Converging lines of evidence implicate the beta-amyloid peptide (Ab) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce Ab production by functionally inhibiting g-secretase, the activity responsible for the carboxy-terminal cleavage required for Ab production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon Ab production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-di¯uoro-phenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP V717F reduces brain levels of Ab in a dose-dependent manner within 3 h. These studies represent the ®rst demonstration of a reduction of brain Ab in vivo. Development of such novel functional g-secretase inhibitors will enable a clinical examination of the Ab hypothesis that Ab peptide drives the neuropathology observed in Alzheimer's disease.

Postmenopausal estrogen depletion is a characterized risk factor for Alzheimer disease (AD), a human disorder linked to high levels of ␤-amyloid peptide (A␤) in brain tissue. Previous studies suggest that estrogen negatively regulates the level of A␤ in the brain, but the molecular mechanism is unknown. Here, we provide evidence that estrogen promotes A␤ degradation mainly through a principal A␤ degrading enzyme, neprilysin, in neuroblastoma SH-SY5Y cells. We also demonstrate that upregulation of neprilysin by estrogen is dependent on both estrogen receptor ␣ and ␤ (ER␣ and ER␤), and ligand-activated ER regulates expression of neprilysin through physical interactions between ER and estrogen response elements (EREs) identified in the neprilysin gene. These results were confirmed by in vitro gel shift and in vivo chromatin immunoprecipitation analyses, which demonstrate specific binding of ER␣ and ER␤ to two putative EREs in the neprilysin gene. The EREs also enhance ER␣-and ER␤-dependent reporter gene expression in a yeast model system. Therefore, the study described here provides a putative mechanism by which estrogen positively regulates expression of neprilysin to promote degradation of A␤, reducing risk for AD. These results may lead to novel approaches to prevent or treat AD.Alzheimer disease (AD) 3 is a progressive neurodegenerative disease characterized by declarative memory impairment and progressive dementia. The level of ␤-amyloid peptide (A␤) is elevated in the brains of AD patients, and A␤ is believed to play a critical role in the pathology of AD (1, 2). Recent studies show that aggregated oligomers of A␤ (protofibrils) play a direct role in neuronal and behavioral deficits in AD patients (3).The rate of A␤ degradation could influence the risk of developing AD, and it has been proposed that stimulation of proteolytic degradation of A␤ could be used as a therapeutic approach for AD (4, 5). Neprilysin is thought to be the primary A␤-degrading enzyme in the brain (6) because degradation of radiolabeled synthetic A␤42 in rat brain is largely inhibited by the neprilysin inhibitor, phosphoramidon (PA) (7, 8) and because neprilysin degrades both monomeric and oligomeric forms of A␤40 and A␤42 in intracellular and extracellular compartments of the brain (9). Moreover, the level of neprilysin mRNA and protein is lower in the hippocampus and temporal gyrus of AD patients (10, 11), which correlates with higher levels of A␤ as A␤ tends to accumulate in these regions (12).Neprilysin activity is also lower in the hippocampus, cerebellum, and caudate of ovariectomized rats than in non-ovariectomized rats, and this effect can be reversed by exogenous 17␤-estradiol (13). These data indicate that 17␤-estradiol positively regulates neprilysin activity in the brain. 17␤-Estradiol was also reported to reduce the generation of A␤ peptides in neuroblastoma cells and neurons (14). The positive regulation of neprilysin by 17␤-estradiol might be a crucial factor in protecting normal adult brain from A␤ damage and in improving...