Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

• hippocampal neurons by apoptotic cell death is a prominent feature of Alzheimer's disease (Cotman and Su, 1996; Li et al., 1997; Smale et al., 1995; Su et al., 1997).
• One potential factor contributing to the susceptibility of these cells to premature death arises from the cytotoxic' effects of amyloid- ⁇ peptide deposition at or near sites of neuronal degeneration.
• Cultured human cells, including neurons undergo apoptotic cell suicide when treated with amyloidogenic A ⁇ peptides (Forloni et al., 1996; LaFerla et al., 1995; Loo et al., 1993).
• apoptotic machinery must contribute either directly or indirectly to the complex proteolytic processing (Haass and Selkoe, 1993; Selkoe et al., 1996; Sisodia and Price, 1995) of the amyloid- ⁇ precursor protein (APP).
• Proteolytic processing of APP leads to elevated A ⁇ peptide formation and to other APP protease cleavage products observed in apoptotic cells.
• apoptotic proteases, specifically the caspases, in APP processing and in the biogenesis of amyloidogenic A ⁇ peptide species is provided. To date there are no direct or indirect methods for specifically evaluating the presence of APP caspase cleavage products.
• agents that can specifically detect the presence of APP caspase cleavage products.
• the presence of the cleavage products can be used as an indication of neuronal apoptosis.
• agents would be useful in recognizing neuronal degeneration, the presence neurodegenrative diseases and in evaluating the progression of such diseases in mammals. More particularly, such agents can be used as a tool in evaluating the effect of candidate inhibitors on neuronal apoptosis and as a possible tool in diagnosing neurodegenerative diseases.
• one object of the present invention was to develop agents capable of specifically detecting APP protease cleavage products.
• Another object was to identify neuronal apoptosis using the agents of the invention.
• An additional object was to use the agents of this invention to distinguish neuronal apoptosis from caspase-independent neuronal necrotic cell death.
• a further object was to use the agents of this invention to detect neuronal apoptosis as an indicator of a- neurodegenerative condition and brain injury.
• the present invention is directed to an antibody that recognizes a neo- epitope created or exposed following a caspase mediated cleavage of APP or APLP and to uses thereof.
• Figure 1A shows that caspase inhibition ablates escalated amyloid- ⁇ peptide production in apoptotic neuronal cells.
• Retinoic acid-differentiated neuronal NT2 cells hNT were cultured in the presence (O) or absence (• , ⁇ ) of serum for 4 days.
• Half of the serum-deprived cell cultures were treated with Z-VAD(OMe)-CH 2 F (100 ⁇ M initial dose, 50 ⁇ M addition at 24 hr intervals) ( ⁇ ).
• a ⁇ (1-40) was measured at the indicated intervals in the resulting cell culture supematants by sandwich ELISA.
• the relative rates of A ⁇ production were calculated once a steady state was achieved (in this case, the slope of the values from days 2-4).
• Figure IB shows the caspase-associated APP proteolysis during apoptosis.
• NT2 cells were induced to undergo apoptosis with 1 ⁇ g/ml camptothecin for 6 hr in the absence or presence of 33 ⁇ M Z-VAD(OMe)-CH 2 F as indicated.
• Apoptosis was assessed by oligonucleosomal DNA fragmentation (left panel).
• Cell lysates were prepared using urea/SDS, resolved on polyacrylamide gels, then immunoblotted for caspase-3 (right, upper panel), poly(ADP)-ribose polymerase (right, middle panel) or APP (right, lower panel).
• a representative experiment (n 5) is shown.
• Figure 1C shows the proteolytic cleavage of amyloid- ⁇ precursor protein by group II caspases in apoptotic cell extracts.
• [ S]Met-labeled APP was generated by coupled in vitro transcription/ translation then incubated with cytosolic extracts from healthy Jurkat cells (lane 1) or extracts from cells that were induced to undergo apoptosis by CD-95 (Fas, APQ-1) immuno-ligation (lane 2).
• Figure ID shows the proteolytic cleavage of amyloid- ⁇ precursor protein by group II caspases in apoptotic cell extracts.
• the cleavage of [ 35 S]Met- labeled APP in apoptotic Jurkat extracts was tested in the presence of the indicated concentrations of tetrapeptide-aldehyde caspase inhibitors (Ac-YVAD-CHO, ⁇ ; Ac- DEVD-CHO, O; Ac-LETD-CHO, •) and quantified by laser densitometry of the resulting fluorogram.
• Figure IE shows the cleavage of amyloid- ⁇ precursor protein by recombinant caspase-3.
• [ S]Met-labeled APP was incubated with the indicated concentrations of recombinant human caspase-3 for 60 min at 37°C.
