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

Abstract

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 . 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 2ϩ 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.

Activation of UTP-Sensitive P2Y2Receptor Induces the Expression of Cholinergic Genes in Cultured Cortical Neurons: A Signaling Cascade Triggered by Ca2+Mobilization and Extracellular Regulated Kinase Phosphorylation

“…1A). P2Y 1 R, P2Y 2 R, P2Y 4 R, and P2Y 6 R were detected in both E18 and adult cortex, consistent with previous reports (Webb et al, 1998;Moore et al, 2001;Siow et al, 2010). Quantitative realtime PCR further revealed that the relative levels of these different P2YR mRNAs increased from embryonic to adult stages (Fig.…”

“…To specifically examine neuronal P2Y 2 R, primary cortical neurons were used as a model (Brewer, 1995;Siow et al, 2010). In preparations of neuron cultures, glial cells can often be found, and astrocytes are known to express P2YR subtypes such as P2Y 2 R and P2Y 4 R (Abbracchio and Ceruti, 2006;Fischer and Krügel, 2007).…”

“…Primary cortical neurons were prepared as previously described (Siow et al, 2010). In brief, the cortex was dissected from brains of embryonic day 18 (E18; mixed sex) rats and incubated with 2.5% trypsin at 37°C for 15 minutes.…”

“…Cultured cortical neurons were collected in lysis buffer containing 150 mM NaCl, 10 mM HEPES pH 7.5, 0.5% Triton X-100, 5 mM EGTA, 5 mM EDTA, 1 mg/ml bacitracin, 1 mg/ml leupeptin, 1 mg/ml aprotinin. For phosphorylation analysis (Siow et al, 2010), cultures were serum-starved for 3 hours before applying reagents. Supernatants of the final lysates prepared from these samples were loaded at 10 ml per gel-lane in differentiation and phosphorylation experiments, while 10 mg of protein was loaded per lane in agonist-treatment assays.…”

“…Studies from our laboratory indicated that ATP, via P2Y 1 R and P2Y 2 R, plays a critical role in regulating gene transcription and in forming postsynaptic specializations at the NMJ (Choi et al, 2001(Choi et al, , 2003Ling et al, 2004;Tung et al, 2004). Moreover, the activation of P2Y 1 R triggered a mitogen-activated protein kinase signaling cascade, which subsequently upregulated the expression of genes encoding acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) (Siow et al, 2010). In comparison, less is known about the extent to which other P2Y receptors are involved in regulating the expression of synaptic genes.…”