Alzheimer's disease (AD) is characterized by the excessive generation and accumulation of p-amyloidpeptides (Ap). y-secretase, the enzyme responsible for immediate release of Ap, is one of the most importantdrug targets in AD therapies. Most current y-secretase inhibitors lack discrimination between y-cleavage ofAPP and other substrates including Notch, and show severe toxicity after chronic administration in animals.We have shown that y-secretase generation of Ap in an N2a cell-free system is ATP dependent. In addition,Gleevec, an Abl kinase inhibitor, which acts by competing at the ATP-binding site of this tyrosine kinase,potently reduces Ap production. Gleevec also reduces Ap production in rat primary neuronal cultures and invivo in guinea pig brains. However, Gleevec does not inhibit y-secretase-catalyzed cleavage of Notch-1. Ourrecent results suggest that ATP analogues and Gleevec enhance the binding of holo-APP and APP-CTF topresenilin-1. Furthermore, Gleevec retards egress of APP- but not Notch-containing vesicles from the ER ina PS1-dependent manner. More importantly, we have identified a novel Gleevec binding protein (GBP). Wehypothesize that Gleevec achieves its actions on trafficking and cleavage of APP through this novel GBP,and propose specific experiments to test this hypothesis. One goal is to use Gleevec and other ATPanalogues as tools to investigate the molecular steps involved in Ap formation. This should prove ofenormous value in Alzheimer's disease research. To achieve this goal, in our revised application, we willcharacterize the effects of ATP analogues and Gleevec on kinetics, substrate binding, and conformationalchanges of y-secretase (Aim I); we will characterize the selective effects of Gleevec on trafficking of APP vsNotch (Aim II); we will examine the action of a newly discovered Gleevec binding protein (GBP) on themodulation of y-secretase activity and APP trafficking (Aim III); we will analyze the effects of Gleevec on ADrelatedpathology, electrophysiology and behavior, using AD transgenic mice (Aim IV); we will characterizethe mechanism by which inhibitor 2, another Abl kinase inhibitor, which also acts by competing at the ATPbindingsite of this tyrosine kinase, modulates y-secretase activity (Aim V). Taken together, these studiesshould elucidate the underlying mechanism by which Gleevec regulates y-secretase activity. Our studyshould accelerate the development of novel therapeutic strategies against Alzheimer's disease.
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