Prion diseases are fatal and incurable neurodegenerative diseases that share many pathological similarities to other neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease. One of the earliest pathological signs commonly detected in all of these diseases is the dysfunction followed by loss of neuronal synapses, spines and eventually dendrites that collectively contribute to disruption of normal brain function. These pathologies tend to progressively accumulate within the brain tissue such that extensive damage typically precedes clinical symptom manifestation and ultimate death of neurons. Clearly, understanding the molecular processes responsible for these pathologies could uncover critical pathway(s) that are responsible for propagating this brain damage and could therefore be exploited for therapy development. However, molecular mechanisms implicated in this early pathology remain unidentified. To address this gap in knowledge, this thesis describes a transcriptional approach coupled with specific isolation of neuronal-enriched tissue which was used to help delineate cellular pathways involved in prion-induced neurodegeneration. Profiling cell bodies of CA1 hippocampal neurons known to be affected during early prion disease revealed temporal alteration in both gene and microRNA (gene regulators) expression throughout disease. On a gene expression level, changes in transcript expression during preclinical disease were reminiscent of an activity-dependent neuroprotective gene signature previously described in the literature. These neuroprotective genes were induced during preclinical disease, diminished as disease progressed and were abolished at clinical disease. In support of this process, upregulation of the phosphorylated form of the neuroprotective transcription factor CREB was detected during preclinical disease in these neurons. Furthermore, several genes known to be induced by CREB were also upregulated at preclinical disease in prion-infected mice. Interestingly, expression of numerous deregulated microRNAs paralleled the neuroprotective gene signature of which several are known to remodel neuronal spines and dendrites. To determine whether other preclinically induced microRNAs were also capable of remodeling neuronal structures, gain-of-function studies were performed in primary mouse hippocampal neurons for the uncharacterized miR-26a-5p. Neurons over-expressing miR-26a-5p had enhanced spine density and dendrite arborization, similar to other preclinically-induced microRNAs. Together, these data suggests that CA1 hippocampal neurons induce a neuroprotective transcriptional signature that is evident early in the course of disease within CA1 hippocampal neurons and is abolished by clinical disease. Reestablishment of key molecules that can induce this neuroprotective signature at a time when these genes begin to dissipate could prolong prion disease onset and delay clinical symptom manifestation. / October 2015

Background and motivation: The event of neural Ca2+ overload is known to have several deleterious effects resulting in cell death caused by ischaemia, hypoglycaemia, hypoxia and several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and AIDS-related dementia. In vitro models for the investigation of the mechanisms involved in Ca2+ overload include brain slice preparations, neuronal cultures as well as acutely isolated neurons, mostly from the hippocampus and cortical brain areas. Additional models for investigating Ca2+ overload may bring about new knowledge to areas of the phenomenon that are still unresolved. Methodology: In this study, several theoretical Ca2+ overload-related interventions were combined aimed at inducing cell death in acutely isolated rat dorsal root ganglia. To elucidate the mechanism/s involved in the cell death observed following exposure to this intervention, the effects of several alterations to the intervention's composition were assessed. This examination was extended by the addition of several recognized and potential protective compounds to the intervention. Cell death was indicated by the trypan blue exclusion assay and recorded after 18 hours exposure to the interventions by counting live and dead neurons under a light microscope. Results and conclusions: The goal was to evaluate the possible application of dorsal root ganglia as a model for neural Ca2+ overload outside the brain. Since Ca2+w as required for cell death to be induced, it is concluded that the observed cell death was indeed primarily due to Ca2+ overload. Besides extracellular Ca2+, KC1-induced depolarization was also required for cell death to be induced, while the antagonists did not demonstrate significant protection against cell death. Based on the results, the mechanism of Ca2+ overload could not be defined beyond doubt, but the voltage activated Ca2+ channels are likely to be involved. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2005.