CA2574727A1 - Biomarkers of neurodegenerative disease - Google Patents

Abstract

Disclosed are biomarkers for neurodegenerative diseases. Methods of identifying such biomarkers and methods of using such biomarkers to, for example, diagnose neurodegenerative disease and monitor disease progression and treatment. Assays, kits, and solid supports related to the biomarkers are also disclosed.

Description

DEMANDE OU BREVET VOLUMINEUX

LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS

THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

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IS16NUAR&S OF NEURODEGENERATIVE DISEASE

1. ACKNOWLEDGEMENTS
1. This work was supported by grants from the National Institute of Aging (LEAD
AG09016, RO1 AG1441 1, and AG00107-15) and Alzheimer's Disease Center AG08665 and a grant from the National Science Foundation (CCR9701911). The U.S. Government has certain rights in this invention.

U. CROSS REFERENCE TO RELATED APPLICATIONS

2. This application claims the benefit of priority to U.S. Provisional Application No.
60/589,318, filed July 19, 2004. U.S. Provisional Application No. 60/589,318 is incorporated by reference herein in its entirety.

III. BACKGROUND

3. Neurodegenerative diseases affect millions of people, greatly reducing their quality of life and, in many, cases, causing death. A relatively common neurodegenerative disease is Parkinson's disease, which affects more than half a million Americans each year. Parkinson's disease is characterized by slowness of movement (bradykinesia), tremor at rest, rigidity of the extremities and neck, stooped posture, minimal facial expressions, problems swallowing (dysphagia), and a paucity of associated movements (e.g., arm swinging). Some patients also experience dementia associated with such abnormalities of motor function.
Parkinson's disease is age-dependant and usually has a gradual onset between the ages of 50 and 70, progressing slowly until death 10 to 20 years later.

4. Alzheimer's disease is another common neurodegenerative disease.
Progression of Alzheimer's disease is associated with gradual changes of consciousness, loss of memory, perception, and orientation as well as loss of personality and intellect. The prevalence of Alzheimer's disease increases dramatically with age.

5. Accurate and easy diagnosis of neurodegenerative diseases prior to autopsy is challenging. Also, the etiology of many neurodegenerative diseases, such as Parkinson's and Alzheimer's disease, is not fully understood. Further, the symptoms associated with one neurodegenerative disease are oftentimes similar to the symptoms of other neurodegenerative diseases, especially at the early stages of disease. Such difficulties can cause confusion and complications with diagnosing and treating patients with such neurodegenerative diseases. For example, the changes that take place in the neural fibers of the Alzheimer's patient are typically positively diagnosed upon histological analysis of the morphological changes that take place in the neurons. It has been shown that gene expression in an Alzheimer's brain changes and that #a ~6ed to identify the onset and progression of an Alzheimer's patient.
However, this type of analysis requires obtaining a brain sample from the patient, and is therefore, most useful in a post mortem setting.

6. As early diagnosis of neurodegenerative disease such as Parkinson's and Alzheimer's can aid in their treatment, a relatively less invasive procedure that is accurate and easier to perform is desirable. As such, needed in the art are compositions and methods for differentiating, diagnosing, and monitoring neurodegenerative diseases. The subject matter disclosed herein addresses these and other needs. For example, disclosed herein are methods and compositions for the diagnosis of neurodegenerative diseases such as Parkinson's and Alzheimer's that involve sampling of the peripheral blood of the patient, rather than sampling neural tissue.

IV. SUMMARY

7. In accordance with the purposes of the disclosed materials, compounds, compositions, articles, and methods, as embodied and broadly described herein, the disclosed subject matter, in one aspect, relates to compounds and compositions and methods for preparing and using such compounds and compositions. In a further aspect, the disclosed subject matter relates to methods for the diagnosis and prognosis of a neurodegenerative disease (e.g., Parkinson's and Alzheimer's) in a particular subject. In a still further aspect, the disclosed subject matter relates to methods of screening for a therapeutic agent for the treatment of a neurodegenerative disease. Still fixrther, disclosed herein are methods of monitoring neurodegenerative disease progression in a subject. In yet a further aspect, the disclosed subject matter relates to methods of monitoring a response to a neurodegenerative disease treatment in a subject, methods of identifying a risk for a neurodegenerative disease in a test subject, and methods of differentially diagnosing a neurodegenerative disease in a test subject. Also, disclosed are diagnostic assays for a neurodegenerative disease and chips, beads, and arrays that can be used in the methods disclosed herein. In many examples, the compositions and methods disclosed herein involve the use of blood from a subject and the analysis of gene expression within the blood cells.

8. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

V. BRIEF DESCRIPTION OF THE FIGURES

9 PCT/US2005/025491 t.. , ~ M~iJg figures, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

10. Figure 1A is a plot of canonical variables 1 and 2 for the first study.
Canonical variables for this plot, and for Figures 1B and 1C, were derived from data for the cell cycle related messages: cyclin D1, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, and protein kinase C alpha. Wald-Wolfowitz runs test p<0.05 for difference between Alzheimer's disease ("AD") and control cases ("Con"). The cases included are shown in Table 1.
Table 1:

Dx N Age Gender MMSE CDR Blessed Duration Fam. Hx AD 8 77.8 3F/5M 21.3 1.3 4.6 4.1 3+/5-Con 7 76.4 3F/5M 30 0 0 NA 2+/5-

11. Figure 1B is a plot of canonical variables 1 and 2 for the second study.
Canonical variables for this plot were derived from data for the same cell cycle related messages as in Figure 1A. Wald-Wolfowitz runs test p<0.05 for difference between AD and control cases. The cases included are shown in Table 2.
Table 2: =

Dx N Age Gender MMSE CDR Blessed Duration Fam. Hx AD 8 78 5F/3M 19 1.5 3.3 3.4 2+/6-Con 8 76 5F/3M 30 0 0 NA 0+/8-

12. Figure 1 C is a plot of canonical variables 1 and 2 for the third study.
Canonical variables for this plot were derived from data for the same cell cycle related messages as in Figure 1A. Wald-Wolfowitz runs test p<0.05 for difference between AD and control cases. The cases included are shown in Table 3.

Table 3:

Dx N Age Gender MMSE CDR Blessed Duration Fam. Hx AD 5 77 4F/1M 21 1.2 3.5 3.5 2+/3-Con 5 62 2F/3M 30 0 0 NA 3+/2-

13. Figure 2A is a plot of canonical variables 1 and 2 for the first study.
Canonical variables for this plot were derived from data for the inflammatory related messages: C5, Cl inhibitor, C5a, complement C3, cyclooxygenase 1, IL17, IL8, LIF, TNF alpha, and IL10r. Wald-Wolfowitz runs test p<0.05 for difference between AD and control cases. The cases included were the same as for Figure 1A.

L-4 1kIV J!of canonical variables 1 and 2 for the second study. Canonical variables for this plot were derived from data for the same inflammatory related messages as in Figure 2A. Wald-Wolfowitz runs test p<0.05 for difference between AD and control cases. The cases included were the same as for Figure 1B.
15. Figure 2C is a plot of canonical variables 1 and 2 for the third study.
Canonical variables for this plot were derived from data for the same inflammatory related messages as in Figure 2A. Wald-Wolfowitz runs test p<0.05 for difference between AD and control cases. The AD and control cases included were the same as for Figure 1 C. Two Parkinson's disease ("PD") cases were added here.
16. Figure 3 is a group of plots of the first canonical variable for the initial set of 8 AD
and 7 control subjects (see Table 1). The transcripts related to cell cycle in the multivariate analysis were: cyclin Dl, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, and protein kinase C alpha. The transcripts related to inflammatory systems in the multivariate analysis were: C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-10r. The transcripts related to cell stress in the multivariate analysis were: Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin. In all three plots Wald-Wolfowitz runs test p<0.05 for difference between early AD and control cases.

17. Figure 4 is a group of plots of the first canonical variable for the second set of subjects (8 AD and 8 controls; see Table 2). The transcripts are the same as in Figure 3. In all three plots Wald-Wolfowitz runs test p<0.05 for difference between early AD
and control cases.
18. Figure 5 is a group of plots of the first canonical variable for the third set of subjects (5 AD, 5 controls, and 2 PD). The transcripts are the same as in Figure 3. In all three plots Wald-Wolfowitz runs test p<0.05 for difference between early AD and control cases.
19. Figure 6 is a silver stained 2D electrophoresis gel. Difference Image of 2 independent human white blood cell samples showing spots present only in one sample but not the other.
20. Figure 7 is a MALDI-TOF mass analysis of isolated differentially expressed protein.
21. Figure 8 is a series of graphs showing differential expression of peripheral leukocyte protein spots from the full cohort of control ("CTL") and Parkinson's disease subjects.

Computer analysis of a subset of spots from the full cohort of control (n=12) and PD (n=12) subjects demonstrates the ability to identify differentially expressed spots of the leukocyte proteome.

12M,-iFWe'q,39S Wi,116tcharts showing the mean baseline characteristics of study participants in the experiments described in Example 4.

23. Figures 10A, l OB, l OC, and 10D are four scanned images of two-dimensional (2D) protein gels that have been silver-stained and dried. Figure l0A is an image showing the levels of proteins expressed in leukocytes obtained from an Alzheimer's patient prior to treatment with valproate (VPA). Figure 10B is an image showing proteins expressed in leukocytes from the same patient four weeks after initiating VPA treatment. Figures 10C and 10D
are enlarged versions of the images of Figures 10A and 10B, respectively, after the images were processed using protein spot-detection software. Spots labeled #278 in Figures 10C and 10D are the spots of a differentially expressed protein before VPA treatment and after VPA
treatment.
24. Figure 11 is a chart listing examples of biomarkers identified using the methods disclosed herein.
25. Figure 12 is a set of four histograms quantifying the effects of the indicated VPA
concentrations on expression of four candidate biomarkers in cultured leukocytes. Three of the four proteins identified as biomarkers in VPA-treated patients also demonstrated changes in expression in response to treatments with increasing doses of VPA.
VI. DETAILED DESCRIPTION
26. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, 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.

A. Definitions 27. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:
28. Throughout the specification and claims the word "comprise" and other forms of the word, such as "comprising" and "comprises," means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

29. As used in the specification and the claims, the singular forms "a," "an,"
and "the"
include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a biomarker" includes mixtures of two or more such biomarkers, reference to "an i~ two or more antibodies, reference to "the subject" includes two or more subjects, and the like.
30. Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself.
For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed then "less than or equal to 10"as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout the application data is provided in a number of different formats and that this data represents endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15.
31. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
32. "Probes" are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art.

33. "Primers" are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur. A primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.

1i.mnE;te 1'M~a~~~s," "elevates," or "raises" refer to levels above control or reference levels. The terms can also include the appearance or occurrence of an event (e.g., a level above a control or reference level that is zero). The terms "decreases,"
"reduces," or "lowers" refer to levels below control or reference levels. These terms can also include the absence or ablation of an event (e.g., a level of zero when a control or reference level is not zero).
35. As used herein, the terms "subject" and "patient" are used interchangeably and mean an individual. Thus, "subject" or "patient" can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. "Subject" or "patient" can also include a mammal, such as a primate. In one particular aspect, a "subject" or "patient" can be a human.
36. As used herein, "sample" refers to any biological material obtained from a subject or patient. In one aspect, a sample can comprise blood, cerebrospinal fluid ("CSF"), or urine. In other aspects, a sample can comprise whole blood, plasma, leukocytes enriched from blood samples, and cultured cells (e.g., leukocytes from a subject). A sample can also include a biopsy or tissue sample including neural tissue. In still other aspects, a sample can comprise whole cells and/or a lysate of the cells. Examples of cells include, but are not limited to, leukocytes such as neutrophils, monocytes, basophils, lymphocytes, eosinophils, or any combination thereof. In another particular aspect, a sample can comprise a leukocyte or substantially pure population of leukocytes or a lysate thereof. The term "substantially pure" with respect to a population of leukocytes or lysates thereof is intended to refer to a sample that contains less than about 1%, less than about 5%, less than about 7%, less than about 10%, less than about 12%, less than about 15%, less than about 20%, less than about 25%, or less than about 30% of cells other than leukocytes, based on the total number of cells in the sample. In a specific example, a sample can comprise lymphocytes, a substantially pure population of lymphocytes, or a lysate of a substantially pure population of lymphocytes. Optionally, the leukocytes can be enriched for a selected type. For example, the leukocyte population can be enriched for lymphocytes and used in the methods described herein. Enrichment can be accomplished using cell sorting techniques like FACS.
37. By "neurodegenerative disease" is meant any disease characterized by the dysfunction and/or death of neurons leading to a loss of neurologic function in the brain, spinal cord, central nervous system, and/or peripheral nervous system. Neurodegenerative diseases can be chronic or acute. Examples of neurodegenerative diseases include, but are not limited to, Parkinson's ~~lteAtias, frontotemporal dementia and Parkinsonism, Alzheimer's disease, Mild Cognitive Impairment, Diffuse Lewy body disease, Dementia with Lewy bodies type, demyelinating diseases such as multiple sclerosis and acute transverse myelitis, amyotrophic lateral sclerosis, Huntington's disease, Creutzfeldt-Jakob disease, AIDs dementia complex, extrapyramidal and cerebellar disorders such as lesions of the corticospinal system, disorders of the basal ganglia, corticobasal ganglionic degeneration, peripheral neuropathy (secondary to diabetes or chemotherapy treatment), progressive supranuclear Palsy, structural lesions of the cerebellum, spinocerebellar degenerations, such as spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and Machado-Joseph), multiple system atrophy, systemic disorders (Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, and mitochondrial multisystem disorder), disorders of the motor unit such as neurogenic muscular atrophies (anterior horn cell degeneration, infantile spinal muscular atrophy, and juvenile spinal muscular atrophy), Down's Syndrome in middle age, subacute sclerosing panencephalitis, Hallervorden-Spatz disease, dementia pugilistica, Pick's disease, and the like. Some examples of acute neurodegenerative disease are stroke, ischemia, and multiple infarct dementia. Sudden loss of neurons may also characterize the brains of patients with epilepsy and those that suffer hypoglycemic insults and traumatic injury of the brain, peripheral nerves, or spinal cord.
38. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.
39. Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, devices, and methods, examples of which are illustrated in the accompanying Examples and Figures.

B. Compositions and methods 40. Disclosed herein are biomarkers, including methods for identifying and using biomarkers. In some specific aspects, the disclosed biomarkers can be used in (i) methods of diagnosing neurodegenerative diseases (e.g., Parkinson's disease and Alzheimer' disease), (ii) methods of tracking the progression of a neurodegenerative disease, (iii) methods of monitoring the response of a subject to a treatment for a neurodegenerative disease, (iv) methods of 6:gic--'fd'~Ã'a"i~i~~44generative disease, (v) methods of distinguishing one neurodegenerative disease from another, and several other methods, as are disclosed herein.
41. By "biomarker" is meant any assayable characteristic or composition that can be used to identify a condition (e.g., a neurodegenerative disease or lack thereof) or the status of a condition in a subject or sample. A biomarker can, in some examples disclosed herein, be a gene whose expression characteristics can be used to identify a condition or status of a condition in a subject or sample. In other examples, a biomarker can be a gene product. By "gene product" is meant a transcript, nucleic acid, or protein. Thus, disclosed herein are biomarkers whose presence, absence, or relative amount can be used to identify a condition or status of a condition in a subject or sample. In one particular example, a biomarker can be a gene product whose presence or absence in a subject is characteristic of a subject having or not having a particular neurodegenerative disease, having a particular risk for developing a neurodegenerative disease, or being at a particular stage of disease. In still another example, a biomarker can be a gene product whose increase or decrease indicates a particular neurodegenerative disease, a particular risk for developing a neurodegenerative disease, or a particular stage of disease. In another example, a biomarker can be a group of various gene products, the presence or absence of which is indicative of a subject having or not having a particular neurodegenerative disease, having a particular risk for developing a neurodegenerative disease, or being at a particular stage of disease. In a further example; a biomarker can be a group of gene products whose pattern of increasing and decreasing expression characterizes a particular neurodegenerative disease or lack thereof. Still further, a biomarker can be a gene product or group of gene products whose pattern of expression is characteristic of the presence or absence of a neurodegenerative disease, or a particular prognosis or outcome of a disease. As used herein, a biomarker can be a surrogate for other clinical tests. Biomarkers identified herein can be measured to determine levels, expression, activity, or to detect variants. As used throughout when detecting levels of expression or activity are discussed, it is understood that this could reflect variants of a given biomarker. Variants include amino acid or nucleic acid variants or post translationally modified variants.
42. Throughout, whenever a protein is discussed, the nucleic acid (e.g., transcript) is also disclosed, unless explicitly stated to the contrary or as would be understood by one of ordinary skill in the art based on the context. Similarly, whenever a nucleic acid is discussed, the protein is also disclosed. In discussions of gene products herein, proteins, nucleic acids, and transcripts ~~.
'~~.eTM collectively, unless explicitly stated to the contrary or as would be understood by one of ordinary skill in the art based on the context.
43. Also, while a biomarker can be a particular gene product, or a particular level or amount of a gene product, a biomarker can also be a particular variable (e.g., a first and/or second canonical variable) obtained when the levels or amounts of such gene products are analyzed in a multivariant canonical analysis.
44. In some examples of the disclosed subject matter, biomarkers for a neurodegenerative disease such as Alzheimer's or Parkinson's are used to diagnose the disease in subjects. And while profiles of message expression by single neurons or homogenates from postmortem human brain can be used to distinguish neurodegenerative diseases from control samples (see e.g., Cheetham JE, et al., J. Neurosci. Methods, 1997;77(1):43-48; Chow N, et al., Proc. Natl. Acad.
Sci. U.S.A., 1998;95:9620-9625), disclosed herein, in one aspect, are methods that combine gene and/or protein profiling and multivariate canonical analysis to differentiate neurodegenerative diseases (e.g., mild AD or sporadic PD) from control blood samples.
45. In one example disclosed herein, blood was drawn from patients diagnosed in an Alzheimer's Disease Center as having probable AD and from an age and sex matched control sample. Messenger RNA was extracted from leukocytes and amplified. The expression level of selected messages was then quantified using low density arrays. Multivariate canonical analyses differentiated Alzheimer's and control leukocytes. The message species studied also differentiated two Parkinson's disease cases from AD and control samples.
These results illustrate the additional accuracy that may be derived by a biomarker that makes use of multiple variables. Of the classes of mRNA species examined, those that best distinguished AD from control leukocytes were those involved in the cell cycle and in inflammatory processes. That members of these same classes also distinguish AD from control brains is consistent with understanding selected phenomena of the brain in Alzheimer's disease without actually invading the brain. There are a variety of applications and patents that discuss the diagnoses of Alzheimer's disease, including U.S. Application Nos. 60/063,274, filed October 24, 1997, 09/178,170, filed October 23, 1997, 09/770,534, filed January 25, 2001, and U.S. Published Application No. 2005-0084875-Al, which are herein incorporated by reference for material at least related to diagnoses of Alzheimer's and methods for same, as well as genes related to the diagnoses of AD.
46. A similar example was performed with patients diagnosed with Parkinson's disease, as is disclosed herein. Specifically, fresh whole blood was drawn from a patient population that 4~Y'~AN"k-lontrol patients. Leukocyte protein concentrations were determined and protein spots that differed in intensity between PD and control patients were identified using Progenesis Discovery software (Nonlinear USA, Inc.; Durham, NC). Difference measurements were subjected to statistical testing. Protein spots were identified as either increasing or decreasing in Parkinson's disease compared with control.
Differentially expressed spots were isolated and identified. The results show that protein analysis from peripheral blood can be used to diagnose neurodegenerative diseases such as PD.
47. The compositions and methods disclosed herein are based on the identification that the expression of certain genes in samples (e.g., blood) from a subject with a neurodegenerative disease is altered. The disclosed methods typically involve comparing the expression of certain genes and sets of genes in the blood of a subject to the expression of the same gene or sets of genes in a control sample. It is understood that the control sample, can be a non-neurodegenerative diseased subject concurrently run, or it can be a standard created by assaying one or more non-neurodegenerative diseased subjects and collecting the expression data. The control sample, thus, can be a standard, which is created and used continuously. For example, a standard could be created by the expression profiles of non-AD or non-PD cases disclosed herein. The standard could include, for example, the average level of expression of a gene or particular set of genes in non-neurodegenerative diseased subject or any other control group.

48. In one particular aspect, disclosed herein are methods of diagnosing a neurodegenerative disease (e.g., Parkinson's or Alzheimer's disease) in a subject. The disclosed methods can comprise assessing a level of expression or activity of one or more selected biomarkers (e.g., gene products) in a sample, for example a sample comprising leukocytes or a lysate thereof, from the subject to be diagnosed and comparing the level of expression or activity of the selected biomarker(s) to a reference standard that indicates the level of expression or activity of the selected biomarker(s) in one or more control subjects. In these methods, a difference or similarity between the level of expression or activity of the selected biomarker(s) and the reference standard can indicate that the subject has or does not have a particular neurodegenerative disease, depending on the particular reference standard.
Methods for assessing and comparing the level of biomarker(s) expression or activity are disclosed herein.
49. By "diagnose" or other forms of the word such as "diagnosing" and "diagnosis" is meant to identify a particular disease. The term also means to distinguish one particular disease from another disease or to distinguish one particular disease from the absence of disease.

l'Di'igiid4143tii~Alirgin to mean to identify a particular stage of a disease, to identify the risk of developing a disease, or to identify a prognosis of a disease.

50. In these particular methods, the subject can be as described herein, for example, any individual, such as a human. In one example, the subject is to be diagnosed for a particular neurodegenerative disease. The subject to be diagnosed can have symptoms of a particular neurodegenerative disease or the subject can be asymptomatic or preclinical for a particular neurodegenerative disease (e.g., Parkinson's or Alzheimer's disease).

51. The control subject can be a subject with a particular neurodegenerative disease (e.g., Parkinson's or Alzheimer's disease), at a particular stage of a disease, with a particular risk of developing a disease, or without a particular neurodegenerative disease. In one example, the subject to be diagnosed and a control subject can be age-matched and/or sex matched. Thus, by comparing the level of expression of one or more selected biomarkers from a subject to the level of expression of the same biomarker(s) in a control subject (i.e., reference standard) one can diagnose the subject for a neurodegenerative disease.
52. A difference or similarity in the level of the biomarker(s) expression or activity can be determined by any quantitative or qualitative comparative analysis between the levels of one or more selected biomarker(s) in the sample and in the reference standard. For example, when the control subject has a particular neurodegenerative disease, then when using the disclosed methods, a similarity between the level of the biomarker(s) in the subject and the control subject can indicate that the subject to be diagnosed also has the particular neurodegenerative disease.
In another example, when the control subject has a particular neurodegenerative disease, then when using the disclosed methods, a difference between the level of the biomarker(s) in the subject and the control subject can indicate that the subject to be diagnosed does not have the particular neurodegenerative disease. Alternatively, when the control subject does not have a particular neurodegenerative disease, then, in this example, a difference between the level of the biomarker(s) in the subject and the control subject can indicate that the subject to be diagnosed has the particular neurodegenerative disease. In still another example, when the control subject does not have a particular neurodegenerative disease, then, in this example, a similarity between the level of the biomarker(s) in the subject and the control subject can indicate that the subject to be diagnosed does not have the particular neurodegenerative disease.
53. In a further example, if a selected gene product was identified as a biomarker by detecting an increase in expression or activity of the gene product in subjects diagnosed with a neurodegenerative disease, for example, Parkinson's or Alzheimer's disease, then an increase in _KpidAkIf QatiQ 4~te'~ ~ei~'erence standard) of the biomarker in a sample from a subject can indicate that the subject has the neurodegenerative disease. On the other hand, if one or more selected gene products were identified as biomarkers by detecting a decrease in expression or activity of the gene products in subjects diagnosed with the neurodegenerative disease, then a decrease (relative to the reference standard) in expression of the protein(s) in a sample from a subject can indicate that the subject has the neurodegenerative disease.

54. Still further, a combination of biomarkers can be use in the disclosed methods. For example, one or more biomarkers can increase and other biomarkers can decrease relative to the reference standard and can thus indicate the presence or absence of a neurodegenerative disease.
1. Assessing levels of expression a) Sample collection 55. As noted, the methods disclosed herein typically involve collecting a sample from a subject. The sample can be of the peripheral blood of a subject, though the uses of other samples are contemplated. A blood sample can be collected in any way that allows for isolation of cells and, subsequently, gene products from these cells. For example, the blood can be collected and via syringe, and then stored at 4 C. Blood samples can be collected in evacuated tubes containing, but not limited to, heparin, EDTA or ADC (yellow tube) or any other anticoagulant.
Another way to collect and store blood can be achieved through the use of PAXgeneTM Blood RNA tubes, which allows the stabilization and the storage of whole blood for up to 5 days at room temperature. The use of any other compound or chemical enhancing eukaryotic mRNA
stability can also be used with this procedure. In one specific exanlple, the pellet can be re-suspended by vortexing at 4 C in 200 L buffer (20 mM Tris, pH 7.5, 0.5%
Nonidet, 1 mM
EDTA, 1 mM PMSF, 0.1 M NaCl, 1X Sigma Protease Inhibitor, and 1X Sigma Phosphatase Inhibitors 1 and 2). The suspension can be kept on ice for 20 minutes with intermittent vortexing. After spinning at 15,000 x g for 5 minutes at 4 C, aliquots of supernatant can be stored at minus 70 C. The collection of other types of samples, e.g., urine, CSF, tissue, etc., can be performed by methods known in the art.

b) Cell collection 56. Typically, once a sample is collected, the various cells contained within the sample can be separated. For example, in certain embodiments, the expression pattern of a gene or set of genes is assayed within a leukocyte population contained within the sample.
Various cells can be isolated in any way, as long as the cells are ultimately preserved such that gene products can be collected from them. For example, one way of isolating leukocytes is lysing the erythrocytes Wdd~~&W'-ie~~Land then collecting the remaining leukocytes, by centrifugation, for example. The assay can be applied to subtypes of leukocytes, such as lymphocytes and their subclasses, monocytes, and other types of blood cells. In addition, non-blood cells can be assayed, such as those from skin, cheek scrapings, muscle, olfactory epithelium, digestive system, urinary system, and reproductive system, for example. Lysing can occur in any way, including, for example, in RNAse free conditions through the use of ammonium chloride or using commercial reagents such as IlVIM[TNOLYSETM and OPTILYSETM (Coulter International Corporation; Miami, FL). Alternatively, HISTOPAQUETM (Sigma; Milwaukee, WI) with or without Ficoll can be used to centrifug.e anticoagulated blood to separate leukocytes from erythrocytes. Another way to achieve this goal is allowing the anticoagulated blood to sediment for 1-2 hours at room temperature and collecting the leukocyte fraction.
Another example of lysing includes centrifuging anticoagulated blood for about 5 minutes at 150-200 g; and then removing the buffy coat. Similar methods for lysing are known in the art and can also be employed.
57. The leukocytes can also be collected using, for example, leukocyte-specific markers for cell sorting. The general leukocyte population can be sorted into subtypes, for example, such as B cells, T cells, basophils, eosinophils, neutrophils, monophils, monocytes, and platelets, and subtypes of these general categories. Markers include, but are not limited to, MHC

glycoproteins, integrins, homing receptors, Fc receptors for IgG, IgE, IgM, IgA, and IgD, complement receptors for lymphokines, interferons, colony stimulating factors, receptors for insulin, receptors for neurotransmitters, chemotactic receptors, membrane enzymes, and transport proteins. These can be sorted, for example, by antibodies that recognize the more than 170 CD antigens.
58. Typically, once the cells, such as leukocytes, are collected the leukocytes themselves will be lysed to collect the nucleic acid and/or proteins. These cells can be lysed in any way.
Leukocyte nucleic acid can be also collected using 4M guanidinium isothiocyanate lysis and cesium chloride ultracentrifugation, for example, or using 4 M guanidine thiocyanate/ 25 mM
sodium citrate/ 0.5% Sarkosyl / 0.1 M,6-mercaptoethanol to lyse the leukocytes and then adding 2M NaOAc prior to centrifugation steps and isopropanol precipitation. Further, nucleic acids can be collected using hydroxy appetite or they can be collected using positively charge magnetic beads. Nucleic acids can also, for example, be precipitated. Total RNA can also be obtained using TRiZOLTM reagent (Gibco Life Technologies, Inc.; Rockville, MD), which is a mono-phasic solution of phenol and guanidine isothiocyanate. Any other reagent that maintains the

-14-t~ ~'; ~ '1'~4611disrupting cells and dissolving cell components can also be used.
Leukocyte proteins and peptides can also be collected by lysing cells in "crack buffer" (50 mM
Tris-HCl (pH 6.8), 100 mM DTT, 100 g/ml PMSF, 2% sodium dodecyl sulfate (SDS), 10%
glycerol, 1 g/ml each of pepstatin A, leupeptin, and aprotinin, and 1 M
sodium orthovanadate), and sheared with a 22 gauge needle. The protein content of the samples can be estimated using the DC protein assay (BioRad). Protein (10-20 g) can be resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% SDS.
59. Typically the total gene product (e.g., biomarker) will be first isolated or collected.
This can be done using any means for collecting nucleic acids and/or proteins.
Once the gene products are collected, they can be washed, and, for example, eluted, if they are to be attached to a type of affinity system, such as magnetic beads. The step of collecting gene products is typically done to acquire the mRNA and/or proteins in the sample, and is not needed if direct collection of the gene product is used. The gene products can be prepared, however, in any way that allows for analysis of gene expression.
c) Preparation of the RNA
60. In some examples, the disclosed methods involve some level of RNA
preparation.
The RNA preparation step is not required to be performed as part of a contiguous method, but in certain methods the RNA should be prepared such that it can be hybridized. In other methods, the RNA can be used to produce cDNA, which can then be used, for example, as a template for a PCR reaction or directly analyzed through, for example, hybridization of a probe. While in theory, the RNA preparation step could be performed far removed from the actual amplification and quantitation steps (e.g., in another laboratory, or at a much earlier time), in many embodiments the RNA isolation and preparation, or amplification of cDNA etc., will occur in conjunction with the amplification and quantitation steps of the methods, such as PCR or llybridization; but this is not required.
61. When an RNA preparation step is included in the disclosed methods, the method of RNA preparation can be any method of RNA preparation that produces enzymatically manipulatable mRNA or analyzable RNA. For example, the RNA can be isolated by using the guanidinium isothiocyanate-ultracentrifugation method, the guanidinium and phenol-chloroform method, the lithium chloride-SDS-urea method or poly A+ / mRNA from tissue lysates using oligo(dT) cellulose method. It is important when isolating the RNA that enough RNA is isolated. Furthermore, typically the quantity of RNA obtained can be determined. For example, typically at least 0.01 ng or 0.5 ng or 1 ng or 10 ng or 100 ng or 1,000 ng or 10,000 ng or

-15-As will be discussed herein, during the amplification of multivariate quantitative PCR it is important that when the amplification is stopped that the amplification of each target product remain be at least about 80% or 85% or 90% or 95% the doubling rate. The number of cycles of PCR that are performed so as to continue to remain at about the doubling rate is related to the amount of total RNA that was used in the cDNA
generation step.

