Therapeutic potential of α7 nicotinic receptor agonists to regulate neuroinflammation in neurodegenerative diseases

Neurodegenerative diseases, such as Alzheimer＇s, Parkinson＇s and Huntington＇s diseases, are all character- ized by a component of innate immunity called neuroinflammation. Neuronal loss and neuroinflammation are two phenomena closely linked. Hence, the neuroinflammation is a relevant target for the management of the neurodegenerative diseases given that, to date, there is no treatment to stop neuronal loss. Several studies have investigated the potential effects of activators of alpha 7 nicotinic acetylcholine receptors in animal models of neurodegenerative diseases. These receptors are widely distributed in the central nervous system. After activation, they seem to mediate the cholinergic anti-inflammatory pathway in the brain. This anti-inflammatory pathway, first described in periphery, regulates activation of microglial cells considered as the resident macrophage population of the central nervous system. In this article, we shortly review the agonists of the alpha 7 nicotinic acetylcholine receptors that have been evaluated in vivo and we focused on the selective positive allosteric modulators of these receptors. These compounds represent a key element to enhance receptor activity only in the presence of the endogenous agonist.

Neuroactive alkaloids that modulate the neuronal nicotinic receptor and provide neuroprotection in an Alzheimer's disease model:the case of Teline monspessulana

Despite the advances in combinatorial or synthetic chemis- try and bioinformatics, recent literature has demonstrated the relevance of nature and biomass as a source of new molecules to treat different pathologies, i.e., bioactive com- pounds obtained from Ecteinascidia turbinate to treat some types of cancer or rapamycin from Streptomyces hygroscop- icus to prevent organ rejection after transplant. This trend will continue simply due to the fact that Mother Nature has been synthesizing molecules for millions of years. In our lab- oratory, we have characterized several compounds obtained from natural sources and that possess important neuronal effects,

Nicotinic receptors modulate antitumor therapy response in triple negative breast cancer cells

BACKGROUND Triple negative breast cancer is more aggressive than other breast cancer subtypes and constitutes a public health problem worldwide since it has high morbidity and mortality due to the lack of defined therapeutic targets.Resistance to chemotherapy complicates the course of patients’treatment.Several authors have highlighted the participation of nicotinic acetylcholine receptors(nAChR)in the modulation of conventional chemotherapy treatment in cancers of the airways.However,in breast cancer,less is known about the effect of nAChR activation by nicotine on chemotherapy treatment in smoking patients.AIM To investigate the effect of nicotine on paclitaxel treatment and the signaling pathways involved in human breast MDA-MB-231 tumor cells.METHODS Cells were treated with paclitaxel alone or in combination with nicotine,administered for one or three 48-h cycles.The effect of the addition of nicotine(at a concentration similar to that found in passive smokers’blood)on the treatment with paclitaxel(at a therapeutic concentration)was determined using the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.The signaling mediators involved in this effect were determined using selective inhibitors.We also investigated nAChR expression,and ATP“binding cassette”G2 drug transporter(ABCG2)expression and its modulation by the different treatments with Western blot.The effect of the treatments on apoptosis induction was determined by flow cytometry using annexin-V and 7AAD markers.RESULTS Our results confirmed that treatment with paclitaxel reduced MDA-MB-231 cell viability in a concentration-dependent manner and that the presence of nicotine reversed the cytotoxic effect induced by paclitaxel by involving the expression of functionalα7 andα9 nAChRs in these cells.The action of nicotine on paclitaxel treatment was linked to modulation of the protein kinase C,mitogen-activated protein kinase,extracellular signal-regulated kinase,and NF-κB signaling pathways,and to an up-regulation of ABCG2 protein expression.We also detected that nicotine significantly reduced the increase in cell apoptosis induced by paclitaxel treatment.Moreover,the presence of nicotine reduced the efficacy of paclitaxel treatment administered in three cycles to MDA-MB-231 tumor cells.CONCLUSION Our findings point to nAChRs as responsible for the decrease in the chemotherapeutic effect of paclitaxel in triple negative tumors.Thus,nAChRs should be considered as targets in smoking patients.

