Prototoxin proteins have been identified as members of the Ly6/uPAR super family whose three-finger motif resembles that of α-bungarotoxin. Though they are known to modify the function of nAChRs, their specificity is still unclear. Our lab identified three prototoxin proteins in the chicken ciliary ganglion: Ch3ly, Ch5ly, and Ch6ly. Ch6ly was later identified as prostate stem cell antigen (PSCA), and specifically decreased the amount of calcium influx through the homomeric α7 nAChR subtype. I then identifiedCh3ly and Ch5ly as LY6E and LYPD6B, respectively. I focused my attention onLYPD6B because of its expression in the brain. This dissertation tests whether LYPD6Bis a prototoxin protein that specifically co-localizes with and modifies the function of the heteromeric α3β4* nAChRs (the other nAChR subtype expressed in the chicken ciliary ganglia). In the first part of my dissertation I performed intracellular two-electrode voltage clamp on Xenopus oocytes co-expressing human LYPD6B and different stoichiometries of the α3β4* nAChR, these included two (α3)2(β4)3 withβ4−α3−β4−β4−α3 and β4−α3−β4−α3−β4 stoichiometries, two (α3)3(β4)2 with stoichiometries β4−α3−α3−β4−α3 and β4−α3−β4−α3−α3, two (α3β4)2(α5D)β4−α3−α5D−β4−α3 and β4−α3−β4−α3−α5D, and (α3β4)2(α5N) with stoichiometries β4−α3−α5N−β4−α3 and β4−α3−β4−α3−α5N. Concatemeric constructs are designed to link nAChR subunits, thus when translated it is done so as a single polypeptide. LYPD6Bincreased the acetylcholine (ACh) potency and desensitization rate, but decreased the maximum current response (Imax) for the (α3)3(β4)2 nAChR subtype. Yet, LYPD6Bonly decreased the Imax for the (α3β4)2α5 D-variant and not the N-variant (associated with increase nicotine consumption). For the second part of my dissertation, I determined if the expression of LYPD6B correlated with nAChRs in an activity dependent manner. Though LYPD6B mRNA expression correlates with nAChR subunit mRNA expression levels, it seemed to be independent of nAChR activity. To determine if fluorescent colocalization occurs between LYPD6B and a specific nAChR subtype, I genetically engineered LYPD6B to express a human influenza hemagglutinin (HA) epitope tag and cloned into a chicken retrovirus. LYPD6B was shown to co-localize only with the α3β4*heteromeric and not the homomeric α7 nAChRs, in a nAChR activity dependent manner. This study adds to the complexity of a prototoxin’s function by suggesting that the specificity is dependent on nAChR type and stoichiometry. It is the first in identifying a prototoxin protein, LYPD6B, which specifically modulates the function of the(α3)3(β4)2 and (α3β4)2(α5 D-variant) heteromeric nAChR subtypes. For the (α3β4)2(α5D-variant) nAChR subtype LYPD6B decreased the Imax. Such observation may be telling of a novel mechanism involved with nicotine dependence. For the(α3)3(β4)2 nAChR subtype LYPD6B increases its ACh sensitivity, desensitization rate, while decreasing Imax. Additionally, the co-localization of LYPD6B and α3β4* nAChRsin the lack of nAChR activity highlights the relevance of the functional effects α3β4*nAChRs exhibit due to LYPD6B. Such relevance may be the utilization of limiting Ach amounts.

