The goal of this experiment was to examine the pattern of ACh release in mPFC and HPC during performance in trace and delay appetitive conditioning.

Upregulation of nicotinic acetylcholine receptors (nAChRs) is a well-documented response to chronic nicotine exposure. Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels consisting of alpha (α2-10) and beta (β2-4) subunits. Nicotine, an agonist of nAChRs, alters trafficking and assembly of some subtypes of nAChRs, leading to an increase in expression of high sensitivity receptors on the plasma membrane. These physiological changes in nAChRs are believed to contribute to nicotine addiction, although the mechanism of these processes has not been resolved. Recently, many studies have converged on the idea that nicotine induces upregulation by an intracellular mechanism. In this dissertation, expression levels of nAChRs were quantified upon exposure to nicotine and its primary metabolite, cotinine. A pH sensitive variant of GFP, super ecliptic pHluorin (SEP), was integrated with a nAChR subunit to study expression and trafficking of nAChRs by differentiating intracellular and plasma membrane inserted receptors. In this work, cotinine is shown to increase the number of α4β2 nAChRs within a cell. Cotinine also affects trafficking of α4β2, evident by a redistribution of intracellular receptors and an increase in single vesicle insertion events on the plasma membrane. This work shows both nicotine and cotinine alter the overall assembly of α4β2 to favor the high sensitivity (α4)2(β2)3 version. Since cotinine and nicotine induce similar physiological changes in nAChRs, the metabolite potentially plays a role in the mechanism of nicotine addiction. Although an intracellular mechanism for upregulation has been supported, a shift in assembly to the high sensitivity (α4)2(β2)3 version exclusively in the endoplasmic reticulum has not previously been detected. In order to study organelle specific changes in stoichiometry, a novel method was developed to isolate single nAChRs in nanovesicles derived from native cell membranes. Separation of nanovesicles originating from the endoplasmic reticulum and plasma membrane, encompassing isolated nAChRs, allows precise changes in stoichiometry to be monitored in subcellular regions. In this work, single molecule bleaching steps of green fluorescent protein (GFP) encoded in each alpha subunit of the pentamer are detected. The number of bleaching steps, or transitions to a nonfluorescent state upon continuous excitation, corresponds to the number of GFP-labeled alpha subunits present. Therefore, the stoichiometry can be deduced by detection of two bleaching steps, as in (α4)2(β2)3, or three bleaching steps, seen in (α4)3(β2)2. Using this method on isolated nAChRs, a shift to assembly of high sensitivity (α4)2(β2)3 receptors is detected definitively within the endoplasmic reticulum. In addition, an increase in (α4)2(β2)3 receptors located on the plasma membrane is shown when nicotine is present. This work provides convincing evidence that nicotine acts intracellularly, within the endoplasmic reticulum, to alter stoichiometry of nAChRs.

des-Formylflustrabromine (dFBr), isolated from the marine organism Flustra foliacea, is the first selective, positive allosteric modulator (PAM) of α4β2 nicotinic acetylcholine receptors that potentiates the action of the neurotransmitter acetylcholine (ACh). Most agonists for this receptor population are not selective and can activate other nACh receptors. A selective PAM, which activates α4β2 nACh receptors only in the presence of ACh, might find application in the treatment of of various neurological diseases such as Alzheimer’s disease or autism. dFBr was examined and found to produce a biphasic dose-response curve over a wide concentration range (i.e., potentiation at low concentration, but inhibition of the ACh-induced response at high concentrations). Our goal was to examine various structural features of dFBr required for potentiation; a secondary goal was to examine the same for inhibition. To understand the structural requirements of dFBr, a systematic ‘deconstruction reconstruction and elaboration’ approach (see p. 48) was employed to determine the contribution of various structural components of dFBr to its activity at α4β2 nACh receptors. Novel compounds were synthesized and characterized. Human α4β2 nACh receptors were expressed in Xenopus oocytes and the actions of dFBr and its analogs were measured using a two-electrode voltage clamp technique. Dose-response curves were obtained for the compounds in the absence and presence of 100 μM ACh. Structural features of dFBr optimal and/or required for PAM action at 42 nACh receptors were identified. A novel reconstructed analog with all the essential features for PAM action was synthesized and submitted for biological testing. Elaborated analogs of dFBr further helped in identification of various structural features important for PAM action and the inhibition of action of ACh. The ‘deconstruction reconstruction and elaboration’ approach (see p.48) identified important structural features of dFBr that modify its actions as a PAM or an antagonist (NAM? or channel blocker?) at α4β2 nACh receptors. This information should be useful for the subsequent design of novel analogs to evaluate their potential for the treatment of neurological disorders associated with ACh.

