Regulation Of Choline Acetyltransferase Research Articles

Enhanced ubiquitination and proteasomal degradation of catalytically deficient human choline acetyltransferase mutants.

Choline acetyltransferase (ChAT) is essential for cholinergic neuron function as it mediates synthesis of the neurotransmitter acetylcholine. ChAT mutations have been linked to the neuromuscular disorder congenital myasthenic syndrome (CMS). One CMS-related ChAT mutation, V18M, reduces enzyme activity and cellular protein levels, and is positioned within a highly conserved proline-rich motif with the sequence 14 PKLPVPP20 . We demonstrate that N-terminal truncation that includes this proline-rich motif, as well as mutation of prolines-17/19 together to alanine (P17A/P19A), dramatically reduces ChAT steady-state protein levels and cellular activity when expressed in cholinergic SN56 neural cells. The invitro activity of bacterially expressed recombinant P17A/P19A-ChAT is also reduced, although this is not caused by changes in protein secondary structure or thermal stability. Treatment of SN56 cells with the proteasome inhibitor MG132 increases cellular P17A/P19A-ChAT steady-state protein levels, and by immunoprecipitation we found that ChAT is ubiquitinated and that polyubiquitination of P17A/P19A-ChAT is increased compared to wild-type (WT) ChAT. Using a novel fluorescent-biorthogonal pulse-chase protocol in SN56 cells, we determined that the protein half-life of P17A/P19A-ChAT (2.2h) is substantially reduced compared to WT-ChAT (19.7h). Lastly, we show that two CMS-related ChAT mutants (V18M and A513T) have enhanced ubiquitination, and that treatment with MG132 can partially restore both the steady-state protein levels as well as cellular activity of some CMS-mutant ChAT. These results identify a novel mechanism for regulation of ChAT through the ubiquitin-proteasome system that is influenced by the conserved N-terminal proline-rich motif of ChAT and may be implicated in CMS pathology. Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons. In this study we find that steady-state protein levels of human 69-kDa ChAT are regulated by the ubiquitin-proteasome system. Mutation of a highly conserved N-terminal proline-rich motif in human 69-kDa ChAT reduces both cellular ChAT protein levels, through enhanced ubiquitination and proteasomal degradation, and enzyme activity. Ubiquitination of catalytically deficient congenital myasthenic syndrome (CMS)-mutant ChAT is increased in cells, and importantly proteasome inhibition partially restores steady-state protein levels as well as cellular activity of some CMS-mutant ChAT proteins.

Paradoxical regulation of ChAT and nNOS expression in animal models of Crohn's colitis and ulcerative colitis

Morphological and functional changes in the enteric nervous system (ENS) have been reported in inflammatory bowel disease. We examined the effects of inflammation on the expression of choline acetyltransferase (ChAT) and nNOS in the muscularis externae of two models of colonic inflammation, trinitrobenzene sulfonic acid (TNBS)-induced colitis, which models Crohn's disease-like inflammation, and DSS-induced colitis, which models ulcerative Colitis-like inflammation. In TNBS colitis, we observed significant decline in ChAT, nNOS, and protein gene product (PGP) 9.5 protein and mRNA levels. In DSS colitis, ChAT and PGP9.5 were significantly upregulated while nNOS levels did not change. The nNOS dimer-to-monomer ratio decreased significantly in DSS- but not in TNBS-induced colitis. No differences were observed in the percentage of either ChAT (31 vs. 33%)- or nNOS (37 vs. 41%)-immunopositive neurons per ganglia or the mean number of neurons per ganglia (55 ± 5 vs. 59 ± 5, P > 0.05). Incubation of the distal colon muscularis externae in vitro with different types of inflammatory mediators showed that cytokines decreased ChAT and nNOS expression, whereas H₂O₂, a component of oxidative stress, increased their expression. NF-κB inhibitor MG-132 did not prevent the IL-1β-induced decline in either ChAT or nNOS expression. These findings showed that TNBS- and DSS-induced inflammation differentially regulates the expression of two critical proteins expressed in the colonic myenteric neurons. These differences are likely due to the exposure of the myenteric plexus neurons to different combinations of Th1-type inflammatory mediators and H₂O₂ in each model.

