Interleukin-1 beta converting enzyme (ICE) is a mammalian homolog of CED-3, a protein required for programmed cell death in the nematode Caenorhabditis elegans. The activity of ICE can be specifically inhibited by the product of crmA, a cytokine response modifier gene encoded by cowpox virus. Microinjection of the crmA gene into chicken dorsal root ganglion neurons was found to prevent cell death induced by deprivation of nerve growth factor. Thus, ICE is likely to participate in neuronal death in vertebrates.

Sympathetic neurons undergo programmed cell death (PCD) upon deprivation of nerve growth factor (NGF). PCD of neurons is blocked by inhibitors of the interleukin‐1beta converting enzyme (ICE)/Ced‐3‐like cysteine protease, indicating involvement of this class of proteases in the cell death programme. Here we demonstrate that the proteolytic activities of the proteasome are also essential in PCD of neurons. Nanomolar concentrations of several proteasome inhibitors, including the highly selective inhibitor lactacystin, not only prolonged survival of NGF‐deprived neurons but also prevented processing of poly(ADP‐ribose) polymerase which is known to be cleaved by an ICE/Ced‐3 family member during PCD. These results demonstrate that the proteasome is a key regulator of neuronal PCD and that, within this process, it is involved upstream of proteases of the ICE/Ced‐3 family. This order of events was confirmed in macrophages where lactacystin inhibited the proteolytic activation of precursor ICE and the subsequent generation of active interleukin‐1beta.

Abstract. To study molecular mechanisms underlying neuronal cell death, we have used sympathetic neurons from superior cervical ganglia which undergo programmed cell death when deprived of nerve growth factor. These neurons have been microinjected with expression vectors containing cDNAs encoding selected proteins to test their regulatory influence over cell death. Using this procedure, we have shown previously that sympathetic neurons can be protected from NGF deprivation by the protooncogene Bcl-2. We now report that the E1B19K protein from adenovirus and the p35 protein from baculovirus also rescue neurons.Other adenoviral proteins, E1A and E1B55K, have no effect on neuronal survival. E1B55K, known to block apoptosis mediated by p53 in proliferative cells, failed to rescue sympathetic neurons suggesting that p53 is not involved in neuronal death induced by NGF deprivation.E1B19K and p35 were also coinjected with Bcl-Xs which blocks Bcl-2 function in lymphoid cells. Although BcI-Xs blocked the ability of Bcl-2 to rescue neurons, it had no effect on survival that was dependent upon expression of E1B19K or p35. p ROGRAMMED cell death plays a key role during development of the nervous system (for review see reference 36), although the molecular mechanisms by which neurons die are unknown. Epigenetic factors, such as neurotrophic factors, seem to promote neuronal survival by blocking an intrinsic cell death program (for review see references 26, 38). Recent identification of proteins that can block apoptosis may be used as tools to unravel pathways of cell death. The Bcl-2 protooncogene (2, 44) is the prototype of these anti-death proteins (1,13,16,24,31,40), and a family of proteins homologous to Bcl-2 is now emerging (for review see reference 49). Among these, the Bcl-X gene is the most homologous to Bcl-2 (6) and encodes two splice variants termed Bcl-X1 and Bcl-Xs. Bcl-Xs lacks a 63-amino acid region that is conserved between different Bel-2 family members. Whereas BcI-X1 has anti-apoptotic function, BclXs inhibits the ability of Bcl-2 to enhance the survival of trophic factor-deprived cells (6).Other anti-apoptotic proteins, with no obvious primary sequence homology with members of the Bcl-2 family, have also been characterized. Among these are the E1B19K and EIB55K proteins from adenovirus and the p35 protein from baculovirus.The first two authors contributed equally to this work.Address all correspondence to Dr. Jean-Claude Martinou, Glaxo Institute for Molecular Biology, 14 Chem des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland. Tel.: (41) 22 706 The EIB gene encodes two major proteins, the 19-kD and 55-kD proteins which cooperate with E1A proteins to allow transformation (3,5,34,45). Although EIA alone is capable of stimulating cell proliferation, this is accompanied by rapid cell degeneration due to apoptosis. The E1B proteins overcome this effect thereby enhancing cell transformation (47). The 19-kD EIB protein can also block the cytotoxic action of tumor necrosis factor or of anti-FAS antibodies (18...

