Corticotropin-Releasing Hormone-Mediated Neuroprotection against Oxidative Stress Is Associated with the Increased Release of Non-amyloidogenic Amyloid ß Precursor Protein and with the Suppression of Nuclear Factor-
B
Frank Lezoualch1,
Stefanie Engert,
Barbara Berning and
Christian Behl
Max Planck Institute of Psychiatry 80804 Munich, Germany
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ABSTRACT
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The neuropeptide CRH is the central regulator of
the hypothalamic-pituitary-adrenal (HPA) stress response system and is
implicated in various stress-related conditions. In the
neurodegenerative disorder Alzheimers disease (AD), levels of CRH are
decreased. AD pathology is characterized by the deposition of the
nonsoluble amyloid ß protein (Aß), oxidative stress, and neuronal
cell death. Employing primary neurons and clonal cells, we demonstrate
that CRH has a neuroprotective activity in CRH-receptor type 1
(CRH-R1)-expressing neurons against oxidative cell death. The
protective effect of CRH was blocked by selective and nonselective
CRH-R1 antagonists and by protein kinase A inhibitors. Overexpression
of CRH-R1 in clonal hippocampal cells lacking endogenous CRH-receptors
established neuroprotection by CRH. The activation of CRH-R1 and
neuroprotection are accompanied by an increased release of
non-amyloidogenic soluble Aß precursor protein. At the molecular
level CRH caused the suppression of the DNA-binding activity and
transcriptional activity of the transcription factor NF-
B.
Suppression of NF-
B by overexpression of a super-repressor mutant
form of I
B-
, a specific inhibitor of NF-
B, led to protection
of the cells against oxidative stress. These data demonstrate a novel
cytoprotective effect of CRH that is mediated by CRH-R1 and downstream
by suppression of NF-
B and indicate CRH as an endogenous protective
neuropeptide against oxidative cell death in addition to its function
in the HPA-system. Moreover, the protective function of CRH proposes a
molecular link between oxidative stress-related degenerative events and
the CRH-R1 system.
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INTRODUCTION
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CRH (1 ) is a 41-amino acid peptide hormone with multiple
biological effects and plays a central regulatory role in the
hypothalamic-pituitary-adrenal stress response system (1 2 3 4 ). Two
different human genes for CRH receptors (CRH-R) have been isolated
encoding CRH-R1 and CRH-R2 (5 6 ). Expression of CRH-R1 mRNA is
distinct from that of CRH-R2 mRNA and most abundant in cerebellar,
neocortical, hippocampal, and sensory relay structures of the rat brain
(3 ). CRH-R1 is a G protein-coupled high-affinity membrane receptor for
CRH using the intracellular stimulation of cAMP as signal transducer
(1 2 3 4 5 6 ). CRH has been implicated in various stress-related disorders
(3 4 ).
Brain areas affected in the progressive neurodegenerative disorder
Alzheimers disease (AD), the most common cause of senile dementia,
show morphological abnormalities in CRH neurons and also dramatic
reductions in the CRH content (7 8 ). Moreover, cognitive impairment in
AD patients is accompanied by decreased concentrations of CRH in
cerebrospinal fluid (9 ). A possible application of CRH and synthetic
CRH-R1 agonists for the treatment of AD is mainly based on the
memory-enhancing effects of CRH in rodents (10 ).
The amyloid ß protein (Aß) and its precursor, the amyloid ß
precursor protein (APP), are believed to play a central role in the
pathogenesis of AD (11 12 13 ). APP is a membrane-spanning glycoprotein
present at high levels in nerve cells. It can be processed via various
pathways, and an alteration in APP processing is the central event in
the formation of Aß deposits in the brain of AD patients (14 ).
During the
-secretase pathway APP holoprotein is cleaved
within the amyloidogenic Aß domain, producing a large amino-terminal
non-amyloidogenic soluble APP (sAPP), which can be detected by
monoclonal antibodies (15 ). The so-called ß- and
-secretase
pathway of APP produces amyloidogenic Aß that can readily form
neurotoxic Aß-aggregates (14 ). Aß itself can be directly neurotoxic
via oxidative stress and can induce lipid peroxidation in cultured
primary and clonal neurons with hydrogen peroxide
(H2O2) as one mediator
(16 ). On the other hand, sAPP resulting from
-secretase processing
and released from the cells has neurotrophic activities. These include
that sAPP increases the synaptic density in mouse brain, protects
neurons against oxidative stress in vitro after
overexpression or after direct addition of sAPP to neuronal cultures,
and confers resistance to apoptosis (17 18 19 20 21 ). Interestingly, the
release of sAPP by neurons can be increased by various G
protein-coupled transmembrane receptors (22 23 ).
During the course of oxidative nerve cell death, the redox-sensitive
transcription factor NF-
B is activated (16 24 25 26 ). NF-
B was the
first eukaryotic transcription factor described to respond directly to
oxidative stress (27 28 ). It was initially identified as a
lymphoid-specific protein that binds to the
-light chain gene
intronic enhancer and predominantly consists of the two subunits p50
and p65. These proteins are members of the NF-
B/Rel family of
transcription factors that are known to control various genes involved
in inflammatory mechanisms. Typically, NF-
B is sequestered in the
cytoplasm by the specific inhibitory protein I
B. Activation and
regulation of NF-
B transition into the nucleus, where it can induce
the transcription of NF-
B-dependent target genes, is tightly
controlled by I
B proteins (29 30 ). Although the primary role for
NF-
B in immune cells has always been thought to be the activation of
defense genes during the inflammatory response, a potential function of
NF-
B during cell death has also been suggested (31 32 33 34 35 ). Neurons
have a baseline activity of NF-
B, and a neuroprotective role for the
suppression of the NF-
B activity has recently been proposed (2426,
36, 37).
Since CRH neurons and CRH levels are affected in AD, the goal of the
present study was to investigate the direct effects of CRH on the
survival of neurons challenged by oxidative stress employing
CRH-R1-expressing rat cerebellar neurons, human neuroblastoma IMR32
(38 39 ) cells, and mouse hippocampal HT22 cells. In addition,
corticotrophic AtT20 cells from mouse pituitary were used, which are
also sensitive to oxidative insults and which are frequently employed
for the investigation of CRH-R1 function. As oxidative challenges Aß,
H2O2, and buthionine
sulfoximine (BSO), an inhibitor of
-glutamylcysteine synthetase
leading to the depletion of the central intracellular antioxidant
glutathione, were used (40 ). We demonstrate here that CRH protects
neurons against oxidative stress and that CRH-R1 activation is
associated with the increased release of non-amyloidogenic sAPP.
