INTRODUÇÃO: A doença de Parkinson é causada pela perda significativa de neurônios dopaminérgicos na substância nigra e perda celular no locus cerúleo, com perda do neurotransmissor dopamina e continuada deposição de inclusões proteicas nos tecidos cerebrais. A doença tem progressão caudo-rostral, iniciando-se no núcleo dorsal do nervo vago e, em grau menor, nos sistema olfativo, com evolução para o mesencéfalo e posteriormente para o prosencéfalo e neocórtex. Cerca de 90% dos casos são idiopáticos e o principal fator de risco é o envelhecimento. Para se compreender a interação genoma-ambiente e o mecanismo molecular nessa doença têm sido conduzidas investigações dos perfis de expressão gênica global em diversos tecidos-alvo. Essa abordagem de genômica funcional utiliza a tecnologia de DNA microarrays para estudo da expressão gênica e ferramentas de bioinformática para análise dos dados gerados. Neste trabalho foi feita uma análise das redes de interação transcricional em tecidos-alvo da doença de Parkinson utilizando-se amostras post mortem de tecidos cerebrais obtidas de pacientes e controles livres da doença. MÉTODOS: Estudo comparativo das redes de interação transcricional no núcleo dorsal do nervo vago, locus cerúleo e substância nigra entre pacientes com doença de Parkinson idiopática nos estágios Braak 4-5 e controles livres da doença utilizando material de necropsia. Foram utilizados DNA microarrays Agilent de 44 K e a análise estatística comparativa de dos transcritos válidos (TMEV) foi feita no vetor paciente X controle para cada região anatômica sob estudo. Para a análise das redes de interação transcricional dos grupos de pacientes e controles em cada região anatômica utilizou-se o software FunNet e as anotações genômicas do Gene Ontology Consortium. RESULTADOS: Os genes com maior número de ligações gene-gene em cada rede transcricional, ou hubs, foram identificados e correlacionados com sua função biológica e possível papel na doença de Parkinson. A análise comparativa entre o perfil de hubs (número de ligações, posição na rede) em cada região anatômica para pacientes e controles revelou que: i) no núcleo dorsal do nervo vago os hubs principais nas redes de controles e pacientes estão relacionados a funções de manutenção da homeostase cerebral e organização neuronal, ii) no locus cerúleo os hubs principais dos controles são genes ligados à manutenção das funções cerebrais, mobilização de células progenitoras (pericitos) e controle de vias inflamatórias, enquanto que na rede de pacientes esses hubs estão ligados aos processos de endo e exocitose, desenvolvimento neuronal e controle do estresse oxidativo e degradação de proteínas; iii) finalmente, na substância nigra os principais hubs da rede de controles estão envolvidos na proteção contra estresse oxidativo e proteínas não dobradas e na manutenção do sistema dopaminérgico mesodiencefálico, enquanto que na rede de pacientes predominam hubs ligados a processos epigenéticos de envelhecimento mitocondrial, transporte vesicular, neurogênese, inflamação e morte neuronal. CONCLUSÕES: Os resultados da análise de redes de interação transcricional de pacientes e controles em diferentes regiões anatômicas são compatíveis com o modelo de progressão caudo-rostral da doença de Parkinson e apontam para mecanismos compensatórios no núcleo dorsal do nervo vago e locus cerúleo / INTRODUCTION: Parkinson\'s disease is caused by a substantial loss of dopaminergic neurons in the substantia nigra and cell loss in locus coeruleus, concomitant loss of dopamine neurotransmitter and continuing deposition of protein within the brain as intracellular inclusions. The disease has a caudal-rostral progression, beginning in the dorsal nucleus of vagus nerve and, in a less extent, in the olfactory system, progressing to the midbrain and finally to the basal forebrain and the neocortex. About 90% of the cases are idiopathic and the main risk factor is ageing. In order to have a better understanding of the genome-environment interactions and of the molecular mechanisms involved in this disease, the investigation of global gene expression in different target tissues has been conducted. This functional genomic approach is based on DNA microarray technology and on the use of bioinformatics for analyzing the data. In the present work an analysis of transcriptional interaction networks in Parkinson\'s disease target tissues was conducted in post mortem cerebral tissue samples obtained from patients and disease-free controls. METHODS: Comparative study of transcriptional interaction networks in the dorsal nucleus of vagus nerve, locus coeruleus, and substantia nigra of idiopathic Parkinson\'s disease patients in Braak stages 4-5 and disease-free controls using post mortem tissue samples. Agilent 44 K DNA microarrays were used and the statistical comparative analysis of valid transcripts was accomplished (TMEV) in the vector patient X control for each anatomic region under study. In order to analyze the transcriptional interaction networks for patient and control groups in each anatomic region the FunNet software and the Gene Ontology genomic annotations were used. RESULTS: The genes with high number of gene-gene connections in each transcriptional network, or hubs, were identified and related to their biological function and putative role in Parkinson\'s disease. The comparative analysis between hub profiles (number of connections, position in the network) in each anatomic region for patients and controls revealed that: i) in the dorsal nucleus of vagus nerve the main hubs in patient and control networks are related to the maintenance of brain homeostasis and to neuronal organization; ii) in the locus coeruleus the main hubs of control network are related to the maintenance of brain functions, mobilization of progenitor cells (pericytes) and control of inflammatory pathways, whereas in the patient network the main hubs are linked to exo and endocytosis processes, neuronal development and control of oxidative stress and protein degradation; iii) finally, in the substantia nigra the main hubs in the control network are related to protection against oxidative stress and unfolded/misfolded proteins and in the maintenance of the midbrain dopaminergic system, whereas in the patient network the main hubs are related to epigenetic processes of mitochondrial ageing, vesicular transport, neurogenesis and inflammation and neuronal death. DISCUSSION: The results of transcriptional interaction networks analyses performed for patient and control groups in different anatomic regions are compatible with the caudal-rostral model of Parkinson\'s disease progression and point out to compensatory mechanisms acting in vagus nerve and locus coeruleus

