the AMSH gene function with the use of recombinase Cre expression vector into mouse blastocytes, and then returning into the womb of a host.

Description

 Description Cell death induction model Non-human animal Technical field

 The present invention relates to a cell death induction model non-human animal in which the gene function of AMSH and / or AMSH-LP is deficient on the chromosome, and a screen for a cell death promoting or suppressing substance using the cell death induction model non-human animal. The present invention relates to a method for diagnosing diseases caused by deficiency of AMSH and / or AMSH-LP, and the like. Background art

Site force-in is an essential factor in cell activation, differentiation, survival, and cell death in many biological events. The effect of the site cytokine on the target cell is obtained by the interaction of the site force-in with a specific receptor that transduces a signal within the target cell. Among many cytokines, interleukin 2 (IL-2) is known as an important soluble ligand that activates T cells in the immune response (Nature 268, 154-156, 1977, Adv. Immunol. 59, 225-277, 1995, Annu. Rev. Immunol. 14, 179-205, 1996). As signal molecules involved in the signaling pathway via IL-12, the present inventors have identified STAM families, STAM 1 and STAM 2, and these STAMs are not only Jak 3 but also Jak 2 It reports that these tyrosine kinases are directly associated and are adapter molecules that play an important role in signaling from cytokine receptors such as IL-12 receptor and GM-CSF receptor to which these tyrosine kinases bind. (FEBS lett. 477, 55-61, 2000, Biochem. Biowhvs. Res. Commun. 225, 1035-1039, 1996, Immunity 6, 449-457, 1997) _P

 In addition, the present inventors have isolated a novel adapter molecule, the associated molecule with the SH3 domain of STAM (AMSH), from human T cells as a molecule associated with the STAM1-SH3 region (Japanese Unexamined Patent Publication No. 2000-2000). No. 1,394,699, J. Biol. Chem. 274, 19129-19135, 1999), SH3 binding motif of AMSH that interacts with both SH3 domains of STAM1 and STAM2 ( SBM) (J. Biol. Chem. 275, 37481-37487, 2000; J. Biol. Chem. 274, 19129-19135, 1999). The AMS H contains, in addition to the binding site of the SH3 domain of STAM, a nuclear localization signal-like sequence and a region similar to the c-Jun activation domain binding protein 1 (JAB1) subdomain. It is known that

 The above-mentioned STAM promotes c-myc induction by IL-12 and granular macrophage stimulating factor (GM-CSF), whereas the SH3-deficient mutant of STAM has a signal that induces cell proliferation. It is known to act as a dominant negative form in transmission (Immunity 6, 449-457, 1997). In addition, a dominant-negative effect on IL-2 or GM-CSF signaling pathways that induces DNA synthesis and c_myc transcription by binding to ST AM1 when the AMSH mutant lacking the C-terminal half (J. Biol. Chem. 274, 19129-19135, 1999). These facts suggest that AMS H is involved in signal transduction of cytokines, in particular, signaling that occurs downstream such as Jak or STAM.

On the other hand, the present inventors have produced STAM1-deficient mice, and have found that hippocampal CA3 pyramidal neurons are deleted in the mice. This indicates that STAM1 is very involved in neuronal cell survival in vivo. In-site site power input In vivo, STAM1 deficiency has little effect on lymphocyte development or the proliferative response to IL-2 or GM-CSF, whereas STAM1 is functionally important in signaling Is known to have no effect. The difference in the functional role of STAM1 between in vitro and in vivo may be due to the compensatory effect of STAM2. In addition, neuronal abnormalities observed in STAM1-deficient mice suggest that AMS H is also involved in the survival of neuronal cells.

Furthermore, the present inventors have identified a novel Grb2 family molecule, G ads / G τ f 40, as a molecule that associates with AΜSΗ (J. Exp. Med. 189, 1383-1390). , 1999). G ads interacts with SLP76 and LAT and is involved in Τ cell receptor (TCR) signaling (J. Exp. Med. 189, 1383-1390, 1999; J. Exp. Med. 189, 1243-1253, 1999). It has been reported that G ads knockout mice and G ads mutant transgenic mice lacking the SH2 domain show impairment in the development of pre-T cells (International Immunology, 13, 777-783, 2001). This indicates that G ads is essential for T cell development. The biological significance of the interaction of G ads with AMS H has not yet been elucidated, but it is likely that AM SH contributes to T cell development by pre-TCR and TCR signaling. ing. Analysis of the cytokine signaling system has been progressing very rapidly since the 1990s, and is one of the fields in which Japan is leading the field of immunology. However, many of the signaling molecules have still unknown functions, and neither AMSH nor {μSH-L} isolated and identified by the present inventors have been elucidated yet. In addition, it was not known that deletion of the AMSΗ or AMSΗ-LΡ gene on the chromosome induces cell death in specific cells. The object of the present invention is to provide hippocampal neuron cells, cerebral cortex AMS that can specifically induce cell death in neural cells such as neurons, and immune cells such as T cells, B cells, and hematopoietic cells, and study and analyze the mechanism of cytokine signaling. A cell death induction model non-human animal in which the gene function of H and Z or AMS H-LP is deficient on the chromosome, and a method for screening a cell death promoting or suppressing substance using the cell death induction model non-human animal. The present invention provides a method for diagnosing a disease caused by deficiency of AMSH and Z or AMSH-LP.

 The present inventors have intensively studied the elucidation of the physiological functions of AM SH and AM SH-LP, and have produced mice in which the gene functions of AMS H and / or AMS H-LP are deficient on the chromosome, In AM SH homo-deficient mice, abnormalities such as growth retardation, abnormal peripheral nerve reflexes, and ptosis occur from 10 days after birth, resulting in 100% death at 3 weeks of age and lymphopathy around 7 days after birth. Other major tissues such as spheroid cells show no obvious abnormalities, but found to specifically induce widespread cell death in cerebral nerve tissues such as cerebral cortex, facial nucleus and hippocampus. In addition, the present inventors have found that the use of cytokines, which play an important role in the survival and differentiation of neuronal cells in vivo and in vitro, has no effect on such cell death, and has completed the present invention. I came to. Disclosure of the invention

That is, the present invention relates to a cell death-inducing model non-human animal (Claim 1) characterized in that the AMSH gene function is deleted on the chromosome, and that the AMS H-LP gene function is deleted on the chromosome. A non-human animal cell death-inducing model (Claim 2) or a non-human animal cell death-inducing model characterized by deletion of AMS H and AMS H-LP gene functions on a chromosome (Claim 2) 3) or the AM Η- LP gene is shown in SEQ ID NO: 3 or 5. The cell death-inducing model non-human animal according to claim 2 or claim 3, wherein the cell death is caused by nervous system cell death and / or immune system cell death. The cell death-inducing model non-human animal according to any one of claims 1 to 4, wherein the nervous system cells are hippocampal neurons or cerebral cortical neurons. The cell death-inducing model non-human animal according to claim 5, wherein the immune system cells are lymphoid cells. (Claim 7), wherein the non-human animal model for cell death induction according to claim 7, wherein the lymphoid cells are T cells, and wherein the non-human animal is a mouse. The cell death according to any one of claims 1 to 8, wherein The present invention relates to an induced model non-human animal (Claim 9).

