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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Cereb Blood Flow Metab. Author manuscript; available in PMC Jan 2, 2014.
Published in final edited form as:
PMCID: PMC3878611
NIHMSID: NIHMS216402
Defective neuropeptide processing and ischemic brain injury: a study on proprotein convertase 2 and its substrate neuropeptide in ischemic brains
Shuqin Zhan,1,3 Hongbo Zhao,1 Aaron J White,1 Manabu Minami,1,4 Giuseppe Pignataro,1,5 Tao Yang,1 Xiaorong Zhu,2 Jingquan Lan,1 Zhigang Xiong,1 Donald F Steiner,2 Roger P Simon,1 and An Zhou1
1 Robert S. Dow Neurobiology Laboratories, Legacy Research, Portland, Oregon, USA
2 Department of Biochemistry, The University of Chicago, Chicago, Illinois, USA
Correspondence: Dr A Zhou, Robert S. Dow Neurobiology Laboratories, Legacy Clinic Research and Technology Center, 1225 NE 2nd Avenue, Portland, OR 97232, USA. azhou/at/downeurobiology.org
3Current address: Xi-An Jiao-Tong University, Xi An, China.
4Current address: Fujieda Heisei Memorial Hospital, Shizouka, Japan.
5Current address: Department of Neuroscience, “Federico II” University of Naples, Naples, Italy.
Using a focal cerebral ischemia model in rats, brain ischemia-induced changes in expression levels of mRNA and protein, and activities of proprotein convertase 2 (PC2) in the cortex were examined. In situ hybridization analyses revealed a transient upregulation of the mRNA level for PC2 at an early reperfusion hour, at which the level of PC2 protein was also high as determined by immunocytochemistry and western blotting. When enzymatic activities of PC2 were analyzed using a synthetic substrate, a significant decrease was observed at early reperfusion hours at which levels of PC2 protein were still high. Also decreased at these reperfusion hours were tissue levels of dynorphin-A(1–8) (DYN-A(1–8)), a PC2 substrate, as determined by radioimmunoassay. Further examination of PC2 protein biosynthesis by metabolic labeling in cultured neuronal cells showed that in ischemic cells, the proteolytic processing of PC2 was greatly attenuated. Finally, in mice, an intracerebroventricular administration of synthetic DYN-A(1–8) significantly reduced the extent of ischemic brain injury. In mice those lack an active PC2, exacerbated brain injury was observed after an otherwise non-lethal focal ischemia. We conclude that brain ischemia attenuates PC2 and PC2-mediated neuropeptide processing. This attenuation may play a role in the pathology of ischemic brain injury.
Keywords: neuropeptide processing, proprotein convertase 2, dynorphin, brain ischemia, neuroprotection, opioid peptide
A critical role of neuropeptide processing in the brain’s response to ischemia has been implicated in an earlier study of carboxypeptidase E (CPE) in the ischemic brains and cultured neurons (Zhou et al, 2004). Carboxypeptidase E, an exoprotease, mediates the posttranslational processing of many neuropeptides in the brain. The study has shown that ischemia causes a delay in the proteolytic conversion of CPE from its proenzyme form into the mature form both in vivo and in vitro. This is a clear indication that neuropeptide processing enzymes are subject to adverse changes after brain ischemia. The study has also shown that mice those lack an active CPE incur exaggerated brain injury after an otherwise non-lethal dosage of ischemia, suggesting an essential role of the neuropeptide processing system in protecting the brain from ischemic injury.
Proprotein convertase 2 (PC2, EC 3.4.21.94) is a calcium- and pH-dependent endoprotease, which is also essential for processing of many neuropeptides in the brain. Preceding the action of CPE or other carboxypeptidases, PC2 performs limited cleavages of precursors in a number of important neuropeptides including, but not limited to, enkephalin, cholecystokinin, VGF (nerve growth factor inducible), nociceptin/orphanin FQ, substance P, somatostatin, α- and γ-melanocyte-stimulating hormone (γ-MSH), and dynorphin (DYN). In PC2-null mice, the production of these neuropeptides is severely attenuated. For example, in the PC2-null brain, the level of DYN-A (1–8), whose cleavage from pro-DYN relies on the presence of active PC2, is undetectable (Berman et al, 2000; Day et al, 1998). PC2-null mice also exhibit vulnerability, or lack of response to certain stresses. Ni et al (2003) have reported that after mild salt loading, PC2-null mice develop hypertension, which is not seen in wild-type mice, because of the lack of γ-MSH. Croissandeau et al (2006), on the contrary, have observed enhanced analgesia toward mechanical or thermal nociceptive stimuli in PC2-null mice.