The cell adhesion molecule L1 regulates the expression of choline acetyltransferase and the development of septal cholinergic neurons

Mutations in the L1 gene cause severe brain malformations and mental retardation. We investigated the potential roles of L1 in the regulation of choline acetyltransferase (ChAT) and in the development of septal cholinergic neurons, which are known to project to the hippocampus and play key roles in cognitive functions. Using stereological approaches, we detected significantly fewer ChAT-positive cholinergic neurons in the medial septum and vertical limb of the diagonal band of Broca (MS/VDB) of 2-week-old L1-deficient mice compared to wild-type littermates (1644 ± 137 vs. 2051 ± 165, P = 0.038). ChAT protein levels in the septum were 53% lower in 2-week-old L1-deficient mice compared to wild-type littermates. ChAT activity in the septum was significantly reduced in L1-deficient mice compared to wild-type littermates at 1 (34%) and 2 (40%) weeks of age. In vitro, increasing doses of L1-Fc induced ChAT activity in septal neurons with a significant linear trend (*P = 0.0065). At 4 weeks of age in the septum and at all time points investigated in the caudate-putamen (CPu), the number of ChAT-positive neurons and the levels of ChAT activity were not statistically different between L1-deficient mice and wild-type littermates. The total number of cells positive for the neuronal nuclear antigen (NeuN) in the MS/VDB and CPu was not statistically different in L1-deficient mice compared to wild-type littermates, and comparable expression of the cell cycle marker Ki67 was observed. Our results indicate that L1 is required for the timely maturation of septal cholinergic neurons and that L1 promotes the expression and activity of ChAT in septal neurons.

Role of cholinergic markers on memory function of rats exposed to hypobaric hypoxia

Hypobaric hypoxia is encountered at high altitude. It has a deleterious effect on cognitive functions. An important cause of memory impairment at high altitude is the impairment of neurotransmission. The present study investigates the role of cholinergic markers in hypobaric hypoxia-induced memory impairment. Rats were exposed to hypobaric hypoxia at 6100 m for 7 days in a simulated-decompression chamber. Memory performance was assessed using the Morris water maze task. Cholinergic markers such as acetylcholine, acetylcholinesterase, choline acetyltransferase, α-7-nicotinic acetylcholine receptor and M 1 muscarinic acetylcholine receptor were also evaluated along with neuronal morphology and DNA fragmentation. We found impairment in memory function along with a decrease in acetylcholine levels, increase in acetylcholinesterase activity, down regulation of choline acetyltransferase, α-7-nicotinic acetylcholine receptor and M 1 muscarinic acetylcholine receptor. We also found that cellular damage is associated with a significant increase in DNA fragmentation. However, administration of acetylcholinesterase inhibitors, such as physostigmine and galantamine, resulted in amelioration of the hypobaric hypoxia induced deleterious effects. It improved acetylcholine level, decreased acetylcholinesterase activity and increased the synthesis of acetylcholine by increasing choline acetyltransferase activity. Also, the acetylcholinesterase inhibitors improved neuronal morphology, perhaps by increasing the expression of α-7-nicotinic acetylcholine receptor and by reducing the acetylcholinesterase level in the cortex and the hippocampus. Therefore, our results suggest cholinergic dysfunction is one of the mechanisms involved in hypobaric hypoxia-induced memory impairment and that acetylcholinesterase inhibitors were able to restore cholinergic function and thus improve memory function.

Exploring the regulation of the expression of ChAT and VAChT genes in NG108-15 cells: implication of PKA and PI3K signaling pathways.

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

Effects of sex hormones, forskolin, and nicotine on choline acetyltransferase activity in human isolated placenta.

The activity of choline acetyltransferase (ChAT) was investigated in the human placenta before and after long-term incubation (24 h) to test the effects of sex hormones, nicotine and forskolin. ChAT activity differed considerably between the amnion (0.03 micromol/mg protein/h) and the villus (0.56). After long-term incubation, ChAT activity persisted in the latter but declined in the amnion. Neither sex hormones (beta-estradiol, testosterone, progesterone; 10 or 100 nM each) nor follicle stimulating hormone and luteinizing hormone (FSH/LH; 8.4 U/ml each) modified ChAT activity. Also nicotine (1 nM-100 microM) did not affect ChAT activity. Forskolin, an activitor of adenylyl cyclase, reduced ChAT activity in the villus but not in amnion. The present model offers the possibility to investigate ChAT regulation in intact tissue under long-term incubation. The risks of maternal smoking during pregnancy cannot be attributed to an effect of nicotine on placental ChAT activity. Differences in the regulation of ChAT appear to exist between neuronal and nonneuronal cells.

