Alzheimer's Disease-Specific Alterations of the Erk1/Erk2 Phosphorylation Ratio-Alzheimer's Disease-Specific Molecular Biomarkers (Adsmb)

Abstract

The present invention relates to methods of diagnosing Alzheimer's Disease as well as to methods of confirming the presence or absence of Alzheimer's Disease in a subject. The present invention is also directed to methods of identifying a lead compound useful for the treatment of Alzheimer's Disease by contacting non-Alzheimer's cells with an amyloid beta peptide, stimulating the cells with a protein kinase C activator, contacting the cells with a test compound, and determining the value of an Alzheimer's Disease-specific molecular biomarker. The present invention is also directed to methods of diagnosing Alzheimer's Disease in a subject by detecting alterations in the ratio of specific phosphorylated MAP kinase proteins in cells after stimulation with a protein kinase C activator. The Alzheimer's Disease-specific molecular biomarkers disclosed herein are useful for the diagnosis of Alzheimer's Disease, monitoring the progression of Alzheimer's Disease in a subject and in screening methods for the identification of compounds for treating or preventing Alzheimer's Disease. The invention is also directed to kits containing reagents for the detection and diagnosis of the presence or absence of Alzheimer's Disease using the Alzheimer's Disease-specific molecular biomarkers disclosed herein.

1 ) A method for determining the absence of Alzheimer's Disease in a subject comprising:
 a) contacting cells from a subject with an amyloid beta peptide;   b) determining whether said contacting step induces an Alzheimer's Disease phenotype in said cells;   wherein the absence of Alzheimer's Disease in said subject is indicated if an Alzheimer's Disease phenotype is induced by said contacting step.   
     
     
         2 ) The method of  claim 1 , wherein the presence of Alzheimer's Disease in said subject is indicated if incubation with said amyloid beta peptide results in no significant alteration or change in an Alzheimer's Disease phenotype in said cells. 
     
     
         3 ) The method of  claim 1 , wherein said amyloid beta peptide is Aβ (1-42). 
     
     
         4 ) The method of  claim 1  further comprising contacting said cells with a protein kinase C activator. 
     
     
         5 ) The method of  claim 4 , wherein said protein kinase C activator is selected from the group consisting of bradykinin, bryostatin, bombesin, cholecystokinin, thrombin, prostaglandin F2α and vasopressin. 
     
     
         6 ) The method of  claim 1 , wherein said cells are peripheral cells. 
     
     
         7 ) The method of  claim 6 , wherein said cells are selected from the group consisting of fibroblasts, saliva, blood, urine, skin cells, buccal mucosa cells and cerebro spinal fluid. 
     
     
         8 ) The method of  claim 1 , wherein said method comprises an in vitro assay. 
     
     
         9 ) The method of  claim 1 , wherein said Alzheimer's Disease phenotype is induced in said cells if the value of an Alzheimer's Disease-specific molecular biomarker is a positive value greater than zero. 
     
     
         10 ) A method for determining the absence of Alzheimer's Disease in a subject comprising:
 a) contacting cells from a subject with Aβ (1-42);   b) contacting said cells with bradykinin;   c) measuring the value of an Alzheimer's Disease-specific molecular biomarker;   wherein the absence of Alzheimer's Disease in said subject is indicated if the value of said Alzheimer's Disease-specific molecular biomarker is a positive value greater than zero.   
     
     
         11 ) The method of  claim 10 , wherein the presence of Alzheimer's Disease in said subject is indicated if incubation with said Aβ (1-42) results in no significant alteration or change in an Alzheimer's Disease phenotype in said cells. 
     
     
         12 ) The method of  claim 10 , wherein said cells are peripheral cells. 
     
     
         13 ) The method of  claim 10 , wherein said cells are selected from the group consisting of fibroblasts, saliva, blood, urine, skin cells, buccal mucosa cells and cerebro spinal fluid. 
     
     
         14 ) A method of identifying a lead compound useful for the treatment of Alzheimer's Disease comprising:
 a) contacting non-Alzheimer's cells with an amyloid beta peptide;   b) contacting said cells from step (a) with an agent that is a protein kinase C activator;   c) contacting said cells from step (b) with a test compound; and   d) determining the value of an Alzheimer's Disease-specific molecular biomarker to identify a lead compound useful for the treatment of Alzheimer's Disease.   
     
     
         15 ) The method of  claim 14 , wherein said amyloid beta peptide is Aβ (1-42). 
     
     
         16 ) The method of  claim 14 , wherein said non-Alzheimer's cells are selected from the group consisting of fibroblasts, saliva, blood, urine, skin cells, buccal mucosa cells and cerebro spinal fluid. 
     
