Choline Acetyltransferase Gene Expression Research Articles

The Discovery of Benzylidene‐Indenone as Selective Butyrylcholinesterase Inhibitor with Choline Acetyltransferase Gene and Neurite Promoting Abilities

AbstractAlzheimer's disease (AD) is a multifactorial and progressive neurodegenerative disease, associated with aging. A total of forty benzylidene‐indenone derivatives were synthesized in search of multi‐target‐directed ligands (MTDL) against AD. Compound 9 was found as the most promising MTDL in this study. It was demonstrated to selectively inhibit human butyrylcholinesterase, increase the gene expression of choline acetyltransferase (CHAT), and promote neurite outgrowth in SH‐SY5Y cells. The compound was also predicted to be able to permeate across the lipid‐infused bilayer, indicating its ability to cross the blood‐brain barrier. The abovementioned findings show compound 9 and its analogs warrant further exploration as potential MTDL anti‐AD agents.

Sevoflurane inhalation has a cognitive impairing effect of aging rats involving the regulation of AChE and ChAT.

Acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) are closely related to the regulation of learning and memory. Nevertheless, whether sevoflurane has influence on cognition through regulating the expression of AChE and ChAT remains unclear. Aging rat model was established by subcutaneously injection of D-galactose for 6 consecutive weeks. To determine the role of AChE and ChAT in sevoflurane-induced cognitive impairment, the Morris water maze (MWM) was used to assess the cognitive and memory function after sevoflurane exposure. Then, the variations of AChE and ChAT was detected by western blotting analysis and quantitative real-time polymerase chain reaction (qRT-PCR) respectively. Our result indicated that aging model rats had showed cognition decline at 2 hours and 1week after exposure to sevoflurane. Moreover, the expression of AChE and ChAT enhanced in rats that had inhaled sevoflurane. Interestingly, our study also found that the increase of oxygen concentration had a positive impact on the gene expression of ChAT. We have identified that the overexpression of AChE and ChAT improved significantly cognitive function after sevoflurane exposure.

Interleukin-4 Promotes Tuft Cell Differentiation and Acetylcholine Production in Intestinal Organoids of Non-Human Primate.

In the intestine, the innate immune system excludes harmful substances and invading microorganisms. Tuft cells are taste-like chemosensory cells found in the intestinal epithelium involved in the activation of group 2 innate lymphoid cells (ILC2). Although tuft cells in other tissues secrete the neurotransmitter acetylcholine (ACh), their function in the gut remains poorly understood. In this study, we investigated changes in the expression of genes and cell differentiation of the intestinal epithelium by stimulation with interleukin-4 (IL-4) or IL-13 in macaque intestinal organoids. Transcriptome analysis showed that tuft cell marker genes were highly expressed in the IL-4- and IL-13-treated groups compared with the control, and the gene expression of choline acetyltransferase (ChAT), a synthesis enzyme of ACh, was upregulated in IL-4- and IL-13-treated groups. ACh accumulation was observed in IL-4-induced organoids using high-performance liquid chromatography-mass spectrometry (HPLC/MS), and ACh strongly released granules from Paneth cells. This study is the first to demonstrate ACh upregulation by IL-4 induction in primates, suggesting that IL-4 plays a role in Paneth cell granule secretion via paracrine stimulation.

Liraglutide inhibits the progression of prediabetes in rats by reducing Raf-1 kinase inhibitor protein.

