AMYLOIDOGENIC PROCESSING OF THE HUMAN AMYLOID PRECURSOR PROTEIN IN PRIMARY CULTURES OF RAT HIPPOCAMPAL-NEURONS

The aim of this study was to investigate the proteolytic processing of the amyloid precursor protein (APP) in polarized primary cultures of hippocampal neurons. We have used the Semliki Forest virus (SFV) vecto r to express human APP695 in hippocampal neurons, sympathetic ganglia, and glial cells. The latter two cells secrete little or no APP, where as hippocampal neurons secrete two forms of APP695, which differ in si alic acid content and in their kinetic appearance in the culture mediu m. In addition, rat hippocampal neurons expressing human APP produced significant amounts of the 4 kDa peptide beta A4. After 3 hr of metabo lic labeling, the relative amount of beta A4 peptide to total cellular APP was 5.3%. Fibroblasts expressing APP695 using the same SFV vector mainly produced a related 3 kDa p3 peptide, a nonamyloidogenic fragme nt. Remarkably, the hippocampal neurons also produced significant amou nts of beta A4-containing C-terminal fragments (10-12 kDa) intracellul arly. Radiosequencing showed that these fragments were created at a pr eviously described beta-secretase cleavage site and at a cleavage site 12 residues from the N terminus of the beta A4 domain (Thr(584) of AP P695), which We named delta-cleavage. Based on the observation that ma ture hippocampal neurons produce two potentially amyloidogenic fragmen ts and secrete substantial amounts of beta A4 when expressing human AP P, our results strengthen the hypothesis that neurons play a central r ole in the process of beta A4 deposition in cases of Alzheimer's disea se and in aged primates.