WO2012037232A2 - Compositions, methods and kits for detecting and treating alzheimer's disease - Google Patents

Abstract

Compositions, kits and methods for detecting AD in a subject at an early stage include in vitro or in vivo analysis of expression of one or more markers of pre-AD. Screening platforms for identifying a therapeutic agent for preventing or treating AD in humans, and kits for detecting the presence of AD in a subject at an early stage are described herein. Compositions and methods for preventing or delaying onset of AD in a subject include a therapeutically effective amount of an inhibitor of at least one of the following proteins: an RIG-1 signaling pathway member, MDA5, VISA, RIG-1, NLRP12, and an HERV protein for inhibiting death and damage of neurons in the subject having pre-AD and a pharmaceutically acceptable carrier.

Description

COMPOSITIONS, METHODS AND KITS FOR DETECTING AND TREATING

ALZHEIMER'S DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of Provisional Application Serial No. 61/383,434 filed September 16, 2010, which is herein incorporated by reference in its entirety.

FIELD OF THE FNVENTION

[0002] The invention relates generally to the fields of molecular genetics, molecular biology, and medicine.

BACKGROUND

[0003] Presently, there is no diagnostic test or in vivo imaging procedure on the market for Alzheimer's disease (AD). Pharmacotherapies are targeted to the neurotransmitter acetylcholine, but do not address disease pathogenesis. In addition to a need for a treatment of this debilitating disease, a need exists for a test to diagnose the disease at an early stage of onset. A major limitation in finding therapeutic solutions for AD has been the lack of reliable methods for early diagnosis of this devastating disease. Identification of a novel mechanism of disease pathogenesis would afford identification of central nervous system (CNS) drug targets for intervention of significance early in the course of disease.

SUMMARY

[0004] Described herein are compositions, methods and kits for early detection, prevention and treatment of AD. The compositions and methods target innate inflammatory mechanisms that contribute to the pathogenesis of AD. Inflammation is associated with many neurodegenerative diseases, including AD. It was discovered that the innate immune system involving RIG- 1 -like receptor (RLR) signaling pathway is activated in patients with pre- symptomatic or early stage AD (referred to herein as "pre-Alzheimer's disease" and "pre -AD"). The RLR family is composed of retinoic acid-inducible gene-I (RIG-1), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). These receptors are localized in the cytoplasm and recognize the genomic RNA of dsRNA viruses and dsRNA generated as the replication intermediate of ssRNA viruses (S. R. Furr, V. S. Chauhan, D. Sterka, Jr. et al, Journal of neurovirology, 1 (2008)). The data described below show that key components of the RLR signaling pathway are significantly elevated in pre-AD patients. These signaling intermediates are RIG-1, MDA5, virus-induced signaling adaptor (VISA) (also known as IFN-P-promoter stimulator (IPS-1), MAVS, CARDIF, or VISA), and the interferon response factor7 (IRF7) and/or interferon response factor3 (IRF3) and/or interferon response factor9 (IRF9). Additionally, the data show that the negative regulator of the innate immune response, NACHT leucine-rich domain and pyrin-containing protein NLRP12, is significantly elevated in brains of patients with pre-AD, indicating that these proteins demonstrate a novel disease process. Also reported herein is data that shows that the expression of RIG-1, VISA, and the NACHT leucine-rich domain and pyrin-containing protein, NLRP12 are significantly elevated in neurons of the temporal cortex and the cerebrospinal fluid (CSF) in presymptomatic AD patients. Increased expression of RIG-1 and VISA were not found in pathologically confirmed cases of amyotrophic lateral sclerosis, Diffuse Lewy Body disease (DLBD) or Huntington's disease (HD), suggesting that activation of RIG-1 signaling may be a specific marker for early AD pathogenesis. Moreover, in vitro results show that stimulation of RIG-1 signaling induces amyloid precursor protein, supporting the idea that activation of the RIG-1 signaling system occurs in incipient AD.

[0005] These studies show for the first time that RIG-1, VISA, MDA5 and NLRP12 may be used as biomarkers in CSF and brain for diagnosis of pre-AD and AD. Uses for this technology include an early diagnostic screen, e.g., a screen for patient selection for AD optimization in clinical trials, and pharmacotherapy, aiding clinicians to assess and treat this devastating neurodegenerative disease.

[0006] Accordingly, described herein is a method of detecting pre-symptomatic or early stage Alzheimer's disease (pre-AD) in at least one subject suspected of having or at risk of having pre-AD. The method includes obtaining a biological sample from the at least one subject; contacting the biological sample with at least a first reagent that detects the presence of RIG-1 protein; measuring the level of RIG-1 protein in the biological sample; and correlating overexpression of RIG-1 in the sample with pre-AD in the at least one subject. The step of contacting the biological sample with at least a first reagent that detects the presence of RIG-1 protein can include contacting the sample with the at least first reagent and a second reagent that detects the presence of at least one of: VISA, IRF-3, IRF-7, IRF-9, RNAseL, MDA5, NLRP12, and a human endogenous retrovirus protein; the step of measuring the level of RIG-1 protein in the biological sample can include measuring the level of RIG-1 protein and the level of the at least one of: VISA, IRF-3, IRF-7, IRF-9, RNAseL, MDA5, NLRP12, and a human endogenous retrovirus protein in the biological sample; and the step of correlating overexpression of RIG- 1 in the sample with pre- AD in the at least one subject can include correlating overexpression of RIG-1 and overexpression of the at least one of: VISA, IRF-3, IRF-7, IRF-9, RNAseL, MDA5, NLRP12, and a human endogenous retrovirus protein in the sample with pre- AD in the at least one subject. The biological sample can be, e.g., cerebral spinal fluid, blood, saliva, serum, plasma, tissue, skin, brain tissue, urine, and epithelial cells from skin, mouth, muscle or other body tissue. In the method, pre-AD can be detected in the at least one subject before the at least one subject displays cognitive impairment due to AD. One or more steps of the method can be performed using one or more of the following assays: enzyme-linked immunosorbent assay, radioimmunoassay, Western blot, flow cytometry, immunofluorescence, immunoprecipitation, protein A, immunoelectrophoretic, immunoscintigraphic detection and protein chip, and PET imaging. In some embodiments, the at least one subject is a plurality of subjects suspected of having pre-AD or who are at risk for AD.

[0007] Also described herein is a a method of detecting pre-AD in a subject suspected of having or at risk of having pre-AD. The method includes: obtaining a biological sample from the subject; contacting the biological sample with at least a first reagent that detects the presence of RIG-1 protein, at least a second reagent that detects the presence of MDA5 protein, at least a third reagent that detects the presence of VISA protein, at least a fourth reagent that detects the presence of NLRP12 protein, and at least a fifth reagent that detects the presence of a human endogenous retrovirus protein; measuring the levels of RIG-1, MDA5, VISA , NLRP12, and human endogenous retrovirus proteins in the biological sample; and correlating overexpression of at least one of the RIG-1, MDA5, VISA, NLRP12, and human endogenous retrovirus proteins in the sample with pre-AD in the subject. The biological sample can be, e.g., cerebral spinal fluid, blood, saliva, serum, plasma, tissue, skin, brain tissue, urine, and epithelial cells from skin, mouth, muscle or other body tissue. In the method, pre-AD can be detected in the subject before the subject displays cognitive impairment due to AD. One or more of the steps of the method can be performed using one or more of the following assays: in vivo brain imaging, including but not limited to single photon emission computed tomography (SPECT), functional magnetic resonance imaging (fMRI), positron emission tomography (PET), FLISA, RT-PCR, qPCR, and DNA chip platforms. [0008] Further described herein is a method of detecting pre- AD in a subject suspected of having or at risk of having pre-AD. The method includes administering a composition including at least a first reagent that detects the presence of RIG-1 protein to the subject, the at least first reagent labeled with or conjugated to a detectable agent; imaging (e.g., PET, fJVIRI, and/or SPECT) the subject's brain to detect the presence and location of the at least first reagent labeled with or conjugated to a detectable agent; measuring the level of RIG-1 protein in a disease- affected area of the subject's brain; and correlating overexpression of RIG-1 in the disease- affected area of the subject's brain with pre-AD in the subject. The at least first reagent can include an antibody or labeled substance that binds to RIG-1 or RIG-1 signaling intermediates and the detectable agent can be, e.g., a radiolabel.

[0009] Yet further described herein is a method of identifying a therapeutic agent for preventing or treating AD in humans. The method includes screening a library of potential therapeutic agents to identify one or more agents that inhibit expression or activity of an RIG-1 signaling pathway member (e.g., RIG-1, MDA5, VISA, IRF7 and NLRP12) or a neurotoxic factor (e.g., beta amyloid, hyperphosphorylated tau, and amyloid precursor protein) involved in AD pathogenesis, and delay or prevent onset of AD in a human. The library can be screened in a high-throughput multi-well format, and the potential therapeutic agents can be small molecules.

[0010] Still further described herein is a composition including a pharmaceutically acceptable carrier and an inhibitor of a protein such as a RIG-1 signaling pathway member (e.g., RIG-1, MDA5, IRF-3, IRF-7, IRF-9, and VISA), NLRP12, VISA, and a human endogenous retrovirus protein, in a therapeutically effective amount for delaying cognitive decline and inhibiting apoptosis and damage of neurons in a patient who has overexpression of at least one of the following: RIG-1, VISA, MDA5 , NLRP12, IRF3 and IRF7, and who displays AD pathology but not cognitive impairment. Typically, the inhibitor decreases expression or activity of the RIG-1 signaling pathway member and is in an amount effective for delaying cognitive decline and damage of neurons in the patient. The composition may further include a vaccine targeted to prevent amyloid beta accumulation or one or more small molecule Bace-modulators. The composition can include a pharmaceutically acceptable carrier and a therapeutically effective amount of an inhibitor of an RIG-1 signaling pathway member (e.g., RIG-1, MDA5, IRF-3, IRF- 7, IRF-9 and VISA), and an inhibitor of one or more of human endogenous retrovirus protein, R Ase L, and NLRP12 in a therapeutically effective amount for inhibiting apoptosis and damage of neurons in the patient.

