The mechanisms and possible sites of acetylcholine release during chick primary sensory neuron differentiation.

AIMS: In this study, we evaluated the ability of differentiating embryonic chick DRG neurons to release and respond to acetylcholine (ACh). In particular, we investigated the neuronal soma and neurites as sites of ACh release, as well as the mechanism(s) underlying this release. MAIN METHODS: ACh release from DRG explants in the Campenot chambers was measured by a chemiluminescent assay. Real-time PCR analysis was used to evaluate the expression of ChAT, VAChT, mediatophore and muscarinic receptor subtypes in DRGs at different developmental stages. KEY FINDINGS: We found that ACh is released both within the central and lateral compartments of the Campenot chambers, indicating that ACh might be released from both the neuronal soma and fibers. Moreover, we observed that the expression of the ChAT and mediatophore increases during sensory neuron differentiation and during the post-hatching period, whereas VAChT expression decreases throughout development. Lastly, the kinetics of the m2 and m3 transcripts appeared to change differentially compared to the m4 transcript during the same developmental period. SIGNIFICANCE: The data obtained demonstrate that the DRG sensory neurons are able to release ACh and to respond to ACh stimulation. ACh is released both by the soma and neurite compartments. The contribution of the mediatophore to ACh release appears to be more significant than that of VAChT, suggesting that the non-vesicular release of ACh might represent the preferential mechanism of ACh release in DRG neurons and possibly in non-cholinergic systems.

beta-Catenin and actin reorganization in HGF/SF response of ST14A cells.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic factor that activates proliferation, differentiation, and migration of various cell types. Its action is mediated by c-Met, a receptor endowed with tyrosine kinase activity that activates complex signaling cascades and mediates diverse cell responses. Although HGF action was first demonstrated in epithelial cells, expression of HGF and c-Met receptor has also been described in developing and adult mammalian brain. In the developing central nervous system, areas of HGF and c-Met expression are coincident with the migratory pathway of precursor cells. In the present article we report that the interaction between c-Met and HGF/SF in striatal progenitor ST14A cells triggers a signaling cascade that induces modification of cell morphology, with decreased cell-cell interactions and increased cell motility; in particular, we analyzed the reorganization of the actin cytoskeleton and the delocalization of beta-catenin and N-cadherin. The testing of other neurotrophic factors (NGF, BDNF, NT3, and CNTF) showed that the observed modifications were peculiar to HGF. We show that phosphoinositide 3-kinase inhibitor treatment, which blocks cell scattering induced by HGF/SF, does not abolish actin and beta-catenin redistribution. The effects of HGF/SF on primary spinal cord cell cultures were also investigated, and HGF/SF was found to have a possible motogenic effect on these cells. The data reported suggest that HGF could play a role in the early steps of neurogenesis as a motogenic factor.

Acetylcholinesterase modulates neurite outgrowth on fibronectin.

Acetylcholinesterase (AChE) has been reported to be involved in the modulation of neurite outgrowth. To understand the role played by different domains, we transfected neuroblastoma cells with three constructs containing the invariant region of AChE, differing in the exon encoding the C-terminus and therefore in AChE cellular fate and localization. All isoforms increased neurite extension, suggesting the involvement of the invariant domain [A. De Jaco, G. Augusti-Tocco, S. Biagioni, Alternative AChE molecular forms exhibit similar ability to induce neurite outgrowth, J. Neurosci. Res. 70 (2002) 756-765]. The peripheral anionic site (PAS) is encoded by invariant exons and represents the domain involved in non-cholinergic functions of AChE. Masking of PAS with fasciculin results in a significant decrease of neurite outgrowth in all clones overexpressing AChE. A strong reduction was also observed when clones were cultured on fibronectin. Treatment of clones with fasciculin, therefore masking PAS, abolished the fibronectin-induced reduction. The inhibition of the catalytic site cannot revert the fibronectin effect. Finally, when clones were cultured on fibronectin in the presence of heparin, a ligand of fibronectin, the inhibitory effect was completely reversed. Our results indicate that PAS could directly or indirectly mediate AChE/fibronectin interactions.

