Abstract
Neural cell adhesion molecule (NCAM) is a membrane protein abundantly expressed in the central nervous system. Recently, it has been reported that dysfunction of NCAM is linked to human brain disorders. Furthermore, NCAM is one of the proteolysis targets of matrix metalloproteinase (MMP), whose activation is implicated in neuronal damage. The aim of this study was to elucidate the involvement of MMP-mediated proteolysis of NCAM in the development of ischemic neuronal damage. Male ddY and MMP-9 knockout (KO) C57BL/6J mice were subjected to 2 h of middle cerebral artery occlusion (MCAO). In MCAO model mice, development of infarction and behavioral abnormality were clearly observed on days 1 and 3 after MCAO. Protein levels of MMP-2 and MMP-9 were significantly increased on days 1 and 3 after MCAO. In addition, full-length NCAM (180 kDa) was significantly decreased, but its metabolite levels increased on day 1 by ischemic stress per se. NCAM small interfering RNA significantly increased the neuronal damage induced by MCAO. MMP inhibition or MMP-9 gene KO attenuated the infarction, behavioral abnormalities, and decrease of NCAM (180 kDa) observed after MCAO in mice. The present findings clearly suggest that MMP-2/MMP-9-mediated NCAM proteolysis is implicated in the exacerbation of ischemic neuronal damage.
Introduction
Focal cerebral ischemia (stroke) is the third most common cause of death in the world (Hankey and Warlow, 1999; Hasegawa et al., 2006). Ischemic neuronal damage is known to be related to mitochondria and endoplasmic reticulum dysfunction, activated oxygen species, and increased expression of inflammatory cytokines and proteolytic enzymes (Heo et al., 1999; Zhao et al., 2004; Sarabi et al., 2008), but the detailed molecular mechanisms are still unknown. In spite of the development of various therapeutic strategies, including thrombolysis with tissue-type plasminogen activator or free radical scavengers (Yepes et al., 2000), it has been difficult to completely cure because of the intractable severe subsequent complications such as paralysis and cognitive dysfunction (Shinohara et al., 2009; Yagi et al., 2009). Thus, clarification of the detailed mechanisms responsible for the development of ischemic neuronal damage is urgently required. The aim of this study was to identify one of the key molecules involved in ischemic neuronal damage.
Neural cell adhesion molecule (NCAM), a member of the Ig superfamily of cell recognition molecules, is expressed on the membranes of neurons and glial cells and promotes cell-cell interaction via homophilic and heterophilic binding (Povlsen et al., 2003; Maness and Schachner, 2007). It is noteworthy that NCAM is known to regulate the processes that influence cell adhesion, cell migration, and neurite outgrowth and to be involved in the development of the nervous system, brain plasticity associated with learning and memory, and neuronal regeneration (Maness and Schachner, 2007). Furthermore, dysfunction of NCAM is reported to contribute to the development of neurological disorders such as schizophrenia, bipolar disorder, and Alzheimer's disease (Vawter, 2000; Todaro et al., 2004; Maness and Schachner, 2007).
NCAM is synthesized in three main membrane-bound isoforms, NCAM-120, NCAM-140, and NCAM-180 (Ronn et al., 1998). It is noteworthy that among these isoforms the proteolysis of NCAM-180 has been reported to relate to psychological disorders in humans (Poltorak et al., 1996; Vawter, 2000; Tanaka et al., 2007). In addition, mice lacking NCAM-180 have deficits in learning, emotional memory, and sensory gating (Bukalo et al., 2004; Maness and Schachner, 2007). Matrix metalloproteinase (MMP), one of the proteolytic enzymes targeting NCAM (Hübschmann et al., 2005; Hinkle et al., 2006), is reported to be activated by ischemic stress (Heo et al., 1999; Zhao et al., 2004). Although some target molecules of MMP such as laminin or interleukin-1β are reported to be involved in neuronal death and/or development of neuronal injury (Gu et al., 2005; Kawasaki et al., 2008), there are no reports indicating that MMP-NCAM interaction is involved in the development of neuronal damage. Here, we focused on the involvement of MMP-mediated proteolysis of NCAM in the development of ischemic neuronal damage using middle cerebral artery occlusion (MCAO) model mice, a popular ischemic model in experimental stroke research that results in prominent ischemic damage as described previously (Harada et al., 2009).
