Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brief "preconditioning" ischemia induces
ischemic tolerance (IT) and protects the animal brain from subsequent
otherwise lethal ischemia. Identification of the signaling
steps most proximal to the development of the IT will allow induction
of the resistance to ischemia shortly after the onset of
stroke. Animal studies demonstrate a key role of tumor necrosis
factor- (TNF-
) in induction of IT. The sphingolipid ceramide is
known as a second messenger in many of the multiple effects of TNF-
.
We hypothesized that ceramide could mediate IT. We demonstrate that
preconditioning of rat cortical neurons with mild hypoxia protects them
from hypoxia and O2-glucose
deprivation injury 24 h later (50% protection). TNF-
pretreatment
could be substituted for hypoxic preconditioning (HP). HP was
attenuated by TNF-
-neutralizing antibody. HP and TNF-
pretreatment cause a two- to threefold increase of intracellular ceramide levels, which coincides with the state of tolerance. Fumonisin
B1, an inhibitor of ceramide
synthase, attenuated ceramide upregulation and HP. C-2 ceramide added
to the cultures right before the hypoxic insult mimicked the effect of
HP. Ceramide did not induce apoptosis. These results suggest that HP is
mediated via ceramide synthesis triggered by TNF-
.
ischemia; hypoxia; tolerance; neurons; tumor necrosis
factor-
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DEVELOPMENT OF AN ISCHEMIC lesion depends on the interaction of many pathophysiological mechanisms, such as release of neurotransmitters, collapse of membrane ion gradients, release of inflammatory cytokines, triggering of apoptotic machinery, failure of the blood-brain barrier, compromised microcirculatory perfusion, generation of reactive oxygen species, and a host of other factors (for reviews see Refs. 8, 10, 16, 25, and 44). These reactions are thought to be counteracted by multiple neuroprotective mechanisms (27). It has become increasingly clear that therapeutic interventions targeting only part of the complex network of mediators that contribute to early progression of ischemic brain damage produce only subtle effects on the outcome of stroke in clinical trials. A solution to this problem could be derived from an understanding of mechanisms by which the cell adapts to ischemic stress.
In animal models of brain ischemia, brief transient episodes of "preconditioning" ischemia induce tolerance and protect the brain from subsequent otherwise lethal ischemia (for review see Ref. 7). Development of the tolerant state takes time, usually 24-72 h (3). A cascade of signaling events initiated by sublethal stress proceeds during the latent period, resulting in a new, stress-resistant, biochemical makeup of brain cells. Identification of the signaling steps most proximal to the development of the tolerant state would allow compression of the latency period and make cells resistant to ischemic stress quickly.
Many deleterious as well as neuroprotective reactions in ischemic brain
are mediated by the pleiotropic cytokines tumor necrosis factor-
(TNF-
) and interleukin-1 (IL-1) (16, 38). Recent studies suggest
that the state of tolerance induced by ischemic preconditioning also
might involve these cytokines. Thus intraperitoneal pretreatment of
gerbils with IL-1 receptor antagonist abolished tolerance to global
ischemia induced by ischemic preconditioning, and injection of
IL-1
and IL-1
mimicked the effect of preconditioning (33).
Similarly, intravenous pretreatment of spontaneously hypertensive rats
with lipopolysaccharide (an agent that is known to induce production of
TNF-
) led to development of ischemic tolerance, and combined
administration with TNF-
-binding protein prevented it (42).
Furthermore, intracisternal pretreatment of mice with TNF-
(32)
protected animals from ischemic injury in a permanent middle cerebral
artery occlusion model. These observations suggest that cellular
resistance to ischemic stress in brain originates at least partially
from engagement of the TNF-
receptor and its downstream messengers.