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ionotropic receptors. When activated, NMDARs let extracellular sodium and calcium ions enter neurons. This calcium influx, depending on its duration, intensity and the presence of nearby signalling proteins can signal to synaptic plasticity. Additionally, physiological NMDAR activity promotes pro-survival cascades and gene transcription, whereas both lack of activation and overactivation of these receptors trigger pro-death signals. Several neurodegenerative pathologies such as stroke/ischemia and Alzheimer’s disease are thought to involve NMDAR overactivation, so-called “excitotoxicity”, but since NMDARs are important for normal neuronal physiology, potential therapeutical approaches needs to go beyond simple antagonism. Here, we studied the receptor subunit composition and the molecular cascades downstream of the receptor activation to try and isolate the pro-death pathways in NMDAR-mediated excitotoxicity. We found that the NR2 subunit composition did not dictate the type of NMDAR-mediated signals, as receptors comprised of NR2B subunits were able to signal to death, survival and plasticity. However, we also found that the intracellular tail of the NR2B subunit was more efficient at triggering neuronal death compared to the NR2A C-terminus, which suggests that different pro-death signalling complexes are associated to each subunit. Two pro-death signals, the p38 and c-Jun N-terminal kinase (JNK) cascades, are key mediators of neuronal excitotoxicity. In a non-neuronal cell line, NMDAR-mediated cell death could be reconstituted but was found to rely solely on JNK and not p38. This was due to the lack of pro-death signals from the NR2B-PDZ domain, a cytoplasmic interacting domain which forms a signalling cassette with the neuronal proteins PSD-95 and neuronal nitric oxide synthase. This PDZ-ligand recruits the p38 cascade in neurons, but was absent in non-neuronal cells. The pro-death p38 pathway could be inhibited in neurons by disrupting the PDZ domain interactions, which protects against excitotoxicity. This disruption was not affecting normal synaptic transmission, potentiation or survival signalling, suggesting that this could be a therapeutically viable avenue. Thus, this work has expanded the understanding of how NMDAR subunits and their cytoplasmic domains mediate signalling leading to a variety of cellular outcomes; a crucial point for the development of a strategy specifically targeting NMDAR- mediated pro-death signalling.

Le vascular endothelial growth factor (VEGF) un facteur de croissance essentiel du système vasculaire exerce des fonctions multiples sur les cellules nerveuses en favorisant la neurogenèse, la plasticité synaptique ou encore l'apprentissage et la mémoire. Cependant, les mécanismes impliqués dans son action régulatrice de la transmission et la plasticité synaptiques restent à élucider. Nous avons récemment mis en évidence une nouvelle interaction entre VEGFR2, le récepteur principal du VEGF, et les récepteurs NMDA (NMDAR) au cours de la migration des neurones pendant le développement du cervelet. Comme les NMDAR sont des acteurs clés de la transmission et de la plasticité synaptique, nous avons exploré le rôle du VEGF dans la régulation de l'expression et de la fonction des NMDAR synaptiques dans l'hippocampe. Nos résultats révèlent que le VEGF et son récepteur sont exprimés dans les régions CA1 et CA3 de l'hippocampe et le domaine extracellulaire de VEGFR2 peut se lier à la sous-unité GluN2B des NMDAR. Le VEGF est capable d'augmenter la transmission synaptique dépendant des NMDAR en régulant l'adressage synaptique des récepteurs exprimant la sous-unité GluN2B. Il se produit également une augmentation du nombre de synapses en présence du VEGF. Ces effets du VEGF requièrent la co-activation des récepteurs VEGFR2 et NMDAR et conduisent à un enrichissement synaptique en récepteurs glutamatergiques de type AMPA qui dépend de l'activation de la CaMKII. Nos travaux démontrent pour la première fois un rôle direct de la signalisation VEGF/VEGFR2 dans la fonction de la synapse excitatrice glutamatergique

Background and motivation: The event of neural Ca2+ overload is known to have several deleterious effects resulting in cell death caused by ischaemia, hypoglycaemia, hypoxia and several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and AIDS-related dementia. In vitro models for the investigation of the mechanisms involved in Ca2+ overload include brain slice preparations, neuronal cultures as well as acutely isolated neurons, mostly from the hippocampus and cortical brain areas. Additional models for investigating Ca2+ overload may bring about new knowledge to areas of the phenomenon that are still unresolved. Methodology: In this study, several theoretical Ca2+ overload-related interventions were combined aimed at inducing cell death in acutely isolated rat dorsal root ganglia. To elucidate the mechanism/s involved in the cell death observed following exposure to this intervention, the effects of several alterations to the intervention's composition were assessed. This examination was extended by the addition of several recognized and potential protective compounds to the intervention. Cell death was indicated by the trypan blue exclusion assay and recorded after 18 hours exposure to the interventions by counting live and dead neurons under a light microscope. Results and conclusions: The goal was to evaluate the possible application of dorsal root ganglia as a model for neural Ca2+ overload outside the brain. Since Ca2+w as required for cell death to be induced, it is concluded that the observed cell death was indeed primarily due to Ca2+ overload. Besides extracellular Ca2+, KC1-induced depolarization was also required for cell death to be induced, while the antagonists did not demonstrate significant protection against cell death. Based on the results, the mechanism of Ca2+ overload could not be defined beyond doubt, but the voltage activated Ca2+ channels are likely to be involved. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2005.

