The present invention provides polypeptides that suppresses neuronal death associated with Alzheimer's disease. Using a neuronal cell system, wherein the expression of familial Alzheimer's disease mutant APP can be induced by ecdysone treatment, a gene that protects the neurons from cell death was successfully isolated. The gene encodes a secretory polypeptide consisting of 24 amino acids, and this polypeptide suppresses neuronal death caused by the expression of APP mutants and presenilin mutants. The polypeptide also suppressed cell death of primary neuronal culture caused by A.beta.. Furthermore, by mutating the amino acids of the polypeptide, the neuronal death suppression activity of the polypwptide was successfully and significantly enhanced. These polypeptides and derivatives thereof are useful as pharmaceuticals to prevent neuronal death associated with Alzheimer's disease, and as seed compounds for developing novel pharmaceuticals for Alzheimer's disease.

Description of the Invention

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide polypeptides that protect neurons from cell death associated with Alzheimer's disease, and use of the same.

The present inventor has previously established a nerve cell line (F11/EcR/V642I), which inductively expresses familial Alzheimer's disease-type mutant V642I amyloid precursor proteins (V642I APP) (see International Publication No. WO 00/14204). According to the system, V642I APP is expressed in F11 neurons in response to ecdysone treatment. Cell death occurred in almost all of the F11/EcR/V642I cells incubated with ecdysone for 2 to 3 days; whereas cell death occurred in only a few cells in the control incubation. The present inventor used the F11/EcR/V642I cells to search for genes that act as antagonists of V642I APP-induced neuronal death.

More specifically, a cDNA library was constructed from the brain of Alzheimer's disease (AD) patient, and was transfected into the F11/EcR/V642I cells mentioned above. Then a death trap screening operation was repeatedly performed to select cells that survived neuronal death induced by V642I APP. As a result, the present inventor succeeded in identifying a novel gene that protect cells against neuronal death induced by V642I APP. It was revealed that the clone, dubbed Humanin (HN) cDNA, encoding a novel polypeptide of 24-amino acids, suppresses neuronal death associated with AD. That is, the clone suppressed neuronal death induced by all of the known types of early-onset familial AD genes [V642I APP, K595N/M596L APP, M146L presenilin (PS)-1, and N1411 PS-2] and by A.beta.1-43. In contrast, the clone had no effect on neurotoxicity of polyglutamine repeat Q79, associated with Huntington's disease (HD)/spinocerebellar ataxia (SCA); and mutants of Cu/Zn-dependent superoxide dismutase (SOD1), associated with amyotrophic lateral sclerosis (ALS). HN mRNA was mainly produced in several organs other than the central nervous system. Transfection of HN cDNA into neurons led to transcription and production of expected peptides, which peptides were secreted into the culture medium up to a level of about 10 .mu.M. The culture supernatant was enough active to demonstrate significant protection of cells from neuronal death induced by V642I APP. Synthetic HN polypeptide also showed neuroprotective action with similar dose-response properties against the four types of AD genes, and its suppression was maximal at 1 to 10 .mu.M. Polypeptides expressed within neurons from a cDNA encoding an HN derivative, lacking secretion ability, failed to protect neurons from cell death. However, the same polypeptide synthesized and added to the culture medium showed protective action, which results indicate that the HN polypeptide acts from outside of the cell. Cys at position 8 and Ser at position 14 were found to be important according to an experiment detecting the activity of polypeptides with modified structure. C8A substitution completely deprived the polypeptide of the cell death rescue activity. On the other hand, S14G substitution remarkably enhanced the rescue activity of the polypeptide. S14G HN polypeptides (HNG) showed complete protective action against all of the four types of FAD genes at low nanomolar concentrations (1 to 10 nM). Anti-AD activity of HN was also observed in primary cultured cortical neurons. Specifically, .mu.M levels of HN and nM levels of S14G derivatives (HNG) protected cells form cell death and cell damage caused by A.beta., whereas C8A (HNA) lacked such activity. Furthermore, upon analysis of detailed structure-function relationship, amino acids from Pro at position 3 to Pro at position 19 were identified to be important for neuroprotective function, and among them, seven residues were identified as essential residues for the activity. In addition, C8 amino acid of S14G HN polypeptide (HNG) could be substituted with basic amino acids, such as His, Arg, or Lys, while maintaining its anti-AD activity. Furthermore, the present inventor succeeded to further enhance the neuroprotective action by introducing two amino acid mutations to the S14G HN polypeptide. Based on these findings, it is possible to develop polypeptides that have higher activity and which are suited for biological administration. These polypeptides open a new path to develop therapeutic drugs for AD, and at the same time, are expected to contribute greatly to the development of AD therapy aiming at protection of neurons from cell death.

The present invention relates to novel polypeptides that protect cells from neuronal death associated with AD, and use of the same. More specifically, the present invention relates to:

(1) a polypeptide that suppresses neuronal death associated with Alzheimer's disease having an amino acid sequence of Formula (I):

Pro-Xn.sub.1-(Cys/bXaa)-(Leu/Arg)-Xn.sub.2-Leu-Thr-(Gly/Ser)-Xn.sub.3-Pro (I) wherein "Cys/bXaa" indicates Cys or a basic amino acid; "(Leu/Arg)" indicates Leu or Arg; "(Gly/Ser)" indicates Gly or Ser; and Xn.sub.1, Xn.sub.2, and Xn.sub.3 independently indicate arbitrary amino acid sequences not more than 10 residues in length, respectively;

(2) a polypeptide according to (a) or (b) shown below: (a) a polypeptide having an amino acid sequence selected from the group of SEQ ID NOs: 5 to 8, 10, 12, 13, 21 to 24, 26 to 29, 32, 33, 37 to 40, 46, 48, 54, and 60; (b) a polypeptide that suppresses neuronal death associated with Alzheimer's disease having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 8, 10, 12, 13, 21 to 24, 26 to 29, 32, 33, 37 to 40, 46, 48, 54, and 60, wherein one or more amino acids have been substituted, deleted, inserted, and/or added;

(3) the polypeptide of (2), which is used to suppress neuronal death;

(4) a fusion polypeptide comprising the polypeptide of any of (1) to (2) fused with other polypeptides;

(5) a DNA encoding the polypeptide of any one of (1) to (4);

(6) a vector into which the DNA of (5) is inserted;

(7) a host cell retaining the vector of (6);

(8) a method for producing the polypeptide of any one of (1) to (4), comprising the steps of culturing the host cell of (7), and recovering the expressed polypeptide from the host cell or culture supernatant thereof;

