WO1992000521A1 - Diagnostic and prognostic methods based on soluble derivatives of the beta amyloid protein precursor - Google Patents

Diagnostic and prognostic methods based on soluble derivatives of the beta amyloid protein precursor Download PDF

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WO1992000521A1
WO1992000521A1 PCT/US1991/004607 US9104607W WO9200521A1 WO 1992000521 A1 WO1992000521 A1 WO 1992000521A1 US 9104607 W US9104607 W US 9104607W WO 9200521 A1 WO9200521 A1 WO 9200521A1
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kilodalton
protein
soluble
patient
amino
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PCT/US1991/004607
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French (fr)
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Steven G. Younkin
Mark R. Palmert
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Case Western Reserve University
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711Alzheimer's disease; Amyloid plaque core protein
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical 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
    • G01N33/6896Neurological disorders, e.g. Alzheimer's disease
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/28Neurological disorders
    • G01N2800/2814Dementia; Cognitive disorders
    • G01N2800/2821Alzheimer

Definitions

  • the present invention relates to methods which can be used to diagnose, prognose, and stage Alzheimer's disease, and monitor response to therapy of Alzheimer's disease. Such methods involve the measurement of the levels in cerebrospinal fluid of the -25 kDa, -105 kDa, and -125 kDa soluble derivatives of the beta amyloid precursor protein.
  • the invention also provides for evaluation and monitoring of neurologic aging.
  • the methods of the invention can also be used to diagnose Down's syndrome and prognose deleterious sequelae of amyloid deposition.
  • Alzheimer's disease is a progressive neurodegenerative disorder of the aged and is a major cause of adult-onset dementia. It is characterized by cerebral depositions of amyloid.
  • the principal proteinaceous component of the amyloid deposited in senile plaques and cerebral blood vessels in Alzheimer's disease is a 39-42 amino acid polypeptide ( ⁇ amyloid protein, /3AP) (Glenner G. G. and Wong, C. W. , 1984, Biochem. Biphys. Res. Commun. 122:1131-1135; Masters et al., 1985, Proc. Natl. Acad. Sci.
  • ⁇ amyloid protein precursor ⁇ APP
  • the 0APP gene produces at least three mRNAs (Kitaguchi et al., 1988, Nature 331:530-532; Ponte et al., 1988, Nature 331:525-527; Tanzi et al., 1988 Nature 331:528-530) through alternative splicing of two exons (Kitaguchi et al., 1988, Nature 331:530-532).
  • One these exons encodes a 19-amino acid domain; the other encodes.
  • a 56-amino acid domain that is highly homologous the Kunitz family of serine protease inhibitors.
  • the 39-to 42-residue / SAP occurs as an internal sequence which extends from the extracellular region into the putative membrane-spanning domain (Kang e al., 1987, Nature 325:733-736; Dyrks et al., 1988, EMBO J. 7:949-947).
  • the full-length forms of this precursor are truncated at their carboxyl-termini to produce -125 and -105 kDa (kilodalton) soluble derivatives (Schubert et al.
  • the -125 kDa derivative which appears to be the same protein as protease nexin-II (Oltersdorf et al., 1989, Nature 341:144-147 and Van Nostrand et al., 1989, Nature 341:546-549), contains the alternatively spliced (Kitaguch et al., 1988, Nature 331:530-532) Kunitz protease inhibito (KPI) domain, whereas the -105 kDa form lacks this insert (Palmert et al., 1989, Proc. Natl. Acad.
  • Alzheimer's type lesions containing amyloid protein are found in adult cases of Down's syndrome (Ball and Nuttall, 1981, Neuropathol. Appl. Neurobiol. 7:13).
  • the present invention relates to methods for the prognosis, diagnosis, and staging of AD. It further relates to methods for monitoring response to therapy in a patient with AD.
  • the methods of the invention involve the measurement of the levels in a sample of cerebrospinal fluid (CSF) of the -25 kDa, -105 kDa, and -125 kDa soluble derivatives of the APP.
  • CSF cerebrospinal fluid
  • detection of an increase in the percentage amount of the ⁇ 25 kDa protein and/or decrease in the percentage amount of the -105 kDa derivative and/or high absolute levels of all three soluble 0APP derivatives, relative to healthy individuals can be used to diagnose or prognose AD.
  • such detection can be used to diagnose Down's syndrome, and to prognose disorders in Down's syndrome patients associated with amyloid deposition.
  • the foregoing can also be used as an indication of neurologic aging.
  • determination of the percentage amounts of the -25 kDa protein and/or the -105 kDa protein can be used to stage AD, or to indicate the extent of mental dementia in a patient.
  • an increase in the percentage amount of -25 kDa protein and/or a decrease in the percentage amount of -105 kDa protein relative to such amount present prior to therapy or in healthy individuals can be deemed a poor response to therapy.
  • the invention is also directed to the soluble -25 kDa amino-ter inal form of the 0APP.
  • FIG. 1 Amino-terminal sequence (single letter code) of the -25 kDa soluble protein found in human CSF. X indicates that the signal was weak and did not permit an amino acid to be assigned. Note that the amino acid sequence predicted from 0APP cDNA (Kang et al., 1987, Nature 325:733-736) begins with a 17-residue signal sequence identified by Dyrks et al. (Dyrks et al., 1988, EMBO J. 7:949-957). Thus the amino-terminal residue of the mature protein is located at position 18 of the predicted sequence. ' FIG. 2. Standard curves from a protein A experiment.
  • FIGURE 3 Derivatives that may be produced when full-length 0APP is cleaved generating soluble forms (adapted from Palmert et al., 1989, Biochem. Biophys. Res. Commun. 165:182-188).
  • the solid bar in each schematic indicates the position of the / 3AP.
  • the relative sizes of the membrane-associated and soluble forms (Palmert et al., 1989, Proc. Natl. Acad. Sci. USA 86:6338-6342) and the observation that the soluble -125 and -105 kDa derivatives in CSF are labeled by antisera to the ⁇ AP (Palmert et al., 1989, Biochem. Biophys. Res. Commun. 165:182-188) indicates that cleavage normally occurs as shown in A or B. The cleavage shown in C may sometimes occur in normal individuals, and it could develop or be enhanced in AD.
  • the present invention is directed to methods of prognosis and diagnosis of AD. It also relates to methods of staging AD and to monitoring response to therapy in a patient with AD.
  • the invention is based upon the measurement-in cerebrospinal fluid (CSF) of a patient of the levels of soluble derivatives of the 0APP, in particular, the approximately (-) 25 kDa, -125 kDa, and -105 kDa soluble forms.
  • the invention is directed to the soluble. -25 kDa amino-terminal form of the APP.
  • the methods provided by the present invention also allow evaluation and monitoring of neurologic aging in an individual.
  • the invention further provides methods for diagnosis of Down's syndrome.
  • soluble as used herein shall mean th which is “not cell-associated, not due to any artificial intervention," as opposed to the term “solubilized,” whic refers to that which is artificially released from a cell surface into solution, e.g., by detergent cell lysis.
  • the methods provided by the present invention involve the measurement of the levels of soluble derivatives of the 0APP in cerebrospinal fluid of a patient.
  • soluble derivatives are selected from the group consisting of the -25 kDa, -105 kDa, and the -125 k ,9APP derivatives, which derivatives consist of a portion o the sequence of the 0APP starting with (as their amino- terminal residue) the amino acid at position 18 of the predicted 0APP sequence (see Kang, 1987, Nature 325:733- 736).
  • the levels of one o more of the above-mentioned soluble derivatives are measured in CSF from a patient.
  • CSF can be obtained by any procedures known in the art.
  • CSF can be obtained by lumbar puncture.
  • Dialysis into desired buffers and concentration before analysis can also be carried out.
  • Measurement of the soluble 3APP derivatives can be by any techniques known in the art.
  • an immunoblotting (Western blotting) procedure with appropriate antisera can be used to quantitate the soluble derivative(s) (see Examples sections, infra) .
  • blots e.g., nitrocellulose
  • denaturing e.g., sodium dodecyl sulfate-polyacrylamide
  • Such an appropriate antibody is one reactive with the soluble derivative being quantitated.
  • antibodies which can be used include but are not limited to an antiserum against amino acids 45-62 in the APP sequence described by Kang et al., 1987, Nature 325:733-736) (anti- ⁇ APP._ _ configure) , and an antiserum to residues 18-35 of the APP. It is expected that antibodies recognizing an epitope within a -25 kDa fragment of the y9APP starting at amino acid 18 should be suitable for such use.
  • the antibody thus employed can be labeled, or it can be reacted with a labeled binding partner of the antibody (e.g. 125I-labeled protein A) for detection and quantitation purposes.
