Raymond Hiu Yeung, Li. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 51-69). / Acknowledgment --- p.iii / Abstract --- p.iv / List of Abbreviations --- p.vii / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1. --- Parkinson's Disease --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Clinical symptoms --- p.2 / Chapter 1.1.3 --- Neuropathology --- p.3 / Chapter 1.2 --- Proposed mechanisms of Neuronal Cell Death in PD / Chapter 1.2.1 --- Oxidative Stress --- p.4 / Chapter 1.2.2 --- Mitochondrial Dysfunction --- p.5 / Chapter 1.2.3 --- Excitotoxicity --- p.6 / Chapter 1.2.4 --- Genetic Factor --- p.8 / Chapter 1.2.5 --- Aging --- p.9 / Chapter 1.3 --- "1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a PD model" / Chapter 1.3.1 --- The discovery of MPTP --- p.10 / Chapter 1.3.2 --- The mechanism of MPTP toxicity --- p.10 / Chapter 1.4 --- Reactive Oxygen Species (ROS) and Antioxidants in CNS / Chapter 1.4.1 --- Superoxide and Superoxide Dismutases --- p.13 / Chapter 1.4.2 --- "Hydrogen Peroxide, Catalase and Glutathione System" --- p.14 / Chapter 1.4.3 --- Hydroxyl Radicals --- p.15 / Chapter 1.4.4 --- Nitric Oxide (NO) --- p.16 / Chapter 1.5 --- Astrocytes / Chapter 1.5.1 --- Characteristics of astrocytes --- p.20 / Chapter 1.5.2 --- The role of astrocytes in PD --- p.21 / Chapter 1.6 --- The aim of this project --- p.24 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Astrocyte cultures --- p.27 / Chapter 2.2 --- MPTP treatment --- p.28 / Chapter 2.3 --- Lactate Dehydrogenase Assay --- p.29 / Chapter 2.4 --- Determination of nitrite and nitrate levels in cultured astrocytes --- p.30 / Chapter 2.5 --- Assay for Cyclic GMP production --- p.32 / Chapter 2.6 --- Inhibition of NO by L-NAME and Dexamethasone --- p.33 / Chapter 2.7 --- NFkB immunostaining --- p.33 / Chapter 2.8 --- Superoxide Dismutase Assay --- p.34 / Chapter 2.9 --- Statistics --- p.36 / Chapter CHAPTER 3: --- RESULTS / Chapter 3.1 --- Lactate dehydrogenase (LDH) activities after MPTP treatment --- p.37 / Chapter 3.2 --- The effects of MPTP on nitrite levels --- p.37 / Chapter 3.2.1 --- Mesencephalic astrocytes --- p.37 / Chapter 3.2.2 --- Striatal astrocytes --- p.38 / Chapter 3.2.3 --- Cortical astrocytes --- p.38 / Chapter 3.3 --- The effects of L-NAME on nitrite levels after MPTP treatment --- p.38 / Chapter 3.4 --- The effects of dexamethasone on nitrite levels after MPTP treatment --- p.39 / Chapter 3.5 --- Change in intracellular cyclic GMP in astrocytes after MPTP treatment --- p.40 / Chapter 3.6 --- The effects of MPTP on NFkB distribution in astrocytes --- p.40 / Chapter 3.7 --- The effects of MPTP on SOD activity in astrocytes --- p.41 / Chapter 3.7.1 --- Mesencephalic astrocytes --- p.41 / Chapter 3.7.2 --- Striatal astrocytes --- p.41 / Chapter 3.7.3 --- Cortical astrocytes --- p.42 / Chapter CHAPTER 4: --- DISCUSSION AND CONCLUSION --- p.43 / REFERENCES --- p.51

Recentemente, com o intuito de identificar genes associados com invasão e proliferação tumoral, identificamos por PCR em tempo real, um aumento de aproximadamente cem vezes da proteína PLP2 em glioblastomas em relação a tecidos normais. Até o momento não há nenhum relato da identificação desta proteína em astrocitomas. Portanto, neste trabalho clonamos e expressamos em bactérias, as alças externas da PLP2 em fusão com a proteína SUMO, com o objetivo de obtermos anticorpos policlonais para serem usados na identificação da PLP2 em tumor humano por western blotting. Realizamos também a expressão da PLP2 fusionada com a EGFP em células de mamífero, para estudar sua distribuição celular, observamos que a PLP2 se concentra em toda a membrana celular e estudos sobre o transito da PLP2 nas células, indicam que ela possa estar envolvida em processos quimiotáticos via CCR1 sugerindo o envolvimento da PLP2 de alguma forma no processo tumorigênico. / Recently, in order to identify genes associated with tumoral invasion and proliferation, identified by real time PCR, an increase of about one hundred times of PLP2 protein in glioblastomas