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.

Ischemic strokes cause excessive release of glutamate, leading to overactivation of N-methyl-D-aspartate receptors (NMDARs) and excitotoxicity-induced neuronal death. For this reason, inhibition of NMDARs has been a central focus in identifying mechanisms to avert this extensive neuronal damage. N-acetyl-aspartyl-glutamate (NAAG), the most abundant neuropeptide in the brain, is neuroprotective in ischemic conditions in vivo. Despite this evidence, the exact mechanism underlying its neuroprotection, and more specifically its effect on NMDARs, is currently unknown due to conflicting results in the literature. Here, we uncover a pH-dependent and subunit specific action of NAAG on NMDARs. Using whole-cell electrophysiological recordings on acute hippocampal slices from adult mice and on HEK293 cells, we found that NAAG increases synaptic GluN2A-containing NMDAR excitatory postsynaptic currents (EPSCs), while effectively decreasing extrasynaptic GluN2B-containing NMDAR EPSCs in physiological pH. Intriguingly, the results of our study further show that in low pH, which is a physiological occurrence during ischemia, NAAG depresses GluN2A-containing NMDAR EPSCs and amplifies its inhibitory effect on GluN2B-containing NMDAR EPSCs, as well as upregulates the surface expression of the GluN2A subunit. Altogether, our data demonstrate that NAAG has differential effects on NMDAR function based on subunit composition and extracellular pH levels. These findings suggest that the role of NAAG as a neuroprotective agent during an ischemic stroke is likely mediated by its ability to reduce NMDAR excitation. The inhibitory effect of NAAG on NMDARs and its enhanced function in acidic conditions makes NAAG a prime therapeutic agent for the treatment of ischemic events.