For afore-mentioned neuropeptides, a change in expression levels and/or a potential role in regulating the brain’s response to ischemia have been reported essentially for all of them. The instrumental role of PC2 in neuropeptide processing and its potential role in regulating the brain’s response to ischemic stroke have prompted us to investigate this processing enzyme in brain ischemia. In this study, we examined changes in gene expression levels, protein levels, and enzymatic activities of PC2 after brain ischemia. We also examined biosynthetic processing of PC2 in cultured neuronal cells under ischemic conditions. To further establish how brain ischemia may affect PC2-mediated neuropeptide processing and its possible consequences, we analyzed levels of DYN-A(1–8) in ischemic brains, compared differences in the severity of ischemia-induced injury between PC2-null and wild-type mice, and tested the effects of DYN-A(1–8) on the extent of ischemic brain injury.
Focal Cerebral Ischemia
Transient focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO), as described earlier (Zhou et al, 2004). The protocols for MCAO for both rats and mice were approved by the Institutional Animal Care and Use Committee of Legacy Research. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the care and use of laboratory animals. Male Sprague–Dawley rats (250 to 300 g) and C57BL/J mice (25 to 30 g) were purchased from Charles River Laboratories (Wilmington, MA, USA). PC2-null mice (Furuta et al, 1997) and their wild-type littermates were bred in-house at the Legacy Research (Portland, OR, USA) using breeding pairs provided by the laboratory of Dr Donald F Steiner at the University of Chicago (Chicago, IL, USA). Briefly, rats were anesthetized with 4% isoflurane in 70% nitrous oxide/30% oxygen, and maintained with 2% isoflurane in 70% nitrous oxide/30% oxygen. Middle cerebral artery occlusion was achieved by introducing a 3-0 silk suture into the lumen of the internal carotid artery, with the external carotid artery and the extracranial branch of the internal carotid artery ligated. After 100-min MCAO, the suture was withdrawn to allow reperfusions up to 24 h. Control animals were sham-operated. Middle cerebral artery occlusion in mice (30 mins) was performed with protocols similar to that for rats, with the use of a coated filament (6.0; Doccol Corp., Redlands, CA, USA) (Pignataro et al, 2007). For both rats and mice, relative regional cerebral blood flow was monitored by a laser-Doppler flowmetry (Transonic Systems Inc., Ethaca, NY, USA).
At the termination of reperfusions, under anesthesia, animals were decapitated. For the use of biochemical analyses, tissues from the ischemic cortical regions of MCA territory, as illustrated in our earlier studies on CPE (Zhou et al, 2004), were immediately dissected, frozen on dry ice, and kept at −80 °C. For the preparation of frozen brain sections, whole brains were frozen on dry ice immediately after decapitation, and 12-μm-thick frozen coronal sections were cut and kept at −80 °C.
In Situ Hybridization and Immunocytochemistry
To construct the plasmid pSPT18.rPC2(1–472), a 0.47-kb fragment from the 3′-end of rat PC2 complimentary DNA was excised from the plasmid pBS.rPC2 (from Dr Richard E Mains, University of Connecticut Health Center, Farmington, MA, USA) by digesting pBS.rPC2 with restriction enzymes BamHI and KpnI (Promega, Madison, WI, USA), and subcloned into the plasmid pSPT18 (Roche Diagnostics, Indianapolis, IN, USA), which was also digested with BamHI and KpnI. Linearized templates for antisense or sense complimentary RNA (cRNA) probes were generated by digesting the plasmid pSPT18.rPC2(1–472) with BamHI or KpnI, respectively. A 0.47-kb digoxigenin (DIG)-UTP-labeled probe was prepared using T7 (antisense) or SP6 (sense) polymerase with a commercial RNA Labeling Kit (Roche Diagnostics). Fluorescence in situ hybridization using DIG-UTP-labeled cRNA probes was performed on frozen brain sections following protocols described earlier for the analysis of CPE (Zhou et al, 2004).
