JP2002542261A - Antibody recognizing APP cleaved by caspase and use thereof - Google Patents

Abstract

(57) Summary Neuronal loss due to amyloid-β peptide (Aβ) formation and apoptosis is a closely related process in the pathogenesis of neurodegenerative diseases, especially Alzheimer's disease. The role of apoptotic proteases and caspases in the cleavage of amyloid-β precursor protein (APP) and the biosynthesis of amyloidogenic Aβ peptide species is described. Antibodies that recognize APP cleaved by caspases and their various potential uses are also described.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The present invention relates to novel antibodies having the ability to recognize a neo-epitope exposed after caspase-mediated cleavage of the amyloid-β precursor protein, and to their novel antibodies. About use.

[0002] Loss of hippocampal neurons due to apoptotic cell death is a hallmark of Alzheimer's disease (Cotman and Su, 1996; Li et al., 1997).
Small et al. Su et al., 1995; , 1997). One potential factor contributing to the susceptibility of these cells to immature death results from the cytotoxic effects of amyloid-β peptide deposition at or near the site of neuronal degeneration. When cultured human cells, including neurons, are treated with the amyloidogenic Aβ peptide, they cause apoptotic cell suicide (Forloni et al., 1).
996; LaFerla et al. Loo et al., 1995; , 1993). At the same time, apoptotic neurons generate increased levels of cytotoxic Aβ peptide species (LeBlanc, 1995). Thus, a possible scenario in the pathology of Alzheimer's disease is that enhanced Aβ peptide formation as a result of genetic predisposition or other physiological factors triggers a partial activation of the endogenous cell suicide pathway in susceptible neurons. It is. Next, the sensitized cells
Elevates the levels of β-peptide, causing an exacerbation of the vicious circle, which leads to progressive neuronal loss that is characteristic of Alzheimer's disease. If this is the case, components of the apoptotic machinery should directly or indirectly contribute to the complex proteolytic processing of the amyloid-β precursor protein (APP) (Haass and Selkoe, 1993; Selkoe). Et al., 19
96; Sisodia and Price, 1995). Proteolytic processing of APP increases Aβ peptide formation and causes other APP protease degradation products to be observed in apoptotic cells. In this study, we provide a role for apoptotic proteases, particularly caspases, in APP processing and biosynthesis of amyloidogenic Aβ peptide species.

[0003] Currently, there is no direct or indirect method to specifically assess the presence of APP caspase cleavage products. Therefore, it would be useful to develop agents that can specifically detect the presence of APP caspase cleavage products. The presence of cleavage products can be used as an indicator of neuronal apoptosis. In particular, such agents would be useful for recognizing the presence of neuronal degeneration, a neuronal degenerative disease, and assessing the progression of such disease in mammals. More specifically, such agents can be used as tools to assess the effects of candidate inhibitors on neuronal apoptosis, and also as potential tools for diagnosing neurodegenerative diseases.

[0004] Accordingly, one object of the present invention was to develop an agent capable of specifically detecting the APP protease cleavage product.

[0005] Another objective was to identify neuronal apoptosis using the agents of the present invention.

A further object was to use the agents of the present invention to distinguish neuronal apoptosis from caspase-independent neuronal mediated necrotic cell death.

[0007] A further object was to use the agents of the present invention to detect neuronal apoptosis as an indicator of neurodegenerative conditions and brain damage.

[0008] These and other objects will be apparent to those skilled in the art from the teachings provided herein.

This application refers to a number of publications, the contents of which are hereby incorporated by reference in their entirety.

SUMMARY OF THE INVENTION [0010] The present invention relates to antibodies that recognize neo-epitope created or exposed after caspase-mediated cleavage of APP or APLP, and uses thereof.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is further described with reference to the accompanying drawings.

FIG. 1A shows that caspase inhibition eliminates enhanced amyloid-β peptide production in apoptotic neuronal cells. Neuron NT2 cells (hNT) differentiated by retinoic acid were incubated in the presence of serum (○).
Alternatively, the cells were cultured in the absence (●, Δ) for 4 days. Half of the serum-depleted cell culture is
-VAD _(OMe) -CH 2 F was treated with (100 [mu] M initial dose, adding 50μM every 24 hours) (■). Aβ (1-40) in the obtained cell culture supernatant was measured at the indicated intervals by sandwich ELISA. After reaching steady state, A
The relative rate of β production was calculated (in this case, the slope of the values on days 2-4). A representative experiment (n = 2) is shown, with each data point being the average from triplicate cultures.

FIG. 1B shows caspase-related APP protein cleavage during apoptosis. As indicated, NT2 cells were induced to undergo apoptosis for 6 hours with 1 μg / ml camptothecin in the absence or presence of 33 μM Z-VAD (OMe) -CH ₂ F, for 6 hours. Apoptosis was investigated by oligonucleosomal DNA fragmentation (left panel). Cell lysates were prepared using urea / SDS, separated on polyacrylamide gels, and then caspase-3 (top right panel),
Immunoblot for poly (ADP) -ribose polymerase (middle right panel) or APP (lower right panel). Representative experiments (n = 5) are shown.

FIG. 1C shows amyloid by group II caspases in apoptotic cell extracts.
FIG. 2 is a diagram showing proteolysis of a do-β precursor protein. Conjugation in vitro
o By transcription / translation³⁵S] Met-labeled APP is generated and then
Extract from cut cells (lane 1) or CD-95 (Fas, A
PO-1) cells induced to undergo apoptosis by immunoligation
Incubated with extracts from vesicles (lane 2). Complete APP (p120 ^APP ) Are three different intermediates (APP) of approximately 85, 88 and 90 kDa
Was cut off. Representative experiments (n = 4) are shown.

FIG. 1D shows the proteolysis of amyloid-β precursor protein by group II caspase in apoptotic cell extracts. Cleavage of [ ³⁵ S] Met-labeled APP in apoptotic Jurkat extracts was performed at the indicated concentrations of tetrapeptide-aldehyde caspase inhibitor (Ac-YVAD-CHO).
, Δ; Ac-DEVD-CHO, ；; Ac-IETD-CHO, ●) and quantified by laser concentration analysis of the obtained fluorogram. Representative experiments (n = 2) are shown.

FIG. 1E shows the degradation of amyloid-β precursor protein by recombinant caspase-3. [ ³⁵ S] Met-labeled APP was incubated with the indicated concentrations of recombinant human caspase 3 at 37 ° C. for 60 minutes. The APP cleavage was quantified by laser densitometric analysis, the relationship ^{_{S t / S 0 = e -kobs}} * t ( here, _{S t} is the concentration of substrate (APP) remaining at time t, _{S 0} is the initial is substrate _{concentration,} was used to determine the value of _{k c} at / _{K m} from _k obs ₌ k _cat ^* [caspase-3 is a / _{K m).} Representative experiments (n = 2) are shown.

FIG. 2 shows increased caspase-3 immunoreactivity in dying pyramidal neurons in the CA3 field of the Alzheimer's brain hippocampus. Paraffin-embedded sections were processed for antigen retrieval and peroxidase neutralization, then paired with immunoaffinity purified antibodies raised against the large subunit of human caspase 3 (MF-R280) and visualized anti-rabbit HRP. (Vectalabs, UK) and hematoxylin counterstaining. a) that the antiserum cannot cross-react with other human caspases; b) that there is no staining with pre-immune serum; c) that antigen preadsorption can eliminate immunostaining; and d) from other rabbits. Staining was specific, as judged by similarities (not shown) to the results using the resulting antibodies. The width of each panel corresponds to 140 μm. Identical results were found in brains from other donors (Alzheimer 3, control 2). Examples shown include an 82-year-old woman clinically and post-mortem diagnosed with Alzheimer's disease, and a brain with no clinical abnormalities that died from pulmonary embolism and was judged neurologically normal It is from a 88 year old man.

FIG. 2A shows paraffin-embedded sections from age-matched neurologically normal individuals. The white arrows indicate healthy caspase-3 negative pyramidal neurons that show little, if any, caspase-3 immunoreactivity.

FIG. 2B shows a paraffin-embedded section from an Alzheimer's disease patient.
Black arrows indicate caspase-3 positive immunoreactive neurons (brown) in Alzheimer samples that are sensitive to degeneration and have degenerated morphology (including "ghosts").

FIG. 3A shows the identification of the site of caspase-mediated proteolytic cleavage of the amyloid-β precursor protein and its removal by mutation. [ ³⁵ S]
Differential mapping with Cys versus [ ³⁵ S] Met and mass spectrometry analysis of the caspase-degraded ΔN-APP deletion construct identified three potential caspase degradation sites (not shown). The putative site was then confirmed by continuous removal of predicted P ₁ Asp residues by site-directed mutagenesis (P ₁
Asp is essential for caspase recognition). [ ³⁵ S] Met indicated
APP mutant polypeptides are generated by coupled in vitro transcription / translation and then in the absence (lanes 1, 3, 5, 7) or presence (lanes 2, 4, 6, 8) of 8 nM caspase-3. Incubated. Representative experiment (n = 3)
It is shown.

FIG. 3B shows the location of the caspase cleavage site within the amyloid-β precursor protein in relation to other structural features. The domains and interaction sites within the amyloid-β precursor protein have been summarized. Proteolytic cleavage site with three caspases are shown below together with the number refers to the Asp residue corresponding to P _1. The region encompassing the Aβ peptide has been expanded above with known mutations shown in parentheses, including the VKMD ^653- VNLD ⁶⁵³ “Swedish” mutations (in circles).

FIG. 4 shows caspase-dependent cleavage of amyloid-β precursor protein in apoptotic intact cells and C-terminal neo-
FIG. 3 shows in situ detection of epitopes.