• S t the concentration of substrate (APP) remaining at time t
• S 0 is the initial substrate concentration
• k obs k cat *[caspase- 3]/K m .
• a representative experiment (n 2) is shown.
• Figure 2 shows increased caspase-3 immunoreactivity in dying pyramidal neurons of CA3 region of Alzheimer's brain hippocampus. Paraffin- embedded sections were processed for antigen retrieval and peroxidase neutralization then probed with an immunoaffinity-purified antibody raised against the large subunit of human caspase-3 (MF-R280) followed by anti-rabbit-HRP visualization (Vectalabs, UK) and hematoxylin counterstaining.
• the staining was specific as judged by: a) inability of the antiserum to cross react with any other human caspase; b) absence of staining with pre-immune serum; c) ability of antigen pre-adsorption to abolish immunostaining; and d) similarity with results using antibodies derived from another rabbit (not shown).
• the width of each panel corresponds to 140 ⁇ m.
• Identical results were found in brains from other donors (3 Alzheimer's, 2 controls). The examples shown are from a 82 yr old female with clinical and post-mortem diagnosis of Alzheimer's disease and an 88 yr old male with no clinical abnormalities who died of a pulmonary embolus and whose brain was judged to be neurologically normal.
• Figure 2A shows the paraffin-embedded sections from the age- matched neurologically normal individual.
• the white arrows indicate healthy, caspase-3 -negative pyramidal neurons showing little, if any, caspase-3 immunoreactivity.
• Figure 2B shows the paraffin-embedded sections from the Alzheimer's patient.
• Black arrows indicate caspase-3 positive immunoreactive neurons (brown) in an area susceptible to degeneration and having degenerative morphology (including 'ghosts') in the Alzheimer's sample.
• Figure 3A shows the identification and mutagenic elimination of sites of caspase-mediated proteolysis of amyloid- ⁇ precursor protein.
• the three potential caspase cleavage sites were identified by [ S]Cys versus [ S]Met differential mapping and mass analysis of caspase-cleaved ⁇ N-APP deletion contracts (not shown). The putative sites were then confirmed by sequential elimination of the predicted Pi Asp residues by site-directed mutagenesis (a Pi Asp is essential for caspase recognition).
• Figure 3B shows a pictogram of the location of caspase cleavage sites within the amyloid- ⁇ precursor protein in relation to other structural features. Domains and interaction sites within the amyloid- ⁇ precursor protein are summarized. The three sites of caspase proteolysis are indicated on the bottom with the number referring to the Asp residue corresponding to P]. The region encompassing the A ⁇ peptide is expanded at the top with known mutations indicated in brackets, including the VKMD 653 - VNLD 653 'Swedish' mutation (circled).
• Figure 4 shows the caspase-dependent cleavage of the amyloid- ⁇ precursor protein in intact cells during apoptosis and in situ detection of a caspase- generated C-terminal neo-epitope.
• Figure 4A shows the C-terminal cleavage of APP during apoptosis in transfected B103 cells.
• Stable B103 cell lines were generated expressing equivalent amounts of the full-length wild-type APP (wt, lanes 1 & 2), APP lacking Ala 721 -
• Figure 4B shows the specific recognition of a caspase-generated APP neo-epitope by oc ⁇ C sp -APP.
• [ S]Met-labeled APP variants were generated by coupled in vitro transcription/ translation (upper panel) then immunoprecipitated (lower panel) using a specific antibody ( ⁇ C sp -APP) that recognizes the caspase- generated neo-epitope that is exposed following cleavage of APP at Asp 720.
• a representative experiment (n 2) is shown.
• Figure 4C shows the C-terminal cleavage of APP during apoptosis in transfected B 103 cells.
• the B103 stable cell line harboring full-length wild-type APP (described in Figure 4A) was treated for 2 hr in the absence (lane 1) or presence (lanes 2 & 3) of 1 ⁇ M staurosporine to induce apoptosis.
• Cells in lane 3 were also treated with 100 ⁇ M of the caspase inhibitor, Z-VAD(OMe)-CH 2 F.
• Cells and apoptotic corpses were harvested and lysed as described above. Lysates were then immunoprecipitated with ⁇ C Csp -APP, resolved by SDS-PAGE and immunoblotted for ⁇ C-APP using 22C11.
• FIG. 4D-4I shows the in situ detection of the caspase-generated ⁇ C- APP neo-epitope during NT2 cell apoptosis.
• Sub-confluent NT2 cells were induced to undergo apoptosis by incubation for 4 hr with 1 ⁇ g/ml camptothecin (E-I) in the absencel (E, H) or presence (F, I) of 50 ⁇ M of the caspase inhibitor, Z-VAD(OMe)- CH 2 F.