when compared to normal tissue. So far, there is no report of identification of this protein in astrocytomas. Therefore in this study, we cloned and expressed in bacteria the external handles of PLP2 fused with SUMO protein in order to obtain polyclonal antibodies for use in identifying the PLP2 in human tumor by western blotting. We also expressing the PLP2 fused with EGFP in mammalian cells to study its cellular distribution, we observed that focuses PLP2 across the cell membrane and studies on the traffic of PLP2 cells, indicate that it may be involved in chemotactic processes via CCR1 suggesting the involvement of PLP2 somehow in the tumorigenic process.

A epilepsia do lobo temporal (ELT) é a um dos casos mais frequente epilepsia em humanos e de maior refratariedade nos pacientes. A maioria dos fármacos antiepilépticos são moduladores da atividade neuronal e atuam sobre canais iônicos do receptor GABAA. Estudos vêm demonstrando o papel das células gliais e da neuroinflamação na epileptogênese e a modulação desta resposta pode ser um alvo potencial para drogas adjuvantes aos fármacos anti-epilépticos. Astrócitos são células gliais participantes da sinapse tripartite, moduladores da atividade neuronal. Os astrócitos são capazes de promover a homeostase de íons e de neurotransmissores, são responsáveis pelo metabolismo energético e da produção de fatores neurotróficos, glutationa, glutamina, S100B e citocinas. Neste trabalho, induzimos status epilepticus (SE) em ratos jovens (PN28) através do modelo lítio-pilocarpina que mimetiza alterações neuronais, bioquímicas e morfológicas similares à ELT em humanos. Os animais foram divididos nos tempos 1, 14 e 56 dias após a indução de status epilepticus (SE). Estes períodos são caracterizados respectivamente como a fase aguda, latente e crônica da epilepsia. Inicialmente, analisamos as mudanças neuroquímicas e astrocitárias ao longo do tempo. Foi observada neuroinflamação inicial e transitória que promove morte neuronal e mudanças ao longo do tempo de astrogliose e disfunção astrocitária. Também foi observado que a proteína S100B, proteína ligante de cálcio, predominantemente astrocitária, pode ser considerado um marcador da disfunção neuronal e astrocitária promovida neste modelo de epilepsia. Em seguida, demonstramos que a modulação da secreção de S100B pelo anti-inflamatório dexametasona um dia após indução de SE reverte a neuroinflamação, astrogliose e disfunção astrocitária à curto e à longo prazo. Por conseguinte, observamos que a modulação do receptor GABAA através de agonistas e antagonistas GABAérgicos altera a secreção de S100B em fatias hipocampais agudas e em cultura de astrócitos. Portanto, pode-se sugerir que as alterações astrogliais e a neuroinflamação dependentes do tempo podem estar ligadas à excitabilidade neuronal e/ou à morte neuronal em ratos jovens em modelo de epilepsia; que a proteína S100B pode ser considerada um marcador deste modelo de epilepsia e que a modulação da sua secreção pode ser um possível alvo farmacológico no tratamento da epilepsia. / Temporal lobe epilepsy (TLE) is the most frequent type of epilepsy in humans and is more associated to refractory to anti-epileptic drugs (AED) in patients. The most AEDs are modulators of neuronal activity and act on ion channels, such as GABAA receptor. Studies have been demonstrating the role of glial cells and neuroinflammation in epileptogenesis. The modulation of this response may be a potential target for adjunctive drugs to anti-epileptic drugs. Astrocytes are glial cells that participated in the tripartite synapse and modulated neuronal activity. Astrocytes are able to promote homeostasis of ions and neurotransmitters, are responsible for energy metabolism and the production of neurotrophic factors, glutathione, glutamine, S100B and cytokines. In this work, we induced status epilepticus (SE) in young rats (PN28) through the lithiumpilocarpine model that mimics neuronal, biochemical and morphological alterations similar to ELT in humans. The animals were