Ceramide selectively inhibits apoptosis-associated events in ngf-deprived sympathetic neurons

ABSTRACT Ceramide manifests both neurotoxic and neuroprotective properties depending on the experimental system. Ito and Horigome previously reported that ceramide delays apoptosis in a

classic model of developmental programmed cell death, i.e. sympathetic neurons undergoing NGF deprivation.1 Here, we investigated the actions of ceramide upon the biochemical and genetic

changes that occur in NGF deprived neurons. We correlate ceramide's neuroprotective actions with the ability of ceramide to antagonize NGF deprivation-induced oxidative stress and

c-_JUN_ induction, both of which contribute to apoptosis in this model. However, ceramide did not block NGF deprivation-induced declines in RNA and protein synthesis, suggesting that

ceramide does not slow all apoptosis-related events. Overall, these results are significant in that they show that ceramide acts early in the death cascade to antagonize two events necessary

for NGF-deprivation induced neuronal apoptosis. Moreover, these results dissociate declines in neuronal function, i.e. macromolecular synthesis, from the neuronal death cascade. SIMILAR

CONTENT BEING VIEWED BY OTHERS APOPTOSIS SIGNALING IS ACTIVATED AS A TRANSIENT PULSE IN NEURONS Article 26 October 2024 DIVERSE MATURITY-DEPENDENT AND COMPLEMENTARY ANTI-APOPTOTIC BRAKES

SAFEGUARD HUMAN IPSC-DERIVED NEURONS FROM CELL DEATH Article Open access 21 October 2022 PROGRAMMED NEURITE DEGENERATION IN HUMAN CENTRAL NERVOUS SYSTEM NEURONS DRIVEN BY CHANGES IN NAD+

METABOLISM Article Open access 17 January 2025 INTRODUCTION Interest in the actions of ceramide as a modulator of apoptosis has grown dramatically in recent years.2,3,4 Ceramide is reported

to modulate apoptosis via several kinase pathways, including MAP kinase, PI-3/AKT kinase, and Jun kinase.5,6,7,8,9 Consistent with the pro-apoptotic role of these pathways in some cell

types, ceramide is often considered pro-apoptotic. However, ceramide also delays apoptosis in primary cultures of sympathetic neurons undergoing NGF deprivation _in vitro_.1 Similarly,

ceramide inhibits apoptosis in other neuronal types or in response to other insults. For example, ceramide also protects sensory neurons from NGF deprivation10 and hippocampal neurons from

excitotoxic and oxidative insults.11 The mechanism(s) underlying the neuroprotective actions of ceramide have not been elucidated. Many of the morphologic and molecular events that accompany

NGF deprivation in sympathetic neurons have been identified. Although few morphologic changes are observed in the first 12 h, the activity of NGF-activated kinases including MAP kinases and

PI-3 kinase decreases sharply within 30 min of deprivation.12,13 This is followed by an interval of oxidative stress at approximately 4 h14,15 and concomitant declines in total RNA and

protein synthesis.16,17 As RNA and protein synthesis declines, several genes are selectively induced, including members of the c-_jun_ and c-_fos_ families.18,19 Members of the caspase

family are then activated leading to degradation of genomic DNA into nucleosomal fragments at approximately 19 h16,17,20 Neuronal death as assessed by staining with vital dyes begins ∼24 h

after deprivation and is largely complete by 48 h. The apoptotic role of several of these events has been elucidated. Inhibitors of oxidative stress and c-Jun at least delay

apoptosis14,18,19 while caspase inhibitors block apoptosis essentially indefinitely.20 In summary, to gain insight into ceramide's ability to delay apoptosis, we evaluated the actions

of ceramide on several of these molecular events. Here, we report that ceramide inhibits oxidative stress and c-_jun_ induction, without altering declines in RNA or protein synthesis or

activating the MAP kinase pathway. These results suggest novel roles for ceramide in delaying neuronal death. RESULTS CERAMIDE ANTAGONIZES NGF DEPRIVATION-INDUCED SYMPATHETIC NEURON DEATH To

begin to evaluate ceramide actions, we confirmed that ceramide antagonizes apoptosis in NGF-deprived sympathetic neurons, as reported by others.1 Neurons were deprived of NGF and

simultaneously treated with ceramide or DMSO vehicle. Apoptosis was visualized as chromatin condensation 24 h later by using Hoechst 33258 staining and fluorescent microscopy.16,17 As

expected, NGF deprivation increased the frequency of neurons manifesting chromatin condensation (Figure 1B) relative to NGF maintained neurons (Figure 1A). In contrast to its pro-apoptotic

effects in many cell types, ceramide did not induce chromatin condensation in the presence of NGF (Figure 1C). Indeed, ceramide reduced the frequency of such neurons in NGF deprived cultures

