Galantamine-induced amyloid-{beta} clearance mediated via stimulation of microglial nicotinic acetylcholine receptors

Reduction of brain amyloid-β (Aβ) has been proposed as a therapeutic target for Alzheimer disease (AD), and microglial Aβ phagocytosis is noted as an Aβ clearance system in brains. Galantamine is an acetylcholinesterase inhibitor approved for symptomatic treatment of AD. Galantamine also acts as an allosterically potentiating ligand (APL) for nicotinic acetylcholine receptors (nAChRs). APL-binding site is located close to but distinct from that for acetylcholine on nAChRs, and FK1 antibody specifically binds to the APL-binding site without interfering with the acetylcholine-binding site. We found that in human AD brain, microglia accumulated on Aβ deposits and expressed α7 nAChRs including the APL-binding site recognized with FK1 antibody. Treatment of rat microglia with galantamine significantly enhanced microglial Aβ phagocytosis, and acetylcholine competitive antagonists as well as FK1 antibody inhibited the enhancement. Thus, the galantamine-enhanced microglial Aβ phagocytosis required the combined actions of an acetylcholine competitive agonist and the APL for nAChRs. Indeed, depletion of choline, an acetylcholine-competitive α7 nAChR agonist, from the culture medium impeded the enhancement. Similarly, Ca(2+) depletion or inhibition of the calmodulin-dependent pathways for the actin reorganization abolished the enhancement. These results suggest that galantamine sensitizes microglial α7 nAChRs to choline and induces Ca(2+) influx into microglia. The Ca(2+)-induced intracellular signaling cascades may then stimulate Aβ phagocytosis through the actin reorganization. We further demonstrated that galantamine treatment facilitated Aβ clearance in brains of rodent AD models. In conclusion, we propose a further advantage of galantamine in clinical AD treatment and microglial nAChRs as a new therapeutic target.     

Functional role of acetylcholine and the expression of cholinergic receptors and components in osteoblasts

Recent studies have indicated that acetylcholine (ACh) plays a vital role in various tissues, while the role of ACh in bone metabolism remains unclear. Here we demonstrated that ACh induced cell proliferation and reduced alkaline phosphatase (ALP) activity via nicotinic (nAChRs) and muscarinic acetylcholine receptors (mAChRs) in osteoblasts. We detected mRNA expression of several nAChRs and mAChRs. Furthermore, we showed that cholinergic components were up-regulated and subunits/subtypes of acetylcholine receptors altered during osteoblast differentiation. To our knowledge, this is the first report demonstrating that osteoblasts express specific acetylcholine receptors and cholinergic components and that ACh plays a possible role in regulating the proliferation and differentiation of osteoblasts.     

Study of nicotinic acetylcholine receptors on cultured antennal lobe neurones from adult honeybee brains

In insects, acetylcholine (ACh) is the main neurotransmitter, and nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission. In the honeybee, nAChRs are expressed in diverse structures including the primary olfactory centres of the brain, the antennal lobes (AL) and the mushroom bodies. Whole-cell, voltage-clamp recordings were used to characterize the nAChRs present on cultured AL cells from adult honeybee, Apis mellifera. In 90% of the cells, applications of ACh induced fast inward currents that desensitized slowly. The classical nicotinic agonists nicotine and imidacloprid elicited respectively 45 and 43% of the maximum ACh-induced currents. The ACh-elicited currents were blocked by nicotinic antagonists methyllycaconitine, dihydroxy-β-erythroidine and α-bungarotoxin. The nAChRs on adult AL cells are cation permeable channels. Our data indicate the existence of functional nAChRs on adult AL cells that differ from nAChRs on pupal Kenyon cells from mushroom bodies by their pharmacological profile and ionic permeability, suggesting that these receptors could be implicated in different functions.     

Nicotinic receptor signaling in nonexcitable cells

The finding that neuronal nicotinic acetylcholine receptors (nAChRs) are present in non-neuronal cells both within and outside the nervous system raises some interesting issues. The mechanisms underlying receptor signaling and its downstream consequences in these cells remain to be elucidated. Factors controlling the release of acetylcholine and the extent of its diffusion are likely to be different for these cells than for traditional neuronal synapses. Recent advances on the physiologic functions of some of these cell types have provided a better insight into possible functional roles for nAChRs in nonexcitable cells. The presence of nAChRs on these cells also implies a broader scope for the actions of nicotine that needs to be considered from a clinical viewpoint. Revealing the potential physiologic roles for nAChRs on nonexcitable cells is likely to provide a more complete understanding of cholinergic signaling.     

