WO2000066145A1

WO2000066145A1 - Method for inducing the cell death of nerve cell line, method for screening compound inhibiting or promoting the cell death of nerve cells, and cell death inhibitor and promoter for nerve cells

Info

Publication number: WO2000066145A1
Application number: PCT/JP2000/002830
Authority: WO
Inventors: Ikuo Nishimoto
Original assignee: Keio University
Priority date: 1999-04-30
Filing date: 2000-04-28
Publication date: 2000-11-09
Also published as: AU4315300A

Abstract

It has been found out that the cell death of a nerve cell line can be conveniently induced by treating the nerve cell line with ApoE4. Further, it has been found out for the first time that ApoE receptor 1 and pertussis toxin (PTX)-sensitive G protein participate in the signal transduction toward cell death of nerve cells responding to ApoE4 stimulus.

Description

A method for inducing cell death of a nerve cell line, a method for screening a compound that inhibits or promotes cell death of a nerve cell, and an inhibitor and a promoter for cell death of a nerve cell

TECHNICAL FIELD The present invention relates to a method for inducing cell death of a neuronal cell line, a method for screening a compound that inhibits or promotes neuronal cell death, and a neuronal cell death inhibitor or cell death promoting agent .

Alzheimer's disease (AD) is one of the most energetically studied neurodegenerative diseases, and has a condition in which brain nerves are progressively lost. Pathologically, extracellular deposits called senile plaques are characteristic of AD, and its main component is A? (Amyloid /?). APP is Α? It has a receptor-like structure with one transmembrane domain (Kang, J. et al., Nature 325: 733-736 (1987)). I is the early onset familial AD (FAD), F, and 3 Tsuno突natural mutation of the G is cage once find, I to V642 of 695 in the amino acid of Arufaroro ₆₉₅ is both a § isoforms APP, It has three mutations, F or G (Hardy, J., Nature Genetics 1: 233-234 (1992)). This type of FAD mutant _c called FAD-APP These mutations were first identified by genetic survey as a genetic cause of AD (Karlinsky, H. et al., Neurology 42: 1445-1453 (1992)). By the way, the present inventors have shown that transient expression of genes encoding three V642 FAD-APPs (V642I / F / G) causes apoptosis in nervous system hybrid cells F11. (Yamatsuji, T. et al., Science 272: 1349-1352 (1996)). Subsequently, similar findings have been reported in PC12 cells (Zhao, B. et al. J. Neurosci. Res. 47: 253-263 (1997)). Apoptosis caused by FAD-APP is pertussis toxin (PTX) sensitive G protein G. And derived therefrom (Yamatsu ji, T. et al., Science 272: 1349-1352 (1996); Yamatsuji, T. et al, EMBO J. 15: 498-509 (1996); Giambarella, U. et al., EMBO J. 16: 4897-4907 (1997)). In addition, FAD-APP is the cytoplasmic region of APP and G. It is known that G protein activation is caused by the interaction of (Nishimoto, I. et al., Nature 362: 75-79 (1993); Okamoto, T. et al., EMBO J. 15: 3769-3777 (1996)). Furthermore, induction of apoptosis of PC12 cells by FAD-APP or presenilin-2 (PS-2) was reported to be PTX-sensitive (Wolozin, B. et al., Science 274: 1710-1713). (1996)). PS-1 behaves like a receptor, via the 39 amino acids at the C terminus. And activates it (Smine, A. et al., J. Biol. Chem. 273: 16281-16288 (1998)). PS-1 and PS-2 are located on chromosomes 14 and 1, respectively, and are encoded by genes involved in the development of familial AD.

As another genetic factor of AD, a gene encoding apolipoprotein E (ApoE) is known. The relationship between ApoE and AD was first proposed by Namba et al. Based on the finding of ApoE immunoreactivity in amyloid deposits in the brain of AD patients (Namba, Y, et al., Brain Res. 541: 163-166 (1991)). ApoE £ 4, located at chromosome 19ql3.2, has late-onset familial and sporadic AD (Saunders, A. M. et al., Neurology 43: 1467-1472 (1993); Corder, EH et al., Science Z 61: 921-923 (1993); Ueki, A. et al., Neurosci. Lett. 163: 166- 168 (1993)) and premature AD, including FAD caused by other genes (Sorbi, S. et al., Ann. Neurol. 38: 124-127 (1995); Isoe, K. et al. , Acta Neurol. Scand. 94:

326-328 (1996)). The ε4 allele is also associated with other neurons such as diffuse Levy body disease with dementia and Parkinson's disease with dementia ^ frontal dementia (Pick's disease). Involved in the onset of dementia in degenerative diseases (Helisalmi, S. et a 1., Neurosci. Lett. 205: 61-64 (1996)), as well as in vascular dementia and ischemic cerebrovascular disorders Dementia, non-AD dementia (Helisalmi, S. et al., J. Neutral. Transm. 104: 913-920 (1997)) and certain alcoholic dementias (Muramatsu, T. et al., J. Neural.

Neurosci. Lett. 205: 61-64 (1996); Ji, Y. et al., Dement. Geriatr. Cogn.

Disord. 9: 243-245 (1998)) and age-related decline in memory (Blesa, R. et al.,

Ann. Neurol. 39: 548-551 (1996)). Therefore, the inheritance of the ApoE ε4 allele can be said to be one of the most important risk factors for the progression of dementia in the elderly and senile, regardless of age. According to a population-based agreement test reported by Slooter et al., It is estimated that approximately 20% of dementia patients are purely ε4 arsenic (Slooter, AJ et al. , Arch. Ne urol. 55: 964-968 (1998)).

There are many polymorphisms in the ApoE gene. The most common alleles are £ 3 and £ 4, encoding the major isoforms of ApoE, ApoE3 and ApoE4, respectively (Zannis, VI et al., J. Lipid. Res. 23: 911 -914 (1982)). ApoE is present on the surface of most major plasma lipoproteins and is known to be involved in the transport of lipids to cells through its action on cell surface receptors (Wilson, C. et al. ., Science 252: 1817-1822 (1991)). Recent studies show that ApoE It was revealed that the system also has important functions. The brain is the tissue with the second highest level of ApoE production and is also abundant in cerebrospinal fluid (Pitas, RE et al., J. Biol. Chem. 262: 14352-14360 (1987)). It is also produced after nerve injury (Mahley, RW, Science 240: 622-630 (1988)). ApoE3 promotes elongation of neuronal axons and ApoE4 is known to suppress it (Nathan, BP et al., Science 264: 850-852 (1994)). Despite these findings, little is known about how ApoE4 is specifically involved in, for example, AD progression. So far, the relationship between amyloid plaques and the pathogenesis of AD has shown that ApoE forms a complex with? And promotes its condensation (Strittmatter, WJ et al., Proc. Natl. Acad. Sci. USA 90: 1977-1981 (1993)), but little is known about the neurotoxicity of ApoE4 without A5. Recently, Raber and colleagues reported transgenic mice overexpressing human ApoE4 using endogenous ApoE-deficient mice, and found that learning was impaired without clear A- deposits ( Raber, J. et al. Proc. Natl. Acad. Sci. USA 95: 10914-10919 (1998)).

