RESUMO
The potential drug target choline acetyltransferase (ChAT) catalyses the production of the neurotransmitter acetylcholine in cholinergic neurons, T-cells, and B-cells. Herein, we show that arylvinylpyridiniums (AVPs), the most widely studied class of ChAT inhibitors, act as substrate in an unusual coenzyme A-dependent hydrothiolation reaction. This in situ synthesis yields an adduct that is the actual enzyme inhibitor. The adduct is deeply buried in the active site tunnel of ChAT and interactions with a hydrophobic pocket near the choline binding site have major implications for the molecular recognition of inhibitors. Our findings clarify the inhibition mechanism of AVPs, establish a drug modality that exploits a target-catalysed reaction between exogenous and endogenous precursors, and provide new directions for the development of ChAT inhibitors with improved potency and bioactivity.
Assuntos
Colina O-Acetiltransferase/antagonistas & inibidores , Inibidores Enzimáticos/química , Ligantes , Acetilcolina/metabolismo , Sítios de Ligação , Biocatálise , Domínio Catalítico , Colina O-Acetiltransferase/metabolismo , Inibidores Enzimáticos/metabolismo , Cinética , Simulação de Dinâmica Molecular , Piridinas/química , Piridinas/metabolismo , Termodinâmica , Temperatura de TransiçãoRESUMO
Hippocampal cholinergic neurostimulating peptide (HCNP) is a secreted undecapeptide produced through proteolytic cleavage of its precursor protein, HCNPpp. Within hippocampal neurons, HCNP increases gene expression of choline acetyltransferase (ChAT), which catalyzes acetylcholine (ACh) synthesis, thereby modulating neural activity. HCNPpp also appears to be expressed in various immune cells. In the present study, we observed that HCNPpp is expressed in U937 human macrophage-like cells and that HCNP exposure suppresses lipopolysaccharide (LPS)-induced gene expression of ChAT. The opposite action is also seen in T lymphocytes, which suggest that HCNP appear to suppress cholinergic system in immune cells. In addition, HCNP suppresses LPS-induced gene expression of inflammatory enzymes including cyclooxygenase 2 (COX2) and inducible nitric oxide (NO) synthase (iNOS). The suppressive effect of HCNP may reflect suppression of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling activated by LPS. Thus, HCNP may have therapeutic potential as an anti-inflammatory drug.
Assuntos
Anti-Inflamatórios/farmacologia , Mediadores da Inflamação/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Neuropeptídeos/farmacologia , Linhagem Celular , Colina O-Acetiltransferase/análise , Colina O-Acetiltransferase/antagonistas & inibidores , Colina O-Acetiltransferase/metabolismo , Ciclo-Oxigenase 2/análise , Ciclo-Oxigenase 2/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Mediadores da Inflamação/análise , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
PURPOSE: To explore genistein, the most active component of soy isoflavones, on viability, expression of estrogen receptor (ER) subtypes, choline acetyltransferase (ChAT), and glutamate receptor subunits in amyloid peptide 25-35-induced hippocampal neurons, providing valuable data and basic information for neuroprotective effect of genistein in Aß25-35-induced neuronal injury. METHODS: We established an in vitro model of Alzheimer's disease by exposing primary hippocampal neurons of newborn rats to amyloid peptide 25-35 (20 µM) for 24 h and observing the effects of genistein (10 µM, 3 h) on viability, expression of ER subtypes, ChAT, NMDA receptor subunit NR2B and AMPA receptor subunit GluR2 in Aß25-35-induced hippocampal neurons. RESULTS: We found that amyloid peptide 25-35 exposure reduced the viability of hippocampal neurons. Meanwhile, amyloid peptide 25-35 exposure decreased the expression of ER subtypes, ChAT and GluR2, and increased the expression of NR2B. Genistein at least partially reversed the effects of amyloid peptide 25-35 in hippocampal neurons. CONCLUSION: Genistein could increase the expression of ChAT as a consequence of activating estrogen receptor subtypes, modulating the expression of NR2B and GluR2, and thereby ameliorating the status of hippocampal neurons and exerting neuroprotective effects against amyloid peptide 25-35. Our data suggest that genistein might represent a potential cell-targeted therapy which could be a promising approach to treating AD.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Morte Celular/fisiologia , Colina O-Acetiltransferase/antagonistas & inibidores , Genisteína/farmacologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/antagonistas & inibidores , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Glutamato/efeitos dos fármacos , Peptídeos beta-Amiloides/fisiologia , Animais , Neurônios/citologia , Neurônios/enzimologia , Neurônios/metabolismo , Fragmentos de Peptídeos/fisiologia , Ratos , Ratos WistarRESUMO
INTRODUCTION: Several pharmacoepidemiological studies indicate that proton pump inhibitors (PPIs) significantly increase the risk of dementia. Yet, the underlying mechanism is not known. Here, we report the discovery of an unprecedented mode of action of PPIs that explains how PPIs may increase the risk of dementia. METHODS: Advanced in silico docking analyses and detailed enzymological assessments were performed on PPIs against the core-cholinergic enzyme, choline-acetyltransferase (ChAT), responsible for biosynthesis of acetylcholine (ACh). RESULTS: This report shows compelling evidence that PPIs act as inhibitors of ChAT, with high selectivity and unprecedented potencies that lie far below their in vivo plasma and brain concentrations. DISCUSSION: Given that accumulating evidence points at cholinergic dysfunction as a driving force of major dementia disorders, our findings mechanistically explain how prolonged use of PPIs may increase incidence of dementia. This call for restrictions for prolonged use of PPIs in elderly, and in patients with dementia or amyotrophic lateral sclerosis.
