Correspondence to: Eugene M. Johnson, Jr., Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110. Tel:(314) 362-3926 Fax:(314) 747-1772 E-mail:ejohnson{at}pcg.wustl.edu.
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Abstract |
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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 inhibitortreated mouse sympathetic neurons maintained mitochondrial membrane poten-tial for 2530 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- 9deficient sympathetic neurons was also coincident with the loss of mitochondrial membrane potential. Thus, caspase inhibition extended commitment to death in trophic factordeprived sympathetic neurons and allowed recovery of neurons arrested after the loss of cytochrome c, but not beyond the subsequent loss of mitochondrial membrane potential.
Key Words: apoptosis, caspase-9, bax, sympathetic neurons, NGF
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Introduction |
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Extensive programmed cell death (PCD)1 occurs during neuronal development. Much of this apoptotic death occurs during the developmental period of target innervation when many neuronal populations are acutely dependent on target-derived trophic factors (
The mechanism by which trophic factor deprivation induces PCD in neurons has been extensively studied in sympathetic neurons that are dependent on NGF for survival (
Recent studies have focused on the importance of mitochondria during apoptosis because experiments with cell-free systems have indicated that the release of cytochrome c from mitochondria represents a critical event during apoptosis (
The exact mechanism by which cytochrome c is released from mitochondria during apoptosis remains unknown. Bcl-2 family proteins, in particular Bax, may regulate the formation of a channel in the outer mitochondrial membrane, allowing components of the intermembrane space, including cytochrome c, to be released ( (
Finally, the events that determine when a cell becomes committed to die during apoptosis remain unclear. Since the mitochondrial events precede caspase activation in many models of apoptosis, cells saved by caspase inhibition may not be able to recover, and may eventually go on to die because mitochondrial oxidative phosphorylation is presumably compromised after cytochrome c loss (
In this paper, we examined the events that permit the NGF-deprived, caspase inhibitortreated sympathetic neurons to be rescued by readdition of NGF. Specifically, we asked the following questions. How long can a cell survive and be capable of rescue with NGF readdition after cytochrome c is released? What events ultimately determine when a cell, treated with caspase inhibitor, becomes committed to death? We also examined the temporal relationship between the loss of cytochrome c and the loss of mitochondrial membrane potential after NGF removal. We found that cytochrome c was released before any significant changes in mitochondrial membrane potential during sympathetic neuronal death. Furthermore, the NGF-deprived sympathetic neurons became committed to die at the time of cytochrome c loss. However, in BAF-treated or caspase-9deficient sympathetic neurons, this commitment to death was extended beyond the time of cytochrome c release, but only until the point at which the mitochondrial membrane potential was lost. Thus, our data indicate that caspase inhibition extends the commitment to death of mouse sympathetic neurons by 2530 h after the loss of cytochrome c, but not beyond the subsequent loss of mitochondrial membrane potential.
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Materials and Methods |
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Reagents
Mitotracker orange CM-H2TMRos was purchased from Molecular Probes. Caspase inhibitor BAF was purchased from Enzyme Systems Products. Collagenase and trypsin were purchased from Worthington Biochemical Corp. All other reagents were purchased from Sigma Chemical Co., unless otherwise stated. Untimed, pregnant ICR mice were purchased from Harlan Sprague Dawley.
