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70 Modulation of neuronal properties by extracellular proteolytic activity
73 ACTIVITY-DEPENDENT REDUCTION IN CULTURE SYSTEM
73 A
SYNAPSE NEUROMUSCULAR
Phillip G. Nelson, Gordon Glaztter and Douglas E. Brenneman Laboratory of Developmenta Neurobiology. NICHD, National Institutes of Health, Bethesda, MD 20892-4480, USA The postnatal reduction in an initially redundant polyneuronal innervation of skeletal muscle is dependent on neuromuscular activity. We have shown that the activity-dependent synapse reduction (ADSR) ts robustly expressed in a tissue culture system. The degree to which the loss is selective for the inactive inputs to the muscle apprears to be related to the amount of stimulation delivered to the preparations. The ADSR can be completely blocked by nanomolar concentrations of hirudin (a specific thrombin inhibitor) and protease nexin 1 (PNI), an endogenous serine protease inhibitor. Exogenous thrombin produces synapse loss in quiescent preparations and this loss is prevented by hirudin. Cholinergic stimulation of cultured mouse my&t&s increases secretion of thrombin activity and levels of prothrombin mRNA in the muscle cells. PNl messagelevels are little, if any, changed by cholinergic stimulation. Neuronal survival in CNS cultures can be influenced by thrombin (iucreased cell death) or PNI (mcre.ased cell survival). We conclude that in both peripheral and central synaptic systems, a balanced expression of thrombiu or thrombin-like proteases and their inhibitors is critically involved in ADSR and neuronal survival and thus in the developing architecture of synaptic circuits.
MODULATION OF NEURONAL PROPERTIES PROTEOLYTIC EXTRACELLULAR BY ACTIVITY Monard, D.*, Luthl, A “. Boucr~, F.*, Mcln\. M.*. Laurent, J.P.“. Schmutz, M.‘. and van dcr Putten, H”. *Frlcdrlch Mieschcr Instttutc. P.O. Box 2543, CH-4002 Bascl, Swnzwland “Pharmn D~v~swn, Preclm,cal Research. F. Hoffmann-LaRochc Ltd.. Ch-4002 Bascl. Swttzerland. ‘0% Dept. Cell. Mol. Biol., Ciba Ltd.. P.0 Box 2543, CH-4002 Basel, Swnrcrland An increasmg amount of cxperirncntal cvidcncc indicates that the balance bctwccn protcases and their bpccific inhibitors ~ntlucnces ncuronal bchaviour. For ~xamplc. Protcasc Ncxln- I (PN-I ). a serpm potently Inhibiting proteases such as thrombtn, plasmmogcn activators or trypsin, has been idcnttficd as a protein promotmg neurue outgrowth in cultured ncuroblastoma or primary neurons. Pi’- I is transiently expressed in many neuronat Populations during dcvclopment and rn a few distinct neurons III the adult. PN-I synthesrs ts induced in glial cells followtng leaton I” horh the pcrtphcral nerwus system and the central nervous system. Transgcmc nnce overexpressing PN-I m neurons or nwc lacking PN-I have been generated to elucidntc the i?r riw funcnonts) of thts serpln. ElcctrophystolognXd srudics pcrformcd &tth htppocampal slices dcrivcd from thcsc transpcnic arnmals Indiwtc that overcxpression of PN-I results tn a significant ~ncrensc in long-tcrrn potcntation (LTP) whereah the abscncc of PN- I leads ,o a decrease tn LTP.
