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Molecular Endocrinology 14 (1):147
Copyright © 2000 by The Endocrine Society

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-{kappa}B

Frank Lezoualc’h1, Stefanie Engert, Barbara Berning and Christian Behl

Max Planck Institute of Psychiatry 80804 Munich, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 Alzheimer’s 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-{kappa}B. Suppression of NF-{kappa}B by overexpression of a super-repressor mutant form of I{kappa}B-{alpha}, a specific inhibitor of NF-{kappa}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-{kappa}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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 Alzheimer’s 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 {alpha}-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 {gamma}-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 {alpha}-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-{kappa}B is activated (16 24 25 26 ). NF-{kappa}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 {kappa}-light chain gene intronic enhancer and predominantly consists of the two subunits p50 and p65. These proteins are members of the NF-{kappa}B/Rel family of transcription factors that are known to control various genes involved in inflammatory mechanisms. Typically, NF-{kappa}B is sequestered in the cytoplasm by the specific inhibitory protein I{kappa}B. Activation and regulation of NF-{kappa}B transition into the nucleus, where it can induce the transcription of NF-{kappa}B-dependent target genes, is tightly controlled by I{kappa}B proteins (29 30 ). Although the primary role for NF-{kappa}B in immune cells has always been thought to be the activation of defense genes during the inflammatory response, a potential function of NF-{kappa}B during cell death has also been suggested (31 32 33 34 35 ). Neurons have a baseline activity of NF-{kappa}B, and a neuroprotective role for the suppression of the NF-{kappa}B activity has recently been proposed (24–26, 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 {gamma}-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-{kappa}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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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. 1AGo). 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. 1BGo). 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 (Student’s t test; toxin + CRH compared with CRH alone). Scale bar in panel A = 50 µm.

 
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 {alpha}-helical CRH9–41 as shown for AtT20 cells (Table 1Go). Moreover, the selective nonpeptide antagonist of CRH-R1 antalarmin (41 ) dose-dependently blocked the protective activity of CRH (Table 1Go). 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 1Go). These data indicate the direct involvement of CRH-R1 and cAMP-signaling pathways in the protective effect of CRH.


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  		Table 1. CRH-R Antagonists and Inhibitors of PKA Inhibit 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. 1CGo). 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. 2Go) and TUNEL-staining (Fig. 3Go). 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. 2Go, A–D and Fig. 3Go, A–D) and quantitatively by counting the number of stained cells (Figs. 2EGo and 3EGo). Hydrogen peroxide induced an increased staining of the nuclei of IMR32 cells with Hoechst, which was prevented by CRH (Fig. 2Go). Consistently, H2O2 induced an apoptotic DNA fragmentation in the IMR32 cells as demonstrated by the increased TUNEL staining, which was prevented by CRH (Fig. 3Go). In both assays the CRH-R antagonist {alpha}-helical CRH9–41 reversed the antiapoptotic activity of CRH indicated by the increased Hoechst and TUNEL staining of the cells (Figs. 2DGo and 3DGo). 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 + {alpha}-helical CRH9–41 ({alpha}-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 + {alpha}-helical CRH9–41 ({alpha}-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).

 
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. 4Go). 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 {alpha}-helical CRH9–41 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. 5AGo).



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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 {alpha}-Helical CRH9–41 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 {alpha}-helical CRH9–41 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.

 
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. 5BGo). 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-{kappa}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-{kappa}B at baseline conditions and after induction of NF-{kappa}B by H2O2 but failed to modify the transcriptional complex of Oct-1 as control (Fig. 6AGo, lanes 1–5). The specificity of the binding activity of NF-{kappa}B is confirmed by the supershift observed when using an antibody specific for the p65 subunit of NF-{kappa}B (Fig. 6AGo, lane 7) and by a diminished DNA-binding activity when an antibody specific for the p50 subunit was employed (Fig. 6AGo, lane 8).



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  		Figure 6. Suppression of the DNA-Binding Activity and the Transcriptional Activity of NF-{kappa}B by CRH

A, The DNA-binding activity of NF-{kappa}B at baseline conditions and after 4 h of stimulation of AtT20 cells with 30 µM H2O2 was decreased by coincubation with 10-8 M CRH as determined by EMSAs while the DNA-binding activity of Oct-1 as control was not altered: untreated control cells (lanes1 and 6); CRH-treated cells (lane 2), H2O2-treated cells (lane 3), coincubation of H2O2 and CRH (lane 4), and 100-fold excess of corresponding nonlabeled probe (NF-{kappa}B or Oct-1, lane 5) are shown. The arrows mark the position of the specific NF-{kappa}B/DNA complex band that was either supershifted using a p65 antibody (lane 7) or diminished using a p50 antibody (lane 8). Open circles depict the position of nonspecific complexes. B, The baseline transcriptional activity (CT) and the H2O2-induced transcriptional activity of NF-{kappa}B are suppressed by 10-8 M CRH in AtT20 cells. Cells were transfected and treated either with CRH alone or in combination with 30 µM H2O2 for 4 h. Luciferase activity of nontreated controls (CT) is arbitrarily shown as 1-fold induction. Results are shown in relative luciferase activity corrected for identical protein amounts. Data are the mean of three independent experiments ± SEM.