Sympathetic Neuronal Cultures
Primary cultures of sympathetic neurons from the superior cervical ganglion (SCG) neurons were prepared from postnatal-day-1old (P1) mice, essentially as described previously for rats (
Culture Conditions and Assessment of Cell Viability after NGF Readdition
Sympathetic neuronal cultures were grown in NGF-containing medium (AM50) for 45 d, and then either maintained in AM50 or treated with various conditions. For NGF deprivation, cultures were rinsed twice with medium lacking NGF (AM0: AM50 medium without NGF), followed by addition of AM0 containing goat antiNGF neutralizing antibody (
Mitochondrial Membrane Potential and Cytochrome c Staining
To assess the status of the mitochondrial membrane potential and cytochrome c in individual cells, sympathetic neurons were loaded with Mitotracker orange and immunostained with cytochrome c antibodies. Sympathetic neurons that were maintained for 45 d in NGF-containing medium were first treated with various conditions and loaded with 1 µM Mitotracker orange (freshly prepared in DMSO) in the appropriate culture medium for 1 h at 37°C in the dark. We found that loading cells with Mitotracker orange before subjecting cells to various experimental conditions was not a reliable method of assessing the mitochondrial membrane potential in sympathetic neurons. For the NGF deprivation experiments, 50 µM BAF was included in the culture medium to eliminate any cell loss that would otherwise affect the quantitation; no BAF was added when the caspase-9deficient cells were analyzed in this assay. Cells were washed four times with PBS and fixed in freshly prepared 4% paraformaldehyde for 30 min at 4°C. After three washes in TBS (100 mM Tris-HCl, pH 7.6, and 0.9% NaCl), the cultures were processed for cytochrome c immunohistochemistry as described previously (
Mitotracker Staining and NGF Rescue in Sympathetic Neurons
The status of Mitotracker orange staining and rescue with NGF readdition in cultures of sympathetic neurons was examined as follows. Sympathetic neuronal cultures were maintained in NGF-containing medium for 5 d, and were deprived of NGF in the presence of 50 µM BAF for 60 h. This time corresponded to the point at which almost all cells had lost cytochrome c staining, and about half the cells had lost their Mitotracker staining. Cells were loaded with Mitotracker by incubating in the same medium containing 250 nM Mitotracker orange (prepared freshly in DMSO) for 1 h at 37°C. Care was taken to ensure minimum exposure of cultures to light in all subsequent manipulations (particularly while taking photographs) because exposure to intense light was found to be toxic to the Mitotracker-labeled cells. The Mitotracker concentration that was used in these rescue experiments (250 nM) was lower than that used in the previous immunostaining experiments (1 µM) to minimize phototoxicity. Since no immunostaining steps with washes were involved in these rescue experiments, the lower Mitotracker concentration was found to be sufficient to provide an adequate signal. After the labeling period, cells were washed four times and incubated in NGF-containing medium. Representative fields of cells (marked from a reference point; day 0 [d0] after NGF rescue) were photographed under phase-contrast microscopy to visualize the cells and under fluorescence microscopy to visualize Mitotracker staining (Fuji Provia film, ASA 1600). Cells were returned to the 37°C incubator, and phase-contrast photographs of the same fields of cells were taken at d2, d4, and d7 after the NGF readdition. For quantitation, the phase-contrast photographs of cells at d0 after NGF rescue were first projected onto a screen and individual cells were numbered. The Mitotracker staining status of these cells was scored as positive or negative. The phase-contrast photographs of the same field of cells at d2, d4, and d7 after NGF rescue were compared to determine which cells were rescued; rescue was defined by the ability of these cells to increase somal diameter after NGF readdition. Only cells whose rescue fate was clearly identifiable were counted. Approximately 200 cells were scored per experiment.
