Meeting Report for: The 14th International Symposium on Cholinergic Mechanisms

Acetylcholinesterase (AChE) is encoded by a single gene, and the alternative splicing at the 3' end produces different isoforms, including tailed (AChET), read-through (AChER), and hydrophobic (AChEH). Different forms of this enzyme exist in different cell types. Each AChE form has been proposed to have unique function, and all of them could be found in same cell type. Thus, the splicing process of different AChE forms remains unclear. Here, we aimed to establish a quantification method in measuring the absolute amount of each AChE splicing variants within a cell type. By using real-time PCR coupled with standard curves of defined copy of AChE variants, the copies of AChET transcript per 100 ng of total RNA were 5.7 x 10(4) in PC12 (rat neuronal cell), 1.3 x 10(4) in Caco-2 (human intestinal cell), 0.67 x 10(4) in TF-1 (human erythropoietic precursor), 133.3 in SH-SY5Y (human neuronal cell), and 56.7 in human umbilical vein endothelial cells (human endothelial cells). The copies of AChEH in these cell types were 0.3 x 10(4), 3.3 x 10(4), 2.7 x 10(4), 133.3, and 46.7, respectively, and AChER were 0.07 x 10(4), 0.13 x 10(4), 890, 3.3, and 2.7, respectively. Furthermore, PC12 and TF-1 cells were chosen for the analysis of AChE splicing pattern during differentiation. The results demonstrated a selective increase in AChET mRNA but not AChER or AChEH mRNAs in PC12 upon nerve growth factor-induced neuronal differentiation. PC12 cells could therefore act as a good cell model for the study on alternative splicing mechanism and regulation of AChET.

The neuroligins are cell adhesion proteins whose extracellular domain belongs to the alpha/beta-hydrolase fold family of proteins, mainly containing enzymes and exemplified by acetylcholinesterase. The ectodomain of postsynaptic neuroligins interacts through a calcium ion with the ectodomain of presynaptic neurexins to form flexible trans-synaptic associations characterized by selectivity for neuroligin or neurexin subtypes. This heterophilic interaction, essential for synaptic differentiation, maturation, and maintenance, is regulated by gene selection, alternative mRNA splicing, and posttranslational modifications. Mutations leading to deficiencies in the expression, folding, maturation, and binding properties of either partner are associated with autism spectrum disorders. The currently available structural and functional data illustrate how these two families of cell adhesion molecules bridge the synaptic cleft to participate in synapse plasticity and support its dynamic nature. Neuroligin partners distinct from the neurexins, and which may undergo either trans or cis interaction, have also been described, and tridimensional structures of some of them are available. Our study emphasizes the partnership versatility of the neuroligin ectodomain associated with molecular flexibility and alternative binding sites, proposes homology models of the structurally non-characterized neuroligin partners, and exemplifies the large structural variability at the surface of the alpha/beta-hydrolase fold subunit. This study also provides new insights into possible surface binding sites associated with non-catalytic properties of the acetylcholinesterase subunit.

The review summarizes evidences from extensive studies suggesting that ascending mesolimbic cholinergic system (AMCS) that terminates in vast areas of forebrain and diencephalic limbic areas is responsible for specific generation of aversive arousal and aversive emotional state. This state is accompanied by emission of threatening and/or alarming vocalizations that served as a quantitative measure of the emotional response. The AMCS originates from the cholinergic neurons within the laterodorsal tegmental nucleus that have widespread and diffuse ascending connections. Activity of the AMCS induced by activation of the muscarinic cholinergic receptors in the terminal fields of this system, or by glutamate stimulation of neurons of the laterodorsal tegmental nucleus, brings about aversive state with alarming vocalizations. It is postulated that release of acetylcholine from the terminals of the AMCS in the vast areas of the forebrain and diencephalon serves as the initiator of the aversive emotional state with concomitant manifestations and alarming vocal signaling. It is concluded that the AMCS serves as a specific physiological, psychological, and social arousing and alarming system.