Moreover, it is shown that the cytoprotective effect of CRH is mediated
by the suppression of the activity of NF-
B. This observed novel
protective activity of CRH may explain some of the beneficial effects
of CRH on cognition and the cellular consequences of decreased CRH
levels in AD patients with respect to the pathogenesis of the
disease.
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RESULTS
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CRH Protects Cells Expressing CRH-R1 against Oxidative Stress
Primary cerebellar neurons, IMR32 cells, and AtT20 cells were
dose-dependently protected by CRH against BSO and Aß, oxidative
stressors that are highly toxic to neuronal cells (Fig. 1
). After a 12-h preincubation with
10-8 M CRH , a protective effect of CRH
against the BSO-induced oxidative cell death of cerebellar granule
neurons was observed (Fig. 1A
). Moreover, there was a dose-dependent
protection by CRH against 20 µM Aß. In all three
cellular systems used (primary cerebellar neurons, IMR32 cells, AtT20
cells), there was a significant protection against Aß toxicity at
nanomolar concentrations (Fig. 1B
). A similar protective effect of CRH
was also observed after challenging the cells with
H2O2 (data not shown). Since 10-8
M CRH caused a robust and significant protection, this
concentration was used for all subsequent studies.

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Figure 1. Neuroprotection against Oxidative Cell Death by CRH
A, Cerebellar neurons were protected against 500 µM
BSO by CRH (10-8 M as detected by
phase-contrast microscopy. B, Cell survival of primary CGC, IMR32
cells, and AtT20 cells was increased after preincubation with
increasing concentrations of CRH upon challenge with Aß (20
µM). C, Stable transfectants of HT22 cells overexpressing
CRH-R1 (HT22-CRH-R1) were dose dependently protected against
H2O2 by CRH as compared with nontransfected
controls (HT22 parental cells; HT22p). Cell survival data of all MTT
tests are presented as the means ± SEM for triplicate
determinations (B and C) with **, P < 0.001 and
*P < 0.01 (Students t test; toxin
+ CRH compared with CRH alone). Scale bar in panel
A = 50 µm.
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The expression of CRH-R1 in the primary and clonal cell systems
employed was initially confirmed, and the expression of CRH-R2 was
excluded by RT-PCR followed by Southern blotting (data not shown). The
dissociation constants (KD) of binding of radiolabeled
ovine-CRH were consistent with those previously published (0.45
nM for cerebellar neurons; 0.06 nM for IMR32;
0.10 nM for AtT20) (3 38 39 ). The intact intracellular
signaling of CRH-R1 via activation of the cAMP pathway was controlled
by transient transfections with a cAMP-response element coupled to a
luciferase gene reporter (CRE-luc) (data not shown).
Neuronal cells lacking functional endogenous CRH-R1 including rat
pheochromocytoma PC12 cells and human neuroblastoma SK-N-MC cells (as
confirmed by RT-PCR-analysis, CRH-binding studies, transfection assays
using a cAMP-response luciferase reporter construct, CRE-luc) were not
protected by CRH (data not shown).
Antagonists of CRH-R1 and Inhibitors of Protein Kinase A (PKA)
Prevent the Protective Effect of CRH
The protective effect of CRH was blocked by the nonselective CRH-R
antagonist
-helical CRH941 as shown for AtT20 cells (Table 1
). Moreover, the selective nonpeptide
antagonist of CRH-R1 antalarmin (41 ) dose-dependently blocked the
protective activity of CRH (Table 1
). The selective inhibitors of
protein kinase A and cAMP antagonist
adenosine-3',5'-monophosphorothioate (Rp-isomer; Rp-cAMPS) (42 ), and
the PKA inhibitor peptide (43 ) also dose-dependently prevented
CRH-mediated protection against
H2O2 (Table 1
). These data
indicate the direct involvement of CRH-R1 and cAMP-signaling pathways
in the protective effect of CRH.
Overexpression of CRH-R1 in Cells Lacking Endogenous CRH-R1
Establishes Protection against Oxidative Stress by CRH
Since the hippocampus is a primary target of
AD-associated neurodegeneration and CRH-R1 is expressed in the
hippocampus, the effect of CRH on the survival of clonal hippocampal
HT22 cells from the mouse was investigated. HT22 cells are frequently
used to study oxidative neuronal cell death (44 45 ), but since in HT22
cells no CRH-R1 expression was detectable as determined by PCR (data
not shown), a HT22 cell line was generated that expresses the human
CRH-R1. Stable hippocampal HT22-CRH-R1 expressing cell clones
(KD = 0.12 nM for ovine-CRH in
HT22-CRH-R1 transfectants) were dose-dependently protected by CRH
against the oxidative insults as induced by
H2O2 compared with
nontransfected parental HT22 cells (HT22p) (Fig. 1C
). Therefore, in the
HT22-CRH-R1 transfectants the protective effect of CRH against
oxidative stress could be established and was achieved at nanomolar
concentrations of CRH.
CRH Prevents
H2O2-Induced
Apoptosis
To determine the pathway of neuronal cell death induced
by oxidative challenges and its prevention by CRH, human neuroblastoma
IMR32 cells were treated with
H2O2 and were subjected to
two different assays to determine apoptosis, Hoechst-staining (Fig. 2
) and TUNEL-staining (Fig. 3
). While TUNEL staining detects the
formation of DNA fragments resulting from the enzymatic activity of
cellular DNAses (46 ), increased staining of the nuclei with Hoechst
33342 indicates changes in the chromatin structure and apoptosis (47 ).