Can plasma glucose and HbA1c predict fetal growth in mothers with different glucose tolerance levels?

To assess whether HbA1c and plasma glucose predicts abnormal fetal growth, 758 pregnant women attending 5 Diabetic Centers were screened for gestational diabetes mellitus (GDM). On glucose challenge (GCT) at 24-27 weeks of gestation (g.w.), negative cases formed the normal control group (N1). Positive cases took an oral glucose tolerance test (OGTT): those found negative were classed as false positives screening test (N2); if they had an OGTT result at least as high as their normal glucose levels, they were classed as having one abnormal glucose value (OAV) at OGTT; two values as GDM. HbA1c was assayed on the day of GCT. We considered fetal macrosomia, large for gestational age (LGA), ponderal index and mean growth percentile. Mean age, pre-pregnancy BMI, fasting plasma glucose (FPG) and HbA1c were progressively higher from N1 to GDM patients. The newborn of N2 mothers were heavier than those with N1 or GDM. The mean growth percentile was significantly higher in N2 than in N1. More LGA babies were born to OAV than to N1 or N2 women. Macrosomia and ponderal index did not differ significantly in the four groups. At logistic regression only plasma glucose at GCT could predict LGA babies and a ponderal index above 2.85. At risk analysis, GDM and OAV significantly predicted LGA babies, and GDM a ponderal index >2.85. In conclusion, FPG at GCT could predict fetal overgrowth and plasma glucose >85mg/dl doubles the risk of LGA infants. HbA1c at 24-27g.w. does not predict fetal overgrowth. Mild alterations in glucose tolerance correlate with fetal overgrowth and needs monitoring and treatment.

Hepatocyte growth factor stimulates cell motility in cultures of the striatal progenitor cells ST14A.

Hepatocyte growth factor/scatter factor (HGF/SF) is a growth factor with pleiotropic effects on different cell types. It acts as a mitogen and motility factor for many epithelial cells. HGF/SF and its receptor Met are present in the developing and adult mammalian brain and control neuritogenesis of sympathetic and sensory neurons. We report that the striatal progenitor ST14A cells express the Met receptor, which is activated after binding with HGF/SF. The interaction between Met and HGF/SF triggers a signaling cascade that leads to increased levels of c-Jun, c-Fos, and Egr-1 proteins, in agreement with data reported on the signaling events evoked by HGF in other cellular types. We also studied the effects of the exposure of ST14A cells to HGF/SF. By time-lapse photography, we observed that a 24-hr treatment with 50 ng/ml HGF/SF induced modification in cell morphology, with a decrease in cell-cell interactions and increase of cell motility. In contrast, no effect on cell proliferation was observed. To investigate which intracellular pathway is primarily involved we used PD98059 and LY294002, two specific inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAP-kinase/ERK-kinase) and phosphoinositide 3-OH kinase (PI3-K), respectively. Cell motility in HGF/SF treated cultures was inhibited by LY294002 but not by PD98059, suggesting that PI3-K plays a key role in mediating the HGF/SF-induced dissociation of ST14A cells. Previous evidence of HGF stimulation of motility in nervous system has been obtained on postmitotic neurons, which have already acquired their specificity. Data reported here of a motogenic response of ST14A cell line, which displays properties of neuronal progenitors, seem of interest because they suggest that HGF could play a role in very early steps of neurogenesis.

Alternative acetylcholinesterase molecular forms exhibit similar ability to induce neurite outgrowth.

