Viral proteins E1B19K and p35 protect sympathetic neurons from cell death induced by NGF deprivation.

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 Bcl-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.

The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis.

Bcl-2 is a mitochondrial- and perinuclear-associated protein that prolongs the lifespan of a variety of cell types by interfering with programmed cell death (apoptosis). Bcl-2 seems to function in an antioxidant pathway, and it is believed that membrane attachment mediated by a COOH-terminal hydrophobic tail is required for its full activity. To identify critical regions in bcl-2 alpha for subcellular localization, activity, and/or interaction with other proteins, we created, by site-directed mutagenesis, various deletion, truncation, and point mutations. We show here that membrane attachment is not required for the survival activity of bcl-2 alpha. A truncation mutant of bcl-2 alpha lacking the last 33 amino acids (T3.1) including the hydrophobic COOH terminus shows full activity in blocking apoptosis of nerve growth factor-deprived sympathetic neurons or TNF-alpha-treated L929 fibroblasts. Confocal microscopy reveals that the T3 mutant departs into the extremities of neurites in neurons and filopodias in fibroblasts. Consistently, T3 is predominantly detected in the soluble fraction by Western blotting, and is not inserted into microsomes after in vitro transcription/translation. We further provide evidence for motifs (S-N and S-II) at the NH2 and COOH terminus of bcl-2, which are crucial for its activity.

Dissecting processing and apoptotic activity of a cysteine protease by mutant analysis.

We have compared the behavior of wild-type mouse NEDD-2, a neural precursor cell-expressed, developmentally down-regulated cysteine protease gene, to various mutant forms of the gene in both apoptotic activity in neuronal cells and proteolytic cleavage in the Semliki Forest virus and rabbit reticulocyte protein expression systems. Our results confirm that NEDD-2 processing and apoptotic activity are linked phenomena. They identify aspartate residues as likely targets for autocatalytic cleavage. They establish that cleavage events only occur at specific sites. Finally, they pinpoint differential effects of individual mutations on the overall proteolytic cleavage patterns, raising interesting questions related to the mechanisms of subunit assembly.

The release of cytochrome c from mitochondria during apoptosis of NGF-deprived sympathetic neurons is a reversible event.

During apoptosis induced by various stimuli, cytochrome c is released from mitochondria into the cytosol where it participates in caspase activation. This process has been proposed to be an irreversible consequence of mitochondrial permeability transition pore opening, which leads to mitochondrial swelling and rupture of the outer mitochondrial membrane. Here we present data demonstrating that NGF-deprived sympathetic neurons protected from apoptosis by caspase inhibitors possess mitochondria which, though depleted of cytochrome c and reduced in size, remained structurally intact as viewed by electron microscopy. After re-exposure of neurons to NGF, mitochondria recovered their normal size and their cytochrome c content, by a process requiring de novo protein synthesis. Altogether, these data suggest that depletion of cytochrome c from mitochondria is a controlled process compatible with function recovery. The ability of sympathetic neurons to recover fully from trophic factor deprivation provided irreversible caspase inhibitors have been present during the insult period, has therapeutical implications for a number of acute neuropathologies.

Prevention of programmed cell death of sympathetic neurons by the bcl-2 proto-oncogene.

Approximately half of the neurons produced during embryogenesis normally die before adulthood. Although target-derived neurotrophic factors are known to be major determinants of programmed cell death--apoptosis--the molecular mechanisms by which trophic factors interfere with cell death regulation are largely unknown. Overexpression of the bcl-2 proto-oncogene in cultured sympathetic neurons has now been shown to prevent apoptosis normally induced by deprivation of nerve growth factor. This finding, together with the previous demonstration of bcl-2 expression in the nervous system, suggests that the Bcl-2 protein may be a major mediator of the effects of neurotrophic factors on neuronal survival.

Inhibition of Bax channel-forming activity by Bcl-2.