  • a labeled binding partner of the antibody e.g. 125I-labeled protein A
  • assays suitable for use will be known to those skilled in the art, including but not limited to assays employing one or more of the following techniques: chromatography (e.g.; ion exchange, immunoaffinity, immunoabsorption, and sizing chromatography such as high pressure liquid chromatography) , centrifugation, electrophoretic procedures, differential solubility, competitive and non- competitive immunoassay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay) , "sandwich” immunoassays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement-fixation as
  • Antibodies against the soluble 0APP derivatives for use in immunodetection assays can be produced by methods known in the art. Such antibodies can be polyclonal or monoclonal.
  • various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of a given soluble ,9APP derivative.
  • various host animals can be immunized by injection with 0APP, or a fragment thereof, or synthetic protein or peptide corresponding to the foregoing, including but not limited to rabbits, mice, rats, etc.
  • Various adjuvants may be used to increase the immunological response, depending on the host species.
  • a monoclonal antibody can be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497), and the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72) and EBV- hybridoma technique (Cole et al., 1985, Monoclonal
  • Antibody fragments which contain the idiotype (binding region) of the molecule can also be used, and ca generated by known techniques.
  • fragmen include but are not limited to: the F(ab'j fragment whic can be produced by pepsin digestion of the antibody molec the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')_ fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • the quantitation of the soluble 3APP derivative( in CSF according to the present invention can be used for diagnosis, staging and/or prognosis of AD. Such quantitat can also be used to monitor therapy of a patient with AD.
  • detection of an. increa in the percentage amount (% of total -25 kDa + -125 kDa + • -105 kDa soluble 0APP derivatives) of the -25 kDa derivati and/or a decrease in the percentage amount of the -105 kDa derivative, relative to healthy individuals can be used a an indication of the presence or impending onset of AD, or predicting the course of AD particularly when serial measurements are made.
  • Increased percentage of the -25 kD protein and/or decreased percentage of -105 kDa protein, and/or high absolute levels of all three soluble 0APP derivatives in non-demented individuals can be a significa risk factor such that a high percentage of the non 7 demente individuals who show such a profile eventually develop AD.
  • the percentage amount of the -25 kDa protein, and/or the percentage amount of the -105 kDa protein can be used to stage AD patients, and as an indication of the mental stat of the AD patients (see. Examples sections, infra) .
  • Such percentage amounts can also be used to monitor therapeutic strategies for AD, such as those aimed at reducing amyloid deposition. For example, an increase in the percentage amount of -25 kDa derivative, and/or decrease in the percentage amount of -105 kDa derivative relative to such amount present prior to therapy or in healthy individuals, can be deemed a poor response to therapy.
  • An increase in the percentage amount of the -25 kD protein, and/or a decrease in the percentage amount of the -105 kDa protein, and/or an increase in the absolute amount(s) of one or more of the soluble ,5APP derivatives ca also be used as an indication of neurologic aging.
  • detection of such an increase for t -25 kDa protein and/or decrease for the -105 kDa protein, o increased absolute amounts, relative to young individuals (e.g., those individuals under age 60), can indicate neurologic aging.
  • the ratios of the level(s) of one or more of the soluble 0APP derivative(s) to the level(s) of one or more different soluble 0APP derivatives can be calculated for use in the methods of the present invention.
  • the ratio o the amount of -25 kDa protein-to the amount of -105 kDa protein in a- sample of CSF can be determined for use in a method of diagnosis, prognosis, staging, or monitoring therapy, as provided by the present invention.
  • kits for use in practicing the methods of the invention comprise, in one or more containers, one or more antibodies directed against th soluble / 5APP derivative(s) being quantitated. 5.3. DIAGNOSIS OF DOWN'S SYNDROME
  • the invention further provides methods for diagnosis of Down's syndrome.
  • methods based on measurements of the soluble / 3APP derivatives including but not limited to detection of an increase in the percentage amount (% of total -25 kDa and
  • -25 kDa derivative and/or a decrease in the percentage amou in CSF of the -105 kDa derivative, relative to healthy individuals, can be used as an indication of the presence o
  • Down's syndrome or as an indication of the presence or impending onset in the Down's syndrome patient of disorders associated with amyloid deposition (e.g., mental dementia).
  • disorders associated with amyloid deposition e.g., mental dementia
  • CSF human cerebrospinal fluid
  • AD Alzheimer's disease
  • the -25 kDa protein was purified from CSF using ammonium sulfate fractionation, separation on a Mono-Q column (Pharmacia) , and preparative SDS/PAGE as previously described for the -125 and -105 kDa proteins (Palmert et al., 1989, Proc. Natl. Acad. Sci. USA 86:6338-6342). Briefly, the / 3APP derivative was separated from the bulk (75%) of the,CSF protein by fractionation at 62.5% of ammonium sulfate saturation (0.41 g/ml) .
  • the resulting pellet was resuspended in 20 mM sodium phosphate (monobasic) buffer ' (pH 6.8), desalted, and loaded onto a Mono Q column (Pharmacia) .
  • a linear 0 to 1 M NaCl gradient in 20 mM sodium phosphate (monobasic) buffer, pH 6 * 8, containing 0.05% Tween 20 was then applied to the column, and the -25 kDa derivative (along with -125 and -105 kDa derivatives) was eluted in a well defined peak at -60% (0.6 M NaCl) of the gradient.
  • Nitrocellulose blots were, then blocked with 5% nonfat dried milk diluted in TBS (10 mM Tris-HCl, pH 8 and 150 M NaCl) ; exposed overnight to anti- ⁇ APP.- g- (Palmert et al., 1988, Biochem. Biophys. Res. Commun. 156:432-437; Palmert et al., 1989, Proc. Natl. Acad. Sci.
  • the size of the excised area corresponded to the typical band size and was uniform from experiment to experiment.
  • CSF CSF was obtained by lumbar puncture 0 with removal of 20 ml of fluid which was then aliquoted into 2 ml containers. Patients were at bed rest for several hours before and after the examination. All psychotropic medications were withdrawn prior to the lumbar puncture. CSF (240 ⁇ l) from each case was exchanged into 1 g mM dibasic/monobasic (1.6/1) phosphate buffer (pH 6.9), concentrated, and analyzed as described above for the standards. Each experiment contained both AD and control samples, which were loaded into alternating.lanes of each gel. 0
  • the -25 kDa protein like the -125 and -105 kDa proteins, is an amino-terminal derivative of the 0APP.
  • the " ⁇ 25 kDa derivative” consists of a major band and 1 or 2 minor bands that migrate at -25 kDa on 5-15% SDS-polyacrylamide gels. All of these bands are specifically labeled by anti- / 3APP.__ 62 and by an antiserum to residues 18-35 of the 0APP.
  • Our sequence and the quantitative data shown below are from the.-.major protein within this complex.
  • AD patients (10 samples were obtained at autopsy and 14 were obtained conventionally from living patients) and 12 non-demented controls (including 3 non-demented patients with Parkinson's disease, 3 with multiple sclerosis, 1 with recurrent encephalitis, 1 with acute psychosis, 1 with hypersomnia, and 3 with no neurological disease) are summarized in Tables I and II.
  • Non-demented controls (30-60 yrs)
  • Non-demented controls (62-79 yrs)
  • Non-demented controls 55-79 yrs) 7 AD (52-73 yrs) 13
  • Non-demented controls (30-60 yrs) 7 45+4 1.5+0.6 6.2+1.6 1.3+0.4 9.0+2.5
  • Non-demented controls (55-79 yrs) 7 65+3 6.8+2.4 10.2+2.1 2.3+0.5 19.3+4.6 AD (52-73 yrs) 13 65+2 6.3+1.0 7.8+3.1 1.6+0.4 15.9+4.3
  • t APP derivatives in CSF indicate that both factors may contribute to amyloid deposition in AD.
  • the percentage of the -105 kDa form is decreased and the percentage of the -25 kDa form is increased in normal aging and, to a greater extent, in AD, indicating that APP processing is altered in these conditions.
  • the absolute levels of soluble 0APP derivatives are increased in elderly control subjects and, to a greater extent, in the least demented AD patients.
  • the altered relative amounts of the -25 and -105 kDa derivatives in CSF correlate remarkably well with amyloid deposition in the sense that they change modestly in the elderly population where there is limited amyloid deposition, show more marked alteration in AD patients where amyloid deposition is pronounced, and change progressively as the severity of dementia increases in the AD population.
