J. Neurochem. (2011) 117, 346–356.
Hypoxic preconditioning (HPC) initiates intracellular signaling pathway to provide protection against subsequent cerebral ischemic injuries, and its mechanism may provide molecular targets for therapy in stroke. According to our study of conventional protein kinase C βII (cPKCβII) activation in HPC, the role of cPKCβII in HPC-induced neuroprotection and its interacting proteins were determined in this study. The autohypoxia-induced HPC and middle cerebral artery occlusion (MCAO)-induced cerebral ischemia mouse models were prepared as reported. We found that HPC reduced 6 h MCAO-induced neurological deficits, infarct volume, edema ratio and cell apoptosis in peri-infarct region (penumbra), but cPKCβII inhibitors Go6983 and LY333531 blocked HPC-induced neuroprotection. Proteomic analysis revealed that the expression of four proteins in cytosol and eight proteins in particulate fraction changed significantly among 49 identified cPKCβII-interacting proteins in cortex of HPC mice. In addition, HPC could inhibit the decrease of phosphorylated collapsin response mediator protein-2 (CRMP-2) level and increase of CRMP-2 breakdown product. TAT-CRMP-2 peptide, which prevents the cleavage of endogenous CRMP-2, could inhibit CRMP-2 dephosphorylation and proteolysis as well as the infarct volume of 6 h MCAO mice. This study is the first to report multiple cPKCβII-interacting proteins in HPC mouse brain and the role of cPKCβII-CRMP-2 in HPC-induced neuroprotection against early stages of ischemic injuries in mice.
two dimensional gel electrophoresis
acetyl-CoA acetyltransferase mitochondrial
collapsin response mediator protein-2
middle cerebral artery occlusion
NADH dehydrogenase [ubiquinone] flavoprotein 2 mitochondrial precursor
neurofilament light polypeptide
protein kinase C
sodium dodecyl sulfate
Trans-Activator of Transcription
terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
ubiquitin carboxyl-terminal hydrolase isozyme L1
It is known that cerebral hypoxic/ischemic insults, such as stroke, cause neuronal damage through the generation of reactive oxygen species, Ca2+ overloading, apoptotic signaling initiation and inflammatory responses (Murphy and Steenbergen 2008; Broughton et al. 2009), but we still lack effective treatments in clinical practice. Fortunately, the understanding of underlying molecular mechanisms in hypoxic preconditioning (HPC), which is described as tissue endurance of lethal injury by adapting to transient noxious insults (Shpargel et al. 2008), may assist in the eventual development of new molecular targets for clinical therapy in cerebral ischemic injuries. Although studies have suggested that multiple protein kinases have been involved in this biological process, such as protein kinase C (PKC) and MAPKs (Hausenloy and Yellon 2006), the precise signaling transduction mechanism of HPC is unclear.
The PKC family, including 10 isoforms of serine/threonine protein kinases, may partake of protective or deleterious effects in an isoform-specific manner (Bright and Mochly-Rosen 2005). Of the various PKC isoforms, we found that the membrane translocation (activation) of conventional PKCβII (cPKCβII) increased in response to HPC both in vivo and in vitro (Li et al. 2005, 2006; Niu et al. 2005). However, questions about the role of cPKCβII in HPC-induced neuroprotection and the components of cPKCβII-mediated signaling pathway in HPC remain unanswered.
Previous studies on the role of PKC isoforms in numerous pathways were limited to a linear paradigm. Because of recent advances in the field of proteomics, researchers can use high-throughput approaches to learn the changes in protein expression, protein modification and protein-protein interaction on a global level. In this study, the role of cPKCβII in HPC-induced neuroprotection and its interacting proteins were determined by functional proteomic techniques. These results would be helpful for understanding the molecular mechanism that underlies cerebral HPC and ischemic injuries.
Material and methods
All general chemicals and chemicals used in isoelectric focusing were purchased either from Sigma-Aldrich (St. Louise, MO, USA) or GE Healthcare, Buckinghamshire, UK. Adult male BABL/c mice (8–10 W, weighing 20–25 g) were purchased from Experimental Animal Center of Chinese Academy of Medical Sciences, PR China. The experimental protocols were approved by the Animal Care and Use Committee of Capital Medical University and consistent with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23).
