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

The direct neurotoxic action of the β‐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 β‐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 β‐amyloid exposure a Ca2+‐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 β‐amyloid neurotoxicity. Acute exposure of rat hippocampal neurons to “aged” β‐amyloid25–35 peptide (5–50 m̈M) induced a slow, concentration‐dependent apoptotic neurotoxicity (25–85%) during a 6 day exposure. Co‐incubation of cultures with β‐amyloid25–35 peptide (25 μM) and inhibitors of nitric oxide synthase and/or xanthine oxidase (NG‐monomethyl‐L‐arginine [1 mM], Nω‐nitro‐L‐arginine [1 mM], oxypurinol [100 m̈M], allopurinol [100 m̈M]), important mediators of nitric oxide, superoxide, and hydroxyl radical formation, did not attenuate β‐amyloid neurotoxicity. Similarly, a reduction in free radical generation by selective inhibition of phospholipase‐A2 cyclooxygenase, and lipoxygenase activities with quinacrine (0.5 m̈M), indomethacin (50 m̈M), and nor‐dihydroguaiaretic acid (0.5 m̈M), respectively, did not reduce the proclivity of β‐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 m̈M), vitamin C (100 m̈M), glutathione (100 m̈M), L‐cysteine (100 m̈M), N‐acetyl‐cysteine (100 m̈M), deferoxamine (5 m̈M), or haemoglobin (35 m̈g/ml) failed to attenuate the neurotoxic action of β‐amyloid. On the other hand, pre‐treatment of cultures with subtoxic concentrations of β‐amyloid peptide significantly increased the vulnerability of neurons to H2O2 exposure and suggest that β‐amyloid peptide renders neurons more sensitive to free radical attack. However, a potential β‐amyloid‐mediated increase in free radical formation is not a proximate cause of the neurotoxic mechanism of β‐amyloid in vitro. © 1994 Wiley‐Liss, Inc.