Breast cancer continues to be the most common diagnosed cancer among women, and radiation or chemotherapy generally fails to provide durable cure, especially in the context of advanced or metastatic disease. Tumours recurrence is believed to be driven by cancer stem cells, which resist anti-cancer therapy and survive to seed relapse after remission in breast cancer patients. Small molecules inhibitors of nicotinic acetylcholine receptors (nAChR) target cancer stem cells, however, the precise nAChR required for breast cancer stem cell activity is unknown. Hence, we propose to test the capacity of shRNAs that target each individual nAChR to inhibit breast cancer stem cell activity. Briefly, we performed a cancer stem cell based pooled lenti-vector shRNA screen, to identify receptors required for the propagation of breast cancer stem cell enriched cultures. Our results demonstrate that the suppression of multiple receptors can be detected and corresponding genes are essential for TIC viability and survival. We anticipate our approach will identify the relevant nAChR receptor required for breast cancer stem cell activity. Such receptors may represent useful drug targets for the development of anti-breast cancer stem cell therapeutics. / Thesis / Master of Science (MSc)

Serotoninergic type 3 receptors (5HT3Rs) are members of the Cys-loop family of ligand-gated ion channels (LGIC), which includes nicotinic ACh, glycine, GABA-A and GABA-C receptors. All members of this family are widely expressed in the central and peripheral nervous systems, where they mostly participate in fast synaptic transmission. Activation of 5HT3Rs on vagal sensory nerve endings affect respiration, circulation, emesis and nociception; and in the central nervous system they are implicated in anxiety, depression, and drug dependence. In contrast, the function of 5HT3Rs in sympathetic neurons has not been fully determined. We discovered that 5HT3Rs interact with nicotinic acetylcholine receptors (nAChRs), the main drivers of the fast cholinergic autonomic synapse, through cross-talk mechanisms. We examined cross-talk by the patch-clamp technique on cultured mouse superior cervical ganglia (SCG) neurons. Co-stimulation of 5HT3Rs and nAChRs resulted in the generation of a combined current that was smaller than arithmetically predicted if the receptors did not interact with one another. This interaction, which we quantified as mean peak amplitude and mean ionic charge, was dependent on activation of 5HT3Rs and nAChRs, and independent of metabotropic receptors, Ca2+ entry and Ca2+ second messenger pathways, and of the direct action of 5HT on nAChRs. Preliminary data using an antibody targeted to the M3-M4 linker region of the 5HT3A subunit revealed that 5HT3Rs and nAChRs possibly cross-talk through physical interactions. These results revealed a potential role of the 5HT3R in the regulation of sympathetic synaptic transmission through cross-talk inhibition of nAChRs.

No description available.