Acetylcholinesterase (AChE) is a key enzyme in the cholinergic nervous system and is one of the most studied proteins in the field of Alzheimer's disease (AD). Moreover, alternative functions of AChE unrelated with the hydrolysis of acetylcholine are suspected. Until now, the majority of investigations on AChE in AD pathology have been focused on the determination of its enzymatic activity level, which is depleted in the AD brain. Despite this overall decrease, AChE activity increases at the vicinity of the two hallmarks of AD, the amyloid plaques and the neurofibrillary tangles (NFT). In fact, AChE may directly interact with Abeta in a manner that increases the deposition of Abeta to form plaques. In the context of protein-protein interactions, we have recently reported that AChE can interact with presenilin-1, the catalytic component of gamma-secretase, influencing its expression level and also its activity. However, the alteration of AChE protein in the AD brain has not been determined. Here, we demonstrated by Western blotting and immunohistochemistry that a prominent pool of enzymatically inactive AChE protein existed in the AD brain. The potential significance of these unexpected levels of inactive AChE protein in the AD brain was discussed, especially in the context of protein-protein interactions with beta-amyloid and presenilin-1.

Acetylcholinesterase (AChE) is a critical enzyme that regulates neurotransmission by degrading the neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for both therapeutic drugs that treat Alzheimer's disease and organophosphate (OP) chemical warfare agents that cripple the nervous system and cause death through paralysis. We are exploring a strategy to design compounds that bind tightly at or near a peripheral or P-site near the mouth of the AChE active site gorge and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. Here, we review our recent reports on two uncharged, natural product inhibitors of AChE, dihydrotanshinone I and territrem B, that have relatively high affinities for the enzyme. We describe an inhibitor competition assay and comment on the structures of these inhibitors in complex with recombinant human acetylcholinesterase as determined by X-ray crystallography. Our results reveal that dihydrotanshinone I binding is specific to only the P-site, while territrem B binding spans the P-site and extends into the acylation or A-site at the base of the gorge.

In neuromuscular and nerve-electroplaque junctions, nerve impulses can be transmitted at high frequencies. This implies that transmission of individual impulses must be very brief. We describe three mechanisms which curtail the time course of individual impulses at these synapses: (1) opening of presynaptic K(+) channels (delayed rectifier) efficiently curtails the presynaptic action potential. Inhibition of K(+) channel by aminopyridines transforms the normally brief postsynaptic potential (2-3 ms) to a long-lasting "giant" potential (exceeding half a second); (2) a low-affinity Ca(2+)/H(+) antiport ensures rapid Ca(2+) sequestration into synaptic vesicles, curtailing the calcium signal and thereby the duration of transmitter release. Indeed vesicular Ca(2+)/H(+) antiport inhibition by bafilomycin or Sr(2+) prolongs the duration of the postsynaptic potential. We recently showed that synaptotagmin-1 is required for this antiport activity; thus the vesicular Ca(2+)/H(+) antiport might be synaptotagmin itself, or regulated by it; and (3) it is recalled that, in these junctions, acetylcholinesterase is highly concentrated in the synaptic cleft and that anticholinesterases lengthen the endplate time course. Therefore, at three different steps of synaptic transmission, an efficient mechanism curtails the local synaptic signal. When one of these three mechanisms is inhibited, the duration of individual impulses is prolonged, but the synapse loses its faculty to fire at high frequencies.

Cholinergic networks have been shown to be involved in generation and modulation of the locomotor rhythmic pattern produced by the mammalian central pattern generators. Here, we show that changes in the endogenous levels of acetylcholine in the sacral segments of the isolated spinal cord of the neonatal rat modulate the locomotor-related output produced by stimulation of sacrocaudal afferents in muscarinic receptor-dependent mechanisms. Cholinergic components we found on sacral relay neurons with lumbar projections through the ventral and lateral funiculi are suggested to mediate this ascending cholinergic modulation. Our findings, possible mechanisms accounting for them, and their potential implications are discussed.

In PNS and CNS remarkable rearrangements occur soon after the connections are laid down in the course of embryonic life. These processes clearly follow the period of developmental cell death and mostly take place during the very beginning of postnatal life. They consist in changes of the peripheral fields of neurons, marked by elimination of many inputs, while others undergo further maturation and strengthening. Along the efforts to uncover the signals that regulate development, it turned out that while the initial construction of the circuits is heavily based on chemical cues, the subsequent rearrangement is markedly influence by activity. Here we describe experiments testing the influence on developmental plasticity of a particular aspect of activity, the timing of nerve impulses in the competing inputs. Two recent investigations are reviewed, indicating strikingly similar developmental features in quite different systems, neuromuscular and visual. A sharp contrast between the effects of synchrony and asynchrony emerges, indicating that Hebb-related activity rules are important not only for learning but also for development.

