NOVEL BETA-SECRETASE CLEAVAGE OF BETA-AMYLOID PRECURSOR PROTEIN IN THE ENDOPLASMIC-RETICULUM INTERMEDIATE COMPARTMENT OF NT2N CELLS

Previous studies have demonstrated that NT2N neurons derived from a hu man embryonal carcinoma cell line (NT2) constitutively process the end ogenous wild-type beta-amyloid precursor protein (APP) to amyloid beta peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generated by intracellular processing must also be present in these cells. Here we show that the NH2-terminal fr agment of APP generated by beta-secretase cleavage (APP beta) is indee d produced from the endogenous full length APP (APP(FL)). Pulse-chase studies demonstrated a precursor-product relationship between APP(FL) and APP beta as well as intracellular and secreted APP beta fragments, In addition, trypsin digestion of intact NT2N cells at 4 degrees C di d not abolish APP beta recovered from the eel lysates. Furthermore, th e production of intracellular APP beta from wild-type APP appears to b e a unique characteristic of postmitotic neurons, since intracellular APP beta was not detected in several non-neuronal cell lines. Signific antly, production of APP beta occurred even when APP was retained in t he ER/intermediate compartment by inhibition with brefeldin A, incubat ion at 15 degrees C, or by expression of exogenous APP bearing the dil ysine ER retrieval motif.