Animal models and experimental protocol
The autohypoxia (acute and repetitive exposure to progressive hypoxia)-induced HPC mouse model was prepared as we reported earlier (Lu and Liu 2001; Li et al. 2005). In brief, mice were placed individually within a 125 mL airtight jar, which was full of fresh air and sealed with a rubber plug to duplicate a progressive hypoxia environment. This procedure is believed to closely simulate the clinical conditions of ‘asphyxia’, which is a combination of hypoxia and hypercarbia. Mice were removed as soon as the first signs of gasping appeared (an indicator to end each hypoxic exposure), then the tolerant time and ending oxygen concentration were recorded. A minimum of 30 min was allowed for recovery in normoxic condition, and then the mice were switched to another hermetically sealed jar of the same volume. Mice exposed to autohypoxia for four times were designated as HPC group (35.6 ± 1.3 min for tolerant time, 3.8 ± 0.4% for ending oxygen concentration), and mice placed in open jars for the same amount of time were used as normoxic control.
The middle cerebral artery occlusion (MCAO)-induced cerebral focal ischemia mouse model was set up using the procedure described previously (Chen et al. 2007) with minor modifications. Mice were anesthetized with pentobarbital sodium (0.06 g/kg i.p.), and then the left common carotid artery and external carotid artery were exposed through a ventral midline incision, upon which the two arteries were ligated. An arteriotomy was made in the common carotid artery allowing the introduction of a 4-0 surgical nylon monofilament with its tip (0.23 mm in diameter) rounded by heat. The filament was gently inserted into the internal carotid artery to occlude the middle cerebral artery. In sham group, mice received the same surgical exposure of carotid artery but without occlusion. During the surgery, body temperature was maintained with the use of a heating lamp and thermal blanket. Mice were placed in a postoperative cage, and kept warm and undisturbed for a minimum of 2 h observation.
The mice were assigned into the following groups: Sham, Ischemia (I, subjected to 6 h MCAO), HPC + I (30 min after HPC treatment) and HPC + Go6983 + I or HPC + LY333531 + I, of which HPC mice were treated with bisindolyl maleimides derived Go6983 (8 nM, 5 μL) or LY333531 (200 nM, 5 μL, cPKCβ-specific inhibitor) by intracerebroventricular injection just before MCAO. Go6983 is a general inhibitor of cPKC, but inhibits cPKCβII activation more specifically at a low concentration. For the specific intervention on collapsin response mediator protein-2 (CRMP-2), 5 μL (10 mg) of Trans-Activator of Transcription (TAT)-CRMP-2 peptide (Sequence: YGRKKRRQRRRGVPRGLY DGPVCEV, California Bioscience Inc., Sacramento, CA, USA), which contains calpain cleavage sequence and can prevent the cleavage of endogenous CRMP-2, was administered by intracerebroventricular injection just before MCAO. The dosages of cPKCβII inhibitors and TAT-CRMP-2 were determined by our preliminary experiments.
For intracerebroventricular injections, all surgery was performed under pentobarbital sodium anaesthesia (0.6 g/kg) with isotonic saline as solvent. Drug administration procedures followed the method described by Alvaro Munoz (Munoz et al. 2003). During anesthesia, animals were positioned in a stereotaxic frame, and a cannula (28-gauge, stainless steel, inner diameter 0.18 mm, outer diameter 0.36 mm) was lowered stereotaxically into the left cerebral ventricle to a position defined by the following coordinates: 0.5 mm posterior to bregma, 1.0 mm lateral to bregma, and 3.5 mm below the skull surface. To ascertain that solutions were administered exactly into the cerebral ventricle, some mice were injected with 5 μL of diluted 1 : 10 India ink and their brains were examined macroscopically after sectioning. The accuracy of the injection technique was evaluated with 95% of injections being correct.
