JP2002356440A - Agent for prevention and treatment of alzheimer's disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an agent for the prevention and treatment of Alzheimer's disease, especially familial Alzheimer's disease. SOLUTION: Insulin-like growth factor I(IGF-I), des(1-3)IGF-1, insulin-like growth factor I receptor (IGF-IR), V922E-IGF-IR, etc., can be used as an agent for the prevention and treatment of Alzheimer's disease based on the findings that IGF-I suppresses the apoptosis of Alzheimer V6421 variant APP through IGF-IR, des(1-3)IGF-I also suppresses the apoptosis in the presence of IGFBP-3 and IGF-IR, especially V922E-IGF-IR suppresses the apoptosis by Alzheimer V642I variant APP even in the absence of IGF-I.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a prophylactic / therapeutic agent for Alzheimer's disease containing insulin-like growth factor I (IGF-I), insulin-like growth factor I receptor (IGF-IR) or the like as an active ingredient.

[0002]

2. Description of the Related Art Alzheimer's disease
e: AD) is the most common of diseases in which neurological function deteriorates, and is characterized by extracellular Aβ amyloid plaques, neurofibrillary tangles in neurons, and extensive neuronal necrosis. Has features. Amyloid precursor protein (APP) is a precursor of Aβ having a one-time transmembrane structure, and V642I / F / G (V642I) in APP ₆₉₅ having 695 amino acids (Nature 325, 733-736, 1987). , V642F,
V642G) mutation is the first established cause of early familial AD (FAD) (Nat. Genet.
1, 233-234, 1992). V, the three mutants of APP ₆₉₅
The cDNA of 642 I / F / G was transformed into a neuron-like transformant of NK1 cells and COS cells (EMBO J. 15, 498-509, 1
996, EMBO J. 16, 4897-4907, 1997, FEBS Lett. 412,
97-101, 1997), neuron / neuroblastoma hybrid F11 cells (Science 272, 1349-1352, 1996, J. Bio).
l. Chem. 275, 34541-34551, 2000), PC12 cells (S
cience 274, 1710-1713, 1996, J. Neurosci. Res. 47,
253-263, 1997), and primary cultured central nervous system (J. N
eurosci. Res. 55, 629-642, 1999) causes cell death. Further studies (EMBO J. 15, 498-509, 1996, Science 272, 1349-135)
2, 1996, Science 274, 1710-1713, 1996, EMBO J. 16,
4897-4907, 1997, J. Biol. Chem. 275, 34541-34551,
2000), V642I-APP, K595N / M5
96L-APP or N141I-presenilin (PS)
-2, which causes FAD, is pertussis toxin (P
TX) in response to neuronal cell death. This is because intracellular signaling
It suggests that these FAD gene products mediate cytotoxicity.

A powerful mechanism underlying the neurotoxicity of the FAD gene is neuronal death caused by cytotoxic Aβ, particularly Aβ1-42, which is secreted by the FAD gene (Proc. Natl. Acad. Sci. USA
90, 7951-7955, 1993, Nature 360, 672-674, 1992, S
science 259, 514-516, 1993, Science 264, 1336-1340,
1994, Science 272, 1349-1352, 1996). It leads to neuronal death even if Aβ causes neurotoxicity and triggers intracellular signaling mechanisms (Brain Res.
756, 205-214, 1997; J. Neurochem. 69, 1601-1611,
1997, Prog. Neurobiol. 62, 633-648, 2000, Nature 4.
05, 360-364, 2000, Nature 403, 98-103, 2000). Therefore, an effective way to suppress neuronal death in FAD is to suppress the signal of death inside neurons expressing the FAD gene, and this method is possible even after Aβ deposition.

[0004] Dore et al. (Proc. Natl. Acad. Sci. USA
94, 4772-4777, 1997) is insulin-like growth factor I
(IGF-I) was found to exert a protective function against Aβ-induced neurotoxicity, suggesting that IGF-I may be a new therapeutic agent for AD. However, information on whether IGF-I effectively suppresses cell death due to the FAD gene is currently not available. Dore et al. Also attributed the IGF-I receptor (IGF-IR) to the protection of nerves by IGF-I based primarily on the poor potency of IGF-II and insulin, but directly No evidence was provided. Identification of a target receptor for the action of IGF-I in the antagonism of AD injury not only elucidates the defense mechanism, but also is very important in producing a low-molecular-weight pseudo-IGF-I having an anti-AD action. is important.

[0005]

An object of the present invention is to provide an agent for preventing or treating Alzheimer's disease, particularly familial Alzheimer's disease.

[0006]

SUMMARY OF THE INVENTION It has been known that insulin-like growth factor I (IGF-I) exerts its power in protecting cells from nerve cell death induced by Aβ amyloid. Precipitation of Aβ amyloid is due to Alzheimer's disease (A
This is one of the pathological features of D). The present inventors have now shown that amyloid precursor protein (APP)
We investigated whether IGF-I exerts a protective function against cell death caused by D (FAD) mutant. In many cell lines, IGF-I Has been found to defend. IG
FBP-3 inhibits the action of IGF-I,
Des (1-3) I in which three N-terminal residues of FI are deleted
It does not inhibit the action of GF-I. des (1-3) IGF
-I acts like IGF-I in the presence of IGFBP-3. In addition, IGF-I receptor (IGF-IR)
Mediates the protective effects of IGF-I. Furthermore, V64 antagonized by IGFBP-3
The antagonistic function of the IGF-I / IGF-IR series against 2I-APP not only has a pathophysiological understanding of AD,
We thought that a powerful treatment for AD could be established. The present inventors have conducted intensive studies to solve the above-mentioned problems, and found that insulin-like growth factor I (IGF-I) was converted from insulin-like growth factor I receptor (IGF-IR) from cell death caused by Alzheimer V642I mutant APP. )
Suppresses cell death through, des (1-3)
Similarly, IGF-I suppresses cell death even in the presence of IGFBP-3, and demonstrates that IGF-IR, particularly V922E-I
It has been confirmed that GF-IR suppresses cell death caused by Alzheimer's V642I mutant APP even in the absence of IGF-I, thereby completing the present invention.

[0007] That is, the present invention relates to a prophylactic / therapeutic agent for familial Alzheimer's disease characterized by using an insulin-like growth factor I (IGF-I) protein as an active ingredient (claim 1). A protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and having an activity of suppressing cell death caused by familial Alzheimer's disease, as an active ingredient Preventive / therapeutic agent for familial Alzheimer's disease characterized by the following (claim 2)
Or a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and having an activity of inhibiting cell death caused by familial Alzheimer's disease The protein is des (1-3) IGF-I, the prophylactic / therapeutic agent for familial Alzheimer's disease according to claim 2 (claim 3), or the protein according to any one of claims 1 to 3. A prophylactic / therapeutic agent for familial Alzheimer's disease, comprising as an active ingredient a vector capable of expressing a protein (claim 4).

[0008] The present invention also relates to insulin-like growth factor I.
A prophylactic / therapeutic agent for Alzheimer's disease characterized by using a receptor (IGF-IR) protein as an active ingredient (Claim 5), or one or several of the proteins having the amino acid sequence shown in SEQ ID NO: 2 A prophylactic / therapeutic agent for Alzheimer's disease, characterized by comprising, as an active ingredient, a protein consisting of an amino acid sequence in which amino acids are deleted, substituted or added and having an activity of suppressing cell death caused by Alzheimer's disease (claim 6). ) Or SEQ ID NO: 2
In a protein consisting of the amino acid sequence shown in
A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added and having an activity of suppressing cell death caused by Alzheimer's disease is V92
The prophylactic / therapeutic agent for Alzheimer's disease according to claim 6, which is 2E-IGF-IR (claim 7),
A prophylactic / therapeutic agent for familial Alzheimer's disease, comprising a vector capable of expressing the protein according to any one of claims 5 to 7 as an active ingredient (claim 8).
The present invention also relates to the preventive / therapeutic agent for Alzheimer's disease according to any one of claims 5 to 8, wherein the Alzheimer's disease is familial Alzheimer's disease (claim 9).

