Author Report for: Xie HQ

Acetylcholinesterase (AChE, EC 3.1.1.7) plays important roles in cholinergic neurotransmission and has been widely recognized as a biomarker for monitoring pollution by organophosphate (OP) and carbamate pesticides. Dioxin is an emerging environmental AChE disruptor and is a typical persistent organic pollutant with multiple toxic effects on the nervous system. Growing evidence has shown that there is a significant link between dioxin exposure and neurodegenerative diseases and neurodevelopmental disorders, most of which involve AChE and cholinergic dysfunctions. Therefore, an in-depth understanding of the effects of dioxin on AChE and the related mechanisms of action might help to shed light on the molecular bases of dioxin impacts on the nervous system. In the past decade, the effects of dioxins on AChE have been revealed in cultured cells of different origins and in rodent animal models. Unlike OP and carbamate pesticides, dioxin-induced AChE disturbance is not due to direct inhibition of enzymatic activity; instead, dioxin causes alterations of AChE expression in certain models. As a widely accepted mechanism for most dioxin effects, the aryl hydrocarbon receptor (AhR)-dependent pathway has become a research focus in studies on the mechanism of action of dioxin-induced AChE dysregulation. In this mini-review, the effects of dioxin on AChE and the diverse roles of the AhR pathway in AChE regulation are summarized. Additionally, the involvement of AhR in AChE regulation during different neurodevelopmental processes is discussed. These AhR-related findings might also provide new insight into AChE regulation triggered by diverse xenobiotics capable of interacting with AhR.

Dioxin exposure is reported to affect nervous system development and increase the risk of neurodegenerative diseases. Generally, dioxin exerts its neurotoxicity via aryl hydrocarbon receptor (AhR). Neurofilament (NF) light (NFL) protein is a biomarker for both neuronal differentiation and neurodegeneration and its expression is controlled by the mitogen-activated protein kinase (MAPK) pathway. However, the effects of dioxin on NFL expression and involved mechanisms are incompletely understood. We aimed to investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on NFL expression and elucidate the underlining signaling pathways and their potential crosstalk, specifically between MAPK and AhR pathway. We employed primary cultured rat cortical neurons to evaluate the effect of TCDD exposure on NFL expression. We also used nerve growth factor (NGF)-treated PC12 cells with specific inhibitors to investigate the involvement of and potential crosstalk between the MAPK pathway and the AhR pathway in mediating the effects of TCDD on NFL expression. After TCDD exposure, NFL mRNA and protein levels were upregulated in cultured neurons. NFL protein was preferentially found in the cell body compared with neurites of the cultured neurons. In PC12 cells, TCDD enhanced both NGF-induced NFL expression and phosphorylation of ERK1/2 and p38. The addition of MAPK-pathway inhibitors (PD98059 and SB230580) partially blocked the TCDD-induced NFL upregulation. CH223191, an AhR antagonist, reversed the upregulation of NFL and phosphorylation of ERK1/2 and p38 induced by TCDD. This study demonstrated TCDD-induced upregulation of NFL in cultured neurons, with protein retained in the cell body. TCDD action was dependent on activation of AhR and MAPK, while crosstalk was found between these two signaling pathways.

Emerging evidence suggests that perinatal dioxin exposure affects neurodevelopment and impairs multiple brain functions, including cognitive, language, learning and emotion, in the offspring. However, the impacts of gestational and lactational exposure to dioxin on behavior and related molecular events are still not fully understood. In this study, female C57BL/6J mice were orally administered three doses of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) (0.1 or 10 mug/kg body weight (bw)) during the pregnancy and lactation periods. The locomotion, exploration and anxiety-related behaviors were examined by an open field test of the young adult female offspring at postnatal day 68. We found that the maternal TCDD exposure, particularly at a low dose, increased movement ability, novelty-exploration and certain anxiety-related behaviors in the offspring. Such hyperactivity-like behaviors were accompanied by the upregulation of certain genes associated with cholinergic neurotransmission or synaptogenesis in the offspring brain. In accordance with the potential enhancement of cholinergic neurotransmission due to the gene upregulations, the enzymatic activity of acetylcholinesterase was decreased, which might lead to excess acetylcholine and consequent hyper-excitation at the synapses. Thus, we found that gestational and lactational TCDD exposure at low dose caused hyperactivity-like behaviors in young adult female offspring and speculated the enhancement of cholinergic neurotransmission and synaptogenesis as potential molecular events underlying the neurobehavioral effects.

