Oxidation of Biomolecules in the Apoptotic Death of Cerebellar Granule Neurons Induced by Potassium Deprivation

Cerebellar granule neurons (CGN) cultured in a 25 mM KCl medium (K25) die apoptotically when they are transferred to a medium containing 5 mM KCl (K5). It has been previously shown that apoptotic death of CGN induced by K5 is mediated by an increase in the levels of reactive oxygen species (ROS). ROS may participate in the apoptotic program either as signaling molecules or as effectors by causing oxidative damage to lipids, DNA and proteins. In this study we evaluated ROS production in CGN treated with K5 for different periods of time and evaluated a possible correlation between ROS production and oxidation of DNA proteins and two lipid peroxidation products, conjugated dienes and malondialdehyde. Under these conditions, we found two episodes of ROS generation, one at an early time (4 h) and another at a later time point (18-24 h). We also identified two peaks in the formation of conjugated dienes, the initial and transient by-products of lipoperoxidation. The first one occurred after 4 h of K5 treatment and the other was observed after 18 h, both of them correlated with the formation of ROS. In contrast, we found significant levels of the late product of lipidperoxidation, malondialdehyde only after 18 h of treatment. Besides, we did not find significant levels of DNA and protein oxidation products that could be correlated with the observed ROS production. These results support the idea that ROS produced early by K5 treatment could act primarily as a signal of the apoptotic cell death and that ROS produced later could be mainly a product of the cell death that could contribute directly to this process.