Nina L. Siow

ATP acts via P2Y1 receptors to stimulate acetylcholinesterase and acetylcholine receptor expression: transduction and transcription control.

At the vertebrate neuromuscular junction ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, the P2Y1 receptor, is localized at the junction, and we propose that this mediates a trophic role for synaptic ATP there. Evidence in support of this and on its mechanism is given here. With the use of chick or mouse myotubes expressing promoter–reporter constructs from genes of acetylcholinesterase (AChE) or of the acetylcholine receptor subunits, P2Y1 receptor agonists were shown to stimulate the transcription of each of those genes. The pathway to activation of the AChE gene was shown to involve protein kinase C and intracellular Ca 2+ release. Application of dominant-negative or constitutively active mutants, or inhibitors of specific kinases, showed that it further proceeds via some of the known intermediates of extracellular signal-regulated kinase phosphorylation. In both chick and mouse myotubes this culminates in activation of the transcription factor Elk-1, confirmed by gel mobility shift assays and by the nuclear accumulation of phosphorylated Elk-1. All of the aforementioned activations by agonist were amplified when the content of P2Y1 receptors was boosted by transfection, and the activations were blocked by a P2Y1-selective antagonist. Two Elk-1 binding site sequences present in the AChE gene promoter were jointly sufficient to drive ATP-induced reporter gene transcription. Thus ATP regulates postsynaptic gene expression via a pathway to a selective transcription factor activation.

Regulation of a Transcript Encoding the Proline-rich Membrane Anchor of Globular Muscle Acetylcholinesterase THE SUPPRESSIVE ROLES OF MYOGENESIS AND INNERVATING NERVES

The transcriptional regulation of proline-rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form acetylcholinesterase (G4 AChE), was revealed in muscle during myogenic differentiation under the influence of innervation. During myotube formation of C2C12 cells, the expression of AChET protein and the enzymatic activity were dramatically increased, but the level of G4 AChE was relatively decreased. This G4 AChE in C2C12 cells was specifically recognized by anti-PRiMA antibody, suggesting the association of this enzyme with PRiMA. Reverse transcription-PCR analysis revealed that the level of PRiMA mRNA was reduced during the myogenic differentiation of C2C12 cells. Overexpression of PRiMA in C2C12 myotubes significantly increased the production of G4 AChE. The oligomerization of G4 AChE, however, did not require the intracellular cytoplasmic tail of PRiMA. After overexpressing the muscle regulatory factors, myogenin and MyoD, the expressions of PRiMA and G4 AChE in cultured myotubes were markedly reduced. In addition, calcitonin gene-related peptide, a known motor neuron-derived factor, and muscular activity were able to suppress PRiMA expression in muscle; the suppression was mediated by the phosphorylation of a cAMP-responsive element-binding protein. In accordance with the in vitro results, sciatic nerve denervation transiently increased the expression of PRiMA mRNA and decreased the phosphorylation of cAMP-responsive element-binding protein as well as its activator calcium/calmodulin-dependent protein kinase II in muscles. Our results suggest that the expression of PRiMA, as well as PRiMA-associated G4 AChE, in muscle is suppressed by muscle regulatory factors, muscular activity, and nerve-derived trophic factor(s).

ATP induces post-synaptic gene expressions in vertebrate skeletal neuromuscular junctions

In vertebrate neuromuscular junctions (nmjs), adenosine 5′-triphosphate (ATP) is stored at the motor nerve terminals and is co-released with acetylcholine during neural stimulation. Several lines of evidence suggest that the synaptic ATP can act as a synapse-organizing factor at the nmjs, mediated by metabotropic P2Y1 receptors. P2Y1 receptor mRNAs in chicken and rat muscles are low in embryo but increases markedly in the adult, and decreased after denervation. The P2Y1 receptor protein is restricted to the nmjs and co-localized with AChRs in adult muscles. The activation of P2Y1 receptor by adenine nucleotides in cultured chick myotubes stimulated the accumulation of inositol phosphates, intracellular Ca2+ mobilization, protein kinase C activity and phosphorylation of extracellular signal-regulated kinases. The receptor activation led to an increase in the expression of transcripts encoding AChE catalytic subunit and AChR subunits. The ATP-induced post-synaptic gene expression is possibly mediated by the activation of signaling cascades of mitogen-activated protein kinase. Therefore, a model is being proposed here that the synaptic ATP has a role of synergy with other regulatory signals, such as neuregulin, which act via their post-synaptic receptors to activate second signaling molecules locally to enhance the transcription of AChR/AChE genes specifically in the adjacent sub-synaptic nuclei during the formation and, especially, the maintenance of post-synaptic specializations at the nmjs.