Fragile-X syndrome (FXS) is the most common form of inherited intellectual impairment. Caused by the transcriptional repression of the Fmr1 gene on the X chromosome, FXS results in the loss of the Fragile-X Mental Retardation Protein (FMRP). Human female patients with FXS are heterozygous for the Fmr1 mutation whereas males are hemizygous. FXS has been studied far less in females than in males due to a generally less severe clinical phenotype. Previous research has implicated the metabotropic glutamate receptor (mGluR) in synaptic plasticity alterations in the cornu ammonis area 1 (CA1) region of the juvenile male Fmr1 knock-out (KO) hippocampus. In contrast, our investigations into the young adult dentate gyrus (DG) subfield of the hippocampus have revealed N-methyl-D-aspartate receptor (NMDAR)-associated impairments in synaptic plasticity. The current study sought to extend these investigations to the young adult female Fmr1 heterozygous (Het) and Fmr1 KO mouse as well as investigate NMDAR- and mGluR-mediated long-term depression (LTD) in the DG and CA1 of the young adult male Fmr1 KO mouse. Input-output curves and paired pulse measures of short-term plasticity were also evaluated in all genotypes. Field electrophysiology revealed a significant impairment in long-term potentiation (LTP) and LTD in male Fmr1 KO and female Fmr1 Het mice that was associated with NMDAR alteration. A more robust synaptic protocol was not able to rescue LTP in the male Fmr1 KO DG. Paired-pulse low-frequency stimulation and (RS)-3,5-dihydroxyphenylglycine (DHPG)-induced mGluR-LTD was intact in all genotypes and brain regions examined. Although further investigation will be required to expand our understanding of FXS and to fully elucidate the mechanisms behind intact synaptic plasticity in the female Fmr1 KO mouse, our results suggest that NMDARs may be poised as important contributors to hippocampal pathophysiology in FXS. / Graduate

A cetamina é uma droga dissociativa utilizada como anestésico humano. Este fármaco é também utilizado como droga de abuso por conter efeitos psicotrópicos. É sabido que a cetamina é um potente antagonista não competitivo do receptor glutamatérgico ionotrópico do tipo N metil-D-aspartato (NMDAr). A diminuição da neurotransmissão do glutamato pelos receptores NMDA está associada com a alteração da percepção, memória e cognição. As neurotrofinas constituem uma família de fatores de crescimento multifuncionais crucial na sobrevivência, diferenciação, proliferação e manutenção de populações neuronais do sistema nervoso e exercem numerosos efeitos em células não neuronais. O fator neurotrófico derivado do cérebro (Brain-derived neurotrophic factor - BDNF) é uma neurotrofina que atua tanto no sistema nervoso central como no sistema nervoso periférico e está relacionada com a sobrevivência dos neurônios, crescimento, diferenciação de novos neurônios e sinapses. O objetivo deste trabalho foi avaliar os efeitos de diferentes doses de cetamina na memória de longa duração no modelo de reconhecimento de novo objeto e analisar os níveis de BDNF no hipocampo dos ratos após injeção intraperitoneal (i.p.) de cetamina. Ratos Wistar machos adultos foram treinados em uma tarefa de reconhecimento de novo objeto e a retenção da memória foi avaliada 24 h após o treino. A administração pós-treino de cetamina prejudicou de forma dose-dependente a retenção da memória de reconhecimento. Experimentos controle mostraram que a cetamina não afetou a memória quando administrada 6 h após o treino ou 24 h antes do treino. Para a dosagem dos níveis de BDNF no hipocampo dorsal, os animais foram treinados e imediatamente receberam salina ou 20 mg/kg de cetamina e foram sacrificados 3 h mais tarde. / Ketamine is a dissociative drug used as a human anesthetic. This drug is also used as a drug of abuse because it contains psychotropic effects. It is known that ketamine is a potent noncompetitive antagonist of glutamatergic ionotropic receptors type N-methyl D-aspartate (NMDAr). The decrease in the neurotransmission of glutamate receptors is associated with the change of perception, memory and cognition. Neurotrophins constitute a family of multifunctional growth factors crucial for the survival and differentiation, proliferation and maintenance of neuronal populations of the nervous system and has numerous effects in non-neuronal cells. The brain-derived neurotrophic factor (BDNF) is a neurotrophin that acts both in the central nervous system and peripheral nervous system and is related to neuron survival, growth, differentiation of new neurons and synapses. The objective of this study was to evaluate the effects of different ketamine doses on long-term memory in the novel object recognition model and analyze the levels of BDNF in the hippocampus of rats after injection (ip) of ketamine. Adult male Wistar rats were trained in a task of recognition of new object and memory retention was evaluated 24h after training. The post-training administration of ketamine impaired in a dose-dependent retention of recognition memory. Control experiments showed that ketamine had no effect on memory when administered 6 h after training or 24 h before training. To measure the levels of BDNF in the dorsal hippocampus, the animals were trained and immediately injected with saline or 20 mg / kg of ketamine and sacrificed 3h later. A kit of sandwich enzyme immunoassay with rabbit monoclonal antibody against BDNF was used to measure the levels of BDNF.