Several groups have reported that acetylcholinesterase (AChE), through a mechanism not involving its catalytic activity, may have a role in fiber elongation. These observations were performed on experimental systems in which acetylcholine synthesis was active. Because neurite outgrowth can be modulated by neurotransmitters, we used the N18TG2 neuroblastoma line, which is defective for neurotransmitter production, to evaluate whether AChE may modulate neurite sprouting in nonenzymatic ways. To avoid the possibility that differences between transfected and mock-transfected clones may be due to the selection procedure, N18TG2 cells were previously subcloned, and the FB5 subclone was used for transfections. We performed transfections of FB5 cells with three distinct constructs encoding for the glycosylphosphoinositol-anchored AChE form, the tetrameric AChE form, and a soluble monomeric AChE form truncated in its C-terminus. A morphometric analysis of retinoic acid-differentiated clones was also undertaken. The results revealed that higher AChE expression following transfection brings about a greater ability of the clones to grow fibers with respect to nontransfected or mock-transfected cells irrespective of the used construct. Having observed no differences between the morphology of the transfected clones, we tested the possibility that the culture substrate can affect the capability of the clones to extend fibers. Also in this case we revealed no differences between the clones cultured on uncoated or collagen-pretreated dishes. These data indicate that alternative AChE molecular forms that differ in their C-teminal region exhibit similar ability to induce fiber outgrowth and suggest that the protein region responsible for this role is located in the invariant portion of the AChE molecule.

Muscarinic acetylcholine receptors induce neurite outgrowth and activate the synapsin I gene promoter in neuroblastoma clones.

The possible role of acetylcholine as a modulator of neuronal differentiation has been tested using a neuroblastoma cell line (N18TG2), which does not synthesize any neurotransmitter. Acetylcholine synthesis has been activated in this line by transfection with a construct containing a choline acetyltransferase (ChAT) cDNA; ChAT-positive clones share a higher ability to grow fibers and an activation of synapsin I expression compared to the parental cells. Atropine, a muscarinic antagonist, abolishes the higher ability to grow fibers of ChAT-positive transfected clones, and the cholinergic agonist carbachol induces higher neurite outgrowth in the parental line. In transient transfections of ChAT-positive clones, the expression of a reporter gene under the control of synapsin I promoter is considerably reduced by atropine, while it is not modified by carbachol; in contrast, in the parental cells, which do not synthesize acetylcholine, the reporter gene expression is induced by carbachol and this effect is abolished by atropine. The data presented provide evidence for the existence of a direct modulation of fiber outgrowth and synapsin I expression by muscarinic receptor activation, which may be related to early growth response gene-1 (EGR-1) levels.

Impairment of cell cycle progression by aflatoxin B1 in human cell lines.

Aflatoxin B1 is a mycotoxin produced by Aspergillus flavus and Aspergillus parasiticum, which may be present as a food contaminant. It is known to cause acute toxic effects and act as a carcinogenic agent. The carcinogenic action has been related to its ability to form unstable adducts with DNA, which represent possible mutagenic sites. On the other hand, the primary cellular target responsible for its toxic action has not yet been clearly identified. Previous data suggested a possible correlation between cell proliferation and responsiveness to aflatoxin toxicity. These observations led us to investigate the effect of the toxin on cell cycle progression of three human cell lines (HepG2, SK-N-MC and SK-N-SH derived from liver and nervous tissue tumours); they were shown to display different responses to toxin exposure and have different growth kinetics. We performed analysis of the cell cycle, DNA synthesis and expression of p21 and p53 in the presence and absence of the toxin in all cell lines exposed. The results of cell cycle cytofluorometric analysis show significant alterations of cell cycle progression as a result of toxin treatment. In all cell lines exposure to a 24 h toxin treatment causes a dose-dependent accumulation in S phase, however, the ability to recover from impairment to traverse S phase varies in the cell lines under study. SK-N-MC cells appear more prone to resume DNA synthesis when the toxin is removed, while the other two cell lines maintain a significant inhibition of DNA synthesis, as indicated by cytofluorimetry and [(3)H]dTR incorporation. The level of p53 and p21 expression in the three cell lines was examined by western blot analysis and significant differences were detected. The ready resumption of DNA synthesis displayed by SK-N-MC cells could possibly be related to the absence of p53 control of cell cycle progression.

Electrospray ionisation tandem mass spectrometry in the characterisation of isomeric benzofurocoumarins.

