RESUMEN
The non-classical function of acetylcholine (ACh) has been reported in neuroinflammation that represents the modulating factor in immune responses via activation of α7 nicotinic acetylcholine receptor (α7 nAChR), i.e., a cholinergic anti-inflammatory pathway (CAP). Acetylcholinesterase (AChE), an enzyme for ACh hydrolysis, has been proposed to have a non-classical function in immune cells. However, the involvement of AChE in neuroinflammation is unclear. Here, cultured BV2 cell, a microglial cell line, and primary microglia from rats were treated with lipopolysaccharide (LPS) to induce inflammation and to explore the regulation of AChE during this process. The expression profiles of AChE, α7 nAChR, and choline acetyltransferase (ChAT) were revealed in BV2 cells. The expression of AChE (G4 form) was induced significantly in LPS-treated BV2 cells: the induction was triggered by NF-κB and cAMP signaling. Moreover, ACh or α7 nAChR agonist suppressed the LPS-induced production of pro-inflammatory cytokines, as well as the phagocytosis of microglia, by activating α7 nAChR and followed by the regulation of NF-κB and CREB signaling. The ACh-induced suppression of inflammation was abolished in AChE overexpressed cells, but did not show a significant change in AChE mutant (enzymatic activity knockout) transfected cells. These results indicate that the neuroinflammation-regulated function of AChE may be mediated by controlling the ACh level in the brain system.
Asunto(s)
Acetilcolinesterasa/metabolismo , Lipopolisacáridos/toxicidad , Microglía/metabolismo , Acetilcolinesterasa/genética , Animales , Línea Celular , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ratones , Microglía/efectos de los fármacos , FN-kappa B/metabolismo , Fagocitosis , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Acetylcholinesterase inhibitors (AChEIs), the most developed treatment strategies for Alzheimer's disease (AD), will be used in clinic for, at least, the next decades. Their side effects are in highly variable from drug to drug with mechanisms remaining to be fully established. The withdrawal of tacrine (Cognex) in the market makes it as an interesting case study. Here, we found tacrine could disrupt the proper trafficking of proline-rich membrane anchor-linked tetrameric acetylcholinesterase (AChE) in the endoplasmic reticulum (ER). The exposure of tacrine in cells expressing AChE, e.g., neurons, caused an accumulation of the misfolded AChE in the ER. This misfolded enzyme was not able to transport to the Golgi/plasma membrane, which subsequently induced ER stress and its downstream signaling cascade of unfolded protein response. Once the stress was overwhelming, the cooperation of ER with mitochondria increased the loss of mitochondrial membrane potential. Eventually, the tacrine-exposed cells lost homeostasis and underwent apoptosis. The ER stress and apoptosis, induced by tacrine, were proportional to the amount of AChE. Other AChEIs (rivastigmine, bis(3)-cognitin, daurisoline, and dauricine) could cause the same problem as tacrine by inducing ER stress in neuronal cells. The results provide guidance for the drug design and discovery of AChEIs for AD treatment. SIGNIFICANCE STATEMENT: Acetylcholinesterase inhibitors (AChEIs) are the most developed treatment strategies for Alzheimer's disease (AD) and will be used in clinic for at least the next decades. This study reports that tacrine and other AChEIs disrupt the proper trafficking of acetylcholinesterase in the endoplasmic reticulum. Eventually, the apoptosis of neurons and other cells are induced. The results provide guidance for drug design and discovery of AChEIs for AD treatment.