Nicotinic acetylcholine receptors (nAChRs) are a major class of ligand-gated ion channels in the brain, with the α7 subtype of nAChRs playing an important role in attention, working memory and synaptic plasticity. Alterations in expression of α7 nAChRs are observed in neurological disorders including schizophrenia and Alzheimer’s disease. Therefore, understanding the fundamentals of how α7 nAChRs are regulated will increase our comprehension of how α7 nAChRs influence neuronal excitability, cognition and the pathophysiology of various neurological disorders. The purpose of this thesis was to investigate how protein kinases modulate the function and trafficking of α7 nAChRs in CNS neurons. In chapter 2, I describe a novel fast agonist applicator that I developed to reliably elicit α7 nAChR currents in both brain slices and cultured cells. In chapter 3, I examined whether an immune protein in the brain, the T-cell receptor (TCR), can modulate α7 nAChR activity. Activation of TCRs decreased α7 nAChR whole-cell recorded currents from layer 1 prefrontal cortical (PFC) neurons. TCR attenuated α7 nAChR currents through the activation of Fyn and Lck tyrosine kinases, which targeted tyrosine 442 in the M3-M4 cytoplasmic loop of α7. The mechanisms of the attenuated α7 current were contributed by a TCR mediated decrease in surface receptor expression and an attenuation of the α7 single-channel conductance. TCR stimulation also resulted in a decrease in neuronal excitability by negatively modulating α7 activity. In chapter 4, I tested whether PKA can modulate α7 nAChR function in CNS neurons. The pharmacological agents PKA agonist 8-Br-cAMP and PKA inhibitor KT-5720, as well as over-expressing dominant negative PKA and the catalytic subunit of PKA, demonstrated that activation of PKA leads to a reduction of α7 nAChR currents in HEK 293T cells and layer 1 cortical interneurons. Serine 365 of the M3-M4 cytoplasmic domain of α7 was necessary for the PKA modulation of α7. The mechanism of down-regulation in α7 receptor function was due to decreased surface receptor expression but not alterations in single-channel conductance nor gating kinetics. The results of this thesis demonstrate that α7 nAChRs constitute a major substrate for modulation via TCR activated tyrosine kinases and the cyclic AMP/PKA pathway. / Graduate / kpragya2000504@gmail.com

Οι α7 και α9 υπομονάδες των νικοτινικών υποδοχέων ακετυλοχολίνης (nAChRs) είναι οι μόνες, ανάμεσα σε μια μεγάλη ποικιλία υπομονάδων του ανθρώπινου nAChR, που σχηματίζουν ομοπενταμερείς υποδοχείς. Για την θεραπεία διαφόρων νευρολογικών διαταραχών αλλά και άλλων ασθενειών, όπου εμπλέκονται οι α7 και α9 nAChRs, απαιτούνται φαρμακευτικές ουσίες που θα στοχεύουν ειδικά σε έναν υπότυπο των nAChRs. Για τον σχεδιασμό τέτοιων φαρμάκων είναι ουσιώδης η διαλεύκανση σε ατομικό επίπεδο της δομής του nAChR. Εντούτοις, η κρυστάλλωση ολόκληρων των διαμεμβρανικών υποδοχέων, αλλά ακόμη και η έκφραση και απομόνωσή τους, σε βαθμό που να επιτρέπει δομικές μελέτες, έχει αποδειχθεί δύσκολος στόχος. Η δυσκολία έγκειται κυρίως στην παρουσία υδρόφοβων διαμεμβρανικών περιοχών και στην μεγάλη ενδοκυττάρια περιοχή που θεωρείται ευκίνητη και ότι δεν έχει σταθερή διαμόρφωση. Στο πλαίσιο αυτό, στοχεύσαμε στην παραγωγή τμημάτων των α7 και α9 υπομονάδων του nAChR, που να είναι κατάλληλα για αναλυτικές δομικές μελέτες, σχεδιάζοντας κατασκευές για την έκφραση των εξωκυτταρικών περιοχών των δύο υπομονάδων ή και κολοβών διαμεμβρανικών μορφών της α7 υπομονάδας. Σε αυτές έχουν απαλειφθεί είτε τμήματα της μεγάλης ενδοκυτταρικής περιοχής, είτε ολόκληρη αυτή η περιοχή και μεγάλα τμήματα της διαμεμβρανικής περιοχής. Στο παρελθόν, είχε εκφραστεί η α7-ΕΚΠ στο ετερόλογο σύστημα έκφρασης Pichia pastoris και είχε οδηγήσει σε συσσωματώματα μεγάλου μοριακού βάρους, ενώ η έκφραση ενός μεταλλάγματος της α7-ΕΚΠ έδειξε σημαντική