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A hipóxia isquemia (HI) pré-natal é uma das principais causas de mortalidade e doenças neurológicas crônicas em neonatos, que podem apresentar déficits remanentes como: retardamento, paralisia cerebral, dificuldade de aprendizado ou epilepsia. Estes prejuízos, provavelmente, estão relacionados com o atraso no desenvolvimento neural, astrogliose e com a perda de neurônios e oligodendrócitos. Déficits funcionais e cognitivos estão associados à degeneração de vias dopaminérgicas e de estruturas hipocampais. A enzima tirosina hidroxilase (TH) é a enzima limitante na síntese de dopamina e seus níveis são alterados em eventos de HI. O óxido nítrico (NO) é um gás difusível que atua modulando diferentes sistemas, participando de eventos como plasticidade sináptica e neuromodulação no sistema nervoso central e é produzido em grandes quantidades em eventos de injúria e inflamação, como é o caso da HI. O presente estudo teve por objetivos avaliar, utilizando o modelo criado por Robinson e colaboradores em 2005, os efeitos da HI sobre o comportamento motor e avaliar o desenvolvimento de estruturas encefálicas relacionadas a este comportamento como a substância negra (SN) e o complexo hipocampal. A HI foi induzida a partir do clampeamento das artérias uterinas da rata grávida, por 45 minutos no décimo oitavo dia de gestação (grupo HI). Em um grupo de fêmeas a cirurgia foi realizada, mas não houve clampeamento das artérias (grupo SHAM). A avaliação do comportamento motor foi realizada com os testes ROTAROD e de campo aberto em animais de 45 dias. Os encéfalos foram processados histologicamente nas idades de P9, P16, P23 e P90, sendo então realizada imunohistoquímica para TH e histoquímica para NADPH diaforase (NADPH-d), para avaliação do NO. Nossos resultados demonstraram redução da imunorreatividade para a TH em corpos celulares na SN aos 16 dias no grupo HI e aumento na imunorreatividade das fibras na parte reticulada aos 23 dias, com a presença de corpos celulares imunorreativos nesta região no grupo HI. Demonstramos também aumento do número de células marcadas para NADPH-d no giro dentado nos animais HI, nas idades analisadas, assim como aumento na intensidade de reação no corno de Ammon (CA1 e CA3) aos 9 dias no grupo HI, e posterior redução nesta marcação aos 23 e 90dias neste mesmo grupo. Nos testes comportamentais, observamos diminuição da atividade motora no grupo HI com uma melhora do desempenho ao longo dos testes no ROTAROD, sem entretanto atingir o mesmo nível do grupo SHAM. Os animais HI não apresentaram maior nível de ansiedade em relação ao grupo SHAM, descartando a hipótese das alterações observadas nos testes de motricidade estarem relacionadas a fatores ansiogênicos. O modelo de clampeamento das artérias uterinas da fêmea se mostrou uma ferramenta importante no estudo das alterações decorrentes do evento de HI pré-natal, por produzir diversos resultados que são similares aos ocorridos em neonatos que passam por este evento. / Perinatal hypoxia-ischemia (HI) is one of the major causes of mortality and chronic neurological diseases in newborns that can show permanent effects such as mental retardation, cerebral palsy, learning difficulty and epilepsy. It is probable that these impairs may be related to a delay in the neural development, astrogliosis and to the death of neurons and oligodendrocytes. Cognitive and functional deficits are related to degeneration of dopaminergic pathways and hippocampus. The enzyme tyrosine hydroxylase (TH) is a limiting step in the dopamine synthesis and its levels are impaired in HI insults. Nitric oxide (NO) is a diffusible gas that acts by modulating different systems and participates in several phenomena such as synaptic plasticity and neuromodulation in the central nervous system and is produced in higher levels in events of injury and inflamation as in the case of HI. This study aimed to evaluate the effects of HI on the motor behavior and to evaluate the development of brain structures related to this behavior as the substantia nigra (SN) and the hippocampal complex, using the model developed by Robinson and colleagues in 2005. HI was induced by clamping the uterine arteries of pregnant rats, for 45 minutes, on the eighteenth day of gestation (group HI). In a group of females, the surgery was performed, but no clamping of the arteries (group SHAM) was made. Assessment of motor behavior was performed with the ROTAROD test and open field test in animals of 45 days (P45) of age. The brains were processed histologically at ages P9, P16, P23 and P90, and then submitted to immunohistochemistry for TH and NADPH diaphorase (NADPH-d) histochemistry for evaluation of NOS. Our results demonstrated an apparent decrease in TH immunoreactivity in cell bodies in the SN at P16 in the HI group and an increase in immunoreactivity of the fibers in the SN pars reticulata at P23 with the presence of TH immunoreactive cell bodies at this same region in the HI group. We also showed an increase in the number of NADPH-d stained cells in the dentate gyrus in the HI group, at all ages, as also an increase in the intensity of staining in the Ammons horn (CA1 and CA3) at P9 in the HI group and, after that, a decrease in this staining at P23 and P90 in this same group. In the behavioral tests we observed a decrease in the motor activity in the HI group with a partial recovery all over the several sessions in the ROTAROD test, however this group did not reach the same performance as the SHAM group. HI animals did not show a higher level of anxiety when compared to SHAM animals, ruling out the hypothesis that anxiogenic factors may be impairing the results in the motor behavior tests. Our results showed that the model of uterine arteries clamping could be an important tool in the study of the effects of perinatal HI, by producing several consequences that are very similar to the effects observed in newborn children who suffered an HI event.

