β-Dystroglycan as a Target for MMP-9, in Response to Enhanced Neuronal Activity

Matrix metalloproteinase-9 has recently emerged as an important molecule in control of extracellular proteolysis in the synaptic plasticity. However, no synaptic targets for its enzymatic activity had been identified before. In this report, we show that β-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 β-dystroglycan. (ii) In neuronal cultures, β-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) β-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) β-Dystroglycan cleavage correlates in time with increased matrix metalloproteinase-9 activity. (v) Finally, β-dystroglycan and matrix metalloproteinase-9 colocalize in postsynaptic elements in the hippocampus. In conclusion, our data identify the β-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. Matrix metalloproteinase-9 has recently emerged as an important molecule in control of extracellular proteolysis in the synaptic plasticity. However, no synaptic targets for its enzymatic activity had been identified before. In this report, we show that β-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 β-dystroglycan. (ii) In neuronal cultures, β-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) β-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) β-Dystroglycan cleavage correlates in time with increased matrix metalloproteinase-9 activity. (v) Finally, β-dystroglycan and matrix metalloproteinase-9 colocalize in postsynaptic elements in the hippocampus. In conclusion, our data identify the β-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. Matrix metalloproteinases (MMPs) 2The 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, γ-aminobutyric acid, Type A.2The 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, γ-aminobutyric acid, Type A. are a family of zinc-dependent endopeptidases acting outside the cells and therefore attributed with digesting extracellular matrix components. These enzymes are produced in a latent form, and after release to extracellular space, they are activated by cleavage off the propeptide (1.Woessner J.F. Nagase H. Matrix Metalloproteinases and TIMPs. Oxford University Press, Oxford and New York2000: 50-129Google Scholar, 2.Van den Steen P.E. Dubois B. Nelissen I. Rudd P.M. Dwek R.A. Opdenakker G. CRC Crit. Rev. Biochem. Mol. Biol. are in a of and tissue and (1.Woessner J.F. Nagase H. Matrix Metalloproteinases and TIMPs. Oxford University Press, Oxford and New York2000: 50-129Google Scholar, 2.Van den Steen P.E. Dubois B. Nelissen I. Rudd P.M. Dwek R.A. Opdenakker G. CRC Crit. Rev. Biochem. Mol. Biol. Scholar, Rev. Biol. Scholar, data show increased and activity of after and in of the Rev. the the of in the recently been In as has been in synaptic plasticity, learning, and Scholar, A. a in and its enzymatic activity in the after seizures has been A. a glutamate produces in the of the the of the data in plasticity, no synaptic targets for its enzymatic activity as been identified in However, that this may the β-dystroglycan to release a the H. H. A. A. Mol. that to the in the that the of the degradation in the hippocampus following with the increased of that the by and of the in Opdenakker G. Finally, cleavage of by and also observed in by A. Biochem. of has proteolysis in the is of that in the postsynaptic in the as by A. is a in the dystrophin-glycoprotein that and the with extracellular matrix and the the is a as a is extracellular and Scholar, is a that its to extracellular matrix such as and and to the in the Biol. in its or and this to Scholar, the role of in the especially in is not is that the of in the produces in neuronal plasticity, to by of A. Scholar, A. R.A. the data to that is a synaptic target for In this report, we of such a we used and also used knock-out A. R.A. and mice. by to the the in the with to and with a the with to of the on and based on and of autoactivating of to the to and the used to that identified by and and by cells with to and of the cells and in after the and on as to the G. for in of New has also been I. adenovector carrying cDNA encoding tissue inhibitor of has been by A. 