There have been several reports on the neurotoxicity of ApoE4. Jordan et al. (Jordan, J. et al., J. Neurosci. 18: 195-204 (1998)) found that 10 μg / ml of ApoE4 was toxic to cultured hippocampal nerves. In addition, Marques et al. (Marques, MA et al., Neuroreport 7: 2529-2532 (1996)) reported that the neurotoxicity of the 22-kDa N-terminal fragment of ApoE3 and ApoE4 generated by thrombin cleavage is ApoE4> ApoE3. Report that there was. Most recently, transgenic mice deficient in endogenous ApoE and overexpressing human ApoE4 caused learning impairment, but not ApoE3 (Raber, J. et al., Proc. Natl. Acad. Sci. USA 95: 10914-10919 (1998)), suggesting that overexpression of ApoE4 also reduces neuronal function in vivo. However, despite the above findings, little is known about the mechanism by which ApoE4 induces neuronal cell death.

Disclosure of the invention

An important step in elucidating the mechanism of neuronal cell death by ApoE4 is the development of a system that can easily induce neuronal cell death in response to ApoE4 stimulation. Such systems can also be very important tools in the development of therapeutics and prophylactics for neurodegenerative diseases such as AD. The present invention has been made in view of such circumstances, and accordingly, it is a first object of the present invention to provide a novel method for inducing neuronal cell death using ApoE4. Further, the present invention provides a novel drug useful for treating a neurological disease and the like, and a method for screening the same, by elucidating the mechanism of cell death of nerve cells in response to ApoE4 stimulation using the method. Is also an issue. The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, by allowing ApoE4 to act on nerve cell lines such as F11 cells and SH-SY5Y cells, the cell death of the nerve cell lines can be easily performed. We have successfully developed a way to guide. Furthermore, the present inventors use this system for inducing cell death of a neuronal cell line, and thereby use ApoE receptor 1 and pertussis toxin (pertussis toxin) in signal transduction leading to neuronal cell death in response to ApoE4 stimulation. (PTX) Sensitive G protein was found to be involved for the first time. The neuronal cell line induction system developed by the present inventors and molecules found to be involved in signal transduction in response to ApoE4 stimulation by the present inventors include neuronal cell death inhibitors and It can be suitably used in screening for cell death promoters. The present invention more specifically relates to

(1) a method for inducing cell death of a nerve cell line, comprising a step of contacting a binding agonist of ApoE receptor 1 with a nerve cell line;

(2) The method according to (1), wherein the binding agonist of ApoE receptor 1 is ApoE4 or a partial peptide thereof.

(3) The method according to (1), wherein the binding agonist of ApoE receptor 1 is a tandem LRP binding peptide.

(4) The method according to any one of (1) to (3), wherein the neural cell line is derived from a neuroblast.

(5) The method according to any one of (1) to (3), wherein the neural cell line is an F11 cell or an SH-SY5Y cell.

(6) A method for screening for a compound that inhibits or promotes neuronal cell death, comprising:

(a) culturing a neural cell line in the presence of a test sample and an ApoE receptor 1 binding agonist,

(b) detecting cell death of the neural cell line, and

(c) selecting a compound having an activity of reducing or increasing the cell death of the neuronal cell line as compared with the case in the absence of the test sample (control).

(7) The method according to (6), wherein the nerve cell line is derived from a neuroblast.

(8) The method according to (6), wherein the nerve cell line is an F11 cell or a SH-SY5Y cell.

(9) Screening for a compound that inhibits or promotes neuronal cell death Law,

(a) contacting ApoE receptor 1 with its binding agonist in the presence of a test sample,

(b) detecting the binding of ApoE receptor 1 to its binding agonist, and

(c) selecting a compound having an activity of inhibiting the binding of ApoE receptor 1 to its binding agonist, a method comprising:

(10) the method according to any one of (6) to (9), wherein the binding agonist of ApoE receptor 1 is ApoE4 or a partial peptide thereof;

(11) The method according to any one of (6) to 9, wherein the binding agonist of ApoE receptor 1 is a tandem IP binding peptide.

(12) A compound that inhibits or promotes neuronal cell death, which can be isolated from the method according to any one of (6) to (11),

(13) The compound according to (12) as an active ingredient, a cell death inhibitor or cell death promoter for nerve cells,

(14) a compound that inhibits binding between ApoE receptor 1 and ApoE4 as an active ingredient, a neuronal cell death inhibitor or cell death promoter,

(15) a neuronal cell death inhibitor comprising ApoE3 or a partial peptide thereof as an active ingredient;

(1 6) shall be the active shed ₂ macroglobulin or active ingredients a partial peptide thereof, of neuronal cell death inhibitors,. The present invention relates to a binding agonist for APOE receptor 1 (also referred to as LDL receptor-related protein (LRP)). The present invention relates to a method for inducing cell death of a nerve cell line, comprising a step of contacting a second strike with a nerve cell line. In the present invention, “ApoE receptor 1 binding agonist” refers to a compound having the ability to act on ApoE receptor 1 to activate the receptor. ApoE receptor 1 binding agonist used in the method of the present invention includes ApoE4 and its partial peptides, but as long as it acts on ApoE receptor 1 to induce cell death of a neural cell line, No restrictions.

