Cholesterol-dependent generation of a unique amyloid β-protein from apically missorted amyloid precursor protein in MDCK cells

To investigate the implications of altered sorting of the beta-amyloid precursor protein (beta APP) in the abnormal generation of amyloid beta-protein (A beta), we characterized A beta secreted from Madin-Darby canine kidney (MDCK) cells which had been stably transfected with a cDNA encoding the human beta-amyloid precursor protein (beta APP695) with a 42 amino acid residue truncation at the carboxyl terminus (Delta C). In Delta C MDCK cells, the intracellular sorting of beta APP is substantially altered to the apical surface. We detected an accumulation of a unique A beta species in the apical compartment of Delta C MDCK cell cultures. This unique A beta was immunoprecipitated with 4G8 (a monoclonal antibody specific fbr A beta 17-24) and detected as a smear on Western blots, but was not immunoprecipitated with BAN50 (a monoclonal antibody raised against A beta 1-16). Interestingly, however, this A beta species was readily immunoprecipitated with BAN50 upon treatment with formic acid. Furthermore, incubation of the Delta C 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 A beta species as follows: first, the A beta was not observed as a smear on Western blots and second, the A beta was immunoprecipitated with BAN50. The present results strongly suggest that an A beta with unique molecular characteristics is generated from the missorted beta APP in vivo in a cholesterol-dependent manner. (C) 1998 Elsevier Science B.V. All rights reserved.