Acetylcholine and acetyl-CoA metabolism in differentiating SN56 septal cell line.

The rate of acetylcholine (ACh) synthesis was found to depend on the activity of choline acetyltransferase (ChAT) and on the concentrations of the two substrates of this enzyme, choline and acetyl-CoA. In SN56 cells treated for 3 days with 1 mM dbcAMP activities of ChAT and acetylcholinesterase (AChE) were elevated. It was accompanied by an increased activity of ATP-citrate lyase (ACL)-an enzyme responsible for provision of part of acetyl-CoA for ACh synthesis in cholinergic neurons. In contrast lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) activities were reduced by dbcAMP. Treatment with 0.001 mM all-trans retinoic acid (RA) elevated ChAT and LDH activities but reduced the activities of AChE and ACL. The combined treatment with db-cAMP and tRA increased ChAT activity in supra-additive fashion. The effects of these two compounds on the other enzymes were not additive. Neither compound altered the activities of carnitine acetyl-transferase, acetyl-CoA synthase, or acetyl-CoA hydrolase. On the other hand, they decreased acetyl-CoA content and rate of ACh release. Overall, the results indicate that tRA upregulates only ChAT expression, whereas dbcAMP upregulates several features of cholinergic neurons including ChAT, AChE, and ACL. Low levels of acetyl-CoA in differentiated cells may result in a low rate of ACh release and resynthesis during their depolarization.     

Nerve growth factor and acetyl-L-carnitine evoked shifts in acetyl-CoA and cholinergic SN56 cell vulnerability to neurotoxic inputs

Different groups of brain cholinergic neurons display variable susceptibility to similar neurotoxic inputs. The aim of this work was to find out whether changes in cholinergic phenotype may alter the availability of acetyl-CoA in mitochondrial compartment and thereby the viability of cholinergic neurons. Cyclic AMP (cAMP) and retinoic acid caused differentiation (DC) of T17 TrkA(+) cholinergic neuroblastoma cells. In addition, it increased the choline acetyltransferase (ChAT) activity, Ca(2+) accumulation and cytoplasmic acetyl-CoA level, but decreased mitochondrial acetyl-CoA and cell resistance to amyloid-beta(25-35) (Abeta) toxicity. Nerve growth factor (NGF) caused similar alterations in the nondifferentiated cells (NC). On the other hand, in DC NGF suppressed ChAT activity and elevated mitochondrial level of acetyl-CoA but also caused a further increase of Ca(2+) content and cell susceptibility to Abeta. The significant inverse correlation was found between ChAT activity and mitochondrial levels of acetyl-CoA. Abeta markedly reduced the expression of cholinergic phenotype, acetyl-CoA content, and viability of DC. These effects were absent or much less pronounced in NC. Acetyl-L-carnitine reversed suppressing effects of Abeta on acetyl-CoA levels and ChAT activity but did not reverse increased mortality in DC. Presented data indicate that increased transmitter activity in highly differentiated cholinergic neurons, decreased acetyl-CoA level in their mitochondrial compartment, and increased Ca(2+) accumulation can make them more prone to neurotoxic conditions. Phenotype-dependent changes in intracellular distribution of acetyl-CoA thus play an important role in regulation of viability and transmitter function in brain cholinergic neurons.     

Aluminum, NO, and nerve growth factor neurotoxicity in cholinergic neurons.

Several neurotoxic compounds, including Al, NO, and beta-amyloid may contribute to the impairment or loss of brain cholinergic neurons in the course of various neurodegenerative diseases. Genotype and phenotypic modifications of cholinergic neurons may determine their variable functional competency and susceptibility to reported neurotoxic insults. Hybrid, immortalized SN56 cholinergic cells from mouse septum may serve as a model for in vitro cholinotoxicity studies. Differentiation by various combinations of cAMP, retinoic acid, and nerve growth factor may provide cells of different morphologic maturity as well as activities of acetylcholine and acetyl-CoA metabolism. In general, differentiated cells appear to be more susceptible to neurotoxic signals than the non-differentiated ones, as evidenced by loss of sprouting and connectivity, decreases in choline acetyltransferase and pyruvate dehydrogenase activities, disturbances in acetyl-CoA compartmentation and metabolism, insufficient or excessive acetylcholine release, as well as increased expression of apoptosis markers. Each neurotoxin impaired both acetylcholine and acetyl-CoA metabolism of these cells. Activation of p75 or trkA receptors made either acetyl-CoA or cholinergic metabolism more susceptible to neurotoxic influences, respectively. Neurotoxins aggravated detrimental effects of each other, particularly in differentiated cells. Thus brain cholinergic neurons might display a differential susceptibility to Al and other neurotoxins depending on their genotype or phenotype-dependent variability of the cholinergic and acetyl-CoA metabolism.     