Extracellular domain nicotinic acetylcholine receptors formed by alpha4 and beta2 subunits

Models of the extracellular ligand-binding domain of nicotinic acetylcholine receptors (nAChRs), which are pentameric integral membrane proteins, are attractive for structural studies because they potentially are water-soluble and better candidates for x-ray crystallography and because their smaller size is more amenable for NMR spectroscopy. The complete N-terminal extracellular domain is a promising foundation for such models, based on previous studies of alpha7 and muscle-type subunits. Specific design requirements leading to high structural fidelity between extracellular domain nAChRs and full-length nAChRs, however, are not well understood. To study these requirements in heteromeric nAChRs, the extracellular domains of alpha4 and beta2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with alpha4beta2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain alpha4beta2 nAChRs formed from subunits with M1 were nearly identical to affinities of alpha4beta2 nAChRs when measured with [3H]epibatidine, cytisine, nicotine, and acetylcholine. Velocity sedimentation suggested that these extracellular domain nAChRs predominantly formed pentamers. The yield of these extracellular domain nAChRs was about half the yield of alpha4beta2 nAChRs. In contrast, [3H]epibatidine binding was not detected from the extracellular domain alpha4 and beta2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both alpha4 and beta2 play an important role for efficient expression of extracellular domain alpha4beta2 nAChRs that are high fidelity structural models of full-length alpha4beta2 nAChRs.     

Neonicotinic analogues: Selective antagonists for α4β2 nicotinic acetylcholine receptors

Nicotine is an agonist of nicotinic acetylcholine receptors (nAChRs) that has been extensively used as a template for the synthesis of α4β2-preferring nAChRs. Here, we used the N-methyl-pyrrolidine moiety of nicotine to design and synthesise novel α4β2-preferring neonicotinic ligands. We increased the distance between the basic nitrogen and aromatic group of nicotine by introducing an ester functionality that also mimics acetylcholine (Fig. 2). Additionally, we introduced a benzyloxy group linked to the benzoyl moiety. Although the neonicotinic compounds fully inhibited binding of both [α-¹²⁵I]bungarotoxin to human α7 nAChRs and [³H]cytisine to human α4β2 nAChRs, they were markedly more potent at displacing radioligand binding to human α4β2 nAChRs than to α7 nAChRs. Functional assays showed that the neonicotinic compounds behave as antagonists at α4β2 and α4β2α5 nAChRs. Substitutions on the aromatic ring of the compounds produced compounds that displayed marked selectivity for α4β2 or α4β2α5 nAChRs. Docking of the compounds on homology models of the agonist binding site at the α4/β2 subunit interfaces of α4β2 nAChRs suggested the compounds inhibit function of this nAChR type by binding the agonist binding site.     

Crystal Structure of Lymnaea stagnalis AChBP Complexed with the Potent nAChR Antagonist DHβE Suggests a Unique Mode of Antagonism

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that belong to the Cys-loop receptor superfamily. These receptors are allosteric proteins that exist in different conformational states, including resting (closed), activated (open), and desensitized (closed) states. The acetylcholine binding protein (AChBP) is a structural homologue of the extracellular ligand-binding domain of nAChRs. In previous studies, the degree of the C-loop radial extension of AChBP has been assigned to different conformational states of nAChRs. It has been suggested that a closed C-loop is preferred for the active conformation of nAChRs in complex with agonists whereas an open C-loop reflects an antagonist-bound (closed) state. In this work, we have determined the crystal structure of AChBP from the water snail Lymnaea stagnalis (Ls) in complex with dihydro-β-erythroidine (DHβE), which is a potent competitive antagonist of nAChRs. The structure reveals that binding of DHβE to AChBP imposes closure of the C-loop as agonists, but also a shift perpendicular to previously observed C-loop movements. These observations suggest that DHβE may antagonize the receptor via a different mechanism compared to prototypical antagonists and toxins.     

Expression of alpha 7 nicotinic acetylcholine receptors by bipolar, amacrine, and ganglion cells of the rabbit retina.