Assuntos
Colina O-Acetiltransferase/antagonistas & inibidores , Demência/epidemiologia , Inibidores da Bomba de Prótons/farmacologia , Simulação por Computador , Demência/induzido quimicamente , Humanos , Incidência , Simulação de Acoplamento Molecular , Inibidores da Bomba de Prótons/efeitos adversos , RiscoRESUMO
For thousands of years, it has been widely believed that walnut is a kind of nut that has benefits for the human body. Walnut oil, accounting for about 70% of walnut, mainly consists of polyunsaturated fatty acids. To investigate the effect of walnut oil on memory impairment in mice, scopolamine (3 mg/kg body weight/d) was used to establish the animal model during Morris Water Maze (MWM) tests. Walnut oil was administrated orally at 10 mL/kg body weight/d for 8 consecutive weeks. The results showed that walnut oil treatment ameliorated the behavior of the memory-impaired mice in the MWM test. Additionally, walnut oil obviously inhibited acetylcholinesterase activity (1.26 ± 0.12 U/mg prot) (p = 0.013) and increased choline acetyltransferase activity (129.75 ± 6.76 U/mg tissue wet weight) in the brains of scopolamine-treated mice (p = 0.024), suggesting that walnut oil could prevent cholinergic function damage in mice brains. Furthermore, walnut oil remarkably prevented the decrease in total superoxide dismutase activity (93.30 ± 5.50 U/mg prot) (p = 0.006) and glutathione content (110.45 ± 17.70 mg/g prot) (p = 0.047) and the increase of malondialdehyde content (13.79 ± 0.96 nmol/mg prot) (p = 0.001) in the brain of scopolamine-treated mice, indicating that walnut oil could inhibit oxidative stress in the brain of mice. Furthermore, walnut oil prevented histological changes of neurons in hippocampal CA1 and CA3 regions induced by scopolamine. These findings indicate that walnut oil could prevent memory impairment in mice, which might be a potential way for the prevention of memory dysfunctions.
Assuntos
Juglans/química , Transtornos da Memória/prevenção & controle , Óleos de Plantas/administração & dosagem , Escopolamina/efeitos adversos , Administração Oral , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Colina O-Acetiltransferase/antagonistas & inibidores , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/enzimologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Óleos de Plantas/farmacologiaRESUMO
Alzheimer disease and related dementias are major challenges, demanding urgent needs for earliest possible diagnosis to optimize the success rate in finding effective therapeutic interventions. Mounting solid scientific premises point at the core acetylcholine-biosynthesizing cholinergic enzyme, ChAT as a legitimate in vivo target for developing positron emission tomography biomarker for early diagnosis and/or monitoring therapeutic responses in the neurodegenerative dementias. Up-to-date, no PET tracer ligands for ChAT are available. Here we report for the first time a novel hierarchical virtual screening approach on a commercial library of ~300,000 compounds, followed by in vitro screening of the hits by a new High-Throughput ChAT assay. We report detailed pharmacodynamic data for three identified selective novel ChAT ligands with IC50 and K i values ranging from ~7 to 26 µM. In addition, several novel selective inhibitors of the acetylcholine-degrading enzymes, AChE and BuChE were identified, with one of the compounds showing an IC50-value of ~6 µM for AChE. In conclusion, this report provides an excellent starting platform for designing and optimizing potent and selective ChAT ligands, with high potential as PET-imaging probe for early diagnosis of AD, and related dementias, such as Down's syndrome and Lewy body disorders.
