The neuroactive steroid pregnanolone glutamate (Pg glu), a synthetic analogue of the naturally occurring pregnanolone sulfate (3alpha5betaS), has neuroprotective properties and a minimum of adverse effects. The subject of my thesis is the influence of selected structural modifications of the molecule Pg glu on biological effects. The first modification involves an increase of lipophilicity, the second involves the attachment of a positively charged group to C3. All these neuroactive steroids are use-dependent inhibitors of NMDA receptors. The first aim of this thesis was to determine the neuroprotective effectiveness of the neuroactive steroids chosen. The second aim was to explore the influence of selected neuroactive steroids on motor coordination, reflexes, anxiety and locomotor activity, as well as the effect of their high doses. The third aim was to create a battery of behavioural tests for screening the biological effects of analogues of Pg glu in laboratory rodents. The neuroprotective effects were evaluated in a model of excitotoxic damage of hippocampus in the rat on the basis of its behavioural consequences. The neuroprotective efficacy of androstane glutamate (And glu) and Pg glu was demonstrated. In the case of positively charged molecules, neuroprotective efficacy was not demonstrated....

NMDA receptors belong to the family of ionotropic glutamate receptors, and are involved in synaptic plasticity, learning and memory. However, overactivation by the agonist glutamate can lead to neuronal death - excitotoxicity. Exitotoxicity is a result of excessive calcium influx into the cell through NMDA receptors, and is associated with many cental nervous system (CNS) diseases. Neurosteroids are endogenous compounds capable of NMDA receptor modulation, thus they may have pharmacological potential in the treatment of CNS disorders. The aim of this work was to investigate how pregnanolone sulfate (PA-S) and pregnanolone hemipimelate (PA-hPim) influence somatic calcium and excitotoxicity. We used fluorescence microscopy for recording changes in somatic calcium concentration. We observed that PA-S had no influence on relative somatic calcium concentration. Synthetic analog PA-hPim increased somatic calcium levels slightly. Next, we used oxygen-glucose deprivation (OGD) in vitro to study the influence of neurosteroids on excitotoxicity. Both PA-S and PA-hPim were neuroprotective in the model of acute OGD in vitro. Moreover, PA-S or PA-hPim pretreatment induced ischemic tolerance to a subsequent OGD episode. Our results suggest that neurosteroids PA-S and PA-hPim are potential candidates for the development...

Indiana University-Purdue University Indianapolis (IUPUI) / Excitotoxicity, a major hallmark of neurodegeneration associated with cerebral ischemia, is a result of accumulation of extracellular glutamate. This excess glutamate leads to hyperactivation of glutamate receptors such as the N-methyl-D-asparate (NMDA) receptors (NMDARs) following the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPARs). Excessive activation of NMDARs causes an influx of calcium, which can eventually activate apoptotic pathways and lead to death of neurons. Regulation of NMDAR subunit composition, localization, surface expression, and activity can balance cell survival via activation of either pro-death or pro-survival pathways after a course of an ischemic insult. Specifically, phosphorylation of different NMDAR subunits defines their activity and downstream signaling pathways. NMDARs are phosphorylated by multiple kinases and dephosphorylated by different phosphatases. Besides phosphatases and kinases, per se, phosphorylation of synaptic proteins that regulate kinase or phosphatase targeting and activity also mediate NMDAR phosphorylation. Spinophilin, a major synaptic scaffolding and protein phosphatase 1 (PP1) targeting protein, mediates substrate phosphorylation via its ability to bind PP1. Our studies focus on delineating the role of spinophilin in the regulation of phosphorylation and function of the GluN2B subunit of the NMDA receptor as well as the role of spinophilin in modulating glutamate-induced neurotoxicity. Interestingly, our data demonstrate that spinophilin sequesters PP1 away from GluN2B thereby enhancing phosphorylation of GluN2B at Ser-1284. These changes impact GluN2B protein interactions, subcellular localization, and surface expression, leading to alterations in the amount of calcium entering the neuron via GluN2B-containing NMDARs. Our data show that spinophilin biphasically regulates GluN2B function. Specifically, Ser-1284 phosphorylation enhances calcium influx through GluN2B containing NMDA receptors, but spinophilin leads to dramatic decreases in the surface expression of the receptor independent of Ser-1284 phosphorylation. Moreover, in spinophilin knockout mice, we observe less PP1 binding to GluN2B and less phosphorylation of Ser-1284, but more surface expression of GluN2B and greater levels of caspase activity. Together, these observations suggest a potential neuroprotective role for spinophilin by decreasing GluN2B-containing NMDA receptor-dependent surface expression and thereby decreasing intracellular calcium and neuronal cell death.

