EXPERIMENTAL NEUROLOGY 105,211-218 (1989) Synaptic Localization of a 66-kDa Soluble Protein from Skeletal Muscle: Evidence for Its Developmental and Neural Regulation JOAN X. COMELLA, JOSEP E. ESQUERDA,~ JOAN RIBERA, AND LIDIA PIEDRAFITA Universitat de Barcelona, Divisi6 de l%studi General de Lleida, Facultat de Medicina, Departament Cie‘ncies MGdiques B&siques, E-25007 Lleida, Catalonia, Spain Soluble proteins from normal, adult denervated, and developing rat muscles were studied in order to identify common molecular species undergoing developmental regulation and nerve dependence. Significant increases in 66- and 30-kDa proteins were found as a conse- quence of 14 days of denervation. Subsequent reinner- vation restores normal adult levels. During develop- ment, high levels of the 66-kDa protein were found in neonatal muscles but slowly decreased concomitant with the following postnatal maturation period; the adult levels were reached at Postnatal Day (P) 2 1. From the immunocytochemical studies it is deduced that both proteins were concentrated mainly at the end-plate re- gion in adult normal muscle. Following denervation, the proteins were found distributed over the entire cell. For the 66-kDa protein, a similar pattern of extensive distribution was seen in immature muscle. Although no data for functional implications for these proteins are available at present, the properties described here make them of interest in understanding nerve-muscle interactions. 0 1989 Academic Press, Inc. INTRODUCTION The biochemical, physiological, and histochemical properties of adult skeletal muscle are under the influ- ence of the (Ymotor neuron, which maintains the mature muscle cell and its differentiated phenotype. The nature of this influence is not well understood, but it appears that neuromuscular activity as well as chemical factors from nerve terminals may be involved (13-l&23,31). To understand better the influence of nerve on muscle cell, a number of experimental approaches have been used and, of these, denervation has been widely em- ployed to block all neurotrophic influences. As a re- sponse to nerve section, denervated muscles undergo several well-defined changes such as (a) the appearance of fibrillation potentials and tetrodotoxin-resistant ac- tion potentials (30); (b) the spread of ACh sensitivity to 1 To whom correspondence should be addressed. extrasynaptic portions of membrane as a consequence of the incorporation of newly synthetized, extrajunctional receptors (1,24); (c) a decrease in the tension generation capacity (28); (d) a loss of the normally well-defined his- tochemical patterns of oxidative and glycolytic enzymes as markers of fiber type (10); (e) an increase in lysosomal and nonlysosomal enzymatic proteolytic activities (18, 32); (f) an increase in the carbohydrate content on the muscle surface (20); (g) a decrease in the total AChE content and more specifically in the collagen-tailed, 16 S form (4); (h) a renewed ability to receive new innerva- tion; and (i) the enhanced synthesis and surface spread- ing of the neural cell adhesion molecule (N-CAM) (6). Some of these changes may be perceived as the reac- quisition of features, some of which are characteristic of the embryonic muscle. At the molecular level, several common features can be identified in denervated and de- veloping skeletal muscles that are absent in their differ- entiated state. In effect, extensive experimentation has revealed similar regulation of phenotypic expression of junctional and extrajunctional AChR molecules in de- nervated or developing muscle (26). The same was found to be true for N-CAM (7), glucose-6-phosphate dehy- drogenase activity (22), and troponin T isoforms (27). Several changes in soluble muscle proteins as a conse- quence of nerve deprivation have been reported (9,12,16). However, none of these changes were implicated further in neurotrophic regulations. Assuming that potential muscle- derived neurotrophic proteins would be present in larger quantities in denervated muscle and, moreover, that they would be submitted to developmental regulation by nerve cells, we have carried out this first study in an attempt to look for soluble muscle proteins with developmental and nerve regulation. In this paper we have identified a 66-kDa protein which has the aforementioned properties. More- over, the immunocytochemical localization of this protein demonstrates that it is evenly distributed along the muscle cell in either undifferentiated or adult denervated muscles, but in adult normal cells this protein is highly restricted at synaptic regions. Part of this work has been reported in a preliminary form (5). 211 0014-4886/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.