Asunto(s)
Acetilcolinesterasa/metabolismo , Apoptosis/efectos de los fármacos , Inhibidores de la Colinesterasa/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Neuronas/efectos de los fármacos , Tacrina/farmacología , Animales , Apoptosis/fisiología , Línea Celular Tumoral , Células Cultivadas , Inhibidores de la Colinesterasa/química , Relación Dosis-Respuesta a Droga , Estrés del Retículo Endoplásmico/fisiología , Células HEK293 , Humanos , Ratones , Simulación del Acoplamiento Molecular/métodos , Neuronas/enzimología , Células RAW 264.7 , Ratas , Ratas Sprague-Dawley , Tacrina/químicaRESUMEN
Stephaniae tetrandrae radix (STR) is a commonly used traditional Chinese medicine in alleviating edema by inducing diuresis. In the clinic, STR extracts or its components are widely used in the treatment of edema, dysuria, and rheumatism for the regulation of water metabolism. Furthermore, STR has been used in treating emotional problems for years by combining with other Chinese herbs. However, the material basis and mechanism of STR on the nervous system have not been revealed. Here, the main components of STR extracts with different extracting solvents were identified, including three major alkaloids, i.e., cyclanoline, fangchinoline, and tetrandrine. The cholinesterase inhibitory activity of STR extracts and its alkaloids was determined using the Ellman assay. Both cyclanoline and fangchinoline showed acetylcholinesterase (AChE) inhibitory activity, demonstrating noncompetitive enzyme inhibition. In contrast, tetrandrine did not show enzymatic inhibition. The synergism of STR alkaloids with huperzine A or donepezil was calculated by the median-effect principle. The drug combination of fangchinoline-huperzine A or donepezil synergistically inhibited AChE, having a combination index (CI) < 1 at Fa = 0.5. Furthermore, the molecular docking results showed that fangchinoline bound with AChE residues in the peripheral anionic site, and cyclanoline bound with AChE residues in the peripheral anionic site, anionic site, and catalytic site. In parallel, cyclanoline bound with butyrylcholinesterase (BChE) residues in the anionic site, catalytic site, and aromatic site. The results support that fangchinoline and cyclanoline, alkaloids derived from STR, could account for the anti-AChE function of STR. Thus, STR extract or its alkaloids may potentially be developed as a therapeutic strategy for Alzheimer's patients.
Asunto(s)
Bencilisoquinolinas/farmacología , Berberina/análogos & derivados , Inhibidores de la Colinesterasa/farmacología , Fármacos Neuroprotectores/farmacología , Stephania tetrandra/química , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Alcaloides/farmacología , Bencilisoquinolinas/aislamiento & purificación , Berberina/aislamiento & purificación , Berberina/farmacología , Sitios de Unión , Butirilcolinesterasa/química , Butirilcolinesterasa/metabolismo , China , Donepezilo/farmacología , Combinación de Medicamentos , Sinergismo Farmacológico , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Extractos Vegetales/química , Plantas Medicinales , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Sesquiterpenos/farmacología , Solventes/químicaRESUMEN
Acetylcholinesterase (AChE) hydrolyzes the neurotransmitter acetylcholine in neurons. However, AChE has been proposed to also have nonneuronal functions in different cell types. Here, we report that AChE is expressed in melanocytes and melanoma cells, and that the tetrameric (G4) form is the major AChE isoform in these cells. During melanogenesis of B16F10 murine melanoma cells, AChE levels decreased markedly. The differentiation of melanoma cells led to (i) an increase in melanin and tyrosinase, (ii) a change in intracellular cAMP levels, and (iii) a decrease in microphthalmia-associated transcription factor (MITF). We hypothesized that the regulation of AChE during melanogenesis is mediated by two transcription factors: cAMP-response element-binding protein (CREB) and MITF. In melanoma cells, exogenous cAMP suppressed AChE expression and the promoter activity of the ACHE gene. This suppression was mediated by a cAMP-response element (CRE) located on the ACHE promoter, as mutation of CRE relieved the suppression. In melanoma, MITF overexpression induced ACHE transcription, and mutation of an E-box site in human ACHE promoter blocked this induction. An AChE inhibitor greatly enhanced acetylcholine-mediated responses of melanogenic gene expression levels in vitro; however, this enhancement was not observed in the presence of agonists of the muscarinic acetylcholine receptor. These results indicate that ACHE transcription is regulated by cAMP-dependent signaling during melanogenesis of B16F10 cells, and the effect of this enzyme on melanin production suggests that it has a potential role in skin pigmentation.
