Methods of diagnosing alzheimer's disease

Abstract

Methods and compositions relating to Alzheimer's disease are provided. Specifically, proteins that are differentially expressed in the Alzheimer's disease state relative to their expression in the normal state are provided. Proteins associated with Alzheimer's disease are identified and described. Methods of diagnosis of Alzheimer's disease using the differentially expressed proteins are also provided, as are methods for the identification and therapeutic use of compounds for the prevention and treatment of Alzheimer's disease.

1 . A method of diagnosing Alzheimer's disease in a subject, the method comprising detecting at least one differentially expressed protein in a tissue sample or body fluid sample from said subject. 
     
     
         2 . A method according to  claim 1 , wherein the nature or degree of the Alzheimer's disease is determined. 
     
     
         3 . A method according to  claim 2 , wherein the progression of the Alzheimer's disease over time is determined. 
     
     
         4 . A method of determining the nature or degree of Alzheimer's disease in a human or animal subject, the method comprising:
 (a) establishing a paradigm in which at least one protein is differentially expressed in relevant tissue or body fluid sample from, or representative of, subjects having differential levels of Alzheimer's disease;   (b) obtaining a sample of the tissue or body fluid from the subject;   (c) determining the presence, absence or degree of expression of the differentially expressed protein or proteins in the sample; and   (d) relating the determination to the nature or degree of the Alzheimer's disease by reference to a previous correlation between such a determination and clinical information.   
     
     
         5 . The method of  claim 4 , wherein the severity of the Alzheimer's disease is determined. 
     
     
         6 . The method of  claim 4  wherein the duration of the Alzheimer's disease is determined. 
     
     
         7 . A method according to  claim 4 , wherein the tissue or body fluid sample is a urine, blood, plasma, serum, saliva or cerebro-spinal fluid sample. 
     
     
         8 . A method according to  claim 4  wherein an increase in the expression of said protein is detected compared to that of a control subject. 
     
     
         9 . A method according to  claim 4  wherein a decrease in the expression of said protein is detected compared to that of a control subject. 
     
     
         10 . A method according to  claim 1  wherein the differentially expressed protein is detected using an antibody specific to said protein, by detecting in the sample an autoantibody specific to said protein, or by mass spectrometry. 
     
     
         11 . A method according to  claim 1  wherein the differentially expressed protein is detected using 2D gel electrophoresis. 
     
     
         12 . A method according to  claim 10  wherein the sample is immobilised on a solid support. 
     
     
         13 . A method according to  claim 1  which comprises detecting more than one differentially expressed protein. 
     
     
         14 . A method according to  claim 13  which comprises detecting four or more differentially expressed proteins. 
     
     
         15 . A method according to  claim 13 , whereby a pattern of said differentially expressed proteins in a tissue sample or body fluid sample of an individual with Alzheimer's disease is used to predict the most appropriate and effective therapy to alleviate the Alzheimer's disease and to monitor the success of that treatment. 
     
     
         16 . A method according to  claim 1  wherein said at least one differentially expressed protein is a protein shown in  FIG. 6 ,  FIG. 7 ,  FIG. 8  or  FIG. 12 . 
     
     
         17 . A method according to  claim 5  wherein at least one of said differentially expressed proteins is Ig lambda chain C region with accession no P01834 found in Spot 177 as shown in  FIG. 6 . 
     
     
         18 . A method according to  claim 6  wherein at least one of said differentially expressed proteins is the serum albumin precursor isoform found in Spot 165 as shown in  FIG. 6 . 
     