FIG. 4A shows A during apoptosis in transfected B103 cells.
FIG. 3 shows the C-terminal truncation of PP. Equal amounts of full-length wild-type APP (wt, lane 1 &
2), Asp⁷²⁰C-terminal residue Ala after caspase cleavage site⁷²¹~ Asn ⁷⁵¹ APP lacking (ΔC-APP, lanes 3 & 4) or Asp⁷²⁰Caspa
Full-length APP (D) containing a point mutation to remove the⁷²⁰A,
A stable B103 cell line expressing lanes 5 & 6) was generated. Induces apoptosis
In the absence (lanes 1, 3, 5) or presence of 1 μM staurosporine
The cell lines were treated under (lanes 2, 4, 6) for 2 hours. Centrifuge cells and
Apoptotic debris is collected, washed with PBS and buffered with Nonidet P-40.
Dissolved in the buffer. The complete APP was conjugated with an α-C21 antibody (C-terminal 21 amino acid of APP).
(Made against acid) and immunodepletion using Protein A-Sepharose collection
Thirsty. The remaining APP fragment was combined with the protein G-Sepharose collection with 6E1
Immunoprecipitation using Antibody 0 (generated against the first 17 residues of Aβ peptide)
Let down. After separation by SDS-PAGE, the C-terminal truncated APP fragment was replaced with (AP
Immunoblotting using 22C11 antibody (made against the amino terminus of P)
And visualized by scanning. The arrow is Asp⁷²⁰Generated by cutting after
APP product (ΔC-APP) or equivalent transfection standard (lane 3)
& 4). Representative experiments (n = 4) are shown.

[0024] Figure 4B, APP neo generated by caspase - epitope is a diagram showing a specific recognition by αΔC ^{Cs p} -APP. [ ³⁵ S] Met-tagged APP variants were generated by coupled in vitro transcription / translation (upper panel), then A
Immunoprecipitation was performed using a specific antibody that recognizes a caspase-generated neo-epitope (αΔC ^Csp- APP), which is exposed after cleavage of APP at sp ⁷²⁰ (lower panel). Representative experiments (n = 2) are shown.

FIG. 4C shows A during apoptosis in transfected B103 cells.
FIG. 4 shows the C-terminal truncation of PP. The B103 stable cell line carrying full-length wild-type APP (described in FIG. 4A) was treated for 2 hours in the absence (lane 1) or presence (lanes 2 & 3) of 1 μM staurosporine to induce apoptosis. . The cells in lane 3 were also treated with 100 μM of the caspase inhibitor Z-VAD (OMe) -CH ₂ F. Cells and apoptotic debris were collected and lysed as described above. The lysate was then immunoprecipitated with αΔC ^Csp- APP and S
Separated by DS-PAGE and immunoblotted for ΔC-APP using 22C11. As a positive control, ΔCA was obtained by stable transfection.
Untreated B103 cells carrying PP were treated similarly (lane 4). Representative experiments (n = 3) are shown.

FIGS. 4D-4I show Δs generated by caspases during NT2 cell apoptosis.
FIG. 2 shows in situ detection of C-APP neo-epitope. Subconfluent NT2 cells were transformed with 50 μM of the caspase inhibitor Z-VAD (OMe)-
Apoptosis was induced by incubating with 1 μg / ml camptothecin (EI) in the absence (E, H) or presence (F, I) of CH ₂ F for 4 hours. Control cells (D, G) were similarly treated with vehicle (DMSO) alone. After cell collection and fixation, all cells were stained with TUNEL (green),
Active caspase -3 (D, E, F; αCsp-3 (MF397); red) - any or [Delta] C-APP neo generated by caspase epitope (red G, H, I;; αΔC C sp -APP) Were co-stained using an antiserum that recognizes All panels give yellow when co-existing, immunoreactive with TUNEL (green) (
(Red). Representative experiments (n = 2) are shown.

FIG. 5 shows in vivo shortening of APP at the C-terminal caspase site during neuronal apoptosis following acute excitotoxicity or ischemic injury. Generation of ΔC-APP in hippocampal neurons undergoing apoptosis
Ex vivo toxicity was investigated in vivo after systemic administration of kainate (AF) or transient global cerebral ischemia (GL).

FIGS. 5A-5F show excitotoxic damage in SV129 mice. Left panels A, C, E are controls and right panels B, D, F are caspase-generated APs of kainic acid treated and euthanized SV129 mice.
P neo-epitope (A and B; αΔC ^Csp- APP), active caspase-3
(C and D; αCsp-3 (MF397)) or adjacent brain sections stained for TUNEL (E and F). The CA3 field of the hippocampus is shown in all panels at the same magnification (bar in panel F = 100 μ). Representative experiments (n = 4) are shown.

5G to 5L are diagrams showing the effect of ischemia-induced damage. Four-vessel occlusion for 12 minutes caused transient global ischemia in rats and reperfusion was performed for the indicated times (control animals (G) underwent a sham surgical procedure without ischemia). . Hippocampal CA
One section was subjected to caspase-generated APP neo-epitope (GJ, L
) Or TUNEL (K). The enlargement ratios of panels G to K are the same (bar of panel K = 400 μ), and panel L is an enlarged image (bar = 75 μ) of the area outlined in panel I. A representative example (n = 4) is shown.

FIG. 6 is a diagram showing the coexistence of ΔC-APP and amyloid-β in senile plaques.
Bar in panel 6C = 100μ.

FIG. 6A shows αΔC ^Csp -AP in the same hippocampal range as shown in (B).
FIG. 3 shows ΔC-APP visualized by P immunoreactivity.

FIG. 6B shows amyloid-β in the hippocampus of a patient diagnosed with Alzheimer's disease.
FIG. 3 shows senile plaques identified by immunoreactivity (arrows).

FIG. 6C shows caspase-generated APP that gives a yellow color when matched.
FIG. 4 is a composite image of the neo-epitope (red) and Aβ (green), illustrating the high degree of overlap between ΔC-APP and amyloid-β immunoreactivity.

In clinically diagnosed Alzheimer's disease patients with n = 7 (males and women aged 77-91 years with an AD diagnosis of 5-14 years; collection 2.3-4.3 hours after death) Similar results were observed, but seven age-matched control patients (79-9
1 year old male and female; collection 2.0-4.0 hours after death).

FIG. 7 shows that caspase-mediated APP production increases amyloid-β peptide formation. Comparable levels of full length APP (wt,
The B103 stable cell line described for FIG. 4A, expressing APP lacking the C-terminal residues (αΔC-APP, columns 2 & 4) after the Asa ^720- Asn ⁷⁵¹ (Asp ⁷²⁰ caspase cleavage site) or Ala ⁷²¹ -Asn ⁷⁵¹ , After culturing for 20 or 48 hours in fresh medium, Aβ peptide levels in the culture medium were quantified by immunoassay using monoclonal antibodies. Representative experiments (n = 4) ± SD are shown.

FIG. 8 shows that “Swedish” familial mutations improve the β-secretase target region of APP as a caspase-6 substrate.

FIG. 8A shows VKMD ⁶⁵³ / A ⁶⁵⁴ of the β-secretase target region of APP (
VKMD-AMC, ○; see Figure 3B), the optimal tetra for "Swedish" dipeptide mutation (V NL D-AMC, caspase predicted by ●) or positional scanning combinatorial substrate libraries -6 FIG. 4 shows a fluorogenic tetrapeptide-aminomethylcoumarin synthesized to correspond to any of the peptides ( VEHD -AMC, □). Their suitability as caspase-6 substrates was tested in reaction mixtures containing recombinant human caspase-6 (2 nM) and various concentrations of fluorogenic ligand. Representative experiment (n = 3)
It is shown.

FIG. 8B shows the removal of the N-terminal caspase cleavage site (ΔN-APP) followed by the wild-type β-secretase region (VKMD ⁶⁵³ / A ⁶⁵⁴ ; columns 1 & 2), the “Swedish” mutation (V NL D ^653). / A ⁶⁵⁴ ; columns 3 & 4) or were engineered to contain either a “Swedish” mutation in which the P ₁ Asp essential for caspase recognition was changed to Ala (V NLA ⁶⁵³ / A ⁶⁵⁴ ; columns 5 & 6). FIG. 4 shows the effect of the APP construct. [ ³⁵ S] Met-tagged proteins are generated from these constructs by coupled in vitro transcription / translation, and then cytosolic extracts from apoptotic cells (solid columns; 1, 3 & 5)
Or recombinant caspase-6 (hatched columns; 2, 4 & 6). Initial AP of APP cleaved at the β-secretase target region for 60 minutes
The ratio to P was determined by fluorography and expressed as ± SD. The results show that the "Swedish" familial mutation caused APP beta against caspase-6.
-Indicates increased sensitivity of the secretase site and that elevated amyloid-β peptide formation in cells carrying “Swedish” APP is dependent on P ₁ Asp required for caspase recognition . Representative experiment (n
= 5) is shown.

FIG. 8C is a diagram showing measurement of cutting. Contains either the wild-type β-secretase sequence (セ) or the “Swedish” dipeptide mutation (ジ) [ ³⁵ S
] Met-labelled ΔN-APP was incubated with the indicated concentrations of recombinant human caspase 6 at 37 ° C for 60 minutes. Phosphor imaging of β-cleaved fragments (
Cleavage was quantified by phosphorimaging and used to determine the value of k _cat / K _m as described in the description of FIG. 1F.

FIG. 8D shows a wild-type VKMD ⁶⁵³ / A ⁶⁵⁴ site, a “Swedish” mutation (V NL D ⁶⁵³ / A ⁶⁵⁴ ) or a “Swedish” mutation (V NLD ⁶⁵³ / A ⁶⁵⁴ ) that changes P ₁ Asp to Ala. FIG. 9 shows immunoreactive products in a stable K562 cell line generated to retain full-length APP with NLA ⁶⁵³ / A ⁶⁵⁴ ). Equal amounts of cells expressing equal amounts of the APP construct were metabolically labeled by culturing for 1 hour in non-methionine-containing medium and then for 5 hours in the presence of [ ³⁵ S] Met. Cells and medium were separated by centrifugation, and then [ ³⁵ S] Aβ peptide was immunoprecipitated (using the 4G8 antibody) from each and SDS-PAGE.
(10-20% polyacrylamide; Tricine gel) and visualized by fluorography. Representative experiments (n = 3) are shown.

DETAILED DESCRIPTION OF THE INVENTION Definitions Unless defined otherwise, scientific and technical terms and nomenclature used herein are defined as follows:
It has the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, cell culture, infection techniques, molecular biology methods, etc. are common methods used in the art. Such standard techniques are described, for example, in Sambrook et al. (1989, Molecular Cloning: A).
Laboratory Manual, Cold Spring Harbo
Laboratories) and Ausubel et al. (199).
4, Current Protocols in Molecular Bio
(Logy, Wiley; New York).