Asunto(s)
Acetilcolinesterasa/metabolismo , Melaninas/biosíntesis , Melanoma Experimental/metabolismo , Factor de Transcripción Asociado a Microftalmía/fisiología , Regulación hacia Arriba/fisiología , Acetilcolina/metabolismo , Acetilcolinesterasa/genética , Animales , Diferenciación Celular , Línea Celular Tumoral , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Melaninas/metabolismo , Melanocitos/citología , Melanocitos/metabolismo , Melanoma Experimental/enzimología , Melanoma Experimental/patología , Ratones , Monofenol Monooxigenasa/metabolismo , Regiones Promotoras GenéticasRESUMEN
Alkaloids having acetylcholinesterase (AChE) inhibitory activity are commonly found in traditional Chinese medicine (TCM); for example, berberine from Coptis chinensis, galantamine from Lycoris radiata, and huperzine A from Huperzia serrata. In practice of TCM, Stephaniae Tetrandrae Radix (STR) is often combined with Coptidis Rhizoma (CR) or Phellodendri Chinensis Cortex (PCC) as paired herbs during clinical application. Fangchinoline from STR and coptisine and/or berberine from CR and/or PCC are active alkaloids in inhibiting AChE. The traditional usage of paired herbs suggests the synergistic effect of fangchinoline-coptisine or fangchinoline-berberine pairing in AChE inhibition. HPLC was applied to identify the main components in herbal extracts of STR, CR, and PCC, and the AChE inhibition of their main components was determined by Ellman assay. The synergism of herb combination and active component combination was calculated by median-effect principle. Molecular docking was applied to investigate the underlying binding mechanisms of the active components with the AChE protein. It was found that fangchinoline showed AChE inhibitory potency; furthermore, fangchinoline-coptisine/berberine pairs (at ratios of 1:5, 1:2, 1:1, and 2:1) synergistically inhibited AChE; the combination index (CI) at different ratios was less than one when Fa = 0.5, suggesting synergistic inhibition of AChE. Furthermore, the molecular docking simulation supported this enzymatic inhibition. Therefore, fangchinoline-coptisine/berberine pairs, or their parental herbal mixtures, may potentially be developed as a possible therapeutic strategy for Alzheimer's patients.
Asunto(s)
Acetilcolinesterasa/metabolismo , Alcaloides/farmacología , Inhibidores de la Colinesterasa/farmacología , Medicamentos Herbarios Chinos/química , Phellodendron/química , Stephania tetrandra/química , Acetilcolinesterasa/química , Alcaloides/química , Bencilisoquinolinas/química , Bencilisoquinolinas/farmacología , Berberina/análogos & derivados , Berberina/química , Berberina/farmacología , Inhibidores de la Colinesterasa/química , Coptis chinensis , Combinación de Medicamentos , Sinergismo Farmacológico , Medicina Tradicional China , Simulación del Acoplamiento Molecular , Extractos Vegetales/químicaRESUMEN
Acetylcholinesterase (AChE) hydrolyzes acetylcholine to terminate cholinergic transmission in neurons. Apart from this AChE activity, emerging evidence suggests that AChE could also function in other, non-neuronal cells. For instance, in bone, AChE exists as a proline-rich membrane anchor (PRiMA)-linked globular form in osteoblasts, in which it is proposed to play a noncholinergic role in differentiation. However, this hypothesis is untested. Here, we found that in cultured rat osteoblasts, AChE expression was increased in parallel with osteoblastic differentiation. Because several lines of evidence indicate that AChE activity in osteoblast could be triggered by Wnt/ß-catenin signaling, we added recombinant human Wnt3a to cultured osteoblasts and found that this addition induced expression of the ACHE gene and protein product. This Wnt3a-induced AChE expression was blocked by the Wnt-signaling inhibitor Dickkopf protein-1 (DKK-1). We hypothesized that the Runt-related transcription factor 2 (Runx2), a downstream transcription factor in Wnt/ß-catenin signaling, is involved in AChE regulation in osteoblasts, confirmed by the identification of a Runx2-binding site in the ACHE gene promoter, further corroborated by ChIP. Of note, Runx2 overexpression in osteoblasts induced AChE expression and activity of the ACHE promoter tagged with the luciferase gene. Moreover, deletion of the Runx2-binding site in the ACHE promoter reduced its activity during osteoblastic differentiation, and addition of 5-azacytidine and trichostatin A to differentiating osteoblasts affected AChE expression, suggesting epigenetic regulation of the ACHE gene. We conclude that AChE plays a role in osteoblastic differentiation and is regulated by both Wnt3a and Runx2.