• Figure 5G to 5L shows the effect of ischemic injury.
• Transient global ischemia was produced in rats by 12 min of four-vessel occlusion followed by reperfusion for the indicated length of time (control animals (G) underwent sham surgical procudures without ischemia).
• Sections through the CA1 region of the hippocampus were stained for the caspase-generated APP neo-epitope (G-J, L) or for TUNEL (K).
• Figure 6 shows the co-localization of ⁇ C-APP with amyloid- ⁇ in senile plaques.
• the bar in panel 6C 100 ⁇ .
• Figure 6A ⁇ C-APP, visualized by ⁇ C Csp -APP immunoreactivity, in the same area of the hippocampus as shown in (B).
• Figure 6B shows senile plaques identified by amyloid- ⁇ immunoreactivity (arrows) in the hippocampus of a patient diagnosed with Alzheimer's disease.
• Figure 6C shows the merged image of the caspase-generated APP neo- epitope (red) and A ⁇ (green), yielding yellow where co-incident, illustrates the high degree of overlap between ⁇ C-APP and amyloid- ⁇ immunoreactivities.
• n 7 clinically-diagnosed Alzheimer's patients (male and female aged 77-91 yrs with 5-14 yrs AD diagnosis; 2.3-4.3 hr post-mortem harvest) but not -in 7 age-matched control patients (male and female aged 79-91 yrs, 2.0-4.0 hr post-mortem).
• FIG. 7 shows that caspase-mediated generation of APP results in elevated amyloid- ⁇ peptide formation.
• the B103 stable cell lines described for Fig. 4A expressing comparable levels of full-length APP (wt, columns 1 & 3) or APP lacking Ala 721 -Asn 751 (the C-terminal residues following the Asp 720 caspase cleavage site ( ⁇ C-APP, columns 2 & 4)), were cultured in fresh medium for 20 or 48 hr after which A ⁇ peptide levels in the culture medium were quantified by immunoassay with monoclonal antibodies.
• Figure 8 shows that the 'Swedish' familial mutation improves the ⁇ - secretase target region of APP as a caspase-6 substrate.
• Figure 8B shows the effect of APP constructs generated to eliminate the N-terminal caspase cleavage sites ( ⁇ N-APP) and then engineered to contain either the wild type ⁇ -secretase region (VKMD 653 /A 654 ; columns 1 & 2), the 'Swedish' mutation (VNLD 653 /A 654 ; columns 3 & 4) or the 'Swedish' mutation with the Pj Asp that is essential for caspase recognition changed to Ala (VNLA /A ; columns 5 & 6).
• [ 35 S]Met-labeled proteins were generated from these constructs by coupled in vitro transcription/ translation then incubated with cytosolic extracts from apoptotic cells (solid columns; 1, 3 & 5) or recombinant caspase-6 (hashed columns; 2, 4 & 6).
• the percentage of the input APP that was cleaved in the ⁇ -secretase target region in 60 min was determined by fluorography and is expressed ⁇ SD.
• the results show an increased susceptibility of the APP ⁇ -secretase site to caspase-6 by the 'Swedish' familial mutation and dependence of elevated amyloid- ⁇ peptide formation in cells harboring 'Swedish' APP on the Pj Asp necessary for caspase recognition.
• FIG. 8C shows a measure of cleavage, [ 35 S]Met-labeled ⁇ N-APP containing either the wild type ⁇ -secretase sequence (O) or the 'Swedish' dipeptide mutation (•) was incubated with the indicated concentrations of recombinant human caspase-6 for 60 min at 37°C. Cleavage was quantified by phosphorimaging of the ⁇ cleavage fragment and used to determine values for k cat K m as described in the legend to Fig. IF.
• Figure 8D shows immunoreactive products in a stable K562 cell lines generated to harbor full-length APP with the wild type VKMD 653 /A 654 site, the 'Swedish' mutation (VNLD 653 /A 654 ) or the 'Swedish' mutation with the P ! Asp changed to Ala (VNLA 653 /A 654 ).
• Identical quantities of cells, expressing equivalent amounts of the APP constructs, were metabolically labeled by culturing in methionine-free medium for 1 hr then for 5 hr in the presence of [ S]Met.
• Nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission (Biochemistry, 1972, 11:1726-1732).
• the present description refers to a number of routinely used recombinant DNA (rDNA) technology terms. Nevertheless, definitions of selected examples of such rDNA terms are provided for clarity and consistency.
• 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 engineering.