Michaelis and Menten found the direct mathematical analysis of their studied enzyme-catalyzed reaction unrealistic 100 years ago, and hence, they avoided this problem by correct adaptation and analysis of the experiment, i.e., differentiation of the progress-curve data into rates. However, the most elegant and ideal simplification of the evaluation of kinetics parameters from progress curves can be performed when the algebraic integration of the rate equation results in an explicit mathematical equation that describes the dynamics of the model system of the reaction. Recently, it was demonstrated that such an alternative approach can be considered for enzymes that obey the generalized Michaelis-Menten reaction dynamics, although its use is now still limited for cholinesterases, which show kinetics that deviate from saturation-like hyperbolic behavior at high concentrations of charged substrates. However, a mathematical approach is reviewed here that might provide an alternative to the decades-old problem of data analysis of cholinesterase-catalyzed reactions, through the more complex Webb integrated rate equation.

Proteins in living organisms have names that are usually derived from their function in the biochemical system their discoverer was investigating. Typical examples are acetylcholinesterase and agrin; however, for both of these, various other functions that are not related to the cholinergic system have been revealed. Our investigations have been focused on the alternative roles of acetylcholinesterase and agrin in the processes of muscle development and regeneration. Previously, we described a role for agrin in the development of excitability in muscle contraction. In this study, we report the effects of agrin on secretion of interleukin 6 in developing human muscle. At the myoblast stage, agrin increases interleukin 6 secretion. This effect seems to be general as it was observed in all of the cell models analysed (human, mouse, cell lines). After fusion of myoblasts into myotubes, the effects of agrin are no longer evident, although agrin has further effects at the innervation stage, at least in in vitro innervated human muscle. These effects of agrin are another demonstration of its non-synaptic roles that are apparently developmental-stage specific. Our data support the view that acetylcholinesterase and agrin participate in various processes during development of skeletal muscle.

For almost 30 years, photoaffinity labeling and protein microsequencing techniques have been providing novel insights about the structure of nicotinic acetylcholine receptors (nAChR) and the diversity of nAChR drug binding sites. Photoaffinity labeling allows direct identification of amino acid residues contributing to a drug binding site without prior knowledge of the location of the binding site within the nAChR or the orientation of the ligand within the binding site. It also distinguishes amino acids that contribute to allosteric binding sites from those involved in allosteric modulation of gating. While photoaffinity labeling was used initially to identify amino acids contributing to the agonist binding sites and the ion channel, it has been used recently to identify binding sites for allosteric modulators at subunit interfaces in the extracellular and the transmembrane domains, and within a subunit's transmembrane helix bundle. In this article, we review the different types of photoaffinity probes that have been used and the various binding sites that have been identified within the structure of nAChR, with emphasis on our recent studies of allosteric modulator binding sites.

The central cholinergic system is believed to be involved in the control of many physiological functions and is an important pharmacological target for numerous neurological pathologies. Here, we summarize our recent observations regarding this topic that we obtained by studying genetically modified mice devoid of particular cholinesterase molecular forms. Our results, collected from mice with deficits of functional cholinesterases in the brain, suggest that the increase in the level of acetylcholine (ACh) has an impact on cognition only in the situation when extracellular ACh is low. Furthermore, we confirmed the central control of movement coordination, which could be of importance for the management of motor problems in patients with Parkinson's disease. At last, we provide clear evidence that while the hypothermic effect of the muscarinic agonist oxotremorine is based on a central mechanism, in contrast, the acetylcholinesterase inhibitor donepezil decreases body temperature by its action in the periphery.

