Caspase inhibition extends the commitment to neuronal death beyond cytochrome c release to the point of mitochondrial depolarization

Nerve growth factor (NGF) deprivation induces a Bax-dependent, caspase-dependent programmed cell death in sympathetic neurons. We examined whether the release of cytochrome c was accompanied by the loss of mitochondrial membrane potential during sympathetic neuronal death. NGF- deprived, caspase inhibitor-treated mouse sympathetic neurons maintained mitochondrial membrane potential for 25-30 h after releasing cytochrome c. NGF- deprived sympathetic neurons became committed to die, as measured by the inability of cells to be rescued by NGF readdition, at the time of cytochrome c release. In the presence of caspase inhibitor, however, this commitment to death was extended beyond the point of cytochrome c release, but only up to the subsequent point of mitochondrial membrane potential loss. Caspase-9 deficiency also arrested NGF-deprived sympathetic neurons after release of cytochrome c, and permitted these neurons to be rescued with NGF readdition. Commitment to death in the NGF-deprived, caspase- 9-deficient sympathetic neurons was also coincident with the loss of mitochondrial membrane potential. Thus, caspase inhibition extended commitment to death in trophic factor-deprived sympathetic neurons and allowed recovery of neurons arrested after the loss of cytochrome c, but not beyond the subsequent loss of mitochondrial membrane potential.     

Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation

We have developed an experimental paradigm to study the mechanism by which nerve growth factor (NGF) allows the survival of sympathetic neurons. Dissociated sympathetic neurons from embryonic day-21 rats were grown in vitro for 7 d in the presence of NGF. Neurons were then deprived of trophic support by adding anti-NGF antiserum, causing them to die between 24 and 48 h later. Ultrastructural changes included disruption of neurites, followed by cell body changes characterized by an accumulation of lipid droplets, changes in the nuclear membrane, and dilation of the rough endoplasmic reticulum. No primary alterations of mitochondria or lysosomes were observed. The death of NGF-deprived neurons was characterized biochemically by assessing [35S]methionine incorporation into TCA precipitable protein and by measuring the release of the cytosolic enzyme adenylate kinase into the culture medium. Methionine incorporation began to decrease approximately 18 h post-deprivation and was maximally depressed by 36 h. Adenylate kinase began to appear in the culture medium approximately 30 h after deprivation, reaching a maximum by 54 h. The death of NGF-deprived neurons was entirely prevented by inhibiting protein or RNA synthesis. Cycloheximide, puromycin, anisomycin, actinomycin-D, and dichlorobenzimidazole riboside all prevented neuronal death subsequent to NGF deprivation as assessed by the above morphologic and biochemical criteria. The fact that sympathetic neurons must synthesize protein and RNA to die when deprived of NGF indicates that NGF, and presumably other neurotrophic factors, maintains neuronal survival by suppressing an endogenous, active death program.     

JNK3 contributes to c-Jun activation and apoptosis but not oxidative stress in nerve growth factor-deprived sympathetic neurons

The stress activated protein kinase pathway culminates in c-Jun phosphorylation mediated by the Jun Kinases (JNKs). The role of the JNK pathway in sympathetic neuronal death is unclear in that apoptosis is not inhibited by a dominant negative protein of one JNK kinase, SEK1, but is inhibited by CEP-1347, a compound known to inhibit this overall pathway but not JNKs per se. To evaluate directly the apoptotic role of the JNK isoform that is selectively expressed in neurons, JNK3, we isolated sympathetic neurons from JNK3-deficient mice and quantified nerve growth factor (NGF) deprivation-induced neuronal death, oxidative stress, c-Jun phosphorylation, and c-jun induction. Here, we report that oxidative stress in neurons from JNK3-deficient mice is normal after NGF deprivation. In contrast, NGF-deprivation-induced increases in the levels of phosphorylated c-Jun, c-jun, and apoptosis are each inhibited in JNK3-deficient mice. Overall, these results indicate that JNK3 plays a critical role in activation of c-Jun and apoptosis in a classic model of cell-autonomous programmed neuron death.     

