Methods of identifying agents that affect cleavage of amyloid-beta precursor protein

Abstract

The present invention provides methods of identifying agents that affect the cleavage of amyloid-β precursor protein (APP) and related vectors, cells and kits, as well as agents identified by the method.

We claim:  
     
         1 . A method of identifying an agent that affects the cleavage of amyloid-β precursor protein (APP) comprising providing a cell containing APP modified in the C-terminal cytoplasmic tail to allow detection of nuclear localization of said cytoplasmic tail; contacting said cell with a candidate agent; and measuring nuclear localization of said C-terminal cytoplasmic tail, wherein an increase or decrease in nuclear localization in the presence of said candidate agent relative to nuclear localization in the absence of said candidate agent is indicative of an agent that affects the cleavage of APP.  
     
     
         2 . The method of  claim 1  wherein APP is modified to comprise a DNA-binding domain of a transcription factor and a transcriptional activator of the same or a different transcription factor, and wherein said cell further contains an indicator gene operably linked to a nucleic acid comprising a binding site for said DNA-binding domain.  
     
     
         3 . The method of  claim 2  wherein said DNA-binding domain is Gal4 or LexA.  
     
     
         4 . The method of  claim 2  wherein said transcriptional activator is VP16.  
     
     
         5 . The method of  claim 1  wherein said APP is modified to comprise a DNA-binding domain of a transcription factor and wherein said cell further comprises an indicator gene operably linked to a nucleic acid comprising a binding site for said DNA-binding domain, and wherein Fe65 is provided to said cell.  
     
     
         6 . The method of  claim 5  wherein said DNA-binding domain is Gal4 or LexA.  
     
     
         7 . The method of  claim 2  wherein nuclear localization is measured by measuring expression of said indicator gene.  
     
     
         8 . The method of  claim 5  wherein nuclear localization is measured by measuring expression of said indicator gene.  
     
     
         9 . The method of  claim 1  wherein said cell is a eukaryotic cell.  
     
     
         10 . The method of  claim 1  wherein said cell is a mammalian cell.  
     
     
         11 . The method of  claim 1  wherein said cell is a human cell.  
     
     
         12 . A method of identifying an agent that affects the cleavage of amyloid-β precursor protein (APP) comprising co-transfecting a cell with (a) a nucleic acid encoding a modified APP, wherein said modified APP comprises Gal4 and VP16 in the C-terminal cytoplasmic tail, and (b) a nucleic acid encoding an indicator gene under the control of one or more copies of a Gal4 regulatory element; contacting said cell with a candidate agent; and measuring expression of said indicator gene, wherein an increase or decrease in expression in the presence of said candidate agent relative to the absence of said agent is indicative of an agent that affects cleavage of APP.  
     
     
         13 . A method of identifying an agent that affects the cleavage of amyloid-β precursor protein (APP) comprising providing a cell containing APP and Tip60 modified to allow detection of nuclear localization of a C-terminal cytoplasmic cleavage product of APP; contacting said cell with a candidate agent; and measuring nuclear localization of said C-terminal cytoplasmic cleavage product, wherein an increase or decrease in nuclear localization in the presence of said agent relative to nuclear localization in the absence of said agent is indicative of an agent that affects the cleavage of APP.  
     
     
         14 . The method of  claim 13  wherein said Tip60 is modified by fusion with the DNA binding domain of a transcriptional activator, and wherein said cell further contains Fe65.  
     
     
         15 . The method of  claim 14  wherein said DNA binding domain is Gal4 or LexA.  
     
     
         16 . The method of  claim 14  wherein said cell further contains an indicator gene operably linked to a nucleic acid comprising a binding site for said DNA binding domain.  
     
     
         17 . The method of  claim 16  wherein said nuclear localization is measured by measuring expression of said indicator gene.  
     
     
         18 . The method of  claim 13  wherein said cell is a eukaryotic cell.  
     
     
         19 . The method of  claim 13  wherein said cell is a mammalian cell.  
     
     
         20 . The method of  claim 13  wherein said cell is a human cell.  
     