Materials and Methods
Animals.
The experiments were performed on male ddY mice (5–6 weeks old; Japan SLC, Osaka, Japan), and MMP-9 KO (background of C57BL/6J) mice were kindly given by the Nagoya University Research Institute of Environmental Medicine (Nagoya, Japan). As described previously (Mizoguchi et al., 2007), MMP-9 homozygous KO mice (10–12 weeks old) were obtained from The Jackson Laboratory (Bar Harbor, ME) and crossed to C57BL/6J mice for eight generations before being made homozygous. Wild-type C57BL/6J mice were obtained from the Japan SLC. The animals were housed at a temperature of 23 to 24°C with a 12-h light-dark cycle (lights on 8:00 AM to 8:00 PM). Food and water were available ad libitum. The present study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, adopted by the Japanese Pharmacological Society. In addition, all experiments were approved by the ethical committee for animals of Kobe Gakuin University (approval number A 060601-10).
Drug Treatments.
GM6001 (N-[(2R)-2(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide), a broad-spectrum MMP inhibitor (Calbiochem, San Diego, CA), and selective MMP-2 and MMP-9 inhibitors, MMP-2 inhibitor III [(2-((isopropoxy)-(1,1′-biphenyl-4-ylsulfonyl)-amino))-N-hydroxyacetamide] (Calbiochem) and MMP-9 inhibitor I [4-methoxyphenyl benzyl(4-((diethylamino)methyl)-2-(hydroxycarbamoyl)-6-methylphenyl)sulfite] (Calbiochem), respectively, were dissolved in 4% DMSO in saline (0.5 μg/μl) and administered in a volume of 5 μg/mouse through the intracerebroventricular route 30 min before and 2 h after ischemia. Control mice were administered 4% DMSO in saline (vehicle) in a volume of 10 μl/mouse i.c.v. NCAM1 ON-TARGETplus SMART pool (NCAM siRNA; Thermo Fisher Scientific, Kanagawa, Japan), ON-TARGETplus Nontargeting Pool (control siRNA; Thermo Fisher Scientific), and in vivo jetPEI + 10% glucose (cationic polymer transfection reagent; Polyplus Transfection, New York, NY) were administrated directly after preparation. Mice were injected with siRNA directed at NCAM (1 μg/mouse i.c.v.) 72 h before MCAO to knock down NCAM protein levels. Intracerebroventricular administrations were performed as described previously (Haley and McCormick, 1957). In brief, a microsyringe with a 27-gauge stainless-steel needle was used for all experiments. Tubing covered all but the terminal 2.5 to 3.0 mm of the needle so as to make a track through the brain and into the lateral ventricle but not through the floor of the lateral ventricle. The needle was inserted unilaterally into the lateral ventricle of the brain (1.0 mm lateral and 1.0 mm posterior to the bregma) as described previously (Franklin and Paxinos, 2008). Verification of needle position in the lateral cerebroventricle was made by intracerebroventricular dye injection and subsequent postmortem brain section verification of dye placement.
Induction of Ischemic/Reperfusion Injury.
The experimental transient focal ischemia mouse model was generated by performing MCAO as described previously (Harada et al., 2009). In brief, mice were anesthetized with 2% isoflurane (Abbott Japan, Osaka, Japan) and maintained in an anesthetized state with 1% isoflurane. The rectal temperature was maintained at 37 ± 0.5°C with the use of a heating blanket (FH-100; Unique Medical, Osaka, Japan) that was feedback-controlled by a rectal temperature probe (PTE-101; Unique Medical) and a small animal heat controller (ATC-101B; Unique Medical). The left common carotid artery (CCA) and external carotid artery underwent a midline pretracheal incision. The vagus nerves were separated carefully from the artery. The CCA and external carotid artery were ligated, and then the internal carotid artery was isolated. Bifurcation of the CCA was made through a small incision, and then a 8-0 nylon monofilament (Shirakawa, Fukushima, Japan) with a 4-mm tip coated in silicon resin (Xantopren Blue and Activator; Heraeus Kulzer, Hanau, Germany) was introduced through a small incision and advanced 9 mm along the internal carotid artery beyond the bifurcation site, thus stopping the blood flow to the middle cerebral artery. After 2 h of ischemia, mice were reanesthetized with isoflurane; then the filament was withdrawn for blood reperfusion. The sham-operated mice were subjected to the procedure mentioned above without MCAO. The operative site was sutured, and mice were allowed to awake from the anesthesia. Mice with brain hemorrhage were eliminated from analysis. The relative cerebral blood flow was measured by laser Doppler flowmetry (TBF-LN1, Unique Medical) to assess the adequacy of the vascular occlusion and reperfusion as described previously (Harada et al., 2009).
