Proteolytic action of thrombin is required for electrical activity-dependent synapse reduction.
Molecular mechanisms of activity-dependent synapse reduction were studied in an in vitro mammalian neuromuscular preparation. Synapse reduction in this model is activity-dependent and is substantially reduced by the broad-spectrum protease inhibitor, leupeptin, suggesting the role of activity-dependent proteolytic action in the process. Our present experiments show that a potent and specific thrombin inhibitor, hirudin, at nanomolar concentration completely blocked the activity-dependent synapse reduction. Furthermore, a naturally occurring serine protease inhibitor, protease nexin I (PNI), which closely colocalizes with acetylcholine receptors at the neuromuscular junction, inhibited the synapse reduction at the same low concentration. In contrast, neither cystatin, a cysteine protease inhibitor, nor aprotinin, a serine protease inhibitor that does not inhibit thrombin, blocked the synapse reduction. Similarly, neither of the inhibitors of the calcium-activated proteases calpain I and II prevented the reduction of synapses. These results strongly suggest that serine proteolytic action by thrombin or thrombin-like molecules is required for synapse reduction in our in vitro model of the mammalian neuromuscular junction.
Liu
Y
Y
Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Fields
R D
RD
Festoff
B W
BW
Nelson
P G
PG
D016564:Amyloid beta-Protein Precursor; D000818:Animals; D000831:Animals, Newborn; D007611:Aprotinin / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors; D002352:Carrier Proteins / Q000494:pharmacology; D002450:Cell Communication; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000502:physiology*; D015891:Cystatins / Q000494:pharmacology; D004558:Electric Stimulation; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D051379:Mice; D009132:Muscles / Q000502:physiology*; D009414:Nerve Growth Factors / Q000494:pharmacology; D016501:Neurites / Q000502:physiology*; D009469:Neuromuscular Junction / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000502:physiology; D011994:Recombinant Proteins / Q000494:pharmacology; D017783:Superior Cervical Ganglion / Q000502:physiology*; D013569:Synapses / Q000187:drug effects / Q000502:physiology*; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D015891:Cystatins; D006629:Hirudins; D009414:Nerve Growth Factors; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins; D007611:Aprotinin; D013917:Thrombin; D002154:Calpain