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

Abstract

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.

Genes Regulated in Neurons Undergoing Transcription-dependent Apoptosis Belong to Signaling Pathways Rather than the Apoptotic Machinery

“…1c) because, at that time, genes necessary to apoptosis induction have already been expressed. Our data were consistent with results reported by others in mouse CGN (17): transcriptional and translational commitment points were ϳ5 h after deprivation. The temporal curves of apoptosis (Fig.…”

R‐Deprenyl and R‐2‐Heptyl‐N‐Methylpropargylamine Prevent Apoptosis in Cerebellar Granule Neurons Induced by Cytosine Arabinoside but Not Low Extracellular Potassium

“…In the present study, we have shown that there is a loss of mitochondrial function (as assessed by CMTMR accumulation) associated with ara C-induced apoptosis but not low K~-induced apoptosis. The latter result appears to contradict the findings of Nardi et al (1997), who reported that there is a rapid loss of ATP and mitochondrial function [assessed by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay] following K + withdrawal from mouse cerebellar granule cells, but there are several major differences between the studies. Nardi et a!.…”

“…1), and in our studies we found that K~withdrawal while serum was maintained produced a slower, less extensive degree of apoptosis. Because Nardi et al (1997) reported that decreases in ATP levels follow but occur later than the loss of neuronal viability and we see no change in CMTMR accumulation in low K + -induced apoptosis, it is likely that the commitment to apoptosis following K + withdrawal does not involve or does not depend on a loss of mitochondrial function. The ara C-induced apoptosis, however, does involve a loss of mitochodrial function early in the apoptotic process (Fig.…”

Contribution of serine racemase/d‐serine pathway to neuronal apoptosis

“…This neuronal death occurs by a sequence of apoptosis-autophagy and necrosis phenomena and is hampered when neurons are grown in medium containing depolarizing levels of potassium (25 mM KCl) or moderate concentration of NMDA. It is assumed that both factors, through different signal transduction pathways, mimic the pro-survival electrical stimulation from afferent synapses (Gallo et al, 1987;D'Mello et al, 1993;Nardi et al, 1997;Xifro et al, 2005). NMDAR-mediated prosurvival activity is, in part, mediated through the activation of PI3K and MAPK pathways (Zhang et al, 1998;Hetman & Kharebava, 2006).…”