This is shown qualitatively by fluorescence microscopy (Fig. 2
, AD
and Fig. 3
, AD) and quantitatively by counting the number of stained
cells (Figs. 2E
and 3E
). Hydrogen peroxide induced an increased
staining of the nuclei of IMR32 cells with Hoechst, which was prevented
by CRH (Fig. 2
). Consistently,
H2O2 induced an apoptotic
DNA fragmentation in the IMR32 cells as demonstrated by the increased
TUNEL staining, which was prevented by CRH (Fig. 3
). In both assays the
CRH-R antagonist
-helical CRH941 reversed the antiapoptotic
activity of CRH indicated by the increased Hoechst and TUNEL staining
of the cells (Figs. 2D
and 3D
). Similar results were also found in
other cellular systems used in this study (e.g. AtT20 cells,
cerebellar granule neurons; data not shown). Next, possible mechanisms
that may mediate the protective activity of CRH against oxidative
stress-induced apoptosis were investigated.

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Figure 2. CRH Prevents Apoptotic Changes in the Chromatin
Structure as Detected by Hoechst 33258 Staining
Changes in the chromatin structure induced by 60 µM
H2O2 in IMR32 cells was prevented by
10-8 M CRH as detected by Hoechst 33258
staining. Staining of the nuclei with Hoechst 33528 after treatment of
the cells with H2O2 alone (B),
H2O2 + CRH (C), and
H2O2 + CRH + -helical CRH941 ( -CRH) (D)
compared with untreated cells as control (A) is shown. For
quantification, Hoechst-positive nuclei were counted in each experiment
(E) and data are presented as the means ± SEM for
triplicate determinations with **, P < 0.001
(H2O2 + CRH compared with
H2O2 alone).
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Figure 3. CRH Prevents Apoptotic DNA Fragmentation as
Detected by TUNEL Staining
DNA fragmentation induced by 60 µM
H2O2 in IMR32 cells was prevented by
10-8 M CRH as detected by TUNEL staining.
Staining of the nuclei with TUNEL after treatment of the cells with
H2O2 alone (B), H2O2 +
CRH (C), and H2O2 + CRH + -helical CRH941
( -CRH) (D) compared with untreated cells as control (A) is shown.
For quantification, TUNEL-positive nuclei were counted in each
experiment (E), and data are presented as the means ±
SEM for triplicate determinations with **,
P < 0.001 (H2O2 + CRH
compared with H2O2 alone).
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CRH Stimulates the Release of Non-amyloidogenic sAPP
Since the activation of several neuronal G protein-coupled
receptors leads to an increased release of neurotrophic
non-amyloidogenic sAPP (22 23 ), we tested whether activation of CRH-R1
by CRH could also enhance the secretion of sAPP. Indeed, as detected
with an N-terminal monoclonal antibody against APP (22C11) (15 22 23 ), the basal release of sAPP is increased in cerebellar neurons
(4-fold), in IMR32 (2.2-fold), and in AtT20 cells (2.0-fold) as well as
in stable HT22-CRH-R1-transfectants (2.3-fold) after a 12-h incubation
with 10-8 M CRH compared with
time-matched untreated controls (Fig. 4
).
Higher concentrations of CRH or an extended incubation time did not
further increase the sAPP release (data not shown). This CRH-driven
enhanced sAPP-release was blocked by the nonselective CRH receptor
antagonist
-helical CRH941 and, moreover, also with the selective
CRH-R1 antagonist antalarmin as shown for AtT20 cells. Antalarmin
reduced the cellular release of sAPP to the control level as quantified
by densitometry (Fig. 5A
).

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Figure 4. CRH Stimulates the Release of Non-amyloidogenic
sAPP
Western blots of APP derivatives secreted by primary cerebellar neurons
(CGC), IMR32 cells, AtT20 cells, HT22-control cells lacking CRH-R1
(HT22 parental cells), and a selected HT22 cell clone stably
overexpressing CRH-R1 (HT22-CRH-R1 stable transfectants) are shown.
Cells were stimulated with 10-8 M CRH, and
Western blots were performed employing the monoclonal antibody 22C11
followed by ECL. Molecular mass markers (MW in kilodaltons) are shown
on the left. CRH was added for 4 h (+) and for
12 h (+), and the sAPP secretion was compared with the
corresponding time-matched controls (-).
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Figure 5. CRH-Stimulated Release of Non-amyloidogenic sAPP Is
Blocked by -Helical CRH941 and by Antalarmin
A, Western blots of APP derivatives secreted by AtT20 cells upon
stimulation with 10-8 M CRH employing the
monoclonal antibody 22C11 followed by ECL are shown. The CRH-R
antagonist -helical CRH941 and the selective CRH-R1 antagonist
antalarmin blocked the release of sAPP induced by CRH. B, Western blots
employing the antiserum R1736, which specifically recognizes
non-amyloidogenic (non-Aß) regions of APP, are presented. The
intensity of the signals was analyzed by densitometer reading of the
autoradiographs of the Western blot and is presented as relative
protein expression. The expression in untreated control cells was
defined arbitrarily as 1.
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To further confirm that the secreted APP after CRH treatment is indeed
non-amyloidogenic, which excludes the formation of potentially
nonsoluble and neurotoxic Aß, we performed Western blots employing an
antiserum to non-Aß regions of APP (R1736) (48 ). As shown for AtT20
cells, CRH selectively stimulated the release of the non-amyloidogenic
sAPP (Fig. 5B
). CRH did not influence the release of APP-like proteins
as detected with monoclonal antibodies against APLP1- and APLP-2 (data
not shown).
CRH Suppresses the DNA-Binding Activity and the Transcriptional
Activity of NF-
B
For the detailed analysis of downstream signal transduction
pathways that may be involved in the protective effect of CRH,
AtT20 cells, which are frequently used as a model system to study the
signaling of CRH-R1, were employed. We found that CRH directly
suppressed the DNA-binding activity and transcriptional activity of the
redox-sensitive transcription factor NF-
B at baseline conditions and
after induction of NF-
B by
H2O2 but failed to modify
the transcriptional complex of Oct-1 as control (Fig. 6A
, lanes 15). The specificity of the
binding activity of NF-
B is confirmed by the supershift observed
when using an antibody specific for the p65 subunit of NF-
B (Fig. 6A
, lane 7) and by a diminished DNA-binding activity when an antibody
specific for the p50 subunit was employed (Fig. 6A
, lane 8).
After transient transfections with NF-
B-reporter plasmids with
an NF-
B-response element coupled to a luciferase gene reporter, we
found that consistent with the DNA-binding data CRH also suppressed the
baseline transcriptional activity of NF-
B and the transcriptional
activity induced by H2O2.