Cloning and Sequence of Nicotinic Acetylcholine Receptor α Subunit from Chilo suppressalis

Nicotinic acetylcholine receptors (nAChRs) play a significant role in excitatory synaptic transmission in insects and are the target for chloronicotinyl and nereistoxin insecticides.In recent years,Chilo suppressalis,an economically important pest of rice,developed high resistance against monosultap,a nereistoxin insecticide acting on nAChR.In order to reveal the hypothesized target insensitive mechanism,studies on the molecular property of nAChR from Chilo suppressalis are required.In this study,the full length cDNA of nAChR α subunit from this pest was cloned by RT-PCR.Sequence analysis shows that it is a novel nAChR α subunit,which was named as Cs α 1(Genbank accession No.AF418987).It contains 1?997?bp nucleotides and involves an open reading frame (ORF) encoding a mature protein of 509 amino acids excluding a signal peptide of 24 amino acids.The deduced amino acid sequence was 52%-94% identical to the reported insect nAChR genes.

Alpha-7 nicotinic acetylcholine receptor agonist treatment in a rat model of Huntington's disease and involvement of heme oxygenase-1

Neuroinflammation is a common element involved in the pathophysiology of neurodegenerative diseases.We recently reported that repeated alpha-7 nicotinic acetylcholine receptor（α7 n ACh R） activations by a potent agonist such as PHA 543613 in quinolinic acid-injured rats exhibited protective effects on neurons.To further investigate the underlying mechanism,we established rat models of early-stage Huntington＇s disease by injection of quinolinic acid into the right striatum and then intraperitoneally injected 12 mg/kg PHA 543613 or sterile water,twice a day during 4 days.Western blot assay results showed that the expression of heme oxygenase-1（HO-1）,the key component of the cholinergic anti-inflammatory pathway,in the right striatum of rat models of Huntington＇s disease subjected to intraperitoneal injection of PHA 543613 for 4 days was significantly increased compared to the control rats receiving intraperitoneal injection of sterile water,and that the increase in HO-1 expression was independent of change in α7 n ACh R expression.These findings suggest that HO-1 expression is unrelated to α7 n ACh R density and the increase in HO-1 expression likely contributes to α7 n ACh R activation-related neuroprotective effect in early-stage Huntington＇s disease.

Antinociceptive effects of novel epibatidine analogs through activation of α4β2 nicotinic receptors

The study of α4β2 nicotinic receptors has provided new indications in the treatment of pain. Efforts have been made to explore new α4β2 nicotinic receptor agonists, including TC-2559, as antinociceptive drugs. In this study, we discovered a set of novel epibatidine analogs with strong binding affinities to the α4β2 nicotinic receptors. Among these compounds, C-159, C-163, and C-9515 attenuated formalin-induced nociceptive responses in mice; C-9515 caused the most potent analgesic effect, which was blocked by mecamylamine, a non-selective nicotinic receptor antagonist. Furthermore, C-9515 potently inhibited chronic constriction injury(CCI)-induced neuropathic pain in rats, which was sensitive to DHβE, a selective α4β2 subtype antagonist,indicating that its analgesic effect was mediated by the activation of the α4β2 nicotinic receptors. In conclusion, the epibatidine analog C-9515 was found to be a potent α4β2 nicotinic receptor agonist with potent analgesic function, which demonstrated potential for the further exploration of its druggability.

