Basal lamina directs acetylcholinesterase accumulation at synaptic sites in regenerating muscle.

Abstract

In skeletal muscles that have been damaged in ways which spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fibers are characterized by junctional folds and accumulations of acetylcholine receptors and acetylcholinesterase (ACHE). The formation of junctional folds and the accumulation of acetylcholine receptors is known to be directed by components of the synaptic portion of the myofiber basal lamina. The aim of this study was to determine whether or not the synaptic basal lamina contains molecules that direct the accumulation of ACHE. We crushed frog muscles in a way that caused disintegration and phagocytosis of all cells at the neuromuscular junction, and at the same time, we irreversibly blocked AChE activity. New muscle fibers were allowed to regenerate within the basal lamina sheaths of the original muscle fibers but reinnervation of the muscles was deliberately prevented. We then stained for AChE activity and searched the surface of the new muscle fibers for deposits of enzyme they had produced. Despite the absence of innervation, AChE preferentially accumulated at points where the plasma membrane of the new muscle fibers was apposed to the regions of the basal lamina that had occupied the synaptic cleft at the neuromuscular junctions. We therefore conclude that molecules stably attached to the synaptic portion of myofiber basal lamina direct the accumulation of AChE at the original synaptic sites in regenerating muscle. Additional studies revealed that the AChE was solubilized by collagenase and that it remained adherent to basal lamina sheaths after degeneration of the new myofibers, indicating that it had become incorporated into the basal lamina, as at normal neuromuscular junctions.

Transgenic engineering of neuromuscular junctions in Xenopus laevis embryos transiently overexpressing key cholinergic proteins.

“…In contrast, neuronal AChE expression in animals transgenic for the human enzyme induced synaptic accumulation of AChE in a manner less subject to host regulation. These observations are consistent with findings demonstrating neuronal contribution ofAChE to the synaptic cleft in adult Xenopus NMJs (20). They also highlight a putative role for muscle AChE in establishing NMJ length as opposed to a neuronal influence on the width of the synaptic cleft.…”

Molecules in Basal Lamina That Direct the Formation of Synaptic Specializations at Neuromuscular Junctions

“…We noted in our studies on AChR and AChE aggregation in regenerating muscles [3,6,18] that regardless of how the muscle was damaged, the new muscle fibers formed folds where they contacted the original synaptic sites on the sheaths ( fig. 2, 3).…”

“…To learn whether the synaptic basal lamina could also direct the accumulation of AChE on regenerating myofibers, the cutaneous pectoris muscles were removed from the frog, pinned out in a Petri dish and crushed, which killed all cells at the neuromuscular junction as in freeze-dam aged muscles [3], The muscles were incu bated in diisopropylfluorophosphate to in hibit irreversibly the original A C hE, some of which persists in the basal lamina for months after damage. The muscles were then re placed in the frog and myofibers were al lowed to regenerate within the sheaths of the original myofibers in the absence of innerva tion.…”

Acetylcholinesterase Expression in Muscle Is Specifically Controlled by a Promoter-Selective Enhancesome in the First Intron

“…The origin of AChE in muscle endplates has been an area of long-standing investigation (Anglister and McMahan, 1985;Grinnell, 1995;Rotundo et al, 1997;Jevsek et al, 2004;Mis et al, 2005). Previous work has shown AChE expression in motor neurons to be present along the axon (Massoulie and Bon, 1982;Rotundo and Carbonetto, 1987).…”