Carboxypeptidase E Protects Hippocampal Neurons During Stress in Male Mice by Up-regulating Pro-survival BCL2 Protein Expression

Abstract

Prolonged chronic stress causing elevated plasma glucocorticoids leads to neurodegeneration. Adaptation to stress (allostasis) through neuroprotective mechanisms can delay this process. Studies on hippocampal neurons have identified carboxypeptidase E (CPE) as a novel neuroprotective protein that acts extracellularly, independent of its enzymatic activity, although the mechanism of action is unclear. Here, we aim to determine if CPE plays a neuroprotective role in allostasis in mouse hippocampus during chronic restraint stress (CRS), and the molecular mechanisms involved. Quantitative RT-PCR/in situ hybridization and Western blots were used to assay for mRNA and protein. After mild CRS (1 h/d for 7 d), CPE protein and mRNA were significantly elevated in the hippocampal CA3 region, compared to naïve littermates. In addition, luciferase reporter assays identified a functional glucocorticoid regulatory element within the cpe promoter that mediated the up-regulation of CPE expression in primary hippocampal neurons following dexamethasone treatment, suggesting that circulating plasma glucocorticoids could evoke a similar effect on CPE in the hippocampus in vivo. Overexpression of CPE in hippocampal neurons, or CRS in mice, resulted in elevated prosurvival BCL2 protein/mRNA and p-AKT levels in the hippocampus; however, CPE^−/− mice showed a decrease. Thus, during mild CRS, CPE expression is up-regulated, possibly contributed by glucocorticoids, to mediate neuroprotection of the hippocampus by enhancing BCL2 expression through AKT signaling, and thereby maintaining allostasis.

In the brain, reversible morphological and chemical changes occur in response to acute and chronic stress in the hippocampus, prefrontal cortex, and amygdala. Long-term chronic restraint stress (CRS) alters synaptic plasticity, impairs hippocampal-dependent spatial learning and memory, induces restructuring of dendritic arbors in hippocampal CA3 neurons, and alters the intra-hippocampal network (1–5). This neuronal allostatic overload is, in part, attributed to excess glucocorticoid (GC) exposure to hippocampal pyramidal neurons (6). Indeed, corticosterone, a key GC stress hormone secreted by the adrenal glands during stress, is known to regulate stress-induced neuronal plasticity (7). GCs show biphasic effects in the nervous system, resulting in neuroprotection or neuronal damage after stress or brain injury; however, both of these effects are dose-dependent and operate within discrete time domains (8). The neuroprotective mechanisms that affect a delay in the allostatic overload remain poorly understood. Identifying the genes and proteins involved in the adaptive response is critical to understanding the neuroprotective mechanisms activated during stress.

Studies on cultured hippocampal neurons have demonstrated that carboxypeptidase E (CPE), a prohormone-processing exopeptidase (9), is a powerful neuroprotective molecule against oxidative stress-induced cell death (10, 11). Recently, this neuroprotective effect was shown to be mediated extracellularly by secreted CPE (12). Moreover, in vivo studies have shown that CPE-knock-out (KO) mice exhibit hippocampal neuronal degeneration in the CA3 region after emotional and physical stress associated with weaning (13). It has also been reported that CPE protein and mRNA increased in the hippocampus of rats subjected to transient global ischemia (14). This elevated expression was correlated with neuronal survival, especially in the CA3 neurons of the hippocampus where CPE levels were most elevated and sustained, as a result of the ischemic stress. In contrast, neurons in the CA1 region showed a transient increase in CPE and were more vulnerable to degeneration (14). Support for a functional role of CPE in the stress response comes also from studies in the Cpe^fat/fat mice. These mice carry a point mutation in the cpe gene that renders the protein unstable and subject to rapid degradation (15); hence, these animals are deficient in CPE. A sublethal episode of focal cerebral ischemia led to severe cell death in the ischemic cortex of the Cpe^fat/fat mice compared to wild-type (WT) mice, suggesting a correlation between CPE expression and neuronal survival. However, although all of these studies strongly indicate a neuroprotective action of CPE in vivo, the molecular mechanism underlying this function remains unclear. Interestingly, defective neuronal cyto-architecture of layer 5 pyramidal neurons of the cortex and of pyramidal neurons of the CA1 hippocampus was observed in CPE-KO mice due to improper dendritic pruning during development (16), an observation independently confirmed by biochemical and cellular studies (17). Both of these results suggest a functional role of CPE in neuronal structure and integrity.

