WO2006108051A2

WO2006108051A2 - Compositions and methods relating to alzheimer's disease

Info

Publication number: WO2006108051A2
Application number: PCT/US2006/012681
Authority: WO
Inventors: Howard Schulman; David Lowe; Christopher H. Becker; Haihong Zhou; Sushmita Mimi Roy
Original assignee: Neurodx, Llc
Priority date: 2005-04-05
Filing date: 2006-04-05
Publication date: 2006-10-12
Also published as: WO2006108051A3

Abstract

The present invention provides compositions, methods and kits useful for the diagnosis and treatment of Alzheimer's disease (AD). In particular, the invention provides polypeptides and metabolites that are markers of AD, polynucleotides that encode the polypeptides and antibodies that specifically bind to the polypeptides. The invention also provides fragments, precursors, successors and modified versions of the foregoing polypeptides, metabolites, polynucleotides and antibodies. The invention also provides compositions comprising the foregoing polypeptides, metabolites, polynucleotides and antibodies. The invention also provides methods for using the polypeptides, metabolites, polynucleotides and antibodies in the diagnosis and treatment of AD, monitoring progression of the disease and screening of candidate therapeutic compounds.

Description

COMPOSITIONS AND METHODS RELATING TO ALZHEIMER'S DISEASE

FIELD OF THE INVENTION The present invention provides compositions, methods and kits useful for the diagnosis and treatment of Alzheimer's disease (AD).

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is the fourth most common cause of death in the United States, after heart disease, cancer and stroke. It currently affects more than four million people in the United States, and this number is expected to double during the next forty years as the population ages. AD is also the most common cause of chronic dementia, and it is estimated that ten percent of the population older than 65 years of age has mild to severe dementia. This high prevalence, combined with the rate of growth of the over 65 demographic, makes dementia and particularly AD, important public health problems.

AD is the third most expensive disease in the United States, costing approximately $100 billion each year. This figure includes both direct costs, such as nursing home care and in-home day care, as well as indirect costs, such as lost subject and caregiver productivity. The early diagnosis and treatment of AD creates economic benefits by slowing the rate of cognitive decline, delaying institutionalization, reducing the burden of care, and improving quality of life.

AD is a complex multigenic neurodegenerative disorder characterized by progressive impairments in memory, behavior, language, and visual-spatial skills, ending ultimately in death. Hallmark pathologies of Alzheimer's disease include granulovascular neuronal degeneration, extracellular neuritic plaques with β-amyloid deposits, intracellular accumulation of Tau protein as neurofibrillary tangles, neurofibrillary degeneration, synaptic loss, and extensive neuronal cell death. It is now known that these histopathologic lesions of AD correlate with the dementia observed in many elderly people.

Considerable human genetic evidence has implicated alterations in production or processing of the human amyloid precursor protein (APP) in the etiology of the disease. However, intensive research has shown that AD is a multifactorial disease with many different, perhaps overlapping, etiologies. Proposed causes for AD include environmental factors (Perl, Environmental Health Perspective 63:149 (1985)), metal toxicity (Perl et al., Science 208:297 (1980)), defects in beta-amyloid protein metabolism (Shijo et al., Science 258:126 (1992); and Kosik, Science 256:780 (1992)), and abnormal calcium homeostasis and/or calcium activated kinases (Mattson et al., J. Neuroscience 12:376 (1992)). However, no single model of AD satisfactorily accounts for all neuropathologic findings or the requirement of aging for disease onset. The mechanisms of disease progression are equally unclear. Early detection and identification of Alzheimer's disease would allow prompt, appropriate treatment and care to be provided. However, there is currently no commercial laboratory diagnostic test for AD. Diagnosing Alzheimer's disease and distinguishing it from other dementias depends primarily on clinical evaluation, and ultimately clinical judgment. This procedure is time consuming and costly, requiring neurological examinations, neuropsychological testing, neuroimaging and laboratory testing of subject samples.

There are no known cures for Alzheimer's disease or treatments that will stop progression of the disease. Rather, the focus of drug treatment is on reducing the symptoms of the disease and potentially slowing its progression. Therefore, there is a need to identify biochemical markers of AD. There is also a need for improved compositions and methods for diagnosing AD and improved compositions and methods for treating AD.

SUMMARY OF THE INVENTION One aspect of the invention provides polypeptides that have been identified as differentially expressed in samples obtained from AD and/or mild cognitive impairment

(MCI) subjects as compared to samples obtained from control subjects, e.g., non-AD and/or non-MCI subjects ("polypeptide markers"). The invention also provides polypeptides that have substantial homology to polypeptide markers, modified polypeptide markers as well as fragments of the polypeptide markers. The invention also includes precursors and successors of the polypeptide markers in biological pathways. The invention also provides molecules that comprise a polypeptide marker, homologous polypeptide, a modified polypeptide marker or a fragment, precursor or successor of a polypeptide marker (e.g., a fusion protein). As used herein, the term "polypeptides of the invention" shall be understood to include all of the foregoing.

Another aspect of the invention provides non-peptide small molecules that have been identified as differentially expressed in samples obtained from AD and/or MCI subjects as compared to samples obtained from control subjects, e.g., non-AD and/or non-MCI subjects ("metabolite markers"). The invention also provides modified metabolite markers as well as fragments of metabolite markers. The invention also includes precursors and successors of the metabolite markers in biological pathways. The invention also provides molecules that comprise a metabolite marker, a modified metabolite marker or a fragment, precursor or successor of a metabolite marker. As used herein, the term "metabolites of the invention" shall be understood to include all of the foregoing.

Another aspect of the invention provides polynucleotides encoding polypeptides of the invention ("polynucleotide markers"). The invention also provides polynucleotides that have substantial homology to polynucleotide markers, modified polynucleotide markers, and fragments of polynucleotide markers. The invention also provides molecules that comprise a polynucleotide marker, homologous polynucleotide, a modified polynucleotide marker or a fragment of a polynucleotide marker (e.g., a vector). The "markers" of the present invention are intended to include analogs, compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not alter the differential expression of the marker. As used herein, the term "polynucleotides of the invention" shall be understood to include all of the foregoing.

Another aspect of the invention provides molecules that specifically bind to a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention. The binding molecule may be an antibody, antibody fragment, or other molecule. The invention also provides methods for producing a binding molecule that specifically recognizes a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention. Another aspect of the invention provides compositions comprising a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention, a binding molecule (e.g., an antibody) that is specific for a polypeptide of the invention, metabolite of the invention or polypeptide of the invention, an inhibitor of a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention, or another molecule that can increase or decrease the level or activity of a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention. Such compositions may be pharmaceutical compositions formulated for use as therapeutics. Another aspect of the invention provides a method for detecting a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention. In one embodiment, the method comprises contacting a biological sample obtained from a subject with a binding molecule (e.g., an antibody) under conditions that permit the formation of a stable complex, and detecting any stable complexes formed. In another embodiment, the method comprises determining the activity of a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention. In another embodiment, the method comprises determining the level of a polypeptide of the invention in a cell obtained from the subject by detecting the presence of a polynucleotide that encodes the polypeptide. Another aspect of the present invention is early identification of cognitive impairment. AD begins very gradually and progresses over many years. There is often a transitional period during which subjects are not cognitively normal but yet do not meet the diagnostic criteria for AD. This transitional stage is termed mild cognitive impairment (MCI). The invention provides methods and compositions of detecting MCI in a subject.

Another aspect of the invention provides a method for diagnosing AD and/or MCI in a subject by detecting a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention in a biological sample. In one embodiment, the method comprises obtaining a sample from a subject suspected of having AD or at risk for AD and comparing the level or activity of a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention in the sample with the level of activity in a sample obtained from a non-AD subject or with a reference range or value. In some embodiments, the method is used for staging or stratifying subjects with AD, monitoring the progression of the disease or response to therapy. In some embodiments, a plurality of polypeptides of the invention, metabolites of the invention, or polynucleotides of the invention are detected. In some embodiments, the method comprises detecting known biomarkers or considering other clinical indicia in addition to detecting one or more polypeptides of the invention, metabolites of the invention or polynucleotides of the invention in a biological sample.

Another aspect of the invention provides methods for treating AD and/or MCI by administering a therapeutic agent to a subject that increases or decreases the level or activity of a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention. For polypeptides of the invention, metabolites of the invention or polynucleotides of the invention that are increased in samples obtained from an AD subject, the method comprises administering a therapeutic agent that decreases (i.e., bring toward the normal range) the level or activity of the polypeptide, metabolite or polynucleotide. Similarly, for polypeptides of the invention, metabolites of the invention or polynucleotides of the invention that are decreased in samples obtained from an AD subject, the method comprises administering a therapeutic agent that increases the level or activity of the polypeptide, metabolite or polynucleotide.

Another aspect of the present invention provides a method for screening a candidate compound for use as a therapeutic agent for treating AD and/or MCI. In one embodiment, the method comprises administering the candidate compound to an AD and/or MCI subject and screening for the ability to modulate the level or activity of a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention. In another embodiment, the method comprises providing the candidate compound to a cell from an AD and/or MCI subject and screening for the ability to modulate the intracellular level of a polypeptide of the invention, a metabolite of the invention or polynucleotide of the invention.

Another aspect of the invention provides a kit for performing the methods described above. In one embodiment, the kit is for the diagnosis of AD and/or MCI by detection of a polypeptide of the invention, metabolite of the invention or polynucleotide of the invention in a biological sample from a subject. A kit for detecting a polypeptide of the invention, metabolite of the invention or polynucleotide of the present invention may include an antibody capable of binding to the polypeptide, metabolite or polynucleotide.

Other features and advantages of the invention will become apparent to one of skill in the art from the following detailed description, the drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic illustration of the analysis of the proteome and metabolome in human CSF used to identify the markers of the present invention.

DETAILED DESCRIPTION OF THE INVENTION The practice of the present invention employs, unless otherwise indicated, conventional methods of analytical biochemistry, microbiology, molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. (See, e.g., Sambrook, J. et al. Molecular Cloning: A Laboratory Manual. 3rd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2000; DNA Cloning: A Practical Approach, Vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition); Transcription and

Translation (B. Hames & S. Higgins, eds., Current Edition); CRC Handbook of Parvoviruses, Vol. I & II (P. Tijessen, ed.); Fundamental Virology, 2nd Edition, Vol. I & II (B. N. Fields and D. M. Knipe, eds.)).

The terminology used herein is for describing particular embodiments and is not intended to be limiting. As used herein, the singular forms "a," "and" and "the" include plural referents unless the content and context clearly dictate otherwise. Thus, for example, a reference to "a marker" includes a combination of two or more such markers. Unless defined otherwise, all scientific and technical terms are to be understood as having the same meaning as commonly used in the art to which they pertain. For the purposes of the present invention, the following terms are defined below: Definitions

As used herein, the terms "AD subject" and "a subject who has AD" are intended to refer to subjects who have been diagnosed with AD or probable AD. The terms "non- AD subject" and "a subject who does not have AD" are intended to refer to a subject who has not been diagnosed with AD or probable AD. A non-AD subject may be healthy and have no other disease, or they may have a disease other than AD.

