Tumor Necrosis Factor Induces Bcl-2 and Bcl-x Expression through
NF
B Activation in Primary Hippocampal Neurons*
Michio
Tamatani
,
Yong Ho
Che,
Hideo
Matsuzaki,
Satoshi
Ogawa,
Haruo
Okado§,
Shin-ichi
Miyake,
Tatsunori
Mizuno, and
Masaya
Tohyama
From the Department of Anatomy and Neuroscience, Osaka University
Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0872 and the
§ Department of Neurobiology, Tokyo Metropolitan Institute
for Neuroscience, 2-6 Musashidai,
Fuchu, Tokyo 183-8526, Japan
 |
ABSTRACT |
Emerging data indicate that tumor necrosis factor
(TNF) exerts a neuroprotective effect in response to brain injury. Here we examined the mechanism of TNF in preventing neuronal death in
primary hippocampal neurons. TNF protected neurons against hypoxia- or
nitric oxide-induced injury, with an increase in the anti-apoptotic
proteins Bcl-2 and Bcl-x as determined by Western blot and reverse
transcriptase-polymerase chain reaction analysis. Treatment of neurons
with an antisense oligonucleotide to bcl-2 mRNA or that
to bcl-x mRNA blocked the up-regulation of Bcl-2 or
Bcl-x expression, respectively, and partially inhibited the neuroprotective effect induced by TNF. Moreover, adenovirus-mediated overexpression of Bcl-2 significantly inhibited hypoxia- or nitric oxide-induced neuronal death. To examine the possible involvement of a
transcription factor, NF
B, in the regulation of Bcl-2 and Bcl-x
expression in TNF-treated neurons, an adenoviral vector capable of
expressing a mutated form of I
B was used to infect neurons prior to
TNF treatment. Expression of the mutant NF
B completely inhibited
NF
B DNA binding activity and inhibited both TNF-induced
up-regulation of Bcl-2 and Bcl-x expression and neuroprotective effect.
These findings indicate that induction of Bcl-2 and Bcl-x expression
through NF
B activation is involved in the neuroprotective action of
TNF against hypoxia- or nitric oxide-induced injury.
 |
INTRODUCTION |
Acute and chronic neurodegenerative disorders are characterized by
increased levels of a variety of inflammatory cytokines. Among them,
the role of tumor necrosis factor
(TNF)1 (1) in injury
responses has been indicated (1, 2). Cell culture studies have shown
that TNF can protect cultured neurons against glucose
deprivation-induced injury and excitatory amino acid toxicity, by
stabilizing intracellular calcium concentration (1). Moreover,
experiments using mice deficient in TNF receptors demonstrated that
endogenous TNF protects neurons against ischemic and excitotoxic
insults, with induction of an antioxidative pathway (2). However, the
exact mechanisms by which TNF protects neurons are largely unknown.
Nuclear factor
B (NF
B) is a ubiquitous transcription factor that
is activated by a variety of cytokines including TNF (3, 4) and is
thought to be a key regulator of genes involved in inflammation,
response to infection, and stress. Conventional NF
B is a heterodimer
that consists of p50 and p65 subunits. The activity of NF
B is
strictly regulated by one of the I
B inhibitory proteins, such as
I
B
or I
B
, which forms a complex with NF
B and keeps
NF
B in the cytoplasm (3, 4). When cells receive signals that
activate NF
B, I
Bs are phosphorylated and degraded through a
ubiquitin/proteasome pathway (5). Coincident with I
B degradation,
activated NF
B translocates to the nucleus and participates in the
transactivation of a variety of genes, including genes for inflammatory
cytokines, chemokines, and cell adhesion molecules (5).
Recently, dominant negative mutations of I
B
that are not
phosphorylated and therefore not proteolyzed have been reported (6, 7).
One such protein is a mutant I
B
that contains serine-to-alanine
mutations at amino acids 32 and 36, which inhibit signal-induced
phosphorylation and subsequent proteasome-mediated degradation of
I
B
. This form of mutant I
B has been used to demonstrate that
inhibition of NF
B induces apoptosis through a variety of cancer
agents (8, 9).
Several pathways are emerging that depend on external stimuli to signal
key molecules in the induction or the prevention of apoptosis. One such
pathway is regulated by the expression of the proto-oncogene
bcl-2 family, whose members include bcl-2, bcl-x, and bax. Bcl-2 is an intracellular protein
that localizes to mitochondria, endoplasmic reticulum, and the nuclear
envelope (10, 11) and has been shown to enhance cell survival by
inhibiting apoptosis under diverse conditions in a variety of cell
types (12-14). The bcl-x gene also functions to regulate
cell death. Bcl-x transcripts are alternatively spliced into a long and
short form or a form lacking the transmembrane domain (15, 16). The
long form (bcl-xL) suppresses cell death, whereas the short form (bcl-xS) acts directly as a dominant
interfering bcl-2 and bcl-xL antagonist,
favoring apoptosis (15, 16). Bax forms a dimer with Bcl-2 or
Bcl-xL and prevents the death repressor activity of these
anti-apoptotic proteins (17, 18). In the nervous system, enforced
expression of Bcl-2 prevents axotomy-induced (19) and ischemia-induced
neuronal death (20), and enhanced expression of Bcl-2 and
Bcl-xL has been documented in neurons that are destined to
survive after ischemic (21) brain injury. On the other hand, Bax
expression is increased in degenerating neurons in the ischemic brain
(22). However, the mechanism by which expression of the
bcl-2 family members is controlled is unknown.
In this report, we found increased expression of Bcl-2 and Bcl-x upon
stimulating primary neurons with TNF. Furthermore, inhibition of NF
B
activation by adenovirus- mediated overexpression of the mutant form of
I
B abolished the up-regulation of Bcl-2 and Bcl-x expression and the
neuroprotective effect induced by TNF.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
Cultures of rat primary hippocampal neurons
were prepared from embryonic day 18 Sprague-Dawley rat embryos as
described previously (23) with slight modifications. Fetal hippocampi
were dissected and digested with calcium/magnesium-free Hank's
balanced salt solution (Life Technologies, Inc., Scotland) containing
0.25% trypsin for 20 min at room temperature. Tissues were dissociated by repeated trituration. The cells were seeded at a density of 1 × 106 cells/cm (2) on poly-L-lysine (10 µg/ml)-coated plates (Falcon, Lincoln Park, NJ) and maintained in
growth medium at 37 °C in a humidified atmosphere of 5%
CO2 and 95% room air. The growth medium consisted of
Dulbecco's modified Eagle's medium supplemented with 10% inactivated
fetal calf serum, 30 mM glucose, and 0.5% (v/v)
penicillin/streptomycin. To prevent growth of glial cells, cytosine
arabinoside (10 µM) was added to the cultures 48 h
after seeding. All experiments were performed in 8-10-day-old cultures.
