Human Choline Acetyltransferase Research Articles

Reduced Density of Cholinergic Interneurons in the Ventral Striatum in Schizophrenia: An In Situ Hybridization Study

The role of the striatum in the pathophysiology of schizophrenia is not understood. In a previous postmortem study, we found a reduction in the density of striatal interneurons that stain immunohistochemically for choline acetyltransferase (ChAT) in schizophrenia. To determine whether this finding represents a specific alteration in ChAT gene expression, we used in situ hybridization to study the striatum of 11 control and 9 schizophrenic subjects with oligonucleotide probes complementary to human ChAT mRNA, preprosomatostatin (PPS) mRNA, and beta-actin mRNA. Densities of ChAT mRNA-positive neurons, ChAT mRNA expression per neuron, PPS mRNA-positive neurons, and beta-actin mRNA expression levels were measured. There were no significant differences between the two groups in densities of PPS mRNA-positive neurons and levels of beta-actin mRNA expression throughout the striatum, or in densities of ChAT mRNA-positive neurons in the caudate nucleus or putamen. However, in the ventral striatum, the mean density of ChAT mRNA-positive neurons was reduced to 26% of control levels in the schizophrenic group. There is a reduction in number or function of the cholinergic interneurons of the ventral striatum in schizophrenia.

Two methods for large-scale purification of recombinant human choline acetyltransferase

Choline acetyltransferase (ChAT) catalyzes the transfer of an acetyl group from acetyl-CoA to choline to produce the neurotransmitter acetylcholine (ACh). We have produced large quantities of pure human ChAT using two different bacterial expression systems. In the first, ChAT is fused to a chitin-binding domain via a self-cleavable linker allowing the release of ChAT without the use of proteases. In the second, ChAT is fused to a hexahistidine (His6) tag at the N-terminus with a linker incorporating a TEV protease cleavage site. In both cases, pure ChAT was produced that has a final specific activity of approximately 50 μmol ACh/min/mg and is suitable for structural characterization. Analysis of purified ChAT by Western blots and mass spectrometry revealed that the C-terminal 15 amino acids were slowly removed by endogenous proteolytic activity, to produce a stable 615 residue protein. Furthermore, we show that purified recombinant human ChAT is highly prone to oxidation, leading to the formation of covalent dimers and/or a loss of catalytic activity. Kinetic parameters of our purified proteins were obtained and, when compared to previously published constants for human placental ChAT, we found that recombinant human ChAT displays lower values for Michaelis and inhibition constants for ACh, which may be due to the complete absence of post-translational modifications.

Protein Kinase C Isoforms Differentially Phosphorylate Human Choline Acetyltransferase Regulating Its Catalytic Activity

Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons; regulation of its activity or response to physiological stimuli is poorly understood. We show that ChAT is differentially phosphorylated by protein kinase C (PKC) isoforms on four serines (Ser-440, Ser-346, Ser-347, and Ser-476) and one threonine (Thr-255). This phosphorylation is hierarchical, with phosphorylation at Ser-476 required for phosphorylation at other serines. Phosphorylation at some, but not all, sites regulates basal catalysis and activation. Ser-476 with Ser-440 and Ser-346/347 maintains basal ChAT activity. Ser-440 is targeted by Arg-442 for phosphorylation by PKC. Arg-442 is mutated spontaneously (R442H) in congenital myasthenic syndrome, rendering ChAT inactive and causing neuromuscular failure. This mutation eliminates phosphorylation of Ser-440, and Arg-442, not phosphorylation of Ser-440, appears primarily responsible for ChAT activity, with Ser-440 phosphorylation modulating catalysis. Finally, basal ChAT phosphorylation in neurons is mediated predominantly by PKC at Ser-476, with PKC activation increasing phosphorylation at Ser-440 and enhancing ChAT activity.

