Parkinson’s disease (PD) is a devastating neurodegenerative disease and the second most prevalent after Alzheimer’s disease. The most characteristic hallmark of Parkinson’s is the presence of Lewy Bodies, clumps of aggregated α-synuclein protein, in the Substantia Nigra. While much has been said and theorized about α-synuclein, mitochondrial dysregulation in neurons of Parkinson’s patients is an equally important consideration due to the role that the mitochondria plays in supplying neurons with their energy needs through ATP. C. elegans is a non-vertebrate animal often used to study aging and neurodegenerative disease due to its simple, well characterized genome. This literature review aims to outline the genetic and some environmental factors that cause mitochondrial dysregulation leading to the progressive neurodegeneration witnessed in Parkinson’s, as modeled in C. elegans. Through a select review of studies done on C. elegans homolog of genes associated with mitochondrial function, this review aims to elucidate the mechanism by which each mutation not only causes the deficits seen in PD on its own but also how it interacts with other genes to worsen or alleviate symptoms. Ultimately, understanding these pathways and mechanism will be crucial to discovering and creating new therapeutic treatments and targets.

L-Dihydroxyphenylalanine (L-DOPA), the anti-parkinsonian drug affording the greatest symptomatic relief of parkinsonian symptoms, is still misunderstood in terms of its neurotoxic potential and the mechanism by which generated dopamine (DA) is able to exert an effect despite the absence of DA innervation of target sites in basal ganglia. This review summaries important aspects and new developments on these themes. On the basis of L-DOPA therapy in animal models of Parkinson's disease, it appears that L-DOPA is actually neuroprotective, not neurotoxic, as indicated by L-DOPAs reducing striatal tissue content of the reactive oxygen species, hydroxyl radical (HO•), and by leaving unaltered the extraneuronal in vivo microdialysate level of HO•. In addition, the potential beneficial anti-parkinsonian effect of L-DOPA is actually increased because of the fact that the basal ganglia are largely DA-denervated. That is, from in vivo microdialysis studies it can be clearly demonstrated that extraneuronal in vivo microdialysate DA levels are actually higher in the DA-denervated vs. the intact striatum of rats - owing to the absence of DA transporter (i.e., uptake sites) on the absent DA nerve terminal fibers in parkinsonian brain. In essence, there are fewer pumps removing DA from the extraneuronal pool. Finally, the undesired motor dyskinesias that commonly accompany long-term L-DOPA therapy, can be viewed as an outcome of L-DOPAs sensitizing DA receptors (D1-D5), an effect easily replicated by repeated DA agonist treatments (especially agonist of the D 2 class) in animals, even if the brain is not DA-denervated. The newest findings demonstrate that L-DOPA induces BDNF release from corticostriatal fibers, which in-turn enhances the expression of D3 receptors; and that this effect is associated with motor dyskinesias (and it is blocked by D3 antagonists). The recent evidence on mechanisms and effects of L-DOPA increases our understanding of this benefical anti-parkinsonian drug, and can lead to improvements in L-DOPA effects while providing avenues for reducing or eliminating L-DOPAs deleterious effects.