Cholinergic differentiation triggered by blocking cell proliferation and treatment with all-trans-retinoic acid

This study determined whether the effect of all-trans-retinoic acid (t-RA) on markers of cholinergic differentiation in a murine septal cell line, SN56.B5.G4, differed depending upon the cell's proliferative status. To develop a model of non-proliferating cells, aphidicolin, a DNA alpha-polymerase inhibitor, was used. Cessation of proliferation by aphidicolin increased intracellular choline and acetylcholine (ACh) levels in the absence of change to choline acetyltransferase (ChAT) activity and mRNA and vesicular ACh transporter (VAChT) mRNA. Importantly, the response to t-RA differed depending upon proliferative status. Consistent with previous reports, t-RA increased ChAT and VAChT mRNA, ChAT activity and intracellular ACh levels in proliferating SN56 cells with no effect on intracellular choline levels. When cells were treated with t-RA while undergoing proliferative arrest, an additive effect of combined treatment was observed on ACh levels; nevertheless, this was only accompanied by an increase in choline levels, VAChT and ChAT mRNAs, but not ChAT activity. Indeed, aphidicolin treatment completely suppressed the t-RA-induced increase in ChAT activity observed in proliferating cells. To explore the response to t-RA in post-mitotic cells, a sequential treatment of aphidicolin and t-RA was employed. t-RA treatment was ineffective in increasing ACh and choline levels, over and above that observed with the aphidicolin treatment alone. Comparable to the combined treatment, sequential treatment lead to an increase in ChAT mRNA without any increase in ChAT activity. In conclusion, both the magnitude and the mechanism(s) of action whereby t-RA enhances the cholinergic phenotype of SN56 cells is dependent upon the cell's proliferative status.     

Experimental immune-mediated damage of septal cholinergic neurons

Degeneration of cholinergic neurons in the medial septum and the diagonal band of Broca is a frequent neuropathological feature of Alzheimer's disease. To determine whether an immune process can injure these basal forebrain cholinergic neurons, we serially immunized guinea pigs with septal cholinergic hybrid cells (SN-56). Following immunization, a relatively selective damage of septal cholinergic neurons, reduction in septal choline acetyltransferase (ChAT) activity and decrease in acetylcholine release in hippocampus were detected. Serum IgG from guinea pigs immunized with SN-56 cells and stereotactically injected into the medial septal region of rats produced a loss of ChAT activity in the medial septum, frontal cortex and hippocampus, together with impairment of learning and long term spatial memory. These data suggest that relatively selective damage to septal cholinergic neurons can be caused by an immune-mediated process in experimental animals.     

Cholinergic differentiation triggered by blocking cell proliferation and treatment with all-trans-retinoic acid

This study determined whether the effect of all-trans-retinoic acid (t-RA) on markers of cholinergic differentiation in a murine septal cell line, SN56.B5.G4, differed depending upon the cell’s proliferative status. To develop a model of non-proliferating cells, aphidicolin, a DNA α-polymerase inhibitor, was used. Cessation of proliferation by aphidicolin increased intracellular choline and acetylcholine (ACh) levels in the absence of change to choline acetyltransferase (ChAT) activity and mRNA and vesicular ACh transporter (VAChT) mRNA. Importantly, the response to t-RA differed depending upon proliferative status. Consistent with previous reports, t-RA increased ChAT and VAChT mRNA, ChAT activity and intracellular ACh levels in proliferating SN56 cells with no effect on intracellular choline levels. When cells were treated with t-RA while undergoing proliferative arrest, an additive effect of combined treatment was observed on ACh levels; nevertheless, this was only accompanied by an increase in choline levels, VAChT and ChAT mRNAs, but not ChAT activity. Indeed, aphidicolin treatment completely suppressed the t-RA-induced increase in ChAT activity observed in proliferating cells. To explore the response to t-RA in post-mitotic cells, a sequential treatment of aphidicolin and t-RA was employed. t-RA treatment was ineffective in increasing ACh and choline levels, over and above that observed with the aphidicolin treatment alone. Comparable to the combined treatment, sequential treatment lead to an increase in ChAT mRNA without any increase in ChAT activity. In conclusion, both the magnitude and the mechanism(s) of action whereby t-RA enhances the cholinergic phenotype of SN56 cells is dependent upon the cell’s proliferative status.     