BackgroundThe cleavage product of Raf-1 kinase inhibitor protein (RKIP), hippocampal cholinergic neurostimulating peptide (HCNP) is involved in the promotion of insulin secretion. Studies have shown that liraglutide can inhibit the progression of prediabetes. This study aims to investigate whether the above effects of liraglutide are related to RKIP and HCNP.MethodsInsulin-1 (INS-1) cells were divided into control group (CON), HCNP group, and HCNP + darifenacin group (H-DAR). The three groups were cultured with Roswell Park Memorial Institute (RPMI) 1640, synthetic HCNP (50 pg/mL) and RPMI 1640, and HCNP + RPMI 1640 + darifenacin respectively. Subsequently, twelve 12- to 14-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats were randomly divided into 2 groups: the placebo group (PBO) and the liraglutide treatment group (LIRA). Six Long Evans Tokushima Otsuka (LETO) rats were used as the control group (CON). The LIRA group was given liraglutide 200 µg/kg intraperitoneally twice a day. After 12 weeks, body weight, fasting blood glucose, 2 hours postprandial blood glucose, and insulin resistance index were recorded. Western blot was used to detect expression level of C-RKIP, N-RKIP, and extracellular signal-regulated kinase of phosphorylation (p-ERK). Real-time quantitative polymerase chain reaction (qRT-PCR) to detect pancreatic tissue choline acetyltransferase (ChAT) and M3 cholinergic receptor (M3R) gene expression levels.ResultsAt glucose concentrations of 5.6 and 16.7 mmol/L, the insulin content in the HCNP group was higher than that in the CON and H-DAR groups (all P<0.01). The body weight and fasting serum insulin (FINS) of rats in the PBO group were higher than those in the LIRA group and the CON group (P<0.01). The relative content of C-RKIP protein in the PBO group was higher than that in the LIRA and CON groups (P<0.01). The relative content of N-RKIP protein and p-ERK protein was lower than that in the LIRA and CON group (P<0.05 and P<0.01, respectively). ChAT and M3R gene expression levels in PBO group were lower than those in LIRA and CON group (P<0.01).ConclusionsLiraglutide promotes the production of HCNP, can increase ChAT activity, activate M3R, and further promote the secretion of insulin.

Hippocampal Cholinergic Neurostimulating Peptide Suppresses LPS-Induced Expression of Inflammatory Enzymes in Human Macrophages.

Hippocampal cholinergic neurostimulating peptide (HCNP) is a secreted undecapeptide produced through proteolytic cleavage of its precursor protein, HCNPpp. Within hippocampal neurons, HCNP increases gene expression of choline acetyltransferase (ChAT), which catalyzes acetylcholine (ACh) synthesis, thereby modulating neural activity. HCNPpp also appears to be expressed in various immune cells. In the present study, we observed that HCNPpp is expressed in U937 human macrophage-like cells and that HCNP exposure suppresses lipopolysaccharide (LPS)-induced gene expression of ChAT. The opposite action is also seen in T lymphocytes, which suggest that HCNP appear to suppress cholinergic system in immune cells. In addition, HCNP suppresses LPS-induced gene expression of inflammatory enzymes including cyclooxygenase 2 (COX2) and inducible nitric oxide (NO) synthase (iNOS). The suppressive effect of HCNP may reflect suppression of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling activated by LPS. Thus, HCNP may have therapeutic potential as an anti-inflammatory drug.

Long-lasting microbial dysbiosis and altered enteric neurotransmitters in adult rats following adolescent binge ethanol exposure.

Human alcoholism and ethanol exposure of adult mice cause acute microbial dysbiosis. Adolescent binge drinking is common, but the effect of adolescent ethanol exposure on the adult microbiome and enteric neurotransmitters has not been studied. In the current study, male Wistar rats received adolescent intermittent ethanol (AIE) treatment, and fecal samples were collected on postnatal day (P)54 and P95 for bacterial 16S rRNA amplicon sequencing. Cecal tissue was collected on P95 for analysis of innate immune and neurotransmitter marker expression. At the genus level, AIE treatment altered the relative abundance of several microbes, including decreased relative abundance of Dehalobacterium and CF231 (a member of the Paraprevotellaceae family) that persisted into adulthood. Across aging, the relative abundance of several microbes was altered in both control- and AIE-treated rats. At P95, AIE exposure was associated with increased cecal serotonin levels and reduced choline acetyltransferase gene expression. Taxonomic shifts at P54 and at P95 suggest that AIE causes both immediate and lasting microbial dysbiosis. The lasting microbial dysbiosis was accompanied by alterations of enteric neurotransmitters.