Experimental Treatments--
The growth medium was replaced with
serum-deprived medium that consisted of Dulbecco's modified Eagle's
medium supplemented with 30 mM glucose and 0.5% (v/v)
penicillin/streptomycin immediately before exposure of cultures to
hypoxia or a NO donor, sodium nitroprusside (SNP; Sigma). Hypoxic
treatment was performed using an incubator attached to a hypoxia
chamber (Coy Laboratory Products, Ann Arbor, MI), which maintained a
humidified atmosphere with low oxygen tension
(pO2; 12-14 torr) as described previously (24).
Human recombinant TNF was purchased from Sigma. Unless otherwise
stated, TNF was added to cultures 24 h prior to the onset of
hypoxia or SNP treatment. Oligodeoxynucleotides (ODN) were purchased
from Yuki Gosei Kogyo Co. (Tokyo, Japan). The sequences of the Bcl-2 and the Bcl-x antisense ODN were 5'-TCCCGGCTTGCGCCAT-3' (25) and
5'-CTGAGACATTTTTAT-3' (26). Three different control ODN were used as
follows: the Bcl-2 sense ODN, the Bcl-x sense ODN, and mismatch control
ODN, 5'-CTGTCGCGCGCTCGACTC-3'.
Recombinant Adenovirus--
The human Bcl-2 cDNA in
pSFFV-neo expression vector, number 3088 (donated by Dr. Korsmeyer,
Washington University), was digested with EcoRI, obtaining
the 1.9-kilobase pair cDNA fragment. The obtained bcl-2
gene was cloned into a cassette cosmid, pAxCALNLw (donated by Dr.
Saito, University of Tokyo) (27). The gene should be kept silent
because of the stuffer of the neo-resistant gene and is
expected to be activated by Cre-mediated excisional deletion of the
stuffer when a sufficient amount of Cre recombinase is expressed. The
cosmid was then transfected to 293 cells together with the
EcoT221-digested adenoviral DNA-terminal protein complex (28) using the
calcium phosphate precipitation method. The desired recombinant
adenovirus (Ad) generated through homologous recombination, designated
as AxCALNLBcl-2, was purified through a CsCl2 gradient followed by extensive analysis.
The recombinant Ads AxCANCre, which efficiently produces a nuclear
localization signal-tagged Cre recombinase under control of the CAG
promoter (29, 30), and AxCALNLZ, which can express LacZ when a
sufficient amount of Cre recombinase is expressed (27), were donated by
Dr. Saito (University of Tokyo).
The recombinant Ad Ad5I
B (donated by Dr. Iimuro, Kyoto University,
Kyoto, Japan) contains the cytomegalovirus promoter, a cDNA of
mutant I
B
that contains serine-to-alanine mutations at amino
acids 32 and 36 (I
B
S32A/S36A) tagged with hemagglutinin (HA)
(31). Ad5LacZ, which contains Escherichia coli
-galactosidase gene, was used as a control virus for Ad5I
B.
All the viruses described above were grown in 293 cells and purified by
CsCl2 gradient centrifugation. Virus titers were determined by plaque assay, and concentrated virus was stored at
80 °C.
Infection was carried out by adding recombinant Ad(s) to
serum-containing medium. The cells were incubated at 37 °C for 60 min with constant agitation. The medium was changed, and the cells were
incubated at 37 °C for 24 h before treatment with TNF or exposure to hypoxia or SNP, unless otherwise stated.
Assessment of Cell Viability--
Neuronal cell viability was
assessed by the release of lactate dehydrogenase (LDH) into the culture
medium, which indicates loss of membrane integrity and cell death. LDH
activity was measured using a commercial kit (Kyokuto Chemical Co.,
Tokyo, Japan), in which a colorimetric assay measures the
pyruvate-mediated conversion of 2,4-dinitrophenylhydrazine into a
visible hydrazone precipitate. Percent neuronal viability was expressed
as (1
experimental value/maximum release) × 100, where the
maximum release was obtained after exposure of untreated control
cultures to 0.2% Triton X-100 for 15 min at 37 °C.
Analysis of DNA Fragmentation in Agarose Gel--
Cortical cells
(3 × 107) were lysed in 1 ml of DNA extraction
solution containing 20 mM Tris-HCl, pH 7.4, 0.1 M NaCl, 5 mM EDTA, and 0.5% sodium dodecyl
sulfate. The lysates were incubated with 100 µg/ml proteinase K at
37 °C for 16 h. After incubation, 1 ml of phenol/chloroform
(1:1, v/v) was mixed well with the cell lysates, which were then
centrifuged at 20,000 × g for 10 min. DNA in the
aqueous phase was incubated with 5 µg/ml DNase-free RNase A at
37 °C for 1 h and extracted with phenol/chloroform again, and
then with chloroform. DNA was collected by precipitation with 2 volumes
of absolute ethanol in the presence of 5 M NaCl. After
centrifugation, the DNA pellets were washed with 70% ethanol and
air-dried. The DNA was dissolved in 10 mM Tris-HCl and 1 mM EDTA, and its concentration was determined at 260 nm by
spectrophotometry. DNA was separated on 1.8% agarose gel containing 1 µg/ml ethidium bromide, and DNA fragments were visualized by exposing
the gel to UV light.
Western Blot Analysis--
Cell extracts for Western blot
analysis were prepared by washing the cells three times with PBS and
lysing them in sample buffer (50 mM Tris-HCl, pH 8.0, 20 mM EDTA, 1% SDS, and 100 mM NaCl). The samples
were boiled for 5 min before subjecting 20 µg of them to
electrophoresis on 12.5% SDS-PAGE gels. After the proteins were
transferred onto polyvinyl difluoride membrane (Millipore Corp.,
Bedford, MA), the membrane was incubated in blocking buffer (1× PBS,
5% nonfat dried milk) for 1 h at room temperature and then probed
with a primary antibody in blocking buffer overnight at 4 °C. The
membrane was washed four times in PBS containing 0.3% Tween 20, probed
with the secondary antibody in blocking buffer for 1 h at room
temperature, and washed again in PBS containing 0.05% Tween 20. Detection of signal was performed with an enhanced chemiluminescence
detection kit (Amersham Corp., Little Chalfont, UK). The primary
antibodies used were anti-rat Bcl-2 monoclonal antibody (MBL, Nagoya,
Japan), rat Bcl-x monoclonal antibody (Transduction Laboratories, Inc.,
Lexington, KY), anti-mouse Bax polyclonal antibody (P-19, Santa Cruz
Biotechnology, Santa Cruz, CA), anti-mouse I
B polyclonal antibody
(Santa Cruz Biotechnology), and anti-mouse
-actin monoclonal
antibody (Sigma).