Chemokines play an important role in the autoimmune diseases. The aim of this study was to investigate the levels of CCL20 and a polymorphism [-786C > T (rs6749704)] in the chemokine gene in patients with multiple sclerosis (MS). The blood samples were collected from 135 MS patients and 135 healthy subjects as a control group. The patients have relapsing-remitting (RRMS; n = 65), primary progressive (PPMS; n = 47), secondary progressive (SPMS; n = 35) or progressive relapsing (PRMS; n = 14) patterns. The serum levels of CCL20 were measured by ELISA. The DNA was analyzed for CCL20 polymorphism using PCR-RLFP. The mean serum levels of CCL20 in the MS group were significantly higher than in the healthy group (P < 0.001). In patients with a SPMS pattern, the frequency of CT genotype at rs6749704 (24.3 %) was significantly lower as compared to patients with other patterns (42.8 %; P < 0.04). No significant differences were observed between subjects with different genotypes in rs6749704 regarding the CCL20 levels. The mean serum levels of CCL20 in both newly diagnosed and previously diagnosed patients was significantly higher than in the healthy group (P < 0.05 and 0.001, respectively). The mean serum levels of CCL20 in patients with RRMS, SPMS and PPMS patterns were significantly higher than in the healthy group (P < 0.004, P < 0.04, and 0.05, respectively). The levels of CCL20 in untreated patients and in patients who received interferon-beta, methylprednisolone or the combination of interferon-beta plus methylprednisolone were higher as compared to the control group (P < 0.05, P < 0.03, P < 0.005, and P < 0.05, respectively). These results showed higher levels of CCL20 in patients that represent that the chemokine may play an important role in the pathogenesis of MS. The rs6749704 polymorphism was an associated SPMS pattern. The levels of CCL20 were not influenced by gender, disease pattern and treatment.

Macromolecular cholinergic pathways are involved in the regulation of addiction, emotions, and motivations, as described at this ISCM. Indeed, in view of the omnipresence in the brain of cholinergic pathways and of their connections with other transmitters' sites and pathways, their involvement in all known human and animal behaviors could be expected and numerous current reports describe such cholinergic correlates. This minireview describes the current status and the future of the cholinergic impact on behavior and emotions, and particularly on one important human phenomenon, the "self" or the "I" (it is only speculative to impute the self to animals).

Nicotinic acetylcholine receptors have been shown to participate in neuroprotection in the aging brain. Lynx protein modulators dampen the activity of the cholinergic system through direct interaction with nicotinic receptors. Although lynx1 null mutant mice exhibit augmented learning and plasticity, they also exhibit macroscopic vacuolation in the dorsal striatum as they age, detectable at the optical microscope level. Despite the relevance of the lynx1 gene to brain function, little is known about the cellular ultrastructure of these age-related changes. In this study, we assessed degeneration in the dorsal striatum in 1-, 3-, 7-, and 13-month-old mice, using optical and transmission electron microscopy. We observed a loss of nerve fibers, a breakdown in nerve fiber bundles, and a loss of neuronal nuclei in the 13-month-old lynx1 null striatum. At higher magnification, these nerve fibers displayed intracellular vacuoles and disordered myelin sheaths. Few or none of these morphological alterations were present in younger lynx1 null mutant mice or in heterozygous lynx1 null mutant mice at any age. These data indicate that neuronal health can be maintained by titrating lynx1 dosage and that the lynx1 gene may participate in a trade-off between neuroprotection and augmented learning.

Recent studies with M3 muscarinic acetylcholine receptor (M3R) mutant mice suggest that drugs selectively targeting this receptor subtype may prove useful for the treatment of various pathophysiological conditions. Moreover, the use of M3R-based designer G protein-coupled receptors (GPCRs) has provided novel insights into how Gq-coupled GPCRs can modulate whole-body glucose homeostasis by acting on specific peripheral cell types. More recently, we succeeded in using X-ray crystallography to determine the structure of the M3R bound to the bronchodilating drug tiotropium, a muscarinic antagonist (inverse agonist). This new structural information should facilitate the development of orthosteric or allosteric M3R-selective drugs that are predicted to have considerable therapeutic potential.

The genetic manipulation of the laboratory mouse has been well developed and generated more and more mouse lines for biomedical research. To advance our science exploration, it is necessary to share genetically modified mouse lines with collaborators between institutions, even in different countries. The transfer process is complicated. Significant paperwork and coordination are required, concerning animal welfare, intellectual property rights, colony health status, and biohazard. Here, we provide a practical example of importing a transgenic mice line, Dynamin 1 knockout mice, from Yale University in the USA to Perking University in China for studying cell secretion. This example including the length of time that required for paper work, mice quarantine at the receiving institution, and expansion of the mouse line for experiments. The procedure described in this paper for delivery live transgenic mice from USA to China may serve a simple reference for transferring mouse lines between other countries too.

