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Endocrinology, doi:10.1210/en.2003-0168
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Endocrinology Vol. 144, No. 9 4051-4060
Copyright © 2003 by The Endocrine Society

Brain Region-Specific Neuroprotective Action and Signaling of Corticotropin-Releasing Hormone in Primary Neurons

Nadhim Bayatti, Jürgen Zschocke and Christian Behl

Independent Research Group Neurodegeneration, Max Planck Institute of Psychiatry, 80804 Munich, Germany; and Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University Mainz, 55099 Mainz, Germany

Address all correspondence and requests for reprints to: Christian Behl, Ph.D., Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University Mainz, 55099 Mainz, Germany. E-mail: cbehl{at}uni-mainz.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CRH regulates the body’s response to stressful stimuli by modulating the activity of the hypothalamic pituitary axis. In primary cultures and cell lines, CRH also acts as a potent neuroprotective factor in response to a number of toxins. Using primary neuronal cultures from the cerebellum, cerebral cortex, and hippocampus, we demonstrate that CRH exerts a brain region-specific neuroprotective effect on amyloid ß 25–35 toxicity. At low CRH concentrations (10-8 M), neuroprotective effects can be observed only in cerebellar and hippocampal cultures, but a higher CRH concentration (10-7 M) additionally led to the protection of cortical neurons. These neuroprotective effects were inhibited by H89, a specific protein kinase A inhibitor. Western blot analysis, carried out using phospho-specific antibodies directed against MAPK, cAMP response element-binding protein (CREB), and glycogen synthase kinase (GSK)3ß also resulted in brain legion-specific differences regarding intracellular signaling. Correlating with cell survival, low CRH concentrations resulted in activation of the CREB pathway and inactivation of GSK3ß in cerebellar and hippocampal cultures, but higher concentrations additionally resulted in activated CREB and inactivated GSK3ß in cortical cultures. In contrast, MAPK activation occurred only in cortical neurons. Differences in signaling were found to be independent of receptor expression levels because RT-PCR analysis indicated no region-specific differences in CRHR1 mRNA expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRH, A 41-AMINO-acid peptide discovered over 20 yr ago, was found to have a key role mediating neuroendocrine effects occurring in response to stressful stimuli through modulation of the hypothalamic-pituitary-adrenal axis (1, 2). CRH also affects synaptic plasticity and promotes memory and learning, and the CRH system is affected by the progression of Alzheimer’s disease (AD) (3, 4, 5, 6, 7). The peptide exhibits neuroprotective properties in clonal cell lines as well as primary cellular models against a wide variety of toxic molecules including the toxic peptide deposited in AD, amyloid ß (Aß), the excitatory neurotransmitter glutamate, and other oxidative insults (8, 9, 10). To date, two CRH receptors (CRHRs) have been identified, and previous studies from our laboratory have shown that the neuroprotective effect of CRH is mediated through its high-affinity cell surface receptor, the CRHR type 1 (CRHR1) (8). Both CRHRs are G protein-coupled receptors linked to number of intracellular signaling pathways including adenylate cyclase-cAMP-protein kinase A (PKA), MAPK, and protein kinase C (PKC) (11). Specific CRH-binding sites are located throughout the brain including the areas particularly affected by AD, such as the hippocampus and cerebral cortex. Intriguingly, CRHR1 receptors are found at particularly high levels in the cerebellum, a region relatively spared from AD pathology (12, 13, 14). The function of the receptors in these areas remains unclear, although they may be associated with a neuromodulatory or developmental role of CRH. In addition, direct activation of hippocampal CRHR1 receptors results in enhanced learning in response to fear conditioning (15), and this process is dependent on the activation of muscarinic glutamate receptors (16). Moreover, when CRH was applied to the lateral septum in the same fear conditioning model, learning was impaired in a CRHR2-dependent fashion, indicating a modulation of this behavior in a region-specific manner depending on receptor subtype (15). Thus, CRH appears to have an important role in modulating the memory-forming processes that are particularly affected by neurological diseases resulting in dementia such as AD. Concentrations of CRH immunoreactivity are reduced in AD tissue, compared with controls, and this is reciprocated by an increase of CRH receptor levels in affected cortical areas (3).

AD is characterized by the deposition of the Aß peptide in amyloid plaques as well as the formation of neurofibrillary tangles (NFTs) composed mainly of the hyperphosphorylated protein tau. A number of protein kinases are known to promote the phosphorylation of tau. Glycogen synthase kinase (GSK)-3ß for example, which is a member of the notch-signaling pathway, can phosphorylate a number of sites on tau and is closely associated to the type observed in AD (17). The activity of GSK-3ß can be regulated at two phosphorylation sites. Phosphorylation of tyrosine216 has been associated with an activation of the molecule, and a phosphorylation of serine9 has been associated with the inactivation of GSK-3ß-mediated tau phosphorylation (for review, see Ref. 18).