Orientador: Alexandre Leite Rodrigues de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T08:32:53Z (GMT). No. of bitstreams: 1 Perez_Matheus_M.pdf: 2859331 bytes, checksum: 541101c0093208622bf70dc9b70cf007 (MD5) Previous issue date: 2013 / Resumo: O sistema nervoso é muito sensível à lesões e sua capacidade regenerativa é limitada. Assim, na maioria dos casos, o reparo tecidual não ocorre ou ocorre de forma incompleta, causando danos irreversíveis. Embora o sistema nervoso do neonato apresente maior capacidade regenerativa do que o adulto, a regeneração também é um processo complexo e limitado. Para melhor compreensão dos mecanismos e limitações da regeneração do SNC, tanto na fase neonatal como na fase adulta, diversos modelos de lesão têm sido propostos por vários autores, inclusive transecção de nervo periférico. Através desses estudos experimentais, diferentes modalidades terapêuticas foram sugeridas, com intuito de evitar as alterações que levam à morte neuronal, seja na fase neonatal ou adulta. Para isso, muitas substâncias, com propriedades neuroprotetoras, têm sido testadas no processo de regeneração, plasticidade sináptica e para evitar a morte neuronal após lesão do sistema nervoso. Dentre a grande variedade de substâncias com potencial neuroprotetor, estão alguns derivados da planta Cannabis sativa. Um deles, o canabidiol (CBD), é o principal componente não-psicotrópico produzido nas glândulas encontradas na superfície das folhas da planta. O presente trabalho teve como objetivo investigar o potencial neuroprotetor do canabidiol e, para isso, foram utilizados ratos neonatos da linhagem Wistar, divididos nos seguintes grupos experimentais: grupo submetido à axotomia do nervo isquiático e tratamento com o canabidiol (grupo CBD), grupo submetido à axotomia e tratamento veículo (grupo PB) e grupo controle, com axotomia sem tratamento (grupo sem tratamento). Assim, cortes histológicos utilizando coloração de Nissl mostraram que os grupos tratados com canabidiol (15 e 30mg/kg) mostraram maior sobrevivência (21%; p<0.0001) de motoneurônios medulares, quando comparados aos grupos que não foram tratados com canabidiol ou tratados com canabidiol (5mg/kg). A análise imunohitoquímica, empregando o anticorpo anti-sinaptofisina revelou, no grupo tratado com canabidiol, preservação de imunoreatividade, refletindo preservação de circuitos sinápticos medulares. Tal manutenção de terminais foi 30% superior nos grupos tratados com canabidiol, comparativamente aos grupos controle (p=0,0003). A imunoistoquímica para os anticorpos anti-GFAP (astrócito) e anti-IBA 1(migroglia) revelou uma menor imunomarcação para o grupo tratado com canabidiol, quando comparado aos grupos não tratados. Tal fato demonstra que o canabidiol reduz astrogliose (anti-GFAP) em 30% (p=0,0149) e reação microglial em 27% (p=0,0086). A análise para detecção de células apoptóticas, através reação de TUNEL (teminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling), mostrou que o tratamento com canabidiol foi neuroprotetor, quando comparado ao grupo axotomia sem tratamento (p=0,0063). Em conjunto, os presentes resultados mostram que o canabidiol apresenta promissora propriedade neuroprotetora, devendo ser melhor investigado, com vistas ao seu emprego clínico futuro / Abstract: The CNS is very sensitive to injury and their regenerative capacity is limited, so in most cases the tissue repair does not occur or occurs incompletely, leading to irreversible damage. Although the nervous system of neonatal rats presents a greater regenerative capacity than adults, the regenerative process is still complex and limited. To better understand the mechanisms and limitations of CNS regeneration, both in the neonatal and adulthood, various injury models have been proposed by several authors, including peripheral nerve transaction. Through these experimental studies several therapeutic modalities have been proposed, in order to prevent the neuronal death caused by injuries and pathological processes. For this reason, many substances with neuroprotective properties have been tested in the regeneration process, synaptic plasticity and to prevent neuronal death after nervous system injury. Among the wide variety of substances with potential neuroprotective action, some are derived from the Cannabis sativa. One, cannabidiol (CBD) is the major non-psychotropic component produced by the glands found on the surface of plant leaves. The present study aimed to investigate the neuroprotective potential of CBD. We used Wistar neonatal rats that were divided into the following experimental groups: sciatic nerve axotomy and CBD treatment (CBD group), axotomy and veículo treatment (PB group) and a control group, without treatment (no-treatment group). Thus, Nissl stainning in spinal cord cross sections showed that the CBD groups (15 e 30mg/kg) had a higher neuronal survival (21%, p <0,0001) when compared to the CBD 5mg/kg group, PLB and no treatment groups. Immunohistochemical analysis, done by ipsi/contralateral ratio of integrated density of pixels quantification, of anti-synaptophysin labeling showed that CBD groups had a greater immunostaining pattern when compared to the other experimental groups (30%, p<0,0003) reflecting a higher preservation of synapses after CBD treatment. Imunolabeling Anti-GFAP and anti-Iba 1 revealed a lower immunostaining in CBD group, showing that CBD treatment decreases the astrocytosis process in 30% (p = 0,0149) and reduce reactive microgliosis in 27% (p=0,0086), when compared to the others experimental groups. Aimed to detect apoptotic cells, we used TUNEL reaction. Quantitative analysis showed that the CBD treatment reduced the apoptotic nuclei detectable, when compared to other experimental groups. These results show that CBD presents promising neuroprotective property and should be better investigated aiming its future clinical use / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