As ApoE4, in addition to naturally-occurring ApoE4, a recombinant ApoE4 prepared by a gene recombination technique can be used in the method of the present invention. Natural ApoE4 can be purified according to the method described in the document “R all et al., 1986, Methods Enzymol. 128: 273-287”. A commercially available product (Cat. # APL100, Chemicon International Inc.) can also be used. In addition, as long as it can act on ApoE receptor 1 and induce cell death in neuronal cell lines, use mutant ApoE4 in which one or more amino acids of natural ApoE4 have been substituted, deleted, added, or inserted. Is also possible. The partial peptide of ApoE4 used in the method of the present invention preferably contains the ApoE receptor 1 binding region of ApoE4 (SEQ ID NO: 1). For example, a peptide consisting of an amino acid sequence in which the binding region overlaps in tandem (tandem LRP-binding peptide / SEQ ID NO: 2) is more efficient than ApoE4, and can induce cell death of a neural cell line more rapidly. It is particularly suitable for the method of the present invention. ApoE receptor 1 binding domain peptides and evening LRP binding peptides can be synthesized by peptide synthesis methods known to those skilled in the art. The neuronal cell line used in the method of the present invention is not particularly limited as long as it can significantly induce cell death in response to stimulation by an ApoE receptor 1 binding agonist, but is derived from neuroblast cells. Those are preferred. Examples of neural cell lines derived from neuroblasts include, for example, F11 cells, which are hybrid cells of primary rat dorsal root ganglion cells and mouse neuroblastoma N18TG2 (D. Platika ¾, 1985, Proc. Natl. Acad. Sci. USA, 82: 3499, T. Yamatsuji et al., 1996, Science, 272: 1349), SH-SY5Y cells, PC12 cells, NTERA2 cells, P19 cells and the like. In NTERA2 cells and P19 cells, those in which neural differentiation is induced by retinoic acid treatment are particularly preferred. In addition, cells expressing the ApoE receptor 1 gene may be applicable to general mammalian cells including CH0 cells and HEK293 cells. To express the ApoE receptor 1 gene in a cell, for example, the ApoE receptor 1 cDNA can be inserted into an appropriate expression vector, introduced into the cell, and expressed. Use a medium suitable for culturing each cell (for example, HamF-12 medium for F11 cells, DMEM medium for SH-SY5Y cells, etc.) under serum-free or serum-containing conditions, eg, 30 / g / ml. Cell death can be induced by adding ApoE4 to the medium and incubating for 72 hours. The present invention also relates to a method for screening a compound that inhibits cell death of a nerve cell. One embodiment of the screening method of the present invention is a method that utilizes the above-described system for inducing neuronal cell death. The above-described neuronal cell death induction system is a suitable model of neuronal cell death in neurodegenerative diseases such as Alzheimer's disease. Compounds that inhibit the induction of cell death in this system are seeds of neurodegenerative disease therapeutic drugs. Is likely to be On the other hand, a compound that promotes cell death in this system is considered to be used as a shade for a therapeutic drug for abnormal occurrence of nerve cells. In the above system, the S / N ratio of cell death is extremely high, and the treatment is simple.By using the above system, prophylactic drugs against neurodegenerative diseases such as Alzheimer's disease and abnormal occurrence of neurons can be obtained. It is possible to perform high-throughput screening to quickly select candidate compounds for therapeutic drugs. The screening method of the present invention is characterized in that, in the above-described system for inducing neuronal cell death, in addition to the ApoE receptor 1 binding agonist, A sample in which it is desired to detect a promoting effect is added, the cell death of a nerve cell is detected, and a compound that reduces or increases the cell death of the nerve cell is selected. That is, (a) a step of culturing a neural cell line in the presence of a test sample and an ApoE receptor 1 binding agonist, (b) a step of detecting cell death of the neural cell line, and (c) Selecting a compound having an activity of reducing or increasing the cell death of the nerve cell line as compared to the case in the absence of the sample (control). Test samples include, for example, purified proteins (including antibodies), expression products of sense and antisense gene libraries, synthetic peptide libraries, cell extracts, cell culture supernatants, and libraries of synthetic low-molecular compounds. Examples include, but are not limited to, solutions containing bacteria-releasing substances such as actinomycete processes, natural materials such as soil, ribozymes, and antisense nucleic acids. Examples of the method for detecting cell death include cell number measurement, live cell measurement by trypan blue exclusion, and biochemical assay using MTT. Furthermore, a method for detecting apoptosis can also be used. The apoptosis detection method, TUNE I _J method, DNA ladder method, a method using an electron microscope, or include a method of detecting a change in the characteristic of the nuclear and cell membrane. Examples of the latter include a method for measuring annexin V and a method for measuring nuclear morphological changes or caspase activity. If the detection results show a significant decrease or increase in the rate of cell death, the test sample used for screening is determined to be a candidate for a neuronal cell death inhibitor or cell death promoter. The inhibitor of the present invention may be any inhibitor that does not completely inhibit cell death, but significantly reduces the cell death ratio as compared to the case where the inhibitor is not added. The promoter of the present invention may be any promoter that significantly increases the cell death ratio as compared with the case where the promoter is not added. Compounds isolated by this screening include ApoE receptor 1 and ApoE receptor Body 1 those that directly act on binding agonists, such as those that directly act on ApoE receptor 1 and inhibit signal transduction by binding of binding agonists (eg, ApoE4) (ApoE receptor 1 angyu gonist), and ApoE In addition to those that act directly on receptor 1 binding agonist and inhibit the activation of ApoE receptor 1 (antagonist of ApoE receptor 1 binding agonist), cells mediated by the binding agonist and / or ApoE receptor 1 Those that act downstream of these in the death signaling pathway (eg, pertussis toxin-sensitive G-protein agonists). In addition, those that directly act on ApoE receptor 1 and Apo E4 to promote cell death signal transduction, and those that act downstream of ApoE receptor 1 to promote signal transduction in signal transduction included. Further, in this example, it was shown that among the ApoE4 receptors, ApoE receptor 1 was particularly involved in cell death of nerve cells in response to ApoE4 stimulation, thereby inhibiting the binding between ApoE4 and ApoE receptor 1. By doing so, it has become possible to suppress cell death of nerve cells. That is, a compound that inhibits the binding between ApoE4 and ApoE receptor 1 may be used as an inhibitor of neuronal cell death. In addition, when a compound that inhibits the binding of ApoE4 to ApoE receptor 1 acts, for example, directly on ApoE receptor 1 and has the ability to activate it, the compound is used as a neuronal cell death promoter. The use of is considered. Therefore, another embodiment of the present invention for screening for a compound having an activity of inhibiting or promoting cell death of a nerve cell is a method using the inhibition of binding between ApoE4 and its receptor as an index. a) contacting ApoE receptor 1 with its binding agonist in the presence of a test sample; (b) detecting binding between ApoE receptor 1 and its binding agonist; and (c) ApoE receptor Selecting a compound having an activity of inhibiting the binding between the compound 1 and its binding agonist. ApoE receptor 1 and its binding agonist used in this method are of natural origin. Or a recombinant prepared by a gene recombination technique. Recombinants are obtained, for example, by introducing DNA constructed to express a fusion protein with an appropriate tag into insect cells, Escherichia coli, or mammalian cells, etc., and converting the expressed protein to an affinity for the tag. It can be used and purified by known biotechnology techniques. Examples of ApoE receptor 1 binding agonists used in the method of the present invention include ApoE4 and partial peptides thereof. There is no particular limitation as long as it can induce cell death of the strain. In addition, a mutant ApoE4 in which one or more amino acids have been substituted, deleted, added, or inserted can be used as long as it can act on ApoE receptor 1 to induce cell death of a neuronal cell line. The partial peptide of ApoE4 used in the method of the present invention preferably contains the ApoE receptor 1 binding region of ApoE4 (SEQ ID NO: 1). For example, a peptide consisting of an amino acid sequence in which the binding region overlaps in tandem (evening LRP binding peptide / SEQ ID NO: 2) can be used. Test samples include, for example, purified proteins (including antibodies), expression products of gene libraries, libraries of synthetic peptides, cell extracts, cell culture supernatants, libraries of synthetic low molecular compounds, actinomycete broth, etc. But also include, but are not limited to, solutions containing bacterial release substances, and natural materials such as soil.

In screening for a cell death inhibitor or promoter based on the inhibition of binding between ApoE receptor 1 and a binding agonist, specifically, for example, cell-engineered ApoE receptor 1 protein or one of them is used. Is fixed to a carrier such as a plate, a bead, or a chip, and ApoE4, other ApoE molecular species, or a part thereof, or one ApoE receptor such as a tandem LRP-binding peptide is immobilized there. The binding between the two is measured using, for example, a BIAC0RE instrument. A test sample is added to the system and a compound that reduces the measured receptor protein-ligand interaction. Search for things and molecules. As a result of the detection, if the addition of the test sample shows significant inhibition of the binding between ΑροΕ receptor 1 and its binding agonist, the test sample used is a neuronal cell death inhibitor or cell death promoter. Is determined to be a candidate. Whether or not the obtained compound actually inhibits or promotes cell death induction is determined by applying these compounds to the above-described neuronal cell death induction system and measuring the cell death ratio. Can be. The compound having an activity of inhibiting the induction of cell death obtained by the screening of the present invention has various medical applications. The disease to be treated or prevented is typically a disease related to cell death of nerve cells, such as Alzheimer's disease. Previous studies have shown that neuronal cell death occurs in Alzheimer's disease (I. Nishimoto et al., 1997, Adv.

