Beta-amyloid protein (A-beta) and a specific heparan sulfate proteoglycan (HSPG) known as perlecan, are two macromolecules which co-accumulates part of fibrillar amyloid deposits in human Alzheimer's disease (AD) brain. Previous attempts to mimic the pathology of AD, including the formation of fibrillar amyloid deposits (as part of amyloid plaques and cardiovascular amyloid accumulation) in transgenic animals by overexpression on only beta- amyloid precursor protein (beta PP) has been unsuccessful. Recent infusion studies now confirm that perlecan may be the essential molecule needed for the ultimate formation, deposition and persistence of fibrillar A-beta deposits in rodent brain (Snow et al, 1994a). The present proposal will therefore test the hypothesis that overexpression of both the A-beta-binding core protein domain of perlecan and beta-PP in transgenic mice is sufficient to produce and animal mimicking all or at least some (i.e. fibrillar and congophillic A-beta amyloid deposits) of the pathology of human AD.
Specific Aim #1 will identify perlecan core protein domain that bind A-beta using a yeast two hybrid system, affinity column chromatography and solid phase binding assays. For the two hybrid will consist of fusion between the Lex-A-binding domain and the C-terminal region of beta- PP, whereas the second hybrid will consist of fusion between a nuclear localized VP 16 activated domain and one of five functional domains of perlecan. Affinity column chromatography and solid phase binding assays will assay the interaction between purified perlecan core protein domains obtained from fusion proteins and A-beta (1-40) or (1-28).
Specific Aim #2 will involve production of transgenic mice overexpression the core protein domains of perlecan which bind A- beta (from aim #1). c Dna for perlecan domains that bind A-beta will be placed under control of a cytomegalovirus enhancer and beta-actin promoter and then microinjected into pronucleoli of fertilized eggs.
Specific Aim# 3 will established transgenic mice that overexpress both a C-terminal region of beta-PP and a specific domain in perlecan core protein. Additionally, perlecan transgenic mice created in specific aim #2, will be mated with beta-PP transgenic mice already established at the University of Washington by the principal investigator. Biochemical, immunochestochemical, histopathological, and electron microscopic analyses will be used to analyze transgenic mice established in these in these aims. A new animal model mimicking aspects of the pathology of AD will provide a needed screening tool for the development of new therapeutic agents fight this disease.