Proteins of the Bcl-2 family are intracellular membrane-associated proteins that regulate programmed cell death (apoptosis) either positively or negatively by as yet unknown mechanisms. Bax, a pro-apoptotic member of the Bcl-2 family, was shown to form channels in lipid membranes. Bax triggered the release of liposome-encapsulated carboxyfluorescein at both neutral and acidic pH. At physiological pH, release could be blocked by Bcl-2. Bcl-2, in contrast, triggered carboxyfluorescein release at acidic pH only. In planar lipid bilayers, Bax formed pH- and voltage-dependent ion-conducting channels. Thus, the pro-apoptotic effects of Bax may be elicited through an intrinsic pore-forming activity that can be antagonized by Bcl-2.

Cholinergic differentiation factor (CDF/LIF) promotes survival of isolated rat embryonic motoneurons in vitro.

We present evidence that the cholinergic differentiation factor (CDF), originally purified from cardiac and skeletal muscle cell-conditioned medium and found to be identical to leukemia inhibitory factor (LIF), promotes survival of embryonic day 14 rat motoneurons in vitro. These neurons were retrogradely labeled with the fluorescent tracer Dil and enriched on a density gradient or purified to homogeneity by fluorescence-activated cell sorting. Subnanomolar concentrations of CDF/LIF supported the survival of 85% of the motoneurons that would have died between days 1 and 4 of culture. The enhanced survival was accompanied by a 4-fold increase in choline acetyltransferase (ChAT) activity per culture. CDF/LIF also increased ChAT activity in dorsal spinal cord cultures, but had no detectable effect on ChAT levels in septal or striatal neuronal cultures. For comparison, other neurotrophic molecules were tested on motoneuron cultures. Ciliary neurotrophic factor had effects on motoneuron survival similar to those of CDF/LIF, whereas basic fibroblast growth factor was somewhat less effective. Nerve growth factor had no effect on the survival of rat motoneurons.

Function and expression of the Bcl-x gene in the developing and adult nervous system.

We have shown that overexpression of Bcl-x can rescue sympathetic neurones from nerve growth factor deprivation in vitro. We have also examined the distribution and expression of Bcl-x mRNA in the developing and adult nervous system using Northern blot and in situ hybridization. Bcl-x mRNA is widespread during development of the nervous system. In embryonic spinal cord, mRNA levels increase at the beginning of the naturally occurring cell death period, suggesting that Bcl-x may be involved in the selection of neurones during this period. In the brain, Bcl-x expression increases after birth to reach a high level in the adult brain. Neurones from the cortex, olfactory bulb, and Purkinje cells are among those expressing the highest levels of Bcl-x mRNA. The widespread expression of Bcl-x during development and in adult brain suggests of a role for Bcl-x beyond simply protecting neurones from developmental cell death.

Bax and Bak proteins require caspase activity to trigger apoptosis in sympathetic neurons.

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.

Multiple promoters of human choline acetyltransferase and aromatic L-amino acid decarboxylase genes.

The promoter regions of human choline acetyltransferase (ChAT) and aromatic L-amino acid decarboxylase (AADC) genes have been analyzed by transient transfection assays. AADC gene is transcribed from two alternative noncoding first exons, 1N and 1NN, expressed in pheochomocytoma and hepatoma cells, respectively. 5' flanking sequences of exon 1 N (from 9000 to 147 bp) display promoter activity in SK-N-BE neuroblastoma cells, but not in MC-I-XC cholinergic neuroepithelioma cells, and in AADC-rich non-neuronal cells. On the contrary, 5' flanking sequences of exon 1 NN (from 1117 to 119 bp) display high promoter activity in human hepatoma cells HepG2, but not in SK-N-BE cells, suggesting high degrees of specificity of promoters N and NN for AADC-expressing neuronal and non-neuronal cells, respectively. Preliminary evidence suggests that leukemia inhibitory factor suppresses the activity of the neuronal promoter in cultured sympathetic neurons. Two alternative first exons, R and M, have been localized in human ChAT gene, and the corresponding promoters characterized in cholinergic PC12 and NG-108-15 cells, and in non-cholinergic neuro2A cells. Several positively or negatively acting cis elements have been localized in the two promoters, as well as a cAMP-inducible, enhancer-like element in the second intron. Among the various cell lines studied, there was no correlation between promoter activities and the expression of the endogenous ChAT gene, suggesting that the fine-tuning of ChAT gene expression is controlled by silencer elements which remain to be localized.