  • the AD and age-matched control populations though significantly different, were overlapping with respect to the absolute and relative levels of 0APP derivatives measured in CSF. These measurements may be useful (i) as part of a series of tests aimed at diagnosing AD, (ii) in predicting the course of AD particularly when serial measurements are made, and (iii) in monitoring therapeutic strategies aimed at reducing amyloid deposition.
  • AD-like profile for these variables is a significant risk factor and that a high percentage of the non-demented individuals who show such a profile eventually develop AD.
  • Various references are cited herein, the disclosures of which are incorporated by reference in the entireties.

Abstract

The present invention relates to methods for the prognosis, diagnosis, and staging of Alzheimer's Disease (AD), and to methods for monitoring response to therapy in a patient with AD. The methods of the invention involve the measurement of the levels in a sample of cerebrospinal fluid (CSF) of the ∩25 kDa, ∩105 kDa, and ∩125 kDa soluble derivatives of the beta amyloid protein precursor (βAPP). In specific embodiments, detection of an increase in the percentage amount of the ∩25 kDa protein and/or decrease in the percentage amount of the ∩105 kDa derivative and/or high absolute levels of all three soluble βAPP derivatives, relative to healthy individuals, can be used to diagnose or prognose AD. The foregoing can also be used to diagnose Down's syndrome, to prognose disorders in Down's syndrome patients associated with amyloid deposition, and as an indication of neurologic aging. In other embodiments, determination of the percentage amounts of the ∩25 kDa protein and/or the ∩105 kDa protein relative to such amount present prior to therapy or in healthy individuals can be deemed a poor response to therapy of AD. The invention is also directed to the soluble ∩25 kDa amino-terminal form of the βAPP.

Description

DIAGNOSTIC AND PROGNOSTIC METHODS BASED ON SOLUBLE DERIVATIVES OF THE BETA AMYLOID PROTEIN PRECURSOR
1. INTRODUCTION The present invention relates to methods which can be used to diagnose, prognose, and stage Alzheimer's disease, and monitor response to therapy of Alzheimer's disease. Such methods involve the measurement of the levels in cerebrospinal fluid of the -25 kDa, -105 kDa, and -125 kDa soluble derivatives of the beta amyloid precursor protein. The invention also provides for evaluation and monitoring of neurologic aging. The methods of the invention can also be used to diagnose Down's syndrome and prognose deleterious sequelae of amyloid deposition.
2. BACKGROUND OF THE INVENTION Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the aged and is a major cause of adult-onset dementia. It is characterized by cerebral depositions of amyloid. The principal proteinaceous component of the amyloid deposited in senile plaques and cerebral blood vessels in Alzheimer's disease is a 39-42 amino acid polypeptide (β amyloid protein, /3AP) (Glenner G. G. and Wong, C. W. , 1984, Biochem. Biphys. Res. Commun. 122:1131-1135; Masters et al., 1985, Proc. Natl. Acad. Sci. USA 82:4245-4249; Selkoe et al., 1986, J. Neurochem. 46:1820-1834) derived from a much larger membrane associated glycoprotein referred to as the β amyloid protein precursor (øAPP) (Goldgaber et al., 1987, Science 235:877-880; Kang et al., 1987, Nature 325:733-736; Robakis et al., 1987, Proc. Natl. Acad. Sci. USA', 84:4190-4194; Tanzi et al., 1987, Science 235:880-884; Kitaguchi et al., 1988, Nature 331:530-532; Ponte et al., 1988, Nature 331:525-527; Tanzi et al., 1988, Nature 331:528-530; Selkoe et al., 1988, Proc. Natl. Acad. Sci. USA 85:7341-7345; Palmert et al., 1988, Biochem. Biophys. Res. Commun. 156:432-437; Takio et al., 1989, Biochem. Biophys. Res. Commun. 160:1296-1303). The 0APP gene produces at least three mRNAs (Kitaguchi et al., 1988, Nature 331:530-532; Ponte et al., 1988, Nature 331:525-527; Tanzi et al., 1988 Nature 331:528-530) through alternative splicing of two exons (Kitaguchi et al., 1988, Nature 331:530-532). One these exons encodes a 19-amino acid domain; the other encodes. a 56-amino acid domain that is highly homologous the Kunitz family of serine protease inhibitors. In each full-length precursor, the 39-to 42-residue /SAP occurs as an internal sequence which extends from the extracellular region into the putative membrane-spanning domain (Kang e al., 1987, Nature 325:733-736; Dyrks et al., 1988, EMBO J. 7:949-947). The full-length forms of this precursor are truncated at their carboxyl-termini to produce -125 and -105 kDa (kilodalton) soluble derivatives (Schubert et al.
1988, Science 241:223-226; Schubert et al., 1989, Proc. Natl. Acad. Sci. USA 86:2066-2069; Weidemann et al., 1989, Cell 57:115-126; Podisny, M. , et al., 1989, Soc. Neurosci. Abstr. 15(2):1379 (Abstr. 541.25); Palmert et al., 1989, Proc. Natl.' Acad. Sci. USA 86:6338-6342; Palmert et al.,
1989, J. Neuropathol. Exp. Neurol. 48:378 (Abstr. 231); Palmert et al., 1988, Biochem. Biophys. Res. Commun. 156:432-437)) that are readily detected in human cerebrospinal fluid (CSF) (Weidemann et al., 1989, Cell 57:115-126; Palmert et al., 1989, Proc. Natl. Acad. Sci. USA 86:6338-6342). Galasko et al. (1989, Soc. Neurosci. Abstr. 15(2):1376 (Abstr. 541.9) disclosed the detection 88 kD and 100 kD proteins in human cerebrospinal fluid reactive with an antibody to the N-terminal region of 0AP The -125 kDa derivative, which appears to be the same protein as protease nexin-II (Oltersdorf et al., 1989, Nature 341:144-147 and Van Nostrand et al., 1989, Nature 341:546-549), contains the alternatively spliced (Kitaguch et al., 1988, Nature 331:530-532) Kunitz protease inhibito (KPI) domain, whereas the -105 kDa form lacks this insert (Palmert et al., 1989, Proc. Natl. Acad. Sci. USA 86:6338- 6342) . The identity of the -105 kDa and -125 kDa proteins in human CSF has been confirmed by purification and direct sequencing of their amino termini, both of which agreed with the sequence predicted from ,9APP cDNAs (Palmert et al., 1989, Proc. Natl. Acad. Sci. USA 86:6338-6342).
There are no definitive diagnostic markers for A other than pathological changes that occur in brain, the most important of which are neurofibrillary tangles, neuritic plaques, and vascular amyloid. Diagnostic techniques less invasive than brain biopsy are greatly needed.
Alzheimer's type lesions containing amyloid protein are found in adult cases of Down's syndrome (Ball and Nuttall, 1981, Neuropathol. Appl. Neurobiol. 7:13).
3. SUMMARY OF THE INVENTION
The present invention relates to methods for the prognosis, diagnosis, and staging of AD. It further relates to methods for monitoring response to therapy in a patient with AD. The methods of the invention involve the measurement of the levels in a sample of cerebrospinal fluid (CSF) of the -25 kDa, -105 kDa, and -125 kDa soluble derivatives of the APP. In specific embodiments, detection of an increase in the percentage amount of the ~25 kDa protein and/or decrease in the percentage amount of the -105 kDa derivative and/or high absolute levels of all three soluble 0APP derivatives, relative to healthy individuals, can be used to diagnose or prognose AD. In another embodiment, such detection can be used to diagnose Down's syndrome, and to prognose disorders in Down's syndrome patients associated with amyloid deposition. The foregoing can also be used as an indication of neurologic aging.
In other embodiments, determination of the percentage amounts of the -25 kDa protein and/or the -105 kDa protein can be used to stage AD, or to indicate the extent of mental dementia in a patient.
In embodiments directed toward monitoring therapy, an increase in the percentage amount of -25 kDa protein and/or a decrease in the percentage amount of -105 kDa protein relative to such amount present prior to therapy or in healthy individuals can be deemed a poor response to therapy.
The invention is also directed to the soluble -25 kDa amino-ter inal form of the 0APP.
4. DESCRIPTION OF THE FIGURES FIG. 1. Amino-terminal sequence (single letter code) of the -25 kDa soluble protein found in human CSF. X indicates that the signal was weak and did not permit an amino acid to be assigned. Note that the amino acid sequence predicted from 0APP cDNA (Kang et al., 1987, Nature 325:733-736) begins with a 17-residue signal sequence identified by Dyrks et al. (Dyrks et al., 1988, EMBO J. 7:949-957). Thus the amino-terminal residue of the mature protein is located at position 18 of the predicted sequence. ' FIG. 2. Standard curves from a protein A experiment. These graphs show the relationship between bound 125I protein A [shown as specific (total minus background) counts per minute (cpm) ] and increasing amounts of APP. This is 1 of 4 such experiments which produced standard curves for the -125, -105, and 25 kDa proteins with average r values of 0.9492 (n=3) , 0.9614 (n=4) , and 0.9409 (n=4) , respectively (see Section 6.1 for details).
