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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-deficient granule cells are not protected from  NMDA-induced excitotoxic cell death. After 10 d in vitro, granule cells were stimulated with 0, 10, 30, and 100 μM NMDA, or  100 μM NMDA with 150 nM MK-801 for 30 min at 37°C. Survival was assessed after 24 h by counting neurons in photomicrographs of calcein AM–stained cultures. Data represent mean ±  range for two independent experiments.
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Figure 4: Bax-deficient granule cells are not protected from NMDA-induced excitotoxic cell death. After 10 d in vitro, granule cells were stimulated with 0, 10, 30, and 100 μM NMDA, or 100 μM NMDA with 150 nM MK-801 for 30 min at 37°C. Survival was assessed after 24 h by counting neurons in photomicrographs of calcein AM–stained cultures. Data represent mean ± range for two independent experiments.

Mentions: Overexpression of BCL-2 is neuroprotective in some models of stroke and excitotoxic injury (Linnik, 1996). Therefore, we tested whether the absence of BAX would be protective in an NMDA model of excitotoxic death in cerebellar granule cells. After 10 d in vitro, cultures from Bax +/+ and Bax −/− were stimulated with 10, 30, or 100 μM NMDA for 30 min and viability was assessed 24 h later by calcein AM staining (Fig. 4). In contrast to K+ deprivation or treatment with an inhibitor of PI-3-K, the absence of Bax was not neuroprotective in this excitotoxic paradigm. The lack of death in cultures simultaneously exposed to 100 μM NMDA and 150 nM MK-801, a highly potent noncompetitive NMDA receptor antagonist (Wong et al., 1986), demonstrated that the death was specific to stimulation of NMDA receptors.


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-deficient granule cells are not protected from  NMDA-induced excitotoxic cell death. After 10 d in vitro, granule cells were stimulated with 0, 10, 30, and 100 μM NMDA, or  100 μM NMDA with 150 nM MK-801 for 30 min at 37°C. Survival was assessed after 24 h by counting neurons in photomicrographs of calcein AM–stained cultures. Data represent mean ±  range for two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139809&req=5

Figure 4: Bax-deficient granule cells are not protected from NMDA-induced excitotoxic cell death. After 10 d in vitro, granule cells were stimulated with 0, 10, 30, and 100 μM NMDA, or 100 μM NMDA with 150 nM MK-801 for 30 min at 37°C. Survival was assessed after 24 h by counting neurons in photomicrographs of calcein AM–stained cultures. Data represent mean ± range for two independent experiments.
Mentions: Overexpression of BCL-2 is neuroprotective in some models of stroke and excitotoxic injury (Linnik, 1996). Therefore, we tested whether the absence of BAX would be protective in an NMDA model of excitotoxic death in cerebellar granule cells. After 10 d in vitro, cultures from Bax +/+ and Bax −/− were stimulated with 10, 30, or 100 μM NMDA for 30 min and viability was assessed 24 h later by calcein AM staining (Fig. 4). In contrast to K+ deprivation or treatment with an inhibitor of PI-3-K, the absence of Bax was not neuroprotective in this excitotoxic paradigm. The lack of death in cultures simultaneously exposed to 100 μM NMDA and 150 nM MK-801, a highly potent noncompetitive NMDA receptor antagonist (Wong et al., 1986), demonstrated that the death was specific to stimulation of NMDA receptors.

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