(9) a method for suppressing neuronal death comprising the step of contacting a neuron with the polypeptide of any one of (1) to (4);

(10) a method for detecting a cell death suppressing activity of the polypeptide of any one of (1) to (4), comprising the steps of: (a) inducing cell death in the presence of the polypeptide of any one of (1) to (4); and (b) detecting level of cell death;

(11) a method for detecting the effect of a chemical compound on neuronal death suppressing activity of a polypeptide of any one of (1) to (4), comprising the steps of: (a) inducing neuronal death in the presence of a test compound and the polypeptide of any one of (1) to (4); and (b) detecting the level of neuronal death;

(12) a method of screening for a chemical compound that regulates the neuronal death suppressing activity of the polypeptide of any one of (1) to (4), comprising the steps of: (a) inducing neuronal death in the presence of a test sample and the polypeptide of any one of (1) to (4); (b) detecting the level of neuronal death; and (c) selecting the compound that enhances or suppresses neuronal death;

(13) a pharmaceutical composition comprising as the effective component the polypeptide of any one of (1) to (4);

(14) the pharmaceutical composition of (13), wherein said composition is a neuronal death suppressant;

(15) the pharmaceutical composition of (13), which is used to prevent or treat diseases that are accompanied by neurodegeneration;

(16) the pharmaceutical composition of (13), which is used to prevent or treat Alzheimer's disease;

(17) an antibody that binds to the polypeptide of any one of (1) to (3);

(18) a DNA for detecting or manipulating DNA encoding the polypeptide of any one of (1) to (4), wherein the DNA comprises at least 15 nucleotides that are complementary to a DNA consisting of the nucleotide sequence of SEQ ID NO: 4 or to a complementary strand thereof; and

(19) a method of screening for a chemical compound that binds to the polypeptide of any one of (1) to (4), comprising the steps of: (a) contacting a test sample with the polypeptide of any one of (1) to (4); (b) detecting the binding activity between the test sample and the polypeptide; and (c) selecting the compound that has the activity to bind to the polypeptide.

The term "polypeptide" herein refers to a peptide or protein consisting of two or more amino acids or amino acid derivatives bound to each other. Peptide isosteres are included as polypeptides of the present invention. The term "polypeptide" normally includes short stranded polypeptides, such as peptides, oligopeptides, and oligomers. It also includes long stranded polypeptides such as proteins. The polypeptide may be naturally modified by post-translational modification and such. They may be also modified by artificial modifications. Modification includes modification of the peptide backbone, amino acid side chain, amino terminus, or carboxyl terminus. The polypeptide may be branched or cyclic. Modification includes acetylation; acylation; ADP ribosylation; amidation; covalent bonding with flavin, nucleotide, nucleotide derivative, lipid, lipid derivative, or phosphatidyl inositol, and such; cross link formulation; cyclization; disulfide bond formation; demethylation; pyroglutamylation; .gamma.-carboxylation; glycosylation; hydroxylation; iodization; methylation; myristoylation; oxidation; phosphorylation; ubiquitination; and so on, but the present invention is not limited to these examples.

The present invention provides polypeptides which protect neurons from cell death associated with Alzheimer's disease. The amino acid sequence of Humanin (HN) polypeptide, isolated by the present inventor, is indicated in SEQ ID NO: 5, and the cDNA sequence of the open reading frame encoding the polypeptide is indicated in SEQ ID NO: 4. Humanin antagonizes neuronal death associated with AD, and shows a saturation activity at a concentration of about 10 .mu.M. In addition, HNG (S14G) (SEQ ID NO: 8), which is a Humanin with an amino acid substitution, showed 100 to 1000 fold higher antagonizing effect compared to Humanin. Further, Cys at position 8 of HNG can be substituted with basic amino acids, such as His, Arg, and Lys, withoutd changes in the activity by the modification of the SH group of the polypeptide. Furthermore, AGA-HNG (SEQ ID NO: 60) which is a derivative of HNG demonstrated an activity several folds higher than that of HNG. The polypeptides of the present invention include Humanin, HNG, AGA-HNG, and substituted forms thereof wherein the Cys residue (referred to as C8) is substituted with a basic amino acid.

Further, the present invention demonstrated that addition of a FLAG tag (DYKDDDDK) (SEQ ID NO: 61) to the C-terminus of Humanin does not affect the neuroprotective action thereof (Example 3). Furthermore, even when the four C-terminal amino acids (KRRA) (SEQ ID NO: 62) of Humanin were substituted with other amino acids, a neuroprotective action equivalent to that of the original Humanin was present in the substituted polypeptide ( Example 6). These facts demonstrate that polypeptides with equivalent or higher neuroprotective action to can be prepared by introducing mutations to the amino acid sequence of Humanin, HNG, AGA-HNG, and substituted forms thereof, wherein the C8 is substituted with a basic amino acid.

The present inventor performed further detailed analysis using deletion mutants of Humanin, and found that even a polypeptide, consisting of 17 amino acids from position 3 to 19 of Humanin (HN-17, SEQ ID NO: 21), is sufficient to protect neurons (Example 13). Furthermore, half or more of the amino acid residues of HNG can be substituted while retaining the activity according to the verification of the neuronal death suppression activity of polypeptides consisting of the 3rd Pro to the 19th Pro of HNG (HNG-17, SEQ ID NO: 24), wherein each amino acid residue of the polypeptide was substituted with another amino acid. This experiment elucidated that 7 amino acids in HNG-17 are essential for neuronal death suppression: Pro at position 1; Cys at position 6; Leu at position 7; Leu-Thr-Gly at positions 10 to 12; and Pro at position 17. Therefore, polypeptides having different amino acid sequences can be prepared by modification, such as substitution, deletion, and/or insertion, of residues other than those described above and retaining the residues above.

What is much more important, is that even the 7 amino acids, mentioned above as essential amino acids for the activity, may be also substituted with other amino acids. For example, the polypeptides retains the neuroprotective activity even when the Gly at position 12 of NHG-17 is Ser (that is NH-17. In addition, a synthetic polypeptide (HNR; SEQ ID NO: 7) wherein the position corresponding to the Leu at position 7 is substituted with Arg, demonstrated a neuroprotective activity similar to that of the synthetic HN (Example 12). Furthermore, as mentioned above, an HNG with basic amino acid, such as His, Arg, or Lys, for the Cys corresponding to position 6 of the HNG-17 shows neuroprotective activity. Particularly, the HNG mutant wherein the Cys is substituted with Arg or Lys demonstrated a neuroprotective activity equivalent to that of the original HNG (Example 14). According to these facts, preparation of polypeptides with equivalent or higher neuronal death suppression activity to the polypeptides, which activities were detected in the Examples of the present invention, are expected by mutating amino acids that are non-essential and/or essential for the activity of these polypeptides.