Bax-/- and Caspase-9-/- Mice
Breeding and genotyping of Bax-deficient mice has been described previously (
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Results |
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Use of Mitotracker Orange as an Indicator of Mitochondrial Membrane Potential in Sympathetic Neurons
To examine the temporal relationship between cytochrome c release and the mitochondrial membrane potential loss in sympathetic neurons after NGF deprivation, cells were loaded with Mitotracker orange and subjected to cytochrome c immunocytochemistry. Mitotracker orange is a mitochondrial membrane potentialsensitive dye that is aldehyde fixable and, therefore, compatible with subsequent immunocytochemical analysis (
NGF-maintained sympathetic neurons loaded with Mitotracker orange showed a punctate staining pattern identical to that of the mitochondria-localized cytochrome c (Fig 1). To confirm that Mitotracker orange was an indicator of mitochondrial membrane potential in sympathetic neurons, we examined Mitotracker staining after treatment with agents that disrupt the mitochondrial membrane potential. NGF-maintained sympathetic neurons were treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) or valinomycin, uncouplers of oxidative phosphorylation (
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An important point to note is that, in these and subsequent experiments, sympathetic neurons were loaded with Mitotracker orange after treatment with various conditions. Loading cells with Mitotracker orange before treatment with CCCP did not result in decreased Mitotracker staining after disruption of the mitochondrial membrane potential (data not shown). Thus, although the uptake of the Mitotracker dye was sensitive to mitochondrial membrane potential, once the Mitotracker dye was taken up by the mitochondria, its signal did not decrease reliably under conditions that disrupted the mitochondrial membrane potential.
Loss of the Mitochondrial Membrane Potential during Sympathetic Neuronal Death Requires Upstream Events that Are Dependent on Macromolecular Synthesis and Bax Function
NGF deprivation induces a series of events that result in the loss of cytochrome c from mitochondria during sympathetic neuronal death. Inhibition of protein synthesis with cycloheximide addition or Bax deficiency blocks the pathway leading to the release of cytochrome c, whereas caspase inhibition does not (
Staining for both cytochrome c and Mitotracker in NGF-maintained sympathetic neurons appeared punctate, as expected for their mitochondrial localization (Fig 2; +NGF). NGF deprivation for 72 h resulted in the loss of both cytochrome c and Mitotracker staining in most cells, indicating that cells had released cytochrome c and lost their mitochondrial membrane potential (Fig 2; -NGF/+BAF). We examined whether the loss of mitochondrial membrane potential seen with NGF deprivation was dependent on events requiring macromolecular synthesis or Bax expression. NGF-deprived, cycloheximide-treated sympathetic neurons maintained both cytochrome c and Mitotracker staining even after 72 h of NGF deprivation (Fig 2; -NGF/+CHX). Likewise, sympathetic neurons from Bax-deficient mice showed intact cytochrome c and Mitotracker staining even after 72 h of NGF deprivation (Fig 2; -NGF/Bax-/-). These results suggest that a common, macromolecular synthesisdependent, Bax-dependent pathway leads to the release of cytochrome c and loss of mitochondrial membrane potential during NGF deprivationinduced sympathetic neuronal death.
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Release of Cytochrome c Precedes the Loss of Mitochondrial Membrane Potential during Sympathetic Neuronal Apoptosis
To understand the mechanism of cytochrome c release during sympathetic neuronal death, we examined whether the loss of cytochrome c preceded or followed the loss of mitochondrial membrane potential in individual sympathetic neurons undergoing apoptosis after NGF deprivation. Sympathetic neurons were deprived of NGF, and at various times after NGF deprivation, the cells were loaded with Mitotracker orange and processed for cytochrome c immunostaining. The status of cytochrome c and Mitotracker staining in individual sympathetic neurons was determined, and the results were grouped into four possible categories: (1) cells that maintained both cytochrome c and Mitotracker staining (CytC+ MitoT+); (2) cells that lost cytochrome c but maintained Mitotracker staining (CytC- MitoT+); (3) cells that maintained cytochrome c but lost Mitotracker staining (CytC+ MitoT-); and (4) cells that lost both cytochrome c and Mitotracker staining (CytC- MitoT-). Examples of cells that were seen in these categories are shown in Fig 3 A. Note that no CytC+ MitoT- cells were observed during NGF deprivationinduced cell death (see below).