(Figure 1D). The biologically ‘inactive’ analog of ceramide, dihydroceramide, had no effect on NGF deprivation (Figure 1E). Quantitation of these effects established that ceramide but not

dihydroceramide was neuroprotective (Figure 1F (_P_<0.05)). This neuroprotection was not as complete as that observed for protein synthesis inhibitors such as cycloheximide21 or caspase

inhibitors,20,21 as ∼8% of neurons still underwent apoptosis in the presence of ceramide. Indeed, we observed that the neuroprotective actions of ceramide were eventually overcome with

increasing duration of NGF deprivation; as reported by Ito and Horigome,1 the neurons underwent apoptosis beginning some 48–72 h after NGF deprivation (data not shown, for quantitation,

see1). In summary, in sharp contrast to the pro-apoptotic actions of ceramide in many situations,2,3,4 ceramide antagonizes apoptosis in NGF deprived sympathetic neurons. CERAMIDE

ANTAGONIZES ‘APOPTOSIS-ASSOCIATED’ GENE EXPRESSION Since we and others previously reported that c-Jun is induced during and is necessary for apoptosis caused by NGF deprivation,18,19 we

began evaluating the actions of ceramide by examining patterns of apoptosis-associated gene expression. We compared the temporal patterns of expression of four ‘baseline’ genes and three

‘apoptosis-associated’ genes. The baseline genes included actin and cyclophilin, which are expressed constitutively in all cell types, and NSE and TOH, which are neuron-specific. The

relative invariability of the baseline genes early in the time course of deprivation demonstrates the uniformity of neuronal plating. The decline in these genes with NGF deprivation reflects

differences in neuronal death, e.g. TOH and NSE decline more slowly in the presence of ceramide (Figure 2A). NGF deprivation led to robust c-_jun_, c-_fos_, and _fosB_ inductions (Figure

2).18,19 Ceramide delayed and decreased each gene induction (Figure 2). For example, ceramide decreased the maximal c-_jun_ induction to 50 and 62% of that of neurons undergoing NGF

deprivation _per se_ (two independent neuronal preparations). Ceramide also delayed and decreased the induction of c-_fos_ and _fosB_, actions which likely reflect that ceramide delays

apoptosis because _in situ_ hybridization studies have shown that at least c-_fos_ is induced coincident with chromatin condensation.18 In summary, ceramide inhibited the induction of

apoptosis-related genes, including that of c-_jun_, which encodes a protein necessary for death in this model.18,19 CERAMIDE BLOCKS NGF DEPRIVATION-INDUCED OXIDATIVE STRESS Oxidative stress

is a result of NGF deprivation, activates c-Jun, and is necessary for apoptosis.14,18,19,22,23 Since ceramide inhibited c-_jun_ induction, we hypothesized that ceramide may modulate

oxidative stress. Hence, we evaluated ceramide effects on oxidative stress, which is a transient event, occurring 3–4 h after NGF deprivation.14 Oxidative stress was quantified by using

H2DCFDA, which is converted to fluorescent DCF upon oxidation and cleavage of its acetate groups by cellular esterases. Neurons were exposed to ceramide or DMSO vehicle in the presence or

absence of NGF for 3.5 h, loaded with H2DCFDA for 30 min, and DCF fluorescence quantified. NGF deprivation induced a significant, twofold increase in DCF fluorescence (Figure 3A), confirming

the work of others.14 Ceramide significantly decreased DCF fluorescence induced by NGF deprivation (_P_<0.05) and had no effect on NGF maintained neurons (Figure 3A). Since DCF

fluorescence can be altered artifactually,24 we evaluated ceramide effects on cells loaded with DCFDA, which is converted directly to DCF by cytosolic esterases, independent of oxidative

stress. Neither ceramide nor NGF deprivation altered the fluorescence of neurons loaded with DCFDA (Figure 3B). In summary, these results indicate that ceramide decreases the oxidative

stress induced by NGF deprivation. This effect may explain ceramide's ability to antagonize apoptosis because oxidative stress is necessary for death.14,25 Moreover, since oxidative

stress likely contributes to c-_jun_ induction in NGF deprived neurons,26 the ability of ceramide to inhibit oxidative stress may be upstream of its actions on c-_jun_. THE MAP KINASE