     
         17 ) The method of  claim 14 , wherein said protein kinase C activator is selected from the group consisting of bradykinin, bryostatin, bombesin, cholecystokinin, thrombin, prostaglandin F2α and vasopressin. 
     
     
         18 ) The method of  claim 14 , wherein the value of said Alzheimer's Disease-specific molecular biomarker is indicative of a compound useful for the treatment of Alzheimer's Disease if said value is less than the value of said Alzheimer's Disease-specific molecular biomarker measured from control cells that have not been contacted with said test compound. 
     
     
         19 ) The method of  claim 18 , wherein said control cells have been contacted with an amyloid beta peptide. 
     
     
         20 ) The method of  claim 19 , wherein said amyloid beta peptide is Aβ (1-42). 
     
     
         21 ) The method of  claim 18  or  19  wherein said control cells have been contacted with an agent that is a protein kinase C activator. 
     
     
         22 ) The method of  claim 14 , wherein said contacting step (c) is performed after said contacting step (b). 
     
     
         23 ) The method of  claim 14 , further comprising the step of modifying said compound identified as being useful for the treatment of Alzheimer's Disease to optimize or improve its safety and efficacy compared to the safety and efficacy of the unmodified compound. 
     
     
         24 ) A modified compound identified by the method of  claim 23  useful for the treatment of Alzheimer's Disease. 
     
     
         25 ) A method of treating Alzheimer's Disease in a subject in need thereof, comprising administering a therapeutically effective amount of a compound of  claim 24  to said subject. 
     
     
         26 ) A method of identifying a compound useful for the treatment of Alzheimer's Disease comprising:
 a) contacting non-Alzheimer's control cells with Aβ (1-42);   b) contacting said cells from step (a) with bradykinin;   c) contacting said cells from step (b) with a test compound; and   d) determining the value of an Alzheimer's Disease-specific molecular biomarker to identify a compound useful for the treatment of Alzheimer's Disease.   
     
     
         27 ) A method of diagnosing Alzheimer's Disease in a subject comprising the steps of contacting cells obtained from said subject with an agent that is a protein kinase C activator and measuring the ratio of specific phosphorylated MAP kinase proteins in said cells to diagnose Alzheimer's Disease in said subject. 
     
     
         28 ) The method of  claim 27 , wherein said steps comprise an in vitro assay. 
     
     
         29 ) The method of  claim 27 , wherein said specific phosphorylated MAP kinase proteins are sequence variants of each other and belong to the same family of proteins. 
     
     
         30 ) The method of  claim 27 , wherein said ratio of specific phosphorylated MAP kinase proteins is the ratio of phosphorylated Erk1 to phosphorylated Erk2 and is calculated by dividing the normalized amount of phosphorylated Erk1 by the normalized amount of phosphorylated Erk2. 
     
     
         31 ) The method of  claim 27 , wherein said protein kinase C activator is selected from the group consisting of bradykinin, bryostatin, bombesin, cholecystokinin, thrombin, prostaglandin F2α and vasopressin. 
     
     
         32 ) The method of  claim 27 , wherein said cells are peripheral cells. 
     
     
         33 ) The method of  claim 32 , wherein said peripheral cells are selected from the group consisting of skin cells, skin fibroblast cells, blood cells and buccal mucosa cells. 
     
     
         34 ) The method of  claim 27 , wherein said cells are not isolated from cerebral spinal fluid. 
     
     
         35 ) The method of  claim 27 , wherein said cells do not comprise cerebral spinal fluid. 
     
     
         36 ) The method of  claim 27 , wherein said cells are not obtained by a spinal tap or lumbar puncture. 
     
     
         37 ) The method of  claim 27 , wherein said protein kinase C activator is contacted with said cells in media comprising serum. 
     
     
         38 ) The method of  claim 27 , wherein said protein kinase C activator is contacted with said cells in serum-free media. 
     
     
         39 ) The method of  claim 27 , wherein said phosphorylated MAP kinase proteins are detected by immunoassay. 
     
     
         40 ) The method of  claim 39 , wherein said immunoassay is a radioimmunoassay, a Western blot assay, an immunofluorescence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, an immunoelectrophoresis assay, a dot blot assay, or a slot blot assay. 
     
     
         41 ) The method of  claim 27 , wherein said measuring is done using a protein array, a peptide array, or a protein micro array. 
     
     
         42 ) The method of  claim 27 , wherein said method further comprises confirming a negative diagnosis of Alzheimer's Disease using the method of  claim 1  or  claim 10 . 
     
     
         43 ) The method of  claim 27 , wherein said method further comprises confirming a positive diagnosis of Alzheimer's Disease using the method of  claim 2  or  claim 11 . 
     
