Predictive Models for Human Organ Toxicity Based on In Vitro Bioactivity Data and Chemical Structure.

Traditional toxicity testing reliant on animal models is costly and low throughput, posing a significant challenge with the increasing numbers of chemicals that humans are exposed to in the environment. The purpose of this investigation was to build optimal prediction models for various human in vivo/organ-level toxicity end points (extracted from ChemIDPlus) using chemical structure and Tox21 in vitro quantitative high-throughput screening (qHTS) bioactivity assay data. Several supervised machine learning algorithms were applied to model 14 human toxicity end points pertaining to vascular, kidney, ureter and bladder, and liver organ systems. Three metrics were used to evaluate model performance: area under the receiver operating characteristic curve (AUC-ROC), balanced accuracy (BA), and Matthews correlation coefficient (MCC). The top four models, with AUC-ROC values >0.8, were derived for endocrine (0.90 ± 0.00), musculoskeletal (0.88 ± 0.02), peripheral nerve and sensation (0.85 ± 0.01), and brain and coverings (0.83 ± 0.02) toxicities, whereas the best model AUC-ROC values were >0.7 for the remaining 10 toxicities. Model performance was found to be dependent on the specific data set, model type, and feature selection method used. In addition, chemical structure and assay data showed different levels of contribution to the prediction of different toxicity end points. Although in vitro assay data, when combined with chemical structure, slightly improved the predictive accuracy for most end points (11 out of 14), a noteworthy finding was the near equal success of the structure-only models, which do not require Tox21 qHTS screening data, and the relatively poor performance of assay-only models. Thus, the top-performing structure-only models from this study could be applied for hazard screening of large sets of chemicals for potential human toxicity, whereas the largest assay contributions to models (i.e., cellular targets) could be used, along with the top-contributing structural features, to provide insight into toxicity mechanisms.

Dioxin induces expression of hsa-miR-146b-5p in human neuroblastoma cells.

Dioxin can cause a series of neural toxicological effects. MicroRNAs (miRs) play important roles in regulating nervous system function and mediating cellular responses to environmental pollutants, such as dioxin. Hsa-miR-146b-5p appears to be involved in neurodegenerative diseases and brain tumors. However, little is known about effects of dioxin on the expression of hsa-miR-146b-5p. We found that the hsa-miR-146b-5p expression and its promoter activity were significantly increased in dioxin treated SK-N-SH cells, a human-derived neuroblastoma cell line. Potential roles of hsa-miR-146b-5p in mediating neural toxicological effects of dioxin may be due to the regulation of certain target genes. We further confirmed that hsa-miR-146b-5p significantly suppressed acetylcholinesterase (AChE) activity and targeted the 3'-untranslated region of the AChE T subunit, which has been down-regulated in dioxin treated SK-N-SH cells. Functional bioinformatic analysis showed that the known and predicted target genes of hsa-miR-146b-5p were involved in some brain functions or cyto-toxicities related to known dioxin effects, including synapse transmission, in which AChE may serve as a responsive gene for mediating the effect.

Identification of differentially expressed genes response to TCDD in rat brain after long-term low-dose exposure.

Several cohort studies have reported that dioxin and dioxin-like polychlorinated biphenyls might impair the nervous system and lead to neurological or neurodegenerative diseases in the elder people, but there is limited research on the involved mechanism. By using microarray analysis, we figured out the differentially expressed genes between brain samples from SD rats after low-dose (0.1µg/(kg▪bw)) dioxin exposure for six months and controls. To investigate the function changes in the course of dioxin exposure, Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on the differentially expressed genes. And the changes of several picked genes have been verified by real-time PCR. A total of 145 up-regulated and 64 down-regulated genes were identified. The metabolic processes, interleukin-1 secretion and production were significantly associated with the differentially expressed genes. And the genes regulated by dioxin also clustered to cholinergic synapse and long-term potentiation. Candidate biomarker genes such as egr1, gad2, gabrb3, abca1, ccr5 and pycard may be toxicological targets for dioxin. Furthermore, synaptic plasticity and neuro-immune system may be two principal affected areas by dioxin.