Brain ischemia events are among the first three mortalities causes in the world associated with high treatments costs and in most cases some degree of permanent functional disability, thus it is necessary with new low cost and effective treatments. In the present study the potential neuroprotective effect of magnesium sulphate was evaluated by quantification of ischemic biomarkers: fluoride resistant acid phosphatase (FRAP), inorganic phosphate (Pi), lactate dehydrogenase (LDH) and nitric oxide (NO) in rats with global ischemia. Adult rats were anaesthetized (sodium thiopental 60mg.kg-1) and magnesium sulphate (1mmol.kg-1) (9 animals) or saline solution (NaCl 0,9%) (11 animals) was infused via jugular vein. Cortical sample was extracted 30 minutes after infusion (normoxic condition), thereafter it was induced global ischemia via respiratory arrest caused by jugular administrated muscular relaxant (Flaxedil, 500μL), and 30 minutes after this state a cortical sample of contralateral hemisphere was taken (hypoxia condition). Samples were preserved with proteases inhibitors (PMFS y NaF) and homogenized. Biomarkers were quantified within 24h from the experiment by the following spectroscopic methods: Gomori (FRAP), Ammonium molybdate (Pi), Pyruvate to lactate reduction (LDH) and Griess (NO). FRAP activity was quantified as an inflammatory biomarker for the first time in the cerebral cortex. Global ischemia increased all biomarkers concentrations of the saline group. During normoxia condition magnesium sulphate reduced Pi (P=0,0002) and LDH activity (P=0,001), while during hypoxia it reduces Pi (P= 0,0002) and LDH activity (P=0,03) compared to saline values. These results strongly suggest the cortical neuroprotectiveeffects of magnesium sulphate in global ischemia induced by respiratory arrest, by reduction of cellular acidosis and energetic deficit.

Stroke increases neurogenesis (birth of new neurons) through upregulation of ciliary neurotrophic factor (CNTF), a potent neurogenic cytokine made almost exclusively in the central nervous system. Previous study found that CNTF is induced and needed to stimulate neurogenesis in the subventricular zone (SVZ) of mouse brain in a stroke model. CNTF also has a neuroprotective function. Focal adhesion kinase (FAK), protein tyrosine kinase 2, is ubiquitously expressed in various cell types and mediates cell adhesion and migration. We previously discovered that systemic inhibition of FAK upregulates CNTF expression in the SVZ, making FAK a pharmacological target to increase CNTF to promote neurogenesis and neuroprotection after stroke. This study examined whether systemic FAK inhibitor treatment after stroke regulates SVZ neurogenesis and neuroprotection using a middle cerebral artery occlusion (MCAO) to induce a stroke in adult male C57BL/6 mice. A filament was inserted in the external carotid artery and then fed through the carotid bifurcation into the internal carotid artery to the base of the middle cerebral artery. After 30 minutes of occlusion, the filament was removed to restore blood flow. Mice were randomly assigned to receive 3 daily doses of saline or FAK inhibitor (FAK14, i.p., 3 mg/kg) and treatment started at 6 hours, 12 days, or 58 days after MCAO. Because CNTF has a neuroprotective function, the amount of tissue damage was analyzed to compare treatment groups. The neuroprotective role of FAK14 was examined by measuring MCAO-induced infarction. The infarct size was measured using the absence of NeuN (neuronal cell marker) and GFAP (activated astrocytes) and presence of CD68 (activated microglia). FAK14 given at 6 hours post-stroke reduced the infarct size to 38% of the uninjured side of the brain compared to 46% with saline. Proliferating cells were labeled by injecting bromodeoxyuridine (BrdU, 50 mg/kg), the mice were processed 2 h after the last BrdU injection, and proliferated cells in the SVZ were counted with unbiased stereology. There were no significant differences in the total numbers of BrdU+ cells between saline and FAK14 at 3, 14 and 60 days. Future studies are needed to confirm the levels of CNTF at the various times of treatment. If there is no difference in CNTF expression or increased expression of counteracting cytokines, no difference in neurogenesis between groups would be expected. The neuroprotective effect of FAK14 during the acute phase following injury could provide novel pharmacological options to stroke patients extending the current therapeutic treatment window.