Possible implications of dysregulated nicotinic acetylcholine receptor diffusion and nanocluster formation in myasthenia gravis

Myasthenia gravis is a rare and invalidating disease affecting the neuromuscular junction of voluntary muscles.The classical form of this autoimmune disease is characterized by the presence of antibodies against the most abundant protein in the neuromuscular junction,the nicotinic acetylcholine receptor.Other variants of the disease involve autoimmune attack of non-receptor scaffolding proteins or enzymes essential for building or maintaining the integrity of this peripheral synapse.This review summarizes the participation of the above proteins in building the neuromuscular junction and the destruction of this cholinergic synapse by autoimmune aggression in myasthenia gravis.The review also covers the application of a powerful biophysical technique,superresolution optical microscopy,to image the nicotinic receptor in live cells and follow its motional dynamics.The hypothesis is entertained that anomalous nanocluster formation by antibody crosslinking may lead to accelerated endocytic internalization and elevated turnover of the receptor,as observed in myasthenia gravis.

Identification of α7 nicotinic acetylcholine receptor on hippocampal astrocytes cultured in vitro and its role on inflammatory mediator secretion

The present study found expressions of a7 nicotinic acetylcholine receptor on hippocampal slices and hippocampal astrocytes using double immunofluorescence stainings. Expression of glial fibdllary acidic protein in the cultured hippocampal slices and hippocampal astrocytes significantly increased, and levels of macrophage inflammatory protein la, RANTES, interleukin-1β, intedeukin-6, and tumor necrosis factor-α increased in the supernatant of cultured astrocytes following exposure to 200 nM amyloid 13 protein 1-42. Preconditioning of 10 μM nicotine, a nicotinic acetylcholine receptor agonist, could attenuate the influence of amyloid β protein 1-42 in inflammatory mediator secretion of cultured astrocytes. Experimental findings indicated that α7 nicotinic acetylcholine receptor was expressed on the surface of hippocampal astrocytes, and activated a7 nicotinic acetylcholine receptor was shown to inhibit inflammation induced by amyloid β protein 1-42.

Targeting α7 nicotinic acetylcholine receptors: a future potential for neuroprotection from traumatic brain injury

Traumatic brain injury （TBI） poses a significant socioeconomic burden in the world. The long lasting consequences in cognitive impairments are often underreported and its mechanisms are unclear. In this perspective, cholinergic dysfunction and thera-peutic strategy targeting this will be reviewed. Novel agents that can target specific subtype of acetylcholine receptors have been developed over the recent years and are at various stages of development, which include AR-R 17779, GTS-21, SSR- 180711A, AR-R17779, and PNU-282987. A detailed review on this topic has been previously published （Shin and Dixon, 2015）.

Neuroprotective and anti-inflammatory effects of a therapy combining agonists of nicotinic α7 and σ1 receptors in a rat model of Parkinson’s disease

To date there is no treatment able to stop or slow down the loss of dopaminergic neurons that characterizes Parkinson’s disease.It was recently observed in a rodent model of Alzheimer’s disease that the interaction between the α7 subtype of nicotinic acetylcholine receptor(α7-nAChR)and sigma-1 receptor(σ1-R)could exert neuroprotective effects through the modulation of neuroinflammation which is one of the key components of the pathophysiology of Parkinson’s disease.In this context,the aim of the present study was to assess the effects of the concomitant administration of N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-furo[2,3-c]pyridine-5-carboxamide(PHA)543613 as an α7-nAChR agonist and 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate(PRE)-084 as aσ1-R agonist in a well-characterized 6-hydroxydopamine rat model of Parkinson’s disease.The animals received either vehicle separately or the dual therapy PHA/PRE once a day until day 14 postlesion.Although no effect was noticed in the amphetamine-induced rotation test,our data has shown that the PHA/PRE treatment induced partial protection of the dopaminergic neurons(15-20%),assessed by the dopamine transporter density in the striatum and immunoreactive tyrosine hydroxylase in the substantia nigra.Furthermore,this dual therapy reduced the degree of glial activation consecutive to the 6-hydroxydopamine lesion,i.e,the 18 kDa translocation protein density and glial fibrillary acidic protein staining in the striatum,and the CD11b and glial fibrillary acidic protein staining in the substantia nigra.Hence,this study reports for the first time that concomitant activation of α7-nAChR andσ1-R can provide a partial recovery of the nigro-striatal dopaminergic neurons through the modulation of microglial activation.The study was approved by the Regional Ethics Committee(CEEA Val de Loire n°19)validated this protocol(Authorization N°00434.02)on May 15,2014.