[0011] Additionally described herein is a A method of preventing or delaying onset of AD in a subject. The method includes administering to the subject a composition includes a pharmaceutically acceptable carrier and a therapeutically effective amount of an inhibitor of an RIG-1 signaling pathway member (e.g., RIG-1, MDA5, IRF-3, IRF-7, IRF-9 and VISA) for inhibiting apoptosis and damage of neurons in the subject.

[0012] Also described herein is a method of preventing or delaying onset of AD in a subject. The method includes administering to the subject a composition including a pharmaceutically acceptable carrier and a therapeutically effective amount of an NLRP12 inhibitor for inhibiting apoptosis and damage of neurons in the subject.

[0013] Further described herein is A method of preventing or delaying onset of AD in a subject. The method includes administering to the subject a composition including a pharmaceutically acceptable carrier, a therapeutically effective amount of an inhibitor of an RIG- 1 signaling pathway member (e.g., RIG-1, MDA5, IRF-3, IRF-7, IRF-9 and VISA) for inhibiting apoptosis and damage of neurons in the subject, and a therapeutically effective amount of an inhibitor of one or more of human endogenous retrovirus protein, RNAse L, and NLRP12 for inhibiting apoptosis and damage of neurons in the subject. In one embodiment, the subject has pre-AD but does not display cognitive impairment due to AD, and administration of the composition delays progression of the pre-AD.

[0014] Yet further described herein is A kit for detecting pre-AD in a subject suspected or at risk of having pre-AD. The kit includes at least a first reagent (e.g., a RIG-1 -specific antibody) for detecting the presence of and quantifying the level of RIG-1 protein in a biological sample from a subject; and instructions for use. The kit can further include a second reagent for detecting the presence of and quantitating the level of one or more of: VISA, IRF-3, IRF-7, IRF- 9, MDA5, NLRP12, RNAse L, and a human endogenous retrovirus protein.

[0015] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0016] As used herein, "protein" and "polypeptide" are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation. [0017] By the terms "RIG-1 protein," "RIG-1 receptor" or "RIG-1 polypeptide" is meant an expression product of a RIG-1 gene (known as DDX58), such as the native human RIG-1 protein (accession no. CAM14168) or a protein that shares at least 65% (but preferably 75, 80, 85, 90,

95, 96, 97, 98, or 99%) amino acid sequence identity with the foregoing and displays a functional activity of a native RIG-1 protein. A "functional activity" of a protein is any activity associated with the physiological function of the protein. For example, functional activities of a native RIG- 1 protein may include recognizing the genomic RNA of dsRNA viruses and dsRNA generated as the replication intermediate of ssRNA viruses, inducing type I interferon (IFN) production in response to RNA viruses, and signaling. RNAseL allows recognition of endogenous RNA by RIG-1 and MDA5. Once either Rig-1 and/or MDA5 are engaged in the signaling pathway, VISA is activated resulting in the production of interferon -inducible genes shared between the IPS-1 and TRIF signaling pathways, production of proinflammatory cytokines, and/or activation of IRF3/7/9.

[0018] By the phrase "overexpression" is meant increased levels of mRNA and/or protein expression as compared to normal tissue.

[0019] As used herein, the phrases "RIG-1 overexpression," "overexpression of RIG-1," "upregulation of RIG-1," and "RIG-1 upregulation" are used interchangeably to mean increased levels of RIG-1 mRNA and protein expression as compared to tissues from a subject not having pre-AD or AD.

[0020] By the terms "VISA protein," "VISA" or " VISA polypeptide" is meant an expression product of a VISA gene such as the native human VISA protein; accession nos. NP 065797 NP_076935 XP_045472), or a protein that shares at least 65% (but preferably 75, 80, 85, 90, 95,

96, 97, 98, or 99%) amino acid sequence identity with the foregoing and displays a functional activity of a native VISA protein. For example, functional activities of a native VISA protein may include mediation of the antiviral response. VISA is also known as IFN-P-promoter stimulator (IPS-1), MAVS, and CARDIF.

[0021] As used herein, the phrases "VISA overexpression," "overexpression of VISA," "upregulation of VISA," and "VISA upregulation" are used interchangeably to mean increased levels of VISA mRNA and protein expression as compared to tissues from a subject not having pre-AD or AD. [0022] By the term "gene" is meant a nucleic acid molecule that codes for a particular protein, or in certain cases, a functional or structural RNA molecule.

[0023] As used herein, a "nucleic acid" or a "nucleic acid molecule" means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid).

[0024] By the terms "RIG-1 gene," "RIG-1 polynucleotide," or "RIG-1 nucleic acid" is meant a native human RIG-1 -encoding nucleic acid sequence, e.g., the native human RIG-1 gene (accession no. CAM14168); a nucleic acid having sequences from which a RIG-1 cDNA can be transcribed; and/or allelic variants and homologs of the foregoing. The terms encompass double- stranded DNA, single-stranded DNA, and RNA.

[0025] By the terms "RNAse-L gene," "RNAse-L polynucleotide," or " RNAse-L nucleic acid" is meant a native human RNAse-L-encoding nucleic acid sequence, e.g., the native human RNAse-L gene (accession no. AAH90934); a nucleic acid having sequences from which a RNAse-L cDNA can be transcribed; and/or allelic variants and homologs of the foregoing. The terms encompass double-stranded DNA, single-stranded DNA, and RNA.

[0026] By the terms "VISA gene," " VISA polynucleotide," or " VISA nucleic acid" is meant a native human VISA -encoding nucleic acid sequence, e.g., the native human VISA gene (accession no. NP_065797 NP_076935 XP_045472); a nucleic acid having sequences from which a VISA cDNA can be transcribed; and/or allelic variants and homologs of the foregoing. The terms encompass double-stranded DNA, single-stranded DNA, and RNA.

[0027] The terms "patient," "subject" and "individual" are used interchangeably herein, and mean a mammalian (e.g., human) subject to be treated and/or to obtain a biological sample from.

[0028] As used herein, "bind," "binds," or "interacts with" means that one molecule recognizes and adheres to a particular second molecule in a sample or organism, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample. Generally, a first molecule that "specifically binds" a second molecule has a binding affinity greater than about 10 to 10¹² moles/liter for that second molecule and involves precise "hand-in-a-glove" docking interactions that can be covalent and noncovalent (hydrogen bonding, hydrophobic, ionic, and van der waals).

[0029] The term "labeled," with regard to a nucleic acid, protein, probe or antibody, is intended to encompass direct labeling of the nucleic acid, protein, probe or antibody by coupling (i.e., physically or chemically linking) a detectable substance (detectable agent) to the nucleic acid, protein, probe or antibody.

[0030] When referring to a nucleic acid molecule or polypeptide, the term "native" refers to a naturally-occurring (e.g., a WT) nucleic acid or polypeptide.

[0031] By the terms "Pre-AD" and "Pre-AD" is meant pre -Alzheimer's disease and is characterized as Braak stages I-III.

[0032] As used herein, the phrase "early onset" is used interchangeably with pre-AD and is characterized as Braak stages I-III

[0033] As used herein, the terms "diagnostic," "diagnose" and "diagnosed" mean identifying the presence or nature of a pathologic condition. When referring to AD, "early diagnosis" and "early detection" refer to identification of onset of disease at Braak stages I-III.

[0034] The term "sample" is used herein in its broadest sense. A sample including polynucleotides, peptides, antibodies and the like may include a bodily fluid, a soluble fraction of a cell preparation or media in which cells were grown, genomic DNA, RNA or cDNA, a cell, a tissue, skin, hair and the like. Examples of samples include saliva, serum, tissue, skin, CSF, blood, plasma, brain (autopsy or biopsy), and epithelial cells from skin, mouth, muscle or other bodily tissue.

[0035] As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent to a patient or subject, or application or administration of the therapeutic agent to an isolated tissue or cell line from a patient or subject, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease, or the predisposition toward disease.

[0036] As used herein, the term "safe and effective amount" refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. By "therapeutically effective amount" is meant an amount of a composition as described herein effective to yield the desired therapeutic response, e.g., an amount effective to block at least one of: innate immunity, inflammation, detrimental cytokine production, amyloid precursor protein production, beta amyloid production, plaque formation, cognitive impairment, and neuronal death/damage, etc.; and prevent or delay onset of AD. The specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.

[0037] Although compositions, kits, and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions, kits, and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1A is a series of immunoblots and a series of graphs showing RIG-1, VISA and NLRP12 are elevated in the temporal cortex of pre -AD patients. Representative immunoblots of temporal brain tissue age-matched controls (Control), presymptomatic Alzheimer disease (EAD) and Alzheimer Disease (AD) were analyzed for RIG-1, MDA5, VISA and NLRP12.

[0039] FIG. IB is a series of immunoblots and a series of graphs showing RIG-1, VISA and NLRP12 are elevated in the temporal cortex of pre- AD patients. Representative immunoblots of occipital cortex of age-matched controls (Control), presymptomatic Alzheimer disease (EAD) and Alzheimer Disease (AD) probed for RIG-1, MDA5, VISA and NLRP12. -actin was used as a protein loading control and internal standard. Data is presented as mean + SEM. N=23 to 25 per group. * p < 0.05.

