Les stratégies thérapeutiques mises en place dans la maladie de Parkinson sont symptomatiques et ne permettent pas de ralentir la progression de la maladie, nécessitant le développement de nouvelles approches neuroprotectrices. La neuroinflammation joue un rôle majeur dans le processus neurodégénératif où elle se manifeste précocement par l’activation de cellules gliales (microglie et astrocytes). En s’appuyant sur l’utilisation de modèles animaux mimant les stades précoces de la maladie, l’élaboration de stratégies thérapeutiques à visée anti-inflammatoire constitue donc une approche thérapeutique prometteuse. Ce travail de thèse a consisté à mettre au point et à caractériser un modèle de lésion partielle à la 6-hydroxydopamine chez le rat afin d’évaluer les effets d’une stratégie thérapeutique originale basée sur l’utilisation en combinaison d’un agoniste des récepteurs nicotiniques α7 et d’un agoniste des récepteurs σ1. En utilisant différentes approches expérimentales, nous avons tout d’abord évalué le processus neurodégénératif et la neuroinflammation dans le modèle que nous avons mis en place. Nos résultats ont montré une dégénérescence partielle et reproductible des neurones dopaminergiques nigro-striataux associée à une importante neuroinflammation. Nos analyses métabolomiques ont également révélé plusieurs altérations spécifiques, apportant ainsi de nouvelles informations sur les mécanismes intervenant dans le processus neurodégénératif. En s’appuyant sur l’utilisation de la tomographie par émission de positrons, nous avons ensuite évalué longitudinalement le profil d’expression des récepteurs nicotiniques α7 dans les structures clés de la voie nigrostriée. Nos résultats ont montré des modifications transitoires de la densité de ces récepteurs pouvant être liées à des réponses microgliales biphasiques en association avec la cinétique de la dégénérescence neuronale. Ainsi, ces résultats renforcent l’idée de cibler spécifiquement les récepteurs nicotiniques α7 dans l’atténuation des processus neuroinflammatoires. Nous avons enfin évalué les effets de notre stratégie thérapeutique dans le modèle et nos résultats ont permis de montrer que ce type de combinaison préserve partiellement l’intégrité des neurones dopaminergiques nigro-striataux et réduit les réactions gliales chez les animaux lésés. Bien qu’il sera nécessaire de confirmer et de compléter ces résultats avec d’autres analyses, ce type de combinaison pourrait constituer une nouvelle entité biochimique prometteuse dans le traitement de la maladie de Parkinson. / Currently, therapeutic strategies in Parkinson’s disease are symptomatic and the progression of the disease is uncontrolled, requiring the development of new neuroprotective approaches. Neuroinflammation plays a major role in the neurodegenerative process where it occurs early through the activation of glial cells (microglia and astrocytes). Based on the use of animal models mimicking the early stages of the disease, the development of anti-inflammatory strategies is therefore a promising therapeutic approach. This thesis work consisted in the development and the characterisation of a partial 6-hydroxydopamine lesion model in rats in order to evaluate the effects of an original therapeutic strategy based on the combined use of a α7 nicotinic receptors agonist and a σ1 receptors agonist. Using different experimental approaches, we first evaluated the neurodegenerative and neuroinflammation processes in the model that we developped. Our results showed a partial and reproductible degeneration of nigro-striatal dopaminergic neurons associated with a marked neuroinflammation. Our metabolic analyses have also revealed several specific alterations, providing new insight on the mechanisms involved in the neurodegenerative process. Using positron emission tomography imaging, we then evaluated longitudinally the expression profile of α7 nicotinic receptors in the key structures of the nigro-striatal pathway. Our results showed transient changes in the density of these receptors that may be linked to biphasic microglial responses in association with the kinetics of neuronal degeneration. Thus, these results reinforce the hypothesis of specifically targeting α7 nicotinic receptors in order to reduce the neuroinflammatory processes. Finally, we evaluated the effects of our therapeutic strategy in the model and our results showed that this type of combination partially preserves the integrity of nigro-striatal dopaminergic neurons and reduces glial reactions in lesioned animals. Although it is necessary to confirm and extend these results, this type of combination could represent a promising new pharmacological approach in the treatment of Parkinson’s disease.

