Alternating metabolic pathways in NGF-deprived sympathetic neurons affect caspase-independent death

Mitochondrial release of cytochrome c in apoptotic cells activates caspases, which execute apoptotic cell death. However, the events themselves that culminate in caspase activation can have deleterious effects because caspase inhibitor-saved cells ultimately die in a caspase-independent manner. To determine what events may underlie this form of cell death, we examined bioenergetic changes in sympathetic neurons deprived of NGF in the presence of a broad-spectrum caspase inhibitor, boc-aspartyl-(OMe)-fluoromethylketone. Here, we report that NGF-deprived, boc-aspartyl-(OMe)-fluoromethylketone-saved neurons rely heavily on glycolysis for ATP generation and for survival. Second, the activity of F0F1 contributes to caspase-independent death, but has only a minor role in the maintenance of mitochondrial membrane potential, which is maintained primarily by electron transport. Third, permeability transition pore inhibition by cyclosporin A attenuates NGF deprivation-induced loss of mitochondrial proteins, suggesting that permeability transition pore opening may have a function in regulating the degradation of mitochondria after cytochrome c release. Identification of changes in caspase inhibitor-saved cells may provide the basis for rational strategies to augment the effectiveness of the therapeutic use of postmitochondrial interventions.     

Biochemical characterization of programmed cell death in NGF-deprived sympathetic neurons

Young sympathetic neurons die when deprived of nerve growth factor (NGF). Under such circumstances, cell death is appropriate to the developing nervous system and requires RNA and protein synthesis. We have hypothesized the existence of an endogenous death program within neurons that is suppressed by trophic factors. The extent and timing of required changes in the synthetic events that comprise the death program are unknown. In an effort to characterize the biochemical events that mediate the death program further, we performed several experiments on embryonic rat sympathetic neurons in vitro. The death program was blocked with cycloheximide when total protein synthesis was inhibited ≥80%. When protein synthesis was inhibited within 22 ± 4 h of NGF deprivation, death was prevented in half the neurons. Hence, we define the commitment point for protein synthesis to be 22 ± 4 h. Analogously, the commitment point for RNA synthesis was 26 ± 4 h and that for NGF rescue, 24 ± 4 h. We tested the ability of a wide variety of chemicals to interfere with the death program. Most compounds tested were unable to prevent neuronal death. Some treatments, however, did save NGF-deprived neurons and were subsequently characterized. These included ultraviolet light and agents that raise intracellular concentrations of cAMP. Finally, we looked for the neuronal expression in vitro and in vivo of genes that have been associated with programmed death in other cell types, including TRPM-2/SGP-2, polyubiquitin, TGFβ-1, c-fos, and c-myc. None of these genes showed significant activation associated with neuronal death. © 1992 John Wiley & Sons, Inc.     

Biochemical characterization of programmed cell death in NGF-deprived sympathetic neurons.

Young sympathetic neurons die when deprived of nerve growth factor (NGF). Under such circumstances, cell death is appropriate to the developing nervous system and requires RNA and protein synthesis. We have hypothesized the existence of an endogenous death program within neurons that is suppressed by trophic factors. The extent and timing of required changes in the synthetic events that comprise the death program are unknown. In an effort to characterize the biochemical events that mediate the death program further, we performed several experiments on embryonic rat sympathetic neurons in vitro. The death program was blocked with cycloheximide when total protein synthesis was inhibited > or = 80%. When protein synthesis was inhibited within 22 +/- 4 h of NGF deprivation, death was prevented in half the neurons. Hence, we define the commitment point for protein synthesis to be 22 +/- 4 h. Analogously, the commitment point for RNA synthesis was 26 +/- 4 h and that for NGF rescue, 24 +/- 4 h. We tested the ability of a wide variety of chemicals to interfere with the death program. Most compounds tested were unable to prevent neuronal death. Some treatments, however, did save NGF-deprived neurons and were subsequently characterized. These included ultraviolet light and agents that raise intracellular concentrations of cAMP. Finally, we looked for the neuronal expression in vitro and in vivo of genes that have been associated with programmed death in other cell types, including TRPM-2/SGP-2, polyubiquitin, TGF beta-1, c-fos, and c-myc. None of these genes showed significant activation associated with neuronal death.     

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.     

