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J. Biol. Chem., Vol. 282, Issue 22, 16036-16041, June 1, 2007
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-Dystroglycan as a Target for MMP-9, in Response to Enhanced Neuronal Activity*
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From the
Department of Molecular and Cellular Neurobiology, Nencki Institute, Pasteura 3, 02-093 Warsaw, Poland, the ¶Department of Neurophysiology, Nencki Institute, Pasteura 3, 02-093 Warsaw, Poland, the Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands, the ||Laboratory of Molecular and Cellular Neurobiology, International Institute of Molecular and Cell Biology, 4 Ks. Trojdena Street, 02-109 Warsaw, Poland, the **Institute of Biomedical and Biomolecular Sciences, School of Pharmacy and Biomedical Science, University of Portsmouth, White Swan Road, Portsmouth PO1 2DT, United Kingdom, and the Centre for Molecular Biology and Neuroscience, Institute of Basic Medical Sciences, Domus Medica (Sognsvannsveien 9), University of Oslo, N-0317 Oslo, Norway
Received for publication, January 23, 2007 , and in revised form, April 6, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-dystroglycan comprises such a neuronal activity-driven target for matrix metalloproteinase-9. This notion is based on the following observations. (i) Recombinant, autoactivating matrix metalloproteinase-9 produces limited proteolytic cleavage of -dystroglycan. (ii) In neuronal cultures, -dystroglycan proteolysis occurs in response to stimulation with either glutamate or bicuculline and is blocked by tissue inhibitor of metalloproteinases-1, a metalloproteinase inhibitor. (iii) -Dystroglycan degradation is also observed in the hippocampus in vivo in response to seizures but not in the matrix metalloproteinase-9 knock-out mice. (iv) -Dystroglycan cleavage correlates in time with increased matrix metalloproteinase-9 activity. (v) Finally, -dystroglycan and matrix metalloproteinase-9 colocalize in postsynaptic elements in the hippocampus. In conclusion, our data identify the -dystroglycan as a first matrix metalloproteinase-9 substrate digested in response to enhanced synaptic activity. This demonstration may help to understand the possible role of both proteins in neuronal functions, especially in synaptic plasticity, learning, and memory. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Despite data implicating MMP-9 in neuronal/synaptic plasticity, no synaptic targets for its enzymatic activity have as yet been identified in neurons. However, recent studies have suggested that this enzyme may digest the 43-kDa -dystroglycan (-DG) to release a 30-kDa product from the full-length subunit. First, Yamada et al. (10) have shown that unidentified MMPs digest -DG to reveal the 30-kDa product in the peripheral tissues. Second, Kaczmarek et al. (11) demonstrated that the appearance of the 30-kDa -DG degradation product in the hippocampus following kainate treatment coincides with the increased levels of MMP-9. Recently, it was reported that -DG expressed at the astrocyte endfeet can be specifically cleaved by macrophage-derived gelatinases (MMP-2 and MMP-9) during leukocyte penetration of the basement membrane in experimental autoimmune encephalomyelitis (12). Finally, selective cleavage of -DG by MMP-2 and MMP-9 was also observed in schwannoma cell line RT4 by Zhong et al. (13). Although, none of those studies has directly addressed -DG proteolysis in neurons or, more specifically, the synapses, it is of note that -DG can be expressed in the postsynaptic membranes in the brain, as shown previously by Zaccaria et al. (14).
Dystroglycan (DG) is a central protein in the dystrophin-glycoprotein complex (DGC) that links dystrophin and the intracellular cytoskeleton with extracellular matrix and anchors the whole complex at the membrane. DG is translated from a single mRNA as a precursor peptide, which is subsequently cleaved into two, non-covalently associated subunits: extracellular (
) and transmembrane () (15, 16). -DG is a highly glycosylated peripheral membrane protein that binds via its carbohydrate side chains to many extracellular matrix ligands such as laminins, agrin, and perlecan (17) and to the presynaptic neurexins in the brain (18). -DG, in turn, through its proline-rich C terminus, binds dystrophin or urotrophin, and this interaction links DGC to F-actin cytoskeleton (17, 19). Although the role of -DG in the brain, especially in neurons, is not known, it is worth mentioning that the specific gene ablation of dystroglycan in the brain produces deficits in neuronal plasticity, similar to those caused by inhibition of MMP-9 (7, 20).