In general, techniques for immunology, such as the generation of antibodies, immunization of animals, isolation of pure antibodies by means including ELISA and affinity chromatography, are commonly used in the art. Is the way. Such standard techniques are:
See, for example, Harlow et al. (1988, Antibodies: AL).
laboratory Manual, Cold Spring Harbor
Laboratories).

As used herein, the nucleotide sequence refers to the IUPAC-IUB Biochemical Nomenclature Committee (Biochemical N) as commonly used in the art.
Omenclature Commission) (Biochemistry)
, 1972, 11: 1726-1732) and are represented as single-stranded, left-to-right, in the 5 'to 3' direction, using the single letter nucleotide notation.

This specification refers to a number of commonly used recombinant DNA (rDNA) technology terms. Nevertheless, definitions of selected examples of such rDNA terms are provided for clarity and consistency.

The term “recombinant DNA” or “recombinant plasmid” as known in the art refers to a DNA molecule resulting from the joining of DNA segments. This is often referred to as genetic manipulation.

The term “DNA segment or molecule or sequence” refers to the deoxyribonucleotide adenine (A), guanine (G), thymine (T) and / or cytosine (C)
Used herein to refer to a molecule composed of These segments, molecules or sequences can be found in nature or derived synthetically. When read in accordance with the genetic code, these sequences may encode a linear stretch or sequence of amino acids, which may be referred to as a polypeptide, protein, protein fragment, and the like.

As used herein, the term “gene” is well-known in the art and relates to a nucleic acid sequence that defines a single protein or polypeptide. The nucleic acid may be all or part of the sequence encoding the polypeptide, as long as the functional activity of the polypeptide is retained.

“Structural gene” is defined as a DNA sequence that is transcribed into RNA and translated into a protein having a specific amino acid sequence, thereby producing a specific polypeptide or protein.

“Restriction endonuclease or restriction enzyme” refers to the recognition of a specific base sequence (usually 4, 5, or 6 base pairs in length) in a DNA molecule, and at all places where this sequence appears. An enzyme that has the ability to cleave DNA molecules. One example of such an enzyme is EcoRI, which recognizes the base sequence GAATTC / CTTAAG and cuts the DNA molecule at this recognition site.

A “restriction fragment” is a DNA molecule produced by digestion of DNA with a restriction endonuclease. Any particular linear genomic or DNA segment can be digested by a particular restriction endonuclease into at least two separate restriction fragment molecules.

“Agarose gel electrophoresis” is an analytical method for fractionating double-stranded DNA molecules based on DNA size. The method is based on the fact that DNA molecules move through the gel as a sieve, so that the smallest DNA molecules have the greatest mobility and travel farther through the gel. Since the sieving characteristics of gels delay the largest DNA molecules, they have the lowest mobility. The fractionated DNA can be visualized by staining a gel, nucleic acid hybridization, or adding a detectable label to the fractionated DNA molecule using methods well known in the art. All of these methods are well known in the art, and specific methods can be found in Ausberg et al. (Supra).

An “oligonucleotide or oligomer” is a molecule consisting of two or more, preferably more than three, deoxyribonucleotides or ribonucleotides. The exact size of a molecule will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. Oligonucleotides can be derived by synthesis, cloning or amplification.

“Sequence amplification” is a method for producing large amounts of a target sequence. In general,
One or more amplification primers are annealed with the nucleic acid sequence. Using appropriate enzymes, sequences found adjacent to or between the primers are amplified. The amplification method used herein is the polymerase chain reaction (PCR).

“Amplification primer” refers to an oligonucleotide that anneals to a DNA region adjacent to the target sequence and can act as a starting primer for DNA synthesis under suitable conditions well known in the art. The synthesized primer extension product is complementary to the target sequence.

The term “plasmid” or “vector” is commonly known in the art, and takes up a nucleotide sequence or a sequence of the present invention and acts as a DNA vehicle into which a DNA of the present invention can be cloned. Gene media, including, but not limited to, plasmid DNA, phage DNA, viral DNA, and the like. Numerous vector types exist and are well known in the art.

The term “DNA construct,” as used herein, refers to a vector or plasmid containing a cloned nucleotide sequence.

The term “expression” refers to the transcription of a structural gene into mRNA (transcription), which is then translated into one or more polypeptides (or proteins) (
Translation) process.

The term “expression vector” is defined as a vector or vehicle as described above, which is designed to allow expression of the inserted sequence after transformation into a host. Usually, a cloned gene (s) (insertion sequence) placed under the control of a regulator sequence, such as a promoter sequence, initiates transcription of the insertion sequence. Such expression control sequences will depend on whether the host for which the vector is designed to express the operably linked gene is prokaryotic, eukaryotic, or both (shuttle vectors). Variations may further include transcription factors such as enhancer elements, termination sequences, tissue specificity factors, and / or translation start and stop sites.

“Eukaryotic expression system” means a combination of a suitable expression vector and a eukaryotic cell system that can be used to express a protein of interest. In some systems, the gene encoding the protein can be inserted into the genome of a virus capable of infecting a particular host cell. Plasmid vectors containing the desired gene may also be used. In all cases, the vector will contain the appropriate regulatory elements (promoters) for expression of the protein in host cells. Additional components, such as a vector or viral genome encoding T7 polymerase, may also be necessary in certain expression systems. Typically used eukaryotic cell types are yeast transfected with a plasmid vector (eg, Saccharomyces cerevisiae).
siae), Piscia pastoris);
Baculovirus (Autographa California)
californica) or Bombyx mori
) (Luckow, Curr. Op. Biotech., 1993, 4: 564).
-572; Griffiths and Page, 1994, Methods
in Molec. biol. 75: 427-440; and Merringt
on et al. , 1997, Molec. Biotech. 8 (3): 283-297
) Infected insect cells (eg SF9, SF21); adenovirus, vaccinia virus, Sindbis virus or Semliki Forest virus infected mammalian cells; and for transient or constitutive (stable) expression Mammalian cells transfected with a DNA vector.

A host cell has been "transfected" by exogenous or heterologous DNA (eg, a DNA construct) when such DNA has been introduced into the cell. The transfection DNA can be integrated into the chromosomal DNA of the host cell (
Covalent bond), and may not be incorporated. For example, in prokaryotes, yeast cells and mammalian cells, the transfection / transforming DNA can be maintained on an episomal factor, such as a plasmid. With respect to eukaryotic cells, an example of a stably transfected cell is one in which the transfection DNA is integrated into the host cell chromosome and is inherited by daughter cells through chromosomal replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones consisting of a population of daughter cells containing the transfection DNA. Transfection methods are well known in the art (Sanbrook et al., 1989, supra; Ausubel et al., 1994, supra).

[0061] Nucleotide sequences and polypeptides useful for practicing the present invention include, but are not limited to, mutants, homologs, subtypes, alleles, and the like. Generally, it is understood that the sequences of the present invention encode a functional protein.
It will be apparent to one skilled in the art that the present invention includes all variants, derivatives or fragments thereof that express a functional protein.

As used herein, the phrase “variant” refers, in the context of the present invention, to a biological activity that is substantially similar (functional or structural) to the initial sequence. Refers to the retained nucleic acid sequence or amino acid sequence. The variants or equivalents may be from the same species or from a different species, may be naturally occurring variants or may be prepared synthetically. Such variants include amino acid sequences having one or more amino acid substitutions, deletions or additions, provided that the biological activity of the protein is conserved. So long as the biological activity of the sequence is generally maintained, so is the variant of the nucleic acid sequence, which may have one or more nucleotide substitutions, deletions or additions.

The term “derivative” is intended to include the variants described above when it contains additional chemical groups that are not normally part of these molecules. These chemical groups can have a variety of purposes, including improving the solubility, absorbability, biological half-life of the molecule, reducing toxicity, and eliminating or reducing undesirable side effects. In addition, these groups may be used for labeling, conjugation purposes, or they may be included in the fusion product (s). Different groups with the ability to mediate the effects described above are described in Remington's Pharmaceutical Sciences and Practice (Remington's The Science and).
Practice of Pharmacy (1995). Methods for coupling such groups to molecules are well known in the art.
The term "fragment" refers to any segment of the identified DNA, RNA or amino acid sequence, and / or any segment of the above variants or derivatives.

The terms “variant”, “derivative” and “fragment” of the present invention may be used herein as isolated / purified or chemically synthesized. Or a protein or nucleic acid molecule, which may be produced by recombinant DNA technology. All of these methods are well known in the art.

The term “APP” is an abbreviation for amyloid-β precursor protein. These are used interchangeably. Different types of APP are known in the art and are named according to the length of the amino acid sequence. Non-limiting examples include APP ⁶⁹⁵ (Genbank accession number CAA31830), APP ⁷⁵¹ (Genbank
Registration number CAA30050) and APP ⁷⁷⁰ (Genbank registration number CAA)
02049) are included. APP ⁷⁵¹ is also called an intermediate APP. For the purposes of this application, the intermediate APP is used, and the amino acid residue numbers are all based on APP ⁷⁵¹ .

The term “APPLP” is an abbreviation for amyloid-β precursor-like protein. These are used interchangeably. APLP is a member of the amyloid precursor protein family. For the purposes of the present invention, APLPs having at least one caspase cleavage site are within the scope of the present invention. APLPs having at least one caspase cleavage site are known in the art and include, but are not limited to, APLP1 (Swissprot accession number P51693) and APLP2 (SLP
wisprot registration number A49321).

The terms “epitope” and “antigenic determinant” are used interchangeably in the conventional sense and refer to a site on an antigen that is recognized by an antibody. A neo-epitope refers to an epitope recognized by an antibody described herein, which recognition occurs as a result of a cleavage event on APP triggered by a caspase enzyme, wherein the epitope It is virtually unrecognized in the absence of an event.

For amino acids, the one-letter code is used, having its standard conventional meaning.

Particular Embodiments Accordingly, the present invention relates to the ability of members of the amyloid precursor protein family to identify degradation products produced by proteolysis by proteases, particularly degradation products produced as a result of degradation by caspase enzymes. The development of drugs having Members belonging to the amyloid precursor protein family and having at least one caspase cleavage site are within the scope of the invention, non-limiting examples of such members include APLP and APP, and more specifically specifically in each APLP1 and APLP2, as well as ^{APP 695, APP 751} and ^{APP 770.}

One of the consequences of caspase cleavage of APP and APLP1 is the creation or exposure of a neo-epitope at the cleavage site of the cleaved protein or as a result of such cleavage. The generation and isolation of antibodies capable of specifically binding to these neo-epitope provides an agent that selectively identifies these cleavage products.

