RESUMO
Nerve growth factor (NGF) is a trophic and survival factor for cholinergic neurons, and it induces the expression of several genes that are essential for synthesis and storage of acetylcholine (ACh), specifically choline acetyltransferase, vesicular ACh transporter (VAChT), and choline transporter. We have found previously that the phosphatidylinositol 3'-kinase pathway, but not the MEK/MAPK pathway, is the mediator of NGF-induced cholinergic differentiation. Here we demonstrate, in the rat pheochromocytoma cell line PC12 and in primary mouse neuronal cultures, that NGF-evoked up-regulation of these three cholinergic-specific genes is mediated by the anti-apoptotic signaling molecule Akt/protein kinase B. Inhibition of Akt activation by the pharmacological inhibitor 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO), or by a peptide fragment derived from the proto-oncogene TLC1, eliminated NGF-stimulated increases in cholinergic gene expression, as demonstrated by RT-PCR and reporter gene assays. Moreover, treatment with HIMO reversed NGF-evoked increases in choline acetyltransferase activity and ACh production. In co-transfection assays with the reporter construct, a dominant-negative Akt plasmid and Akt1-specific small interfering RNA also attenuated NGF-induced cholinergic promoter activity. Our data indicate that, in addition to its well-described role in promoting neuronal survival, Akt can also mediate signals necessary for neurochemical differentiation.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Fator de Crescimento Neural/farmacologia , Proteína Oncogênica v-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Acetilcolina/metabolismo , Análise de Variância , Animais , Células Cultivadas , Colina O-Acetiltransferase/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteína Oncogênica v-akt/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , Ratos , Septo do Cérebro/citologia , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
Synthesis, storage and release of acetylcholine (ACh) require the expression of several specialized enzymes, including choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and the high-affinity choline transporter (CHT). Extracellular factors that regulate CHT expression and their signaling pathways remain poorly characterized. Using the NSC-19 cholinergic cell line, derived from embryonic spinal cord, we compared the effects of the second messenger cAMP on the expression of CHT and the cholinergic locus containing the ChAT and VAChT genes. Treatment of NSC-19 cells with dbcAMP and forskolin, thus increasing intracellular cAMP levels, significantly reduced CHT mRNA expression, while it upregulated ChAT/VAChT mRNA levels and ChAT activity. The cAMP-induced CHT downregulation was independent of PKA activity, as shown in treatments with the PKA inhibitor H-89. The alternative Epac-Rap pathway, when stimulated by a specific Epac activator, led to significant downregulation of CHT and ChAT, and, to a lesser extent, VAChT. In contrast, the PKA activator 6-BNZ-cAMP stimulated the expression of all three genes, but with varying concentration-dependence profiles. Our results indicate that elevations of intraneuronal cAMP concentration have differential effects on the cholinergic phenotype, depending on the involvement of different downstream effectors. Interestingly, although CHT is expressed predominantly in cholinergic cells, its regulation appears to be distinct from that of the cholinergic locus.
Assuntos
Acetilcolina/metabolismo , Encéfalo/metabolismo , Fibras Colinérgicas/metabolismo , AMP Cíclico/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Neurônios/metabolismo , Animais , Bucladesina/farmacologia , Linhagem Celular Tumoral , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hibridomas , Proteínas de Membrana Transportadoras/genética , Camundongos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/genéticaRESUMO
An important feature of cholinergic neurons is high-affinity choline transport, which allows them to reuse choline for the synthesis of ACh needed to support cholinergic neurotransmission. The choline transporter, designated CHT, was recently cloned. We applied RT/PCR to monitor the expression of CHT in the developing mouse CNS from embryonic day 14 (E14) to postnatal day 30 (P30). We found that CHT was expressed early in development, predominantly in the regions containing cholinergic neurons. In the spinal cord, CHT mRNA was present at close to adult levels at the earliest time point examined (E14) and showed almost no changes after birth. In the striatum and the septum, CHT mRNA increased steadily during embryonic stages and leveled off after birth. Surprisingly, CHT mRNA expression was also detected in other brain regions, notably in the cerebellum, where it peaked on E19, and then rapidly declined during postnatal development. CHT protein was detected by Western blotting as a band of apparent molecular weight of 70 kDa. The accumulation of this protein during development lagged behind mRNA accumulation in all tissues. We also examined the effects of NGF and BMP-4, the potent inducers of choline acetyltransferase and vesicular acetylcholine transporter genes, on CHT expression. Both factors increased CHT mRNA accumulation in primary septal cultures. The effect of NGF was dependent on the PI3K signaling, as it was abolished by the PI3K inhibitor LY294002. This result indicates that some of the signals regulating other cholinergic-specific genes also control CHT expression.