In the present study, we examined the role of CPE in the maintenance of neuronal allostasis (18) in mouse hippocampus during CRS and investigated the molecular basis for this function. We found that CPE expression was increased in the hippocampus after 7days of CRS. Bioinformatics and in vitro studies demonstrated that the synthetic GC, dexamethasone, was able to directly up-regulate CPE expression via the binding of the GC receptor to the CPE promoter. The increased CPE expression correlated with an increase in expression of the antiapoptotic/prosurvival Bcl2 gene, and BCL2 protein in the hippocampus, an increase of which could impart a neuroprotective effect on the hippocampal neurons. The up-regulation of Bcl2 expression appeared to involve the AKT signaling pathway. Furthermore, CRS resulted in decreased BCL2 and increased BAX expression in CPE-KO mice. These findings suggest that CPE can mediate neuroprotection of the hippocampal neurons during mild CRS and may play an important role in maintaining allostasis.

Materials and Methods

Ethics statement

All animal procedures described herein were approved by the National Institute of Child Health and Human Development Animal Care and Use Committee, National Institutes of Health. Due to the nature of the experimental design, the animals were subjected to a restraint stress procedure. All other times the mice were monitored in their home cage in a stress-free environment where they were given food and water ad libitum in a humidity- and temperature-controlled room under a 12-hour light-dark cycle. After experimentation, to minimize pain without drugs, the mice were rapidly euthanized by cervical dislocation and decapitation by an experienced animal handler.

Animals and restraint paradigm

Male C57BL/6 mice (12 wk old) were obtained from Taconic Farms (Derwood, Maryland) and allowed to acclimatize to the animal facility and routine for 5 days. CPE-KO mice and WT litter mates (12 wk old) were raised in our animal facility. For experimentation, mice were restrained in transparent 50-mL plastic conical tubes with air holes for ventilation. For acute stress, mice were killed immediately after 1 hour of restraint (0 h) or were returned to their home cages and killed 1, 2, 6, or 24 hours after restraint (2 mice per time point, n). Two naïve mice were included as the control, unstressed group. This experiment was performed 3 times, N. For chronic stress, mice were restrained for 1 hour per day for 7 consecutive days and killed immediately after restraint on day 7 or 24 hours later (the recovery group). This experiment was performed 4 times with the C57BL/6 mice (3–4 mice per time point) and once with the CPE-KO mice (3 CPE-KO and 3 WT littermates). Five CPE-KO mice and 4 WT littermates were analyzed as naïve controls. Control mice were left in their home cages throughout the experimental paradigm. Brains of stressed and control mice were rapidly removed and either frozen directly on dry ice or dissected to remove the hippocampus, which was frozen on dry ice. All tissues were stored at −80°C until processed. N represents number of experiments performed; n represents number of samples per experiment.

In situ hybridization

Twelve coronal brain sections across the hippocampal region (10- to 14-μm-thick) from 3 mice from each group (naïve, chronic, recovery) were cut and mounted on Superfrost Plus slides and stored at −80°C until processing for in situ hybridization. The cRNA probe used to detect murine Cpe mRNA was 980 bp, corresponding to nucleotides (nos. 540–1520) of GenBank NCBI Reference Sequence: NM_013494 and labeled with ³⁵S-uridine-5′-triphosphate. A standard protocol was used to perform the labeling and hybridization (19, 20). The signal was visualized by autoradiography using X-ray film (Kodak, Biomax-MR, Rochester, New York). Quantification of the Cpe mRNA was performed using the Simple PCI imaging software (Hamamatsu Corporation, Herrsching am Ammersee, Germany).