'Excitotoxicity' represents the excitatory amino acid mediated degeneration of neurons. Glutamate is the major excitatory neurotransmitter in the brain. Glutamate excitotoxicity has been implicated in a number of neurodegenerative disorders like Stroke, Epilepsy, Alzheimer's disease and traumatic brain injury. This neurotoxicity is summed up by the 'glutamate hypothesis' which describes the cause of neuronal cell death as an excessive release of glutamate causing over excitation of the glutamate receptors and subsequent increase in influx of calcium leading to cell death. An effort to counteract this neurotoxicity has lead to the development of glutamate receptor antagonists that can effectively serve as neuroprotective agents. Nevertheless, the downside to these drugs has been the side effects observed in clinical trial patients due to their disruptive action on the physiological function of these receptors like learning and memory. This work was undertaken to identify targets that can effectively be used to treat excitotoxicity without affecting any normal physiological functions. In one approach, (chapter I) we have identified the KATP channels as an effective modulator of epileptogenesis. In another approach, (Chapter II) we show that targeting the AMPA receptor subunit GluR2 is a practical strategy for stroke therapy. KATP channels that are gated by intracellular ATP/ADP concentrations are a unique subtype of potassium channels and play an essential role in coupling intracellular metabolic events to electrical activity. Opening of KATP channels during energy deficits in the central nervous system (CNS) induces efflux of potassium ions and in turn hyperpolarizes neurons. Thus, activation of KATP channels is thought to be able to counteract excitatory insults and protect against neuronal death. Here, we show that, functional Kir6.1 channels are located at excitatory pre-synaptic terminals as a complex with type-1 Sulfonylurea receptors (SUR1) in the hippocampus. The mutant mice with deficiencies in expressing the Kir6.1 or the SUR1 gene are more vulnerable to generation of epileptic form of seizures, compared to wild-type controls. Whole-cell patch clamp recordings demonstrate that genetic deletion of the Kir6.1/SUR1 channels enhances glutamate release at CA3 synapses. Hence, expression of functional Kir6.1/SUR1 channels inhibits seizure responses and possibly acts via limiting excitatory glutamate release. In addition to epilepsy, ischemic stroke is a leading cause of death in developed countries. A critical feature of this disease is a highly selective pattern of neuronal loss; certain identifiable subsets of neurons, particularly CA1 pyramidal neurons in the hippocampus are severely damaged, whereas others remain intact. A key step in this selective neuronal injury is Ca2+/Zn2+ entry into vulnerable neurons through [alpha]-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels, a principle subtype of glutamate receptors. AMPA receptor channels are assembled from glutamate receptor (GluR) -1, -2, -3, and -4 subunits. Circumstance data have indicated that the GluR2 subunits dictate Ca2+/Zn2+ permeability of AMPA receptor channels and gate injurious Ca2+/Zn2+ signals in vulnerable neurons. Here we show that ischemic insults induce toxic Ca2+ entry through AMPA receptors into vulnerable neurons by modification of GluR2 RNA editing. Thus, targeting of GluR2 subunit can be considered as a promising target for stroke therapy.