A cholinesterase activity can be found in all kingdoms of living organism, yet cholinesterases involved in cholinergic transmission appeared only recently in the animal phylum. Among various proteins homologous to cholinesterases, one finds neuroligins. These proteins, with an altered catalytic triad and no known hydrolytic activity, display well-identified cell adhesion properties. The availability of complete genomes of a few metazoans provides opportunities to evaluate when these two protein families emerged during evolution. In bilaterian animals, acetylcholinesterase co-localizes with proteins of cholinergic synapses while neuroligins co-localize and may interact with proteins of excitatory glutamatergic or inhibitory GABAergic/glycinergic synapses. To compare evolution of the cholinesterases and neuroligins with other proteins involved in the architecture and functioning of synapses, we devised a method to search for orthologs of these partners in genomes of model organisms representing distinct stages of metazoan evolution. Our data point to a progressive recruitment of synaptic components during evolution. This finding may shed light on the common or divergent developmental regulation events involved into the setting and maintenance of the cholinergic versus glutamatergic and GABAergic/glycinergic synapses.

Alzheimer's disease is associated with genetic risk factors, of which the apolipoprotein E (ApoE) is the most prevalent, and is affected by environmental factors that include education early in life and exposure to metals. The industrial and military use of depleted uranium (DU) resulted in an increase of its deposition in some areas and led to a possible environmental factor. The present study aims to ascertain the effects on the behaviour and the metabolism of cholesterol and acetylcholine of ApoE-/- mice exposed to enriched environment (EE) and exposed to DU (20 mg/L) for 14 weeks. Here we show that ApoE-/- mice were unaffected by the EE and their learning and memory were similar to those of the non-enriched ApoE-/- mice. ApoE-/- mice showed a significant decrease in total (-16 %) and free (-16 %) cholesterol in the entorhinal cortex in comparison to control wild-type mice. Whatever the housing conditions, the exposure to DU of ApoE-/- mice impaired working memory, but had no effect on anxiety-like behaviour, in comparison to control ApoE-/- mice. The exposure of ApoE-/- mice to DU also induced a trend toward higher total cholesterol content in the cerebral cortex (+15 %) compared to control ApoE-/- mice. In conclusion, these results demonstrate that enriched environment does not ameliorate neurobehaviour in ApoE-/- mice and that ApoE mutation induced specific effects on the brain cholesterol. These findings also suggested that DU exposure could modify the pathology in this ApoE model, with no influence of housing conditions.

Mice deficient in acetylcholinesterase (AChE; EC3.1.1.7) exhibited significant phenotypical and biochemical changes when compared with wild-type littermates. They showed a delay of growth in weight and size, immature external ears, and persistent body tremor, and they circled when walking. The molecular mechanisms underlying these changes have not been investigated yet. Here, we studied the profiles of both the messenger RNA (mRNA) and protein expression in the brain of AChE-deficient mice using mRNA microarray, quantitative PCR, and two-dimensional difference gel electrophoresis (2D DIGE) coupled to protein identification with matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Analysis of gene expression profile was conducted by DAVID ( http:\/\/david.abcc.ncifcrf.gov ) and Ingenuity Pathway Analysis (IPA, http:\/\/www.ingenuity.com ). Previous results implicated that there is a close relationship between lipid metabolisms which were associated with central nervous system development. Here, we demonstrated that the mRNA expressions of brain specific fatty acid protein 7 (fabp-7) and phospholipase A2 group IV (pla2g4) were significantly downregulated in AChE-deficient mice. These results suggested that AChE may play a role in neurogenesis and neurodegeneration by specifically regulating lipid metabolism in the brain.

The locus coeruleus (LC) is an important brainstem area involved in cocaine addiction. However, evidence to elucidate how cocaine modulates the activity of LC neurons remains incomplete. Here, we performed whole recordings in brain slices to evaluate the effects of cocaine on the sodium (Na(+)), potassium (K(+)), calcium (Ca(2+)) channels, and glutamatergic synaptic transmission in the locus coeruleus neurons. Local application of cocaine significantly and reversibly reduced the spontaneous firing rate but did not affect action potential amplitude, rising time, decay time, or half width of noradrenergic locus coeruleus neurons. Moreover, cocaine attenuated the sodium current but did not affect potassium and calcium currents. The N-methyl-D-aspartate receptor mediated excitatory postsynaptic currents were reduced by neuropeptide galanin but not cocaine. All those data demonstrate that cocaine has inhibitory effect on the spontaneous activities and sodium current in locus coeruleus neurons. Therefore, neuromodulation of sodium channel in locus coeruleus neurons may play an important role in drug addiction.