The ICE family of cysteine proteases as effectors of cell death

Programmed cellular suicide follows a set of distinct morphological events involving profound cytoplasmic and nuclear changes. The recent discovery of a family of mammalian homologues of the Caenorhabditis elegans cell death protein CED-3 is now providing insight into how these events might be brought about. These mammalian proteins encode cysteine proteases with homology to the interleukin-1beta converting enzyme (ICE). CED-3 and seven of its currently known mammalian homologues cleave their substrates after an aspartate residue, a property shared only by the cytotoxic T cell (CTL) protease granzyme B which is necessary for the CTL-mediated killing of target cells. A number of proteins previously known to be cleaved in cells undergoing apoptosis have now been shown to be targeted by ICE-like proteases. Although many questions remain, it is becoming increasingly clear that this unique group of proteases play a central effector role in the process of physiological cell death. This article reviews various aspects of the ICE family of proteases.     

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.     

Cyclosporin A inhibits caspase-independent death of NGF-deprived sympathetic neurons: a potential role for mitochondrial permeability transition

Opening of the permeability transition pore (PTP) has been implicated as an important mitochondrial event that occurs during apoptosis. We examined the role of the PTP in the well-characterized cell death of rat sympathetic neurons deprived of nerve growth factor (NGF) in vitro. Removal of NGF causes these neurons to undergo either a classic apoptotic cell death or, when treated with a broad-spectrum caspase inhibitor such as boc-aspartyl(OMe)-fluoromethylketone (BAF), a delayed, nonapoptotic cell death. The PTP inhibitor, cyclosporin A (CsA), blocked commitment-to-die in the presence of BAF, as defined by the ability of NGF readdition to rescue cells, but had little effect on commitment-to-die in the absence of BAF. CsA did not have trophic effects on BAF-saved cells, but did block the decrease in mitochondrial membrane potential. These data suggest that PTP opening is a critical event in caspase-independent, nonapoptotic (but not caspase-dependent, apoptotic) death of NGF-deprived rat sympathetic neurons.     

Caspase-4 and caspase-5, members of the ICE/CED-3 family of cysteine proteases, are CrmA-inhibitable proteases

Proteases of the caspase family are implicated in mammalian apoptosis and constitute a protease cascade. We characterized caspase-4 (TX/ICH-2/ICErelII) and caspase-5 (ICErelIII/TY), which are most closely related to caspase-1 (ICE) among the caspase family. Although overexpression of caspase-4 and caspase-5 induced apoptosis, confirming previous observations, this apoptosis was not inhibited by a caspase-1-specific tetrapeptide inhibitor (Ac-YVAD-CHO), suggesting that caspase-4 and caspase-5 have different substrate specificities from caspase-1 and also that caspase-4- and caspase-5-induced apoptosis is not mediated by caspase-1. CrmA, a cowpox virus-derived caspase-1 inhibitor that prevents apoptosis induced by various stimuli, was cleaved by caspase-4 and caspase-5, and inhibited their proteolytic activity as assessed by cleavage of pro-caspase-3 (pro-CPP32/Yama/apopain). Thus, caspase-4 and caspase-5 are CrmA-inhibitable proteases like caspase-1 and might be involved in apoptosis.     

High-affinity inhibition of a family of Plasmodium falciparum proteases by a designed adaptive inhibitor

Drug development against viral or microbial targets is often compounded by the existence of naturally occurring polymorphisms or drug resistant mutations. In the case of Plasmodium falciparum, the etiological agent of malaria, four related and essential proteases, plasmepsin I, II, and IV and the histo-aspartyl protease (HAP), have been identified in the food vacuole of the parasite. Since all of these enzymes are involved in the hemoglobin degradation of infected victims, the simultaneous inhibition of the four enzymes can be expected to lead to a faster starvation of the parasite and to delay the onset of drug resistance, since four enzymes will need to mutate in a concerted fashion. This study describes the design of an adaptive inhibitor intended to inhibit the entire plasmepsin family. Adaptive inhibitors bind with extremely high affinity to a primary target within the family and maintain significant affinity against the remaining members. This objective is accomplished by engineering the strongest and most specific interactions of the inhibitor against conserved regions of the binding site and by accommodating target variations by means of flexible asymmetric functional groups. Using this approach, we have designed an inhibitor with subnanomolar affinity (0.5 nM) against the primary target, plasmepsin II, and with no loss or a very small loss of affinity against plasmepsin IV, I, and HAP (K(i) ratios of 0.4, 7.1, and 17.7, respectively). The core of the inhibitor is defined by an allophenylnorstatine scaffold. Adaptability is provided by an asymmetric amino indanol functional group facing one of the key variable regions in the binding site. Adaptive inhibitors, which display high affinity against several variations of a primary target, are expected to play an important role in the chemotherapy of infectious diseases.     

