ATP and P2Y1 nucleotide receptor in cortical neurons: Localization, signal transduction and transcriptional regulation

In vertebrate nervous system, adenosine 5'-triphosphate (ATP) is co-stored and co-released at central and peripheral cholinergic and bioaminergic neuronal synapses. At the neuromuscular junctions, ATP and P2Y1 receptor have been demonstrated to play roles in activating post-synaptic gene expressions of acetylcholine receptor (AChR) and acetylcholinesterase (AChE). However, the trophic role of ATP in the central nervous system (CNS) is still not fully understood. In CNS, ATP is released from both neurons and glia; the existence of extracellular ATP further expands the potential functional significance of the nucleotide-mediated signaling.; To study the functional role of ATP and P2Y1 receptor in the CNS, primary culture of cortical neurons from rat embryos was established and used as a model system in my study. The expression of P2Y1 receptor from both cerebrum of adult rat and cultured cortical neuron was determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The P2Y1 transcript, among other P2Y receptor subtypes, were predominantly expressed in both cerebrum and cultured neurons. During the differentiation of cultured cortical neurons, the expression of P2Y1 receptor, recognized by an anti-P2Y1 receptor antibody at ∼63 kDa, was increased over 5-fold from day in vitro (DIV) 5 to the maximal expression on DIV 20. By using specific antibodies, the immunohistofluorescent staining studies revealed that P2Y1 receptor was highly expressed in the neurons rather than in the glia. In addition, P2Y1 receptor was partially co-localized with a post-synaptic marker, PSD-95, in the cultured cortical neurons. To demonstrate the interaction between P2Y1 receptor and PSD-95, a co-immunoprecipitation approach was applied. The receptor was shown to be associated with PSD-95 via a DTSL-PDZ interaction. This P2Y1-PSD-95 association recruited a membrane localization of PSD-95. By mutation or deletion in the C-terminal of P2Y1 receptor, the specific interaction with PSD-95 was abolished and, subsequently, PSD-95 was no longer localized at the membrane.; In cultured cortical neurons, the cell signaling mechanism(s) induced by P2Y1 receptor was revealed by application of different P2Y 1 receptor agonists. Activation of the receptor elevated intracellular Ca2+ level and activated mitogen-activated protein (MAP) kinase signaling cascade, which resulted in the phosphorylations of its downstream signaling molecules including extracellular signal-regulated kinase (ERK) and cAMP-responsive element binding protein (CREB). The P2Y1-induced cell signaling could lead to the activation of specific gene transcription in cultured cortical neurons. Representative genes from both cholinergic and glutamatergic systems were chosen to study in this P2Y1 receptor-mediated effect. The activation of P2Y1 receptor up-regulated the gene expressions of AChE and NR2A (a NMDA receptor subunit). In addition, the promoters of human ACHE gene and mouse NR2A gene were tagged with a luciferase reporter, respectively; the activities of these promoters were induced by the activation of P2Y1 receptor in the cultured neurons. The gene activations were considered to be mediated by two distinctive transcription factors, Elk-1 and CREB. In accord to this notion, a mutation in either of these regulatory factor binding sites on the promoters abolished the P2Y1-mediated response.; In summary, the results showed the expression and the restricted cellular localization of P2Y1 receptors in cultured cortical neurons. Activation of the P2Y1 receptors contributes to the control of the gene expression of several synaptic effector proteins, such as AChE and NR2A. These findings further illustrate the trophic role of ATP during the formation and maintenance of the neuron to neuron synapses in the CNS.