T2R receptors are the classical bitter taste receptors which detect and transduce bitter taste in a subset of taste receptor cells (TRCs). The TRPM5-dependent T2Rs are G-protein coupled receptors (GPCRs) and are linked to G protein, gustducin to initiate an intracellular signaling cascade for the transduction of bitter tastants. Nicotine is bitter. However, at present the transduction mechanisms for the detection of nicotine in are poorly understood. Previous studies from our laboratory using TRPM5 knockout (KO) mice demonstrated that the T2R pathway is insufficient in explaining the taste perception of nicotine. TRPM5 KO mice elicited chorda tympani (CT) taste nerve responses to nicotine, albeit significantly smaller than the wild type (WT) mice and still responded to nicotine as an aversive stimulus. Following addition of mecamylamine (Mec), a non-specific blocker of neuronal nicotinic acetylcholine receptors (nAChRs), CT responses to nicotine were partially inhibited in both WT and TRPM5 KO mice. Mec also decreases the aversive response to nicotine in both WT and TRPM5 KO mice. These studies led to the hypothesis that both a TRPM5-independent and TRPM5-dependent pathways are responsible for the detection and transduction of the bitter taste of nicotine in TRCs. The TRPM5-independent pathway most likely utilizes the nAChRs expressed in TRCs and function as bitter taste receptors for nicotine. We have subsequently demonstrated the expression of nAChRs in a subset of TRPM5-positive TRCs. However, this mechanism is not well understood in other cell types, particularly undifferentiated epithelial cells, such as HEK293T cells. The specific aims of this project were: (i) To identify which components of T2R-dependent taste reception as well as components of nAChRs are expressed in HEK293T cells; (ii) To determine if HEK293T cells co-express these components; (iii) To identify if exposure to nicotine modulates the expression of T2R and nAChR dependent components in HEK293T cells; (iv) To determine if TRCs express functional nAChR ion channels; and (v) To determine if nAChRs are involved in the release of neuropeptides, such as brain-derived neurotrophic factor (BDNF) in HEK293T cells. The data obtained in HEK293T cells was compared with parallel studies on adult cultured human fungiform taste cells (HBO) done independently by Dr. Jie Qian, a postdoctoral fellow in Dr. Vijay Lyall’s lab. The results of combined studies on HBO and HEK293T cells indicates that TRPM5-positive cells also co-express ionotropic nAChRs, comprising a and β subunits. The nAChRs are capable of forming ion pores and when stimulated by nicotine and create a parallel TRPM5-independent pathway for the detection of nicotine. Using molecular and immunocytochemical techniques, our results demonstrate that mRNAs and proteins for bitter taste receptors and downstream intracellular signaling components as well as subunits necessary for the formation of nAChRs are expressed in HBO and HEK293T cells. Results demonstrated that TRPM5-positive HEK293T cells co-expressed nAChR subunits throughout the entire population. Nicotine increased the influx of Ca2+ in a dose dependent manner, which was somewhat reduced by the addition of TRPM5 blocker, triphenylphosphine oxide (TPPO). Both mRNA and protein expression were altered in a biphasic pattern with a maximum increased observed at 0.5 µM nicotine with a decrease in expression at higher concentrations. The synthesis of neurotrophic factor BDNF, required for maturation of taste bud cells and their innervating nerves, increased in HEK293T cells exposed to nicotine, however, nicotine did not trigger the release of BDNF. These results were then compared and contrasted with HBO cells to better understand the comparative effects of nicotine on both undifferentiated and differentiated cells. The data on HBO cells is presented in the Appendix.

Abstract The broad aim of this thesis is to structurally and functionally explore two α-conotoxins, from venomous sea snails, Vc1.1 and RgIA, in the hope of improving their journey to becoming analgesic therapeutics (introduction to conotoxins in Chapter 1). Vc1.1 is a two-disulfide peptide that is of interest as a potential therapeutic for the treatment of neuropathic pain. Despite investigations, limited structure-activity relationships have been conducted on this α-conotoxin. Consequently there is restricted insight into the interaction of this peptide with one of its analgesic targets, the α9α10 nicotinic acetylcholine receptor (nAChR). Late in this PhD project, the GABAB receptor was implicated as the possible target for conotoxins in neuropathic pain relief. However, there is still debate in the literature with regard to the true target of Vc1.1 and the α9α10 nAChR is still believed to be the target by some groups. This thesis predominantly focuses on the α9α10 nicotinic acetylcholine receptor. Chapter 4 of this thesis presents an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp or Lys (materials and methods described in Chapter 3) and examined using NMR spectroscopy (theory of NMR presented in Chapter 2), to determine the effects of the mutations on the structure of Vc1.1. The structural fold was found to be preserved in all peptides except where Pro was substituted. Chapter 5 explores the effect of these mutations on the blocking of acetylcholine (ACh)-evoked membrane currents at the α9α10 nAChR. Electrophysiological studies showed that the key residues for Vc1.1’s activity are Asp5-Arg7 and Asp11-Ile15, as changes at these positions resulted in the loss of activity at the α9α10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. Hence, Chapter 6 describes a second generation of mutants that was synthesized, namely N9G, N9I, N9L, S4R and S4K+N9A, all of which were more potent than Vc1.1 at both the rat α9α10 and the human α9/rat α10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the α3β2, α3β4 and α7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the α9α10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the α9α10 nAChR subtype and will help in the further development of Vc1.1 or its analogs as drugs. However, despite peptides exhibiting high degrees of potency and selectivity, such as Vc1.1 and RgIA, they are potentially hindered in their development as drugs due to their stability and bioavailability limitations, leading to invasive delivery techniques. Chapter 7 presents a range of cyclic RgIA analogs, tested at their targets the α9α10 nAChR and the GABAB receptor, that retain their activity and increase their stability in human serum relative to non-cyclic RgIA. NMR spectroscopy was used to determine the structure of the non-cyclic peptide and the cyclic peptide to confirm similarities in the global fold of the peptide. Structural perturbations and reduced activities were observed for cyclic RgIA analogs cyclized via linkers composed of three and four residues. Analogs with five, six and seven residues showed no structural perturbations, but differences in their activities at the different receptors. Because cRgIA-6 showed high potency for the GABAB receptor and lower potency for the α9α10 nAChR, this study has identified a GABAB selective peptide. Additionally, because the cRgIA-7 showed high potency for the α9α10 nAChR and low potency for the GABAB receptor, a α9α10 nAChR selective analog has also been identified. With improvements in these peptides against enzymatic attack, they show great potential on their path to becoming orally available analgesics as they may be able to withstand enzymatic conditions in the stomach.

