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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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c-Jun phosphorylation increases in both Bax +/+ and Bax −/− granule cells after K+ deprivation. Bax +/+ (a–d) and Bax −/−  (e–h) cultures were switched to K25+S (a, b, e, and f) or K5+S (c, d, g, and h) medium for 6 h and immunostained with an antibody that  specifically recognizes the ser-63 phosphorylated form of c-Jun (a, c, e, and g) and stained with the nuclear dye bisbenzimide (b, d, f, and  h). Bar, 5 μm.
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Figure 7: c-Jun phosphorylation increases in both Bax +/+ and Bax −/− granule cells after K+ deprivation. Bax +/+ (a–d) and Bax −/− (e–h) cultures were switched to K25+S (a, b, e, and f) or K5+S (c, d, g, and h) medium for 6 h and immunostained with an antibody that specifically recognizes the ser-63 phosphorylated form of c-Jun (a, c, e, and g) and stained with the nuclear dye bisbenzimide (b, d, f, and h). Bar, 5 μm.

Mentions: In addition to increases in mRNA levels, another indication of an increase in c-Jun activity is the phosphorylation of c-Jun on serines 63 and 73 of the transactivation domain (Binetruy et al., 1991; Smeal et al., 1991, 1992). Therefore, we determined the phosphorylation status of the c-Jun transactivation domain during PCD by immunostaining with an antibody that specifically recognizes the serine 63– phosphorylated form of c-Jun (Fig. 7). Cells maintained in K25+S medium (Fig. 7, a and e) had almost no staining. After 6 h of K+ deprivation, wild-type granule cells showed significant phosphorylation of c-Jun on serine 63 (Fig. 7 c). Similar to the parallel increases in c-jun mRNA levels in Bax +/+ and Bax −/− cultures, phospho-Jun staining also increased in Bax −/− granule cells deprived of K+ for 6 h (Fig. 7 g). Staining was not increased in the few nonneuronal cells in these cultures (data not shown). In nuclei counterstained with bisbenzimide (Fig. 7, b, d, f, and h), some K+-deprived, wild-type cells showed nuclear margination and chromatin condensation at 6 h (Fig. 7 d). The increase in c-Jun phosphorylation and c-jun mRNA in both wild-type and Bax −/− cultures indicates that BAX functions downstream of the increase in c-Jun activity that is associated with PCD.


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)

c-Jun phosphorylation increases in both Bax +/+ and Bax −/− granule cells after K+ deprivation. Bax +/+ (a–d) and Bax −/−  (e–h) cultures were switched to K25+S (a, b, e, and f) or K5+S (c, d, g, and h) medium for 6 h and immunostained with an antibody that  specifically recognizes the ser-63 phosphorylated form of c-Jun (a, c, e, and g) and stained with the nuclear dye bisbenzimide (b, d, f, and  h). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: c-Jun phosphorylation increases in both Bax +/+ and Bax −/− granule cells after K+ deprivation. Bax +/+ (a–d) and Bax −/− (e–h) cultures were switched to K25+S (a, b, e, and f) or K5+S (c, d, g, and h) medium for 6 h and immunostained with an antibody that specifically recognizes the ser-63 phosphorylated form of c-Jun (a, c, e, and g) and stained with the nuclear dye bisbenzimide (b, d, f, and h). Bar, 5 μm.
Mentions: In addition to increases in mRNA levels, another indication of an increase in c-Jun activity is the phosphorylation of c-Jun on serines 63 and 73 of the transactivation domain (Binetruy et al., 1991; Smeal et al., 1991, 1992). Therefore, we determined the phosphorylation status of the c-Jun transactivation domain during PCD by immunostaining with an antibody that specifically recognizes the serine 63– phosphorylated form of c-Jun (Fig. 7). Cells maintained in K25+S medium (Fig. 7, a and e) had almost no staining. After 6 h of K+ deprivation, wild-type granule cells showed significant phosphorylation of c-Jun on serine 63 (Fig. 7 c). Similar to the parallel increases in c-jun mRNA levels in Bax +/+ and Bax −/− cultures, phospho-Jun staining also increased in Bax −/− granule cells deprived of K+ for 6 h (Fig. 7 g). Staining was not increased in the few nonneuronal cells in these cultures (data not shown). In nuclei counterstained with bisbenzimide (Fig. 7, b, d, f, and h), some K+-deprived, wild-type cells showed nuclear margination and chromatin condensation at 6 h (Fig. 7 d). The increase in c-Jun phosphorylation and c-jun mRNA in both wild-type and Bax −/− cultures indicates that BAX functions downstream of the increase in c-Jun activity that is associated with PCD.

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