Cholesterol-dependent generation of a unique amyloid beta-protein from apically missorted amyloid precursor protein...

To investigate the implications of altered sorting of the beta-amyloid precursor protein (betaAPP) in the abnormal generation of amyloid beta-protein (Abeta), we characterized Abeta secreted from Madin-Darby canine kidney (MDCK) cells which had been stably transfected with a cDNA encoding the human beta-amyloid precursor protein (betaAPP695) with a 42 amino acid residue truncation at the carboxyl terminus (DeltaC). In DeltaC MDCK cells, the intracellular sorting of betaAPP is substantially altered to the apical surface. We detected an accumulation of a unique Abeta species in the apical compartment of DeltaC MDCK cell cultures. This unique Abeta was immunoprecipitated with 4G8 (a monoclonal antibody specific for Abeta17-24) and detected as a smear on Western blots, but was not immunoprecipitated with BAN50 (a monoclonal antibody raised against Abeta1-16). Interestingly, however, this Abeta species was readily immunoprecipitated with BAN50 upon treatment with formic acid. Furthermore, incubation of the DeltaC MDCK cells with compactin, an inhibitor of de novo cholesterol synthesis, or with filipin, a cholesterol-binding drug, resulted in marked changes in the characteristics of this Abeta species as follows: first, the Abeta was not observed as a smear on Western blots and second, the Abeta was immunoprecipitated with BAN50. The present results strongly suggest that an Abeta with unique molecular characteristics is generated from the missorted betaAPP in vivo in a cholesterol-dependent manner.