Assuntos
Acetilcolina/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Sistema Nervoso Central/metabolismo , Fibras Colinérgicas/metabolismo , Proteínas de Membrana Transportadoras/genética , Fator de Crescimento Neural/metabolismo , Animais , Animais Recém-Nascidos , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/farmacologia , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Células Cultivadas , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/crescimento & desenvolvimento , Colina O-Acetiltransferase/metabolismo , Fibras Colinérgicas/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Imuno-Histoquímica , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Fator de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Medula Espinal/embriologia , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
A characteristic feature of several encephalopathies is preferential impairment of cholinergic neurons. Their particular susceptibility to cytotoxic insults may result from the fact that they utilise acetyl-CoA both for energy production and acetylcholine synthesis. In addition, phenotypic modifications of cholinergic neurons are likely to influence their susceptibility to specific harmful conditions. SN56 cholinergic cells were differentiated by the combination of dibutyryl cAMP and retinoic acid. Al and sodium nitroprusside (SNP, NO donor) exerted direct additive inhibitory effects on mitochondrial aconitase activity. However, NO, Al, or amyloid beta (Abeta)(25-35) caused none or only slight changes of choline O-acetyl transferase (ChAT) and pyruvate dehydrogenase (PDH) activity and relatively small loss of non-differentiated cells (NCs). On the other hand, in differentiated cells (DCs) these neurotoxins brought about marked decreases of these enzyme activities along with greater than in non-differentiated ones increase of cell-death rate. Abeta(35-25) had no effect on these cell parameters. NO and other compounds aggravated detrimental effect of each other particularly in differentiated cells. Thus, differential vulnerability of brain cholinergic neurons to various degenerative signals may result from their phenotype-dependent ratios of acetylcholine to acetyl-CoA synthesising capacities.
Assuntos
Alumínio/toxicidade , Peptídeos beta-Amiloides/toxicidade , Óxido Nítrico/toxicidade , Sistema Nervoso Parassimpático/citologia , Septo do Cérebro/citologia , Aconitato Hidratase/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Colina O-Acetiltransferase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Sistema Nervoso Parassimpático/efeitos dos fármacos , Complexo Piruvato Desidrogenase/metabolismo , Septo do Cérebro/efeitos dos fármacosRESUMO
Eosinophils are observed to localize to cholinergic nerves in a variety of inflammatory conditions such as asthma, rhinitis, eosinophilic gastroenteritis, and inflammatory bowel disease, where they are also responsible for the induction of cell signaling. We hypothesized that a consequence of eosinophil localization to cholinergic nerves would involve a neural remodeling process. Eosinophil co-culture with cholinergic IMR32 cells led to increased expression of the M2 muscarinic receptor, with this induction being mediated via an adhesion-dependent release of eosinophil proteins, including major basic protein and nerve growth factor. Studies on the promoter sequence of the M2 receptor indicated that this induction was initiated at a transcription start site 145 kb upstream of the gene-coding region. This promoter site contains binding sites for a variety of transcription factors including SP1, AP1, and AP2. Eosinophils also induced the expression of several cholinergic genes involved in the synthesis, storage, and metabolism of acetylcholine, including the enzymes choline acetyltransferase, vesicular acetylcholine transferase, and acetylcholinesterase. The observed eosinophil-induced changes in enzyme content were associated with a reduction in intracellular neural acetylcholine but an increase in choline content, suggesting increased acetylcholine turnover and a reduction in acetylcholinesterase activity, in turn suggesting reduced catabolism of acetylcholine. Together these data suggest that eosinophil localization to cholinergic nerves induces neural remodeling, promoting a cholinergic phenotype.
Assuntos
Acetilcolina/metabolismo , Eosinófilos/metabolismo , Neurônios/metabolismo , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Técnicas de Cocultura , Proteínas Granulares de Eosinófilos/metabolismo , Regulação da Expressão Gênica , Humanos , Fator de Crescimento Neural/metabolismo , Neurônios/enzimologia , Comunicação Parácrina , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Receptor Muscarínico M2/biossíntese , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Sítio de Iniciação de Transcrição , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
Nerve growth factor (NGF) exerts anti-apoptotic, trophic and differentiating actions on sympathetic neurons and cholinergic cells of the basal forebrain and activates the expression of genes regulating the synthesis and storage of the neurotransmitter acetylcholine (ACh). We have been studying the intracellular signaling pathways involved in this process. Although, in the rat pheochromocytoma cell line PC12, NGF strongly activates the mitogen-activated protein kinase (MAPK) pathway, prolonged inhibition of MAPK kinase (MEK) activity by PD98059 or U0126 did not affect the ability of NGF to up-regulate choline acetyltransferase (ChAT) or to increase intracellular ACh levels. In contrast, the treatment with the phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002, but not with its inactive analogue LY303511, completely abolished the NGF-induced production of ACh. Inhibition of PI3K also eliminated the NGF effect on the intracellular ACh level in primary cultures of septal neurons from E18 mouse embryos. Blocking the PI3K pathway prevented the activation of cholinergic gene expression, as demonstrated in RT/PCR assays and in transient transfections of PC12 cells with cholinergic locus promoter-luciferase reporter constructs. These results indicate that the PI3K pathway, but not the MEK/MAPK pathway, is the mediator of NGF-induced cholinergic differentiation.