The present invention also provides a method for administering a test substance to a non-human animal model for inducing cell death according to any one of claims 1 to 9, or a method for preparing a tissue, organ, or cell derived from the animal. And a cell death-inducing model non-human animal and wild-type non-human animal according to any one of claims 1 to 9. 10. The method for screening a substance promoting or suppressing cell death according to claim 10 (claim 11), wherein the method is characterized by comparing and evaluating the case of a non-human animal. 11. The method for screening for a cell death promoting or inhibiting substance according to claim 11, wherein the non-human animal is a wild-type non-human animal of the same litter as the non-human animal model for cell death induction according to any one of claims 9 to 9. And the non-human animal is a mouse. A method for screening a cell death promoting or suppressing substance according to any one of claims 10 to 12 (Claim 13), and a screening method for a cell death promoting or suppressing substance according to any one of Claims 10 to 13. The cell death promoting or inhibiting substance obtained by the method (Claim 14) and the cell death promoting or suppressing substance according to Claim 14 AMSH and Z or AMSH characterized by containing a cell death inhibitor as an active ingredient. A therapeutic agent for a disease caused by LP deficiency (Claim 15), and AMSH and / or AMSH- A method for diagnosing a disease caused by AMSH and / or AMSH-LP deficiency, characterized by extracting the LP gene and examining the presence or absence of the gene abnormality (Claim 16). A probe for diagnosing a disease caused by AMSH and / or AMSH-LP deficiency consisting of all or a part of the antisense strand of DNA or RNA encoding the protein of Z or AMS H-LP (claim 1) 7) The method according to claim 17, which comprises a probe for diagnosing a disease caused by the deficiency of AMSH and Z or AMSH_LP, which is characterized by deficiency of AMSH and / or AMSH-LP. The present invention relates to a kit for diagnosing a disease (claim 18).

Further, the present invention provides a DNA encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (b) an amino acid sequence shown in SEQ ID NO: 2; A protein comprising an amino acid sequence in which several amino acids are deleted, substituted or added, and having AMS H-LP activity (claim 19), the base sequence shown in SEQ ID NO: 1 or a sequence complementary thereto, or It hybridizes with a DNA comprising a sequence containing a part or all of these sequences (Claim 20) or a DNA constituting the gene according to Claim 20 under stringent conditions, and has an AMSH-LP activity. DNA encoding a protein having the following (a) or (b): (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 4; Shown in 4 A protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and having AMS H-LP activity (claim 22); Nucleotide sequence or its complementary sequence or a sequence containing part or all of these sequences And DNA encoding a protein having an AMSH-LP activity, which hybridizes with the DNA constituting the gene according to claim 23 under stringent conditions. 4) or a DNA encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 6; (b) a protein comprising the amino acid sequence shown in SEQ ID NO: 6 A protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and having AMSH-LP activity (Claim 25); the nucleotide sequence represented by SEQ ID NO: 5; A DNA comprising a complementary sequence or a sequence containing a part or all of these sequences (claim 26) or a DNA constituting the gene according to claim 26 under stringent conditions, and AMS H—with LP activity One or several amino acids in the DNA encoding the protein (claim 27), the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (claim 28), or the amino acid sequence shown in SEQ ID NO: 2 A protein having an AMS H-LP activity (claim 29), a protein comprising the amino acid sequence shown in SEQ ID NO: 4 (claim 30), A protein comprising the amino acid sequence shown in SEQ ID NO: 4 in which one or several amino acids have been deleted, substituted or added, and having AMS H-LP activity (Claim 31); A protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (claim 32) or one or several amino acids in the amino acid sequence shown in SEQ ID NO: 6 Or addition of one amino acid sequence, and proteins with AMS H- LP activity for (claim 3 3). BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a diagram showing the gene maps of the AMS H knockout mouse and the wild-type mouse of the present invention, the PCR method and the Southern blot method in each mouse, and the results of Northern blot analysis in each mouse. .

 FIG. 2 is a diagram showing the results of the survival curves of AMS H knockout mice, heterozygous mice, and wild-type mice of the present invention, and the results of the average weight of each mouse.

FIG. 3 is a diagram showing the results of T cell development and cell growth response by cytokines in the AM SH knockout mouse and wild type mouse of the present invention ₍ FIG. 4 shows the results of the AM SH knockout mouse and wild type mouse). 4 is a photograph showing the results of tissue analysis in the brain of a wild-type mouse.

 FIG. 5 is a photograph showing the results of neuronal cell death in the posterior hippocampal coronal section of the AMS H knockout mouse and wild type mouse of the present invention. FIG. 6 is a photograph showing the AM SH knockout mouse and wild type mouse of the present invention. 1 is a photograph showing the result of AMSH mRNA expression in the brain of a mouse.

 FIG. 7 is a photograph showing the results of cell viability when the hippocampal neurons of the AMS H knockout mouse and wild-type mouse of the present invention were initially cultured in vitro.