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G. we that is in G. and in of by in that the are we a β-dystroglycan in to of β-dystroglycan and is with the that to the extracellular space, for a proteolysis of is neuronal we neuronal that of Biol. in with and the by in glutamate to a in the of the of This of stimulation and by a the is after neuronal stimulation of neuronal with cells cleavage by proteolysis of the β-dystroglycan is after the of the are in with after in with glutamate for and as a control used as a of is cleavage of after neuronal stimulation is we the neuronal with the is to with and to its enzymatic H. Biol. after the the with and the by in the glutamate in β-dystroglycan cleavage following with but not following with used as a control In the of glutamate no and cells with to the of the degradation of β-dystroglycan after stimulation is matrix metalloproteinase-9 neuronal with and a control adenovector cDNA of after the for with glutamate and the of cleavage by in the of β-dystroglycan after the stimulation with glutamate is in the with with with a control a of with limited proteolysis of and this is not to cleavage of β-dystroglycan a the with of with cleavage of and this is by of seizures by of β-dystroglycan cleavage after the of seizures in but not in knock-out and used as of are either or In the we extracellular on the of the neuronal for with cleavage that not increased by glutamate stimulation with an of the degradation of that in the of the to also proteolysis of in and this cleavage is and with PTZ, a and that is to increased synaptic stimulation in vivo A. G. after the the and to the a in the of the in the and no such in the knock-out we cleavage of in neuronal with the used this an increased glutamate activity in a neuronal A. I. I. that the not activity is increased of stimulation and to the of the observed to enhanced activity of cleavage after bicuculline proteolysis of β-dystroglycan is synaptic and the release of stimulation and the of the neuronal with and and with activity of in the of release of to the after the bicuculline This is as after the stimulation the of is not the propeptide latent of the β-dystroglycan cleavage after the bicuculline as after the is a in the of the of the with cleavage of β-dystroglycan after bicuculline stimulation in with with a control adenovector used as a this report, we that is a target of neuronal in the in vivo as as in neuronal in following of this (i) Recombinant, produces limited proteolytic cleavage of in in as as in the vivo (ii) In neuronal cultures, proteolysis occurs in response to synaptic with either glutamate or proteolysis is blocked by a metalloproteinase inhibitor. (iii) of degradation is also observed in the hippocampus in vivo in response to and is in the knock-out mice. (iv) in the and in the hippocampus in cleavage correlates in time with increased and and colocalize in postsynaptic elements in the that by to is with its identified in Scholar, data also the notion that the in postsynaptic to the A. In the of the we also observed However, is that the is in the latent to its enzymatic activity outside the cells (1.Woessner J.F. Nagase H. Matrix Metalloproteinases and TIMPs. Oxford University Press, Oxford and New York2000: 50-129Google Scholar, 2.Van den Steen P.E. Dubois B. Nelissen I. Rudd P.M. Dwek R.A. Opdenakker G. CRC Crit. Rev. Biochem. Mol. Biol. and is not that we not observed of with the synaptic this by our data with neuronal as as that is outside the cells A. our a role of in for such a role has been in in limited proteolysis in the neuronal as as in the hippocampus in This to a a after the neuronal either in or in we observed of the of the of is the that is also a as after the a in the of the had been This by a of the also that of occurs activity as is blocked by and this is on and not on not in knock-out mice. of both neuronal and with increased the of the of that is for cleavage of in in as as in the vivo and in we observed of the of These may to activity of either or as both Opdenakker G. Scholar, A. Biochem. However, in knock-out the of is not increased in response to enhanced neuronal that is for of the neuronal with bicuculline that after the but not are increased in the This also that of synaptic is for to extracellular as as after the also A. and is by limited proteolysis of this time of the of cleavage after the synaptic is the as following glutamate stimulation of the that this demonstration of the first target in response to enhanced synaptic activity the may help to understand the possible role of both and proteins in neuronal functions, especially in synaptic plasticity, learning, and memory. that is activated and learning, and its by either or the of as as Scholar, A. in increased the to In A. R.A. that in the of in the hippocampus. of the is observed in of such as and are with H. H. Scholar, H. I. I. Scholar, I. H. In in the encoding proteins such as extracellular as as with the are with and we in the of encoding and of following and in vivo A. Scholar, A. Mol. This a role for the and in plasticity. that either or with extracellular as as with