Taken together, the aforementioned data lead us to hypothesize that -DG is a synaptic target for MMP-9. In this report, we provide several lines of evidence supporting such a notion.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Recombinant Autoactivating MMP-9 (aaMMP-9)—Expression of previously described (22) autoactivating mutant of MMP-9 was performed according to the Bac-to-Bac baculovirus expression system manual (Invitrogen). Briefly, G100L MMP-9 mutant (generous gift from Katherine Fisher, Pfizer) was cloned to pFastBac1, and the resulting recombinant plasmid was used to transform DH10Bac-competent cells. Colonies that performed transposition were identified by blue-white selection, and recombinant bacmid was isolated and verified by PCR. The Sf21 insect cells were transfected with recombinant bacmid using Cellfectin® reagent (Invitrogen) to obtain recombinant baculovirus. After amplification and titration of the recombinant baculovirus, Sf21 cells were infected and incubated in Sf-900IISFM serum-free medium (Invitrogen). Forty-eight hours after infection, the culture medium was collected, and recombinant aaMMP-9 was purified on gelatin-SepharoseTM 4B (GE Healthcare) as described previously (23).
Adenoviral Vectors—Viral stocks were prepared according to the procedure described before (24). The recombinant Ad--galactosidase has also been described previously (25). The recombinant adenovector carrying cDNA encoding tissue inhibitor of metalloproteinases-1 (Ad-TIMP-1) has been described by Okulski et al. (26).
Pentylenetetrazole (PTZ) Stimulation—Pharmacological stimulation was done by the intraperitoneal PTZ injection (50 mg/kg), and animals were sacrificed 5 min after the seizure onset. Hippocampi were isolated and homogenized in ice-cold homogenization buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 5 mM CaCl2, and 10 µl/ml protease inhibitor mixture, Sigma). Homogenates were then analyzed by the Western blot approach.
Hippocampal Extracts and Their Treatment with aaMMP-9—Wistar rats were sacrificed, and hippocampi were isolated and homogenized in homogenization buffer (20 mM Tris-Cl, pH 7.4, at 4 °C; 137 mM NaCl; 25 mM -glycerophosphate; 2 mM NaPPi; 1 mM Na3VO4; 1% Triton X-100; 10% glycerol; 2 mM benzamidine; 0.5 mM dithiothreitol; 1 mM phenylmethylsulfonyl fluoride). One hundred micrograms of hippocampal extracts were incubated at 37 °C for an hour with 100 ng of recombinant MMP-9 in reaction buffer (50 mM Tris-Cl, pH 7.5; 10 mM CaCl2; 1 µM ZnCl2), also in the presence of 10 mM 1,10-phenanthroline. Homogenates were then analyzed by Western blot.
Cell Culture—Cortical neurons were cultured from newborn (postpartum day 0) Wistar rats as described previously (27). Briefly, rats were decapitated, and cortices were dissected and digested for 30 min in papain (Worthington PAP) at 37 °C. The reaction was stopped by triple wash in trypsin inhibitor (Sigma) and followed by trituration. Cells were seeded on 6-well plates covered with poly-DL-lysine (50 µg/ml; Sigma) in a concentration of 2 x 106 cells/well in basal medium Eagle (Cambrex) in the presence of 10% bovine calf serum (Hyclone), 35 mM glucose (Sigma), 1 mM L-glutamine (Invitrogen), and 0.5% penicillin/streptomycin (Invitrogen). On the second day in vitro (DIV), cytosine--D-arabino-furanoside (Sigma) was added to medium (at a final concentration of 2.5 µM) to prevent the growth of glia. Cells were used for experiments on 7 DIV. In the case of bicuculline stimulation, on 2 DIV, the culture medium was changed for Neurobasal medium (Invitrogen) in the presence of 2% B27 supplement (Invitrogen), 35 mM glucose (Sigma), 1 mM L-glutamine (Invitrogen), and 0.5% penicillin/streptomycin (Invitrogen), and in these conditions, cells were used for experiment on 14 DIV.
Cell Culture Stimulation—The cultures were stimulated as described (27) with slight modifications. Cells were treated with either 50 µM glutamate (in basal medium Eagle) on 7 DIV or 10 µM bicuculline (cultured in Neurobasal medium) on 14 DIV. For the cells treated with glutamate, 6-cyano-7-nitroquinoxaline-2,3-dione (40 µM) was added to all plates the night before the stimulation to reduce endogenous synaptic activity, and 5 µM nimodipine was added 30 min before the stimulation.