The RNA can be isolated from any desired cell or cell type and from any organism, including mammals, such as mouse, rat, rabbit, dog, cat, monkey, and human, as well as other non-mammalian animals, such as fish or amphibians, as well as plants and even prokaryotes, such as bacteria.

d) RNA expression analysis 62. Once the cells or cell type, such as leukocytes, are collected, the expressed messenger RNA contained within these cells can be assayed. This can be done using any of a number of means, such as hybridization, Northern blot, RT-PCR, real-time RT-PCR, single channel quantitative multiplex RT-PCR, oligo- and/or cDNA arrays or any technology that can lead to nucleotide quantification. An example of such an approach can be derived from automated instruments based on the use of DNA-chip teclmologies, an example of which can be found at Integrated Nano-Technologies LLC, http://www.integratednano.coin/. For example, one way of assaying the total mRNA is to collect all of the nucleic acids contained within the cells, by for example, lysing the cells, and precipitating the nucleic acids. Messenger RNA
can be collected in any way, such as using a polyT oligonucleotide, which will specifically hybridize with the polyA tail contained on messenger RNA transcripts. This method, however, relies on the presence of the 3'-polyA tail, but under certain conditions, this tail may be degraded. Thus, another way of directly analyzing all messenger RNA, including fragments, can be to use message specific primers with reverse transcriptase to make cDNA. This cDNA
can then be assayed directly or amplified and assayed by, for example, using quantitative PCR discussed herein. It is understood that the ultimate goal is the analysis of gene expression which can be accomplished in any way which analyzes the expression of genes and compares their expression.
It is understood that direct hybridization or other means of identification of mRNA is considered, as well as means where the mRNA is manipulated to form, for example, cDNA
which is directly analyzed through, for example, hybridization or other identification, or which itself can be amplified producing, for example, a PCR product, which itself can be directly

-16-or otherwise identified or manipulated. As long as the end goal of identification of gene expression is realized it is contemplated herein.

63. The analysis of the expression can be through, for example, hybridization of probes.
For example, probes for the specific genes to be analyzed can be contained on, for example, a chip, and the mRNA can be analyzed for binding to the chip. These chips are typically referred to as arrays, such as microarrays or macroarrays.

(1) Microarrays 64. An array is an orderly arrangement of samples, providing a medium for matching known and unknown DNA samples based on base-pairing rules. Typically the process of identifying the unknowns is automated. An array experiment can make use of common assay, systems such as microplates or standard blotting membranes and can be created by hand or make use of robotics to deposit the sample. In general, arrays are described as macroarrays or microarrays, the difference being the size of the sample spots. Macroarrays contain sample spot sizes of about 300 m or larger and can be easily imaged by existing gel and blot scanners. The sample spot sizes in a microarray can be 300 m or less, but typically less than 200 m in diameter and these arrays usually contain thousands of spots. Microarrays typically require specialized robotics and/or imaging equipment that are generally constructed for each unique application of a microarray. Terminologies that have been used in the literature to describe this technology include, but are not limited to, biochip, DNA chip, DNA microarray, GENECHIP TM

(Affymetrix's high density, oligonucleotide-based DNA array product (Affymetrix, Inc.; Santa Clara, CA)), and gene array.
65. DNA microarrays typically are fabricated by high-speed robotics, generally on glass or nylon substrates, for which probes with known identity are used to determine complementary binding, thus allowing massively parallel gene expression and gene discovery studies. An experiment with a single DNA chip can provide information on thousands of genes simultaneously. It is herein contemplated that the disclosed microarrays can be used for any purpose, including monitoring gene expression, disease diagnosis, gene discovery, drug discovery (pharmacogenomics), and toxicological research or toxicogenomics.

66. There are two variants of the DNA microarray technology, in terms of the property of arrayed DNA sequence with known identity. The main difference between Type I
and Type II
arrays is that in a Type I array there is typically a single sequence or set of related sequences, such as a set of allelic sequences, and in Type II microarrays there are many different sequences attached to the surface.

-17-=x ''.i[ ~,~j:u-~ ~t-Rt A3tcrb "a~-"r~fa~~t=comprise a probe, typically a cDNA
(500 to about 5,000 bases long) that is immobilized to a solid surface such as glass using robot spotting and exposed to a set of targets either separately or in a mixture. This method is traditionally referred to as a DNA
microarray. With Type I microarrays, localized multiple copies of one or more polynucleotide sequences, preferably copies of a single polynucleotide sequence are immobilized on a plurality of defined regions of the substrate's surface. A polynucleotide refers to a chain of nucleotides ranging typically from 5 to 10,000 nucleotides. These immobilized copies of a polynucleotide sequence are suitable for use as probes in hybridization experiments. The immobilized sequences are then probed with a number of different samples, typically at different regions of the chip, such that samples which contain nucleotides that hybridize to the immobilized sample can be identified.
68. To prepare beads coated with immobilized probes, beads are immersed in a solution containing the desired probe sequence and then immobilized on the beads by covalent or noncovalent means. Alternatively, when the probes are immobilized on rods, a given probe can be spotted at defined regions of the rod. Typical dispensers include a micropipette delivering solution to the substrate with a robotic system to control the position of the micropipette with respect to the substrate. There can be a multiplicity of dispensers so that reagents can be delivered to the reaction regions simultaneously. In one embodiment, a microarray is formed by using ink-jet technology based on the piezoelectric effect, whereby a narrow tube containing a liquid of interest, such as oligonucleotide synthesis reagents, is encircled by an adapter. An electric charge sent across the adapter causes the adapter to expand at a different rate than the tube and forces a small drop of liquid onto a substrate (see Baldeschweiler, et al., PCT
publication W095/251116).
69. Samples can be any sample containing polynucleotides (polynucleotide targets) of interest and obtained from any bodily fluid (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. DNA or RNA can be isolated from the sample according to any of a number of inethods well known to those of skill in the art. For example, methods of purification of nucleic acids are described in Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization with Nucleic Acid Probes.
Part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed. Elsevier (1993). In one embodiment, total RNA is isolated using the TRIZOLTM total RNA isolation reagent (Gibco Life Technologies, Inc., Rockville, MD) and mRNA is isolated using oligo d(T) colurnn chromatography or glass beads.

-18-j~~e'r' ~i~~~ '~~~~tidi"~t~ ~'WR Aing, the hybridization signals obtained should reflect accurately the amounts of control target polynucleotide added to the sample.

70. The plurality of defined regions on the substrate, immobilized polynulcoeotide, can be arranged in a variety of formats. For example, the regions may be arranged perpendicular or in parallel to the length of the casing. Furthermore, the targets do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group. The linker groups may typically vary from about 6 to 50 atoms long. Suitable linker groups include ethylene glycol oligomers, diamines, diacids and the like. Reactive groups on the substrate surface react with one of the terminal portions of the linker to bind the linker to the substrate.
The other terminal portion of the linker is then functionalized for binding the probes.
71. Sample polynucleotides may be labeled with one or more labeling moieties to allow for detection of hybridized probe/target polynucleotide complexes. The labeling moieties can include compositions that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means. The labeling moieties include radioisotopes such as 32P, 33P, or 35S, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers such as fluorescent markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, biotin, and the like.
72. Labeling can be carried out during an amplification reaction, such as polymerase chain reaction and in vitro or in vivo transcription reactions. Alternatively, the labeling moiety can be incorporated after hybridization once a probe-target complex is formed.
In one preferred embodiment, biotin is first incorporated during an amplification step as described above. After the hybridization reaction, unbound nucleic acids are rinsed away so that the only biotin remaining bound to the substrate is that attached to target polynucleotides that are hybridized to the polynucleotide probes. Then, an avidin-conjugated fluorophore, such as avidin-phycoerythrin, that binds with high affinity to biotin is added.

73. Hybridization causes a polynucleotide probe and a complementary target to form a stable duplex through base pairing. Hybridization methods are well known to those skilled in the art, and stringent conditions for hybridization can be defined by salt concentration, temperature, and other chemicals and conditions as discussed herein. Varying additional parameters, such as hybridization time, the concentration of detergent (sodium dodecyl sulfate, SDS) or solvent (formamide), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Additional variations on these conditions will be readily apparent to

-19-and Berger SL, Methods Enzymol., 1987; 152:399-407;
Kimmel AR, Methods Enzymol., 1987; 152:507-511; Ausubel FM, et al., Short Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1997; and Sambrook J, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989).
74. Methods for detecting complex formation are well known to those skilled in the art.
In one example, the polynucleotide probes are labeled with a fluorescent label and measurement of levels and patterns of complex fonnation is accomplished by fluorescence microscopy, such as confocal fluorescence microscopy. An argon ion laser excites the fluorescent label, emissions are directed to a photomultiplier, and the amount of emitted light detected and quantitated. The detected signal should be proportional to the amount of probe/target polynucleotide complex at each position of the microarray. The fluorescence microscope can be associated with a computer-driven scanner device to generate a quantitative two-dimensional image of hybridization intensities. The scanned image is examined to determine the abundance/expression level of each hybridized target polynucleotide.
75. Tii a differential hybridization experiment, polynucleotide targets from two or more different biological samples are labeled with two or more different fluorescent labels with different emission wavelengths. Fluorescent signals are detected separately with different photomultipliers set to detect specific wavelengths. The relative abundances/expression levels of the target polynucleotides in two or more samples are obtained. Typically, microarray fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one microarray is used under similar test conditions. In one example, individual polynucleotide probe/target complex hybridization intensities are normalized using the intensities derived from internal normalization controls contained on each microarray.
76. Type II microarrays comprise an array of oligonucleotides (e.g., from about 20 to about 80-mer oligos) or peptide nucleic acid (PNA) probes that is synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. The array is exposed to labeled sample DNA, hybridized, and the identity/abundance of complementary sequences is determined. This method, "historically" called DNA chips, was developed at Affymetrix, Inc., (Santa Clara, CA), which sells its photolithographically fabricated products under the GENECHIP trademark.
77. The basic concept behind the use of Type II arrays for gene expression is simple:
labeled cDNA or cRNA targets derived from the mRNA of an experimental satnple are hybridized to nucleic acid probes attached to the solid support. By monitoring the amount of

-20-t~lab~l Cie-location, it is possible to infer the abundance of each mRNA
species represented. Although hybridization has been used for decades to detect and quantify nucleic acids, the combination of the miniaturization of the technology and the large and growing amounts of sequence information, have enormously expanded the scale at which gene expression can be studied.
78. Microarray manufacturing can begin with a 5-inch square quartz wafer.
Initially the quartz is washed to ensure uniform hydroxylation across its surface. Because quartz is naturally hydroxylated, it provides an excellent substrate for the attachment of chemicals, such as linker molecules, that are later used to position the probes on the arrays.
79. The wafer is placed in a bath of silane, which reacts with the hydroxyl groups of the quartz, and forms a matrix of covalently linked molecules. The distance between these silane molecules determines the probes' packing density, allowing arrays to hold over 500,000 probe locations, or features, within a mere 1.28 square centimeters. Each of these features harbors millions of identical DNA molecules. The silane film provides a uniform hydroxyl density to initiate probe assembly. Linker molecules, attached to the silane matrix, provide a surface that may be spatially activated by light.
80. Probe synthesis occurs in parallel, resulting in the addition of an A, C, T, or G
nucleotide to multiple growing chains simultaneously. To define which oligonucleotide chains will receive a nucleotide in each step, photolithographic masks, carrying 18 to 20 square m windows that correspond to the dimensions of individual features, are placed over the coated wafer. The windows are distributed over the mask based on the desired sequence of each probe.
When ultraviolet light is shone over the mask in the first step of synthesis, the exposed linkers become deprotected and are available for nucleotide coupling.
81. Once the desired features have been activated, a solution containing a single type of deoxynucleotide with a removable protection group is flushed over the wafer's surface. The nucleotide attaches to the activated linkers, initiating the synthesis process.
82. Although each position in the sequence of an oligonucleotide can be occupied by 1 of 4 nucleotides, resulting in an apparent need for 25 x 4, or 100, different masks per wafer, the synthesis process can be designed to significantly reduce this requirement.
Algorithms that help minimize mask usage calculate how to best coordinate probe growth by adjusting synthesis rates of individual probes and identifying situations when the same mask can be used multiple times.
83. Some of the key elements of selection and design are common to the production of all microarrays, regardless of their intended application. Strategies to optimize probe hybridization,

-21-RWJ' i"ded in the process of probe selection. Hybridization under particular pH, salt, and temperature conditions can be optimized by taking into account melting temperatures and using empirical rules that correlate with desired hybridization behaviors.

84. To obtain a complete picture of a gene's activity, some probes are selected from regions shared by multiple splice or polyadenylation variants. In other cases, unique probes that distinguish between variants are favored. Inter-probe distance is also factored into the selection process.
85. A different set of strategies is used to select probes for genotyping arrays that rely on multiple probes to interrogate individual nucleotides in a sequence. The identity of a target base can be deduced using four identical probes that vary only in the target position, each containing one of the four possible bases.
86. Alternatively, the presence of a consensus sequence can be tested using one or two probes representing specific alleles. To genotype heterozygous or genetically mixed samples, arrays with many probes can be created to provide redundant information, resulting in unequivocal genotyping. In addition, generic probes can be used in some applications to maximize flexibility. Some probe arrays, for example, allow the separation and analysis of individual reaction products from complex mixtures, such as those used in some protocols to identify single nucleotide polymorphisms (SNPs).
87. In certain examples, the disclosed genes or gene sets whose expression are related to the diagnosis of a particular neurodegenerative disease (e.g., Alzheimer's or Parkinson's) in a sample (e.g., peripheral blood) are attached. to a microarray. In certain embodiments, for example, the chip can be divided into sections each of which contain a variety of regions and alleles for one of the genes in the diagnosis set, and another region of the chip can contain a variety of regions and alleles for another of the genes in the diagnosis set.
In yet another example, each region of the chip could contain all of the alleles and regions of all of the genes in the set. There are many variations on this type example, which include the use of all or any subset of the disclosed neurodegenerative disease diagnostic genes, and it is understood that any region of these disclosed genes, from 3 bases to the full-length sequence can be used as a probe region for immobilization. Furthermore, any allelic or variant can also be used.
(2) Multivariate single channel quantitative RT-PCR
88. One particularly useful means for assaying the expression levels is through the use of multivariate single channel quantitative RT-PCR. These methods are disclosed in U.S. Patent Applications 60/336,095, filed November 30, 2001, 60/397,475, filed July 19, 2002, 10/496,626,

-22-1229, filed March 12, 2001, and 10/096,710, filed March 12, 2002, which are herein incorporated by reference at least for material related to methods and compositions related to multivariate single channel quantitative RT-PCR.
Briefly, the method utilizes a PCR-based high-throughput method for simultaneously analyzing the expression of multiple genes. The method can use minute quantities of starting material and reach single copy levels of efficiency, for example, where only a single target nucleic acid was available, such as a single copy of transcript from a single target cell. For example, for the analysis of 20 transcripts in triplicate for 4 subjects, less than 1 g total RNA per subject is needed.
The disclosed methods are capable of simultaneously analyzing multiple genes. The disclosed methods use gene-specific primers in particular ways. The disclosed methods can quantitate multiple genes with the use of a single signal reagent, such as a fluorescent probe.
89. hi general, the method is useful for obtaining quantitative information about the expression of many different genes in a sample that can contain as little as a single cell. Since the disclosed methods are quantitative, comparisons of the expression patterns at a quantitative level between a variety of different cell states or cell types can be achieved. In general, total RNA can be isolated from the target sample using any isolation procedure. This RNA can then be used to generate first strand copy DNA (cDNA) using any procedure, for example, using random primers, oligo-dt primers, or random-oligo-dt primers, which are oligo-dt primers coupled on the 3'-end to short stretches of specific sequence covering all possible combinations, so the primer primes at the junction between the polyA tract and non-poly A
tract associated with messenger RNA (mRNA). The cDNA is then used as a template in a PCR
reaction. This PCR reaction is performed with primer sets, a forward and a reverse primer, that are specific for the expressed genes, which are to be tracked. This reaction can contain as many different primer sets as desired, but typically would contain between 5 and 100 different sets of primers, each specific for a single gene or single isoform (including any specific number between 5 and 100).
Typically all of the primers will be in about equimolar concentration. After performing a number of PCR cycles, for example 15 cycles, such that the DNA is still amplifying at about greater than 80% or 85% or 90% or 95% the doubling rate, the PCR is stopped.
Typically, in the first round of PCR, if quantitative PCR (real time PCR) was performed, one does not reach the threshold cycle of amplification. However, the disclosed methods in certain embodiments can still work if amplification proceeds for about less than 9, 8, 7, 6, 5, 4, 3, 2, or 1 cycle(s) past the threshold cycle. The number of cycles in the first round depends on the amount of starting materials. For example, 20 cycles can be used for single cell experiments. The PCR reaction is

-23-qh''~p~f ~''1~~A~c~ int~i~FriwP'r~e~e~ion tubes for a (new) second round of PCR. Each of the tubes contains a fraction of the previous PCR reaction mixture which contains all of the products produced from all of the specific primers present in the first PCR mixture. In the second PCR
mixture, containing the fraction of the first PCR mixture, typically only one of the specific primer sets or a new primer set is added, in addition to the universal primer which has the molecular beacon attached, and the PCR is performed. Typically this second round of PCR is performed using quantitative real time PCR protocols, which, for example, rely on increases in fluorescence at each cycle of PCR through (for example, probes that hybridize to a portion of one of the amplification probes) the release of fluorescence from a quencher sequence while the uniprimer (universal primer) binds to the DNA sequence. Fluorescence approaches used in real-time quantitative PCR are typically based on a fluorescent reporter dye such as SYBR green, FAM, fluorescein, HEX, TET, TAMRA, etc. and a quencher such as DABSYL, Black Hole, etc.
When the quencher is separated from the probe during the extension phase of PCR, the fluorescence of the reporter can be measured. Systems like Molecular Beacons, Taqman Probes, Scorpion Primers or Sunrise Primers and others use this approach to perform real-time quantitative PCR. Examples of methods and reagents related to real time probes can be found in U.S. Patent Nos. 5,925,517; 6,103,476; 6,150,097, and 6,037,130, which are incorporated by reference herein at least for material related to detection methods for nucleic acids and PCR
methods. In addition to performing the above steps, the generation of a standard curve for the primer sets, and typically for each individual primer set, should be made so that data obtained from the second round of PCR can be accurately correlated with an absolute copy number of the original starting material in the target sample, containing for example, the target cell or cells.
Each of these steps of the general method, as well as the reagents and variations of the method, are discussed in detail herein. A key aspect to understanding the disclosed methods is the combination of a first PCR containing the multiple different primer sets in a batch PCR mixture in which all target gene products or fragments of gene products are amplified with a second PCR
panel in which the specific amplification reaction occurs in which a portion of the batch PCR
mixture is amplified with specific primer sets. Quantitation is typically achieved by reference to a standard curve that is generated for the complete primer sets or each individual primer set.

e) Preparation of protein 90. In some examples, the disclosed methods can involve some level of protein or peptide preparation. The protein or peptide preparation step is not required to be performed as part of a contiguous method, but in certain methods the protein or peptide should be prepared

-24-f if;;,(f as disclosed herein. While in theory, the protein or peptide preparation step could be performed far removed from the actual analysis steps (e.g., in another laboratory, or at a much earlier time), in many embodiments the protein or peptide isolation and preparation, e.g., electrophoresis, will occur in conjunction with the quantitation steps of the methods; but this is not required.
91. When a protein or peptide preparation step is included in the disclosed methods, the method of protein or peptide preparation can be any method of protein or peptide preparation that produces analyzable protein or peptide. For example, the sample cells can be lysed in "crack buffer" (50 mM Tris-HCl (pH, 6.8), 100 mM DTT, 100 g/ml PMSF, 2% SDS, 10%
glycerol, 1 g /ml each of pepstatin A, leupeptin, and aprotinin, and 1 m sodium orthovanadate), and sheared with a 22 gauge needle. The protein content of the samples can be estimated using the DC protein assay (BioRad). Protein (10-20 g) can be resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% SDS. Typically the quantity of protein or peptide obtained can be determined.
The proteins or peptides can be isolated from any desired cell or cell type and from any organism, including mammals, such as mouse, rat, rabbit, dog, cat, monkey, and human, as well as other non-mammalian animals, such as fish or amphibians, as well as plants and even prokaryotes, such as bacteria.
f) Protein expression analysis 92. In certain embodiments of the disclosed methods, assessing the level of expression of one or more proteins or peptides can be performed. The level of protein or peptide expression can be assessed in addition to the nucleic acid analysis disclosed herein, or as an alternative to the nucleic acid analysis. Assessing a level of expression of one or more proteins in a sample can be performed by various techniques known in the art. For example, assessing the level of expression can involve analyzing one or more proteins by two-dimensional gel electrophoresis, mass spectroscopy (MS), matrix-assisted laser desorption/ionization-time of fligllt-MS (MALDI-TOF), surface-enhanced laser desorption ionization-time of flight (SELDI-TOF), high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS), protein chip expression analysis, gene chip expression analysis, and laser densitometry, including combinations of these techniques. In another example of a technique for analyzing protein expression levels, one can assay the amount of mRNA that encodes for a particular protein or proteins.

-25-3- ques an antibody or other agent that selectively binds to a protein can be used to detect the amount of that protein expressed in a sample. For example, the level of expression of a protein can be measured using methods that include, but are not limited to, Western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent activated cell sorting (FACS), or a combination thereof. Also, antibodies, aptamers, or other ligands that specifically bind to a protein can be affixed to so-called "protein chips" (protein microarrays) and used to measure the level of expression of a protein in a sample. In other methods, immunofluorescence techniques can be used to visually assess the expression level of a protein iin a sample. In immunofluorescence techniques, antibodies that specifically bind to a protein are visualized to indirectly detect the presence of a protein on the cell surface of intact leukocytes, and/or throughout permeabilized leukocytes.
94. The term "antibodies" is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term "antibodies" are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof.
Monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U.S. Patent No.
4,816,567 and Morrison, et al., Proc. Natl. Acad. Sci. U.S.A. 1984;81:6851-6855).
95. In one example, an antibody to alpha synuclein or a conformer thereof can be used to identify the level of alpha-synuclein or various conformers thereof.
Specifically, antibodies to native-alpha synuclein, dopamine-adducted alpha-synuclein, and oligomeric or aggregated alpha-synuclein can be used. These antibodies and methods for their preparation and'isolation are disclosed in U.S. Patent Application entitled "Alpha-Synuclein Antibodies and Methods Related Thereto," filed on July 19, 2005, to Federoff et al.
96. Non-antibody ligands that selectively bind to a protein can also be used to detect the presence, the absence, and/or to quantify the expression of a protein. For example, ligands can be fluorescently labeled (e.g. conjugated to fluorescent molecule, such as green fluorescent protein (GFP)) or ligands can be radiolabeled. Labeled ligands can be contacted with a sample,

-26-~'lig~a~i't~~ ~ ~rotein can be assessed. The amount of labeled ligand that binds to proteins in the sample is an indication of the amount of a particular protein present in the sample. When the protein is a cell surface molecule, a protein ligand can be contacted to intact cellular sample to detect the expression level of the protein at the cell surface. Alternatively, the integrity of leukocytes in a sample can be compromised by permeabilizing or lysing the cells, and subsequently assessing the amount of labeled ligand that binds to proteins in the sample of lysed leukocytes.
97. Labels can be directly or indirectly attached to antibodies or non-antibody ligands.
Direct labeling includes, for example, attaching a label directly to the antibody or non-antibody ligand. Indirect labeling includes, for example, attaching a label to a second or third antibody or non-antibody ligand.
98. In cases where the level of expression of a protein is regulated at the genetic level, expression levels of the protein can be indirectly monitored by detecting the expression level of the gene that encodes the protein. Methods suitable for detecting and/or quantifying genetic expression that can be used include, but are not limited to, Northern blot, RNAse protection analyses, reverse transcription-polymerase chain reaction (RT-PCR), and gene-chip (e.g., nucleotide expression microarray) technologies.