Targeting acetylcholinesterase to membrane rafts: a function mediated by the proline-rich membrane anchor (PRiMA) in neurons.

In the mammalian brain, acetylcholinesterase (AChE) is anchored in cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor). We present evidence that at least part of the PRiMA-linked AChE is integrated in membrane microdomains called rafts. A significant proportion of PRiMA-linked AChE tetramers from rat brain was recovered in raft fractions; this proportion was markedly higher at low rather than at high concentrations of cold Triton X-100. The detergent-resistant fraction increased during brain development. In NG108-15 neuroblastoma cells transfected with cDNAs encoding AChE(T) and PRiMA, PRiMA-linked G(4) AChE was found in membrane rafts and showed the same sensitivity to cold Triton X-100 extraction as in the brain. The association of PRiMA-linked AChE with rafts was weaker than that of glycosylphosphatidylinositol-anchored G(2) AChE or G(4) Q(N)-H(C)-linked AChE. It was found to depend on the presence of a cholesterol-binding motif, called CRAC (cholesterol recognition/interaction amino acid consensus), located at the junction of transmembrane and cytoplasmic domains of both PRiMA I and II isoforms. The cytoplasmic domain of PRiMA, which differs between PRiMA I and PRiMA II, appeared to play some role in stabilizing the raft localization of G(4) AChE, because the Triton X-100-resistant fraction was smaller with the shorter PRiMA II isoform than that with the longer PRiMA I isoform.

The PRiMA-linked cholinesterase tetramers are assembled from homodimers: hybrid molecules composed of acetylcholinesterase and butyrylcholinesterase dimers are up-regulated during development of chicken brain.

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE(T) and BChE(T) with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q(N-GPI)). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q(N-GPI) always consist of AChE(T) and BChE(T) homodimers. The dimer formation of AChE(T) and BChE(T) depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal "t-peptides" in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE(T) or BChE(T) homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.

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.

Quality assessment of a formulated Chinese herbal decoction, Kaixinsan, by using rapid resolution liquid chromatography coupled with mass spectrometry: A chemical evaluation of different historical formulae.

Kaixinsan is an ancient Chinese herbal decoction mainly prescribed for patients suffering from mental depression. This decoction was created by Sun Si-miao of Tang Dynasty (A.D. 600) in ancient China, and was composed of four herbs: Radix and Rhizome Ginseng, Radix Polygalae, Rhizoma Acori Tatarinowii and Poria. Historically, this decoction has three different formulations, each recorded at a different point in time. In this study, the chemical compositions of all three Kaixinsan formulae were analyzed. By using rapid resolution LC coupled with a diode-array detector and an ESI triple quadrupole tandem MS (QQQ-MS/MS), the Radix and Rhizome Ginseng-derived ginsenosides including Rb(1), Rd, Re, Rg(1), the Radix Polygalae-derived 3,6'-disinapoyl sucrose, the Rhizoma Acori Tatarinowii-derived α- and ß-asarone and the Poria-derived pachymic acid were compared among the three different formulations. The results showed variations in the solubility of different chemicals between one formula and the others. This systematic method developed could be used for the quality assessment of this herbal decoction.

Estrogenic and neuroprotective properties of scutellarin from Erigeron breviscapus: a drug against postmenopausal symptoms and Alzheimer's disease.

Besides the classical hormonal effect, estrogen possesses neuroprotective effects in the brain, which leads to the searching of novel treatments for neurodegenerative diseases such as Alzheimer's disease. Scutellarin is a major flavone derived from Herba Erigerontis, a Chinese medicine derived from Erigeron breviscapus, which has been shown here to possess both estrogenic and neuroprotective properties. Scutellarin showed the estrogenic effects by activating the estrogen responsive elements and phosphorylation of estrogen receptor alpha in cultured MCF-7 cells: the activation was in a dose-dependent manner. On the other hand, scutellarin inhibited the aggregation of beta-amyloid in vitro, and prevented the cell death mediated by beta-amyloid when applied to cultured neuronal PC12 cells. These results therefore suggested that Herba Erigerontis and its component scutellarin might have therapeutic effects against postmenopausal symptoms and Alzheimer's disease.