Alzheimer's disease is a multi-factorial neurodegenerative disorder devastatingly affecting the aged population worldwide. Previous studies have shown that medicinal herbs used in traditional Chinese medicine might be benefit to Alzheimer's disease patients. Berberine and palmatine, two isoquinoline alkaloids found in several medicinal herbs, were used for memory enhancement in China. In this study, the inhibitory effects of combined berberine and palmatine on acetylcholinesteras were evaluated using recombinant human acetylcholinesterase. Our results showed that the combination of berberine and palmatine inhibited acetylcholinesterase in a mixed competitive pattern. By the median-effect principle, the calculated combination index of the combination was less than 1, suggesting that berberine and plamatine inhibited acetylcholinesterase synergistically. Furthermore, the drug-reducing index of berberine and palmatine were 2.98 and 2.66, respectively. Taken together, the results showed that the combination of the two alkaloids might potentially be developed as a more effective therapeutic strategy for Alzheimer's disease patients.

While the functional implications of AChE-T, PRiMA and ColQ have been firmly established, those of glypiated AChE remain uncertain. Insights into the physiological meaning of glycosylphosphatidylinositol (GPI)-linked AChE-H were gained by comparing nervous and non-nervous tissues for the amount of AChE mRNA variants they contained. PCR showed that AChE-T mRNA prevailed in the mouse brain, spinal cord, sciatic nerve and muscle, and AChE-H mRNA in the bone marrow and thymus, as well as in the human gut. The similar levels of AChE-T and AChE-H mRNAs in mouse liver and human kidney contrasted with the almost exclusive presence of catalytically active AChE-H in both organs. The absence of PRiMA mRNA in liver suggested that the tetramers made of AChE-T fail to bind to the cell membrane and are secreted due to the lack of PRiMA in non-nervous organs. In contrast, glypiated AChE-H is largely and lastingly bound to the cell membrane. Thus, non-synaptic glypiated AChE-H seems to be the counterpart of synaptic PRiMA-linked AChE-T, the former designed for clearing ACh waves, the latter for confronting ACh bursts, and both for helping to protect cells against the harmful effects of durable nicotinic and muscarinic activation.

Cocaine hydrolase gene transfer of mutated human butyrylcholinesterase (BChE) is evolving as a promising therapy for cocaine addiction. BChE levels after gene transfer can be 1,500-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. Because mutated BChE is approximately 70 % as efficient in hydrolyzing acetylcholine as wild-type enzyme, it is important to examine the impact on cholinergic function. Here, we focused on memory and cognition (Stone T-maze), basic neuromuscular function (treadmill endurance and grip strength), and coordination (Rotarod). BALB/c mice were given adeno-associated virus vector or helper-dependent adenoviral vector encoding mouse or human BChE optimized for cocaine. Age-matched controls received saline or luciferase vector. Despite high doses (up to 10(13) particles per mouse) and high transgene expression (1,000-fold above baseline), no deleterious effects of vector treatment were seen in neurobehavioral functions. The vector-treated mice performed as saline-treated and luciferase controls in maze studies and strength tests, and their Rotarod and treadmill performance decreased less with age. Thus, neither the viral vectors nor the large excess of BChE caused observable toxic effects on the motor and cognitive systems investigated. This outcome justifies further steps toward an eventual clinical trial of vector-based gene transfer for cocaine abuse.

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ) in the form of asymmetric AChE (AChE/ColQ). We exploited the proprietary NMJ-targeting signals of ColQ to treat congenital myasthenia and to explore the mechanisms of autoimmune myasthenia gravis (MG). Mutations in COLQ cause congenital endplate AChE deficiency (CEAD). First, a single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq-/- mice normalized motor functions, synaptic transmission, and partly the NMJ ultrastructure. Additionally, injection of purified recombinant AChE/ColQ protein complex into gluteus maximus accumulated AChE in non-injected forelimbs. Second, MuSK antibody-positive MG accounts for 5-15 % of MG. In vitro overlay of AChE/ColQ to muscle sections of Colq-/- mice, as well as in vitro plate-binding of MuSK to ColQ, revealed that MuSK-IgG blocks binding of ColQ to MuSK in a dose-dependent manner. Passive transfer of MuSK-IgG to wild-type mice markedly reduced the size and intensity of ColQ signals at NMJs. MuSK-IgG thus interferes with binding of ColQ to MuSK. Elucidation of molecular mechanisms of specific binding of ColQ to NMJ enabled us to ameliorate devastating myasthenic symptoms of Colq-/- mice and also to reveal underlying mechanisms of anti-MuSK-MG.