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.

TGF-beta 2 attenuates the injury-induced death of mature motoneurons.

The distributions of transforming growth factor-betas (TGF-betas) and their receptors suggest that the TGF-betas regulate motoneuron survival. This hypothesis was tested by avulsing the hypoglossal nerve of adult rats and perfusing either TGF-beta 2 or vehicle adjacent to the hypoglossal nucleus. By 4 weeks, half of the avulsed motoneurons had died. Infusion of 6 ng of TGF-beta 2 adjacent to the avulsed motor nucleus caused a significant attenuation of this death. This dose of TGF-beta 2 is low compared to that used with GDNF or BDNF in previous studies of avulsed motoneurons, indicating that TGF-beta 2 may be one of the more potent survival factors for adult motoneurons. TGF-beta 2 was, however, unable to prevent or reduce the axotomy-induced down regulation of choline acetyltransferase. Other motoneuron survival factors also have a narrow-spectrum of actions, suggesting that the homeostasis of motoneurons is regulated by a cocktail of growth factors with distinct but partially overlapping actions.

Choline Acetyltransferase from the electric organ of Electrophorus electricus (L.)--physicochemical characterization and immunochemical identification.

It is well known that the regulation of choline acetyltransferase (ChAT) activity, under physiological conditions, is important for the development and neuronal activities of cholinergic systems. The purification of ChAT has been obtained from many sources such as electric organs of fishes, Drosophila melonogaster, and mammals. We have prepared choline acetyltransferase from a pool of supernatants obtained by differential centrifugation of electric organ homogenates from Electrophorus electricus (L.) in Tris-phosphate buffer, 0.05 M, pH 7.6. The first step of the enzyme purification was performed by ammonium sulfate precipitation at 40% and 80%. The precipitate at 80% was solubilized with sodium-phosphate buffer 0.05 M, pH 7.6, dialyzed, chromatographed on DEAE-52 column and the active fraction submitted to FPLC system columns (Mono-Q: ion exchange- Superose-12: gel filtration). ChAT activity from the eluates was estimated by Fonnun's method [Fonnun, 1975], with Acetyl-Coenzyme A tritium labelled ([3H]AcCoA) as substrate, and the synthesis of 3HACh formed was measured. The peak from gel filtration showed a relative molecular mass of 80 offkDa with highest activity in the order of 77,42 nmoles ACh/min/mg protein. This fraction was analyzed by SDS-PAGE and a band of 42 kDa was detected with Coomassie blue stain, indicating that the enzyme is formed by two subunits. Employing an antibody, the presence of ChAT was confirmed with the Western blotting technique. Isoelectrofocusing analysis demonstrated two isoforms with pI of 6,49 and 6,56, respectively.

Choline acetyltransferase: celebrating its fiftieth year.

It is well known that the regulation of choline acetyltransferase (ChAT) activity under physiological and pathological conditions is important for the development and neuronal activities of cholinergic systems involved in many fundamental brain functions. This review focuses on recent progress in understanding the regulation of ChAT at the levels of both the protein and the mRNA. A deficiency in ChAT activity has been reported for neurodegenerative conditions such as Alzheimer's disease, amyotrophic lateral sclerosis, and schizophrenia. Although a major feature of ChAT regulation is likely to involve the spatial and temporal control of transcription, regulation of expression can also be at the level of RNA processing, transport/translocation, turnover, or translation. In addition, there is increasing evidence that ChAT might be regulated at the posttranslational level by compartmentation and/or covalent modification, i.e., phosphorylation, as well as noncovalent modification (protein-protein interaction, etc.). Synaptic activity and the state of neuronal transmission may also involve the regulation of ChAT at different levels via both positive and negative feedback loops, as was demonstrated in the characterization of two ChAT mutant Drosophila strains. Clearly, identification of cholinergic-specific elements and the characterization of the trans-acting factors that bind to them represent an important area of future research. Equally important is research on the mechanisms governing ChAT as an enzymatic entity. The future should be an exciting time during which we look forward to the elucidation of the cholinergic signal and its regulation as well as the determination of the three-dimensional structure of the enzyme.