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION As a preventive / therapeutic agent for familial Alzheimer's disease of the present invention, insulin-like growth factor I is used.
In addition to (IGF-I), in a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 such as des (1-3) IGF-I, an amino acid sequence in which one or several amino acids have been deleted, substituted or added And proteins having an activity of suppressing cell death caused by familial Alzheimer's disease, and vectors capable of expressing these proteins (the above-mentioned substances are collectively referred to as “the IGF-I
There is no particular limitation as long as it is a prophylactic / therapeutic agent containing an active ingredient as the active ingredient. In addition to insulin-like growth factor I receptor (IGF-IR), V922E-IGF-
A protein comprising an amino acid sequence represented by SEQ ID NO: 2, such as IR, comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and having an activity of suppressing cell death caused by Alzheimer's disease And vectors capable of expressing these proteins (the above substances are collectively referred to as “the IGF
-IR-related substance) is not particularly limited as long as it is a prophylactic / therapeutic agent containing, for example, an active ingredient.

[0010] The IGF-I-related substance and the IGF
-When an IR-related substance is used as a prophylactic / therapeutic agent for Alzheimer's disease, a normal pharmaceutically acceptable carrier, binder, stabilizer, excipient, diluent, pH buffer, disintegrant,
Various compounding ingredients for preparations such as solubilizers, solubilizers, and isotonic agents can be added. Such a prophylactic / therapeutic agent can be administered orally or parenterally. That is, it can be orally administered in a commonly used dosage form, for example, a powder, granule, capsule, syrup, suspension or the like, or, for example, a solution, emulsion, suspension or the like. The preparation can be administered parenterally and topically in the form of an injection, or can be administered intranasally in the form of a spray.

When the present IGF-I-related substance or the expression vector is used as the IGF-IR-related substance, it is effective for gene therapy. When such a vector is administered locally, the present IGF-I-related substance other than the vector is effective. Compared to the case where a therapeutic agent containing a substance as an active ingredient is locally administered, the stable expression of the substance makes it possible to supply the active ingredient to the local area more stably. IGF-I, IGF-IR
While such proteins are unstable, stable expression for a predetermined period can be obtained by introducing a gene into a nerve cell using an expression vector. As such a vector, a virus vector such as a herpes virus (HSV) vector, an adenovirus vector, and a human immunodeficiency virus (HIV) vector can be preferably mentioned. Among these virus vectors, H.V.
SV vectors are preferred. The HSV vector is safe because HSV is not integrated into chromosomal DNA, and the administration period can be adjusted because the expression does not extend for a long time. The above expression vector using a virus vector can be prepared by a conventional method.

[0012]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the technical scope of the present invention is not limited to these examples. Example A [Materials and Methods] (A-1: Materials) V642I variant of a wild-type APP ₆₉₅ (wtAPP) and APP ₆₉₅ (V642I-APP)
Was prepared by the method described in the literature (EMBO J. 15, 498-509, 1996). In addition, wild-type human IGF-IR (w
tIGF-IR) and IGF-IR variants were described in the literature (J. B.
iol. Chem. 270, 19041-19045, 1995). Recombinant human IGF-I and des (1-3)
IGF-I was purchased from Fujisawa Pharmaceutical (Osaka, Japan) and Peninsula Laboratories (San Carlos, CA), respectively. IGF-I was also purchased and used from Boehringer Mannheim (Mannheim, Germany) and Becton-Dickinson (Franklin Lake, NJ).
-I showed similar results. Insulin and EGF are Sigma (St. Louis, Mo.) and PDGF and IGFBP-3 are upstate.
Biotechnology (Lake Placid, NY), NGF and IGF-II were purchased from Becton-Dickinson.

(A-2: Northern blot analysis) NK1
Total RNA was extracted from cells or F11 cells, and 10 μg of total RNA was subjected to agarose gel electrophoresis, followed by Hybo.
After transfer to an nd-N nylon membrane, Northern blotting was performed. Hybridization conditions for Northern blots were 65 ° C. overnight for cDNA probes and 37 ° C. overnight for oligonucleotide probes. The following probe [EGF receptor (E
GFR) cDNA: BamHI fragment (about 1 kb, RIKEN gene bank), insulin receptor (IR) cDNA: XhoI
Fragment (about 1 kb), IGF-I receptor (IGF-IR)
cDNA: NcoI fragment (about 1.8 kb, J. Biol. Chem. 26
7, 25958-25965, 1992, J. Biol. Chem. 270, 29224-29
228, 1995), IGF-II / mannose-6-phosphate receptor cDNA: HindIII fragment (about 1.5 kb, J. Bio
l. Chem. 267, 25958-25965, 1992, J. Biol. Chem. 27
0, 29224-29228, 1995), p75 low affinity NG
F receptor (LNGFR) cDNA: HindIII / XbaI fragment
(About 1.6 kb), TrkA cDNA: BamHI / NheI fragment
(Approx. 1.2 kb, courtesy of Dr. Andy A. Welcher: Proc.
Natl. Acad. Sci. USA 89, 2374-2378, 1992), PDG
F receptor (PDGFR) β cDNA: sense (5'-GAT
GGA AGG TGA TTG AGT CTG TGA GCT CTG ACG GCC ATG A
GT ACA TCT-3 ') (SEQ ID NO: 3: primer 1) and antisense (3'-CTA CCT TCC ACT AAC TCA GAC ACT CGA
GAC TGC CGG TAC TCATGT AGA-5 ') (SEQ ID NO: 4: primer 2) (DNA polymerase I in the presence of [α- ³² P] dCTP
Annealed with Klenow fragment of the above) was used.

(A-3: One-step RT-PCR) r
Tth DNA polymerase (RT-PCR High Plus, Toyobo) and total RNA of NK1 cells or F11 cells
Was used to perform one-step RT-PCR. After performing reverse transcription reaction at 60 ° C. for 30 minutes to synthesize cDNA,
Using the primers shown below, 94 ° C for 1 minute, 60 ° C
Forty cycles of the 1.5 minute polymerase chain reaction were performed. As primers (for IR, a sense primer: 5′-GCG ATA TGG TGA TGA GGA GCT GCA-3 ′ (SEQ ID NO: 5: primer 3) and an antisense primer: 5′-
GGT CTC TGC CTC ACC CTT GAT GAT-3 '(SEQ ID NO: 6: primer 4) and IGF-IR have a sense primer: 5'-ACA GAG TAC CCT TTCTTT GAG AGC-3' (SEQ ID NO: 7: primer 5 ), And antisense primer 5'-A
GAPDH using AG AAC ACA GGA TCT GTC CAC GAC-3 '(SEQ ID NO: 8: primer 6) as a control
Has a sense primer: 5'-TCC ACC ACC CTGTTG CTG
TA-3 '(SEQ ID NO: 9: primer 7) and antisense primer: 5'-ACC ACA GTC CAT GCC ATC AC-3' (SEQ ID NO: 10: primer 8) were used.

(A-4: Transfection of NK1 cells and TUNEL, ELISA) Reference (EMBO J. 15,
498-509, 1996) 4 × 10 ⁴ (cells / well) in a 24-well plate or 10 ^{6 in a} 100 mm dish.
(Cells / dish) to provide NK1 cells for transfection and 2% in DME supplemented with 10% bovine serum (CS) and penicillin / streptomycin.
The cells were cultured for 4 hours. cDNA and Lipofectamine (Li
pofectAMINE: GibcoBRL, Gaithersburg, MD :; ELISA, TUNEL, and immunohistochemical analysis: cDNA in a 24-well plate (0.5 μg)
And LipofectAMINE (1 μl), immunoblot analysis:
CDNA (10 μg) in a 100 mm dish and LipofectAM
With INE (20 μl), in DME without serum
Cells were transfected. In the above two protocols (ELISA, TUNEL, and immunohistochemical analysis and immunoblot analysis), cDNA expression showed the same results. Further, after culturing in the absence of serum for 24 hours, the medium was changed to a DME medium supplemented with 1% CS. Various growth factors were added to these media and the cells were fixed after 24 hours of culture. APP-positive cells and their nuclei were separately stained with anti-APP monoclonal antibody 22C11 and acridine orange. APP-positive cells and their nuclei were stained with anti-APP monoclonal antibody 22C11 and acridine orange, respectively.
And the measurement of the rate of V642I-APP-expressing cell death by compaction is described in the literature (EMBO J. 15, 498-509, 199).
6) Performed according to the method described. In addition, nucleosome fragmented DNA was in situ analyzed by TUNEL using the method described in the literature (EMBO J. 15, 498-509, 1996).
u and visualized and quantified by ELISA.

