Marie-Françoise Diebler

Coregulation of Two Embedded Gene Products, Choline Acetyltransferase and the Vesicular Acetylcholine Transporter

Abstract: The gene encoding the vesicular acetylcholine transporter (VAChT) has recently been localized within the first intron of the gene encoding choline acetyltransferase (ChAT) and is in the same transcriptional orientation. These two genes, whose products are required for the expression of the cholinergic phenotype, could therefore be coregulated. We thus tested the effects on VAChT gene expression of the cholinergic differentiation factor/leukemia inhibitory factor and retinoic acid, both of which induce ChAT activity and increase ChAT mRNA levels in cultured sympathetic neurons. These factors increased both the number of binding sites for vesamicol, a specific ligand of VAChT, and VAChT immunoreactivity. This increase in the number of VAChT molecules resulted from an increase in the amount of VAChT mRNA, as assessed by reverse transcription‐PCR and which paralleled that of ChAT mRNAs. These data suggest a functional role for ChAT and VAChT gene organization and are consistent with the existence of a coregulatory mechanism for the embedded ChAT and VAChT genes.

Cloning and expression of the vesamicol binding protein from the marine ray Torpedo. Homology with the putative vesicular acetylcholine transporter UNC-17 from Caenorhabditis elegans.

Complementary DNA clones corresponding to a messenger RNA encoding a 56 kDa polypeptide have been obtained from Torpedo marmorata and Torpedo ocellata electric lobe libraries, by homology screening with a probe obtained from the putative acetylcholine transporter from the nematode Caenorhabditis elegans. The Torpedo proteins display approximately 50% overall identity to the C. elegans unc‐17 protein and 43% identity to the two vesicle monoamine transporters (VMAT1 and VMAT2). This family of proteins is highly conserved within 12 domains which potentially span the vesicle membrane, with little similarity within the putative intraluminal glycosylated loop and at the N‐ and C‐termini. The ~ 3.0 kb mRNA species is specifically expressed in the brain and highly enriched in the electric lobe of Torpedo. The Torpedo protein, expressed in CV‐1 fibroblast cells, possesses a high‐affinity binding site for vesamicol (K d = 6 nM), a drug which blocks in vitro and in vivo acetylcholine accumulation in cholinergic vesicles.

Differences in the developmental expression of the vesicular acetylcholine transporter and choline acetyltransferase in the rat brain

The neurotransmitter acetylcholine (ACh) is synthesized by the enzyme choline acetyltransferase (ChAT) and then transported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Since the VAChT gene is located within the first intron of the ChAT gene, it is likely that expression of the two genes is coregulated. We compared the developmental expression of VAChT and ChAT mRNA and protein in rat brain. ChAT mRNA and enzyme activity increased by almost 10-fold from embryonic day 19 to adulthood, with the most pronounced increase occurring after birth. In contrast, VAChT mRNA increased by only about 2-fold from late embryonic stages to adult levels. However, VAChT protein followed the developmental pattern of ChAT activity, revealing a large excess of VAChT mRNA over VAChT protein during early stages of development. The results are suggestive of differential mechanisms of ChAT and VAChT regulation during brain development, and of possible translational control of VAChT expression.

Expression of the vesicular acetylcholine transporter in mammalian cells

Publisher Summary This chapter focuses on the cloning of the Torpedo vesicular acetylcholine transporter and its functional identification by expression in mammalian cells and addresses the issue of the cellular biology of vesicular acetylcholine transporter (VAChT) through its immunological characterization and with regard to the control of the expression of a cholinergic phenotype. The advent of new techniques within molecular biology has recently enabled rapid progress in the molecular identification of vesicular neurotransmitter transporters. The protein was suggested to be the cholinergic vesicular transporter. Starting with this observation, the advantage of the cholinergic nature of the Torpedo electric lobe motoneurons was taken to identify the homologous protein. It was shown that it was the vesamicol binding protein and that it was able to accumulate acetylcholine (ACh) into intracellular organelle. The chapter demonstrates that the coregulation of choline acetyltransferase (ChAT) and VAChT mRNA and protein expression by two classes of factors, which induce differentiation of noradrenergic neurons toward a cholinergic phenotype. Because of the conserved embedded organization of the VAChT and ChAT genes, it is tempting to envision that common enhancer elements permit regulatory factors to ensure the coordinated expression of both the enzyme responsible for the synthesis of acetylcholine and the protein allowing its storage in synaptic vesicles.