Western Blotting—After the stimulation, the cells were lysed in the lysis buffer (20 mM Tris-Cl, pH 7.4, at 4 °C; 137 mM NaCl; 25 mM -glycerophosphate; 2 mM NaPPi; 2 mM EDTA; 1 mM Na3VO4; 1% Triton X-100; 10% glycerol; 2 mM benzamidine; 0.5 mM dithiothreitol; 1 mM phenylmethylsulfonyl fluoride; and 10 µl/ml protease inhibitor mixture, Sigma), the protein concentration in each sample was measured using the Bradford method (Sigma), and samples were brought to equal protein concentration by H2O dilution. Lysates were mixed with 5 x SDS sample buffer and denatured, and 20 µg of total protein samples were loaded on 12% SDS-polyacrylamide gels. The samples were electrotransferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore), which were blocked 2 h at room temperature with 10% nonfat milk in Tris-buffered saline with 0.1% Tween 20. After blocking, the membranes were incubated at 4 °C overnight with the following antibodies: anti--dystroglycan (NCL-b-DG, Novocastra, 1:500), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (MAB374, Chemicon, 1:2000), and anti--actin (A5441, Sigma, 1:6000), all diluted in 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20. Membranes were than incubated 2 h at room temperature with peroxidase-labeled secondary antibody diluted 1:10,000 in 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20. After washing, peroxidase activity were visualized with ECLplus reagent (GE Healthcare).
Gel Zymography—Samples of the culture medium were mixed with 3 x SDS sample buffer without dithiothreitol and loaded on 8% SDS-polyacrylamide gels containing 2 mg/ml gelatin. After electrophoresis, the gels were washed twice with 2.5% Triton X-100 for 30 min at room temperature and incubated overnight in developing buffer (50 mM Tris-Cl, pH 7.5; 10 mM CaCl2; 1 µM ZnCl2; 1% Triton X-100; 0.02% NaN3) at 37 °C with moderate shaking. Gels were stained with Coomassie Brilliant Blue and shortly destained.
Electron Microscopic Immunocytochemistry—This was performed according to the previously described procedures (28, 29). In brief, normal Wistar rats were perfused transcardially with a fixative consisting of 4% paraformaldehyde plus 0.1% glutaraldehyde. The brains were postfixed in the same fixative, cut into 0.5–1.0-mm slices, cryoprotected, snap-frozen in liquid propane (–170 °C), and subjected to freeze substitution (in Leica EM AFS apparatus). Specimens were then embedded in Lowicryl HM20 resin, polymerized by UV light at –45 °C to 0 °C, and cut into ultrathin sections. The immunoreactions consisted of sequential incubations with a mixture of primary antibodies: (i) a mouse anti--dystroglycan (NCL-b-DG, Novocastra) (undiluted) and (ii) rabbit anti-MMP-9 (Torrey Pines Scientific) diluted 1:50 followed by species-specific goat secondary antibodies coupled to 6- or 10-nm gold particles (GE Healthcare). The specimens were examined with a Philips CM 10 electron microscope at 60 kV.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-dystroglycan (6 nm) were found in close vicinity to gold particles signaling MMP-9 (10 nm) (Fig. 1). The subcellular colocalization of -dystroglycan and MMP-9 is consistent with the idea that MMP-9 could be secreted to the extracellular space, allowing for a focal, MMP-9-mediated proteolysis of membrane-bound -dystroglycan.
To examine whether -DG is cleaved upon neuronal stimulation, we employed cortical primary neuronal cultures that include minimal components of glia (around 10%) (32). The cultures were stimulated in vitro with 50 µM glutamate, and the whole cell lysates were analyzed by Western blotting. As shown in Fig. 2, glutamate exposure led to a significant increase in the level of the cleaved, 30-kDa form of -DG. This increase was notable at 10 min of stimulation and was followed by a decrease over the next 10-min period.