Myocyte enhancer factor 2 mediates acetylcholine-induced expression of acetylcholinesterase-associated collagen ColQ in cultured myotubes.

The collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase (AChE) in vertebrate muscles. Two ColQ transcripts, ColQ-1 and ColQ-1a, driven by two distinct promoters are expressed differentially in mammalian slow- and fast-twitch muscles, respectively. Such expression patterns are determined by the contractile activity in different muscle fiber types. To reveal the regulatory role of muscular activity on ColQ expression, acetylcholine and nicotine were applied onto C2C12 muscle cells: the challenge increased the expression of ColQ-1/ColQ-1a mRNAs. The agonist challenge induced the phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In parallel, over expression of an active mutant of CaMKII enhanced both ColQ-1/ColQ-1a mRNA levels in cultured C2C12 myotubes. Moreover, the over expression of myocyte enhancer factor 2 (MEF2), a downstream mediator of CaMKII, in the myotubes potentiated the CaMKII-induced ColQ expression. The current results reveal a signaling cascade that drives the expression profiles of ColQ in responding to activity challenge in cultured myotubes.

Regulation of PRiMA-linked G(4) 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.

The promoter activity of proline-rich membrane anchor (PRiMA) of globular form acetylcholinesterase in muscle: suppressive roles of myogenesis and innervating nerve.

The tetrameric globular form of acetylcholinesterase (G(4) AChE) is present and precisely controlled in muscles. The assembly and membrane targeting of G(4) AChE are directed by a proline-rich membrane anchor (PRiMA). It has been demonstrated that in muscle cells, the expression of PRiMA mRNA, as well as the level of G(4) AChE was suppressed by myogenesis and innervating nerve. A human PRiMA promoter-driven luciferase reporter was employed in this study to further reveal the activity of PRiMA transcription during myogenic differentiation and the influence of innervation. In parallel with PRiMA mRNA, the PRiMA promoter activity was suppressed by both myogenic regulatory factor(s) (MRFs) and nerve-derived factor(s). These results suggest that the regulation of PRiMA mRNA expression in muscle by MRFs and nerve-derived factors is due to a control system at the transcriptional level.

Transcriptional control of different subunits of AChE in muscles: signals triggered by the motor nerve-derived factors.

Acetylcholinesterase (AChE) is a highly polymorphic enzyme. Alternative splicing in the 3' region of the primary transcript generates different subunits that contain the same catalytic domain but with distinct carboxyl termini. In mammals, the AChE(R) variant produces a soluble monomer that is up-regulated in the brain during stress. The AChE(H) variant produces a GPI-anchored dimer that is mainly expressed in blood cells, while AChE(T) variant is largely predominant in the brain and muscle. AChE(T) subunits associate with a collagen tail subunit (ColQ) forming asymmetric AChE species (A(4), A(8), and A(12) AChE) in muscle, and also form amphiphilic tetramers associated with a proline-rich membrane anchor (PRiMA) as globular AChE (G(4) AChE) in brain and muscle. The formation of these AChE forms depends on the physiological status of the muscles, and on the innervating nerves. The motor nerves achieve this regulation by two distinct mechanisms: release of the trophic factor calcitonin gene-related peptide (CGRP) and nerve-evoked electrical activity, which differentially regulate the expression levels of AChE(T), PRiMA and ColQ via different downstream signaling cascades. The regulatory mechanisms provided by the nerve are important to account for the different expression patterns of AChE and associated proteins in fast- and slow-twitch muscles.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces alterations in myogenic differentiation of C2C12 cells.

