Acetylcholine Synthesis Research Articles

Neuromodulatory potential of Asparagus racemosus and its bioactive molecule Shatavarin IV by enhancing synaptic acetylcholine level and nAChR activity

Cholinergic dysfunction has been commonly known to be associated with plethora of neurodegenerative disorders and also serves as a biomarker. Recently, cholinergic system demonstrated that acetylcholine has major role in regulation of its function therefore the main therapeutic regimens towards disease management have been focused on increasing acetylcholine levels. The current study explores the potential of Asparagus racemosus extract (ARE) and its bioactive molecule Shatavarin IV (SIV) in improving cholinergic transmission via utilizing Caenorhabditis elegans considering as a model system. Observations and results obtained through this study have clearly showed significant modulation in cholinergic function by increasing acetylcholine (ACh) levels and the nicotinic acetylcholine receptors (nAChRs) activity. Further exploration on mechanistic facet pointed towards ARE and SIV modulatory potential through increased synaptic ACh level by blocking acetyl cholinesterase at enzyme level and by regulating increment in transcript level of cha-1, and cho-1 that are directly responsible for the synthesis of ACh. Further, the up-regulation of unc-38 and unc-50 transcripts could be the reason for enhanced nAChR activity and investigation on stress modulator activity showed excellent efficiency of ARE and SIV in diminishing ROS thereby lowering the oxidative damage.

Combined therapy with Medotilin/Elfunat for the prevention and treatment of neuropsychiatric complications after open-heart surgery

After open-heart surgery, postoperative neuropsychiatric complications often develop: stroke — in 4.8 % of cases, diffuse encephalopathy — in 12 %, behavior changes — in 5–40 %, cognitive impairment — in 12–79 %, mental disorders — in 13–64 %. The causes of their development are acetylcholine deficiency with subsequent dysfunction of the cholinergic system and oxidative stress, which result from hypoxia, hypoperfusion, and a systemic inflammatory reaction. In this regard, the appointment of drugs that restore the acetylcholine synthesis – choline alfoscerate (Medotilin), and drugs that suppress oxidative stress – mexidol (Elfunate) is considered a promising direction for the prevention and treatment of neuropsychiatric complications. The study conducted on the basis of the regional cardiac surgery center of the Odessa Regional Hospital demonstrated that the combined use of Medotilin and Elfunate can reduce the frequency of neuropsychiatric complications, as well as reduce the duration of treatment in the hospital.

The miR-182-5p/FGF21/acetylcholine axis mediates the crosstalk between adipocytes and macrophages to promote beige fat thermogenesis.

A dynamically regulated microenvironment, which is mediated by crosstalk between adipocytes and neighboring cells, is critical for adipose tissue homeostasis and function. However, information on key molecules and/or signaling pathways regulating the crosstalk remains limited. In this study, we identify adipocyte miRNA-182-5p (miR-182-5p) as a crucial antiobesity molecule that stimulated beige fat thermogenesis by promoting the crosstalk between adipocytes and macrophages. miR-182-5p was highly enriched in thermogenic adipocytes, and its expression was markedly stimulated by cold exposure in mice. In contrast, miR-182-5p expression was significantly reduced in adipose tissues of obese humans and mice. Knockout of miR-185-5p decreased cold-induced beige fat thermogenesis whereas overexpression of miR-185-5p increased beiging and thermogenesis in mice. Mechanistically, miR-182-5p promoted FGF21 expression and secretion in adipocytes by suppressing nuclear receptor subfamily 1 group D member 1 (Nr1d1) at 5′-UTR, which in turn stimulates acetylcholine synthesis and release in macrophages. Increased acetylcholine expression activated the nicotine acetylcholine receptor in adipocytes, which stimulated PKA signaling and consequent thermogenic gene expression. Our study reveals a key role of the miR-182-5p/FGF21/acetylcholine/acetylcholine receptor axis that mediates the crosstalk between adipocytes and macrophages to promote beige fat thermogenesis. Activation of the miR-182-5p–induced signaling pathway in adipose tissue may be an effective approach to ameliorate obesity and associated metabolic diseases.

