The transcriptional regulation of acetylcholinesterase during the formation and maintenance of neuromuscular junctions

In vertebrate neuromuscular junctions (NMJs), acetylcholine receptor (AChR) and acetylcholinesterase (AChE) are co-localized to form one of post-synaptic specializations; their expression and localization are precisely regulated in order to provide the effective and efficient muscle contraction. Several lines of evidence suggest that nerve-derived factors secreted by motor neuron regulate the expression of post-synaptic genes. Two candidates, calcitonin gene-related peptide (CGRP) and adenosine 5’-triphosphate (ATP) were chosen to investigate their roles in regulating AChE expression in the post-synaptic muscle. To investigate the molecular mechanisms of AChE gene transcription in muscle, a human AChE promoter tagged with a luciferase gene (pAChE-Luc) was used as a reporter to probe the regulatory elements that could be responsible for CGRP- and ATP-mediated AChE expression. In cultured chick myotubes, the treatment of CGRP induced the expression of endogenous chick AChE catalytic subunit, while it reduced the human AChE promoter-driven Iuciferase activity. The application of Bt2-cAMP and forskolin, as well as the over expression of G-protein α-subunits, produced similar responses of the CGRP treatment. Thus, an intracellular cAMP level increased chick AChE expression but decreased human AChE promoter activity, and vice versa. The over expression of cAMP-responsive element binding protein (CREB) in pAChE-Luc transfected myotubes markedly enhanced the cAMP-mediated AChE expression in up- and down-regulated chick and human enzymes, respectively. Purified CREB bound the CRE sequence that was derived from human AChE promoter, while the mutation on the CRE site blocked the binding of CREB, and therefore, it markedly enhanced the expression of the promoter-driven luciferase; however, its response to cAMP inhibition in cultured myotubes was partially retained. Transcript encoding an ATP receptor subtypes called P2Y1 receptor was developmental expressed in muscles and spinal cords, and the receptors were co-localized with AChRs in adult NMJs. Stimulation of P2Y1 receptor by its agonist induced the chick AChE expression and human AChE promoter-driven luciferase activity. The signalling mechanisms of P2Y1 receptor-induced gene expression was elucidated in cultured myotubes. The enzymatic activity of calcium-dependent protein kinase (PKC) was stimulated by P2Y1 receptor, while the receptor-induced AChE gene expression was blocked by PKC inhibitors. The phosphorylation of extracellular signal-regulated kinase (Erk) in myotubes was stimulated in a transient manner by P2Y1 receptor activation. The expression of constitutive active mutant of Raf increased AChE expression, while the dominant negative mutant of Raf blocked the receptor-induced gene activation in cultured myotubes. The phosphorylation of transcription factor Elk-1 in myotubes was induced by the activation of P2Y1 receptor; this activation subsequently induced the transcription activity of human AChE promoter. In summary, my findings here suggest that a cAMP-dependent signalling pathway utilized by CGRP, and a mitogen-activated protein kinase (MAP kinase)-dependent signalling pathway utilized by ATP, could regulate the AChE gene transcription in the post-synaptic muscle, via different responsive elements on the AChE promoter. These findings support the notation that the nerve-derived factors such as CGRP and ATP could play critical roles in regulating the AChE expression in post-synaptic muscle during the formation and maintenance of the NMJs