• DNA segment or molecule or sequence refers to molecules comprised of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C). These segments, molecules or sequences can be found in nature or synthetically derived. When read in accordance with the genetic code, these sequences can encode a linear stretch or sequence of amino acids which can be referred to as a polypeptide, protein, protein fragment and the like.
• the term "gene” is well known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide.
• the nucleic acid can be full-length or a partial sequence encoding a polypeptide, so long as the functional activity of the polypeptide is retained.
• a "structural gene” defines a DNA sequence transcribed into RNA and translated into a protein having a specific amino acid sequence thereby giving rise to a specific polypeptide or protein.
• Restriction endonuclease or restriction enzyme is an enzyme that has the capacity to recognize a specific base sequence (usually 4, 5 or 6 base pair in length) in a DNA molecule, and to cleave the DNA molecule at every place where this sequence appears.
• An example of such an enzyme is EcoRI, which recognizes the base sequence GAATTC/CTTAAG and cleaves a DNA molecule at this recognition site.
• Restriction fragments are DNA molecules produced by the digestion of DNA with a restriction endonuclease. Any given linear genome or DNA segment can be digested by a particular restriction endonuclease into at least two discrete molecules of restriction fragments.
• Oligonucleotide or oligomer is a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three. The exact size of the molecule will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide .
• An oligonucleotide can be derived synthetically, by cloning or by amplification.
• Sequence amplification is a method for generating large amounts of a target sequence. In general, one or more amplification primers are annealed to a nucleic acid sequence. Using appropriate enzymes, sequences found adjacent or in between the primers are amplified.
• DNA construct refers to a vector or plasmid comprising a cloned nucleotide sequence.
• expression defines the process by which a structural gene is transcribed into mRNA (transcription), the rnRNA is then translated (translation) into one polypeptide (or protein) or more.
• SF9, SF21 infected with baculoviras (Autographa califomica or Bombyx mori) (Luckow, Curr. Op. Biotech., 1993, 4:564-572; Griffiths and Page, 1994, Methods in Molec. biol. 75:427-440; and Merrington et al, 1997, Molec. Biotech. 8(3):283-297); mammalian cells infected with adenoviras, vaccinia virus, Sindbis viras, or semliki forest virus; and mammalian cells transfected with DNA vectors for transient or constitutive (stable) expression.
• a host cell is "transfected" by exogenous or heterologous DNA (e.g. a
• nucleotide sequences and polypeptides useful to practice the invention include without being limited thereto, mutants, homologs, subtypes, alleles, and the like. It is understood that generally, the sequences of the present invention encode a functional protein. It will be clear to a person skilled in the art that the present invention comprises all variants, derivatives or fragments thereof, that express a functional protein.
• derivative is intended to include any of the above described variants when comprising additional chemical moiety not normally a part of these molecules.
• chemical moieties can have varying purposes including, improving a molecule's solubility, absorption, biological half life, decreasing toxicity and eliminating or decreasing undesirable side effects.
• these moieties can be used for the purpose of labeling, binding, or they may be comprised in fusion product(s). Different moieties capable of mediating the above described effects can be found in Remington 's The Science and Practice of Pharmacy (1995).
• APP is an abbreviation for Amyloid- ⁇ Precursor Protein. These are used interchangeably. Different forms of APPs are known in the art and are designated according to the length of the amino acid sequence, non-limiting examples include APP 695 (Genbank accession number CAA31830), APP 751 (Genbank accession number CAA30050) and APP 770 (Genbank accession number CAA02049). APP 751 , can be referred to as the intermediate form APP. For the purpose of the present application the intermediate form APP is used, all the referred to amino acid residues
• APLP is an abbreviation for Amyloid- ⁇ Precursor Like Protein. These are used interchangeably. APLPs are members of the amyloid precursor protein family. For the purpose of the present invention APLPs having at least one caspase cleavage site are within the scope of the instant invention. Forms of APLPs having at least one caspase cleavage site are known in the art, non-limiting examples include APLP1 (Swissprot accession number P51693 and APLP2 (Swissprot accession number A49321).
• Neo-epitope refers to an epitope that is recognized by the antibodies described herein, this recognition is subsequent to a cleavage event on APP caused by a caspase enzyme, said epitope being substantially unrecognized in the absence of such a cleavage event.
• Single letter abbreviations for amino acids are used and have their standard conventional meanings.
• the present invention thus concerns the development of agents capable of identifying cleavage products generated by protease proteolysis of members belonging to the amyloid precursor protein family, particularly the cleavage products generated as a result of cleavage by a caspase enzyme.
• Members belonging to the amyloid precursor protein family and having at least one caspase cleavage site are within the scope of this invention, non-limiting examples of such members include APLPs and APPs, more particularly, APLP1 and APLP2, and APP 695 , APP 751 and APP 770 , respectively.