A set of aminoalkoxy-substituted, differently annullated furocoumarins, differing in the position of the aminoalkoxy chain and in the unsaturation level of the fused ring, has been subjected to electron impact and electrospray ionisation (ESI) experiments. In order to achieve a distinct characterisation of isomeric compounds, which partially failed under electron impact conditions, collision-induced dissociation experiments were performed on protonated molecules. The breakdown curves obtained by varying the tickle voltage on an ion trap ESI instrument led to the desired characterisation.

In vitro and in vivo inhibition of SK-N-MC neuroblastoma growth using cyclic nucleotide phosphodiesterase inhibitors.

The effect of cyclic nucleotide phosphodiesterase (PDE) inhibitors Zaprinast and DC-TA-46 has been tested on SK-N-MC neuroblastoma growth. Antiproliferative activity of the tested drugs was assayed both in vitro and in the xenograft model of nude mice. In clonal density experiments, the IC50 value was 3.3 microM for Zaprinast and 1.9 microM for DC-TA-46, while 7.5 microM BCNU alkylating agent was required to obtain the same effect. SK-N-MC cells xenografted in the nude mouse showed that the administration of Zaprinast and DC-TA-46 caused a significant 50% decrease of the tumour weight. These data demonstrate that PDE inhibitors may be useful for at least reducing tumour growth; they may be of interest for further evaluation as alternative molecules in the design of multiple agent protocols for neuroblastoma treatment.

Acetylcholine synthesis and neuron differentiation.

Development of the nervous system is dependent on the co-operation between cell determination events and the action of epigenetic factors; in addition to well known factors, e.g. growth factors, neurotransmitters have been assigned a role as "morphogens" and modulators of neuronal differentiation in an early developmental phase. The possible role of acetylcholine as a modulator of neuronal differentiation has been considered in two experimental systems. A neuroblastoma cell line, which does not synthesise any neurotransmitter, has been transfected with a choline acetyltransferase construct; activation of acetylcholine synthesis, thus achieved, is followed by a higher expression of neuronal specific traits. The presence in these cells of muscarinic receptors is consistent with the existence of an autocrine loop, which may be responsible for the more advanced differentiation state observed in the transfected cells. Expression of cholinergic markers appears as a common feature of DRG sensory neurons, independently of the neurotransmitter used. Choline acetyltransferase can be detected in DRG at early developmental stages. The distribution of muscarinic receptors in DRG has suggested that activation of acetylcholine synthesis may be related in an early developmental phase to the interaction between neurons and nonneuronal cells and to modulation of cell differentiation. Both systems suggest that acetylcholine may have a role as a modulator of neuronal differentiation.

Modulation of cholinergic marker expression by nerve growth factor in dorsal root ganglia.

The presence of cholinergic markers in sensory ganglia has suggested a possible functional role of acetylcholine both as a cofactor of morphogenesis in embryonic life and in sensory transduction during adult life. Acetylcholine, in fact, is able to excite cutaneous nociceptors and to modulate noxious stimuli. Nerve growth factor (NGF) overexpression induces the survival of nociceptive neurons, the expression of their specific markers, and hyperalgesia. On the other hand, NGF modulate the levels of cholinergic markers in several area of nervous system. Considering these observations, the present work aims to investigate whether NGF is able also to control the expression of cholinergic markers in chick sensory neurons in culture. We selected three developmental stages (E8, E12, and E18) representative of different phases of chick embryo development and performed observations on culture in which NGF was omitted at the plating time, withdrawn after the initial 24 hr of culture or maintained for 48 hr. In the experimental protocol devised, NGF did not significantly affect cell survival. At E12 a 48 hr treatment with NGF causes a significant but limited increase in acetylcholinesterase activity; activity increase was not observed when NGF was removed after 24 hr. No changes in acetylcholinesterase activity were observed at E8 and E18 stages. NGF appears to be more effective in the modulation of choline acetyltransferase activity. At E12, in fact, about a doubling of enzyme activity was measured after 24 or 48 hr of treatment with NGF. A response was also found at E18, when a 50% increase in choline acetyltransferase activity was observed just after 24 hr treatment. The behavior of muscarinic receptors in response to NGF differs compared to the two cholinergic enzymes. At E8 and E12 a profound increase in muscarinic receptor expression was observed. Conversely, at E18 NGF produces a 50% reduction of receptors. Considering these observations and the demonstrated role of muscarinic receptors in the desensitization of nociceptors, the reduction of muscarinic receptors in DRG after NGF treatment is in agreement with the proposed algogenic action of NGF in the skin.

