Trophic Factor Deprivation Research Articles

G1/S Cell Cycle Blockers and Inhibitors of Cyclin-Dependent Kinases Suppress Camptothecin-Induced Neuronal Apoptosis

Previous studies have demonstrated that G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases (CDKs) prevent the death of nerve growth factor (NGF)-deprived PC12 cells and sympathetic neurons, suggesting that proteins normally involved in the cell cycle may also serve to regulate neuronal apoptosis. Past findings additionally demonstrate that DNA-damaging agents, such as the DNA topoisomerase (topo-I) inhibitor camptothecin, also induce neuronal apoptosis. In the present study, we show that camptothecin-induced apoptosis of PC12 cells, sympathetic neurons, and cerebral cortical neurons is suppressed by the G1/S blockers deferoxamine and mimosine, as well as by the CDK-inhibitors flavopiridol and olomoucine. In each case, the IC50 values were similar to those reported for inhibition of death induced by NGF-deprivation. In contrast, other agents that arrest DNA synthesis, such as aphidicolin and N-acetylcysteine, failed to block death. This suggests that the inhibition of DNA synthesis per se is insufficient to provide protection from camptothecin. We find additionally that the cysteine aspartase family protease inhibitor zVAD-fmk inhibits apoptosis evoked by NGF-deprivation but not camptothecin treatment. Thus, despite their shared sensitivity to G1/S blockers and CDK inhibitors, the apoptotic pathways triggered by these two causes of death diverge at the level of the cysteine aspartase. In summary, neuronal apoptosis induced by the DNA-damaging agent camptothecin appears to involve signaling pathways that normally control the cell cycle. The consequent death signals of such deregulation, however, are different from those that result from trophic factor deprivation.

Flunarizine enhances survival and regeneration of sensory and motor neurons after peripheral nerve injury

The diphenylpiperazine, flunarizine, partially prevents apoptosis after trophic factor deprivation in neural crest-derived neurons. Flunarizine protects dorsal root ganglion neurons (DRG) after nerve growth factor (NGF) withdrawal in vitro and after peripheral nerve injury in newborn rats in vivo. We have further studied the mechanisms of neuronal protection by flunarizine. Oligosomal DNA fragmentation, a hallmark of apoptosis, was significantly decreased by treatment of DRG neurons with flunarizine after NGF deprivation. We examined the effect on survival of the timing of administration of flunarizine to DRG neurons both in vitro and in vivo. Flunarizine effectively rescued dissociated DRG neurons if administered up to six hours after NGF withdrawal. In vivo, flunarizine prevented DRG neuronal death after sciatic axotomy in newborn rats if given soon after injury. Long-term experiments were done to test the ability of flunarizine to protect neurons and enhance regeneration after sciatic nerve injury. Newborn rats were subjected to peripheral nerve injury and administered flunarizine for four weeks; no further treatment was given for an additional 12 weeks. The group treated with flunarizine demonstrated a significantly increased number of DRG and spinal motor neurons that had regenerated axons into the distal sciatic nerve as determined by retrograde labeling with HRR Myelinated axons in the sural nerve in the group treated with flunarizine increased by nearly two-fold compared to control animals. Thus, flunarizine was able to enhance survival and promote long-term regeneration of sensory and motor spinal neurons after peripheral nerve injury.

Increased cerebellar Purkinje cell numbers in mice overexpressing a human bcl-2 transgene.

