Acetylcholine and regulation of gene expression in developing systems.

One of the major questions related to nervous system development is the identification of signals directing neuronal populations to specific phenotypes (e.g., cholinergic, adrenergic, or peptidergic neurons) and involved in cell-to-cell interactions. Although neurotrophins have long been known for their function in development, the neurotransmitter role as modulator of gene expression and differentiation has been recognized only recently. Evidence for the ability of various neurotransmitter molecules to influence various cellular events during neuron differentiation has been reported in several systems (Lauder and Schambra, 1999). We have focused our interest on acetylcholine (ACh) and its possible role in the regulation of neuron-specific gene expression, using different experimental systems: (1) neuroblastoma cell lines, as a model of cholinergic neuron differentiation; (2) dorsal root ganglia (DRG) sensory neurons, which activate the expression of a cholinergic system early in development, in spite of their peptidergic or aminoacidergic neurotransmission; and (3) primary cultures of Schwann cells. Data obtained on each system will be described briefly

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. (C) 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved

Alternative AChE molecular forms exhibit similar ability to induce neurite outgrowth in transfected neuroblastoma clones

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. Since neurite outgrowth can be modulated by neurotrasmitter, we used the N18TG2 neuroblastoma line which is defective for neurotransmitter production in order to evaluate whether AChE may modulate neurite sprouting in non-enzymatic ways. We performed transfections of FB5 cells (a subclone of N18TG2) with three distinct constructs encoding for: i) the glycosylphosphoinositol- anchored AChE form, ii) the tetrameric AChE form and iii) a soluble monomeric AChE form truncated in its C-terminal. A morphometric analysis of retinoic acid differentiated clones was then undertaken. The results revealed that higher AChE expression following transfection brings about a higher ability of the clones to grow fibers with respect to non-transfected or mock-transfected cells, irrespective of the used construct