Activities of nicotinic acetylcholine receptors modulate neurotransmission and synaptic architecture

The cholinergic system is involved in a broad spectrum of brain function, and its failure has been implicated in Alzheimer＇s disease. Acetylcholine transduces signals through muscarinic and nicotinic acetylcholine receptors, both of which influence synaptic plasticity and cognition. However, the mechanisms that relate the rapid gating of nicotinic acetylcholine receptors to persistent changes in brain function have remained elusive. Recent evidence indicates that nicotinic acetylcholine receptors activities affect synaptic morphology and density, which result in persistent rearrangements of neural connectivity. Further investigations of the relationships between nicotinic acetylcholine receptors and rearrangements of neural circuitry in the central nervous system may help understand the pathogenesis of Alzheimer＇s disease.

Computational Determination of the Binding Mode of α-Conotoxin to Nicotinic Acetylcholine Receptor

Abstract Conotoxins belong to the large families of disulfide-rich peptide toxins from cone snail venom, and can act on a broad spectrum of ion channels and receptors. They are classified into different subtypes based on their targets. The a-conotoxins selectively inhibit the current of the nicotinic acetylcholine receptors. Because of their unique selectivity towards distinct nAChR subtypes, a-conotoxins become valuable tools in nAChR study. In addition to the X-ray structures of a-conotoxins in complex with acetyleholine-binding protein, a homolog of the nAChR ligand-binding domain, the high-resolution crystal structures of the extracellular domain of the al and a9 subunits are also obtained. Such structures not only revealed the details of the configuration of nAChR, but also provided higher sequence identity templates for modeling the binding modes of a-conotoxins to nAChR. This mini-review summarizes recent modeling studies for the determination of the binding modes of a-conotoxins to nAChR. As there are not crystal structures of the nAChR in complex with conotoxins, computational modeling in combination of mutagenesis data is expected to reveal the molecular recognition mechanisms that govern the interactions between a-conotoxins and nAChR at molecular level. An accurate determination of the binding modes of a-conotoxins on AChRs allows rational design of a-conotoxin analogues with improved potency or selectivity to nAChRs.

Nicotinic Acetylcholine Receptor Gene Family of the Pea Aphid,Acyrthosiphon pisum

The nicotinic acetylcholine receptors （nAchRs） are cholinergic receptors that form ligand-gated ion channels by ifve subunits in insect and vertebrate nervous systems. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Here, we identiifed and cloned 11 candidate nAChR subunit genes in Acyrthosiphon pisum using genome-based bioinformatics combined modern molecular techniques. Most A. pisum nAChRs including α1, α2, α3, α4, α6, α8, and β1 show highly sequence identities with the counterparts of other insects examined. Expression proifles analysis showed that all subunit genes were expressed in adult head. At least two subunits have alternative splicing that obviously increase A. pisum nicotinic receptor diversity. This study will be invaluable for exploring the molecular mechanisms of neonicotinoid-like insecticides in sucking pests, and for ultimately establishing the screening platform of novel insecticides.

