Cooperation between glutathione depletion and protein synthesis inhibition against naturally occurring neuronal death

doi:10.1016/S0306-4522(98)00110-9

Neuroscience

Volume 86, Issue 3, 8 June 1998, Pages 895-902

https://doi.org/10.1016/S0306-4522(98)00110-9 Get rights and content

Abstract

It is generally agreed that naturally-occurring neuronal death in developing animals is dependent on the synthesis of proteins. Oxidative stress, as when intracellular concentrations of free radicals are raised or when cell constituents such as membrane lipids or protein thiols are oxidized, is also involved in various types of neuronal death. In the present report, we show that the number of naturally dying retinal cells in the chick embryo can be reduced by intraocular injections of cycloheximide, an inhibitor of protein synthesis. l-buthionine-[S,R]-sulfoximine, an inhibitor of glutathione synthesis, can either enhance or diminish the cell death, depending on the conditions of treatment. Moreover, when the two inhibitors are combined, l-buthionine-[S,R]-sulfoximine potentiates the neuroprotective effects of cycloheximide. Measurements of retinal glutathione concentration and protein synthesis show the specificity of the treatments: buthionine-sulfoximine diminishes glutathione concentrations but not protein synthesis whereas cycloheximide inhibits protein synthesis without decreasing glutathione concentrations.

Naturally-occurring neuronal death thus seems to involve the synthesis of proteins, and is also influenced by oxidative phenomena. Our results extend previous data in tectal-lesioned embryos, and suggest that a moderate, non-lethal oxidative stress can enhance the resistance of ganglion cells that might otherwise have died (spontaneously or following axotomy) owing to insufficient retrograde trophic support.

Section snippets

Animals and products

White Leghorn chick embryos were purchased from Pulfer Geflügefarm (Thörishaus, Switzerland), BSO and cycloheximide from Sigma, l-[4,5-³H]leucine from Amersham Life Science, and Reagents for biochemical assays from Sigma or Fluka (Buchs, Switzerland).

The embryos received an injection into the right eye of BSO, cycloheximide, or vehicle (0.9% NaCl), the volume injected was adjusted to 5 μl, as described previously.[8]Injections were performed after 12 embryonic days of incubation (E12, E0 being

Effects of intraocular injections on glutathione retinal concentrations

Intraocular injection of BSO at E12, E12+8h, or E12+16h did not modify the survival of the embryos as compared to NaCl-treated embryos (i.e. 80% of the embryos survived until E13). The concentration of GSH in NaCl-treated embryos ranged between 15.77 and 23.65 nmol/mg of protein; comparable concentrations have been reported in bovine,[54]guinea-pig,[32]and human retinas.[47]BSO induced a dose-dependent reduction of GSH retinal concentrations (Table 1). When injected at E12, the lowest dose of

The nature of the cells counted as pyknotic

The present report describes the effects on naturally-occurring neuronal death of BSO, a GSH depleting agent, and of cycloheximide, a protein synthesis inhibitor. The data confirm and extend our previous report of the synergist effects of the two drugs on axotomized ganglion cells.[9]

The ganglion cell layer of the chick embryo contains the ganglion cells, and the displaced amacrine cells. Displaced amacrine cells represent 22 to 24% of the neurons in E10–E15 chick embryos[34]and 30–35% after

Conclusions

We have previously shown that axotomy-induced ganglion cell death can be reduced by antioxidants[8]and cycloheximide,8, 9suggesting that ganglion cells die through mechanisms involving oxidative stress and protein synthesis.6, 31Moreover, axotomy-induced ganglion cell death could be nearly totally inhibited by combinations of cycloheximide and BSO,[9]suggesting cell death-regulating interactions between oxidative processes and the protein synthesis machinery.[58]Our present data show that, as

Acknowledgements

This study was supported by the Swiss National Foundation for Scientific Research, grants 31-40709.94 and 31-50598.97 and by the Swiss Federal Office for Education and Science (EU Bio4 CT 96-0649). We thank L. Grollimund and N. Turrian for their skilful technical support.