Proprotein convertase 2 protein or DIG on brain sections that underwent in situ hybridization procedures was analyzed by fluorescent immunocytochemistry using standard protocols (Zhou et al, 2004). A rabbit polyclonal antibody against PC2 (used at 1:1,000) and a mouse monoclonal antibody against DIG (used at 1:500) were purchased from Affinity BioReagents (Golden, CO, USA) and Roche Diagnostics, respectively. Cy3-conjugated goat-anti-rat or mouse IgG and 4′, 6-diamidino-2-phenylindole (DAPI)-containing mounting fluid (to stain nuclei) were from Vector Co. (Burlingame, CA, USA). After staining, fluorescent signals on sections were examined with a Leica epifluorescence microscope (Leica Microsystems Inc., Bannockburn, IL, USA) attached to a digital camera, and analyzed with the assistance of the Bioquant program (Bioquant Image Analysis, Nashville, TN, USA).
Western Blotting
Cortices were homogenized with a buffer consisting of 50 mmol/L Tris-HCl, pH 7.5, 1% Triton X-100, 10% glycerol, and protease inhibitors (Berman et al, 2000). Protein concentrations in cleared supernatants were quantified by the Bradford method (Sigma-Aldrich, St Louis, MO, USA). In the following previous described protocols (Zhou et al, 2004), 50 μg proteins were fractionated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and blotted onto an Immobilon-P membrane (Millipore, Billerica, MA, USA). Blots were then incubated with a rabbit polyclonal antibody against PC2 (gift of Dr Steiner; used at 1:1,000), followed by the detection with the enzyme-catalyzed chemiluminescence (ECL) method (NEN Life Science Products, Boston, MA, USA) using an HRP-conjugated secondary antibody. The equality of protein loadings among different samples was verified by staining the blots with Coomassie Blue after antibody incubation and ECL detection.
Proprotein Convertase 2 Activity Assay
Analyses of PC2 activity followed protocols described by Berman et al (2000). Briefly, 20 μg of proteins from tissue homogenates were incubated with 200 μmol/L L-pGlu-Arg-Thr-Lys-Arg-7-amino-4-methylcoumarin (Peptides International, Louisville, KY, USA) in 100 mmol/L sodium acetate, pH 5.0, and 1 mmol/L CaCl2 at 37 °C for 2 h. In parallel incubations, 1 μmol/L CT peptide (Sigma-Genosys, The Woodlands, TX, USA), a PC2-specific inhibitor (Zhu et al, 1996), was added to reactions. The release of 7-amino-4-methylcoumarin (AMC) was measured using a Spectra Max GEMINI spectrofluorimeter (Molecular Devices, Union City, CA, USA; excitation 360 nm/emission 480 nm). The amount of product formed was calculated using free AMC as a standard. The activity inhibited by the presence of CT peptide was considered as a PC2-specific activity.
Radioimmunoassay
Tissues from cortical regions, as described earlier, were homogenized and then boiled in 0.1 mol/L hydrochloric acid for 15 mins, followed by centrifugation of boiled homogenates at 13,000 g for 30 mins at 4 °C. Proteins (55 μg) from cleared supernatants were dried on a SpeedVac and analyzed for DYN-A(1–8) by radioimmunoassay using a commercial kit (Bachem AG, Torrance, CA), following the procedures described by the manufacturer. Radioactivity was counted on a Cobra 5000 gamma-counter (Packard Instrument, Meriden, CT, USA). Synthetic, non-radioactive DYN-A(1–8) was used to establish a standard curve. All samples were analyzed in duplicates.
In Vitro Simulated Ischemia in Cultured Neuronal Cells and Metabolic Labeling Analyses
NS20Y neuroblastoma cells were maintained in DMEM (Dulbecco’s Modified Eagle’s medium; Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum and differentiated with 8-(4-chlorophenylthio)adenosine 3′/5′-cyclic monophosphate (Sigma) according to Sirianni et al (1999). Overexpression of recombinant rat PC2 was achieved by transient transfection of the plasmid pCMV6c.rPC2 (Zhou et al, 1998) with Lipofectamine 2000 (Invitrogen). Simulated ischemia in transfected and differentiated NS20Y cells were induced by oxygen and glucose deprivation (OGD), as described earlier (Zhou et al, 2004), by incubating cells in glucose-free, serum-free, and glutamine-free media in an anaerobic chamber (Forma Scientific, Marietta, OH, USA) equilibrated with 85% N2/5% CO2/10% H2 for desired periods of time. After the OGD treatment, cells were allowed to recover in complete media under normal conditions for 1 h before metabolic labeling analyses according to the previously described protocols (Zhou et al, 2004). Briefly, cells were pulse-labeled with [35S]methionine (1 mCi/mL, 1,000 Ci/mmol; Perkin Elmer, Waltham, MA, USA) suspended in methionine-free DMEM media for 30 mins with or without a subsequent 2-h chase incubation in nonradioactive complete media. At the end of incubation, media were collected and cellular proteins were extracted, and both media and cell extracts were subjected to immunoprecipitation with an anti-PC2 antibody (the same antibody that was used for western blotting) and protein A-argarose beads (Sigma-Aldrich). Immuno-precipitated PC2 proteins were fractionated on 7.5% SDS-PAGE slab gels and detected by autoradiography.