PATHWAY IS NOT ACTIVATED BY CERAMIDE Two lines of evidence suggest that ceramide may modulate oxidative stress by activating the MAP kinase pathway. First, ceramide activates this kinase

pathway in some cell types.7 Second, adding NGF back to NGF-deprived cultures inhibits oxidative stress via the MAP kinase pathway because the MAP kinase inhibitor PD098059 blocks this NGF

action.15 Therefore, we examined whether ceramide activates the MAP kinase pathway in sympathetic neurons. To reduce the NGF stimulated level of MAP kinase activity, neurons were deprived of

NGF for 14 h, which is insufficient to induce apoptosis. The neurons were then either left unstimulated for 30 min, or were treated with ceramide or NGF for 30 min. Activation of the MAP

kinase pathway was assessed by two means. First, ERK activation was assessed by detecting ERK phosphorylation by using a phospho-ERK antibody to label neurons (Figure 4A–C), which were

counterstained with Hoechst 33258 to show the presence of neurons (Figure 4D–F). NGF stimulation caused a robust increase in ERK phosphorylation (Figure 4B), relative to the unstimulated

cells (Figure 4A). That neurons were present in each case is documented by the Hoechst 33258 staining in parallel (Figure 4D,E). In contrast to this robust NGF effect, ceramide treatment did

not increase phospho-ERK labeling (Figure 4C,F). We also assessed whether ceramide activated the MAP kinase pathway by using Western blots to examine ERK phosphorylation (Figure 4G). NGF

again induced a clear increase in ERK phosphorylation (lane 2) while ceramide treated cells (lane 3) were similar to unstimulated cells (lane 1). That similar amounts of protein were loaded

into each lane was demonstrated by labeling with an antibody against total ERKs. Hence, ceramide does not appear to activate the MAP kinase pathway in sympathetic neurons. CERAMIDE DOES NOT

BLOCK NGF DEPRIVATION-INDUCED METABOLIC DORMANCY Since the actions of ceramide might be attributable to an overall slowing in cellular metabolism, we examined the effects of ceramide on

macromolecular synthesis. RNA and protein synthesis was quantified by [5,6-3H]uridine and [35S]L-methionine incorporation, respectively. NGF deprivation _per se_ caused a profound decline in

macromolecular synthesis well before neuronal death (Figure 5), as reported by others.16,17 However, ceramide had no effect on this metabolic decline (Figure 5). In the presence of NGF,

ceramide showed only a modest and delayed inhibition of RNA and protein synthesis, as reported by others (1, data not shown). Hence, the apoptosis-delaying actions of ceramide do not involve

an exacerbated decline in cellular metabolism. DISCUSSION The primary findings reported here are that ceramide selectively antagonizes certain events associated with NGF deprivation-induced

apoptosis in sympathetic neurons. More specifically, ceramide antagonized oxidative stress, c-_jun_ induction, and apoptosis, but had no effect on metabolic decline. We interpret these

results as suggesting that the currently unknown pathway leading to metabolic dormancy is divergent from the postulated pathway involving oxidative stress, c-_jun_ induction, and apoptosis.