• the present invention provides antibodies that are capable of binding specifically and selectively to an APP and APLP caspase cleaved product.
• a first embodiment of the present invention is directed to an antibody that is capable of binding to a neo-epitope, created or exposed following a caspase mediated cleavage of APP and APLP.
• the caspase is selected from caspase 3, caspase 6 and caspase 8.
• caspase 3 has more than one cleavage site along the APPs and at least one along the APLPs.
• cleavage site along the APLP1 and APLP2 are at the amino acid residue number 620 and 732, respectively.
• the cleavage sites of the intermediate APP are at the amino acid residue number 197, 219 and 720, these are sites designated: APP 197 , APP 219 and APP 720 , respectively.
• an antibody raised with an antigenic determinant comprising a peptide sequence corresponding to the amino acid residues APP 714 to APP 720 , based on the APP 751 .
• the antigenic determinant further comprises an additional amino acid residue for coupling to the carrier protein KLH (Keyhole Limpet Hemocyanin).
• KLH Keyhole Limpet Hemocyanin
• the coupling amino acid is cysteine and the antigenic determinant comprises KLH-cysteine- APP to APP
• the antibodies are, purified polyclonal antibodies that specifically bind to the target, the created or exposed epitope.
• Monoclonal antibodies raised in accordance with the antigenic determinant of the present invention for the purpose of recognizing caspase cleaved APLP and APP products, using conventional methods, are also within the scope of the present invention.
• an antigenic determinant for immunizing a mammal in order to furnish an anti-seram containing an antibody that is capable of binding to a neo-epitope, created or exposed following caspases mediated cleavage of APLP1 and APP.
• the antigenic determinant comprises a peptide having at least four amino acid residues and containing the caspase consensus sequence V/DXXD.
• a method for generating an antibody capable of specifically binding to a neo-epitope, created or exposed following a caspase mediated cleavage of APLP and APP comprising: immunizing a mammal with a peptide having at least four amino acid residues and containing the caspase consensus sequence V/DXXD to provide antisera containing antibodies against said peptide; collecting said antisera; and selecting for an antibody specific to a caspase generated neo-epitope.
• APP and amyloid ⁇ -peptide have been studied in the CSF of individuals by Carroll et al., and in multiple tissues including plasma in transgenic mice by Fukuchi et al. These references show that APP and cleaved species of APP are found in tissues other than brain. It is believed that APLP and APLP caspase cleavage products can be similarly found in tissue other than brain. Thus, the presence of APLP and APP caspase cleavage products in tissues such as blood, plasma and CSF would allow easy access to a clinical specimen in which neuronal apoptosis can be detected and evaluated.
• the antibodies of the present invention are useful in a diagnostic method in which APLP and APP cleaved products can be detected in a clinical specimen. More specifically the antibodies of the present invention provide a diagnostic tool for the detection of at least one caspase cleaved APP product in a clinical specimen.
• the antibodies of this invention capable of specifically recognising neo-epitopes exposed following caspase mediated cleavage of APP, are useful in a method for diagnosing conditions in which there is neuronal apoptosis.
• Examples of such conditions include neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive multiple sclerosis, head trauma, prion related conditions, Creutzfeldt-Jacob disease, spongiform encephalopathy, Friedreich's ataxia, fatal familial insomnia, Pelizaeus- Merzbacher disease, schizophrenia, dentatorubropallidoluysian atrophy, spinocerebellar atrophy type 3, spinal bulbar muscular atrophy, spinal cord injury, stroke and brain injury.
• caspase cleaved APP fragments can be detected in mammals using tissues other than brain, detection thereof can be used as an indicator in disease detection and disease progression.
• tissues that are easily accessible for use in a diagnostic method in a mammal include plasma and cerebrospinal fluid.
• the antibodies of the present invention can be used in the detection of apoptotic neuronal cells.
• the antibodies described herein it is possible to follow the evolution of neuronal cells undergoing apoptosis.
• Modulating compounds include inducers and inhibitors of apoptosis.
• neuronal cells can be artificially induced into apoptosis and the progression of the apoptotic process followed using the antibodies of the present invention.
• the antibodies can be used to study the evolution of neuronal apoptosis.
• the progression of apoptosis can be evaluated in the presence or absence of test compounds and the results compared.
• an assay system for selecting modulators of neuronal apoptosis in a specific aspect of the present application, there is provided an assay system for selecting modulators of neuronal apoptosis.
• an assay for identifying inhibitors of neuronal apoptosis is provided. Such an assay can be scaled- up to a high throughput system.
