Mechanisms of caspase-independent neuronal death: energy depletion and free radical generation.

Cultured rat embryonic cortical neurons undergo apoptosis when treated with the topoisomerase-I inhibitor camptothecin. Pharmacological or molecular caspase inhibition prevents apoptosis, but the neurons still die in a delayed nonapoptotic manner. Here we examine the mechanisms leading to such caspase-independent death, focusing on events related to mitochondrial malfunction, which accompanies this delayed death. Given that mitochondria are the major source of ATP in primary neurons, we examined the cellular energy state. Mitochondrially generated ATP was specifically reduced in neurons treated with camptothecin and Boc-aspartyl-fluoromethylketone. Augmentation of cellular ATP by manipulation of the glucose content in the cultures led to an increase in survival specifically in delayed caspase-independent but not early caspase-dependent death. As another possible consequence of mitochondrial malfunction, we found an induction of reactive oxygen species in delayed death. The free radical scavenger Tempol, but not other classes of antioxidants, reduced oxidative stress and promoted survival. Other potential events known to be a direct or indirect consequence of mitochondrial dysfunction, such as the induction of autophagy, release of apoptosis-inducing factor, or opening of the mitochondrial permeability transition pore, were not found to play a significant role in caspase-independent neuronal death. Combining the strategies of increasing intracellular ATP and reducing free radicals led to an additive increase in neuronal survival. We conclude that energy failure and free radical generation contribute to caspase-independent neuronal death. Both could represent potential targets for therapeutic interventions complementary to caspase inhibition.