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Regulation of antioxidant metabolism by translation initiation factor 2alpha.

Tan S, Somia N, Maher P, Schubert D - J. Cell Biol. (2001)

Bottom Line: The phosphorylation of eIF2alpha also results in resistance to oxidative stress.In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca(2+).Therefore, eIF2alpha is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Cellular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

ABSTRACT
Oxidative stress and highly specific decreases in glutathione (GSH) are associated with nerve cell death in Parkinson's disease. Using an experimental nerve cell model for oxidative stress and an expression cloning strategy, a gene involved in oxidative stress-induced programmed cell death was identified which both mediates the cell death program and regulates GSH levels. Two stress-resistant clones were isolated which contain antisense gene fragments of the translation initiation factor (eIF)2alpha and express a low amount of eIF2alpha. Sensitivity is restored when the clones are transfected with full-length eIF2alpha; transfection of wild-type cells with the truncated eIF2alpha gene confers resistance. The phosphorylation of eIF2alpha also results in resistance to oxidative stress. In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca(2+). In contrast, the resistant clones maintain high GSH levels and show no elevation in peroxides or Ca(2+) when stressed, and the GSH synthetic enzyme gamma-glutamyl cysteine synthetase (gammaGCS) is elevated. The change in gammaGCS is regulated by a translational mechanism. Therefore, eIF2alpha is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.

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ROS and Ca2+ levels after exposure to glutamate. ROS levels were measured by flow cytometry using the fluorescent dye DCF. (A) ROS levels increase 72-fold (gray line) in wild-type HT22 cells exposed to 5 mM glutamate for 10 h. However, ROS levels do not increase in resistant clones 8 and 15 after 10-h exposure to glutamate. (B) ROS levels after exposure to glutamate are increased (gray line) in wild-type HT22 cells and in HT22 cells infected with empty vector or the S51A mutant of eIF2α. ROS levels do not increase in HT22 cells expressing the eIF2α mutant S51D when exposed to 2 mM glutamate for 10 h. 10,000 live cells were assayed, and the experiment was repeated two times with similar results. (C) Cytosolic Ca2+ levels were measured using flow cytometry and the ratiometric dye Indo-1. HT22 cells exposed to 5 mM glutamate for 10 h have a large increase in cytosolic Ca2+ compared with untreated cells. After glutamate exposure, resistant clones 8 and 15 maintain cytosolic Ca2+ levels similar to the wild-type untreated control. (D) HT22 cells exposed to 2 mM glutamate for 10 h have a large increase in cytosolic Ca2+. HT22 cells infected with the empty vector or the S51A mutant of eIF2 also show similar increases in Ca2+. The S51D mutant of eIF2α prevents the glutamate-induced increase in Ca2+ when stably expressed in HT22 cells. All samples were prepared in duplicate. 10,000 live cells were assayed in each experiment, and the study was repeated twice with similar results.
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Figure 6: ROS and Ca2+ levels after exposure to glutamate. ROS levels were measured by flow cytometry using the fluorescent dye DCF. (A) ROS levels increase 72-fold (gray line) in wild-type HT22 cells exposed to 5 mM glutamate for 10 h. However, ROS levels do not increase in resistant clones 8 and 15 after 10-h exposure to glutamate. (B) ROS levels after exposure to glutamate are increased (gray line) in wild-type HT22 cells and in HT22 cells infected with empty vector or the S51A mutant of eIF2α. ROS levels do not increase in HT22 cells expressing the eIF2α mutant S51D when exposed to 2 mM glutamate for 10 h. 10,000 live cells were assayed, and the experiment was repeated two times with similar results. (C) Cytosolic Ca2+ levels were measured using flow cytometry and the ratiometric dye Indo-1. HT22 cells exposed to 5 mM glutamate for 10 h have a large increase in cytosolic Ca2+ compared with untreated cells. After glutamate exposure, resistant clones 8 and 15 maintain cytosolic Ca2+ levels similar to the wild-type untreated control. (D) HT22 cells exposed to 2 mM glutamate for 10 h have a large increase in cytosolic Ca2+. HT22 cells infected with the empty vector or the S51A mutant of eIF2 also show similar increases in Ca2+. The S51D mutant of eIF2α prevents the glutamate-induced increase in Ca2+ when stably expressed in HT22 cells. All samples were prepared in duplicate. 10,000 live cells were assayed in each experiment, and the study was repeated twice with similar results.