In this study we investigate the possibility as to whether a brain region-specific neuroprotective action of CRH against the toxic fragment of Aß (Aß 25–35) exists by carrying out cellular survival assays on rat primary cultures of neurons established from three brain regions, the cerebral cortex, hippocampus, and cerebellum. Any putative differences in neuroprotection may be mirrored by differences in the signaling profile of the peptide. CRH-mediated activation on two previously known targets, cAMP response element-binding protein (CREB) and MAPK were examined in these cultures. The intracellular signaling pathways responsible for CRH-mediated neuroprotection are analyzed using specific inhibitors for a number of these pathways as well as focusing on the possible involvement of CRH in preventing the process of tau phosphorylation by analyzing putative effects on GSK3ß.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and primary cell culture
Primary mixed neuronal cultures were established as described previously with some minor modifications (19). Timed mated Sprague Dawley (Charles River, Sulzfeld, Germany) rats were maintained at a 12-h dark, 12-h light cycle and were allowed to access food and water ad libitum. Pregnant dams were killed by CO2 inhalation, and newborns were physically decapitated. Neocortical and hippocampal tissue was dissected from embryonic d 18 rat brains while cerebellar tissue was removed from postnatal d 3 animals. After dissection, tissue pieces were incubated for 20 min in Ca2+-, Mg2+-free Dulbecco’s PBS (Life Technologies, Inc., Karlsruhe, Germany) containing 0.1% trypsin and 0.02% EDTA. Cells were then transferred to Ca2+-, Mg2+-free Hanks’ balanced salt solution (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies, Inc.) and dissociated gently by titration. Undissociated matter was filtered through a 50-µm pore-sized Nybolt mesh (Eckardt, Waldkirch, Germany), and cells were centrifuged at 200 x g for 4 min. The pellet was resuspended in MEM (Life Technologies, Inc.) supplemented with 10% horse serum (Life Technologies, Inc.), and the number of viable cells excluding trypan blue were counted. Cells were seeded in either 6-well or 96-well cluster plates (TPP, Trasadingen, Switzerland) that had been previously coated with poly-L-ornithine (0.1 mg/ml; molecular mass 100–200 kDa; Sigma, Munich, Germany) at a density of 150,000 cells/cm2 and 50,000 cells/well, respectively. In all experiments, culture medium was changed after 1 d in vitro to serum-free neurobasal medium (Life Technologies, Inc.) supplemented with B27 without antioxidants (Life Technologies, Inc.). Rat/human CRH (10-6 to 10-8 M; Calbiochem, Schwalbach, Germany) was used throughout, Urocortin (10-7 to 10-8 M; Calbiochem), Aß (2–40 µM; Bachem, Heidelberg, Germany), H89 (10 µM; Calbiochem), forskolin (2 x 10-6 M; Tocris, Ellisville, MO) were added to the cultures as specified in the text.

Western blotting
Cells were scraped off dishes in the presence of lysis buffer consisting of 60 mM Tris-HCl, 2% sodium dodecyl sulfate (SDS), 10% sucrose, 2 mM phenylmethylsulfonyl fluoride (Merck, Darmstadt, Germany), 1 mM sodium orthovanadate (Sigma), 10 µg/ml aprotinin (Bayer, Leverkusen, Germany). Lysates were quickly sonified, boiled 5 min at 95 C, and stored at -80 C until used. Protein concentrations were measured using a standard BCA assay (Perbio, Bonn, Germany), and samples were diluted in sample buffer [250 mM Tris-HCl (pH 6.8), containing 4% SDS, 10% glycerol, 2% ß-mercaptoethanol, and 0.002% bromophenol blue] and boiled for a further 5 min. Proteins from each sample (10 µg/well) were separated by SDS-PAGE (10%) and subsequently transferred to nitrocellulose membranes by electroblotting. Membranes were blocked in 5% nonfat milk in TBS/Tween 20 (0.5%) for 30 min and incubated in the presence of the primary antibody overnight at 4 C. The following antibodies were used: anti-ERK1/2, antiphospho-ERK1/2, antiphospho-GSK3ß, GSK3ß (all from Cell Signal, Frankfurt, Germany; all 1:500), antiphospho-CREB (Upstate Biotechnologies, Lake Placid, NY; 1:500), anti-CREB (Calbiochem; 1:500), and CRHR (1:100; Santa Cruz Biotechnologies, Santa Cruz, CA). Proteins were detected using the enhanced chemiluminescence kit (Amersham, Freiburg, Germany) after incubation of blots for 2 h at room temperature with corresponding horseradish peroxidase-conjugated antibodies (Dianova, Hamburg, Germany). ODs of bands were calculated using Scion software (Frederick, MD); in all cases levels of phosphorylated proteins were normalized to total unphosphorylated levels and depicted as a ratio in bar diagrams.