The Purkinje cell is a primary organizer in the development of the cerebellum. Purkinje cells may provide positional information cues that regulate afferent innervation, and Purkinje cell target size controls the adult number of afferent olivary neurons and granule cells. While Purkinje cells are necessary for the survival of olivary neurons and granule cells during periods of programmed cell death, little is known about the survival requirements of Purkinje cells in vivo. To determine if Purkinje cells are subject to programmed cell death during development we have analyzed Purkinje cell numbers in two lines of transgenic mice that overexpress a human gene for bcl-2 (Hu-bcl-2). Bcl-2 is a protooncogene that inhibits apoptosis in many cell types. Overexpression of bcl-2 in vitro and in vivo rescues neurons from trophic factor deprivation or naturally occurring cell death. In the mice analyzed in this study, transgene expression is driven by the neuron-specific enolase promoter that is first expressed embryonically in most regions of the brain in one line and postnatally in the second line. We have counted Purkinje cells in three adult control mice, five early overexpressing transgenics, and three late expressing transgenics. The number of Purkinje cells in the Hu-bcl-2 transgenic mice is significantly increased above control numbers, with an increase of 43% in the embryonically overexpressing line and an increase of 27% in the postnatally overexpressing line. Because bcl-2 overexpression has been shown to rescue other neurons from programmed cell death, the increase in Purkinje cell numbers in overexpressing bcl-2 transgenics suggests that Purkinje cells undergo a period of cell death during normal development.

The expression of calretinin in transfected PC12 cells provides no protection against Ca 2+-overload or trophic factor deprivation

To address the question whether calretinin (CR) may protect cells against Ca 2+ overload or trophic factor deprivation, PC12 cells were transfected with plasmids containing a CR coding region under control of a cytomegalovirus promoter. Nerve growth factor (NGF) treatment induced differentiation, increased transfection efficiency (at least 10-fold) and activated the CR gene (as found by RNase protection method and immunohistochemistry). Exogenous CR expression was identified either in living cells by fluorescence of green fluorescent protein (when the CR coding region was fused to this protein) or in fixed cells by CR immunoreactivity. Undifferentiated and NGF-differentiated populations of transfected cells were incubated in the presence of a Ca 2+-ionophore or in media deprived of serum or NGF. Expression of exogenous CR in undifferentiated or NGF-treated cells (due to transfection) or endogenous CR (due to gene activation by NGF) did not render PC12 cells more resistant to insults such as Ca 2+-overload and trophic factor deprivation.

BAX Is Required for Neuronal Death after Trophic Factor Deprivation and during Development

Members of the BCL2-related family of proteins either promote or repress programmed cell death. BAX, a death-promoting member, heterodimerizes with multiple death-repressing molecules, suggesting that it could prove critical to cell death. We tested whether Bax is required for neuronal death by trophic factor deprivation and during development. Neonatal sympathetic neurons and facial motor neurons from Bax-deficient mice survived nerve growth factor deprivation and disconnection from their targets by axotomy, respectively. These salvaged neurons displayed remarkable soma atrophy and reduced elaboration of neurites; yet they responded to readdition of trophic factor with soma hypertrophy and enhanced neurite outgrowth. Bax-deficient superior cervical ganglia and facial nuclei possessed increased numbers of neurons. Our observations demonstrate that trophic factor deprivation–induced death of sympathetic and motor neurons depends on Bax.

Immediate early gene expression in response to cerebral ischemia. Friend or foe?

Cerebral ischemia is a potent modulator of gene expression. Immediate early genes undergo rapid induction after both global and focal cerebral ischemia. Many immediate early genes code for transcription factors. Additional genes, including those encoding for neurotrophic factors and neurotransmitter systems, are induced in a delayed fashion after cerebral ischemia. The functional significance of early and late gene regulation after cerebral ischemia requires further investigation. These changes may be beneficial (friend) or detrimental (foe). Many of the genes are likely neuroprotective and important for recovery, but others may be involved in ischemic cell death mediated by apoptosis. We review evidence that supports the hypothesis that cell death after cerebral ischemia occurs through the dual pathways of ischemic necrosis and apoptosis. Gene regulation, including immediate early genes, is required for programmed neuronal death after trophic factor deprivation and is predicted to be involved in apoptosis triggered by cerebral ischemia. Novel therapies following cerebral ischemia may be directed at genes mediating either recovery or apoptosis.

