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

Abstract

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-amyloid_25–35 peptide (5–50 m̈M) induced a slow, concentration-dependent apoptotic neurotoxicity (25–85%) during a 6 day exposure. Co-incubation of cultures with β-amyloid_25–35 peptide (25 μM) and inhibitors of nitric oxide synthase and/or xanthine oxidase (N^G-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.