G. G. Scholar, and both are important for neuronal the of also recently in cells that the of has a for the of for the in has to G. A. been in is that in may in and for for Matrix metalloproteinases (MMPs) 2The 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, γ-aminobutyric acid, Type A.2The 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, γ-aminobutyric acid, Type A. are a family of zinc-dependent endopeptidases acting outside the cells and therefore attributed with digesting extracellular matrix components. These enzymes are produced in a latent form, and after release to extracellular space, they are activated by cleavage off the propeptide (1.Woessner J.F. Nagase H. Matrix Metalloproteinases and TIMPs. Oxford University Press, Oxford and New York2000: 50-129Google Scholar, 2.Van den Steen P.E. Dubois B. Nelissen I. Rudd P.M. Dwek R.A. Opdenakker G. CRC Crit. Rev. Biochem. Mol. Biol. are in a of and tissue and (1.Woessner J.F. Nagase H. Matrix Metalloproteinases and TIMPs. Oxford University Press, Oxford and New York2000: 50-129Google Scholar, 2.Van den Steen P.E. Dubois B. Nelissen I. Rudd P.M. Dwek R.A. Opdenakker G. CRC Crit. Rev. Biochem. Mol. Biol. Scholar, Rev. Biol. Scholar, data show increased and activity of after and in of the Rev. the the of in the recently been In as has been in synaptic plasticity, learning, and Scholar, A. a in and its enzymatic activity in the after seizures has been A. a glutamate produces in the of the the of the data in plasticity, no synaptic targets for its enzymatic activity as been identified in However, that this may the β-dystroglycan to release a the H. H. A. A. Mol. that to the in the that the of the degradation in the hippocampus following with the increased of that the by and of the in Opdenakker G. Finally, cleavage of by and also observed in by A. Biochem. of has proteolysis in the is of that in the postsynaptic in the as by A. is a in the dystrophin-glycoprotein that and the with extracellular matrix and the the is a as a is extracellular and Scholar, is a that its to extracellular matrix such as and and to the in the Biol. in its or and this to Scholar, the role of in the especially in is not is that the of in the produces in neuronal plasticity, to by of A. Scholar, A. R.A. the data to that is a synaptic target for In this report, we of such a we used and also used knock-out A. R.A. and mice. by to the the in the with to and with a the with to of the on and based on and of autoactivating of to the to and the used to that identified by and and by cells with to and of the cells and in after the and on as to the G. for in of New has also been I. adenovector carrying cDNA encoding tissue inhibitor of has been by A. Biol. stimulation by the and after the and in and inhibitor by the and with and and in of for an with of in also in the of by day as H. and and digested for in by in inhibitor and by on with in a of in in the of and the day in to a of to the of used for on In the of bicuculline on DIV, the for in the of and and in cells used for on as H. with with either glutamate on or bicuculline in on the cells with to the the stimulation to synaptic and the the the cells in the and inhibitor the in the and to by with and and of on blocked with in with the with the following and in in with with in in with activity with of the with and on the with for and in with with and to the A. Scholar, In with a of in the in and to in by to and of with a of (i) a and (ii) by to or with a we used and also used knock-out A. R.A. and mice. by to the the in the with to and with a the with to of the on and based on and of autoactivating of to the to and the used to that identified by and and by cells with to and of the cells and in after the and on as to the G. for in of New has also been I. adenovector carrying cDNA encoding tissue inhibitor of has been by A. Biol. stimulation by the and after the and in and inhibitor by the and with and and in of for an with of in also in the of by day as H. and and digested for in by in inhibitor and by on with in a of in in the of and the day in to a of to the of used for on In the of bicuculline on DIV, the for in the of and and in cells used for on as H. with with either glutamate on or bicuculline in on the cells with to the the stimulation to synaptic and the the the cells in the and inhibitor the in the and to by with and and of on blocked with in with the with the following and in in with with in in with activity with of the with and on the with for and in with with and to the A. Scholar, In with a of in the in and to in by to and of with a of (i) a and (ii) by to or with a to in the A. Scholar, G. G. we