The sphingolipid ceramide has been implicated as a second messenger in
many of the multiple signaling pathways initiated on binding of TNF-
to its p55 receptor (13, 22). Although most of these studies have
focused on the role of ceramide in induction of apoptosis and cell
cycle control (34, 39), there is also evidence that ceramide can cause
cytoprotection (15). We hypothesized that ceramide could play a role in
induction of ischemic tolerance. This hypothesis was tested in an in
vitro model of tolerance. We present evidence for the first time that
preconditioning of cortical neurons with mild hypoxia results in
TNF-
-mediated upregulation of intracellular ceramide levels that is
necessary and sufficient for induction of the tolerant state.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cortical neuronal cultures. Cortical neuronal cultures were established from 2-day-old Sprague-Dawley rats. Cerebral cortices without meninges were placed into a dissection medium [0.3% (wt/vol) glucose, 0.75% (wt/vol) sucrose, and 28 mM HEPES in Hanks' balanced salt solution, pH 7.3, osmolarity 320 mosmol/kg], cut into small pieces, treated with 0.25% trypsin for 20 min at 37°C, and then resuspended in DMEM-high-glucose (4,500 mg/l), 2 mM glutamine, 1% antibiotic/antimycotic (all from GIBCO Life Technology), 10% fetal bovine serum (Summit Biotechnology), and 40 µg/ml DNase (Boerhinger Mannheim) and triturated 20 times in culture medium. The cell suspension was centrifuged at low speed (1,000 rpm) to eliminate cell debris, resuspended in culture medium [Neurobasal-A with 2% B27 supplement, 1 mM L-glutamine (all from GIBCO Life Technology), and 0.2% horse serum (Sigma Chemical)] at 5.5-5.8 × 105/ml (1 brain yielded a 24-ml cell suspension), and plated in 500 µl/well in 24-well plates (Costar) precoated with 2.5 µg/cm2 of poly-L-lysine. The glucose concentration in Neurobasal-A was 4,500 mg/l. Nonneuronal cells were eliminated by changing the medium 20 min after plating and by adding 15 µg/ml 5'-fluoro-2'-deoxyuridine (Sigma Chemical) to the culture medium. Immunostaining of neurons with antibody against neuron-specific enolase (Chemicon International, Temecula, CA) and astrocytes with glial fibrillary acidic protein-specific antibody (Boerhinger Mannheim) demonstrated that astrocyte contamination was <5%.
Hypoxic pretreatment. Hypoxic pretreatment was performed on day 4 in vitro after withdrawal of 200 µl (of 400 µl/well) of culture medium. Neuronal cultures were placed in modular incubator chambers (Billups Rothenberg, Del Mar, CA) and flushed with a gas mixture of 5% CO2-95% N2 for 20 min at room temperature (when the cells were grown in 60-mm dishes, flushing time was 15 min). The chambers were sealed and incubated at 37°C for 20 min. O2 concentration in the culture medium was monitored with an O2 meter (Microelectrodes, Bedford, NH) and reached 8% at the end of pretreatment. After pretreatment, 200 µl/well of normoxic medium were added back to the cultures, and they were incubated in normoxic conditions (5% CO2, 100% humidity at 37°C) for 24 h and then subjected to severe hypoxia.
Severe hypoxic treatment. Cells were subjected to severe hypoxic treatment on day 5 in vitro. Culture medium was completely removed from naïve and preconditioned cells and substituted with 200 µl/well of Neurobasal-A medium plus 1 mM L-glutamine (no B27 supplement and horse serum). The plates were flushed with 5% CO2-95% N2 in hypoxic chambers until O2 concentrations dropped to 2% (~40 min). Chambers were agitated every 5 min to ensure maximal gas exchange in the culture medium. The chambers were sealed and incubated for 2.5 h at 37°C (O2 concentration in the medium was 5% at the end of incubation). For reoxygenation, 200 µl/well of normoxic culture medium containing double concentrations of B27 supplement and horse serum were added to the cells, cells were placed in a regular tissue culture incubator, and cell viability was measured at indicated times. Control cells were subjected to the same washing and feeding procedures with normoxic medium. For glucose deprivation studies, cells were incubated in 200 µl/well of DMEM containing no glucose, and 200 µl/well of DMEM containing double concentrations of glucose were added to the cultures on reoxygenation.
Pretreatment of neuronal cultures with TNF- and C-2
ceramide and blocking reagents.
TNF-
was added to neuronal cultures on day
4 at 25 ng/ml in Neurobasal-A plus 1 mM
L-glutamine and 2% B27
supplement and 0.2% horse serum for 24 h (it was washed out just
before the severe hypoxic treatment).
N-acetylceramide (C-2 ceramide) was
added to the cultures at the beginning of severe hypoxia
(day 5 in vitro) at 10 µM and
remained in the medium during the entire reoxygenation period.
Anti-TNF-
neutralizing antibody (8 µg/ml at
ND50 = 3-6 µg/ml; R & D Systems) or fumonisin
B1 at 50 µM (Alexis
Biochemicals, San Diego, CA) was added to neuronal cultures at the time
of preconditioning. Both reagents were washed out just before
incubation of cells in severe hypoxia.