Ο νικοτινικός υποδοχέας της ακετυλοχολίνης είναι ο σημαντικότερος στόχος στην αυτοάνοση νόσο βαριά μυασθένεια. Στο μεγαλύτερο ποσοστό των ασθενών (85%) ανιχνεύονται αυτοαντισώματα έναντι του μυϊκού υποδοχέα της ακετυλοχολίνης, τα οποία μπορούν να οδηγήσουν σε διαταραχή της μεταγωγής του κινητικού ερεθίσματος. Σε αυτή την μελέτη στόχος είναι η κατασκευή και έκφραση των εξωκυτταρικών περιοχών των υπομονάδων α και β του ανθρώπινου μυϊκού υποδοχέα της ακετυλοχολίνης σε κύτταρα εντόμων μετά από επιμόλυνση με βακιλοϊό. Στα ίδια πλαίσια πραγματοποιήθηκε η σύνδεση των υπομονάδων α και β με πεπτιδικό συνδέτη για την δημιουργία του συγκαταμερούς β-α. Τα ανασυνδυασμένα αυτά πεπτίδια απομονώθηκαν και χαρακτηρίστηκαν σε διαλυτή μορφή. Σε επόμενο στάδιο έγινε η ακινητοποίηση τους σε υπόστρωμα σεφαρόζης και ο έλεγχος της ικανότητας πρόσδεσης αυτοαντισωμάτων από ορό μυασθενών με απώτερο στόχο τηv χρήση τους ως πιθανοί ανοσοπροσροφητές σε μια καινούρια θεραπευτική προσέγγιση για την μυασθένεια. / Myasthenia gravis (MG) is an autoimmune disease caused in the majority of patients (85%) by autoantibodies against the human muscle acetylcholine receptor (AChR),a post-synaptic ligand-gated ion-channel located at the neuromuscular junction.Autoantibodies against the AChR cause loss of the available and functional AChRs leading to muscle weakness and fatigability.An attractive therapeutic approach is the extracorporeal specific removal of the pathogenic autoantibodies using AChR-based immunoadsorbents. In this study, the N-terminal extracellular domains (ECD) of AChR the subunits α1 and β1 were cloned into baculovirus expression vectors and heterologously expressed using insect SF9 cells.Additionally, the two subunits were linked by the use of a flexible peptide linker to form the β1-α1 concatamer.The recombinant proteins were expressed as soluble polypeptides, purified and characterized.Furthermore, they were immobilised on sepharose beads in order to test them as immunoadsorbents using sera from MG patients. They were all found to bind anti-AChR autoantibodies, albeit to varying degrees. They, thus, pose as potential candidates for the therapeutic antigen-specific clearance of MG sera.