Ordering the Cell Death Pathway: DIFFERENTIAL EFFECTS OF BCL2, AN INTERLEUKIN-1-CONVERTING ENZYME FAMILY PROTEASE INHIBITOR, AND OTHER SURVIVAL AGENTS ON JNK ACTIVATION IN SERUM/NERVE GROWTH FACTOR-DEPRIVED PC12 CELLS

Previous studies indicate that activation of c-Jun kinase (JNK) is necessary for apoptosis of trophic factor-deprived PC12 cells and that death in this system is suppressed by multiple agents, including BCL2, inhibitors of the interleukin-1-converting enzyme (ICE) family of proteases, blockers of transcription, and a variety of small molecules with differing modes of action. Here, we determine the order in which these agents block apoptosis relative to JNK activation. Overexpression of BCL2 promotes PC12 cell survival and blocks JNK activation caused by trophic factor withdrawal. Similarly, the survival-promoting agents aurintricarboxylic acid, N-acetylcysteine, the nitric oxide generator diethylenetriamine nitric oxide, 8-bromo-cGMP, and 8-(4-chlorophenylthio)-cAMP act upstream to inhibit JNK activation. In contrast, zVAD-fluoromethylketone (a permeant ICE family inhibitor), actinomycin D, and the G1/S cell cycle inhibitor deferoxamine, all promote survival after trophic factor withdrawal, but do not affect JNK activation. These findings are consistent with the presence of an ordered cell death pathway triggered by trophic factor deprivation in which 1) BCL2 and a number of survival-promoting agents act upstream of JNK, 2) ICE family protease actions, regulated genes required for cell death, and certain cell cycle blockers lie either downstream of JNK or on independent pathways required for apoptotic death.

The d4 Gene Family in the Human Genome

The d4 domain, a novel zinc finger-like structural motif, was first revealed in the rat neuro-d4 protein. Here we demonstrate that the d4 domain is conserved in evolution and that three related genes form a d4 family in the human genome. The human neuro-d4 is very similar to rat neuro-d4 at both the amino acid and the nucleotide levels. Moreover, the same splice variants have been detected among rat and human neuro-d4 transcripts. This gene has been localized on chromosome 19, and two other genes, members of the d4 family isolated by screening of the human genomic library at low stringency, have been mapped to chromosomes 11 and 14. The gene on chromosome 11 is the homolog of the ubiquitously expressed mouse gene ubi-d4/requiem, which is required for cell death after deprivation of trophic factors. A gene with a conserved d4 domain has been found in the genome of the nematodeCaenorhabditis elegans.The conservation of d4 proteins from nematodes to vertebrates suggests that they have a general importance, but a diversity of d4 proteins expressed in vertebrate nervous systems suggests that some family members have special functions.

Functional role of interleukin 1 beta (IL-1 beta) in IL-1 beta-converting enzyme-mediated apoptosis.

Prointerleukin-1 beta (pro-IL-1 beta) is the only known physiologic substrate of the interleukin-1 beta (IL-1 beta)-converting enzyme (ICE), the founding member of the ICE/ced-3 cell death gene family. Since secreted mature IL-1 beta has been detected after apoptosis, we investigated whether this cytokine, when produced endogenously, plays a role in cell death. We found that hypoxia-induced apoptosis can be inhibited by either the IL-1 receptor antagonist (IL-1Ra) or by neutralizing antibodies to IL-1 or to its type 1 receptor. IL-1Ra also inhibits apoptosis induced by trophic factor deprivation in primary neurons, as well as by tumor necrosis factor alpha in fibroblasts. In addition, during the G1/S phase arrest, mature IL-1 beta induces apoptosis through a pathway independent of CrmA-sensitive gene activity. We also demonstrate that Ice, when expressed in COS cells, requires the coexpression of pro-IL-1 beta for the induction of apoptosis, which is inhibited by IL-1Ra. Interestingly, we found that mature IL-1 beta has antiapoptotic activity when added exogenously before the onset of hypoxia, which we found is caused in part by its ability to downregulate the IL-1 receptor. Our findings demonstrate that pro-IL-1 beta is a substrate of ICE relevant to cell death, and depending on the temporal cellular commitment to apoptosis, mature IL-1 beta may function as a positive or negative mediator of cell death.

The contrasting roles of ICE family proteases and interleukin-1beta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation.