Asunto(s)
Acetilcolinesterasa/genética , Diferenciación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación Enzimológica de la Expresión Génica , Osteoblastos/citología , Osteoblastos/metabolismo , Proteína Wnt3A/metabolismo , Acetilcolinesterasa/metabolismo , Animales , Diferenciación Celular/genética , Células Cultivadas , Humanos , RatasRESUMEN
Acetylcholinesterase (AChE; EC 3.1.1.7) is known to hydrolyze acetylcholine at cholinergic synapses. In mammalian erythrocyte, AChE exists as a dimer (G2 ) and is proposed to play role in erythropoiesis. To reveal the regulation of AChE during differentiation of erythroblast, erythroblast-like cells (TF-1) were induced to differentiate by application of erythropoietin (EPO). The expression of AChE was increased in parallel to the stages of differentiation. Application of EPO in cultured TF-1 cells induced transcriptional activity of ACHE gene, as well as its protein product. This EPO-induced event was in parallel with erythrocytic proteins, for example, α- and ß-globins. The EPO-induced AChE expression was mediated by phosphorylations of Akt and GATA-1; because the application of Akt kinase inhibitor blocked the gene activation. Erythroid transcription factor also known as GATA-1, a downstream transcription factor of EPO signaling, was proposed here to account for regulation of AChE in TF-1 cell. A binding sequence of GATA-1 was identified in ACHE gene promoter, which was further confirmed by chromatin immunoprecipitation (ChIP) assay. Over-expression of GATA-1 in TF-1 cultures induced AChE expression, as well as activity of ACHE promoter tagged with luciferase gene (pAChE-Luc). The deletion of GATA-1 sequence on the ACHE promoter, pAChEΔGATA-1 -Luc, reduced the promoter activity during erythroblastic differentiation. On the contrary, the knock-down of AChE in TF-1 cultures could lead to a reduction in EPO-induced expression of erythrocytic proteins. These findings indicated specific regulation of AChE during maturation of erythroblast, which provided an insight into elucidating possible mechanisms in regulating erythropoiesis.
Asunto(s)
Acetilcolinesterasa/metabolismo , Diferenciación Celular/efectos de los fármacos , Eritroblastos/efectos de los fármacos , Eritroblastos/enzimología , Eritropoyetina/farmacología , Acetilcolinesterasa/genética , Bencenamina, 4,4'-(3-oxo-1,5-pentanodiil)bis(N,N-dimetil-N-2-propenil-), Dibromuro/farmacología , Línea Celular , Inmunoprecipitación de Cromatina , Cromonas/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Lípidos de la Membrana/metabolismo , Morfolinas/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Tiempo , TransfecciónRESUMEN
Corydalis Rhizoma (CR) is a commonly used traditional Chinese medicine for its potency in activating blood circulation and analgesia. In clinic, CR extracts or components are commonly used in the treatment of myocardial ischemia, rheumatism, and dysmenorrhea with different types of inflammation. However, due to different mechanism of pain and inflammation, the anti-inflammatory property of CR has not been fully revealed. Here, the major chromatographic peaks of CR extracts in different extracting solvents were identified, and the anti-inflammatory activities of CR extracts and its major alkaloids were evaluated in LPS-treated macrophages by determining expressions of proinflammatory cytokines, IκBα and NF-κB. The most abundant alkaloid in CR extract was dehydrocorydaline, having >50% of total alkaloids. Besides, the anti-inflammatory activities of dehydrocorydaline and its related analogues were demonstrated. The anti-inflammatory roles were revealed in LPS-treated cultured macrophages, including (i) inhibiting proinflammatory cytokines release, for example, TNF-α, IL-6; (ii) suppressing mRNA expressions of proinflammatory cytokines; (iii) promoting IκBα expression and suppressing activation of NF-κB transcriptional element; and (iv) reducing the nuclear translocation of NF-κB. The results supported that dehydrocorydaline was the major alkaloid in CR extract, which, together with its analogous, accounted the anti-inflammatory property of CR.
