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. 2001 Jan 1;21(1):169-75.
doi: 10.1523/JNEUROSCI.21-01-00169.2001.

Bcl-X(L)-caspase-9 interactions in the developing nervous system: evidence for multiple death pathways

A U Zaidi  1 , C D'Sa-Eipper, J Brenner, K Kuida, T S Zheng, R A Flavell, P Rakic, K A Roth
Affiliations

Bcl-X(L)-caspase-9 interactions in the developing nervous system: evidence for multiple death pathways

A U Zaidi et al. J Neurosci. .

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(L)-deficient embryos was completely prevented by concomitant Caspase-9 deficiency. In contrast, bcl-x(-/-)/caspase-9(-/-) 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.

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Figures

Fig. 1.
Fig. 1.
Caspase-9 deficiency blocks the increased immature neuron apoptosis caused by Bcl-XL deficiency.A–C, Hematoxylin and eosin-stained sections from the E12.5 brainstem show normal healthy neurons in a wild-type embryo (A) and large numbers of apoptotic neurons in a Bcl-XL-deficient embryo (B; examples of apoptotic nuclei are indicated by arrows).Bcl-x−/−/caspase-9−/−brainstem (C) shows no evidence of increased apoptosis. D–F, A bisbenzimide-stained section from a Bcl-XL-deficient embryo shows frequent abnormal condensed nuclei in the anterior spinal cord (D; examples indicated by arrows), and simultaneous labeling for activated Caspase-3 immunoreactivity (E, red) reveals numerous positive cells. Dual staining for activated Caspase-3 immunoreactivity and nuclei in abcl-x−/−/caspase-9−/−E12.5 spinal cord (F) shows neither activated Caspase-3 immunoreactivity nor condensed nuclei. Scale bar, 25 μm.
Fig. 2.
Fig. 2.
Bcl-XL deficiency fails to prevent neurodevelopmental abnormalities in Caspase-9-deficient embryos. Toluidine blue-stained horizontal sections from the E12.5 telencephalon show well organized neuroepithelium in wild-type (A) but neithercaspase-9−/−(B) norbcl-x−/−/caspase-9−/−(C) embryos. In both Caspase-9-deficient embryos, the telencephalic vesicle is thickened, and supernumerary cells fill the lateral ventricle (V).a, Anterior. Scale bar, 25 μm.
Fig. 3.
Fig. 3.
Quantification of Caspase-3 activation and cell death in wild-type, Caspase-9-deficient, Bcl-XL-deficient, and double-deficient telencephalic cells. E12.5 telencephalic cells were grown for 48 hr in DMEM; activated Caspase-3 immunoreactive cells were detected with CM1 antiserum (A), and cell death (B) was determined by propidium iodide labeling. Bcl-XL-deficient cells had increased Caspase-3 activation and cell death; concomitant Caspase-9 deficiency inhibited the death-promoting effects of Bcl-XL in this in vitro paradigm. No effect of gene dosage on Caspase-3 activation or cell viability was observed for eitherbcl-x or caspase-9, and therefore, data from wild-type and heterozygous mutants were pooled (+/o denotes +/+ and +/− mice). Each data point represents the mean ± SEM. *p < 0.05 compared withbcl-x+/o/caspase-9+/ocells.
Fig. 4.
Fig. 4.
Effect of proapoptotic and antiapoptotic gene disruptions on AraC-induced telencephalic neuron Caspase-3 activation and cell death. Primary telencephalic neuron cultures were prepared from control and homozygous mutant embryos. After 48 hr of AraC exposure, Caspase-3 activation and cell death was assessed using CM1 antiserum and SYTOX Green labeling. In Bcl-XL-deficient neurons, AraC treatment produced a significant increase in the number of cells exhibiting activated Caspase-3 immunoreactivity (A) and in dead cells (B). Although Bax-, Caspase-9-, Caspase-3-, and p53-deficient neurons all exhibited reduced Caspase-3 activation compared with control neurons after AraC exposure, only Bax- and p53-deficient neurons exhibited significant neuroprotection. Heterozygous bax,caspase-9, and caspase-3 mutant neurons behaved identically to wild-type neurons and therefore, data from wild-type and +/− cultures were pooled (+/o denotes +/+ and +/− mice). In contrast, p53+/− neurons exhibited an intermediate degree of neuroprotection (data not shown) and therefore, only wild-type littermates were used to compare the effects of p53 deficiency. Each data point represents mean ± SEM (n = 5–20 for the different genotypes).
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