[0040] FIG. 2 is a series of micrographs of neurons showing that RIG-1 signaling proteins are expressed in neurons in the temporal cortex of EAD patients. Immunohistochemical staining showing neurons in the temporal cortex of EAD individuals (middle column) express increased RIG-1 (row 1), MDA5 (row 2), VISA (row 3) and NLRP12 (row 4). Age-matched controls (left column) and LAD (right column).

[0041] FIG. 3 is a series of graphs showing RLR protein expression in the CSF of AD patients. CSF samples were obtained from age-matched controls (Control), presymptomatic Alzheimer disease (EAD) and Alzheimer Disease (AD) cases and were analyzed by immunoblotting for protein expression levels of (A) RIG-1, (B) MDA5, (C) VISA and (D) NLRP12 (E) RNaseL. Data is presented as mean + SEM. N=7-18 per group. * p < 0.05.

[0042] FIG. 4 A is a series of photographs of immunoblots and a series of graphs showing that patients with amyotrophic lateral sclerosis (ALS), DLBD or HD have similar levels of VISA as compared to age-matched controls. *p <0.05 compared to age-matched control.

[0043] FIG. 4B is a series of photographs of immunoblots and a series of graphs showing that patients with ALS (A), DLBD (B) or HD (C) have similar levels of NLRP12 as compared to age-matched controls. *p <0.05 compared to age-matched control.

[0044] FIG. 5 is a series of immunoblots and graphs showing that inflammasome protein expression is not altered in AD. Representative immunoblots of temporal and occipital cortices of age-matched controls (Control), presymptomatic Alzheimer disease (EAD) and Alzheimer Disease (AD) cases immunob lotted for NLRP1, ASC, caspase-1, caspase-5, IL-Ιβ and IL-18. β- actin was used as a protein loading control and internal standard. Data is presented as mean + SEM. N=5 per group. * p < 0.05..

[0045] FIG. 6 is a series of photographs of immunoblots and a series of graphs showing that human astrocytes treated with poly(LC) have significant increases in the levels of Rig-1, VISA and amyloid precursor protein (APP) when compared to controls. *p < 0.05, #p< 0.1 compared to control.

[0046] FIG. 7 is a series of photographs of immunoblots and a series of graphs showing that RLR proteins were found in plasma of AD patients.

[0047] FIG. 8 is a series of photographs of immunoblots and a series of graphs showing that RLR proteins were found in serum of AD patients.

DETAILED DESCRIPTION

[0048] Described herein are compositions, methods and kits for detecting, preventing and treating AD. One embodiment of a method and/or kit for detecting AD at an early stage is a specific test for a diagnostic marker panel in blood, CSF, saliva, serum, plasma, skin, or other biological sample from a subject, that measures RIG signaling components in patients with pre- cognitive impairment AD (i.e., pre-AD). Another embodiment of a method and/or kit for detecting AD at an early stage includes in vivo imaging agents that target key members of the RIG signaling pathway. Identification of key members of the RIG signaling pathway provides a platform for identifying molecules that interfere with or inhibit this signaling pathway. A high- throughput platform will find particular use for the development of therapeutics to treat pre- AD and AD. Based on the experimental results described below, RIG-1, VISA, MDA5 and NLRP12 may be used as biomarkers in CSF and brain for diagnosis of pre -AD and AD. In one embodiment of a method of delaying onset of AD in a subject, administering an inhibitor of activity or expression of one of RIG-1, MDA5, VISA at therapeutically effective concentrations may be used to delay the onset of AD and provide neuroprotection. In another embodiment, IFN delivery at therapeutically effective concentrations may be used to delay the onset of AD and provide neuroprotection. This new technology may be synergistic with other treatment strategies in development, including AD vaccines targeted to amyloid beta clearance and small molecule Bace-modulators.

Biological Methods

[0049] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology, ed. Ausubel et al, Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Conventional methods of gene transfer and gene therapy can also be adapted for use in the present invention. See, e.g., Gene Therapy: Principles and Applications, ed. T. Blackenstein, Springer Verlag, 1999; Gene Therapy Protocols (Methods in Molecular Medicine), ed. P.D. Robbins, Humana Press, 1997; and Retro-vectors for Human Gene Therapy, ed. CP. Hodgson, Springer Verlag, 1996. Immunology techniques are generally known in the art and are described in detail in methodology treatises such as Advances in Immunology, volume 93, ed. Frederick W. Alt, Academic Press, Burlington, MA, 2007; Making and Using Antibodies: A Practical Handbook, eds. Gary C. Howard and Matthew R. Kaser, CRC Press, Boca Raton, Fl, 2006; Medical Immunology, 6^th ed., edited by Gabriel Virella, Informa Healthcare Press, London, England, 2007; and Harlow and Lane ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988.

Methods of Alzheimer's Disease Early Detection

[0050] Described herein are methods of detecting AD in a subject (e.g., human) using biomarkers to identify early onset of disease, before symptoms appear. Typically, the subject is suspected of or at risk of having AD. The presence of CSF biomarkers of AD prior to any clinical symptoms as shown herein demonstrates that the pathology of AD precedes the clinical symptoms and further demonstrates that the methods described herein enable the detection of those changes prior to the appearance of cognitive dysfunction. A method of detecting AD in a subject can include identifying one or more of the following proteins in a biological sample from the subject or in vivo (i.e., in the subject): RIG-1, VISA, MDA5 signaling proteins, NLRP12, and all human endogenous retrovirus (HERVs) proteins (e.g., gag, pol, env). The method can alternatively or additionally include identifying a gene in vivo (i.e., in a subject) or a biological sample from a subject that encodes one of the following proteins: RIG-1, VISA, MDA5, signaling proteins, MDA5 signaling proteins, IFN-stimulated gene factor 3 (ISGF3), STAT1, STAT2, IRF 9 and/or 3/7, protein kinase R (PKR), 2'5'-oligoandenylate synthase (OAS), NLR 12, and human HERVs (e.g., gag, pol and env genes).

[0051] One embodiment of a biomarker method of detecting pre-symptomatic AD (pre-AD) in at least one subject suspected of having or at risk of having pre-AD includes obtaining a biological sample from the at least one subject; contacting the sample with at least a first reagent that detects the presence of RIG-1 protein; measuring the level of RIG-1 protein in the biological sample; and correlating overexpression of RIG-1 in the sample with pre-AD in the at least one subject. In such an embodiment, the method can further include detecting the presence of and measuring the level of one or more additional proteins. For example, the step of contacting the sample with at least a first reagent that detects the presence of RIG-1 protein can include contacting the sample with the at least first reagent as well as a second reagent that detects the presence of one or more of the following proteins: VISA, RNAseL, IRF-7, IRF-3, IRF-9, MDA5, NLR 12, and an HERV protein. In this example, the step of measuring the level of RIG-1 protein in the biological sample includes measuring the level of RIG-1 protein and the level of one or more of VISA, RNAseL, IRF-7, IRF-3, IRF-9, MDA5, NLRP12, and an HERV protein in the biological sample, and the step of correlating overexpression of RIG-1 in the sample with pre-AD in the at least one subject includes correlating overexpression of RIG-1 and overexpression of one or more of VISA, RNAseL, IRF-7, IRF-3, IRF-9, MDA5, NLRP12 and an HERV protein in the sample with pre-AD in the at least one subject.

[0052] Any suitable biological sample can be used in the methods. Examples of biological samples include CSF, blood, saliva, serum, plasma, tissue, skin, brain (autopsy or biopsy), urine, and epithelial cells from skin, mouth, muscle or other body tissue. In a typical method, pre-AD is detected in a subject before the subject displays clinical manifestations of pre- AD. Examples of clinical manifestations include cognitive impairment, loss of intellectual function, loss of memory, behavior and personality changes, deterioration of activities in daily life, and muteness. The steps of the method can be performed using any suitable protocol or assay. Examples of suitable assays include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays, Western blots, flow cytometry assays, immunofluorescence assays, immunoprecipitation assays, protein A assays, immunoelectrophoretic assays, protein chip assays (arrays), and other related techniques. However, any suitable method or assay can be used to measure the level of RIG- 1 protein (or other protein described herein) in a biological sample from a subject. In some embodiments, biological samples from a plurality of subjects suspected of having or at risk of having pre- AD can be analyzed simultaneously, e.g., in a high-throughput format.

[0053] Any suitable reagent for detecting expression of and quantitating levels of one or more proteins including RIG-1 can be used. In a typical embodiment, an antibody (e.g., monoclonal, polyclonal, Fab fragment, etc.) specific for the protein whose presence and expression level is being analyzed is used. In some embodiments, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the compositions, kits and methods described herein. Antibodies specific for RIG-1 (or other proteins described herein) may be provided in a diagnostic kit that incorporates at least one of these procedures to detect RIG-1 expression. The kit may contain other components, packaging, instructions, or other material to aid the detection of the protein and use of the kit.

[0054] In some methods of detecting pre- AD in a subject, the presence of one or more genes as described herein is detected. For example, a gene signature can be used as a biomarker panel. Expression profiling of mRNA levels can be used to detect pre-AD in a subject. Protein levels can be examined by immunoblotting or ELISA of CSF samples or by brain imaging or radiolabeled compounds. Early stage (pre-AD) affected individuals will have higher levels of the referred proteins than late stage (AD) subjects. Examples of techniques by which such genes and gene products can be identified and analyzed include but are not limited to: in vivo brain imaging, including but not limited to single photon emission computed tomography (SPECT), functional magnetic resonance imaging (fMRI), and positron emission tomography (PET), FLISA, RT-PCR, qPCR, and DNA chip platforms (as well as any suitable imaging method or device). Numerous antibody-based detection formats are well known in the art, and include ELISA, radioimmunoassays, immunoblots, Western blots, flow cytometry, immunofluorescence assays, immunoprecipitation, protein A assays, Immunoelectrophoresis assays, enzymatic assays, fluorometric assays, colorometric assays, photometric assays, and other related techniques. DNA chip platforms. In an embodiment in which the presence of a gene is being analyzed, specific real-time PCR, for example, can be used to diagnose early onset AD in a subject. In another embodiment in which the presence of a gene(s) is being analyzed, a DNA chip platform can be used. Genetic analysis of mRNA of the proteins described herein may be used to predict pre- AD.