The inhibitor profiling of the caspase family of proteases using substrate-derived peptide glyoxals

A series of substrate-based α-keto-β-aldehyde (glyoxal) sequences have been synthesised and evaluated as inhibitors of the caspase family of cysteine proteases. A number of potent inhibitor sequences have been identified. For example, a palmitic acid containing sequence pal-Tyr-Val-Ala-Asp-glyoxal was demonstrated to be an extremely effective inhibitor of caspase-1, inhibiting not only the action of the protease against synthetic fluorogenic substrates (K_i = 0.3 nM) but also blocking its processing of pro-interleukin-1beta (pro-IL-1β). In addition, the peptide Ac-Asp-Glu-Val-Asp-glyoxal, which is based on the consensus cleavage sequence for caspase-3, is a potent inhibitor of this protease (K_i = 0.26 nM) yet only functions as a comparatively modest inhibitor of caspase-1 (K_i = 451 nM). Potent inhibitor sequences were also identified for caspases-6 and -8. However, the degree of discrimination between the family members is limited. The ability of Ac-Asp-Glu-Val-Asp-glyoxal to block caspase-3 like activity in whole cells and to delay the development of apoptosis was assessed. When tested against caspase-3 like activity in cell lysates, Ac-Asp-Glu-Val-Asp-glyoxal displayed effective inhibition similar to that observed against recombinant caspase-3. Treatment of whole cells with this potent caspase-3 inhibitor was however, not sufficient to significantly stall the development of apoptosis in-vitro.     

Transmitter status in cultured rat sympathetic neurons: plasticity and multiple function

Considerable recent study of the development of transmitter status in sympathetic principal neurons, both in vivo and in culture, has produced several surprising findings. In this paper we review work on cultured immature and adult principal neurons dissociated from the superior cervical ganglia of rats. The main points are; 1) Immature principal neurons that display adrenergic properties during the first postnatal week in culture can be shifted to cholinergic status, including formation of functional cholinergic synapses, by coculture with nonneuronal cells (e.g., dissociated heart cells) or by medium conditioned by such cells. Through the use of microcultures that contain only a single neuron grown on heart cells, it has been possible to demonstrate the transition from adrenergic to cholinergic function directly by serial physiological assays of the same neuron at intervals of days or weeks. 2) During this transition, the cultured neurons display adrenergic/cholinergic dual function. This dual function has also been observed in principal neurons isolated from ganglia of adult rats. 3) Some cultured neurons secrete a third transmitter, probably adenosine or a phosphorylated derivative. This purinergic function is expressed with adrenergic or cholinergic function, or with both (triple function). In some cases, the main effect exerted by a neuron on cocultured cardiac myocytes is purinergic.     

Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases.

The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3 (glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1 beta-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades.     

Physiological properties of newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons

We have examined the physiological properties of transmission at newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons in vitro. Chick neurons were labeled with fluorescent carbocyanine dyes before they were placed into culture (Honig and Hume, 1986), and were studied by making intracellular recordings during the first 2 weeks of coculture. Evoked monosynaptic excitatory postsynaptic potentials (EPSPs) were not observed until 48 h of coculture. Beyond this time, the frequency with which connected pairs could be found did not vary greatly with time. With repetitive stimulation, the evoked monosynaptic EPSPs fluctuated in amplitude from trial to trial and showed depression at frequencies as low as 1 Hz. To gain further information about the quantitative properties of transmission at newly formed synapses, we analyzed the pattern of fluctuations of delayed release EPSPs. In mature systems, delayed release EPSPs are known to represent responses to single quanta, or to the synchronous release of a small number of quanta. For more than half of the connections we studied, the histograms of delayed release EPSPs were extremely broad. This result suggested that either quantal reponses are drawn from a continuous distribution that has a large coefficient of variation or that there are several distinct size classes of quantal responses. The pattern of fluctuation of monosynaptic EPSPs was consistent with both of these possibilities, and was inconsistent with the possibility that monosynaptic EPSPs are composed of quantal subunits with very little intrinsic variation. Although variation in the size of responses to single quanta might arise in a number of ways, one attractive explanation for our results is that the density and type of acetylcholine receptors varies among the different synaptic sites on the surface of developing sympathetic ganglion neurons.     

Physiological properties of newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons.

