Pen2 and Presenilin-1 Modulate the Dynamic Equilibrium of Presenilin-1 and Presenilin-2 γ-Secretase Complexes

Abstract

␥-Secretase is known to play a pivotal role in the pathogenesis of Alzheimer disease through production of amyloidogenic A␤42 peptides. Early onset familial Alzheimer disease mutations in presenilin (PS), the catalytic core of ␥-secretase, invariably increase the A␤42:A␤40 ratio. However, the mechanism by which these mutations affect ␥-secretase complex formation and cleavage specificity is poorly understood. We show that our in vitro assay system recapitulates the effect of PS1 mutations on the A␤42:A␤40 ratio observed in cell and animal models. We have developed a series of small molecule affinity probes that allow us to characterize active ␥-secretase complexes. Furthermore we reveal that the equilibrium of PS1-and PS2-containing active complexes is dynamic and altered by overexpression of Pen2 or PS1 mutants and that formation of PS2 complexes is positively correlated with increased A␤42:A␤40 ratios. These data suggest that perturbations to ␥-secretase complex equilibrium can have a profound effect on enzyme activity and that increased PS2 complexes along with mutated PS1 complexes contribute to an increased A␤42:A␤40 ratio.

Modulation of γ-secretase specificity using small molecule allosteric inhibitors

“…blue indicated CS-1 had little to no effect on cell viability up to 30 M. In addition, we found that CS-3 exhibited an identical inhibitory profile with a slightly increased EC 50 for A␤42 inhibition (approximately 5 M). Compound E inhibited the production of all 3 ␤-amyloid species with equal potency (Fig.…”

“…The majority of mutations within each of these genes cause an increase in the ratio of A␤42 to A␤40 in biochemical, cellular, and animal models. Recent studies suggest that alteration of ␥-secretase complex dynamics and/or formation of ␥-secretase complexes with mutated components can affect the enzymatic cleavage specificity (30,31). Despite these advances in our understanding, little is known regarding the molecular mechanisms that control the specificity of ␥-secretase-mediated cleavage at the A␤40, A␤42, or Notch1 cleavage locations.…”

Familial Alzheimer Disease Presenilin-1 Mutations Alter the Active Site Conformation of γ-secretase

“…1B). All three probes only photolabel PS1-NTF but not PS1-CTF or other subunits (18,22,23,25). Membranes isolated from N2a cells stably expressing WT, M146L, E280A, FIGURE 1.…”

“…The PS1 M146V knock-in mice have been well characterized for memory formation and A␤ production (18,27,28). Membranes isolated from the M146V knock-in and PS1 WT mouse brain were labeled with the photoprobes JC8 and L646.…”

“…Little is known about the molecular mechanism of these mutations in altering the specificity of ␥-secretase. A previous study showed that expression of PS1 FAD mutants in PS1-and PS2-null cells led to the formation of ␥-secretase complexes with different molecular weights (17), whereas another study showed that the overexpression of PS1 FAD mutants can also alter the equilibrium of wild-type PS1 and PS2 in ␥-secretase complexes (18). Furthermore, we have demonstrated that proteoliposomes containing PS1 mutants alone are sufficient to alter the specificity of ␥-secretase for the production of A␤40 and A␤42 (16).…”