Biomarkers for neurodegenerative disorders

[0001] This application claims the benefit of U.S. Provisional Application No. 60/731,339, filed Oct. 27, 2005, which application is incorporated herein by reference in its entirety.

[0002] This invention was made with government support under federal grant nos. R01AG025327 and R01ES012703 awarded by National Institutes of Health. The United States Government may have certain rights in this invention.

[0003] The present specification incorporates herein by reference, each in its entirety, the sequence information on the Compact Disks (CDs) labeled Copy 1 and Copy 2. The CDs are formatted on IBM-PC, with operating system compatibility with MS-Windows. The files on each of the CDs are as follows:

[0004] Neurodegenerative disorders, e.g. Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy body (DLB) diseases, are diagnosed primarily by clinical presentations, limited laboratory investigations and, more recently, structural and functional neuroimaging analysis (Bacskai et al., J Cereb Blood Flow Metab, 2002. 22(9): p. 1035-41; Klunk et al., J Neuropathol Exp Neurol, 2002. 61(9): p. 797-805; and Small et al., J Mol Neurosci, 2002. 19(3): p. 323-7). However the diagnosis based on these approaches is unsatisfactory. As determined by pathological examination, diagnostic accuracy of various neurodegenerative diseases varies between 50% to 85% depending on the disease involved, the experience of physicians and the stages of the diseases (Jankovic et al., Arch Neurol, 2000. 57(3): p. 369-72; Hughes et al., Brain, 2002. 125(Pt 4): p. 861-70; Litvan et al., Arch Neurol, 1998. 55(7): p. 969-78; Rajput et al., Can J Neurol Sci, 1991. 18(3): p. 275-8; Hughes et al., J Neurol Neurosurg Psychiatry, 1992. 55(3): p. 181-4; and McKeith et al., Semin Clin Neuropsychiatry, 2003. 8(1): p. 46-57). The fact that the diagnosis cannot be made with reasonable certainty until the latter stages of the diseases possibly underlies the current state of clinical management, i.e. none of the available therapies, particularly those aimed at preventing disease's progression, is effective; this could simply be due to the fact that most neurons are already degenerated by the time diagnosis is made. It is also noteworthy that it is common for patients with various neurodegenerative diseases to go undetected using current approaches (Love et al., Histopathology, 2004. 44(4): p. 309-17).

[0005] Biomarkers are biological characteristics used to indicate or to measure disease risk, presence, and progression. Ideally, an optimal biomarker should be precise, reliable, inexpensive, as well as reflect the pathophysiological mechanisms of neurodegenerative diseases. Presently, no established diagnostic biomarkers can confirm AD, PD or DLB or monitor their progression with high sensitivity at high specificity. Furthermore, markers are most useful if they can detect at an early or even preclinical stages of diseases. In searching for biochemical markers in body fluids, including plasma, urine, and cerebrospinal fluid (CSF), only limited success has been achieved despite decades of research. It has been felt recently that this is largely due to the heterogeneity of all neurodegenerative diseases, i.e. several markers may be needed to detect subpopulations of patients (Olsson et al., Clin Chem, 2005. 51(2): p. 336-45).

[0006] The development of genomics, proteomics, and metabolomics has greatly enhanced the ability to discover multiple markers that are not only useful for diagnosis of AD, PD and DLB but also shed more lights on their pathogenesis. However, these studies are limited, as none has taken other neurodegenerative diseases into consideration, and in addition, very few studies have been performed using cases with pathological verification. The present invention addresses this need.

Relevant Literature

[0007] Bacskai et al., J Cereb Blood Flow Metab, 2002. 22(9): p. 1035-41; Klunk et al., J Neuropathol Exp Neurol, 2002. 61(9): p. 797-805; Small et al., J Mol Neurosci, 2002. 19(3): p. 323-7; Jankovic et al., Arch Neurol, 2000. 57(3): p. 369-72; Hughes et al., Brain, 2002. 125(Pt 4): p. 861-70; Litvan et al., Arch Neurol, 1998. 55(7): p. 969-78; Rajput et al., Can J Neurol Sci, 1991. 18(3): p. 275-8; Hughes et al., J Neurol Neurosurg Psychiatry, 1992. 55(3): p. 181-4; McKeith et al., Semin Clin Neuropsychiatry, 2003. 8(1): p. 46-57; Love et al., Histopathology, 2004. 44(4): p. 309-17; Olsson et al., Clin Chem, 2005. 51(2): p. 336-45; Zhang et al., Neurobiol Aging, 2005. 26(2): p. 207-27; and Zhang et al., J Alzheimers Dis, 2005. 7(2): p. 125-33.

[0008] The present invention provides methods for diagnosing neurodegenerative disease, such as Alzheimer's Disease, Parkinson's Disease, and dementia with Lewy body disease by detecting a pattern of gene product (e.g., protein) expression in a cerebrospinal fluid sample and comparing the pattern of gene product expression from the sample to a library of gene product expression pattern known to be indicative of the presence or absence of a neurodegenerative disease. Also provided are kits, systems and devices for practicing the subject methods.

[0009] The present invention provides a method for detecting presence or absence of a neurodegenerative disease in a subject by detecting a pattern of gene product expression present in a cerebrospinal fluid sample obtained from a subject; and comparing the pattern of gene product expression from the cerebrospinal fluid sample to a library of gene product expression pattern known to be indicative of the presence or absence of a neurodegenerative disease, wherein the comparing indicates the presence or absence of a neurodegenerative disease.

[0010] In some embodiments, the gene product is a polypeptide. In some embodiments, the detecting is by mass spectrometry. In other embodiments, the detecting is by immunoassay. In certain embodiments, the immunoassay is enzyme linked immunosorbent assay (ELISA). In other embodiments, the detecting by a Luminex xMAP system. In certain embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or dementia with Lewy body disease.

[0011] The present invention also provides a method for monitoring progression of a neurodegenerative disease in a subject by detecting a first pattern of expression of gene products present in a cerebrospinal fluid sample obtained from a subject at a first time point, wherein said first pattern is indicative of a neurodegenerative disease; detecting a second pattern of expression of gene products present in a cerebrospinal fluid sample obtained from a subject at a second time point; and comparing the first and second patterns of expression of gene products from the cerebrospinal fluid samples, wherein the comparing provides for monitoring of the progression of the neurodegenerative disease from the first time point to the second time point.

[0012] In some embodiments, the gene product is a polypeptide. In some embodiments, the detecting is by mass spectrometry. In other embodiments, the detecting is by immunoassay. In certain embodiments, the immunoassay is enzyme linked immunosorbent assay (ELISA). In other embodiments, the detecting by a Luminex xMAP system. In certain embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or dementia with Lewy body disease.

[0013] The present invention also provides a method of providing a differential diagnosis between Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy body disease (DLB) in a subject by detecting a pattern of gene product expression present in a cerebrospinal fluid sample obtained from a subject; and comparing the pattern of gene product expression from the cerebrospinal fluid sample to a library of gene product expression patterns known to be indicative of the presence or absence of AD, PD and DLB, wherein the comparing providing a differential diagnosis between AD, PD, and DLB.

[0014] In some embodiments, the gene product is a polypeptide. In some embodiments, the detecting is by mass spectrometry. In other embodiments, the detecting is by immunoassay. In certain embodiments, the immunoassay is enzyme linked immunosorbent assay (ELISA). In other embodiments, the detecting by a Luminex xMAP system.

[0015] The present invention also provides a system, including a computing environment; an input device, connected to the computing environment, to receive data from a user, wherein the data received includes a pattern of gene product expression from a cerebrospinal fluid sample obtained from a subject; an output device, connected to the computing environment, to provide information to the user; and a computer readable storage medium having stored thereon at least one algorithm to provide for comparing the pattern of gene product expression from the cerebrospinal fluid sample to a library of gene product expression pattern known to be indicative of the presence or absence of a neurodegenerative disease. In some embodiments, the computing environment includes a local computer local to the user and a remote computer at a site remote to the user, wherein the local computer and the remote computer are connected through a network, and wherein the computer readable storage medium is provided on the remote computer.

[0016] The present invention also provides a computer readable medium including a program stored thereon, wherein the program provides for execution of one or more algorithms to provide for comparing a pattern of gene product expression from a cerebrospinal fluid sample obtained from a subject to a library of gene product expression pattern known to be indicative of the presence or absence of a neurodegenerative disease.

[0017] These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.

[0026] A “neurodegenerative disease”, as used in the current context, is readily understood by one of ordinary skill in the art to include any abnormal physical or mental behavior or experience where the death or dysfunction of neuronal cells is involved in the etiology of the disorder, or is affected by the disorder. As used herein, neurodegenerative diseases encompass disorders affecting the central and peripheral nervous systems, and include such afflictions as memory loss, stroke, dementia, personality disorders, gradual, permanent or episodic loss of muscle control. Examples of neurodegenerative diseases for which the current invention can be used preferably include, but are not limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Dementia with Lewy Body, amyotrophic lateral sclerosis, epilepsy, myasthenia gravis, neuropathy, ataxia, dementia, chronic axonal neuropathy and stroke.

[0027] As used herein “Parkinson's disease” or “PD” refer to a condition of disturbance of voluntary movement in which muscles become stiff and sluggish, movement becomes clumsy and difficult and uncontrollable rhythmic twitching of groups of muscles produces characteristic shaking or tremor. The condition is believed to be caused by a degeneration of pre-synaptic dopaminergic neurons in the brain. The absence of adequate release of the chemical transmitter dopamine during neuronal activity thereby leads to the Parkinsonian symptomatology.

[0028] As used herein “Alzheimer's disease” or “AD” refers to a condition characterized by the abnormal deposition of amyloid in the brain of a patient in the form of extra-cellular plaques and intra-cellular neurofibrillary tangles. The rate of amyloid accumulation is a combination of the rates of formation, aggregation and egress from the brain. It is generally accepted that the main constituent of amyloid plaques is the 4 kD amyloid protein (βA4, also referred to as Aβ, β-protein and βAP) which is a proteolytic product of a precursor protein of much larger size. The symptoms of Alzheimer's disease are similar to those of other dementias. They include memory loss, changes in personality, problems using language, disorientation, difficulty doing daily activities, and disruptive behavior.

[0029] As used herein “dementia with Lewy body” or “DLB” refers to a condition characterized by widespread neurodegeneration with formation of Lewy bodies not only in the dopaminergic system but also in other brain regions. The major symptoms of DLB are fluctuating cognition, visual hallucinations and parkinsonian signs. This is a disease considered by some as a collision between AD and PD; its clinical diagnosis is extremely challenging.

[0030] A “gene product” is a biopolymeric product that is expressed or produced by a gene, such as a peptide or protein. A gene product may be, for example, an unspliced RNA, an mRNA, a splice variant mRNA, a polypeptide, a post-translationally modified polypeptide, a splice variant polypeptide etc. Also encompassed by this term are biopolymeric products that are made using an RNA gene product as a template (i.e., cDNA of the RNA). A gene product may be made enzymatically, recombinantly, chemically, or within a cell to which the gene is native. In many embodiments, if the gene product is proteinaceous, it exhibits a biological activity. In many embodiments, if the gene product is a nucleic acid, it can be translated into a proteinaceous gene product that exhibits a biological activity.

[0031] The terms “polypeptide” and “protein”, interchangeably used herein, refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

[0032] The term “polynucleotide” refers to polymeric forms of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, these terms include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. These terms further include, but are not limited to, mRNA or cDNA that comprise intronic sequences (see, e.g., Niwa et al. (1999) Cell 99(7):691-702). The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidites and thus can be an oligodeoxynucleoside phosphoramidate or a mixed phosphoramidate-phosphodiester oligomer. Peyrottes et al. (1996) Nucl. Acids Res. 24:1841-1848; Chaturvedi et al. (1996) Nucl. Acids Res. 24:2318-2323. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars, and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support. The term “polynucleotide” also encompasses peptidic nucleic acids (Pooga et al Curr Cancer Drug Targets. (2001) 1:231-9).

[0033] A composition (e.g. a polynucleotide, polypeptide, antibody, or host cell) that is “isolated” or “in substantially isolated form” refers to a composition that is in an environment different from that in which the composition naturally occurs. For example, a polynucleotide that is in substantially isolated form is outside of the host cell in which the polynucleotide naturally occurs, and could be a purified fragment of DNA, could be part of a heterologous vector, or could be contained within a host cell that is not a host cell from which the polynucleotide naturally occurs. The term “isolated” does not refer to a genomic or cDNA library, whole cell total protein or mRNA preparation, genomic DNA preparation, or an isolated human chromosome. A composition which is in substantially isolated form is usually substantially purified.

[0034] As used herein, the term “substantially purified” refers to a compound (e.g., a polynucleotide, a polypeptide or an antibody, etc.,) that is removed from its natural environment and is usually at least 60% free, preferably 75% free, and most preferably 90% free from other components with which it is naturally associated. Thus, for example, a composition containing A is “substantially free of” B when at least 85% by weight of the total A+B in the composition is A. Preferably, A comprises at least about 90% by weight of the total of A+B in the composition, more preferably at least about 95% or even 99% by weight. In the case of polynucleotides, “A” and “B” may be two different genes positioned on different chromosomes or adjacently on the same chromosome, or two isolated cDNA species, for example.

[0035] The present invention provides methods for diagnosing neurodegenerative disease, such as Alzheimer's Disease, Parkinson's Disease, and dementia with Lewy body disease by detecting a pattern of gene product (e.g., protein) expression in a cerebrospinal fluid sample and comparing the pattern of gene product expression from the sample to a library of gene product expression pattern known to be indicative of the presence or absence of a neurodegenerative disease. Also provided are kits and devices for practicing the subject methods.

[0036] Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0037] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0039] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the marker” includes reference to one or more markers and equivalents thereof known to those skilled in the art, and so forth.

[0040] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only” and the like in connection with the recitation of claim elements, or the use of a “negative” limitation.

[0041] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Overview

[0042] The present invention is based on the identification and quantification of cerebrospinal fluid (CSF) proteins using an unbiased quantitative proteomic approach called iTRAQ (isobaric Tagging for Relative and Absolute protein Quantification) to label pre-fractionated human CSF, and followed by MudPIT (Multidimensional Protein Identification Technology), prior to mass spectrometry (MS) analysis. This multiplex format allowed simultaneous comparison of the proteome of CSF in AD, PD, DLB patients and healthy controls. This analysis not only identified 1,540 CSF proteins (see FIGS. 6A-6T and FIGS. 7A-7V), thereby greatly expanding the current knowledge about the human CSF proteome, but also detected 136, 73, and 100 proteins that displayed quantitative changes unique to AD, PD, and DLB, respectively. Finally, the sensitivity at 95% specificity of each of eight exemplary markers or composite markers was calculated, demonstrating that the combination of several markers could distinguish between AD, PD and DLB patients not only from controls, but also from each other with high sensitivity at 95% specificity.

[0043] In addition, several exemplary panels of unique makers are capable of distinguishing AD, PD and DLB patients from each other as well as from controls with high sensitivity at 95% specificity (see e.g., FIGS. 5A-5YY).

Methods of the Invention

[0044] The invention features methods for diagnosing neurodegenerative disease, such as Alzheimer's Disease, Parkinson's Disease, and dementia with Lewy body disease by detecting a pattern of gene product (e.g., proteins/peptides) expression in a cerebrospinal fluid sample and comparing the pattern of gene product expression from the sample to a library of gene product expression pattern (e.g., FIGS. 5A-5YY) known to be indicative of the presence or absence of a neurodegenerative disease. In general, the detection of a pattern of gene product expression in a CSF sample obtained form a subject as described herein can be accomplished using any acceptable methodology.

[0045] The term's “neurodegeneration” and “neurodegenerative condition or disease” as used in the present application stand for the same and are used interchangeable throughout the application. These terms include any condition of the brain that is associated with a neuronal malfunctioning. Various diseases associated with neurodegeneration include Alzheimer's disease, Parkinson disease, dementia with Lewy Body, Huntington' disease, Creutzfeld Jacob disease, frontal temporal lobe dementia, normal Pressure Hydrocephalus, and amyotrophic lateral sclerosis. However, this list is not complete. Other diseases known to be associated with neuronal malfilrictioning are included as well. In certain embodiments of the present invention, the neurodegenerative disease or condition to be specifically detected, monitored, quantified and/or differentially diagnosed is chosen from the group consisting of Alzheimer's disease, and dementia with Lewy Body.

[0046] In general, the method for detecting the presence or absence of a neurodegenerative disease in a subject includes detecting a pattern of gene product expression present in a cerebrospinal fluid sample obtained from a subject; and comparing the pattern of gene product expression from the cerebrospinal fluid sample to a library of gene product expression pattern known to be indicative of the presence or absence of a neurodegenerative disease, wherein the comparing indicates the presence or absence of a neurodegenerative disease.

[0047] Any possible combination of gene product(s), such as proteins and peptides, that have an altered level in a CSF sample obtained form a subject under a certain neurological condition can be used for the detection of the presence or absence of a neurological disease, the monitoring of a neurological disease, including assessing therapeutic effects of a treatment regimen (e.g., administration of a therapeutic drug), or the differential diagnosis of AD, PD, or DLB. An exemplary list of candidate gene products that are suitable for use in the detection, monitoring, and differential diagnosis methods of the present invention are summarized in FIGS. 5A-5YY.

