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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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mRNA levels of  c-jun and CPP32 increase in  both Bax +/+ and Bax −/−  granule cells after K+ deprivation. Cultures were  switched to K5+S and  cDNA was prepared from  granule cells after 1, 3, 6, 9,  12, 18, 24, 48, or 72 h. Control cultures were maintained  in K25+S medium. cDNA  from ∼4,000 cells was used in  a 50-μl PCR reaction as described in Materials and  Methods. Identical results  were obtained in an independent neuronal preparation.  (A) PCR products. (B) PhosphorImager quantitation of  Bax +/+ PCR products. (C)  PhosphorImager quantitation of Bax −/− PCR products. Time 0 was used as control level for B and C.
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Figure 6: mRNA levels of c-jun and CPP32 increase in both Bax +/+ and Bax −/− granule cells after K+ deprivation. Cultures were switched to K5+S and cDNA was prepared from granule cells after 1, 3, 6, 9, 12, 18, 24, 48, or 72 h. Control cultures were maintained in K25+S medium. cDNA from ∼4,000 cells was used in a 50-μl PCR reaction as described in Materials and Methods. Identical results were obtained in an independent neuronal preparation. (A) PCR products. (B) PhosphorImager quantitation of Bax +/+ PCR products. (C) PhosphorImager quantitation of Bax −/− PCR products. Time 0 was used as control level for B and C.

Mentions: Whether the increase in c-jun mRNA and presumably the action of c-jun is downstream or upstream of BAX is unknown. To address this issue and examine other message levels, cultures from Bax +/+ and Bax −/− animals were switched to K5+S for 1, 3, 6, 9, 12, 18, 24, 48, or 72 h and mRNA was isolated, reverse transcribed, and selectively amplified by PCR. mRNA levels of cyclophilin declined rapidly in wild-type granule cells (Fig. 6, A and B); actin and neuron-specific enolase were similar to cyclophilin (data not shown). mRNA levels of these same messages in Bax −/− cultures declined far less than in wild-type cultures reflecting the lack of death of Bax −/− cells (Fig. 6, A and C). Similar to results seen in rat cerebellar granule cells (Miller and Johnson, 1996), mRNA levels for c-jun increased approximately fourfold in K+-deprived wild-type granule cells (Fig. 6 B). c-jun mRNA also increased in K+-deprived, Bax-deficient granule cells (Fig. 6 C), demonstrating that increases in c-jun were upstream of Bax.


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)

mRNA levels of  c-jun and CPP32 increase in  both Bax +/+ and Bax −/−  granule cells after K+ deprivation. Cultures were  switched to K5+S and  cDNA was prepared from  granule cells after 1, 3, 6, 9,  12, 18, 24, 48, or 72 h. Control cultures were maintained  in K25+S medium. cDNA  from ∼4,000 cells was used in  a 50-μl PCR reaction as described in Materials and  Methods. Identical results  were obtained in an independent neuronal preparation.  (A) PCR products. (B) PhosphorImager quantitation of  Bax +/+ PCR products. (C)  PhosphorImager quantitation of Bax −/− PCR products. Time 0 was used as control level for B and C.
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Related In: Results  -  Collection

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Figure 6: mRNA levels of c-jun and CPP32 increase in both Bax +/+ and Bax −/− granule cells after K+ deprivation. Cultures were switched to K5+S and cDNA was prepared from granule cells after 1, 3, 6, 9, 12, 18, 24, 48, or 72 h. Control cultures were maintained in K25+S medium. cDNA from ∼4,000 cells was used in a 50-μl PCR reaction as described in Materials and Methods. Identical results were obtained in an independent neuronal preparation. (A) PCR products. (B) PhosphorImager quantitation of Bax +/+ PCR products. (C) PhosphorImager quantitation of Bax −/− PCR products. Time 0 was used as control level for B and C.
Mentions: Whether the increase in c-jun mRNA and presumably the action of c-jun is downstream or upstream of BAX is unknown. To address this issue and examine other message levels, cultures from Bax +/+ and Bax −/− animals were switched to K5+S for 1, 3, 6, 9, 12, 18, 24, 48, or 72 h and mRNA was isolated, reverse transcribed, and selectively amplified by PCR. mRNA levels of cyclophilin declined rapidly in wild-type granule cells (Fig. 6, A and B); actin and neuron-specific enolase were similar to cyclophilin (data not shown). mRNA levels of these same messages in Bax −/− cultures declined far less than in wild-type cultures reflecting the lack of death of Bax −/− cells (Fig. 6, A and C). Similar to results seen in rat cerebellar granule cells (Miller and Johnson, 1996), mRNA levels for c-jun increased approximately fourfold in K+-deprived wild-type granule cells (Fig. 6 B). c-jun mRNA also increased in K+-deprived, Bax-deficient granule cells (Fig. 6 C), demonstrating that increases in c-jun were upstream of Bax.

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