Emerging data indicate that prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could interfere with myogenic differentiation in vivo. Acetylcholinesterase (EC3.1.1.7; AChE), an enzyme critical for cholinergic neurotransmission, is abundantly expressed in neurons and mature myotubes, and we recently found that muscle AChE expression was suppressed in parallel with the inhibition of myogenic differentiation upon TCDD treatment in mouse C2C12cells. This TCDD-induced suppression of muscle AChE was proposed to involve an aryl hydrocarbon receptor (AhR)-independent mechanism, but the precise underlying mechanism remains unclear. Considering the widely recognized role of muscular activity in AChE expression and its potential crosstalk with the AhR signaling pathway, we sought to investigate the effect of TCDD on muscle AChE expression in the presence of muscular activity. Therefore, we employed a highly contractile rat primary skeletal muscle culture system in which AChE activity and the expression of genes related to it (AChE T subunit and collagen Q (ColQ)) were increased during the myogenic differentiation process. Although TCDD treatment successfully induced the expression of genes regulated by AhR activation, the treatment exerted no notable effects on myogenic differentiation. Moreover, muscle AChE enzymatic activity and mRNA level remained unchanged following TCDD treatment, and only ColQ mRNA expression was slightly increased after 4-day treatment with TCDD (10(-10)M). The compensatory role of muscle-contraction-related signaling pathways in this newly identified unresponsiveness of muscle AChE to TCDD warrants further investigation.

Acetylcholinesterase (EC3.1.1.7; AChE) is a key enzyme in the cholinergic system. Emerging evidence has shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a typical persistent organic pollutant, suppressed neuronal AChE activity via dysregulation of different biosynthesis processes in human and rat neuronal cells. In the nervous system, astrocytes protect neurons from environmental pollutants. As a known target cell of TCDD, the astrocyte might be involved in TCDD effects on neuronal AChE. Therefore, in the present study, we found astrocyte-derived conditioned medium (ACM) could induce AChE activity preferentially in mature neurons in the absence of TCDD. The enzymatic activity of AChE was generally decreased in cultured cortical neurons upon direct treatment with TCDD (0.003-0.01nM). This trend of changes in AChE activity was not significantly altered in immature neurons exposed to ACM produced in the presence of TCDD (TACM group), but reversed in mature neurons. Compared with effects of treatment with ACM plus TCDD (ACMT), a significant differential effect on AChE activity was found in the TACM group in response to TCDD treatment specifically in immature neurons, suggesting the presence of a TCDD-specific active component derived from the astrocyte. Inconsistent alterations in expression and enzymatic activities of the AChE T subunit (AChET) and the proline-rich membrane anchor (PRiMA) were found, suggesting that a mechanism of action beyond the transcriptional level might be involved. These data indicate that the astrocyte might play a protective role in TCDD-induced alterations of neuronal AChE in certain stages of differentiation.

Acetylcholinesterase (AChE, EC 3.1.1.7) is a classical biomarker for monitoring contamination and intoxication of organophosphate (OP) and carbamate pesticides. In addition to these classical environmental AChE inhibitors, other organic toxic substances have been found to alter AChE activity in various species. These emerging organic AChE disruptors include certain persistent organic pollutants (POPs), polycyclic aromatic hydrocarbons (PAHs), and wildly used chemicals, most of which have received considerable public health concern in recent years. It is necessary to re-evaluate the environmental significances of AChE in terms of these toxic substances. Therefore, the present review is aiming to summarize correlations of AChE activity of certain organisms with the level of the contaminants in particular habitats, disruptions of AChE activity upon treatment with the emerging disruptors in vivo and in vitro, and action mechanisms underlying the effects on AChE. Over 40 chemicals belonging to six main categories were reviewed, including 12 POPs listed in the Stockholm Convention. AChE activity in certain organisms has been found to be well correlated with the contamination level of certain persistent pesticides and PAHs in particular habitats. Moreover, it has been documented that most of the listed toxic chemicals could inhibit AChE activity in diverse species ranging from invertebrates to mammals. Besides directly inactivating AChE, the mechanisms in terms of interference with the biosynthesis have been recognized for some emerging AChE disruptors, particularly for dioxins. The collected evidence suggests that AChE could serve as a potential biomarker for a diverse spectrum of organic environmental pollutants.