We compare here the mechanisms of apoptotic death of PC12 cells induced by down-regulation of Cu2+,Zn2+ superoxide dismutase (SOD1) and withdrawal of trophic support (serum/nerve growth factor). Our previous results indicated that the initiating causes of death are different in each paradigm. However, bcl-2 rescues cells in either paradigm, suggesting common downstream elements to the cell death pathway. To determine whether the ICE [interleukin 1beta converting enzyme] family of proteases, which is required for apoptosis on trophic factor withdrawal, is also required for apoptosis induced by oxidative stress, we have developed a novel peptide inhibitor that mimics the common catalytic site of these enzymes and thereby blocks their access to substrates. This differs from the more usual pseudosubstrate approach to enzyme inhibition. Blockade of ICE family proteases by either this inhibitor or by a permeant competitive ICE family antagonist rescues PC12 cells from apoptotic death following apoptosis induced by down-regulation of SOD1, as well as from trophic factor/nerve growth factor deprivation. SOD1 down-regulation results in an increase in interleukin 1beta (IL- 1beta) production by the cells, and cell death under these conditions can be prevented by either blocking antibodies against IL-1beta or the IL-1 receptor antagonist (IL-1Ralpha). In contrast, trophic factor withdrawal does not increase IL-1beta secretion, and the blocking antibody failed to protect PC12 cells from trophic factor withdrawal, whereas the receptor antagonist was only partially protective at very high concentrations. There were substantial differences in the concentrations of pseudosubstrate inhibitors which rescued cells from SOD1 down-regulation and trophic factor deprivation. These results suggest the involvement of different members of the ICE family, different substrates, or both in the two different initiating causes of cell death.

Intracellular Calcium Levels Influence Apoptosis in Mature Sensory Neurons after Trophic Factor Deprivation

Embryonic dorsal root ganglion (DRG) neurons require nerve growth factor (NGF) for survivalin vitro.Withdrawal of NGF results in an apoptotic death in these immature DRG neurons. After time in culture, DRG neurons become progressively less dependent upon NGF for survival. Immature embryonic DRG neurons remain highly dependent upon NGF during their first 14 days in cell culture but by Day 21 the majority lose their NGF dependence for survival. During this period of maturation, the intracellular calcium concentration ([Ca2+]i) progressively increases from the lower levels found in immature DRG neurons to the higher levels that are characteristic of older or mature DRG neurons. By changing the cell culture medium to one with very low calcium, we were able to lower [Ca2+]iin the mature neurons to levels similar to those found in immature neurons. These mature neurons (e.g., E-15 DRG neurons grown for 21 days in culture), normally NGF independent, became highly dependent upon NGF for survival. The onset of DNA fragmentation is a marker of apoptotic cell death. We measured the onset of DNA fragmentation in apoptotic neurons with use of the fluorescent dye, Hoechst, in neurons maintained in either standard calcium medium (1800 mM) or in the low calcium medium (0.35 mM). A higher percentage of neurons with lowered [Ca2+]ishowed initial signs of apoptosis, i.e., DNA condensation, at earlier times after NGF deprivation. This work provides further evidence to support a “set-point” hypothesis regarding the relationship between intracellular calcium concentration and NGF dependence for survival in DRG neurons.

Peptide inhibitors of the ice protease family arrest programmed cell death of motoneurons in vivo and in vitro

Members of the CED-3/interleukin-1β-converting enzyme (ICE) protease family have been implicated in cell death in both invertebrates and vertebrates. In this report, we show that peptide inhibitors of ICE arrest the programmed cell death of motoneurons in vitro as a result of trophic factor deprivation and in vivo during the period of naturally occurring cell death. In addition, interdigital cells that die during development are also rescued in animals treated with ICE inhibitors. Taken together, these results provide the first evidence that ICE or an ICE-like protease plays a regulatory role not only in vertebrate motoneuron death but also in the developmentally regulated deaths of other cells in vivo.