Identification of a Novel Nuclear Localization Signal Common to 69- and 82-kDa Human Choline Acetyltransferase

We demonstrated previously that 69- and 82-kDa human choline acetyltransferase are localized predominantly to the cytoplasm and the nucleus, respectively. We have now identified a nuclear localization signal common to both forms of enzyme using confocal microscopy to study the subcellular compartmentalization of choline acetyltransferase tagged with green fluorescent protein in living HEK 293 cells. To identify functional nuclear localization and export signals, portions of full-length 69-kDa choline acetyltransferase were cloned into the vector peGFP-N1 and the cellular distribution patterns of the fusion proteins observed. Of the nine constructs studied, one yielded a protein with nuclear localization and another produced a protein with cytoplasmic localization. Mutation of the critical amino acids in this novel putative nuclear localization signal in the 69- and 82-kDa enzymes demonstrated that it is functional in both proteins. Moreover, 69-kDa choline acetyltransferase but not the 82-kDa enzyme is transported out of the nucleus by the leptomycin B-sensitive Crm-1 export pathway. By using bikaryon cells expressing both 82-kDa choline acetyltransferase and the nuclear protein heterogeneous nuclear ribonucleoprotein with green and red fluorescent tags, respectively, we found that the 82-kDa enzyme does not shuttle out of the nucleus in measurable amounts. These data suggest that 69-kDa choline acetyltransferase is a nucleocytoplasmic shuttling protein with a predominantly cytoplasmic localization determined by a functional nuclear localization signal and unidentified putative nuclear export signal. For 82-kDa choline acetyltransferase, the presence of the unique amino-terminal nuclear localization signal plus the newly identified nuclear localization signal may be involved in a process leading to predominantly nuclear accumulation of this enzyme, or alternatively, the two nuclear localization signals may be sufficient to overcome the force(s) driving nuclear export.

Phosphorylation of 69-kDa Choline Acetyltransferase at Threonine 456 in Response to Amyloid-β Peptide 1–42

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons. In the brain, these neurons are especially vulnerable to effects of beta-amyloid (A beta) peptides. Choline acetyltransferase is a substrate for several protein kinases. In the present study, we demonstrate that short term exposure of IMR32 neuroblastoma cells expressing human choline acetyltransferase to A beta-(1-42) changes phosphorylation of the enzyme, resulting in increased activity and alterations in its interaction with other cellular proteins. Using mass spectrometry, we identified threonine 456 as a new phosphorylation site in choline acetyltransferase from A beta-(1-42)-treated cells and in purified recombinant ChAT phosphorylated in vitro by calcium/calmodulin-dependent protein kinase II (CaM kinase II). Whereas phosphorylation of choline acetyltransferase by protein kinase C alone caused a 2-fold increase in enzyme activity, phosphorylation by CaM kinase II alone did not alter enzyme activity. A 3-fold increase in choline acetyltransferase activity was found with coordinate phosphorylation of threonine 456 by CaM kinase II and phosphorylation of serine 440 by protein kinase C. This phosphorylation combination was observed in choline acetyltransferase from A beta-(1-42)-treated cells. Treatment of cells with A beta-(1-42) resulted in two phases of activation of choline acetyltransferase, the first within 30 min and associated with phosphorylation by protein kinase C and the second by 10 h and associated with phosphorylation by both CaM kinase II and protein kinase C. We also show that choline acetyltransferase from A beta-(1-42)-treated cells co-immunoprecipitates with valosin-containing protein, and mutation of threonine 456 to alanine abolished the A beta-(1-42)-induced effects. These studies demonstrate that A beta-(1-42) can acutely regulate the function of choline acetyltransferase, thus potentially altering cholinergic neurotransmission.

Functional regulation of choline acetyltransferase by phosphorylation.

Choline acetyltransferase (ChAT) catalyzes synthesis of acetylcholine (ACh) in cholinergic neurons. ACh synthesis is regulated by availability of precursors choline and acetyl coenzyme A or by activity of ChAT; ChAT regulates ACh synthesis under some conditions. Posttranslational phosphorylation is a common mechanism for regulating the function of proteins. Analysis of the primary sequence of 69-kD human ChAT indicates that it has putative phosphorylation consensus sequences for multiple protein kinases. ChAT is phosphorylated on serine-440 and threonine-456 by protein kinase C and CaM kinase II, respectively. These phosphorylation events regulate activity of the enzyme, as well as its binding to plasma membrane and interaction with other cellular proteins. It is relevant to investigate differences in constitutive and inducible patterns of phosphorylation of ChAT under physiological conditions and in response to challenges that cholinergic neurons may be exposed to, and to determine how changes in phosphorylation relate to changes in neurochemical transmission.