 FIG. 8 is a graph showing the results of cell viability when neurons derived from the AMSH knockout mouse and wild-type mouse of the present invention were stimulated in the presence of various cytokines. BEST MODE FOR CARRYING OUT THE INVENTION

The cell death-inducing model non-human animal of the present invention refers to a hippocampal neuron cell, a cerebral cortical neuron cell after birth or with aging due to deficiency of the gene function of AMSH and / or AMSHLP on the chromosome Neural cells such as glia cells, lymphoid cells such as T cells or B cells, monocytes, A non-human animal that specifically induces cell death in immune system cells such as macrophages, basophils, eosinophils, mast cells, neutrophils, megakaryocytes, and hematopoietic progenitor cells such as erythrocytes. In addition, the non-human animal in which the gene function of AM SH and / or AM SH-LP is chromosomally deficient is defined as all or one of the endogenous genes of non-human animal encoding AMS H and / or AMS H-LP. Part is inactivated by gene mutation such as disruption / deletion / substitution, and refers to a non-human animal that has lost the function of expressing AMS H and Z or AM SH-LP. Specific examples include rodents such as mice and rats, but are not limited thereto. The gene sequence and amino acid sequence of AMSH are disclosed in JP-A-2000-139469, and the DNA sequence of AMS H-LP is shown in SEQ ID NO: 4 or 6. DNA encoding a protein consisting of an amino acid sequence, consisting of an amino acid sequence shown in SEQ ID NO: 4 or 6 in which one or several amino acids have been deleted, substituted or added, and — DNA encoding protein having LP activity, consisting of the nucleotide sequence shown in SEQ ID NO: 3 or 5 (GenBa.nk Accession number AB010121) or its complementary sequence, or a sequence containing part or all of these sequences Examples of the DNA include a DNA, a nucleotide sequence represented by SEQ ID NO: 3 or 5 (GenBank Accession number AB010122), a complementary sequence thereof, or a DNA comprising a sequence containing part or all of these sequences. These can be prepared by known methods from, for example, a mouse gene library, a mouse cDNA library, a human gene library, a human cDNA library, etc., based on the DNA sequence information and the like.

The wild-type non-human animal in the present invention means an animal of the same species as the non-human animal deficient in AMS H and / or AMS HLP gene function, Above all, litters can be suitably exemplified. AMSH deficient, AMS H-LP deficient, or AMSH.AMSH_LP double deficient in these homozygous non-human animals born according to Mendelian law It is preferable to use the wild type at the same time, since accurate comparison experiments can be performed at the individual level. Preferred examples of the cell death induction model non-human animal of the present invention include AMS H knockout mouse, AMS H-LP knockout mouse, and AMS H · AMS H-LP double knockout mouse as wild-type non-human animals. Specific examples of such knockout mice include wild-type mice of the same litter. Hereinafter, a case where the non-human animal is a mouse will be described as an example.

As a method for producing the above-mentioned AMS H knockout mouse, any method can be used as long as it can produce a knockout mouse that has lost the function of expressing AMS H, but AM SH can be prepared at a desired time. A production method using a switching expression system capable of losing the expression function is more preferable. As such a switching expression system, a Cre / 1oxP expression system (J. Molecular Biology 150, 467-486, 1981) , J. Molecular Biology 150, 487-507, 1981, "Mainly on the introduction and analysis of mouse rapomanual gene", p. 245-250) and the yeast Saccharomyces cerevisiae FL PZF RT recombination system (J. Mol. Biol. 284, 363-384, 1998) and the like, but are not limited thereto. Specifically, the AMS H gene was screened using gene fragments obtained by a method such as PCR from a mouse gene library, and the screened AM SH gene was screened using recombinant DNA technology for the AM SH gene. All or part of the gene is replaced with a chimera gene such as the neomycin resistance gene, and the diphtheria toxin A fragment (DT-A) gene or simple herpes virus thymidine kinase (HS V—tk) Genes such as genes are introduced to create a targeting vector, and the produced targeting vector is linearized, introduced into ES cells by an electoral poration (electroporation) method, etc., and homologous recombination is performed. And selecting ES cells that show resistance to antibiotics such as G418 and ganciclovir (GANC) from the homologous recombinants. It is preferable to confirm whether or not the selected ES cell is the desired recombinant by a Southern blot method or the like.

The above-mentioned recombinant ES cells are microinjected into mouse blastocysts, and the blastocysts are returned to the foster mother mice to produce chimeric mice. When this chimeric mouse is mated with a wild-type mouse, a heterozygous mouse can be obtained. By mating this heterozygous mouse (AMSH ^{+ /} ), an AMS H knockout mouse (AMS H — / One) can be obtained. As a method for confirming that the AMSH gene in such an AMSH knockout mouse is deficient on the chromosome, for example, DNA is isolated from the tail of the mouse obtained by the above method, and the Southern blot is performed. And the like. Further, the AMS H-LP knockout mouse (AMS H_LP ^{_ /-} ) of the present invention can be produced by the same method as the above-mentioned method for producing the AMSH knockout mouse.

A method for producing a non-human animal in which the gene function of AMS H and AMS H-LP on the chromosome is deficient on the chromosome, that is, an AMS H • AMS H-LP double knockout mouse of the present invention expresses AM SH and AMSH-LP. Any method can be used as long as it can produce a double knockout mouse that has lost function. For example, the AMS H heterozygous mouse (AMS H ⁺ ) can be replaced with an AM SH-LP heterozygous mouse. Somatic mouse (AM SH— LP ^{+ /} ) or AMS H—LP homozygous mouse (AMS H— LP One z I) and are mated, resulting ^{AM SH + / - · AMS H-} LP + / - mice or ^{AMS H + / - · AMS H-} LP- Bruno - AMS H · by mating with the mouse A method for producing AMS H—LP double knockout mice (AMS H-AMS HL P-/-) can be mentioned. As described above, the Cre / 1 o XP expression system and the yeast Saccharomyces cerevisiae FLP It may be a production method in which a switching expression system such as a / FRT recombination system is introduced.

 The cell death-inducing model non-human animal of the present invention includes neural cells such as hippocampal neurons, cerebral cortical neurons, and glial cells, lymphoid cells such as T cells or B cells, monocytes, macrophages, and basophils. , Eosinophils, mast cells, neutrophils, megakaryocytes, models for specifically inducing cell death in immune system cells such as erythrocytes, and models for studying and analyzing cytokine signaling Model to study the mechanism of T cell differentiation and proliferation, and to study the pathogenesis, analysis, treatment, etc. of diseases caused by deficiency of AMSH and / or AMSH-LP gene function, for example, diseases in the cerebral nervous system Useful for models. In addition, since these mice lacking gene function lack the ability to eat, they are useful in the development of treatment methods for diseases associated with central nervous system abnormalities related to eating, and such findings are provided by the present invention. It was first revealed. Using such a cell death-inducing model non-human animal, a disease caused by a defect in the gene function of AMS H and Z or AMS H-LP, for example, growth retardation, abnormal peripheral nerve reflex, abnormalities such as ptosis, etc. And drugs useful for the treatment of diseases in the cerebral nervous system such as cell death of cells specific to cerebral nerve tissue such as the cerebral cortex, facial nucleus, and hippocampus, ie, substances that promote or suppress cell death.