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-DG after neuronal stimulation is MMP-dependent, we infected the cortical neuronal cultures with the adenoviral vector Ad-TIMP-1. TIMP-1 is known to bind MMP-9 with high affinity and to block its enzymatic function (33). Twenty-four hours after the infection, the cultures were stimulated with 50 µM glutamate, and the cell lysates were analyzed by Western blotting. As shown in Fig. 3, the glutamate stimulation-dependent increase in -dystroglycan cleavage was abolished following infection with Ad-TIMP-1 but not following infection with Ad--galactosidase, used as a control (Fig. 3A). In the absence of glutamate exposure, there was no difference between TIMP-1-infected and -galactosidase-infected cells with respect to the level of the -DG degradation product.
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-DG on the cultured. Indeed, treatment of the neuronal cultures for 30 min with aaMMP-9 caused -DG cleavage that could not be significantly increased by subsequent glutamate stimulation (Fig. 3B). Furthermore, when hippocampal homogenates were incubated with recombinant aaMMP-9 (Fig. 3C), there was an accumulation of the degradation (30-kDa) product of -DG that was completely abolished in the presence of 1,10-phenanthroline, the Zn2+ chelator, known to inhibit MMP activity.
We have also investigated whether rapid activity-induced proteolysis of -DG can occur in vivo, and if so, whether this cleavage is MMP-9 dependent. MMP-9 knockout and wild-type mice were injected intraperitoneally with PTZ, a proconvulsant and GABAA receptor antagonist that is known to provoke increased synaptic stimulation in vivo (34). Five minutes after the seizure onset, the hippocampi were isolated and subjected to the Western blot analysis. Fig. 3D shows a significant increase in the level of the 30-kDa -DG form in the PTZ-treated wild-type mice and no such increase in the PTZ-treated MMP-9 knock-out mice.
Finally, we recorded cleavage of -DG in neuronal cultures treated with bicuculline. At the concentration used (10 µM), this GABAA receptor blocker induces an increased glutamate receptor-dependent activity in a functional neuronal network (35). Fig. 4A shows that the MMP-9 (but not MMP-2) activity is strongly increased at 5 min of stimulation and returns to basal level within the next 5 min. An accumulation of the 30-kDa -DG form was observed to follow directly enhanced MMP-9 activity (Fig. 4B). Expression of TIMP-1 effectively prevented -DG cleavage after bicuculline administration (Fig. 4C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-DG is a target of neuronal MMP-9 in the brain in vivo as well as in neuronal cultures in vitro. The following lines of evidence support this notion. (i) Recombinant, autoactive MMP-9 produces limited proteolytic cleavage of -DG in brain neurons cultured in vitro as well as in the hippocampal ex vivo extracts. (ii) In neuronal cultures, -DG proteolysis occurs in response to synaptic activation with either glutamate or bicuculline. The -DG proteolysis is blocked by TIMP-1, a metalloproteinase inhibitor. (iii) The same phenomenon of -DG degradation is also observed in the hippocampus in vivo in response to seizures, and it is absent in the MMP-9 knock-out mice. (iv) Both in the cultured brain neurons and in the hippocampus in vivo, -DG cleavage correlates in time with increased MMP-9 activity, and -DG and MMP-9 colocalize in postsynaptic elements in the hippocampus.
The fact that -DG was found by electron microscopic immunocytochemistry to be membrane-bound is consistent with its previously identified localization in various cell types, including neurons (17, 36). Our data also support the notion that the protein can be present in asymmetric postsynaptic specializations, which belong to the excitatory neurons (14). In the close vicinity of the -DG, we have also observed MMP-9. However, it is known that the enzyme is released in the latent form to acquire its enzymatic activity outside the cells (1, 2), and thus it is not very probable that MMP-9 could digest -DG intracellularly. Notably, we have not observed colocalization of MMP-9 with -DG within the synaptic cleft; however, this result can be explained by our data obtained with neuronal cultures (Fig. 4A), as well as literature indicating that MMP-9 is available outside the cells only very transiently (37). Thus our colocalization results indicated only a potential role of MMP-9 in -DG proteolysis. The extensive evidence for such a role has been obtained in subsequent functional studies, carried out mostly in vitro.
We demonstrated limited -DG proteolysis in the neuronal cultures as well as in the hippocampus in vivo. This phenomenon was found to be a very rapid process since already within a few minutes after the neuronal stimulation, either in vitro or in vivo, we observed appearance of the truncated form of the protein. Furthermore, of special note is the finding that -DG processing is also a very transient phenomenon as already within 20 min after the stimulation, a decrease in the level of the 30-kDa form had been observed. This result could be explained by a rapid turnover of the molecule.