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 C2C12cells 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.

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.

Acetylcholinesterase (AChE; EC 3.1.1.7) is a vital functional enzyme in cholinergic neurotransmission which can rapidly hydrolyze the neurotransmitter, acetylcholine, in the central and peripheral nervous systems. Emerging evidence showed that in addition to classical environmental AChE inhibitors, such as 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 affect gene expression through multiple 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 alterations, additional potential signaling cascades and epigenetic mechanisms are proposed for dioxin-induced changes in neuronal AChE. Mitogen activated protein (MAP) kinase, 3'-5'-cyclic adenosine monophosphate (cAMP) and calcium-related 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 in 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.

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.

Recently, acetylcholinesterase (AChE, EC 3.1.1.7) has received increased attention in the field of environmental sciences. Evaluation of the effects of environmental contaminants on AChE enzymatic activity not only can reflect, to some extent, the interference with the nervous system, but also can be used for monitoring pollution. Our previous study showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) suppressed neuronal AChE enzymatic activity via transcriptional downregulations mediated by aryl hydrocarbon receptor. In the present study, the effects of several other dioxin-like compounds (DLCs) on neuronal AChE activity were determined, including 1,2,3,7,8-pentachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, 2,3,4,7,8-pentachlorodibenzofuran, and 2,3,7,8-tetrabromodibenzo-p-dioxin. The results showed that the enzymatic activity of AChE was significantly decreased by approximately 15-30 % after exposure to a certain concentrations of the DLCs, whereas incubating neuronal cell lysates directly with these DLCs did not inhibit AChE enzyme. Subsequent molecular mechanism study showed that these chemicals could decrease ACHE promoter activity, as well as AChE T mRNA expression, thereby suggesting the involvements of transcriptional regulation in these effects. These findings on DLCs are similar with those on 2,3,7,8-TCDD, pointing to the possibility that exposure to dioxin and DLCs, which frequently coexist in the contaminated environments, may concurrently interfere with the cholinergic functions via AChE.

Cholinesterases (ChEs) have been identified in vertebrates and invertebrates. Inhibition of ChE activity in invertebrates, such as bivalve molluscs, has been used to evaluate the exposure of organophosphates, carbamate pesticides, and heavy metals in the marine system. The golden apple snail (Pomacea canaliculata) is considered as one of the worst invasive alien species harmful to rice and other crops. The ChE(s) in this animal, which has been found recently, but poorly characterized thus far, could serve as biomarker(s) for environmental surveillance as well as a potential target for the pest control. In this study, the tissue distribution, substrate preference, sensitivity to ChE inhibitors, and molecular species of ChEs in P. canaliculata were investigated. It was found that the activities of both AChE and BChE were present in all test tissues. The intestine had the most abundant ChE activities. Both enzymes had fair activities in the head, kidney, and gills. The BChE activity was more sensitive to tetra-isopropylpyrophosphoramide (iso-OMPA) than the AChE. Only one BChE molecular species, 5.8S, was found in the intestine and head, whereas two AChE species, 5.8S and 11.6S, were found there. We propose that intestine ChEs of this snail may be potential biomarkers for manipulating pollutions.

Background: Deficits in cognitive functioning have been reported in humans exposed to dioxins and dioxin-like compounds. Evidence suggests that dioxins induce cholinergic dysfunction mediated by hypothyroidism. However, little is known about direct effects of dioxins on the cholinergic system.Objectives: We investigated the action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on acetylcholinesterase (AChE), a key enzyme in cholinergic neurotransmission.Methods: We used SK-N-SH human-derived neuronal cells to evaluate the effect of dioxin exposure on AChE.Results: We consistently found a significant decrease in enzymatic activity of AChE in cultured neurons treated with TCDD. We also found that, unlike organophosphate pesticides that directly act on the catalytic center of AChE, the suppressive effect of dioxin was through transcriptional regulation. The addition of CH223191, an inhibitor of the aryl hydrocarbon receptor (AhR)-dependent pathway, counteracted the TCDD-induced suppression of AChE, suggesting involvement of the AhR-dependent pathway. The existence of putative dioxin-responsive element (DRE) consensus sequences in the human ACHE promoter region further supported this hypothesis. Consistent with the absence of DRE elements in mouse or rat ACHE promoter regions, suppression of AChE by TCDD did not occur in rat neuronal cells, indicating a potential species-specific effect.Conclusions: In SK-N-SH cells, dioxin suppressed the activity of neuronal AChE via AhR-mediated transcriptional down-regulation. This is the first study to report direct interference by dioxin with the cholinergic neurotransmission system.