The polypeptide of the present invention includes polypeptides that suppress neuronal death associated with Alzheimer's disease (AD) and having an amino acid sequence consisting of Formula (I): Pro-Xn.sub.1-(Cys/bXaa)-(Leu/Arg)-Xn.sub.2-Leu-Thr-(Gly/Ser)-Xn.sub.3-Pro (1) (SEQ ID NO: 63). Herein, "Cys/bXaa" indicates Cys or a basic amino acid; "(Leu/Arg)" indicates Leu or Arg; "(Gly/Ser)" indicates Gly or Ser; and Xn.sub.1, Xn.sub.2, and Xn.sub.3 independently indicate arbitrary amino acids not more than 10 residues, respectively. A polypeptide that has the amino acid sequence as above may be also expressed as: Pro-(Xaa).sub.1-10-(Cys/bXaa)-(Leu/Arg)-(Xaa).sub.1-10-Leu-Thr-(Gly/Ser)-- (Xaa).sub.1-10Pro (SEQ ID NO: 64) (II) (wherein Xaa indicates an arbitrary amino acid; "(Xaa).sub.m-n" indicates m to n residues of arbitrary amino acids; "bXaa" indicates a basic amino acid; "Cys/bXaa" indicates Cys or a basic amino acid; "(Leu/Arg)" indicates Leu or Arg; and "(Gly/Ser)" indicates Gly or Ser).

Basic amino acids refer to amino acids in which its R group (side chain) is positively charged at pH7.0. Examples of natural basic amino acids include Arg, Lys, and His. The amino acid sequences of a polypeptide of this invention that has Arg, Lys, or His as the basic amino acids can be represented, for example, as: Pro-Xn.sub.1-(Cys/Arg/Lys/His)-(Leu/Arg)-Xn.sub.2-Leu-Thr-(Gly/Ser)-Xn.su- b.3-Pro (SEQ ID NO: 65) (III) (wherein "(Cys/Arg/Lys/His)" indicates Cys, Arg, Lys, or His; "(Leu/Arg)" indicates Leu or Arg; "(Gly/Ser)" indicates Gly or Ser; and Xn.sub.1, Xn.sub.2, and X.sub.n3 independently indicate arbitrary amino acids not more than 10 residues, respectively). Herein, Arg and Lys are particularly preferable as the basic amino acid at this position.

Preferably, Xn.sub.1, Xn.sub.2, and Xn.sub.3 are independently arbitrary amino acids of 2 to 6, 0 to 4, and 2 to 6 residues, respectively (that is, Xn.sub.1=(Xaa).sub.2-6, Xn.sub.2=(Xaa).sub.0-4, and Xn.sub.3=(Xaa).sub.2-6); more preferably 3 to 5, 1 to 3, and 3 to 5 residues, respectively (that is, Xn.sub.1=(Xaa).sub.3-5, Xn.sub.2=(Xaa).sub.1-3, and Xn.sub.3=(Xaa).sub.3-5); and most preferably 4, 2, and 4 residues, respectively (that is, Xn.sub.1=(Xaa).sub.4, Xn.sub.2=(Xaa).sub.2, and Xn.sub.3=(Xaa).sub.4). Added amino acids of about 6 residues sometimes forms an .alpha.-helix and behaves like a single amino acid residue. A polypeptide of the present invention may be a polypeptide wherein arbitrary amino acids with no more than 6 residues are added to all or any one of Xn.sub.1, Xn.sub.2, and Xn.sub.3 consisting of arbitrary amino acids of 4 residues, 2 residues, and 4 residues, respectively.

Such polypeptide may be prepared according to known peptide synthesis techniques, and also by the expression of a DNA that encodes such polypeptides.

Preferably, the sequence of Xn.sub.1 includes, for example, sequences consisting of (Arg/Ala)-(Gly/Ala)-(Phe/Ala)-(Ser/Ala) (SEQ ID NO: 66), and sequences with conservative substitution thereof. Herein, for example, "Arg/Ala" indicates Arg or Ala ("/" indicates that it is either one of the residues; the same is indicated throughout the description herein). Examples of such sequences include Arg-Gly-Phe-Ser (SEQ ID NO: 67), Ala-Gly-Phe-Ser (SEQ ID NO: 68), Arg-Ala-Phe-Ser (SEQ ID NO: 69), Arg-Gly-Ala-Ser (SEQ ID NO: 70), Arg-Gly-Phe-Ala (SEQ ID NO: 71), and so on. Other examples include Arg-Gly-Ala-Ala (SEQ ID NO: 72), Arg-Ala-Phe-Ala (SEQ ID NO: 73), Arg-Ala-Ala-Ser (SEQ ID NO: 74), Arg-Ala-Ala-Ala (SEQ ID NO: 75), Ala-Gly-Phe-Ala (SEQ ID NO: 76), Ala-Gly-Ala-Ser (SEQ ID NO: 77), Ala-Gly-Ala-Ala (SEQ ID NO: 78), Ala-Ala-Phe-Ser (SEQ ID NO: 79), Ala-Ala-Phe-Ala (SEQ ID NO: 80), Ala-Ala-Ala-Ser (SEQ ID NO: 81), Ala-Ala-Ala-Ala (SEQ ID NO: 82), and such. Conservative substitution can be exemplified by substitution within a group of amino acids, corresponding to conservative substitution, which will be described later. On the other hand, the sequence of Xn.sub.2 preferably includes, for example, sequences consisting of (Leu/Ala)-(Leu/Ala), and sequences with conservative substitution thereof. Such sequences include Leu-Leu, Ala-Leu, Leu-Ala, and such. Ala-Ala can be also exemplified as such sequences. Furthermore, the sequence of Xn.sub.3 preferably includes, for example, sequences consisting of (Glu/Ala)-(Ile/Ala)-(Asp/Ala)-(Leu/Ala) (SEQ ID NO: 83), and sequences with conservative substitution thereof. Such examples include Glu-Ile-Asp-Leu (SEQ ID NO: 84), Ala-Ile-Asp-Leu (SEQ ID NO: 85), Glu-Ala-Asp-Leu (SEQ ID NO: 86), Glu-Ile-Ala-Leu (SEQ ID NO: 87), Glu-Ile-Asp-Ala (SEQ ID NO: 88), and so on. Other examples are Glu-Ile-Ala-Ala (SEQ ID NO: 89), Glu-Ala-Asp-Ala (SEQ ID NO: 90), Glu-Ala-Ala-Leu (SEQ ID NO: 91), Glu-Ala-Ala-Ala (SEQ ID NO: 92), Ala-Ile-Asp-Ala (SEQ ID NO: 93), Ala-Ile-Ala-Leu (SEQ ID NO: 94), Ala-Ile-Ala-Ala (SEQ ID NO: 95), Ala-Ala-Asp-Leu (SEQ ID NO: 96), Ala-Ala-Asp-Ala (SEQ ID NO: 97), Ala-Ala-Ala-Leu (SEQ ID NO: 98), Ala-Ala-Ala-Ala (SEQ ID NO: 99), and so on. The sequences of Xn.sub.1, Xn.sub.2, and Xn.sub.3 may be selected from arbitrary combinations.