• candidate inhibitors identified using the antibodies of the present invention may be comprised in a pharmaceutical composition for treating mammals including humans in need of such treatment.
• the antibodies generated to detect caspase mediated APP cleavage products can be included in a kit.
• a kit would comprise at least one antibody capable of binding to at least one neo-epitope, exposed following caspase mediated cleavage of APP. This could have applications for research or diagnostic purposes.
• APP clones were derived from human APP 751 . Clone designations below are in the following format: [construct]: [vector]: [insert site/ insert release]: [sense orientation]: [identifier]. Clones were generated, as indicated, by PCR- mediated template modification and were fully sequenced before use. Clones for in vitro transcription/ translation include: ⁇ [wtAPP 751 ; Met !
• Mammalian expression clones include:
• B103 cells a rat neuronal cell line
• K562 cells a human erythroleukemia cell line
• Expression levels were determined during the course of each experiment by immunoblotting to verify that each cell line expressed comparable levels of their respective APP constructs.
• Cell line designations below are in the following format: [designation]: [expressed protein]: [transfection clone]: [selection]: [identifier].
• Stable cell lines include:
• Two rabbits were immunized with the peptide that includes the seven amino acids in APP that precede the caspase cleavage site at aspartic acid 720 (N terminal APP 714 to C terminal APP 720 , inclusive) as well as an amino-terminal cysteine residue to allow coupling to KLH.
• the rabbits were boosted three times with the immunizing peptide over a 10 week period.
• the antisera were pooled and affinity purified by three successive chromatographic steps: (1) the antisera was depleted of immunoglobulins recognizing intact APP by adsorption to a bridging peptide that spans the caspase cleavage site (N terminal APP 713 to C terminal APP 726 , inclusive) immobilized on Sepharose 4B (Pharmacia) by cyanogen bromide activation; (2) the flow through from step (1) was applied to a Sepharose 4B column containing the immunizing peptide (immobilized to the support as described in (1)); the column was washed and the specific antibodies were eluted via a pH gradient and placed in a borate buffer (0.125 M borate); (3) the eluant from the second step was absorbed against the immobilized bridging peptide as described in (1) and the flow through was collected.
• the antisera was depleted of immunoglobulins recognizing intact APP by adsorption to a bridging peptide
• Subconfluent NT2 cells were incubated with 1 ⁇ g/ml campthothecin in the presence or absence of 50 ⁇ M Z-VAD(OMe)-CH 2 F for 4 hours at 37°C with 5% CO 2 .
• the final concentration of DMSO used to dilute the camptothecin and Z- VAD(OMe)-CH 2 F was 0.3% (v/v).
• As a control cells were incubated with 0.3% (v/v) DMSO alone. The cells were detached from the plate with Cell Dissociation Solution (Sigma) and pooled with the culture media (to retain floating apoptotic cells).
• the cells were fixed in 10% neutral buffered formalin for 10 min at room temperature, washed twice in phosphate-buffered saline, and then centrifuged using a Cytospin centrifuge onto poly-L-lysine-coated glass slides (Sigma) and dried overnight. The cells were then co-stained for TUNEL and either ⁇ C Csp -APP at a dilution of 1/4000 or Cs ⁇ -3(MF397) at a dilution of 1/3000, essentially as described previously (Black et al., 1998) except that the proteinase K treatment was omitted.
• EXAMPLE 7 DNA laddering Extraction of DNA to visualize olignucleosomal fragmentation was performed as follows. Subconfluent NT2 cells were incubated with 1 ⁇ g/ml campthothecin in the presence or absence of 33.3 ⁇ M Z-VAD(OMe)-CH2F for 6 hours at 37°C with 5% CO2- As a control, cells were incubated with vehicle (0.2 % (v/v) DMSO) alone. Cells were removed from the dish by scraping, pooled with the culture medium, and pelleted at 10,000 x g for 10 min.
• Cell pellets (2 x 10 cells/pellet) were resuspended in 0.5 ml of 0.6% (w/v) SDS, 10 mM EDTA (pH 7.5) followed by the addition of NaCl to a final concentration of 1M. After overnight incubation at 4°C, the supematants were clarified by centrifugation at 14,000 x g for 20 min at 4°C and treated with 7 ⁇ g/ml DNase-free RNase for 45 min at 37°C.
• Z-VAD(OMe)-CH2F a pan-caspase inhibitor (Garcia-Calvo et al., 1998), was purchased from Enzyme Systems Products.
• NT2 cells human neuronal precursor
• B 103 cells rat neuronal
• FBS fetal bovine serum
• Retinoic acid-differentiated NT2 neurons hNT were maintained in neuron conditioned medium (Stratagene) except during the course of seram-deprival experiments when they were cultured in DMEM (-/+ 10% (v/v) FBS).
