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Schizophrenia is characterized by positive and negative symptoms along with cognitive symptoms that include impairment in working memory (WM). WM is the storage of relevant information for short intervals of time to guide thoughts and actions. The neural correlate of WM is thought to be the persistent activity exhibited during the retention interval of WM tasks. Persistent activity is hypothesized to be mediated by the activation of NMDA receptors (NMDAR) within recurrent neuronal circuits. Consistent with this hypothesis, studies with healthy humans and monkeys have shown that the administration of the NMDAR antagonist ketamine induces memory-load dependent deficits in WM, along with increasing response time. In parallel to this, the pathophysiology of schizophrenia has been hypothesized to rest on the hypofunction of NMDAR. Previous studies in humans indicate that blockade of NMDAR induces schizophrenia-like symptoms. In addition, symptoms of schizophrenia patients are alleviated with sub-chronic treatments focusing on the activation of the NMDAR co-agonist site. Based on these observations, I tested the hypothesis that increasing the activation of NMDAR with co-agonist stimulation has beneficial effects on WM. D-serine (100mg/kg/day-6 weeks) was orally administered to two female macaque monkeys performing a visual sequential comparison task (VCST), which allows the manipulation of memory load. In this task, the monkeys had to identify the location of a colour change within an array of 2 to 5 coloured stimuli following a retention interval of 1 second. I hypothesized that sub-chronic treatment with D-serine produces a gradual improvement in the monkeys’ performance on the VSCT. Specifically, I predicted that the improvement would scale with memory load due to increased demands on WM resources at higher loads. Contrary to my hypothesis, D-serine produced minute changes in response accuracy, which were not memory load-dependent. Also, the response latency of the monkeys was found to increase, which is commonly observed following NMDAR antagonist treatments. These findings suggest that D-serine has a limited role in increasing the activation of NMDARs to improve WM per se. The beneficial effects reported by NMDAR co-agonists in schizophrenic patients could be a general reduction in cognitive symptoms, not specifically related to WM. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2013-10-01 17:45:20.643

GRIN2A and GRIN2B encode the GluN2A and GluN2B subunits of the NMDA receptor, a subtype of ionotropic glutamate receptor that displays voltage-dependent block by Mg2+ and a high permeability to Ca2+. These receptors play important roles in synaptogenesis, synaptic transmission and synaptic plasticity, as well as contributing to neuronal loss and dysfunction in several neurological disorders. Recently, individuals with a range of childhood onset epilepsies, intellectual disability and other neurodevelopmental abnormalities have been found to carry heterozygous gene-disrupting or protein-altering point mutations in GRIN2A and GRIN2B. This thesis addresses the hypothesis that these point mutations cause key functional disturbances to NMDA receptor properties that contribute to neurodevelopmental disorders. To test this hypothesis, a group of related mutations were selected for functional assessment in heterologous systems: four missense mutations affecting residues in or near the subunit pore regions, all of which are associated with epilepsy and intellectual disability. To model the impact of gene disrupting mutations in GRIN2A, a preliminary analysis of the functional consequences of GluN2A haploinsufficiency in a genetically modified rat was also performed. Three of the four missense mutations were found to be associated with profound alterations in fundamental NMDA receptor properties: compared to wild type, GluN2AN615K was found to reduce Mg2+ block, GluN2BN615I and GluN2BV618G to cause potentiation by Mg2+, and GluN2AN615K and GluN2BN615I showed reduced conductance. GluN2AR586K was not found to influence the parameters assessed. When GluN2AN615K was expressed alongside wild type subunits in the same NMDA receptor, it was found to have a dominant negative effect. Finally, I established successful gene targeting in a new rat Grin2A knock-out model, and observed that heterozygous neurons had lower GluN2A protein expression and current density, making a good model to study human epilepsies associated with loss of a GRIN2A allele. This thesis provides evidence that three missense mutations in GRIN2A and GRIN2B affect physiologically important properties of the NMDA receptor, and that GluN2A haploinsufficiency influences important neural properties in vitro. This is consistent with these mutations causing disease and highlights these and related mutations as potential therapeutic targets in the future.

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.