Nanoscale interactions between the nicotinic acetylcholine receptor and cholesterol

Cholesterol is a major lipid in biological membranes.It not only plays a structural role but also modulates a wide range of functional properties of neurotransmitter and hormone receptors and ion channels.The membraneembedded segments of the paradigm neurotransmitter receptor for acetylcholine(nAChR)contain linear sequences of amino acids with the capacity to recognize cholesterol.These cholesterol consensus domains have been designated as“CARC”and its mirror sequence“CRAC”.CARC preferentially occurs in the exoplasmic-facing membrane leaflet,and CRAC,in the cytoplasmic-facing hemilayer.Both motifs are highly conserved among ion-channel and neurotransmitter receptor proteins in vertebrate nervous systems,where they recognize cholesterol,and in prokaryotic homologues in bacteria,where they recognize hopanoids.This phylogenetically conserved trait is an indication that the hopanoids in some bacteria and cholesterol in eukaryotes subserve analogous functions,probably contributing to the stability of membrane-embedded protein domains.Structural studies from our laboratory using superresolution optical microscopy(“nanoscopy”)have disclosed other interrelated functional and structural properties exerted by cholesterol on the nAChR.The neutral lipid content at the cell surface influences both the macromolecular organization of the receptor and its translational mobility(diffusion)in the plane of the membrane.

Homology Model and Ligand Binding Interactions of the Extracellular Domain of the Human α4β2 Nicotinic Acetylcholine Receptor

Addiction to nicotine, and possibly other tobacco constituents, is a major factor that contributes to the difficulties smokers face when attempting to quit smoking. Amongst the various subtypes of nicotinic acetylcholine receptors (nAChRs), the α4β2 subtype plays an important role in mediating the addiction process. The characterization of human α4β2-ligand binding interactions provides a molecular framework for understanding ligand-receptor interactions, rendering insights into mechanisms of nicotine addiction and may furnish a tool for efficiently identifying ligands that can bind the nicotine receptor. Therefore, we constructed a homology model of human α4β2 nAChR and performed molecular docking and molecular dynamics (MD) simulations to elucidate the potential human α4β2-ligand binding modes for eleven compounds known to bind to this receptor. Residues V96, L97 and F151 of the α4 subunit and L111, F119 and F121 of the β2 subunit were found to be involved in hydrophobic interactions while residues S153 and W154 of the α4 subunit were involved in the formation of hydrogen bonds between the receptor and respective ligands. The homology model and its eleven ligand-bound structures will be used to develop a virtual screening program for identifying tobacco constituents that are potentially addictive.

Nicotine alpha 4 beta 2 receptor-mediated free calcium in an animal model of facial nucleus injury

Previous studies have demonstrated that the cholinergic system, via nicotinic receptors, regulates intracellular free calcium levels in the facial nucleus under normal physiological conditions. However, the regulation of nicotinic receptors on free calcium levels following facial nerve injury remains unclear. In the present study, an animal model of facial nerve injury was established, and changes in nicotinic receptor expression following facial nerve injury in rats were detected using reverse transcription polymerase chain reaction. Nicotinic receptor-mediated changes of free calcium levels following facial nucleus injury were determined by laser confocal microscopy. Results showed no significant difference in nicotinic receptor expression between the normal group and the affected facial nerve nucleus. The nicotinic receptor a4132 subtype increased free calcium levels following facial nerve injury by promoting calcium transmembrane influx, and L-type voltage-gated calcium channel-mediated influx of calcium ions played an important role in promoting calcium transmembrane influx. The nicotinic receptor-mediated increase of free calcium levels following facial nerve injury provides an important mechanism for the repair of facial nerve injury.

Receptor variability-driven evolution of snake toxins

Three-finger toxins （TFTs） are well-recognized non- enzymatic venom proteins found in snakes. However, although TFTs exhibit accelerated evolution, the drivers of this evolution remain poorly understood. The structural complexes between long-chain α-neurotoxins, a subfamily of TFTs, and their nicotinic acetylcholine receptor targets have been determined in previous research, providing an opportunity to address such questions. In the current study, we observed several previously identified positively selected sites （PSSs） and the highly variable C-terminal loop of these toxins at the toxin/receptor interface. Of interest, analysis of the molecular adaptation of the toxin-recognition regions in the corresponding receptors provided no statistical evidence for positive selection. However, these regions accumulated abundant amino acid variations in the receptors from the prey of snakes, suggesting that accelerated substitution of TFTs could be a consequence of adaptation to these variations. To the best of our knowledge, this atypical evolution, initially discovered in scorpions, is reported in snake toxins for the first time and may be applicable for the evolution of toxins from other venomous animals.