Synergistic activation of the human choline acetyltransferase gene by c-Myb and C/EBPβ

To elucidate regulatory mechanisms at the transcriptional level of the human choline acetyltransferase gene (hChAT) we performed cotransfections assays in NG108-15 and SN56 cells using ChAT-CAT reporter plasmids with c-Myb and C/EBPβ expression plasmids. The hChAT gene has several promoters, one of which (promoter P2 or M-type) is both c-Myb and C/EBPβ inducible as 3–4-fold trans-activation was obtained in both cell lines when using either c-Myb or C/EBPβ expression vectors alone. The simultaneous expression of c-Myb and C/EBPβ in the absence or presence of NGFI-C (egr4) leads respectively to a 15-fold and 32-fold synergistic transcriptional activation of promoter P2. In the region upstream of exon M (P2) we identified a functional composite element including a c-Myb next to a C/EBP binding site. An oligonucleotide containing the composite element confers c-Myb and C/EBPβ responsiveness to a heterologous promoter which is reduced after mutation of the c-Myb binding site. We also show that the coactivators CBP/p300 are required for c-Myb and C/EBPβ trans-activation function and that RARα, RXRα and T3R have an inhibitory action on the synergistic transcriptional activity of c-Myb and C/EBPβ and propose a model to explain the phenomena. Taken together, the results suggest that the synergistic effect of c-Myb and C/EBPβ, previously observed in the hematopoietic system, functions equally in the neuronal system.

A novel untranslated 'exon H' of the human choline acetyltransferase gene in placenta.

To investigate the existence of 5'-region(s) of human choline acetyltransferase (hChAT) mRNA in placenta we analyzed the presence or absence of ChAT 5'-untranslated regions (UTR) in human neuronal and non-neuronal cells. Total RNA from human spinal cord, placenta, cultured choriocarcinoma JEG-3 and neuroblastoma CHP126 and MC-IXC cells was reverse transcribed and used for polymerase chain reaction amplification (RT-PCR). We used a sense primer located in the 5'-flanking region, in the previously defined intronic sequence and an anti-sense primer located in the common coding exon 2 of the hChAT gene. An amplified product of 567 bp in size was obtained only in human placenta and in JEG-3 cells whereas it was absent in spinal cord, CHP126 and MC-IXC cells. It was designated 'H-type' of ChAT mRNA. Whereas CHP126 produced the R- and N-type of ChAT mRNAs, no transcript of the N-and R-type was detected in JEG-3 and human placenta. In addition, CHP126 and JEG-3 cells and placenta showed the expression of the M-type of ChAT mRNA. The identity of the amplified 567 bp product (H-type) was confirmed by Southern hybridization and sequencing. The nucleotide sequence of the amplified fragment in placenta revealed the existence of a previously unknown type of ChAT mRNA produced by alternative splicing. Using primer extension we further determined the transcription initiation site of the H-type hChAT mRNA in placenta. These results demonstrate the expression of a novel ChAT mRNA isoform in human placenta in addition to the M-type. These data may be possibly explained by the presence of a placenta specific promoter in the ChAT gene, which might be the proximal promoter P1.

Functional Characterization of Phosphorylation of 69-kDa Human Choline Acetyltransferase at Serine 440 by Protein Kinase C

Choline acetyltransferase, the enzyme that synthesizes the transmitter acetylcholine in cholinergic neurons, is a substrate for protein kinase C. In the present study, we used mass spectrometry to identify serine 440 in recombinant human 69-kDa choline acetyltransferase as a protein kinase C phosphorylation site, and site-directed mutagenesis to determine that phosphorylation of this residue is involved in regulation of the enzyme's catalytic activity and binding to subcellular membranes. Incubation of HEK293 cells stably expressing wild-type 69-kDa choline acetyltransferase with the protein kinase C activator phorbol 12-myristate 13-acetate showed time- and dose-related increases in specific activity of the enzyme; in control and phorbol ester-treated cells, the enzyme was distributed predominantly in cytoplasm (about 88%) with the remainder (about 12%) bound to cellular membranes. Mutation of serine 440 to alanine resulted in localization of the enzyme entirely in cytoplasm, and this was unchanged by phorbol ester treatment. Furthermore, activation of mutant enzyme in phorbol ester-treated HEK293 cells was about 50% that observed for wild-type enzyme. Incubation of immunoaffinity purified wild-type and mutant choline acetyltransferase with protein kinase C under phosphorylating conditions led to incorporation of [(32)P]phosphate, with radiolabeling of mutant enzyme being about one-half that of wild-type, indicating that another residue is phosphorylated by protein kinase C. Acetylcholine synthesis in HEK293 cells expressing wild-type choline acetyltransferase, but not mutant enzyme, was increased by about 17% by phorbol ester treatment.

