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.

Neurotoxicity induced by overstimulation of N-methyl-D-aspartate (NMDA) receptors is due, in part, to a sustained rise in intracellular Ca 2؉ ; however, little is known about the ensuing intracellular events that ultimately result in cell death. Here we show that overstimulation of NMDA receptors by relatively low concentrations of glutamate induces apoptosis of cultured cerebellar granule neurons (CGNs) and that CGNs do not require new RNA or protein synthesis. Glutamate-induced apoptosis of CGNs is, however, associated with a concentration-and time-dependent activation of the interleukin 1␤-converting enzyme (ICE)͞CED-3-related protease, CPP32͞Yama͞apopain (now designated caspase 3). Further, the time course of caspase 3 activation after glutamate exposure of CGNs parallels the development of apoptosis. Moreover, glutamate-induced apoptosis of CGNs is almost completely blocked by the selective cell permeable tetrapeptide inhibitor of caspase 3, Ac-DEVD-CHO but not by the ICE (caspase 1) inhibitor, Ac-YVAD-CHO. Western blots of cytosolic extracts from glutamate-exposed CGNs reveal both cleavage of the caspase 3 substrate, poly(ADP-ribose) polymerase, as well as proteolytic processing of pro-caspase 3 to active subunits. Our data demonstrate that glutamateinduced apoptosis of CGNs is mediated by a posttranslational activation of the ICE͞CED-3-related cysteine protease caspase 3.

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