Nicotine exerts a therapeutic effect in ulcerative colitis (UC) but the mechanism underlying this effect, is not clear. However, this effect may imply that nicotine has some, as yet to be discovered, effect on the immune system. The aim of the work described in this thesis was to characterise the nicotinic acetylcholine receptors (nAChRs) on human peripheral blood lymphocytes in term of receptor subtype. To achieve this, a combination of radioligand binding assays, pharmacological and molecular biological techniques were used. The data obtained from the binding studies suggested that the presence of one binding site for (-)- nicotine on human peripheral blood lymphocytes with a Kd 15 ± 5.759 nM (1.5 ± 5.759 x 10-8 M) and Bmax 2253 ± 409 sites/cell. The competition studies showed that ligands competing with [3H]-(-)-nicotine were (-)-nicotine, epibatidine and α-bungarotoxin, while others ligands for nAChRs displaced radiolabelled nicotine in insignificant quantities. Thus, radioligand-binding experiments suggest that the binding site for nicotine on human peripheral blood lymphocytes is a nAChR containing α7 and possibly α4 or/and b2 containing nAChR subunits. No evidence was obtained to suggest the presence of a non-cholinergic nicotine receptor. Furthermore, considerable subject to subject variation in the specific binding of radiolabelled nicotine was observed. Because of this only tentative conclusions could be drawn from radioligand binding data. Polymerase chain reaction (RT-PCR) was then used to demonstrate mRNA for the subunits of nAChRs suggested by radioligand binding studies. Data obtained show that the human peripheral blood lymphocytes tested, expressed mRNAs for α4, α5, α7, β2 neuronal nAChRs subunits and β1 muscle nAChR subunit. Expression of the α5 mRNA subunit of nAChR was observed in the lymphocytes in each sample of lymphocytes tested. In contrast, the expression pattern of mRNAs for α4, α7, β1, and β2 mRNAs subunits of nAChRs, varied between individuals. Finally, Western blot analysis was used to confirm that mRNA expression resulted in the expression of protein for nAChR subunits in human peripheral lymphocytes using monoclonal antibodies against α4, α5, α7, and β2 nAChR subunits, which had been detected by RT-PCR. The results obtained from the Western blot analysis show that protein for α4, α5, and α7 nAChR subunits was expressed in most, but not all of the human peripheral blood lymphocyte samples tested and some of the bands obtained were faint. In contrast, protein for the β2 nAChR subunit was observed in a few samples tested and the bands were faint. From the results obtained in this study, it is possible to conclude that human peripheral blood lymphocytes may contain nAChRs with subunit compositions of α4β2, α4β2α5, and/or α7. However, further studies are necessary to show whether or not the single binding site for nicotine demonstrated by radioligand binding experiments is due to one or all of these nAChRs. Thus, the findings of the present study suggest the presence of nAChR on human peripheral blood lymphocytes. Nicotine and its effect may occur through these non- neuronal nAChRs mechanisms. Such a mechanism of action could account for the beneficial of nicotine in ulcerative colitis. Furthermore, a compound that acts on these receptors, but not on nAChRs found on other cells may have therapeutic utility in the treatment of inflammation.

α7 nicotinic acetylcholine receptors (nAChR) are highly permeable to Ca2+ and are clinical targets for Alzheimer’s disease and schizophrenia. The aim of this work was to examine α7 nAChR-mediated Ca2+ signalling in neuronal cells using three different methods, and to evaluate the effects of the desensitizing agonist and prototypical smoking-cessation drug sazetidine-A on α7 nAChRs. Initial studies used 96-well plate assays with SH-SY5Y cells to characterize responses evoked by the α7 nAChR-selective agonist PNU-282987 and positive allosteric modulator PNU-120596. This was complemented by live-imaging of cortical cultures, where the compounds evoked robust Ca2+ responses from 12 % of cells. Co- application with Cd2+, ryanodine and xestospongin-C significantly inhibited these responses, suggesting the involvement of voltage-gated Ca2+ channels and Ca2+- induced Ca2+-release. CNQX and MK801 also significantly inhibited α7 nAChR mediated Ca2+ elevations, indicating a role for glutamate release. A high-content screening assay was developed to further examine these phenomena. Exploratory experiments using KCl, AMPA and NMDA validated a protocol that could be used to image Ca2+ elevations in large cell populations. Inconsistent responses to PNU-120596 and PNU2-282987 were also observed, reflecting the scarcity of α7 nAChRs in cortical cultures and the need for assay optimization. Combination with immunofluorescent labelling revealed α7 nAChR mediated Ca2+ elevations in a subpopulation of astrocytes and neurons, some of which were GABAergic. PNU-120596 potentiated the effects of sazetidine-A in SH-SY5Y cells (EC50 0.4 μM) eliciting responses in 14 % of cells in cortical cultures in a methyllycaconitine- sensitive manner, consistent with α7 nAChR activation. Pre-incubation with sazetidine-A concentration-dependently attenuated subsequent α7 nAChR-mediated responses in SH-SY5Y cells (IC50 476 nM) and cortical cultures, suggesting that α7 nAChRs could play a role in the behavioural effects of sazetidine-A. These comparative experiments enhance our understanding of α7 nAChR signalling and provide a new method to study them further.