Quantitation of neuronal injury. Quantitation of neuronal injury was performed by means of an ethidium homodimer fluorescence exclusion test. Ethidium homodimer and other DNA-binding fluorescent dyes are commonly used for cytotoxicity tests. These dyes are polar and, therefore, cell impermeant, unless the integrity of the cell membrane is compromised by a cytotoxic reagent. Ethidium homodimer has been specifically designed for live/dead assays, and it is superior to other stains because of its higher DNA affinity, very low membrane permeability, and very low background fluorescence. It has been used in cytotoxicity assays since 1995; however, its application for neuronal cultures has been limited, although it has been documented in several studies (26, 31). One of the reasons is that neuronal cultures do not form monolayers, and the number of cells per culture or per certain area of the culture varies. This variability becomes even greater when cells are subjected to stress, such as hypoxia, because many cells disintegrate or detach from the dish. Thus it was necessary to measure not only the number of cells that fail to exclude the dye but also the number of cells that remained in the culture after treatment. Our modification of the assay was to add a mild detergent at the end of the assay to make all the cells in the culture permeable to the dye, which allowed us to estimate the total number of cells remaining in the culture wells after each experimental condition.
This is illustrated by a representative experiment presented in Fig. 1. There were only a few dead cells in control cultures (Fig. 1A) compared with hypoxia-treated wells (Fig. 1C). To estimate how many cells remained in the well, all the cells were made permeable to the dye by addition of the detergent saponin. This procedure revealed a significant cell loss in hypoxia-treated cultures (Fig. 1D) compared with control (Fig. 1B). To quantify fluorescence changes, neurons were plated in 24-well plates and subjected to the treatments mentioned above. At the end of each experiment, culture medium was withdrawn and cells were incubated with 6 µM ethidium homodimer (Molecular Probes, Eugene, OR) in Hanks' buffer at 300 µl/well for 30 min at 37°C. Cell fluorescence was measured with a CytoFluor 4000 fluorescent plate reader (PerSeptive Biosystems, Framingham, MA) at excitation/emission wavelengths of 530/620 nm. Background fluorescence was measured on each plate and subtracted. The percentage of dead cells was calculated by means of the following formula
![]() |
![]() |
|
Intracellular ceramide levels.
Intracellular ceramide levels were measured in neurons that were grown
in 60-mm culture dishes and subjected to preconditioning hypoxia or to
TNF- pretreatment. At 0, 16, 20, 24, 28, and 32 h after
reoxygenation, cells were washed twice with cold PBS, scraped off, and
pelleted. Cell pellets were subjected to lipid extraction, and
intracellular ceramide was quantitated by means of reverse-phase HPLC
according to Santana et al. (37), as described in detail elsewhere
(13). Ceramide values were normalized per lipid phosphate, as described
previously (13).
Measurements of TNF- concentrations.
Neuronal cultures in 24-well plates were covered with 0.4 ml of culture
medium and subjected to hypoxic pretreatment. Aliquots of culture
medium were withdrawn at 4, 8, and 24 h after preconditioning, and
TNF-
levels were measured in these samples with an ELISA kit for rat
TNF-
(Endogen, Woburn, MA) according to the manufacturer's instructions.
Visualization and quantitation of apoptotic cells. Visualization and quantitation of apoptotic cells were performed with the In Situ Cell Death Detection Kit (POD, Boehringer Mannheim). Staining of control and C-2 ceramide-treated neuronal cultures was performed according to the manufacturer's protocol. The samples were analyzed with a Zeiss Axiovert 10 light microscope (magnification ×40). Digitized images of 20 microscopic fields per experimental condition were generated using a DAGE MTI DEI-750 attached camera. The same microscope and camera settings were used for all samples. The number of apoptotic cells within each image was determined by means of a Scion Image computer program and expressed as a sum in pixels of all positively stained areas. The size of one apoptotic nucleus was 80-145 pixels [113 ± 22.5 (SD), n = 10]. Areas <45 pixels were considered "debris" and were excluded from the measurements.
Immunostaining for TNF- receptor.
Neurons were subjected to preconditioning hypoxia and 24 h later were
fixed with Bouin's solution and immunostained with goat polyclonal
antibody directed against TNF-
type 1 receptor/p55 (Santa Cruz) at
1:100 dilution. Digitized images of the samples were generated as
described above for evaluation of apoptosis. TNF-
type 1 receptor/p55 expression was quantified by means of Scion Image and
expressed as a mean intensity of staining. A background intensity was
measured for each image and subtracted from mean intensity; 12-14
images were analyzed per experimental condition.
Statistical analysis. Statistical analysis was carried out by two-factor ANOVA and by paired t-test by use of Excel software.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Description of the model of hypoxic-ischemic tolerance in vitro.
Analysis of morphological changes of cortical neurons subjected to 2.5 h of hypoxic treatment demonstrated significant cell death (Fig.
2B)
compared with control untreated cultures (Fig. 2A). Cultures preconditioned with
mild hypoxia were more resistant to hypoxic treatment, with fewer dead
cells (Fig. 2C). The effect of
hypoxic treatment on neuron viability was quantified by means of the
ethidium homodimer assay (see MATERIALS AND
METHODS). Measurements of dead cell number
demonstrated that hypoxia-induced injury of neuronal cells had already
begun during hypoxic treatment and then progressed after reoxygenation.