A Ewing's Sarcoma Cell Line Showing Some, but Not All, of the Traits of a Cholinergic Neuron

Abstract: The Ewings sarcoma cell line ICB 112 was examined in detail for a cholinergic phenotype. Choline acetyltransferase activity (12.3 ± 2.9 nmol/h/mg of protein) was associated with the presence of multiple mRNA species labeled with a human choline acetyltransferase riboprobe. Choline was taken up by the cells by a high‐affinity, hemicholinium‐3‐sensitive transporter that was partially inhibited when lithium replaced sodium in the incubation medium; the choline taken up was quickly incorporated into both acetylcholine and phosphorylcholine. High‐affinity binding sites for vesamicol, an inhibitor of vesicular acetylcholine transport, were also present. The mRNAs for synaptotagmin (p65) and the 15‐kDa proteolipid were readily detected and were identical in size to those observed in cholinergic regions of the human brain. Cumulative acetylcholine efflux was increased by raising the extracellular potassium level or the addition of a calcium ionophore, but the time course of stimulated efflux was slow and persistent. These results show that this morphologically undifferentiated cell line is capable of acetylcholine synthesis and expresses markers for synaptic vesicles as well as proteins implicated in calcium‐dependent release but lacks an organized release mechanism.

Compared effects of two vesicular acetylcholine uptake blockers, AH5183 and cetiedil, on cholinergic functions in Torpedo synaptosomes: acetylcholine synthesis, choline transport, vesicular uptake, and evoked acetylcholine release

Abstract: We examined the effects of two drugs, AH5183 and cetiedil, demonstrated to be potent inhibitors of acetylcholine (ACh) transport by isolated synaptic vesicles on cholinergic functions in Torpedo synaptosomes. AH5183 exhibited a high specificity toward vesicular ACh transport, whereas cetiedil was shown to inhibit both high‐affinity choline uptake and vesicular ACh transport. Choline acetyltransferase was not affected by either drug. High external choline concentrations permitted us to overcome cetiedil inhibition of high‐affinity choline transport, and thus synthesis of [14C]ACh in treated preparations was similar to that in controls. We then tested evoked ACh release in drug‐treated synaptosomes under conditions where ACh translocation into the vesicles was blocked. We observed that ACh release was impaired only in cetiedil‐treated preparations; synaptosomes treated with AH5183 behaved like the controls. Thus, this comparative study on isolated nerve endings allowed us to dissociate two steps in drug action: upstream, where both AH5183 and cetiedil are efficient blockers of the vesicular ACh translocation, and downstream, where only cetiedil is able to block the ACh release process.

Control of the calcium concentration involved in acetylcholine release and its facilitation: an additional role for synaptic vesicles?

2,5-Diterbutyl-1,4-benzohydroquinone, a specific blocker of Ca2+-ATPase pumps, increased acetylcholine release from an identified synapse of Aplysia, as well as from Torpedo and mouse caudate nucleus synaptosomes. Because 2,5-diterbutyl-1,4-benzohydroquinone does not change the presynaptic Ca2+ influx, the enhancement of acetylcholine release could be due to an accumulation of Ca2+ in the terminal. This possibility was further checked by studying the effects of 2,5-diterbutyl-1,4-benzohydroquinone on twin pulse facilitation, classically attributed to residual Ca2+. While preventing the fast sequestration of Ca2+ by presynaptic organelles, 2,5-diterbutyl-1,4-benzohydroquinone magnified both twin pulse facilitation observed under low extracellular Ca2+ concentration and twin pulse dysfacilitation observed under high extracellular Ca2+ concentration. Thus, it is concluded that 2,5-diterbutyl-1,4-benzohydroquinone, by preventing Ca2+ buffering near transmitter release sites, modulates acetylcholine release. As 2,5-diterbutyl-1,4-benzohydroquinone was also shown to decrease by 50% the uptake of 45Ca2+ by isolated synaptic vesicles, we propose that synaptic vesicles can control the presynaptic Ca2+ concentration triggering the release of neurotransmitter.

