Alzheimer's disease diagnosis based on mitogen-activated protein kinase phosphorylation

Abstract

A method of diagnosing Alzheimer's disease in a patient comprises determining whether the phosphorylation level of an indicator protein in cells of the patient after stimulus with an activator compound is abnormally elevated as compared to a basal phosphorylation level, the indicator protein being e.g. Erk1/2 and the activator compound being e.g. bradykinin.

What is claimed is: 
     
         1 . A method for screening compounds to identify a compound useful for treatment or prevention of Alzheimer's disease, the method comprising:
 (a) contacting test cells from an Alzheimer's disease subject with at least one compound for screening;   (b) stimulating the test cells with at least one agent that triggers intracellular calcium release via the inositol 1,4,5-trisphosphate (IP-3) receptor, wherein step (b) is performed before, during, or after step (a);   (c) measuring the amount of phosphorylation of at least one mitogen activated protein kinase (MAPK) protein in the test cells at one or more time points after stimulating the test cells; and   (d) comparing the amount of phosphorylation of the at least one MAPK protein in the test cells with the amount of phosphorylation in control cells from an Alzheimer's disease subject that are not contacted with the at least one compound at the same one or more time points;   wherein the at least one compound is accepted if it inhibits or prevents the increased phosphorylation.   
     
     
         2 . The method of  claim 1 , wherein the at least one agent is chosen from bradykinin and a bradykinin receptor agonist. 
     
     
         3 . The method of  claim 1 , wherein the at least one MAPK protein is Erk1/2. 
     
     
         4 . The method of  claim 1 , wherein step (c) comprises measuring the amount of phosphorylation at a single time point after step (a). 
     
     
         5 . The method of  claim 1 , wherein the cells are skin fibroblasts. 
     
     
         6 . The method of  claim 1 , wherein step (c) comprises detecting phosphorylation in a lysate of the test cells. 
     
     
         7 . A method. of screening compounds for use as activator compounds in a stimulus-response assay, the method comprising:
 measuring the effect of at least one test compound on phosphorylation of at least one indicator protein in cells of an Alzheimer's disease subject and cells of a non-Alzheimer's disease control; and   selecting a compound that increases the amount or duration, or both the amount and duration of phosphorylation of the at least one indicator protein in the Alzheimer's disease cells as compared to the non-Alzheimer's disease control cells.   
     
     
         8 . A diagnostic test kit for Alzheimer's disease comprising anti-phospho-mitogen activated protein kinase (MAPK) protein antibody and bradykinin. 
     
     
         9 . A method for selecting medication for treating or preventing Alzheimer's disease in a patient, the method comprising:
 selecting a possible therapeutic compound for treating Alzheimer's disease,   administering the possible therapeutic compound to the patient,   contacting cells obtained from the patient with at least one compound that stimulates phosphorylation of at least one indicator protein in the cells; and   detecting the presence or absence of an abnormally elevated level of the phosphorylated at least one indicator protein   wherein the presence of an abnormally elevated level indicates that the possible therapeutic compound is not therapeutic for the patient, and the absence of an abnormally elevated level indicates that the possible therapeutic compound is therapeutic for the patient.   
     
     
         10 . The method of  claim 9 , further comprising treating or preventing Alzheimer's disease in the subject by administering to the subject the compound shown to be therapeutic for the patient. 
     
     
         11 . A method of treating or preventing Alzheimer's disease in a subject, the method comprising administering an effective amount of a medicament, wherein the medicament
 (a) inhibits or prevents abnormally elevated phosphorylation of at least one MAPK protein in cells of the subject as compared to non-Alzheimer's disease control cells; and/or   (b) inhibits at least one event caused by abnormally elevated phosphorylation of the at least one MAPK protein.   
     
     
         12 . The method of  claim 11 , wherein the medicament inhibits Erk1/2 phosphorylation. 
     
     
         13 . The method of  claim 11 , wherein the medicament comprises at least one inhibitor chosen from an inhibitor of protein kinase C activity, an inhibitor of PI-3 kinase activity, an inhibitor of c-src protein tyrosine kinase activity, and an inhibitor of the IP-3 receptor. 
     
     
         14 . A method for screening compounds to identify a compound useful for treatment or prevention of Alzheimer's disease, the method comprising:
 (a.) obtaining test cells from skin fibroblast cells of an Alzheimer's disease subject, and contacting the test cells with a compound for screening;   (b) obtaining control cells from skin fibroblast cells of the subject, and contacting the control cells with a control agent for the compound, or incubating the control cells in the absence or either the compound or the control agent;   (c) stimulating the test cells and the control cells with an amount of bradykinin effective for stimulating phosphorylation, wherein step (c) is performed before, during, or after steps (a) and (b);   (d) measuring, the amount of phosphorylated Erk1/2 in the test cells and in the control cells at one or more time points chosen from about 2 minutes, about 5 minutes, about 10 minutes, about 20 minutes, and about 30 minutes after contacting the cells with bradykinin by Western blotting using an antibody specific for phospho-Erk1/2, wherein the amount of phosphorylated Erk1/2 in the test cells and the control cells is normalized, respectively, to the total amount of protein present in the test cells and the control cells;   (e) comparing the amount of phosphorylated Erk1/2 in the test cells with the amount of phosphorylated Erk1/2 in the control cells to determine whether the compound inhibits and/or prevents bradykinin-induced increase in phosphorylation Erk1/2 in the test cells compared to the control cells;   wherein the compound is identified as useful for the treatment or prevention of Alzheimer's disease if the compound inhibits and/or prevents the increased phosphorylation.   
     
     
         15 . A method of reducing at least one of proteolysis of amyloid precursor protein, secretion of amyloid protein β, and phosphorylation of tau protein in a human cell, wherein the cell shows increased IP-3 receptor mediated phosphorylation of MAPK protein compared to a control human cell, the method comprising contacting the cell with at least one inhibitor of phosphorylation of MAPK in an amount effective to reduce the phosphorylation to the level in the control cell.