According to the present invention, a method for screening a substance for promoting or suppressing cell death is provided. Examples of the method include a method of administering a test substance to the cell death-inducing model non-human animal of the present invention, and a method of contacting a test substance with a tissue, organ, or cell derived from the cell death-inducing model non-human animal. it can. The method of contacting a tissue, organ, or cell derived from a cell death-inducing model non-human animal with a test substance includes a cell death-inducing model non-human animal, for example, a tissue, organ, or tissue derived from the AMSH knockout mouse. Specific examples of the method of contacting cells with a test substance and measuring / evaluating cell viability in the cells can be given.The method of administering the test substance to a cell death-inducing non-human animal can also be exemplified. For example, for example, a method of administering a test substance to the AMS H knockout mouse and measuring and evaluating the cell viability in a tissue, organ, or cell derived from the non-human animal, or a method of testing the AMS H knockout mouse Specific examples of methods for administering the substance and measuring and evaluating the degree of change in life span, growth, tissue morphology, and peripheral nerve reflex in the non-human animal But can be, but not limited to. At the time of the above-mentioned screening, it is preferable to compare and evaluate the cell death-inducing model non-human animal with a wild-type mouse of the same litter.

 Examples of the cell death promoting or suppressing substance obtained by the screening method of the present invention, such as a cell death promoting or suppressing substance, include a neurotrophic factor-like substance, a glial cell-like neurotrophic factor (GDNF) family member molecule, and TNF. Apoptosis-inducing molecules such as family molecules can be exemplified. In addition, the therapeutic agent for a disease caused by deficiency of AM SH and Z or AM SH-LP of the present invention is not particularly limited as long as it is a therapeutic agent containing the above-mentioned cell death inhibitor or the like as an active ingredient. In addition, by administering such a therapeutic agent to mammals or the like in an appropriate amount and method, it is possible to treat the disease caused by the deficiency of AMSH and / or AMSH-LP.

 Furthermore, the AMSH and / or AMSH-LP protein of the present invention

Three The whole or a part of the antisense strand of DNA or RNA can be used as a diagnostic probe for a disease caused by AMS H and Z or AMS H-LP deficiency. The use of AM SH and a diagnostic kit for a disease caused by a Z or AM SH-LP deficiency, including a probe for diagnosing a disease caused by a Z or AMS H-LP deficiency, can be used, for example, to reduce growth, peripheral nerves, etc. AMS H and / or AMSH-LP for diseases in the cerebral nervous system such as abnormal reflexes, abnormalities such as ptosis, and cell death of cells specific to cranial nerve tissues such as the cerebral cortex, facial nucleus, and hippocampus It is possible to diagnose a disease caused by the deficiency of the protein. The above-mentioned diagnostic probe is the whole or a part of the antisense strand of DNA (cDNA) or RNA (cRNA) encoding AMS H and / or AMS H-LP, and has a sufficient degree as a probe. Those having a length (at least 20 bases or more) are preferred. Specific examples of the above AM SH-LP include proteins consisting of the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 (GenBank Accession number AB010120, AB010121, AB010122, respectively). Specific examples of the DNA encoding LP include a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5 (GenBank Accession number AB010120, AB010121, AB010122). Samples used for such detection include, but are not limited to, genomic DNA, RNA or cDNA obtained from a biopsy of a subject's cells, such as blood, urine, saliva, and tissue. When such a sample is used instead of the sample, a sample amplified by PCR or the like may be used.

Examples of the protein of the present invention include human AMSH_LP represented by SEQ ID NO: 2, mouse AMS H-LP represented by SEQ ID NO: 4 or 6, and amino acid sequence represented by SEQ ID NO: 2, 4 or 6. One or several keys Proteins having an amino acid sequence in which amino acids have been deleted, substituted or added and having AMS H-LP activity, such as proteins having AMS H-LP activity, can be mentioned. The protein of the present invention can be prepared by a known method based on the DNA sequence information and the like, and its origin is not particularly limited.

The DNA to be used in the present invention may be any DNA that encodes the above-mentioned protein. For example, DNA encoding human AMS H-LP shown in SEQ ID NO: 2 and DNA encoding SEQ ID NO: 4 Or the DNA encoding mouse AMS H-LP shown in 6, or the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 in which one or several amino acids have been deleted, replaced or added. And DNA encoding a protein having AMS H-LP activity, the base sequence shown in SEQ ID NO: 1, 3 or 5, or a complementary sequence thereof, and a DNA containing a part or all of these sequences. Can be mentioned. These can be prepared based on the DNA sequence information and the like, for example, from a human or mouse gene library or cDNA library by a known method. In addition, hybridization was carried out under stringent conditions against various DNA libraries by using the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5 or its complementary sequence and part or all of these sequences as probes. By isolating the DNA that hybridizes to the probe, DNA encoding a protein having AMS H-LP activity, which is the same as human AMS H-LP and mouse AMSH-LP, is obtained. You can also. The DNA thus obtained is also within the scope of the present invention. Hybridization conditions for obtaining the DNA of the present invention include, for example, hybridization at 42 ° C., and 42 ° C. with a buffer containing 1 × SSC and 0.1% SDS. Cleaning process In this case, a hybridization treatment at 65 ° C. and a washing treatment at 65 ° C. with a buffer containing 0.1 XSSC and 0.1% SDS can be more preferably mentioned. The factors affecting the stringency of the hybridization include various factors other than the above-mentioned temperature conditions, and those skilled in the art can appropriately combine the various factors and use the above-described hybridizing method. It is possible to achieve the same stringency as that of the application.

 Hereinafter, the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited to these examples.

Example 1 (Generation of AMSH-deficient mouse)

 Using the human AMS H cDNA as a probe, a genomic DNA clone of the AMS H gene was isolated by screening a single mouse / Sv mouse gene library. The genomic sequences of the three isolated clones were partially overlapping and had at least six exons, including the 5'-noncoding exon of AMS H. The evening-targeting vector is located between the 4.1 kb (Smal-Scal) and 4.8 kb (BamHI-BamHI) gene sequences from the 129 / SV mouse gene library. 4 The 0.6-k Hindlll-BamHI gene fragment of the intron was replaced with a neomycin-resistant (PGK_neo) cassette (Fig. 1B), and neomycin resistance containing exons 3 and 4 upstream of exon 5 The targeting vector for the mouse AMSH gene was inserted by inserting 1 oxP into the side of the cassette and inserting a diphtheria toxin A chain gene cassette without a poly A region as a negative selection marker. (Figures 1A and 1B :).