Orientador: Fabio Augusto Barbieri / Resumo: A locomoção e o controle postural são duas tarefas muito presentes no cotidiano e que impactam diretamente no nível de funcionalidade e na qualidade de vida. Sabemos que em pessoas com DP apresentam desordens nessas tarefas e que a medicação dopaminérgica melhora os parâmetros motores na locomoção e no controle postural em pessoas com DP. Entretanto, o tempo para o início da eficácia da medicação após sua ingestão na locomoção e no controle postural em pessoas com DP, especialmente em tarefas desafiadoras, ainda é controverso, assim como a duração desta eficácia. Assim, o objetivo geral desta dissertação é analisar o comportamento da ação da medicação dopaminérgica na locomoção e no controle postural em pessoas com DP. Para isso foram desenvolvidos dois estudos, no primeiro estudo, foi avaliado o comportamento temporal da medicação dopaminérgica no andar e no segundo estudo, no controle postural. Participaram dos estudos 15 pessoas com DP (sendo 6 do gênero feminino; 69 ±7 anos; H&Y 1 - 3; UPDRS-III 27,1 ± 12), avaliadas em dois dias distintos. No primeiro dia foram realizadas avaliações clínicas dos sintomas motores e do equilíbrio estático e dinâmico No segundo dia foi avaliado o andar e o controle postural. Os participantes foram avaliados no estado “OFF” da medicação, logo após a ingestão da medicação e a cada 30 minutos, até o período de três horas após a ingestão da medicação. Para responder aos objetivos dos estudos, os dados referentes a locomoção e ao controle postur... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Gait and postural control are two tasks that are present in daily life and that directly affects the level of functionality and quality of life. We know that in people with PD have disorders in these tasks and that dopaminergic medication improves motor parameters in gait and postural control. However, the time to onset of medication efficacy after ingestion in gait and postural control in people with PD, especially in challenging tasks, is still controversial, as is the duration of this efficacy. Therefore, the general objective of this dissertation is to analyze the behavior of the action of dopaminergic medication on gait and postural control in people with PD. For this, two studies were developed, in the first study, we evaluated the temporal behavior of dopaminergic medication in gait and in the second study, in postural control. Participated in the study 15 people with PD (6 females; 69 ± 7 years; H&Y 1 - 3; UPDRS-III 27.1 ± 12), evaluated on two different days. In the first, he performed clinical evaluations of motor symptoms and static and dynamic balance. On the second day, we evaluated walking and postural control. Participants have evaluated the “OFF” state of medication, immediately after the ingestion of medication and every 30 minutes, up to three hours after the ingestion of medication. In order to meet the objectives of the studies, the data related to gait and postural control will be compared by ANOVAs with factors according to the independent variables of e... (Complete abstract click electronic access below) / Mestre

Human neurosphere stem cells offer promising potential for the treatment of neurodegenerative diseases. Their well characterized multi-potency of differentiating into neurons, astrocytes, and oligodendrocytes when exposed to the optimum exogenous growth factors make them an exciting area of study (38). Finding novel endogenous methods of modulating stem cell metabolism will allow for the safer treatment of various brain disorders (34). In this experiment, melatonin, N-acetylserotonin, L-tryptophan, and L-DOPA are added in three different concentrations to neurospheres suspended in HNSC/GBM media with less than optimal concentrations of exogenous epidermal growth factor (EGF) and fibroblast growth factor (FGF). The alamarBlue assay (resazurin) was chosen as the most suitable assay for measuring neurosphere metabolism. Metabolic neural stem cells would cause the greatest reduction of the oxidized alamarBlue reagent (resazurin?resorufin), which was detected by a fluorescent plate reader (39-41). The percent reduction in alamarBlue was calculated for all four molecules at three different concentrations and compared to controls without any molecule. Our results illustrate that there was no statistically significant difference at p<0.05 between the biological molecules and the control group except for two exceptions (labeled with asterisks on figures 3 and 5) L-DOPA at a 40 micromolar concentration after 4 hours of incubation and melatonin at a 40 micromolar concentration after 52 hours of incubation.

Part A of two Project Reports; Part B can be found at http://hdl.handle.net/11375/17660 / <p> The recent development of a technique for the synthesis of the molecule 5-(¹⁸F) fluoro-dopa has opened a vast field of research into the study of the intracerebral metabolism. In order to take full advantage of this new tracer compound it will be necessary to use an imaging system which will be capable of providing three dimensional information concerning the rapid changes in activity as the ¹⁸F travels through the brain. This report describes the results os investigations into a possible design for a device which would be capable of this type of dynamic imaging. The design in question employs multiwire proportional chambers (MWPC) as detectors of the .511 MeV gamma rays which result from the annihilation of the positron emitted in the decay of ¹⁸F. </p> <p> For comparison purposes, a breif review of various other types of positron tomographic systems which are presently in use or under development is presented. This review emphasises the resolution, data aquisition speed, and instalation cost for each of these designs. </p> <p> In order to eliminate the need for costly research into the various design aspects of multiwire proportional chambers for use with positron annihilation radiation, the design presented here relies exclusively on methods and designs developed and proven feasible by other groups. These are incorporated into a system to suit the present needs. </p> <p> The design presented uses two pairs of 50x50 cm² MWPC's at 180° to each other and separated by approximately 50 cm. The chambers utilize electromagnetic delay-line readout techniques for the anode and wound bi-filar cathode planes. They are filled with a "magic gas" mixture at a slightly positive pressure. The effeciency of the chambers for .511 MeV photons is increased by employing "sandwich" type converters. Signals from the detectors are transfered to a small computer where they are stored for later tomographic reconstruction off-line. </p> <p> On the basis of the working designs from which the present design has been drawn, one would expect the positron camera to have a sensitivity of better than 1000 counts/second-microcurie. The total costs of development and construction leading up to a working device suitable for clinical use, not including the cost of a dedicated computer, are estimated to be less than seventy five thousand dollars. </p> / Thesis / Master of Engineering (ME)

No description available.

