Human Choline Acetyltransferase Research Articles

Multiple promoters of human choline acetyltransferase and aromatic L-amino acid decarboxylase genes

The promoter regions of human choline acetyltransferase (ChAT) and aromatic L-amino acid decarboxylase (AADC) genes have been analyzed by transient transfection assays. AADC gene is transcribed from two alternative non-coding first exons, 1N and 1NN, expressed in pheochomocytoma and hepatoma cells, respectively. 5′ flanking sequences of exon 1 N (from 9000 to 147 bp) display promoter activity in SK-N-BE neuroblastoma cells, but not in MC-I-XC cholinergic neuroepithelioma cells, and in AADC-rich non-neuronal cells. On the contrary, 5′ flanking sequences of exon 1 NN (from 1117 to 119 bp) display high promoter activity in human hepatoma cells HepG2, but not in SK-N-BE cells, suggesting high degrees of specificity of promoters N and NN for AADC-expressing neuronal and non-neuronal cells, respectively. Preliminary evidence suggests that leukemia inhibitory factor suppresses the activity of the neuronal promoter in cultured sympathetic neurons. Two alternative first exons, R and M, have been localized in human ChAT gene, and the corresponding promoters characterized in cholinergic PC12 and NG-108-15 cells, and in non-cholinergic neuro2A cells. Several positively or negatively acting cis elements have been localized in the two promoters, as well as a cAMP-inducible, enhancer-like element in the second intron. Among the various cell lines studied, there was no correlation between promoter activities and the expression of the endogenous ChAT gene, suggesting that the fine-tuning of ChAT gene expression is controlled by silencer elements which remain to be localized.

Enhancer containing unusual GC box-like sequences on the human choline acetyltransferase gene

The minimum requirement of an enhancer for the human choline acetyltransferase (ChAT) gene was analyzed by mutagenesis and protein-DNA interaction studies. A series of deletion and site-specific mutants were expressed transiently in a rat cholinergic neuroblastoma cell, NS20Y. The results revealed that the distal region between −970 and −941 base pairs (bp) from the transcription start site is essential for efficient transcription of the human ChAT gene. Two GC box-like sequences were located within this region, and they were shown to bind purified Sp1 transcription factor by footprinting and gel retardation analyses. This sequence, however, has an orientational effect and is located approximately 1000 bp from the transcription start site, unusual for the conventional GC box sequence. Furthermore, the sequence between these GC box-like sequences is shown to bind another novel trans-acting factor by gel retardation assay and to be necessary for efficient enhancer activity. On the other hand, gel retardation assays using a nuclear extract from Drosophila SL2 cells suggested that non-Sp1 trans-acting factors could also participate in the enhancing activity of this element. Taken together, the results demonstrate that two GC box-like sequences and another trans-acting factor-binding sequence located between −970 and −941 bp act coordinately and are essential for efficient transcription of this low expressed human ChAT gene.

Cholinergic neuron-specific expression of the human choline acetyltransferase gene is controlled by silencer elements.

Choline acetyltransferase (ChAT) is specifically expressed in cholinergic neurons. To identify control mechanisms regulating the cell-specific expression of the gene encoding ChAT, transient expression of the luciferase gene driven by human ChAT gene 5'flanking sequences was compared in cholinergic and noncholinergic cell lines. Analysis of the gene indicated the presence of two regulatory elements with selective silencing activity. These elements, located between nucleotides -2043 to -3347 and nucleotides -3347 to -6550, act cooperatively to repress promoter activity > 10-fold in a human adrenergic neuroblastoma cell line, SHSY5Y, and a human osteosarcoma cell line, 143 TK-, while exhibiting less than a two-fold effect in cholinergic cell lines. Deletion of either nucleotides -2043 to -3347 or nucleotides -3348 to -6550 reduced cell-specific repression by approximately half. Such differential repression appears to be responsible for the selective expression of the ChAT component of the cholinergic phenotype.