     
         19 . A method according to  claim 16  wherein said at least one differentially expressed proteins is one of the following proteins shown in  FIG. 6 ,  FIG. 7 ,  FIG. 8  or  FIG. 12  or a fragment thereof: apolipoprotein A-IV precursor, apolipoprotein C-III precursor, transthyretin, galectin 7, complement C4 precursor, complement factor H, S100 calcium binding protein or ceruloplasmin, histone 2B, Ig lambda chain C region, fibrinogen gamma chain precursor, inter-alpha-trypsin heavy chain H4 precursor, complement C3 precursor, clusterin precursor, gamma or beta actin, haptoglobin precursor or the serum albumin precursor isoform found in spot ID no 2, 14, 15, 123, 165, 176 or 184 of  FIG. 6 . 
     
     
         20 . A method according to  claim 19 , wherein said fragment comprises; residues 270-309 of apolipoprotein A-IV; resides 680-1446-1744 of complement C4; or wherein said fragment is an N-terminal fragment of apolipoprotein A-IV which migrates as a 28 kD fragment in SDS-PAGE. 
     
     
         21 . A method according to  claim 19  wherein said at least one differentially expressed proteins is one of the following proteins shown in  FIG. 6 ,  FIG. 7  or  FIG. 12  or a fragment thereof: alpha-2-macroglobulin, Ig alpha-1 chain C, apolipoprotein A-IV, complement factor H or serum albumin precursor found in Spot 2 of  FIG. 6   
     
     
         22 . The method of  claim 1  which further comprises determining an effective therapy for treating the Alzheimer's disease. 
     
     
         23 . A method of treatment by the use of an agent that will restore the expression of one or more differentially expressed proteins in the Alzheimer's disease state to that found in the normal state in order to prevent the development or progression of Alzheimer's disease. 
     
     
         24 . A method of screening an agent to determine its usefulness in treating Alzheimer's disease, the method comprising:
 (a) obtaining a sample of relevant tissue or body fluid taken from, or representative of, a subject having Alzheimer's disease symptoms, who or which has been treated with the agent being screened;   (b) determining the presence, absence or degree of expression of a differentially expressed protein or proteins in the tissue from, or representative of, the treated subject; and,   (c) selecting or rejecting the agent according to the extent to which it changes the expression, activity or amount of the differentially expressed protein or proteins in the treated subject having Alzheimer's disease symptoms.   
     
     
         25 . A method according to  claim 24 , which method further comprises, prior to step (a), the step of establishing a paradigm in which at least one protein is differentially expressed in relevant tissue or body fluid from, or representative of, subjects having Alzheimer's disease symptoms and normal subjects. 
     
     
         26 . The method of  claim 24 , wherein the agent is selected if it converts the expression of the differentially expressed protein or proteins towards that of a normal subject. 
     
     
         27 . The method of  claim 24 , wherein the agent is selected if it converts the expression of the protein or proteins to that of the normal subject. 
     
     
         28 . A method of screening an agent to determine its usefulness in treating Alzheimer's disease, the method comprising:
 (a) obtaining over time samples of relevant tissue or body fluid taken from, or representative of, a subject having Alzheimer's disease symptoms, who or which has been treated with the agent being screened;   (b) determining the presence, absence or degree of expression of a differentially expressed protein or proteins in said samples; and,   (c) determining whether the agent affects the change over time in the expression of the differentially expression protein in the treated subject having Alzheimer's disease symptoms.   
     
     
         29 . A method according to  claim 28 , which method further comprises, prior to step (a), the step of
 establishing a paradigm in which at least one protein is differentially expressed in relevant tissue or body fluid from, or representative of, subjects having Alzheimer's disease symptoms and normal subjects; and   establishing that expression of said differentially expressed protein diverges over time in subjects having Alzheimer's disease symptoms and normal subjects.   
     
     
         30 . The method of  claim 25 , wherein the subjects having differential levels of protein expression comprise:
 (a) normal subjects and subjects having Alzheimer's disease symptoms; and,   (b) subjects having Alzheimer's disease which have not been treated with the agent and subjects Alzheimer's disease symptoms which have been treated with the agent.   
     
     
         31 . The method of  claim 30 , wherein the differential levels of protein expression are not observed in normal subjects who have and have not been treated with the agent. 
     