Bien que la maladie d’Alzheimer (MA) soit la cause de démence la plus fréquente, lesmécanismes qui sous-tendent les déficits cognitifs chez les patients restent mal connus.Cependant, les peptides amyloïdes (Aβ) semblent être un acteur majeur impliqué dansl’apparition des troubles mnésiques au cours de l’évolution de la maladie, notamment de parleur capacité à induire un hypofonctionnement du système cholinergique associé au déclinmnésique. Sur la base de ces observations, le rôle joué par les récepteurs cholinergiquesnicotiniques α7 (α7-nAChRs) a été largement étudié, au vue de leur capacité à interagir avecles Aβ, sans toutefois dégager un consensus quant à l’implication de ces récepteurs dans lesdéficits mnésiques induits par les Aβ.Afin d’améliorer notre compréhension quant aux mécanismes sous-tendant les effetsdélétères induits par les Aβ dans les déficits mnésiques, notre travail visait à identifier le rôlejoué par les récepteurs α7-AChRs via une approche comportementale, pharmacologique etmoléculaire. Ainsi, nous avons utilisé un modèle « souris » basé sur des injections de formesoligomériques d’Aβ(1-42) (Aβo(1-42)) dans la région CA1 de l’hippocampe dorsal (dCA1),structure cérébrale impliquée dans les processus mnésiques, atteinte de manière précoce dansla MA et exprimant fortement les récepteurs α7-nAChRs.La première partie de cette étude a consisté à mettre au point et à valider notre modèleanimal d’étude des effets induits par les Aβo(1-42) dans le dCA1 par une approchecomportementale et moléculaire. Nous montrons que les injections répétées d’Aβo(1-42) dans ledCA1 induisent une perturbation spécifique de la mémoire de travail alors que la mémoirespatiale est préservée lorsque les performances mnésiques sont évaluées 7 jours après ladernière injection. Nous avons également montré que cette perturbation de la mémoire detravail est associée à une absence d’activation/phosphorylation de ERK1/2 au sein du réseauhippocampo-frontal et septo-hippocampique. Ces données nous ont permis de valider notremodèle expérimental permettant d’étudier spécifiquement l’impact des Aβo(1-42) dansl’hippocampe dorsal.Dans une seconde partie, nous nous sommes focalisés sur le rôle joué par lesrécepteurs α7-nAChRs dans les perturbations mnésiques induites par les Aβo(1-42). Nosrésultats montrent que (1) les souris KOα7 ne présentent pas de déficits de mémoire de travailconsécutivement aux injections intra-dCA1 d’Aβo(1-42), (2) les déficits mnésiques ainsi que lala perturbation de l’activation de ERK1/2 induits par les Aβo(1-42) sont compensés par destraitements pharmacologiques agoniste partiel et antagoniste des récepteurs α7-nAChRs, (3)le traitement par un agoniste complet des récepteurs α7-nAChRs ne permet pas de prévenir lesdéficits mnésiques. Au regard de ces résultats, le récepteur α7-nAChRs semble être essentielau développement des déficits mnésiques induits par les Aβo(1-42), et l’utilisationd’antagonistes de ces récepteurs pourraient être une cible potentielle pour le développementde nouvelles stratégies thérapeutiques. / Although Alzheimer’s disease (AD) has been considered as one of the major causesfor dementia, the mechanisms by which cognitive decline appear still remain unclear.However, amyloid-β peptides (Aβ) seem to play a central role in the appearance of memoryimpairments in the time course of the disease, inducing down-regulation of the cholinergicsystem which is associated with cognitive decline. Based on these observations, the role of α7nicotinic receptors (α7-nAChRs) which can interact with Aβ was widely studied withoutconsensus about the involvement of these receptors in memory deficits induced by Aβ.In order to improve our knowledge about the mechanisms involved in Aβ side effects,our work aims at identify the role of α7-nAChRs via behavioral and molecular approaches.Thus, we used a mice model based on injections of oligomeric assemblies of Aβo(1-42) (Aβo(1-42)) in the CA1 field of the dorsal hippocampus (dCA1) which is a brain structure stronglyinvolved in memory processes, precociously affected in the AD and with a high density of α7-nAChRs.The first part of this study was to develop and validate this animal model to studythe effects induced by Aβo(1-42) in the dCA1 by behavioral and molecular approaches. Weshow that repeated injections of Aβo(1-42) in the dCA1 induce a specific disruption of workingmemory 7 days after the last injection whereas spatial memory is spared. We also showed thatworking memory disturbance is associated with decreased activation / phosphorylation ofERK1 / 2 in the hippocampo-frontal and septo-hippocampal networks. These data allowed usto validate our experimental model to specifically study the impact of Aβo(1-42) into the dorsalhippocampus.In the second part, we focused on the role played by the α7- nAChRs receptors inmemory disturbances induced by Aβo(1-42). Our results show that (1) KOα7 mice do notexhibit working memory deficits consecutively to intra-dCA1 Aβo(1-42) injections, (2) thememory deficits and decreasing activation of ERK1/2 induced by Aβo(1-42) are offset bypharmacological treatments partial agonist and antagonist of α7-nAChRs receptors, (3)treatment with a full agonist of α7-nAChRs receptors does not prevent memory deficits .Given these results, the α7-nAChRs receptor appears to be essential to the development ofmemory deficits induced by Aβo(1-42), and the use of antagonists of these receptors might be apotential target for developing new therapeutic strategies for AD.