The regulation of choline acetyltransferase by interleukin-3 on septal cholinergic cell line

The regulation of choline acetyltransferase by interleukin-3 on septal cholinergic cell line

Effect of cholinesterase inhibitor and exercise on choline acetyltransferase and acetylcholinesterase activities in rat brain regions

This study sought to determine whether the choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) enzymes in the brain were affected in a regionally selective manner by chemical and physical stressors: 1) subacute administration of physostigmine (Phy); 2) exercise; and 3) the combination of these two stressors. ChAT and AChE activities in corpus striatum were significantly decreased due to Phy as well as Phy + exercise. This suggests that corpus striatum is affected by chemical stressors but more so by the combination of chemical and physical stressors. The brainstem is the only region which showed inhibition of ChAT activity due to exercise. Subacute Phy also inhibited brainstem ChAT activity. The hippocampus showed significant decrease in ChAT activity due to Phy + exercise but not due to Phy alone. These results suggest that the brain regions involved with control of motor, autonomic and cognitive functions were affected by subacute Phy and exercise. These data are consistent with the hypothesis that the responsiveness of these brain regions to different stressors is a function of the level of ongoing cholinergic activity and that elevations in ACh levels due to AChE inhibition may have long-term effects on the regulation of ChAT and AChE activities through a negative feedback mechanism.

Influence of soluble environmental factors on the development of fetal brain acetylcholinesterase-positive neurons cultured in a chemically defined medium: comparison with the effects of l-triiodothyronine ( l-T 3)

In cerebral hemisphere neuronal cultures derived from 15-day-old rat embryos, the addition of l-triiodothyronine ( l-T 3) or nerve growth factor (NGF) enhanced the expression of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in a dose-dependent manner. When cultures were supplemented with both agents at maximal effective concentrations, the stimulation in ChAT and AChE activities was significantly greater than the sum of the individual effects. Conversely, when the cultures were exposed to astrocyte conditioned medium grown in the presence or absence of l-T 3 ( CM + l- T 3 or CM − l- T 3 ), laminin and fibroblast growth factor (FGF), ChAT and AChE activities were not stimulated above those of control cultures when added alone or in combination with l-T 3. Furthermore, l-T 3, NGF, CMs, laminin and FGF did not affect AChE + cell survival, but significantly increased neurite outgrowth and branching with NGF and l-T 3 being the most powerful agents followed by CMs, laminin and FGF. Additionally, the simultaneous addition of l-T 3 with either laminin or FGF in culture, caused an additive effect to the effect of l-T 3 in the neurite density of AChE + cells with both agents. This study shows that (1) thyroid hormones do not act through the regulation of soluble neurotrophic factors produced by astroglial cells, (2) thyroid hormones interact with the effect of NGF on ChAT and AChE activities, (3) the regulation of ChAT and AChE activities and the neurite outgrowth are independently regulated, and (4) the regulation of ChAT and AChE activities is very specific compared with that of neurite outgrowth. Thus, our recent immunocytochemical demonstration of l-T 3 nuclear receptors (TNR), in a subpopulation of AChE + cells correlated with l-T 3 morphological effects suggests that some effects of l-T 3 on the maturation of a subpopulation of AChE + cells may result from a direct effect of this hormone with its specific nuclear receptors.

Regulation of Choline Acetyltransferase

This chapter presents the studies related to choline acetyltransferase (ChAT) and cholinergic biology into a more comprehensive neurobiological context. The recent results of biochemical, immunological, molecular biological, arid immunocytochemical studies of ChAT and its regulation are discussed. The phylogenetic distribution of ChAT in the nervous systems of all animals indicates a long and distinguished evolutionary history for the production of the protein and the utility of cell-cell communication via acetylcholine. ChAT activity, like that of any enzyme, can be regulated by a number of molecular mechanisms at many different levels. A variety of these potential control points are investigated using most available techniques and intellectual approaches. These approaches, including short-term regulation of enzyme activity, regulation of ChAT gene transcription and translation, and intracellular compartmentalization are outlined. The two substrates used for the catalytic production of ACh by ChAT are acetyl Coenzyme A and choline. A number of other pharmacological approaches have been used to identify potentially important steps for regulatory control of ChAT activity. Various second messenger systems have been implicated in influencing the properties of cholinergic cells in general and ChAT activity in particular.