Neuronal death associated with AD is induced by the expression of APP, PS-1, or PS-2 mutants (for example, V642I/F/G APP, NL-APP, M146L PS-1, and N141I PS-2) in established neuronal cell lines (for example, F11 cells) and primary neuronal cultures (for example, rat brain cortical primary culture); and also by the addition of A.beta. (for example, A.beta.1-43) to primary neuronal cultures. The term "suppression of neuronal death associated with Alzheimer's disease" herein defines to suppress at least one of the neuronal deaths associated with AD including those mentioned above. Specifically, the polypeptides of the present invention include those, that suppress at least any one of these neuronal deaths associated with AD. The suppression of cell death doesn't have to be a complete suppression so long as the suppression is significant. The activity of proteins to suppress neuronal death can be detected according to the method described in the Examples, or by other published methods (see for example, International Publication No. WO 00/14204).

More specifically, a method as follows can be exemplified: (1) transfect neurons (for example, F11 cells) with vectors expressing FAD genes, such as V642I/F/G APP, NL-APP, M146L PS-1, and N141I PS-2, alone or in combination with a vector expressing a polypeptide to be examined; (2) cultivating the cells for a defined period (for example, 72 hours); and (3) detecting level of cell death by trypan blue exclusion assay. Alternatively, a polypeptide to be examined is prepared in advance, and cell death may be measured upon transfection of FAD genes into cells in the presence or absence of the polypeptide. FAD genes may be also conditionally expressed using an inductive promoter. A polypeptide is determined to suppress neuronal death associated with AD, when the cell death under the existence of the protein is significantly decreased in comparison to those induced in the absence of the polypeptide to be examined. Additionally, other cells such as primary cultured neurons may be used, and induction of cell death can be also carried out by the addition of A.beta.. Cell death can be measured by detecting morphological changes, LDH release, or apoptosis (morphological changes of the nucleus, fragmentation of DNA, and such) in addition to trypan blue exclusion.

Further, the polypeptides of the present invention include polypeptides having an amino acid sequence selected from the group of SEQ ID NOs: 5 to 8, 10, 12, 13, 21 to 24, 26 to 29, 32, 33, 37 to 40, 46, 48, 54, and 60; and polypeptides that suppress neuronal death associated with Alzheimer's disease (AD), wherein one or more amino acids of the polypeptide selected from the group of SEQ ID NOs: 5 to 8, 10, 12, 13, 21 to 24, 26, to 29, 32, 33, 37 to 40, 46, 48, 54, and 60 have been substituted, deleted, inserted, and/or added.

Although there are no particular limitations on the number of amino acid residues to be mutated, it is considered that the number of the residues to be mutated by substitution, deletion, and/or insertion in a amino acid sequence, is generally 15 residues or less, preferably 12 residues or less, more preferably 10 residues or less, and even more preferably 8 residues or less (for example, 5 residues or less). There is no limitation on the number of the amino acids to be added, so long as the activity to suppress neuronal death associated with AD is maintained. Artificially produced amino acid sequences and naturally occurring polypeptide sequences are included in the amino acid sequence wherein the amino acids have been substituted, deleted, inserted, and/or added.

The original activity of a polypeptide is expected to be retained by artificial substitution of amino acids between amino acids with similar characteristics. The polypeptides of the present invention include polypeptides that suppress neuronal death associated with AD, having an amino acid sequence selected from the group of SEQ ID NOs: 5 to 8, 10, 12, 13, 21 to 24, 26 to 29, 32, 33, 37 to 40, 46, 48, 54, and 60 with conservative substitution to the amino acids of the polypeptides. Conservative substitutions are considered to be important for substituting amino acids essential for suppressing neuronal death (for example, the 7 amino acids essential for HNG-17, mentioned above). Such conservative substitution of amino acids is well known to those skilled in the art. Group of amino acids between which conservative substitution can be exemplified by: (1) basic amino acids (for example, lysine, arginine, and histidine); (2) acidic amino acids (for example, aspartic acid, and glutamic acid); (3) uncharged polar amino acids (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine); (4) non-polar amino acids (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan); (5) .beta. branched amino acids (for example, threonine, valine, and isoleucine); (6) aromatic amino acids (for example, tyrosine, phenylalanine, tryptophan, and histidine), and such. Alternatively, the activity to suppress neuronal death, stability, tissue localization, and such of the polypeptide can be enhanced or decreased by non-conservative substitution.

The polypeptide of this invention may be produced as a synthetic polypeptide by known peptide synthesis techniques (Japanese Biochemical Society edition, "Shin Seikagaku Jikken Koza Tanpakushitu (New Course on Biochemistry Experiments, Proteins) VI," pp. 3-74, Tokyo Kagakudojin, 1992). The method for peptide synthesis may be either solid-phase synthesis or liquid-phase synthesis. Further, polypeptides with arbitrary amino acid mutations can be prepared through the introduction of mutation to Humanin cDNA (for example, SEQ ID NO: 4) by the production of synthetic DNA or by site directed mutagenesis; and then, expressing the mutated cDNA in a host cell. There are no limitations on the number and position of the amino acids to be modified so long as the obtained polypeptide suppresses neuronal death associated with AD.

Although there is no limitation regarding the number of amino acid residues in the polypeptides of this invention, however, for example, when the polypeptide is used as a pharmaceutical composition, polypeptides of smaller molecular size are generally preferred. Absence of portions (for example, amino acid residues or functional groups) unnecessary for the activity decreases antigenicity, and non-specific interactions with other molecules can be avoided which as a result is expected to reduce unfavorable side effects. The polypeptides of the present invention consist of preferably 500 amino acid residues or less, more preferably 100 residues or less, much more preferably 50 residues or less, and even more preferably 30 residues or less. The average molecular weight of the polypeptides is preferably 60 kDa or less, more preferably 15 kDa or less, more preferably 6 kDa or less, and even more preferably 4 kDa or less.

