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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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Inhibition of PI-3-K does not induce apoptosis in Bax  −/− granule cells. After 7 d in vitro, granule cells were switched  to K25+S medium in the presence of 30 μM LY 294002, an inhibitor of PI-3-K. Control cells were switched to K25+S medium.  Survival was determined by counting neurons in photomicrographs of calcein AM–stained cultures 48 h after treatment. Data  represent mean ± range for two independent experiments.
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Figure 3: Inhibition of PI-3-K does not induce apoptosis in Bax −/− granule cells. After 7 d in vitro, granule cells were switched to K25+S medium in the presence of 30 μM LY 294002, an inhibitor of PI-3-K. Control cells were switched to K25+S medium. Survival was determined by counting neurons in photomicrographs of calcein AM–stained cultures 48 h after treatment. Data represent mean ± range for two independent experiments.

Mentions: We have previously shown that PI-3-K activity is increased by K+ depolarization (Miller et al., 1997). This increase appears critical to the survival of depolarized neurons as shown by the ability of two inhibitors of PI-3-K to produce death indistinguishable from that caused by removal of high K+ (Miller et al., 1997). Because BCL-2 family members have been recently implicated in signaling pathways (Gajewski and Thompson, 1996; Wang et al., 1996; Zha et al., 1996), we directly tested the ability of Bax −/− cells to survive in the absence of PI-3-K activity. Neurons maintained for 7 d in vitro were switched to K25+S with 30 μM LY 294002, an inhibitor of PI-3-K activity. Viability was assessed after 48 h by calcein AM staining (Fig. 3). Similar to previous results (Miller et al., 1997), LY 294002 blocked the survival-promoting activity of K+ in Bax +/+ cells. Results from cultures of animals heterozygous for Bax were similar to those from wild type. In contrast, neurons from Bax −/− cultures survived in the presence of LY 294002, implying that Bax deficiency blocked apoptosis downstream of PI-3-K.


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)

Inhibition of PI-3-K does not induce apoptosis in Bax  −/− granule cells. After 7 d in vitro, granule cells were switched  to K25+S medium in the presence of 30 μM LY 294002, an inhibitor of PI-3-K. Control cells were switched to K25+S medium.  Survival was determined by counting neurons in photomicrographs of calcein AM–stained cultures 48 h after treatment. Data  represent mean ± range for two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Inhibition of PI-3-K does not induce apoptosis in Bax −/− granule cells. After 7 d in vitro, granule cells were switched to K25+S medium in the presence of 30 μM LY 294002, an inhibitor of PI-3-K. Control cells were switched to K25+S medium. Survival was determined by counting neurons in photomicrographs of calcein AM–stained cultures 48 h after treatment. Data represent mean ± range for two independent experiments.
Mentions: We have previously shown that PI-3-K activity is increased by K+ depolarization (Miller et al., 1997). This increase appears critical to the survival of depolarized neurons as shown by the ability of two inhibitors of PI-3-K to produce death indistinguishable from that caused by removal of high K+ (Miller et al., 1997). Because BCL-2 family members have been recently implicated in signaling pathways (Gajewski and Thompson, 1996; Wang et al., 1996; Zha et al., 1996), we directly tested the ability of Bax −/− cells to survive in the absence of PI-3-K activity. Neurons maintained for 7 d in vitro were switched to K25+S with 30 μM LY 294002, an inhibitor of PI-3-K activity. Viability was assessed after 48 h by calcein AM staining (Fig. 3). Similar to previous results (Miller et al., 1997), LY 294002 blocked the survival-promoting activity of K+ in Bax +/+ cells. Results from cultures of animals heterozygous for Bax were similar to those from wild type. In contrast, neurons from Bax −/− cultures survived in the presence of LY 294002, implying that Bax deficiency blocked apoptosis downstream of PI-3-K.

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