that is in G. and in of by in that the are we a β-dystroglycan in to of β-dystroglycan and is with the that to the extracellular space, for a proteolysis of is neuronal we neuronal that of Biol. in with and the by in glutamate to a in the of the of This of stimulation and by a the cleavage of after neuronal stimulation is we the neuronal with the is to with and to its enzymatic H. Biol. after the the with and the by in the glutamate in β-dystroglycan cleavage following with but not following with used as a control In the of glutamate no and cells with to the of the degradation of β-dystroglycan after stimulation is matrix metalloproteinase-9 neuronal with and a control adenovector cDNA of after the for with glutamate and the of cleavage by in the of β-dystroglycan after the stimulation with glutamate is in the with with with a control a of with limited proteolysis of and this is not to cleavage of β-dystroglycan a the with of with cleavage of and this is by of seizures by of β-dystroglycan cleavage after the of seizures in but not in knock-out and used as of are either or In the we extracellular on the of the neuronal for with cleavage that not increased by glutamate stimulation with an of the degradation of that in the of the to also proteolysis of in and this cleavage is and with PTZ, a and that is to increased synaptic stimulation in vivo A. G. after the the and to the a in the of the in the and no such in the knock-out we cleavage of in neuronal with the used this an increased glutamate activity in a neuronal A. I. I. that the not activity is increased of stimulation and to the of the observed to enhanced activity of cleavage after bicuculline proteolysis of β-dystroglycan is synaptic and the release of stimulation and the of the neuronal with and and with activity of in the of release of to the after the bicuculline This is as after the stimulation the of is not the propeptide latent of the β-dystroglycan cleavage after the bicuculline as after the is a in the of the of the with cleavage of β-dystroglycan after bicuculline stimulation in with with a control adenovector used as a to in the A. Scholar, G. G. we that is in G. and in of by in that the are we a β-dystroglycan in to of β-dystroglycan and is with the that to the extracellular space, for a proteolysis of β-dystroglycan. is neuronal we neuronal that of Biol. in with and the by in glutamate to a in the of the of This of stimulation and by a the cleavage of after neuronal stimulation is we the neuronal with the is to with and to its enzymatic H. Biol. after the the with and the by in the glutamate in β-dystroglycan cleavage following with but not following with used as a control In the of glutamate no and cells with to the of the degradation we extracellular on the of the neuronal for with cleavage that not increased by glutamate stimulation with an of the degradation of that in the of the to activity. also proteolysis of in and this cleavage is and with PTZ, a and that is to increased synaptic stimulation in vivo A. G. after the the and to the a in the of the in the and no such in the knock-out mice. Finally, we cleavage of in neuronal with the used this an increased glutamate activity in a neuronal A. I. I. that the not activity is increased of stimulation and to the of the observed to enhanced activity of cleavage after bicuculline this report, we that is a target of neuronal in the in vivo as as in neuronal in following of this (i) Recombinant, produces limited proteolytic cleavage of in in as as in the vivo (ii) In neuronal cultures, proteolysis occurs in response to synaptic with either glutamate or proteolysis is blocked by a metalloproteinase inhibitor. (iii) of degradation is also observed in the hippocampus in vivo in response to and is in the knock-out mice. (iv) in the and in the hippocampus in cleavage correlates in time with increased and and colocalize in postsynaptic elements in the that by to is with its identified in Scholar, data also the notion that the in postsynaptic to the A. In the of the we also observed However, is that the is in the latent to its enzymatic activity outside the cells (1.Woessner J.F. Nagase H. Matrix Metalloproteinases and TIMPs. Oxford University Press, Oxford and New York2000: 50-129Google Scholar, 2.Van den Steen P.E. Dubois B. Nelissen I. Rudd P.M. Dwek R.A. Opdenakker G. CRC Crit. Rev. Biochem. Mol. Biol. and is not that we not observed of with the synaptic this by our data with neuronal as as that is outside the cells A. our a role of in for such a role has been in in limited proteolysis in the neuronal as as in the hippocampus in This to a a after the neuronal