After linearizing the above targeting vector, it was introduced into J1 ES cells derived from 129 / SV by the electroporation method, and G418 resistant E S clones were selected (Mol. Cell. Biol. 20, 9346-9355, 2000, Exp. Med. 191, 365-374, 2000, Blood 87, 956-967, 1996), and were subjected to Southern blot hybridization. Homologous recombinants were identified. The resulting G418-resistant ES clone having the three mutant alleles was transiently transfected with the pCXN2-Cre expression vector, a recombinase Cre-expressing vector induced by actin-opening motor, and then neomycin-resistant. The G418-sensitive ES clones from which the sex cassette has been removed are selected and the 3.7 kb fragment of the PGK-neo cassette between the two 1o XP sequences is removed by Southern blot hybridization and PCR. Confirmed that it was done. The resulting two G418-sensitive ES clones were injected into C57BL / 6 blastocysts and transplanted into foster mothers to produce chimeric mice. Such a male chimeric mouse is bred to a female C57B LZ6 mouse, and an F1 heterozygous mouse having an AMSH mutation is identified by the Southern blot hybridization method. F2 homozygotes were produced by crossing body mice.

 F2 mice prepared above [Wild-type mice (+ / +), homozygous mice (1 no), and heterozygous mice (_—) obtained by mating F1 heterozygous mice] The genotype was determined by Southern blot hybridization and PCR using a biopsy specimen obtained from the tail of an F2 mouse as described above. As the oligonucleotide primer for the PCR, 5 AM (5'-TCCCACCTCCTCTTGCTATTTCATACCC-3 ': SEQ ID NO: 7) and 3 L (5'-ACTTGACAGACTTTAGAATCACCCAGAA-3': SEQ ID NO: 8) were used (FIG. 1B). ). The results are shown in FIG. 1C. The bands of 8. Okb and 13.0 kb in FIG. 1C indicate the mutant allele and the wild-type allele, respectively. This suggests that chimeric mice generated from one of the two G4 18-sensitive ES clones described above would have mutant alleles as offspring.

7 It was confirmed that it was inherited.

 Next, in wild-type mice (+ / +) and homozygous mice (1-no)

To examine AMSH expression, Northern blot analysis was performed using total; NA from the brain of each mouse. Total RNA derived from the brain of each mouse was extracted using TRIzol (manufactured by Life Technologies, Inc.), and 20 g of the obtained RNA was subjected to electrophoresis and electrophoresis to obtain a Hybond-N Nylon membrane (Amersham). Pharmacia Biotech). Then, I advance Random primer DNA labeling kit Ver.2 (Takara Biomedicals Co. Bruno [child stranded, - performing Haiburidize one Chillon with [α ^{3 2} P] probe labeled with d CTP (Amersham Pharmacia Biotech Inc.) As a probe, a 1.6 kbp fragment of AMS HcDNA shown in Fig. 1B and a j3-actin probe described in a literature (J. Biol. Chem. 274, 19129-19135, 1999) were used. The hybridization was performed using a hybridization solution [50% formamide, 5 XDenhai'dt solution, 5 XSSC, 0.1% SDS, 20 mM Tris-HC 1 (pH 7.5) and sonication and denaturation of 200 ^ g / m1 salmon sperm DNA] for 20 hours at 42 ° C, and the hybridized membrane is washed with a washing solution [0.2 XS 30 And 0.1% 303] at 65 ° C. After washing, a Bio Image Analyzer-MacBAS1500 (manufactured by Fuji Film Co., Ltd.) The results are shown in Fig. 1 D. As a result, no expression of AMSH was observed in the homozygous mouse, and the AMSH mRNA was mutated due to mutation of AMSH mRNA. (Fig. 1D) It is evident from the above that homologous mutations in the AMSH gene cause AMSH deficiency in mice.

Example 2 (AMS H-deficient mouse genotype)

Morphology of neonatal AM SH-deficient mice compared to littermates of wild-type mice No abnormalities were found. When the genotype of neonatal mice (n = 237) born by crossing AMS H ^{+ /} — was analyzed, the Mendel ratio was AMS H + / + mice (24.0%), AM SH ^{+ /} - The results were as expected for mice (52.3%) and AM SH-/-mice (23.6%). This indicates that AMS H is not important for embryonic development. However, all AMS H-no mice (1-Z-; 〇) died between the days of birth (P) 19 and P 23 (20.8 ± 1.1) (Fig. 2A). . When the mouse was dissected, the stomach was empty, and it was considered starved. In addition, it was confirmed that the AMS H-I mouse showed a considerable growth delay at P7, and began to lose weight after P16 (Fig. 2B). The AM SH 1/1 mouse at P15 showed a neurological abnormality in which the hind limbs contracted in the trunk direction when the body was suspended using the tail. At P16, a drooping of the upper eyelid (eyelid droop) was observed in the AMS H-Normal mouse of No.3. Histopathological analysis of 12-day-old AM SH-/-mice revealed that, among the tested tissues, tissues other than the brain (thymus, spleen, liver, lung, heart, kidney, intestinal tract, colon, and Stomach) did not show any abnormality. The survival rate and growth rate of AMS H ^{+ /} one mouse (+ no;; ■) were not clearly different from that of the wild-type littermate mouse (+ / +; see). (Figures 2A and 2B).

 Previously, AMSH was cloned as a downstream signaling molecule at site force-in receptors such as IL_2 and GM-CSF (J. Biol. Chem. 274, 19129-19135, 1999). We examined whether AMS H deficiency affected the development of T and B lymphocytes and the proliferation of T cells in response to site force-in or stimulation with anti-CD3 antibodies. . 15-day-old AMSH——Thymocytes or spleen cells from mouse or wild-type mouse are suspended in PBS supplemented with 3% FCS, and fluorescein

9 Monoclonal antibody (anti-CD4 monoclonal antibody, anti-CD8 monoclonal antibody, anti-B220 monoclonal antibody, anti-CD3 monoclonal antibody) labeled with isothiocyanate (FITC) or phycoerythrin (PE) To prevent non-specific binding on the cell surface, pre-incubate in normal mouse serum, and use anti-CD4 monoclonal antibody and anti-CD8 monoclonal antibody (both from Pharmingen) for thymocytes. The spleen cells were double-stained for 30 minutes at 4 ° C. using an anti-B220 monoclonal antibody and an anti-CD3 monoclonal antibody (both from Pharmingen). After staining, the cell surface stained with mAbs was set in a mode of two or three colors using CellQuest Software, and analyzed with a FACSCalibur flow cytometer (Becton Dickinson immunocytometry Systems, Inc.). The results are shown in FIG. 3A. As a result, in thymocytes or spleen cells derived from AMS H— / one mouse (AMSH——), CD4, CD8, B220 were similar to those of wild-type mouse (AMSH ^{+ / +} ). , And CD3 expression indicated that there was no difference in the thymic T-cell subpopulation or spleen B-cell population between AM S Η- ζ_ mice and wild-type mice (Figure 3A).

