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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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Diagrammatic representation of the events associated  with programmed cell death in cerebellar granule cells.
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Figure 12: Diagrammatic representation of the events associated with programmed cell death in cerebellar granule cells.

Mentions: In this study, we demonstrated that PCD in cerebellar granule cells induced by K+ deprivation, K+/serum deprivation, or inhibition of PI-3-K requires BAX. In contrast, BAX was not required for excitotoxic cell death in granule cells since we found that Bax −/− granule cells were not protected from NMDA-induced cell death. Because Bax −/− granule cells did not die in response to K+ deprivation, we were able to use this model to define where BAX is acting in the pathway leading to apoptosis. We analyzed several metabolic and genetic events associated with PCD in granule cells and found that Bax deficiency did not affect these early events. On the other hand, we found that BAX was required for the activation of caspases since Bax +/+, but not Bax −/− cells cleaved a fluorogenic DEVD substrate in response to K+ deprivation. Our data suggest that while likely to be involved in cell death, caspases may not be solely responsible for causing cell death in granule cells since blocking these proteases with peptide inhibitors delayed, but did not significantly block, the loss of cells. These studies of Bax-knockout cells are consistent with BAX or BAK induction studies in which cell death also proceeded in the presence of caspase inhibitors (Xiang et al., 1996; McCarthy et al., 1997). From these results and previous studies, we are now able to define a sequence of events associated with PCD in granule cells (Fig. 12).


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)

Diagrammatic representation of the events associated  with programmed cell death in cerebellar granule cells.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 12: Diagrammatic representation of the events associated with programmed cell death in cerebellar granule cells.
Mentions: In this study, we demonstrated that PCD in cerebellar granule cells induced by K+ deprivation, K+/serum deprivation, or inhibition of PI-3-K requires BAX. In contrast, BAX was not required for excitotoxic cell death in granule cells since we found that Bax −/− granule cells were not protected from NMDA-induced cell death. Because Bax −/− granule cells did not die in response to K+ deprivation, we were able to use this model to define where BAX is acting in the pathway leading to apoptosis. We analyzed several metabolic and genetic events associated with PCD in granule cells and found that Bax deficiency did not affect these early events. On the other hand, we found that BAX was required for the activation of caspases since Bax +/+, but not Bax −/− cells cleaved a fluorogenic DEVD substrate in response to K+ deprivation. Our data suggest that while likely to be involved in cell death, caspases may not be solely responsible for causing cell death in granule cells since blocking these proteases with peptide inhibitors delayed, but did not significantly block, the loss of cells. These studies of Bax-knockout cells are consistent with BAX or BAK induction studies in which cell death also proceeded in the presence of caspase inhibitors (Xiang et al., 1996; McCarthy et al., 1997). From these results and previous studies, we are now able to define a sequence of events associated with PCD in granule cells (Fig. 12).

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