(A-5: Transfection of F11 cells) Transfection was performed using F11 cells grown by culturing in Ham's F-12 medium supplemented with 18% fetal bovine serum (FBS) and antibiotics. Was performed in the following manner. F11 cells (18% fetal bovine serum (F
7x10 ⁴ (cells / well) in a 6-well plate cultured for 12-16 hours in Ham's F-12 medium supplemented with BS) plus lipofection [V642I-APP] for 3 hours in the absence of serum. 0.5μ cDNA
g, 1 μl of LipofectAMINE, plus reagent (GibcoBRL)
Was transfected with V642I-APP, and cultured in Ham's F-12 medium supplemented with 18% fetal bovine serum (FBS) for 2 hours. The medium was further supplemented with or without inhibitor or αIR3 (Oncohene Science, Cambridge, Mass.) For 10 minutes.
% Of fetal bovine serum (FBS) and ham F-12 medium supplemented with IGF-I, and the cells were further cultured for 67 hours. 72 hours after transfection, cell mortality was measured by the trypan blue exclusion assay as follows.

(A-6: Trypan blue exclusion assay and TUNEL) Pipette 50 μl of trypan blue 0.4% solution (Sigma) and 200 μl of cell suspension (final concentration 0.08%). After gentle mixing, the number of cells stained was measured within 3 minutes and cell mortality was calculated. The cell death rate by this method indicates the number of dead cells in all cells including floating cells and adherent cells at the end of the experiment. Cell mortality by this method is described in the literature (J. Neurosci. 20, 8401-8409, 2000).
WST-8 [2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- using the described cell count kit-8 (Wako Pure Chemical Industries, Tokyo, Japan) (2,4-disulfophenyl)-
2H-tetrazolium, monosodium salt]. Furthermore, F1 was obtained by the method described in the literature (EMBO J. 15, 498-509, 1996).
TUNEL experiments were performed on one cell. V642I
-Transfection of APP and 24 hours of culture in the absence of serum, IGF-I or des (1-
3) Cells were treated with IGF-I.

Twenty-four hours after the start of the above processing, TUNE
L was performed. F11 / EcR / V642I cells were isolated from the literature (Biochem. Biophys. Res. Commun. 274, 445-454, 200).
0) It was prepared by the method described below. F11 / EcR /
V642I cells were transferred to medium with 10% FBS and cultured for 24 hours. Further, the cells were treated with 40 μM EcD in the presence of 10% FBS and cultured for 48 hours. IGF-I, IGF-I to which anti-IGF-I antibody was added, or des (1-3) IGF-I was added to EcD.
And cultured. In addition, anti-IGF-I antibody is IGF-
I (Ab-2: a goat polyclonal antibody that antagonizes human IGF-I) and was purchased from Oncogene Research Products (Cambridge, Mass.).

(A-7: CN-procaspase cDNA)
Construction and immunoblot) Inactive procaspase-
The construction of cDNA of No. 3 is performed using a kit (Clontech, Cambridge, UK) and 5'-ATT ATT CAG GCC TCA CGT GGT ACA GAA
CTG-3 '(SEQ ID NO: 11: primer 9 (TCA at bases 13 to 15 corresponds to a mutated sequence)) and cDNA of complete procaspase-3 having activity (from Dr. Miura Masayuki of Osaka University) Donation). The inactive procaspase-3 (CN-procaspase-
The cDNA of 3) is Lipofectamine Plus (Lipofemin).
ctAMINE Plus: 0.4 μg CN-procaspase-3
cDNA, 0.6 μg V642I-APP cDN
A, 2 μl Lipofectamine, 4 μl plus reagent) using 10 nM IGF-I or 100 μM Ac
-DEVD-CHO (Peptide Institute Inc., Japan)
In the presence or absence of pcDNA or V642I-
F11 cells were transfected with APP cDNA. 48 hours after the start of transfection, the cells were collected, lysed, and subjected to immunoblot analysis using an anti-caspase-3 antibody, an anti-APP antibody, or an anti-actin antibody. Further Ac-DEVD-C
HO-dependent cleavage of CN-procaspase-3 was examined.

[0020] Monoclonal anti-IGF-IR antibody αIR
3 precipitated IGF-IR. After serum starvation for 12 hours, NK1 cells (10 ⁶ (cells / well)) were treated with 10 nM IGF-I or insulin for 3 hours.
Treated at 7 ° C for 2 minutes. The NK1 cells were placed in an ice-cold buffer [50 mM Tris / HCl (pH 7.4), 1
mM EDTA, 1% NP-40, 1 mM DTT,
After dissolving with 300 μl of 1 mM PMSF, 1 mM Na ₃ VO ₄ ] and centrifuging, protein G Sepharose (Am
ercham Pharmacia Biotech, Uppsala, Sweden)
The supernatant was cultured together with 2 μg of αIR3 bound to and.
The precipitate of the culture solution was subjected to electrophoresis on a 7.5% SDS polyacrylamide gel (SDS-PAGE), and then blotted on a PVDF sheet, followed by 10 mM Tris / HCl (pH 7.4), 2% Was blocked with skim milk and 1% BSA, and RC20H [HRP
Labeled anti-phosphotyrosine antibody, dilution ratio 1/5000; Tran
sduction Lab (Lexington, KY)] for 1 hour at room temperature and visualized by ECL.

(A-8: Measurement of Antagonism of IGF-IR) In order to examine the antagonism of mutation-activated IGF-IR against cell death induced by V642I-APP, reference was made to the literature (J. Biol. Chem. 270, 19041-19045, 1995)
Similarly to the method described, 0.5 μg of V642I-APP cDNA was replaced with V922E-IGF-IR and V922I-IR.
Cells were transfected by lipofection with IGF-IR or wtIGF-IR cDNA, and the number of dead cells in V642I-APP-expressing cells was counted.

Example B [Results] B-1 (IG against cell death by V642I-APP
Antagonistic Effect of FI) Cell death is caused by three V642 mutants, which are FAD mutants of APP, which are transiently expressed in NK1 cells (EMBO J. 15, 498-509, 1996, EMBOJ. 16, 48
97-4907, 1997). In this system, cell death is associated with FAD characteristics, as the FAD variant has the highest incidence of cell death out of a total of 19 possible V642 variants and the lowest toxicity of wtAPP It is suggested that you do. V642I-APP and known growth factors (PDGF in glial cells of rat optic nerve) that have been reported to date to inhibit cell death using this system
(Cell 70, 31-46, 1992) and NGF in rat sympathetic neurons and PC12 cells (EMBO J. 6, 1145-1154, 198).
7, J. Cell Biol. 115, 461-471, 1991, J. Cell Biol.
119, 1669-1680, 1992), FGF in vascular endothelial cells (Bio
chem. Biophys. Res. Commun. 168, 1194-1200, 199
0), EGF in embryonic cells (J. Cell Biol. 113, 671-680,
1991), and IGF-I, IGF-II, and PDGF in c-myc-induced cell death of rat-1 cells (EMBO J.
13, 3286-3295, 1994)), factors having an antagonistic effect, EGF, PDGF, IGF-I, IGF-II, insulin and NGF were examined in various systems.

The effects of cell growth factors on NK1 cells are shown below (FIG. 1, lower panel). Each lane in the lower diagram of FIG. 1 shows various growth factors (lane 1: 100 ng / ml PDGF,
Lane 2: 10 nM EGF, lane 3: 10 nM N
GF, lane 4: 10 nM IGF-II, lane 5
7: 0.1, 1, 10 nM IGF-I, lanes 8-1
0: 0.1, 1, 10 nM insulin). Transfection was performed three times individually, and the percentage of cell death is shown as mean ± standard error (Stud
ent's t test: P <0.01). 1 nM IGF-I is V
Although cell death by 642I-APP was significantly suppressed (FIG. 1, lower panel, lane 6), 1 nM insulin did not show much inhibition (FIG. 1, lower panel, lane 9). Also, 1
The addition of 0 nM IGF-I or 10 nM insulin caused strong suppression of cell death (FIG. 1, lower panel lanes 7, 10), but the addition of 10 nM IGF-II did not show much suppression (FIG. 1 lower figure lane 4). Other growth factors had no effect until the concentration reached 100 nM. IGF-I and insulin
In each concentration-dependent manner, an antagonistic effect on cell death by V642I-APP was shown (FIG. 1, lower panel, lanes 5 to 7, 8).
10), the 50% inhibition value (IC ₅₀ ) was about 0.5 nM for IGF-I and about 5 nM for insulin. Also V
642I-APP expression is not affected by IGF-I, suggesting that V642I-APP expression is not a target for action by IGF-I. Therefore, IGF-I
The family was found to suppress cell death of NK1 cells expressing V642I-APP.

