A Molecular Mechanism for Synapse Elimination: Novel Inhibition of Locally Generated Thrombin Delays Synapse Loss in Neonatal Mouse Muscle

Abstract

Activity-dependent, polyneuronal synapse elimination (ADPSE) is a programmed, regressive event in the development of the nervous system and readily studied at the neuromuscular junction, where it is complete 15-20 days after birth. Local excess, or imbalanced, protease activity is one of several possible underlying mechanisms. In this regard, thrombin mediates activity-dependent synapse loss in an in vitro model of ADPSE. To test the involvement of thrombin in vivo, we locally applied the leech thrombin-specific inhibitor, hirudin. We monitored neuromuscular behavior, correlated with acetylcholinesterase and silver nitrate histochemistry at endplates, for changes in the timecourse of in vivo synapse elimination and assayed both thrombin activity and prothrombin expression in developing muscle. Hirudin retarded elimination, without altering motor performance, uniquely at Postnatal Day 5 (P5) and maximally at P9. Reverse transcription-polymerase chain reaction (PCR) showed that neonatal muscle was a source of local prothrombin, with peak expression during the first week after birth. A specific chromogenic assay revealed that local thrombin, activated from muscle-derived prothrombin, peaked during maximal synapse remodeling.

Synapse Number and Synaptic Efficacy Are Regulated by Presynaptic cAMP and Protein Kinase A

“…An alternative hypothesis suggests that the active synapse releases an unknown "poisonous" signal, which renders the inactive synapse nonfunctional and causes it to retract (Liu et al, 1994a(Liu et al, , 1994bZoubine et al, 1996;Sanes and Lichtman, 1999). Our data presented in this study do not support this notion, because if either a VD4 or LPeD1a synapse were to poison the supernumerary LPeD1b cell, which was excluded or eliminated from the synaptogenic program, then it would have failed to receive innervation by the second VD4b (Fig.…”

Thrombin downregulates muscle acetylcholine receptors via an IP3 signaling pathway by activating its G‐protein‐coupled protease‐activated receptor‐1

“…This effect could alternatively result from the inhibition of thrombin possibly produced by the muscle. Indeed, thrombin activity has been detected in muscle cells in vitro (Glazner et al, 1997) and in vivo (Zoubine et al, 1996). The fact that hirudin increases AChRn in the absence of added thrombin is a good argument in favor of the proposition that downregulation of AChR by thrombin is a physiologically relevant event.…”

“…The serine protease thrombin, first described as cleaving fibrinogen into fibrin in blood clotting, also regulates multiple extravascular cellular processes by activating its receptor PAR-1 (Grand et al, 1996). Thrombin has been shown to stimulate proliferation of primary myoblasts (Suidan et al, 1996) and to favor synapse elimination at the neuromuscular junction (Zoubine et al, 1996;Jia et al, 1999). Furthermore, gene inactivation of PAR-1 and of the thrombin precursor prothrombin partially results in embryonic death (Connolly et al, 1996;Xue et al, 1998).…”

“…During skeletal muscle development, PAR-1 is expressed both in vitro and in vivo . Thrombin activity has been detected in muscle cells in vitro (Glazner et al, 1997) and in vivo (Zoubine et al, 1996), and prothrombin and PAR-1 transcript levels are maximum during embryonic skeletal muscle development . Moreover, prothrombin mRNA is expressed in myotubes but not in myoblasts and thrombin activity is regulated in tissues by its inhibitor protease nexin-1 (PN-1) which is expressed only after myoblast fusion into myotubes (Verdière-Sahuqué et al, 1996;Akaaboune et al, 1998), suggesting that a balance between thrombin and its inhibitor PN-1 may be important for myotube differentiation.…”

“…Moreover, prothrombin mRNA is expressed in myotubes but not in myoblasts and thrombin activity is regulated in tissues by its inhibitor protease nexin-1 (PN-1) which is expressed only after myoblast fusion into myotubes (Verdière-Sahuqué et al, 1996;Akaaboune et al, 1998), suggesting that a balance between thrombin and its inhibitor PN-1 may be important for myotube differentiation. Because previous reports have shown that thrombin is involved in neuromuscular synaptic plasticity (Liu et al, 1994;Zoubine et al, 1996), we have focused our study on the acetylcholine receptor (AChR), a key molecule in the formation of the neuromuscular synapse and the best characterized marker of neuromuscular synapse differentiation (Sanes and Lichtman, 2001). We report that thrombin decreases AChR a-subunit gene expression and the number of surface AChR (AChRn) molecules upon activation of its receptor PAR-1 and that the specific thrombin inhibitor, hirudin, reverses this effect by inhibiting the thrombin present in the cultures.…”