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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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GSH levels in resistant cells. GSH levels were measured in control, untreated cells, and cells exposed to 5 or 2 mM glutamate for 10 h. (A) 5 mM glutamate. Clones 8 (white bars) and 15 (hatched bars) have higher basal GSH levels than wild-type HT22 and only deplete to 72 ± 4 and 56 ± 1% of their basal GSH levels, respectively, with glutamate exposure. (B) 2 mM glutamate. Wild-type HT22 cells (black bars), empty vector infected cells (white bars), and S51A mutant infected cells (hatched bars) show GSH levels that are depleted to 20–30% of basal levels. In the S51D mutant-expressing cells (narrow hatched bars), GSH depleted to only ∼50% of the basal level. 100% GSH is defined as the GSH level assayed in the untreated control cells. The numbers above the glutamate exposed bars indicate the percentage of GSH relative to the basal level in the same cell line (n = 3).
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Figure 5: GSH levels in resistant cells. GSH levels were measured in control, untreated cells, and cells exposed to 5 or 2 mM glutamate for 10 h. (A) 5 mM glutamate. Clones 8 (white bars) and 15 (hatched bars) have higher basal GSH levels than wild-type HT22 and only deplete to 72 ± 4 and 56 ± 1% of their basal GSH levels, respectively, with glutamate exposure. (B) 2 mM glutamate. Wild-type HT22 cells (black bars), empty vector infected cells (white bars), and S51A mutant infected cells (hatched bars) show GSH levels that are depleted to 20–30% of basal levels. In the S51D mutant-expressing cells (narrow hatched bars), GSH depleted to only ∼50% of the basal level. 100% GSH is defined as the GSH level assayed in the untreated control cells. The numbers above the glutamate exposed bars indicate the percentage of GSH relative to the basal level in the same cell line (n = 3).

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)

GSH levels in resistant cells. GSH levels were measured in control, untreated cells, and cells exposed to 5 or 2 mM glutamate for 10 h. (A) 5 mM glutamate. Clones 8 (white bars) and 15 (hatched bars) have higher basal GSH levels than wild-type HT22 and only deplete to 72 ± 4 and 56 ± 1% of their basal GSH levels, respectively, with glutamate exposure. (B) 2 mM glutamate. Wild-type HT22 cells (black bars), empty vector infected cells (white bars), and S51A mutant infected cells (hatched bars) show GSH levels that are depleted to 20–30% of basal levels. In the S51D mutant-expressing cells (narrow hatched bars), GSH depleted to only ∼50% of the basal level. 100% GSH is defined as the GSH level assayed in the untreated control cells. The numbers above the glutamate exposed bars indicate the percentage of GSH relative to the basal level in the same cell line (n = 3).
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Figure 5: GSH levels in resistant cells. GSH levels were measured in control, untreated cells, and cells exposed to 5 or 2 mM glutamate for 10 h. (A) 5 mM glutamate. Clones 8 (white bars) and 15 (hatched bars) have higher basal GSH levels than wild-type HT22 and only deplete to 72 ± 4 and 56 ± 1% of their basal GSH levels, respectively, with glutamate exposure. (B) 2 mM glutamate. Wild-type HT22 cells (black bars), empty vector infected cells (white bars), and S51A mutant infected cells (hatched bars) show GSH levels that are depleted to 20–30% of basal levels. In the S51D mutant-expressing cells (narrow hatched bars), GSH depleted to only ∼50% of the basal level. 100% GSH is defined as the GSH level assayed in the untreated control cells. The numbers above the glutamate exposed bars indicate the percentage of GSH relative to the basal level in the same cell line (n = 3).
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