Sigma-1 receptors (σ-1Rs) are chaperone-like proteins that are broadly distributed throughout the central nervous system and in other tissues. They have been implicated in several physiological and pathological processes, primarily by their ability to modulate certain voltage- and ligand-gated ion channels. Growing evidence suggests that σ-1Rs regulate the functions of ion channels, such as voltage-gated K+ 1.2 (Kv 1.2) and the human Ether-à-go-go-Related Gene (hERG) ion channels, by modulating their expression, trafficking, and targeting. While it is well documented that σ-1Rs enhance the function of N-methyl-D-aspartate receptors (NMDARs), the mechanisms of this enhancement remain poorly understood. Using biochemical methods, we show that 90 minutes after intraperitoneal (i.p.) injection of σ-1R agonists such as (+)-SKF 10,047 (SKF) or (+)-Pentazocine (PTZ) (2 mg/kg), there is an increase in the expression of GluN2 subunits of NMDARs and postsynaptic density protein-95 (PSD-95) in the rat hippocampus. Following activation of σ-1Rs, co-immunoprecipitation (Co-IP) experiments reveal an increased interaction between σ-1Rs and NMDAR subunits; sucrose gradient centrifugation demonstrates an increase in the protein levels of GluN2 subunits in vesicular compartment; and biotinylation shows an increase in the surface levels of GluN2A-containing NMDARs. Taken together, our results suggest σ-1Rs may enhance NMDARs function by increasing their expression, trafficking, and surface levels. This σ-1R-mediated increase in NMDAR expression and surface levels might be involved in several physiological processes such as learning and memory. Our findings also suggest that σ-1Rs could form a potential target for designing novel antipsychotics.

The sigma-1 receptors (σ-1Rs) are endoplasmic reticulum (ER) resident proteins shown to have chaperone-like functions, and are widely distributed throughout the central nervous system (CNS). They reside at a specialized membrane called mitochondria- associated ER-membrane (MAM) and can modulate numerous voltage- and ligand-gated ion channels. One of these channels is the N-methyl-D-aspartate receptor (NMDAR), and σ-1R ligands are able to enhance the potentiation of NMDARs, but the mechanism involved remains poorly understood. Using various biochemical techniques, we show that 90 min following an i.p. injection of σ-1R agonists ((+)-SKF 10,047 (SKF), (+)- Pentazocine (PTZ), or PRE-084 (PRE), there is an increase in the expression of GluN2- containing NMDARs in the rat hippocampus. These results suggest that σ-1R activation is able to enhance NMDAR function by modulating protein expression levels both in the cytosol and on the cell surface. This suggests that σ-1Rs could be excellent therapeutic targets for many neurological disorders, and for the development of novel antipsychotics.

Sigma-1 receptors (σ-1Rs) are chaperone-like proteins that are broadly distributed throughout the central nervous system and in other tissues. They have been implicated in several physiological and pathological processes, primarily by their ability to modulate certain voltage- and ligand-gated ion channels. Growing evidence suggests that σ-1Rs regulate the functions of ion channels, such as voltage-gated K+ 1.2 (Kv 1.2) and the human Ether-à-go-go-Related Gene (hERG) ion channels, by modulating their expression, trafficking, and targeting. While it is well documented that σ-1Rs enhance the function of N-methyl-D-aspartate receptors (NMDARs), the mechanisms of this enhancement remain poorly understood. Using biochemical methods, we show that 90 minutes after intraperitoneal (i.p.) injection of σ-1R agonists such as (+)-SKF 10,047 (SKF) or (+)-Pentazocine (PTZ) (2 mg/kg), there is an increase in the expression of GluN2 subunits of NMDARs and postsynaptic density protein-95 (PSD-95) in the rat hippocampus. Following activation of σ-1Rs, co-immunoprecipitation (Co-IP) experiments reveal an increased interaction between σ-1Rs and NMDAR subunits; sucrose gradient centrifugation demonstrates an increase in the protein levels of GluN2 subunits in vesicular compartment; and biotinylation shows an increase in the surface levels of GluN2A-containing NMDARs. Taken together, our results suggest σ-1Rs may enhance NMDARs function by increasing their expression, trafficking, and surface levels. This σ-1R-mediated increase in NMDAR expression and surface levels might be involved in several physiological processes such as learning and memory. Our findings also suggest that σ-1Rs could form a potential target for designing novel antipsychotics.