Inhibition of cytosolic human forebrain choline acetyltransferase activity by phospho-l-serine: A phosphomonoester that accumulates during early stages of alzheimer's disease

There is no satisfactory explanation for the cholinergic deficit characteristic of Alzheimer's disease. We have performed a series of experiments which demonstrate that (a) an inhibitor of cytosolic human brain choline acetyltransferase is present in the cytosol of Alzheimer brain tissue, (b) human brain cytosolic choline acetyltransferase activity is inhibited by phospho-L-serine in a competitive manner. Cytosol was prepared from human forebrain or amygdala and the K m for choline and acetyl CoA of the choline acetyltransferase were 750 μM and 12.5 μM, respectively. Phospho-L-serine was found to be a competitive inhibitor of this enzyme with respect to choline but not with respect to acetyl CoA with a Ki 750 μM for the human forebrain and 3 μM for human amygdala. These concentrations of phospho-L-serine are present in brain tissue at early stages of Alzheimer's disease. Several other phosphomonoesters and phosphodiesters that are increased in Alzheimer's disease were either less inhibitory or without effect. The addition of heat denatured and non-heat denatured cytosol from Alzheimers forebrain inhibited the choline acetyltransferase activity present in control human brain cytosol. The inhibitory activity of the Alzheimers cytosol was retained in TCA deproteinized samples and removed by dialysis or by alkaline phosphatase treatment. Dialysis of the cytosol increased the choline acetyltransferase activity of 5 of 8 Alzheimer's disease samples from 21 to 118% with p values of < 0.025 or < 0.001, respectively. These observations provide evidence that an endogenous non-proteinaceous, dialyzable, phosphomonoester, present in Alzheimers brain inhibits the choline acetyltransferase of both control and Alzheimers brain. Therefore, an alteration in membrane phospholipid metabolism could secondarily contribute to the hypocholinergic state observed in Alzheimer's disease.

Identification and analysis of the human choline acetyltransferase gene promoter.

Choline acetyltransferase (ChAT) is the key enzyme responsible for the synthesis of the neurotransmitter acetylcholine and is reduced in various central neurodegenerative diseases. From a previously selected 12.6 kb human choline acetyltransferase (hChAT) genomic clone, we have identified and characterized a promoter region of 895 bp. Sequence analysis revealed the presence of a TATA-like box, a CAAT box and several putative regulatory responsive elements. Three transcription initiation sites were determined by primer extension analysis. The Northern blot of poly(A)+ RNA, showed a single band of 2300 Nt in the human nucleus accumbens and facial nucleus. By using transient transfections into NE-1-115 and COS-1 cells of the 5' flanking region of the hChAT gene we identified a sequence of 66 bp upstream of the transcription start site which confers responsiveness to proto-oncogenes c-Fos/c-Jun. These data suggest that the hChAT gene may be a physiological target of c-Fos/c-Jun and therefore may play a role in neuronal responses to various stimuli.

Cyclic AMP regulation of the human choline acetyltransferase gene.

Regulation of the human choline acetyltransferase gene by 8-bromo-cAMP was investigated by transfecting into NS-20Y cells choline acetyltransferase promoter sequences fused to the reporter gene firefly luciferase. Promoter activity was localized to nucleotides -163 to +73. This region of the gene responded to 8-bromo-cAMP in a similar fashion as the endogenous enzyme. However, nearly the same induction was observed for constructs containing unrelated promoters. Furthermore, fusion of the choline acetyltransferase gene to the thymidine kinase promoter yielded no increased induction by 8-bromo-cAMP. These results suggest that cAMP regulates choline acetyltransferase gene transcription through an indirect mechanism.

A complementary DNA for human choline acetyltransferase induces two forms of enzyme with different molecular weights in cultured cells

Complementary DNA (cDNA) clones containing the entire coding region of human choline acetyltransferase (ChAT) were isolated from cDNA libraries prepared from the autopsied spinal cord. In the human cDNA, the ATG codon assigned to the putative initiation codon for pig, rat and mouse ChAT cDNAs was replaced by ACG. The human cDNA contained an in-frame ATG codon 324 nucleotides upstream of the ACG codon. Therefore, human ChAT cDNA should code for a 748 amino acid polypeptide of 82.6 kDa. This deduced molecular weight was larger than that of ChAT protein purified from the human brain and placenta (64–70 kDa). The human ChAT cDNA containing the entire coding region was ligated to an expression vector and introduced into African green monkey kidney (COS) cells and Chinese hamster ovary (CHO) cells. The cells expressed high ChAT activity and produced two protein bands immunostained with an antibody to monkey ChAT. The molecular weight of the proteins was estimated to be approximately 70 and 80 kDa by polyacrylamide-SDS gel electrophoresis. When partial cDNAs that lacked the first ATG but contained the replaced ACG codon were introduced into COS cells, the cells expressed moderate ChAT activity and an immunoreactive protein band of 70 kDa. These results indicate that translation of human ChAT mRNA starts at two sites and produces two enzyme proteins with different molecular weights. It might be that the larger form of ChAT molecule is an enzyme precursor for processing or that the N-terminal extrapeptide is needed for subcellular localization of the enzyme.