INTRODUÇÃO: A doença de Parkinson é causada pela perda significativa de neurônios dopaminérgicos na substância nigra e perda celular no locus cerúleo, com perda do neurotransmissor dopamina e continuada deposição de inclusões proteicas nos tecidos cerebrais. A doença tem progressão caudo-rostral, iniciando-se no núcleo dorsal do nervo vago e, em grau menor, nos sistema olfativo, com evolução para o mesencéfalo e posteriormente para o prosencéfalo e neocórtex. Cerca de 90% dos casos são idiopáticos e o principal fator de risco é o envelhecimento. Para se compreender a interação genoma-ambiente e o mecanismo molecular nessa doença têm sido conduzidas investigações dos perfis de expressão gênica global em diversos tecidos-alvo. Essa abordagem de genômica funcional utiliza a tecnologia de DNA microarrays para estudo da expressão gênica e ferramentas de bioinformática para análise dos dados gerados. Neste trabalho foi feita uma análise das redes de interação transcricional em tecidos-alvo da doença de Parkinson utilizando-se amostras post mortem de tecidos cerebrais obtidas de pacientes e controles livres da doença. MÉTODOS: Estudo comparativo das redes de interação transcricional no núcleo dorsal do nervo vago, locus cerúleo e substância nigra entre pacientes com doença de Parkinson idiopática nos estágios Braak 4-5 e controles livres da doença utilizando material de necropsia. Foram utilizados DNA microarrays Agilent de 44 K e a análise estatística comparativa de dos transcritos válidos (TMEV) foi feita no vetor paciente X controle para cada região anatômica sob estudo. Para a análise das redes de interação transcricional dos grupos de pacientes e controles em cada região anatômica utilizou-se o software FunNet e as anotações genômicas do Gene Ontology Consortium. RESULTADOS: Os genes com maior número de ligações gene-gene em cada rede transcricional, ou hubs, foram identificados e correlacionados com sua função biológica e possível papel na doença de Parkinson. A análise comparativa entre o perfil de hubs (número de ligações, posição na rede) em cada região anatômica para pacientes e controles revelou que: i) no núcleo dorsal do nervo vago os hubs principais nas redes de controles e pacientes estão relacionados a funções de manutenção da homeostase cerebral e organização neuronal, ii) no locus cerúleo os hubs principais dos controles são genes ligados à manutenção das funções cerebrais, mobilização de células progenitoras (pericitos) e controle de vias inflamatórias, enquanto que na rede de pacientes esses hubs estão ligados aos processos de endo e exocitose, desenvolvimento neuronal e controle do estresse oxidativo e degradação de proteínas; iii) finalmente, na substância nigra os principais hubs da rede de controles estão envolvidos na proteção contra estresse oxidativo e proteínas não dobradas e na manutenção do sistema dopaminérgico mesodiencefálico, enquanto que na rede de pacientes predominam hubs ligados a processos epigenéticos de envelhecimento mitocondrial, transporte vesicular, neurogênese, inflamação e morte neuronal. CONCLUSÕES: Os resultados da análise de redes de interação transcricional de pacientes e controles em diferentes regiões anatômicas são compatíveis com o modelo de progressão caudo-rostral da doença de Parkinson e apontam para mecanismos compensatórios no núcleo dorsal do nervo vago e locus cerúleo / INTRODUCTION: Parkinson\'s disease is caused by a substantial loss of dopaminergic neurons in the substantia nigra and cell loss in locus coeruleus, concomitant loss of dopamine neurotransmitter and continuing deposition of protein within the brain as intracellular inclusions. The disease has a caudal-rostral progression, beginning in the dorsal nucleus of vagus nerve and, in a less extent, in the olfactory system, progressing to the midbrain and finally to the basal forebrain and the neocortex. About 90% of the cases are idiopathic and the main risk factor is ageing. In order to have a better understanding of the genome-environment interactions and of the molecular mechanisms involved in this disease, the investigation of global gene expression in different target tissues has been conducted. This functional genomic approach is based on DNA microarray technology and on the use of bioinformatics for analyzing the data. In the present work an analysis of transcriptional interaction networks in Parkinson\'s disease target tissues was conducted in post mortem cerebral tissue samples obtained from patients and disease-free controls. METHODS: Comparative study of transcriptional interaction networks in the dorsal nucleus of vagus nerve, locus coeruleus, and substantia nigra of idiopathic Parkinson\'s disease patients in Braak stages 4-5 and disease-free controls using post mortem tissue samples. Agilent 44 K DNA microarrays were used and the statistical comparative analysis of valid transcripts was accomplished (TMEV) in the vector patient X control for each anatomic region under study. In order to analyze the transcriptional interaction networks for patient and control groups in each anatomic region the FunNet software and the Gene Ontology genomic annotations were used. RESULTS: The genes with high number of gene-gene connections in each transcriptional network, or hubs, were identified and related to their biological function and putative role in Parkinson\'s disease. The comparative analysis between hub profiles (number of connections, position in the network) in each