Cholinergic agents affect the light responses of many ganglion cells (GCs) in the mammalian retina by activating nicotinic acetylcholine receptors (nAChRs). Whereas retinal neurons that express beta2 subunit-containing nAChRs have been characterized in the rabbit retina, expression patterns of other nAChR subtypes remain unclear. Therefore, we evaluated the expression of alpha7 nAChRs in retinal neurons by means of single-, double-, and triple-label immunohistochemistry. Our data demonstrate that, in the rabbit retina, several types of bipolar cells, amacrine cells, and cells in the GC layer express alpha7 nAChRs. At least three different populations of cone bipolar cells exhibited alpha7 labeling, whereas glycine-immunoreactive amacrine cells comprised the majority of alpha7-positive amacrine cells. Some GABAergic amacrine cells also displayed alpha7 immunoreactivity; alpha7 labeling was never detected in rod bipolar cells or rod amacrine cells (AII amacrine cells). Our data suggest that activation of alpha7 nAChRs by acetylcholine (ACh) or choline may affect glutamate release from several types of cone bipolar cells, modulating GC responses. ACh-induced excitation of inhibitory amacrine cells might cause either inhibition or disinhibition of other amacrine and GC circuits. Finally, ACh may act on alpha7 nAChRs expressed by GCs themselves.     

Electrophysiology of a chick neuronal nicotinic acetylcholine receptor expressed in Xenopus oocytes after cDNA injection

Brain nicotinic acetylcholine receptors (nAChRs) are made up of protein subunits that differ from those constituting muscle nAChRs. To characterize the physiological properties of one class of avian brain nicotinic receptor, we injected the nuclei of Xenopus oocytes with full-length cDNAs for the ligand binding (alpha 4) and structural (n alpha) subunits. Injected oocytes had large ACh-induced currents in the microampere range that were insensitive to alpha-bungarotoxin, as expected for neuronal nAChRs. We found that these brain nAChRs incorporate at least two alpha 4 subunits and that their functional properties differ from muscle nAChRs in at least two respects: the elementary conductance is considerably smaller (20 pS), and channels in outside out patches stop functioning within a few minutes.     

Calcium modulation and high calcium permeability of neuronal nicotinic acetylcholine receptors.

Two properties were found to distinguish neuronal from muscle nicotinic acetylcholine receptors (nAChRs). First, neuronal nAChRs have a greater Ca2+ permeability. The high Ca2+ flux through neuronal nAChRs activates a Ca(2+)-dependent Cl- conductance, and the Ca2+ to Cs+ permeability ratio (PCa/PCs) is 7 times greater for neuronal than for muscle nAChRs. A second difference between the receptor types is that neuronal nAChRs are potently modulated by physiological levels of external Ca2+. Neuronal nAChR currents are enhanced by external Ca2+ in a dose-dependent manner. The results indicate that changes in extracellular Ca2+ modulate neuronal nAChRs and may modulate cholinergic synapses in the CNS. Also, activation of neuronal nAChRs produces a significant influx of Ca2+ that could be an important intracellular signal.     

IgE-induced degranulation of mucosal mast cells is negatively regulated via nicotinic acetylcholine receptors

The autonomic nervous system is known to mediate mast cell activation. We investigated expression of nicotinic acetylcholine receptors (nAChRs) in mucosal-type mast cells and their contribution to the regulation of mast cell activation. Expression of mRNA of nAChR α4, α7, and β2 subunits were detected in specially differentiated mucosal-type murine bone marrow-derived mast cells (mBMMCs). Pretreatment with non-specific nAChRs agonists, acetylcholine, nicotine and epibatidine and a specific α7 subunit agonist GTS-21 significantly inhibited antigen-induced degranulation of mBMMCs in a dose-dependent manner and GTS-21-induced inhibition was significantly blocked by α7 subunit antagonist, α-bungarotoxin. Furthermore, confocal microscopy also demonstrated surface binding of α-bungarotoxin on mBMMCs. Our findings indicate that mucosal mast cell activation may be negatively regulated mainly through nAChR α7 subunit, suggesting that nAChRs are involved in neuronal-mucosal mast cell interactions.     

A motif present in the main cytoplasmic loop of nicotinic acetylcholine receptors and catalases

A motif containing five conserved amino acids (RXPXTH(X)14P) was detected in 111 proteins, including 82 nicotinic acetylcholine receptor (nAChR) subunits and 20 catalases. To explore possible functional roles of this motif in nAChRs two approaches were used: first, the motif sequences in nAChR subunits and catalases were analysed and compared; and, second, deletions in the rat alpha2 and beta4 nAChR subunits expressed in Xenopus oocytes were analysed. Compared to the three-dimensional structure of bovine hepatic catalase, structural coincidences were found in the motif of catalases and nAChRs. On the other hand, partial deletions of the motif in the alpha2 or beta4 subunits and injection of the mutants into oocytes was followed by a very weak expression of functional nAChRs; oocytes injected with alpha2 and beta4 subunits in which the entire motif had been deleted failed to elicit any acetylcholine currents. The results suggest that the motif may play a role in the activation of nAChRs.     