This suppression caused by CRH was approximately 70% for baseline and
approximately 50% for the
H2O2-induced
transcriptional activity of NF-
B as compared with the corresponding
controls (Fig. 6B
).
Overexpression of an I
B-
-Super-repressor Protects Cells
against Oxidative Stress
To elucidate the exact role of the suppression of NF-
B in
the cytoprotective activity of CRH, we investigated the effect of the
suppressed activity of NF-
B on cell survival upon challenge with
oxidative stress. Therefore, the activity of NF-
B was blocked
independently from CRH by the transfection of AtT20 cells with a
super-repressor form of I
B-
. This I
B-
-super-repressor is
resistant to both phosphorylation and proteolytic degradation and
therefore prevents the nuclear translocation of NF-
B (49 ). The
transient overexpression of this construct reduced the transcriptional
activity of NF-
B by approximately 50% (Fig. 7A
). In toxicity assays performed in
combination with the transfection assays, we found a significant
protection against H2O2
afforded by the suppression of NF-
B by I
B-
(Fig. 7B
).
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DISCUSSION
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Here we showed that the endogenous neuropeptide CRH protects
neuronal cells expressing CRH-R1 against oxidative insults. This
protective effect 1) was blocked by CRH-R1 antagonists and by
inhibitors of PKA signaling, 2) was accompanied by an enhanced release
of the AD-associated APP in its non-amyloidogenic soluble form (sAPP),
and 3) was mediated by the suppression of the activity of the
transcription factor NF-
B.
The pathology of AD is associated with neuronal cell loss,
neurofibrillary tangle formation, deposition of Aß forming so-called
plaques in susceptible brain regions and with increased oxidative
stress (11 12 13 40 ). In addition, there are pathological changes in CRH
neurons and dramatic reductions in the CRH content (7 8 9 ). By providing
evidence for a neuroprotective activity of CRH against oxidative
stress-induced apoptotic cell death, the presented data argue for a
possible molecular link between AD-associated oxidative neuronal cell
death, the depletion in CRH during AD pathogenesis, and the potent
cognition-enhancing effects of CRH (10 ). Oxidative challenges as,
e.g. induced by
H2O2, caused chromatin
changes and DNA fragmentations that are typical features of apoptosis
and that were prevented by CRH (Figs. 2
and 3
). Indeed,
H2O2 is a potent inducer of
apoptosis also in other cell systems (50 51 ). The antiapoptotic
protective activity of CRH appears to be specific for the activation of
CRH-R1 and through its proximal signaling pathways in the cells
studied, since 1) it appeared only in cells that specifically express
the CRH-R1, 2) it could be established by overexpression of CRH-R1 in
neuronal cells lacking this receptor, 3) it was blocked by the specific
CRH-R1 antagonist antalarmin, and 4) it was prevented by antagonists of
the cAMP-signaling pathway such as PKA inhibitors.
The protective activity of CRH is associated with increased release of
non-amyloidogenic sAPP (Figs. 4
and 5
), which may have trophic effects
for the cells. Indeed, sAPP can serve neuroprotective functions against
oxidative challenges in various cellular models as repeatedly shown
(18 19 ), and it confers resistance against p53-mediated apoptosis
(21 ). Moreover, sAPP can increase the synaptic density in the mouse
brain (20 ) and has memory-enhancing effects (52 ), which may explain
also the long-term memory-enhancing effects of CRH in vivo
(10 ). Loss of synaptic function and deficits in cognition and memory
are central pathological events in AD. Although intriguing, on the
basis of our data we cannot conclude that the protection is directly
mediated by the increased amount of protective sAPP in the cell
culture.
However, the finding of an increased release of non-amyloidogenic sAPP
driven by CRH may have some important long-term implications concerning
AD pathology, since the processing of APP is believed to be the central
step during the pathogenesis of AD (13 14 ). The proteolytic cleavage
of full-length APP through the
-secretase pathway that prevents the
formation of potentially neurotoxic Aß by the release of
non-amyloidogenic sAPP can be induced by various agents, including the
female sex hormone estrogen (53 ), which has been shown to be an
effective neuroprotectant (44 ). Release of sAPP can also be enhanced by
several neuronal receptors that are linked to G protein signaling
including the muscarinic acetylcholine receptor (22 ). CRH-R1 is also a
G protein-coupled receptor (2 3 ) and, on the basis of our data, its
high-affinity agonist CRH may therefore be added to the group of
promotors of the release of non-amyloidogenic sAPP. In addition to the
use of a monoclonal antibody against sAPP (22C11) (15 22 ) in this
paper, also an antiserum specifically recognizing non-Aß
regions has been employed (48 ), clearly indicating the
non-amyloidogenic nature and, therefore,
-secretase cleavage of APP.
The extent of the CRH-driven increased sAPP release found in the
present study is comparable to those reported previously for the
activation of muscarinic acetylcholine receptors transfected into human
embryonic kidney cells and after long-term treatment of primary
cerebrocortical neurons with estrogen (22 53 ). By stimulating the
release of non-amyloidogenic sAPP, CRH may, therefore, affect the
long-term deposition of Aß during AD pathogenesis.
CRH influences the intracellular signal transduction network, since
CRH-R1 activation did not only activate cAMP-mediated transcriptional
pathways, but did also induce a cross-talk with the activity of the
transcription factor NF-
B. Here, CRH caused a substantial
suppression of the constitutive as well as the oxidative stress-induced
DNA-binding activity and transcriptional activity of NF-
B (Fig. 6
).
This observation is consistent with studies from Parry and Mackman
(54 ), who showed an inhibition of NF-
B by cAMP in immune cells and
proposed that the NF-
B activity is inhibited by the activation of
the protein kinase A-signaling pathway. The fact that PKA inhibitors
inhibited the protective effect of CRH (Table 1
) underlines the
importance of PKA downstream signaling of CRH-R1 in this model.