Furthermore, the present invention relates to fusion polypeptides of the above-mentioned polypeptides of the present invention with other polypeptides. A fusion polypeptide is a polypeptide in which at least two polypeptides that are not bound in nature are joined, and can be produced by peptide synthesis, or by expressing nucleic acids wherein the polypeptide encoding regions are ligated in frame. Examples of other polypeptides that is fused to the protein of this invention include arbitrary polypeptides comprising short peptides with few residues, such as tags, and long polypeptides, such as proteins. Specifically, such examples include His tag, HA tag, GFP, maltose binding protein, and glutathione S-transferase (GST). Additionally, antibody fragments (Fc fragment), and such may be also used. Other examples include leader sequence, secretion signal, and preprotein or proprotein sequences, but the present invention is not limited to these examples. Further, a group of polypeptides, that facilitates the polypeptide of this invention to effectively pass the blood-brain barrier, can be fused to the protein of the present invention.

Furthermore, the polypeptides of this invention include salts thereof. Such salts are derived from acids or bases of the polypeptides. Specifically, such salts can be exemplified by salts formed with inorganic acids (for example, hydrochloride, phosphate, hydrobromide, hydrosulfate, nitrate, etc.); salts formed with organic acids (for example, acetate, lactate, formate, butyrate, glycolate, propionate, fumarate, maleate, succinate, tartrate, citrate, malate, oxalate, benzoate, methane sulfonate, benzene sulfonate, etc.); and salts formed with bases (for example, ammonium salt, alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and salts formed with organic bases, and salts formed with amino acids such as arginine and lysine).

Furthermore, the polypeptide of the present invention includes derivatives thereof. Herein, the term "derivatives" refers to molecules that have a form, which has been altered by modification, addition, mutation, substitution, or deletion of functional groups of the polypeptide of this invention according to conventional methods. Such alterations of functional groups are carried out, for example, to protect functional groups of the polypeptides, to regulate the stability or histological localization of the polypeptides, or to regulate the activity of the polypeptides, and so on. The polypeptides of the present invention are exemplified by those polypeptides wherein any one of the N-terminus, C-terminus, and functional groups of the polypeptides constituting amino acid side chains are modified by substituents, such as protecting groups. The substituents include, for example, various alkyl groups, acyl groups, amide groups, phosphate groups, amino groups, carboxyl groups, and ester groups; however, the present invention is not limited to these examples.

Furthermore, the polypeptides of the present invention include polymers, such as dimers wherein the polypeptides are bound to each other; branched molecules; and cyclized molecules. Further, the polypeptides may be bound to a carrier. For example, the polypeptides of this invention may be bound to polyethylene glycol (PEG), dextran, other polymers, and so on.

Amino acids that constitute the polypeptides of the present invention may be in the L form and/or D form. The use of D amino acids is effective for lowering degradation by peptidases. Additionally, the amino acids are not limited to natural amino acids, and may be also unnatural amino acids. Unnatural amino acids are exemplified by homoserine, .beta.-hydroxyvaline, 0-4-hydroxyphenyl tyrosine, .alpha.-t-butyl glycine, 2-amine butyrate, .alpha.-cyclohexyl glycine, .alpha.-phenyl glycine, and such. Further, the peptide bonds of the polypeptides may be appropriately substituted with covalent bonds other than peptide bonds. The sensitivity to peptidases of the polypeptides can be lowered by the substitution to non-peptide bonds, which enhances drug efficacy duration and which offers a wide selection of administration routes. The non-peptide bonds are exemplified by imino bonds, ester bonds, hydrazine bonds, semicarbazide bonds, and azo bonds, but the present invention is not limited to these examples.

Further, chemical compounds, that mimic the structure of the polypeptides of the present invention, may be designed. For example, based on the physical and chemical properties (which may be analyzed by conventional methods including active site modification, NMR, and X-ray crystallography) relating to the structure of the polypeptides of this invention a map of physical and chemical functions, that are important for neuroprotective action of the polypeptides, is constructed. Then, molecules that simulate these functions are designed and synthesized. Alternatively, the polypeptides of the present invention are expected to bind to a receptor due to its high activity, and thus, compounds that bind to the same receptor may be designed. Whether molecules derived in this manner possess a neuroprotective action or not can be assayed according to the method described in the Examples.

The present invention also provides DNA encoding a polypeptide of this invention. There is no particular limitation on the origin of the DNA of the present invention, and includes synthetic DNA, genomic DNA, cDNA, and such. The DNA of this invention includes a cDNA that encodes Humanin, described in SEQ ID NO: 4. A DNA having any nucleotide sequence based on the degeneracy of genetic code may be included so long as it encodes the amino acids described in SEQ ID NOs: 5 to 8, 10, 12, 13, 21 to 24, 26 to 29, 32, 33, 37 to 40, 46, 48, 54, or 60. In addition to the coding region, a DNA of the present invention may include non-coding sequences (including non-transcriptional sequence, non-translational sequence, promoter, enhancer, suppressor, transcription factor binding sequence, splicing sequence, poly(A) addition sequence, IRES, mRNA stabilizing/destabilizing sequence, and such) at the 5' and 3' ends.

The DNA of the present invention may be used to produce a polypeptide of this invention by inserting the DNA into a vector. Furthermore, it is also possible to use the DNA for application to gene therapy as described below.

The host to produce a polypeptide of the present invention is not limited in any way, and cells or unities such as Escherichia coli, yeast, mammalian cells, plant cells, insect cells, and so on may be used. The host-vector system may be, for example, the baculovirus-Sf cell line (Okamoto et al., J. Biol. Chem. 270: 4205-4208, 1995); the pcDNA-CHO cell line (Takahashi et al., J. Biol. Chem. 270:19041-19045, 1995); the CMV promoter plasmid-COS cell line (Yamatsuji et al., EMBO J. 15:498-509, 1996); and such, but are not limited thereto.

The polypeptides of the present invention may be secreted from host cells. As described in the Examples, Humanin, HNG, and such were secreted from cells, wherin the polypeptides were expressed to the extracellular region, and the secreted polypeptides antagonized neuronal death. When secreted to the cell exterior, the polypeptides of the present invention can be conveniently recovered from the culture supernatant of the host cells.