anatomic region for patients and controls revealed that: i) in the dorsal nucleus of vagus nerve the main hubs in patient and control networks are related to the maintenance of brain homeostasis and to neuronal organization; ii) in the locus coeruleus the main hubs of control network are related to the maintenance of brain functions, mobilization of progenitor cells (pericytes) and control of inflammatory pathways, whereas in the patient network the main hubs are linked to exo and endocytosis processes, neuronal development and control of oxidative stress and protein degradation; iii) finally, in the substantia nigra the main hubs in the control network are related to protection against oxidative stress and unfolded/misfolded proteins and in the maintenance of the midbrain dopaminergic system, whereas in the patient network the main hubs are related to epigenetic processes of mitochondrial ageing, vesicular transport, neurogenesis and inflammation and neuronal death. DISCUSSION: The results of transcriptional interaction networks analyses performed for patient and control groups in different anatomic regions are compatible with the caudal-rostral model of Parkinson\'s disease progression and point out to compensatory mechanisms acting in vagus nerve and locus coeruleus

Ce travail de thèse porte sur l'étude de la réaction microgliale et des interactions microglie/neurones dans un modèle murin de neurodégénérescence dopaminergique induit par l'injection de 6‐hydroxydopamine (6‐ OHDA). Dans ce modèle, nous décrivons tout d'abord les cinétiques d'activation microgliale, de perte neuronale et d'altérations comportementales en relation avec le déficit dopaminergique. Dans la substance noire lésée ont été observées une perte progressive des neurones dopaminergiques TH+ (Tyrosine Hydroxylase) ainsi qu'une activation microgliale précoce mais transitoire. Le rôle délétère de cette activation microgliale est fortement suggéré par la mise en évidence d'une protection partielle contre la toxicité induite par la 6‐OHDA dans des souris génétiquement modifiées DAP12 Knock‐In, dont la densité microgliale est constitutivement diminuée. Par ailleurs, nous avons identifié différents types de contacts intercellulaires entre les neurones et la microglie de la substance noire lésée. Ces interactions physiques sont matérialisées entre autres sous la forme de contacts intimes entre le corps cellulaire des cellules microgliales et le soma des neurones dopaminergiques. De façon intéressante, ce type d'interaction se met en place quelques jours avant le pic de mort neuronale et dans la grande majorité des cas, concerne des neurones présentant des signes morphologiques d'apoptose. Finalement, nous avons également identifié un nouveau type d'interaction physique entre neurones et microglie sous la forme de ramifications microgliales pénétrant le soma des neurones. Ces interactions s'apparentent aux "tunelling nanotubes" décrits dans la littérature et représentent un type particulier de ramifications microgliales perforantes que nous avons nommées "tunelling ramifications". La présence de vacuoles TH+ dans le cytoplasme de nombreuses cellules microgliales suggère que les ramifications microgliales pénétrantes sont le support d'un processus de microphagocytose ciblant le cytoplasme des neurones dopaminergiques. La fonction précise de ces interactions et les mécanismes moléculaires qui les suscitent restent à définir. Toutefois, ce travail de thèse apporte un ensemble de données originales sur le dialogue microglie/neurones dans un modèle murin de la maladie de Parkinson / This thesis work is aimed to study microglial reaction and microglia/neuron interactions in a murine model of dopaminergic neurodegeneration induced by the injection of 6‐hydroxydopamine (6‐OHDA). In this model, we first describe the kinetics of microglial activation, neuronal cell loss and behavioral alterations in relation with the dopaminergic defect. In the injured substantia nigra, we observed a progressive loss of TH+ (Tyrosine Hydroxylase ‐positive) dopaminergic neurons and an early but transient microglial activation. The deleterious role of microglial activation is strongly suggested by the observation of a partial neuroprotection against 6‐OHDA‐induced toxicity in genetically DAP12 Knock‐In mice, in which microglial cells are defective in regard to their number and function. In addition, we identified various types of cell‐tocell contacts between neurons and microglia in the injured substantia nigra. Such physical interactions were established between microglia and neuronal cell bodies several days before the peak of neuronal death and in the majority of cases in neurons showing morphological signs of apoptosis. Finally, we also identified a new type of physical interactions consisting in microglial ramifications penetrating the soma of TH+ neurons. These interactions present similarities with the so‐called « tunelling nanotubes » previously described in the literature and represent a particular type of penetrating microglial ramifications the we named "tunelling ramifications.". Interestingly, in the injured substantia nigra, the presence of TH+ vacuoles in the cytoplasm of numerous microglial cells strongly suggests that microglial ramifications support microphagocytosis targeted toward the cytoplasm of dopaminergic neurons. The precise function and molecular mechanisms of such unique interactions need to be further assessed. However, our work provides a set of original data that deepens our knowledge on the dialogue between microglia and neurons in a mouse model of Parkinson's disease