Dioxin-induced toxicities that affect the development of the motor system have been proposed since many years. However, cellular evidence and the molecular basis for the effects are limited. In this study, a cultured mouse myoblast cell line, C2C12, was utilized to examine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on myogenic differentiation and expression of acetylcholinesterase (AChE), a neuromuscular transmission-related gene. The results showed that TCDD exposure at 10-10 M repressed the myotube formation of C2C12 cells by disturbing the fusion process and suppressing the expression of myosin heavy chain, a myobute structural protein, and not by induction of cytotoxicity. Furthermore, TCDD dose dependently suppressed the transcriptional expression and enzymatic activity of AChE during the myogenic differentiation, particularly in the middle stage. However, the administration of aryl hydrocarbon receptor antagonists, CH223191 and alpha-naphthoflavone, did not completely reverse the TCDD-induced downregulation of muscular AChE during myogenic differentiation. These findings suggest that low dose exposure to dioxin may result in disturbances of muscle differentiation and neuromuscular transmission.

CDC42 expression is altered by dioxin exposure and mediated by multilevel regulations via AhR in human neuroblastoma cells.

Emerging evidence has shown that dioxin causes dysregulation of microRNAs (miRs) in a variety of tissues or cells. However, little is known about dioxin effects on neuronal miRs expression. In the present study, 277 differentially expressed miRs were identified by miRs microarray analysis in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, at 10-10 M) treated SK-N-SH neuroblastoma cells. Among them, 53 miRs exhibited changes of more than 0.4-fold. Consistent with the microarray data, we verified the induction effect of TCDD on hsa-miR-608 expression, which is a primate-specific miR associated with brain functions. Bioinformatics analysis showed involvement of hsa-miR-608 in cytoskeleton organization, in which one of the hsa-miR-608 target genes, Cell Division Cycle 42 (CDC42), might play a role. We also confirmed induction of CDC42 expression by TCDD in SK-N-SH cells. TCDD induced the expression of CDC42 mRNA in hsa-miR-608 inhibitor transfected cells more obviously than in control cells, suggesting involvement of both transcriptional and post-transcriptional mechanisms in the TCDD-induced CDC42 regulation. Furthermore, CH223191, an antagonist of the aryl hydrocarbon receptor (AhR), counteracted TCDD-induced hsa-miR-608 and CDC42 expression. These results indicated that AhR not only mediates transcriptional induction of CDC42, but also hsa-miR-608-induced post-transcriptional regulation of CDC42 in dioxin treated neuroblastoma cells.

Blockade of IDO-kynurenine-AhR metabolic circuitry abrogates IFN-γ-induced immunologic dormancy of tumor-repopulating cells.

Interactions with the immune system may lead tumorigenic cells into dormancy. However, the underlying molecular mechanism is poorly understood. Using a 3D fibrin gel model, we show that IFN-γ induces tumour-repopulating cells (TRCs) to enter dormancy through an indolamine 2,3-dioxygenase 1 (IDO1)-kynurenine (Kyn)-aryl hydrocarbon receptor (AhR)-p27 dependent pathway. Mechanistically, IFN-γ signalling triggers differentiated tumour cell apoptosis via STAT1; however, when IDO1 and AhR are highly expressed as in TRCs, IFN-γ results in IDO1/AhR-dependent p27 induction that prevents STAT1 signalling, thus suppressing the process of cell death and activating the dormancy program. Blocking the IDO/AhR metabolic circuitry not only abrogates IFN-γ-induced dormancy but also results in enhanced repression of tumour growth by IFN-γ-induced apoptosis of TRCs both in vitro and in vivo. These data present a previously unrecognized mechanism of inducing TRC dormancy by IFN-γ, suggesting a potential effective cancer immunotherapeutic modality through the combination of IFN-γ and IDO/AhR inhibitors.

Chick acetylcholinesterase promoter regulation.