βελτίωση της υδροφιλικότητας του μορίου και σχηματισμό ολιγομερών κυρίως πενταμερών μορίων (Zouridakis et al. 2009). Σε αυτήν την εργασία, έχουμε επιτύχει να απομονώσουμε τα σχηματιζόμενα πενταμερή μόρια αυτού του μεταλλάγματος, εκμεταλλευόμενοι την ιδιότητα τους να εκλούονται σε μεγάλο εύρος συγκέντρωσης ιμιδαζολίου, κατά την χρωματογραφία συγγένειας. Ακόμη, η ενζυμική απογλυκοζυλίωση του μεταλλάγματος αυτού, βοήθησε στην περαιτέρω μείωση της ετερογένειας των απομονωμένων πενταμερών μορίων. Αν και ο αρχικός έλεγχος συνθηκών κρυστάλλωσης των γλυκοζυλιωμένων πενταμερών μορίων οδήγησε στον σχηματισμό μικροκρυστάλλων, δεν στάθηκε δυνατή η βελτιστοποίηση της ανάπτυξής τους. Η έκφραση της αγρίου τύπου εξωκυτταρικής περιοχής της α9 υπομονάδας του ανθρώπινου nAChR (α9wt) στο ετερόλογο σύστημα έκφρασης P. pastoris, οδήγησε στην παραγωγή κυρίως μονομερών μορίων που διαχωρίζονται εύκολα από τα σχηματιζόμενα ολιγομερή. Η μονομερής μορφή της α9wt έδειξε αξιοσημείωτη διαλυτότητα, σταθερότητα και ομοιογένεια καθώς και ικανότητα πρόσδεσης της α-μπουγκαροτοξίνης, έναν ειδικό ανταγωνιστή του μυικού και των ομοπενταμερών νευρικών nAChRs. Προκειμένου να υποβοηθηθεί η συναρμολόγηση των εκφραζόμενων α7 και α9 ΕΚΠ προς τον σχηματισμό πενταμερών μορίων, σχεδιάσαμε μεταλλάξεις που στηρίχθηκαν σε τρισδιάστατα μοντέλα ομολογίας (3D homology modelling) αυτών, χρησιμοποιώντας ως πρότυπο την κρυσταλλική δομή της ομόλογης, διαλυτής πρωτεΐνης δεσμεύσεως της ακετυλοχολίνης (AChBP) από το μαλάκιο Lymnaea stagnalis. Οι μεταλλαγές αυτές έγιναν είτε σε υδρόφοβα επιφανειακά αμινοξικά κατάλοιπα, με στόχο να αυξήσουμε την υδροφιλικότητα του μορίου, είτε σε κατάλοιπα που εντοπίζονται στις διεπιφάνειες μεταξύ δύο πρωτομερών, ώστε να ενισχύσουμε τις διαμοριακές αλληλεπιδράσεις και να υποβοηθήσουμε την συναρμολόγηση τους προς πενταμερή μόρια. Τα προκύπτοντα μεταλλάγματα έχουν ταύτιση αμινοξικής αλληλουχίας 70-95% με την αντίστοιχη του αγρίου τύπου και σε ορισμένες περιπτώσεις η έκφρασή τους στην P. pastoris οδήγησε στον σχηματισμό αλλά και την απομόνωση πενταμερών μορίων. Η σάρωση των μεταλλαγμάτων απέτυχε στην ανεύρεση κάποιας συνθήκης κρυστάλλωσής τους. Ωστόσο, ο αρχικός έλεγχος συνθηκών κρυστάλλωσης των μονομερών μορίων της α9wt είχε ως αποτέλεσμα τον προσδιορισμό διαφορετικών συνθηκών όπου σχηματίζονται πολλαπλοί κρύσταλλοι, τόσο για την γλυκοζυλιωμένη, όσο και την απογλυκοζυλιωμένη μορφή της. Επιπλέον, η βελτιστοποίηση αυτών των κρυστάλλων στην περίπτωση της γλυκοπρωτεΐνης, οδήγησε στο σχηματισμό μονοκρυστάλλων, που περιθλούν ακτίνες-Χ σε ικανοποιητική ανάλυση, καθιστώντας αυτούς τους κρυστάλλους ως υποσχόμενο υλικό για την επίλυση της δομής της άγριου τύπου α9 ΕΚΠ. Τέλος, η έκφραση στην κυτταρική σειρά εντόμων Sf9 με το σύστημα των βακιλοϊών της ολόκληρης α7 υπομονάδας του nAChR και των «κολοβών» διαμεμβρανικών μορφών της οδήγησε στην ορθή στόχευση των σχηματιζόμενων υποδοχέων στην κυτταροπλασματική μεμβράνη, ενώ οι φαρμακολογικές τους ιδιότητες προσεγγίζουν αυτές του φυσικού α7 υποδοχέα. Παρά το γεγονός ότι η έκφραση των κατασκευών αυτών είχε χαμηλή απόδοση και παρά τις δυσκολίες διαλυτοποίησης και απομόνωσής τους, ανάλυση με χρωματογραφία μοριακού αποκλεισμού, για τουλάχιστον δύο από τα διαμεμβρανικά μεταλλάγματα, δείχνει ότι οι διαλυτοποιημένες πρωτεΐνες έχουν σχηματίσει έναν πληθυσμό ολιγομερών της πρωτεΐνης ο οποίος πιθανότατα αντιστοιχεί σε πενταμερή μόρια. Τα παραπάνω, σε συνδυασμό με την απουσία της εύκαμπτης ενδοκυττάριας περιοχής, καθιστούν αυτά τα α7 μεταλλάγματα, εφόσον ξεπεραστούν οι δυσκολίες της απόδοσης της έκφρασης και της απομόνωσης τους, κατάλληλα για λειτουργικές και δομικές μελέτες. / The neuronal α7 and α9 subunits of the nicotinic acetylcholine receptor (nAChR) are the only amongst the known human nAChR subunits to form homopentamers, with five cholinergic ligand-binding sites. Elucidation of their crystal structure