[0048] Detection of an alerted marker expression pattern(s) in a CSF sample obtained from a subject as compared to that of a normal subject (e.g., a subject known to not have a neurodegenerative disease) is an indicator of neurodegenerative disease, such as AD, PD, or DLB. As with all controls mentioned herein, the control is preferably derived from CSF of subjects without any neurological diseases or taking any medicines for any conditions that might influence neurological functions.

[0049] In general, at least enough gene products from FIGS. 5A-5YY are selected for the subject methods that provide for the specific detection of the presence or absence of a neurodegenerative disease. In most embodiments, at least two ore more gene products from FIGS. 5A-5YY are selected for determining the presence or absence of a neurodegenerative disease. In some embodiments, a least three or more genes are selected, including about four or more gene products, and about five or more gene products.

[0050] The present invention also provides a method for differential diagnosis between Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy body disease (DLB) in a subject by detecting a pattern of gene product expression present in a cerebrospinal fluid sample obtained from a subject; and comparing the pattern of gene product expression from the cerebrospinal fluid sample to a library of gene product expression patterns known to be indicative of the presence or absence of AD, PD and DLB, wherein said comparing providing a differential diagnosis between AD, PD, and DLB.

[0051] It will be appreciated that the number of gene products selected for use in the present methods will be in part dictated by the specific gene products that are selected for the analysis and whether a general diagnosis of neurodegenerative disease is desired or a differential diagnosis of PD, AD, or DLB is desired. As will be readily apparent to one having skill in the art, the expression level of certain gene products will be modulated as compared to a control in certain conditions and will not be modulated (i.e., decrease or increased) in other conditions as compared to a control. For example, as shown in FIG. 5A, a decrease in expression of BDNF1 is witnessed in AD, while no change (NC) in expression as compared to a control is witnessed in PD or DLB. Likewise, as shown in FIG. 5B, a decrease in expression of Chromogranin B is witnessed in AD, while an increase in expression is witnessed in PD and no change in expression is witnessed in DLB.

[0052] As such, in some embodiments, the pattern of gene product expression will be detected and compared to the library of gene product expression patterns known to be indicative of the presence or absence of a neurodegenerative disease. In certain embodiments, the assessment of gene product expression of a single gene product will provide a preliminary result and will be followed up with the assessment of at least a second gene product expression.

[0053] The present invention also provides a method for monitoring progression of a neurodegenerative disease in a subject by detecting a first pattern of expression of gene products present in a cerebrospinal fluid sample obtained from a subject at a first time point, wherein said first pattern is indicative of a neurodegenerative disease; detecting a second pattern of expression of gene products present in a cerebrospinal fluid sample obtained from a subject at a second time point; and comparing the first and second patterns of expression of gene products from the cerebrospinal fluid samples, wherein the comparing provides for monitoring of the progression of the neurodegenerative disease from the first time point to the second time point.

[0054] In certain embodiments, the method of monitoring progression of a neurodegenerative disease in a subject will include detecting a pattern of expression of gene products present in a CSF sample obtained from a subject at more than two time points, such as three or more. In general, the time points for detecting a pattern of expression of gene products can be separated by any amount of time that is desired. For example, the first time point and second time point can be separated by about 3 months, about 6 months, or about 1 year or more, such as about 3 or more years.

[0055] In general, it will be appreciated by one of skill in the art that the duration of time between the first time point and the second time point must be sufficient to provide for a monitoring of the progression of the neurodegenerative disease.

[0056] In certain embodiments, the monitoring of the neurodegenerative disease in the subject will be conducted in parallel with a treatment regimen for the neurodegenerative disease. In such embodiments, the method of monitoring the neurodegenerative disease during treatment will provide information of whether the treatment is improving the condition, or having no effect or an adverse effect on the condition. In such embodiments, the first time point may be either just before, concurrent with, or just after the in initiation of a treatment regimen and the second time point may be a time point following a desired treatment period. For example, in such embodiments, the second time point may be about 6 month or more following initiation of treatment, including about 1 year, about 2 years, or more. For example, the detection of the pattern of expression of gene products present in a CSF sample obtained from the subject may be determined about once every 6 months to monitor progression of the disease and efficacy of the treatment regimen.

[0057] In general, methods of the invention involving detection of a gene product (e.g., proteins or polypeptides). In one embodiment, the methods involve contacting a sample with a probe specific for the gene product of interest (e.g., marker polypeptide). “Probe” as used herein in such methods is meant to refer to a molecule that specifically binds a gene product of interest (e.g., the probe binds to the target gene product with a specificity sufficient to distinguish binding to target over non-specific binding to non-target (background) molecules). “Probes” include, but are not necessarily limited to, antibodies (e.g., antibodies, antibody fragments that retain binding to a target epitope, single chain antibodies, and the like), or other polypeptide, peptide, or molecule (e.g., receptor ligand) that specifically binds a target gene product of interest.

[0058] The probe and sample suspected of having the gene product of interest are contacted under conditions suitable for binding of the probe to the gene product. For example, contacting is generally for a time sufficient to allow binding of the probe to the gene product (e.g., from several minutes to a few hours), and at a temperature and conditions of osmolarity and the like that provide for binding of the probe to the gene product at a level that is sufficiently distinguishable from background binding of the probe (e.g., under conditions that minimize non-specific binding). Suitable conditions for probe-target gene product binding can be readily determined using controls and other techniques available and known to one of ordinary skill in the art.

[0059] The probe can be an antibody or other polypeptide, peptide, or molecule (e.g., receptor ligand) that specifically binds a target polypeptide of interest.

[0060] The detection methods can be provided as part of a kit. Thus, the invention further provides kits for detecting the presence/absence and/or a level of expression of a marker of the invention, and/or a polypeptide in a human CSF sample. The kits of the invention for detecting a marker polypeptide generally comprise a moiety that specifically binds the polypeptide, which may be a specific antibody. The kit may optionally provide additional components that are useful in the procedure, including, but not limited to, buffers, developing reagents, labels, reacting surfaces, means for detection, control samples, standards, instructions, and interpretive information.

Detecting a Marker Polypeptide in Diagnosing Neurodegenerative Disease

[0061] The gene products according to the methods of the present invention can be detected by any suitable method. Detection paradigms that can be employed to this end include enzymatic methods, including immunological-based methods, optical methods, electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g., multipolar resonance spectroscopy. It is to be understood that the present invention is not limited to a particular detection method. However, in some embodiments detection is by, for example, fluorescent detection, spectrometric detection, chemiluminescent detection, matrix assisted laser desorption-time-of flight (MALDI-TOF) detection, high pressure liquid chromatographic detection, charge detection, mass detection, radio frequency detection, and light diffraction detection. Exemplary detection methods that are suitable for use with the subject methods are described herein.

[0062] Detection by Capture Agent

[0063] In some embodiments, detection of gene products is by use of capture reagents specific to the gene products (e.g., polypeptides). In general, the biospecific capture reagent is bound to a solid phase, such as a bead, a plate, a membrane or a chip. Methods of coupling biomolecules, such as antibodies, to a solid phase are well known in the art. They can employ, for example, bifunctional linking agents, or the solid phase can be derivatized with a reactive group, such as an epoxide or an imidizole, that will bind the molecule on contact. Biospecific capture reagents against different gene products can be mixed in the same place, or they can be attached to solid phases in different physical or addressable locations. For example, one can load multiple columns with derivatized beads, each column able to capture a single gene product. Alternatively, one can pack a single column with different beads derivatized with capture reagents against a variety of gene products, thereby capturing all the analytes in a single place. Accordingly, antibody-derivatized bead-based technologies, such as Multi-Analyte Profiling (xMAP™) technology of Luminex (Austin, Tex.) can be used to detect the gene products.

[0064] Luminex xMAP™ is based on polystyrene particles (microspheres) that are internally labeled with two different fluorophores. When excited by a 635-nm laser, the fluorophores emit light at different wavelengths, e.g., 658 and 712 nm. By varying the 658-nm/712-nm emission ratios, the beads are individually classified by the unique Luminex 100 IS analyzer. A third fluorophore coupled to a reporter molecule allows for quantification of the interaction that has occurred on the microsphere surface. The Luminex xMAP™ technology is described, for example, in U.S. Pat. Nos. 5,736,330, 5,981,180, and 6,057,107, all of which are specifically incorporated by reference.

[0065] In yet another embodiment, the surfaces of biochips can be derivatized with the capture reagents directed against specific gene products (e.g., selected from FIGS. 5A-5YY). Biochips generally comprise solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Frequently, the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound there.

[0066] Detection by Mass Spectrometry

[0067] In some embodiments, the gene products (e.g., polypeptides) are detected by mass spectrometry, a method that employs a mass spectrometer to detect gas phase ions. Examples of mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these. In such embodiments, the relative levels of gene products in each sample can be determined with mass spectrometry where a standard curve can be generated using corresponding synthetic peptides without isotope labeling. Alternatively, the gene products (e.g., polypeptides) in the sample can be identified and quantified when the identical synthetic peptides are isotope labeled and spiked in the sample.

[0068] In certain embodiments the mass spectrometer is a laser desorption/ionization mass spectrometer. In laser desorption/ionization mass spectrometry, the analytes are placed on the surface of a mass spectrometry probe, a device adapted to engage a probe interface of the mass spectrometer and to present an analyte to ionizing energy for ionization and introduction into a mass spectrometer. A laser desorption mass spectrometer employs laser energy, typically from an ultraviolet laser, but also from an infrared laser, to desorb analytes from a surface, to volatilize and ionize them and make them available to the ion optics of the mass spectrometer.

[0069] In general, a probe with an adsorbent surface is contacted with the CSF sample obtained from a subject for a period of time sufficient to allow gene products (e.g., peptides) that may be present in the sample to bind to the adsorbent surface. After an incubation period, the substrate is washed to remove unbound material. Any suitable washing solutions can be used; such as an aqueous solution. The extent to which molecules remain bound can be manipulated by adjusting the stringency of the wash. The elution characteristics of a wash solution can depend, for example, on pH, ionic strength, hydrophobicity, degree of chaotropism, detergent strength, and temperature. An energy absorbing molecule is then applied to the substrate with the bound gene products.

[0070] The gene products bound to the substrate are then detected in a gas phase ion spectrometer such as a time-of-flight mass spectrometer or an ion trap mass spectrometer. The gene products are ionized by an ionization source such as a laser, the generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions. The detector then translates information of the detected ions into mass-to-charge ratios. Detection of a gene product typically will involve detection of signal intensity. Thus, both the quantity and mass of the gene product can be determined.

[0071] In another mass spectrometry method, the gene product(s) (e.g., polypeptides) can be first captured on a chromatographic resin that binds the target molecules. For example, the resin can be derivatized with anti-gene product proteins antibodies. Alternatively, this method could be preceded by chromatographic fractionation before application to the bio-affinity resin. After elution from the resin, the sample can be analyzed by MALDI, electrospray, or another ionization method for mass spectrometry. In another alternative, one could fractionate on an anion exchange resin and detect by MALDI or electrospray mass spectrometry directly. In yet another method, one could capture the gene product(s) on an immuno-chromatographic resin that comprises antibodies that bind the target molecules, wash the resin to remove unbound material, elute the bound molecules from the resin and detect the eluted proteins by MALDI, electrospray mass spectrometry or another ionization mass spectrometry method.

[0072] Detection by Immunoassay

[0073] Any of a variety of known immunoassay methods can be used for detection, including, but not limited to, immunoassay, using an antibody specific for the encoded polypeptide, e.g., by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and the like; and functional assays for the encoded polypeptide, e.g., binding activity or enzymatic activity.

[0074] For example, an immunofluorescence assay can be easily performed on fractionated or non-fractioned human CSF. It is also possible to perform such assays in plasma if sufficient markers are diffused from human CSF to plasma.

[0075] To increase the sensitivity of the assay, the immunocomplex may be further exposed to a second antibody, which is labeled and binds to the first antibody, which is specific for the encoded polypeptide. Typically, the secondary antibody is detectably labeled, e.g., with a fluorescent marker. The cells which express the encoded polypeptide will be fluorescently labeled and easily visualized under the microscope. See, for example, Hashido et al. (1992) Biochem. Biophys. Res. Comm. 187:1241-1248.

[0076] As will be readily apparent to the ordinarily skilled artisan upon reading the present specification, the detection methods and other methods described herein can be varied. Such variations are within the intended scope of the invention. For example, in the above detection scheme, the probe for use in detection can be immobilized on a solid support, and the test sample (e.g., human CSF or plasma) contacted with the immobilized probe. Binding of the test sample to the probe can then be detected in a variety of ways, e.g., by detecting a detectable label bound to the test sample.

[0077] Thus generally the methods comprise: a) contacting the sample with an antibody specific for a gene product (e.g., a marker selected from FIGS. 5A-5YY); and b) detecting binding between the antibody and molecules of the sample. The level of antibody binding (either qualitative or quantitative) indicates the susceptibility of the patient to a neurodegenerative disease. For example, where the marker polypeptide is present at a level greater than that associated with a negative control level, then the patient is susceptive to neurodegenerative disease.

[0078] Suitable controls include a sample known not to contain the marker polypeptide; a sample contacted with an antibody not specific for the marker polypeptide; a sample having a level of polypeptide associated with neurodegenerative disease. A variety of methods to detect specific antibody-antigen interactions are known in the art and can be used in the method, including, but not limited to, standard immunohistological methods, immunoprecipitation, an enzyme immunoassay, and a radioimmunoassay.

[0079] In general, the specific antibody will be detectably labeled, either directly or indirectly. Direct labels include radioisotopes; enzymes having detectable products (e.g., luciferase, β-galactosidase, and the like); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals, e.g., ¹⁵²Eu, or others of the lanthanide series, attached to the antibody through metal chelating groups such as EDTA; chemiluminescent compounds, e.g., luminol, isoluminol, acridinium salts, and the like; bioluminescent compounds, e.g., luciferin, aequorin (green fluorescent protein), and the like.

[0080] The antibody may be attached (coupled) to an insoluble support, such as a polystyrene plate or a bead. Indirect labels include second antibodies specific for antibodies specific for the encoded polypeptide (“first specific antibody”), wherein the second antibody is labeled as described above; and members of specific binding pairs, e.g., biotin-avidin, and the like. The biological sample may be brought into contact with and immobilized on a solid support or carrier, such as nitrocellulose, that is capable of immobilizing cells, cell particles, or soluble proteins. The support may then be washed with suitable buffers, followed by contacting with a detectably-labeled first specific antibody. Detection methods are known in the art and will be chosen as appropriate to the signal emitted by the detectable label. Detection is generally accomplished in comparison to suitable controls, and to appropriate standards.

Polypeptide Arrays

[0081] Polypeptide arrays provide a high throughput technique that can assay a large number of polypeptides in a sample. This technology can be used as a tool to test for expression of a marker polypeptide and assessment of neurodegenerative disease. Of particular interest are arrays which comprise a probe for detection of one or more of the gene products selected from FIGS. 5A-5YY.

[0082] A variety of methods of producing arrays, as well as variations of these methods, are known in the art and contemplated for use in the invention. For example, arrays can be created by spotting polypeptide probes onto a substrate (e.g., glass, nitrocellulose, etc.) in a two-dimensional matrix or array having bound probes. The probes can be bound to the substrate by either covalent bonds or by non-specific interactions, such as hydrophobic interactions.

[0083] Samples of polypeptides can be detectably labeled (e.g., using radioactive or fluorescent labels) and then hybridized to the probes. Alternatively, the polypeptides of the test sample can be immobilized on the array, and the probes detectably labeled and then applied to the immobilized polypeptides. In most embodiments, the “probe” is detectably labeled. In other embodiments, the probe is immobilized on the array and not detectably labeled. In such embodiments, the sample is applied to the polypeptide array and bound gene products (e.g., peptides) are detected using secondary labeled probes

[0084] Examples of such protein arrays are described in the following patents or published patent applications: U.S. Pat. No. 6,225,047; PCT International Publication No. WO 99/51773; U.S. Pat. No. 6,329,209, PCT International Publication No. WO 00/56934 and U.S. Pat. No. 5,242,828.

Computer-Based Systems and Methods

[0085] The invention also provides a variety of computer-related embodiments. Specifically, the automated means for performing the methods described above may be controlled using computer-readable instructions, i.e., programming. Accordingly, in some embodiments the invention provides computer programming for analyzing and comparing a pattern of gene product expression present in a CSF sample obtained from a subject to a library of gene product expression patterns known to be indicative of the presence or absence of a neurodegenerative disease, wherein the comparing indicates the presence or absence of a neurodegenerative disease.

[0086] In another embodiment, the invention provides computer programming for analyzing and comparing a first and a second pattern of expression of gene products from CSF samples takes from a subject in at least two different time points, wherein the first pattern is indicative of a neurodegenerative disease. In such embodiments, the comparing provides for monitoring of the progression of the neurodegenerative disease from the first time point to the second time point.

[0087] In yet another embodiment, the invention provides computer programming for analyzing and comparing a pattern of gene product expression from CSF sample to a library of gene product expression patterns known to be indicative of the presence or absence of AD, PD and DLB, wherein the comparing providing a differential diagnosis between AD, PD, and DLB.