Delayed-rectifier potassium channels comprised of the Kv1.2 subunit are critical in maintaining appropriate neuronal excitability and determining the threshold for action potential firing. This is attributed in part to the interaction of the Kv1.2 subunit with an unidentified molecule that confers bimodal channel activation gating, allowing neurons to adapt to repetitive trains of stimulation and protecting against hyperexcitability. It is well established that the Sigma-1 receptor (Sig-1R) regulates members of the Shaker K+ channel family at baseline and upon Sig-1R ligand-activation. While an interaction between Kv1.2 and Sig-1R has been previously demonstrated, the biophysical nature of this interaction has not been elucidated. We hypothesized that Sig-1R may regulate the Kv1.2 biophysical properties and may further act as the unidentified modulator of Kv1.2 activation gating. To explore the interaction between Kv1.2 and Sig-1R, whole-cell voltage-clamp electrophysiology and apFRET imaging experiments were performed in recombinant HEK293 cells transiently transfected with Kv1.2 and Sig-1R. It was found that ligand-activation of Sig-1R decreases Kv1.2 current amplitude, likely due to a ligand-dependent change in Sig-1R activity rather than increased association of Sig-1R with Kv1.2. Further, we show that Sig-1R interacts with Kv1.2 in baseline conditions to modulate bimodal activation gating. We show that Sig-1R modulation of Kv1.2 is abolished both in the presence of Kvβ2, a known auxiliary subunit of Kv1.2, and following expression of the Sig-1R mutation underlying ALS16 (Sig-1R-E102Q). These data respectively suggest that Kvβ2 physically occludes the interaction of the Sig-1R with Kv1.2, and that E102 may be a residue critical for efficient Sig-1R modulation of Kv1.2. Taken together, this data provides novel insights regarding the modulation of neuronal delayed-rectifier potassium channels by Sig-1R. This work provides a new role for Sig-1R in the regulation of neuronal excitability and introduces a mechanism of pathophysiology in Sig-1R dysfunction.

Cognitive dysfunction is a core symptom of schizophrenia, which is poorly treated by current antipsychotic medication. Deficits in the GABAergic system, as demonstrated by convergent genetic and [125I]-iomazenil imaging evidence from patients, are thought to underlie these cognitive deficits. The sub-chronic PCP rodent model was used as it shows cognitive and behavioural parallels to schizophrenia and therefore provides a translational model for some aspects of the disease. However the neurobiological mechanisms responsible for the behavioural alterations in this model have not been fully elucidated. The main aim of the studies presented in this thesis was to investigate the construct validity of the sub-chronic PCP model in relation to the GABAergic and sigma-1 (σ1) receptor systems. Transcriptional changes in gene markers were studied using qRT-PCR and proteomic alterations were investigated using radioligand binding, autoradiography and Western blotting. Finally, the cognitive enhancing potential of σ1 receptor modulators was tested using the novel object recognition (NOR) task. Data presented in chapter 3 shows that sub-chronic PCP treatment in rats produces an increase in GABAA receptor α5-subunit mRNA and a decrease in α3 and δ subunit mRNA levels. No differences were observed in the mRNA levels of the other studied GABAA receptor subunits (α1, α2, α4 or γ2). No alterations in benzodiazepine site- or α5-subunit-containing GABAA receptors were seen following a 7-day washout period, although increased frontal cortical levels of α5-subunit protein were observed prior to the washout period. This suggests that sub-chronic PCP treatment affects extrasynaptic cortical GABAA receptor expression, as shown by the alterations in α5- and δ-subunits, which may contribute to the cognitive deficits observed in this model. Studies in chapter 4 showed that sub-chronic PCP administration causes frontal cortical reductions in parvalbumin, GAD67, GABA transporter-1 and calretinin mRNA levels. No alterations were observed for somatostatin, GAD65, or GABA transporter-3 mRNA, although changes in the mRNA levels for the astrocytic marker glial fibrillary acidic protein were observed in the cerebellum, frontal cortex and hippocampus of sub-chronic PCP-treated animals. No differences in the frontal cortical protein levels of GAD67, GAT-1 and calretinin were observed, suggesting that any proteomic differences in these markers which are present in the sub-chronic PCP model, they are limited in a layer- or cell-type-specific manner. The NOR task is a translational cognitive test that measures recognition memory, which is known to be impaired in schizophrenia. Data in chapter 5 of this thesis showed that sub-chronic PCP-induced and delay-induced recognition memory deficits were ameliorated by acute administration of the σ1receptor agonist (PRE-084) at 1 and 3mg/kg and by the σ1receptor antagonist (NE-100) at 1mg/kg. NE-100 at 3mg/kg proved effective at ameliorating delay-, but not PCP-induced memory deficits. No procognitive effect was observed at lower concentrations of either compound or by co-administration of both compounds. These observations suggest that the improvement of recognition memory deficits is mediated, in part, by σ1 receptors in female rats. The overall results of these studies suggest that sub-chronic PCP administration causes frontal cortical transcriptional alterations in GABAergic neuronal markers which correlate to clinical findings in schizophrenia patients, although these alterations were not observed at the proteomic level following the washout period. These findings also suggest that the σ1 receptor is a potential therapeutic target for recognition memory deficits in schizophrenia, as well as other disorders.