The p75 neurotrophin receptor: effects on neuron survival in vitro and interaction with death domain-containing adaptor proteins.

The p75 neurotrophin receptor (p75NTR) is a death domain (DD) containing receptor of the TNF/FAS(APO-1) family. p75NTR has recently been shown to mediate apoptosis in certain types of neurons as well as in oligodendrocytes. The molecular mechanisms by which p75NTR stimulates apoptosis are still unknown. Here, we have tested whether overexpression of p75NTR could modulate survival of sympathetic neurons cultured in the presence or absence of NGF. Moreover, using the yeast two-hybrid system, we tested whether p75NTR intracellular domain was able to dimerize or interact with known DD-containing proteins including FADD, RIP, RAIDD and TRADD. We found that over-expression of p75NTR had no effect on the survival of sympathetic neurons cultured in the presence of NGF but instead delayed neuronal death following NGF deprivation. These results strongly support the finding that p75NTR is not involved in the apoptosis process induced by NGF deprivation in sympathetic neurons. We also foun d that the intracellular domain of p75NTR failed to associate either with itself or with other known DD-containing proteins. This suggests that the mechanisms by which p75NTR triggers apoptosis in certain cell types are different from those used by other receptors of the TNF/FAS family.

Dissection of functional domains in Bcl-2 alpha by site-directed mutagenesis.

Bcl-2 alpha is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. How Bcl-2 confers cell survival is unknown, although antioxidant and antiprotease functions have been proposed. In addition, protein structures of Bcl-2 that are crucial for its survival activity are still ill-defined. Bcl-2 can occur as Bcl-2 alpha or Bcl-2 beta, two alternatively spliced forms which solely differ in their carboxyl termini. The finding that Bcl-2 alpha is active and membrane bound, but Bcl-2 beta is inactive and cytosolic, indicates that the carboxyl terminus contributes to the survival activity of Bcl-2. This region contains two subdomains, a domain X with unknown function and a hydrophobic stretch reported to mediate membrane association of Bcl-2 alpha. Recently Bcl-2-related proteins have been identified. These include Bax that heterodimerizes with Bcl-2 and, when overexpressed, counteracts Bcl-2. Bax contains two highly conserved regions of sequence homology with Bcl-2, referred to as Bcl-2 homology 1 and 2 (BH1 and BH2) domains. Site-directed mutagenesis studies have revealed that both domains are not only novel dimerization motifs for the interaction of Bax with Bcl-2 but also crucial for the survival activity of Bcl-2. Interestingly, the C-terminal end of BH2 encompasses the Bcl-2 alpha/beta splice site, as well as part of domain X in Bcl-2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

p53 protein in sympathetic neurons: cytoplasmic localization and no apparent function in apoptosis.

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. Over-expression 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.

MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies a new class of mitogen-activated protein kinase phosphatase.

MKP-1 (also known as CL100, 3CH134, Erp, and hVH-1) exemplifies a class of dual-specificity phosphatase able to reverse the activation of mitogen-activated protein (MAP) kinase family members by dephosphorylating critical tyrosine and threonine residues. We now report the cloning of MKP-3, a novel protein phosphatase that also suppresses MAP kinase activation state. The deduced amino acid sequence of MKP-3 is 36% identical to MKP-1 and contains the characteristic extended active-site sequence motif VXVHCXXGXSRSXTXXXAYLM (where X is any amino acid) as well as two N-terminal CH2 domains displaying homology to the cell cycle regulator Cdc25 phosphatase. When expressed in COS-7 cells, MKP-3 blocks both the phosphorylation and enzymatic activation of ERK2 by mitogens. Northern analysis reveals a single mRNA species of 2.7 kilobases with an expression pattern distinct from other dual-specificity phosphatases. MKP-3 is expressed in lung, heart, brain, and kidney, but not significantly in skeletal muscle or testis. In situ hybridization studies of MKP-3 in brain reveal enrichment within the CA1, CA3, and CA4 layers of the hippocampus. Metrazole-stimulated seizure activity triggers rapid (<1 h) but transient up-regulation of MKP-3 mRNA in the cortex, piriform cortex, and some amygdala nuclei. Metrazole stimulated similar regional up-regulation of MKP-1, although this was additionally induced within the thalamus. MKP-3 mRNA also undergoes powerful induction in PC12 cells after 3 h of nerve growth factor treatment. This response appears specific insofar as epidermal growth factor and dibutyryl cyclic AMP fail to induce significant MKP-3 expression. Subcellular localization of epitope-tagged MKP-3 in sympathetic neurons reveals expression in the cytosol with exclusion from the nucleus. Together, these observations indicate that MKP-3 is a novel dual-specificity phosphatase that displays a distinct tissue distribution, subcellular localization, and regulated expression, suggesting a unique function in controlling MAP kinase family members. Identification of a second partial cDNA clone (MKP-X) encoding the C-terminal 280 amino acids of an additional phosphatase that is 76% identical to MKP-3 suggests the existence of a distinct structurally homologous subfamily of MAP kinase phosphatases.