99. Optionally, the level of expression of multiple proteins can be determined simultaneously or nearly simultaneously. For example, two-dimensional (2D) gel electrophoresis can be used to simultaneously or nearly simultaneously assess the expression level of thousands of proteins in a sample. (See e.g., Vietor and Huber, Biochim. Biophys.
Acta., 1997;1359:187-99, which is incorporated by reference herein for at least its teachings of methods to assess levels of protein expression). In one aspect, the disclosed methods can include 2D gel electrophoresis, where a mixture of proteins are prepared from the sample, e.g., by lysing leukocytes and mixing the protein lysate with sample buffer. The protein mixture can be loaded onto a gel slab, electrophoresed in two dimensions, and then the gel slab can be dried.
After resolution by 2D electrophoresis, expression levels of individual proteins or groups of proteins can be assessed. Protein levels can be assessed by silver staining or Coomassie staining.
If the proteins in a sample are labeled, then measuring the amount of label can be used to assess the amount of protein.

g) Levels of gene product expression and canonical variables 100. The detection of the levels of expression of the target genes, the genes disclosed herein as related to a particular neurodegenerative disease (e.g., Parkinson's and Alzheimer's)

-27-the presence of a neurodegenerative disease in a sample (e.g., blood) of a subject with the disease, i.e., biomarkers, can occur in any way as disclosed herein.

In some examples, what is typically required is the detection of nucleic acid (e.g., transcripts). In other examples, what is typically required is the detection of proteins. There are many means for detection of gene products, such as radioactivity or fluorescence or any other methods as disclosed herein. Any means can be used.
101. Typically any data that is collected can be normalized for general expression levels in the cell. This can be done in variety of ways, for example, by comparing all transcripts to that of 0-actin expression, which is present in all cells. Other methods of normalization can be based on approaches other than expression of any single gene. For quantitative PCR, a standard curve should be attained for each message assayed. These standard curves then become the basis for deriving absolute copy numbers. Internal controls include, but are not limited to, 0-actin, GAPDH, tubulin, etc. or external controls such as but not limited to cytoplasmic or nuclear LacZ, agamous, or known labeled cRNAs spiked into each hybridization. Using bio-chip approaches, data normalization can also be obtained through averaging samples of interest to the overall array background.
102. In certain examples related to hybridization, there can be a stripping and reprobing step that increases the specificity of the readings. For example, samples can be stripped and reprobed for the T7 promoter.
103. Typically once the expression pattern of a gene product or set of gene products is obtained, the expression pattern must be analyzed. The analysis typically involves performing some type of statistical analysis of the relative expression levels between the gene, genes, or gene sets themselves, as well as the comparison to the control or standard gene, genes, or gene sets. Such methods of analysis are disclosed herein.
104. In one example, a level of expression of a biomarker (e.g., a nucleic acid or protein gene product) can be subject to a univariant and/or multivariant canonical analysis to produce a first and/or second canonical variable. The univariate tests can be the well known T-test or the N-test. One N-test that is suitable for use herein is disclosed in Technical Report 04/01 at http=//www urmc rochester.edulsmd/biostat/people/techreports.html, which is incorporated by reference herein at least for its teaching of the statistical analysis via the N-test.
105. There are a number of methods of multivariate analysis. Any of these may be applied. In one example, multivariant analysis can be performed using commercially available software, such as SAS/STAT Software, available from SAS Institute, Inc.
(Cary, NC).

-28-'(9d$ rih ",~Il components analysis can be used, for example. These both deal with methods of analyzing matrices of data. Typically the canonical variables consist of a first canonical and a second canonical variable. The multivariant analysis can be the essentially non-parametric test for multiple testing inference. Such multivariate statistical testing relies on canonical discriminant analysis. This analysis determines the variables (messages) that best distinguish groups and assigns weights to each variable. The first canonical variable generally provides the best distinction between groups. The second canonical variable operates on the residual variance that remains unaccounted for by canonical variable 1.
Additional iterations are possible with diminishing effect.
106. Canonical discriminant analysis is equivalent to canonical correlation analysis between the quantitative variables and a set of diumny variables coded from the class variable.
In the following notation the dummy variables can be denoted by y and the quantitative variables by x. The total sample covariance matrix for the x and y variables is:

S S,x SXy SYX Sn, 107. When c is the number of groups, nt is the number of observations in group t, and St is the sample covariance matrix for the x variables in group t, the within-class pooled covariance matrix for the x variables is SP = E ~_cF(~,t -1)St 108. The canonical correlations, p;, are the square roots of the eigenvalues, N. of the following matrix. The corresponding eigenvectors are v;.
S,pl iz SXYSri 1S'Sp'/z 109. Letting V be the matrix with the eigenvectors v; that correspond to nonzero eigenvalues as columns, the raw canonical coefficients are calculated as follows.
R=Sp'zV

110. The pooled within-class standardized canonical coefficients are:
P = diag(Sp)'ZR

111. And the total sample standardized canonical coefficients are:
T = diag(SXX)"R

112. It may be calculated by any of the following:

-29-XaZ
X,diag(Sp)'P
,diag(Sxx)Y7 X, 113. For the Multhiariate tests based oi E-1H, where n is the total number of observations.
E = (n -1)(Syy - SS,,-1SXy) H = (n -1)SyxS,{,-1SXy 114. The above described multivariant analysis can, as previously noted, be performed with commercially available software such SAS/STAT Software, available from SAS Institute, Inc. (Cary, NC). In the methods disclosed herein, one can input the levels of expression from a set of gene products into such a program. The set of gene products can be any set of genes, such as the ones disclosed herein. In some examples, one set of gene products can correspond to a control sample or group of controls and another set of gene products can correspond to a sample or group of samples with disease. The levels of gene products can be inputted as absolute or relative amounts or concentrations. The levels can also be signal intensities, for example, from radio- or fluoroanalyses of the gene products. As noted, a result of the multivariant analysis is a first and/or second canonical variable.
115. This first and/or second canonical variable produced from the multivariant analysis can be used, as is disclosed herein, in the disclosed methods as a substitute for or in addition to the level of expression for a biomarker. That is, in some examples disclosed herein, the first and/or second canonical variable obtained from analyzing the levels of expression of one or more biomarkers from a subject can be compared to a reference standard that comprises a first and/or second canonical variable obtained from a multivariant canonical analysis of levels of expression of biomarkers from a control or group of control subjects.
116. For example, as shown herein, inultivariant canonical analysis was used herein as a diagnostic of Alzheimers disease. For example, when using genes related to inflammation, the range for control values for canonical variable 1 can be from about -0.5 to about -3.1 and the range for AD can be from about 0 to +4.4. For genes related to cell stress, the range for control values for canonical variable 1 can be from about -4.8 to about -0.1 and the range for AD can be from about +0.1 to about +4.1. For genes related to the cell cycle/cell death, the range for control values for canonical variable 1 can be from about +2.6 to about -3.1 and the range for AD can be from about +3.0 to about -2.3. Each of these three classes of gene products

-30-giia4~~ri crllrol to a degree that is statistically significant by the Wald-Wolfowitz Runs test, even though there may be some overlap. In terms of overlap between AD
and control, cell cycle/cell death has the most, inflammation has a one case overlap and cell stress shows no overlap.
117. It is noted that the results of the multivariant analysis will, of course, depend on the particular input (e.g., the particular gene products, the particular levels of those gene products, and the particular sample sets). As such the addition or substitution of other genes or the use of different diseases can alter the ranges for canonical variables.
This is illustrated by comparing Figure 4 and Figure 5, where the canonical variable 1 for cell stress is different in Figure 5 due to the inclusion of two PD patients in the control. Dispite variability in the particular value of canonical variable 1 and/or 2, distinction of diseases is still possible, as would be recognized by one of skill in the art (as is illustrated in Figure 5 for example).

2. Message class 118. At the core of the disclosed compositions and methods is the analysis of certain messages that are correlated with a neurodegenerative disease such as Parkinson's and Alzheimer's. In certain embodiments these messages can be single messages, but typically classes of messages, and sets of messages will be analyzed because they can provide more accurate assessment than for any one of the genes contained within the class or set by itself.
Table 4 shows exemplary targets that can be analyzed. Multiple gene products, whether mRNA

or protein, analyzed simultaneously, allows a neurodegenerative disease such as Parkinson's or Alzheimer's to be diagnosed.
119. For example, one class of genes that are useful in diagnosing a neurodegenerative disease such as Alzheimer's is the class of cell cycle transcripts. These can include, for example, cyclin D1, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, and protein kinase C alpha.
120. In another example, a class of genes that are useful in diagnosing a neurodegenerative disease such as Alzheimer's is the class of inflammatory response transcripts.
These can include, for example, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr.

121. In still another example, a class of genes that are useful in diagnosing a neurodegenerative disease such as Alzheimer's is the class of cell stress transcripts. These can include, for example, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin.
122. In yet another example, any combination or subset of cyclin Dl, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, Cl inhibitor, IL-17r, IL-8,

-31-i'p"tk1-l antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin can be used.
123. In still other examples, other genes related to cell cycle/death, inflammation, and stress, as would be know to those of skill in the art, can be used.
124. In another example, a class of genes that are useful in diagnosing a neurodegenerative disease such as Parkinson's disease include, for example, HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 epsilon, Prohibitin, Phospoglycerate mutase 1, Apoliporotein AI, Superoxide dismutase, RNA-binding protein regulatory subunit, Chain A
thioredoxin peroxidase B, RAS-related protein RAP1B, Tumor rejection antigen, Haptoglobin, Fibrin beta, actin-interacting protein 1(AIP1), mitogen activated protein kinase I(MAPKI), actin or a fragment thereof, glutaraldehyde-3-phosphate dehydrogenase (GAPDH), transforming protein RhoA, acidic leucine-rich nuclear phosphoprotein 32 family member B
(ANP32B or APRIL), peroxiredoxin II, an amyloid precursor protein (APP), a-secretase, 0-secretase, y-secretase, Ao peptide, Fe65, Tip60, SERCA, PS 1/2, nectin-la, and non-amyloid 0 component of senile plaque (NACP/ a-synuclein).
125. There can be any number of gene products in the sets or classes. For example, there can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or 100 different genes or gene products within a set or class. Furthermore, it is understood that there could be multiple alleles of a particular gene which could also make up a set. In certain embodiments, there will be 7 or 8 genes within a set of transcripts to be used for analysis.
126. In certain embodiments one gene can be used for analysis, such as gene products related to alpha-1 antichymotrypsin, crystalline, and cycloogygenase I[.

Table 4: Exemplary targets for neurodegenerative diseases, such as Alzheimer's and Parkinson's, which can be analyzed. General classes of these targets are also provided, though targets can have functions that impact a variety of cell processes other than those identified.
SEQ ID Gene Name NCBI Acc No Function Comments Class NO:
Alpha-l- Inflamin-1 Alphal-ACT J05176 antichymotrypsin ation / cell mRNA cycle / stress Alpha-l- cDNA Inflamm 2 Alphal-ACT T40002 antichymotrypsin, ation / cell precursor mRNA clone cycle / stress

- 32 -~..~ waE w Activates Thiol 3 GAPDH M17851 group during Metabolic catalysis cAMP response 4 CREB M34356 element binding Transcrip-4 factor protein Cell specific marker distinguishes Astrocyte .5 GFAP J04569 astrocytes from other marker glial cell during development of CNS
Developmental-upon terminal neural differentiation, Marker of 6 Nestin X65964 nestin is down DNA early regulated and neurons replaced by neurofilaments Involved in proteolytical 7 PS 1/S 182 AF416717 processing of APP Proteolytic and NOTCH, enzyme regulates epithelial-cadherin function Involved in stress 8 HSP27 NM001540 resistance and actin Stress organization Molecular 9 HSP90-alpha NM 005348 chaperone, has Stress ATPase activity by similarity Molecular HSP90-beta NM 007355 chaperone, has Stress - ATPase activity by similarity Contain intermediate filament proteins N, Neuron 11 NF-M Y00067 M and H involved in DNA structure maintenance of neuronal caliber Contain intermediate filament proteins N, not Neuron 12 NF-L X05608 M and H involved in complete structure maintenance of cds, DNA
neuronal caliber 13 C-jun AY217548 Transcription factor DNA Transcrip-AP-1 tion factor Tuberin/549 and Titberin/4B2 For these 2 tuberins listed, see Cell TSC1 and TSC2 below: cycle/death

- 33 -P.
Tuberous sclerosis 2 protein: Implicated as tumor suppressor, may have function in vesicular transport, may also play role in regulation of cell growth arrest and in regulation of transcription mediated by steroid receptors, interaction of hamartin and mRNA, tuberin may not Cell 14 TSC2 X75621 facilitate vesicular complete cycle/death docking, specifically cds stimulates the intrinisic GTPase activity of Ras-related protein RAPlA and RAB5, suggests possible mechanism for role in regulating cell growth, mutations in tuberin lead to constitutive activation of RAP1A
in tumors Hamartin, tuberous sclerosis protein 1, implicated as tumor suppressor, may 15 TSC1 NM 000368 have function in Cell - vesicular transport, cycle/death interaction between hamartin and tuberin may facilitate vesicular docking 16 IAP homo B NM 001166 Apoptotic Cell - suppressor cycle/death 17 Kinesin light NM182923 May play a role in Molecular chain 1 - organelle transport motor Force producing 18 Kinesin light NM022822 protein, may play a clone Molecular chain 2 - role in organelle motor transport,

-34-Intracellular Stress and inflamm-molecule, stores inflamm-19 Ferritin L NM000146 iron in a nontoxic, ation / ROS
soluble, readily (reactive available form oxygen s ecies) Functional molecule is roughly spherical-polymeric ferric iron Stress and 20 Ferritin H NM 002032 inflamm-- core is deposited into it's central ation / ROS
cavity Destroys radicals normally produced 21 SOD-1 NM_000454 within cells, and Stress and which are toxic to ~~ation biological systems 22 Alpha tubulin N1VI 006082 Major constituent of Cell - microtubules structure Suppresses apoptosis in variety of cell systems, regulates cell death by controlling mitochondrial 23 Bcl-2 M13995 membrane Cell death permeability, appears to function in feedback loop system with caspases, inhibits caspase activity Activation 24 ICE-rel-II U28014 cascade/apoptosis Cell death execution, cleaves caspace I
Interleulin 1 beta converting enzyme, thiol protease cleaves IL-1 beta between an ASP and an ALA, releasing Cell death /
25 ILIBCE M87507 mature cytokine inflamYna-which is involved in tion a variety of inflammatory processes, specifically inhibited by cowpox virus CRMA protein

-35-... E.._ Accelerates programmed cell death by binding 26 BAK NM 001188 to/antagonizing the Cell death - A suppressor BCL1-2 or its adenovirus homolog ElB 19K
protein Retards apoptosis 27 Bfl-1 NM_004049 induced by IL-3 Cell death de rivation GTP-binding nuclear protein RAN, involved in nucleocytoplasmic 28 RAN TC4 N1VI_006325 transport, required Stress for the import of proteiui into the nucleas and for RNA
export 29 Ras-L, TC25 XM 171081 Regulates cellular Stress - responses Involved in control 30 Cdk4 AF507942 of cell cycle, cell DNA
division protein kinase 4 31 Cyclin B1 P14635 Control of cell cycle clones Cell cycle/
in G2/1VI transition only death Role in growth regulation, 32 Cyclin Gl NM 004060 associated with Cell cycle/
- G2/M phase arrest in death response to DNA
damage Regulates CDK7, involved in cell 33 Cyclin H NM 001239 cycle control and in Cell cycle/
- RNA transcription death by RNA polymerase II
May be involved In 34 Cyclin Al NM 003914 cell cycle at G1/S Cell cycle/
- and G2/M death transitions Essential in cell Cell cycle/
35 Cyclin A2 AF518006 cycle at Gl/S and DNA
G2/M transitions death

36 Cyclin E1 AF518727 Control of cell cycle DNA Cell cycle/
at G1/S transition death Essential for control

37 Cyclin E2 AF106690 of cell cycle at late Cell cycle/
death Gl and eary S phase Role in growth

38 Cyclin G2 NM. 004354 regulation, negative Cell cycle/
- regulation of cell death cycle progression Essential for control Cell cycle/

39 Cyclin Dl NM_053056 of cell cycle at Gl/S death transition Human heat shock

40 HSP70 M11717 protein (hsp 70) DNA Stress gene, complete Heat shock 70 kDa protein 1, in cooperation of other chaperones, HSP70S
stabilize pre-existent proteins against aggregation and mediate the folding of newly translated polypeptides,

41 HS71 NM_005345 HSP70S in Stress mitochondria and endoplasmic reticulum provide driving force for protein translocation, are involved in signal transduction pathways with Heat shock-related 70 kDa protein 2, HSP70S stabilize pre-existent proteins

42 HS72 NM_021979 against aggregation Stress and mediate the folding of newly translated ol e tides

43 HS74 NM 002154 Heat shock 70 kDa Stress rotein 4

44 HS76 X51757 Heat shock 70 kDa DNA Stress protein 6 Heat shock 70 kDa DNA,

45 HS77 M11236 gene Stress protein (fragment) segment Regulates the development of cells destined to form and maintain skeleton, Cell

46 c-fos K00650 thought to have role DNA
cycle/death in signal transduction, cell proliferation, and differentiation May act as negative Cell

47 Weel X62048 regulator of entry into G2/M transition cycle/death

48 Fral X16707 Fos-related antigen 1 DNA Cell cycle/death Transcriptional repressor of genes that require BHLH

49 Hes1 AF264785 protein for Transcrip-transcription, may tion factor act as negative regulator of myogenesis see 16 CR genes Stress crystallin below inflamma-ion Alpha crystallin A
chain, may Stress contribute to the

50 CRAA NM 000394 inflamma-transparency and ion refractive index of the lens Rosenthal fiber protein, Alpha crystallin B chain, Stress

51 CRAB NM 001885 may contribute to inflamma-- the transparency and ion refractive index of the lens- Acc#

Alpha crystallin C Stress

52 CRAC NM_014365 chain, protein kinase inflamma-H11 ion Breta crystallin B 1, Stress

53 CRB1 U35340 dominant structural inflamma-components of vertebrate eye lens ion !I ' .N... "~ .. f. ,::!~ ~...(t .. ~ !,::w. ".':::,t ;-.. Beta crystallin B2, dominant structural Stress

54 CRB2 NM 000496 inflamma-components of vertebrate eye lens ion Beta crystallin B3, dominant structural Stress

55 CRB3 P26998 inflamma-components of vertebrate eye lens ion Beta crystallin A3, Stress

56 CRBA P05813 dominant structural inflamma-components of vertebrate eye lens ion Beta crystallin A2, Stress

57 CRBB AF166331 dominant structural inflamma-components of ion vertebrate eye lens Beta crystallin A4, Stress

58 CRBD NM 001886 dominant structural inflamma-components of ion vertebrate eye lens Beta crystallin S, Gamma crystallin S, Stress

59 CRBS NM017541 dominant structural inflamma-components of ion vertebrate eye lens Gamma crystallin A, gamma crystallin 5, Stress

60 CRGA P11844 dominant structural inflamma-components of ion vertebrate eye lens Gamina crystalllin B, dominant Stress

61 CRGB M19364 structural DNA inflamma-components of ion vertebrate eye lens Gamma crystallin C, Stress

62 CRGC NM 020989 dominant structural inflamma-components of ion vertebrate eye lens Gamma crystallin D, Stress

63 CRDG NM 006891 doininant structural inflamma-components of ion vertebrate eye lens Lamda crystallin Stress

64 CRYL NM 015974 homolog, inflatnma-ion P' ' 77,j .::I L.., mu-crystallin homolog, binds thyroid hormone, presumably involved in regulation of free Stress

65 CRYM NM 001888 intracellular concentration of tri- inflamma-iodothyronine and ion access to its nuclear receptors, expressed in neural tissue, muscle and kidney Required in higher Cell

66 Cdc2 AF512554 cells for entry into S DNA
phase and mitosis cycle/death

67 hTR2-11 M29960 Human steroid unknown receptor May have regulatory role in membrane interactions during

68 Synaptotagmin NM_005639 trafficking of Synapse synaptic vesicles at active zone of synapse Believed to interact

69 AP180 AB014556 with cytoplasmic Synapse tails of membrane proteins Promotes survival of

70 BDNF M61176 neuronal populations Cell death located in CNS or directly connected

71 bcl-xl U72398 Potent inhibitor of DNA Cell death cell death Suppresses

72 bcl-2 P10415 apoptosis, regulates Cell death cell death, inhibits caspase activity Buffers cytosolic hlflamma-

73 calbindin D2 X06661 calcium tion / cell cycle / death Destroys radicals

74 SOD-1 NM_000454 normally produced Stress / cell death and which are toxic Reduces low molecular weight Stress /

75 Glutaredoxin NM002064 disulfides and inflamma-proteins defends tion against ROS

w. !. 7Kinesin aa .;~..,~k r.,,,~ Microtubule-associated force- Not Molecular

76 H X65873 producing protein, complete may play a role in cds motor organelle transport

77 PKC-type beta X07109 type beta I Cell II cycle/death Ras family small Cell

78 Rit U71203 GTP binding protein RIT proliferation

79 Rin U71204 Also see 3 Rin genes Cell death below Ras effector protein, may serve as

80 Rinl L36463 inhibitory modulator Cell death of neuronal plasticity in aversive memory formation Ras effector protein, may function as upstream activator and/or downstream

81 Rin2 NM_018993 effector for RAB5B, Cell death may function as guanine nucleotide exchange(GEF) of Potential Ras effector protein, may

82 Rin3 AL159141 function as guanine Cell death nucleotide exchange (GEF) Tyrosine-preotein kinase receptor, may

83 Protein tyrosine D50479 be involved in cell Cell death kinase adhesion processes, particularly central nervous system

84 NAIP NM 004536 Prevents motor Cell death nueron a o tosis Key role in synaptic plasticity,synaptogen esis, excitotoxicity, NMDA Rec memory aquisition

85 (zetal) L13266 and learning, Synapse mediates neuronal functions in glutamate neurotransmission : (I__, IT Human alpha-actin

86 Alpha actin J05192 (ACTA) mRNA, Cell structure complete cds

87 Beta actin X00351 ~volved in various DNA Cell es of cell motility structure leads to the

88 Topoisomerase NM003286 conversion of one Cell I - topological isomer cycle/death of DNA to another Makes double-strand breaks, transient

89 Topoisomerase ~_001067 breakage and Cell II subsequent rejoining cycle/death of DNA strands CDK4 inhibitor p16 see the three CDK Cell below cycle/death Cyclin-dependent kinase 4 inhibitor A, p 161NK4, interacts strongly with CDK4

90 CDKN2 L27211 and CDK6, inhibits Cell its ability to act with cycle/death cyclins D, could act as negative regulator in proliferation of normal cells Cyclin-dependent kinase 4 inhibitor B, interacts strongly with CDK4 and Cell

91 CDKN2B U17075 CDK6, potent cycle/death inhibitor, potential effector of TBF-beta induced cell cycle arrest Cyclin-dependent kinase 4 inhibitor D, Cell

92 CDKN2D U49399 interacts strongly cycle/death with CDK4 and Attach integral membrane proteins

93 Ankyrin 2, Z26634 to cytoskeletal not Cell Brain elements, also bind complete structure to cytoskeletal proteins ..t Attach integral membrane proteins Cell

94 Erythrocyte M28880 to cytoskeletal structure elements, bind to cytoskeletal proteins

95 PIG3 AF010309 Cell death Accelerates

96 BaxA L22473 programmed cell Cell death death, membrane isoform alpha Accelerates

97 BaxB L22474 programmed cell Cell death death, cytoplasmic isoform beta C 1 q see the three C 1 Q Inflamma-genes below tion Complement Clq subcomponent. A
chain (precursor);
associates with

98 C1QA NM015991 coenzymes C1R and Inflamma-1 S to yield C l-the tion first component of the serum complement system Complement Clq subcomponent. B
chain (precursor);
associates with not

99 C1QB X03084 coenzymes C1R and complete ~flarruna-C 1 S to yield C l-the cds tion first component of the serum complement system Complement Clq Inflamma-

100 C 1 QC NM_l 72369 subcomponent C Clone tion ( recursor

101 C1RF NM 006688 C1Q related factor Inflamma-- tion Splicing factor, arginine/serine-rich

102 SF2 Flag NM_006924 1, pre-mRNA Splicing splicing factor SF2, factor P33 subunit, plays a role in exon skipping

103 MCIF AF273052 CTCL tumor antigen Cell death se70-2 May play a role in post synaptic

104 APP1 rTM005166 function, can Cell death regulate neurite outgrowth May play a role in the regulation of hemostasis, may have inhibitory Organelle

105 APP2 NM_001642 properties towards trafficing coagulation factors, may interact with G-protein signaling pathways Possibly involved in structural functions as organizing other

106 Synaptophysin P08247 membrane synapse components or in targeting vesicles to plasma membrane Promotes

107 Tau J03778 microtubule Cell assembly and structure stability Acid protease active in intracellular protein break down,

108 Cathepsin D NM_001909 involved in Stress pathogenesis such as breast cancer and Alzheimer's Disease

109 GAP 43 NM 002045 Associated with Cell growth - nerve owth Glutathione S-transferase Mul, conjugation of reduced glutathione Stress / cell

110 pGTH4 J03817 to a wide number of death exogenous and endogenous hydrophobic electrophiles Microsomal glutathione S-transferase 1, conjugation of reduced glutathione

111 pHMGST J03746 to a wide number of Stress / cell exogenous and death endogenous hydrophobic electrophiles, has wide substrate specificity Arginine/serine rich splicing factor 10, sequence-specific

112 Tra2-C2 U61267 RNA-binding Splicing protein which factor participates in control of pre-mRNA splicing

113 MCD-C2 NM 012470 Transporin-SR unknown Involved in ATP-dependent selective degradation of proteins,

114 Ubiquitin X1VI 055013 maintenance of Stress / cell - chromatin structure, death regulation of gene expression, stress response, ribosomal biogenesis Putative pre-mRNA
splicing factor RNA

115 Pht6 AB011149 helicase, probable Splicing ATP-binding RNA factor Helicase involved in pre-mRNA splicing Facilitative glucose

116 pGHT1 P11166 transporter, glucose Stress / cell transporter type 1, death erythrocyte/brain Epidermal growth Stress /

117 CR3 AF251550 factor-like cripto DNA inflamm-rotein CR3 ation .. . ..{ . "!t õd Implicated in various adhesive interactions of monocytes, macs Stress /

118 CR3 alpha NM000632 and granulocytes, as inflamm-chain well as mediating uptake of ation complement-coated particles

119 C5 rlM 001735 Complement C5 Stress /
precursor inflamm-ation Mitochondrial ene See list below:
Component of mitochondrial

120 MPR-S12 015235 ribosome small unit Cell energy which comprises a 12S rRNA and 30 distint proteins 28S ribosomal protein S 16.
Homosapiens mitochondrial

121 MRP-16 NM 016065 ribosomal protein Cell energy S16 (MRPS16) nuclear gene encoding mitochondrial protein, mRNA
Cytochrome C
oxidase polypeptide I, Homo sapiens mitochondrial DNA, 16559 bp, component of

122 COI D38112 respiratory chain that DNA Cell energy catalyzes the reduction of oxygen to water.
Cytochrome C is subunits 1-3 form functional core for enzyme com lex Cytochrome C
oxidase polypeptide II, subunits 1-3 form fi.inctional core for

123 COII M90100 enzyme coinplex, Cell energy homosapiens cyclooxygenase-2 (Cox-2) mRNA, complete cds Cytochrome C
oxidase polypeptide III, subunits 1-3 form functional core

124 COIII J01415 fH~an e complex. DNA Cell energy mitochondrion, complete genome, 16569 bp, DNA
circular PRI
H. sapiens mitochondrial genome, 16569 bp DNA circular PRI
mitochondrial gene includes: 12S, 16S
ribosomal RNA, 22 tRNA, ATPase subunit 6 8, (NADH
dehydrogenase

125 V00662 subunits 1, 2, 3, 4, DNA Cell energy 4L, 5, 6), cytochrome b, (cytochrome c oxidase subuits I, II, III) (tranfer RNA:
Arg, Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val) ... ,.,.. ,. ~.., , z .. ,,,.. ,..., . .,.. .,.,.,. , õ õ
Complement factor D precursor. Factor D cleaves factor B
when latter is Conlplement complexed with Stress /