RNA Isolation and RT-PCR--
Total RNA (derived from 2 × 107 cells) was extracted from rat primary neurons by means
of the acid guanidinium-thiocyanate phenol chloroform method (32). For
RT-PCR, 1 µg of total RNA was reverse-transcribed using oligo(dT)
(Pharmacia Biotech, Uppsala, Sweden) and Moloney murine leukemia virus
reverse transcriptase (Applied Biosystems, Foster City, CA) in a volume
of 25 µl. For PCR amplification, specific oligonucleotide primer
pairs (0.5 µM each) were incubated with 5 µl of
cDNA template in a 50-µl PCR reaction mixture containing 1.5 mM MgCl2, 25 mM KCl, 10 mM Tris, pH 9.2, 1 µl of deoxynucleotides (1 mM each), and 1 unit of Taq polymerase. The
sequences of primers used in this study were as follows: Bcl-2 sense
primer, 5'-CTGGTGGACAACATCGCTCTG-3', and Bcl-2 antisense primer,
5'-GGTCTGCTGACCTCACTTGTG-3'; Bcl-x sense primer,
5'-AGGCTGGCGATGAGTTTGAA-3', and Bcl-x antisense primer,
5'-CGGCTCTCGGCTGCTGCATT-3'; Bax sense primer,
5'-TGGTTGCCCTTTTCTACTTTG-3', and Bax antisense primer,
5'-GAAGTAGGAAAGGAGGCCATC-3';
-actin sense primer,
5'-TGCCCATCTATGAGGGTTACG-3', and
-actin antisense primer,
5'-TAGAAGCATTTGCGGTGCACG-3'. Dilutions of cDNA were amplified for
26-28 cycles at 94 °C for 30 s, 55 °C for 30 s, and
72 °C for 30 s. The amplified PCR products were analyzed by 5%
polyacrylamide gel electrophoresis and ethidium bromide staining. The
product of constitutively expressed
-actin mRNA served as the
control. All the products were assayed in the linear response range of the RT-PCR amplification process; the cycle number used was determined by finding the midpoint of linear amplification on a sigmoid curve for
both amplification products with cycle numbers of 24-30 plotted against band density. The identity of each PCR product was confirmed by
subcloning the amplified cDNAs into the pGEM-T vector (Promega) and sequencing.
Electrophoretic Mobility Shift Assay--
Nuclear extracts were
prepared from primary neuronal cultures according to published methods
(33, 34) with some modifications. In brief, cells were harvested by
scraping and washing in 0.5 volumes of cold PBS. The cells were then
washed once in 0.1 volumes of cold buffer A (10 mM HEPES,
pH 7.9, 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM dithiothreitol). The washed cell pellets were
then suspended in 50 µl of buffer A plus 0.1% Nonidet P-40
supplemented with 1 µg/ml leupeptin and aprotinin and were incubated
on ice for 10 min. After incubation, the pellets were mixed briefly by vortexing and were centrifuged at 10,000 rpm at 4 °C for 5 min in a
microcentrifuge. The supernatant was carefully removed, and the nuclear
pellet was resuspended in 20 µl of cold buffer C (20 mM
HEPES, 25% glycerol, 0.42 M NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM
dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride) containing 1 µg/ml leupeptin and aprotinin and incubated on ice for
15 min with intermittent vortexing. The extracts were then centrifuged
at 10,000 rpm at 4 °C for 10 min, and the supernatant was divided
into aliquots and frozen at
70 °C. Protein concentrations were
determined using the Bio-Rad protein assay kit.
A double-stranded oligonucleotide containing the sequence corresponding
to the classical NF
B consensus site (5'-AGTTGAGGGGACTTTCCCAGCC-3', Santa Cruz Biotechnology) was end-labeled with
[
-32P]ATP using T4 kinase (Life Technology, Inc.).
Unincorporated nucleotides were removed using two Sephadex G-50 columns
(Amersham Pharmacia Biotech). Binding reactions were carried out in a
final volume of 25 µl consisting of 10 mM HEPES, pH 7.9, 4 mM Tris-HCl, 60 mM KCl, 1 mM
EDTA, 1 mM dithiothreitol, 10% glycerol, 1.5 mg/ml bovine
serum albumin, 2 µg of poly(dI-dC), 2-10 µg of nuclear extract,
and 0.5 ng of 32P-labeled oligonucleotide probe (50,000 cpm). Reactions were incubated for 20 min at room temperature. Binding
reactions were subjected to nondenaturing polyacrylamide
electrophoresis through 4% gels in a 1× Tris borate-EDTA buffer
system. Gels were dried and subjected to autoradiography.
Statistical Analysis--
Results are presented as mean ± S.E. Experimental groups were compared by one-way or two-way ANOVA,
followed by Scheffe's post hoc test. p values less than
0.05 were considered significant.
 |
RESULTS |
TNF Protects Neurons against Hypoxia or Nitric Oxide--
Previous
studies have shown that cultured neurons are vulnerable to killing by
hypoxia or nitric oxide (NO), with features of apoptosis (35, 36).
Exposure for 24 h to hypoxia or 50 µM sodium
nitroprusside (SNP) resulted in neuronal survival of 30-50% (Fig.
1), in agreement with previous reports
(35, 36). To examine the effect of TNF, neurons were pretreated with
increasing concentrations of TNF for 24 h and then exposed for
24 h to hypoxia or 50 µM SNP. Pretreatment with TNF
dose-dependently increased survival of neurons exposed to
hypoxia or SNP; 100 ng/ml TNF rescued 54 or 50% of cells from death
induced by hypoxia or SNP, respectively (Fig. 1A).
Consistent with these results, pretreatment with TNF (100 ng/ml)
attenuated DNA laddering induced by hypoxia or SNP (Fig.
1B).

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 1.
TNF protects cultured hippocampal neurons
against hypoxia and SNP. A, cultured neurons were
preincubated for 24 h with vehicle (saline) or the indicated
concentrations of TNF and then were exposed to either normoxia
(control), hypoxia, or 50 µM SNP. Neuronal survival was
examined 24 h later by LDH assay. Data are mean ± S.E. for
three independent experiments performed in triplicate.