Modulation of acetylcholinesterase and voltage-gated Na(+) channels in choline acetyltransferase- transfected neuroblastoma clones.

Neurotransmitters appear early in the developing embryo and may play a role in the regulation of neuronal differentiation. To study potential effects of acetylcholine production in neuronal differentiation, we used the FB5 subclone of N18TG2 murine neuroblastoma cells stably transfected with cDNA for choline acetyltransferase. We tested whether the forced acetylcholine production can modify the expression or the cellular localization of different neuronal markers. We studied the activity, localization, and secretion of acetylcholinesterase in view of its possible role in the modulation of the morphogenetic action of acetylcholine and of its proposed role of a regulator of neurite outgrowth. FB5 cells are characterized by a high level of acetylcholinesterase, predominantly released into the culture medium. Acetylcholinesterase secretion into the medium was lower in choline acetyltransferase-transfected clones than in nontransfected and antisense-transfected controls. Moreover, sequential extraction of acetylcholinesterase revealed that detergent-extracted, i.e., membrane-associated, activity was higher in the transfected clones expressing choline acetyltransferase activity than in both control groups. These observations suggest that a shift occurs in the utilization of acetylcholinesterase in choline acetyltransferase-transfected clones from a secretion pathway to a pathway leading to membrane localization. In addition, the choline acetyltransferase-positive clones showed higher densities of voltage-gated Na(+) channels and enhanced high-affinity choline uptake, suggesting the accomplishment of a more advanced differentiated neuronal phenotype. Finally, binding experiments demonstrated the presence of muscarinic acetylcholine receptors in all examined clones. This observation is consistent with the proposed existence of an autocrine loop, which may be important for the enhancement in the expression of neurospecific traits.

A poly(ethylene glycol)-supported quaternary ammonium salt: An efficient, recoverable, and recyclable phase-transfer catalyst

A quaternary ammonium salt readily immobilized on a soluble poly(ethylene glycol) polymer support efficiently catalyzes different reactions carried out under phase-transfer catalysis conditions; the catalyst, easily recovered by precipitation and filtration, shows no appreciable loss of activity when recycled three times.

Muscarinic receptors modulate intracellular calcium level in chick sensory neurons.

In the present work we have studied the variation of intracellular calcium levels induced by muscarinic agonists in chick dorsal root ganglia neurons. Muscarinic agonists such as muscarine and oxotremorine cause an increase of intracellular calcium levels in fura-2AM-loaded DRG neurons of E18 chick embryos. This increase was abolished following treatment with 1 microM atropine but not by 1 microM mecamylamine, indicating that the observed intracellular calcium increase, was dependent on muscarinic receptor activation. Stimulation in absence of external calcium or pre-incubation of the DRG cultures with thapsigargin or Mn(2+) demonstrated that [Ca(2+)](i) increase is mainly due to its release from intracellular stores. The use of selective antagonists of muscarinic receptor subtypes also indicated that M(1) and to a lesser extent M(3) receptor subtypes are responsible for the observed intracellular calcium mobilization. Finally pre-treatment of DRG cultures with pertussis toxin showed that the variation of [Ca(2+)](i) levels was dependent on PTX-insensitive G-protein. Moreover muscarinic agonists induce in DRG also the increase of IPs level, suggesting that the variations of intracellular calcium levels may be due at least in part to the activation of the phosphoinositide transduction pathway. In conclusion the reported observations demonstrate the activity of muscarinic receptors in sensory neurons, suggesting a functional role for acetylcholine in sensory transduction.

Activation of neurospecific gene expression by antennapedia homeobox peptide.