Dysfunction in the Sigma-1 receptor (Sig-1R) is implicated in many neurodegenerative diseases such as Alzheimer’s Disease (AD). Recently, agonists of the Sig-1R have been found to be neuroprotective in AD and provide significant improvements in symptoms. The hallmarks of AD are aggregation of amyloid-β (Aβ) plaques and development of neurofibrillary tau tangles in the brain, which are thought to be correlated with progressive neuronal cell death in AD. Aβ leads to increased endoplasmic reticulum (ER) stress, decreased autophagy, and increased apoptosis, all of which may be contributing to the neuronal cell death that is seen in AD. The Sig-1R is known to reduce ER stress, increase autophagy, and decrease apoptosis. However, as of yet there is little research on the ability of the Sig-1R to specifically reduce Aβ toxicity through these pathways. Therefore, through the use of in vitro and ex vivo models, this study examined the pathways through which activation of the Sig-1R may exert its protective effects against Aβ toxicity. Here, it is shown that activation of the Sig-1R reduces neuronal cell apoptosis in vitro, and reduces tissue death in the CA3 region of the hippocampus ex vivo. Furthermore, this reduction in cell and tissue death may be a result of reduction of ER stress and a return towards baseline levels of autophagy. Together, this research provides insight as to how the Sig-1R may be an important therapeutic target in AD through protection against apoptosis and tissue death.

Le récepteur sigma-1 est une protéine chaperonne active dans des tissus lésés. Le récepteur sigma-1 est principalement exprimé dans le cerveau et joue un rôle neuroprotecteur dans l’ischémie ou les maladies neurodégénératives. Le récepteur sigma-1 est également exprimé dans des lignées cellulaires cancéreuses et des travaux récents suggèrent sa participation dans la prolifération et l’apoptose. Cependant, son rôle dans la carcinogenèse reste à découvrir. Les canaux ioniques sont impliqués dans de nombreux processus physiologiques (rythme cardiaque, influx nerveux, …). Ces protéines membranaires émergent actuellement comme une nouvelle famille de cibles thérapeutiques dans les cancers. Au cours de ma thèse, j’ai montré que le récepteur sigma-1 régule l’activité du canal potassique voltage-dépendent hERG et du canal sodique voltage-dépendent Nav1.5 respectivement dans des cellules leucémiques et des cellules issues de cancer du sein. J’ai également montré que le récepteur sigma-1, à travers son action sur l’adressage du canal hERG, augmente l’invasivité des cellules leucémiques en favorisant leur interaction avec le microenvironnement tumoral. Ces résultats mettent en évidence le rôle du récepteur sigma-1 sur la plasticité électrique des cellules cancéreuses et suggèrent l’intérêt de cette protéine chaperonne comme cible thérapeutique potentielle pour limiter la progression tumorale. / The sigma-1 receptor is a chaperone protein active in damaged tissues. The sigma-1 receptor is mainly expressed into brain and have a neuroprotective role in ischemia and neurodegenerative diseases. The sigma-1 receptor is also expressed into cancer cell lines and recent investigations suggest its involvement into proliferation and apoptosis. However, its role in carcinogenesis remains to delineating. Ion channels are involved in numerous physiological processes (heart beating, nervous influx, …). These membrane proteins currently emerge as a new class of therapeutic targets in cancer. During my thesis, I observed that the sigma-1 receptor regulates voltage-dependent potassium channel hERG and voltage-dependent sodium channel Nav1.5 activities respectively into leukemic and breast cancer cell lines. I also demonstrated that the sigma-1 receptor, through its action on hERG channel, increases leukemia invasiveness by promoting interaction with tumor microenvironment. These results highlight the role of the sigma-1 receptor on cancer cell electrical plasticity and suggest this chaperone protein as a potential therapeutic target to limit tumor progression.