Predatory shallow-water tropical marine snails within the genus Conus are estimated to consist of up to 700 species. These carnivorous mollusks have devised efficient venom harpoon-like radular teeth that allow them to predominantly incapacitate polychaete annelids (vermivores), in some cases fish (piscivores), or other mollusks (molluscivores) as an envenomation survival strategy for feeding, defense, and competitor deterrence. The venom of each Conus species contains a distinctive assortment of over 50 diversified disulfide-rich conotoxins with varied pharmacological specificities that selectively inhibit the function of ion channels (Ca2+, Na+, K+) or nicotinic acetylcholine receptors (nAChRs) involved in the animal neurotransmission. Across the genus Conus, the conotoxins represent an extensive array of ion channel blockers each showing an exquisite selectivity to distinguish between channels / receptors and even particular their subtypes. Novel conotoxins detected in the molecular neurobiological approach, providing chemists and pharmacologists a vast library (>50,000 individual toxins) of conotoxins have been further screened for their abilities to modify the responses of tissues to pain stimuli as a first step in describing their potential as lead compounds for novel drugs. In this article, we present the natural history of the Conus biology as well as the nomenclature, classification, structure, neurotoxicological mechanisms, post-translational modification, and pharmaceutical applications of conotoxins. In addition, we also set up the bioinformatic database and search engine about hitherto-identified name and distribution of Conus species and neuropharmacological mechanism, accession number, sequence, and 3D structure of conotoxins and provide researchers advantageous tools for further investigation.

The striatum is the initial input nuclei of the basal ganglia, and it serves as an integral processing center for action selection and sensorimotor learning. Glutamatergic projections from the cortex and thalamus converge with dense dopaminergic axons from the midbrain to provide the primary inputs to the striatum. Striatal output is then relayed to downstream basal ganglia nuclei by GABAergic medium – sized spiny neurons, which comprise at least 95% of the population of neurons in the striatum. The remaining population of local circuit neurons is dedicated to regulating the activity of spiny projection neurons, and although spiny neurons form a weak lateral inhibitory network among themselves via local axon collaterals, feedforward modulation exerts more powerful control over spiny neuron excitability. Of the striatal interneurons, only one class is not GABAergic. These neurons are cholinergic and correspond to the tonically active neurons (TANs) recorded in vivo, which respond to specific environmental stimuli with a transient depression, or pause, of tonic firing. Striatal cholinergic interneurons account for less than 2 % of the striatal neuronal population, yet their axons form an extensive and complex network that permeates the entire striatum and significantly shapes striatal output by acting at numerous targets via varied receptor types. Indeed, the persistent level of ambient striatal acetylcholine as well as changes to that basal acetylcholine level underlie the major mechanisms of cholinergic signaling in the striatum, however regulation of this system by the local striatal microcircuitry is not well understood. This dissertation finds that activation of intrastriatal cholinergic fibers elicits polysynaptic