The enteric nervous system (ENS) is an incredibly complex neural network that is extensively integrated within the neuroimmunoendocrine system through countless signalling pathways that have yet to be fully characterized. In the last decade we have discovered that many more neurotransmitters are at work in the ENS than was originally thought. This opens up new avenues of research into physiological phenomena traditionally thought to be associated only with the central nervous system, such as NMDA receptor-induced excitotoxicity, and how these may influence immune interactions. In particular, the kynurenine pathway of the tryptophan catabolism produces many neuro-active and immuno-active constituents whose effects are unknown in the ENS but are of great consequence in many neurodegenerative disorders of the CNS. Our study hypothesized that co-culture of the enteric neurons with mast cells would increase neuronal survival through kynurenic acid production in quinolinic acid (QUIN)-induced excitotoxic conditions. This study developed a novel in vitro co-culture system of enteric neurons and glia grown from murine longitudinal muscle-myenteric plexus tissue and bone marrow-derived mast cells. In addition, a pipeline in image analysis software CellProfiler was designed and optimized in order to reduce human bias and error in subsequent immunocytochemical image analysis. Furthermore, we identified the genetic expression of subunits of the NMDA glutamate receptor in cultured enteric neurons via PCR, which suggests that these cultured neurons may be susceptible to excitotoxicity. PCR analysis of cultured mast cells seemed to indicate that our cultured mast cells do not express KAT-III, the enzyme needed to produce the neuroprotective KYNA. Overall, co-culture with mast cells seemed to decrease neuronal survival. This project developed a novel methodology for the in vivo study of mast cell-nerve interactions, and lays the groundwork for future studies in excitotoxicity in the ENS. / Thesis / Master of Science (MSc) / The enteric nervous system is a vast web of nerves and immune cells that innervates the gut and interacts with the central nervous system through the gut-brain axis. An important mediator in this system is the mast cell, a type of immune cell often involved in protective responses to venoms and allergens. Intriguingly, in normal physiological conditions these cells are in close contact with nerves in the periphery, despite their potential to release damaging constituents. While mast cells are well-known for inciting inflammation and releasing toxic granules, they can also synthesize and release potentially beneficial neuroactive compounds, such as neurotransmitters or growth factors. The aim of this study was to characterize mast cell-nerve interactions in neurotoxic conditions, to see if the proximity of mast cells to nerves might serve a neuroprotective purpose.

No

The overall aim of this thesis was to establish strategies to minimize secondary damage to the injured spinal cord. Secondary damage that follows spinal cord injury (SCI) involves inflammatory and excitotoxic pathways. Regulation of these pathways using immunomodulatory and neuroprotective substances potentially protects the injured spinal cord from further damage. We also developed and studied resorbable biomaterials to be used as carriers for potential neuroprotectants to the injured spinal cord. We used transversal spinal cord slice cultures (SCSCs) derived from postnatal mice as a model. SCSCs were maintained on different biomaterials and were studied after treatment with immunomodulatory and/or neurotrophic factors. They were further excitotoxically injured and subsequently treated with interleukin-1 receptor antagonist (IL1RA) or by neural crest stem cell (NCSC)-transplantation. The results show that biocompatible and resorbable hydrogels based on hyaluronic acid (HA) preserved neurons in SCSCs to a much higher extent than a conventional collagen-based biomaterial or standard polyethylene terephthalate (PET) membrane inserts. Glial activation was limited in the cultures maintained on HA-based hydrogel. The anti-inflammatory factor IL1RA protected SCSCs from degenerative mechanisms that occur during in vitro incubation, and IL1RA also protected SCSCs from excitotoxic injury induced by N-Methyl-d-Aspartate (NMDA). IL1RA specifically protected neurons that resided in the ventral horn, while other neuronal populations such as dorsal horn neurons and Renshaw cells did not respond to treatment. Finally, transplantation of NCSCs onto excitotoxically injured SCSCs protected from neuronal loss, apoptosis and glial activation, while NCSCs remained undifferentiated. The results presented in this thesis indicate that carriers based on HA seem to be more suitable than conventional collagen-based biomaterials since they enhance neuronal survival per se. The observed neuroprotection is likely due to biomechanical properties of HA. IL1RA protects SCSCs from spontaneous degeneration and from NMDA-induced injury, suggesting that excitotoxic mechanisms can be modulated through anti-inflammatory pathways. Different neuronal populations are affected by IL1RA to various degrees, suggesting that a combination of different neuroprotectants should be used in treatment strategies after SCI. Finally, NCSCs seem to protect SCSCs from excitotoxic injury through paracrine actions, since they remain undifferentiated and do not migrate into the tissue during in vitro incubation. It seems that combinations of neuroprotectants and carrier substances should be considered rather than one single strategy when designing future treatments for SCI. Incorporation of neuroprotectants such as IL1RA combined with stem cells in injectable biocompatible carriers based on HA is the final goal of our group in the treatment of SCI.