From the results of the Northern blot, it was found that EGF receptor (5.8 kb), IGF-II
/ Mannose-6-phosphate receptor (9.0k
b) and IGF-I receptor (IGF-IR) (1
1 kb) of mRNA was confirmed to be transcribed, but TrkA (expected size was 3.2 kb)
b), p75 low affinity NGF receptor (LNGFR)
(3.8 kb), PDGFβ receptor (5.6 kB)
b) and insulin receptor (IR) (5.5k)
b, 6.0 kb, and 7.0 kb) mRNA were not transcribed. The results of transcription of IGF-IR and IR confirmed by RT-PCR in NK1 cells are shown in the upper part of FIG. IR cDNA (lanes 1 and 2), I
GF-IR cDNA (lanes 3, 4) and total RNA
For samples (lanes 5 to 7), IR or IGF-I bands were obtained by RT-PCR using IGF-IR primers (lanes 2, 4, 6) or IR primers (lanes 1, 3, 5). Was detected. In lanes 5 and 6, a 490 bp IR, 4
This shows that a 48 bp IGF-IR band was detected. GAPDH of 450 bp is used as a control marker (lane 7). Further, the band shown in the upper diagram of FIG.
Hybridization using the cDNA probe of R confirmed that IGF-IR was transcribed (lanes 4 and 6 in the middle part of FIG. 1). From the above results, the action of insulin on cells was determined by IGF-IR
It is suggested that this is done via

B-2 (IGF-I Inhibits cell death)
Effect of GFBP-3) Serum regulates the effects of IGF-I and IGF-II.
GFBP is reported to be included (Baxter RC (1
991) Physiological roles of IGF binding protein.I
n: Modern Concepts of Insulin-Like Growth Factors
(Spencer EM, ed), pp371-380. New York: Elsevie
r.). At least six IGFs of the IGFBP family
Of the BPs subtypes, IGFBP-3 is known to have the highest IGF binding activity in serum and to affect the pleiotropic action of IGF-I (Growth Regul. 2, 8
0-87, 1992). From the above, the effect of IGFBP-3 on cell death suppression of IGF-I was examined. Note that wtA
Cells transfected with PP or V642I-APP were transfected with 10 nM IGFBP-3 (FIG. 2A), des (1-3) IGF-I at various concentrations (FIG. 2B), or 10nM.
M des (1-3) IGF-I (FIG. 2C) in the presence (+) or absence (−) of 10 nM IGF-I.
The results of treatment with I or des (1-3) IGF-I are shown. In addition, transfection was performed three times individually, and the percentage of cell death was shown as the mean ± standard error. As a result, 10 nM of IGFBP-3 and IGF-I were V642
Although cell death by I-APP was suppressed, IGFBP-3
Alone did not have the effect of suppressing cell death by V642I-APP (FIG. 2A). Therefore, since IGFBP-3 did not act on insulin, the observed IGF
The effect of -I is not non-specific and IGFBP-3 is
42I-APP was found to block the inhibitory effect of IGF-I on cell death.

B-3 (des (1-3) inhibitory action on cell death of IGF-I) des (1-3) which does not have three N-terminal residues of IGF-I which is essential for binding to IGFBP-3 Using IGF-I, the cell death inhibitory effect of V642I-APP was examined. Note that Mock in FIGS. 2B and 2C shows a result obtained by transfecting only the vector as a control. 10 nM des (1-3) IGF-I
Inhibited cell death by V642I-APP almost completely, and its intensity was similar to that of IGF-I (FIG. 2B).
Furthermore, the cell death inhibitory effect of des (1-3) IGF-I was not affected by IGFBP-3 (FIG. 2A). From these facts, it is considered that IGFBP-3 directly inhibits the cell death inhibitory action of IGF-I.

As a result of performing two different assays (ELISA assay and TUNEL assay) on the nucleosomal DNA fragment, d
The es (1-3) IGF-I inhibitory action was confirmed. An ELISA assay of the fragmented DNA revealed that 10 nM des (1-3) IGF-I blocked V642I-APP-induced oligonucleosomal DNA fragmentation (FIG. 2C). As previously reported (Science 272, 1349-1352, 1996), T
In the UNEL assay, expression of V642I-APP resulted in fragmentation within the nucleosomes of NK1 cells,
In situ labeled DNA increased sharply (FIG. 3). Furthermore, 10 nM des (1-3) IGF-I completely suppressed the fragmentation of DNA by V642I-APP (FIG. 3). Therefore, the cell death-suppressing effect of des (1-3) IGF-I on V642I-APP resulted in a result consistent with the nuclear morphological assay for cell death.

B-4 (IGF-I suppresses cell death in neurons transfected with V642I-APP) Two different neuronal systems (F11 cells or F11 cells)
/ EcR / V642I cells) and IGF-I and d
The cell death inhibitory effect of es (1-3) IGF-I was confirmed. One neuronal system is the immortalized neuronal cell line F11 cells (Science 272, 1349-1352, 1996). F11 cells are hybrid cells of primary cultured neurons of 13-day-old rat embryos and mouse neuroblastoma NTG18, one of the best primary cultured neuronal immortalized cell lines. In addition, F11 cells have many neuronal features such as maintenance of neuronal gangliosides and action potential generation (under no differentiation factor treatment) (P
roc. Natl. Acad. Sci. USA 82, 3499-3503, 1985). As in previous reports (Science 272, 1349-1352, 1996)
By transfecting the cDNA of V642I-APP into F11 cells, a large amount of DNA fragments
Detected by NEL (FIG. 4, left panel, showing one of three similar results). In addition, in F64 cells transfected with V642I-APP, 10%
nM IGF-I or 10 nM des (1-3) I
In the medium to which GF-I was added, DNA fragmentation was rapidly suppressed.

Procaspase-3 stimulated by transfection of V642I-APP using inactive procaspase-3 (CN-procaspase-3)
Was examined for its inhibition by cleavage of IGF-I.
CN-procaspase-3 was added to F11 cells by V642I-
When transfected with APP cDNA, in the presence of Ac-DEVD-CHO, CN-
Procaspase-3 was cleaved at 67.8 ± 8.7% (mean ± standard deviation, n = 3). Ac-DEVD-CHO
Is an inhibitor specific to caspases, and Ac-DEVD- of CN-procaspase-3, which has no self-cleaving function.
CHO sensitive cleavage reflects the intracellular action of caspase-3. Caspase-3 fragment having activity could not be detected by transfection of V642I-APP. V642 could not be detected
This suggests that the expression of I-APP is slow over one day or more, and that the rapid disappearance of active caspase-3 fragments. Also, CN-procaspase-3
Was co-transfected into F11 cells together with V642I-APP cDNA and treated with 10 nM IGF-I. Only 8.4 ± 5.8% of CN-procaspase-3 was cleaved. This means that IGF-
I shows that V642I-APP stimulates the cleavage of procaspase-3, a process essential for inducing cell death.