Ischemic strokes are a major cause of death and disability, yet efficacious pharmacotherapies remain limited. Although neuronal cell death during stroke is primarily induced via excessive Ca2+ influx through NMDARs following overactivation by uncontrolled glutamate release, antagonism of these receptors has been shown to be ineffective due to intolerable side effects. This thesis highlights a novel therapeutic strategy for stroke wherein NMDAR-mediated excitotoxicity is temporarily and dynamically mitigated via the initiation of a process termed “glycine induced NMDAR internalization” (GINI). While GINI occurs in vitro following application of high doses of glycine, achieving these levels of glycine in vivo has long been thought impossible as glycine transporters (GlyT1) maintain synaptic glycine levels well below saturating concentrations. Here, we show that GINI can be triggered in vivo when mice are administered a glycine transporter-1 antagonist (GlyT1-A) prior to stroke and that this strategy provides neuroprotection. Mice pre-treated with a GlyT1-A, which elevates glycine levels, exhibited significantly smaller stroke volumes, reduced cell death, and significantly minimized behavioural deficits following stroke induction by either photothrombosis (PT) or endothelin-1 (ET-1). Moreover, we observed preservation of vasculature function and morphology in the peri-infarct area. These data strongly suggest that elevating brain glycine levels with GlyT1-As should be considered as a novel pharmacotherapy for ischemic stroke.

Stroke is serious neurological disease and is a major cause of death as well as disability throughout the globe. Stroke has a complex pathophysiology that involves inflammatory pathways, excitotoxicity mechanisms, oxidative damage, apoptosis, ionic imbalances, angiogenesis and neuroprotection. 85% of strokes are ischemic and occurs when a cerebral vessel, or any vessel supplying the brain, narrows or loses pressure resulting in subsequent brain ischemia and infarction downstream to the site of obstruction depriving tissues of vital oxygen and nutrients. This may be caused by either atherosclerotic thrombi or distant emboli defined as a mass of clotted blood or other material. It is estimated that over a billion people currently smoke cigarettes or use other tobacco products, seeing as smoking is a major risk factor for stroke this is of major concern. Platelets are hematopoietic cells produced by bone marrow megakaryocytes. Platelets play a role in the development of ischemic stroke primarily by means of their participation in the formation of thromboemboli, the presence of abnormal platelet function may predispose patients to a pro-thrombotic, pro-inflammatory state. The reorganization of the cytoskeleton in platelets is an important factor in the complex mechanisms found in thrombosis and haemostasis. The platelet membrane contains a large number of receptors which specifically bind agonists that stimulate the physiological platelet response. Oxidative stress is one of the mechanisms involved in the neuronal damage of stroke. Oxidative stress is a state of imbalance between free radical production, in particular, reactive oxygen species (ROS), and the ability of the organism to neutralize them, leading to progressive oxidative damage. Smoking is known to result in the generation of various free radicals. Flow cytometric analysis of the platelets of thrombo-embolic ischemic stroke patients and smokers revealed that the membranes of the two groups were altered in some form as well as an increased activation in both groups when compared to healthy individuals. Superoxide levels in the platelets were higher in smokers when compared to stroke patients, while hydrogen peroxide levels were elevated in the platelets of both groups. Superoxide was elevated in the whole blood samples of both groups. The production and subsequent reactions of reactive oxygen species appear to be influential in stroke and smoking and may likely be a crucial factor in the development of a pro-thrombotic, pro-inflammatory state which may prove to be a hallmark in the pathophysiology of stroke and smoking. Confocal microscopy and Scanning electron microscopy showed that platelets of stroke patients and smokers appear to be more activated and more prone to form tight clots. Furthermore an increased amount of superoxide is present in the platelets of stroke patients and smokers, specifically in the centre of clots. This may be an indication that once platelets have aggregated and started to fuse together, the mitochondria are expelled from the platelets and “trapped” within the clot. Atomic force microscopy also indicated both the stroke patients and smoker‟s platelets appear to be in a more activated state than the control group. Here it is apparent that some form of cytoskeletal rearrangement takes place to a more severe extent in the stroke group than in the smokers. Necrosis may be present in the platelets of stroke patients while neither apoptosis nor necrosis can be identified in the platelets of smokers however some form of membrane alteration is likely present. All the techniques used showed an increase in platelet activation in stroke patients and smokers, necrotic platelets may be present in the stroke patients while the platelet membrane of smokers seems to be altered. ROS is present and alters the platelet function of smokers and stroke patients in some way. It appears as if thrombo-embolic ischemic stroke patients and smokers‟ platelets have similar trends in activation but the processes involved to achieve this differ as there are structural differences present. These differences may prove a useful tool to further understand the pathophysiology behind thrombo-embolic ischemic stroke as well as to discover new therapeutic pathways. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Physiology / Unrestricted

