CHOLESTEROL-DEPENDENT GENERATION OF A UNIQUE AMYLOID BETA-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 be ta-protein (A beta), we characterized A beta secreted from Madin-Darby canine kidney (MDCK) cells which had been stably transfected with a c DNA encoding the human beta-amyloid precursor protein (beta APP695) wi th a 42 amino acid residue truncation at the carboxyl terminus (Delta C). In Delta C MDCK cells, the intracellular sorting of beta APP is su bstantially 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 m onoclonal antibody specific fbr A beta 17-24) and detected as a smear on Western blots, but was not immunoprecipitated with BAN50 (a monoclo nal antibody raised against A beta 1-16). Interestingly, however, this A beta species was readily immunoprecipitated with BAN50 upon treatme nt with formic acid. Furthermore, incubation of the Delta C MDCK cells with compactin, an inhibitor of de novo cholesterol synthesis, or wit h filipin, a cholesterol-binding drug, resulted in marked changes in t he characteristics of this A beta species as follows: first, the A bet a was not observed as a smear on Western blots and second, the A beta was immunoprecipitated with BAN50. The present results strongly sugges t that an A beta with unique molecular characteristics is generated fr om the missorted beta APP in vivo in a cholesterol-dependent manner. ( C) 1998 Elsevier Science B.V. All rights reserved.