     
         44 ) A method of diagnosing Alzheimer's Disease in a subject comprising the steps of:
 a) contacting cells from said subject with an agent that is a protein kinase C activator;   b) measuring the ratio of a phosphorylated first MAP kinase protein to a phosphorylated second MAP kinase protein, wherein said phosphorylated first and second MAP kinase proteins are obtained from said cells after said contacting step;   c) measuring the ratio of phosphorylated first MAP kinase protein to phosphorylated second MAP kinase protein in cells from said subject that have not been contacted with the agent of step (a);   d) subtracting the ratio obtained in step (c) from the ratio obtained in step (b);   e) diagnosing the presence of Alzheimer's Disease in said subject based on the difference calculated in step (d).   
     
     
         45 ) The method of  claim 44 , wherein the difference obtained in step (d) is diagnostic of Alzheimer's Disease in said subject if the difference is a positive value. 
     
     
         46 ) The method of  claim 44 , wherein the difference obtained in step (d) indicates the absence of Alzheimer's Disease in said subject if the difference is a negative value or zero. 
     
     
         47 ) The method of  claim 44 , wherein said steps comprise an in vitro assay. 
     
     
         48 ) The method of  claim 44 , wherein said first and second MAP kinase proteins are sequence variants of each other. 
     
     
         49 ) The method of  claim 44 , wherein said first MAP kinase protein is Erk1 and said second MAP kinase protein is Erk2. 
     
     
         50 ) The method of  claim 44 , wherein said ratios are calculated by dividing the normalized amount of phosphorylated Erk1 by the normalized amount of phosphorylated Erk2. 
     
     
         51 ) The method of  claim 44 , wherein said protein kinase C activator is selected from the group consisting of bradykinin, bryostatin, bombesin, cholecystokinin, thrombin, prostaglandin F2α and vasopressin. 
     
     
         52 ) The method of  claim 44 , wherein said cells are peripheral cells. 
     
     
         53 ) The method of  claim 52 , wherein said peripheral cells are selected from the group consisting of skin cells, skin fibroblast cells, blood cells and buccal mucosa cells. 
     
     
         54 ) The method of  claim 44 , wherein said cells are not isolated from cerebral spinal fluid. 
     
     
         55 ) The method of  claim 44 , wherein said cells do not comprise cerebral spinal fluid. 
     
     
         56 ) The method of  claim 44 , wherein said cells are not obtained by a spinal tap or lumbar puncture. 
     
     
         57 ) The method of  claim 44 , wherein said protein kinase C activator is contacted with said cells in media comprising serum. 
     
     
         58 ) The method of  claim 44 , wherein said protein kinase C activator is contacted with said cells in serum-free media. 
     
     
         59 ) The method of  claim 44 , wherein said phosphorylated MAP kinase proteins are detected by immunoassay. 
     
     
         60 ) The method of  claim 59 , wherein said immunoassay is a radioimmunoassay, a Western blot assay, an immunofluorescence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, an immunoelectrophoresis assay, a dot blot assay, or a slot blot assay. 
     
     
         61 ) The method of  claim 44 , wherein said measuring is done using a protein array, a peptide array, or a protein micro array. 
     
     
         62 ) The method of  claim 44 , wherein said method further comprises confirming a negative diagnosis of Alzheimer's Disease using the method of  claim 1  or  claim 10 . 
     
     
         63 ) The method of  claim 44 , wherein said method further comprises confirming a positive diagnosis of Alzheimer's Disease using the method of  claim 2  or  claim 11 . 
     
     
         64 ) A kit for diagnosing the presence or absence of Alzheimer's Disease in a subject comprising:
 a) an agent that is a protein kinase C activator;   b) an antibody specific for a phosphorylated first MAP kinase protein; and   c) an antibody specific for a phosphorylated second MAP kinase protein.   
     
     
         65 ) The kit of  claim 64 , wherein said protein kinase C activator is selected from the group consisting of bradykinin, bryostatin, bombesin, cholecystokinin, thrombin, prostaglandin F2α and vasopressin. 
     
     
         66 ) The kit of  claim 64 , wherein said first MAP kinase protein and said second MAP kinase protein are sequence variants of each other. 
     
     
         67 ) The kit of  claim 64 , wherein said first MAP kinase protein is Erk1 and said second MAP kinase protein is Erk2. 
     
     
         68 ) The kit of  claim 64 , wherein said antibody specific for a phosphorylated first MAP kinase protein is an anti-phospho-Erk1 antibody. 
     
     
         69 ) The kit of  claim 64 , wherein said antibody specific for a phosphorylated second MAP kinase protein is an anti-phospho-Erk2 antibody. 
     