The pentameric ligand gated ion channel super family plays a central role in fast synaptic communication between neurons and at the neuromuscular junction. Extensive studies on the prototypic pLGIC, the Torpedo nicotinic acetylcholine receptor (nAChR) have revealed an exquisite lipid sensitivity, with the nAChR adopting a novel uncoupled conformation in membranes lacking activating anionic and neutral lipids. The lipid-exposed transmembrane alpha-helix, M4, in each homologous subunit likely acts as a lipid sensor. One model proposes that activating lipids promote M4 “binding” to the adjacent alpha-helices, M1 and M3, to enhance interactions between the M4 C-terminus and the Cys-loop of the agonist-binding domain, with such interactions promoting coupling between the agonist site and channel gate. The first part of my thesis indirectly tests this hypothesis by exploring the effects of membrane hydrophobic thickness on nAChR function. Specifically, I tested the hypothesis that thicker membranes, which should promote alignment of M4 parallel to M1/M3 and thus helix-helix interactions, favor a coupled conformation. Although I found that the nAChR is uncoupled in all membranes tested, regardless of hydrophobic thickness, thicker membranes promote transitions from uncoupled to ultimately the desensitized state over the minutes to hours time frame. In contrast to anionic lipids, which influence function primarily via a conformational selection mechanism, membrane hydrophobic thickness influences function via a kinetic mechanism - thick membranes lower the activation energy between uncoupled and coupled conformations to promote conformational transitions. In the second part of my thesis, I used the two prokaryotic homologs, GLIC and ELIC, to explore how amino acid interactions at the interface between M4 and M1/M3 influence channel activity. Alanine scanning mutagenesis of this interface shows that disruption of almost any interaction in GLIC leads to a loss of folding and/or function, while analogous mutations in ELIC typically lead to no change or produce gains in function. Sequence comparisons with other members of the pLGIC superfamily suggest that the transmembrane domains of GLIC and ELIC represent two distinct archetypes. Each archetype may strike a different balance between the need for strong M4 binding to M1/M3 to promote folding and pentamer assembly, and the need for weaker interactions that allow for greater conformational flexibility during function.

Nicotine's rewarding effects are mediated through distinct subunits of nAChRs, encoded by different nicotinic cholinergic receptor (CHRN) genes and expressed in discrete regions in the brain. In the present study, we aimed to test the associations between rare variants at CHRN genes and nicotine dependence (ND), and alcohol dependence (AD). A total of 26,498 subjects with nine different neuropsychiatric disorders in 15 independent cohorts, which were genotyped on Illumina, Affymetrix, or PERLEGEN microarray platforms, were analyzed. Associations between rare variants (minor allele frequency (MAF) <0.05) at CHRN genes and nicotine dependence, and alcohol dependence were tested. The mRNA expression of all Chrn genes in whole mouse brain and 10 specific brain areas was investigated. All CHRN genes except the muscle-type CHRNB1, including eight genomic regions containing 11 neuronal CHRN genes and three genomic regions containing four muscle-type CHRN genes, were significantly associated with ND, and/or AD. All of these genes were expressed in the mouse brain. We conclude that CHRNs are associated with ND (mainly) and AD, supporting the hypothesis that the full catalog of ND/AD risk genes may contain most neuronal nAChRs-encoding genes.