Pharmacol., 41: 337-368). As mentioned earlier, it has been suggested that ApoE is involved in this cell death (Namba, Y. et al., Brain Res. 541: 163-166 (1991); Saunders, AM et al. al., Neurology 43: 1467-1472 (1993); Corder, EH et al., Science 261: 921-923 (1993); Ueki, A, et al., Neurosci. Lett. 16 3: 166-168 (1993). Sorbi, S. et al., Ann. Neurol. 38: 124-127 (1995); Isoe,

K. et al., Acta Neurol. Scand. 94: 326-328 (1996)). Therefore, the compound isolated by the screening method of the present invention is expected to be used as an agent for inhibiting cell death of neuronal cells in Alzheimer's disease. In addition to Alzheimer's disease, other nerves such as diffuse levy body disease with dementia, Parkinson's disease with dementia, and frontal dementia Degenerative diseases (Helisalmi, S. et al.,

Neurosci. Lett. 205: 61-64 (1996)), vascular dementia, dementia associated with ischemic cerebrovascular disease, and certain alcoholic dementias (Muramatsu, T. et al., J. Neural. Transm.

104: 913-920 (1997)) and other non-AD dementias (Helisalmi, S. et al., Neurosci. Lett. 205: 61-64 (1996); Ji, Y. et al., Dement. Geriatr. Cogn. Disord. 9:

243-245 (1998)) and age-related memory loss (Blesa, R. et al., Ann. Neurol. 39: 548-551 (1996)) are also targets for treatment and prevention. Recently, nerves, nerve cells, or adrenal medulla cells from different individuals have been transplanted due to Parkinson's disease, etc., but due to the cause of neuronal death in the diseased individual (in the above case, an individual with Parkinson's disease), transplantation was performed. Nerve cells may die in a short period of time. To prevent this, co-administration with transplantation can be used, such as ex vivo treatment of the neuronal cells for transplantation with a nerve cell death inhibitor of the present invention or expression of a gene expressing an inhibitory effect, followed by transplantation. It is possible to do. Further, the nerve cell death inducer is useful as a therapeutic agent for killing abnormally generated nervous system cells, for example, neuroblastoma, adrenal medulla tumor, brain tumor and the like. Further, the present inventors, cell death of neuronal cell lines in response to ApoE4 stimulus, to be inhibited by ₂ macro globulin shed ApoE3 and active known as well as ApoE receptor ligands 1 and ApoE4 Indicated. Therefore, these ApoE receptor 1 ligands can be used as cell death inhibitors for neuronal cell lines, and can also be applied to therapeutic or prophylactic drugs for the above diseases. Natural ApoE3 can be purified according to the method described in the document “Rall et al., 1986, Methods Enzymol. 128: 273-287”. Further, ApoE3 is Chemicon Internati onal Inc. than (Catalog No. # APL90), Matahi ₂ macroglobulin are available from Yagi Res earch Center (catalog # YU- 60002). Activation of shed ₂ Makuroguropuri down may be performed by Mechiruamin process. In addition, as long as it has a cell death inhibitory effect on a neuronal cell line, natural forms of ApoE3 (Zannis, VI et al., 1984, J. Biol. Chem. 259: 5495-5499) and hihi ₂ macroglobulin (P oiler, E et al., 1992, Hum. Genet. 88: 313-319). Mutants in which amino acids have been substituted, deleted, added, or inserted can also be used. In addition, these partial peptides can be used as long as they have a cell death inhibitory effect on a nerve cell line.

The amino acid sequences of ΑροΕ3 and ApoE4 differ only in amino acid position 112, suggesting that Cys at position 112 in ApoE3 is important for inhibiting cell death. Therefore, the partial peptide of ApoE3 is preferably a peptide comprising a region containing Cys at position 112. Examples of such a peptide include a peptide corresponding to positions 104 to 121 of ApoE3 (SEQ ID NO: 3). When compounds that can inhibit or promote neuronal cell death are used as pharmaceuticals, these compounds themselves can be directly administered to patients, or they can be formulated and administered by known pharmaceutical methods. It is possible. For example, it is formulated and administered in an appropriate combination with a pharmacologically acceptable carrier or vehicle, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, etc. It is possible. Administration to the patient can be, for example, transdermal, intranasal, transbronchial, intramuscular, intravenous, intrathecal, intraventricular, or oral, depending on the nature of the compound. The dose varies depending on the patient's age, body weight, symptoms, administration method and the like, but those skilled in the art can appropriately select an appropriate dose. If the compound can be encoded by DNA, the DNA may be incorporated into a gene therapy vector to perform gene therapy. The dose and administration method vary depending on the patient's weight, age, symptoms, etc., but can be appropriately selected by those skilled in the art. When used as a reagent, other components such as cholesterol, VLDL (ultra low density lipoprotein), LDL (low density lipoprotein), HDL (high density lipoprotein), and / or DMS0 are mixed. Can be used. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the neurotoxicity of ApoE4. Panel A shows the results of treating F11 cells with 30 zg / ml ApoE4, ApoE3, or carrier (PBS) alone (control) for 72 hours and determining the cell death ratio. Panel B is a diagram showing the results of determining the cell death ratio by treating F11 cells for 72 hours with increasing concentrations of ApoE4 as shown. Panel C shows the results of incubating F11 cells with 30 zg / ml ApoE4 for the indicated times and determining the cell death ratio. The numbers represent the SE of at least five independent experiments. FIG. 2 is a diagram showing the control of cell death by an ApoE region-specific polypeptide. Panel A shows the results of treating F11 cells for 16 hours with 25 タン M tandem LRPBP, 25〃M control peptide [ApoE3 peptide (LGAMEDVCGRLVQYRGE), ApoE4 peptide (LGADMEDVRGRLVQYRGE)], or carrier alone (control). Show. Panel B shows the results of treating Π1 cells with 25 〃M tandem LRPBP for the times indicated. As shown at the top of the figure, evening dem LRPBP is a peptide consisting of tandem repeats of L141 to L149 of ApoE common to ApoE3 and ApoE4, and the control ApoE3 and ApoE4 peptides are ApoE3 and ApoE4, respectively. Corresponding to L104 to E121. Panel C shows the results of measuring cell death by treating F11 cells for 72 hours with increasing concentrations of ApoE3 peptide in the presence or absence of 30 μg / ml ApoE4. Panel D shows the results obtained by treating F11 cells with 100 / M ApoE4 peptide in the presence or absence of 30 μg / ml ApoE4 for 72 hours and measuring cell death. Each number represents the average SE from at least five independent experiments. Figure 3 is a photograph showing the cell death induced by ApoE4, the inhibition by shed ₂ M.