Expression, purification and characterization of recombinant human choline acetyltransferase: phosphorylation of the enzyme regulates catalytic activity

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms. In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases. Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme. Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively. Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h. By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus. Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and α-calcium/calmodulin-dependent protein kinase II (α-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity. The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or α-CaM kinase, with only PKC yielding increased enzyme activity. Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity. These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity.

Expression, purification and characterization of recombinant human choline acetyltransferase: phosphorylation of the enzyme regulates catalytic activity.

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms. In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases. Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme. Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively. Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h. By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus. Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity. The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or alpha-CaM kinase, with only PKC yielding increased enzyme activity. Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity. These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity.

Nuclear Localization of the 82-kDa Form of Human Choline Acetyltransferase

Choline acetyltransferase is the enzyme catalyzing synthesis of the neurotransmitter acetylcholine in cholinergic neurons. In human, transcripts encoding two forms of the enzyme with apparent molecular masses of 69 and 82 kDa are found in brain and spinal cord; the 82-kDa form differs from the 69-kDa enzyme only in terms of a 118-amino acid extension on its amino terminus. Using green fluorescent protein-tagged choline acetyltransferase, we show that the 82-kDa enzyme is targeted to nuclei of cells, whereas the 69-kDa protein is found in cytoplasm. Expression of site-directed and deletion mutants of the 82-kDa isoform reveals that the extended amino terminus contains a nuclear localization signal in the first nine amino acids which targets the protein to nucleus. This represents the first report of a neurotransmitter-synthesizing enzyme that is localized to the cell nucleus.

Cooperation between phosphorylation and acetylation processes in transcriptional control.

We previously reported that the activation of the M promoter of the human choline acetyltransferase (ChAT) gene by butyrate and trapoxin in transfected CHP126 cells is blocked by PD98059, a specific mitogen-activated protein kinase kinase (MEK) inhibitor (E. Espinos and M. J. Weber, Mol. Brain Res. 56:118-124, 1998). We now report that the transcriptional effects of histone deacetylase inhibitors are mediated by an H7-sensitive serine/threonine protein kinase. Activation of the ChAT promoter by butyrate and trapoxin was blocked by 50 microM H7 in both transient- and stable-transfection assays. Overexpression of p300, a coactivator protein endowed with histone acetyltransferase activity, stimulated the ChAT promoter and had a synergistic effect on butyrate treatment. These effects were blocked by H7 and by overexpressed adenovirus E1A 12S protein. Moreover, both H7 and PD98059 suppressed the activation of the Rous sarcoma virus (RSV) and simian virus 40 promoters by butyrate in transfection experiments. Similarly, the induction of the cellular histone H1(0) gene by butyrate in CHP126 cells was blocked by H7 and by PD98059. Previous data (L. Cuisset, L. Tichonicky, P. Jaffray, and M. Delpech, J. Biol. Chem. 272:24148-24153, 1997) showed that the induction of the H1(0) gene by butyrate is blocked by okadaic acid, an inhibitor of protein phosphatases. We now show that the activation of the ChAT and RSV promoters by butyrate in transfected CHP126 cells is also blocked by 200 nM okadaic acid. Western blotting and in vivo metabolic labeling experiments showed that butyrate has a biphasic effect on histone H3 phosphorylation, i.e., depression for up to 16 h followed by stimulation. The data thus strongly suggest that the transcriptional effects of histone deacetylase inhibitors are mediated through the activation of MEK1 and of an H7-sensitive protein kinase in addition to protein phosphatases.