Cell fractionation
Membrane fractions were prepared as previously described (20). Briefly, cells were scraped off in sucrose buffer (250 mM sucrose, 5 mM Tris, 5 µg/ml aprotinin, and 10 mM phenylmethylsulfonyl fluoride), lysed by vigorous shaking, and spun at 720 x g for 5 min. The pellet was discarded and the supernatant was spun at 100,000 x g for 45 min, 4 C. Resulting supernatant was discarded, and the pellet was resuspended in Western blotting lysis buffer and labeled as membrane fraction. Proteins were stored at -80 C until used.

Cellular survival assays
Cell survival was assayed by two methods previously established (21): 1) colorimetric assays of metabolic activity using tetrazole 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT; Sigma), and 2) counting total cell number in the presence of the fluorescent DNA intercalator propidium iodide (PI) (Sigma). In the case of the former, cells were incubated in the presence of 0.5 mg/ml of the MTT reagent for 4–8 h, after which the cells were lysed by the addition of 1 volume of solubilization solution (40% dimethylformamide, 10% SDS, pH 4.0). ODs in wells were read after 24 h with a Dynatech plate reader with a 550 nm cut-off filter (DynaTech Corp., Eningen, Germany). Diagrams indicated the extent of cellular survival expressed as a percentage of control. In the case of the PI staining, cells were rinsed in PBS before incubation with 1 mg/ml PI for 5 min. Cultures were then rinsed again with PBS, and the total cell number in one visual field of the microscope (magnification, x200) was counted blind for treatment through one diameter of each well. In both cases experiments were repeated at least three times with similar results.

RT-PCR
Total RNA from primary hippocampal, cerebellar, and cortical neurons was isolated using the Absolutely RNA RT-PCR miniprep kit according to the manufacturer’s instructions (Stratagene, Amsterdam, The Netherlands). Synthesis of cDNA was performed at 37 C for 60 min on 0.75 µg of denatured (5 min at 75 C) total RNA. The 20-µl reaction mixture contained 10x reverse transcriptase buffer (QIAGEN, Hilden, Germany), 2 µl of 10 µM oligo(dT)23 primer (Sigma), 2 µl of 5 mM deoxynucleotide triphosphate (QIAGEN), 10 U RNasin (Promega, Mannheim, Germany), and 4 U Omniscript reverse transcriptase (QIAGEN). A mock control without reverse transcriptase was carried out to exclude DNA contamination. PCR amplifications were performed on 2 µl cDNA in a final volume of 25 µl including 2.5 µl 10x PCR buffer (Invitrogen, Karlsruhe, Germany), 0.75–1.5 µl 50 mM MgCl2, 0.5 µl of 10 mM deoxynucleotide triphosphates (peqLab, Erlangen, Germany), 0.5 µl of 15 pmol sense and antisense primers, and 0.5 U Taq polymerase (Invitrogen). PCR conditions were 30 sec at 94 C, 30 sec at 55–57 C, and 45 sec at 72 C for 30–31 cycles. These cycles were preceded by a 5-min denaturation step at 95 C and followed by a 5-min elongation step at 72 C. Total PCR samples were run on agarose gels and detected by ethidium bromide staining. The PCR primers were provided by ThermoHybaid (Ulm, Germany). A PCR was performed with primers specific for ß-actin, 5'-CTACAATGAGCTGCGTGTGGC-3' (sense), and 5'-CAGGTCCAGACGCAGGATGGC-3' (antisense) as control for cDNA quality and quantity; this primer set amplified a 275-bp PCR product. Specific primers for the CRHR isoforms have been designed as follows: 5'-CTGAGTGCCAGGAGATTCTC-3' (sense) and 5'-CGTAGTGCAGCTTCCCAATG-3' (antisense) for CRHR1 (462-bp PCR product); 5'-TGATCCACTGGAACCTCATC-3' (sense) and 5'-TGGAGTACGTCATGACGATG-3' (antisense) for CRHR2 (208-bp product). To ensure that the PCR products fall within the linear range, cycle dependency was carried out for actin (24–30 cycles), CRHR1 (26–32 cycles), and CRHR2 (28–36 cycles). PCR samples were run on agarose gels, ODs were measured using Scion Image software (Scion Corp.), and plotted on graphs. In each case the correlation coefficient, R2 was 0.95 or greater.