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Cited by (14)

Expression of cystatin C prevents oxidative stress-induced death in PC12 cells
2005, Brain Research Bulletin
Cystatin C, an inhibitor of cysteine proteinases, is suggested to be involved in oxidative stress-induced apoptosis of cultured CNS neurons and various neuronal diseases in vivo; however, little is known about its mechanism of action. To address the role cystatin C plays in oxidative stress-induced neuronal cell death, we established PC12 cell lines that stably expressed rat cystatin C. These cystatin C-expressing PC12 cells showed remarkable resistance to high (50%) oxygen atmosphere. This resistance correlate with expression levels of cystatin C, demonstrating that cystatin C has a protective effect on high oxygen-induced cell death. In contrast, in a normal (20%) oxygen atmosphere neither control nor cystatin C-expressing PC12 cells showed a significant change in the number of living cells, indicating that cystatin C does not play an important role in the regulation of cellular proliferation. Furthermore, the cystatin C-expressing cell line also resisted other oxidative stresses, including glutamate- and 13-l-hydroperoxylinoleic acid (LOOH)-induced cell death. These results demonstrate that cystatin C has protective effects against various oxidative stresses that induce cell death.
Dual role of the NF-κB transcription factor in the death of immature neurons
2001, Neuroscience
We have previously shown that the extent of axotomy-induced death of retinal ganglion cells is reduced by cycloheximide, an inhibitor of protein synthesis, and that an earlier sublethal oxidative insult induced by buthionine sulfoximine, a glutathione synthesis inhibitor, enhances the protective effects of cycloheximide. Thus, axotomy-induced ganglion cell death seems to involve an interaction between the redox status and genetic expression. The redox-sensitive transcription factor nuclear factor-κB (NF-κB) is a logical candidate for providing this interaction. In the present study, we injected intraocularly selective inhibitors of NF-κB in chick embryos either unlesioned, or after a unilateral tectal lesion, which axotomizes ganglion cells. The number of dying cells in the retina contralateral to the lesion was reduced in embryos receiving NF-κB inhibitors as compared with vehicle-injected controls. In contrast, the same NF-κB inhibitors administered as pretreatment before intraocular injection of buthionine sulfoximine and cycloheximide drastically raised neuronal death and induced fulgurant degenerative changes in the retina.
The most parsimonious interpretation of our results is that in axotomized retinal ganglion cells of chick embryos NF-κB may have either death-promoting or death-inhibiting effects. We propose a theoretical model to explain these dual effects assuming the existence of parallel death pathways differently affected by NF-κB. These results may have implications for future redox-based therapeutic strategies for neuroprotection.
Relationships between neuronal death and the cellular redox status. Focus on the developing nervous system
1999, Progress in Neurobiology
During the development of the nervous system, a large number of neurons are eliminated through naturally occurring neuronal death. Many morphological and biochemical properties of such dying neurons are reminiscent of apoptosis, a type of death involving the action of genetically-programmed events but also epigenetic phenomena including oxidative stress.
The following review contains three parts focusing respectively on basic knowledge of neuronal death and redox regulation, the mechanisms involved in neuronal death which are ordered in three sequential phases, and on the complex relations between neuronal fate and the redox status.
Finally, we point out that oxidants are not always detrimental for neuronal survival. On the one hand, dying neurons often display signs of oxidative stress, including an elevation of their intracellular concentration of free radicals.
Antioxidants may reduce the extent of neuronal death, suggesting a causal implication of free radicals in the death-process. On the other hand, at high concentrations antioxidants may lose their protective effects on developing neurons, and a non-lethal oxidative stress may potentiate the protective effects of other agents.
These data suggest that free radicals, perhaps through their effects on cellular signalling pathways, may have positive effects on neuronal survival, provided that their intraneuronal concentrations are maintained at low levels.
Much evidence suggests that the neuronal redox status must be maintained within a narrow range of values compatible with survival. Antioxidants may protect neurons subjected to an oxidative stress following axotomy or trophic factor-deprivation; but excessive reduction may become equally detrimental for neurons.
Inhibitors of mitogen-activated protein kinases protect axotomized developing neurons
1999, Brain Research
Axotomy kills developing neurons by mechanisms dependent on protein synthesis and influenced by the redox status. Amongst the redox-regulated transduction systems regulating gene expression are the mitogen-activated protein kinases (MAPKs). In the chick embryo, inhibitors of two different MAPK pathways, including notably the p38 kinase pathway, reduce the number of dying axotomized retinal ganglion cells. The regulation of the genetic events associated to axotomy-induced death thus seems to involve MAPKs.
Protection of axotomized ganglion cells by salicylic acid
1999, Brain Research
Neuronal survival is influenced by the redox environment, and it has been shown that antioxidants protect developing neurons from the effects of axotomy. Here, we show that the intraocular injection of salicylic acid (SA) reduces the number of dying axotomized ganglion cells in the chick embryo. The antioxidant properties of SA are probably responsible for its protective effects, whose U-shaped dose-dependency matches that of several other antioxidants. We conclude that SA protects axotomized neurons by maintaining the redox status near an optimal set-point.
An optimal redox status for the survival of axotomized ganglion cells in the developing retina
1999, Neuroscience
The neuronal redox status influences the expression of genes involved in neuronal survival. We previously showed that antioxidants may reduce the number of dying ganglion cells following axotomy in chick embryos. In the present study, we show that various antioxidants, including the new spin trap azulenyl nitrone and 1,3-dimethyl-2-thiourea, protect axotomized ganglion cells, confirming that neuronal death involves an imbalance of the cellular redox status towards oxidation. However, high concentrations of antioxidants did not protect ganglion cells, suggesting that excessive reduction is detrimental for neurons. Simultaneous injections of two different antioxidants gave results only partly supporting this view. Combinations of azulenyl nitrone and N-acetyl cysteine in fact gave greater protection than either antioxidant alone, whereas N-acetyl cysteine lost its neuroprotective effects and diminished those of α-phenyl-N-tert-butyl nitrone when the two compounds were injected simultaneously. The results of the combined treatments suggest that azulenyl nitrone and α-phenyl-N-tert-butyl nitrone do not have the same chemical effects within the ganglion cells. Moreover, N-acetyl cysteine's own antioxidant properties enhance the spin trapping effects of azulenyl nitrone but potentiate the toxicity of α-phenyl-N-tert-butyl nitrone.
Our main conclusion is that neuronal survival requires the maintenance of the redox status near an optimal set-point. “Reductive stress” may be as dangerous as oxidative stress.