As used herein, the terms "MCI subject" and "a subject who has MCI" are intended to refer to subjects who have been diagnosed with MCI or probable MCI. MCI diagnosis can be based on the Petersen clinical criteria for MCI (Petersen RC, Smith GE,

Waring SC et al. (1999), "Mild Cognitive Impairment: Clinical Characterization and Outcome. "Arch Neurol 56(3):303-308.). The Petersen criteria for MCI diagnosis include 1) a memory complaint by the subject, since most people in this transitional stage of impairment are aware of their difficulties; 2) relatively normal general cognitive function, i.e., other cognitive domains such as language, attention, executive function and visuospatial skills are relatively normal; 3) abnormal memory for the age of the subject; 4) not demented, i.e., do not meet the standard Diagnostic and Statistical Manual of Mental Disorders, Vol. IV criteria for dementia; and 5) absence of psychiatric symptoms. The terms "non-MCI subject" and "a subject who does not have MCI" are intended to refer to a subject who does not meet the diagnostic criteria for MCI or probable MCI. A non-MCI subject may be healthy and have no other disease, or they may have a disease other than MCI and AD.

The term "subject," as used herein, refers to any living organism capable of eliciting an immune response. The term subject includes, but is not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. As used herein, the term "antibody" refers to an immunoglobulin molecule capable of binding an epitope present on an antigen. The term is intended to encompasses not only intact immunoglobulin molecules such as monoclonal and polyclonal antibodies, but also bi-specific antibodies, humanized antibodies, chimeric antibodies, anti-idiopathic (anti-ID) antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, fusion proteins and any modifications of the foregoing that comprise an antigen recognition site of the required specificity. As used herein, the term "biological sample" includes a sample from any body fluid or tissue (e.g., serum, plasma, blood, cerebrospinal fluid, urine, sputum, thin cortical slice, brain tissue homogenate).

As used herein, a component (e.g., a marker) is referred to as "differentially expressed" in one sample as compared to another sample when the method used for detecting the component provides a different level or activity when applied to the two samples. A component is referred to as "increased" in the first sample if the method for detecting the component indicates that the level or activity of the component is higher in the first sample than in the second sample (or if the component is detectable in the first sample but not in the second sample). Conversely, a component is referred to as "decreased" in the first sample if the method for detecting the component indicates that the level or activity of the component is lower in the first sample than in the second sample (or if the component is detectable in the second sample but not in the first sample). In particular, marker is referred to as "increased" or "decreased" in a sample (or set of samples) obtained from an AD and/or MCI subject (or a subject who is suspected of having AD and/or MCI, or is at risk of developing AD and/or MCI) if the level or activity of the marker is higher or lower, respectively, compared to the level of the marker in a sample (or set of samples) obtained from anon- AD and/or non-MCI subject, or a reference value or range.

As used herein, the terms "significant difference" and "significantly different" are intended to refer to a difference in the molecule that permits the molecule be resolved using a detection assay, such as LC-MS, GC-MS, immunoassays, hybridization and enzyme assays, 2-D gel separations, binding assays (e.g., immunoassays), and competitive inhibition assays.

As used herein, the terms "fold increase" and "fold decrease" refer to the relative increase or decrease of in the level of a marker in one sample (or set of samples) compared to another sample (or set of samples). A positive fold change indicates an increase in the level of a marker while a negative fold change indicates a decrease in the level of a marker. The increase or decrease may be measured by any method or technique known to those of skill in the art. As will be appreciated by one of skill in the art, the observed increase or decrease may vary depending on the method or technique that is used. As used herein, the term "marker" includes polypeptide markers, metabolite markers and polynucleotide markers that are differentially expressed in one sample compared to another sample. For clarity of disclosure, aspects of the invention will be described with respect to "polypeptide markers," "metabolite markers" and "polynucleotide markers." However, statements made herein with respect to "polypeptide markers" are intended to apply to other polypeptides of the invention.

Likewise, statements made herein with respect to "metabolite markers" and "polynucleotide" markers are intended to apply to other metabolites and polynucleotides of the invention, respectively. Thus, for example, a polynucleotide described as encoding a "polypeptide marker" is intended to include a polynucleotide that encodes a polypeptide marker, a polypeptide that has substantial homology to a polypeptide marker, modified polypeptide markers, fragments, precursors and successors of a polypeptide marker, and molecules that comprise a polypeptide marker, homologous polypeptide, a modified polypeptide marker or a fragment, precursor or successor of a polypeptide marker (e.g., a fusion protein). As used herein, the term "polypeptide" refers to a single amino acid or a polymer of amino acid residues. A polypeptide may be composed of two or more polypeptide chains. A polypeptide includes a protein, a peptide, an oligopeptide, and an amino acid. A polypeptide can be linear or branched. A polypeptide can comprise modified amino acid residues, amino acid analogs or non-naturally occurring amino acid residues and can be interrupted by non-amino acid residues. Included within the definition are amino acid polymers that have been modified, whether naturally or by intervention, e.g., formation of a disulfide bond, glycosylation, lipidation, methylation, acetylation, phosphorylation, or by manipulation, such as conjugation with a labeling component. As used herein, a "fragment" of a polypeptide refers to a plurality of amino acid residues comprising an amino acid sequence that has at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 20 contiguous amino acid residues or at least 30 contiguous amino acid residues of a sequence of the polypeptide. As used herein, a "fragment" of polynucleotide refers to a single nucleic acid or to a polymer of nucleic acid residues comprising a nucleic acid sequence that has at least 15 contiguous nucleic acid residues, at least 30 contiguous nucleic acid residues, at least 60 contiguous nucleic acid residues, or at least 90% of a sequence of the polynucleotide. In the present invention, the terms "fragments," "analogs," or "derivatives" are used interchangeably to mean a chemical substance that is related to another substance (i.e., marker). The fragment can be, for example, differentially expressed in one sample compared to another sample. In a preferred embodiment, the fragment is differentially expressed in samples from AD subjects when compared to non-AD subjects. As used herein, a compound is referred to as "isolated" when it has been separated from at least one component with which it is naturally associated. For example, a polypeptide can be considered isolated if it is separated from contaminants including metabolites, polynucleotides and other polypeptides. Isolated molecules can be either prepared synthetically or purified from their natural environment. Standard quantification methodologies known in the art can be employed to obtain and isolate the molecules of the invention.

As used herein, the term "polynucleotide" refers to a single nucleotide or a polymer of nucleic acid residues of any length. The polynucleotide may contain deoxyribonucleotides, ribonucleotides, and/or their analogs and may be double-stranded or single stranded. A polynucleotide can comprise modified nucleic acids (e.g., methylated), nucleic acid analogs or non-naturally occurring nucleic acids and can be interrupted by non-nucleic acid residues. For example a polynucleotide includes a gene, a gene fragment, cDNA, isolated DNA, mRNA, tRNA, rRNA, isolated RNA of any sequence, recombinant polynucleotides, primers, probes, plasmids, and vectors. Included within the definition are nucleic acid polymers that have been modified, whether naturally or by intervention.

In some embodiments, a polypeptide or metabolite marker is a member of a biological pathway. As used herein, the term "precursor" or "successor" refers to molecules that precede or follow the polypeptide marker, metabolite marker or polynucleotide marker. Thus, once a polypeptide marker, metabolite marker or polynucleotide marker is identified as a member of one or more biological pathways, the present invention can include additional members of the biological pathway that come before or follow the polypeptide marker, metabolite marker or polynucleotide marker. Such identification of biological pathways and their members is within the skill of one in the art.

As used herein, the term "specifically binding," refers to the interaction between binding pairs (e.g., an antibody and an antigen) with an affinity constant of at most 10^"6 moles/liter, at most 10^'7 moles/liter, or at most 10^"8 moles/liter.

As used herein, two polypeptides are "substantially homologous" when there is at least 70% homology, at least 80% homology, at least 90% homology, at least 95% homology or at least 99% homology between their amino acid sequences, or when polynucleotides encoding the polypeptides are capable of forming a stable duplex with each other. Likewise, two polynucleotides are "substantially homologous" when there is at least 70% homology, at least 80% homology, at least 90% homology, at least 95% homology or at least 99% homology between their amino acid sequences or when the polynucleotides are capable of forming a stable duplex with each other. In general, "homology" refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis of similarity and identity, such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5 Suppl. 3:353-358, National biomedical Research Foundation, Washington, D.C., which adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math. 2:482-489, 1981 for peptide analysis. Programs for determining nucleotide sequence similarity and identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent similarity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded- specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.

Polypeptide and Metabolite Markers The invention is based in part on the discovery that certain polypeptides and metabolites are differentially expressed in cerebrospinal fluid (CSF) samples obtained from AD subjects and MCI subjects compared to CSF samples obtained from control subjects, e.g., non-AD and/or non-MCI subjects. The markers of the present invention may have diagnostic and/or therapeutic use in other neurological disorders in addition to AD. The terms "neurological disorder" or "CNS disorder," refer to an impairment or absence of a normal neurological function or presence of an abnormal neurological function in a subject. For example, neurological disorders can be the result of disease, injury, and/or aging. As used herein, neurological disorder also includes neurodegeneration, which causes morphological and/or functional abnormality of a neural cell or a population of neural cells. Non-limiting examples of morphological and functional abnormalities include physical deterioration and/or death of neural cells, abnormal growth patterns of neural cells, abnormalities in the physical connection between neural cells, under- or over production of a substance or substances, e.g., a neurotransmitter, by neural cells, failure of neural cells to produce a substance or substances which it normally produces, production of substances, e.g. , neurotransmitters, and/or transmission of electrical impulses in abnormal patterns or at abnormal times. Neurological disorders include, but are not limited to, memory disorders, dementia, memory loss, epilepsy, and ischemia. Neurological disorders also include neurodegenerative diseases. Neurodegeneration can occur in any area of the brain of a subject and is seen with many disorders including, but not limited to, Amyotrophic

Lateral Sclerosis (ALS), multiple sclerosis, Huntington's disease, and Parkinson's disease. CSF samples obtained from AD subjects and/or MCI subjects and non-AD and/or non-MCI subjects were separated into a high molecular weight fraction, containing proteins with molecular weights greater than about 5-kDa, and a low molecular weight fraction containing free floating peptides and small molecules having a molecular weight of less than about 5-kDa. After removal of high abundance proteins, the high molecular weight fraction was digested with trypsin. Each fraction was separated by chromatographic means and analyzed by mass spectrometry. The high molecular weight fraction was submitted to proteolysis before analysis by mass spectrometry as discussed in the Example. The resulting spectra were compared to identify individual markers that showed significant association with AD and/or MCI.

CSF samples from AD subjects were analyzed against non-AD subjects resulting in statistically significant (p<0.05) markers of AD, which were either increased (see Tables IA, 5A, and 9A) or decreased (see Tables IB, 5B, and 9B) compared to the non- AD levels.

CSF samples from MCI subjects were analyzed against non-MCI subjects resulting in statistically significant (p<0.05) markers of MCI, which were either increased (see Tables 2A, 6A, and 10A) or decreased (see Tables 2B, 6B, and 10B) compared to the non-MCI levels. MCI markers can be used to identify subjects in early stages of AD.

CSF samples from AD subjects and MCI subjects were analyzed against non-AD and non-MCI control subjects resulting in statistically significant (p<0.05) markers of cognitive impairment, which were either increased (see Tables 3A, 7A, and HA) or decreased (see Tables 3B, 7B, and 1 IB) compared to the control levels. In this comparison, AD and MCI subjects were treated as a single cohort and compared to the control group. The identified molecules are markers for an cognitive impairment that is biased toward markers associated with both AD and MCI.