Ce travail de thèse décrit et analyse la réaction neuro-inflammatoire accompagnant la mort cellulaire neuronale dans un modèle murin de la maladie de Parkinson. Dans ce modèle, induit par l’injection intrastriatale d'un analogue toxique de la dopamine, la 6-hydroxydopamine (6-OHDA), nous décrivons les caractéristiques et la cinétique de l’activation microgliale, de l'infiltration lymphocytaire T, de la perte de neurones dopaminergiques TH+ (Tyrosine Hydroxylase) et des altérations du comportement moteur. Nos observations sont complétées par une étude neuropathologique de la substance noire chez des patients atteints de maladie de Parkinson. Les résultats montrent que, chez l'homme comme chez la souris, la mort de neurones dopaminergiques induit une infiltration T de faible intensité, limitée à la substance noire et s'accompagnant d'une activation microgliale. Dans un deuxième temps, nous analysons l'impact d'une déficience lymphocytaire T génétiquement déterminée sur les paramètres histologiques et fonctionnels caractérisant le modèle 6-OHDA. Nos résultats montrent que, comparées à des souris contrôles immunocompétentes, les souris immunodéficientes de souche Foxn1 KO, CD3 KO, NOD SCID ou RAG1 KO présentent toutes, à des degrés divers, une susceptibilité significativement accrue aux effets neurotoxiques de la 6-OHDA. L'aggravation observée de la perte neuronale s'accompagne d'une accentuation majeure des troubles du comportement moteur et de l'activation microgliale. Ce travail démontre l'importance de la neuro-inflammation et de l'immunité adaptative dans la physiopathologie du modèle 6-OHDA. Il suggère également que les LyT infiltrant la substance noire des patients atteints de maladie de Parkinson exercent un rôle inhibiteur sur l'activation microgliale et pourraient par ce mécanisme ralentir l'évolution de la perte neuronale dopaminergique. En résumé, ce travail de thèse apporte un ensemble de données originales sur les interactions entre LyT, microglie et neurones dopaminergiques dans le contexte de la maladie de Parkinson et du modèle murin 6-OHDA / This thesis work describes and analyzes the neuroinflammatory reaction that accompanies neuronal cell death in a murine model of Parkinson's disease. In this model, induced by the intrastriatal injection of 6-hydroxydopamine (6-OHDA), a toxic dopamine analog, we report on the main features and kinetics of microglial activation, T-cell infiltration, loss of TH+ (Tyrosine Hydroxylase) dopaminergic neurons and motor behavior alterations. We also assessed the presence of T-cells in the susbstantia nigra of Parkinson's disease patients and found that, as observed in the 6-OHDA murine model, the neuronal cell death of dopaminergic neurons triggers a low-grade T-cell infiltration that accompanies microglial activation. We then studied the impact of genetically-determined T-cell immunodeficiency on histological and functional outcomes in the 6-OHDA model. Our results show that, as compared to immunocompetent control mice, immunodeficient strains consisting in Foxn1 KO, CD3 KO, NOD SCID or RAG KO mice consistently presented, at varied levels, a highest susceptibility to 6-OHDA induced dopaminergic neurodegeneration. The observed accentuation of neuronal cell loss was accompanied by a marked increase of microglial activation and motor behavior alterations. Our work demonstrates the pathophysiological role of neuroinflammation and adaptative immunity in the 6-OHDA model. It also suggests that T-cells infiltrating the substantia nigra of Parkinson's disease patients dampen microglial activation and could, via this inhibitory effect, slow the progression of dopaminergic cell loss. Overall this thesis work provides original data on the interactions between T-cells, microglia and dopaminergic neurons in the context of Parkinson's disease and the murine 6-OHDA model

Ce travail porte sur l’étude fonctionnelle de deux gènes préférentiellement exprimés dans deux régions du cerveau touchées par des pathologies neurodégénératives : Capucine, un marqueur du striatum, structure qui dégénère au cours de la maladie de Huntington et Agpat4, un marqueur de l’aire tegmentaire ventrale et de la substance noire compacte, dont les neurones dopaminergiques sont sélectivement atteints lors de la maladie de Parkinson. Des lignées de souris invalidées pour ces gènes ont été générées au laboratoire et au cours de ma thèse j’ai procédé à leur caractérisation. L’expression striatale du gène de la Capucine étant significativement diminuée dans des modèles murins de la maladie de Huntington, nous avons souhaité évaluer son rôle éventuel dans la pathogenèse de cette maladie. Pour ce faire, nous avons examiné, dans le cadre d’une collaboration, l’effet du knock-out et de la surexpression du gène de la Capucine sur la vulnérabilité des neurones striataux à un fragment de la Huntingtine mutée dans un modèle murin de la maladie de Huntington. Les données montrent que la Capucine n’a pas d’effet significatif sur la toxicité du fragment de la Huntingtine mutée dans le modèle étudié.La protéine Agpat4 présente des homologies de séquence avec des acyltransférases impliquées dans le métabolisme des phosphoglycérides. J’ai réalisé des études d’expression par différentes techniques de biologie moléculaire qui montrent que le gène d’Agpat4 