Assuntos
Colina O-Acetiltransferase/antagonistas & inibidores , Acetilcolinesterase/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Síndrome de Down/metabolismo , Humanos , Corpos de Lewy/metabolismo , Doenças Neurodegenerativas/metabolismoRESUMO
The autonomic nervous system consists of sympathetic and parasympathetic nerves, which functionally antagonize each other to control physiology and homeostasis of organs. However, it is largely unexplored how the autonomic nervous system is established during development. In particular, early formation of parasympathetic network remains elusive because of its complex anatomical structure. To distinguish between parasympathetic (cholinergic) and sympathetic (adrenergic) ganglia, vesicular acetylcholine transporter (VAChT) and choline O-acetyltransferase (ChAT), proteins associated with acetylcholine synthesis, are known to be useful markers. Whereas commercially available antibodies against these proteins are widely used for mammalian specimens including mice and rats, these antibodies do not work satisfactorily in chickens, although chicken is an excellent model for the study of autonomic nervous system. Here, we newly raised antibodies against chicken VAChT and ChAT proteins. One monoclonal and three polyclonal antibodies for VAChT, and one polyclonal antibody for ChAT were obtained, which were available for Western blotting analyses and immunohistochemistry. Using these verified antibodies, we detected cholinergic cells in Remak ganglia of autonomic nervous system, which form in the dorsal aspect of the digestive tract of chicken E13 embryos. The antibodies obtained in this study are useful for visualization of cholinergic neurons including parasympathetic ganglia.
Assuntos
Anticorpos/metabolismo , Colina O-Acetiltransferase/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Animais , Embrião de Galinha , Galinhas , Colina O-Acetiltransferase/antagonistas & inibidores , Neurônios Colinérgicos/metabolismo , Trato Gastrointestinal/embriologia , Proteínas Vesiculares de Transporte de Acetilcolina/antagonistas & inibidoresRESUMO
The present study investigated the neuroprotective effects of taurine, an essential amino acid for growth and development of central nervous system. Intracerebroventricular streptozotocin (ICV-STZ) model of cognitive impairment was used in male Wistar rats (270 ± 20 g). Morris water maze, elevated plus maze and passive avoidance paradigm were used to assess cognitive performance. Taurine (40, 60 and 120 mg/kg) was administered orally for 28 days following STZ administration on day 1. Oxidative stress parameters (malondialdehyde, glutathione, nitric oxide and superoxide dismutase) and cholinesterases (acetylcholinesterase and butyrylcholinesterase) activity were measured at end of the study in the cortex and hippocampus. Levels of TNF-α, IL-1ß, expression of rho kinase-II (ROCK-II), glycogen synthase kinase-3ß (GSK-3ß) and choline acetyltransferase (ChAT) were studied in cortex and hippocampus. STZ caused significant cognitive impairment as compared to normal control. Chronic administration of taurine attenuated STZ-induced cognitive impairment. Increased oxidative stress and increased levels of TNF-α, IL-1ß induced by STZ were also significantly attenuated by taurine. Taurine significantly (p < 0.05) decreased the STZ-induced increased expression of ROCK-II in cortex and hippocampus. Further, STZ-induced increased activity of cholinesterases was significantly (p < 0.001) mitigated by taurine. STZ decreased the expression of ChAT in hippocampus which was significantly (p < 0.05) reversed by taurine. However, GSK-3ß expression was not altered by either STZ or taurine. The present study indicates that taurine exerts a neuroprotective role against STZ-induced cognitive impairment in rats. This effect is probably mediated by modulating oxidative stress, cholinesterases, inflammatory cytokines and expression of ROCK-II. Thus, this study suggests a potential of chronic taurine administration in cognitive impairment of Alzheimer's type.