Thus, the present invention provides antibodies that have the ability to specifically and selectively bind the caspase cleavage products of APP and APLP.

Thus, a first embodiment of the present invention relates to an antibody having the ability to bind to neo-epitope created or exposed after caspase-mediated cleavage of APP and APLP.

In certain aspects of this embodiment, the caspase is selected from caspase-3, caspase-6, and caspase-8.

[0074] In another aspect of the invention, caspase-3 has more than one cleavage site on APP and at least one on APLP. In certain aspects, A
The cleavage sites on PLP1 and APLP2 are located at amino acid residues 620 and 732, respectively. The cleavage site for the intermediate APP is based on the amino acid number of APP ⁷⁵¹ , located at amino acid residues ¹⁹⁷ , ²¹⁹ and ⁷²⁰ , which are the sites designated as APP ¹⁹⁷ , APP ²¹⁹ and APP ⁷²⁰ , respectively.

In one aspect of the invention, the ability to bind to a neoepitope created or exposed by caspase-3 cleavage at the named cleavage site of APP ⁷⁵¹ , including APP ¹⁹⁷ , APP ²¹⁹ and APP ⁷²⁰ An antibody is provided.

[0076] In a preferred aspect of the present invention, Neo either or exposure is produced by caspase-3 cleavage in the cleavage site APP ^{72 0,} designated the APP ⁷⁵¹ - antibodies capable of binding to an epitope are provided.

In another aspect, there is provided an antibody made using an antigenic determinant consisting of a peptide comprising at least 4 amino acid residues and comprising the caspase consensus sequence V / DXXD. This antibody is capable of recognizing a neo-epitope created or exposed after caspase-mediated cleavage of members of the amyloid precursor protein family, including APLP and APP. Preferably,
Members of the amyloid precursor protein family ^is a APP containing APP 695, ^{APP 7 51} and ^{APP 770.} More preferably, the member of the family is APP ⁷⁵¹ .

In a preferred aspect, based on APP ⁷⁵¹ , amino acid residues APP ^714- A
Antibody prepared using an antigenic determinant comprising the peptide sequence corresponding to PP ⁷²⁰ is provided.

In certain aspects, for the purpose of enhancing antigenicity, the antigenic determinant further comprises additional amino acid residues for coupling with the carrier protein KLH (keyhole limpet hemocyanin). This is a method well known in the art,
Any other means that can enhance antigenicity are within the scope of the invention.

In a more specific aspect, the coupling amino acid is at the amino terminus of the antigenic determinant.

In the most specific aspect, the coupling amino acid is cysteine and the antigenic determinants include KLH-cysteine-APP ^714- APP ⁷²⁰ .

In another aspect of the invention, the antibody is a purified, polyclonal antibody that specifically binds to the target, ie, the created or exposed epitope. Monoclonal antibodies made with the antigenic determinants of the present invention for the purpose of recognizing caspase cleavage products of APLP and APP using conventional methods are also within the scope of the present invention.

In another aspect, the invention provides a method for providing an antiserum comprising an antibody capable of binding to a neo-epitope created or exposed following caspase-mediated cleavage of APLP1 and APP. An antigenic determinant for immunizing is provided.

[0084] In another aspect of this embodiment, the antigenic determinant comprises a peptide having at least four amino acid residues and containing the caspase consensus sequence V / DXXD.

In another aspect of the invention, a caspase consensus sequence V having at least four amino acid residues
Immunizing a mammal with a peptide containing / DXXD, providing an antiserum containing an antibody to the peptide, collecting the antiserum, and specific for a neo-epitope generated by caspases Methods for generating antibodies having the ability to specifically bind to neo-epitope created or exposed after caspase-mediated cleavage of APLP and APP, comprising selecting a specific antibody Is done.

The presence of APP and amyloid β-peptide has been studied in a number of tissues, including the plasma of transgenic mice by Carroll et al. In individual CSF and by Fukuchi et al. These references indicate that APP and APP degraded species are found in tissues other than the brain. APLP and APLP caspase breakdown products are also believed to be found in tissues other than the brain. Therefore, A in tissues such as blood, plasma and CSF
The presence of caspase cleavage products of PLP and APP will allow easy access to clinical specimens that can detect and assess neuronal apoptosis. Accordingly, the antibodies of the present invention are useful in diagnostic methods that can detect cleavage products of APLP and APP in clinical specimens. More specifically, the antibodies of the present invention provide a diagnostic tool for detecting at least one caspase-cleaved APP product in a clinical specimen.

The antibodies of the invention having the ability to specifically recognize neo-epitope exposed after caspase-mediated cleavage of APP are useful in methods for diagnosing conditions in which neuronal apoptosis is present. Examples of such conditions include Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, progressive multiple sclerosis, head injury, prion-related conditions, Creutzfeldt-
Jacob's disease, spongiform encephalopathy, Friedreich's
Ataxia, fatal familial insomnia, Pelizeus-Merzbacher (Peli
neurodegenerative diseases such as zaeus-Merzbacher's disease, schizophrenia, dentate nucleus pallidum pallidum atrophy, spinal cord cerebellar atrophy type 3, spinal medulla muscular atrophy, spinal cord injury, stroke and brain injury. included. Since caspase-cleaved fragments of APP can be detected in mammals using tissues other than brain, their detection can be used as an indicator of disease detection and disease progression. Non-limiting examples of readily available tissues for use in diagnostic methods in mammals include plasma and cerebrospinal fluid.

[0088] In certain embodiments, the antibodies of the invention can be used in the detection of apoptotic neuronal cells.

More specifically, it is possible to follow the progression of apoptotic neuronal cells by using the antibodies described herein.

In one application of the invention, there is provided the use of these antibodies in the selection of a test agent as a modulator of apoptosis. Modulatory compounds include inducers and inhibitors of apoptosis.

In one aspect of the present application, neuronal cells can be artificially induced into apoptosis, and the antibodies of the invention can be used to follow the progress of the apoptotic process. In particular, antibodies can be used to study the development of neuronal apoptosis.

In another aspect of the present application, the progression of apoptosis in the presence or absence of a test compound can be assessed and the results compared.

In a specific aspect of the present application, an assay system for selecting a modulator of neuronal apoptosis is provided.

[0094] In a more specific aspect of the present application, an assay is provided for identifying an inhibitor of neuronal apoptosis. Such assays can be extended to high-throughput systems.

In one aspect of the present application, a candidate inhibitor identified using an antibody of the invention comprises:
Such treatment may be included in a pharmaceutical composition for treating a mammal, including a human, in need thereof.

In a further aspect of the invention, antibodies generated to detect the product of caspase-mediated cleavage of APP may be included in a kit. Such a kit would include at least one antibody capable of binding at least one neo-epitope exposed after caspase-mediated cleavage of APP. It has applications for research or diagnostic purposes.

The present invention is further illustrated in connection with the following examples.

Example 1 Recombinant Engineering All APP clones were derived from human APP ⁷⁵¹ . The following clone notation is: [construct]: [vector]: [insertion site / insertion release]: [sense direction]: [
Identifier]. Clones were cloned into P
Generated by CR-mediated template modification and fully sequenced before use. in vit
ro clones for transcription / translation include:

[0099]

Embedded image Is included.

The mammalian expression clones include:

[0101]

Embedded image Is included.

Example 2 Stable Cell Lines To generate stable cell lines, B103 cells (rat neuronal cell line) or K562 cells (human erythroleukemia cell line) were transfected with the indicated pCEP4 plasmid and then Continuous selection with mycin. The level of expression during each course of the experiment was determined by immunoblotting to confirm that each cell line expressed comparable levels of each APP construct. The following cell line notation:

[0103]

Embedded image It is in the format. Stable cell lines include:

[0104]

Embedded image Is included.

Example 3 An antibody that specifically recognizes the APP neo-epitope generated by caspase (
Generation of αΔC ^Csp- APP) 7 amino acids (N-terminal APP ^{714 to} C-terminal APP ⁷²⁰ (inclusive)) and KLH of APP followed by a caspase cleavage site in aspartic acid 720
A peptide containing an amino-terminal cysteine residue that allows coupling with
One rabbit was immunized. Rabbits were boosted three times with the immunizing peptide over 10 weeks. At 10 weeks, titer against the immunizing peptide (determined by ELISA, using the chromogenic substrate AΒTS against horseradish peroxidase, maximal dilution giving a positive reaction optionally set at OD ₄₅₀ = 0.2 Was> 1: 204,800 and 1: 12,520. Antisera were pooled and affinity purified by three successive chromatography steps. (
1) Sepharose 4B (Pharmacia (Pharmacia)
ia)) by immobilization on the bridging peptide containing a caspase cleavage site (N-terminal APP ^{713 to} C-terminal APP ⁷²⁶ (inclusive))
Immunoglobulins that recognize intact APP were depleted from the antiserum. (2) Step (1)
)) (Immobilized on a support as described in (1))
It was applied to a Sepharose 4B column containing the immunizing peptide, the column was washed, the specific antibody was eluted by a pH gradient and placed in borate buffer (0.125 M borate). (3) The eluate from the second step was adsorbed on the immobilized bridging peptide as described in (1) and the flow-through was collected. The ELISA titer of this antibody preparation is <1: 142,000 (<5 ng / ml) for the immunizing peptide corresponding to the neo-epitope generated by caspases and the bridge corresponding to the complete APP > 1:71 (>
10 μg / ml).

Example 4 Immunocytochemistry of caspase-cleaved fragments of APP in apoptotic NT2 cells
Sub-confluent NT2 cells were isolated from 50 μM Z-VAD (OMe) -CH ₂ 37 ° C., 5% CO 2 in the presence or absence of F₂For 4 hours at 1 μg / ml
Incubated with camptothecin. Can
Putthecin and Z-VAD (OMe) -CH₂D used to dilute F
The final concentration of MSO was 0.3% (v / v). As a control, 0.3% (v
/ V) Cells were incubated with DMSO only. Cell dissociation solution (Cell
 Plate cells with Dissociation Solution (Sigma)
And pooled with the culture medium (retaining floating apoptotic cells).