est exprimé dans la plupart des tissus catécholaminergiques. Pour déterminer l’activité endogène d’Agpat4 et son rôle physiologique dans les tissus où elle est exprimée, j’ai comparé le métabolome de tissus de souris invalidées pour le gène d’Agpat4 et sauvages par chromatographie en phase liquide couplée à la spectrométrie de masse. Mes résultats indiquent que l’invalidation du gène d’Agpat4 perturbe le métabolisme non seulement de différentes classes de lipides, notamment les lysophosphatidyléthanolamines, mais aussi celui des catécholamines. / This work concerns the functional study of two genes preferentially expressed in two brain regions affected by neurodegenerative diseases: Capucine, a marker of the striatum, a structure that degenerates in Huntington's disease and Agpat4, a marker of the ventral tegmental area and the substantia nigra pars compacta, whose dopaminergic neurons are selectively affected in Parkinson's disease. Mouse lines deficient for Capucine and Agpat4 have been generated in the laboratory and during my PhD thesis I carried out their characterization.As the striatal gene expression of Capucine is significantly reduced in mouse models of Huntington's disease, we wished to evaluate its possible role in the pathogenesis of this disease. In a collaborative work, we examined the effect of the knockout and overexpression of the Capucine gene on the vulnerability of striatal neurons to a mutant Huntingtin fragment in a mouse model of Huntington’s disease. The data show that Capucine has no significant effect on the toxicity of the mutant Huntingtin fragment in the considered model.The Agpat4 protein has sequence homologies with acyltransferases involved in the metabolism of phosphoglycerides. I conducted expression studies using different molecular biology techniques, which showed that the Agpat4 gene is expressed in most catecholaminergic tissues. To determine the endogenous activity of Agpat4 and its physiological role in the tissues where it is expressed, I compared the metabolomes of Agpat4-deficient and wild-type mice tissues by liquid chromatography coupled with mass spectrometry. My results indicate that Agpat4 deficiency alters not only the metabolism of different lipid classes, in particular lysophosphatidylethanolamines, but also the metabolism of catecholamines.

No description available.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, characterized pathologically by a marked loss of dopaminergic nigrostriatal neurons and clinically by disabling movement disorders. PD can be treated by inhibiting monoamine oxidase (MAO), specifically MAO–B, since this is a major enzyme involved in the catabolism of dopamine in the substantia nigra of the brain. Inhibition of MAO–B may conserve the dopamine supply in the brain and may therefore provide symptomatic relief for PD patients. Selegiline is an irreversible MAO–B inhibitor and is currently used for the treatment of PD. Irreversible inhibitors inactivate enzymes by forming stable covalent complexes. The process is not readily reversed either by removing the remainder of the free inhibitor or by increasing the substrate concentration. Even dilution or dialysis does not dissociate the enzyme inhibitor complex and restore enzyme activity. From a safety point of view it may therefore be more desirable to develop reversible inhibitors of MAO–B. In this study, caffeine was used as lead compound to design, synthesize and evaluate new reversible inhibitors of MAO–B. This study is based on the finding that C–8 substituted caffeine analogues are potent MAO inhibitors. For example, (E)–8–(3–chlorostyryl)caffeine (CSC) is an exceptionally potent competitive inhibitor of MAO–B with an enzyme–inhibitor dissociation constant (Ki value) of 128 nM. In this study caffeine was similarly conjugated at C–8 to various side–chains. The effect that these chosen side–chains had on the MAO–B inhibition activity of C–8 substituted caffeine analogues will then be evaluated. The caffeine analogues were also evaluated as human MAO–A inhibitors. For the purpose of this study, saturated C–8 side chains were selected with the goal of discovering new C–8 side chains that enhance the MAO–A and ?B inhibition potency of caffeine. As mentioned above, the styryl side chain is one example of a side chain that enhances the MAO–B inhibition potency of caffeine. Should a side chain with promising MAO inhibition activity be identified in this study, the inhibition potency will be further optimized in a future study by the addition of a variety of substituents to the C–8 side chain ring. For example, halogen substitution of (E)–8– styrylcaffeine enhances the MAO–B inhibition potency by up to 10 fold. The saturated side chains selected for the present study included the phenylethyl (1), phenylpropyl (2), phenylbutyl (3) and phenylpentyl (4) functional groups. Also included are the cyclohexylethyl (8), 3–oxo–3– phenylpropyl (5), 4–oxo–4–phenylbutyl (6) moieties. A test compound containing an unsaturated linker between C–8 of caffeine and the side chain ring, the phenylpropenyl analogue 7, was also included. This study is therefore an exploratory study to discover new C–8 moieties that are favorable for MAO– inhibition. All the target compounds were synthesized by reacting 1,3–dimethyl–5,6–diaminouracil with an appropriate carboxylic acid in the presence of a carbodiimide dehydrating agent. Following ring closure and methylation at C–7, the target inhibitors