*p < 0.05 versus TNF = 0 value; two-way ANOVA followed by Scheffe's post hoc test.
B, agarose gel electrophoresis of oligonucleosomal DNA
fragments (DNA laddering). Neurons pretreated with either vehicle
or TNF (100 ng/ml) were exposed to hypoxia or 50 µM SNP
for 24 h, and then DNA extracted from neuron cultures was
subjected to conventional agarose gel electrophoresis.
MK, 1-kilobase pair ladder molecular weight marker.
|
|
TNF Induces Expression of Bcl-2 and Bcl-x--
We previously
characterized neuronal death induced by hypoxia and nitric oxide and
demonstrated decreased Bcl-2 protein levels in neurons accompanied by
neuronal apoptosis (35, 36). In addition, Furukawa et al.
(25) demonstrated that cycloheximide exerted a neuroprotective action
by inducing Bcl-2 expression. Therefore, we examined whether the
neuroprotective effect of TNF involves induction of Bcl-2 and related
proteins, Bcl-x and Bax. Western blot analysis showed that TNF induced
a concentration-dependent increase in Bcl-2 and Bcl-x
protein levels, whereas the expression of Bax protein was not induced
by TNF (Fig. 2A). Bcl-x
immunoreactivity was evident as a single band of 26 kDa, the predicted
size of Bcl-xL protein, and the antibody did not detect bands at the corresponding size of Bcl-xS or Bcl-xbo protein
in any sample tested. RT-PCR analysis revealed increased expression of
bcl-2 and bcl-x mRNA upon stimulation with
TNF in a dose-dependent manner, whereas expression of
bax mRNA was unchanged (Fig. 2B). As for the
isoform of bcl-x in rat hippocampal neurons, only a fragment
337 base pairs in size, corresponding to the bcl-xL isoform, was obtained by PCR at 30 cycles in the presence or absence of
TNF stimulation. A fragment 150 base pairs in size, corresponding to
bcl-xS, was not detected (data not shown). The time course of changes in Bcl-2, Bcl-x, and Bax protein levels after exposure to 100 ng/ml TNF is shown in Fig. 2C. Both the
Bcl-2 and Bcl-x protein levels increased within 6 h, peaked by
18 h, and remained elevated at 24 h. The Bax protein level
was unchanged by 100 ng/ml TNF up to 24 h of examination (Fig.
2C).

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 2.
TNF induces expression of Bcl-2 and Bcl-x but
not Bax. Hippocampal cultures were exposed for 24 h to the
indicated concentrations of TNF (A and C) or to
100 ng/ml TNF for the indicated times (B). A and
B, extracts of neurons were subjected to SDS-PAGE, and the
blots were probed with antibodies to Bcl-2, Bcl-x, Bax or -actin.
Visualization of the proteins was performed with ECL. Arrow indicates
Bax immunoreactivity at the predicted molecular size. C,
total RNA was isolated, and bcl-2, bcl-x,
bax and -actin mRNA were amplified by RT-PCR.
Resulting PCR products were separated by 5% polyacrylamide gel
electrophoresis and visualized by ethidium bromide staining. Results
were confirmed in three repeated assays.
|
|
Antisense Oligonucleotides against bcl-2 and/or bcl-x mRNA
Inhibit Neuroprotective Effect of TNF--
To explore the role of
TNF-induced up-regulation of Bcl-2 and Bcl-x expression in neuronal
survival, antisense ODN for Bcl-2 and Bcl-x was employed. We showed
that treatment of neurons with 10 µM Bcl-2 or Bcl-x
antisense ODN for 24 h in the presence of TNF almost completely
blocked TNF-induced up-regulation of Bcl-2 or Bcl-x expression,
respectively, as determined by Western blot analysis (Fig.
3A). Addition of both Bcl-2
and Bcl-x antisense ODN at a concentration of 10 µM
inhibited the up-regulation of both Bcl-2 and Bcl-x expression induced
by TNF (Fig. 3A). Then the effect of Bcl-2 and/or Bcl-x
antisense ODN(s) or mismatch control ODN on cell survival in
TNF-treated neurons exposed for 24 h to hypoxia or SNP was
examined. The Bcl-2 or Bcl-x antisense ODN (10 µM)
significantly reduced the neuroprotective effect of TNF-
in cells
exposed for 24 h to hypoxia or 50 µM SNP (Fig. 3B). In addition, treatment with both the Bcl-2 and Bcl-x
antisense ODN more effectively inhibited TNF-induced neuroprotection
than either of the antisense ODN alone (Fig. 3B).
Administration of mismatch control ODN (Fig. 3, A and
B) or Bcl-2 or Bcl-x sense ODN (data not shown) at 10 µM, which did not affect the up-regulation of Bcl-2 and
Bcl-x expression induced by TNF, had no effect on the neuroprotective
effect of this cytokine.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of Bcl-2 and Bcl-x antisense
oligonucleotide on Bcl-2 and Bcl-x expression and cell survival in
TNF-treated hippocampal neurons. A, cultures were
exposed for 24 h to 10 µM Bcl-2 antisense ODN
(B2AS), 10 µM Bcl-x antisense ODN
(BXAS), 10 µM Bcl-2 antisense ODN, plus 10 µM Bcl-x antisense ODN (B2AS + BXAS), or 10 µM mismatch control ODN (MC) in the presence
or absence of 100 ng/ml TNF. Extracts from the cells were subjected to
SDS-PAGE, and the blots were probed with antibodies to Bcl-2, Bcl-x, or
-actin. B, hippocampal cultures pretreated for 24 h
with Bcl-2 antisense ODN (B2AS), Bcl-x antisense ODN
(BXAS), Bcl-2 antisense ODN plus Bcl-x antisense ODN
(B2AS + BXAS), or mismatch control ODN (MC) in
the presence or absence of TNF were exposed for 24 h to hypoxia or
50 µM SNP, and then neuronal survival was determined by
LDH assay. Data are mean ± S.E. for five independent experiments
performed in triplicate. *, p < 0.05 versus
vehicle and , p < 0.05 versus TNF in each
of the three groups (control, hypoxia, and SNP); ANOVA followed by
Scheffe's post hoc test.
|
|
Adenovirus-mediated Overexpression of Bcl-2 Protects Neurons
against Hypoxia or Nitric Oxide--
Adenoviral vectors capable of
expressing Bcl-2 in hippocampal neurons were obtained to analyze
further the effect of Bcl-2 expression on hypoxia- or NO-mediated
neurotoxicity. We attempted to create a Bcl-2-expressing adenoviral
vector under the control of CAG promoter; however, we were unable to
obtain such a vector, perhaps because Bcl-2 expressed in 293 cells
after homologous recombination might inhibit cell death of 293 cells
and thereby suppress proliferation of the virus. Therefore,
AxCALNLBcl-2, which is expected to express Bcl-2 only when a sufficient
amount of Cre recombinase is expressed, was generated. Primary
hippocampal neurons were infected with AxCANCre and AxCALNLBcl-2 or
AxCALNLZ, a control virus carrying
-galactosidase instead of
bcl-2, and whole cell extracts were prepared for Western
blot analysis. As shown in Fig.
