FAD mutations in presenilin-1 or amyloid precursor protein decrease the efficacy of a γ-secretase inhibitor: Evidence for direct involvement of PS1 in the γ-secretase cleavage complex

To investigate the mechanism of regulation of Aβ production by familial Alzheimer's disease linked presenilin 1 (PS1), we used a cell-free system that allows de novo Aβ generation to examine whether PS1 participates directly in the γ-secretase reaction. Optimal Aβ generation in vitro was achieved at mildly acidic pH and could be inhibited by the aspartyl protease inhibitor pepstalin A, consistent with the suggestion that γ-secretase is an aspartyl protease. Dominant negative mutations of the critical transmembrane aspartates in PS1 or full deletion of PS1 did not alter the maturation of APP in the secretory pathway. Instead, PS1 had a direct effect on the inhibition of Aβ production by a designed peptidomimetic inhibitor: the inhibition was significantly less effective in cells expressing FAD-causing mutations in either APP or PS1 than in cells expressing the wild-type proteins. Taken together, these findings suggest that PS1 participates physically in a complex with APP during the γ-secretase cleavage event.