Measurement of Infarct Volume.
Mice were euthanized by cervical dislocation on days 1 and 3 after MCAO. The brains were cut into 2-mm-thick coronal slices (−2, 0, +2, and +4 mm from the bregma) using a brain slicer. The brain slices were then incubated in normal saline containing 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma, St. Louis, MO) for 10 min at 37°C. After staining, the brain slices were fixed with 4% paraformaldehyde (Sigma) for 2 h, and then stored in phosphate-buffered saline. Areas not stained red with TTC were considered to be infarctions. The brain slices were scanned. Unstained areas (infarct areas) were measured using image analysis software (Image J; National Institutes of Health, Bethesda, MD) and Adobe Photoshop Elements 5.0 (Adobe Systems Incorporated, Tokyo, Japan). The infarct volume (mm3) was calculated by multiplication of infarct volume (mm3) and intensity (intensity = intensity of left hemisphere − intensity of right hemisphere).
Measurement of Brain Edema.
Brain edema was assessed by measuring the brain water content according to the wet-dry method as described previously (Ding-Zhou et al., 2003; Liu et al., 2008). In brief, after decapitation, the brain was quickly removed and dissected along the interhemispheric fissure into the ischemic and nonischemic cerebral hemispheres. Tissues were weighed with a scale to within 0.1 mg. Dry weight of the brain was determined after heating the tissue for 3 days at 100°C in a drying oven. Brain water content was then calculated as [(wet weight − dry weight)/wet weight] × 100.
Neurological Examination.
Neurological examination was performed using the neurological deficit score (NDS) comprising consciousness (0, normal; 1, restless; 2, lethargic; 3, stuporous; 4, seizures; 5, death), walking (0, normal; 1, paw; 2, unbalanced walking; 3, circling; 4, unable to stand; 5, no movement), and limb tone (0, normal; 1, spastic; 2, flaccid), and pain reflex was scored after reperfusion as described previously (Harada et al., 2009). Pain reflex was assessed using the tail-flick test (pain reflex = latency after MCAO − latency before MCAO). A cutoff time of 10 s was used to prevent any injury to the tail.
Western Blot Analysis.
On the indicated days after reperfusion, mice were decapitated and brains were removed. Ipsilateral cortex was dissected and homogenized with homogenize buffer (20 mM Tris-HCl, pH 7.5, 120 mM NaCl, 4% Tween 20, 2 mM β-mercaptoethanol, 1 mM Na3VO4, 5 mM benzamidine, 20 mM NaF, 1 mM p-nitrophenyl phosphate, 5 mM imidazole, 50 mg/ml trypsin inhibitor, 50 mg/ml leupeptin, 50 mg/ml aprotinin, 5 mg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride). After centrifugation at 15,000g, the supernatant was collected and immediately lysed in SDS-sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol), boiled, and reduced with β-mercaptoethanol. To investigate protein expression patterns in control and ischemic cortex, equal amounts (20 μg) of total protein extracts were prepared. After mixing with 2× sample buffer, each sample was separated by nonreducing gel in native condition for NCAM, MMP-2, and MMP-9 (120 V, 90 min). Markers used were Precision Plus Protein Standards Kaleidoscope (Bio-Rad Laboratories, Hercules, CA). After separation, proteins were transferred to nitrocellulose membranes (15 V, 50 min). All blots were blocked with 10% skim milk (blocking agent; GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) in PBS, pH 7.4, or TBS containing 0.1% Tween 20 (PBS-T or TBS-T, respectively) at room temperature for 1 h. Then, the membranes were incubated with the primary antibodies diluted in blocking buffer at 4°C overnight. The dilution rates of the primary antibodies were 1:200, 1:1000, 1:1000, and 1:20,000 for NCAM (65 and 180 kDa) (Sigma), MMP-2 (Abcam, Tokyo, Japan), MMP-9 (Cell Signaling, Tokyo, Japan), and GAPDH (Millipore Bioscience Research Reagents, Temecula, CA), respectively. After washing with PBS-T or TBS-T for 30 min, the membrane was incubated with secondary antibody [horseradish peroxidase-labeled, affinity-purified antibody to rabbit IgG+IgM (H+L) (1:1000; Kirkegaard and Perry Laboratories, Gaithersburg, MD) for MMP-2 and MMP-9, horseradish peroxidase-labeled, affinity-purified antibody to mouse IgG+IgM (H+L) (1:10,000; Kirkegaard and Perry Laboratories for NCAM (65 and 180 kDa) and GAPDH] at room temperature for 40 min. After washing with PBS-T or TBS-T for 30 min, antigen was detected by using the standard chemical luminescence method (GE Healthcare). Detection of band was used by Light-Capture II (ATTO, Tokyo, Japan) and Cs-Analyzer (version 3.0; ATTO).