GABAA inhibitory postsynaptic currents (IPSCs) in cholinergic interneurons recorded in brain slices. Excitation of striatal GABAergic neurons via nicotinic acetylcholine receptors (nAChRs) mediates this polysynaptic inhibition in a manner independent of dopamine. Moreover, activation of a single cholinergic interneuron is capable of eliciting polysynaptic GABAA IPSCs onto itself and nearby cholinergic interneurons. These findings provide an important insight into the striatal microcircuitry controlling cholinergic neuron excitability. / text

Οι υποδοχείς της ακετυλοχολίνης (AChRs) είναι διαμεμβρανικές πρωτεΐνες ενεργοποιούμενες με τη δέσμευση της ακετυλοχολίνης (ACh). Με κριτήρια, όπως η χημική συγγένεια που εμφανίζουν για σηματοδότικά μόρια και οι φαρμακολογικές τους ιδιότητες, ταξινομούνται στην ομάδα των νικοτινικών AChRs και στην ομάδα των μουσκαρινικών AChRs. Οι νικοτινικού τύπου υποδοχείς δημιουργούνται από τη συναρμογή πέντε ομόλογων υπομονάδων και υποδιαιρούνται σε μυϊκού τύπου, ευρισκόμενους κυρίως στους σκελετικούς μύες των σπονδυλωτών και σε νευρικού τύπου, απαντώμενους κατά κύριο λόγο στο κεντρικό και περιφερικό νευρικό σύστημα. Οι AChRs σχετίζονται με σειρά παθολογικών καταστάσεων, μεταξύ των οποίων και η βαρειά μυασθένεια (Myasthenia Gravis-MG). Η μυασθένεια χαρακτηρίζεται από χρόνια μυϊκή αδυναμία, προκαλούμενη από τη δράση αντισωμάτων υψηλής συγγένειας έναντι του μυϊκού τύπου AChR. Με απώτερο σκοπό τη διερεύνηση της παθογονικότητας των αυτοαντισωμάτων έναντι μεμονωμένων υπομονάδων του AChR, προχωρήσαμε στην παραγωγή ανασυνδυασμένων πολυπεπτιδικών τμημάτων των υπομονάδων στο ζυμομύκητα Pichia pastoris. Τα πολυπεπτίδια χρησιμοποιήθηκαν στην παρασκευή χρωματογραφικών-ανοσοπροσροφητικών στηλών, που εφαρμόστηκαν ακολούθως για την απομόνωση αυτοαντισωμάτων από επιλεγμένους ορούς μυασθενικών ατόμων. Η παθογόνος δράση των απομονωμένων αυτοαντισωμάτων ελέχθηκε μέσω της προκαλούμενης απώλειας υποδοχέων (αντιγονική τροποποίηση-antigenic modulation) σε κυτταρική σειρά (ΤΕ671) που εκφράζει τον AChR και μέσω της χορήγησή τους σε πειραματόζωα και τον έλεγχο της εμφάνισης χαρακτηριστικών συμπτωμάτων της νόσου. Εκτενής συγκριτική μελέτη μεταξύ τεσσάρων επιλεγμένων ορών και αντισωμάτων έναντι της α1 και της β υπομονάδας του υποδοχέα, που απομονώθηκαν από τους συγκεκριμένους ορούς, έδειξαν πως τα αυτοαντισώματα ευθύνονται για δράση των ορών στους υποδοχείς των κυττάρων. Τόσο οι ολικοί οροί όσο και τα απομονωμένα-καθαρά αυτοαντισώματα έναντι των υπομονάδων α1 και β, προκάλεσαν δοσοεξαρτώμενη απώλεια υποδοχέων στα κύτταρα και μάλιστα τα αντι-α1 αντισώματα εμφανίστηκαν περίπου τέσσερις φορές δραστικότερα από τα αντι-β. Η ικανότητα των μερικώς απαλλαγμένων από αυτοαντισώματα έναντι του υποδοχέα ορών να προκαλούν απώλεια υποδοχέων στα κύτταρα, φάνηκε να ποικίλλει και να συσχετίζεται άμεσα με το είδος των αντισωμάτων που έχουν παραμείνει στον ορό, υποστηρίζοντας μια διαφορετικότητα στην παθογονικότητα των επιμέρους αντισωμικών κλασμάτων. Με σκοπό την επιβεβαίωση και ενίσχυση των αποτελεσμάτων που προκύπτουν από τα in vitro πειράματα, ακολούθησαν προσπάθειες για την πρόκληση πειραματικής μυασθένειας σε πειραματόζωα, με τη χορήγηση ορών μυασθενικών και καθαρών αυτοαντισωμάτων έναντι διαφόρων υπομονάδων του υποδοχέα. Η χορήγηση σε ζώα τόσο του ολικού ορού, όσο και καθαρών αντισωμάτων έναντι της α1-υπομονάδας του υποδοχέα, προκάλεσαν σημαντική απώλεια βάρους και εμφάνιση έντονων συμπτωμάτων μυϊκής αδυναμίας, μέχρι και το θάνατο. Πειραματόζωα που ενέθηκαν με το κλάσμα του ορού από το οποίο έχουν απομακρυνθεί τα συγκεκριμένα αντισώματα εμφάνισαν