Neonicotinoid insecticides display partial and super agonist actions on native insect nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are present in high density in insect nervous tissue and are targeted by neonicotinoid insecticides. Improved understanding of the actions of these insecticides will assist in the development of new compounds. Here, we have used whole-cell patch-clamp recording of cholinergic neurons cultured from the central nervous system of 3rd instar Drosophila larvae to examine the actions of acetylcholine (ACh) and nicotine, as well as the neonicotinoids imidacloprid, clothianidin and P-CH-clothianidin on native nAChRs of these neurons. Dose-response data yield an EC(50) value for ACh of 19 microm. Both nicotine and imidacloprid act as low efficacy agonists at native nAChRs, evoking maximal current amplitudes 10-14% of those observed for ACh. Conversely, clothianidin and P-CH-clothianidin evoke maximal current amplitudes up to 56% greater than those evoked by 100 microm ACh in the same neurons. This is the first demonstration of 'super' agonist actions of an insecticide on native insect nAChRs. Cell-attached recordings indicate that super agonism results from more frequent openings at the largest (63.5 pS) conductance state observed.     

Possible Effect of Activation of α7-Nicotinic Acetylcholine Receptors in the Mitochondrial Membrane on the Development of Apoptosis

Using flow cytometry and sandwich-immunoenzyme assay, we showed that nicotinic acetylcholine receptors with a subunit α7 (nAChRs α7) expressed in the outer mitochondrial membrane are involved in the control of mitochondria-dependent apoptosis. Pre-incubation of the mitochondria with an nAChRs α7 agonist, choline, decreased dissipation of the membrane potential of these organelles induced by the action of 0.5 mM hydrogen peroxide (H₂O₂) but did not influence the analogous effect of a high Ca²⁺ concentration (90 μM). Agonists of nAChRs α7 (choline, acetylcholine, and PNU 282987), or an inhibitor of voltage-dependent anion channels, DIDS, prevented the release of cytochrome c from the intermembrane mitochondrial space under the action of H₂O₂. In contrast, an antagonist of nAChRs α7, methyllycaconitine, promoted the release of cytochrome c and prevented the effects of agonists. The obtained data confirm the active involvement of nAChRs α7 and voltage-dependent anion channels in the process of formation of mitochondrial pores. In this case, agonists of mitochondrial nAChRs α7 subunits exert an antiapoptotic effect, while antagonists of mitochondrial nAChRs α7 subunits manifest a proapoptotic action.     

Endogenous Cholinergic Inputs and Local Circuit Mechanisms Govern the Phasic Mesolimbic Dopamine Response to Nicotine

Nicotine exerts its reinforcing action by stimulating nicotinic acetylcholine receptors (nAChRs) and boosting dopamine (DA) output from the ventral tegmental area (VTA). Recent data have led to a debate about the principal pathway of nicotine action: direct stimulation of the DAergic cells through nAChR activation, or disinhibition mediated through desensitization of nAChRs on GABAergic interneurons. We use a computational model of the VTA circuitry and nAChR function to shed light on this issue. Our model illustrates that the α4β2-containing nAChRs either on DA or GABA cells can mediate the acute effects of nicotine. We account for in vitro as well as in vivo data, and predict the conditions necessary for either direct stimulation or disinhibition to be at the origin of DA activity increases. We propose key experiments to disentangle the contribution of both mechanisms. We show that the rate of endogenous acetylcholine input crucially determines the evoked DA response for both mechanisms. Together our results delineate the mechanisms by which the VTA mediates the acute rewarding properties of nicotine and suggest an acetylcholine dependence hypothesis for nicotine reinforcement.     

Discovery of non-peptide, small molecule antagonists of α9α10 nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonic inflammatory pain

A series of azaaromatic quaternary ammonium analogs has been discovered as potent and selective α9α10 nicotinic acetylcholine receptor (nAChR) antagonists. The preliminary structure-activity relationships of these analogs suggest that increased rigidity in the linker units results in higher potency in inhibition of α9α10 nAChRs and greater selectivity over α7 nAChRs. These analogs represent a new class of analgesic for the treatment of neuropathic and tonic inflammatory pain.     