Recently, NF-
B has been suggested to be implicated in
glutamate-induced neurotoxicity (24 36 37 ). Consistent with the
present study, the suppression of the glutamate-induced activation of
NF-
B achieved by aspirin led to neuroprotection (24 ). In another
toxicity paradigm, we observed that the oxidative cell death of HT22
cells induced by the dopamine D2 receptor antagonist haloperidol was
prevented after the suppression of NF-
B by the I
B-
super-repressor of NF-
B activity, the same construct we used also in
the present study to block NF-
B (26 ). Interestingly, an increased
NF-
B activity has been found to be associated with Aß deposits in
affected AD brain regions (25 ), suggesting a participation of NF-
B
and NF-
B-driven genetic programs in the AD-associated pathological
events. Indeed, the suppression of NF-
B by I
B-
mimicked the
cytoprotective effect of CRH (Fig. 7
). This strongly suggests that the
suppressive effect of CRH on NF-
B was directly mediating its
protective activity. The block of NF-
B by CRH may either directly
lead to the suppression of NF-
B-driven proapoptotic genetic programs
or indirectly to the activation of intrinsic cellular protective
programs. Considering the suggested role of this transcription factor
in nerve cell death and in AD (36 37 ). This effect of CRH on the
activity of NF-
B may be of central importance.
Consistent with our data, CRH has been found to function as an
endogenous neuroprotective peptide to prevent neurodegeneration during
hypoxia in rat brain (55 ). In other experimental neurodegenerative
paradigms, such as in vivo models of cerebral ischemia,
antagonists of CRH-R, such as
-helical CRH941, exerted some
neuroprotective activities (56 57 ). This contradictory result may be
due to basic differences in the neurodegenerative paradigms studied and
may also reflect partial agonistic activities of this particular
antagonist. Moreover, it must be stressed that
-CRH941 is a
nonselective CRH-R1 and CRH-R2 receptor blocker and may cause a variety
of effects not related to CRH-R1 function. Therefore, additional
in vivo studies using CRH and selective CRH-R1 antagonists
in animal models of neurodegeneration are needed.
With respect to AD pathology, it is of further interest that the CRH-R1
is expressed at high levels in brain regions that are among those areas
least affected during AD-associated neurodegeneration, such as the
cerebellum (3 ). Here, we show that, indeed, primary neurons from the
cerebellum and other neuronal cells with functional CRH-R1 are highly
protected against oxidative cell death by CRH (
Figs. 13

). On the
basis of the presented data, it can be speculated that a permanent
activation of CRH-R1 by CRH could serve as a basic protective stimulus
rendering CRH-R1-expressing neurons more resistant against exogenous
oxidative insults. This view would also explain a detrimental role for
the observed decrease in CRH levels in AD. With a reduction in overall
CRH a basic neuronal protection may be lost, rendering the neurons more
vulnerable to accumulating exogenous insults. Therefore, future
therapeutic approaches targeting the CRH-R1 system may prove useful for
neuroprotection and, ultimately, for the treatment of AD and other
oxidative stress-related neurological conditions.
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MATERIALS AND METHODS
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Cell Culture
Cerebellar neurons (cerebellar granule cells; CGC) were prepared
from 8-day-old Sprague Dawley rat pups as previously described (58 ) and
were grown in basal modified Eagles medium containing 10%
heat-inactivated FCS, KCl, glutamine, and gentamicin. Proliferation of
glial cells was prevented by treatment with cytosine
arabino-furanoside. After 7 days in vitro, cerebellar
cultures containing 95% neurons (as defined by neuron-specific
immunocytochemical stainings) were used for the different assays. IMR32
is a human neuroblastoma and AtT20 is a mouse pituitary tumor cell line
from ATCC (Manassas, VA). Both cell lines (as well as the
rat primary cerebellar granule neurons) express functional CRH-R1 and
were cultured as described previously (38 ). HT22 is a clonal
hippocampal cell line from mouse that is frequently used to study
oxidative nerve cell death (44 45 ). The selective protein kinase A
inhibitor adenosine-3',5'-monophosphorothioate (Rp-isomer; Rp-cAMPS)
(42 ) was from BIOLOG (Bremen, Germany) and the PKA inhibitor peptide
(17-residue synthetic peptide corresponding to PKA inhibitor) (43 ) was
from Upstate Biotechnology, Inc. (Lake Placid, NY). All
media, media supplements, and sera were from Life Technologies, Inc. (Eggenstein, Germany). Aß used in this study was the
toxic fragment Aß25-35 (16 ) and was purchased
from Bachem (Hannover, Germany). All other chemicals were
from Sigma (Deisenhofen, Germany) unless otherwise stated.
The nonpeptide CRH-R1 antagonist antalarmin was from Dr. G. P.
Chrousos (NIH, Bethesda, MD) (41 ).
PCR and CRH-Binding Studies
PCRs for the detection of the expression of CRH-R1 mRNA-followed
by Southern blottings were performed according to standard procedures
using PCR-primers specific for CRH-R1 mRNA and CRH-R2 mRNA (Biognostik;
Göttingen, Germany). CRH binding studies were performed using
[125I]Tyro-ovine CRH (Du
Pont, NEN Life Science Products, Boston, MA) as described
(38 ). Binding data (triplicate determinations) were analyzed and KD
values were determined with the EBDA and LIGAND program which provides
a non-linear, least-square regression analysis.
Transfection Experiments
To test the cAMP-driven transcription induced by CRH a
luciferase reporter construct containing a cAMP-response element
(CRE-luc) (59 ) was transiently transfected into the clonal and primary
cells using polyethyleneimine (PEI; Aldrich, Germany). Transfection
conditions and luciferase data evaluations were performed exactly as
described (26 ).
For stable overexpression of the CRH-R1 in the clonal mouse hippocampal
HT22 cells, the human CRH-R1 cDNA was permanently introduced using PEI.
Before transfection the CRH-R1 cDNA was subcloned into the polylinker
site (HindIII/XbaI) of the pcDNA3 mammalian
expression vector containing the neomycin resistance gene for selection
of transfected cell clones (Invitrogen; Germany).
To assay the transcriptional activity of NF-
B transient
transfections were performed using a NF-
B reporter plasmid
containing 6 NF-
B-binding DNA consensus sites linked to a luciferase
reporter gene (NF-
B-Luc) exactly as described (26 ). As control
vector Tk-Luc containing only the thymidine kinase promoter linked to a
luciferase construct was employed. The activity of NF-
B was
specifically suppressed employing transfections with the I
ß
super-repressor-construct.