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All NGF-maintained sympathetic neurons had intact cytochrome c and Mitotracker staining (Fig 3 B; 0 h). Upon NGF deprivation, this population of cells decreased gradually, such that by 24 h after NGF removal, only 50% of cells were CytC+ MitoT+ and, by 120 h after NGF removal, none of the cells had intact cytochrome c or Mitotracker staining (Fig 3 B). Conversely, the population of cells that had lost both cytochrome c and Mitotracker staining increased gradually after NGF removal and by 120 h after NGF removal, all cells were CytC- MitoT-. We also observed a transient increase in the population of cells that had lost cytochrome c but still maintained Mitotracker staining (CytC- MitoT+) after NGF removal. More important however, we did not observe any cells that maintained cytochrome c, but lost Mitotracker staining (CytC+ MitoT-) at any time point after NGF deprivation (Fig 3 B). Since we observed cells that were CytC- MitoT+ but never cells that were CytC+ MitoT- after NGF deprivation, our data indicate that individual sympathetic neurons first released cytochrome c and subsequently lost mitochondrial membrane potential.
The time course of the loss of cytochrome c and the loss of mitochondrial membrane potential in NGF-deprived, BAF-treated sympathetic neurons (data taken from Fig 3 B) is shown in Fig 3 C. Consistent with our previous results, 50% percent of the mouse (ICR) sympathetic neurons released cytochrome c by 24 h after NGF removal (30 h after release of cytochrome c. As discussed above, these experiments were done in the presence of caspase inhibitor. Without caspase inhibition, mitochondrial depolarization presumably follows the loss of cytochrome c relatively rapidly, since cells activate caspases and become committed to die soon after releasing cytochrome c (see below).
Caspase Inhibition Allows Sympathetic Neurons to Recover after Cytochrome c Release, but Not Beyond the Subsequent Loss of Mitochondrial Membrane Potential
Recent studies indicate that NGF-deprived, BAF-treated sympathetic neurons that have released cytochrome c are capable of recovering with subsequent NGF readdition (
NGF-maintained sympathetic neurons were deprived of NGF either in the absence or presence of the caspase inhibitor BAF. At various times after NGF deprivation, NGF was added back to the cultures and the neurons were allowed to recover for 7 d. The number of surviving cells after this 7-d rescue period was quantitated. This long-term rescue assay was needed to determine unambiguously whether caspase inhibition extended the commitment to death. Examples of BAF-saved sympathetic neurons that were rescued with NGF readdition are shown in Fig 4 A. Control cells deprived of NGF for 72 h without any BAF show extensive cell death (Fig 4 A; -NGF 72 hr). Caspase inhibition with BAF addition blocks cell death in these neurons. Note that all seven cells marked in the -NGF + BAF condition appear saved at 72 h by the criterion of being phase-bright. However, after 7 d of NGF readdition, only three of the seven cells in that field were able to be recovered with NGF (Fig 4 A; NGF rescue 7 d). Thus, after 7 d of NGF rescue, it was possible to distinguish unequivocally between those cells that were rescued with NGF readdition (cells 2, 5, and 6), and those cells that had already become committed to die and were not able to recover with NGF readdition (cells 1, 3, 4, and 7).
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Using this NGF rescue assay, we determined the percentage of cells that became committed to die at various times after NGF deprivation, either in the absence or presence of BAF. The rescue data (Fig 4 B) were compared with the time course of loss of cytochrome c and Mitotracker staining (from Fig 3 C) since internally controlled parallel cultures that were deprived of NGF at the same time were used in both these assays. Without BAF addition, 50% of mouse sympathetic neurons became committed to die by 25 h after NGF deprivation (Fig 4 B). The time course of commitment after NGF deprivation is almost identical to the time course of the loss of cytochrome c (Fig 4 B). BAF addition extended the commitment of sympathetic neuronal death by
2530 h. 50% of the NGF-deprived, BAF-saved sympathetic neurons became committed to die by 5560 h of NGF deprivation (Fig 4 B). Remarkably, the time course of commitment to death in the presence of caspase inhibitor was indistinguishable from the time course of loss of Mitotracker staining, which marks the loss of mitochondrial membrane potential in these neurons (Fig 4 B). We define commitment to death in the absence of caspase inhibitor as Commitment 1 (coincident with cytochrome c release) and commitment to death in the presence of caspase inhibitor as Commitment 2 (coincident with mitochondrial membrane potential loss).