[0055] As mentioned above, a method of detection as described herein can include in vivo imaging. In one example of such an embodiment, a reagent that detects the presence of RIG- 1 protein or other protein as described herein (e.g., VISA, RNAseL, IRF-7, IRF-3, IRF-9, MDA5, IPS, human endogenous retrovirus proteins, etc.) is labeled or otherwise made detectable. When detectably labeled, such a reagent is useful as an imaging agent and as a reagent in other diagnostic procedures that are used to detect pathological signs of pre -AD. Using standard imaging techniques, the site and extent of pathology can be detected. A typical method of detecting pre- AD in a subject suspected of having or at risk of having AD includes administering a composition including at least a first reagent that is labeled with or conjugated to a detectable agent and that detects the presence of RIG- 1 protein to the subject; imaging the subject's brain to detect the presence and location of the at least first reagent labeled with or conjugated to a detectable agent; measuring the level of RIG-1 protein in the subject's brain (e.g., in the temporal cortex or other disease-affected area); and correlating overexpression of RIG-1 in the subject's brain with pre- AD in the subject. In this method, the detectably labeled reagent for detecting the presence of RIG-1, for example, in the subject's brain is typically an antibody or ligand. However, any suitable reagent can be used. Monoclonal antibodies that specifically bind RIG-1 or other proteins described herein may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497, 1975; U.S. Pat. No. 4,376,110; Ausubel et al, eds., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1987, 1992); Harlow and Lane ANTIBODIES: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Colligan et al, eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), the contents of which are incorporated entirely herein by reference. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof. A hybridoma producing a monoclonal antibody of the present invention may be cultivated in vitro, in situ or in vivo.

[0056] In an in vivo imaging method as described herein, any suitable detectable agent can be used. A detectable agent is capable of producing, either directly or indirectly, a detectable

3 14 32 35 125 signal. For example, the detectable agent may be a radioisotope, such as H, C, P, S, or I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugating the at least first reagent to the detectable agent may be employed. Methods for conjugating antibodies to detectable agents are described by, for example, Hunter et al, Nature, 144:945 (1962); David et al, Biochemistry, 13: 1014-1021 (1974); Pain et al, J. Immunol. Meth., 40:219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30:407-412 (1982).

[0057] Whether or not RIG-1 (and one or more additional proteins as described herein, e.g., VISA, MDA5, RIG-1 signaling proteins, MDA5 signaling proteins, ISGF3, STAT1, STAT2, IRF-3, IRF-7, IRF-9, PKR, 2'5'-OAS, NLRP12, and HERVs) is overexpressed in a biological sample or in a subject's brain can be determined by comparing the level of RIG-1 expression in the biological sample to a baseline level (also known as a control level) of expression of RIG-1. A "baseline level" is a control level, and in some embodiments a normal level or a level not observed in subjects having pre- AD. Therefore, it can be determined, based on the control or baseline level of RIG-1 expression, whether a sample to be evaluated for pre -AD development has a measurable increase (i.e., overexpression, upregulation) in expression of RIG-1, as compared to the baseline level. In certain embodiments, the baseline level can be established from a previous sample from the subject being tested, so that the disease state of the subject can be monitored over time and/or so that the efficacy of a given therapeutic protocol can be evaluated over time.

Screening Platform for Therapeutics for AD [0058] Described herein are methods and platforms for screening molecules, compounds, etc., for AD therapeutics. Such methods and platforms can include use of animal models and cellular assays as a screening platform for novel therapeutics aimed at key pathways and neurotoxic factors that are involved in pathogenesis of AD. A typical method of identifying a therapeutic agent for preventing or treating AD in humans includes screening a library of potential therapeutic agents (e.g., small molecules) to identify one or more agents that inhibit expression or activity of an RIG-1 signaling pathway member or a neurotoxic factor involved in AD pathogenesis, and delay or prevent onset of AD in a human. Examples of RIG-1 signaling pathway members include selected RIG-1, MDA5, VISA and IRF7/3/9 (i.e., IRF-3, IRF-7, IRF- 9), and examples of neurotrophic factors involved in AD pathogenesis include, e.g., beta amyloid, hyperphosphorylated tau, and amyloid precursor protein. In a typical embodiment, the library is screened in a high-throughput multi-well format.

Compositions For Delaying or Preventing Onset of AD In a Subject

[0059] Compositions for delaying or preventing onset of AD (e.g., pre-AD) in a subject are described herein. In one embodiment, a composition includes a pharmaceutically acceptable carrier and a therapeutically effective amount of an inhibitor of at least one of the following proteins: an RIG-1 signaling pathway member (e.g., RIG-1, MDA5, IRF-7 and VISA), NLRP12, and a human endogenous retrovirus protein. The amount of the inhibitor in the composition is a therapeutically effective amount for inhibiting inflammation or cell death in the subject having pre-AD (e.g., inhibiting apoptosis of and damage of neurons in the subject). An inhibitor of an RIG-1 signaling pathway member, NLR 12, or an HERV protein decreases expression or activity of the RIG-1 signaling pathway member, NLR 12, or a human endogenous retrovirus protein. Generally, the composition is administered to a subject that displays inherent (in the brain) AD pathology, but mild to no evident cognitive impairment. Typically, what is meant by AD pathology is AD autopsy diagnosis. In one embodiment, a composition for inhibiting apoptosis and damage of neurons in the subject having pre-AD also includes a vaccine targeted to amyloid beta clearance or one or more small molecule Bace-modulators.

[0060] In the compositions described herein, any suitable inhibitor can be used. An inhibitor of RIG-1, for example, reduces the level of RIG-1 in a cell and/or reduces the activity of RIG-1 in a cell. Any agent that reduces the level of RIG-1 (or other protein described herein) in a cell and/or reduces the activity of RIG-1 (or other protein described herein) in a cell can be used. An inhibitor of RIG-1, for example, active to reduce the level of RIG-1 protein in the cell may be an inhibitor of transcription and/or translation of RIG-1. In addition, an inhibitor of RIG-1 active to reduce the level of RIG-1 protein in the cell may stimulate degradation of the RIG-1 protein and/or RIG-1 -encoding RNA. An inhibitor of RIG-1 transcription and/or translation may be a nucleic acid-based inhibitor such as antisense oligonucleotides or siRNAs complementary to a target RIG-1 mRNA, as well as ribozymes and DNA enzymes which are catalytically active to cleave the target mRNA. Another example of an inhibitor of RIG-1 activity is a small molecule inhibitor that inhibits RIG-1 activity by altering its protein conformation or by interfering with essential protein-protein interactions. Additional examples of suitable inhibitors include antibodies, and known inhibitors of the RLR pathway such as BX795. When formulated for delivery to the brain in therapeutically effective amounts, the compositions can be used as a treatment or prophylactic approach to block or inhibit the signaling pathways described herein. Preventing or delaying onset of AD includes preventing plaque formation, protecting neurons, preventing neural apoptosis, necrosis, and other types of cell death.

Effective Doses

[0061] The compositions described above are preferably administered to a mammal (e.g., non-human primate, bovine, canine, rodent, human) in an effective amount, that is, an amount capable of producing a desirable result in a treated subject (e.g., delaying or preventing onset of pre- AD or AD in the subject). Toxicity and therapeutic efficacy of the compositions utilized in methods described herein can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary arts, dosage for any one animal depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently.

[0062] The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the pathology of the disease. A composition as described herein is typically administered at a dosage that inhibits RIG-1 (or other protein described herein) biological activity and/or expression, as assayed by identifying levels of RIG-1 signaling intermediates or downstream effector molecules of this signaling pathway, or using any that assay that measures the expression or the biological activity of RIG-1 or other protein described herein. Methods of Delaying or Preventing Onset of AD

[0063] Described herein are methods of preventing or delaying onset of AD which include administering a therapeutically effective amount of a pharmaceutical composition including a pharmaceutically acceptable carrier and a therapeutically effective amount of an inhibitor of R Ase L, and/or an RIG-1 signaling pathway member such as RIG-1, MDA5, IRF-3, IRF-7, IRF-9 or VISA for inhibiting cell death and damage of neurons to the subject (e.g., a mammal such as a human). A typical embodiment is a method of treating a subject suffering from early- onset AD (pre-AD), or disorder or symptom thereof. In this embodiment, administration of a pharmaceutical composition including a therapeutically effective amount of an inhibitor of RNAseL and/or an RIG-1 signaling pathway member such as RIG-1, MDA5, IRF-3, IRF-7, IRF- 9 or VISA for inhibiting cell death and damage of neurons is sufficient to treat the disease or disorder or symptom thereof and prevent further disease progression. In another embodiment, a method of preventing onset of AD or pre-AD involves administering a pharmaceutical composition including a pharmaceutically acceptable carrier and a therapeutically effective amount of an inhibitor of RNAseL, and/or an RIG-1 signaling pathway member such as RIG-1, MDA5, IRF-3, IRF-7, IRF-9 or VISA for inhibiting cell death and or inflammation to a subject that does not exhibit any signs or symptoms of pre-AD or AD but who is suspected of having a predisposition to AD sufficient to prevent onset of pre-AD or AD.