Quantification of cholinergic and select non-cholinergic mesopontine neuronal populations in the human brain

The pars compacta and pars dissipata of the pedunculopontine nucleus contain cholinergic cell group Ch5, and the laterodorsal tegmental nucleus contains cholinergic cell group Ch6. The pedunculopontine nucleus has been implicated in a variety of functions, including mediation of rapid eye movement sleep and in extrapyramidal motor function, although the role of cholinergic and non-cholinergic neurons is unclear. Quantitative neuroanatomical techniques were used to map the distribution of cholinergic neurons in the mesopontine nuclei of the adult human brain. In addition, the number and distribution of comparably sized non-cholinergic neurons at selected anatomical levels were compared. An antibody raised against human choline acetyltransferase was used to stain immunohistochemically the mesopontine neurons in six brains, ranging in age from 28 to 60 years. The rostrocaudal length of the Ch5/Ch6 cell complex was approximately 10 mm. The estimated total number of cells was similar for all brains, and varied by less than 7%. The estimated average number of cholinergic cells in the combined pedunculopontine and laterodorsal tegmental nuclei was approximately 20 000, with 30% of the cells in the pedunculopontine nucleus pars compacta, 57% in the pedunculopontine nucleus pars dissipata and 13% in the laterodorsal tegmental nucleus. There was no correlation between cell number and age. Within areas of mesopontine tegmentum occupied by the Ch5 cholinergic neurons, there were often more non-cholinergic neurons than comparably sized cholinergic neurons. The present study provides detailed maps of the distribution and number of mesopontine cholinergic neurons in the normal human brain. Many non-cholinergic neurons are intermixed with the cholinergic pedunculopontine neurons. One region of the pedunculopontine nucleus pars dissipata containing few cholinergic neurons, located adjacent to the ventral border of the pedunculopontine nucleus pars compacta, may correspond to the midbrain–extrapyramidal area as defined previously in rodent and in non-human primate. These data will be useful for quantitative neuropathological studies concerning the role of both cholinergic and non-cholinergic mesopontine neurons in diseases proposed to affect these neurons, including Parkinson's disease, schizophrenia and progressive supranuclear palsy.

Neurons with choline acetyltransferase immunoreactivity and mRNA are present in the human cerebral cortex.

We examined the cerebral cortex of five autopsied individuals without neurological and psychiatric diseases by immunohistochemistry using an anti-human recombinant choline acetyltransferase (ChAT) polyclonal antibody and in situ hybridization with 35S-labeled human ChAT riboprobes. The immunohistochemistry detected positive neurons which were medium-sized or large pyramidal neurons located predominantly in layers III and V. The density of such neurons was higher in the motor and secondary sensory areas than in other cortical areas; the immunoreactive neurons in layer V were more densely distributed in the motor area and those in layer III were distributed in the secondary sensory areas. Positively stained, non-pyramidal neurons were observed in the superficial layer of the cingulate gyrus and parahippocampus. No immunoreactive neurons were found in the primary sensory areas. The in situ hybridization detected some neurons with signals for ChAT mRNA in the cerebral cortex, most of which were distributed in layer V of the motor area and in layer III of the secondary visual area. These results indicate that the human cerebral cortex contains cholinergic neurons and displays regional and laminal variations in their distribution.

30 Regulation of human choline acetyltransferase gene by inhibitors of histone deacetylase

30 Regulation of human choline acetyltransferase gene by inhibitors of histone deacetylase

In situ mRNA hybridization study of the distribution of choline acetyltransferase in the human brain

We examined the distribution of choline acetyltransferase (ChAT) mRNA in the brain of six autopsied individuals by in situ hybridization with 35 S -labeled human ChAT riboprobes. Neurons containing hybridization signal for ChAT mRNA were observed in the nucleus of the diagonal band of Broca, the basal nucleus of Meynert, the caudate nucleus, the putamen, the pedunculopontine tegmental nucleus, the laterodorsal tegmental nucleus, the parabigeminal nucleus, the oculomotor nucleus and the trochlear nucleus. These findings were in good agreement with previous ChAT-immunohistochemical data. In contrast, labeled neurons were not observed in the medial septal and medial habenular nuclei, in which previously ChAT-immunoreactive neurons have been identified in many mammalian species, including the human. An unexpected result of the present study was the demonstration of neurons with ChAT mRNA signal in restricted areas of the human cerebral cortex.