• Acute apoptotic induction of the neuronal precursor cell line NT2 with camptothecin resulted in oligonucleosomal DNA fragmentation and concomitant maturation of procaspase-3, cleavage of caspase-3 substrates (such as PARP; poly(ADP-ribose) polymerase) and a ca. 3.5 kDa reduction in the mass of endogenous APP (lanes 2).
• PRECURSOR PROTEIN Owing to the increase in APP proteolysis and A ⁇ peptide formation that accompanies neuronal apoptosis, we sought to determine whether any of the biochemical components of the cell death pathway might contribute to this process.
• Apoptosis itself is dependent on a family of cysteine proteases, the caspases, which manifest the apoptotic phenotype by cleaving a discrete subset of cellular polypeptides at Asp-x bonds (Alnemri et al., 1996; Nicholson and Thornberry, 1997; Thornberry et al., 1997).
• Jurkat cells were initially chosen because they contain multiple caspase family members (caspases-2, -3, -4, -6, -7, -8 and -9) that are readily activated during apoptosis (unpublished).
• caspases-2, -3, -4, -6, -7, -8 and -9 that are readily activated during apoptosis (unpublished).
• the 120 kDa APP was rapidly processed to a triad of smaller polypeptides of approximately 85-90 kDa.
• caspase-3 predominantly accounts for APP proteolysis during apoptosis.
• caspase-3 was the most efficient enzyme to cleave APP, although minor cleavage also occurred with caspases-6 and -8 (not shown).
• caspase-3 If caspase-3 is involved in APP processing and neuronal apoptosis, it should be present at the sites of A ⁇ production and neurodegeneration.
• Caspase-3 (CPP32, apopain, Yama)(Femandes-Alnemri et al., 1994; Nicholson et al., 1995; Tewari et al., 1995) is one of the key effector caspases in mammalian cell suicide.
• 35 recombinant caspase-3 generated two small [ S]Cys-containing fragments (23 and 25 kDa) and a larger polypeptide (85 kDa), indicating that two of the caspase cleavage sites were within the first N-terminal 220 amino acids of APP and a third site was present at about residue 700 (not shown). The position of the latter site was further refined by cleaving an APP constract lacking the N-terminal 220 amino acids ( ⁇ N- APP).
• caspase consensus sites (Thornberry et al., 1997) were present at: APP 194 to APP 197 , APP 216 to APP 219 and APP 717 to APP 720 , inclusive, these sites are designated (P 4 )DNVD 197 (P,)/S, (P 4 )DYAD 219 (P,)/G and (P 4 )VEVD 720 (P ⁇ )/A, respectively (Fig. 3B).
• Cleavage at the VEVD 720 A site is consistent with the approx. 3.5 kDa reduction in APP observed during apoptosis in NT2 cells (see Fig IB, lane 2) and appears, at least with the cell lines tested, to be the predominant site of caspase proteolysis in intact cells (although APP fragments corresponding to cleavage at the two upstream sites have been observed by others (Zhang et al., 1997)). Positive identification of this C- terminal cleavage event was confirmed using a neo-epitope antibody that we generated for immunohistochemical detection of caspase-generated ⁇ C-APP in situ.
• the antisera was raised against a synthetic peptide corresponding to the living C- terminus of ⁇ C-APP, [KLH-cysteine- APP 714 to APP 720 ], then purified by immunoadsorption to the same peptide and two cycles of immunodepletion with a bridging peptide corresponding to intact APP straddling the cleavage site comprising the amino acid residues APP 714 to APP 726 , inclusive.
• This antibody designated ⁇ C sp -APP, was confirmed to be highly specific for the caspase-generated neo-epitope in APP by three criteria: a) its titre by ELISA was > 2000-fold selective for the immunizing peptide (corresponding to ⁇ C-APP) versus the bridging peptide harboring the same sequence (which corresponds to intact APP) (see Methods), b) the antibody could immunoprecipitate biosynthetic [ 35 S]APP that was truncated at the
• caspase-mediated APP proteolysis measured by ⁇ C sp -APP immunoreactivity
• caspase-mediated APP proteolysis was not detectable in the CA3 region of the hippocampus of control mice (panel A) whereas intense oc ⁇ C Csp -APP immunoreactivity was observed in CA3 neurons of SN129 mice 48 hr after administration of kainic acid (25 mg/kg, s.c.) (panel B).
• kainic acid 25 mg/kg, s.c.