Assuntos
Acetilcolina/biossíntese , Colina O-Acetiltransferase/biossíntese , Fibras Colinérgicas/enzimologia , Regulação da Expressão Gênica/fisiologia , Fator de Crescimento Neural/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Acetilcolina/genética , Animais , Células Cultivadas , Colina O-Acetiltransferase/antagonistas & inibidores , Fibras Colinérgicas/efeitos dos fármacos , Cromonas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Morfolinas/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Células PC12 , Inibidores de Fosfoinositídeo-3 Quinase , RatosRESUMO
High susceptibility of cholinergic neurons to neurotoxic signals may result from their utilization of acetyl-CoA for both energy production and acetylcholine synthesis. SN56 cholinergic cells were transfected stably with cDNA for choline acetyltransferase. Transfected cells (SN56ChAT2) expressed choline acetyltransferase activity and acetylcholine content, 17 times and 2 times higher, respectively, than did nontransfected cells. Transfection did not change pyruvate dehydrogenase but decreased the acetyl-CoA level by 62%. Differentiation by cAMP and retinoic acid caused an increase of choline acetyltransferase activity and decrease of acetyl-CoA levels in both cell lines. Negative correlation was found between choline acetyltransferase activity and acetyl-CoA level in these cells. SN56ChAT2 cells were more susceptible to excess NO than were native SN56 cells, as evidenced by the thiazolyl blue reduction assay. Thus, the sensitivity of cholinergic neurons to pathologic conditions may depend on the cholinergic phenotype-dependent availability of acetyl-CoA.
Assuntos
Acetilcoenzima A/metabolismo , Acetilcolina/metabolismo , Colina O-Acetiltransferase/metabolismo , Neurônios/fisiologia , ATP Citrato (pro-S)-Liase/metabolismo , Acetilcoenzima A/genética , Acetilcolina/genética , Animais , Diferenciação Celular , Sobrevivência Celular , Colina O-Acetiltransferase/genética , AMP Cíclico/metabolismo , Células Híbridas , Camundongos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo , Fenótipo , Complexo Piruvato Desidrogenase/metabolismo , Ratos , Septo Pelúcido/fisiologia , Transfecção , Tretinoína/metabolismo , Células Tumorais CultivadasRESUMO
Nerve growth factor (NGF) is a peptide displaying multiple cholinotropic activities. The aim of this work was to explain mechanisms of the positive and negative effects of NGF on phenotypic properties and viability of cholinergic cells. To discriminate these effects we used two p75NTR receptor-positive lines of cholinergic neuroblastoma cells, SN56 and T17 that are devoid of or express high affinity NGF (TrkA) receptors, respectively. cAMP and retinoic acid caused differentiation of both cell lines. In addition to the morphologic maturation, the increase of choline acetyltransferase activity, acetylcholine, Ca and cytoplasmic acetyl-CoA levels and decrease of mitochondrial acetyl-CoA and cell viability were observed. NGF caused similar effects in non-differentiated T17 cells but had no influence on non-differentiated SN56 cells. On the contrary, in both cAMP/all-trans-retinoic acid (RA) differentiated cell lines, NGF resulted in a similar suppression of cholinergic phenotype along with an increase of mitochondrial acetyl-CoA and cell susceptibility to nitric oxide and amyloid-beta25-35. These effects of NGF were prevented by an antibody against the p75NTR receptor. Data indicate that: (i) positive cholinotrophic effects of NGF required activation of both TrkA and p75NTR receptors; (ii) cAMP/RA-evoked differentiation inhibited NGF effects mediated by TrkA receptors and activated its p75NTR-dependent suppressing influences and (iii) a differentiation-evoked decrease of mitochondrial acetyl-CoA and an elevation of mitochondrial Ca could augment impairment of cholinergic neurons by neurotoxic signals.