Fo Shou San (FSS) is an ancient herbal decoction comprised of Rhizoma Chuanxiong (RC; Chuanxiong) and Radix Angelicae Sinensis (RAS; Danggui) in a ratio of 2 : 3. It is mainly prescribed for patients having a blood deficiency. This combination is considered the most popular herb pair among Chinese medicines; however, the rationale of having these two chemically similar herbs within the decoction has historically not been made clear. Here, we attempted to reveal the chemical and biological properties of this decoction as a means to deduce its mechanism of action. The effects of FSS were determined in different cell culture models. With respect to stimulation of blood circulation, FSS inhibited ADP-mediated platelet aggregation in a dose-dependent manner. In order to reveal the hematopoietic effect of this decoction, FSS was applied onto cultured K562 human leukemia cells and Hep3B human hepatocellular carcinoma cells. Application of FSS in cultured K562 cells inhibited cell proliferation and subsequently induced the production of hemoglobin. Additionally, the mRNA expression of erythropoietin (EPO) was induced in a dose-dependent manner when FSS was applied to Hep3B cells. The current results reveal the effects of FSS in different cell models, paving a direction for mechanistic studies.

Heat shock response, an induced transcription of a set of genes in response to high temperature, occurs in all organisms. In neurons, the catalytic subunit of acetylcholinesterase (AChE(T)) interacts with proline-rich membrane anchor (PRiMA) to form a globular tetrameric form (G(4) form). In this study, we examined the effects of heat shock on the transcription and protein assembly of AChE(T) in cultured NG108-15 cells. The transcription of AChE(T) was rapidly induced by heat shock at 40 degrees C, reaching a 15-fold increase in 3h and decreasing thereafter. On the other hand, the level of PRiMA mRNA was not affected after the heat shock. In parallel with AChE(T) mRNA, the enzymatic activity of cellular AChE, in terms of G(1) and G(2) forms, was increased after heat shock; however, the PRiMA-linked G(4) remained unchanged. These results suggest that heat shock can induce the expression level of AChE(T) by the regulation of AChE(T) transcripts in NG108-15 cells.

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.

Acetylcholinesterase (AChE) inhibitors are widely used for the treatment of Alzheimer's disease (AD). Several AChE inhibitors, e.g. rivastigmine, galantamine and huperzine are originating from plants, suggesting that herbs could potentially serve as sources for novel AChE inhibitors. Here, we searched potential AChE inhibitors from flavonoids, a group of naturally occurring compounds in plants or traditional Chinese medicines (TCM). Twenty-one flavonoids, covered different subclasses, were tested for their potential function in inhibiting AChE activity from the brain in vitro. Among all the tested flavonoids, galangin, a flavonol isolated from Rhizoma Alpiniae Officinarum, the rhizomes of Alpiniae officinarum (Hance.) showed an inhibitory effect on AChE activity with the highest inhibition by over 55% and an IC(50) of 120 microM and an enzyme-flavonoid inhibition constant (K(i)) of 74 microM. The results suggest that flavonoids could be potential candidates for further development of new drugs against AD.

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.

Acetylcholinesterase (AChE) is well known to process different molecular forms via the distinct interacting partners. Proline-rich membrane anchor (PRiMA)-linked tetrameric globular AChE (G4 AChE) is mainly found in the vertebrate brain; however, recent studies from our laboratory have suggested its existence at neuromuscular junctions (nmjs). Both muscle and motor neuron express AChE at the nmjs. In muscle, the expression of PRiMA-linked AChE is down-regulated during myogenic differentiation and by motor neuron innervation. As compared with muscle, spinal cord possessed higher total AChE activity and contained PRiMA-linked AChE forms. The spinal cord expression of this form increased during development. More importantly, PRiMA-linked G4 AChE identified as aggregates localized at nmjs. These findings suggest that the restricted localization of PRiMA-linked G4 AChE at the nmjs could be contributed by the pre-synaptic motor neuron and/or the post-synaptic muscle fiber.