Sigma receptors originally thought to be an opioid receptor is now categorized as a distinct class of receptor. There are two main subtypes, the sigma-1 receptor and an uncharacterised binding site, named the sigma-2 binding site. The presence of the sigma-2 binding site shows high correlation with proliferation of cells and is associated with cancer. I have categorized sigma-1 and sigma-2 binding sites in 11 human tumour cell lines. I have demonstrated that tumour cell lines from a range of tissues express both sigma-1 and sigma-2 binding sites. One exception is the MCF7 breast cancer cell line, which lacks sigma-1 receptors. I show that the quantitation of sigma-2 binding sites using the "masking" protocols are flawed, significantly overestimating levels of sigma-2 binding sites. I propose novel protocols to determine levels of sigma-1 receptors and sigma-2 binding sites in cell lines and tissue. Using radioligand binding assays in MCF7 cells, I have characterised novel sigma-2 ligands. These ligands are simple ammonium salts containing a single nitrogen atom. They are simpler than the previously recognised pharmacophore for the sigma-2 site. I have shown that these simple ammonium salts show graded affinity for the sigma-2 binding site. The highest affinity ligands were dihexylammonium (pKi 7.58) and dioctylammonium (pKi 7.9). I have used these ammonium salts and previously characterised ligands to determine sigma-2 binding site biology. I have shown that the biological activity of these drugs is related neither to their hydrophobicity nor their ability to effect calcium signalling in cells. I propose that the Hill slope of binding is inversely related to the efficacy of a ligand to inhibit metabolic activity of cancer cells. Furthermore, I offer an explanation as to why concentrations of sigma-2 ligands far higher than their determined binding affinities are required to inhibit metabolic activity.

The sigma-1 receptor, although originally classified as an opioid receptor is now thought of as distinct receptor class, sharing no homology with any other known mammalian protein. The receptor has been implicated with a number of diseases including cancer and depression. Modulation of the receptors activity with agonists has potential antidepressant activity whereas antagonists lead to death of cancer cells. Using radioligand binding assays, utilizing the cancer cell line MDA-MB-468, which highly expresses the sigma-1 receptor, a series of novel specific, high affinity, sigma-1 receptor ligands have been characterised. These ligands differed from any previous sigma- 1 receptor ligand in that they are very simple ammonium salts, containing a single nitrogen atom and either straight or branched carbon chains. The binding studies revealed that the straight-chain ammonium salts gave nH values of 1 whereas the branched-chain ammonium salts had statistically significant lower nH values. The ammonium salts were tested for sigma-1 receptor activity in vitro using ratiometric Fura-2 calcium assays and the MTS cell proliferation assay. Branched-chain ammonium salts appeared to have sigma-1 receptor antagonist like effects on cytoplasmic calcium and cell proliferation, whereas the straight-chain ammonium salts behaved as sigma-1 receptor agonists. Three ammonium salts stood out as potential effective sigma-1 receptor drugs, the straight-chain ammonium salt dipentylammonium, and two branched-chain ammonium salts, bis(2-ethylhexyl)ammonium and triisopentylammonium. The ammonium salts were then tested in vivo. Dipentylammonium showed significant antidepressant properties when tested in behavioural models for depression and bis(2-ethylhexyl)ammonium and triisopentylammonium were able to significantly inhibit the growth of tumours implanted in mice. Finally I looked at the coupling of the sigma-1 receptor with G-proteins and show that sigma-1 receptor antagonists dose dependently reduce G-protein activity and inhibition of G-proteins enhanced the sigma-1 antagonists' effects of calcium signalling.