πολύ ηπιότερα ή και καθόλου συμπτώματα, ενώ απουσία συμπτωμάτων καταγράφηκε και κατά τη χορήγηση ορού που περιείχε αποκλειστικά αντισώματα έναντι της β υπομονάδας, αλλά και απομονωμένων αντι-β αντισωμάτων. Η παρούσα μελέτη υπέδειξε τα αυτοαντισώματα έναντι του AChR ως τον μοναδικό παθογόνο παράγοντα στον ορό μυασθενικών ατόμων, συμβάλλοντας στην κατανόηση της παθοφυσιολογίας της νόσου. Επιβεβαίωσε την υπεροχή των αντι-α1 αντισωμάτων έναντι των αντι-β, ως πρός την παθογονικότητά τους, τόσο in vitro όσο και in vivo, με την επιφύλαξη βέβαια που επιβάλλει ο μικρός αριθμός δειγμάτων που μελετήθηκαν. Η δυνατότητα λήψης αντισωμάτων που στοχεύουν σε συγκεκριμένη υπομονάδα μπορεί να συμβάλλει στη λεπτομερή μελέτη της δραστικότητας του κλάσματος και να οδηγήσει στη συσχέτισή του με την εμφάνιση συγκεκριμένων συμπτωμάτων της νόσου. / Acetylcholine receptors (AChRs) are integral membrane proteins that respond to the binding of acetylcholine (ACh), which is synthesized, stored and finally released by cholinergic neurons. Like other transmembrane receptors, AChRs have been classified according to either their pharmacological properties or their relative affinities for various molecules, and can therefore be further divided into: i) nicotinic AChRs, which are particularly responsive to nicotine and ii) muscarinic AChRs, which are particularly responsive to muscarine. AChRs are involved in myasthenia gravis (MG) and many other physiological disorders, mainly affecting the central and peripheral nervous system. In MG, autoantibodies are directed against the nicotinic AChR at the neuromuscular junction. The disease is characterized by various symptoms, including muscle weakness and fatigability, due to defective neuromuscular transmission. To obtain an insight into the role of the various anti-AChR antibody specificities in MG, we isolated and studied the in vitro and in vivo activity of autoantibodies targeting individual AChR subunits. Using recombinant proteins corresponding to extracellular domains (ECDs) of individual AChR subunits as immunoadsorbents; we isolated autoantibodies which specifically bind to these subunits. We then used the well established TE671 human muscle cell line to examine the in vitro functions of subunit-specific autoantibody populations through their ability to induce nAChR antigenic modulation. Isolated subunit-specific autoantibodies were also used to determine their capacity to passively transfer experimental MG into lab animals. Our results clearly demonstrated that autoantibodies against the α1 or β subunit can cause AChR loss via antigenic modulation in a dose-dependent manner, the anti-α1 autoantibodies being much more effective than the anti-β autoantibodies. Furthermore, we showed that the autoantibody-depleted sera were much less effective, or were completely inactive, at causing AChR loss. In in vivo experiments, the administration of MG sera derivatives to lab animals showed that sera enriched in anti-α1 autoantibodies, as well as the corresponding pure anti-α1 autoantibodies from two individuals, are efficient in inducing MG like symptoms to the animals. A single serum contained almost 100% anti-β antibodies and the corresponding purified antibodies did not cause any clinical MG symptoms. The depleted fraction of MG sera tested, induced mild symptoms or no symptoms were observed, and this is in agreement with the in vitro results, strongly suggesting that the anti-AChR autoantibodies in MG sera and mainly the anti-α1 specificities are the sole pathogenic factor in anti-AChR antibody-seropositive MG.