Acetylcholine metabolism by cultured neurons from rat nodose ganglia: regulation by a macromolecule from muscle-conditioned medium

Acetylcholine metabolism has been studied in primary cultures of neurons dissociated from newborn rat nodose ganglia. Neither nerve growth factor nor muscle-conditioned medium had any detectable effect on the long-term survival of these neurons, which appeared well differentiated upon phase-contrast and electron microscopic examination. [ 3H]Acetylcholine synthesis and accumulation by 2–3-week old nodose cultures and choline acetyltransferase activity were increased (2.0 ± 0.15)-fold and (2.0 ± 0.48)-fold, respectively, upon growth with muscle-conditioned medium, whereas acetylcholinesterase was decreased (1.5 ± 0.1)-fold (means ± SEM, n = 5–9). The same effects were observed when comparing nodose cultures grown in the presence of proteins purified from conditioned medium in four fractionation steps. This purified material had no effects on the protein content of the cultures, or on lactate dehydrogenase activity, and thus did not affect the overall growth of the cultures. We demonstrate that this factor copurifies with a factor(s) involved in the regulation of choline acetyltransferase and acetylcholinesterase in neuron cultures from newborn rat superior cervical ganglia [ Swerts, Le Van Thai, Vigny and Weber (1983) Devl Biol. 100, 1–111 and from embryonic day 13 rat embryo spinal cord [ Giess and Weber (1984) J. Neurosci. 4, 1442–1452]. Although the cholinergic factor from muscle-conditioned medium has not been purified to homogeneity, the data suggest that the same extracellular, macromolecular factor may be involved in the regulation of acetylcholine metabolism in derivatives from the neural crest, the neural tube and the epibranchiai placodes.

Regulation of choline acetyltransferase in primary cell cultures of spinal cord by neurotransmitter l-norepinephrine

Neurotransmitter L-norepinephrine increased up to 8-fold the activity of choline acetyltransferase (CAT), the enzyme responsible for the synthesis of acetylcholine, in mouse spinal cord cells in culture grown for several days. The increase of CAT activity by L-norepinephrine was mediated by a beta-adrenergic receptor in the same manner as the response of intracellular cyclic AMP. Derivatives of cyclic AMP caused an increase of CAT activity to the level similar to that of L-norepinephrine. A cyclic AMP phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine (IBMX), enhanced the elevation of CAT activity by L-norepinephrine. These results indicate that L-norepinephrine stimulated the synthesis of CAT molecules via the action of cyclic AMP. The pretreatment of cells with 5-fluoro-2'-deoxyuridine (FdU) markedly diminished the numbers of satellite cells and, in parallel, the responses of CAT activity to L-norepinephrine. The increase of cyclic AMP by L-norepinephrine was also reduced by pretreatment of the cells with FdU. In contrast, co-cultures of spinal cord with heart muscle markedly (30-fold) stimulated CAT activity both with and without pretreatment of FdU. The addition of L-norepinephrine and co-cultures with heart muscle showed an additive effect. These observations indicate that the stimulatory effect of L-norepinephrine on CAT activity is mostly, if not only, mediated via the interaction with satellite cells, and that the increase of CAT activity by L-norepinephrine is based on a mechanism different from that of co-cultures with heart muscle cells.

Distribution of acetylcholine in the normal and denervated pigeon ciliary ganglion.

A gas chromatograph/mass spectrometer has been used for the positive identification and estimation of acetylcholine (ACh) in normal and denervated adult pigeon ciliary ganglia. The measurements for the denervated ganglia were done 1–15 days after surgical section of the oculomotor nerve. Ganglionic transmission is blocked from 2 to 13 days after nerve section 21 . Histological observation of the cellular elements was done with electron microscopic techniques. ACh (as chloride) is distributed in the normal ganglia as follows: 3 ng/mm in the intraganglionic branches of the oculomotor nerves; 20–22 ng in the presynaptic nerve terminals; 8 ng in the ganglion cells, and 2 ng/mm in the intraganglionic branches of the postganglionic nerves. It has been estimated that 1.2 pg of ACh is in each cell soma. The level of ACh in presynaptic elements (intraganglionic branches of the oculomotor nerve and nerve terminals) decreases to a residual value of 1–2 ng/mm of ACh after 5 days of denervation. This value was measured in the distal stump of the degenerating oculomotor nerve. ACh also decreases in the postsynaptic elements (ganglion cells and nerves) especially after 5 days, reaching levels of less than 1 ng after 10 days. ACh levels tend to increase after 15 days when reinnervation occurs. It is postulated that the amount of ACh present in the postsynaptic elements depends upon the integrity of the presynaptic element and that probably this transsynaptic effect in the ACh synthesis is mediated through regulation of choline acetyltransferase. It is also speculated that the ACh measured in the denervated stump is present in the Schwann cell.