Death commitment point is advanced by axotomy in sympathetic neurons

Axotomized neurons have several characteristics that are different from intact neurons. Here we show that, unlike established cultures, the axotomized sympathetic neurons deprived of NGF become committed to die before caspase activation, since the same proportion of NGF-deprived neurons are rescued by NGF regardless of whether caspases are inhibited by the pan-caspase inhibitor Boc-Asp(O-methyl)-CH(2)F (BAF). Despite prolonged Akt and ERK signaling induced by NGF after BAF treatment has prevented death, the neurons fail to increase protein synthesis, recover ATP levels, or grow. Within 3 d, all the mitochondria disappear without apparent removal of any other organelles or loss of membrane integrity. Although NGF does rescue intact BAF-treated 6-d cultures after NGF deprivation, rescue by NGF fails when these neurons are axotomized before NGF deprivation and BAF treatment. Moreover, cytosolic cytochrome c rapidly kills axotomized neurons. We propose that axotomy induces signals that make sympathetic neurons competent to die prematurely. NGF cannot repair these NGF-deprived, BAF-treated neurons because receptor signaling (which is normal) is uncoupled from protein renewal, and the mitochondria (which are damaged) go on to be eliminated. Hence, the order of steps underlying neuronal death commitment is mutable and open to regulation.     

SM-20 is a novel mitochondrial protein that causes caspase-dependent cell death in nerve growth factor-dependent neurons

Sympathetic neurons undergo protein synthesis-dependent apoptosis when deprived of nerve growth factor (NGF). Expression of SM-20 is up-regulated in NGF-deprived sympathetic neurons, and ectopic SM-20 is sufficient to promote neuronal death in the presence of NGF. We now report that SM-20 is a mitochondrial protein that promotes cell death through a caspase-dependent mechanism. SM-20 immunofluorescence was present in the cytoplasm in a punctate pattern that colocalized with cytochrome oxidase I and with mitochondria-selective dyes. Analysis of SM-20/dihydrofolate reductase fusion proteins revealed that the first 25 amino acids of SM-20 contain a functional mitochondrial targeting sequence. An amino-terminal truncated form of SM-20 was not restricted to mitochondria but instead localized throughout the cytosol and nucleus. Nevertheless, the truncated SM-20 retained the ability to induce neuronal death, similar to the wild type protein. SM-20-induced death was accompanied by caspase-3 activation and was blocked by a general caspase inhibitor. Additionally, overexpression of SM-20, under conditions where cell death is blocked by a general caspase inhibitor, did not result in widespread release of cytochrome c from mitochondria. These results indicate that SM-20 is a novel mitochondrial protein that may be an important mediator of neurotrophin-withdrawal-mediated cell death.     

Comparison Between the Timing of JNK activation, c-Jun Phosphorylation, and Onset of Death Commitment in Sympathetic Neurones

Abstract: We have investigated the relationship between c-Jun N-terminal kinase (JNK) activity, apoptosis, and the potential of survival factors to rescue primary rat sympathetic neurones deprived of trophic support. Incubation of sympathetic neurones in the absence of nerve growth factor (NGF) caused a time-dependent increase in JNK activity, which became apparent by 3 h and attained maximal levels that were three- to fourfold higher than activity measured in neurones maintained for the same periods with NGF. Continuous culture in the presence of either NGF or the cyclic AMP analogue 4-(8-chlorophenylthio) cyclic AMP (CPTcAMP) not only prevented JNK activation from occurring, but also suppressed JNK activity that had been elevated by prior culture of the neurones in the absence of trophic support. When either NGF or CPTcAMP was added to cultures that had been initially deprived of neurotrophic support for up to 10 h, this resulted in complete suppression of total JNK activity, arrest of apoptosis, and rescue of >90% of the neurones that did not display apoptotic morphology by this time. However, when either agent was added after more protracted periods of initial neurotrophin deprivation (≥ 14 h), although this also resulted in near-complete suppression of total JNK activity and short-term arrest of apoptosis, not all of the neurones that appeared to be nonapoptotic at the time of agent addition were rescued. The lack of death commitment after 10 h of maintained JNK activity was not due to a late induction of c-Jun expression, because the majority of newly isolated sympathetic neurones had already been expressing high levels of c-Jun in their nuclei for several hours, yet were capable of being rescued by NGF. Elevation of JNK activity as a result of neurotrophic-factor deprivation was also associated with enhanced phosphorylation of c-Jun, assessed by immunoblot analysis and immunocytochemistry, and addition of NGF to cultures previously deprived of neurotrophic support resulted in a reversion of the state of phospho-c-Jun to that observed in cultures that had been maintained in the continuous presence of trophic support. We conclude that activation of JNK and c-Jun phosphorylation are not necessarily rate-limiting for apoptosis induction. In some neurones undergoing prolonged NGF deprivation, suppression of JNK activity and c-Jun dephosphorylation by NGF may be insufficient to effect their rescue. Thus, if c-Jun mediates death by increasing the expression of “death” genes, these must become effective very close to the death commitment point.     

