Bcl-X_L–Caspase-9 Interactions in the Developing Nervous System: Evidence for Multiple Death Pathways

Abstract

Programmed cell death is critical for normal nervous system development and is regulated by Bcl-2 and Caspase family members. Targeted disruption of bcl-x L , an antiapoptotic bcl-2 gene family member, causes massive death of immature neurons in the developing nervous system whereas disruption of caspase-9, a proapoptotic caspase gene family member, leads to decreased neuronal apoptosis and neurodevelopmental abnormalities. To determine whether Bcl-X L and Caspase-9 interact in an obligate pathway of neuronal apoptosis, bcl-x/ caspase-9 double homozygous mutants were generated. The increased apoptosis of immature neurons observed in Bcl-X Ldeficient embryos was completely prevented by concomitant Caspase-9 deficiency. In contrast, bcl-x Ϫ /Ϫ /caspase-9 Ϫ /Ϫ embryonic mice exhibited an expanded ventricular zone and neuronal malformations identical to that observed in mice lacking only Caspase-9. These results indicate both epistatic and independent actions of Bcl-X L and Caspase-9 in neuronal programmed cell death.To examine Bcl-2 and Caspase family-dependent apoptotic pathways in telencephalic neurons, we compared the effects of cytosine arabinoside (AraC), a known neuronal apoptosis inducer, on wild-type, Bcl-X L -, Bax-, Caspase-9-, Caspase-3-, and p53-deficient telencephalic neurons in vitro. AraC caused extensive apoptosis of wild-type and Bcl-X L -deficient neurons. p53-and Bax-deficient neurons showed marked protection from AraC-induced death, whereas Caspase-9-and Caspase-3-deficient neurons showed minimal or no protection, respectively. These findings contrast with our previous investigation of AraC-induced apoptosis of telencephalic neural precursor cells in which death was completely blocked by p53 or Caspase-9 deficiency but not Bax deficiency. In total, these results indicate a transition from Caspase-9-to Bax-and Bcl-X L -mediated neuronal apoptosis.

p53 deficiency fails to prevent increased programmed cell death in the Bcl-XL-deficient nervous system

“…Consistent with our previous studies, p53 7/7 neurons showed a marked attenuation and bcl-x 7/7 neurons a significant exacerbation of the effects of AraC on both caspase-3 activation and cell death ( Figure 1A,B). 18,21 Telencephalic neurons derived from bcl-x 7/7 /p53 7/7 embryos exhibited dramatic resistance to AraC-induced caspase-3 activation and death, equivalent to that observed in bcl-x +/+ /p53 7/7 telencephalic cultures ( Figure 1A,B). These findings suggest that AraC triggers a p53-dependent death pathway in telencephalic neurons that engages downstream Bcl-2 family mediated events including caspase-3 activation.…”

“…Consistent with our previous in vitro studies of DNA damage-induced neuronal death, Bcl-X L -deficient neurons showed increased, and p53-deficient neurons decreased, susceptibility to the death promoting effects of AraC, a known genotoxic agent. 18,21 p53 deficiency completely blocked the increased apoptotic susceptibility of Bcl-X L -deficient neurons to AraC and there was no difference in either caspase-3 activation or extent of death between bcl-x +/+ /p53 7/7 and bcl-x 7/7 /p53 7/7 neurons. These findings suggest that Bcl-X L lies downstream of p53 in the apoptotic pathway of DNA damage-induced death of telencephalic neurons.…”

“…19 ± 21 We have also shown that addition of the nucleoside analog, AraC to telencephalic neurons in vitro results in extensive caspase-3 activation and cell death over the subsequent 24 ± 48 h period. 18,21 This effect is exacerbated in Bcl-X L -deficient and attenuated in p53-deficient telencephalic cultures. 18,21 p53 has both Bcl-2 family dependent and independent effects; 22 ± 24 we therefore examined the relationship between p53 and Bcl-X L in untreated and AraC exposed (25 mM) telencephalic neuron cultures prepared from control, p53 7/7 , bcl-x 7/7 , and bcl-x 7/7 /p53 7/7 E12.5 mice.…”

“…18,21 This effect is exacerbated in Bcl-X L -deficient and attenuated in p53-deficient telencephalic cultures. 18,21 p53 has both Bcl-2 family dependent and independent effects; 22 ± 24 we therefore examined the relationship between p53 and Bcl-X L in untreated and AraC exposed (25 mM) telencephalic neuron cultures prepared from control, p53 7/7 , bcl-x 7/7 , and bcl-x 7/7 /p53 7/7 E12.5 mice. In untreated cells, there was no significant difference in baseline viability (93 ± 96%) or CM1 immunoreactivity (1.5 ± 2.7%) between groups (data not shown).…”

“…17 In a previous study of primary telencephalic neuronal cultures, we found increased caspase-3 activation and cell death in cytosine arabinoside (AraC)-exposed Bcl-X L -deficient cells; in contrast, p53-deficient neurons were remarkably resistant to the effects of AraC. 18 In an attempt to identify the apoptotic signals regulating naturally-occurring neuronal cell death and to define the molecular pathway of AraC-induced telencephalic neuron death, we generated bcl-x 7/7 /p53 7/7 mice and examined the effect of p53 deficiency on Bcl-X Ldeficient neurons in vivo and in vitro. Our data suggest that under normal developmental conditions, cell cycle dysregulation and inadequate DNA repair are relatively minor apoptotic stimuli in the central nervous system and that Bcl-X L expression is critical for preventing immature neuron apoptosis caused by an undefined developmental signal(s) through a p53-independent pathway.…”