Mentions: To understand the role of eIF2α in oxidative glutamate toxicity, several parameters of the glutamate response were measured in the resistant clones and the S51A and S51D mutant-expressing cell lines and compared with the wild-type HT22 cells. HT22 cells undergo a rapid depletion of GSH upon exposure to glutamate (Tan et al. 1998b). After 8 h of exposure to glutamate, GSH levels drop below 20% of their normal levels. Comparison of wild-type HT22 cells to glutamate-resistant clones 8 and 15 after 10 h of exposure to 5 mM glutamate revealed that the GSH levels in the resistant cells do not go below 50% of the GSH levels in untreated resistant clones. Furthermore, before glutamate exposure, both cell lines have higher GSH levels than untreated wild-type HT22 cells (Fig. 5 A). The maximal difference in survival between the S51D mutant-expressing cell line and the control HT22 cells is detected at 2 mM glutamate (Fig. 3). When GSH levels in wild-type cells infected with the S51A or S51D mutants or the empty vector are measured after 10 h of exposure to 2 mM glutamate, the S51D mutant cell line shows a decrease to ∼50% of the original level compared with the 70% decrease in the wild-type and empty vector–infected cells. On the other hand, the S51A mutant cell line shows a decrease in GSH to ∼20% of control levels (Fig. 5 B). This pattern of GSH depletion is consistent with the survival data which demonstrate that although the S51D-expressing HT22 cells are still healthy and dividing after 24 h of glutamate exposure, the other cell lines are dead (Fig. 3). HT22 cells exposed to glutamate for 10 h show a very large increase in ROS which follows the drop in GSH (Tan et al. 1998b). The fluorescent dye DCF was used to determine the levels of ROS production by flow cytometry in the resistant and mutant cell lines after exposure to toxic levels of glutamate. The level of ROS in wild-type HT22 cells after exposure to 5 mM glutamate for 10 h is increased >70-fold (Fig. 6 A). In contrast, the glutamate-resistant cell lines 8 and 15 do not show an increase in ROS above normal levels, and the cells survive and continue to divide (Fig. 6 A). When HT22 cells are exposed to 2 mM glutamate for 10 h, the cells die and there is an increase in ROS, although the DCF intensity is more diffuse than with 5 mM glutamate (Fig. 6 B). The same pattern of increased DCF is seen in cells expressing empty vector and the mutant S51A. However, HT22 cells expressing the mutant S51D have low levels of ROS and were able to survive glutamate treatment (Fig. 6 B). Finally, Ca2+ influx was measured in wild-type, resistant, and phosphorylation mutant-expressing cells. Ca2+ levels were determined by FACS® analysis using the ratiometric dye Indo-1 (Tan et al. 1998b). After 10 h of exposure to 5 mM glutamate, HT22 cells have much higher levels of Ca2+ than untreated controls, whereas resistant cell lines 8 and 15 maintained intracellular Ca2+ levels similar to those of the wild-type HT22 cells (Fig. 6 C). The same experiment was performed on the phosphorylation mutant-expressing cell lines exposed to 2 mM glutamate. Ca2+ levels increase significantly in the wild-type HT22 cells as well as the empty vector and S51A-expressing HT22 cells. The intracellular Ca2+ level in the S51D-expressing cells remained similar to the HT22 cells that were not exposed to glutamate (Fig. 6 D). These data show that both the downregulation of eIF2α in clones 8 and 15 and the expression of the dominant negative phosphorylation mutant S51D all prevent the decrease in GSH and the increases in ROS and Ca2+ normally associated with oxidative stress–induced cell death.


Regulation of antioxidant metabolism by translation initiation factor 2alpha.