Mes travaux de thèse concernent le rôle du récepteur D1 de la dopamine dans les dyskinésies L-dopa induites, effets secondaires extrêmement handicapants du traitement de la maladie de Parkinson. En condition de dénervation striatale mimant l’environnement de la maladie de Parkinson, le traitement chronique par la L-dopa entraine des altérations majeures du trafic intraneuronal et de la signalisation du récepteur D1 de la dopamine dans les principaux neurones cibles de la dopamine, les neurones épineux de taille moyenne du striatum. Il existe en particulier une hypersensibilisation des récepteurs D1 dans les neurones striataux, avec une abondance accrue à la membrane plasmique et une diminution du niveau d’expression de la protéine GRK6 (Protéine kinase des Récepteurs Couplés aux Protéines G 6), un des acteurs clefs des phénomènes de désensibilisation, en relation directe avec l’apparition des dyskinésies.C’est dans ce contexte que se situe mon travail de thèse qui a eu pour objectif de mettre à profit et/ou de développer différents modèles expérimentaux et outils « in vivo » et « in vitro ». Nous avons associé des techniques d’imagerie cellulaire et tissulaire à des approches comportementales, afin d’explorer certains des événements cellulaires et moléculaires à l’échelle du neurone striatal et des réseaux neuronaux, reliant le niveau d’expression du récepteur D1, sa compartimentation cellulaire, son trafic intraneuronal et les dyskinésies ou des conditions pharmacologiques équivalentes.Nous avons confirmé dans le modèle du rat lésé unilatéralement à la 6-OHDA, traité par la L-dopa et développant des mouvements anormaux analogues aux dyskinésies chez l’homme, que le récepteur D1 est anormalement abondant à la membrane plasmique des neurones du striatum, alors qu’il devrait être internalisé après stimulation par son ligand naturel, la dopamine. Nous avons mis en évidence que les mécanismes d’internalisation après stimulation par un agoniste restent néanmoins fonctionnels. Après administration de l’agoniste D1, chez les animaux dyskinétiques, l’abondance des récepteurs D1 augmente dans les compartiments notamment impliqués dans les mécanismes d’internalisation et de transport (vésicules) et de dégradation (corps multivésiculaires). Nous avons apporté une explication possible à cette abondance anormale et à ce défaut d’internalisation, en montrant qu’ils pourraient être dus à une hétérodimérisation entre les récepteurs D1 et D3. La co-activation des récepteurs D1 et D3 par la L-dopa favoriserait l’ancrage du récepteur D1 à la membrane plasmique des neurones striataux.Dans ce cadre, l’abord de l’étude de l’implication du protéasome dans la régulation de l’expression du récepteur D1 de la dopamine nous a semblé particulièrement important, sur la base des premières études soulignant l’implication de ce système catalytique dans le contrôle de l’activité et du métabolisme des récepteurs aux neurotransmetteurs. Nous avons révélé pour la première fois des liens entre l’activité catalytique du protéasome et la dynamique intraneuronale du récepteur D1 et plus particulièrement nous avons montré que son activité chymotrypsine-like est réduite de façon spécifique dans le striatum d’animaux dyskinétiques, comme une conséquence directe d’une déplétion en dopamine associée à une hyperstimulation dopaminergique.Nous avons testé en situation expérimentale une stratégie « thérapeutique » nouvelle en restaurant le mécanisme de désensibilisation homologue du récepteur D1 de la dopamine, par correction du déficit de la kinase GRK6 par transfert du gène correspondant via l’injection intrastriatale d’un vecteur lentiviral. Nous avons montré que cette approche permet de réduire considérablement la sévérité des dyskinésies dans les modèles rat et primate non-humain, analogues des dyskinésies chez l’homme et qu’elle restaure les effets thérapeutiques de la L-dopa. Ces effets sont la conséquence de la restauration des mécanismes de désensibilisation homologue : la surexpression de GRK6 entraîne l’internalisation spécifique des récepteurs D1. L’ensemble de nos résultats s’inscrit dans une démarche de recherche translationnelle menée depuis plusieurs années au laboratoire allant de la cellule au patient, avec pour but de