Intracerebroventricular Administration of Peptides
In adult C57BL/J mice (25 to 30 g), 15 mins before MCAO, 20 nmol DYN-A(1–8) peptide (Bachem AG) suspended in 0.5 μL artificial CSF (aCSF), or aCSF alone (control) was administered through ICV injection using protocols previously described (Pignataro et al, 2007). After 30-min MCAO followed by 24 h reperfusion, animals were killed under anesthetization. Whole brains were removed, and frozen coronal sections were prepared for determination of ischemic injury.
Analyses of Ischemic Brain Injury
Ischemia-induced brain cell injury was analyzed with the TdT-mediated dUTP nick end labeling (TUNEL) method using a commercial kit (Roche Diagnostics). TUNEL-positive cells were counted in four × 20 cortical fields, with two sections from each brain counted and results were summed for each brain (Zhou et al, 2004). Numbers of DAPI-stained cells were documented for the same fields and summed for each brain. Percentages of TUNEL-positive cells were used to compare the extent of injury between two appropriate groups of animals. When desired, ischemic injury was also determined by estimating tissue infarction with the cresyl violet (CV) staining method (Pignataro et al, 2007; Tureyen et al, 2004), in which relative volumes of ipsilateral tissues devoid of CV staining were estimated by analyzing five coronal sections taken between bregma + 2.58 mm and + 0.74 mm at an interval of 20 sections, and calculated with the assistance of the Bioquant program (Pignataro et al, 2007; Tureyen et al, 2004).
Statistics
Unless stated otherwise, changes at multiple reperfusion hours were compared by one-way analysis of variance followed by Newmann–Keul’s test. Differences between two animal groups or cell culture conditions were compared by unpaired or paired, respectively, t-test. A P-value ≤0.05 was accepted as significant.
In adult rat brains, detectable levels of PC2 mRNA and protein have been described for most brain regions (Billova et al, 2007; Schafer et al, 1993; Winsky-Sommerer et al, 2000). In both the cerebral cortex and striatum, including regions directly affected in the ischemic model under study, PC2 is expressed in all populations of neuronal cells, with the strongest signals detected at layers IV and V of the cortex, as well as in projection and interneurons of the striatum. In progression following our earlier studies on CPE in the ischemic cortex, with a focus on PC2 and PC2-mediated neuropeptide processing under ischemic conditions, results introduced below primarily describe changes in ischemic cortices.
Changes in Proprotein Convertase 2 Expression and Biosynthesis after Ischemia
After 100 mins MCAO, at 4-h reperfusion, enhanced intensities of signals for both mRNA and protein of PC2 were observed in the ischemic cortex when compared with those in the control cortex (Figures 1A and 1B), with changes in signal intensities for the PC2 mRNA more prominent than those of the PC2 protein. At 24-h reperfusion, intensities of these signals decreased substantially in the ischemic cortex to levels lower than those of the control cortex (Figures 1A and 1B).
Figure 1
Figure 1
Changes in PC2 expression after brain ischemia in rats. (A) In situ hybridization analysis of PC2 mRNA. Frozen brain sections were first incubated with a DIG-labeled cRNA probe for PC2, followed by the immunocytochemical detection of DIG. (B) Immunocytochemistry (more ...)