[0088] The methods and systems described herein can be implemented in numerous ways. In one embodiment of particular interest, the methods involve use of a communications infrastructure, for example the internet. Several embodiments of the invention are discussed below. It is also to be understood that the present invention may be implemented in various forms of hardware, software, firmware, processors, or a combination thereof. The methods and systems described herein can be implemented as a combination of hardware and software. The software can be implemented as an application program tangibly embodied on a program storage device, or different portions of the software implemented in the user's computing environment (e.g., as an applet) and on the reviewer's computing environment, where the reviewer may be located at a remote site (e.g., at a service provider's facility).

[0089] For example, during or after data input by the user, portions of the data processing can be performed in the user-side computing environment. For example, the user-side computing environment can be programmed to provide for defined test codes to denote platform, carrier/diagnostic test, or both; processing of data using defined flags, and/or generation of flag configurations, where the responses are transmitted as processed or partially processed responses to the reviewer's computing environment in the form of test code and flag configurations for subsequent execution of one or more algorithms to provide a results and/or generate a report in the reviewer's computing environment.

[0090] The application program for executing the algorithms described herein may be uploaded to, and executed by, a machine comprising any suitable architecture. In general, the machine involves a computer platform having hardware such as one or more central processing units (CPU), a random access memory (RAM), and input/output (I/O) interface(s). The computer platform also includes an operating system and microinstruction code. The various processes and functions described herein may either be part of the microinstruction code or part of the application program (or a combination thereof) which is executed via the operating system. In addition, various other peripheral devices may be connected to the computer platform such as an additional data storage device and a printing device.

[0091] As a computer system, the system generally includes a processor unit. The processor unit operates to receive information, which generally includes test data (e.g., specific gene products assayed), and test result data (e.g., the pattern of gene product expression for a sample). This information received can be stored at least temporarily in a database, and data analyzed in comparison to a library of gene product expression patterns known to be indicative of the presence or absence of a neurodegenerative disease, including PD, AD, and DLB, as described above.

[0092] Part or all of the input and output data can also be sent electronically; certain output data (e.g., reports) can be sent electronically or telephonically (e.g., by facsimile, e.g., using devices such as fax back). Exemplary output receiving devices can include a display element, a printer, a facsimile device and the like. Electronic forms of transmission and/or display can include email, interactive television, and the like. In an embodiment of particular interest, all or a portion of the input data and/or all or a portion of the output data (e.g., usually at least the library of gene product expression patterns known to be indicative of the presence or absence of a neurodegenerative disease) are maintained on a server for access, preferably confidential access. The results may be accessed or sent to professionals as desired.

[0093] A system for use in the methods described herein generally includes at least one computer processor (e.g., where the method is carried out in its entirety at a single site) or at least two networked computer processors (e.g., where gene product expression data for a CSF sample obtained from a subject is to be input by a user (e.g., a technician or someone performing the activity assays)) and transmitted to a remote site to a second computer processor for analysis (e.g., where the pattern of gene expression is compared to a library of gene product expression patterns known to be indicative of the presence or absence of a neurodegenerative disease), where the first and second computer processors are connected by a network, e.g., via an intranet or internet). The system can also include a user component(s) for input; and a reviewer component(s) for review of data, and generation of reports, including detection of neurodegenerative disease, differential diagnosis of PD, AD, and DLB, or monitoring the progression of a neurodegenerative disease. Additional components of the system can include a server component(s); and a database(s) for storing data (e.g., as in a database of report elements, e.g., a library of gene product expression patterns known to be indicative of the presence or absence of a neurodegenerative disease, or a relational database (RDB) which can include data input by the user and data output. The computer processors can be processors that are typically found in personal desktop computers (e.g., IBM, Dell, Macintosh), portable computers, mainframes, minicomputers, or other computing devices.

[0094] The networked client/server architecture can be selected as desired, and can be, for example, a classic two or three tier client server model. A relational database management system (RDMS), either as part of an application server component or as a separate component (RDB machine) provides the interface to the database.

[0095] In one embodiment, the architecture is provided as a database-centric user/server architecture, in which the user application generally requests services from the application server which makes requests to the database (or the database server) to populate the activity assay report with the various report elements as required, especially the assay results for each activity assay. The server(s) (e.g., either as part of the application server machine or a separate RDB/relational database machine) responds to the user's requests.

[0096] The input components can be complete, stand-alone personal computers offering a full range of power and features to run applications. The user component usually operates under any desired operating system and includes a communication element (e.g., a modem or other hardware for connecting to a network), one or more input devices (e.g., a keyboard, mouse, keypad, or other device used to transfer information or commands), a storage element (e.g., a hard drive or other computer-readable, computer-writable storage medium), and a display element (e.g., a monitor, television, LCD, LED, or other display device that conveys information to the user). The user enters input commands into the computer processor through an input device. Generally, the user interface is a graphical user interface (GUI) written for web browser applications.

[0097] The server component(s) can be a personal computer, a minicomputer, or a mainframe and offers data management, information sharing between clients, network administration and security. The application and any databases used can be on the same or different servers.

[0098] Other computing arrangements for the user and server(s), including processing on a single machine such as a mainframe, a collection of machines, or other suitable configuration are contemplated. In general, the user and server machines work together to accomplish the processing of the present invention.

[0099] Where used, the database(s) is usually connected to the database server component and can be any device which will hold data. For example, the database can be any magnetic or optical storing device for a computer (e.g., CDROM, internal hard drive, tape drive). The database can be located remote to the server component (with access via a network, modem, etc.) or locally to the server component.

[0100] Where used in the system and methods, the database can be a relational database that is organized and accessed according to relationships between data items. The relational database is generally composed of a plurality of tables (entities). The rows of a table represent records (collections of information about separate items) and the columns represent fields (particular attributes of a record). In its simplest conception, the relational database is a collection of data entries that “relate” to each other through at least one common field.

[0101] Additional workstations equipped with computers and printers may be used at point of service to enter data and, in some embodiments, generate appropriate reports, if desired. The computer(s) can have a shortcut (e.g., on the desktop) to launch the application to facilitate initiation of data entry, transmission, analysis, report receipt, etc. as desired.

Computer-Readable Storage Media

[0102] The invention also contemplates a computer-readable storage medium (e.g. CD-ROM, memory key, flash memory card, diskette, etc.) having stored thereon a program which, when executed in a computing environment, provides for implementation of algorithms to carry out all or a portion of the methods described herein, including detection of neurodegenerative disease, differential diagnosis of PD, AD, and DLB, or monitoring the progression of a neurodegenerative disease. Where the computer-readable medium contains a complete program for carrying out the methods described herein, the program includes program instructions for collecting, analyzing and comparing a pattern of gene product expression patterns from a CSF sample obtained from a subject to a library of gene product expression patterns known to be indicative of the presence or absence of a neurodegenerative disease, and generally includes computer readable code devices for interacting with a user as described herein, processing that data in conjunction with analytical information, and generating unique printed or electronic media for that user.

[0103] Where the storage medium provides a program which provides for implementation of a portion of the methods described herein (e.g., the user-side aspect of the methods (e.g., data input, report receipt capabilities, etc.)), the program provides for transmission of data input by the user (e.g., via the internet, via an intranet, etc.) to a computing environment at a remote site. Processing or completion of processing of the data may be carried out at the remote site to provide for detection of neurodegenerative disease, differential diagnosis of PD, AD, and DLB, or monitoring the progression of a neurodegenerative disease. The computer-readable storage medium can also be provided in combination with one or more reagents for carrying out one or more of the activity assays (e.g., control compounds, cells, probes, arrays, or other activity assay test kit components).

Kits

[0104] Also provided by the subject invention are kits for practicing the subject methods, as described above, including detection of neurodegenerative disease, differential diagnosis of PD, AD, and DLB, or monitoring the progression of a neurodegenerative disease. The subject kits include at least one or more of: a probe or primer for detection of a marker polynucleotide, a marker polypeptide, or an anti-marker polypeptide antibody. Other optional components of the kit include: restriction enzymes, control primers and plasmids; nucleic acid or polypeptide standards; buffers; reaction mixtures (e.g., for carrying out the assay); enzymes (e.g., DNA polymerase, reverse transcriptase, and the like); cells; and the like. The various components of the kit may be present in separate containers or certain compatible components may be precombined into a single container, as desired.

[0105] In addition to above-mentioned components, the subject kits typically further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0106] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Methods and Materials

[0107] The following methods and materials are used in the examples below.

[0108] Chemicals and Antibodies

[0109] All reagents were purchased from Sigma Aldrich (St. Louis, Mo.) unless otherwise specified. Antibody list: Apolipoprotein (Apo)CI (goat anti-human, Biodesign International, Kennebunkport, Me.); ApoD (mouse monoclonal; Vision Biosystems, Norwell, Mass.); ApoH (rabbit polyclonal, Accurate Chemical & Scientific Corporation, Westbury, N.Y.); calcium/calmodulin-dependent protein kinase IIB isoform 8 (Ca/CaMKIIB; rabbit polyclonal, Stratagen, Cedar Creek, Tex.); ceruloplasmin (sheep polyclonal, Abcam, Cambridge, Mass.), chromogranin B (rabbit polyclonal, Abcam); Cu/Zn superoxide dismutase (Cu/Zn SOD; mouse anti-human, Calbiochem, La Jolla, Calif.); β-fibrinogen (goat polyclonal, Santa Cruz Biotechnology, Santa Cruz, Calif.); furin convertase (MON-148; mouse monoclonal, Alexis Biochemicals, San Diego, Calif.); α-1B-glycoprotein (A1BG; rabbit polyclonal, Aviva Systems Biology, San Diego, Calif.); haptoglobin (chicken polyclonal, Abcam), osteonectin (SPARC; mouse anti-human, Haematologic Technologies, Essex Junction, Vt.); semaphorin 7A (CDW108; mouse monoclonal, Chemicon International, Temecula, Calif.); T-cadherin (H-126; rabbit polyclonal, Santa Cruz Biotechnology); and vitamin D binding protein ((VitD BP) or Gc-globulin (chicken polyclonal; GenWay Biotech, San Diego, Calif.). Secondary antibodies included rabbit anti-chicken IgG-HRP, rabbit anti-sheep IgG-HRP, rabbit anti-goat IgG-HRP, and goat anti-rabbit IgG-HRP (Sigma-Aldrich). Rabbit anti-mouse IgG-HRP was purchased from Abcam.

[0110] Patients

[0111] All individuals underwent evaluation that consisted of medical history, physical and neurologic examinations, laboratory tests, and neuropsychological assessment. Laboratory evaluation included complete blood count: serum electrolytes, blood urea nitrogen, creatinine, glucose, vitamin B12, and thyroid stimulating hormone; all results were within normal limits. A brief summary on inclusion and exclusion criteria is provided below for normal controls as well as patients with AD, PD or DLB. Demographic information is listed in Table 1 for all subjects/patients.

[0112]

TABLE 1
Characteristics of patients and age-matched controls
	M:F	Age	MMSE	Time of CSF Tap to
	Ratio	(Mean ± SD)	(Mean ± SD)	Autopsy (years)
Control	10	7:3	67 ± 6	29.3 + 0.68	NA
AD	10	6:4	72 ± 9	13.1 + 6.87	2.38 + 1.52
PD	10	7:3	63 ± 7	29.7 + 0.36	NA
DLB	5	5:0	69 ± 11	19.9 + 5.47	2.51 + 0.71

[0113] Normal aged controls: The control subjects were community volunteers in good health. Neuropsychological evaluation included: the Mini-Mental State Exam (MMSE) (Folstein et al., J Psychiatr Res, 1975. 12(3): p. 189-98), Trail-Making Tests A and B (Reitan et al., Percept Mot Skills, 1958. 8: p. 271-276). Clinical Dementia Rating Scale (CDR (Morris, Int Psychogeriatr, 1997. 9(Suppl 1): p. 173-6; discussion 177-8)), the Mattis and Coblentz Dementia Rating Scale score (DRS (Mattis et al., , S. and J. Coblentz, Mental status examination for organic mental syndrome in the elderly patient. Geriatric psychiatry: A handbook for psychiatrists and primary care physicians, ed. L. Belleck and T. Karasu. 1976, New York: Grune and Stratton. 77-121)) and -the New York University (NYU) version of the Logical Memory II subscale (Immediate and Delayed Paragraph Recall) from the Wechsler Memory Scale—Revised (Flicker et al., Neurology, 1991. 41(7): p. 1006-9). Control subjects had no signs or symptoms suggesting cognitive decline or neurologic disease; all subjects had a MMSE score between 28 and 30; a CDR score of 0, and NYU paragraph recall scores (immediate and delayed)>6. Exclusion criteria also included heavy cigarette smoking (more than 10 packs/year), alcohol use other than socially, and any psychotherapeutic use. Finally, it should be emphasized that although no pathological confirmation had been obtained in any of these subjects, all of them had been followed for approximately three years without demonstrating any symptoms or signs of neurological disorders, including mild cognitive impairment (MCI).

[0114] AD: Patients were diagnosed with probable AD according to NINDS-ADRDA criteria confirmed by a clinical team consensus conference at the Oregon Aging and Alzheimer's Disease Research Center and concurred by investigators at the UW-Alzheimer's Disease Search Center. One important aspect of this study was that only subjects with post-mortem pathological confirmation of AD according to NIA-Ragan criteria (high) were included in this study. CSF was collected during life and maintained at −70° C. until analysis. The average time from CSF collected to autopsy was 2.8 years (also see Table 1).

[0115] PD: Only clinically probable PD patients defined with NINDS criteria, which is based on those described by Drs. Calne (Calne et al., Ann Neurol, 1992. 32(Suppl): p. S125-7) and Gelb (Gelb et al., Arch Neurol, 1999. 56(1): p. 33-9), were included. Essentially, patients were required to have three Group A signs, i.e. resting tremor, bradykinesia, rigidity and asymmetric onset, and have sustained response to levodopa or a DA agonist. Patients with the following features (Group B signs) were excluded: 1) prominent postural instability in the first three years after symptom onset, 2) freezing phenomenon in the first three years, 3) hallucinations unrelated to medications in the first three years, 4) dementia preceding motor symptoms or in the first year, 5) supranuclear gaze palsy (other than restriction of upward gaze) or slowing of vertical saccades, 6) severe, symptomatic dysautonomia unrelated to medications, and 7) documentation of a condition known to produce parkinsonism and plausibly connected to the patient's symptoms (such as suitably located focal brain lesions or neuroleptic use within the past six months). Please note, like the control patients, all of these patients were still alive at the time of proteomic analysis, i.e. no pathological confirmation of PD had been obtained yet. Nonetheless, all patients included in this study had sustained response to DA drugs for at least three years and there was no need to revise clinical diagnosis on any of these patients after follow-up evaluation when this manuscript was written.

[0116] DLB: These patients were initially diagnosed with probable AD according to NINDS-ADRDA criteria, but each developed parkinsonism, fluctuation cognition, and visual hallucinations, characteristic of DLB, shortly after CSF was obtained, yielding a revised diagnosis clinically. As mentioned early, as sensitivity and specificity of clinical criteria for DLB diagnosis are not high (McKeith et al., Semin Clin Neuropsychiatry, 2003. 8(1): p. 46-57), only subjects with post-mortem pathological confirmation of DLB were included in this study. More specifically, all subjects had AD pathology in addition to cortical Lewy bodies. One caveat is that some pathologists may classify this entity as a Lewy body variant of AD (LBV-AD). Another major variant of DLB is dementia cases with diffuse Lewy bodies in the cortex in the absence of AD pathology, which overlaps with PD plus dementia both clinically and pathologically. Finally, it should be noted that this group of patients was hardest to obtain, particularly when the quality of CSF was taken into consideration (see below), and thus this study was limited to include only five DLB cases.

[0117] Collection of CSF and Quality Control

[0118] Following written informed consent, individuals were placed in the lateral decubitus position and the L4-5 interspace was infiltrated with 1% lidocaine. Lumbar puncture (LP) was performed with a 20 g or 24 g spinal needle. Individuals remained at bed rest for one hour following LP. All CSF for proteomic analysis was taken from the 15^th to 25^th ml collected to limit variations arising from rostral-caudal gradient. In addition, all LP was performed in the morning to limit potential circadian fluctuation of CSF proteins and metabolites.

[0119] The protein concentration in CSF is relatively low compared to plasma (CSF:plasma= 1/20), and in addition, the protein profiles in CSF are similar to those in plasma (Blennow et al., Eur Neurol, 1993. 33(2): p. 129-33), suggesting that even a minor contamination of CSF with blood could significantly confound the interpretation of quantitative proteomic analysis of CSF. To minimize blood contamination in the CSF samples, only CSF samples with <10 RBCs/ml and a serum:CSF ApoB (a protein not generated in CNS) ratio >6000 were included in this study. This approach has been utilized successfully in previous CSF proteomics studies (Zhang et al., Neurobiol Aging, 2005. 26(2): p. 207-27; Zhang et al., J Alzheimers Dis, 2005. 7(2): p. 125-33).