Quantitative RT-PCR of Cpe and Bcl2

Total RNA (500 ng) was subjected to first-strand cDNA synthesis (Improm-II Reverse Transcription System, Promega, Madison, Wisconsin; or Transcriptor First Strand cDNA Synthesis Kit, Roche, Mannheim, Germany) followed by real-time quantitative PCR amplification of the cDNA (12.5 ng) in an ABI 7500 Sequence Detector using Power SYBR green I Master Mix (Applied Biosystems, Foster City, California). Forward and reverse primers of 100 nM (18 S rRNA, as internal normalization control) or 300 nM (Cpe and Bcl2) were used. The cycling conditions were: 10 minutes denaturation at 95°C and 40 cycles of DNA synthesis at 95°C for 15 seconds and 60°C for 1 minute. Primer sequences for Cpe were fwd: 5′-CTCATCAGCTACCTGGAGCA-3′, rev: 5′-AGCAAGCAATCGCCAGTAAT-3′; for Bcl2 fwd: 5′-AAGCTGTCACAGAGGGGCTA-3′, rev: 5′-CAGGCTGGAAGGAGAAGATG-3′; for 18 S fwd: 5′-CTCTTAGCTGAGTGTCCCGC-3′, rev: 5′-CTGATCGTCTTCGAACCTCC-3′. All reactions were performed in triplicate and relative amount of target mRNA was normalized to an internal control, 18S rRNA.

Western blot for CPE, BCL2, BAX, and pAKT

Protein extracts from whole hippocampal tissue and neurons in culture were prepared by homogenizing with T-protein extraction reagent (Pierce, Rockford, Illinois), supplemented with Complete Inhibitor Cocktail (Roche). Twenty or forty micrograms of protein were run on 12% SDS-PAGE gels and transferred onto nitrocellulose membrane (Invitrogen, Carlsbad, California), according to standard protocols. After blocking with 5% nonfat milk, CPE, BCL2, BAX, or p-AKT on the membrane was detected using mouse anti-CPE monoclonal antibody (BD Biosciences, Franklin Lakes, New Jersey), anti-BCL2 monoclonal antibody, anti-BAX monoclonal antibodies, and anti-p-AKT monoclonal antibodies (Cell Signaling, Beverly, Massachusetts) at 1:5000, 1:2000, 1:3000, and 1:2000 dilutions, respectively. After incubating with fluorophore-conjugated antimouse secondary antibodies (Li-COR, Lincoln, Nebraska), the bands were visualized by the Odyssey infrared imaging system version 2.1 (Li-COR, Inc) and quantified as arbitrary units by the Odyssey software. The expression levels were normalized to β-actin and expressed as the mean ± SEM of arbitrary units. All samples were analyzed, quantified, and normalized from 3 separate experiments for both acute and chronic stress paradigms. The fourth chronic paradigm was analyzed by in situ hybridization. SeeBlue Plus2 protein standards (Invitrogen) were run on every gel as markers for the relative molecular mass of proteins detected by Western blot.

Neuronal cultures

Hippocampal neurons, prepared from rat embryos (E18) as described previously (21), were seeded on polyethyleneimine or poly-l-lysine-coated plates.

Dexamethasone treatment

Neuronal cultures were incubated with 10% fetal bovine serum, 90% DMEM containing 100 mg/L Primocin (Lonza, Cologne GmbH, Germany) for 1 day, and then replaced with Neurobasal medium (Life Technologies [Invitrogen]). After 3 days, neurons were treated with dexamethasone (1 μM in ethanol) for 24 hours. RNA and protein were extracted from neurons for quantitative RT-PCR of Cpe mRNA and Western blot as described above.

Transduction with cpe

Neurons were incubated for 3 days in Neurobasal medium and then transduced with adenovirus (150 multiplicity of infection) carrying LacZ or WT CPE. The adenovirus vector for WT CPE was custom generated by Vector Biolabs (Philadelphia, Pennsylvania). After 4 days, RNA and protein were extracted from the neurons for quantitative RT-PCR and Western blot analysis of Bcl2.

Cpe-promoter analysis and luciferase assay

The cpe promoter sequence (−2000/+500 bp) was analyzed using the Genomatix software suite (Genomatix GmbH, Munich, Germany). Three putative glucocorticoid receptor-binding sites (GRBS) were identified: −1460 to −1442, −1283 to −1265, and −388 to −370. CPE promoter fragments of different sizes comprising all 3 GRBS, 2 GRBS, only 1 GRBS or no GRBS were initially cloned into a pGL3-basic luciferase vector and assessed for dexamethasone promoter activity (data not shown). The GRBS at position −1460/−1442 was the only one found to be responsive to GCs in this system and used for further characterization. A 300-bp fragment of the cpe promoter containing only this GRBS (−1460/−1442) was custom cloned (GenScript, Piscataway, New Jersey) into the pGL3-basic luciferase vector (Promega). Using this construct as a template, the GRBS was mutated at locations −1455 and −1447. These point mutations disrupted the GRBS without affecting other potential transcription factor-binding sites located in the same area based on in silico analysis. Hippocampal neurons were transfected using the Amaxa Rat Neuron Nucleofector Kit (Lonza), with the luciferase reporter comprising the WT GRBS or the impaired GRBS and a pGL4.73 vector (Renilla luciferase reporter vector) as a control for transfection efficiency. Luciferase assays were performed using the Dual Luciferase Reporter assay kit, according to the manufacturer's instructions (Promega). Luminescence was measured using the TD-20/20 Glomax luminometer (Promega). Results were expressed as percentage of luciferase activity compared to the controls.