The polypeptides of the present invention or DNAs that encodes the polypeptides of the present invention can be made into reagents for suppressing neuronal death. A DNA of the present invention can be appropriately inserted into a vector, and the vector can be used as a reagent. In addition to using a polypeptide or DNA itself as the reagent, a reagent that includes a polypeptide of this invention or a DNA that encodes a polypeptide of this invention may be appropriately combined with sterilized water, saline solution, buffer, salts, stabilizers, preservatives, detergents, other proteins (BSA, etc.), transfection reagents (including lipofection reagent), and such. These may be mixed in advance, or may be kept separately until they are mixed before use.

Cell death of neurons can be suppressed by contacting a polypeptide of the present invention to the neuron. A polypeptide of this invention is contacted to the outside of a cell. Specifically, a polypeptide of the invention is added to the culture medium in a cell culture system. For use in vivo, a polypeptide of the invention is administered so that the polypeptide will be contacted to the cell, which is the target, to suppress cell death. Although the concentration of a polypeptide of the invention depends on the strength of the activity to suppress neuronal death and the purpose of the use thereof, the maximum activity of the polypeptide is reached at a concentration of about 10 .mu.M or less if the polypeptide possesses an activity equivalent to that of HN, and at about 10 nM or less if the polypeptide possesses an activity equivalent to that of HNG. If the polypeptides are secretory polypeptides, the polypeptides will be secreted to the cell exterior, even when it is introduced or expressed intracellularly, and thus, cell death can be suppressed. For example, to suppress cell death with secretory polypeptides, a DNA encoding a polypeptide of the invention is expressed intracellularly. Cell death of neurons can be suppressed in cell culture systems or in vivo by introducing a vector that expresses a polypeptide of the present invention into a cell. Additionally, co-culturing with cells expressing a secretory polypeptide of this invention, or ex vivo administration of the secretory polypeptides enables to suppress cell death of surrounding cells.

Accordingly, a polypeptide of the present invention, a DNA encoding the polypeptide, and a vector containing the DNA may be used to suppress neuronal death. These act as neuronal death suppressant. Furthermore, the present invention provides use of the polypeptides of the invention, DNAs encoding the polypeptides, and vectors containing the DNAs for suppressing neuronal death.

The present invention also provides pharmaceutical compositions containing a polypeptide of this invention, or a vector, wherein a DNA encoding a polypeptide of this invention has been inserted, as its active ingredient. A polypeptide of the present invention can protect cells from neurodegeneration by adding the polypeptide extracellularly, or by intracellular expression of a secretory form of the polypeptide. Therefore, a polypeptide of this invention is useful as a pharmaceutical composition particularly active against diseases associated with neurodegeration.

As described in the Examples (see Original Patent), chemically synthesized Humanin (HN) polypeptide suppresses neuronal death at a concentration of about 10 nM or more in the extracellular solution, and a maximum suppression is achieved at a concentration of 1 to 10 .mu.M. On the other hand, HNG and AGA-HNG polypeptides showed significant or sufficient neuroprotective action at about 1 nM. The neuroprotective action is presented by introducing and expressing a DNA encoding the polypeptides in the cell. Therefore, a vector expressing a polypeptide of this invention as a medicament may be used to perform gene therapy. Secretor types of the polypeptides, or polypeptides, modified with secretion signal attachment, may be expressed for the gene therapy. Administration methods for the vectors include in vivo and ex vivo methods. Vector systems for gene therapy include: adenovirus vector; AAV (adenovirus-associated virus) vector; herpesvirus vector (all refer to Robbins and Ghivizzani, Pharmacol. Ther. 80:35-47, 1998); retrovirus vector (Engel and Kohn, Front. Biosci. 4:e26-33, 1999); lentivirus vector (Lundstrom, K., 1999, J. Recept. Signal. Transduct. Res. 19:673-686); and such, but are not limited thereto.

The target diseases to be prevented or treated using a polypeptide of the present invention, or using a vector that expresses the polypeptide is not limited in any way, so long as the used polypeptide of the present invention is effective for treating the disease. Examples of preferred target diseases include neuron-related diseases, in particular Alzheimer's disease. Previous studies having revealed that cell death of neurons occurs in Alzheimer's disease (T. Nishimoto et al., 1997, Adv. Pharmacol., 41:337-368). Some sort of activation of APP (I. Nishimoto et al., 1998, Neurobiol. Aging., 19:S33-S38) and presenilin (Nishimura et al., 1999, Clin. Genet. 55:219-225) are suggested to be associated with the cell death. Therefore, pharmaceutical compositions of this invention are expected to be applicable as medicament for protection against neurodegeneration that occurs in Alzheimer's disease. In addition to Alzheimer's disease, for example, diseases caused by cell death of neurons due to cerebral ischemia (T. Kirino, 1982, Brain Res., 239:57-69) can be prevented by the use of a pharmaceutical composition of the present invention. Further, Parkinson's disease that accompanies dementia (M. H. Polymeropoulos et al., 1997, Science, 276:2045-2047); diffuse Lewy bodies disease (M. G. Spillantini et al., 1998, Proc. Natl. Acad. Sci. USA, 95:6469-6473); dementia that accompanies Down's disease; and such are also targets of the treatment and prevention using a protein of the invention. Furthermore, since APLP1, which is an APP analogue, is said to be the causative gene for congenital nephrotic syndrome (Lenkkeri, U. et al., 1998, Hum. Genet. 102:192-196), renal diseases, such as nephrotic syndrome, is also the target for the treatment and prevention.

In addition to the direct administration of the active ingredient to a patient, a pharmaceutical composition of this invention may be formulated following conventional drug implementations. For example, the composition may be administered after appropriately formulating it with pharmacologically acceptable carriers or medium, specifically, sterilized water or saline, vegetable oils, emulsifiers, suspending agents, detergents, stabilizers, sustained-release preparations, and such. A pharmaceutical composition of this invention may be in the form of an aqueous solution, tablet, capsule, troche, buccal tablet, elixir, suspension, syrup, nasal drop, inhalant solution, and such. The content of the polypeptide in these preparations makes a suitable dosage acquirable.

Administration to patients may be carried out depending on the properties of the used active ingredient. Example of suitable administration methods include percutaneous, intranasal, transbronchial, intramuscular, intraperitoneal, intravenous, intraspinal, intracerebroventricular, or oral administrations, but are not limited thereto. When using the pharmaceutical composition in the treatment of cerebral neurodegenerative diseases, it is preferable to introduce the pharmaceutical composition to the central nervous system by an appropriate arbitrary route including a intravenous, intraspinal, intracerebroventricular, or intradural injection. The dosage varies according to the age, body weight, condition of a patient, method of administration, and such, but one skilled in the art can suitably select them. The dosage and administration method varies depending on the histological localization of the active ingredient of the pharmaceutical composition of the present invention, therapeutic purpose, body weight, age, and condition of a patient, and such, but can be selected suitably by those skilled in the art.