Neurological deficits evaluation
Neurological deficits of mice were assessed 6 h after MCAO, at which time point the infarct volume and neurological deficits may not be fully installed, but peri-infarct region (penumbra) persists more apparently. The procedure used is an adaptation of that validated by Rodriguez et al. (2005). It involves an initial phase of undisturbed observation (hypomobility, lateralized posture, flattened posture, hunched back, piloerection, ataxic gait, circling, tremors, twitches, convulsions and respiratory distress) and a later manipulative phase (passivity, hyperreactivity, irritability, ptosis, urination, decreased body tone, forelimb flexion, decreased muscle strength, body rotation and motor incoordination). Based on the results of above observation, neurological behavior was scored on a 10-point scale. In all cases, when the criteria for the precise grade were not met, the nearest appropriate number was utilized: 1, 3, 5, 7 and 9.
Measurement of infarct size, edema ratio and TUNEL staining
Immediately after the evaluation of neurological deficits, double blind measurements of the infarct volume were taken. Some mice were killed at 6 h MCAO, and then the brain was quickly removed and cut into 1.5-mm thickness coronal sections. Brain sections were incubated for 20 min in a solution of 0.5% 2,3,5-triphenyltetrazolium chloride (TTC) in 10 mM phosphate buffered saline at 37°C, and then the slices were scanned into a computer. According to the evaluation procedure reported by Wexler et al. (2002), the infarct size was analyzed. Edema ratio (E) was calculated using the following equation: ; ∑VL and ∑VR are the volume of left (ischemic) and right (non-ischemic) hemisphere volume, respectively.
Part of the mice brains were cut into 10-μm thickness sections for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). The TUNEL staining kit (DeadEnd™Fluorometric TUNEL system, Promega, Madison, WI, USA) was used to assess cell apoptosis. For apoptosis assessment, the brain sections were placed in equilibration buffer and incubated with nucleotide mix and rTdT enzyme at 37°C for 1 h. The reaction was stopped by using 2× saline-sodium citrate buffer. Hoechst 33258 was used to stain neural nuclei. The images were visualized by fluorescence microscope (Leica DM4000B, Wetzlar, Germany).
Sample preparation and Western blot analysis
As we reported previously (Li et al. 2005), the mice brain were removed at 6 h after MCAO and immediately placed into ice-cold artificial CSF (in mM: NaCl 125.0, KCl 2.5, CaCl2 2.0, NaHCO3 26.0, NaH2PO4 1.25, MgCl2 1.0, glucose 5.0, pH 7.4) bubbling with 95% O2 and 5% CO2. The cortex from indicated regions were collected according to the previously reported (Ashwal et al. 1998). In brief, 2 mm from the anterior tip of the frontal lobe were cut, while the left brain was sectioned to four 2-mm thickness slices. A longitudinal cut approximately 1 mm from the midline through each hemisphere was made to remove the tissue supplied by anterior cerebral artery. And then we made a transverse diagonal cut at approximately the 45 degree angle position, to separate the ischemic core and the peri-infarct region (penumbra). The corresponding regions from the non-ischemic hemisphere were dissected as contralateral control. All the tissues were frozen in liquid nitrogen, and kept frozen at −70°C for later analysis.
The frozen samples were rapidly thawed and homogenized at 4°C in buffer A (5 mM Tris–Cl, pH 7.5, containing 2 mM dithiothreitol (DTT), 2 mM EDTA, 1 mM EGTA, 5 μg/mL each of leupeptin, aprotinin, pepstatin A and chymostatin, 50 mM potassium fluoride, 50 μM okadaic acid, 5 mM sodium pyrophosphate). Homogenates were centrifuged at 30 000 g for 30 min at 4°C. The supernatants were collected as cytosolic fraction. The pellets were re-suspended in buffer B [Buffer A containing 0.5% Nonidet P-40 (Sigma-Aldrich Corp., St. Louis, MO, USA)] before being sonicated and centrifuged at 30 000 g for 30 min at 4°C again. The resulting supernatants were obtained as the particulate fraction. In order to get whole tissue homogenate, frozen samples were homogenized at 4°C in buffer C [Buffer A mixed with 2% sodium dodecyl sulfate (SDS)] and sonicated to dissolve the tissue completely. Protein concentration was determined by BCA kit (Pierce Company, Rockford, IL, USA) with albumin diluted in lysis buffer as standard.