Gelatin Zymography.
Similarly prepared protein samples (as in Western blot analysis) were incubated with 50% gelatin Sepharose 4B (GE Healthcare) for 4 h. After washing with wash buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM CaCl2, 0.05% BRIJ-35, 0.02% NaN3), samples were eluted by elution buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM CaCl2, 0.05% BRIJ-35, 0.02% NaN3, 10% DMSO). After centrifugation for 30 min at 4°C, supernatant was loaded and separated in a precast gel by use of a gelatin zymography kit (Primary Cell, Hokkaido, Japan). After separation by electrophoresis, the gel was renatured and then incubated with developing buffer at 37°C over 60 h. After developing, the gel was stained with 0.5% Coomassie blue R-250 for 30 min and then destained appropriately in methanol and acetic acid in water (30:5:65). Detection of band was used by Light-Capture II (ATTO) and Light Viewer SLIM 57 (ATTO). Semiquantified analysis of band intensity was performed using Image J (National Institutes of Health).
Statistical Analysis.
Data are presented as means ± S.E.M. Comparisons of parameters at a given time point among multiple treatment groups, as well as within treatment group comparisons across more than two time points, were conducted using a one-way analysis of variance, followed by a post hoc Dunnett test or Scheffe's test. Comparisons of parameters measured in the absence versus the presence of treatment were conducted using unpaired Student's t test. The data for NDS were analyzed using a Steel-Dwass test of post hoc nonparametric multiple comparison tests or a Wilcoxon-Mann-Whitney U test. Data are presented as medians (25th–75th percentile). Before statistical post hoc test, we checked normality and equal variance test for all data. The differences were regarded as statistically significant when the p value was less than 0.05.
Results
Development of Neuronal Damage and Protein Levels or Activity of MMP-2 and MMP-9 in the Cerebral Cortex after Cerebral Ischemic Stress.
As shown in Fig. 1A, the infarct area in the cortex, hippocampus, and striatum was gradually enlarged on day 3 compared with day 1 after MCAO (Fig. 1, A and B). In addition, the increase of brain water content, indicating the development of edema, was significantly observed and gradually enlarged on days 1 and 3 after MCAO (Fig. 1C). In addition, NDS was more significantly increased in the MCAO group than in the sham group on days 1 and 3 (Fig. 1D). In the Western blot analysis, we detected a 72-kDa band for MMP-2 and a 108-kDa band for MMP-9, and they were significantly increased on days 1 and 3 after MCAO compared with the sham group (Fig. 1, E and F). The gelatin zymograms clearly demonstrated the increase of MMP-2 and MMP-9 activity on day 3 after MCAO. Semiquantified data showed the significant differences, in a GM6001, a broad-range MMP inhibitor, reversible manner (Fig. 1G).
Effect of NCAM siRNA on Ischemic Neuronal Damage.
To confirm the role of NCAM in MCAO-induced neuronal damage, we investigated whether NCAM knockdown increases the ischemic neuronal damage. Three days after NCAM siRNA administration, NCAM (180 kDa) protein levels were significantly decreased in the cortex in naive mice (Fig. 2A). In addition, we confirmed the NCAM (180 kDa) protein levels were kept lower until 4 days after siRNA administration (data not shown). Thus, we performed MCAO on day 3 after NCAM siRNA administration.