Trophic effect of granulocyte-macrophage colony-stimulating factor on central cholinergic neurons in vitro

Granulocyte-macrophage colony-stimulating factor (GM-CSF) elevated choline acetyltransferase (ChAT) activities of mouse septal neurons as well as of cholinergic hybridoma line cells SN6.10.2.2 in vitro. It augmented ChAT activities and neurite extension of interleukin 3-activated cholinergic neurons. Thus, GM-CSF should be added as a trophic factor for central cholinergic neurons.     

Stimulation of choline acetyltransferase by C3d, a neural cell adhesion molecule ligand

Septal cholinergic neurons project to the hippocampus and release acetylcholine, a neurotransmitter involved in learning and memory. The enzyme choline acetyltransferase (ChAT) is responsible for synthesizing acetylcholine. Promoting ChAT activity and acetylcholine release can lead to new treatments for neurodegenerative diseases with cholinergic deficits, such as Alzheimer's disease. We present evidence that the synthetic molecule C3d, which is a peptide mimetic of the neural cell adhesion molecule (NCAM), promotes ChAT activity in cultures of rat embryonic septal neurons. Our data demonstrate that ChAT activity triggered by C3d is dependent on the fibroblast growth factor receptor (FGFR) and the mitogen-activated protein kinase (MAPK) pathway. C3d did not affect the number of cholinergic neurons in culture, indicating that NCAM homophilic binding enhances ChAT activity, without affecting cholinergic cell survival. In conclusion, the NCAM mimetic peptide C3d promotes ChAT activity in septal neurons through FGFR and MAPK. These findings are relevant to the design of new strategies aimed at stimulating cholinergic function and improving cognition in disorders such as Alzheimer's disease.     

Coexistence of cholinergic, catecholaminergic, serotonergic, and glutamatergic neurotransmitter markers in mouse clonal hybrid neurons derived from the septal region.

Two clonal immortalized neurons designated SN6.1b and SN6.2a were isolated by limiting dilution from a mouse embryonic septal cholinergic neuronal hybrid cell line SN6 (Hammond et al., 1986). In the serum-containing medium without extra differentiating agents, one-third of SN6.1b cells stably exhibited a morphology of differentiated neurons with extensive elaborate neurites, while a majority of SN6.2a cells, along with the parent cell line SN6, were round in shape with poorly branched short processes. Neurochemical studies showed that both clones synthesized choline acetyltransferase (ChAT), dopamine, norepinephrine, serotonin, and glutamate. Immunocytochemically, they expressed a number of neuronal antigens, such as 200-kDa neurofilament protein, neuron-specific enolase, microtubule-associated protein 2, tau protein, tubulin, neural cell adhesion molecule, Thy-1.2, saxitoxin-binding sodium channel protein, ChAT, tyrosine hydroxylase, serotonin, and glutamate. The coexistence of cholinergic, catecholaminergic, serotonergic, and glutamatergic neurotransmitter markers in the clonal hybrid septal neurons that express a variety of immunocytochemical properties of differentiated neurons suggests that embryonic septal cholinergic neurons are potentially multiphenotypic with respect to neurotransmitter synthesis.     