[0064] In yet another embodiment, a method of preventing or delaying onset of pre-AD or AD in a subject includes administering to the subject a composition including a pharmaceutically acceptable carrier and a therapeutically effective amount of an NLRP12 inhibitor for inhibiting apoptosis and damage of neurons in the subject. In some embodiments, inhibitors of two or more proteins are included within a single composition, or are administered to a subject in two or more compositions. For example, such a method can include administering to the subject a composition including a pharmaceutically acceptable carrier and a therapeutically effective amount of an inhibitor of an RIG-1 signaling pathway member (e.g., RIG-1, MDA5, IRF-3, IRF- 7, IRF-9 and VISA) as well as a therapeutically effective amount of an inhibitor of another protein such as an HERV protein, RNAse L, or NLRP12 for inhibiting apoptosis and damage of neurons in the subject. In this method, the subject typically has pre-AD, and administration of the composition delays progression of the pre-AD. [0065] The therapeutic methods of the invention (which include prophylactic treatment) in general include administration of a therapeutically effective amount of the compositions described herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for AD, or symptom thereof. Determination of those subjects "at risk" can be made by the diagnostic tests described herein, or opinion of a subject or health care provider.

[0066] In another aspect, a method of monitoring treatment progress is described herein. The method includes determining a level of diagnostic marker such as RIG-1 (e.g., any target delineated herein modulated by a composition or agent described herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with AD in which the subject has been administered a therapeutic amount of a composition as described herein sufficient to treat or alleviate the disease or symptoms thereof. The level of marker determined in the method can be compared to known levels of marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In some embodiments, a second level of marker (e.g., RIG-1) in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain embodiments, a pre -treatment level of marker (e.g. RIG-1) in the subject is determined prior to beginning treatment according to the methods described herein; this pre- treatment level of marker can then be compared to the level of marker in the subject after the treatment commences, to determine the efficacy of the treatment. In some embodiments, a subject who is not suspected of or at risk of having pre-AD is tested for pre-AD using the methods described herein.

[0067] Therapeutic compositions described herein can be administered to a subject by any suitable delivery vehicle and route. Examples of delivery vehicles and means for delivering compositions include antibody (vaccine) delivery, gene therapy including viral vectors, liposomes, aptamers, and other biologies. The administration of a composition including a therapeutically effective amount of an inhibitor of at least one of the following proteins: an RIG- 1 signaling pathway member, VISA, MDA5, NLRP12, and an HERV protein for the treatment of pre-AD and AD may be by any suitable means that results in a concentration of the therapeutic that is effective in blocking RLR signaling, preventing cell death, or preventing inflammation. An inhibitor as described herein may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for local or systemic administration (e.g., parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, intracranially). The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[0068] Compositions as described herein including small molecules may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

[0069] Compositions for parenteral use may be provided in unit dosage forms (e.g., in single- dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that delays or prevents onset of AD, the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing agents.

[0070] As indicated above, the pharmaceutical compositions described herein may be in a form suitable for sterile injection. To prepare such a composition, the suitable active therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.

[0071] Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactin, poly-(isobutyl cyanoacrylate), poly(2- hydroxyethyl-L-glutam- nine) and, poly(lactic acid). Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be nonbiodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(gly colic acid) or poly(ortho esters) or combinations thereof).

[0072] At least two anti-AD therapeutics (e.g., an inhibitor of at least one of the following proteins: an RIG-1 signaling pathway member, NLRP12, RNAse L, and an HERV protein and an AD vaccine or small molecule Bace-modulator, VISA, Rig-1, MDA5) may be mixed together in a single composition, or may be administered separately. For example, an anti-AD therapeutic may be administered in combination with any other standard or newly emerging anti-AD therapy; such methods are known to the skilled artisan and described in Remington's Pharmaceutical Sciences by E. W. Martin.

[0073] Formulations for oral use include tablets containing the active ingredient(s) (e.g., inhibitor of at least one of the following proteins: an RIG-1 signaling pathway member, NLRP12, RNAse L, and an HERV protein) in a mixture with non-toxic pharmaceutically acceptable excipients. Such formulations are known to the skilled artisan. Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

[0074] The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period. The coating may be adapted to release the active drug in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active drug until after passage of the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose). Furthermore, a time delay material, such as, e.g., glyceryl monostearate or glyceryl distearate may be employed.

[0075] The solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes, (e.g., chemical degradation prior to the release of the active therapeutic substance). The coating may be applied on the solid dosage form in a similar manner as that described in Encyclopedia of Pharmaceutical Technology, supra.

[0076] Formulations for oral use may also be presented as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders and granulates may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

Kits [0077] Described herein are kits for detecting the presence of AD in a subject (e.g., human) at an early stage (pre- AD). A typical kit for detecting pre- AD in a subject suspected or at risk of having pre- AD includes at least a first reagent for detecting the presence of and quantitating the level of RIG- 1 protein (or other protein described herein) in a biological sample from the subject, and instructions for use. In one embodiment, a kit includes a monoclonal or polyclonal antibody to RIG-1, a detectable label, and instructions for use. The at least one reagent can be, for example, an RIG-1 -specific antibody. A kit can further include a second reagent for detecting the presence of and quantitating the level of another protein such as VISA, IRF-3, IRF-9, IRF-7, MDA5, NLRP12, RNAse L, or an HERV protein. A kit may include a well plate to carry the mixture of the different reagents, as well as one or more washing buffers. Optionally, kits may also contain one or more of the following: containers which include positive controls, containers which include negative controls, photographs or images of representative examples of positive results and photographs or images of representative examples of negative results.

EXAMPLES

[0078] The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way.

Example 1 - Regulation of Pattern Recognition Receptors in AD

[0079] Infection of cells by viruses and microorganisms activates innate immune inflammatory responses (Furr et al., Journal of Neurovirology, 1 :2008). The initial sensing of infection is mediated by pattern recognition receptors, which include Toll-like receptors, RIG-1- like receptors, NOD-like receptors, and C-type lectin receptors (C. Zipfel, Current Opinion in Plant Biology, 12(4):414, 2009). The intracellular signaling pathways triggered by these receptors initiate expression of inflammatory mediators that eliminate pathogens and infected cells (C. Zipfel, Current Opinion in Plant Biology, 12(4):414, 2009). Aberrant activation of these signaling systems leads to disease and induction of autoimmunity. Recent evidence has accumulated that AD is associated with a central nervous system (CNS) inflammatory response. For example, Αβ fibrils trigger inflammatory responses through TLR4/TLR6 in the presence of CD36 (Salminen et al, Progress in Neurobiology vol. 87(3): 181, 2009). Moreover, a polymorphism in the TLR4 extracellular domain has been reported to be associated with protection against late -onset AD in an Italian population (Minoretti et al., Neuroscience Letters vol. 391(3): 147, 2006), suggesting a sterile inflammatory response could influence AD pathology through TLR4 signaling. Activation of innate immune cells in the CNS appears to be a universal component of neuroinflammation. Viewed from the perspective of inducers, sensors, transducers and effectors of innate immunity, AD may be distinguished by a disease-specific mechanism for induction of inflammatory responses. Distinct pathways for production of inducers of inflammation and the specific anatomical localizations at which these processes occur are likely determinants of the specific pathological features of AD.

[0080] The RIG- 1 -like receptors (RLRs) family represents another sensing system and are composed of RIG-1, melanoma differentiation-associated gene 5 (MDA5), and LPG2. RIG-1 recognizes relatively short dsRNA (up to 1 kb) whereas MDA5 detects long dsRNA (more than 2 kb) to activate synthesis of type I interferons (IFNs), including multiple forms of IFN-a and single forms of IFN-β, IFN-ω (Honda and Taniguchi, Nature Reviews vol. 6(9):644, 2006). Type I IFNs play central roles in antiviral responses by inducing apoptotic cell death in virally infected cells, rendering cells resistant to virus infection, activating acquired immunity, and stimulating hematopoietic stem cell turnover and proliferation (Bekisz et al, Growth Factors (CHur, Switzerland) vol. 22(4): 243, 2004). Secreted type I IFNs alert surrounding cells via type I IFN receptors by triggering a signaling cascade that leads to phosphorylation and nuclear translocation of IFN-stimulated gene factor 3 (ISGF3). ISGF3 induces expression of IFN- inducible antiviral genes such as protein kinase R (PKR) and 2'5'-oligoadenylate synthase. PKR suppresses the proliferation of virus-infected cells and 2'5'-OAS activates RNaseL, which cleaves viral nucleotides in order to inhibit virus production. However, little information is available about the role of RLRs in AD pathology.

[0081] RLRs are localized in the cytoplasm and recognize the genomic RNA of dsRNA viruses and dsRNA generated as the replication intermediate of ssRNA viruses and also act as sensors of cellular damage (Takeuchi and Akira Cell vol. 140(6):805 2010). RLRs activate downstream signaling proteins such as N-terminal CARD-containing adaptor IFN-P-promoter stimulator 1 (IPS-1) (also known as VISA, MAVS, or CARDIF) (Kawai and Akira Nature Immunology vol. 7(2): 131, 2006), TRAF3, TRADD (Michallet et al, Immunity vol. 28(5):651, 2008) evoking type 1 IFN production. To compare the expression patterns of key components of RIG- 1 -like signaling in normal, pre-AD (early) and AD (late-stage) subjects, a panel of anti-RLR signaling antibodies was used to characterize expression in these two experimental groups compared to age-matched patients with normal cognition and no evidence of AD pathology at autopsy. These studies show that pre-AD involves activation of RIG- 1 -like signaling pathway and NLRP12 in temporal cortex in pre-AD and in the CSF.