divided at times 1, 14 and 56 days after the induction of SE. These periods are characterized respectively as the acute, latent and chronic phase of epilepsy. Initially, we analyzed neurochemical and astrocytic changes over time. Initial and transient neuroinflammation was observed and promoted over time neuronal death, astrogliosis and astrocytic dysfunction. It has also been observed that the protein S100B, a calcium-binding protein, predominantly astrocytic, can be considered a marker of neuronal and astrocytic dysfunction promoted by this model of epilepsy. Next, we demonstrate that the modulation of S100B secretion by the antiinflammatory dexamethasone one day after SE induction reverses neuroinflammation, astrogliosis and astrocytic dysfunction in the acute and chronic time. Therefore, we analyzed that modulation of the GABAA receptor through GABAergics agonists and antagonists alters the secretion of S100B in acute hippocampal slices and in astrocyte culture. Therefore, it may be suggested that astroglial changes and time dependent neuroinflammation may be related to neuronal excitability and/or neuronal death in young rats in this epilepsy model; that S100B protein can be considered a marker of this epilepsy model and that the modulation of its secretion may be a possible pharmacological target in the treatment of epilepsy.

Afecções musculoesqueléticas crônicas são um problema de saúde pública devido à sua alta prevalência, seu alto custo econômico e por seu impacto negativo na qualidade de vida de pacientes e seus familiares. Diante disso, pesquisadores têm estudado lesões musculares em modelos animais para melhor compreensão dos mecanismos envolvidos na iniciação e manutenção de distúrbios musculoesqueléticos. Na tentativa de esclarecer os mecanismos nociceptivos envolvidos neste processo e encontrar terapias efetivas, nosso grupo de pesquisa vem utilizando modelos experimentais de dor e potenciais tratamentos farmacológicos e não farmacológicos como objeto de estudo. O presente estudo teve como finalidade avaliar se o tratamento farmacológico com gabapentina é eficaz em reverter a dor muscular de ratos com miosite crônica induzida pela injeção de Adjuvante de Freund Completo (CFA) no músculo gastrocnêmio, e ainda, analisar a influência da gabapentina em células gliais e citocinas pró e anti-inflamatórias no sistema nervoso central e periférico destes animais. Nossos resultados demonstram o efeito da gabapentina em células gliais. Esta ação induz uma diminuição na expressão de astrócitos e microglias, levando a um aumento de citocinas anti-inflamatórias e inibição de citocinas pró-inflamatórias e consequentemente uma melhora do quadro nociceptivo em nosso modelo experimental. / Chronic musculoskeletal disorders are a public health problem due to its high prevalence, high economic cost and negative impact on patients and their relatives quality of life. Due to this, researchers have been studying muscle injury in animal models to better understand the mechanisms involved in the initiation and maintenance of musculoskeletal disturbs. To elucidate the nociceptive mechanisms involved in this process and seek for effective therapies, our research group has been using experimental models of pain and potential pharmacological and non-pharmacological treatments as object of study. The aim of this study was to assess whether the treatment with gabapentin is effective in reversing the muscle pain injury in rats with chronic myositis induced by the injection of Complete Freund\'s Adjuvant (CFA) in the gastrocnemius muscle. Also to analyze the influence of gabapentin on glial cells and pro-and anti-inflammatory cytokines in the central and peripheral nervous system of these animals. Our results demonstrate the effect of gabapentin on glial cells. This action are able to decrease expression of astrocytes and microglia, leadind to an increase in anti-inflammatory cytokines and inhibition of proinflammatory cytokines and an improvement of the nociceptive picture in our experimental mode.