Neurotrophic effect of hematopoietic cytokines on cholinergic and other neurons in vitro

We examined the effects of interleukin-3 (IL-3) and other hematopoietic cytokines on the neurotransmitters, neurite formation, and differentiation in cholinergic and other types of neurons. IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor, granulocyte colony-stimulating factor and erythropoietin (Epo) elevated choline acetyltransferase (ChAT) activity in septal cholinergic cell line SN6 as well as in primary cultured septal neurons without increasing protein contents of the cells. These effects were dose-dependent and the optimal doses were not different from those for blood cells. IL-3 had neurite-promoting activity but GM-CSF had no such effect. Both IL-3 and GM-CSF decreased intracellular acetylcholine concentration, and elevated glutamic acid decarboxylase and intracellular GABA in septal neuronal cultures. Epo elevated monoamines in PC12 cells. These effects are thought to result from direct action through their specific receptors in neurons, because (i) anti-IL-3-receptor antibody abolished the ChAT activity in septal neurons increased by IL-3; (ii) mRNA and immunoreactivity for β subunits of IL-3 receptors were expressed in septal cholinergic neurons and (iii) presence of receptos for GM-CSF and Epo in neurons has been reported. Our observation and others strongly support that neural-immune interactions are important not only in the defense mechanism in the nervous system but also in the development, differentiation and function of neurons.     

Cholinergic markers are expressed in developing and mature neurons of chick dorsal root ganglia

The presence of acetylcholinesterase has been reported in chick dorsal root ganglia at early developmental stages although acetylcholine is not known to play a role in these ganglia. Recently, we reported that during development the level of acetylcholinesterase increases continuously and the enzyme becomes gradually expressed in all sensory neurons. These observations prompted the study of the developmental pattern of expression of other cholinergic markers, such as choline acetyltransferase (ChAT) and the high affinity transport mechanism for choline. ChAT activity is barely detectable at early developmental stages (E7) and increases markedly thereafter, with an activity profile similar to that described for acetylcholinesterase. A similar increase in enzyme activity is also observed when ChAT is measured in dorsal root ganglia explants and in dissociated cells in culture. The study of ChAT activity in cultured cells shows an increase over a period of 3 days, thus ruling out the hypothesis that motor fibers, still associated to the ganglia, may represent a possible source of the enzyme. Immunostaining of whole ganglia or cultured cells shows that ChAT immunoreactivity is not restricted to a specific neuronal subpopulation but appears as a common marker of sensory neurons. High affinity choline uptake, blocked by hemicholinium, is present in sensory neurons cultured from E7 dorsal root ganglia. Observations on cultured neurons from later stages (E18) indicate that choline transport is not a transient property of sensory neurons. These observations show a similar pattern of expression of several cholinergic markers during development. Such a pattern is maintained at significant levels also in mature ganglia. © 1994 Wiley-Liss, Inc.     

Retinoic acid and nerve growth factor induce differential regulation of nicotinic acetylcholine receptor subunit expression in SN56 cells

Retinoic acid (RA) and nerve growth factor (NGF) have multiple functions in the regulation of neuronal development. In the present study, we characterized the expression of different nicotinic acetylcholine receptor (nAChR) subtypes in the cholinergic SN56 cell line and investigated the roles of RA and NGF in the expression of choline acetyltransferase (ChAT) and different nAChR subtypes. The nAChR agonist [(3)H]epibatidine was bound to two sites, with apparent affinities of 13 and 380 pM. RT-PCR analysis revealed expression of alpha3, alpha4, alpha5, alpha7, beta2, and beta4 nAChR subunits. RA treatment induced morphological changes, and the mRNA level of ChAT was maximally elevated after 4 days of exposure. The density of [(3)H]epibatidine binding sites and the mRNA and protein level of the alpha3 and beta2 nAChR subunits were also increased by RA-induced differentiation. RA down-regulated the mRNA and protein level of the alpha4 nAChR subunit, whereas no significant change was observed in the mRNA and protein level of the alpha7 nAChR subunit. NGF treatment increased the mRNA and protein level of the alpha3 and beta2 nAChR subunits. No morphological effects of NGF were observed, and the mRNA level of ChAT and mRNA and protein level of the alpha4 and alpha7 nAChR subunits were not significantly altered. Validation was performed with real-time RT-PCR. The present results show that RA and NGF have different effects on the expression of ChAT and the morphology and the expression pattern of different nAChR subunits in cholinergic SN56 cells.     