126 NM_001928 factor C3B, inflamma-factor D activating the tion / death C3BBB complex, which then becomes the C3 convertase of alternate pathway Activation of Cl complex under control of Cl Stress /

127 Cl inhibitor M13656 inhibitor; may play inflamma-important role in tion / death physiological pathways Function not yet Stress /

128 Clusterin NM 001831 clear, associated inflamma-- with programmed tion / death cell death Complement factor 1 precursor, responsible for cleaving alpha- Stress /

129 Factor 1 NM 000204 chains of C4B and inflamma-- C3B in the presence tion of cofactors C4-binding protein and Factor H
respectively

130 S-protein NM 001264 Expressed in skin Plays a central role Stress /
C3a in the activation of

131 ~aphylatoxin ~-000064 the complement inflamma-anaphylatoxin / death system Activation of C5 by a C5 convertase initiates the spontaneous Stress /

132 C5a ~ 001735 assembly of the late inflarnma-anaphylatoxin - complement tion / death components C5-C9 into the membrane attack complex ~ ~ .. = . t Mediates cellular binding of particles Stress /

133 Complement Y00816 and immune inflatruna-receptor typel complexes that have tion / death activated complement Receptor for complement 3CDd and for Epstein-Barr Stress /

134 Complement NM001877 virus on human B- inflamma-receptor type2 - and T-cells, tion / death participates in B
lymphocytes activation May play important Stress /

135 COXl M59979 role in regulating or inflainina-promoting cell tion roliferation Prostaglandin G/H
synthase 2 precursor, may have role as Stress /

136 COXII NM 000963 major mediator of inflamma-- inflamnlation and/or tion role for prostanoid signaling in activity-de endent plasticity Mouse ATP-specific succinyl-CoA synthase: see below:
phosphorylation of

137 Q9P2R7 the tricarboxylic acid Cell energy cycle Interleukin-I
Interleukin I receptor, type I Stress /

138 NM 000877 precursor, receptor inflamma-receptor - for IL-1A, IL-1B, IL- tion Basic transcription

139 transcription Q02446 factor factor SP4 Interleukin 17 Interleukin-17 Stress /

140 NM 014339 receptor precursor inflainma-receptor tion Pf =K .::F= ~ -- =~:~ = ~'~" ~ ~' ''' !~ ~E Chemotactic factor attracts neutrophils, basophils, and T-cells; involved in Stress /

141 Interleukin 8 NM_000584 neutrophil inflamma-activation, released tion in response to inflammatory stimulus Stimulates Stress /

142 Interleukin 15 U14407 proliferation of T- inflamma-lym hocytes tion Interleukin 15 Interleukin 15 Stress /

143 receptor U31628 receptor alpha chain inflamma-recursor tion Stimulates migratory response CD4+
lymphocytes, monocytes, and Stress /

144 Interleukin 16 AF053412 eosinphils; induces inflamma-T-lymphocyte tion expression of interleukin 2 receptor; ligand for Interleukin 10 Receptor for IL-10; Stress /

145 NM001558 binds IL-10 with inflamma-receptor alpha high affinity tion Receptor for IL-10 and IL-22, serves as accessory chain Interleukin 10 essential for active Stress /

146 inflamma-receptor beta Z17227 IL-10 receptor inflamma-complex and to tion initiate IL- 10-induced signal transduction events Leukemia inhibitory factor, precursor, has Stress /

147 LIF NM 002309 the capacity to inflamma-- induce tenninal tion differentiation in leukemic cells Can induce cell death of certain tumor lines; potent

148 TNF-alpha M10988 pyrogen; under Inflamma-certain conditions tion / death can stimulate cell proliferation; induce cell differentiation Controls proliferation,

149 TGF-Betal NM 000660 differentiation; Growth regulates actions of factor many other growth factors Interleukin 12 Can act as growth Stress /

150 alpha chain NM_000882 factor for activated T inflamma-and NK cells tion Receptor with high affinity for TNFSF2/TNF-alpha,

151 TNF-R2 NM_001066 TRAF1/TRAF2 Cell death complex recruits apoptotic suppressors Tuinor necrosis factor receptor family superfamily member 11B
(precursor), acts as Osteoclastogene decoy receptor for

152 sis inhibitory NM 002546 RANKI' thereby Cell death factor - neutralizes its function in osteoclastogenesis, inhibits activation of osteoclasts and promotes osteoclast apoptosis in vivo Endothelium-

153 Endothelin-2 NM 001956 derived Vasoconstric vasoconstrictor -tion peptides Endothelium-

154 Endothelin-3 NM 000114 derived Vasoconstric vasoconstrictor -tion peptides Non-specific

155 Endothelin M74921 receptor for Cell death receptor Endothelin 1, 2 and U 7 -_ r. : u 1 ...[ :' :,! mRNA, Stress Cytokine receptor not /

156 CCR5 X68149 binds to BLC coiuplete inflanima-tion cds homo sapiens Human chromosome 17

157 chromosome 17 AC005837 sequence, clone sequence hRPK.318_A_15,co mplete sequence Human DNA
sequence for clone RP5-1104E15 on chromosome 22 q12.3-13.1, contains MGAT3 gene for mannosyl (beta-1,4-)-glycoprotein beta-1,4-N-acetylglucosaminyl transferase, the gene for a predicted Human protein, the ATF4

158 chromosome 22 AL022312 gene for activating Cell energy sequence transcription factor 4(tax-responsive enhancer element B67), and 5' end of CACNAII gene for voltage-dependent calcium channel, alpha II subunit, contains ESTs, STSs, GSSs, and 5 putative CpG
islands, complete sequence Required for T-cells ni.A' Stress /

159 CD45 Y00062 activation through inflamma-the antigen receptor c dmplete tion c 1 ,! _ ,. 3175i ~ .. Low affinity immunoglobulin gamma Fc region receptor II-C
precursor; involved Fc gamma R in variety of effector Stress / cell

160 type II C U90938 and regulatory death functions such as phagocytosis of immune complexes and modulation of antibody production by B-cells

161 LOX-1 or OLR- NM002543 Stress / cell 1 - death Involved in plasma clearance of chylomicron remnants and activated alpha 2-macroglobulin, also Inflamma-

162 LRP-1 NM_002332 in the local tion metabolism of complexes between plasminogen activators and their endogenous irihibitors Expressed by macs in chronic mRNA

163 MRP(8) Y00278 inflammations, not Inflamma-cystic fibrosis complete tion antigen, calgranulin cds A
ATP-binding

164 Q9BX80 also: AF352582 cassette transporter Cell energy Is the beta-chain of major Inflamma-

165 B2 NM 004048 histocompatibility microglobulin - tion complex class I
molecules

166 ENA-78 U12709 Involved in DNA Inflamma-neutro hil activation tion Plays a critical role Inflamma-

167 CD74 NM 004355 in MHC Class II
tion antigen processing

168 Ribosomal AA366442 Cell death rotein S 4 See also 5 clathrin

169 Clathrin AA361745 genes listed below: synapse Synaptosomal-associated protein, Clathrin coat assembly protein AP180, adaptins are components of adapter complexes that link clathrinto Synapse /

170 A180 NM_014841 receptors in coated cell cycle /
vesicles, protein death complexes are believed to interact with cytoplasmic tails of membrane proteins, leads to their selection and concentration Clathrin light chain A, is major protein Synapse /

171 CLCA M20471 of polyhedral coat of cell cycle /
coated pits and death vesicles Clathrin light chain B; major protein of Synapse /

172 CLCB M20469 polyhedral coat of cell cycle /
coated pits and death vesicles Clathrin heavy chain 1; major protein of polyhedral coat of coated pits and Synapse /

173 CLH1 NM_004859 vesicles, two different adapter link cell cycle /
death clathrin lattice to either plasma membrane or to trans golgi network n~ .,R++:... . õr. . T. . a ua. :. m.,.. .R ..
.. . .. ,.
"!i , ... .t .. . ,.., t .., ..
Protein complexes link clathrin lattice to either plasma membrane or to trans golgi network;
Clathrin heavy chain 2, major protein of

174 CLH2 U41763 polyhedral coat of DNA
coated pits and replication vesicles, two different adapter protein complexes link clathrin lattice to either plasma membrane or to trans golgi network Is auxiliary protein of DNA polymerase

175 PCNA M15796 delta and is involved Cell cycle in control of eukaryotic DNA
replication

176 P55cdc NM 001255 CDC20 cell division Cell cycle /
cycle 20 homolog death Functions as dosage-dependent inducer in mitotic control, is a tyrosine protein phosphatase required for progression of cell cycle, it directly Cell cycle /

177 cdc25A (MPI1) NM_001789 dephosphorylates death CDC2 and activate its kinase activity, also dephosphorylates CDK2 in complex with cyclin E, in vitro Functions as dosage-dependentinducerin mitotic control, is a tyrosine protein

178 Cdc25B (MPI2) M81934 phosphatase required Cell cycle /
for progression of death cell cycle, it directly dephosphorylates CDC2 and activate its kinase activity ,. e ,,.. :.m . ! ... . ~I a li ... ... !E :Ic ...... .
Apoptotic adapter molecule recruits caspase-8 or caspase-10 to the activated FAS
(CD95) or TNFR-1

179 FADD NM_003824 receptors, resulting Cell death aggregate called DISC-death inducing signaling complex- performs caspase-8 proteolytic activation Essential conlponent of SCF ubiquitin ligase complex (serves as adapter to links F-box protein to CULl) which Cell cycle

180 Skpl U33760 mediates death ubiquitination of proteins involved in cell cycle progression, signal transduction, transcription Involved in activation cascade of caspases responsible for apoptosis execution, recruited to both FAS and Cell cycle /

181 Mch4 U60519 TNFR-1 I; n FADD death dependent manner;
cleaves, activates several caspases and hydrolyzes some small molecule substrates Participates in Wnt signaling pathway, implicated in hormonal control of

182 GSK-3B NM 002093 several regulatory Cell cycle /
- proteins including death glycogen synthase, myb, and transcription factor c jun li~'' ~w;: 4I~" ~.1~ :t=.!i ~~ f~ ~~ ,,E ~ ' ii ~' !!;,:it f~.,il.. :::i-.:C.. Involved of preferential repair of

183 ERCC6 NM 000124 active genes, DNA repair - presumed DNA or RNA unwinding function May play a role in terminal differentiation of Cell

184 SKI X15218 skeletal muscle cells differentia-but not in tion determination of cells to myogenic lineage Microtubule associated protein RP/EB family

185 EB1: NM 012325 member 1, maybe Cell involved in structure microtubule polymerization and spindle function Cytochrome C

186 or NM 004718 oxidase VII-a related Cell energy protein, mitochondrial Phosphorylates serine- and arginine-rich (SR) proteins of spliceosomal complex, may be

187 CLK3 L29217 constituent of Splicing network of regulatory mechanisms that enable SR proteins to control RNA
splicing Transferrin receptor protein 1; transferrin

188 Transferrin R NM 003234 receptor is necessary Stress / ROS
- for development of erythrocytes and nervous system Transferrin receptor protein 2; mediates

189 Transferrin R NM 003227 cellular uptake of Stress / ROS
- transferrin-bound iron in non-iron dependent manner ,ttq Calcium-activated, phospholipid-dependent serine-and threonine-specific enzyme, PKC is activated by diacylglycreol which Stress / cell

190 PKC-a X52479 in turns /
phosphorylates a cycle death range of cellular proteins; also serves as the receptor for phorbol esters, a class of tumor promotors CDK activating kinase, transcription factor, CDK's are

191 CAK NM 001799 activated by binding Cell cycle - to a cyclin, mediate the progression through the cell cycle Tyrosine kinase of the non-receptor type, involved in the IFN- Inflamma-

192 JAK-1 NM_002227 alpha/beta/gamma signal pathway, tion / stress kinase partner for the interleukin (IL)-2 receptor Mitogen-activated

193 MAPKAP NM 004635 protein kinase Cell cycle /
- activated protein death kinase 3 Glutathione S
transferase P, conjugation of reduced glutathione

194 GSTP NM_000852 to a wide number of Cell energy exogenous and endogenous homophobic electrophiles 1~ . '. ~ ::Sf . ~~:.:~ ..::~t Involved in the activation cascade of caspases responsible for apoptosis execution, cleaves

195 Apopain U26943 and activates sterol Cell death regulatory element binding proteins (SREBPS), involved in the cleavage of Huntingtin Single stranded DNA endonuclease involved in DNA

196 ERCC5 NM 000123 DNA repair excision repair, makes the 3' incision in the repair ATP-dependent 3'-5' DNA helicase, component of the core-TFIIH basal transcription factor,

197 ERCC3 NM 000122 involved in DNA repair - nucleotide incision repair of DNA, and, when complexed to CAK in RNA
transcription by RNA polymerase II
CCAAT-BP see 9 genes below transcription Stimulates transcription of various genes by

198 CBFA NM 006166 recognizing/binding transcription - to CCAAT motif in promotors, CCAAT-binding transcription factor subunit A
Stimulates transcription of various genes by

199 NFYA M59079 recognizing/binding transcription to CCAAT motif in promotors, CCAAT-binding transcription factor subunit B

Stimulates transcription from

200 CBF NM_005760 the HSP70 Stress /
promotor, CCAAT- transcription box-binding transcription factor DNA binding protein recognizes CCAAT homolgy common to many promotors and

201 CEBA U34070 enhanced core DNA Transcrip-homology common tion to many enhancers, CCAAT/ enhancer binding protein alpha Important transcriptional activator in the regulation of genes Transcrip-

202 in the tion /
202 CEBB X52560 immune and DNA inflamma-inflammatory tion response, CCAAT/enhancer binding protein beta DNA binding protein recognizes CCAAT homolgy common to many promotors and enhanced core Transcrip-

203 CEBD NM 005195 homology common tion /
to many enhancers, inflamma-involved in immune tion and inflammatory response, CCAAT/binding enhancer protein delta a} %.: ,. L DNA binding protein recognizes CCAAT homolgy common to many promotors and Transcrip-

204 CEBE U48865 enhanced core DNA tion homology common to many enhancers, CCAAT/enhancer binding protein e silon Transcription factor binds to enhancer elemnt PRE-lof IL4 Transcrip-

205 CEBG NM_001806 gene, tion CCAAT/enhancer binding protein gamma Probably has role as repressor of developmentally regulated gene expression,may act Transcrip-

206 CUT1 M74099 by preventing tion / death binding of positively-avtiving factors to promotors, CCAAT
displacement protein

207 HLHP Id2 NM 002166 DNA-binding Cell cycle /
- protein inhibitor ID2 death Guanine nucleotide-binding protein G(S), alpha subunit (adenylate cyclase Cell energy I
G-S alpha stimulating G alpha death / many

208 subunit P04895 protein), involved as other modulators or ftmctions transducers in various transmembrane signaling systems aE T.~. . ::::u : ,w-, .w:;~: . i~;::.. õ i~ ' :s .,, .. Involved in chromatin remodeling, part of complex that opens Transcrip-

209 1NI1 NM_003073 the chromatin to tion facilitate transcriptional machinery to access their targets Activates erythroid

210 LCR-F1 U08853 specific, globin gene expression Tumor necrosis factor receptor superfamily member 16, low affinity Cell owth

211 Low affmity ~_002507 receptor can bind to death NGF, BDNF, NT-3, NT-4, mediates cell survival and cell death of neural cells Receptor for EGF, is

212 EGF-R U48722 involed in control of Cell growth cell growth and differentiation Insulin receptor,

213 Insulin R NM 000208 binds insulin, has Cell energy - tyrosine-protein kinase activity Alpha-l-catenin, cadherin associated protein, associates with cytoplasmic domain of variety of cadherins,

214 A-Catenin NM 001903 association of Cell cycle /
- catenins to death cadherons produces complex linked to actin filament network, may play crucial role in cell differentiation Alpha-2-catenin, Cell cycle /

215 NM_004389 alpha catenin related death rotein Receptor for

216 Integrin a-3 M59911 fibronectin, laminin, Inflanuna-collagen, epilegrin, tion / death thrombospondin Integrin alpha-5 precursor, Integrin alpha-5Beta1 is Fibronectin R receptor for hiflamma-

217 alpha P08648 fibrinogen and tion / death fibronectin, it recognizes the sequence R-G-D and its ligands Cytokine stimulates growth and

218 GM-CSF M11734 differentiation of Growth hematopoietic factor precursor cells from various lineages

219 Glu-6-P A1250347 Cell energy isomerase Chemotactic factor that attracts monocytes and

220 MCP-1 M24545 basophils, augments ~flamma-monocyte anti-tumor tion / death activity, binds to CCR2 and CCR4 Heparin binding

221 Pleiotrophin NM 002825 mitogenic protein, Cell cycle - has neurite extension activity Important role in organization of cytoskeleton-binds, Cell

222 Thymosin b-10 NM021103 sequesters actin structure monomers (G actin), therefore inhibits actin polyinerization Highly basic Soares fetal liver

223 protein R94142 spleen 1NFLS homo Unknown sapiens clone Required for the biosynthesis of the tetrasaccharide

224 XGPT NM 007255 linkage region of Cell energy - proteoglycans, specially for small proteoglycans in skin fibroblasts i..F31 Promotes cell death, appears to act as a

225 BAD AF031523 link between growth Cell death factor receptor signaling and the a o totic pathways Involved in activation cascade of

226 Mch3 U39613 caspases responsible Cell death for apoptosis execution Involved in activation cascade of

227 Mch6 U56390 caspases responsible Cell death for apoptosis execution

228 MPP2 L16783 Putative M phase Cell cycle ho ho rotein 2

229 Mek2 L11285 Cell cycle Band 4.1-like protein 1, (neuronal protein 4.1), may

230 4.1N Q9H4G0 function to confer Cell stability and structure plasticity to neuronal membrane via multiple interactions Gamma enolase, Neuron

231 NSE Full length NM_001975 neuro specific marker enolase, Dynein see 9 genes below Dynein light chain 2A, cytoplasmic, may be involved in assembly and motor Cell cycle /

232 DL2A AF161511 function of dynein, organelle which plays central transport role in cell division and intracellular transport Dynein light chain 2B, cytoplasmic, may be involved in assembly and motor Cell cycle /

233 DL2B NM_130897 function of dynein, organelle which plays central transport role in cell division and intracellular transport E ;i 11.. ..:: .. iE "i Dynein light chain mRNA, 4A, axonemal, force not Cell cycle /

234 DL4A AL035366 generating protein in complete organelle respiratory cilia cds transport Ciliary dynein heavy Cell cycle /
chain 9 force

235 DYH9 AF257737 generating protein in organelle respiratory cilia transport Dynein light chain 1, cytoplasinic, maybe involved in some dynein-related intracellular Cell cycle /

236 DYLl N1VI 003746 transport and organelle - motility, may play a role in changing or transport maintaining spatial distribution of cytoskeletal structures Ciliary dynein heavy Cell cycle /

237 DYHB Q96DT5 chain 11, force organelle generating protein in respiratory cilia transport Dynein heavy chain, cytosolic;
cytoplasmic dynein acts as a motor for Cell cycle /

238 DYHC Q14204 intracellular organelle retrograde motility of vesicles and transport organelles along tubules, dynein has ATPase activity Dynein intermediate chain 1, cytosolic;
intermediate chains seem to help dynein bind to dynactin 150 kDa component, Cell cycle /

239 DYI1 AF063228 may play role in organelle mediating transport interaction of cytoplasmic dynein with membranous organelles and kinetochores Dynein intermediate chain 2, cytosolic; Cell cycle /

240 DYI2 MVI 001378 intermediate chains organelle - seem to help dynein bind to dynactin 150 transport kDa component 127. Some additional examples of biomarkers, whose level of expression can be assessed and compared to a reference standard for example, include human transformer 2-beta, hTra2-beta, human SAF-b, Mainclone, pht6, MIF, mainclone interacting factor, pp 17, ESAF, hnRNPG, cd2like kinases clkl-4. Further examples of biomarkers include those listed in Table 5. Specific examples of these biomarkers include, but are not limited to, HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 epsilon, Prohibitin, Phospoglycerate mutase 1, Apoliporotein Al, Superoxide dismutase, RNA-binding protein regulatory subunit, Chain A
thioredoxin peroxidase B, RAS-related protein RAP1B, Tumor rejection antigen, Haptoglobin, Fibrin beta, including combinations thereof. In other examples, suitable biomarkers include, but are not limited to, proteins having a molecular weight (MW) of 27,100 and isoelectric point (pI) of 7.58, a MW of 25,400 and pI of 6.2, and a MW of 27,600 and pI of 5.92.

Table 5: Identified proteins that differ between Parkinson's disease patients and control subjects.
Protein MW (molecular weight pI (isoelectric point) 1 66,204 5.67 2 63,131 7.59 3 62,052 5.98 4 59,332 7.42 5 54,879 5.42 6 36,106 7.58 7 32,567 5.10 8 29,785 5.80 9 29,559 7.60 10 26,908 5.65 11 25,546 7.64 12 24,376 6.36 153 ' :r. 4,3 7 5.82 14 21,719 5.92 15 145,916 5.29 16 42,839 5.42 17 60,376 6.72 18 27,100 7.58 19 25,400 6.2 20 27,600 5.92 128. Other specific examples of biomarkers, whose level of expression can be assessed and compared to a reference standard, as described herein, include actin-interacting protein 1 (AIP1), mitogen activated protein kinase I(MAPKI), actin or a fragment thereof, annexin Al, 14-3-3 protein epsilon, glutaraldehyde-3-phosphate dehydrogenase (GAPDH), transforming protein RhoA, acidic leucine-rich nuclear phosphoprotein 32 family member B
(ANP32B or APRIL), peroxiredoxin II, an amyloid precursor protein (APP), cx-secretase, 0-secretase, -y-secretase, A(3 peptide, Fe65, Tip60, SERCA, PS1/2, nectin-la, or non-amyloid (3 component of senile plaque (NACP/ a-synuclein).
3. Comparing levels of expression 129. In some examples of the disclosed methods, when the level of expression of a biomarker(s) is assessed (and optionally a first and/or second canonical variable obtained), the level (or canonical variable) can be compared with the level of expression of the biomarker(s) (or canonical variable obtained therefrom) in a reference standard. By "reference standard" is meant the level of expression of a particular biomarker(s) from a sample or subject lacking a neurodegenerative disease, at a different stage of a disease, or in the absence of a particular variable such as a therapeutic agent. Alternatively, the reference standard can comprise a known amount of biomarker. Such a known amount can correlate with an average level of subjects lacking a neurodegenerative disease, at a different stage of the disease, or in the absence of a particular variable such as a therapeutic agent. A reference standard can also include the expression level of one or more biomarkers from one or more different samples or subjects as described herein. For example, a reference standard can include an assessment of the expression level of one or more biomarkers in a sample from a subject that does not have a neurodegenerative disease, is at a different stage of progression of a neurodegenerative disease, or has not received treatment for a neurodegenerative disease. Another exemplary reference 6'&&. of the expression level of one or more biomarkers in samples taken from multiple subjects that do not have a neurodegenerative disease, are at a different stage of progression of a neurological disease, or have not received treatment for a neurological disease.
130. When the reference standard includes the level of expression of one or more biomarkers in a sample or subject in the absence of a therapeutic agent, the control sample or subject can be the same sample or subject to be tested before or after treatment with a therapeutic agent or can be a different sample or subject in the absence of the therapeutic agent.
Alternatively, a reference standard can be an average expression level calculated from a number of subjects without a particular neurodegenerative disease. A reference standard can also include a known control level or value known in the art. In one aspect of the methods disclosed herein, it can be desirable to age-match a reference standard with the subject diagnosed with a neurodegenerative disease. A reference standard can also be a first or second canonical variable obtained from a multivariant canonical analysis of levels of expression of a biomarker(s) from a control or group of control subjects.
131. In one technique to compare levels of expression of gene products from two different samples (e.g., a sample from a subject diagnosed with a neurodegenerative disease and a reference standard), each sample can be separately subjected to 2D gel electrophoresis.
Alternatively, each sample can be differently labeled and both samples can be loaded onto the same 2D gel. See e.g., Unlu et al., Electrophoresis, 1997;18:2071-2077, which is incorporated by reference herein for at least its teachings of methods to assess and compare levels of gene product expression. The same gene product or group of gene products in each sample can be identified by the relative position within the pattern of gene products resolved by 2D
electrophoresis. The expression levels of one or more gene products in a first sample can then be compared to the expression level of the saine gene product(s) in the second sample, thereby allowing the identification of a gene product or group of gene products that is expressed differently between the two samples (e.g., a biomarker). This comparison can be made for subjects before and after they are suspected of having a neurodegenerative disease, before and after they begin a therapeutic regimen, and over the course of that regimen.
132. In another technique, the expression level of one or more gene products can be in a single sample as a percentage of total expressed gene products. This assessed level of expression can be compared to a preexisting reference standard, thereby allowing for the ~~~nti~icat~~rr eh~ t are differentially expressed in the sample relative to the reference standard.
133. Gene products whose expression levels vary from a reference standard can be identified by, for example, extracting those gene products from a 2D gel and employing an identification technology such as mass spectroscopy (MS), which includes techniques such as or matrix-assisted laser desorption/ionization-time of flight-MS (MALDI-TOF).
Accordingly, in one aspect, disclosed herein are methods of identifying biomarkers relevant to various stages of a neurodegenerative disease (e.g., its onset and progression) by examining gene product expression in samples (e.g., samples comprising leukocytes or lysates thereof).
134. Other methods can be used instead of 2D electrophoresis to identify the level of gene product expression in a sample and compare that level to a reference standard, and can be used in the methods disclosed herein. Some of these methods utilize spectroscopic techniques such as surface-enhanced laser desorption ionization-time of flight (SELDI-TOF). Other methods rely on chromatographic techniques such as high performance liquid chromatography (HPLC), or fast protein liquid chromatography (FPLC). Multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) can separate and identify multiple peptides. See Link, et al., Nat. Biotechnol., 1999;17:676-82.
Additional chromatographic methods for identifying multiple proteins are described in U.S. Patent No.
6,908,740. In still other methods, chips (e.g., arrays of protein binding antibodies, ligands, or aptamers) can be used to identify gene products that are expressed differently in a sample than in a reference standard. See, e.g., Glokler and Angenendt, J. Chromatogr. B
Analyt. Technol.
Biomed. Life Sci., 2003;797:229-240. These references are incorporated by reference herein at least for their teachings of methods to assess and compare gene product expression levels.
135. When differential gene expression causes the expression of one or more gene products to be different in the sample and the reference standard, these one or more gene products can be further identified using methods that identify differentially expressed gene transcripts, e.g., gene chip (nucleotide expression microarrays) or differential display technologies (e.g. differential display kits from Clontech, Palo Alto, CA or GenHunter, Nashville, TN). These references are incorporated by reference herein at least for their teachings of methods to assess and compare protein expression levels.
136. When comparing the level of expression of a gene product with the reference standard an increase in the level of expression of the gene product, as compared to the reference standard, can identify the gene product as a biomarker for diagnosing the neurodegenerative aises6, disease. Alternatively, a decrease in the level of expression of the gene product, as compared to the reference standard, can also identify the gene product as a biomarker for diagnosing the neurodegenerative disease. Finally, a combination of increased gene products and decreased gene products as compared to a reference standard can identify the gene products as biomarkers for diagnosing the neurodegenerative disease.
137. Biomarkers identified by the disclosed methods can be used in a variety of other methods. For example, biomarkers can be used to diagnose a particular neurodegenerative disease. In another example, biomarkers can be used monitor the progression of a disease since the level of expression of some biomarkers can become more pronounced (or less pronounced) as a particular neurodegenerative disease progresses. In yet another example, a biomarker can be used to monitor a subject's response to treatment for a disease. These and other uses are disclosed herein.
4. Specific methods 138. Disclosed are methods of diagnosing a neurodegenerative disease such as Alzheimer's or Parkinson's disease comprising collecting a sample (e.g. blood or leukocytes) from a subject, assaying the expression of a set of genes in the sample, and comparing this expression to a control.
139. Also disclosed are methods of diagnosing a neurodegenerative disease, the method comprising, collecting a blood sample from a subject, assaying the expression of a set of genes in the sample, and comparing this expression to a control.
140. Also disclosed are methods of diagnosing a neurodegenerative disease, the method coinprising, collecting leukocytes from a subject, assaying the expression of a set of genes in the leukocytes, and comparing this expression to a control.
141. Disclosed are methods, wherein the subject has also been diagnosed with a clinical dementia test, wherein the clinical dementia test is the NINCDS or DSM-IV test.
142. Disclosed are methods, wherein the control would have a score of above 27 and an AD patient has a score below 22 in the Mini-Mental Status Examination (MMSE).