FIGURE 3. Derivatives that may be produced when full-length 0APP is cleaved generating soluble forms (adapted from Palmert et al., 1989, Biochem. Biophys. Res. Commun. 165:182-188). The solid bar in each schematic indicates the position of the /3AP. The relative sizes of the membrane-associated and soluble forms (Palmert et al., 1989, Proc. Natl. Acad. Sci. USA 86:6338-6342) and the observation that the soluble -125 and -105 kDa derivatives in CSF are labeled by antisera to the βAP (Palmert et al., 1989, Biochem. Biophys. Res. Commun. 165:182-188) indicates that cleavage normally occurs as shown in A or B. The cleavage shown in C may sometimes occur in normal individuals, and it could develop or be enhanced in AD.
5. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to methods of prognosis and diagnosis of AD. It also relates to methods of staging AD and to monitoring response to therapy in a patient with AD. The invention is based upon the measurement-in cerebrospinal fluid (CSF) of a patient of the levels of soluble derivatives of the 0APP, in particular, the approximately (-) 25 kDa, -125 kDa, and -105 kDa soluble forms. In another embodiment, the invention is directed to the soluble. -25 kDa amino-terminal form of the APP. The methods provided by the present invention also allow evaluation and monitoring of neurologic aging in an individual. The invention further provides methods for diagnosis of Down's syndrome. The term "soluble" as used herein shall mean th which is "not cell-associated, not due to any artificial intervention," as opposed to the term "solubilized," whic refers to that which is artificially released from a cell surface into solution, e.g., by detergent cell lysis.
As detailed in the Examples section, infra, we have quantitated the -25 kDa, -105 kDa, and -125 kDa soluble N-terminal 0APP derivatives, and demonstrate (i) that in AD, there is a significant decrease in the relati amount of the -105 kDa form and a corresponding significa increase in the relative amount of the -25 kDa form (ii) that these changes correlate with the mental status of th AD patients, and (iii) that the same changes occur to a lesser extent in elderly as compared with young control patients.
5.1. MEASUREMENT OF SOLUBLE DERIVATIVES OF THE 0APP IN CEREBROSPINAL FLUID
The methods provided by the present invention involve the measurement of the levels of soluble derivatives of the 0APP in cerebrospinal fluid of a patient. Such soluble derivatives are selected from the group consisting of the -25 kDa, -105 kDa, and the -125 k ,9APP derivatives, which derivatives consist of a portion o the sequence of the 0APP starting with (as their amino- terminal residue) the amino acid at position 18 of the predicted 0APP sequence (see Kang, 1987, Nature 325:733- 736).
According to the invention, the levels of one o more of the above-mentioned soluble derivatives are measured in CSF from a patient.
For the purpose of such measurement, CSF can be obtained by any procedures known in the art. For example, CSF can be obtained by lumbar puncture. Dialysis into desired buffers and concentration before analysis can also be carried out. Measurement of the soluble 3APP derivatives can be by any techniques known in the art. In a preferred embodiment, an immunoblotting (Western blotting) procedure with appropriate antisera can be used to quantitate the soluble derivative(s) (see Examples sections, infra) . For example, blots (e.g., nitrocellulose) of denaturing (e.g., sodium dodecyl sulfate-polyacrylamide) electrophoretic gels of separated soluble CSF proteins can be reacted with the appropriate antibody for detection and quantitation purposes. Such an appropriate antibody is one reactive with the soluble derivative being quantitated. For example, to quantitate all three -25 kDa, -105 kDa, and -125 kDa derivatives with one antibody, antibodies which can be used include but are not limited to an antiserum against amino acids 45-62 in the APP sequence described by Kang et al., 1987, Nature 325:733-736) (anti-θAPP._ _„) , and an antiserum to residues 18-35 of the APP. It is expected that antibodies recognizing an epitope within a -25 kDa fragment of the y9APP starting at amino acid 18 should be suitable for such use. The antibody thus employed can be labeled, or it can be reacted with a labeled binding partner of the antibody (e.g. 125I-labeled protein A) for detection and quantitation purposes. It is envisioned that other assays suitable for use will be known to those skilled in the art, including but not limited to assays employing one or more of the following techniques: chromatography (e.g.; ion exchange, immunoaffinity, immunoabsorption, and sizing chromatography such as high pressure liquid chromatography) , centrifugation, electrophoretic procedures, differential solubility, competitive and non- competitive immunoassay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay) , "sandwich" immunoassays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and immunoelectrophoresis assays, to name but a few; for example, immunoassay of HPLC fractions of CSF.
Antibodies against the soluble 0APP derivatives for use in immunodetection assays can be produced by methods known in the art. Such antibodies can be polyclonal or monoclonal.
For example, various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of a given soluble ,9APP derivative. For the production of antibody, various host animals can be immunized by injection with 0APP, or a fragment thereof, or synthetic protein or peptide corresponding to the foregoing, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species.
A monoclonal antibody can be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497), and the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72) and EBV- hybridoma technique (Cole et al., 1985, Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77- 96) and others. Antibody fragments which contain the idiotype (binding region) of the molecule can also be used, and ca generated by known techniques. For example, such fragmen include but are not limited to: the F(ab'j fragment whic can be produced by pepsin digestion of the antibody molec the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')_ fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
5.2. DIAGNOSIS, PROGNOSIS, AND MONITORING THERAPY OF AD
The quantitation of the soluble 3APP derivative( in CSF according to the present invention can be used for diagnosis, staging and/or prognosis of AD. Such quantitat can also be used to monitor therapy of a patient with AD.
In a specific embodiment, detection of an. increa in the percentage amount (% of total -25 kDa + -125 kDa + -105 kDa soluble 0APP derivatives) of the -25 kDa derivati and/or a decrease in the percentage amount of the -105 kDa derivative, relative to healthy individuals, can be used a an indication of the presence or impending onset of AD, or predicting the course of AD particularly when serial measurements are made. Increased percentage of the -25 kD protein and/or decreased percentage of -105 kDa protein, and/or high absolute levels of all three soluble 0APP derivatives in non-demented individuals can be a significa risk factor such that a high percentage of the non7demente individuals who show such a profile eventually develop AD. The percentage amount of the -25 kDa protein, and/or the percentage amount of the -105 kDa protein, can be used to stage AD patients, and as an indication of the mental stat of the AD patients (see. Examples sections, infra) . Such percentage amounts can also be used to monitor therapeutic strategies for AD, such as those aimed at reducing amyloid deposition. For example, an increase in the percentage amount of -25 kDa derivative, and/or decrease in the percentage amount of -105 kDa derivative relative to such amount present prior to therapy or in healthy individuals, can be deemed a poor response to therapy.
An increase in the percentage amount of the -25 kD protein, and/or a decrease in the percentage amount of the -105 kDa protein, and/or an increase in the absolute amount(s) of one or more of the soluble ,5APP derivatives, ca also be used as an indication of neurologic aging. Thus, i particular embodiments, detection of such an increase for t -25 kDa protein and/or decrease for the -105 kDa protein, o increased absolute amounts, relative to young individuals (e.g., those individuals under age 60), can indicate neurologic aging.
In other specific embodiments of the invention, the ratios of the level(s) of one or more of the soluble 0APP derivative(s) to the level(s) of one or more different soluble 0APP derivatives can be calculated for use in the methods of the present invention. For example, the ratio o the amount of -25 kDa protein-to the amount of -105 kDa protein in a- sample of CSF can be determined for use in a method of diagnosis, prognosis, staging, or monitoring therapy, as provided by the present invention.
In yet another embodiment, the absolute level of one or more of the soluble 0APP derivatives can be determine for use in the methods of the present invention. Kits for use in practicing the methods of the invention are also provided. Such kits comprise, in one or more containers, one or more antibodies directed against th soluble /5APP derivative(s) being quantitated. 5.3. DIAGNOSIS OF DOWN'S SYNDROME
The invention further provides methods for diagnosis of Down's syndrome. In specific embodiments, methods based on measurements of the soluble /3APP derivatives, including but not limited to detection of an increase in the percentage amount (% of total -25 kDa and
-125 kDa and -105 kDa soluble 0APP derivatives) in CSF of t
-25 kDa derivative and/or a decrease in the percentage amou in CSF of the -105 kDa derivative, relative to healthy individuals, can be used as an indication of the presence o
Down's syndrome or as an indication of the presence or impending onset in the Down's syndrome patient of disorders associated with amyloid deposition (e.g., mental dementia).