L’utilisation intensive des insecticides pour lutter contre les insectes ravageurs de culture et vecteurs demaladies, a conduit à des polémiques sur le mode d’action des insecticides. Ces polémiques sont liéesau fait que le mode d’action des insecticides, notamment des néonicotinoïdes est mal connu. Ils agissentprincipalement sur les récepteurs à l’acétylcholine (ACh) de type nicotinique (nAChR) qui jouent un rôlefondamental dans la transmission synaptique cholinergique. Bien que ces récepteurs soient bien décritschez les mammifères, très peu d’études ont évalué l’effet des néoniotinoïdes sur un récepteur natifd’insecte.Au cours de cette thèse, nous avons pour la première fois exprimé en ovocytes de xénope un récepteurhomomérique ⍺7 de blatte et étudié ces propriétés pharmacologiques vis-à-vis des néonicotinoïdes,comparativement au récepteur a7 de rat. Nos résultats révèlent un récepteur atypique qui est insensibleà l’⍺-bungarotoxine et qui n’est pas activé par les néonicotinoïdes. Ainsi, bien que les gènes codantpour les sous-unités α7 de blatte et de rat forment un groupe monophylétique distinct des autres sousunitésd’insectes et de mammifères, les récepteurs homomériques obtenus semblent avoir despropriétés pharmacologiques différentes. Parallèlement, nous avons étudié les propriétéspharmacologiques des nAChR natifs et notamment l’effet modulateur d’un pyréthrinoïde, la permethrine,sur les courants induits par le dinotefurane. Ce travail a permis d’évaluer le mode d’action d’unantiparasitaire, le Vectra 3D. Enfin, nous avons également entrepris de développer la techniqued’extraction membranaire afin de l’utiliser comme alternative pour étudier le mode d’action desinsecticides.Pour conclure, cette thèse a permis une avancée sur l’étude de la caractérisation des propriétéspharmacologiques des récepteurs nicotiniques neuronaux des insectes et sur l’étude du mode d’actiondes insecticides néonicotinoïdes. / The intensive use of insecticides against crop pests and vectors of human and animal leads to several polemics about their mode of action. All these controversies are related to the fact that the mode of action of insecticides in insects is poorly unknown, in particular neonicotinoids which act on nicotinic acetylcholine (ACh) receptors (nAChR).During this PhD thesis, we characterized for the first time the pharmacological properties of a cockroach ⍺7 homomeric receptor in a xenopus oocyte. Our results revealed that cockroach ⍺7 in an atypical receptor that is insensitive to ⍺-bungarotoxin and not activated by neonicotinoids. Cockroach and rat ⍺7 receptors which are included in the same cluster have distinct pharmacological properties. We then studied the pharmacological properties of native receptors, in particular, the modulatory effect of permethrin on dinotefuran-induced currents. This work was included in the study of Vectra 3D. We also evaluated the use of insect central nervous system membrane extraction as a strategy to study the pharmacological properties of insect native nAChRs.To conclude, this PhD contribute to the study of the pharmacological properties of insect nAChRs and the study of the mode of action of neonicotinoids insecticides.

Consumption of alcohol and tobacco is common all around the world and these drugs are frequently consumed concomitantly. It has been estimated that 70-80 % of alcoholics are smokers and non-alcoholic drinkers are more often smokers than teetotallers. Alcohol and tobacco may affect the risk of developing neurological diseases and might influence this risk differently when combined compared to when only one of these compounds is consumed. Some in vitro-research have shown that non-toxic concentrations of ethanol and nicotine, in combination, can exert toxicity, and might do so in a synergistic way. In this work, investigations were made to see if the neuronal nicotinic acetylcholine receptors (nAChRs) and NMDA receptors are involved in this interactive behaviour between ethanol and nicotine. A human neuroblastoma SH-SY5Y cell line was treated with ethanol and nicotine at different concentrations and cell viability was measured through an MTT-assay. A significant reduction in cell viability was induced by chronic treatment with a low-dose combination of ethanol and nicotine. The cell viability reduction was completely inhibited by pretreatment with the non-specific nAChR antagonist mecamylamine. This suggests that nAChRs are involved in low-dose ethanol-nicotine interactions. The NMDA receptor antagonist memantine did not affect the ethanol-nicotine effect, which implies that NMDA receptors are not involved in low-dose ethanol-nicotine interactions in SH-SY5Y cells. However, it is unclear if the SH-SY5Y cell line expresses fully functional NMDA receptors. The expression of NMDA receptors might vary with cell passage number. Further research has to be done to uncover the contribution of specific nAChR subtypes to the ethanol-nicotine interaction. There also remains to be revealed if human neuroblastoma SH-SY5Y cells express fully functional NMDA receptors and how cell passage number affects the expression of these receptors.