Assuntos
Colina O-Acetiltransferase/metabolismo , Disfunção Cognitiva/metabolismo , Mediadores da Inflamação/metabolismo , Estresse Oxidativo/fisiologia , Estreptozocina/toxicidade , Taurina/administração & dosagem , Animais , Colina O-Acetiltransferase/antagonistas & inibidores , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/tratamento farmacológico , Esquema de Medicação , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Mediadores da Inflamação/antagonistas & inibidores , Injeções Intraventriculares , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
The effect of ß-amyloid aggregates on activity of choline acetyltransferase (ChAT) which is responsible for synthesizing acetylcholine (ACh) in human brain is investigated through the two-enzyme/two-compartment (2E2C) model where the presynaptic neuron is considered as compartment 1 while both the synaptic cleft and the postsynaptic neuron are considered as compartment 2 through suggesting three different kinetic mechanisms for the inhibition effect. It is found that the incorporation of ChAT inhibition by ß-amyloid aggregates into the 2E2C model is able to yield dynamic solutions for concentrations of generated ß-amyloid, ACh, choline, acetate, and pH in addition to the rates of ACh synthesis and ACh hydrolysis in compartments 1 and 2. It is observed that ChAT activity needs a high concentration of ß-amyloid aggregates production rate. It is found that ChAT activity is reduced significantly when neurons are exposed to high levels of ß-amyloid aggregates leading to reduction in levels of ACh which is one of the most significant physiological symptoms of AD. Furthermore, the system of ACh neurocycle is dominated by the oscillatory behavior when ChAT enzyme is completely inhibited by ß-amyloid. It is observed that the direct inactivation of ChAT by ß-amyloid aggregates may be a probable mechanism contributing to the development of AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Colina O-Acetiltransferase/metabolismo , Modelos Neurológicos , Acetilcolina/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Colina/metabolismo , Colina O-Acetiltransferase/antagonistas & inibidores , Neurônios Colinérgicos/metabolismo , Simulação por Computador , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Agregados Proteicos/fisiologia , Agregação Patológica de Proteínas/etiologia , Agregação Patológica de Proteínas/metabolismoRESUMO
Diabetes mellitus is a chronic metabolic disorder and has been associated with cognitive dysfunction. In our earlier study, chronic Urtica dioica (UD) treatment significantly ameliorated diabetes induced associative and spatial memory deficit in mice. The present study was designed to explore the effect of UD leaves extract on muscarinic cholinergic system, which has long been known to be involved in cognition. Streptozotocin (STZ) (50 mg/kg, i.p., consecutively for 5 days) was used to induce diabetes followed by treatment with UD extract (50 mg/kg, oral) or rosiglitazone (5 mg/kg, oral) for 8 weeks. STZ-induced diabetic mice showed significant reduction in hippocampal muscarinic acetylcholine receptor-1 and choline acetyltransferase expressions. Chronic diabetes significantly up-regulated the protein expression of acetylcholinesterase associated with oxidative stress in hippocampus. Besides, STZ-induced diabetic mice showed hypolocomotion with up-regulation of muscarinic acetylcholine receptor-4 expression in striatum. Chronic UD treatment significantly attenuated the cholinergic dysfunction and oxidative stress in the hippocampus of diabetic mice. UD had no effect on locomotor activity and muscarinic acetylcholine receptor-4 expression in striatum. In conclusion, UD leaves extract has potential to reverse diabetes mediated alteration in muscarinic cholinergic system in hippocampus and thereby improve memory functions.
Assuntos
Colina O-Acetiltransferase/biossíntese , Diabetes Mellitus Experimental/metabolismo , Hipocampo/metabolismo , Extratos Vegetais/uso terapêutico , Receptor Muscarínico M1/biossíntese , Urtica dioica , Animais , Colina O-Acetiltransferase/antagonistas & inibidores , Diabetes Mellitus Experimental/tratamento farmacológico , Hipocampo/efeitos dos fármacos , Masculino , Camundongos , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Folhas de Planta , Receptor Muscarínico M1/antagonistas & inibidores , EstreptozocinaRESUMO
Acetylcholine (ACh) is well known to induce persistent γ-oscillations in the hippocampus when applied together with physostigmine, an inhibitor of the ACh degrading enzyme acetylcholinesterase (AChE). Here we report that physostigmine alone can also dose-dependently induce γ-oscillations in rat hippocampal slices. We hypothesized that this effect was due to the presence of choline in the extracellular space and that this choline is taken up into cholinergic fibers where it is converted to ACh by the enzyme choline-acetyltransferase (ChAT). Release of ACh from cholinergic fibers in turn may then induce γ-oscillations. We therefore tested the effects of the choline uptake inhibitor hemicholinium-3 (HC-3) on persistent γ-oscillations either induced by physostigmine alone or by co-application of ACh and physostigmine. We found that HC-3 itself did not induce γ-oscillations and also did not prevent physostigmine-induced γ-oscillation while washout of physostigmine and ACh-induced γ-oscillations was accelerated. It was recently reported that ChAT might also be present in the extracellular space (Vijayaraghavan et al., 2013). Here we show that the effect of physostigmine was prevented by the ChAT inhibitor (2-benzoylethyl)-trimethylammonium iodide (BETA) which could indicate extracellular synthesis of ACh. However, when we tested for effects of extracellularly applied acetyl-CoA, a substrate of ChAT for synthesis of ACh, physostigmine-induced γ-oscillations were attenuated. Together, these findings do not support the idea that ACh can be synthesized by an extracellularly located ChAT.
