DEVELOPMENTAL REGULATION OF THE SERPIN, PROTEASE NEXIN-I, LOCALIZATION DURING ACTIVITY-DEPENDENT POLYNEURONAL SYNAPSE ELIMINATION IN MOUSE SKELETAL-MUSCLE

During vertebrate neuromuscular development, all muscle fibers are tra nsiently innervated by more than one neuron. Among the numerous factor s shown to potentially influence the passage from poly- to mononeurona l innervation, serine proteases and their inhibitors appear to play im portant roles. In this regard, protease nexin I (PNI), a potent inhibi tor of the serine protease, thrombin, is highly localized to the neuro muscular junction (NMJ). In turn, thrombin is responsible for activity -dependent synapse elimination both in an in vitro model, and in vivo. In the present study, we used a monospecific anti-PNI polyclonal anti body to study the developmental kinetics of PNI expression in mouse le g skeletal muscle. By using immunoblotting, we detected PNI at embryon ic day 16 (E16), as a 48-kDa band. This 48-kDa PNI band became promine nt in leg muscle extracts at postnatal day 5 (P5) and remained so in e xtracts from adult muscle. In contrast, a higher molecular weight immu noreactive PNI band, which was sodium dodecyl sulfate- and beta-mercap toethanol-resistant, was first detected at E16, increased at birth (P0 ), and then decreased at P15, i.e., after the wave of polyneuronal syn apse elimination had occurred in these muscles. The results of an enzy me-linked immunosorbent assay, measuring active, complexed, and trunca ted PNI, correlated with Western blot data. We used immunocytochemistr y to probe the localization of PNI at the NMJ and found that PNI was p resent in the cytoplasm of myotubes at E16, but neither then nor at bi rth did it colocalize with acetylcholine receptors. PNI became localiz ed at NMJs by P5 and increased by P15, after which it remained stably concentrated there in the adult. Finally, we studied the gene expressi on of PNI mRNA, by using Northern blotting, and showed that PNI mRNA w as present in skeletal muscle and remained stable throughout the time- course studies, suggesting that developmental regulation of muscle PNI occurs principally at the translational and/or post-translational lev els. These results suggest that the localization of PNI, through a bin ding site or ''receptor'' may play an important role in differentiatio n and maintenance of synapse. J. Comp. Neurol. 397:572-579, 1998. (C) 1998 Wiley-Liss, Inc.