L’acétylcholine (ACh) est un important neurotransmetteur qui est produit dans le système nerveux. Cependant, cette molécule est aussi produite par d’autres cellules non-neuronales du corps humain. Cette dernière est produite en abondance par les lymphocytes T CD4+, qui sont la cible principale du virus de l’immunodéficience humaine (VIH). ACh exerce ses effets sur les cellules par l’intermédiaire de ses récepteurs nicotiniques (n) et muscariniques (m) qui sont exprimés à la fois sur les cellules immunitaires et non immunitaires dans le corps. Il est bien connu que l’ACh a des effets anti inflammatoires sur les cellules immunitaires, et c’est le récepteur nicotinique qui est un joueur indispensable de cet effet. SLURP-1 (Secreted Ly6/uPAR-related Protein-1), est une autre molécule secrétée par les cellules T activées et d’autres cellules. Elle agit comme un ligand allostérique pour le récepteur α7, et module les effets de l'ACh sur les lymphocytes T. Il est peu connu comment ce système cholinergique extra-neuronal (ENCS) est régulé chez les individus infectés par le VIH. Nos résultats démontrent que le taux d'ACh et de SLURP-1 en circulation ne change pas significativement chez les sujets infectés par le VIH comparé aux témoins sains. Cependant, le niveau de ces médiateurs est plus élevé chez les sujets infectés à long termes non progresseur (LTNP), qui contrôlaient la réplication virale, depuis plus que sept ans, sans aucune thérapie. Il est tentant de spéculer que le niveau élevé de ces deux composantes de l'ENCS peut jouer un rôle dans leur capacité à contrôler la réplication du VIH. Les résultats de cette étude montrent que l’agoniste du récepteur α7 diminue, et que l’antagoniste de ce même récepteur augmente la réplication virale in vitro, dans les cellules activées par le phytohaemagglutinin (PHA). En outre, l'hémicholinium (HC-3), un composé qui inhibe la capacité des cellules à produire ACh en compétition avec leur absorption de choline, augmente la réplication virale. L'expression du récepteur α7 sur les lymphocytes T CD4 + provenant du sang périphérique, mais pas sur les monocytes, était significativement réduite (p <0,01) chez les individus infectés par le VIH, et elle n'a pas été entièrement restaurée par le traitement antirétrovirale (TAR). Tandis que l'expression du récepteur adrénergique β-2 a diminué significativement (p <0,01) sur les monocytes et les lymphocytes T CD4 + chez des individus infectés par le VIH. Ces cellules répondent à la norépinephrine via ce récepteur et l’ACh secrété. Dans l'ensemble, les résultats cette étude suggèrent que le VIH provoque une modulation significative des différentes composantes de l'ENCS chez les individus infectés par le virus. Ce système pourrait être manipulé pour réduire la réplication virale et l’inflammation chez ces patients. / Acetylcholine (ACh) is an important neurotransmitter produced in the nervous system. However, the molecule is also produced by non-neuronal cells in the body. CD4+ T cells, the main targets of HIV-1, produce it abundantly. ACh exerts its effects on cells via its nicotinic (n) and muscarinic (m) receptors that are expressed on both immune and non-immune cells in the body. ACh is well known to exert anti-inflammatory effects on immune cells. The main receptor that is indispensable for the anti-inflammatory effects of ACh is the α7 nicotinic receptor. Another molecule, secreted by activated T cells and by other cells is SLURP-1 (Secreted Ly6/uPAR-related Protein-1), which acts as an allosteric ligand for α7 and fine tunes the effects of ACh on T cells. Little is known as to how this extra-neuronal cholinergic system (ENCS) is regulated in HIV-infected individuals. Our results show that the circulating levels of ACh and SLURP-1 do not change significantly in HIV-infected individuals, as compared to the circulating levels in healthy controls. Interestingly, higher levels of these soluble mediators were detected in HIV-infected long-term non-progressors (LTNP) who control the viral