 
After transient transfections with NF-{kappa}B-reporter plasmids with an NF-{kappa}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-{kappa}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-{kappa}B as compared with the corresponding controls (Fig. 6BGo).

Overexpression of an I{kappa}B-{alpha}-Super-repressor Protects Cells against Oxidative Stress
To elucidate the exact role of the suppression of NF-{kappa}B in the cytoprotective activity of CRH, we investigated the effect of the suppressed activity of NF-{kappa}B on cell survival upon challenge with oxidative stress. Therefore, the activity of NF-{kappa}B was blocked independently from CRH by the transfection of AtT20 cells with a super-repressor form of I{kappa}B-{alpha}. This I{kappa}B-{alpha}-super-repressor is resistant to both phosphorylation and proteolytic degradation and therefore prevents the nuclear translocation of NF-{kappa}B (49 ). The transient overexpression of this construct reduced the transcriptional activity of NF-{kappa}B by approximately 50% (Fig. 7AGo). In toxicity assays performed in combination with the transfection assays, we found a significant protection against H2O2 afforded by the suppression of NF-{kappa}B by I{kappa}B-{alpha} (Fig. 7BGo).



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  		Figure 7. Suppression of the Transcriptional Activity of NF-{kappa}B by the Overexpression of the I{kappa}B-{alpha}-Super-Repressor Protects against Oxidative Cell Death

A, Overexpression of the I{kappa}B{alpha} super-repressor suppresses the baseline activity of NF-{kappa}B in AtT20 cells compared with cells transfected with the control vector. Cells were cotransfected with CMV-I{kappa}B{alpha} super-repressor and 6xNF-{kappa}B-tk-Luc vector or with the CMV-control-vector missing the I{kappa}B{alpha} cDNA (control vector) and 6xNF-{kappa}B-tk-Luc vector. The transcriptional activity of NF-{kappa}B is shown. ***, P < 0.0001 for the luciferase activity for the transfection with super-repressor compared with the transfection with control vector. B, AtT20 cells were transfected with CMV-I{kappa}B{alpha} super-repressor or with the CMV-control vector. The cell viability as assessed by the MTT reduction is shown after 60 µM H2O2 challenge for 20 h. **, P < 0.001 cells transfected with the super-repressor + H2O2 compared with cells transfected with the control vector + H2O2.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
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-{kappa}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. 2Go and 3Go). 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. 4Go and 5Go), 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 {alpha}-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, {alpha}-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-{kappa}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-{kappa}B (Fig. 6Go). This observation is consistent with studies from Parry and Mackman (54 ), who showed an inhibition of NF-{kappa}B by cAMP in immune cells and proposed that the NF-{kappa}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 1Go) underlines the importance of PKA downstream signaling of CRH-R1 in this model. Recently, NF-{kappa}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-{kappa}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-{kappa}B by the I{kappa}B-{alpha} super-repressor of NF-{kappa}B activity, the same construct we used also in the present study to block NF-{kappa}B (26 ). Interestingly, an increased NF-{kappa}B activity has been found to be associated with Aß deposits in affected AD brain regions (25 ), suggesting a participation of NF-{kappa}B and NF-{kappa}B-driven genetic programs in the AD-associated pathological events. Indeed, the suppression of NF-{kappa}B by I{kappa}B-{alpha} mimicked the cytoprotective effect of CRH (Fig. 7Go). This strongly suggests that the suppressive effect of CRH on NF-{kappa}B was directly mediating its protective activity. The block of NF-{kappa}B by CRH may either directly lead to the suppression of NF-{kappa}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-{kappa}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 {alpha}-helical CRH9–41, 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 {alpha}-CRH9–41 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. 1–3GoGoGo). 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
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 Eagle’s 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-{kappa}B transient transfections were performed using a NF-{kappa}B reporter plasmid containing 6 NF-{kappa}B-binding DNA consensus sites linked to a luciferase reporter gene (NF-{kappa}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-{kappa}B was specifically suppressed employing transfections with the I{kappa}ß{alpha} 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 {alpha}-helical CRH9–41 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-{kappa}B (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were performed exactly as described (26 ) employing radioactively labeled oligonucleotide probes for NF-{kappa}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-{kappa}B-Luc and Tk-Luc plasmids, and D. W. Ballard for the I{kappa}ß{alpha} super-repressor- and I{kappa}ß{alpha}-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. Back