Accumulating evidence suggests parasympathetic dysfunction and elevated inflammation as underlying processes in multiple peripheral and neurological diseases. Acetylcholine, the main parasympathetic neurotransmitter and inflammation regulator, is hydrolyzed by the two closely homologous enzymes, acetylcholinesterase and butyrylcholinesterase (AChE and BChE, respectively), which are also expressed in the serum. Here, we consider the potential value of both enzymes as possible biomarkers in diseases associated with parasympathetic malfunctioning. We cover the modulations of cholinesterase activities in inflammation-related events as well as by cholinesterase-targeted microRNAs. We further discuss epigenetic control over cholinesterase gene expression and the impact of single-nucleotide polymorphisms on the corresponding physiological and pathological processes. In particular, we focus on measurements of circulation cholinesterases as a readily quantifiable readout for changes in the sympathetic/parasympathetic balance and the implications of changes in this readout in health and disease. Taken together, this cumulative know-how calls for expanding the use of cholinesterase activity measurements for both basic research and as a clinical assessment tool.

Selective serotonin reuptake inhibitors (SSRIs) show anti-inflammatory effects, suggesting a possible interaction with both Toll-like-receptor 4 (TLR4) responses and cholinergic signaling through as yet unclear molecular mechanism(s). Our results of structural modeling support the concept that the antidepressant fluoxetine physically interacts with the TLR4-myeloid differentiation factor-2 complex at the same site as bacterial lipopolysaccharide (LPS). We also demonstrate reduced LPS-induced pro-inflammatory interleukin-6 and tumor necrosis factor alpha in human peripheral blood mononuclear cells preincubated with fluoxetine. Furthermore, we show that fluoxetine intercepts the LPS-induced decreases in intracellular acetylcholinesterase (AChE-S) and that AChE-S interacts with the nuclear factor kappa B (NFkappaB)-activating intracellular receptor for activated C kinase 1 (RACK1). This interaction may prevent NFkappaB activation by residual RACK1 and its interacting protein kinase PKCbetaII. Our findings attribute the anti-inflammatory properties of SSRI to surface membrane interference with leukocyte TLR4 activation accompanied by intracellular limitation of pathogen-inducible changes in AChE-S, RACK1, and PKCbetaII.

Recently, acetylcholinesterase (AChE, EC 3.1.1.7) has received increased attention in the field of environmental sciences. Evaluation of the effects of environmental contaminants on AChE enzymatic activity not only can reflect, to some extent, the interference with the nervous system, but also can be used for monitoring pollution. Our previous study showed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) suppressed neuronal AChE enzymatic activity via transcriptional downregulations mediated by aryl hydrocarbon receptor. In the present study, the effects of several other dioxin-like compounds (DLCs) on neuronal AChE activity were determined, including 1,2,3,7,8-pentachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, 2,3,4,7,8-pentachlorodibenzofuran, and 2,3,7,8-tetrabromodibenzo-p-dioxin. The results showed that the enzymatic activity of AChE was significantly decreased by approximately 15-30 % after exposure to a certain concentrations of the DLCs, whereas incubating neuronal cell lysates directly with these DLCs did not inhibit AChE enzyme. Subsequent molecular mechanism study showed that these chemicals could decrease ACHE promoter activity, as well as AChE T mRNA expression, thereby suggesting the involvements of transcriptional regulation in these effects. These findings on DLCs are similar with those on 2,3,7,8-TCDD, pointing to the possibility that exposure to dioxin and DLCs, which frequently coexist in the contaminated environments, may concurrently interfere with the cholinergic functions via AChE.