Disrupted in Schizophrenia 1 (DISC1) is a well supported risk factor for schizophrenia, bipolar disorder and major recurrent depression. DISC1 is a multifunctional multicompartmentalised scaffold protein with essential roles in neuronal proliferation, differentiation, migration and integration. DISC1 also modulates pathways of vital importance for neuronal signalling and plasticity. One of the major hypotheses for the cause of psychiatric illness is N-methyl-D-aspartate (NMDA) receptor hypofunction. It was observed that NMDA receptor antagonists can induce symptoms of schizophrenia in unaffected individuals, and exacerbate symptoms in patients with schizophrenia. Recent work in our laboratory showed that DISC1 complexes with NMDA receptors within the cell body and at synapse of neurons. Here I studied whether DISC1, or DISC1 missense variants, affect the trafficking of NMDA receptors. This was done by quantifying surface NMDA receptor expression in the presence of DISC1 or variant DISC1. I found that one common variant, 607F, causes a significant reduction in surface expressed NMDA receptors. I went on to show that DISC1 reduces the number of internalised receptors associating with early RAB5-containing endosomes. This indicates that DISC1 may be involved in the trafficking and recycling of NMDA receptors, a process that may be affected by the missense DISC1 variant 607F. Further to this I studied the effects on NMDA receptor trafficking of DISC1 pathway partners Nuclear Distribution Element 1 (NDE1) and Trafficking-protein kinesin binding 1 (TRAK1), both regulators of neuronal intracellular trafficking. Phosphorylation of NDE1 at T131 has been shown to be modulated by DISC1. Using phospho-mimic and phospho-dead NDE1 expression constructs I observed a significant reduction in the surface-expressed NMDA receptors in cells expressing the phospho-mimic form of NDE1. NDE1 may therefore be involved in the trafficking of NMDA receptors, and this role may be modulated by phosphorylation of NDE1. Finally, TRAK1 was shown to associate robustly with the GluN2B subunit, and to decrease the surface expression of NMDA receptors, most likely by sequestering them. The TRAK1-induced GluN2B sequestration may be an artefact, but the association of the trafficking molecule TRAK1 with this subunit may point towards a role in NMDA receptor trafficking. These proteins have been shown to associate with each other and may form a complex in order to traffic NMDA receptors. Disruption of this complex by defective DISC1 expression may affect NMDA receptor trafficking. In the brain this could conceivably contribute to NMDA receptor hypofunction and the development of psychiatric illness.

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.

NMDA glutamate receptor is a ligand-gated ion channel that mediates a major component of excitatory neurotransmission in the central nervous system (CNS). NMDA receptors are activated by simultaneous binding of two different agonists, glutamate and glycine/ D-serine1. With aging, glutamate concentration gets altered, giving rise to glutamate toxicity that contributes to age-related pathologies like Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and dementia88,95. Some treatments for these conditions include NMDA receptor blockers like memantine130. However, when completely blocking the receptors, there is a restriction of the receptor's normal physiological function59. A different approach to regulate NMDAR receptors is thorough allosteric modulators that could allow cell type or circuit-specific modulation, due to widely distributed GluN2 expression, without global NMDAR overactivation59,65,122. In one study, we hypothesized that the compound CNS4 selectively modulates NMDA diheteromeric receptors (GluN2A, GluN2B, GuN2C, and GluN2C) based on (three) different glutamate concentrations. Electrophysiological recordings carried out on recombinant NMDA receptors expressed in xenopus oocytes revealed that 30μM and 100μM of CNS4 potentiated ionic currents for the GluN2C and GluN2D subunits with 0.3μM Glu/100μM Gly. However, when using 300μM Glu/100μM Gly, CNS4 inhibited the relative response in the GluN2D subunit and had no effect on the remaining subunits. CNS4 reduced the response to glutamate alone for GluN2A but increased it for GluN2B and did not appear to replace glutamate. Another set of electrophysiological recordings measuring current-voltage relationship was made in order to understand ion flow across the channel in the presence of CNS4. 100μM CNS4 numerically increased the ionic inward current through the channel pore with more positive membrane potential, reflected by a significant difference in reversal potential values, in the GluN2C and GluN2D subunits. CNS4 also exhibited a non-voltage dependent activity and it did not appear to compete with magnesium which naturally blocks the receptor. Finally, the effect of CNS4 on calcium uptake and cellular viability was study in neurons from primary rat brain culture. Cortial and striatal neurons were given excessive doses of synthetic agonist NMDA in order to hyperactivate native NMDAR. In the calcium assay, 100µM of CNS4 significantly increased calcium upatake when given with 300µM NMDA compared with NMDA alone in cortex and when given with 100µM and 300µM NMDA in striatum. In the MTS assay, CNS4 did not alter neuronal viability in either cortical or striatal neurons compared with NMDA alone. Also, when CNS4 was used in non treated neurons it did not alter neuronal viability. Findings from the primary brain culture let us conclude that CNS4 could facilitate calcium influx and possibly be non toxic for neurons. / Master of Science / NMDA ionotropic glutamate receptors are predominately expressed in the central nervous system (CNS). These receptors are activated by glutamate and glycine/ D-serine1. With aging, glutamate concentration in the synapse gets altered giving rise to toxic environments for neurons that can contribute to age-related pathologies like Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and dementia88,95. Some treatments of these conditions include the receptor blockers like memantine130. However, when completely blocking the receptors, there is a restriction of the receptor's normal physiological function59. A different approach to regulate NMDAR is through allosteric modulators that are compounds that modulate the receptor function without competing with endogenous agonists59,65,122. In this study, we hypothesized that the compound CNS4 selectively modulates NMDAR based on glutamate concentration. Electrophysiological recordings on stage four xenopus oocytes helped us to identify the dose-dependent activity of CNS4 and we found that 30 and 100μM of CNS4 selectively potentiates ionic currents for GluN2C and GluN2D subunits with 0.3μM Glu/100μM Gly but inhibited currents for only GluN2D with 300μM Glu/100μM Gly. Following this, a current-voltage plot was made to examine the channel activity of CNS4. We found a numerical increase of ionic inward current through the channel pore with more positive membrane potential values in the GluN2C and GluN2D subunits. Also, the effect of CNS4 on the ion current activity changed based on glutamate concentration, and CNS4 did not exhibit a voltage-dependent activity, which is a positive feature for compounds that target the receptor133. Finally, to better understand the effect of the compound CNS4 in primary neurons in a toxic environment, a rat brain neuronal culture was made. Increasing doses of NMDA with constant 100µM CNS4 increased cellular Ca2+ influx in a dose-dependent manner. Particularly, 100µM CNS4 with 300µM NMDA exhibited a significant increase in Ca2+ influx in both cortical and striatal neurons compared with 300µM NMDA alone. However, when used alone, 100µM CNS4 did not have an effect on the amount of Ca2+ influx. In addition, CNS4 plus NMDA did not increase viability compared to NMDA alone, and CNS4 alone did not proportionally reduce neuronal viability.