4A, an increased level of
Bcl-2 was detected in neurons infected simultaneously with AxCANCre at
multiplicity of infection (m.o.i.) of 2 (2 m.o.i.) and AxCANBcl-2 (10 m.o.i.) at 24 h after infection, whereas cells infected with
AxCANCre (2 m.o.i.) and AxCALNLZ (10 m.o.i.) did not show increased
protein levels. Twenty-four hours after viral infection, the cultures were exposed to hypoxia or 50 µM SNP for 24 h, and
then neuronal viability was determined. Infection with AxCANCre and
AxCALNLZ did not affect cell viability in neurons exposed to either
normoxia, hypoxia, or SNP (Fig. 4B). In contrast, neurons
infected with AxCANCre and AxCALNLBcl-2 exhibited a 47% reduction of
hypoxia-induced cell death and a 38% reduction of cell death induced
by SNP, although it did not affect the survival of normoxic neurons
(Fig. 4B).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 4.
Overexpression of Bcl-2 protects hippocampal
neurons against hypoxia- and nitric oxide-induced toxicity.
Primary hippocampal neurons were infected with AxCANCre (2 m.o.i.) and
AxCALNLBcl-2 (10 m.o.i.) or AxCALNLZ (10 m.o.i.). A,
extracts from neuronal cultures 24 h after infection were
subjected to gel electrophoresis, and the blots were probed with
antibodies to Bcl-2. B, at 24 h after viral infection,
the cultures were exposed to hypoxia or 50 µM SNP for
24 h, and then the neuronal viability was determined by LDH assay.
Data are mean ± S.E. for three independent experiments performed
in triplicate. *, p < 0.05 versus vehicle
in each of the three groups (control, hypoxia, and SNP); ANOVA followed
by Scheffe's post hoc test.
|
|
Assessment of Recombinant Adenovirus Expressing I
B Super
Repressor--
The super repressor I
B contains serine-alanine
mutations in residues 32 and 36, which inhibit its phosphorylation and
proteosome-mediated degradation (8, 9). An HA-tagged version of this
B S32A/S36A driven by the cytomegalovirus promoter enhancer into the
replication-deficient adenovirus Ad5 (Ad5I
B) was generated by Iimuro
et al. (31). Primary cortical neurons were infected with
Ad5I
B for 24 h at various m.o.i., and whole cell extracts were
prepared for Western blot analysis. HA-tagged I
B was identified by
its slower mobility compared with endogenous I
B by Western blotting
using an anti-I
B antibody, documenting the expression of this
cDNA by the adenoviral vector (Fig.
5A).

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 5.
Expression of I Ba
S32A/S36A inhibits TNF-induced up-regulation of Bcl-2 expression and
the protective effect of TNF against neurotoxicity induced by hypoxia
or SNP. A, extracts from neuronal cultures 24 h
after infection with Ad5I B at various m.o.i. were subjected to gel
electrophoresis, and the blots were probed with antibodies to I B.
HA-tagged I B was identified by its slower mobility compared with
endogenous I B . B, NF B DNA binding activity was
assessed by electrophoretic mobility shift assay using NF B
oligonucleotide probe with nuclear extracts prepared from neurons
incubated for 1 h with or without 100 ng/ml TNF. Neurons were
infected or not infected with Ad5I B or Ad5LacZ at m.o.i. 20 at
24 h before TNF treatment. C, cultures were infected or
not infected with Ad5I B or Ad5LacZ (m.o.i. 20) and exposed for
24 h to vehicle or 100 ng/ml TNF. Extracts from the cells were
subjected to SDS-PAGE, and the blots were probed with antibodies to
Bcl-2, Bcl-x, or -actin. D, at 24 h after infection
with Ad5I B or Ad5LacZ (m.o.i. 20), cultures were treated for 24 h with vehicle (saline) or 100 ng/ml TNF in the presence or absence of
Bcl-2 and Bcl-x antisense ODN at 10 µM each (B2AS + BXAS), and then exposed for 24 h to hypoxia or 50 µM
SNP. Neuronal survival was then determined by LDH assay. Data are
mean ± S.E. for five independent experiments performed in
triplicate. *, p < 0.05 versus vehicle and
, p < 0.05 versus TNF in each of the
three groups (control, hypoxia, and SNP); ANOVA followed by Scheffe's
post hoc test.
|
|
In order to demonstrate that expression of mutant I
B effectively
inhibited NF
B DNA binding activity in TNF-treated neurons, electrophoretic mobility shift assays were performed on crude nuclear
extracts derived from cells that were infected with Ad5I
B or control
adenovirus Ad5LacZ at m.o.i. of 20 and then 24 h later were
treated with TNF for 1 h. Inhibition of NF
B DNA binding activity was demonstrated with Ad5I
B, but not with Ad5LacZ (Fig. 5B).
These results indicate that expression of the mutant I
B protein in
neurons potently inhibits TNF-induced NF
B DNA binding activity.
I
B Super Repressor Inhibits TNF-
-induced Up-regulation of
Bcl-2 and Bcl-x Expression and Neuroprotective Effect--
In order to
determine whether NF
B activation involves TNF-induced up-regulation
of Bcl-2 and Bcl-x expression, we examined the expression of these
anti-apoptotic proteins in neurons expressing the mutant I
B in
response to treatment with TNF. Neurons that were infected for 24 h with Ad5I
B or Ad5LacZ at m.o.i. of 20 were treated with 50 ng/ml
TNF-
for 24 h. Cells were then analyzed for Bcl-2 expression by
Western blot. Western blot analysis showed that in neurons expressing
mutant I
B, TNF-induced up-regulation of Bcl-2 and Bcl-x protein
levels was inhibited to a similar level as observed in control
untreated neurons (Fig. 5C). In neurons infected with
Ad5LacZ, however, TNF up-regulated Bcl-2 and Bcl-x protein levels to a
similar level as that in control TNF-treated cells (Fig.