Agrin and neuregulin are neurotrophic molecules well known for their roles at synapses in the peripheral and central nervous systems. The expression of these two molecules is not restricted to these sites however, as they are broadly expressed across multiple organ systems. What roles do agrin and neuregulin play within these alternate systems; what is the function of these molecules outside the nervous system? Here I investigate potential roles for agrin and neuregulin in inflammation. Inflammation is an immediate response by innate immune cells, primarily macrophages, to infection and is characterized by the synthesis of pro-inflammatory mediators. The innate immune system possesses multiple redundant mechanisms to locally control inflammation. The neuro-immune axis is one means of control. Often called the cholinergic anti-inflammatory pathway, it acts to regulate local inflammation via nerve-secreted acetylcholine signaling through the homopentameric α7 nicotinic acetylcholine receptors (α7nAChR) present on macrophages. Both agrin and neuregulin have been independently described to share an intricate relationship with acetylcholine receptors (AChR) in the nervous system. Agrin is best known for its role in AChR aggregation at the neuromuscular junction while neuregulin has related roles in AChR transcription, cell survival, communication and differentiation. Based on the common characteristics of synapses in the nervous and immune systems we were curious to see if agrin and neuregulin played analogous roles on macrophages. Here we show that agrin and its receptor dystroglycan are expressed on RAW264.7 macrophages. In addition, agrin treated macrophages demonstrate increased endogenous agrin and α7nAChR expression. By blocking α-dystroglycan (α-DG), a receptor for agrin, with an anti-α-DG antibody we further saw a reduction in agrin expression. We also show that agrin is able to aggregate surface α7nAChRs and transmembrane agrin co-localizes with α7nAChRs therein. Agrin appears to induce approximately a 15-fold increase in anti-inflammatory cytokine IL-10 in macrophages but does not increase pro-inflammatory cytokine TNF-α or IL-6 synthesis. Agrin-treated macrophages challenged with LPS, a potent activator of inflammation, exhibit a 57% decrease in IL-6. Macrophages treated with agrin also exhibit a 4-fold increase in STAT3, a regulator of anti-inflammatory action. The potential anti-inflammatory effects of agrin in the periphery parallel previous work describing the effects of neuregulin in the brain. Previous work completed by our lab suggests a role for neuregulin in augmenting the expression of α7nAChRs on microglia, the macrophages of the brain, but not in peripheral macrophages. Here we show that treatment of LPS challenged microglia with neuregulin produces an 88% decrease in IL-6 and a 33% decrease in TNF-α. These results indicate both agrin and neuregulin are able to induce an increase in α7nAChRs and augment the synthesis of pro- and anti-inflammatory cytokines in their respective systems. These results also further the support the evidence of neuro-immune crosstalk in the immune system. Taken together these results present two novel players in inflammatory regulation by macrophages in the periphery and CNS.