Statistical analysis

Data were analyzed by Student t test or ANOVA. Significance was set at P < .05.

Results

Increased Cpe mRNA and CPE expression in hippocampus after CRS

A 1-hour acute restraint stress in mice resulted in an immediate down-regulation of Cpe mRNA (n = 2, N = 3) in the hippocampus at 0-, 1-, and 2-hour time points. This reduced expression remained low for up to 6 hours after the stress but was fully recovered by 24 hours (t test, P < .05) (Figure 1A) (ANOVA for 0, 1, 2, 6, and 24-h groups: CPE, F[5, 30] = 2.701, P < .05). However, the reduced mRNA did not translate into a significant reduction of CPE protein (Figure 1, B and C) even though there was a trend similar to the mRNA expression (ANOVA for 0, 1, 2, 6, and 24-h groups: CPE, F[5, 30] = 1.2018, P = .332). In contrast to the acute restraint stress, CRS for 1 hour for 7 days and with a 24-hour recovery showed an upward trend in Cpe mRNA expression, although not statistically significant when assayed by quantitative RT-PCR of the whole hippocampus (n = 3, N = 3) (Figure 1B) (ANOVA: CPE, F[2, 24] = 1.94, P = .164). However, CPE protein levels were increased by 50% immediately after the seventh day (t test for chronic group, P < .01) and by 70% in the subsequent 24-hour recovery group (t test, P < .001) (Figure 1, E and F) (ANOVA: CPE, F[2, 24] = 44.059, P < .001).

Figure 1.

Effects of stress on CPE expression in hippocampus. Cpe mRNA levels in the hippocampus of mice were reduced immediately and up to 6 hours after acute restraint stress (A) but tended to be elevated after chronic stress (D). Representative Western blots and graphical representation showing expression of CPE protein in tissue lysates of mouse hippocampus of the acute stress (B and C) and chronic stress groups including the 24-hour recovery and naïve groups (E and F). Total protein (20 μg) was loaded per lane. Blots were probed for CPE and β-actin with respective monoclonal antibodies. There was a significant increase in CPE protein in the chronic and recovery groups compared to naïve controls (±SEM; n = 6). Bar graphs, percentage of Cpe mRNA normalized to housekeeping gene 18S rRNA, and CPE protein normalized to β-actin. The average ± SEM of the ratios is plotted. Statistical analysis was performed using the Student t test. *, P < .05; **, P < .001; ***, P < .0001. M, markers (kDa).

We used in situ hybridization to characterize the distribution and expression patterns of Cpe in the rostral/caudal-dorsal/ventral extent of the hippocampus in naïve and stressed groups. Cpe mRNA showed high levels of expression in the pyramidal neurons of all hippocampal subfields (CA1-CA3) except the dentate gyrus (Figure 2). An especially high expression of Cpe mRNA was found in the CA3 region. Significant increases in Cpe mRNA were observed in the CA1, CA2, and CA3 regions of the hippocampus of the stressed animals compared to the naïve group (ANOVA, Section A, F[2, 9] = 9.20, P < .01; Section B, F[2, 9] = 44.41, P < .001; Section C, F[2, 9] = 50.25, P < .001) (Figure 2). Of note is that the mRNA remained elevated in the 24-hour recovery group (Figure 2, bar graphs).

Figure 2.