Binocular Diplopia After A Snakebite

A snakebite is an injury caused by a bite from a snake, often resulting in puncture wounds inflicted by animal’s fangs and sometimes resulting in envenomation. Venom may cause a complex condition that can be local damage, neuromuscular dysfunction, or systemic vascular damage leading to hemolysis. The main cause of mortality and morbidity in snake bites is neurotoxicity, and the main cause of neurotoxicity is sudden neuromuscular paralysis. Synthesis, packaging, transport, and release of the neurotransmitter acetylcholine in the presynaptic field, blockade of postsynaptic nicotinic acetylcholine receptors, acetylcholine esterase inhibition, muscarinic effects of some snake toxins, and inhibition of voltage-gated calcium gates, changes in the neuromuscular junction after snakebite can explain neuromuscular paralysis secondary to the snake bite. We present a case, with bilateral diplopia and ptosis after snakebite, that was recovered after the use of anticholinesterase therapy and antivenom.

Change in the neurochemical signature and morphological development of the parvocellular isthmic projection to the avian tectum

Neurons can change their classical neurotransmitters during ontogeny, sometimes going through stages of dual release. Here, we explored the development of the neurotransmitter identity of neurons of the avian nucleus isthmi parvocellularis (Ipc), whose axon terminals are retinotopically arranged in the optic tectum (TeO) and exert a focal gating effect upon the ascending transmission of retinal inputs. Although cholinergic and glutamatergic markers are both found in Ipc neurons and terminals of adult pigeons and chicks, the mRNA expression of the vesicular acetylcholine transporter, VAChT, is weak or absent. To explore how the Ipc neurotransmitter identity is established during ontogeny, we analyzed the expression of mRNAs coding for cholinergic (ChAT, VAChT, and CHT) and glutamatergic (VGluT2 and VGluT3) markers in chick embryos at different developmental stages. We found that between E12 and E18, Ipc neurons expressed all cholinergic mRNAs and also VGluT2 mRNA; however, from E16 through posthatch stages, VAChT mRNA expression was specifically diminished. Our ex vivo deposits of tracer crystals and intracellular filling experiments revealed that Ipc axons exhibit a mature paintbrush morphology late in development, experiencing marked morphological transformations during the period of presumptive dual vesicular transmitter release. Additionally, although ChAT protein immunoassays increasingly label the growing Ipc axon, this labeling was consistently restricted to sparse portions of the terminal branches. Combined, these results suggest that the synthesis of glutamate and acetylcholine, and their vesicular release, is complexly linked to the developmental processes of branching, growing and remodeling of these unique axons.

Physiology of cholinergic and adrenergic synapses. Role in pharmacology and practical significance

The transmission of signals between cholinergic neurons and from neurons to muscle cells (neuro-neuronal and neuromuscular transduction) occurs through synapses. They are formed by the membranes of two contacting cells, presynaptic and postsynaptic, which are separated by a narrow synaptic gap. The review article provides up-to-date information about the physiological processes in cholinergic and adrenergic synapses. The role of these synapses in pharmacology and their practical significance are presented. The transmission of excitation in cholinergic synapses occurs with the help of acetylcholine. The stages of synthesis, storage and release of acetylcholine are the same in all cholinergic neurons. The specific effects of acetylcholine mediated through cholinergic synapses depend mainly on the type of synaptic cholinergic receptors. In the system of efferent innervation, adrenergic synapses are formed by the endings of postganglionic sympathetic (adrenergic) fibers and cells of effector organs.

Mass Spectrometric Enzyme Histochemistry for Choline Acetyltransferase Reveals De Novo Acetylcholine Synthesis in Rodent Brain and Spinal Cord

Choline acetyltransferase (ChAT), responsible for the synthesis of acetylcholine, plays an important role in neurotransmission. However, no method to visualize the ChAT activity in tissues has been reported to date. In this study, mass spectrometry imaging (MSI) was used to visualize ChAT activity in situ, which is difficult with conventional enzyme histochemistry. By using choline chloride-trimethyl-d9 (choline-d9) as a substrate and simultaneously supplying an inhibitor of cholinesterase to tissues, we succeeded in directly visualizing the ChAT activity in the rodent brain and spinal cord. The findings revealed heterogeneous ChAT activity in the striatum of the mouse brain and in the spinal lower motor neurons that connect the anterior horn to the ventral root. Furthermore, extending the developed method to spinal cord injury (SCI) model mice revealed the site-specific effect of primary and secondary injury on ChAT activity. This study shows that the MSI-based enzyme histochemistry of ChAT could be a useful tool for studying cholinergic neurons.