Assuntos
Região CA3 Hipocampal/enzimologia , Colina O-Acetiltransferase/metabolismo , Espaço Extracelular/enzimologia , Ritmo Gama/fisiologia , Acetilcoenzima A/administração & dosagem , Acetilcoenzima A/metabolismo , Acetilcolina/administração & dosagem , Acetilcolina/análogos & derivados , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Animais , Região CA3 Hipocampal/efeitos dos fármacos , Colina/metabolismo , Colina O-Acetiltransferase/antagonistas & inibidores , Colinérgicos/farmacologia , Inibidores da Colinesterase/farmacologia , Relação Dose-Resposta a Droga , Ritmo Gama/efeitos dos fármacos , Hemicolínio 3/farmacologia , Masculino , Microeletrodos , Inibidores da Captação de Neurotransmissores/farmacologia , Fisostigmina/farmacologia , Ratos Wistar , Técnicas de Cultura de TecidosRESUMO
Acetylcholine is not only a neurotransmitter, but is found in a variety of non-neuronal cells. For example, the enzyme choline acetyltransferase (ChAT), catalyzing acetylcholine synthesis, is expressed by the colonic epithelium of different species. These cells release acetylcholine across the basolateral membrane after luminal exposure to propionate, a short-chain fatty acid. The functional consequence is the induction of chloride secretion, measurable as increase in short-circuit current (Isc) in Ussing chamber experiments. It is unclear how acetylcholine is produced and released by colonic epithelium. Therefore, the aim of the present study was the identification (on mRNA and protein level) and functional characterization (in Ussing chamber experiments combined with HPLC detection of acetylcholine) of transporters/enzymes in the cholinergic system of rat colonic epithelium. Immunohistochemical staining as well as RT-PCR revealed the expression of high-affinity choline transporter, ChAT, carnitine acetyltransferase (CarAT), vesicular acetylcholine transporter (VAChT), and organic cation transporters (OCT 1, 2, 3) in colonic epithelium. In contrast to blockade of ChAT with bromoacetylcholine, inhibition of CarAT with mildronate did not inhibit the propionate-induced increase in Isc, suggesting a predominant synthesis of epithelial acetylcholine by ChAT. Although being expressed, blockade of VAChT with vesamicol was ineffective, whereas inhibition of OCTs with omeprazole and corticosterone inhibited propionate-induced Isc and the release of acetylcholine into the basolateral compartment. In summary, OCTs seem to be involved in regulated acetylcholine release by colonic epithelium, which is assumed to be involved in chemosensing of luminal short-chain fatty acids by the intestinal epithelium.
Assuntos
Acetilcolina/metabolismo , Colina O-Acetiltransferase/metabolismo , Colo/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Propionatos/farmacologia , Animais , Colina O-Acetiltransferase/antagonistas & inibidores , Colina O-Acetiltransferase/genética , Cromatografia Líquida de Alta Pressão , Colo/enzimologia , Colo/metabolismo , Feminino , Imuno-Histoquímica , Técnicas In Vitro , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Masculino , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/genética , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Multiple sclerosis (MS) is a progressive T cell-mediated autoimmune demyelinating inflammatory disease of the central nervous system (CNS). Although it is recognized that cognitive deficits represent a manifestation of the disease, the underlying pathogenic mechanisms remain unknown. Here we provide evidence of spatial reference memory impairments during the pre-motor phase of experimental autoimmune encephalomyelitis (EAE) in mice. Specifically, these cognitive deficits were accompanied by down-regulation of choline acetyltransferase (ChAT) mRNA expression on day 5 and 11 post-immunization, and up-regulation of inflammatory cytokines in the hippocampus and prefrontal cortex. Moreover, a marked increase in B1R mRNA expression occurred selectively in the hippocampus, whereas protein level was up-regulated in both brain areas. Genetic deletion of kinin B1R attenuated cognitive deficits and cholinergic dysfunction, and blocked mRNA expression of both IL-17 and IFN-γ in the prefrontal cortex, lymph node and spleen of mice subjected to EAE. The discovery of kinin receptors, mainly B1R, as a target for controlling neuroinflammatory response, as well as the cognitive deficits induced by EAE may foster the therapeutic exploitation of the kallikrein-kinin system (KKS), in particular for the treatment of autoimmune disorders, such as MS, mainly during pre-symptomatic phase.