Beauty of the beast: anticholinergic tropane alkaloids in therapeutics

Tropane alkaloids(TAs)are among the most valued chemical compounds known since pre-historic times.Poisonous plants from Solanaceae family(Hyoscyamus niger,Datura,Atropa belladonna,Scopolia lurida,Mandragora officinarum,Duboisia)and Erythroxylaceae(Erythroxylum coca)are rich sources of tropane alkaloids.These compounds possess the anticholinergic properties as they could block the neurotransmitter acetylcholine action in the central and peripheral nervous system by binding at either muscarinic and/or nicotinic receptors.Hence,they are of great clinical impor-tance and are used as antiemetics,anesthetics,antispasmodics,bronchodilator and mydriatics.They also serve as the lead compounds to generate more effective drugs.Due to the important pharmacological action they are listed in the WHO list of essential medicines and are available in market with FDA approval.However,being anticholinergic in action,TA medication are under the suspicion of causing dementia and cognitive decline like other medications with anticholinergic action,interestingly which is incorrect.There are published reviews on chemistry,biosynthesis,phar-macology,safety concerns,biotechnological aspects of TAs but the detailed information on anticholinergic mecha-nism of action,clinical pharmacology,FDA approval and anticholinergic burden is lacking.Hence the present review tries to fill this lacuna by critically summarizing and discussing the above mentioned aspects.

Exploring the Nicotinic Acetylcholine Receptor-associated Proteome with iTRAQ and Transgenic Mice

Neuronal nicotinic acetylcholine receptors （nAChRs） containing Gt4 and 132 subunits are the principal receptors in the mammalian central nervous system that bind nicotine with high affin- ity. These nAChRs are involved in nicotine dependence, mood disorders, neurodegeneration and neuroprotection. However, our understanding of the interactions between a4β2-containing （a4β2） nAChRs and other proteins remains limited. In this study, we identified proteins that inter- act with ct4β2 nAChRs in a gene-dose dependent pattern by immunopurifying β2 nAChRs from mice that differ in ct4 and β2 subunit expression and performing proteomic analysis using isobaric tags for relative and absolute quantitation （iTRAQ）. Reduced expression of either the a4 or the β2 subunit results in a correlated decline in the expression of a number of putative interacting proteins. We identified 208 proteins co-imrnunoprecipitated with these nAChRs. Furthermore, stratified lin- ear regression analysis indicated that levels of 17 proteins was correlated significantly with expres- sion of at4β2 nAChRs, including proteins involved in cytoskeletal rearrangement and calcium signaling. These findings represent the first application of quantitative proteomics to produce a β2 nAChR interactome and describe a novel technique used to discover potential targets for pharma- cological manipulation of a4β2 nAChRs and their downstream signaling mechanisms.

Tobacco-specific Carcinogen 4-(Methylnitrosoamino)-1-(3-pyridyl)-1-butanone(NNK) Activating ERK1/2 MAP Kinases and Stimulating Proliferation of Human Mammary Epithelial Cells

Cigarette smoking is correlated with the development of various cancers. 4- （Methylnitresoamino） -1- （3-pyridyl） - 1-butanone（NNK） is one of the major tobacco-specific carcinogens in the cigarette smoke, which increases the risk of breast cancer. In the present study, it was demonstrated that NNK rapidly activated ERK1 and ERK2 MAP kinases in human normal mammary epithelial cells. It was found that there are two different routes for the activation of ERK1/2 with NNK. One is from nicotinic receptor nAchR to MEK1/2, and the other is from tyrosine kinase containing receptor to MEK1/2. The tobacco-specific carcinogen NNK shows a strong proliferative effect on normal human mammary epithelial cells and cancer mammary epithelial cells.