replication for more than seven years without any chemotherapy. It is tempting to speculate that the increase in levels of these two soluble mediators of the ENCS present in HIV-infected LTNPs may play a role in their ability to control HIV replication. The results from this study show that an α7 agonist decreased HIV replication, whereas a receptor antagonist increased its replication in vitro in human PHA blasts. Furthermore, hemicholinium (HC-3), a compound that inhibits the ability of the cells to produce ACh, by competing with their uptake of choline, increases the viral replication. The expression of the α7 receptor on peripheral blood CD4+ T cells, but not on monocytes, was significantly reduced (p<0.01) in HIV-infected individuals, and it was not fully restored by antiretroviral therapy (ART). Interestingly, the expression of the β2 adrenergic receptor was decreased significantly (p<0.01) on both monocytes and CD4+ T cells in HIV-infected individuals. These cells respond to norepinephrine via this receptor and secrete ACh. Overall, the results of this study suggest that HIV causes significant modulation of different components of the ENCS in virus-infected individuals. This system could be manipulated to reduce viral replication and inflammation in these patients.

Neuroinflammation is a major driving force in the progression of neurodegenerative disorders. Nicotinic acetylcholine receptors, as well as cannabinoid CB2 receptors, have been shown to have strong anti-inflammatory properties when activated. These effects are shown, in vivo, to be a result of stimulation of α7 nAChRs and CB2 cannabinoid receptors. Microglia cells, an immune cell in the brain, are shown to express both of these receptor subtypes. The studies detailed herein, investigated the ability of two compounds, nicotine and Δ9-THC, in modulation of inflammatory processes. Stimulation of these receptors on microglia using nicotine and Δ9-THC blocked the activation of these cells, observed through reductions in pro-inflammatory cytokine production. Reductions in inflammation as well as pathology in the PSAPP mouse model of Alzheimer’s Disease were also observed following nicotine and Δ9-THC administration. These data raise the possibility that α7 nAChRs and CB2 cannabinoid receptors may prove to be viable and effective strategy for reducing neuroinflammation observed in neurodegenerative disease.

-- / 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.

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.

The exact mechanism and progression of Alzheimer's disease (AD) at present is not fully understood. In patients suffering from AD, damage to the hippocampal region and impairment of learning and memory is present. It is also known that a buildup of β-amyloid plaques occur in AD patients and that β-amyloid interacts with some subtypes of neuronal nicotinic acetylcholine receptors (neuronal nAChRs). These receptors are composed of five subunits. The most prevalent nAChR subunit composition through the brain as a whole is α7. Previous data produced from our lab suggests that α7 nAChRs are also one of the most prevalent subunits expressed by interneurons within the hippocampal region, a part of the brain known to be involved in memory and learning. It is hypothesized that one mechanism through which learning and memory becomes impaired in AD is through the interaction of β-amyloid with these nAChRs. It has previously been established that nanomolar amounts of β-amyloid inhibit the peak currents of α7 nAChRs. However, concentrations of β-amyloid in the picomolar range, in some studies show an activation of α7 nAChRs, while other studies no activation is seen. In this experiment we show that human α7 subunit nAChRs are not activated by β-amyloid42 at 1 pM- 30 nM concentrations. We also show that short, seven-second applications of β-amyloid interact with the α7 nAChRs to alter the kinetics of the channel, however, the exact mechanism and pattern by which it effects the channel is still unclear.