     
         70 ) The kit of  claim 64 , further comprising an amyloid beta peptide. 
     
     
         71 ) The kit of  claim 70 , wherein said amyloid beta peptide is AD (1-42). 
     
     
         72 ) A kit for diagnosing the presence or absence Alzheimer's Disease in a subject comprising:
 a) an amyloid beta peptide;   b) an antibody specific for a phosphorylated first MAP kinase protein; and   c) an antibody specific for a phosphorylated second MAP kinase protein.   
     
     
         73 ) The kit of  claim 72 , wherein said amyloid beta peptide is Aβ (1-42). 
     
     
         74 ) A method for screening a test compound useful for the treatment of Alzheimer's Disease comprising:
 a) contacting cells isolated from a subject diagnosed with Alzheimer's Disease with an agent that is a protein kinase C activator, wherein said contacting is in the presence of the test compound;   b) measuring the ratio of a phosphorylated first MAP kinase protein to a phosphorylated second MAP kinase protein, wherein said phosphorylated first and second MAP kinase proteins are obtained from said cells after said contacting in step (a);   c) measuring the ratio of phosphorylated first MAP kinase protein to phosphorylated second MAP kinase protein in cells from said subject that have not been contacted with the test compound of step (a);   d) subtracting the ratio obtained in step (c) from the ratio obtained in step (b);   e) identifying a test compound useful for the treatment of Alzheimer's Disease based on the difference calculated in step (d).   
     
     
         75 ) The method of  claim 74 , wherein the difference calculated in step (d) is indicative of an test compound useful for the treatment of Alzheimer's Disease if said difference is a negative value or zero. 
     
     
         76 ) The method of  claim 74 , wherein said steps comprise an in vitro assay. 
     
     
         77 ) The method of  claim 74 , wherein said first and second MAP kinase proteins are sequence variants of each other. 
     
     
         78 ) The method of  claim 74 , wherein said first MAP kinase protein is Erk1 and said second MAP kinase protein is Erk2. 
     
     
         79 ) The method of  claim 74 , wherein said ratios are calculated by dividing the normalized amount of phosphorylated Erk1 by the normalized amount of phosphorylated Erk2. 
     
     
         80 ) The method of  claim 74 , wherein said protein kinase C activator is selected from the group consisting of bradykinin, bryostatin, bombesin, cholecystokinin, thrombin, prostaglandin F2α and vasopressin. 
     
     
         81 ) The method of  claim 74 , wherein said cells are peripheral cells. 
     
     
         82 ) The method of  claim 74 , wherein said peripheral cells are selected from the group consisting of skin cells, skin fibroblast cells, blood cells and buccal mucosa cells. 
     
     
         83 ) The method of  claim 74 , wherein said cells are not isolated from cerebral spinal fluid. 
     
     
         84 ) The method of  claim 74 , wherein said cells do not comprise cerebral spinal fluid. 
     
     
         85 ) The method of  claim 74 , wherein said cells are not obtained by a spinal tap or lumbar puncture. 
     
     
         86 ) The method of  claim 74 , wherein said protein kinase C activator is contacted with said cells in media comprising serum. 
     
     
         87 ) The method of  claim 74 , wherein said protein kinase C activator is contacted with said cells in serum-free media. 
     
     
         88 ) The method of  claim 74 , wherein said phosphorylated MAP kinase proteins are detected by immunoassay. 
     
     
         89 ) The method of  claim 88 , wherein said immunoassay is a radioimmunoassay, a Western blot assay, an immunofluorescence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, an immunoelectrophoresis assay, a dot blot assay, or a slot blot assay. 
     
     
         90 ) The method of  claim 74 , wherein said measuring is done using a protein array, a peptide array, or a protein micro array. 
     
     
         91 ) A method of monitoring Alzheimer's Disease progression in a subject comprising measuring an Alzheimer's Disease-specific molecular biomarker. 
     
     
         92 ) The method of  claim 91  further comprising measuring said Alzheimer's Disease-specific molecular biomarker at more than one time point. 
     
     
         93 ) The method of  claim 91 , wherein a decrease in the numerical value of said Alzheimer's Disease-specific molecular biomarker is indicative of Alzheimer's Disease progression in said subject. 
     
     
         94 ) A composition useful for the treatment of Alzheimer's Disease comprising a compound identified by the method of  claim 74 . 
     
     
         95 ) A pharmaceutical composition for treating Alzheimer's Disease in a subject in need thereof comprising a therapeutically effective amount of a compound identified by the method of  claim 74 . 
     
     
         96 ) A method of treating Alzheimer's Disease comprising administering a therapeutically effective amount of a pharmaceutical composition of  claim 95  to a subject in need thereof.