Acetylcholinesterase (AChE), a highly polymorphic enzyme with various splicing variants and molecular isoforms, plays an essential role in the cholinergic neurotransmission by hydrolyzing acetylcholine into choline and acetate. The AChE(T) variant is expressed in the brain and muscle: this subunit forms non-amphiphilic tetramers with a collagen tail (ColQ) as asymmetric AChE (A(12) AChE) in muscle, and amphiphilic tetramers with a proline-rich membrane anchor (PRiMA) as globular AChE (G(4) AChE) in the brain and muscle. During the brain development, the expression of amphiphilic G(4) AChE is up regulated and becomes the predominant form of AChE there. This up-regulation of G(4) AChE can be attributed to the increased expressions of both AChE(T) and PRiMA. A significant portion of this membrane-bound G(4) AChE is localized at the membrane rafts of the cell membranes derived from the brain. This raft association could be directed by PRiMA via its CRAC (cholesterol recognition/interaction amino acid consensus) motif and C-terminus. In cultured cortical neurons and muscles, the PRiMA-linked AChE was clustered and partially co-localized with synaptic proteins. The restricted localizations suggest that the raft association of PRiMA-linked AChE could account for its synaptic localization and function.

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.

ETHNOPHARMACOLOGICAL EVIDENCE: Danggui buxue tang (DBT), a Chinese medicinal decoction that is being commonly used as hematopoietic medicine to treating woman menopausal irregularity, contains two herbs: radix Astragali and radix Angelicae Sinensis. Pharmacological results indicate that DBT can stimulate the production of erythropoietin (EPO), a specific hematopoietic growth factor, in cultured cells. AIM OF THE STUDY: In order to reveal the mechanism of DBT's hematopoietic function, this study investigated the activity of the DBT-induced EPO expression and the upstream regulatory cascade of EPO via hypoxia-induced signaling in cultured kidney fibroblasts (HEK293T). MATERIALS AND
METHODS: DBT-induced mRNA expressions were revealed by real-time PCR, while the change of protein expressions were analyzed by Western blotting. For the analysis of hypoxia-dependent signaling, a luciferase reporter was used to report the transcriptional activity of hypoxia response element (HRE).
RESULTS: The plasmid containing HRE, being transfected into HEK293T, was highly responsive to the challenge of DBT application. To account for the transcriptional activation of HRE, DBT treatment was shown to increase the mRNA and protein expressions of hypoxia-inducible factor-1alpha (HIF-1alpha). In addition, the activation of Raf/MEK/ERK signaling pathway by DBT could also enhance the translation of HIF-1alpha, suggesting the dual actions of DBT in stimulating the EPO expression in kidney cells. CONCLUSION: Our study indicates that HIF pathway plays an essential role in directing DBT-induced EPO expression in kidney. These results provide one of the molecular mechanisms of this ancient herbal decoction for its hematopoietic function.

Danggui Buxue Tang (DBT), a herbal decoction composed of Radix Astragali (RA) and Radix Angelica sinensis (RAS), has been used for treating menopausal irregularity in women for more than 800 years in China. According to the old tradition, RAS had to be processed with yellow wine before DBT preparation, which markedly reduced the amount of ligustilide in RAS and DBT, as well as enhanced the bioactivities of DBT. Here, we hypothesized that ligustilide would be an ingredient that possessed suppressive effects on DBT's functions. In the presence of ligustilide, the amount of astragaloside IV, calycosin, formononetin, and total polysaccharides extracted from RA were decreased. An increase of ligustilide caused a decrease of DBT's osteogenic activity in stimulating proliferation and differentiation of cultured bone cells. In addition, in the presence of a high level of ligustilide, DBT caused a side effect inducing the proliferation of breast MCF-7 cells. The current results strongly suggest that ligustilide is a negative regulator that hinders DBT to achieve its biological efficacy, which supports the traditional practice of preparing DBT using the ethanol-treated RAS.

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 alpha- and beta-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.