Moreover, since ceramide did not activate the MAP kinase pathway, which is the only pathway known to block oxidative stress in this system, ceramide may act via a novel mechanism to block

the oxidative stress induced by NGF deprivation. Overall, ceramide is emerging as a second messenger with multipotent actions, the final result of which depend on cell type and cellular

environment. Although ceramide is frequently considered as an inducer of apoptosis in non-neuronal cells, our results are in agreement with other reports suggesting that ceramide can inhibit

neuronal apoptosis in response to several stimuli.1,10,11 Our results contribute to the understanding of ceramide mechanisms by revealing unexpected and possibly cell-type specific actions

of ceramide, that is, inhibition of oxidative stress and c-_jun_ induction. The ability of ceramide to inhibit oxidative stress may account for its ability to antagonize c-_jun_ induction

and apoptosis because oxidative stress occurs prior to and is required for c-_jun_ induction and apoptosis.14,23 Recently, we reported that the superoxide producing enzyme NADPH oxidase is

present in sympathetic neurons and contributes to their oxidative stress and apoptosis after NGF deprivation.27 Hence, one possible target for the antioxidant actions of ceramide is NADPH

oxidase. Although the mechanism(s) activating NADPH oxidase and a possible role for ceramide in this pathway are unclear, these two pathways intersect at the point of Rac1. A small

GTP-binding protein, Rac1 is activated after NGF deprivation28 and is known to maximize NADPH oxidase activation in other cell types.29 Rac1 is activated after ceramide treatment in

lymphocytic cells.30 Hence, ceramide may inhibit oxidative stress via a competition for second messengers that are involved in both pathways. Although the activation of the MAP kinase

pathway is required for NGF to suppress reactive oxygen species generation,15 this pathway was not activated by ceramide. That ceramide but not dihydroceramide antagonized apoptosis suggests

that ceramide acts through one of the three enzymes that have been associated with direct ceramide actions, which include PKC-zeta, ceramide activated protein kinase (CAPK), and

ceramide-activated protein phosphatase (CAPP) (reviewed in Mathias _et al_.4). Little is known regarding PKC-zeta and apoptosis. While CAPK has been implicated in phosphorylation of the EGF

receptor,31 ceramide likely does not act via the NGF high affinity receptor Trk in a similar fashion as ceramide did not increase ERK phosphorylation. If oxidative stress is separable from

c-_jun_ activation, ceramide-induced CAPP activation may inhibit c-_jun_ induction by inhibiting the JNK pathway, which we and others have shown is activated in NGF-deprived sympathetic

neurons.3233,34 In summary, our observation that ceramide inhibits oxidative stress induced by NGF deprivation, and reports that oxidative stress is sufficient to activate the JNK pathway

and c-_jun_ expression22,23 suggests the most parsimonious explanation of ceramide actions focuses on the pathways leading to oxidative stress. Considering the actions of ceramide more

generally, the cell cycle may play a role in the ceramide's neuroprotective effects. As part of ceramide's actions as a cellular ‘biostat’, ceramide has been reported to induce

cell cycle arrest at the G0/G1 boundary in multiple cell types (reviewed in2). Since we and other have reported that neurons undergoing apoptosis manifest aspects of cell cycle re-entry,

including the induction of genes necessary for cell cycle progression through the Go/G1 boundary such as _cyclinD1_,35,36 the anti-apoptotic effects of ceramide may reflect arrested cell

cycle progression. Consistent with this possibility, oxidative stress in some cell types has been shown to contribute to cell proliferation, and indeed may mediate the effects of at least

some proliferative agents (reviewed in37). Hence, the actions of ceramide in blocking oxidative stress may be relevant to cell cycle re-entry as well. In summary, neuronal apoptosis has been

implicated in chronic neurodegenerative diseases such as Alzheimers disease,38,39,40 and acute conditions such as stroke.41,42,43 Hence, elucidating the neuroprotective mechanisms of agents

such as ceramide may lead to therapeutically relevant insights. Here, the demonstration that ceramide antagonizes apoptosis without supporting RNA and protein synthesis suggests that the

ceramide-modulated pathways are probably not strong candidate targets for chronic therapy. However, agents such as ceramide that promote neuronal survival in the short-term may lead to

useful therapies in acute situations. MATERIALS AND METHODS NEURONAL CULTURES Primary cultures of sympathetic neurons were prepared and maintained with NGF (50 ng/ml) for 5–7 days as

described previously.18 NGF deprivation was performed by replacing the NGF-containing medium with an identical medium except that NGF was replaced with an NGF neutralizing antibody (Sigma).

