Transcriptional regulation of acetylcholinesterase by muscle activity

Abstract

Neurotransmission at the neuromuscular junction involves the release of acetylcholine from the presynaptic nerve terminal, and following its action at the postsynaptic receptor, termination by the serine hydrolase, acetylcholinesterase (AChE). AChE is tightly regulated by muscle and nerve activity, in part through elicitation of muscle contractions. Denervation increases AChE activity in chicken muscle but decreases it in rat muscle. The aim of this dissertation was to delineate the role of transcriptional activation in the regulation of AChE by identifying genomic elements involved in the transcriptional regulation of AChE by muscle contraction in vitro and in vivo . The in vitro paradigm used rat primary muscle cultures that were transfected with genomic fragments containing flanking sequences of the AChE promoter, driving the expression of a luciferase reporter gene, and either electrically stimulated to enhance contractile activity or treated with the Na+ channel blocker, tetrodotoxin, to inhibit contractions. One cis-element within intron I, an N-box, was necessary for the response to contraction and its mutagenesis or co-transfection with a dominant negative form of GABP, a transcription factor that binds to the N-box, eliminated the response to contraction. Another cis-element involved in regulation of AChE was identified in the region upstream of the basal promoter as a binding site for NFAT, a transcription factor implicated in the regulation of gene expression by contractile activity. Overexpression of NFAT in muscle cultures transfected with AChE reporter constructs containing this element increased reporter activity and probes containing this element interacted with NFAT in an in vitro binding assay. Studies in vivo employed transient transfection by intramuscular plasmid injection in innervated and denervated muscle of mice and chicks. These studies demonstrated that intron I is necessary for decreased reporter activity in response to denervation and that the differential response between mice and chicks is due to different cis-elements. Together, these findings provide clear support that AChE transcription is regulated by muscle contraction and that elements within intron I as well as the upstream regulatory region play a role in the regulation.