is essential in order to design highly specific drugs for treatment of several neurological diseases and disorders related to them and will serve as the prototype for understanding the structure of all other members of the ligandgated ion channel superfamily. Crystallisation of the intact receptors is a difficult task to fulfil, partially due to their hydrophobic transmembrane regions. Therefore, we aim at the expression of crystallisable human α7 and α9 extracellular domains (ECDs) or truncated α7 forms lacking either only their large and probably unordered intracellular domain or large parts of its transmembrane domain. Regarding the α7 ECD, expression of the wild type form in yeast Pichia pastoris led into formation of aggregates (Avramopoulou et al. 2004). Yet, a previously described mutant of this ECD (α7m10, Zouridakis et al. 2009) succeeded in the formation of oligomers, mostly corresponding to pentamers, due to improved solubility and subunit assembly of this mutant. In this study, we managed to isolate apparently pentameric assemblies of the various expressed oligomeric states, by optimizing its first-step purification procedure (metal affinity chromatography), using a narrow stepwise increase of imidazole concentrations. In order to further improve the protein homogeneity, we proceeded to the isolation of its deglycosylated pentameric form. The relatively low polydispersity of both the glycosylated and deglycosylated α7m10 ECDs, allowed for crystallization trials, which have resulted in microcrystallic formations. Further optimization of these microcrystals failed. As to the α9 ECD, expression of the wild type form in yeast Pichia pastoris led to the formation of both monomers and a variety of oligomers. The monomeric α9 ECD showed significant monodispersity, solubility and stability and exhibited binding ability of α- bungarotoxin, a specific nAChR antagonist. In order to facilitate the pentameric assembly and enhance the solubility of these α7 and α9 ECDs, we designed several mutants based on generated 3D homology models, using as template the crystal structure of the homologous soluble molluscan acetylcholine binding protein (AChBP). Several solvent accessible hydrophobic residues were replaced with more hydrophilic ones and some interface-located residues were mutated so as to facilitate the formation of additional inter-subunit interactions. The resulting mutants shared moderate and considerably high sequence identities (70-95%) with the wild type ECDs and in some cases, formation of pentamers was accomplished. Crystallisation screening for mutant ECDs failed in producing any hit. However, the pilot crystallisation trials of monomeric wild-type α9 ECD resulted the formation of plate-like multi crystals for both its glycosylated and deglycosylated forms. Further optimisation of these crystals succeeded in producing single crystals of the glycoprotein, to produce single crystals, which diffracted X-rays to satisfactory resolution, in a home source X-ray generator. Therefore, these crystals seem to be a promising material for solving the wild type α9 ECD structure. The intact and truncated α7 nAChRs under study were expressed in the Sf9/baculovirus system and showed surface receptor expression, while presenting near-native ligand-binding affinities for characteristic nAChR agonists and antagonists. Despite the low expression yield and solubilisation and purification difficulties, gel filtration analysis for at least two truncated mutants revealed the presence of a monodispersed oligomeric population, probably corresponding to pentamers. All these, taken together with the lack of the flexible large intracellular domain, render these α7 mutants, after overcoming the expression yield and purification difficulties, a suitable material for performing both functional and structural studies.