• TU ⁇ EL labeling confirmed that the CA3 neurons which displayed ⁇ C Csp -APP and active caspase-3 immunoreactivity were apoptotic (panel F).
• caspase-mediated APP cleavage occurs in vivo, we determined whether the apoptotic death of CA1 neurons produced by a brief episode of global ischemia was also accompanied by increased production of ⁇ C-APP (Fig. 5G-L).
• ⁇ C-APP fragment is derived by caspase-mediated proteolysis of APP
• increased production of ⁇ C-APP in vivo accompanied the generation of active caspase-3 in apoptotic neurons.
• Caspase-mediated alteration of APP processing after acute brain injury may in part account for the increased susceptiblity to Alzheimer's disease in human patients as well.
• She-like PTB domain of XI 1 interacts with the NPTY motif at the C-terminus of APP and reduces A ⁇ peptide formation by binding to and slowing APP processing (Borg et al., 1998). It is likely that removal of this and other key components of the C-terminus of APP biases its route of cellular degradation along an amyloidogenic pathway (Russo et al., 1998).
• APP polypeptide with the dominant processing site being at Asp (Fig. 3B), at least one documented familial Alzheimer's disease (FAD) mutation introduces an additional caspase-susceptibility site to the molecule.
• FAD familial Alzheimer's disease
• the 'Swedish' mutation of APP is associated with an inherited form of Alzheimer's disease with an earlier onset and elevated production of 4 kDa amyloidogenic A ⁇ peptides (Cai et al., 1993; Citron et al., 1992; Haass et al., 1995; Holcomb et al., 1998).
• the mutation changes the VXMD 653 sequence (amino acid residues corresponding to APP 650 to APP 653 ) at the ⁇ - secretase site to VNLD , a motif with substantially improved caspase recognition elements.
• the 'Swedish' mutation generates a site that would be preferentially recognized by group ID. caspases, especially caspase-6, as predicted by our positional-scanning combinatorial substrate library (Thornberry et al., 1997).
• fluorogenic tetrapeptides corresponding to wild type and 'Swedish' sequences were synthesized and tested for their ability to be cleaved by recombinant caspase-6.
• VNLD-AMC VNLD-AMC
• VKMD-AMC wild type fluorogenic peptide
• Fig. 8A a differential also occurred in the context of the full-length APP polypeptide with cytosolic extracts from apoptotic cells as well as with recombinant caspase-6 (Fig. 8B).
• the 'Swedish' mutation accelerated the rate of proteolysis at the ⁇ secretase site (columns 3 & 4) and this cleavage was entirely abolished by mutation of the P !
• caspases may contribute to ⁇ -secretase activity, particularly in the 'Swedish' Alzheimer's disease mutation.
• caspases may contribute to cleavage at the V (K or N)(M or L) D 653 /A 654 caspase-6 site(Wang et al., 1996).
• Caspases contribute to the complex proteolytic processing of the amyloid- ⁇ precursor protein and appear to contribute to amyloidogenic A ⁇ peptide formation by at least two distinct mechanisms.
• the cleavage of APP at endogenous caspase consensus sites ((P 4 )DNVD 197 (P ! )/S, (P 4 )DYAD 219 (Pj)/G and,
• (P )VEVD P ⁇ /A probably corrupts the normal intracellular processing of APP that would otherwise preclude it from A ⁇ peptide formation or accumulation.
• a 'vicious cycle', occuring over a protracted period of time, may therefore proceed via the following steps in susceptible neurons: a) caspase-3- mediated truncation of APP at the C-terminus, b) adulteration of normal APP processing with shunting of the residual polypeptide towards an amyloidogenic pathway, c) generation of elevated levels of cytotoxic A ⁇ peptide species, resulting in A ⁇ -induced neuronal stress, d) progressive up-regulation and or activation of endogenous caspases, and e) exacerbated APP proteolysis.
• caspases In addition to APP cleavage, caspases have recently been implicated in alternative presenilin proteolysis (Kim et al., 1997; Vito et al., 1997). Caspases may thus play an important role in both the generation of neurotoxic A ⁇ peptides as well as in the ultimate death of neurons by apoptosis in Alzheimer's disease.
• CPP32 a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta- converting enzyme. J Biol Chem 269, 30761-4.
• Alzheimer's A beta peptide induces neurodegeneration and apoptotic cell death in transgenic mice. Nat Genet 9, 21-30.
• Apoptosis is induced by beta-amyloid in cultured central nervous system neurons. Proc Natl Acad Sci U S A 90, 7951-5.
• the four-vessel occlusion rat model method for complete occlusion of vertebral arteries and control of collateral circulation. Stroke 19, 913-4.
• Yama/CPP32 beta a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 57, 801-9.

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