Two mRNAs are transcribed from the human gene for choline acetyltransferase.

The product of the choline acetyltransferase (ChAT) gene is the enzyme that synthesizes the neurotransmitter acetylcholine. A 14.4-kb portion of the human ChAT gene contains 7 exons, which are estimated to comprise approximately one-third of the human protein coding sequence by comparison with porcine ChAT mRNA. Two of the exons were used to identify polyadenylated human ChAT gene transcripts on Northern blots. An exon with 84% identity to the region of porcine ChAT mRNA that codes for the amino terminus of the corresponding protein detected 6,000- and 2,300-nucleotide mRNAs in RNA isolated from human CHP134 neuroblastoma cells. Only the 2,300-nucleotide mRNA was detected by a second probe containing an exon with 96% identity to porcine ChAT mRNA in the domain that encodes amino acids 204-263 of the predicted porcine ChAT protein. Further evidence that two species of human mRNA are produced from the human ChAT gene was obtained from nuclease protection assays using an antisense RNA probe prepared from a human ChAT cDNA clone. Total RNA isolated from either CHP134 cells or adult human nucleus basalis protected 525- and 400-nucleotide fragments of this probe, confirming the presence of two species of RNA that differ by the inclusion of an internal exon. cDNA clones of each of these transcripts have been isolated. Their sequences suggest that the 2,300-nucleotide mRNA encodes enzymatically active human ChAT, while translation of the 6,000-nucleotide mRNA would be terminated prematurely by a shift in the reading frame. These results indicate that a complex pattern of transcription produces two mRNAs with different coding potentials from the human ChAT gene.

Nerve growth factor regulation of choline acetyltransferase gene expression in rat embryo basal forebrain cultures.

Nerve growth factor (NGF) increases the activity of choline acetyltransferase (ChAT), the synthetic enzyme for acetylcholine, in rat basal forebrain neurons both in vivo and in vitro. In poly(A)+ RNA isolated from cultures prepared from the embryonic (E15) rat basal forebrain, radiolabeled probes from the human ChAT gene detected a 3,700 nt and a less abundant 2,300 nt transcript. After growth in the presence of NGF, the abundance of both mRNAs was increased approximately twofold, paralleling the increase in ChAT enzyme activity. In vivo, the human ChAT probes detected a single 3,700 nt form of ChAT mRNA in both embryonic and adult rat basal forebrain. These results suggest that the NGF-mediated increase in ChAT activity in basal forebrain cultures is regulated at the transcriptional level.

Human placental choline acetyltransferase activity at parturition

Choline acetyltransferase (ChAT) activity measured in human placentae obtained during labour, or after labour of spontaneous onset, was significantly lower than that measured in placentae obtained before the onset of labour. The reduction in ChAT activity may partly account for the reduction in placental acetylcholine (ACh) content and release into maternal vessels at this time. When cycloheximide (10 μ/ml) was infused for 3 h into both fetal and maternal vessels of the perfused placental lobule, it did not significantly alter placental ChAT activity or ACh output from the fetal vessels, though it significantly reduced placental βHCG release from the maternal side of the perfused lobule. These results suggest that regulation of placental CHAT activity at the time of human parturition may be due to endogenous modulators of its activity, rather than to acute changes in the synthesis of this enzyme.

Alzheimer's disease: Is the decrease of the cholinergic innervation of the hippocampus related to intrinsic hippocampal pathology?