The infarct volume was significantly increased on day 1 after MCAO in the NCAM siRNA-treated group compared with the control group (Fig. 2, B and C). NDS was also more significantly increased in the NCAM siRNA-treated group than in the control group (Fig. 2D). Because the development of infarction accomplishes maximum on day 3 after MCAO, it was hard to compare the difference between control and siRNA-treated groups on day 3 after MCAO.
Protein Levels of NCAM (180 kDa) and NCAM Cleavage Product (65 kDa) after Cerebral Ischemic Stress.
Because NCAM is one of the proteolysis targets of MMP, the levels of NCAM (180 kDa) and NCAM cleavage product (65 kDa) after cerebral ischemic stress were determined. NCAM (180 kDa) protein levels were more significantly decreased on day 1 after MCAO compared with the sham group, whereas it returned to the sham levels on day 3 (Fig. 3A). On the other hand, NCAM cleavage product levels (65 kDa) were more significantly increased on days 1 and 3 compared with the sham group (Fig. 3B).
Effect of a Nonselective MMP Inhibitor, GM6001, on Ischemic Neuronal Damage, NCAM Protein Levels, and NCAM Cleavage Product Levels.
We investigated whether MMP inhibitor suppresses the development of ischemic neuronal damage. As shown in Fig. 4, A and B, the infarct volume observed on day 3 after MCAO was significantly decreased by treatment of GM6001. Furthermore, the ischemic stress-induced increase of NDS was also significantly inhibited by GM6001 treatment (Fig. 4C). In addition, the decrease in NCAM (180 kDa) protein levels observed on day 1 was completely recovered to the control level by GM6001 (Fig. 4D). In addition, the increment of NCAM cleavage product levels (65 kDa) observed on days 1 and 3 was significantly suppressed by GM6001 (Fig. 4E).
Effect of MMP Isozyme-Specific Inhibitors on Ischemic Neuronal Damage, NCAM Protein Levels, and NCAM Cleavage Product Levels.
To determine which MMP plays a critical role in MCAO, we used selective inhibitors for MMP-2 and MMP-9. The development of infarction and increase of NDS observed on day 3 was significantly decreased by MMP-2 inhibitor III (Fig. 5, A–C). The decrease in NCAM (180 kDa) protein levels observed on day 1 was completely recovered to the control level by MMP-2 inhibitor III (Fig. 5D). In addition, the increment of NCAM cleavage product levels (65 kDa) observed on days 1 and 3 were significantly suppressed by this inhibitor (Fig. 5E). Furthermore, treatment with the selective MMP-9 inhibitor I showed the same results as the selective MMP-2 inhibitor III (Fig. 6).
Ischemic Neuronal Damage, NCAM Protein Levels, and NCAM Cleavage Product Levels after Cerebral Ischemic Stress in MMP-9 KO Mice.
To confirm the role of MMP-9 in MCAO, we investigated whether MCAO shows ischemic neuronal damage by using MMP-9 KO mice. Because the C57BL/6 strain is known to be more susceptible to cerebral ischemia compared with ddY mice (Yang et al., 1997), we investigated only on day 1 after MCAO. The development of infarction was significantly reduced in the MMP-9 KO mice compared with WT mice on day 1 after MCAO (Fig. 7, A and B). The NDS on day 1 was also more significantly decreased in the MMP-9 KO mice compared with WT mice (Fig. 7C). In addition, the decreases in NCAM (180 kDa) protein levels and increment of NCAM cleavage product levels (65 kDa) in WT mice were not observed in the MMP-9 KO mice on day 1 after MCAO (Fig. 7, D and E).
Discussion
NCAM, one of the members of the Ig superfamily of CAMs, is encoded by a single gene located in chromosome 11 in humans. As a result of alternative splicing, three types of isoforms are known: NCAM-180 (180 kDa), NCAM-140 (140 kDa), and NCAM-120 (120 kDa), which differ in their C terminals (intracellular part) (Tacke and Goridis, 1991; Doherty et al., 2000). NCAM is widely expressed in the nervous system where it plays a pivotal role in proliferation, migration, axonal outgrowth, fasciculation, and synaptic plasticity (Ronn et al., 1998; Berezin et al., 2000; Povlsen et al., 2003; Maness and Schachner, 2007). Furthermore, NCAM is found on glial and neuronal cells and participates in binding these cells together (Vawter, 2000). It has been suggested that neuron-glia interactions are involved in the physiological function of neurons (Vawter, 2000; Chadi et al., 2009). Aside from its role in cell-cell interactions, an important role for NCAM in the regulation of intracellular signaling pathways has been revealed (Povlsen et al., 2003; Kiryushko et al., 2004).