The muscarinic acetylcholine type 2 receptor (M2 mAChR), a member of the GPCR superfamily, is found throughout the parasympathetic nervous system where it controls pulmonary, urinary, and cardiac function, and neurotransmission. The molecular mechanisms that regulate M2 mAChR availability at the cell surface are an important component in controlling these physiological events. Since beta-arrestin proteins are known to regulate the activity of other GPCRs, we sought to identify their role in regulating M2 mAChR activity, a topic that remains contentious in the field. To achieve this goal we utilized mouse embryonic fibroblasts (MEFs) derived from beta-arrestin knockout mice lacking one or both isoforms (MEF KO1, KO2, or KO1/2 cells) in addition to exogenous expression of beta-arrestin mutants. This study demonstrates that agonist-induced internalization of M2 mAChR is beta-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with beta-arrestin in early endosomal vesicles suggesting it behaves as a class B receptor. Next, we sought to identify beta-arrestin s function in regulating the post-endocytic trafficking (down-regulation) of the M2 mAChR. MEF KO1/2 cells were unable to down-regulate M2 mAChRs whereas MEF KO1 or KO2 cells retained the ability to do so. In MEFwt cells, both M2 mAChR and beta-arrestin exhibited basal ubiquitination that increased following agonist stimulation. Receptor degradation appeared to be regulated by the ubiquitination status of beta-arrestin 2, since expression of a chimeric â-arrestin 2 form fused to ubiquitin increased both constitutive and agonist-promoted down-regulation, whereas expression of a beta-arrestin 2 mutant lacking putative ubiquitination sites, beta-arrestin 2K18R, K107R, K108R, K207R, K296R, significantly blocked degradation while internalization and stable association remained intact. Upon further analysis, the beta-arrestin 2K18R, K107R, K108R, K207R, K296R mutant blocked delivery of M2 mAChR to the late endosome/lysosome, presumably where degradation occurs. Inhibition of proteasome-dependent recycling of ubiquitin blocked receptor down-regulation without affecting internalization or the ubiquitination state of the M2 mAChR while ubiquitination of beta-arrestin 2 diminished significantly. These results support a role for ubiquitinated beta-arrestin in mediating M2 mAChR sorting and degradation in the lysosome. Collectively, these studies give us new insight on the function of beta-arrestin in regulating the activity of the M2 mAChR.

Οι νικοτινικοί υποδοχείς της ακετυλοχολίνης (nAChRs) είναι πενταμερή διαμεμβρανικά γλυκοπρωτεϊνικά μόρια τα οποία ανήκουν στην υπερ-οικογένεια των συνδεόμενων με προσδέτη ιοντικών καναλιών και ανάλογως με τη θέση τους στα σπονδυλωτά διακρίνονται σε νευρικού τύπου και μυϊκού τύπου. Ο μυϊκός nAChR συναντάται στη νευρομυΪκή σύναψη και έχει στοιχειομετρία (α1)2β1γδ ή (α1)2β1εδ. Στους νευρικού τύπου nAChRs, μεταξύ άλλων ανήκει και ο α4β2 υποδοχέας ο οποίος συναντάται σε υψηλά επίπεδα στον εγκέφαλο του ανθρώπου και εμφανίζεται με τη στοιχειομετρία (α4)2(β2)3 ή (α4)3(β2)2. Αποτελέσματα μελετών έχουν δείξει ότι ο υποδοχέας αυτός εμπλέκεται σε νευροεκφυλιστικές νόσους – Alzheimer, Parkinson, σχιζοφρένεια – καθώς και στον εθισμό στο κάπνισμα. Για το λόγο αυτό ο α4β2 υποδοχέας αποτελεί σημαντικό στόχο για το σχεδιασμό φαρμάκων και συνεπώς οι πληροφορίες που αφορούν τη δομή του και κυρίως το εξωκυτταρικό τμήμα του (ECD) – όπου συναντώνται οι θέσεις πρόσδεσης των προσδετών – είναι σημαντικές. Στο εργαστήριό μας έχει κατασκευαστεί και εκφράζεται στο ζυμομύκητα Pichia pastoris ένα συγκαταμερές το οποίο αποτελείται από τα ECDs των υπομονάδων β2 και α4 συνδεδεμένα σε σειρά μέσω ενός πεπτιδίου 24 αμινοξικών καταλοίπων (β2-α4). Η υψηλή υδροφιλικότητα και οι καλές ιδιότητες πρόσδεσης συνδετών αποτελούν σπουδαία πλεονεκτήματα που καθιστούν το συγκαταμερές αυτό σημαντικό μόριο για προσπάθειες κρυσταλλογραφικής ανάλυσης. Στηριζόμενοι σε