L'inflammation chronique relayée par la glie activée contribue à la dégénérescence des neurones dopaminergiques (ND) au cours de la maladie de Parkinson (MP). L'étendue des dégâts cellulaires provoqués par la réaction inflammatoire dépend de l'efficacité des mécanismes régulateurs de l'inflammation. Les glucocorticoïdes endogènes sont des régulateurs puissants de l'inflammation agissant via le récepteur des glucocorticoïdes (GR). Notre équipe a récemment montré le rôle central du GR microglial dans la régulation de la mort neuronale dont la sévérité est corrélée à l'intensité et la durée de l'inflammation. Mon projet de thèse a été d'étudier le rôle des GR microglial et astrocytaire dans la régulation des réponses inflammatoires au cours de la dégénerescence des ND. Dans la première partie de ma thèse, nous avons effectué une analyse transcriptomique comparative de microglie ex vivo isolée de souris traitées au MPTP (modèle de parkinsonisme) et avons identifié des gènes régulés par le GR microglial, potentiellement impliqués dans l'inflammation chronique. Dans la deuxième partie de ma thèse, nous avons mis en évidence la régulation par le GR microglial de la mort neuronale induite par l'activation de TLR9. L'ADN mitochondrial endogène peut engendrer la mort neuronale en activant le TLR9, en cas de dysfonction du GR microglial. Dans la troisième partie de mon travail, nous avons démontré que le GR astrocytaire régule la survie des ND en modulant l'expression de gènes pro-inflammatoires et l'activité excessive des hémicanaux à connexine 43. Globalement, les GR microglial et astroglial jouent des rôles essentiels dans la régulation de l'inflammation aigue et chronique. / Chronic inflammation, mounted by activated glia, contributes to dopamine neuron (DN) loss, a major hallmark of Parkinson’s disease. It can be postulated that the extent of DN injury inflicted by inflammation is affected by the efficacy of regulatory mechanisms. The activation of hypothalamic–pituitary–adrenal axis results in release of glucocorticoids, which activate glucocorticoid receptor (GR). GR exerts adaptive responses including resolution of inflammation to restore the homeostatic state. We previously demonstrated the role of microglial GR in regulating the intensity and duration of inflammation, which influences DN survival. My thesis was centered on dissecting the roles of microglial and astrocytic GR during DN degeneration in experimental Parkinsonism. In the first part of my thesis, we conducted comparative transcriptome experiments of ex vivo microglia acutely isolated from mice treated with MPTP (model of parkinsonism) and identified genes and pathways in microglia regulated by GR, potentially involved in chronic inflammation in PD. In the second part of my thesis, we found that microglial GR regulates Toll-Like Receptor 9-induced DN loss by regulating the lysosomal compartment and demonstrated that diminished sensitivity of GR in microglia creates a permissive environment for TLR9 activation by endogenous mitochondrial DNA to become lethal for DNs. In the third part of my work, we showed that during DN degeneration, astrocytic GR regulates inflammatory gene expression and prevents connexin-43 hemichannel activity that contributes to DN loss. Overall, both microglial and astrocytic GR play essential roles in regulating chronic and acute inflammation.

D'après le postulat de Hebb, les réseaux neuronaux adaptent leur connectivité sous l'influence des activités pré- et post-synaptiques. La " spike-timing-dependent plasticity " (STDP) est une règle d'apprentissage synaptique de type Hebbien, qui repose sur la structure temporelle précise des patrons d'activités appariées de part et d'autre de la synapse. La plasticité cortico-striatale serait le substrat biologique de l'apprentissage procédural effectué par les ganglions de la base. Les neurones de sortie du striatum agissent comme des détecteurs de coïncidence des activités corticales et thalamiques. La STDP cortico-striatale pourrait donc jouer un rôle crucial dans les processus d'encodage de l'apprentissage et la mémoire procédurale. Nous avons exploré les conditions d'émergence et d'expression de la STDP cortico-striatale. / According to Hebbian theory, neural networks refine their connectivity by patterned firing of action potentials in pre- and postsynaptic neurons. Spike-timing-dependent plasticity (STDP) is a synaptic Hebbian learning rule relying on the precise order and the millisecond timing of the paired activities on either side of the synapse. Temporal coding via STDP may be essential for the role of the striatum in learning of motor sequences in which sensory and motor events are associated in a precise time sequence. Corticostriatal long-term plasticity provides a fundamental mechanism for the function of the basal ganglia in procedural learning. Striatal output neurons act as detectors of distributed patterns of cortical and thalamic activity. Thus, corticostriatal STDP should play a major role in information processing in the basal ganglia, which is based on a precise time-coding process. Here, we explored the conditions required for the emergence of corticostriatal STDP.