Materials and Methods:

[0082] Patients and brain samples: Small pieces (3 mm) of fresh-frozen human temporal and occipital cortex were obtained from the University of Miami Brain Endowment Bank. Samples were obtained from three groups of patients diagnosed with early AD, late-stage AD and age- matched controls. Patients under the pre-AD symptom category had a Braak stage between 1 and 4. Patients under the category of AD were between 4/5 to 6. The age range is between 60 to 89 years old (Table 1). Brain samples of patients with HD were from the caudate; samples from patients with ALS were from the motor cortex, and samples of patients with DLBD were from the temporal cortex. Postmortem CSF samples were drawn from clinically diagnosed patients with neuropatho logical disease status. Human astrocytes were maintained in culture and treated with synthetic ligand poly(I:C) or untreated (C) for 21 hours. Astrocytes were lysed and immunob lotted for RIG-1, VISA and amyloid precursor protein.

[0083] Immunoblotting. Occipital and temporal cortices were homogenized in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VC>4) with protease cocktail inhibitor (Sigma). Proteins were resolved in 10-20% Tris-HCl Criterion precasted gels (Bio-Rad), transferred to polyvinylidene difluoride membranes (Applied Biosystems) and placed in blocking buffer (PBS, 0.1% Tween-20, 0.4%> I-Block (Applied Biosystems) and then incubated for 1 h with the following antibodies at a dilution of 1 : 1000: anti-RIG-1 (Anaspec), anti-MDA5 (Abeam), anti-VISA (Anaspec), anti-RNaseL (Abeam), anti-IL-Ιβ (Cell Signaling), anti-IL18 (Abeam), anti-NLRP12 (Calbiochem), anti-caspase-1 (Imgenex) and anti-caspase-5 (Imgenex). Membranes were incubated for 1 h with primary antibodies followed by appropriate secondary horseradish peroxidase (HRP)-linked antibodies (Cell Signaling). Visualization of signal was enhanced by chemiluminescence using a phototope-HRP detection kit (Cell Signaling). To control for protein loading, immunoblots were stripped with Restore, Western blot stripping buffer (Pierce) and blotted for β-actin using monoclonal anti-P-actin antibody (1 :8000, Sigma). Quantification of band density was performed using the UN-SCAN-IT gel software, and data was normalized to β-actin.

[0084] Statistical Analysis. Data are expressed as mean +/- standard error of the mean (+/- s.e.m.). Statistical comparisons between treated and untreated groups were made using one-tailed Student's t-test. P-value of significance was *P < 0.05, # < 0.1.

Results

[0085] The data indicate that the RIG- 1 -like signaling intermediates RNase L, RIG-1, MDA5, and VISA are significantly upregulated in the temporal cortex of patients with pre- AD (Figure 1A). Additionally, the levels of NLRP12, a negative regulator of innate immunity were significantly elevated in pre -AD. Immunoblots of occipital cortex, a brain area not affected in AD did not show significant increases in these signaling proteins demonstrating region specific regulation (Figure IB). These data show that cases of apparent pre- AD (pathologic but not cognitive impairments) are associated with the activation of the RIG-like-receptor signaling pathway suggesting a viral-like etiology in AD.

[0086] Next, a panel of anti-RLR and inflammasome signaling antibodies was used to characterize biomarkers in CSF samples of patients diagnosed with pre-AD, AD and normal age- matched controls. The clinical phenotypes of the fifteen patients in this study are summarized in Table 2. It is noteworthy that five patients are normal age-matched controls (controls), five patients who have been diagnosed as pre-AD (Braak stages I-III, preAD) and five patients diagnosed with various advanced stages (Braak III-V) of AD (AD). As shown in Figure 3, significant increases in RNaseL, RIG-1, and VISA were present in CSF of pre-AD patients compared to age-matched controls. Moreover, significant increases in RNaseL and RIG-1 were also present in CSF from AD patients. These studies show for the first time that RNaseL, RIG-1 and VISA may be used as biomarkers in CSF for diagnosis of pre-AD and AD.

[0087] In order to establish whether other innate immune signaling pathways may be activated in pre-AD and later in the course of AD, the temporal (T) and occipital (O) cortical samples from control, pre-AD and AD patients were analyzed for expression of NALP1 inflammasome proteins including: NALP1, ASC, caspase-1, caspase-5, IL-Ιβ and IL-18. As shown in Figure 5, no significant changes were observed between the groups in the levels of expression of these NALP1 inflammasome-signaling proteins. Therefore, AD appears to involve increased expression of the RIG-1 pathway and not the NALP1 signaling pathway. [0088] In order to establish whether the RIG-1 signaling pathway was modulated in other neurodegenerative diseases, analyzed brain samples of patients with ALS, DLBD and HD were analyzed for expression of VISA (Figure 4A) and NLRP12 (Figure 4B). No significant changes in the levels of these proteins were observed in ALS and DLBD, however HD subjects showed a significant reduction in the levels of VISA and NLRP12 when compared to control brain samples (ALS, DLB and HD). Thus, alterations in RIG-1 signaling appear to be specific for AD and HD and not other neurodegenerative diseases, including ALS, and DLBD.

[0089] In order to provide evidence that RIG-1 ligands stimulate amyloid precursor protein, human astrocytes were maintained in culture and treated with synthetic ligand poly(LC) for 21 hours whereas controls (C) were left untreated. As shown in Figure 6, poly (I:C) significantly increased levels of RIG-1, VISA, and the pi 10 and p70 subunits of APP. Thus, human astrocytes may be used as a screening platform for drug therapies to inhibit APP.

[0090] AD is one of the most common age-related neurodegenerative diseases with approximately 7% of people older than 65 years and 40% of people older than 80 years being affected in industrialized countries. AD is a neurodegenerative disorder characterized by a progressive cognitive impairment, the consequence of neuronal dysfunction and ultimately the death of neurons. The amyloid hypothesis proposes that neuronal damage results from the accumulation of insoluble, hydrophobic, fibrillar peptides such as amyloid-β. These peptides activate enzymes resulting in a cascade of second messengers including prostaglandins and platelet-activating factor. Apoptosis of neurons is thought to follow as a consequence of the uncontrolled release of second messengers. Biochemical, histopathological and genetic studies suggest that pro-inflammatory cytokines play a role in neurodegeneration during AD. In the current study, the regulation of pattern recognition receptors, (Toll-like receptors, RIG-1 -like receptors, NOD-like receptors, and C-type lectin receptors) and the intracellular signaling pathways and inflammatory mediators that eliminate pathogens and infected cells, were examined. The results described herein demonstrate for the first time that aberrant activation of the RIG-1 like receptor signaling systems in pre -AD and components in this pathway may be used as diagnostic markers as an early event in AD pathology in human brain and CSF postmortem. Example 2 - Innate Immune Response Proteins as Biomarkers of Presymptomatic Alzheimer's Disease

[0091] Reported herein is data that shows that the expression of two PRRs, RIG-1 and VISA, and NLRP12, are significantly elevated in neurons of the temporal cortex and the CSF in presymptomatic AD patients. Increased expression of RIG-1 and VISA were not found in pathologically confirmed cases of amyotrophic lateral sclerosis, Diffuse Lewy Body disease or HD, suggesting that activation of RIG-1 signaling may be a specific marker for early AD pathogenesis. Moreover, in vitro results show that stimulation of RIG-1 signaling induces amyloid precursor protein, supporting the idea that activation of the RIG-1 signaling system occurs in incipient AD.

RESULTS

[0092] RIG-1 signaling proteins are elevated in Presymptomatic AD. A panel of anti-RLR and inflammasome signaling antibodies were used to characterize biomarkers of inflammation in CSF and brain samples of patients pathologically confirmed with presymptomatic Alzheimer's disease (EAD), advanced end-stage (LAD) and normal age-matched controls. The clinical phenotypes and neuropathologic findings of the patients are summarized in Table 1. The cohort consisted of 25 age-matched neurologically normal controls (controls), 23 patients who were clinically non-demented, but had pathologic evidence of senile plaques and neurofibrillary tangles consistent with possible or probable AD (Braak stages I-IV), and 23 patients who met clinical diagnostic criteria and had more advanced postmortem evidence of end-stage AD (Braak V-VI). Except for the borderline presymptomatic cases, all AD patients met Alzheimer's Disease and Related Disorders Association criteria for the clinical diagnosis of AD and Consortium to Establish a Registry for Alzheimer's Disease and National Institute of Aging- Reagan Institute neuropathology criteria for the diagnosis of AD.

[0093] Table 1 - The clinical phenotypes and neuropathologic findings of patients pathologically confirmed with EAD, LAD and normal age-matched controls

Control

Gender Age at Death

Male 67

Male 67

Male 76

Female 78

Female 91 Male 61

Male 61

Male 82

Male 76

Female 91

Male 64

Male 83

Male 59

Male 58

Female 95

Male 56

Male 65

Male 69

Female 70

Male 61

Male 67

Female 56

Female 51

Male 87

Male 56

EAD

Gender Age at Death Braak Stage

Male 61 1

Male 74 1

Female 68 3

Male 68 2

Female 88 1

Female 89 3

Male 67 3

Female 78 3

Male 64 1

Male 83 1

Male 59 3

Male 58 2

Female 95 1

Male 56 2

Male 65 2

Male 69 3

Female 70 3 Male 61 3

Male 67 3

Female 56 2

Female 51 3

Male 87 2

Male 66 3

[0094] Table 2 - The clinical phenotypes and neuropathologic findings of patients who provided CSF and pathologically confirmed with EAD, LAD and normal age-matched controls

Male 82 0

Male 82 0

Female 87 0

Female 109 0

Male 93 0

Female 85 0

EAD

Age at Braak

Gender Death Stage

Female 90 1

Female 92 2

Female 100 1

Female 84 1

Female 81 2

Female 80 1

Male 81 2

Female 76 2

Female 83 2

Male 83 1

Female 97 3

Male 80 2

Male 77 4

Female 92 3

Female 95 4

Female 69 4

Female 90 3

Male 96 4

LAD

Age at Braak

Gender Death Stage

Male 85 5

Male 78 6

Male 85 6

Female 84 6

Male 85 6

Male 80 6

Male 87 6

Male 73 6 Female 81 6

Female 83 6

Female 91 5

[0095] Figure 1A shows that RIG-1, VISA and NLRP12 are significantly increased in the temporal cortex of presymptomatic AD as compared to age-matched controls, whereas the levels of MDA-5 did not. However, the level of these RIG-1 signaling intermediates in the occipital cortex, a region not affected by AD did not show significant changes between the various groups (Fig. IB).