Dans le cadre de la recherche d'antagonistes du NMDA, nous avons synthétisé une librairie de nouveaux composés dérivés du Selfotel (CGS 19755). Ces phosphonates aromatiques ont été obtenus par réaction de couplage croisé utilisant une catalyse au palladium (couplage d'Hirao). Leur hydrolyse a conduit aux acides phosphoniques correspondants. Dans une second temps, la série pipéridine a été obtenue par hydrogénation complète des motifs pyridiniques en utilisant de l'oxyde de platine comme catalyseur.Lors de l'analyse de ces composés, des propriétés spectroscopiques intéressantes en RMN ont été mises en évidence. Des monocristaux ont été obtenus sur certains des acides, ce qui a permis de confirmer leurs structures par diffraction des rayons X. Ces experiences ont clairement montré que l'hydrogénation du cycle aromatique est un processus de cis-hydrogénation.Les études biologiques préliminaires effectuées sur des cultures de neurones ont indiqué une activité certaine sur les récepteurs NMDA. Une partie d'entre eux agissait comme antagonistes du NMDA tandis que l'autre montrait des activités de neuroprotection. Des expériences complémentaires par un test de fluorescence, ont été mises en place. Dans ce contexte, les outils de génie génétique ont été utilisés pour créer une lignée cellulaire de mammifère exprimant de façon stable le récepteur NMDA. Toutefois, ces expériences n'ont pas conduit à des résultats permettant d'expliquer les résultats biologiques obtenus. / The library of novel compounds being derivatives of NMDA antagonists Selfotel (CGS 19755) was synthesized. The series of aromatic esters were obtained in palladium catalyzed cross-coupling reaction (Hirao coupling), followed by their hydrolysis and then the series of aliphatic acids was obtained upon their hydrogenation over PtO2. Majority of designed compounds have not been synthesized before. What is worth emphasizing, none of this compound was studied as potential NMDA receptor antagonist. During the analysis of compounds, interesting spectroscopic properties were observed as well as few of compounds were obtained as monocrystals, which enabled to obtain their crystal structures. Crystal structures of aliphatic acids unequivocally identified that hydrogenation of aromatic ring is a cis process and yields predominantly only one diastereomeric mixture.Initial biological studies performed on cultures of neurons indicated that the compounds posses activity towards NMDA receptors – part of them acting as antagonists and part as protectants or facilitators.More advanced investigation of biological activity by means of fluorescence screening assay, was planned to be performed. Consequently, genetic engineering tools were used in order to create mammalian cell line stably expressing NMDA receptor what would allow to perform these studies. However, these experiments were not successful. Therefore, further investigation should be performed in order to confirm the obtained biological results.