     
         21 . A method of identifying an agent that affects the cleavage of amyloid-β precursor protein (APP) comprising co-transfecting a cell with (a) a nucleic acid encoding APP, (b) a nucleic acid encoding Fe65, (c) a nucleic acid encoding a fusion protein of Tip60 and Gal4, and (d) a nucleic acid encoding an indicator gene under the control of one or more copies of a Gal4 regulatory element; contacting said cell with a candidate agent; and measuring expression of said indicator gene, wherein an increase or decrease in expression in the presence of said candidate agent relative to the absence of said agent is indicative of an agent that affects the cleavage of APP.  
     
     
         22 . A vector comprising a first nucleic acid encoding amyloid-β precursor protein (APP) operably linked to a promoter, wherein a second nucleic acid encoding a heterologous DNA-binding domain of a transcription factor is contained within the region of the first nucleic acid that encodes the C-terminal cytoplasmic tail of APP.  
     
     
         23 . A vector-comprising a nucleic acid encoding amyloid-β precursor protein (APP) operably linked to a promoter wherein a nucleic acid module encoding a heterologous DNA-binding domain of a transcription factor and a transcriptional activator of the same or a different transcription factor is contained within the region of the nucleic acid that encodes the C-terminal cytoplasmic tail of APP.  
     
     
         24 . The vector of  claim 28  wherein the DNA-binding domain is Gal4 or LexA.  
     
     
         25 . The vector of  claim 23  wherein the DNA-binding domain is Gal4 or LexA and the transcriptional activator is VP16.  
     
     
         26 . A vector comprising a nucleic acid encoding Tip60 and a heterologous DNA binding domain of a transcription factor.  
     
     
         27 . The vector of  claim 26  wherein said DNA binding domain is Gal4.  
     
     
         28 . A cell comprising the vector of  claim 22 .  
     
     
         29 . A cell comprising the vector of  claim 23 .  
     
     
         30 . A cell comprising the vector of  claim 24 .  
     
     
         31 . An agent that affects the cleavage of amyloid-β precursor protein (APP) identified by a method comprising providing a cell containing APP modified in the C-terminal cytoplasmic tail to allow detection of nuclear localization of said cytoplasmic tail; contacting said cell with a candidate agent; and measuring nuclear localization of said C-terminal cytoplasmic tail, wherein an increase or decrease in nuclear localization in the presence of said candidate agent relative to nuclear localization in the absence of said candidate agent is indicative of an agent that affects the cleavage of APP.  
     
     
         32 . An agent that affects the cleavage of amyloid-β precursor protein (APP) identified by a method comprising providing a cell containing APP and a protein that interacts with APP to activate transcription, wherein the protein is modified to allow detection of nuclear localization of a C-terminal cytoplasmic cleavage product of APP; contacting said cell with a candidate agent; and measuring nuclear localization of said C-terminal cytoplasmic cleavage product, wherein an increase or decrease in nuclear localization in the presence of said agent relative to nuclear localization in the absence of said agent is indicative of an agent that affects the cleavage of APP.  
     
     
         33 . A composition comprising the agent of  claim 31 .  
     
     
         34 . A composition comprising the agent of  claim 32 .  
     
     
         35 . A kit comprising a first compartment containing cells comprising a vector wherein said vector comprises a nucleic acid encoding amyloid-β precursor protein (APP) operably linked to a promoter wherein a nucleic acid module encoding a heterologous DNA-binding domain of a transcription factor and a transcriptional activator of the same or a different transcription factor is contained within the region of the nucleic acid that encodes the C-terminal cytoplasmic tail of APP.  
     
     
         36 . The kit of  claim 35  wherein said cells further contain a nucleic acid encoding an indicator gene under the control of a regulatory element for said DNA-binding domain.  
     
     
         37 . A kit comprising a first compartment containing a vector comprising a nucleic acid encoding amyloid-β precursor protein (APP) operably linked to a promoter wherein a nucleic acid module encoding a heterologous DNA-binding domain of a transcription factor and a transcriptional activator of the same or a different transcription factor is contained within the region of the nucleic acid that encodes the C-terminal cytoplasmic tail of APP.  
     
     
         38 . The kit of  claim 37  further comprising a second compartment containing a reporter plasmid comprising a nucleic acid encoding an indicator gene under the control of a regulatory element for said DNA-binding domain.