At the end of 2.5 h of hypoxic incubation, ~13% of the cells were
dead (P = 0.004, n = 4; Fig. 3A). The
number of dead cells doubled during 8 h of reoxygenation (P = 0.0001, n = 6). Progression of
hypoxia-initiated cell death continued for up to 24 h of observation.
No significant cell death was observed in sham-washed control cultures
maintained in normoxia during the entire period of observation (Fig.
3A). Hypoxic preconditioning of
neuronal cultures 24 h before the hypoxic insult inhibited cell death
during the period of incubation in the hypoxic environment by 50%
(P = 0.044, n = 4) and during progression of
neuronal injury after reoxygenation by 52 and 39% at 8 h
(P = 0.0003, n = 7) and 24 h
(P = 0.0013, n = 7) after reoxygenation,
respectively (Fig. 3A). Measurements
of ethidium fluorescence in hypoxia-preconditioned cultures 24 h after
preconditioning (immediately before the main insult) revealed no cell
death and no cell loss as a result of preconditioning (data not shown).
|
|
Role of TNF- in hypoxic preconditioning of neurons.
Experiments in animal models have demonstrated that intracisternal
administration of TNF-
had a protective effect in the middle
cerebral artery occlusion model of brain ischemia (32). On the
basis of these observations, we sought to investigate whether TNF-
can mimic the protective effect of preconditioning in neuronal cells in
our model. TNF-
was added to neuronal cultures at 25 ng/ml 24 h
before hypoxic treatment. Immediately before placement of the cells
into hypoxic chambers, the culture medium with TNF-
was completely
exchanged for the culture medium containing no TNF-
, and cells were
subjected to 2.5 h of hypoxia. The results of this experiment are
presented in Fig.
4A. The
number of dead cells at 0, 8, and 24 h after cell reoxygenation
demonstrated that pretreatment with TNF-
protected neurons from a
hypoxic insult to the same degree as did hypoxic
preconditioning: 40% (P = 0.004, n = 3), 53%
(P = 0.002, n = 5), and 44%
(P = 0.002, n = 5), respectively.
|
Preconditioning does not change surface expression of
TNF- type 1 receptor (p55).
Neurons are not only capable of TNF-
synthesis in response to
ischemic stress (16, 25), but they also express TNF-
receptors and
amplify this response through paracrine and autocrine mechanisms (41).
Studies of lipopolysaccharide- and TNF-induced tolerance in macrophages
and monocytes (18) suggest that unresponsiveness of preconditioned
cells to the second stimulation with these agents could result from
downregulation of the respective receptors caused by the
preconditioning treatment. Because our data demonstrated that hypoxic
preconditioning was mediated by TNF-
and because TNF-
is also
known to mediate cytotoxic effects during ischemia, we wanted
to rule out the possibility that the small amount of TNF-
that is
released during preconditioning could downregulate expression of
TNF-
type 1 receptor by shedding or by endocytosis, and this would
make preconditioned cells more resistant to cytotoxic amounts of
TNF-
, which are released during the severe hypoxia.
|
Ceramide is a messenger of ischemic tolerance.
The results of the experiments presented above strongly suggested that
TNF- release in neurons preconditioned with mild hypoxia initiated a
signaling cascade responsible for cellular resistance to subsequent
hypoxic insult. The role of a sphingolipid, ceramide, as a mediator of
TNF-
effects has been demonstrated in many cellular models. It has
been shown that cell-permeable ceramide analogs, when added to the
neuronal cultures, mimic TNF-
effects such as apoptosis (6) or
cytoprotection (15, 20). Accordingly, we sought to investigate whether
ceramide was a mediator of tolerance.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A molecular mechanism(s) that is switched on at the end of the lag period after ischemic preconditioning and confers cell resistance to ischemic stress should meet the following requirements. First, it should affect various types of brain cells as well as their multiple responses to ischemia. A mechanism affecting expression of multiple genes would suit this requirement. Second, the release or synthesis of such a "tolerizing" factor(s) should be delayed after onset of preconditioning and reach optimal levels when tolerance is demonstrated in the model system.
We have presented evidence that ischemic tolerance is mediated by de
novo synthesis of ceramide triggered by TNF-. Ceramide kinetics in
preconditioned cells, its key role in TNF-
receptor signaling, and
its pleiotropic effects on cells (including neuroprotection) conform to
the above requirements well. This new function of ceramide is supported
by the following observations.
First, exogenous TNF- and exogenous C-2 ceramide were able to
substitute for hypoxic pretreatment in making cortical neurons resistant to a subsequent hypoxic insult. Under these conditions, TNF-
and C-2 ceramide protected neurons to the same degree as did
hypoxic preconditioning.