Statistics
Experiments with statistics were carried out three to five times with similar results. ODs of bands from Western blots were measured with Scion Image software (Scion Corp.) using the ratios of phosphorylated:unphosphorylated bands. Values were subsequently calculated and plotted as fold increase of untreated controls. Representative blots are shown. In both Western blots and cell survival assays, one-way ANOVA was carried out using Sigma Stat software (SPSS Science, Chicago, IL) using data pooled from independent experiments, and significance between treatment groups was further analyzed using the post hoc Tukey test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Brain region-specific neuroprotective action of CRH against Aß-induced toxicity in primary neuronal cultures
The cell death accompanying the pathological processes underlying AD has been reported to commence in the transentorhinal region of the cerebral cortex, spreading to limbic system, before extending to the association areas of the cerebral cortex (14). Later other brain regions including the hippocampus also become affected (22), although the cerebellum remains relatively spared. Interestingly, the cerebellum contains a high level of specific CRH-binding sites, and we analyzed whether pathological brain region-specific differences in AD also extend to the protective action of CRH in the brain. Primary neurons were established from cortical, hippocampal, and cerebellar rat brain tissue and initially pretreated with CRH (10-8 M, 24 h) (8). This concentration and time period was previously known to be optimally protective in both primary neuronal and clonal cell line cultures in response to a number of toxins including Aß (8). Subsequently cells were incubated for 48 h with Aß 25–35. Previous studies showed that cotreatment of neurons with both CRH and toxin are ineffective in affording neuroprotection (8). MTT cell survival assays (Fig. 1Go) indicated that CRH has a protective effect against Aß 25–35 toxicity in cerebellar and hippocampal cultures, yet this effect seems to be lacking in cortical cells, in which no differences in survival between CRH pretreated or untreated cells were observed after incubation with Aß 25–35. The protective effect of 24-h CRH pretreatment against Aß 25–35-induced toxicity was confirmed in cell counts of hippocampal primary cultures incubated with PI, a marker of dead cells (Fig. 2Go). The number of CRH pretreated cells positively stained by PI after 48 h incubation with Aß 25–35 was significantly lower than cells incubated with Aß 25–35 alone.



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  		FIG. 1. CRH-mediated neuroprotection of primary cultures from different brain regions using MTT assays. Primary cells from cerebellum, hippocampus, and cortex were treated with CRH (10-8 M) 24 h before administration of Aß (25 26 27 28 29 30 31 32 33 34 35 ). MTT assays were carried out after a further 24 h. Graphs represent neuronal survival calculated as percentage of controls. Statistically significant differences between CRH-treated and untreated cells are indicated (*): P < 0.05 as calculated using the post hoc Tukey test (n = 3–5 for each region).

 


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  		FIG. 2. CRH-mediated neuroprotection in primary hippocampal cell cultures. Primary hippocampal cultures were treated with CRH (10-8 M) 24 h before administration of Aß (25 26 27 28 29 30 31 32 33 34 35 ). Subsequently, live cells were incubated with PI (1 mg/ml) and photographed with a green UV fluorescent filter (PI) or under phase contrast. The graph shows cell counts for PI-positive stained cell nuclei found in each visual field. Statistically significant differences between CRH-treated and untreated cells are indicated (*): P < 0.05 as calculated using the post hoc Tukey test (n = 3). Scale bars, 50 µM.

 
Brain region-specific activation of intracellular pathways by CRH in primary neuronal cultures
CRH binds specifically to CRHR1, a G protein-linked seven-transmembrane domain spanning protein associated with a number of putative neuroprotective intracellular signaling pathways (11). These have been reported to include MAPK and cAMP/PKA/CREB. Analyzing CRH-induced activation of these pathways by Western blotting using phospho-specific antibodies, we investigated whether the brain region-specific differences exhibited by CRH in the cell survival assays correlated with the activation of any particular pathway(s). Using antibodies directed against the phosphorylated (active) forms of MAPK 1/2 (pERK 1/2), and of CREB (pCREB), we also observed region-specific differences (Fig. 3Go). CRH (10-8 M), induced robust levels of pCREB in both cerebellar and hippocampal cultures with no observable effect on pCREB levels in cortical cultures. In contrast, pERK levels increased only transiently (within 30 min) in the cerebral cortex, and no such increases were observed in either hippocampal or cerebellar cultures.



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  		FIG. 3. Identification of CRH signaling profile in primary neurons from various brain regions. Primary neurons from cerebellum, hippocampus and cerebral cortex were treated with CRH (10-8 M) for the indicated times (positive controls: S, 10% horse serum; F, forskolin 5 x 10-6 M, both 30 min). Western blot analysis was carried out using phospho-specific and total antibodies for CREB, MAPK 1/2 (ERK 1/2), and GSK3ß. ODs of the bands were measured, and graphs show the fold increase in activated proteins for each time point, compared with controls. Statistically significant differences between CRH-treated and untreated cells are indicated (*): P < 0.05 as calculated using the post hoc Tukey test (n = 3–5 for each set of blots).