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/fisiopatologia , Sistema Calicreína-Cinina/imunologia , Transtornos da Memória/imunologia , Transtornos da Memória/fisiopatologia , Transtornos dos Movimentos/imunologia , Transtornos dos Movimentos/fisiopatologia , Comportamento Espacial , Animais , Colina O-Acetiltransferase/antagonistas & inibidores , Colina O-Acetiltransferase/biossíntese , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Encefalomielite Autoimune Experimental/genética , Feminino , Hipocampo/enzimologia , Hipocampo/imunologia , Hipocampo/patologia , Interferon gama/antagonistas & inibidores , Interferon gama/genética , Interleucina-17/antagonistas & inibidores , Interleucina-17/genética , Sistema Calicreína-Cinina/genética , Transtornos da Memória/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transtornos dos Movimentos/genética , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Distribuição Aleatória , Receptor B1 da Bradicinina/deficiência , Receptor B1 da Bradicinina/genética , Receptor B2 da Bradicinina/agonistas , Receptor B2 da Bradicinina/deficiência , Receptor B2 da Bradicinina/genética , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
The biological function of histone deacetylases (HDACs), namely, repression of gene expression by removing an acetyl group from a histone N-terminal tail, plays an important role in numerous biological processes such as cell cycle, differentiation, and apoptosis in the development of individual tissues, including the brain. We previously showed the possible role of HDAC activity in the regulation of gene expression of choline acetyltransferase (ChAT), a specific marker for cholinergic neurons and their function, in NG108-15 neuronal cells as an in vitro model of cholinergic neurons. The objectives of the present study were to specify key HDACs and investigate the essential role of HDACs in ChAT gene regulation in NG108-15 cells. The experiments using different types of HDAC inhibitors indicated that class IIa HDACs substantially participate in the regulation of ChAT gene expression. In addition, HDAC9, a class IIa enzyme, was dramatically decreased at the protein levels, and dissociated from the promoter region of ChAT gene during neuronal differentiation. Furthermore, knockdown of HDAC9 by siRNA increased ChAT gene expression in undifferentiated cells. These findings demonstrate that HDAC9 is responsible for repressing ChAT gene expression in NG108-15 neuronal cells, and thus plays an important role in cholinergic differentiation.
Assuntos
Acetilcolina/biossíntese , Colina O-Acetiltransferase/antagonistas & inibidores , Colina O-Acetiltransferase/genética , Neurônios Colinérgicos/enzimologia , Regulação para Baixo/genética , Histona Desacetilases/fisiologia , Proteínas Repressoras/fisiologia , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Colina O-Acetiltransferase/biossíntese , Neurônios Colinérgicos/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação Enzimológica da Expressão Gênica/genética , Histona Desacetilases/genética , Hibridomas , Camundongos , Neurogênese/genética , Ratos , Proteínas Repressoras/genéticaRESUMO
In the heart, acetylcholine (ACh) slows pacemaker activity, depresses contractility and slows conduction in the atrioventricular node. Beside these cardiovascular effects, ACh has also been associated with an anti-inflammatory and anti-apoptotic pathway. There is no evidence for ACh synthesis and excretion in other cell types than neuronal cells in the heart. Therefore, this study investigates whether cardiomyocytes are able to synthesize, transport and excrete ACh in the heart. We chose a rat model of different aged rats (neonatal, 6-8 week = young, 20-24 month = old). By real-time PCR, Western blot and immunofluorescence experiments we could demonstrate that adult, but not neonatal cardiomyocytes, express the choline acetyltransferase (ChAT). The expression level of ChAT is down-regulated in old cardiomyocytes. Furthermore, we found that young and old cardiomyocytes express the ACh transport proteins choline transporter-1 (CHT-1) and the vesicular acetylcholine transporter (VAChT). The amount of ACh excretion detected by high performance liquid chromatography (HPLC) is significantly down-regulated in old cardiomyocytes. Bromo-acetylcholine (BrACh), a specific ChAT inhibitor, significantly decreased ACh concentrations in cardiomyocyte supernatants demonstrating that ChAT is the main ACh synthesizing enzyme in cardiomyocytes. In conclusion, we could demonstrate that adult, but not neonatal, cardiomyocytes are able to synthesize, transport and excrete ACh in the rat heart. The expression level of ChAT and the ACh excretion amount are significantly down-regulated in old cardiomyocytes. This finding may provide new physiological/pathological aspects in the communication between cardiomyocytes and other cell types in the myocardium, e.g. fibrocytes, neurocytes or endothelial cells.