Next, the proliferative response of T cells to stimuli such as cytokines or anti-CD3 antibody was examined. 1 of 2-day-old AM SH- Bruno - mice or wild type mice - derived spleen cells (2 X 1 0 ⁵ cells; Fig. 3 B), splenic T cells (2 X 1 0 ⁵ cells; Fig. 3 C) or thymus Cells (5 × 10 ⁵ cells; FIG. 3D) were reconstituted with 200 1 medium per well (10% FCS, 50 / M 2-mercaptomethanol, 50 M penicillin, And RPMI 1640 medium supplemented with 50 M streptomycin), and seeded on a 96-well plate containing rhIL-2 (manufactured by Ajinomoto Co.), recombinant murine IL-4 (manufactured by PeproTech), Anti-CD3 antibody (145.2C11) or concanapalin A (ConA) For 42 hours in the presence of After incubation, such cells ^[3 H] - thymidine down labeled with recovered after 6 hours, ^[3 H] - measured by thymidine MicroBeta liquid scintillation counter ^ Amersham Pharmacia Biotech Co. intake). The results are shown in FIGS. Based on these findings, spleen cells, spleen T cells, or thymic cells derived from AMS H-no mice, as well as cells derived from wild-type mice, have ConA, IL-14, IL-12, Proliferation response to the CD3 antibody or a stimulus obtained by combining them was confirmed. From the above, it became clear that AMSH was not important in the development of T cells and B cells, and in the T cell proliferation response to stimuli such as cytokine or anti-CD3 antibody.

Example 3 (AMS H — / — Brain histopathological abnormalities)

 Next, the brain specimens of AMS H—Z— mice were histopathologically analyzed and compared with those of AMSH + / + mice using a commercially available kit (TACS2 TdT-Blue).

TUN EL staining was performed according to the protocol attached to the Label In Situ Apoptosis Detection Kit; TREVIGEN Instructions). A brain section was prepared from each of the above mice at embryonic age of 19.5 days, 6 days, and 16 days, and each brain section was deparaffinized and rehydrated. The decomposition reaction was performed in a PBS solution containing K for 15 minutes, and the reaction was terminated by adding tap water (tap H ₂ O). Such brain sections were treated with 1 XT dT (terminal transferase) labeling buffer for 5 minutes, followed by 37 times in 1 XT dT labeling buffer containing TdT, biotinylated dUTP, and manganese chloride. Incubation was performed for 1 hour in a humidified room at ° C. 1 After terminating the reaction with XT dT stop buffer, visualize AMS H DNA by treating this section with streptavidin-conjugated horseradish peroxidase and TACS Blue Label (manufactured by TREVIGEN). It has become. In addition, the above sections were prepared using Nuclear Fast Red (Sigma). Counterstained. Fig. 4 shows the results.

Differences in brain sections containing hippocampus and cerebral cortex from AM SH − / − mouse embryos at embryonic day 19.5 (E19.5) were different from those of wild-type mice (AMS H ^{+ / +} ) Could not be confirmed (Figs. 4A and 4B). However, hippocampal CA1 neurons were significantly abolished in P6 AM SH— ^κ —mice, despite normal cerebral cortex (FIGS. 4C-4F). In P16 AMS H1 /-mice, part of hippocampal cA1 was completely lost and the number of neurons in the cerebral cortex was drastically reduced (Fig. 4G-4J). On the other hand, in the brain section containing the cerebellar cortex of P16 (Fig. 4K and 4L) and the olfactory bulb, AMS H was expressed in large amounts, but no histopathological abnormality was observed.

 A portion of hippocampal CA1 from wild-type and AM SH-no-P6 mice was compared at high magnification and compared. As a result, several atrophied neurons were clearly observed in AMSH-ha mice. (Fig. 5, upper panel). To investigate the mechanism of neuronal loss, a part of the hippocampus CA1 of each mouse was stained with TUNEL. TUN EL-positive cells were found in the hippocampus C A1 subunit of the AMSH——mouse, but not in wild-type mice (FIG. 5, middle and bottom). This suggests that the degeneration and loss of neurons in AMS H − / − mice is due to cell death. Example 4 (Expression of AMS H mRNA in wild-type mouse brain)

In order to examine the expression of AMS H mRNA in the brain, each C57BLZ from embryonic day 18.5 days (E.18.5) to postnatal day 56 (P56) shown in Fig. 6A was used. Whole brains of 6 mice were collected, and Northern plot analysis was performed in the same manner as in Example 1 (FIG. 6A). As a result, the expression level of AMS H mRNA was highest in E18.5, then gradually decreased until P15, and the expression of AMSH mRNA was maintained at a constant level until P56. . This indicates that AMS H functions at the embryonic stage and immediately after birth. I found out.

Next, in order to examine the expression and localization of AMS H by transcription in the brain, reference was made to the literature (Brain Res. Mol. Brain Res. 25, 364-368, 1994; Brain Res. Mol. Brain Res. 54, 311). -315, 1998) with modifications to the method described, and in-site hybridization of mouse AMS H was performed. Whole-frozen wild-type mouse embryos at various developmental stages, or brain tissue from wild-type mice at various times after birth, are cut on a cryostat to a thickness of 30 / zm, and each section is divided into 4 sections. After fixation with 0.1% paraformaldehyde / 0.1 M sodium phosphate buffer (PB; pH 7.2), 0.1 M triethanolamine containing 0.25% acetic anhydride (p H 8.0), pre-hybridization solution [50% deionized formamide, 4XSSC, 0.02% ficoll, 1% sarkosyl (sodium N-lauryl sarcosine), 0% Buffer containing 1 M PB and 100 g of Zm1 t-RNA] for 1 hour. After that, 1 0% dextran sulfate, 1 0 0 m M Jichiotorei Torr, at及Beauty ^{3 5} S-labeled AMSH Purehai Buridaize one Deployment buffer supplemented with oligonucleotides pro part, the Haiburidize one Chillon 4 2 ° C, and the sections were washed four times with 0.1 XSSC / 0.1% sarkosyl at 50 ° C for 30 minutes. Exposure to Hyperfilm 3 -max (Amersham Pharmacia Biotech) was performed at room temperature for 2 weeks, and radiography was performed at 4 ° C for 3 weeks using NTB2 nuclear track emulsion (Kodak). The results are shown in FIG. 6B. From these results, AM SH mRNA expression was observed in a wide range of the cerebral cortex and ventricle in the brain of a wild-type mouse embryo at 14 days of age (E 14). In P10 wild-type mice, its expression was also observed in the olfactory bulb, cerebral cortex, hippocampus and cerebellum. Transcription of AMS H in P56 wild-type mice is similar to that of P10 mouse brain. The expression level was confirmed, but the expression level was significantly reduced. In addition, the localization of AMS H transcript was consistent with the localization of neuronal loss seen in the brain of AMSH-deficient mice. These suggest that AM SH is involved in the survival of newborns in the hippocampus and cerebrum.