Protein (50 μg) from each sample per lane were loaded on 10% SDS–polyacrylamide gel electrophoresis. The gels were electrophoresed, and then transferred onto polyvinylidene difluoride membrane (GE Healthcare) at 4°C. After several rinses with TTBS (20 mM Tris–Cl, pH 7.5, 0.15 M NaCl and 0.05% Tween-20), the transferred polyvinylidene difluoride membrane was blocked with 10% non-fat milk in TTBS for 1 h and incubated with the corresponding primary antibodies for 4 h. The horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Stressgen Biotechnologies Corporation, Victoria, BC, Canada) was used as second antibodies. Following incubation with the primary and secondary antibodies, the Enhanced Chemiluminescence (ECL) kit (GE Healthcare) was employed to detect the signals. To verify equal loading of protein, the blots were reprobed with primary monoclonal antibody against β-actin (Sigma-Aldrich Company).
Immunoprecipitation, two dimensional gel electrophoresis, silver staining and image analysis
Protein (500 μg) from cytosolic or particulate fractions were immunoprecipitated with 2 μg of polyclonal antibodies against cPKCβII (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After 3 h incubation, protein G sepharose was added for 12 h at 4°C and centrifuged for 1 min at 12 000 g. The precipitates were rinsed with immunoprecipitation buffer (0.5% NP-40, Tris–Cl pH 8.0, 0.15 M NaCl) four times to remove non-specific interaction products. For the control experiments, anti-cPKCβII antibody was replaced with IgG at the same volume in parallel. The co-immunoprecipitates were resolved in thiourea buffer (urea 6 M, thiourea 2 M, CHAPS 4%) for two dimensional gel electrophoresis (2-DE) or loading buffer for SDS–polyacrylamide gel electrophoresis. Equal amount of protein samples from three independent mice were pooled to reduce interindividual variation. Triplicate gels were run for each group to obtain statistical significance for protein differences.
First dimensional isoelectric focusing was performed on an Ettan IPGphor System by using 11 cm Immobilized pH gradient (IPG) strips with a linear pH gradient from 3 to 11 (GE Healthcare) to cover a wide range of cPKCβII-interacting proteins. Sample was loaded and active rehydration was performed at 30 V for 12 h at 20°C. Gels were run as follows: 200 V for 1 h, step; 500 V for 1 h, step; 1000 V for 1 h, step; 8000 V for 1 h, gradient; 8000 V for 3 h. The strips were then equilibrated with a solution containing 6 M urea, 30% glycerol, 2% SDS, 50 mM Tris–Cl, pH 8.8 and 1% DTT for 15 min, and then the strips were treated with the same solution containing 4% w/v iodoacetamide instead of DTT. The strips were over-layered onto 10% homogeneous polyacrylamide gels and electrophoresed at 20 mA per gel. Gels were stained using a silver staining kit (GE Healthcare) according to the manufacture’s procedure. Images were digitized with an image scanner and analyzed by using ImageMasterTM 2D Platinum Software version 5.0 (GE Healthcare). All spots were used to do gel-to-gel comparison. The total density in a gel image was used to normalize each spot volume and minimize inter-gel variation. The amount of each protein spot was expressed in terms of its volume. To reflect the quantitative variations in the protein spot volumes, the spot volumes were normalized as a percentage of the total volume of all the spots present in a gel. An average number of fold changes was calculated from three repeated experiments.
In-gel tryptic digestion, protein identification and database searches
Protein spots were excised and prepared for mass spectrometry (MS) after the process of destaining, reduction, alkylation, tryptic digestion and peptides extraction. Peptides were added to a metal plate with saturated α-cyano-4-hydroxycinnamic acid and analyzed by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS using a PrOTOF 2000 mass spectrometer (PerkinElmer, Waltham, MA, USA). Trypsin autocleavage peaks were used as internal standards for the mass calibration. The above data were all subjected to database searches for protein identification using a Matrix-Science Mascot search engine. Two databases, NCBInr and Swiss-Prot, were searched to yield more comprehensible results. These searches were performed with restriction to tryptic peptides: cysteine carbamidomethylation was selected as variable modification; a maximum of one trypsin miscleavage was allowed; peptide mass tolerance was set to 0.2 Da. Protein scores greater than 54 are significant (p < 0.05).