Alzheimer's disease (AD) is a neurodegenerative disorder that manifests in the form of deficiencies in cognitive processes such as memory and learning. The pathological features of AD include hyperphosphorylated tau proteins that form neurofibrillary tangles as well as senile plaques composed primarily of the peptide β-amyloid (Aβ). When present in high concentrations in the brain, Aβ inhibits certain subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) in the hippocampus. The effects of Aβ in the hippocampus have proven to be neurotoxic, resulting in reduced functionality of nAChRs and the subsequent death of neurons in the cholinergic pathway. The early stages of AD are characterized by reduction of nAChR density and by degeneration of the cholinergic neurons that provide input to the hippocampus. Because the hippocampus plays a critical role in memory formation and other cognitive processes, dysfunction in this brain region results in significant cognitive deficiencies. Understanding the interaction between Aβ and the structurally and functionally diverse nAChR subtypes and possible downstream effects in signaling cascades that might result from that interaction are important steps in comprehending AD pathogenesis. Comprehension of this interaction and factors that might influence it could lead to the development of pharmaceutical agents useful in the treatment of AD.

Introduction: Cholinergic network modulation is carried out through the neurotransmitter acetylcholine (ACh) and expression of α4β2 nicotinic acetylcholine recetors (nAChRs) in central brain regions responsible for the detection of external sensory stimuli through thalamic and basal forebrain circuits but also within mesolimbic reward signaling. Alterations in α4β2 availability could therefore contribute to pathologically increased eating behavior leading to obesity. Investigations of task-related cholinergic neurotransmission in vivo in human obesity comparing baseline versus stimulus conditions have yet to be established. Objective: Aim of this exploratory study was to investigate the neurobiological mechanisms of cholinergic signaling and its ramifications on eating behavior to possibly identify α4β2 as a pharmacological target in obesity therapy approaches. Primary outcome measure was the distribution volume calculated from PET data by VOI-based analyses. We compared α4β2 nAChR availability in OB (participants with obesity) with NW (normal weight participants) under baseline and stimulus conditions. Secondary, we explored whether changes in eating behavior measured by VAS (visual analogue scores) are correlated with changes in α4β2 nAChR availability. We also hypothesized that this relationship differs between resting state and stimulus conditions in both NW as well as OB. Materials and Methods: Study population consisted of 16 study participants with OB (N=16; mean BMI 37.8±3.18 kg/m2; 10 females; mean age 40.6±14.0; range from 20-62 years) and 14 NW (N=14; mean BMI 21.8±1.90 kg/m2; 11 females; mean age 28.1±7.58; range from 19-45 years), all mentally healthy and non-smokers. Every participant underwent simultaneous PET-MR imaging (mMR Siemens) under baseline and stimulus conditions, applying a standard set of salient food items. Calculations of VT was based on the bolus-infusion protocol. This includes investigation of VT as the ratio between mean (-)-[18F]flubatine in brain tissue and mean plasma (-)-[18F]flubatine in venous blood samples at 120 until 165 minutes post injection. During each visit VAS data were obtained. Results: No significant group differences in VT between NW and OB under baseline conditions were found, while OB showed a trend towards lower VT in the