CSF samples from AD subjects were analyzed against MCI subjects resulting in statistically significant (p<0.05) markers, which were either increased (see Tables 4A, 8A, and 12A) or decreased (see Tables 4B, 8B, and 12B) compared to the MCI levels.

These markers can be used to monitor the progression from MCI to AD.

The markers set forth in the Tables are each identified by a plurality of the following: the mass to charge ratio (m/z), chromatographic retention time (RT), the charge state of a molecular ion (z), protonated parent mass (M+H), expression ratio (exp. ratio), which is a ratio of mean group intensities indicating the relative normalized signal for disease group compared to control, retention index (RI), which is a linear measure of a component's elution time in comparison to a straight-chain alkane series injected independent of the sample, fold change (an expression change factor where positive indicates an intensity increase and negative indicates a decrease versus the control) and where available, identification number from NCBFs reference sequence database (Accession # and gi #) and additional information (e.g., the name or sequence of the peptide marker as contained in the NCBI queried database and database searching using the TurboQUEST program). As one of skill in the art will appreciate, the physical and chemical properties presented in the Tables are sufficient to distinguish the component from other materials; e.g., the components are uniquely identified by the mass to charge ratio (m/z) and the retention time (RT). A number of comparison studies were performed to identify the polypeptide and metabolite markers listed using various groups of AD and/or MCI subjects and non-AD and/or non-MCI subjects as controls. The Tables list markers that were found to be differentially present with statistical significance. Where a polypeptide or metabolite marker was found to be statistically significant in a plurality of studies, the data associated with the observations of highest statistical significance is presented.

Some variation is inherent in the measurements of the physical and chemical characteristics of the markers. The magnitude of the variation depends to some extent on the reproductively of the separation means and the specificity and sensitivity of the detection means used to make the measurement. Preferably, the method and technique used to measure the markers is sensitive and reproducible.

The retention time and mass to charge ratio may vary to some extent depending on a number of factors relating to the protocol used for the chromatography and the mass spectrometry parameters (e.g., solvent composition, flow rate). Preferably, sample preparation and analysis conditions are carefully controlled. However, one of skill in the art will appreciate that the possibility of contamination or measurement of artifacts can never be completely eliminated. The data set forth in the Tables reflects the method that was used to detect the markers, When a sample is processed and analyzed as described in the Example, the retention time of the marker is about the value stated for the marker (within about 10% of the value stated, within about 5% of the value stated, within about 1% of the value stated) and has a mass to charge ratio of about the value stated for the marker (within about 10% of the value stated, within about 5% of the value stated, within about 1% of the value stated).

The polypeptide, metabolite and polynucleotide markers of the invention are useful in methods for diagnosing AD and/or MCI, determining the extent and/or severity of the disease, monitoring progression of the disease and/or response to therapy. The markers are also useful in methods for treating AD and/or MCI and for evaluating the efficacy of treatment for the disease. The markers may also be used as pharmaceutical compositions or in kits. The markers may also be used to screen candidate compounds that modulate their expression. The markers may also be used to screen candidate drugs for treatment of AD and/or MCI.

In one aspect, the invention provides polypeptides and metabolites. In one embodiment, the invention provides an isolated component described in Table 1A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B,Table 8 A&B, Table 9A&B, Table 10 A&B, Table 11 A&B, or Table

12A&B. In another embodiment, the invention provides an isolated molecule that comprises a foregoing polypeptide or metabolite.

In another embodiment, the invention provides a polypeptide or metabolite having substantial homology with a component set forth in Table 1 A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B, Table 8A&B,

Table 9A&B, Table 10A&B, Table 11 A&B, or Table 12A&B. In another embodiment, the invention provides a molecule that comprises a foregoing polypeptide.

In another embodiment, the invention provides a polypeptide or metabolite having (i) a mass-to-charge value and (ii) an RT value of about the values stated, respectively, for a component described in Table 1A&B, Table 2A&B, Table 3A&B,

Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B, Table 8 A&B, Table 9A&B, Table 10A&B, Table 11 A&B, or Table 12A&B. In another embodiment, the invention provides a molecule that comprises a foregoing polypeptide or metabolite. In another embodiment, the invention provides a polypeptide or metabolite having (i) a mass-to-charge value within 10% (more particularly within 5%, more particularly within 1%) and (ii) an RT value within 10% (more particularly within 5%, more particularly within 1%) of the m/z and RT values stated, respectively, for a component described in Table 1A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B, Table 8A&B, Table 9A&B, Table 10A&B, Table 11A&B, or Table 12A&B. In another embodiment, the invention provides a molecule that comprises a foregoing polypeptide or metabolite. In another embodiment, the invention provides a polypeptide that is a fragment, precursor, successor or modified version of a marker described in Table 1 A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B, Table 8 A&B, Table 9A&B, Table 10 A&B, Table 11 A&B, or Table 12A&B. In another embodiment, the invention provides a molecule that comprises the foregoing polypeptide.

In another embodiment, the invention provides a polypeptide or metabolite that is structurally different from the components specifically identified in Table 1A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B, Table 8A&B, Table 9A&B, Table 10A&B, Table 11 A&B, or Table 12A&B but has the same (or nearly the same) function or properties. For example, such a polypeptide may have amino acid sequence that is changed only in nonessential amino acid residues. In another embodiment, the invention provides a molecule that comprises a foregoing polypeptide or metabolite.

Polypeptide and metabolite markers may be isolated by any suitable method known in the art. Native polypeptide and metabolite markers can be purified from natural sources by standard methods known in the art (e.g., chromatography, centrifugation, differential solubility, immunoassay). In one embodiment, polypeptide and metabolite markers may be isolated from a CSF sample using the chromatographic methods disclosed herein. In another embodiment, polypeptide and metabolite markers may be isolated from a sample by contacting the sample with substrate-bound antibodies that specifically bind to the marker. Metabolite makers may be synthesized using the techniques of organic and inorganic chemistry. Given the amino acid sequence or the corresponding DNA, cDNA, or mRNA that encodes them, polypeptides markers may be synthesized using recombinant or chemical methods. For example, polypeptide markers can be produced by transforming a host cell with a nucleotide sequence encoding the polypeptide marker and cultured under conditions suitable for expression and recovery of the encoded protein from the cell culture. (See, e.g., Hunkapiller et al., 1984 Nature 310:105-111).

Polynucleotides Encoding Polypeptide Markers

In one aspect, the invention provides polynucleotides that encode the polypeptides of the invention. The polynucleotide may be genomic DNA, cDNA, or mRNA transcripts that encode the polypeptides of the invention.

In one embodiment, the invention provides polynucleotides that encode a polypeptide described in Table 1A&B, Table 2A&B, Table 3A&B, Table 4A&B,

Table 5A&B, Table 6A&B, Table 7A&B, Table 8A&B, Table 9A&B, Table 10A&B, Table 11A&B, or Table 12A&B, or a molecule that comprises such a polypeptide.

In another embodiment, the invention provides polynucleotides that encode a polypeptide having substantial homology with a component set forth in Table 1 A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B,

Table 8A&B, Table 9A&B, Table 10A&B, Table 11 A&B, or Table 12A&B, or a molecule that comprises such a polypeptide.

In another embodiment, the invention provides polynucleotides that encode a polypeptide having (i) a mass-to-charge value and (ii) an RT value of about the values stated, respectively, for a marker described in Table 1 A&B, Table 2A&B, Table 3 A&B,

Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B, Table 8A&B, Table 9A&B, Table 10 A&B, Table 11 A&B, or Table 12 A&B, or a molecule that comprises such a polypeptide.

In another embodiment, the invention provides polynucleotides that encode a polypeptide having (i) a mass-to-charge value within 10% (more particularly within 5%, more particularly within 1%) and (ii) an RT value within 10% (more particularly within 5%, more particularly within 1%) of the m/z and RT values stated, respectively, for a component described in Table 1A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B, Table 8A&B, Table 9A&B, Table 10A&B, Table 11 A&B, or Table 12A&B, or a molecule that comprises such polypeptide.

In another embodiment, the invention provides polynucleotides that encode a polypeptide that is a fragment, precursor, successor or modified version of a marker described in Table 1 A&B, Table 2A&B, Table 3 A&B, Table 4A&B, Table 5 A&B, Table 6A&B, Table 7A&B, Table 8A&B, Table 9A&B, Table 10A&B, Table 11 A&B, or Table 12A&B, or a molecule that comprises such polypeptide.

In another embodiment, the invention provides polynucleotides that encode a polypeptide that is structurally different from a polypeptide specifically identified in

Table 1A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B, Table 8 A&B, Table 9A&B, Table 10A&B, Table 11 A&B, or Table 12A&B but has about the same (or nearly the same) function or properties, or a molecule that comprises such polypeptide. In some embodiments, the polynucleotides described may be used as surrogate markers of AD or of MCI. Thus, for example, if the level of a polypeptide marker is increased in AD-subjects, an increase in the mRNA that encodes the polypeptide marker may be interrogated rather than the polypeptide marker (e.g., to diagnose AD in a subject). Many of the polypeptides listed in Table 1A&B, Table 2A&B, Table 3A&B,

Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B, Table 8A&B, Table 9A&B, Table 10A&B, Table 11 A&B, and Table 12A&Bare fragments of complete proteins ("parent proteins"), either because they were present as fragments in the sample or as a result of the trypsin digestion that was performed during the processing of certain fractions of the sample (see Example). The parent proteins are included as polypeptide markers. In many cases, the sequence of the parent protein can be ascertained from the amino acid sequence of the fragment by searching a protein sequence database.

Polynucleotides encoding the polypeptides markers listed in Tables 1-12 can be used to screen existing genomic, cDNA or expression libraries to find the gene that encodes the polynucleotide of the invention. A library is typically screened using a probe that is complementary either to the polynucleotide that encodes a polypeptide in Tables 1-12, or to its complement, under hybridization conditions. Hybridization is monitored by any suitable method known in the art. Once located, the gene can be cloned. The protein product of a gene that encodes a fragment of a polynucleotide marker is also included as a polypeptide marker. Alternatively, the sequence of the polynucleotide that encode a polypeptide listed in Tables 1-12 can be used to search databases such as SWISS-PROT and GenBank, which will provide the gene sequence(s) comprising the nucleic acid sequence, and the amino acid sequence of the gene product. Polynucleotide markers may be isolated by any suitable method known in the art.

Native polynucleotide markers may be purified from natural sources by standard methods known in the art (e.g., chromatography, centrifugation, differential solubility, immunoassay). In one embodiment, a polynucleotide marker may be isolated from a mixture by contacting the mixture with substrate bound probes that are complementary to the polynucleotide marker under hybridization conditions.

Alternatively, polynucleotide markers may be synthesized by any suitable chemical or recombinant method known in the art. In one embodiment, for example, the makers can be synthesized using the methods and techniques of organic chemistry. In another embodiment, a polynucleotide marker can be produced by polymerase chain reaction (PCR).

Binding Molecules In one aspect, the invention provides molecules that specifically bind to a polypeptide marker, a metabolite marker or a polynucleotide marker. The binding molecules include antibodies and antibody fragments.

In one aspect, the invention provides antibodies that specifically bind to a component described in Table 1A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B, Table 8A&B, Table 9A&B, Table 10A&B,

Table 11 A&B, or Table 12A&B, or to a molecule that comprises a foregoing component.