Another cell line of the two different neuronal lines is ecdysone (EcD) -induced V642I-
F11 / EcR / V642I cells prepared by transfecting APP cDNA into F11 / EcR cells. F11 / EcR cells are stable F11 cells that express both EcD receptor (EcR) and retinoid X receptor (J. Biol. Chem. 275, 34541).
-34551, 2000). F11 / EcR / V642I cells are E
Treatment with cD causes the clonal characteristic of cell death (Biochem. Biophys. Res. Commun. 274, 445).
-454, 2000). EcD-induced toxicity in this cell line has been shown to cause cell death by V642I-APP expression (Biochem. Biophys. Res. Commu.
n. 274, 445-454, 2000). F11 / EcR / V642I
In cells, cell death due to V642I-APP expression is IG
It is suppressed depending on the amount of FI used (the upper right figure in FIG. 4).
The cell death-suppressing effect of GF-I was blocked by adding an anti-IGF-I antibody (FIG. 4, middle right diagram). The inhibition of the cell death inhibitory action of IGF-I is described in des (1-3)
The same was true when IGF-I was added. Also, E
Cell viability of V642I-APP expressing cells damaged by cD induction was 1 nM des (1-3) IG
It was completely recovered by the addition of FI (FIG. 4, lower right panel), and all morphological changes of cell death (sphering, atrophy, detachment from the plate, etc.) were eliminated. The numerical values in the graph on the right side of FIG. 4 are the average value ± standard deviation of six processes. From the above results, low concentrations (nM level) of IGF-I and / or de
s (1-3) IGF-I was shown to block neuronal cell death by V642I-APP.

B-5 (anti-V642I-AP of IGF-I)
Involvement of IGF-IR in P action) The observed strong potency of IGF-I
It has been shown to exhibit a mediating role for IGF-IR, as well as negative expression of IR in cells and F11 cells. We will investigate the involvement of IGF-IR by (i) whether signal inhibitors of IGF-IR block the anti-cell death effect of IGF-I;
(Ii) whether the anti-IGF-IR blocking antibody affects the anti-cell death effect of IGF-I; (iii) the mutatedly activated IGF-IR mimics the anti-cell death effect of IGF-I Or not; approach was made from three directions.

(I) Effect of IGF-IR signal inhibitor In NK1 cells, 100 μM of genistein and 1
0nM wortmannin is V642I-APP
Significantly blocked the cell death-suppressing effect of IGF-I on (Fig. 5A). In F11 cells, the cell death inhibitory effect was similarly blocked (FIG. 5B). However, in NK1 cells and F11 cells, 100 μM genistein or 10 nM wortmannin was used as a control mortality (NK1 cell: mock, F11 cell: N11).
oT and pcDNA) or V642I-APP did not affect cytotoxicity (FIGS. 5A, 5B). Under the above experimental conditions, the transfection efficiency is 60% to 70%.
% (J. Biol. Chem. 275, 34541-34551, 200
0), 70% to 80% of cells transfected with V642I-APP died in 72 hours.
%Met. FIG. 5A shows the results of three individual transfections as mean ± standard error (Student's t-
test: P <0.01), FIG. 5B shows the mean of three individual results ±
The standard deviation is shown (Student's t-test: P <0.01).
Treatment of F11 cells with 10 nM IGF-I completely suppressed cell death due to V642I-APP. Furthermore, the inhibitory effect of IGF-I on cell death by V642I-APP was not complete by the effect of genistein, but 10 nM wortmanni.
It could be significantly attenuated by n or 100 μM genistein (FIG. 5B).

Wortmannin and genistein are inhibitors that block tyrosine kinase and PI-3 kinase, and IGF-IR activates in a ligand-dependent manner. Genistein and wortmannin are tyrosine kinases (J. Biol. Chem. 262, 5592-5595, 1987).
And PI-3 kinase (J. Biol. Chem. 267, 2157-2163,
1992) were significantly low at 100 μM and 10 nM, respectively.
Further, treatment of F11 cells with PD98059, an inhibitor specific to MEK, results in V642I
Genistein did not inhibit the cell death inhibitory effect of IGF-I in F11 cells transfected with -APP (FIG. 5B). This result indicates that in the cell death inhibitory effect of IGF-I, genistein is not MEK / MAP kinase further downstream of signal transduction, but IGF-I
This suggests the possibility of directly inhibiting R kinase.

(Ii) Effect of Anti-IGF-IR Antibody Three similar experiments were performed on the blocking effect of αIR3 on the cell death inhibitory effect of IGF-I. The representative result is shown in the right diagram of FIG. 5C. Vector only (lane 1), 10 nM IGF-I (lane 3), or 10 nM
Cells treated with M insulin (lane 4) resulted in αIR3 precipitation. Non-specific mouse I
In immunoblot with gG, 10 nM IG
Precipitation occurred in the lysate of FI-treated NK1 cells (lane 2). Arrow indicates IGF-IR β chain, about 100 kD
It corresponds to the size of a. αIR3 is the IGF of IGF-I
-It is known that it specifically inhibits binding to IR and has little binding to IR (J. Biol. Chem. 2
65, 9458-9463, 1990; J. Biol. Chem. 268, 2655-266
1,1993). Also, αIR3 is known to completely block the cell division promoting action of IGF-I (J. Bio.
l. Chem. 265, 9458-9463, 1990). Also in this example, a low concentration of αIR3 was detected in IGF-
Blocked the cell death inhibitory effect of I, but had no effect with normal IgG. Note that αIR3 itself is V642
It did not affect cell death induced by I-APP. The αIR3 antibody is a human and rodent IGF-I
Although it was thought that only R was recognized, αIR3
Recognition of monkey IGF-IR in one cell was confirmed. In the presence of 10 nM IGF-IR, αIR3
As a result, a phosphotyrosine protein (about 100 kDa) was precipitated from the NK1 cell lysate (FIG. 5C, right panel). This precipitated protein is suggested to be an IGF-IRβ chain precipitated together with the IGF-IRα chain by αIR3. In addition, in the case of ordinary IgG, no precipitation occurred even when the same experiment was performed.

IGF-I of αIR3 in F11 cells
Antagonism was investigated. 0.5 μg of V642IA
PP cDNA or pcDNA was transfected, and untransfected F11 cells (NoT) were cultured in the presence or absence of 10 nM IGF-I and / or 10 nM αIR3 (FIG. 5D, left) 10 columns). Also, cDNA of V642I-APP
Transfected into F11 cells (FIG. 5D
Right 7 columns), αIR3 (concentration 0, 1, from left to right)
(3, 10, 20, 50, 100 nM were performed) and 10 nM of IGF-I, and cell death was measured by trypan blue exclusion assay. FIG. 5D shows the average value ± standard deviation of three individual experiments. In this system, the cytotoxicity of V642I-APP was completely suppressed by 10 nM of IGF-I which was present at the same time, and the cell death inhibitory effect of IGF-I was αIR.
3 showed an antagonistic effect in a dose-dependent manner (FIG. 5D).
ΑIR3 showed a complete antagonistic effect at about 10 nM, which is the same low concentration used for IGF-I. A high concentration (about 100 nM) of αIR3 promotes growth, which is a pseudo-IGF-I action (J. Biol. Chem. 268,
2655-2661, 1993), but as far as suppression of cell death by IGF-I was concerned, high concentrations of αIR3 did not show cell growth promotion. F
The fact that transcription of IGF-IR mRNA was observed in 11 cells and almost no transcription of IR mRNA was observed was consistent with the results of the Northern blot, and these data indicate that IGF-I was not expressed in F11 cells. I
GF-IR suggests that V642I-APP suppresses cell death.

(Iii) IGF-IR activated by mutation
Next, V922E-IG in the absence of IGF-I
The function of F-IR was examined. The IGF-IR mutant has a point mutation V922E in the transmembrane region and its β
Show constitutively increased tyrosine kinase activity in the chain and serve as a constitutively active IGF-IR (J. Biol.
Chem. 270, 19041-19045, 1995). V922I-IGF-IR, in which tyrosine kinase is inactive, was also used for comparison. The experiments were performed three times individually, and the mean ± standard error is shown in FIG. 5E (Student's t-test: P <0.01).
By co-transfecting the cDNA of the activated mutant IGF-IR, V642I-AP
Cell death by P is drastically suppressed (FIG. 5E), whereas mutant tyrosine kinase constitutively inactive V92
2I-IGF-IR (J. Biol. Chem. 270, 19041-1904)
5, 1995) did not have a cell death inhibitory effect. V922E-IGF-IR also showed that receptor tyrosine kinase action, phosphorylation of IRS-1, and PI3-kinase activation were constitutively active. Furthermore, in co-transfection using wtIGF-IR, an inhibitory effect on cell death can be observed (Student's t-test: P <0.03), w
It is suggested that tIGF-IR originally has a cell death inhibitory activity (absent in V922I-IGF-IR).
V642 in the co-transfection experiments described above.
There was no difference in I-APP expression. These data suggest that IGF-IR can block cell death induced by V642I-APP.