Example 5 (Impaired survival function of primary neurons from AMS H-deficient mice) Hippocampal CA1 neurons are known to induce cell death by stress such as hypoglycemia, anoxia or metabolism (Exp. Brain Res. 88, 91-105, 1992, Exp. Neurol. 162, 1-12, 2000, Neuroscience 90, 1325-1338, 1999, J. Nerosci. 18, 5151-5159, 1998, Brain Res. 671, 305 -308, 1995, J. Neurosci. 15, 1001-1011, 1995). Thus, to rule out the possibility that the loss of hippocampal neurons in AMS H-deficient mice was caused by the above-mentioned stress, we examined the expression of wild-type or AM SH- embryos at the age of 18.5 days. Primary culture of the isolated hippocampal neurons was performed by the following method. Embryo age 18.5 days wild-type mouse (AMS H ^{+ / +} ) or AMS H- _ mouse (AMS H-I) Embryo-derived hippocampus is dissected and minced finely with scissors, free of calcium and magnesium Primary hippocampal neurons were isolated by milling in a Hank's balanced salt solution using a 9-in siliconized Pasteur pipet. NEUROBASAL MEDIUM containing 0.5 mM L-glutamine and B27 supplement (Life Technologies, Inc.) on a plate coated with poly-D-lysine (Falcon) Technologies, Inc, Inc.) were placed, and the cells were seeded in a cell density of 6 0 0-8 0 0 / mm ^2, 5% C_〇 ₂ and 95% humidified environment and indoor air As a result, hippocampus neurons derived from AMSH-deficient mouse embryos died immediately after culture in vitro, but hippocampal neurons derived from wild-type mouse embryos did not die, at least 8 For days, they had differentiated into neuronal cells (Figure 7). this These results indicate that AMS H plays an important role in neuronal cell survival.

Nerve growth factor (NGF) (Neurobiol. 10, 381-391, 2000, Brain Res. Mol. Brain Res. Rev. 30, 176-188, 1999, Cell 77, 627-638, 1994), transformation transformation Factor / 3 (TG F jS) (J. Neurochem. 75, 2227-2240, 2000, Nat. Neurosci. 3, 1085-1090, 2000, Proc. Natl. Acad. Sci. USA 91, 12599-12603, 1994) , Tumor necrosis factor α (Neuron 12, 139-153, 1994, J. Biol. Chem. 274, 8531-8538, 1999, Eur. J. Neurosci. 12, 3863-3870, 2000), It is known that cytokines such as brain-derived neurotrophic factor (BDNF) (Cell 77, 627-638, 1994) play an important role in the survival and differentiation of neuronal cells in in vivo and in vitro. Thus, in order to elucidate the mechanism of AMSH function in the survival of neuronal cells, we examined whether the above-mentioned cytokines had an effect on cell death of neurons in AMSH-deficient mice in vitro. The primary culture cells of hippocampal neurons derived from the wild type mouse (AM SH ^{+ / +} ) or AMS H-no-mouse (AMS H-no-) at the embryonic age of 18.5 days were converted to murine NGF ( Life Technologies, Inc.), recombinant human brain-derived neurotrophic factor (BDNF, PeproTech, Inc.), recombinant human TGF j31 (PeproTech, Inc.), or recombinant murine TNFα (PeproTech. , Inc.) in the same manner as described above. Then, according to the protocol manual of Fluoroskan Ascent (manufactured by Labsystems), add the Alamar blue fluorescent dye (Alamar Biosciences) to each culture well at the time shown in Fig. 8 so that the final concentration becomes 10%. After incubating various cells for 120 minutes at 37 ° C. in the dark, the cell viability and mitochondrial activity were measured. In addition, the viability index (average intensity of the experimental column minus average intensity of the blank) was measured at 544 nm light. The fluorescence intensity (590 nm) of each excited well was measured by Fluoroskan Ascent (Labsystems), and the survival index was determined by the following equation. (Formula 1) Viability index at each period _{時期 1 0 0 (%)}

 Viability Index on Day 0 The results are shown in FIG. From these results, TGF 3, TNF a,

When NGF and BDNF were used to stimulate hippocampal neurons derived from AMS H ^{+ / +} mice, it was confirmed that the survival index increased. However, the survival index of hippocampal neurons from AMS H − / − mice did not increase with any of the above cytokines. From the above, it was found that the above-mentioned cytokines had no effect on the cell death of hippocampal neurons in AMS H− / mouse.

Example 6 (AMS H—LP deficient mouse production)

Another molecule with high homology to AMSH, AMSH-LP (AMSH-like protein), was identified from a database search, and full-length cDNA was isolated from a mouse cDNA library. The DNA sequence of the identified human AMSH-LP gene is shown in SEQ ID NO: 1, the DNA sequence of the mouse-derived AMS H-LP gene is shown in SEQ ID NOs: 3 and 5, and the AMS H-LP Are shown in SEQ ID NOs: 2, 4 and 6, respectively. The AM SH cDNA expresses a protein having a molecular weight of about 55 kD, and has a nuclear localization signal-like sequence and a STAM-SH3 binding sequence like AM SH. From this, the present inventors speculated that AMS H-LP had a function overlapping with AMSH, compensated for the function of AMSH, and did not recognize abnormalities other than the nervous system in AMSH-deficient mice. AMS H-LP-deficient mice were prepared in the same manner as described in Example 1. First, Using the AMS H-LP cDNA as a probe, the AMS H-LP gene was isolated from the 129 / Sv mouse gene library, and a targeting vector was prepared using the obtained AMSH-LP gene. AMS H-LP hetero ES cells were obtained by screening after introduction into the cells. A chimeric mouse is prepared using the obtained hetero ES cells, the male chimeric mouse is bred to a female C57BL / 6 mouse, and an F1 heterozygous mouse having an AMS H-LP mutation is Identification was performed by the hybridization method, and F2 homozygotes (AMSH-LP- /) were prepared by crossing F1 heterozygous mice (AMSH-LP ^+/- ).

