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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 Kconcentration ([K~] 0),a significant part of the genomic DNA was already cleaved to highmolecular-weight fragments. This phenomenon was intensified with the progression of the death process. Addition of cycloheximide to the neurons 4 h after high [K~]0 deprivation resulted in no cell loss and complete recovery of the damaged DNA. DNA margination and nuclearfragmentation as assessed by 4,6-diaminodiphenyl-2-phenylindole staining were observable in a few cells beginning .-~4 h after the removal of high [K~]0and developed to nuclear condensation 4 h later. Six hours after high [K~]0 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~]0. Similar to high [K~]0 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~]0 removal. An~-~40% decrease in RNA and protein synthesis was detected by 6 h of [K~]0removal 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 activityevents that occurred within the critical time-may indicate that these processes have an important part in the mechanism that committed the neurons to die.

1. The monoamines dopamine (DA), norepinephrine (NE), epinephrine (E), and serotonin (5-HT) serve as endogenous neurotransmitters in the nervous system. We recently reported that the neurotransmitter DA can trigger apoptosis (programmed cell death; PCD) in cultured, postmitotic chick embryo sympathetic neurons, suggesting a role for apoptosis in degenerative processes such as Parkinson's disease (PD). However, PD is also associated with involvement of other monoaminergic (MA) neuronal systems (noradrenergic and serotoninergic), though to a lesser extent. 2. We therefore tested the apoptosis-triggering potential of NE, E, and 5-HT in comparison to the DA effect, in cultured postmitotic nerve growth factor (NGF)-dependent chick embryo sympathetic neurons and mouse cerebellar granule cells. 3. In both model systems MA induced neuronal attrition characteristic of apoptosis. MA caused marked morphological alterations: severe neuronal soma shrinkage, membrane blebbing, nuclear condensation and fragmentation, and axonal disintegration. Flow-cytometric analysis of propidium iodide-stained cell nuclei revealed characteristic apoptotic nuclear fragmentation. MA-induced apoptosis could be blocked by SH-group containing antioxidants but not by inhibitors of transcription and translation. 4. Comparison between the two model systems revealed that the cerebellar granule neurons were distinctly more sensitive to the neurotoxic potential of the MA than sympathetic neuronal cells. Significant differences in the dose dependencies and time course of the apoptotic effect were observed among the examined MA, graded as DA > NE approximately E > 5-HT. 5. We conclude that the apoptosis triggering potential, probably mediated by oxidative metabolites, is shared by all MA tested, but with differential time course and dose dependencies. A correlation can be drawn between the effects of DA vs NE vs 5-HT and the relative involvement of dopaminergic/noradrenergic/serotoninergic pathways in PD, which may suggest a common multisystem underlying abnormality in neuronal apoptosis-control mechanisms.

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