C6-ceramide (Cayman Chemicals, Ann Arbor, MI, USA) stock solutions (25 mM) were made in DMSO and then added to the culture dishes to a concentration similar to that shown to be

neuroprotective by others, i.e. 12–18 μM;11 similar results were obtained with each concentration. Control cultures were treated with DMSO vehicle only. CELL DEATH ASSAYS Cultures were

exposed to ceramide with or without NGF for 24 h, fixed with 4% paraformaldehyde in PBS for 20 min, and stained with Hoechst 33258 at a concentration of 1 μg/ml in PBS for 10 min. The

frequency of neurons manifesting condensed or punctate chromatin was then scored by an observer ‘blinded’ as to neuronal treatments. The experiments were performed in triplicate, with at

least 250 neurons being scored in each dish. The effects of ceramide were quantified in four independent experiments, with the effects of dihydroceramide being quantified in the last two

experiments in parallel. After correction for background neuronal death, i.e. the frequency of death in NGF-maintained dishes, differences in neuronal death were analyzed by ANOVA for an

incomplete block design, with a _post-hoc_ Fishers PLSD comparison of the means. GENE EXPRESSION ASSAYS RT–PCR quantitation of gene expression was performed as described previously.18

Briefly, poly-A+ RNA isolated from ∼1,000 neurons was converted to cDNA by using random hexamers to prime reverse transcriptase (Superscript II, Life Technologies, Gaithersburg, MD, USA).

Stock PCR reaction mixtures (50 μl) were prepared on ice and contained 50 μM dCTP, 100 μM each of dGTP, dATP and dTTP, 10 μCi [32P]dCTP (3000 Ci/mmole), 1×reaction buffer (Life

Technologies), 1 μM each primer, 1 U of Taq polymerase (Life Technologies) and 3% of the cDNA synthesized in the reverse transcription. The stock solutions were then separated into three

aliquots that were covered with mineral oil and subjected to various numbers of cycles of PCR. The use of multiple cycles allows us to determine the minimum number of cycles necessary to

detect PCR product, and thereby stay within the linear region of PCR amplification. Typical reaction conditions were one min at 94°C, 1 min at 55°C and 2 min at 72°C. After amplification,

the cDNAs were separated by polyacrylamide gel electrophoresis and visualized by using phosphorimaging technology. The sequences of the primers used were published previously.44,45 This

assay was validated by showing that PCR product yields were linear with respect to input RNA and that the technique detected known gene inductions in neuronal cultures, i.e. HSP70 in a heat

shock paradigm.44 OXIDATIVE STRESS MEASUREMENTS Oxidative stress was quantified by using the indicator 2′,7′-dichlorodihydrofluoroscein diacetate (H2DCFDA, Molecular Probes, Eugene, OR, USA)

which is converted by oxidative stress and cellular esterases to the fluorescent 2′,7′-dichlorofluoroscein (DCF). We also quantified the effects of ceramide on the fluorescence of cells

treated with 2′,7′-dichlorofluoroscein diacetate (DCFDA, Molecular Probes), which is an already oxidized version of H2DCFDA. Neurons were plated onto 96 well tissue culture plates at a

density of ∼1000 neurons/well. After 5–6 days in culture, neurons were either maintained or deprived of NGF, in the presence or absence of ceramide for 3.5 h. H2DCFDA or DCFDA was then added

to a final concentration of 160 μM. Samples were treated in triplicate or greater. The dishes were returned to the 37°C incubator for 30 min. Fluorescence emission at 535 nm after

excitation at 485 nm was quantified by using a plate reader (Perkin Elmer HTS-7000). Because of potential oxidation by the excitation laser, samples were scanned only once. Differences

between ceramide and untreated samples were analyzed by using a Students _t_-test. ERK IMMUNOFLUORESCENCE Neurons were deprived of NGF for 14 h and then retreated with NGF (50 ng/ml) or with

C6-ceramide for 30 min. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in TBS, blocked with antibody buffer (TBS+5% goat serum+0.3% Triton X-100) and then

labeled overnight with an antibody against phosphorylated MAP kinase (Promega) diluted 1 : 200 in antibody buffer. After two TBS washes, the cells were exposed to cy-3 conjugated,

anti-rabbit secondary antibody (Jackson Immunochemicals) for 2 h. The plates were washed twice with TBS and stained with Hoechst 33,258 (Molecular Probes). Staining was detected with

fluorescence microscopy. To standardize the images, film exposure times and subsequent processing procedures were identical among the various samples and images. WESTERN BLOTTING ANALYSIS