In another embodiment, the invention provides antibodies that specifically bind to a polypeptide having substantial homology with a component set forth in Table 1A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B,

Table 7A&B, Table 8 A&B, Table 9A&B, Table 10A&B, Table 11 A&B, or Table

12A&B, or to a molecule that comprises a foregoing polypeptide. In another embodiment, the invention provides antibodies that specifically bind to a component having (i) a mass-to-charge value and (ii) an RT value of about the values stated, respectively, for a marker described in Table 1 A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B, Table 8A&B,

Table 9A&B, Table 10 A&B, Table 11 A&B, or Table 12 A&B, or to a molecule that comprises a foregoing component.

In another embodiment, the invention provides antibodies that specifically bind to a component having (i) a mass-to-charge value within 10% (more particularly within 5%, more particularly within 1%) and (ii) an RT value within 10% (more particularly within 5%, more particularly within 1%) of the m/z and RT values stated, respectively, for a component described in Table 1A&B, Table 2A&B, Table 3 A&B, Table 4A&B, Table 5 A&B, Table 6A&B, Table 7 A&B, Table 8 A&B, Table 9 A&B, Table 10A&B, Table 11 A&B, or Table 12A&B, or to a molecule that comprises a foregoing component.

In another embodiment, the invention provides antibodies that specifically bind to a component that is a fragment, modification, precursor or successor of a marker described in Table 1 A&B, Table 2A&B, Table 3 A&B, Table 4A&B, Table 5 A&B, Table 6A&B, Table 7A&B, Table 8A&B, Table 9A&B, Table 10A&B, Table 11 A&B, or Table 12A&B, or to a molecule that comprises a foregoing component.

In another embodiment, the invention provides antibodies that specifically bind to a polypeptide marker, a metabolite marker or a polynucleotide marker that is structurally different from a component specifically identified in Table 1A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B, Table 8A&B, Table 9A&B, Table 10A&B, Table 11 A&B, or Table 12A&B but has the same (or nearly the same) function or properties, or to a molecule that comprises a foregoing component.

Certain antibodies that specifically bind polypeptide markers, metabolite markers or polynucleotide markers of the invention already may be known and/or available for purchase from commercial sources. In any event, the antibodies of the invention may be prepared by any suitable means known in the art. For example, antibodies may be prepared by immunizing an animal host with a marker or an immunogenic fragment thereof (conjugated to a carrier, if necessary). Adjuvants (e.g., Freund's adjuvant) optionally may be used to increase the immunological response. Sera containing polyclonal antibodies with high affinity for the antigenic determinant can then be isolated from the immunized animal and purified. Alternatively, antibody-producing tissue from the immunized host can be harvested and a cellular homogenate prepared from the organ can be fused to cultured cancer cells. Hybrid cells which produce monoclonal antibodies specific for a marker can be selected. Alternatively, the antibodies of the invention can be produced by chemical synthesis or by recombinant expression. For example, a polynucleotide that encodes the antibody can be used to construct an expression vector for the production of the antibody. The antibodies of the present invention can also be generated using various phage display methods known in the art.

Antibodies that specifically bind markers of the invention can be used, for example, in methods for detecting components described in Table 1 A&B, Table 2A&B, Table 3 A&B, Table 4A&B, Table 5 A&B, Table 6A&B, Table 7 A&B, Table 8 A&B,

Table 9A&B, Table 10 A&B, Table 11 A&B, or Table 12 A&B using methods and techniques well-known in the art. In some embodiments, for example, the antibodies are conjugated to a detection molecule or moiety (e.g., a dye, and enzyme) and can be used in ELISA or sandwich assays to detect markers of the invention. In another embodiment, antibodies against a polypeptide marker, metabolite marker or polynucleotide marker of the invention can be used to assay a tissue sample (e.g., a thin cortical slice) for the marker. The antibodies can specifically bind to the marker, if any, present in the tissue sections and allow the localization of the marker in the tissue. Similarly, antibodies labeled with a radioisotope may be used for in vivo imaging or treatment applications.

Compositions

Another aspect of the invention provides compositions comprising a polypeptide, metabolite or polynucleotide marker of the invention, a binding molecule that is specific for a polypeptide, metabolite or polynucleotide marker (e.g., an antibody), an inhibitor of a polypeptide, metabolite or polynucleotide marker, or other molecule that can increase or decrease the level or activity of a polypeptide marker, metabolite marker or polynucleotide marker. Such compositions may be pharmaceutical compositions formulated for use as a therapeutic.

In one embodiment, the invention provides a composition that comprises a polypeptide, metabolite or polynucleotide marker of the invention, such as a component described in Table 1A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B, Table 8A&B, Table 9A&B, Table 10A&B, Table 11A&B, or Table 12A&B, a polypeptide having substantial homology with a component or having (i) a mass-to-charge value and (ii) an RT value of about the values, respectively, for a component, or a molecule comprising such a component.

Alternatively, the invention provides a composition that comprises a component that is a fragment, modification, precursor or successor of a marker described in Table 1A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B, Table 7A&B, Table 8A&B, Table 9A&B, Table 10A&B, Table 11A&B, or Table 12A&B, or to a molecule that comprises a foregoing component.

In another embodiment, the invention provides a composition that comprises a polypeptide or metabolite that is structurally different from a component specifically identified in Table 1 A&B, Table 2A&B, Table 3A&B, Table 4A&B, Table 5A&B, Table 6A&B, Table 7 A&B, Table 8 A&B, Table 9A&B, Table 10 A&B, Table 11 A&B, or Table 12A&B but has the same function or properties, or a molecule that comprises a foregoing component.

In another embodiment, the invention provides a composition that comprises a polynucleotide that binds to a polypeptide or metabolmic marker, or a molecule that comprises a foregoing polynucleotide. In another embodiment, the invention provides a composition that comprises an antibody that specifically binds to a polypeptide or metabolomic marker, or a molecule that comprises a foregoing antibody.

In another embodiment, the invention provides a composition that comprises a modulator of the level or activity of a polypeptide marker (e.g., an inhibitor of a polypeptide marker, an antisense polynucleotide which is complementary to a polynucleotide that encodes a polypeptide marker), or a molecule that comprises a foregoing modulator. Such compositions may be pharmaceutical compositions. Typically, a pharmaceutical composition comprises a therapeutically effective amount of an active agent and is formulated with a suitable excipient or carrier. The invention also provides pharmaceutical compositions for the treatment of AD and/or MCI. These compositions may include a marker protein and/or nucleic acid of the invention (e.g., for those markers which are decreased in quantity or activity in AD samples versus non-AD samples), and can be formulated as described herein. Alternately, these compositions may include an antibody which specifically binds to a marker protein of the invention and/or an antisense polynucleotide which is complementary to a polynucleotide marker of the invention (e.g., for those markers which are increased in quantity or activity in AD samples versus non-AD samples), and can be formulated as described herein. The pharmaceutical compositions of the invention can be prepared in any suitable manner known in the pharmaceutical art. The carrier or excipient may be a solid, semisolid, or liquid material that can serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are well known in the art and include, but are not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical compositions may be adapted for oral, inhalation, parenteral, or topical use and may be administered to the subject in the form of tablets, capsules, aerosols, inhalants, suppositories, solutions, suspensions, powders, syrups, and the like. As used herein, the term "pharmaceutical carrier" may encompass one or more excipients. In preparing formulations of the compounds of the invention, care should be taken to ensure bioavailability of an effective amount of the agent. Suitable pharmaceutical carriers and formulation techniques are found in standard texts, such as Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,

Pennsylvania.

Methods for Detecting

The markers of the invention may be detected by any method known to those of skill in the art, including without limitation LC-MS, GC-MS, immunoassays, hybridization and enzyme assays. The detection may be quantitative or qualitative. A wide variety of conventional techniques are available, including mass spectrometry, chromatographic separations, 2-D gel separations, binding assays (e.g., immunoassays), competitive inhibition assays, and so on. Any effective method in the art for measuring the present/absence, level or activity of a metabolite, polypeptide or polynucleotide is included in the invention. It is within the ability of one of ordinary skill in the art to determine which method would be most appropriate for measuring a specific marker.

Thus, for example, a ELISA assay may be best suited for use in a physician's office while a measurement requiring more sophisticated instrumentation may be best suited for use in a clinical laboratory. Regardless of the method selected, it is important that the measurements be reproducible. The markers of the invention can be measured by mass spectrometry, which allows direct measurements of analytes with high sensitivity and reproducibility. A number of mass spectrometric methods are available. Electrospray ionization (ESI), for example, allows quantification of differences in relative concentration of various species in one sample against another; absolute quantification is possible by normalization techniques (e.g., using an internal standard). Matrix-assisted laser desorption ionization

(MALDI) or the related SELDI® technology (Ciphergen, Inc.) also could be used to make a determination of whether a marker was present, and the relative or absolute level of the marker. Mass spectrometers that allow time-of-flight (TOF) measurements have high accuracy and resolution and are able to measure low abundant species, even in complex matrices like serum or CSF.

For protein markers, quantification can be based on derivatization in combination with isotopic labeling, referred to as isotope coded affinity tags ("ICAT"). In this and other related methods, a specific amino acid in two samples is differentially and isotopically labeled and subsequently separated from peptide background by solid phase capture, wash and release. The intensities of the molecules from the two sources with different isotopic labels can then be accurately quantified with respect to one another. In addition, one- and two-dimensional gels have been used to separate proteins and quantify gels spots by silver staining, fluorescence or radioactive labeling. These differently stained spots have been detected using mass spectrometry, and identified by tandem mass spectrometry techniques.

In a preferred embodiment, the markers are measured using mass spectrometry in connection with a separation technology, such as liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry. In particular, coupling reverse- phase liquid chromatography to high resolution, high mass accuracy ESI time-of-flight (TOF) mass spectroscopy allows spectral intensity measurement of a large number of biomolecules from a relatively small amount of any complex biological material. Analyzing a sample in this manner allows the marker (characterized by a specific RT and m/z) to be determined and quantified.

As will be appreciated by one of skill in the art, many other separation technologies may be used in connection with mass spectrometry. For example, a wide selection of separation columns is commercially available. In addition, separations may be performed using custom chromatographic surfaces (e.g., a bead on which a marker specific reagent has been immobilized). Molecules retained on the media subsequently may be eluted for analysis by mass spectrometry.

Analysis by liquid chromatography-mass spectrometry produces a mass intensity spectrum, the peaks of which represent various components of the sample, each component having a characteristic mass-to-charge ratio (m/z) and retention time (RT).

The presence of a peak with the m/z and RT of a marker indicates that the marker is present. The peak representing a marker may be compared to a corresponding peak from another spectrum (e.g., from a control sample) to obtain a relative measurement. Any normalization technique in the art (e.g., an internal standard) may be used when a quantitative measurement is desired. "Deconvoluting" software is available to separate overlapping peaks. The retention time depends to some degree on the conditions employed in performing the liquid chromatography separation. The preferred conditions, those used to obtain the retention times that appear in the Tables, are set forth in the Example. The mass spectrometer preferably provides high mass accuracy and high mass resolution. The mass accuracy of a well-calibrated Micromass TOF instrument, for example, is reported to be approximately 2 mDa, with resolution m/Δm exceeding 5000.

In other preferred embodiments, the level of the markers may be determined using a standard immunoassay, such as sandwiched ELISA using matched antibody pairs and chemiluminescent detection. Commercially available or custom monoclonal or polyclonal antibodies are typically used. However, the assay can be adapted for use with other reagents that specifically bind to the marker. Standard protocols and data analysis are used to determine the marker concentrations from the assay data. A number of the assays discussed above employ a reagent that specifically binds to the marker. Any molecule that is capable of specifically binding to a marker is included within the invention. In some embodiments, the binding molecules are antibodies or antibody fragments. In other embodiments, the binding molecules are non- antibody species. Thus, for example, the binding molecule may be an enzyme for which the marker is a substrate. The binding molecules may recognize any epitope of the targeted markers.