La supervivencia de los organismos superiores depende de que sus células se organicen y actúen de manera sincronizada para cumplir funciones específicas, para lo cual es fundamental la comunicación intercelular. Este proceso es básico para la vida de todas las células, pero es la razón de ser en las neuronas que están especializadas en recibir información, procesarla y comunicarla a otras células. En el sistema nervioso, la principal forma de comunicación se realiza a través de la sinapsis química, en la que una neurona libera un mensajero químico, el neurotransmisor, que es reconocido por un receptor presente en otra célula permitiéndole responder al mensaje. La acetilcolina es uno de los principales neurotransmisores utilizados por las neuronas, y sus receptores, tanto metabotrópicos como ionotrópicos, están expresados en muchos tipos celulares. Los receptores ionotrópicos de acetilcolina, llamados receptores nicotínicos, son canales catiónicos pentaméricos que pertenecen a la familia de receptores Cys-loop. El receptor nicotínico de acetilcolina α7 es un homopentámero que exhibe propiedades funcionales particulares fundamentales para su rol neuromodulador, incluyendo la elevada permeabilidad al Ca2+ y la capacidad para transformar respuestas ionotrópicas transitorias en eventos más sostenidos de señalización metabotrópica. Es uno de los receptores nicotínicos más abundantes en el sistema nervioso, aunque también se encuentra presente en otros tejidos. En el sistema nervioso central cumple un rol importante en procesos de cognición, atención y memoria, al regular la liberación de neurotransmisores, mediar la transmisión sináptica rápida y modular la excitabilidad neuronal. Una disminución de su actividad se ha asociado con diversos desórdenes neurológicos y neurodegenerativos, incluyendo esquizofrenia, autismo y enfermedad de Alzheimer. El receptor α7 también se expresa en células no neuronales, tales como -entre otras-, los astrocitos, la microglía, los linfocitos B y T, las células epiteliales, los macrófagos, cumpliendo un rol importante en inmunidad, inflamación y neuroprotección. Las acciones neuromoduladoras, neuroprotectoras y antinflamatorias sistémicas del receptor nicotínico de acetilcolina α7 junto a sus propiedades únicas de activación, desensibilización, permeabilidad al Ca2+ y rol dual ionotrópico-metabotrópico, lo han convertido en un blanco farmacológico emergente muy importante en diversos desórdenes neurológicos, neurodegenerativos e inflamatorios. Este trabajo de tesis se basó en el estudio de los aspectos moleculares relacionados a diferentes tipos de modulación de las funciones ionotrópicas y metabotrópicas del receptor nicotínico de acetilcolina α7 humano. Para ello se utilizaron principalmente técnicas electrofisiológicas a nivel de canal único y de corrientes macroscópicas, en conjunto con análisis de proteínas por western blot y ensayos de movimiento de Ca2+ intracelular. El capítulo I se centró en el estudio de la modulación de las funciones ionotrópicas y metabotrópicas del receptor α7 por eventos de fosforilación/desfosforilación. Se demostró que favorecer el estado desfosforilado de las tirosinas del dominio intracelular de α7 potencia la actividad ionotrópica del receptor. A nivel de corrientes unitarias, el efecto potenciador involucró un aumento en la frecuencia y duración de los episodios de activación, mientras que a nivel de corrientes macroscópicas se manifestó por una disminución en la velocidad de decaimiento de la corriente, y un aumento en la tasa de recuperación desde el estado desensibilizado. Por el contrario, la desfosforilación de las tirosinas tuvo un efecto negativo en la actividad metabotrópica del receptor, estudiada por western blot a partir de la vía de ERK 1/2. Además, a diferencia de lo observado para las tirosinas, las alteraciones en el estado de fosforilación de serinas y treoninas del dominio intracelular no ocasionaron cambios importantes en la actividad ionotrópica de α7 en las condiciones experimentales aquí utilizadas. En síntesis, los resultados presentados en este capítulo ponen en evidencia que la fosforilación de las tirosinas, si bien es absolutamente necesaria para la actividad metabotrópica de α7 mediada por la vía ERK 1/2, actúa como un modulador negativo de la actividad ionotrópica del receptor. El capítulo II abordó el estudio de la asociación funcional entre un fragmento peptídico de la glicoproteína S del SARS-CoV-2 (Y674-R685) y el receptor nicotínico de acetilcolina α7 humano. La asociación entre SARS-CoV-2 y los receptores nicotínicos fue propuesta en forma de hipótesis al comienzo de la pandemia. Más adelante, simulaciones de dinámica molecular mostraron que el fragmento Y674-R685 no solo tiene afinidad por α7, sino que penetra profundamente en el bolsillo de unión a agonista del receptor. En este capítulo, en primer lugar, se demostró que el fragmento Y674-R685 actúa como un agonista silente de α7, ya que es capaz de provocar corrientes unitarias y macroscópicas del receptor, pero solo en presencia de un