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 P2Y(1) 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 P2Y(1) 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 P2Y(1) (G-protein-coupled) receptors induced the elevation of intracellular Ca(2+) and activation of a mitogen-activated protein kinase/Raf-1 signaling cascade. This led to distinct up-regulation of the genes encoding acetylcholinesterase (AChE(T) variant), choline acetyltransferase, and the N-methyl-d-aspartate receptor subunit NR2A. This was confirmed, in the example of AChE, to arise from P2Y(1)-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 P2Y(1) receptor activation. The combined findings reveal that ATP, via its P2Y(1) receptor, can act trophically in brain neurons to regulate the gene expression of direct effectors of synaptic transmission.

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).

cDNA encodes Xenopus P2Y(1) nucleotide receptor: expression at the neuromuscular junctions.

A cDNA encoding P2Y(1) receptor was isolated by cross-hybridization with chicken homolog. The deduced amino acid sequence of P2Y(1) receptor with 361 amino residues is 80-85% identical to human, rodent and avian homologs. When the cDNA was expressed in mammalian cells, the activation of P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphate, and adenosine 3',5'-bismonophosphate (A3P5P) or other antagonists blocked its action; these pharmacological properties showed resemblance of P2Y(1) receptor family in higher vertebrate. A transcript encoding P2Y(1) receptor at approximately 3.2 kb was revealed in the brain, spinal cord and muscle of adult, and it is strongly expressed in developing brain, spinal cord and myotomal muscles of the embryos by hybridization. P2Y(1) receptor was shown to be restricted to the neuromuscular junctions and co-localized with AChRs in adult muscle. These results support the notion that ATP and its P2Y(1) receptor subtype are effectors in organizing the post-synaptic apparatus.

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 (G(4) AChE), was revealed in muscle during myogenic differentiation under the influence of innervation. During myotube formation of C2C12 cells, the expression of AChE(T) protein and the enzymatic activity were dramatically increased, but the level of G(4) AChE was relatively decreased. This G(4) 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 G(4) AChE. The oligomerization of G(4) 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 G(4) 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 G(4) AChE, in muscle is suppressed by muscle regulatory factors, muscular activity, and nerve-derived trophic factor(s).

ATP potentiates agrin-induced AChR aggregation in cultured myotubes: activation of RhoA in P2Y1 nucleotide receptor signaling at vertebrate neuromuscular junctions.

At vertebrate neuromuscular junctions, 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, P2Y(1) receptor, is localized at the nmjs, and we propose that this mediates a trophic role for synaptic ATP there. In cultured myotubes, the activation of P2Y(1) receptors modulated agrin-induced acetylcholine receptor (AChR) aggregation in a potentiation manner. This potentiation effect in agrin-induced AChR aggregation was reduced by antagonizing the P2Y(1) receptors. The guanosine triphosphatase RhoA was shown to be responsible for this P2Y(1)-potentiated effect. The localization of RhoA in rat and chicken skeletal muscles was restricted at the neuromuscular junctions. Application of P2Y(1) agonists in cultured myotubes induced RhoA activation, which showed an additive effect with agrin-induced RhoA activation. Over-expression of dominant-negative mutant of RhoA in cultured myotubes diminished the agrin-induced AChR aggregation, as well as the potentiation effect of P2Y(1)-specific agonist. Application of UTP in the cultures also triggered similar responses as did 2-methylthioadenosine 5'-diphosphate, suggesting the involvement of other subtypes of P2Y receptors. These results demonstrate that RhoA could serve as a downstream mediator of signaling mediated by P2Y(1) receptor and agrin, which therefore synergizes the effects of the two neuron-derived trophic factors in modulating the formation and/or maintenance of post-synaptic apparatus at the neuromuscular junctions.