In control cortices, two major forms of PC2 protein were detected by western blot analyses—a 75-kDa form and a 64-kDa form, presumably representing the pro-enzyme form and the mature, active form, respectively, of PC2 (Figure 2A). To capture possible changes in PC2 proteins at an earlier post-ischemia time, an 1-h reperfusion time point was included in the western blot analysis of PC2 in ischemic cortices. After 100-min MCAO, levels of total PC2-immuno-reactive proteins at 1- and 4-h reperfusion times were not statistically different from those of control brains, as determined by densitometric analyses of PC2 protein bands revealed by western blotting. At 24-h reperfusion, the level of PC2 protein decreased significantly (P = 0.008). Interestingly, at 1-h reperfusion, although the level of total PC2 protein remained high, there appeared an increased presence of the pro-PC2-sized form of PC2 protein in the ischemic cortex, as measured by the ratio of the pro-PC2-sized form over total PC2 proteins. Although differences in such ratios were not statistically significant among different groups (P = 0.287), a trend of increase implicates that brain ischemia may cause changes in the proteolytic processing or activation of PC2, notably at an early time point after ischemia. As western blot analyses cannot distinguish newly synthesized and preexisting proteins, and to investigate whether ischemia may attenuate the proteolytic conversion of pro-PC2 into PC2, we conducted metabolic labeling analyses on cultured NS20Y cells shortly after the OGD treatment. This cell line, though neuroblastoma in nature, can be readily differentiated into a neuronal phenotype, and is known for having suitable intracellular conditions that allow efficient processing of neuropeptides (Sirianni et al, 1999). As shown in Figure 2B, under normal conditions, pro-PC2 can be effectively converted into PC2. In ischemic cells, the most abundant PC2 proteins were in sizes of pro-PC2 and its biosynthetic intermediate. There was a significant decrease in the production of the 64-kDa PC2 protein in ischemic cells when compared with that in control cells (P = 0.009). Also noticeable in ischemic cells was the appearance of several PC2-immunor-eactive proteins in sizes that are not of properly processed PC2 protein under normal conditions, suggesting an increased degradation of PC2 protein under ischemic conditions.
Figure 2
Figure 2
Proprotein convertase 2 protein and its biosynthesis in ischemic brains and cultured ischemic neuronal cells. (A) After 100-min MCAO, proteins were extracted from control and ischemic cortices at 1-, 4-, and 24-h reperfusions, respectively (Control, 1, (more ...)
Decreased Proprotein Convertase 2 Activities and Levels of DYN-A(1–8) in Ischemic Brains at Early Reperfusion Hours
The afore-described ischemia-induced changes in PC2 expression and biosynthesis have directed us to further investigate whether ischemia may result in a change of PC2 enzymatic activities and the production of its substrate neuropeptides.
In the control cortex, the specific PC2 activity (pmol/h per mg protein; mean±s.e.) was 0.35±0.06. In ischemic cortices at 1- and 4-h reperfusions, PC2 activities were 0.17±0.03 and 0.16±0.04, respectively, which were significantly lower than those in the control cortex (P = 0.006) (Figure 3A). Under normal conditions, a number of DYN peptides including DYN-A(1–17) and DYN-B(1–13) are excised from pro-DYN through the action of PCs. In the presence of an active PC2, DYN-A(1–17) is further cleaved to release DYN-A(1–8) (Berman et al, 2000; Day et al, 1998). The results of radioimmunoassay (Figure 3B) showed that levels of DYN-A(1–8) (pmol per g protein; mean±s.e.) in ischemic cortices at 1- and 4-h reperfusions after 100-min MCAO were significantly lower than those in the control cortex (9.9±1.6 and 10.6±0.5, respectively, versus 23.5±4.9; P = 0.002). Hence, after brain ischemia, there were substantial decreases in both PC2 activities and DYN-A(1–8) at early reperfusion hours.
Figure 3
Figure 3
Decreased PC2 activity and levels of DYN-A(1–8) after brain ischemia. (A) The specific PC2 activity in tissue homogenates (mean±s.e.) of control and ischemic cortices was determined using a synthetic substrate. At both 1- and 4-h reperfusions, (more ...)