143. Also disclosed are methods, wherein the AD subject would have a score of above 1.2 or 1.5 in the Clinical Dementia Rating scale (CDR).
144. Disclosed are methods, wherein the control would be determined using the Blessed Dementia Rating Scale (BDRS).
145. Disclosed are methods of diagnosing a neurodegenerative disease, the method comprising collecting peripheral blood sample from a subject, lysing erythrocytes contained NPIr'~naining leukocytes, lysing the leukocytes producing a lysed sample, collecting total nucleic acids in the lysed sample forming a nucleic acid sample, isolating the RNA in the nucleic acid sample, extracting the RNA in the nucleic acid sample, collecting the polyA RNA, and identifying the presence of a set of RNA transcripts.
146. Also disclosed are methods of diagnosing a neurodegenerative disease, the method comprising collecting a sample (e.g., a leukocyte sample) from a subject, collecting the mRNA within the sample, hybridizing the mRNA with a collection of nucleic acids, wherein the collection of nucleic acids comprises one or more genes found in Table 4, such as cyclin D1, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, and protein kinase C
alpha, which are related to cell cycle, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, which are related to inflammatory systems, and Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, which are related to cell stress, including any combination thereof.
147. Disclosed are methods of diagnosing a neurodegenerative disease, the method comprising collecting peripheral blood sample from a subject, lysing erythrocytes contained within this sample, collecting the remaining leukocytes, lysing the leukocytes producing a lysed sample, collecting total nucleic acids in the lysed sample forming a nucleic acid sample, isolating the RNA in the nucleic acid sample, extracting the RNA in the nucleic acid sample, collecting the polyA RNA, and identifying the presence of a set of RNA transcripts.
148. Also disclosed are methods of diagnosing a neurodegenerative disease, the method comprising collecting a peripheral blood sample from a subject, collecting leukocytes from the peripheral blood sample, wherein collecting the leukocytes comprises lysing erythrocytes in the peripheral blood sample and centrifuging, lysing the leukocytes, collecting a total nucleic acid sample from the lysed leukocytes, wherein the collection of the nucleic acid comprises adsorption of the nucleic acids on magnetic beads, collecting a total RNA sample from the nucleic acid sample, collecting a polyA mRNA sample from the total RNA sample, hybridize the total mRNA sample with a set of diagnostic genes, wherein the set of diagnostic genes comprises one or more genes from Table 4, such as cyclin D1, cyclin B, cyclin G1, weel, hTR2, CDC25b, GSK3 beta, and protein kinase C alpha, which are related to cell cycle, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, which are related to inflammatory systems, and Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, which are related to cell stress, including any combination thereof, analyzing which diagnostic genes are hybridized by an mRNA in the mRNA sample.

õõ, . ...,. .
iused, the data can be normalized by normalizing a housekeeping gene such as GapDH, cyclophilin, or actin. Other methods include spiking samples, normalizing to the average or sum of signal intensity over the whole array.

a) Method of Screening for a Therapeutic Agent 150. In yet another aspect, disclosed herein are methods for screening for a therapeutic agent for the treatment of a neurodegenerative disease (e.g., Parkinson's or Alzheimer's disease).
The disclosed methods comprise contacting a leukocyte or population of leukocytes with the agent to be screened and detecting a level of expression or activity of a biomarker for the neurodegenerative disease. Alternatively, instead of leukocytes, neuronal cells or populations thereof can be used. In these methods, an increase or decrease in the level of expression or activity of the biomarker can indicate a therapeutic agent for the treatment of the neurodegenerative disease.
151. In one aspect, the disclosed methods can be utilized to screen agents that are nucleic acids, antibodies, polypeptides, or small molecules, including any therapeutic mixtures or combinations thereof.
152. Contacting the leukocyte or lysate thereof can be accomplished by any technique.
For example, the cells or lysate can be submerged or immersed in the agent or solution containing the agent. In another example, the cells or lysate can be coated or sprayed with the agent or solution containing the agent. In still another example, the cells or lysate can be contacted with a medium, such as a culture medium, that contains the agent or solution containing the agent. In a further example, the cells or lysate can be infused with the agent or solution containing the agent. The particular method of contacting the leukocytes or lysate thereof with the agent to be screened will be readily apparent to one of ordinary skill in the art and will depend on such factors as the size of the sample, the particular agent to be screened, convenience, preference, and the like.
153. The biomarker whose level of expression or activity is detected can be one or more genes or proteins that are down-regulated in the neurodegenerative disease. In this example, when the agent increases the level of expression or activity of the gene or protein biomarkers, this can indicate a therapeutic agent for the treatment of the particular neurodegenerative disease. Alternatively, the biomarker can be one or more genes or proteins that are up-regulated in the neurodegenerative disease. In this example, a therapeutic agent for the treatment of the particular neurodegenerative disease can be indicated when the agent decreases the level of expression or activity of the gene or protein biomarkers. Still further, a .., .. ,,.,, ,,,, ~ ., ~u$~g~aed while another biomarker(s) can be down-regulated in the neurodegenerative disease. In this example, when the agent decreases the level of expression or activity of the gene or protein biomarker(s) that is up-regulated in the neurodegenerative disease and/or increases the level of expression or activity of the gene or protein biomarker(s) that is down regulated in the neurodegenerative disease, this can indicate a therapeutic agent for the treatment of the particular neurodegenerative disease.
154. In an additional aspect of the disclosed methods, one can further determine whether the therapeutic agent alters the level of expression or activity of the biomarker in neurons. In one aspect, the neurons can be dopaminergic neurons.
155. In yet another aspect, the disclosed methods can further comprise determining whether the therapeutic agent prevents the development of or slows the progression of a neurodegenerative disease in an animal model of the disease. For example, when the neurodegenerative disease is Parkinson's disease, suitable animal models include, but are not limited to, a MPTP model, a 6-OHDA model, a paraquat model, or a rotenone model.
b) Method of Monitoring Neurodegenerative Disease Progression 156. In still another aspect, disclosed herein are methods of monitoring neurodegenerative disease (e.g., Parkinson's or Alzheimer's disease) progression in a subject.
The disclosed methods comprise comparing a level of expression or activity of a biomarker for a neurodegenerative disease in a sample comprising leukocytes or a lysate thereof obtained from the subject at multiple time points.
157. Also, disclosed herein are methods of monitoring a response to a neurodegenerative disease (e.g., Parkinson's disease) treatment in a subject.
The disclosed methods can comprise comparing a level of expression or activity of a biomarker for the neurodegenerative disease in a sample comprising leukocytes or a lysate thereof obtained from the subject at multiple time points during treatment of the subject.
158. In these methods, the subject can be as disclosed above (e.g., human).
Also, the subject can be asymptomatic or preclinical for neurodegenerative disease at one or more of the multiple time points. In another example, the subject has not received treatment for the neurodegenerative disease at one or more of the multiple time points.
159. By "treatment" is meant any medical intervention that the subject received or undergoes for the purpose of curing, preventing, or alleviating the disease.
Treatment can include, but is not limited to, pharmacological therapy (e.g., the administration of pharmaceuticals), nutritional therapy (e.g., the administration of vitamins, hormones, ... ,, i : rlaitra~~~bi~~~Lg~ tra~~z~il~'i'6~t' ; or supplements, or the alteration of diet), physical therapy, surgical treatment, non-pharmacological therapy, behavioral modification, and the like.
Optionally, the subject receives treatment for a neurodegenerative disease at one or more of the multiple time points. Optionally, the subject is treated with a neuroprotective agent at or before one of the inultiple time points. Optionally, the subject is treated with a dopamine agonist (e.g., levodopa) at one or more of the multiple time points. In another specific example, the subject is treated with a neuroprotective agent at one or more of the multiple time points.
160. Examples of neuroprotective agents which can be used to treat a subject include, but are not limited to, an acetylcholinesterase inhibitor, a glutamatergic receptor antagonist, kinase inhibitors, HDAC inhibitors, anti-flammatory agents, divalproex sodium, or any combination thereof. Examples of other neuroprotective agents can include, but are not limited to, Obidoxime Chloride; Pralidoxime Chloride; Pralidoxime Iodide; Pralidoxime Mesylate, Alverinc Citrate; Anisotropine Methylbromide; Atropine; Atropine Oxide Hydrochloride;
Atropine Sulfate; Belladonna; Benapryzine Hydrochloride; Benzetimide Hydrochloride;
Benzilonium Bromide; Biperiden; Biperiden Hydrochloride; Biperiden Lactate;
Clidinium Bromide; Cyclopentolate Hydrochloride; Dexetimide;.Dicyclomine Hydrochloride;
Dihexyverine Hydrochloride; Domazoline Fumarate; Elantrine; Elucaine;
Ethybenztropine;
Eucatropine Hydrochloride; Glycopyrrolate; Heteronium Bromide; Homatropine Hydrobromide;
Homatropine Methylbromide; Hyoscyamine; Hyoscyamine Hydrobromide; Hyoscyamine Sulfate; Isopropamide Iodide; Mepenzolate Bromide; Methylatropine Nitrate;
Metoquizine;
Oxybutynin Chloride; Parapenzolate Bromide; Pentapiperium Methylsulfate;
Phencarbamide;
Poldine Methylsulfate; Proglumide; Propantheline Bromide; Propenzolate Hydrochloride;
Scopolamine Hydrobromide; Tematropium Methylsulfate; Tiquinamide Hydrochloride;
Tofenacin Hydrochloride; Toquizine; Triampyzine Sulfate; Trihexyphenidyl Hydrochloride;
Tropicamide. Further examples include, but are not limited to, Albutoin;
Ameltolide; Atolide;
Buramate; Carbamazepine; Cinromide; Citenamide; Clonazepam; Cyheptamide;
Dezinamide;
Dimethadione; Divalproex Sodium; Eterobarb; Ethosuximide; Ethotoin; Flurazepam Hydrochloride; Fluzinamide; Fosphenytoin Sodium; Gabapentin; Ilepcimide;
Lamotrigine;
Magnesium Sulfate; Mephenytoin; Mephobarbital; Methetoin; Methsuximide;
Milacemide Hydrochloride; Nabazenil; Nafimidone Hydrochloride; Nitrazeparn; Phenacemide;
Phenobarbital; Phenobarbital Sodium; Phensuximide; Phenytoin; Phenytoin Sodium; Primidone;
Progabide; Ralitoline; Remacemide Hydrochloride; Ropizine; Sabeluzole;
Stiripentol;
Sulthiame; Thiopental Sodium; Tiletamine Hydrochloride; Topirarnate;
Trimethadione;

;-%l~~bhf&[ S:6Hju&j:7-VTd''~~kcid; Vigabatrin; Zoniclezole Hydrochloride;
Zonisamide. Still other examples of anti-imflammatory agents include, but are not limited to, Alclofenac;
Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal;
Amcinafide;
Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen;
Apazone;
Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride;
Bromelains;
Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;
Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate;
Cormethasone Acetate;
Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate;
Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;
Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;
Endrysone; Enlimomab;
Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole;
Fenbufen;
Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone;
Fluazacort; Flufena.mic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin Butyl;
Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate;

Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; Halopredone Acetate; Ibufenac;
Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin;
Indomethacin Sodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate; Isoxepac;
Isoxicam;
Ketoprofen; Lofemizole Hydrochloride; Lomoxicam; Loteprednol Etabonate;
Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine;

Meseclazone; Methylprednisolone Suleptanate; Momiflumate; Nabumetone;
Naproxen;
Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein; Orpanoxin;
Oxaprozin;
Oxyphenbutazone; Paranyline Hydrochloride; Pentosan Polysulfate Sodium;
Phenbutazone Sodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine;
Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole;
Proxazole Citrate;
Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate; Sanguinarium Chloride;
Seclazone;
Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate;
Talosalate; Tebufelone;
Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac;
Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin; or Zomepirac Sodium.
161. In other examples, the subject can be treated with a non-pharmacological treatment, i.e., treatments that do not primarily involve drugs. Examples of such non-pharmacological drugs include, but are not limited to, brain stimulation, which is typically used in PD, ventricular shunt and transposition of omentum, which has been used in AD. In still other ~
f- e~anri0l6 9 yii~i~~~ulij~'t ca[~ f~~ t~eated by behavioral modification.
Still further examples of treatments involve gene therapy, transplants, and stem cells.
162. In the disclosed methods, a level of biomarker(s) expression or activity assessed at one point in time can be the same as the level assessed at another point in time. This can indicate that the particular neurodegenerative disease has not changed (e.g., the disease has not gotten worse or better). In another example, a level of biomarker(s) expression or activity at an earlier point in time can be more or less than the level at a later point in time. This can indicate that the neurodegenerative disease is progressing. If a biomarker's level of expression has been previously shown to increase from an earlier point in time to a later point in time and to correlate with (a) worsening or (b) improvement of the symptoms of a disease, then a lower amount of biomarker present in the earlier sample relative to the later sample can be considered to be an indication that the subject's condition is (a) worsening or (b) improving, respectively. On the other hand, if a biomarker's level of expression has been shown to decrease from an earlier point in time to a later point in time and to correlate with (a) worsening or (b) improvement of the symptoms of a disease, then a higher amount of biomarker present in the earlier sample relative to the later sample can be an indication that the subject's condition is (a) worsening or (b) improving, respectively. In another example, a combination of biomarkers, where some biomarkers in the combination increase from an earlier point in time to a later point in time during disease progression and other biomarkers decrease, can be used.
163. Also, the level of a biomarker can be correlated with a worsening or an improveinent in one or more symptoms of a neurodegenerative disease in response to the therapy. Gene product(s) whose expression levels are different between a sample taken prior to treatment or at an earlier point in time during treatment and a sample taken at a later point in time during treatment or after treatment can identify a biomarker for the response of a subject to a treatment for a neurodegenerative disease.
164. In these methods, a difference in a level of expression or activity of a biomarker between various samples can be indicative of the subject's responsiveness to the administered treatment for the neurodegenerative disease. If a biomarker's expression has been previously shown to increase in subjects that (a) respond or (b) fail to respond to the treatment for a neurodegenerative disease, then a larger amount of biomarker in a later sample relative to an earlier sample can be an indication that the subject is (a) responding or (b) not responding, respectively, to the treatment. Alternatively, if a biomarker's expression has been previously shown to decrease in subjects that (a) respond or (b) fail to respond to a treatment for a It : aur'ougiarlvaf'dis~~a~'e;=~~h~n a smaller amount of biomarker in a later sample relative to an earlier sample can be considered to be an indication that the subject is (a) responding or (b) not responding, respectively to the treatment. Alternatively, if a combination of biomarkers are used and one or more biomarkers have been previously shown to decrease in subjects that (a) respond or (b) fail to respond to a treatment for a neurodegenerative disease and one or more other biomarkers have been previously shown to increase, then a change in the amount of biomarkers in the combination of biomarkers from a later sample relative to an earlier sample can be considered to be an indication that the subject is (a) responding or (b) not responding, respectively to the treatment.

c) Method of Identifying a Risk for a Neurodegenerative Disease in a Subject 165. In yet a further aspect, disclosed herein are methods for identifying a risk for a neurodegenerative disease (e.g., Parkinson's or Alzheimer's disease) in a test subject. The disclosed methods comprise determining a level of expression or activity of a biomarker for a neurodegenerative disease from a sample obtained from the test subject, wherein the sample comprises leukocytes or a lysate thereof; and correlating the level of expression or activity level of the biomarker determined for the test subject with the levels for a reference subject. The method can further comprise determining the level of the biomarker(s) from a population of reference subjects diagnosed with the neurodegenerative disease and/or from a population of reference subjects without the neurodegenerative disease. In the disclosed methods, a correlation between levels for a reference population without the neurodegenerative disease and the levels for the test subject can identify a low risk for the particular neurodegenerative disease in the test subject. Also, a correlation between the levels determined for the reference population with the neurodegenerative disease and the levels for the test subject can identify a high risk for the neurodegenerative disease in the test subject.

166. By "correlation" is meant any relationship between data. For example, a correlation can be determined through a statistical analysis of the levels of biomarker expression or activity (e.g., standard deviation, degree of confidence, etc.). A
correlation can also be an empirical determination based on the levels of biomarker expression or activity.

167. Gene product data (e.g., transcript and/or proteomic data) can be subjected to the following statistical analyses. To account for technical variability, each subject sample can be run in triplicate on 2D gels (e.g., 52 subjects x 3 gels equals 156 gels).
Averaged spot intensities can then be used for further analysis. Gender, basic clinical diagnosis, and clinical indices can ih'tllepot~~ohIs data. Primary comparisons using simple univariate statistical methods such as two-sample t-tests can proceed to identify those proteins whose expression in leukocytes are different between disease subjects and the control group.
Proteomic data from 2D
gel electrophoresis and MALDI-TOF mass spectroscopy can be first analyzed using the statistical tools, including t-tests and ANOVAs, contained within the Progenesis Workstation Image Analysis and Informatics software program (Nonlinear USA, Inc.; Durham, NC). In cases of heavy dependence in these data a step-down multivariate resampling algorithm can be used to address the multiplicity of tests, as disclosed in Troendle, A permutational step-up method of testing multiple outcomes, Biometrics, 1996;52:846-859, which is incorporated by reference herein at least for its teachings of statistical methods.
168. In some aspects, multivariate statistical methods can be more appropriate and powerf-ul in all classifications and associations being considered in such studies. Canonical discriminant analysis can be performed to use the profiling of all gene products together for the disclosed methods. Logistic discrimination between groups based on multivariate observations can be used since it generally out-performs the normal-theory-based linear discriminant analysis (see McLachlan, Discriminant analysis and statistical pattern recognition, Wiley, New York, 1992, which is incorporated by reference herein at least for its teaching of statistical methods).
To identify those proteins associated to behavioral indices, linear models and generalized linear models (e.g., those disclosed in Nelder and McCullagh, Generalized Linear Models, CRC Press, Boca Raton, FL, 1999, which is incorporated by reference herein at least for its teaching of linear models) can be fitted to profile proteins differentially expressed in the neurodegenerative disease and non-neurodegenerative disease leukocytes and to identify those proteins whose expression changes are related to the severity of disease. These models can also take in consideration the confounding issues of some clinical factors. Missing values can be handled as suggested by Little and Little (Applications of Modern Missing Data Methods, CRC Press, Boca Raton, FL, 2002, which is incorporated by reference herein at least for its teaching of statistical methods).
In cases of unexpected complication of data analysis, the skill artisan can pursue appropriate statistical methods and even develop and program statistical methods to serve these specific aims, including the use of nonparametric empirical Bayesian analysis proposed by Efron and Tibshirani, Empirical bayes methods and false discovery rates for microarrays, Genet Epidemiol.
2002;23:70-86, which is incorporated by reference herein at least for its teaching of statistical methods.

Hfteihod of Distinguishing One Neurodegenerative Disease from Another Disease 169. In another aspect, disclosed herein are methods of differentially diagnosing a neurodegenerative disease, (e.g., Parkinson's or Alzheimer's disease) in a test subject. The disclosed methods comprise assessing a level of expression or activity of one or more selected biomarker(s) in a sample comprising leukocytes or a lysate thereof from the test subject and comparing the level of expression or activity of the selected biomarker(s) to a reference standard that indicates the level of expression or activity of the selected biomarker(s) in one or more populations of neuropathologic control subjects with one or more neuropathological control diseases. In the disclosed methods, a difference or similarity between the level of expression or activity of the selected biomarker(s) and the reference standard can indicate a differential diagnosis of one neurodegenerative as compared to the neuropathological control diseases.
170. By "differentially diagnosing" is meant to identify the presence of one particular disease in a subject and/or identify the absence of another disease in a subject. The phrase also means to distinguish one particular disease from another disease or from the absence of a disease. "Differentially diagnose" is also used herein to mean to identify a particular stage of one disease, to identify the risk of developing a particular disease, or to identify a prognosis of a particular disease.
171. By "neuropathologic control subject" is meant a subject (e.g., human) or group of subjects that have one or more neurodegenerative diseases, as described herein. For example, the neuropathologic control subject can be one or more subjects with Alzheimer's disease, frontal-temporal dementia, mild cognitive impairment, and Parkinson's disease, plus disorders that comprise additional symptoms (e.g., multiple system atrophy, corticobasal ganglionic degeneration, Parkinson's disease with Alzheimer's). The neuropathologic control subject can also be a subject or group of subjects that does not have a particular disease. Still further, neuropathologic control subject can also be a subject or group of subjects that has a particular risk or predisposition of developing a disease.
172. In these methods, the test subject and the reference populations can be age or sex matched or both.
e) Methods of Identifying a Biomarker 173. Biomarkers for a neurodegenerative disease, such as Parkinson's or Alzheimer's disease, can be identified by the methods disclosed herein. In one aspect, methods for identifying a biomarker for a particular neurodegenerative disease can comprise assessing a level qf o~t~i~ir~1&6~~flgene products in a sample comprising leukocytes or a lysate thereof from at least one subject (e. g., . human) diagnosed with the neurodegenerative disease, and comparing the level of expression of the gene products to a reference standard. In this method, an increase or decrease in the level of expression of the gene products, as compared to the reference standard, can identify the gene products as biomarkers for the particular neurodegenerative disease.
174. In some cases, the same biomarkers can be used for diagnosing, monitoring disease progression, and/or monitoring the response of a subject to a therapy for a disease, are the same biomarker. The altered expression of the biomarker (under the conditions of the two or more different uses) can reflect the same fundamental biochemical and metabolic pathways that underlie the pathology of the particular neurodegenerative disease. For example, the same biological pathways can cause both a gene product to be expressed at a particular level in a healthy subject that is free of the neurodegenerative disease and the same gene product to be expressed at a similar level in a in a subject responding to a treatment for the disease.
175. Furthermore, because the altered expression of certain biomarkers can be a due to changes in biochemical/inetabolic pathways that underlie the pathology of a disease, these biomarkers also represent therapeutic targets. If a change in expression of a biomarker causes one or more symptoms of a neurodegenerative disease, then the biomarker can be a therapeutic target. Therapeutic targets can be used in methods for discovering compounds that modulate the expression or activity of one or more candidate biomarkers and/or improve one or more symptoms of the neurodegenerative disease (i.e., candidate therapeutic agents).

5. Clinical features of Alzheimer's 176. One hundred years ago Alois Alzheimer described the major behavioral and neuropathological features of the neurodegenerative disorder bearing his name.
AD is characterized clinically/behaviorally by progressive impairment of memory and cognition.
Neuropathological and neurobiological changes associated with this slow progression of clinical symptoms include accumulation of amyloid plaques and neurofibrillary tangles (NFTs) (Gearing M, et al., The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part X.
Neuropathology confirmation of the clinical diagnosis of Alzheimer's disease.
Neurology.

1995;45(3 Pt 1):461-466) gliosis (Unger JW, Microscopy Res. Technique, 1998;43:24-28), reduced dendritic plasticity relative to normal aged (Buell and Coleman, Science, 1979;206(4420):854-856; Flood DG, et al., Brain Research, 1985;345(2):366-368;
Flood DG, et al., Brain Research, 1987;402(2):205-216), and reduced density of neurons (Coleman PD, et al., [ W6iiro~=~~ilm~~:t-t~~~' ~g~,1~9~~8 /,19(6):521-545; TerryRD, et al., Annals of Neurology, 1987;21:530-539; West MJ, et al., Lancet, 1994;344:769-772) and synapses (Scheff SW, et al., Neurobiology of Aging, 1990;11(1):29-37).

6. Gene expression in Alzheimer's 177. Studies of altered gene expression in Alzheimer's disease brain tissue have shown a general reduction of message level estimated at about 35% (Doebler JA, et al., J.
Neuropathology & Experimental Neurology, 1987;46(l):28-39), (Griffin WS, et al., Alzheimer Disease & Associated Disorders, 1990;4(2):69-78), (Harrison PJ, et al., Psychological Medicine, 1991;21:855-866). Against this background of a general reduction of mRNA, selected studies have demonstrated increased as well as decreased expression of a wide variety of genes. Some gene classes affected in Alzheimer's disease are expressed in a neuron specific manner. These especially include decreased expression of selected genes that are related to synaptic structure and function and the neuronal cytoskeleton (Ginsberg SD, et al., Annals of Neurology, 2000;48(1):77-87; Yao P, et al., J. Neuroscience, 1998;18(7):2399-2411). Other classes of genes whose expression is altered in AD include those related to the cell cycle (Arendt T, Neurobiology of Aging, 2000;21(6):783-796; Husseman JW, et al., Neurobiology of Aging, 2000;21(6):815-828; Nagy Z, et al., Neurobiology of Aging, 2000;21(6):761-769;
Vincent I, et al., J. Neuroscience, 1997;17:3588-3598) and inflammatory/stress responses (for a review, see Akiyama H, et al., Neurobiology of Aging, 2000;21(3):383-421). These gene classes are expressed in a variety of cell types that reside outside the nervous system including leukocytes (Wakutani Y, et al., Dementia, 1995;6(6):301-305), monocytes (Jung SS, et al., Neurobiology of Aging, 1999;20(3):249-257), and epitllelial cells (Schmitz A, et al., Histochemistry & Cell Biology, 2002;117(2):171-180) as well as other cell types.

178. Multivariate analysis of profiles of expression of multiple gene products (messages) by single neurons or homogenates from postmortem human brain can be used to distinguish neurodegenerative disease (e.g., Parkinson's and Alzheimer's disease) from control samples (Cheetham JE, et al., J. Neuroscience Methods, 1997;77(l),:43-48, Chow N, et al., Proc.
Natl. Acad. Sci. U.S.A., 1998;95:9620-9625).

179. As disclosed herein, data relating to using multiple genes to diagnose of neurodegenerative disease (e.g., Parkinson's and Alzheimer's disease) is obtained from samples such as peripheral blood and blood leukocytes. For example, varying sets of genes are used and genes related to the inflammatory response as well as the cell cycle are predicative. Blood was drawn from patients diagnosed in our Alzheimer's Disease Center as having probable (mild) AD

ed control sample. Message was extracted from peripheral blood leukocytes and amplified (Eberwine J, et al., PNAS U.S.A. 1992;89(7):3010-3014). The expression level of selected messages was then quantified. Multivariate statistical analyses differentiated Alzheimer's and control white blood cells. It was found that expression levels of genes related to the cell cycle and to inflammatory responses distinguish blood samples of AD
cases from samples from non-demented control cases. These specific gene sets and classes were also shown to be classes of genes that are also differentially expressed in AD
brain. This study was repeated three times with 3 different sets of cases.
180. As disclosed herein, the expression of genes related to the cell cycle and to inflammatory responses is affected in peripheral leukocytes from AD cases, a finding that parallels altered expression of these gene classes in the AD brain. There are two major conclusions to be reached regarding the data presented here: (1) expression profiles of multiple genes are effective at distinguishing mild AD (average CDR 1.2-1.5) from non-demented control cases and, (2) the gene classes described as distinguishing AD from control peripheral blood samples are similar to gene classes whose expression has been shown to be altered in the brain in AD. This is consistent with the concept of AD as a systemic disease or a disease with major systemic consequences.