F11 cells were treated with ΙΟΟηΜ methylamine treated ₂ M (panel A), 30 〃g / ml ApoE4 (panel B), or ΙΟΟηΜ methylamine treated ₂ M and 30 〃g / ml ApoE4 (Panel C) for 72 hours. Each panel is a typical phase contrast microscope image. As a result of performing the same experiment three times, essentially the same results were obtained. FIG. 4 is a view showing the induction of cell death by sodium fluoride. Panel A shows the results of determining the percentage of cell death by treating F11 cells for 24 hours with increasing concentrations of NaF or NaCl as shown. Panel B in the presence or absence of A1C1 ₃ of 100 / M, the time shown, cells were treated with NaF of LOMM, is a graph showing the results obtained by determining the ratio of cell death. Panel C in the presence or absence of A1C1 ₃ in 100〃M a view to increase as shown the concentration of NaF, 12 hours cells were processed, the results obtained by determining the ratio of cell death is there. Panel D shows the results of F11 cells treated with 10 mM NaF for 12 hours and then exposed to 2 mM deferoxamine for 24 hours. `` NaF '' represents the result of NaF treatment after sham pretreatment, and `` D + NaF '' represents the result of NaF treatment after pretreatment with dexoxamine. “D only” indicates the result of pretreatment with deferoxamine and then treatment with the carrier alone). Each value represents the average soil SE from 4 to 6 independent experiments. Fig. 5 is a photograph showing the typical results of cell death induction by NaF treatment and inhibition by dextroxamine in panel D of Fig. 4 (A: control after sham pretreatment and treatment with carrier only). , B represents the results of NaF treatment after the false pretreatment, and C represents the results of pretreatment with deferoxamine and treatment with the carrier alone). FIG. 6 is a photograph showing DNA ladder formation in cell death induced by ApoE4. F11 cells were treated with 30 g / ml ApoE4 (lane 2) or 30 μg / ml ApoE3 (lane 3) for 24 hours. Cells were also cultured in the presence of Ac-DEVD-CH0 (lane 4), l〃g / ml PTX (lane 5), or 30〃g / ml ApoE3 (lane 6) for 30Λ ^ / ΠΙ The cells were treated with 1 ApoE4 for 24 hours. The DNA was extracted and 2 μg of DNA in each lane was separated on a 2% agarose gel. DNA extracted from F11 cells grown in complete growth medium was used as another control in the DNA ladder generation test (lane 1). "MK" denotes the molecular weight Ma one car, a product obtained by digesting a human _{(c I 8 5 7 Sam7)} DNA with Styl. This force produces bands in the lower half of the gel at 500-600 bp intervals. Three similar independent experiments were performed and typical results are illustrated. FIG. 7 is a photograph showing the results of TUNEL Atssei on cell death induced by ApoE4. F11 cells were treated with 30 μg / ml ApoE3 (panel A) or 30 μg / ml ApoE4 (panel for 24 hours and stained with the TUNEL method. Panel C shows cells with l〃g / ml Panel D shows the results of treatment with 30 g / ml ApoE4 for 24 hours in the presence of PTX, and Panel D shows the results of treatment of F11 cells with lOmM NaF for 6 hours. The fluorescence image of TUNEL staining is superimposed on the image, and the fragmented DNA is taken up by FITC-labeled dUTP by terminal deoxynucleotidyl transferase (TdT) and stained by green fluorescence. (The white cells indicated by triangles in the figure) In the cells that underwent apoptosis, DNA leaked from the nucleus, and DNA staining was seen not only in the nucleus but also in the cytoplasm. Figure 8 shows the effect of ApoE on glial cells. Bu695 glial cells were treated with 30 μg / ml of ApoE3, ApoE4, or carrier only (control) for 72 hours and cell viability was measured (right three columns). The experiment was performed under the same conditions (the three columns on the left), and the numbers represent the average of three independent experimental results S. E. Fig. 9 shows the promotion of ApoE3 neurotoxicity in SH-SY5Y cells. The figure shows the effect of ApoE4 (30 μg / ml), ApoE4 (30 μg / ml), ApoE3 (30 / g) on pure neuroblastoma cells SH-SY5Y. / ml) and ApoE4 (30 zg / ml) or nothing (control) were treated for 48 hours, and the percentage of cell death was measured. It represents the average SE of the experimental results.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples. The cell culture conditions and reagents are as follows. F11 cells (Yamatsuji, T. et al., Science 272: 1349-1352 (1996)) were cultured in HamF-12 medium (Gibco BRL) containing 18% fetal serum (FBS HyClon e Laboratories) and antibiotics. Propagated. Bu695 cells [(Hayashi, Y. et al., Biochem. Biophys. Res. Commun. 187: 1249-1255 (1992)); provided by Dr. K. Yoshikawa (Osaka University, Osaka, Japan)], 10% FBS And in DME (Gibco BRL) medium with antibiotics. SH-SY5Y cells (M.

Morishima and Y. Ihara (provided by The University of Tokyo, Tokyo, Japan)] were cultured in DMEM containing 10% fetal calf serum. Recombinant ApoE3 (Cat. # APL90) and ApoE4 (Cat. # APL100) were purchased from Chemicon International Inc. PTX was purchased from Calbiochem-Nova biochem (Cat. # 516560). In the following examples, unless otherwise specified, recombinant ApoE protein was used. Heat inactivation of PTX was performed by incubation at 90 ° C for 1 hour. NaF (Cat. # S-7920) and Deferoxamine (C at. # D-9533) were purchased from Sigma. Ac-DEVD-CH0 was purchased from Peptide Inc. (Cat. # 317 2-V). Shed ₂ - macroglobulin (ratio _₂ -Macroglobulin; _{α 2 Μ)} was purchased from Yagi Research Center (Cat IYU-60002 .). For activation of ₂ M, first treat with 200 mM methylamine in 50 mM Tris / HCl (pH 8.0) and 150 mM NaCl for 16 to 18 hours at room temperature in the dark, and add 20 mM Hepes / NaOH (pH 7.4). Dialysis was performed for 48 hours while changing the dialysate consisting of 150 mM NaCl five times to remove unreacted methylamine. The spleen ₂ M thus prepared was further dialyzed against serum-free HamF-12 medium for 4 hours. Dialysis Saretahi ₂ M is sterilized by filtration microfiltration evening one 0.22 / M, and stored at 4 ° C, used up within 2 weeks.

[Example 1] ApoE3 and ApoE4 treatment of nervous system hybrid cells F11

Incubation of F11 cells with 30 zg / ml recombinant ApoE4 or a carrier containing recombinant ApoE3 (phosphate-buffered saline; PBS) or carrier alone (control) for 72 hours induces cell death Was measured by Atsushi using trypan blue exclusion as an index. A trypable exclusion assay for cell death was performed as follows. The cells were not washed but suspended in a serum-free medium by gentle pipetting. A 0.1% trypan solution in PBS was added to the cell suspension to a final concentration of 0.02% and incubated at 37 ° C for 15 minutes. Cell viability was determined by measuring the percentage of cells that did not stain with Tribble monocell in the total cells (cell mortality = 100-cell viability). Thus, cell mortality by this method represents the ratio of dead cells to total cells, including adherent cells and cells floating in culture. The experiment was repeated at least three times independently. Assay showed that when F11 cells were incubated with 30 Aig / ml recombinant ApoE4 for 72 hours, about 70% of the cells died (Fig. 1A). In contrast, at the same concentration of ApoE3, cell death of F11 cells was not observed even after incubation for 72 hours. Purified natural ApoE3 and ApoE4 were purified according to the method described in the document “Rall et al., 1896, Methods Enzymol. 128: 273-287j. However, the neurotoxicity of native ApoE4 was more rapid than with recombinant ApoE4 (30 μg / ml purified native ApoE4, ApoE3, or carrier alone (control ), The cell death rate when F11 cells were incubated for 24 hours was 87.6 ± 4.4, 10.6 ± 4.2, or 7.2 ± 1.2. (%; Average person SD)). Since the efficiency of ApoE4 cell death increased in a dose-dependent manner in the range of 0.3 to 30 µg / ml, this cell death was considered to be mediated by the ApoE receptor (Fig. 1B). In addition, F11 cells were incubated with 30 g / ml ApoE4 for various times, and the cell death ratio was measured. The cell death ratio increased unidirectionally up to 72 hours (Fig. 1C ).

