Inhibitors of free radical formation fail to attenuate direct beta-amyloid25-35 peptide-mediated neurotoxicity in rat hippocampal cultures.

Abstract

The direct neurotoxic action of the beta-amyloid protein, the major constituent of senile plaques, may represent the underlying cause of neuronal degeneration observed in Alzheimer's disease. The apoptotic-mediated neuronal death induced by beta-amyloid appears to reside in its ability to form Ca(2+)-permeable pores in neuronal membranes resulting in an excessive influx of Ca2+ and the induction of neurotoxic cascades. It is possible that during beta-amyloid exposure a Ca(2+)-mediated increase in free radical generation may exceed the defensive capacity of cells and thus lead to cell death. Consequently, in the present study we have investigated the effect of a panoply of antioxidants and inhibitors of free radical formation on the development of beta-amyloid neurotoxicity. Acute exposure of rat hippocampal neurons to "aged" beta-amyloid25-35 peptide (5-50 microM) induced a slow, concentration-dependent apoptotic neurotoxicity (25-85%) during a 6 day exposure. Co-incubation of cultures with beta-amyloid25-35 peptide (25 microM) and inhibitors of nitric oxide synthase and/or xanthine oxidase (NG-monomethyl-L-arginine [1 mM), N omega-nitro-L-arginine [1 mM], oxypurinol [100 microM], allopurinol [100 microM]), important mediators of nitric oxide, superoxide, and hydroxyl radical formation, did not attenuate beta-amyloid neurotoxicity. Similarly, a reduction in free radical generation by selective inhibition of phospholipase-A2 cyclooxygenase, and lipoxygenase activities with quinacrine (0.5 microM), indomethacin (50 microM), and nor-dihydroguaiaretic acid (0.5 microM), respectively, did not reduce the proclivity of beta-amyloid to induce cell death. Exposure of cultures to catalase (25 U/ml) and/or superoxide dismutase (10 U/ml) as well as the free radical scavengers vitamin E (100 microM), vitamin C (100 microM), glutathione (100 microM), L-cysteine (100 microM), N-acetyl-cysteine (100 microM), deferoxamine (5 microM), or haemoglobin (35 micrograms/ml) failed to attenuate the neurotoxic action of beta-amyloid. On the other hand, pre-treatment of cultures with subtoxic concentrations of beta-amyloid peptide significantly increased the vulnerability of neurons to H2O2 exposure and suggest that beta-amyloid peptide renders neurons more sensitive to free radical attack. However, a potential beta-amyloid-mediated increase in free radical formation is not a proximate cause of the neurotoxic mechanism of beta-amyloid in vitro.