Statistics and analysis
We performed quantitative analysis for the immunoblot by using GelDoc-2000 Imagine System. For membrane translocation, the ratio of cPKCβII (band density in particulate/bands densities in both particulate and cytosol) in the Control group was expressed and normalized as 100%. For protein expression level, the protein ratio (band density of protein/band density of β-actin) was also expressed as 100% in the Control group. The data from other group were expressed as percentage of that from Control group. The presented values were expressed as mean ± SE. Statistical analysis was conducted by one-way anova followed by all pairwise multiple comparison procedures using Bonferroni test, and the significance was regarded as p < 0.05.
cPKCβII mediated HPC-induced neuroprotection against cerebral ischemic injuries of MCAO mice
Middle cerebral artery occlusion-induced cerebral ischemic injuries were evaluated by neurological deficits evaluation, 2,3,5-triphenyltetrazolium chloride and TUNEL staining. We found that 6 h MCAO-induced cerebral focal ischemia significantly changed the neurological behavior of mice, such as the occurrence of circling, forelimb flexion and hypomobility (8.5 ± 0.2, n = 10), while HPC could attenuate the neurological deficits in HPC + I mice (6.5 ± 0.4, n = 10) when compared with that of ischemia group (p < 0.05). However, intracerebroventricular injection of 5 μL cPKCβII inhibitor Go6983 (8 nM) or LY333531 (200 nM) could abolish this HPC-induced neuroprotective effect (8.9 ± 0.3 or 9.0 ± 0.5, p < 0.05 vs. HPC + I, n = 10, Fig. 1a).
Similarly, Fig. 1(b–d) showed that HPC could significantly decrease the infarct volume (26.8 ± 1.00%) and edema ratio (5.9 ± 0.4%) when compared with those of ischemia group (p < 0.05 vs. infarct volume: 36.8 ± 1.0%; edema ratio: 11.2 ± 0.5%, n = 10 per group). cPKCβII inhibitor Go6983 or LY333531 application before ischemia blocked HPC-induced reductions of infarct volume (36.3 ± 1.3% or 34.0 ± 1.3, n = 10) and edema ration (9.4 ± 0.3% or 7.2 ± 1.5, n = 10). Consistent with the results of neurological score, infarct volume and edema ration, HPC also reduced significantly (p < 0.05, n = 3 per group) the percentage of apoptotic cells (4.5 ± 1.1%) in the peri-infarct region when compared with that of ischemia (9.6 ± 1.2%), HPC + Go6983 + I (12.4 ± 2.3%) and HPC + LY333531 + I (10.7 ± 0.6%). The typical results of TUNEL staining were shown in Fig. 1(e).
To further reveal the involvement of cPKCβII activation in HPC-induced neuroprotection, we detected the cPKCβII membrane translocation and protein expression both in ischemic core and peri-infarct region of HPC mice. As shown in Figure S1, we found that cPKCβII membrane translocation in ischemic core and peri-infarct region decreased significantly at 6 h MCAO, but HPC could inhibit the decrease of cPKCβII membrane translocation in the ischemic cortex (p < 0.05, n = 5 per group). Protein expression levels didn’t undergo significant changes in the ischemic core and peri-infarct region of both control and HPC mice.
Proteomic analysis of the cPKCβII-interacting proteins in brain of HPC mice
The typical 2-DE results were showed in Fig. 2(a and c) (n = 3). A total of 49 proteins, including 34 proteins in particulate and 15 proteins in cytosolic fractions, were identified by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS (Table S1). According to Protein ANalysis THrough Evolutionary Relationships (PANTHER) classification system (http://www.pantherdb.org/), these proteins were further classified by categories of molecular functions and biological processes. The results suggested that cPKCβII may interact with proteins that belong to ubiquitin-protein ligase, cysteine protease, ATP synthase, amino acid activation and large G-protein hydrolase (Figure S2).