Previous studies have implicated that two at-risk haplotypes (HapA and HapB) of gene-encoding 5-lipoxygenase-activating protein (ALOX5AP) were significantly associated with stroke. The aim of this study was to explore the association between haplotypes of ALOX5AP gene and risk for ischemic stroke (IS) in Chinese Han population. A total of 492 patients with IS and 490 matched control subjects were recruited. Six ALOX5AP SNPs (SG13S377, SG13S114, SG13S41, SG13S89, SG13S32 and SG13S35) were genotyped by SNaPshot minisequence technique. A common genetic variant SG13S114/AA in the ALOX5AP gene was associated with IS in this Chinese cohort (OR = 2.514, 95 % CI = 1.667 ~ 3.790). HapA (TGA) and HapB (AAAG) had no significant difference in the patients (36.3 and 18.5 %, respectively) and controls (37.6 and 16.3 %, respectively) (P = 0.631 and P = 0.375, respectively). But, the frequency of Hap (GAAG) was significantly higher in the patients than that in the controls after Bonferroni's adjustment (P = 0.006). To conclude, SG13S114/AA of the ALOX5AP gene was associated with an increased risk for IS. A novel risk haplotype, Hap (GAAG) was a genetic risk factor for IS in this Chinese population.

MiR-132 is enriched in the central nerve system and is thought to be involved in neuronal development, maturation and function, and to be associated with several neurological disorders including Alzheimer's disease. In addition to its documented neuronal functions, an emerging role for miR-132 in tumorigenesis has been suggested. Recently, hsa-miR-132 was shown to be modulated in different tumor types. However, its role in non-small cell lung cancer (NSCLC) remains unclear. Here, we show that hsa-miR-132 can initiate apoptosis in NSCLC cells to dramatically attenuate tumor formation in nude mice independent of its effect on the proliferation/apoptosis-associated gene, acetylcholinesterase (AChE). Interestingly, hsa-miR-132 has no pro-apoptotic effect in normal pulmonary trachea epithelium. Taken together, these results suggest that hsa-miR-132 represses NSCLC growth by inducing apoptosis independent of AChE.

Transgenic mouse has shown great advantages in the study of Alzheimer's disease (AD) and drug screening as AD develops rapidly resent years, while more detail information of these transgenic mice and experience of application are needed. To obtain the basic background information of the B6C3-Tg (APPswe/PSEN1dE9) double-transgenic mouse, which was reported with early onset AD, three- to ten-month-old B6C3-Tg AD mice and normal C57BL/6 mice were selected randomly to test the ability of learning memory by Morris water maze, the brain acetylcholinesterase (AChE) activity by AChE kit, and beta amyloid protein level by immunohistochemistry staining. Compared with the control group, the escape latency time of B6C3-Tg AD mice at 9 and 10 months of age is significantly longer (P < 0.05) in Morris maze test, and the activity of brain AChE is higher. beta-Amyloid plaques were observed at 3 months of age and developed rapidly. Statistical analysis showed a positive correlation between the area of these plaques and the ages of B6C3-Tg AD mouse (y = 0.0355e(0.5557x), R = 0.9557). The model's behavior is conformed to simulate behaviors of human Alzheimer's disease at the early stage and may provide detail background information a new choice when transgenic mice are needed in the research of AD.

Cholinesterases (ChEs) have been identified in vertebrates and invertebrates. Inhibition of ChE activity in invertebrates, such as bivalve molluscs, has been used to evaluate the exposure of organophosphates, carbamate pesticides, and heavy metals in the marine system. The golden apple snail (Pomacea canaliculata) is considered as one of the worst invasive alien species harmful to rice and other crops. The ChE(s) in this animal, which has been found recently, but poorly characterized thus far, could serve as biomarker(s) for environmental surveillance as well as a potential target for the pest control. In this study, the tissue distribution, substrate preference, sensitivity to ChE inhibitors, and molecular species of ChEs in P. canaliculata were investigated. It was found that the activities of both AChE and BChE were present in all test tissues. The intestine had the most abundant ChE activities. Both enzymes had fair activities in the head, kidney, and gills. The BChE activity was more sensitive to tetra-isopropylpyrophosphoramide (iso-OMPA) than the AChE. Only one BChE molecular species, 5.8S, was found in the intestine and head, whereas two AChE species, 5.8S and 11.6S, were found there. We propose that intestine ChEs of this snail may be potential biomarkers for manipulating pollutions.