5C).
Next, the effect of mutant I
B expression on TNF-induced neuronal
survival in comparison with the effect of simultaneous treatment with
Bcl-2 and Bcl-x antisense ODN was examined. Infection with Ad5I
B,
but not with Ad5LacZ, almost completely inhibited the neuroprotective
effect of TNF against hypoxia- and NO-induced toxicity (Fig.
5D). The inhibition of TNF-induced neuroprotective effect by
treatment with Ad5I
B was significantly more potent than that with
Bcl-2 and Bcl-x antisense ODN (p < 0.05) (Fig. 5D).
 |
DISCUSSION |
Previous studies have demonstrated that overexpression of Bcl-2 in
neurons can prevent or delay cell death induced by a variety of stimuli
in vitro (37, 38) and in vivo (19, 20).
Overexpression of Bcl-xL also protects sympathetic neurons from
apoptosis induced by nerve growth factor deprivation (39). In
addition, we previously showed decreased levels of Bcl-2 expression in
hypoxia- and nitric oxide-induced neuronal apoptosis (35, 36).
Furthermore, Bcl-2 and Bcl-x are induced primarily in neurons destined
to survive following focal ischemia (22) and global ischemia (21).
Thus, it is likely that the endogenous levels of Bcl-2 and Bcl-x
regulate neuronal survival. Our present data demonstrated that TNF
protects neurons against hypoxia- and nitric oxide-induced toxicity and up-regulates Bcl-2 and Bcl-x expression. Pretreatment of neurons with
Bcl-2 or Bcl-x antisense ODN which inhibits up-regulation of Bcl-2 or
Bcl-x expression, respectively, significantly inhibits the
neuroprotective effect induced by TNF, indicating that either Bcl-2 and
Bcl-x induction is involved in the neuroprotective action of this
cytokine. Simultaneous addition of both the antisense ODN, which
inhibits up-regulation of Bcl-2 and Bcl-x expression, seems to
additively but not synergistically inhibit TNF-induced neuroprotection.
Evidence taken from previous studies suggests that Bax actively
promotes cell death, unless it is bound by either Bcl-2 or
Bcl-xL (17, 18). Thus, the ratio of Bax to Bcl-2 and Bcl-x
seems to be critical for cell survival. Therefore, TNF-induced
expression of Bcl-2 and Bcl-x might antagonize the pro-apoptotic
function of Bax, whose expression was unchanged by TNF. Moreover, the
present finding that overexpression of Bcl-2 protects neurons supports
this anti-apoptotic protein as a critical factor for neuronal
protection against hypoxia- or nitric oxide-induced injury. Production
of TNF has been investigated in an ischemic brain injury model (40), in
which the cytokine is rapidly synthesized in the brain. Enhanced
expression of Bcl-2 and Bcl-x has also been documented in neurons that
are destined to survive after ischemic brain injury (21). Therefore, it
might be that TNF produced by resident brain cells up-regulates Bcl-2
and Bcl-x expression in neurons and thereby protects them from ischemic injury.
Most cells including neurons express specific receptors for TNF. Two
receptors have been characterized and designated TNF receptor 1 (TNFR1)
and TNF receptor 2 (TNFR2). In the present study, human TNF, which
activates TNFR1 but not TNFR2 in rodent cells (41), induced Bcl-2 and
Bcl-x expression and protected hippocampal neurons against hypoxia- and
NO-induced cytotoxicity. TNFR1 is linked to a signal transduction
pathway involving a sphingomyelinase that induces release of ceramide,
which then results in activation of the transcription factor NF
B
(42). We here demonstrated that TNF-
induced NF
B activity in
cultured hippocampal neurons as determined by gel shift assay, in
agreement with a previous report by Mattson et al. (43). Our
experiments using Ad5I
B showed that inhibition of NF
B activation
completely abolished the neuroprotective effect and inhibited the
induction of Bcl-2 and Bcl-x expression in TNF-treated cells. These
results indicate that the neuroprotective effect and up-regulation of
Bcl-2 and Bcl-x expression induced by TNF are mediated by activation of NF
B.
The relation between NF
B and apoptosis was elucidated recently by
studies demonstrating that inhibition of NF
B, either by the I
B
super repressor (8, 9) or in Rel A (p65) knock-out cells (44), results
in increased apoptosis. Contrary to these reports, it has been shown
that NF
B activation induces apoptosis in certain cells (45, 46)
including bone marrow cells and leukemia cells. It is not known at
present if NF
B activation is involved in promoting apoptosis in some
cells and if it is responsible for preventing apoptosis in other cells.
Regarding neuronal cells, it has been reported that activation of
NF
B protected hippocampal neurons against oxidative stress-induced
apoptosis, using
B decoy DNA approach (43), and that inhibition of
NF
B in PC12 cells by exposure to
B decoy DNA or
pyrrolidinedithiocarbamate induced apoptosis that could not be
prevented by nerve growth factor (47). These findings, together with
our present observations, indicate that the NF
B signaling cascade is
closely involved in prevention of apoptosis in neuronal cells.
Based on our current understanding of the roles of NF
B activation in
the regulation of apoptosis, gene expression of proteins that exert
anti-apoptotic effects may be involved. We demonstrated here that
up-regulation of Bcl-2 and Bcl-x expression induced by TNF is
completely inhibited by I
B super repressor, indicating that Bcl-2
and Bcl-x are among the gene targets of NF
B. The bcl-2 and bcl-x genes have complex structures, and the promoters
have not been well characterized. Recently, the presence of NF
B site has been demonstrated in the promoters of human bcl-2 and
bcl-x (48). Therefore, the regulation of Bcl-2 and Bcl-x
expression that we have observed to be mediated by NF
B in neurons is
probably related to an analogous site in the rat bcl-2 and
bcl-x promoter, respectively.
In addition to bcl-2 and bcl-x, expression of
other genes may be induced by TNF through NF
B activation. Recently,
it has been reported that the expression of manganese superoxide
dismutase is induced by TNF, which is abolished by
B decoy DNA, in
hippocampal neurons (43). Moreover, in the present study, the
neuroprotective effect of TNF was blocked more potently by I
B super
repressor than by antisense ODN against bcl-2 and
bcl-x, in both of which TNF-induced up-regulation of Bcl-2
and Bcl-x expression was completely blocked. This suggests that other
proteins that exert a cytoprotective effect may be induced by NF
B
activation in TNF-treated neurons.
