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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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BAX is required for increases in caspase activity. Cultures were switched to K5+S medium, lysed after 4, 8, 12, 24, 48,  or 72 h, and cleavage of DEVD-AMC was determined. Control  cultures were switched to K25+S medium and treated identically.  Data represent mean ± SD for triplicate measurements from one  experiment and are representative of two additional independent  experiments.
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Figure 8: BAX is required for increases in caspase activity. Cultures were switched to K5+S medium, lysed after 4, 8, 12, 24, 48, or 72 h, and cleavage of DEVD-AMC was determined. Control cultures were switched to K25+S medium and treated identically. Data represent mean ± SD for triplicate measurements from one experiment and are representative of two additional independent experiments.

Mentions: Caspases have been implicated in several cell death models (for review see Henkart, 1996; Schwartz and Milligan, 1996). We examined whether caspases 2, 3, and 7 were activated after K+ deprivation. The activity of this subset of proteases can be measured by monitoring the cleavage of the fluorogenic substrate DEVD-AMC. In Bax +/+ and Bax +/− cells, DEVD-selective caspase activity increased to 18 times control levels by 8 h after K+ deprivation and then decreased as the cells died over the next 64 h. In stark contrast, granule cells from Bax −/− animals did not increase DEVD-specific caspase activity at any time after K+ deprivation (Fig. 8). These results indicate that BAX is required for activation of this subset of caspases in response to removal of potassium.


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)

BAX is required for increases in caspase activity. Cultures were switched to K5+S medium, lysed after 4, 8, 12, 24, 48,  or 72 h, and cleavage of DEVD-AMC was determined. Control  cultures were switched to K25+S medium and treated identically.  Data represent mean ± SD for triplicate measurements from one  experiment and are representative of two additional independent  experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139809&req=5

Figure 8: BAX is required for increases in caspase activity. Cultures were switched to K5+S medium, lysed after 4, 8, 12, 24, 48, or 72 h, and cleavage of DEVD-AMC was determined. Control cultures were switched to K25+S medium and treated identically. Data represent mean ± SD for triplicate measurements from one experiment and are representative of two additional independent experiments.
Mentions: Caspases have been implicated in several cell death models (for review see Henkart, 1996; Schwartz and Milligan, 1996). We examined whether caspases 2, 3, and 7 were activated after K+ deprivation. The activity of this subset of proteases can be measured by monitoring the cleavage of the fluorogenic substrate DEVD-AMC. In Bax +/+ and Bax +/− cells, DEVD-selective caspase activity increased to 18 times control levels by 8 h after K+ deprivation and then decreased as the cells died over the next 64 h. In stark contrast, granule cells from Bax −/− animals did not increase DEVD-specific caspase activity at any time after K+ deprivation (Fig. 8). These results indicate that BAX is required for activation of this subset of caspases in response to removal of potassium.

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