Intracellular site of gamma-secretase cleavage for A beta 42 generation inNeuro 2a cells harbouring a presenilin 1 mutation

Previously, we reported that mutations in presenilin 1 (PS1) increased the intracellular levels of amyloid beta-protein (A beta)42. However, it is sti ll not known at which cellular site or how PS1 mutations exert their effect of enhancing A beta 42-gamma-secretase cleavage. In this study, to clarify the molecular mechanisms underlying this enhancement of A beta 42-gamma-se cretase cleavage, we focused on determining the intracellular site of the c leavage. To address this issue, we used APP-C100 encoding the C-terminal be ta-amyloid precursor protein (APP) fragment truncated at the N terminus of A beta (C100); C100 requires only gamma-secretase cleavage to yield A beta. Mutated PS1 (M146L)-induced Neuro 2a cells showed enhanced A beta 1-42 gen eration from transiently expressed C100 as well as from full-length APP, wh ereas the generation of A beta 1-40 was not increased. The intracellular ge neration of A beta 1-42 from transiently expressed C100 in both mutated PS1 -induced and wild-type Neuro 2a cells was inhibited by brefeldin A. Moreove r, the generation of A beta 1-42 and A beta 1-40 from a C100 mutant contain ing a di-lysine endoplasmic reticulum retention signal was greatly decrease d, indicating that the major intracellular site of gamma-secretase cleavage is not the endoplasmic reticulum. The intracellular generation of A beta 1 -42/40 from C100 was not influenced by monensin treatment, and the level of A beta 1-42/40 generated from C100 carrying a sorting signal for the trans -Golgi network was higher than that generated from wild-type C100. These re sults using PS1-mutation-harbouring and wild-type Neuro 2a cells suggest th at A beta 42/40-gamma-secretase cleavages occur in the Golgi compartment an d the trans-Golgi network, and that the PS1 mutation does not alter the int racelluar site of A beta 42-gamma-secretase cleavage in the normal APP prot eolytic processing pathway.