Fll cells were incubated with 30 μg / ml ApoE4 for 72 hours in the presence or absence of some of the reagents shown in Table 1. Thereafter, cell death was measured using exclusion of tripan blue as an index. The results are shown in Table 1. PTX was used at l〃g / ml, and ApoE3 at 30Adg / ml. PTX was inactivated by heat treatment as described above. The numbers represent the average SE from four independent experiments. “*” Indicates a significant difference with respect to the effect of ApoE4 at p <0.01, and “**” indicates no significant difference. As shown in Table 1, cell death by ApoE4 was significantly inhibited by the coexistence of ApoE3. This suggested that the reason that ApoE3 did not induce cell death was not due to inactivity of ApoE3. After 72 hours of treatment with ApoE4, most of the cells that underwent cell death condensed and became rounded and eventually detached from the plate (see Figure 3B), indicating that ApoE4 caused apoptosis of F11 cells. (See below).

table 1

Inhibition of ApoE4-induced cell death by PTX or ApoE3

Cell death ratio (%)

ApoE3 10.2 ± 5.1

ApoE4 74.7 ± 2.2 ApoE4 + PTX 25.1 ± 6.6 ApoE4 + Inactivated PTX 73.5 ± 11.6 ** ApoE4 + ApoE3 30.4 ± 10.0 *

[Example 2] Effect of ApoE receptor binding peptide

To investigate whether the neurotoxicity of ApoE4 is mediated by binding to cell surface receptors, we tested the effects of L141-L149 (LRKLRKRLL / SEQ ID NO: 1), a peptide in the ApoE receptor binding domain. did. This peptide is known to act on ApoE receptor 1 (ApoERl), also called LDL receptor-related protein (LRP) (Wang, XS and Gruenstein, E., J. Cell. Physiol. 173: 73). -83 (1997)). In order for this peptide (LRKLMRLL) to act on ApoE receptor 1, a concentration on the order of mM is required, whereas LMLRKRLL is an 18-residue peptide (L MLRKRLLLRKLRKRLL / SEQ ID NO: 2, referred to as "tandem LRPBP (tandem LRP binding peptide)") is also known to have similar effects at tens of M (Wang, XS and Gruenstein, E., J. Cel Physiol. 173: 73-83 (1997)). Therefore, an experiment was performed in which Fll cells were incubated with 25 / M evening LRPBP. As a result, cell death was induced in almost 100% of the cells after 16 hours of incubation (FIG. 2A). In contrast, 18 amino acid control peptides corresponding to the ApoE3 and ApoE4 specific regions (L104-E121) (each "ApoE3 peptide" (LGADMEDVC GRLVQYRGE / SEQ ID NO: 3) and "ApoE4 peptide" (LGADMEDVRGRLVQYRGE / (SEQ ID NO: 4)) did not induce cell death (Fig. 2A).

LRPBP was incubated with FlI cells for varying treatment times and cell death was measured. As a result, it was found that the effect of tandem LRPBP was more immediate than ApoE4 full length (Fig. 2B). Cell death by this peptide was observed 3 hours after treatment, reaching almost 100% within 16 hours. This result indicates that the cell death signal of ApoE receptor 1 is rapidly transmitted into cells, suggesting that ApoE4 is not taken up and metabolized by ApoE4. Therefore, the slower action of ApoE4 may be due to the slower interaction between ApoE4 and the ApoE4 receptor, or a region other than the ApoE4 LRP-binding domain may delay the onset of cell death. . Another possibility is that the action of ApoE4 is mediated by a different receptor than the action of tandem LRPBP. The rapid cell death by tandem LRPBP is consistent with the observation that this peptide is causing apoptosis. Indeed, cell death by this peptide has the typical characteristics of apoptosis, with most of the peptide-treated cells becoming small and rounded and eventually detaching from the plate within 24 hours. These data suggest that ApoE receptor 1 (LHP) or other ApoE receptors mediate the neurotoxic effects of ApoE4. Although a neuroprotective effect was observed in ApoE3, it was not observed in ApoE4, so this effect is due to whether residue 112 at the position of ApoE protein is Cys (for ApoE3) or Arg (for ApoE4). It is suggested that a mechanism for recognizing whether or not this is involved. Thus, an experiment was conducted on the effect of an ApoE3-specific polypeptide (ApoE3 peptide) corresponding to residues 104 to 121 of ApoE3. As a result, this peptide was found to inhibit ApoE4-induced neuronal cell death in a dose-dependent manner (Fig. 2C). Cell death was almost completely inhibited by 100〃M of the peptide. In contrast, ApoE4-specific polypeptides (ApoE4 peptides) differing only by one residue showed no inhibitory effect at concentrations up to 100 / M (Fig. 2D). This fact suggests that ApoE3 mediates the neuroprotective effect of ApoE3 by binding to an ApoE3-specific receptor through a region containing residues 104 to 121.

[Example 3] Effect of spike ₂ -macroglobulin (spike ₂ M) on the action of ApoE4

Action of ApoE4 is to further demonstrate that through the ApoE receptor 1, examined the effect of shed ₂ M. As with the ApoE4 allele, shed ₂ M deletion polymorphism has been reported to be a risk factor of frequently seen AD (Blacker, D. et al, Nature Genetics 19:. 357-360 (1998 )). Shed ₂ M is another known ligand of the ApoE receptor 1, also referred to as ApoE receptor 1 Wahi ₂ M receptor. The shed ₂ M binds to ApoE receptors 1, it is necessary to shed ₂ M is activated by Mechiruamin. The interaction of the flight ₂ M by Protea one peptidase or Mechiruamin, shed ₂ M is activated, causing a structural change, to bind to the LRP binding site. Konohi _{2 Μ (ΙΟΟηΜ)} the result of the action 72 hours F11 cells, regardless of whether treatment with Mechiruamin, shed ₂ M alone did not cause little cell death F11 (Fig. 3 A). However, Mechiruamin treated Tahi ₂ M _{(activation-2} M) is clearly inhibited cell death induction by ApoE4 (3 Ous / ml) (Table 2), the typical morphological changes of apoptosis associated with cell death Inhibited (Figure 3C). With ₂ M without treatment with methylamine Did not show such an effect (Table 2). These results, Fei of ₂ M LRP binding type is shown to act suppressively to neuronal cell death induced by ApoE4, the ApoE4 neurotoxicity simultaneously are indeed through the ApoE receptor 1 Has been demonstrated. Table 2 shows the results. The F11 cells in the presence or absence ApoE4 of 30 zg / ml, and 72 hours untreated shed ₂ M or activation of Iotaomikuron'omikuron'itamyu (Mechiruamin processing) alpha ₂ Micromax. Thereafter, cell death was measured using trypan blue exclusion as an index. The numbers represent the average soil SE from at least five independent experiments. “*” Indicates a significant difference in the effect of ΑροΕ4 at ρ <0.01, and “**” indicates no significant difference. Table 2