As described above, the binding molecules may be identified and produced by any method accepted in the art. Methods for identifying and producing antibodies and antibody fragments specific for an analyte are well known. Examples of other methods used to identify the binding molecules include binding assays with random peptide libraries (e.g., phage display) and design methods based on an analysis of the structure of the marker. The markers of the invention, especially the metabolite markers, also may be detected or measured using a number of chemical derivatization or reaction techniques known in the art. Reagents for use in such techniques are known in the art, and are commercially available for certain classes of target molecules.

Finally, the chromatographic separation techniques described above also may be coupled to an analytical technique other than mass spectrometry such as fluorescence detection of tagged molecules, NMR, capillary UV, evaporative light scattering or electrochemical detection.

Measurement of the relative amount of an RNA or protein marker of the invention may be by any method known in the art (see, e.g., Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring

Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Typical methodologies for RNA detection include RNA extraction from a cell or tissue sample, followed by hybridization of a labeled probe (e.g., a complementary polynucleotide) specific for the target RNA to the extracted RNA, and detection of the probe (e.g., Northern blotting). Typical methodologies for protein detection include protein extraction from a cell or tissue sample, followed by hybridization of a labeled probe (e.g., an antibody) specific for the target protein to the protein sample, and detection of the probe. The label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Detection of specific protein and polynucleotides may also be assessed by gel electrophoresis, column chromatography, direct sequencing, or quantitative PCR (in the case of polynucleotides) among many other techniques well known to those skilled in the art.

Detection of the presence or number of copies of all or a part of a marker gene of the invention may be performed using any method known in the art. Typically, it is convenient to assess the presence and/or quantity of a DNA or cDNA by Southern analysis, in which total DNA from a cell or tissue sample is extracted, is hybridized with a labeled probe (e.g., a complementary DNA molecule), and the probe is detected. The label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor. Other useful methods of DNA detection and/or quantification include direct sequencing, gel electrophoresis, column chromatography, and quantitative PCR, as is known by one skilled in the art.

Polynucleotide similarity can be evaluated by hybridization between single stranded nucleic acids with complementary or partially complementary sequences. Such experiments are well known in the art.

Methods for Diagnosing Alzheimer's Disease and/ or Mild Coginitive Impairment

In another aspect, the invention provides methods for diagnosing AD in a subject. In one embodiment, the invention provides a method for determining whether a subject has AD. In another embodiment, the invention provides a method for determining whether a subject has MCI. These methods comprise obtaining a biological sample from a subject suspected of having AD and/or MCI, or at risk for developing AD, detecting the level or activity of a marker of the invention in the sample, and comparing the result to the level or activity of the marker in a sample obtained from anon- AD and/or non-MCI subject, or to a reference range or value. In one embodiment, an increased level or activity of the marker in a sample obtained from a subject suspected of having AD, or at risk for developing AD, is indicative that the subject has or is at risk for developing AD. Markers appropriate for this embodiment include those that have been identified as increased in samples obtained from AD subjects compared with samples fromnon-AD subjects (e.g., markers described in Table IA, Table 3A, Table 4A, Table 5 A, Table 7A, Table 8A, Table 9A, Table 1 IA, and Table 12A). Other markers appropriate for this embodiment include fragments, precursors, successors and modified versions of such markers, polypeptides having substantial homology to such markers, components having an m/z value and RT value of about the values set forth for the markers described in Table IA, Table 3 A, Table 4A, Table 5 A, Table 7 A, Table 8 A, Table 9A, Table 1 IA, and Table 12A, and molecules comprise one of the foregoing. Other appropriate markers for this embodiment will be apparent to one of skill in the art in light of the disclosure herein.

In another embodiment, an increased level or activity of the marker in a sample obtained from a subject suspected of having MCI, or at risk for developing MCI, is indicative that the subject has or is at risk for developing AD. Markers appropriate for this embodiment include those that have been identified as increased in samples obtained from MCI subjects compared with samples from non-MCI subjects (e.g., markers described in Table 2A, Table 3 A, Table 6A, Table 7A, Table 1OA, and Table 1 IA). Other markers appropriate for this embodiment include fragments, precursors, successors and modified versions of such markers, polypeptides having substantial homology to such markers, components having an m/z value and RT value of about the values set forth for the markers described in Table 2A, Table 3 A, Table 6A, Table 7A,

Table 1OA, and Table 1 IA, and molecules comprise one of the foregoing. Other appropriate markers for this embodiment will be apparent to one of skill in the art in light of the disclosure herein.

In another embodiment, a decreased level or activity of the marker in a sample obtained from a subject suspected of having AD, or at risk for developing AD, is indicative that the subject has or is at risk for developing AD. Markers appropriate for this embodiment include those that have been identified as decreased in samples obtained from AD subjects compared with samples fromnon-AD subjects (e.g., markers described in Table IB, Table 3B, Table 4B, Table 5B, Table 7B, Table 8B, Table 9B, Table HB, and Table 12B). Other markers appropriate for this embodiment include fragments, precursors, successor or modified versions of such markers, polypeptides having substantial homology to such markers, components having an m/z value and RT value of about the values set forth for the markers described in Table IB, Table 3B, Table 4B, Table 5B, Table 7B, Table 8B, Table 9B, Table HB, and Table 12B, and molecules comprise one of the foregoing. Other appropriate markers for this embodiment will be apparent to one of skill in the art in light of the disclosure herein In another embodiment, a decreased level or activity of the marker in a sample obtained from a subject suspected of having MCI, or at risk for developing AD, is indicative that the subject has or is at risk for developing MCI. Markers appropriate for this embodiment include those that have been identified as decreased in samples obtained from MCI subjects compared with samples from non-MCI subjects (e.g., markers described in Table 2B, Table 3B, Table 6B, Table 7B, Table 10B, and Table

1 IB). Other markers appropriate for this embodiment include fragments, precursors, successor or modified versions of such markers, polypeptides having substantial homology to such markers, components having an m/z value and RT value of about the values set forth for the markers described in Table 2B, Table 3B, Table 6B, Table 7B, Table 1OB, and Table HB, and molecules comprise one of the foregoing. Other appropriate markers for this embodiment will be apparent to one of skill in the art in light of the disclosure herein

In addition to diagnosing AD and/or MCI, the foregoing methods can be used to determine whether a subject is more likely than not to have AD, that the subject is more likely to have AD than another disease, or that the subject has an increased likelihood of having AD. The foregoing methods can also be used to confirm a diagnosis of AD.

The invention also provides a method for determining a subject's risk of developing AD, the method comprising obtaining a biological sample from a subject, detecting the level or activity of a marker in the sample, and comparing the result to the level or activity of the marker in a sample obtained from a non-AD subject, or to a reference range or value wherein an increase or decrease of the marker is correlated with the risk of developing AD.

The invention also provides methods for determining the stage or severity of AD, the method comprising obtaining a biological sample from a subject, detecting the level or activity of a marker in the sample, and comparing the result to the level or activity of the marker in a sample obtained from a non-AD subject, or to a reference range or value wherein an increase or decrease of the marker is correlated with the stage or severity of the disease. In another aspect, the invention provides methods for monitoring the progression of the disease in a subject who has AD, the method comprising obtaining a first biological sample from a subject, detecting the level or activity of a marker in the sample, and comparing the result to the level or activity of the marker in a second sample obtained from the subject at a later time, or to a reference range or value wherein an increase or decrease of the marker is correlated with progression of the disease.

Each marker may be considered individually, although it is within the scope of the invention to provide combinations of two or more markers for use in the methods and compositions of the invention. The use of such combinations typically will increase the confidence of the analysis. For example, a panel of markers may include markers that are increased in level or activity in AD subject samples as compared to non-AD subject samples, markers that are decreased in level or activity in AD subject samples as compared to non-AD subject samples, or a combination thereof. A panel of makers may include one or more markers of the invention as well as one or more known biomarkers of AD (e.g., Aβ). The panel of markers may also be evaluated with other clinical indicia of AD (e.g., ADAS-Cog, MRI/CT imaging).

The marker may be detected in any biological sample obtained from the subject, or in some cases, from a relative of the subject, by any suitable method known in the art (e.g., immunoassays, hybridization assay) see supra.

Methods for Treating Alzheimer's Disease and/or Mild Cognitive Impairment

The invention also provides methods for treating AD and/or MCI, as well as other diseases or conditions, by providing a therapeutic agent to a subject that increases or decreases the level or activity of at least one marker of the invention.

In one embodiment, the method comprises administering a therapeutic agent to a subject that increases level or activity of at least one polypeptide, metabolite or polynucleotide marker of the invention that is decreased in samples obtained from AD subjects compared to samples obtained from non-AD subjects or to a reference range or value.

In another embodiment, the method comprises administering a therapeutic agent to a subject that decreases the level of at least one polypeptide, metabolite or polynucleotide marker of the invention that is increased in samples obtained from AD subjects compared to samples obtained from non-AD subjects or to a reference range or value.

In another embodiment, the method further comprises first obtaining a sample from an AD subject, determining the presence, level or activity of at least one marker of the invention in the sample compared to samples obtained from a non-AD subject or to a reference range or value. If the marker is increased in the sample obtained from the AD subject, a therapeutic agent that decreases the level of the marker is administered to the subject. If the marker is decreased in the sample obtained from the AD subject, a therapeutic agent that increases the level of the marker is administered to the subject.

In another embodiment, the method comprises administering a therapeutic agent to a subject that increases level or activity of at least one polypeptide, metabolite or polynucleotide marker of the invention that is decreased in samples obtained from MCI subjects compared to samples obtained from non-MCI subjects or to a reference range or value.

In another embodiment, the method comprises administering a therapeutic agent to a subject that decreases the level of at least one polypeptide, metabolite or polynucleotide marker of the invention that is increased in samples obtained from MCI subjects compared to samples obtained from non-MCI subjects or to a reference range or value.

In another embodiment, the method further comprises first obtaining a sample from a MCI subject, determining the presence, level or activity of at least one marker of the invention in the sample compared to samples obtained from a non-MCI subject or to a reference range or value. If the marker is increased in the sample obtained from the MCI subject, a therapeutic agent that decreases the level of the marker is administered to the subject. If the marker is decreased in the sample obtained from the MCI subject, a therapeutic agent that increases the level of the marker is administered to the subject.

Therapeutic agents include but are not limited to polypeptide markers, metabolite markers, polynucleotide markers, molecules comprising a polypeptide marker, metabolite marker or polynucleotide marker, antibodies to polypeptide marker, metabolite marker or polynucleotide marker, modulators of the level or activity a polypeptide or polynucleotide marker (e.g., an inhibitor, anti-sense polynucleotides) or compositions comprising one or more of the foregoing. Generally, the therapeutic agents used in the invention are administered to the subject in an effective amount. An "effective amount" is typically the amount that is sufficient to obtain beneficial or desired clinical results. The effective amount is generally determined by a physician with respect to a specific subject and is within the skill of one in the art. Factors that may be taken into account in determining an effective amount include those relating to the condition being treated (e.g., type, stage, severity) as well as those relating to the subject (e.g., age, sex, weight).