In situ hybridization study of Cpe mRNA expression in hippocampus. Mouse brain coronal sections of naïve (top), chronic (middle), and recovery (bottom) groups were probed with a Cpe RNA probe and hippocampal areas recorded as pseudo bright field images. Images in Section A, B, and C represent the hippocampus at different coronal depths as illustrated in Figures 41, 43, and 49 from ref. (39) and shown as diagrams here. Cpe mRNA levels were quantified as total gray signal within the CA1–CA3 region of interest adjusted for the background signal of the slide (nontissue area) and represented as bar graphs ± SEM from the respective groups, n = 3 per group. Cpe mRNA levels were increased in the CA1–CA3 regions in mice subjected to CRS compared to control mice (Naïve). No expression was observed in the dentate gyrus (DG). Statistical analysis was performed using independent-sample Student t test. *, P < .05; **, P < .01 vs control.

CPE expression in cultured hippocampal neurons is up-regulated by dexamethasone

Because the levels of GCs are increased during stress in vivo, we investigated if CPE expression could be modulated by the GC receptor agonist, dexamethasone, in primary cultures of rat hippocampal neurons. Cpe mRNA expression in dexamethasone-treated neurons was found to be significantly increased by ∼40% compared to control cells (t test, P < .001) (Figure 3A), concomitant with an increase in CPE protein levels (∼20%), analyzed by Western blot (t test P < .05) (Figure 3B). Bioinformatic analysis using the Genomatix software suite identified 3 putative glucocorticoid receptor-binding sites (GRBS) located within 2000 bp before the first transcription start site of the cpe gene (Figure 3C). Using luciferase constructs comprising those 3 GRBS, 2 GRBS, only 1 GRBS, or no GRBS at all, we determined that the GRBS located at position −1460/−1442 was the only one activated by GCs in hippocampal neurons (data not shown). To confirm this finding, a 300-bp fragment of the cpe promoter comprising this GRBS was cloned into a luciferase vector. We used this construct as a template to perform site-directed mutagenesis that disrupted the GRBS. A 24-hour treatment by dexamethasone of primary hippocampal neurons transfected with the reporter containing the WT GRBS resulted in a significant ∼40% increase (t test, P < .01) in luciferase activity, whereas no increase was observed when neurons were transfected with the reporter containing impaired GRBS (Figure 3D). This result indicates that GCs can directly regulate the expression of CPE in hippocampal neurons through the cpe promoter.

Figure 3.

CPE expression is regulated by GC. Twenty-four-hour treatment of rat primary hippocampal neurons with the synthetic GC, dexamethasone, significantly increased Cpe mRNA (A) and protein (B) levels. (C) Bioinformatic analysis of the cpe promoter revealed the presence of 3 putative GRBS. A 300-bp fragment of the cpe promoter comprising the active −1460/−1442 GRBS was inserted into a luciferase vector to produce the “wild-type GRBS” construct. Directed mutagenesis was used to create 2 point mutations at positions −1455 and 1447 to disrupt the GRBS and create the “impaired GRBS” construct. Black arrows indicate transcription start sites. Gray arrows represent point mutations. (D) Twenty-four-hour treatment by dexamethasone significantly increased the luciferase activity in rat hippocampal neurons transfected with the “wild-type GRBS” construct, compared to cells transfected with the “impaired GRBS” construct, showing that GC can directly regulate CPE expression through the cpe promoter. Statistical analysis was performed using the Student t test. *, P < .05; **, P < .01; ***, P < .001.

Coordinate expression of BCL2, BAX, and CPE in hippocampus after CRS

To show that increased CPE expression correlates with expression of a prosurvival protein, we analyzed the expression of BCL2, a prosurvival/antiapoptotic protein known to play a critical role in protecting cells against death under stress conditions (22). In hippocampi of WT mice subjected to CRS, BCL2 protein levels were elevated by 60% to 90% (n = 3, N = 3, t test chronic, P < .01; recovery, P < .001) (Figure 4, A and B) compared to control (naïve) animals. Parallel analysis of the hippocampal tissue for BAX, a proapoptotic protein, showed a decrease in the WT mice subjected to the CRS compared to the controls (Figure 4, C and D) (ANOVA: BCL2, F[2, 24] = 11.91, P < .001; BAX, F[2, 24] = 10.39, P < .001); however, although the recovery group showed a downward trend in expression, it was not statistically significant. We further showed that overexpression of CPE in primary hippocampal neurons up-regulated the expression of Bcl2 mRNA by 2.5 fold (t test P < .01) compared to control cells transfected with an empty vector (Figure 5A). In addition, BCL2 protein also showed an increase (t test, P < .001) and BAX protein expression was reduced (P < .01) (Figure 5, B and C) (ANOVA: BCL2, F[2, 9] = 53.04, P < .001; BAX, F[2, 9] = 10.63, P < .01). Furthermore, in the absence of CPE, the CPE-KO mice subjected to CRS showed significantly reduced levels of BCL2 protein expression in the hippocampus compared to naïve CPE-KO mice (n = 3, N = 1, naïve CPE-KO vs chronic KO, t test, P < .05, Figure 6A). As expected, the levels of BAX were increased by ∼50% (n = 3, N = 1, t test, P < .01) in the CPE-KO mice subjected to the CRS (Figure 6B), whereas it decreased in the WT littermates after CRS (P < .05) (ANOVA between naïve CPE-WT, CPE-KO, chronic CPE-WT and CPE-KO groups; BCL2, F[3, 30] = 51.41, P < .001; BAX, F[3, 30] = 21.21, P < .001).