For example, to protect cerebral neurons against degeneration in Alzheimer's disease treatment, it is preferable to administer a polypeptide of the present invention so that the concentration around the target cells is sufficient to effectively suppress neurodegeneration. Specifically, Humanin polypeptide or compounds, that have equivalent protective action against neuronal death with Humanin, should be administered at a concentration of at least 1 nM or more, preferably 10 nM or more, more preferably 100 nM or more, and much more preferably 1 .mu.M or more. HNG or comounds, that have equivalent protective action against neuronal death with HNG, should be administered at a concentration of at least 1 pM or more, preferably 10 pM or more, more preferably 100 pM or more, and much more preferably 1 nM or more. On the other hand, a comparable effect with HNG can be expected by the use of AGA-HNG, at a concentration of a tenth of the HNG concentration. The dosage to achieve these concentrations can be appropriately determined taking the administration route into consideration.

The present invention also provides antibodies binding to a polypeptide of the invention. The antibodies of this invention include polyclonal antibodies and monoclonal antibodies. Polyclonal antibodies can be prepared, for example, as follows: a polypeptide of the invention, such as HN and HNG, or partial peptides thereof are prepared; rabbit, goat, sheep, and such are sensitized with these peptides as the antigen. Antigenic peptides can be bound to other proteins according to needs. For example, they can be bound with carrier proteins, such as key-hole limpet hemocyanin and albumin for immunization. Monoclonal antibodies can be prepared using splenocytes of immunized mice and rats to obtain hybridomas that produce monoclonal antibodies. Production of antibodies can be carried out according to conventional methods (Ed. Harlow and David Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).

Using conventional biochemical techniques, such as ammonium sulfate fractionation, protein G sepharose column, and affinity column with immobilized antigens, polyclonal antibodies can be purified from serum, and monoclonal antibodies can be purified from hybridoma culture supernatant or from ascites of animals inoculated with hybridoma.

Additionally to the use of the antibodies prepared in this manner to absorb the polypeptide of this invention, the antibodies may be utilized, for example, to test and diagnose structural alterations of a polypeptide of this invention, and to detect the expression level of a polypeptide of this invention.

Decrease in blood or interstitial concentration of Humanin or Humanin-like peptide, including concentration in the nerve tissue, may be used to diagnose or prognosticate degenerative diseases of the nerves, including AD, and degenerative diseases of other organs. For example, it is possible that the progress of the disease of AD patients with low HN activity in blood is faster with bad prognosis compared to patients of the same kind of AD with high HN activity. Conceivable methods for testing are exemplified by measuring the concentrations of a pepetide of the invention in the blood or tissue samples by RIA using anti-Humanin antibody, or by testing biopsy samples by immunohistological staining. Furthermore, for example, the polypeptide level can be monitored during the treatment which includes administration of a polypeptide of this invention.

The antibodies of the present invention may be antibody fragments thereof, so long as it binds to a protein of the invention. For example, the antibody fragment may be Fab, F(ab').sub.2, Fv, or modified antibodies thereof. Additionally, humanized antibodies or human antibodies, and such are also included in the antibodies of the present invention. An antibody of the present invention can be used as "a test reagent for a polypeptide of this invention", and additionally, it can be appropriately combined with sterilized water, saline solution, buffer, salts, stabilizers, preservatives, detergents, other proteins (BSA, etc.), and such. These may be mixed in advance or may be kept separately until they are mixed at the time of use.

The present invention also provides a DNA comprising at least 15 nucleotides, which is a complementary to a DNA (SEQ ID NO: 4) encoding Humanin or complementary strand thereof, and are used to detect or manipulate a gene encoding a polypeptide of this invention. The term "detection of a gene" refers to a detection based on a gene including the detection of the existence, mutations, expression, and such of a gene. The term "manipulation of a gene" refers to gene manipulations, such as introduction of mutation(s) to a gene; amplification of a gene; and inhibition of the expression of a gene. The term "detection or manipulation of a gene" includes detection and regulation of the expression of a gene. Herein, the term "complementary strand" is defined as one strand of a double strand DNA composed of A:T and G:C base pair to the other strand. Also, "complementary" is defined as not only those completely matching within a continuous region of at least 15 nucleotides, but also those having a homology of 70% or more, preferably 50% or more, more preferably 80% or more, even more preferably 90% or more (for example, 95% ore more) within that region. The homology may be determined, for example, according to a method described in the literature (Altschul, S. F. et al., 1990, J. Mol. Biol. 215:403-410).

Such DNA includes, probes and primer for detecting or amplifying DNAs or RNAs encoding a peptide of the invention; as well as nucleotide and nucleotide derivatives (for example, antisense oligonucleotides, DNAs encoding ribozymes, and such) for suppressing the expression of a polypeptide of this invention. When used as a primer, such a DNA is complementary at the 3'-end, and restriction enzyme recognition sequences or tags can be added to the 5'-end.

Further, the present invention provides a method for detecting the activity of a polypeptide of this invention to suppress cell death. The method comprises: (a) inducing cell death in the presence of a polypeptide of this invention; and (b) detecting level of cell death. Specific manipulation of the method can be carried out according to the method described herein. The method may be used to determine whether a polypeptide of the invention has the effect to suppress cell death in various cells, and to quantify the suppressive effect. There are no particular limitations on the cells to be used in the method, and various cells that may encounter cell death can be used. Further, induction of cell death can be carried out using cell death induction system for respective cells known in the art. Neurons may be used to detect the effect of a polypeptide of the invention on various conditions, such as neuronal death-inducing stimuli, environmental changes, and gene expression. The detection method can also detect differences of sensitivity against a polypeptide of the invention in neuronal death existing among biological species or subspecies, and between individuals. This enables to analyze the effectiveness of a polypeptide of the invention, for example, among ethnic groups, race, or individuals. According to the method, for example, detailed analysis of conditions for clinical application of a polypeptide of the invention can be carried out.