were obtained. Inhibition potencies were determined using recombinant human MAO–A and MAO–B as enzyme sources. The inhibitor potencies were expressed as IC50 values. The most potent MAO–B inhibitor was 8–(5– phenylpentyl)caffeine (4) with an IC50 value of 0.656 ?M. In contrast, all the other test inhibitors were moderately potent MAO–B inhibitors. In fact the next best MAO–B inhibitor, 8–(4– phenylbutyl)caffeine (3) was approximately 5 fold less potent than 4 with an IC50 value of 3.25 ?M. Since the 5–phenylpentyl moiety is the longest side chain evaluated in this study, this finding demonstrates that longer C–8 side chains are more favorable for MAO–B inhibition. Interestingly, compound 5 containing a cyclohexylethyl side chain (IC50 = 6.59 ?M) was approximately 4 fold more potent than the analogue containing the phenylethyl linker (1) (IC50 = 26.0 ?M). This suggests that a cyclohexyl ring in the C–8 side chain of caffeine may be more optimal for MAO–B inhibition and should be considered in future studies. The caffeine analogues containing the oxophenylalkyl side chains (5 and 6) were weak MAO–B inhibitors with IC50 values of 187 ?M and 46.9 ?M, respectively. This suggests that the presence of a carbonyl group in the C–8 side chain is not favorable for the MAO–B inhibition potency of caffeine. The unsaturated phenylpropenyl analogue 7 was also found to be a relatively weak MAO–B inhibitor with an IC50 value of 33.1 ?M. In contrast to the results obtained with MAO–B, the test caffeine analogues were all weak MAOA inhibitors. With the exception of compound 5, all of the analogues evaluated were selective inhibitors of MAO–B. The most potent MAO–B inhibitor, 8–(5–phenylpentyl)caffeine (4) was the most selective inhibitor, 48 fold more potent towards MAO–B than MAO–A. This study also shows that two selected analogues (5 and 3) bind reversibly to MAO–A and ?B, respectively, and that the mode of MAO–A and –B inhibition is competitive for these representative compounds. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, characterized pathologically by a marked loss of dopaminergic nigrostriatal neurons and clinically by disabling movement disorders. PD can be treated by inhibiting monoamine oxidase (MAO), specifically MAO–B, since this is a major enzyme involved in the catabolism of dopamine in the substantia nigra of the brain. Inhibition of MAO–B may conserve the dopamine supply in the brain and may therefore provide symptomatic relief for PD patients. Selegiline is an irreversible MAO–B inhibitor and is currently used for the treatment of PD. Irreversible inhibitors inactivate enzymes by forming stable covalent complexes. The process is not readily reversed either by removing the remainder of the free inhibitor or by increasing the substrate concentration. Even dilution or dialysis does not dissociate the enzyme inhibitor complex and restore enzyme activity. From a safety point of view it may therefore be more desirable to develop reversible inhibitors of MAO–B. In this study, caffeine was used as lead compound to design, synthesize and evaluate new reversible inhibitors of MAO–B. This study is based on the finding that C–8 substituted caffeine analogues are potent MAO inhibitors. For example, (E)–8–(3–chlorostyryl)caffeine (CSC) is an exceptionally potent competitive inhibitor of MAO–B with an enzyme–inhibitor dissociation constant (Ki value) of 128 nM. In this study caffeine was similarly conjugated at C–8 to various side–chains. The effect that these chosen side–chains had on the MAO–B inhibition activity of C–8 substituted caffeine analogues will then be evaluated. The caffeine analogues were also evaluated as human MAO–A inhibitors. For the purpose of this study, saturated C–8 side chains were selected with the goal of discovering new C–8 side chains that enhance the MAO–A and ?B inhibition potency of caffeine. As mentioned above, the styryl side chain is one example of a side chain that enhances the MAO–B inhibition potency of caffeine. Should a side chain with promising MAO inhibition activity be identified in this study, the inhibition potency will be further optimized in a future study by the addition of a variety of substituents to the C–8 side chain ring. For example, halogen substitution of (E)–8– styrylcaffeine enhances the MAO–B inhibition potency by up to 10 fold. The saturated side chains selected for the present study included the phenylethyl (1), phenylpropyl (2), phenylbutyl (3) and phenylpentyl (4) functional groups. Also included are the cyclohexylethyl (8), 3–oxo–3– phenylpropyl (5), 4–oxo–4–phenylbutyl (6) moieties. A test compound containing an unsaturated linker between C–8 of caffeine and the side chain ring, the phenylpropenyl analogue 7, was also included. This study is therefore an exploratory study to discover new C–8 moieties that are favorable for MAO– inhibition. All the target compounds were synthesized by reacting 1,3–dimethyl–5,6–diaminouracil with an appropriate carboxylic acid in the presence of a carbodiimide dehydrating agent. Following ring closure and methylation at C–7, the target inhibitors were obtained. Inhibition potencies were determined using recombinant human MAO–A and MAO–B as enzyme sources. The inhibitor potencies were expressed as IC50 values. The most potent MAO–B inhibitor was 8–(5– phenylpentyl)caffeine (4) with an IC50 value of 0.656 ?M. In contrast, all the other test inhibitors were moderately potent MAO–B inhibitors. In fact