RESUMEN
Acetylcholine (ACh) regulates inflammation via α7 nicotinic acetylcholine receptor (α7 nAChR). Acetylcholinesterase (AChE), an enzyme hydrolyzing ACh, is expressed in immune cells suggesting non-classical function in inflammatory responses. Here, the expression of PRiMA-linked G4 AChE was identified on the surface of macrophages. In lipopolysaccharide-induced inflammatory processes, AChE was upregulated by the binding of NF-κB onto the ACHE promotor. Conversely, the overexpression of G4 AChE inhibited ACh-suppressed cytokine release and cell migration, which was in contrast to that of applied AChE inhibitors. AChEmt, a DNA construct without enzymatic activity, was adopted to identify the protein role of AChE in immune system. Overexpression of G4 AChEmt induced cell migration and inhibited ACh-suppressed cell migration. The co-localization of α7 nAChR and AChE was found in macrophages, suggesting the potential interaction of α7 nAChR and AChE. Besides, immunoprecipitation showed a close association of α7 nAChR and AChE protein in cell membrane. Hence, the novel function of AChE in macrophage by interacting with α7 nAChR was determined. Together with hydrolysis of ACh, AChE plays a direct role in the regulation of inflammatory response. As such, AChE could serve as a novel target to treat age-related diseases by anti-inflammatory responses.
RESUMEN
Flavonoids, considered as phytoestrogen mainly deriving from fruit and vegetable, are known to have beneficial effects in brain functions. The role of flavonoids in induction of a cholinergic enzyme, acetylcholinesterase (AChE), was being explored here. In cultured PC12â¯cells, twenty-four commonly found flavonoids were tested for its induction on AChE activity. Fourteen flavonoids showed induction, and five of them had robust effect, i.e. daidzin, alpinetin, irisflorentin, cardamonin and lysionotin. The induction of AChE was fully blocked by pre-treatment of G15 (a selective G protein-coupled receptor 30 [GPR 30] antagonist), suggesting a direct involvement of a membrane-bound estrogen receptor, named as GPR 30, in the cultures. In addition, daidzin was further identified to induce expression of tetrameric globular form of proline-rich membrane anchor (PRiMA)-linked AChE. In parallel, application of daidzin in cultured PC12â¯cells significantly induced expression of neurofilaments, markers for neuronal differentiation. Taken together, flavonoids could induce the expression of AChE via GPR 30 in cultured PC12â¯cells, which could be a good candidate for possible treatment of the brain diseases.
Asunto(s)
Acetilcolinesterasa/genética , Flavonoides/farmacología , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Acetilcolinesterasa/metabolismo , Animales , Benzodioxoles/farmacología , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Flavonoides/antagonistas & inhibidores , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Regulación Enzimológica de la Expresión Génica , Células PC12 , Quinolinas/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
Acetylcholinesterase (AChE) hydrolyzes acetylcholine at cholinergic synapses, and which has various isoforms of AChE, i.e. AChER, AChEH and AChET, deriving from single gene. AChEH exists as a glycophosphatidylinositol (GPI)-linked dimer (G2), presents mainly in plasma membrane of mammalian erythrocyte. Transgenic mice with ACHE gene depletion were employed here to investigate the possible role of AChE in blood cell formation. ACHE knock-out mice were found to suffer normocytic anemia. In erythrocyte of ACHE-/- mice, the amount of hemoglobin, especially α-globin, was found to be markedly reduced. In addition, the number of erythrocyte and hematocrit of ACHE-/- mice were significantly lowered. To probe the role of AChE isoforms in erythroid differentiation, erythroblast-like cells (TF-1) over-expressed with different AChE isoforms were induced to differentiate by erythropoietin (EPO): this differentiation induced the expression of each AChE isoform. Only in the TF-1â¯cells over-expressed with AChEH, the EPO-induced transcriptions and protein expressions of α- and ß-globins could be significantly enhanced, which therefore suggested that AChEH might regulate the responsiveness of TF-1â¯cells to EPO. The alternation of EPO-induced downstream signaling might be accounted by association of AChE with EPO receptor in cell surface. The findings indicated the significance of AChE in erythroblast maturation, which provided an insight in elucidating possible mechanisms in regulating erythropoiesis.