In summary, our findings demonstrated that induction of Bcl-2 and Bcl-x
expression through NF
B activation is involved in the neuroprotective
effect of TNF. In addition, our finding that inhibition of NF
B
activation by expression of a mutant form of I
B more potently
abolishes the neuroprotective effect of TNF than inhibition of
TNF-induced up-regulation of Bcl-2 and Bcl-x expression by the
antisense ODN suggests that other genes responsible for anti-apoptotic
activity are induced by NF
B activation. Identification of the genes
will certainly help to understand the regulation of neuronal survival
and to design new therapeutic strategies for acute and chronic
neurodegenerative disorders.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Y. Iimuro (Kyoto University,
Japan) for providing Ad5I
B; Drs. Saito and Kanegae (University of
Tokyo, Japan) for providing pAxCALNLw, AxCANCre, and AxCALNLZ;
Dr. Miwa (Tokyo Metropolitan Institute for Neuroscience) for
purification of adenovirus and determining virus titers; and Dr.
Korsmeyer (Washington University) for providing the human Bcl-2
cDNA vector, number 3088.
 |
FOOTNOTES |
*
This work was supported by a grant-in-aid from the Ministry
of Education, Science and Culture of Japan.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. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Anatomy and
Neuroscience, Osaka University Medical School, 2-2 Yamadaoka, Suita,
Osaka 565, Japan. Tel.: 81-6-879-3221; Fax: 81-6-879-3229; E-mail:
tama{at}anat2.med.osaka-u.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
TNF, tumor necrosis
factor;
Ad, adenovirus;
HA, hemagglutinin;
LDH, lactate dehydrogenase;
m.o.i., multiplicity of infection;
NO, nitric oxide;
NF
B, nuclear
factor
B;
PAGE, polyacrylamide gel electrophoresis;
PBS, phosphate-buffered saline;
RT-PCR, reverse transcriptase-polymerase
chain reaction;
SNP, sodium nitroprusside;
TNFR1, tumor necrosis factor
receptor 1;
TNFR2, tumor necrosis factor receptor 2;
ANOVA, analysis of
variance;
ODN, oligodeoxynucleotides.
 |
REFERENCES |
-
Cheng, B.,
Christakos, S.,
and Mattson, M. P.
(1994)
Neuron
12,
139-153[Medline]
[Order article via Infotrieve]
-
Bruce, A. J.,
Boling, W.,
Kindy, M. S.,
Peschon, J.,
Kraemer, P. J.,
Carpenter, M. K.,
Holtsberg, F. W.,
and Mattson, M. P.
(1996)
Nat. Med.
2,
788-794[Medline]
[Order article via Infotrieve]
-
Siebenlist, U.,
Franzoso, G.,
and Brown, K.
(1994)
Annu. Rev. Cell Biol.
10,
405-455[CrossRef]
-
Baldwin, A. S.
(1994)
Annu. Rev. Immunol.
14,
649-681[CrossRef][Medline]
[Order article via Infotrieve]
-
Baeurle, P. A.,
and Baltimore, D.
(1996)
Cell
87,
13-20[Medline]
[Order article via Infotrieve]
-
Brockmann, J. A.,
Scherr, D. C.,
McKinsy, T. A.,
Hall, S. M.,
Qi, X.,
Le, W. Y.,
and Ballard, D. W.
(1995)
Mol. Cell. Biol.
15,
2809-2818[Abstract]
-
Traenckner, E. B. M.,
Pahl, H. L.,
Henkel, T.,
Schmidt, K. N.,
Wilk, S.,
and Baeuerle, P. A.
(1995)
EMBO J.
14,
2876-2883[Abstract]
-
Wang, C. Y.,
Mayo, M. W.,
and Baldwin, A. S., Jr.
(1996)
Science
274,
784-787[Abstract/Free Full Text]
-
Van Antwerp, D. J.,
Martin, S. J.,
Kafri, T.,
Green, D. R.,
and Verma, I. M.
(1996)
Science
274,
787-789[Abstract/Free Full Text]
-
Monaghan, P.,
Robertson, D.,
Amos, T. A. S.,
Dyer, M. J. S.,
Mason, D. Y.,
and Greaves, M. F.
(1992)
J. Histochem. Cytochem.
40,
1819-1825[Abstract/Free Full Text]
-
Krajewski, S.,
Tanaka, S.,
Takayama, S.,
Schreiber, R. D.,
and Korsmeyr, S. J.
(1993)
Cancer Res.
53,
4701-4714[Abstract]
-
Vaux, D. L.,
Cory, S.,
and Adams, J. M.
(1988)
Nature
335,
440-442[CrossRef][Medline]
[Order article via Infotrieve]
-
Hockenbery, D. M.,
Nunez, G.,
Milliman, C.,
Schriber, R. D.,
and Korsmeyer, S. J.
(1990)
Nature
348,
334-336[CrossRef][Medline]
[Order article via Infotrieve]
-
Sentman, C. L.,
Shutter, J. R.,
Hockenberry, D.,
Kanagawa, O.,
and Korsmeyer, S. J.
(1991)
Cell
67,
879-888[Medline]
[Order article via Infotrieve]
-
Boise, L. H.,
Gonzalez-Garcia, M.,
Postema, C. E.,
Ding, L.,
Lindsten, T.,
Turka, L. A.,
Mao, X.,
Nunez, G.,
and Thompson, C. B.
(1993)
Cell
74,
597-608[Medline]
[Order article via Infotrieve]
-
Fang, W.,
Rivard, J. J.,
Mueller, D. L.,
and Behrens, T. W.
(1994)
J. Immunol.
153,
4388-4398[Abstract/Free Full Text]
-
Sato, T.,
Hanada, M.,
Bodrug, S.,
Iries, S.,
Iwama, N.,
Boise, L. H.,
Thompson, C. B.,
Golemis, E.,
Fong, L.,
Wang, H. C.,
and Reed, J. C.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
94,
5113-5118[Abstract/Free Full Text]
-
Yin, X. M.,
Oltvai, Z. N.,
and Korsmeyer, S. J.
(1994)
Nature
369,
321-323[CrossRef][Medline]
[Order article via Infotrieve]
-
Dubois-Dauphin, M.,
Frankowski, H.,
Tsujimoto, Y.,
Huarte, J.,
and Martinou, J. C.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
3309-3313[Abstract]
-
Martinou, J. C.,
Dubois-Dauphin, M.,
Staple, J. K.,
Rodriguez, I.,
Frankowski, H.,
Missotten, M.,
Albertini, P.,
Talabot, D.,
Catsicas, S.,
Pietra, C.,
and Huarte, J.