modulador alostérico positivo. Por otro lado, se demostró que Y674-R685 también ejerce una modulación negativa de α7, que se evidenció por una profunda disminución, dependiente de la concentración, en la duración de los episodios de activación de los canales potenciados y en la amplitud de las respuestas macroscópicas provocadas por la acetilcolina. De esta manera, utilizando distintos enfoques electrofisiológicos, se develó la existencia de una interacción funcional entre el fragmento Y674-R685 de la glicoproteína S del SARS-CoV-2 y el receptor α7 que proporciona las bases moleculares para seguir explorando la participación de los receptores nicotínicos en la fisiopatología de la COVID-19. El capítulo III se basó en el estudio del receptor α7 como blanco del cannabidiol, lo cual resulta de gran interés debido al uso expandido de este fitocannabinoide para tratar diferentes condiciones patológicas gracias a sus propiedades terapéuticas y a la ausencia de efectos psicoactivos. Para ello se exploró el efecto del cannabidiol en las funciones ionotrópicas y metabotrópicas de α7 mediante técnicas electrofisiológicas y ensayos de movimiento de Ca2+ intracelular. En lo que respecta a las funciones ionotrópicas, se demostró que el cannabidiol produce una rápida disminución de la actividad del canal a nivel de corrientes unitarias evidenciada por la reducción en la frecuencia de los episodios de activación. Este efecto fue dependiente de la concentración y se dio con una CI50 en el rango submicromolar, lo que indica una potente modulación negativa. Por otra parte, el cannabidiol también produjo una modulación negativa en la función metabotrópica de α7 que se evidenció por una marcada disminución en las respuestas de Ca2+ intracelular tras la activación del receptor. Estos resultados demuestran que el cannabidiol ejerce una modulación negativa de α7 de relevancia farmacológica que debe tenerse en cuenta a la hora de evaluar los posibles usos terapéuticos del fitocannabinoide. En conjunto, los resultados presentados en esta tesis amplían el entendimiento de los aspectos moleculares relacionados con la modulación de las funciones ionotrópicas y metabotrópicas del receptor nicotínico de acetilcolina α7 en distintas condiciones, a saber, fisiológicas (eventos de fosforilación/desfosforilación), patológicas (fragmento peptídico derivado de la glicoproteína S del SARS-CoV-2) y terapéuticas (cannabidiol). / The survival of higher organisms depends on the ability of their cells to be well organized and to behave in a synchronized manner to fulfill specific functions for which intercellular communication is of pivotal importance. This process is basic for the life of all cells, but it is the raison d'être in neurons that are specialized in receiving information, processing it, and communicating it to other cells. In the nervous system, the main form of communication is carried out via the chemical synapse, in which a neuron releases a chemical messenger, the neurotransmitter, which is identified by a receptor present in another cell, allowing it to respond to the message. Acetylcholine is one of the main neurotransmitters used by neurons, and its receptors, both metabotropic and ionotropic, are expressed in many cell types. Ionotropic acetylcholine receptors, called nicotinic receptors, are pentameric cation channels belonging to the Cys-loop receptor family. The α7 nicotinic acetylcholine receptor is a homopentamer with particular functional properties critical to its neuromodulatory role, including high Ca2+ permeability and the ability to transform transient ionotropic responses into more sustained metabotropic signaling events. It is one of the most abundant nicotinic receptors in the nervous system, although it is also present in other tissues. In the central nervous system, it plays an important role in cognition, attention, and memory, by regulating the release of neurotransmitters, mediating rapid synaptic transmission, and modulating neuronal excitability. A decrease in α7 activity has been associated with various neurological and neurodegenerative disorders, such as schizophrenia, autism, and Alzheimer's disease. The α7 receptor is also expressed in non-neuronal cells; namely, astrocytes, microglia, B and T lymphocytes, epithelial cells, and macrophages, and plays an important role in immunity, inflammation, and neuroprotection. The neuromodulatory, neuroprotective, and systemic anti-inflammatory actions of α7, together with its unique activating, desensitizing, Ca2+ permeability, and dual ionotropic-metabotropic properties, have made the receptor a very important emerging drug target in various neurological, neurodegenerative, and inflammatory disorders. This P.D. thesis work was based on the study of molecular aspects related to different types of modulation of the ionotropic and metabotropic functions of the human α7 nicotinic acetylcholine receptor. To this end, electrophysiological techniques at the single channel and macroscopic current levels were mainly used, as well as protein analysis by