Ameliorative effect of fisetin against lipopolysaccharide and restraint stress-induced behavioral deficits via modulation of NF-κB and IDO-1.

Fisetin, a plant active polyphenol, is well known for its antioxidant and free radical scavenging activities. The present study was designed to explore the detailed molecular mechanism underlying its neuroprotective effects. The young male mice were either administered a single dose of lipopolysaccharide (0.83 mg/kg) or subjected to restraint stress (6 h per day for 28 days) to induce behavioral deficits in different groups. Fisetin (15 mg/kg) was orally administered for the last 14 days of the study. Lipopolysaccharide (LPS) as well as restraint stress (RS) exposure caused behavioral alterations (anxiety and depressive-like behavior). Gene expression analysis showed upregulation of nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) and indoleamine 2,3-dioxygenase (IDO)-1 gene expression along with downregulation of Nrf-2 (nuclear factor erythroid 2-related factor 2), HO-1 (heme oxygenase-1), and ChAT (choline acetyltransferase) gene expression level in RS and RS+LPS groups. Fisetin administration significantly ameliorated behavioral and neurochemical deficits in LPS, RS, and RS+LPS groups. These findings clearly indicated that fisetin administration improved behavioral functions and suppressed the NF-κB and IDO-1 (indoleamine 2,3-dioxygenase) activation along with their antioxidant effect, suggesting fisetin as an intriguing nutraceutical for the management of inflammation-associated neurological disorders.

A transient insulin system dysfunction in newborn rat brain followed by neonatal intracerebroventricular administration of streptozotocin could be accompanied by a labile cognitive impairment

The early postnatal period is a critical period of hippocampus development, which is highly dependent on insulin receptor (IR) signaling and very important in cognitive function. The present study was conducted in order to present a model of neonatal transient brain insulin system dysfunction through finding an appropriate dose of injection of streptozotocin (STZ) during the neonatal period.Sixty male Wistar rat pups were divided into 4 groups of 15 and received intracerebroventricular saline or STZ (icv-STZ) (15, 20 and 25μg/kg) on postnatal day 7. Gene expression of IR and target genes for IR signaling (choline acetyltransferase (ChAT) and Tau) were measured at the ages of 2 and 7 weeks. Behavioral tests were performed at the ages of 3 and 6 weeks to assess short- and long-term cognitive function. 20μg/kg dose of icv-STZ was estimated as the optimal dose causing transient alteration in gene expression of IR, ChAT and Tau. Additionally, cognitive function of the animals restored to normal level at the age of 6 weeks. Therefore, 20μg/kg dose of icv-STZ is proposed as a new approach to generating transient brain insulin system dysfunction associated with transient cognitive impairments at a critical postnatal period of brain development.

Effects of filgrastim on gene expression of cholinergic system in the rat hippocampus

Background: Filgrastim is a granulocyte colony-stimulating factor (G-CSF), which also showed effects on memory and cognation. Although recent studies have begun to explore G-CSF-related mechanisms of action in memory, little is known about its effects on cholinergic system. Since the cholinergic system has a key role in memory and some studies reported effects of G-CSF on cholinergic activity. Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) gene expression was examined in the rat frontal cortex and hippocampus. Aims and Objectives: This study was designed to investigate the effect of G-CSF treatment on gene expression of AChE and ChAT in the frontal cortex and hippocampus during learning. Materials and Methods: In this study, male Wistar rats were treated for 2 week with filgrastim intraperitoneally at doses of 10, 50, 70 μg/kg/day. All rats received Morris water maze training (4 trials/day for 5 days). Moreover, ChAT and AChE gene expression was assessed by real-time polymerase chain reaction in the rat frontal cortex and hippocampus. Results: Over 5 days of training, filgrastim at doses 10, 50 and 70 μg/kg/day did not change the gene expression of ChAT and AChE in frontal cortex and hippocampus although at dose of 50 ug/kg a little changes but not significant observed. Conclusions: The findings observed in this study propose that G-CSF memory enhancer effects doesn’t only cholinergic and other mechanism may be involve.

