Alzheimer's disease is characterized by clinical dementia in association with pathologic alterations in brain proteins. Structural lesions include extracellular beta/A4-amyloid deposits. Beta/A4-amyloid is derived through proteolysis of a large, transmembrane precursor, the beta/A4-amyloid precursor protein (APP). Mutations in the coding sequence of APP are associated with familial cerebral amyloidoses, pointing to the importance of APP in the pathogenesis of amyloidosis. The association of different APP-mutation genotypes with the cerebral amyloidosis phenotype strongly suggests that alternative amyloidogenic proteolysis may be a final common pathway in both familial and sporadic cerebral amyloidotic diseases, including Alzheimer's disease. A standard pathway for proteolysis of about 30% of APP molecules (in PC-12 cells) cleaves within the beta/A44- amyloid domain, precluding amyloid formation. The generation of beta/A4-amyloid must therefore occur via an alternative proteolytic pathway. Evidence for the existence of alternative pathways has emerged from several laboratories, in studies of human cerebral vessels, brain, and cells in continuous culture. Cell culture systems which generate microheterogeneous proteolysis of APP include the rat PC-12 line (under conditions of supraphysiological protein phosphorylation), the monkey fibroblast (following overexpression of human APP in recombinant vaccinia virus), the human APP-transfected human 293 cell, and the recombinant human APP-baculovirus infected Sf9 cell. The Sf9 system is particularly attractive because of the extraordinarily high-level expression of recombinant protein, providing convenience for purification and sequencing of species of interest. Sf9 cells faithfully recapitulate many of the biological properties of mammalian cells. When human APP is expressed in Sf9 cells, a portion of APP molecules is cleaved in a position exactly identical to the major cleavage site for proteolyzing APP in human cells, thus providing validity to the use of the Sf9 cell as a model system for APP proteolysis. At high multiplicities-of-infection, in addition to the cleavage of APP at this major conserved site (which generates a 14-15 Kda carboxyl-terminal fragment), Sf9 cells produce a discrete and limited number of other APP carboxyl-terminal fragments, including species of 16-, 17- and 25-Kda. By antigenic analysis, the 17-Kda species has been demonstrated to incorporate amino-terminal epitopes of the beta/A4-amyloid domain and is thus a putative amyloidogenic fragment. While there is mounting immunochemical evidence for such putative amyloidogenic fragments, direct protein sequencing of such a species has not been achieved, and is the primary goal of this proposal, using the baculoviral/Sf9 expression system. In addition, putative amyloidogenic mutant APP molecules (from familial cerebral amyloidoses) will be overexpressed in baculoviruses, and their proteolytic fragments characterized, purified and sequenced. The definitive identification of amyloidogenic pathways for APP proteolysis is crucial to the successful dissection of amyloidogenesis and to the design of strategies for in vitro models of amyloidogenesis.