After centrifugation, cells were fixed in 10% neutral buffered formalin for 10 minutes at room temperature, washed twice with phosphate buffered saline, and then polystained using a Cytospin centrifuge. -Glass slides coated with L-lysine (Sigma (
Sigma)) and dried overnight. Then, TUNEL and αΔC ^Csp- APP or 1/3 of a 1/4000 dilution were performed essentially as described previously (Black et al., 1998) except that the proteinase K treatment was omitted.
Cells were co-stained for any of the 000 dilutions of αCsp-3 (MF397).

Example 5 In Vivo Model of Acute Brain Injury All animal manipulations were performed by the Canadian Medical Research Council (Medical Research).
h. Guide for the Care and Use for Laboratory Animal Care and Use, as recommended by the Council of Canada.
of Expertial Animals) (Canadian C
original on Animal Care, 1993).

For the treatment with kainate, adult male mice (C57 / B6 × XJL; 25-30 g)
Charles River, Montreal (Mon)
treal)), a 35 mg / kg kainic acid was injected intraperitoneally by bolus injection. 4
Eight hours later, animals were euthanized with an overdose of pentobarbital (100 mg / kg, ip). Brains were harvested, dissected and analyzed as described below.

For transient forebrain ischemia, adult male Wistar rats (250-
275 g; Charles River, Montreal) was used for all experiments. Temporary forebrain ischemia is based on the 4-vessel occlusion (4-VO) procedure (Pulsinelli et al., 1982).
) (Pulsinelli and Buchan, 19).
88). Animals that had convulsions during 4-VO or after reperfusion were excluded from the study, along with animals that did not fully develop a defect in righting reflex during ischemia. In the sham-treated animals, the carotid artery was exposed but no occlusion was performed.

Example 6 TUNEL and Fluorescent Immunohistochemistry of Rodent and Human Brain Fresh frozen brain sections (12 μm thick) were prepared with 0.1% (v / v) Triton X-100.
Was pretreated with PBS containing Then, a primary antiserum that selectively recognizes active caspase-3 (αCsp-3 (MF397)); (p17 / p12) rabbit polyclonal antiserum raised against ₂ caspase 3; Sections with purified immunoglobulin recognizing the ΔC-APP neo-epitope used or generated by caspases (αΔC ^Csp- APP (see above) used at 1/2000 dilution) at 4 ° C. for 48 hours Was incubated. After washing three times with PBS (5 minutes each), sections CY ³ labeled donkey anti-rabbit IgG (
1/500, Amersham) for 2 hours at room temperature to visualize the immunoreactivity of active caspase-3 or ΔC-APP. DNA fragmentation characteristic of apoptosis was detected by TUNEL as previously described (
Xu et al. , 1997).

Example 7 DNA Ladder Formation DNA extraction for visualizing oligonucleosome fragmentation was performed as follows. Subconfluent NT2 cells were isolated from 33.3 μM Z-VA.
Incubated with 1 μg / ml camptothecin for 6 hours at 37 ° C., 5% CO ₂ in the presence or absence of D (OMe) -CH ₂ F. As a control, cells were incubated with vehicle only (0.2% (v / v) DMSO). Cells are removed from the dish by detachment, pooled with culture medium, and 10,000 xg
For 10 minutes. Add cell pellet (2 × 10 ⁵ cells / pellet) to 0.
The cells were resuspended in 5 ml of 0.6% (w / v) SDS, 10 mM EDTA (pH 7.5), and NaCl was added to a final concentration of 1M. After overnight incubation at 4 ° C., cells were clarified by centrifugation at 4 ° C., 14,000 × g, 20 minutes, and treated with RNase containing 7 μg / ml non-DNase at 37 ° C. for 45 minutes. The DNA was phenol / chloroform / isoamyl alcohol (25: 24: 1
, V / v / v), isopropanol precipitated, loading buffer (30% (v / v) glycerol, 10 mM EDTA pH 7.5, 0.05
% (W / v) bromophenol blue) and 1 × TAE (40 mM
Analyzed by electrophoresis on a 1.2% agarose gel in Tris acetic acid, 1 mM EDTA pH 8.3). After ethidium bromide staining, bands were visualized.

Example 8 Other Procedures Recombinant human caspases were generated as previously described (Roto).
nda et al. Thornbury et al., 1996; , 1997), 50 mM HEPES / KOH (pH 7.0), 10% (w / v) sucrose, 2 mM EDTA, 0.
In vitro cleavage assays were performed in a buffer composed of 1% (w / v) CHAPS, 5 mM dithiothreitol. Peptide aldehyde and fluorogenic aminomethyl coumarin were prepared by solid phase synthesis essentially as described previously (Rano et al., 1997). _{Z-VAD (OMe) -CH 2} F, pan - caspase inhibitor (. Garcia-Calvo et al., 1998) were purchased from Enzyme Systems Products (Enzyme Systems Products). NT2 cells (human neuron progenitor cells) and B103 cells (rat neuron cells) were treated with antibiotics and 10% (v / v) fetal bovine serum (FBS).
Was maintained in DMEM containing Retinoic acid differentiated NT2 neurons (hNT
) Were maintained in neuronal conditioned medium (Stratagene) except for the course of serum deprivation experiments cultured in DMEM (-/ + 10% (v / v) FBS). K562 cells (human erythroleukemia) and Jurkat cells (human T lymphocytes) were maintained in RPMI-1640 containing antibiotics and 10% (v / v) FBS. Apoptosis was indicated by 1 μM staurosporine (whole cell line) or 1 μg / m 2 as indicated.
Induced by CD95 (Fas / APO-1) ligation by using 1 μg / ml anti-Fas antibody (Jurkat only) with 1 camptothecin (whole cell line). Cell extracts were prepared as previously described (Nichol
son et al. , 1995).

Results Neuronal NT2 cells showed elevated levels of amyloid-β during apoptosis.
Produces a peptide, which is attenuated by caspase inhibition.

Apoptotic primary human neurons have been shown to increase the rate of amyloid-β peptide formation by 3- to 4-fold (LeBlanc, 19).
95). Therefore, whether this can be reproduced in cultured human neuronal cell lines,
And whether apoptosis inhibition prevented Aβ production. Retinoid-differentiated human neuronal NT2 cells (hNT) induced to undergo apoptosis by serum deprivation have an essentially increased rate of Aβ peptide formation (approximately 4
(Fold) (FIG. 1A), this increase in apoptosis-related Aβ formation was attenuated by a non-selective, irreversible caspase inhibitor. The rate of Aβ production in the presence of caspase inhibitors was restored to the steady-state levels found in non-apoptotic hNT cells, but was not completely abolished, which indicates that apoptosis enhances the pathway to Aβ production But not essential for its occurrence. Induction of acute apoptosis of the neuronal progenitor cell line NT2 by camptothecin (FIG. 1B) results in oligonucleosomal DNA fragmentation, while concomitantly maturating procaspase-3, (PAR
P; cleaved caspase-3 substrate (such as poly (ADP-ribose) polymerase) and resulted in an approximately 3.5 kDa decrease in the mass of endogenous APP (lane 2). The degree of proteolysis of all three polypeptides was nearly identical (procaspase-3 = 37 ± 4%; PARP = 66 ± 9%; APP = 44 ± 4%; n =
5), in all cases, disappeared by co-incubation with caspase inhibitor (lane 3). This indicates that APP is processed by a caspase-dependent event during apoptosis, suggesting that caspase cleavage may directly contribute to the relatively high propensity to form Aβ peptides observed in apoptotic cells. Is shown.

Caspase-3 cleaves amyloid-β precursor protein directly and efficiently.

Neuronal apoptosis includes APP protein cleavage and Aβ peptide formation
And the biochemical components of the cell death pathway contribute to this process
Or not. Apoptosis itself is a sub-component of cellular polypeptides.
Cleavage of the set at the Asp-x junction exhibits an apoptotic phenotype
A family of cysteine proteases that depend on caspases (Alne
mori et al. , 1996; Nicholson and Thornbury,
1997; Thornberry et al. , 1997). Core group for caspases
The quality recognition motif is a tetrapeptide (P₄ ~ P₁), And P₁Asp is an absolute requirement for cleavage and P ₄ Residues are key specificity determinants. Caspases are usually found in healthy cells
Exists as an inactive proenzyme, but in response to various apoptotic stimuli,
It is converted to active protease by proteolysis. First,³⁵S] Sign AP
P with extracts from healthy Jurkat cells and apoptotic Jurkat cells
Together, they allow for potential caspase potential in APP processing.
The direct role was tested (FIG. 1C). The first choice for Jurkat cells was
Several members of the caspase family that are readily activated during
Because it contains Sparses-2, -3, -4, -6, -7, -8 and -9)
Yes (unpublished). The 120 kDa APP is relatively small, approximately 85-90 kDa.
It was rapidly processed into triads of polypeptides. These polypeptides
Show that primary human neurons that have depleted trophic support (LeBlanc, 19
95; LeBlanc et al., 1996), or H₂O₂Humans during induced apoptosis
APP observed in neuron cell lines (Zhang et al., 1997).
The mass is consistent with the processing intermediate, indicating that AP
In vitro proteolysis of P represents a phenomenon in neuronal cells
Suggests that A similar APP cleavage product is the NT2 human neuronal precursor
Apoptotic extracts from other cell types, including cells and B103 neuroblastoma cells
Therefore, it was also generated (not shown). In complete NT2 cells during apoptosis
The observation of a single APP cleavage event (FIG. 1B) indicates that the APP is transmembrane.
Limits the proteolytic susceptibility of the cleavage sites, and the three cleavage sites
One of them suggested that it may be more vulnerable in vivo
ing. The protease inhibitor profile shows that all three cleavage products
Consequences of a non-E64-sensitive cysteine protease that is characteristic of zeolites
(Not shown). This is because the caspase-specific peptide-aldehyde
Confirmed by being able to block APP processing to 85-90 kDa triad
(FIG. 1D). The caspase inhibitor Ac-DEVD-CHO is an APP process.
Is a potent inhibitor of₅₀= 1 nM), Ac-IETD-CHO and
The effects of Ac-YVAD-CHO were all substantially lower. Jurkat cells
Contains all three caspase subgroup members,
Results include caspase-3, a key mediator of neuronal apoptosis
Group II caspases are the major source of APP cleavage observed in apoptotic cells
Is the cause. The same result was obtained in NT2 neuron progenitor cells.
As a result, caspase-3 was the major caspase in this cell line.
([Caspase-3] about 55 pM; all other caspases <14 pM; unpublished
), It is not surprising. Overall, these results indicate that caspase-3
Is a major cause of APP proteolysis during apoptosis.
To support this, the 120 kDa APP has been converted to 85-90 kDa triads.
Protein degradation was also reproduced by recombinant caspase-3 (FIG. 1E) and
Analysis of the dynamics showed that APP protein cleavage by caspase-3 was poly (ADP-
Ribose) polymerase (k in the same experiment)_cat/ K_m= 15.6 × 10⁵ M^-1s^-1; Not shown).
Sharp dynamics (k_cat/ K_m= 5.14 × 10⁵M^-1s^-1Happens in)
It is shown that. The tested human caspases (caspase-1-10 and
Granzyme B; each tested at equimolar concentrations under the same conditions
Caspase-3 was the most efficient enzyme to cleave APP
However, slight cleavage occurred also by caspase-6 and -8 (not shown).
No).