Acute and chronic effects of aluminum on acetyl-CoA and acetylcholine metabolism in differentiated and nondifferentiated SN56 cholinergic cells

Mechanisms of preferential loss of cholinergic neurons in the course of neurodegenerative diseases are unknown. Therefore, we investigated whether differentiation-evoked changes in acetyl-CoA and acetylcholine metabolism contribute to the susceptibility of cholinergic neuroblastoma to cytotoxic effects of Al. In SN56 cells differentiated with retinoic acid and dibutyryl cAMP (DC), pyruvate utilization and acetyl-CoA content were lower and acetylcholine level higher than in nondifferentiated cells (NC), respectively. In DC Al and Ca accumulations were 50% and 100%, respectively higher than in NC. Acute Al addition caused inhibition, whereas its chronic application had no effect on pyruvate utilization both in NC and in DC. On the other hand, in both experiments, Al evoked a greater decrease of acetyl-CoA level in DC than in NC. Acute addition of Al depressed acetylcholine release from DC to two times lower values than in NC. On the other hand, chronic addition of Al increased ACh release from DC over twofold, being without effect on its release from NC. These findings indicate that higher accumulation of Ca, along with low levels of acetyl-CoA, could make DC more susceptible to neurotoxic inputs than NC. Excessive acetylcholine release, evoked by Al, is likely to increase acetyl-CoA utilization for resynthesis of the neurotransmitter pool and cause deficit of this metabolite in DC. On the other hand, NC, owing to lower Ca accumulation, slower ACh metabolism, and higher level of acetyl-CoA, would be less prone to these harmful conditions.     

Changes in the septohippocampal cholinergic system following removal of molar teeth in the aged SAMP8 mouse

We investigated the effect of dysfunctional teeth on age-related changes in the septohippocampal cholinergic system by assessing acetylcholine (ACh) release and choline acetyltransferase (ChAT) activity in the hippocampus and ChAT immunohistochemistry in the medial septal nucleus and the vertical limb of the diagonal band in young-adult and aged SAMP8 mice after removal of their upper molar teeth (molarless condition). Aged molarless mice showed decreased ACh release and ChAT activity in the hippocampus and a reduced number of ChAT-immunopositive neurons in the medial septal nucleus compared to age-matched control mice, whereas these effects were not seen in young-adult mice. The results suggest that the molarless condition in aged SAMP8 mice may enhance an age-related decline in the septohippocampal cholinergic system.     

Cholinergic function in cultures of mouse spinal cord neurons

Cholinergic synapses formed in cultures of fetal mouse spinal cord (SC) and superior cervical ganglion (SCG) were studied using intracellular and extracellular stimulation and recording as well as immunohistochemical staining for choline acetyltransferase (ChAT). Dissociated SC neurons and SC explants exhibited cholinergic terminals on SCG and SC neurons as demonstrated by ChAT immunoreactivity. Intracellular recordings showed that cholinergic inputs to SCG neurons were relatively common and that these synaptic inputs were blocked by the nicotinic acetylcholine (ACh) receptor blocker, tubocurarine. A comparison of three preparations indicated that the incidence of cholinergic activity recorded in SCG neurons was significantly higher in co-cultures of SCG with spinal cord ventral horn (VH) neurons grown on a substrate of non-neuronal cells from cerebral cortex, than in co-cultures with VH alone or with SC and dorsal root ganglion cells. Consistency between cholinergic physiology and staining for ChAT-positive terminals on SCG neuronal somata was obtained in cultures of SC explants grown with dissociated SCG. Application of acetylcholine, muscarine, and/or vasoactive intestinal polypeptide (VIP) produced slow excitation of SC neurons. Fast excitatory cholinergic interactions between SC neurons were not observed. Excitatory synaptic interactions between SC neurons were augmented by ACh or muscarine, while inhibitory synaptic interactions were diminished. Both types of synaptic modulation probably were produced by a presynaptic mechanism. Acetylcholine or muscarine affected synaptic interactions between SC neurons in only one-third of the synaptic connections tested, suggesting that the incidence of presynaptically cholinoceptive SC neurons is low in dissociated cell cultures. The experimental results show that a culture system incorporating dissociated fetal mouse SC neurons or explants of SC with sympathetic ganglion neurons expresses both nicotinic and muscarinic cholinergic function.     

Characterization of a neurotrophic factor produced by cultured astrocytes involved in the regulation of subcortical cholinergic neurons.