αποτελέσματα μελετών που έχουν δείξει ότι μόρια που δεν κρυσταλλώνονται εύκολα μόνα τους, μπορούν να κρυσταλλωθούν ευκολότερα αν συνδεθούν με άλλα πρωτεϊνικά μόρια, έγινε η παραγωγή μονοκλωνικών αντισωμάτων (mAbs) έναντι του β2α4 ώστε τμήματα των mAbs που θα προκύψουν από πέψη αυτών με παπαΐνη (Fab τμήματα) να συγκρυσταλλωθούν μελλοντικά με το β2-α4. Στο πρώτο μέρος της εργασίας πραγματοποιήθηκε η παραγωγή mAbs έναντι του β2-α4. Για το σκοπό αυτό χρησιμοποιήθηκε η τεχνική της κυτταρικής σύντηξης μυελωματικών κυττάρων και σπληνικών κυττάρων αρουραίου ανοσοποιημένου έναντι του β2-α4. Αποτέλεσμα της μεθόδου αυτής είναι η παραγωγή υβριδωμάτων καθένα από τα οποία εκκρίνει ένα συγκεκριμένο mAb. Στη συνέχεια αυτής της διαδικασίας έγινε η επιλογή έξι υβριδώματων από τα οποία εκκρίνονταν αντίστοιχα έξι mAbs (mAbNR1-mAbNR6) με διαφορετικές ικανότητες πρόσδεσης. Πέντε από τα έξι mAbs αποδείχθηκε ότι προσδένουν είτε στη β2 είτε στην α4 υπομονάδα ενώ ένα από αυτά (mAbNR6) φαίνεται να προσδένει στη διεπιφάνεια των δύο υπομονάδων. Τα αντισώματα mAbNR2 και mAbNR3 παρουσιάζουν υψηλή ικανότητα πρόσδεσης αυστηρά για στην β2 και α4 υπομονάδα αντίστοιχα, ενώ τα υπόλοιπα αντισώματα πραγματοποιούν διασταυρούμενες αλληλεπιδράσεις και με άλλες υπομονάδες. Πειράματα με ολόκληρο τον ανθρώπινο υποδοχέα α4β2 έδειξαν ότι το mAbNR2 προσδένει και σε αυτόν, γεγονός που οδηγεί στο συμπέρασμα ότι το αντίσωμα αυτό θα μπορούσε να αποτελέσει χρήσιμο εργαλείο και για τον εντοπισμό του α4β2 υποδοχέα σε ανθρώπινο νευρικό ιστό. Στο δεύτερο μέρος της εργασίας πραγματοποιήθηκε απομόνωση και στη συνέχεια πέψη του mAbNR2 καθώς και άλλων δύο μονοκλωνικών αντισωμάτων του εργαστηρίου mAb73 (έναντι της β1 υπομονάδας του μυϊκού nAChR) και mAb198 (έναντι της α1 υπομονάδας του μυϊκού nAChR). Τα αντισώματα αυτά απομονώθηκαν από καλλιέργειες υβριδωμάτων και στη συνέχεια πραγματοποιήθηκε πέψη αυτών για τη δημιουργία Fab τμημάτων. Τα τμήματα Fab χρησιμοποιήθηκαν στη συνέχεια για τη δημιουργία συμπλόκων με τις αντίστοιχες υπομονάδες με σκοπό τη συγκρυστάλλωση. Τελικός σκοπός αυτής της διαδικασίας είναι η μελέτη της δομής των nAChRs και των υπομονάδων τους καθώς και η διευρεύνηση του τρόπου αλληλεπίδρασης αυτών με τα αντισώματα. / Nicotinic acetylcholine receptors (nAChRs) are pentameric transmembrane glycoproteins which belong to the super-family of ligand-gated ion channels. Depending on the location of the nAChRs they are categorized into two groups: muscle type and neuronal type. The muscle type nAChR is present in the neuromuscular junction with the stoichiometry (α1)2β1γδ or (α1)2β1εδ. The α4β2 receptor subtype belongs to the neuronal group, it is abundant in the human brain and its stoichiometry is (α4)2(β2)3 or (α4)3(β2)2. The α4β2 receptor is thought to be implicated in addiction to nicotine and in several neurological diseases including Alzheimer’s and Parkinson’s. For this reason this subtype is an attractive target for drug design and information concerning its extracellular domain (ECD) structure – where the ligand binding site is located – is invaluable. In our laboratory, the yeast Pichia pastoris expression system has been used for the expression of linked ECDs of α4 and β2 nAChR subunits (concatamer β2-α4). We managed to produce a hydrophilic molecule with near-native pharmacological profile for structural studies. Since several published data indicate that crystals of a molecule can be easier obtained when it is co-crystallized with an interaction partner, we produced monoclonal antibodies (mAbs) against β2-α4. Following mAb digestion with papain enzyme the produced Fab fragments will be co-crystallized with β2-α4. In the first part, mAbs against β2-α4 were produced. Rats were immunized against this molecule and their spleen cells were fused with myeloma cells. The result of this process was the production of hybridomas which secreted specific mAbs. Six hybridomas were selected for production of mAbs. These six mAbs (mAbNR1-mAbNR2) had