(Discussion) The following can be considered from the above experimental results. IGF-I is expressed as A in NK1, F11 and F11 / EcR / V642I cells.
It showed an effect of protecting cells from the toxicity of the FAD V642I mutant of PP. At present, IGF-I has been shown to act as an anti-cell death factor in a number of systems (Proc. Natl. Acad. Sci. USA 90, 10989-10993, 19).
93, EMBO J. 13, 3286-3295, 1994, Cancer Res. 55, 3
03-306, 1995). In the present invention, the antagonism of IGF-I against cell death caused by the FAD gene has been clarified for the first time. The above examples also show that IGF-IR acts as a target for cytoprotection by IGF-I against AD-related injury. Based on the relatively strong potency of IGF-I compared to IGF-II, Dore et al. (Proc. Natl. Acad. Sci. USA 94, 4772-4777, 1997).
Speculated that IGF-IR was involved in the effects of IGF-I on Aβ-induced neuronal death. IGF-
As the first report illustrating the anti-cell death function of IR, Se
(Cancer Res. 55, 303-306, 1995) selected IGF-IR for its antagonism of etoposide-induced cell death in fibroblasts.

In the above examples, the downstream target of IGF-I is the intracellular signaling pathway of IGF-IR involving tyrosine kinase and PI-3 kinase, and their involvement is in V922E-IGF-IR. Supported by action. This mutant IGF-IR shows structurally increased receptor tyrosine kinase activity,
Activates the IRS-1 / PI-3 kinase cascade and glucose uptake without binding to
It does not activate the s / MAP kinase pathway, so it is entirely impossible to provide a stimulus for cell growth in the absence of IGF-I (J. Biol. Chem. 270,
19041-19045, 1995). Therefore, Akt's variable action, cytochrome c release inhibition (Mol. Cell Biol. 1
9, 5800-5810, 1999) and Serk / Thr kinase downstream of PI-3 kinase, suppression of BAD, caspase-9, or Forkhead transcription factor by phosphorylation (Cell 87, 619-628, 1996, Cell 91). , 231-241, 199
7, Science 282, 1318-1321, 1998, Cell 96, 857-868,
1999), post-translational modification of cytosolic factors downstream of cytochrome c and upstream of caspase-9 (J. Cell Biol.
151, 483-494, 2000), transcriptional activity of p53, or B
Activation by transcription of cl-x (FeBS Lett. 437, 112-11)
6, 1998, Cell Death Differ 6, 290-296, 1999), the cytoprotection by V922I-IGF-IR was observed. -It is suggested that APP is essential for exerting an anti-cell death function through a Ras-dependent mechanism. This idea is
PI, not Ras / Raf / MEK / MAP kinase
Many reports have reported that the activity of -3 kinase is essential for the receptor tyrosine kinase to exert an antagonistic effect on cell death in a wide variety of cells (for example, Sci.
ence 267, 2003-2006, 1995, Biochem. Biophys. Res.
Commun. 273, 322-327, 2000)
The anti-cell death signal by GF-IR is PI-3 kinase and A
kt mediated by Kulik et al. (Mol. Cel.
l Biol. 17,1595-1606, 1997).

The causes of AD have heretofore been discussed mainly in terms of aggressive factors, ie, injuries that can explain AD pathologically, such as Aβ and excitotoxins. However, recent studies have shown that I
GF-I (Proc. Natl. Acad. Sci. USA 94, 4772-477
7, 1997; and this example), activity-dependent neurotrophic factors (J. Mol. Neurosci. 7, 235-244, 1996, Proc. Nat.
Acad. Sci. USA 96, 4125-4130, 1999) and basic FGF (Proc. Natl. Acad. Sci. USA 96, 4125-
4130, 1999), beginning to identify the protective effects of neurotrophic factors, (i) the human body has a defense mechanism against the AD process, (ii) the pathological findings of the AD It suggests that it may not be a simple result, but a result of a collision between the attack and defense mechanisms. IGF-IR is a circulating polypeptide normally contained in plasma and produced mainly in the liver.
In the e manner, it is also produced in many tissues. IGF-IR produced by the brain is mainly des (1-3) IGF
-I (Proc. Natl. Acad. Sci. USA 83,
4904-4907, 1986). IGF-IR is expressed in various tissues including the brain. Therefore, IGF-I and des (1
-3) IGF-I can act as an in vivo defense mechanism through IGF-IR. V in APP
For patients with a mutation of 642, such mutations present in APP throughout life induce acute neurotoxicity in vitro (EMBO J. 15, 498-509, 1996, Science 27
2, 1349-1352, 1996, EMBO J. 16, 4897-4907, 1997, B
iochem. Biophys. Res. Comm. 274, 445-454, 2000, J.
Despite the fact that Biol. Chem. 275, 34541-34551, 2000), AD develops around the age of 50 (Ann. NY Ac)
ad. Sci. 695, 198-202, 1993). If simply interpreted based on the antagonism of IGF-I and IGF-IR, neurotoxicity does not occur while the defense system is functioning,
When I was in my 50s and IGF-I function was impaired, AP
In individuals in whom V642 in P is mutated, AD may develop clinically. In support of this, the biological activity of plasma IGF-I gradually decreases with age (Johns. Hopkins. Med. J. 149, 115-117,
1981, J. Clin. Invest. 67, 1361-1369, 1981, J. Ger
ontology 40, 2-7, 1985, J. Clin. Invest. 86, 952-96
1, 1990). Also, an abnormal decrease in IGF-I production,
May increase or increase the risk of developing FAD. Mustafa et al., Who agreed with this idea,
It was reported that reduced production of GF-I was associated with FAD caused by the K595N / M596L mutant APP (Geriatr. Cogn. Disord. 10, 446-451, 1999).

This example also recognizes that IGF-I, as previously discussed by Qurion et al. (Pharm. Acta. Helv. 74, 273-280, 2000), is the basis for developing therapies for AD. It also suggests that it could be done. The observed inhibition by IGFBP-3 of the protective effect of IGF-I suggests that the potential usefulness of IGF-I itself may be limited.
Several IGFBPs, including IGFBP-3 as a major one, are circulating in plasma. IG
FBPs are also produced locally. Thus, intravenously administered IGF-I is captured by IGFBPs,
As a result, its cytoprotective effect may be weakened. However, this example further focuses on the fact that IGF-I in an unbound form may be an effective alternative to therapeutic dosages. While this idea is appealing, it should be emphasized that the non-IGFBP bound form of IGF-I has only a short half-life in the body (Am. J. Physiol. 271, E649-657, 199
6). There is another potential therapeutic treatment that has the ability to avoid suppression by IGFBP. It is IGF
-V922E that functions independently of I and IGF-IR-
Virus-mediated transfer of IGF-IR. In addition, V9
22E-IGF-IR has an additional advantage over IGF-I. As a major effect, IGF-I stimulates cells to proliferate in various tissues. IGF
Overproduction of -I causes gigantism and acromegaly due to overgrowth of skeletal tissue. Furthermore, IGF-I is found in the adult nervous system in neuronal progenitors (J. Neurosci. 20,
2896-2903, 2000) and astrocytic brain tumors (J. Neuroonco
l, 35, 315-325, 1997).
In contrast, V922E-IGF-IR does not stimulate cells to proliferate, but instead works on glucose uptake and cytoprotection (J. Biol. Chem. 19041-19045, 1995; and this example). Therefore, V922E-IGF-I
R gene transfer is more relevant with the goal of selectively suppressing cell death by minimizing unwanted effects of IGF-I. The present study, thus,
Provides a molecular clue not only for understanding pathologically but also for establishing a powerful treatment for AD.

[0041]

According to the present invention, AD can be understood pathologically and pathologically, and a powerful treatment for AD can be established. In particular, the IGF-I, I
GF-IR, des (1-3) IGF-I, V922E
-A prophylactic / therapeutic agent for Alzheimer's disease such as IGF-IR is useful for Alzheimer's disease, particularly familial Alzheimer's disease. In addition, such therapeutic agents can be expected to be applied to gene therapy and the like.