Example 7 (Production of AMS H * AMS H—LP double deficient mouse)

AM SH homo-deficient mice are born normally, but die at three weeks of age and cannot leave offspring. Accordingly AM SH heterozygous mice (A MS H ^{+ /} I) and AMS H- LP heterozygous mice ^{(AMS H- LP + / -)} AMS H + / by mating - · AMS H- LP ^{+ /} A mouse was prepared, and the mouse was intercrossed to prepare an AMSH · AMS H—LP double-deficient mouse (AMS H—z—AMS H—LP 1/1). Industrial applicability

The cell death-inducing model non-human animal of the present invention includes neural cells such as hippocampal neurons, cerebral cortical neurons, and glial cells, lymphoid cells such as T cells or B cells, monocytes, macrophages, and basophils. Research on models that specifically induce cell death in immune system cells such as hematopoietic progenitor cells such as eosinophils, mast cells, neutrophils, megakaryocytes, and erythrocytes, and on site force-in signaling Researching models to analyze and mechanisms in T cell differentiation and proliferation Of AM SH and / or AM SH-LP gene function, for example, a model for studying the pathogenesis, analysis, and treatment of diseases in the cerebral nervous system. When model non-human animals are used, diseases caused by a defective gene function of AMS H and Z or AMS H_LP, for example, growth retardation, abnormal peripheral nerve reflex, abnormalities such as ptosis, cerebral cortex, facial nucleus, etc. It is also possible to screen for a drug useful for treating diseases in the cerebral nervous system such as cell death of cells specific to brain nerve tissue such as the hippocampus, that is, a substance that promotes or suppresses cell death.

Claims

The scope of the claims I. A non-human animal model for cell death induction, wherein the function of the AMSH gene is deleted on the chromosome. 2. A cell death-inducing model non-human animal characterized by the deficiency of the AMSH-LP gene function on the chromosome.  3. A non-human animal model for cell death induction, wherein the gene function of AMSH and AMSH_LP has been deleted on the chromosome.  4. The non-human animal model for cell death induction according to claim 2 or 3, wherein the AMSH-LP gene is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 3 or 5.  5. The cell death-inducing model non-human animal according to any one of claims 1 to 4, wherein the cell death is nervous system cell death and / or immune system cell death. 6. The cell death-inducing model non-human animal according to claim 5, wherein the nervous system cells are hippocampal neurons or cerebral cortical neurons. 7. The non-human animal model for cell death induction according to claim 5, wherein the immune system cells are lymphocyte cells.  8. The non-human animal model for cell death induction according to claim 7, wherein the lymphoid cells are T cells. 9. The non-human animal model for cell death induction according to any one of claims 1 to 8, wherein the non-human animal is a mouse.  10. A method of administering a test substance to the cell death-inducing model non-human animal according to any one of claims 1 to 9, or contacting a tissue, organ, or cell derived from the animal with the test substance. Screening method for a cell death promoting or inhibiting substance. II. A cell death-inducing model non-human animal and a field according to any one of claims 1 to 9. 10. The method for screening a substance promoting or suppressing cell death according to claim 10, wherein the method is compared with a case of a live non-human animal.  12. The cell according to claim 11, wherein the wild-type non-human animal is a wild-type non-human animal of the same litter as the model non-human animal according to any one of claims 1 to 9. A method for screening a death promoting or suppressing substance.  13. The method for screening a substance for promoting or suppressing cell death according to any one of claims 10 to 12, wherein the non-human animal is a mouse.  1 4. A cell death promoting or inhibiting substance obtained by the method for screening a cell death promoting or inhibiting substance according to any one of claims 10 to 13.  15. A therapeutic agent for a disease caused by AMSH and Z or AMSH-LP deficiency, comprising the cell death inhibitor according to claim 14 as an active ingredient.  1 6. Extract the AMSH and / or AMSH-LP gene from the sample and examine the presence or absence of the gene abnormality to detect the disease caused by AMSH and / or AMSH_LP deficiency. Diagnostic method.  1 7. Diagnosis of disease caused by deficiency of AM SH and / or AMS H-LP consisting of all or a part of antisense strand of DNA or RNA encoding AMS H and / or AMS H-LP protein probe.  1 8. Caused by AM SH and / or AM SH-LP deficiency, comprising a diagnostic probe for a disease caused by AM SH and / or AM SH-LP deficiency according to claim 17. A diagnostic kit for illnesses.  1 9. DNA encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (b) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted or added, and having AMSH-LP activity 20. A DNA consisting of the base sequence shown in SEQ ID NO: 1 or a sequence complementary thereto, or a sequence containing a part or all of these sequences. 2 1. A DNA encoding a protein that hybridizes with DNA constituting the gene according to claim 20 under stringent end conditions and has AMSH-LP activity.  2 2. DNA encoding the following protein (a) or (b):  (a) a protein consisting of the amino acid sequence of SEQ ID NO: 4  (b) a protein consisting of the amino acid sequence of SEQ ID NO: 4 in which one or several amino acids have been deleted, substituted or added, and having AMSH-LP activity  2 3. A DNA consisting of the base sequence shown in SEQ ID NO: 3 or a sequence complementary thereto, or a sequence containing a part or all of these sequences. 24. A DNA encoding a protein that hybridizes with DNA constituting the gene according to claim 23 under stringent end conditions and has AMSH-LP activity.  2 5. DNA encoding the following protein (a) or (b):  (a) a protein consisting of the amino acid sequence of SEQ ID NO: 6  (b) a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 in which one or several amino acids have been deleted, substituted or added, and having AMSH_LP activity  2 6. A DNA consisting of the nucleotide sequence shown in SEQ ID NO: 5, its complementary sequence, or a sequence containing a part or all of these sequences. 27. A DNA encoding a protein that hybridizes with DNA constituting the gene according to claim 26 under stringent conditions and has AMSH-LP activity. 2 8. A protein consisting of the amino acid sequence shown in SEQ ID NO: 2. 2 9. A protein comprising the amino acid sequence shown in SEQ ID NO: 2 with one or several amino acids deleted, substituted or added, and having AMS H-LP activity.  30. A protein consisting of the amino acid sequence of SEQ ID NO: 4.  3 1. A protein comprising the amino acid sequence of SEQ ID NO: 4 with one or several amino acids deleted, substituted or added, and having AMS H-LP activity.  3 2. A protein consisting of the amino acid sequence shown in SEQ ID NO: 6. 3 3. A protein consisting of the amino acid sequence shown in SEQ ID NO: 6 with one or several amino acids deleted, substituted or added, and having AMS H-LP activity.