Amyloid beta-mediated oxidative and metabolic stress in rat cortical neurons: no direct evidence for a role for H2O2 generation.

H2O2 and free radical-mediated oxidative stresses have been implicated in mediating amyloid beta (1-40) [A beta (1-40)] neurotoxicity to cultured neurons. In this study, we confirm that addition of the H2O2-scavenging enzyme catalase protects neurons in culture against A beta-mediated toxicity; however, it does so by a mechanism that does not involve its ability to scavenge H2O2. A beta-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 A beta treatment: (a) glyceraldehyde-3-phosphate dehydrogenase activity, (b) hexose monophosphate shunt activity, and (c) glucose oxidation via the tricarboxylic acid cycle. lonspray mass spectra of A beta in the incubation medium indicated that A beta 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 A beta. Our results challenge a pivotal role for H2O2 generation in mediating A beta toxicity, and we suggest that impairment of energy homeostasis may be a more significant early factor in the neurodegenerative process.

Impairment of integrin-mediated cell-matrix adhesion in oxidant-stressed PC12 cells.

Oxidants are believed to play an important and complex role in neuronal injury and death in the aging process and various neurode generative diseases. We studied the effect of oxidative stress on integrin-mediated cell-extracellular matrix (ECM) interactions using the PC12 neuronal cell line. In assays in which attachment was measured between 30 and 90 min, addition of hydrogen peroxide (H2O2) to the attachment medium resulted in a dose-dependent inhibition of initial cell attachment to collagen. Addition of H2O2 also caused previously attached cells to detach from collagen. The inhibition by H2O2 was specific for integrin-mediated adhesion, since attachment to substrata coated with non-ECM molecules was much less affected. Exposure of cells to H2O2 resulted in a rapid and profound reduction of intracellular ATP, accompanied by only a slight increase in intracellular free Ca2+ concentration ([Ca2+]i). Treatment of cells with the microfilament-disrupting agent, cytochalasin B, like that with H2O2, inhibited cell adhesion to collagen. We propose that integrin-mediated cell adhesion, which requires interactions between cytoplasmic portions of integrin subunits and cytoskeletal microfilaments, is impaired by oxidative stress as a result of the depletion of intracellular ATP and that such depletion is an early event in the process of oxidant-induced neuronal injury.

Human cortical neuronal (HCN) cell lines: a model for amyloid beta neurotoxicity.

Human cortical neuronal cell lines HCN-1A and HCN-2 are killed for following exposure of the differentiated cells to amyloid beta-peptide(1-40), a component of senile plaques and other amyloid deposits in brains from Alzheimer's patients. We present a model of A beta toxicity uncomplicated by the presence of other cell types that can be used to address the mechanism of A beta neurotoxicity. This model will be useful in the evaluation of neuroprotective compounds which may attenuate cortical neuronal loss in Alzheimer's disease.