A transmembrane site determines sensitivity of neuronal nicotinic acetylcholine receptors to general anesthetics

Neuronal nicotinic acetylcholine receptors (nAChRs) are potential targets for a wide variety of general anesthetics. We recently showed that alpha(4)beta(2) nAChRs are more sensitive than alpha(4)beta(4) receptors to the gaseous anesthetics nitrous oxide and xenon. The present study examines chimeric and point mutant rat nAChRs expressed in Xenopus oocytes and identifies a single amino acid residue (beta(2)-Val(253) or beta(4)-Phe(255)) near the middle of the second transmembrane segment (TM2) that determines gaseous anesthetic sensitivity. Mutations of this residue in beta subunits and the homologous residue of alpha(4) subunits (alpha(4)-Val(254)) showed that this position also determines sensitivities of nAChRs to acetylcholine, isoflurane, pentobarbital, and hexanol. In contrast, these mutations did not affect actions of ketamine. The positively charged sulfhydryl-specific reagent methanethiosulfonate ethylammonium reacted with a cysteine introduced at alpha(4)-Val(254) or beta(2)-Val(253), and irreversibly reduced anesthetic sensitivities of nAChRs. Propyl methanethiosulfonate is an anesthetic analog that covalently binds to a TM2 site of gamma-aminobutyric acid(A) and glycine receptors and irreversibly enhances receptor function. However, propyl methanethiosulfonate reversibly inhibited cysteine-substitution mutants at alpha(4)-Val(254) or beta(2)-Val(253) of nAChRs, and did not affect anesthetic sensitivity. Thus, residues alpha(4)-Val(254) and beta(2)-Val(253) alter channel gating and determine anesthetic sensitivity of nAChRs, but are not likely to be anesthetic-binding sites.     

Transport of multiple nicotinic acetylcholine receptors in the rat optic nerve: high densities of receptors containing alpha6 and beta3 subunits

Neuronal nicotinic acetylcholine receptors (nAChRs) are abundant in the rat retina and at least seven heteromeric subtypes have been detected. Axons of retinal ganglion cells form the optic nerve and innervate areas of the brain important for visual processing, including the lateral geniculate nucleus, the superior colliculus, and the pretectal nucleus. Development of eye-specific layers in these projection areas are dependent upon retinal waves which are initially mediated by nAChRs [ Feller et al. , Science 272 (1996), 1182 ; Penn et al. , Science 279 (1998), 2108 ; Bansal et al. , J. Neurosci. 20 (2000), 7672 ]. Unilateral eye-enucleation studies in the rat indicate that nAChRs are on the terminals of optic nerve axons, where they may mediate influences of acetylcholine on visual pathways. In this study, we use radioligand binding and immunoprecipitation with subunit-selective antibodies to investigate the subunit composition of nAChRs in the rat optic nerve. We found multiple nAChR subtypes in the optic nerve, all of which contain the β2 subunit. Most of these receptors are mixed heteromeric subtypes, composed of at least three different subunits. Included among these subtypes is the highest percentage and density of α6- and β3-containing nAChRs of any area of the rat CNS that has been reported.     

A nicotinic acetylcholine receptor mutation (Y151S) causes reduced agonist potency to a range of neonicotinoid insecticides

Neonicotinoid insecticides are potent selective agonists of insect nicotinic acetylcholine receptors (nAChRs). Since their introduction in 1991, resistance to neonicotinoids has been slow to develop, but it is now established in some insect field populations such as the planthopper, Nilaparvata lugens, a major rice pest in many parts of Asia. We have reported recently the identification of a target-site mutation (Y151S) within two nAChR subunits (Nlalpha1 and Nlalpha3) from a laboratory-selected field population of N. lugens. In the present study, we have examined the influence of this mutation upon the functional properties of recombinant nAChRs expressed in Xenopus oocytes (as hybrid nAChRs, co-expressed with a rat beta2 subunit). The agonist potency of several nicotinic agonists has been examined, including all of the neonicotinoid insecticides that are currently licensed for either crop protection or animal health applications (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam). The Y151S mutation was found to have no significant effect on the maximal current (I(max)) observed with the endogenous agonist, acetylcholine. In contrast, a significant reduction in I(max) was observed for all neonicotinoids (the I(max) for mutant nAChRs ranged from 13 to 81% of that observed on wild-type receptors). In addition, nAChRs containing the Y151S mutation caused a significant rightward shift in agonist dose-response curves for all neonicotinoids, but of varying magnitude (shifts in EC(50) values ranged from 1.3 to 3.6-fold). The relationship between neonicotinoid structure and their potency on nAChRs containing the Y151S target-site mutation is discussed.