The number of neurons in the mammalian brain is determined by a balance between cell proliferation and programmed cell death. Recent studies indicated that Bcl-XL prevents, whereas Caspase-3 mediates, cell death in the developing nervous system, but whether Bcl-X L directly blocks the apoptotic function of Caspase-3 in vivo is not known. To examine this question, we generated bcl-x͞caspase-3 double mutants and found that caspase-3 deficiency abrogated the increased apoptosis of postmitotic neurons but not the increased hematopoietic cell death and embryonic lethality caused by the bcl-x mutation. In contrast, caspase-3, but not bcl-x, deficiency changed the normal incidence of neuronal progenitor cell apoptosis, consistent with the lack of expression of Bcl-X L in the proliferative population of the embryonic cortex. Thus, although Caspase-3 is epistatically downstream to Bcl-XL in postmitotic neurons, it independently regulates apoptosis of neuronal founder cells. Taken together, these results establish a role of programmed cell death in regulating the size of progenitor population in the central nervous system, a function that is distinct from the classic role of cell death in matching postmitotic neuronal population with postsynaptic targets. P rogrammed cell death (apoptosis) is an important mechanism in mammalian nervous system development (1, 2). First, programmed cell death adjusts postmitotic neuron number to match the size of their peripheral targets (3). Second, early brain-region-specific apoptosis such as cell death in the lateral edges of the hindbrain neural fold is essential for normal neural tube closure (4). Finally, the incidence of apoptosis within the proliferative ventricular zones (VZ) suggests a potential role of programmed cell death in regulating the size of the progenitor pool (5). Given its importance in normal brain development, the mechanism of apoptosis has been a subject of active investigation.Recent gene-targeting studies identified Bcl-X L as a critical antiapoptotic factor and Bax, Apaf-1, Caspase-9, and Caspase-3 as key proapoptotic molecules during normal brain development (6-12). Mice lacking bcl-x die as embryos and experience massive death of hematopoietic cells and postmitotic neurons (6). In addition, Bcl-X L -deficient neurons are markedly susceptible to trophic factor withdrawal in vitro (13). In contrast, mice lacking Bax show decreased cell death without obvious malformations (14), whereas targeted disruptions of apaf-1, caspase-9, or caspase-3 lead to decreased neuronal apoptosis in the embryonic nervous system and gross structural abnormalities (8-12).In the nematode Caenorhabditis elegans, the homologues of Bax (EGL-1), Bcl-X L (CED-9), Apaf-1 (CED-4), and caspases (CED-3) have analogous effects on programmed cell death and form a linear cell death pathway (15). Thus, it is an intriguing possibility that Bax, Bcl-X L , Apaf-1, Caspase-9, and Caspase-3 constitute an evolutionary conserved cell death pathway during brain development in mammals. Consistent with this ...
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.
Programmed cell death (apoptosis) of both proliferating neuroblasts and postmitotic neurons is essential for normal nervous system development. To study the molecular regulation of apoptosis in neuronal progenitor cells, we developed a flow cytometric assay capable of distinguishing between viable, apoptotic, and necrotic cell populations. Incubation of freshly dissociated telencephalic cells from gestational day 12–13 mouse embryos with either cytosine arabinoside (AraC) or staurosporine caused a marked increase in the percentage of apoptotic cells. Both drugs induced caspase-3 activation, as determined by in vitro cleavage of a caspase-3 substrate and immunocytochemical detection of activated caspase-3. Treatment of telencephalic cells with the broad caspase inhibitor BAF, blocked caspase-3 activation and protected cells against both AraC and staurosporine-induced apoptotic death. These results indicate that neuronal progenitors possess a caspase-dependent apoptotic pathway, the activation of which may regulate neuronal progenitor cell numbers in vivo.
We have identi®ed a C. elegans protein, ceBNIP3, homologous to the human BCL-2/EIB-19K interacting BCL-2 family pro-apoptotic protein BNIP3. In transiently transfected mammalian cells, ceBNIP3 complexes with CED-9, the worm homolog of BCL-2. CeBNIP3 also eciently heterodimerizes with the cell death protease proCED-3 by direct binding via the prodomain. Transfection of ceBNIP3 and CED-3 results in enhanced proteolytic processing of the CED-3 zymogen and in cooperative induction of apoptosis. Coexpression of CED-9 suppresses the cooperative cell death induced by ceBNIP3 and CED-3. In cells coexpressing CED-9, ceBNIP3 and CED-3, all three proteins exist as a ternary complex suggesting that CED-9 may suppress cooperative apoptosis induced by CED-3 and ceBNIP3 by simultaneous complex formation with CED-3 and ceBNIP3. Our results suggest that ceBNIP3 may be a novel component of the C. elegans apoptosis paradigm and may initiate apoptosis by recruiting CED-3 to mitochondria and other cytoplasmic membranes.
The N-terminal 48 amino acids of the Tat protein of human immunodeficiency virus type (HIV)-1 constitute its activation region. This region can autonomously activate transcription when targeted to the HIV-1 long terminal repeat or certain heterologous promoters either through DNA binding sites located upstream of the transcription initiation site or via downstream RNA binding sites in mammalian cells. To determine whether the Tat activation region can function in yeast, we have assayed the effect of a chimeric gene (GAL-Tat48) expressing the DNA binding domain of the yeast transcription factor Gal4 (residues 1-147) and the activation region of Tat on GAL1 promoter-directed expression of the lacZ reporter gene in Saccharomyces cerevisiae. Our results indicate that the Gal-Tat48 fusion protein can induce significant activation of the GAL1 promoter. Analysis of a number of Tat mutants located within the activation region indicate that the amino acid residues of Tat essential for trans-activation in mammalian cells are also required for transactivation in yeast. Our results suggest that Tat-mediated transcriptional activation may involve a mechanism conserved among yeast and mammalian cells.
Programmed cell death (apoptosis) is critical for normal brain morphogenesis and may be triggered by neurotrophic factor deprivation or irreparable DNA damage. Members of the Bcl2 and caspase families regulate neuronal responsiveness to trophic factor withdrawal; however, their involvement in DNA damage-induced neuronal apoptosis is less clear. To define the molecular pathway regulating DNA damage-induced neural precursor cell apoptosis, we have examined the effects of drug and gamma-irradiation-induced DNA damage on telencephalic neural precursor cells derived from wild-type embryos and mice with targeted disruptions of apoptosis-associated genes. We found that DNA damage-induced neural precursor cell apoptosis, both in vitro and in vivo, was critically dependent on p53 and caspase 9, but neither Bax nor caspase 3 expression. Neural precursor cell apoptosis was also unaffected by targeted disruptions of Bclx and Bcl2, and unlike neurotrophic factor-deprivation-induced neuronal apoptosis, was not associated with a detectable loss of cytochrome c from mitochondria. The apoptotic pathway regulating DNA damage-induced neural precursor cell death is different from that required for normal brain morphogenesis, which involves both caspase 9 and caspase 3 but not p53, indicating that additional apoptotic stimuli regulate neural precursor cell numbers during telencephalic development.
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