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Cooperation between glutathione depletion and protein synthesis inhibition against naturally occurring neuronal death

Abstract

Section snippets

Animals and products

Effects of intraocular injections on glutathione retinal concentrations

The nature of the cells counted as pyknotic

Conclusions

Acknowledgements

Devl Brain Res.

Meth. Cell Biol.

Eur. J. Pharmac.

Comp. Biochem. Physiol.

Neuron

J. biol. Chem.

Expl Neurol.

Expl Eye Res.

Neurosci. Lett.

J. biol. Chem.

Neuroscience

J. biol. Chem.

Meth. Enzym.

Devl Biol.

Free Radic. Biol. Med.

Neuroscience

Brain Res.

Expl Eye Res.

Free Radic. Biol. Med.

Biochem. Pharmac.

Expl Eye Res.

Neurosci. Lett.

Regulation of cyclin-dependent kinase inhibitor p21(WAF1/Cip1/Sdi1) gene expression in hepatic regeneration

Hepatology

Effect of nonprotein thiols on protein synthesis in isolated rat hepatocytes

Experientia

Removal of serum from primary cultures of cerebellar granule neurons induces oxidative stress and DNA fragmentation: protection with antioxidants and glutamate receptor antagonists

J. Neurosci. Res.

Hyperthermia protects against light damage in the rat retina

Science

Limits to the dependence of developing neurons on protein synthesis in their axonal target territory

Anat. Embryol.

Programmed cell death in the developing nervous system

Brain Pathol.

Hyperthermia and hypoxia increase tolerance of retinal ganglion cells to anoxia and excitotoxicity

Invest. Ophthal. vis. Sci.

Axotomy-induced retinal ganglion cell death in development: its time-course and its diminution by antioxidants

Proc. R. Soc. Lond.

Neuronal death during development in the isthmo-optic nucleus of the chick: sustaining role of afferents from the tectum

J. comp. Neurol.