Structural Determinants of Neurotrophin Action

Five decades of research on NGF have led to the discovery of a small family of evolutionarily conserved proteins, which have vital functions in the survival and neuronal development of specific neuronal populations. The generation of mice lacking neurotrophin expression has recapitulated classic experiments using anti-NGF antibodies to dissect the physiological effects of trophic factor deprivation (73). Very similar outcomes resulted from both the NGF immunodepletion experiments and the transgenic mouse experiments. The genetic results also verify the structural predictions made from binding results in heterologous cells. The findings in cell culture and animal experiments clearly indicate the efficacy of neurotrophic factors for promoting the survival of prominent neuronal populations such as sensory and motor neurons. The high degree of conservation of neurotrophin structure is accompanied by a surprising variation in the amino acid contacts used by each neurotrophin with p75 and the trk receptor family members. It is this variation that may provide specificity for each ligand-receptor complex. The future challenge will be to make use of this knowledge to design effective therapeutic strategies to treat neurodegeneration and nerve injury.

Glia modulate the response of murine cortical neurons to excitotoxicity: glia exacerbate AMPA neurotoxicity

We have developed "pure" neuronal cultures (< 1% astrocytes) from mouse neocortex to study the effect of glial cells on the response of neurons to injury. Cortical neurons were found to require glial-conditioned medium to survive. Immature neurons, 2-4 d in vitro, deprived of glial-conditioned medium, underwent apoptosis over 48 hr, as suggested by condensed nuclear morphology, DNA fragmentation, and protection by inhibition of macromolecular synthesis. Apoptosis induced by trophic factor deprivation has been described for other neuronal populations, such as superior cervical ganglion and dorsal root ganglion cells. Cortical neurons in pure culture provide another neuronal population for the study of apoptosis induced by trophic factor deprivation. We then studied the interaction of neurons and glia under excitotoxic conditions. Experiments on mature cultures showed that pure neuronal cultures were at least 10-fold more sensitive to acute glutamate exposure than were neuronal-glial ("mixed") cocultures. The difference in sensitivity between pure neurons and mixed cultures was reduced when mixed cultures were treated with the glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC). In 24 hr exposure to N-methyl-D-aspartate (NMDA), or oxygen, glucose deprivation, pure neurons were more sensitive than mixed cultures; trans-PDC again increased the sensitivity of mixed cultures to nearly that of pure neuronal cultures. In contrast, mixed and pure neuronal cultures exposed to NMDA for 10 min, or to kainate for 24 hr, had similar injury dose-response curves, suggesting that glial glutamate uptake is a less important protective mechanism in these excitotoxic injuries. Surprisingly, pure neurons were less sensitive than mixed cultures to (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) toxicity at concentrations up to 100 microM. This does not reflect astrocyte toxicity, as AMPA at concentrations to 1 mM did not injure astrocyte cultures. Glial cultures showed increased levels of glutamate in the extracellular medium in response to exposure to AMPA, but not NMDA or kainate. However, pure neuronal and mixed cultures exposed to the same concentration of AMPA did not have elevated levels of glutamate in the media. We found that glia were generally neuroprotective under excitotoxic conditions, likely through their ability to clear extracellular glutamate. However, the presence of glia exacerbated AMPA neurotoxicity.

N-acetylcysteine (D- and L-stereoisomers) prevents apoptotic death of neuronal cells

In the present study we tested whether N-acetyl-L-cysteine (LNAC) affects apoptotic death of neuronal cells caused by trophic factor deprivation. LNAC, an antioxidant, elevates intracellular levels of glutathione. We used serum-deprived PC12 cells, neuronally differentiated PC12 cells deprived of serum and NGF, and NGF-deprived neonatal sympathetic neurons. In each case LNAC prevents apoptotic DNA fragmentation and maintains long-term survival in the absence of other trophic support. Unlike NGF, LNAC does not induce or maintain neurite outgrowth or somatic hypertrophy. To rule out actions of LNAC metabolic derivatives, we assessed N-acetyl-D-cysteine (DNAC). DNAC also prevents death of PC12 cells and sympathetic neurons. However, other antioxidants were ineffective in this regard. Since it has been hypothesized that trophic factors prevent neuronal death by either preventing or coordinating cell cycle progression, we tested whether LNAC or DNAC treatment can affect cell cycle. We found that both (but not other antioxidants) suppress proliferation and DNA synthesis by PC12 cells and do so at concentrations similar to those at which they prevent apoptotic death. Although the abilities of LNAC and DNAC to rescue cells from apoptosis triggered by trophic factor deprivation could derive from their direct influences on cellular responsiveness to oxidative stress, our observations raise the possibility of a mechanism involving cell cycle regulation.