 
Besides the activation of putative neuroprotective pathways, an alternative activity of CRH may involve the inhibition of pathways leading to cell death. Therefore, the effect of CRH on GSK3ß activation was also carried out. GSK3ß is a proapoptotic signaling kinase that mediates the toxic effects that result as a consequence of neurofibrillary tangle formation by promoting tau phosphorylation (23). In a manner similar to CREB phosphorylation, a low CRH concentration (10-8 M) led to the phosphorylation of GSK3ß at ser9 (indicating a deactivation of the protein) in cultures from both cerebellar and hippocampal brain regions but not in cultures from the cerebral cortex (Fig. 3Go). Interestingly, the temporal patterns of both CREB and GSK3ß phosphorylation differ between regions. Phosphorylation is more sustained in hippocampal cultures (activation peak 60 min) as compared with cerebellar cultures (activation peak 15–30 min).

In conclusion, brain-region specific differences in CRH signaling exist, and the activation of the CREB and inactivation of GSK3ß pathways correlate with the survival-promoting effect of CRH.

CRH-induced survival in cortical cultures occurs at higher concentrations and is also paralleled by activation of CREB and inactivation of GSK3ß
Further clarification of the brain-region specific differences in CRH-mediated neuroprotection was carried out in cortical cultures. CRHR1 receptors may alter in receptor density or be differentially linked to multiple G proteins in different brain regions, leading to differential intracellular signaling pathway response to CRHR activation (24). Therefore, higher CRH concentrations may be required to induce the activation of cell survival-promoting intracellular pathways in the cerebral cortex. Pretreatment of cortical cultures with a higher concentration of CRH (10-7 M) for 24 h results in a significant increase of cell survival after subsequent Aß 25–35-induced toxicity as measured with MTT (Fig. 4AGo). Furthermore, a robust increase in CREB phosphorylation, as well as GSK3ß inactivation (ser9 phosphorylation), was observed after 30 min CRH exposure to the cultures, only at CRH concentrations of 10-7 M or more (Fig. 4Go, B and C). The observation that higher concentrations of CRH are required to promote protection against Aß 25–35-induced toxicity in cortical neurons may be due to effects that CRH exerts on the CRHR2, which shows lower affinity. Therefore, MTT assays for Aß 25–35 toxicity (5 µM) were carried out in cortical cultures using the potent CRHR1 antagonist R121919 (25), with CRH (10-6 to 10-8 M) and urocortin (10-7 to 10-8 M), a high-affinity CRHR2 and CRHR1 ligand (Fig. 4DGo). Cotreatment of neurons with R121919 (100 ng/ml) in combination with neuroprotective concentrations of CRH (10-7 to 10-8 M), or urocortin (10-7 to 10-8 M) 24 h before toxic insult resulted in the inhibition of the protective response. R121919 alone had no effect on cell viability. In conclusion, brain-region differences observed are concentration dependent, and these data further support the hypothesis that CRH activation of CRHR1 and subsequent intracellular pathways leading to CREB phosphorylation or inactivation of GSK3ß correlate with the neuroprotective function of this peptide.



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  		FIG. 4. CRH-mediated CREB phosphorylation and neuroprotection in cortical cultures. Survival assays (A and D) and Western blots (B and C) were carried out using cortical cultures to analyze the effect of higher concentrations of CRH. A, Cortical neurons were treated with CRH (10-7 M) 24 h before administration of Aß 25–35. MTT assays were carried out after a further 24 h. The graph represents neuronal survival calculated as percentage of controls. Statistically significant differences between CRH-treated and untreated cells are indicated (*): P < 0.05 as calculated using the post hoc Tukey test (n = 4). B, Primary cells from cortex were treated with CRH (10-8 to 10-6 M), forskolin (F), or dbcAMP (cAMP) for 30 min. Western blots were then carried out with phospho-specific and total CREB antibodies. Statistically significant differences between CRH-treated and untreated cells are indicated (*): P < 0.05 as calculated using the post hoc Tukey test (n = 3). C, Western analysis using phospho-specific and total GSK3ß antibodies after cortical neurons were treated with CRH (10-7 M) for the indicated time periods. Statistically significant differences between CRH-treated and untreated cells are indicated (*): P < 0.05 as calculated using the post hoc Tukey test (n = 4). D, Cortical neurons were treated with CRH or urocortin at the indicated concentrations, alone or in combination with a potent CRHR1 antagonist, R121919 (100 ng/ml), 24 h before administration of Aß 25–35. MTT assays were carried out after a further 24 h. The bar diagram represents neuronal survival calculated as percentage of controls. R121919 alone is represented by a gray bar. Statistically significant differences between cells treated with hormone alone and cells treated with a combination of hormone and R121919 are indicated (*): P < 0.05 as calculated using the post hoc Tukey test (n = 4).