Caspase-3 levels are elevated in moribund neurons in the human Alzheimer's brain.

If caspase-3 is involved in APP processing and neuronal apoptosis, it must be at the site of Aβ production and neurodegeneration.

Caspase-3 (CPP32, apopain, Yama) (
Fernandes-Alnemri et al. , 1994; Nicholson et al.
, 1995; Tewari et al. , 1995) is one of the important effector caspases in mammalian cell suicide. Of particular importance in neuronal cell death is that caspases with severe defects in developing neurons and apoptosis, leading to brain enlargement (2-3 times), excessive cell weight and double brain structure. 3 deficient mouse phenotype (Kuida et al., 199).
6). The present inventors have shown that in patients with Alzheimer's disease, caspase-3 immunoreactivity is increased in moribund pyramidal neurons in the hippocampus including the CA3 field, which is one of the main sites of neuronal deficiency in Alzheimer's disease. Found (FIG. 2). This contextual evidence indicates that caspase-3 mediates or contributes to these cell deaths in neurodegeneration, and that caspase-3 mediated proteolytic cleavage of APP indicates that human Alzheimer's disease It suggests that this process is involved as a component of the pathogen.

Identification of Three Major Sites of Caspase-Mediated APP Protein Cleavage The sites of caspase-induced proteolysis within the 120 kDa APP polypeptide were determined. First, this was facilitated by placing a non-existent Cys residue at the amino terminus of APP. [ ³⁵ S] C by recombinant caspase-3
Treatment of the ys-tagged APP produced two small [ ³⁵ S] Cys-containing fragments (23 and 25 kDa) and a relatively large polypeptide (85 kDa), indicating that two of the caspase cleavage sites were the first of APP. Located at the N-terminal 220 amino acids, a third site was shown to be near residue 700 (not shown). The latter site was further localized by truncating the APP construct lacking the N-terminal 220 amino acids (ΔN-APP). ^[35 S] The Cys APP and ^[35 S] 3 one of the regions predicted to contain caspase cleavage site by analysis of fragment sizes ^{Met APP, APP 194 ~APP 197,} APP 21 6 ~APP 219 and ^{APP 717} ~APP to ⁷²⁰ (inclusive), good caspase-consensus site (Thornberry et al., 1997) was present. These sites are (P ₄ ) DNVD ¹⁹⁷ (P ₁ ) / S, (
_{^{P 4) DYAD 219 (P 1}} ) / G and _{^{(P 4) VEVD 720 (P}} 1) is named / A (FIG. 3B). Site-directed mutagenesis of the essential P ₁ Asp at each site confirmed that these were in fact proteolytic sites by caspases (FIG. 3A). For example, the APP triple mutant (APP D ¹⁹⁷ A,
D ²¹⁹ A, D ⁷²⁰ A) can also be produced by recombinant caspases (lanes 7 & 8).
It could not be degraded by extracts from apoptotic cells (not shown). (Equivalent results were also observed for the low molecular weight counterpart of the fragment described in FIG. 3A (not shown).)

[0122] Caspase-mediated APP proteolysis, in whole cells,
Occurs mainly in Asp ⁷²⁰ and rises during apoptosis.

Next, stable transfection of B103 neuron cells with APP containing either wild-type or P ₁ Asp to Ala mutation,
Cleavage at the above sites was tested in whole cells. (B103 cells were selected because of the extremely low level of endogenous APP and ease of analysis of transfected site-specific mutants.) B103 retaining stably transfected wild-type APP When we induced the cells to undergo apoptosis,
Caspase-mediated carboxy-terminal (P ₄ ) VEVD ⁷²⁰ (P ₁ ) /
A cleavage at A was observed (FIG. 4A, lanes 1 & 2). This disconnect event
Triple mutant (not shown) or ^D720A point mutant (lanes 5 & 6)
Completely demonstrated that caspases were directly involved in APP processing in whole neuronal cells. Non-apoptotic cells also cleave APP at the carboxy terminus at approximately 15% of the rate that occurs in apoptotic cells (lanes 1 vs. 2), also due to a mutation of P ₁ Asp required for caspase recognition (D ⁷²⁰ A). It was removed (lane 5). VE
Cleavage at the VD ^720A site is consistent with the approximately 3.5 kDa decrease in APP observed during apoptosis in NT2 cells (see FIG. 1B, lane 2).
At least in the cell lines tested, it is likely to be the major site of proteolysis by caspases in whole cells (although APP fragments corresponding to cleavage at two upstream sites have been observed (Zhang et al., 1988). 1997
)). The definitive identification of this C-terminal truncation event is due to the Δ
It was confirmed in situ using a neo-epitope antibody generated by the inventors for immunohistochemical detection of C-APP. The antiserum was composed of a synthetic peptide [KLH-cysteine-APP ^714- AP
P ⁷²⁰ ] and then immunoadsorbed to the same peptide (immunoa
purified by two cycles of immunodepletion using a bridging peptide corresponding to the complete APP, including the cleavage site, including the amino acid residues APP ⁷¹⁴ -APP ⁷²⁶ (inclusive). . This antibody, termed αΔC ^Csp- APP, is based on three criteria:
It was confirmed to be highly specific for neo-epitope generated by caspases in APP. a) The titer by ELISA indicates that the immunizing peptide (ΔC
-Equivalent to APP), the bridging peptide (complete A)
> 2000 fold more selective (equivalent to PP) (see method). b) The antibody was able to immunoprecipitate biosynthetic [ ³⁵ S] APP truncated at the ^D720 caspase site, but not complete APP (FIG. 4).
B). And c) the antibody failed to immunoprecipitate complete APP from healthy NT2 cells, but subsequent apoptosis induction could immunoprecipitate the ΔC-APP cleavage product (FIG. 4C). In the latter case, the formation of immunoprecipitable ΔC-APP in NT2 cells was abolished by the presence of a caspase inhibitor (lane 3 vs. 2). Comparable results were also obtained by immunohistochemical staining of NT2 cells (FIGS. 4D-I). NT2 cells induced to undergo apoptosis by camptothecin became TUNEL positive, and at the same time, stained for active caspase-3 (panel E) and APP neo-epitope generated by caspase (panel H). All three of these apoptosis-related events were abolished by the presence of caspase inhibitors (panels F and I). Collectively, these experiments demonstrate that caspase-mediated proteolysis of APP at the C-terminal VEVD ⁷²⁰ A site occurs during apoptosis in whole cells. Further, the αΔC ^Csp- APP neo-epitope antibody is
The ability to recognize the end generated by caspase proteolysis in situ makes it a useful tool for tracking this cleavage event in vivo.

[0124] Caspase-mediated APP proteolysis is critical for acute brain injury
The early loss of hippocampal neurons that occurs in hippocampal neurons n vivo is a hallmark of Alzheimer's disease pathogenesis. Therefore, we decided to determine if caspase-mediated APP protein cleavage could be detected in vivo during hippocampal cell death. To investigate these events, two experimental acute brain injury models resulting in apoptotic hippocampal neuronal loss were used. That is, a
A) kainic acid (excitotoxicity) -induced seizures, in which CA3 neurons die mainly, and b) global cerebral ischemia, in which CA1 neurons disappear. These models have the additional advantage that acute brain injury is an established risk factor for Alzheimer's disease susceptibility, and may thus elucidate the molecular basis for this increased vulnerability. Excitotoxicity model of neuronal damage (FIGS. 5A-F)
In caspase-mediated cleavage of APP protein, as measured by αΔC ^Csp- APP immunoreactivity, was not detectable in the CA3 field of the hippocampus of control mice (panel A), but kainic acid administration (25 mg / kg, sc) 4
Eight hours later, CA129 neurons of SV129 mice had strong αΔC ^Csp- APP.
Immunoreactivity was observed (Panel B). Consistent with the production of this fragment by a caspase-3-dependent mechanism, a marked increase in active caspase-3 immunoreactivity was observed in the CA3 field of adjacent hippocampal tissue sections from animals injected with kainate. However, it was not observed in the vehicle injected animals (Panel D) (Panel C). TUNEL
The labeling confirmed that CA3 neurons showing immunoreactivity of αΔC ^Csp- APP and active caspase-3 were undergoing apoptosis (panel F). To further establish that caspase-mediated cleavage of APP proteins occurs in vivo, we have shown that apoptotic CA1 neuronal death caused by short-term global ischemia also reduced the production of ΔC-APP. It was determined whether an increase was involved (FIGS. 5G-L). No basal αΔC ^Csp- APP immunoreactivity was observed in the CA1 field of rats that underwent the sham surgical procedure (panel G).
Twenty-four hours after transient global ischemia caused by four-vessel occlusion for 12 minutes, αΔC ^Csp- APP immunoreactivity was clearly visible in fragile CA1 neurons (
Panel H). At 48 hours, αΔC ^Csp- APP immunolabeling was more intense compared to the 24 hour time point (panel I) and after 72 hours, a small amount of residual CA1
Immunoreactivity was seen only in neurons (panel J). In CA1 neurons following this brief global ischemia, the time course of generation of active caspase-3 immunoreactivity and αΔC ^{C sp} -APP immunoreactivity was similar (data not shown). In line with previous findings indicating that CA1 neurons undergo apoptosis after transient global ischemia, TUNE
L label was elevated (panel K). At high magnification, αΔC ^Csp- APP immunoreactivity was evident in both dendrites and cell bodies of dying CA1 neurons (
Panel L). Taken together, these results indicate that neuronal apoptosis in the rodent hippocampus caused by either excitotoxicity or ischemia damage indicates that ΔC-AP
It is shown to be associated with an increase in P formation. Consistent with our in vitro findings indicating that the ΔC-APP fragment is derived from caspase-mediated cleavage of APP proteins, increased in vivo ΔC-APP production is associated with apoptotic neurons. This was accompanied by the production of active caspase-3. Alterations in caspase-mediated APP processing following acute brain injury may also be part of the increased susceptibility to Alzheimer's disease in human patients. APP cleaved by caspase coexists with senile plaques in the human Alzheimer's brain.