[0120] Sample Preparation Before Proteomic Analysis

[0121] Previous experience has shown that extensive analysis of well-characterized pooled samples is more productive than analyzing individual samples. This is largely due to the limitation of current MS technology, i.e. a low reproducibility when an identical sample is analyzed multiple times (Zhang et al., Neurobiol Aging, 2005. 26(2): p. 207-27; Zhou et al., J Biol Chem, 2004. 279(37): p. 39155-64). For example, if profiling is done with an individual sample, when a marker is identified in one individual (e.g. an AD patient) but not the other, there is no way of telling whether it is due to the nature of the subject/patient or variation in ionization of MS unless an independent validation process is performed, which is not currently available in a high throughput manner. To circumvent this difficulty, the following strategy was adopted: discovering potential biomarkers with pooled samples (diseased vs. controls) with extensive chromatographic separation of peptides and multiple injections to reach the “bottom of the iceberg”. After potential biomarkers are identified, individual samples were confirmed and/or validated to achieve information related to the sensitivity and specificity of each marker (Zhang et al., Neurobiol Aging, 2005. 26(2): p. 207-27; Zhang et al., J Alzheimers Dis, 2005. 7(2): p. 125-33). Hence, in the current study, in discovery phase CSF samples were pooled from 10 AD, 10 PD, 5 DLB, and 10 controls before proteomic analysis.

[0122] The other issue related to CSF proteomics has to do with its unique profiles, i.e. overtly enriched in albumin and immunoglobulins (IgGs) (Blennow et al., Eur Neurol, 1993. 33(2): p. 129-33) with a dynamic range of protein concentrations ˜10⁹ as opposed to a dynamic range of ˜10⁸ for typical cell lysates (Corthals et al., Electrophoresis, 2000. 21(6): p. 1104-15). Because all current proteomic techniques are inheritably biased toward abundant proteins (Yuan et al., Electrophoresis, 2002. 23(7-8): p. 1185-96), fractionation of CSF is required before detailed proteomic analysis of CSF can be achieved. Thus, a graduated organic fractionation approach was followed that was recently developed to process CSF before standard MudPIT analysis of CSF proteins (Zhang et al., Neurobiol Aging, 2005. 26(2): p. 207-27). Briefly, pooled CSF was mixed with 1.5 volume of acetonitrile (ACN) first to generate the first pellet (P1), and then the supernatant was further mixed with final 3.0 volume of ACN to generate the second pellet (P2) and a supernatant (S2), which was dialyzed with a porous (500 D) membrane to desalt. With this approach, more than 90% of albumin and IgGs are found in the first pellet (Zhang et al., Neurobiol Aging, 2005. 26(2): p. 207-27).

[0123] iTRAQ Labeling and Two Dimensional Liquid Chromatography

[0124] Three fractions from each pooled CSF sample, i.e. P1, P2, and S2, were matched across all four groups of patients/subjects, forming three iTRAQ experiments. Briefly, 100 μg protein from each corresponding fraction (e.g. P1 fraction from AD, PD, DLB and controls) was digested in parallel with trypsin and then labeled with one of the four-iTRAQ™ reagents following the manufacturer's instructions. Next, four samples labeled with iTRAQ reagents were combined (a total of 400 g proteins), and loaded onto a strong cation exchange (SCX) column (0.5 mm×200 mm) that had been equilibrated in 0.05% formic acid/20% ACN and pH 3.0 (buffer A) at a flow rate of 200 μl/min. Peptides were eluted by applying a linear gradient from 0 to 100% buffer B (500 mM ammonium formiate/20% ACN, pH 3.0). 11 fractions were collected from each sample and dried down in a SpeedVac (Thermo Savant, Holbrook, N.Y.).

[0125] SCX fractionated peptides from each sample were then dissolved in 0.5% trifluoroacetic acid (TFA) and separated using reverse phase (RP) chromatography. Nano-capillary liquid chromatography (LC) was performed using the LC Packings UltiMate™ with Famos™ autosampler and Switchos™ automated switching valve (LC Packings, Sunnyvale, Calif.). Samples were loaded onto a capillary precolumn cartridge (Dionex, Sunnyvale, Calif.). The trap column was washed with mobile phase A containing 2% ACN and 0.1% TFA in HPLC water. The flow rate was set at 0.4 μl/min. The sample was then loaded onto a 15 cm×100 μm ID Magic C18 3 μm, 100-angstrom packing capillary LC column (Michrome BioResources Inc., Auburn, Calif.). The gradient run was from 5% mobile phase B (80% ACN, 20% HPLC water, 0.08% TFA) to 90% mobile phase B for 85 minutes. The eluted gradient was mixed with 7 mg/ml re-crystallized α-cyano-4-hydroxycinnamic acid (Sigma) in 60% ACN, 2.6% (5 mg/ml) ammonium citrate with internal standard (AB's 4700 Mass Standard Kit) and spotted onto a stainless steel MALDI plate with the Probot™ (LC Packings). Samples were spotted at 5-seconds intervals using a 24×24 array pattern for a total of 576 spots per plate. In total, 36 LC MALDI plates were spotted and analyzed by a 4700 Proteomic System.

[0126] MS Analysis and Protein Identification

[0127] Quantitative MS analysis was carried out using the 4700 Proteomics Analyzer with TOF/TOF Optics (Applied Biosystems or AB, Foster City, Calif.). MS reflector positive ion mode with automated acquisition of 800-4000 m/z range was used with 1000 shots per spectrum. A maximum of 15 peaks were selected per spot, with a minimum signal-noise (S/N) ratio of 75 and cluster area of 500. Greater than 36000 precursors were selected and were submitted for MS/MS, where a positive ion mode with CID cell on and 1 kV collision energy were used, and 3000 shots accumulated per spectrum. For each spotted plate, a total of 576 MS, and more than 1200 MS/MS spectra, were acquired. Identification of proteins was achieved using Mascot (Matrix Science, Boston, Mass.) algorithm and searched against the International Protein Index (IPI; Version 3.01), a database also used in one of the recent studies of human CSF (Xu et al., Int. Rev. of Neurobiol, 2005). In addition, protein identification was determined with a newer version of the IPI database (3.10) as well as with the Celera Discovery System™ database (20050302) that is typically used by AB's 4700 Proteomic System. Finally, identified proteins were further filtered by ProteinProphet, a program routinely used in the lab to enhance the accuracy of protein identification. Protein quantification was achieved by averaging ratios of all peptides of each identified protein; normalization, assuming a Gaussian distribution with median of 1 when all peptides were considered between control and experimental groups, was performed before ratios were calculated.

[0128] Western Blot

[0129] Western blot analysis was performed as described previously (Andreasen, et al., Clin Neurol Neurosurg, 2005. 107(3): p. 165-73) with minor modifications. In brief, equal amounts of human CSF proteins (and equal volumes as well for pooled samples) were run on SDS/PAGE Tris-HCl Criterion Gels (Bio-Rad Laboratories, Hercules, Calif.) under reducing conditions, transferred to PVDF membranes (Bio-Rad), blocked, and probed overnight at 4° C. with primary antibodies of ApoC1 (1:2000), ApoD (1:10000), ApoH (1:1000), A1BG (1:10000), chromogranin B (1:10000), Ca/CaMKIIB (1:500), ceruloplasmin (1:2000), β-fibrinogen (1:2000), furin (1:5000), haptoglobin (1:2000), semaphorin 7A (1:500), SPARC (1:5000), Cu/Zn-SOD (1:10000), T-cadherin (1:250), or VitD BP (1:4000). The secondary antibodies were added, and detected by enhanced chemiluminescence or by ECL plus western blotting detection system (Amersham Biosciences, N.J.). Relative levels of each protein were quantified by measuring optical densities (OD) of the corresponding bands compared to a pooled sample containing all cases. Protein concentration of the CSF was determined by the Bradford method with bovine serum albumin (Pierce, Ill.) as the standard.

[0130] Quantifying the Diagnostic Ability of Candidate Markers

[0131] Quantifying the diagnostic ability of a single marker: The performance of each of the eight confirmed candidate markers was evaluated both graphically and statistically with receiver operating characteristic (ROC) curve methods. ROC curves associate the sensitivity of a diagnostic test to the entire range of the possible false positive rate (FPR). The FPR is equal to one minus the test specificity. The area under the ROC curve (AUC) indicates the average sensitivity of a marker over the entire ROC curve. The sensitivity of each marker was computed at 95% specificity. Establishing statistical significance of a single marker was performed by the Wilcoxon rank-sum test, which evaluates the significance of the entire ROC curve. To aid interpretation of the data when comparing markers, raw data was transformed with the natural log so their behavior among healthy subjects more accurately reflected a normal distribution with a mean of 0 and unit standard deviation (McIntosh et al., Gynecol Oncol, 2004. 95(1): p. 9-15). Standardization of the markers, which leaves the ROC curves unchanged, also facilitates the comparison of two different markers because the units of measurement are now similar, i.e. the number of standard deviations above the average normal subject.

[0132] Combining markers: After the eight candidate markers were ranked based on the sensitivity at 95% specificity, p-value from Wilcoxon rank-sum test, and the area under curve (AUC) value, the top five markers were chosen for calculation of composite markers (CM). This was accomplished by evaluating a linear combination, which can be easily interpreted, or by the weighting of any two standardized markers from the top five markers identified. Logistic regression was used to optimize marker combination and estimate the weights. Logistic regression has several theoretical properties that make it convenient for applied biomarker research (McIntosh et al., Biometrics, 2002. 58: p. 657-664). For instance, its P values evaluate whether the marker combination, compared to the single marker, significantly increases the “distance” between cases and controls. If the model is correctly specified, the sensitivity of the resulting CM is maximized at all specificities simultaneously, although the theoretically correct model cannot ever be known in practice. After establishing significance, resulting ROC curves were then examined to evaluate the quality of the composite marker.

[0133] Using pooled, well characterized CSF samples and multidimensional peptide separation techniques, followed by 4700 TOF-TOF analysis, a total of 1,540 proteins were identified (FIGS. 6A-6T and FIGS. 7A-V). Of these, 804 were called single hits (FIGS. 7A-V). Single-hits refers to the fact that a protein is identified from the MS/MS spectrum of a single peptide and is therefore judged as being less reliably identified than those proteins identified with multiple peptide tandem mass spectra. Nonetheless, all protein identification was based on meeting the criteria of having at least one peptide whose individual score was above the 95% confidence interval threshold (p<0.05) and also identified as the top-ranked matching sequence for that spectrum. Furthermore, all proteins also had a probability of being more than 95% correct as determined by ProteinProphet.

[0134] When the list of identified proteins was compared to the previous analysis of human CSF, where close to 1,000 proteins were identified (Xu et al., Int. Rev. of Neurobiol, 2005) using the same database, 449 of those proteins were identified again in the current study. To state it differently, 1,091 new proteins were identified in the current study, thereby increasing the total identified CSF proteins to 1,883. Of note was also the observation that 51 proteins identified previously by a single peptide were now identified by more than two peptides. Examples included testican-1 precursor, ApoM, neuroligin 2 precursor, xylosyltransferase 1, and sortilin 1 preprotein. On the other hand, 90 proteins identified previously by more than two peptides were now identified by only a single peptide. These included cathepsin L precursor, collagen alpha 1(III) chain precursor, Mn-SOD, mitochondrial precursor, gelsolin precursor, and peroxiredoxin 2. Thus, technically, the proteins identified by more than two peptides were 1,097 when all of the studies are combined.

[0135] Classification of the 1,540 proteins identified in this study, shown in FIG. 1, is based on a modified scheme developed in previous publications (Zhang et al., Neurobiol Aging, 2005. 26(2): p. 207-27; Zhang et al., J Alzheimers Dis, 2005. 7(2): p. 125-33); it includes cell cycle/death, cell structure/motility/transport/traffic, extracellular matrix/adhesion, developmental process, immunity/defense, metabolism, neuronal activities/signal transduction, and unknown functions. However, as demonstrated in Table 2, the database used for protein identification can have a significant role in not only the number of proteins identified but also classifications of proteins (Xu et al., Int. Rev. of Neurobiol, 2005). As a result, IPI 3.01 (a database used in the recent CSF study) was used rather than an updated version IPI 3.10 in this study to make sure the data can be compared meaningfully. However, to further illustrate the contribution of databases on the outcome of protein identification, the identical MS data generated by 10 plates of 1.5P fraction were searched against the updated IPI version (3.10) as well as the newest Celera Discovery System (CDS) database (20050302) that is provided by Applied Biosystems, the manufacturer of the 4700 proteomic station. The results for database comparison are listed in Table 2 with several major points noted below. First, the overlap was only 26.0% between IPI 3.01 and CDS database if common protein names were used, but improved to 86.8% when peptides were considered regardless of protein names. Second, the overlap was much higher between two different versions of IPI database; and, consistent with previous results, the overlap was higher for proteins identified by more than two peptides than those by single peptide. Finally, although the IPI database appears to be maturing, the difference in protein identification between 3.01 and an earlier version (Xu et al., Int. Rev. of Neurobiol, 2005) vs. 3.01 and 3.10 was about the same, i.e. a change in the database when an identical MS data set was used resulted in about 10% difference in protein identification.

[0136]

TABLE 2
Identification of proteins with the same MS data
against different database
	Proteins Identified	Overlap
	IPI	IPI		IPI	Celera vs.
	(3.01)	(3.10)	Celera	3.10 vs. 3.01	IPI 3.01
Common names	567	559	782	62.9%	26.0%
Two peptides	250	291	569	65.4%	30.5%
Single peptide	317	268	213	58.9%	23.2%
Peptides	1584	1759	1506	96.7%	86.8%
regardless of
protein name

[0137] Compared with previous results, 1,091 new proteins were identified, expanding the total CSF proteome to 1,883 proteins, the most. extensive characterization of human CSF proteins today. The significant increase in the number of protein identification in this study largely resulted from two major factors, namely better separation of peptides by extensive chromatography and utilization of a more advanced MS instrument. The advantage of using better instrumentation is obvious, regardless of proteomic platforms used. Extensive chromatography is essential in LC based proteomics, even when MudPIT is used, as complex samples usually yield hundreds of thousands of peptides after proteins are digested. This issue is especially challenging in proteomic analysis of CSF or plasma, where albumin and IgGs constitute more than 75% of total proteins (Blennow et al., Eur Neurol, 1993. 33(2): p. 129-33), i.e. unless peptides are separated well, protein identification by MS will be centered on abundant proteins. Here, extensive peptide separation was achieved by utilizing two consecutive processes: 1) perform RP separation with a nano-capillary LC system that increases sensitivity by at least 10 fold as compared to a conventional micro-capillary LC system, resulting in less peptides eluted onto each MALDI plate; and 2) spot each LC run to 24×24 (576) arrays on an MALDI plate instead of a standard 198 spot array, thereby further separating peptides. Good peptide separation is evidenced by the fact that more than 400 (417 to be precise) proteins were identified in 1.5P fraction, notwithstanding it was overtly enriched in albumin and IgGs (Zhang et al., Neurobiol Aging, 2005. 26(2): p. 207-27).

[0138] The significance of extensive identification of the human CSF proteome is apparent, as it not only expands substantially the current knowledge regarding human CSF proteins, but also supplies the necessary information to appropriately interpret protein biomarkers of age-related neurodegenerative diseases. In addition, the impact of this data goes beyond neurodegenerative diseases because intense interest has also been expressed in other CNS diseases, including multiple sclerosis (Hammack et al., Mult Scler, 2004. 10(3): p. 245-60), acute brain injury (Siman et al., Neurobiol Dis, 2004. 16(2): p. 311-20), and CNS tumors (Zheng et al., J Neuropathol Exp Neurol, 2003. 62(8): p. 855-62).

[0139] Individual quantification of the identified peptides were based on the individual ratios from signature ion peak areas of the iTRAQ reagents tags of the identified peptides from AD, PD and DLB samples compared with the healthy individuals' signature ion peak areas. Peptide ratios from each protein were grouped and averaged together to give protein level modulations ratios for 1,520 identified proteins. Modulated proteins were found to be involved in several biological processes such as cellular metabolism, immunity and defense, signal transductions and neural activities, and synaptic transmissions. Some of these modulations exceeded two or three folds up or down. As the first step towards selecting candidate proteins for further study, changes with more than 50% increase or decrease were defined as significant. Changes that were less than <20% and >20% but less than 50% were defined as non-significant and ambiguous, respectively.

[0140] With these criteria, AD, PD, and DLB patients had a total of 388, 282, and 380 proteins that displayed significant changes from controls. Next, the focus was further narrowed on the protein markers that were unique only to AD, PD, or DLB. For instance, a protein marker, e.g. calreticulin precursor (IPI00020599), was excluded if it were significantly increased not only in AD vs. controls but also PD vs. controls. It should be noted, however, if a marker, e.g. DJ977L11.1 (IPI00478622), displayed a significant increase in AD vs. controls, but a decrease in PD vs. controls, the marker was considered not only unique to AD but also to PD. With this approach, 154, 81, and 113 proteins were identified that were uniquely altered with AD, PD and DLB, respectively (FIGS. 5A-5YY).