Cell Survival and Apoptosis Assays
For the analysis of the CRH effects on neuronal survival, CRH
(human/rat from Bachem; Germany) was added 12 h
before toxin addition. After additional 20 h cell survival was
determined using the reduction of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), a
first indicator of oxidative cell death (16 ), exactly as described and
was confirmed by trypan blue exclusion and cell countings (using
phase-contrast microscopy and morphological criteria and trypan blue
exclusion) (16 ). DNA fragmentation indicative of apoptosis was detected
using the TUNEL assay (Roche Molecular Biochemicals;
Germany). As an additional assay for apoptosis staining of the cells
with Hoechst 33342 was performed as described (60 ). Briefly, for both
apoptosis assays cells were plated and pretreated with CRH and or
-helical CRH941 or left untreated. Then the cells were challenged
with H2O2 for 12 h.
After incubation with Hoechst 33342 (final concentration 1 mg/ml) for
10 min, cells were washed with PBS, resuspended in DMEM (lacking
phenol-red) and visualized using fluorescence microscopy. Nuclei
stained with TUNEL (TUNEL-positive nuclei) or with Hoechst 33342
(Hoechst-positive nuclei) were counted investigating optical fields of
>200 cells in at least three separate experimental sets.
Western Blot Analysis
Subconfluent cell cultures (primary cerebellar neurons, IMR32,
HT22, AtT20 cell clones) were cultivated in DMEM without serum and CRH
was added for the indicated time periods. Conditioned supernatants
containing secreted proteins were collected and size selected using
Sephadex G-25M columns (Pharmacia Biotech,
Freiburg, Germany). Concentrations of the lyophilized proteins were
determined with a standard Bradford assay (Bio-Rad Laboratories, Inc., München, Germany) and equal amounts were subjected
to SDS-PAGE. After electroblotting the following primary antibodies
were used: monoclonal APP antibody (22C11; Roche Molecular Biochemicals; Germany), monoclonal antibodies against APP-like
protein 1 and -2 (kindly provided by Dr. Gerd Multhaup; ZMBH,
Heidelberg, Germany), and the antiserum designated R1736 (kindly
provided by Dr. Dennis Selkoe, Harvard Medical School, Boston, MA).
Specific antibody binding was detected using ECL (Amersham Pharmacia Biotech, Braunschweig, Germany). For
quantification, autoradiographs of representative experiments were
scanned using a photometer (Beckman, Fullerton, CA)
and fold induction of sAPP-release compared with corresponding
controls was calculated. Western blotting experiments were repeated at
least three times with identical results.
Electrophoretic Mobility Shift Assay (EMSA)
Nuclear extracts for the EMSA were prepared after a
miniextraction protocol (61 ). EMSAs and supershift analysis using
antibodies specific for the p50 and p65 subunits of NF-
B (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA) were performed exactly as
described (26 ) employing radioactively labeled oligonucleotide probes
for NF-
B or Oct-1 of the EMSA kit from Promega Corp.
(Heidelberg; Germany).
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to C. W. Liaw for human CRH-R1 cDNA, P.
Maher for providing HT22 cells, G. Multhaup for APLP1/2 antibodies, D.
Selkoe for R1736 antiserum, D. Spengler for the CRE-Luc construct, P.
Baeuerle for the NF-
B-Luc and Tk-Luc plasmids, and D. W.
Ballard for the I
ß
super-repressor- and I
ß
-control
construct. G. P. Chrousos, who provided the selective CRH-R1
antagonist antalarmin, is also gratefully acknowledged. In addition, we
thank B. Lutz for critical reading of the manuscript and E. Guell for
secretarial help.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Christian Behl Ph.D., Independent Research Group Neurodegeneration, Max Planck Institute of Psychiatry, 80804 Munich, Germany;
This work was supported in part by the Deutsche Hirnliga e.V. (
C.B.).
1 Present Address: INSERM U 446, Université de Paris-Sud,
Faculty of Pharmacy, 92296 Chatenay-Malabry, France. 
Received for publication February 22, 1999.
Revision received September 18, 1999.
Accepted for publication September 22, 1999.
 |
REFERENCES
|
---|
-
Vale W, Spiess J, Rivier C, Rivier J 1981 Characterization of a 41-residue ovine hypothalamic peptide that
stimulates secretion of corticotropin and ß-endorphin. Science 213:13941397[Medline]
-
Owens MJ, Nemeroff CB 1991 Physiology and pharmacology of
corticotropin-releasing factor. Pharmacol Rev 43:425473[Medline]
-
Behan DP, Grigoriadis DE, Lovenberg T, Chalmers D, Heinrichs
S, Liaw C, De Souza EB 1996 Neurobiology of corticotropin releasing
factor (CRH) receptors and CRH-binding protein: implications for the
treatment of CNS disorders. Mol Psychol 1:265277
-
Holsboer F, Barden N 1996 Antidepressants and
hypothalamic-pituitary-adrenocortical regulation. Endocr Rev 17:187205[Medline]
-
Chen R, Lewis KA, Perrin MH, Vale WW 1993 Expression cloning
of a human corticotropin-releasing-factor receptor. Proc Natl Acad Sci
USA 90:89678971[Abstract]
-
Liaw CW, Lovenberg TW, Barry G, Oltersdorf T, Grigoriadis DE,
de Souza EB 1996 Cloning and characterization of the human
corticotropin-releasing factor-2 receptor complementary
deoxyribonucleic acid. Endocrinology 137:7277[Abstract]
-
Bissette G, Reynolds GP, Kilts CD, Widerlöv W, Nemeroff
CB 1985 Corticotropin- releasing factor-like immunoreactivity in senile
dementia of the Alzheimer type. Reduced cortical and striatal
concentrations. JAMA 254:30673069[Abstract]
-
De Souza EB, Whitehouse PJ, Kuhar MJ, Price DL, Vale WW 1986 Reciprocal changes in corticotropin-releasing factor (CRH)-like
immunoreactivity and CRH receptors in cerebral cortex of Alzheimers
disease. Nature 319:593595[Medline]
-
Pomara N, Singh RR, Deptula D, LeWitt PA, Bissette G, Stanley
M, Nemeroff CB 1989 CSF corticotropin-releasing factor (CRH) in
Alzheimers disease: its relationship to severity of dementia and
monoamine metabolites. Biol Psychiatry 26:500504[CrossRef][Medline]
-
Behan DP, Heinrichs SC, Troncoso JC, Liu XJ, Kawas CH, Ling N,
De Souza EB 1995 Displacement of corticotropin releasing factor from
its binding protein as a possible treatment for Alzheimers disease.
