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. 1999 Sep 15;19(18):7860-9.
doi: 10.1523/JNEUROSCI.19-18-07860.1999.

Bax-dependent caspase-3 activation is a key determinant in p53-induced apoptosis in neurons

S P Cregan  1 , J G MacLaurin, C G Craig, G S Robertson, D W Nicholson, D S Park, R S Slack
Affiliations

Bax-dependent caspase-3 activation is a key determinant in p53-induced apoptosis in neurons

S P Cregan et al. J Neurosci. .

Abstract

p53 is a pivotal molecule regulating the death of neurons both after acute injury and during development. The molecular mechanisms by which p53 induces apoptosis in neuronal cells, however, are not well understood. We have shown previously that adenovirus-mediated p53 gene delivery to neurons was sufficient to induce apoptosis. In the present study we have examined the molecular mechanism by which p53 evokes neuronal cell death. Adenovirus-mediated delivery of p53 to cerebellar granule neurons resulted in caspase-3 (CPP32) activation followed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining and loss of viability as determined by an MTT survival assay. To determine whether Bax is essential for caspase-3 activation, p53 was expressed in Bax-deficient cells. Bax null neurons did not exhibit caspase-3 activation in response to p53 and were protected from apoptosis. To determine whether Bax-dependent caspase-3 activation was required in p53-mediated neuronal cell death, caspase-3-deficient neurons were examined. Our results indicate that caspase-3-deficient neurons exhibit a remarkable delay in apoptosis and a dramatic decrease in TUNEL-positive cells. These studies demonstrate that p53-induced cell death in postmitotic neurons involves a Bax-dependent caspase-3 activation, suggesting that these molecules are important determinants in neuronal cell death after injury.

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Figures

Fig. 1.
Fig. 1.
β-Galactosidase expression in cerebellar granule neurons. Neurons were infected at the time of plating and fixed after 72 hr in culture. Cells infected with AdLacZ were stained with X-gal for the detection of β-galactosidase activity (A, 10 MOI LacZ; B, 50 MOI LacZ). Scale bar, 20 μm.
Fig. 2.
Fig. 2.
p53 induces apoptosis in cerebellar granule neurons. Cerebellar granule neurons were uninfected (A–C) or infected at 50 MOI with AdLacZ (D–F) or Adp53 (G–I). After 72 hr cells were fixed in 4% paraformaldehyde and stained for TUNEL (C, F, I) and counterstained with Hoechst (B, E,H). A, D,G, Corresponding phase-contrast micrographs. LacZ-infected cells retain morphology identical to uninfected controls, with no detectable increase in TUNEL staining: uninfected (C) versus LacZ-infected (F). In contrast, cells carrying Adp53 exhibit a significant increase in TUNEL staining (I). Scale bar, 20 μm.
Fig. 3.
Fig. 3.
p53 expression in cerebellar granule neurons. Neurons infected at 50 MOI with either AdLacZ (A,B), as controls, or Adp53 (C,D) were fixed in 4% paraformaldehyde after 72 hr and stained with an antibody directed against human p53 (B,D) and counterstained with Hoechst (A,C). Scale bar, 20 μm.
Fig. 4.
Fig. 4.
p53 and LacZ protein levels in cerebellar granule neurons. Neurons were either uninfected or infected at 50 MOI at the time of plating with either AdLacZ or Adp53. After 48 and 72 hr, protein was extracted and Western blot analysis was performed to visualize transgene expression.
Fig. 5.
Fig. 5.
p53 induces caspase-3-like activity in cerebellar granule neurons. Neurons were infected at 50 MOI with AdLacZ or Adp53 at the time of plating. After 48 and 72 hr, protein was extracted, and caspase activity was measured. A, Caspase-3 like activity determined by measurement of AcDEVD-pNA cleavage.B, Caspase-1 like activity as determined by AcYVAD-pNA cleavage. Activity is shown as fold increase over control (uninfected replicate dishes), and bars represent the mean with error bars indicating SD; n = 3. Each nrepresents a cell culture derived from a separate experiment.
Fig. 6.
Fig. 6.
p53 induces the cleavage of caspase-3 protein in cerebellar granule neurons. Cells were either uninfected or infected with AdLacZ or Adp53 at 50 MOI, and protein was harvested at 48 and 72 hr for Western blot analysis. After SDS-PAGE, protein was transferred to nitrocellulose filters and probed with an antibody specific for mouse caspase-3, and loading was standardized with actin. Note the disappearance of the 32 kd band 72 hr after infection with Adp53.
Fig. 7.
Fig. 7.
Bax deficiency renders cerebellar granule neurons resistant to p53-induced cell death. A, Cells were either uninfected or infected with AdLacZ or Adp53 at 50 MOI, and an MTT survival assay was conducted at 48, 72, 96, and120 hr. Control, uninfected cells were considered 100%, and results are reported as the percentage of control. Points are the average of cultures derived from three separate brains with matching wild-type littermates (n = 3), with error bars showing SD. B, Phase-contrast photomicrographs of Bax +/+ and −/− neurons 72 hr after infection at 50 MOI with either AdLacZ or Adp53. Scale bar, 60 μm.
Fig. 8.
Fig. 8.
Bax-deficient neurons are resistant to p53-mediated apoptosis. Cerebellar granule neurons were infected at 50 MOI with AdLacZ or Adp53 at the time of plating. A, After 72 hr in culture, Bax +/+ (a–d) or Bax −/− (e–h) neurons were fixed and stained with TUNEL (b, d, f,h) and counterstained with Hoechst (a,c, e, g). Scale bar, 100 μm. B, Quantitation of TUNEL-positive cells. Data represent the mean and SD from three independent experiments.
Fig. 9.
Fig. 9.
p53-induced caspase-3-like activity is dependent on Bax. Neurons were infected at 50 MOI with AdLacZ or Adp53 at the time of plating. After 72 hr in culture, protein was extracted, and AcDEVD-AFC cleavage activity was measured in Bax-deficient and wild-type cells. Bars represent the average of three separate experiments, and error bars show SD.
Fig. 10.
Fig. 10.
Absence of caspase-3 expression in CPP32 mutant mice. Protein was extracted from brains derived from wild-type (+/+), heterozygous (−/+), and CPP32 null (−/−) mice, and Western blot analysis was performed. Filters were probed with mouse monoclonal antibodies directed against caspase-3 and actin.
Fig. 11.
Fig. 11.
Caspase-3-deficient neurons exhibit a delay in p53-induced cell death. A, Cells were either uninfected or infected with AdLacZ or Adp53 at 50 MOI, and an MTT survival assay was conducted at 48, 72, 96, and 120 hr. Control, uninfected cells were considered 100%, and results are reported as the percentage of control. Points are the average of cultures derived from three separate brains with matching wild-type littermates (n = 3), with error bars showing SD.B, Phase-contrast photomicrographs of CPP32 +/+ and −/− neurons 72 hr after infection at 50 MOI with either AdLacZ or Adp53. Scale bar, 60 μm.
Fig. 12.
Fig. 12.
Caspase-3-deficient neurons expressing p53 exhibit decreased TUNEL staining. Cerebellar granule neurons were infected at 50 MOI with AdLacZ or Adp53 at the time of plating.A, After 72 hr in culture, CPP32+/+ (a–d) or CPP32−/− (e–h) neurons were fixed and stained with TUNEL (b, d,f, h) and counterstained with Hoechst (a, c, e,g). Scale bar, 100 μm. B, Quantitation of TUNEL-positive cells. Data represent the mean and SD from three independent experiments.
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