[0042]

[Sequence List] SEQUENCE LISTING <110> KEIO UNIVERSITY <120> Drugs of Alzheimer's disease <130> 000000296 <140> <141> <150> JP 2001/65721 <151> 2001-03-08 <160> 11 <170> PatentIn Ver. 2.1 <210> 1 <211> 153 <212> PRT <213> Homo sapiens <400> 1 Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe 1 5 10 15 Cys Asp Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His Leu 20 25 30 Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala 35 40 45 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 50 55 60 Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly 65 70 75 80 Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys 85 90 95 Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 100 105 110 Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp 115 120 125 Met Pro Lys Thr Gln Lys Glu Val His Leu Lys Asn Ala Ser Arg Gly 130 135 140 Ser Ala Gly Asn Lys Asn Tyr Arg Met 145 150 <210> 2 <211> 1337 <212> PRT <213> Homo sap iens <400> 2 Glu Ile Cys Gly Pro Gly Ile Asp Ile Arg Asn Asp Tyr Gln Gln Leu 1 5 10 15 Lys Arg Leu Glu Asn Cys Thr Val Ile Glu Gly Tyr Leu His Ile Leu 20 25 30 Leu Ile Ser Lys Ala Glu Asp Tyr Arg Ser Tyr Arg Phe Pro Lys Leu 35 40 45 Thr Val Ile Thr Glu Tyr Leu Leu Leu Phe Arg Val Ala Gly Leu Glu 50 55 60 Ser Leu Gly Asp Leu Phe Pro Asn Leu Thr Val Ile Arg Gly Trp Lys 65 70 75 80 Leu Phe Tyr Asn Tyr Ala Leu Val Ile Phe Glu Met Thr Asn Leu Lys 85 90 95 Asp Ile Gly Leu Tyr Asn Leu Arg Asn Ile Thr Arg Gly Ala Ile Arg 100 105 110 Ile Glu Lys Asn Ala Asp Leu Cys Tyr Leu Ser Thr Val Asp Trp Ser 115 120 125 Leu Ile Leu Asp Ala Val Ser Asn Asn Tyr Ile Val Gly Asn Lys Pro 130 135 140 Pro Lys Glu Cys Gly Asp Leu Cys Pro Gly Thr Met Glu Glu Lys Pro 145 150 155 160 Met Cys Glu Lys Thr Thr Ile Asn Asn Glu Tyr Asn Tyr Arg Cys Trp 165 170 175 Thr Thr Asn Arg Cys Gln Lys Met Cys Pro Ser Thr Cys Gly Lys Arg 180 185 190 Ala Cys Thr Glu Asn Asn Glu Cys Cys His Pro Glu Cys Leu Gly Ser 195 200 205 Cys Ser Ala Pro Asp Asn Asp Thr Ala Cys Val Ala Cys Arg His Tyr 210 215 220 Tyr Tyr Ala Gly Val Cys Val Pro Ala Cys Pro Pro Asn Thr Tyr Arg 225 230 235 240 Phe Glu Gly Trp Arg Cys Val Asp Arg Asp Phe Cys Ala Asn Ile Leu 245 250 255 Ser Ala Glu Ser Ser Asp Ser Glu Gly Phe Val Ile His Asp Gly Glu 260 265 270 Cys Met Gln Glu Cys Pro Ser Gly Phe Ile Arg Asn Gly Ser Gln Ser 275 280 285 285 Met Tyr Cys Ile Pro Cys Glu Gly Pro Cys Pro Lys Val Cys Glu Glu 290 295 300 Glu Lys Lys Thr Lys Thr Ile Asp Ser Val Thr Ser Ala Gln Met Leu 305 310 315 320 Gln Gly Cys Thr Ile Phe Lys Gly Asn Leu Leu Ile Asn Ile Arg Arg 325 330 335 Gly Asn Asn Ile Ala Ser Glu Leu Glu Asn Phe Met Gly Leu Ile Glu 340 345 350 Val Val Thr Gly Tyr Val Lys Ile Arg His Ser His Ala Leu Val Ser 355 360 365 Leu Ser Phe Leu Lys Asn Leu Arg Leu Ile Leu Gly Glu Glu Gln Leu 370 375 380 Glu Gly Asn Tyr Ser Phe Tyr Val Leu Asp Asn Gln Asn Leu Gln Gln 385 390 395 400 Leu Trp Asp Trp Asp His Arg Asn Leu Thrile Ile Lys Ala Gly Lys Met 405 410 415 Tyr PheAla Phe Asn Pro Lys Leu Cys Val Ser Glu Ile Tyr Arg Met 420 425 430 Glu Glu Val Thr Gly Thr Lys Gly Arg Gln Ser Lys Gly Asp Ile Asn 435 440 445 445 Thr Arg Asn Asn Gly Glu Arg Ala Ser Cys Glu Ser Asp Val Leu His 450 455 460 Phe Thr Ser Thr Thr Thr Ser Lys Asn Arg Ile Ile Ile Thr Trp His 465 470 475 480 Arg Tyr Arg Pro Pro Asp Tyr Arg Asp Leu Ile Ser Phe Thr Val Tyr 485 490 495 Tyr Lys Glu Ala Pro Phe Lys Asn Val Thr Glu Tyr Asp Gly Gln Asp 500 505 510 Ala Cys Gly Ser Asn Ser Trp Asn Met Val Asp Val Asp Leu Pro Pro 515 520 525 Asn Lys Asp Val Glu Pro Gly Ile Leu Leu His Gly Leu Lys Pro Trp 530 535 540 Thr Gln Tyr Ala Val Tyr Val Lys Ala Val Thr Leu Thr Met Val Glu 545 550 555 560 Asn Asp His Ile Arg Gly Ala Lys Ser Glu Ile Leu Tyr Ile Arg Thr 565 570 575 Asn Ala Ser Val Pro Ser Ile Pro Leu Asp Val Leu Ser Ala Ser Asn 580 585 590 Ser Ser Ser Gln Leu Ile Val Lys Trp Asn Pro Pro Ser Leu Pro Asn 595 600 605 Gly Asn Leu Ser Tyr Tyr Ile Val Arg Trp Gln Arg Gln Pro Gln Asp 610 615 620 Gly Tyr Leu Tyr Arg His Asn Tyr Cys Ser Lys Asp Lys Ile Pro Ile 625 630 635 640 Arg Lys Tyr Ala Asp Gly Thr Ile Asp Ile Glu Glu Val Thr Glu Asn 645 650 655 Pro Lys Thr Glu Val Cys Gly Gly Glu Lys Gly Pro Cys Cys Ala Cys 660 665 670 Pro Lys Thr Glu Ala Glu Lys Gln Ala Glu Lys Glu Glu Ala Glu Tyr 675 680 685 Arg Lys Val Phe Glu Asn Phe Leu His Asn Ser Ile Phe Val Pro Arg 690 695 700 Pro Glu Arg Lys Arg Arg Asp Val Met Gln Val Ala Asn Thr Thr Met 705 710 710 715 720 Ser Ser Arg Ser Arg Asn Thr Thr Ala Ala Asp Thr Tyr Asn Ile Thr 725 730 735 Asp Pro Glu Glu Leu Glu Thr Glu Tyr Pro Phe Phe Glu Ser Arg Val 740 745 750 Asp Asn Lys Glu Arg Thr Val Ile Ser Asn Leu Arg Pro Phe Thr Leu 755 760 765 Tyr Arg Ile Asp Ile His Ser Cys Asn His Glu Ala Glu Lys Leu Gly 770 775 780 Cys Ser Ala Ser Asn Phe Val Phe Ala Arg Thr Met Pro Ala Glu Gly 785 790 795 800 Ala Asp Asp Ile Pro Gly Pro Val Thr Trp Glu Pro Arg Pro Glu Asn 805 810 815 Ser Ile Phe Leu Lys Trp Pro Glu Pro Glu Asn Pro Asn Gly Leu Ile 820 825 830 Leu Met Tyr Glu Ile Lys Tyr Gly Ser Gln Val Glu Asp Gln Arg Glu 835 840 845 Cys Val Ser Arg Gln Glu Tyr Arg Lys Tyr Gly Gly Ala Lys Leu Asn 850 855 860 Arg Leu Asn Pro Gly Asn Tyr Thr Ala Arg Ile Gln Ala Thr Ser Leu 865 870 875 880 880 Ser Gly Asn Gly Ser Trp Thr Asp Pro Val Phe Phe Tyr Val Gln Ala 885 890 895 Lys Thr Gly Tyr Glu Asn Phe Ile His Leu Ile Ile Ala Leu Pro Val 900 905 910 Ala Val Leu Leu Ile Val Gly Gly Leu Val Ile Met Leu Tyr Val Phe 915 920 925 His Arg Lys Arg Asn Asn Ser Arg Leu Gly Asn Gly Val Leu Tyr Ala 930 935 940 940 Ser Val Asn Pro Glu Tyr Phe Ser Ala Ala Asp Val Tyr Val Pro Asp 945 950 955 960 Glu Trp Glu Val Ala Arg Glu Lys Ile Thr Met Ser Arg Glu Leu Gly 965 970 975 Gln Gly Ser Phe Gly Met Val Tyr Glu Gly Val Ala Lys Gly Val Val 980 985 990 Lys Asp Glu Pro Glu Thr Arg Val Ala Ile Lys Thr Val Asn Glu Ala 995 1000 1005 Ala Ser Met Arg Glu Arg Ile Glu Phe Leu Asn Glu Ala Ser Val Met 1010 1015 1020 Lys Glu Phe Asn Cys His His Val Val Arg Leu Leu Gly Val Val Ser 1025 1030 1035 1040 G ln Gly Gln Pro Thr Leu Val Ile Met Glu Leu Met Thr Arg Gly Asp 1045 1050 1055 Leu Lys Ser Tyr Leu Arg Ser Leu Arg Pro Glu Met Glu Asn Asn Pro 1060 1065 1070 Val Leu Ala Pro Pro Ser Leu Ser Lys Met Ile Gln Met Ala Gly Glu 1075 1080 1085 Ile Ala Asp Gly Met Ala Tyr Leu Asn Ala Asn Lys Phe Val His Arg 1090 1095 1100 Asp Leu Ala Ala Arg Asn Cys Met Val Ala Glu Asp Phe Thr Val Lys 1105 1110 1115 1120 Ile Gly Asp Phe Gly Met Thr Arg Asp Ile Tyr Glu Thr Asp Tyr Tyr 1125 1130 1135 Arg Lys Gly Gly Lys Gly Leu Leu Pro Val Arg Trp Met Ser Pro Glu 1140 1145 1150 Ser Leu Lys Asp Gly Val Phe Thr Thr Tyr Ser Asp Val Trp Ser Phe 1155 1160 1165 Gly Val Val Leu Trp Glu Ile Ala Thr Leu Ala Glu Gln Pro Tyr Gln 1170 1175 1180 Gly Leu Ser Asn Glu Gln Val Leu Arg Phe Val Met Glu Gly Gly Leu 1185 1190 1195 1200 Leu Asp Lys Pro Asp Asn Cys Pro Asp Met Leu Phe Glu Leu Met Arg 1205 1210 1215 Met Cys Trp Gln Tyr Asn Pro Lys Met Arg Pro Ser Phe Leu Glu Ile 1220 1225 1230 Ile Ser Ser Ile Lys Glu Glu Glu Met Glu Pro Gly Phe Arg Glu Val Ser 1235 1240 1245 Phe Tyr Tyr Ser Glu Glu Asn Lys Leu Pro Glu Pro Glu Glu Leu Asp 1250 1255 1260 Leu Glu Pro Glu Asn Met Glu Ser Val Pro Leu Asp Pro Ser Ala Ser 1265 1270 1275 1280 Ser Ser Ser Leu Pro Leu Pro Asp Arg His Ser Gly His Lys Ala Glu 1285 1290 1295 Asn Gly Pro Gly Pro Gly Val Leu Val Leu Arg Ala Ser Phe Asp Glu 1300 1305 1310 Arg Gln Pro Tyr Ala His Met Asn Gly Gly Arg Lys Asn Glu Arg Ala 1315 1320 1325 Leu Pro Leu Pro Gln Ser Ser Thr Cys 1330 1335 <210> 3 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer 1 <400> 3 gatggaaggt gattgagtct gtgagctctg acggccatga gtacatct 48 <210> 4 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer 2 <400> 4 ctaccttcca ctaactcaga cactcgagac tgccggtact catgtaga 48 <210> 5 < 211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer 3 <400> 5 gcgatatggt gatgaggagc tgca 24 <210> 6 <211> 24 <212> D NA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer 4 <400> 6 ggtctctgcc tcacccttga tgat 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer 5 <400> 7 acagagtacc ctttctttga gagc 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer 6 <400> 8 aagaacacag gatctgtcca cgac 24 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer 7 <400> 9 tccaccaccc tgttgctgta 20 <210> 10 <211> 20 <212 > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer 8 <400> 10 accacagtcc atgccatcac 20 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer 9 <400> 11 attattcagg cctcacgtgg tacagaactg 30