Cloning of a bcl-2 homologue by interaction with adenovirus E1B 19K.

A number of DNA viruses carry apoptosis-inhibiting genes which enable the virus to escape from the host response. The adenovirus E1B 19K protein can inhibit apoptosis induced by E1A, tumour-necrosis factor-alpha, FAS antigen and nerve growth factor deprivation. The molecular basis of this inhibition remains poorly understood, but the fact that protection is seen in the absence of other viral proteins suggests that E1B 19K targets cellular proteins. We report here the identification of three cellular proteins that bind E1B 19K. One of these is a new member of the bcl-2 family, which we have called bak (for bcl-2 homologous antagonist/killer). This protein, which is expressed in a wide variety of cell types, binds to E1B 19K and to the Bcl-2 homologue Bcl-XL (ref. 17) in yeast. In addition, overexpression of bak in sympathetic neurons deprived of nerve growth factor accelerates apoptosis and blocks the protective effect of co-injected E1B 19K.

Molecular cloning and functional characterization of a novel mitogen-activated protein kinase phosphatase, MKP-4.

Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38/RK/CSBP (p38) mitogen-activated protein (MAP) kinases are target enzymes activated by a wide range of cell-surface stimuli. Recently, a distinct class of dual specificity phosphatase has been shown to reverse activation of MAP kinases by dephosphorylating critical tyrosine and threonine residues. By searching the expressed sequence tag data base (dbEST) for homologues of known dual specificity phosphatases, we identified a novel partial human sequence for which we isolated a full-length cDNA (termed MKP-4). The deduced amino acid sequence of MKP-4 is most similar to MKP-X/PYST2 (61% identity) and MKP-3/PYST1 (57% identity), includes two N-terminal CH2 domains homologous to the cell cycle regulator Cdc25 phosphatase, and contains the extended active site sequence motif VXVHCXAGXSRSXTX3AYLM (where X is any amino acid) conserved in dual specificity phosphatases. MKP-4 produced in Escherichia coli catalyzes vanadate-sensitive breakdown of p-nitrophenyl phosphate as well as in vitro inactivation of purified ERK2. When expressed in COS-7 cells, MKP-4 blocks activation of MAP kinases with the selectivity ERK > p38 = JNK/SAPK. This cellular specificity is similar to MKP-3/PYST1, although distinct from hVH-5/M3-6 (JNK/SAPK = p38 >>> ERK). Northern analysis reveals a highly restricted tissue distribution with a single MKP-4 mRNA species of approximately 2.5 kilobases detected only in placenta, kidney, and embryonic liver. Immunocytochemical analysis showed MKP-4 to be present within cytosol although punctate nuclear staining co-localizing with promyelocytic protein was also observed in a subpopulation (10-20%) of cells. Chromosomal localization by analysis of DNAs from human/rodent somatic cell hybrids and a panel of radiation hybrids assign the human gene for MKP-4 to Xq28. The identification and characterization of MKP-4 highlights the emergence of an expanding family of structurally homologous dual specificity phosphatases possessing distinct MAP kinase specificity and subcellular localization as well as diverse patterns of tissue expression.

Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons.

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.