Exacerbated Ischemic Brain Injury in PC2-Null Mice
Despite being deficient in processing of a number of neuropeptides, the cortical morphology of the PC2-null brain, when examined by DAPI and CV staining, appears comparable to that of the wild-type brain (data not shown). For this experiment, we chose 30-min MCAO instead of 60-min MCAO, as the latter will result in total infarction of both cortical and striatal tissues in mice. In our earlier studies on CPE in ischemic brains, we observed that, in the wild-type mice and under normal conditions, 30-min MCAO would cause substantial damage to the striatum and little damage to the cortex. Hence, applying a 30-min MCAO will allow an evaluation of the extent of cortical injury, which may otherwise be indistinguishable between the wild-type mice and PC2-null mice after a 60-min MCAO. When PC2-null and wild-type mice were subjected to 30-min MCAO, a significantly higher number of TUNEL-positive cells were detected in the ischemic cortex of PC2-null mice than those in wild-type mice (25.72%±12.10% and 2.33%±2.27%, respectively; P = 0.025) (Figure 4). This result strongly suggests that an active PC2 is essential for brain cell survival after ischemic stress.
Figure 4
Figure 4
Exacerbated ischemic brain injury in PC2-null mice after MCAO. Frozen brain sections were prepared from PC2-null mice (PC2-null; n = 8) and wild-type littermates (WT; n = 4) after 30-min MCAO and 24-h reperfusion. Cell damages were analyzed by the TUNEL (more ...)
A Protective Role of DYN-A(1–8)
Synthetic DYN-A(1–8) (20 nmol) was administered to mice before 30-min MCAO, which was followed by 24-h reperfusion. When brain injury was examined by the TUNEL method, a limited number of TUNEL-positive cells (2.1%±1.0%) were found in the ischemic cortices of animals receiving aCSF (control)—an expected outcome for a 30-min MCAO in mice—whereas few were found in animals receiving DYN-A(1–8). This suggests a protective role of DYN-A(1–8) against ischemic brain injury. Such an effect of DYN-A(1–8) was more clearly shown when the striatal region was included in analyses. Figure 5A shows that the percentages of TUNEL-positive cells on the ipsilateral hemisphere of the brain were 20.8%±5.4% in control animals versus 2.9%±1.4% in animals receiving DYAN-A(1–8); the difference was significant (P = 0.014). Reduced ischemic brain injury in animals treated with DYAN-A(1–8) was further indicated by CV staining. As shown in Figure 5B, relative tissue volumes devoid of CV staining were 7.0%±2.3% and 1.2%±0.6% for control and DYAN-A(1–8)-receiving animals, respectively; the difference was also significant (P = 0.046).
Figure 5
Figure 5
Administration of DYN-A(1–8) reduces ischemic brain injury. Fifteen minutes before 30-min MCAO, each animal of two groups received aCSF (control; n = 5) or 20 nmol DYN-A(1-8) peptide (n = 4), respectively, through ICV injection. At 24 h after (more ...)
The studies reported here address the role of neuropeptide processing in the brain’s response to ischemia. We first examined PC2, an enzyme that performs endoproteolytic processing of neuropeptide precursors in the secretory pathway, in ischemic cortices. Results of fluorescent in situ hybridization analyses of PC2 mRNA levels showed a transient upregulation after 100-min MCAO in rats (Figure 1A). This is not surprising, as it is known that the promoter region of the PC2 gene contains several elements that can potentially respond to ischemic stress (Jansen et al, 1997; Yan et al, 2000), and elevated PC2 mRNA levels in the brain have been reported for several pathophysiological conditions (Bhat et al, 1993; Noel et al, 1998; Oyarce et al, 1996). Of particular interest is whether the PC2 protein in the brain could be processed properly to its active form after ischemia, when its expression levels were still high. The proteolytic activation of PC2, in addition to a dependence on 7B2—a chaperone-like protein (Westphal et al, 1999; Zhu and Lindberg, 1995)—is regulated by calcium concentrations in the secretory pathway (Guest et al, 1997). Depletion of the ER calcium pool in ischemic cells has been well described (Hayashi and Abe, 2004; Paschen and Doutheil, 1999). Although there is still a lack of detailed information concerning changes in calcium concentrations in different compartments of the secretory pathway after brain ischemia, we suspect that the overall disruption of intraluminal homeostasis of the secretory pathway would not favor PC2 for either its processing from pro-PC2 to its matured form or its enzymatic activity, or both. Indeed, in ischemic brains, we found both a trend of accumulation of pro-PC2-sized protein and a decrease in PC2 activity in ischemic brains (Figures 2 and and3)3) at reperfusion time points at which total protein levels of PC2 remained high (Figure 1A). The observation of attenuated proteolytic processing of PC2 in ischemic NS20Y cells (Figure 2B) provides a mechanistic explanation for changes of PC2 in ischemic brain tissues. The substantial loss of PC2 enzymatic activity, when its protein levels were still high, after brain ischemia (Figure 3A) is also important to note. As discussed below, an ischemia-induced decrease in PC2 activity may have a significant impact on brain cell survival after ischemia.