western blot and intracellular Ca2+ movement assays. Chapter I focused on the study of α7 receptor ionotropic and metabotropic function modulation by phosphorylation/dephosphorylation events. It was shown that favoring the dephosphorylated state of α7 intracellular domain tyrosine residues potentiates its ionotropic activity. At the single-channel level, this potentiating effect involved an increase in the frequency and duration of activation episodes, while at the macroscopic level it was manifested by a decrease in the rate of current decay and by an increase in the rate of recovery from the desensitized state. In contrast, tyrosine dephosphorylation had a negative effect on receptor metabotropic activity, studied by western blot from ERK 1/2 pathway. In addition, unlike what was observed for tyrosine residues, alterations in the phosphorylation status of serine and threonine residues present in the intracellular domain did not cause any significant changes in α7 ionotropic activity under the experimental conditions used. Summing up, the results collected in this chapter show that tyrosine phosphorylation, although it is absolutely necessary for α7 metabotropic activity mediated by ERK 1/2 pathway, acts as a negative modulator of receptor ionotropic activity. Chapter II focused on the study of the functional association between a peptide fragment of SARS-CoV-2 S glycoprotein (Y674-R685) and human α7 nicotinic acetylcholine receptor. The association between SARS-CoV-2 and nicotinic receptors was hypothesized at the beginning of the pandemic. Later, molecular dynamics simulations showed that the Y674-R685 fragment not only has affinity for α7 but also penetrates deep into its agonist-binding pocket. In this chapter, it was firstly stated that the Y674-R685 fragment acts as a silent α7 agonist, since it is capable of triggering single-channel and macroscopic currents, but only in the presence of a positive allosteric modulator. On the other hand, it was shown that Y674-R685 also exerts α7 negative modulation, which was evidenced by a profound concentration-dependent decrease in the duration of acetylcholine-induced activation episodes from potentiated channels and macroscopic responses. In this way, using different electrophysiological approaches, the existence of functional interaction between SARS-CoV-2 S glycoprotein Y674-R685 fragment and α7 receptor was revealed, which provides the molecular bases to further explore nicotinic receptor participation in COVID-19 pathophysiology. Chapter III was based on the study of the α7 receptor as a target of cannabidiol, which is of great interest due to the expanded use of this phytocannabinoid to treat different pathological conditions thanks to its therapeutic properties and the absence of psychoactive effects. To this end, the effect of cannabidiol on α7 ionotropic and metabotropic functions was explored using electrophysiological techniques and intracellular Ca2+ movement assays. Regarding ionotropic functions, it was shown that cannabidiol produces a rapid decrease in single-channel activity evidenced by the reduction in activation episodes frequency. This concentration-dependent effect occurred with an IC50 in the submicromolar range, indicating a potent negative modulation. On the other hand, cannabidiol also produced a negative modulation in α7 metabotropic function that was evidenced by a marked decrease in intracellular Ca2+ responses after receptor activation. These results demonstrate that cannabidiol exerts α7 negative modulation of pharmacological relevance that must be taken into account when evaluating possible therapeutic uses of the phytocannabinoid. Taken together, the results presented in this thesis broaden the understanding of molecular aspects related to the modulation of α7 nicotinic acetylcholine receptor ionotropic and metabotropic functions under different conditions, namely physiological (phosphorylation/dephosphorylation events), pathological (SARS-CoV-2 S glycoprotein fragment) and therapeutic (cannabidiol).

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

y / The α7 nicotinic acetylcholine receptors (nAChRs) have been highlighted as a target for cognitive enhancement in schizophrenia. Adult female hooded Lister rats received sub-chronic phencyclidine (PCP) (2mg/kg) or vehicle i.p. twice daily for 7 days, followed by 7 days’ washout. PCP-treated rats then received PNU-120596 (10mg/kg; s.c.) or saline and were tested in the attentional set-shifting task. Sub-chronic PCP produced a significant cognitive deficit in the extra-dimensional shift (EDS) phase of the task (p < 0.001, compared with vehicle). PNU-120596 significantly improved performance of PCP-treated rats in the EDS phase of the attentional set-shifting task (p < 0.001). In conclusion, these data demonstrate that PNU-120596 improves cognitive dysfunction in our animal model of cognitive dysfunction in schizophrenia, most likely via modulation of α7 nACh receptors. / This work was partially funded by Johnson & Johnson Pharmaceutical Research and Development.