The role of TNF- and its downstream messenger ceramide as mediators
of hypoxic preconditioning is further supported by the fact that
hypoxic preconditioning resulted in no tolerance if it was performed in
the presence of TNF-
-neutralizing antibody (but not in the presence
of nonspecific antibody) or in the presence of the inhibitor of
ceramide de novo synthesis, fumonisin
B1. Double the
ND50 of antibody was used in the
experiments. According to the manufacturer, this dose was able to
neutralize 0.025 ng/ml of TNF-
. There were ~2.5 × 105 cells/well, which means that,
during preconditioning, 100 pg of TNF-
could be released per
106 neurons. TNF-
ELISA
demonstrated that the amount of TNF-
in culture medium increased by
6 pg/well, which contained ~2.5 × 105 neurons, suggesting that
30
pg/106 cells were released. These
calculations do not include cell membrane- and plastic surface-bound
TNF-
. In fact, the amount of TNF-
in the medium peaked at 8 h
after preconditioning and decreased by 24 h, probably because of
TNF-
binding to these surfaces. Our observation that preconditioned
neurons expressed high levels of TNF-
p55 receptor further suggests
engagement of TNF-
signaling pathways in induction of tolerance.
Because the majority of TNF-
effects have been attributed to p55
receptor (1, 35), expression of p75 receptor has not been tested,
although one cannot exclude its role in preconditioning.
The inhibitory effect of fumonisin B1 on neuronal ceramide synthase (sphingosine-N-acyltransferase) has been previously demonstrated (30). The ability of fumonisin B1 to block hypoxic preconditioning suggests that de novo synthesis of ceramide contributes to induction of tolerance.
Further evidence for the ceramide being a TNF- messenger in
induction of ischemic tolerance is derived from the measurements of
ceramide levels in cells preconditioned with hypoxia or pretreated with
TNF-
. Both treatments resulted in a delayed increase of intracellular ceramide levels, which coincided with development of
resistance to severe hypoxic insult. These data are consistent with our
previous observations which demonstrated that astrocytes and brain
endothelial cells preconditioned with TNF-
also exhibited delayed
ceramide responses, which coincided with a tolerant state (14). The
ability of fumonisin B1 to abolish
ceramide synthesis parallels its effect on ischemic tolerance and
strongly argues for the role of ceramide as a mediator of tolerance.
Different culture conditions and the older age of neuronal cells could explain the different temporal parameters required to achieve tolerance in our model compared with the model designed by Bruer and co-workers (5). Nevertheless, our study confirms their findings that a minimal lag period of ~24 h is required for cells to become tolerant after hypoxic preconditioning. Employment of fluorescent techniques for rapid quantitation of dead cells has allowed us to detect neuronal damage and protection immediately after hypoxic treatment even before reoxygenation, which has been impossible with traditional lactate dehydrogenase measurements because of the latency of the enzyme leakage (21). We demonstrate that hypoxic pretreatment attenuates neuronal injury that occurs during the hypoxic insult and neuronal injury that develops during reoxygenation. The fact that preconditioning did not result in cell loss or cell death rules out the possibility that selection of "stronger" cells during hypoxic pretreatment explains the data.
Ceramide effects in neuronal cells range from protective to apoptotic.
Cell-permeable ceramide analogs induced apoptosis in embryonic chick
hemisphere neurons (43) and in mesencephalic neurons (6). In different
models, the same ceramide derivatives added exogenously exhibited a
protective effect. Thus exogenous ceramide induced protection of
hippocampal neurons against glutamate, FeSO4, and amyloid -peptide
toxicity. This protective effect of ceramide was blocked by inhibitors
of RNA and protein synthesis (15). Controversial results on the role of
ceramide in apoptosis signaling in neurons have been obtained when
endogenous ceramide levels were manipulated. Increase of intracellular
ceramide levels by blocking ceramide catabolism in sensory neurons (36)
and inhibition of intracellular ceramide levels with the inhibitor of
its de novo synthesis (12) were found to promote apoptosis. In one
study, both effects of ceramide (protective and proapototic) have been
demonstrated in the same cells (spinal motoneurons), depending on
ceramide concentrations (19). We previously reported that the C-2
ceramide effect on cell viability depends on its dose and demonstrated
that 10 µM ceramide was not harmful to astrocytes and brain
microvascular endothelial cells (14). According to measurements of
apoptosis presented here, ~20% of cells in neuronal cultures were
apoptotic, most probably due to culture conditions, to low numbers of
astrocytes in these cultures, and to constitutive apoptosis, which
occurs in developing brain. Addition of 10 µM C-2 ceramide did not
cause an increase in the percentage of apoptotic cells.