Superoxide dismutase delays neuronal apoptosis: A role for reactive oxygen species in programmed neuronal death

Sympathetic neurons in culture die by apoptosis when deprived of nerve growth factor (NGF). We used this model of programmed cell death to study the mechanisms that mediate neuronal apoptosis. Cultured sympathetic neurons were injected with copper/zinc superoxide dismutase protein (SOD) or with an expression vector containing an SOD cDNA. In both cases apoptosis was delayed when the neurons were deprived of NGF. The delay was similar to that seen when a bcl-2 expression vector was injected. SOD, injected 8 hr after NGF deprivation, provided no protection, indicating that superoxide production may occur early in response to trophic factor deprivation. We have demonstrated, with a redox-sensitive dye, an increase in reactive oxygen species (ROS) that peaked at 3 hr after sympathetic neurons were deprived of NGF. If NGF was added back to the culture medium after the period of peak ROS generation, apoptosis was completely prevented, suggesting that ROS production serves as an early signal, rather than a toxic agent, to mediate apoptosis.

Neurotoxin-induced cell death in neuronal PC12 cells is mediated by induction of apoptosis

Death of neuronal cells during development and following deprivation of trophic factors is known to occur via an active mechanism requiring RNA and protein synthesis, known as apoptosis. Apoptosis is a form of cell “suicide” whereby the cell decides its own fate by activating a genetic programme of cell death. In contrast, necrosis is a passive uncontrolled form of cell death often observed in response to a toxic insult. Although it is known that neuronal cell death during development occurs by apoptosis, the mechanisms underlying neurotoxin-induced neuronal cell death remain poorly understood. In this study we have examined the mechanism by which 6-hydroxydopamine, a specific neurotoxin for catecholaminergic cells, induces neuronal cell death in vitro. We report that 6-hydroxydopamine induces cell death in the neuronal PC12 cell line via a mechanism which has the characteristic morphological and biochemical hallmarks of apoptosis. PC12 cells induced to die by 6-hydroxydopamine treatment exhibited cell shrinkage, classical chromatin condensation and membrane blebbing. Analysis of DNA integrity from 6-hydroxydopamine-treated cells revealed cleavage of DNA into regular sized fragments, a biochemical hallmark of apoptosis. 6-Hydroxydopamine-induced apoptosis of PC12 cells was suppressed by desipramine, a monoamine uptake inhibitor, suggesting that 6-hydroxydopamine is initiating apoptosis via a specific intracellular mechanism. Aurintricar☐ylic acid, a general inhibitor of nucleases, also suppressed 6-hydroxydopamine-induced apoptosis, suggesting the involvement of an endonuclease in the death pathway. The aetiology of idiopathic Parkinson's disease remains uncertain, although evidence suggests that endogenous and/or exogenous toxins may initiate neuronal cell death in this disease. The dopaminergic neurotoxin 6-hydroxydopamine is used to generate animal models of Parkinson's disease in vivo. We have demonstrated that this neurotoxin kills neuronal cells in vitro by an active process of apoptosis. Thus, the possibility exists that cell death in neurodegenerative diseases such as Parkinsonism also occurs in an active manner initiated by as yet unidentified environmental or metabolic toxins. Cell death that involves activation of an apoptotic programme can be modulated by addition of extracellular trophic factors, and is also controlled by the levels of intracellular factors. If neurotoxin-induced apoptosis plays a role in Parkinson's disease the implication is that the neuronal degeneration may be prevented by pharmacological manipulations.

Proliferative inhibition by dominant-negative Ras rescues naive and neuronally differentiated PC12 cells from apoptotic death.