Doctor of Philosophy (Medicine) / Glutamate transporters play a role in removing extracellular excitatory neurotransmitter, L-glutamate into the cells. The rate of the uptake depends on the density of the transporters at the membrane. Some studies claimed that glutamate transporters could transit between the cytoplasm and the membrane on a time-scale of minutes. The present study examined the distribution of glutamate transporter GLAST predominantly expressed in rat cortical cultured astrocytes between the membrane and the cytoplasm by using deconvolution microscopy and then analyzing the images. The regulation of the distribution of GLAST was studied in the presence of glutamate transporter substrate (D-aspartate), purinergic receptor activators (α,β-methylene ATP, adenosine), neuroleptic drugs (clozapine, haloperidol), ammonia (hyperammonia) and Na+/K+-ATPase inhibitors (ouabain, digoxin and FCCP). It was demonstrated that the translocation of GLAST towards the plasma membrane was induced by D-aspartate, α,β-methylene ATP, adenosine, clozapine and ammonia (at 100 μM and very high concentrations of 10 mM). However, the inhibition of Na+/K+-ATPase activity had an opposite effect, resulting in redistribution of GLAST away from the membrane. It has previously been claimed that the membrane-cytoplasm trafficking of GLAST was regulated by phosphorylation catalysed by protein kinase C delta (PKC-delta). Involvement of this mechanism has, however, been put to doubt when rottlerin, a PKC-delta inhibitor, used to test the hypothesis showed to inhibit Na+/K+-ATPase-mediated uptake of Rb+, suggesting that rottlerin influenced the activity of Na+/K+-ATPase. As Na+/K+-ATPase converts ATP to energy and pumps Na+, K+ ions, thus helping to maintain normal electrochemical and ionic gradients across the cell membrane. Its inhibition also reduced D-aspartate transport and could impact on the cytoplasm-to-membrane traffic of GLAST molecules. Furthermore, rottlerin decreased the activity of Na+/K+-ATPase by acting as a mitochondrial inhibitor. The present study has focused on the inhibition of Na+/K+-ATPase activity by rottlerin, ouabain and digoxin in homogenates prepared from rat kidney and cultured astrocytes. The activity of Na+/K+-ATPase was measured by the absorption of inorganic phosphate product generated from the hydrolysis of ATP and the fluorescent transition of the dye RH421 induced by the movement of Na+/K+-ATPase. This approach has a potential to test whether the rottlerin effect on Na+/K+-ATPase is a direct inhibition of the enzyme activity. Rottlerin has been found to block the activity of Na+/K+-ATPase in a dose-dependent manner in both rat kidney and astrocyte homogenates. Therefore, rottlerin inhibited the activity of Na+/K+-ATPase directly in a cell-free preparation, thus strongly indicating that the effect was direct on the enzyme. In parallel experiments, ouabain and digoxin produced similar inhibitions of Na+/K+-ATPase activity in rat kidney while digoxin blocked the activity of Na+/K+-ATPase to a greater extent than ouabain in rat cortical cultured astrocytes. In a separate set of experiments, Na+/K+-ATPase in the astrocytic membrane was found to be unsaturated in E1(Na+)3 conformation in the presence of Na+ ions and this could explain the differences between the effects of digoxin and ouabain on the activity of Na+/K+-ATPase in rat astrocytes. In addition, it was found that at low concentrations of rottlerin, the activity of Na+/K+-ATPase was increased rather than inhibited. This effect was further investigated by studying rottlerin interactions with membrane lipids. The activity of Na+/K+-ATPase has been reported to be regulated by membrane lipids. The enzyme activity can be enhanced by increasing fluidity of the lipid membrane. I have, therefore, proposed that rottlerin binds to the membrane lipids and the effects of rottlerin on Na+/K+-ATPase are mediated by changes in the properties (fluidity) of the membrane. The hypothesis was tested by comparing rottlerin and a detergent, DOC (sodium deoxycholate), for their binding to the lipids by using a DMPC (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine) monolayer technique. DOC has been shown to both increase and inhibit activity of Na+/K+-ATPase in a manner similar