6. EXAMPLE: SOLUBLE DERIVATIVES IN THE β AMYLOID PROTEIN PRECURSOR IN CEREBROSPINAL FLUID: ALTERATIONS IN NORMAL AGING AND IN ALZHEIMER'S DISEASE
As described herein we isolated and sequenced a soluble -25 kDa amino-terminal derivative of the β amyloid protein precursor (0APP) that can be detected in human cerebrospinal fluid (CSF) . In CSF samples from 24
Alzheimer's disease (AD) patients and 12 controls, we then quantitated -this -25 kDa form as well as the -125 and -105 kDa derivatives that we have previously identified (Palmert et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6338-6342).
Our analysis shows (i) that, in AD, there is a significant decrease in the relative amount of the -105 kDa form and a corresponding significant increase in the relative amount of the -25 kDa form, (ii) that these changes correlate with the mental status of the AD patients, and (iii) that the same changes occur to a lesser exent in elderly as compared with young control patients. These observations indicate that processing of the /SAPP changes in normal individuals as they age and to a greater extent in those who develop AD.
6.1. METHODS 6.1.1. ANTISERUM Production of antiserum to amino acids 45-62 ( APP45_62) in the /3APP sequence (Rang et al., 1987, Nature 325:733-736) has been described (Palmert et al., 1988, Biochem. Biophys. Res. Commun. 156:432-437), as has the characterization of this antiserum (Palmert et al., 1988, Biochem. Biophys. Res. Commun. 156:432-437; Palmert et al., 1989, Proc. Natl. Acad. Sci. USA 86:6338-6342). Briefly, a synthetic peptide corresponding to j9APP.g_g2 was conjugated to keyhole limpet hemocyanin for immunization of rabbits.
6.1.2. PURIFICATION AND SEQUENCING The -25 kDa protein was purified from CSF using ammonium sulfate fractionation, separation on a Mono-Q column (Pharmacia) , and preparative SDS/PAGE as previously described for the -125 and -105 kDa proteins (Palmert et al., 1989, Proc. Natl. Acad. Sci. USA 86:6338-6342). Briefly, the /3APP derivative was separated from the bulk (75%) of the,CSF protein by fractionation at 62.5% of ammonium sulfate saturation (0.41 g/ml) . The resulting pellet was resuspended in 20 mM sodium phosphate (monobasic) buffer' (pH 6.8), desalted, and loaded onto a Mono Q column (Pharmacia) . A linear 0 to 1 M NaCl gradient in 20 mM sodium phosphate (monobasic) buffer, pH 6*8, containing 0.05% Tween 20 was then applied to the column, and the -25 kDa derivative (along with -125 and -105 kDa derivatives) was eluted in a well defined peak at -60% (0.6 M NaCl) of the gradient. The fractions comprising this peak were pooled, desalted, concentrated, and subjected to preparative SDS/PAGE on a 5-15% gradient gel. To concentrate the -25 kDa derivative for sequencing, it was eluted from the preparative gel, electrophoresed in a single lane of another 5-15% SDS/PAGE gel, and transferre to Immobilon (Millipore Corp.). Sequence data was obtain using an Applied Biosystems 477A Sequenator.
6.1.3. PREPARATION AND QUANTITATION OF STANDARDS
Standards were prepared by dialyzing 25 ml of C from a control case (age = 74 yrs, postmortem interval = hrs) against 20 mM sodium phosphate (monobasic, pH 6.8) a loading it onto a Mono-Q column. Bound proteins were eluted with a linear 0 to 1 M NaCl gradient in 20 mM sodi phosphate (monobasic, pH 6.8). The void volume and the portion of the gradient between 0 and 47.8%, which contai none of the three 0APP derivatives, were combined to produce a constant background of 0APP-free CSF protein. Those proteins that eluted between 47.8 and 100% of the gradient, including the /3APP derivatives, were then addedt the "background" protein in varying amounts to make standards that had a total protein content equal to that i our average CSF sample and that contained enough of the /9APP derivatives to span the range present in our CSF samples. The standards were exchanged twice into 1 mM dibasic/monobasic (1.6/1) phosphate buffer (pH 6.9) using Centricon-10 microconcentrators (Amiσon) according to manufacturer's specifications. The concentrated standards were dried under vacuum and resuspended for SDS/PAGE on 5- 15% gradient gels. Nitrocellulose blots were, then blocked with 5% nonfat dried milk diluted in TBS (10 mM Tris-HCl, pH 8 and 150 M NaCl) ; exposed overnight to anti-øAPP.- g- (Palmert et al., 1988, Biochem. Biophys. Res. Commun. 156:432-437; Palmert et al., 1989, Proc. Natl. Acad. Sci. USA 86:6338-6342) diluted 1:100 in TBST (TBS (Tris-buffered saline) and 0.5% Tween-20) ; washed with TBST 4x for 15 minutes; exposed for 1 hour to 0.4 μCi/ml of [125]I protein A (ICN, specific activity 7.71 μCi/μq) diluted in TBST containing 5% nonfat dried milk; and washed with TBST 4x for 15 minutes. Autoradiograms were obtained so that labeled proteins could be visualized by superimposing the autoradiogram over the blot. Quantitation was achieved by excising each area of the nitrocellulose blot that contained the -125, -105, or major -25 kDa protein. The size of the excised area corresponded to the typical band size and was uniform from experiment to experiment. We defined the specific cpm for each protein as the total counts in the excised area minus those present in an area of equal size cut from a lane which contained only the "background" CSF proteins.
6.1.4. INDIVIDUAL CSF SAMPLES The clinical diagnosis of AD for the autopsied cases was established by a postmortem review of available medical records by an experienced clinician using standardized research criteria modified from the Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association, 1986, The Diagnostic and
Statistical Manual of Mental Disorders. Third edition- revised. Washington) . Pathological criteria for the diagnosis in these cases were similar to those suggested by a joint committee under the direction of' the National Institute of Aging and recently validated (Tierney et al., 1988, Neurology 38:359-364). The diagnosis of AD in the living patients was established clinically by reviewing criteria suggested by the National Institute of Neurological and Communicative Disorders and Stroke with the Alzheimer's Disease and Related Disorders Association (McKhann et al., 1984, Neurology 34:939-944). This method is valid and reliable (Morris et al., 1988, Ann. Neurol. 24:17-22). All patients included were classified as probable AD. Three of the subjects from whom CSF was obtained during life have subsequently died and AD was confirmed at postmortem examination in all three. The clinical diagnosis of Parkinson's disease (PD) required the presence of at least 2 of the 4 major features of the 0 idiopathic form of PD: resting tremor, stooped posture, shuffling gait, or muscular rigidity. No patients with secondary parkinsonism were included. Most of the living patients from whom CSF was obtained had received the 5 "Mini-Mental Status Examination" (Folstein et al., 1975, J. Psychiat. Res. 12:189-198). This brief examination of mental function is highly reliable and valid; a score of 24 or below is compatible with a diagnosis of dementia. For the living patients, CSF was obtained by lumbar puncture 0 with removal of 20 ml of fluid which was then aliquoted into 2 ml containers. Patients were at bed rest for several hours before and after the examination. All psychotropic medications were withdrawn prior to the lumbar puncture. CSF (240 μl) from each case was exchanged into 1 g mM dibasic/monobasic (1.6/1) phosphate buffer (pH 6.9), concentrated, and analyzed as described above for the standards. Each experiment contained both AD and control samples, which were loaded into alternating.lanes of each gel. 0
6.2. RESULTS
Using ammonium sulfate fractionation, fast protein liquid chromatography on a Mono Q column, and preparative SDS/PAGE (SDS-polyacrylamide gel 5 electrophoresis) , we have isolated a -25 kDa protein that is present in human CSF and that we previously showed (Palmert et al., 1989, Proc. Natl. Acad. Sci. USA 86:6338- 6342) to be specifically labeled by an antiserum against amino acids 45-62 in the APP (anti-0APP4g_62) . As shown in FIG. 1, we have sequenced 25 amino acids at the terminus of this -25 kDa protein, 23 residues were detectable, and all 23 were identical to those predicted from the published cDNA sequence. The amino-terminal residue of the -25 kDa protein, like that of the -125 and -105 kDa derivatives (Palmert et al., 1989, Proc. Natl. Acad. Sci. USA 86:6338- 6342) , is located at position 18 of the predicted sequence (Kang et al., 1987, Nature 325:733-736) after a 17-residue signal sequence (Dyrks et al., 1988, EMBO J. 7:949-957). On this basis we conclude that the -25 kDa protein, like the -125 and -105 kDa proteins, is an amino-terminal derivative of the 0APP. It should be noted that the "~25 kDa derivative" consists of a major band and 1 or 2 minor bands that migrate at -25 kDa on 5-15% SDS-polyacrylamide gels. All of these bands are specifically labeled by anti-/3APP.__62 and by an antiserum to residues 18-35 of the 0APP. Our sequence and the quantitative data shown below are from the.-.major protein within this complex. To assess the -125, -105, and -25 kDa /9APP derivatives in control and AD CSF, we have used I- labeled protein A to quantitate anti-0APP.g_62 immunoblots of CSF samples containing the three derivatives. Standard curves, which were generated by adding increasing amounts of the -125, -105, and -25 kDa 0APP derivatives to a constant background of øAPP-free CSF protein, showed a satisfactory relationship between the added /9APP and the amount of 125I-labeled protein A bound to immunoblots
(Figure 2) . Thus, this method is useful for assessing the absolute amount of each 3APP derivative in CSF samples (measured as cpm 125I-protem A bound to the immunolabeled protein in 240 μl of crude CSF) . Moreover, it is particularly well suited for determining the relative amounts of the three βkPP derivatives because these proteins in each CSF sample are processed together and compared in a single gel lane.