Tan S, Somia N, Maher P, Schubert D - J. Cell Biol. (2001)

ROS and Ca2+ levels after exposure to glutamate. ROS levels were measured by flow cytometry using the fluorescent dye DCF. (A) ROS levels increase 72-fold (gray line) in wild-type HT22 cells exposed to 5 mM glutamate for 10 h. However, ROS levels do not increase in resistant clones 8 and 15 after 10-h exposure to glutamate. (B) ROS levels after exposure to glutamate are increased (gray line) in wild-type HT22 cells and in HT22 cells infected with empty vector or the S51A mutant of eIF2α. ROS levels do not increase in HT22 cells expressing the eIF2α mutant S51D when exposed to 2 mM glutamate for 10 h. 10,000 live cells were assayed, and the experiment was repeated two times with similar results. (C) Cytosolic Ca2+ levels were measured using flow cytometry and the ratiometric dye Indo-1. HT22 cells exposed to 5 mM glutamate for 10 h have a large increase in cytosolic Ca2+ compared with untreated cells. After glutamate exposure, resistant clones 8 and 15 maintain cytosolic Ca2+ levels similar to the wild-type untreated control. (D) HT22 cells exposed to 2 mM glutamate for 10 h have a large increase in cytosolic Ca2+. HT22 cells infected with the empty vector or the S51A mutant of eIF2 also show similar increases in Ca2+. The S51D mutant of eIF2α prevents the glutamate-induced increase in Ca2+ when stably expressed in HT22 cells. All samples were prepared in duplicate. 10,000 live cells were assayed in each experiment, and the study was repeated twice with similar results.
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Related In: Results  -  Collection