The protein levels of NCAM and its post-translational modified form polysialylated NCAM have been reported to be highly susceptible to modulation by stress (Bisaz et al., 2009). In regard to cerebral ischemic stress, the increase of polysialylated NCAM was observed in the subventricular zone that is involved in the proliferation of endogenous neural progenitor cells (Macas et al., 2006), although there are no reports about the alteration of NCAM per se. We focused on NCAM-180, the largest isoform of NCAM that possesses the full size of cytoplasmic termini. It is noteworthy that we found that ischemic stress per se induced down-regulation of full-length NCAM (180 kDa) in the cortex. It is possible that down-regulation of full-length NCAM (180 kDa) becomes one of the triggers of neuronal damage under ischemic stress.
In this study, we clearly confirmed that down-regulation of NCAM by siRNA significantly exacerbated the neuronal damage in MCAO model mice. These findings suggest that NCAM possesses a surviving effect on the central nervous system. Previous reports have demonstrated that NCAM deficiency causes morphological alterations in the central nervous system, including a reduction in brain weight and the size of the olfactory bulb (Cremer et al., 1994). These results may correlate with our observations that show the importance of NCAM in neuronal survival. Furthermore, many reports have indicated that NCAM KO mice lead to impaired long-term potentiation (Cremer et al., 1998) and severe cognitive impairments and emotional alterations (Cremer et al., 1994; Bisaz et al., 2009), suggesting the importance of NCAM in brain function. As we have reported previously, the postischemic memory dysfunction could be developed in this MCAO model (Harada et al., 2009, 2010). It is possible that down-regulation of full-length NCAM (180 kDa) may be involved in postischemic memory dysfunction.
The ischemic stress induced not only the decrease of full-length NCAM (180 kDa), but also the increase of NCAM cleavage product (65 kDa), which is considered a proteolytic fragment of NCAM (Tanaka et al., 2007) by extracellular proteases. MMP is a family of zinc-dependent endopeptidases that can be released or activated in a neuronal activity-dependent manner (Mizoguchi et al., 2007) and are attractive targets in the field of inflammatory and psychological diseases (Yong et al., 2001; Kawasaki et al., 2008; Mizoguchi et al., 2008). They cleave all constituents of the extracellular matrix including collagens and laminins (Yong et al., 2001). It has been well known that MMPs can also target a variety of nonmatrix proteins including varied soluble molecules, cell surface receptors, and synaptic cell adhesion molecules including NCAM (Vawter, 2000; Conant et al., 2010). The soluble extracellular domain of NCAM has been identified in normal brain, potentially arising from ectodomain shedding of the extracellular domain of transmembrane NCAM by MMPs (Hübschmann et al., 2005; Hinkle et al., 2006).
There are at least 25 members of the MMP family, and their activity is tightly regulated. Although MMPs are expressed in both glial and neuronal cells (Yong et al., 2001; Szklarczyk et al., 2002), ischemic stress is known to induce MMP-2 and MMP-9, major isozymes in the MMP family, in glial cells such as reactive microglia and astrocytes (Svedin et al., 2007). Ischemic stress accompanies the progression of neuronal death via cleavage of precursors of inflammatory cytokines by MMP-2 and MMP-9 (Amantea et al., 2007; del Zoppo et al., 2007).
In this study, MMP-2 and MMP-9 were significantly up-regulated on days 1 and 3 after ischemic stress. The increased enzymatic activity of MMP-2 and MMP-9 was also detected on day 3 after ischemic stress, corresponding with previous studies (Asahi et al., 2001; Lee et al., 2004; Koistinaho et al., 2005). Because the increase of full-length NCAM (180 kDa) was accompanied by the decrease of NCAM cleavage product (65 kDa) by an inhibition of MMP-2 and MMP-9, these changes seem to be caused by the transmembrane proteolysis of NCAM via activation of these enzymes. Furthermore, the present results using MMP-9 null mice obviously support this hypothesis at least for the involvement of MMP-9.
