Potassium Deprivation-Induced Apoptosis of Cerebellar Granule Neurons: A Sequential Requirement for New mRNA and Protein Synthesis, ICE-Like Protease Activity, and Reactive Oxygen Species

Abstract

Potassium (K ⁺) deprivation-induced apoptosis of cerebellar granule neurons requires new mRNA and protein synthesis. Using a fluorogenic substrate for interleukin-1β converting enzyme (ICE), we show that K ⁺ deprivation of cerebellar granule neurons induces cycloheximide-sensitive ICE-like protease activity. A peptide inhibitor of ICE-like protease activity, Ac-YVAD-chloromethylketone (Ac-YVAD-CMK), prevents K ⁺ deprivation-induced apoptosis. Further, reactive oxygen species (ROS) are essential mediators of K ⁺ deprivation-induced apoptosis of cerebellar granule neurons because neuronal death is also blocked by superoxide dismutase, N-acetyl- l-cysteine, and free radical spin traps. Using fluorescent assays, we show that ROS production after K ⁺ deprivation is blocked by actinomycin D, cycloheximide, and Ac-YVAD-CMK, suggesting that ROS act downstream of gene transcription, mRNA translation, and ICE activation. Taken together, we show that new mRNA and protein synthesis, activation of ICE-like proteases, and ROS production are sequential events in K ⁺ deprivation-induced apoptosis of cerebellar granule neurons.