PROTEOLYTIC ACTION OF THROMBIN IS REQUIRED FOR ELECTRICAL ACTIVITY-DEPENDENT SYNAPSE REDUCTION

Molecular mechanisms of activity-dependent synapse reduction were stud ied in an in vitro mammalian neuromuscular preparation. Synapse reduct ion in this model is activity-dependent and is substantially reduced b y the broad-spectrum protease inhibitor, leupeptin, suggesting the rol e of activity dependent proteolytic action in the process. Our present experiments show that a potent and specific thrombin inhibitor, hirud in, at nanomolar concentration completely blocked the activity-depende nt synapse reduction. Furthermore, a naturally occurring serine protea se inhibitor, protease nexin I (PNI), which closely colocalizes with a cetylcholine receptors at the neuromuscular junction, inhibited the sy napse reduction at the same low concentration. In contrast, neither cy statin, a cysteine protease inhibitor, nor aprotinin, a serine proteas e inhibitor that does not inhibit thrombin, blocked the synapse reduct ion. Similarly, neither of the inhibitors of the calcium-activated pro teases calpain I and II prevented the reduction of synapses. These res ults strongly suggest that serine proteolytic action by thrombin or th rombin-like molecules is required for synapse reduction in our in vitr o model of the mammalian neuromuscular junction.