Based on image analysis, the expressions of four cPKCβII-mediated proteins in cytosol (Fig. 2b, n = 3) and eight proteins in particulate fractions (Fig. 2d, n = 3) changed significantly (more than 1.5-fold). As shown in Table 1, cPKCβII-interacted acetyl-CoA acetyltransferase mitochondrial (Acat1) and cysteine-rich hydrophobic domain 1 protein experienced a apparent down-regulation in cytosolic fraction; CRMP-2 was down-regulated in cytosolic fraction but up-regulated in particulate fraction; the protein levels of neurofilament light polypeptide (NF-L), 14-3-3γ, ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), glutamine synthetase, ATP synthase subunit δ and NADH dehydrogenase [ubiquinone] flavoprotein 2 mitochondrial precursor (Ndufv2) increased in the particulate fraction. In addition, cPKCβII-interacted α-enolase decreased both in cytosolic and particulate fractions of HPC mouse cortex.
|Protein name||Accession number||MW (kDa)||PI||Fold change|
|Collapsin response mediator protein-2 (CRMP-2)||O08853||62.24||5.95||+1.73|
|ATP synthase subunit δ, mitochondrial||Q9DCX2||18.38||5.52||+3.19|
|Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1)||Q9R0P9||24.82||5.14||+1.60|
|NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial precursor (Ndufv2)||Q9D6J6||27.61||7.00||+2.05|
|Neurofilament light polypeptide (NF-L)||P08551||61.47||4.62||+4.04|
|14-3-3 protein gamma (14-3-3γ)||P61982||28.29||4.80||+3.25|
|Glutamine synthetase (GS)||P15105||42.09||6.64||+4.15|
|Cysteine-rich hydrophobic domain 1 protein (Chic1)||Q8CBW7||25.77||4.56||−1.55|
|Acetyl-CoA acetyltransferase, mitochondrial (Acat1)||Q8QZT1||44.79||8.71||−1.90|
Furthermore, the interactions between cPKCβII and four proteins, including CRMP-2, NF-L, 14-3-3γ and UCHL1, were validated by reciprocal immunoprecipitation with commercially available antibodies (Fig. 3a). The changes of these cPKCβII-interacted proteins were also confirmed by Western blot (Fig. 3b), and its quantitative results (Fig. 3c–f, n = 3 per group) were consistent with that of 2-DE analysis: CRMP-2 decreased in cytosol but increased significantly in particulate fraction (p < 0.05); NF-L, 14-3-3γ and UCHL1 increased significantly in particulate fraction (p < 0.05) of HPC mice. However, the questions about whether ischemia could induce dissociation of cPKCβII and its interacting proteins or HPC could rescue these interactions still need further investigations.
CRMP-2 proteolysis and phosphorylation levels were associated with cPKCβII activation in ischemic cortex of MCAO mice
As one of the cPKCβII-interacting proteins, CRMP-2 belongs to the CRMP family and is involved in growth cone collapse and axon outgrowth (Arimura et al. 2005; Uchida et al. 2005). Therefore, we determined the changes of CRMP-2 proteolysis and phosphorylation in ischemic cortex of mice following 6 h MCAO. As shown in Fig. 4, CRMP-2 phosphorylation level reduced, while a 55-kDa breakdown product (proteolysis level), not total CRMP-2, increased significantly (p < 0.05, n = 4 per group) both in ischemic core and peri-infarct region. Interestingly, HPC could inhibit (p < 0.05, n = 4 per group) both the decrease of phosphorylated CRMP-2 level and increase of breakdown product production, but cPKCβII inhibitor Go6983 (8 nM) abolished this HPC-induced inhibitory effect on CRMP-2 dephosphorylation and proteolysis in ischemic core of MCAO mice (p < 0.05, n = 4 per group). In addition, TAT-CRMP-2 peptide, which prevents the cleavage of endogenous CRMP-2, could also inhibit (p < 0.05, n = 5 per group) CRMP-2 dephosphorylation and proteolysis in ischemic core as well as infarct volume of 6 h MCAO treated mice (Fig. 5).