[0141] As demonstrated by data presented in Table 2, the numbers, as well as the types of proteins identified changed significantly when the database was altered. Given that none of the current databases is complete, it is imperative to confirm candidate protein markers not only for their identifications but also for their quantifications as determined by proteomics with alternative means before extensively pursuing their utilities in clinical diagnosis. Currently, there is no high throughput method available for this purpose, so consequently, Western blot analysis was used achieve this goal. Several criteria were used in selecting candidate proteins for further confirmation, including: 1) proteins had to be identified by more than two unique peptides; 2) markers should be unique to each disease, i.e. a marker common to two diseases was not considered; 3) markers with known biological functions were preferred; 4) markers identified by both IPI and Celera database were preferred with exception of those with appealing biological functions; and 5) commercial antibodies needed to be available. With these caveats in mind, 15 antibodies were purchased and tested initially with pooled samples that were also used for proteomic analysis. The antibodies chosen were A1BG, ApoCI, ApoC-III, ApoD, ApoH, Ca/CaMKIIB, ceruloplasmin, chromogranin B, β-fibrinogen, furrin, haptoglobin, semaphorin 7A precursor, sparc (osteonectin), Cu/Zn-SOD, T-cadherin, and VitD BP.

[0142] Among the 15 antibodies tested with pooled samples, only 8 of them were confirmed not only with respect to their identification, i.e. a distinct band was observed in human CSF at appropriate molecular weight for each marker, but also their quantification, meaning that quantitative changes as determined by Western blot were consistent with proteomic assessment for at least one of the diseases. These markers were ApoCI, ApoH, ceruloplasmin, chromogranin B, β-fibrinogen, haptoglobin, T-cadherin, and VitD BP. Western blot results are shown for β-fibrinogen as an example in FIG. 2, panel A, where quantification was performed with samples being normalized to the amount of protein as well as CSF volume.

[0143] To calculate the sensitivity/specificity for each marker in their ability of differentiating one disease from controls or from each other, all eight antibodies were then studied in individual samples. Of note, these samples were saved before pooled samples were generated for proteomic analysis and initial confirmation with Western blot analysis mentioned above. The results on Western blot analysis on individual samples is summarized in Table 3, and an actual gel blot is shown in FIG. 2 panel B, again for β-fibrinogen as an example with the samples being normalized to the amount of proteins loaded only. Notably, data shown in Table 3 was obtained initially by correcting the OD value of each band to a pooled sample containing all testing samples and run on the same gel. Next, the data was transformed to percent of controls for the sake of ease of comparison with proteomic data. As seen in the Table 3, Western quantification of each marker correlated with proteomic analysis reasonably well, with exception of chromogranin B and T-cadherin, meaning that the results obtained in pooled samples were not replicated in individual ones when tested with Western blot using these two antibodies. It should be emphasized, though, that a 20% decrease in a protein, e.g. ApoH, in PD patients does not necessarily disagree with a 50% decrease as determined by proteomic analysis. This is for the reason that the dynamic range of a Western blot is not as good as MS analysis, particularly when there is no purified antigen to optimize the Western blot run and establish a standard curve from which each band can be quantified more reasonably.

[0144]

TABLE 3
Measurements of each markers in individual cases
Protein	AD	PD	DLB	Control
Apo H	1.29	1.07	1.33	1.30 ± 0.036
	[0.99]	[0.82]	[1.02]
	(↑)	(↓↓)	(*)
Apo C1	1.83	1.89	1.49	2.06 ± 0.49
	[0.89]	[0.92]	[0.72]
	(*)	(*)	(↓↓)
Ceruloplasmin	1.07	0.90	1.00	1.06 ± 0.031
	[1.01]	[0.85]	[0.94]
	(*)	(↓↓)	(*)
Chromogranin B	1.14	1.20	1.01	1.18 ± 0.071
	[0.97]	[1.02]	[0.86]
	(↓↓)	(↑)	(*)
β-Fibrinogen	1.28	1.07	1.05	0.95 ± 0.035
	[1.35]	[1.13]	[1.11]
	(↑↑)	(*)	(*)
Haptoglobin	2.40	1.75	1.16	0.95 ± 0.033
	[2.53]	[1.84]	[1.22]
	(↑↑)	(↑)	(*)
T-Cadherin	1.28	1.10	1.13	1.12 ± 0.077
	[1.14]	[0.98]	[1.01]
	(*)	(*)	(↓↓)
Vit-D BP	1.45	1.03	0.98	1.19 ± 0.082
	[1.22]	[0.87]	[0.82]
	(*)	(↓↓)	(*)
Values (mean ± SE) for each marker are calculated first by correcting OD of each distinct band with the OD of the same band derived from a pooled sample containing all cases and run on the same gel.
Value expressed in [ ] are derived from the raw data divided by the mean of control cases shown in the last column, i.e. expressed as percent of controls. The rationale behind data transformation was to replicate the way that proteomic data were obtained.
↑↑: Proteomic changes greater than 50% as compared to controls;
↑: Proteomic changes greater than 20% but less than 50% as compared to controls;
*Proteomic changes less than 20% as compared to controls.

[0145] Table 4 summarizes the overall discrimination ability of each marker (its AUC) to classify different diseases and controls, their ROC Curves, and the P values from Wilcoxon sum-rank tests. It appeared that two markers, i.e. β-fibrinogen and VitD BP, can differentiate AD from controls as well as other diseases with AUC as 78% and 88%, respectively, and 40% to 50% sensitivity at 95% specificity. Both Wilcoxon p values are less than 0.05. Similarly, ApoH and ceruloplasmin appeared to be able to segregate PD from controls and other diseases very well. ApoH has the largest AUC and the smallest P value over the eight markers: AUC=87%, P value=0.004, and sensitivity at 95% specificity=67%. Ceruloplasmin was the next best maker with AUC=77%, P value=0.03 and sensitivity at 95% specificity=56%. However, none of the eight markers are statistically significant predictors of DLB over control or other diseases. Their Wilcoxon P values range from 0.07 to 0.9, probably because the small sample size tested for DLB cases (five for discovery and four for confirmation).

[0146]

TABLE 4
Summaries of ROC curves for the eight markers
	Case Group
	AD	PD	DLB
				Non-			Non-		Non-
Control Group	PD	DLB	CT	AD	DLB	Cont	PD	CT	DLB
Apo C1
AUC	0.478	0.625	0.633	0.536	0.667	0.605	0.495	0.667	0.656
Sense (0.95)	0.000	0.100	0.000	0.000	0.222	0.000	0.000	0.000	0.250
Wilcoxon P value	0.659	0.536	0.360	0.984	0.412	0.489	0.740	0.412	0.354
Apo H
AUC	0.806	0.550	0.525	0.610	0.861	0.944	0.869	0.391	0.593
Sense (0.95)	0.600	0.400	0.300	0.100	0.778	0.778	0.667	0.250	0.250
Wilcoxon P value	0.038*	0.835	0.896	0.329	0.078	0.008*	0.004*	0.461	0.580
Chromogranin B
AUC	0.611	0.750	0.574	0.525	0.847	0.525	0.621	0.806	0.801
Sense (0.95)	0.111	0.556	0.000	0.111	0.667	0.000	0.111	0.000	0.000
Wilcoxon P value	0.438	0.214	0.603	0.779	0.101	0.862	0.315	0.131	0.073
Ceruloplasmin
AUC	0.772	0.708	0.457	0.672	0.667	0.809	0.765	0.722	0.440
Sense (0.95)	0.444	0.444	0.111	0.444	0.556	0.556	0.556	0.000	0.000
Wilcoxon P value	0.811	0.336	0.728	0.168	0.413	0.041*	0.030*	0.270	0.620
Fibrinogen
AUC	0.733	0.725	0.844	0.777	0.556	0.756	0.493	0.722	0.531
Sense (0.95)	0.500	0.500	0.600	0.500	0.333	0.444	0.000	0.250	0.000
Wilcoxon P value	0.111	0.251	0.023*	0.020*	0.821	0.095	0.967	0.270	0.888
Haptoglobin
AUC	0.594	0.675	0.811	0.702	0.667	0.815	0.600	0.722	0.545
Sense (0.95)	0.300	0.500	0.600	0.300	0.333	0.667	0.000	0.250	0.000
Wilcoxon P value	0.496	0.375	0.038*	0.087*	0.413	0.041	0.364	0.270	0.800
T-cadherin
AUC	0.622	0.625	0.589	0.609	0.556	0.549	0.578	0.500	0.554
Sense (0.95)	0.200	0.300	0.400	0.300	0.222	0.111	0.000	0.250	0.000
Wilcoxon P value	0.402	0.535	0.548	0.347	0.821	0.794	0.507	0.940	0.932
Vit D
AUC	0.950	0.975	0.767	0.880	0.555	0.685	0.758	0.778	0.777
Sense (0.95)	0.800	0.900	0.400	0.400	0.222	0.222	0.222	0.000	0.000
Wilcoxon P value	0.005	0.021*	0.071	0.002*	0.821	0.233	0.037*	0.168	0.092
AUC denotes the area under ROC curve whereas Sense (0.95) denotes the sensitivity at 95% specificity of the ROC curve. The Wilcoxon sum-rank test with P value less than 0.05 was marked by an asterisk.

[0147] The fact that none of the single markers could detect AD, PD or DLB with 100% sensitivity at 95% specificity is expected, simply because all neurodegenerative diseases, including AD, PD, and DLB, are heterogeneous in nature, i.e. subgroups of patients may show different markers. Consequently, whether higher sensitivity could be achieved by combining individual markers was next investigated, and the results, shown in Table 5, appeared to show that this was indeed the case. Several conclusions can be drawn from the results presented in Table 5. First, the combination of two markers could achieve a higher sensitivity than a single marker alone. For instance, at 95% specificity, β-fibrinogen and VitD BD had 50% and 40% sensitivity, respectively, when tested alone in differentiating AD from controls and other diseases; but the sensitivity increased to 100% when the two markers were combined. Second, a better single marker does not necessarily mean it will perform better when the combination approach is taken. This can be illustrated in PD markers, where both ApoH (67%) and ceruloplasmin (56%) had better sensitivity than chromogranin B (11%) when tested alone; but when ApoH was combined with ceruloplasmin and chromogranin B, respectively, the sensitivity remained the same for ceruloplasmin, but improved to 78% for ApoH when combined with chromogranin B. In addition, both p values were now at or lower than 0.05 after two markers were combined, showing that chromogranin B, not ceruloplasmin, helps ApoH outperform ApoH alone. Furthermore, when VitD BP and ApoC were combined, the sensitivity for differentiating DLB from other diseases also increased to 50% at 95% specificity and with p value for VitD BP as 0.04 and for ApoC as 0.09. Last, but not least, no overt improvement was seen when a third maker was added to composite marker panel. Combination of more than three markers was not pursued to avoid the over-fitting problem in a data set that is underspecified.

[0148]

TABLE 5
Summaries for composite markers
				Sense	P-value for	P-value for
	Marker 1	Marker 2	AUC	(0.95)	marker 1	marker 2
AD versus all	Vit D BP	β-Fibrinogen	0.99	1.00	0.0635	0.0207
others	Ceruloplasmin	β-Fibrinogen	0.94	0.89	0.0142	0.0199
PD versus all	ApoH	Chromogranin B	0.92	0.78	0.0086	0.0560
others	Ceuloplasmijn	Chromogranin B	0.93	0.22	0.0052	0.0203
DLB versus all	ApoC1	Chromogranin B	0.92	0.50	0.0737	0.086
others	ApoC!	Vit D BP	0.86	0.50	0.0394	0.0890
AUC denotes the area under ROC curve whereas Sense (0.95) represents the sensitivity at 95% specificity of the ROC curve for CM of marker 1 and 2. P value for marker 1 represents the likelihood ratio P value from logistic regression for marker 1 given marker 2, whereas P value for marker 2 represents the P value of marker 2 given marker 1 in the logistic regression model.

[0149] Results on the performance of CM as well as ROC curves for both single and CM are also shown graphically in FIG. 3 and FIG. 4, where the joint behaviors of standardized markers among disease and control groups were displayed. As clearly shown in FIG. 3 and FIG. 4, the ability of CM to separate AD or PD from other diseases or healthy controls with higher sensitivity at high specificity was better when two dimensions instead of one was used. Similarly, both ROC curve plots show the improvement of sensitivity over all the ranges of specificity of CM compared to each individual marker. The statistical significance of the logistic regression proves that both markers are significant and important contributors to the resulting CM ROC curve.

[0150] Many candidate markers were discovered for AD patients in this study. As many groups, including us, have investigated AD CSF markers in the past with proteomic approaches(Zhang et al., J Alzheimers Dis, 2005. 7(2): p. 125-33; Puchades et al., Brain Res Mol Brain Res, 2003. 118(1-2): p. 140-6; Carrette et al., Proteomics, 2003. 3(8): p. 1486-94; Blennow et al., Expert Rev Mol Diagn, 2005. 5(5): p. 661-72; and Choe et al., Electrophoresis, 2002. 23(14): p. 2247-51), one would wonder how the current results compare with those reported in the literature. A fair comparison between the results with those of other groups is very hard. This is because there are many variables involved in proteomic studies, including difference in sample preparation, quality control of CSF samples (potential blood supplement in particular), patient population, proteomic platforms used, criteria used for protein identification, whether identified proteins have been confirmed or validated, and type of database used, which is a critical issue as demonstrated in this and previous studies (Xu et al., Rev. of Neurobiol, 2005). Nonetheless, all of the results generated by all platforms of proteomics were compiled in Table 7, demonstrating, as expected, that only a very small fraction of proteins change in the same direction, whether increase or decrease in AD vs. controls, among all or most studies. The increased proteins are albumin precursor, amyloid beta A4 protein precursor, α-1- antitrypsin precursor, ApoA-II precursor, complement C4 precursor, a hypothetical protein, β-2-microglobulin (isoforms) (with one exception), neuronal pentraxin I precursor, retinol binding protein (with one exception), and thioredoxin (except in the previous study where this protein was not quantified). There is only one consistently decreased protein, i.e. β-1,3-N-acetylglucosaminyltransferase bGnT-6, EWI2, when AD patients are compared to controls.

[0151]

