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

doi:10.1016/S0306-4522(02)00179-3

Neuroscience

Volume 113, Issue 2, 12 August 2002, Pages 331-338

https://doi.org/10.1016/S0306-4522(02)00179-3 Get rights and content

Abstract

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.

Section snippets

Neuroblastoma cell cultures

N18TG2 cells, derived from mouse C-1300 neural crest tumor (Nelson et al., 1976), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS). ChAT-transfected clones were obtained as previously described (Bignami et al., 1997) using the pcDL1-ChAT expression plasmid (Ishii et al., 1990) which contains a 2.1-kb insert for the complete coding sequence of rat ChAT. The transfected clones were cultured in DMEM supplemented with 10% FCS and 400 μg/ml

ChAT activity

The stable expression of ChAT gene was detected by biochemical enzyme assay. ChAT specific activity was measured in N18TG2 cells and in ChAT-transfected clones. As expected ChAT activity was undetectable in the N18TG2 cells while the enzyme activity levels in the transfected clones were about 3 and 17 nmol of ACh synthesized/min/mg of protein for ChAT-positive clones 3/1 and 2/4 respectively (Table 1), higher than in a cholinergic cell line such as the hybrid 108CC15 neuroblastoma line (Bignami

Discussion

Two major events of the neuronal differentiation program have been considered in the described experiments: neurite outgrowth, which is a necessary step for reaching the target cells, and expression of synapsin I, a protein involved in neurotransmitter release. The possible role of ACh synthesis activation on the regulation of the two events has been addressed in an experimental system represented by a neurotransmitter silent neuroblastoma clone and upon activation of ACh synthesis by

Conclusion

The data reported here are consistent with the hypothesis that the forced expression of ChAT in neurotransmitter-inactive neuroblastoma cells brings about the activation of an autocrine loop via muscarinic receptors, which is responsible for the modulation of neuron-specific trait expression. It is pertinent to recall that muscarinic receptor activation is known to induce the transient expression of immediate early genes, as c-jun and c-fos (Treyo and Brown, 1991). At the same time the data

Acknowledgements

This work was supported by funds from MURST Cofin and Institute Pasteur-Fondazione Cenci-Bolognetti.

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

Abstract

Section snippets

Neuroblastoma cell cultures

ChAT activity

Discussion

Conclusion

Acknowledgements

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Int. Rev. Cytol.

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Neurosci. Lett.

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

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A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine

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