Mitotracker Staining Predicts Rescue with NGF Readdition in Individual Sympathetic Neurons Undergoing Apoptosis
The population-based study shown above demonstrates a good correlation between loss of Mitotracker staining and failure to be rescued with NGF readdition (Fig 4). To test further the hypothesis that loss of mitochondrial membrane potential marks the commitment to death in caspase inhibitortreated neurons (Commitment 2), we examined whether the correlation between the maintenance of mitochondrial membrane potential and rescue with NGF readdition could also be seen in individual sympathetic neurons undergoing apoptosis. Sympathetic neurons were deprived of NGF in the presence of BAF for 60 h (2.5 d). NGF deprivation for 60 h corresponds to a time when >90% of cells had lost cytochrome c and 50% of cells had lost Mitotracker staining (Fig 3 C). Cells were loaded with Mitotracker orange (250 nM for 1 h) and photographed to document which cells had lost or maintained Mitotracker staining (Fig 5 A, -NGF + BAF 2.5 d). As expected,
50% of cells had lost Mitotracker staining at that time (data not shown). NGF was added to the cultures, and the neurons were allowed to recover for 7 d. Phase-contrast photographs of the identical field were taken at 2, 4, and 7 d after NGF readdition to follow the status of individual sympathetic neurons (Fig 5 A; +NGF rescue d2, d4, and d7). More than 90% of the cells that were Mitotracker-positive were rescued with NGF readdition (Fig 5 B), in contrast to <15% of the Mitotracker-negative cells that were capable of rescue with NGF readdition (Fig 5 B). The photograph in Fig 5 A show examples of the correlation seen between Mitotracker staining and rescue with NGF addition. In the field shown, all Mitotracker-positive cells (4 out of 10 cells, numbered 14) were rescued with NGF readdition, whereas the Mitotracker-negative cells were not (Fig 5 A).
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Caspase-9deficient Sympathetic Neurons Are Arrested after the Loss of Cytochrome c and Can Be Rescued with NGF Readdition
One prediction from our results is that any genetic manipulation that arrests NGF-deprived sympathetic neurons after the release of cytochrome c would allow these neurons to be rescued with NGF readdition but only until the point of loss of mitochondrial membrane potential. We tested this prediction on sympathetic neurons from caspase-9deficient mice. We chose to examine caspase-9deficient sympathetic neurons because caspase-9 is thought to be the apical caspase that becomes activated once cytochrome c is released from mitochondria (
Sympathetic neurons from caspase-9deficient mice were deprived of NGF for 72 h to determine first the effect of caspase-9 deletion on sympathetic neuronal death. Cultures from the wild-type littermates were examined in parallel. Caspase-9deficient neurons appeared phase-bright and had intact neurites even after 72 h of NGF deprivation, whereas those from wild-type littermates appeared degenerated and dead at this time (Fig 6 A). These results indicate that caspase-9 deficiency prevented sympathetic neurons from undergoing apoptosis after NGF deprivation. The NGF-deprived, caspase-9-/- neurons appeared atrophic presumably because these neurons, like the NGF-deprived, BAF-saved neurons are arrested after the metabolic changes that occur after NGF removal (
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We next examined whether the NGF-deprived, caspase-9deficient sympathetic neurons were capable of being rescued with NGF readdition. Caspase-9-/- neurons were deprived of NGF and, at various times after NGF deprivation, NGF was added back for 7 d to assess the number of rescued neurons. Cultures from wild-type littermates were examined in parallel. The status of cytochrome c and Mitotracker staining in caspase-9-/- neurons was also examined in parallel at identical times after NGF deprivation. Caspase-9 deficiency inhibited sympathetic neuronal death after the point of cytochrome c release; virtually all caspase-9deficient cells had lost cytochrome c by 24 h after NGF deprivation (Fig 6 B). Cytochrome c release in these cells is complete by 24 h, as reported previously for this genetic background (C57BL/6) (