The association of caspase-mediated cleavage of APP with senile plaque formation was investigated in human patients diagnosed with late-stage Alzheimer's disease (FIG. 6). Senile plaque and αΔ
C ^{Csp -APP} immunoreactivity, not suffering from Alzheimer's disease, age has not been observed only rarely in control patients matched. In contrast, a number of amyloid-β positive plaques were observed in hippocampal sections from patients suffering from Alzheimer's disease (panel B), and αΔC ^Csp- APP immunoreactivity was frequently observed in senile plaques ( > 9
0%) (Panel A). These deposits had a spot-like appearance and were localized in senile plaques (Panel C). However, it was not possible to morphologically define the phenotype of cells in which αΔC ^Csp- APP immunoreactivity is localized. These findings are consistent with the hypothesis that ΔC-APP produced by caspases contributes to plaque formation.

Caspase-mediated cleavage of APP protein increases the rate of amyloid-β peptide formation in neuronal cells.

Since APP is cleaved by caspases during neuronal injury in vivo, we next determined whether it had an effect on the endogenous APP processing pathway leading to Aβ peptide formation. ^{This, C-terminal,} truncated APP constructs corresponding to the product of proteolysis by caspases in ^{D 720} site (
(ΔC-APP) was tested in stably transfected B103 cells (FIG. 7). Aβ peptide production from these cells is based on full-length APP
And increased 5-fold compared to cells expressing equivalent levels of full length APP (column 2 to 1 at 20 h, 4 to 3 at 48 h).
One possible explanation for this effect is that given that caspase proteolysis is not a direct cause of Aβ excision from APP, caspase proteolysis is a contaminant to normal endogenous APP metabolism and probably complete Separation of the key interaction domain contained at the C-terminus of the APP polypeptide results in truncated molecules being more prone to be metabolized by the amyloidogenic pathway (summarized in FIG. 3B). For example, the Shc-like PTB domain of X11 is APP
Has been recently demonstrated to interact with the NPTY motif at the C-terminus of A. and reduce Aβ peptide formation by binding APP and delaying APP processing (Borg et al., 1998). It is likely that removal of this and other key components at the C-terminus of APP will bias the cytolytic pathway through the amyloidogenic pathway (Russo et al., 1998).

The “Swedish” familial mutation of APP improves an inadequate caspase consensus site in the β-secretase region. The three caspase cleavage sites (primarily is such processing sites in addition to an Asp ⁷²⁰⁾ (FIG. 3B), at least one proven familial Alzheimer's disease (FAD) mutation to introduce additional caspase sensitive sites in the molecule. "Swedish" mutations in APP are associated with early onset and 4k
It is associated with genotype Alzheimer's disease with increased production of Da amyloidogenic Aβ peptide (Cai et al., 1993; Citron et al., 1992; Ha).
ass et al. Holcomb et al., 1995; , 1998). The mutation is β
- ^{VKMD 653} sequence at secretase site ^(amino acid residue corresponding to ^APP 650 ~APP ^653), is changed to V NL ^{D 653} is a motif having a caspase recognition factor is substantially improved. In particular, the “Swedish type” mutations, as predicted by our positional scanning combinatorial substrate library (Thornberry et al., 1997), were identified as II.
Generate sites that will be preferentially recognized by group I caspases, especially caspase-6. To test this possibility, fluorogenic tetrapeptides corresponding to wild-type and "Swedish" sequences were synthesized and tested for their ability to be cleaved by recombinant caspase-6. In of VNL D-AMC (Swedish type)
vitro cleavage was lower than peptide V EH D-AMC containing optimal caspase-6 primary recognition sequence predicted by the combinatorial library, VKMD-AMC significantly than (wild-type) fluorogenic peptide High (Fig. 8
A). Differences also occurred for cytosolic extracts from apoptotic cells and full-length APP polypeptide using recombinant caspase-6 (FIG. 8B). In each case, the “Swedish” mutation accelerates the rate of proteolysis at the β-secretase site (columns 3 & 4), which is a mutation of P ₁ Asp essential for caspase recognition (D ⁶⁵³ A). Completely disappeared (column 5 &
6). The β-cleaving activity observed in apoptotic cell extracts was caspase-6
Is consistent with the fact that it is due to group III caspases (including caspase-6), its inhibitor profile (Ac-IETD-CHO potency>Ac-DEVD-CHO> Ac-YVAD- (CHO) supported (
Not shown). Measurement of the rate of caspase-6 mediated proteolysis of the β-secretase site (FIG. 8C) indicated that the “Swedish” mutation
As a caspase-6 site in the PP polypeptide, it was shown to improve it three-fold (k _cat / K _m = 0.8 × 10 ⁴ M ⁻¹ s ⁻¹ for wild-type APP, “Swedish type K _cat / K _m = 2 for APPs containing mutations
. 2 × 10 ⁴ M ⁻¹ s ⁻¹ ). Both wild-type APP and those containing the "Swedish" mutation, caspase-1, -3, -7 and -8, or granzyme B
Other caspases such as could not be cleaved at this site (not shown), indicating that this event was restricted to caspase-6.

Enhanced amyloid-β peptide formation occurs in cells carrying the “Swedish” mutation of APP, which is dependent on protein cleavage by caspases at the β-secretase site.

The result of the “Swedish” mutation in stably transfected K562 cells is a substantial increase in amyloid-β peptide production by the cells (FIG. 8).
D), including those reported previously (Citron et al., 199).
2; Citron et al. , 1995). The formation of the amyloidogenic 4 kDa Aβ peptide was dramatically reduced in K562 cells containing “Swedish” APP in which the P ₁ Asp required for proteolysis by caspases was removed from the β-secretase region (D ⁶⁵³ A, Lanes 3: 2, 6: 5), 3 kDa
Aβ peptide formation did not decrease. Aβ (1-40) sandwich ELI
SA gave the same results (substantially elevated Aβ production in cells carrying the “Swedish” mutation (65 ± 4 arbitrary units, whereas levels in cells carrying wild-type APP were undetectable) and P ₁ D ⁶⁵³ A Mutation Attenuation (1
6 ± 4 arbitrary units)). Similar results were seen in B103 neuron cells expressing the same construct (similar to that observed in K562 cells, the intracellular 4 kDa Aβ peptide formation was eliminated in the D ^{65 3} A cell culture supernatant). However, another cleavage producing an <4 kDa extracellular Aβ peptide (probably a cleavage at Phe ⁶⁵⁶ as previously reported (Citron et al., 1995)) occurred (not shown). Taken together, these findings indicate that caspases are directly involved in the enhancement of Aβ production that occurs in cells carrying the “Swedish” mutation of APP. Other evidence indicates that caspases (especially caspase-6), especially in “Swedish” Alzheimer's disease mutations,
It supports that it contributes to β-secretase activity. First, although there is heterogeneity at the amino terminus of the Aβ peptide, some of the resulting peptides are:
V (K or N) (M or L) corresponds to cleavage at the ^D653 / ^A654 caspase-6 site (Wang et al., 1996). Second, analysis of the amino acid residues in APP that make up the minimal recognition region of the “major protease” responsible for β-secretase activity (determined as Val ^{650 to} Ala ⁶⁵⁴ ), revealed a P ₄ -P ₁ 'caspase- 6 exactly coincides with the recognition site (Citron et al., 1995). Finally, caspase-6 is present in the hippocampus and frontal cortex of human Alzheimer brain samples (and age-matched controls) at levels that can be easily detected by immunoblotting (not shown). Taken together, these findings support a role for caspase-6 in APP cleavage at the β-secretase region and explain why Aβ peptide formation is elevated in patients with “Swedish” familial mutations. , Suggesting a mechanism assisted by caspases.

[0131] Caspases contribute to the complex proteolytic processing of the amyloid-β precursor protein and amyloidogenic A by at least two separate mechanisms.
It is thought to contribute to β peptide formation. Endogenous caspase consensus site ((P ₄ ) DNVD ¹⁹⁷ (P ₁ ) / S, (P ₄ ) DYAD ²¹⁹ (P ₁ )
Cleavage of APP at / G and the major (P ₄ ) VEVD ⁷²⁰ (P ₁ ) / A interferes with the normal intracellular processing of APP, which will probably prevent the formation or accumulation of Aβ peptides. Therefore, in a susceptible neuron, the following steps may cause a long-running “vicious cycle”. a) truncation of APP at the C-terminus mediated by caspase-3; b) contamination of normal APP processing by diverting the remaining polypeptide into the amyloidogenic pathway;
A) generation of elevated levels of cytotoxic Aβ peptide species resulting in Aβ-induced neuronal stress; d) progressive up-regulation and / or activation of endogenous caspases; and e) exacerbated APP protein cleavage. In addition, elevated levels of APP (susceptible to caspase cleavage) have been reported in moribund motoneurons, which could put additional APP in the cycle (Barnes et al., 1998). The caspase proenzyme, although at a lower level compared to the mature counterpart, is catalytic and does not necessarily require an apoptosis initiation event for this cycle to proceed (Muzio).
Et al. Srinivasula et al., 1998; , 1998; Yang et al. , 19
98). The second mechanism involves caspase-6 mediated proteolysis in the β-secretase region. The process is accelerated by residue changes at this site, such as "Swedish" dipeptide mutations that improve sensitivity to this enzyme. Caspases have recently been shown to be involved in other presenilin proteolysis in addition to APP cleavage (Kim et al., 1997; Vito et al., 1997). Thus, caspases may play an important role both in the production of neurotoxic Aβ peptides in Alzheimer's disease and in the eventual death of neurons by apoptosis.

[0132]

[Table 1]

[0133] All references cited herein are fully incorporated herein by reference.

[Brief description of the drawings]

FIG. 1A shows that caspase inhibition eliminates enhanced amyloid-β peptide production in apoptotic neuronal cells.