     
         32 . The method of  claim 25 , wherein the subjects having Alzheimer's disease symptoms are human subjects with Alzheimer's disease. 
     
     
         33 . The method of  claim 25 , wherein the subjects having Alzheimer's disease symptoms are mutant amyloid precursor protein (APP) transgenic mice, presenilin-1 (PS-1) transgenic mice, double transgenic APP/PS-1 transgenic mice and/or glycogen synthase kinase transgenic mice, and the normal subjects are wild-type mice. 
     
     
         34 . The method of  claim 33 , wherein the tissue or body fluid samples are brain tissue samples. 
     
     
         35 . The method of  claim 33 , wherein the tissue or body fluid samples are urine, blood, plasma, serum, saliva or cerebro-spinal fluid samples. 
     
     
         36 . The method of  claim 4 , wherein the paradigm is established using two-dimensional gel electrophoresis carried out on the relevant tissue or a protein-containing extract thereof. 
     
     
         37 . The method of  claim 4 , wherein the paradigm is established using SELDI analysis of the relevant tissue or a protein-containing extract thereof. 
     
     
         38 . The method of  claim 24 , wherein the differentially expressed protein or proteins comprise at least one of the proteins shown in  FIG. 6 ,  FIG. 7 ,  FIG. 8  and  FIG. 12 , or a rodent equivalent thereof. 
     
     
         39 . A method according to  claim 38  wherein at least one of said differentially expressed proteins is one of the following proteins shown in  FIG. 6 ,  FIG. 7 ,  FIG. 8  or  FIG. 12  or a fragment thereof: apolipoprotein A-IV precursor, apolipoprotein C-III precursor, transthyretin, galectin 7, complement C4 precursor, histone 2B, Ig lambda chain C region, fibrinogen gamma chain precursor, complement factor H, inter-alpha-trypsin heavy chain H4 precursor, complement C3 precursor, clusterin precursor, gamma or beta actin, haptoglobin precursor or the serum albumin precursor isoform found in spot ID no 2, 14, 15, 123, 165, 176 or 184 of  FIG. 6 , or a rodent equivalent thereof. 
     
     
         40 . A method according to  claim 40 , wherein said fragment comprises amino acid residues 270-309 of apolipoprotein A-IV; or residues 1446-1744 of complement C4; or a rodent equivalent thereof. 
     
     
         41 . A method according to  claim 39  wherein at least one of said differentially expressed proteins is one of the following proteins shown in  FIG. 6 ,  FIG. 7  or  FIG. 12 , or a fragment thereof: alpha-2-macroglobulin, Ig alpha-1 chain C apolipoprotein A-IV, complement factor H or serum albumin precursor found in Spot 2 of  FIG. 6 ; or a rodent equivalent thereof. 
     
     
         42 . A method of making a pharmaceutical composition which comprises having identified an agent using the method of  claim 24 , the further step of manufacturing the agent and formulating it with an acceptable carrier to provide the pharmaceutical composition. 
     
     
         43 . A method of identifying a protein which is differentially expressed in relevant tissue or body fluid sample from subjects with Alzheimer's disease and normal subjects, comprising:
 i) immobilising a tissue sample or body fluid sample or protein-containing extract thereof on a solid support   ii) analysing the immobilised proteins by surface enhanced laser desorption time of flight mass spectroscopy   iii) comparing the spectra obtained to detect differences in protein expression between Alzheimer's subjects and normal subjects.   
     
     
         44 . The method of  claim 43 , wherein the tissue or body fluid samples are blood, serum or cerebro-spinal fluid samples. 
     
     
         45 . The method of  claim 43 , further comprising the step of isolating a differentially expressed protein identified in the method. 
     
     
         46 . The method of  claim 45 , further comprising the step of characterising the isolated protein. 
     
     
         47 . The method of  claim 28 , wherein the agent is selected if it prevents or slows the change over time in the expression of the differentially expressed protein.