Immune mechanisms are thought to be involved in the pathological disease process in a number of childhood epileptic syndromes and encephalitis. Of particular interest is the occurrence of autoantibodies to essential neuronal proteins, for example the N-methyl-D-aspartate receptor (NMDAR), in the blood and spinal fluid in some of these patients. The aims of this study were: to examine the sera of newly diagnosed paediatric epilepsy patients for specific neuronal autoantibodies, correlate to epilepsy phenotype and disease outcomes; to investigate the pathogenicity and epileptogenicity of central nervous system autoantibodies (CNS) in vivo; and to test new therapies in vitro and in vivo based on the potential pathogenic mechanisms. In 290 paediatric patients with new-onset epilepsy and seizures tested for CNS autoantibodies, 11.4% were positive (33/290 versus 8/112 in controls; p=0.01, Fisher's exact test). Previously unreported contactin-2 antibody positive and contactin-associated-protein 2 (CASPR2) antibody positive epilepsy patients were described. Patients with 'focal epilepsy of unknown cause' were more likely to be antibody positive. To test the pathogenicity and epileptogenicity of these antibodies, a novel in vivo telemetry system was used to continuously record electroencephalogram (EEG) in mice injected into the cerebral lateral ventricle with NMDAR antibody (NMDAR-Ab) positive immunoglobulin (IgG). Although no spontaneous seizures were seen, mice challenged with the pro-convulsant pentylenetetrazole (PTZ) had increased seizure susceptibility, and more epileptiform "spikes" in the EEG after PTZ compared to healthy control (HC) IgG injected mice. Seizure susceptibility strongly correlated with binding intensity of NMDAR-Ab IgG analysed in post-mortem tissue. Given the hypothesis this epileptogenic effect was mediated by NMDAR-Abs internalising cell surface NMDARs, and to try and rescue this deficit, a neurosteroid, pregnenolone sulphate (PregS) known to increase NMDAR cell surface expression, was therapeutically used. This approach worked in vitro, and although in vivo effects were not yet established, treatment with neurosteroids may be beneficial for autoantibody mediated neurological disease.

A cetamina é uma droga dissociativa utilizada como anestésico humano. Este fármaco é também utilizado como droga de abuso por conter efeitos psicotrópicos. É sabido que a cetamina é um potente antagonista não competitivo do receptor glutamatérgico ionotrópico do tipo N metil-D-aspartato (NMDAr). A diminuição da neurotransmissão do glutamato pelos receptores NMDA está associada com a alteração da percepção, memória e cognição. As neurotrofinas constituem uma família de fatores de crescimento multifuncionais crucial na sobrevivência, diferenciação, proliferação e manutenção de populações neuronais do sistema nervoso e exercem numerosos efeitos em células não neuronais. O fator neurotrófico derivado do cérebro (Brain-derived neurotrophic factor - BDNF) é uma neurotrofina que atua tanto no sistema nervoso central como no sistema nervoso periférico e está relacionada com a sobrevivência dos neurônios, crescimento, diferenciação de novos neurônios e sinapses. O objetivo deste trabalho foi avaliar os efeitos de diferentes doses de cetamina na memória de longa duração no modelo de reconhecimento de novo objeto e analisar os níveis de BDNF no hipocampo dos ratos após injeção intraperitoneal (i.p.) de cetamina. Ratos Wistar machos adultos foram treinados em uma tarefa de reconhecimento de novo objeto e a retenção da memória foi avaliada 24 h após o treino. A administração pós-treino de cetamina prejudicou de forma dose-dependente a retenção da memória de reconhecimento. Experimentos controle mostraram que a cetamina não afetou a memória quando administrada 6 h após o treino ou 24 h antes do treino. Para a dosagem dos níveis de BDNF no hipocampo dorsal, os animais foram treinados e imediatamente receberam salina ou 20 mg/kg de cetamina e foram sacrificados 3 h mais tarde. / Ketamine is a dissociative drug used as a human anesthetic. This drug is also used as a drug of abuse because it contains psychotropic effects. It is known that ketamine is a potent noncompetitive antagonist of glutamatergic ionotropic receptors type N-methyl D-aspartate (NMDAr). The decrease in the neurotransmission of glutamate receptors is associated with the change of perception, memory and cognition. Neurotrophins constitute a family of multifunctional growth factors crucial for the survival and differentiation, proliferation and maintenance of neuronal populations of the nervous system and has numerous effects in non-neuronal cells. The brain-derived neurotrophic factor (BDNF) is a neurotrophin that acts both in the central nervous system and peripheral nervous system and is related to neuron survival, growth, differentiation of new neurons and synapses. The objective of this study was to evaluate the effects of different ketamine doses on long-term memory in the novel object recognition model and analyze the levels of BDNF in the hippocampus of rats after injection (ip) of ketamine. Adult male Wistar rats were trained in a task of recognition of new object and memory retention was evaluated 24h after training. The post-training administration of ketamine impaired in a dose-dependent retention of recognition memory. Control experiments showed that ketamine had no effect on memory when administered 6 h after training or 24 h before training. To measure the levels of BDNF in the dorsal hippocampus, the animals were trained and immediately injected with saline or 20 mg / kg of ketamine and sacrificed 3h later. A kit of sandwich enzyme immunoassay with rabbit monoclonal antibody against BDNF was used to measure the levels of BDNF.