The results of our assessment of CSF samples from
24 AD patients (10 samples were obtained at autopsy and 14 were obtained conventionally from living patients) and 12 non-demented controls (including 3 non-demented patients with Parkinson's disease, 3 with multiple sclerosis, 1 with recurrent encephalitis, 1 with acute psychosis, 1 with hypersomnia, and 3 with no neurological disease) are summarized in Tables I and II.
For Table I, to assess changes in the relative amounts of the 3 ,5APP derivatives, the relative percentages were determined in each CSF sample and averaged to give the mean values tabulated here. The mean percentages calculated in this way give a true indication of the relative amounts of the /9APP derivatives present in the CSF of each patient group. These means cannot, on purely mathematical- grounds, be determined reliably from the average absolute amounts shown in Table II. Statistical significance was verified using unpaired Student's t tests.
Samples from the living and autopsy AD patients both showed a reduction in the percentage of -105 kDa form and an increase in the percentage of the' -25 kDa although the changes were slightly more pronounced in the samples obtained at autopsy. ω ω t ι -» _* TABLE I
RELATIVE AMOUNTS OF 3APP DERIVATIVES IN CONTROL AND AD CSF APP measured in CSF
Group
Young vs. Elderly Controls
Non-demented controls (30-60 yrs) Non-demented controls (62-79 yrs)
Figure imgf000020_0001
AD Cases by Mental Status
Mini-mental:17-24 Mini-mental:11-16 Mini-mental:0
Figure imgf000020_0002
All AD and Control Cases
Non-demented controls All AD
Figure imgf000020_0003
Age-matched Cases'
Non-demented controls (55-79 yrs) 7 AD (52-73 yrs) 13
Figure imgf000020_0004
For Table II, the absolute amounts of the three
/9APP derivatives in control and AD CSF are expressed in terms of cpm X 10 -3 of 125I-protem A bound to the immunolabeled protein and normalized for the amount of protein present in 240 μl of CSF. Because the protein content of the CSF samples showed little variation among the subgroups we examined, the same relationships were see whether the absolute numbers were normalized for volume
(240 μl) or protein content. Statistical significance was verified using unpaired Student's t tests (* denotes comparison between the least demented group of AD patients and the 12 controls) . The protein content of the CSF samples showed little variation among the subgroups we examined. Thus, the same relationships were seen whether the absolute numbers were normalized for volume (240 μl) o protein content. The specific cpm values for each experiment have been corrected for the radioactive decay that occurred during the course of experimentation. N.B.
If one calculates the relative amounts of each form from t average absolute data shown above, one does not obtain the values shown in Table I. These differences are not due to errors in compiling the data. They occur because on purel mathematical-grounds the relative amounts calculated from the average absolute values do not reliably depict the average relative amounts that are present in the populatio
To accurately assess changes in the relative amounts of th β PP derivatives, the relative percentages must be determined in each CSF sample and then averaged as we have done in Table I. O t o 01 o 01 Ul
TABLE II
ABSOLUTE AMOUNTS OF j9APP DERIVATIVES IN CONTROL AND AD CSF j8APP measured in CSF
Group No. Age 25kDa 105kDa 125kDa Total
Young vs. Elderly Controls
Non-demented controls (30-60 yrs) 7 45+4 1.5+0.6 6.2+1.6 1.3+0.4 9.0+2.5 Non-demented controls (62-79 yrs) 5 68+3 9.3+2.6 12.4+2.2 2.7+0.6 24.4+4.5
(p=0.006) (p=0.04) (p=0.06) (p=0.009)
AD Cases by Mental Status
Mini-mental: 17-24 6 71+5 7.8+2.2* 25.0+8.3* 3.2+1.2* 36.6+11.4* Mini-mental: 11-16 4 74+3 3.6+1.0 3.6+0.5 1.0+0.1 8.2+1.0 Mini-mental:0 4 68+3 6.4+1.3 3.7+2.7 0.9+0.5 11.0+3.9 *(p=0.28) *(p=0.02) *(p=0.18) *(p=0.03)
All AD and Control Cases
Non-demented controls 1.9+0.4* 15.4+3.2* All AD
Figure imgf000022_0001
1.8+0.4 17.7+3.7
Age-matched Cases
Non-demented controls (55-79 yrs) 7 65+3 6.8+2.4 10.2+2.1 2.3+0.5 19.3+4.6 AD (52-73 yrs) 13 65+2 6.3+1.0 7.8+3.1 1.6+0.4 15.9+4.3
Comparison of CSF samples from the elderly contro (62-79 years, mean + SE = 68 + 3) with those from the young controls (30-60 years, mean = 45 + 4) showed significant changes in both the relative and absolute amounts of the 3 βhPP derivatives. In the seven young controls, 14% of tota ,5APP was the -25 kDa form, 70% was the -105 kDa form, and 1 was the -125 kDa form. In the five elderly controls, the relative amount of the -25 kDa form was significantly highe at 35% (p-0.02), the relative amount of the -105 kDa form w significantly lower at 53% (p=0.04), and the relative amoun of the -125 kDa was reduced non-significantly to 11% (p=0.2 (see Table I) . The elderly controls also showed a more tha 6-fold increase in the absolute amount of the -25 kDa derivative (p=0.006), a 2-fold increase in both the -105 kD (p=0.04) and the -125 kDa (p=0.06) forms, and a more than two-fold increase in total βAPP derivatives (p=0.009) (Tabl II).
The shift toward a higher percentage of the -25 k form and a lower percentage of the -105 kDa form that we observed in elderly controls occurred to an even greater extent in AD patients. In 24 AD patients as compared to th 12 controls, the relative amount of the -25 kDa protein increased significantly from 23 to 47% (p=0.003), the relative amount of the -105 kDa protein decreased significantly from 63 to 42% (p=0.004), and the relative amount of the -125 kDa form decreased non-significantly fro 14 to 11% (p=0.11) of the total 0APP (Table. I). Since the A patients were on average 18 years older than the controls, compared an age matched subset that consisted of samples fro the 7 oldest controls (55-79 years, mean = 65 + 3) and the 1 youngest AD patients (52-73 years, mean = 65 + 2) . These age-matched AD cases also showed a significant increase in the percentage of the -25 kDa protein (p=0.03), a significan reduction in the percentage of the -105 kDa form (p=0.04), and a non-significant reduction in the percentage of the -12 kDa form (p-0.33) (Table I).
Of the 24 AD CSF samples analyzed, 10 were obtaine at autopsy and 14 were obtained conventionally from living patients whose mental status was assessed. When the results from the 14 living AD patients were grouped according to the results of Mini-mental testing, a striking trend became apparent (see Table I) . As dementia became more severe, the percentage of the -25 kDa form rose progressively from 25% i patients with a Mini-mental score of 17-24, to 42% in patients with a score of 11-16, and 70% in patients with a score of 0. Conversely, the percentage of the -105 kDa for decreased progresively from 65% in patients with a Mini- mental score of 17-24, to 45% in patients with a score of 11-16, and 23% in patients with a score of 0.