Further, the present invention provides a method for detecting the effect of chemical compounds on the suppression of neuronal death by a polypeptide of the invention. The method comprises the steps of: (a) inducing neuronal death in the presence of a polypeptide of the present invention and a test compound; and (b) detecting the level of neuronal death. The method may be used to assay chemical compounds that enhance or suppress neuronal death by a polypeptide of the invention. A polypeptide of the present invention is suggested to exhibit a cell death suppressing effect by acting to the neuronal surface. The action of a candidate compound that inhibits, or on the contrary enhances contact of a polypeptide of the invention to the cell surface can be investigated according to the method. Alternatively, the use of the detection method enables screening of chemical compounds that regulate the suppression of neuronal death by a polypeptide of the invention. The method comprises the steps of: (a) inducing neuronal death in the presence of a test compound and a polypeptide of the present invention; (b) detecting the level of neuronal death; and (c) selecting the chemical compound that enhances or suppresses neuronal death. In step (c), the result can be compared to that with arbitrary control. More specifically, for example, one can select in step (c) the chemical compound that enhances or suppresses neuronal death in the presence of a test sample compared to those detected in the absence of a test sample. Chemical compounds that enhance neuronal death serve as candidate compounds, inhibiting the suppression of neuronal death by a polypeptide of the invention; whereas chemical compounds that suppress neuronal death serve as candidate compounds, further enhancing the suppression of neuronal death by a polypeptide of the invention. Alternatively, a different compound other than the test sample can be used as a control in the above-mentioned screening method. Specifically, cell death are detected using compounds, other than the test sample, that regulate the suppression of neuronal death by a polypeptide of the invention; selecting in step (c) the chemical compounds from the test samples in step (a), which compounds suppresses or enhances neuronal death compared to the result obtained with the other compound. According to the screening method, chemical compounds having stronger effects compared to existing compounds can be screened with respect to their ability to regulate the suppression of neuronal death by a polypeptide of the invention.

Test samples for the screening include, for example, purified proteins (including antibodies); expression products of gene libraries; synthetic peptide libraries; cell extracts; cell culture supernatants; libraries of low-molecular weight synthetic compounds; natural materials, such as soil; solutions containing substances released from bacteria such as Actinomyces broth; and so on, but are not limited thereto.

Induction on neuronal death and administration of a polypeptide of the invention can be carried out according to the Examples. There is no particular limitation on the timing of the application of a test sample to cells, and they may be applied before, after, or simultaneously with the application of a polypeptide of the invention. Further, there is no limitation on the method for the application of a test sample, and for example, the sample is added to the medium of a cultured cell line. If the sample is a nucleic acid, it can be introduced into a cell. In addition to the methods above, the test sample can be applied by arbitrary administration methods.

Chemical compounds evaluated by the action of the compounds in the above-mentioned test, or compounds obtained by the screening serve as candidate compounds that regulate the activity of a polypeptide of the invention. These compounds may be applied to prevent or treat diseases associated with Alzheimer's disease.

Further, the present invention provides a method of screening for compounds that bind to a polypeptide of the invention. Such screening can be performed by a method comprising the steps of: (a) contacting a polypeptide of the present invention with a test sample; (b) detecting the binding activity between the polypeptide of the invention and the test sample; and (c) selecting the sample that bind to the polypeptide of this invention.

Depending on the screening method, a polypeptide of the invention may be used in the screening as a soluble polypeptide, or in a form bound to a support. A polypeptide of the invention may be labeled. Examples of labeling include labeling by radioactive isotopes, fluorescent substances, and biotin or digoxigenin; tag sequence addition; and such.

Test samples for the screening may be, for example, purified proteins (including antibodies); expression products of gene libraries; synthetic peptide libraries; cell extracts; cell culture supernatants; libraries of low-molecular weight synthetic compounds; natural materials, such as soil; solutions containing substances released from bacteria, such as Actinomyces broth; and so on, but are not limited thereto. Test samples to be used in the screening may be appropriately labeled according to needs. The labels include, for example, radioactive labels, fluorescent labels, and such, but are not limited thereto.

For example, screening for proteins, that bind to a polypeptide of the present invention, can be carried out by applying cell extract of tissues or cells, expected to express proteins that bind to a polypeptide of this invention, to an affinity column to which a polypeptide of this invention is immobilized; and purifying the proteins that specifically bind to the column.

Alternatively, a cDNA library is prepared from tissues or cells, expected to express proteins that bind to a polypeptide of the invention (for example, brain cortical tissue; and neurons, such as F11) using phage vectors; then plaques are formed on agarose; and screening by Western blotting is carried out using labeled polypeptides of this invention. The screening can be also conducted by a "two hybrid system", and so on. Specifically, a method utilizing a "two hybrid system" is conducted as follows: (1) a DNA-binding peptide, such as GAL4 DNA-binding region, and a transcription activating peptide, such as GAL4 transcription activation region, is expressed as a fusion protein with a polypeptide of the present invention and a test protein, respectively; and (2) the binding of the protein of the present invention and the test protein is detected as the expression of a reporter gene attached downstream of a promoter, having a binding sequence of the DNA binding peptide.

Furthermore, receptors of a polypeptide of the present invention can be cloned by the screening method of this invention. In case of screening receptors, it is preferable to prepare the test samples from tissues or cells, expected to express receptors (for example, brain cortical tissue, nerve cell line, neuroblastoma cells, and teratocarcinoma cells). Examples of nerve cell lines include F11 cells; PC12 cells (L. A. Green and A. S. Tischler, 1976, Proc. Natl. Acad. Sci. USA, 73:2424-2428); NTERA2 cells (J. Skowronski and M. F. Singer, 1985, Proc. Natl. Acad. Sci. USA, 82:6050-6054); SH-SY5Y cells (L. Odelstad et al., 1981, Brain Res., 224:69-82); and so on.

Alternatively, molecules binding to a polypeptide of the present invention can be screened by contacting synthetic compounds; natural product bank; and random phage peptide display libraries to an immobilized polypeptide of the invention. Further, screening by detecting the binding utilizing surface plasmon resonance imaging (for example, manufactured by BIAcore) is possible. These screening methods may be performed by high-throughput screening utilizing combinatorial chemistry techniques.

Compounds, that bind to a polypeptide of the present invention, obtained according to the screening method of this invention, serve as candidate compounds, regulating the activity of a polypeptide of the invention. Thus, these compounds are applicable to prevent or treat diseases associated with Alzheimer's disease.
 

Claim 1 of 21 Claims

1. An isolated polypeptide that suppresses neuronal death associated with Alzheimer's disease having an amino acid sequence of Formula (I): Pro-Xn.sub.1-(Cys/bXaa)-(Leu/Arg)-Xn.sub.2-Leu-Thr-(Gly/Ser)-Xn.sub.3-Pro (I) (SEQ ID NO: 63) wherein "Cys/bXaa" indicates Cys or a basic amino acid; "(Leu/Arg)" indicates Leu or Arg; "(Gly/Ser)" indicates Gly or Ser; and Xn.sub.1, Xn.sub.2, and Xn.sub.3 independently indicate arbitrary amino acid sequences not more than 10 residues in length, respectively.

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