Methods for diagnosing and treating Alzheimer's disease

Abstract

Methods for evaluating the risk of an individual to develop Alzheimer's disease using cultured neural crest-derived melanocytes are described. Also described are methods of therapy for Alzheimer's disease using peptides that bind to the neurotrophin receptor (p75 NTR ) and competitively inhibit the binding of β-amyloid to the p75 NTR .

1 - 12 . (canceled)  
     
     
         13 . A peptide that inhibits β-amyloid binding to neural-crest-derived cells or β-amyloid mediated apoptosis of neural-crest-derived cells, wherein the peptide comprises amino acid sequence lysine-glycine-lysine or lysine-glycine-alanine, wherein the peptide is identified by: 
 a) culturing neural crest-derived cells that express the p75 nerve growth factor receptor;  
 b) introducing into the cell culture of step a) β-amyloid protein, or peptide, in a concentration sufficient to bind to and activate the p75 nerve growth factor receptor, and the substance to be tested, thereby producing a test-culture;  
 c) maintaining the test-culture of step b) under conditions sufficient for the β-amyloid protein or peptide or test-substance to bind to the p75 nerve growth factor receptor expressed on the neural crest-derived cells;  
 d) determining β-amyloid activation of the p75 nerve growth factor receptor; and  
 e) comparing β-amyloid activation of the p75 nerve growth factor receptor of neural crest-derived cells cultured in step b) with β-amyloid activation of the p75 nerve growth factor receptor of neural crest-derived cells in a control culture comprising cells cultured in the absence of test-substance, wherein a decrease of β-amyloid activation of the p75 nerve growth factor receptor of neural crest-derived cells in the test culture compared to β-amyloid activation of the p75 nerve growth factor receptor of neural crest-derived cells in the control culture is indicative of a substance that inhibits β-amyloid binding to the p75 nerve growth factor receptor or indicative of neural crest-derived cells a substance that inhibits β-amyloid-mediated apoptosis in neural crest-derived cells.  
 
     
     
         14 - 23 . (Canceled)  
     
     
         24 . The peptide of  claim 13 , wherein the peptide is a cyclic peptide.  
     
     
         25 - 27 . (Canceled)  
     
     
         28 . A method of evaluating the risk of an individual to develop Alzheimer's disease associated with β-amyloid protein or β-amyloid peptide activation of the p75 nerve growth factor receptor, comprising the steps of: 
 a) culturing growing epidermal melanocytes expressing the p75 nerve growth factor receptor obtained from the individual;  
 b) introducing β-amyloid protein or β-amyloid peptide into the melanocyte culture of step a) in a concentration sufficient for the β-amyloid protein or peptide to activate the p75 nerve growth factor receptor;  
 c) determining whether the p75 nerve growth factor receptor is activated by β-amyloid protein or β-amyloid peptide; and  
 d) comparing the activation of the p75 nerve growth factor receptor of melanocytes cultured in step b) with the activation of the p75 nerve growth factor receptor of control melanocytes also cultured in the presence of β-amyloid protein or peptide, wherein activation of the p75 nerve growth factor receptor of the melanocytes of step b) greater than activation of p75 nerve growth factor receptor in the control melanocytes is an indication that the individual is at risk to develop Alzheimer's disease.  
 
     
     
         29 . A method according to  claim 28 , wherein determining whether the p75 nerve growth factor receptor is activated further comprises an assay method selected from the group consisting of: an assay to determine melanocyte cell yield, an assay to determine the inducement of Bax protein expression; an assay to determine the onset of melanocyte apoptosis and an assay to determine the presence of plaque-like structures in the melanocyte culture.  
     
     
         30 . A method according to  claim 29 , wherein the assay to determine the onset of melanocyte apoptosis is an assay selected from the group consisting of: measurement of propidium iodine incorporation into nuclear fragments, the TUNEL reaction and demonstration of fragmented DNA.  
     
     
         31 . A method according to  claim 28 , wherein the β-amyloid peptide is a peptide selected from the group consisting of: β-amyloid 1-40 peptide; β-amyloid 1-42 peptide; β-amyloid 25-36 peptide or β-amyloid 28-30 peptide.  
     
     
         32 . A method according to  claim 28 , wherein the β-amyloid protein is β-amyloid precursor protein.  
     