Assuntos
Acetilcolina/metabolismo , Envelhecimento/metabolismo , Miócitos Cardíacos/metabolismo , Neurônios , Acetilcolina/biossíntese , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Colina O-Acetiltransferase/antagonistas & inibidores , Colina O-Acetiltransferase/metabolismo , Masculino , Miocárdio/enzimologia , Miocárdio/metabolismo , Miócitos Cardíacos/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , Neurônios/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Congenital myasthenic syndromes (CMSs) are a group of clinically and genetically heterogeneous inherited disorders of the neuromuscular junction. Mutations in the acetylcholine transferase (CHAT) gene cause a pre-synaptic CMS, typically associated with episodic apnoea and worsening of myasthenic symptoms during crises caused by infections, fever or stress. Between crises symptoms may be mild and variable. Acetylcholinesterase - inhibitor therapy is reported to improve clinical symptoms and reduce crises. PATIENTS AND METHODS: We present data on the long-term follow-up of 11 patients with a congenital myasthenic syndrome due to nine different CHAT mutations; ten of the patients have not been previously reported. RESULTS AND CONCLUSIONS: Manifestation varied from the neonatal period to the age of two years, follow-up time from nine months to 12 years. This cohort of CHAT patients studied here enabled us to describe two distinct phenotypes: The neonatal-onset group suffers from apnoeic crises, respirator dependency and bulbar weakness. Apnoea should be carefully distinguished from seizures; a CMS should be taken into account early to start appropriate therapy. Infantile-onset patients show mild permanent weakness, but experience apnoeic crises and worsening which resolve with Acetylcholinesterase - inhibitor treatment. However, after several years of treatment proximal muscle strength may decrease and lead to wheelchair dependency despite the continuation of Acetylcholinesterase - inhibitor therapy.
Assuntos
Colina O-Acetiltransferase/genética , Predisposição Genética para Doença/genética , Mutação/genética , Síndromes Miastênicas Congênitas/etiologia , Síndromes Miastênicas Congênitas/genética , Arginina/genética , Colina O-Acetiltransferase/antagonistas & inibidores , Estimulação Elétrica/métodos , Eletroencefalografia/métodos , Inibidores Enzimáticos/uso terapêutico , Feminino , Testes Genéticos , Glicina/genética , Histidina/genética , Humanos , Estudos Longitudinais , Imageamento por Ressonância Magnética/métodos , Masculino , Músculo Esquelético/patologia , Síndromes Miastênicas Congênitas/tratamento farmacológico , Síndromes Miastênicas Congênitas/fisiopatologia , Receptores Proteína Tirosina Quinases/imunologia , Receptores Colinérgicos/imunologia , Receptores Colinérgicos/metabolismoRESUMO
PURPOSE: The aim of the present study was to use an in vivo model of retinal excitotoxicity to investigate the neuroprotective effect of somatostatin (SRIF)-ergic agents. METHODS: Adult Sprague-Dawley rats (weight range, 250-300 g) intravitreally received (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA; 21, 42, 84 nmol/eye) or PBS (50 mM). Time-dependent responses were examined in animals that received AMPA (42 nmol/eye). Animals received AMPA (42 nmol) alone or in combination with SRIF (10(-5), 10(-4) M) or the sst-selective ligands lanreotide (sst(2),10(-5),10(-4) M), L-779976 (sst(2,)10(-6),10(-5), 10(-4) M), L-797591 (sst(1),10(-4) M), and L-803087 (sst(4),10(-4) M). Immunohistochemistry and TUNEL studies were used to examine retinal cell loss and protection. Immunochemistry, Western blot analysis, and radioimmunoassay assessed the viability of sst(2A) receptors and SRIF levels, respectively, in control and AMPA-treated tissue. RESULTS: AMPA (42 nmol) treatment resulted in total and major loss of ChAT and bNOS immunoreactivity, respectively, 24 hours after its administration. This loss was sustained up to 30 days for ChAT- and 8 days for bNOS-expressing amacrine cells. SRIF and the sst(2) receptors were not affected by AMPA. SRIF and the sst(2) analogs protected the retina from the AMPA insult in a dose-dependent manner, whereas activation of the sst(1) and sst(4) subtypes had no effect. TUNEL staining confirmed AMPA-induced retinal ischemia and L-779976 neuroprotection. CONCLUSIONS: These results demonstrate for the first time that SRIF and the sst(2) analogs, administered intravitreally, protect the retina from excitotoxicity. Further studies are essential to ascertain the therapeutic relevance of these results.
