Biochemical and Temporal Analysis of Events Associated with Apoptosis Induced by Lowering the Extracellular Potassium Concentration in Mouse Cerebellar Granule Neurons

Abstract: We analyzed biochemically and temporally the molecular events that occur in the programmed cell death of mouse cerebellar granule neurons deprived of high potassium levels. An hour after switching the neurons to a low extracellular K⁺ concentration (K⁺]_o), a significant part of the genomic DNA was already cleaved to high-molecular-weight fragments. This phenomenon was intensified with the progression of the death process. Addition of cycloheximide to the neurons 4 h after high K⁺]_o deprivation resulted in no cell loss and complete recovery of the damaged DNA. DNA margination and nuclear fragmentation as assessed by 4,6-diaminodiphenyl-2-phenylindole staining were observable in a few cells beginning ～4 h after the removal of high K⁺]_o and developed to nuclear condensation 4 h later. Six hours after high K⁺]_o deprivation, the DNA was fragmented into oligonucleosome-sized fragments. Within 6 h after removal of the extracellular K⁺, 50% of the neurons were committed to die and lost their ability to be rescued by readministration of 25 mM K⁺]_o. Similar to high K⁺]_o deprivation, inhibition of RNA or protein synthesis failed to halt neuronal degeneration of a similar percentage of cells 6 h after the onset of the death process. Mitochondrial function steadily decreased after K⁺]_o removal. An ～40% decrease in RNA and protein synthesis was detected by 6 h of K⁺]_o removal during the period of cell death commitment; rates continued to decline gradually thereafter. The temporal characteristics of the DNA damage and recovery, DNA cleavage to oligonucleosome-sized fragments, and the reduction in mitochondrial activity—events that occurred within the critical time—may indicate that these processes have an important part in the mechanism that committed the neurons to die.