Asunto(s)
Acetilcolinesterasa/metabolismo , Receptores de Eritropoyetina/metabolismo , Acetilcolinesterasa/química , Acetilcolinesterasa/inmunología , Animales , Anticuerpos/inmunología , Diferenciación Celular , Línea Celular , Dimerización , Eritroblastos/citología , Eritroblastos/metabolismo , Eritropoyetina/farmacología , Expresión Génica/efectos de los fármacos , Hemoglobinas/metabolismo , Humanos , Ratones , Ratones Noqueados , Receptores de Eritropoyetina/inmunologíaRESUMEN
Genistein, 4',5,7-trihydroxyisoflavone, is a major isoflavone in soybean, which is known as phytestrogen having known benefit to brain functions. Being a common phytestrogen, the possible role of genistein in the brain protection needs to be further explored. In cultured PC12 cells, application of genistein significantly induced the expression of neurofilaments (NFs), markers for neuronal differentiation. In parallel, the expression of tetrameric form of proline-rich membrane anchor (PRiMA)-linked acetyl-cholinesterase (G4 AChE), a key enzyme to hydrolyze acetylcholine in cholinergic synapses, was induced in a dose-dependent manner: this induction included the associated protein PRiMA. The genistein-induced AChE expression was fully blocked by the pre-treatment of H89 (an inhibitor of protein kinase A, PKA) and G15 (a selective G protein-coupled receptor 30 (GPR30) antagonist), which suggested a direct involvement of a membrane-bound estrogen receptor (ER), named as GPR30 in the cultures. In parallel, the estrogen-induced activation of GPR30 induced AChE expression in a dose-dependent manner. The genistein/estrogen-induced AChE expression was triggered by a cyclic AMP responding element (CRE) located on the ACHE gene promoter. The binding of this CRE site by cAMP response element-binding protein (CREB) induced ACHE gene transcription. In parallel, increased expression levels of miR132 and miR212 were found when cultured PC12 cells were treated with genistein or G1. Thus, a balance between production and destruction of AChE by the activation of GPR30 was reported here. We have shown for the first time that the activation of GPR30 could be one way for estrogen or flavonoids, possessing estrogenic properties, to enhance cholinergic functions in the brain, which could be a good candidate for possible treatment of neurodegenerative diseases.
RESUMEN
Adenosine 5'-triphosphate (ATP), a neurotransmitter and a neuromodulator, has been shown to be co-stored and co-released with acetylcholine (ACh) at the pre-synaptic vesicles in vertebrate neuromuscular junction (nmj). Several lines of studies demonstrated that binding of ATP to its corresponding P2Y1 receptors (P2Y1R) in muscle and neuron regulated the post-synaptic gene expressions. Indeed, the expression of acetylcholinesterase (AChE) in muscle was markedly decreased in P2Y1R-/- (P2Y1R knock-out) mice. In order to search for possible role of P2Y1R in cholinergic function of the brain, the expression of globular form AChE was determined in the brain of P2Y1R-/- mice. In contrast to that in muscle, the amounts of AChE activity, AChE catalytic subunit, structure subunit PRiMA and the amount of ACh, in the brain were not, significantly, altered, suggesting the role of P2Y1R in neuron could have different function as that in muscle. However, the expressions of a series of neuronal development markers, i.e. neurofilaments, were reduced in P2Y1R-/- mouse brain, indicating P2Y1R may be involved in neuronal development process.