In the least demented group of AD patients, the percentages of the 3 forms were essentially the same as in the 12 non-demented controls. There was, however, a strikin increase in the absolute amount of the -105 kDa form (p=0.02 that, along with non-significant increases in the -125 (p=0.18) and -25 kDa (p=0.28) forms, resulted in a significant-increase in total 0APP (p==0.03) in the least demented AD patients as compared to the 12 controls (see Table II) . These changes, which were primarily due to extraordinarily high levels of 0APP derivatives in 3 of the patients examined, could be due to an overall increase in 0APP production or to an increase in the' rate at which soluble derivatives are produced from the full-length forms. 6.3. DISCUSSION Two factors that contribute significantly to amyloid deposition in other amyloidoses are aberrant catabolism and increased levels of the various amyloidogeni precursors (Cohen, A.S., and Connors, L.H., 1987, J. Pathol. 151:110; Castano, E.M. and Fragione, B.F., 1988, 58:122-132 and Kisilevsky, R. , 1988, 65:1805-1815). Our analysis of t APP derivatives in CSF indicates that both factors may contribute to amyloid deposition in AD. First, the percentage of the -105 kDa form is decreased and the percentage of the -25 kDa form is increased in normal aging and, to a greater extent, in AD, indicating that APP processing is altered in these conditions. Second, the absolute levels of soluble 0APP derivatives are increased in elderly control subjects and, to a greater extent, in the least demented AD patients.
If one considers young controls, elderly controls, slightly demented, moderately demented, and severely demente AD patients as a continuum, then our data show (i) a continuous increase in the relative amount of the -25 kDa form (with overlap of the elderly control and slightly demented patients) (lines 1-5, Table I) and (ii) an increasing level of the soluble derivatives (particularly th -105 kDa form) that peaks in the slightly demented group ahd then declines in the moderately and severely demented AD patients (lines i-5. Table II). These changes may be due to (i) accelerated proteolysis of the large soluble /3APP derivatives (particularly the -105 kDa KPI-free form) that leads to an increase in the relative amount of the -25 kDa form and (ii) increased production of soluble derivatives du either to an overall increase in 3APP production or to an increase in the rate at which soluble forms are produced fro full-length forms. Changes in the relative rates of these 2 opposing processes (production and degradation of APP derivatives) would readily account for the changes we observed in our comparison of young controls, elderly controls, and AD patients with dementia of increasing severity. Moreover, these changes could play an important role in amyloid deposition either by increasing the turnover of normal amyloidogenic intermediates or by generating aberrant, øAP-bearing forms from which amyloid could be generated.
The altered relative amounts of the -25 and -105 kDa derivatives in CSF correlate remarkably well with amyloid deposition in the sense that they change modestly in the elderly population where there is limited amyloid deposition, show more marked alteration in AD patients where amyloid deposition is pronounced, and change progressively as the severity of dementia increases in the AD population. The AD and age-matched control populations, though significantly different, were overlapping with respect to the absolute and relative levels of 0APP derivatives measured in CSF. These measurements may be useful (i) as part of a series of tests aimed at diagnosing AD, (ii) in predicting the course of AD particularly when serial measurements are made, and (iii) in monitoring therapeutic strategies aimed at reducing amyloid deposition. In addition, it is possible that an AD-like profile for these variables (increased % -25 kDa and decreased % -105 kDa, as was observed in the moderately and severely demented AD patients, or high absolute levels of APP, as was observed in the least demented AD patients) is a significant risk factor and that a high percentage of the non-demented individuals who show such a profile eventually develop AD. Various references are cited herein, the disclosures of which are incorporated by reference in the entireties.
The present invention is not to be limited in s by the embodiments disclosed in the examples which are intended as illustrations of a few aspects of the inventi and any embodiments which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown described herein will become apparent to those skilled in art and are intended to fall within the scope of the appe claims.

Claims

WHAT IS CLAIMED IS:
1. A substantially purified protein consisting of an amino acid sequence identical to a portion of the beta amyloid precursor protein, which portion (a) has a molecular weight of approximately 25 kilodaltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis; and (b) comprises the following sequence at its amino- terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is the leucine.
2. A method for diagnosing Alzheimer's disease in a patient comprising: measuring the amounts in a sample of cerebrospinal fluid obtained from the patient, of a soluble -25 kilodalton protein, a soluble -105 kilodalton protein, and a soluble -125 kilodalton protein, and calculating therefrom the percentage amount among the -25, -105, and -125 kilodalton proteins of the -25 kilodalton protein, in which the -25, -105, and -125 kilodalton proteins each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (b) comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in which the kilodalton molecular weights are determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and in which an increased percentage amount of the soluble -25 kilodalton protein relative to those percentage amounts present in healthy individuals or in the patient at an earlier time indicates the presence of Alzheimer's disease i the patient.
3. The method according to claim 2 which further comprises calculating the percentage amount, among the -25, -105, and -125 kilodalton proteins, of the -105 kilodalton protein, in which a decreased percentage amount of the soluble -105 kilodalton protein relative to those percentage amounts present in healthy individuals or in the patient at an earlier time indicates the presence of Alzheimer's disease in the patient.
10
4. A method for diagnosing Alzheimer's disease in a patient comprising: measuring the amounts in a sample of cerebrospinal fluid obtained from the patient, of an -25 kilodalton soluble protein, an -105 kilodalton soluble protein, and an -125 kilodalton soluble protein, and
15 calculating therefrom the percentage amount among the -25, -105, and -125 kilodalton proteins of the -105 kilodalton protein, in which the -25, -105, and -125 kilodalton proteins each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (b)
20 comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in which the kilodalton molecular weights are as determined by sodium
__ dodecyl sulfate-polyacrylamide gel electrophoresis, and in which a decreased percentage amount of the soluble -105 kilodalton protein relative to those percentage amounts present in healthy individuals or in the patient at an earlier time indicates the presence of Alzheimer's disease in
30 the patient.
5. A method for prognosing Alzheimer's disease in a patient comprising: measuring the amounts in a sample of cerebrospinal fluid obtained from the patient, of a soluble 35 -25 kilodalton protein, a soluble -105 kilodalton protein, and a soluble -125 kilodalton protein, and calculating therefrom the percentage amount among the -25, -105, and -125 kilodalton proteins of the -25 kilodalton protein, in which the -25, -105, and -125 kilodalton proteins each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (b) comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in which the kilodalton molecular weights are determined by sodium dodecyl sulfate-polyacryla ide gel electrophoresis; and in which an increased percentage amount of the soluble -25 kilodalton protein relative to those percentage amounts present in healthy individuals or in the patient at an earlier time indicates the impending onset or progression of Alzheimer's disease in the patient.
6. The method according to claim 5 which further comprises calculating the percentage amount, among the -25, -105, and -125 kilodalton proteins, of the -105 kilodalton protein, in which a decreased percentage amount of the soluble -105- ilodalton protein relative to those percentage amounts present in healthy individuals or in the patient at an earlier time indicates the impending onset or progression of Alzheimer's disease in the patient.
7. A method for prognosing Alzheimer's disease in a patient comprising: measuring the amounts in a sample of cerebrospinal fluid obtained from the patient, of an -25 kilodalton soluble protein, an -105 kilodalton soluble protein, and an -125 kilodalton soluble protein, and calculating therefrom the percentage amount among the -25, -105, and -125 kilodalton proteins of the -105 kilodalton protein, in which the -25, -105, and -125 kilodalton prote each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (b) comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in whi the kilodalton molecular weights are as determined by sodi dodecyl sulfate-polyacrylamide gel electrophoresis, and in which a decreased percentage amount of the soluble -105 kilodalton protein relative to those percentage amounts present in healthy individuals or in the patient at an earlier time indicates the impending onset or progression Alzheimer's disease in the patient.
8. A method for staging Alzheimer's disease comprising: measuring the amounts in a sample of cerebrospinal fluid obtained from the patient, of a soluble -25 kilodalton protein, a soluble -105 kilodalton protein, and a soluble -125 kilodalton protein, and calculating therefrom the percentage amount among the -25, -105, and -125 kilodalton proteins of the -25 kilodalton protein, in which the -25* -105, and -125 kilodalton proteins each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (b) comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in whic the kilodalton molecular weights are determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and in which the percentage amount of the soluble -25 kilodalton protein is indicative of the stage of Alzheimer's disease.
9. The method according to claim 8 which further comprises calculating the percentage amount, among the -25, -105, and -125 kilodalton proteins, of the -105 kilodalton protein, in which the percentage amount of the soluble -105 kilodalton protein is indicative of the stage of Alzheimer's disease.
10. A method for staging Alzheimer's disease comprising: measuring the amounts in a sample of cerebrospinal fluid obtained from the patient, of an -25 kilodalton soluble protein, an -105 kilodalton soluble protein, and an -125 kilodalton soluble protein, and calculating therefrom the percentage amount among the -25, -105, and -125 kilodalton proteins of the -105 kilodalton protein, in which the -25, -105, and -125 kilodalton protein each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (b) comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in which the kilodalton molecular weights are as determined by sodiu dodecyl sulfate-polyacrylamide gel electrophoresis, and in which the percentage amount of the soluble -105 kilodalton protein is indicative of the stage of Alzheimer's disease.