In vertebrate neuromuscular junction, acetylcholinesterase (AChE) is colocalized with acetylcholine receptor (AChR). This synaptic expression of AChE requires precise regulation of the AChE gene. However, the gene regulation pattern has species variation. Previous studies (Massoulié, 2002) indicated that AChE activities in muscles decreased in rat but increased in chicken after denervation. The spatial arrangement of regulatory elements in promoters among animals therefore might be varied. The genomic structures of AChE have been analyzed in Torpedo, mouse, rat, and human but not in chick, and the molecular mechanism(s) responsible for contrary regulation of AChE between chick and mammal has been proposed (Choi et al., 2001) but not fully understood. Here, we report the cloning of the chick AChE promoter, the regulation of which is being characterized.

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).

New perspectives for multi-level regulations of neuronal acetylcholinesterase by dioxins.

Acetylcholinesterase (AChE; EC 3.1.1.7) is a vital functional enzyme in cholinergic neurotransmission which can rapidly hydrolyze neurotransmitter, acetylcholine, in the central and peripheral nervous systems. Emerging evidence showed that in addition to classical environmental AChE inhibitors, e.g. organophosphate and carbamate pesticides, dioxins are a new type of xenobiotic causing impairment of AChE. Dioxin can transcriptionally or post-transcriptionally suppress AChE expression in human neuroblastoma cells or mouse immune cells via the aryl hydrocarbon receptor (AhR) pathway, respectively. Dioxins can affect gene expression through other mechanisms, such as cross-talk with other signaling cascades and epigenetic modulations. Therefore, in this review, by summarizing the known mechanisms of AChE regulation and dioxin-induced gene alteration, potential signaling cascades and epigenetic mechanisms are proposed for dioxin-mediated AChE regulation. Mitogen activated protein (MAP) kinase, 3', 5'-cyclic adenosine monophosphate (cAMP) and calcium-related singaling pathways, as well as potential epigenetic mechanisms, such as DNA methylation, and post-transcriptional regulation via microRNAs, including hsa-miR-132, hsa-miR-212 and hsa-miR-25-3p are discussed here. These proposed mechanisms may be invaluable not only to promote comprehensive understanding of the action mechanisms for dioxin, but to illustrate the molecular basis of dioxin-induced health impacts.

2,3,7,8-Tetrachlorodibenzo-p-dioxin suppress AChE activity in NGF treated PC12 cells.

PC12 is a well studied cell model for neuronal differentiation. AChE is also considered as a marker for neuronal differentiation. In this study, we detected the change of AChE activity during the NGF induced differentiation of PC 12 cells, and targeted on the ratio of the activity of AChE on the cell surface, and found that NGF mainly increased the intracellular AChE activity. Dioxin is a kind of persistent organic pollutants which have extreme impact on human health and widely distributed all over the world. Recently, AChE was reported as a target of the toxicity of dioxin. Here we investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on AChE activity in the PC12 cells, and found that at the later stage of differentiation, TCDD could decrease the AChE activity. This down regulation might not related to transcriptional regulation.

ATP induces the post-synaptic gene expression in neuron-neuron synapses: Transcriptional regulation of AChE catalytic subunit.

Adenosine 5'-triphosphate (ATP) is an important trophic factor, which is co-stored and co-released at central and peripheral cholinergic synapses. The synaptic ATP induces post-synaptic gene transcription during the formation and maintenance of vertebrate neuromuscular junction (nmj) via a mitogen-activaton protein (MAP) kinase signaling pathway and subsequently activates acetylcholinesterase (AChE) and acetylcholine receptor (AChR) genes. However, the role of ATP in the central nervous system is still not clear. Primary culture of rat cortical neurons was used as a model system to study the biological functions of ATP in neuron-neuron synapses. During the differentiation of cultured cortical neurons, the protein levels of AChE and one of the ATP receptor subtypes, P2Y1 receptor, were increased. By using a human AChE promoter tagged with a luciferase-reporter gene, the transcriptional regulation of AChE gene by ATP could be monitored. The activation of P2Y1 receptors could regulate the AChE promoter activity in cultured cortical neurons. These results suggested the activation of P2Y receptors may play role(s) in synaptic gene expression of neuron-neuron synapses in the brain.