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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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Inhibitors of caspases do not block death, but do block  DEVD-AMC cleavage. Cultures were switched to K5+S, K5+S  plus 100 μM BAF, or K5+S plus 100 μM ZVAD-FMK. Control  cultures were switched to K25+S medium. (A) After 12, 24, or 48 h  neuronal survival was determined by calcein AM staining. (mean ±  SD, N = 3 experiments) (B) After 8, 12, 18, 24, or 48 h cultures  were lysed and assayed for DEVD-AMC cleavage. Fluorescence  was measured after 20 min at room temperature (mean ± range,  N = 2 experiments).
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Figure 9: Inhibitors of caspases do not block death, but do block DEVD-AMC cleavage. Cultures were switched to K5+S, K5+S plus 100 μM BAF, or K5+S plus 100 μM ZVAD-FMK. Control cultures were switched to K25+S medium. (A) After 12, 24, or 48 h neuronal survival was determined by calcein AM staining. (mean ± SD, N = 3 experiments) (B) After 8, 12, 18, 24, or 48 h cultures were lysed and assayed for DEVD-AMC cleavage. Fluorescence was measured after 20 min at room temperature (mean ± range, N = 2 experiments).

Mentions: Inhibiting caspases with peptide inhibitors blocks PCD in several models (Henkart, 1996; Schwartz and Milligan, 1996). For example, BAF or ZVAD-FMK, two inhibitors of caspases, have dramatic effects on saving sympathetic neurons from PCD induced by trophic factor deprivation (Deshmukh et al., 1996). We examined the effect of these compounds on K+ deprivation–induced cell death in granule cells. Neither compound was toxic to granule cells. After 3 d exposure to either 100 μM ZVAD or 100 μM BAF in K25+S medium, survival was 96 ± 1 and 103 ± 3%, respectively. In K5+S medium, while both BAF and ZVAD were able to delay PCD in granule cells at 12 h, neither afforded significant protection at later time points (Fig. 9 A). One possible explanation for the lack of protection was that BAF and ZVAD did not function as caspase inhibitors in these cells, perhaps because they did not effectively cross the plasma membrane. To rule out this possibility, we deprived cells of K+ in the presence of 100 μM BAF or 100 μM ZVAD and then measured caspase activity. Cells treated with either compound showed no increase in DEVD-AMC cleavage after K+ deprivation (Fig. 9 B), demonstrating that BAF and ZVAD did indeed penetrate granule cells and function as caspase inhibitors in these cells.


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)

Inhibitors of caspases do not block death, but do block  DEVD-AMC cleavage. Cultures were switched to K5+S, K5+S  plus 100 μM BAF, or K5+S plus 100 μM ZVAD-FMK. Control  cultures were switched to K25+S medium. (A) After 12, 24, or 48 h  neuronal survival was determined by calcein AM staining. (mean ±  SD, N = 3 experiments) (B) After 8, 12, 18, 24, or 48 h cultures  were lysed and assayed for DEVD-AMC cleavage. Fluorescence  was measured after 20 min at room temperature (mean ± range,  N = 2 experiments).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139809&req=5

Figure 9: Inhibitors of caspases do not block death, but do block DEVD-AMC cleavage. Cultures were switched to K5+S, K5+S plus 100 μM BAF, or K5+S plus 100 μM ZVAD-FMK. Control cultures were switched to K25+S medium. (A) After 12, 24, or 48 h neuronal survival was determined by calcein AM staining. (mean ± SD, N = 3 experiments) (B) After 8, 12, 18, 24, or 48 h cultures were lysed and assayed for DEVD-AMC cleavage. Fluorescence was measured after 20 min at room temperature (mean ± range, N = 2 experiments).
Mentions: Inhibiting caspases with peptide inhibitors blocks PCD in several models (Henkart, 1996; Schwartz and Milligan, 1996). For example, BAF or ZVAD-FMK, two inhibitors of caspases, have dramatic effects on saving sympathetic neurons from PCD induced by trophic factor deprivation (Deshmukh et al., 1996). We examined the effect of these compounds on K+ deprivation–induced cell death in granule cells. Neither compound was toxic to granule cells. After 3 d exposure to either 100 μM ZVAD or 100 μM BAF in K25+S medium, survival was 96 ± 1 and 103 ± 3%, respectively. In K5+S medium, while both BAF and ZVAD were able to delay PCD in granule cells at 12 h, neither afforded significant protection at later time points (Fig. 9 A). One possible explanation for the lack of protection was that BAF and ZVAD did not function as caspase inhibitors in these cells, perhaps because they did not effectively cross the plasma membrane. To rule out this possibility, we deprived cells of K+ in the presence of 100 μM BAF or 100 μM ZVAD and then measured caspase activity. Cells treated with either compound showed no increase in DEVD-AMC cleavage after K+ deprivation (Fig. 9 B), demonstrating that BAF and ZVAD did indeed penetrate granule cells and function as caspase inhibitors in these cells.

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