Nucleus basalis of Meynert (NBM; NW: mean VT= 11.6; OB: mean VT=10.2; mean difference= 1.35; p= 0.119). Under stimulus conditions, OB demonstrate higher thalamic VT (Thalamus; NW: mean VT= 25.1; OB: mean VT= 28.8; mean difference: -3.63; p= 0.028). Additionally, OB showed a tendency to greater VT mean differences between resting state and stimulus conditions compared with NW. Correlational analyses revealed statistically significant positive correlation (r= 0.61) between HPT and VAS “satiety” in NW and a significant negative correlation (r= -0.59) between NAc and VAS “disinhibition” in OB. Conclusion: These first in-human data suggest substantial changes in cholinergic signaling in brain circuits that process external sensory stimuli with high-incentive properties such as visual food cues in obesity. If confirmed in an extended population with larger sample size and including seed-based fMR imaging investigations, the α4β2 nAChR represent a promising target for pharmacological intervention as a non-invasive alternative to surgical procedures to combat the obesity epidemic.:2. Table of Contents 2. TABLE OF CONTENTS 2 3. ABBREVIATIONS 2 LIST OF FIGURES 4 LIST OF TABLES 6 4. INTRODUCTION 6 4.1 OBESITY AND THE CENTRAL CHOLINERGIC SYSTEM 7 4.2 CHOLINERGIC NEUROTRANSMISSION 9 4.3 STRUCTURE AND RECEPTOR KINETICS OF NACHR 11 4.4 TOPOGRAPHY OF CENTRAL NACHR 13 4.5 CHOLINERGIC NEUROMODULATION 15 4.5.1 Cholinergic Neuromodulation and Cognitive Processes 16 4.5.2 Cholinergic Neuromodulation and Reward 19 4.5.3 Cholinergic Neuromodulation and Eating Behavior 21 4.6. POSITRON EMISSION TOMOGRAPHY (PET) AS A MOLECULAR IMAGING TECHNIQUE FOR MEASURING NACHR IN VIVO 23 4.6.1 PET Imaging 23 4.6.2 Imaging of α4β2 Nicotinic Acetylcholine Receptors 23 4.6.3 (-)-[18F]flubatine: a specific α4β2 nAChR radiotracer 25 4.7 VOLUMES OF INTEREST (VOI) 33 5. OBJECTIVE 34 6. MATERIALS AND METHODS 35 6.1 ETHICS STATEMENT 35 6.2 STUDY DESIGN 35 6.3 STUDY PARTICIPANTS 36 6.4 VISUAL ANALOGUE SCALE (VAS) 38 6.5 PET/MR IMAGING 39 6.6 IMAGING DATA AND BLOOD PLASMA ANALYSIS 43 6.7 STATISTICAL ANALYSIS 45 7. RESULTS 46 7.1 EPIDEMIOLOGICAL DATA 46 7.2 BASELINE AND STIMULUS VT CALCULATIONS 47 7.3 INTRA-INDIVIDUAL VT ASSESSMENT BETWEEN BASELINE AND STIMULUS CONDITIONS 53 7.4 CORRELATIONAL ANALYSES OF VAS VERSUS VT 57 8. DISCUSSION 63 9. SUMMARY 70 10. REFERENCES 72 11. ANLAGEN 81

α-conotoxins are 13–19 amino acid toxin peptides that bind various nicotinic acetylcholine receptor (nAChR) subtypes. α-conotoxin Mr1.7c (MrIC) is a 17 amino acid peptide that targets α7 nAChR. Although MrIC has no activating effect on α7 nAChR when applied by itself, it evokes a large response when co-applied with the type II positive allosteric modulator PNU-120596, which potentiates the α7 nAChR response by recovering it from a desensitized state. A lack of standalone activity, despite activation upon co-application with a positive allosteric modulator, was previously observed for molecules that bind to an extracellular domain allosteric activation (AA) site at the vestibule of the receptor. We hypothesized that MrIC may activate α7 nAChR allosterically through this site. We ran voltage-clamp electrophysiology experiments and in silico peptide docking calculations in order to gather evidence in support of α7 nAChR activation by MrIC through the AA site. The experiments with the wild-type α7 nAChR supported an allosteric mode of action, which was confirmed by the significantly increased MrIC + PNU-120596 responses of three α7 nAChR AA site mutants that were designed in silico to improve MrIC binding. Overall, our results shed light on the allosteric activation of α7 nAChR by MrIC and suggest the involvement of the AA site.