Consistent with the time course of cytochrome c loss, >95% of wild-type sympathetic neurons were committed to die (i.e., could not be rescued with NGF readdition) by 24 h after NGF deprivation (Fig 6 B). In contrast, at that time, >80% of caspase-9deficient sympathetic neurons were capable of rescue with NGF readdition (Fig 6 B). Thus, caspase-9-/- sympathetic neurons were arrested at a point after the loss of cytochrome c and could be rescued be with NGF readdition. Longer periods of NGF deprivation resulted in the rescue of fewer caspase-9deficient cells. 50% of caspase-9deficient sympathetic neurons were not capable of recovery by 50 h of NGF deprivation, and none were capable of rescue after 5 d of NGF deprivation (Fig 6 B). As predicted from our data with BAF (Fig 4), the time course of commitment to death of caspase-9deficient sympathetic neurons was found to be similar to the time course of loss of Mitotracker staining in these neurons (Fig 6 B).
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Discussion |
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In this study, we examined the molecular events that determine when sympathetic neurons become committed to die after trophic factor withdrawal. We asked whether caspase inhibition, which inhibits NGF deprivationinduced apoptosis after release of cytochrome c, extends the commitment to death and examined what events might ultimately commit the caspase inhibitor-treated neurons to die. Three main conclusions from this study are as follows. First, NGF-deprived sympathetic neurons released cytochrome c before loss of mitochondrial membrane potential. The NGF-deprived, BAF-treated sympathetic neurons maintained the mitochondrial membrane potential 2530 h after release of cytochrome c. Second, NGF-deprived sympathetic neurons became committed to die at the time of cytochrome c loss (defined as Commitment 1). Third, in the presence of a caspase inhibitor or in caspase-9deficient sympathetic neurons, this commitment point of death was extended beyond cytochrome c release but only until the later point of loss of mitochondrial membrane potential (defined as Commitment 2). Once cells lost mitochondrial membrane potential, they were no longer capable of recovery with NGF readdition. Thus, caspase inhibition allowed NGF-deprived sympathetic neurons to recover with NGF readdition after cytochrome c release but not beyond the subsequent loss of mitochondrial membrane potential.
Cytochrome c Release Precedes the Loss of the Mitochondrial Membrane Potential during Sympathetic Neuronal Apoptosis
NGF deprivation induces the loss of cytochrome c, before caspase activation, during sympathetic neuronal death (
We cannot exclude the possibility that subtle changes in mitochondrial membrane potential undetectable with Mitotracker may have occurred during the release of cytochrome c in sympathetic neurons. Although several reagents are available for measuring mitochondrial membrane potential, a lack of consensus exists regarding which is the best reagent to use on intact cells (
How are NGF-deprived sympathetic neurons able to maintain their mitochondrial membrane potential after cytochrome c release? One possibility is that glycolytic pathways may generate enough ATP, such that mitochondrial membrane potential can be maintained by reversal of the F0F1-ATPase. For example, - cells that lack mitochondrial DNA use glycolytic ATP to maintain mitochondrial membrane potential (
Loss of Mitochondrial Membrane Potential Marks the Commitment to Death in the Caspase Inhibitorsaved Sympathetic Neurons
We have used the criterion of NGF rescue (the ability to respond to NGF readdition with an increase in somal diameter) to demonstrate that caspase inhibition extended the commitment to death in NGF-deprived sympathetic neurons from the point of cytochrome c release to the later point of mitochondrial depolarization (Fig 4). This study extends previous observations showing that NGF-deprived, BAF-saved sympathetic neurons are capable of increasing somal diameter (
The ability of NGF-deprived, BAF-saved sympathetic neurons to be rescued with NGF readdition correlated well with their ability to maintain a mitochondrial membrane potential after cytochrome c release (Fig 4). This correlation held remarkably well, even in individual sympathetic neurons (Fig 5). These results are consistent with the hypothesis that once cells lose their ability to generate sufficient ATP to maintain their mitochondrial membrane potential, they can no longer be rescued with NGF readdition.