Consistent findings in the hippocampi of patients with Alzheimer's disease are the presence of neurofibrillary tangles in pyramidal neurons and the loss of choline acetyltransferase activity due to degeneration of hippocampal cholinergic terminals. The present study sought to clarify, in the brains of five patients with Alzheimer's disease and four controls, whether the loss of cholinergic terminals in the hippocampal stratum pyramidale in Alzheimer's disease is related to degenerative changes in hippocampal pyramidal cells. A polyclonal antibody to human choline acetyltransferase was employed to visualize immunohistochemically cholinergic terminals. Hippocampal neurons were stained with Cresyl Violet, neurofibrillary tangles with thioflavin S and a monoclonal antibody against phosphorylated neurofilament (RT97). Quantification of the stained structures was performed in CA4, CA1 and the subiculum, on five sections selected from the entire anteroposterior extent of each hippocampus. In the group of Alzheimer patients, the densities of cholinergic terminals were homogeneously diminished in the three hippocampal subregions in comparison with the controls (32–33%). In contrast, a significant loss of pyramidal neurons was found only in CA1, and the density of neurofibrillary tangles was markedly increased only in CA1 and the subiculum in Alzheimer's disease. These findings suggest that there is no relationship between the loss of cholinergic terminals and the degeneration of pyramidal cells in the hippocampus of patients with Alzheimer's disease.

Characterization of Mono‐ and Polyclonal Antibodies Against Highly Purified Choline Acetyltransferase: A Monoclonal Antibody Shows Reactivity in Human Brain

Choline acetyltransferase (ChAT) from porcine brain was purified by immunoaffinity chromatography, and the highly purified enzyme was subsequently used for immunization of mice and rabbits. After fusion of mouse spleen cells, 32 cultures producing monoclonal antibodies directed against ChAT were detected by an enzyme-linked immunosorbent assay (ELISA) with immunoaffinity-purified ChAT. Of these original 32, the most active 11 cultures were cloned and used for ascites production. The 11 clones generated monoclonal antibodies of the immunoglobulin (Ig) M class (three), the IgG1 subclass (seven), and the IgG2b subclass (one). The isoelectric points of the antibodies of the IgG class were different in each case. The monoclonal antibodies exhibited different binding characteristics in the above ELISA and on western blots. Two monoclonal antibodies demonstrated excellent immunohistological results with neurons of rat brain and spinal cord. One of them reacted well immunohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA.

Human choline acetyltransferase (CHAT): Partial gene sequence and potential control regions

Choline acetyltransferase (CHAT) (EC2.3.1.6) is the biosynthetic enzyme for the neurotransmitter acetylcholine in the central and peripheral nervous systems. In this study, a human CHAT genomic clone has been isolated and partially sequenced at its 5′ end. This fragment contains the first four exons with an ACG initiator codon and potential control regions including TATA, CAAT, GC boxes, and several transcription control sequences. A comparison of the primary structure of CHAT among pig, rat, mouse, and Drosophila is presented.

Isolation and sub-chromosomal localization of a DNA fragment of the human choline acetyltransferase gene

A DNA fragment of 219 bp was obtained by polymerase chain reaction (PCR) on human genomic DNA using two oligonucleotide mixtures derived from peptide sequences of human placenta choline acetyltransferase (ChAT) and from partially conserved amino acid sequences of rat, porcine and Drosophila ChAT. Sequence homology with porcine ChAT demonstrated that this fragment is part of the human ChAT gene. This gene was assigned to chromosome 10 by hybridization of the 219 bp DNA probe with DNA from human-hamster somatic cell hybrids, and to region 10q11.2-10qter by PCR experiments.

1(2-Benzoylethyl)pyridinium chloride: A new potent and selective inhibitor of bovine brain and human placental choline acetyltransferase

A newly synthesized 1(2-benzoylethyl)pyridinium (BEP), an analog of the choline acetyltransferase (ChA) inhibitor (2-benzoylethyl)trimethylammonium (BETA), was evaluated for its ability to inhibit ChA from bovine brain and human placenta. Its ChA inhibitory properties were compared with that of BETA. BEP was found to be an effective inhibitor of ChA (I 50: 10–18 μM). BEP, as well as BETA, was a linear noncompetitive inhibitor of ChA with respect to both substrates, acetylcoenzyme A and choline. BEP and BETA were poor inhibitors of electric eel acetylcholinesterase. These observations indicate that BEP is a potent and selective inhibitor of ChA. Furthermore, because of the possible delocalization of the positive charge at the N atom of the BEP molecule throughout the pyridine ring, it is anticipated that BEP would have a higher potential for lipid solubility, stability and selectivity than BETA.