 
Differential expression of CRHR1 and CRHR2 in primary neuronal cultures
Any region-specific differences observed in CRH signaling may depend on the levels of CRHRs expressed. Therefore, RT-PCR was carried out to characterize the relative amounts of CRHR transcripts in our cell culture models (Fig. 5AGo). The receptor responsible for the neuroprotective effects of CRH, CRHR1, is expressed in cerebellar, hippocampal, and cortical cultures at equal levels, as characterized by the presence of prominent PCR products specifically amplified from CRHR1 mRNA. Conversely, the lower-affinity CRHR2 is expressed only weakly in hippocampal and cortical cultures, and cerebellar neurons exhibited no detectable levels of this receptor. PCR was carried out in each case within the linear range of cycles for each particular primer pair.



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  		FIG. 5. Differential expression of CRHR1 and CRHR2 in primary neurons from different brain regions. A, RT-PCR was carried out with total RNA extracted from neuronal cell cultures initiated from the cerebellum (CER), hippocampus (HIPP), and cerebral cortex (CTX), using primers specific for the CRHR1 and CRHR2 receptors. As loading controls, PCR using ß-actin-specific primers was also carried out (M, 100-bp ladder; N, negative control). B, Western blot showing expression of CRHRs in membrane fractions from CER, HIPP, and CTX. Blotting for actin was also carried out as loading control.

 
Western blot analysis was also carried out on membrane fractions of cerebellar, hippocampal, and cortical cultures using an antibody that recognizes both CRHR1 and CRHR2 (Fig. 5BGo). A band was detected migrating at approximately 58 kDa, consistent with the molecular mass of brain CRHR (11). Hippocampal and cortical neurons appear to express CRHRs at higher levels than in the cerebellar neurons.

In conclusion, differences in mRNA and protein expression of the CRHRs in different brain regions do not correlate with the different intracellular signaling pathways activated by CRH.

CRH-mediated neuroprotection of hippocampal neurons occurs through a PKA-dependent mechanism
CRH, by binding to its receptor, can induce production of cAMP through G protein-dependent activation of adenylate cyclase (11). This in turn leads to the activation of PKA and further targets downstream such as CREB. In addition to PKA, activation of other intracellular signaling pathways such as PKC and MAPK also channel to pCREB (26). Closer examination of the pathways involved in CRH-mediated neuroprotection was carried out in MTT cell survival assays using H89, Gö6983, and PD98059, specific inhibitors of PKA, PKC, and MAPK, respectively. In primary hippocampal cultures, both MTT toxicity assays and total cell counts indicated that only H89 (10 µM; Fig. 6AGo) and not Gö6983 (1 µM) or PD98059 (20 µM) (data not shown) inhibited CRH-mediated neuroprotection against Aß 25–35. H89 completely inhibited CRH (30 min, 10-8 M)-induced activation of CREB, as demonstrated by Western blotting using phospho-specific antibodies (Fig. 6BGo). Therefore, CRH-induced activation of CREB appears to be PKA mediated. In conclusion, the neuroprotective effect of CRH on primary neurons occurs in a PKA-dependent, PKC-independent, and MAPK-independent manner and is in agreement with previous studies (10).



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  		FIG. 6. Mechanism of CRH-mediated neuroprotection. A, MTT assay from hippocampal neurons pretreated for 24 h with CRH (10-8 M), H89 (10 µM) alone, or in combination, followed by 24 h Aß 25–35 (40 µM) treatment. Diagram represents neuronal survival calculated as percentage of untreated controls. Statistically significant differences between CRH-treated vs. untreated cells as well as between CRH- vs. CRH \ H89-treated cells are indicated: * and **, respectively. In both cases P < 0.05 as calculated using the post hoc Tukey test (n = 4). B, Western blot showing efficacy of H89-mediated inhibition of CRH-induced CREB in hippocampal neurons. Cells were treated with CRH (10-8 M), H89 (10 µM) alone, or in combination for 30 min. Statistically significant differences between CRH-treated and untreated cells are indicated (*): P < 0.05 as calculated using the post hoc Tukey test (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using primary neuronal cultures, we demonstrated in this study that CRH exhibits brain region-specific differences in neuroprotection against Aß toxicity as well as activation of intracellular signaling pathways. Although lower CRH concentrations (10-8 M) promote neuronal survival in cerebellar and hippocampal cultures, higher CRH (<=10-7 M) concentrations are required for cortical neurons. Moreover, CRH protects neurons from Aß toxicity through a common pathway involving activation of PKA, and additionally, inactivation of GSK3ß may be involved in inhibiting processes such as tau phosphorylation.