Little is known about the molecular mechanisms mediating TNF- and
ceramide-induced cytoprotection. Protection of sympathetic neurons
against nerve growth factor (NGF) deprivation with exogenous ceramide
has been associated with NGF binding to its low-affinity receptor (20).
Ceramide was also shown to decrease the levels of
microtubule-associated protein
and to increase the calpain-derived spectrin breakdown product by modifying the activity of calpain 1 proteinase in PC-12 cells. It was suggested that the latter effect of
ceramide might increase neuronal regeneration and remodeling. We have
noticed that pretreatment with C-2 ceramide not only rescued hypoxia-injured cultures but also made control cultures look
"healthier," which might be related to described effects of C-2
ceramide on calpain. Much evidence has been accumulated that implicates
activation of nuclear factor-
B (NF-
B) in mechanisms controlling
apoptosis/survival in neuronal cells through stabilization of
intracellular Ca2+ concentration,
increase in density of the outward
K+ currents, and induction of
antioxidants (2, 11, 28). Which of these mechanisms is involved in
ceramide-mediated hypoxic preconditioning in neuronal cells is not
clear. Recent studies of myocardial preconditioning (29, 45) suggest a
participation of NF-
B in adaptation to ischemia. In support
of this hypothesis, loss of NF-
B activity was observed during brain
ischemia and inhibition of NF-
B sensitized brain cells to
cytotoxic effects of TNF-
in vitro (4).
More experiments are needed to elucidate the signaling events downstream from ceramide that make neurons tolerant to ischemic injury. The recently identified ceramide-activated proline-directed serine-threonine kinase (24) and a serine-threonine phosphatase (9) could be suggested as attractive initial targets for these studies. Sphingosine or sphingosine 1-phosphate could also be involved in downstream signaling (40).
![]() |
ACKNOWLEDGEMENTS |
---|
J. Liu and I. Ginis contributed equally to this work.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: I. Ginis, Stroke Branch, NINDS, Bldg. 36, Rm. 4A03, National Institutes of Health, Bethesda, MD 20892-4128 (E-mail: ginis{at}codon.nih.gov).
Received 29 June 1999; accepted in final form 22 August 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barbara, J. A.,
X. Van Ostad,
and
A. Lopez.
Tumour necrosis factor- (TNF-
): the good, the bad and potentially very effective.
Immunol. Cell Biol.
74:
434-443,
1996[ISI][Medline].
2.
Barger, S. W.,
D. Horster,
K. Furukawa,
Y. Goodman,
J. Krieglstein,
and
M. P. Mattson.
Tumor necrosis factors- and -
protect neurons against amyloid
-peptide toxicity: evidence for involvement of a
B-binding factor and attenuation of peroxide and Ca2+ accumulation.
Proc. Natl. Acad. Sci. USA
92:
9328-9332,
1995[Abstract].
3.
Barone, F. C.,
R. F. White,
P. A. Spera,
J. Ellison,
R. W. Currie,
X. Wang,
and
G. Z. Feuerstein.
Ischemic preconditioning and brain tolerance: temporal histological and functional outcomes, protein synthesis requirement, and interleukin-1 receptor antagonist and early gene expression.
Stroke
29:
1937-1951,
1998
4.
Botchkina, G. I.,
E. Geimonen,
M. L. Bilof,
O. Villarreal,
and
K. J. Tracey.
Loss of NF-B activity during cerebral ischemia and TNF cytotoxicity.
Mol. Med.
5:
372-381,
1999[ISI][Medline].
5.
Bruer, U.,
M. K. Weih,
N. K. Isaev,
A. Meisel,
K. Ruscher,
A. Bergk,
G. Trendelenburg,
F. Wiegand,
I. V. Victorov,
and
U. Dirnagl.
Induction of tolerance in rat cortical neurons: hypoxic preconditioning.
FEBS Lett.
414:
117-121,
1997[ISI][Medline].
6.
Brugg, B.,
P. P. Michel,
Y. Agid,
and
M. Ruberg.
Ceramide induces apoptosis in cultured mesencephalic neurons.
J. Neurochem.
66:
733-739,
1996[ISI][Medline].
7.
Chen, J.,
and
R. Simon.
Ischemic tolerance in brain.
Neurology
48:
306-311,
1997[ISI][Medline].
8.
Del Zoppo, G. J.
Microvascular responses to cerebral ischemia/inflammation.
Ann. NY Acad. Sci.
823:
132-147,
1997[Abstract].
9.
Dobrowsky, R. T.,
C. Kamibayashi,
M. C. Mumby,
and
Y. A. Hannun.
Ceramide activates heterotrimeric protein phosphatase 2A.
