WO2000066145A1 - Procede permettant d'induire la mort de cellules de ligne de cellules nerveuses, procede de criblage de compose inhibant ou favorisant la mort de cellules nerveuses, et inhibiteur de mort de cellules et promoteur de cellules nerveuses - Google Patents

Procede permettant d'induire la mort de cellules de ligne de cellules nerveuses, procede de criblage de compose inhibant ou favorisant la mort de cellules nerveuses, et inhibiteur de mort de cellules et promoteur de cellules nerveuses Download PDF

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WO2000066145A1
WO2000066145A1 PCT/JP2000/002830 JP0002830W WO0066145A1 WO 2000066145 A1 WO2000066145 A1 WO 2000066145A1 JP 0002830 W JP0002830 W JP 0002830W WO 0066145 A1 WO0066145 A1 WO 0066145A1
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cell death
apoe4
cells
cell
binding
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PCT/JP2000/002830
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English (en)
Japanese (ja)
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Ikuo Nishimoto
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Keio University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Definitions

  • 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
  • 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 .
  • AD Alzheimer's disease
  • senile plaques extracellular deposits called senile plaques are characteristic of AD, and its main component is A? (Amyloid /?).
  • APP is ⁇ ? 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 ⁇ 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)).
  • FAD-APP This type of FAD mutant c called FAD-APP
  • V642I / F / G V642 FAD-APPs
  • PTX pertussis toxin
  • 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)).
  • PS-1 serotonin-2
  • ApoE apolipoprotein E
  • 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.
  • the ⁇ 4 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.
  • ApoE 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.
  • 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.
  • 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 Pertussis toxin
  • 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
  • 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;
  • a method for screening for a compound that inhibits or promotes neuronal cell death comprising:
  • nerve cell line is an F11 cell or a SH-SY5Y cell.
  • a neuronal cell death inhibitor comprising ApoE3 or a partial peptide thereof as an active ingredient
  • the present invention relates to a binding agonist for APOE receptor 1 (also referred to as LDL receptor-related protein (LRP)).
  • LRP LDL receptor-related protein
  • 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.
  • 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.
  • 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.
  • 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).
  • SEQ ID NO: 1 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 3 ⁇ 4, 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.
  • cells expressing the ApoE receptor 1 gene may be applicable to general mammalian cells including CH0 cells and HEK293 cells.
  • the ApoE receptor 1 cDNA can be inserted into an appropriate expression vector, introduced into the cell, and expressed.
  • a medium suitable for culturing each cell for example, HamF-12 medium for F11 cells, DMEM medium for SH-SY5Y cells, etc.
  • serum-free or serum-containing conditions eg, 30 / g / ml.
  • 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.
  • 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.
  • 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.
  • a method for detecting apoptosis can also be used.
  • 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.
  • 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
  • binding agonists eg, ApoE4
  • ApoE receptor 1 angyu gonist binding agonists
  • ApoE ApoE receptor 1 angyu gonist
  • ApoE an 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).
  • 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.
  • 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).
  • SEQ ID NO: 1 the ApoE receptor 1 binding region of ApoE4
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.,
  • 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.
  • 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.
  • 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.
  • natural forms of ApoE3 Zannis, VI et al., 1984, J. Biol. Chem. 259: 5495-5499
  • 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.
  • these partial peptides can be used as long as they have a cell death inhibitory effect on a nerve cell line.
  • 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).
  • a pharmacologically acceptable carrier or vehicle specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, etc.
  • a pharmacologically acceptable carrier or vehicle specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, etc.
  • 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.
  • the compound can be encoded by DNA, the DNA may be incorporated into a gene therapy vector to perform gene therapy.
  • 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 ⁇ ⁇ 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 ⁇ 1 cells with 25 ⁇ M tandem LRPBP for the times indicated.
  • 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.
  • 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 ⁇ g / ml ApoE4.
  • Panel D shows the results obtained by treating F11 cells with 100 / M ApoE4 peptide in the presence or absence of 30 ⁇ g / 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.
  • 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
  • ⁇ D + NaF '' represents the result of NaF treatment after pretreatment with dexoxamine.
  • 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
  • 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 ⁇ g / ml ApoE3 (lane 3) for 24 hours.
  • FIG. 7 is a photograph showing the results of TUNEL Atssei on cell death induced by ApoE4.
  • F11 cells were treated with 30 ⁇ g / ml ApoE3 (panel A) or 30 ⁇ g / 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
  • Panel D shows the results of treatment of F11 cells with lOmM NaF for 6 hours.
  • FIG. 9 shows the promotion of ApoE3 neurotoxicity in SH-SY5Y cells.
  • the figure shows the effect of ApoE4 (30 ⁇ g / ml), ApoE4 (30 ⁇ g / 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.
  • 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; ⁇ 2 ⁇ ) 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.
  • Fll cells were incubated with 30 ⁇ g / 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.
  • L141-L149 LRKLRKRLL / SEQ ID NO: 1
  • ApoE receptor 1 ApoE receptor 1
  • LRP LDL receptor-related protein
  • LMLRKRLL is an 18-residue peptide (L MLRKRLLLRKLRKRLL / SEQ ID NO: 2, referred to as "tandem LRPBP (tandem LRP binding peptide)"
  • Tandem LRPBP tandem LRP binding peptide
  • LRPBP 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. .
  • ApoE4-specific polypeptides 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.
  • Action of ApoE4 is to further demonstrate that through the ApoE receptor 1, examined the effect of shed 2 M.
  • 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.
  • NaF sodium fluoride
  • 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.
  • 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 ⁇ g / 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.
  • F11 cells were treated with 30 ⁇ g / 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 ⁇ g / 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
  • 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 ⁇ g / 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.
  • tests using ⁇ ⁇ ⁇ 4 and ⁇ in tandem LR ⁇ ⁇ ⁇ ⁇ 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.
  • 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)).
  • 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.
  • PTX pertussis toxin

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Abstract

Selon l'invention, on peut induire de manière appropriée la mort de cellules d'une ligne de cellules nerveuses en traitant ladite ligne par l'apolipoprotéine E4, ApoE4. En outre, le récepteur 1 d'ApoE et la protéine G sensible à la toxine coquelucheuse (PTX) participent à la transduction de signal vers la mort de cellules nerveuses répondant à la stimulation d'ApoE4.
PCT/JP2000/002830 1999-04-30 2000-04-28 Procede permettant d'induire la mort de cellules de ligne de cellules nerveuses, procede de criblage de compose inhibant ou favorisant la mort de cellules nerveuses, et inhibiteur de mort de cellules et promoteur de cellules nerveuses WO2000066145A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005082399A2 (fr) * 2004-02-27 2005-09-09 Ai2 Limited Traitement d'infections bacteriennes
WO2005082399A3 (fr) * 2004-02-27 2005-12-22 Univ Manchester Traitement d'infections bacteriennes
AU2005216703B2 (en) * 2004-02-27 2011-06-23 Ai2 Limited Treatment of bacterial infections
WO2006004201A1 (fr) * 2004-07-01 2006-01-12 Eisai R & D Management Co., Ltd. Promoteur de la régénérescence nerveuse

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