Antennapedia homeobox peptide has been reported to enhance neurite outgrowth and branching. Thus it is of interest to investigate whether antennapedia peptide is capable of modulating the expression of genes related to different events of neuronal development. In this paper we report the enhancement of a 68 KDa neurofilament subunit, choline acetyltransferase and acetylcholinesterase expression in spinal cord neurons, elicited by antennapedia peptide. Modulation of gene expression is different with respect to each gene product analyzed, suggesting a specific action of the peptide on diverse genes controlling different events of neuronal differentiation.

Neuronal glycolytic pathway impairment induced by HIV envelope glycoprotein gp120.

Neurological impairment is a common feature of Acquired Immunodeficiency Syndrome (AIDS); functional alterations have been reported both in central and peripheral nervous system and the Human Immunodeficiency Virus (HIV) envelope glycoprotein gp120 has been proposed as a neurotoxin acting through a calcium-dependent mechanism. On the other hand it has been reported that gp120 treatment also induce about a 20% decrease in the cerebral glucose utilization and in the cellular ATP levels. The reported observations were performed on experimental system where also non-neuronal cells where present; in order to evaluate whether a direct interaction between HIV proteins and neuronal cells takes place, we used a neuroblastoma cultures where only neuronal cells are present. We analysed the effects of gp120 on the N18TG2 neuroblastoma clone. Treatments were performed both on growing and confluent cultures. Short time treatment with gp120 of confluent cultures causes a 25% reduction in the level of neuron-specific enolase, resulting in a similar decrease of oxygen consumption. Long time exposure of growing cells also causes a reduction in cell survival. Furthermore, using a membrane-specific fluorescent probe we observed that gp120 produces an increase of membrane trafficking. These observations suggest a direct interaction between the viral envelope protein and neuronal cells, which results in an alteration of glycolytic metabolism. This alteration may be related to the neurologic impairments observed in AIDS patients.

Variations of synaptotagmin I, synaptotagmin IV, and synaptophysin mRNA levels in rat hippocampus during the estrous cycle.

Periodic changes in ovarian steroid levels during fertility cycles affect learning both in humans and in rats in parallel with electrophysiological and morphological fluctuations in selective neuronal populations. In particular, during the estrous cycle of the female rat, hippocampal CA1 region undergoes cyclic modifications in synaptic density. To investigate the molecular mechanisms involved in synaptic remodeling during the estrous cycle, we analyzed the expression of three presynaptic markers, synaptotagmin I, synaptotagmin IV, and synaptophysin, in the female adult rat brain by in situ hybridization. Relative abundance in mRNA for these three markers was quantified at four phases of the estrous cycle: diestrus, proestrus (AM and PM), and estrus. mRNA levels for syt1 exhibited cyclic variations in pyramidal neurons of the CA3 region of hippocampus during the estrous cycle, while mRNA levels for syt4 and SYN were relatively invariant in this or other regions of the hippocampus. Because CA3 pyramidal neurons make synaptic contacts in CA1, modulation of syt1 expression in CA3 may participate in the changes in synaptic density observed in CA1 during the estrous cycle. Furthermore, both syt1 and SYN mRNA varied cyclically in layer II, but not in layer III of entorhinal cortex, while syt4 remained unchanged throughout the cycle. These data suggest that regular variations in steroid hormone levels during fertility cycles may alter the properties of several networks involved in information processing and learning and memory through altered levels of presynaptic proteins.

Expression of neuron-specific enolase in adult rat brain following status epilepticus.

Increased levels of neuron-specific enolase (NSE), a key glycolytic enzyme, in either the cerebrospinal fluid or the serum is correlated with both the duration and the outcome of status epilepticus. To further understand the molecular basis of seizure-induced elevations in NSE protein, we investigated NSE mRNA expression in the adult rat brain following systemic administration of kainic acid. The findings demonstrated either no change or a decrease in NSE gene expression during, and following, status epilepticus, suggesting that posttranscriptional mechanisms are responsible for seizure-induced increases in NSE protein.