TABLE 7
Comparison of AD CSF markers across all proteomic studies
			Previous	Peer	Current
No.	Name (IPI)	Common Name	study [13]	Literature	study
1	IPI00244477	Similar to fem-1 homolog a	↑↑	NI	NI
2	IPI00171473	Spondin 1, (f-spondin)	↑↑	NI	NC
		extracellular matrix protein
3	IPI00181232	Ca2+-dependent activator	↑↑	NI	NI
		protein for secretion 2
4	IPI00001508	Proinsulin precursor	↑↑	NI	NI
5	IPI00245370	Insulin-like growth factor	↑↑	NI	NI
		binding protein 2 (36 kD)
6	IPI00032220	Angiotensinogen precursor	↑↑	NI	NC
7	IPI00006608	Amyloid beta A4 protein	↑↑	NI	NC
	IPI00219182	precursor		NI	↑
	IPI00219189			NI	NI
8	IPI00233778	Complement component 1, r	↑↑	NI	NI
		subcomponent
9	IPI00300241	Hypothetical protein	↑↑	NI	↑↑
10	IPI00032258	Complement C4 precursor	↑↑	NI	↑
11	IPI00234495	Cathepsin B preproprotein*	↑↑	NI	NI
12	IPI00332161	Ig gamma-1 chain C region	↑↑	NI	NI
	IPI00328111	Factor VII active site mutant	↑↑	NI
		immunoconjugate
13	IPI00064607	MEGF10 protein*	↑↑	NI	NI
14	IPI00013299	Neuroblastoma, suppression of	↑↑	NI	NI
		tumorigenicity 1
15	IPI00333982	Hypothetical protein	↑↑	NI	NI
	IPI00168728	FLJ00385 protein	↑↑	NI	NI
16	IPI00219020	a. Splice isoform 1 of Q13748	↑↑	NI	NI
		Tubulin alpha-2 chain
	IPI00177441	b. Similar to Tubulin alpha-		NI	NI
		3/alpha-7 chain
	IPI00180675	c. Hypothetical protein		NI	NI
	IPI00179709			NI	NI
	IPI00183040			NI	NI
	IPI00166768
	IPI00216005	d. Tubulin alpha-8 chain	↑↑	NI	NI
	IPI00218345	e. Tubulin, alpha 2 isoform 2	↑↑	NI	NI
17	IPI00022371	Histidine-rich glycoprotein	↑↑	NI	NC
		precursor
18	IPI00220562	Neuronal pentraxin I precursor	↑↑	NI	↑
19	IPI00004656	a. Alpha-2-microglobulin	↑↑	NI	NI
		precursor
	IPI00182398	b. Hypothetic protein	↑↑	NI	NI
20	IPI00021854	Apolipoprotein A-II precursor #	↑↑	NI	↑↑
21	IPI00009997	Beta-1,3-N-	↓↓	NI	↓
		acetylglucosaminyltransferase
		bGnT-6
22	IPI00298853	Vitamin D-binding protein	↓↓	NI	NC
		precursor
23	IPI00257600	Cell adhesion molecule with	↓↓	NI	NI
		homology to L1CAM precursor
24	IPI00027425	Prion protein	↓↓	NI	NC
	IPI00022284
25	IPI00293592	Hypothetical protein	↓↓	NI	NI
		AF447587*
	IPI00168464	Hypothetical protein*	↓↓	NI	NI
26	IPI00056478	EWI2	↓↓	NI	↓
	IPI00186736	LIR-D1	↓↓	NI	NC
27	IPI00020984	Calnexin*	↓↓	NI	NI
28	IPI00232736	Similar to RIKEN cDNA	↓↓	NI	NI
		2410146L05*
29	IPI00027547	Dermcidin precursor	↓↓	NI	↑↑
30	IPI00025456	LJ00053 protein	↓↓	NI	NI
31	IPI00045498	a. Hypothetical protein,	↓↓	NI	NI
		JKTBP1delta6 ( )
	IPI00011274	b. Heterogeneous nuclear	↓↓	NI	NI
		ribonucleoprotein D-like
32	IPI00183616	a. Hypothetic protein	↓↓	NI	NI
	IPI00218719	b. Splice isoform 2 of P78527		NI	NI
		DNA-dependent protein kinase
		catalytic subunit
	IPI00233252	c. DNA-dependent protein		NI	NI
		kinase catalytic subunit
33	IPI00155723	a. Leukophysin	↓↓	NI	NI
	IPI00215638	b. DEAD/H (Asp-Glu-Ala-	↓↓	NI
		Asp/His) box polypeptide 9
		isoform 1
34	IPI00032292	Metalloproteinase inhibitor 1	↓↓	NI	NC
		precursor*
35	IPI00027381	Lymphocyte antigen 75	↓↓	NI	NI
		precursor
36	IPI00033086	a. Disks large-associated	↓↓	NI	NI
		protein 2*
	IPI00221115	b. Splice isoform 2 of Q9P1A6		NI	NI
		Disks large-associated protein 2
	IPI00221116	c. Splice isoform 3 of Q9P1A6		NI	NI
		Disks large-associated protein 2
37	IPI00170706	KIAA1412 protein*	↓↓	NI	NI
38	IPI00170548	PRO2000 protein*	↓↓	NI	NI
39	IPI00479805	ApoA4	NI	↓ [53]	↑↑
40		7.7 kDa unknown protein	NI	↑ [35]	NI
41	IPI00305457	Alpha-1-antitrypsin precursor	↑↑	↑ [34]	↑
42	IPI00216722	Alpha-1β glycoprotein	NC	↓ [34, 54]	NI
43	IPI00022431	Alpha-2-HS glycoprotein	↑	↓ [34]	NI
44	IPI00022434	Albumin precursor	↑	↓ [34]	↑↑
45	NCBI	ApoA1	Identified	↓ [34]	NI
	178775*
46	NCBI	ApoE	Identified	↓ [34, 53]	NI
	4557325;
	178848*
47	NCBI	ApoJ	Identified	↓ [34]	NI
	178855*
48	GenBank ID	Apolipoprotein H precursor	NI	↓ [54]	NI
	4557327*
49	*	Beta-amyloid (1-42)	NI	↓ [37]	NI
				ELISA
				↓ [55] non-
				protemics
50	NCBI	Cell cycle progression 8 protein	NI	↓ [34]	NI
	4758048*
51	GenBank ID	Chitinase 3-like 1	NI	↑ [54]	NI
	4557018*
52	GenBank ID	Cystatin C	NC	↑ [35, 54]	NI
	181387*
53	*	Kininogen precursor	Identified	↓ [34]	NI
54	*	Phospho-tau	NI	↑ [55] non-	NI
				proteomics
55	178775*	Proapolipoprotein	NI	↓ [53]	NI
56	NCBI 730305;	Protaglandin D2 synthase	NC	↓ [34] ↑	NI
	GenBank ID			[54]
	455962672*
57	IPI00479848	Retinol binding protein	↑	↓ [34] ↑	↑
				[53]
58	*	Tau	NI	↓ [37] WB	NI
				↑ [55] non-
				proteomics
59	IPI00216298	Thioredoxin	NI	↑ [54]	↑
60	NCBI	Transferrin precursor	↑	↓ [34]	NI
	4557871*
61	IPI00022432	Transthyretin	↑	↓ [34] ↑	NC
				[53]
62	IPI00383014	VGF protein	Identified	↓ [35]	↑
63	4699583*	Zn-a-2 glycoprotein	NI	↑ [53]	NI
64	IPI00004656	β-2-Microglobulin (isoforms)	↑↑	↑ [35, 53,	↑
				54]
				↓ [34]
↑↑: Increase (Ratio of AD vs. control >1.5)
↓↓: Decrease (Ratio of AD vs. control <0.67)
↑: Increase (Ratio of AD vs. control >1.2 but <1.5)
↓: Decrease (Ratio of AD vs. control >0.67 but <0.83)
NC: No change (Ratio of AD vs. control between 1.2~0.83)
NI: Not Identified.
*No IPI number as they are identified by others using different database.
#: Protein unique to AD as listed in FIGS. 5A-5YY.
Cited References:
[13] - Zhang et al., J Alzheimers Dis, 2005. 7(2): p. 125-33
[34] - Puchades et al., Brain Res Mol Brain Res, 2003. 118(1-2): p. 140-6
[35] - Carrette et al., Proteomics, 2003. 3(8): p. 1486-94
[37] - Choe et al., Electrophoresis, 2002. 23(14): p. 2247-51
[53] - Davidsson et al., Neuroreport, 2002. 13(5): p. 611-5
[54] - Hu et al., Mol Cell Proteomics, 2005.
[55] - Davidsson et al., Dis Markers, 2005. 21(2): p. 81-92
Multiple entries in a box without letter designation signify one protein with multiple names.
Multiple entries designated by letters indicate multiple possible protein candidates from the sequenced peptides, typically isoforms and precursors.
IPI: International Protein Index.

[0152] The concordance between the previous and current studies is higher, as in addition to the proteins mentioned above, the following proteins also demonstrate similar quantitative changes: afamin precursor, chemokine (C-X-C motif) ligand 16, β-galactosidase binding lectin precursor, GM2 activator precursor, ganglioside, α-2-macroglobulin precursor, and selenium-binding protein 1. Finally, 7 proteins displayed significant changes in the current study (FIGS. 5A-5YY) in the same direction, whether increased or decreased in AD vs. controls, as those listed in the previous publication where they have changes >20% but <50% alternations (Zhang et al., J Alzheimers Dis, 2005. 7(2): p. 125-33), and consequently are not listed in Table 7. These proteins are: dystroglycan precursor, haptoglobin, hemopexin precursor, insulin-like growth factor binding protein 2 precursor, ribonuclease 6 precursor, mimecan precursor, and tetranectin precursor.

[0153] What is most remarkable is that among all of the proteins with consistent changes in most experiments, only very few are unique to AD, i.e. most previous “candidate” markers were also changing more or less in the same direction in PD or DLB cases. Because it is not difficult for an experienced clinician to diagnose demented subjects from controls, the utility of these “non-unique” markers diminishes significantly. The only unique marker that has been consistently found in all studies is ApoA-II precursor. The other close possibility is haptoglobin, which is why this protein was selected for further confirmation even though it also displayed more than 20% changes in PD vs. controls. These results emphasize again that it is imperative to include other disease controls in addition to age-matched controls when the goal is to identify unique disease markers.

[0154] Candidate markers unique to AD fall into four major categories: immune/inflammation, transportation related proteins, e.g. ApoII-A, ApoC1 and ApoH, cellular metabolism, and neural transmission (FIGS. 5A-5YY). It is not practical to discuss each of the candidate proteins in detail; thus, the discussion will be focused on three confirmed markers demonstrating relatively high sensitivity, i.e. β-fibrinogen, haptoglobin, and VitD BP. β-Fibrinogen, best known for its role in coagulation and inflammation, has at least two isoforms, α and β, and it is not clear whether this protein is synthesized in the brain or transported via the blood brain barrier (BBB) (Strohmeyer et al., Brain Res Mol Brain Res, 2000. 81(1-2): p. 7-18). Nonetheless, it has been recognized for some time now that the activity of fibrinogen is increased in the plasma of AD patients (Gupta et al., Int J Clin Pract, 2005. 59(1): p. 52-7). The role of increased β-fibrinogen in AD CSF is not clear, but it can be at least hypothesized that it could potentially enhance microglial activation, a process implicated as one of the major mechanisms of cell death in PD (Shie et al., Am J Pathol, 2005. 166(4): p. 1163-72). On the other hand, an increase in haptoglobin in CSF has been associated with a subpopulation of AD patients, and it is initially (Pantoni et al., Acta Neurol Scand, 1995. 91(3): p. 225) thought to be due to an abnormal penetration of haptoglobin in AD patients secondary to compromised BBB (Alafuzoff et al., Acta Neuropathol (Berl), 1987. 73(2): p. 160-6; Tomimoto et al., Stroke, 1996. 27(11): p. 2069-74). Some studies have also associated with haptoglobin with increased risk in some AD patients, although contradictory results have also been reported (Matsuyama et al., Hum Hered, 1986. 36(2): p. 93-6). Nonetheless, several studies preformed with conventional methods also show that the level of haptoglobin increases in AD patients (Johnson et al., Appl Theor Electrophor, 1992. 3(2): p. 47-53). However, as demonstrated in this study, an increase in haptoglobin alone is not sufficient to differentiate AD from other neurological diseases. VitD BP has been classically associated with calcium metabolism and bone remodeling, although recently it has been noted that mRNA levels for this protein are decreased in the hippocampus in Alzheimer's patients (Sutherland et al., Brain Res Mol Brain Res, 1992. 13(3): p. 239-50). Nevertheless, like haptoglobin, the role of VitD BP in the pathogenesis of AD is largely unknown.

[0155] Prior to this study, very little was known about markers unique to PD and DLB. Three confirmed good candidate markers for PD are ceruloplasmin, chromogranin B and ApoH. Ceruloplasmin is an interesting protein because it has been implicated to play a central role in PD pathogenesis owing to two observations: 1) iron deposition in PD substantia nigra correlates with the severity of the disease (Hochstrasser et al., Neurology, 2004. 63(10): p. 1912-7); and 2) ceruloplasmin, an important protein for iron transportation, is decreased in the blood of PD patients (Torsdottir et al., Pharmacol Toxicol, 1999. 85(5): p. 239-43). Finally, It should be emphasized that this protein did not decrease in all PD patients in an earlier study (Loeffler et al., Alzheimer Dis Assoc Disord, 1994. 8(3): p. 190-7), consistent with the facts that this disease is heterogeneous in nature and that it is probably not sufficient by itself to detect PD patients with high sensitivity at high specificity. The influence of chromogranin B, a non-significant marker by itself in confirmation studies, on the overall performance of ApoH is also remarkable, as it significantly improved the sensitivity of ApoH to differentiate PD from controls as well as other diseases. Notably, a study performed years ago has suggested that although chromogranin B cannot differentiate AD or PD from controls by itself, the ratio between chromogranin A and B may be a correcting factor for neuropeptides seen in human CSF (Eder et al., J Neural Transm, 1998. 105(1): p. 39-51). The third marker that might be important in PD is ApoH, a protein clearly reported to be present in human CSF (Koch et al., J Lipid Res, 2001. 42(7): p. 1143-51), although its role in PD or in neurodegenerative diseases in general remains to be defined.

[0156] The markers unique to DLB also appeared to be related to two lipoproteins, i.e. ApoC1 and ApoH. Again, the role of ApoH in DLB or neurodegenerative disease in general is largely unknown, although given the fact that it is also increased in PD, one might argue its role in Lewy body disease. Very little is known about the role of ApoC in Lewy body disease, including PD and DLB, or dementia. However, several issues are worth commenting. First, one does not need to know the function of a protein in order for it to be a diagnostic tool; a good example of this type of use is the presence of oligoclonal bands in the CSF in the absence of identical bands in serum, which has been widely used clinically to aide the diagnosis of multiple sclerosis (McLean et al., Brain, 1990. 113(Pt 5): p. 1269-89). Second, all of these novel proteins should be studied further not only for their diagnostic use, but also for their roles in the pathogenesis of neurodegenerative diseases. Finally, given the limited DLB cases studied, caution needs to be excised with respect to the significance of these proteins.

[0157] Accordingly, the results show that examples of potential good combination of markers included β-fibrinogen plus Vit D BP or ceruloplasmin for AD, chromogranin B plus ceuloplamin or ApoH for PD and ApoC1 plus chormgranin B or VitD BP for DLB (Table 5).

[0158] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and not Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in angiotensinogen precursor and enolase 2, an increase in ADAM 10 precursor, an increase in Hect domain and RLD 4, and an increase in KIAA1291 protein. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) both angiotensinogen precursor and enolase 2 decrease in AD, do not change in PD, but increase in DLB; (2) ADAM 10 precursor increases in AD, but does not change in PD or DLB; and (3) Hect domain and RLD 4 increase in AD, decrease in PD, but do not change in DLB.

[0159]

Change in
Expression	Protein Name
↓	Angiotensinogen precursor
↑	ADAM 10 precursor
↓	Enolase 2
↑	Hect domain and RLD 4
↑	KIAA1291 protein

[0160] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and not Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in IL-17RC and PLXDC2 protein, an increase in Golgi phosphoprotein 2, an increase in spondin 1 precursor, and an increase in ZNF627 protein. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) Golgi phosphoprotein 2 increases in AD, does not change in PD, but decreases in DLB; (2) IL-17RC decreases in AD but does not change in PD or DLB; (3) PLXDC2 protein decreases in AD, does not change in PD but increases in DLB; and (4) spondin 1 precursor and ZNF627 protein increase in AD, but do not change in PD or DLB.

[0161]

Change in
Expression	Protein Name
↑	Golgi phosphoprotein 2
↓	IL-17RC
↓	PLXDC2 protein
↑	Spondin 1 precursor
↑	ZNF627 protein

[0162] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and not Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in chromogranin A and divalent cation tolerant protein CUTA, an increase in apolipoprotein D, an increase in haptoglobin precursor, and an increase in reticulocalbin 2 precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) apolipoprotein D precursor and reticulocalbin 2 precursor increase in AD but do not change in PD or DLB; (2) chromogranin A decreases in AD but does not change in PD or DLB; (3) divalent cation tolerant protein CUTA decreases in AD, does not change in PD, but increases in DLB; and (4) haptoglobin precursor increases in AD and PD, but does not change in DLB.

[0163]

Change in
Expression	Protein Name
↑	Apolipoprotein D precursor
↓	Chromogranin A
↓	Divalent cation tolerant protein CUTA
↑	Haptoglobin precursor
↑	Reticulocalbin 2 precursor

[0164] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and not Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in secretogranin I precursor, a decrease in splice isoform 2 of insulin receptor, an increase in AMBP protein precursor, an increase in kallikrein 6 precursor, and an increase in TRIF-related adapter molecular. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) AMBP protein precursor and TRIF-related adapter molecule increase in AD but do not change in PD or DLB; (2) kallikrein 6 precursor increases in AD, decreases in PD, but does not change in DLB; and (3) secretogranin I precursor and splice isoform 2 of insulin receptor precursor decrease in AD but do not change in PD or DLB.

[0165]

Change in
Expression	Protein Name
↑	AMBP protein precursor
↑	Kallikrein 6 precursor
↓	Secretogranin I precursor
↓	Splice isoform 2 of insulin receptor precursor
↑	TRIF-related adapter molecule

[0166] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and not Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in brain-derived neurotrophic factor BDNF1, a decrease in interleukin-1 receptor-associated kinase-like 2, a decrease in neurexin 1-alpha precursor, and an increase in alpha 1 type XIII collagen isoform 3. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) alpha 1 type XIII collagen isoform 3 increases in AD, decreases in PD but does not change in DLB; (2) brain-derived neurotrophic factor BDNF1 and interleukin-1 receptor-associated kinase-like 2 decrease in AD but do not change in PD or DLB; (3) latent transforming growth factor beta binding protein 2 increases in AD, does not change in PD, but decreases in DLB; and (4) neurexin 1-alpha precursor decreases in AD, increases in PD but does not change in DLB.

[0167]

Change in
Expression	Protein Name
↑	Alpha 1 type XIII collagen isoform 3
↓	Brain-derived neurotrophic factor BDNF1
↓	Interleukin-1 receptor-associated kinase-like 2
↑	Latent transforming growth factor beta binding protein 2
↓	Neurexin 1-alpha precursor

[0168] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and not Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in cadherin-13 precursor, an increase in ADAM 10 precursor, an increase in fibrinogen beta chain precursor, an increase in HLA class I histocompatibility antigen, an increase in E alpha chain precursor and an increase in Zinc finger protein 95. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) ADAM 10 precursor, fibrinogen beta chain precursor, HLA class I histocompatibility antigen, E alpha chain precursor and zinc finger protein 95 homolog increase in AD but do not change in PD or DLB; and (2) cadherin-13 precursor decreases in AD but does not change in PD or DLB.

[0169]

Change in
Expression	Protein Name
↑	ADAM 10 precursor
↓	Cadherin-13 precursor
↑	Fibrinogen beta chain precursor
↑	HLA class I histocompatibility antigen,
	E alpha chain precursor
↑	Zinc finger protein 95 homolog

[0170] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and not Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in PPIB protein, a decrease in prostatic binding protein, a decrease in SAYY8238, a decrease in transcriptional activator SRCAP, and an increase in inter-alpha-trypsin inhibitor heavy chain HI precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) inter-alpha-trypsin inhibitor heavy chain HI precursor increases in AD but does not change in PD or DLB; and (2) PPIB protein, prostatic binding protein, SAYY8238, and transcriptional activator SRCAP decrease in AD but do not change in PD or DLB.

