Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain.

Converging lines of evidence implicate the beta-amyloid peptide (Ass) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce A beta production by functionally inhibiting gamma-secretase, the activity responsible for the carboxy-terminal cleavage required for A beta production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon A beta 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-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP(V717F) reduces brain levels of Ass in a dose-dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain A beta in vivo. Development of such novel functional gamma-secretase inhibitors will enable a clinical examination of the A beta hypothesis that Ass peptide drives the neuropathology observed in Alzheimer's disease.

Regulation of amyloid precursor protein processing by Abeta in human glioma cells.

Amyloid precursor protein (APP) is cleaved to neurotoxic/proinflammatory amyloid beta protein (Abeta) or to the neuroprotective secreted alpha-APPs. A balance in APP metabolism may influence the outcome between toxicity and protection to central nervous system (CNS) neurons in Alzheimer's disease. Treatment of U-373 MG astrocytoma cells with aggregated Abeta (1-40) decreases APP secretion into the medium to 10-30% of control values. This decreased secretion appears to be specific for APP since Abeta treatment causes an approximately 2-fold increase in interleukin-8 (IL-8) secretion. Abeta treatment also causes a 4- to 9-fold increase in total cell-associated APP. This increase is due to cellular retention of alpha secretase-cleaved APP and a 2-fold increase in mature full-length APP. These data suggest that deposition of aggregated Abeta may contribute to Alzheimer's-associated neurotoxicity by altering the metabolism of the APP protein. Abeta may exert harmful effects by decreasing the secretion of neuroprotective or neurotrophic APP and, in addition, by increasing intracellular full-length APP; thereby providing increased substrate for generation of amyloidogenic peptide within astrocytes.

Suppression of an amyloid beta peptide-mediated calcium channel response by a secreted beta-amyloid precursor protein.

Secreted isoforms of the beta-amyloid precursor protein potently enhance neuronal survival in cell cultures exposed to toxic amyloid beta peptide. Lowering of intracellular calcium levels to offset the increases in intraneuronal calcium caused by amyloid beta peptide is thought to underly this neuroprotection. Because we have shown previously that an amyloid beta peptide-mediated potentiation of calcium channel currents may contribute to this cytosolic calcium overload, the present study examined the effects of a secreted beta-amyloid precursor protein on the calcium channel response to amyloid beta peptide. When compared with untreated cultured rat hippocampal neurons, cells that underwent a 24 h preincubation with beta-amyloid precursor protein 751 displayed decreases in the relative size of the calcium channel response to amyloid beta peptide. A membrane-permeable analog of cyclic GMP, a second messenger believed to be involved in the calcium regulation process mediated by beta-amyloid precursor proteins, also attenuated the modulatory calcium channel response. Co-application of beta-amyloid precursor protein 751 with amyloid beta peptide did not alter calcium channel response to amyloid beta peptide. Taken together, these findings suggest that secreted beta-amyloid precursor proteins can suppress a calcium channel response to amyloid beta peptide that is potentially injurious to the cell, and as such, may define a neuroprotective mechanism that is specific for amyloid beta toxicity.

Regulation of cytokine secretion and amyloid precursor protein processing by proinflammatory amyloid beta (A beta).

Neurodegenerative processes in Alzheimer's disease (AD) are thought to be driven, in part, by the deposition of amyloid beta (A beta), a 39-43-aminoacid peptide product resulting from an alternative cleavage of amyloid precursor protein (APP). In addition to its neurotoxic properties, A beta may influence neuropathology by stimulating glial cell cytokine and acute phase protein secretion in affected areas of the brain (e.g., cortex, hippocampus). Using an in vitro human astrocyte model (U-373 MG astrocytoma cells), the effects of A beta treatment on acute phase protein (APP and alpha-1-antichymotrypsin [alpha 1-ACT]) and interleukin-8 (IL-8) were examined. U-373 MG cells secreted increased levels of alpha 1-ACT and neurotrophic/neuroprotective alpha-cleaved APP (alpha APP) after exposure to interleukin-1 beta (IL-1 beta) for 24 hours. A beta treatment resulted in a similar, but modest increase in alpha 1-ACT secretion, a two- to threefold stimulation of IL-8 production, and, conversely, a profound reduction in the levels of secreted alpha APPs. A beta inhibited alpha APP secretion by U-373 MG cells in a concentration- and conformation-dependent manner. Moreover, the reduction in alpha APP secretion was accompanied by an increase in cell-associated APP. Another proinflammatory amyloidogenic peptide, human amylin, similarly affected APP processing in U-373 astrocytoma cells. These data suggest that A beta may contribute to Alzheimer's-associated neuropathology by lowering the production of neuroprotective/neurotrophic alpha APPs. Moreover, the concomitant increase in cell-associated APP may provide increased substrate for the generation of amyloidogenic peptides within astrocytes.

Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons.

Neurotoxicity induced by overstimulation of N-methyl-D-aspartate (NMDA) receptors is due, in part, to a sustained rise in intracellular Ca2+; 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 1beta-converting enzyme (ICE)/CED-3-related protease, CPP32/Yama/apopain (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 glutamate-induced apoptosis of CGNs is mediated by a posttranslational activation of the ICE/CED-3-related cysteine protease caspase 3.

Neuronal membrane conductance activated by amyloid beta peptide: importance of peptide conformation.

Whole-cell voltage-clamp recording and circular dichroism (CD) spectroscopy were used to assess the importance of amyloid beta peptide (A beta) conformation for eliciting homeostatically disruptive membrane conductances in embryonic rat hippocampal neurons. A beta that assumed a random coil conformation when freshly dissolved in water did not alter cell resting ('leak') membrane conductances. In contrast, after several days incubation ('aging'), the same peptide samples became capable of activating a large, rapid onset and potentially toxic increase in leak membrane conductance that coincided temporally with a transition in peptide conformation from random coil to beta-sheet. Interestingly, this membrane activity was not mimicked with chemically equivalent A beta s that immediately adopted a beta-sheet conformation in water ('pre-aged'). These findings suggest that, under conditions that allow for a gradual transition of random coil A beta to beta-sheet structures, peptide conformation may be an important determinant of the toxic consequences of A beta-mediated membrane conductances.