Previous studies from our laboratory identified CRH as a neuroprotective molecule counteracting the toxic effects of a number of stressors including Aß in cerebellar primary neurons as well as clonal cell lines (8). In addition, using a specific CRHR1 antagonist, antalarmin, these studies identified CRHR1 as the specific receptor subtype responsible for mediating these neuroprotective effects. Moreover, CRH is able to induce the increased turnover of the nonamyloidogenic Aß precursor protein by promoting the activity of the nontoxic {alpha}-secretase pathway (8). Here we show that CRH is able to promote neuronal survival in cortical and hippocampal neurons, the classical primary sites of degeneration during AD. Recent reports have confirmed the protective nature of CRH against Aß toxicity as well as the requirement of CRHR1 to mediate the effect (10, 27).

We have also shown a brain region-specific difference in the neuroprotective action of CRH that is concentration dependent. Cortical neurons require CRH at a high nanomolar or low micromolar concentration to promote survival, but cerebellar and hippocampal cultures require CRH in the nanomolar concentration range. This is in contrast to the study of Pedersen et al. (10) in which CRH protects cortical and hippocampal neurons against Aß 25–35 (1 µM) toxicity already at picomolar levels. These discrepancies may be due to cell-culture differences because we required higher concentrations of Aß 25–35 to induce cell death (5–40 µM). Nevertheless, in our study, the application of CRH in the nanomolar range is physiologically relevant because previous published studies show that CRH stimulation of cAMP by adenyl cyclase activation, proopiomelanocortin-peptide secretion, and activation of cAMP-dependent protein kinase in anterior pituitary cells occurs at EC50 values in the nanomolar range (28, 29). The pharmacological profile (concerning EC50 values) of CRH is similar in the brain, pituitary, and spleen (11, 30, 31, 32). Brain-region specific differences were also observed concerning the signaling profile of CRH in neuronal cultures from the three brain regions studied. We demonstrated, in a manner correlating with the survival-promoting effects of CRH, that CREB activation and GSK3ß inactivation occurs in cerebellar and hippocampal cultures already at low CRH concentrations, but cortical cultures require higher CRH concentrations.

Activation of the MAPK pathway was observed only in cortical cultures. Interestingly, the temporal patterns of both CREB and GSK3ß phosphorylation differ between regions, i.e. CRH induces a more sustained phosphorylation in hippocampal cultures (activation peak 60 min), compared with cerebellar cultures (activation peak 15 min). The observed regional and temporal differences in signaling may be due to differential receptor densities and/or differentially linked G proteins because of receptor activation of both CRHR1 and CRHR2 subtypes, regional expression of specific G proteins, or even differential intracellular compartmentalization of G proteins. In the cerebellum, CRHR1 mRNA and protein has been localized to granular cell layer and is exclusively the only CRHR subtype present. Similarly in the neocortex, CRHR1 is the predominant subtype present. In the hippocampus CRHR1 can be found in the CA1 and CA3 regions of the hippocampus as well as the dentate gyrus. In contrast to the cerebellum, CRHR2, the low-affinity CRHR, is additionally present throughout the hippocampal formation (33, 34, 35). Characterization of the mRNA levels of CRHRs in our cellular models are in agreement with these previous observations. The level of CRHR1 transcripts appears to be constant throughout cultured cerebellar, cortical, and hippocampal neurons, but CRHR2 transcripts are present at low levels in the cerebral cortex and hippocampus. Therefore, considering the similar effects of CRH on cerebellar and hippocampal cultures in terms of both neuroprotection and signaling, it is most likely that regional differences and/or intracellular membrane distribution of G proteins account for the observed differences. A recent report demonstrated that stimulated CRHRs in rat cortical membranes are linked to the activation of at least five different G proteins including multiple Gs subtypes (36). The authors were uncertain as to which Gs or combinations of Gs proteins result in the activation of adenylate cyclase and concluded that tissue-specific differences exist.