C. Compositions 181. Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein.
For example, if a particular method of diagnosing a neurodegenerative disease is disclosed and discussed and a number of modifications that can be made to a number of molecules including the method of diagnosing a neurodegenerative disease are discussed, specifically contemplated is each and every combination and permutation of the method of diagnosing the neurodegenerative disease and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination I-ORTUs, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions.
Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be perfonned with any specific embodiment or combination of embodiments of the disclosed methods.
1. Sequence similarities 182. It is understood that as discussed herein the use of the terms homology and identity mean the same thing as similarity. Thus, for example, if the use of the word homology is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their nucleic acid sequences. Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not.
183. In general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the variants and derivatives in terms of homology to specific known sequences. This identity of particular sequences disclosed herein is also discussed elsewhere herein. In general, variants of genes and proteins herein disclosed typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level. ' 184. Another way of calculating homology can be performed by published algorithms.
Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math, 1981;2:482, by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 1970;48:443, by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A., 1988;85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

&VOel.of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker M, Science, 1989;244:48-52; Jaeger, et al., Proc. Natl. Acad. Sci.
U.S.A., 1989;86:7706-7710; Jaeger, et al., Methods Enzymol., 1989;183:281-306, which are herein incorporated by reference for at least material related to nucleic acid alignment. It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein.
186. For example, as used herein, a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above: For example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods. As another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods. As yet another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages).

2. Hybridization/selective hybridization 187. The term hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene.
Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G
interacting with C or A
interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide. The hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For ct~nc~rit~ 1Is, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.
188. Parameters for selective hybridization between two nucleic acid molecules are well known to those of skill in the art. For example, in some embodiments selective hybridization conditions can be defined as stringent hybridization conditions.
For example, stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps. For example, the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X
SSC or 6X SSPE) at a temperature that is about 12-25 C below the T. (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5 C to about 20 C below the Tm. The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA
immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations. The conditions can be used as described above to achieve stringency, or as is known in the art. (Sambrook, et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989; Kunkel, et al. Methods Enzymol., 1987;154:367, which is herein incorporated by reference for material at least related to hybridization of nucleic acids). A preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68 C (in aqueous solution) in 6X SSC
or 6X SSPE
followed by washing at 68 C. Stringency of hybridization and washing, if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for.

Likewise, stringency of hybridization and washing, if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T
richness of any area wherein high homology is desired, all as known in the art.
189. Another way to define selective hybridization is by looking at the amount (percentage) of one of the nucleic acids bound to the other nucleic acid. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid. Typically, the non-limiting primer is in for example, 10 or 100 or 1000 fold excess. This type of assay can re~~per1'e1W-iRaWtofi'iWns where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their kd, or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their kd.
190. Another way to define selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer molecules are extended. Preferred conditions also include those suggested by the manufacturer or indicated in the art as being appropriate for the enzyme performing the manipulation.
191. Just as with homology, it is understood that there are a variety of methods herein disclosed for determining the level of hybridization between two nucleic acid molecules. It is understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated meeting the parameters of any of the methods would be sufficient. For example if 80% hybridization was required and as long as hybridization occurs within the required parameters in any one of these methods it is considered disclosed herein.
192. It is understood that those of skill in the art understand that if a composition or method meets any one of these criteria for determining hybridization either collectively or singly it is a composition or method that is disclosed herein.

3. Nucleic acids 193. There are a variety of molecules disclosed herein that are nucleic acid based, including, for example, the nucleic acids that encode, for example, any of the genes disclosed herein as being associated with the onset or progression of a neurodegenerative disease (e.g., Parkinson's and Alzheimer's disease), as well as any other proteins disclosed herein, as well as various functional nucleic acids. The disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and It is understood that, for example, when a vector is expressed in a cell, the expressed mRNA will typically be made up of A, C, G, and U. Likewise, it is understood that if, for example, an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery it is advantageous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment.
a) Nucleotides and related molecules 194. A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The base moiety of a nucleotide can be adenine-9-yl (A), cytosine-l-yl (C), guanine-9-yl (G), uracil,l-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. A non-limiting example of a nucleotide would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).
195. A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties.
196. Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA).
Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to confonn to a double helix type structure when interacting with the appropriate target nucleic acid.
197. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl.
Acad. Sci. USA, 1989,86, 6553-6556).
198. A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleoticle substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a hie1d'btide; C~ii%Iotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.
199. A Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face includes the N7 position and reactive groups (NH2 or 0) at the C6 position of purine nucleotides.
(1) Primers and probes 200. Disclosed are compositions including primers and probes, which are capable of interacting with the genes disclosed herein. In certain embodiments the primers are used to support DNA amplification reactions. Typically the primers will be capable of being extended in a sequence specific manner. Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer. Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA
sequencing, DNA
extension, DNA polymerization, RNA transcription, or reverse transcription.
Techniques and conditions that amplify the primer in a sequence specific manner are preferred. In certain embodiments the primers are used for the DNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner. Typically the disclosed primers hybridize with the nucleic acid or region of the nucleic acid or they hybridize with the complement of the nucleic acid or compleinent of a region of the nucleic acid.
201. The size of the primers or probes for interaction witli the transcripts listed in Table 4, such as transcripts related to cell cycle such as cyclin Dl, cyclin B, cyclin Gl, wee1, hTR2, CDC25b, GSK3 beta, and protein kinase C alpha, transcripts related to inflammatory systems such as C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-10r, and transcripts related to cell stress such as Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, and transcripts of proteins listed in Tables 5 and 6. In certain embodiments, the primers or probes can be any size that supports the desired enzymatic manipulation of the primer, such as DNA amplification or the simple hybridization of the probe or primer. A typical primer or probe for the genes listed in Table 4, such as cyclin D 1, ~-ac~11n~~;"~~~~'~~'~';K~~*2, CDC25b, GSK3 beta, and protein kinase C alpha, which are related to cell cycle, C5, C1 inhibitor, IL-17r, IL-8, L1F, TNF-alpha, and IL-lOr, which are related to inflammatory systems, and Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, which are related to cell stress, and genes of proteins listed in Tables 5 and 6, would be at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.
202. In other embodiments a primer or probe for the genes listed in Table 4, such as cyclin D1, cyclin B, cyclin Gl, wee1, hTR2, CDC25b, GSK3 beta, and protein kinase C alpha, which are related to cell cycle, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, which are related to inflammatory systems, and Alpha-1 antichymotrypsin, HSP 27, HSP
90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, which are related to cell stress, and genes of proteins listed in Tables 5 and 6, can be less than or equal to 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.
203. In certain embodiments the primers and probes are designed such that they are outside primers whose nearest point of interaction with the genes found in Table 4, such as cyclin Dl, cyclin B, cyclin G1, weel, hTR2, CDC25b, GSK3 beta, and protein kinase C alpha, which are related to cell cycle, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, which are related to inflammatory systenls, and Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, which are related to cell stress, and genes of proteins listed in Tables 5 and 6, is within 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 99, 100, 125, 150, 175, or 200 nucleotides of the outermost defining nucleotide of the genes listed in Table 4, such as cyclin D1, cyclin B, cyclin Gl, wee 1, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and 1L-lOr, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, region or complement of the genes listed in Table 4, such as cyclin D1, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-10r, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, and genes of proteins listed in Tables 5 and 6.
204. In certain embodiments the primers and probes are designed such that they are outside primers whose nearest point of interaction with the genes listed in Table 4, such as cyclin Dl, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C
alpha, C5, Cl inliibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, and genes of proteins listed in Tables 5 and 6, is at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, or 200 nucleotides away from the outermost defining nucleotide of the genes listed in Table 4, such as cyclin D1, cyclin B, cyclin G1, weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, region or complement of the genes listed in Table 4, such as cyclin D1, cyclin B, cyclin G1, wee1, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, and genes of proteins listed in Tables 5 and 6.
205. The primers for the genes listed in Table 4, such as cyclin D1, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5. Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-l Or, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, and genes of proteins listed in Tables 5 and 6, typically will be used to produce an amplified DNA product that contains a specific region of the 16:16ize of the product will be such that the size can be accurately determined to within 1, or 2, or 3 nucleotides.
206. In certain embodiments this product is at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.
207. In other embodiments the product is less than or equal to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long.
(2) Functional Nucleic Acids 208. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction.
Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting. For example, functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, and external guide sequences. The functional nucleic acid molecules can act as affectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.
209. Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functional nucleic acids can interact with the mRNA of the genes listed in Table 4, such as cyclin D1, cyclin B, cyclin G1, wee 1, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, and genes of proteins listed in Tables 5 and 6, or the genomic DNA of the genes listed in Table 4, such as cyclin D1, cyclin B, cyclin Gi, weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-1al~~ia U&-~~~~~f~r;"'"~''alchymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, and genes of proteins listed in Tables 5 and 6, or they can interact with the polypeptide product of the genes listed in Table 4, such as cyclin D1, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, and the proteins listed in Tables 5 and 6. Often functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.
210. Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation.
Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC. It is preferred that antisense molecules bind the target molecule with a dissociation constant (kd) less than or equal to 10-6, 10-8, 10-10, or 10-12. A
representative sample of methods and techniques which aid in the design and use of antisense molecules can be found in the following non-limiting list of U.S. Patents: 5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437.
211. Aptamers are molecules that interact with a target molecule, preferably in a specific way. Typically aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind small molecules, such as ATP (U.S. Patent No. 5,631,146) and theophiline (U.S. Patent No. 5,580,737), as well as large molecules, such as reverse transcriptase (United States patent (44.S. Patent No. 5,543,293). Aptamers can bind very tightly with kds from the target molecule of less than 10-12 M. It is preferred that the aptamers bind the target molecule with a kd less than 10-6, 10, 10-", or 10"12. Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule (U.S. Patent No. 5,543,293). It is preferred that the aptamer have a kd with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower than the ka with a background binding molecule. It is preferred when doing the comparison for a polypeptide for example, that the background molecule be a different polypeptide. For example, when determining the specificity of aptamers of the genes listed in Table 4, such as cyclin Dl, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, or genes of proteins listed in Tables 5 and 6, the background protein could be serum albumin. Representative examples of how to make and use aptamers to bind a variety of ' different target molecules can be found in the following non-limiting list of U.S. Patent Nos.
5,476,766, 5,503,978, 5,631,146, 5,731,424, 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660, 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and 6,051,698.

212. Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid.
It is preferred that the ribozymes catalyze intermolecular reactions. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes, (for example, but not limited to, the following U.S. Patent Nos. 5,334,711, 5,436,330, 5,616,466, 5,633,133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, WO 9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO 9718312 by Ludwig and Sproat) hairpin ribozymes (for example, but not limited to the following U.S. Patent Nos.
5,631,115, 5,646,031, 5,683,902, 5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), and tetrahymena ribozymes (for exarnple, but not limited to the following U.S. Patent Nos.
5,595,873 and 5,652,107). There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo (for example, but not limited =tb'tli~ . .... eos. 5,580,967, 5,688,670, 5,807,718, and 5,910,408).
Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA
substrates.
Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence. Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in the following non-limiting list of U.S.
Patent Nos. 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855, 5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and 6,017,756.
213. Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a kd less than 10"6, 10-8, 10-10, or 10-12. Representative examples of how to make and use triplex forming molecules to bind a variety of different target molecules can be found in the following non-limiting list of U.S. Patent Nos. 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773, 5,834,185, 5,869,246, 5,874,566, and 5,962,426.
214. External guide sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, and this complex is recognized by RNAse P, which cleaves the target molecule. EGSs can be designed to specifically target a RNA molecule of choice. RNAse P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 by Yale, and Forster and Altman, Science, 1990;238:407-409).
215. Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukaryotic cells. (Yuan, et al., Proc. Natl.
Acad. Sci. U.S.A., 1992;89:8006-8010; WO 93/22434 by Yale; WO 95/24489 by Yale; Yuan and Altman, EMBO
J., 1995;14:159-168, and Carrara, et al., Proc. Natl. Acad. Sci. U.S.A., 1995;92:2627-2631).
Representative examples of how to make and use EGS molecules to facilitate cleavage of a 11~7zeqaal~~e~~rit be found in the following non-limiting list of U.S.
Patents:
5,168,053, 5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162.

4. Peptides 216. Also disclosed herein are compositions that are amino acid based, such as proteins, peptides, and polypeptides. By "protein," "peptide," or "polypeptide" is meant an amino-acid based polymer, including variants, derivatives, and modifications, as described herein and as are well understood by those of skill in the art. Amino-acid sequence modifications typically fall into one or more of three classes:
substitutional, insertional, or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once;
insertions usually will be on the order of about from 1 to 10 amino-acid residues; and deletions will range about from 1 to 30 residues. Substitutions, deletions, insertions or any combination thereof can be present in the proteins disclosed herein. The tenns "protein," "peptide," and "polypeptide" are used interchangeably herein.
217. Exemplary proteins that can be used in the methods disclosed herein include HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 protein epsilon, Prohibitin, Phospoglycerate mutase 1, Apoliporotein AI, Superoxide dismutase, RNA-binding protein regulatory subunit, Chain A thioredoxin peroxidase B, RAS-related protein RAP1B, Tumor rejection antigen, Haptoglobin, Fibrin beta, actin-interacting protein 1(AIP1), mitogen activated protein kinase I(MAPY-T), actin or a fragment thereof, glutaraldehyde-3-phosphate dehydrogenase (GAPDH), transforming protein RhoA, acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B or APRIL), peroxiredoxin II, an amyloid precursor protein (APP), an cx secretase, a,6-secretase, a-y-secretase, an A,6 peptide, Fe65, Tip60, SERCA, PS 1/2, nectin-1 a, and non-amyloid 0 component of senile plaque (NACP/
a-synuclein).
5. Variants 218. It is understood that there are numerous variants and alleles of the genes used herein for analysis of neurodegenerative diseases, such as the genes listed in Table 4, such as Cy. cI~~~ .,--weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, Alpha-1 antichymotrypsin, HSP 27, HSP
90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin.
These variants and alleles can be used to detect neurodegenerative diseases (e.g., Parkinson's and Alzheimer's) as disclosed herein. As discussed herein there are numerous variants of the gene products from the genes listed in Table 4, such as cyclin D1, cyclin B, cyclin Gl, wee 1, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-lOr, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, that are known and herein contemplated. Typically these variants will manifest themselves in changes in the related nucleic acid or gene, and thus, variants of the disclosed diagnostic and prognostic genes, such as the genes listed in Table 4 (e.g., cyclin D1, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-l Or, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin) produced, by for example, as alleles or strain differences, are disclosed. Protein and nucleic acid variants and derivatives and alleles are well understood to those of skill in the art and in can involve amino acid sequence modifications. It is understood that modifications in the methods or compositions can be accomplished to deal with, for example particular alleles.

6. Sequences 219. There are a variety of sequences related to the, for example, genes listed in Table 4, such as cyclin Dl, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C
alpha, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-10r, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, and Table 5, such as HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 protein epsilon, Prohibitin, Phospoglycerate mutase 1, Apoliporotein AI, Superoxide dismutase, RNA-binding protein regulatory subunit, Chain A thioredoxin peroxidase B, RAS-related protein RAP1B, Tumor rejection antigen, Haptoglobin, Fibrin beta, actin-interacting protein 1(AIP1), mitogen activated protein kinase I(1VIAPKI), actin or a fragment thereof, glutaraldehyde-3-phosphate dehydrogenase (GAPDH), transforming protein RhoA, acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B or APRIL), peroxiredoxin II, an amyloid precursor protein (APP), an a-secretase, a,6-secretase, ay-secretase, an A,13 peptide, Fe65, Tip60, SERCA, PS1/2, nectin-la, or non-amyloid 0 component of senile plaque (NACP/ a-synuclein), ~ ~. ...1..,. i1~F ti:~a:,' rÃr It ".. iIn ,. il, ~~as ~vell as~~~~ ot4~~r ~
tei~ slosed herein that are disclosed on Genbank, and these sequences and others are herein incorporated by reference in their entireties as well as for individual subsequences contained therein.
220. A variety of sequences are provided herein and these and others can be found in Genbank, at www.pubmed.gov. Those of skill in the art understand how to resolve sequence discrepancies and differences and to adjust the compositions and methods relating to a particular sequence to other related sequences. Primers and/or probes can be designed for any sequence given the information disclosed herein and known in the art.

7. Alternative embodiments 221. It is understood that post-transcriptional as well as post-translational processes can take place with any of the variants presented here. Disclosed are technologies that can measure such post-transcriptional or post translational processes such as and not limited to post-transcriptional silencing.
8. Methods of Validating Biomarkers 222. Biomarkers for a neurodegenerative disease can be validated in a variety of ways.
For example, the expression of a biomarker can be assessed in one or more subjects diagnosed with a neurodegenerative and in one or more subjects who do not have the neurodegenerative disease. A biomarker whose expression varies between the two groups can be a validated biomarker. The larger the two groups of subjects are, the more reliable the validation. The expression level of a biomarker can also be assessed in a model system for neurodegenerative disease. For example, the level of expression can be tested in leukocyte-containing samples of an animal model for a neurodegenerative disease. Expression of a biomarker (or its homolog) can be assessed in an animal model of a neurodegenerative disease and in a control group. A
biomarker, or homolog thereof, whose expression varies between the model animals and the control animals can be a validated biomarker.
9. Solid Supports 223. Disclosed herein are solid supports (including, stable and mobile forms) wherein at least one address is a biomarker or ligand as disclosed herein. Also disclosed are solid supports wherein at least one address is the sequences, portion of the sequences, or variant of the sequences set forth in any of the nucleic acid sequences or peptide sequences disclosed herein or a ligand for said sequences. Disclosed are chips where at least one address is the sequences or part of the sequences set forth in any of the nucleic acid sequences disclosed herein or a nucleic acid that hybridizes thereto. Also disclosed are chips where at least one address is the sequences ghh in any of the peptide sequences disclosed herein or a ligand for said sequence.
224. Also disclosed are chips where at least one address is a variant of the sequences or part of the sequences set forth in any of the nucleic acid sequences disclosed herein or a nucleic acid that hybridizes to said nucleic acid variant. Also disclosed are chips where at least one address is a variant of the sequences or portion of sequences set forth in any of the peptide sequences disclosed herein or a ligand that binds to said variant peptide.

225. Solid supports include stable supports like slides, chips, microarrays, and nanoarrays comprising any of the biomarkers or antibodies or non-antibody ligands for the biomarkers disclosed herein. Solid supports also include mobile supports like beads comprising any of the biomarkers or antibodies or non-antibody ligands for the biomarkers disclosed herein.
10. Computer readable mediums 226. It is understood that the disclosed nucleic acids and proteins can be represented as a sequence consisting of the nucleotides or amino acids. There are a variety of ways to display these sequences, for example the nucleotide guanosine can be represented by G
or g. Likewise the amino acid valine can be represented by Val or V. Those of skill in the art understand how to display and express any nucleic acid or protein sequence in any of the variety of ways that exist, each of which is considered herein disclosed. Specifically contemplated herein is the display of these sequences on computer readable mediums, such as, commercially available floppy disks, tapes, chips, hard drives, compact disks, and video disks, or other computer 'readable mediums. Also disclosed are the binary code representations of the disclosed sequences. Those of skill in the art understand what computer readable mediums. Thus, computer readable mediums on which the nucleic acids or protein sequences are recorded, stored, or saved.
227. Disclosed are computer readable mediums comprising the sequences and information regarding the sequences set forth herein.

11. Kits 228. Disclosed herein are kits that are drawn to reagents that can be used in practicing the methods disclosed herein. The kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods. For example, the kits could include primers to perform the amplification reactions discussed in certain embodiments of the methods, as well as the buffers and enzymes required to use the primers as intended. In other examples, the kits could include one or more of ~ ~ i~,.. lõ
~Eu~h~~ . ~,,., ._ ~_... ,. ..,..ar~d~ 1 ~~,~
~t ... .blo~at~~~s~~9r= 'Yit s~'~tlf~c~~sed herein, as well as the buffers, labels, enzyme5, secondary or tertiary antibodies, etc. required to use the biomarkers or ligands as intended. In a further exarnple, disclosed is a kit for diagnosing a subject for a neurodegenerative disease (e.g., Parkinson's or Alzheimer's), comprising one or more of the oligonucleotides set forth in Table 4.

12. Diagnostic Assays 229. Also disclosed are diagnostic assays for neurodegenerative diseases. The disclosed assays comprise contacting a sample comprising a leukocyte or a lysate thereof with one or more antibodies or fragments thereof for a biomarker for a neurodegenerative disease.
Antibodies for the disclosed biomarkers can be made my methods known in the art and as disclosed herein.

D. Methods of making the compositions 230. The compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted.
1. Nucleic acid synthesis 231. For example, the nucleic acids, such as, the oligonucleotides to be used as primers can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System 1Plus DNA
synthesizer (for example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B). Synthetic methods useful for making oligonucleotides are also described by Ikuta et al., Ann. Rev. Biochem., 1984;53:323-356, (phosphotriester and phosphite-triester methods), and Narang, et al., Methods Enzyinol., 1980;65:610-620, (phosphotriester method). Protein nucleic acid molecules can be made using known methods such as those described by Nielsen, et al., Bioconjug. Chem., 1994;5:3-7.

2. Peptide synthesis 232. One method of producing the disclosed proteins is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc or Boc (tert butyloxycarbonoyl) chemistry (Applied Biosystems, Inc., Foster City, CA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to the disclosed proteins, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a peptide or protein can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. (Grant, Synthetic Peptides: A User Guide. WH
Freeman and Co., N.Y., 1992; Bodansky and Trost, Ed. Principles of Peptide Synthesis. Springer-Verlag Inc., N.Y., 1993, which are herein incorporated by reference at least for material related to peptide synthesis).
233. Alternatively, the peptide or polypeptide is independently synthesized in vivo as described herein. For example, advances in recombinant glycoprotein production methods, which allow more cost effective production of human glycoproteins by colonies of transgenic rabbits (www.bioprotein.com) or by yeast strains carrying human N-glycosylation system enzymes (Hamilton, et al., Science, 2003;301:1244-6; Gerngross, Nature Biotechnology, 2004;22:1409) can be used.
234. Once isolated, independent peptides or polypeptides may be linked, if needed, to form a peptide or fragment thereof via similar peptide condensation reactions.
For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen, et al., Biochemistry, 1991;30:4151). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson, et al., Science, 1994; 266:776-9). The first step is the chemoselective reaction of an unprotected synthetic peptide thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site (Baggiolini, et al., FEBS Lett. 1992;307:97-101; Clark-Lewis, et al., J. Biol.
Chem., 1994;269:16075; Clark-Lewis, et al., Biochemistry, 1991;30:3128; Rajarathnam, et al., Biochemistry, 1994;33:6623-30).

fWNSf~ ET 11.1.
1lterlia~'iIV 4~;=~Wprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non peptide) bond (Schnolzer, et al., Science, 1992;256:221). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton, et al., Techniques in Protein Chemistry IV. Academic Press, New York, N.Y., pp. 257-67, 1992).

3. Antibodies 236. The disclosed antibodies can be made using any procedure which produces antibodies. For example, disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 1975;256:495.
In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro, e.g., using the HIV Env-CD4-co-receptor complexes described herein.
237. The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patents Nos.
5,804,440 and 6,096,441, which are incorporated by reference herein at least for their teachings of antibody preparation.
238. In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 and U.S.
Patent No.
4,342,566, which are incorporated by reference herein at least for their teachings of antibody preparation. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.

239. The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or ~f 'VW~t~'~L:I~rovided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment.
These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, Curr.
Opin. Biotechnol., 1992;3:348-354, which is incorporated by reference herein at least for its teachings of antibody preparation).
240. The disclosed human antibodies can be prepared using any technique.
Examples of techniques for human monoclonal antibody production include those described by Cole, et al.
(Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boemer, et al.
(J. Immunol., 1991;147(1):86 95), which are incorporated by reference herein at least for their teachings of antibody preparation. Human antibodies (and fragments thereof) can also be produced using phage display libraries (Hoogenboom, et al., J. Mol. Biol., 1991;227:381; Marks, et al., J. Mol. Biol. 1991;222:581, which are incorporated by reference herein at least for their teachings of antibody preparation). The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits, et al., Proc. Natl. Acad. Sci. U.S.A., 1993;90:2551-5; Jakobovits, et al., Nature, 1993;362:255-8; Bruggermann, et al., Year in Immunol., 1993;7:33, which are incorporated by reference herein at least for their teachings of antibody preparation).

241. Methods for huma.nizing non-human antibodies are well known in the art.
For example, humanized antibodies can be generated according to the methods of Winter and co-workers (Jones, et al., Nature, 1986;321:522-5, Riechmann, et al., Nature, 1988;332:323-7, Verhoeyen et al., Science 1988;239:1534-6), by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody. Methods that can be used to produce humanized antibodies are also described in U.S. Patent Nos. 4,816,567, 565,332, 5,721,367, 5,837,243, 5,939,598, 6,130,364, and 6,180,377, wliich are incorporated by reference herein at least for their teachings of antibody preparation.

..~.~ ~~o16Q!"s for making the compositions

242. Disclosed are processes for making the compositions as well as making the intermediates leading to the compositions. For example, disclosed are nucleic acids and proteins in SEQ IDNOs:l-257. There are a variety of methods that can be used for making these compositions, such as synthetic chemical methods and standard molecular biology methods. It is understood that the methods of making these and the other disclosed compositions are specifically disclosed.

243. Disclosed are nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid comprising the sequence set forth in Table 4, such as cyclin D1, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, protein kinase C
alpha, C5, Cl inhibitor, IL-17r, IL-g, LIF, TNF-alpha, and IL-lOr, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin, and genes of proteins listed in Tables 5 and 6, and a sequence controlling the expression of the nucleic acid.

244. Also disclosed are nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence having 80% identity to a sequence set forth in Table 4, and a sequence controlling the expression of the nucleic acid.

245. Disclosed are nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence that hybridizes under stringent hybridization conditions to a sequence set forth in Table 4 and a sequence controlling the expression of the nucleic acid.

246. Disclosed are nucleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence encoding a protein such as HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 epsilon, Prohibitin, Phospoglycerate mutase 1, Apoliporotein AI, Superoxide dismutase, RNA-binding protein regulatory subunit, Chain A thioredoxin peroxidase B, RAS-related protein RAP 1B, Tumor rejection antigen, Haptoglobin, Fibrin beta, actin-interacting protein 1(AIP1), mitogen activated protein kinase I
(MAPKI), actin or a fragment thereof, glutaraldehyde-3-phosphate dehydrogenase (GAPDH), transforming protein RhoA, acidic leucine-rich nuclear phosphoprotein 32 family member B

(ANP32B or APRIL), peroxiredoxin II, an arnyloid precursor protein (APP), a-secretase, ,6-secretase, y-secretase, Ao peptide, Fe65, Tip60, SERCA, PS 1/2, nectin-la, and non-amyloid component of senile plaque (NACP/ a-synuclein), or proteins set forth in Tables 5 or 6, or proteins of genes and a sequence controlling an expression of the nucleic acid molecule.