Assuntos
Fármacos Neuroprotetores/farmacologia , Neurotoxinas/farmacologia , Retina/efeitos dos fármacos , Somatostatina/análogos & derivados , Somatostatina/farmacologia , Células Amácrinas/enzimologia , Amidas/farmacologia , Animais , Colina O-Acetiltransferase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Feminino , Imuno-Histoquímica , Indóis/farmacologia , Isquemia/induzido quimicamente , Masculino , Fármacos Neuroprotetores/administração & dosagem , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Receptores de Somatostatina/agonistas , Retina/enzimologia , Vasos Retinianos , Somatostatina/administração & dosagem , Fatores de Tempo , Corpo Vítreo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Zinc is a trace element necessary for proper development and function of brain cells. However, excessive accumulation of zinc exerts several cytotoxic effects in the brain. The aim of this work was to see whether cytotoxic effects of zinc are quantitatively correlated with changes in acetyl-CoA metabolism. The zinc levels up to 0.20 mmol/L caused concentration-dependent inhibition of pyruvate dehydrogenase (PDH) activity that correlated with the increase in trypan blue-positive fraction and the decrease in cultured cell number (r = 0.96, p = 0.0001). Chronic exposure of cells to 0.15 mmol/L zinc decreased choline acetyltransferase and aconitase activities, cytoplasmic acetyl-CoA and whole cell ATP level by 38%, 57%, 35%, and 62%, respectively but caused no change in mitochondrial acetyl-CoA level and activities of other enzymes of glycolytic and tricarboxylic acid cycle. dl-alpha-lipoamide when added simultaneously with zinc to cultured cells or their homogenates attenuated its chronic or acute suppressive effects. In homogenates of chronically Zn-treated cells, lipoamide overcame PDH but not aconitase inhibition. Presented data indicate that acute-transient elevation of zinc caused reversible inhibition of PDH, aconitase activities and acetyl-CoA metabolism, which when prolonged could lead to irreversible enzyme inactivation yielding decrease in cell viability and secondary suppression of their cholinergic phenotype.
Assuntos
Acetilcoenzima A/efeitos dos fármacos , Acetilcolina/biossíntese , Metabolismo Energético/efeitos dos fármacos , Degeneração Neural/induzido quimicamente , Neurônios/efeitos dos fármacos , Zinco/toxicidade , Acetilcoenzima A/metabolismo , Aconitato Hidratase/antagonistas & inibidores , Aconitato Hidratase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Colina O-Acetiltransferase/antagonistas & inibidores , Colina O-Acetiltransferase/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/fisiologia , Relação Dose-Resposta a Droga , Metabolismo Energético/fisiologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/toxicidade , Glicólise/efeitos dos fármacos , Glicólise/fisiologia , Camundongos , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Neuroblastoma , Neurônios/metabolismo , Neurotoxinas/toxicidade , Complexo Piruvato Desidrogenase/efeitos dos fármacos , Complexo Piruvato Desidrogenase/metabolismo , Ácido Tióctico/análogos & derivados , Ácido Tióctico/farmacologiaRESUMO
The effects of a potent inhibitor of choline acetyltransferase (ChAT), BW813U, on timing behavior in mature (6-10 months) and aged (26-30 months) male rats were assessed. Twenty rats were trained on a discrete trial 20-s peak-interval (PI) procedure. During baseline (non-drug) training, the time of the maximum response rate (peak time) for mature rats was approximately at the time of scheduled reinforcement, but peak time for aged rats was reliably later. A single administration of BW813U (100 mg/kg, ip) produced a long-lasting increase in peak time for both mature and aged rats that occurred gradually and was synergistic with age. These horizontal shifts in peak time indicate a change in the content of reference memory for the remembered time of reinforcement that is similar for both aging processes and BW813U administration. When a 5-s gap was imposed in the signal, PI-GAP procedure, control rats of both ages summed the signal durations before and after the gap, whereas rats given BW813U showed no retention of the signal duration prior to the gap. This loss of trial-specific temporal information suggests a drug-induced working memory dysfunction. Taken together, these results demonstrate that both working and reference memory for temporal information are sensitive to choline acetyltransferase inhibition in rats.
Assuntos
Envelhecimento/fisiologia , Encéfalo/enzimologia , Colina O-Acetiltransferase/metabolismo , Transtornos da Memória/enzimologia , Memória/fisiologia , Acetilcolina/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Colina O-Acetiltransferase/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Inibidores Enzimáticos/farmacologia , Masculino , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/fisiopatologia , Testes Neuropsicológicos , Ratos , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Fatores de TempoRESUMO
BACKGROUND: Alzheimer's disease (AD) is a devastating neurodegeneration with a characteristic deficit in cholinergic neurotransmission. Treatment with acetylcholinesterase (AChE) inhibitors aims to reverse this deficit and does ameliorate the decline in cognition in some AD patients, although response is variable. OBJECTIVE: To examine whether sequence variation in the gene encoding choline acetyltransferase (CHAT), which encodes the major catalytic enzyme of the cholinergic pathway, predicts response to AChE inhibitors. METHODS: Alzheimer's disease patients (121) were treated with cholinesterase inhibitors and the effect of treatment on cognition was measured using the Mini Mental State Examination (MMSE). Six polymorphisms in CHAT were analysed for association with change in MMSE score. RESULTS: After correction for multiple testing, we found one SNP, rs733722, in a promoter region of CHAT, is associated with response of AD patients to cholinesterase inhibitors (P = 0.03) and accounts for 6% of the variance in response to AChE inhibitors. CONCLUSION: Rs733722 represents a putative marker of response to AChE inhibitors in AD patients.