The level or activity of a polypeptide marker may be increased or decreased by any suitable technique or method known in the art. The level of a polypeptide marker may be increased by providing the polypeptide marker to a subject. Alternatively, the level of a polypeptide marker may be increased by providing a polynucleotide that encodes the polypeptide marker (e.g., gene therapy). For those polypeptide markers with enzymatic activity, compounds or molecules known to increase that activity may be provided to the subject.

The level of a polypeptide marker may be decreased by providing antibodies specific for the polypeptide marker to the subject. Alternatively, the level of a polypeptide marker may be decreased by providing a polynucleotide that is "anti-sense" to the polynucleotide that encodes the polypeptide marker, or that encodes dysfunctional proteins. For those polypeptide markers with enzymatic activity, compounds or molecules known to decrease that activity (e.g., inhibitor or antagonist).

The level of a metabolite marker may be increased or decreased by any suitable technique or method known in the art. The level of a metabolite marker may be increase by providing the metabolite marker to the subject. Conversely, the level of a metabolite marker may be decreased by providing antibodies specific for the metabolite marker to the subject.

The therapeutic compounds described herein may be administered alone or in combination with another therapeutic compound, or other form of treatment. The compounds may be administered to the subjects in any suitable manner known in the art (e.g., orally, topically, subcutaneously, intradermally, intramuscularly, intravenously, intra-arterially, intrathecally). Metabolites may be combined with an excipient and formulated as tablets or capsules for oral administration. Polypeptides may be formulated for parenteral administration to avoid denaturation by stomach acids. For polynucleotides, vectors may be constructed for administration to the subject by a virus or other carrier. In a typical embodiment, cDNA is delivered to target cells (e.g., bone marrow cells) that are later reintroduced into the subject for expression of the encoded protein. A therapeutic composition can be administered in a variety of unit dosage forms depending upon the method of administration.

Methods for Screening Candidate Compounds

In another aspect, the invention provides methods for screening candidate compounds for use as therapeutic compounds. In one embodiment, the method comprises screening candidate compounds for those that bind to a polypeptide, metabolite or polynucleotide molecule of the invention. Candidate compounds that bind to markers can be identified using any suitable method or technique known in the art.

In one embodiment, a candidate compound or a control is contacted with marker and the ability of the candidate compound to form stable complexes is determined (e.g., flow cytometry, immunoprecipitation). The candidate compound, the marker, or an antibody that specifically binds either may be labeled to facilitate detection. The candidate molecule or marker may be immobilized on a solid support (e.g., a bead).

In another embodiment, cells expressing a polypeptide marker are contacted with a candidate compound or a control and the ability of the candidate compound to form stable complexes with the cells is determined. The candidate compound or the marker may be labeled to facilitate detection.

In another embodiment, the method comprises screening candidate compounds for those that have a stimulatory or inhibitory effect on the activity of a marker comprising comparing the activity of the marker in the presence of the candidate molecule with the activity of the marker in the absence of the candidate molecule (e.g., in the presence of a control).

In another embodiment, the method comprises screening candidate compounds for those that have the ability to increase or decrease the level of a polypeptide, metabolite or polynucleotide marker in a biological sample obtained from a subject, obtaining a first sample from the subject, providing the candidate compound or a control to the subject, at a later time obtaining a second sample from the subject, and comparing the respective activities, and then comparing the respective activities or levels of the marker in the first and second sample. Candidate compounds for which the level or activity of the marker is changed (either increased or decrease) are selected. This embodiment can be used in a clinical trial where a plurality of subjects is evaluated and the results are statistically significant.

Kits

In another aspect, the invention provides a kit for detecting a polypeptide, metabolite, or polynucleotide marker. In another aspect, the invention provides a kit for diagnosing AD in a subject by detecting at least one polypeptide, metabolite or polynucleotide marker in a biological sample from the subject.

In another aspect, the invention provides a kit for diagnosing MCI in a subject by detecting at least one polypeptide, metabolite or polynucleotide marker in a biological sample from the subject.

In another aspect, the invention provides a kit for screening candidate compounds by detecting stable complexes between the candidate compound and a polynucleotide, metabolite or polynucleotide marker.

The kits of the invention may comprise one or more of the following: an antibody, wherein the antibody specifically binds with a polypeptide or metabolite marker, a labeled binding partner to the antibody, a solid phase upon which is immobilized the antibody or its binding partner, a polynucleotide probe that can hybridize to a polynucleotide marker, pairs of primers that under appropriate reaction conditions can prime amplification of at least a portion of a polynucleotide marker or a polynucleotide encoding a polypeptide marker (e.g., by PCR), instructions on how to use the kit, and a label or insert indicating regulatory approval for diagnostic or therapeutic use.

EXAMPLES Markers of the invention were identified through a cross-sectional, no treatment studies, designed to examine potential biomarkers in MCI and AD subjects relative to controls, according to the Petersen Clinical Criteria for MCI (1999) and the NINCDS- ADRDA criteria, respectively. CSF samples were obtained from age and gender matched controls. (McKhann G, Drachman D, Folstein M, Katzman R, Price D, and Stadlan EM (1984). "Clinical diagnosis of Alzheimer's disease: report of the NINCDS- ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease." Neurology 34, 939-944; Petersen RC, Smith GE,

Waring SC et al. (1999), "Mild Cognitive Impairment: Clinical Characterization and Outcome. "Arch Neurol 56(3):303-308.)

Subject Selection. Cerebrospinal samples were obtained from 25 control, 35 MCI, and 35 AD subjects. Clinical evaluation was performed in a standardized way, and diagnostic evaluation of all subjects included clinical examination, consisting of medical history, physical, neurological and psychiatric examination, screening laboratory tests, an electrocardiogram, a chest radiograph, an electroencephalogram and a computed tomographic scan of the brain. The diagnosis of probable AD was made according to the National Institute of Neurological and Communicative Disorder and Stroke and Alzheimer's Disease and Related Disorders Association criteria. The severity of dementia was evaluated using the Mini Mental State Examination (MMSE). The average MMSE score was 22.8 in the AD group. The AD group consisted of 35 individuals, 10 men and 25 women, and the average age was 80.6 years. Mild cognitive impairment (MCI) was diagnosed in subjects with memory impairment but no other symptoms of dementia. The average MMSE score was 27.8. The MCI group consisted of 35 individuals, 14 men and 21 women, and the average age was 69.3 years. The control group consisted of 25 individuals, 8 men and 17 women, with an average age of 67.7 years. The MMSE score was above 28, and these individuals did not have a history, symptoms, or signs of psychiatric or neurological disease, malignant disease, or systemic disorders.

Sample collection. CSF samples were obtained were obtained from AD, MCI and control subjects using lumbar puncture and stored for analysis. CSF samples were obtained in accordance with a clinical protocol and informed consent that were approved by an institutional review board (IRB) and with procedures that adhere to Good Clinical Practice.

Sample Analysis. CSF from control, AD, and MCI subjects was subjected to mass spectrometric analysis for differential expression of proteins and metabolites, and for their identification. The present study generated data from the three types of fractions as shown in Figure 1— the metabolomic or low molecular weight components of CSF (both HPLC-MS and GC-MS), and the proteomic or high molecular weight component of CSF (HPLC-MS). CSF Proteome. The proteomic, high-molecular-weight (HMW) fraction contains a number of abundant proteins, e.g. albumin and IgG, which may preclude the detection of lower abundance proteins in the fluid analyzed. A protein removal method using an Agilent column was used to substantially deplete the most abundant proteins in order to increase the effective dynamic range of the measurements, i.e. to detect more of the lower abundance molecules. This method is based on high affinity antibody-antigen interactions of more proteins and is specifically designed to remove six high-abundant proteins in a single column: albumin, IgG, antitrypsin, IgA, transferrin, and haptoglobin. Hence, this method depletes a greater fraction of the undesired high abundance proteins, such as albumin and reduces suppression of the signal from the remaining low abundance proteins. This selective immuno-depletion provides an enriched pool of low- abundant proteins for downstream proteomics analysis allowing for enhanced sensitivity of detection of desired proteins.

Digestion of proteins. The eluted proteins were denatured, disulfide bonds reduced, and sulfhydryl groups carboxymethylated prior to digestion by modified trypsin. During this process, low molecular weight molecules were excluded during a buffer exchange step with a 5 kDa cut-off filter. The tryptic peptides were then profiled (individual molecules tracked across samples and their differential expression determined) by liquid chromatography-electrospray ionization-mass spectrometry (LC- ESI-MS) on high-resolution time-of-flight (TOF) instruments using a capillary chromatography column (300 micrometer internal diameter). The chromatography used was on-line reverse phase chromatography for one-dimensional (1-D) chromatography with a water/acetonitrile 100 minute gradient, and 0.1% formic acid added to aid in ionization efficiency and chromatographic behavior. Each unit of MS analysis consists of 20 samples, 18 study samples and 2 QC samples. The typical injection amount for 1- D analysis is 20 micrograms of protein.

Identification of peptides (proteins). Identification of proteins occurs via identification of peptides. Peptides of interest (significantly changing) in expression level are linked to tandem mass spectrometry (MS/MS) experiments on quadrupole- time-of-flight (Q-TOF) and ion-trap mass spectrometers using extra sample material. The resulting MS/MS spectra contain fragmentation patterns with characteristic peptide backbone cleavages. Each MS/MS raw spectrum from an isolated precursor ion is compared using commercially available software with in silico protein digestion and fragmentation using NCBFs RefSeq database to find a match, and hence identification. A match-quality score is reported. This identification approach also applies to peptides found in the LC-MS metabolomic fraction.

The process of identification initially involves a linking of accurate mass/charge ratio and elution time on LC of each component to a previously built library or database that has identified the peptide in human CSF. Approximately 1/4* of the molecules that are differentially expressed are rapidly identified by this process with the current buildup of the new library. Those components present at lowest concentrations are less likely to be already present in the library. A "directed" identification of those molecules of interest that are not identified by linking, a process in which MS/MS is performed on extra sample material for each component based on its mass/charge ratio and elution time on LC. Because of the effort needed to identify those components that are neither linked nor identified by directed identification, highly selectivity (i.e., low p values and/or large fold changes) is used in selecting the remaining unidentified molecules. This additional identification involves larger scale purification of fractions from the same type of fluid followed by directed MS/MS -based identification as above. The basic study data presented here is largely based on a linked library.

CSF Metabolome. The metabolomic, LMW fraction was obtained from a few hundred microliters of the raw fluid by first removing proteins by precipitation with the addition of an organic solution. The supernatant containing the LMW fraction was further divided into two fractions. One metabolomic fraction consists of volatile or volatilizable small molecule components analyzed by gas chromatography-electron- impact ionization-mass spectrometry (GC-EI-MS). Volatilization was enhanced by trimethylsilyl derivatization of active hydrogens. The carrier gas was helium. The second metabolomic fraction consists of nonvolatile components analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) using reverse phase (RP) chromatography. This fraction contains low molecular weight free-floating peptides and non-volatile metabolites. LC-MS for the metabolome is performed in essentially the same manner as for the proteome. For both the GC-MS and LC-MS, high-resolution (R > 5,000) time-of-flight mass spectrometers were used for profiling.