The NGF-deprived, BAF-treated sympathetic neurons that could not be rescued with NGF readdition eventually died without exhibiting any characteristics of apoptosis. For example, dying cells did not immunostain with CM1 antibodies that label active caspase-3 (
Caspase-9 Is Required for Sympathetic Neuronal Death
Our results show that caspase-9 is important in mediating sympathetic neuronal death (Fig 6). NGF-deprived, caspase-9deficient sympathetic neurons were capable of rescue with NGF readdition even after the point of cytochrome c loss. As with the BAF-treated cells, the eventual commitment to death in the NGF-deprived, caspase-9deficient sympathetic neurons also correlated with the loss of mitochondrial membrane potential (Fig 6 B). The observation that caspase-9 deficiency or BAF addition provided similar neuroprotection in NGF-deprived sympathetic neurons suggests that caspase-9 is the critical apical caspase activated in these cells once cytochrome c is released from the mitochondria. Fig 7 depicts our current model of the sympathetic neuronal death pathway after NGF deprivation.
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What Criteria May Permit Rescue of Cells Arrested after Release of Cytochrome c
The ability of caspase inhibitors to keep postmitotic sympathetic neurons alive and capable of rescue with NGF readdition contrasts with the results in primary fibroblasts and several cell lines in which caspase inhibition merely changes the phenotype of death but ultimately has no effect on the commitment to death. Specifically, caspase inhibition did not allow these mitotic cells to maintain clonogenecity even with serum readdition (
Alternatively, mitochondrial sequestered, death-promoting proteins, such as AIF, that are released along with cytochrome c (
The time by which commitment to death is extended with caspase inhibition (time between Commitment 1 and Commitment 2) varies not only among different cell types, but also among different species for the same cell type. For example, rat sympathetic neurons lose cytochrome c in 50% of neurons by 2224 h after NGF removal. Yet, >50% of NGF-deprived, BAF-treated rat sympathetic neurons are rescued with NGF readdition, even after 4 d of NGF removal (72 h after cytochrome c release, in contrast to a period of
30 h in mouse sympathetic neurons (Fig 4). Whether differences in cell sizes of sympathetic neurons from rat versus mouse contribute to the window of time between Commitment 1 and Commitment 2 is unclear. Consistent with this hypothesis, the survival of mouse cerebellar granule neurons, which are much smaller than sympathetic neurons, is extended for only a few hours after potassium deprivation with caspase inhibition (
From a therapeutic perspective, the ability of caspase inhibition to promote survival (Fig 4 and Fig 6) and functional recovery (
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Footnotes |
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1 Abbreviations used in this paper: BAF, boc-aspartyl(OMe)-fluoromethylketone; CCCP, carbonyl cyanide m-chlorophenylhydrazone; MPT, mitochondrial permeability transition; PCD, programmed cell death.
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Acknowledgements |
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We thank Kevin A. Roth and Barbara Klocke for supplying us with the caspase-9 knockout mice and Stanley Korsmeyer for the Bax-deficient mice. We also thank Louis Chang, Charles Harris, Krista Moulder, Patricia A. Osborne, Girish Putcha, and Brian Tsui-Pierchala for critical review of this manuscript.
This work was supported by the National Institutes of Health grants AG 12947 and NS 38651 (to E.M. Johnson Jr.) and Paralyzed Veterans of America Spinal Cord Research Foundation grant 1786 (to M. Deshmukh).
Submitted: 28 April 2000
Revised: 8 June 2000
Accepted: 8 June 2000
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References |
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