[0096] The levels of RIG-1, MDA5 and VISA were significantly lower in AD temporal cortices when compared to EAD samples, but in occipital cortex of AD patients the level of RIG-1 was significantly elevated when compared to EAD. Immunohistochemical staining revealed that increased RIG-1, VISA and NLRP12 immunoreactivity was present in neurons of the temporal cortex in EAD individuals when compared to age-matched controls and LAD (Fig. 2). In addition, it was found that these signaling proteins were present in CSF samples (Fig. 3, Table 2) in which the levels of RIG-1, VISA, RNase L and NLRP12 were significantly lower in AD compared to EAD. Moreover, the levels of MDA5 in CSF were higher in EAD compared to control, whereas the levels of NLRP12 were higher in control samples compared to LAD. Thus, these studies show that RIG-1 and VISA may be used as biomarkers for diagnosis of EAD.

[0097] VISA and NLRP12 signaling proteins are not elevated in amyotrophic lateral sclerosis, DLBD and HD. To establish whether other neurodegenerative diseases resulted in alterations of innate immune signaling proteins, the motor cortex of ALS, the temporal cortex of DLBD and caudate region from HD patients (Table 3) were analyzed. As shown in Figures 4A and 4B, neither the level of VISA or NLRP12 was altered in ALS and DLBD. However, in HD samples the expression of these proteins was significantly decreased. Therefore, VISA and NLRP12 do not demonstrate a similar pattern of protein expression as observed in EAD in which these proteins are elevated in the temporal cortex.

[0098] Table 3 - Gender and age at death in ALS, DLBD, and HD patients

DLBD ALS

Gender Age at Death Gender Age at Death

Male 74 Male 79

Male 86 Male 55 Male 76 Male 66

Female 85 Male 83

Female 83 Male 70

HD

Gender Age at Death

Female 66

Male 47

Male 54

Male 77

Female 85

[0099] NLRP1 inflammasome components are not altered in brain of AD patients. Previous studies in rodents suggest that the inflammasome is involved in the pathology of AD (Halle, A. et al. Nat Immunol 9, 857-865 (2008)). To determine if inflammasome protein expression is altered in the brain of AD patients, the occipital and temporal cortex were analyzed for expression levels of inflammasome signaling proteins, caspase-1, caspase-5, NLRP1, ASC, IL- 1β and IL-18 proteins. As shown in Figure 5, protein levels of caspase-1, caspase-5, ASC and IL- 18 did not show significant alterations in protein expression among controls, EAD and AD cases in either the temporal and the occipital cortices. Significant changes were found in NLRP1 in the temporal cortex and IL-Ιβ in the occipital cortex. Therefore, it does not appear that inflammasome signaling contributes to the pathology of AD.

[00100] RIG-1 ligand increases APP in human astrocytes. Human astrocytes were stimulated with poly(LC) of low molecular weight for 18 hrs to induce RIG-1 signaling and assayed cell cultures for production of RIG-1, VISA and APP. Treatment with synthetic double stranded RNA significantly elevated VISA and APP (Figure 6). Therefore, RIG-1 ligands stimulate APP production in human astrocytes that may contribute to underlying pathomechanisms of AD.

[00101] Referring to Figure 2, RIG-1 signaling proteins are expressed in neurons in the temporal cortex of EAD patients. Immunohistochemical staining showing neurons in the temporal cortex of EAD individuals (middle column) express increased RIG-1 (row 1), MDA5 (row 2), VISA (row 3) and NLRP12 (row 4). Age-matched controls (left column) and LAD (right column). Immunohistochemical staining revealed that increased RIG-1, VISA and NLRP12 immunoreactivity was present in neurons of the temporal cortex in EAD individuals when compared to age-matched controls and LAD (Fig. 2). Regarding the immunohistochemical staining procedures, paraffin-embedded sections (10 μΜ) were stained with anti-RIG-1 (AnaSpec), anti-MDA5 (Abeam), anti-VISA (AnaSpec) and anti-NLRP12 (Calbiochem) as described (de Rivero Vaccari et al. 2008) using diaminobenzidine (DAB) as the chromophore followed by hematoxylin staining.

METHODS

[00102] Immunoblotting and cell culture experiments. Brain or astrocyte lysates were prepared for immunoblotting as described in de Rivero Vaccari et al. (J Neurosci 28, 3404-3414 (2008)). Brains were obtained from the University of Miami Brain Endowment Bank™ and from the University of Kentucky Alzheimer's Disease Center Brain Bank. Brains corresponded to patients diagnosed with AD, presymptomatic cases of pathologically confirmed AD, HD, ALS and DLB and age-matched control subjects. Lysates and CSF samples were immunoblotted for RLR signaling proteins as well as inflammasome proteins. Primary human astrocytes (Lonza) were stimulated with poly(LC). (Invivogen) for 18 h to activate RLR signaling and assayed by immunoblots analysis for protein levels of APP expression.

[00103] Patients and brain samples. Pathologic specimens (3 mm) of fresh-frozen human temporal (BA38) and occipital cortex (BA17) were obtained from the University of Miami Brain Endowment Bank™ and from the University of Kentucky Alzheimer's Disease Center Brain Bank. Samples were obtained from three groups of patients non-demented but diagnosed at brain autopsy with possible to probable AD (Braak stage I - IV; N = 23), late-stage AD (Braak stage V and VI (N = 23) and age-matched non-neurologic controls (N=25). Patients in the presymptomatic AD group met neuropathologic criteria for incipient AD with Braak stage 1 through 4. All AD cases were clinically demented with Braak stages 5 or 6 (Tables 3, 4 and 5).

[00104] Immunoblotting. Occipital and temporal cortices were homogenized in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃V0₄) with protease cocktail inhibitor (Sigma). Proteins were resolved in 10-20% Tris-HCl Criterion precasted gels (Bio-Rad), transferred to polyvinylidene difluoride membranes (Applied Biosystems) and placed in blocking buffer (PBS, 0.1 %> Tween-20, 0.4%> I-Block (Applied Biosystems) and then incubated for 1 h with the following antibodies at a dilution of 1 : 1000: RIG-1 (Anaspec), MDA5 (Abeam), VISA (Anaspec), RNase L (Immgenex) NLRP12 (Calbiochem), APP (Abeam), NLRP1 (Bethyl laboratories as described previously in de Rivero Vaccari et al, J Neurosci 28, 3404-3414 (2008)), ASC (Santa Cruz), Caspase-1 (Imgenex), Caspase-5 (Imgenex), IL-Ιβ (Cell Signaling) and IL-18 (Abeam). Membranes were incubated for 1 h with primary antibodies followed by appropriate secondary horseradish peroxidase (HRP)-linked antibodies (Cell Signaling). Visualization of signal was enhanced by chemilluminescence using a phototope-HRP detection kit (Cell Signaling). To control for protein loading, immunoblots were stripped with Restore, Western blot stripping buffer (Pierce) and blotted for β-actin using monoclonal anti- -actin antibody (1 :8000, Sigma). Quantification of band density was performed using the UN-SCAN- IT gel software, and data was normalized to β-actin. Similar procedures were carried to perform immunoblotting of CSF samples that were blotted for RIG-1, MDA5, VISA, RNase L and NLRP12.

[00105] Immunohistochemical staining procedures. Paraffin-embedded sections (10 μΜ) were stained with anti-RIG-1 (AnaSpec), anti-MDA5 (Abeam), anti-VISA (AnaSpec) and anti- NLRP12 (Calbiochem) as described (de Rivero Vaccari et al, J Neurosci 28, 3404-3414 (2008)) using diaminobenzidine (DAB) as the chromophore followed by hematoxylin staining.

[00106] Astrocyte culture preparation and RLR stimulation. Primary astrocytes (Lonza) were plated and grown in culture for 7d prior to experimentation. Poly(I:C)Lyovec of low molecular weight (Invivogen) was used to stimulate Rigl at 6 μg/ml. Controls used were Lyovec vector alone (Invivogen) and no treatment. Stimulation with ligands was carried for 18 h according to manufacturer's instructions.

[00107] Statistical Analysis. Data are expressed as mean +/- standard error of the mean (+/- s.e.m.). Statistical comparisons between treated and untreated groups were made using one-tailed Student's t-test. P-value of significance was * p < 0.05.

SUMMARY

[00108] Here, evidence demonstrating that the RIG-1 signaling system is activated in the innate immune response in disease-affected brain areas of EAD patients is provided. It was found that RIG-1, VISA and NLRP12 are significantly increased in neurons of the temporal cortex of EAD and that the level of these RIG-1 signaling intermediates in the occipital cortex, a region not affected by AD did not show significant change. Increased expression of RIG-1 and VISA were not found in disease-affected brain tissues of patients with ALS, DLBB or HD, indicating that activation of RIG-1 signaling does not appear to play a role in these neurodegenerative conditions. Moreover, in vitro results reported here show that stimulation of RIG-1 signaling induces APP production, also supporting the idea that aberrant activation of the RIG-1 signaling system is an early event in AD pathology in human brains, postmortem. The results further show that NLRP12 is elevated in EAD patients. NLRP12 is expressed in monocytes/macrophages and granulocytes and inhibits the activation of noncanonical NF-κΒ by associating with and inducing proteasome-mediated degradation of NF-KB-inducing kinase. The in vitro data further demonstrate that short synthetic viral RNA stimulates RIG-1 signaling in human astrocytes leading to increased production of APP.

