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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 −/− cerebellar granule cells do not undergo apoptosis in response to K+ deprivation. Cerebellar granule cells from Bax  +/+ (a, b, e, and f) and Bax −/− (c, d, g, and h) were maintained for 7 d in vitro and then switched to K25+S medium (a–d) or K5+S  medium (e–h). Photomicrographs of phase contrast (a, c, e, and g) and the corresponding calcein AM–stained images (b, d, f, and h)  were taken 72 h after the media were switched. Bar, 20 μm.
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Figure 1: Bax −/− cerebellar granule cells do not undergo apoptosis in response to K+ deprivation. Cerebellar granule cells from Bax +/+ (a, b, e, and f) and Bax −/− (c, d, g, and h) were maintained for 7 d in vitro and then switched to K25+S medium (a–d) or K5+S medium (e–h). Photomicrographs of phase contrast (a, c, e, and g) and the corresponding calcein AM–stained images (b, d, f, and h) were taken 72 h after the media were switched. Bar, 20 μm.

Mentions: Dissociated cerebellar granule cells maintained in 25 mM K+ and serum undergo a PCD that is apoptotic if they are deprived of both potassium and serum (K5−S medium) (D'Mello et al., 1993). To determine whether BAX, a proapoptotic BCL-2 family member, was important in cerebellar granule cell apoptosis, we used cultures of dissociated cerebellar granule cells from P7 wild-type and Bax-deficient mice (Knudson et al., 1995). After 7 d in vitro, cultures of Bax +/+, and Bax −/− cerebellar granule cells were switched to medium containing low potassium and serum (K5+S) or maintained in high potassium and serum (K25+S). After 72 h, cultures were stained with calcein AM, which stains living cells. Phase-contrast images and the corresponding calcein AM photomicrographs are presented in Fig. 1. While virtually all of the Bax +/+ cells died by 72 h (compare Fig. 1, a and b with e and f ), granule cells from Bax −/− mice did not undergo apoptosis (compare Fig. 1, c and d with g and h). To determine the number of surviving cells, we counted the number of neurons on photomicrographs of cultures stained with calcein AM after 0, 12, 24, 48, or 72 h in K5+S medium (Fig. 2). All the granule cells from Bax −/− mice were protected from cell death (Fig. 2, open triangles) while more than 90% of the wild-type granule cells died by 72 h (Fig. 2, open circles). Granule cells from heterozygous animals died completely by 72 h (Fig. 2, open squares), though the time course of death was slightly slower. Nuclei from Bax −/− cultures deprived of K+ for 72 h were indistinguishable from control cultures maintained in K25+S medium (data not shown), while Bax +/+ and Bax +/− cultures displayed characteristic apoptotic nuclear changes 6 h after switching to K5+S medium (data not shown).


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 −/− cerebellar granule cells do not undergo apoptosis in response to K+ deprivation. Cerebellar granule cells from Bax  +/+ (a, b, e, and f) and Bax −/− (c, d, g, and h) were maintained for 7 d in vitro and then switched to K25+S medium (a–d) or K5+S  medium (e–h). Photomicrographs of phase contrast (a, c, e, and g) and the corresponding calcein AM–stained images (b, d, f, and h)  were taken 72 h after the media were switched. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Bax −/− cerebellar granule cells do not undergo apoptosis in response to K+ deprivation. Cerebellar granule cells from Bax +/+ (a, b, e, and f) and Bax −/− (c, d, g, and h) were maintained for 7 d in vitro and then switched to K25+S medium (a–d) or K5+S medium (e–h). Photomicrographs of phase contrast (a, c, e, and g) and the corresponding calcein AM–stained images (b, d, f, and h) were taken 72 h after the media were switched. Bar, 20 μm.
Mentions: Dissociated cerebellar granule cells maintained in 25 mM K+ and serum undergo a PCD that is apoptotic if they are deprived of both potassium and serum (K5−S medium) (D'Mello et al., 1993). To determine whether BAX, a proapoptotic BCL-2 family member, was important in cerebellar granule cell apoptosis, we used cultures of dissociated cerebellar granule cells from P7 wild-type and Bax-deficient mice (Knudson et al., 1995). After 7 d in vitro, cultures of Bax +/+, and Bax −/− cerebellar granule cells were switched to medium containing low potassium and serum (K5+S) or maintained in high potassium and serum (K25+S). After 72 h, cultures were stained with calcein AM, which stains living cells. Phase-contrast images and the corresponding calcein AM photomicrographs are presented in Fig. 1. While virtually all of the Bax +/+ cells died by 72 h (compare Fig. 1, a and b with e and f ), granule cells from Bax −/− mice did not undergo apoptosis (compare Fig. 1, c and d with g and h). To determine the number of surviving cells, we counted the number of neurons on photomicrographs of cultures stained with calcein AM after 0, 12, 24, 48, or 72 h in K5+S medium (Fig. 2). All the granule cells from Bax −/− mice were protected from cell death (Fig. 2, open triangles) while more than 90% of the wild-type granule cells died by 72 h (Fig. 2, open circles). Granule cells from heterozygous animals died completely by 72 h (Fig. 2, open squares), though the time course of death was slightly slower. Nuclei from Bax −/− cultures deprived of K+ for 72 h were indistinguishable from control cultures maintained in K25+S medium (data not shown), while Bax +/+ and Bax +/− cultures displayed characteristic apoptotic nuclear changes 6 h after switching to K5+S medium (data not shown).

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