FIG. 1B shows caspase-related APP protein cleavage during apoptosis.

FIG. 1C shows proteolysis of amyloid-β precursor protein by group II caspase in apoptotic cell extracts.

FIG. 1D shows proteolysis of amyloid-β precursor protein by group II caspase in apoptotic cell extracts.

FIG. 1E shows the degradation of amyloid-β precursor protein by recombinant caspase-3.

FIG. 2A shows increased caspase-3 immunoreactivity in dying pyramidal neurons in the CA3 area of the Alzheimer's brain hippocampus. FIG. 2 shows paraffin-embedded sections from age-matched neurologically normal individuals.

FIG. 2B shows increased caspase-3 immunoreactivity in dying pyramidal neurons in the CA3 field of the Alzheimer's brain hippocampus. It is a figure which shows the paraffin embedding section from a patient with Alzheimer's disease.

FIG. 3A shows the identification of sites of caspase-mediated proteolytic cleavage of the amyloid-β precursor protein and the removal by mutation.

FIG. 3B shows the location of caspase cleavage sites within the amyloid-β precursor protein in relation to other structural features.

FIG. 4A shows caspase-dependent cleavage of amyloid-β precursor protein in intact cells undergoing apoptosis, and in situ detection of C-terminal neo-epitope generated by caspase. FIG. 4 shows C-terminal truncation of APP during apoptosis in transfected B103 cells.

FIG. 4B shows caspase-dependent cleavage of amyloid-β precursor protein in intact cells undergoing apoptosis, and in situ detection of C-terminal neo-epitope generated by caspase. FIG. 3 shows the specific recognition of APP neo-epitope generated by caspase by αΔC ^Csp- APP.

FIG. 4C shows caspase-dependent cleavage of amyloid-β precursor protein in intact cells undergoing apoptosis and in situ detection of C-terminal neo-epitope generated by caspase. FIG. 4 shows C-terminal truncation of APP during apoptosis in transfected B103 cells.

FIG. 4D shows in situ detection of ΔC-APP neo-epitope generated by caspases during NT2 cell apoptosis.

FIG. 4E shows in situ detection of ΔC-APP neo-epitope generated by caspases during NT2 cell apoptosis.

FIG. 4F shows in situ detection of ΔC-APP neo-epitope generated by caspases during NT2 cell apoptosis.

FIG. 4G shows in situ detection of ΔC-APP neo-epitope generated by caspases during NT2 cell apoptosis.

FIG. 4H shows in situ detection of ΔC-APP neo-epitope generated by caspases during NT2 cell apoptosis.

FIG. 41 shows in situ detection of ΔC-APP neo-epitope generated by caspases during NT2 cell apoptosis.

FIG. 5A shows excitotoxic damage in SV129 mice.

FIG. 5B shows excitotoxic damage in SV129 mice.

FIG. 5C shows excitotoxic damage in SV129 mice.

FIG. 5D shows excitotoxic damage in SV129 mice.

FIG. 5E shows excitotoxic damage in SV129 mice.

FIG. 5F shows excitotoxic damage in SV129 mice.

FIG. 5G shows the effect of ischemia damage.

FIG. 5H shows the effect of ischemia damage.

FIG. 5I shows the effect of injury due to ischemia.

FIG. 5J illustrates the effect of ischemia damage.

FIG. 5K is a graph showing the effect of injury due to ischemia.

FIG. 5L shows the effect of ischemia damage.

FIG. 6A is a view showing the coexistence of ΔC-APP and amyloid-β in senile plaques. FIG. 7 shows ΔC-APP visualized by αΔC ^Csp -APP immunoreactivity in the same hippocampal range as shown in (B).

FIG. 6B is a diagram showing the coexistence of ΔC-APP and amyloid-β in senile plaques. FIG. 2 shows senile plaques identified by amyloid-β immunoreactivity (arrow) in the hippocampus of a patient diagnosed with Alzheimer's disease.

FIG. 6C shows the coexistence of ΔC-APP and amyloid-β in senile plaques. FIG. 4 is a synthetic image of caspase-generated APP neo-epitope (red) and Aβ (green), giving a yellow color when matched, illustrating the high degree of overlap between ΔC-APP and amyloid-β immunoreactivity. ing.

FIG. 7 shows that caspase-mediated APP production increases amyloid-β peptide formation. Comparable levels of full length APP (wt, column 1
& 3) or the B103 stable cell line described for FIG. 4A expressing APP lacking the C-terminal residue after the Asp ⁷²⁰ caspase cleavage site (αΔC-APP, columns 2 & 4) or Ala ^{721 to} Asn ⁷⁵¹ After culturing for 20 or 48 hours in an appropriate medium, the level of Aβ peptide in the culture medium was quantified by immunoassay using a monoclonal antibody. Representative experiments (n = 4) ± SD are shown.

FIG. 8A shows that the “Swedish” familial mutation improves the β-secretase target region of APP as a caspase-6 substrate. VKMD ⁶⁵³ / A ⁶⁵⁴ (VKMD-AMC, ；; see FIG. 3B), β-secretase target region of APP (see FIG. 3B), “Swedish” dipeptide mutation (V NL D-AMC, ●) or positional scanning combinatorial substrate live FIG. 4 shows a fluorogenic tetrapeptide-aminomethylcoumarin synthesized to correspond to one of the optimal tetrapeptides for caspase-6 ( VEHD -AMC, □) predicted by Larry.

FIG. 8B. “Swedish type” familial mutations indicate that APP as a caspase-6 substrate
FIG. 3 shows that the β-secretase target region is improved. N-terminal caspase cutting
The cleavage site was removed (ΔN-APP) and then the wild-type β-secretase region (VKM
D⁶⁵³/ A⁶⁵⁴Columns 1 & 2), "Swedish" mutation (VNLD ⁶⁵³ / A⁶⁵⁴Column 3 & 4) or P essential for caspase recognition₁ A
"Swedish" mutation in which sp is changed to Ala (VNLA ⁶⁵³/ A ⁶⁵⁴ Efficacy of an APP construct engineered to contain any of columns 5 &6);
It is a figure showing a result.

FIG. 8C shows that the “Swedish” familial mutation improves the β-secretase target region of APP as a caspase-6 substrate. It is a figure showing measurement of cutting.

FIG. 8D shows that the “Swedish” familial mutation improves the β-secretase target region of APP as a caspase-6 substrate. Wild-type ^{VKMD 65} 3 ^{/ A 654} sites, "Swedish" mutation ^{(V NL D 653 / A 654} )
Or stable _P 1 Asp was shown created to hold the full length APP with a change to have "Swedish" mutation ^{(V NL A 653 / A 654} ) to Ala K56
FIG. 2 shows immunoreactive products in a two-cell system.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme court ゛ (Reference) G01N 33/566 G01N 33/566 (72) Inventor Roy, Sofie Ashe 9 Aseyu, Quebec, Canada・ 3 ・ 1 ・ 1, Kirkland, Trans-Canada Highway 16711 (72) Inventor Nicholson, Donald Dubrill Canada, Quebec-Aciyu ・ 9 ・ Ashiu ・ 3 ・ 1 ・ 1, Kirkland, Trans- Canada Highway 16711 (72) Inventor Shiyu, Daiken Canada, Quebec Ashyu 9 Ashyu 3 El, Kirkland, Trans-Canada Highway 16711 (72) Inventor Robertson, George Quebec, Ashyu, 9, Ash, Canada • 3 El 1, Kirkland, Trans-Canada Highway 16711 (72) Inventor Juan, Ching Chi, Canada, Quebec Ashyu 9 Ashyu 3 El 1, Kirkland, Trans-Canada Highway 16711 F term (reference) 4B064 AG27 CA10 CD20 CE12 DA13 4H045 AA11 AA20 BA42 CA40 DA76 EA50 FA71 GA26

Claims (16)

[Claims] 1. An antibody that recognizes a neo-epitope created or exposed after caspase-mediated cleavage of APP and APLP. 2. An antigenic determinant having a consensus sequence for a caspase cleavage site consisting of at least four amino acids, wherein the consensus sequence is V
2. The antibody of claim 1, comprising / DXXD. 3. The antibody according to claim 1, wherein the APP is cleaved by a caspase selected from caspase-3, caspase-6, and caspase-8. 4. The antibody according to claim 3, wherein APP and APLP are cleaved by caspase-3. 5. The antibody of claim 4, wherein the neo-epitope created or exposed after caspase-mediated cleavage of APP is cleaved by caspase-3. 6. APP ^is 197,2 based on the amino acid number of the ^{APP 751}
6. The antibody of claim 5, comprising three caspase-3 cleavage sites designated 19 and 720. 7. The antibody of claim 6, which is capable of binding to a neo-epitope created or exposed after caspase-3 cleavage at the designated cleavage site 720. 8. The method according to claim 8, comprising at least four amino acid residues, and
An antibody produced using an antigenic determinant comprising a peptide containing the consensus sequence V / DXXD. 9. The antibody according to claim 8, wherein the antibody is produced using an antigenic determinant containing a peptide having an amino acid sequence of APP ^{714 to} APP ⁷²⁰ . 10. The antibody of claim 9, wherein the antigenic determinant is coupled to an antigenic enhancer. 11. The coupled antigenic determinant is KLH-cysteine-A
Including PP ⁷¹⁴ ~APP ^720, antibody of claim 10. 12. The antibody according to claim 1, which is polyclonal. 13. The antibody according to claim 1, which is monoclonal. 14. An antiserum comprising an antibody capable of binding a neo-epitope exposed after caspase-mediated cleavage of APP and APLP. 15. A method for generating an antibody capable of binding to a neo-epitope created or exposed after caspase-mediated cleavage of APP and APLP, comprising at least four amino acid residues And a caspase-consensus sequence V
Immunizing a mammal with a peptide containing / DXXD to provide an antiserum containing an antibody to the peptide, collecting the antiserum, and specific for a neo-epitope generated by caspases A method comprising selecting a suitable antibody. 16. A method for detecting or characterizing the level of caspase-mediated apoptosis in a mammalian patient, comprising obtaining a tissue sample from a patient suspected of having neuronal apoptosis. AP cleaved with caspase by contacting with the antibody described in
A method comprising detecting the presence or amount of P and APLP, and comparing the amount of caspase-cleaved APP and APLP to a standard.