76
75
MECHANISM OF ACTION OF CNS MYELINASSOCIATED NEURITE GROWTH INHIBITORS NI-3.51250
MOLECULAR INTERACTIONS THAT FACILITATE THE GROWTH OF COMMISSURAL AXONS ACROSS THE MIDLINE
Christine
E. Bandtlow,
Jens Fritsche
and Martin
E. Schwab
1. lmdmesser. Department ol~h’eurosctences.Case Western Rescr~c Llnivcrsity, Cleveland, OH 41106
Brnm Rcscarch lnstttute. Utuversity of Zurtch August-Forel-Str I X029 Zurtch. Swrzrhnd
Commissural axons of the chick lumbosacral spinal cord grw to the floor plate in response to a gradtent of nctnn. Our laboratory has investigated the subsequentmolecular stgrtals that cause commissural growth cones to first cross the midline and to then turn to grow rostrally along the contralateral border of the Boor plate. Blocking the function of spectftc adheston molecules by the injection of antibodies resulted in stgnificaut pathfiindmg errors and led us to Propose thal growth across the midhne is facthutted by an interacttoo between axonin-l (TAG 1) on the growth cones and NrCAM on the floor plate Jn the absence of this interaction, growth cones stall upon reaching the floor plate and many turn erroneously on the ipsilateral side. They nou appear to find the floor plate inhibitory This idea was tested m culture by observing the behavior of comtmssural growth cones co-cultured with floor plate explants Under control condittous, &cy rapidly entered ffoor plate rxphmts and preferred these IOa laminum substratum However when the funcuon of either axonin- or NtCAM was blocked, growth cones failed to enter the Boor plate explants, and were observed m time lapse imaging to collapse upon filopodial contact tvith the floor plate. We propose a model in which the floor plate contains a mixture of growth promoting and inhibiting signals and the balance between these determines whether an axon will cross the midline Supprtcd by YIH grant NS 19640 from NINDS
The most potent ncurttc growth mhrbttors arc found ln CNS m~cltn of htgher vetlebrates (NI-351250) and have been dcmonstratcd to be ctuctally tnvolved m the lack of regencratton of CNS Fabertracts upon inJuty We have previously addressed the question about the tntracellular mechanisms whtch are acti\.ated by these tnhtbttors Long-lastmg paralysts of e~tendmg neuntes was seen upon contact wtth ohgodendrocytes resulttng m collapse of gro%th cone structures Subsequent studies showd that growth cone collapse ts causally mcdtated by the transtcnt rtsc of mtracellular calcmm concentrattons due to the rclcasc of calcmm from caf felnei~anodine-sensltl~c tntracellular stores The questton how changes tn tntracellular calcutm or other second messengersmight lead to the cytoskclctal changes that undcrhe growth cone collapse ts poorly understood The pnmaty events leading to grolrth cone collapse may bc depolymertzation of actm filaments m growth cones Based on the fact that many acttn-assoctatcd molecules arc present m the leadtng edge of the growth cone. many of which have then acttvtttes modified by calcmm, one may spcculatc that the spcctiic modulation of such protems contnbutc to the morphological changes of the gro\+th cone structure Smce many of these protcms arc phosphoprotems. we have mvesttgatcd tf NI-35/250 modulates the level of phospho~latron of these protcms by dtrect or mdtrect acttvatton of otosoltc protctn ktnases or phosphatases
68
73 A
SYNAPSE NEUROMUSCULAR
Phillip G. Nelson, Gordon Glaztter and Douglas E. Brenneman Laboratory of Developmenta Neurobiology. NICHD, National Institutes of Health, Bethesda, MD 20892-4480, USA The postnatal reduction in an initially redundant polyneuronal innervation of skeletal muscle is dependent on neuromuscular activity. We have shown that the activity-dependent synapse reduction (ADSR) ts robustly expressed in a tissue culture system. The degree to which the loss is selective for the inactive inputs to the muscle apprears to be related to the amount of stimulation delivered to the preparations. The ADSR can be completely blocked by nanomolar concentrations of hirudin (a specific thrombin inhibitor) and protease nexin 1 (PNI), an endogenous serine protease inhibitor. Exogenous thrombin produces synapse loss in quiescent preparations and this loss is prevented by hirudin. Cholinergic stimulation of cultured mouse my&t&s increases secretion of thrombin activity and levels of prothrombin mRNA in the muscle cells. PNl messagelevels are little, if any, changed by cholinergic stimulation. Neuronal survival in CNS cultures can be influenced by thrombin (iucreased cell death) or PNI (mcre.ased cell survival). We conclude that in both peripheral and central synaptic systems, a balanced expression of thrombiu or thrombin-like proteases and their inhibitors is critically involved in ADSR and neuronal survival and thus in the developing architecture of synaptic circuits.