(1994)
Neuron
13,
1017-1030[Medline]
[Order article via Infotrieve]
-
Chen, J.,
Graham, S. H.,
Nakayama, M.,
Zhu, R. L.,
Jin, K.,
Stetler, A.,
and Simon, R. P.
(1997)
J. Cereb. Blood Flow Metab.
17,
2-10[Medline]
[Order article via Infotrieve]
-
Isenmann, S.,
Stoll, G.,
Schroeter, M.,
Krajewski, S.,
Reed, J. C.,
and Bahr, M.
(1998)
Brain Pathol.
8,
49-63[Medline]
[Order article via Infotrieve]
-
Murphy, T. H.,
Schnaar, R. L.,
and Coyle, J. T.
(1990)
FASEB J.
4,
1624-1633[Abstract/Free Full Text]
-
Ogawa, S.,
Herwig, G.,
Esposito, C.,
Macaulay, A. P.,
Brett, J.,
and Stern, D.
(1990)
J. Clin. Invest.
85,
1090-1098[Medline]
[Order article via Infotrieve]
-
Furukawa, K.,
Estus, S.,
Fu, W.,
Mark, R. J.,
and Mattson, M. P.
(1997)
J. Cell Biol.
136,
1137-1149[Abstract/Free Full Text]
-
Wang, Z.,
Karras, J. G.,
Howard, R. G.,
and Rothstein, T. L.
(1995)
J. Immunol.
155,
3722-3725[Abstract]
-
Kanegae, Y.,
Lee, G.,
Sato, Y.,
Tanaka, M.,
Nakai, M.,
Sakai, T.,
Sugano, S.,
and Saito, I.
(1995)
Nucleic Acids Res.
23,
3816-3821[Abstract]
-
Miyake, S.,
Makimura, M.,
Kanegae, Y.,
Harada, S.,
Sato, T.,
Takamori, K.,
Tokuda, C.,
and Saito, I.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
1320-1324[Abstract/Free Full Text]
-
Niwa, H.,
Yamamura, K.,
and Miyazaki, J.
(1991)
Gene (Amst.)
108,
193-200[CrossRef][Medline]
[Order article via Infotrieve]
-
Okuyama, T.,
Fujino, M.,
Li, X. L.,
Funeshima, N.,
Kosuga, M.,
Saito, I.,
Suzuki, S.,
and Yamada, M.
(1998)
Gene Ther.
5,
1047-1053[CrossRef][Medline]
[Order article via Infotrieve]
-
Iimuro, Y.,
Nishiura, T.,
Hellerbrand, C.,
Behrns, K.,
Schoonhoven, R.,
Grisham, J. W.,
and Brenner, D. A.
(1998)
J. Clin. Invest.
101,
802-811[Abstract/Free Full Text]
-
Chomczynski, P.,
and Sacchi, N.
(1987)
Anal. Biochem.
162,
156-159[CrossRef][Medline]
[Order article via Infotrieve]
-
Dignam, J. D.,
Lebovitz, R. M.,
and Roeder, R. G.
(1983)
Nucleic Acids Res.
11,
1475-1489[Abstract]
-
Osborn, L.,
Kunkel, S.,
and Nabel, G. J.
(1989)
Proc. Natl. Acad. Sci. U. S. A.
86,
2336-2340[Abstract]
-
Tamatani, M.,
Ogawa, S.,
Nunez, G.,
and Tohyama, M.
(1998)
Cell Death Differ.
5,
911-919[CrossRef][Medline]
[Order article via Infotrieve]
-
Tamatani, M.,
Ogawa, S.,
and Tohyama, M.
(1998)
Mol. Brain Res.
58,
27-39[CrossRef][Medline]
[Order article via Infotrieve]
-
Garcia, I.,
Martinou, I.,
Tsujimoto, Y.,
and Martinou, J. C.
(1992)
Science
258,
302-304[Medline]
[Order article via Infotrieve]
-
Allsopp, T. E.,
Wyatt, S.,
Paterson, H. F,
and Davies, A. M.
(1993)
Cell
73,
295-307[Medline]
[Order article via Infotrieve]
-
Frankowski, H.,
Missotten, M.,
Fernandez, P. A.,
Martinou, I.,
Michel, P.,
Sadoul, R.,
and Martinou, J. C.
(1995)
Neuroreport
6,
1919-1921
-
Uno, H.,
Matsuyama, T.,
Akita, H.,
Nishimura, H.,
and Sugita, M.
(1997)
J. Cereb. Blood Flow Matab.
17,
491-499
-
Barbara, J. A.,
Smith, W. B.,
Gamble, J. R.,
Ostade, X., V.,
Vandenable, P.,
Tavernier, J.,
Fiers, W.,
Vadas, M., A.,
and Lopez, A. F.
(1994)
EMBO J.
13,
843-850[Abstract]
-
Kolesnick, R.,
and Golde, D, W.
(1994)
Cell
77,
325-328[Medline]
[Order article via Infotrieve]
-
Mattson, M. P.,
Goodman, Y.,
Luo, H.,
Fu, W.,
and Furukawa, K.
(1997)
J. Neurosci. Res.
49,
681-697[CrossRef][Medline]
[Order article via Infotrieve]
-
Beg, A. A.,
and Baltimore, D.
(1996)
Science
274,
782-784[Abstract/Free Full Text]
-
Bessho, R.,
Matsubara, K.,
Kubota, M.,
Kuwakado, K.,
Hirota, H.,
Wakazono, Y.,
Lin, Y. W.,
Okuda, A.,
Kawai, M.,
Nishikomori, R.,
and Heike, T.
(1994)
Biochem. Pharmacol.
48,
1883-1889[CrossRef][Medline]
[Order article via Infotrieve]
-
Abbadie, C.,
Kabrun, N.,
Bouali, F.,
Smardova, J.,
Stehelin, D.,
Vandenbunder, B.,
and Enrietto, P. J.
(1993)
Cell
75,
899-912[Medline]
[Order article via Infotrieve]
-
Taglialatela, G.,
Robinson, R.,
and Perez-Polo, J. R.
(1997)
J. Neurosci. Res.
47,
155-162[CrossRef][Medline]
[Order article via Infotrieve]
-
Dixon, E. P.,
Stephenson, D. T.,
Clemens, J. A.,
and Little, S. P.
(1997)
Brain Res.
776,
222-229[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.