Prolyl hydroxylase inhibitors delay neuronal cell death caused by trophic factor deprivation

Nerve growth factor (NGF) serves a critical survival-promoting function for developing sympathetic neurons. Following removal of NGF, sympathetic neurons undergo apoptosis characterized by the activation of c-Jun N-terminal kinases (JNKs), up-regulation of BH3-only proteins including BcL-2-interacting mediator of cell death (BIM)_EL, release of cytochrome c from mitochondria, and activation of caspases. Here we show that two small-molecule prolyl hydroxylase inhibitors frequently used to activate hypoxia-inducible factor (HIF) – ethyl 3,4-dihydroxybenzoic acid (DHB) and dimethyloxalylglycine (DMOG) – can inhibit apoptosis caused by trophic factor deprivation. Both DHB and DMOG blocked the release of cytochrome c from mitochondria after NGF withdrawal, whereas only DHB blocked c-Jun up-regulation and phosphorylation. DHB, but not DMOG, also attenuated the induction of BIM_EL in NGF-deprived neurons, suggesting a possible mechanism whereby DHB could inhibit cytochrome c release. DMOG, on the other hand, was substantially more effective at stabilizing HIF-2α and inducing expression of the HIF target gene hexokinase 2 than was DHB. Thus, while HIF prolyl hydroxylase inhibitors can delay cell death in NGF-deprived neurons, they do so through distinct mechanisms that, at least in the case of DHB, are partly independent of HIF stabilization.     

Identification of a novel kinase-like gene induced during neuronal cell death.

When deprived of neurotrophic factors, neuronal cells undergo a form of programmed cell death that involves a cascade of gene expression. To better understand this cascade, we screened the genes induced during programmed cell death evoked in neuronal PC6-3 cells by NGF-depletion and discovered a novel gene, NIPK (Neuronal cell death Inducible Putative Kinase), that contains a kinase-like domain. Expression of NIPK was also induced in cultured sympathetic neurons by NGF deprivation and in cortical neurons exposed to the Ca2+ ionophore, A23187. In contrast, NIPK was not induced during non-neuronal cell death evoked by serum or growth factor deprivation, or by treatment with methyl methanesulfonate, an agent that causes cell death by damaging DNA. Taken together, these findings suggest that NIPK is involved in programmed cell death via a pathway that is present in neurons but is absent in non-neurons.     

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.     

Inhibition of protein synthesis prevents cell death in sensory and parasympathetic neurons deprived of neurotrophic factor in vitro

Shortly after neurons begin to innervate their targets in the developing vertebrate nervous system they become dependent on the supply of a neurotrophic factor, such as nerve growth factor (NGF) for survival. Recently, Martin et al. (1988) have shown that inhibiting protein synthesis prevents the death of NGF-deprived sympathetic neurons, suggesting that NGF promotes neuronal survival by suppressing an active cell death program. To determine if other neurotrophic factors may regulate neuronal survival by a similar mechanism we examined the effects of inhibiting protein and RNA synthesis in other populations of embryonic neurons that require different neurotrophic factors, namely: 1) trigeminal mesencephalic neurons, a population of proprioceptive neurons that are supported by brain-derived neurotrophic factor; 2) dorsomedial trigeminal ganglion neurons, a population of cutaneous sensory neurons that are supported by NGF; 3) and ciliary ganglion neurons, a population of parasympathetic neurons that are supported by ciliary neuronotrophic factor. Blocking either protein or RNA synthesis rescued all three populations of neurons from cell death induced by neurotrophic factor deprivation in vitro. Thus, at least three different neurotrophic factors appear to promote survival by a similar mechanism that may involve the suppression of an endogenous cell death program.