Analysis of Caenorhabditis elegans acetylcholine synthesis mutants reveals a temperature-sensitive requirement for cholinergic neuromuscular function.

In Caenorhabditis elegans, the cha-1 gene encodes choline acetyltransferase (ChAT), the enzyme that synthesizes the neurotransmitter acetylcholine. We have analyzed a large number of cha-1 hypomorphic mutants, most of which are missense alleles. Some homozygous cha-1 mutants have approximately normal ChAT immunoreactivity; many other alleles lead to consistent reductions in synaptic immunostaining, although the residual protein appears to be stable. Regardless of protein levels, neuromuscular function of almost all mutants is temperature-sensitive, i.e., neuromuscular function is worse at 25° than at 14°. We show that the temperature effects are not related to acetylcholine release, but specifically to alterations in acetylcholine synthesis. This is not a temperature-dependent developmental phenotype, because animals raised at 20° to young adulthood and then shifted for 2 h to either 14° or 25° had swimming and pharyngeal pumping rates similar to animals grown and assayed at either 14° or 25°, respectively. We also show that the temperature-sensitive phenotypes are not limited to missense alleles; rather, they are a property of most or all severe cha-1 hypomorphs. We suggest that our data are consistent with a model of ChAT protein physically, but not covalently, associated with synaptic vesicles; and there is a temperature-dependent equilibrium between vesicle-associated and cytoplasmic (i.e., soluble) ChAT. Presumably, in severe cha-1 hypomorphs, increasing the temperature would promote dissociation of some of the mutant ChAT protein from synaptic vesicles, thus removing the site of acetylcholine synthesis (ChAT) from the site of vesicular acetylcholine transport. This, in turn, would decrease the rate and extent of vesicle-filling, thus increasing the severity of the behavioral deficits.

Enzymes of acetylcholine metabolism in the rat inferior colliculus

Although there is strong evidence for cholinergic projections to the rat inferior colliculus, especially from the pedunculopontine tegmental nucleus (Noftz et al., 2020), there is a lack of information about the quantitative prevalence of the enzymes of acetylcholine metabolism in its various portions. We have used microdissection of freeze-dried sections combined with radiometric assays to map the distributions in the rat inferior colliculus of the activities of choline acetyltransferase (ChAT), which catalyzes synthesis of acetylcholine, and acetylcholinesterase (AChE), which catalyzes its breakdown by hydrolysis. Both enzyme activities were present throughout the inferior colliculus. Average ChAT activity was consistently somewhat higher in the external cortex, excluding its most superficial layer, than in the dorsal cortex or central nucleus. Within the external cortex, ChAT activity was about half as high laterally in its most superficial layer as elsewhere. The distribution of AChE activity was more uniform than that of ChAT. Overall, ChAT activity in the rat inferior colliculus was relatively low, about a fifth of that in whole brain of rat and lower than in other central auditory regions, whereas AChE activity was about two-thirds that of rat whole brain and about average for central auditory regions. The results are compared to previous measurements for cat and hamster inferior colliculus. They are consistent with a modest role for cholinergic neurotransmission in the inferior colliculus, to modulate the activity of its major neuronal types.

Role of Serine 263 in the Human Choline Transporter 1 and Congenital Myasthenic Syndrome

Disruptions of the neuromuscular junction (NMJ) result in congenital myasthenic syndromes (CMS). CMS is characterized by a range of phenotypes including muscle weakness, apnoeas, akinesia or even death. CMS type 20 is caused by mutations in the SLC5A7 gene which encodes for the choline transporter (CHT1). CHT1 re-uptake of choline is a requirement for acetylcholine synthesis and NMJ function. The 13 transmembrane domain CHT1 protein is also thought to be phosphorylated at several sites but the role of phosphorylation remains unknown. We have recently reported the missense recessive variant c.C788>T in SLC5A7 (NM_021815.4), in a family with a history of CMS using whole exome sequencing. This variant encodes a p.S263F substitution in CHT1 and results in a complete loss of transport function despite a normal plasma membrane localization. To assess whether a change in phosphorylation and/or charge/size of position 263 results in altered transport function, three substitutions were introduced into CHT1: p.S263A, and phosphomimetics p.S263D and p.S263E. Upon expression in HEK293 cells and the utilization of cell surface biotinylation experiments, we found that WT, p.S263F and p.S263A mutants were present at the plasma membrane. Radioactive transport assays indicated that the p.S263A mutant retained a high level of transport activity while the p.S263D and p.S263E mutants were non-functional. Immobilized metal affinity electrophoresis suggests that wild-type CHT1 may be more phosphorylated than the p.S263F CHT1 mutant. These results support the essential role of the Serine 263 residue in CHT1 choline transport possibly via altering its phosphorylation status as well as size/charge at this position.

