Vesicular Acetylcholine Transporter Gene Research Articles

Developmental age-dependent upregulation of choline acetyltransferase and vesicular acetylcholine transporter mRNA expression in neonatal rat septum by nerve growth factor

We examined the effect of intraventricular injection of nerve growth factor (NGF) on the choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) mRNA expression in the septa of neonatal rats. Rat pups were injected with 2.5 S NGF or cytochrome-c (control) on postnatal days (PN) 4 and 18, and sacrificed 3 days after injections for analysis of ChAT and VAChT mRNA levels by dot-blot hybridization of total septal RNA. In the NGF-treated pups, the ChAT and VAChT mRNA levels were elevated 3- and 2-fold, respectively, at PN7, and 1.8- and 1.3-fold at PN21. These results indicate that (1) NGF upregulates the expression of both ChAT and VAChT genes, (2) NGF has a greater effect on the expression of ChAT mRNA than VAChT mRNA, and (3) the effect of exogenous NGF on the expression of both genes diminishes with developmental age.

Chapter 6 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.

Specific Vesicular Acetylcholine Transporter Promoters Lie within the First Intron of the Rat Choline Acetyltransferase Gene

The sequence encoding the vesicular acetylcholine transporter (VAChT) has recently been localized within the first intron of the choline acetyltransferase (ChAT) gene in various species. In rat, we previously identified a class of VAChT mRNAs that may originate from the same promoter region as two ChAT mRNAs. Here, we demonstrate by a detailed analysis of the 5'-noncoding region of the VAChT gene, that two specific VAChT promoters lie within the first intron of the ChAT gene. Two VAChT mRNAs are generated from these promoters. These results demonstrate that the promoter regions of these two genes are intermingled, which highlight the unique organization of the ChAT/VAChT gene locus.

Coordinated Up-regulation of Choline Acetyltransferase and Vesicular Acetylcholine Transporter Gene Expression by the Retinoic Acid Receptor α, cAMP, and Leukemia Inhibitory Factor/Ciliary Neurotrophic Factor Signaling Pathways in a Murine Septal Cell Line

The proteins responsible for acetylcholine (ACh) synthesis (choline acetyltransferase, ChAT) and storage (vesicular ACh transporter, VAChT) are encoded by two closely linked genes in vertebrates, with the VAChT coding sequence contained within the first intron of the ChAT gene. This unusual genomic organization suggests that the transcription of these two genes is coordinately regulated. Using Northern analysis we studied the modulation of ChAT and VAChT expression in a murine septal cell line (SN56) by three groups of agents: retinoids, trophic factors belonging to the leukemia inhibitory factor/ciliary neurotrophic factor (LIF/CNTF) family, and cAMP. All-trans-retinoic acid increased both ChAT and VAChT mRNA levels in SN56 cells up to 3.5-fold, and elevated intracellular ACh levels by 2.5-fold. This effect was mimicked by a retinoic acid receptor alpha (RAR alpha) agonist (Ro 40-6055) and prevented by a specific antagonist (Ro 41-5253), indicating that it was mediated by RAR alpha. ChAT- and VAChT-specific transcripts were also induced (up to 3-fold) by treatment with CNTF or LIF (20 ng/ml, 48 h), as well as by dibutyryl cAMP (1 mM). All these agents increased the ACh level in the cells (up to 2.5-fold). Dibutyryl cAMP had a greater effect on the level of VAChT mRNA (4-fold induction) than on the level of ChAT mRNA (2-fold induction), suggesting a quantitatively differential transcriptional regulation of the two genes by the cAMP pathway. The effects of the three groups of agents studied on ChAT and VAChT mRNA levels were additive, pointing to several independent mechanisms by which the cholinergic properties of septal neurons can be modulated.

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

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.

Functional identification of a vesicular acetylcholine transporter and its expression from a “cholinergic” gene locus.

The vesicular acetylcholine transporter (VAChT) has been identified and characterized based on the acquisition of high affinity vesamicol binding and proton-dependent, vesamicol-sensitive acetylcholine accumulation by a fibroblast cell line transfected with a clone from a rat pheochromocytoma cDNA library encoding this protein. The distribution of VAChT mRNA coincides with that reported for choline acetyltransferase (ChAT), the enzyme required for acetylcholine biosynthesis, in the peripheral and central cholinergic nervous systems. A human VAChT cDNA was used to localize the VAChT gene to chromosome 10q11.2, which is also the location of the ChAT gene. The entire sequence of the human VAChT cDNA is contained uninterrupted within the first intron of the ChAT gene locus. Transcription of VAChT and ChAT mRNA from the same or contiguous promoters within a single regulatory locus provides a previously undescribed genetic mechanism for coordinate regulation of two proteins whose expression is required to establish a mammalian neuronal phenotype.