[Brief description of the drawings]

FIG. 1 shows the effect of growth factors on cell death by V642I-APP in NK1 cells.

FIG. 2 shows that IGFBP-3 inhibits IGF-I cell death.
It is a figure showing the effect of.

FIG. 3 is a graph showing the results of a cell death inhibitory effect of des (1-3) IGF-I.

FIG. 4. I against neurotoxicity due to V642I-APP.
It is a figure which shows the effect of GF-I and des (1-3) IGF-I.

FIG. 5 is a diagram showing the involvement of IGF-IR in the inhibitory effect of IGF-I on cell death on V642I-APP.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) A61P 43/00 105 A61K 37/36 // C07K 14/62 ZNA 37/02 C12N 15/09 C12N 15/00 A F term (Reference) 4B024 AA01 BA02 CA04 EA02 EA04 HA17 4C084 AA02 AA03 AA07 AA13 AA17 BA35 CA01 CA53 DB58 DB63 MA13 MA17 MA22 MA23 MA35 MA37 MA41 MA43 MA52 MA55 MA59 MA66 NA14 ZA162 ZB212 4C087 AA01 AA02 BC23 MA23 MA52 MA55 MA59 MA66 NA14 ZA16 ZB21 ZC41 4H045 AA30 BA10 CA40 DA38 EA20 FA74

Claims (9)

[Claims] 1. Insulin-like growth factor I (IGF-
I) A prophylactic / therapeutic agent for familial Alzheimer's disease, comprising a protein as an active ingredient. 2. A protein consisting of the amino acid sequence shown in SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added,
A prophylactic / therapeutic agent for familial Alzheimer's disease, comprising a protein having an activity of suppressing cell death caused by familial Alzheimer's disease as an active ingredient. 3. A protein comprising the amino acid sequence shown in SEQ ID NO: 1, comprising one or several amino acids deleted, substituted or added,
And a protein having an activity of suppressing cell death caused by familial Alzheimer's disease is des (1-3) IGF-I
The preventive / therapeutic agent for familial Alzheimer's disease according to claim 2, characterized in that: 4. A prophylactic / therapeutic agent for familial Alzheimer's disease, comprising a vector capable of expressing the protein according to claim 1 as an active ingredient. 5. An insulin-like growth factor I receptor (IG)
An agent for preventing or treating Alzheimer's disease, which comprises an F-IR) protein as an active ingredient. 6. A protein comprising an amino acid sequence represented by SEQ ID NO: 2, wherein the protein comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added,
A prophylactic / therapeutic agent for Alzheimer's disease, comprising a protein having an activity of suppressing cell death caused by Alzheimer's disease as an active ingredient. 7. A protein consisting of the amino acid sequence shown in SEQ ID NO: 2, comprising one or several amino acids deleted, substituted or added,
7. The prophylactic / therapeutic agent for Alzheimer's disease according to claim 6, wherein the protein having an activity of suppressing cell death caused by Alzheimer's disease is V922E-IGF-IR. 8. A prophylactic / therapeutic agent for familial Alzheimer's disease, comprising a vector capable of expressing the protein according to claim 5 as an active ingredient. 9. The preventive / therapeutic agent for Alzheimer's disease according to claim 5, wherein the Alzheimer's disease is familial Alzheimer's disease.