We have also demonstrated that activity-dependent processing of -DG occurs via MMP activity as it is blocked by TIMP-1, and furthermore, this processing is critically dependent on MMP-9 and not on other MMPs since it does not take place in MMP-9 knock-out mice. Moreover, incubation of both neuronal culture and hippocampal homogenate with recombinant active MMP-9 significantly increased the level of the 30-kDa form of -DG. Altogether, these results suggest that MMP-9 is responsible for activation-dependent cleavage of -DG. Interestingly, however, in non-stimulated neurons in culture, as well as in the hippocampi ex vivo and in vivo, we observed noticeable levels of the truncated form of -DG. These may be due to basal activity of either MMP-2 or MMP-9 as both proteases can cleave -DG (12, 13). However, in MMP-9 knock-out mice, the level of cleaved -DG is not increased in response to enhanced neuronal activity, indicating that MMP-9 is critical for stimulation-driven -DG cleavage. Furthermore, treatment of the neuronal cultures with 10 µM bicuculline shows that after the stimulation, only MMP-9 but not MMP-2 levels are increased in the cell medium. This result also indicates that activation of synaptic receptors is sufficient for releasing MMP-9 to extracellular environment as quickly as 5 min after the stimulus (see also Ref. 37) and is immediately followed by limited proteolysis of -DG. Importantly, this time frame of the accumulation of 30-kDa -DG cleavage product after the synaptic activation is the same as following glutamate stimulation of the cultured neurons.
Finally, it should be emphasized that this demonstration of the first specific MMP-9 target in response to enhanced synaptic activity at the central nervous system synapses, reported herein, may help to understand the possible role of both MMP-9 and -DG proteins in neuronal functions, especially in synaptic plasticity, learning, and memory. Recent studies have shown that MMP-9 is activated during long term potentiation and learning, and its inhibition by either genetic or pharmacological means prevents the formation of long-lasting plastic changes as well as long term memory (6, 7). Also, in humans, increased MMP-9 activity, resulting from the specific gene polymorphism, was found to ameliorate dementia symptoms (38). In turn, Moore et al. (20) have shown that mutant mice selectively deficient in the brain dystroglycan suffer from late phase of long term potentiation deficits in the hippocampus. Furthermore, functional disruption of the DGC is observed in several forms of inherited muscular dystrophies such as muscle-eye-brain disease, Walker-Warburg syndrome, and Fukuyama congenital muscular dystrophy, which are all associated with cognitive deficits (39–41). In addition, mutations in the genes encoding dystroglycan-binding proteins such as laminin (its extracellular ligand) as well as dystrophin (linking it with the F-actin) are associated with mental retardation (congenital and Duchenne muscular dystrophy, respectively). Notably, we have previously demonstrated specific changes in the hippocampal expression patterns of transcripts encoding dystrophin and neurexins (the presynaptic interacting partners of DG) following kainate and PTZ treatment in vivo (42, 43). This suggests a functional role for the entire DGC complex at central synapses and in their plasticity. Moreover, it was shown that -DG can either directly or indirectly interact with extracellular signal-regulated kinases (ERK) as well as with focal adhesion kinase (FAK) (30, 44), and both those kinases are important for neuronal plasticity including the induction of long term potentiation (45). It was also shown recently in Schwann cells that the 30-kDa form of -DG has a greater affinity for the short isoform of utrophin (Up71) than for the dystrophin isoform (Dp116), which in turn has greater affinity to full-length -DG (46). Although these interactions have been found in glia, it is probable that similar change in affinity may influence actin cytoskeleton in neurons and allow for changes in, for example, dendritic spine morphology.
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FOOTNOTES |
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1 To whom correspondence should be addressed. Tel.: 48-22-659-3001; Fax: 48-22-822-5342; E-mail: l.kaczmarek{at}nencki.gov.pl.
2 The abbreviations used are: MMP, matrix metalloproteinase; TIMP, tissue inhibitors of matrix metalloproteinases; aaMMP-9, autoactivating MMP-9; Ad-TIMP-1, adenovector carrying cDNA encoding TIMP-1; DG, dystroglycan; DGC, dystrophin-glycoprotein complex; PTZ, pentylenetetrazole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DIV, day in vitro; GABAA, 
-aminobutyric acid, Type A.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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