Human choline acetyltransferase gene maps to region 10q11–q22.2 by in situ hybridization

Human choline acetyltransferase gene maps to region 10q11–q22.2 by in situ hybridization

Differential vulnerability of cholinergic projections to the mediodorsal nucleus of the thalamus in senile dementia of Alzheimer type and progressive supranuclear palsy

The cholinergic innervation of the mediodorsal nucleus of the thalamus, which is thought to originate primarily in the laterodorsal tegmental nucleus and the substantia innominata, was studied by acetylcholinesterase histochemistry and immunohistochemistry with a polyclonal antiserum against human choline acetyltransferase on autopsy tissue from eight control subjects, five patients with progressive supranuclear palsy and four patients with senile dementia of Alzheimer type. In controls, cholinergic innervation of the mediodorsal nucleus of the thalamus was distributed heterogeneously in densely labelled patches surrounded by less heavily stained matrix. In patients with progressive supranuclear palsy, the density of choline acetyltransferase-positive varicosities decreased by 75% in the matrix and 60% in the patches. The number of choline acetyltransferase-positive cell bodies decreased by 84% in the laterodorsal tegmental nucleus, but more moderately (−33%) in the substantia innominata. In patients with senile dementia of Alzheimer type, choline acetyltransferase-positive varicosities decreased by 34% in the matrix, but 46% in the patches. Choline acetyltransferase-labelled cell bodies were spared in the laterodorsal tegmental nucleus, whereas severe loss (− 80%) was observed in the substantia innominata. These results suggest that cholinergic innervation of mediodorsal nucleus matrix derives mainly from the laterodorsal tegmental nucleus and mediodorsal nucleus patches from the substantia innominata. Differential loss of innervation to the matrix and patches in progressive supranuclear palsy and senile dementia of Alzheimer type may in turn differentially affect mediodorsal nucleus innervation of the frontal cortex, resulting in dissimilar symptomatologies.

Localization of a 900-bp-long fragment of the human choline acetyltransferase gene to 10q11.2 by nonradioactive in situ hybridization

Localization of a 900-bp-long fragment of the human choline acetyltransferase gene to 10q11.2 by nonradioactive in situ hybridization

Compartmental ordering of cholinergic innervation in the mediodorsal nucleus of the thalamus in human brain

The cholinergic innervation of the mediodorsal (MD) nucleus of the thalamus was visualized immunohistochemically in human brain postmortem, using an antibody against human choline acetyltransferase (ChAT). The ChAT staining of the MD nucleus was more intense than in the surrounding thalamic nuclei but weaker than that of the striatum. No ChAT-positive cell bodies were detected. The ChAT-positive neuropil was unevenly distributed, with patches of dense immunoreactivity with a weaker surrounding matrix. In adjoining sections stained for ChAT immunoreactivity and for acetylcholinesterase (AChE) activity, the zones enriched in AChAT-immunostained neuropil corresponded to AChE-rich regions. The three-dimensional reconstruction of the richest zone in AChE/ChAT activity evidenced a cylindrical organization throughout the rostrocaudal axis of the MD nucleus. Counts of ChAT-positive varicosities confirmed an inhomogeneous Distribution; the density of varicosities was 30% higher in ChAT-rich regions than in surrounding matrix. These findings suggest that the activity of intrinsic neurons within the nucleus may be differentially regulated by cholinergic systems.

Localization of the choline acetyltransferase (CHAT) gene to human chromosome 10

A cDNA clone encoding the complete sequence of porcine choline acetyltransferase (CHAT) isolated by S. Berrard et al. (1987, Proc. Natl. Acad. Sci. USA 84: 9280–9284) was hybridized to TaqI digests of a panel of 25 human-rodent somatic cell hybrids and to a complementary panel of 10 human-rodent hybrids in order to determine the chromosomal localization of human CHAT. To enhance the detection of the human signal, hybridization and washings were performed under low stringency conditions on membranes presaturated with sonicated DNA from parental rodent strains. All informative human fragments had the same distribution among the hybrids, mapping CHAT to a single human chromosome. CHAT was assigned to chromosome 10 because all other chromosomes were eliminated by exclusion based on the analysis of the signal segregation. This result indicates that mutation of the CHAT gene cannot be responsible for the primary defect in familial Alzheimer's disease.