L'excitotoxicité est un mécanisme pathologique qui résulte généralement du relargage massif de glutamate par les neurones en souffrance et qui conduit à la destruction des réseaux neuronaux ainsi qu'à des pertes cellulaires qui peuvent sévèrement impacter les fonctions cognitives et motrices. Au niveau du système nerveux central la séquence d'évènements qui supportent ce processus a bien été décrite et sert aujourd'hui de base au développement d'approches thérapeutiques ciblés pour limiter les conséquences du phénomène. Au niveau de l'oreille interne, les processus excitotoxiques pourraient également être impliqués dans différents types d'atteintes des fonctions auditives et vestibulaires tels que les traumas acoustiques, les surdités brusques, la neurite vestibulaire ou encore les vertiges d'origines ischémiques. Bien que les mécanismes moléculaires qui supportent ce type d'atteinte aient été bien explorés au niveau cochléaire, ils restent peu documentés au niveau du vestibule. De récentes études réalisées par notre équipe ont mis en évidence la participation des récepteurs du glutamate à la neurotransmission vestibulaire en conditions normales et ont également démontré les conséquences histologiques et fonctionnelles d'une atteinte excitotoxique du vestibule. Elles ont par ailleurs mis en évidence le fort potentiel de réparation spontané des synapses vestibulaires après déafférentation. Dans le présent travail de thèse, nous avons voulu identifier les effecteurs cellulaires qui contrôlent les phénomènes de déafférentation et de réafférentation. Dans ce but, nous avons mis au point un modèle d'étude original, qui permet un suivi histologique et fonctionnel de la séquence des évènements biologiques mis en jeu dans ces processus. En combinant des approches en immunohistochimie, microscopie électronique, électrophysiologie moléculaire et pharmacologie nous démontrons que les récepteurs glutamatergiques de types AMPA et NMDA sont tous deux impliqués dans les processus de déafférentation. Les récepteurs NMDA sont également essentiels au processus de réparation synaptique. Les résultats de ce travail apportent une nouvelle lumière sur le rôle des récepteurs du glutamate dans le processus de lésion/réparation des synapses vestibulaires. Cette nouvelle donne pourrait impacter directement sur les stratégies pharmacologiques de protection en cours de développement dans le domaine de la pathologie vestibulaire, mais également par extension à celui de la pathologie auditive. En outre le modèle de culture organotypique de tranche d'organe vestibulaire pourrait trouver application pour le screening de nouveaux composés à propriétés protectrices ou régénératrices. / Excitotoxicity is a pathological mechanism that usually results from the massive release of glutamate by suffering neurons and that leads to destruction of neural networks as well as cell losses that may severely impact cognitive and motor functions. In the central nervous system the sequence of events that supports this process has been extensively studied and is now the basis for the development of targeted therapeutic approaches to limit the consequences of the phenomenon. In the inner ear, excitotoxic damages may also support in different types of auditory and vestibular disorders such as acoustic traumas, sudden hearing loss, vestibular neuritis or dizziness of ischemic origin. Though the molecular mechanisms that support this type of injury have been well explored at cochlear level, they remain poorly documented in the vestibule. Recent studies by our team have confirmed the involvement of glutamate receptors in the vestibular calyx neurotransmission in normal conditions and also showed histological and functional consequences of excitotoxic damage in the vestibule. They also revealed the potential for spontaneous repair of the vestibular synapses after deafferentation. In present thesis, we aimed at identifying the cellular effectors that control the phenomena of deafferentation and reafferentation. For this purpose, we developed an original study model, which allows tracking histological and functional assessment of biological events involved in these processes. By combining approaches in immunohistochemistry, electron microscopy, molecular electrophysiology and pharmacology we demonstrate that both AMPA and NMDA type glutamate receptors are mainly involved in the process of deafferentation. NMDA receptors are also essential for synaptic repair process. The results of this work provide a new light on the role of glutamate receptors in the process of injury / repair of vestibular synapses. The novel observations could directly impact on the ongoing pharmacological protection strategies in the field of the vestibular pathology, and by extension to that of the hearing pathology. In addition, the organotypic culture model of vestibular organ slices may find application for screening new compounds with protective or regenerative properties.