ATP potentiates the formation of AChR aggregate in the co‐culture of NG108‐15 cells with C2C12 myotubes

The role of adenosine 5′‐triphosphate (ATP) and P2Y1 nucleotide receptor in potentiating agrin‐induced acetylcholine receptor (AChR) aggregation is being demonstrated in a co‐culture system of NG108‐15 cell, a mouse neuroblastoma X rat glioma hybrid cell line that resembles spinal motor neuron, with C2C12 myotube. In the co‐cultures, antagonized P2Y1 receptors showed a reduction in NG108‐15 cell‐induced AChR aggregation. Parallel to this observation, cultured NG108‐15 cell secreted ATP into the conditioned medium in a time‐dependent manner. Enhancement of ATP release from the cultured NG108‐15 cells by overexpression of active mutants of small GTPases increased the aggregation of AChRs in co‐culturing with C2C12 myotubes. In addition, ecto‐nucleotidase was revealed in the co‐culture, which rapidly degraded the applied ATP. These results support the notion that ATP has a role in directing the formation of post‐synaptic apparatus in vertebrate neuromuscular junctions.

Calcitonin gene-related peptide induces the expression of acetylcholinesterase-associated collagen ColQ in muscle : a distinction in driving two different promoters between fast-and slow-twitch muscle fibers

The presence of a collagenous protein (ColQ) characterizes the collagen‐tailed forms of acetylcholinesterase at vertebrate neuromuscular junctions (nmjs). Two ColQ transcripts as ColQ‐1 and ColQ‐1a, driven by two promoters: pColQ‐1 and pColQ‐1a, were found in mammalian slow‐ and fast‐twitch muscles, respectively, which have distinct expression pattern in different muscle fibers. In this study, we show the differential expression of CoQ in different muscles is triggered by calcitonin gene‐related peptide (CGRP), a known motor neuron‐derived factor. Application of CGRP, or dibutyryl‐cAMP (Bt2‐cAMP), in cultured myotubes induced the expression of ColQ‐1a transcript and promoter activity; however, the expression of ColQ‐1 transcript did not respond to CGRP or Bt2‐cAMP. The CGRP‐induced gene activation was blocked by an adenylyl cyclase inhibitor or a dominant negative mutant of cAMP‐responsive element (CRE) binding protein (CREB). Two CRE sites were mapped within the ColQ‐1a promoter, and mutations of the CRE sites abolished the response of CGRP or Bt2‐cAMP. In parallel, CGRP receptor complex was dominantly expressed at the nmjs of fast muscle but not of slow muscle. These results suggested that the expression of ColQ‐1a at the nmjs of fast‐twitch muscle was governed by a CGRP‐mediated cAMP signaling mechanism.

ATP Induces Synaptic Gene Expressions in Cortical Neurons: Transduction and Transcription Control via P2Y1 Receptors

Studies in vertebrate neuromuscular synapses have revealed previously that ATP, via P2Y receptors, plays a critical role in regulating postsynaptic gene expressions. An equivalent regulatory role of ATP and its P2Y receptors would not necessarily be expected for the very different situation of the brain synapses, but we provide evidence here for a brain version of that role. In cultured cortical neurons, the expression of P2Y1 receptors increased sharply during neuronal differentiation. Those receptors were found mainly colocalized with the postsynaptic scaffold postsynaptic density protein 95 (PSD-95). This arises through a direct interaction of a PDZ domain of PSD-95 with the C-terminal PDZ-binding motif, D-T-S-L of the P2Y1 receptor, confirmed by the full suppression of the colocalization upon mutation of two amino acids therein. This interaction is effective in recruiting PSD-95 to the membrane. Specific activation of P2Y1 (G-protein-coupled) receptors induced the elevation of intracellular Ca2+ and activation of a mitogen-activated protein kinase/Raf-1 signaling cascade. This led to distinct up-regulation of the genes encoding acetylcholinesterase (AChET variant), choline acetyltransferase, and the N-methyl-d-aspartate receptor subunit NR2A. This was confirmed, in the example of AChE, to arise from P2Y1-dependent stimulation of a human ACHE gene promoter. That involved activation of the transcription factor Elk-1; mutagenesis of the ACHE promoter revealed that Elk-1 binding at its specific responsive elements in that promoter was induced by P2Y1 receptor activation. The combined findings reveal that ATP, via its P2Y1 receptor, can act trophically in brain neurons to regulate the gene expression of direct effectors of synaptic transmission.

The cAMP-dependent protein kinase mediates the expression of AChE in chick myotubes

Calcitonin gene-related peptide (CGRP), a neuropeptide synthesized by motor neurons, stimulates the expression of AChR and AChE at the vertebrate neuromuscular junctions. The signaling mechanism of CGRP-induced AChE expression in muscle was determined both in vitro and in vivo. In cultured chick myotubes, the intracellular cAMP-dependent protein kinase (PKA) activity increased to ∼2-fold after the application of CGRP or PKA activators; the induction was blocked by PKA inhibitors. in vivo transfection analysis on chick gastrocnemius muscles showed that the transfection of cDNA encoding constitutively active mutant Gαs increased the expression of AChE mRNA and protein to ∼2-fold, while the constitutively active mutant Gαi cDNA transfection showed an opposite effect. The induced catalytic subunit of AChE at ∼105 kDa was determined by specific antibody. These findings indicate that the CGRP-induced AChE expression in chick muscle is mediated by a PKA-dependent pathway.