+ E
'"~1;: id ~Ijis~l~ 'i ~'~ii~~Cleic acid molecules produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence encoding a protein having 80% identity to a protein such as HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 epsilon, Prohibitin, Phospoglycerate mutase 1, Apoliporotein AI, Superoxide dismutase, RNA-binding protein regulatory subunit, Chain A thioredoxin peroxidase B, RAS-related protein RAP1B, Tumor rejection antigen, Haptoglobin, Fibrin beta, actin-interacting protein 1(AIPl), mitogen activated protein kinase I(MAPKI), actin or a fragment thereof, glutaraldehyde-3-phosphate dehydrogenase (GAPDH), transforming protein RhoA, acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B or APRIL), peroxiredoxin II, an amyloid precursor protein (APP), a-secretase, (3-secretase, 7-secretase, A(3 peptide, Fe65, Tip60, SERCA, PS1/2, nectin-la, and non-amyloid 0 component of senile plaque (NACP/ a-synuclein), or a protein set forth in Tables 5 or 6, and a sequence controlling an expression of the nucleic acid molecule.
248. Disclosed are nucleic acids produced by the process comprising linking in an operative way a nucleic acid molecule comprising a sequence encoding a peptide having 80%
identity to a protein set forth in Tables 5 or 6, wherein any change from the Table 5 or 6 are conservative changes and a sequence controlling an expression of the nucleic acid molecule.
249. Disclosed are cells produced by the process of transforming the cell with any of the disclosed nucleic acids. Disclosed are cells produced by the process of transforming the cell with any of the non-naturally occurring disclosed nucleic acids.
250. Disclosed are any of the disclosed peptides produced by the process of expressing any of the disclosed nucleic acids. Disclosed are any of the non-naturally occurring disclosed peptides produced by the process of expressing any of the disclosed nucleic acids. Disclosed are any of the disclosed peptides produced by the process of expressing any of the non-naturally disclosed nucleic acids.
251. Disclosed are animals produced by the process of transfecting a cell within the animal with any of the nucleic acid molecules disclosed herein. Disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules disclosed herein, wherein the animal is a mammal. Also disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules disclosed herein, wherein the mammal is mouse, rat, rabbit, cow, sheep, pig, or primate.

[I::JAd df ~~~bf~6't~JE animals produced by the process of adding to the animal any of the cells disclosed herein.

E. Methods of using the compositions 1. Methods of using the compositions as research tools 253. The disclosed compositions can be used in a variety of ways as research tools.
For example, the disclosed compositions, such as SEQ ID NOs: 1-257 can be used to study the effects of various therapies on a neurodegenerative disease.
254. The compositions can be used for example as targets in conibinatorial chemistry protocols or other screening protocols to isolate molecules that possess desired functional properties related to a neurodegenerative disease (e.g., Alzheimer's and Parkinson's disease).
255. The disclosed compositions can also be used diagnostic tools related to neurodegenerative diseases such as Alzheimer's and Parkinson's disease.
256. The disclosed compositions can be used as discussed herein as either reagents in micro arrays or as reagents to probe or analyze existing microarrays. The disclosed compositions can be used in any known method for isolating or identifying single nucleotide polymorphisms. The compositions can also be used in any known method of screening assays, related to chip/micro arrays. The compositions can also be used in any known way of using the computer readable embodiments of the disclosed compositions, for example, to study relatedness or to perform molecular modeling analysis related to the disclosed compositions.

VII. EXAMPLES
257. 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 the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in C or is at ambient temperature, and pressure is at or near atmospheric.

A. Example 1 Molecular distinction of Alzheimer's disease from analysis of leukocyte RNA
1. Methods:

a) Patient Recruitment 258. Patients were recruited through the Geriatric Neurology and Psychiatry Clinic at Monroe Community Hospital. Following entrance into this study with informed consent, 1~nf~rrn~ti~oi a'bat~'c' 1o~ cical dementia tests was gathered from patients.
Blood samples were stored at 4 C until processed for RNA isolation (less than 12 hours).
259. Data presented here were obtained from three independent samples from three different groups of people. RNA from each of these samples was extracted;
hybridized and analyzed at different times. Sample 1 consisted of 8 AD and 7 control cases.
Sample 2 consisted of 8 AD and 8 control cases. Sample 3 consisted of 5 AD, 4 control and 2 PD cases.
Sample characteristics are summarized in Tables 1-3.

2. Subjects 260. AD subjects included in the study were diagnosed with probable or possible AD
on the basis of NINCDS (McKhann G, et al., Neurology, 1984;34(7):939-944) and DSM IV
criteria for AD. Examination by a neurologist was performed to confirm diagnosis and to measure disease severity. Disease severity was assessed using the Mini-Mental Status Examination (MMSE) (Folstein MF, et al., J. Psychiatric Res., 1975;12(3):189-198), the Clinical Dementia Rating scale (CDR) (Hughes CP, et al., British J. Psychiatry, 1982;140:566-572), and the Blessed Dementia Rating Scale (BDRS) (Blessed G, et al., British J.
Psychiatry, 1968;114(512):797-811). Control subjects included in the study scored above 27 on the MMSE, wliile AD cases scored below 22. The average CDR of AD cases ranged from 1.2 to 1.5. Any subject with a history of bleeding diathesis or coagulopathy was excluded.

3. Isolation of RNA from whole blood samples 261. To extract polyA-RNA from leukocytes, an inRNA isolation kit for blood was utilized (Roche). In brief, erythrocytes were selectively lysed and leukocytes were collected by centrifugation. The leukocytes were then lysed and the total nucleic acids were collected by non-specific adsorption to magnetic glass beads and magnetic separation. Following a series of washes and elution of the nucleic acids from the magnetic glass beads, the mRNA was specifically captured by the use of biotin-labeled oligo(dT) and streptavidin-coated magnetic particles. After removal of other nucleic acids (DNA, rRNA, tRNA) by washing, mRNA
samples were collected and stored at minus 80 C until later use. The concentration and purity of samples were checked by OD260i280. It is understood that any RNA isolation procedure could be used.
4. Construction and hybridization of cDNA arrays 262. The cDNA clones used are listed in Table 4. The dbEST database of the National Center for Biotechnology Information was searched for relevant 3'-cDNA clones and the clones were either purchased from distributors or gifts from various laboratories.
The cDNA clones '' ~~=us~d i~..~~~s~4~~t~t~~'vi~~~~~~~~~i~i~~is gifts from many investigators:
CREB, tuberin, nestin, cyclin Dl and GAD from Jim Eberwine; BDNF, bcl-2, bcl-xs, bcl-xl, calbindin, and SOD-1 from Denise Figlewicz; hTR2 from Chawnshang Chang, GAP-43, APP, and PS 1 from Rachel Neve;
P19 from Bert Vogelstein; synaptotagmin I from T. Sudhof; GTH, pGTH4, and pHMGST from Dr. Davi;
ubiquitin from Dr. Roharaker; cdc2 from Inez Vincent; and SF2flag, Pht6, and tra2-C2 from Stefan Stamm. The rest of the cDNA clones were dbEST clones from distributors of I.M.A.G.E.
Consortium cDNA clones.
263. All of the cDNA clones were sequenced to confirm their identity. One microgram of each linearized cDNA was denatured in 0.2 N NaOH/0.2 mM EDTA at 37 C for 30 minutes.
The sample volume was neutralized with 0.3 M NaOAc, pH 4.5. The cDNAs were immediately printed as previously described (Chow N, et al., Proc. Natl. Acad. Sci. U.S.A.
1998;95:9620-9625) on a nylon membrane (Micron Separations) using a 96 pin replicator (Nalge Nunc) with each cDNA spotted four times. The membranes were prehybridized at 42 C in hybridization solution (50% fonnamide/5X SSPE/5X Denhardt's solution/0. 1% SDS/ 10% dextran sulfate/50, g/ml denatured salmon sperm DNA/100 g/ml tRNA) for 3 hours before adding the RNA
probes. After oveniight incubation at 42 C, blots were washed in 2X SSC/0.1%
SDS at 55 C
for 1 hour, 2X SSC/0.1% SDS/10 g/ml RNAse A at 37 C for 1 hour, and 2X
SSC/0.1% SDS at 37 C for 1 hour. Membranes were then exposed to a storage phosphor screen.

5. Data acquisition and analysis 264. Hybridization intensity of each dot was detected by laser densitometric scanning (Phosphoimager, Molecular Dynamics). Values (counts) for each spot obtained by phosphoimager analysis were corrected using local background. The amount of cDNA deposited on each spot in the array was quantified by stripping and reprobing the membrane with an oligonucleotide specific for the T7 promoter present in all vectors. These data provided a correction for potential spot-to-spot differences in deposition of cDNA on the membrane. To ensure accurate comparisons across arrays, signals were normalized using the average of all markers (cDNAs) in an array for each RNA sample. The resulting standardized data were analyzed by canonical analysis. This analysis determines the variables (messages) that best distinguish groups and assigns weights to each variable. As is true for multivariate analyses, the number of variables must not exceed the number of cases (Kshirsager AM, Multivariate Analysis, Dekker M, New York, N.Y., 1972). The first canonical variable provides the best distinction between groups. The second canonical variable operates on the residual variance that remains unaccounted for by canonical variable 1. Additional iterations are possible with If'~'''~iiri~i~isl~i~'=~~~~~-'"T-N-iffi~~ihalal significance of the separation between AD and control cases resulting from canonical analysis was assessed by the Wald-Wolfowitz runs test (Siegel S, Nonparametric Statistics, McGraw-Hill, New York, N.Y., 1-312, 1956).

B. Results 1. Message classes that distinguish AD from control blood samples.
265. Since valid results from multivariate canonical analyses require that the number of variables (messages in this case) be less than the number of cases (subjects) used (Kshirsager AM, Multivariate Analysis, Dekker M, New York, N.Y., 1972) messages were formed into subsets of 7 or 8 messages out of the total of 64 messages studied. Only two out of the 5 subsets of messages examined across a113 samples produced consistent separation of AD
from control cases. These two subsets were messages related to the cell cycle and messages related to inflammatory responses. Figures 2A, 2B, and 2C plot canonical variable 1 vs.
canonical variable 2 for those messages related to the cell cycle. Figures 2A, 2B, and 2C plot canonical variable 1 vs. canonical variable 2 for those messages related to the inflammatory responses.
266. These 6 plots demonstrate that generally distinction of disease categories can be achieved with little overlap between groups. Note that the categorization of 41 (43 including 2 Parkinson's disease cases) cases on the basis of leukocyte expression of cell cycle messages agreed with the clinical classification in 38/41 (40/43 counting PD) of the cases with 3 control cases in the AD space and no AD cases in the control space. Genes related to the inflammatory system placed no control cases in the AD space and one AD case in the control space. The inflammatory genes correctly distinguished the 2 PD cases from both control and AD spaces.
267. It should be noted that a very small number of individual messages among those we sampled yielded statistically significant (or close to significant) differences between AD and control cases. These were alpha-1 antichymotrypsin, crystallin and cyclooxygenase II.

However, they were not sufficient in themselves to distinguish AD from control without significant overlap.
268. Similar plots of canonical variables for other sets of messages were not as successful in distinguishing AD from control leukocyte message profiles (data not shown). This provides evidence that the positive results illustrated in Figures 1 and 2 are specific to the two gene sets presented in Figures 1 and 2.
269. There are two major conclusions regarding the data presented here: (1) expression profiles of multiple genes are effective at distinguishing mild AD
(average CDR 1.2-1.5) from non-demented control cases and, (2) the gene classes we describe as distinguishing AD

1~5Walfs&nples are similar to gene classes whose expression has been shown to be altered in the brain in AD. Blood cells can be used to conduct basic research on the molecular mechanisms of AD. Data resulting from such studies can lead to design of therapeutic molecules. Additionally blood cells can used to monitor therapeutic efficacy in clinical trials of therapeutic agents as well as the efficacy of treatment of individual patients.

2. Expression profiles of multiple genes are effective at distinguishing Alzheimer's disease 270. The most comprehensive clinical diagnosis of AD is a complex, expensive process involving many assessments (McKhann G, et al., Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology, 1984;34(7):939-944). Even in expert hands the diagnosis is considered provisional, subject to neuropathological confirmation at autopsy. In AD centers, the clinical diagnosis reaches an autopsy confirmation rate of 85-90% (Gearing M, et al., Neurology, 1995;45(3 Pt 1):461-466). In less expert hands, the rate of autopsy confirmed diagnosis is lower. The expense and difficulties of diagnosing AD
have led to a search for an easily sampled and successful biomarker of disease. Some of the biomarkers that have been described include those that are invasive (e.g., spinal tap (Davidson P, et al., J. Neural Transmission-General Section, 1997;104(6-7):711-720)) or require expensive equipment and expertise (Killiany RJ, et al., Neurology, 2002;58(8):1188-1196). Although these procedures are extremely useful in investigative studies, they do not offer promise for routine, large scale diagnostic use. Other tests have been described that draw on easily obtained peripheral samples from, for example, blood cells (Nagy Z, et al., Neuroscience Letters, 2002;317(2):81-84), (Padovani A, et al., Archives of Neurology, 2002;59:71-75), skin (Ikeda K, et al., Dementia & Geriatric Cognitive Disorders, 2000; 11(5):245-25 0) and urine (Pratico D, et al., Archives of Neurology, 2002;59:972-976). Additional studies have described tests for AD
that utilize responses to pharmacological intervention (Scinto LF, et al., Neurology, 1999;52(3):673-674). Many of these studies directed at distinguishing AD from control samples on the basis of peripheral tissues have been successful at yielding statistically significant differences between AD and control samples. However, the clinical utility of the tests described in these studies has been limited by significant overlap between AD and control samples. The disclosed data indicate that this overlap may be diminished by making use of multiple variables.
271. Expression profiling in brain has shown that multivariate analysis of the expression of multiple messages can distinguish AD from control brain (Chow N, et al., Proc.

31 ]4;' 5:9620-9625). More recently, multivariate analysis of expression of multiple genes has been successful in distinguishing malignant from non-malignant samples of tissues other than brain (Welsh JB, et al., Proc. Nat. Acad. Sci. U.S.A., 2001;98(3):1176-1181). In the data presented herein, applying analysis to quantitative data on expression levels of multiple message species extracted from peripheral blood leukocytes has yielded promising separation of AD from control samples. In addition, two samples from PD cases have been distinguished from both AD and control samples, suggesting that the distinction being made between AD and control samples is distinguishing properties other than those of general neurodegeneration.
272. The data do however "misclassify" a very few number of cases. The nature of the misclassification is that 3 out of 19 cases that were clinically defmed as non-demented yielded values that fell among the AD cases. The data herein is consistent with these cases being "preclinical" cases in which there exists some degree of AD pathology. Such a suggestion is consistent with the Braaks' data demonstrating that brains of as many as 20%
of persons in their 20s exhibited AD pathology. (Braak H, et al., Neurobiology of Aging, 1997;18(4):351-357).
These data provide a compelling argument for the existence of an extended preclinical stage of AD during which there is frank pathology.
273. In addition to these control cases that gave values consistent with AD, there was one clinically defined AD case (out of 21) that fell among the control cases.
A search of the existing clinical and pathological data for this case has not revealed distinguishing characteristics that would rationally separate this from the other AD cases in our sample.
274. Other sets of genes selected either randomly or on the basis of other properties (e.g., growth factors) do not distinguish AD from control samples to the extent that is possible with the gene sets related to the cell cycle and to the inflammatory system (data not shown).
This suggests that the disclosed finding of segregation on the basis of selected gene sets is not an artifact of the methods of analysis we employed.
275. The data presented here suggest that the gene sets disclosed herein have can be clinically useful and, as discussed herein, also suggest a relationslZip between molecular events in brain and in peripheral blood cells.
3. Gene classes that distinguish AD brain also distinguish AD blood samples.
276. It is notable that the classes of gene products shown herein to distinguish AD
from control peripheral blood leukocytes are also among those classes of gene products that have altered expression in AD brain. Certainly, selected neuron specific gene products that are 9~fE~ct6cfL Jn related to the synapse (Callahan LM, et al., J.
Neuropathology & Experimental Neurology, 2002;61(5):384-395; Yao P, et al., Neurobiology of Disease, 2003;12:97-109), are not to be expected to play a role in leukocytes.
However, there are gene products that are not neuron specific that have also been shown to have altered expression in AD brain. Of particular interest here are molecules related to the cell cycle (Arendt T, Neurobiology of Aging, 2000;21(6):783-796; Vincent I, et al., J.
Neurosci., 1997;17:3588-3598; Nagy Z, et al., Neuroscience Letters, 2002;317(2):81-84;
Harris PL, et al., Neurobiology of Aging, 2000;21(6):837-841; Wu Q, et al., Neurobiology of Aging, 2000;21(6):797-806; Zhu, et al., Neurobiology of Aging, 2000;21(6):837-841) and molecules related to inflammatory systems (Akiyama H, et al., Neurobiology of Aging, 2000;21(3):383-421 and Bamberger ME, and Landreth GE, Microscopy Research & Technique, 2001;54(2):59-70).
In fact, a recent suminary of the inflammatory system in AD brain states "A
virtual textbook of inflammatory mediators has been observed in the Alzheimer's disease brain over the last 15 years" (Akiyama H, et al., Neurobiology of Aging, 2000;21(3):383-421). Indeed, studies of AD

tissues other than brain have demonstrated altered expression of cell cycle (Nagy Z, et al., Neuroscience Letters, 2002;317(2):81-84; Stieler JT, et al., NeuroReport, 2001;12(18):3969-3972) as well as inflammatory systein (Scali C, et al., Neurobiology of Aging, 2002;23(4):523-530; De Luigi A, et al., Mechanisms of Ageing & Development, 2001;122(16):1985-1995;
Kusdra L., et al., Immunobiology, 2000;202(1):26-33; Lombardi VR, et al., J.
Neuroimmunology, 1999;97(1-2):163-171; Remarque EJ, et al., Experimental Gerontology, 2001;36:171-176) genes in several tissues, including blood cells (Nagy Z, et al.,. Neuroscience Letters, 2002;317(2):81-84; Scali C, et al., Neurobiology of Aging, 2002;23(4):523-530) and epithelial cells (Schmitz A, et al., Histochemistry & Cell Biology, 2002;117(2):171-180).
277. The above studies do not prove that selected common changes in gene expression in both peripheral and brain cells may be consistent with the concept of a common, systemic disease mechanism or common links between blood and brain. (Webster S, et al., Biochemical & Biophysical Research Communications, 1995;217:869-875; Kimberly WT, et al., J. Biol.
Chem., 2001;276(43):40288-40292; Cao X, et al., Science, 2001;293(5527):115-120).
278. The data suggest that disordered expression of cell cycle and inflammatory genes may play a central role in the pathophysiology of AD in many cell types, both within and outside the nervous system. Additional neuropathophysiological aspects of AD may be consequences of these pivotal events, including tau phosphorylation by kinases also related to the cell cycle (Busciglio J, et al., Neuron, 1995;14(4):879-888; Ferreira A, et al., Molecular & Cellular "I~Tei~rosc~~~ri~#es;~ 7t;0~~'1F~' -J~4; Greenberg SM, et al., Proc. Nat.
Acad. Sci. U.S.A., 1994;91(15):7104-7108) and consequent cytoskeletal disruption, a variant of programmed cell death as a response to the inability of post mitotic neurons to successfully complete the cell cycle as well as cell death as a response to inflammatory challenge.
279. RNA profiling of the expression of multiple genes by peripheral leukocytes followed by canonical discriminant analysis can be used both as a biological tool for the analysis of molecular alterations in disease, as well as a tool for differentiation between Alzheimer's and control patients. These methods also suggest a potential to differentiate numerous other diseases. It is not the significant difference between individual genes (although a few genes have significant correlations) that provides a clear discrimination between patients with a specific disease and others, but rather an analysis based on weighted sums of many genes.
280. The finding that gene expression by peripheral blood leukocytes is affected in AD
reinforces the concept of AD as a systemic disease. Furthermore, that the cell cycle and inflammatory gene classes affected in peripheral leukocytes are similar to gene classes affected in brain in AD is a commonality that suggests and is consistent with parallel molecular mechanisms of AD in brain and blood cells, perhaps initiated by APP peptides.

C. Example 2 Inflammatory, cell cycle, and stress transcripts and molecular distinction of Alzheimer's disease from peripheral blood leukocytes 1. Methods:
a) Patient Recruitment 281. Patients were recruited through the Geriatric Neurology and Psychiatry Clinic at Monroe Community Hospital. Following entrance into this study with inÃormed consent, information on a battery of clinical dementia tests was gathered from patients.

282. Data presented here were obtained from three independent sets of samples from three different groups of people. Leukocyte RNA from each of these samples was extracted, hybridized to custom cDNA arrays and analyzed at different times. Sample 1 consisted of 8 early AD and 7 control cases. Sample 2 consisted of 8 new early AD and 8 new control cases.
Sample 3 consisted of 5 new early AD, 4 new control and 2 (new) Parkinson's disease (without dementia) cases. A total of 21 early AD, 19 control, and 2 PD cases were investigated.

2. Subjects 283. AD subjects included in the study were diagnosed with probable or possible AD
on the basis of NINCDS (McKhann G, et al., Neurology, 1984;34(7):939-944) and DSM IV
criteria for AD. Examination by a neurologist was performed to confirm diagnosis and to ~I~D7 ~a~ ~ , ,t,.. ..
r~ea~urE ~c~~s~~fs~ ~e veti~ ~t e severity was assessed using the Mini-Mental Status Examination (MMSE; Folstein et al., J. Psychiatric Res., 1975;12(3):189-198), the Clinical Dementia Rating scale (CDR; Hughes et al., British J. Psychiatry, 1982;
140:566-572), and the Blessed Dementia Rating Scale (BDRS; Blessed et al., British J. Psychiatry, 1968;114(512):797-811). Control subjects included in the study scored above 27 on the MMSE, while AD cases scored below 22. The mean CDR of AD cases in each of the three samples ranged from 1.2 to 1.5. Since these were not autopsy confirmed cases, the assignment of each case to a specified disease category relies on the accuracy of the clinical diagnosis. Any subject with a history of bleeding diathesis or coagulopathy was excluded. Blood samples were drawn by a phlebotomist and stored at 4 C until processed for RNA isolation (less than 8 hours).
3. Isolations of RNA from whole blood samples 284. To extract polyA-RNA from leukocytes, an inRNA isolation kit for blood was utilized (Roche). In brief, erythrocytes were selectively lysed and leukocytes were collected by centrifugation. The leukocytes were then lysed and the total nucleic acids were collected by non-specific adsorption to magnetic glass beads and magnetic separation. Following a series of washes and elution of the nucleic acids from the magnetic glass beads, the inRNA was captured by the use of biotin-labeled oligo(dT) and streptavidin-coated magnetic particles. After removal of other nucleic acids (DNA, rRNA, tRNA) by washing, mRNA samples were collected and stored at minus 80 degree Celsius until later use. The concentration and purity of samples were checked by OD260i280= It is understood that any RNA isolation procedure could be used.
4. Construction of cDNA arrays 285. The cDNA clones represented in the arrays were selected based on previous microarray studies (e.g., Chow, et al., Proc. Natl. Acad. Sci. USA.
1998;95:9620-9625) and a subset of those of interest in the field of AD research. The dbEST database of the National Center for Biotechnology Information was searched for relevant 3' cDNA clones.
The cDNA
clones used in this study were gifts from many investigators or were from distributors of I.M.A.G.E. Consortium cDNA clones. 172 transcripts were represented in the arrays.
286. All of the cDNA clones were sequenced in house to confirm their identity.
One microgram of each linearized cDNA was denatured in 0.2 N NaOH/0.2 mM EDTA at 37 C for 30 minutes. The sample volume was neutralized with 0.3 M NaOAc, pH 4.5. The cDNAs were immediately printed as previously described (Chow, et al., Proc. Natl. Acad.
Sci. USA.
1998;95:9620-9625) on a nylon membrane (Micron Separations) using a 96 pin replicator (Nalge Nunc) with each cDNA spotted four times. The membranes were prehybridized at 42 C in Ml- ~u2cNri10641;='forinamide/5X SSPE/5X Denhardt's solution/0. 1% SDS/ 10%
~' '' gtlo dextran sulfate/50, g/ml denatured salmon sperm DNA/100 g/ml tRNA) for 3 hours before adding the RNA probes. After overnight incubation at 42 C, blots were washed in 2X
SSC/0.1% SDS at 55 C for 1 hour, 2X SSC/0.1% SDS/10 g/ml RNAse A at 37 C for 1 hour, and 2X SSC/0.1% SDS at 37 C for 1 hour. Membranes were then exposed to a storage phosphor screen.
5. Data acquisition and analysis 287. Hybridization intensity of each dot was detected by laser densitometric scanning (Phosphoimager, Molecular Dynamics). Values (counts) for each spot obtained by phosphoimager analysis were corrected using local background. The amount of cDNA deposited on each spot in the array was quantified by stripping and reprobing the membrane with an oligonucleotide specific for the T7 promoter present in all vectors. These data provided a correction for potential spot-to-spot differences in deposition of cDNA on the membrane. To ensure accurate comparisons across arrays, signals were normalized using the average of all markers (cDNAs) in an array for each RNA sample. The resulting standardized data were analyzed by two univariate tests and one multivariate test. The univariate tests were the t-test and the N-test. The latter N-test is a newly devised, essentially non-parametric test for multiple testing inference. (Technical Report 04/01 at http://www.urmc.rochester.edu/smd/biostat/people/techreports.html).
Multivariate statistical testing relied on canonical discriminant analysis. The multivariant analysis was performed on SAS/STATTM software from SAS Institute, Inc. (Cary, NC). This analysis determines the variables (messages) that best distinguish groups and assigns weights to each variable. The first canonical variable provides the best distinction between groups. The second canonical variable operates on the residual variance that remains unaccounted for by canonical variable 1.
Additional iterations are possible with diminishing effect.
D. Results 288. Since valid results from multivariate canonical analyses requires that the number of variables analyzed (messages in this case) be less than the number of cases (subjects) used (Kshirsager, AM. Multivariate Analysis. New York: Dekker, M., 1972) messages were formed into 5 subsets of 7 or 8 messages out of the total of 172 transcripts studied.
Three of the subsets formed were based on hypotheses that specified systems known to be affected in AD brain would also be affected in AD leukocytes. These three sets of transcripts were those related to either cell stress (especially oxidative stress), inflammatory system, or cell cycle/apoptosis. Two ~~==~t~Et ~~~I~{~E ~~~~e b ~~t~riie composed of transcripts that approached significance in the t-test and one composed of transcripts chosen at random as a control for spurious results from the analytical methods used. These transcript sets were then utilized to analyze the data from three independent samples of early AD and control subjects. The statistical significance of the separation between AD and control cases resulting from canonical analysis was assessed by the Wald-Wolfowitz runs test (Siegel S, Nonparametric Statistics, McGraw-Hill, New York, N.Y.
1956:1-312).
289. Univariate analysis by t-test revealed three transcripts to be statistically significantly different between AD and control samples. These were: alpha-1 antichymotrypsin, crystallin and cyclooxygenase II. Univariate analysis by N-test analysis revealed one transcript to approach statistically significant difference between AD and control. This was ERCC6. None of the transcripts revealed by either of these univariate statistics were sufficient in themselves to distinguish early AD from control without significant overlap.
290. Multivariate canonical discriminant analysis was, however, able to deliver excellent distinction between peripheral blood leukocytes of early Alzheimer's disease and control subjects. The first study established that canonical discriminant analysis of the sets of transcripts related to cell stress, inflammatory system and cell cycle/apoptosis was able to distinguish AD from control samples with minimal overlap. Each point in each plot represents a composite "score" [canonical variable 1] for one individual. This score was determined by multivariate canonical discriminant analysis of the 8-10 transcripts selected to represent cell stress (e.g., Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, and transferrin) (see Fig. 3A), inflannnation (e.g., C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, and IL-10r) (see Fig. 3B), or cell cycle (e.g., cyclin Dl, cyclin B, cyclin Gl, weel, hTR2, CDC25b, GSK3 beta, and protein kinase C alpha) (see Fig. 3C). Two transcript sets, chosen on the basis of t-tests of significance or selected randomly, did not cleanly distinguish early AD fiom control cases (data not shown).
291. A repetition with new, non-overlapping sets of subjects confirmed that transcripts sets related to cell stress, inflammatory system and cell cycle/apoptosis were again able to distinguish early AD from control cases (Figure 4). Subsequently, a new, additional, set of subjects was recruited, leukocytes collected, mRNA extracted and hybridized to new arrays.
This replicate again confirmed that transcript sets related to cell stress, inflammatory system and cell cycle/apoptosis were able to distinguish early AD from control cases (Figure 5).