Acetylcholine signaling genes are required for cocaine-stimulated egg laying in Caenorhabditis elegans.

The toxicity and addictive liability associated with cocaine abuse are well-known. However, its mode of action is not completely understood, and effective pharmacotherapeutic interventions remain elusive. The cholinergic effects of cocaine on acetylcholine receptors, synthetic enzymes, and degradative enzymes have been the focus of relatively little empirical investigation. Due to its genetic tractability and anatomical simplicity, the egg laying circuit of the hermaphroditic nematode, Caenorhabditis elegans, is a powerful model system to precisely examine the genetic and molecular targets of cocaine in vivo. Here, we report a novel cocaine-induced behavioral phenotype in C. elegans, cocaine-stimulated egg laying. In addition, we present the results of an in vivo candidate suppression screen of synthetic enzymes, receptors, degradative enzymes, and downstream components of the intracellular signaling cascades of the main neurotransmitter systems that control C. elegans egg laying. Our results show that cocaine-stimulated egg laying is dependent on acetylcholine synthesis and synaptic release, functional nicotinic acetylcholine receptors, and the C. elegans acetylcholinesterases.

Potential Role of L-Carnitine in Autism Spectrum Disorder.

L-carnitine plays an important role in the functioning of the central nervous system, and especially in the mitochondrial metabolism of fatty acids. Altered carnitine metabolism, abnormal fatty acid metabolism in patients with autism spectrum disorder (ASD) has been documented. ASD is a complex heterogeneous neurodevelopmental condition that is usually diagnosed in early childhood. Patients with ASD require careful classification as this heterogeneous clinical category may include patients with an intellectual disability or high functioning, epilepsy, language impairments, or associated Mendelian genetic conditions. L-carnitine participates in the long-chain oxidation of fatty acids in the brain, stimulates acetylcholine synthesis (donor of the acyl groups), stimulates expression of growth-associated protein-43, prevents cell apoptosis and neuron damage and stimulates neurotransmission. Determination of L-carnitine in serum/plasma and analysis of acylcarnitines in a dried blood spot may be useful in ASD diagnosis and treatment. Changes in the acylcarnitine profiles may indicate potential mitochondrial dysfunctions and abnormal fatty acid metabolism in ASD children. L-carnitine deficiency or deregulation of L-carnitine metabolism in ASD is accompanied by disturbances of other metabolic pathways, e.g., Krebs cycle, the activity of respiratory chain complexes, indicative of mitochondrial dysfunction. Supplementation of L-carnitine may be beneficial to alleviate behavioral and cognitive symptoms in ASD patients.

Acetylcholine production by group 2 innate lymphoid cells promotes mucosal immunity to helminths.

Innate lymphoid cells (ILCs) are critical mediators of immunological and physiological responses at mucosal barrier sites. Whereas neurotransmitters can stimulate ILCs, the synthesis of small-molecule neurotransmitters by these cells has only recently been appreciated. Group 2 ILCs (ILC2s) are shown here to synthesize and release acetylcholine (ACh) during parasitic nematode infection. The cholinergic phenotype of pulmonary ILC2s was associated with their activation state, could be induced by in vivo exposure to extracts of Alternaria alternata or the alarmin cytokines interleukin-33 (IL-33) and IL-25, and was augmented by IL-2 in vitro. Genetic disruption of ACh synthesis by murine ILC2s resulted in increased parasite burdens, lower numbers of ILC2s, and reduced lung and gut barrier responses to Nippostrongylus brasiliensis infection. These data demonstrate a functional role for ILC2-derived ACh in the expansion of ILC2s for maximal induction of type 2 immunity.