Captopril modifies angiotensin-converting enzyme but not choline acetyltransferase gene expression in the frontal cortex of renovascular hypertensive rats

Background: The classic renin-angiotensin system (RAS) has a role in the cardiovascular system and water homeostasis in the body, but recently, the existences of this system with all of its components including angiotensinogen, angiotensin converting enzyme (ACE), and angiotensin receptors have been shown in the mammalian brain. Recent clinical studies suggest that treatment of hypertensive patients with the RAS affecting drugs, such as captopril improves their cognitive function. Aims and Objectives: This study was conducted to evaluate the effects of captopril on the frontal cortex levels of ACE and choline acetyltransferase (ChAT) in renovascular hypertensive rats receiving captopril. Materials and Methods: The rats were randomly divided into 3 groups of 8 animals; control, Goldblatt two kidney one clip (2K1C) hypertensive rats and Goldblatt 2K1C hypertensive rats received 5 mg/kg captopril. After 8 days of treatment; the rats were sacrificed and expression of ACE and ChAT in the frontal cortex were assessed by real-time polymerase chain reaction. The results were analyzed with two-way ANOVA test using SPSS software and the level of significance was set at P < 0.05. Results: Captopril treatment decreased the levels of ACE mRNA in the frontal cortex of renovascular hypertensive rats but has no significant effect in the ChAT mRNA levels. Conclusion: Captopril as a nootropic drug has no significant effect on the ChAT gene expression in the frontal cortex of renovascular hypertensive rats and it may exert its memory enhancing effect on the frontal cortex via a non-cholinergic mechanism.

A silk peptide fraction restores cognitive function in AF64A-induced Alzheimer disease model rats by increasing expression of choline acetyltransferase gene

This study investigated the effects of a silk peptide fraction obtained by incubating silk proteins with Protease N and Neutrase (SP-NN) on cognitive dysfunction of Alzheimer disease model rats. In order to elucidate underlying mechanisms, the effect of SP-NN on the expression of choline acetyltransferase (ChAT) mRNA was assessed in F3.ChAT neural stem cells and Neuro2a neuroblastoma cells; active amino acid sequence was identified using HPLC-MS. The expression of ChAT mRNA in F3.ChAT cells increased by 3.79-fold of the control level by treatment with SP-NN fraction. The active peptide in SP-NN was identified as tyrosine-glycine with 238.1 of molecular weight. Male rats were orally administered with SP-NN (50 or 300mg/kg) and challenged with a cholinotoxin AF64A. As a result of brain injury and decreased brain acetylcholine level, AF64A induced astrocytic activation, resulting in impairment of learning and memory function. Treatment with SP-NN exerted recovering activities on acetylcholine depletion and brain injury, as well as cognitive deficit induced by AF64A. The results indicate that, in addition to a neuroprotective activity, the SP-NN preparation restores cognitive function of Alzheimer disease model rats by increasing the release of acetylcholine.

Aluminum-Induced Cholinergic Deficits in Different Brain Parts and Its Implications on Sociability and Cognitive Functions in Mouse.

Aluminum is associated with etiology of many neurodegenerative diseases specially Alzheimer's disease. Chronic exposure to aluminum via drinking water results in aluminum deposition in the brain that leads to cognitive deficits. The study aimed to determine the effects of aluminum on cholinergic biomarkers, i.e., acetylcholine level, free choline level, and choline acetyltransferase gene expression, and how cholinergic deficit affects novel object recognition and sociability in mice. Mice were treated with AlCl3 (250mg/kg). Acetylcholine level, free choline level, and choline acetyltransferase gene expression were determined in cortex, hippocampus, and amygdala. The mice were subjected to behavior tests (novel object recognition and social novelty preference) to assess memory deficits. The acetylcholine level in cortex and hippocampus was significantly reduced in aluminum-treated animals, as compared to cortex and hippocampus of control animals. Acetylcholine level in amygdala of aluminum-treated animals remained unchanged. Free choline level in all the three brain parts was found unaltered in aluminum-treated mice. The novel object recognition memory was severely impaired in aluminum-treated mice, as compared to the control group. Similarly, animals treated with aluminum showed reduced sociability compared to the control mice group. Our study demonstrates that aluminum exposure via drinking water causes reduced acetylcholine synthesis in spite of normal free choline availability. This deficit is caused by reduced recycling of acetylcholine due to lower choline acetyltransferase level. This cholinergic hypofunction leads to cognitive and memory deficits. Moreover, hippocampus is the most affected brain part after aluminum intoxication.