Nicotinic acetylcholine receptor (nAChR) activation is neuroprotective and nicotine is a cognitive enhancer. Loss of nAChRs, deposition of tau neurofibrillary tangles, cleavage of amyloid precursor protein (APP) and inflammation are well documented in the pathogenesis of Alzheimer’s disease (AD). Sequential cleavage of APP by β- and γ-secretase enzymes generates soluble Aβ peptides, with oligomeric forms of Aβ implicated in both the control of synaptic excitability and dysregulation of synaptic transmission and induction of neuronal death in AD. Aβ production is inhibited by calcium-dependent recruitment of α-secretase, as exemplified by activation of N-methyl-D-aspartate receptors (NMDAR). All neurodegenerative diseases are associated with inflammation, arising from altered homeostasis of the innate immune system, resulting in heightened activation of immune cells and induction of a pro-inflammatory environment. Stimulation of the α7 subtype of nAChR is anti-inflammatory and also enhances cognition and promotes neuronal survival. This work addressed the hypotheses that stimulation of highly calcium-permeable α7nAChR inhibits Aβ production by promoting α-secretase-mediated processing of APP and also modulates inflammatory cellular behaviour of microglia. Thus, this study assessed the role of α7nAChR at glutamatergic synapses, through probing effects on APP processing and phagocytosis in primary cortical neurons and microglia, respectively. Primary cortical neurons expressed functional α7nAChR and glutamate receptors, and through a number of experimental approaches, including immunoblotting and a cleavage reporter assay, results indicated α7nAChR activation with the α7nAChR-selective agonist PNU-282987 and positive allosteric modulator PNU-120596 had no effect on APP and Tau, in contrast to NMDAR activation that significantly modulated these proteins. Data suggest low expression of α7nAChR, coupled with distinct localisation of presynaptic α7nAChR and postsynaptic APP could explain the lack of effect. In addition, primary microglia were highly responsive to lipopolysaccharide and possessed functional α7nAChR that coupled to ERK phosphorylation. Microglial α7nAChR activation promoted neuroprotective phagocytic behaviour, in agreement with the ‘cholinergic anti-inflammatory pathway’. This study supports the hypothesis that α7nAChR are modulators of anti-inflammatory behaviour, thus α7nAChR-selective ligands are viable candidates for the treatment of AD and promoting cognitive enhancement.

Neuronal nicotinic acetylcholine receptors (nAChRs) are key players in both cognitive and autonomic processes. In the cognitive domains of the brain, destruction of cholinergic inputs or disruption of nAChR function result in cognitive deficits as observed in Alzheimer's disease, schizophrenia, brain trauma and aging. By contrast, moderate activation of nAChRs supports neuroprotection and improves cognitive functions. In addition, neuronal nAChRs are also expressed in important autonomic centers such as the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus (DMV) that support autonomic visceral reflexes and homeostasis. In this study, the underlying mechanisms of nAChR activation and its pharmacology were investigated in the hippocampus and the NTS, critical brain regions supporting cognitive and autonomic functions, respectively. Specific Aim 1 of this study was to determine the capacity of physiological levels of choline to activate α7 nAChRs in hippocampal CA1 pyramidal neurons and interneurons. A weak persistent activation of α7 nAChRs can be neuroprotective. These levels of activation can be achieved by selective or non-selective α7 nAChR agonists or inhibitors of ACh esterase (AChEI). However, nicotinic agonists desensitize α7 nAChRs while AChEI produces side effects limiting their overall clinical and pre-clinical effectiveness. These limitations can be avoided by using a novel class of drugs; type-II positive allosteric modulators of α7 nAChRs (α7-PAMs) such as PNU-120596 (i.e., PNU). At physiological levels, choline alone is ineffective as an α7 agonist because of its low concentration in the cerebrospinal fluid (~10 µM) and low potency for α7 activation (EC50~1.5 mM). However, the results pertaining to Specific Aim 1 demonstrate that in the presence of PNU (1-5 µM) , 10 µM choline produces persistent α7 activation expressed on CA1 pyramidal and interneurons which may be fine-tuned to achieve optimal neuroprotection and cognitive benefits. Specific Aim 2 was to test the novel concept that PNU mediated changes in α7 receptor kinetics can alter the biophysical properties of α7 channel-drug interactions and thereby increase the probability and the apparent affinity of open channel block. The results of this study suggest that the compounds (e.g., Bicuculline) that do not potently interact with α7 ion channels in the absence of PNU begin to interact potently in its presence. These emergent properties of α7 channel-drug interactions in the presence of PNU need to be recognized in drug development as they may lead to unanticipated side effects and serious misinterpretation of data. Specific Aim 3 investigated the pharmacology and mechanisms of action of pre-synaptic non-α7 and α7 nAChRs in the caudal NTS neurons. Although, activation of nAChRs is known to enhance pre-synaptic release of glutamate in subsets of caudal NTS neurons, its mechanism of action has been elusive. However, the results from this study demonstrated that nicotine-mediated enhancement of glutamate release requires Ca2+ influx via nAChRs but does not require any contribution from voltage-gated Ca2+ ion channels (VGCCs) and presynaptic Ca2+ stores. Moreover, both functional α7 and non-α7 nAChRs were found to contribute to the presynaptic effects of nicotine in subsets of NTS neurons. However, co-expression of α7 and non-α7 nAChRs on the same glutamatergic presynaptic terminals was not detected. Collectively, these studies may help in developing new therapeutic strategies to selectively target nAChR-associated pathways that support cognitive and autonomic functions in health and disease.

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.

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 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.