[0171]

Change in
Expression	Protein Name
↑	Inter-alpha-trypsin inhibitor heavy chain H1 precursor
↓	PPIB protein
↓	Prostatic binding protein
↓	SAYY8238
↓	Transcriptional activator SRCAP

[0172] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and not Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in cell growth regulator with EF hand domain 1, a decrease in metallothionein-III, a decrease in neuronal pentraxin receptor, an increase in heat shock 10 kDa protein 1 (chaperonin 10), and an increase in integral membrane protein 2B. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) cell growth regulator with EF hand domain 1 decreases in AD, increases in PD, but does not change in DLB; (2) heat shock 10 kDa protein 1 and integral membrane protein 2B increase in AD but do not change in PD or DLB; and (3) metallothionein-III and neuronal pentraxin receptor decrease in AD but do not change in PD or DLB.

[0173]

Change in
Expression	Protein Name
↓	Cell growth regulator with EF hand domain 1
↑	Heat shock 10 kDa protein 1 (chaperonin 10)
↑	Integral membrane protein 2B
↓	Metallothionein-III
↓	Neuronal pentraxin receptor

[0174] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and not Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in apolipoprotein E precursor, a decrease in glucosidase II beta subunit precursor, a decrease in neural proliferation differentiation, a decrease in control protein-1 precursor, an increase in cytokine-like protein C17 precursor, and an increase in voltage-dependent calcium channel gamma-6 subunit. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) apolipoprotein E precursor, glucosidase II beta subunit precursor, neural proliferation differentiation and control protein-1 precursor decrease in AD, but do not change in PD or DLB; and (2) cytokine-like protein C17 precursor and voltage-dependent calcium channel gamma-6 subunit increase in AD, but do not change in PD or DLB.

[0175]

Change in
Expression	Protein Name
↓	Apolipoprotein E precursor
↑	Cytokine-like protein C17 precursor
↓	Glucosidase II beta subunit precursor
↓	Neural proliferation differentiation and control
	protein-1 precursor
↑	Voltage-dependent calcium channel gamma-6 subunit

[0176] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and not Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in inhibin beta A chain precursor, a decrease in neurofascin isoform 2, a decrease in receptor-type tyrosine-protein phosphatase-like N precursor, an increase in antigen MLAA-20, an increase in HLA class I histocompatibility antigen, and an increase in B-27 alpha chain precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) antigen MLAA-20, HLA class I histocompatibility antigen and B-27 alpha chain precursor increase in AD but do not change in PD or DLB; and (2) inhibin beta A chain precursor, neurofascin isoform 2 and receptor-type tyrosine-protein phosphatase-like N precursor decrease in AD but do not change in PD or DLB.

[0177]

Change in
Expression	Protein Name
↑	Antigen MLAA-20
↑	HLA class I histocompatibility antigen, B-27 alpha chain
	precursor
↓	Inhibin beta A chain precursor
↓	Neurofascin isoform 2
↓	Receptor-type tyrosine-protein phosphatase-like N precursor

[0178] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and not Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in chromogranin B, a decrease in neuronal pentraxin I precursor, an increase in alpha-1-acid glycoprotein1 precursor, an increase in KARCA1 protein, and an increase in sortilin 1 preprotein. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) alpha-1-acid glycoprotein 1 precursor increases in AD, decreases in PD, but does not change in DLB; (2) chromogranin B decreases in AD, increases in PD, but does not change in DLB; (3) KARACA1, and sortilin I preprotein increase in AD, do not change in PD, but decrease in DLB; and (4) neuronal pentraxin 1 precursor decreases in AD, does not change in PD, but increases in DLB.

[0179]

Change in
Expression	Protein Name
↑	Alpha-1-acid glycoprotein 1 precursor
↓	Chromogranin B
↑	KARCA1 protein
↓	Neuronal pentraxin I precursor
↑	Sortilin 1, preproprotein

[0180] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and not Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in G protein-coupled sphingolipid receptor, a decrease in superoxide dismutase 1, soluble, an increase in apolipoprotein H, an increase in mosaic serine protease, and an increase in myosin-reactive immunoglobulin heavy chain variable region. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) apolipoprotein H increases in AD, decreases in PD, but does not change in DLB; (2) G protein-coupled sphingolipid receptor decreases in AD, but does not change in PD or DLB; (3) mosaic serine protease and myosin-reactive immunoglobulin heavy chain variable region increase in AD, but do not change in PD or DLB; and (4) superoxide dismutase 1, soluble decreases in AD, does not change in PD, but increases in DLB.

[0181]

Change in
Expression	Protein Name
↑	Apolipoprotein H
↓	G protein-coupled sphingolipid receptor
↑	Mosaic serine protease
↑	Myosin-reactive immunoglobulin heavy chain variable
	region
↓	Superoxide dismutase 1, soluble

[0182] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and not Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in vacuolar ATP synthase subunit S1 precursor, an increase in apolipoprotein C-1 precursor, an increase in matrix Gla-protein precursor, an increase in SAA1 protein, and an increase in splice isoform 2 of insulin-like growth factor II precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) apolipoprotein C-1 precursor and SAA1 protein increase in AD, decrease in PD, but do not change in DLB; (2) matrix Gla-protein precursor and splice isoform 2 of insulin-like growth factor II precursor increase in AD, but do not change in PD or DLB; and (3) vacuolar ATP synthase subunit S1 precursor decreases in AD, but does not change in PD or DLB.

[0183]

Change in
Expression	Protein Name
↑	Apolipoprotein C-1 precursor
↑	Matrix Gla-protein precursor
↑	SAA1 protein
↑	Splice isoform 2 of insulin-like growth factor II precursor
↓	Vacuolar ATP synthase subunit S1 precursor

[0184] A patient's CSF providing the following protein expression pattern is diagnosed as having Parkinson's disease (PD) and not Alzheimer's disease (AD) or dementia with Lewy body (DLB): a decrease in amyloid-like protein 1 precursor, a decrease in extracellular matrix protein 1, a decrease in HRPE773, a decrease in selenoprotein M precursor, and an increase in Golgi autoantigen, golgin subfamily B member 1. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) amyloid-like protein 1 precursor and extracellular matrix protein 1 decrease in PD, but do not change in AD or DLB; (2) Golgi autoantigen, golgin subfamily B member 1 increases in PD, decreases in AD, but does not change in DLB; (3) HRPE773 decreases in PD, does not change in AD, but increases in DLB; and (4) selenoprotein M precursor decreases in PD but does not change in AD or DLB.

[0185]

Change in
Expression	Protein Name
↓	Amyloid-like protein 1 precursor
↓	Extracellular matrix protein 1
↑	Golgi autoantigen, golgin subfamily B member 1
↓	HRPE773
↓	Selenoprotein M precursor

[0186] A patient's CSF providing the following protein expression pattern is diagnosed as having Parkinson's disease (PD) and not Alzheimer's disease (AD) or dementia with Lewy body (DLB): a decrease in alpha 1 type XIII collagen isoform 3, a decrease in prothrombin precursor, a decrease in retinol binding protein 4, a decrease in plasma and vitamin D-binding protein precursor, and an increase in putative 4 repeat voltage-gated ion channel. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) alpha 1 type XIII collagen isoform 3 decreases in PD, increases in AD, but does not change in DLB; (2) prothrombin precursor, retinol binding protein 4, plasma and vitamin D-binding protein precursor decrease in PD but do not change in AD or DLB; and (3) putative 4 repeat voltage-gated ion channel increases in PD, decreases in AD, but does not change in DLB.

[0187]

Change in
Expression	Protein Name
↓	Alpha 1 type XIII collagen isoform 3
↓	Prothrombin precursor
↑	Putative 4 repeat voltage-gated ion channel
↓	Retinol binding protein 4, plasma
↓	Vitamin D-binding protein precursor

[0188] A patient's CSF providing the following protein expression pattern is diagnosed as having Parkinson's disease (PD) and not Alzheimer's disease (AD) or dementia with Lewy body (DLB): a decrease in cochlin precursor, a decrease in cystatin C precursor, a decrease in KRT8 protein, a decrease in metabotropic glutamate receptor 3 precursor, and an increase in neurexin 1-alpha precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) cochlin precursor decreases in PD, increases in AD, but does not change in DLB; (2) cystatin C precursor and KRT8 protein decrease in PD, but do not change in AD or DLB; (3) metabotropic glutamate receptor 3 precursor decreases in PD, does not change in AD, but increases in DLB; and (4) neurexin 1-alpha precursor increases in PD, decreases in AD, but does not change in DLB.

[0189]

Change in
Expression	Protein Name
↓	Cochlin precursor
↓	Cystatin C precursor
↓	KRT8 protein
↓	Metabotropic glutamate receptor 3 precursor
↑	Neurexin 1-alpha precursor

[0190] A patient's CSF providing the following protein expression pattern is diagnosed as having Parkinson's disease (PD) and not Alzheimer's disease (AD) or dementia with Lewy body (DLB): a decrease in alpha-1-acid glycoprotein 1 precursor, a decrease in Hook homolog 3, a decrease in Kallikrein 6 precursor, an increase in ATP-binding cassette, an increase in sub-family A, member 1 and an increase in pyruvate kinase 3 isoform 2. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) alpha-1-acid glycoprotein 1 precursor and Kallikrein 6 precursor decrease in PD, increase in AD, but do not change in DLB; (2) ATP-binding cassette, sub-family A, member 1 and pyruvate kinase 3 isoform 2 increase in PD, but do not change in AD or DLB; and (3) Hook homolog 3 decreases in PD, but does not change in AD or DLB.

[0191]

Change in
Expression	Protein Name
↓	Alpha-1-acid glycoprotein 1 precursor
↑	ATP-binding cassette, sub-family A, member 1
↓	Hook homolog 3
↓	Kallikrein 6 precursor
↑	Pyruvate kinase 3 isoform 2

[0192] A patient's CSF providing the following protein expression pattern is diagnosed as having Parkinson's disease (PD) and not Alzheimer's disease (AD) or dementia with Lewy body (DLB): a decrease in ceruloplasmin precursor, a decrease in heparin-binding EGF-like growth factor precursor, a decrease in SAA1 protein, an increase in cell growth regulator with EF hand domain 1 and an increase in selenium binding protein 1. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) cell growth regulator with EF hand domain 1 increases in PD, decreases in AD, but does not change in DLB; (2) ceruloplasmin precursor and heparin-binding EGF-like growth factor precursor decrease in PD, but do not change in AD or DLB; (3) SAA1 protein decreases in PD, increases in AD, but does not change in DLB; and (4) selenium binding protein 1 increases in PD but does not change in AD or DLB.

[0193]

Change in
Expression	Protein Name
↑	Cell growth regulator with EF hand domain 1
↓	Ceruloplasmin precursor
↓	Heparin-binding EGF-like growth factor precursor
↓	SAA1 protein
↑	Selenium binding protein 1

[0194] A patient's CSF providing the following protein expression pattern is diagnosed as having Parkinson's disease (PD) and not Alzheimer's disease (AD) or dementia with Lewy body (DLB): a decrease in apolipoprotein A-II precursor, a decrease in mammalian ependymin related protein 1, a decrease in splice isoform 1 of lysosomal trafficking regulator, a decrease in splice isoform 2 of integrin alpha-7 precursor, and an increase in C-type natriuretic peptide precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) apolipoprotein A-II precursor decreases in PD, increases in AD, but does not change in DLB; (2) C-type natriuretic peptide precursor increases in PD, but does not change in AD or DLB; (3) mammalian ependymin related protein 1 decreases in PD, does not change in AD, but increases in DLB; and (4) splice isoform 1 of lysosomal trafficking regulator and splice isoform 2 of integrin alpha-7 precursor decrease in PD, but do not change in AD or DLB.

[0195]

Change in
Expression	Protein Name
↓	Apolipoprotein A-II precursor
↑	C-type natriuretic peptide precursor
↓	Mammalian ependymin related protein 1
↓	Splice isoform 1 of lysosomal trafficking regulator
↓	Splice Isoform 2 of integrin alpha-7 precursor

[0196] A patient's CSF providing the following protein expression pattern is diagnosed as having Parkinson's disease (PD) and not Alzheimer's disease (AD) or dementia with Lewy body (DLB): a decrease in apolipoprotein C-I precursor, a decrease in insulin-like growth factor binding protein 5 precursor, a decrease in splice isoform 1 of transcription factor E2-alpha, an increase in ribonuclease 4 precursor and an increase in splice isoform 1 of basigin precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) apolipoprotein C-I precursor and insulin-like growth factor binding protein 5 precursor decrease in PD, increase in AD, but do not change in PD; (2) ribonuclease 4 precursor increases in PD, but does not change in AD or DLB; (3) splice isoform 1 of basigin precursor increases in PD, does not change in AD, but decreases in DLB; and (4) splice isoform 1 of transcription factor E2-alpha decreases in PD, does not change in AD, but increases in DLB.

[0197]

Change in
Expression	Protein Name
↓	Apolipoprotein C-I precursor
↓	Insulin-like growth factor binding protein 5 precursor
↑	Ribonuclease 4 precursor
↑	Splice isoform 1 of basigin precursor
↓	Splice isoform 1 of transcription factor E2-alpha

[0198] A patient's CSF providing the following protein expression pattern is diagnosed as having Parkinson's disease (PD) and not Alzheimer's disease (AD) or dementia with Lewy body (DLB): a decrease in activating receptor pilrbeta, a decrease in apolipoprotein M, a decrease in polymeric-immunoglobulin receptor precursor, an increase in CD99L2 protein and an increase in chromogranin B. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) activating receptor pilrbeta decreases in PD, does not change in AD, but increases in DLB; (2) apolipoprotein M decreases in PD, but does not change in AD or DLB; (3) CD99L2 protein increases in PD, does not change in AD, but decreases in DLB; (4) chromogranin B increases in PD, decreases in AD, but does not change in DLB; and (5) polymeric-immunoglobulin receptor precursor decreases in PD, does not change in AD, but increases in DLB.

[0199]

Change in
Expression	Protein Name
↓	Activating receptor pilrbeta
↓	Apolipoprotein M
↑	CD99L2 protein
↑	Chromogranin B
↓	Polymeric-immunoglobulin receptor precursor

[0200] A patient's CSF providing the following protein expression pattern is diagnosed as having Parkinson's disease (PD) and not Alzheimer's disease (AD) or dementia with Lewy body (DLB): a decrease in KIAA1265 protein, a decrease in ribosomal protein L3-like, an increase in laminin gamma-1 chain precursor, an increase in prion protein, and an increase in protein tyrosine phosphatase, non-receptor type substrate 1 precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) KIAA1265 protein decreases in PD, does not change in AD, but increases in DLB; (2) laminin gamma-1 chain precursor and protein tyrosine phosphatase, non-receptor type substrate 1 precursor increase in PD, do not change in AD, but decrease in DLB; (3) prion protein increases in PD, but does not change in AD or DLB; and (4) ribosomal protein L3-like decreases in PD, but does not change in AD or DLB.

[0201]

Change in
Expression	Protein Name
↓	KIAA1265 protein
↑	Laminin gamma-1 chain precursor
↑	Prion protein
↑	Protein tyrosine phosphatase, non-receptor type substrate 1
	precursor
↓	Ribosomal protein L3-like

[0202] A patient's CSF providing the following protein expression pattern is diagnosed as having Parkinson's disease (PD) and not Alzheimer's disease (AD) or dementia with Lewy body (DLB): a decrease in apolipoprotein H, a decrease in DJ977L11.1, a decrease in serine/threonine-protein kinase PLK2, an increase in Ig kappa chain V-I region HK102 precursor and an increase in Rho-GTPase activating protein 10. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) apolipoprotein H and DJ977L11.1 decrease in PD, increase in AD, but do not change in DLB; (2) Ig kappa chain V-I region HK102 precursor and serine/threonine-protein kinase PLK2 increase in PD, but do not change in AD or DLB; and (3) Rho-GTPase activating protein 10 increases in PD, does not change in AD, but does decrease in DLB.

[0203]

Change in
Expression	Protein Name
↓	Apolipoprotein H
↓	DJ977L11.1
↑	Ig kappa chain V-I region HK102 precursor
↑	Rho-GTPase activating protein 10
↓	Serine/threonine-protein kinase PLK2

[0204] A patient's CSF providing the following protein expression pattern is diagnosed as having dementia with Lewy body (DLB) and not Alzheimer's disease (AD) or Parkinson's disease (PD): a decrease in Rho-GTPase activating protein 10, a decrease in somatostatin precursor, a decrease in sortilin 1, preprotein, an increase in angiotensinogen precursor and an increase in myosin. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) angiotensinogen precursor and myosin increase in DLB, but do not change in AD or PD; (2) Rho-GTPase activating protein 10 decreases in DLB, does not change in AD, but increases in PD; (3) somatostatin precursor decreases in DLB, but does not change in AD or PD; and (4) sortilin 1, preprotein decreases in DLB, increases in AD, but does not change in PD.

[0205]

Change in
Expression	Protein Name
↑	Angiotensinogen precursor
↑	Myosin
↓	Rho-GTPase activating protein 10
↓	Somatostatin precursor
↓	Sortilin 1, preproprotein

[0206] A patient's CSF providing the following protein expression pattern is diagnosed as having dementia with Lewy body (DLB) and not Alzheimer's disease (AD) or Parkinson's disease (PD): a decrease in coagulation factor V, a decrease in SCMH1 protein, an increase in KIAA1265 protein, an increase in neuronal pentraxin I precursor and an increase in profilin 2 isoform. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) coagulation factor V and SCMH1 protein decrease in DLB, but do not change in AD or PD; (2) KIAA1265 protein increases in DLB, does not change in AD, but decreases in PD; (3) neuronal pentraxin I precursor increases in DLB, decreases in AD, but does not change in PD; and (4) profilin 2 isoform increases in DLB, but does not change in AD or PD.

