Oxidative stress is implicated in neuronal apoptosis that occurs in physiological settings and in neurodegenerative disorders. Superoxide anion radical, produced during mitochondrial respiration, is involved in the generation of several potentially damaging reactive oxygen species including peroxynitrite. To examine directly the role of superoxide and peroxynitrite in neuronal apoptosis, we generated neural cell lines and transgenic mice that overexpress human mitochondrial manganese superoxide dismutase (MnSOD). In cultured pheochromocytoma PC6 cells, overexpression of mitochondria-localized MnSOD prevented apoptosis induced by Fe2+, amyloid beta-peptide (Abeta), and nitric oxide-generating agents. Accumulations of peroxynitrite, nitrated proteins, and the membrane lipid peroxidation product 4-hydroxynonenal (HNE) after exposure to the apoptotic insults were markedly attenuated in cells expressing MnSOD. Glutathione peroxidase activity levels were increased in cells overexpressing MnSOD, suggesting a compensatory response to increased H2O2 levels. The peroxynitrite scavenger uric acid and the antioxidants propyl gallate and glutathione prevented apoptosis induced by each apoptotic insult, suggesting central roles for peroxynitrite and membrane lipid peroxidation in oxidative stress-induced apoptosis. Apoptotic insults decreased mitochondrial transmembrane potential and energy charge in control cells but not in cells overexpressing MnSOD, and cyclosporin A and caspase inhibitors protected cells against apoptosis, demonstrating roles for mitochondrial alterations and caspase activation in the apoptotic process. Membrane lipid peroxidation, protein nitration, and neuronal death after focal cerebral ischemia were significantly reduced in transgenic mice overexpressing human MnSOD. The data suggest that mitochondrial superoxide accumulation and consequent peroxynitrite production and mitochondrial dysfunction play pivotal roles in neuronal apoptosis induced by diverse insults in cell culture and in vivo.

Many cases of autosomal dominant early onset Alzheimer's disease (AD) result from mutations in the gene encoding presenilin-1 (PS-1). PS-1 is an integral membrane protein expressed ubiquitously in neurons throughout the brain in which it is located primarily in endoplasmic reticulum (ER). Although the pathogenic mechanism of PS-1 mutations is unknown, recent findings suggest that PS mutations render neurons vulnerable to apoptosis. Because increasing evidence indicates that mitochondrial alterations contribute to neuronal death in AD, we tested the hypothesis that PS-1 mutations sensitize neurons to mitochondrial failure. PC12 cell lines expressing a PS-1 mutation (L286V) exhibited increased sensitivity to apoptosis induced by 3-nitropropionic acid (3-NP) and malonate, inhibitors of succinate dehydrogenase, compared with control cell lines and lines overexpressing wild-type PS-1. The apoptosis-enhancing action of mutant PS-1 was prevented by antioxidants (propyl gallate and glutathione), zVAD-fmk, and cyclosporin A, indicating requirements of reactive oxygen species (ROS), caspases, and mitochondrial permeability transition in the cell death process. 3-NP induced a rapid elevation of [Ca2+]i, which was followed by caspase activation, accumulation of ROS, and decreases in mitochondrial reducing potential and transmembrane potential in cells expressing mutant PS-1. The calcium chelator BAPTA AM and agents that block calcium release from ER and influx through voltage-dependent channels prevented mitochondrial ROS accumulation and membrane depolarization and apoptosis. Our data suggest that by perturbing subcellular calcium homeostasis presenilin mutations sensitize neurons to mitochondria-based forms of apoptosis that involve oxidative stress.