The principal roles of PKC in I/HPC development have been reported in cultured neurons and brain slices (Selvatici et al. 2003). However, because of the biological complexity and the dual involvement of PKC isoforms during I/HPC, there is a lack of detailed information regarding PKC isoforms in response to neuronal protection or damage. Among the 10 isoforms of PKC, we have reported that the membrane translocation (activation) of cPKCβII, γ and nPKCε increased in response to HPC both in vivo and in vitro (Li et al. 2005, 2006; Niu et al. 2005). Furthermore, this study is the first to report multiple cPKCβII-interacting proteins in HPC mouse brain and the role of cPKCβII-CRMP-2 in HPC-induced neuroprotection against ischemic injuries.
nPKCε activation has been shown to protect neurons and astrocytes from ischemia-induced damage by oxygen-glucose deprivation in neuronal or neuron-astrocyte mixed cultured cell lines (Wang et al. 2004). Using a rat MCAO model, Bright et al. found that systemic administration of ψεRACK, an nPKCε-selective peptide activator, confers neuroprotection against subsequent cerebral ischemic injury through 24 h post-stroke (Bright et al. 2008). This nPKCε-initiated ischemic preconditioning or neuroprotection might be mediated by targeting the respiratory chain of synaptic mitochondria, mitochondrial K(+) (ATP) channel or altering cerebral blood flow (Raval et al. 2007; Dave et al. 2008, 2009; la-Morte et al. 2011). In addition, we also found cPKCγ signaling molecules, especially the cPKCγ-synapsin pathway is responsible for HPC-induced neuroprotection against cerebral ischemic injuries of mice (about to be reported).
The detrimental role of cPKCβ has been reported in cardiac and lung ischemia/reperfusion by using cPKCβ-null or wild type mice fed with the cPKCβ inhibitor LY333531 (Fujita et al. 2004; Yan et al. 2006; Kong et al. 2008). However, we found that HPC could reduce 6 h MCAO-induced neurological deficits, cerebral infarct volume and cell apoptosis via the activation of cPKCβII in the peri-infarct region of ischemic cortex. The causes of the discrepancy about the role of cPKCβII in ischemic lung, heart and brain may ascribe to the various cell types and signaling pathways in different organs.
So far, little is known about the intracellular cPKCβII-specific signaling networks in brain. Yan et al. reported that cPKCβ may be an early trigger to the activation of c-Jun N-terminal kinase and p38 MAPK that occurs in response to ischemia/reperfusion (Yan et al. 2006). In this study, we identified 49 proteins which could be co-immunoprecipitated with cPKCβII from cytosolic and particulate fractions of mouse cortex. According to the PANTHER classification system, theses cPKCβII-interacting proteins are involved in cell structure, protein folding and protein acetylation; after activation in HPC, cPKCβII would expand its interactions with some proteins, such as ATP synthase and Ndufv2 for oxidative stress, Acat1 and α-enolase for energy metabolism, CRMP-2 for axon guidance, UCHL1 for ubiquitin proteasome, GRP78 for protein folding and assembly, and 14-3-3γ for neural development.
Among these identified proteins, most of them were initially reported as cPKCβII- interacting protein. 14-3-3γ is a brain-specific protein and has the ability to bind a multitude of functionally diverse signaling proteins including PKC, phosphatases and transmembrane receptors (Dougherty and Morrison 2004). Recent study showed that ischemia could up-regulate 14-3-3γ in astrocytes through c-Jun N-terminal kinase/c-Jun/activator protein 1 (AP-1) pathway (Dong et al. 2009). In addition, the possible binding sites of 14-3-3γ with PKC isoforms have been reported by using bioinformatic survey (Johnson et al. 2010). In this study, we found that cPKCβII might form complex with 14-3-3γ and this interaction was enhanced during HPC.
Neurofilament proteins are made up by the copolymerization of the neurofilament light, medium and heavy chain proteins. NF-L alterations have been described in brain ischemic injury, neural development and neurodegenerative diseases (Mink and Johnston 2000; Lariviere and Julien 2004). In this study, we observed that the interaction of NF-L with cPKCβII increased in particulate fraction of cerebral cortex in response to HPC.
