Intracellular site of γ‐secretase cleavage for Aβ42 generation in Neuro 2a cells harbouring a presenilin 1 mutation

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Abstract
Previously, we reported that mutations in presenilin 1 (PS1) increased the intracellular levels of amyloid β-protein (Aβ)42. However, it is still not known at which cellular site or how PS1 mutations exert their effect of enhancing Aβ42–γ-secretase cleavage. In this study, to clarify the molecular mechanisms underlying this enhancement of Aβ42–γ-secretase cleavage, we focused on determining the intracellular site of the cleavage. To address this issue, we used APP–C100 encoding the C-terminal β-amyloid precursor protein (APP) fragment truncated at the N terminus of Aβ (C100); C100 requires only γ-secretase cleavage to yield Aβ. Mutated PS1 (M146L)-induced Neuro 2a cells showed enhanced Aβ1–42 generation from transiently expressed C100 as well as from full-length APP, whereas the generation of Aβ1–40 was not increased. The intracellular generation of Aβ1–42 from transiently expressed C100 in both mutated PS1-induced and wild-type Neuro 2a cells was inhibited by brefeldin A. Moreover, the generation of Aβ1–42 and Aβ1–40 from a C100 mutant containing a di-lysine endoplasmic reticulum retention signal was greatly decreased, indicating that the major intracellular site of γ-secretase cleavage is not the endoplasmic reticulum. The intracellular generation of Aβ1–42/40 from C100 was not influenced by monensin treatment, and the level of Aβ1–42/40 generated from C100 carrying a sorting signal for the trans-Golgi network was higher than that generated from wild-type C100. These results using PS1-mutation-harbouring and wild-type Neuro 2a cells suggest that Aβ42/40–γ-secretase cleavages occur in the Golgi compartment and the trans-Golgi network, and that the PS1 mutation does not alter the intracelluar site of Aβ42–γ-secretase cleavage in the normal APP proteolytic processing pathway.