Figure 4.

Expression of BCL2 and BAX in chronic restraint stress mice and hippocampal neurons. Representative Western blots (A and C) and bar graph representation (B and D) of the quantification of BCL2 and BAX protein in total tissue lysates from hippocampus of WT mice after chronic stress alone or chronic stress with a 24-hour recovery, compared to the naïve group. Statistical analysis was performed using the Student t test. *, P < .05; ***, P < .001; M, markers (kDa).

Figure 5.

Coordinate regulation of CPE, BCL2, and BAX expression in primary hippocampal cultured neurons. (A) The mRNA level of Bcl2 in cultured hippocampal neurons overexpressing CPE is elevated. Representative Western blots (B) and bar graph representation (C) of the quantification of CPE, BCL2, and BAX proteins in total cell lysates of primary hippocampal neurons expressing WT CPE. Statistical analysis was performed using the Student t test. **, P < .001; ***, P < .001; M, markers (kDa).

Figure 6.

BCL2 and BAX protein levels in the hippocampus of WT and CPE-KO mice subjected to chronic restraint stress. Representative Western blots and bar graph representation of the quantification of BCL2 (A and B) and BAX (C and D) protein in total tissue lysates from hippocampus of WT and CPE-KO mice after mild chronic restraint stress compared to the naïve group. Statistical analysis was performed using the Student t test. *, P < .05; **, P < .001. M, markers (kDa). Blots in (A) were from 2 separate gels that were run and analyzed at the same time.

Phosphorylation of AKT in the hippocampus of mice after CRS

Given that expression of BCL2 can be up-regulated through the activation of the AKT signaling pathway (23) and the known relationship between AKT and neuroprotection (24), we examined whether AKT phosphorylation was changed in the hippocampus after CRS. We found that there was an increase in AKT phosphorylation in the mice subjected to the CRS compared to the naïve group (n = 3, N = 2, naïve vs chronic, t test, P < .05; naïve vs recovery, P = .85; chronic vs recovery, P = .01; Figure 7, A and B) and this increase returned to baseline levels in the recovery group (ANOVA: p-AKT, F[2, 15] = 8.99, P < .01). In the study of the CPE-KO mice, phospho-AKT levels were not statistically different between those that were subjected to CRS and the naïve animals (naïve CPE-KO vs chronic KO, P = .76; Figure 7, C and D) whereas, similar to the results of Figure 7, A and B, phospho-AKT levels were increased after CRS in the WT littermates (naïve CPE-WT vs chronic CPE-WT, P < .05) (ANOVA between naïve CPE-WT, CPE-KO, chronic CPE-WT and CPE-KO groups; p-AKT, F[3, 20] = 7.32, P < .01).

Figure 7.

P-AKT expression is increased in the hippocampus of mice subjected to chronic restraint stress and reduced in CPE-KOs. Representative Western blots (A and C) and bar graph (B and D) representation of the quantification of phospho-AKT in WT mice after chronic stress alone or chronic stress with a 24-hour recovery (Rec), compared to the naïve group and WT and CPE KO mice after mild chronic restraint stress compared to the naïve group. Statistical analysis was performed using the Student t test. *, P < .05; M, markers (kDa).