Nature 378:284287[CrossRef][Medline]
-
Masters CL, Simms G, Weidenman NA, Multhaup G, McDonald BL,
Beyreuther K 1985 Amyloid plaque core protein in Alzheimer disease and
Down syndrome. Proc Natl Acad Sci USA 82:42454249[Abstract]
-
Glenner GG 1988 Alzheimers disease: its proteins and genes.
Cell 52:307308[Medline]
-
Hardy J 1997 Amyloid, the presenilins and Alzheimers
disease. Trends Neurosci 20:154159[CrossRef][Medline]
-
Haass C, Selkoe DJ 1993 Cellular processing of ß-amyloid
precursor protein and the genesis of amyloid ß-peptide. Cell 75:10391042[Medline]
-
Weidemann A, Konig G, Bunke D, Fischer P, Salbaum JM, Masters
CL, Beyreuther K 1989 Identification, biogenesis, and localization of
precursors of Alzheimers disease A4 amyloid protein. Cell 57:11526[Medline]
-
Behl C, Davis JB, Lesley R, Schubert D 1994 Hydrogen peroxide
mediates amyloid ß protein toxicity. Cell 77:817822[Medline]
-
Saitoh T, Sundsmo M, Roch JM, Kimura N, Cole G, Schubert D,
Oltersdorf T, Schenk DB 1989 Secreted form of amyloid ß protein
precursor is involved in the growth regulation of fibroblasts. Cell 58:615622[Medline]
-
Mattson MP, Cheng B, Culwell AR, Esch FS, Lieberburg I, Rydel
RE 1993 Evidence for excitoprotective and intraneuronal
calcium-regulating roles for secreted forms of the ß-amyloid
precursor protein. Neuron 10:243254[Medline]
-
Schubert D, Behl C 1993 The expression of amyloid ß protein
precursor protects nerve cells from ß-amyloid and glutamate toxicity
and alters their interaction with the extracellular matrix. Brain Res 629:275282[CrossRef][Medline]
-
Mucke L, Masliah E, Johnson WB, Ruppe MD, Alford M,
Rockenstein EM, Forss-Petter S, Pietropaolo M, Mallory M, Abraham CR 1994 Synaptotropic effects of human amyloid ß protein precursors in
the cortex of transgenic mice. Brain Res 666:151167[CrossRef][Medline]
-
Xu X, Yang D, Wyss-Coray T, Yan J, Gan L, Sun Y, Mucke L 1999 Wild-type but not Alzheimer-mutant amyloid precursor protein confers
resistance against p53-mediated apoptosis. Proc Natl Acad Sci USA 96:75477552[Abstract/Free Full Text]
-
Nitsch RM, Slack BE, Wurtman RJ, Growdon JH 1992 Release of
Alzheimer amyloid precursor derivatives stimulated by activation of
muscarinic acetylcholine receptors. Science 258:304307[Medline]
-
Slack BE, Nitsch RM, Livneh E, Kunz GM Jr, Breu J, Eldar H,
Wurtman RJ 1993 Regulation by phorbol esters of amyloid precursor
protein release from Swiss 3T3 fibroblasts overexpressing protein
kinase C
. J Biol Chem 268:2109721101[Abstract/Free Full Text]
-
Grilli M, Pizzi M, Memo M, Spano P 1996 Neuroprotection by
aspirin and sodium salicylate through blockade of NF-
B
activation. Science 274:13831385[Abstract/Free Full Text]
-
Kaltschmidt B, Uherek M, Volk B, Baeuerle P, Kaltschmidt C 1997 Transcription factor NF-
B is activated in primary neurons
by amyloid ß peptides and in neurons surrounding early plaques from
patients with Alzheimer disease. Proc Natl Acad Sci USA 94:26422647[Abstract/Free Full Text]
-
Post A, Holsboer F, Behl C 1998 Induction of NF-
B
activity during haloperidol-induced oxidative toxicity in clonal
hippocampal cells-suppression of NF-
B and neuroprotection by
antioxidants. J Neurosci 18:82368246[Abstract/Free Full Text]
-
Schreck R, Zorbas H, Winnacker EL, Bäuerle PA 1991 Reactive oxygen intermediates as apparently widely used messengers in
the activation of the NF-
B transcription factor and HIV-1. EMBO
J 10:22472258[Abstract]
-
Schmidt KN, Amstad P, Cerutti P, Bäuerle PA 1995 The
roles of hydrogen peroxide and superoxide as messengers in the
activation of transcription factor NF-
B. Chem Biol 2:1322[Medline]
-
Baeuerle PA, Henkel T 1994 Function and activation of
NF-
B in the immune system. Annu Rev Immunol 12:141179[CrossRef][Medline]
-
Baldwin AS 1996 The NF-
B and I
B proteins: new
discoveries and insights. Annu Rev Immunol 14:649681[CrossRef][Medline]
-
Wu M, Lee HY, Bellas RE, Schauer SL, Arsura M, Katz D,
Fitzgerald MJ, Rothstein TL, Sherr DH, Sonenshein GE 1996 Inhibition of
NF-
B/rel induces apoptosis of murine B cells. EMBO J 15:46824690[Abstract]
-
Beg AA, Baltimore D 1996 An essential role for NF-
B in
preventing TNF-
-induced cell death. Science 274:782784[Abstract/Free Full Text]
-
Liu Z, Hsu H, Goeddel DV, Karin M 1996 Dissection of TNF
receptor 1 effector functions: JNK activation is not linked to
apoptosis while NF-
B activation prevents cell death. Cell 87:565576[Medline]
-
Van Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM 1996 Suppression of TNF-
-induced apoptosis. Science 274:787789[Abstract/Free Full Text]
-
Wang CY, Marty MW, Baldwin Jr AS 1996 TNF- and cancer
therapy-induced apoptosis: potentiation by inhibition of apoptosis.