Cereboost™, an American ginseng extract, improves cognitive function via up-regulation of choline acetyltransferase expression and neuroprotection

In Alzheimer disease (AD), amyloid-beta (Aβ) peptides induce the degeneration of presynaptic cholinergic system, in which decreased activity of enzyme choline acetyltransferase (ChAT) responsible for acetylcholine synthesis is observed. Cereboost™, an extract of American ginseng extract, contains a high concentration of Rb1 ginsenoside which is a well-known ingredient improving human cognitive function. We investigated the effects of Cereboost™ on learning and memory function of mice challenged with an Aβ1-42 peptide and the underlying mechanisms in vitro. Cereboost™ protected against Aβ1-42-induced cytotoxicity in F3.ChAT stem cells, and enhanced the ChAT gene expression. Aβ1-42 injection into the mouse brain impaired the cognitive function, which was recovered by oral administration of Cereboost™. In addition, Cereboost™ restored brain microtubule-associated protein 2 and synaptophysin as well as acetylcholine concentration. The results demonstrate that Cereboost™ administration recovered the cognitive function of AD model animals by enhancing acetylcholine level via ChAT gene expression and neuroprotection.

Acetylcholine induces fibrogenic effects via M2/M3 acetylcholine receptors in non-alcoholic steatohepatitis and in primary human hepatic stellate cells.

The parasympathetic nervous system (PNS), via neurotransmitter acetylcholine (ACh), modulates fibrogenesis in animal models. However, the role of ACh in human hepatic fibrogenesis is unclear. We aimed to determine the fibrogenic responses of human hepatic stellate cells (hHSC) to ACh and the relevance of the PNS in hepatic fibrosis in patients with non-alcoholic steatohepatitis (NASH). Primary hHSC were analyzed for synthesis of endogenous ACh and acetylcholinesterase and gene expression of choline acetyltransferase and muscarinic ACh receptors (mAChR). Cell proliferation and fibrogenic markers were analyzed in hHSC exposed to ACh, atropine, mecamylamine, methoctramine, and 4-diphenylacetoxy-N-methylpiperidine methiodide. mAChR expression was analyzed in human NASH scored for fibrosis. We observed that hHSC synthesize ACh and acetylcholinesterase and express choline acetyltransferase and M1-M5 mAChR. We also show that M2 was increased during NASH progression, while both M2 and M3 were found upregulated in activated hHSC. Furthermore, endogenous ACh is required for hHSC basal growth. Exogenous ACh resulted in hHSC hyperproliferation via mAChR and phosphoinositide 3-kinase and Mitogen-activated protein kinase kinase (MEK) signaling pathways, as well as increased fibrogenic markers. We show that ACh regulates hHSC activation via M2 and M3 mAChR involving the phosphoinositide 3-kinase and MEK pathways in vitro. Finally, we provide evidence that the PNS may be involved in human NASH fibrosis.