transposer la compréhension des données expérimentales concernant les anomalies de l’expression du récepteur D1 de la dopamine en stratégies thérapeutiques dans les dyskinésies L-dopa induites. Nos investigations montrent qu’il est possible d’agir sur l’expression du récepteur D1 à la membrane plasmique des neurones striataux de manière indirecte, en manipulant trois co-activateurs de son métabolisme, pour espérer réduire « in fine » la sévérité des dyskinésies. / In my thesis work, I studied the role of dopamine D1 receptor in L-dopa induced dyskinesia, a debilitating complication of Parkinson's disease’s treatment. In condition of striatal denervation, that mimics the Parkinson's disease environment, chronic treatment with L-dopa leads to major alterations of intraneuronal trafficking and dopamine D1 receptor signaling in the major target of dopamine neurons, the striatal medium spiny neurons. In particularly, there is a D1 receptor hypersensitivity in striatal neurons, with an increased abundance of D1 receptor at the plasma membrane and a decreased level of GRK6 protein expression, a key actor in desensitization mechanism, directly related with the apparition of dyskinesia.In this context, I used different in vitro and in vivo experimental models and tools. I have associated cell and tissue imaging techniques and behavioural approaches in order to explore cellular and molecular events in striatal neuron and neuronal networks, linking the D1 receptor expression level, its cellular compartmentalization, its intraneuronal trafficking and the dyskinesia behaviour or equivalent pharmacological conditions.We confirmed in the rat analog of L-dopa-induced dyskinesia, i.e., the L-dopa-induced abnormal involuntary movements in unilaterally 6-hydroxydopamine (6-OHDA)-lesioned animals, that D1 receptor is abnormally abundant in the plasma membrane of neurons in the striatum, whereas it should be internalized after stimulation by its natural ligand, the dopamine. We showed that nevertheless the internalization mechanisms after agonist stimulation remains functional. After D1 agonist administration in dyskinetic animals, D1 receptor abundance increases in the cytoplasmic compartments involved in the internalization and transport (vesicles) and degradation (multivesicular bodies) mechanisms. Based on D3 receptor antagonist experiment, we propose that this abnormal abundance and this lack of internalization could be due to heterodimerization between the D1 and D3 receptors. D1 and D3 receptors co-activation by L-dopa might anchor D1 receptor at the plasma membrane of striatal neurons.In this context, analysis of proteasome involvement in the regulation of dopamine D1 receptor expression seemed particularly important, on the basis of the first studies underlying proteasome involvement in the activity and metabolism of neurotransmitter receptors. We demonstrated for the first time links between the proteasomal catalytic activity and D1 receptor intraneuronal dynamics and more particularly we showed that the proteasome chymotrypsin-like activity is reduced specifically in the striatum of dyskinetic animals, as a direct consequence of dopamine depletion associated with dopaminergic hyperstimulation.We tested in experimental condition, a new "therapeutic"strategy in order to restore the dopamine D1 receptor homologous desensitization mechanism, correcting the GRK6 kinase deficit by gene transfer through the intrastriatal injection of a lentiviral vector. We showed that this approach reduces significantly the dyskinesia severity in rat and non-human primate models and restores the L-dopa therapeutic effects. These effects are a consequence of the homologous desensitization mechanisms restoration : indeed GRK6 overexpression provokes specific D1 receptor internalization.Our results are part of a translational research conducted over several years in the laboratory from cell to patient, in order to translate our increased understanding of D1 receptor function abnormalities into therapeutic strategies for L-dopa induced dyskinesia. Our investigations show that it is possible to act on D1 receptor expression at the plasma membrane of striatal neurons via various routes, all resulting into diminished dyskinesia severity.