the next best MAO–B inhibitor, 8–(4– phenylbutyl)caffeine (3) was approximately 5 fold less potent than 4 with an IC50 value of 3.25 ?M. Since the 5–phenylpentyl moiety is the longest side chain evaluated in this study, this finding demonstrates that longer C–8 side chains are more favorable for MAO–B inhibition. Interestingly, compound 5 containing a cyclohexylethyl side chain (IC50 = 6.59 ?M) was approximately 4 fold more potent than the analogue containing the phenylethyl linker (1) (IC50 = 26.0 ?M). This suggests that a cyclohexyl ring in the C–8 side chain of caffeine may be more optimal for MAO–B inhibition and should be considered in future studies. The caffeine analogues containing the oxophenylalkyl side chains (5 and 6) were weak MAO–B inhibitors with IC50 values of 187 ?M and 46.9 ?M, respectively. This suggests that the presence of a carbonyl group in the C–8 side chain is not favorable for the MAO–B inhibition potency of caffeine. The unsaturated phenylpropenyl analogue 7 was also found to be a relatively weak MAO–B inhibitor with an IC50 value of 33.1 ?M. In contrast to the results obtained with MAO–B, the test caffeine analogues were all weak MAOA inhibitors. With the exception of compound 5, all of the analogues evaluated were selective inhibitors of MAO–B. The most potent MAO–B inhibitor, 8–(5–phenylpentyl)caffeine (4) was the most selective inhibitor, 48 fold more potent towards MAO–B than MAO–A. This study also shows that two selected analogues (5 and 3) bind reversibly to MAO–A and ?B, respectively, and that the mode of MAO–A and –B inhibition is competitive for these representative compounds. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.

The midbrain dopamine system is involved in many aspects of purposeful behaviour and, when compromised, can have devastating effects on movement and cognition as seen in diseases like Parkinson's. In the healthy brain, dopamine neurons are thought to play particularly important roles in learning by signalling errors in reward prediction. The objective of this thesis was to investigate the diversity in the functional properties of the midbrain dopamine system, and how this is altered through genetic variation of relevance to Parkinson's and development of cell phenotype. This objective was addressed with a combination of behavioural experiments, in vivo single-cell recording and labelling (both in anaesthetised as well as awake rodents), immunofluorescence labelling, retrograde tracing and stereology. In a first set of experiments, it was demonstrated that chronic as well as acute genetic challenges can alter the firing patterns of midbrain dopamine neurons. Using a novel bacterial artificial chromosome-transgenic rat model, it was shown that the R1441C mutation in human leucine-rich repeat kinase 2, which is linked to Parkinson's, leads to motor deficits and an age-dependent reduction in the in vivo firing variability and burst firing of substantia nigra pars compacta (SNc) dopamine neurons. These findings help reveal processes of early, pre-degenerative dysfunction in dopamine neurons in Parkinson's. Similar effects on firing variability and burst firing of SNc dopamine neurons were found in a mouse model with conditional knock- out of the transcription factors Forkhead box A1 and A2 (FoxA1/2) in midbrain dopamine neurons. These findings indicate that FoxA1/2 are not only crucial for the early development of dopamine neurons, but also their function in the mature brain. In a second set of experiments in wildtype mice, it was demonstrated that midbrain dopamine neurons (located in SNc and ventral tegmental area) show diverse expression of the molecular markers Calbindin, Calretinin, Aldh1a1, Sox6, Girk2, SatB1 and Otx2. It was found that selective expression of these markers is of use for discriminating between midbrain dopamine neurons that project to dorsal striatum or nucleus accumbens. To elucidate whether the diverse molecular marker expression would map onto dopamine neurons whose firing correlates with distinct behavioural events, midbrain dopamine neurons were recorded and labelled in head-fixed awake mice either exposed to neutral sensory stimuli or performing a classical conditioning paradigm. The population activity of midbrain dopamine neurons was not modulated by neutral sensory stimuli. Interestingly, fewer than 50% of identified dopamine neurons showed phasic firing increases following reward- predicting cue and/or reward delivery, despite the common assumption that most (if not all) midbrain dopamine neurons signal reward prediction errors. Instead, firing was modulated by other explanatory factors, such as licking, or showed no modulation during the task. Response types of midbrain dopamine neurons were not correlated with their anatomical location nor the selective or combinatorial expression of the markers Aldh1a1, Calbindin and Sox6. In conclusion, the first set of experiments identified how different genetic burdens can alter the in vivo firing of midbrain dopamine neurons, and provide new insights into how circuits can change in pathological or compensatory ways at early disease stages in Parkinson's. The second set of experiments revealed striking heterogeneity of midbrain dopamine neurons in the intact system, and established further a functional diversity in the response types of identified midbrain dopamine neurons that is only partially consistent with canonical reward prediction error signalling.