MODULATION OF NEURONAL PROPERTIES PROTEOLYTIC EXTRACELLULAR BY ACTIVITY Monard, D.*, Luthl, A “. Boucr~, F.*, Mcln\. M.*. Laurent, J.P.“. Schmutz, M.‘. and van dcr Putten, H”. *Frlcdrlch Mieschcr Instttutc. P.O. Box 2543, CH-4002 Bascl, Swnzwland “Pharmn D~v~swn, Preclm,cal Research. F. Hoffmann-LaRochc Ltd.. Ch-4002 Bascl. Swttzerland. ‘0% Dept. Cell. Mol. Biol., Ciba Ltd.. P.0 Box 2543, CH-4002 Basel, Swnrcrland An increasmg amount of cxperirncntal cvidcncc indicates that the balance bctwccn protcases and their bpccific inhibitors ~ntlucnces ncuronal bchaviour. For ~xamplc. Protcasc Ncxln- I (PN-I ). a serpm potently Inhibiting proteases such as thrombtn, plasmmogcn activators or trypsin, has been idcnttficd as a protein promotmg neurue outgrowth in cultured ncuroblastoma or primary neurons. Pi’- I is transiently expressed in many neuronat Populations during dcvclopment and rn a few distinct neurons III the adult. PN-I synthesrs ts induced in glial cells followtng leaton I” horh the pcrtphcral nerwus system and the central nervous system. Transgcmc nnce overexpressing PN-I m neurons or nwc lacking PN-I have been generated to elucidntc the i?r riw funcnonts) of thts serpln. ElcctrophystolognXd srudics pcrformcd &tth htppocampal slices dcrivcd from thcsc transpcnic arnmals Indiwtc that overcxpression of PN-I results tn a significant ~ncrensc in long-tcrrn potcntation (LTP) whereah the abscncc of PN- I leads ,o a decrease tn LTP.
76
75
MECHANISM OF ACTION OF CNS MYELINASSOCIATED NEURITE GROWTH INHIBITORS NI-3.51250
MOLECULAR INTERACTIONS THAT FACILITATE THE GROWTH OF COMMISSURAL AXONS ACROSS THE MIDLINE
Christine
E. Bandtlow,
Jens Fritsche
and Martin
E. Schwab
1. lmdmesser. Department ol~h’eurosctences.Case Western Rescr~c Llnivcrsity, Cleveland, OH 41106
Brnm Rcscarch lnstttute. Utuversity of Zurtch August-Forel-Str I X029 Zurtch. Swrzrhnd
Commissural axons of the chick lumbosacral spinal cord grw to the floor plate in response to a gradtent of nctnn. Our laboratory has investigated the subsequentmolecular stgrtals that cause commissural growth cones to first cross the midline and to then turn to grow rostrally along the contralateral border of the Boor plate. Blocking the function of spectftc adheston molecules by the injection of antibodies resulted in stgnificaut pathfiindmg errors and led us to Propose thal growth across the midhne is facthutted by an interacttoo between axonin-l (TAG 1) on the growth cones and NrCAM on the floor plate Jn the absence of this interaction, growth cones stall upon reaching the floor plate and many turn erroneously on the ipsilateral side. They nou appear to find the floor plate inhibitory This idea was tested m culture by observing the behavior of comtmssural growth cones co-cultured with floor plate explants Under control condittous, &cy rapidly entered ffoor plate rxphmts and preferred these IOa laminum substratum However when the funcuon of either axonin- or NtCAM was blocked, growth cones failed to enter the Boor plate explants, and were observed m time lapse imaging to collapse upon filopodial contact tvith the floor plate. We propose a model in which the floor plate contains a mixture of growth promoting and inhibiting signals and the balance between these determines whether an axon will cross the midline Supprtcd by YIH grant NS 19640 from NINDS
The most potent ncurttc growth mhrbttors arc found ln CNS m~cltn of htgher vetlebrates (NI-351250) and have been dcmonstratcd to be ctuctally tnvolved m the lack of regencratton of CNS Fabertracts upon inJuty We have previously addressed the question about the tntracellular mechanisms whtch are acti\.ated by these tnhtbttors Long-lastmg paralysts of e~tendmg neuntes was seen upon contact wtth ohgodendrocytes resulttng m collapse of gro%th cone structures Subsequent studies showd that growth cone collapse ts causally mcdtated by the transtcnt rtsc of mtracellular calcmm concentrattons due to the rclcasc of calcmm from caf felnei~anodine-sensltl~c tntracellular stores The questton how changes tn tntracellular calcutm or other second messengersmight lead to the cytoskclctal changes that undcrhe growth cone collapse ts poorly understood The pnmaty events leading to grolrth cone collapse may bc depolymertzation of actm filaments m growth cones Based on the fact that many acttn-assoctatcd molecules arc present m the leadtng edge of the growth cone. many of which have then acttvtttes modified by calcmm, one may spcculatc that the spcctiic modulation of such protems contnbutc to the morphological changes of the gro\+th cone structure Smce many of these protcms arc phosphoprotems. we have mvesttgatcd tf NI-35/250 modulates the level of phospho~latron of these protcms by dtrect or mdtrect acttvatton of otosoltc protctn ktnases or phosphatases
68