Tem sido demonstrado o envolvimento do glutamato, através de diferentes receptores, nos mecanismos excitotóxicos que levam à morte neuronal na maioria das doenças neurodegenerativas do Sistema Nervoso Central (SNC). Adicionalmente, a Parawixina 10 (Pwx 10) tem sido demonstrada possuir efeito neuroprotetor em modelos de lesão atuando sobre o transporte de glutamato. Os objetivos gerais deste trabalho foram: i) estudar, em um curso temporal (24h, 1, 2 e 4 semanas), as alterações na expressão dos receptores AMPA no hipocampo de ratos induzidas pela injeção local de NMDA e ii) estudar o efeito neuroprotetor da Pwx 10 neste modelo. Foram utilizados ratos Wistar machos, submetidos à cirurgia estereotáxica para a microinjeção de salina ou NMDA no hipocampo dorsal. Alguns grupos de animais foram tratados com Pwx 10 a partir de 1h ou 24h após NMDA. O teste comportamental no labirinto aquático de Morris (LAM) e a coloração de Nissl foram realizados para verificar a extensão e eficácia da lesão por NMDA e o efeito neuroprotetor da Pwx 10. A expressão dos receptores foi estudada através do método de imunoistoquímica. Foram também realizados experimentos de imunofluorescência para GFAP e NeuN para avaliação da gliose e presença de neurônios na área lesada. Foi observado comprometimento das funções de aprendizado e memória no LAM, além de intensa perda de células neuronais e proliferação glial na região do CA1 que recebeu o NMDA, comprovando a eficiência da lesão pelo agonista. Observamos um curso temporal de diferentes alterações na expressão das subunidades GluR1 e GluR2 dos receptors AMPA no hipocampo, que podem ser relacionadas ao complexo mecanismo que ocorre em resposta à microinjeção de NMDA resultando em uma lesão local e na ativação da plasticidade neuronal. O tratamento com Pwx 10 apresentou efeito neuroprotetor, sendo este mais pronunciado quando a toxina foi administrada a partir de 1h após o agonista. / It has been shown the involvement of glutamate, through different receptors, on the excitotoxic mechanisms which result on the neuronal death reported in most neurodegenerative disorders of the CNS. In addition, Parawixina 10 (Pwx 10) has been demonstrated to act as neuroprotective in models of injury regulating the glutamatergic neurotransmission through glutamate transporters. The aims of this work were: i) to study, in a time course (24h, 1, 2 and 4 weeks), the changes on the expression of AMPA receptors in rat hippocampus induced by NMDA intrahippocampal injection, and ii) to study the neuroprotective effect of Pwx 10 in this moldel. Male Wistar rats has been used, submitted to stereotaxic surgery for saline or NMDA microinjection into dorsal hippocampus. Some groups of animals were treated with Pwx 10 from 1h or 24h after NMDA. The behavioral test on Morris water maze (MWM) and the Nissl staining were performed for evaluating the extension and efficacy of the NMDA injury and the neuroprotective effect of the Pwx 10 . The expression of the receptors was analyzed by immunohistochemistry. The expression of GFAP and NeuN on the lesioned area has also been investigated by immunofluorescency. It was observed the impaiment of learning and memory functions in the MWM, and intense loss of neuronal cells and glial proliferation in CA1 that received the NMDA, confirming the efficiency of the injury by the agonist. We observed a time course of distinct changes on the expression of GluR1 and GluR2 subunits of AMPA receptors in hippocampus, which may be related to the complex mechanism triggered in response to NMDA injection resulting in a local injury and on the activation of neuronal plasticity. The treatment with Pwx 10 showed neuroprotective effect, being most pronounced when the toxin was administrated from 1h after NMDA.