We have examined the physiological properties of transmission at newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons in vitro. Chick neurons were labeled with fluorescent carbocyanine dyes before they were placed into culture (Honig and Hume, 1986), and were studied by making intracellular recordings during the first 2 weeks of coculture. Evoked monosynaptic excitatory postsynaptic potentials (EPSPs) were not observed until 48 h of coculture. Beyond this time, the frequency with which connected pairs could be found did not vary greatly with time. With repetitive stimulation, the evoked monosynaptic EPSPs fluctuated in amplitude from trial to trial and showed depression at frequencies as low as 1 Hz. To gain further information about the quantitative properties of transmission at newly formed synapses, we analyzed the pattern of fluctuations of delayed release EPSPs. In mature systems, delayed release EPSPs are known to represent responses to single quanta, or to the synchronous release of a small number of quanta. For more than half of the connections we studied, the histograms of delayed release EPSPs were extremely broad. This result suggested that either quantal responses are drawn from a continuous distribution that has a large coefficient of variation or that there are several distinct size classes of quantal responses. The pattern of fluctuations of monosynaptic EPSPs was consistent with both of these possibilities, and was inconsistent with the possibility that monosynaptic EPSPs are composed of quantal subunits with very little intrinsic variation. Although variation in the size of responses to single quanta might arise in a number of ways, one attractive explanation for our results is that the density and type of acetylcholine receptors varies among the different synaptic sites on the surface of developing sympathetic ganglion neurons.     

(p-Amidinophenyl)methanesulfonyl fluoride, an irreversible inhibitor of serine proteases

p-(Amidinophenyl)methanesulfonyl fluoride (p-APMSF) has been synthesized and shown to be a specific, irreversible inhibitor of the class of plasma serine proteases which demonstrate substrate specificity for the positively charged side chains of the amino acid lysine or arginine. In equimolar concentration, this compound causes immediate and complete irreversible inhibition of bovine trypsin and human thrombin. A 5-10-fold molar excess of reagent over enzyme is required to achieve complete irreversible inhibition of bovine Factor Xa, human plasmin, human C1-r, and human C1-s. the Ki of p-APMSF for all of the above-mentioned proteases is between 1 and 2 microM. In contrast, p-APMSF in large molar excess does not inactivate chymotrypsin or acetylcholinesterase. The unique reactivity of p-APMSF has been further shown in comparison with the related compound p-nitrophenyl (p-amidinophenyl)methanesulfonate which is an active-site titrant for thrombin but reacts poorly with Factor Xa, C1-r, and C1-s and is not hydrolyzed by bovine trypsin or human plasmin. Similarly, (p-amidinophenyl)methanesulfonate has a Ki of 30 microM for thrombin but is a poor inhibitor of trypsin, Factor Xa, C1-r, C1-s, and plasmin. Studies with bovine trypsin have demonstrated that the inhibitory activity of p-APMSF is the result of its interaction with the diisopropyl fluorophosphate reactive site. The unique reactivity of this inhibitor classifies it as one of the most effective active site directed reagents for this class of serine proteases. Collectively, these results suggest that the primary substrate binding site of these enzymes, which share a high degree of structural homology, do in fact significantly differ from each other in their ability to interact with low molecular weight inhibitors and synthetic substrates.     

Mu-conotoxin SmIIIA,a potent inhibitor of tetrodotoxin-resistant sodium channels in amphibian sympathetic and sensory neurons

Mu-conotoxins are a family of peptides from the venoms of predatory cone snails. Previously characterized mu-conotoxins preferentially block skeletal muscle voltage-gated sodium channels. We report here the discovery (via cloning), synthesis, and electrophysiological characterization of a new peptide in this family, mu-conotoxin SmIIIA from Conus stercusmuscarum. Although mu-conotoxin SmIIIA shares several biochemical characteristics with other mu-conotoxins (the arrangement of cysteine residues and a conserved arginine believed to interact with residues near the channel pore), it has distinctive features such as the absence of hydroxyproline. In voltage-clamped dissociated neurons from frog sympathetic and dorsal root ganglia, the peptide inhibited the majority of tetrodotoxin-resistant sodium currents irreversibly; in contrast, tetrodotoxin-sensitive sodium currents were largely unaffected by the peptide. We believe that mu-conotoxin SmIIIA is the first specific antagonist of tetrodotoxin-resistant voltage-gated sodium channels to be discovered. Thus, the peptide provides a new and potentially useful tool to investigate the functional roles of tetrodotoxin-resistant voltage-gated sodium channels, including those that are found in sensory nerves that convey nociceptive information.     

Squamous cell carcinoma antigen 1 is an inhibitor of parasite-derived cysteine proteases

The physiological significance of the squamous cell carcinoma antigens 1 (SCCA1) and SCCA2, members of the ovalbumin serpin family, remains unresolved. In this study, we examined whether SCCA1 or SCCA2 inhibits protozoa- or helminth-derived cysteine proteases. SCCA1, but not SCCA2, potently inhibited the cysteine protease activities of CPB2.8 from Leishmania mexicana, cruzain from Trypanosoma cruzi, rhodesain from Trypanosoma brucei rhodesience, and cathepsin L2 from Fasciola hepatica. The inhibitory activities of SCCA1 were due to its resistance to cleavage by the cysteine proteases. The findings indicate that induction of cysteine protease inhibitors might be a novel defense mechanism against parasite development.