Science 274:784787[Abstract/Free Full Text]
-
Lipton SA 1997 Janus faces of NF-
B: neurodestruction
versus neuroprotection. Nat Med 3:2022[Medline]
-
Lezoualch F, Behl C 1998 Transcription factor NF-
B:
friend or foe of neurons? Mol Psychiatry 3:152[CrossRef][Medline]
-
Dieterich KD, DeSouza EB 1996 Functional
corticotropin-releasing factor receptors in human neuroblastoma cells.
Brain Res 733:113118[CrossRef][Medline]
-
De Souza EB 1995 Corticotropin-releasing factor receptors:
physiology, pharmacology, biochemistry and role in central nervous
system and immune disorders. Psychoneuroendocrinology 20:789819[CrossRef][Medline]
-
Coyle JT, Puttfarcken P 1993 Oxidative stress, glutamate, and
neurodegenerative disorders. Science 262:689695[Medline]
-
Webster EL, Lewis DB, Torpy DJ, Zachman EK, Rice KC, Chrousos
GP 1996 In vivo and in vitro
characterization of antalarmin, a nonpeptide corticotropin releasing
hormone (CRH) receptor antagonist: suppression of pituitary ACTH
release and peripheral inflammation. Endocrinology 137:57475750[Abstract]
-
Wang LY, Salter MW, MacDonald JF 1991 Regulation of kainate
receptors by cAMP- dependent protein kinase and phosphatases. Science.
253:11321135
-
Glass DB, Cheng HC, Mende-Mueller L, Reed J, Walsh DA 1989 Primary structural determinants essential for potent inhibition of
cAMP-dependent protein kinase by inhibitory peptides corresponding to
the active portion of the heat-stable inhibitor protein. J Biol
Chem 264:88028810[Abstract/Free Full Text]
-
Behl C, Skutella T, Lezoualch F, Post A, Widmann M, Newton
CJ, Holsboer F 1997 Neuroprotection against oxidative stress by
estrogens: structure-activity relationship. Mol Pharmacol 51:535541[Abstract/Free Full Text]
-
Li Y, Maher P, Schubert D 1997 A role for 12-lipoxygenase in
nerve cell death caused by glutathione depletion. Neuron 19:453463[CrossRef][Medline]
-
McCarthy NJ, Evan GI 1998 Methods for detecting and
quantifying apoptosis. Curr Top Dev Biol 36, 259278
-
Dive C, Gregory CD, Phipps DJ, Evans DL, Milner AE, Wyllie AH 1992 Analysis and discrimination of necrosis and apoptosis (programmed
cell death) by multiparameter flow cytometry. Biochim Biophys Acta.
1133:27585
-
Podlisny MB, Tolan DR, Selkoe DJ 1991 Homology of the amyloid
ß protein precursor in monkey and human supports a primate model for
beta amyloidosis in Alzheimers disease. Am J Pathol 8:14231435
-
Brockman JA, Scherer DC, Mckinsey TA, Hall SM, Qi XX, Lee WY,
Ballard DW 1995 Coupling of a signal response domain in IkBa to
multiple pathways for NF-
B activation. Mol Cell Biol 15:28092818[Abstract]
-
Goel R, Khanduja KL 1998 Oxidative stress-induced
apoptosisan overview. Curr Sci 75:13381345
-
Ikeda K, Kajiwara K, Tanabe E, Tokumaru S, Kishida E, Masuzawa
Y, Kojo S 1999 Involvement of hydrogen peroxide and hydroxyl radical in
chemically induced apoptosis of HL-60 cells. Biochem Pharmacol 57:13611365[CrossRef][Medline]
-
Meziane H, Dodart JC, Mathis C, Little S, Clemens J, Paul SM,
Ungerer A 1998 Memory-enhancing effects of secreted forms of the
ß-amyloid precursor protein in normal and amnestic mice. Proc Natl
Acad Sci USA 95:1268312688[Abstract/Free Full Text]
-
Xu H, Gouras GK, Greenfield JP, Vincent B, Naslund J,
Mazzarelli L, Fried G, Jovanovic JN, Seeger M, Relkin NR, Liao F,
Checler F, Buxbaum JD, Chait BT, Thinakaran G, Sisodia SS, Wang R,
Greengard P, Gandy S 1998 Estrogen reduces neuronal generation of
Alzheimer ß-amyloid peptides. Nat Med 4:447451[Medline]
-
Parry GC, Mackman N 1997 Role of cyclic AMP response
element-binding protein in cyclic AMP inhibition of NF-
B-mediated
transcription. J Immunol 159:54505456[Abstract]
-
Fox MW, Anderson RE, Meyer FB 1993 Neuroprotection by
corticotropin releasing factor during hypoxia in rat brain. Stroke 24:10721077[Abstract]
-
Lyons MK, Anderson RE, Meyer FB 1991 Corticotropin releasing
factor antagonist reduces ischemic hippocampal neuronal injury. Brain
Res 545:339342[CrossRef][Medline]
-
Strijbos PJLM, Relton JK, Rothwell NJ 1994 Corticotropin-releasing factor antagonist inhibits neuronal damage
induced by focal cerebral ischaemia or activation of NMDA receptors in
the rat brain. Brain Res 656:405408[CrossRef][Medline]
-
Sparapani M, Buonamici L, Ciani E, Battelli MG,
Ceccarelli G, Stirpe F, Contestabile A 1997 Toxicity of ricin and
volkensin, two ribosome-inactivating proteins, to microglia, astrocyte,
and neuron cultures. Glia 20:203205[CrossRef][Medline]
-
Spengler D, Rupprecht R, Van LP, Holsboer F 1992 Identification and characterization of a 3',5'-cyclic adenosine
monophosphate-responsive element in the human corticotropin-releasing
hormone gene promotor. Mol Endocrinol 6:19311941[Abstract]
-
Lezoualch F, Skutella T, Widmann M, Behl C 1996 Melatonin
prevents oxidative stress-induced cell death in hippocampal cells.
Neuroreport 7:20712077[Medline]
-
Schreiber E, Matthias P, Müller M, Schaffner W 1989 Rapid detection of octamer binding proteins with "mini-extracts,"
prepared from a small number of cells. Nucleic Acids Res 17:6419[Medline]