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.

The peripheral and highly lipid-exposed M4 α-helix, although distant from the agonist binding site, channel gate, and other important gating structures, is involved in modulating function of the nicotinic acetylcholine receptor. M4 "senses" changes in the surrounding lipid environment and may consequently affect receptor function by altering specific interactions between the M4 C-terminus and the Cys-loop. An example of this lipid sensing ability is demonstrated by a lipid-facing Cys418 to Trp substitution on αM4 (αM4 C418W) of the muscle-type receptor, which subtly alters protein-lipid interactions and potentiates channel function 16-fold, leading to a slow-channel congenital myasthenic syndrome. Through the use of mutational studies and mutant cycle analysis, I determine that, contrary to previous studies, M4–Cys-loop interactions are not critical to wild-type muscle-type receptor function, nor are they involved in C418W-induced potentiation. Instead, C418W potentiates channel activity by enhancing local M4-M1 interactions mediated by three polar side-chains, which are absolutely critical to potentiation. I show that altered M4-M1 interactions are ultimately translated to two important gating structures, which work in tandem to stabilize the open conformation of the receptor. These studies highlight how altered protein-lipid interactions can affect channel function and contribute to our understanding of the underlying gating mechanism of the muscle-type receptor.

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.

Numerous studies suggest that modulation of the cholinergic system through the use of nicotinic agonists can improve motor function in humans or animals with motor disorders. Specifically, although there are no approved therapeutics for patients with ataxia, the nicotinic receptor agonist varenicline has demonstrated efficacy to improve coordination and gait in several groups of patients with different subtypes of ataxia. Importantly, the mechanism underlying the varenicline’s mechanism of action to improve motor function remains to be elucidated. Thus, the purpose of these experiments was to first quantify gait impairments in rats with olivocerebellar ataxia utilizing an objective treadmill-based system to investigate temporospatial aspects of animals’ gait. These results were used to calculate an index that characterizes deviations from ‘normal’ gait, as similarly employed in clinical studies. The translational validity of this method of gait assessment was investigated by comparing gait impairments between these animals and those reported for humans with ataxia. It was next investigated whether varenicline could attenuate any gait impairments and thus improve motor functioning in these animals, as suggested by clinical findings. Finally, varenicline’s mechanism of action was investigated by attempting to block its effects by pretreating animals with the nicotinic antagonist mecamylamine. Thus, these studies demonstrate the involvement of nicotinic acetylcholine receptors in the mechanism of varenicline’s effects to improve motor functioning. Moreover, these results provide translational methods by which the efficacy of other, more selective nicotinic agonists to improve motor functioning can be tested preclinically prior to their use in humans with ataxia.

Proper synaptic connectivity is critical for communication between cells and information processing in the brain. Neurons are highly interconnected, forming synapses with multiple partners, and these connections are often refined during the course of development. While decades of research have elucidated many molecular players that regulate these processes, understanding their specific roles can be difficult due to the large number of synapses and complex circuitry in the brain. In this thesis, I investigate mechanisms that establish neural circuits in the simple organism C. elegans, allowing us to address this important problem with single cell resolution in vivo. First, I investigate remodeling of excitatory synapses during development. I show that the immunoglobulin domain protein OIG-1 alters the timing of remodeling, demonstrating that OIG-1 stabilizes synapses in early development but is less critical for the formation of mature synapses. Second, I explore how presynaptic excitatory neurons instruct inhibitory synaptic connectivity. My work shows that disruption of cholinergic neurons alters the pattern of connectivity in partnering GABAergic neurons, and defines a time window during development in which cholinergic signaling appears critical. Lastly, I define novel postsynaptic specializations in GABAergic neurons that bear striking similarity to dendritic spines, and show that presynaptic nrx-1/neurexin is required for the development of spiny synapses. In contrast, cholinergic connectivity with their other postsynaptic partners, muscle cells, does not require nrx-1/neurexin. Thus, distinct molecular signals govern connectivity with these two cell types. Altogether, my findings identify fundamental principles governing synapse development in both the developing and mature nervous system.

The high rate of relapse often seen in breast cancer patients has been suggested to be the result of a small subset of chemotherapy-resistant cancer stem cells (CSCs), believed to be responsible for initiating tumor formation. These CSCs possess the capability to self-renew and give rise to a hierarchy of cells which makes up the bulk of a tumor. Neurotransmitters have been suggested to influence CSC self-renewal and proliferation capabilities, and antagonists of neurotransmission pathways have been implicated as possible treatment methods for chemo-resistant tumors. Using nicotinic acetylcholine receptor (nAChR) antagonists in sphere-forming assays, we have identified a very promising candidate compound: MG624. We found this compound to have a high selectivity for sphere-forming cells over non-sphere-forming cells in vitro, in a dose-dependent relationship, across a panel of cell lines as well as in patient-derived xenograft cells. This was validated in two ex vivo assays, where tumor formation was significantly delayed in mice injected with MG624-treated HCC1954 cells at both the IC50 and IC90 of the compound, indicating that MG624 does indeed target functional BTICs. MG624 was also found to synergize with both taxotere and doxorubicin chemotherapies in vitro, and shrink tumors in NOD/SCID mice when combined with taxotere in vivo. MG624 in combination with taxotere was found to induce apoptosis, and prevent cells from entering into the M-phase of the cell cycle. Interestingly, MG624 was found to eliminate intratumoral fibroblasts in combination with taxotere, despite taxotere being found to recruit fibroblasts to the tumor site when used on its own. Most importantly, the combination of MG624 and taxotere was found to significantly delay tumor progression/relapse in mice, indicating that MG624 may be an excellent candidate compound to one day be combined with chemotherapy to provide durable remission to breast cancer patients. / Thesis / Master of Science (MSc)

Myasthenia gravis (MG) is an autoimmune disease whose name means "grave muscular weakness". MG is a rare disease affecting only 200-400 persons per million and the characteristic symptoms include muscle weakness, particularly in highly active voluntary muscles. MG affects the neuromuscular junction in an antibody-mediated manner, resulting in impaired nerve-muscle cell communication in affected individuals. Specifically, two main proteins are targeted: nicotinic acetylcholine receptors (ACh receptors) and a muscle-specific tyrosine kinase (MuSK). Previous studies have discovered the mechanism of MG pathogenesis but the exact mechanisms which cause the failure to maintain self-tolerance have not been discovered. Based on current knowledge of MG, this paper will explore potential causes of the disease and provide numerous hypotheses directed at future research opportunities.