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.

Amylin, a 37-amino-acid amyloidogenic peptide, bears biophysical similarities to the amyloid-beta peptide (A beta) deposited in Alzheimer's disease. Using embryonic rat hippocampal cultures we tested whether amylin induces neurotoxicity similar to that previously observed with A beta(1-40). Treatment with human amylin(1-37) resulted in prominent toxicity as assessed by phase-contrast microscopy and quantification of lactate dehydrogenase in the medium. Amylin-induced neurotoxicity was morphologically similar to that induced by A beta(1-40). In contrast, the nonamyloidogenic rat amylin showed negligible neurotoxicity despite having 95% sequence similarity to human amylin. Only full-length human amylin was toxic; various amylin peptide fragments including amino acid residues 20-29 were nontoxic at similar concentrations. These studies suggest that unrelated amyloidogenic peptides like human amylin and A beta can adopt a similar neurotoxic conformation in vitro. Similar conformation-dependent neurotoxicity may drive the prominent neurite degeneration around compacted but not diffuse deposits of A beta in Alzheimer's disease.

Clusterin is a secreted glycoprotein that is markedly induced in many disease states and after tissue injury. In the CNS, clusterin expression is elevated in neuropathological conditions such as Alzheimer's disease (AD), where it is found associated with amyloid-/3 (A/I) plaques. Clusterin also coprecipitates with A/I from CSF, suggesting a physiological interaction with A/I. Given this interaction with A/I, the goal of this study was to determine whether clusterin could modulate A/I neurotoxicity. A mammalian recombinant source of human clusterin was obtained by stable transfection of hamster kidney fibroblasts with pADHC-9, a full-length human cDNA clone for clusterin. Recombinant clusterin obtained from this cell line, as well as a commercial source of native clusterin purified from serum, afforded dosedependent neuroprotection against A/I(1-40) when tested in primary rat mixed hipppocampal cultures. Clusterin afforded substoichiometric neuroprotection against several lots of A/3(1 -40) but not against H 202 or kainic acid excitotoxicity. These results suggest that the elevated expression of clusterin found in AD brain may have effects on subsequent amyloid-/3 plaque pathology.

H2O2 and free radical‐mediated oxidative stresses have been implicated in mediating amyloid β(1–40) [Aβ(1–40)] neurotoxicity to cultured neurons. In this study, we confirm that addition of the H2O2‐scavenging enzyme catalase protects neurons in culture against Aβ‐mediated toxicity; however, it does so by a mechanism that does not involve its ability to scavenge H2O2. Aβ‐mediated elevation in intracellular H2O2 production is suppressed by addition of a potent H2O2 scavenger without any significant neuroprotection. Three intracellular biochemical markers of H2O2‐mediated oxidative stress were unchanged by Aβ treatment: (a) glyceraldehyde‐3‐phosphate dehydrogenase activity, (b) hexose monophosphate shunt activity, and (c) glucose oxidation via the tricarboxylic acid cycle. Ionspray mass spectra of Aβ in the incubation medium indicated that Aβ itself is an unlikely source of reactive oxygen species. In this study we demonstrate that intracellular ATP concentration is compromised during the first 24‐h exposure of neurons to Aβ. Our results challenge a pivotal role for H2O2 generation in mediating Aβ toxicity, and we suggest that impairment of energy homeostasis may be a more significant early factor in the neurodegenerative process.