Ce travail s’est focalisé sur l’implication, longtemps négligée, du complexe centremédian/parafasciculaire (CM/Pf) du thalamus dans le fonctionnement physiopathologique des ganglions de la base (GB), avec pour objectif principal d’évaluer le potentiel thérapeutique du ciblage de ce noyau dans le traitement chirurgical par stimulation cérébrale profonde (SCP) des mouvements anormaux. Notre étude a porté dans un premier temps sur un modèle de la maladie de Parkinson (MP) chez le rat. La SCP à haute fréquence (SHF) du CM/Pf réduit différents troubles parkinsoniens (akinésie et négligence sensorimotrice) ainsi que les dyskinésies L&#8208;Dopa induites. Cependant, la mise en évidence d’une action négative du traitement dopaminergique sur les effets anti&#8208;parkinsoniens de cette stimulation compromet l’intérêt de cette cible dans le cadre de la MP dont la L&#8208;Dopa reste le traitement de référence. Au niveau cellulaire, la SHF&#8208;CM/Pf interfère très largement avec les effets de la lésion dopaminergique dans le réseau des GB, confirmant la position clé de ce complexe dans la modulation de l’activité des GB. De plus, la comparaison des effets comportementaux et cellulaires de la SHF de cette cible avec celle du noyau subthalamique, cible actuellement privilégiée dans le traitement de la MP, montre des spécificités en relation notamment avec une action sélective de la manipulation du CM/Pf sur le noyau entopédonculaire (EP), homologue chez le rongeur du globus pallidus interne dont l’implication dans les dyskinésies est bien documentée. Notre étude électrophysiologique in vivo confirme la relation entre activité du Pf et manifestation des dyskinésies en mettant en évidence une réactivité du Pf au traitement chronique à la L&#8208;Dopa. Nous avons également montré que la lésion dopaminergique entraine une diminution de l’expression d’un marqueur métabolique de l’activité neuronale dans le Pf qui est complètement normalisée par la SHF, suggérant pour la première fois que la SHF pourrait corriger une diminution d’activité du noyau ciblé. Enfin, la relation étroite entre l’EP et le CM/Pf nous a encouragé à évaluer les effets de la SCP du Pf sur les dystonies sachant que celles&#8208;ci sont traitées par SCP du globus pallidus interne. Nous avons montré, sur le modèle de hamster dtsz, que l’induction des dystonies est favorisée par la SCP à basse fréquence du Pf alors qu’elle est retardée par la SHF appliquée de façon subchronique (9 jour), ce qui met en évidence l’implication du CM/Pf dans cette autre affection motrice liée aux GB. / The involvement of the centremedian/parafascicular (CM/Pf) thalamic complex has long been neglected in the pathophysiological functioning of basal ganglia (BG). However, this complex forms a functional loop with the BG suggesting that it could be a new target for the surgical treatment by deep brain stimulation (DBS) of BG&#8208;related disorders such as Parkinson's disease (PD). In this context, we evaluated the therapeutic potential of CM/Pf&#8208;DBS in a rat PD model. DBS at high frequency (HFS) of CM/Pf alleviates PD symptoms (akinesia, sensorimotor neglect) as well as L&#8208;Dopa&#8208;induced dyskinesias. However, our observation that chronic L&#8208;Dopa suppresses the antiparkinsonian benefits provided by CM/Pf&#8208;HFS compromises the interest of this target for PD. At cellular level, CM/Pf&#8208;HFS widely impacts the dopaminergic denervation&#8208;induced changes in the BG, showing that CM/Pf is a key node for modulating BG function. Comparison of the behavioral and cellular effects of HFS of CM/Pf versus subthalamic nucleus, the currently preferred target for PD treatment, led us to suggest that their differential impact on akinesia and dyskinesia may be due to a selective action of CM/Pf&#8208;HFS on entopeduncular nucleus (EP), the rodent homologous of internal globus pallidus, whose involvement in dyskinesia is already documented. In vivo electrophysiological recordings of CM/Pf neurons provided further support for the relationship between CM/Pf and dyskinesia. We also showed that the dopaminergic lesion resulted in a decreased gene expression of a metabolic marker of neuronal activity in the CM/Pf, which is completely normalized by HFS, providing the first evidence that HFS may be able to correct a decrease in activity of the targeted nucleus. Finally, given the close relationship between EP and CM/Pf and knowing that internal globus pallidus is a target for DBS in dystonia, we evaluated the effects of CM/Pf&#8208;DBS in an animal model of this disorder, the dtsz hamster. We found that the stress induction of dystonia in this model is delayed by subchronic CM/Pf HFS whereas it is favored by low frequency stimulation providing evidence forthe involvement of CM/Pf in another BG&#8208;related movement disorder.