Inhibition by Fei ₂ Micromax in cell death induced by ΑροΕ4

Cell death ratio (%)

Untreated Λ ₂ Μ 8.9 ± 3.5 Activated ₂ Μ 13.5 ± 5.1 ΑροΕ4 76.1 ± 3.0 ΑροΕ4 + Untreated ₂ Μ 70.8 ± 5.2 ** ΑροΕ4 + Activated ₂ Μ 12.5 ± 2.6 * [Example 4] Effect of PTX and sodium fluoride (NaF)

To determine whether G protein is involved in cell death induced by ApoE4, F11 cells were treated with 30 μg / ml ApoE4 in the presence of 1 μg / ml PTX. As a result of 72 hours of incubation, the action of ApoE4 was significantly inhibited (Table 1). In contrast, PTX inactivated by heat treatment could not inhibit ApoE4 from inducing cell death of F11 cells, and the inhibitory effect of PTX is due to the enzymatic activity of PTX toxin. It is suggested that it is not due to chemicals or contaminants in the PTX solution. These results indicated that ApoE4-induced cell death was related to PTX-sensitive G proteins. If the PTX-sensitive G protein could mediate the cell death effect of ApoE4, it would be possible to induce cell death without ApoE4-mediated activation by directly activating the PTX-sensitive G protein. Therefore, the effect of sodium fluoride (NaF), which is an established activator for heterotrimeric G proteins and is known to have little effect on small G proteins, was examined.

NaF or NaCl was added at varying concentrations to the culture supernatant of F11 cells and after 24 h incubation, the percentage of cell death was determined. The experiment was performed 4-6 times. Figure 4A shows the results.

Treatment of F11 cells with 10 mM NaF induced cell death in most cells (Figure 4A). The effect of NaF was as rapid as tandem LRPBP, with almost all cells dying within 24 hours after NaF treatment (FIG. 4B). Almost all cells condensed within 24 hours, rounded and then detached from the dish. These results support that NaF induces apoptosis. The effect of NaF was dose-dependent, ranging from 1 to 10 mM (FIG. 4A). In contrast, NaCl had no such effect under the same conditions. In order for NaF to activate intracellular G proteins, 1 A concentration of 2020 mM is required (Kelly, E. et al., Br. J. Pharmacol. 100: 223-230 (1990); Funakoshi, A. et al., Regul. Pept. 37: 1-7 (1992); De Boer, AH et al., Eur.J. Biochem. 219: 1023-1029 (1994)) suggest that NaF activates heterotrimeric G proteins. Supports the induction of cell death.

When NaF enters the cell, it forms a complex with the intracellular aluminum and activates the G protein by mimicking the phosphoric acid of GTP (Sternweis, PC and Gi et al., AG, Proc. Natl. Acad. Sci. USA 79: 4888-4891 (1982)). Therefore, an sharpness of aluminum chloride (A1C1 ₃₎ or aluminum Isseki Defuerokisami emissions (deferoxamine) has verified the effect on the action of NaF. in the presence or absence of A1C1 ₃ in Loo zM, adding 10mM of NaF in the culture supernatant, by changing between time and incubated with the cells was determined the percentage of cell death (Fig. 4B). Also, in the presence or absence of A1C1 ₃ of 100〃M, by changing the concentration of NaF, it processes the 12-hour cells was determined the percentage of cell death (Fig. 4C). These results indicated that aluminum clearly enhanced the action of NaF. A1C1 ₃ effects that appear stronger in the measurement of the time course of NaF toxicity was observed. That, A1C1 ₃ in the absence, in inducing cell death of NaF approximately 100% of the cells LOMM, became necessary 24 hours, the 100〃M A1C1 ₃ presence, in the same NaF concentration Cell death was induced earlier, with 10 mM NaF causing cell death in almost all cells in just 12 hours. Slope at time zero of the curve showing the time course of cell death in the A1C1 ₃ presence than under A1C1 ₃ absence higher than twice. At different time scales, these curves show the time course of binding of GTPaS, a non-hydrolyzable GTP analog that can mimic NaF, to purified G protein, as well as low and high concentrations. Similar to the time course of activation of G proteins by NaF in the presence of aluminum concentrations (Sternweis, PC and Gilman, AG, Proc. Natl. Acad. Sci. USA 79: 4888-4891 (1982)). This is in good agreement with the fact that NaF induces cell death of F11 cells by directly activating G proteins. In addition, aluminum deferoxamine, which is an all-day sharp, inhibited the action of NaF. Pretreatment of F11 cells with 2 mM deferoxamine significantly inhibited the neurotoxicity of NaF (Fig. 4D) and almost completely inhibited NaF-induced cell condensation and spheroidization (Fig. 5). From these data, it was confirmed that the cell death effect of NaF was mediated by the trimeric G protein. Taken together with the inhibitory effects of PTX, we conclude that the PTX-sensitive trimeric G protein mediates ApoE4 neurotoxicity.

The PTX-sensitive G protein, a trimeric G protein, is thought to be involved in neuronal apoptosis (Yan, GM et al., J. Neurochem. 65: 242 5-2431 (1995); Yamatsuji, T. et al., Science 272: 1349-1352 (1996a); olo zin, B. et al., Science 274: 1710-1713 (1996)). G ₂ Although the § ₂ in cultured cells transfected Hue transfected is induced DNA cleavage, G _ai or G. a non Sa Buyunidzuto the G protein which is PTX-sensitive . Is not induced by the expression of ApoE4 (Giambarella, U. et al., EMBO J. 16: 4897-4907 (1997)), indicating that cell death induced by ApoE4 is probably mediated by the PTX-sensitive G protein G. Conceivable.

[Example 5] Analysis of cell death induced by ApoE4

5-1) MA ladder Atsushi DNA ladder generated when cell death due to apoptosis occurs was detected under the following conditions. F11 cells were treated with 30 μg / ml ApoE4 (lane 2) or 30 μg / ml Apo E3 (lane 3) for 24 hours. Cells were also grown at 30 μg / ml in the presence of 10 μM Ac-DEVD-CH0 (lane 4), 1 zg / ml PTX (lane 5), or 30 / g / ml ApoE3 (lane 6). ApoE4 for 24 hours. The DNA was extracted and 2 μg of DNA in each lane was separated on a 2% agarose gel. No treatment, just grown in complete growth medium DNA extracted from F11 cells was also used as a control in the MA ladder generation test (lane 1). DNA ladder assay was performed using a kit (TaKaRa ApopLadder Experimental Kit; Cat. # MK600) according to the attached manual. The experiment was repeated at least three times independently. Figure 6 shows the results. As shown in the figure, treatment with ApoE4 at 30 μg / ml for 24 hours induced induction of 180 bp ladder formation of DNA, but ApoE3 almost did not produce DNA ladder as in the case of untreated. The generation of DNA ladders of the size of an oligonucleotide is one of the fundamental features that define cell death by apoptosis. Treatment of cells with 30 / g / ml ApoE4 in the presence of l〃g / ml PTX dramatically inhibited induction of DNA ladder (Figure 6, lane 5). This is consistent with PTX inhibiting ApoE-induced cell death (Table 1). When cells were treated with ApoE4 at 30 μg / ml in the presence of 10 μM Ac-DEVD-CH0, production of DNA ladder was also suppressed (FIG. 6, lane 4). This result suggests that ApoE4-induced oligonucleosome-sized DNA cleavage is caused by caspase-activated DNase. It was also found that ApoE4-induced DNA cleavage of F11 cells was suppressed by the coexistence of ApoE3 (Fig. 6, lane 6). Consistent with the previous conclusion that NaF causes apoptosis, a 180 bp DNA ladder was observed with NaF treatment. Treatment of F11 cells with NaC1 did not induce DNA ladder formation, but treatment of the cells with 10 mM NaF caused oligonucleosomal-sized DNA breaks 6 hours after treatment. . This MA ladder formation was suppressed by the coexistence of 10-M Ac-DEVD-CH0.

