The objectives of the proposed research are 1) to understand the manner in which Alzheimer's amyloid precursor proteins (APP) interact with extracellular matrix components (ECM); 2) to determine the precise molecular sites of interaction between APP and ECM components; and 3) to use this information to design specific agents to interfere with these interactions i.e. potential therapies for Alzheimer's amyloid. The definitive histologic criteria for the diagnosis of Alzheimer's disease (AD) are amyloids, in the form of congophilic angiopathy, neuritic plaques or neurofibrillary tangles. The beta protein and APP in two of these forms of amyloid coexist and interact tightly with a specific ECM component. These interactions may be involved mechanistically in the genesis of these amyloid deposits. Whether these amyloids are primary or secondary events in the pathogenesis of AD is unresolved. Nevertheless as learned from other forms of amyloid the effects of the deposits themselves can be devastating. Neurotoxic and neurotrophic properties of APP fragments and neurotrophic effects of ECM components support the idea that these constituents are responsible for neuronal cell death and altered neuron-neuron connectivity at sites of neuritic plaques. Understanding of, and interference with, amyloid development and evolution, are respectively logical scientific goals and therapeutic targets in AD. A modified ELISA technique is used to study the binding of the components of interest. One component is immobilized on a solid phase, and a second in solution, binds to the first. The quantity of the bound second component is determined with specific antisera. Defined peptides of APP are used to determine which peptides inhibit the binding of the two components, thus defining the binding site(s) on APP for an ECM component. Similarly, fragments of the glycosaminoglycan side chains of heparan sulphate proteoglycan, one ECM component involved in this binding, are used to define carbohydrate structures involved in these bindings. These data will provide an understanding of the sites involved in APP:ECM interactions, and define the structure of agents which may interfere with this process. An additional important question is that of the influence of ECM components on APP proteolysis. APP, or APP:ECM complexes generated in solution, will be subjected to proteolysis. Using 2D-PAGE and Western blotting a comparison of APP peptides generated from the initial conditions will provide information about the influence of ECM on APP processing.
