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Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death.

Miller TM, Moulder KL, Knudson CM, Creedon DJ, Deshmukh M, Korsmeyer SJ, Johnson EM - J. Cell Biol. (1997)

Bottom Line: Wild-type granule cells in 5 mM K+ increased cleavage of DEVD-aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC.These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death.However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 microM NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax -/- neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K+ deprivation. Wild-type granule cells in 5 mM K+ increased cleavage of DEVD-aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.

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Cell death in the presence of BAF is TUNEL-negative. Cultures were switched to K5+S (b and e) or K5+S plus 100 μM  BAF (c and f). Control cultures were switched to K25+S medium (a and d). After 24 h, cultures were fixed, stained with the TUNEL reagent (d–f), and stained with bisbenzimide (a–c) as described in Materials and Methods. The percent TUNEL-positive cells is the number of TUNEL-positive cells divided by the number of bisbenzimide-stained cells (g). Data represent the mean ± range for two independent experiments.
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Figure 10: Cell death in the presence of BAF is TUNEL-negative. Cultures were switched to K5+S (b and e) or K5+S plus 100 μM BAF (c and f). Control cultures were switched to K25+S medium (a and d). After 24 h, cultures were fixed, stained with the TUNEL reagent (d–f), and stained with bisbenzimide (a–c) as described in Materials and Methods. The percent TUNEL-positive cells is the number of TUNEL-positive cells divided by the number of bisbenzimide-stained cells (g). Data represent the mean ± range for two independent experiments.

Mentions: Further evidence that BAF was able to function inside cells was found in a TUNEL assay of K+-deprived granule cells. TUNEL serves as an in situ marker of DNA fragmentation (Gavrieli et al., 1992), one of the hallmarks of apoptosis. The number of TUNEL-positive cells was determined in cells switched to K25+S, K5+S, or K5+S with 100 μM BAF (Fig. 10, d–f, respectively). To facilitate visualization of the total number of cells, the nuclei were stained with bisbenzimide (Fig. 10, a–c). As expected, the percent of TUNEL-positive cells increased markedly after switching cells from K25+S to K5+S (Fig. 10, d and e) from 14 to 84% (Fig. 10 g). Although BAF did not block cell death (Fig. 9 A), BAF blocked the increase in TUNEL positivity associated with depriving granule cells of potassium (Fig. 10, f and g).


Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death.

Miller TM, Moulder KL, Knudson CM, Creedon DJ, Deshmukh M, Korsmeyer SJ, Johnson EM - J. Cell Biol. (1997)

Cell death in the presence of BAF is TUNEL-negative. Cultures were switched to K5+S (b and e) or K5+S plus 100 μM  BAF (c and f). Control cultures were switched to K25+S medium (a and d). After 24 h, cultures were fixed, stained with the TUNEL reagent (d–f), and stained with bisbenzimide (a–c) as described in Materials and Methods. The percent TUNEL-positive cells is the number of TUNEL-positive cells divided by the number of bisbenzimide-stained cells (g). Data represent the mean ± range for two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139809&req=5

Figure 10: Cell death in the presence of BAF is TUNEL-negative. Cultures were switched to K5+S (b and e) or K5+S plus 100 μM BAF (c and f). Control cultures were switched to K25+S medium (a and d). After 24 h, cultures were fixed, stained with the TUNEL reagent (d–f), and stained with bisbenzimide (a–c) as described in Materials and Methods. The percent TUNEL-positive cells is the number of TUNEL-positive cells divided by the number of bisbenzimide-stained cells (g). Data represent the mean ± range for two independent experiments.
Mentions: Further evidence that BAF was able to function inside cells was found in a TUNEL assay of K+-deprived granule cells. TUNEL serves as an in situ marker of DNA fragmentation (Gavrieli et al., 1992), one of the hallmarks of apoptosis. The number of TUNEL-positive cells was determined in cells switched to K25+S, K5+S, or K5+S with 100 μM BAF (Fig. 10, d–f, respectively). To facilitate visualization of the total number of cells, the nuclei were stained with bisbenzimide (Fig. 10, a–c). As expected, the percent of TUNEL-positive cells increased markedly after switching cells from K25+S to K5+S (Fig. 10, d and e) from 14 to 84% (Fig. 10 g). Although BAF did not block cell death (Fig. 9 A), BAF blocked the increase in TUNEL positivity associated with depriving granule cells of potassium (Fig. 10, f and g).

Bottom Line: Wild-type granule cells in 5 mM K+ increased cleavage of DEVD-aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC.These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death.However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 microM NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax -/- neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K+ deprivation. Wild-type granule cells in 5 mM K+ increased cleavage of DEVD-aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.

Show MeSH
Related in: MedlinePlus