The activation of the MAPK pathway by CRH has been previously reported in cortical cultures (37) as well as hippocampal slices (9). Although CRHR1-dependent activation of MAPK by CRH is thought to occur through a Gq-mediated mechanism that involves PKC and Raf-1 phosphorylation in endometrial cells (24), no direct evidence is available to suggest that this also occurs in neuronal tissue. CRH receptors are linked to a number of other receptors including those for neurotransmitters. {gamma}-Aminobutyric acid, opioid, muscarinic, and N-methyl-D-aspartate glutamate receptors as well as D2 dopamine receptors have all been reported to be linked to CRHR action and future studies must focus on the involvement of such receptors on any intracellular CRH response (4, 16, 38, 39, 40). The function of CRH-induced MAPK activation in neuronal cells is unclear. CRH-induced MAPK activation accompanies neuroprotection against glutamate toxicity in hippocampal slices (9) but is not thought to be involved in protecting against Aß toxicity (10). Additionally, CRH-mediated induction of long-term depression in the cerebellum is PKC dependent, and induction of long-term potentiation in the hippocampus may also require activation of this pathway (6, 7). MAPK-mediated effects by CRH on neurite outgrowth of a catecholaminergic cell line have been reported (41), and it has been speculated that peptides such as CRH may play an important role during neural development (42). This may particularly be the case in the cerebellum in which CRHR1 is present in the embryonic cerebellar anlage (43) and follows a defined temporal and spatial expression pattern during cerebellar development (44).

The intracellular downstream mechanisms responsible for CRH-mediated neuroprotection were further analyzed. Neuroprotection against Aß toxicity was observed only at CRH concentrations that led to an activation of the transcription factor CREB. Therefore, the involvement of the intracellular signaling pathways leading to the phosphorylation of this transcription factor were studied in more detail. CREB phosphorylation occurs through both MAPK- and PKA-dependent mechanisms (26). In agreement with a previous report, CRH-mediated neuroprotection was found to be PKA dependent using the specific inhibitor H89, indicating that the adenylate cyclase-cAMP-PKA pathway is responsible for the observed effects (10). Conversely, the MAPK inhibitor PD98059 and the PKC inhibitor Gö6983 had no effect on CRH-mediated neuroprotection.

The classical hallmarks of AD are the formation of amyloid plaques, composed mainly of Aß protein, and also the production of NFTs, which contain high levels of the hyperphosphorylated protein tau. Located primarily in axons and associated to mictrotubules, tau undergoes phosphorylation by a number of kinases including GSK3ß (40). Hyperphosphorylation of tau leads to disassociation from microtubules and subsequent abnormal and/or overproduction, causing aggregation of the molecule (45, 46). In addition, higher levels of GSK3ß have been found in Alzheimer brains, compared with controls, and GSK3ß also associates with NFTs in affected tissue (47, 48, 49). Therefore, the observation that CRH promotes phosphorylation of serine9, and hence the inactivation of basal GSK3ß in all brain regions analyzed is particularly striking. Future experiments should address whether CRH or other specific CRHR1 ligands may actually influence tau (hyper)phosphorylation itself both in vitro and in vivo.

In conclusion, the presented results demonstrate that CRH induces concentration-dependent, brain region-specific differences in neuroprotection and signaling in a primary neuronal model. CRH is able to activate neurprotective-promoting pathways in response to Aß toxicity in all regions analyzed, and this occurs through a PKA-dependent pathway. Considering the effects of CRH on mediating the stress response, this molecule may not be directly relevant in the treatment of AD; nevertheless, the CRH system is strikingly affected by the progression of the disease in the cerebral cortex and striatum (3, 50, 51). Studies have reported that CRH levels fall from approximately 10–20 pg/mg wet weight tissue in the cerebral cortex of control brains to less than 5 pg/mg wet weight tissue in AD-affected cerebral cortices. Reciprocal increases in the CRHR1 are also observed through the cerebral cortex in affected brains (3). Conversely in the hippocampus, CRH levels show little or no difference between control and AD-affected brains (52).

Taken together with previous reports, activation of CRHR1-dependent pathways seem to be particularly beneficial against the pathological processes occurring in AD. CRH can also direct the processing of Aß precursor protein to the potentially nontoxic {alpha}-secretase pathway (8) as well as inactivate GSK3ß, a kinase involved in neurofibrillary tangle formation. Future studies must therefore focus on the development of compounds that increase the amount of free circulating CRH in the brain, a process that leads to improved memory and learning in rats (53).


   Acknowledgments
 
We thank Dr. F. Ohl for supplying R121919.


   Footnotes
 
Current address for N.B., J.Z., and C.B.: Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University Mainz, 55099 Mainz, Germany.

This work was supported by the Verum Foundation.

Abbreviations: AD, Alzheimer’s disease; Aß, amyloid ß; CREB, cAMP response element-binding protein; CRHR, CRH receptor; CRHR1, CRHR type 1; GSK, glycogen synthase kinase; MTT, tetrazole 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide; NFT, neurofibrillary tangle; pCREB, phosphorylated (active) form of CREB; pERK, phosphorylated (active) form of MAPK; PI, propidium iodide; PKA, protein kinase A; PKC, protein kinase C; SDS, sodium dodecyl sulfate.

Received February 3, 2003.

Accepted for publication June 2, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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