Received for publication February 22, 1999. Revision received September 18, 1999. Accepted for publication September 22, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. 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 213:1394–1397[Free Full Text]
  2. Owens MJ, Nemeroff CB 1991 Physiology and pharmacology of corticotropin-releasing factor. Pharmacol Rev 43:425–473[Medline]
  3. 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 1:265–277
  4. Holsboer F, Barden N 1996 Antidepressants and hypothalamic-pituitary-adrenocortical regulation. Endocr Rev 17:187–205[CrossRef][Medline]
  5. Chen R, Lewis KA, Perrin MH, Vale WW 1993 Expression cloning of a human corticotropin-releasing-factor receptor. Proc Natl Acad Sci USA 90:8967–8971[Abstract/Free Full Text]
  6. 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 137:72–77[Abstract]
  7. 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 254:3067–3069[Abstract]
  8. 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 Alzheimer‘s disease. Nature 319:593–595[CrossRef][Medline]
  9. Pomara N, Singh RR, Deptula D, LeWitt PA, Bissette G, Stanley M, Nemeroff CB 1989 CSF corticotropin-releasing factor (CRH) in Alzheimer‘s disease: its relationship to severity of dementia and monoamine metabolites. Biol Psychiatry 26:500–504[CrossRef][Medline]
  10. 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 Alzheimer‘s disease. Nature 378:284–287[CrossRef][Medline]
  11. 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 82:4245–4249[Abstract/Free Full Text]
  12. Glenner GG 1988 Alzheimer’s disease: its proteins and genes. Cell 52:307–308[CrossRef][Medline]
  13. Hardy J 1997 Amyloid, the presenilins and Alzheimer‘s disease. Trends Neurosci 20:154–159[CrossRef][Medline]
  14. Haass C, Selkoe DJ 1993 Cellular processing of ß-amyloid precursor protein and the genesis of amyloid ß-peptide. Cell 75:1039–1042[CrossRef][Medline]
  15. Weidemann A, Konig G, Bunke D, Fischer P, Salbaum JM, Masters CL, Beyreuther K 1989 Identification, biogenesis, and localization of precursors of Alzheimer’s disease A4 amyloid protein. Cell 57:115–26[CrossRef][Medline]
  16. Behl C, Davis JB, Lesley R, Schubert D 1994 Hydrogen peroxide mediates amyloid ß protein toxicity. Cell 77:817–822[CrossRef][Medline]
  17. 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 58:615–622[CrossRef][Medline]
  18. 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 10:243–254[CrossRef][Medline]
  19. 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 629:275–282[CrossRef][Medline]
  20. 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 666:151–167[CrossRef][Medline]
  21. 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 96:7547–7552[Abstract/Free Full Text]
  22. Nitsch RM, Slack BE, Wurtman RJ, Growdon JH 1992 Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors. Science 258:304–307[Abstract/Free Full Text]
  23. 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 {alpha}. J Biol Chem 268:21097–21101[Abstract/Free Full Text]
  24. Grilli M, Pizzi M, Memo M, Spano P 1996 Neuroprotection by aspirin and sodium salicylate through blockade of NF-{kappa}B activation. Science 274:1383–1385[Abstract/Free Full Text]
  25. Kaltschmidt B, Uherek M, Volk B, Baeuerle P, Kaltschmidt C 1997 Transcription factor NF-{kappa}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 94:2642–2647[Abstract/Free Full Text]
  26. Post A, Holsboer F, Behl C 1998 Induction of NF-{kappa}B activity during haloperidol-induced oxidative toxicity in clonal hippocampal cells-suppression of NF-{kappa}B and neuroprotection by antioxidants. J Neurosci 18:8236–8246[Abstract/Free Full Text]
  27. Schreck R, Zorbas H, Winnacker EL, Bäuerle PA 1991 Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-{kappa}B transcription factor and HIV-1. EMBO J 10:2247–2258[Medline]
  28. 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-{kappa}B. Chem Biol 2:13–22[CrossRef][Medline]
  29. Baeuerle PA, Henkel T 1994 Function and activation of NF-{kappa}B in the immune system. Annu Rev Immunol 12:141–179[Medline]
  30. Baldwin AS 1996 The NF-{kappa}B and I{kappa}B proteins: new discoveries and insights. Annu Rev Immunol 14:649–681[CrossRef][Medline]