Ecklonia cava Attenuates PM2.5-Induced Cognitive Decline through Mitochondrial Activation and Anti-Inflammatory Effect.

To evaluate the effects of Ecklonia cava (E. cava) on ambient-pollution-induced neurotoxicity, we used a mouse model exposed to particulate matter smaller than 2.5 µm in aerodynamic diameter (PM2.5). The intake of water extract from E. cava (WEE) effectively prevented the learning and memory decline. After a behavioral test, the toll-like receptor (TLR)-4-initiated inflammatory response was confirmed by PM2.5 exposure in the lung and brain tissues, and the WEE was regulated through the inhibition of nuclear factor-kappa B (NF-κB)/inflammasome formation signaling pathway and pro-inflammatory cytokines (IL-6 and IFN-γ). The WEE also effectively improved the PM2.5-induced oxidative damage of the lungs and brain through the inhibition of malondialdehyde (MDA) production and the activation of mitochondrial activity (mitochondrial ROS content, mitochondria membrane potential (MMP), adenosine triphosphate (ATP) content, and mitochondria-mediated apoptotic molecules). In particular, the WEE regulated the cognition-related proteins (a decreased amyloid precursor protein (APP) and p-Tau, and an increased brain-derived neurotrophic factor (BDNF)) associated with PM2.5-induced cognitive dysfunction. Additionally, the WEE prevented the inactivation of acetylcholine (ACh) synthesis and release as a neurotransmitter by regulating the acetylcholinesterase (AChE) activity, choline acetyltransferase (ChAT), and ACh receptor (AChR)-α3 in the brain tissue. The bioactive compounds of the WEE were detected as the polysaccharide (average Mw; 160.13 kDa) and phenolic compounds including 2′-phloroeckol.

Competitive inhibition of the high-affinity choline transporter by tetrahydropyrimidine anthelmintics

The high-affinity choline transporter CHT1 mediates choline uptake, the rate-limiting and regulatory step in acetylcholine synthesis at cholinergic presynaptic terminals. CHT1-medated choline uptake is specifically inhibited by hemicholinium-3, which is a type of choline analog that acts as a competitive inhibitor. Although the substrate choline and the inhibitor hemicholinium-3 are well-established ligands of CHT1, few potent ligands other than choline analogs have been reported. Here we show that tetrahydropyrimidine anthelmintics, known as nicotinic acetylcholine receptor agonists, act as competitive inhibitors of CHT1. A ligand-dependent trafficking assay in cell lines expressing human CHT1 was designed to search for CHT1 ligands from a collection of biologically active compounds. We found that morantel as well as other tetrahydropyrimidines, pyrantel and oxantel, potently inhibits the high-affinity choline uptake activity of CHT1 in a competitive manner similar to the inhibitor hemicholinium-3. They also inhibit the high-affinity choline transporter from the nematode Caenorhabditis elegans. Finally, tetrahydropyrimidines potently inhibit the high-affinity choline uptake in rat brain synaptosomes at a low micromolar level, resulting in the inhibition of acetylcholine synthesis. The rank order of potency in synaptosomes is as follows: morantel > pyarantel > oxantel (Ki = 1.3, 5.7, and 8.3 μM, respectively). Our results reveal that tetrahydropyrimidine anthelmintics are novel CHT1 ligands that inhibit the high-affinity choline uptake for acetylcholine synthesis in cholinergic neurons.

Possible correlated variation of GABAA receptor α3 expression with hippocampal cholinergic neurostimulating peptide precursor protein in the hippocampus

Cholinergic neural activation from the medial septal nucleus to hippocampus plays a crucial role in episodic memory as a regulating system for glutamatergic neural activation in the hippocampus. As a candidate regulating factor for acetylcholine synthesis in the medial septal nucleus, hippocampal cholinergic neurostimulating peptide (HCNP) was purified from the soluble fraction of young adult rat hippocampus. HCNP is released from its precursor protein (HCNP-pp), also referred to as phosphatidylethanolamine-binding protein 1. We recently reported that HCNP-pp conditional knockout (KO) mice, in which the HCNP-pp gene was knocked out at 3 months of age by tamoxifen injection, display no significant behavioral abnormalities, whereas HCNP-pp KO mice have a diminished cholinergic projection to CA1 and a decreased of theta activity in CA1. In this study, to address whether HCNP-pp reduction in early life is associated with behavioral changes, we evaluated the behavior of HCNP-pp KO mice in which HCNP-pp was downregulated from an early phase (postnatal days 14–28). As unexpected, HCNP-pp KO mice had no behavioral deficits. However, a significant positive correlation between HCNP-pp and gamma-aminobutyric acid A (GABAA) receptor α3 subunit mRNA expression was found in individuals. This finding suggests involvement of HCNP-pp in regulating GABAA receptor α3 gene expression.

