INTRACELLULAR GENERATION AND ACCUMULATION OF AMYLOID BETA-PEPTIDE TERMINATING AT AMINO-ACID-42

Amyloid beta-peptide (A beta) is known to accumulate in senile plaques of Alzheimer's disease (AD) patients and is now widely believed to pl ay a major role in the disease. Two populations of peptides occur term inating either at amino acid 40 or at amino acid 42 (A beta 1-40 and A beta 1-42). Alternative N-terminal cleavages produce additional heter ogeneity (A beta x-40 and A beta x-42), Peptides terminating at amino acid 42 are believed to be the major player in sporadic AD as well as familial AD (FAD). Whereas the cellular mechanism for the generation o f AP terminating at amino acid 40 is well understood, very little is k nown about the cleavage of A beta after amino acid 42, By using two in dependent methods we demonstrate intracellular A beta 1-42 as well as A beta x-42 but less A beta x-40 and A beta 1-40 in kidney 293 cells s tably transfected with wild type beta-amyloid precursor protein (beta APP) or the FAD-associated Val/Gly mutation, Moreover, retention of be ta APP within the endoplasmic reticulum (ER) by treatment with brefeld in A does not block the cleavage at amino acid 42 but results in an in creased production of all species of A beta terminating at amino acid 42. This indicates that the cleavage after amino acid 42 can occur wit hin the ER. Treatment of cells with monensin, which blocks transport o f (beta APP) within the Golgi causes a marked accumulation of intracel lular A beta x-42 and A beta x-40. Therefore these experiments indicat e that the gamma-secretase cleavage of A beta after amino acid 42 can occur within the ER and later within the secretory pathway within the Golgi. Moreover inhibition of reinternalization by cytoplasmic deletio ns of beta APP as well as inhibition of intracellular acidification by NH4Cl does not block intracellular A beta 1-42 or A beta x-42 product ion.