Regulation of PRiMA-linked G4 AChE by a cAMP-dependent signaling pathway in cultured rat pheochromocyoma PC12 cells

The catalytic subunit of acetylcholinesterase (AChE(T)) interacts with proline-rich membrane anchor (PRiMA) to form PRiMA-linked G(4) AChE on membrane surface for its cholinergic function. Cultured PC12 cells expressed the transcripts encoding AChE(T) and PRiMA I, but the expression of PRiMA II transcript was below detection. Upon the treatment of dibutyryl-cAMP (Bt(2)-cAMP) and forskolin in cultured cells to stimulate the cAMP-dependent signaling pathway, the mRNA expressions of both AChE(T) and PRiMA I, as well as the enzymatic activity were up-regulated. More importantly, sucrose density gradient analysis revealed that both G(1) and G(4) AChE isoforms were increased in the Bt(2)-cAMP-treated cultures. These results suggest that the regulation of PRiMA-linked G(4) AChE in terms of gene transcription and molecular assembly in the cultured PC12 cells could be mediated by a cAMP-dependent signaling mechanism.

Activation of UTP-Sensitive P2Y2 Receptor Induces the Expression of Cholinergic Genes in Cultured Cortical Neurons: A Signaling Cascade Triggered by Ca2+ Mobilization and Extracellular Regulated Kinase Phosphorylation

ATP functions as an extracellular signaling molecule that is costored and coreleased with neurotransmitters at central and peripheral neuronal synapses. Stimulation by ATP upregulates the expression of synaptic genes in muscle—including the genes for nicotine acetylcholine receptor (α-, δ-, and ε-subunits) and acetylcholinesterase (AChE)—via the P2Y receptor (P2YR), but the trophic response of neurons to the activation of P2YRs is less well understood. We reported that cultured cortical neurons and the developing rat brain expressed different types of P2YRs, and among these the UTP-sensitive P2Y2R was the most abundant. P2Y2R was found to exist in membrane rafts and it colocalized with the postsynaptic protein PSD-95 in cortical neurons. Notably, agonist-dependent stimulation of P2Y2R elevated the neuronal expression of cholinergic genes encoding AChE, PRiMA (an anchor for the globular form AChE), and choline acetyltransferase, and this induction was mediated by a signaling cascade that involved Ca2+ mobilization and extracellular regulated kinases 1/2 activation. The importance of P2Y2R action was further shown by the receptor’s synergistic effect with P2Y1R in enhancing cholinergic gene expression via the robust stimulation of Ca2+ influx. Taken together our results revealed a developmental function of P2Y2R in promoting synaptic gene expression and demonstrated the influence of costimulation of P2Y1R and P2Y2R in neurons.

Transcriptional control of different acetylcholinesterase subunits in formation and maintenance of vertebrate neuromuscular junctions

Acetylcholinesterase (AChE; EC 3.1.1.7) is a highly polymorphic enzyme (Massoulié, 2002). Asingle ACHE gene produces several types of catalytic subunits by alternative splicing, but a single splice variant, called type T (AChET), is expressed in adult mammalian muscle and brain. Catalytic subunits of AChET produce amphiphilic monomers and dimers, nonamphiphilic homotetramers, as well as heteromeric associations with anchoring proteins, ColQ (collagenous subunit) and PRiMA (proline-rich membrane anchor), which allow their functional localization in cholinergic synapses (Massoulié, 2002). ColQ characterizes the collagen-tailed forms (Aforms) of AChE and butyrylcholinesterase (BChE), which are localized in the basal lamina at neuromuscular junctions (NMJs) of vertebrates (Krejci et al., 1999); in these molecules (A4, A8, A12), one, two, or three tetramers of catalytic subunits are disulfide-linked to the strands of a triple helix of ColQ collagen. The cDNAs encoding ColQ, which have two transcripts, have been cloned: ColQ-1a predominantly in fast-twitch muscle, and ColQ-1 predominantly in slow-twitch muscle. The tetrameric globular (G4) form of AChE is characterized by linkage to PRiMA. PRiMAcDNA encodes a single-pass approximately 20-kDa type-I transmembrane protein and, similar to that of ColQ, contains a short PRAD (proline-rich attachment domain) that is able to organize AChE catalytic subunits into tetramers and anchor the enzyme at the surface of neuron and muscle (Massoulié, 2002).