Regulatory mechanisms of acetylcholine synthesis and release by T cells

AimsMuscarinic and nicotinic acetylcholine (ACh) receptors are expressed in immune cells. ACh synthesized by choline acetyltransferase (ChAT) and released in T cells binds to these receptors. Furthermore, we have recently demonstrated the involvement of mediatophore, a homooligomer of a 16-kDa proteolipid subunit of vacuolar H+-ATPase, in ACh release from T cells. In this study, we investigated the effects of phorbol 12-myristate 13-acetate (PMA), dibutyryl cAMP (dbcAMP) and FK506, an immunosuppressant calcineurin inhibitor, on lymphocytic cholinergic activity in T cells. Main methodsWe determined the content and release of ACh in human leukemic T cell line MOLT-3 cells using a sensitive and specific radioimmunoassay for ACh. In addition, expression of ChAT mRNA and ChAT activity were investigated using reverse-transcription-polymerase chain reaction and Fonnum method, respectively. Key findingsPhytohemagglutinin (PHA), a T-cell activator, up-regulated ChAT mRNA expression, synthesis and release of ACh. PMA, a protein kinase C (PKC) activator, and dbcAMP, a protein kinase A (PKA) activator, also increased ChAT activity and ACh synthesis by up-regulating ChAT gene expression. FK506 inhibited PHA-induced up-regulation of ChAT mRNA expression, suggesting the involvement of calcineurin-mediated pathways in ChAT gene transcription. SignificanceActivation of PKC and PKA up-regulates ACh synthesis in T cells, and immunological activation triggers ChAT gene transcription through calcineurin-mediated pathways.

Histone deacetylase 9 as a negative regulator for choline acetyltransferase gene in NG108-15 neuronal cells

The biological function of histone deacetylases (HDACs), namely, repression of gene expression by removing an acetyl group from a histone N-terminal tail, plays an important role in numerous biological processes such as cell cycle, differentiation, and apoptosis in the development of individual tissues, including the brain. We previously showed the possible role of HDAC activity in the regulation of gene expression of choline acetyltransferase (ChAT), a specific marker for cholinergic neurons and their function, in NG108-15 neuronal cells as an in vitro model of cholinergic neurons. The objectives of the present study were to specify key HDACs and investigate the essential role of HDACs in ChAT gene regulation in NG108-15 cells. The experiments using different types of HDAC inhibitors indicated that class IIa HDACs substantially participate in the regulation of ChAT gene expression. In addition, HDAC9, a class IIa enzyme, was dramatically decreased at the protein levels, and dissociated from the promoter region of ChAT gene during neuronal differentiation. Furthermore, knockdown of HDAC9 by siRNA increased ChAT gene expression in undifferentiated cells. These findings demonstrate that HDAC9 is responsible for repressing ChAT gene expression in NG108-15 neuronal cells, and thus plays an important role in cholinergic differentiation.

American ginseng improves neurocognitive function in senescence‐accelerated mice: Possible role of the upregulated insulin and choline acetyltransferase gene expression

To investigate the effects of American ginseng on neurocognitive function and glucose regulation in senescence-accelerated mice. Male senescence-resistant inbred strains (SAMR1) and senescence-prone inbred strains (SAMP10) mice were divided into five groups and fed either a control diet or an American ginseng-supplemented diet (1% or 2% g/g) from 6 weeks to 10 months of age. Bodyweight, levels of fasting plasma glucose (FPG) and grading scores were monitored every month and neurocognitive functions were evaluated at 9 months of age with a KUROBOX apparatus using a stress-free positive cue task. Gene expressions of peroxisome proliferator-activated receptor delta (PPAR-δ), insulin, choline acetyltransferase (ChAT) and amyloid precursor protein (APP) in the brain were measured by real-time quantitative reverse transcription polymerase chain reaction assays. American ginseng decreased FPG in SAMR1 mice, but increased FPG in SAMP10 mice. Correct visit ratios were higher in both SAMR1 and SAMP10 strains consuming an American ginseng-supplemented diet. Gene upregulation of insulin and ChAT in the brain, but not of PPAR-δ or APP, was evident in American ginseng-fed groups. Daily consumption of American ginseng induced an enhancement in neurocognitive function in senescence-accelerated mice, which could be related to the upregulation of insulin and ChAT gene expression in the brain. Geriatr Gerontol Int 2012; 12: 123-130.