[00109] In search of new biomarkers for early detection and differential diagnosis, these findings indicate that RIG-1, VISA, RNase L and NLRP12 may be reliable CSF biomarkers for the early diagnosis of AD. Because the development of early diagnostic neuroimaging tools is critical, since almost 30 million people suffer from this disease worldwide, molecular tracers to tag RIG-1, VISA and NLRP12 in pathological lesions may afford a novel diagnostic tool for detection of early AD.

Example 3 - RLR Proteins Found in Plasma of AD Patients

[00110] As shown in FIG. 9, RLR proteins were found in plasma of AD patients. RLR protein expression in the plasma of AD patients is shown in FIG. 7. In this experiment, CSF samples were obtained from age-matched controls (Braak 0-1), presymptomatic Alzheimer disease (Braak 2-4) and Alzheimer Disease (Braak 5-6) cases and were analyzed by immunoblotting for protein expression levels of RIG-1, MDA5, VISA and NLRP12. Data is presented as mean + SEM. N=7-18 per group. * p < 0.05.

Other Embodiments

[00111] Any improvement may be made in part or all of the compositions, kits, and method steps. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. Any statement herein as to the nature or benefits of the invention or of the preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context.

Claims

What is claimed is:

1. A method of detecting pre-symptomatic or early stage Alzheimer's disease (pre- AD) in at least one subject suspected of having or at risk of having pre- AD comprising:

(a) obtaining a biological sample from the at least one subject;

(b) contacting the biological sample with at least a first reagent that detects the presence of RIG- 1 protein;

(c) measuring the level of RIG- 1 protein in the biological sample; and

(d) correlating overexpression of RIG- 1 in the sample with pre- AD in the at least one subject.

2. The method of claim 1, wherein step (b) comprises contacting the sample with the at least first reagent and a second reagent that detects the presence of a protein selected from the group consisting of: VISA, IRF-3, IRF-7, IRF-9, RNAseL, MDA5, NLRP12, and a human endogenous retrovirus protein, step (c) comprises measuring the level of RIG- 1 protein and the level of the protein selected from the group consisting of: VISA, IRF-3, IRF-7, IRF-9, RNAseL, MDA5, NLRP12, and a human endogenous retrovirus protein in the biological sample, and step (d) comprises correlating overexpression of RIG- 1 and overexpression of the protein selected from the group consisting of: VISA, IRF-3, IRF-7, IRF-9, RNAseL, MDA5, NLRP12, and a human endogenous retrovirus protein in the sample with pre- AD in the at least one subject.

3. The method of claim 2, wherein the biological sample is selected from the group consisting of: cerebral spinal fluid, blood, saliva, serum, plasma, tissue, skin, brain tissue, urine, and epithelial cells from skin, mouth, muscle or other body tissue.

4. The method of claim 1 , wherein pre- AD is detected in the at least one subject before the at least one subject displays cognitive impairment due to AD.

5. The method of claim 1, wherein steps b) and c) are performed using one of the assays selected from the group consisting of: enzyme-linked immunosorbent assay, radioimmunoassay, Western blot, flow cytometry, immunofluorescence, immunoprecipitation, protein A, immunoelectrophoretic, immunoscintigraphic detection and protein chip, and PET imaging.

6. The method of claim 1, wherein the at least one subject comprises a plurality of subjects suspected of having pre- AD or who are at risk for AD.

7. A method of detecting pre -AD in a subject suspected of having or at risk of having pre- AD comprising:

(a) obtaining a biological sample from the subject;

(b) contacting the biological sample with at least a first reagent that detects the presence of RIG- 1 protein, at least a second reagent that detects the presence of MDA5 protein, at least a third reagent that detects the presence of VISA protein, at least a fourth reagent that detects the presence of NLRP12 protein, and at least a fifth reagent that detects the presence of a human endogenous retrovirus protein;

(c) measuring the levels of RIG- 1, MDA5, VISA , NLRP12, and human endogenous retrovirus proteins in the biological sample; and (d) correlating overexpression of at least one of the RIG-1, MDA5, VISA, NLRP12, and human endogenous retrovirus proteins in the sample with pre- AD in the subject.

8. The method of claim 7, wherein the biological sample is selected from the group consisting of: cerebral spinal fluid, blood, saliva, serum, plasma, tissue, skin, brain tissue, urine, and epithelial cells from skin, mouth, muscle or other body tissue, and wherein pre -AD is detected in the subject before the subject displays cognitive impairment due to AD.

9 The method of claim 7, wherein steps b) and c) are performed using one of the assays selected from the group consisting of: in vivo brain imaging, including but not limited to single photon emission computed tomography (SPECT), functional magnetic resonance imaging (fMRI), positron emission tomography (PET), FLISA, RT-PCR, qPCR, and DNA chip platforms.

10. A method of detecting pre -AD in a subject suspected of having or at risk of having pre- AD comprising:

(a) administering a composition comprising at least a first reagent that detects the presence of RIG-1 protein to the subject, the at least first reagent labeled with or conjugated to a detectable agent;

(b) imaging the subject's brain to detect the presence and location of the at least first reagent labeled with or conjugated to a detectable agent;

(c) measuring the level of RIG-1 protein in a disease-affected area of the subject's brain; and (d) correlating overexpression of RIG-1 in the disease-affected area of the subject's brain with pre- AD in the subject.

11. The method of claim 10, wherein the at least first reagent comprises an antibody or labeled substance that binds to RIG-1 or RIG-1 signaling intermediates and the detectable agent comprises a radiolabel.

12. The method of claim 10, wherein imaging of the subject's brain consists of PET, fMRI, and/or SPECT.

13. A method of identifying a therapeutic agent for preventing or treating AD in humans, the method comprising screening a library of potential therapeutic agents to identify one or more agents that a) inhibit expression or activity of an RIG-1 signaling pathway member or a neurotoxic factor involved in AD pathogenesis, and b) delay or prevent onset of AD in a human.

14. The method of claim 13, wherein the RIG-1 signaling pathway member is selected from the group consisting of: RIG-1, MDA5, VISA, IRF7 and NLRP12, and the neurotrophic factor involved in AD pathogenesis is selected from the group consisting of: beta amyloid, hyperphosphorylated tau, and amyloid precursor protein.

15. The method of claim 13, wherein the library is screened in a high-throughput multi- well format, and the potential therapeutic agents are small molecules.

16. A composition comprising a pharmaceutically acceptable carrier and an inhibitor of a protein selected from the group consisting of: an RIG-1 signaling pathway member, NLRP12, VISA, and a human endogenous retrovirus protein in a therapeutically effective amount for delaying cognitive decline and inhibiting apoptosis and damage of neurons in a patient who has overexpression of at least one of the following: RIG-1, VISA, MDA5 , NLRP12, IRF3 and IRF7, and who displays AD pathology but not cognitive impairment.

17. The composition of claim 16, wherein the RIG-1 signaling pathway member is selected from the group consisting of: RIG-1, MDA5, IRF-3, IRF-7, IRF-9, and VISA, and wherein the inhibitor decreases expression or activity of the RIG-1 signaling pathway member and is in an amount effective for delaying cognitive decline and damage of neurons in the patient.

18. The composition of claim 16, further comprising a vaccine targeted to prevent amyloid beta accumulation or one or more small molecule Bace-modulators.

19. The composition of claim 16, wherein the composition comprises a pharmaceutically acceptable carrier and a therapeutically effective amount of an inhibitor of an RIG-1 signaling pathway member selected from the group consisting of: RIG-1, MDA5, IRF-3, IRF-7, IRF-9 and VISA, and an inhibitor of a protein selected from the group consisting of: human endogenous retrovirus protein, RNAse L, and NLRP12 in a therapeutically effective amount for inhibiting apoptosis and damage of neurons in the patient.

20. A method of preventing or delaying onset of AD in a subject, the method comprising administering to the subject a composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an inhibitor of an RIG-1 signaling pathway member selected from the group consisting of: RIG-1, MDA5, IRF-3, IRF-7, IRF-9 and VISA for inhibiting apoptosis and damage of neurons in the subject.

21. A method of preventing or delaying onset of AD in a subject, the method comprising administering to the subject a composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an NLRP12 inhibitor for inhibiting apoptosis and damage of neurons in the subject.

22. A method of preventing or delaying onset of AD in a subject, the method comprising administering to the subject a composition comprising a) a pharmaceutically acceptable carrier, b) a therapeutically effective amount of an inhibitor of an RIG-1 signaling pathway member selected from the group consisting of: RIG-1, MDA5, IRF-3, IRF-7, IRF-9 and VISA for inhibiting apoptosis and damage of neurons in the subject, and c) a therapeutically effective amount of an inhibitor of a protein selected from the group consisting of: human endogenous retrovirus protein, RNAse L, and NLRP12 for inhibiting apoptosis and damage of neurons in the subject.

23. The method of claim 22, wherein the subject has pre -AD but does not display cognitive impairment due to AD, and administration of the composition delays progression of the pre- AD.

24. A kit for detecting pre-AD in a subject suspected or at risk of having pre- AD, the kit comprising:

(a) at least a first reagent for detecting the presence of and quantifying the level of RIG-1 protein in a biological sample from a subject; and

(b) instructions for use.

25. The kit of claim 24, wherein the at least one reagent is a RIG-1 -specific antibody.

26. The kit of claim 24, further comprising a second reagent for detecting the presence of and quantitating the level of a protein selected from the group consisting of: VISA, IRF-3, IRF-7, IRF-9, MDA5, NLRP12, RNAse L, and a human endogenous retrovirus protein.