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Figure 6: ROS and Ca2+ levels after exposure to glutamate. ROS levels were measured by flow cytometry using the fluorescent dye DCF. (A) ROS levels increase 72-fold (gray line) in wild-type HT22 cells exposed to 5 mM glutamate for 10 h. However, ROS levels do not increase in resistant clones 8 and 15 after 10-h exposure to glutamate. (B) ROS levels after exposure to glutamate are increased (gray line) in wild-type HT22 cells and in HT22 cells infected with empty vector or the S51A mutant of eIF2α. ROS levels do not increase in HT22 cells expressing the eIF2α mutant S51D when exposed to 2 mM glutamate for 10 h. 10,000 live cells were assayed, and the experiment was repeated two times with similar results. (C) Cytosolic Ca2+ levels were measured using flow cytometry and the ratiometric dye Indo-1. HT22 cells exposed to 5 mM glutamate for 10 h have a large increase in cytosolic Ca2+ compared with untreated cells. After glutamate exposure, resistant clones 8 and 15 maintain cytosolic Ca2+ levels similar to the wild-type untreated control. (D) HT22 cells exposed to 2 mM glutamate for 10 h have a large increase in cytosolic Ca2+. HT22 cells infected with the empty vector or the S51A mutant of eIF2 also show similar increases in Ca2+. The S51D mutant of eIF2α prevents the glutamate-induced increase in Ca2+ when stably expressed in HT22 cells. All samples were prepared in duplicate. 10,000 live cells were assayed in each experiment, and the study was repeated twice with similar results.
Mentions: To understand the role of eIF2α in oxidative glutamate toxicity, several parameters of the glutamate response were measured in the resistant clones and the S51A and S51D mutant-expressing cell lines and compared with the wild-type HT22 cells. HT22 cells undergo a rapid depletion of GSH upon exposure to glutamate (Tan et al. 1998b). After 8 h of exposure to glutamate, GSH levels drop below 20% of their normal levels. Comparison of wild-type HT22 cells to glutamate-resistant clones 8 and 15 after 10 h of exposure to 5 mM glutamate revealed that the GSH levels in the resistant cells do not go below 50% of the GSH levels in untreated resistant clones. Furthermore, before glutamate exposure, both cell lines have higher GSH levels than untreated wild-type HT22 cells (Fig. 5 A). The maximal difference in survival between the S51D mutant-expressing cell line and the control HT22 cells is detected at 2 mM glutamate (Fig. 3). When GSH levels in wild-type cells infected with the S51A or S51D mutants or the empty vector are measured after 10 h of exposure to 2 mM glutamate, the S51D mutant cell line shows a decrease to ∼50% of the original level compared with the 70% decrease in the wild-type and empty vector–infected cells. On the other hand, the S51A mutant cell line shows a decrease in GSH to ∼20% of control levels (Fig. 5 B). This pattern of GSH depletion is consistent with the survival data which demonstrate that although the S51D-expressing HT22 cells are still healthy and dividing after 24 h of glutamate exposure, the other cell lines are dead (Fig. 3). HT22 cells exposed to glutamate for 10 h show a very large increase in ROS which follows the drop in GSH (Tan et al. 1998b). The fluorescent dye DCF was used to determine the levels of ROS production by flow cytometry in the resistant and mutant cell lines after exposure to toxic levels of glutamate. The level of ROS in wild-type HT22 cells after exposure to 5 mM glutamate for 10 h is increased >70-fold (Fig. 6 A). In contrast, the glutamate-resistant cell lines 8 and 15 do not show an increase in ROS above normal levels, and the cells survive and continue to divide (Fig. 6 A). When HT22 cells are exposed to 2 mM glutamate for 10 h, the cells die and there is an increase in ROS, although the DCF intensity is more diffuse than with 5 mM glutamate (Fig. 6 B). The same pattern of increased DCF is seen in cells expressing empty vector and the mutant S51A. However, HT22 cells expressing the mutant S51D have low levels of ROS and were able to survive glutamate treatment (Fig. 6 B). Finally, Ca2+ influx was measured in wild-type, resistant, and phosphorylation mutant-expressing cells. Ca2+ levels were determined by FACS® analysis using the ratiometric dye Indo-1 (Tan et al. 1998b). After 10 h of exposure to 5 mM glutamate, HT22 cells have much higher levels of Ca2+ than untreated controls, whereas resistant cell lines 8 and 15 maintained intracellular Ca2+ levels similar to those of the wild-type HT22 cells (Fig. 6 C). The same experiment was performed on the phosphorylation mutant-expressing cell lines exposed to 2 mM glutamate. Ca2+ levels increase significantly in the wild-type HT22 cells as well as the empty vector and S51A-expressing HT22 cells. The intracellular Ca2+ level in the S51D-expressing cells remained similar to the HT22 cells that were not exposed to glutamate (Fig. 6 D). These data show that both the downregulation of eIF2α in clones 8 and 15 and the expression of the dominant negative phosphorylation mutant S51D all prevent the decrease in GSH and the increases in ROS and Ca2+ normally associated with oxidative stress–induced cell death.

Bottom Line: The phosphorylation of eIF2alpha also results in resistance to oxidative stress.In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca(2+).Therefore, eIF2alpha is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Cellular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

ABSTRACT
Oxidative stress and highly specific decreases in glutathione (GSH) are associated with nerve cell death in Parkinson's disease. Using an experimental nerve cell model for oxidative stress and an expression cloning strategy, a gene involved in oxidative stress-induced programmed cell death was identified which both mediates the cell death program and regulates GSH levels. Two stress-resistant clones were isolated which contain antisense gene fragments of the translation initiation factor (eIF)2alpha and express a low amount of eIF2alpha. Sensitivity is restored when the clones are transfected with full-length eIF2alpha; transfection of wild-type cells with the truncated eIF2alpha gene confers resistance. The phosphorylation of eIF2alpha also results in resistance to oxidative stress. In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca(2+). In contrast, the resistant clones maintain high GSH levels and show no elevation in peroxides or Ca(2+) when stressed, and the GSH synthetic enzyme gamma-glutamyl cysteine synthetase (gammaGCS) is elevated. The change in gammaGCS is regulated by a translational mechanism. Therefore, eIF2alpha is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.

Show MeSH
Related in: MedlinePlus