  31. Wu M, Lee HY, Bellas RE, Schauer SL, Arsura M, Katz D, Fitzgerald MJ, Rothstein TL, Sherr DH, Sonenshein GE 1996 Inhibition of NF-{kappa}B/rel induces apoptosis of murine B cells. EMBO J 15:4682–4690[Medline]
  32. Beg AA, Baltimore D 1996 An essential role for NF-{kappa}B in preventing TNF-{alpha}-induced cell death. Science 274:782–784[Abstract/Free Full Text]
  33. 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-{kappa}B activation prevents cell death. Cell 87:565–576[CrossRef][Medline]
  34. Van Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM 1996 Suppression of TNF-{alpha}-induced apoptosis. Science 274:787–789[Abstract/Free Full Text]
  35. Wang CY, Marty MW, Baldwin Jr AS 1996 TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of apoptosis. Science 274:784–787[Abstract/Free Full Text]
  36. Lipton SA 1997 Janus faces of NF-{kappa}B: neurodestruction versus neuroprotection. Nat Med 3:20–22[CrossRef][Medline]
  37. Lezoualc’h F, Behl C 1998 Transcription factor NF-{kappa}B: friend or foe of neurons? Mol Psychiatry 3:15–2[CrossRef][Medline]
  38. Dieterich KD, DeSouza EB 1996 Functional corticotropin-releasing factor receptors in human neuroblastoma cells. Brain Res 733:113–118[CrossRef][Medline]
  39. De Souza EB 1995 Corticotropin-releasing factor receptors: physiology, pharmacology, biochemistry and role in central nervous system and immune disorders. Psychoneuroendocrinology 20:789–819[CrossRef][Medline]
  40. Coyle JT, Puttfarcken P 1993 Oxidative stress, glutamate, and neurodegenerative disorders. Science 262:689–695[Abstract/Free Full Text]
  41. 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 137:5747–5750[Abstract]
  42. Wang LY, Salter MW, MacDonald JF 1991 Regulation of kainate receptors by cAMP- dependent protein kinase and phosphatases. Science. 253:1132–1135
  43. 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 264:8802–8810[Abstract/Free Full Text]
  44. Behl C, Skutella T, Lezoualc’h F, Post A, Widmann M, Newton CJ, Holsboer F 1997 Neuroprotection against oxidative stress by estrogens: structure-activity relationship. Mol Pharmacol 51:535–541[Abstract/Free Full Text]
  45. Li Y, Maher P, Schubert D 1997 A role for 12-lipoxygenase in nerve cell death caused by glutathione depletion. Neuron 19:453–463[CrossRef][Medline]
  46. McCarthy NJ, Evan GI 1998 Methods for detecting and quantifying apoptosis. Curr Top Dev Biol 36, 259–278
  47. 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. 1133:275–85
  48. 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 Alzheimer’s disease. Am J Pathol 8:1423–1435
  49. 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-{kappa}B activation. Mol Cell Biol 15:2809–2818[Abstract]
  50. Goel R, Khanduja KL 1998 Oxidative stress-induced apoptosis–an overview. Curr Sci 75:1338–1345
  51. 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 57:1361–1365[CrossRef][Medline]
  52. 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 95:12683–12688[Abstract/Free Full Text]
  53. 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 4:447–451[CrossRef][Medline]
  54. Parry GC, Mackman N 1997 Role of cyclic AMP response element-binding protein in cyclic AMP inhibition of NF-{kappa}B-mediated transcription. J Immunol 159:5450–5456[Abstract]
  55. Fox MW, Anderson RE, Meyer FB 1993 Neuroprotection by corticotropin releasing factor during hypoxia in rat brain. Stroke 24:1072–1077[Abstract/Free Full Text]
  56. Lyons MK, Anderson RE, Meyer FB 1991 Corticotropin releasing factor antagonist reduces ischemic hippocampal neuronal injury. Brain Res 545:339–342[CrossRef][Medline]
  57. 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 656:405–408[CrossRef][Medline]
  58. 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 20:203–205[CrossRef][Medline]
  59. 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 6:1931–1941[Abstract]
  60. Lezoualc’h F, Skutella T, Widmann M, Behl C 1996 Melatonin prevents oxidative stress-induced cell death in hippocampal cells. Neuroreport 7:2071–2077[Medline]
  61. 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[Free Full Text]



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