Stimuli that induce a cholinergic neuronal phenotype of NG108-15 cells upregulate ChAT and VAChT mRNAs but fail to increase VAChT protein.

The vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) are encoded by genes organized in a single gene locus, and coregulation of the transcription of the two genes has been repeatedly reported in cholinergic tissues. In the present study, different stimuli were used to induce the differentiation of the hybridoma cells NG108-15 and we examined their effects on the modulation of VAChT and ChAT expression at the mRNA and protein levels. All agents upregulated the VAChT and ChAT mRNA levels, but to a different extent. ChAT activity was increased by retinoic acid, dexamethasone, and dibutyrylcyclic AMP (dbcAMP), and a synergistic effect was observed with a combined dexamethasone and dbcAMP treatment. Nonetheless, no changes in the VAChT protein level could be observed, as judged from ligand binding studies as well as from immunochemical detection. Hemicholinium-3-sensitive choline uptake, hemicholinium-3 binding, and acetylcholine content were increased by differentiating agents, with a rank order of potency comparable to their effects on ChAT activity. Prominent changes were observed in the expression of vesicular protein markers, particularly with the associated treatment dexamethasone and dbcAMP. Thus, it appears that although the different stimuli we have been using are able to stimulate neuronal features and activate the transcription of cholinergic genes, they did not contrive to increase the level of VAChT protein in these cells.

Exploring the Regulation of the Expression of ChAT and VAChT Genes in NG108-15 Cells: Implication of PKA and PI3K Signaling Pathways

Involvement of different protein kinases regulated by cAMP and implication of muscarinic receptors in the regulation of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) mRNA levels and ChAT activity has been studied in NG108-15 cells. Dibutyryl cAMP enhanced both ChAT and VAChT mRNA levels and stimulated ChAT activity. Muscarinic stimulation or inhibition did not change ChAT activity or the receptor subtype mRNA pattern. MEK1/2 did not affect the regulation of ChAT and VAChT mRNA levels. However, PKA plays a major role in regulating ChAT and VAChT mRNA levels, because H89 decreased both. Strikingly, inhibition of PI3K by LY294002 had two opposite effects: ChAT mRNA level was decreased and VAChT mRNA level was increased. Such a result consolidates the observation that ChAT and VAChT genes, despite their unusual organization in a single “cholinergic locus,” can be differentially or synergistically regulated, depending on the activated signaling pathways.

More than one way to toy with ChAT and VAChT

Expression of choline acetyltransferase (ChAT) and of the vesicular acetylcholine transporter (VAChT) is required for the acquisition and the maintenance of the cholinergic phenotype. The ChAT and VAChT genes have been demonstrated to share a common gene locus and this suggests a coordinate regulation of their expression. In the present work, we examined the effects of several differentiating treatments on the modulation of ChAT and VAChT expression at the mRNA and protein levels in growing and differentiating NG108-15 cells. In cells grown in the presence of serum, all the agents tested-retinoic acid, dexamethasone and dibutyrylcyclicAMP (dbcAMP)-induced an increase of ChAT and VAChT mRNA levels but with different efficacy. Treatment with dbcAMP plus dexamethasone resulted in the largest increase of VAChT mRNA amount while retinoic acid mostly enhanced ChAT mRNA level. However, while ChAT activity was increased by all agents, no change in the VAChT protein level was detected. In cells differentiated by serum deprivation, only retinoic acid was effective in inducing an increase of VAChT and ChAT mRNA and ChAT activity, while we observed a downregulation by dbcAMP and dexamethasone. Treatment with the antimitotic agent cytosine arabinoside led to an increase of ChAT activity which was further largely enhanced by the addition of dbcAMP plus dexamethasone, but to only a slight change in VAChT activity. We further showed that complex glycosylation processes which might play a role in targeting and/or stability of the membrane protein VAChT are deficient in these cells. Indeed, in transient transfection assays with the reporter soluble enzyme luciferase placed under regulatory and promoter regions of the VAChT gene, we observed a modulation of luciferase expression by differentiating agents. These data illustrate the complexity of the processes which participate to the expression of the ChAT and VAChT genes, both at the transcriptional and posttranslational levels.