MM ea~~ A&' -study the other two transcript sets, chosen on the basis of t-test significance and selected randomly did not cleanly distinguish early AD from control cases.
These 9 plots of Figures 3-5 demonstrate that generally excellent distinction of disease categories can be achieved with little overlap between groups (The differentiation of early AD

from control is significant beyond the p<0.01 level (Wald-Wolfowitz runs test). Note that the analysis distinguished 2 Parkinson's disease cases from early AD and control cases.

1. Gene classes that distinguish AD in brain also distinguish AD blood samples 293. Like Example 1, the classes of gene products shown here to distinguish AD
from control peripheral blood leukocytes are also among those classes of gene products that are known to have altered expression in AD brain. The three classes of transcripts investigated do not appear to have performed equally well. Although transcripts related to cell stress and inflammatory system were quite consistent in their distinction of disease state, cell cycle transcripts were less consistent. Nevertheless, the data suggest that multivariate analysis of expression profiles of peripheral cells can be used in the study of molecular phenomena of Alzheim.er's disease.
294. Selected neuropathological/neurobiological aspects of AD can be consequences of altered expression of cell cycle, cell stress, and inflamination events, all of which are pivotal in the life of a cell. As one example, activation of cell cycle kinases by A
beta leads to tau ph.osphorylation (Busciglio et al., Neuron. 1995;14(4):879-888; Greenberg et al., Proc. Nat.
Acad. Sci. U.S.A., 1994;91(15):7104-7108), which produces the cytoskeletal disassembly required by cell division. Cytoskeleton disassembly then would have consequences for maintenance of neuronal processes, transport and the support of synapses.
295. The data presented here suggest that the gene classes used can be become clinically useful in a variety of ways including diagnosis (perhaps even early, preclinical or antecedent, diagnosis) and monitoring therapeutic efficacy. Furthermore, the evidence that similar classes of gene products are affected in both brain and blood allows that peripheral blood cells can serve as surrogates for brain cells in the study of selected fundamental molecular and cell biological mechanisms of Alzheimer's or neurodegenerative diseases in general. The data presented here indicate that in AD and PD similar systems are affected in both brain and blood does not require that they behave perfectly equivalently in both classes of cells.

i ~"~is'o~ ~ 1ray Data 296. The meanings that may be extracted from array data are dependent on the array specifics and the analysis methods used. The combination of targeted arrays and analytical methods used was designed to test the hypothesis that selected molecular systems related to fundamental cell biology known to be affected in AD brain would also be found to be affected in peripheral blood leukocytes. Focused was specifically on transcripts related to inflammatory systems, cellular stress, and cell cycle/apoptosis. The targeted arrays used herein were, therefore, designed to include multiple probes related to each of these three systems. Rather than emphasize analytical methods designed to test AD/control differences of individual transcripts, a method of analysis that was able to test differences between AD and control profiles of gene expression by making simultaneous use of multiple transcripts was used. The classical method of canonical discriminant analysis was selected. This method makes use of knowledge of the existence of two (or more) groups to find the set of variables (transcripts) that best differentiates the groups. The analysis assigns a weight to each transcript, and these weights are used to calculate a "score" (e.g., canonical variable) for each person. The analysis is as described hereinbefore and was performed with the SAS/STATTM software. It is this score that is presented in Figures 3-5.
297. The value of multivariate statistical methods in distinguishing effects of disease on profiles of gene expression has been demonstrated in a variety of studies, including distinguishing both rion-brain (ovarian cancer, Welsh et al., Proc. Nat. Acad.
Sci. U.S.A., 2001;98(3):1176-1181), as well as brain diseases (Tang et al., 2004). This latter study differentiated three neurological conditions (tuberous sclerosis complex 2, neurofibromatosis type 1, and Down's syndrome) on the basis of expression profiles of transcripts derived from blood samples. They state "Each disease was associated with a unique gene expression pattern in blood that can be accurately distinguished by a classifier." The present data emphasize that differing expression levels of individual genes are often not as powerful in discriminating disease state as an analysis based on weighted sums of many gene products. The interpretation of array data continues to be a developing enterprise.

3. Mechanism of Common Effects on Blood and Brain Cells 298. The fact that gene expression by peripheral blood leukocytes is altered in Alzheimer's disease in ways that are suggestive of events in brain can be explained by communication between peripheral blood elements and the brain (e.g. Hickey WF, et al., 1991;
see Hickey, 1999 for review). Furthermore, the permeability of the blood-brain barrier to such ''&ci 'f ~~a~~=b~ri~~s~tov~'ri"~o be~~eiihanced by a variety of factors, including A beta peptides (e.g.
~
Farkas IG, et al., 2003) and selected A beta peptides have been shown to be increased in the peripheral circulation in AD (see Irizarry MC, 2004, for review). It is not necessary to rely on blood-brain cominunication alone as a source of A beta peptides in the blood since blood cells themselves, especially platelets, express APP, BACE and other components differentially in AD
(e.g. Colciaghi et al., 2004; Baskin et al., 2000; Li et al., 1999; Rosenberg et al., 1997). This suggests that mechanisms other than (or in addition to) blood-brain communication may play important roles in modulating AD effects on peripheral cells. Although the presence of A beta peptides in the circulatory system may be pertinent, other peripheral cells outside the vasculature, including fibroblasts, express components of the APP system and show differences between AD and control samples (Emiliani et al., 2003; Ikeda et al., 2000;
Zhao et al., 2002;
Scali et al., 2002; see Etcheberrigaray et al., 1996; Gibson et al., 1996 and Gibson and Huang, 2002, for reviews). These data are consistent with the concept of AD as a systemic disease whose major clinical manifestations arise from its effects on the brain.
299. As disclosed herein, multivariate analyses of transcripts related to inflammation, cell stress and cell cycle/apoptosis expressed by peripheral blood leukocytes are able to distinguish early AD from control cases. Several other conclusions can be made: (1) The classes of transcripts shown herein to be able to distinguish AD from control leukocytes are similar to classes of transcripts known to be affected in the brain in AD. (2) These data may have implications for the early (perhaps preclinical, antecedent) diagnosis of AD and for monitoring disease progression and therapeutic efficacy.

E. Example 3 molecular distinction of Parkinson's disease from peripheral blood leukocytes 300. Parkinson's disease protein analysis identified a patient population which included 13 Parkinson's disease (PD) patients and 9 age-matched control patients. Fresh whole blood was drawn, red blood cells lysed and leukocytes harvested. Leukocyte protein concentrations were determined and protein integrity determined by SDS-PAGE
electrophoresis followed by Coomassie blue staining. Equal amounts of leukocyte protein lysates were subjected to 2D-gel electrophoresis. Gels were silver stained, dried and scanned with a laser densitometer followed by limited computerized comparisons (Figure 6). Protein spots that differ in intensity between PD and control patients were identified using Progenesis Discovery software (Nonlinear USA, Inc.; Durham, NC). Difference measurements subjected to statistical testing. Seventeen spots were identified as either increasing or decreasing in Parkinson's disease 1;n~p~r~a 3''fi~A ~~btitiEa~~a~~~r Differentially expressed spots were punched from duplicate Coomassie blue stained gels and subject to in-gel trypsin digestion. Isolated proteins were identified following MALDI-TOF mass spectrometry followed by comparison with public protein databases (Table 6 and Figure 6).

Table 6: Provisionally identified proteins that differ between Parkinson's disease patients and control subjects.

SEQ ID NO: MW pI Accession Number Mass Spec ID
241 66,204 5.67 N1V1 002147 HSP60 242 63,131 7.59 AAH18648 Dihydrolipoamide dehydrogenase 243 62,052 5.98 JC5704 ER-60 protease 244 59,332 7.42 AAH00337 Glucose-6-phosphate dehydrogenase 245 54,879 5.42 NP001677 ATP-synthase beta chain 246 36,106 7.58 NP_000691 Annexin I

247 32,567 5.10 NP 006752 14-3-3 epsilon

248 29,785 5.80 NP_002625 Prohibitin

249 29,559 7.60 AAH62302 Phospoglycerate mutase 1

250 26,908 5.65 AAA51747 Apoliporotein AI

251 25,546 7.64 1MSD A Superoxide dismutase

252 24,376 6.36 NP_009193 RNA-binding protein regulatory subunit

253 23,327 5.82 1QMV A Chain A, thioredoxin peroxidase B

254 21,719 5.92 NP_056461 RAS-related protein RAP1B

255 145,916 5.29 NP003290 Tumor rejection antigen

256 42,839 5.42 AAC27432 Haptoglobin

257 60,376 6.72 P02675 Fibrin beta iftnpi~'~ ~f'~6lli~ication of Differentially Expressed Biomarkers in Patients Undergoing Treatment for Alzheimer's Disease 301. A study was carried out to identify valproate-responsive proteins. 15 patients with mild to moderate Alzheimer's disease were examined before (baseline) and following four weeks of divalproex sodium treatment. Figure 9 shows some characteristics of the patients that participated in the study. Peripheral blood samples were collected into 10 ml lavender top tubes using standard venipuncture at baseline and four weeks following initiation of treatment.
Samples were processed for leukocyte proteins by selective lysis of red blood cells, followed by centrifugation,of the sample, retention of the leukocyte pellet, and storage at minus 80 C.
302. Prior to two-dimensional (2D) gel analysis, samples were quickly thawed on ice and lysed using a standard solubilization buffer. Protein concentration was determined and protein integrity assayed by SDS-PAGE electrophoresis followed by Coomassie-blue staining.
Equal amounts of leukocyte protein lysates were subjected to 2D gel electrophoresis and gels were silver-stained, dried, and scanned with a laser densitometer followed by limited computerized comparisons. See Figure 10. Differentially expressed protein spots were identified using the Progenesis Discovery software and statistical testing (from Nonlinear Dynamics, Durham, NC). Nine spots were identified as either increasing or decreasing in patients on valproate therapy. Differentially expressed spots were punched from duplicate Coomassie-blue stained gels, subjected to in-gel trypsin digestion, and identified using MALDI-TOF mass spectrometry (see Figure 11) and comparisons with public protein databases. The nine biomarkers identified are listed and described in Figure 12. These nine biomarkers include actin-interacting protein 1(A1P 1), mitogen activated protein kinase I(1VIAPKI), actin or a fragment thereof, annexin Al, 14-3-3 protein epsilon, glutaraldehyde-3-phosphate dehydrogenase (GAPDH), transfonning protein RhoA, acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B or APRIL), or peroxiredoxin II.
303. These results show that biological leukocyte-containing biological samples can be used to identify biomarkers that are useful for monitoring a patient's response to treatment for Alzheimer's disease.
G. Example 5 Confirmation of Differentially Expressed Proteins Using in vitro Leukocytes.
304. Three of the nine targets were validated using human leukocytes cultured in the presence of valproate at two days in vitro. Briefly, human leukocytes were cultured from non-demented (control) subjects and subjected to varying concentrations of valproate (0-5 mM) egrziii~r~tifaEt'~~ire:-='I;eukocyte proteins were subjected to SUS-PAGE
electrophoresis and Western blotting using antibodies to the targets. For three of the proteins, Valproate treatment recapitulated the change observed in the initial 2D gel study (Figure 12).
305. Expression levels of annexin Al and APRIL in cultured leukocytes decreased in a dose-dependent manner in response to Valproate treatment. These results are consistent with the observed decrease of annexin Al and APRIL expression in patients treated with valproate (VPA). Again, consistent with the results of Example 4, expression levels of peroxiredoxin II
increased in cultured Leukocytes in a dose-dependent manner in response to VPA
treatment, with expression peaking in response to approximately 1.0 mM of VPA. Of the biomarkers tested, only actin expression in cultured leukocytes failed to track the change in expression patterns observed in VPA-treated patients.
306. These results use indicate that the expression patterns (of at lest some, and perhaps most) of the candidate biomarkers identified in VPA-treated Alzheimer's patients were not artifactual, since they correlated with the expression pattern of the biomarkers in cultured cells, as assessed using a different quantitative technique.
307. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

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Claims (74)

IX. CLAIMS

What is claimed is:

1. A method of diagnosing a neurodegenerative disease in a subject, the method comprising:
a. assessing a level of expression or activity of one or more selected biomarkers in a sample comprising leukocytes or a lysate thereof from the subject to be diagnosed; and b. comparing the level of expression or activity of the selected biomarker to a reference standard that indicates the level of expression or activity of the selected biomarker in one or more control subjects, wherein when the control subject has the neurodegenerative disease, a similarity between the level of expression of the selected biomarker and the reference standard indicates that the subject to be diagnosed has the neurodegenerative disease, and wherein when the control subject does no have the neurodegenerative disease, a difference between the level of expression of the selected biomarker and the reference standard indicates that the subject to be diagnosed has the neurodegenerative disease.

2. The method of claim 1, wherein the subject to be diagnosed is a human.

3. The method of claim 1, wherein the sample is a blood sample.

4. The method of claim 3, wherein the sample comprises a substantially pure population of leukocytes or a lysate thereof.

5. The method of claim 4, wherein the leukocytes are neutrophils, monocytes, basophils, lymphocytes, eosinophils, or any combination thereof.

6. The method of claim 1, wherein the selected biomarker is one or more proteins comprising HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 epsilon, Prohibitin, Phospoglycerate mutase 1, Apoliporotein AI, Superoxide dismutase, RNA-binding protein regulatory subunit, Chain A thioredoxin peroxidase B, RAS-related protein RAP IB Tumor rejection antigen, Haptoglobin, Fibrin beta, or combinations thereof.

7. The method of claim 1, wherein the selected biomarker is one or more transcripts comprising cyclin D1, cyclin B, cyclin G1, weel, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, Cl inhibitor, IL-17r, IL-8, LIF, TNF-alpha, IL-10r, Alpha-antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, transferrin, or combinations thereof.

8. The method of claim 1, wherein the selected biomarker is one or more proteins comprising a protein having a molecular weight of 27,100 and isoelectric point of 7.58, a molecular weight of 25,400 and isoelectric point of 6.2, a molecular weight of 27,600 and isoelectric point of 5.92, or combinations thereof.

9. The method of claim 1, wherein assessing the level of expression or activity comprises analyzing one or more selected biomarkers by one or more techniques comprising Western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent activated cell sorting (FACS), two-dimensional gel electrophoresis, mass spectroscopy (MS), matrix-assisted laser desorption/ionization-time of flight-MS (MALDI-TOF), surface-enhanced laser desorption ionization-time of flight (SELDI-TOF), high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS), protein chip expression analysis, gene chip expression analysis, or laser densitometry.

10. The method of claim 1, wherein assessing the level of expression or activity comprises conducting a multivariant canonical analysis.

11. The method of claim 1, wherein the subject to be diagnosed and the control subject are age-matched.

12. A method of screening for a therapeutic agent for the treatment of a neurodegenerative disease, the method comprising:

a. contacting a leukocyte or population of leukocyte or population of leukocytes with the agent to be screened;
and b. detecting a level of expression or activity of a biomarker for the neurodegenerative disease, an increase or decrease in the level of expression or activity of the biomarker indicating a therapeutic agent for the treatment of the neurodegenerative disease.

13. The method of claim 12, wherein the biomarker is one or more genes or proteins that are down-regulated in the neurodegenerative disease and wherein the agent increases the level of expression or activity of genes or proteins.

14. The method of claim 12, wherein the biomarker is one or more genes or proteins that are up-regulated in the neurodegenerative disease and the agent decreases the level of expression or activity of the genes or proteins.

15. The method of claim 12, further comprising determining whether the therapeutic agent alters the level of expression or activity of the biomarker in neurons.

16. The method of claim 12, wherein the neurons are dopaminergic neurons.

17. The method of claim 12, further comprising determining whether the therapeutic agent prevents the development of or slows the progression of the neurodegenerative disease in an animal model of the neurodegenerative disease.

18. The method of claim 12, wherein the animal model is a MPTP model.

19. The method of claim 12, wherein the animal model is a 6-OHDA model.

20. The method of claim 12, wherein the leukocytes are neutrophils, monocytes, basophils, lymphocytes, eosinophils, or any combination thereof.

21. The method of claim 12, wherein the biomarker is one or more proteins comprising HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate dehydrogenate, ATP-synthase beta chain, Annexin I, 14-3-3 epsilon, Prohibitin, Phospoglycerate mutase 1, Apoliporotein A1, Superoxide dismutase, RNA-binding protein regulatory subunit, Chain A thioredoxin peroxidase B, RAS-related protein RAP1B, Tumor rejection antigen, Haptoglobin, Fibrin beta, or combinations thereof.

22. The method of claim 12, wherein the biomarker is one or more transcripts comprising cyclin D1, cyclin B, cyclin G1, wee 1, hTR2, CDC25b, GSK3 beta, protein kinase C
alpha, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, IL-10r, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, transferrin, or combinations thereof.

23. The method of claim 12, wherein the biomarker is one or more proteins comprising a protein having a molecular weight of 27,100 and isoelectric point of 7.58, a molecular weight of 25,400 and isoelectric point of 6.2, a molecular weight of 27,600 and isoelectric point of 5.92, or combinations thereof.

24. The method of claim 12, wherein the agent is a nucleic acid, an antibody, polypeptide, or a small molecule.

25. A method of monitoring a neurodegenerative disease progression in a subject, the method comprising comparing a level of expression or activity of a biomarker for the neurodegenerative disease in a sample comprising leukocytes or a lysate thereof obtained from the subject at multiple time point.

26. The method of claim 25, wherein the subject is a human.

27. The method of claim 25, wherein the subject is asymptomatic or preclinical for the neurodegenerative disease at one or more of the multiple time points.

28. The method of claim 25, wherein the subject has not received treatment for the neurodegenerative disease at or before one or more of the multiple time points.

29. The method of claim 25, wherein the subject has received treatment for the neurodegenerative disease at or before one or more of the multiple time points.

30. The method of claim 25, wherein the subject has been treated with a dopamine agonist.

31. The method of claim 29, wherein the subject has been treated with levodopa at or before one or more of the multiple time points.

32. The method of claim 29, wherein the subject has been treated with a neuroprotective agent at or before one or more of the multiple time points.

33. The method of claim 32, wherein the neuroprotective agent is an acetylcholinesterase inhibitor, a glutamatergic receptor antagonist, HDAC inhibitors, an anti-inflammatory agent, or divalproex sodium.

34. The method of claim 25, wherein the biomarker is one or more proteins comprising HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 epsilon, Prohibitin, Phospoglycerate mutase 1, Apoliporotein A1, Superoxide dismutase, RNA-binding protein regulatory subunit, Chain A thioredoxin peroxidase B, RAS-related protein RAP1B, Tumor rejection antigen, Haptoglobin, Fibrin beta, or combinations thereof.

35. The method of claim 25, wherein the biomarker is one or more transcripts comprising cyclin D1, cyclin B, cyclin G1, wee1, hTR2, CDC25b, GSK3 beta, protein kinase C
alpha, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, IL-10r, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, transferrin, or combinations thereof.

36. The method of claim 25, wherein the biomarker is one or more proteins comprising a protein having a molecular weight of 27,100 and isoelectric point of 7.58, a molecular weight of 25,400 and isoelectric point of 6.2, a molecular weight of 27,600 and isoelectric point of 5.92, or combinations thereof.

37. The method of claim 25, wherein the sample is a blood sample.

38. The method of claim 25, wherein the leukocytes are neutrophils, monocytes, basophils, lymphocytes, eosinophils, or any combination thereof.

39. A method of monitoring a response to a neurodegenrative disease treatment in a subject, the method comprising comparing a level of expression or activity of a biomarker for the neurodegenrative disease in a sample comprising leukocytes or a lysate thereof obtained from the subject at multiple time points during treatment of the subject.

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

41. The method of claim 39, wherein the subject is asymptomatic or preclinical for Parkinson's disease at one or more of the multiple time points.

42. The method of claim 39, wherein the subject is treated with a neuroprotective agent at or before one of the multiple time points.

43. The method of claim 39, wherein the neuroprotective agent is an acetylcholinesterase inhibitor, a glutamatergic receptor antagonist, an anti-inflammatory agent, a kinase inhibitor, or divalproex sodium.

44. The method of claim 39, wherein the biomarker is one or more proteins comprising HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 epsilon, Prohibitin, Phospoglycerate mutase 1, Apoliporotein AI, Superoxide dismutase, RNA-binding protein regulatory subunit, Chain A thioredoxin peroxidase B, RAS-related protein RAP1B, Tumor rejection antigen, Haptoglobin, Fibrin beta, or combinations thereof.

45. The method of claim 39, wherein the biomarker is one or more transcripts comprising cyclin D1, cyclin B, cyclin G1, wee1, hTR2, CDC25b, GSK3 beta, protein kinase C
alpha, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, IL-10r, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, transferrin, or combinations thereof.

46. The method of claim 39, wherein the biomarker is one or more proteins comprising a protein having a molecular weight of 27,100 and isoelectric point of 7.58, a molecular weight of 25,400 and isoelectric point of 6.2, a molecular weight of 27,600 and isoelectric point of 5.92, or combinations thereof.

47. The method of claim 39, wherein the sample is a blood sample.

48. The method of claim 39, wherein the leukocytes are neutrophils, monocytes, basophils, lymphocytes, eosinophils, or any combination thereof.

49. A method of identifying a risk for a neurodegenerative disease in a test subject, the method comprising:

a. determining a level of expression or activity of a biomarker for the neurodegenerative disease from a sample obtained from the test subject, wherein the sample comprises leukocytes or a lysate thereof; and b. correlating the level of expression or activity level of the biomarker determined for the test subject with the levels for a reference subject, a correlation between levels determined for the reference subject without the neurodegenerative disease and the levels for the test subject identifying a low risk for the neurodegenerative disease in the test subject and a correlation between the levels determined for the reference subject with the neurodegenerative disease and the levels for the test subject identifying a high risk for the neurodegenerative disease in the test subject.

50. The method of claim 49, further comprising determining the level of the biomarkers from a population of reference subjects diagnosed with the neurodegenerative disease or from a population of reference subjects without the neurodegenerative disease.

51. The method of claim 49, wherein the test subject is a human.

32. The method of claim 49, wherein the test subject is asymptomatic. or preclinical for the neurodegenerative disease.

53. The method of claim 49, wherein the test subject and the reference populations are age-matched.

54. The method of claim 49, wherein the biomarker is one or more proteins comprising HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 epsilon, Prohibitin, Phospoglycerate mutase 1, Apoliporotein AI, Superoxide dismutase, RNA-binding protein regulatory subunit, Chain A thioredoxin peroxidase B, RAS-related protein RAP1B, Tumor rejection antigen, Haptoglobin, Fibrin beta, or combinations thereof.

55. The method of claim 49, wherein the biomarker is one or more transcripts comprising cyclin D1, cyclin B, cyclin G1, wee1, hTR2, CDC25b, GSK3 beta, protein kinase C
alpha, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, IL-10r, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, transferrin, or combinations thereof.

56. The method of claim 49, wherein the biomarker is one or more proteins comprising a protein having a molecular weight of 27,100 and isoelectric point of 7.58, a molecular weight of 25,400 and isoelectric point of 6.2, a molecular weight of 27,600 and isoelectric point of 5.92, or combinations thereof.

57. The method of claim 49, wherein the sample is a blood sample.

58. The method of claim 49, wherein the leukocytes are neutrophils, monocytes, basophils, lymphocytes, eosinophils, or any combination thereof.

59. A method of differentially diagnosing a neurodegenerative disease in a test subject, the method comprising:
a. assessing a level of expression or activity of one or more selected biomarkers in a sample comprising leukocytes or a lysate thereof from the test subject; and b. comparing the level of expression or activity of the selected biomarker to a reference standard that indicates the level of expression or activity of the selected biomarker in one or more populations of neuropathologic control subjects with one or more neuropathological control diseases, wherein a difference or similarity between the level of expression or activity of the selected biomarker and the reference standard indicating a differential diagnosis of the neurodegeneraive disease as compared to the neuropathological control diseases.

60. The method of claim 59, wherein the subject is a human.

61. The method of claim 59, wherein the subject is asymptomatic or preclinical for the neurodegenerative disease.

62. The method of claim 59, wherein the populations of neuropathologic control subjects are selected from the group consisting of one or more subjects with Alzheimer's disease, frontal-temporal dementia, mild cognitive impairment, and Parkinson's disease.

63. The method of claim 59, wherein the subject and the control subjects are age-matched.

64. The method of claim 59, wherein the sample is a blood sample.

65. The method of claim 59, wherein the sample comprises a substantially pure population of leukocytes or a lysate thereof.

66. The method of claim 65, wherein the leukocytes are neutrophils, monocytes, basophils, lymphocytes, eosinophils, or any combination thereof.

67. The method of claim 59, wherein the selected biomarker is one or more proteins comprising HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 epsilon, Prohibitin, Phospoglycerate mutase 1, Apoliporotein AI, Superoxide dismutase, RNA-binding protein regulatory subunit, Chain A thioredoxin peroxidase B, RAS-related protein RAP1B, Tumor rejection antigen, Haptoglobin, Fibrin beta, or combinations thereof.

68. The method of claim 59, wherein the biomarker is one or more transcripts comprising cyclin D1, cyclin B, cyclin G1, wee1, hTR2, CDC25b, GSK3 beta, protein kinase C
alpha, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, IL-10r, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, transferrin, or combinations thereof.

69. The method of claim 59, wherein the biomarker is one or more proteins comprising a protein having a molecular weight of 27,100 and isoelectric point of 7.58, a molecular weight of 25,400 and isoelectric point of 6.2, a molecular weight of 27,600 and isoelectric point of 5.92, or combinations thereof.

70. The method of claim 59, wherein assessing the level of expression or activity comprises analyzing one or more selected biomarkers by one or more techniques comprising Western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent activated cell sorting (FACS), two-dimensional gel electrophoresis, mass spectroscopy (MS), matrix-assisted laser desorption/ionization-time of flight-MS (MALDI-TOF), surface-enhanced laser desorption ionization-time of flight (SELDI-TOF), high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS), protein chip expression analysis, gene chip expression, or laser densitometry.

71. A solid support comprising one or more biomarkers, wherein the biomarker is one or more proteins comprising HSP60, Dihydrolipoamide dehydrogenase, ER-60 protease, Glucose-6-phosphate dehydrogenase, ATP-synthase beta chain, Annexin I, 14-3-3 epsilon, Prohibitin, Phospoglycerate mutase 1, Apoliporotein AI, Superoxide dismutase, RNA-binding protein regulatory subunit, Chain A thioredoxin peroxidase B, RAS-related protein RAP1B, Tumor rejection antigen, Haptoglobin, Fibrin beta, or combinations thereof.

72. The solid support of claim 71, wherein the biomarker is one or more proteins chosen from a protein having a molecular weight of 27,100 and isoelectric point of 7.58, a molecular weight of 25,400 and isoelectric point of 6.2, a molecular weight of 27,600 and isoelectric point of 5.92, or combinations thereof.

73. A solid support comprising one or more biomarkers, wherein the biomarker is one or more transcripts comprising cyclin D1, cyclin B, cyclin G1, wee1, hTR2, CDC25b, GSK3 beta, protein kinase C alpha, C5, C1 inhibitor, IL-17r, IL-8, LIF, TNF-alpha, IL-10r, Alpha-1 antichymotrypsin, HSP 27, HSP 90, crystalline, GAPDH, ferritin H, ferritin L, cox 1, cox 2, transferrin, or combinations thereof.

74. The solid support of claim 71, wherein the solid support is chosen from a chip, microarray, nanoarray, or bead.