Identification of Volatile and Non-volatile Metabolites. For molecular identification of the volatile LMW molecules, electron-impact ionization provides characteristic fingerprint fragmentation patterns that can lead to identification if the molecule has been analyzed previously in pure form and entered into a database such as the database provided by National Institute of Standards and Technology (NIST). Otherwise, use of accurate mass to constrain the elemental composition is also useful. Finally, tandem mass spectrometry (MS/MS) is available with a triple-quadruple instrument. For those molecules tracked and deemed to be of interest due to their significant differential expression, it often is possible to later obtain identification with further effort, sometimes requiring an isolation from the complex mixture.

Identification of endogenous peptides in the LC-metabolome is performed by MS/MS (described above, for the case of digested proteins) but typically involves more de novo sequencing. Non-volatile metabolites observed from LC-MS profiling are typically identified through a combination of accurate mass measurements, tandem mass spectrometric analysis and chemical database searching. Accurate masses, usually within 2 to 3 mDa, are obtained through averaging over several scans, smoothing and centering from the original LC-MS profiling raw data. The elemental composition is then generated from each accurate mass with constraints such as number of elements allowed and mass tolerance. All possible elemental compositions are searched in chemical databases to find candidate structures. The fragmentation patterns are obtained from a separate tandem mass spectrometric experiment, where a list of metabolites is subject to high-energy collision on a Quadruple Time-of-Flight (Q-TOF) mass spectrometer, yielding characteristic fragment ions. Metabolite fragmentation patterns will help determine the right metabolite structure.

Post-Translational Modification (PTM). A number of methods were used to detect PTM of the identified polypeptides. Using the known fixed mass that a PTM adds, Turbo SEQUEST software can identify up to three PTMs on a peptide. In addition to such informatics approaches, a number of biochemical methods can be used to detect PTMs. For example, mixtures of phosphopeptides can be extracted using anti-phospho- tyrosine and anti-phospho-serine/threonine antibodies. Another approach is the use of an activated metal surface or column to capture phosphorylated proteins. A third tool is the use of dephosphorylating enzyme alkaline phosphatase and a re-analysis to examine changes. In addition to phosphorylation, glycosylation may also play a role in AD. The analysis of the O- and N-glycosylation can be performed by tryptic digestion of the protein, isolation of glycopeptides by lectin chromatography and mass measurement before and after enzymatic deglycosylation. Carbohydrate structures are calculated from the mass difference between glycosylated and deglycosylated peptide plus the use of tandem MS (MS/MS). Differential Quantification Strategy. The differential quantification method used relies on the changes in analyte signal intensities directly reflecting their concentrations in one sample relative to another. Samples are not mixed nor are the samples otherwise manipulated beyond that required for the LC-MS analysis itself. The sample preparation and LC-MS conditions need to be carefully controlled, however, for optimal results, and frequent quality control samples are analyzed to assure stable, reproducible performance.

This quantification technology employs overall spectral intensity normalization by employing signals of molecules that do not change concentration from sample to sample. In this way, a simple correction can be applied for any drift over time in overall LC-MS response and/or differences in sample concentrations. The computer application, called MassView™ 2 software, performs normalization by determining the median of the ratios for a large number of molecular components, requiring no operator intervention. The MassView™ 2 software also performs the following automated functions: spectral smoothing, baseline subtraction, noise evaluation, isotopic analysis, peak identification, intensity evaluation, inter-scan evaluation to construct chromatographic peaks, inter-file (inter-sample) evaluation to establish molecular components for analysis, normalization (mentioned above), and finally, quantification for the thousands of components.

Quantification for GC-MS is done by referencing the intensity of all molecular components to one or two isotopically labeled and spiked components in the complex mixture. The simpler chromatography and ionization, relative to LC-MS, makes this a feasible approach for quantification. Peak identification is performed via the AMDIS program published by NIST; this program deconvolutes electron-impact ionization mass spectra over chromatographic time. Components are tracked using a library with an entry for each component constrained by a tight chromatographic time window and mass fingerprint pattern. Determination ofp-Value. Univariate hypothesis tests for each mass spectrometry component were used for the comparisons of means between control, MCI and AD groups. Parametric or non-parametric tests were used, depending on the normality of the data. If the data were approximately normally distributed, the parametric statistic was used (t-test); if not, the nonparametric statistic (Wilcoxon test) was used. Goodness-of-fit statistics (Shapiro-Wilk) and tests of skewness and kurtosis were performed to assess the normality of each biometric component. The results of these tests are presented in form of a p-value per component. The p-value represents the probability of a false positive on a univariate level.

Results. The results were combined with the results of studies performed on different groups of AD subjects, MCI subjects and normal controls. These results are presented in the Tables.

Those skilled in the art will appreciate, or be able to ascertain using no more than routine experimentation, further features and advantages of the invention based on the above-described embodiments. Accordingly, the invention is not to be limited by what has been particularly shown and described, except as indicated by the appended claims.

All publications and references are herein expressly incorporated by reference in their entirety.

Certain illustrative claims of the invention are defined as follows:

Table 1A. 1D LC Proteome: AD vs. Control. Positive Fold Change.

Table 1B. 1D LC Proteome: AP vs. Control. Negative Fold Change.

Table 2A. 1 D LC Proteome: MCI vs. Control. Postive Fold Change.

o

Table 3A. 1D LC Proteome AD & MCI versus Control. Positive Fold Change.

Table 3B. 1D LC Proteome AD & MCI versus Control. Negative Fold Change-

Table 4A. 1D LC Proteome: AD vs. MCI. Positive Fold Change.

Table 4B. 1D LC Proteome: AD vs. MCI. Negative Fold Change.

Table 5A. GC Metabolome: AD vs. Control Positive Fold Change.

Table 5B. GC Metabolome: AD vs. Control. Negative Fold Change.

Table 6A. GC Metabolome: MCl vs. Control. Positive Fold Change

Table 6B. GC Metabolome: MCI vs. Control. Negative Fold Change

Table 7A. GC Metabolome: AP and MCl vs. Control. Positive Fold Change.

Table 7B. GC Metabolome: AD and MCI vs. Control. Negative Fold Change.

Table 8A. GC Metabolome: AD vs. MCI. Positive Fold Change.

Table 8B. GC Metabolome: AD vs. MCI. Negative Fold Change.

Table 9A. LC Metabolome: AD vs Control. Positive Fold Change.

Table 9B. LC Metabolome: AD vs Control. Negative Fold Change.

Table 1OA. LC Metabolome: MCl vs. Control. Positive Fold Change.

O

Table 1OB. LC Metabolome: MCI vs. Control. Negative Fold Change.

Table 11 A. LC Metabolome: AD & MCI vs. Control. Positive Fold Change.

Table 11 B. LC Metabolome: AD & MCI vs. Control. Negative Fold Change.

Table 12A. LC Metabolome: AD vs. MCI. Positive Fold Change.

Table 12B. LC Metabolome: AD vs. MCI. Negative Fold Change.

Claims

CLAIMS:

1. A method for diagnosing neurodegenerative disease in a subject, the method comprising: obtaining a biological sample from the subject; determining the level of a marker in the sample; comparing the level of the marker in the sample to a reference value.

2. The method of claim I, wherein the marker is selected from the group consisting of the molecules identified in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, Table 5B, Table 6A, Table 6B, Table 7A, Table 7B, Table 8A, Table 8B, Table 9A, Table 9B, Table 1OA, Table 10B, Table HA, Table HB, Table 12A, and Table 12B.

3. The method of claim 2, wherein the neurodegenerative disease is Alzheimer's Disease.

4. The method of claim 2, wherein the neurodegenerative disease is Mild Cognitive Impairment (MCI).

5. The method of claim 2, wherein the biological sample is a body fluid.

6. The method of claim 5, wherein the body fluid is selected from the group consisting of blood, serum, plasma, cerebrospinal fluid, urine, and saliva

7. The method of claim 2, wherein the marker comprises a polypeptide or fragment thereof.

8. The method of claim 2, wherein the marker comprises a metabolite or fragment thereof.

9, The method of claim 2, wherein the reference value is the level of the marker in at least one sample from a non-AD subject.

10. A method for diagnosing Alzheimer's Disease in a subject, the method comprising: obtaining one or more biological samples from the subject; determining the level of a plurality of markers in the one or more biological samples, wherein at least one of the plurality of markers is selected from the group consisting of the molecules disclosed in Table IA, Table IB,

Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, Table 5B, Table 6A, Table 6B, Table 7A, Table 7B, Table 8A, Table 8B, Table 9 A, Table 9B, Table 1OA, Table 1OB, Table HA, Table HB, Table 12A, and Table 12B; comparing the level of at least one of the plurality of markers to a reference value.

11. The method of claim 10, wherein the biological sample is a body fluid.

12. The method of claim 11 , wherein the body fluid is selected from the group consisting of blood, serum, plasma, cerebrospinal fluid, urine, and saliva.

13. The method of claim 10, wherein at least one of the plurality of markers is a polypeptide or a fragment thereof.

14. The method of claim 10, wherein at least one of the plurality of markers is a metabolite or a fragment thereof

15. The method of claim 10, wherein at least one of the plurality of markers is a metabolite or a fragment thereof and at least one of the plurality of markers is a protein or a fragment thereof.

16. The method of claim 10, wherein at least two of the plurality of markers are selected from the group consisting of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, Table 5B, Table 6A, Table 6B, Table 7A, Table 7B, Table 8A, Table 8B, Table

9A, Table 9B, Table 10A, Table 10B, Table HA, Table HB, Table 12A, and Table 12B.

17. The method of claim 10, wherein at least ten of the plurality of markers are selected from the group consisting of the molecules set forth in Table IA, Table

IB, Table 2A, Table 2B, Table 3 A, Table 3B, Table 4A, Table 4B, Table 5A, Table 5B, Table 6A, Table 6B, Table 7A, Table 7B, Table 8A, Table 8B, Table 9A, Table 9B, Table 10A, Table 10B, Table HA, Table HB, Table 12A, and Table 12B.

18. The method of claim 10, wherein at least one of the plurality of markers is selected from the group consisting of molecules set forth in Table IA, Table 2A, Table 3A, Table 4A, Table 5 A, Table 6A, Table 7A,Table 8A, Table 9A, Table 10A, Table 1 IA, or Table 12A.

19. The method of claim 18, wherein the reference value is the level of at least one of the plurality of markers in at least one sample from a non-AD subject, and wherein the level of the at least one of the plurality of markers is increased by at least one fold with respect to the reference value.

20. The method of claim 19, wherein the level of the at least one of the plurality of markers is increased by at least two fold with respect to the reference value.

21. The method of claim 10, wherein at least one of the plurality of markers is selected from the group consisting of the molecules set forth in Table IB,

Table 2B, Table 3B, Table 4B, Table 5B, Table 6B, Table 7B,Table 8B, Table 9B, Table 10B, Table 11B, or Table 12B.

22. The method of claim 21, wherein the reference value is the level of the at least one of the plurality of markers in at least one sample from a non-AD subject, and wherein the level of the at least one of the plurality of markers is increased by at least one fold with respect to the reference value.

23. The method of claim 22, wherein the level of the at least one of the plurality of markers is increased by at least two fold with respect to the reference value.

24. The method of claim 2, wherein the marker is not expressed in non-AD subjects.

25. The method of claim 2, wherein the level of the marker is determined by detecting the presence of a polypeptide.

26. The method of claim 25, wherein the polypeptide is the marker.

27. The method of claim 25, wherein the polypeptide shares 70% homology with the marker.

28. The method of claim 25, wherein the polypeptide is a modified form of the marker.

29. The method of claim 25, wherein the polypeptide is a precursor to the marker.

30. The method of claim 25, wherein the polypeptide is a metabolite of the marker.

31. The method of claim 25, wherein the method further comprises detecting the presence of the polypeptide using a reagent that specifically binds to the polypeptide or a fragment thereof.