Activation of the MAP kinase cascade by histone deacetylase inhibitors is required for the stimulation of choline acetyltransferase gene promoter

We previously described that the major promoter (M) of human choline acetyltransferase (ChAT) gene is activated by three inhibitors of histone deacetylase, butyrate, trichostatin and trapoxin, in transfected CHP126 neuroepithelioma cells. We now show that trapoxin and butyrate triggered a rapid and transient phosphorylation of ERK1/2 kinases, that was suppressed by PD98059, a highly specific inhibitor of MAP kinase kinase MEK1. The stimulation of ChAT promoter activity by trapoxin or butyrate did not require ongoing protein synthesis, and was suppressed by PD98059. The overexpression of dominant negative mutants of H-ras or ERK2 proteins depressed ChAT promoter activation by trapoxin in transient transfection assays. Conversely, the overexpression of constitutively active mutants of H-ras or MEK1 proteins had little or no effect on ChAT promoter activity, but strongly synergized with trapoxin. These data thus suggest that the activation of the MEK/ERK kinase cascade plays a necessary, but not sufficient, role in the regulation of ChAT promoter by inhibitors of histone deacetylase.

Transcriptional activation of human choline acetyltransferase by AP2- and NGF-induced factors

ChAT (choline acetyltransferase) is the enzyme responsible for acetylcholine synthesis and is specifically expressed in cholinergic neurons. To further characterize the transcriptional regulation of the hCHAT (human ChAT) gene by NGF, we examined the effects upon ChAT promoter activity of a family of transcription factors which are activated by NGF and several extracellular stimuli and encoded by immediate-early genes. These include NGFI-A (Egr1, zif268), NGFI-C (Egr2), Krox-20 and NGFI-B (Nurr77). Two fragments of the hChAT gene were used for functional analysis carrying 944 bp (P1) and 4000 bp (P1+P2) of the 5′ flanking region in front of the chloramphenicol acetyltransferase (CAT) reporter gene. They were transiently co-transfected with NGFI-A, NGFI-C, Krox-20 and NGFI-B expression vectors in NG108-15, SN6 and COS-1 cells. CAT activity after transfection of the p4000 ChAT-CAT reporter into both neuronal cell lines (NG108-15 and SN6 cells) was increased up to 5-fold in the presence of co-transfected NGFI-A and up to 5- and 12-fold after co-transfection of NGFI-C expression vector in NG108-15 and SN6 cells, respectively. In NG108-15 cells, dbcAMP excerted a strong enhancing activity on the transactivation properties of NGFI-C while this was not observed when cells were transfected with NGFI-A. These trans-activation effects were specific for neuronal cells. When NG108-15 cells were treated with dbcAMP in the presence of H89, a specific PKA inhibitor, the increase of transcriptional activity of NGFI-C was abolished, indicating that a signalling transduction mechanism through PKA plays a role in NGFI-C-induced trans-activation. Electrophoretic mobility-shift assays showed that the sequence GCCCGGGGAG (NGFRE) located 1205 bp upstream of the first coding ATG (E1) can bind NGFI-A but not NGFI-C. Several possibilities explaining the observed results are discussed. Finally, transfections of ChAT-CAT reporters including the P1+P2 region or a minimal ChAT enhancer present in the P2 region in front of a heterologous promoter indicated the presence of a regulatory element which conferred AP2-dependent trans-activation with homologous as well as with heterologous promoter constructs.

Immunization of guinea pigs with human choline acetyltransferase induces selective lower motoneuron destruction

Prior studies have demonstrated that guinea pigs immunized with bovine spinal motoneurons develop immune-mediated lower motoneuron disease. In the present experiments, guinea pigs immunized with choline acetyltransferase (ChAT) from human placenta develop lower motoneuron destruction and striated muscle atrophy. In this model, increased IgG was detected in lower motoneurons and at the motor end-plate by immunocytochemistry. Ultrastructural analysis revealed an increase in calcium content and in the density of synaptic vesicles in axon terminals at neuromuscular junctions. Similar morphological changes could be induced in mice following passively transfer of IgG from ChAT-immunized guinea pigs. The increased IgG uptake and raised calcium content in motor axon terminals as well as the selective lower motoneuron damage, suggest that a similar final common pathway can lead to motoneuron injury following immunization with human placental ChAT or bovine spinal motoneurons.