The mitochondrial F1F0 ATP synthase is a critical enzyme for generating sufficient ATP for sustenance of life in mammals, and an ATP hydrolase under ischemic conditions (Grover et al. 2008). To prevent ATP wastage in ischemia, the ATP hydrolysis activity of ATP synthase should be inhibited selectively without affecting its ATP synthesis activity. We found that cPKCβII could bind to δ subunit of ATP synthase, and this subunit protein levels in particulate fraction increased after HPC. However, whether cPKCβII could regulate the ATP hydrolysis or ATP synthesis activities of the F1F0 ATP synthase needs to be further studied. Ndufv2 is one subunit of the NADH ubiquinone oxidoreductase which belongs to the mitochondrial oxidative phosphorylation system and plays a part in oxidative stress (Sharma et al. 2009). Acat1 and α-enolase are key enzymes involved in protein acetylation (Chang et al. 2009) and glycolysis (Pancholi 2001). The enhanced or weakened interaction between cPKCβII and these enzymes suggested a potential role of cPKCβII in regulation of oxidative stress and energy metabolism during cerebral HPC development.
As a member of the ubiquitin C-terminal hydrolases, it was reported that S18Y mutation of UCHL1 conferred a specific antioxidant when expressed at physiological levels in human neuroblastoma cells and primary cortical neurons (Kyratzi et al. 2008). UCHL1 over-expression may contribute to the ubiquitin-mediated stress response to ischemic injury in rabbits, and it may also serve as a neuron-enriched protein marker for brain injury in humans (Yamauchi et al. 2008; Siman et al. 2009). The interaction between cPKCβII and UCHL1 was first observed in our experiments, but whether cPKCβII could regulate the activity of UCHL1 needs to be further confirmed.
Collapsin response mediator protein-2, which also be reported as a substrate of Rho, Cyclin-dependent kinase 5 (CDK5) and Glycogen synthase kinase 3 beta (GSK3β), is involved in growth cone collapse, axon outgrowth, ischemia and Alzheimer’s disease (Arimura et al. 2005; Uchida et al. 2005; Yoshimura et al. 2005). However, the physiological and pathological functions of CRMP-2 are still largely unknown. Recent studies showed that CRMP-2 could interact with neurofibromin in rat brain, which suggested a novel role of CRMP-2 in neuronal morphogenesis (Lin and Hsueh 2008). Several studies also described the changes of CRMP-2 in cerebral ischemic, neurotoxic and traumatic injuries. Chen et al. (2007) reported the up-regulation of CRMP-2 protein expression in ischemic cortex of MCAO rat. Zhou et al. (2008) demonstrated the hypophosphorylation of CRMP-2 in neonatal mouse brain following hypoxia-ischemia treatment hypoxia-ischemia, which might be caused by inactivation of Cyclin-dependent kinase 5 (CDK5) kinase. After neurotoxin treatment or traumatic brain injury, CRMP-2 was found to be degraded in calpain-2 dependent manner (Zhang et al. 2007). In this study, we first revealed the interaction between cPKCβII and CRMP-2, and found that HPC and TAT-CRMP-2 peptide could inhibit CRMP-2 dephosphorylation and proteolysis in ischemic core as well as infarct volume of 6 h MCAO mice. According to the report that increased phoshphorylation level and reduced cleavage of CRMP-2 could enhance axonal resistance to neurotoxicity (Hou et al. 2009), we suggested that cPKCβII activation might function as an axonal anti-collapse factor through phosphorylation of CRMP-2.
In summary, we first identified multiple cPKCβII interacting proteins in HPC mouse brain. Of particular interest, the cPKCβII-CRMP-2 pathway might be responsible for the HPC-induced neuroprotection against early stages of ischemic injuries in mice. The further studies on the identified cPKCβII-interacting proteins will expand our understanding of the molecular mechanisms underlying cerebral HPC, and assist in the eventual development of new molecular targets for clinical therapy in stroke.
This work was supported by the following grants: National Natural Science Foundation of China (30871219, 31071048), China 973 Program (2006CB504100), Key Scientific Developing Program of Beijing Municipal Commission of Education (KZ200810025012), Beijing Municipal Program for Hundred-Thousand-Ten Thousand Excellent Talents of New Century (Li J), Funding Project for Academic Human Resources Development in Institutions of Higher Learning under the Jurisdiction of Beijing Municipality (PHR200906116), and Ph.D. Programs Foundation of Ministry of Education of China (20091107110001).