ILC3-derived acetylcholine promotes protease-driven allergic lung pathology

ILC3-derived acetylcholine promotes protease-driven allergic lung pathology

Choline Acetyltransferase Induces the Functional Regeneration of the Salivary Gland in Aging SAMP1/Kl -/- Mice.

Salivary gland dysfunction induces salivary flow reduction and a dry mouth, and commonly involves oral dysfunction, tooth structure deterioration, and infection through reduced salivation. This study aimed to investigate the impact of aging on the salivary gland by a metabolomics approach in an extensive aging mouse model, SAMP1/Klotho -/- mice. We found that the salivary secretion of SAMP1/Klotho -/- mice was dramatically decreased compared with that of SAMP1/Klotho WT (+/+) mice. Metabolomics profiling analysis showed that the level of acetylcholine was significantly decreased in SAMP1/Klotho -/- mice, although the corresponding levels of acetylcholine precursors, acetyl-CoA and choline, increased. Interestingly, the mRNA and protein expression of choline acetyltransferase (ChAT), which is responsible for catalyzing acetylcholine synthesis, was significantly decreased in SAMP1/Klotho -/- mice. The overexpression of ChAT induced the expression of salivary gland functional markers (α–amylase, ZO-1, and Aqua5) in primary cultured salivary gland cells from SAMP1/Klotho +/+ and -/- mice. In an in vivo study, adeno-associated virus (AAV)-ChAT transduction significantly increased saliva secretion compared with the control in SAMP1/Klotho -/- mice. These results suggest that the dysfunction in acetylcholine biosynthesis induced by ChAT reduction may cause impaired salivary gland function

Нейрохимические особенности нейропептид-Y-ергических энтеральных нейронов подслизистого сплетения тонкой кишки в постнатальном онтогенезе

Neuropeptide Y (NPY) plays an important role in the nervous system, including the regulation of vascular tone and gastrointestinal secretion, has a direct inhibitory effect on intestinal motility and secretion. Colocalization of NPY with the enzyme for the synthesis of acetylcholine - choline acetyltransferase (ChAT), neuronal NO synthase (nNOS), vasoactive intestinal peptide (VIP) and calcium-binding protein calbindin (CB) was detected in the neurons of the submucous plexus of the small intestine of rats from the moment of birth until old age using immunohistochemical method of double labeling with antibodies and fluorescence microscopy. From the moment of birth, all NPY-immunoreactive neurons colocalize ChAT. Most of NPY-containing neurons also contain VIP and CB. In aged rats, the percentage of NPY-immunoreactive neurons containing CB, VIP, and ChAT decreases. In young rats from newborns to 20-day-old as well as in aged rats, nNOS is detected in NPY-positive neurons. Thus, at the early stages of ontogenesis and in the senescence, the enteric metasympathetic NPY-immunoreactive submucous neurons contain a wider range of neurotransmitters when compared to adult animals.

The unsolved mystery of hippocampal cholinergic neurostimulating peptide: A potent cholinergic regulator

Cholinergic efferent networks located from the medial septal nucleus to the hippocampus play a pivotal role in learning and memory outcomes by generating regular theta rhythms that enhance information retention. Hippocampal cholinergic neurostimulating peptide (HCNP), derived from the N-terminus of HCNP precursor protein (HCNP-pp), promotes the synthesis of acetylcholine in the medial septal nuclei. HCNP-pp deletion significantly reduced theta power in CA1 possibly due to lower levels of choline acetyltransferase-positive axons in CA1 stratum oriens, suggesting cholinergic disruptions in the septo-hippocampal system. This review also explores HCNP as a potent cholinergic regulator in the septo-hippocampal network while also examining the limitations of our understanding of the neurostimulating peptide.