Analyses of tissue levels of DYN-A(1–8), a cleavage product of pro-DYN by PC2 activity, revealed in decreased levels of this neuropeptide in ischemic cortices (Figure 3B). DYN-related peptides vary in their affinities to receptors and in their interactions with other proteins (Hauser et al, 2005). In general, DYN-A-related neuropeptides preferably bind to the κ-opioid receptor. Involvement of κ-receptor in neuroprotection after focal ischemia has long been known (Chen et al, 2004; Hall and Pazara, 1988). DYN-A(1–8), in addition to the κ-receptor, also binds to δ- and μ-receptors, and may have a higher affinity to the δ-receptor (Bell and Traynor, 1998; Schulz et al, 1984), whose activation is anti-apoptotic and whose inactivation induces cell death in stressed cells (Cao et al, 2003; Hayashi et al, 2002; Ma et al, 2005). Hence, our present finding that brain ischemia attenuated the production of DYN-A(1–8), especially at early reperfusion hours, may have a pathologic significance.
Our observation of reduced ischemic brain injury in animals receiving exogenous DYN-A(1–8) (Figure 5) is the first in describing a protective role of this neuropeptide under ischemic conditions. This result agrees well with a recent report by Charron et al (2008) that the administration of κ- or δ-receptor agonist protects CA1 neurons from ischemic cell death in a global ischemia model in rats. Although it remains to be established whether the protective effect of DYN-A(1–8) is exerted through κ- or δ-receptor, or both, the results, nevertheless, show its potential role in preventing cell death after ischemia. The results also suggest an important role of PC2-mediated processing of DYN peptides in determining the brain’s response to ischemic stress.
Cpefat/fat mice are a mouse strain that expresses an inactive CPE protein because of a spontaneous mutation in the CPE gene ((Fricker et al, 1996; Naggert et al, 1995). Similar to our previous observation on Cpefat/fat mice (Zhou et al, 2004), PC2-null mice showed exacerbated ischemic brain injury after 30-min MCAO (Figure 5). Although PC2-null and Cpefat/fat mice share certain abnormalities in neuropeptide and peptide hormone processing, they differ in their neuroendocrine conditions. For example, adult male Cpefat/fat mice will become severely obese and hyperglycemic at about 12 weeks of age (Leiter et al, 1999), whereas PC2-null mice, in general, maintain a normal body weight and become hypoglycemic under fasting conditions (Furuta et al, 1997). The exacerbating effect of hyperglycemia on ischemic brain injury is well established, whereas the effects of hypoglycemia have been less studied and it may be beneficial. A similar outcome after brain ischemia in these two different processing-deficient strains suggests that the increased vulnerability to brain ischemia in them is likely a consequence of defective neuropeptide processing, rather than that of chronic metabolic disorders. It would be interesting to see in future studies whether the exacerbated ischemic brain injury in PC2-null mice can be prevented by administrating DYN-A(1–8) to the animals.
It remains to be determined whether brain ischemia may also attenuate the processing of other PC2 substrate neuropeptides, and the consequences of it. It should be noted that functional roles of many opioid-like neuropeptides can be paradoxical in respect to pro- or anti-cell death after brain ischemia. Their possible excitotoxic effects through glutamate receptors, especially under pathophysiological conditions, as well as possible toxic effects of their precursor forms whose function are still poorly understood, are all important issues that must be studied in details in future work.
In summary, this study has shown a high responsiveness of PC2-mediated neuropeptide processing system to brain ischemia, adverse changes of this system under ischemic conditions, and the importance of sustaining the normal function of this system in protecting the brain from ischemic injury.
Acknowledgments
The authors thank Chelsea Piper for general lab assistance and Sue Crawford for administrative assistance.
This study was supported by the grants from National Institutes of Health (NS046560) and the American Heart Association (0450142Z) to A Zhou. S Zhan was partially supported by a scholarship from Xi-An Jiao-Tong University (Xi An, China).
Footnotes
Disclosure
The authors state no conflict of interest.
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