A cetamina é uma droga dissociativa utilizada como anestésico humano. Este fármaco é também utilizado como droga de abuso por conter efeitos psicotrópicos. É sabido que a cetamina é um potente antagonista não competitivo do receptor glutamatérgico ionotrópico do tipo N metil-D-aspartato (NMDAr). A diminuição da neurotransmissão do glutamato pelos receptores NMDA está associada com a alteração da percepção, memória e cognição. As neurotrofinas constituem uma família de fatores de crescimento multifuncionais crucial na sobrevivência, diferenciação, proliferação e manutenção de populações neuronais do sistema nervoso e exercem numerosos efeitos em células não neuronais. O fator neurotrófico derivado do cérebro (Brain-derived neurotrophic factor - BDNF) é uma neurotrofina que atua tanto no sistema nervoso central como no sistema nervoso periférico e está relacionada com a sobrevivência dos neurônios, crescimento, diferenciação de novos neurônios e sinapses. O objetivo deste trabalho foi avaliar os efeitos de diferentes doses de cetamina na memória de longa duração no modelo de reconhecimento de novo objeto e analisar os níveis de BDNF no hipocampo dos ratos após injeção intraperitoneal (i.p.) de cetamina. Ratos Wistar machos adultos foram treinados em uma tarefa de reconhecimento de novo objeto e a retenção da memória foi avaliada 24 h após o treino. A administração pós-treino de cetamina prejudicou de forma dose-dependente a retenção da memória de reconhecimento. Experimentos controle mostraram que a cetamina não afetou a memória quando administrada 6 h após o treino ou 24 h antes do treino. Para a dosagem dos níveis de BDNF no hipocampo dorsal, os animais foram treinados e imediatamente receberam salina ou 20 mg/kg de cetamina e foram sacrificados 3 h mais tarde. / Ketamine is a dissociative drug used as a human anesthetic. This drug is also used as a drug of abuse because it contains psychotropic effects. It is known that ketamine is a potent noncompetitive antagonist of glutamatergic ionotropic receptors type N-methyl D-aspartate (NMDAr). The decrease in the neurotransmission of glutamate receptors is associated with the change of perception, memory and cognition. Neurotrophins constitute a family of multifunctional growth factors crucial for the survival and differentiation, proliferation and maintenance of neuronal populations of the nervous system and has numerous effects in non-neuronal cells. The brain-derived neurotrophic factor (BDNF) is a neurotrophin that acts both in the central nervous system and peripheral nervous system and is related to neuron survival, growth, differentiation of new neurons and synapses. The objective of this study was to evaluate the effects of different ketamine doses on long-term memory in the novel object recognition model and analyze the levels of BDNF in the hippocampus of rats after injection (ip) of ketamine. Adult male Wistar rats were trained in a task of recognition of new object and memory retention was evaluated 24h after training. The post-training administration of ketamine impaired in a dose-dependent retention of recognition memory. Control experiments showed that ketamine had no effect on memory when administered 6 h after training or 24 h before training. To measure the levels of BDNF in the dorsal hippocampus, the animals were trained and immediately injected with saline or 20 mg / kg of ketamine and sacrificed 3h later. A kit of sandwich enzyme immunoassay with rabbit monoclonal antibody against BDNF was used to measure the levels of BDNF.