     
         33 . A method according  claim 28 , wherein the concentration of β-amyloid protein or β-amyloid peptide in step b) is from about 25 nM to about 100 μM.  
     
     
         34 . (Canceled)  
     
     
         35 . A method of evaluating the risk of an individual to develop Alzheimer's disease associated with β-amyloid protein or β-amyloid peptide activation of the p75 nerve growth factor receptor, comprising the steps of: 
 a) culturing epidermal melanocytes expressing the p75 nerve growth factor receptor obtained from the individual, thereby producing a test culture, and culturing a control cell line of epidermal melanocytes expressing the p75 nerve growth factor receptor, thereby producing a control culture, under conditions suitable for maintaining the melanocyte cultures;  
 b) determining the amount of β-amyloid protein or β-amyloid precursor protein produced by the test culture and control culture; and  
 c) comparing the amount of β-amyloid precursor protein or β-amyloid proteins produced, wherein production of the β-amyloid precursor protein, or protein in the test culture greater than production of β-amyloid precursor protein, or peptide on the control culture is an indication that the individual is at risk for Alzheimer's disease.  
 
     
     
         36 . A method according to  claim 35 , wherein the production of β-amyloid precursor protein or β-amyloid protein is determined by Northern blot analysis to measure the amount of β-amyloid precursor protein mRNA, or β-amyloid protein mRNA secreted by the melanocytes.  
     
     
         37 . A method according to  claim 35 , wherein the production of β-amyloid precursor protein or amount of β-amyloid protein secreted is determined by Western blot analysis using an antibody specific for the β-amyloid precursor protein or β-amyloid protein.  
     
     
         38 . A method according to  claim 35 , wherein after step a), the cultures are exposed to UV irradiation.  
     
     
         39 . A method of evaluating the risk of an individual to develop Alzheimer's disease associated with β-amyloid protein or β-amyloid peptide activation of the p75 nerve growth factor receptor, comprising the steps of: 
 a) culturing epidermal melanocytes obtained from the individual;  
 b) measuring the amount of p75 nerve growth factor and p140 tyrosine kinase A receptor expressed on the surface of the cultured melanocytes; and  
 c) determining the ratio of the amount of p75 nerve growth factor expressed relative to the amount of p140 tyrosine kinase A expressed.  
 
     
     
         40 . A method of treating Alzheimer's disease in an individual, comprising inhibiting the binding of β-amyloid protein or β-amyloid peptide to the p75 nerve growth factor receptor expressed on central nervous system neuronal cells, comprising administering to the individual a peptide comprising the amino acid sequence lysine-glycine-alanine, whereby the peptide binds to the p75 nerve growth factor receptor and inhibits the binding of β-amyloid protein or peptide to the p75 nerve growth factor receptor.  
     
     
         41 - 42 . (Canceled)  
     
     
         43 . The method of  claim 40  wherein the peptide is selected from the group consisting of: SEQ ID NO.: 1; SEQ ID NO.: 3 and SEQ ID NO.: 4.  
     
     
         44 . A method of treating Alzheimer's disease in an individual, comprising inhibiting expression of the p75 NTR  on central nervous system neuronal cells comprising contacting the cells with an anti-p75 NTR  anti-sense nucleic acid hybridizes to the p75 NTR  and inhibits expression of the receptor.  
     
     
         45 . A method of evaluating the risk of an individual to develop Alzheimer's disease associated with β-amyloid protein or β-amyloid peptide activation of the p75 nerve growth factor receptor, comprising the steps of: 
 a) obtaining a skin specimen containing melanocytes from the individual  
 b) quantifying the level of p75 NTR  in the specimen, wherein an increase in the level of p75 NTR  as compared to an age-matched control specimen is an indication that the individual is at risk for Alzheimer's disease.  
 
     
     
         46 . The method of  claim 45 , wherein the specimen is contacted with a labeled nucleic acid probe comprising a nucleic acid sequence encoding the p75 NTR  under conditions suitable for hybridization of the probe with p75 NTR  present in the melanocytes the specimen, such that the level of hybridized probe is indicative of the level of p75 NTR .  
     
     
         47 . The method of  claim 45 , wherein the specimen is contacted with nucleic acid primers comprising a nucleic acid sequence encoding the p75 NTR  under conditions suitable for the amplification the p75 NTR  present in the melanocytes such that the level of amplified p75 NTR  is indicative of the level of p75 NTR  in the specimen.