Les maladies neurodégénaratives (MNDs) sont les troubles neurologiques les plus fréquents chez l’homme et touchent des millions de personnes à travers le monde. Elles affectent le fonctionnement du système nerveux de façon chronique et progressive et conduisent souvent au décès du malade. L’évolution de ces maladies est très variable et les traitements disponibles actuellement ne permettent pas de modifier leur progression mais seulement d’atténuer les manifestations symptomatiques.Les récepteurs σ1 correspondent à une classe unique de récepteurs transmembranaires du réticulum endoplasmique. Exprimés au niveau du SNC et en particulier dans les neurones, les lymphocytes et les oligodendrocytes, ces récepteurs sont connus pour être impliqués dans la régulation de nombreux neurotransmetteurs. Même si les mécanismes de transduction après leur activation ne sont pas complètement élucidés, de plus en plus d’études mettent en évidence le potentiel thérapeutique de ces récepteurs. En effet, depuis leur découverte, les récepteurs σ1 ont été impliqués dans de nombreuses pathologies dont des MNDs.Ces travaux de thèse s’inscrivent donc dans ce contexte. Le projet consistait à concevoir, synthétiser et évaluer de nouveaux ligands σ1 pour une utilisation en neuroprotection. Les dérivés tétrahydroquinolin-hydantoïnes préalablement conçus au laboratoire, ont montré des affinités nanomolaires envers la protéine σ1, une bonne sélectivité, une faible cytotoxicité et des propriétés ADME compatibles avec un développement thérapeutique. Evalués dans différents modèles, ces composés ont montrés des propriétés anti-inflammatoires associées à une action neuroprotective. Cependant, bien que les Tic-hydantoïnes soient des composés chimiquement stables, ils ont montré une faible stabilité métabolique. Trois nouvelles familles de composés ont donc été conçues et synthétisées afin de pallier à ces problèmes. Leurs affinités, sélectivités, cytotoxicités et propriétés ADME ont été évaluées. Des tests comportementaux ont également été réalisés sur les composés les plus intéressants afin de déterminer leur profil agoniste ou antagoniste. Finalement, le meilleur candidat, évalué dans un modèle in vivo de sclérose en plaques, a montré des propriétés neuroprotectrices intéréssantes. / Neurodegenerative diseases are the most common neurological disorders in humans, affecting millions of people worldwide. They affect the nervous systems in chronic and progressive way and often lead to the death of the patient. The evolution of these diseases is highly variable and currently available treatments do not alter their growth but only moderate symptomatic manifestations.Sigma-1 receptors represent a structurally unique class of transmembrane receptors of the endoplasmic reticulum. Expressed in the central nervous system and especially in neurons, lymphocytes and oligodendrocytes, these receptors are known to be involved in the regulation of numerous neurotransmitters. Even if the signal transduction pathway after activation of σ1 receptors is not completely understood, more and more evidences suggest that they represent a potential therapeutic target in many diseases. Indeed, since their discovery, the σ1 receptors have been implicated in various pathologies including neurodegenerative disorders. Thus, it is in this context that our interest is focused on the conception and synthesis of novel σ1 receptors ligands for the treatment of neurodegenerative diseases. Fused and optimized tetrahydroquinoline-hydantoin derivatives designed in our laboratory showed nanomolar σ1 affinity, σ2 /σ1 selectivity, very low cytotoxicity and ADME properties compatible with therapeutic development. Evaluated in different models, these compounds showed an anti-inflammatory activity associated with a neuroprotective action. However, while the Tic-Hydantoin derivatives are chemically stable, they showed a low metabolic stability. Thus, three novel families of compounds were synthesized in order to compensate for these problems. Their affinities, selectivities, cytotoxicities and their ADME properties were evaluated. Behavioural testing was carried out on the most interesting compounds to determine the agonist or antagonist profil. Finally, evaluated in in vivo model of multiple sclerosis, the best compound showed interesting neuroprotective properties.