Intracellular Generation and Accumulation of Amyloid β-Peptide Terminating at Amino Acid 42

Amyloid β-peptide (Aβ) is known to accumulate in senile plaques of Alzheimer’s disease (AD) patients and is now widely believed to play a major role in the disease. Two populations of peptides occur terminating either at amino acid 40 or at amino acid 42 (Aβ1–40 and Aβ1–42). Alternative N-terminal cleavages produce additional heterogeneity (Aβ x -40 and Aβ 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 of Aβ terminating at amino acid 40 is well understood, very little is known about the cleavage of Aβ after amino acid 42. By using two independent methods we demonstrate intracellular Aβ1–42 as well as Aβ x -42 but less Aβ x -40 and Aβ1–40 in kidney 293 cells stably transfected with wild type β-amyloid precursor protein (βAPP) or the FAD-associated Val/Gly mutation. Moreover, retention of βAPP within the endoplasmic reticulum (ER) by treatment with brefeldin A does not block the cleavage at amino acid 42 but results in an increased production of all species of Aβ terminating at amino acid 42. This indicates that the cleavage after amino acid 42 can occur within the ER. Treatment of cells with monensin, which blocks transport of (βAPP) within the Golgi causes a marked accumulation of intracellular Aβ x -42 and Aβ x -40. Therefore these experiments indicate that the γ-secretase cleavage of Aβ after amino acid 42 can occur within the ER and later within the secretory pathway within the Golgi. Moreover inhibition of reinternalization by cytoplasmic deletions of βAPP as well as inhibition of intracellular acidification by NH 4 Cl does not block intracellular Aβ1–42 or Aβ x -42 production.