Histone deacetylase 9 regulates choline acetyltransferase gene expression in NG108‐15 cells

Histone deacetylases (HDACs) catalyze the removal of acetyl groups from N‐terminal lysine residues of histone tails, resulting in chromatin condensation and transcriptional repression. We have previously shown that the inhibition of HDAC activity by a general and specific inhibitor of HDACs, trichostatin A, markedly increased the gene expression of choline acetyltransferase (ChAT), the synthetic enzyme for neurotransmitter acetylcholine and a specific marker for cholinergic phenotype, in NG108‐15 cells as a model of cholinergic neurons. In the present study, we examined which HDACs are involved in the regulation of ChAT gene. In experiments using class‐specific HDAC inhibitors, class IIa HDACs were a strong candidate for involvement in the regulation of ChAT gene in NG108‐15 cells. In addition, the expression of HDAC9, a class IIa enzyme, was dramatically decreased at the level of mRNA and protein during dibutyryl cyclic‐AMP‐induced cholinergic differentiation, whereas class I and other class IIa HDACs were not. Furthermore, the knockdown of HDAC9 by siRNA induced ChAT gene expression in proliferating cells. These results suggest that HDAC9 plays a key regulatory role in the acquisition of cholinergic phenotypes in NG108‐15 cells. This study was supported, in part, by a Research Fellowship of the Japan Society for the Promotion of Science to SA.

Asymmetric regulation by estrogen at the cholinergic gene locus in differentiated NG108-15 neuronal cells

Aims Estrogen acts as a neurogenerative and neuroprotective factor in the cholinergic system. Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are regarded as markers of cholinergic neurons. The genes coding these proteins are located at a common locus, the cholinergic gene locus. However, few details concerning activation of the locus have been obtained. We examined the effect of estrogen on the activation pattern of the locus using a cholinergic cell line. Main methods NG108-15 neuronal cells, as a model of cholinergic neurons, were used. Dose-dependent effects of estradiol (E 2) on the gene expression of ChAT and VAChT were quantitatively determined by a real-time RT-PCR. The expression of ChAT mRNA variants was qualitatively evaluated by RT-PCR using specific primers. Key findings The expression of ChAT and VAChT mRNA was strongly enhanced with the induction of differentiation. The enhanced expression of ChAT mRNA was further increased dose-dependently by E 2 (10 − 10 to 10 − 7 M), while that of VAChT mRNA did not respond to E 2. The up-regulation of ChAT mRNA expression by E 2 was abolished by co-treatment with a pure-antagonist of estrogen receptors. A qualitative analysis of ChAT mRNA variants revealed the R types, which share a common sequence with the VAChT gene, and type M ChAT mRNA to mainly be expressed, and that the appearance of these variants was not altered by E 2. Significance The cholinergic gene locus in differentiated NG108-15 neuronal cells is further activated by E 2, but the effect is restricted to the transcription of ChAT gene.

Involvement of histone acetylation in the regulation of choline acetyltransferase gene in NG108-15 neuronal cells

Post-translational modification of histone such as acetylation of N-terminal of lysine residues influences gene expression by modulating the accessibility of specific transcription factors to the promoter region, and is essential for a wide variety of cellular processes in the development of individual tissues, including the brain. However, few details concerning the acquisition of specific neurotransmitter phenotype have been obtained. In the present study, we investigated the possible involvement of histone acetylation in the gene expression of choline acetyltransferase (ChAT), a specific marker for cholinergic neuron and its function, in NG108-15 neuronal cells as an in vitro model of cholinergic neuron. Treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA), which induces global histone hyper-acetylation of the cells, resulted in marked increase in the expression of ChAT gene in proliferating NG108-15 cells. Furthermore, RT-PCR analysis using primer pairs for individual variants of ChAT mRNA (R1-4, N1, and M type) revealed that M type, not R1-4 and N1 type, ChAT mRNA were mainly transcribed, and chromatin immunoprecipitation assay indicated that the promoter region of M type ChAT gene was highly acetylated, in the dibutyryl cyclic AMP-induced neuronal differentiation of NG108-15 cells. The present findings demonstrate that the acquisition of neurotransmitter phenotype is epigenetically, at least the hyper-acetylation on the core promoter region of ChAT gene, regulated in NG108-15 neuronal cells.