different binding properties. Five of them (mAbNR1-mAbNR5) were anti-β2 or anti-α4 and one (mAbNR6) seemed to bind at the interface of the two subunits. mAb-NR2 and mAb-NR3 were highly specific for β2 and α4 respectively, whereas the other four mAbs exhibited some cross-reactivity with other nAChR subunits. Also, mAbNR2 could be useful for the detection of α4β2 subtype in human neuronal tissue as it shows high specificity for the human wild type α4β2 receptors. The second part of this project involved mAb purification and digestion to Fab. mAbNR2 and two other antibodies that have been previously produced in our lab (mAb73 and mAb198) were used. mAb73 binds to the β1 subunit of the muscle nAChR and mAb198 binds to α1 subunit of neuronal nAChR. These mAbs were isolated from hybridoma cultures and then digested to Fab fragments. The Fabs were then used to obtain complexes with the corresponding subunits for co-crystallization trials. The final aim of this process is to investigate the structure of nAChRs and its subunits as well as their interaction with the corresponding mAbs.

-- / The nicotinic acetylcholine receptors (nAChRs) are transmembrane proteins, composed of five subunits and belong to the superfamily of ligand gated ion channels The nAChRs are distinguished according to their topological and pharmacological characteristics in muscle and nervous type. Both the muscle and the nervous type are involved in the execution of many physiological functions (eg, nerve impulse transmission) but respectively in the pathogenesis of many diseases (eg Myasthenia Gravis,Parkinson's,Alzheimer's).This makes imperative the need to design drugs that target specific to each type of receptor. A prerequisite for achieving this objective is to study the structure of the extracellular regions of the receptor, as it is known that the specific areas are recognised by the cholinergic ligands and the abnormal antibodies. The α7 subunit of the human nicotinic acetylcholine receptor, can be used as a model for this study as It is expressed as a homopentamer. Wanting therefore to avoid the large and hydrophobic transmembrane regions of the receptor that would hinder the achievement of the objective, we focused on the extracellular domain (ECD) of the receptor .So, according to the above, a recombinant form of the extracellular region of the receptor was constructed and expressed previously in our laboratory (Zouridakis et al., 2009). The recombinant protein was (α7-mut10-myc-His), expressed in soluble form, in sufficient concentration and showed about three times greater affinity for I125-a-bgtx compared to the wild type (α7-ΔCDwt). Furthermore, studies of dynamic light scattering and electron microscopy confirmed the formation of homopentamer molecules. Moreover, the deglycosylated form of the protein displayed all these enhanced features, allowing the entry of crystallization experiments with both the glycosylated and the deglycosylated form. In order to further improve the specific mutant, new recombinant forms of the extracellular region of the α7 subunit of the nAChR were constructed. The recombinant forms were expressed with different expression tags in their N-or C-terminal in order to improve the folding of the molecule. The FLAG-α7-mut10-myc-His was produced in greater quantity and Ηts deglycosylated form differs significantly, indicating probably a more homogeneous protein population. Also, analysis of the molecule bygel filtration showed the predominant formation of a homopentamer molecule and the absence of high molecular weight aggregates. This protein, has enhanced features compared to the α7-mut10-myc-His and thus can proceed to crystallization trials. The second part of the study refers to the construction concateremers of the α7ECD. Σwo peptide linkers varying in their length were used. The mutant which carried the smaller linker (AGS)8, showed greater solubility compared to the more extended one (AGS)11.