Sympathetic neuronal cultures (20 000 cells/plate) were deprived of NGF for 14 h and then treated with either NGF or C6-ceramide for 30 min. Proteins were electrophoresed on 15% SDS–PAGE

gels, transferred onto PVDF membranes, blocked in 5% milk, and probed with a total MAP kinase antibody (New England Biolabs; 1 : 1000 dilution) or a phospho-MAP kinase antibody (Promega; 1 :

 20 000 dilution) overnight. The membranes were washed with 0.1% Tween in TBS, labeled with HRP-conjugated anti-rabbit secondary antibodies (Amersham, 1 : 2000 dilution) for 2 h and then

washed again in 0.1% Tween in TBS. Labeled proteins were detected by using ECL-PLUS (Amersham). METABOLIC PARAMETERS RNA and protein synthesis rates were determined by using a protocol

described by others.16 Neuronal cultures were maintained in 24-well plates (approximately 2500 cells/well). The culture medium was then replaced with medium containing 10 μM L-methionine,

with or without NGF and/or ceramide or DMSO vehicle. Parallel cultures that were NGF deprived upon neuronal plating, and therefore consisting only of non-neuronal cells, were used to provide

background values. At the indicated times, cultures were exposed to 10 μCi/ml [35S]L-methionine (>1000 Ci/mmol) and 10 μCi/ml [5,6-3H]uridine (35–50 Ci/mmol) for 4 h at 37°C. The cells

were washed with medium and lysed with 500 μl buffer containing 0.5% SDS, 1 mM EDTA, and 10 mM Tris (pH 7.5). Ten μg each of BSA and yeast tRNA were added to each sample and the protein and

RNA precipitated simultaneously by adding cold 10% trichloroacetic acid and 1% sodium pyrophosphate on ice. Precipitates were captured on a 0.45 μm nitrocellulose filter, washed with cold

10% TCA, water, and finally with 70% ethanol. The filters were dried and radioactivity quantified by liquid scintillation counting. Radioactivity in each sample was normalized by subtracting

the radioactivity in the background sample and then dividing by the average c.p.m. observed in parallel culture samples maintained in NGF. In preliminary work, we demonstrated that

radioactivity incorporation was linear with respect to labeling time for at least 4 h (data not shown). ABBREVIATIONS * CAPK: ceramide activated protein kinase * CAPP: ceramide-activated

protein phosphatase * DCF: 2′, 7′-dichlorofluoroscein * DCFDA: 2′, 7′-dichlorofluoroscein diacetate * H2DCFDA: 2′, 7′-dichlorodihydrofluoroscein diacetate * TBS: Tris buffered saline

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Neurosci._ 17: 7736–7745 Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS The authors would like to acknowledge the support of NIH (S Estus, grant NS-34370 and SP

Tammariello, AG-00242) and HM Tucker, for critical discussions of the manuscript and for preparing the figures. We also acknowledge R Kyruscio for statistical analysis and JP McGillis for

critical comments on the manuscript. AUTHOR INFORMATION Author notes * P Nair Present address: Department of Surgery, UTHSC at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284,

Texas, USA AUTHORS AND AFFILIATIONS * Department of Physiology, Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40536, Kentucky, USA P Nair, S P Tammariello & S

Estus Authors * P Nair View author publications You can also search for this author inPubMed Google Scholar * S P Tammariello View author publications You can also search for this author

inPubMed Google Scholar * S Estus View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to S Estus. ADDITIONAL INFORMATION

Edited by R Lockshin RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Nair, P., Tammariello, S. & Estus, S. Ceramide selectively inhibits

apoptosis-associated events in NGF-deprived sympathetic neurons. _Cell Death Differ_ 7, 207–214 (2000). https://doi.org/10.1038/sj.cdd.4400628 Download citation * Received: 14 July 1999 *

Revised: 16 October 1999 * Accepted: 25 October 1999 * Published: 07 March 2000 * Issue Date: 01 February 2000 * DOI: https://doi.org/10.1038/sj.cdd.4400628 SHARE THIS ARTICLE Anyone you

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Springer Nature SharedIt content-sharing initiative KEYWORDS * neuronal apoptosis * c-_jun_ * oxidative stress