We have used the nerve growth factor (NGF)-responsive PC12 cell line as a model to examine the role of cell cycle progression in apoptotic neuronal cell death triggered by withdrawal of trophic support. Because p21 Ras plays a key role in mitogenic signaling, we tested whether interference with the activity of this protein would affect cell cycle progression and thereby apoptotic death after trophic factor deprivation. For this purpose, we exploited PC12 cells transfected with an inducible form of dominant-inhibitory Ras. In contrast to non-transfected and uninduced cells, which continue to synthesize DNA when deprived of trophic support, PC12 cells induced to express dominant-inhibitory Ras showed little thymidine incorporation. When non-transfected and uninduced cells were deprived of trophic support, these underwent rapid apoptotic death that could be prevented by NGF. However, cells in which dominant-inhibitory Ras was induced and which were consequently quiescent did not die upon withdrawal of trophic support and showed long-term survival in the absence of NGF or other trophic factors. Moreover, induction of dominant-inhibitory Ras also rescued non-dividing, neuronally differentiated PC12 cells from death caused by NGF withdrawal. These findings suggest a relationship between proliferative capacity and neuronal apoptosis and raise the hypothesis that following withdrawal of trophic support, neurons undergo an unsuccessful and fatal attempt to re-enter the cell cycle.

Neurites Can Remain Viable after Destruction of the Neuronal Soma by Programmed Cell Death (Apoptosis)

During the development of the nervous system extensive programmed neuronal death occurs that is regulated by neurotrophic factors. Invariably, degeneration and death of the neuronal soma as a result of trophic factor deprivation is accompanied by concurrent degeneration of the neurites. By examining the degeneration of sympathetic neurons after deprivation of their physiological trophic factor nerve growth factor, we show that the "slow Wallerian degeneration" allele (Wlds) expressed by homozygous mutant C57BL/Ola mice alters the normal time course of programmed neuronal death by selectively and dramatically delaying the onset of neurite disintegration. In contrast, degenerative events affecting the neuronal soma are not altered: Atrophy of the soma, apoptotic disintegration of the nucleus, commitment to die, and loss of viability occur normally. The enucleate neurites remaining after death of the soma have an intact plasma membrane, are metabolically active, and require an active metabolism for physical integrity. We suggest that the degeneration of neurites during developmentally occuring neuronal death is controlled by events confined to the neurites and occurs autonomously from the neuronal soma. Furthermore, programmed neuronal death of the soma proceeds independent from any influence exerted by degenerating neurites.

The Ability of Diphenylpiperazines to Prevent Neuronal Death in Dorsal Root Ganglion Neurons In Vitro After Nerve Growth Factor Deprivation and In Vivo After Axotomy

The mechanism of neuroprotection by the calcium channel antagonist flunarizine against neuronal death is unknown. We investigated the ability of other calcium channel antagonists (cinnarizine, nimodipine, nicardipine, diltiazem, and verapamil), calmodulin antagonists, and calpain inhibitors to prevent neuronal death in rat dorsal root ganglion neurons in vitro after nerve growth factor (NGF) deprivation and the ability of cinnarizine and diltiazem to protect in vivo after axotomy. In vitro, only neurons treated with cinnarizine or flunarizine were protected from death after withdrawal. In vivo, cinnarizine, but not diltiazem, protected dorsal root ganglion neurons in rats after unilateral sciatic nerve crush. Intracellular calcium concentration ([Ca2+]i) was evaluated with fura 2 after NGF deprivation in vitro. Neurons "committed to die" 24 h after NGF deprivation displayed a decline in [Ca2+]i before visible morphological deterioration consistent with cell death. The influx of extracellular calcium was not necessary to produce neuronal death. Neurons deprived of NGF gradually lost the ability to respond to elevated external potassium with an increase in [Ca2+]i during the first 24 h after trophic factor deprivation. After 24 h, neurons deprived of NGF could not be rescued by readministration of NGF. Neurons protected from cell death with diphenylpiperazines maintained their response to high external potassium, suggesting continued membrane integrity. We speculate that diphenylpiperazines may protect sensory neurons via an unknown mechanism that stabilizes cell membranes.