J. Biol. Chem.
268:
15523-15530,
1993
10.
Feuerstein, G. Z.,
X. Wang,
and
F. C. Barone.
The role of cytokines in the neuropathology of stroke and neurotrauma.
Neuroimmunomodulation
5:
143-159,
1998[ISI][Medline].
11.
Furukawa, K.,
and
M. P. Mattson.
The transcription factor NF-B mediates increases in calcium currents and decreases in NMDA- and AMPA/kainate-induced currents induced by tumor necrosis factor-
in hippocampal neurons.
J. Neurochem.
70:
1876-1886,
1998[ISI][Medline].
12.
Furuya, S.,
J. Mitoma,
A. Makino,
and
Y. Hirabayashi.
Ceramide and its interconvertible metabolite sphingosine function as indispensable lipid factors involved in survival and dendritic differentiation of cerebellar Purkinje cells.
J. Neurochem.
71:
366-377,
1998[ISI][Medline].
13.
Ghosh, S.,
J. C. Strum,
and
R. M. Bell.
Lipid biochemistry: functions of glycerolipids and sphingolipids in cellular signaling.
FASEB J.
11:
45-50,
1997
14.
Ginis, I.,
U. Schweizer,
M. Brenner,
J. Liu,
N. Azzam,
M. Spatz,
and
J. M. Hallenbeck.
TNF- pretreatment prevents subsequent activation of cultured brain cells with TNF-
and hypoxia via ceramide.
Am. J. Physiol. Cell Physiol.
276:
C1171-C1183,
1999
15.
Goodman, Y.,
and
M. P. Mattson.
Ceramide protects hippocampal neurons against excitotoxic and oxidative insults and amyloid -peptide toxicity.
J. Neurochem.
66:
869-872,
1996[ISI][Medline].
16.
Hallenbeck, J. M.
Significance of the inflammatory response in brain ischemia.
Acta Neurochir. Suppl.
66:
27-31,
1996[Medline].
17.
Hallenbeck, J. M.,
A. J. Dutka,
S. N. Vogel,
E. Heldman,
D. A. Doron,
and
G. Feuerstein.
Lipopolysaccharide-induced production of tumor necrosis factor activity in rats with and without risk factors for stroke.
Brain Res.
541:
115-120,
1991[ISI][Medline].
18.
Higuchi, M.,
and
B. B. Aggarwal.
TNF induces internalization of the p60 receptor and shedding of the p80 receptor.
J. Immunol.
152:
3550-3558,
1994
19.
Irie, F.,
and
Y. Hirabayashi.
Application of exogenous ceramide to cultured rat spinal motoneurons promotes survival or death by regulation of apoptosis depending on its concentrations.
J. Neurosci. Res.
54:
475-485,
1998[ISI][Medline].
20.
Ito, A.,
and
K. Horigome.
Ceramide prevents neuronal programmed cell death induced by nerve growth factor deprivation.
J. Neurochem.
65:
463-466,
1995[ISI][Medline].
21.
Juurlink, B. H.,
and
L. Hertz.
Ischemia-induced death of astrocytes and neurons in primary culture: pitfalls in quantifying neuronal cell death.
Brain Res. Dev. Brain Res.
71:
239-246,
1993[ISI][Medline].
22.
Kolesnick, R.,
and
D. W. Gold.
The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling.
Cell
77:
325-328,
1994[ISI][Medline].
23.
Levesque, A.,
A. Paquet,
and
M. Page.
Improved fluorescent bioassay for the detection of tumor necrosis factor activity.
J. Immunol. Methods
178:
71-76,
1995[ISI][Medline].
24.
Liu, J.,
S. Mathias,
Z. Yang,
and
R. N. Kolesnick.
Renaturation and tumor necrosis factor- stimulation of a 97-kDa ceramide-activated protein kinase.
J. Biol. Chem.
269:
3047-3052,
1994
25.
Maiese, K.
From the bench to the bedside: the molecular management of cerebral ischemia.
Clin. Neuropharmacol.
21:
1-7,
1998[ISI][Medline].
26.
Mark, R. J.,
K. Hensley,
D. A. Butterfield,
and
M. P. Mattson.
Amyloid -peptide impairs ion-motive ATPase activities: evidence for a role in loss of neuronal Ca2+ homeostasis and cell death.
Neuroscience
15:
6239-6249,
1995[Medline].
27.
Mattson, M. P.
Neuroprotective signal transduction: relevance to stroke.
Neurosci. Biobehav. Rev.
21:
193-206,
1997[ISI][Medline].
28.
Mattson, M. P.,
Y. Goodman,
H. Luo,
W. Fu,
and
K. Furukawa.
Activation of NF-B protects hippocampal neurons against oxidative stress-induced apoptosis: evidence for induction of manganese superoxide dismutase and suppression of peroxynitrite production and protein tyrosine nitration.