11. A method for determining the extent of dementia in a patient with Alzheimer's disease comprising: measuring the amounts in a sample of cerebrospinal fluid obtained from the patient, of a soluble -25 kilodalton protein, a soluble -105 kilodalton protein, and a soluble -125 kilodalton protein, and calculating therefrom the percentage amount among the -25, -105, and -125 kilodalton proteins of the -25 kilodalton protein, in which the -25, -105, and -125 kilodalton proteins each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (b) comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in whic the kilodalton molecular weights are determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and in which the percentage amount of the soluble -25 kilodalton protein is indicative of the extent of dementia.
12. The method according to claim 11 which furth comprises calculating the percentage amount, among the -25, -105, and -125 kilodalton proteins, of the -105 kilodalton protein, in which the percentage amount of the soluble -105 kilodalton protein is indicative of the extent of dementia.
13. A method for determining the extent of dementia in a patient with Alzheimer's disease comprising: measuring the amounts in a sample of cerebrospinal fluid obtained from the patient, of an -25 kilodalton soluble protein, an -105 kilodalton soluble protein, and an -125 kilodalton soluble protein, and calculating therefrom the percentage amount among the -25, -105, and -125 kilodalton proteins of the -105 kilodalton protein, in which the -25, -105, and -125 kilodalton proteins each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (b) comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in which the kilodalton molecular weights are as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in which the percentage amount of the soluble -105 kilodalton protein is indicative of the extent of dementia.
14. A method for monitoring the effect of a therapeutic treatment on a patient with Alzheimer's disease comprising: measuring the amounts in a sample of cerebrospinal fluid obtained from the patient, of a soluble -25 kilodalton protein, a soluble -105 kilodalton protein, and a soluble -125 kilodalton protein, and calculating therefrom the percentage amount among the -25, -105, and -125 kilodalton proteins of the -25 kilodalton protein, in which the -25, -105, and -125 kilodalton proteins each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and -(b) comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in which the kilodalton molecular weights are.determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and in which an increased percentage amount of the soluble -25 kilodalton protein relative to those percentage amounts present in healthy individuals or in the patient prior to the treatment indicates a poor response to the therapeutic treatment.
.
15. The method according to claim 14 which further comprises calculating the percentage amount, among the -25, -105, and -125 kilodalton proteins, of the -105 kilodalton protein, in which a decreased percentage amount of the soluble -105 kilodalton protein relative to those percentage amounts present in healthy individuals or in the patient prior to the treatment indicates a poor response to the therapeutic treatment.
16. A method for monitoring the effect of a therapeutic treatment on a patient with Alzheimer's disease comprising: measuring the amounts in a sample of cerebrospinal fluid obtained from the patient, of an -25 kilodalton soluble protein, an -105 kilodalton soluble protein, and an -125 kilodalton soluble protein, and calculating therefrom the percentage amount among the -25, -105, and -125 kilodalton proteins of the -105 kilodalton protein, in which the -25, -105, and -125 kilodalton proteins each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (b) comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in which the kilodalton molecular weights are as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in which a decreased percentage amount of the soluble -105 kilodalton protein relative to those percentage amounts present in healthy individuals or in the patient prior to the treatment indicates a poor response to the therapeutic treatment.
17. A method for diagnosing an early stage of or prognosing Alzheimer's disease in a patient comprising: measuring the total amount in a sample of cerebrospinal fluid obtained from the patient, of a soluble -25 kilodalton protein, a soluble -105 kilodalton protein, and a soluble -125 kilodalton protein, in which the -25, -105, and -125 kilodalton proteins each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precurs protein, and (b) comprise the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in whic the kilodalton molecular weights are determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and in which an increased total amount of the soluble -25, -105, a -125 kilodalton proteins relative to those amounts present healthy individuals of the patient's approximate age, indicates the presence of an early stage of or impending onset of Alzheimer's disease.
18. A method for the detection of neurologic agi in a patient comprising: measuring the total amount in a sample of cerebrospinal fluid obtained from the patient, of soluble -25 kilodalton protein, a soluble -105 kilodalton protein, and a soluble -125 kilodalton protein, in which th -25, -105, and -125 kilodalton proteins each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (b) comprise the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in whic the kilodalton molecular weights are determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and in which an increased total amount of the soluble -25, -105, a -125 kilodalton proteins relative to those amounts present young individuals indicates neurologic aging.
19. A method for the detection of neurologic agi in a patient comprising measuring the amount of a soluble protein in a sample of cerebrospinal fluid obtained from th patient, in which the protein (a) has a molecular weight of approximately 25 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (b) consists of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (c) comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly, in which the amino-terminal residue is the leucine; and in which an elevated level of the soluble protein relative to those levels present in young individuals indicates neurologic aging.
20. A method for diagnosing Down's syndrome in a patient comprising: measuring the amounts in a sample of cerebrospinal fluid obtained from the patient, of a soluble -25 kilodalton protein, a soluble -105 kilodalton protein, and a soluble -125 kilodalton protein, and calculating therefrom the percentage amount among the -25, -105, and -125 kilodalton proteins of the -25 kilodalton protein, in which the -25, -105, and -125 kilodalton proteins each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (b) comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in which the kilodalton molecular weights are determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and in which an increased percentage amount of the soluble -25 kilodalton protein relative to those percentage amounts present in healthy individuals indicates the presence of Down's syndrome in the patient.
21. The method according to claim 20 which further comprises calculating the percentage amount, among the -25, -105, and -125 kilodalton proteins, of the -105 kilodalton protein, in which a decreased percentage amount of the soluble -105 kilodalton protein relative to those percentage amounts present in healthy individuals indicates the presence of Down's syndrome in the patient.
22. A method for diagnosing Down's syndrome in a patient comprising: measuring the amounts in a sample of cerebrospinal fluid obtained from the patient, of an -25 kilodalton soluble protein, an -105 kilodalton soluble protein, and an -125 kilodalton soluble protein, and calculating therefrom the percentage amount among the -25, -105, and -125 kilodalton proteins' of the -105 kilodalton protein, in which the -25, -105, and -125 kilodalton proteins each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (b) comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in which the kilodalton molecular weights are as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in which a decreased percentage amount of the soluble -105 kilodalton protein relative to those percentage amounts present in healthy individuals indicates the presence of Down's syndrome in the patient.
23. A method for prognosing a disorder associated with amyloid deposition in a patient with Down's syndrome comprising: measuring the amounts in a sample of cerebrospinal fluid obtained from the patient, of a soluble -25 kilodalton protein, a soluble -105 kilodalton protein, and a soluble -125 kilodalton protein, and calculating therefrom the percentage amount among the -25, -105, and -125 kilodalton proteins of the -25 kilodalton protein, in which the -25, -105, and -125 kilodalton proteins each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (b) comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in which the kilodalton molecular weights are determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and in which an increased percentage amount of the soluble -25 kilodalton protein relative to those percentage amounts present in healthy individuals or in the patient at an earlier time indicates the impending onset or progression of a disorder associated with amyloid deposition in the patient.
24. The method according to claim 23 which further comprises calculating the percentage amount, among the -25, -105, and -125 kilodalton proteins, of the -105 kilodalton protein, in which a decreased percentage amount of the soluble -105 kilodalton protein relative to those percentage amounts present in healthy individuals or in the patient in an earlier time indicates the impending onset or progression of a disorder associated with amyloid deposition in the patient.
25. A method for prognosing a'disorder associated with amyloid deposition in a patient with Down's syndrome comprising: measuring the amounts in a sample of cerebrospinal fluid obtained from the patient, of an -25 kilodalton soluble protein, an -105 kilodalton soluble protein, and an -125 kilodalton soluble protein, and calculating therefrom the percentage amount among the -25, -105, and -125 kilodalton proteins of the -105 kilodalton protein, in which the -25, -105, and -125 kilodalton proteins each (a) consist of an amino acid sequence identical to a portion of the beta amyloid precursor protein, and (b) comprises the following sequence at its amino-terminus: leu-glu-val-pro-thr-asp-gly-asn-ala-gly in which the amino-terminal residue is leucine; and in which the kilodalton molecular weights are as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in which a decreased percentage amount of the soluble -105 kilodalton protein relative to those percentage amounts present in healthy individuals or in the patient at an earlier time indicates the impending onset or progression of a disorder associated with amyloid deposition in the patient.
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