32. The method of claim 31, wherein the reagent is selected from the group consisting of an antibody, an antibody derivative, and an antibody fragment.

33. The method of claim 2, wherein the level of the marker is determined by detecting the presence of a metabolite.

34. The method of claim 33, wherein the metabolite is the marker.

35. The method of claim 33, wherein the metabolite is a modified form of the marker.

36. The method of claim 33, wherein the metabolite is a precursor to the marker.

37. The method of claim 33, wherein the metabolite is a metabolic product of the marker.

38. The method of claim 2, wherein the subj ect is a lab animal.

39. The method of claim 2, wherein the subject is a human subject.

40. A method for monitoring the progression of Alzheimer's Disease in a subject, the method comprising: obtaining a first biological sample from the subject; measuring the level of a marker in the first sample, wherein the marker is selected from the group consisting of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, Table 5B, Table 6A, Table 6B, Table 7 A, Table 7B, Table 8A, Table 8B, Table 9A, Table 9B, Table 1OA, Table 1OB,

Table HA, Table HB, Table 12A, and Table 12B; obtaining a second biological sample from the subject; measuring the level of the marker in the second sample; and comparing the level of the marker measured in the first sample with the level of the marker measured in the second sample.

41. A method of assessing the efficacy of a treatment for Alzheimer's Disease in a subject, the method comprising comparing:

(i) the level of a marker measured in a first sample obtained from the subject before the treatment has been administered to the subject, wherein the marker is selected from the group consisting of the molecules set forth in Table IA,

Table 2A, Table 3A, Table 4A, Table 5A, Table 6A, Table 7A,Table 8A, Table

9 A, Table 10A, Table HA, and Table 12 A; and

(ii) the level of the marker in a second sample obtained from the subject after the treatment has been administered to the subject, wherein a decrease in the level of the marker in the second sample relative to the first sample is an indication that the treatment is efficacious for treating

Alzheimer's Disease in the subject.

42. A method of assessing the efficacy of a treatment for Alzheimer' s Disease in a subject, the method comprising comparing:

(i) the level of a marker in a first sample obtained from the subject before the treatment has been administered to the subject, wherein the marker is selected from the group consisting of the molecules set forth in Table IB, Table 2B, Table 3B, Table 4B, Table 5B, Table 6B, Table 7B,Table 8B, Table 9B, Table

1OB, Table 11B, and Table 12B; and

(ii) the level of the marker in a second sample obtained from the subject after the treatment has been administered to the subject, wherein an increase in the amount of the marker in the second sample, relative to the first sample, is an indication that the treatment is efficacious for inhibiting

Alzheimer's Disease in the subject.

43. A method of treating Alzheimer's Disease (AD) in a subject, the method comprising inhibiting expression of a gene corresponding to a marker selected from the group consisting of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, Table 5B, Table 6A, Table 6B,

Table 7 A, Table 7B, Table 8A, Table 8B, Table 9 A, Table 9B, Table 1OA, Table 1OB, Table 11A, Table HB, Table 12A, and Table 12B.

44. A method for diagnosing Alzheimer's Disease in a subject, the method comprising: obtaining a biological sample from a subject; determining a first amount of a first marker in the biological sample, wherein the first marker is increased in subjects with Alzheimer's disease; determining a second amount of a second marker in the biological sample, wherein the second marker is decreased in subjects with Alzheimer's Disease; comparing the first amount to a first reference value and comparing the second amount to a second reference value, wherein a significantly difference exists between both (i) the first amount and the first reference value and (ii) second amount and the second reference value, and wherein the differences are indicative that the subject has Alzheimer's Disease.

45. The method of claim 44, wherein the first marker is a molecule selected from the group consisting of the molecules set forth in Table 1A Table 2A, Table 3A, Table 4A, Table 5 A, Table 6A, Table 7A,Table 8 A, Table 9A, Table 1OA, Table 11 A and Table 12A.

46. The method of claim 44, wherein the second marker is a molecule selected from the group consisting of the molecules set forth in Table IB, Table 2B, Table 3B, Table 4B, Table 5B, Table 6B, Table 7B,Table 8B, Table 9B, Table 1OB, Table HB, and Table 12B.

47. A method for diagnosing Alzheimer's Disease in a subject, the method comprising: obtaining a sample from the subject; determining the amount of at least one first marker in the sample, wherein the at least one first marker is selected from the group consisting of the molecules set forth in Table IA, Table 2A, Table 3A, Table 4A, Table 5A, Table 6A, Table 7A,Table 8A, Table 9A, Table 10A, Table HA, and Table 12A; determining the amount of at least one second marker in the sample, wherein the at least one second marker is selected from the group consisting of the molecules set forth in Table IB, Table 2B, Table 3B, Table 4B, Table 5B, Table 6B, Table 7B,Table 8B, Table 9B, Table 1OB, Table HB, and Table 12B; comparing the amounts of the at least one first marker and at least one second marker in the sample from the subject to the amounts of the at least one first marker and at least one second marker in at least one sample from a subject not suspected of having

Alzheimer's Disease, wherein a measurable difference exists between the amounts measured for at least 50% of the markers.

48. An isolated molecule selected from the group consisting of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table

4B, Table 5A, Table 5B, Table 6 A, Table 6B, Table 7 A, Table 7B, Table 8A, Table 8B, Table 9A, Table 9B, Table 1OA, Table 1OB, Table HA, Table HB, Table 12A, and Table 12B.

49. A composition comprising a molecule selected from the group consisting of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 5 A, Table 5B, Table 6A, Table 6B, Table 7A, Table 7B, Table 8A, Table 8B, Table 9 A, Table 9B, Table 10A, Table 10B, Table HA, Table HB, Table 12A, and Table 12B.

50. A method for aiding in the diagnosis of Alzheimer's Disease in a subject, the method comprising: obtaining a biological sample from the subject; determining the level of a marker in the sample, wherein the marker is selected from the group consisting of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 5 A, Table 5B, Table 6A, Table 6B, Table 7A, Table 7B, Table 8A, Table 8B, Table 9A, Table 9B, Table 1OA, Table 1OB, Table HA, Table HB, Table 12A, and Table 12B; comparing the level of the marker in the sample to a reference value; and determining from the results of the comparison whether the subject is more or less likely to have Alzheimer's Disease.

51. A method for determining the type, stage or severity of Alzheimer's Disease in a subject, the method comprising: obtaining a biological sample from the subject; determining the level of a marker in the sample, wherein the marker is selected from the group consisting of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 5 A, Table 5B, Table 6A, Table 6B, Table 7 A, Table 7B, Table 8 A, Table 8B, Table 9 A, Table 9B, Table 1OA,

Table 1OB, Table HA, Table HB, Table 12A, and Table 12B; comparing the level of the marker in the sample to a reference value; and determining from the results of the comparison the type, stage or severity of Alzheimer's Disease in the subject.

52. A method for determining the risk of developing Alzheimer's Disease in a subject, the method comprising: obtaining a biological sample from the subject; determining the level of a marker in the sample, wherein the marker is selected from the group consisting of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, Table 5B, Table 6A, Table 6B, Table 7 A, Table 7B, Table 8 A, Table 8B, Table 9A, Table 9B, Table 1OA, Table 1OB, Table 11 A, Table HB, Table 12A, and Table 12B; comparing the level of the marker in the sample to a reference value; and determining from the results of the comparison that the subject has an increased or decreased risk of developing Alzheimer's Disease.

53. A method for diagnosing mild cognitive impairment in a subject, the method comprising: obtaining a biological sample from the subject; determining the level of a marker in the sample, wherein the marker is selected from the group consisting of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3 A, Table 3B, Table 4A, Table 4B, Table 5 A, Table 5B, Table 6A, Table 6B, Table 7 A, Table 7B, Table 8 A, Table 8B, Table 9 A, Table 9B, Table 1 OA,

Table 1OB, Table HA, Table HB, Table 12A, and Table 12B; comparing the level of the marker in the sample to a reference value.

54. The method of claim 1, wherein the marker shares 70% homology with one or more of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B,

Table 3A, Table 3B, Table 4A, Table 4B, Table 5 A, Table 5B, Table 6A, Table 6B, Table 7A, Table 7B, Table 8A, Table 8B, Table 9A, Table 9B, Table 10A, Table 1OB, Table HA, Table HB, Table 12A, and Table 12B.

55. The method of claim 25, wherein the marker is a modified form of one or more of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, Table 5B, Table 6A, Table 6B, Table 7A, Table 7B, Table 8A, Table 8B, Table 9A, Table 9B, Table 10A, Table 1OB, Table 1 IA, Table 1 IB,

Table 12A, and Table 12B.

56. The method of claim 25, wherein the marker is a precursor to one or more of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 5 A, Table 5B, Table 6A, Table 6B, Table 7A, Table 7B,

Table 8A, Table 8B, Table 9A, Table 9B, Table 10A, Table 10B, Table 11A, Table HB, Table 12A, and Table 12B.

57. The method of claim 25, wherein the marker is a metabolite of one or more of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B,

Table 4A, Table 4B, Table 5A, Table 5B, Table 6A, Table 6B, Table 7A, Table 7B, Table 8A, Table 8B, Table 9A, Table 9B, Table 10A, Table 10B, Table HA, Table HB, Table 12A, and Table 12B.

58. The method of claim 25, wherein the marker is a compound in a known metabolic pathway including one or more of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, Table 5B, Table 6A, Table 6B, Table 7A, Table 7B, Table 8A, Table 8B, Table 9A, Table 9B, Table 10A, Table 10B, Table HA, Table HB, Table 12A, and Table 12B.

59. The method of claim 25, wherein the marker regulates a known metabolic pathway including one or more of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, Table 5B, Table 6A, Table 6B, Table 7A, Table 7B, Table 8A, Table 8B, Table 9A, Table 9B, Table 10A, Table 10B, Table 1 IA, Table 1 IB, Table 12A, and Table 12B.

60. A kit comprising a molecule selected from the group consisting of the molecules set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 5A, Table 5B, Table 6A, Table 6B, Table 7A, Table 7B, Table 8A, Table 8B, Table 9A, Table 9B, Table 1OA, Table 1OB, Table 1 IA, Table 1 IB, Table

12A, and Table 12B.

61. A kit comprising a reagent that specifically binds to a molecule selected from the group consisting of the molecules set forth in Table IA, Table IB, Table 2 A, Table 2B, Table 3 A, Table 3B, Table 4A, Table 4B, Table 5 A, Table 5B, Table 6A, Table 6B,

Table 7 A, Table 7B, Table 8 A, Table 8B, Table 9A, Table 9B, Table 10A, Table 1OB, Table HA, Table HB, Table 12A, and Table 12B.

62. A method for diagnosing Alzheimer's Disease in a subject, the method comprising: obtaining a biological sample from the subject; determining the level of a protein in the sample that specifically binds to a marker, wherein the marker is selected from the group consisting of set forth in Table IA, Table IB, Table 2A, Table 2B, Table 3 A, Table 3B, Table 4A, Table 4B, Table 5A, Table 5B, Table 6A, Table 6B, Table 7A, Table 7B, Table 8A, Table 8B, Table 9A,

Table 9B, Table 10A, Table 10B, Table HA, Table HB, Table 12A, and Table 12B; comparing the level of the protein marker in the sample to a reference value.