Discussion

In this study, we showed 1) that treatment of hippocampal neurons in culture with dexamethasone increased CPE expression (Figure 3) and is consistent with our bioinformatic analysis and luciferase reporter assays demonstrating the presence of a functional GRBS in the cpe promoter region; 2) overexpression of CPE in hippocampal neurons resulted in an up-regulation of Bcl2 mRNA and protein (Figure 5). Our studies with mice showed 1) that Cpe mRNA and protein levels were increased in the hippocampus as a consequence of mild CRS and remained elevated during the 24-hour recovery group (Figures 1 and 2); 2) the CRS-induced elevation of CPE protein levels correlated with the up-regulation of expression of the prosurvival factor, BCL2, and a reduction in the proapoptotic protein BAX (Figure 4); 3) CPE-KO mice had decreased levels of BCL2 and increased levels of BAX after mild CRS, in contrast to WT littermates (Figure 6), suggestive of hippocampal neurons undergoing degeneration in vivo in the absence of CPE as a result of the stress; 4) during CRS, p-AKT protein in the hippocampus was increased (Figure 7), suggesting that the increase in BCL2 expression is mediated through AKT signaling triggered by CPE.

Stress has dramatic effects in the brain including neuronal cell death. CRS for 21 days results in neuronal remodeling, such as retraction and simplification of apical dendrites of hippocampal CA3 pyramidal neurons, which modifies the intra-hippocampal network (1), impairment of hippocampal-dependent spatial learning and memory (25), and neurodegeneration (26). However, in our 7-day CRS paradigm, there was no obvious evidence of gross degeneration of the hippocampus (Figure 2), allowing us to investigate the potential neuroprotective role of CPE within the physiologic time frame prior to allostatic overload. On a molecular level, even though the allostatic effect is a complex interchange between many molecules, we focused on CPE in light of other studies, suggesting that its increased expression is associated with neuronal survival and its absence is associated with neurodegeneration. Our experiments are the first to establish that in the whole animal, restraint stress regulated the expression of CPE in the hippocampus. The acute restraint stress caused a transient reduction in Cpe mRNA that returned to normal after 24 hours (Figure 1A), a result that may represent an initial phase response to this kind of stress. This is similar to a report showing that acute social isolation stress of nonweaned piglets for 15 minutes also resulted in a decrease in Cpe mRNA expression in the frontal lobe (27). However, in contrast to the acute short-term response, mild CRS caused an increase in Cpe mRNA (Figure 2) and protein (Figure 1, E and F), which was maintained 24 hours after the stress ended. Increases in Cpe mRNA were observed in the CA1, CA2, and CA3 regions of the hippocampus, with the highest elevation in the CA3 region of the stressed compared to control (naïve) animals. The differential responses to acute versus chronic stress may represent an initial response, followed by a secondary response in the regulation of Cpe expression in the neurons and represents an intriguing level of regulation in that opposite modes of expression are employed in a time-dependent manner.

Other studies have shown that CPE expression in the brain is modulated during different types of stress. Both transient global (14) and focal cerebral ischemia (28) caused transient changes in the expression of CPE mRNA and protein, depending on the brain areas studied, demonstrating that CPE levels responded to ischemic stress. For the transient global ischemia, the elevated expression of CPE in the hippocampus was correlated with neuronal survival, especially in the CA3 neurons of the hippocampus where CPE levels were most elevated and sustained. In studies of the CPE-KO mice (29, 30), the hippocampal CA3 region was completely degenerated by 6 weeks of age (10). However, this was not a developmental defect, because the CA3 region was normal at 3 weeks of age (13). Instead, recent studies showed that the neurodegeneration in these CPE-KO mice was caused by the emotional and physical stress associated with weaning, which involved maternal separation, tail snips for genotyping, and ear tagging at 3 weeks of age (13). Hence in the complete absence of CPE, CA3 pyramidal neurons degenerate as a consequence of the stress from weaning. Parenthetically, it has been reported that in fear response stress, an increase in Cpe mRNA was observed in the amygdala of male rats subjected to cat (predator) odor that correlated with avoidance and anxiety-like behaviors (31). Our observations that CRS resulted in an increase in CPE expression in the hippocampus of mice corroborates with these previous studies, indicating that CPE may play a neuroprotective role in different types of stress in hippocampal and extrahippocampal areas. Indeed, in a mouse model of the human neurodegenerative disease, neuronal ceroid lipofuscinoses, CPE is increased >10-fold, although its role in this animal model is unknown (32).