either in or in we observed of the of the of is the that is also a as after the a in the of the had been This by a of the also that of occurs activity as is blocked by and this is on and not on not in knock-out mice. of both neuronal and with increased the of the of that is for cleavage of in in as as in the vivo and in we observed of the of These may to activity of either or as both Opdenakker G. Scholar, A. Biochem. However, in knock-out the of is not increased in response to enhanced neuronal that is for of the neuronal with bicuculline that after the but not are increased in the This also that of synaptic is for to extracellular as as after the also A. and is by limited proteolysis of this time of the of cleavage after the synaptic is the as following glutamate stimulation of the that this demonstration of the first target in response to enhanced synaptic activity the may help to understand the possible role of both and proteins in neuronal functions, especially in synaptic plasticity, learning, and memory. that is activated and learning, and its by either or the of as as Scholar, A. in increased the to In A. R.A. that in the of in the hippocampus. of the is observed in of such as and are with H. H. Scholar, H. I. I. Scholar, I. H. In in the encoding proteins such as extracellular as as with the are with and we in the of encoding and of following and in vivo A. Scholar, A. Mol. This a role for the and in plasticity. that either or with extracellular as as with G. G. Scholar, and both are important for neuronal the of also recently in cells that the of has a for the of for the in has to G. A. been in is that in may in and for for In this report, we that is a target of neuronal in the in vivo as as in neuronal in following of this (i) Recombinant, produces limited proteolytic cleavage of in in as as in the vivo (ii) In neuronal cultures, proteolysis occurs in response to synaptic with either glutamate or proteolysis is blocked by a metalloproteinase inhibitor. (iii) of degradation is also observed in the hippocampus in vivo in response to and is in the knock-out mice. (iv) in the and in the hippocampus in cleavage correlates in time with increased and and colocalize in postsynaptic elements in the hippocampus. that by to is with its identified in Scholar, data also the notion that the in postsynaptic to the A. In the of the we also observed However, is that the is in the latent to its enzymatic activity outside the cells (1.Woessner J.F. Nagase H. Matrix Metalloproteinases and TIMPs. Oxford University Press, Oxford and New York2000: 50-129Google Scholar, 2.Van den Steen P.E. Dubois B. Nelissen I. Rudd P.M. Dwek R.A. Opdenakker G. CRC Crit. Rev. Biochem. Mol. Biol. and is not that we not observed of with the synaptic this by our data with neuronal as as that is outside the cells A. our a role of in for such a role has been in in limited proteolysis in the neuronal as as in the hippocampus in This to a a after the neuronal either in or in we observed of the of the of is the that is also a as after the a in the of the had been This by a of the also that of occurs activity as is blocked by and this is on and not on not in knock-out mice. of both neuronal and with increased the of the of that is for cleavage of in in as as in the vivo and in we observed of the of These may to activity of either or as both Opdenakker G. Scholar, A. Biochem. However, in knock-out the of is not increased in response to enhanced neuronal that is for of the neuronal with bicuculline that after the but not are increased in the This also that of synaptic is for to extracellular as as after the also A. and is by limited proteolysis of this time of the of cleavage after the synaptic is the as following glutamate stimulation of the Finally, that this demonstration of the first target in response to enhanced synaptic activity the may help to understand the possible role of both and proteins in neuronal functions, especially in synaptic plasticity, learning, and memory. that is activated and learning, and its by either or the of as as Scholar, A. in increased the to In A. R.A. that in the of in the hippocampus. of the is observed in of such as and are with H. H. Scholar, H. I. I. Scholar, I. H. In in the encoding proteins such as extracellular as as with the are with and we in the of encoding and of following and in vivo A. Scholar, A. Mol. This a role for the and in plasticity. that either or with extracellular as as with G. G. Scholar, and both are important for neuronal the of also recently in cells that the of has a for the of for the in has to G. A. been in is that in may in and for for for with cDNA of the