As shown in the present study, the development of edema was significantly observed after ischemic stress. Because many reports have also demonstrated that the proteolytic degradation by MMP of critical blood-brain barrier contributes to the development of postischemic edema (Asahi et al., 2001; Lee et al., 2004; Svedin et al., 2007), it is possible that NCAM is involved not only in the MMP-mediated development of ischemic neuronal damage but also in the MMP-mediated development of postischemic edema. Although we could not distinguish which MMP (MMP-2 or MMP-9) is more important in ischemic stress-induced NCAM degradation in this study, our results indicate that their specificity against NCAM and the characteristic differences between MMP-2 and MMP-9 in the NCAM degradation may be small. Clinical reports showing that proteolytic product of NCAM by MMP (65- to 70-kDa bands) increases under neuropathological conditions (Tanaka et al., 2007) are in agreement with our findings in the present study. It is noteworthy that this is the first study demonstrating the MMP-mediated proteolysis of NCAM in a cerebral ischemic stress model animal. As demonstrated, the full length of NCAM (180 kDa) was significantly decreased on day 1 after MCAO and it recovered to normal levels on day 3, whereas the protein levels of NCAM cleavage product (65 kDa) kept increasing until day 3. These results suggest that some physiological compensatory reaction against the down-regulation of NCAM (180 kDa) may occur after ischemic stress, while its cleavage reaction by MMP-2 and MMP-9 into NCAM cleavage product (65 kDa) continued. Furthermore, the transient decrease of full-length NCAM (180 kDa) in the early phase of ischemic stress may be one of the important triggers in the development of neuronal damage. Note that MMP-9 inhibitor I is a potent and selective inhibitor of MMP-9, but it also inhibits and MMP-13 and MMP-1 at higher concentrations. Likewise, MMP-2 inhibitor III is a potent and selective inhibitor of MMP-2, but also inhibits MMP-9 and MMP-3 at higher concentrations, suggesting the possible involvement of MMPs other than MMP-2 and MMP-9, but that remains to be determined.
The increase of a fragment of NCAM (65–70 kDa) and the other fragment of NCAM (105–115 kDa) has been reported in patients with schizophrenia or Alzheimer's disease (Vawter, 2000; Todaro et al., 2004; Tanaka et al., 2007). Taken together, proteolysis of NCAM by MMP-dependent shedding (Hübschmann et al., 2005; Hinkle et al., 2006) may occur in neurodegenerative disorders. Although we did not determine the alterations of NCAM fragment (105–115 kDa) under ischemic stress, the present findings indicate that the MMP-mediated NCAM proteolysis may be implicated in the exacerbation of ischemic neuronal damage. Furthermore, it is possible that the accumulation of NCAM cleavage product is related to a deteriorative or degenerative process in ischemic neuronal damage as described in the case of schizophrenia (Vawter, 2000); however, it remains to be determined.
In conclusion, we found that MMP-mediated decrease of full-length NCAM (180 kDa) is a new mechanism for the development of ischemic neuronal damage. These findings may help the development of new drugs or therapeutic strategies against stroke in the future.
Authorship Contributions
Participated in research design: Shichi, Fujita-Hamabe, and Tokuyama.
Conducted experiments: Shichi, Fujita-Hamabe, Harada, and Tokuyama.
Contributed new reagents or analytic tools: Mizoguchi, Yamada, Nabeshima, and Tokuyama.
Performed data analysis: Shichi, Fujita-Hamabe, Harada, and Tokuyama.
Wrote or contributed to the writing of the manuscript: Shichi, Fujita-Hamabe, Nabeshima, and Tokuyama.
Footnotes
This work was supported by grants-in-aid and special coordination funds from the Academic Frontier Project, Cooperative Research Center of Life Sciences.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.178079.
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ABBREVIATIONS:
- NCAM
- neural cell adhesion molecule
- MMP
- matrix metalloproteinase
- MCAO
- middle cerebral artery occlusion
- CCA
- common carotid artery
- TTC
- 2,3,5-triphenyltetrazolium chloride
- NDS
- neurological deficit score
- ANOVA
- analysis of variance
- WT
- wild type
- KO
- knockout
- siRNA
- small interfering RNA
- DMSO
- dimethyl sulfoxide
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- PBS
- phosphate-buffered saline
- PBS-T
- PBS containing 0.1% Tween 20
- TBS
- Tris-buffered saline
- TBS-T
- TBS containing 0.1% Tween 20
- GM6001
- N-[(2R)-2(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide.
- Received December 9, 2010.
- Accepted May 19, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics