The current project examined the ability of a novel series of guandine and amidine-containing nicotine analogs to interact with several native and recombinantlyexpressed mammalian neuronal nicotinic-acetylcholine receptor (nAChR) subtypes. Rational drug design methods and parallel organic synthesis was used to generate a library of guanidine-containing nicotine (NIC) analogs (AH compounds). A smaller series of amidine-containing nicotine analogs (JC compounds) were also synthesized. In total, >150 compounds were examined. Compounds were first assayed for affinity in a high-throughput [3H]epibatidine radioligand-binding screen. Lead compounds were evaluated in subtype-selective binding experiments to probe for affinity at the α4β2* and α7* neuronal nAChRs. Several compounds were identified which possess affinity and selectivity for the α4β2* subtype [AH-132 (Ki=27nm) and JC-3-9 (Ki=11nM)]. Schild analysis of binding suggests a complex one-site binding interaction at the desensitized high-affinity nAChR. Whole-cell functional fluorescence (FLIPR) assays revealed mixed subtype pharmacology. AH-compounds were identified which act as activators and inhibitors at nAChR subtypes, while lead JC-compounds were found which possess full agonist activity at α4β2* and α3β4* subtypes. Compounds were identified as partial agonists, full agonists and inhibitors of multiple nAChR subtypes. Several SAR-based, ligand-receptor pharmacophore models were developed to guide future ligand design. Second-generation lead compounds were identified.

La Broncho-pneumopathie Chronique Obstructive (BPCO) est une maladie respiratoire grave caractérisée par une inflammation chronique entrainant des lésions irréversibles de l’épithélium respiratoire et du parenchyme pulmonaire. Le principal facteur de risque est le tabagisme mais des études d’association génétique pangénomiques ont montré que certains polymorphismes nucléotidiques simples (SNP) des récepteurs nicotiniques (nAChRs) sont associés à l’incidence de la BPCO. Un de ces polymorphisme est le variant rs16969968 dans le 5ème exon du gène CHRNA5 codant la sous-unité α5. Le but de ce travail a été d’évaluer in vivo l’implication du SNP α5 dans les lésions pulmonaires caractérisant la BPCO et d’étudier l’impact fonctionnel du polymorphisme sur les voies de signalisation mises en jeu en aval des nAChRs. A l’aide de différents modèles in vivo murins et humains, nous avons pu montrer qu’indépendamment du tabagisme, le SNPα5 est associé à une inflammation cellulaire plus marquée, une sécrétion de cytokines pro-inflammatoires, des lésions emphysémateuses, une hyperplasie des cellules mucipares et des cellules Club moins fréquentes par rapport au génotype sauvage. Le SNPα5 est associé à une altération de la perméabilité calcique des cellules épithéliales et une modulation de la voie de signalisation AC3-PKA/C. Cette étude apporte pour la première fois une explication biologique à l’association entre le SNPα5 et la BPCO décrite dans les études d’association génétique pangénomiques à travers un rôle pro-inflammatoire du SNPα5 au niveau pulmonaire. / Chronic Obstructive Pulmonary Disease (COPD) is a critical respiratory disease characterized by a chronic inflammation leading to irreversible epithelial and parenchymal injuries. The main risk factor is tobacco consumption but several genome-wide association studies (GWAS) described some single-associated polymorphisms (SNP) on nicotinic acetylcholine receptors (nACHR) genes associated with COPD incidence. One of these polymorphisms is the rs16969968 variant in the 5th exon of CHRNA5 gene coding the α5 subunit (SNP α5). The aim of this study was to determine in vivo the involvement of SNPα5 in COPD-associated lung injuries and to deciphere the functional impact of the polymorphism on nAChR signaling pathways. Thanks to several in vivo models (mouse and human), we describe here that the SNPα5 is associated, irrespective of the tobacco consumption, to an increased inflammation, pro-inflammatory cytokines secretion, emphysema, goblet cell hyperplasia, and Club cell diminution compared to the wild-type genotype. The SNPα5 is associated with a decreased calcium influx and a modulation of AC3-PKA/C pathway in airway epithelial cells. Our study describe for the first time a biological explanation for the association between SNPα5 and COPD shown in GWAS with a pro-inflammatory role of SNPα5 in the lung.

<p>Impulses coming from peripheries are modified in the spinal cord and transmitted to the brain. Several neurotransmitters have been involved in the processing of impulses in the spinal dorsal horn. Acetylcholine (ACh) is one of many neurotransmitters involved in the regulation of nociception in the spinal cord. In this study we investigated the role of nicotinic, muscarinic, serotonergic and GABA receptors in the regulation of spinal ACh release since these receptors are reported to be involved in spinal nociceptive processes.</p><p>Different receptor ligands were infused intraspinally via microdialysis and the spinal ACh release was measured by on-line HPLC. Receptor-ligand binding studies were performed with spinal cord homogenates as well as receptors expressed in cells.</p><p>In the first study, we found that nicotine and some of the nicotinic antagonists used increased ACh release suggesting that spinal ACh release is regulated by different nAChRs. Nicotine and nicotinic agonists may act on different types of receptors with different affinity to produce the observed net effect of increased ACh release. We propose the possibility of an involvement of three different nicotinic receptor subtypes in the regulation of spinal ACh release. </p><p>The effect of epibatidine, which is regarded as a nicotinic agonist, on muscarinic receptors was investigated in the second study. We propose that epibatidine, in μM concentrations, is a partial muscarinic receptor agonist that may interact with spinal muscarinic receptors to increase ACh release. The dual action on both nAChRs and mAChRs may explain the potent analgesic effect observed after intra-spinal epibatidine administration.</p><p>In the third study, we investigated the role of serotonin receptor involvement in ACh release control. The results suggest that only 5-HT_1A and 5-HT_2A receptors are involved in spinal ACh release. Considering current knowledge, the most probable location of 5-HT_2A receptors is on cholinergic neurones. On activation of the 5-HT_2A receptors the cellular excitability of cholinergic neurones is increased which results in an increasing ACh release. The 5-HT_1A receptors might be located on cell bodies of GABA neurones which inhibit the firing rate of the GABA neurones when activated by serotonin. </p><p>In the fourth study, we investigated the GABA receptor involvement in the regulation in spinal ACh release. We found that GABA_A receptors are tonically inhibiting spinal ACh release. The results further suggest that GABA_B receptors also are involved in the regulation of spinal ACh release. However, unlike GABA_A antagonists, GABA_B antagonists do not increase ACh release. This suggests that GABA_B receptors are not tonically regulating the spinal ACh release. </p>

Impulses coming from peripheries are modified in the spinal cord and transmitted to the brain. Several neurotransmitters have been involved in the processing of impulses in the spinal dorsal horn. Acetylcholine (ACh) is one of many neurotransmitters involved in the regulation of nociception in the spinal cord. In this study we investigated the role of nicotinic, muscarinic, serotonergic and GABA receptors in the regulation of spinal ACh release since these receptors are reported to be involved in spinal nociceptive processes. Different receptor ligands were infused intraspinally via microdialysis and the spinal ACh release was measured by on-line HPLC. Receptor-ligand binding studies were performed with spinal cord homogenates as well as receptors expressed in cells. In the first study, we found that nicotine and some of the nicotinic antagonists used increased ACh release suggesting that spinal ACh release is regulated by different nAChRs. Nicotine and nicotinic agonists may act on different types of receptors with different affinity to produce the observed net effect of increased ACh release. We propose the possibility of an involvement of three different nicotinic receptor subtypes in the regulation of spinal ACh release. The effect of epibatidine, which is regarded as a nicotinic agonist, on muscarinic receptors was investigated in the second study. We propose that epibatidine, in μM concentrations, is a partial muscarinic receptor agonist that may interact with spinal muscarinic receptors to increase ACh release. The dual action on both nAChRs and mAChRs may explain the potent analgesic effect observed after intra-spinal epibatidine administration. In the third study, we investigated the role of serotonin receptor involvement in ACh release control. The results suggest that only 5-HT1A and 5-HT2A receptors are involved in spinal ACh release. Considering current knowledge, the most probable location of 5-HT2A receptors is on cholinergic neurones. On activation of the 5-HT2A receptors the cellular excitability of cholinergic neurones is increased which results in an increasing ACh release. The 5-HT1A receptors might be located on cell bodies of GABA neurones which inhibit the firing rate of the GABA neurones when activated by serotonin. In the fourth study, we investigated the GABA receptor involvement in the regulation in spinal ACh release. We found that GABAA receptors are tonically inhibiting spinal ACh release. The results further suggest that GABAB receptors also are involved in the regulation of spinal ACh release. However, unlike GABAA antagonists, GABAB antagonists do not increase ACh release. This suggests that GABAB receptors are not tonically regulating the spinal ACh release.

Rett Syndrome (RTT) is a developmental disorder that affects numerous neuronal systems that underlie problems with breathing, movement, cognition and sleep. RTT is caused by mutations in the methyl-CpG-binding protein 2 (Mecp2) gene. MeCP2 is a ubiquitous protein that is found in all mature neurons and binds to methylated DNA to repress transcription; thus regulating protein expression levels in neurons. The mutations in Mecp2 affect a large number of proteins that are crucial for regulating neuronal activity. Despite the abnormal expression of many of these proteins, mice with a total loss of MeCP2 can live to adulthood and some people with RTT can live to a very late age as well. It is possible that mutations in the Mecp2 gene not only cause widespread defects, but also elicit neuroadaptive processes that may limit the impact of the MeCP2 dysfunction. To test this hypothesis we performed these studies in which we focused on how synaptic and membrane currents were altered to maintain normal neuronal activity in Mecp2-null mice. We show two examples from different neurons where neuroadaptations of ion channel expression allowed the neuron to remain viable. First, the properties of the nicotinic acetylcholine receptor (nAChR) current were altered in LC neurons in Mecp2-null mice. This was caused by changes in the nicotinic receptor subunit expression. Despite the changes in the nAChR current, the cholinergic modulation of LC neuron activity in WT and Mecp2-null mice were similar. Secondly, we show that the fast Na+ voltage-gated and the hyperpolarization-activated currents were altered in mesencephalic trigeminal V (Me5) propriosensory neurons. The changes in the hyperpolarization-activated current caused a smaller sag and post-inhibitory rebound. Opposite to what we expected, these cells were hyperexcitable. The hyperexcitability was due to changes in the fast Na+ voltage-gated current causing a decreased action potential threshold. Alterations in the ionic currents in Me5 neurons seem to be due to changes in subunit expression patterns. These results indicate that despite the complications caused by defects in the Mecp2 gene, neurons respond by rearranging receptor / ion channel expression. This reorganization allows neurons to remain viable despite the MeCP2 deficiency.

One of the etiological theories of schizophrenia is dysregulation of the immune system. Autoantibodies specific for the α7 subunit of the nicotinic receptor could potentially contribute to the pathophysiology of the disease. In this study, positive antibodies specific for the receptor were found to exist in 23% of the patients diagnosed with schizophrenia (n = 21). On the average, levels for the antibody were elevated in the schizophrenia patient population than in controls. The data also suggests that there is a significant correlation between antibody titer and age, lending support to the neurodegenerative nature of the disease.

No description available.

Yes / Cholinergic dysfunction has been shown to be central to the pathophysiology of Alzheimer’s disease and has also been postulated to contribute to cognitive dysfunction observed in various psychiatric disorders, including schizophrenia. Deficits are found across a number of cognitive domains and in spite of several attempts to develop new therapies, these remain an unmet clinical need. In the current study we investigated the efficacy of donepezil, risperidone and selective nicotinic α7 and α4β2 receptor agonists to reverse a delay-induced deficit in recognition memory. Adult female Hooded Lister rats received drug treatments and were tested in the novel object recognition (NOR) task following a 6 h inter-trial interval (ITI). In all treatment groups, there was no preference for the left or right identical objects in the acquisition trial. Risperidone failed to enhance recognition memory in this paradigm whereas donepezil was effective such that rats discriminated between the novel and familiar object in the retention trial following a 6 h ITI. Although a narrow dose range of PNU-282987 and RJR- 2403 was tested, only one dose of each increased recognition memory, the highest dose of PNU-282987 (10 mg/kg) and the lowest dose of RJR-2403 (0.1 mg/kg), indicative of enhanced cognitive performance. Interestingly, these compounds were also efficacious when administered either before the acquisition or the retention trial of the task, suggesting an important role for nicotinic receptor subtypes in the formation and retrieval of recognition memory. / This work was conducted at the University of Bradford and was funded by b-neuro. However all our recent studies mentioned in the discussion section have been conducted at the University of Manchester (UoM), and funded by b-neuro, Autifony, Innovate UK (formerly TSB) and UoM

Purpose: To establish a tool, termed as antimuscarinic activity (AMA), to predict the incidence of antimuscarinic adverse events (AMAEs).Methods: A literature review, focused on drugs having off-target interaction with muscarinic receptors, was performed. Prototypical drugs olanzapine, diphenhydramine, paroxetine were selected for the analysis. Scopolamine and darifenacin were included as positive and negative controls, respectively. Physiochemical properties, pharmacokinetic data, and clinical incidence of AMAEs for the selected drugs were collected from reported literature. Extrapolation of literature data was carried-out to obtain exposure data. To determine the drugs muscarinic affinity (Ki values), experiments were performed using 3H-QNB and membrane suspensions of M1, M2, and M3. Cmax, values were combined with Ki values to generate the relevant AMA. Validation of the AMA biomarker was carried-out against the reported AMAEs incidence. Results: With the exclusion of scopolamine and olanzapine for CNS and peripheral AMAEs, respectively, AMA ranking was related to the drugs AMAEs.

Stroke is the leading cause of long-term disability in the developed world and can have devastating effects on the health and everyday functioning of individuals. In most cases stroke is ischemic and is caused by the obstruction of blood flow due to a clot in the brain blood vessels. This initiates a cascade of events that result in tissue death and loss of behavioural function associated with the damaged region. The peri-infarct cortex is a region surrounding the infarct core that survives the ischemic event and is most susceptible to pharmacological treatments and rehabilitation. α4ß2 nicotinic acetylcholine receptor (nAChR) signalling has been implicated as a mechanism that affects cell survival and cell death in the acute response after stroke. Nicotinic receptor signalling is also involved in modulating brain excitability, which can affect neural plasticity and restoration of cortical circuits and lead to recovery of lost function after stroke. In order to elucidate the role of α4ß2 nAChRs on acute and chronic recovery after stroke, we tested two hypotheses: (1) blocking α4ß2 nAChRs triggers acute neuroprotection and (2) α4ß2 nAChRs play a role in regulating plasticity and long-term functional recovery. In the first set of experiments a new model of targeted photothrombotic stroke was induced in a distal branch of the middle cerebral artery (MCA) in awake and anaesthetized mice. Mice treated with the α4ß2 nAChR antagonist dihydro-ß-erythroidine (DHßE) showed smaller lesion sizes relative to vehicle controls and this effect was greater in mice that were awake during stroke induction. To determine the mechanism of α4ß2 nAChRmediated neuroprotection, changes in collateral flow were measured using Evans bluestained surface angiograms and laser Doppler flowmetry. Contrary to what was expected, DHßE did not appear to induce neuroprotection by altering collateral flow. In the second set of experiments, we first used confocal imaging to quantify and characterize the expression of α4ß2 nAChRs after stroke. Next, mice were induced with a targeted photothrombotic stroke in the forelimb somatosensory cortex. Mice were then chronically treated with DHßE to determine if α4ß2 nAChR antagonism could improve recovery of function. Behavioural tests showed that blocking α4ß2 nAChRs chronically had no effect on forelimb function after stroke. Voltage-sensitive dye imaging was used to measure forelimb-evoked responses in the somatosensory cortex and revealed no differences in cortical responsiveness between treated and non-treated groups. Altogether, these results show that changes in α4ß2 nAChR signalling that occur after stroke mediate ischemic cell death but do not have an effect on long-term recovery and plasticity. Moreover, they present a novel pathway for investigating stroke pathophysiology and the development of acute neuroprotective treatments. / Graduate / 0317 / aseto@uvic.ca