Molecular basis underlying the neuroprotective function of CPE

To understand the molecular basis of the neuroprotective function of CPE, we analyzed the changes in the prosurvival protein, BCL2, which regulates cell death by controlling mitochondria membrane permeability (33). Cell death induced by mitochondrial leakage due to pore formation is regulated by a balance between pro- and antiapoptotic proteins in the BCL2 family. We found that overexpression of Cpe up-regulated the expression of Bcl2 mRNA in cultured hippocampal neurons (Figure 5C) and indeed these proteins were also elevated in the hippocampi of mice after CRS (Figure 4, A and B). This suggests that CPE may exert its neuroprotective effect by increasing BCL2 expression. The increase in BCL2 protein in the hippocampus of WT mice after the CRS could presumably counteract the effects of proapoptotic proteins produced by cells under stress. In contrast to the WT mice, BCL2 levels in the CPE-KO mice decreased after CRS, suggesting a correlation between the lack of CPE and the reduction of BCL2 in the hippocampus. It is possible however that, similar to the neurodegeneration of CA3 neurons in the CPE-KO mice after the stress associated with weaning, further degeneration may occur during the CRS, contributing to the lower BCL2 levels. Interestingly, the proapoptotic protein, BAX, was decreased in WT mice after CRS and likely contributed to the neuroprotection effect by preventing mitochondrial leakage. In contrast, the increase of BAX in the CPE-KO hippocampus suggests at the very least that in combination with the decreased BCL2, these cells exhibit the molecular signature of cells undergoing the initial stages of neurodegeneration. CRS also increased AKT phosphorylation in the hippocampus, suggesting the effect of CPE may involve activation of the AKT signal transduction pathway. Indeed, studies have shown that p-AKT can up-regulate BCL2 expression (23). Because CPE is secreted from hippocampal neurons and can act extracellularly to protect these neurons from oxidative stress (12), we propose that in vivo, CPE activates the AKT signaling pathway to increase BCL2 expression for neuroprotection during CRS in mice.

How might the CPE neuroprotective response be activated to achieve allostasis?

Activation of the CPE neuroprotective response during stress may be through different pathways. However, GCs are the key modulators of the stress response. Similar to the dual response of CPE expression in neurons after acute versus CRS, GCs can be neuroprotective or cause neurodegeneration depending on its levels and length of time in circulation (7). Studies have shown that GCs can increase expression of neuroprotective molecules such as NGF, FGF2, and Lipocortin-1 (34–36); however, acute (34, 37) or chronic (38) levels of GCs leads to reduced expression of brain-derived neurotrophic factor in the hippocampus, contributing to neurodegeneration. Our 24-hour treatment of hippocampal neurons in culture with dexamethasone up-regulated Cpe expression at the mRNA (Figure 3A) and protein (Figure 3B) levels. Furthermore, our luciferase reporter studies showed that dexamethasone increased the luciferase activity in neurons transfected with a construct containing the −1460/−1442 GRBS, but not in neurons transfected with a construct containing an impaired version of this GRBS (Figure 3D). These findings suggest that the GC receptor could potentially bind to the Cpe promoter and up-regulate Cpe expression in the hippocampus during stress to initiate the neuroprotective response, similar to that of NGF or FGF2 mentioned above. However, it is premature at this point to conclude GC involvement in CPE expression in vivo as other signals likely play a role as well.

GCs are released from the adrenal cortex in response to ACTH from corticotrophs in the anterior pituitary and elicit a negative feedback loop on the corticotrophs to reduce secretion of ACTH and transcription of pro-opiomelanocortin, the prohormone precursor of ACTH. We have demonstrated that dexamethasone could also reduce the expression of CPE in AtT20 cells, a mouse corticotroph cell line (data not shown); hence, the reduction of CPE expression during acute restraint stress in the hippocampus mimics that of endogenous Cpe in corticotrophs. It would appear therefore that neurons respond to GCs differently than AtT20 cells with respect to CPE expression, possibly contributed by different transactivating factors present in different cell types.

In conclusion, we have demonstrated that during mild chronic stress CPE expression is up-regulated in the hippocampus, possibly but not exclusively due to the increase in GC secretion and its action on the cpe promoter. The increase in CPE expression contributes to an increase in expression of BCL2, an antiapoptotic protein/prosurvival factor that inhibits mitochondrial membrane permeability, to promote neuronal survival, possibly through AKT signaling, during CRS. This study provides a mechanism by which CPE could play a critical role in neuroprotection to maintain allostasis in hippocampal neurons during mild CRS.