The expression and localization of the proline-rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form acetylcholinesterase (G(4) AChE), were studied at vertebrate neuromuscular junctions. Both muscle and motor neuron contributed to this synaptic expression pattern. During the development of rat muscles, the expression of PRiMA and AChE(T) and the enzymatic activity increased dramatically; however, the proportion of G(4) AChE decreased. G(4) AChE in muscle was recognized specifically by a PRiMA antibody, indicating the association of this enzyme with PRiMA. Using western blot and ELISA, both PRiMA protein and PRiMA-linked G(4) AChE were found to be present in large amounts in fast-twitch muscle (e.g. tibialis), but in relatively low abundance in slow-twitch muscle (e.g. soleus). These results indicate that the expression level of PRiMA-linked G(4) AChE depends on muscle fiber type. In parallel, the expression of PRiMA, AChE(T) and G(4) AChE also increased in the spinal cord during development. Such expression in motor neurons contributed to the synaptic localization of G(4) AChE. After denervation, the expression of PRiMA, AChE(T) and G(4) AChE decreased markedly in the spinal cord, and in fast- and slow-twitch muscles.

The mammalian protein CutA was first discovered in a search for the membrane anchor of mammalian brain acetylcholinesterase (AChE). It was co-purified with AChE, but it is distinct from the real transmembrane anchor protein, PRiMA. CutA is a ubiquitous trimeric protein, homologous to the bacterial CutA1 protein that belongs to an operon involved in resistance to divalent ions ("copper tolerance A"). The function of this protein in plants and animals is unknown, and several hypotheses concerning its subcellular localization have been proposed. We analyzed the expression and the subcellular localization of mouse CutA variants, starting at three in-frame ATG codons, in transfected COS cells. We show that CutA produces 20-kDa (H) and 15-kDa (L) components. The H component is transferred into the secretory pathway and secreted, without cleavage of a signal peptide, whereas the L component is mostly cytosolic. We show that expression of the longer CutA variant reduces the level of AChE, that this effect depends on the AChE C-terminal peptides, and probably results from misfolding. Surprisingly, CutA increased the secretion of a mutant possessing a KDEL motif at its C terminus; it also increased the formation of AChE homotetramers. We found no evidence for a direct interaction between CutA and AChE. The longer CutA variant seems to affect the processing and trafficking of secretory proteins, whereas the shorter one may have a distinct function in the cytoplasm.

Proline-rich membrane anchor (PRiMA) is a molecule to organize acetylcholinesterase (AChE) into tetrameric globular form (G(4)) that anchors onto the plasma membrane in brain and muscle. In mammal, PRiMA is encoded by a single gene with two splicing variants, PRiMA I and PRiMA II: PRiMA II is different to PRiMA I by its absence of a C-terminal cytoplasmic domain. The existence of these isoforms has not been revealed in avian specie. By using RT-PCR and bioinformatic analyses, two splicing variants of PRiMA were identified in chicken cerebrum. One variant contains very similar domains as compared to mammalian PRiMA I. The other variant, named as PRiMA II, has a very distinct cytoplasmic C-terminus of having 26 amino acids. Both forms of chicken PRiMA were able to organize the formation of G(4) AChE when that was over expressed together with AChE(T) subunit in cultured cells. The level of PRiMA mRNA, mainly PRiMA I, was higher in slow-twitch muscle than that of in fast-twitch muscle of chicken. This finding suggests that the muscle fiber type-specific expression of G(4) AChE in chicken could be a result of the different expression pattern of PRiMA in fast- and slow-twitch muscles.

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.

The toxicity of aggregated beta-amyloid (A beta) has been implicated as a critical cause in the development of Alzheimer's disease (AD). Hibifolin, a flavonol glycoside derived from herbal plants, possessed a strong protective activity against cell death induced by aggregated A beta. Application of hibifolin in primary cortical neurons prevented the A beta-induced cell death in a dose-dependent manner. In cultured cortical neurons, the pre-treatment of hibifolin abolished A beta-induced Ca(2+) mobilization, and also reduced A beta-induced caspase-3 and caspase-7 activation. Moreover, DNA fragmentation induced by A beta could be suppressed by hibifolin. In addition to such protection mechanisms, hibifolin was able to induce Akt phosphorylation in cortical neurons, which could be another explanation for the neuroprotection activity. These results therefore provided the first evidence that hibifolin protected neurons against A beta-induced apoptosis and stimulated Akt activation, which would be useful in developing potential drugs or food supplements for treating AD.

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 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.

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.

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.

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 (Bt(2)-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 Bt(2)-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 Bt(2)-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.

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).

Acetylcholinesterase (AChE; EC 3.1.1.7) is a highly polymorphic enzyme (Massoulie, 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 (Massoulie, 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 (Massoulie, 2002).

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 (Massoulie, 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.