J. Neurosci. Res.
49:
681-697,
1997[ISI][Medline].
29.
Maulik, N.,
H. Sasaki,
and
N. Galang.
Differential regulation of apoptosis by ischemia-reperfusion and ischemic adaptation.
Ann. NY Acad. Sci.
874:
401-411,
1999
30.
Merrill, A. H., Jr.,
G. van Echten,
E. Wang,
and
K. Sandhoff.
Fumonisin B1 inhibits sphingosine (sphinganine) N-acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ.
J. Biol. Chem.
268:
27299-27306,
1993
31.
Mukhin, A. G.,
S. A. Ivanova,
J. W. Allen,
and
A. I. Faden.
Mechanical injury to neuronal/glial cultures in microplates: role of NMDA receptors and pH in secondary neuronal cell death.
J. Neurosci. Res.
51:
748-758,
1998[ISI][Medline].
32.
Nawashiro, H.,
K. Tasaki,
C. A. Ruetzler,
and
J. M. Hallenbeck.
TNF- pre-treatment induces protective effects against focal cerebral ischemia in mice.
J. Cereb. Blood Flow Metab.
17:
483-490,
1997[ISI][Medline].
33.
Ohtsuki, T.,
C. A. Ruetzler,
K. Tasaki,
and
J. M. Hallenbeck.
Interleukin-1 mediates induction of tolerance to global ischemia in gerbil hippocampal CA1 neurons.
J. Cereb. Blood Flow Metab.
16:
1137-1142,
1996[ISI][Medline].
34.
Pena, L. A.,
Z. Fuks,
and
R. Kolesnick.
Stress-induced apoptosis and the sphingomyelin pathway.
Biochem. Pharmacol.
53:
615-621,
1997[ISI][Medline].
35.
Peschon, J. J.,
D. S. Torrance,
K. L. Stocking,
M. B. Glaccum,
C. Otten,
C. R. Willis,
K. Charrier,
P. J. Morrissey,
C. B. Ware,
and
K. M. Mohler.
TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation.
J. Immunol.
160:
943-952,
1998
36.
Ping, S. E.,
and
G. L. Barrett.
Ceramide can induce cell death in sensory neurons, whereas ceramide analogues and sphingosine promote survival.
J. Neurosci. Res.
54:
206-213,
1998[ISI][Medline].
37.
Santana, P.,
L. A. Pena,
A. Haimovitz-Friedman,
S. Martin,
D. Green,
M. McLoughlin,
C. Cordon-Cardo,
E. H. Schuchman,
Z. Fuks,
and
R. N. Kolesnick.
Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis.
Cell
86:
189-199,
1996[ISI][Medline].
38.
Shohami, E.,
I. Ginis,
and
J. M. Hallenbeck.
Dual role of tumor necrosis factor- in brain injury.
Cytokine Growth Factor Rev.
10:
119-130,
1999[ISI][Medline].
39.
Smyth, M. J.,
L. M. Obeid,
and
Y. A. Hannun.
Ceramide: a novel lipid mediator of apoptosis.
Adv. Pharmacol.
41:
133-154,
1997[Medline].
40.
Spiegel, S.
Sphingosine 1-phosphate: a prototype of a new class of second messengers.
J. Leukoc. Biol.
65:
341-344,
1999[Abstract].
41.
Tartaglia, L. A.,
and
D. V. Goeddel.
Two TNF receptors.
Immunol. Today
13:
151-153,
1992[ISI][Medline].
42.
Tasaki, K.,
C. A. Ruetzler,
T. Ohtsuki,
D. Martin,
H. Nawashiro,
and
J. M. Hallenbeck.
Lipopolysaccharide pre-treatment induces resistance against subsequent focal cerebral ischemic damage in spontaneously hypertensive rats.
Brain Res.
748:
267-270,
1997[ISI][Medline].
43.
Wiesner, D. A.,
and
G. Dawson.
Staurosporine induces programmed cell death in embryonic neurons and activation of the ceramide pathway.
J. Neurochem.
66:
1418-1425,
1996[ISI][Medline].
44.
Wityk, R. J.,
and
B. J. Stern.
Ischemic stroke: today and tomorrow.
Crit. Care Med.
22:
1278-1293,
1994[ISI][Medline].
45.
Xuan, Y. T.,
X. L. Tang,
S. Banerjee,
H. Takano,
R. C. Li,
H. Han,
Y. Qiu,
J. J. Li,
and
R. Bolli.
Nuclear factor-B plays an essential role in the late phase of ischemic preconditioning in conscious rabbits.
Circ. Res.
84:
1095-1099,
1999