No description available.

La mélanogenèse engage 3 enzymes. La tyrosinase catalyse l’oxydation des 2 premiers substrats pour former la DOPAquinone. Celle-ci, en présence de cystéine peut s’y conjuguer pour produire des pheomélanines (pigments clairs). En son absence, la DOPAquinone est convertie en DOPAchrome, précurseur de la voie de synthèse des eumélanines (pigments foncés). Leur formation implique deux autres enzymes : TRP-1 et TRP-2. Un dérèglement de ce processus entraîne des dermatoses de type hyperpigmentations chez les phototypes IV à VI. Traiter ces problématiques efficacement sans effets secondaires nécessite d'identifier de nouvelles molécules actives capables de réguler la synthèse des eumélanines.Pour atteindre l'objectif, nous avons évalué l’effet inhibiteur de 100 venins d’animaux sur l’activité DOPA oxydase de la tyrosinase de champignon. Des analyses HPLC et spectrophotométriques alternées, ont permis de localiser la sous-fraction active conte-nant la molécule d'intérêt. La détermination du poids moléculaire par spectrométrie de masse conjuguée à l’analyse d'AA après hydrolyse acide a permis d'identifier l’Argiotoxine-636 (ARGTX-636). Elle est un inhibiteur mixte de la diphénolase. Des études préliminaires de SAR ont également été réalisées. En parallèle, une méthode analytique par HPLC/MS a été mise au point pour confirmer l'activité inhibitrice de la DHICA oxydase de l'enzyme par l'ARGTX-636.Pour la première fois est reportée l’isolation et la caractérisation d’un nouveau composé extrait d’un venin d’araignée capable de réguler la mélanogenèse. Cette approche peut avoir un impact majeur dans la recherche de nouvelles molécules actives dans le domaine de la dermo-cosmétique. / Melanogenesis involves three enzymes. Tyrosinase catalyses the oxidation of the two first substrates and leads to the formation of DOPAquinone, which may be conjugated with cysteine to produce pheomelanins (red-yellow pigments). In absence of cys-teine, DOPAquinone is spontaneously converted in DOPAchrome, the precursor of eumelanin products (brown-black pig-ments). The formation of eumelanin molecules partially relies on the activity of two other enzymes called Tyrosinase related protein 2 and Tyrosinase related protein 1. A dysregulation of this process engenders hyperpigmentation tasks into skin IV to VI phototypes. To treat these issues effectively without side effects requires the identification of new active molecules able to regulate the eumelanin synthesis.To achieve the objective, we evaluated the inhibitory effect of 100 animals venoms, on the DOPA oxidase activity of mushroom tyrosinase. HPLC and spectrophotometric analysis alternating, we allowed us to localize the active sub-fraction containing the interest molecule. The molecular weight determination by mass spectrometry combined with the AA analysis after acid hy-drolysis identified argiotoxin-636 (ARGTX-636). It is a mixed-type inhibitor on diphenolase activity. SAR preliminary stu-dies have also been conducted. In parallel, an analytical method by HPLC / MS was developed to confirm the DHICA oxy-dase activity inhibitory effect by ARGTX-636.For the first time is reported the isolation and characterization of a novel compound extracted from a spider venom able to regulate melanogenesis process. This approach can have a major impact in the search for new active compounds in the dermo-cosmetics field.