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We show that the pro-apoptotic proteins Bax and Bak trigger apoptosis when over-expressed in sympathetic neurons cultured in the presence of NGF. This effect can be blocked with z-VAD-fmk, a peptide inhibitor of caspases, but not with anti-apoptotic chemical compounds such as antioxidants or proteasome inhibitors. These results demonstrate that in sympathetic neurons Bax and Bak are sufficient to induce apoptosis in the absence of any other apparent cell death stimulus and that their effect is mediated by caspases but does not require reactive oxygen species nor activity of the proteasome.

Background: The human NECDIN gene is involved in a neurodevelopmental disorder, PraderWilli syndrome (PWS). Previously we reported a mouse Necdin knock-out model with similar defects to PWS patients. Despite the putative roles attributed to Necdin, mainly from in vitro studies, its in vivo function remains unclear. In this study, we investigate sensory-motor behaviour in Necdin deficient mice. We reveal cellular defects and analyse their cause.

Apoptosis and necrosis are two types of cell death with different morphologic features. We report here the isolation of a monoclonal antibody, BV2, that specifically recognize cells undergoing developmental programmed cell death in different tissues of the chicken and zebra-finch embryos. The antigen recognized by BV2 monolonal antibody is detected in vitro in primary chicken embryonic fibroblasts induced to die by actinomycin D, as well as fibroblasts induced to die by chemical anoxia. The expression of this specific antigen during necrosis appears to require active protein synthesis. These dinps provide evidence that cells from different embryonic tissues undergoing prgrammed cell death during vertebrate development express simlr antigens and indicate that apoptosis and necrosis may share similar biochemical features.The morphological concept of apoptosis, defined as a physiological type of cell death, has progressively been used as a synonym with the term programmed cell death, originally described during normal development (1) and implying the existence of a genetic program of cell death (2). Apoptosis is believed to account for most cell death during development and in normal adult tissue turnover, and it can also be induced experimentally by various biological, chemical, or physical agents (3). Necrosis, in contrast, has been defined as a passive degenerative phenomenon and is observed in a tissue subjected to direct toxic or physical injury-such as hyperthermia, hypoxia, and ischemia or to complement-mediated lysis (4-7). To investigate the mechanism of cell death, we have identified a monoclonal antibody (mAb), BV2, that specifically recognizes cells undergoing programmed cell death in developing chicken embryos, and we used BV2 mAb as a specific marker of chicken programmed cell death to study the relationship between apoptosis and necrosis.MATERIALS AND METHODS Generation of mAbs. Chicken eggs were purchased from Spafas, Norwich, CT. Zebra finches (Poephila guttata) were grown in the avian facilities of the California Institute of Technology. Chicken embryos were staged according to V. Hamburger and H. L. Hamilton (8). The generation of the cell-death-specific mAbs by neonatal tolerization (9) is described in detail elsewhere (10). BV2 hybridoma was screened on sections of day 7 chicken hind limb foot plates, expanded, and assayed several times for antibody reactivity and stabilized by three cycles of cloning by limiting dilution. The immunoglobulin (IgM) type was determined by ELISA using rat mAb anti-mouse immunoglobulin classes from Amersham. BV2 hybridoma cells (2-5 x 106) were injected into 7-to 8-week-old F1 BALB/cJ x C57/BL male mice (The Jackson Laboratory) primed with incomplete Freund's adjuvant (Cappel), and 2 weeks later, ascites fluid was collected and purified by E-Z-Sep (Middlesex Sciences, Foxborough, MA).Immunofluorescence. Whole embryos and limbs were rinsed in cold phosphate-buffered saline (PBS) and fixed in 4% periodate/lysine/paraformaldehyde prepared in PBS for 12 hr at 4...

The p53 tumour suppressor gene plays a major role in controlling cell cycle and apoptosis in many different cell types. Here we have examined the status and the potential apoptosis inducing activity of p53 in sympathetic neurons. The p53 protein is expressed in rat sympathetic neurons cultured in the presence of NGF. The protein is not upregulated when these neurons are induced to die upon NGF deprivation. Overexpression of wild‐type human p53 in neurons cultured in the presence of NGF does not trigger apoptosis nor does it accelerate apoptosis when the neurons are deprived of NGF. Finally endogenous p53 expression is not necessary for neuronal cell death triggered by NGF deprivation since neurons prepared from p53 knockout mice undergo normal cell death upon NGF deprivation. Our results suggest that p53 may have an unknown function in post‐mitotic neurons which is distinct from its well described roles in apoptosis or cell cycle control. © 1996 Wiley‐Liss, Inc.