to that displayed by rottlerin. The effects of rottlerin and DOC on the DMPC monolayers were studied by measuring the surface pressure of DMPC monolayers and surface area per DMPC molecule. I established that both rottlerin and DOC decreased the surface pressure of DMPC monolayers and increased the surface area per DMPC molecule. This indicates that both rottlerin and DOC penetrated into the DMPC monolayers. If rottlerin can interact with the lipids, changes in fluidity of the lipid membrane cannot be ruled out and should be considered as a possible factor contributing to the effects of rottlerin on the activity of Na+/K+-ATPase. Overall, the study demonstrates that rottlerin is not only a PKC-delta inhibitor but can have additional effects, both on the enzyme activities (Na+/K+-ATPase) and/or on lipid-containing biological structures such as membranes. The findings have implication not only for studies where rottlerin was used as a supposedly specific PKC-delta inhibitor but also for mechanisms of its toxicity.

<p>The symptoms of Parkinson's disease occur due to degeneration of dopamine neurons in substantia nigra. It has been demonstrated that glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor when it comes to protect and enhance survival of dopamine neurons in animal models of Parkinson's disease. The aim of this study was to evaluate short- and long-term effects of GDNF on survival and nerve fiber outgrowth of dopamine cells and astrocytic migration in mouse fetal ventral mesencephalic (VM) tissue. Primary tissue cultures were made of mouse fetal VM tissue and evaluated at 7 and 21 days in vitro (DIV) in terms of dopaminergic nerve fiber outgrowth and astrocytic migration when developed with GDNF present, partially, or completely absent. The results revealed that VM tissue cultured in the absence of GDNF did not exhibit any significant differences in migration of astrocytes or dopaminergic nerve fiber outgrowth neither after 7 DIV nor after 21 DIV, when compared with tissue cultured with GDNF present. Migration of astrocytes and dopaminergic nerve fiber outgrowth reached longer distances when tissue was left to develop for 21 DIV in comparison with 7 DIV. In order to study the long-term effects of GDNF, mouse fetal dopaminergic tissue was transplanted into the ventricles of adult mice and evaluated after 6 months. No surviving dopamine neurons were present in the absence of GDNF. In contrast dopamine neurons developed with GDNF did survive, indicating that GDNF is an essential neurotrophic factor when it comes to long-term dopamine cell survival. More cases have to be assessed in the future in order to strengthen the findings. Thus, transplanted dopamine neurons will be assessed after 3 and 12 months in order to map out when dopamine neurons deprived of GDNF undergo degeneration.</p>

<p>Stroke is the major cause of serious long-term disability with a sufficient acute treatment for only a very limited number of patients. Limited recovery of neurological functions occurs and can be elevated by a permissive post-stroke milieu. Housing animals in an enriched environment modulates regenerative mechanisms in the nonischemic peri-infarct area which might be an attractive target for pharmacological treatments to promote recovery.</p><p>Upon ischemia, cellular lipids are released due to massive cell damage and free lipids significantly contribute to the progression of acute and delayed cell death. The aim of this study was to evalute the effect of enriched environment on lipid metabolism. In particular we characterize the activation of the transcription factor liver X receptor (LXR) in glial scar formation and regulation of cholesterol balance of relevance for functional recovery following stroke.                                      Brain tissues from animals subjected to permanent occlusion of middle cerebral artery (pMCAo) were analysed for LXRα and β protein expression. We found an upregulation and an increased transcriptional activity of LXRβ in the peri-infarct area of rats housing in an enriched environment following pMCAO. Our data anticipate that enriched environment may have positive effects on lipid recycling in the ischemic hemisphere following experimental stroke.<strong></strong></p>

Graduation date: 1993