5-2) TUNEL staining

ApoE4-induced neurotoxicity was further verified by staining with the TUNEL method.

F11 cells were treated with 30 μg / ml ApoE3 or 30 g / ml ApoE4 for 24 hours, and TUNE Cells were stained by the L method. Further, F11 cells were treated with 30 μg / ml of ApoE4 for 24 hours in the presence of l ^ g / ml of PTX, and subjected to TUNEL staining. In addition, F11 cells were treated with 10 mM NaF for 6 hours, and the same experiment was performed. TUNEL method kit (Boehringer Mannheim In

This was performed using the Situ Cell Death Detection Kit with Fluorescein; Cat. # 1684795) according to the attached manual. Cells were spread on slide glass precoated with poly-D-lysine and incubated in HamF_12 medium containing 18% FBS and antibiotics. The experiment was repeated at least three times independently. As shown in Fig. 7, treatment with 30 g / ml ApoE4 for 24 hours increased the proportion of F11 cells stained by the TUNEL method, but with ApoE3 treatment, almost all cells stained as untreated I couldn't see it. In the TUNEL method, the fragmented DNA is stained (Gavrieli, Y. et al., J. Cell. Biol. 119: 493-501 (1992)). When cells were treated with 30 g / ml ApoE4 in the presence of lzg / ml PTX, almost all cells stained by the TUNEL method were similar to the results obtained by trypable exclusion (Table 1). Not observed (Figure 7, C). As expected, when F11 cells were treated with 10 mM NaF, many cells were stained by the TUNEL method 6 hours after treatment (Figure 7, D).

5-3) Effect of ApoE4 on glial cells

The toxicity induced by ApoE4 was reduced by the glial cell Bu695 (Hayashi, Y. et al., Biochem. Biophys. Res. Commun. 187: 1249-1255), which is a neural cell but not a neuroblast cell line. (1992)). Bu695 glial cells were treated with 30 μg / ml ApoE3, ApoE4, or carrier (control) for 72 hours, and cell viability was measured. As a control, an experiment was performed on F11 cells under the same conditions. Figure 8 shows the results.

ApoE4 treatment at 30 / g / ml induced a large number of cell deaths in F11 cells, but in Bu695 cells, treatment with 30 / g / ml ApoE4 or ApoE3 None induced cell death. In addition, tests using タン ροΕ4 and ΑροΕ in tandem LR において were performed on SH-SY5Y cells (pure neuroblastoma) and CH0 cells (non-neuronal cells). -SY5Y cells induce a large number of cell deaths, but CH0 cells hardly induce cell deaths (ApoE4 (30 zg / ml) or tandem LRPBP (25 / M) In SH-SY5Y cells, they were 77.9 ± 7.7 or 86.7 ± 6.3%, and in CH0 cells, they were 7.8 ± 2.7 or 17.0 ± 2.0%, respectively. It represents the average SE of the experimental result)]]. In addition, inhibition of cell death by ApoE3 was also observed in SH-SY5Y cells (FIG. 9). Taken together, these data suggest that the toxicity induced by ApoE4 is specific to cells derived from neuroblasts. The fact that glial cells were resistant to ApoE4-induced cell death is consistent with reports that glial cells are a major site of ApoE production in the brain (Srivastava, RA et al Biochem. Mol. Biol. Int. 38: 91-101 (1996)).

INDUSTRIAL APPLICABILITY The present invention has revealed that ApoE4 induces cell death of nerve cell lines with high efficiency. Furthermore, it was found that ApoE receptor 1 and pertussis toxin (PTX) -sensitive G protein are involved in signal transduction leading to neuronal cell death in response to ApoE4 stimulation. Using this cell death inducing system and targeting the molecule involved in signal transmission, it has become possible to screen a compound that suppresses cell death of nerve cells. Compounds that suppress such neuronal cell death are expected to be used as drugs for treating diseases involving neuronal death, including Alzheimer's disease, in particular, dementia in general.

Claims

The scope of the claims

1. A method for inducing cell death of a neuronal cell line, comprising the step of contacting a binding agonist of ApoE receptor 1 with a neuronal cell line.

2. The method of claim 1, wherein the binding agonist of ApoE receptor 1 is ApoE4 or a partial peptide thereof.

3. The method of claim 1, wherein the binding agonist of ApoE receptor 1 is a tandem LRP binding peptide.

4. The method according to any one of claims 1 to 3, wherein the neural cell line is derived from a neuroblast.

5. The method according to any one of claims 1 to 3, wherein the neural cell line is an F11 cell or a SH-SY5Y cell.

6. A method for screening for a compound that inhibits or promotes neuronal cell death,

(a) culturing a neuronal cell line in the presence of a test sample and an ApoE receptor 1 binding agonist,

(b) detecting cell death of the neural cell line, and

(c) selecting a compound having an activity of reducing or increasing the cell death of the nerve cell line as compared to the case in the absence of the test sample (control).

7. The method of claim 6, wherein the neuronal cell line is derived from a neuroblast.

8. The method according to claim 6, wherein the nerve cell line is an F11 cell or a SH-SY5Y cell.

9. A method of screening for a compound that inhibits or promotes neuronal cell death,

(b) detecting the binding of ApoE receptor 1 to its binding agonist, and

(c) selecting a compound having an activity of inhibiting the binding of ApoE receptor 1 to its binding agonist.

10. The method according to any one of claims 6 to 9, wherein the binding agonist of ApoE receptor 1 is ApoE4 or a partial peptide thereof.

11. The method according to any one of claims 6 to 9, wherein the binding agonist of ApoE receptor 1 is a tandem LRP binding peptide.

12. A compound that inhibits or promotes neuronal cell death, which can be isolated by the method according to any one of claims 6 to 11.

13. A neuronal cell death inhibitor or cell death promoter comprising the compound according to claim 12 as an active ingredient.

14. An inhibitor of neuronal cell death or a cell death promoter comprising, as an active ingredient, a compound that inhibits the binding between ApoE receptor 1 and ApoE4.

15 5. A neuronal cell death inhibitor comprising ApoE3 or a partial peptide thereof as an active ingredient.

1 6. And active shed ₂ macroglobulin or active ingredients that part peptide, cell death inhibitors of neuronal cells.