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....

Following a cortical injury, neurons in areas near and connected to the site of injury begin to degenerate. The observed neuronal death may contribute to the severity of the observed behavioral impairments. The purpose of the present study was to examine if progesterone, a hormone known for its effectiveness at reducing cerebral edema, could protect against secondary neuronal death and facilitate the acquisition of an avoidance learning task in an ablation model of cortical injury. Rats served as sham controls or received bilateral ablation of the medial prefrontal cortex followed by a 10-day regimen of progesterone (4 mg/kg) or oil vehicle (1 ml/kg) beginning 1 h after cortical lesions. Progesterone-treated lesion rats showed a significant facilitation of avoidance learning compared to oil- treated lesion controls. In addition, progesterone-treated lesion animals did not differ from either progesterone- or oil-treated sham controls in avoidance learning. Anatomical analysis revealed that progesterone treatment decreased the amount of neuronal death seen in the striatum and the mediodorsal nucleus of the thalamus. The findings are consistent with the notion that progesterone is an effective neuroprotective agent and suggest that the hormone can reduce the behavioral impairments associated with frontal cortical ablation injury.

Resistance to tissue hypoxia is a robust fundamental adaptation to low oxygen supply, and represents a novel neuroscience problem with significance to mammalian physiology as well as human health. With the underlying mechanisms strongly conserved in evolution, the ability to resist tissue hypoxia in natural systems has recently emerged as an interesting model in mammalian physiology research to understand mechanisms that can be manipulated for the clinical management of stroke. The extraordinary ability to resist tissue hypoxia by the naked mole rat (NMR) indicates the presence of a unique mechanism that underlies the remarkable healthy life span and exceptional hypoxia resistance. This opens an interesting line of research into understanding the mechanisms employed by the naked mole rat (. Heterocephalus glaber) to protect the brain during hypoxia. In a series of studies, we first examined the presence of neuroprotection in the brain cells of naked mole rats (NMRs) subjected to hypoxic insults, and then characterized the expression of such neuroprotection in a wide range of time intervals. We used oxygen nutrient deprivation (OND), an in vitro model of resistance to tissue hypoxia to determine whether there is evidence of neuronal survival in the hippocampal (CA1) slices of NMRs that are subjected to chronic hypoxia. Hippocampus neurons of NMRs that were kept in hypoxic condition consistently tolerated OND right from the onset time of 5. h. This tolerance was maintained for 24. h. This finding indicates that there is evidence of resistance to tissue hypoxia by CA1 neurons of NMRs. We further examined the effect of hypoxia on metabolic rate in the NMR. Repeated measurement of metabolic rates during exposure of naked mole rats to hypoxia over a constant ambient temperature indicates that hypoxia significantly decreased metabolic rates in the NMR, suggesting that the observed decline in metabolic rate during hypoxia may contribute to the adaptive mechanism used by the NMR to resist tissue hypoxia. This work is aimed to contribute to the understanding of mechanisms of resistance to tissue hypoxia in the NMR as an important life-sustaining process, which can be translated into therapeutic interventions during stroke.