Tem sido demonstrado o envolvimento do glutamato, através de diferentes receptores, nos mecanismos excitotóxicos que levam à morte neuronal na maioria das doenças neurodegenerativas do Sistema Nervoso Central (SNC). Adicionalmente, a Parawixina 10 (Pwx 10) tem sido demonstrada possuir efeito neuroprotetor em modelos de lesão atuando sobre o transporte de glutamato. Os objetivos gerais deste trabalho foram: i) estudar, em um curso temporal (24h, 1, 2 e 4 semanas), as alterações na expressão dos receptores AMPA no hipocampo de ratos induzidas pela injeção local de NMDA e ii) estudar o efeito neuroprotetor da Pwx 10 neste modelo. Foram utilizados ratos Wistar machos, submetidos à cirurgia estereotáxica para a microinjeção de salina ou NMDA no hipocampo dorsal. Alguns grupos de animais foram tratados com Pwx 10 a partir de 1h ou 24h após NMDA. O teste comportamental no labirinto aquático de Morris (LAM) e a coloração de Nissl foram realizados para verificar a extensão e eficácia da lesão por NMDA e o efeito neuroprotetor da Pwx 10. A expressão dos receptores foi estudada através do método de imunoistoquímica. Foram também realizados experimentos de imunofluorescência para GFAP e NeuN para avaliação da gliose e presença de neurônios na área lesada. Foi observado comprometimento das funções de aprendizado e memória no LAM, além de intensa perda de células neuronais e proliferação glial na região do CA1 que recebeu o NMDA, comprovando a eficiência da lesão pelo agonista. Observamos um curso temporal de diferentes alterações na expressão das subunidades GluR1 e GluR2 dos receptors AMPA no hipocampo, que podem ser relacionadas ao complexo mecanismo que ocorre em resposta à microinjeção de NMDA resultando em uma lesão local e na ativação da plasticidade neuronal. O tratamento com Pwx 10 apresentou efeito neuroprotetor, sendo este mais pronunciado quando a toxina foi administrada a partir de 1h após o agonista. / It has been shown the involvement of glutamate, through different receptors, on the excitotoxic mechanisms which result on the neuronal death reported in most neurodegenerative disorders of the CNS. In addition, Parawixina 10 (Pwx 10) has been demonstrated to act as neuroprotective in models of injury regulating the glutamatergic neurotransmission through glutamate transporters. The aims of this work were: i) to study, in a time course (24h, 1, 2 and 4 weeks), the changes on the expression of AMPA receptors in rat hippocampus induced by NMDA intrahippocampal injection, and ii) to study the neuroprotective effect of Pwx 10 in this moldel. Male Wistar rats has been used, submitted to stereotaxic surgery for saline or NMDA microinjection into dorsal hippocampus. Some groups of animals were treated with Pwx 10 from 1h or 24h after NMDA. The behavioral test on Morris water maze (MWM) and the Nissl staining were performed for evaluating the extension and efficacy of the NMDA injury and the neuroprotective effect of the Pwx 10 . The expression of the receptors was analyzed by immunohistochemistry. The expression of GFAP and NeuN on the lesioned area has also been investigated by immunofluorescency. It was observed the impaiment of learning and memory functions in the MWM, and intense loss of neuronal cells and glial proliferation in CA1 that received the NMDA, confirming the efficiency of the injury by the agonist. We observed a time course of distinct changes on the expression of GluR1 and GluR2 subunits of AMPA receptors in hippocampus, which may be related to the complex mechanism triggered in response to NMDA injection resulting in a local injury and on the activation of neuronal plasticity. The treatment with Pwx 10 showed neuroprotective effect, being most pronounced when the toxin was administrated from 1h after NMDA.