When dissociated subcortical cells were cultured in the presence of conditioned medium of relatively differentiated astrocytes (ACM), a marked increase was observed in the expression of choline acetyltransferase (ChAT), an enzyme required for the synthesis of the neurotransmitter acetylcholine. Astrocytes from the target regions of subcortical neurons, the hippocampus and the cerebral cortex, produced neurotrophic factor consistently more than those derived from the nontarget region, the cerebellum. The production of cholinergic trophic activity was increased with the maturation of astrocytes. Even though, nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF) are known cholinergic trophic compounds produced by astrocytes in vitro, a large part of the neurotrophic activity in our ACM was not related to either of these 2 factors. This is because (i) ACM and NGF produced an additive effect on ChAT activity, (ii) only a small proportion of the cholinergic trophic activity in ACM was abolished by anti-NGF antibody, and (iii) treatment with CNTF had no effect on ChAT activity of basal forebrain cholinergic neurons. On the other hand, when cholinergic neurons are cultured on a preformed layer of astrocytes, addition of basal fibroblast growth factor (bFGF) failed to increase further the ChAT activity. Similarly the effects of ACM and bFGF were not additive. A large proportion of the cholinergic trophic activity in ACM was neutralized by anti-bFGF antibody. These findings would suggest that the trophic activity on septal cholinergic neurons in our ACM was due to bFGF or a bFGF-like compound.     

The effect of selective lesions on vestibular nuclear complex choline acetyltransferase activity in the rat

Biochemical, physiological and behavioral evidence suggests that acetylcholine (ACh) may play a neurotransmitter role in central vestibular function. However, the anatomic basis for a possible cholinergic influence on the vestibular nuclear complex (VNC) is unknown. To investigate vestibular cholinergic anatomy, we have made selective lesions of neurons intrinsic to the VNC, and of most known afferents to the VNC, and we have measured the activity of choline acetyltransferase (ChAT), a specific marker for cholinergic neurons, following such lesions. We found that a kainic acid lesion of the VNC, and lesions of vestibular afferents, including the VIIIth cranial nerve, cerebellum, spinal cord, vestibular commissure and the interstitial nucleus of Cajal, did not affect VNC ChAT activity. We conclude that there are no cholinergic neurons intrinsic to the VNC, and that these lesioned afferents are not cholinergic. It is likely, therefore, that a cholinergic projection to the VNC arises from a region other than those lesioned; possibilities include the nuclei of the reticular formation, the upper cervical cord and local pontomedullary nuclei.     

Cholinergic neurotoxicity induced by ethylcholine aziridinium (AF64A) in neuron-enriched cultures

The sequence of events in neuronal changes induced by the cholinotoxin ethylcholine aziridinium (AF64A) was studied. Neuron-enriched cultures derived from 8-day-embryonic chick cerebra were treated with AF64A at concentrations of 10(-5), 10(-4) and 10(-3) M. Choline acetyltransferase (ChAT) was used as an index of cholinergic neurons. Changes in cell morphology, the immunocytochemical and biochemical presence of ChAT, and DNA and protein content were assessed. Neuron-enriched cultures exposed to AF64A showed a dose-dependent response; after 24 h of exposure to 10(-3) M toxin all cells were dead, whereas a concentration of 10(-5) M did not alter culture morphology or DNA and protein contents. Despite the lack of cytological changes and the presence of ChAT immunoreactivity, biochemically assessed ChAT activity was reduced 36% in 10(-5) M treated cultures. Thus, the implicated decrease in acetylcholine synthesis in these cells cannot entirely account for the neuronal degeneration. Simultaneous exposure of cultures to both AF64A and 10 times higher concentrations of choline chloride delayed or diminished the neurotoxic changes. The protective effect of high choline concentrations was interpreted as evidence of competition between choline and AF64A for the high affinity choline transport system and as constituents in the cell membrane. Examination of the temporal sequence of cytotoxic changes in 10(-4) M exposed cultures revealed that disruption of neuronal aggregates and fragmentation of neurites occurred between 4 and 8 hours of exposure. After 24 h, some neurons survived but with attenuated arbors; in contrast, astrocytes appeared intact, suggesting that glial cells are more resistant than neurons to the toxic effects of AF64A. These findings suggest this culture model may be useful to further elucidate the mechanisms of AF64A drug action and study differentiation of cultured neuronal populations in the absence of cholinergic cells.