DESCRIPTION The long-range objective of this research is the precise biochemical elucidation of the structural components and mechanisms of formation of the initial amyloid beta-peptide deposits in the brain. In this pursuit several observations have been made that are the foundation of this proposal. First, the formation of parenchymal amyloid plaques in Down's syndrome (DS), is preceded by the presence of increased concentration of soluble, aggregated amyloid beta-peptide (A-beta), that ends at residue 42. Second, three major, N-terminally truncated A-beta peptides beginning at residues 1, 3 and 11 are the invariant components of the soluble A-beta aggregate and, subsequently, senile plaques. Third, all three A-beta forms are extensively modified at the N-termini: aspartate-1 is either isomerized or racemized, glutamates-3 and -11 are cyclized to pyroglutamates. Fourth, the same truncated A-beta peptides, although not aggregated, are also present in the cerebrospinal fluid. Fifth, brain concentration of the water-soluble, pyroglutamate-modified A-beta increases, relative to the full-length peptide, with age in DS and is dramatically increased in some familial Alzheimer's disease (AD), brains. Sixth, water-soluble A-beta aggregate is partially resistant to unspecific proteolysis. All these observations point to a critical role of the truncated A-beta peptides in the initiation of amyloid deposits. Therefore, it is proposed to determine the structural basis of the generation of major A-beta peptides present in the brain. Specifically we will a) determine the primary structure, molecular organization and quantity of major modified A-beta forms in non-AD aging brain, DS brain, and in transgenic animals; b) investigate if the N-terminally truncated A-beta are formed from A-beta aggregates and/or A-beta precursor protein (APP); C-terminal fragments (CTFs), isolated from brain; c) investigate if the truncated A-beta, in the form found in the brain, can be proteolytically generated in vitro from recombinant CTFs. The proposed studies will involve biochemical and enzymological techniques, mass spectrometric analysis and development of specific immunological and molecular biological reagents.