␥-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.

Although ␥-secretase is recognized as a therapeutic target for Alzheimer's disease, side effects associated with strong inhibition of this aspartyl protease raised serious concerns regarding this therapeutic strategy. However, it is not known whether moderate inhibition of this enzyme will allow dissociation of beneficial effects in the CNS from mechanism-based toxicities in the periphery. We tested this possibility by using a series of mice with genetic reduction of ␥-secretase (levels ranging from 25 to 64% of control mice). Here, we document that even 30% reduction of ␥-secretase can effectively ameliorate amyloid burden in the CNS. However, global reduction of this enzyme below a threshold level increased the risk of developing squamous cell carcinoma as well as abnormal proliferation of granulocytes in a ␥-secretase dosage-dependent manner. Importantly, we demonstrate that there exists a critical ␥-secretase level that reduces the risk of amyloidosis in the CNS and limits tumorigenesis in epithelia. Our findings suggest that moderate inhibition of ␥-secretase represents an attractive anti-amyloid therapy for Alzheimer's disease.

Alzheimer disease (AD) is an age-related disorder. Aging and female gender are two important risk factors associated with sporadic AD. However, the mechanism by which aging and gender contribute to the pathogenesis of sporadic AD is unclear. It is well known that genetic mutations in γ-secretase result in rare forms of early onset AD due to the aberrant production of Aβ42 peptides, which are the major constituents of senile plaques. However, the effect of age and gender on γ-secretase has not been fully investigated. Here, using normal wild-type mice, we show mouse brain γ-secretase exhibits gender- and age-dependent activity. Both male and female mice exhibit increased Aβ42∶Aβ40 ratios in aged brain, which mimics the effect of familial mutations of Presenilin-1, Presenlin-2, and the amyloid precursor protein on Aβ production. Additionally, female mice exhibit much higher γ-secretase activity in aged brain compared to male mice. Furthermore, both male and female mice exhibit a steady decline in Notch1 γ-secretase activity with aging. Using a small molecule affinity probe we demonstrate that male mice have less active γ-secretase complexes than female mice, which may account for the gender-associated differences in activity in aged brain. These findings demonstrate that aging can affect γ-secretase activity and specificity, suggesting a role for γ-secretase in sporadic AD. Furthermore, the increased APP γ-secretase activity seen in aged females may contribute to the increased incidence of sporadic AD in women and the aggressive Aβ plaque pathology seen in female mouse models of AD. In addition, deceased Notch γ-secretase activity may also contribute to neurodegeneration. Therefore, this study implicates altered γ-secretase activity and specificity as a possible mechanism of sporadic AD during aging.