Preparations of gamma-secretase activity

Abstract

The present invention provides cell-free γ-secretase activity. The method of the invention utilizes a membrane source of APP/γ-secretase mixture in the assay to determine factors that may enhance or decrease enzymatic activity affecting β-amyloid peptide production. The cell membranes used in the assay may be from cells expressing an endogenous APP or, preferably, cells expressing a recombinant APP. The APP may be full-length or a fragment capable of being proteolytically cleaved by γ-secretase. In addition, the APP expressed in the cells may have one or more mutation, such as a point mutation, small deletion, etc.

What is claimed is:  
     
         1 . An isolated preparation of γ-secretase activity comprising a APP/γ-secretase mixture, wherein said activity is characterized by the ability to produce Aβ in vitro.  
     
     
         2 . The preparation of  claim 1 , wherein said preparation comprises a solubilized APP/γ-secretase mixture.  
     
     
         3 . The preparation of  claim 1 , wherein the preparation comprises membranes having an associated APP/γ-secretase mixture.  
     
     
         4 . The preparation of  claim 1 , wherein the preparation comprises isolated γ-secretase activity reconstituted with APP or an APP proteolytic product.  
     
     
         5 . The preparation of  claim 1 , wherein the APP/γ-secretase mixture comprises CT99.  
     
     
         6 . The preparation of  claim 1 , wherein the APP/γ-secretase mixture comprises an APP having a mutation which increases γ-secretase activity.  
     
     
         7 . The preparation of  claim 6 , wherein the APP mutation is the “Swedish”, mutation of APP (K595N/M596L).  
     
     
         8 . A method of identifying a compound characterized by an ability to alter γ-secretase activity, said method comprising the steps of: 
 contacting an APP/γ-secretase mixture with a candidate compound; and  
 determining levels of APP/γ-secretase proteolytic products;  
 wherein levels of APP proteolytic products are indicative of the effect of the compound on in vivo secretion activity.  
 
     
     
         9 . The method of  claim 8 , wherein the levels of APP proteolytic products are compared to a predetermined standard level.  
     
     
         10 . The method of  claim 8 , wherein background levels of γ-secreatse/APP proteolytic products have been removed prior to contact with the candidate compound.  
     
     
         11 . The method of  claim 8 , wherein the levels of APP proteolytic products are determined using antibodies that recognize a protein selected from the group consisting of: Aβ, γCTF, and p3.  
     
     
         12 . A method of identifying a compound characterized by the ability to alter γ-secretase activity, said method comprising the steps of: 
 providing an APP/γ-secretase mixture;  
 removing background levels of APP γ-secretase proteolytic products from said mixture;  
 contacting the mixture with a compound; and  
 determining levels of APP γ-secretase proteolytic products;  
 wherein the level of APP proteolytic products is indicative of the effect of the compound on γ-secretase activity.  
 
     
     
         13 . The method of  claim 12 , wherein the APP/γ-secretase mixture is provided by isolating a membrane preparation from cells expressing APP or an APP proteolytic  
     
     
         14 . The method of  claim 12 , wherein the APP/γ-secretase mixture is solubilized.  
     
     
         15 . The method of  claim 12 , wherein the APP/γ-secretase mixture is provided by reconstituting γ-secretase activity with APP or an APP proteolytic product.  
     
     
         16 . The method of  claim 12  wherein the effect of the compound is determined by comparing the effect with control APP/γ-secretase mixture not in contact in the compound.  
     
     
         17 . The method of  claim 12 , further comprising contacting the mixture with exogenous APP or APP proteolytic product.  
     
     
         18 . The method of  claim 12 , further comprising: 
 treating the membrane preparation with a compound to alter γ-secretase activity.    
     
     
         19 . The method of  claim 18 , wherein the compound increases γ-secretase activity, and wherein the compound is a phospholipid.  
     
     
         20 . The method of  claim 19 , wherein the phospholipid is selected form the group consisting of cardiolipin, L-alpha-phosphatidylserine, L-alpha-phosphotidylcholine, and 1,2 dipalmitoyl-5-glycerol-3-phosphocholine.  
     
     
         21 . The method of  claim 20 , wherein the membranes are isolated from cells having a mutation in a gene involved in APP/γ-secretase processing.  
     
     
         22 . The method of  claim 21 , wherein the cells have a mutation in APP.  
     
     
         23 . The method of  claim 21 , wherein the cells have a mutation in a presenilin gene.  
     
     
         24 . A method for reducing β-amyloid plaque formation in a subject, comprising administering a compound isolated according to the method of  claim 8  in an amount sufficient to alter in vivo γ-secretase activity; 
 wherein the altered γ-secretase activity results in reduced APP/γ-secretase processing and reduced release of β-amyloid peptide in brain tissue.  
 
     
     
         25 . The method of  claim 24 , wherein the compound is an inhibitor of γ-secretase activity, and 
 wherein the reduced γ-secretase activity results in decreased levels of C-terminal proteolysis of APP; and  
 wherein the γ-secretase activity is between 30 and 80% below normal, and the β-amyloid release levels are 20-80% of normal.  
 
     
     
         26 . The method of  claim 24 , wherein the patient is diagnosed with Alzheimer's disease.  
     
     
         27 . The method of  claim 24 , wherein the patient is at risk for Alzheimer's disease, and wherein the compound is administered to prevent β-amyloid plaque formation.  
     
     
         28 . A method of inhibiting the formation of β-amyloid plaque, comprising: 
 contacting cells subject to β-amyloid plaque formation with the isolated preparation of  claim 1 ,  
 allowing the cells to remain in contact with the isolated preparation for a period of time and under conditions such that β-amyloid plaque formation is inhibited.