[0207]

Change in
Expression	Protein Name
↓	Coagulation factor V
↑	KIAA1265 protein
↑	Neuronal pentraxin I precursor
↑	Profilin 2 isoform
↓	SCMH1 protein

[0208] A patient's CSF providing the following protein expression pattern is diagnosed as having dementia with Lewy body (DLB) and not Alzheimer's disease (AD) or Parkinson's disease (PD): a decrease in neurexophilin 4, a decrease in neuronal pentraxin receptor isoform 1, an increase in enolase 2, an increase in N-acetyllactosaminide beta-1,3,-N-acetylglucosaminyltransferase and an increase in parvalbumin. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) enolase 2 increases in DLB, decreases in AD, but does not change in PD; (2) N-acetyllactosaminide beta-1,3,-N-acetylglucosaminyltransferase and parvalbumin increase in DLB, but do not change in AD or PD; and (3) neurexophilin 4 and neuronal pentraxin receptor isoform 1 decrease in DLB, but do not change in AD or PD.

[0209]

Change in
Expression	Protein Name
↑	Enolase 2
↑	N-acetyllactosaminide
	beta-1,3-N-acetylglucosaminyltransferase
↓	Neurexophilin 4
↓	Neuronal pentraxin receptor isoform 1
↑	Parvalbumin

[0210] A patient's CSF providing the following protein expression pattern is diagnosed as having dementia with Lewy body (DLB) and not Alzheimer's disease (AD) or Parkinson's disease (PD): a decrease in Fas apoptotic inhibitory molecule 2, a decrease in protein tyrosine phosphatase, non-receptor type substrate 1 precursor, an increase in brain abundant, membrane attached signal protein 1, an increase in divalent cation tolerant protein CUTA, and an increase in superoxide dismutase 1, soluble. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) brain abundant, membrane attached signal protein 1, divalent cation tolerant protein CUTA and superoxide dismutase 1, soluble increase in DLB, decrease in AD, but do not change in PD; (2) Fas apoptotic inhibitory molecule 2 decreases in DLB, but does not change in AD or PD; and (3) protein tyrosine phosphatase, non-receptor type substrate 1 precursor decreases in DLB, does not change in AD, but increases in PD.

[0211]

Change in
Expression	Protein Name
↑	Brain abundant, membrane attached signal protein 1
↑	Divalent cation tolerant protein CUTA
↓	Fas apoptotic inhibitory molecule 2
↓	Protein tyrosine phosphatase, non-receptor type substrate 1
	precursor
↑	Superoxide dismutase 1, soluble

[0212] A patient's CSF providing the following protein expression pattern is diagnosed as having dementia with Lewy body (DLB) and not Alzheimer's disease (AD) or Parkinson's disease (PD): a decrease in latent transforming growth factor beta binding protein 2, a decrease in T-cadherin, a decrease in transcription elongation regulator 1, an increase in apolipoprotein C-III precursor and an increase in lysozyme C precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) apolipoprotein C-III precursor increases in DLB, does not change in AD, but decreases in PD; (2) latent transforming growth factor beta binding protein 2 and transcription elongation regulator 1 decrease in DLB, increase in AD, but do not change in PD; (3) lysozyme C precursor increases in DLB, but does not change in AD or PD; and (4) T-cadherin decreases in DLB, but does not change in AD or PD.

[0213]

Change in
Expression	Protein Name
↑	Apolipoprotein C-III precursor
↓	Latent transforming growth factor beta binding protein 2
↑	Lysozyme C precursor
↓	T-Cadherin
↓	Transcription elongation regulator 1

[0214] A patient's CSF providing the following protein expression pattern is diagnosed as having dementia with Lewy body (DLB) and not Alzheimer's disease (AD) or Parkinson's disease (PD): a decrease in apolipoprotein C1, a decrease in dermatopontin precursor, a decrease in proenkephalin A precursor, a decrease in Rho-associated protein kinase 1 and an increase in tetranectin precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) apolipoprotein C1, dermatopontin precursor, proenkephalin A precursor and Rho-associated protein kinase 1 decrease in DLB, but do not change in AD or PD; and (2) tetranectin precursor increases in DLB, decreases in AD, but does not change in PD.

[0215]

Change in
Expression	Protein Name
↓	Apolipoprotein C1
↓	Dermatopontin precursor
↓	Proenkephalin A precursor
↓	Rho-associated protein kinase 1
↑	Tetranectin precursor

[0216] A patient's CSF providing the following protein expression pattern is diagnosed as having dementia with Lewy body (DLB) and not Alzheimer's disease (AD) or Parkinson's disease (PD): a decrease in splice isoform 1 of basigin precursor, a decrease in splice isoform 2 of glutaryl-CoA dehydrogenase, mitochondrial precursor, a decrease in splice isoform 2 of sodium/potassium/calcium exchanger 2 precursor, an increase in metabotropic glutamate receptor 3 precursor and an increase in polymeric-immunoglobulin receptor precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) metabotropic glutamate receptor 3 precursor and polymeric-immunoglobulin receptor precursor increase in DLB, do not change in AD, but decrease in PD; (2) splice isoform 1 of basigin precursor decrease in DLB, do not change in AD, but increase in PD; and (3) splice isoform 2 of glutaryl-CoA dehydrogenase, mitochondrial precursor and splice isoform 2 of sodium/potassium/calcium exchanger 2 precursor decrease in DLB, but do not change in AD or PD.

[0217]

Change in
Expression	Protein Name
↑	Metabotropic glutamate receptor 3 precursor
↑	Polymeric-immunoglobulin receptor precursor
↓	Splice isoform 1 of basigin precursor
↓	Splice isoform 2 of glutaryl-CoA dehydrogenase,
	mitochondrial precursor
↓	Splice isoform 2 of sodium/potassium/calcium exchanger 2
	precursor

[0218] A patient's CSF providing the following protein expression pattern is diagnosed as having dementia with Lewy body (DLB) and not Alzheimer's disease (AD) or Parkinson's disease (PD): a decrease in 2′-phophodiesterase, a decrease in laminin gamma-1 chain precursor, a decrease in splice isoform 3 of reelin precursor, an increase in apolipoprotein C-II precursor and an increase in splice isoform 3 of integrin alpha-7 precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) 2′-phosphodiesterase and splice isoform 3 of reelin precursor decrease in DLB, but do not change in AD or PD; (2) apolipoprotein C-II precursor and splice isoform 3 of integrin alpha-7 precursor increase in DLB, but do not change in AD or PD; and (3) laminin gamma-1 chain precursor decreases in DLB, does not change in AD, but increases in PD.

[0219]

Change in
Expression	Protein Name
↓	2′-phosphodiesterase
↑	Apolipoprotein C-II precursor
↓	Laminin gamma-1 chain precursor
↑	Splice isoform 3 of integrin alpha-7 precursor
↓	Splice isoform 3 of reelin precursor

[0220] A patient's CSF providing the following protein expression pattern is diagnosed as having dementia with Lewy body (DLB) and not Alzheimer's disease (AD) or Parkinson's disease (PD): a decrease in MGAT3 protein, a decrease in sulfatase 2 isoform b precursor, an increase in activating receptor pilrbeta, an increase in nucleobindin 1 precursor and an increase in selenoprotein P precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) activating receptor pilrbeta increases in DLB, does not change in AD, but decreases in PD; (2) MGAT3 protein and sulfatase 2 isoform b precursor decrease in DLB, but do not change in AD or PD; and (3) nucleobindin 1 precursor and selenoprotein P precursor increase in DLB, but do not change in AD or PD.

[0221]

Change in
Expression	Protein Name
↑	Activating receptor pilrbeta
↓	MGAT3 protein
↑	Nucleobindin 1 precursor
↑	Selenoprotein P precursor
↓	Sulfatase 2 isoform b precursor

[0222] A patient's CSF providing the following protein expression pattern is diagnosed as having dementia with Lewy body (DLB) and not Alzheimer's disease (AD) or Parkinson's disease (PD): a decrease in protein C20 orf 98, a decrease in SH3-domain GRB2-like 1, an increase in hemopexin precursor, an increase in latent transforming growth factor-beta-binding protein 2 precursor and an increase in transthyretin precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) hemopexin precursor, latent transforming growth factor-beta-binding protein 2 precursor and transthyretin precursor increase in DLB, but do not change in AD or PD; and (2) protein C20 orf98 and SH3-domain GRB2-like 1 decrease in DLB, but do not change in AD or PD.

[0223]

Change in
Expression	Protein Name
↑	Hemopexin precursor
↑	Latent transforming growth factor-beta-binding protein 2
	precursor
↓	Protein C20 orf98
↓	SH3-domain GRB2-like 1
↑	Transthyretin precursor

[0224] A patient's CSF providing the following protein expression pattern is diagnosed as having dementia with Lewy body (DLB) and not Alzheimer's disease (AD) or Parkinson's disease (PD): a decrease in Kelch/ankyrin repeat containing cyclin A1 interacting protein, a decrease in splice isoform 1 of SWI/SNF-related, matrix associated, actin-dependent regulator, a decrease in splice isoform 2 of metabotropic glutamate receptor 8 precursor, an increase in neuroendocrine convertase 2 precursor and an increase in sortilin-related receptor precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) Kelch/ankyrin repeat containing cyclin A1 interacting protein and splice isoform 1 of SWI/SNF-related, matrix associated, actin-dependent regulator decrease in DLB, increase in AD, but do not change in PD; (2) neuroendocrine convertase 2 precursor and sortilin-related receptor precursor increase in DLB, do not change in AD or PD; and (3) splice isoform 2 of metabotropic glutamate receptor 8 precursor decreases in DLB, but does not change in AD or PD.

[0225]

Change in
Expression	Protein Name
↓	Kelch/ankyrin repeat containing cyclin A1 interacting
	protein
↑	Neuroendocrine convertase 2 precursor
↑	Sortilin-related receptor precursor
↓	Splice isoform 1 of SWI/SNF-related, matrix associated,
	actin-dependent regulator
↓	Splice isoform 2 of metabotropic glutamate receptor 8
	precursor

[0226] A patient's CSF providing the following protein expression pattern is diagnosed as having dementia with Lewy body (DLB) and not Alzheimer's disease (AD) or Parkinson's disease (PD): a decrease in neural cell adhesion molecule 1, 140 kDa isoform precursor, a decrease in splice isoform 2 of collagen alpha 2(VI) chain precursor, an increase in DNA-directed RNA polymerase I largest subunit, an increase in latent transforming growth factor-beta binding protein 4 and an increase in protein FAM3C precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) DNA-directed RNA polymerase I largest subunit increases in DLB, decreases in AD, but does not change in PD; (2) latent transforming growth factor-beta binding protein 4 and protein FAM3C precursor increase in DLB, but do not change in AD or PD; and (3) neural cell adhesion molecule 1, 140 kDa isoform precursor and splice isoform 2 of collagen alpha 2(VI) chain precursor decrease in DLB, but do not change in AD or PD.

[0227]

Change in
Expression	Protein Name
↑	DNA-directed RNA polymerase I largest subunit
↑	Latent transforming growth factor-beta binding protein 4
↓	Neural cell adhesion molecule 1, 140 kDa isoform
	precursor
↑	Protein FAM3C precursor
↓	Splice isoform 2 of collagen alpha 2(VI) chain precursor

[0228] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and/or Parkinson's disease (PD) and not dementia with Lewy body (DLB): a decrease in apolipoprotein A-II precursor, a decrease in cochlin precursor, a decrease in serine/threonine-protein kinase PLK2, a decrease in splice isoform 3 of integrin alpha-7 precursor, and an increase in hepatocellular carcinoma associated protein TB6. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) apolipoprotein A-II precursor and cochlin precursor increase in AD, decrease in PD, but do not change in DLB; (2) hepatocellular carcinoma associated protein TB6 increases in AD, but does not change in PD or DLB; and (3) serine/threonine-protein kinase PLK and splice isoform 3 of integrin alpha-7 precursor do not change in AD, decrease in PD, but do not change in DLB. The presence of quantitative changes in unique markers of two diseases indicates either an overlap of AD and PD or an uncharacterized disease having a CSF protein profile featuring both AD and PD.

[0229]

Change in
Expression	Protein Name
↓	Apolipoprotein A-II precursor
↓	Cochlin precursor
↑	Hepatocellular carcinoma associated protein TB6
↓	Serine/threonine-protein kinase PLK2
↓	Splice isoform 3 of integrin alpha-7 precursor

[0230] A patient's CSF providing the following protein expression pattern is diagnosed as having Parkinson's disease (PD) and/or dementia with Lewy body (DLB) and not Alzheimer's disease (AD): a decrease in apolipoprotein C-III precursor, a decrease in CD99L2 protein, a decrease in HRPE773, a decrease in polymeric-immunoglobulin receptor precursor and an increase in activating receptor pilrbeta. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) activating receptor pilrbeta, apolipoprotein C-III precursor, HRPE773 and polymeric-immunoglobulin receptor precursor do not change in AD, decrease in PD, but increase in DLB; and (2) CD99L2 protein does not change in AD, increases in PD, but decreases in DLB.

[0231]

Change in
Expression	Protein Name
↑	Activating receptor pilrbeta
↓	Apolipoprotein C-III precursor
↓	CD99L2 protein
↓	HRPE773
↓	Polymeric-immunoglobulin receptor precursor

[0232] A patient's CSF providing the following protein expression pattern is diagnosed as having Alzheimer's disease (AD) and/or dementia with Lewy body (DLB) and not Parkinson's disease (PD): a decrease in brain abundant, membrane attached signal protein 1, a decrease in KARCA1 protein, an increase in golgi phosphoprotein 2, an increase in tetranectin precursor and an increase in transcription elongation regulator 1. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) brain abundant, membrane attached signal protein 1 and tetranectin precursor decrease in AD, do not change in PD, but increase in DLB; and (2) Golgi phosphoprotein 2, KARCAI protein, and transcription elongation regulator 1 increase in AD, do not change in PD, but decrease in DLB.

[0233]

Change in
Expression	Protein Name
↓	Brain abundant, membrane attached signal protein 1
↑	Golgi phosphoprotein 2
↓	KARCA1 protein
↑	Tetranectin precursor
↑	Transcription elongation regulator 1

[0234] A patient's CSF providing the following protein expression pattern is diagnosed as having a neurodegenerative disease in general and not specifically Alzheimer's disease (AD) ,Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in hect domain and RLD4, a decrease in secretogranin III precursor, a decrease in splice isoform 2 of ephrin type-A receptor 5 precursor, an increase in apolipoprotein C-III precursor and an increase in splice isoform 7 of amyloid beta A4 protein precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) apolipoprotein C-III precursor does not change in AD, decreases in PD, but increases in DLB; (2) hect domain and RLD 4 increase in AD, decrease in PD, but do not change in DLB; (3) secretogranin III precursor decreases in AD, but does not change in PD or DLB; (4) splice isoform 2 of ephrin type-A receptor 5 precursor does not change in AD or PD, but decreases in DLB; and (5) splice isoform 7 of amyloid beta A4 protein precursor does not change in AD, increases in PD, but does not change in DLB.

[0235]

Change in
Expression	Protein Name
↑	Apolipoprotein C-III precursor
↓	Hect domain and RLD 4
↓	Secretogranin III precursor
↓	Splice isoform 2 of ephrin type-A receptor 5 precursor
↑	Splice isoform 7 of amyloid beta A4 protein precursor

[0236] A patient's CSF providing the following protein expression pattern is diagnosed as having a neurodegenerative disease in general and not specifically Alzheimer's disease (AD) ,Parkinson's disease (PD) or dementia with Lewy body (DLB): a decrease in golgi autoantigen, golgin subfamily B member 1, a decrease in reticulon 4, isoform D, a decrease in splice isoform 1 of transcription factor E2-alpha, an increase in collagen alpha 2(I) chain precursor, and an increase in splice isoform 1 of neuroendocrine protein 7B2 precursor. This determination is based on the results provided in FIGS. 5A-5YY, which show that (1) collagen alpha 2(I) chain precursor increases in AD, but does not change in PD or DLB; (2) Golgi autoantigen, golgin subfamily B member 1 decreases in AD, increases in PD, but does not change in DLB; (3) reticulon 4, isoform D does not change in AD, decreases in PD, but does not change in DLB; (4) splice isoform 1 of neuroendocrine protein 7B2 precursor does not change in AD or PD, but increases in DLB; and (5) splice isoform 1 of transcription factor E2-alpha does not change in AD, decreases in PD, but increases in DLB.

[0237]

Change in
Expression	Protein Name
↑	Collagen alpha 2(I) chain precursor
↓	Golgi autoantigen, golgin subfamily B member 1
↓	Reticulon 4, isoform D
↑	Splice isoform 1 of neuroendocrine protein 7B2 precursor
↓	Splice isoform 1 of transcription factor E2-alpha

[0238] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof; are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.